Horse Feeding and Management 2020 MDPI

Horse Feeding
and Management
Edited by
Markku Saastamoinen
Printed Edition of the Special Issue Published in Animals
www.mdpi.com/journal/animals
Horse Feeding and Management
Horse Feeding and Management
Special Issue Editor

Markku Saastamoinen
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

Markku Saastamoinen
Natural Resources Institute
Finland (LUKE)
Finland
Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland
This is a reprint of articles from the Special Issue published online in the open access journal Animals
(ISSN 2076-2615) from 2019 to 2020 (available at: https://www.mdpi.com/journal/animals/special
issues/horse feeding and management).
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c 2020 by the authors. Articles in this book are Open Access and distributed under the Creative
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Contents
About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface to ”Horse Feeding and Management” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Tayler L. Hansen, Elisabeth L. Chizek, Olivia K. Zugay, Jessica M. Miller, Jill M. Bobel,
Jessie W. Chouinard, Angie M. Adkin, Leigh Ann Skurupey and Lori K. Warren
Digestibility and Retention Time of Coastal Bermudagrass (Cynodon dactylon) Hay by Horses
Reprinted from: Animals 2019, 9, 1148, doi:10.3390/ani9121148 . . . . . . . . . . . . . . . . . . . . 1
Markku Saastamoinen and Susanna Särkijärvi

Effect of Linseed (Linum usitatissimum) Groats-Based Mixed Feed Supplements on Diet Nutrient
Digestibility and Blood Parameters of Horses
Reprinted from: Animals 2020, 10, 272, doi:10.3390/ani10020272 . . . . . . . . . . . . . . . . . . . 20
Markku Saastamoinen, Susanna Särkijärvi and Elisa Valtonen

The Effect of Diet Composition on the Digestibility and Fecal Excretion of Phosphorus in Horses:
A Potential Risk of P Leaching?
Joaquı́n Bull, Fernando Bas, Macarena Silva-Guzmán, Hope Helen Wentzel,

Juan Pablo Keim and Mónica Gandarillas
Characterization of Feeding, Sport Management, and Routine Care of the Chilean Corralero
Horse during Rodeo Season
Malin Connysson, Marie Rhodin and Anna Jansson

Effects of Horse Housing System on Energy Balance during Post-Exercise Recovery
Sara Ringmark, Anna Skarin and Anna Jansson

Impact of Year-Round Grazing by Horses on Pasture Nutrient Dynamics and the Correlation
with Pasture Nutrient Content and Fecal Nutrient Composition
Dominique-Marie Votion, Anne-Christine François, Caroline Kruse, Benoit Renaud,

Arnaud Farinelle, Marie-Catherine Bouquieaux, Christel Marcillaud-Pitel and Pascal Gustin
Answers to the Frequently Asked Questions Regarding Horse Feeding and Management
Practices to Reduce the Risk of Atypical Myopathy
Katrin M. Lindroth, Astrid Johansen, Viveca Båverud, Johan Dicksved, Jan Erik Lindberg
and Cecilia E. Müller
Differential Defecation of Solid and Liquid Phases in Horses—A Descriptive Survey
Katrina Merkies, Chloe Ready, Leanne Farkas and Abigail Hodder

Eye Blink Rates and Eyelid Twitches as a Non-Invasive Measure of Stress in the Domestic Horse
v
Béke Nivelle, Liesbeth Vermeulen, Sanne Van Beirendonck, Jos Van Thielen and
Bert Driessen
Horse Transport to Three South American Horse Slaughterhouses: A Descriptive Study
Nicoletta Miraglia, Elisabetta Salimei and Francesco Fantuz

Equine Milk Production and Valorization of Marginal Areas—A Review
Agata Rzekeć, Céline Vial and Geneviève Bigot

Green Assets of Equines in the European Context of the Ecological Transition of Agriculture
vi
About the Special Issue Editor
Markku Saastamoinen, Ph.D., Senior Scientist, graduated at the University of Helsinki in 1987
in Animal Science (animal nutrition and animal genetics) with a doctorial thesis ‘Genetic and
Environmental Parameter for Measures of Racing Performance in Standadrbred and Finnhorse
Trotters’ at the University of Helsinki in 1997. He is currently working as a senior scientist in Natural
Resources Institute Finland (LUKE) on several topics related to horses and horse production, and
as a lecturer in equine nutrition and management at the University of Helsinki. He has published
scientific publications on equine nutrition, genetics, management exercise physiology, and equine
economy. He has held several national and European positions in scientific and horse organizations
and committees and was president of the Equine Workshop on Equine Nutrition (EWEN) as well as a
secretary and vice president of the Horse Commission of the European Association of Animal Science
(EAAP).
vii
Preface to ”Horse Feeding and Management”
Horses perform variety of roles in our society, serving people in several ways. Thus, the links
between equine health and good dietary treatment must be recognized to increase our understanding
of the needs of the horse. Proper nutrition and feeding management are the main objectives to fulfil
the ethological and physiological needs and to ensure the well-being and performance of horses. They
influence the growth, reproduction, performance capacity, and health of the horse. Horse feeding
is challenging for many horse owners as well as trainers and breeders. Many horses suffer from
overweight as well as many diseases associated with nutrition. Other management issues, including
stable and environmental conditions and feeding systems, have a major impact on the health and
well-being of horses. Horses’ management also influences their environment. In addition, there are
many innovations in horse feeding and management. Large amounts of evidence- and science-based
knowledge support good nutrition and management practices in horse husbandry. It is important
to ensure these data are available to all stakeholders and people working in the horse industry.
Consequently, the objective of this Special Issue and book is to publish research papers dealing
with horse nutrition and management and the interrelations between management, nutrition, health,
wellbeing, and environment to strengthen the knowledge about nutrition and management of all
horse categories.
Markku Saastamoinen
ix
animals
Article
Digestibility and Retention Time of Coastal
Bermudagrass (Cynodon dactylon) Hay by Horses
Tayler L. Hansen, Elisabeth L. Chizek, Olivia K. Zugay, Jessica M. Miller, Jill M. Bobel,
Jessie W. Chouinard, Angie M. Adkin, Leigh Ann Skurupey and Lori K. Warren *
Department of Animal Sciences, University of Florida, Gainesville, FL 32611, USA;
[email protected] (T.L.H.); [email protected] (E.L.C.); ozugay@ufl.edu (O.K.Z.);
[email protected] (J.M.M.); jbrides2@ufl.edu (J.M.B.); jessie23@ufl.edu (J.W.C.);
aadkin@ufl.edu (A.M.A.); [email protected] (L.A.S.)
* Correspondence: lkwarren@ufl.edu
Received: 30 October 2019; Accepted: 13 December 2019; Published: 14 December 2019
Simple Summary: Longer retention of forages with increased fiber concentrations may be a
compensatory digestive strategy in horses. We investigated the digestive characteristics of
bermudagrass hay, a prominent warm-season grass in the southeast United States that has greater
fiber concentrations than other common forages fed to horses. The morphological structure and
photosynthetic pathway of warm-season grasses differ from cool-season grasses and legumes which
may have important impacts on equine digestion and digesta transit through the gastrointestinal
tract. The retention time of Coastal bermudagrass was longer than alfalfa or orchardgrass hay.
The digestibility of Coastal bermudagrass decreased with increasing maturity, but the fiber digestibility
of alfalfa and orchardgrass was similar to the earliest maturity of Coastal bermudagrass hay.
The chemical composition of the plant cell wall influences diet digestibility and is a major difference
between warm-season and cool-season forages. The increased retention time of Coastal bermudagrass
allows for microbial fermentation to occur longer, adapting to more difficult-to-digest plant cell walls
in warm-season forages. The decrease in diet digestibility when horses consume warm-season forages
can be reduced by feeding early maturity forage, by harvesting hay at an earlier stage of growth or
managing pastures in a vegetative state.
Abstract: Bermudagrass (Cynodon dactylon) and other warm-season grasses are known for their
increased fiber concentrations and reduced digestibility relative to cool-season grasses and legumes.
This study investigated the digestive characteristics and passage kinetics of three maturities of Coastal
bermudagrass hay. A 5 × 5 Latin square design experiment was used to compare the digestion
of five hays: alfalfa (Medicago sativa, ALF), orchardgrass (Dactylis glomerata, ORCH), and Coastal
bermudagrass harvested at 4 (CB 4), 6 (CB 6), and 8 weeks of regrowth (CB 8). Horses were fed
cobalt-ethylenediaminetetraacetic acid (Co-EDTA) and ytterbium (Yb) labeled neutral detergent fiber
(NDF) before an 84-h total fecal collection to determine digesta retention time. Dry matter digestibility
was greatest for ALF (62.1%) and least for CB 6 (36.0%) and CB 8 diets (36.8%, SEM = 2.1; p < 0.05).
Mean retention time was longer (p < 0.05) for Coastal bermudagrass (particulate 31.3 h, liquid 25.3 h)
compared with ORCH and ALF (28.0 h, SEM = 0.88 h; 20.7 h, SEM = 0.70 h). Further evaluation
of digesta passage kinetics through mathematical modeling indicated ALF had distinct parameters
compared to the other diets. Differences in digestive variables between forage types are likely a
consequence of fiber physiochemical properties, warranting further investigation on forage fiber and
digestive health.
Keywords: alfalfa; equine; fiber; forage maturity; mathematical modeling; mean retention time;
orchardgrass; rate of passage; warm-season grass
Animals 2019, 9, 1148; doi:10.3390/ani9121148 1 www.mdpi.com/journal/animals

Animals 2019, 9, 1148
1. Introduction
Bermudagrass (Cynodon dactylon) is one of the most prominent forages in the southeast United
States; however, some horse owners and equine professionals assume that bermudagrass, particularly
the Coastal variety, is a lower quality hay due to increased fiber concentrations. Furthermore, feeding
Coastal bermudagrass hay in this region has been implicated as a cause of ileocecal impaction in
horses [1]. The increased fiber concentrations of Coastal bermudagrass and fine, soft texture have been
hypothesized to contribute to impaction [2], but greater fiber concentration is a common characteristic
among warm-season grasses. Bermudagrass and other grasses common to subtropical and tropical
climates (e.g., bahiagrass, millet, sorghum) possess a series of anatomical and biochemical modifications
for C4 photosynthesis that distinguish them from C3 plants. The Kranz anatomy of C4 plants features
tightly bundled mesophyll cells that form a ring around bundle-sheath cells. The proximity of
mesophyll and bundle-sheath cells allows for carbon concentrating mechanisms in photosynthesis,
reducing photorespiration in C4 plants. Plants using C4 carbon fixation are more efficient than C3
carbon fixation in areas of drought, high temperatures, and low nutrient inputs [3]. However, C4
plants tend to a have lower nutritive value via greater fiber concentrations that can lead to decreased
animal performance [4].
Greater forage fiber concentrations have long been associated with decreased diet digestibility [5].
Forage digestibility by horses decreases by half a percentage unit for every one percentage unit increase
in NDF concentration [6]. Using equine fecal inoculum, Lowman et al. [7] reported that time to
reach total gas production took longer for oat (Avena sativa) straw and wheat (Triticum aestivum) straw
compared with alfalfa (Medicago sativa) hay and grass haylage. Furthermore, the specific type of dietary
fiber (insoluble vs. soluble) alters in vitro digestibility measurements [8]. Not only fiber concentration,
but the specific composition of hemicellulose, cellulose, and lignin in the plant cell may alter digestion
by horses.
The degradation of forage fiber in the equine gastrointestinal tract may be influenced by digesta
rate of passage (ROP); however, a consistent relationship between fiber concentration and digesta
mean retention time (MRT) has not been shown in horses. Low nutritional value forages have a
longer retention time than high-quality legumes [9], but no difference in MRT was observed when
horses were fed similar forage species differing in fiber concentration [10,11]. The influence of fiber
concentration on digesta MRT may be confounded by factors such as the level of intake and feed
particle size [12]. Furthermore, low-fermentable dietary fibers alter ROP through changes in digesta
viscosity in the small intestine [13]. Such changes may not be detectable in total tract mean retention
time (TTMRT) calculations.
Several mathematical models have been used to describe digesta passage in ruminants that
improve understanding of passage kinetics by estimating retention time in the rumen from fecal marker
excretion [14–16]. These models have been applied to equine fecal marker excretion with the hopes
of increasing the understanding of digesta ROP in horses [10,11,17–20]. The models described by
Dhanoa et al. [14] and Pond et al. [15] have been used most frequently to describe digesta passage
in horses. The Dhanoa et al. [14] model is a mechanistic model based on first order kinetics. Digesta
flows through an unspecified number of compartments with decreasing compartment retention
times [14]. In contrast, the stochastic model described by Pond et al. [15] increases the passage rate of an
age-dependent compartment to account for an increased probability of digesta leaving a compartment
based on previous residence time in the compartment. These models have not been compared with the
same data, due in part to the model equations failing to converge with experimental data collected
from horses. With more advanced computer applications, a thorough investigation of model fit can be
conducted while also exploring the effect of dietary characteristics on passage parameters in the horse.
We hypothesized that the greater hemicellulose concentration of Coastal bermudagrass would
alter digestive characteristics. The objective of this study was to compare the digestibility and MRT of
Coastal bermudagrass to alfalfa and orchardgrass (Dactylis glomerata) hays, which are other common
forages fed to horses. Mean retention time was measured using liquid and particulate phase external
2
Animals 2019, 9, 1148
markers, and fecal marker excretion was modeled using previously developed equations for marker
excretion by ruminants [14,15]. We hypothesized that the use of mathematical modeling would
provide a greater understanding of ROP variables than TTMRT alone. Differences in total tract MRT of
Coastal bermudagrass compared with alfalfa and orchardgrass hay indicate fiber chemical composition
alters digesta movement in the gastrointestinal tract of the horse. Longer digesta retention of Coastal
bermudagrass may be an important compensation strategy to maximize the available nutrients from
slowly degraded fibers in warm-season grasses.
2. Materials and Methods

All animal protocols were approved by the University of Florida Institutional Animal Care and
Use Committee (201509618) under the FASS Guide for the Care and Use of Agricultural Animals in
Research and Teaching [21]. This study took place from 1 July 2015 to 9 September 2015, in Gainesville,
FL, USA. The mean temperature was 26.3 ◦ C and relative humidity was 88.5% during the study period.
Five mature Quarter Horse geldings (8 ± 3 years, 552 ± 14 kg, BCS 6.0 ± 0.4 [22], mean ± SEM)
housed at the University of Florida’s Horse Teaching Unit in Gainesville, FL were used in this study.
Before the start of the study, horses were fed Coastal bermudagrass hay or kept in warm-season grass
pastures. Horses received routine vaccinations and anthelmintic treatment before entering the study.
Farrier care was maintained during the study according to standard operating procedures of the Unit.
During the study, horses were individually housed in 3.7 m × 3.7 m stalls bedded with wood shaving
and provided access to 7.4 m × 18.3 m outdoor, grass-free paddocks with sand footing for 3 h each day
for voluntary exercise.
Five hays (Table 1) were used to evaluate 5 forage-based diets (Table 2). Hay was fed at 1.6% body
weight (BW) (dry matter (DM) basis). Alfalfa (ALF) and orchardgrass (ORCH) hays were purchased
from a commercial hay dealer (Larson Farms; Ocala, FL). Coastal bermudagrass hays were harvested
in Alachua, FL at 4 weeks (CB 4), 6 weeks (CB 6), and 8 weeks (CB 8) of regrowth under similar
management conditions. The CB 4 and CB 6 were second cuttings, whereas the CB 8 was a first cutting.
Based on producer harvesting schedules and study timeline, 8 weeks of regrowth as a second cutting
was not feasible for this study. The orchardgrass hay had a high electrolyte concentration, therefore,
sodium chloride and potassium chloride were added to each diet to better balance electrolyte intake
between diets. Horses were fed a vitamin/mineral pellet (0.1 to 0.125% BW, DM basis) during the
evening meal to meet micronutrient requirements [23].
Diets were evaluated in a 5 × 5 Latin square design experiment. A standard 5 × 5 Latin square
was randomly selected from Fisher and Yates [24]. Horses were randomly assigned to different rows
and each period was considered a column. Each period lasted 14 days and consisted of a 10.5-day
restricted intake phase when the ration was split into two equal-sized meals fed at 0730 and 1930 h
(Table 2). On day 7, an 84-h total fecal collection that began during the evening meal was conducted
to determine diet digestibility and retention time. As part of a companion study [25], horses had ad
libitum access to hay for the remaining 3.5 days before the start of the next period.
Table 1. Nutrient composition of feedstuffs.
Coastal 4 Coastal 6 Coastal 8 Vit/Min Vit/Min

Nutrient a Alfalfa Orchardgrass
Weeks Weeks Weeks Suppl 1 b Suppl 2 c
DM, % 88.4 90.9 90.0 91.8 91.6 89.4 90.5
DE d , Mcal/kg 2.50 2.09 1.95 1.90 1.85 2.76 3.31
CP, % 23.2 11.5 18.5 12.7 12.6 15.3 37.2
NDF, % 37.7 57.2 67.5 70.9 73.3 43.3 16.9
ADF, % 29.5 42.0 32.7 34.7 35.1 25.9 8.4
ADL, % 8.3 2.8 4.6 5.0 6.0 n.m. n.m.
Starch, % 1.3 0.2 1.6 1.6 2.5 n.m. n.m.
ESC, % 5.9 9.6 4.3 4.4 4.4 n.m. n.m.
WSC, % 6.3 12.5 3.6 4.3 4.8 n.m. n.m.
3
Animals 2019, 9, 1148
Table 1. Cont.
Coastal 4 Coastal 6 Coastal 8 Vit/Min Vit/Min

Nutrient a Alfalfa Orchardgrass
Weeks Weeks Weeks Suppl 1 b Suppl 2 c
Ca, % 1.58 0.32 0.57 0.39 0.38 1.31 3.12
P, % 0.24 0.23 0.30 0.27 0.18 1.78 1.19
Na, % 0.067 0.44 0.067 0.023 0.12 0.23 0.40
K, % 2.33 2.12 1.58 1.74 0.82 1.03 1.60
Cl, % 0.93 1.56 0.45 0.33 0.17 0.60 0.75
uNDFom, % e 20.9 11.6 21.4 25.8 38.6 n.m. n.m.
aNutrient composition of forages analyzed by NIRS at Dairy One Inc. (Ithaca, NY, USA). b Gro-n-Win alfa (Buckeye
Nutrition, Dalton, OH, USA) analyzed by wet chemistry at Dairy One Inc. (Ithaca, NY, USA). c Equalizer (Seminole
Feed, Ocala, FL, USA) analyzed by wet chemistry at Dairy One Inc. (Ithaca, NY, USA). d Digestible energy calculated
according to Pagan [26]. e Undigestible NDF (ash-free) determined after 240-h in vitro incubation by Dairyland
Laboratories (Arcadia, WI, USA). All values are on a 100% DM basis except DM. n.m. not measured.
Table 2. Diet composition and nutrient intake of experimental diets 1 .
Item ALF ORCH CB 4 CB 6 CB 8

Ingredient, % DMI
Alfalfa 93.7
Orchardgrass 92.8
Coastal Bermuda, 4 weeks 91.8
Vit/Min Suppl 1 a 5.9
Vit/Min Suppl 2 b 7.2 7.2 7.2 7.1
Sodium Chloride 0.4 0.3 0.4 0.2
Potassium Chloride 0.7 0.6 1.3
Daily Intake
DM, % BW 1.71 1.73 1.74 1.74 1.75
DE c , Mcal/kg BW 0.043 0.038 0.035 0.35 0.034
CP, g/kg BW 3.87 2.31 3.43 2.50 2.48
NDF, g/kg BW 6.47 9.36 11.01 11.56 11.94
ADF, g/kg BW 4.98 6.83 5.34 5.65 5.72
Ca, mg/kg BW 265.9 90.23 130.2 101.4 99.82
P, mg/kg BW 56.2 51.69 62.89 58.09 43.69
K, mg/kg BW 383.1 359.2 332.8 348.4 261.2
Na, mg/kg BW 39.3 75.03 37.92 30.88 35.6
Cl, mg/kg BW 197.8 359.2 174.5 145.9 159.4
1 Abbreviations. ALF, alfalfa; ORCH, orchardgrass; CB 4, Coastal bermudagrass 4-weeks regrowth; CB 6, Coastal
bermudagrass 6-weeks regrowth; CB 8, Coastal bermudagrass 8-weeks regrowth. a Gro-n-Win alfa (Buckeye
Nutrition, Dalton, OH, USA). b Equalizer (Seminole Feeds, Ocala, FL, USA). c Digestible energy calculated according
to Pagan [26].
External markers were prepared and used to determine digesta MRT for each gelding. A lithium
salt of Co-EDTA was prepared according to the methods of Udén et al. [27] as a marker for the liquid
phase of digesta. For the particulate marker, Yb-acetate was bound to neutral detergent fiber residue
according to Ringler and Lawrence [28]. Bermudagrass hay was chopped by a hammer mill until
it passed through a 1.27-cm screen and then boiled in neutral detergent solution for 1 h (60 g of
bermudagrass hay per liter of neutral detergent solution). Neutral detergent fiber residue was labeled
at a concentration of 100 g of NDF residue/L of 0.007 M Yb solution (prepared by dissolving 2.96 g of
Yb (III) acetate tetrahydrate in 1 L of distilled water) [28]. The prepared Co-EDTA was 13.7% Co (DM
basis) and Yb-labeled NDF residue was 7304 mg Yb/kg DM.
On day 7 of each period, horses were fed 1.5 mg of each marker per kilogram BW with the evening
meal of vitamin/mineral pellets. Marker intake was monitored and spilled feed was immediately
returned to the feed bucket to ensure complete marker consumption. On average, horses consumed
the markers in 14.7 min (range 9 to 30 min).
4
Animals 2019, 9, 1148
Immediately before and during fecal collections, stalls were stripped of bedding and swept clean.
All voided feces were collected directly from the floor of rubber-matted stalls. In order to minimize
contamination of feces with hay, dirt, and other debris and to prevent the horse from stepping in the
feces, stalls were checked for fresh excreta every 15 min. Horses were removed from their stalls in 2 to
4-h intervals and temporarily placed in a stall bedded with pine shavings to allow horses to comfortably
urinate. If a horse urinated in their primary stall, urine was removed with a wet-dry vacuum. Horses
were hand-walked for two 15-min periods (06:00 and 20:00) each day during fecal collections.
Feces were compiled in 2-h intervals for the first 60-h following marker dosing and then in 4-h
intervals from 60 to 84 h post marker dosing. Excreted feces were weighed and homogenized after each
time interval with 10% of the feces retained for a 24-h composite sample and a 200-g subsample saved
for marker concentration determination. Feces collected the first 12 h post marker dosing were only
retained for marker concentration analysis, and feces collected from 12 h to 84 h post marker dosing
were used for both marker concentration analysis and 24-h composite samples. During fecal collections,
orts were collected prior to the next feeding. Orts were time-matched to 24-h fecal composites to
determine diet digestibility. Fecal samples were stored at −20 ◦ C until analysis.
Frozen fecal samples were thawed at 4 ◦ C for 48 h. Fecal samples, representative feed samples
from each total fecal collection, and orts were dried in a 60 ◦ C forced air oven until achieving a constant
weight. Samples were ground to pass a 1-mm screen using a Wiley Mill prior to laboratory analysis.
Twenty-four-hour fecal composite samples, representative feed samples, and orts were used
to determine DM, organic matter (OM), NDF, and ADF digestibility (DMD, OMD, NDFD, ADFD,
respectively). Samples were dried in triplicate at 60 ◦ C until a constant weight and then ashed at 600
◦ C for 8 h to calculate OM concentration. Fiber concentrations were sequentially determined using an
ANKOM 200 Fiber Analyzer [29]. Heat-stable α-amylase was used in the NDF analysis of all samples.
Digestibility was determined as ((Nutrient Intake − Nutrient Output)/Nutrient Intake × 100).
Marker concentrations were determined on fecal samples composited in 2- and 4-h intervals
following marker dosing. Fecal samples were dried in triplicate in a 60 ◦ C forced-air oven until a
constant weight to determine DM concentration. A 0.500 g subsample was weighed and placed into a
Teflon digestion vessel with 8 mL of 15.8 N nitric acid. Samples were sealed and digested for 15 min at
180 ◦ C using a microwave-assisted acid digestion procedure (Anton-Paar, Ashland, VA, USA). Samples
were allowed to cool and diluted to 25 mL. Samples were centrifuged at 1050× g for 15 min and the
supernatant collected for determination of marker concentrations using inductively coupled plasma
spectrometry (Perkin-Elmer, Inc., Shelton, CT, USA) [30,31]. The minimum element detection limit
was 0.1 mg/L. Marker recovery was calculated as (Marker Excreted/Marker Dosed × 100).
Total tract MRT was calculated arithmetically according to Blaxter et al. [32] and
Thielemans et al. [33]. Total tract MRT calculated according to Blaxter et al. [32] is

mt
MRT = i i (1)
mi
where mi = the amount of marker in the ith sample (g) and ti = time from dosage of the marker to
the middle of the ith sampling interval (h). The equation described by Thielemans et al. [33] uses the
concentration of the marker in the sample and MRT is calculated as

t C Δt
MRT = i i i (2)
Ci Δti
where ti = time from dosage of the marker to the middle of the ith sampling interval (h),
Ci = concentration of marker in the ith sample (mg/kg DM), and Δti = time interval between
the middle of the ith and ith − 1 sample (h).
5
Animals 2019, 9, 1148
Fecal marker excretion data were fit with compartment models described by Dhanoa et al. [14] and
Pond et al. [15]. The multicompartment model derived by Dhanoa et al. [14] is a mechanistic model
based on first order kinetics where marker concentration (mg/kg DM) of the feces can be modeled as
−Δt
Marker Concentration = Ae−k1 t e−(N−2)e (3)
where A is a scaling parameter, k1 = rate constant for the first compartment (h−1 ), t = time from marker
dosage (h), Δ = k2 − k1 where k2 is the rate constant for the second compartment (h−1 , assuming k2 >
k1 ), and N = the number of exponentially distributed compartments. The rate constants do not change
over time; therefore, the compartments are considered age-independent (the rate digesta leaves a
compartment is not influenced by past residence time). The exponentially distributed compartments
described by Dhanoa et al. [14] can represent multiple sub-compartments within a larger mixing
compartment. The two-compartment model featuring a γ-distribution described by Pond et al. [15]
was also fit to fecal marker excretion data (mg/kg DM) as
⎡ ⎤
⎢⎢
n
δi (λ1 t)n−i ⎥⎥⎥
Marker Concentration = C2 ⎢⎢⎣⎢δn e−k2 (t) − e−λ1 t ⎥⎥ (4)
(n − i)! ⎦
i=1
where C2 = the initial concentration in the second compartment if the marker dose had been
introduced into the compartment and instantaneously mixed, n = order of the γ-distribution in
the first compartment, k2 = rate parameter for exponentially distributed residence times (h−1 ), t = time
after dosing of marker (h), λ1 = rate parameter for γ-distributed residence times (h−1 ), and δ = λ1 /(λ1 −
k2 ). Time delay was incorporated into the Pond et al. [15] equation by substituting t for t-TT, where t is
the time from marker dosing (h) and TT is transit time. Six orders of γ-distribution were analyzed (n = 1,
2, 3, 4, 5, 6) to test the G1G1, G2G1, G3G1, G4G1, G5G1, and G6G1 model described by Pond et al. [15].
If marker residence time is exponentially distributed (n = 1) in a compartment, the compartment is
age-independent, indicating that the rate the marker leaves is not dependent on past residence time.
However, if the ROP of a marker in a compartment changes over time, the compartment is considered
age-dependent. Marker concentration in an age-dependent compartment can be modeled with a
γ-distribution of order 2 or greater. Increasing the order of the γ-distribution alters the shape of the
curve such that the emergence of marker from the compartment is slowed [15]. Curves were fit using
nonlinear least squares methods in MATLAB (Version R2015a, Mathworks, Natick, MA, USA) with
model parameter start values randomly assigned (Computer Code S1). Bounds for rate parameters
were set between 0 and 1.
Model parameters were used to determine total tract mean retention time (TTMRT) for each fitted
equation to fecal marker excretion. For the Dhanoa et al. [14] model, TTMRT (h) was calculated as
1 1
N−1
1
MRT = + + , k2 > k1 (5)
k1 k2 k2 + (i − 2)(k2 − k1 )
i=3
where k1 and k2 are rate parameters (h−1 ) of the first and second compartments and N is the number of

exponentially distributed compartments. The term N−1 1
i = 3 k2 +(i−2)(k2 −k1 ) is said to represent the transit
time (TT) of digesta markers or the time from dosing the marker to the first appearance of marker in
the collected sample. Total tract MRT (h) for the Pond et al. [15] model was calculated as
n 1
MRT = + + TT (6)
λ1 k2
where λ1 is the age-dependent compartment rate constant (h−1 ), k2 is the age-independent compartment
rate constant (h−1 ), n is the order of the γ-distribution, and TT is the transit time (h). The age-dependent
compartment MRT (CMRT1 ) was determined by n/λ1 and the age-independent compartment MRT
6
Animals 2019, 9, 1148
(CMRT2 ) was determined by 1/k2 . When n = 1, λ1 is replaced by k1 , and CMRT1 is an age-independent

compartment.
Unless otherwise noted, data are presented as means ± SEM. Data were checked for normality
using the Kolmogorov–Smirnov test and the Shapiro–Wilk test. Data were analyzed as a Latin square
design using a mixed model ANOVA in SAS (v 3.8 SAS Studio, Cary, NC, USA). Fixed effects included
dietary treatment and period, and the random effect was horse. The influence of feeding Coastal
bermudagrass (CB 4, CB 6, and CB 8) compared with other hays (alfalfa and orchardgrass) on digestive
variables was determined using contrasts. Statistically significant means were separated by Scheffe’s
method. Model derived TTMRT was compared to arithmetic calculations using both two one-sided
tests of equivalence and regression analysis. For equivalence testing, the acceptable difference was
10%. Statistical trends were defined as p < 0.1 and differences at p < 0.05.
3. Results
3.1. Diet Digestibility

One factor that affects digestibility measurements is feed refusal. Orts were collected during 26 of
the 75 daily measurements of intake, most frequently when horses were fed the CB 8 diet. The mean
weight of orts was 0.07 kg (DM basis). Ash concentration of hay orts ranged from 24.6% to 66.8%,
indicating contamination with sand from the environment. Thus, hay ort weight was corrected by
multiplying ort weight by the ratio of ort ash concentration to forage ash concentration. Orts were
analyzed for nutrient composition and subtracted from nutrient intake to correct for any feed not
consumed by the horses.
Differences in fecal excretion were related to variations in diet digestibility. Horses fed Coastal
bermudagrass hay diets defecated 1.4 times more frequently (p < 0.05) than when fed alfalfa hay
(Table 3). Horses fed CB 6 and CB 8 excreted more feces (p < 0.05) than horses consuming ALF, ORCH,
or CB 4. Dry matter and OM digestibilities were greatest (p < 0.05) for ALF, whereas a reduction in
DMD and OMD was observed when horses were fed CB 6 and CB 8. There was a 32.0% reduction
(p < 0.05) in NDFD and a 47.1% decrease (p < 0.05) in ADFD digestibility for the CB 6 and CB 8 diets
compared with the other diets.
Table 3. Fecal excretion and diet digestibility of five experimental diets 1 (n = 5).
Diet 2 Contrast 3
Variable ALF ORCH CB 4 CB 6 CB 8 SEM
p-Value p-Value
Defecation
10.0 c 11.5 b,c 14.1 a,b 15.3 a 14.0 a,b 0.5 <0.001 <0.001
Frequency, times/d
Fecal Excretion, kg
3.55 d 4.41 c 5.04 b 5.84 a 5.87 a 0.20 <0.001 <0.001
DM/d
Fecal DM, % 19.7 20.9 20.0 20.5 22.5 0.42 0.074 0.255
Urination
10.6 10.6 8.7 8.3 10.7 0.58 0.324 0.161
Frequency, times/d
Digestibility, %
DM 62.1 a 51.2 b 47.2 b 36.0 c 36.8 c 2.1 <0.001 <0.001
OM 63.1 a 52.3 b 46.8 c 37.3 d 37.6 d 2.1 <0.001 <0.001
NDF 43.1 a 42.4 a 46.2 a 31.1 b 31.8 b 1.7 <0.001 <0.001
ADF 40.2 a 39.8 a 39.8 a 23.9 b 24.3 b 1.9 <0.001 <0.001
bermudagrass 6-weeks regrowth; CB 8, Coastal bermudagrass 8-weeks regrowth; SEM, standard error of the mean.
2 Main effect of diet. 3 Contrast between Coastal bermudagrass (CB 4, CB 6, CB 8) and other diets (ALF, ORCH).
a,b,c,d Means with unlike superscripts differ (p < 0.05).
7
Animals 2019, 9, 1148
3.2. Fecal Marker Excretion
3.2.1. Marker Excretion and Recovery

Mean fecal marker excretion is presented in Figure 1. External marker concentrations were
detected in feces between 5 to 13 h after feeding horses external markers. Element concentrations
were below instrument detection limits by 60 h post marker dosing, thus, fecal samples were only
analyzed for marker concentrations to 72 h post marker dosing. A pulsatile pattern was observed in
some individual fecal marker excretion curves (Supplementary Figures S1–S5).

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Figure 1. Two period moving average of fecal marker excretion of (a) Yb and (b) Co after dosing
external markers (SEM = 12.4 and 13.2 mg/kg DM, respectively). Abbreviations. ALF, alfalfa; ORCH,
orchardgrass; CB 4, Coastal bermudagrass 4-weeks regrowth; CB 6, Coastal bermudagrass 6-weeks
regrowth; CB 8, Coastal bermudagrass 8-weeks regrowth.
Marker recovery ranged from 73.3 to 97.6% for Yb and 73.9 to 115% for Co. Particulate marker
recovery did not differ by diet (Table 4). Liquid marker recovery tended to differ among diets (p = 0.075)
with mean Co recovery greatest in the ORCH diet and lowest in the CB 4 diet. Particulate and liquid
marker recovery did not differ within a horse for each period.
Table 4. Particulate (Yb) and liquid (Co) marker recovery 1 (n = 5).
Diet 2 Contrast 3
Variable ALF ORCH CB 4 CB 6 CB 8 SEM
p-Value p-Value
Particulate, % 80.6 86.3 85.5 82.5 85.0 1.44 0.549 0.948
Liquid, % 85.5 95.1 79.4 80.3 82.6 2.17 0.075 0.025
2 Main effect of diet. 3 Contrast between Coastal bermudagrass (CB 4, CB 6, CB 8) and other diets (ALF, ORCH).
8
Animals 2019, 9, 1148
3.2.2. Modeling Fecal Marker Excretion

Seventy six percent of model equations fit fecal excretion data for each horse within a period
using initial parameter ranges and start values defined in the program code. When the model did
not converge using the code, model parameter ranges were adjusted using curve fitting software in
MATLAB to obtain an acceptable fit (as indicated by the R2 value being non-negative).
Mean model result from all data is depicted for all equations in Figure 2. Mean model fit, parameter
values, and retention time from fecal marker excretion of each observation are summarized in Table 5.
Model parameters were nonzero (p < 0.05) for 47% of the fitted equations. The scaling parameter
was less than 0 for the G1G1 model. As the order of the γ-distribution increased for the equations
described by Pond et al. [15], TT, CMRT2 , and TTMRT decreased, whereas CMRT1 increased. The root
mean square error (RMSE) ranged from 2.932 to 42.23 and 3.369 to 29.85 for particulate and liquid
fecal marker excretion, respectively (Table 5). The model described by Dhanoa et al. [14] had the
lowest RMSE and Akaike’s information criterion (AIC) for the particulate and liquid phases of digesta.
Among the six two-compartment γ-distributed equations described by Pond et al. [15], the G5G1
model best fit particulate marker excretion and the G4G1 equation best fit liquid marker excretion
based on AIC values (Table 5). Because the AIC values increased once the order 5 and order 4 γ-gamma
distributions were fit to the particulate and liquid marker excretion, fitting marker excretion to the
two-compartment model was terminated at the order 6 γ-gamma distribution.

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Figure 2. Model result derived from equations described by Dhanoa et al. [14] and Pond et al. [15]
applied to all experimental data of (a) Yb and (b) Co after dosing external markers. Abbreviations.
G1G1, first order two-compartment model; G2G1, second order two-compartment model; G3G1,
third order two-compartment model; G4G1, fourth order two-compartment model; G5G1, fifth order
two-compartment model; G6G1, sixth order two-compartment model according to Pond et al. [15].
9
Table 5. Model goodness of fit, parameters, and retention time of equations used to fit particulate (Yb) and liquid (Co) fecal marker excretion (diets combined,
N = 25; means ± SE).
Variable Dhanoa a G1G1 b G2G1 b G3G1 b G4G1 b G5G1 b G6G1 b

Particulate
RMSE 11.70 20.48 16.30 14.83 14.02 12.43 12.91
Animals 2019, 9, 1148
AIC 128.8 163.1 148.1 142.3 138.7 134.3 137.0

Models with Nonzero Rate
4 0 20 72 76 84 92
Parameters, %
A 2.68 × 1011 ± 2.65 × 1011
C, mg Yb/kg digesta −476 ± 26.6 636 ± 39.6 761 ± 49.6 856 ± 57.6 901 ± 56.3 916 ± 63.0
k1 , h−1 0.265 ± 0.017 0.133 ± 0.007
k2 , h−1 0.355 ± 0.011 0.142 ± 0.008 0.187 ± 0.006 0.223 ± 0.008 0.253 ± 0.008 0.271 ± 0.009 0.277± 0.012
λ1 , h−1 0.192 ± 0.007 0.235 ± 0.011 0.279 ± 0.014 0.321 ± 0.018 0.370 ± 0.024
N 48 ± 12.9
TT, h 25.8 ± 1.15 17.5 ± 0.71 15.6 ± 0.64 13.4 ± 0.69 11.9 ± 0.66 10.6 ± 0.62 9.50 ± 0.61
CMRT1 , h 4.20 ± 0.29 8.01 ± 0.40 10.7 ± 0.34 13.3 ± 0.47 15.0 ± 0.56 16.4 ± 0.63 17.4 ± 0.77
CMRT2 , h 2.89 ± 0.096 7.48 ± 0.33 5.46 ± 0.15 4.61 ± 0.17 4.07 ± 0.17 3.82 ± 0.18 3.87 ± 0.26
TTMRT, h 32.9 ± 1.00 33.0 ± 0.99 31.7 ± 0.93 31.3 ± 0.92 31.0 ± 0.90 30.8 ± 0.90 30.8 ± 0.88
Liquid
10
RMSE 13.88 17.51 15.57 14.95 14.04 12.43 14.01
AIC 141.1 154.6 147.2 145.0 142.6 143.0 143.5
Models with Nonzero Rate
16 0 52 76 80 84 84
Parameters, %
A 8.00 × 108 ± 4.33 × 108
C, mg Co/kg digesta −470 ± 26.1 527 ± 21.4 582 ± 33.7 630 ± 42.6 642 ± 47.6 639 ± 66.3
k1 , h−1 0.201 ± 0.020 0.202 ± 0.031
k2 , h−1 0.443 ± 0.045 0.144 ± 0.006 0.166 ± 0.008 0.184 ± 0.011 0.202 ± 0.015 0.206 ± 0.017 0.213 ± 0.020
λ1 , h−1 0.333 ± 0.055 0.430 ± 0.072 0.517 ± 0.086 0.593 ± 0.097 0.655 ± 0.105
N 8.34 × 104 ± 83191
TT, h 17.3 ± 1.27 11.8 ± 0.48 10.1 ± 0.41 8.49 ± 0.38 7.47 ± 0.39 6.32 ± 0.43 5.41 ± 0.46
CMRT1 , h 6.18 ± 0.53 6.20 ± 0.40 8.12 ± 0.64 9.81 ± 0.85 10.9 ± 0.97 12.0 ± 1.10 12.8 ± 1.17
CMRT2 , h 2.57 ± 0.15 7.23 ± 0.29 6.39 ± 0.34 6.09 ± 0.44 5.85 ± 0.51 5.86 ± 0.52 5.87 ± 0.55
TTMRT, h 26.0 ± 0.92 25.2 ± 0.79 24.6 ± 0.71 24.4 ± 0.68 24.3 ± 0.65 24.1 ± 0.63 24.1 ± 0.63
aDhanoa et al. [14] b Pond et al. [15]. Abbreviations. G1G1, first order two-compartment model; G2G1, second order two-compartment model; G3G1, third order two-compartment
model; G4G1, fourth order two-compartment model; G5G1, fifth order two-compartment model; G6G1, sixth order two-compartment model according to Pond et al. [15]; RMSE, root
mean square error; AIC, Akaike’s information criterion; TT, transit time; CMRT, compartment mean retention time; TTMRT, total tract mean retention time.
Animals 2019, 9, 1148
3.3. Digesta Mean Retention Time
3.3.1. Mean Retention Time Calculated from Model Parameters

The best fitting models to describe marker excretion were used to compare digesta ROP between
diets. The Dhanoa et al. [14] and Pond et al. [15] G5G1 models were used to analyze particulate phase
ROP. The Dhanoa et al. [14] and Pond et al. [15] G4G1 models were used to analyze liquid digesta ROP.
Particulate TTMRT differed by diet (p = 0.020 and p = 0.022, respectively; Table 6) when Yb marker
excretion was fit to both the Dhanoa et al. [14] and Pond et al. [15] G5G1 model. Rates of passage, k1 and
k2 , did not differ among diets when fecal excretion was fit to equations described by Dhanoa et al. [14];
however, CMRT1 tended to be longer (p = 0.084) when horses were fed ALF compared with CB 6.
Transit time calculated according to Dhanoa et al. [14] tended to be longer (p = 0.074) in CB 8 than
ALF. Modeling particulate fecal marker excretion using the G5G1 model [15], λ1 tended to differ by
diet (p = 0.069) with λ1 trending towards being quicker (p = 0.102) in ALF than CB 8. The other
model parameters k2 and TT did not differ by diet. Age-dependent compartment mean retention time
(CMRT1 ) was shorter (p = 0.014) in ALF than CB 8 and tended to be shorter (p = 0.065) than CB 4. There
was no difference in the age-independent compartment mean retention time (CMRT2 ).
Table 6. Model parameters and compartment retention times for particulate (Yb) and liquid (Co)
marker fecal excretion 1 (n = 5).
Diet 2 Contrast 3
Item ALF ORCH CB 4 CB 6 CB 8 SEM
p-Value p-Value
Particulate
Dhanoa A
A 2.4 × 109 3.0 × 109 1.3 × 1012 3.1 × 109 3.5 × 109 4.3 × 108
k1 , h−1 0.210 0.287 0.248 0.294 0.259 0.0536 0.223 0.239
k2 , h−1 0.357 0.380 0.336 0.372 0.328 0.0450 0.338 0.312
N 107 34 29 36 33 83191 0.179 0.054
TT, h 22.0 y 23.5 x,y 27.9 x,y 26.9 x,y 28.7 x 1.27 0.035 0.004
CMRT1 , h 5.59 x 3.77 x,y 3.82 x,y 3.63 y 4.17 x,y 0.532 0.049 0.051
CMRT2 , h 2.92 2.68 3.03 2.70 3.12 0.148 0.303 0.479
TTMRT, h 30.5 y 30.0 y 34.7 x,y 33.2 x,y 35.9 x 0.921 0.020 0.003
G5G1 B
C, mg Yb/kg
1134 1007 906 780 681 56.3
digesta
λ1 , h−1 0.396 0.342 0.277 0.319 0.272 0.018 0.069 0.014
k2 , h−1 0.249 0.279 0.280 0.274 0.272 0.009 0.751 0.489
TT, h 11.4 9.02 10.3 11.1 11.4 0.616 0.362 0.948
CMRT1 , h 13.6 b,y 15.4 a,b 18.2 a,b,x 16.1 a,b 18.5 a 0.634 0.008 0.002
CMRT2 , h 4.47 3.64 3.59 3.71 3.71 0.182 0.508 0.218
TTMRT, h 29.5 a,b 28.1 b 32.1 a,b 30.9 a,b 33.6 a 0.900 0.022 0.007
Liquid
Dhanoa A
A 1919 3.0 × 109 1.5 × 109 1.3 × 109 9.8 × 107 2.7 × 1011
k1 , h−1 0.106 x 0.234 0.145 0.111 y 0.120 0.0165 0.042 0.023
k2 , h−1 0.708 x 0.440 0.373 0.376 0.320 y 0.0108 0.042 0.016
N 4.2 × 105 50 49 33 31.2 13 0.661 0.075
TT, h 11.3 b 13.6 a,b 19.9 a 20.7 a 20.7 a 1.14 0.005 <0.001
CMRT1 , h 9.52 a 6.14 b 5.57 b 4.0 b 5.58 b 0.291 <0.001 <0.001
CMRT2 , h 1.75 b 2.48 a,b 2.75 a,b 2.70 a,b 3.17 a 0.096 0.009 0.005
TTMRT, h 22.6 c,z 22.3 b,c,y,z 28.2 a,b,x,y 27.5 a,b,c,x 29.5 a 1.00 0.002 <0.001
11
Animals 2019, 9, 1148
Table 6. Cont.
Diet 2 Contrast 3
Item ALF ORCH CB 4 CB 6 CB 8 SEM
p-Value p-Value
G4G1 B
C, mg Yb/kg
465 801 614 704 565 43
digesta
λ1 , h−1 1.095 a,x 0.491 a,b,y 0.359 b 0.329 b 0.310 b 0.0856 0.002 0.001
k2 , h−1 0.107 b,y 0.216 a,b,x 0.203 a,b 0.247 a 0.236 a,b,x 0.0152 0.010 0.012
TT, h 8.42 x 5.45 y 8.15 7.34 8.00 0.391 0.042 0.194
CMRT1 , h 4.71 b 10.3 a 12.6 a 13.4 a 13.6 a 0.973 <0.001 <0.001
CMRT2 , h 9.44 a 5.32 b 5.61 b 4.20 b 4.69 b 0.513 0.001 0.002
TTMRT, h 22.6 b,c 21.0 c 26.4 a 25.0 a,b 26.3 a 0.650 <0.001 <0.001
1Abbreviations. ALF, alfalfa; ORCH, orchardgrass; CB 4, Coastal bermudagrass 4-weeks regrowth; CB 6, Coastal
bermudagrass 6-weeks regrowth; CB 8, Coastal bermudagrass 8-weeks regrowth; SEM, standard error of the
mean; RMSE, root mean square error; AIC, Akaike’s information criterion; TT, transit time; CMRT, compartment
mean retention time; TTMRT, total tract mean retention time. 2 Main effect of diet. 3 Contrast between Coastall
bermudagrass (CB 4, CB 6, CB 8) and other diets (ALF, ORCH). A Dhanoa et al. [14]. B Pond et al. [15]. a,b,c Means
with unlike superscripts differ (p < 0.05). x,y,z Means with unlike superscripts tend to differ (p < 0.1).
3.3.2. Arithmetically Calculated Mean Retention Time

Arithmetically calculated particulate digesta MRT (Table 7) using the equation described by
Blaxter et al. [32] was longer (p = 0.019) than when calculated according to Thielemans et al. [33], but
the mean difference was only 0.2 h (30.0 ± 0.88 vs. 29.8 ± 0.88 h). Th method of calculation did not
affect liquid MRT (23.4 ± 0.70 vs. 23.5 ± 0.67 h). For both equations, particulate MRT was longer
(p < 0.001) than liquid MRT.
Particulate MRT differed by diet (p < 0.001) and was longer (p < 0.05) when horses were fed CB 8
compared with ORCH (Table 7). Liquid MRT differed by diet (p < 0.001). Horses fed ORCH had a
shorter (p < 0.01) liquid MRT than when fed the Coastal bermudagrass diets. Liquid MRT differed
(p < 0.011) between horses fed ALF compared with CB 4 and CB 8 and tended to be shorter (p < 0.079)
than horses fed the CB 6 diet.
Table 7. Mean retention time (h) of particulate digesta measured with Yb-NDF and Co-EDTA external
markers 1 (n = 5).
Diet 2 Contrast 3
Equation ALF ORCH CB 4 CB 6 CB 8 SEM
p-Value p-Value
Blaxter et al. [32]
Particulate 29.2 a,b 26.9 b 31.1 a,b 30.0 a,b 32.7 a 0.88 0.010 0.006
Liquid 21.3 b,c,y 20.1 c 25.7 a 24.3 a,b,x 25.9 a 0.70 <0.001 <0.001
Thielemans et al. [33]
Particulate 28.9 a,b 27.0 b 30.4 a,b 29.6 a,b 32.6 a 0.88 0.010 0.008
Liquid 21.2 b,c,y 20.6 c 25.4 a 24.1 a,b,x 25.9 a 0.67 <0.001 <0.001
2 Main effect of diet. 3 Contrast between Coastal bermudagrass (CB 4, CB 6, CB 8) and other diets (ALF, ORCH). a,b,c
Means with unlike superscripts differ (p < 0.05). x,y Means with unlike superscripts tend to differ (p < 0.10).
3.3.3. Comparing Model-Derived and Arithmetically Calculated Mean Retention Time

The mean retention times calculated from Dhanoa et al. [14] and Pond et al. [15] model parameters
were compared with arithmetically calculated MRT using two one-sided test of equivalence (TOST)
with a 10% difference and regression analysis. Model-derived TTMRT was similar to (p < 0.05) MRT
calculated according to the equation described by Blaxter et al. [32] or Thielemans et al. [33] for the
G2G1, G3G1, G4G1, G5G1 models for the particulate phase of digesta. For the liquid phase of digesta,
models described by Pond et al. [15] were similar to (p < 0.05) arithmetic MRT, but TTMRT calculated
according to Dhanoa et al. [14] was over 2 h shorter than arithmetic MRT.
12
Animals 2019, 9, 1148
Arithmetically calculated MRT and model-derived TTMRT are plotted in Figure 3. For
the particulate phase of digesta (Figure 2a), the equation relating TTMRT to MRT according to
Blaxter et al. [32] was MRT = 0.7929x + 3.903 (RMSE = 1.980; r2 = 0.8071; p < 0.001) for TTMRT
calculated according to Dhanoa et al. [14] and MRT = 0.9737x − 0.05589 (RMSE = 0.6256; r2 = 0.9807;
p < 0.001) when TTMRT according to the G5G1 model described by Pond et al. [15]. When MRT was
calculated according to Thielemans et al. [33], MRT = 0.7935x + 3.680 (RMSE = 1.960; r2 = 0.8105;
p < 0.001) for TTMRT calculated according to Dhanoa et al. [14] and MRT = 0.9743x − 0.2772
(RMSE = 0.5592; r2 = 0.9846; p < 0.001) when TTMRT according to the G5G1 model described by
Pond et al. [15]. For the liquid phase of digesta (Figure 2b), the equation relating TTMRT to MRT
according to Blaxter et al. [32] was MRT = 0.6329x + 6.956 (RMSE = 2.017; r2 = 0.6852; p < 0.001)
for TTMRT calculated according to Dhanoa et al. [14] and the y-intercept differed (p = 0.007) from
zero. Mean retention time calculated according to Blaxter et al. [14] was related to TTMRT calculated
according to the G4G1 model described by Pond et al. [15] as MRT = 1.064x − 2.408 (RMSE = 0.6661;
r2 = 0.9657; p < 0.001) and the y-intercept differed (p = 0.028) from zero. When TTMRT was calculated
according to Thielemans et al. [33], MRT = 0.5326x + 9.984 (RMSE = 2.565; r2 = 0.4879; p < 0.001)
for TTMRT calculated according to Dhanoa et al. [14] and MRT = 0.8692x + 2.748 (RMSE = 2.129;
r2 = 0.6474; p < 0.001) when TTMRT according to the G4G1 model described by Pond et al. [15].
For the regression equation relating TTMRT from the G4G1 model to MRT calculated according to
Thielemans et al. [33], the y-intercept differed from zero (p = 0.003).
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Figure 3. Comparison of arithmetically calculated mean retention time (MRT) calculated according to
Blaxter et al. [32] and Thielemans et al. [33] compared with total tract mean retention time (TTMRT)
calculated according to Dhanoa et al. [14] (×) and the best fitting two-compartment model described by
Pond et al. [15] (•) for the particulate (a) and liquid (b) phase of digesta. The 1 to 1 line is denoted by
the dashed line.
13
Animals 2019, 9, 1148
4. Discussion
Increasing the retention time of Coastal bermudagrass may be an important digestive strategy
in horses to adapt to the more difficult-to-digest fiber particles of warm-season grasses. Greater
retention time of highly fibrous, warm-season forages allows for a lengthened exposure of digesta to
microbial degradation. Forage fiber composition may also influence digesta passage rate within the
gastrointestinal tract, as observed when mathematically modeling fecal marker excretion when horses
were fed alfalfa hay, which has an increased concentration of pectin compared to grasses.
Forage type affected DMD, with the greatest digestibility observed when horses were fed
alfalfa. Several other studies have reported greater digestibility of legume hays than cool-season or
warm-season grass hays [34–40]. The DMD of alfalfa hay in this study falls within the 54–66% range of
alfalfa hay DMD reported in the literature [34–40]. Chemically, legume forages have greater protein
and pectin concentrations and decreased insoluble fibers, allowing for a faster rate of digestion [41].
Legumes also contain a greater proportion of more easily digested mesophyll cells compared to
grasses. The accumulation of lignin in alfalfa cell walls occurs primarily in alfalfa stems, whereas
lignin accumulates in both grass stems and leaves during maturity [42,43]. Thus, there is a greater
extent of cell wall digestion of alfalfa leaves compared to grasses [44].
Longer intervals of growth before harvest increased Coastal bermudagrass fiber and lignin
concentrations in hay and resulted in reduced dry matter, organic matter, and fiber digestibilities.
The internal girder structure of C4 forages firmly links the epidermis to vascular bundles, reducing
the rate of digestion [45,46]. Akin et al. [47] reported that even typically highly digestible plant
mesophyll cells were only partially degraded with increasing plant maturity in bermudagrass samples.
The reported digestibility of Coastal bermudagrass hay ranges from 41–53% [34,35,48–50]. Although
the DMD of CB 4 fell within the reported range, the CB 6 and CB 8 had reduced digestibility compared
with reported values. Because fiber concentration is negatively correlated with digestibility [6],
the comparatively low digestibility of CB 6 and CB 8 to other published data is surprising as the Coastal
bermudagrass used in those studies often had a greater detergent fiber concentration than the hays
utilized in the current study [34,48–50]. Although the Coastal bermudagrass harvested at four weeks
regrowth had greater NDF and hemicellulose concentrations than orchardgrass and alfalfa hays, fiber
digestibility was similar. However, a reduction in NDFD and ADFD was observed in the CB 6 and
CB 8 diets. Phenolic compounds and hemicellulose composition changes with increasing plant cell
maturity [47,51,52] likely leading to the decreased digestibility of Coastal bermudagrass harvested at
longer intervals of growth. The results of the current study indicate that differences beyond plant fiber
concentration, such as hemicellulose composition and lignin concentration also affect the digestibility
of Coastal bermudagrass hay.
One strategy to adapt to the lower digestibility of high fiber C4 grasses such as Coastal
bermudagrass is for the digesta retention time to increase. Other high fiber forages, such as oat
straw, have also been reported to have increased MRT in equines [9]. Longer exposure to microbial
fermentation in the equine hindgut can increase cell wall digestibility and could compensate for a
slower rate of degradation of fibers in warm-season grasses [46,53,54]. This selective retention of
Coastal bermudagrass hay in the gastrointestinal tract (GIT) may be due to differences in the particle
size of digesta, rate of intake, or changes in GIT motility. How fiber affects gastrointestinal transit is
not clearly understood in horses, people, or other mammals. One theory is that the fiber can trap water
in the gastrointestinal tract, altering the way bacteria and solutes interact in the GIT [55]. In addition
to luminal contents, gut motility is also driven by a range of neurohormones. Peptide YY (PYY)
and glucagon-like peptide-1 (GLP-1) are important neurohormones regulating colonic motility [56],
and the secretion of PYY and GLP-1 increases with the addition of fiber to diets [57,58]. Gut motility,
neurotransmitters, and digesta characteristics warrant future investigation for potential mechanisms
of regulating digesta transit in horses.
Differences in ROP were identified between forage types that were not apparent in TTMRT,
indicating that mathematically modeling fecal marker excretion can advance the study of digesta
14
Animals 2019, 9, 1148
passage in horses. Alfalfa ROP parameters derived from Dhanoa et al. [14] and Pond et al. [15] differed
from other diets, indicating differences in digesta passage kinetics for a legume hay compared to
grasses. For the particulate phase of digesta, λ1 was quicker and CMRT1 shorter in ALF than CB 8,
even though no total tract differences were determined when TTMRT was calculated according to the
G5G1 model. When particulate CMRT1 was calculated according to Dhanoa et al. [14], CMRT1 was
longer in ALF compared with CB 6, a result also not reflected in TTMRT. When the liquid phase of
digesta was modeled according to the G4G1 model, the age-dependent compartment (CMRT1 ) was
shorter than the age-independent compartment when horses were fed alfalfa. In contrast, all the grass
forages had longer age-dependent CMRT than age-independent CMRT. Alfalfa and other legumes
have higher pectin (a soluble fiber) concentrations compared to grasses. Soluble fiber has been shown
to increase retention time in the small intestine and delay gastric emptying in other species [59,60].
The effect of fiber type on digesta ROP in the horse may be better elucidated using mathematical
models than total tract MRT.
In the current study, compartment models described by both Dhanoa et al. [14] and Pond et al. [15]
adequately fit equine marker excretion using non-linear least squares methods by modifying model
parameter start values and bounds of rate parameters. Both one- and two-compartment models have
been used in the recent literature, with the best fitting model depending on the study. Equations
described by both Dhanoa et al. [14] and Pond et al. [15] have failed to produce a solution of acceptable
model fit due to lack of convergence between experimental data and the model [10,19,61]. However,
computing power and collaboration with computer scientists and modelers can greatly reduce the
likelihood of models failing to converge with experimental data. Future studies incorporating
mathematical modeling into digesta ROP studies will help to identify the best ways to describe digesta
passage in the equine GIT.
Although using the equation described by Dhanoa et al. [14] resulted in an improved fit compared
with the Pond et al. [15] models based on AIC values, TTMRT from the best fitting Pond et al. [15]
models were more similar to arithmetically calculated MRT. The mathematical basis of these two
models differ. The equation described by Dhanoa et al. [14] represents an unspecified number of
exponentially distributed compartments, whereas the Pond et al. [15] equations used in the current
study represented two distinct compartments plus transit time. The first two compartments in the
Dhanoa et al. [14] model (CMRT1 and CMRT2 ) have the longest retention time, and the remaining
compartments are summed to determine transit time. Transit time is often defined as the amount of
time from marker dosing to the first appearance of marker in the feces, but Ellis et al. [62] described
transit time in the Dhanoa et al. [14] model as the sum of time in the remaining system of mixing
compartments. The difference in transit time definition may be part of the discrepancy between transit
time calculated from the different model equations. Additionally, the Dhanoa et al. [14] model may
represent a set of exponentially distributed compartments that are within a larger mixing structure.
The lack of agreement between arithmetic MRT and TTMRT calculated by the Dhanoa et al. [14] model
may be explained by these differences.
The use of mathematical models could be further enhanced if theoretical compartments could
be correlated to anatomical sections of the GIT. Moore-Colyer et al. [10] hypothesized that the two
compartments of the Pond et al. [15] model represented the large colon for the age-dependent
compartment and the cecum for the age-independent compartment because retention time in the colon
is longer than the retention time in the cecum [63]. Transit time was hypothesized to represent residence
time in the remaining structures of the GIT (i.e., stomach and small intestine). In a previous study
using similar methodology, modeling fecal marker excretion when horses were fed mainly forage diets
resulted in a CMRT similar to MRT in the cecum and colon [64,65]. However, Murray et al. [19] rejected
the hypothesis that the age-dependent compartment was the colon because they observed a longer
retention time in the age-independent compartment. The longer retention time of the age-independent
compartment by Murray et al. [19] may be due to the diet (alfalfa and sugar beet pulp, which are high
in soluble fiber), as observed in this study. Thus, diet may have a greater influence on compartment
15
Animals 2019, 9, 1148
mean retention time than connections with anatomical compartments. Overall, mathematical models
show promise to describe passage kinetics in horses, but the physiological relevance of compartment
retention times remains unclear.
5. Conclusions
In conclusion, the horse appears to adopt a digestive strategy to decrease the rate of passage of
digesta when fed warm-season grass forages. Increasing the retention time allows for fiber particles
with greater hemicellulose and lignin concentrations to be exposed to microbial fermentation longer.
Because warm-season forages have slower rates of degradation, this change in retention time allows
the horse to maximize potential nutrients obtained from the diet. Using mathematical models further
characterized differences in digesta ROP between forages which were not apparent when evaluating
total tract MRT alone. Minor discrepancies between models and arithmetically calculated MRT were
observed and should be resolved for future use. Nonetheless, mathematical modeling should be
incorporated into future equine nutrition research to expand knowledge on digesta passage and equine
science in general.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2615/9/12/1148/s1,

Computer Code S1: Matlab Model Fit; Supplementary Figures S1–S5: Individual Horse Marker Excretion
Within Diet.
Author Contributions: Conceptualization, L.K.W., T.L.H., and E.L.C.; methodology, L.K.W., T.L.H., and E.L.C.;
validation, L.K.W., and T.L.H.; formal analysis, T.L.H.; investigation, T.L.H., E.L.C., O.K.Z., J.M.M., J.M.B., L.A.S.,
J.W.C., A.M.A. and L.K.W.; resources, L.K.W.; writing—original draft preparation, T.L.H.; writing—review and
editing, L.K.W., T.L.H.
Funding: This research received no external funding.
Acknowledgments: Seminole Feed Inc. (Ocala, FL, USA) provided a vitamin, mineral, protein pellet to feed in
this study. The authors are grateful for the staff and students at the University of Florida/Institute of Food and
Agricultural Sciences Horse Teaching Unit to complete this study. The authors would like to thank K. Bissinger,
K. Bush, S. Chewing, M. Di-Lernia, B. Eubanks, T. Fraguela, J. Gerbert, K. Irvine, A. Mesa, N. Oliver, H.
Miller, V. Robbins, D. Van Camp for animal care, sample collection, and laboratory assistance. J. Bauman, B.
d’Angelo, and N. Wilkinson were an essential team for marker analysis on fecal samples. We additionally
would like to acknowledge the thoughtful discussion provided by J. Brendemuhl, A.L. Fowler, T.J. Hackmann,
and L. Sollenberger about this data.
Conflicts of Interest: The authors declare no conflict of interest.
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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
19
animals
Article
Effect of Linseed (Linum usitatissimum) Groats-Based
Mixed Feed Supplements on Diet Nutrient
Digestibility and Blood Parameters of Horses
Markku Saastamoinen * and Susanna Särkijärvi
Production Systems, Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland;
susanna.sarkijarvi@luke.fi
* Correspondence: markku.saastamoinen@luke.fi
Received: 28 January 2020; Accepted: 7 February 2020; Published: 10 February 2020
Simple Summary: In this study, the effect of linseed groat-based fibrous feed supplements on diet
digestibility was studied. In addition, possible detrimental health effects due to continuous feeding
of such supplemental feeds containing linseed were examined by evaluating blood parameters.
The supplemented diets had statistically significantly higher digestibility of crude protein compared
to the control diet. In addition, the digestibility of fat (ether extract) was higher in the supplemented
diets than in the basal feeding. There were no statistically significant differences or trends in the
blood parameters between the treatments. It is concluded that linseed by-products (linseed groats
0.8 g/kg BW/d) combined with other fibre sources can be safely used, for example, in feeding strategies
replacing grains in the horses’ rations in order to reduce the intake of starch.
Abstract: Linseed (Linum usitatissimum) and its by-products are common supplements used in equine
diets and are claimed to have beneficial health effects. In this study, the effect of linseed groat-based
fibrous feed supplements on diet digestibility was studied. Also, possible detrimental health effects
due to continuous feeding of supplemental feeds containing linseed were examined by evaluating
blood parameters. The experimental design was arranged as two balanced 3 × 3 Latin Squares.
The horses were individually fed at the maintenance energy level, the forage-to-concentrate ratio being
70:30, with three diets: (A) Control diet consisting of dried hay and whole oats; (B) Control diet + Feed
1; and (C) Control diet + Feed 2. Feed 1 contained 70% of linseed groats, 15% dried carrot, 10% dried
garlic and 5% molasses. Feed 2 contained 65% linseed groats, 15% molassed sugar-beet pulp, 10%
dried garlic, 5% dried carrot and 5% molasses. Digestibility data were obtained by using chromium
mordanted straw as an indigestible external marker for the estimation of apparent digestibility. Blood
samples were collected from the jugular vein at the end of each feeding period to evaluate the possible
effects of the supplemented diets B and C on the health of the horses. Diets B and C had a higher
digestibility of crude protein compared to the control diet A (p < 0.05). In addition, the digestibility of
ether extract was higher in the supplemented diets than in the basal feeding (p < 0.01). There were no
statistically significant differences or trends (p > 0.05) in the blood parameters between the treatments.
It is concluded that linseed groat-based supplements (offering approximately 6.3%–6.7% linseed
groats in the diet’s dry matter (DM), or 0.8 g/kg BW/d), and feed containing soluble fibre sources
(sugar-beet pulp, dehydrated carrot), improved the crude protein and fat digestibility of hay-oats
diets of horses, and can be used, for example, in feeding strategies replacing grains in the horse
rations in order to reduce the intake of starch without any adverse effects on the blood parameters
and health of the horses.
Keywords: feeding; haematology; flax seed; fibre

Animals 2020, 10, 272
1. Introduction
Linseed (Linum usitatissimum) or by-products (groats, cakes, meals) of linseed oil pressing have
been used in human and animal nutrition for decades because they are believed to have numerous
beneficial effects, many times without any scientific evidence. The “basic” horse nutrition literature in
different countries [1–3] has recommended the feeding of linseed in various amounts for a long time as
a supplemental feed to promote gut and skin health as well as coat quality. Thus, linseed products
are commonly used in equine diets [4,5]. However, there is a knowledge gap on the nutritional and
health effects of feeding linseed products to horses, because scientific research about this is scarce.
For example, proper and safe supplementation levels are not given or known.
Linseed meal is high in crude fibre, acid detergent fibre (ADF) and neutral detergent fibre (NDF) [6].
Pectins and other dietary fibres of linseed have been proved to promote the health of the gastrointestinal
tract in humans and dogs (e.g., [7,8]). The hull fraction (outer seed coat) contains 2%–7% polysaccharide
mucilage [6,9]. Mucilage is readily water dispersible and forms a viscous slime, which is believed to
have positive effects on the stomach and gut [10]. Further, in our preliminary study [11], linseed-based
feed enhanced sand removal from the digestive tract of the horses.
Linseed oil is a good source of valuable fatty acids (omega-3) [12]. Groats and meal from
cold-pressing may have an oil content of up to 20% [13]. Thus, linseed can be viewed as a cost-effective
and economical way to boost omega-3 fatty acids in the feed [14]. In one study [15], a significant
improvement in a skin test response to Culicoides spp. was reported due to linseed supplementation.
Improved hair coat and skin condition scores have also been obtained in dogs after one month of
linseed supplementation [16]. Horses have low fatty acid elongation activity, which is important for
the inflammatory response, and there is speculation that linseed as a source of omega-3 PUFA may
decrease signs of laminitis by inhibiting inflammatory mediators [17]. In a quite recent study [18],
increased concentrations of red blood cells, haemoglobin and haematocrit, as well as improved n-3
fatty acid profiles, were reported as a result of linseed oil supplementation. Vineyard et al. [19] found
that supplementing horses with milled linseed resulted in pronounced early inflammatory responses
to phytohaemagglutinin injections. Both studies also showed increased fatty acid contents of red blood
cell membranes. In addition, fats are an important source of energy for horses [20] and can be applied
to reduce the starch content of the diet. However, the ether extract digestibility of linseed observed for
horses was lower compared to oats and bran [21].
Linseed by-products are rich in protein [6], but a comparison of linseed meal to blended milk
products showed that the growth and feed/gain were much better for milk products, the main reason
being their better lysine content [22]. Linseed by-products have not been successfully used as protein
sources in chicks either [23]. Instead, conflicting results on the effects on growth and health have been
reported for pigs [24,25].
Consequently, there are several reasons for the interest in including linseed meal or oily linseed
by-products in horse diets by horse owners [5]. However, linseed is known to contain compounds that
may be toxic or have anti-nutritive properties [6,26], when the enzyme linase releases cyanide from the
glycoside and diclucosides of the seeds [27]. Cyanide levels in linseed are below the level hazardous to
humans [6], but there is some concern about the possibility of cyanide poisoning in horses, which are
fed linseed [3]. However, intoxications or studies on this matter and where the daily intakes are given
have not been reported in horses.
Williams and Lamprecht [28] reviewed studies where linseed oil has been fed to horses, but the
feeding of linseed by-products has rarely been the subject of controlled studies with horses, or any
other species for that matter. In addition, data regarding the effects (beneficial or detrimental) on diet
digestibility and/or animal health when large amounts are fed is scarce. Instead of this, rather small
amounts (only 50–120 g/d) are recommended to horse diets [2]. Science-based levels are not given,
but recently Lindinger [29] concluded in his article in a veterinary science journal, based on a trade
blog [4]), that the highest recommended amount for horses is 454 grams/d (1 pound). Neither research
data exists regarding feeds in which linseed is combined with botanically diverse fibre sources.
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Therefore, the objectives of this study were to: (1) investigate the effect of two linseed groat-based
mixed feed supplements containing other fibrous ingredients on diet digestibility; (2) evaluate the
possible detrimental health effects due to continuous feeding of linseed groats supplements in terms
of blood parameters. The hypothesis was that there will be no detrimental effects due to the linseed
supplementation on the diet digestibility and the haematological values of the horses. The results
can be applied in the practical feeding of horses, or by the feed industry utilising linseed groats as a
feed ingredient.
2.1. Horses and their Management

The influence of two linseed-based feed supplements on diet digestibility was examined with six
Finnhorse mares (5–14 years; mean initial BW 636 kg, s.d. 37.8 kg), owned by MTT Agrifood Research
Finland (currently Natural Resources Institute Luke). The experiment was conducted in the facilities
of Luke. The experimental horses were individually housed in stalls (3 × 3 m) with peat as bedding.
The horses had free access to water and a salt block and they were de-wormed before the experiment,
and dental care and vaccinations had also been carried out regularly prior to the experiment. During
the experiment, they were freely exercised daily in outdoor paddocks for four hours, and one hour by
riding at a slow walk, to fulfil their needs of exercise and ensure their wellbeing.
The experimental design was arranged as two balanced 3 × 3 Latin Squares. Each experimental
period consisted of 21 days: 16 days of adaptation to the new diet followed by a five-day period of
collecting representative spot faecal samples. The BW (electronic animal scale Lahden Vaaka/Lahti
Precision Ltd., Lahti, Finland) of the horses was monitored after each collection period to control
possible changes and to adjust the individual energy intakes if necessary.
In animal handling and sample collection, the European Union recommendation directives
(1999/275/EU) and national animal welfare and ethical legislation set by the Ministry of Agriculture
and Forestry of Finland were followed carefully. The experimental procedures were evaluated and
approved by The Animal Care Committee of MTT (Permit 9/2001) before the study was started.
2.2. Experimental Feeds and Feeding

The horses were randomly allotted to three dietary treatments: (A) Control diet consisting of
dried hay dominated by timothy grass and whole oats; (B) Control diet + Feed 1; and (C) Control
diet + Feed 2. Feed 1 contained 70% of linseed groats, 15% dried carrot, 10% dried garlic and 5%
molasses. Feed 2 contained 65% linseed groats, 15% molassed sugar-beet pulp, 10% dried garlic, 5%
dried carrot and 5% molasses. The hay and oats for this experiment were produced by Luke. Feed 1
and Feed 2 were manufactured in a single batch for this experiment by a Finnish medical and food
factory (Neomed Ltd., Somero, Finland) and were in granulated form (granulated in 70–80 ◦ C heat for
5 to 6 min). The linseed groats in the experimental feeds were by-products of cold-pressing of linseed
oil with an average fat (oil) content of 20%. The other raw materials were included in the experimental
feeds in order to improve the palatability as well as owing to their technological properties [30]. They
are also common supplemental feeds included in horse diets. The average chemical composition of the
feeds is presented in Table 1.
The horses were individually fed at the maintenance energy level according to the Finnish
Feed Tables and Feeding Recommendations [13], the forage-to-concentrate ratio being 70:30.
Each experimental feed ration was formulated and adjusted to be as isocaloric and isonitrogenous as
possible. The average daily allowances of hay, oat and experimental feeds in each dietary treatment
are presented in Table 2. About 8% of the oats was substituted with the experimental feeds in the
treatments B and C.
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Table 1. Average chemical composition of the experimental feeds (g/kg dry matter).
Composition Hay Oats Feed 1 Feed 2

Dry matter g kg-1 870.7 883.1 905.2 885.4
Organic matter 934.7 971.4 930.6 924.3
Crude protein 95.0 112.8 211.5 209.8
Ether extract 15.6 61.0 185.7 172.7
NDF 687.7 263.3 183.3 228.0
Crude fibre 339.0 89.0 96.0 105.3
Ash 63.5 28.6 69.4 75.7
NFE 485.1 708.6 437.5 436.5
ME MJ/kg DM 9.10 12.6 14.3 13.9
NDF = neutral detergent fibre; NFE = nitrogen free extract; ME = metabolisable energy.
Table 2. Daily allowances of hay, oats and experimental feeds (Feed 1 or Feed 2) fed in each dietary
treatment (DM kg/day) (with ranges).
Feed Diet A (Control) Diet B (Feed 1) Diet C (Feed 2)

5.89 5.44 5.43
Hay
(5.66–6.36) (5.22–5.92) (5.22–5.75)
1.85 1.74 1.74
Oats
(1.80–2.12) (1.68–1.89) (1.68–1.84)
0.757 0.745
Experimental feed —
(0.710–0.780) (0.730–0.750)
The change in rations between periods was made gradually during the first five days of the
adaptation period. Feeds were offered in equal meal sizes three times a day at 06:30, 12:30 and 17:30.
The grain ration was given about 30 min after the hay ration. The experimental feeds (Feeds 1 and 2)
were aimed to be fed at a level of approximately 10% of the total dry matter (DM) intake, the average
daily portion being 765 g DM/horse divided into three equal portions that were fed separately after the
intake of oats. They were soaked in warm water (45–50 ◦ C) before feeding to ensure their palatability.
Mineral intakes were balanced with a commercial vitamin–mineral mixture (Suomen Rehu Ltd.,
Seinäjoki, Finland).
2.3. Feed and Faeces Sampling

Digestibility data were obtained by using chromium mordanted straw (68 mg Cr/g DM) with a daily
dose of 1.6 g/kg feed DM as an indigestible external marker for the estimation of apparent digestibility.
The chromium mordanted straw was prepared according to Udèn et al. [31]. The chromium dosage
was calculated separately for every feed portion and served on the top of the concentrate three times a
day, as described in detail by Särkijärvi and Saastamoinen [32]. Samples of hay and oats were collected
for analysis over the last seven days of each period, and stored until the end of the five-day collection
period. Faecal grab samples (500 g) were taken from each horse twice a day after the morning and
mid-day feeding, during the five-day collection period. Samples were collected from the floor of the
pen from a freshly produced pile. Daily faecal samples were stored at –24 ◦ C until mixed, sub-sampled
and dried (at 100 ◦ C for 1 h + at 60 ◦ C for 72 h) for laboratory analysis [32].
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Feed and faeces samples were analysed in the feed laboratory of Luke (Luke Laboratories,
Jokioinen, Finland) for dry matter (DM), organic matter (OM), neutral detergent fibre (NDF), crude
fibre (CF), crude protein (CP), ether extract (EE) and ash with standard wet chemical methods as
described by Särkijärvi et al. [33]. The nitrogen-free extract (NFE) was calculated: (100–CP–CF–EE–ash).
The digestible CP (DCP) was calculated: DCP (g/kg DM) = CP (g/kg DM) × CP digestibility
(g/kg CP)/1000, where the CP digestibility was taken from the Finnish Feed Tables and Feeding
Recommendations [11]. The metabolisable energy value (ME) was calculated according to the British
energy evaluation system [34].
2.4. Blood Sampling

Blood samples were collected at the end of each period to evaluate the possible effects of the diets
on the health of the horses. The samples (2 × 10 mL) were collected 90 min after the morning meal from
the jugular vein to heparinised blood collection tubes, and centrifuged. The blood analysis consisted of
the contents of red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), haematocrit (HcT),
and fibrinogen, as well as liver enzymes alanine aminotransferase (ALT) and γ-glutamyltransferase
(GT), to indicate possible detrimental effects of linseed (cyanogenic glucosides) on the liver. All samples
were analysed in the clinical laboratory of Luke.
2.5. Statistical Analysis

The data were analysed with a linear mixed model using the MIXED procedure of the SAS system
using the REML estimation method. The following statistical model was applied:
Yijk = μ + ai + tj + p(sq)k + εijk (1)
where Yijk is the observation, μ is the overall mean, ai is the random effect of ith animal (i = 1, . . . 6), tj
is the fixed effect of jth dietary treatment (j = 1, . . . 3), p(sq)k is the fixed effect of kth period within the
square (k = 1, . . . 3) and εijk is the normally distributed error with a mean of 0 and the variance δ2 .
Residuals were tested for normality. The differences were tested with Tukey’s test, and the level of
significance was set at the 5% level.
3. Results
3.1. Feed and Nutrient Intakes

The palatability of all the diets was good and there were no feed refusals. There were only very
minor changes (± 0.8–2.2% between the measurements) in the body weights of the horses during
the study. Diet intake was isocaloric and isonitrogenous between study periods (all diets combined;
Table 3), but the intakes differed between the diets (Table 4). The average intakes of fat (EE) and CP
of the supplemented horses (Diets B and C) were 58.0% and 14.1% higher than in the control group.
Concerning ME, CF and NDF intakes, the differences were much smaller with +2.0%, –4.0% and
–4.5%, respectively.
Table 3. Average daily ME, CP and DM intakes (± s.d.) for each period.
Intake Period I Period II Period III

ME MJ 80.3 ± 4.8 81.8 ± 3.9 80.1 ± 3.2
CP g 832.4 ± 68.9 850.4 ± 73.1 835.2 ± 50.6
DM kg 7.86 ± 0.37 7.98 ± 0.29 7.86 ± 0.31
ME = metabolisable energy; CP = crude protein; DM = dry matter.
The proportions of linseed groats (on a DM basis) in diets B and C were 6.7% and 6.3%, respectively.
On the BW basis, the intakes of linseed groats were approximately 0.8 g DM/kg BW/d.
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The supplementation of the experimental feeds improved only the digestibilities of total diet
fat (ether extract, EE) and crude protein (CP) (p = 0.0012 and 0.0182, respectively) compared to
the control diet (Table 5). In addition, the digestibility of ash (minerals) seemed to be numerically
(but not statistically, p = 0.2093) somewhat higher in the supplemented diets than in the control
diet. None of the digestibility values differed between the supplemental diets (Diet B versus Diet C)
(p-values = 0.47–0.80).
Table 4. Average daily dry matter, metabolisable energy and nutrient intakes (±s.d.) in each
dietary treatment.
Intake Diet A (Control) Diet B (Feed 1) Diet C (Feed 2)

DM kg 7.77 ± 0.39 7.94 ± 0.28 7.91 ± 0.27
ME MJ 77.3 ± 3.9 82.9 ± 3.0 81.5 ± 2.6
EE g 206.4 ± 10.8 332.8 ± 9.7 318.9 ± 6.5
CP g 763.9 ± 40.2 877.5 ± 29.6 867.3 ± 26.5
DCP g 518.5 ± 26.5 618.2 ± 18.2 612.0 ± 23.2
NDF g 4556.3 ± 209.1 4338.6 ± 156.8 4358.9 ± 166.6
CF 2164.8 ± 108.0 2072.0 ± 74.4 2073.9 ± 83.0
DM = dry matter; ME = metabolisable energy; EE = ether extract (fat); DCP = digestible crude protein; CP = crude
protein; NDF = neural detergent fibre; CF = crude fibre.
Table 5. Average apparent digestibility coefficients (%) and standard deviations (s.d.) for the total diet
nutrients in the control and experimental diets.
Diet B Diet C p Value

Composition Diet A (Control) (Feed 1) (Feed 2) (B and C vs. Control Diet)
Dry matter 54.8 (5.29) 55.3 (2.54) 57.0 (3.96) 0.5065
Organic matter 56.9 (5.05) 57.2 (3.24) 58.9 (3.92) 0.5710
Crude protein 61.4 (6.20) 64.0 (4.46) 65.7 (5.83) 0.0182
Ether extract 56.2 (7.99) 68.0 (5.02) 68.8 (5.46) 0.0012
NDF 47.2 (6.01) 47.8 (4.17) 47.9 (4.16) 0.8139
Crude fibre 44.6 (5.27) 43.9(4.45) 46.3 (4.87) 0.8484
Ash 19.7 (20.08) 25.1 (8.74) 27.4 (10.68) 0.2093
NFE 62.8 (5.09) 62.0 (2.83) 63.5 (3.32) 0.9601
NDF = neutral detergent fibre; NFE = nitrogen free extract.
3.3. Blood Parameters

There were no statistically significant differences or trends (p > 0.05) in the blood parameters
between the diets (Table 6). The variation in the blood parameters (except ALT) was largest when the
diet C was fed to the horses. γ -glutamyltransferase (GT) activity was numerically (but not statistically)
somewhat higher, and the ALT activity lower, in the linseed supplemented diets. The average number
of WBC and concentration of fibrinogen were also numerically higher in linseed supplemented horses
but were within the reference values as well. Compared to the other horses, one horse had an
exceptionally high GT activity (23–37 U/l), and another individual a low ALT activity (2.0 U/l) during
the study period. The Hb and RBC values in all horses were low.
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Table 6. Average values of blood parameters and standard deviations (s.d.) for the horses in the control
and experimental diets.
Diet B Diet C Reference Values

Blood Parameters Diet A (Control)
(Feed 1) (Feed 2) [35,36]
Glutamyltransferase, U/l 17.0 (4.46) 19.7 (6.66) 18.0 (7.10) 10–70
Alanine aminotransferase, U/l 6.33 (2.02) 5.67 (2.57) 5.83 (1.95) 5–45
Haemoglobin, g/l 127.7 (7.27) 124.2 (5.64) 126.0 (11.03) 120–155
Haematocrit, % 34.7 (2.15) 34.3 (1.88) 34.4 (3.51) 34–43
Red blood cells, ×1012 /l 6.99 (0.55) 6.86 (0.55) 6.92 (0.89) 7.0–9.0
White blood cells, × 109 /l 7.35 (1.02) 7.25 (0.69) 7.23 (1.72) 4.6–9.5
Fibrinogen, g/l 2.67 (0.28) 2.78 (0.25) 2.86 (0.34) 1.2–4.0
None of the differences between the groups were statistically significant (all p > 0.05; p-values 0.6–1.0).
4. Discussion

Maintaining BW showed that the feeding level used equalled the energy needs [11] of the horses.
By period, intakes were isocaloric and isonitrogenous. However, the average fat (EE) intake of the
supplemental diets (B and C) was more than 1.5-fold greater compared to the control diet, and the CP
was 14.1% higher. In contrast, the differences were minor for energy and fibre components.
The palatability of the studied supplemental feeds was good. In our earlier unpublished study,
we observed that the palatability of plain linseed groats was not good when fed in large (more than 6%
in DM) portions. Delobel et al. [37] found no effects of flaxseed oil supplementation on diet palatability
in horses. The other ingredients (sugar-beet pulp, carrot and molasses) of the linseed supplements fed
in the present study likely improved the palatability of the supplemental feeds. Because of the 10%
content of dried garlic in the supplemented diets, the horses had to ingest approximately 120 mg/kg
BW dried garlic, which has been reported to be within the safe limits of garlic intake given by The
National Academies [38].

The improved digestibilities of dietary CP and EE when the supplemental feeds were fed is most
likely due to the high concentrations and intakes of those nutrients in the feeds, and is supported
by previous studies for CP [32,39,40] and EE [21,41]. In addition, Reitnour and Salsbury [42] found
that the caecal administration of linseed meal increased the digestibility of total diet protein. There is,
however, no evidence that fat and protein of linseed groats are more digestible than those of the control
diet. The improvements may also be partly attributed to the dilution of endogenous faecal nitrogen
and fat at higher intakes, which enhances their apparent digestibility [41,43–46].
Supplementing the diets with the experimental feeds caused a minor decrease in the NDF and
CP intakes. The supplemental feeds contained properly digestible fibre sources, carrot and molassed
sugar-beet pulp. Both of those ingredients are rich in dietary fibre of a soluble form, and have
approximately the same amount of CF, but the NDF content of sugar-beet pulp is approximately
two times that of carrot [30,47]. Sugar-beet pulp containing a lot of soluble and highly fermentable
fibre [48] has been reported to be well utilised by horses [49–51]. No data on the digestibility of
carrot is available. In addition, Snel et al. [52] (in their review of studies with different animal
species) and Dongowski et al. [53] (in rats) have reported that the dietary fibre in sugar-beet pulp may
have a prebiotic effect on intestinal flora improving the microbial activity and, thus, the digestibility.
Murray et al. [54] reported in horses that sugar-beet pulp enhanced total diet digestibility fed together
with forages. In addition, Lindberg and Palmgren Karlsson [44] explained their results with a positive
effect of soluble and fermentable fibre in horses when sugar-beet pulp and fat was added to horse diets.
As a potentially contributing factor, Clauss et al. [55] reported that nutrient supply to gut bacteria is
the major digestive constraint in horses.
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Because the experimental feeds contained many ingredients (being combinations of botanically
diverse fibrous feeds), comparisons with previous studies including different diet compositions,
and where linseed or variety of its by-products was used, are difficult. It is likely that the other
components have their influences too, and that there are confounding and synergistic effects of the diet
ingredients. Reitnour and Salsbury [42] found that the caecal administration of linseed meal decreased
diet DM digestibility. In our unpublished study, we found that the supplementation of plain linseed
groats from 0% to 10% (in the diet DM) gradually decreased the digestibility of the diet nutrients.
This has also been reported in dogs [8], and may be due to the poor digestibility of linseed husks
and the mucilage content of the hull. Linseed meal is also high in lignin [48], and most of the dietary
fibre in linseed meal is in an insoluble form [8]. These findings are supported by Takagi et al. [21]
(intakes were not given) who reported very low (21.8) digestibility for the crude fibre of linseed in
horses. In one study, the inclusion of extruded linseed (20% in DM) in the diets of horses decreased the
digestibility of nutrients compared to hay-only and hay/wheat bran diets [56]. In agreements with
the results of the present study, Smolders et al. [57] found increased digestibility values of the diet
nutrients when horses were fed compound feed containing (16%) linseed expeller plus more digestible
ingredients (cereals). In the present study, the intake of linseed groats was 6.3%–6.7% in DM, and when
combined with digestible fibre sources, also improved the diet’s digestibility. Inconsistency of the
results between studies is likely due to the different methods and processing of adding linseed or
by-products, and different compositions of the diets.
Concerning other animal species, low levels (8%–10% in the feed) of linseed meal in pig feed may
improve digestibility and growth rate, but 12% inclusion caused adverse effects [58]. Sled dogs can
utilise up to 4.2% linseed cake as a source of fibre without severe reductions in nutrient digestibility or
feed consumption [8]. In dairy cows, linseed supplementation improved total tract nutrient utilisation
without any adverse effects on ruminal fermentation [59].
Regarding the method applied in determining digestibility, Palmgren Karlsson [60] suggested that
chromium mordanted fibre could be an alternative to the administration of chromium, but may result
in underestimated digestibility values. In Särkijärvi et al. [33], however, chromium mordanted silage
gave quite precise digestibility values in horses (of a similar breed, gender and age as in this study).
4.3. Haematology Parameters

The serum concentrations of the liver enzymes alanine aminotransferase (ALT) and
γ-glutamyltransferase (GT) were within the reference values of Finnhorses, but close to their lower
limit [61]. Elevated ALT and GT values might have indicated possible detrimental effects of linseed
(cyanogenic glucosides) on the liver. Additionally, the average number of WBC and concentration
of fibrinogen were within the reference values. It can be concluded that there were no inflammatory
reactions in the bodies of the horses due to the diet. The large variation in the haematology parameters
when the Feed 2 diet was fed to the horses was likely due to the small number of horses used in
this study.
The Hb, HcT and RBC values in all horses were low and close to the lower limit of the reference
values of Finnhorses [61,62] regardless of the feeding group. The generally low Hb might be a result of
the low feeding and exercise intensity of the horses [63].
Based on the blood analyses, we concluded that no adverse health effects in horses were caused
from the supplementation of the diet with linseed groat-based feeds (offering approximately 6.3%–6.7%
linseed groats in the diet DM or 0.8 g/kg BW/d) during the nine-week experimental period. This is
supported by Vineyard et al. [19] who fed milled flaxseed for 70 days without any health problems.
No negative effects on health or performance of sport horses were found when linseed cake
(990 g/d) was fed for 60 days [62], which agreed with O’Neil et al. [15] who observed no negative
side effects when milled flaxseed was fed (1g/kg BW). The latter researchers concluded that stomach
acid can inactivate enzymes within the seeds, which are required to interact with glycosides to form
cyanide. Most glycosidases have a pH-optimum of around 7, so in herbivores with acid digestion, e.g.,
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horses, they are usually inactivated [26]. In pigs, Batterham et al. [24] report lighter kidneys, pancreas
and spleens for those animals given linseed, but no effects on the weight of livers were observed. They
concluded that this may be a result of the anti-nutritional factors of linseed. Mazza and Oomah [26]
concluded that most herbivores excrete the unhydrolysed cyanogenic compounds without harm to
the animal.
The content of the anti-nutritive compounds in seeds depends on the cultivar, location and
year of production, with the cultivar having the most significant effect [6,26]. The current and new
L. usitatissimum varieties for human nutrition are rather low in toxic and detrimental compounds [26],
as were the cultivars used in the present study (Neomed Ltd., Somero, Finland). According to
Abraham et al. [63], in case of missing or inactivated glucosidase, the hazard potential (to humans) is
low. Boiling is usually recommended to remove the potentially toxic cyanide components, and heat
processing of linseed reduces its content of cyanogenic glycosides. Thus, in the present study, both the
manufacturing process in the temperatures of 75–80 ◦ C, and soaking into 45–50 ◦ C water before feeding,
might also have decreased the content of possible harmful compounds of the linseed groats [6,26].
In addition, HCN content is reduced when linseed is mixed with several ingredients and when the
product is pelleted [64] as in the present study.
5. Conclusions
Linseed groat-based supplements (offering approximately 6.3%–6.7% linseed groats in the diet
DM or 0.8 g/kg BW/d), and containing soluble fibre sources (sugar-beet pulp, dehydrated carrot),
improved the crude protein and fat digestibility of hay/oats diets of horses, and had no effects on
fibre digestibility. No adverse or anti-nutritional effects were observed on the availability of any
component of the diet or the haematologic parameters and health of the horses. Linseed by-products
combined with other fibre sources can be used, for example, in feeding strategies replacing grains in the
horse rations in order to reduce the intake of starch. There is a need to investigate the synergetic and
confounding effects of diet ingredients of different sources, especially botanically diverse fibrous feeds.
Author Contributions: M.S. and S.S. contributed methodology, investigation and data curation; S.S. contributed
formal analysis and data analyzing; M.S. contributed resource, writing—original draft preparation, supervision
and project administration. All authors have read and agreed to the published version of the manuscript.
Funding: This research was carried out by an independent governmental research institute and partly (30%)
funded by Neomed Ltd.
Conflicts of Interest: The authors declare no conflict of interest. The funder had no role in the design of the study;
in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish
the results.
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31
animals
Article
The Effect of Diet Composition on the Digestibility
and Fecal Excretion of Phosphorus in Horses: A
Potential Risk of P Leaching?
Markku Saastamoinen 1, *, Susanna Särkijärvi 1 and Elisa Valtonen 2
1 Production Systems, Natural Resources Institute Finland (Luke), FI-31600 Jokioinen, Finland;
susanna.särkijärvi@luke.fi
2 Department of Animal Science, University of Helsinki, FI-00790 Helsinki, Finland;
[email protected]
* Correspondence: markku.saastamoinen@luke.fi
Received: 30 October 2019; Accepted: 12 January 2020; Published: 15 January 2020
Simple Summary: This study aimed to examine phosphorus utilization and excretion in feces when
typical feeds and forage-based diets are fed. The hypothesis was that feeding regimes might influence
phosphorus digestibility and excretion in feces, and therefore the environmental impact of horse
husbandry. We also studied the nutrient digestibilities of the diets, as well as the proportion of the
soluble fraction of P of the total phosphorus. Horse dung may pose a potential risk of P run-off into
the environment if not properly managed. Supplementation with inorganic P should be controlled in
the diets of mature horses in light work to decrease the excretion of P in feces.
Abstract: The main horse phosphorus excretion pathway is through the dung. Phosphorus originating
from animal dung and manure has harmful environmental effects on waters. The number of horses has
increased in many countries, and several studies have pointed that leaching of P from horse paddocks
and pastures are hotspots for high P leaching losses. The hypothesis was that feeding regimes might
influence phosphorus digestibility and excretion in feces, and therefore the environmental impact of
horse husbandry. A digestibility experiment was conducted with six horses fed six forage-based diets
to study phosphorus utilization and excretion in feces. The study method was a total collection of
feces. The experimental design was arranged as an unbalanced 6 × 4 Latin Squares. Phosphorus
intake increased with an increasing concentrate intake. All studied diets resulted in a positive P
balance and, the P retention differed from zero in all except the only-hay diet, in which the intake was
lower compared to the other diets. The digestibility of P varied from 2.7 to 11.1%, and supplementing
forage-diets with concentrates slightly improved P digestibility (p = 0.024), as it also improved the
digestibilities of crude protein (p = 0.002) and organic matter (p = 0.077). The horses excreted an
average of 20.9 ± 1.4 g/d P in feces. Excretion was smallest (20.0 g) in horses on a hay-only diet (p =
0.021). The average daily phosphorus excretion resulted in 7.6 kg P per year. The soluble P part of
the total P in feces accounted for about 88% of the P excreted in feces, and is vulnerable to runoff
losses and may leach into waters. Thus, horse dung may pose a potential risk of P leaching into the
environment if not properly managed, and is not less harmful to the environment than that from
other farm animals. Supplementation with inorganic P should be controlled in the diets of mature
horses in light work to decrease the excretion of P in feces.
Keywords: environment; horse nutrition; phosphorus loss; phosphorus supplementation;

phosphorus retention

Animals 2020, 10, 140
1. Introduction
Macro-mineral phosphorus plays an important role in bone formation as a constituent of
phosphoproteins, phospholipids, and nucleic acids, and in energy and fat metabolism [1–3]. In animals,
80–85% of phosphorus is stored in the bones and teeth, and the remainder in soft tissues and body
fluids [3]. In the skeleton, phosphorus forms hydroxyapatite with calcium. In growing animals,
the need for phosphorus is greater than in adult animals, since developing bones require more
phosphorus than already developed bones in adult animals [4].
Phosphorus deficiency is found throughout the world in areas with soil poor in phosphorus.
Deficiency symptoms include, but are not limited to, rice disease, osteomalacia, nervous system
symptoms, stiff joints, muscle weakness, poor fertility, impaired ovarian function and consequent
irregular rotation, poor growth in juvenile animals, and impaired weight gain in adult animals [1].
To avoid these detrimental effects of phosphorus malnutrition and ensure efficient intake, phosphorus is
usually routinely supplemented in horses’ diets.
Phosphorus absorption is influenced by the intake, source, and composition of the feed ration [5].
In adult horses, which mainly eat roughage, absorption efficiency is 35%, and in lactating mares and
growing horses it is 45% as their diets are often supplemented by larger amounts of concentrates [1].
There may be some improvement in phosphorus absorption as the need for phosphorus increases,
for example, through exercise or when the phosphorus content of the diet increases [6]. Fowler et al. [7]
concluded that yearlings can utilize organic P as well as mature horses.
The main site of the gastrointestinal tract of phosphorus absorption in horses is the dorsal colon,
but some phosphorus is also absorbed from the small intestine [8,9]. Fowler et al. [7] suggested
that degradation either occurs after the site of P absorption, or liberated P is recycled back into the
gastrointestinal tract.
The main phosphorus excretion pathway is through the dung. The phosphorus content of
feces is directly proportional to the phosphorus content of the diet [10]. Especially in a diet rich in
forage, the horse often gets too much supplemented phosphorus to meet its needs, and excess P is
excreted from the body in the feces [11]. A very low proportion (about 1%) of the phosphorus is
excreted in the urine [8,10,12], and is thus usually ignored in studies dealing with P digestibility.
In the gastrointestinal tract, endogenous phosphorus secretion occurs in the small intestine and in the
cecum [8,13]. Endogenous phosphorus secretion is due to the presence of phosphorus compounds in
gastrointestinal fluids such as saliva and gastric, pancreatic, and biliary fluids [8,14].
The digestibility of phosphorus is influenced by its form and amount in feed, and its interaction
with other feed components and minerals, e.g., Ca and Ca:P ratio [1,15]. Cereals are good sources
of phosphorus [3], but a significant proportion of the phosphorus in cereals is bound to phytic acid,
which is poorly digestible in monogastric animals [16]. However, the horse is able to digest phytate
phosphorus [7,17,18]. The content of phosphorus in grasses approximately equates to that in cereals
(about 3 g/kg DM), but the phosphorus content of grasses is significantly influenced by the age of the
crop at the time of harvest [19,20]. Mineral supplements usually contain inorganic forms of phosphorus
like monocalcium phosphate, dicalcium phosphate, or phosphorite as phosphorus sources [4].
Phosphorus and nitrogen originating from animal manure are the main environmental and water
pollutants from agriculture. There is imbalance of N and P in the manure, and P is considered more
harmful, because when it is in excess of crop requirements, soil becomes saturated resulting in P
runoff [21]. Because the number of horses has increased in many countries, and horses are kept in
paddocks and pastures there is risk of P leaching to the environment also from horse husbandry.
In addition, horse manure is widely utilized in agriculture. However, the NRC [1] considers horse
feces to be less risky to the environment compared to that from other farm animals because it assumes
that horse manure contains less water-soluble phosphorus, prone to runoff. However, later studies
have pointed that leaching of P from horse paddocks and pastures are hotspots for high P leaching
losses [22–25].
33
Animals 2020, 10, 140
In general, dietary strategies have been developed for many animal species to effectively reduce
the total P concentration in manure. As we were interested in the possible detrimental environmental
impact of horses, we studied phosphorus utilization and excretion in feces applying typical feeds and
forage-based diets fulfilling the current P -intake recommendations [1,19]. We also studied the nutrient
digestibilities of the diets, as well as the proportion of the soluble fraction of P of the total phosphorus.
The hypothesis was that feeding regimes might influence phosphorus digestibility and excretion in
feces, and therefore the environmental impact of horse husbandry.

A digestibility experiment was conducted with six forage-based diets typically fed to horses
in Finland. The study was conducted at the facilities of the Natural Resources Institute Finland
(Luke) in Southwest Finland. In animal handling and sample collection, the European Union
recommendation directives (2010/63/EU) and national animal welfare and ethical legislation set by
the Ministry of Agriculture and Forestry of Finland were followed carefully. The experimental
procedures were evaluated and approved by the national ethical committee for animal experiments
(https://www.avi.fi/web/avi/elainkoelautakunta-ella) (ESAVI/8331/04.10.07/2013).
2.1. Horses and their Management

Six adult Finnhorse mares (5–13 years; initial BW 552 ± 32 kg, mean BCS 6 = moderately fleshy)
owned by Luke were used in the study. All the experimental horses had the same managing and
feeding history before the trial. The horses were individually housed in stalls (3 m × 3 m) with peat
as bedding. The horses were de-wormed before the experiment. Dental care and vaccinations had
been carried out regularly prior to the experiment. The horses were freely exercised daily in groups in
outdoor paddocks (with sand grounds) for 2–4 h, except during the collection period, when they were
led in walk by a rope in the stable corridors (consisting of two connected 32 m long corridors with
concrete and asphalt floors) for 15 min. In the paddocks, the horses had masks to prevent sand eating.
The study method was a total collection of feces. The experimental design was arranged as
an unbalanced 6 × 4 Latin Squares. The study consisted of six treatments and four 21-day periods.
Each period started with a five-day feed change period followed by 12 days of adaptation to the new
diet, and four-day period of collecting feces samples. During the collection period, the peat bedding
was changed to rubber mats. The body weight (BW) (electronic animal scale Lahden Vaaka/Lahti
Precision Ltd., Lahti, Finland) and body condition score (BCS) [26] of the horses was monitored after
each collection period.
2.2. Experimental Feeds and Feeding

The diets were formulated and adjusted to be as isocaloric as possible. The horses were individually
fed at a level of 65–75 g DM kg −1 W0.75 , corresponding to the feeding and energy level recommended in
light work in accordance with the Finnish Feed Tables and Feeding Recommendations [19]. The regularly
obtained BCS and BW were used to control their possible changes, and the individual energy intakes
were adjusted if necessary. The horses were fed three times per day (at 6:00 a.m., 12:00 noon and
6:00 p.m.), except in the mornings of blood sampling days, when the forages were fed at 7:30 and
concentrates at 8:00 o’clock.
The diets were (dry matter basis) (A) hay 100%; (B) haylage 100%; (C) hay 80% + whole oats 20%;
(D) hay 65% + whole oats 35%; (E) hay 80% + commercial pelleted complete feed 20% (Lantmännen,
Malmö, Sweden) and (F) hay 65% + commercial pelleted complete feed 35%. All diets except those
including the complete feed were balanced with a mineral mixture (Ca:P = 3.57) in which P was in the
form of monocalcium phosphate (Vilomix Ltd., Paimio, Finland) according to the P and Ca needs of the
horses. The complete feed (the added P was in the form of monocalcium phosphate, Ca:P 1.43) covered
the mineral requirements of the horses. Forages were fed from special hay-boxes to avoid dropping,
and the concentrates were fed from feed mangers. Free water was available from float valve drinkers.
34
Animals 2020, 10, 140
The dried hay was produced by a local farmer in Ypäjä (60◦ 48 34 N, 23◦ 16 35 E). The haylage
(Prohay Ltd., Punkalaidun, Finland, 61◦ 06 40 N, 23◦ 06 20 E) was packed in 20 kg air tight plastic
packages and purchased from the producer. The oats were produced by Luke.
Feed samples were collected daily and stored at –20 ◦ C until analysis. The samples were analyzed
at the Luke Laboratories (Jokioinen, Finland) for dry matter (DM), crude protein (CP), NDF, ADF,
crude fiber (CF), and ash with standard wet chemical methods e.g., [27], as well as for P and Ca content
using the method by Huang and Schulte [28]. The chemical composition of the feeds is presented in
Table 1.
Table 1. Average chemical composition (g/kg DM) and energy value (ME MJ/kg DM) of the
experimental feeds.
Composition Hay Haylage Oats Complete Feed Mineral Mixture 1

Dry matter 83.6 59.4 86.1 86.9 96.0
Crude protein 82.1 122.5 107.0 124.4
Crude fiber 318.7 326.7 103.9 113.5
NDF 596.0 615.0 260.5 305.4
ADF 317.5 326.6 104.3 123.3
Ash 62.3 67.6 32.2 74.0 737.8
Calcium 2.4 2.9 0.5 7.0 156.4
Magnesium 1.2 1.3 1.3 4.2 45.0
Phosphorus 2.2 2.7 3.6 4.9 43.6
Energy ME 9.7 9.6 12.0 11.0
NDF = neutral detergent fiber; ADF = acid detergent fiber; 1 Inorganic mineral sources: monocalcium phosphate,
limestone meal, magnesium oxide; ME = metabolisable energy.
2.3. Feces Sampling and Analysis

Feces were collected for four days (Monday–Friday). Before the start of the collection period,
the peat bedding was removed from the boxes and replaced with rubber mats. The feces collected
overnight (every day at 9:00 a.m.) was thoroughly mixed and weighed, and a representative sample
was taken. Partial samples were pooled throughout the collection period and stored at −20 ◦ C. The daily
amount of sampled feces was 12% of the total daily amount collected. Feces that were entangled with
foreign substances such as urine were defined as waste. The amount of waste feces was weighed but
not utilized in the analysis.
Fecal samples were analyzed at the Luke Laboratories for dry matter (DM), nitrogen, NDF, ADF,
CF ash as well as total and (water) soluble P content. Nitrogen was analyzed with the Khjeldal
method (AOAC-984.13) using Foss Khjeltec 2400 analyzer (Foss Analytical AB, Höganäs, Sweden),
and the CP content was calculated as 6.25 × N. NDF and ADF were analyzed with ANKOM 220
fiber analyzer (ANKOM Technology, Macedon, NY, USA) using 25 μm nylon bags [29,30]. Total P
was analyzed spectrometrically (ICP-OES, Thermo Jarrel Ash Iris advantage, Franklin, MA, USA).
The proportion of soluble P (phospahte-P, PO4 -P) was analyzed from 1:60 water extracts using a
continuous photometric flow analyzer (Aquakem 250, Thermo Fisher Scientific Inc., Vantaa, Finland)
as described by Keskinen et al. [31]. Because only a very low proportion (about 1%) of the phosphorus
is excreted in the urine [8,10,12], urine was not collected in this experiment.
2.4. Blood Samples and Analysis

Blood samples were collected 90 min after the morning meal every Wednesday during the
collection period. A blood sample was drawn from the jugular vein into two sample tubes (2 × 10 mL).
The samples were analyzed for inorganic P and Ca photometrically using wavelengths 660 nm and
340 nm, respectively. The analyses were performed in a clinical authorized laboratory (Ellab Ltd.,
Ypäjä, Finland).
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Animals 2020, 10, 140

Differences in digestibility, as well as intake, excretion and retention parameters between the diets
were statistically analyzed using the SAS (SAS 9.3, 2008) GLM procedure (SAS Institue, Cary, NC, USA)
applying the following statistical model:
Yijk = μijk + ai + pj + dk + eijk (1)
where μijk is the overall mean, ai is the random effect of the animal (i = 1 . . . 6), pj is the fixed effect of
the period (j = 1 . . . 4), dk is the fixed effect of the diet (k = 1 . . . 6), and eijk is the normally distributed
error with a mean of 0 and variance δ2 . The differences between the diets were tested with orthogonal
contrasts: (1) B vs. A and C-F; (2) A vs. C-F; (3) C and D vs. E and F; (4) C vs. E and D vs. F; and (5)
the interactions between the type of concentrate and concentrate level C and D vs. E and F, C vs. E,
and D vs. F. Concerning the retention values, it was also tested if they differ from zero.
Differences in the proportion of soluble P of the excreted P were not studied because the diets
were composed of different ingredients containing various sources of P (inorganic and organic sources,
phytate P) that were not analyzed.
3. Results

The feed, energy and nutrient intakes for each diet are presented in Table 2. The DM intake was
smallest in horses eating only haylage (p < 0.001), but same time they had the largest CP intake (p < 0.001).
Concerning the concentrate supplemented diets (hay + oats or complete feed), the concentrate level
did not affect the DM intake. The horses maintained their BW and BCS during the experiment (mean
initial BW = 552 ± 32 kg, mean final BW = 558 ± 32 kg).
Table 2. Mean daily energy (MJ ME), dry matter (g) and nutrient intakes (g) for the experimental diets.
Diet A B C D E F Statistical Significance (p-Values)

Pooled
SEM Haylage Hay vs. Oats vs. ConT ×
Forage Hay Haylage Hay Hay Hay Hay ConL
vs. Others ConS Comp ConL
ConL 0 0 O20 035 C20 C35
ME 74.3 73.3 79.1 82.6 77.6 78.7 0.4 <0.001 <0.001 <0.001 <0.001 0.009
DM 7536 6702 7568 7734 7601 7595 17.0 <0.001 0.038 0.037 0.439 0.338
OM 6981 6186 7148 7250 7081 7075 47.3 <0.001 0.033 0.027 0.368 0.272
CP 607 812 647 705 685 744 17.3 <0.001 0.014 0.047 0.009 0.979
CF 2366 2155 2079 1852 2102 1873 16.4 <0.001 0.001 0.196 <0.001 0.946
NDF 4427 4060 3992 3647 4073 3753 33.9 0.055 <0.001 0.018 <0.001 0.715
ADF 2361 2155 2076 1845 2109 1891 18.8 <0.001 <0.001 0.058 <0.001 0.746
Ash 556 516 510 485 519 521 17.0 0.901 0.033 0.202 0.519 0.446
ME = metabolisable energy MJ/day; DM = dry matter; OM = organic matter; CP = crude protein; CF = crude fiber;
NDF = neutral detergent fiber; ADF = acid detergent fiber; O = Oats; C = Complete feed; ConL = Concentrate
level (20 or 35% of oats O or complete feed C); ConS = Concentrate supplementation; ConT = concentrate type
(oats/complete feed); Comp = complete feed.
3.2. Intake, Fecal Excretion and Digestibility of Phosphorus

The average P intake was 22.0 ± 2.0 g/d. The P intake increased with an increasing concentrate
intake (Table 3). Horses ingesting oats had larger daily intake of P (22.8–24.8 g) compared to those fed
with the complete feed (20.9–24.1 g) (p < 0.001) and horses fed with hay only had smaller intakes than
horses supplemented with concentrates (20.6 vs. 20.9–24.8 g) (p < 0.001). Horses fed with haylage had
somewhat smaller P intake than those who ate hay (p = 0.036).
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Animals 2020, 10, 140
Table 3. Daily intake (g), fecal excretion (g), digestibility (%) and retention (g) of phosphorus (P).
A B C D E F Statistical Significance (p-Values)
Diet/Forage Pooled Haylage ConT

Hay Oats
Hay Haylage Hay Hay Hay Hay SEM vs. ConL ×
vs.ConS vs.Comp
Others ConL
ConL 0 0 O20 O35 C20 C35
Intake P 20.6 22.1 22.8 24.8 20.9 24.1 0.21 0.036 <0.001 <0.001 <0.001 0.027
Excretion P 20.0 20.2 21.5 22.1 19.9 21.5 0.42 0.125 0.021 0.025 0.033 0.251
Digestibility P 2.7 8.0 5.6 11.1 4.9 10.6 1.98 0.652 0.037 0.761 0.024 0.974
Retention 0.6 1.9 1.0 2.8 0.9 1.9 0.45 0.354 0.075 0.25 0.014 0.379
O = oats; C = complete feed; ConL = concentrate level (20 or 35% of oats O or complete feed C); ConS = concentrate
supplementation; ConT = concentrate type (oats/complete feed); Comp = complete feed.
The average daily quantity of dung was 15.6 ± 2.5 kg/horse. The horses excreted an average
of 20.9 ± 1.4 g/d P in feces. Excretion was smallest (20.0 g) in horses on a hay-only diet (p = 0.021)
(Table 3). Horses supplemented with oats excreted somewhat more P (21.5–22.1 vs. 19.9–21.5 g) than
those supplemented with the complete feed (p < 0.025), and the excretion increased with increasing
concentrate intake (p = 0.033).
The horses were on a positive P balance in all diets (Table 3). The retention of P was largest in the
diet D (with the highest complete feed level) being 2.8 g/d. The retention values were different from
zero for the diets B (p = 0.002), C (p = 0.05), D (p <0.001), E (p = 0.08), and F (p = 0.002). The P retention
increased (p = 0.0145) with the increasing concentrate level. Feeding concentrates slightly improved P
digestibility (p = 0.024). The amount of water-soluble phosphorus of the P excreted in feces was 18.3 ±
2.5 g/d, on average. This corresponds to 87.6% of the P in feces.
3.3. Intake, Fecal Excretion and Digestibility of Calcium and Magnesium

The intake of calcium was largest in the horses fed with haylage only (p > 0.001) (Table 4).
Ingestion of concentrates decreased Ca intake, depending on type of concentrate fed. Mg intake
was smallest in the horses on a haylage-only diet. The intake increased when concentrates were fed.
The increase was largest with complete feed (interaction p < 0.001).
Table 4. Daily intake (g), excretion (g) and digestibility (%) of calcium (Ca) and magnesium (Mg).

Diet/Forage Pooled
Hay Haylage Hay Hay Hay Hay ConL
ConL 0 0 O20 O35 C20 C35
Intake
Ca 32.6 34.2 29.6 28.3 24.7 30.4 0.45 <0.001 <0.001 0.010 0.001 <0.001
Mg 9.31 8.71 9.25 9.74 13.9 17.3 0.26 <0.001 <0.001 <0.001 <0.001 <0.001
Excretion
Ca 19.1 17.7 17.5 17.8 19.4 20.2 0.98 0.330 0.757 0.048 0.610 0.797
Mg 10.4 10.1 10.1 10.4 10.6 12.9 0.28 0.030 0.107 <0.001 0.002 0.006
Digestibility
Ca 41.6 47.1 29.3 38.1 22.6 31.7 7.10 0.096 0.187 0.371 0.265 0.987
Mg −13.1 −19.0 -9.0 -7.0 23.4 25.3 4.73 0.001 0.002 <0.001 0.702 0.993
O = Oats; C = Complete feed; ConL = Concentrate level (20 or 35% of oats O or complete feed C); ConS = Concentrate
Concerning the excretion of minerals, horses on the haylage-only diet excreted somewhat less Mg
than fed with hay only (p = 0.03). Comparing the concentrates, horses supplemented with complete
feed excreted more both Ca and Mg than those supplemented with oats (p = 0.048 and p < 0.001,
respectively). The digestibility of Ca did not differ between the diets. Concerning Mg, digestibility was
lowest in the haylage-only diet, and lower when oats was fed compared with feeding the complete
feed. The variation in Mg digestibility values was large (Table 4).
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3.4. Digestibility of the Diet Nutrients

The dry matter digestibility of the haylage-only diet was lower compared with the other diets
(p < 0.001) (Table 5). Supplementing the forage diets with concentrates improved the digestibilities of
crude protein (p = 0.002) and organic matter (p = 0.077) but the concentrate level fed did not affect the
digestibility of the fiber fractions. The CP digestibility of the haylage-only diet was better (p = 0.009)
compared to the other diets. Correspondingly, the CP digestibility of the hay-only diet was the lowest
(p = 0.004).
Table 5. Apparent digestibility coefficients (%) of the diet nutrients.

Diet/Forage Pooled
Hay Haylage Hay Hay Hay Hay ConL
ConL 0 0 O20 O35 C20 C35
DM 55.0 49.9 56.6 59.3 57.5 60.1 1.33 <0.001 0.046 0.543 0.090 0.998
Ash 38.4 31.6 29.8 29.6 34.9 37.7 2.73 0.437 0.105 0.033 0.666 0.591
OM 56.3 51.4 58.5 61.3 59.1 61.8 1.29 <0.001 0.023 0.699 0.077 0.972
CP 50.9 63.6 51.7 61.8 57.3 63.6 1.83 0.009 0.004 0.066 0.002 0.310
CF 49.5 45.7 46.1 43.0 49.6 56.5 2.61 0.685 0.291 0.206 0.294 0.997
NDF 49.5 46.3 46.3 42.5 49.0 47.5 2.44 0.811 0.278 0.144 0.334 0.635
ADF 47.2 41.6 41.7 41.4 45.5 40.5 3.40 0.668 0.224 0.669 0.488 0.497
DM = dry matter; OM = organic matter; CP = crude protein; CF = crude fiber; NDF = neutral detergent fiber;
ADF = acid detergent fiber. O = Oats; C = Complete feed; ConL = Concentrate level (%); ConS = Concentrate
3.5. Blood Concentrates of P and Ca

The between diet variation of the blood serum P and Ca concentrations was small. The average
blood serum P concentration was 1.16 ± 0.04 mmol/L. Comparing concentrate types, the concentration
was larger when oats was fed than when the complete feed was fed (1.21 vs. 1.13 mmol/L) (p < 0.031).
The mean blood Ca concentration of the horses was 3.14 ± 0.04 mmol/L. It was larger when the horses
were on a hay-only diet compared with the diets containing concentrates (3.22 vs. 3.12 mmol/L)
(p < 0.001).
4. Discussion
The main goal of this experiment was to find the differences between the diets for P utilization,
rather than the actual values for specific diets. Because studying digestibilities of other nutrients was
only a secondary aim of this study, the results are discussed only briefly.
4.1. Feed Values and Nutrient Intakes

The feed values corresponded to the analyzed values presented for Finnish forages produced
for horses [32], the hay being of “medium nutritional quality” and the haylage of “high nutritional
quality”. Concerning oats, the CP content was lower than that presented in the Finnish Feed Tables and
Feeding Recommendations (10.4. vs. 12–13%) [19]. The NDF, ADF, and CF values were also lower than
the values presented for average Finnish oats [19]. The P, Ca, and Mg content of the forages and oats
was lower than the values presented for hays and haylages [19]. The mineral content was also clearly
lower than reported for Norwegian and Swedish haylage samples collected from horse farms [20].
The nutrient intakes naturally varied because of the differences in the composition of the feeds
and actual intakes, although the individual diets were initially formulated and balanced to correspond
the needs of each horse [19] and be as isocaloric as possible. The smaller DM intake of the horses in the
haylage-only diet resulted from restriction of haylage intake because of its high CP content. This led
naturally also to smaller energy (ME) intake. In addition, the size of the horse affected the individual
daily portion such that larger horses had larger portions. The horses maintained their BW and BCS
during the experiment, indicating that the feeds, feeding regime, and intakes applied covered the
nutritional requirements of the horses (energy and protein) in the course of the experimental period
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Animals 2020, 10, 140
The mean daily energy intake of 77.5 MJ ME/d during the course of the experiment agreed with the
recommendation for horses in light work [19].
4.2. Intake, Fecal Excretion and Digestibility of Phosphorus

The differences in the P intake between the diets were due to the differences in the feeds and diet
compositions, but the forms of P were not analyzed. P concentrations in oats and complete feed were
higher than in the forages. In addition, the oat supplemented diets were balanced for minerals (P and
Ca) with a mineral supplement mixture which also increased the P intake. The intakes of P were in
accordance with the current recommendations [1,19], except on the highest concentrate levels where
they exceeded the recommendations.
The excretion of phosphorus observed here was within the ranges presented in the
literature [7,12,18,33,34]. P excretion is linearly related to its intake, and the intake increases with the
increasing concentrate ingestion [10,12,33]. Van Doorn et al. [35] concluded that horses can regulate P
digesting and thus P balance. The extra P can be excreted in the feces.
In the present study, the digestibility of P in adult horses varied from 2.7 to 11.1%, improving
with an increased concentrate intake. This is well in line with the results of van Doorn et al. [18] for
adult horses (2.4–15.4%). However, higher values (4.2–28.7%) have also been reported [15,34,36] for
adult horses (with a range between 4 and 25 years). In many studies the digestibilities show impaired
values with increasing age. The largest digestibilities (37–42%) have been reported for young horses
(8-months-olds), but they decline quickly (to 2.0–7.7%) when the horses are between one and two
years old [7,12]. Elzinga et al. [36] reported digestibility of 4.2% for aged (19–28 years) horses. P
digestibility is therefore influenced by the age of the horse. In this study, the horses were between
5 and 13 years old, and P digestibility seems to accord well with previous studies when the age of
the horses is considered. In previous studies, higher values have also been reported when hay +
concentrates were fed e.g., [6,7,15,18,34,35] compared with forage-only diets [12,28]. The digestibility
of P may also improve somewhat with increasing P intake [6]. Furthermore, digestibility is affected by
the components (feeds) of the diet [1].
Diets with the highest concentrate levels (35% oats or complete feed) had better digestibility
compared to the other diets. All diets resulted in a positive P balance and the P retention differed
from zero in all except the only-hay diet, in which the intake was lower compared to the other
diets. According to previous studies [10,18], the P retention increased with P intake in adult animals.
The P retention values observed in our study were smaller (less than half) than reported for adult
(appr. 6-year-olds) Standardbred horses [18] fed mixed hay + concentrate diets, but in the same time,
the intakes were also correspondingly smaller. According to that study, the reasons for the P retention
in adult horses are not known. However, Buchholz-Bryant [37] reported a higher P retention in mature
horses (7 to 11 year old) at rest compared to exercised horses. In the present study the horses were
only freely exercised daily in outdoor paddocks for 2–4 h, and during the collection period, they were
walked manually in the stable corridors for 15 min In the study of Van Doorn et al. [18], the horses
were given 1-h walk on a treadmill, and during the collection period they walked manually for 10 min
indicating rather light work level. Thus, the light work load of the horses may be one reason for
the P retention in our study. In addition, because phosphorus is stored in many body tissues and
fluids [1–3], some P can be accumulated to these, too. The retention values presented here includes
also the urine P, because urine was not collected and analyzed for P. This was done, because according
to studies [8,10,12], only a very low proportion (about 1%) of the phosphorus is excreted in the urine.
The Ca:P ratio of the feed or diet affects not only the digestibility of phosphorus but also the
digestibility of calcium [15]. A high calcium intake can impair phosphorus digestibility at a Ca:P
ratio of 2.58 or more [1,18]. However, the dietary Ca:P ratios in the current study were much lower,
1.14–1.58. Concerning fecal Ca:P ratios, Böswald et al. [38] found that in horses (and other large
hindgut fermenters), the fecal Ca:P ratio is lower than the dietary Ca:P ratio. The present study was in
accordance with this, the mean fecal ratios ranging from 0.80 to 0.95.
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Animals 2020, 10, 140
4.3. Intake, Fecal Excretion and Digestibility of Calcium and Magnesium

Calcium and magnesium intakes also differed between the diets because of the differences in
the mineral content of the feeds. The intakes of Ca and Mg agreed with the recommendations [1,19],
with the exception that Mg intake was above the recommended values when complete feed was fed.
Intakes of Ca from the concentrates were small. Oats was low in Ca, and the increased proportion of
oats and decreased proportion of forage in the diet resulted in a decline in Ca intake. Concerning the
intakes of Mg, the larger Mg content of the complete feed compared to that of oats explains the
differences in Mg intake.
The excretion of Ca in the present study was smaller than reported in the literature e.g., [7,18]
because the intake was smaller. It is also likely that different Ca sources have an influence. The Mg
excretion agreed with previous studies [7,18].
The amounts of excreted Ca and Mg in feces are related to intakes [39,40], but no effect of intake
was observed by Nielsen et al. [33]. Meyer et al. [41,42] reported that diets containing more roughage
resulted in a higher renal excretion of Ca and Mg.
The observed digestibilities of Ca not differing between the diets agreed with the literature
values [2,7,18,35]. Van Doorn et al. [18] have reported that high amounts of P and phytate P may
decrease Ca digestibility. The larger excretion of Mg compared with its intake in all diets, except those
including the complete feed, explains, the poor Mg digestibility in these diets. The digestibility of Mg
has been reported to be largely varying, and negative values have also been reported [2,7,18,35], as in
the present study. Because Ca and Mg are excreted in large quantities in urine [9,40], the observed
digestibilities here may be underestimated.
4.4. Digestibility of the Diet Nutrients

It is very likely that the lower DM digestibility of the haylage-only diet was due to its larger fiber
content (NDF, ADF, CF) compared to the other diets. The better CP digestibility of the haylage-only diet
was due to its larger CP content, and correspondingly, the poor CP digestibility of the hay-only diet was
due to its low CP content. These results are supported for example by Särkijärvi and Saastamoinen [27]
and Ragnarsson and Lindberg [43,44]. The positive effect of including concentrates in the forage diets
agrees with previous studies, being e.g., due to the lower NDF content of the diet e.g., [45,46]. The NDF
content also explains the digestibility value differences between the forage diets. The digestibility
values observed in the present study are comparable with those reported previously for Finnhorses of
the same age e.g., [27,47].
4.5. Blood Concentrates of P and Ca

The blood phosphorus levels are affected by phosphorus intake [48,49], which may explain
why blood levels were highest in the horses whose diets were supplemented with oats.
Greiwe-Crandell et al. [50] suggested that mares fed an all-forage diet marginal or low in phosphorus
may mobilize P from bone.
The blood Ca concentration was largest in the horses on the hay-only diet. Meyer et al. [42] reported
higher plasma Ca levels for forage fed horses than concentrate fed horses. Some other studies, however,
pointed out that the blood Ca concentration does not depend on the Ca intake [48,49]. Regarding all
diets, the average blood serum Ca and P concentrations were within the normal ranges used for
Finnhorses (https://www.movet.fi/laboratoriokasikirja/). To maintain physiologic Ca and P blood levels,
mammals can absorb them from the gastrointestinal tract or change their bone turnover [38].
4.6. Impact of Horse Diets on P Leaching

The daily quantity of dung produced by the horses was in line with the literature values [7,51,52],
depending, however, on diet and feed intake. The average daily phosphorus excretion of about 21 g in
feces in this study, when typical diets and current recommendations [1,19] were applied, resulted in
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Animals 2020, 10, 140
7.6 kg P per year. If the P is not properly absorbed in stable beddings, or if the dung in paddocks is
altered by rain and water from melting snow, P in feces and manure may present an environmental
risk when leaching into waters.
The soluble P part of the total P in feces available for the utilization of plants accounted for about
88% of the P excreted in feces in this study. Ögren et al. [12] reported a proportion of 80% of soluble
P. The P that is unavailable is vulnerable to runoff losses. According to Chapuis-Lardy et al. [53],
excess dietary P is excreted in feces in water-soluble forms. Dougherty et al. [54] pointed out that
around 90% of P losses occurred in water-soluble form. The leaching P from the dung of horses is
mainly inorganic [55]. Consequently, the argument of the NRC [1] that horse manure is less harmful
to the environment compared with that from other farm animals because of its low proportion of
water-soluble P, is not correct in the light of the results of this and previous studies.
It is possible that the composition of the diet affects the solubility of P, as reported for dairy
cows and pigs [56,57]. However, in this study it was irrelevant to compare the diets because of their
composition, i.e., the inclusion of various P sources in the same diet. Further studies can be suggested
to be carried out concerning this issue also in horses. Ögren et al. [12] concluded that soluble P has a
strong positive relationship to P intake in horses.
As P loss is linearly related to its intake in various animal species [12,53,58], it is impossible to
conclude how polluting horse industry is compared with other forms of animal production. However,
in the study of Ögren et al. [12], the high proportion of inorganic P in horse feces indicated that P
overfeeding of horses might be more harmful to the environment than P overfeeding of dairy cows.
Previously, several other authors have also reported that horse paddocks may pose a high risk of
extensive P loss [22–25,55,59]. Regular removal of dung from paddocks is recommended to minimize
this risk [25,55,60,61]. How often this should be done naturally depends on the time the horses spend
in the paddocks and livestock density/ha. Phosphorus sorbing materials (e.g., Fe containing) [22,59],
filtering materials (geotextile-gravel) [61], or organic (bedding) materials [62] can also be used on
paddock surfaces to reduce leaching loss.
Ögren et al. [12] concluded that an increase in the P requirement for growing horses is not
justified. The present study shows that it is unnecessary to supplement the diets of mature horses,
especially those in light work, with inorganic phosphorus, when the diets are supplemented with
concentrates. According to Fowler et al. [7], the organic P in feeds may fulfill the needs of horses
in light work, and no supplementation with inorganic P is needed. Balancing the diets for P intake
can be estimated to save both money and environment in dairy production [63]. There may also be
economic motives to catch the P in feces and absorb it in bedding materials, because the use of horse
manure may reduce fertilizing costs. When horse manure is composted, its nutrients can be recycled
and utilized [31], which reduces the use of inorganic fertilizers.
In addition, optimizing the proportions of the diet components, for example by supplementing
the forage diets with concentrates, may improve the digestibility of phosphorus. However, it is
necessary to analyze the feeds for the mineral concentrations because of the large variation [20,32,64].
In complete feeds for horses, P (and other mineral) concentrations are usually formulated to cover the
requirements of an “average horse” when “medium-quality” forages are fed. They thus do not take
into account the true mineral concentrations in the other components of the diet. This may result in
over- or undernutrition in practical feeding. As in this study, they also contain the added P in inorganic
form. In addition, when increasing the proportion of concentrates in the daily ration, the possible
detrimental effects of starch [65] have to be considered. In the present study, the concentrate levels fed
were not very large, and the diets were based on forages.
5. Conclusions
Horse dung may pose a potential risk of P leaching into the environment, and is not less harmful
to the environment than that from other farm animals if not properly managed, because most of the
P in feces is in soluble form. Supplementation with inorganic P should be controlled in the diets of
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Animals 2020, 10, 140
adult horses in light work to decrease the excretion of P in feces. Supplementing forage diets with
concentrates may improve the digestibility of phosphorus and, thus, improves the availability of P to
horses. More research especially into cost effective feeding strategies and their applications for horses
is essential, e.g., concerning diet composition and ingredients, to reduce horse industry’s harmful
impacts on and risks to water quality.
Author Contributions: M.S. and S.S. contributed methodology and investigation; S.S. contributed formal analysis;
M.S. contributed resource, writing—original draft preparation, supervision; E.V. contributed experimental part and
investigation, and data analyzing. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Finnish Ministry of the Environment (YM 126/481/2012).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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45
animals
Article
Characterization of Feeding, Sport Management,
and Routine Care of the Chilean Corralero Horse
during Rodeo Season
Joaquín Bull 1 , Fernando Bas 1 , Macarena Silva-Guzmán 2 , Hope Helen Wentzel 3 ,
Juan Pablo Keim 4 and Mónica Gandarillas 1,4, *
1 Departamento de Ciencias Animales, Facultad de Agronomía e Ingeniería Forestal, Pontificia Universidad
Católica de Chile, Avda. Vicuña Mackenna 4860, Santiago 7820436, Chile; [email protected] (J.B.); [email protected] (F.B.)
2 Private statistical consultant, Guardia Vieja 441, Santiago 7510318, Chile; [email protected]
3 Escuela de Graduados, Facultad de Ciencias Agrarias, Universidad Austral de Chile, Valdivia 5110566, Chile;
[email protected]
4 Instituto de Producción Animal, Facultad de Ciencias Agrarias, Universidad Austral de Chile,
Independencia 641, Valdivia 5110566, Chile; [email protected]
* Correspondence: [email protected]
Received: 1 August 2019; Accepted: 11 September 2019; Published: 17 September 2019
Simple Summary: The Chilean corralero horse holds great cultural importance due to its use
in Chilean rodeo, the national sport. However, information regarding this breed is sparse, especially
husbandry, feeding, and training recommendations, which could present challenges for their proper
care. A survey of horse farms in several regions from central to southern Chile was conducted in order
to document current management of the Chilean corralero horses which participated in the 2014–2015
Chilean Rodeo Federation season. In the survey, horse owners and trainers were asked about horse
gender and size, daily routine, exercise and competition regimen, and feeding practices. All horses
in the study were kept in stalls for at least 12 h daily and spent the rest of the day either tied or loose
in pens or paddocks. Horses were in moderate- to high-intensity exercise programs, with workouts six
days/week and two rodeos per month. Feeding practices varied greatly among farms but most horses
received forage (alfalfa or grass hay) and an energy feed (oats, corn, or concentrate), while protein
and lipid supplements were less common. The goal of this characterization of current management
of the Chilean corralero horse is to contribute to information available about this breed to improve
husbandry practices.
Abstract: The aim of this study was to characterize the routine care, training, feeding, and nutritional
management of Chilean corralero horses that participated in the rodeos of the Chilean Rodeo
Federation. Forty-nine horse farms between the Metropolitan (33◦ 26 16” south (S) 70◦ 39 01” west
(W)) and Los Lagos Regions (41◦ 28 18” S 72◦ 56 12” W), were visited and a survey was conducted on
the management and feeding of the Chilean horse. Of the horses which participated in at least one
official rodeo in the 2014–2015 season, 275 horses were included in the study. The survey consisted of
five questions about general data on the property and the respondent, four questions on the animal
characteristics, five questions about where the horses were kept during the day, seven questions to
characterize the amount of exercise done by the horse, and 18 questions about feeding practices;
additionally, the amount of feed offered was weighed. All horses in this study were in training and
kept in their stall for at least 12 h and remained tied or loose for the rest of the day. The intensity
of daily exercise of the rodeo Chilean horse could be classified as moderate to heavy and consisted
of being worked six days/week and participating in two rodeos/month. Ninety-eight percent of
respondents had watering devices in the stables. The diet of the Chilean corralero horse during
the training season is based on forages, mainly alfalfa hay, plus oats as an additional energy source.
Protein supplements such as oil seed by-products are used less frequently. A wide variation was

Animals 2019, 9, 697
observed in the diets and quantities of feed offered, which suggests that the feeding management of
these individuals is not formulated according to their requirements.
Keywords: Chilean corralero horse; rodeo; feeding practices
1. Introduction
Horses served humans for centuries as a source of food, as well as for military, agricultural
labor, and sport purposes [1]. The International Federation for Equestrian Sports (FEI) recognizes
eight disciplines for competition (dressage, jumping, vaulting, endurance, reining, combined driving,
eventing, and para-equestrian). Nevertheless, there are several other widely known equestrian sport
competitions worldwide, such as polo and racing.
In Chile, “rodeo” was declared the national sport in 1962 (Decree 269 of the National Council of
Sports), when it became part of the Olympic Committee of Chile [2]. This sport is one of the culturally
rich activities that take place in the Chilean countryside. It symbolizes traditional cattle-working and
is part of the traditional folklife with its own customs [2,3]. After football (soccer), rodeo is the second
most popular sport in Chile due to its massive following and because it became an important cultural
symbol throughout national history.
The breeding aim for the Chilean corralero horse is to produce a horse that is suitable and
functional for saddle and stock work, as well as practicing rodeo [4].
The rodeo is performed in an oval-shaped area and consists of a pair of riders (each rider is called
a “huaso”) and their horses (“collera”) running half laps around the arena while working a steer and
attempting to pin it against a large 12-m cushion (“quincha”). Horses run a total of approximately
400 m at 6.95 m/s in each round of the competition [5]. The rodeo is highly regulated by the Chilean
Rodeo Federation, and there are 311 officially registered arenas throughout the national territory [6],
with 350 competitions per season, which begins in August each year and ends with the national
championship in April of the following year.
Despite the national importance of rodeo, there is little information available and easily accessible
about the Chilean corralero horse, specifically related to equine numbers, competing animals, feeding
type, and routine management. Moreover, unlike other equestrian sports like show-jumping [7],
racing [8], and three-day eventing [9], there is no known characterization of feeding management
and schedule or dietary ingredients. Since competition horses may develop nutritional and digestive
problems such as gastric ulcers, decreased appetite, and weight loss [10], it is important to establish
feeding practices of sport horses undergoing hard work. Considering that the rodeo sport season
lasts eight months every year, with some horses competing every weekend or every other weekend,
a properly designed training routine and sound feeding management is crucial. Moreover, since the
Chilean corralero horse is considered a small horse (height at the withers 99.86–136.4 cm) and huasos
weigh an average of 80.78 + 10.02 kg, some of these horses may be overburdened [5], thus requiring
additional energy.
The lack of information on Chilean corralero horses also makes it difficult for breeders, trainers,
riders, veterinarians, and owners to develop management and feeding standards. Therefore, this work
seeks to contribute with relevant information on diet composition and feed ingredients utilized daily
in the diet of Chilean corralero horses actively participating in the national sport. Hence, the objective
of this study was to characterize the feeding and nutritional management, routine care, and training of
Chilean corralero horses that participate in the rodeos of the Chilean Rodeo Federation.
47
Animals 2019, 9, 697
2.1. Animals
The target population for this study was healthy Chilean corralero horses that participated in the
2014–2015 season of the Chilean national rodeo competition and that were in training at the time of
the visit. The data to identify individuals that participated during that season were provided by the
Chilean Rodeo Federation.
The horses’ owners were contacted by telephone or e-mail to request their participation in the study,
and an interview was scheduled if they were willing to participate. As a result, a personal visit was carried
out between October and December of 2015 to 49 farms (275 horses) located between the Metropolitan and
the Los Lagos regions (33◦2616” south (S), 70◦391” west (W) to 41◦2818” S, 72◦5612” W). Farms were
located in the following regions: Metropolitan (n = 5), Valparaiso (n = 3), O’Higgins (n = 6) Maule (n = 5),
Bío-Bío (n = 10), Araucanía (n = 8), Los Ríos (n = 5), and Los Lagos (n = 7).
2.2. Survey Design

The interview consisted of a questionnaire with five questions regarding general farm and
respondent information (farm name, location, farm administrator, position, date of the visit),
three questions to characterize the animal (registration number, sex, date of birth), five questions
about housing conditions and activity (hours spent within the stall, outdoor pen, paddock, and tied),
and four questions characterizing the type and amount of daily exercise of the horse (minutes spent
walking, trotting, cantering, and in the “bumping activity”, on a weekly and daily basis). “Bumping”
(topeo) is when the horse performs lateral canter half-passes while maintaining contact at a 45◦ angle
between the horse’s chest and the steer’s body, using a lateral movement to push slightly on the steer
and maintain him at a 45◦ angle to the horse’s body.
The questionnaire also included 18 questions about nutritional management with regard to feeding
(feed ingredient type, feeding frequency, and schedule), water availability and source (automatic
drinker, water bucket, etc.), access to pasture, hay type, grains, protein supplements, other commercial
concentrate or byproduct, minerals, oils, if feedstuffs were moisturized, nutritional advisor, and other
information (de-wormer type and frequency, incidence of colic and laminitis per year).
The daily amount of each ingredient offered was weighed with a digital scale. The girth and
scapula–ischial length (SIL) were measured using a measuring tape to estimate live body weight,
using the formula of Carroll and Huntington [11], where
weight (kg) = (girth (cm2 ) × SIL (cm))/11,877. (1)
The girth corresponds to the thoracic perimeter (cm) and was measured by wrapping a 3-m
flexible measuring tape around the girth, just behind the withers, of each horse. The scapula–ischial
length (SIL) corresponds to the length from scapula (point of the shoulder) to ischial tuberosity (point
of the buttock) in centimeters [12].
The unique registration number and date of birth of the horses were obtained from the owners or
the animal’s registration certificate.
2.3. Statistical Analyses

Data obtained from the surveys were consolidated in an Excel spreadsheet and reported as
a descriptive analysis (average ± standard deviation, frequency, average, minimum, maximum).
One-way ANOVA between horses was conducted to determine significant differences among treatments,
and a Tukey honestly significant difference (HSD) test was performed whenever there were significant
differences. Significance was declared with a p-value lower than 0.05. Pearson correlations were
performed between feedstuffs (hay, corn, oats) utilized and incidence of colic and laminitis. The Pearson
48
Animals 2019, 9, 697
correlation was performed among different exercises types (walk, trot, canter). Data were processed
using Statistica V 7.0.
3. Results
Measurements were collected from 275 horses located on 49 farms, and general characterizations
about gender, age, and physical characteristics are presented in Table 1. The evaluated farms had
geldings, mares, and stallions, which respectively accounted for 30.6%, 32.7%, and 36.7% of the entire
sampled population. The average age per horse was 9.9 ± 2.6 years old. Geldings in this survey were
older than mares and stallions (p < 0.05).
Table 1. Characterization of horses included in the survey, concerning gender, age, and estimated
weight calculated from the girth and scapula–ischial length (SIL).
Parameter
Estimated
Age (Years) Girth (cm) SIL (cm)
Gender N % Weight (kg)
(Mean ± SD) (Mean ± SD) (Mean ± SD)
(Mean ± SD)
Geldings 84 30.6 10.9 ± 2.4 a 379.3 ± 27.9 b 166.4 ± 4.3 b 150.6 ± 5.1 b
Mares 90 32.7 9.7 ± 2.6 b 394.2 ± 23.8 a 168.3 ± 3.7 a 153.1 ± 5.7 a
Stallions 101 36.7 9.4 ± 2.6 b 377.8 ± 25.8 b 165.5 ± 4.4 b 151.7 ± 4.9 ab
All 275 100.0 9.9 ± 2.6 383.7 ± 26.8 166.7 ± 4.3 151.8 ± 5.3
a,b Values within a column with different superscript letters differ significantly at p ≤ 0.05.
The estimated weight of mares was greater than males (geldings and stallions) (p < 0.05), but there
was no significant difference between gelding and stallions. When all horses were considered,
the average estimated weight was 383.7 ± 26.8 kg. The averages of girth and SIL per horse were
166.7 ± 4.3 cm and 151.8 ± 5.3 cm, respectively, with mares having a larger girth compared to stallions
and geldings (p < 0.05) and a larger SIL compared to geldings (p < 0.05).
Routine care did not follow a common pattern among farms; however, all horses were kept
in individual stalls during the night and for part of the day. Eighty-six horses (31.3%) spent the entire
day in stalls. Of the rest, 163 horses spend part of the day tied, 24 in pens, and two in paddocks (Table 2).
Table 2. Routine management of Chilean corralero horses.
Number and Percentage of Horses

Hours/Day
Stalls Tied Pens Paddock
0–5 0 17 (6.2%) ‡ 0 0
6–10 0 2 (0.7%) † 0 0
172 144 (52.4%) 24 (8.7%)
11–15 † † 2 (0.7%) †
(62.5%)
15–19 17 (6.2%) 0 0 0
20–24 86 (31.3%) 0 0 0
275
Total 163 (59.3%) 24 (8.7%) 2 (0.7%)
(100.0%)
† ‡
Animals that spent less than 15 h in a stall; animals that spent within 15 to 19 h in a stall.
The sport and exercise management routine of the Chilean corralero horses is presented in Table 3.
Each farm studied had a unique routine for each of their horses. On average, horses trotted for
8.6 ± 6.6 min/day and cantered for 22.3 ± 6.1 min/day. All farms utilized bumping as an exercise, usually for
durations of between 10 and 20 min and two times/week. Lunging was not a common practice among
trainers; only 6.5% of horse were lunged for more than 15 min. A Pearson correlation (r = −0.44) was
detected between trot and canter exercises, indicating that the time spent on one of the two exercises is
49
Animals 2019, 9, 697
inversely correlated to the other. There was no correlation detected between exercise routine and number of
rodeos annually. The average number of rodeos during the season was 8.6 ± 4.7 per horse; 20 (41.0%), 16
(33.0%), and five (10.0%) farms participated in two, three, and four rodeos per month, respectively. Eight
farms participated in one or fewer than one rodeo per month during the 2014–2015 season.
Table 3. Exercise routine (minutes/workout) of Chilean corralero horses.
Trot Canter Lunge Bumping † Tournaments/Season

N 275 275 275 275
Mean 8.6 22.3 2.5 16.3
SD 6.6 6.1 8.7 6.4
Time spent per activity (minutes/workout)
0–15 78.5 6.9 93.5 37.8
15–30 20.4 61.8 2.5 54.2
30–45 1.1 31.3 3.3 8.0
>45 0 0.0 0.7 0.0
Activity (days per week): trot/canter/lunge
Mean 5.6 2.4 8.6
SD 0.6 0.7 4.7
† Bumping refers to the exercise in which the horse performs lateral canter half-passes while maintaining contact at
a 45◦ angle between the horse’s chest and the steer’s body.
Feeding and nutritional management of the Chilean corralero horse during the rodeo season is
summarized in Table 4. Results showed that 81.1% of the horses were fed twice daily (morning and
noon/night), and 18.9% received three meals (morning, noon and afternoon/evening). The horses that
had two rations per day ate during the morning and night (212 horses from 37 farms) or noon and
night (11 horses from two farms). None of the horses surveyed had access to pasture.
Table 4. Feedstuffs by type and quantity that were offered to every horse on a daily basis, as fed.
Horses N (%) Farms N Daily feed allowance SD Max Min

Forages (kg/d)
Alfalfa hay 122 (44.4) 22 8.8 2.3 19.1 4.9
Grass hay 67 (24.3) 13 9.2 1.7 14.3 5.6
Alfalfa cubes and/or pellets 64 (23.3) 8 8.6 0.9 11.3 7.1
Mix 22 (8.0) 6 10.7 2.6 15.7 7.0
275 49
Cereal grains (kg/d)
Oats 57 (77.0) 14 2.6 1.6 6.4 0.6
Corn 17 (33.0) 4 1.3 1.3 3.6 0.2
74 18
Wheat by-products (kg/d)
Wheat bran 93 (60.4) 19 1.9 1.2 5.3 0.5
Wheat middlings 61 (39.6) 11 2.4 1.3 6.0 0.7
154 30
Oilseed by-products (kg/d)
Soybean meal 54 (90.0) 10 0.8 0.4 0.9 0.1
Canola meal 6 (10.0) 1 0.9 0.0 0.9 0.9
60 11
Comercial concentrate (kg/d)
Locally produced 75 (83.3) 17 2.3 2.1 9.0 0.6
Imported 15 (16.6) 4 2.0 1.3 3.8 0.5
90 21
Oils (ml/d)
None 226 39 0
Corn oil 2 1 200 †
50
Animals 2019, 9, 697
Table 4. Cont.
Horses N (%) Farms N Daily feed allowance SD Max Min

Sunflower oil 9 1 45
Olive oil 2 1 50
Linseed oil 26 4 60
Soybean oil 5 1 40
Fish oil 5 1 30
Minerals blocks
No addition 94 17 (34.7%)
Addition 181 32 (65.3%)
Suplements (any kind)
No addition 53 12 (24.5%)
Addition 222 37 (75.5%)
† Only at one farm during a limited period.
A total of 265 horses were fed hay. Each studied farm used more than one hay type to feed their
horses. The average amount of hay offered per horse was 9.0 ± 1.9 kg/day as fed. Alfalfa hay was
the most commonly used forage, followed by a pasture hay and alfalfa cubes/pellets. Twenty-seven
percent of horses received some type of cereal grains. Oats were the most commonly used grain,
followed by corn which was offered fine, ground, or rolled.
Wheat milling by-products were also part of the feed used, where 61.2% of the farms used
middlings or bran. The delivered amount of wheat bran ranged from 0.5 kg/day to 5.3 kg/day,
whereas wheat middlings were offered at a range of 0.7 to 6.0 kg/day.
A total of 38 farms (77.6% of farms surveyed) did not use oilseed by-products (soybean and canola
meal) to feed any of their horses. Eleven farms utilized oilseed by-products in their ration, and only
one fed all their horses with oilseed by-products.
Commercial concentrates (based on mixtures of different energetic ingredients such as oats, corn,
soybean meal, molasses, wheat middling, and vegetable oils) were used in the Chilean corralero horse
diets, with 42.9% (21 of the 49) of the owners including 2.2 ± 2.0 kg daily. Mineral premix and vitamins
were used in 181 horses from 32 of the studied farms, whereas 93 horses from 16 farms were not fed
supplements. Finally, only 41 horses from 10 farms were fed different oils or fats, with linseed oil being
the most popular.
One hundred and ninety-seven horses from 27 farms were dewormed between two and six times
per year.
Of 275 horses, 149 horses (54.2%) did not suffer colic during the previous year, whereas 47 (17.1%)
and nine (3.3%) horses suffered colic one or two times during the year before, respectively. There was
a negative correlation (r = −0.72) between the numbers of colic episodes that the horses suffered per
year and the amount of hay (other than alfalfa hay) offered, whereas the number of colic episodes that
the horses suffered per year was positively correlated with corn grain (r = 0.6) and wheat middlings
(r = 0.7).
Around 63% did not suffer laminitis disease the previous year, and 12% suffered laminitis during
that period. The remaining interviewees did not know the answer. A negative correlation (r = −0.72)
between the incidence of laminitis and the amount of hay was observed.
4. Discussion
Unfortunately, there is little scientific literature describing the characteristics of the Chilean
corralero horse. In 1950, Denhardt [13] described the horse as a muscular, strongly built animal,
with a broad chest and a good distance between its shoulders. Years later, a review of morphological
characteristics of this breed during the rodeo sport season was reported by García et al. [14]. The girth
of the Chilean corralero horse usually ranges between 162 and 182 cm for males and 164 and 184 cm
for females [15]. In this study, the girth data fell within these ranges. Mares had a larger girth
51
Animals 2019, 9, 697
(168.3 ± 3.7 cm) than both stallions (165.5 ± 4.4 cm) and geldings (166.4 ± 4.3 cm), which was similar
to the values obtained by García et al. [14], who reported 170.3 ± 7.1 cm for females and 168.8 ± 5.3
and 169.6 ± 6.4 for stallions and geldings, respectively. However, there is no established reference
parameter for the scapula–ischial length (SIL) of this breed. In this study, the average SIL for mares
was 153.1 ± 5.7 cm, while it was 150.6 ± 5.1 cm for geldings and 151.7 ± 4.9 cm for stallions. In the
case of García et al. [14], mares had an SIL of 147.9 ± 6.3 cm, geldings had an SIL of 148.2 ± 7.9 cm,
and stallions were shorter at a length of 145.6 ± 6.4 cm.
In this study, the wither height was not measured but information obtained from the study of Muñoz
et al. [5] showed that the standard height of the withers of the breed established at 138–148 cm [15].
The morphological characteristics mentioned above indicate that Chilean corralero horse is considered
a small horse. To estimate the average liveweight, the formula proposed by Carroll and Huntington [11]
was used, and the estimated weight of the surveyed horses was 383.66 + 26.8 kg. Mares had the greatest
estimated average weight of 394.16 ± 23.8, followed by geldings and stallions, with average weights of
379.34 ± 27.9 and 377.8 ± 25.8, respectively. The standard of the Chilean corralero horse establishes that
mares are 2 cm longer in SIL than males due to their reproductive anatomy [16]. Several other studies
about morphometric measurements of the Chilean horse are available for further information [5].
Stabling is a common practice in sport horse management and is part of the evolution of the horse
throughout history, as it changed from being utilized primarily for agricultural and military purposes
to use in sports and leisure [17]. This trend to confine the animal allows for a better control of the
daily routine, feed intake, reproduction, and health management, among other benefits. To our best
knowledge, there is limited information about Chilean corralero horse breeding and management,
even during the reproductive or training phases. This research was conducted focusing on the sport
phase of those horses exclusively focused on rodeo training. In the rodeo, as in any other equine sport
discipline, horses are kept within single stalls which limit locomotion, making horse care easier and
more economical [18]. However, the restricted motion and the lack of freedom to express characteristic
behaviors may lead to stress and vices [19]. Upon the results of this study, it is clear from the routine
handling management questions of Chilean corralero horses that 100% of the horses spent some time of
the day within the stall (Table 2), usually more than 12 h a day. Another large percentage (59.3%) of the
horses spent between a few hours to up to half of their day tied. Only a small percentage of the horses
were left free in pens (8.7%) or paddocks (0.7%). These extensive confinement periods correspond to
abnormal behavior and vices in the Chilean corralero horse and, according to Muñoz et al. [20], 10%
of the horses studied exhibited these behaviors. Prolonged stabling can result in several undesirable
behaviors such as “cribbing” (biting hard, often wooden surfaces, frequently swallowing air in the act)
which can generate small fractures (less than 5 mm) in the horses’ teeth [21].
Daily exercise routines of the Chilean corralero horse were quite variable among farms. It seems
that every trainer and/or owner managed their horses without following a common pattern within
the discipline. Overall, Chilean corralero horses exercised six days per week, and the daily exercise
consisted of walking, trotting, cantering, and bumping. Total daily exercise lasted 49.6 ± 27.8 min.
The NRC [22] categorizes exercise as light, moderate, heavy, and very heavy for horses, depending on
the mean heart rate, description, and types of event. In this case, the Chilean corralero horse training
can best be categorized as heavy exercise, since, on average, the horse worked 4–5 h/week, during
which time the activity was composed of 30% walk, 50–65% trot or canter, and 5% canter, jumping,
or other skill. In this case, since there are no data on mean heart rate, bumping may be considered
similar to jumping or cantering [22].
Horses are non-ruminant herbivores with an enormous capacity to digest and obtain energy
from fibrous ingredients [23]. However, they have a small stomach (8% of the total gastrointestinal
tract volume) and, thus, must eat small quantities many times throughout the day. In free-ranging
conditions, horses spend between 16 and 20 h per day grazing and, thus, in nature, can consume small
amounts of feed throughout the day [24]. Nevertheless, for practical reasons, owners of confined
horses may not be able to simulate this feeding delivery [25]. Feeding frequency affects horse health
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Animals 2019, 9, 697
and, thus, performance. In this study, a major portion of the animals surveyed (81.1%) were fed twice
a day, whilst the remaining 18.9% were fed three times per day, which is within the recommendations
for feeding horses to avoid colic [26]. Furthermore, the number of meals provided for the horses in this
study was the same as the number of meals reported in a survey study of show-jumping horses [7],
horses in the United Kingdom (UK) with high energy requirements [17], but differed from eventing
horses [9] and racehorses [27], which received between one and five meals per day.
With regard to the type and amount of feed offered, this study found a wide variation in the diets
and quantities of food offered, which suggests that the feeding management of these individuals is not
formulated according to their requirements but rather according to the managers’ personal criteria.
All horses surveyed had access to some kind of forage in concordance with recommendations by
the NRC [22]. An average of 9.0 kg/horse (2.3% live bodyweight, “bwt”) of either alfalfa hay and/or
pasture hay was offered daily; however, the exact amount varied greatly, between 5.6 and 19.1 kg/horse
(as fed). More recently published recommendations for horses with high energy requirements indicate
that hay intake should be 20 g dry matter (DM)/kg bwt/day [25]. Considering a 400 kg bwt for the
Chilean corralero horse, the minimum forage intake should be 8 kg of DM daily. In contrast to
high fiber feeding, 59% of the sampled horses received grains (oats or corn) and/or a commercial
concentrate (locally produced or imported). These energetic ingredients were offered from 2.6, 1.3,
2.3, and 2.0 kg per day as fed for oats, corn, local, and imported commercial concentrate, respectively
(Table 4). The average grain or concentrate offered was 2.1 kg/horse daily, which represents 0.5%
of the horse liveweight. This value is in accordance with the recommendations of Owens [28] and
the NRC [22]. Wheat by-products (wheat middlings and wheat bran) were also offered to horses as
part of the daily diet as a replacement or partial replacement of energetic feedstuffs. The average
offered was 2.1 kg/horse; nevertheless, the NRC [22] lists both by-products as concentrates (3.2 and
3.4 Mcal DE/kg, respectively) even although both are considerably higher in fiber than corn (ADF
15.5 and 12.1% respectively, compared to 3.4% ADF in corn) but similar to oat ADF content (13.5%).
As the partial or total replacement of grain with wheat by-products did not follow a common pattern
among farms, it is suggested that every owner follows their own criteria when establishing feeding
practices for the Chilean corralero horse. Finally, when compared to the 400 kg bwt horses from the
NRC tables [22], Chilean corralero horses are fed 2.8% of their bwt, which is slightly greater than the
recommendation for horses in heavy exercise. It is worth noting that NRC recommendations do not
account for breed-type behavior. Chilean corralero horses have a nervous and alert temperament which
may increase the average energy requirements compared with a regular 400-kg horse [29]. Rosselot et
al. [29] found that Chilean corralero horses tend to demonstrate proactive avoidance behaviors when
presented with a challenge in a handling test; thus, this increased activity in response to environmental
stressors could also impact nutrient requirements. Another factor that must be considered is the total
rider and saddle weight that the horse supports on its back during competition and daily training.
Muñoz et al. [5] conducted a study to determine the weight supported by Chilean corralero horses
during a rodeo competition. The estimated back load maximum capacity of these Chilean corralero
horses was 115.0 ± 6.1 kg, and the riders weighed on average 80.8 kg with a saddle weight of 11.6 kg;
therefore, while the horse is not overloaded, the weight of the rider and saddle corresponds to 25% of
the horse’s weight and, thus, has a lesser impact on its relative energy expenditure (25.7 Mcal/d DE) as
compared to other sports with a lower rider/horse weight ratio such as show jumping and endurance,
where the sum of weights for riders and saddle represents approximately 15% of the horses’ weight
and the energy requirements.
In terms of nutrition, the use of fats and oils was not common, with just 17.8% of horses fed with
some oil source. The amount varied greatly but averaged 30–60 mL/day. In general, corn oil was
preferred for horse feeding since it was proven that, among vegetable and animal oils, it is the most used
and accepted [30]. In contrast, mineral supplementation and the use of other additives/supplements
are common practice within the industry. Sixty-five percent of horses were supplemented with mineral
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Animals 2019, 9, 697
blocks, and 75.5% were given some supplement (liver protectors, vitamins premix, creatine sources,
and joint supplements, among others).
In this study, colic events were negatively correlated to hay consumption and positively correlated
to grain and wheat middling intake. Colic is a complex multifactorial condition and it has some
association with feeds and feeding [31]. Grain overloading and, thus, high-starch diets along with
low-fiber diets may predispose horses to colic [32]. In this study, grains were included in the diet with
a high variation: 2.6 ± 1.6 kg/day of oats on average, ranging from 0.6–6.4 kg/day and 1.3 ± 1.3 kg/day
of corn ranging from 0.2 to 3.6 kg/day. As mentioned before, the recommended amount of grain offered
to a horse daily is 500 g/100 kg of liveweight. Thus, the Chilean corralero horse (400 kg liveweight) is
expected to consume 2.0 kg of grains to supply the energy requirements (20–25 Mcal/d DE) according to
NRC guidelines [22]. This recommendation comes from the limited ability of the enzymes in the horses’
small intestine to digest starch from cereal grains. When grain, which is high in starch, is overfed,
part of the carbohydrates mentioned above are incompletely digested and escape absorption in the
small intestine, traveling to the large intestine and promoting a disruption in the microbial population,
leading to abnormal pH conditions which compromise intestine health [32]. These alterations may
increase the risk of colic, laminitis, and other pathologies.
A characterization of the animal and its routine care was carried out through this study. This information
complements all physics hypsometry studies that were done. To reach a better understanding of this breed’s
nutritional requirements, more data should be taken in situ, such as time dedicated to working in each
activity, as well as heart rate, blood metabolites, and other physiological variables.
Chilean legislation for animal welfare only establishes that owners should provide adequate
feeding and healthcare to their animals, regardless the animal species. Therefore, the results of this
study may provide information which could be considered when creating or enforcing legislation
regarding horse care and training.
5. Conclusions
The average Chilean corralero horse spends approximately 12 h daily within a stall, and spends its
remaining hours tied, loose in a pen, and/or training. The horse’s workload can be classified as moderate
to heavy intensity according to the NRC [22], and includes six workouts weekly (walk/trot/canter) with
two bumping workouts per week and two competitions per month.
During the training season, the diet of the Chilean corralero horse is based on forages, mainly
alfalfa hay, plus oats as an additional energy source. Protein supplements such as oilseed by-products
are used less frequently.
Additional studies are required to provide further information on nutritional requirements,
feeding management, and exercise in order to improve the raising and management of this breed.
Author Contributions: Conceptualization, J.P.K. and M.G.; data curation, J.B., M.S.-G., and J.P.K.; funding
acquisition, F.B. and M.G.; investigation, J.B., J.P.K., and M.G.; methodology, J.P.K. and M.G.; project administration,
J.B., J.P.K., and M.G.; resources, F.B.; software, M.S.-G.; supervision, M.G.; validation, M.S.-G.; visualization,
M.S.-G. and H.H.W.; writing—original draft, J.B. and M.G.; writing—review and editing, F.B., H.H.W., and J.P.K.
Funding: This research received no external funding. The Animal Sciences Department of the Facultad de
Agronomía e Ingeniería Forestal de la Pontificia Universidad Católica de Chile funded minor expenses related to
the investigation.
Conflicts of Interest: The authors declare no conflicts of interest.
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animals
Article
Effects of Horse Housing System on Energy Balance
during Post-Exercise Recovery
Malin Connysson 1, *, Marie Rhodin 2 and Anna Jansson 2
1 Wången National Center for Education in Trotting, Vången 110, S-835 93 Alsen, Sweden
2 Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences,
SE-75007 Uppsala, Sweden; [email protected] (M.R.); [email protected] (A.R.)
* Correspondence: [email protected]; Tel.: +46-(0)640-17431
Received: 29 October 2019; Accepted: 8 November 2019; Published: 14 November 2019
Simple Summary: Horse management aims to keep horses healthy and ensure good performance
and animal welfare. Many horses are currently kept in individual box stalls indoors, a housing
system that limits free movement, exploration, and social interaction, and may also subject horses to
lower air quality. The alternative is a free-range housing system where horses are kept in groups
outdoors. Anecdotal information indicates concerns among sports horse trainers that lack of rest
in such systems delays recovery and impairs performance. This study examined whether recovery
after competition-like exercise in Standardbred trotters was affected by housing system. The results
showed that a free-range housing system did not delay recovery in Standardbred trotters, and in fact
had positive effects on appetite and recovery of energy balance.
Abstract: This study examined the effects of two housing systems (free-range and box stalls) on
recovery of energy balance after competition-like exercise in Standardbred horses. Eight adult
geldings (mean age 11 years) were used. The study had a change-over design, with the box stall
(BOX) and free-range group housing (FreeR) treatments each run for 21 days. The horses were fed
forage ad libitum and performed two similar race-like exercise tests (ET), on day 7 and day 14 in each
treatment. Forage intake was recorded during the last 6–7 days in each period. Blood samples were
collected before, during, and until 44 h after ET. Voluntary forage intake (measured in groups with
four horses in each group) was higher in FreeR horses than BOX horses (FreeR: 48, BOX: 39, standard
error of the mean (SEM) 1.7 kg (p = 0.003)). Plasma non-esterified fatty acids (NEFA) was lower at
20–44 h of recovery than before in FreeR horses (p = 0.022), but not in BOX horses. Housing did not
affect exercise heart rate, plasma lactate, plasma urea, or total plasma protein concentration. Thus the
free-range housing system hastened recovery in Standardbred trotters, contradicting anecdotal claims
that it delays recovery. The free-range housing also had positive effects on appetite and recovery of
energy balance.
Keywords: NEFA; Standardbred trotters; feed intake
1. Introduction
Many horses are currently housed in individual box stalls in stables [1–4]. Box stalls facilitate
supervision, individual feeding and grooming of the horses, but obviously limit their scope for free
movement, exploration and social interaction. An alternative is a modern housing system where horses
are kept in groups in paddocks with shelters and lying areas and with individual feeding controlled
by transponders. Anecdotal information indicates concerns among sports horse trainers that lack of
rest in such systems delays recovery and impairs competition performance. However, unpublished
data [5] indicate that picky-eater Standardbred trotters kept in a group housing system have better
body condition than when housed in box stalls, indicating better appetite and higher feed intake.

Animals 2019, 9, 976
Environmental factors such as space allowance, group size and feeder characteristics have been shown
to affect feed intake in pigs [6]. Little is known about how physical environment affects feed intake in
horses. Keeping horses in groups may affect eating; which has been shown to be highly synchronized
in group-housed horses [7,8].
In the short term, recovery involves decreasing muscle temperature, compensating for oxygen
debt, and regulating acid–base balance. In the longer term, it also involves energy replenishment, fluid
balance recovery, and tissue re-synthesis. Energy balance can be monitored by measuring body weight,
energy expenditure (using heart rate), body condition score (BCS), and substrate usage (non-esterified
fatty acids (NEFA) and urea). NEFA, originating from lipolysis of adipose tissue, have been widely
shown to increase in ponies and horses during periods of insufficient energy intake [9–13]. When
amino acids are used as energy substrate there is a degradation that starts with deamination, where the
amino group is removed and converted into ammonia. Ammonia released by this process is removed
from the body by forming urea in the liver.
This study examined whether recovery of energy balance after competition-like exercise in
Standardbred horses fed ad libitum was affected by housing system. Two different systems were
compared: free-range group housing (FreeR) and an individual box-stall housing system (BOX) in
which activity in groups was possible for only 4–5 h/day. The hypothesis was that free-range housing
hastens recovery compared with box stall housing.

Umeå local ethics committee approved the study (A 54-13) and it was performed in compliance
with European Union directives on animal experiments (2010/63/EU; European Union, 2010) and with
laws (Swedish Constitution, 1988:534) and regulations (Swedish Board of Agriculture Constitution,
2012:26) governing experiments on live animals in Sweden.
2.1. Horses and Management

Eight adult Standardbred geldings in training (mean age 11 years, range 9–13 years) were used.
The study was performed during May–June 2015 and all horses had raced during the period 2005–2015
and had average earnings of 146,553 SEK (range 26,500–495,798 SEK). Mean bodyweight at the
beginning of the trial was 509 kg (range 410–562 kg). The horses were trained under the supervision of
professional trainers (licensed by the Swedish Trotting Association) according to a training program
similar to that used by Swedish trotting trainers [14].
2.2. Experimental Design

The horses were randomly allocated to two groups of four and kept in the box stall (BOX) or
free-range group housing system (FreeR) for 21 days, followed by a complete change-over to the other
treatment. Between treatments, the horses had a three-day transition period in the new housing system.
All horses performed two similar exercise tests (ET), on day 7 (ET1) and day 14 (ET2) in each treatment
period. On days 3, 11, and 18 of each treatment, the horses were exercised on a track (5000 m warm-up,
3000 m at a speed of 11.1 m/s).
The horses in treatment BOX were housed individually in 3 m × 3 m boxes with wood shavings
and were let out into a sand paddock (2000 m2 ) together with the other horses in their group for 4–5 h
every day. In the boxes, the horses had ad libitum access to water in buckets. The horses in treatment
FreeR were kept together in a group housing area that consisted of a paved paddock (3200 m2 ) with a
shelter with rubber matting and automatic feeding stations. They were offered water ad libitum in
water barrels.
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2.3. Diet
In treatment BOX, the horses were fed forage ad libitum in their stall, but no feed was offered
during the paddock stay. Feed was offered four times per day (07.00, 12.30, 17.00, and 20.00 h) and
there had to be left-overs at every meal to provide ad libitum access.
In treatment FreeR, the horses were fed from automatic feeding stations that recognized the
individuals by transponders. With this technique, feed allowance was regulated by time and all horses
had access to the feeding station for more time than they used it (free access). All horses but one had
400 min eating time/day, while in one horse the access was 500 min/day to ensure free access.
The same forage was used in both housing systems, a haylage (dry matter (DM) 78%, 11.2 MJ
metabolizable energy (ME)/kg DM, 14.3% crude protein (CP), calcium (Ca) 5.6 g/kg DM, phosphorus
(P) 2.2 g/kg DM, and magnesium (Mg) 1.7 g/kg DM). The horses were also given 0.5 kg concentrate
(Krafft Sport, Malmö, Sweden (12 MJ ME/kg, 11% CP)) and 80 g mineral and vitamin supplement
(Krafft Miner Vit, Malmö, Sweden) (Ca 55 g/kg, P 65 g/kg, Mg 60 g/kg, salt (NaCl) 125 g/kg, copper (Cu)
900 mg/kg, selenium (Se) 15 mg/kg, vitamin A 100,000 IU/kg, vitamin D3 10,000 IU/kg, and vitamin E
5000 mg/kg) every day. In the BOX system this was fed once a day, while in FreeR the automatic feed
station divided the total allowance equally between every hour of the day.
The horses were offered ad libitum access to salt blocks during the whole study and they also had
extra salt mixed with beet pulp after the exercise on days 3, 11, and 18 (250 g beet pulp and 20 g NaCl).
2.4. Exercise Test

On the day of the exercise test (ET), the horses were transported 50 km to an official 1000-m oval,
banked, gravel racetrack (Östersunds race track, Sweden). The horses performed the ET in groups
of four, two from each treatment. The same driver drove the same horse in a harness race sulky on
all test occasions and the horses raced in the same group at the same time on all occasions. The ET
started with a warm-up consisting of 4000 m slow trot (6.3–6.7 m/s) and 500 m trot (10 m/s). After
the warm-up, the horses walked the track for 10 min. They then trotted 2140 m in the same race field
position, at 11.8–12.2 m/s for the first 1640 m and as fast as they could (free positioning) for the last
500 m. This was followed by a cool-down of 1000 m slow trot (6.3–6.7 m/s). Approximately one hour
after crossing the finish line in the ET, the horses were transported back home, and three hours after
the ET they were put back into their housing system.
2.5. Measurements, Sampling, and Analysis

The horses were weighed on a scale (Tru-test E2000S2, Auckland, New Zealand) every morning.
Body weight measurements on the day before ET day and on the morning of ET day were used as
“Rest” values for bodyweight. Rectal temperature was measured every day, at 07.00 h. Before and after
each experimental period, the body condition score (BCS) of all horses was evaluated as specified by
Henneke et al. [15]. During days 15–22 (in period 1) and days 14–19 (in period 2), forage intake was
measured by weighing the allowance and left-overs. In treatment FreeR, the horses were fed forage
from two feeding stations (four feeding places) and intake had to be measured for the whole group.
Mean ambient temperature on ET days and the three following days was 8.4 ◦ C (range 1.3–9.8 ◦ C).
During ET days, blood samples were collected via a catheter inserted in vena jugularis after local
cutaneous anesthesia (Tapin (lidocaine 25 mg/g, prilocaine 25 mg/g), Orifarm Generics, Stockholm,
Sweden). The catheter was inserted approximately one hour before the first blood sampling. Blood
samples were collected in 6-mL lithium-heparinized tubes (102 IU) and kept on ice until centrifuging
(10 min, 920 × g, 18 ◦ C), after which the plasma was frozen (−20 ◦ C). Blood samples were collected at
rest in the stable (Rest), at the racetrack 1 min after the finish line (FL) and after 10, 180, 240, 300, 360,
and 420 min of recovery. At 20 and 44 h after the ET, blood samples were collected by venipuncture
from the jugular vein.
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Animals 2019, 9, 976
Total plasma protein concentration (TPP) was measured in all plasma samples using a
handheld refractometer (Atago, Tokyo, Japan). Plasma lactate concentration was analyzed in
samples Rest, FL and after 10, 180, and 420 min of recovery, using an enzymatic (L-lactate
dehydrogenase and glutamate-pyruvate transaminase) and spectrophotometric method (Boehringer
Mannheim/R-Biopharm, Darmstadt, Germany), with intraassay coefficient of variation (CV) 2.2%
in this study. Plasma NEFA concentration was analyzed in samples taken at Rest, FL, and after 10,
180, 240, and 420 min of recovery and also after 20 and 44 h, by quantitative determination using an
enzymatic colorimetric method (Wako Chemicals GmbH, Neuss, Germany), with intraassay CV 1.8%
in this study. Plasma urea concentration was analyzed with a spectrophotometric method (Urea Assay
Kit, Cell Biolabs Inc., San Diego, CA, USA), with intraassay CV 1.3% in this study.
Heart rate was continuously recorded during the race and up to 420 min of recovery using a heart
rate recorder (Polar CS600X Polar Electro, Kempele, Finland) and the data were analyzed using Polar
ProTrainer 5 Equine Edition Software (Polar Electro, Kempele, Finland). Mean recovery heart rate was
calculated using recordings from 270–410 min of recovery.

Analysis of variance was performed with the MIXED procedure in SAS (version 9.4; SAS Institute
Inc., Cary, NC, USA), using an autoregressive (AR(1)) structure. Plasma sample results were pooled
into recovery 3–7 h (180–420 min), and recovery 20–44 h (22 h and 44 h). Statistical analysis was
performed with a statistical model including fixed effects of housing, sample, and the interaction
between them. The model for an observed variable of horse i in housing j, sample k, was:
Yijk = μ + ηi + πj + γk + (πγ)ik + eijk , (1)
where μ is the overall mean, ηi is the effect of horse, πj is the effect of housing, γk is the effect of
sample, (πγ)ik is the effect of the interaction between housing and sample, and eijk is the random
error. The random part included horse, horse × housing, and period. Observations within each horse
× period × housing combination were modeled as repeated measurements. For urea, race had a
significant effect and was included in the model.
Forage intake, bodyweight, BCS, heart rate, and velocity data were analyzed by a statistical model
including fixed effects (housing, period, and the interaction between housing and period). The model
for an observed variable of horse i in period j, in housing k was:
Yijk = μ + ηi + πj + γk + (πγ)ik + eijk , (2)
where μ is the overall mean, ηi is the effect of horse, πj is the effect of housing, γk is the effect of
sample, (πγ)ik is the effect of the interaction between housing and sample, and eijk is the random error.
The random part included horse and horse × housing. Observations within each horse were modeled
as repeated measurements.
Post-hoc comparisons were adjusted for multiplicity using the Bonferroni method. Values are
presented as least square means (LSM) with the standard error of the mean (SEM) in a parenthesis.
Differences were considered statistically significant at p < 0.05.
3. Results
One of the horses was excluded due to a hoof crack, although it showed no clinical signs of pain.
Training and ET were still performed with that horse and it was included in feed intake measurements,
since these were done by group. Group forage intake was higher in treatment FreeR than treatment
BOX (FreeR: 48 (1.7), BOX: 39 (1.7) kg (p = 0.003)) and the mean bodyweight during the whole treatment
periods (21 days) tended to be higher in FreeR horses than in BOX horses (FreeR: 505 (13), BOX: 500
(13) kg (p = 0.07)). There was no difference in BCS between the housing systems (FreeR: 4.8 (0.4), BOX:
4.7 (0.4) (p = 0.93)).
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3.1. Rest
Body weight was higher in FreeR horses than BOX horses at Rest (morning day before ET +
morning before ET) (Table 1). Housing did not affect plasma NEFA, urea, or TPP concentration on the
morning before ET (Tables 1 and 2).
Table 1. Body weight and total plasma protein in Standardbred horses kept in free-range group housing
(FreeR) or box housing (BOX). SEM = standard error of the mean.
Variable Sample FreeR SEM BOX SEM p-Value

Body weight (kg)
Rest 509 13 504 13 0.038
20−44 h recovery 504 a 13 498 a 13 0.020
Total plasma protein (g/L)
Rest 59.5 0.8 60.4 0.8 0.996
Finish line 73.5 a 0.8 74.3 a 0.8 1.000
10 min recovery 67.0 a 0.8 67.4 a 0.8 1.000
3−7 h recovery 61.0 0.8 62.3 a 0.8 0.405
20−44 h recovery 61.8 a 0.8 62.6 a 0.8 1.000
a Significantly different (p < 0.05) from Rest.
Table 2. Plasma concentrations of non-esterified fatty acids (NEFA), urea and lactate in Standardbred
horses kept in free-range group housing (FreeR) or box housing (BOX). SEM = standard error of
the mean.
Variable Sample FreeR SEM BOX SEM p-Value

Plasma NEFA (mmol/L)
Rest 0.26 0.04 0.21 0.04 1.000
Finish line 0.33 0.04 0.34 a 0.04 1.000
10 min recovery 0.54 a 0.04 0.55 a 0.04 1.000
3–7 h recovery 0.24 0.04 0.22 0.04 1.000
20–44 h recovery 0.16 a 0.04 0.25 0.04 0.019
Plasma urea (mmol/L)
Rest 5.0 0.3 4.6 0.3 0.481
Finish line 5.1 0.3 4.9 b 0.3 1.000
10 min recovery 5.2 0.3 4.9 a 0.3 0.818
3–7 h recovery 5.6 a 0.3 5.3 a 0.3 0.718
20–44 h recovery 4.8 0.3 4.7 0.3 1.000
Plasma lactate (mmol/L)
Rest 0.9 0.8 1.3 0.8 1.000
Finish line 20.8 a 0.8 20.6 a 0.8 1.000
10 min recovery 16.0 a 0.8 16.0 a 0.8 1.000
3–7 h recovery 1.0 0.7 1.1 0.7 1.000
a Significantly different (p < 0.05) from Rest. b Tendency for significant difference (p ≤ 0.09) from Rest.
3.2. Finish Line and 10 min Recovery

Housing system did not affect peak heart rate (FreeR: 224 (2), BOX: 221 (2) beats/min), plasma lactate
concentration (Table 2), or velocity during the simulated race (FreeR: 12.0 (0.1), BOX: 12.0 (0.1) m/s).
Plasma lactate concentrations and TPP were higher than Rest values at the finish line and after
10 min of recovery during both FreeR and BOX (Tables 1 and 2). Plasma NEFA concentrations were
higher than Rest values at 10 min of recovery during both FreeR and BOX, and for BOX treatment also
at the finish line (Table 2). Plasma urea concentrations were higher than Rest values at the finish line
(tendency p = 0.071) and after 10 min of recovery during BOX treatment (Table 2). There was no effect
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Animals 2019, 9, 976
of FreeR compared with BOX on plasma NEFA, urea, or TPP concentration at finish line or after 10 min
of recovery (Tables 1 and 2).
3.3. Short-Term Recovery (3–7 h)

As mentioned, heart rate, plasma lactate, NEFA, urea, and TPP concentrations all increased from
rest to race, and therefore recovery was necessary. There was a tendency during 3–7 h recovery for
higher heart rate in FreeR horses compared with BOX horses (47 (1) vs. 43 (2) beats/min; p = 0.100)
(Figure 1). The different housing systems did not significantly affect plasma lactate, plasma NEFA,
urea, or TPP concentration during 3–7 h recovery (Tables 1 and 2). During short-term recovery, plasma
urea concentrations were greater than the Rest values (Table 2). During short-term recovery, TPP were
higher than the Rest values during BOX housing (Table 1).
Figure 1. Box-plot of 3–7 h recovery heart rates in Standardbred horses kept in free-range group
housing (FreeR) or box housing (BOX).
3.4. Long-Term Recovery (20–44 h)

Body weight did not return to the Rest values during long-term recovery (Table 1). Plasma NEFA
returned to the Rest value during 20–44 h of recovery in BOX horses, while in FreeR horses it not only
returned to the Rest value but fell below it (Table 2). Plasma NEFA was lower in FreeR horses than in
BOX horses during 20–44 h of recovery (Table 2) and bodyweight was higher in FreeR horses than in
BOX horses during this period (Table 1). Housing system had no significant effect on plasma urea
or TPP concentration during 20–44 h of recovery (Tables 1 and 2). Plasma TPP concentration during
20–44 h of recovery was significantly higher than the Rest value (Table 1).
4. Discussion
In this study, there was little or no difference in short-term (3–7 h) metabolic recovery in horses
kept in the free-range and box stall housing systems evaluated. In fact, the results indicated that
in the long-term perspective, the free-range system may be beneficial. The lack of differences in
NEFA, urea, and TPP responses during short-term recovery indicates that water and feed intake
were similar in the two housing systems during the first hours after ET. During long-term recovery,
the NEFA levels were very low in FreeR horses, even lower than before the ET. This indicates quick
and efficient recovery of energy balance by horses in this housing system, an indication supported by
the higher daily feed consumption observed in the FreeR housing system. The hormone insulin plays
an important role in regulating lipolysis in adipose tissue, as increased insulin concentration results in
decreased lipolysis and thereby decreased release of NEFA. An increased insulin response in FreeR
(due to higher feed intake) may have lowered the NEFA response but the response may also have been
influenced by different feeding intervals in the housing systems. Low post-exercise appetite has been
observed in athletic horses (box-housed) and is suggested to be associated with the hormone’s active
ghrelin, adiponectin, and leptin, and/or gastric ulcers [16,17]. Low BCS and periods of low appetite are
conditions seen in some horses during periods of intense training and racing, and our results indicate
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that a group housing system might counteract these problems. Our findings indicate that physical
environment is important for feed intake in horses, as reported earlier for pigs [6]. The lower feed
intake in box-housed horses could have been due to the daily 4–5 h without feed in the paddock,
but that is close to the time span without eating observed in wild-living horses and was probably
compensated for by increased feed intake. Compensatory increases in feed consumption after a period
of feed restriction have been reported in ponies [18].
Although plasma NEFA concentrations indicated that energy balance was restored in the horses,
bodyweight was still not back to resting values at 44 h of recovery. In French Trotters, the post racing
decrease in bodyweight is reported to be on average 9.4 kg (range 0–26 kg) and bodyweight requires on
average 3.3 days to recover [19]. An exercise-induced decrease in body weight also seems to be affected
by whether the horses are transported [20]. It has been suggested that sweat losses are responsible
for 90% of post-exercise weight losses [21], which are therefore not affected by energy balance but by
water and electrolyte intake. Since total plasma protein concentrations remained elevated from resting
values at the end of the study period, it is likely that the horses did not recover fluid balance within
44 h. The salt intake in the present study might have been too low to compensate for losses, since salt
blocks have been shown to be an inadequate sole source of salt for athletic horses [22] and the extra
salt offered (20 g) to the horses on the day after ET might not have been sufficient.
There were no differences in plasma lactate responses between the housing systems, which was
probably due to events in the immediate recovery period after exercise (i.e., 1000 m slow-down trot,
walk, and road transport back home) being the same in both treatments. It has been shown previously
that, during the first 30 min after intense exercise, lactate removal can be increased two-fold by light
exercise [23]. Interestingly, there was no significant difference in HR between the housing treatments
during short-term recovery indicating that energy expenditure was similar in this period [24]. In both
systems, HR seemed to be slightly higher than earlier observed [25] in box-stalled Standardbred horses
at night (36–40 bpm vs. 43–47 bpm in our study). We expected HR to be higher when horses were
kept in group housing compared to box housing, due to more physical activity (walking around).
Earlier studies have shown that physical activity in stabled or partly stabled horses is lower than in
horses housed in free-range systems [26]. In addition, keeping horses in groups in paddocks seems to
increase physical activity compared with keeping them in individual paddocks [27]. In BOX horses,
an elevation of HR from expected levels at rest is difficult to explain and accordingly also the lack of
difference in HR between treatments. One possible reason for the lack of difference in HR is because
HR was elevated in BOX horses due to horses being more alert, excited, or stressed in this system (by
people and horses moving around in the stable). This assumption contradicts anecdotal claims that
horses kept in a free-range group housing system are less relaxed than stabled horses.
5. Conclusions
This study found that a free-range housing system hastened recovery in Standardbred trotters,
rather than delaying it as suggested by anecdotal claims. The free-range housing system also had
positive effects on appetite and recovery of energy balance.
Author Contributions: Conceptualization, M.C. and A.J.; data curation, M.C., M.R., and A.J.; formal analysis,
M.C. and M.R.; funding acquisition, A.J.; investigation, M.C.; methodology, M.C. and A.J.; project administration,
M.C.; resources, M.C. and A.J.; supervision, M.R. and A.J.; validation, M.C., M.R., and A.J.; writing—original
draft, M.C., M.R., and A.J.; writing—review and editing, M.C., M.R., and A.J.
Funding: This research was funded by the Swedish–Norwegian Foundation for Equine Research (Stiftelsen
Hästforskning) grant number H-16-47-194 and HIT (Hinrichs Innovation and Technology) Active Stable. Horses
and facilities were provided by Wången National Centre for Education in Trotting.
Acknowledgments: Thanks also to Ulf Hedenström, Lars-Åke Svärdfeldt, Sara Sandqvist, Emma Folkesson, and
the students at Wången for assistance during the study.
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to
publish the results.
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animals
Article
Impact of Year-Round Grazing by Horses on Pasture
Nutrient Dynamics and the Correlation with Pasture
Nutrient Content and Fecal Nutrient Composition
Sara Ringmark 1, *, Anna Skarin 2 and Anna Jansson 1
1 Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences,
SE-75007 Uppsala, Sweden
2 Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences,
SE-75007 Uppsala, Sweden
* Correspondence: [email protected]; Tel.: +46-18-671422
Received: 26 June 2019; Accepted: 19 July 2019; Published: 29 July 2019
Simple Summary: Horse grazing may benefit biodiversity. This study compared the effect of
horses grazing year-round to that of mowing on pasture quality in a forest-grassland landscape in
Sweden. Twelve Gotlandsruss stallions were kept in three enclosures (~0.35 horse/hectare) without
supplementary feeding for 2.5 years. Each enclosure contained three exclosures where pasture was
not grazed, but mown monthly. Horse grazing increased the diversity of pasture nutrient content.
Moreover, energy and protein concentrations and grass availability increased in areas grazed by
horses, but decreased where grass was mown. This indicates that year-round grazing can be used
to increase biodiversity, a suggestion supported by botanical observations. Nutrient content in
horses’ droppings was found to correlate with nutrient content in pasture, so analysis of droppings
may be used to roughly estimate the quality of pasture consumed by horses. Under the conditions
studied, pasture protein content was sufficient to meet horse requirements year-round, while energy
content and pasture availability may have been limited in winter. Monthly data presented here on
the nutritive value of pasture can help guide the management of year-round grazing systems in the
Nordic countries.
Abstract: Horse grazing may benefit biodiversity, but the impact of year-round grazing on nutrient
dynamics has not been evaluated previously. This study compared pasture quality in a forest-grassland
landscape grazed year-round by horses with that in exclosed mown areas. Twelve Gotlandsruss
stallions were kept without supplementary feeding in three enclosures (~0.35 horse/ha) outside
Uppsala, Sweden, from May 2014 to September 2016. Each enclosure contained three mown exclosures,
where grass sward samples were collected monthly and analyzed for chemical composition and
vegetation density. Fecal grab samples were collected and analyzed for crude protein (CP) and
organic matter (OM) content. There were no differences in exclosure pasture energy or CP content
between enclosures (p > 0.05). In grazed areas, there were differences in grass energy and CP content
(p > 0.05) between enclosures. During the three summers studied, energy and CP content increased
in the enclosures, but decreased in the exclosures. By the end, biomass content/ha was greater in the
enclosures than in the exclosures. Fecal OM and CP content showed moderate to strong correlations
with pasture nutrient content (r = 0.3–0.8, p < 0.05). Thus, in contrast to monthly mowing, horse
grazing diversified pasture chemical composition and increased its nutritive value.
Keywords: pasture; horse nutrition; crude protein; exclosures

Animals 2019, 9, 500
1. Introduction
Year-round grazing by cattle, sheep, and horses is common in many European countries, but not
in Sweden. To our knowledge, the effects on pasture quality and quantity of keeping horses year-round
on extensive grazing have not been evaluated previously in the Scandinavian countries. Reasons
for this might include the comparatively short growing season, the need for shelter to meet animal
welfare legislation, and expected low nutrient content of pasture during winter. Lack of validated
methods for monitoring horse nutrient intake on pasture may be another reason. However, studies in
Germany have shown that the nutrient content of pastures grazed year-round can meet or exceed the
requirements of adult cattle and horses, even in winter [1]. In contrast, a study on year-round grazing
horses in France indicated that crude protein intake was very low six months per year, and that adult
maintenance requirement was met only in April–September [2]. This estimation was based on fecal
analyses of crude protein and an observed positive correlation between dietary and fecal crude content.
The use of fecal crude protein analysis to monitor pasture crude protein content and intake needs,
however, to be further validated.
Year-round grazing systems may have the potential to reduce feed costs, but may also support
horses’ natural behaviors and contribute to increased biological diversity. Abandonment of natural
and semi-natural grasslands and forest encroachment, induced by lack of large herbivores in open
landscapes, has caused loss of flora and fauna biodiversity in Sweden [3]. Studies in European
countries, including Sweden, indicate that grazing horses can be used instead of cattle and sheep to
promote biological diversity [4–9]. Horses remove more vegetation per unit body mass than cattle [10],
create mosaic patches of short and tall grass, and leave more broad-leaved plants than cattle [10]. Horses
prefer grasses [2], but their intake of forbs and shrubs may increase during periods of intense grazing in
winter and spring [11,12], and they may also perform bark-stripping [13]. Use of horses in a year-round
grazing system could therefore have great impacts on the landscape and biological diversity [14].
The overall aim of this study was to describe the seasonal and land-to-land variation in pasture
quality in a Swedish forest-grassland landscape grazed year-round by horses, and compare it with
that in adjacent exclosure areas mown monthly. A second aim was to investigate fecal sampling as a
measure of pasture quality. The results are discussed in relation to whether the fodder quality was
acceptable to meet energy and protein requirements in horses, and to the possible impact on pasture
diversification. The hypotheses tested were that pasture energy and protein content can meet animal
requirements but with differences between land areas; that horse grazing alters pasture energy and
nutrient composition compared with mowing; and that fecal crude protein (CP) content is correlated
with pasture nutrient concentration.

The study was carried out between May 2014 and September 2016 in Krusenberg, Uppsala, Sweden
(59◦ 44 8” N, 17◦ 38 58” E). During the 15 years preceding the study, mean daily temperature April–October
was 12.4 ± 5.0 ◦ C (±SD) and mean precipitation was 1.7 ± 4.3 mm/day, while in November–March the
values were −0.6 ± 5.4 ◦ C and 1.4 ± 2.7 mm/day, respectively (Swedish Meteorological and Hydrological
Institute (SMHI) weather station Uppsala Aut, https://www.smhi.se/klimatdata).
In our study, the summer was defined to start after the first four consecutive days with mean
temperature >+5 ◦ C in spring and to end after the first four days with <+5 ◦ C in fall, which defined
the start of winter. Based on this definition, the summer season started on 11 April 2015 and on
30 March 2016, while the winter season started on 9 November 2014 and on 10 October 2015. The study
was approved by Uppsala animal welfare ethics committee (license number: C28/14). Data on daily
temperature and monthly precipitation during the study period were retrieved from the Department
of Earth Sciences, Uppsala University, Sweden (www.geo.uu.se).
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2.1. Horses and Management

Twelve one-year old Gotlandsruss stallions (mean body weight 185 ± 21 kg at the start) from
six different breeders were used in the study. Gotlandsruss is a native Swedish horse breed that has
been present on the island of Gotland from at least the seventeenth century [15] and probably the
thirteenth century. The horses were divided into three groups of four and allocated to three enclosures
at the start of the experiment on 21 May 2014. The horses were kept without supplementary feeding
throughout the study. To avoid the grazing preferences of an individual horse or group affecting
pasture composition, the groups were rotated between the enclosures on 27 May 2015 and 20 May
2016, i.e., each group grazed each enclosure for one growing season. In January 2016, one individual
was excluded from the study due to an injury. Each enclosure contained a 16 m2 shelter (Figure 1).
Water was offered in automatic water troughs, located in the forest, during summer, spring, and fall.
During winter, when the temperature was below 0 ◦ C, water was offered once/day in plastic troughs.
In all enclosures, water was also available in streams in the forest, even during winter. A salt block
with trace minerals (May 2014–August 2014: Ab Hansson & Möhring, Halmstad, Sweden, content
(mg/kg): Zinc 300, manganese 200, copper 80, iodine 50, selenium 20, cobalt 12; August 2014–September
2016: Standard, KNC, Netherlands, content (mg/kg): Zinc 810, copper 220, iodine 100, selenium 20)
was provided in all enclosures. Horses were dewormed five times during the study period, using
Banminth (Pyrantel, Zoetis Finland Oy, Helsinki, Finland), Equimax (Ivermectin and Praziquantel,
Sofarimex Indústria Química e Farmacêutica Ltd., Cacém, Portugal), or Cydectin (Moxidectin, Zoetis
Manufacturing & Research Spain, Gerona, Spain). Once per month, fresh grab sample (approximately
300 g) of faces were collected immediately after defecation from a minimum of two horses/enclosure
(in total a minimum of six samples). These samples were stored at −20 ◦ C until analysis.
Figure 1. Aerial view of the three enclosures (1, 2, and 3) used in the study, showing position of
shelters, water troughs, mineral blocks, exclosures, and pasture transects. Photo taken on 24 May 2016
at 150 m altitude.
2.2. Enclosures and Exclosures

The three enclosures (En1–En3) were 13, 11, and 10 ha in size, respectively, and consisted of
approximately 1/3 fields and 2/3 forest (Table 1). Each was surrounded by electric fencing. Dominating
vegetation types, according to the classification used by the Swedish land survey (lantmateriet.se),
were recorded in plots 15 m2 placed at 35 m spacing in a grid and located with GPS [16]. The vegetation
was then merged into three different vegetation classes: grassland, forest, and semi-forest. Forest was
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defined as forested areas with >30% crown coverage and semi-forest with 10–30% crown coverage,
while grassland had <10% crown coverage. In addition, plant species and species coverage were
recorded in a 20 × 20 cm square in the plots (Table 1). The fields had not been grazed by horses for at
least 10 years, but En1 and En2 had been grazed by cattle and En3 had been used for production of
conserved forage.
In each enclosure, three exclosures each measuring 42.5 m × 5 m were fenced off using electric
fencing (Figure 1). All exclosures were placed in the edge zone between forest and open field, with
20 m of the exclosure in the forest and 22.5 m in the open field.
Table 1. Area (ha) of vegetation types within each enclosure and dominant plant species identified in a
vegetation inventory performed in all three enclosures in May 2014 (study start).
Vegetation Type, ha Enclosure 1 Enclosure 2 Enclosure 3

Grassland 2.7 3.3 2.7
Forest 10.7 5.8 6.8
Semi-forest a 0 1.3 0.2
Total area 13.4 10.3 9.7
Dominant Plant Species in
Grassland, %
Grasses 60 57 26
Yarrow (Achillea millefolium) 10 14
Dandelion (Taraxacum spp.) 7 10 47
Hempnettle (Galeopsis tetrahit) 6
White clover (Trifolium repens) 5
Dominant Ground Cover in
Forest, %
Mosses 46 29 55
Grasses 11 26 12
Bilberry (Vaccinium myrtillus) 12 7 10
Lingonberry (Vaccinium vitis-idaea) 5 4 3
a Semi-forest includes forest areas dominated by deciduous forest with a large proportion of grass in the ground cover.
2.3. Pasture Sampling

The pasture in the open field areas was sampled in the second week of each month all year round,
except when the ground was covered with snow (0–29 cm, December 2014–March 2015, January 2016,
and March 2016). Four types of sample were collected: Forage, volume, graze, and exclosure.
Forage samples were collected by clipping a grab sample of vegetation 5 cm from the ground
every 10 m along a transect crossing the open fields. In En2 and En3, samples were collected along that
one transect, but due to the shape of En1, the sampling line was L-shaped and longer, and samples
were collected every 20 m to retrieve the same amount of samples representing the approximate same
size of grassland.
Volume samples were taken along the same transects as the forage samples, but every 50 m (100 m
in En1), collecting all vegetation 5 cm from the ground within a 30 cm × 30 cm square (0.09 m2 ). The
volume samples were placed in plastic bags and weighed later for determination of pasture quantity.
Graze samples were collected in the area where horses were grazing at the time of sampling.
Vegetation was cut close to the ground, i.e., the height at which the horses were assumed to graze.
Exclosure samples were taken within a 50 cm × 50 cm quadrat (0.25 m2 ) in the open field part of
the exclosures, 2 m from the fence (Figure 1). All vegetation above 5 cm from the ground within the
quadrat was collected by mowing with a scissor. Grassland production was assessed by weighing
the Exclosure samples, determining the dry matter (DM) content, and calculating the amount of DM
per hectare.
In February and April 2016, only graze samples were collected, due to small sample size/no
sample for the other three sample types. At all sampling sites except those for the graze samples, grass
height was measured using a herbometer (Herbometre, AGRO-Systémes, La membrolle sur Chosille,
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France), with a 30 cm × 30 cm square plate placed on top of the vegetation. On occasion, horses were
observed eating bilberry plants (Vaccinium myrtillus L.) and in December 2014, random samples of
bilberry, without berries or leaves, were collected at the time of grazing/browsing. All samples were
stored at −20 ◦ C until analysis.
2.4. Analyses of Chemical Composition

The exclosure samples from the three exclosures within each enclosure were pooled to one sample
before analysis of nutrient content. To determine DM content, pasture and feces samples were dried
at 60 ◦ C for 24 h and milled in a 1 mm hammer mill (Kamas, Slagy 200 B, Malmö, Sweden). A 2 g
subsample was then dried at 103 ◦ C for 16 h. Ash content was determined by incinerating a 2 g sample
at 550 ◦ C for 3 h, after which the residue was cooled and weighed. Organic matter (OM) content was
calculated by subtracting the content of ash from the DM content. Digestibility coefficient of organic
matter (VOS) and metabolizable energy (ME) content were determined in vitro according to Lindgren
163 [17]. The ME content is, however, based on the ME for ruminants, so was adjusted for horses using
the following equation derived by Jansson et al. [18]:
MEhorse = 1.12(MEruminant ) − 1.1
The concentration of neutral detergent fiber (NDF) was determined according to Chai and
Uden [19]. Analysis of CP was performed according to Kjeldahl [20], where ammonia nitrogen
concentration was determined by direct distillation with a Kjeltec 2460 analyser (Foss, Hilleröd,
Denmark) and N content was multiplied by 6.25 to give the CP content. To estimate the amount of
digestible CP (dCP) [19], the following equation used was:
dCP = 0.939-31.1/g CP kg/DM
The ratio between digestible CP and ME (RdCPME) was also calculated, since this is an
established measure of horse feed quality in Sweden [18]. In samples retrieved in June 2016 (see
Table S1), macronutrient concentrations were analyzed by inductively coupled plasma-atomic emission
spectroscopy (ICP-AES) using Spectro Flame equipment (SPECTRO Analytical Instruments, Kleve,
Germany). Due to small sample size, values for exclosures in En2 and En3 in October 2014 are missing.
2.5. Statistical Analyses

All statistical analyses were performed in Statistical Analysis Systems package 9.4 (SAS Institute
Inc., Cary, NC, USA). Differences were considered significant at p < 0.05. Values presented are least
square means (LSmeans) ± standard error (SE).
To study if climate differed significantly between years during the study period, effects of year on
air temperature and precipitation were estimated in a mixed model including an interaction between
season and year.
Test of differences in pasture nutrient content between samples retrieved in exclosures and the
samples retrieved in the enclosures, as well as possible differences between the different enclosure
sample types (Forage, Volume, and Graze), were performed using a mixed model, with enclosure
as repeated measurement and an effect of interaction between enclosure and time period (year and
month). When the p-value for the interaction effect between enclosure and time period was <0.1, a
separate analysis without the interaction effect was run. If no interaction effect is reported, values
refer to the latter analysis. As analysis of differences in pasture nutrient content showed no significant
difference between forage and volume samples, these were pooled before further analysis.
To test if different land areas, i.e., enclosures, responded differently in terms of nutrient content,
as well as pasture quantity on horse grazing and mowing, a mixed model with enclosure as repeated
measurement was used. The same model was also used to test if season (summer/winter) and year
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affected the nutritional content and pasture quantity. An analysis including the effect of interaction
between year and season was also performed.
3. Results
Mean air temperature and precipitation did not differ between the three study years (p > 0.05).
During summer, mean temperature was 13.3 ± 5.8 ◦ C and mean precipitation was 1.8 ± 4.3 mm/day.
During winter, the corresponding values were 1.3 ± 5.1 ◦ C and 1.0 ± 2.2 mm/day.
3.1. Variation in Pasture Nutrient Content Between Exclosures and Enclosures, and Between Sample Types
The exclosure samples showed lower DM and higher CP and ME contents than the forage and
volume samples from the enclosures, but there was no difference in NDF content (Table 2). The graze
samples showed higher nutrient contents than the other three sample types (with the exception of
energy content in exclosure samples, which was similar) (Table 2). There was a significant interaction
between sample type and time period for ME per kg OM and CP as a percentage of OM (p < 0.05),
where ME and CP remained at a high concentration in the period May–September in the graze samples,
while decreasing from August onwards in the other sample types.
The bilberry shrubs sampled in the forest had the following composition: DM 45%, CP 7% of DM,
digestible CP 32 g/kg DM, ME 3.2 MJ/kg DM, and OM 97% of DM.
Table 2. Content of dry matter (DM), metabolizable energy (ME) per kg organic matter (OM), crude
protein (CP) as % of OM, and neutral detergent fiber (NDF) as % of OM in three different types of pasture
samples collected in three enclosures grazed by horses, and in three exclosures per enclosure, monthly
between May 2014 and September 2016, except for December 2014–March 2015 and January-April 2016.
Enclosures p
Sample Exclosures
Forage Volume Graze
ME, MJ/kg OM 10.0 ± 0.2 a 10.0 ± 0.2 a 10.9 ± 0.2 b 10.5 ± 0.2 b 0.0002
CP, % of OM 11.9 ± 0.4 a 11.6 ± 0.5 a 17.0 ± 0.4 b 13.7 ± 0.4 c <0.0001
NDF, % of OM 57.2 ± 0.7 a 56.7 ± 0.9 a 51.0 ± 0.7 b 56.8 ± 0.8 a <0.0001
DM, % 34 ± 1 a 34 ± 1 a 29 ± 1 b 27 ± 1 b <0.0001
a,b,c Different superscript letters indicate significant differences within rows (p < 0.05).
3.2. Variation in Pasture Quantity

Mean grass sward height was lower and DM and ME content/ha were higher in enclosures
compared with exclosures, but there were also differences between the enclosures (Table 3). During
the summer in 2016, grass sward height decreased in both enclosures and exclosures compared with in
2015, but DM and ME content/ha only decreased in exclosures (Table 4).
Table 3. Mean grass sward height and content of dry matter (DM) and metabolizable energy (ME)
in the enclosures grazed by horses and in the exclosures within each enclosure mown monthly from
May 2014 to September 2016. Exclosures were mown at the same spots as sward height measurements
were made.
Enclosures Exclosures
p Means
En1 En2 En3 Mean En1 En2 En3 Mean
Grass sward
5.5 ± 0.1 a 7.7 ± 0.2 b 5.5 ± 0.2 a 5.3 ± 0.1 4.3 ± 0.4 a 4.9 ± 0.4 a,b 6.0 ± 0.4 b 5.6 ± 0.4 <0.05
height, cm
DM, kg/ha 957 ± 114 a
1393 ± 114 b 741 ± 114 a 886 ± 73 525 ± 111 487 ± 111 721 ± 111 326 ± 76 <0.0001
ME, MJ/ha 8494 ± 895 a 11,204 ± 928 b 7351 ± 895 a 7470 ± 636 5208 ± 1171 4912 ± 1171 7760 ± 1171 3048 ± 701 <0.0001
a,b,c Different superscript letters indicate significant differences (p < 0.05) between enclosures (En1, En2, En3).
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Table 4. Mean summer season grass sward height and content of dry matter (DM) and metabolizable
energy (ME) per year in the enclosures grazed by horses and in the exclosures within each enclosure
mown monthly from May 2014 to September 2016. Exclosures were mown at the same spots as sward
height measurements were made.
Enclosures Exclosures
2014 2015 2016 2014 2015 2016
Grass sward
8.9 ± 0.21 a 5.9 ± 0.2 b 5.2 ± 0.2 c - 7.1 ± 0.4 a 5.4 ± 0.4 b
height, cm
DM, kg/ha 1560 ± 160 a
863 ± 176 b 770 ± 176 b 714 ± 96 a 760 ± 105 a
260 ± 105 b
ME, MJ/ha 13,858 ± 1293 a
8133 ± 1416 b 8176 ± 1466 b 7201 ± 1044 a 7869 ± 1144 a 2810 ± 1144 b
a,b,c Different superscript letters indicate significant differences between years (p < 0.05).
3.3. Variation in Pasture Nutrient Content between Enclosures

In exclosure samples, there was no general effect of the different enclosures on any of the nutritional
parameters analyzed (Table 5). In the pooled forage + volume samples and in graze samples, the
content of ME, CP, and NDF/kg OM showed differences (p < 0.05) between enclosures (Table 5).
Table 5. Content of dry matter (DM), metabolizable energy (ME) per kg organic matter (OM), crude
protein (CP) as % of OM, and neutral detergent fiber (NDF) content as % of OM in four types of pasture
samples collected in three enclosures (En1–En3) grazed all year round by Gotlandsruss and in three
exclosures per enclosure. Samples were collected monthly between May 2014 and September 2016,
except for December 2014–March 2015 and January–April 2016. LSmeans ± SE, p-values indicate the
general effect of enclosure.
Sample Type En1 En2 En3 p

Exclosure
ME, MJ/kg OM 10.8 ± 0.2 10.9 ± 0.2 11.2 ± 0.2 0.2925
CP, % of OM 14.9 ± 0.4 14.3 ± 0.4 13.9 ± 0.4 0.1955
NFD, % of OM 55.0 ± 1.1 a 54.7 ± 1.1 a,b 51.8 ± 1.1 b 0.0924
DM, % 27 ± 1 29 ± 1 26 ± 1 0.2722
Enclosure Graze
ME, MJ/kg OM 10.4 ± 0.2 a 10.7 ± 0.2 a 11.5 ± 0.2 b 0.0029
CP, % of OM 16.9 ± 0.8 a,b 16.0 ± 0.8 a 18.8 ± 0.8 b 0.0427
NFD, % of OM 52.7 ± 1.4 a 53.9 ± 1.3 a 45.6 ± 1.3 b 0.0001
DM, % 30 ± 1 a,b 31 ± 1 a 27 ± 1 b 0.0159
Enclosure Forage + Volume
ME, MJ/kg OM 10.1 ± 0.1 a 9.3 ± 0.1 b 10.6 ± 0.1 c <0.0001
CP, % of OM 11.5 ± 0.4 a 11.9 ± 0.4 a,b 12.5 ± 0.4 b 0.1405
NFD, % of OM 54.7 ± 1.1 a 59.9 ± 1.1 b 54.2 ± 1.1 a 0.0007
DM, % 34 ± 1 a,b 37 ± 1 a 31 ± 1 b 0.0083
Different superscript letters within rows indicate significant differences (p < 0.05) between enclosures (En1,
a,b,c
En2, En3).
3.4. Variation in Pasture Quality Between Years, Seasons, and Months

As pasture was not sampled in all months throughout the year, only differences within seasons
between years are presented (Figure 2). During the study period, summer pasture NDF concentration
decreased in the enclosures, while ME and CP concentrations increased (Figure 2). In the exclosures,
summer pasture CP remained unchanged between years, while ME concentration increased and NDF
concentration showed a decrease.
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73
Figure 2. Content of metabolizable energy (ME) per kg organic matter (OM), crude protein (CP) as % of OM, and neutral detergent fiber (NDF) as % of OM in three
types of pasture samples (graze, forage + volume, exclosure) collected monthly in three enclosures grazed by 12 Gotlandsruss between May 2014 and September
2016. One year is defined as start of summer season to end of winter season. Each enclosure contained three exclosures. An asterisk indicates significant difference
between seasons within year (p < 0.05). Different lowercase letters (a, b) indicate differences between years within winter season, while different uppercase letters (A,
B) indicate differences between years within summer season (p < 0.05).
Animals 2019, 9, 500
Mean monthly nutrient composition and RdCPME in forage + volume and graze samples in all
three enclosures during the whole study period are shown in Tables S2 and S3. Mean ME content
ranged from 4.9 ± 0.5 to 12.0 ± 0.6 MJ per kg DM, mean CP content ranged from 7 ± 1 % to 24 ± 3 %,
mean digestible CP per kg DM ranged from 37 ± 12 g to 190 ± 26 g, and mean RdCPME ranged from
6.4 ± 1.1 to 15.9 ± 2.6 (Table S2).
3.5. Fecal Composition and Correlation with Pasture Nutrient Content

The OM and CP content in feces, but not the DM content, were dependent on the individual horse,
but overall the OM content in feces was lower in En3 than in En1 and En2 (Table 6). Content of CP,
both as % of OM and as % of DM, was lowest in En1 and highest in En3 (Table 6). Concentrations of
CP in feces were lower in winter than in summer (Figure 3). Within season, fecal CP concentration
increased with year.
Table 6. Content of dry matter (DM), organic matter (OM), and crude protein (CP) as % of DM and as
% of OM in feces from Gotlandsruss, divided equally between three enclosures (En1–En3) and grazing
all year round.
En1 En2 En3 p

DM, % 20 ± 0.3 a 19 ± 0.3 b 21 ± 0.3 a <0.0001
OM, % 79 ± 0.5 a 80 ± 0.5 a 75 ± 0.5 b <0.0001
CP, % of DM 8.4 ± 0.2 a 9.1 ± 0.2 b 9.7 ± 0.2 c <0.0001
CP, % of OM 10.8 ± 0.3 a 11.5 ± 0.3 b 13.1 ± 0.3 c <0.0001
Figure 3. (A) Monthly variation and (B) seasonal and between-year variation in crude protein (CP)
content (LSmeans ± SE) as % of organic matter (OM) in feces from Gotlandsruss grazing without
supplementary feeding from May 2014 to September 2016. One year is defined as start of summer
season to end of winter season.
The content of DM, OM, and CP in feces showed moderate to strong correlations with the DM,
NDF, OM, and CP concentrations in graze samples and in forage + volume samples (Tables 7 and 8).
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Table 7. Correlation coefficient (r, p-value) between nutrient concentrations in graze samples and
in fecal samples from 12 Gotlandsruss kept in three enclosures between May 2014 and September
2016 without supplementary feeding. DM = dry matter, OM = organic matter, CP = crude protein,
VOS = digestibility coefficient of organic matter, NDF = neutral detergent fiber.
Fecal Samples
Graze Samples
DM OM CP, % of DM CP, % of OM
r 0.37703 −0.30271 −0.28796
DM ns
p 0.0032 0.0198 0.0270
r −0.58502 −0.32107 0.62955 0.64978
VOS
p <0.0001 0.0132 <0.0001 <0.0001
r 0.36083 0.53560 −0.69221 −0.75195
NDF
p 0.0050 <0.0001 <0.0001 <0.0001
r −0.41346 0.36627
OM ns ns
p 0.0011 0.0043
r −0.35722 −0.48196 0.60511 0.66090
CP, % of DM
p 0.0055 0.0001 <0.0001 <0.0001
r −0.55235 0.49911 0.57349
CP, % of OM ns
p <0.0001 <0.0001 <0.0001
r 0.48350 0.46697 −0.75952 −0.80312
NDF, % of OM
p 0.0001 0.0002 <0.0001 <0.0001
Table 8. Correlation coefficient (r, p-value) between nutrient concentrations in forage + volume samples
and in fecal samples from 12 Gotlandsruss kept in three enclosures between May 2014 and September
2016 without supplementary feeding. DM = dry matter, OM = organic matter, CP = crude protein,
VOS = digestibility coefficient of organic matter, NDF = neutral detergent fiber.
Fecal Samples
Volume + Forage Samples
DM OM CP, % of DM CP, % of OM
r 0.58646 −0.45184 −0.44157
DM, % ns
p <0.0001 0.0003 0.0005
r −0.62653 −0.44629 0.75445 0.76465
VOS
p <0.0001 0.0004 <0.0001 <0.0001
r 0.57023 0.48945 −0.84249 −0.86310
NDF, %
p <0.0001 <0.0001 <0.0001 <0.0001
r
OM, % ns ns ns ns
p
r −0.38627 −0.43487 0.64359 0.67240
CP, % of DM
p 0.0025 0.0006 <0.0001 <0.0001
r −0.38872 −0.42891 0.64552 0.67353
CP, % of OM
p 0.0023 0.0007 <0.0001 <0.0001
r 0.56563 0.50105 −0.84228 −0.86426
NDF, % of OM
p <0.0001 <0.0001 <0.0001 <0.0001
4. Discussion
The results obtained in this three-year study showed that horse grazing altered pasture nutrient
composition and diversified pasture chemical composition (between enclosures) to a greater extent
than mowing. This indicates that horses can manage pasture and are therefore suitable for year-round
grazing in Sweden, as a means to increase pasture diversity. To our knowledge, this is the first study
at Nordic latitudes to evaluate the effect on pasture chemical composition of year-round grazing by
horses without supplementary feeding. Year-round grazing is currently not practiced in the region
because of the lack of vegetation growth in winter (i.e., temperatures below 5 ◦ C for more than four
days). However, the study area is within a zone suggested to be suitable with respect to climate
conditions for rewilding of horses, although local biotic factors, land cover, and soil type may influence
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the degree of suitability [21]. The study was conducted between 2014 and 2016, under temperature
and precipitation conditions typical for the region, and the results are therefore of general relevance for
this form of horse management.
4.1. Pasture Quality and Effects of Sample Type

One likely explanation for the diversified nutrient content of grazed pasture is the grazing behavior
of horses. Horses perform selective grazing [22,23], and also create mosaic landscape patterns [24],
as some areas are frequently grazed close to the ground while others are avoided. In the present
study, this was reflected in the higher content of ME and CP and lower content of NDF in the graze
samples, compared with the forage and volume samples, indicating an earlier botanical stage. An
additional explanation for the altered nutrient content may be the change in botanical composition
reported previously for the study area [4]. For example, grazing favored prostrate plant species (low
plant height at maturity).
The three enclosures all had different qualities. En1 and En2 showed a different response to En3
with respect to sward height and nutrient content, for example. The reason is unclear, but En3 had
previously been cultivated (forage production) and En1 and En2 had been grazed by cattle. En2 was
also the enclosure with the lowest grazing pressure, as it had a larger grass area (3.3 ha, compared
with 2.7 ha in En1 and En3), and the plant composition differed (Table 1). Mowing of the exclosures
was performed without selection, creating similar plant stress between enclosures, which might have
evened out initial differences in species and chemical composition. Horses, on the other hand, graze
selectively [22,23] and might have favored different species and areas in the three enclosures, as well
as putting more stress on the plants by grazing them shorter than mowing (i.e., <5 cm). In addition,
the trampling effect of horses might have affected the botanical composition and, therefore, also the
nutrient content.
Compared with the exclosure areas, from which grazing horses were excluded, grazed pasture
showed both lower (volume and forage samples) and higher (graze samples) concentrations of CP,
depending on sample type. The difference in composition between sample types may be due to the
forage + volume samples and the exclosure samples including only plant parts >5 cm. These plant
parts may be of a later botanical stage [25], as reflected by the higher content of NDF compared with
graze samples collected close to the ground. Both the graze and forage + volume samples showed
relatively high correlations with fecal NDF and CP content, indicating that they may both be valid
sampling options when measuring the nutrient content of pasture consumed by horses.
Observations from Sweden indicate that heavily grazed grasses may have higher CP and energy
content in October than grasses subjected to lower grazing pressure [26]. This is likely due to old
biomass being replaced by new, nitrogen-rich leaves as grazing increases defoliation [27], and is
presumably the reason for the higher nitrogen content in the graze samples in the present study.
During summer seasons, ME and CP increased in graze and forage + volume samples, while they
were unchanged in exclosure samples and remained high in the graze samples for longer than in the
other sample types. These results are in accordance with observations in sheep pastures that frequent
grazing during the vegetation period improves nutrient quality compared with mowing [28]. The
results confirm that grazing managed at the right intensity can enhance the quality of pasture [29].
4.2. Pasture Quantity

Pasture dry matter production in the study area was within the range reported for other horse and
ruminant year-round grazed pastures in Germany [1], and also similar to that reported for natural pastures
in Sweden grazed by cattle and sheep [30]. In addition, based on the 10-year mean for forage harvest from
cultivated grassland, the years included in the study seem to be representative for the region [31].
The pasture quantity values determined in the exclosures could be regarded as a measure of
overall pasture production, while the volume samples collected in the enclosures could be considered
a measure of the amount of pasture available to the horses. The exclosures were mown at the same
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Animals 2019, 9, 500
sites as the exclosure samples were taken. In contrast, the volume samples were collected by walking
along a straight line, and with this method, sampling sites may have varied slightly between sampling
occasions. Comparisons of pasture and energy quantity between enclosures and exclosures should
therefore be made with caution. However, the lack of differences in grass sward height between
enclosures and exclosures implies that mowing to 5 cm every month resulted in a similar rate of
vegetation removal as horse grazing within the enclosures.
Pasture quantity decreased over the study years in both enclosures and exclosures (2015 and 2016).
This may therefore be an effect of annual variation, rather than an effect of horse grazing, on pasture
production. To evaluate the long-term impact of horse grazing on pasture production, much longer
studies are required.
4.3. Ability of Pastures to Meet the Nutritional Requirements of Horses

Despite the cold winters in the region, CP content during winter (7–11% of DM) was within the
range reported for other European winter pastures [1]. Assuming a maximum DM intake capacity
of 3% of body weight [32–34], a 250 kg adult stallion with a CP requirement of 432 g/day [35] would,
in theory, manage with a CP concentration in pasture of 6% of DM, which is just below the lowest
value recorded in the present study. However, as found for other European pastures [1], the amount of
pasture available and the ME content during winter were insufficient to maintain body condition in
horses. In addition, a snow layer of >10 cm covered the ground for 14–31 days/winter in our study,
making the pasture more difficult to access. At the beginning of winter, when snow was still absent,
the mean energy content of the pasture was estimated to be 6200 MJ ME/ha in the grasslands. Assuming
a winter season lasting five months, and that all vegetation sampled here could be consumed by the
horses, this would supply each horse with 10 MJ ME/day, which represents 30% of the estimated daily
requirements [35]. However, in reality the samples would probably contain plants generally not consumed
by the horses and the energy requirement of the horses may have been higher during cold spells.
The insufficient levels of energy and CP in the pasture were reflected in loss of body weight and
body condition in the horses during the winter months (unpublished data). On the other hand, during
the growing season, pasture provided a surplus of energy great enough for the horses to store body
fat, compensating for energy deficiency in winter. En2 was the only enclosure where no horse at any
time would have required supplementary feeding to maintain a functional body condition. An energy
content in pasture of at least 12,000 MJ ME/ha in November therefore seems sufficient to avoid horses
becoming underweight at the given animal density (approximately 1000 kg horse on 3 ha of grassland
and 7 ha forest) and summer conditions. However, the lack of need for supplementary feeding in En2
could also be due to this enclosure containing a rather large area (1.3 ha) of semi-forest with some grass
in the understory.
Interestingly, during wintertime, horses spent more time in the forest (unpublished data). Therefore,
tree materials, some grass, and bilberry plants probably comprised a greater proportion of the horses’
diet during winter. The chemical analysis of bilberry plants showed rather low contents of ME and CP.
However, it should be noted that the analytical methods used are designed for grasses and legumes,
and may be less relevant for shrubs.
4.4. Correlation of Pasture Quality and Fecal Composition

The correlations between fecal and pasture concentrations of CP (as % of DM and OM) were
moderate to strong (r = 0.57–0.67). These were similar to correlations reported for stabled horses fed a
forage-only diet [36]. The fairly high correlation implies that a fecal grab samples could be used to give a
rough estimate of CP intake in grazing horses. However, the method may not apply if the horses are
growing or mares are lactating, as their nutrient requirements are higher than those of adult horses [35].
The trend for fecal CP concentration to be correlated with season is similar to that reported for
bachelor horses in Camargue [2] and feral horses in Canada [37]. However, the seasonal variation
in the present study was greater, ranging from 5.6% of DM in January to 17.0% of DM in May. The
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Animals 2019, 9, 500
increment in fecal CP content with year reflected the pasture composition, as CP content in pasture
vegetation was also higher in the second and third summer than at the start of the study.
Surprisingly, the correlation with fecal CP concentration was slightly stronger with forage +
volume samples than with graze samples. The highest CP values were observed in Graze samples and
the CP content was periodically much higher than the requirement. However, this was not reflected
in higher CP content in feces compared with the forage + volume samples, probably because more
of the easily digestible nitrogen was excreted with urine and not with feces [38]. The intention with
collecting the graze samples was to get a more accurate estimate of the nutritional composition of the
pasture actually consumed by the horses. However, both the graze samples and the grab sample of
the feces were spot-samples taken at random times and the graze samples represented conditions at
those times, while fecal samples would consist of digesta ingested hours to days before sampling [38].
This may be another reason why the CP content of forage + volume samples correlated better with CP
concentration in feces.
4.5. Practical Implications

Our study provides practical data on the quality and quantity of Swedish pasture grazed
year-round by horses. The area of semi-natural pastures in Sweden is decreasing [39]. At the same time,
the number of horses in Sweden is increasing, from 85,000 in 1970 [40] to now approaching 355,000,
which makes horses more common than dairy cows [31]. There is, thus, great potential for using horses
in landscape conservation in Sweden. However, most horses are stabled for most of the year and the
main roughage fed is hay or haylage harvested from cultivated leys. The results in the present study
indicate that pasture grazed year-round south of latitude 60◦ N in Sweden can have a sufficient energy
and nutrient content to meet the nutritional recommendations of adult horses for at least 10 months per
year (Table S3, no data for January and March) and that the amount of pasture may be a limitation. The
results presented here could be used as the basis for recommendations on utilization of semi-natural
pastures by horses even outside the growing season, as an alternative to feeding hay or haylage, for
example. Compared with feeding conserved forage, pasture provides increased opportunities for
horses to express their natural behavior and requires less resources than harvesting, conservation, and
transportation of hay/haylage. The low content of energy in shrubs such as bilberry means that forest
pastures could be suitable to meet the feeding behavior requirement of obese horses, although this
would require further evaluation. Increased grazing of semi-natural pastures would also increase
biological diversity [4] and help preserve agricultural landscapes. Moreover, our data on the nutrient
composition of Swedish semi-natural pastures support the suggestion [21] that they could be suitable
for future rewilding of horses at this latitude.
5. Conclusions
Compared with mowing, year-round grazing by horses in Sweden increased pasture nutrient
quality and diversity. This indicates that year-round grazing by horses in Sweden could be used as
a general tool to increase biodiversity. Pasture sampling method affected the pasture quality results
but, overall, CP content was sufficient to meet the horses’ requirements year-round, while the energy
content and pasture availability may be a limitation during winter. Fecal grab samples proved to give
a fairly good estimate of CP intake in grazing horses, but should be complemented with analysis of
pasture quality for pregnant, lactating, and young horses with high CP requirements.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2615/9/8/500/s1:

Table S1. Content of macronutrients in pasture; Table S2. Yearly variation in pasture nutrient content in forage +
volume samples; Table S3. Yearly variation in pasture nutrient content in graze samples.
Author Contributions: S.R. contributed to the methodology, validation, formal analysis, investigation, data
curation, and original draft manuscript preparation. A.S. contributed to the methodology, investigation, review and
editing, and visualization. A.J. contributed to the conceptualization, methodology, formal analysis, investigation,
resources, reviewing and editing, project administration, and funding acquisition.
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Animals 2019, 9, 500
Funding: This work was supported by the Swedish World Wildlife Fund, the Helge Ax:on Johnsson Foundation,
the Swedish University of Agricultural Sciences, and contributors to a crowd-funding campaign carried out in
2016 by the Swedish University of Agricultural Sciences.
Acknowledgments: We would like to thank our colleague Carl-Gustaf Thulin for contributing to the
conceptualization and for commenting on the manuscript, and students Oceane Martinét, Elin Mattsson, and
Karin Näslund for contributing to data collection. We also thank staff at the feed laboratory, Department of Animal
Nutrition and Management, SLU.
Conflicts of Interest: The authors declare no conflict of interest. The funders played no role in the design of the
study; in the collection, analyses, or interpretation of data; in writing the manuscript; or in the decision to publish
the results.
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animals
Article
Answers to the Frequently Asked Questions
Regarding Horse Feeding and Management Practices
to Reduce the Risk of Atypical Myopathy
Dominique-Marie Votion 1 , Anne-Christine François 2, *, Caroline Kruse 3 , Benoit Renaud 2 ,
Arnaud Farinelle 4 , Marie-Catherine Bouquieaux 1 , Christel Marcillaud-Pitel 5 and Pascal Gustin 2
1 Equine Pole, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary
Medicine, University of Lieège, 4000 Liège 1 (Sart Tilman), Belgium; [email protected] (D.-M.V.);
[email protected] (M.-C.B.)
2 Department of Functional Sciences, Faculty of Veterinary Medicine, Pharmacology and Toxicology,
Fundamental and Applied Research for Animals & Health (FARAH), University of Liège, 4000 Liège
1 (Sart Tilman), Belgium; [email protected] (B.R.); [email protected] (P.G.)
3 Department of Functional Sciences, Faculty of Veterinary Medicine, Physiology and Sport Medicine,
Fundamental and Applied Research for Animals & Health (FARAH), University of Liège, 4000 Liège
1 (Sart Tilman), Belgium; [email protected]
4 Fourrages Mieux asbl, 6900 Marloie, Belgium; [email protected]
5 Réseau d’Epidémio-Surveillance en Pathologie Équine (RESPE), 14280 Saint-Contest, France;
[email protected]
Received: 1 February 2020; Accepted: 14 February 2020; Published: 24 February 2020
Simple Summary: Equine atypical myopathy is a severe intoxication of grazing equids resulting
from the ingestion of samaras or seedlings of trees from the Acer species. The sycamore maple (Acer
pseudoplatanus) is involved in European cases whereas the box elder (Acer negundo) is recognized
as the cause of this seasonal pasture myopathy in the Unites States of America. In Europe, young
and inactive animals with a thin to normal body condition and no feed supplementation, except for
hay in autumn, are at higher risk. The risk is also associated with full time pasturing in a humid
environment. Indeed, dead leaves piling up in autumn as well as, the presence of trees and/or woods
presumably exposes the horses to the sycamore maple. This manuscript answers the most frequently
asked questions arising from the equine field about feeding and management of equines to reduce the
risk of atypical myopathy. All answers are based on data collected from 2006 to 2019 by the “Atypical
Myopathy Alert Group” (AMAG, Belgium) and the “Réseau d’épidémiosurveillance en Pathologie
équine” (RESPE, France) as well as on a review of the most recent literature.
Abstract: In 2014, atypical myopathy (AM) was linked to Acer pseudoplatanus (sycamore maple) in
Europe. The emergence of this seasonal intoxication caused by a native tree has raised many questions.
This manuscript aims at answering the five most frequently asked questions (FAQs) regarding (1)
identification of toxic trees; reduction of risk at the level of (2) pastures and (3) equids; (4) the risk
associated with pastures with sycamores that have always been used without horses being poisoned
and (5) the length of the risk periods. Answers were found in a literature review and data gathered by
AM surveillance networks. A guide is offered to differentiate common maple trees (FAQ1). In order
to reduce the risk of AM at pasture level: Avoid humid pastures; permanent pasturing; spreading of
manure for pasture with sycamores in the vicinity and avoid sycamore maple trees around pasture
(FAQ2). To reduce the risk of AM at horse level: Reduce pasturing time according to weather
conditions and to less than six hours a day during risk periods for horses on risk pasture; provide
supplementary feeds including toxin-free forage; water from the distribution network; vitamins and
a salt block (FAQ3). All pastures with a sycamore tree in the vicinity are at risk (FAQ4). Ninety-four
percent of cases occur over two 3-month periods, starting in October and in March, for cases resulting
from seeds and seedlings ingestion, respectively (FAQ5).

Animals 2020, 10, 365
Keywords: equine atypical myopathy; Acer spp.; risk factors; environment
1. Introduction
Equine atypical myopathy (AM) is a severe pasture-associated intoxication that may occur in
autumn and spring following the ingestion of certain species of maple (Acer) seeds and seedlings,
respectively. This environmental intoxication is linked to Acer pseudoplatanus (sycamore maple) in
Europe and Acer negundo (box elder) in the US [1,2]. These trees may contain several toxins [3].
The ingestion of samaras or seedlings of the incriminated trees goes with the ingestion of two
cyclopropylamino acids, hypoglycin A (HGA) and methylenecyclopropylglycine (MCPG) [4]. These
toxins have been confirmed to be implicated in European AM cases. Long before the discovery of the
cause of AM, Fowden and Pratt (1973) [3], reported the presence of cyclopropyl derivates in seeds of
the different representatives of the Acer’s species. Both Acer pseudoplatanus and Acer negundo seeds
have been found to contain HGA and MCPG. On the contrary, other maple trees commonly found in
Europe as Acer platanoides (Norway maple) and Acer campestre (field maple) tested negative for these
compounds [3].
In fact, HGA and MCPG are not toxic per se but need to be converted into their active metabolites, i.e.,
methylenecyclopropylacetyl-CoA (MCPA-CoA) and methylenecyclopropylformyl-CoA (MCPF-CoA),
respectively [4,5]. Both MCPA-CoA and MCPF-CoA inhibit enzymes that participate in β-oxidation
and thus energy production from lipid metabolism [5,6]. The typical sign of intoxication is an
acute rhabdomyolysis syndrome unrelated to exercise. This clinical picture may be seen on several
horses within a group [7–10]. In more than 50% of the cases, the following clinical signs were
observed: Weakness, recumbency, myoglobinuria, full bladder, stiffness, depression, muscle tremors or
fasciculation, reluctance to move, sweating, normothermia, and congested mucous membranes [10–12]
Atypical myopathy has a high mortality rate (i.e., 74%) that varies between countries and years (from
43% [11] to 97% [13]). The overall mortality rate of 74% average data among countries included in the
study of van Galen et al., (2012) and does not take into account the different sources of variability [11].
For example, a study reports a lower mortality rate (i.e., 44%) in hospitalized animals [14]. It is
hypothesized that less critical cases are driven to a hospital where appropriate symptomatic treatment
is easier to provide. These two factors may contribute to the higher survival rate in an equine hospital
than in the field. In any case, only the administration of vitamins and antioxidants has proven to be
beneficial for survival [12,15].
In 2004, an alert group named “Atypical Myopathy Alert Group” (AMAG) was launched to warn
horse practitioners and owners of the risk peaks. The alerts are released following case declarations
and the AMAG regularly updates its data with the latest number of cases. Additionally, to its
disease surveillance role, AMAG collects epidemiologic data about AM that has emerged in several
European countries since 2006. French cases are gathered in close collaboration with the Réseau
d’Épidémio-Surveillance en Pathologie Équine (RESPE) which monitors equine diseases through a
network of French sentinel practitioners. Thanks to the European surveillance network, we now know
that several Père David’s deer (Elaphurus davidianus) have succumbed from this intoxication in different
zoos in Germany. This indicates that ruminants pasturing in the vicinity of sycamore trees may also be
intoxicated [16].
In light of the high mortality rate and the absence of specific treatment, prevention is the key to
avoid intoxication of animals. Before discovering the cause of AM, epidemiological studies revealed
risk factors associated with management practices of horse and pasture [15,17] In 2014, the cause of AM
was discovered and linked to Acer pseudoplatanus in Europe [1]. Despite the cause of the intoxication
being known, outbreaks have continued to occur. The development of a condition caused by a native
tree has raised many questions among all actors attached to the equine sector.
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Animals 2020, 10, 365
The most frequently asked question (FAQ) concerns the identification of toxic trees. Other
commonly-asked questions involve the feeding and management practice of equids in order to reduce
the risk of intoxication. These main FAQs can be summarized as being: (FAQ1) “Which maples are
toxic? Is this tree a maple and if so, is it toxic?”; (FAQ2) “How can AM be prevented (at pasture level)?”;
(FAQ3) “How can AM be prevented (at horse level)?”; (FAQ4) “Our pasture is surrounded by sycamore
maple trees, but no case of AM ever occurred in our grazing horses. Does this mean the pasture is safe
for our animals?” and (FAQ5), “When does the risk of AM start and stop in autumn and spring?”.
This manuscript answers to these FAQs regarding horse nutrition and management practices in
order to prevent AM both by reviewing the most recent literature and by analyzing epidemiological
data gathered since 2006.
2.1. Literature Review

A systematic review was performed using on the electronic databases PubMed and Scopus with
« atypical myopathy » AND « horse » as keywords. In addition, abstracts of proceedings of meetings
dedicated to horses were consulted (e.g., AAEP—American Association of Equine Practitioners
Annual Convention, AVEF—Association des Vétérinaires Équins Français, BEPS—Belgian Equine
Practitioners Society Study Days, BEVA—British Equine Veterinary Association and, WEVA—World
Equine Veterinary Association Congress).
2.2. Epidemiological Data

Information regarding AM cases in Europe over a 13-year period (2006–2019) was collected via
standardized questionnaires available on the AMAG (http://www.myopathie-atypique.be) and the
RESPE (https://respe.net) websites. These forms were completed via email or phone contact with the
owners or veterinarian whenever possible. Additionally, cases were also gathered by direct contact via
mail or phone between owner/veterinarian and the principal investigator of this study. Information
about the management and environment of diseased equines was obtained from the animals’ owners
whereas clinical data was collected from veterinary surgeons. Cases occurring between the 1 September
to the end of February were classified as “autumnal cases” and those from the 1 March up to the end of
August as “spring cases”.
3. Results
3.1. Literature Review

Among 68 records identified in PubMed, five were rejected as they did not concern AM. From
the search in Scopus, 514 results were obtained that were refined by selecting only research articles.
From the 82 remaining studies, 63 were out of the scope and were discarded. In total, more than 127
documents (research articles and abstracts) were obtained.
3.2. Epidemiological Data

Epidemiologic data from 3039 cases were recorded in 14 different countries from autumn 2006
to 30 November 2019 (Figure 1, Table 1). This data set includes all cases communicated to the
surveillance networks with a tentative diagnosis of AM. During this period, 14 autumnal outbreaks
were encountered with a mean (± S.D.) of 164.6 ± 153.3 (median 79.5) reported cases. For spring,
12 outbreaks were recorded with a mean (± S.D.) of 56.5 ± 77.3 (median 24.0) diseased horses per
outbreak. For all outbreaks together, the mean (± S.D.) number of cases is 112.5 ± 132.4 (median
41). Parts of these data (n = 824) have already been analyzed to define risk (Table 2) and protective
(Table 3) factors on cases in Belgium [17], in the UK [12] and at a European level [11,15]. Equines
particularly at risk for AM were found to be young (i.e., less than 3 years of age) and inactive animals
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with normal body condition score and receiving hay in autumn [17]. The risk of intoxication was also
associated with full time pasturing in a humid environment where dead leaves pile up in autumn,
with the presence of trees and/or woods and thus presumably exposed to the above-mentioned maple
trees [12,15,17]. During the ten years that have passed since the last epidemiological study performed
at an European level [11,15], 2433 new cases have been reported, reaching a total number of 3039
cases available for the current study. The whole database (i.e., 2006–2019) was cleaned by removing
all equids that were not at pasture at the onset of clinical signs or within the week preceding these
signs, equids that were diagnosed with another disease as well as equids having a low probability of
intoxication (e.g., no pigmenturia and serum creatine kinase activities <10.000 IU/L; normal values
50–200: IU/L) according to van Galen et al., 2012 [11]. As opposed to the study of van Galen et al.,
2012 [11], cases too poorly documented to make a definitive diagnosis have been retained in the study
group. A total of 2371 cases was included in this study. The age distribution of these cases over the
years are presented in Figure 2. The weekly occurrence of AM during the spring and autumnal seasons
may be found in Figure 3a,b, respectively.
Figure 1. European distribution of atypical myopathy cases notified to the disease surveillance networks
from autumn 2006 to November 2019.
84
Table 1. Total number of atypical myopathy cases in Europe notified to the disease surveillance networks from autumn 2006 to November 2019 (n = 3039).
Year 2006 2007 2008 2009 2010 2011 2012 2013 2014 * 2015 2016 2017 2018 2019
Total/
Countries Country
Spring
Spring
Spring
Spring
Spring
Spring
Spring
Spring
Spring
Spring
Spring
Spring
Spring
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Autumn
Animals 2020, 10, 365
Austria 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 4 9 0 0 17
Belgium 46 7 18 0 5 0 69 13 14 3 18 0 1 0 141 8 51 5 6 8 52 20 5 1 191 49 1 732
Czech Republic 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 0 2 17 0 0 19 2 0 49
Denmark 2 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 2 0 0 8
France 32 1 11 18 11 0 134 106 32 10 40 16 4 1 64 13 71 9 24 8 194 181 31 18 114 47 26 1216
Germany 7 0 3 5 0 0 93 21 2 2 59 2 0 0 24 1 8 0 0 0 21 20 4 0 33 4 2 311
Ireland 0 0 0 0 0 0 2 0 0 0 0 0 0 0 2 0 38 0 0 0 1 2 0 0 1 0 0 46
Luxembourg 1 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3
Portugal 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 2
Spain 0 0 0 0 0 0 0 0 0 0 52 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 52
Sweden 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 7
Switzerland 0 0 9 0 0 0 31 3 0 0 6 0 0 1 12 0 0 0 0 0 9 3 0 0 7 0 0 81
The Netherlands 13 0 3 0 2 0 34 7 0 0 0 0 0 0 18 1 4 0 0 0 1 2 0 2 9 10 1 107
United Kingdom 1 0 13 0 0 0 39 20 3 0 33 6 2 2 52 13 154 20 2 1 11 11 3 1 13 3 5 408
Total/season 102 8 57 23 18 0 406 171 51 16 213 24 7 4 322 41 326 34 34 17 292 256 43 26 398 115 35
3039
85
Total/year 102 65 41 406 222 229 31 326 367 68 309 299 424 150
Comments: This last counting replaces all previously published data; the word “autumn” should not be taken strictly since clinical series continue into early winter; (*) in 2014, atypical
myopathy was linked to Acer pseudoplatanus (sycamore maple) in Europe.
Table 2. Risk factors identified in from former epidemiological studies [12,15,17].
Category Risk Factors Odds Ratio 95% Cl for Odds Ratio

Young horses (<3 years)
Demographic data
3.08 [17] (b) 1.01–9.39 [17] (b)
Thin (or normal weight *)
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(3.85 [15]/2.20 [17] (b)) (1.77–8.37 [15]/1.01–4.79 [17] (b))

5.42 [15] 2.577–11.42 [15]
3.07 [17] (b) in winter 1.45–6.50 [17] (b) in winter
At pasture 24 h a day all year round 3.78 [17] (b) in spring 1.49–9.59 [17]] (b) in spring
Horse management
23.2 [17] (b) in summer 1.41–382 [17] (b) in summer
10.9 [17] (b) in autumn 3.56–33.4 [17]] (b) in autumn
Not physically active 11.8 [17] (b) 5.02–27.8 [17] (b)
Feeding practice Hay given in autumn 4.09 [17]] (a) 1.18–14.1 [17] (a)
History of previous death of horse(s) on the pasture 4.45 [17]] (b) 1.61–12.29 [17] (b)
Lush pasture in winter 3.95 [17] (b) 1.49–10.46 [17] (b)
3.43 [15] 1.52–7.77 [15]
Sloping pasture/steep slope
86
3.70 [17] (b) 1.58–8.68 [17] (b)
11.11 [15] 4.82–25.59 [15]
Access to dead leaves piled up in autumn
10.47 [17] (b) 2.82–40.88 [17] (b)
Pasture
Presence of trees at pasture * 7.82 [15] 1.99–30.73 [15]
Dead wood at pasture 3.12 [15] 1.42–6.84 [15]
Humid pasture 2.63 [17] (b) 1.29–5.36 [17] (b)
Pasture surrounded by or containing a stream/river 2.78 [17] (b) 1.24–6.19 [17] (b)
Spreading of manure 5.73 [17] (b) 2.40–13.69 [17] (b)
(*) Parameters that are not consistent among studies [15] cases with a high probability of or confirmed atypical myopathy vs. and the cases with a low probability of having AM or with
another diagnosis [17] (a) confirmed cases vs. clinically healthy co-grazing equidae [17] (b) confirmed cases vs. control horses.
Table 3. Protective factors identified in former epidemiological studies [12,15,17].
Category Protective Factors Odds Ratio 95% Cl for Odds Ratio

Demographic data Overweight 0.25 [17] (b) 0.09–0.69 [17] (b)
0.11 [17] (a) 0.01–0.67 [17] (a)
Frequent deworming
Animals 2020, 10, 365
0.05 [17] (b) 0.01–0.16 [17] (b)

Regular vaccination 0.10 [17] (b) 0.05–0.21 [17] (b)
Regular physical activity 0.08 [17] 0.03–0.19 [17]
Horse management
Weather-dependent pasturing time in spring 0.24 spring [17] 0.06–0.89 spring [17]
and in autumn 0.10 autumn [17] 0.02–0.56 autumn [17]
0.04 [15] 0.01–0.19 [15]
<6 h at pasture per day
0.62 [17] 0.16–2.36 [17]
No access to pasture 0.03 [15] 0.00–0.22 [15]
Supplementary feeds all year round 0.17 [15] 0.05–0.59 [15]
0.20 [17] (a) for silage 0.04–0.94 [17] (a) for silage
Silage and concentrate feed in autumn + corn
0.19 [17] (a) for concentrate feed 0.04–0.87 [17] (a) for concentrate food
87
in winter
0.22 [17] (a) for corn 0.05–0.93 [17] (a) for corn
Feeding practice
and water supply 3.52 [12] 1.08–11.47 [12]
Salt block (all year)
0.20 [17] 0.09–0.40 [17]
Water provision in tank/bathtub 0.25 [15] 0.09–0.69 [15]
Water supplied by the distribution network 0.39 [17] (b) 0.17–0.88 [17] (b)
Pasture Gentle slope 0.34 [17] (b) 0.14–0.84 [17] (b)
[12] Survivors vs. nonsurvivors [15] cases with a high probability of or confirmed atypical myopathy vs. the cases with a low probability of having AM or with another diagnosis [17] (a),
confirmed cases vs. clinically healthy co-grazing equidae [17] (b), confirmed cases vs. control horses.
Animals 2020, 10, 365
Figure 2. Frequency distribution of equids with age categories: <3 years, >3 years and <20 years and
>20 years old (n = 1510) over the study period (2006–2019).
(a )
Figure 3. Cont.
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( b)
Figure 3. (a). Spring cases: weekly occurrence of atypical myopathy cases from week 9 (1 March) up
to week 20 (31 May) over the study period (2006–2019); (b). Autumnal cases: weekly occurrence of
atypical myopathy cases from week 41 (1 October) up to week 52 (31 December) over the study period
(2006–2019).
4. Discussion
The origin of the results consists of previous epidemiological studies modified and completed by
the analysis of the newest data. The results contribute to answer the FAQs regarding horse feeding
and management practices to reduce the risk of AM.
4.1. FAQ1: “Which Maples Are Toxic? Is this Tree a Maple and If So, Is It Toxic?”
The question about which maple trees are toxic is often associated with a request to identify
trees on the pasture. The database consultation indicates that 99% of pastures contain or are directly
bordered by tree. However, looking at the data from 2014 up to now, it is observed that 20% (92/456)
of AM horse owners could not answer if seeds and/or seedlings of sycamore trees were present in
their meadow. Despite the educational material available on the Internet (https://en.wikipedia.org),
horse owners and veterinarians are still struggling to recognize the different maple species (personal
observation). This phenomenon is accentuated due to the numerous erroneous descriptions in the
literature [18]. A guide from Renaud et al., (2019) is available [19], helping the different actors to
differentiate the three Acer species commonly found in European pastures where cases have been
declared [20].
The maple genus includes approximately 561 species [21]. Some of them are extensively planted
as ornamental trees because of their autumnal color. As a result, there is not only a demand to
distinguish non-toxic trees (Acer platanoides (Norway maple) and Acer campestre (Field maple)) from
Acer pseudoplatanus, but also many questions regarding the potential toxicity of other maple species.
Even though up to now, not all maple trees have been tested, it is one of note that almost 50 years
before the discovery of the cause of AM, the incriminated toxins had already been tested in many
Acer species [3]. Among the tested species, the following species have tested positive for HGA and/or
MCPG (non-exhaustive list): Acer palmatum, Acer japonicum, Acer macrophyllum, Acer spicatum, Acer
saccharinurn, and Acer saccharum. These exotics species may be found in ornamental gardens and may
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spread to the neighboring regions [22]. Therefore, these Acer species might ultimately represent a risk
of intoxication for equids.
4.2. FAQ2: “How Can AM Be Prevented (at Pasture Level)?”

Atypical myopathy occurs seasonally with outbreaks starting in autumn that may continue in
early winter. On the contrary, spring outbreaks usually cease before summer. At pasture level, the
risk can be decreased (1) by avoiding contact with toxic plant material and (2) by favoring low-risk
meadows for pasturing during autumn and spring.
4.2.1. Avoid Contact with Toxic Plant Materials

Former epidemiological studies identified access to dead leaves piled up in autumn, the presence
of trees and dead wood on pastures as risk factors for AM [15,17]. This observation is presumably
due to the presence of Acer pseudoplatanus and the ingestion of samaras and seedlings in autumn and
spring, respectively [23–25]. It is therefore important to be able to recognize Acer pseudoplatanus, its
samaras and seedlings. When in doubt, professional expertise should be sought to identify the tree
(botanists and/or forestry agents might be of help). Recently, it has been suggested that flowers falling
from sycamore trees after heavy rainfall and/or wind could be an additional source of intoxication [26].
Depending on weather conditions, samaras of Acer species may be able to spread their seeds up to
several hundred meters [27]. Therefore, pasture contamination with seeds or seedlings is not necessarily
linked to the presence of a tree on the pasture. In early autumn, especially after windy weather has
dispersed sycamore samaras, it is recommended to equids’ owners to identify contaminated areas
in their pasture. The removal of seeds may help to prevent AM [28]. However, when samaras are
too abundant and/or too widely dispersed within the premise, grazing in the affected area must be
prohibited. Another way to limit grazing to areas free of fallen seeds and/or flowers and/or seedlings is
to create parcels within the pasture [26].
Additionally, the spreading of manure and/or harrowing of pastures was found to increase the
risk of AM [17]. This practice might favor the dispersal of the toxic material throughout the pasture
and subsequent intoxication of horses.
Regarding prevention at the pasture level, there is growing interest in the disposal of seedlings. It is
worth noting that seedlings still contain HGA after herbicidal spraying or mowing [29]. These techniques
are therefore ineffective regarding the destruction of toxic material.
4.2.2. Use or Create Low-Risk Pastures

Permanent pasturing was found to be a risk factor. This is most probably due to the associated
decrease in grass quantity, which leads equids to ingest the etiological agent [12,15,17]. Our database
indicates that, in 64% of our cases, the pasture grass was bare or absent of grass. This observation
correlates with previous epidemiological studies [11]. A good pasture management (for example
pasture rotation) is advised in order to offer lush pastures. Indeed, a green meadow will limit the
ingestion of seeds and seedling by horses allowing them to eat mainly grass [11,17].
Pastures particularly at risk for AM have Acer pseudoplatanus in their vicinity. Grazing on these
meadows should be avoided during the risky seasons (see FAQ4). Furthermore, humid pastures are of
particular risk for AM and grazing should therefore also be avoided on these pastures [17]. HGA is
a water-soluble toxin that may pass from plants to water by direct contact [26,30]. This solubility
might explain the risk associated with humidity and the protective factor linked to drinking water
provided via the distribution network. For this reason, only pastures that do not contain rivers and/or
freestanding water should be used during the risky seasons.
4.3. FAQ3: “How Can AM Be Prevented (at Horse Level)?”

All type of equids were affected by AM including donkeys (1.6% of the cases) and zebras
(3 cases from zoological parks) with no highlighted risk factor associated with any species. The first
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epidemiological study highlighted that young horses, especially those <3 years of age were the primary
affected group [17]. Later on, van Galen et al. (2012) [11] found that all age groups were represented.
Our data suggest a gradual change in age distribution of cases over the years (Figure 2). In 2006, 71%
of affected equids were less than 3 years old, whereas now this age group represents only 36% of
individuals. This finding is unlikely to be explained by acquisition of immunity to the toxins since some
survivors of AM did succumb to a second episode of the disease (unpublished data). An explanation
could be that the population at risk is increasing because toxic pressure has increased over the years.
The practical usefulness of this information is that all equids must be considered at risk, whatever their
age. At the animal level, the risk can be decreased by (1) management and (2) feeding practices.
4.3.1. Management of Grazing Time

The intoxication is intimately linked to pasturing as van Galen et al., (2012) reported that 98%
of affected horses were at pasture at onset of clinical signs [11]. Our data confirms this information
with 99.8% of horses being at pasture when clinical signs declared. The few remaining cases had been
stabled for less than a week which implies these animals may have been in contact with the toxin
before. Up to now, not a single case of our database has been confirmed in horses that had no access to
pasture and/or paddock based on HGA and MCPA-carnitine detection (unpublished data). Thus, it
is advised to stable horses during autumn and spring outbreaks if seeds or seedlings are, or may be,
present at pasture [26]. However, keeping a horse in the stable day and night may be difficult and is
not considered as good practice in animal welfare [31,32]. Interestingly, the limitation of grazing time
to less than six hours a day was found to be a protective factor [15]. Consistent with a previous study
of UK cases [12], 97.5% of equids of our database had spent more than six hours per day at pasture.
This observation suggests that the length of exposure to the toxins appears to be a determining factor
in the risk of AM.
Specific weather conditions have been linked with AM outbreaks [9,10,33,34] whereas
weather-dependent pasturing time (i.e., stabling horses when inclement weather is forecast) reduces the
risk of AM [17]. Reducing pasturing time according to weather conditions was not a recommendation
implemented by owners in our cases since less than 1% of them were in compliance with this preventive
measure. Our data reinforces the value of this preventive measure.
4.3.2. Feed and Water Supply

Receiving supplementary feeds (hay, straw, complete mix, oats, barley, and/or corn) throughout
the year decreases the risk of AM [17]. Atypical myopathy results from an energetic imbalance
subsequent to HGA and MCPG poisoning. Feed provides energy substrates (especially carbohydrates)
that supports the energetic metabolism and also vitamins and antioxidants known to increase the
chance of survival [15]. The mitochondrial enzymes inhibited by HGA are flavin adenine dinucleotide
(FAD) dependent. This cofactor originates from riboflavin (vitamin B2) suggesting that it would be
useful to give this vitamin [35]. Alfalfa is an excellent natural source of riboflavin as well as, to a lesser
extent, the hay from common grass [36]. In addition, well-nourished horses might be less tempted
to ingest samaras and/or seedlings. Among horses receiving supplementary feeds in their daily diet
(64%; n = 665), our data indicates that 61% received concentrated feed (complete mix, oats, and/or
barley) and 50% had access to a salt block providing minerals. However, a salt block did not prevent
these animals to be intoxicated.
Giving hay in autumn was identified as a risk factor [17] (and 40% of our cases receiving
supplementary feed were fed with hay only). Indeed, hay may contain seeds and seedlings with
detectable HGA concentration after several months [29] and even years of storage [30]. Gonzales et al.
(2019) suggests that AM might occur in stabled horses [29] but this hypothesis is not sustained by our
data since, as above-mentioned, not a single case has been confirmed by blood testing in equids with no
access to pasture and/or paddock. However, we do agree that giving hay produced from contaminated
pasture would increase the risk of AM in horses kept at pasture. It is probably wise not to produce
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hay/haylage and/or silage from pasture areas in the vicinity of sycamore trees [29]. Providing hay in
autumn is controversial. Indeed, this practice was found to be a risk factor in a case-control study [17]
but that appears nevertheless as a protective factor when comparing management practice in pasture
of cases versus pastures of controls [15]. From this result, we can suggest that forages free from toxins
should be given at libitum, but hay should neither be placed on the ground, nor under sycamore trees,
since both practices could increase the risk of ingesting toxic material.
4.3.3. Drinking Water

Water supplied by the distribution network [17] or stored in a tank or in an bathtub [15] are
protective factors. These observations suggest that water from other sources may be contaminated by
the toxins. This hypothesis is reinforced by the study of Renaud et al., (2019) [30], which showed that
HGA is released by stagnant flowers or seeds from Acer pseudoplatanus in contact with water. On the
other hand, when water is dripping off flowers, no HGA is detected in collected water. However, this
latest observation can be modulated by the fact that (1) the concentration of HGA can be below the
limit detection threshold of the quantification method or (2) that HGA might be degraded in water.
To the authors knowledge, there is no published study about the stability of HGA in water.
4.4. FAQ4: “ Our Pasture Is Surrounded by Sycamore Maple Trees, but No Case of AM ever Occured in Our
Grazing Horses. Does this Mean the Pasture Is Safe for Our Animals?”
Our data and former epidemiological studies indicate that unexplained sudden deaths of horse(s)
had been noted on 22% of the pastures where cases were grazing [11]. In other words, 80% of cases
were grazing in pastures that had no history of previous death of equid(s) (regardless of the suspected
cause). Atypical myopathy is an emerging disease and a pasture surrounded by sycamore trees should
not be considered as safe for pasturing horses.
4.5. FAQ5: “When Does the Risk of AM Start and Stop in Autumn and Spring?”
As previously reported, cases of AM occur more frequently in autumn (76%; n = 1801) than in
spring (24%; n = 570). The expressions “autumn” and “spring” should not be taken stricto senso, since
the autumnal clinical series are continuing in early winter and some spring cases are occurring after
the 21 June. It is worth noting that 94% of “spring” cases occurred between the 1 March and the 31 May
and 94% of “autumnal” cases occurred between the 1 October up to the 31 December (Figure 3a,b).
The cause of autumnal outbreaks cessation is not precisely known. Before discovering the etiology
of AM, it was hypothesized that severe frost might destroy the etiological agent since outbreaks tend
to cease after several days of deep freezing [10]. Now that HGA has been described as incriminated
toxin, this hypothesis can be refuted since laboratory investigation showed that HGA is unaltered after
several freeze–thaw cycles [37]. In our laboratory (unpublished data), we have collected samaras from
the environment on a weekly basis since 2016 from now and, with very few exceptions, HGA has
always been detected in seeds of sycamore tree. However, clinical series of AM usually fully stop in
winter and resume with the germination of the samaras (personal observation). The analysis of HGA
concentration over time in samaras fallen on the ground showed a high variability from tree to tree and
from week to week thus impeding an easy interpretation of the evolution of toxicity. These field studies
were complicated by the fact that the samaras collected on the ground had fallen at very different times.
Therefore, the cause of the ceasing “autumnal” outbreaks in winter is not known but could result from
a reduction in accessibility (e.g., adheration to the ground following rain and frost) and/or a decrease
in toxins’ concentration).
Regarding spring outbreaks, horse owners wonder if the case series stops because the seedlings
have lost in toxicity. Actually, the end of spring outbreaks may not be explained by the disappearance
of the toxicity since the seedlings remain toxic [26]. It is however hypothesized that spring outbreaks
cease following a relative decrease in risk of intoxication by grazing. This reduced risk of intoxication
might result from (1) a lusher meadow, (2) the observed decrease in toxicity of seedlings with their
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growth [26], (3) a decrease in palatability of older plants [26], (4) less frequently encountered weather
conditions favoring toxicity and (5), a significant natural disappearance of seedlings. Regarding the
latter, only a small percentage (<20%) of seedlings recorded in early spring on heavily contaminated
pastures are still present in early summer (unpublished data). This observation added to the fact that
herbicidal spraying do not reduce HGA concentration in sycamore seedlings [29] questions the benefit
of herbicide treatments.
5. Conclusions
As there is no specific treatment for AM yet, prevention is the key. The risk of developing AM
results from the combinations of protective and risk factors. In order to reduce the risk of AM, it
is advised to avoid humid pastures, permanent pasturing, spreading of manure, and contact with
sycamore plant material. During the risky periods pasturing time should be modulated according
to weather conditions and limited to less than six hours a day. Grazing equids should receive
supplementary feeds, with preferences for feeds containing riboflavin. When hay or silage are fed,
it is necessary to exercise caution ensuring the forages are toxin-free. Also, it is advised to supply a
salt block and provide drinking water from the distribution network. It is worth noting that AM is an
emerging disease and equids of any age and all pastures with a sycamore tree in the vicinity must
be considered at risk. These preventive measures should be implemented for a period of 3 months
twice yearly, starting in March for “spring cases” then again in October to prevent “autumnal cases”.
As mentioned before, these are the critical seasons and samaras or seedlings are likely to be present on
the pasture. A French version of this paper with additional illustrations can be found in supplementary
materials (Supplementary S1).

Supplementary S1: A French version of this paper with additional illustrations.
Author Contributions: Data curation, M.-C.B.; Funding acquisition, D.-M.V.; Investigation, D.-M.V.; Resources,
A.F. and C.M.-P.; Supervision, D.-M.V. and P.G.; Writing—original draft, D.-M.V. and A.-C.F.; Writing—review and
editing, B.R. and C.K. All authors have read and agreed to the published version of the manuscript.
Funding: The study was supported by the la Wallonie agriculture SPW (Service public de Wallonie; Belgique),
l’“Institut français du cheval et de l’équitation (Ifce)” of France and by “Les Fonds Spéciaux pour la Recherche
(FSR)” of Liege University (Belgium).
Acknowledgments: The authors thank all communicating veterinarians and owners of affected horses for
their collaboration.
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35. Westermann, C.M.; Dorland, L.; Votion, D.M.; De Sain-van der Velden, M.G.M.; Wijnberg, I.D.; Wanders, R.J.A.;
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animals
Article
Differential Defecation of Solid and Liquid Phases in
Horses—A Descriptive Survey
Katrin M. Lindroth 1, *, Astrid Johansen 2 , Viveca Båverud 3 , Johan Dicksved 1 ,
Jan Erik Lindberg 1 and Cecilia E. Müller 1
1 Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences,
P.O. Box 7024, 750 07 Uppsala, Sweden; [email protected] (J.D.); [email protected] (J.E.L.);
[email protected] (C.E.M.)
2 NIBIO, Norwegian Institute of Bioeconomy Research, P.O. Box 115, 1431 Ås, Norway; [email protected]
3 National Veterinary Institute, 751 89 Uppsala, Sweden; [email protected]
* Correspondence: [email protected]; Tel.: +46-1867-1781
Received: 30 October 2019; Accepted: 28 December 2019; Published: 1 January 2020
Simple Summary: Free faecal liquid is a condition in horses where faeces are voided in one solid
and one liquid phase. The presence of free faecal liquid may cause management problems in equine
husbandry and is potentially contributing to impaired equine welfare. Causes of free faecal liquid
are not known, but nutritional factors such as the feeding of specific forages have been suggested
to be of importance. Characterization of horses showing free faecal liquid and their feeding and
management was, therefore, performed via an internet-based survey in order to map the condition
further. Results showed that horses with free faecal liquid included a large variety of different breeds,
ages, disciplines, coat colours, housing systems and feeding strategies, meaning that almost any
type of horse could be affected. Horses that were reported to show free faecal liquid did so with
all types of feeding strategies, but changes from wrapped forage to hay, to pasture, or to another
batch of wrapped forage often resulted in diminished signs of free faecal liquid. Horses were also
reported to have a comparably high incidence of colic in relation to published data for other horse
populations. The results indicated that more detailed studies are required for a further understanding
of the underlying cause of free faecal liquid.
Abstract: Free faecal liquid (FFL) is a condition in horses where faeces are voided in one solid and
one liquid phase. The liquid phase contaminates the tail, hindlegs and area around the anus of the
horse, resulting in management problems and potentially contributing to impaired equine welfare.
The underlying causes are not known, but anecdotal suggestions include feeding wrapped forages or
other feed- or management-related factors. Individual horse factors may also be associated with the
presence of FFL. This study, therefore, aimed to characterize horses showing FFL particularly when
fed wrapped forages, and to map the management and feeding strategies of these horses. Data were
retrieved by a web-based survey, including 339 horses with FFL. A large variety of different breeds,
ages, disciplines, coat colours, housing systems and feeding strategies were represented among the
horses in the study, meaning that any type of horse could be affected. Respondents were asked to
indicate if their horse had diminished signs of FFL with different changes in forage feeding. Fifty-eight
percent (n = 197) of the horse owners reported diminished signs of FFL in their horses when changing
from wrapped forages to hay; 46 (n = 156) of the horse owners reported diminished signs of FFL
in their horses when changing from wrapped forages to pasture; 17% (n = 58) reported diminished
signs of FFL when changing from any type of forage batch to any other forage. This indicated that
feeding strategy may be of importance, but cannot solely explain the presence of FFL. The results also
showed that the horses in this study had a comparably high incidence of previous colic (23%, n = 78)
compared to published data from other horse populations. In conclusion, the results showed that
FFL may affect a large variety of horse types and that further studies should include detailed data on

Animals 2020, 10, 76
individual horse factors including gastrointestinal diseases as well as feeding strategies, in order to
increase the chance of finding causes of FFL.
Keywords: colic; equine; free faecal liquid; faecal water syndrome; feed changes; nutrition
1. Introduction
Free faecal liquid (FFL) is a condition in horses where faeces are voided in two physical phases;
one solid and one liquid phase. The solid phase can be typical equine faecal balls, or more watery and
similar to cowpat faeces. The liquid phase is a brown-coloured liquid that can be voided separately or
together with the solid phase. The condition has previously been referred to as free faecal water and/or
free faecal water syndrome (FWS), and cases have been described in Germany [1–3], Denmark [4] and
Italy [5], but the overall incidence of FFL is not known. Horse owners in Sweden and Norway have
anecdotally reported cases of FFL in horses, and have referred to the condition as “haylage intolerance”
due to an assumed association with feeding wrapped forages (including grass conserved as silage
and/or haylage with dry matter concentrations from 300–840 g per kg [6–8]). During the latest 25 years,
wrapped forages such as grass silage and haylage have partially or totally replaced hay in equine feed
rations in Nordic countries [9–11].
Horses affected with FFL may show discomfort when voiding faeces and/or faecal liquid, such
as nervous trampling with hindlegs and extensive tail swishing, but no symptoms of disease have
been described [1,5]. The faecal liquid may, however, cause lesions in the skin around the anus and
on the inside of the hindlegs, as well as dirty tail and hindlegs of affected horses. The causes of FFL
are unknown, but feeding wrapped forages instead of hay, feeding high amounts of alfalfa, being
over 20 years of age, having poor dentition and endoparasitic infections have been suggested [1]. In a
German study [1], associations between the presence of FFL and intrinsic horse factors such as being a
gelding, paint-coloured and low in the social hierarchy in a group of horses were found. Improvement
of the condition have been reported in association to changes in the diet in a one-horse case study [5]
and after faecal transplantation performed in a study including 10 horses with FFL and twelve horses
assessed to be clinically healthy [4]. However, no clear associations to feed- or management-related
factors [1,3,12] has been reported. Systematic collection of data on horse characteristics as well as on
feeding and management of horses affected with FFL is scarce in the scientific literature. The aim of the
present study was, therefore, to characterize horses showing FFL (when fed wrapped forages) and to
map the feeding and management strategies of these horses. Such information is required for further
studies of causes for the condition.

An online survey directed to owners and/or caretakers of horses showing FFL when fed wrapped
forages was performed. The inclusion criteria were that horses should be >2 years old, showing FFL
when fed wrapped forages and be located in Sweden or Norway (two countries in close proximity
and with similar conditions for horse feeding and management). The inclusion criteria were given
on the start page of the survey, and 3 questions in the survey were control questions ensuring
that the inclusion criteria were met. The survey was created using the tool Netigate (Netigate,
Stockholm) and was advertised through the website of the Department of Animal Nutrition and
Management, SLU (http://www.slu.se/sv/institutioner/husdjurens-utfodring-vard/), Norsk hestesenter
(http://www.nhest.no), Norwegian institute of Bioeconomy Research (http://www.nibio.no) and the
website Hästsverige (http://www.hastsverige.se), a Swedish platform communicating equine research
to the public. The full survey is available in Table S1 (Supplementary Materials).
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Animals 2020, 10, 76
2.1. Data Collection

The survey was open from March 2016 to March 2017 and was available in both Swedish and
Norwegian language. Respondents were instructed to give information about one horse per entry,
even if they owned more than one horse showing FFL. If so, it was possible for the same respondent to
answer the survey several times. The respondents were asked to answer questions about the horse, how
it was kept and managed, current and previous feeding and history of gastrointestinal disturbances
including FFL. The survey contained 50 questions in total, divided into horse characteristics (e.g., age,
sex, breed, colour, body condition score [13] and temper as judged by the respondent); training
(e.g., discipline, intensity); management (e.g., type of housing system, paddock use); current feeding
(type and amount of feeds), feeding and watering strategies (e.g., number of feedings, time between
feedings, how feed was offered in stable and paddock, type of water source and access to salt); presence
of FFL (e.g., presence and symptoms of FFL, changes in faecal appearance due to feed changes, number
of affected horses kept in the same housing system); and previous history of gastrointestinal tract
(GIT) diseases.
2.2. Data Treatment

The data on horse breeds contained over 30 different breeds but with very few individuals
in several breeds. Therefore, this variable was transformed to breed type. The reported breeds
were divided into 4 breed groups; warmblood type horses (Appaloosa (n = 3), crossbred horses of
warmblood type (n = 64), European warmblood riding horses (n = 82), Lusitano (n = 1), Standardbreds
(n = 15), Paint horse (n = 2), Pura Raza Espaniola (n = 11) and Quarter horse (n = 2)); cold-blood
type horses (Ardennais (n = 4), Clydesdale (n = 1), Cold-blooded trotter (n = 13), crossbred horses of
cold-blood type (n = 19), Dølehorse (n = 2), Friesian horse (n = 3), Haflinger (n = 3), North-Swedish
draught horse (n = 8), Norwegian Fjord Horse (n = 12), Shire (n = 1) and Tinker (n = 1)); Hot-blooded
horses (Angloarabian (n = 1), Arabian (n = 8), and Thoroughbreds (n = 2)); and native pony breeds
(Connemara (n = 10), crossbred ponies (n = 25), Gotland pony (n = 6), Icelandic horse (n = 13), New
Forest (n = 10), Shetland pony (n = 6), Welsh cob (n = 3) and Welsh pony (n = 8)).
2.3. Calculations and Statistical Analysis

Statistical analysis was performed using SAS (Statistical Analysis System Institute Inc., Cary, NC,
USA) version 9.4 for Windows. For continuous variables, minimum, maximum, quartiles (Q1, Q2
(median) and Q3), mean and standard deviation was calculated. The reported bodyweight (BW) of the
horse and reported feeding levels were used to calculate the daily intake of feed (g or kg) per 100 kg BW
and day. Descriptive analysis was performed using PROC FREQ. During data treatment, it was found
that 23% (n = 78) of the horses had a history of colic. The horses were, therefore, further divided into
one colic and one non-colic group for comparisons of type of clinical signs during FFL episodes. Each
clinical sign was compared separately between the groups using a Chi2 -test (with expected model).
Level of significance was set at p < 0.05.
3. Results
In total, 780 responses to the survey were obtained. Out of these, 12 responses represented horses
younger than 2 years of age, 234 responses were for horses that did not show FFL but other types of
problems when fed wrapped forages, and 195 responses were incomplete. These 441 responses were
excluded from the dataset, leaving 339 full responses for further evaluation.
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Animals 2020, 10, 76
3.1. Horses and Signs of Free Faecal Liquid

The age of the horses in the study ranged from 2.5 to 28 years (average 11 ± 5.9 years). The majority
of the horses were geldings (57%, n = 193) (Table 1) and of warmblood breed type (53%, n = 180)
(Figure 1). Thirty-seven percent (n = 123) of the horses had bay coat colour followed by chestnut
(19%, n = 64), grey (14%, n = 47) and black (8%, n = 27). Body condition scores (BCS) ranged from 1 to
4 (on a scale of 0 to 5 [13]), with a normal distribution around BCS 3 as median (55%, n = 186) (Table 1).
The most frequently reported disciplines horses were used for were leisure riding (82%, n = 278),
dressage (37%, n = 125) and show jumping (34%, n = 115) (Figure 2). A majority of the horses
(63%, n = 215) were reported to perform a low-intensity exercise (Table 1). Extended information on
horse characteristics is reported in Appendix A (Table A1). Twenty-nine percent (n = 98) of the horses
were reported to show distinct irritation manifested by extensive tail swishing and nervous trampling
of hindlegs while voiding faecal liquid and/or faeces, whereas 35% (n = 118) did not show any signs
other than FFL (Figure 3). A bloated abdomen was reported in 29% (n = 98) of the horses during
episodes of FFL (Figure 3). Fifty-two percent (n = 170) of the respondents reported that only their horse
in the stable showed FFL, while 48% (n = 159) of the respondents stated that there were more horses in
the stable that showed FFL.
Table 1. Characteristics of horses showing free faecal liquid (n = 339).
Item No. of Horses % of Horses

Country (stabled in)
Sweden 191 56
Norway 148 44
Gender
Mare 134 40
Gelding 194 57
Stallion 11 3
Coat colour
Bay 123 37
Chestnut 64 19
Grey 47 14
Black 27 8
Paint 24 7
Palomino/Isabelline 21 6
Cremello 19 6
Other (Leopard pattern/buckskin) 14 4
Body condition score 1
<3 75 22
3 188 55
>3 76 22
Training intensity
Low 215 63
Medium 63 19
High 23 7
Breaking in 23 7
No training 2 15 4
1 According to the scale of Carroll and Huntington, 1988. 2 No training includes horses kept as pets or for company.
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Animals 2020, 10, 76
60
50
Percentage of horses (%)
40
30
20
10
0
Warmblood horses Cold-blood horses Hot-blooded horses Native pony breeds
(and crossbreds) (and crossbreds)
Breed types
Figure 1. Distribution of breed types for horses showing free faecal liquid (n = 339).
90
80
70
60
50
40
30
20
10
0
Disciplines performed
Figure 2. Distribution of disciplines performed by horses with free faecal liquid (n = 339), as reported
by respondents. Multiple-choice question resulting in that the sum could exceed 100 percent.
100
Animals 2020, 10, 76
40
35
30
25
20
15
10
5
0
No clinical Colic Skin Swollen legs Bloated Irritation
signs ¹ problems abdomen while
voiding
faeces ²
Figure 3. Percentage of horses showing different clinical signs associated with episodes of free faecal
liquid, as reported by respondents (n = 339). Multiple-choice question resulting in a sum that could
exceed 100%. 1 No clinical signs mean no signs other than free faecal liquid. 2 Including extensive tail
swishing and/or trampling with hindlegs while voiding faeces and/or faecal liquid.
3.2. Management
The majority of the horses (79%, n = 271) were kept in individual boxes at night and outside
in paddocks during the daytime, while 19% (n = 61) were kept in loose-housing systems (Table 2).
The bedding materials used in stables and loose housing systems were a combination of straw and
shavings (37%, n = 125), straw only (20%, n = 68) and shavings only (17%, n = 58) (Table 2). Horses that
spent their daytime in paddocks were generally kept outside for 8–12 h per day (48%, n = 163), while
29% (n = 98) were kept outside for more than 12 h per day and 21% (n = 71) were kept outside for less
than 4 h per day (Table 2). The type of paddocks were soil paddocks (39%, n = 132), grass paddocks
(old grass during winter) (28%, n = 94) or sand/gravel paddocks (23%, n = 78) (Table 2). Forty-eight
percent (n = 163) of the horses were kept on pasture for 9–12 weeks, while 21% (n = 71) were on
pasture for ≤8 weeks and 8% (n = 27) were kept on pasture for >12 weeks (Table 2). The majority
(55%, n = 186) of the horses were dewormed if faecal egg counts showed sufficiently high numbers
to indicate deworming according to national guidelines (www.sva.se) (Table 2). Other deworming
procedures included regular deworming more than one time per year (36%, n = 122), dewormed if
considered necessary (7%, n = 25) or not dewormed (1%, n = 4) (Table 2). Horses were reported to
have access to water by tubs, buckets or automatic waterers when kept in the stable, paddock and
at pasture. At pasture, horses were also reported to have access to water by natural water sources
(Table A1). Extended information on management factors is presented in Table A1 (Appendix A).
3.3. Feeding
The majority (74%, n = 250) of horses were fed forage in meals, while 26% (n = 89) were fed
forages ad libitum. Grass haylage (defined as in Table S1, Supplementary Materials) was offered to
95% (n = 322) of the horses, whereas 5% (n = 17) of the horses were fed grass silage (Figure 4). Hay
was fed to 50% (n = 170) of the horses (Figure 4). In general, horses were fed roughage-dominated
feed rations with on average 90% roughage, and 7% concentrates in the daily feed ration (Table 3).
Daily amounts of different feedstuffs are reported in Table 3. Most of the horses (67%, n = 227) fed
forage in meals were fed forage 3 to 4 times daily, and the time between two forage feedings seldom
exceeded 8 h (Table A1). Horses that were fed in their paddocks were served forage in tubs or haynets
(60%, n = 204), or on the ground (45%, n = 153) (Table A1). Eight percent (n = 27) were not fed forage in
their paddocks (Table A1). The majority (66%, n = 224) purchased their forage from a producer outside
101
Animals 2020, 10, 76
the farm, while the remaining proportion used forage produced on the farm (Table A1). About half
(48%, n = 163) of the respondents stated that they did not know the forage nutritive contents (Table A1).
Table 2. Description of the management of horses showing free faecal liquid (n = 339, if not otherwise
mentioned. Deviances in N were due to missing responses for that particular question).
Item No. of Horses % of Horses

Housing system (n = 337)
Individual box 271 79
Loose housing system 64 19
Group housing 2 1
Bedding (n = 336)
Straw 67 20
Shavings 57 17
Combination of straw and shavings 125 37
Sawdust 40 12
Wood pellets 26 8
Straw pellets 11 3
Other (paper, mix of sawdust and peat, rubber mat, raw sawdust) 10 3
Time spent per day in paddock during winter (n = 245)
<4 h 5 2
4–7 h 67 20
8–12 h 163 48
>12 h 10 30
Paddock ground (n = 332)
Grass (old grass during winter) 94 28
Sand/Gravel 79 24
Soil 133 40
Other 26 8
Annual time spent on pasture
<4 weeks 5 2
4–8 weeks 67 20
9–12 weeks 163 48
>12 weeks 100 30
No pasture 4 1
Anthelmintic routines
Regularly dewormed ≥ 1 times per year 122 36
Dewormed due to high 1 egg counts ≥ 1 times per year 154 45
Dewormed due to high 1 egg counts < 1 times per year 34 10
Dewormed if considered necessary 25 7
Not dewormed 4 1
1 According to national guidelines (www.sva.se).
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Animals 2020, 10, 76
100
90
80
70
60
50
40
30
20
10
0
Grass hay Grass haylage ¹ Grass silage ² Straw Lucerne ³
Roughage type
Figure 4. Different types of roughage fed to horses showing free faecal liquid (n = 339). Multiple
roughages could be assigned in the survey, resulting in a sum of percentages exceeding 100. 1 Wrapped
forage with ≥50% DM. 2 Wrapped forage with <50% DM. 3 Includes both pelleted lucerne and lucerne
chaff in a dried format.
Table 3. Daily amounts of different feedstuffs (kg per 100 kg bodyweight (BW) per day) and proportion
(%) of roughage and concentrate in the diet offered to horses showing free faecal liquid (n = 339).
Item No. of Horses Min Q1 Q2 Q3 Max Mean SD

Roughage and concentrate feeding, Kg/100 kg
BW/d 1
Grass hay 165 0.1 0.2 0.2 0.3 0.5 0.2 0.06
Grass haylage 251 0.2 1.7 2.0 2.3 6.0 2.0 0.67
Grass silage 4 1.2 1.2 2.9 3.8 4.7 2.9 1.73
Straw 14 0.1 0.2 0.3 0.6 2.3 0.4 0.45
Lucerne 2 10 0.01 0.1 0.1 0.2 0.7 0.1 0.13
Total amount of roughage 217 0.3 1.5 2.0 3.1 4.8 1.8 2.17
Total amount of concentrate 190 0.01 0.1 0.2 0.3 1.0 0.2 0.18
Roughage proportion of total feed ration (%) 3 249 20 90 100 100 100 90 0.14
Concentrate proportion of total feed ration (%) 3,4 107 0 1 5 10 80 7 0.14
Mineral supplementation, g/100 kg BW 218 0.1 6.0 10.8 17.8 83.3 13.5 11.43
1 Horses reported to have ad libitum access to roughage, forage or having straw as bedding material were not
included. 2 Horses reported to have access to roughage ad libitum without concentrates in the diet were included. 3
Horses reported to have access to roughage ad libitum without concentrates in the diet were included. 4 Horses
reported not to be fed concentrate were excluded. Min = Minimum value. Q1–Q3: First-, second- (=median) and
third quartile. Max = Maximum value. SD = Standard deviation.
More than half of the horses (56%, n = 190) were fed concentrates, and the most common type was
commercial concentrates (n = 118) followed by vegetable oil (n = 104) and molassed sugar beet pulp
(n = 22) and (Table A1). Supplemental feeds were used for 84% (n = 285) of the horses in the study and
mostly comprised mineral and vitamin feed (Table A1). For horses reported to be fed concentrates,
217 horses were fed concentrates 1–2 times per day and the remaining proportion was fed concentrates
more often (Table A1). The presence of FFL was reported to diminish when changing from wrapped
forages to hay (58%, n = 197), from wrapped forage to pasture (46%, n = 156) and from one batch of
wrapped forage to another batch (17%, n = 58) (Table 4). However, not all horses showed any change
in the presence of FFL with feed changes (7%, n = 24) and not all horses had been subjected to all feed
changes (2%, n = 7) (Table 4).
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Table 4. Changes in the presence of free faecal liquid in the horses in the study (n = 339) with diet
changes as reported by respondents. “Less loose” refer to the absence and/or reduced amount of liquid
phase in faeces compared to before the feed change, as reported by respondents. Not all respondents
had tried all response alternatives.
No. of % of
Item
Horses Horses
Faecal appearance less loose when changing from wrapped forage to hay 198 58
Faecal appearance less loose when changing from wrapped forage to pasture 157 46
Faecal appearance less loose when changing to another batch of wrapped forage 56 17
No change in faecal appearance with any change in feeding 24 7
Faecal appearance more loose in association to changing feeds 20 6
Faecal appearance less loose when changing from primary to regrowth harvest 1 16 5
Faecal appearance less loose when using feed additives 2 8 4
Have not tried any change in feeding 5 2
1Wrapped forages. 2 Feed additives reported included yeast, linseed, psyllium seed, thiamine and various types of
commercial pro- and prebiotics.
3.4. Gastrointestinal Health

Eighty-seven percent (n = 295) of the horses in the study had not been treated for any gastrointestinal
disease within the 3 previous months prior to responding to the survey. Twenty-two percent (n = 76) of
the horses in the study were reported to have been examined for stomach ulcers, however, only 14 were
reported to have been examined with gastroscopy. Of these, 9 were diagnosed with gastric ulcers. Nearly
one-quarter of the horses in the study (23%, n = 78) were reported to have had a previous history of colic.
Therefore, horses were divided into two groups, one with a previous history of colic and one with no
previous history of colic, and compared within each type of symptom they were showing during episodes
of FFL (Figure 5). For horses reported to show no clinical signs during episodes of FFL (n = 119), there was
a higher proportion of horses with no previous history of colic (74%, n = 88) compared to horses with a
previous history of colic (26%, n = 31) (p < 0.001). For horses reported to show signs of colic during episodes
of FFL (n = 71) there was a higher proportion of horses with a previous history of colic (87%, n = 62)
compared to horses with no previous history of colic (13%, n = 9) (p < 0.001).
100
90
80
70
Number of horses
60
50
40
30
20
10
0
(n=119) (n=71) (n=14) (n=10) (n=98) (n=98)
No clinical Colic Skin Swollen legs Bloated Irritation

signs ¹ problems abdomen while
voiding
faeces ²
Previous history of colic No previous history of colic
Figure 5. The horse owner reported clinical signs during episodes of free faecal liquid in horses with
(n = 77) and without (n = 256) a previous history of colic. Multiple signs could be selected in the survey,
resulting in the numbers of horses for all symptoms exceeding the total number of horses in the study.
1 No clinical signs mean no signs other than FFL. 2 Including extensive tail swishing and/or trampling
with hindlegs.
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Animals 2020, 10, 76
For horses reported to show a bloated abdomen (n = 98), there was a tendency (p = 0.08) for a
higher proportion of horses with a previous history of colic (67%, n = 66) compared to horses with no
previous history of colic (33%, n = 32).
3.5. Stereotypic Behaviour

Nineteen percent (n = 64) of the horses in the study were reported to show stereotypic behaviour.
The reported stereotypic behaviours included crib biting (15%, n = 8), wind sucking (4%, n = 2),
weaving (7%, n = 4), box walking (11%, n = 6), wood chewing (60%, n = 33) and tongue rolling
(7%, n = 4).
4. Discussion
4.1. Horses
In the present study, the typical horse showing FFL was reported to be of warmblood type, have
bay coat colour, be a gelding, on average 12 years old and be used for leisure riding, dressage or
show jumping. A similar distribution of horse characteristics has been described within other horse
populations in Sweden and Norway [14,15], which indicates that the population in the present study is
a reflection of the normal horse population. As a large variety of breeds, ages and disciplines were
represented in the population in the present study, it is evident that FFL could appear in almost any
type of horse. The proportion of geldings was larger compared to the proportions of mares and
stallions in the current study, which is in agreement with the findings in a previous study on FFL [1]
where a larger proportion of geldings was found in the case group compared to controls. However,
it may be more common to keep geldings as leisure horses [16,17], compared to mares and stallions,
and this could explain the higher proportion of geldings in both studies. The reported BCS was in
relation to what was expected. The majority of horses were reported to have a BCS of 3 (normal BCS),
which is in agreement with previous descriptions of FFL [1,5] where horses were not reported to show
weight loss or loss of BCS. It has also been reported that horse owners are commonly underestimating
the body condition score of their horses compared to a trained professional scorer [18]. This may
indicate that the horses in the present study were not underweight or in lower than normal BCS. In a
previous study, horses with the coat colour paint were over-represented (29%, n = 12) in a group of
horses showing FFL compared to horses in two control groups, which comprised 10% (n = 4) and 8%
(n = 3) paint coloured horses, respectively) [1]. In the current study, the proportion of paint horses
was lower than the proportions of the bay, chestnut, grey and black horses. Therefore, an association
between the coat colour paint and the presence of FFL cannot be confirmed from the results in the
current study. It has been suggested that grey horses may be more prone to show FFL due to the
higher risk of melanoma [19], which may cause defecation difficulties, but no such association has
been identified in the literature or in this study. Social stress has been suggested to play a role for the
presence of FFL, as the majority of FFL-affected horses did not defend their feed against other horses
and were judged as low in the hierarchy in a previous study [1]. However, in the same study [1], high
ranked horses also showed FFL, indicating that stress from being low in the hierarchy is not a sole
explanation for the presence of FFL.
4.2. Management
The majority of the horses in this study were kept outside in paddocks for 4–8 h in soil or
sand/gravel paddocks and almost half the horses were fed forage on the ground. Feeding forage on the
ground could lead to increased ingestion of soil and sand particles causing irritation of the intestinal
mucosa, which has been associated with gastrointestinal conditions such as diarrhoea, including
voiding of loose and watery faeces [20], and colic [21]. Whether or not this management factor plays a
role in the presence of FFL remains to be further elucidated.
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Animals 2020, 10, 76
4.3. Feeding
Despite the fact that about half of the horses were reported to have less loose faeces when feeding
was changed from wrapped forages to hay (58%) or to pasture (46%), this was not the case for all
horses in the study. In addition, about one-quarter of the horses improved by changing from one batch
to another batch (including from primary to regrowth harvest) of wrapped forage. This indicates
that the occurrence of FFL cannot be generally attributed to feeding wrapped forages. This finding is
supported by results from a previous study in which horses displaying FFL predominantly were fed
hay [1]. In addition, FFL has not been reported in controlled feeding studies with healthy horses fed
silage, haylage and hay from the same grass sward and harvest [22,23]. As there may be individual
variation in the gut microbiota of horses [24–26], it is possible that individuals respond differently to
the same feed. Further study within this area is highly interesting and may provide more insight into
factors contributing to the presence of both FFL and colic.
Although the forage conservation method may influence both chemical and microbial composition
in forage [22], forages differ in a number of other factors as well. One important factor is plant
maturity at harvest, which greatly influences overall digestibility in the equine GIT and the nutritive
value of the forage [27]. Valle et al. [5] reported a horse with FFL to maintain a reduced or absent
production of FFL with gradual changes in the nutritional plan to meet the theoretical nutritional
requirements of the horse. Changes included reducing energy content in the feed ration by excluding
concentrate feeds and decreasing the amount of forage and changing batch of forage in combination
with increased exercise. However, simultaneously with the nutritional changes, the horse was treated
with sulfasalazine, making it difficult to evaluate the effect of the changes in the feeding. Only half of
the horse owners in the current study reported that they knew the nutritive content in their forage
(through forage analytical reports), indicating that half of the horses may have been under- or overfed
in relation to their nutritional requirements. Whether this is a factor of importance for the presence of
FFL is currently not known.
The composition of the total feed ration and the ratio between forage and concentrate may be of
importance for the physical appearance of faeces, as two-phase separation (liquid and solid phase)
of both digesta and faeces has been reported in horses fed hay ad libitum with inclusion of grains
(4.55 kg every 12 h) but not when fed only the same hay ad libitum [28]. In horses fed hay only, a clear
separation between solid and liquid phases was present in the contents of the right dorsal colon (RDC),
but faecal balls were well-formed and with no separation in liquid and solid phases. No or minimal
gas bubbles were present in RDC content when horses were fed hay [28]. When horses were fed hay
with the inclusion of grains, RDC contents were more homogenous and foamy, with less separation of
phases, and the liquid phase was more viscous than in horses fed hay only. Faeces of horses fed hay and
grains were, however, less formed and had a clear separation, where the liquid phase had noticeable
gas bubbles and was more viscous compared to faeces of horses fed hay only [28]. One explanation
for this result could be differences in the hydrophilic properties of ingesta components, which may
differ when horses were fed hay only or hay with grain inclusion. The hydrophilic properties of the
ingesta have been suggested as a cause of osmotic diarrhoea [29,30]. In the current study, horses were
fed much smaller proportions of concentrates compared to what was described by Lopes et al. [28].
However, smaller amounts of concentrates fed daily (2.5–5 kg) have been reported to increase the risk
of colic [31–33] and may affect the ingesta and its transit as well. As horses displaying FFL seldom
show clinical signs of disease, FFL may be a type of osmotic diarrhoea. Further insights in causes of
FFL may be provided by investigations of forage and concentrate proportions in the total feed ration of
horses with FFL.
4.4. Gastrointestinal Health

In previous studies, no symptoms of disease, such as fever or weight loss or loss of body condition
has been described in horses with FFL [1,5]. Nine horses were reported to have been diagnosed
with gastric ulcers, but overall very few horses (n = 14) had been examined for gastric ulcers with
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Animals 2020, 10, 76
gastroscopy. From the results of the current study, it cannot be ruled out that gastric ulcers may be
associated with the presence of FFL, even though the incidence of gastric ulcers was lower than what
has been described in other studies of gastric ulcers in leisure horses (17% to 58%, [34–36]).
In the present study, almost one-quarter of the horses were reported to have had a previous
history of colic. A colic incidence between 3.5% and 10.6% has been reported for general horse
populations [10,32,37,38] and of 4.8% within a German population of horses showing FFL [3].
This indicates that the incidence of colic was higher for Swedish and Norwegian horses showing FFL.
In addition, in the present study, 55% of the horses did not show any clinical signs other than faecal
liquid during FFL episodes, whereas the remaining proportion of horses were reported to have one or
a combination of several clinical signs including e.g., colic symptoms. The latter proportion also had
a higher number of horses with a previous history of colic. This indicates that causes of FFL could
differ among different horses, or that FFL is a generic symptom from several different conditions of a
similar nature. The number of clinical signs during an FFL episode could also depend on the severity
of the condition, which could not be assessed in this study. Further studies of FFL should preferably
include detailed descriptions of duration, intensity and severity of FFL episodes as well as previous
disturbances in the GIT of FFL-affected horses. It is possible that the hindgut microbiota of FFL-horses
is responsible for the clinical signs. Transplantation of faecal microflora in affected horses has been
reported to decrease the severity of FFL in a controlled study [4]. However, in the same study [4],
horses were also treated with omeprazole and psyllium seeds in addition to faecal transplant, making
it difficult to evaluate the effects.
4.5. Stereotypic Behaviour

The reported incidence of stereotypic behaviour among FFL horses in the study was 19% when
wood chewing was included. Tree-wood chewing may not always be a stereotypy but could be related
to low-fibre diets [39], and when excluded, the incidence of stereotypies was approximately 8%, which
is comparable to the previously reported incidence of 7.1–12.3% [40–44] in other horse populations.
Factors reported to be associated with an increased risk of stereotypic behaviour in previous studies
include low levels of social interactions with other horses [44–46], low forage availability [45,46] and
low number of horses kept in the same and/or adjacent paddocks [45]. The majority of horses in the
current study were kept in individual boxes when stabled and fed forage in predetermined portions
during the day, which may have contributed to the incidence of stereotypic behaviour among horses in
this study.
4.6. Survey Response and Limitations of the Study

The advantages of performing online surveys are the ease and low cost of data collection, the
automation in data input and handling, which reduce errors, and the flexibility in survey design to
make it easier for participants to respond to the questionnaire. The disadvantage includes the absence of
an interviewer, and that data are reported by the horse owner, which could result in misinterpretations
among respondents for some of the questions. Some of the variables have previously been shown to
be difficult to estimate correctly for horse-owners, such as BCS [18]. It should be recognized that all
conclusions drawn from this study were based on the perceptions of the respondents, which may vary
in their knowledge of equine feeding and management.
In order to control that the respondents of the survey were within the intended group, control
questions based on the inclusion criteria, such as the age of the horse, and if the horse had problems
with FFL when fed wrapped forages, were asked. This resulted in the elimination of 441 responses to
the present study, indicating that such controls could be of high importance to enhance the quality of
data input in online surveys.
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5. Conclusions
There was a large variety of horse characteristics, including breed type, age and coat colour,
in horses with FFL. Many, but not all horses in this study were reported to show less separation
of solid and liquid phases in their faeces when changing from wrapped forages to hay, pasture or
another batch of wrapped forage. Horses with FFL were also reported to have a higher incidence of a
previous history of colic compared to reports from other horse populations. Further research on FFL in
horses is of interest and should include details on feeding (such as forage nutritive values, feed ration
composition), and gastrointestinal tract health and function (such as the presence of stomach ulcers,
colic, gastrointestinal tract response to different feedstuffs), as well as detailed descriptions of severity
and duration of FFL episodes.

Table S1, The survey distributed to Swedish and Norwegian horse owners having horses showing FFL when fed
wrapped forages. Modified (Translated from Swedish and Norwegian language) for the purpose of publication.
Bulleted points indicate the response of the questions and different response alternatives are comma-separated.
Space was provided for alternative answers where necessary.
Author Contributions: The authors have contributed to the following sections of this work: Conceptualization,
K.M.L. and C.E.M.; data curation, K.M.L.; formal analysis, K.M.L., A.J., V.B., J.D., J.E.L., and C.E.M.; funding
acquisition, A.J. and C.E.M.; investigation, K.M.L. and C.E.M.; methodology, K.M.L. and C.E.M.; project
administration, K.M.L., A.J., and C.E.M.; writing—original draft preparation, K.M.L.; writing—review and
editing, K.M.L., A.J., V.B., J.D., J.E.L., and C.E.M. All authors have read and agreed to the published version of
the manuscript.
Funding: The Swedish-Norwegian Foundation funded this research for equine research, grant number H-15-47-063,
2015 and the Swedish University of Agricultural Science.
Acknowledgments: The authors would like to thank all horse-owners who contributed to the data. In addition,
thanks to Professor Ulf Olsson, Biostokastikum, Department of Energy and Technology, Swedish University of
Agricultural Sciences, for very valuable and helpful statistical support and advice.
study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript, or in the decision
to publish the results.
Abbreviations
The following abbreviations are used in this manuscript:
BCS Body Condition Score

DM Dry matter
FFL Free faecal liquid
FMT Faecal microbiota transplantation
GIT Gastrointestinal tract
RDC Right dorsal colon
Appendix A
Horse owner responses to the survey for variables not presented in tables or figures in the main text. The
number (n) and proportion (%) of horses presented are presented in Table A1.
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Table A1. Extended information on management factors and feeding strategies for horses with free
faecal liquid (n = 339), as reported by respondents.
Variables Total Number of Horses % of All Horses

Horses showing FFL when fed wrapped forages 339 100
Region of stable
Southern 126 37
Central 81 24
North 39 12
Western 1 23 7
Eastern 1 70 21
Horse imported
No 253 75
Do not know 6 2
Yes 80 24
Ability of horse to keep desired BCS
Easy keeper 97 29
Normal 187 55
Hard keeper 55 16
Type of water source in stable/loose housing system
Frostless waterer 49 14
Frostless tub 43 13
Waterer 31 9
Tub 31 9
Bucket 78 23
Natural water source 4 1
Combination of bucket and waterer 103 30
Type of water source in paddock during winter
Frostless tub 80 24
Waterer 4 1
Tub 124 37
Bucket 37 11
Other (Combination of tub and bucket, bucket and waterer) 55 16
Type of pasture
Pasture on arable land 106 31
Natural pasture 135 40
Forest 6 2
No pasture 30 9
Other (Combination of different pasture types) 61 18
Type of water source on pasture
Frostless tub 12 4
Waterer 10 3
Tub 165 49
Bucket 8 2
Other (Combination of bucket and tub) 66 19
Access to saltlick while on pasture
Yes 220 65
No 119 35
Saltlick in stable/loose housing system
Yes 296 87
No 43 13
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Table A1. Cont.

Time from last deworming
Not dewormed 11 3
0–3 months ago 75 22
>1 year ago 59 17
Origin of the forage
Bought 226 67
Produced on farm, but not by the owner 42 12
Produced on farm by the owner 69 20
Other 1 0
Forages analysis
Yes 144 42
No 163 48
Do not know 37 9
Number of feedings of forage per day
1 time 1 0
2 times 14 4
3 times 122 36
4 times 108 32
>4 times 33 10
Free access 89 26
Storage of forage
Indoors 223 66
Outdoors 114 34
Outdoors, covered 65 19
Outdoors, uncovered 49 14
Hay indoors, wrapped forages outdoors uncovered 1 0
Hay indoors, wrapped forages outdoors covered 1 0
Maximum time between two feedings of roughage
0–2 h 2 1
2–4 h 35 10
4–8 h 115 34
8–12 h 101 30
>12 h 8 2
Don’t know 78 24
Feeding strategy for roughage in paddock 2
Forage not fed in the paddock 27 8
On the ground 113 33
In the feeding rack 79 23
In a haynet 19 6
In a tub or similar 62 18
Other (combination of ground and feeding rack/haynet) 39 12
Type of concentrate fed 2
Grains 16 5
Molassed sugar beet pulp 22 6
Linseed/Linseed cake 4 1
Soybean meal 14 4
Potato protein 33 10
Wheat bran 3 1
Vegetable oil 104 31
No concentrate fed 149 44
Commercial concentrate 118 63
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Animals 2020, 10, 76
Table A1. Cont.

Number of concentrate feedings per day
0 times 149 44
1 time 116 34
2 times 107 32
3 times 49 14
4 times 7 2
>4 times 1 0
Type of supplemental feeds fed 2
Mineral feeds 237 70
Multivitamins 58 17
B-vitamins (and Biotin) 10 3
Selenium and Vitamin E 9 3
Garlic 13 4
Herbs 8 2
Other (Yeast, magnesium) 93 27
Not fed supplemental feeds 51 16
Storage of concentrates
In covered/closed containers indoors 255 66
In uncovered/open containers indoors 11 3
In paper bags/original package indoors 18 5
No concentrate 51 15
Other 4 1
Previous treatment of other gastro-intestinal diseases
No 296 87
Don’t know 11 3
Yes 32 9
1 Only for Norwegian respondents. 2 Multiple choice question.
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113
animals
Article
Eye Blink Rates and Eyelid Twitches as a
Non-Invasive Measure of Stress in the
Domestic Horse
Katrina Merkies 1,2, *, Chloe Ready 1 , Leanne Farkas 1 and Abigail Hodder 1
1 Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada
2 Campbell Centre for the Study of Animal Welfare, University of Guelph, Guelph, ON N1G 2W1, Canada
Received: 12 July 2019; Accepted: 13 August 2019; Published: 15 August 2019
Simple Summary: Eye blink rate has been used as an indicator of stress in humans and, due to its
non-invasive nature, could be useful to measure stress in horses. Horses exhibit both full and half
blinks as well as eyelid twitches. We exposed 33 horses to stressful situations such as separation from
herdmates, denied access to feed and sudden introduction of a novel object, and determined that
full and half eye blinks decrease in these situations. Feed restriction was the most stressful for the
horse as indicated by increased heart rate, restless behaviour and high head position. The decrease
in eye blink rate during feed restriction was paralleled with an increase in eyelid twitches. There
was no increase in eyelid twitches or heart rate with the other treatments indicating that the horses
did not find these overly stressful, but they did focus their attention more during these situations.
Observation of eye blinks and eyelid twitches can provide important information on the stress level
of horses with a decrease in eye blinks and an increase in eyelid twitches in stressful environments.
Abstract: Physiological changes provide indices of stress responses, however, behavioural measures
may be easier to determine. Spontaneous eye blink rate has potential as a non-invasive indicator of
stress. Eyelid movements, along with heart rate (HR) and behaviour, from 33 horses were evaluated
over four treatments: (1) control—horse in its normal paddock environment; (2) feed restriction—feed
was withheld at regular feeding time; (3) separation—horse was removed from visual contact with
their paddock mates; and (4) startle test—a ball was suddenly thrown on the ground in front of the
horse. HR data was collected every five s throughout each three min test. Eyelid movements and
behaviours were retrospectively determined from video recordings. A generalized linear mixed
model (GLIMMIX) procedure with Sidak’s multiple comparisons of least squares means demonstrated
that both full blinks (16 ± 12b vs. 15 ± 15b vs. 13 ± 11b vs. 26 ± 20a full blinks/3 min ± SEM; a,b
differ p < 0.006) and half blinks (34 ± 15ab vs. 27 ± 14bc vs. 25 ± 13c vs. 42 ± 22a half blinks/3
min ± SEM; a,b,c differ p < 0.0001) decreased during feed restriction, separation and the startle test
compared to the control, respectively. Eyelid twitches occurred more frequently in feed restriction
(p < 0.0001) along with an increased HR (p < 0.0001). This study demonstrates that spontaneous blink
rate decreases while eyelid twitches increase when the horse experiences a stressful situation.
Keywords: spontaneous blink rate; eyelid twitches; stress; horse; behaviour; welfare
1. Introduction
Stress is defined as the response of an organism to environmental stimuli that threatens its internal
equilibrium [1]. As a prey species, the domestic horse (Equus caballus) has developed adaptive fear and
flight responses when faced with external stressors [2]. However, modern husbandry practices routinely
subject horses to aversive stimuli such as transportation, social isolation and medical intervention.

Animals 2019, 9, 562
Identifying indicators of stress in the horse is fundamental for the welfare of the animal itself and the
safety of the handler [3].
Various physiological measures can be used to assess stress responses in animals including heart
rate and heart rate variability [4], blood or salivary cortisol [5]. However, these measures have their
limitations, including the increase in stress due to the invasive nature of drawing blood, for example [6].
As a result, researchers have explored behavioural indicators to augment physiological data. For
example, horses exposed to various stressors demonstrate higher head carriage [7], focused orientation
of the ears [8], increased vocalizations [9] and increased mouth movements [10]. Assessing stress
responses in animals appears more accurate when using a combination of both behavioural and
physiological indicators [11,12]. A novel scale developed to identify stress-related behaviours subjected
32 horses to known stressful husbandry practices including the sound of electric coat clippers, social
isolation and grooming procedures [13]. Moderate to high levels of stress showed an increase in oral
behaviours, flared nostrils and flattened or pinned ears which correlated with an increase in heart rate
(HR) and salivary cortisol [13].
However, evidence of behaviours associated with stress in horses is conflicting. Horses subjected to
two stressful handling tasks—walking across a tarpaulin and walking through streamers attached to an
overhead pole—displayed an increase in heart rate variability and eye temperature [14]. The time taken
or willingness to complete each task was not associated with physiological indicators, showing that the
horses did experience stress even when not overtly displaying stress behaviours [14]. Further, horses
undergoing a hair clipping procedure, a known aversive management practice, showed compliant
behaviour while displaying an increase in HR, salivary cortisol and eye temperature [15]. These studies
suggest that a horse’s level of compliance and/or ability to tolerate stressors is not indicative of their
level of arousal, and influences such as training may overshadow emotional responses [15].
Understanding the response of an animal to external stressors through valid behavioural indicators
can be challenging and subjective, however behaviour is an easily observable and non-invasive
measurement [16]. Identifying valid indicators of stress is essential to understanding the animal and
ultimately improving welfare.
Eye-blink rate has been used as a non-invasive measure of arousal to predict stress levels in
humans [17]. Blinking is defined as a quick movement of the eyelid that opens and closes the palpebral
fissure and is composed of three different blinks: spontaneous, reflex and voluntary [18]. The levator
palpebrae superioris muscle of the upper eyelid is primarily responsible for opening the eyelid,
whereas the orbicularis oculi muscle encircling the palpebral fissure works to close the eyelid [19].
Upon close observation, different eyelid movements are noticeable, ranging from full blinks (complete
closure of both eyelids with concomitant suppression of vision) to partial blinks (incomplete closure of
the eyelids) and eyelid twitches (movement of the upper eyelid through innervation of the levator
palpebrae superioris muscles with no movement of the orbicularis oculi muscles) [20]. Partial blinks
have been observed in humans focused on computer terminal displays [21] and have been used as a
diagnostic for dry eye disease [22]. Partials blinks have also been documented in both dogs [23] and
cats [24]. Due to the large size and lateral placement of a horse’s eyes, identifying eyelid movements
is easily observable. Although little investigation has been done on half blinks in horses, it has been
incorporated into the Equine Facial Action Coding System (EquiFACS) as its own action unit [25].
Spontaneous blinks are uniquely different from voluntary and reflexive blinks, as they can
represent a range of information processing functions spanning attention and working memory [26].
Humans subjected to stressful stimuli through social and emotional recollection tests exhibited an
increase in spontaneous blink rate and a similar trend has been demonstrated in guinea pigs that
are in states of emotional arousal following handling [27]. Although an increase in spontaneous
blink rate has been observed in humans subjected to stressful and neurologically-demanding stimuli,
spontaneous blink rate has also been found to decrease when the subject is most attentive while
performing demanding tasks or exposed to stressful visual stimuli [28,29]. This suggests that humans
reduce their spontaneous blink rate when perceiving visual stimuli in order to maximize the amount
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Animals 2019, 9, 562
of information entering the nervous system; thus, increased spontaneous blink rate potentially hinders
attention in humans and the ability to perceive immediate stimuli [28]. Therefore, the influence of
specific stressors such as visual stimuli, emotional anxiety and/or neurological levels of arousal that
initiate a fight or flight response must be considered when investigating behavioural responses in
horses [17,28].
While spontaneous blink rate has been used as a non-invasive measure of stress in humans, little
research has been applied to using spontaneous blink rate as a behavioural indicator in horses, and no
research has differentiated between different eyelid movements. This study aims to investigate the
use of spontaneous blink rate and eyelid movements as a non-invasive measure of stress in domestic
horses (Equus caballus) in response to induced, external stressors. Based on the limited and contrasting
evidence reviewed, sources of stressors including feed restriction and separation from herdmates were
selected to induce social and neurological states of arousal. In comparison, the startle test was chosen
to induce a stress response from visual stimuli. We hypothesized that spontaneous blink rates in horses
would significantly increase during feed restriction and separation from conspecifics and decrease
during the startle test in comparison to the control.

This project was approved by the University of Guelph’s Animal Care Committee (AUP #3143) in
compliance with the Canadian Council on Animal Care guidelines for the use of animals in research.
2.1. Subjects and Housing

This study used 33 riding lesson horses (Equus caballus) from three different facilities in Eastern
Ontario, Canada. The horses had a mean age of 11 ± 6 years, and all horses were in good health with
no documented instances of digestive issues or ulcers that might impact the feed restriction treatment.
As a variety of breeds were represented, they were categorized into four categories: Thoroughbred and
Thoroughbred crosses (n = 10), warmbloods (e.g., Hanoverian; n = 8), stock (e.g. Quarter Horse; n = 7)
and ponies (e.g., Welsh; n = 8). Before and after each treatment, the horses were housed in their usual
stalls and followed their regular regimes, including being turned out with their normal herdmates
and maintaining their normal exercise routines. Their diets and feeding schedules remained the same
throughout the study, with the exception of the feed restriction treatment when feed was withheld.
2.2. Treatments
Each horse was exposed to each of the four treatments in randomized order.
(i) Control: the horse was observed individually for three minutes in their normal turnout
environment, with the exception of the presence of the handler and observer. The horse was
surrounded by, or within sight of, their paddock mates. Observations occurred during quiet times at
the facility with no expectation of predictable events such as riding or feeding.
(ii) Feed restriction: the horse was tied individually in their stall during their regular afternoon
feeding time, and was observed while feed was withheld for three minutes, during which time the
horse watched their neighbouring conspecifics being fed.
(iii) Separation: the horse was led individually from their normal environment to an isolated
testing arena. Once there, the horse was asked to stand and was observed for three minutes. There was
no visual contact with their conspecifics although auditory contact was still possible. Observations
occurred during quiet times at the facility with no expectation of predictable events such as riding
or feeding.
(iv) Startle test: the horse was led individually from their normal environment to an isolated
testing arena where they were unable to see conspecifics although auditory contact was still possible.
Once there, the horse was asked to stand while a ball was thrown suddenly on the ground 2 m in front
of the horse (Figure 1), and the horse was observed for three minutes. Observations occurred during
quiet times at the facility with no expectation of predictable events such as riding or feeding.
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Figure 1. Horse with heart rate (HR) monitor (Polar RS800) during the startle test. The ball was tossed
approximately 2 m in front of the horse. The handler maintained a fairly loose lead. The observer
(not visible in this photo) was positioned about 3 m from the horse’s right eye.
2.3. Data Collection

All data at any one facility were collected over two or three days, and all treatments for any one
horse were tested on a single day. At least 10 min prior to each treatment, each horse was outfitted
with a heart rate monitor (Polar RS800, Lachine, QC, USA; Figure 1) to allow them to acclimatize to the
monitors. Heart rate (HR) was collected every five s throughout each treatment.
During each treatment, a handler held the horse by a lead rope attached to the halter, and the same
handler was used throughout the study. The handler held the horse in place on a fairly loose lead (1 m),
with just enough contact to maintain the head relatively still without restricting movement. A single
observer was used throughout the study and maintained a position about 3 m perpendicular to the
right eye of the horse. The observer videotaped all treatments using a Panasonic 2MOS video camera,
with focus maintained on the right eye of the horse. One individual coded all the behaviours (Table 1)
retrospectively from the videos using Observer XT (Version 12.0, Noldus, Leesburg, VA, USA). All
occurrences of eye blinks and eyelid twitches were tallied, while the total duration of ear movement,
head movement, mouth movement and restlessness was calculated over each 3 min treatment and
reported as a percentage of total time.
2.4. Data Analysis

The data was exported from Observer XT to Microsoft Excel 2011 (Supplementary File SBrate
data.xlsx). All statistical analyses were carried out using SAS (Version 9.4, Toronto, ON), and significance
was considered to be p < 0.05.
As there was no missing data, n = 33 for all analyses. Residuals were graphically inspected to
determine the fit of the model, and horse HRs were log transformed since they did not achieve a normal
distribution (Kolmogorov–Smirnov test, p < 0.01). A generalized linear mixed model procedure was
used to analyse the effect of treatment on behaviour and HR, with location, age, breed and treatment
as the independent variables and horse as the random factor. Sidak’s methodology was used to test
multiple comparisons of least squared means for each behaviour across treatments.
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Animals 2019, 9, 562
Table 1. Ethogram of behaviours observed in horses (n = 33) during each of the four treatments—control,
feed restriction, separation from conspecifics and the startle test. Adapted from [13,25,30,31].
Behaviour Description
Eye—full blink The right eye becomes momentarily but completely closed
The right upper lid moves toward the lower lid of the eye but does not
Eye—half blink
cover the eye completely
Fine fibrillar movement of the skin involving the levator palpebrae
Eyelid—twitch
superioris muscle of the upper eyelid
Ears—forward The right ear points forward in an attentive manner
Ears—sideways The right ear is angled to the side
Ears—back The right ear is turned backward
Head—above withers The right eye level goes above the height of the withers
Head—even with withers The right eye is even with the height of the withers
Head—below withers The right eye level drops below the height of the withers
The lips are in motion, either with mouth shut, with the tongue licking
Oral behaviour
or coming out of the mouth, or chewing
Any movement made by the legs, including movement that causes the
Restlessness
horse to move out of view of the camera
3. Results
On average, horses performed full blinks 8–9 times/min in the absence of any stressors. This rate
decreased to 5 blinks/min in the presence of any external stressors. Conversely, eyelid twitches increased
from about 2/min in the control situation to 6/min during feed restriction. Full eye blinks occurred more
often during control than during any other treatment (F(3,95) = 9.88, p < 0.0001; Figure 2). Half blinks
occurred most often during control and feed restriction treatments, and least often during separation or
startle test (F(3,95) = 10.65, p < 0.0001; Figure 2). Eyelid twitches were more evident during the feed
restriction treatment than during any other treatment (F(3,95) = 9.46, p < 0.0001; Figure 2).
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Figure 2. Total number of observations of full eye blinks, half blinks and eyelid twitches (±SD) in horses
(n = 33) over a 3 min observation period during control, feed restriction, separation from conspecifics
or a startle test. a,b,c differ across treatments p < 0.0001.
118
Animals 2019, 9, 562
Horse heart rate was higher during feed restriction (44 ± 13 beats per minute (bpm)) and lower
during separation (37 ± 7 bpm) and the startle test (37 ± 8 bpm) compared to the control (39 ± 8 bpm)
(F(3,92) = 306.12, p < 0.0001). There was no effect of facility (p > 0.05) on the behaviours or HR.
The horses’ right ear was forward more often during separation and the startle test (F(3,95) =
8.29, p < 0.0001; Figure 3), whereas it was more often sideways during feed restriction and the control
(F(3,95) = 22.53, p < 0.0001). There was no difference among treatments for the percentage of time the
horses had their ears back (F(3,95) = 0.82, p > 0.49).
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Figure 3. Percentage of time that horses’ (n = 33) ears were forward, sideways or back (±SD) over a 3
min observation period during feed restriction, separation from conspecifics, a startle test or control.
a,b,c differ across treatments p < 0.0001.
Horses held their head raised more frequently during feed restriction (F(3,95) = 30.02, p < 0.0001;
Figure 4) and held their head low more often during the control treatment and startle test (F(3,95) =
7.15, p = 0.0002).
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Figure 4. Percentage of time that horses’ (n = 33) heads were held high, neutral or low (±SD) over a 3
min observation period during feed restriction, separation from conspecifics, a startle test or control.
a,b,c differ across treatments p < 0.0001.
Oral behaviours were most evident during the feed restriction, with significantly fewer during
separation and the startle test (F(3,95) = 11.42, p < 0.0001). Horses were more often restless during feed
restriction than separation or the startle test (F(3,95) = 6.78, p = 0.0003).
119
Animals 2019, 9, 562
4. Discussion
The aim of this study was to determine changes in eyelid movement in horses during exposure to
stressful stimuli. It was expected that spontaneous blink rate would increase during exposure to mental
stressors such as feed restriction and separation from conspecifics and decrease during exposure to
visual stimuli via the startle test. However, our results showed a decrease in both full and half blinks
in response to each of the test situations.
Feed restriction was clearly the most stressful situation for the horse demonstrated by an increase
in HR, restlessness, oral behaviours and percentage of time the head was held high. However, as HR
increased only slightly, it may be concluded that this was only a mildly stressful situation. During
separation from conspecifics and the startle test, horses had their right ear forward for more of the
time, indicating focused attention in front of them that presumably maximized visual information
processing. The horses may also have been attentive to sounds emanating outside of the test arena,
although such sounds could come from any direction. Heart rate during these two situations was
slightly decreased, showing that while attention was focused, physiologically the horses did not appear
stressed. It could be that the presence of the human handler and observer may have had an appeasing
effect on the horse, as Merkies et al. [7,10] demonstrated a decrease in HR when any human was
present rather than the horse being completely alone.
Like full blinks, half blinks were most evident in the control environment, with the lowest
frequency in the startle test where visual stimulation is important for the horse to process information
about their environment. Similarly, in humans, the number of both full and partial blinks decreased
when they were asked to perform a reading test requiring visual concentration [32]. Also shown in
guinea pigs, eye blink rate decreased during handling compared to the control, although this study
did not verify the stressful effects of handling [27]. Conversely, both full and half blinks characterized
in cats increased under conditions of induced fear [24].
A recent study investigating stress responses in horses observed a decrease in mean spontaneous
blink rate only during the first minute of a 10 min sham clipping procedure [33]. This decline preceded
the onset of a significant increase in spontaneous blink rate for the following nine min, suggesting
that the initial reduction may be characteristic of a fight or flight response, allowing the horse to
visually fixate on the stimulus before responding accordingly. In our current study, blink rate was
calculated as a mean over three min. It could be that a longer time frame may have noted a subsequent
increase in spontaneous blink rate similar to Mott et al.’s [33] study. It would have been interesting if
Mott et al. [33] had collected data on differing eyelid movements.
Our results showed an increase in eyelid twitches during feed restriction, which the horses found
stressful. Conversely, separation from conspecifics and the startle test did not evoke an increase in
eyelid twitches, suggesting that eyelid twitches are more prevalent during stressful situations as the
horses did not appear to find these two latter situations stressful.
Research investigating horse facial expressions led to the development of the equine Facial Action
Coding System (EquiFACS) for use in determining behavioural indicators of a horse’s emotional
state [25]. In accordance with EquiFACS, the Equine Pain Face and Horse Grimace Scale (HGS)
were created after observing changes in equine facial expressions when horses were subjected to
painful procedures. These scales identified notable changes in eye expression associated with horses
experiencing negative situations [31,34]. An angled eye was found to be indicative of stress by
comparing the shape of the eye with the appearance of eye-white during treatments associated with
negative arousal [35]. The appearance of “worry wrinkles”, defined as a contraction of the levator
anguli oculi medialis muscle and the corrugator supercilii muscle, are prominent during situations
involving negative arousal such as food competition, in contrast to relaxed eyes during positive stimuli
such as grooming [35]. These “worry wrinkles” may be similar to eyelid twitches that appear to
increase under stressful situations. As Hintze et al.’s [35] study analysed photographs, what they may
have witnessed was an eyelid twitch in a static moment. Furthermore, a more recent study showed
that worry wrinkles could be assessed systematically regardless of horse sex, age or breed, with the
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exception of angle. In this instance, thoroughbred horses displayed less contraction of the eyelid
muscles than ponies, with coldbloods displaying the strongest contraction [36].
In horses, an increase in spontaneous blink rate was associated with a more anxious temperament
accompanied by more movement, while a decrease in spontaneous blink rate was associated with more
docile behaviour [37]. The authors proposed that these differences in temperament may be directly
related to striatal dopamine levels, with anxious horses having elevated dopamine while docile horses
have lower levels of dopamine. In humans, spontaneous blink rate has been positively associated
with striatal dopamine production. For example, it is well known that spontaneous blink rate is
decreased in humans experiencing Parkinson’s disease, an affliction that causes reduced functioning
due to a decrease in dopamine production, whereas patients with schizophrenia (a hyperdopaminergic
condition) exhibit increased anxiety and increased blink rate [38]. Additionally, Colzato et al. [39]
demonstrated that both high and low spontaneous blink rates caused a decrease in performance in
humans undergoing a start–stop task whereas those with average spontaneous blink rates performed
best. These results emphasize the effect of tonic dopamine levels on spontaneous blink rate, which
is an important baseline to understand changes in blink rates as a result of induced environmental
stressors. It may be that a reduced spontaneous blink rate could indicate underlying pathologies such
as depression, or alternatively that activity level may not necessarily reflect the level of arousal.
To our knowledge, this is the first report of the significance of eyelid twitches in horses. Blinking,
and even more so eyelid twitches, are relatively easy measures to examine in horses, although the
observer must be fairly close to the horse in question. Monitoring changes in blink rate, and in
particular eyelid twitches, could alert the observer to changes in the level of arousal of the horse.
A decrease in spontaneous blink rate concomitant with an increase in eyelid twitches may indicate
a stressful situation for the horse, whereas a decrease in spontaneous blink unaccompanied by an
increase in eyelid twitches may indicate an environment that is engaging but not stressful to the horse.
Further research investigating specific eyelid movements in relation to level of arousal could give us
insight into the emotional responses of horses. For example, in humans, facial electromyography has
been successfully used to correlate facial muscle activation to positive or negative emotions. Since we
cannot ask horses to self-report how they are feeling, physiological measures that differentiate between
pleasant and unpleasant experiences may allow us to infer underlying emotions. Further investigation
of changes in spontaneous blink rate and eyelid twitches over varying time spans is needed to identify
patterns and temporal trends in response to stressful stimuli.
5. Conclusions
Horses exposed to stressful environments decrease their spontaneous eye blink rate and increase
the frequency of eyelid twitches. However, if the environment is simply visually stimulating, eyelid
twitches do not appear to increase even if eye blink rate decreases. Monitoring spontaneous blink rate
is a sensitive metric of neural activity and differentiating eye blinks from eyelid twitches may provide
insight on the level of arousal of the horse.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2615/9/8/562/s1.

Author Contributions: Conceptualization, K.M.; methodology, K.M. and C.R.; formal analysis, K.M.; investigation,
K.M., C.R. and L.F.; resources, K.M.; data curation, K.M. and L.F.; writing—original draft preparation, L.F. and
A.H.; writing—review and editing, K.M.; supervision, K.M.; project administration, K.M.; funding acquisition,
K.M.
Acknowledgments: The authors would like to thank the three stables who volunteered the use of their school
horses for data collection. Also, thanks to Amelia Garnett for coding many of the videos.
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Animals 2019, 9, 562
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Article
Horse Transport to Three South American Horse
Slaughterhouses: A Descriptive Study
Béke Nivelle 1,2, *, Liesbeth Vermeulen 3 , Sanne Van Beirendonck 4 , Jos Van Thielen 4,5 and
Bert Driessen 1,2
1 Laboratory of Livestock Physiology, Department of Biosystems, KU Leuven, 3001 Heverlee, Belgium;
[email protected]
2 Dier&Welzijn vzw, 3583 Paal, Belgium
3 Westvlees NV, 8840 Westrozebeke, Belgium; [email protected]
4 Bioengineering Technology TC, KU Leuven, 2440 Geel, Belgium;
[email protected] (S.V.B.); [email protected] (J.V.T.)
5 Thomas More, 2440 Geel, Belgium
Received: 4 March 2020; Accepted: 30 March 2020; Published: 1 April 2020
Simple Summary: In the western world, the number of slaughtered horses is decreasing, but still
about 5 million horses are slaughtered worldwide each year. The conditions in which horses are
transported to the slaughterhouses are a topic of discussion. This study intended to investigate
the circumstances of commercial slaughter horse transport and to detect possible risk factors for
horse welfare. Therefore, 23 commercial horse transports to three South American slaughterhouses
were monitored. During transport, a camera was mounted in each loading space so that horse
behaviour could be analysed after transport. Fighting behaviour could not be explained by stocking
density, environmental parameters, trailer characteristics, duration and distance of the journey. The
temperature and relative humidity were recorded every five minutes in all loading spaces. Average
temperatures exceeded the thermoneutral zone in six transports, but it is not clear if and to what
extent horse welfare was impaired. Overall, loading and transporting of the horses went well, but
the infrastructure of the loading area did not always promote smooth loading and can therefore be
improved. At later visits, we noted that this issue was addressed.
Abstract: Between November 2016 and October 2017, 23 horse transports from 18 collection points
to two slaughterhouses in Argentina and one in Uruguay were monitored. The goal of this study
was to characterize the current practices in commercial horse transports and to detect potential
threats to horse welfare. A total of 596 horses were transported over an average distance of 295 ±
250 km. Average transport duration was 294 ± 153 min. The infrastructure did not always promote
smooth loading, but the amount of horses that refused to enter the trailers was limited. In each
loading space, a camera was mounted to observe horse behaviour during the journey. Ambient
temperature and relative humidity (RH) were recorded every five minutes in each loading space. In
14 of the 23 transports, the maximum temperature rose above 25 ◦ C and the average temperature
was over 25 ◦ C during six transports. The average temperature humidity index (THI) exceeded
72 during six transports. The average stocking density was 1.40 ± 0.33 m2 per horse, or 308 ±
53 kg/m2 . The degree of aggression differed between the front and rear loading space. Stocking
density, environmental parameters, trailer characteristics, and transport duration and distance did
not influence aggressiveness.
Keywords: horses; transport; slaughter

Animals 2020, 10, 602
1. Introduction
In the USA, Australia and Europe, the number of slaughtered horses is decreasing. In Belgium
for example, the number of slaughtered horses decreased from 21,390 slaughtered horses in 2001 to
only 5895 horses in 2018 [1]. Annually, about 5 million horses are slaughtered worldwide [2]. In 2018,
China was the country that slaughtered the most horses worldwide—more specifically, about 1.59
million [2]. In the same year, 389,153 horses were slaughtered in South America [2]. However, to this
day, the circumstances in which horses are transported give rise to discussion [3].
Animals should be handled as carefully as possible at all times, including during the transportation
process to the slaughterhouse. The animals should undergo as little stress as possible, on the one hand
for welfare reasons, but also to prevent any deterioration in the quality of the horse meat. Quality loss
can occur as a result of excessive stress, bruises or injuries [4–7]. Suboptimal ambient parameters such
as temperature, relative humidity, ammonia and carbon dioxide concentration in the air can cause
stress, but also inappropriate infrastructure and psychological stressors. Examples of psychological
stressors are the determination of dominance rank, and transport activities such as loading, unloading
and the transport itself. In addition, activities that are part of the management of the animals, such as
the weaning of young animals, weighing of animals or changing housing, can also cause stress [8].
The conditions of (non-commercial) transports of sport and company horses and the response of
these horses to transport are well studied [9,10]. On the other hand, commercial transport of horses
is studied to a lesser extent. Furthermore, most studies in which horses are transported untethered
in groups involve healthy horses that are used to being transported [9,11–14]. Studies investigating
slaughter horse transports or horses that were sold as slaughter horses are less numerous [13,15–19].
However, from those studies it is clear that a number of transport-related factors influence horse
welfare. Journey distance and time [20,21], loading density [7,15,22,23], handling [7,24,25], new
environments [24], potentially re-grouping or mixing with unfamiliar animals [24], fasting and
deprivation of water [24], the myriad of trailer designs [7,23], driving behaviour [7], road type and
quality [7,22,24], traffic conditions [24], suspension systems and building materials of the trailers [15,23],
environmental conditions in the trailer [7,22], and weather conditions [15,23,26] all affect horse welfare.
According to Morgan [27], the thermoneutral zone of a horse is on average between 5 ◦ C and
25 ◦ C. Another study defines the thermoneutral zone between −1 ◦ C and 24 ◦ C [27]. The differences
in estimation of the thermoneutral zone are, among other things, probably due to acclimatization,
body condition and climate [28]. For example, the upper limit of the thermoneutral zone lowers with
increasing humidity. At a relative humidity (RH) of more than 50%, it gets harder for the animals to
dissipate heat to the environment [29]. The temperature humidity index (THI) is a useful parameter to
estimate the thermal comfort of organisms [30].
Legislation involving animal welfare differs between countries. At the same time, meat is traded
from countries with less stringent welfare requirements, like Argentina [31–33] and Uruguay [34], to
countries with higher welfare requirements, like members of the European Union. The European Union
(EU) sets welfare requirements at the time of killing for companies willing to export animal products
to the EU (Council Regulation (EC) No 1099/2009) [35], but does not impose direct requirements on
the transport of those animals to the slaughterhouses in third countries. The international Horse
Meat Federation [36], on the other hand, expects its members to meet the requirements set in their
“Manual for the Animal Welfare of horses during transport and slaughtering”, which is based on
existing legislation and international guidelines [36].
The first aim of this study was to characterize the current practices of the commercial horse
transport from collection points to slaughterhouses in Argentina and Uruguay. Secondly, potential risk
factors for horse welfare were detected.

Between November 2016 and October 2017, a total of 23 horse transports in Argentina and Uruguay
were monitored from loading at a collection point to one of the three selected horse slaughterhouses. A
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Animals 2020, 10, 602
total of 596 half-bred horses with an average weight of 415 (±38) kg were picked up at 18 collection
points and were transported to Lamar (Argentina), Frigorífico General Pico (Argentina) and Sarel
(Uruguay). The transports were spread throughout a year, so that a number of transports were
monitored in each season. A total of six transports took place in the spring (November 2016), six
in the summer (March 2017), six in the fall (June 2017) and five in the winter (October 2017). Each
season two transports per slaughterhouse were monitored, except for October 2017. At that time,
only one transport to Frigorífico General Pico was followed up. The transports were carried out with
different types of transport vehicles. In this study, three types of vehicles are distinguished, namely
trucks, tractor–trailers and truck–trailers (Figure 1). A truck is a pulling vehicle with one inseparable
loading space and is therefore a single unit. In the case of a tractor–trailer, the towing vehicle can be
(dis)connected from the trailer via a fifth-wheel coupling. Finally, a truck–trailer is a truck, as defined
above, with a trailer connected to it through a drawbar. This transport combination therefore exists of
two separated loading spaces.
Figure 1. Example of different transport combinations. (a) A truck–trailer: a truck pulling a trailer
and (b) A tractor–trailer: a pulling vehicle (without loading space) hauling a trailer through a
fifth-wheel coupling.
The transports were monitored and supervised by the same researcher. The researcher also
mounted the sensors and cameras in the trailers and recorded specific transport data. In each trailer
the horses were filmed. The camera (Trophy Cam model 119437, Bushnell, China) filmed fragments
of one minute at intervals of about 100 seconds. On average, 38.9% (±3.8)% of each transport was
filmed. After the transports, the footage was viewed, and the behaviour and interactions of the horses
were analysed. During the analysis of the videos, it was noted per trailer how many horses fell and
whether the animals fought ‘hardly or not’, ‘averagely’ or ‘a lot’. A fall was considered to be a loss of
balance in which parts of the body other than the hooves touched the ground. If the horse could restore
equilibrium without other body parts touching the ground, this was called stumbling. Furthermore,
the temperature, relative humidity (RH) and dew point were automatically recorded (EL-USB-2, Lascar
Electronics, Wiltshire, UK) every five minutes in each loading space. These data were automatically
written to an excel file. For the analysis of the environmental parameters, the thermoneutral zone
used by Morgan [27], namely between 5 ◦ C and 25 ◦ C, is taken as the starting point. In addition, the
temperature humidity index (THI) is calculated using the following formula [37], with T, temperature
in ◦ C, RH expressed as a number between 0 and 1:
THI = 0.8T + RH × (T − 14.4) + 46.4 (1)
The timing of various operations such as loading and unloading, the duration and distance of
the transport, the number of intermediate stops for (police) checks, as well as the stocking density,
dimensions and characteristics of the loading spaces were recorded. The observer noted what tools
the drivers, which are the persons that handle the horses, used. Furthermore, the characteristics and
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Animals 2020, 10, 602
dimensions of loading docks were registered. The openness of the side walls was categorised as ‘open’
when the surroundings could be seen easily through the wall, ‘half open’ when the view through the
side walls was limited, and ‘solid’ when the horses could not see anything through the side walls of
the loading dock. All the parameters that were considered during this investigation are listed in the
Appendix A in Tables A1 and A2.
The data were processed using SAS Enterprise Guide and SAS 9.4. Averages and standard
deviations were calculated using the PROC UNIVARIATE procedure. Correlations between the
environmental parameters in the front and rear trailer were calculated using the regression (PROC
REG) procedure. The frequency procedure (PROC FREQ) was used for all frequency calculations
and generalized linear mixed models (PROC GLIMMIX) were used to identify which parameters
influenced the degree of aggression and falling of horses during transport.
3. Results
3.1. Loading Dock

In 17 of the 18 collection points, a loading dock was present to facilitate the loading of the horses.
From two collection points, horses were picked up more than once. However, only one loading dock
was used for two transports. In one collection point, the loading dock did not have a slope, since the
transport vehicle could be parked so that surface of the loading area was at the same level as the floor
of the trailer. Therefore, this loading deck was excluded from the averages (Table 1). The slope of the
loading docks was on average 17.4◦ (±3.6)◦ , which equals 31.5% (±7.0)% (Table 1). Seven out of 21
loading docks had a slope steeper than 20.0◦ (36.4%) and the slope of all but one loading dock was
steeper than 10.0◦ (17.6%). The length of the loading dock (measured on the surface of the loading
dock) was 4.01 (±0.90) m and the height was 1.18 (±0.17) m (Table 1).
Table 1. Dimensions and slope of the loading docks in the 18 collection points in degrees and
in percentages.
Average ± Standard
Parameter N Minimum Maximum Median
Deviation
Length (m) 4.01 ± 0.90 21 2.60 6.50 3.89
Height (m) 1.18 ± 0.17 21 0.93 1.74 1.17
Height side
1.59 ± 0.22 19 1.16 1.94 1.60
walls (m)
Slope (◦ ) 17.4 ± 3.6 21 9.3 23.6 17.1
Slope (%) 31.5 ± 7.0 21 16.4 43.6 30.7
The surface of the loading docks consisted of only soil (26.1%), a combination of soil and wood
(26.1%), only wood (13.0%), concrete (partly) covered with soil (8.7%), a combination of soil and grit
(8.7%) or straw (4.4%). The side walls of the loading docks were, on average, 1.59 (±0.22)-m-high and
constructed from wood—mostly planks (82.6%), but round wooden beams (4.3%) in one instance. The
side walls of three (13.0%) loading docks were categorised as ‘open’, seven (30.4%) as ‘half open’ and 10
(43.5%) as ‘solid’. Of three (13.0%) loading docks, the kind of side wall construction was not registered.
3.2. Loading
Loading the horses took an average of 12.2 (±7.1) minutes per transport and 0.49 (±0.27) minutes
per horse (Table 2). Spread over three transports, five horses (0.84%) had to be led into the trailer with
a halter: three horses in one loading did not want to enter the trailer and twice one horse refused to
enter the trailer. In the end, two of these horses could not be loaded at all. The trucks left the collection
points 8.3 (±4.4) minutes after the loading process was completed. In total, 73.9% of the transports
departed in the morning, on average at 11:15 a.m. (±1:31; between 9:25 a.m. and 3:20 p.m.). Figure 2
shows the arrival and departure times of the transports.
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Animals 2020, 10, 602
Table 2. Duration of loading and standstill after loading before departure.
Average ± Standard
Parameter N Minimum Maximum Median
Deviation
Loading time
12.2 ± 7.1 23 2.0 32.0 10.0
(minutes)
Loading time per horse
0.49 ± 0.27 23 0.19 1.23 0.42
(minutes/horse)
Duration standstill
before departure 8.26 ± 4.28 23 2.00 19.00 8.00
(minutes)
Figure 2. Arrival and departure times of the monitored transports. The duration of the transports is
mentioned in the coloured bars. The transport numbers are the same as used in Tables A3 and A4 in
the Appendix A.
Tools such as a flag, whip or stick were used to drive the horses on the trailers. We define a stick
as a narrow, long and little or not flexible object to drive the horses with. A whip is a thin, not very
flexible stick, with or without a handle on it. In this context, a flag is a stick with a piece or ribbons of
textile or plastic, so that the movements of the object are more visible to the horses. Flags were used in
22 (95.7%) of the 23 transports. Four times (17.4%) a whip or a stick was used and three times (13.0%)
a rider on horseback drove the slaughter horses onto the trailer. The tools were not used to hit or
poke animals on sensitive body parts, but to give visual signals. Occasionally, the horses were gently
touched with the stick, whip or flag, but not to the extent that the touch could cause pain or discomfort.
Divided over 13 loads, a total of 23 (3.86%) horses stumbled during loading, with a maximum of
five horses during one loading. Five falling horses were noted, spread over three loadings. During a
loading in which three horses stumbled, also three horses fell and during two other loads, in which
respectively three and five horses stumbled, one horse fell each time. No falls were noted in other
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loadings. The falls were caused by pushing and/or fights among horses. One of the fallen horses was
ran over by the other horses when the group abruptly turned around in the narrowing space towards
the loading ramp. The abrupt turn of the group was caused by the directions of the drivers.
Horses enter the transport vehicles through trapdoors, which are guillotine-type doors. Internal
doors were also trapdoors. During 13 of the 23 transports, at least one horse bumped its head against
the trapdoor. In five transports, three or more horses (maximum eight) bumped their heads against the
trapdoor. The height of the trapdoors where horses hit their heads varied between 1.51 and 1.81 m.
3.3. Environmental Parameters

The lowest trailer temperature observed during transport was 6.0 ◦ C, while the maximum
temperature was 35.5 ◦ C (Table 3). In the trailers, the temperature never dropped below the lower
limit of the thermoneutral zone, being 5.0 ◦ C. The upper limit of the thermoneutral zone, 25.0 ◦ C,
was exceeded during 14 of the 23 (60.9%) transports. During six (26.1%) transports, the average
temperature in the trailer was above 25.0 ◦ C and during five (21.7%) transports, the minimum
temperature in the trailer exceeded 25.0 ◦ C. For seven (30.4%) transports, the maximum temperature
was above or equal to 30.0 ◦ C. There was a strong correlation between the front and rear load for both
average, minimum and maximum temperatures (Table 4).
Table 3. The average, minimum and maximum temperature, RH and THI in the loading spaces. The
average, minimum and maximum of each parameter was first calculated per transport and then per
time period.
Average ± Standard
Parameter N Minimum Maximum
Deviation
Temperature 22.0 ± 5.0 38 6.0 35.5
Spring 25.4 ± 3.7 11 15.0 35.5
Summer 24.2 ± 2.2 11 17.5 32.5
Autumn 15.3 ± 2.3 9 6.0 26.0
Winter 21.7 ± 4.2 7 14.0 32.0
RH 57.4 ± 13.5 38 27.5 99.0
Spring 52.6 ± 14.9 11 27.5 97.0
Summer 59.9 ± 12.9 11 42.5 96.5
Autumn 60.3 ± 6.2 9 43.5 80.0
Winter 57.5 ± 16.4 7 31.5 99.0
THI 67.8 ± 6.5 37 45.5 83.0
Spring 72.0 ± 4.6 11 59.0 83.0
Summer 71.4 ± 2.7 11 62.6 81.5
Autumn 59.1 ± 3.3 9 45.5 72.8
Winter 66.5 ± 4.9 6 57.3 78.0
The RH varied between 28.0% and 99.0% (Table 3). There was a strong correlation between the
front and rear loading space for both average, minimum and maximum humidity (Table 4). There was
no correlation between temperature and RH within the same loading space.
Due to the lack of a reference framework with limit values for heat stress in horses [38,39], the THI
is tested against the values used for dairy cattle. A THI of 72–78 is labelled as mild heat stress, while a
THI between 79 and 89 stands for severe heat stress in dairy cattle [40–42] (Appendix A, Figure A1).
The THI ranged from 45.5–83.0 during transports (Table 3). In 13 (57%) transports, the maximum THI
exceeded 72, which is the lower limit for mild heat stress in cattle. For six (26%) transports, the average
THI was above or equal to 72 and for four (17.4%) transports, the minimum THI was at least equal to
72. During six (26.1%) transports, the maximum THI value was between 78 and 89, the standard for
severe heat stress in cattle. However, the average THI value always remained below 78. There was a
strong correlation between the front and rear loading space for both average, minimum and maximum
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humidity (Table 4). The average temperature, RH, dew point temperature and THI are shown per
transport in Appendix A Table A4.
Table 4. Correlations between temperature (T), relative humidity (RH) and temperature humidity
(THI) in front and rear loading space. The p-value of “a” is the p-value of the correlation coefficient.
TAF = average temperature in the front loading space; TAR = average temperature in the rear loading
space; TMinF = minimum temperature in the front loading space; TMinR = minimum temperature rear
loading space; TMaxF = maximum temperature front loading space; TMaxR = maximum temperature
rear loading space. RHAF = average RH front in front loading space; RHAR = average RH in rear
loading space; RHMinF = minimum RH in front loading space; RHMinR = minimum RH in rear loading
space; RHMaxF = maximum RH in front loading space; RHMaxR = maximum RH in rear loading space.
THIAF = average THI in the front loading space; THIAR = average THI in the front loading space;
THIMinF = minimum THI in the front loading space; THIMinR = minimum THI in the rear loading
space; THIMaxF = maximum THI in the front loading space; THIMaxR = maximum THI in the rear
loading space.
Parameter Equation (Y = aX + b) N r2 p-Value a p-Value b

TAF and TAR TAF = 1.01710 × TAR − 0.26312 14 0.9847 <0.0001 0.7465
TMinF and TMinR TMinF = 1.04965 × TMinR − 0.77318 14 0.9840 <0.0001 0.3104
TMaxF and TMaxR TMaxF = 0.85989 × TMiaxR + 3.92441 14 0.8111 <0.0001 0.2244
RHAF and RHAR RHAF = 1.00838 × RHAR + 0.22294 14 0.9861 <0.0001 0.9020
RHMinF and RHMinR RHMinF = 0.86383 × RHMinR + 6.00346 14 0.9246 <0.0001 0.0668
RHMaxF and RHMaxR RHMaxF = 0.99305 × RHMiaxR + 0.78603 14 0.9676 <0.0001 0.8120
THIAF and THIAR THIAF = 1.01061 × THIAR − 0.64323 14 0.9878 <0.0001 0.7704
THIMinF and THIMinR THIMinF = 1.04821 × THIMinR − 3.03323 14 0.9831 <0.0001 0.2441
THIMaxF and THIMaxR THIMaxF = 0.92326 × THIMiaxR + 5.81791 14 0.8453 <0.0001 0.4859
3.4. Trucks
Different types of transport vehicles were used to carry out the 23 transports (see Materials and
Methods). Some vehicles were used for multiple transports. Table A3 in the Appendix A shows the
type of vehicle used for each transport and the frequency of use of the vehicle during the monitoring.
Only one of the trucks had a roof consisting of a black sail. The average dimensions of the loading
spaces are shown in Table A3 in the Appendix A. In 10 transports (43.5%) the front loading space was
divided into several compartments: eight times (34.8%) into two compartments and twice (39.1%)
into three. The rear loading space was divided into two (six times; 40.0%) or three (two times; 13.3%)
compartments in eight (53.3%) of the 15 transports with two loading spaces.
The floor in all loading spaces was provided with wire mesh to prevent slipping of the horses.
Different types of wire mesh could be distinguished. The most common were the standard wire mesh
(Figure 3a), where the rods are on top of each other and the rods do not bend between the crossings.
This type of wire mesh was used in 20 (87.0%) transports. The curved wire mesh (Figure 3b), which is
bent between the crossings, was used in two transports, just like the diamond-shaped wire mesh with
connections in one plane (Figure 3c). In the Appendix A Table A3 shows which type of wire mesh was
found per transport and per trailer. The average mesh size was 26.0 ± 4.5 cm by 23.7 ± 4.8 cm. On
average, the wire mesh was 1.29 ± 0.38 cm thick and mounted at a height of 2.60 ± 0.78 cm. In our
observations, both on the spot and afterwards, no shoed horses were detected in any of the transports.
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Figure 3. Different types of wire mesh that were used for the 23 observed slaughter horse transports.
(a) Standard wire mesh—here addressed as “standard”; (b) standard wire mesh, which is curved
between connection points—here called “curved”; (c) diamond shaped wire mesh, with connections in
one plane—here called “diamond shaped”.
3.5. Trailer Density

The average density of the trailers was 1.40 (±0.33) m2 /horse or 308 (±53) kg/m2 . Table 5 shows
the average density in m2 /horse and kg/m2 per trailer and per compartment. Average density varied
between 0.94 m2 /horse and 2.45 m2 /horse. Stallions were not always separated from mares and geldings
during transport. During at least five transports, one or more stallions were loaded. In at least two
of these transports, the stallions were not separated from the other horses. Once, a stallion standing
between mares was moved to another compartment before departure, because of his aggressive
behaviour. In the other compartment however, the stallion was not separated from the other horses
behind a fence or ropes either. To prevent further biting, a rope was tied tightly in the mouth. The
stallion then stopped his aggressive behaviour. Several times a pony or a young horse was transported
in the same compartment with significantly larger horses.
Table 5. Average available space per horse (m2 /horse) and average density (kg/m2 ). T1 = front loading
space; T2 = rear loading space.
Average ±
Parameter Standard N Minimum Maximum Median
Deviation
Surface area of loading
34.70 ± 9.50 23 13.50 41.91 39.45
space (m2 )
Average density of full
loading space 1.40 ± 0.33 23 0.94 2.45 1.38
(m2 /horse)
Density T1 (m2 /horse) 1.40 ± 0.36 23 0.80 2.45 1.33
Compartment 1 1.52 ± 0.53 8 1.03 2.68 1.46
Compartment 2 1.38 ± 0.44 7 0.97 2.25 1.33
Compartment 3 1.25 ± 0.35 2 1.00 1.50 1.25
Density T2 (m2 /horse) 1.38 ± 0.22 15 0.99 1.88 1.38
Compartment 1 1.36 ± 0.27 6 1.06 1.74 1.36
Compartment 2 1.53 ± 0.49 6 1.15 2.48 1.37
Compartment 3 1.86 ± 0.53 2 1.49 2.23 1.86
Average density
308 ± 53 21 191 402 327
(kg/m2 )
Density T1 (kg/m2 ) 312 ± 63 21 191 473 308
Compartment 1 283 ± 65 6 175 352 304
Compartment 2 322 ± 57 6 208 365 338
Compartment 3 362 1 362 362 362
Density T2 (kg/m2 ) 309 ± 42 14 227 354 326
Compartment 1 334 ± 74 5 253 446 344
Compartment 2 291 ± 67 5 191 362 322
Compartment 3 212 1 212 212 212
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3.6. Aggression and Falling during Transport

The degree of aggression was assessed per loading space during 22 transports, of which 15
transports with a truck–trailer, together accounting for 38 loading spaces. A loading space refers to the
space in one transport component. A truck therefore has one loading space just like a tractor–trailer,
while a truck–trailer has two loading spaces. In 13 (34.2%) loading spaces, hardly any or no fights were
registered. In 15 (39.5%) loading spaces, an average amount of fights was recorded, and a lot of fights
were recorded in 11 (28.9%) loading spaces. No relation was found between the degree of aggression
and the density (front loading space: p = 0.78; rear loading space: p = 0.25), the thickness of the wire
mesh on the floor (front loading space: p = 0.23; rear loading space: p = 0.20), the transport duration
(front loading space: p = 0.90; rear loading space: p = 0.98) or distance (front loading space: p = 0.93;
rear loading space: p = 0.78), the average temperature (front loading space: p = 0.33; rear loading
space: p = 0.79), the average dew point temperature (front loading space: p = 0.18; rear loading space:
p = 0.99) and the average THI (front loading space: p = 0.28; rear loading space: p = 0.80). However,
the degree of aggression differed between the front and rear loading spaces (p = 0.05). Splitting the rear
loading space into two or more compartments resulted in less fighting than when the rear loadings
spaces consisted of one compartment (p = 0.05). In the case of the front loading space (22 transports),
no relation was found between compartmentalisation and the degree of fighting (p = 0.73). It should
be noted, however, that the fighting behaviour in the rear loading space could only be monitored for
14 transports, since only 14 out of 15 truck–trailers had the rear loading spaces successfully filmed.
In only one journey did a horse fall twice. In the 22 other journeys, no horses fell, except once
before departure. That horse was then removed from the truck and not taken to the slaughterhouse.
No connection could be found between the degree of fighting and the falls of the horses, but this is
likely due to the limited number of horses that fell during the journey.
3.7. Transport Distance and Duration

The transport distances from collection point to slaughterhouse ranged from 37–700 km (Table 6).
The transport time varied from 73–632 min (Table 6). One transport lasted 480 min or eight hours,
while two (8.7%) other transports lasted longer, more specifically 500 and 632 min. The horses were not
unloaded during the journey and had no ability to eat or drink. The transport duration and distance
were strongly correlated (r2 = 0.94; p < 0.0001) for the 23 observed transports: with increasing distance,
the transport duration increased according to:
Y = 49.7 + 0.8X (2)
(with Y = the transport duration in minutes and X = the distance in km)
Table 6. Average transport duration, distance and average number of stops and police checks
per transport and per 100 km. Duration of loading, unloading and standstill after loading and
before unloading.
Parameter Average ± Standard Deviation N Minimum Maximum Median

Transport duration (minutes) 296 ± 150 23 73 632 268
Distance (km) 295 ± 177 23 37 700 250
Number of stops per transport 2.74 ± 1.33 23 0.0 5.0 3.0
Number of stops per 100 km 1.14 ± 0.90 23 0.00 4.35 1.04
Number of police checks per transport 0.87 ± 1.00 23 0.00 3.00 1.00
Number of police checks per 100 km 0.39 ± 0.58 23 0.00 1.97 0.25
Standstill between arrival and unloading (minutes) 14.8 ± 12.6 22 3.0 45.0 10.0
Duration of unloading (minutes) 12.5 ± 9.5 20 2.0 36.0 9.0
Duration of unloading per horse (minutes/horse) 0.473 ± 0.285 20 0.115 0.947 0.388
An average of 2.74 (±1.33) stops were inserted per transport, of which an average of 0.87 (±1.00)
were inserted for police checks (Table 6).
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3.8. Unloading
After arrival, transporters had to wait for an average of 14.8 (±12.6) minutes before unloading
(Table 6). The unloading of the horses took on average 12.5 (±9.5) min per transport and 0.473 (±0.285)
per horse (Table 6). There was no correlation between the duration of loading and unloading (r2 = 0.11;
p = 0.08).
4. Discussion
4.1. Loading Docks

Besides good handling, the professional federation of the international Horse Meat Sector
(HoMeFe) [36] as well as the European Consortium of the Animal Transport Guides Project (CATGP) [7]
stresses the importance of good loading dock design, construction and maintenance to minimize the
risk of slipping, falling, injuries and stress to animals while (un)loading. Therefore, CATGP [7] sums
up a number of ‘good’ and ‘better’ practices in its guidelines for transport of slaughter horses. The
good practices are derived from the Council Regulation 1/2005 of 22 December 2004 on the protection
of animals during transport and related operations [7,43].
The loading area should be constructed in a way that prevents distress, excitement and injury
as much as possible [7,36,43]. Good practices include that the slope of the loading dock does not
exceed 36.4% (or 20.0◦ ) [7,36,43,44], better practices demand a maximum slope of no more than 10.0◦
(17.3%) [7]. When the slope is steeper than 10.0◦ or 17.6%, the loading dock must be equipped with
some sort of system that improves passage of animals without the risk of slipping [36,43], such as
stair steps or foot battens [7,36,45]. Argentinian legislation states that the slope of the loading dock
should not exceed 30.0◦ (57.7%) and should be equipped with foot battens [33]. Good loading practices
also include using a slip-resistant and anti-sliding surface on the loading ramp [7,36,43]. To prevent
animals from falling off or escaping from the (un)loading dock, side walls should be provided [7,36,43].
Side walls which limit the view on the environment prevent animals from being distracted by what is
happening around the loading dock and might thereby simplify (un)loading [7,36,44]. Grandin [46]
also mentions that solid side walls are more efficient in preventing escape attempts due to the blocked
vision of the animals.
4.2. Loading and Unloading

Loading duration is considered to be an indicator of the ease of loading. Since no loading times of
slaughter horses are available for comparison, the observed loading durations can only be compared
to these of beef cattle. María et al. [47] noted an average loading time of 1.20 (±0.86) minutes per beef
bull. However, horses and cattle cannot be compared in terms of the leniency of their movements;
these data support the observation that the loading of the horses in general went quite smoothly.
However, infrastructure did not always promote smooth loading. Horses regularly bumped
their heads against trapdoors, clearly indicating that the trapdoors were too low. Furthermore, when
stressed, for example due to rushed driving, horses carry their head higher [48] and thereby the chance
of head-bumping is increased, especially when trapdoor height is rather low. Argentinian legislations
states that the trapdoors should be at least 1.60 m high, which was not always the case, to prevent the
horses from hurting their heads and backs [33].
In a total of seven loadings, the observing researcher noted that horses hesitated or reacted
anxiously to specific elements of the infrastructure; for example, low or not fully opened trapdoors,
a bar hanging too low over the passageway, a steep slope of a loading dock, or an uneven or muddy
ground on or before the loading dock. Falling horses were only observed in loadings in which multiple
horses stumbled. The falls were caused by pushing or fights between horses, which was clearly caused
by the directions of the drivers in one instance. The directions of the drivers were not necessarily
wrong in this case, but with a calmer approach the fall might not have happened. Furthermore,
drivers must ensure that they do not give conflicting signals to the horses, for example by standing too
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close to the passageway of the horses when another driver is directing the horses to go there. These
conflicting signals create confusion and thereby chaos. However, the presence of the investigators was
probably—to a certain extent—an additional stress factor for the horses and loading crew.
For all transports, unloading went quite well. However, better communication between the
slaughterhouse and the transporter may reduce the standstill before unloading the horses on arrival.
According to Friend [23], all horses experience stress during transport. Loading and unloading
might even be more stressful to animals than the transport itself [47], but horses that were loaded
before and did not have any negative experiences with loading experienced less stress than animals
that were loaded for the first time [49]. It is not known whether the horses observed in this study had
been loaded and transported before, and how any previous transports were perceived. It is possible
that the horses that did not want to enter the trailer had had previously negative experiences during a
transport, or had other bad experiences [13]. If the person who loads the horses has a good relationship
with the horses, the stress level during loading decreases [49]. Since the horses do not have much
contact with people during rearing, there is little evidence that there is a relationship of trust between
the caretakers and the horses. Due to the lack of (positive) transport experiences of the horses and
the absence of a trust relationship with the drivers, the importance of efficient and knowledgeable
driving increases, in order to make loading as smooth as possible [49]. The authors of [47] state that
education and training of the personnel is likely to be one of the most effective measures to improve
horse welfare during loading and transport. HoMeFe demands that everyone involved in the transport
of slaughter horses is trained regularly [36]. However, it is not clear whether or not the drivers and
horse transporters of the observed transports had training in the handling and transporting of horses.
HoMeFe prohibits the use of electric driving aids, sticks and dogs [36]. As well as the World
Organisation for Animal Health (OIE) [50], Grandin [51] mentions that the use of an electric prod
should be avoided if possible, since the electric prod might cause the animals to become agitated and
therefore sometimes dangerous. Furthermore, drivers should not scream, flap their arms or make
sudden movements to keep the animals as calm as possible [36,50,51]. After all, besides their adverse
effects on animal welfare, multiple studies demonstrate the adverse effects of the use of electric prods
and incompetent handling on meat quality in pork and beef [52–54].
No electric prods were used and handlers stayed calm. Flags were used in all but one loading.
Against the requirements of HoMeFe [36], a whip or stick was used in four loadings, but never to
hit horses.
4.3. Environmental Parameters

About three-quarters of the transports left before noon, implying that the transports were often
being carried out during the hottest moments of the day.
When the environmental temperature rises above the upper limit of the thermoneutral zone,
the animal has to invest energy to keep its body temperature constant [7,26]; for example, by sweating,
peripheral vasodilation, and by increasing the respiratory rate [7,26]. However, it is not clear from
what point welfare is compromised. The temperature regularly rose above 25 ◦ C, which is the upper
limit of the thermoneutral zone of horses [27], and RH regularly increased above 50%. Above 50%,
heat is dissipated less efficiently [29].
Based on the THI framework for producing dairy cows (Appendix A Figure A1) [42], mild and
sometimes severe heat stress occurred during transports. However, THI must be interpreted
carefully, since this parameter does not take solar load and wind speed into account [55]. Especially
during transport in an open trailer, air displacement might enhance heat dissipation. On the
other hand, the horses are standing close to each other during transport, which might limit heat
dissipation. Moreover, the lack of a reference framework with limit values for heat stress in horses
complicates interpretation.
Older horses are known to be more prone to heat stress than younger horses. When exposed to the
same level of exercise, older horses overheat in a much shorter time than younger horses, indicating
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that their ability to dissipate excess heat is less compared to that of younger horses [56]. However, the
age of the slaughter horses in this study was not known.
4.4. Trucks and Trailer Density

The floor of all transport vehicles was provided with wire mesh, as demanded by Argentinian
legislation [33]. Different authorities and guidelines have determined the minimum and maximum
density for horse transports, which explains the differences in thresholds. According to the CATGP [7],
the density may vary between 1.00 m2 per horse and 1.75 m2 per horse, depending on the size and age
of the horse (Appendix A Table A5). The available space per horse may deviate by a maximum of
10% from the directive, depending on the physical condition, the weather conditions and the probable
transport time [7]. HoMeFe [36] has drafted guidelines in its specifications for the density during
transport. The available space must be between 1.1 m2 per horse and 1.4 m2 per horse. Depending on
the physical condition of the horses, the weather conditions, the travel time, the weight and the height
of the horses, the actual density may deviate a maximum of 20% from the guidelines. This means
that the available surface area per horse may vary between 0.88 m2 and 1.68 m2 [36]. In one or more
compartments of six transports, the average surface area per animal deviated more than 20% from the
HoMeFe guideline [36]: the surface area per animal was too large in five instances, and it was too small
in one instance (Appendix A Table A6). It should be noted that the allowed stocking densities differ
substantially between the CATGP [7] and HoMeFe [36] guidelines. However, comparing stocking
densities expressed as surface area per horse is not evident, since adult horses can differ substantially
in size. Two trailers with the same loading density expressed in m2 /horse can be, in reality, quite a
different stocking density in kg/m2 for these two loadings. On the other hand, determining the number
of horses that can be loaded based on the estimated average weight of a group of horses might be
prone to estimation errors.
Stull [57] compared some physiological parameters and the increase in injuries between horses
transported at low (1.40–1.54 m2 /horse) and high stocking densities (1.14–1.31 m2 /horse). She concluded
that it is better to provide at least 1.40 m2 /horse during transport, depending on the weight, conformation
and size of the horses. Extra attention must be paid to the design of the trailer in order to prevent
injuries to the horses [57].
A few times a pony or a young horse was transported in the same compartment with other,
significantly larger horses. This is contrary to European Regulation EC 1/2005 [43] and the Argentinian
Resolución 581/2014 [33], which states that animals of significantly different sizes or ages and sexually
mature mares and stallions, must be handled and transported separately, unless the animals have
been reared together, are accustomed to each other, or when the separation would cause distress.
Similarly, mares accompanied by their dependent foals are not subject the above provisions. Finally,
animals ’hostile to each other’ and tied and untied animals should not be transported in the same
compartment [43]. In at least five transports, one or more stallions were loaded. The stallions were not
separated from the other horses in at least two transports, which is contrary to the abovementioned
European Regulation [43], Argentinian legislation [33], and guidelines from HoMeFe [36]. One stallion
that was not separated from the other horses was aggressive and was therefore placed in another
compartment and had a rope tied tightly in the mouth. After putting on the rope, the stallion stopped
behaving aggressively, but this was probably due to the inconvenience caused by the rope.
4.5. Aggression and Falling during Transport

In this study, no relationships could be demonstrated between environmental parameters and
aggression. This suggests that the environmental parameters are not the most important factors that
may or may not provoke aggression. As well as the current study (Appendix A Table A6), Iacono
and colleagues [18] could not demonstrate a relationship between the degree of aggression and the
density or fatigue of the horses during the transport of untied horses. According to the authors of [18],
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aggression is more likely to be a consequence of individual horses than stocking density. The current
observations confirm this assumption (Appendix A Table A6).
4.6. Transport Distance and Duration

Argentinian legislation allows for a transport duration up to 36 hours without feeding, watering
or rest [31]. In Uruguayan law, no maximum transport duration is mentioned [34]. According to the
specifications of HoMeFe [36], a transport may take up to 12 h. In exceptional cases, the transport may
last up to 14 h, for example if the destination can be reached by continuing the ride for a maximum
of two hours [36]. This requirement differs substantially from the specifications of the European
Regulation EC 1/2005 [43], that states that a transport may only take up to eight hours. In exceptional
cases, the transport of trained horses may last up to 24 h if they are watered and fed every eight
hours and if the transport vehicle meets some extra requirements for roof construction, presence and
quality of litter, feeding and watering regime, partitions, ventilation, climate control and navigation
system. However, for unbroken horses, the European Regulation does not allow any extension of the
transport duration of eight hours, regardless of the transport vehicle in which the unbroken horses are
transported [43].
Friend [14] claims that it is advisable to regularly provide the horses with water on the truck
during long-distance transport in warm conditions, in order to reduce dehydration, stress and fatigue.
CATGP [7] recommends to water horses every 4.5 h, while the European Council Regulation EC
1/2005 [43] states that Domestic Equidae have to be watered every eight hours. Notwithstanding,
Friend [14] also mentions that water consumption can be highly variable among different horses in the
same situation [11]. Likewise, other studies question the watering of horses during transport [17,58].
After all, it often takes some time before the horses start drinking, about 20 min to an hour after the
water is offered. Some horses did not drink, possibly for fear of the new water source or because of
stress associated with the transport. Furthermore, the difference in weight loss between horses that did
and did not drink suggested that the horses probably did not drink a large amount of water [17,59].
Therefore, it seems especially important that the horses are sufficiently hydrated before departure and
have access to sufficient fresh water immediately after arrival [14,36,60].
5. Conclusions
Our study identified the current practices of the commercial horse transport from collection points
to slaughterhouses in Argentina and Uruguay. Some risk factors have been detected and could be
improved. The loading and unloading of the horses generally went quite smoothly. Better training of
drivers and optimized infrastructure (a level ground surface before and on the loading dock, sufficiently
high trapdoors, steepness of the loading docks, provision of steps or foot battens on the loading dock,
etc.) can prevent a lot of confusion and chaos for the horses, and thereby improve welfare. Driving
aids were always used correctly. Most journeys started before noon, implying that the horses were
often transported during the hottest moments of the day.
Still, on the one hand, interpretation of THI values is difficult because of a lack of reference
framework for horses. On the other hand, not all parameters that affect thermal comfort are included
in the THI. Therefore, it is not clear from what point on welfare is compromised. Stocking densities
were not always according to relevant guidelines and significantly smaller horses or stallions were
not always separated from the other horses. No influence of environmental parameters or transport
characteristics on the degree of fighting behaviour could be demonstrated. On the contrary, the
degree of aggression differed between the front and rear loading spaces of the same transport vehicle,
suggesting that animal-specific factors, rather than environmental factors, determine the occurrence of
aggressive behaviour. The willingness of all actors involved—slaughterhouses, transporters, loading
crew, etc.—to conduct this study and to address shortcomings, underscores the growing awareness of
animal welfare issues in Argentina and Uruguay.
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Author Contributions: Conceptualization, B.D.; methodology, B.D. and L.V.; formal analysis, L.V. and B.N.;
investigation, L.V.; data curation, L.V. and B.N.; writing—original draft preparation, B.N. and B.D.; writing—review
and editing, L.V., S.V.B. and J.V.T.; supervision, B.D. and J.V.T.; project administration, L.V and B.N. All authors
have read and agreed to the published version of the manuscript.
Acknowledgments: The staff of the slaughterhouses and all drivers are acknowledged for their collaboration.
Appendix A
Table A1. Observed parameters of the transport and transport vehicle.
Parameter Unit
Transport date
Slaughterhouse
Date of slaughter
Address of the collection point
License plate of all parts of the transport combination
Presence of a roof on the loading spaces Yes/no
Does the roof cover the whole loading space?? Yes/no
Colour of the roof
Internal dimensions of the loading space
m
- length, width, height and height of the solid wall
Surface area of the loading spaces m2
Number of compartments in the front and rear loading space
Dimensions of the compartments
m
- length and width
Loading density (averagely, per loading space and per m2 /horse
compartment) kg/m2
Presence of tread plates in the front and rear loading space Yes/no
Number of tread plates
Length and width of the tread plates m
Connecting method of the trailers: connected (yes) or not
connected (no). If the trailers are connected, both trailers can
Yes/no
be loaded through the hind gate. If trailers are not connected,
both trailers should be (un)loaded separately.
Dimensions of the gates to enter the vehicle, between loading
m
spaces, compartments or separate trailers (width and height)
Presence of wire mesh on the floor of the loading spaces Yes/no
Standard;
Type of wire mesh
Standard, bent; Diamond shaped
Mesh dimensions (length and width), height of the top of the
m
wire mesh and thickness of the wire mesh
Presence of fences in the loading space to create different
Yes/no
compartments
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Table A2. Observed parameters during loading and unloading.
Parameter Unit
Dimensions of the loading ramp
- length, height, width m
- slope degrees
Material that covers loading ramp (soil, wood, . . . )
Point of time on which loading and unloading started and ended (total, per
trailer and per compartment)
Duration of loading and unloading (total, per trailer and per compartment) Minutes
Number of horses per transport, per trailer and per compartment
Hygiene of the loading space: is the loading space clean? Yes/no
Number of horses that bumped their head against the upside of the gates
while (un)loading
Number of horses that stumbled during (un)loading
Number of horses that fell during (un)loading
Number of horses that had leg injuries at the moment of (un)loading
Number of horses that had a belly injury at the moment of (un)loading
Number of horses that limped while (un)loading
Number of horses that held the mouth opened while (un)loading
Number of horses that made noise while (un)loading (long and loud,
screamy whinnying)
Number of horses that had the nostrils widely opened while (un)loading
Number of horses that fled back from the trailer during loading
Number of horses that had a head injury while (un)loading
Number of horses that heavily snort during (un)loading
Number of horses that sweat heavily during (un)loading
Number of horses that scrape the floor during (un)loading
Number of horses that fight during (un)loading
Colour of the flags (and other driving tools) at (un)loading
Material used for (un)loading the horses
Did the drivers leave the gate of the loading spaces opened when going
back to drive the other horses on the vehicle?
Was the gate fully opened when (un)loading the horses? Yes/no
Were there any remarks during the (un)loading?
Weather conditions at (un)loading
- sunny Yes/no
- cloudy Yes/no
- rainy Yes/no
- windy Yes/no
- misty Yes/no
- moist Yes/no
- CO2 -concentration ppm
- light intensity lux
Point of time of departure, arrival, starting and finishing loading and
unloading (total, per trailer and per compartment) and point of time of the
end of weighing
Duration of standstill before departure, standstill before unloading,
minutes
(un)loading (total, per loading space and per compartment) and weighing
Distance of the transport km
Average speed km/u
Duration of transport minutes
Number of stops during transport
Number of police controls during transport
Degree of aggression/fighting during transport (per loading space) Not or barely, averagely or a lot
Number of fallen horses during transport (per trailer)
Temperature in the loading space during transport (measurement every five ◦C
minutes, calculated average, minimum and maximum) per loading space
RH in the loading space during transport (measurement every five minutes,
%
calculated average, minimum and maximum) per loading space
Dew point temperature in the loading space during transport (measurement
every five minutes, calculated average, minimum and maximum) per ◦C
loading space
Dimensions of the (un)loading ramp
- length, width, height m
- slope degrees
Is the gate completely opened when unloading? Yes/no
138
Animals 2020, 10, 602
Figure A1. Temperature humidity index (THI) with classification in comfort zones for dairy cattle [42].
139
Table A3. Some details of the 23 observed transports. A letter in the column “Same transport vehicle” indicates that this (part of) the transport combination was also
used in the other transport where the same letter is mentioned. In the column “Number of trailers” is mentioned of how many the loading spaces the transport
combination consists. The internal dimensions of the loading spaces and the height of the solid wall are given in the last four columns. “Solid wall” refers to the part
of the side wall of the loading space which is not made up of trellis. The function of the solid wall is to block the view of the horses and more importantly to prevent
the horses from getting stuck (with their legs) in the trellis.
Animals 2020, 10, 602
Same Length of Width Height

Transport Number Type of Number Duration Distance Type of Wire Height Solid
Number Season Slaughterhouse Transport Loading Loading Loading
Date of Trailers Vehicle of Horses (Minutes) (km) Mesh Wall (m)
Vehicle Space (m) Space (m) Space (m)
1 Spring 1/11/’16 Lamar 1 Tractor–trailer 25 a 191 206 Standard 15.76 2.48 2.05 1.30
9 Standard 5.81 2.45 2.15 1.13
2 Spring 3/11/’16 Lamar 2 Truck–trailer 139 152
16 Standard 10.20 2.52 2.20 1.13
3 Spring 7/11/’16 General Pico 1 Truck 8 110 37 Standard 5.50 2.50 1.83 1.13
11 b Standard 5.92 2.50 2.03 1.16
4 Spring 9/11/’16 General Pico 2 Truck–trailer 455 461
18 c Standard 10.15 2.50 2.03 1.16
21 Standard, bent 8.34 2.54 1.90 1.30
5 Spring 14/11/’16 Sarel 2 Truck–trailer 73 63
21 Standard, bent 8.16 2.54 1.90 1.30
21 Standard, bent 6.70 2.50 2.05 1.40
17 Standard, bent 7.56 2.50 2.02 1.40
16 d Standard 7.83 2.36 1.80 1.40
7 Summer 7/03/’17 Sarel 2 Truck–trailer 175 154
16 Standard 7.53 2.40 1.80 1.40
8 Summer 9/03/’17 Sarel 1 Tractor–trailer 38 425 407 Standard 15.00 2.67 1.83 1.35
10 e Standard 6.15 2.50 2.20 1.30
9 Summer 14/03/’17 Lamar 2 Truck–trailer 209 192
18 Standard 10.00 2.48 2.10 1.28
10 f Standard 4.74 2.45 2.00 1.21
140
20 Standard 10.48 2.45 2.00 1.21
11 Summer 20/03/’17 General Pico 1 Truck 11 419 470 Standard 5.40 2.50 2.00 1.30
6 Standard 5.30 2.50 1.78 1.15
12 Summer 20/03/’17 General Pico 2 Truck–trailer 335 289
14 Standard 10.50 2.50 1.90 1.14
12 Standard 6.25 2.50 1.81 1.20
13 Fall 1/06/’17 Sarel 2 Truck–trailer 268 228
20 Standard 10.42 2.49 1.83 1.19
Diamond
14 Fall 2/06/’17 Sarel 1 Truck 12 201 185 shaped in one 6.20 2.30 1.89 1.30
surface
15 Fall 6/06/’17 General Pico 1 Tractor–trailer 15 480 585 Standard 14.70 2.50 2.05 1.20
9 b Standard 5.92 2.50 2.03 1.16
16 Fall 7/06/’17 General Pico 2 Truck–trailer 500 569
17 c Standard 10.15 2.50 2.03 1.16
11 Standard 6.65 2.40 1.95 1.24
17 Fall 12/06/’17 Lamar 2 Truck–trailer 331 338
16 Standard 10.00 2.37 1.95 1.24
10 Standard 6.00 2.50 1.77 1.18
18 Fall 13/06/’17 Lamar 2 Truck–trailer 263 323
19 Standard 10.00 2.50 2.10 1.19
10 Standard 6.00 2.34 1.80 1.17
19 Winter 13/10/’17 Sarel 2 Truck–trailer 461 380
Diamond
16 shaped in one 10.73 2.34 1.81 1.23
surface
20 Winter 13/10/’17 Sarel 1 Truck 16 d 370 380 Standard 7.83 2.36 1.80 1.40
11 f Standard 4.74 2.45 2.00 1.21
21 Winter 17/10/’17 General Pico 2 Truck–trailer 632 700
23 Standard 10.48 2.45 2.00 1.21
11 e Standard 6.15 2.50 2.20 1.30
22 Winter 23/10/’17 Lamar 2 Truck–trailer 175 160
16 Standard 10.00 2.48 2.10 1.28
23 Winter 24/10/’17 Lamar 1 Tractor–trailer 26 a 200 180 Standard 15.76 2.48 2.05 1.30
Table A4. Some details of the transport conditions. If in the column “weather conditions” “rain” is mentioned, this does not necessarily mean that it rained
continuously during loading, transport and/or unloading.
Transport Number Type of Number of Same Duration Distance Density Weather Average T in Average Averagera Dew Point Average
Number Season Slaughterhouse Transport Loading Space
Date of Vehicle Horses (Minutes) (km) Conditions RH (%) Temperature (◦ C) THI
Trailers Vehicle m2 /horse kg/m2 (◦ C)
Cloudy, rainy,
1 Spring 1/11/’16 Lamar 1 Tractor–trailer 25 a 191 206 1.58 307.8 23.9 60.1 15.5 71.0
misty
Animals 2020, 10, 602
9 1.58 247.3 Sunny, windy 26.8 35.0 10.1 72.2

2 Spring 3/11/’16 Lamar 2 Truck–trailer 139 152
16 1.61 243.5 Standard 26.3 35.2 9.7 71.6
3 Spring 7/11/’16 General Pico 1 Truck 8 110 37 1.72 231.6 Cloudy, rainy 15.9 92.2 14.6 60.5
11 b 1.33 346.1 Sunny, windy 23.8 46.8 11.0 69.4
4 Spring 9/11/’16 General Pico 2 Truck–trailer 455 461
18 c 1.43 320.6 Standard 24.2 45.9 11.2 69.9
21 1.01 333.8 Sunny 28.5 47.4 16.1 75.8
21 0.99 341.2 Standard, bent 29.1 46.4 16.3 76.5
Cloudy, sunny at
21 0.80 473.1 28.8 54.7 18.8 77.3
6 Spring 15/11/’16 Sarel 2 Truck–trailer 87 69 arrival
17 1.11 339.4 Standard, bent 28.8 54.8 18.8 77.3
16 d 1.15 336.1 Sunny 26.9 56.2 17.4 75.0
7 Summer 7/03/’17 Sarel 2 Truck–trailer 175 154
16 1.13 341.8 Standard 26.4 51.1 17.2 74.4
Cloudy, rainy,
8 Summer 9/03/’17 Sarel 1 Tractor–trailer 38 425 407 1.05 343.3 22.7 86.3 19.9 71.3
moist
10 e 1.54 308.5 Sunny, moist 27.2 49.6 15.7 74.5
18 1.38 344.2 Standard 27.0 49.6 15.5 74.2
Cloudy, sunny at
10 f 1.16 391.5 21.5 51.6 11.1 67.3
10 Summer 16/03/’17 Lamar 2 Truck–trailer 279 250 arrival
20 1.28 354.2 Standard 21.4 52.0 11.1 67.2
141
Alternately sunny
11 Summer 20/03/’17 General Pico 1 Truck 11 419 470 1.23 357.0 24.6 56.1 15.2 71.8
and cloudy, moist
Alternately sunny
6 2.21 192.9 22.9 59.8 14.5 69.5
12 Summer 20/03/’17 General Pico 2 Truck–trailer 335 289 and cloudy
14 1.88 227.2 Standard 22.8 59.7 14.4 69.4
12 1.30 306.0 Sunny 18.2 66.0 11.7 63.5
13 Fall 1/06/’17 Sarel 2 Truck–trailer 268 228
20 1.30 307.2 Standard 18.1 66.1 11.6 63.2
14 Fall 2/06/’17 Sarel 1 Truck 12 201 185 1.19 335.9 Sunny 18.3 56.8 9.6 63.3
15 Fall 6/06/’17 General Pico 1 Tractor–trailer 15 480 585 2.45 191.0 Sunny, windy 14.5 56.8 5.8 57.8
Alternately sunny
9 b 1.67 267.7 15.8 61.5 8.3 59.8
16 Fall 7/06/’17 General Pico 2 Truck–trailer 500 569 and cloudy, windy
Table A4. Cont.
Transport Number Type of Number of Same Duration Distance Density Weather Average T in Average Averagera Dew Point Average
Number Season Slaughterhouse Transport Loading Space
Date of Vehicle Horses (Minutes) (km) Conditions RH (%) Temperature (◦ C) THI
Trailers Vehicle m2 /horse kg/m2 (◦ C)
17 c 1.47 303.4 Standard
Alternately sunny
11 1.45 285.9 12.0 52.0 2.4 54.5
17 Fall 12/06/’17 Lamar 2 Truck–trailer 331 338 and cloudy
Animals 2020, 10, 602
16 1.48 280.0 Standard 12.9 50.4 2.8 55.8

Alternately sunny
10 1.50 295.6 14.2 67.6 8.2 57.4
18 Fall 13/06/’17 Lamar 2 Truck–trailer 263 323 and cloudy
19 1.32 337.0 Standard 13.9 65.9 7.6 56.8
Cloudy, windy,
10 1.40 285.8 15.9 70.9 10.5 60.0
19 Winter 13/10/’17 Sarel 2 Truck–trailer 461 380 sunny at arrival
Diamond shaped
16 1.57 255.7
in one surface
Cloudy, moist,
20 Winter 13/10/’17 Sarel 1 Truck 16 d 370 380 1.16 326.8 16.5 67.3 10.3 60.9
sunny at arrival
Cloudy, rainy,
11 f 1.16 351.0 21.4 87.0 19.1 69.6
21 Winter 17/10/’17 General Pico 2 Truck–trailer 632 700 moist
23 1.28 332.0 Standard
Alternately sunny
11 e 1.40 22.4 47.5 10.5 68.0
22 Winter 23/10/’17 Lamar 2 Truck–trailer 175 160 and cloudy, rainy
16 1.55 Standard 21.2 48.6 9.8 66.6
Cloudy, rainy,
23 Winter 24/10/’17 Lamar 1 Tractor–trailer 26 a 200 180 1.48 27.2 40.7 12.7 74.0
sunny at arrival
142
Animals 2020, 10, 602
Table A5. Required space per horse for road transport in Europe [7,43].
Type of Horse Available Space

Adult horses 1.75 m2 (0.7 × 2.5 m)
Young horses (6–24 months) (transport <48 hours) 1.2 m2 (0.6 × 2.0 m)
Young horses (6–24 months) (transport >48 hours) 2.4 m2 (1.2 × 2.0 m)
Ponies (<1.44 m) 1.0 m2 (0.6 × 1.8 m)
Foals (0–6 months) 1.4 m2 (1.0 × 1.4 m)
Table A6. Density per compartment of the transports with deviating density in one or more
compartments. Densities in bold deviate from the norm. * = unknown distribution. T1 = front
loading space; T2 = hind loading space; C1 = first compartment; C2 = second compartment;
C3 = third compartment.
Transport Slaughter Number of Density

Slaughter-House Compartment Fighting
Date Date Trailers (m2 /Horse)
7/11/2016 8/11/2016 General Pico 1 T1 1.72 Average
T1 0.80 Average
15/11/2016 16/11/2016 Sarel 2
T2 1.11 High
T1* 1.54 Barely or not
T2, C1 1.06
14/3/2017 15/3/2017 Lamar 2
T2, C2 2.48 Average
T2, C3 2.23
T1 2.21 Barely or not
20/3/2017 21/3/2017 General Pico 2
T2 1.88 Barely or not
T1 2.45
6/6/2017 8/6/2017 General Pico 1 T1, C1 2.68 High
T1, C2 2.25
T1, C1 1.46
High
T1, C2 1.33
23/10/2017 25/10/2017 Lamar 2 T2, C1 1.74
T2, C2 1.49 High
T2, C3 1.49
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animals
Review
Equine Milk Production and Valorization of Marginal
Areas—A Review
Nicoletta Miraglia 1 , Elisabetta Salimei 1, * and Francesco Fantuz 2
1 Dipartimento Agricoltura, Ambiente e Alimenti, Università degli Studi del Molise, Campobasso 86100, Italy;
[email protected]
2 Scuola di Bioscienze e Medicina Veterinaria, Università degli Studi di Camerino, Camerino MC 62032, Italy;
[email protected]
* Correspondence: [email protected]; Tel.: +39-0874-404-850
Received: 3 December 2019; Accepted: 19 February 2020; Published: 22 February 2020
Simple Summary: The revaluation of equine milk for human consumption is showing an increased
interest from a scientific point of view. As practical relapse of the peculiar characteristics of horse
and donkey milk, and their potentialities as food products, the dairy equine enterprise is developing
worldwide. The milk production can therefore contribute to the whole equine industry, but crucial
factors still need to be elucidated. Aiming to promote advances of knowledge on the dairy equine
enterprise, aspects of management of the dairy horse and donkey are reviewed in the frame of
marginal areas, with a special focus on dam and foal feeding, and welfare, besides milk quality.
Abstract: The equine dairy chain is renewing the interest toward horse and donkey breeding for the
production of milk with potential health promoting properties. The dairy equine chain for human
consumption could contribute to the rural eco-sustainable development for the micro-economies
of those areas threatened by marginalization. As a part of the whole equine industry, and its
possible impact in the modern and future society, the main traits of the equine dairy enterprise are
reviewed with a special focus on management of animals and milk. Equine milk compositional and
nutritional peculiarities are described as also related to milk hygiene and health issues. Scientific
and technical aspects of the feeding management are considered in the frame of the emerging dairy
equine enterprise, where pasture is an essential element that allows to match production goals for
horses and donkeys, biodiversity preservation, as well as landscape safeguard.
Keywords: equine milk; dairy equine chain; dairy equine management and feeding; biodiversity;
landscape; pasture
1. Introduction
Equine breeding represents one of the most promising activities in rural development, which
is considered a key strategy for restructuring the agriculture sector by means of diversification and
innovation [1]. The equine species are involved not only in activities concerning their use for work and
tourism, but also in niche activities related to the production of food and non-food products [2,3]. The
high versatility of the equine species represents a strong argument for the conservation of endangered
equine breeds and populations [4,5]. Many breeds occupy special niches and contribute to the
biodiversity due to their own genetic characteristics, coming from adaptive mechanisms developed in
centuries of evolution in specific local environments [6,7]. Consequently, policies for the safeguard of
endangered equine breeds and autochthonous populations have been developed, also considering the
recovery of the relationship among humans, animals, and territory, as a ’system integrator’ of the rural
eco-sustainable development [8,9]. The renewed interest toward equine milk and derivatives is today

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sustained by the emerging dairy equine enterprise, which is developing in France, Italy, Mongolia,
China, Kazakhstan, Kirgizstan, Greece, Germany, and many other countries [10–12].
As a part of the whole equine industry and its potential impact in the modern and future society,
the equine dairy enterprise is described in its main traits. Based on peculiarities of equine milk for
human consumption, the essential features of management of animals and milk are reviewed in the
context of marginal areas. Aspects of nutrition of the dairy equids are examined in the frame of those
areas where pasture and natural meadows represent the main land use, as a further contribution to
landscape safeguard.
2. Equine Milk: Properties, Potentials, and Benefits

The nutritional and therapeutic peculiarities of equine milk are known since ancient times, as
Hippocrates [13] and Herodotus [14] described in the 5th century BC. Moreover, the consumption of
koumiss (or airag), i.e., a traditional drink made in Central Asia, according to a nomads’ recipe [15], is
reported in literature, not only as an ingredient of the traditional “white diet” of the Mongolian steppes
population [16], but also as a popular remedy for a variety of diseases [17,18]. The traditional use of
donkey milk is also reported in China and South America for the treatment of many illnesses [19].
Recent scientific findings on the equine milk compositional peculiarities and their potential
health promoting properties have increased interest toward its use for human consumption,
especially for sensitive consumers, such as children with allergies to cow’s milk protein, as well
as immunocompromised or debilitated people [10,20]. In Europe, the dairy equine enterprise started
up in France as part of a project on animal diversity preservation [21], and spread out in many marginal
areas of the world where these monogastric herbivores are well adapted to difficult environments,
with scarce availability of forages, often of poor-quality. Today, equine milk is mainly marketed for
human consumption as raw, pasteurized, or freeze-dried [22], and as fermented derivatives [15]. In
Italy, the price of donkey milk ranges from 9 to 15 €/L of raw milk, 14 to 17.5 €/L of pasteurized milk,
and 27.5 to 36 €/100 g of powdered milk, either spray dried or lyophilized [23,24]. Equine milk is
also used in the non-food sector, as an ingredient in cosmetic products [25]. Data on the worldwide
production of equine milk are not available, but equine milk has been reported to be consumed by
30 million people [26]. It should also be considered that consumer cognizance of equine milk and
derivatives is so far limited, as well as common awareness of its local availability [18,27]. Besides
communication gaps to be overcome, the emerging niche market of equine milk raises questions on
appropriate management strategies of dam and foals, as mainly related to animal nutrition as well as
environmental issues, besides food security and animal welfare.
2.1. Equine Milk Compositional and Nutritional Features

Table 1 summarizes the average horse and donkey milk gross composition and energy content
from the recent literature. Values from human and cow milk are given for comparison. It should be
considered that equine milk components are mainly affected by nutrition, length of lactation, and
health status of the mammary gland, besides genetics. Equine milk has a high water content and
shows a lower fat content than human and cow milk (Table 1). The milk fat globules diameter, likely
related to lipid digestibility, is reported to be lower than in human and bovine milk [28].
Horse and donkey milk are closer to human milk in terms of both protein and lactose content than
cow milk (Table 1). On this regard, it is worth noting that, although about 50% of the world population
is lactose intolerant, the daily intake of 14 g of lactose is usually well-tolerated [28]. Moreover, the
content of lactose is lower in fermented milk. From a nutritional point of view, it is also important
to highlight the hypocaloric content (Table 1) that makes equine milk an inadequate food for infants,
when not supplemented with vegetal oil (about 40g L−1 ) [28].
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Table 1. Average milk gross composition and energy content from different species 1 .
Item Horse Donkey Human Cow

Total solids, g kg−1 103.1 95.3 125 127
Fat, g kg−1 10.3 7 35 41
Protein, g kg−1 16.8 16 12 34
Lactose, g kg−1 63 66 64 48
Ash, g kg−1 4.2 4.1 1.9 7
Gross energy, MJ kg−1 1.98 1.75 2.69 3.19
1 Sources: [15,26,28–31].
The ash content of equine milk (Table 1), which is intermediate between human and cow milk,
shows a decline throughout the lactation consistent with Ca and P concentrations in milk [15,20].
Although the absolute values of Ca and P in equine milk are reported variable, and in average higher
than in human milk, the Ca:P ratio is reported to be in average 1.3 and 1.72, respectively, for donkey
and horse milk, while it accounts for 1.7 and 1.23 in human and bovine milk, respectively [31–33]. As
a further dietary consideration, the mineral content of milk is not reported to be influenced by the
maternal diet in mammalians, except for Se and I [34,35].
Pieszka et al. [36] reviewed the level of fat-soluble vitamins (A, D, E) in mare milk and found
them consistent with values reported for bovine milk. Donkey milk is reported to contain a higher
level of vitamin D [37] but it displays very low contents of vitamins A and E, as probably related to the
low-fat content [28]. Among the water-soluble vitamins, pyridoxine, pantothenic acid, cobalamin, and
vitamin C have so far been detected at high levels only in mare milk [28,36].
After the first clinical evidences on the successful use of equine milk in children with multiple
food allergies, reported in 1992 and 2000, respectively, for donkey and horse milk [38,39], donkey
milk has mainly been the subject of numerous studies about its use in the diets of children affected
by cow’s milk protein allergy, thanks to its high palatability, due to the high lactose content, and
low allergenicity, related to the nitrogenous components [28,40]. Equine milk with high hygiene
characteristics, and properly supplemented from a nutritional point of view, has been confirmed as
a promising alternative in the dietary treatment of children affected not only by Immunoglobulin
E-mediated cow’s milk protein allergy, but also by food protein-induced enterocolitis, occurring in the
first six months of life [40,41]. However, findings on the efficacy of equine milk use in the fulfilment of
nutrient requirements of children cannot be so far considered conclusive, as they need to be confirmed
by larger studies [27]. For these reasons, the use of donkey milk is nowadays considered an ingredient
in a solid-food diet, or after the first year of life for children [42].
In regards to the allergenicity of equine milk, the proteomic profile of equine milk has been
extensively studied in recent years [43,44], and microheterogeneity is displayed due to genetic variants
and post-translational modification [28]. A significant effect of the breed and stage of lactation on
gene expression and milk composition, and the association among genetic polymorphisms, gene
expression, and milk protein and fat contents have also been observed in mare milk [45–47]. This
leads to the relevant role of the dairy equine enterprise in the survival of equine breeds, e.g., Lipizzan,
Icelandic, German Warmblood, Akhal-Teke, Franches-Montagnes, Comtois, Italian Heavy Draught,
Russian Heavy Draft, and Polish Coldblood among horses; and Poitou, Zamarano Leonés, Burro de
Miranda, Ragusano, and Amiata among donkeys, and in the preservation of environment, landscape,
and vegetal diversity of areas where they are adapted to live [2,24].
2.2. Functional and Bioactive Compounds

Milk, besides allergens, is a source of many bioactive and functional compounds, i.e. metabolites,
enzymes, hormones, trophic, and protective factors that are involved in proper growth and nutrition
in newborns [48], or in proper secretion of the mammary gland [49].
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Among the bioactive and functional proteins detected in milk, there are enzymes active in
protection against protozoa, bacteria, and viruses, e.g., lysozyme and lactoferrin [48]. Lysozyme
accounts for 10.5% and 21% of whey proteins, respectively, in horse and donkey milk, but only 5.5% of
whey protein in human milk; on the contrary, a higher level of lactoferrin (26.6% of whey protein) is
detected. Lactoferrin in horse and donkey milk accounts, on average, for only 7% and 4.48% of whey
protein, respectively [11,50,51]. Lysozyme activity was found unaffected by thermal treatment at 72 ◦ C
up to 3 min [52,53].
Other enzymes in milk are of technological relevance, such as alkaline phosphatase representing
an index of pasteurization efficiency, with activity reported to be about 100 mU L−1 in thermal treated
equine milk [22].
In mammalian milk, hormones and growth factors have also been detected and classified as
bioactive peptides [54] derived from the maternal metabolism. Among them, there are leptin, insulin,
ghrelin, Insulin-like growth factor 1 (IGF-1), and thyroid hormones that are involved in the central
regulation of food intake and in the maintenance of energy balance. Their role in milk may be related
to the regulation of growth, to the development and maturation of the neonatal gut, and of the immune
and neuroendocrine system of the newborn [48]. Considering the species-specificity of many proteins,
it is worth noting that leptin has been measured as human equivalent in both horse and donkey
milk, and human-like ghrelin, IGF-1, and triiodothyronine (T3) were measured in donkey milk [15,20].
It is worth noting that the milk T3 content was affected by the diet in lactating donkeys [55]. The
role of variations in the maternal hormone status of equids, as related to both physiological status,
and how intensive husbandry strategies might interact with their adaptive capacities in the farming
environment, deserves attention and needs to be further considered.
Bioactive peptides are also encrypted in the sequence of milk proteins and are released from them
following enzymatic proteolysis, under gastrointestinal digestion or during fermentation. These dietary
components exert health promoting, i.e., antimicrobial, antihypertensive, antioxidant, antithrombotic,
immunomodulatory, antiproliferative, and opioid activities in the organism, beyond their nutritive
value [56–59]. It should be noted, however, that technological treatments carried out to prolong milk
shelf life could considerably affect structure, as well as the functional and nutritive properties of milk
components, especially peptides and proteins, that might lead to a greater susceptibility to infection
and/or the development of allergies [60].
In immunonutrition, the antioxidant properties of nutrients, such as alpha-tocopherol and
beta-carotene are known, but increasing scientific evidence suggests the role of dietary lipids in the
regulation of neonatal immune function and in the severity of symptoms of allergies [61]. Recent
studies show the interesting free fatty acids profile of equine milk, with saturated fatty acids content
(50%) lower than that reported for goat and sheep milk, and a higher proportion of monounsaturated
fatty acids and polyunsaturated fatty acids (PUFA) than ruminant milk [28]. A balanced ratio between
n3PUFA and n6PUFA, respectively considered anti-inflammatory and pro-inflammatory nutrients, is
reported for horse and donkey milk [62]. Moreover, in regards to the variability of the lipid fraction,
Martini et al. [63] observed an increased content of oleic, palmitoleic, and vaccenic acids considered
with a positive effect on human health, and a lowered concentration of stearic acid in donkey milk
samples collected in the winter.
The atherogenic and thrombogenic indices, calculated on fatty acid composition, candidate equine
milk as an interesting food for people with allergic and inflammatory conditions [28,64]. Moreover, the
fatty acids profile detected after in vitro digestion shows significant differences depending on the milk
sources, with a prevalence of saturated fatty acids released from both human and donkey milk [65].
Heat damages have been observed in donkey’s milk on functional lipid compounds, which may also
directly and indirectly influence gut environment and immunoinflammatory functions [66,67].
The recent advances of knowledge on the claimed nutraceutical properties, here summarized,
suggest that, when scientifically demonstrated, the added value of the equine milk should be properly
exploited in the dairy equine enterprise.
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3. Dairy Equine Management and Nutrition
3.1. Equine Milk Yield and Management of the Dairy Equine Enterprise
The core of the dairy equine enterprise is related to the management of dams and foals, and
of the milking practice, showing important differences from the conventional dairy species. Firstly,
dams and foals live together until weaning, which occurs at 7 months (for foals) or later; dams won’t
start to be milked before 20 d from foaling [10,68]. Secondly, since the equine mammary gland is
characterized by small volume, and milk is mainly alveolar [69], milk harvesting can be carried out
many times per day. In the Steppes of Central Asia, mares are milked 4–5 times per day [70], while in
more intensive dairy farms located in Europe, mares and jennies are frequently milked depending on
consumer demand, up to eight times a day [10,62,71]. Milking is carried out at least 2 hours after foal
separation from the mother [70,72]. This distinctive trait of the dairy equine enterprise introduced the
neologism “milking session”, i.e., the interval from foal separation up to the end of each milking [62].
It must be noted that milk ejection is not reported to be affected by the presence of the foal during
milking in the dairy donkey farm [68], while it is recommended in the dairy horse farm for a complete
oxytocin release [10]. In this regard, the selection for milkability of mares would greatly improve the
milking routine, reducing the labor costs [73].
Milk harvested per milking session is reported to range within 500–2000 mL and 200–900 mL for
mares and jennies, respectively [62,70,74–77], regardless of the milking technique used (mechanical or
manual).
The available literature data on daily equine milk yield have been obtained under different
methodological approaches, which partially explains the high variability of values reported in Table 2.
The daily milk production is estimated to be 15–35 g kg−1 bodyweight [10,29,78,79]. However,
literature data are inconclusive, as the value recently estimated for the dairy donkey, i.e., 12 g milk
kg−1 body weight, shows [80]. Todini et al. [81] reported an average milk yield per milking of 2.68 mL
kg−1 bodyweight.
Table 2. Daily milk yield (kg/d) reported in literature for horse and donkey from d30 to d180 of
lactation 1 .
Item Horse Donkey

Mean value 11.66 2.68
s.d.2 5.3 1.96
Min 3.9 0.72
Max 17.2 6
1 Sources: Horse: [15,80,82,83], Donkey: [23,25,30,31,72,76–80,84]; 2 standard deviation.
Milk yield is affected by many factors, including the farming system, nutrition and feeding,
strategy and type of milking (manual or mechanical), individual milkability, stage of lactation, and size
and body condition of animals, besides genetics [11]. Because of the lack of standardized methodologies
in equine milking studies, the effect of the breed on dairy performances of mares and jennies is not
currently defined. According to Doreau and Martin Rosset [10], any breed can be milked, provided the
animals accept the milking procedure.
The farming system is a major cause of the observed variability in equine milk production, as
reported for pastoralist areas of the Steppes of Central Asia [70,85], or for more intensive systems,
described for both koumiss and dairy donkey farms. In the latter, shelters are available on pasture, and
milking is usually carried out in dedicated areas or facilities [72,83]. Donkeys raised under temperate
conditions are reported to need more protection in rainy and windy weather than horses, as the
results of the adaptation of donkeys to semi-arid environments of Africa vs. continental climate, and
Eurasian Steppe environments where horses evolved [86]. The grazed area must be close to the milking
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site [70,79] so that the proximity of pasture represents a constraint in the dairy equine enterprise and
management of milking influences the feeding strategy.
In intensive farming systems, the dairy mare and jenny are milked in ad hoc facilities equipped
with sheep milking machines adapted to the equine mammary characteristics [15,68]. With trained
animals and skilled operators, no difference was observed in the amount of milk harvested manually
or mechanically per milking session, but milk microbial contamination can be reduced by the proper
use of milking machine. This introduces a crucial aspect of the equine milk production and its
commercialization, related to consumer safety.
3.2. Equine Milk: Hygiene and Health Issues

In Europe, equine milk is mainly commercialized at farm or by means of vending machines (raw
milk), but it is also available at shops and supermarkets (pasteurized milk) or online (pasteurized and
powdered milk) [22,71,87,88].
The risk associated with equine milk consumption is considered reasonably low when compared
to bovine milk. The presence of pathogens, such as Escherichia coli O157, Salmonella spp., Campylobacter
spp., Yersinia enterocolitica, Brucella spp., Mycobacterium spp., Bacillus cereus, Cronobacter sakazakii,
Streptococcus equi subsp. zooepidemicus, Rhodococcus equi, Streptococcus dysgalactiae subsp. equisimilis,
Clostridium difficile, and Burkholderia mallei is reported to be low [87]. However, the variable level of
microbial contamination of equine raw milk, ranging from 3.0 to 5.87 log CFU mL−1 milk, warns
against ineffective sanitization of equipment and facilities, as well as packaging and storing conditions
of milk, even after thermal treatments [22,29,87].
For these reasons, while alternative processing for equine milk sanitation and shelf life extension
are studied [22], thermal treatment is always recommended before consumption [87].
In regards to the mammary gland health status, the somatic cell count is reported to be below
50,000 cells mL−1 milk and mastitis is rarely observed in the dairy equine farm [29,87,89]. However,
injuries or improper milking procedures reported for more intensive farming systems can affect the
mammary health status [31,87].
Equine milk is gaining interest as an alternative food for sensitive consumers, so that high hygiene
standards represents an important issue in the dairy equine enterprise, and it affects the labor costs for
cleanliness of facilities, and areas frequented by the animals.
3.3. Feeding the Dairy Equine and Pasture Management

The nutritive value and the potential health-promoting properties of equine milk are related
to the horse and donkey’s metabolic utilization of the diet. These monogastric species and hindgut
fermenter herbivores are reported to be better utilizers of metabolizable dietary energy than ruminants,
at high levels of cell wall [90]. It is also well known that the dietary influence on milk composition
is more direct in the equine species than in ruminants [10,90]. Regardless of the farming system,
as already mentioned, the common denominator in the diet is the presence of forages and pasture,
whose management is crucial for dairy equine production and welfare [71,72,74,91,92]. Because of the
evolutionary history of the two equine species, their different feeding behavior and metabolism should
also be considered in relation to nutrient requirements, management of feeding, and their impact on
land preservation.
3.3.1. Feeding the Dairy Horse

According to Doreau and Martin-Rosset [10], no different approaches are required in feeding the
dairy or nursing horse, as far as the energy and nitrogen requirements are concerned. The nutritional
requirements of the lactating mare (600 kg body weight) are summarized in Table 3 [93]. To sustain the
milk production, forages account on average for 50 to 80 percent of the dry matter of the diet, and
they can supply 40 to 70 percent of the mare’s annual nutritional requirements [5,94]. The dry matter
intake of mares (Table 3) depends on the quality of the diet at foaling [93]. At the onset of lactation, the
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voluntary intake of mares is reported to be high (20–30 g dry matter per kg body weight). However,
the dry matter intake is scarcely a limiting factor for the mare to meet nutritional requirements [93].
Table 3. Recommended nutrient requirements and intake for lactating mares (600 kg body weight) [93].
Lactation, Milk Yield, Horse Feed Units *, Horse Digestible Crude Dry Matter Intake,
Month kg d−1 n d−1 Protein **, g d−1 kg d−1
1st 18 10.1 1131 13.5–18.0
2nd 19.8 10.3 1091 15.0–19.0
3rd 19.2 9.6 1030 15.0–19.0
4th 17.4 9.1 844 13.5–18.0
5th 13.2 7.9 629 12.5–15.0
6th 12 7.6 603 10.5–13.0
* Horse Feed Units (UFC) = 9.42 MJ Net Energy; ** Horse Digestible Crude Protein (MADC).
The diet composition varies according to quality and availability of pasture and forages. In case
of good grassland conditions, dairy mare foals generally in spring, just before they turn out, and use
natural or sown pasture during the grazing season [95]. They are generally dried up in autumn (early
October), after 190–210 days of lactation. During winter (110–120 days), the mares are fed a limited
amount of hay of medium quality (organic matter digestibility, OMD = 50–55%) [93], and cereals, or a
mixed diet based on straw, ad libitum, and hay of good quality (OMD = 55–60%). In case of harsh
conditions, mare foals generally in early spring, one month before turning out. They graze pastures of
uplands. They are dried off in autumn (late October), and grazed resources meet the requirements of
animals over 9 months of lactation. In case of low productive areas, mares graze for about 60–70% of
the total grazing season. In late autumn and early winter, mares graze refusals of cattle and sheep in
the lowlands [93].
In the dairy horse enterprise, the strategy of the feeding system is based on pasture availability
throughout the year and consists, generally, in matching the highest requirements of the animals
with the maximum biomass production [96]. It must also include provisions of preserved feedstuffs
to be used in case of particularly adverse climatic conditions. The main aim of the feeding strategy,
notwithstanding the horse breed, is that dairy mares gain body weight in early lactation to nurse
adequately the foal and to be rebred as soon as possible, to achieve a 12-month interval between two
subsequent foalings [94]. Foals live with their mothers at pasture and they are allowed to nurse when
mares are not milked. Table 4 shows the nutrient requirements of foals (600 kg of adult body weight)
performing an optimal or moderate growth rate.
Table 4. Recommended nutrient requirements and intake for foals (600 kg adult body weight) at 3–6
months of age with a growth rate optimal or moderate [93].
Body Weight, Horse Feed Units *, Horse Digestible Crude Dry Matter Intake,
Gain, g d−1
kg n d−1 Protein **, g d−1 kg d−1
249 1000–1200 6 647 6.0–8.0
207 800–900 4.8 497 5.5–7.5
* Horse Feed Units (UFC) = 9.42 MJ Net Energy; ** Horse Digestible Crude Protein (MADC).
Mares should be managed at pasture with the aim to recover body weight and a proper body
condition at drying off in late summer or fall, to ensure good nutritional conditions in pregnancy during
winter [93]. Mares increase their body weight (+6–8%) during the last three months of pregnancy, as
they are usually fed from 100% to 120% of their energy requirements, and during the first month after
foaling (+3%) when they turn out in spring [93].
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In extensive farming systems, grazing dairy mares should meet 80% of the total requirements
over the 7-month lactation period. The animals use the vegetation regrowth from September until
December. As already mentioned, in more intensive farming systems, hays or maize silage (30–35%
dry matter content, 0.80–0.84 Horse Feed Unit per kg dry matter) and low concentrate supplementation
are offered during winter.
3.3.2. Feeding the Dairy Donkey

Nutrient requirements and suggested allowances, nowadays available specifically for donkeys, are
mainly devoted to working animals, i.e., used for transportation, small agricultural works, and equine
therapy, and to animals at maintenance, i.e., companion animals, often castrated, hosted in international
animal rescue charities, e.g., Donkey Sanctuary [72,79,97,98]. The available nutrient requirements of
the dairy donkey are either based on results from one study on foal growth, or they are derived from
domestic horse data, whose behavior and physiology are known to differ from those of the donkey.
Consequently, they cannot be considered conclusive and need further investigation [20,72,79,98,99].
As reported by the US Research Council on equids, donkeys maximize their dry matter intake when
good quality hay is offered [100]. For donkeys at maintenance, Raspa et al. [79] report a maximum dry
matter intake per kg of body weight, declining from 32 to 12 g, with ad libitum diets based on either
legume forages or barley straw. In order to prevent nutritional diseases, e.g., hyperlipemia and obesity,
a diet high in fiber is suggested for companion donkeys at maintenance [99].
Because the mentioned lack of information in the specific literature on protein and energy
requirements of lactating donkeys [20,72,79,98], the common strategy is represented by ad libitum
administration of forage-based diets associated to a monthly evaluation of the body condition
score [31,79].
In the dairy equine farming system, pasture should be always available for its positive effect on
animal welfare and milk quality [79]; however, when jennies are milked, grazing time and quanti-
qualitative availability of grazed areas are limited, as also observed for dairy mares [70]. According
to preliminary results on the grazing behavior of Miranda breed jennies in mountain pastures,
Couto et al. [101] observed that the activities in searching and prehension lasted, on average, 16 hours
per day with a preferential intake of herbaceous species. However, up to 30% of the intake was
represented by shrubs, probably because of a low grass availability. This suggests the interesting role
of these autochthonous donkeys in preserving the pasture areas from degradation and fire risks [101].
It is also worth considering that grazing time does not significantly affect the daily dry matter intake of
donkeys at maintenance, when they also have free access to preserved forages [102].
Results of a survey carried out on 12 dairy donkey farms in Italy confirm the inclusion of pasture
in the lactating donkey diets always associated to hay administration [72], likely due to the limited
availability of pasture. Cereals and/or mixed feeds, commercial or not, are also administered to
lactating donkeys, and diets are frequently salt supplemented [31,72].
Other data from on field studies about milk production report a high feed intake (30–32 g dry
matter per kg body weight) of dairy jennies at the first 3–4 months of lactation. Moreover, diets are
characterized on average (on a dry matter basis) by a 70:30 forage-to-concentrate ratio, a protein content
of 10–13 g per 100 g, and a digestible energy value of 8.5–10.0 MJ per kg [20].
After digestion, dietary fats, soluble carbohydrates, and proteins are mainly absorbed by the small
intestine of equids. Due to the negligible biohydrogenation before absorption, the direct influence
of the diet on the fatty acid composition of milk is expected. In this regard, the supplementation
of the mares’ diet in late pregnancy, and early lactation with eicosapentaenoic acid (EPA) and/or
docosahexaenoic acid (DHA) did not affect the linoleic and linolenic milk content, but it increased the
arachidonic acid, EPA, and DHA milk concentrations [103]. However, in jennies, the transfer of n3
polyunsaturated fatty acids (PUFAs) from blood to milk is reported to be more efficient than that of n6
PUFAs [20]. For a nutritionally correct ratio of n3:n6 PUFA in equine milk, dietary lipid sources should
be evaluated with regard to the fatty acid profile. Dietary factors can also influence the palatability
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of donkey milk. ‘Green’ aromatic notes and related compounds have been identified in milk when
jennies were fed fresh forage [20].
The survey by Dai et al. [72] reported that the diet always includes hay (100% of farms) and pasture
(about 92% farms) in non-lactating jennies. Concentrates and salt supplements are also administered,
but in a lower percentage than during lactation [72]. Stallions, which are either grouped with females or
housed individually, are mainly fed hay supplemented with mixed feeds, cereals, and salt or additives.
Pasture availability is reported for about 70% of farms [72].
Nutrient requirements for foals are not defined and only rare data are available on growing rates
in donkeys [104]. The administration of milk replacement formulas to foals is not common in the dairy
donkey farm [72]. However, a highly digestible creep feed is usually distributed to nursing foals until
one month of age, when dams are not milked [20]. Later, complementary feeds are administered to
foals until weaning (7–12 months of age) [72]. Constant access to clean water and salt blocks is highly
recommended for both foals and dams [20].
The welfare status of the animals needs to be constantly monitored by recommended indicators,
such as body condition score, and hydration score [105]. Vaccinations and deworming are also
recommended in all animals, along with regular hoof, dental, and health care treatments [105], even if
they are not reported to be common practices in dairy donkey farms [72].
3.4. Pasture in the Dairy Equine Enterprise

Different systems of grazing management are possible: extensive vs. semi-extensive, associated
or not with ruminants [106]. Depending on the grazing species and their nutrient requirements, the
correspondence between animals and characteristics of the forage availability (in quality and quantity)
is crucial for a sustainable use of the landscape [107]. As herbivore species, horses and donkeys have
the ability to exploit large amounts of fibrous forages, often of low nutritive value in less favored areas,
available for grazing and/or foraging. The ability of the equids to produce in high forage feeding
systems is mainly explained by their distinctive features in selecting, consuming, and digesting forages
and grazed resources [102], so that seasonal variations in grazing behavior and diet selection have
been observed in mares [108]. In particular, equids show several adaptive abilities in harsh conditions
when the total nutrient requirements can be achieved on a long-term period [102].
In free-ranging conditions, horses spend up to 70% of their time to consume available food
resources and only 30% for other activities. This ingestive activity is usually distributed over several
meals during the day and grazing also occurs during the night [70,109]. Moreover, grazing time can
increase in autumn and in winter, and the length of grazing is in relation to the cell wall content of the
sward [110].
In high forage systems, pasture is the major source of nutrients for dams and foals along the
breeding cycle. Based on the type and composition of the grazing species, as well as on the carrying
capacity of the pasture, the sustainable grazing period ranges from 100 to 130 days (Northern Europe),
to 230 days (Central Europe) [5]. Especially in marginal areas, the most relevant management of horses
and donkeys at pasture implies the evaluation of the nutritive value of forages available for the optimal
animal response to match economic profits, technical feasibility, and ecological sustainability [107].
Pasture productivity varies according to the geographical zone and the climatic conditions. In
Europe, the grazing period is usually limited in Northern countries by short summers. In countries
of Central Europe, generally characterized by extensive grazing lands and high quality forages, long
grazing periods are observed, while the grass growth is usually depressed by summer dryness in
Southern Europe [5]. Table 5 shows the average chemical composition of pasture in selected areas
of Europe.
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Table 5. Chemical components and estimated energy content of pasture in European areas. Values
expressed on a dry matter basis [5,111].
Country Crude Protein, g kg−1 Crude Fiber, g kg−1 Horse Feed Units *, n kg−1
Finland 200–230 180–200 0.69–0.73
France, lowlands 131–168 244–276 0.76–0.82
France, uplands 111–166 223–304 0.66–0.92
Italy, lowlands 85–159 242–325 0.67–0.90
Italy, uplands 117–155 285–345 0.63–0.85
* Horse Feed Units (UFC) = 9.42 MJ Net Energy.
In addition, it should be noted that climate changes may affect the forage population dynamics,
its nutritive value, as well as the growing and grazing seasons, so that different approaches in the
management of land, animals, and forage resources may be required [112]. The forage intake depends
on the quality of plant resources and their ingestibility, the time of grazing, the grazing activity,
and the stocking rate, especially in multispecies herding situations. On this purpose, practical and
flexible models have been studied for the assessment of a grazing pressure compatible with the
conservation of pasture in less favored areas [113]. Moreover, the adoption of appropriate strategies is
also recommended, such as rotational grazing, control of infesting species, safeguard from parasites
diffusion in the sward, and fertilization (180 kg N ha−1 ) [107,108,114].
In extensive systems, characterized by low quality and poor productivity of natural pastures,
the total requirements can be met using low grazing intensity, with a stocking rate of 0.3–0.7 animal
ha−1 , depending on the grass availability [106,114]. In more intensive systems of Central Europe, a
concentrate supplementation is offered to horses, depending on their activity. Grass is plentiful until
the beginning of July (beginning of the third vegetation cycle), then the production declines from
mid-July to the end of August [94]. In Mediterranean regions, depending on the geographical area, the
grazing season starts between April and May. A considerable reduction of the grass production is
observed, depending on the variable rainfall in July, August, and early September. Then, up to the end
of October, a regrowth of the grass can occur, offering the availability of fresh forages to foals in the
weaning period [5].
The average growth of pasture grass during the grazing period in Italy is shown in Figures 1
and 2, respectively, for Central and Southern Italy pastures. It is interesting to note that in the pasture
area located in Southern Italy, and in a mixed grazing system, including cattle, sheep goat, and horse,
the sustainable stocking rate simulated in two subsequent years varied from 1.14 to 1.35 Adult Bovine
Unit ha−1 , due to different climatic conditions and carrying capacities of the pasture [107].

NJGU\PDWWHUKD G

PRQWKV0D\2FW
*UDVVJURZWK
Figure 1. The average growth of pasture grass in Central Italy during the grazing period (modified
from [94]).
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Animals 2020, 10, 353

NJGU\PDWWHUKD G

PRQWKV0D\2FW
*UDVVJURZWK
Figure 2. The average growth of pasture grass in Southern Italy (modified from [94]).
In order to achieve biodiversity and production goals in sustainable grazing systems of less
favored areas, further management strategies include the reduction of the stocking rate, the periodic
exclusion of the more degraded areas from grazing, the administration of complementary hay and
concentrates, as well as the use of autochthonous breeds [115,116].
As a final consideration, the dairy equine enterprise, here described in its essential and promising
traits, is the result of different environmental conditions, management strategies, and socio-economical
aspects. Furthermore, no data on the evaluation of the economic impact of the dairy equine milk
production are available in literature. However, besides labor, feeding, housing, and milking facilities,
the evaluation of costs should also include those related to availability of infrastructures on pastures
and marginal areas, and social costs of labor and bureaucracy, whose incidence can be relevant and
different among countries [92]. Moreover, among the immaterial benefits, the impact of the dairy
equine enterprise to environmental issues, such as landscape safeguard and biodiversity preservation,
should also be included in a costs-to-benefits ratio evaluation [117], as also reported for horses used for
tourism and work [3]. Alternatively, a price premium, based on environmental standards and labels,
should be recognized to the products of the dairy equine enterprise.
4. Conclusions
The dairy enterprise involving equids, here discussed in its essential traits, represents a promising
activity for the micro-economies of marginal areas around the world, because of its potentialities
in human nutrition, biodiversity, and landscape preservation. Notwithstanding the advances of
knowledge on milk nutritional and safety characteristics, as well as the improvement of technical
skills in milk management, in depth studies are still required, especially in terms of animal nutrition
and feeding. A better understanding on nutrient requirements of the dairy equid at pasture in
heterogeneous and marginal areas will boost the interest toward endangered equine breeds, their
milk, and their habitat. Positive relapses would in fact include the protection of plant diversity in
the achievement of a productive and sustainable use of the landscape. Among the innovations for
sustainable agriculture, the production of equine milk and derivatives with high nutritional value
and health promoting properties should be therefore considered a promising extension of the equine
industry for the modern and future society.
Author Contributions: Conceptualization, N.M., E.S. and F.F.; methodology, E.S., N.M. and F.F.; writing—original
draft preparation, E.S., F.F. and N.M.; writing—review and editing, E.S. and F.F.; supervision, E.S., F.F. and N.M.
All authors have read and agreed to the published version of the manuscript.
157
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animals
Review
Green Assets of Equines in the European Context of
the Ecological Transition of Agriculture
Agata Rzek˛eć 1 , Céline Vial 1,2, * and Geneviève Bigot 3
1 Research Unit MOISA (Marchés, Organisations, Instituts et Stratégies d’acteurs)-French National Research
Institute for Agriculture, Food and Environment (INRAE), CIHEAM-IAMM, CIRAD, Montpellier Supagro,
Univ Montpellier, 34060 Montpellier, France; [email protected]
2 Pôle Développement, Innovation, Recherche-French Institute for Horse and Horse Riding (Ifce),
61310 Exmes, France
3 Université Clermont Auvergne, AgroParisTech, French National Research Institute for Agriculture,
Food and Environment (INRAE), VetAgro Sup, Research Unit Territoires, 63000 Clermont-Ferrand, France;
[email protected]
Received: 30 October 2019; Accepted: 2 January 2020; Published: 8 January 2020
Simple Summary: Equines have a peculiar place in our society. From livestock to sport, through to
landscape managers and leisure partners, equines show a wide range of little-known environmental
advantages and assets. Today’s wake-up calls about the environment are progressively putting
pressure on stakeholders of the agricultural sector, including the equine industry. This study focusses
on the main environmental consequences of equine use and possession in Europe based on scientific
and technical sources under the lens of five leading sectors where equines show unique impacts as
green assets. Now, more than ever before, it is important to highlight the role of equines as a green
alternative in political debates and management practices to give them the place equines deserve in
the ecological transition of agriculture.
Abstract: Despite the decline of equine populations in the middle of the 20th century, the European
horse industry is growing again thanks to economic alternatives found in the diversification of the
uses of equines (sports, racing, leisure, etc.). Equines have many environmental advantages, but the
fragmentation of the sector and the lack of synthetic knowledge about their environmental impacts
do not enable the promotion of these assets and their effective inclusion in management practices and
European policies. To highlight the equine environmental impacts, a literature review was carried out
to cover the main European stakes. This work led to the identification of five “green assets”, fields
where equines show unique environmental advantages compared to other agricultural productions.
These green assets are linked to the nature of equines (grazing and domestic biodiversity), to their
geographical distribution (land use), and to their use by human beings (tourism and work). Today,
when searching for sustainable solutions to modern environmental issues, the use of equines is a
neglected green alternative. Better knowledge and use of equine green assets could partly respond to
more ecological agricultural needs and contribute to the development of this animal industry, which
has a place in regional development and in Europe’s sustainable transition.
Keywords: equine; horse; environment; green assets; land use; equine grazing; domestic biodiversity;
equine and equestrian tourism; equine work; multifunctional review
1. Introduction
In the European Union (EU), after World War II, equine numbers declined drastically because of
the motorization of transport (estimates generally agree that horse numbers decreased approximately

Animals 2020, 10, 106
90% in Europe by the 1950s [1]). For example, in France, the total number of equines was evaluated
to be three million at the beginning of the 20th century but was less than half a million at the end
of this century (Figure 1). Before 1950, horses were largely used for agriculture, transportation, and
the army. This was particularly the case for heavy (or draft horses) (represented by the light color
in Figure 1), but also for saddle horses (represented by the dark color in Figure 1). The European
community was built after the Second World War to maintain peace and ensure the autonomy of its
inhabitants regarding basic necessities, particularly food products. To this end, a Common Agricultural
Policy (CAP) (which still exists today) was created to improve agricultural production, first to improve
cereal yields and then to improve animal productions. However, equines were not included in the
CAP’s plans. In this post-war period, the market situation was geared toward productivism, where the
disinterest in equines as a source of power and their absence in development policies led the European
equine population to collapse. In France, the decline of heavy horses led to the construction of national
programs in 1980 to develop meat production [2,3]. After 1970, saddle horses began to be used for
other purposes (sport and leisure), which explains the progressive increase in their numbers (Figure 1).
This trend was similar in Sweden. In 1920, there were 700,000 horses; then, their numbers decreased to
around 95,500 in 1980 before increasing again [4]. However, today, the equine population is still lower
(48%) than it was in 1920 (362,700 heads in 2010) [5]. However, the recent increases in equine livestock
have not been observed in Mediterranean countries: 87% of horses were lost in Greece between 1983
and 2000, and 31% and 36% were lost in Spain and Portugal, respectively, between 1987 and 2000 [6].
This population decline could be linked to difficulties in national economies during this period.
Today, Europe has 88.4 million cattle, 150 million pigs, 86.8 million sheep, and 12.7 million goats to
ensure the animal protein needs of the European population (Eurostat, 2017), but Europe has only six
million equines [7], according to the European Horse Network (a non-profit network of stakeholders
acting at the world, European, national, or regional level within the European equine sector).
Figure 1. Evolution of the horse population in France from 1800 to 2010 (translated from French [8]).
Since the end of the 20th century, the equine industry has undergone significant evolutions linked
to the diversification of equine uses: first, in terms of sports, racing, and leisure; and second, in terms
of meat and milk productions, traction, therapy, and even companionship. This gave rise to debates
on the status of equines (as a farm animal or pet) between European countries but also inside each
country. For example, in the United Kingdom, horses are seen as companion animals, whereas in other
European countries—France, Germany, or Sweden for example—equines are considered livestock [9].
These issues are particularly problematic because the European agricultural census could be
a powerful tool to quantify the equine population at the European level, but today, this census
underestimates the equine population because only equines kept by farmers are counted (EU Regulation
(EC) No 1166/2008 19.11.2008), whereas, for example, in France, only half of the equine livestock was
kept on farms in 2010 [10]. This problem is similar in other European countries (in Germany, for
example) [11]. No other database exists at this scale, so the current official figures are misleading.
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To counter this, the European Union required the creation of a central national database for each Member
State that would identify all equines. This requirement was presented in the EU Regulation (EC) No
262/2015 3.03.2015, and the creation of these databases remains a work in progress in some member
states today [9]. This lack of data concerning equine numbers in Europe complicates descriptions
about their importance and impacts.
At present, the diversity of equine uses leads to a large variety of impacts on the environment,
especially when activities are exclusive to equines, such as sports or racing. This creates difficulties
in listing and evaluating the environmental impacts of the entire equine industry. However, in
today’s European context, local authorities aim to maintain rural activities and to support agriculture
in its sustainable transition. These terms, in the EU context, include all policies that search for the
transformation of current societal systems to minimize negative effects on the environment and promote
innovative projects [12]. A variety of new uses of equines could meet these challenges. In particular,
the European Horse Network has expressed the need for a foundation of scientific resources to build
arguments and promote equines in European policies and debates. Consequently, this study describes
equines not only as animal producers, but also as ecosystem service providers, especially for land use
and biodiversity conservation. This choice consolidates the fact that equines are not only seen as a
source of agricultural goods (leisure diversification or meat production, for example), but generate a
wide range of other externalities.
The aim of this review is to highlight the most important services provided by equines for the
environment at the European level. In order to answer public and professional stakeholders’ questions
about the inclusion of equines in public policies according to priority, we first met with stakeholders to
understand their main issues. Then, a literature review of the available knowledge on the green assets
of equines was conducted. Consequently, this paper examines the environmental assets (and limits) of
equines that appear to be most important in the context of European policies about agriculture and
rural development.
2. Choice and Definition of the Main Green Assets

To achieve this goal, the reflection about equine environmental assets started with fifteen interviews
among key stakeholders coming from various institutes at the French and European level: the European
Commission, the European Parliament, the French Ministry of Agriculture, the International Federation
of Equestrian Tourism, the French Permanent Representation in Brussels, and the European Federation
of Working Horses. The aims of the interviews were also to estimate the main issues of the equine
industry toward environmental challenges. The interviews were semi-directed and were conducted
from May 2019 to July 2019. The choice of respondents was made in order to have a large panel of
expertise scales (France or Europe) and professional functions (researchers, institutional stakeholders,
and professional representatives). The distribution of these 15 interviews was as equal as possible in
function and level of decision (Table 1). The high number of interviews from European institutional
stakeholders was undertaken to have a better view of the diversity of the issues at this level. Put another
way, it was not easy to find professional representatives concerned with the green assets of equines at
the European level.
Table 1. Distribution of interviewees according to the scale of expertise and the function.
Scale of Expertise/Function * Researchers Institutional Stakeholders Professional Representatives

France 3 3 3
Europe 1 4 1
* Respondents who answered questions about the French equine industry are counted in the group “Scale of expertise:
France.” Those who answered questions with a European point of view are counted in “Scale of expertise: Europe”.
These interviews entailed a reflection based on sectors where equines have particular and specific
impacts on the environment, here called “green assets”.
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Globally, the most cited impact is grazing, which was described positively, e.g., in terms of pasture
maintenance, complementarity with other livestock, and as a carbon sink, even if some respondents
insisted on the destructive nature of equine grazing when mismanaged. Equine work and tourism
were seen as green alternatives even if the dynamism of equine work was perceived quite negatively,
at least in Western Europe. Another point was the key role of buildings and infrastructures related
to equines. These buildings are, in most cases, made from wood, which is a renewable material
with positive technical characteristics, e.g., isolation, and favorable effects on the landscape. Manure
methanization was also mentioned. Indeed, recycling manure by producing energy is a promising
way to improve the environmental image of the equine industry. More generally, according to the
respondents, equines have quite a positive impact on the environment since they do not eject as much
methane as cattle do, they graze, there is a very rich domestic biodiversity that permits a wide range
of uses, even as alternatives for engines in agriculture, for example. Often, no negative effects were
spontaneously mentioned by stakeholders when asked about environmental impacts.
This process led to an evaluation of the whole European equine industry that considered the
current European context and challenges. Green assets were directly or indirectly linked to European
rural policies: the maintenance of open areas through grazing, agritourism, and the maintenance of
endangered breeds [7].
Green assets are generally directly linked to:
• The inherent nature of equines (as non-ruminant herbivores whose species presents a high
biodiversity of breeds).
• The geographical repartition of equines and their particular land use.
• Their use by humans, which can offer environmental benefits. Even if all human activities with
equines benefit from a green image linked to the use of an animal, only some of these uses have
positive environmental impacts. Of course, the impacts of equine breeding, whatever the equine’s
future use, are considered when examining green assets for land use, breed biodiversity, and
grazing. We decided to focus on two equine uses that generate specific environmental advantages:
equine work and tourism.
This initial information was completed through a literature review of the newest sources possible
to build the first state-of-the-art overview of this subject. This review mainly focused on Europe,
even though some sources also concerned other continents. In addition to international references,
many countries, especially in Europe, developed studies published only in their national language.
Consequently, this study was supplemented by French-language references. From March 2019 until
November 2019, we used two main databases, Web of Science and Google Scholar to search for articles
and reports. To select documents, we searched for the terms “equid*”, ”equine*,” “environment*,”
“horse*,” and all words related to green assets, in both French and English.
This literature review highlighted key arguments for five green assets:
• Equine grazing: Equine grazing incidence is unique because of this animal’s morphology and its
physiology specificities, especially regarding ruminants (the main herbivores raised on grasslands).
In particular, equine grazing is done on different patches made of lawns and high grasses.
• Domestic biodiversity: Human and environmental selection has led to a rich diversity of equine
breeds all over the world. Some of these breeds are currently endangered and their conservation
is an important issue, which could be introduced in European policies.
• Land use: Equines are present in various areas, especially where other livestock is presently
absent. This land use is directly linked to the place of equines in society; as livestock, it is possible
to find equines in farms and large areas, but as family pets, equines can be encountered near
houses, sometimes on small plots of lands that are not usable for agriculture.
• Tourism: Equines can be used as a means of transport but also as travel companions to discover
wild countries and landscapes.
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• Equine work: Equines are also used in tourism, cities, and agriculture as a source of energy,
whereas other livestock are not, at least in Europe.
3. State of the Current Scientific Knowledge Concerning the Five Equine Green Assets
3.1. How does the Inherent Nature of Equines Impact the Environment?
Equines are non-ruminant herbivores. Equine grazing impacts pastures differently than cattle,
sheep, or goat grazing thanks to the particular physiology and morphology of equines, who have a
double row of incisors and a high capacity for ingestion linked to the absence of a rumen. They adapt
their diet easily according to the available forage. Their behavior also differs from that of cattle in
their feed preferences and greater movement when grazing (as there is no rumination rest). These
differences induce various impacts on grasslands according to the whether horses graze alone or are
associated with ruminants.
3.1.1. Equine Grazing
Grasslands in Europe and Their Maintenance

Meadows are lands covered by grasses and legumes and mainly aim to be a feed source for
livestock through grazing or mowing. Meadows are known to be a carbon sink: they can stock 60 to
70 tons of carbon per hectare in temperate areas [13,14]. Moreover, the presence of legumes allows for
the fixation of nitrogen from the air to the ground. There are different types of meadows: permanent
grasslands (retained over 5 years, with possible reseeding after 5 years); semi-natural grasslands (a
particular kind of permanent grassland because they exist for more than five years and are known
to be among the most species-rich habitats in Europe [15]); cultivated grasslands (seeded each year
(or more often than 5 years), and they are the most commonly used in current breeding systems [15]);
and other less productive grasslands present in arid or rugged areas, which are called rangelands
(shrub lands, steppes, alpine communities, marshes, tundras, etc.) [16]. In Europe, grasslands cover
21% of the agricultural land in the European Union, while croplands cover 22% and woodlands cover
38% (Eurostat, 2015). However, there is a decrease in permanent grasslands in Europe [15], while
pastures are known to have different positive impacts: they are seen as pleasant and aesthetic [16] and
provide multifunctional goods [15] that produce agricultural commodities and maintain biodiversity,
soil, and water quality, even in suburban areas [17], in addition to meeting the needs of herbivorous
productions. Animal grazing presents three general consequences: (1) it maintains a certain level
of vegetal biomass [18] with the control of invasive species (through the intake of plants [19] and
trampling [19]); (2) it has effects on plant metabolism (defence, resistance, and avoidance) [19]; and (3)
it enables the creation of ecological niches [20].
Equine Grazing Specificities

Equines show specificities that are morphological, physiological, and behavioral. First, equines
have two rows of incisors that allow them to graze on short grasses [16] lower than cattle are able
to and reach young plants that are easily digestible [20]. This leads to a panel of feed preferences.
Equines are graminoids feeders [16] and make less use of forbs and legumes than cattle [15]. Equines’
preferences change when there is feed shortage. Equines move more easily toward less palatable
grasses species than cattle [21], particularly in winter [16]. This may be explained by their physiology.
The absence of rumination permits more time for feed intake [22]; less methane emissions compared to
cattle (100 kg/CH4 /dairy cow in Western Europe [23], more specifically: 117.9 kg CH4 /dairy cow/year
in France [24], and 18.0 kg CH4 /horse/year in Western Europe [23] and 20.7 kg CH4 /horse/year in
France [24]); and no limitation of intake capacity due to rumen volume [25]. Consequently, the global
intake capacity of equines can be bigger than that of cattle [16] as equines graze longer than cattle [26].
Most equines (except high-performance horses like racehorses) do not need high-quality feed; instead,
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they are able to live on low-nutrition feed [27]. Moreover, their physiology explains their adaptation to
a low starch diet because of the low activity of amylase [15]. They also seem not to be able to digest
the secondary metabolites of some plants (such as shrubs) [26]. Because of this, there should be a
lower control of shrubs when equines graze them compared to other herbivorous species. However, in
extensive Mediterranean conditions with a low stocking rate, shrubs are controlled by local equines [16].
Equines do not appear to be affected by shrubs’ defences, such as thorns [26].
As a consequence of these specificities, equines can adapt their intake through a reduction of
feed resources during harsh environmental periods [16]. Some primitive equine breeds are better
able to mobilize the bodily reserves they gain during summer to survive during the winter if grass
is not sufficient [16] than some specialized equine breeds. For example, in Iceland, despite the cold
climate, equine grazing occurs even during winter and is supplemented with the hay refused by cattle
or sheep [16]. Likewise, equines can be raised in Camargue, where plants are halophilic and resources
are scarce [16].
Equines impact pasture differently than other herbivores because their grazing behavior induces
a particular heterogeneity of plant cover [28]. Some areas with high plants are not grazed by equines
and could be used as latrines [15], but they could be grazed if there were a lack of resources [27].
Nevertheless, there is also the risk of overgrazing and soil erosion in equine pastures [15]. Equines can
impact a pasture, for example, by trampling during periods of exercise [29] or if they are concentrated
in small areas. Finally, gnawing on trees in semi-natural pastures was reported [20] due to the lack of
minerals [15], but this can be reduced with supplementation [30].
Studies on exclusive equine grazing show the positive impacts on flora, including an increase
of legumes in France [20], a control of competitive grass [15,20], and control of some shrub species
in Camargue [20] (e.g., Vaccinium myrtillus by trampling [16,31]), alongside negative impacts, such
as the limited control of fast forest regeneration in boreal conditions [15] and an increase of foams in
Iceland [20]. In particularly harsh conditions, some lands are maintained by equines to decrease fire
risks, for example, by reducing the aerial biomass of gorse in Galicia [16]). Equines are able to preserve
and maintain pastoral biodiversity [15] by grazing in areas abandoned by agriculture. This process is
identified as a specific threat to habitats and species by the European Union, as the invasion of some
plants left non-grazed by livestock leads to landscape closure [25].
Equine grazing also impacts fauna:
• The populations of small herbivores increased due to high-quality vegetative regrowth.

• Insectivorous birds, such as the spoonbill, appeared in pastures grazed by equines in the
Netherlands (as with ducks in French wetlands) [20].
• The wolf population in Galicia, Spain, was maintained partly thanks to ponies bred in semi-natural
conditions. These one were the preferential feed source of wolves [27], which consequently did
not attack other livestock. As a result, farmers felt less disposed to shoot them down. Finally,
ponies were an indirect way to conserve the wolf population.
Pastures can be grazed by equines only, by two or more herbivorous species, or mowed. All these
types of pasture management methods allow animals to feed and exert their own effects on the
environment (biomass, grassland evenness, and effects on butterflies) depending on the area (grazing
seems to be a better option in Central Europe and mowing may be more suitable in Southern
Europe) [32]. In boreal conditions, continuous equine grazing seems to be less beneficial to biodiversity
than alternative grazing regimes (late grazing, years without grazing, and mowing) [15]. In Sweden,
year-round equine grazing increased the pasture quality and diversity compared to mowing [28].
The Impact of Cattle and Horse Mixed Grazing on Meadows

The heterogeneity of plant cover may be beneficial for flora and fauna but can lead to pasture
destruction when it is mismanaged. Indeed, non-grazed areas can be invaded by shrubs, while grazed
areas can be overgrazed. To prevent this situation, mixed grazing with two herbivorous species could
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be a solution. Mixed grazing is “a practice of stocking two or more species of grazing or browsing
animals on the same land unit, not necessarily at the same time but within the same grazing season” [16].
Cattle and equines graze preferentially in similar habitats, such as grasslands [26]. This can lead to
a competition for resources but also complementarity because of their different foraging behaviors.
A survey carried out on farmers who raised both (dairy and/or beef) cattle and horses in the French
central mountains highlighted the equines’ ability to exploit the grasses refused by cattle (total surveyed
farmers: 25) [33]. In Massif Central (French mountains), when equines were introduced on cattle
pastures, their nutritive value increased because of the development of higher nutritive plants (within
the following conditions: 350–440 kg live weight per hectare and as many horses as cattle) [16,20].
When bred with cattle, equines could graze in poorer pastures not useable by cattle because of their
low nutritive requirements [33]. Moreover, in winter, equines can remain on pastures and continue
maintaining them while cattle are held in stables [16]. However, mixing cattle and equine grazing can
generate problems, especially when increasing the number of animals in the pasture. For example,
when the equine stocking rate is too high, cattle may be disadvantaged because they are unable to eat
enough [27,34]. The main consequences of mixed grazing on grassland management are:
• A lower workload: Because their grazing behavior is different from and complementary to
cattle, equine replace the use of machines for mowing grass refused by cattle and for crushing
wastelands [22,33]. In the absence of equines, farmers would need to use the roller chopper more
frequently [22].
• A decrease of the parasitic burden: This may be explained by different host sensitivities and
improved nutritional status (intake of various plant species) [16,35].
• A better control of woody species: In Massif Central, woody species were better controlled when
pastures were grazed by cattle and horses than by cattle only [20].
In France, equines are often raised with cattle in grassland areas [10]. A study of 51 farms located
in the highlands, breeding cattle (for beef or milk production) and heavy horses together, highlighted
the common practices and confirmed the preceding results about the advantages of mixed grazing [22]:
• Equine grazed mostly after dairy cattle but grazed simultaneously with suckling cows and heifers.
• Equine grazing helped to remove grasses refused by milked cows.
• Equine were present on small plots, fields far from stables, and on poor pastures.
In this survey, equines comprised on average about 10% of the total livestock in terms of livestock
units. Heavy horses were bred for meat production mainly. This breeding process is seen as a
complement to the production of cattle for use in grasslands but is not considered a significant source
of income. On the other hand, thanks to equine pasture management, the mechanical maintenance of
meadows decreased, which can be seen as an indirect contribution to the greater efficiency of forage
systems in the highlands.
Beyond grazing specificities, the inherent nature of equines leads to another environmental asset:
equine species present high biodiversity among their breeds, which is shaped by the environment and
human beings.
3.1.2. Domestic Biodiversity

Several equine breeds are well adapted to poor grasslands and semi-wild breeding systems [18,26,27]
because their format and size seem to ease their growth in these specific areas [36].
For a long time, and still today, natural and human selection have affected breeds, including those
that live in semi-natural or particularly harsh conditions [25]. Human beings selected the most suitable
breeds for different uses (traction, meat production, racing, and sport), leading to a high variety of sizes,
formats, and phenotypes of equines sorted into breeds. A breed may be defined as a breeding pool of
individuals that share a common phenotype (which is typically purely morphological, i.e., coat color,
height, etc.) [25]. Worldwide, in 2011, there were 397 equine breeds according to the Universal Equine
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Life Number (the international lifelong identification number for equine, www.ueln.net). Germany
(46 breeds), France (37), and the United States (34) were the countries with the greatest numbers of
breeds. Of all these breeds, three-quarters were saddle horses (sport, recreation, and ponies), 15% were
heavy horses (France had the highest heavy breeds number with 9), and racing breeds represented
only 5% of breeds [37]. Today, uses of equines are diversifying, and competition between studbooks
is increasing because of the internationalization of the equine industry. Consequently, some equine
populations collapse when they are not useful anymore. For example, worldwide, 60 donkey breeds
are known, but only 28 have had their morphologies described [38]. Only one donkey breed out of 28
is considered to be not threatened by the Food and Agriculture Organization of the United Nations, as
80% of the donkey population disappeared over 20 years in Europe [ibid.].
Being part of a breed means being identified in a studbook, which is a book of genealogy where
breed standards are established. A closed studbook does not allow foals whose parents are not
identified in the book; this keeps the breed “pure.” However, to improve the traits of interest, studbooks
may be opened to some other breeds that will shape the breed. As an example, the pure-bred Arabian
and thoroughbred breeds underlie 25% of the genetic variability of 500,000 saddle horses from 55
different breeds, born between 2002 and 2011 in Europe [39]. A consequence of this intensive direct
selection is the specialization of breeds toward modern uses. This specialization is a threat to the
versatile, multi-skilled, and generalized breeds. Conversely, these breeds can be seen as an emblem of
a region that needs to find new approaches to remain competitive. Finding economically sustainable
alternative activities in environmental conservation projects (e.g., animal traction, equestrian tourism,
and meat and milk production) would be an interesting way to conserve local and endangered breeds.
European aids for endangered breeds help breeders maintain their activity, even if equine
production is no longer economically competitive (Regulation (EU) 2016/1012 of the European
Parliament and of the Council of 8 June 2016). This underlines a contradiction in common European
programs that give funds based on a small number of animals to preserve animal biodiversity and not
just their skills [40]. This kind of breed development is not sufficient to ensure economic viability [41].
However, these programs conserve invaluable environmental services and cultural heritage [25,42].
Equines, because of their essential nature, directly impact the environment. Nevertheless, these
impacts may differ depending on the location of the animals.
3.2. How Does the Spatial Repartition of Equines Impact Land Use?
Equines require forage areas for grazing and for preserved fodder harvesting. Equine grazing
may lead to the maintenance of open areas and a possible improvement in the agronomic quality of
grasslands [43]. Breeding farms, riding schools, racecourses, and trails are other kinds of indirect land
uses by equines. A French region typology of equine farms highlighted that equines are present in 91%
of all cantons (exclusive of Corsica) [44], which means that the equine industry extends to various areas.
Equines are also present in suburban areas: 75% of equines are encountered in the most inhabited
areas in Sweden, for example [45]. This is also the case in Scotland [46], Germany [47], France [48],
and Belgium [17]. Equines are also present in Polish post-agricultural lands and forests [49], Spanish
heathlands [27], and British grasslands [25]. Studies across Europe agree that equines, whatever their
use, are located in various kinds of lands: (a) suburban areas, (b) rural areas, and (c) sensitive areas,
such as mountains [17,44–48].
3.2.1. Suburban Areas

Pastures in suburban areas are seen as an extensive method of farming and a good way to
conserve water quality; however, these pastures are disadvantageous for agriculture [30] because of
their small available surface, the presence of housing and non-rural neighbors, and land conflicts.
Equines are kept in suburban areas thanks to urban demand [47]; equines are not only seen as a source
of agricultural income [50] but as a leisure activity [51] or even as a family “member” whose place is
near the home [43]. As a link between urbanization and rurality [51], equines are present in transitional
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areas that have been abandoned by agriculture but have not yet been developed by urbanization [43].
This kind of “sub-agriculture” [43] may be called “soft urbanization” [17] because equines are a spatial
and functional link between residential areas and agriculture, and are also of concern in land conflicts
with other agricultural productions. In fact, these conflicts for land force equines to reach the edges of
urban areas [43]. Nevertheless, equines remaining in these areas mobilize a large array of services,
including veterinarians, feed industries, trainers, equipment sellers, and transporters [47].
Some studies quantify the presence of equines in suburban areas, underlying their growing
importance. In France, research conducted on 49 municipalities showed that equines use between 1%
and 3.5% of suburban areas depending on the region [43]. In Sweden, it was shown that the density
of equines can increase up to 6 horses/km2 near urban areas but that specialized equine farms near
suburban areas keep more equines than farms in rural areas [51].
In terms of environmental impacts, the presence of equines is comparable to the introduction
of nature in the city, which improves landscapes [51]. Equines can graze on small plots near forests
and gardens (for example, in Flanders (Belgium) and the Netherlands [17]). Their presence yields
positive changes, such as positive land management, added landscape value, and job creation [43,45].
Equine grazing is perceived as being a positive element of landscape management in Belgian suburban
areas [17] and in Sweden, where the terms used by inhabitants to describe the equine presence were
globally positive: ecology, landscape managing, and useful [51]. The presence of equines in these areas
opens the possibility to include equines in reflections about urban planning [43,51]. Nevertheless, the
high density of equines in small areas could pose a threat to the environment [47] through problems,
such as overgrazing, droppings concentration, and destruction of landscapes, because of the creation
of mismanaged infrastructures (overgrazed paddocks, and horse-riding rings) [45]. Equines are also a
source of odor, insects, and lack of safety [43,45], thereby leading to land conflicts. In Belgium, because
of the population density of the country, farmers and equine owners fight for land [52]. Indeed, only
10% of land is considered to be rural (depending on the density of inhabitants) in this country, but
agriculture is present in 45% of the total national area, and 1/3 of grasslands are grazed by equines,
underlying their high presence in suburban grasslands [17]. The presence of equines in these areas
does not always imply grazing because equines can be fed with cereals and preserved forage. In some
cases, the available area does not allow grazing, and equines are kept in small unproductive plots or
stables [34]. For example, in the Berlin suburban region, there is no grazing in specialized and intensive
farms [47]. This raises specific issues related to overgrazing and manure management. More studies
about grazing in these specific areas are needed [17].
It is important to note that the presence of equines in suburban areas depends on urban sprawl
(horse riding schools near cities), but sometimes urban sprawl depends on the presence of equines
(owners who want to live closer to their horses) [43]. This kind of agriculture in suburban areas could
respond to sustainable issues according to some authors [47], but communication actions should be
deployed in order to raise awareness about the risks of overgrazing, droppings concentration, manure
management, safety, and land laws.
3.2.2. Rural Areas

Rural areas are the main production areas for animal feed and for other derived products, such
as straw. In France, equines use from 1.5–6% of the total rural areas. Equine numbers depend on
the type of agriculture: the numbers are lower if there is more professional agriculture (e.g., if the
lands are used for food production) and they are higher if non-professional agriculture prevails (e.g.,
retired persons or multi-active land owners) [43]. The presence of equines may be combined with other
local agricultural production, thereby maintaining pastures even in intensive agricultural areas [44].
Farmers can experience benefits from the presence of equines on their land, such as selling equine feed,
letting the equines graze on unproductive pastures, or receiving manure.
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3.2.3. Sensitive Areas

A sensitive area has a long-term capacity to maintain and enhance natural resources, such as
soil and water quality, biodiversity, and the landscape. In such areas, agriculture is more constrained
than in the lowlands. Mountains are part of this definition. Other kinds of sensitive areas include
natural rangelands in harsh climates and abandoned agriculture areas invaded by shrub, wetlands,
or heathlands.
Some sensitive areas produce difficulties when using machines because of the slope or soil depth.
Consequently, in these areas, animal husbandry seems to be the most adapted solution to preserve
landscapes and maintain economic activity. For example, in French plains, farms breed mostly saddle
and race horses with high economic value [10], whereas few heavy horses are associated with cattle
farming in the uplands, in an attempt to improve grassland management despite their low economic
value [22]. In this study, up to 15% of farmers said that they would abandon some parcels of land if they
did not possess equines [22]. In Poland, ponies named Konik Polski use forests and post-agricultural
areas, exerting several impacts on them, including a reduction of shrubs and bushes through trampling,
an increase in coprophagic insects, an increase in birds (thanks to greater food resource diversity
and hiding place availability), and an increase in the interest and awareness of nature and ecology
for tourists and local inhabitants [49]. In Galicia, Spain, where transhumance was abandoned, the
presence of equines raised in semi-wild conditions helped to restore heathlands [16]. Heathlands
and grasslands in the United Kingdom are usually grazed by sheep, but there have also been studies
on the reintroduction of ponies to these areas, where the authors recognized the value of equines
as conservation grazers [25]. Rewilding areas abandoned by agriculture with large mammalians,
such as equines, are a proposed solution to maintain the important functional links between plants
and pollinators in grassland ecosystems in Sweden [18]. Some releases of equines may be a threat
to ecosystems, as sometimes breeds are not indigenous to the region. Specific attention should be
given [25] to wisely choosing breeds that are to be introduced in natural areas, as well as the density
of the released equines. In the “Parc des Volcans d’Auvergne” (an environmentally protected area
in France), some authors prescribed the use of mixed grazing with equines and cattle to restore the
area [53]. Rangelands are also affected by the definition of sensitive areas. Mediterranean zones show
the possibility to access a diversity of pastures (salty pastures, marshes, and rice stubbles), which
complement each other and permit local equine breeders to lengthen the grazing season [16]. In areas
where the soil is fragile, like in low mountainous olive groves, donkeys are usually used to control
grass growth because they weigh less than other heavy animals, such as cattle or horses [38].
In highlands, preserving natural biodiversity when maintaining human activity is possible
through agriculture, and more specifically, through animal husbandry. Indeed, the maintenance of the
open landscape is particularly interesting for its biodiversity, as explained above, to preserve vegetal
resources in non-arable lands, but also to manage areas for human activities, such as hiking or skiing.
Because of their slopes and peculiar climates, arable lands are scarce, and pasture meadows are favored
as a source of food for livestock, thereby spearheading agricultural products, such as meat and milk.
To conclude, equines take part in the problems of land pressure but have complementarities to
agriculture and urbanization in terms of their functionality for land use and maintenance [43]. Other
environmental advantages of equines come from their use by humans for working or leisure activities.
3.3. Animal Uses Serving Environmental Issues

Equines have been used by humans since their domestication. Activities with equines are often
seen as “natural.” However, not every use of equines is environmentally virtuous; for example, horses
travelling by plane for international competitions or races. According to our findings in the literature
review, two uses present interesting environmental assets: equine work and tourism.
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3.3.1. Equine Work

In this study, a working equine refers to an equine who is used to work with humans; provides
energy that can be substituted by other sources of energy, other machines, or types of transport;
and generates earnings [54]. Sport and leisure horses, equines used for therapy, race horses, and
equines bred for meat or milk productions are not affected by this definition. There exist four
primary non-exclusive types of equine work: agriculture (mostly vineyards and market gardening
in organic production systems), forests (logging), human transportation (such as equines drawing a
carriage for tourists or schoolchildren), and public service missions (watering, garbage collection, and
mounted police).
In the world, there are ten times more animals used as sources of traction energy than motorized
tractors. In “developed” countries, 26% of the land area is managed by animal traction (versus 52% in
“developing” countries) [54], especially in sensitive areas [55] or in mountains [56], where plot structures
(slope and soil quality) make mechanization difficult. However, these numbers have decreased over
time in Europe. For example, in Poland, the percentage of horses used in agriculture compared to all
sources of energy decreased from 93.8% in 1950 to 1.73% in 2009 [57]. In the context of productivity
gains in post-war Europe, it was necessary to work on bigger areas in less time. Machines seem to
be more adapted to this aim than animals, as their use increases sowing, treatment, and harvesting
speeds, along with work efficiency, as well as decreases the time dedicated to crops. Nevertheless, a
full replacement of animals used in traction by machines may be perceived as a heritage loss [56] and a
threat to the environment (soil quality, for example).
Equines as a Potential Source of Renewable Energy

Equines consume fodder, which is considered a renewable source of energy because it does not
involve fossil energy in the narrowest sense (unlike fossil fuels or biofuels) [58]. In fact, biofuels may
be considered, in some cases, to be a renewable source of energy, but they need the same arable lands
as crops and are the focus of land conflict debates. Grasslands used to feed equines could be located in
non-arable parts of the territory, as noted above. A one-day harvest allows for enough forage to feed
an equine for one year in Switzerland [54]. A total of 0.6 ha of alfalfa, 0.5 ha of oats, and 0.5 ha of wheat
for straw are enough to feed two horses working on 14 hectares for 140 days a year in Croatia; for
the remainder of the year, these horses stay in stables, where they are fed the by-products of crops, or
graze on roadside vegetation or in orchards [58]. Moreover, grasslands and areas worked with equines
can be fertilized with their manure. In the case of biofuels, nitrogen must be imported or manufactured
and is spread on plots where it evaporates into the atmosphere, providing the main source for N2 O
emissions [58]. The animals must be fed all year, whereas machines can be used occasionally and
refuelled infrequently. Despite this disadvantage, equine work allows farmers to attain better feed and
energy autonomy [54], to highlight a traditional vision, to be appreciated by urban inhabitants [58],
and to maintain a diversified gene pool through the use of local equine breeds [55]. Finally, equine
work is considered by some authors as a form of sustainable agriculture [58].
On Arable Lands
On arable lands, soil compaction is known to be the most severe form of degradation in conventional
agriculture [55,58]. There is a difference between the paths made by machines (continuous, because
of their tyres, with deep soil compaction) and equines (intermittent, because of their hooves, with
superficial soil compaction) [58]. The soil porosity was higher after using donkeys or cattle compared
to a motorized machine [55]. A comparison between the use of a donkey and a motorized machine for
the ploughing, fertilization, and preparation of rapeseed in the context of the high hills in northern
Italy was made thanks to the life-cycle assessment (LCA) approach for which inventory data were
taken from the GaBi 4.0 database. All aspects related to the life spans of animals were considered,
except the end of life: pregnancy, growing and maintenance (health care, feed, keep, and equipment),
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and work. For machines, material acquisition, manufacturing, utilization, transport, and disposal
were considered. This information was acquired through interviews with animal owners, field
measurements, and technical reports from manufacturers. The results showed that, for the same
amount of carbon emissions (1 kg eqCO2 ), a donkey was able to prepare 330.63 m2 of land, whereas
the machine prepared only 18.69 m2 (three operational stages were considered on a 1000 m2 functional
unit: ploughing, application of the fertilizer, and seedbed preparation (harrowing and opening seed
furrows). Manure from donkeys was assumed to be applied as a fertilizer, so environmental impacts
from fertilizer production were avoided in the case of animal traction) [56]. If the fossil fuel used for
machines had been replaced by biofuels, the relative effects on the environment could have been 9%
lower. When comparing classic machines and donkeys, these effects were 97% lower [56]. In Ireland,
yields were greater when animal traction was chosen after the long-term use of tractors [58]. It is
important to note that equine traction is well adapted to small areas. Finally, there are not enough
studies about equine work in mountainous areas, where they could have particular assets [55].
In Forest Areas
Equines are known to be more drivable than machines in forests, or on rugged or narrow fields.
Thanks to this skill, there is less damage to residual trees [55] because machines need more space to
access fields [55] and create disturbances [56]. Without counting trail development costs, the use of an
equine was more profitable up to 50 m [55]. This distance increased up to 200 m when trail development
costs were considered [55]. A comparison between the use of a mule and a motorized machine in a
one-kilometer distant forest was made thanks to the LCA approach and showed that, for the same
amount of carbon emissions (1 kg eqCO2 ), a mule was able to bring 311.30 kg of wood, whereas the
machine brought only 79.64 kg of wood (three stages were considered: (i) felling (individual cutting of
trees), limbing (removing branches), and bucking (cutting into logs); (ii) yarding (collection of logs);
and (iii) transport from the forest to the farm (1 km). The functional unit was set to 100 kg of wood at
the warehouse. In the animal traction scenario, a mule was used in stages (ii) and (iii)) [56]. If the fossil
fuel used for the machines was replaced by biofuels, the relative effects on the environment could have
been 26% lower. When comparing a classic machine and a mule, these effects were 74% lower [56].
Other Agricultural and Territorial Works

Equines can also be used in:
• Old vineyards, because their drivability permits work in narrow rows and on terraced or steeped
fields (Douro River Valley, Portugal; Bordeaux, France; Sibeira Sacra region, Spain [55]).
• Greenhouses, because their drivability allows for precise work [54] and can be highlighted in
ecological production.
• Natural areas, where they are less noisy, degrade the soil less, and frighten local fauna less [54],
thereby enabling them to work in protected and sensitive areas. It is possible to compare this
to the consequences of equestrian tourism on wild fauna, which are perhaps less frightened by
equines than by pedestrians or bikers [59]. Mules are still present in some European areas, such as
national parks, where it is impossible or forbidden to use motorized tractors [38].
• Cities where they decrease the carbon footprint and are used as “city pacification” agents [60].
3.3.2. Tourism
According to the International Federation of Equestrian Tourism (FITE), the term “equestrian
tourism,” which emerged in the 1950s [61], concerns all outdoor activities with equines outside of
residential areas. Indeed, it is necessary to distinguish between equestrian tourism and equine tourism:
• Equestrian tourism comprises itinerant journeys with a ridden or hitched equine or on foot
supported by a pack equine.
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• Equine tourism concerns all activities devoted to equines, in their presence or not, that attract
tourists, including sport events, cultural events, races, fairs, museums dedicated to these animals,
riding courses, etc.
In addition, the two kinds of tourism linked to equines can be local (i.e., tourists move inside their
region of origin) or non-local (inter-regional, international, etc.).
France is the third-largest country in terms of rider numbers in Europe (behind the United
Kingdom and Germany), but is considered to be the leader in equestrian tourism and the first travel
destination, with 60,000 kilometres of equestrian trails in 2011 [61]. Other countries highlighted in the
European report on the equine industry in 2001 are Greece and Portugal, where donkeys are still used
for tourism [62]. Equestrian tourism has expanded through farm diversification called agritourism,
which is affected by the European development policies for 2014–2020 [36]. This kind of tourism can
involve farmers who want to promote local breeds to preserve culture and tradition, as is the case of
Camargue horses related to specific bull farming in marshes of the Rhone River estuary in the south of
France (Figure 2) [61] or Icelandic ponies in Iceland [63], where tourists want to find a link between
nature, animals, and local culture.
Figure 2. Presentation of the Camargue region and the use of the local equine breeds in this area.
Impacts of Equestrian Tourism

Equestrian tourism is a form of sustainable leisure [61], though there are very few studies on
the direct impacts of using equines in tourism; these studies are mainly American (12 in total, as of
2019) and Australian (six in total, as of 2019) [64]. This is why it is important to contextualize the
results presented below, as climate and cultural history are not the same in every region. Europe
has always been a host to large mammals, whereas Australia never housed such animals before
1800 [64]. A comparison between hiking, cycling, and horse riding [64] shows that the impacts on
the environment were the same between these means of transport but they differed in the degree of
impact (e.g., soil compaction and erosion, loss of organic matter biomass, and biodiversity losses).
The two most severe impacts of equestrian tourism are: (1) nitrification of rivers and soils because of
the overconcentration of phosphor in poor soils, and (2) zoochory through fur and manure, which
raises the risk of invasive plants being spread in protected areas [64]. On the other hand, this spread
may be beneficial for the flora diversity of poor soils.
Equestrian tourism is also an illustration of soft roaming. Indeed, nature-based recreation activities
impact wild fauna (most of them, even if non-motorized, have negative impacts on birds in terms
of their metabolisms, behaviors, and habitat disturbances [59]). There are no studies concerning
equestrian travel, but it is possible to consider a softer approach for wild fauna; wild animals are,
perhaps, less frightened by horses than by pedestrians. In addition, the trails used for horse-riding
are a softer way to adapt land to tourism than roads. Moreover, in sensitive and protected areas [61],
horseback tourism also creates and maintains trails in a useful state for other users.
The negative impacts of equestrian tourism must also be mentioned. Infrastructures are not always
adapted to equestrian tourism, and this shortfall can present destructive impacts on the environment.
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Indeed, during a long-term journey, a horse must rest every 20–40 kilometres but this is not always
accomplished [65]. In Poland, for example, on the longest national equestrian trail (2100 km in 2012),
there are only 36 liveries; this means one stable every 41 km [65]. When searching for a campsite or
pasture, horse-riders can destroy sensitive or protected areas [64]. Moreover, from surveys carried out
on equestrian tourists, security, comfort, and conviviality were more important when travelling than
ecosystem conservation [64].
Equine Activities and Tourism

Because of the wide definitions of equine tourism, it is difficult to list all ecological effects
of such tourism. However, some reflections about this issue can be highlighted. In France, for
example, the professional organization of the horse industry provides awards to infrastructures that
put efforts toward improving their environmental impacts, such as riding schools in Camargue [61]
or the numerous stud farms and boarding stables all over the country, in the form of environmental
labels (www.label-equures.com, accessed 27.11.2019). This is also the start for reflecting upon the
environmental impacts of equestrian events (the transport of horses and persons, the use of natural
resources, etc.) in France through an evaluation of the most impacting positions, like wash areas [66].
These efforts provide an early ecological wake-up call for all events, including equine-related ones,
and need further research.
4. Discussion
4.1. Links between Green Assets and Specific Issues

The green assets highlighted above are not completely independent but are mostly linked to
each other.
This fact can be first illustrated through the example of the Camargue horse, which plays a key
role in land planning and tourist development. This equine breed is known to be a representative of
local cultural heritage (Figure 3). Raised in the wetlands, Camargue horses are robust and largely
participate in the maintenance of this sensitive area through shrub grazing. They are also hardy enough
to be used to control and move cattle herds in these vast swamps. The Camargue region attracts
tourists because of its particular fauna (such as flamingos, horses, bulls, etc.) and flora (halophilic
plants). The Camargue equine breed is also well adapted to be ridden by tourists to discover the
natural environment of this area. Finally, Camargue horses can also be found in suburban areas, such
as in riding schools or pasture areas near houses.
Figure 3. The Camargue horse breed as an image of the link between the five green assets.
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Each green asset has its own specificities and issues but some of these issues are common to several
green assets. For example, because of the task intensity, animal traction sometimes must be assisted by
another source of energy (in cities for example) [60]. To avoid soil compaction, innovative projects have
emerged to improve machines, but the use of equines is not always proposed as a solution [58]. Finally,
there is a need to improve the equipment for animal traction [55] and to inform stakeholders about
this need. Presently, the use of equine work depends on the geographical and market opportunities
created thanks to these animals [56]. These opportunities should also be developed for threatened
breeds whose population have collapsed since the middle of the 20th century. Regardless of the species,
these communities must face common challenges [41], such as how to ensure their competitiveness
on the international market while maintaining the appearance of an iconic breed that is illustrative
of regional culture and heritage, and how to conciliate the development programs and conservation
programs for an already threatened breed. The uniqueness of some breeds is used to bring tourism
into the breeds’ native region, such as Icelandic ponies in Iceland [63], even if tourism may be a source
of conflicts with other users and even sometimes a source of the deterioration of protected areas [61].
These conflicts may also appear in land use. Equine owners are often disadvantaged toward more
“agricultural” productions [17]. In suburban areas, equines stand on transitional plots, which are
rented or borrowed [43], within the urban network. This presence may lead to disturbances, such as
smells and noises. However, in Sweden, a survey made in the suburban areas where equines were
encountered showed that the inhabitants were less annoyed by equines than by other disturbances,
such as noisy roads or mowers [51]. Urban planning does not consider the specificities of the equine
industry. Equine owners want to keep their animals close to their houses and urban centers but are
restrained because of land conflicts. If urban planning undertook a multifunctional approach for every
activity [17], there would be fewer conflicts of interest [51]. Conversely, intensive equine holdings
on small plots, as is often done in suburban areas, can be harmful to the landscape. This is why
“horsiculture” is sometimes seen as degrading for the environment [45] as it is directly linked to the
specificities of equine grazing that leave areas overgrazed if badly managed. In addition, in most
cases, only broodmares and growing horses graze, while stallions or equines in training do not [22].
Moreover, the workload increases when horses and donkeys graze if they are trained daily because,
sometimes, it is necessary to pick them up from distant plots [22].
Facing this context, each member state of the European Union has specificities concerning equines.
Some countries orient their equine industry more toward sports and racing (e.g., England and Germany)
and do not consider equines to be a source for rural development [67], whereas others highlight
their equine sector through native breeds and tourism, such as Iceland [68]. Thus, impacts on the
environment will be as diverse as the uses of equines and farming in each area. In some countries,
equine husbandry is perceived as an intensive process (no grazing, many individuals on small plots,
main outlet exported, etc.), for example, in Belgium, the Netherlands, or Luxembourg, because of the
lack of available land. In other countries, equines are still used as a source of energy or entertainment,
with grazing on large lands and sometimes in semi-wild conditions, such as in Poland [49]. In occidental
Europe, land use by equines depends mainly on the type of area (rural, suburban, or sensitive), as
discussed in the aforementioned studies. This grouping may be different in other countries, such as
Romania, where horses are still used in agriculture [69]. Thus, the numbers of equines may still be
anecdotal in suburban areas.
Consequently, it is important to determine how to best take advantage of the green assets
of equines.
4.2. How to Better Use Equine Green Assets
4.2.1. For Equine Keepers

This knowledge is partially taught in agricultural training. Indeed, there is a lack of training courses
and technical information for small equine owners to teach the specificities of equines’ relationship to
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the environment. Difficulties arise from the atomization of horse owners and the diversity of horse
keeping. Moreover, owners mostly raise horses in small areas with low productive value, so the technical
improvements of pastures are difficult to apply. The risks of injury or escape have also been is raised
by equine breeders, even if equine grazing itself is not to be feared. Advice for management practices
needs to be adapted to the specific conditions of each horse keeper according to the type of meadow, the
herd size, the physiological and nutritional needs of the equines, and welfare requirements. To improve
this situation, communication and teaching about pasture management practices, related risks, and
threats are necessary. Raising awareness and improving management practices could improve equine
environmental impacts, highlight their green assets, and better use these assets in everyday practice.
It is already possible to propose some examples of practical recommendations to equine owners:
• In order to avoid the overconcentration of manure in suburban areas, equine owners may build
reliable partnerships with local farmers, who can use manure as fertilizer. Manure can also be
recycled and rapidly composted to improve soils in city parks. Another solution is to transport
manure to methanation firms for energy production [70].
• Equine grazing has interesting characteristics in pastures and shows complementarities with other
herbivores, such as cattle. Thus, associating these animals could be a first step toward improving
pasture quality and maintenance.
• When searching to buy an equine for leisure or tourism, looking for a native breed could be a
good option if the future owner wants a hardy equine that is well adapted to the local climate.
These breeds may value local feed resources better and more cheaply than other breeds. At the
same time, this act would help to conserve threatened breeds, facilitating a cultural development
of the region and maintaining the genetic diversity of equine species.
• When travelling on horseback, it is important to follow trails that are dedicated to horse riding, to
avoid protected areas, and to take care of the paths.
• Equine work represents a diversification opportunity for riding schools. This diversification can
be achieved using equines that are already present in the structures for maintenance tasks, either
on site or in collaboration with local municipalities (service provision).
Despite these few examples, it is necessary to develop further recommendations for equine
keepers in order to establish clear aims, build a reliable argument, and ensure adequate follow-up.
These recommendations could be spread by local authorities, teaching centers, and the professional or
public institutes responsible for the horse industry. As a contribution to advancing knowledge, this
literature review is a first step that will need regular updates to enhance the advisors’ arguments.
4.2.2. For Institutional and Political Stakeholders

Equines are often forgotten in political debates as they are seen as both livestock and pets.
This duality is exacerbated by the large diversity of stakeholders responsible for the equine industry
within Europe, including ministries of agriculture, sports, and tourism; technical institutes; research
institutes; national and regional associations; breed associations; and equestrian federations. Moreover,
there is a lack of practical information and courses on equine green assets that are usable by stakeholders.
Promoting equine grazing and communicating about its benefits on the environment can improve
different situations. This could also help integrate equines into political debates and help develop
research on this topic. Hence, it is possible to propose some practical recommendations to institutional
and political stakeholders:
• Equines are an interesting alternative for the maintenance of small abandoned lands in suburban
areas that could be promoted by local authorities.
• In regions where grasslands or rangelands are important, local development policies could include
aids in favor of the equine industry, for example, subsides for cattle farmers to also hold equines,
or for the creation of numerous platforms to help horse owners meet farmers for feed purchases,
pasture grazing, or the use of manure.
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• Regional subsidies could support the breeding and keeping of local breeds. These could also be
integrated in local tourist events or as a vector for job-creation.
• The trails used for equestrian tourism and camping sites should be framed well to keep tourists
from disturbing natural areas. Moreover, every trail should clearly indicate whether it is adapted for
equines to ensure that equestrian tourists use the trails safely. Further, linking equestrian tourism
stakeholders with stakeholders from the tourism sector or those in charge of protected areas could
be an interesting way to develop collaborative actions to support sustainable regional development.
• When discussing new sustainable projects concerning ecological farming or public service missions
in cities, equines could be included in the list of suggested alternatives based on the assets presented
in this paper if all economic, social, and welfare conditions are fulfilled.
• The new 2020 CAP is in process. At a national level, its measures could better support the equine
industry through new agri-environmental measures for equines, such as the use of animal traction,
the practice of mixed grazing, the use of local threatened breeds, and the use of equines to maintain
vacant suburban plots of land.
Finally, by gathering the available scientific knowledge about equine green assets, this paper offers
some common reflections and issues about the place of equines in a sustainable regional development.
5. Conclusions
The equine industry is constantly evolving according to changes in society. One of the next steps
is linked to the growing environmental awareness. This issue concerns citizens but also the political
spheres, thereby putting pressure on the stakeholders of all economic sectors, including the equine
industry. Indeed, in most European countries, environmental issues are not yet considered to be
important enough by stakeholders in the equine industry. However, through their green assets, equines
can have an active role in ecological transition and debates, both alone and as a complement to other
economic productions and services. In the future, it could be interesting to support knowledge exchange
in order to progress equine research, thus making this industry more visible and understandable, and
to include equines in political debates about the environment and raise awareness about equine uses to
avoid radical actions from animal activists. Creating and publishing all kinds of communication media,
such as articles, photos, videos, websites, and podcasts, could be a way to reach a larger audience and
make equine owners adapt their management practices to better use equine green assets.
From European organizations to society, everyone should be aware of the potential place of
equines during the ecological and agronomic transition toward a greener future.
Author Contributions: Conceptualization, A.R.; investigation, A.R.; writing—original draft, A.R.; visualization,
A.R.; supervision, C.V.; project administration, C.V.; writing—review and editing, C.V. and G.B.; validation, G.B.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the French Institute for Horse and Horse Riding (Ifce) and the European
Horse Network (EHN).
Acknowledgments: The work has been carried out with the help of the National Research Institute for Environment
and Agriculture (IRSTEA). The authors thank Pascale Heydemann, Amandine Julien, Agnès Orsoni, and Claire
Cordilhac from IFCE and Florence Gras from EHN for their input in this study.
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Horse Feeding

Special Issue Editor

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

ISBN 978-3-03928-552-5 (Pbk)

About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface to ”Horse Feeding and Management” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Markku Saastamoinen and Susanna Särkijärvi

Markku Saastamoinen, Susanna Särkijärvi and Elisa Valtonen

Joaquı́n Bull, Fernando Bas, Macarena Silva-Guzmán, Hope Helen Wentzel,

Malin Connysson, Marie Rhodin and Anna Jansson

Sara Ringmark, Anna Skarin and Anna Jansson

Dominique-Marie Votion, Anne-Christine François, Caroline Kruse, Benoit Renaud,

Katrina Merkies, Chloe Ready, Leanne Farkas and Abigail Hodder

Nicoletta Miraglia, Elisabetta Salimei and Francesco Fantuz

Agata Rzekeć, Céline Vial and Geneviève Bigot

Received: 30 October 2019; Accepted: 13 December 2019; Published: 14 December 2019

Animals 2019, 9, 1148; doi:10.3390/ani9121148 1 www.mdpi.com/journal/animals

2. Materials and Methods

Table 1. Nutrient composition of feedstuﬀs.

Coastal 4 Coastal 6 Coastal 8 Vit/Min Vit/Min

Coastal 4 Coastal 6 Coastal 8 Vit/Min Vit/Min

Table 2. Diet composition and nutrient intake of experimental diets 1 .

Item ALF ORCH CB 4 CB 6 CB 8

(CMRT2 ) was determined by 1/k2 . When n = 1, λ1 is replaced by k1 , and CMRT1 is an age-independent

3.1. Diet Digestibility

3.2. Fecal Marker Excretion

3.2.1. Marker Excretion and Recovery

Table 4. Particulate (Yb) and liquid (Co) marker recovery 1 (n = 5).

3.2.2. Modeling Fecal Marker Excretion

Variable Dhanoa a G1G1 b G2G1 b G3G1 b G4G1 b G5G1 b G6G1 b

AIC 128.8 163.1 148.1 142.3 138.7 134.3 137.0

3.3. Digesta Mean Retention Time

3.3.1. Mean Retention Time Calculated from Model Parameters

3.3.2. Arithmetically Calculated Mean Retention Time

3.3.3. Comparing Model-Derived and Arithmetically Calculated Mean Retention Time

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2615/9/12/1148/s1,

Received: 28 January 2020; Accepted: 7 February 2020; Published: 10 February 2020

Keywords: feeding; haematology; ﬂax seed; ﬁbre

Animals 2020, 10, 272; doi:10.3390/ani10020272 20 www.mdpi.com/journal/animals

2. Materials and Methods

2.1. Horses and their Management

2.2. Experimental Feeds and Feeding

Composition Hay Oats Feed 1 Feed 2

Feed Diet A (Control) Diet B (Feed 1) Diet C (Feed 2)

2.3. Feed and Faeces Sampling

2.4. Blood Sampling

2.5. Statistical Analysis

Yijk = μ + ai + tj + p(sq)k + εijk (1)

3.1. Feed and Nutrient Intakes

Intake Period I Period II Period III

3.2. Diet Digestibility

Intake Diet A (Control) Diet B (Feed 1) Diet C (Feed 2)

Diet B Diet C p Value

3.3. Blood Parameters

Diet B Diet C Reference Values

4.1. Feed and Nutrient Intakes

4.2. Diet Digestibility

4.3. Haematology Parameters

Received: 30 October 2019; Accepted: 12 January 2020; Published: 15 January 2020

Keywords: environment; horse nutrition; phosphorus loss; phosphorus supplementation;