Animal 18 (2024) 101203

Contents lists available at ScienceDirect

Animal
The international journal of animal biosciences

Effect of combining the methanogenesis inhibitor 3-nitrooxypropanol

and cottonseeds on methane emissions, feed intake, and milk production
of grazing dairy cows
C. Muñoz a,⇑, I.A. Muñoz a, R. Rodríguez a, N.L. Urrutia a, E.M. Ungerfeld b
a
Centro Regional de Investigación Remehue, Instituto de Investigaciones Agropecuarias, Ruta 5 km 8 norte, 5290000 Osorno, Región de Los Lagos, Chile
b
Centro Regional de Investigación Carillanca, Instituto de Investigaciones Agropecuarias, Camino Cajón-Vilcún km 10, 4880000 Temuco, Región de La Araucanía, Chile

a r t i c l e i n f o a b s t r a c t

Article history: No single enteric CH4 mitigating strategy has been consistently effective or is readily applicable to rumi-
Received 21 December 2023 nants in grassland systems. When CH4 mitigating strategies are effective under grazing conditions, mit-
Revised 16 May 2024 igation is mild to moderate at best. A study was conducted to evaluate the potential of combining two
Accepted 17 May 2024
CH4 mitigation strategies deemed feasible to apply in grazing dairy cows, the methanogenesis inhibitor
Available online 24 May 2024
3-nitrooxypropanol additive (3-NOP) and cottonseed supplementation (CTS), seeking to enhance their
individual CH4 mitigating potential. Forty-eight dairy cows were evaluated in a continuous grazing study
Keywords:
and supplemented with either a starch-based concentrate (STA) or one that contained cottonseeds
Lipids
Pasture
(1.75 kg DM/d; CTS), and with either 19 g/d of 10% 3-NOP (BovaerÒ) or the additive’s carrier (placebo),
Pulse-dosing in a 2
2 factorial arrangement of treatments. Treatments were supplied mixed with a concentrate sup-
Supplementation plement (5 kg/d as fed) and offered in two equal rations at milking. Methane emissions were measured
3-nitrooxypropanol on weeks 4 and 8 using the sulphur hexafluoride tracer gas technique over a 5-d period. The 3-NOP and
CTS treatments tended to interact on absolute CH4 such that 3-NOP decreased CH4 by 13.4% with STA, but
there was no mitigation with 3-NOP and CTS. Treatment interactions were also obtained for CH4 yield,
where 3-NOP tended to decrease CH4 when supplied with STA, and tended to increase it with CTS. The
increase in CH4 yield with the CTS diet was driven by a numerical decrease in DM intake. Methane inten-
sity was not affected by the 3-NOP or CTS treatments. Total volatile fatty acids in ruminal fluid were not
affected by 3-NOP supplementation, but a reduction in acetate and an increase in propionate proportion
occurred, resulting in decreased acetate: propionate. The 3-NOP additive decreased grass intake; how-
ever, energycorrected milk yield and milk composition were largely unaffected. Milk urea increased
with 3-NOP supplementation. Combining twice daily supplementation of 3-NOP and CTS did not enhance
their CH4 mitigation potential when fed to grazing dairy cows. The relatively low inhibition of CH4 pro-
duction by 3-NOP compared to studies with total mixed rations may result from the mode of delivery
(pulse dosed twice daily) and time gap caused by experimental handling and moving of animals to pas-
ture after 3-NOP supplementation in the milking parlour, which could have impaired the synchrony
between the additive presence in the rumen and grass intake in paddocks.
Ó 2024 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium. This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Implications likely reduced due to the time gap between its administration in
the milking parlour and the cows’ grass ingestion in the paddocks,
No methane mitigating strategy is currently applicable to graz- allowing 3-nitrooxypropanol metabolism in the rumen, and the
ing dairy cows. We proposed combining twice-daily supplementa- higher lipid and fibre content of cottonseeds. Continuous delivery
tion with 3-nitrooxypropanol and cottonseeds to enhance their alternatives for 3-nitrooxypropanol into the rumen are crucial to
effectiveness. We learned that 3-nitrooxypropanol tended to boost effectiveness in grazing systems.
mildly decrease methane emissions with a starch-based concen-
trate, but not with cottonseeds. The additive’s effectiveness was
Introduction

⇑ Corresponding author. In the last decade, several enteric CH4 mitigation strategies for
E-mail address: [email protected] (C. Muñoz). ruminants have been evaluated globally with varying degrees of

https://doi.org/10.1016/j.animal.2024.101203
1751-7311/Ó 2024 The Author(s). Published by Elsevier B.V. on behalf of The Animal Consortium.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

success (see meta-analysis by Arndt et al., 2022; and review by between them  be those synergistic, antagonistic, or wholly addi-
Beauchemin et al., 2022). Most of the work with dairy cows has tive (i.e., lack of interaction). Based on their differing mechanisms
been carried out in intensive confined production systems with for inhibiting CH4 production, we hypothesised that the combina-
high genetic merit cows and total mixed rations (TMR) with rela- tion of 3-NOP and cottonseed supplemented twice daily to grazing
tively high concentrate input (i.e. about 30–50% DM). Less work dairy cows would have an additive effect at decreasing CH4 yield
has taken place on pasture-based systems with dairy cattle of compared to those not given 3-NOP or cottonseed. The aim of this
lower production levels maintained mostly outdoors throughout study was to evaluate the effects of combining the supplementa-
the year and fed diets based on grazed grass, conserved forages, tion of the methanogenesis inhibitor 3-NOP and cottonseeds sup-
and lower concentrate supply levels. Since the 1950 ’s, only 15% plemented twice daily on CH4 emissions, feed intake, and milk
of the published in vivo experimental work on CH4 mitigation production of grazing dairy cows.
has been conducted under grazing conditions (Vargas et al.,
2022). In addition, effective CH4 mitigation strategies that are rel-
Material and methods
evant and have been validated for pasture systems provide only
mild to moderate mitigation, e.g. 17% CH4 intensity with increas-
The experiment was conducted in spring between October 25th
ing feeding level, 13% CH4 intensity with decreasing grass matu-
and December 19th of 2021, at INIA Remehue experimental farm
rity and 12% absolute CH4 when including tanniferous forages
(40°310 S; 73°030 W, Osorno, Chile).
(Arndt et al., 2022). And, they often decrease CH4 intensity but
increase absolute CH4 production. Two CH4 abatement strategies
that appear feasible to apply under grazing conditions and are clos- Animals
est to practical application by farmers are the CH4 mitigating addi-
tive 3-nitrooxypropanol (3-NOP) and cottonseed supplementation Forty-eight Holstein Friesian cows, 12 primiparous and 36 mul-
(Arndt et al., 2022; Hristov et al., 2022). tiparous, were used in this grazing study. Cows were blocked into
The additive 3-NOP is a compound that targets the enzyme 12 groups balanced according to parity (three groups of primi-
methyl coenzyme-M reductase, key for CH4 formation in the parous cows, and nine groups of multiparous cows with
rumen, inhibiting methanogenesis. Meta-analyses indicate that 2.7 ± 1.16 lactations; mean ± SD), lactation stage (73.0 ± 8.7
this compound is effective at decreasing the CH4 yield of dairy DIM), milk production (25.3 ± 3.2 kg) and BW (418 ± 29 kg for
cows compared to diets non-supplemented with 3-NOP in the primiparous and 467 ± 48 kg for the multiparous cows) at
confined-total mixed ration-based systems (38.8%; Dijkstra the beginning of the experiment, and within blocks, randomly allo-
et al., 2018; 30.9%; Kebreab et al., 2023), and its efficacy is both cated to one of four treatments.
dose- and diet-dependent (Dijkstra et al., 2018; Jayanegara et al.,
2018; Kebreab et al., 2023). Almost all 3-NOP studies have been Experimental design and diet
undertaken in intensive systems (tie stalls, free stalls or feedlots),
with a few reporting CH4 reductions with high forage diets fed to The study was conducted over 8 continuous weeks and had a
confined animals (Miller et al., 2023). In confinement systems, 3- 2
2 factorial arrangement of treatments under a completely ran-
NOP continuous supply to the rumen relies on the additive being domised block design.
well mixed with forages and concentrate in TMR and delivered The concentrate treatments were as follows: (i) a starchbased
continuously in every mouthful of feed, thus promoting a consis- supplement without cottonseed and with a 3-NOP placebo (STA,
tent fermentation and CH4 mitigating pattern. In grazing systems, PBO); (ii) a STA supplement with 19 g/d of the 3-NOP-containing
supplements are delivered usually once or twice daily, or even additive BovaerÒ 10 (DSM Nutritional Products AG, Kaiseraugst,
once every few days in more extensive systems where animal Switzerland) (STA, 3-NOP); (iii) a supplement with 1.75 kg DM/d
management is less frequent. Few studies with 3-NOP have been of whole cottonseed with lint and PBO (CTS, PBO); and (iv) a CTS
conducted providing the additive in a supplement that the animal supplement with 3-NOP (CTS, 3-NOP). The 3-NOP additive con-
may consume only once or twice a day. Reynolds et al. (2014) tained a minimum of 10% (w/w) 3-NOP carried in propylene glycol
dosed cows fed TMR twice daily with 3-NOP administered through and silicon dioxide (so cows were supplied 1.9 g 3-NOP daily) to
the rumen cannula, and Muetzel et al. (2019) dosed cows fed grass achieve a 3-NOP target dose rate of 95 mg/kg DM calculated con-
in confinement twice daily with 3-NOP administered mixed into a sidering an estimated intake of 20 kg DM/cow d1. A relatively
supplement. To our knowledge, no published studies at the time of higher dose than the widely reported 3-NOP inclusion target of
writing have evaluated the use of 3-NOP under grazing conditions. 60–80 mg/kg DM with TMR was chosen in this study to account
Supplementation of dietary lipids is an effective CH4 mitigation for the additive being fed only twice daily instead of continuously.
strategy (Beauchemin et al., 2020; Arndt et al., 2022; Beauchemin The 3-NOP dose targeted was also based on previous reports of
et al., 2022) that acts through lowering the amount of fermentable decreased CH4 from cattle that were fed high-forage diets (36.4%
organic matter in the rumen, having toxic effects on methanogens NDF; Vyas et al., 2016). The placebo contained the 3-NOP carrier
and protozoa, inhibiting H2 producing bacteria, and by offering an at the same amount as the 3-NOP treatment.
alternative ruminal electron sink via biohydrogenation of unsatu- Diets consisted of ad libitum grazed grass supplemented with
rated fatty acids (Beauchemin et al., 2009). In the grazing systems 5 kg/d (as fed basis) of a concentrate according to treatments
of southern Chile, cottonseed is a feed ingredient that is reasonably offered in two equal rations in the milking parlour (Table 1). Both
accessible to dairy farmers. Compared to whole linseed and rape- the STA and CTS supplements were based on ground corn. In the
seed, cottonseed as a source of lipids was more effective in STA supplement (which was used as a control for the CTS treat-
decreasing the CH4 yield of dairy cows fed TMR (Muñoz et al., ment), ground barley and rapeseed meal replaced cottonseeds to
2019) and grazed grass (Muñoz et al., 2021). Others have also obtain at formulation isoenergetic (13.4 MJ estimated metabolis-
reported moderate decreases in CH4 yield and intensity with cot- able energy/kg DM) and isonitrogenous (14% CP) supplements.
tonseed (Grainger et al., 2008b; Grainger et al., 2008a; Grainger Both STA and CTS concentrates included a vitamin and mineral
et al., 2010). premix. Prior to the beginning of the study, the four concentrate
One alternative to increase the effectiveness of 3-NOP and cot- supplements were manufactured by homogenously mixing all
tonseed supplemented separately to mitigate CH4 is to evaluate the ingredients (including the 3-NOP additive and the placebo) and
effects of their combination and identify possible interactions stored in 25-kg feed bags. One exception to this was the inclusion
2
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

Table 1
Ingredient and chemical composition of pasture and treatment supplements1 offered to grazing dairy cows.

Pasture STA concentrate CTS concentrate

Items Week 4 Week 8 PBO 3-NOP PBO 3-NOP
Ingredient, % DM
Ground corn – – 56.4 56.4 55.7 55.7
Whole cottonseed with lint – – – – 39.1 39.1
Rapeseed meal – – 15.2 15.2 – –
Ground barley – – 23.1 23.2 – –
Placebo2 – – 0.39 – 0.39 –
Formulated 3-NOP product3 – – – 0.39 – 0.39
Mineral and vitamin premix4 – – 4.88 4.88 4.82 4.82
Chemical composition
DM, % 14.1 16.8 88.7 88.9 90.7 90.7
Ash, % DM 11.5 10.9 7.05 7.22 6.22 5.60
CP, % DM 21.3 21.4 13.5 13.3 14.5 15.5
Starch, % DM – – 49.3 50.2 28.8 25.4
Ether extract, % DM 3.26 2.98 1.44 1.33 14.6 13.7
NDF, % DM 40.7 40.6 16.2 16.5 24.1 25.1
ADF, % DM 25.4 26.6 – – – –
In vitro digestibility, % DM 81.4 76.6 89.4 89.3 74.6 72.6
1
Concentrates without cottonseed (STA), with whole cottonseed with lint (CTS), and with a placebo (PBO) or the methanogenesis inhibitor 3-nitrooxypropanol (3-NOP).
Mean values from composite samples of concentrates from weeks 1–4 and 6–8 of the study.
2
3-NOP carrier: SiO2 and propylene glycol.
3
BovaerÒ 10 (DSM Nutritional Products AG, Kaiseraugst, Switzerland) composition: 10% (w/w) 3-NOP carried in SiO2 and propylene glycol.
4
Containing per kg: Ca 200 g, P 50 g, Mg 50 g, Cl 90 g, Na 60 g, S 20 g, Cu 1 200 mg, Mn 2 000 mg, Zn 5 500 mg, I 100 mg, inorganic Se 25 mg, Co 10 mg, organic Se 10 mg,
vitamin A 100 000 IU, vitamin D 50 000 IU, vitamin E 1 000 IU (Vetersal Lechería Alta Producción, Veterquímica, Osorno, Chile).

of the whole cottonseed with lint in the CTS supplements which atmospheric pressure (around 120 kPa) with N2, left to rest for at
could not be homogenously mixed at the manufacturing plant least 1 h, prior to sub-sampling into four evacuated chromatogra-
and was added to rations daily using a cement mixer. phy vials. Daily samples were analysed in duplicate by gas chro-
Cows grazed perennial ryegrass-based pasture as their main matography with auto-sampler (Perkin Elmer Clarus 600;
diet component. A grazing area of 16 ha divided into three main Waltham, MA, USA). Methane was separated using a Carboxen-
paddocks was used in the study. Each paddock was subdivided into 1010 plot column, 15 m
0.32-mm ID (Supelco, Sigma-Aldrich,
four equal sub-paddocks and assigned to treatment groups which St. Louis, MO, USA) and a flame ionisation detector operating at
strip-grazed separately. Daily, following the morning milking, each 250 °C. The SF6 gas was separated using an Elite GS Molesieve col-
group was offered a grazing area with a fresh strip of grass. Electric umn, 30 m
0.53-mm ID
50-lm film thickness (Perkin Elmer,
fences in front and behind each group limited access to the area Waltham, MA, USA) and an electron captor detector operating a
grazed the previous day. There were 2.5 grazing rotations during 300 °C. Methane emissions were calculated as the product of the
the study i.e. cows went back to plots previously grazed. The dis- SF6 permeation tube release rate and the ratio of CH4:SF6 concen-
tance covered by the cows between the milking parlour, manage- tration in samples, adjusted for background gas concentrations
ment chute and furthest grazing paddock was 1.1 km. Cows had (Muñoz et al., 2015). Individual daily CH4 emission was averaged
access to fresh clean water, supplied by mobile troughs in each over the 5 days of measurement, prior to statistical analysis.
grazing strip throughout the study. Cows were milked twice daily The CH4 conversion factor was calculated as: [Ym; CH4 produc-
at 0630 and 1600 h. tion (g/d)
1.396 (g/L)
0.03954 (MJ/L)  gross energy intake
(MJ/d)
100)].
Sampling and measurements Grass intake was measured on the same 5 days as CH4 produc-
tion (weeks 4 and 8), using the double n-alkane technique (Mayes
Methane measurements were done in weeks 4 and 8 of the et al., 1986). Twice daily (after milking) cows were orally dosed
study using the sulphur hexafluoride (SF6) tracer gas technique using a balling gun with cellulose bungs containing the synthetic
as described by Muñoz et al. (2021). Permeation tubes were filled alkane C32. An exception to this was that, on the first day of dosing,
by National Institute of Water and Atmospheric Research (Auck- cows received two boluses in the morning and one bolus in the
land, New Zealand) in October 2020 with an initial SF6 charge of afternoon, and on the last day of dosing, cows received only the
2733 ± 73 mg. The SF6 release rates after 8 weeks of calibration morning bolus. Cellulose bungs contained on average 414 ± 36.9
at 39 °C were 4.2 ± 0.86 mg/d. The permeation tubes were dosed and 431 ± 40.3 mg of alkane C32 in periods 1 and 2, respectively.
orally to cows in week 3 of the study. On the first measurement Alkane C32 was dosed for 12 d, 7 d to reach a steady state and 5
day, after the morning milking and treatment supplementation, d for measurement where twice daily faeces collection was carried
the CH4 measurement equipment was placed on each cow. Each out after milking by faeces voiding or rectal grab. At the end of the
sampling unit consisted of one evacuated V-shaped PVC canister collection period, the refrigerated faeces samples were combined
positioned on the cows’ neck connected to a sampling line contin- per cow and period, dried at 60 °C for 48 h, and ground through
ually sampling the eructed and exhaled gases from near to the a 1-mm sieve prior to alkane concentration determination. On fae-
cow’s muzzle. The air flow to the canister was restricted by a cal collection days, samples of the concentrate and grass per treat-
crimped capillary tube fitted within an in-line air filter to a sam- ment were collected daily and combined per treatment, dried, and
pling rate of 0.36 SCCM. In addition, four sampling units were ground as described for faeces. N-alkanes C31, C32, C33 and C35 con-
placed outside the grazing area to measure ambient SF6 and CH4 centration in feed and faeces samples were determined using gas
gases. All canisters were replaced after 24 h, and this was repeated chromatography as by Martínez et al. (2017). Briefly, samples were
daily for 5 days. The samples in canisters were pressurised to above ground to 10 lm, and the n-alkanes were extracted and quantified
3
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

using GC (GC 2010 Plus; Shimadzu, Kyoto, Japan) fitted to a flame manually separating them into ryegrass, clover, dead material,
ionisation detector using helium as the carrier gas. The column was and other species, on a DM basis (60 °C for 48 h).
a 30 m
0.53 mm (i.d.) capillary RTX-1 (RestekÒ, Bellefonte, Penn- Grass and concentrate samples for chemical analysis were col-
sylvania, USA) with 1.50 lm film thickness. Grass intake was calcu- lected daily in CH4 measurement weeks, pooled weekly per treat-
lated according to equation by Mayes et al. (1986): ment, dried at 60 °C for 48 h, and ground through a 1-mm sieve
Fi

using a Wiley mill, prior to chemical analysis. The nutrient compo-
Fj
Dj þ I c
C j  I c
C j sition of the samples was analysed based on the following meth-
Grass intake ðkg DM=dÞ ¼
Gi  ðFF i
Gj Þ ods: nitrogen (N) analysed in duplicate and CP calculated as
j
N
6.25 (Method 2001.11; AOAC International, 2016); NDF
where Gi and Fi are the respective concentrations (mg/kg DM) of the expressed including residual ash as by Van Soest et al. (1991);
alkane C33 in grass and faeces; Gj and Fj are the respective concen- ADF expressed including residual ash as by Goering and Van
trations of the alkane C32 in grass and faeces; Dj is the amount of Soest (1970); ash as by Bateman (1970); ether extract determined
alkane j dosed (mg/d). using a Soxtec system HT6 following the manufacturer manual;
Using alkanes C31, C33 and C35 as internal markers, apparent DM starch based on AOAC Official Method 996.11; AOAC
digestibility estimates were calculated by subtracting to one the International (2016), and in vitro digestibly (IVD) as by Goering
alkane concentration in feed over the alkane concentration in fae- and Van Soest (1970). Gross energy was calculated based on chem-
ces. Faecal alkane concentrations were corrected for incomplete ical composition as per FAO (2003).
recovery using the recovery rates published by Dillon (1993) which
were 0.777 (C31), 0.844 (C33) and 0.891 (C35). The digestibility esti- Statistical analyses
mates were obtained as an average of the three alkanes.
Twice daily individual milk yield was automatically recorded at In week 4, one cow from the STA 3-NOP treatment died sud-
each milking and cow BW was measured using an automatic scale denly at pasture without apparent clinical signs and, as determined
when leaving the milking parlour. These individual measurements by necropsy, by causes unrelated to treatment, and was removed
were averaged weekly per cow throughout the study (DeLaval from the dataset. Also, a cow from the CTS PBO treatment devel-
Alpro MM15; DeLaval International, Tumba, Sweden). On weeks oped and received treatment for mastitis in weeks 7 and 8 of the
1, 3, 4, 6 and 8 of the study, individual milk samples were collected study and her records for those weeks were excluded. Animal data
at two consecutive milkings, combined for each cow according to from the first 2 weeks of the experiment were omitted from the
yield, and stored refrigerated using bronopol as a preservative until analysis as considered for adaptation to treatments.
analyses. Milk fat, milk protein, and milk urea concentrations were Repeated measurements (CH4, DM intake, DM digestibility, milk
determined using IR spectroscopy (Milkoscan FT6000, Foss Electric, yield and composition, rumen fermentation and BW) were initially
Hillerød, Denmark). To estimate energycorrected milk (ECM) analysed using a mixed model including the main effects of supple-
yield, milk production was adjusted for energy using the equation mentation with 3-NOP and cottonseed, their interaction, the main
by Tyrrell and Reid (1965). effects of week and parity (primiparous or multiparous) and their
In week 6 of the study, rumen fluid was collected from all cows double and triple interactions with 3-NOP and cottonseed
after the morning and evening milkings at 0830 and 1730 h using a supplementation:
stomach tube (Ruminator; profs-products.com (accessed on 06
response ¼ ov erall mean þ 3NOP þ cottonseed þ ð3NOP
March 2022)) and the first 150 mL discarded to avoid saliva con-
tamination. The individual collection time was recorded. Rumen
cottonseedÞ þ week þ ðweek
3NOPÞ þ ðweek
samples were filtered, and rumen pH was immediately measured
cottonseedÞ þ ðweek
3NOP
cottonseedÞ þ parity
(ExStik pH Meter, Extech Instruments, Boston, U.S.). Samples were
frozen (20 °C) until analysed. Volatile fatty acid concentration
þ ðparity
3NOPÞ þ ðparity
cottonseedÞ þ ðparity
was determined from a 1 mL sample aliquot preserved with
3NOP
cottonseedÞ þ cow ðrandomÞ þ error
0.2 mL of 20% (m/v) meta-phosphoric acid by gas chromatography
(Perkin Elmer Clarus 580, PerkinElmer, Waltham, US) using a cap- For each treatment factor, n = 12. Because parity only interacted
illary column (Elite-FFAP; PerkinElmer, Shelton, CT, USA) and flame with 3-NOP or cottonseed on total volatile fatty acid (VFA) concen-
ionisation detector (Ungerfeld et al., 2019). tration (P = 0.003), parity was left in the model for rumen variables
All pasture characteristics were assessed by measurements con- only, which also included time of sampling (am or pm), and the
ducted in samples cut at 3 cm from the ground (Pérez-Prieto et al., double and triple interactions between 3-NOP and cottonseed with
2012). Pregrazing herbage mass was measured per treatment twice time of the day and with parity:
weekly by cutting a 1
5 m strip of grass with a motor scythe in
response ¼ ov erall mean þ 3NOP þ cottonseed þ time þ parity
the area due to be grazed, collecting, and weighing all mown pas-
ture, and determining its DM (subsample dried at 60 °C for 48 h). þ ð3NOP
cottonseedÞ þ ð3NOP
timeÞ þ ð3NOP
Four measurements of grass height were made per strip with a ris-
parityÞ þ ðcottonseed
timeÞ þ ðcottonseed
parityÞ
ing platemeter (F200 Farmworks, Feilding, New Zealand) before
and after cutting. Pasture density was determined by dividing
þ ð3NOP
cottonseed
timeÞ þ ð3NOP
cottonseed
the pregrazing mass by the cutting depth. Estimates of forage mass
parityÞ þ cow ðrandomÞ þ error
pregrazing and postgrazing were carried out daily by multiplying
the grass height (measured with a rising platemeter over 60 times For all other animal response variables, a reduced model was used
crisscrossing the area) by the grass density. The daily grazing area including the fixed main effects and double and triple interactions
of each treatment group was calculated based on the daily esti- of 3-NOP, cottonseed, and week, and the random effect of cow:
mate of available forage adjusted according to an initial predeter-
response ¼ ov erall mean þ 3NOP þ cottonseed þ week
mined allowance of 22 kg DM/cow. As the study progressed, the
grazing areas were adjusted to maintain a target postgrazing þ double interactions þ triple interaction
height of around 6 cm compressed herbage. Botanical composition þ cow ðrandomÞ þ error
was determined on measurement weeks through samples of pas-
ture obtained by cutting at ground level 15 handfuls of grass and
4
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

Milk yield and BW in the 2-wk period immediately prior to the DM digestibility (P < 0.001), where with the CTS concentrate, 3-
beginning of the trial were used as covariables of milk yield and NOP decreased DM digestibility (P < 0.001), but not with the STA
BW change, respectively. The effect of treatments on pasture char- concentrate (P = 0.11).
acteristics was determined including the fixed main effects and
interactions of 3-NOP, cottonseed and measurement date. For all Methane emissions
variables, significance for fixed effects and interactions was
declared at P < 0.05 and tendencies at 0.05  P < 0.10. When inter- Treatments 3-NOP and CTS tended to interact on absolute CH4
actions were significant or tendencies identified, the PBO and 3- (P = 0.050), where with the STA concentrate, 3-NOP numerically
NOP treatments were separately compared within the STA and decreased CH4 by 13.4%, but not with the CTS concentrate (Table 4).
CTS treatments. There was an interaction between 3-NOP and CTS on CH4 yield
Outliers were identified as those observations falling outside of (P = 0.011), where 3-NOP tended to decrease CH4 yield by 9.7%
the 99% distribution of studentised residuals. Outliers so identified compared to PBO with the STA concentrate (P = 0.067) and tended
were examined for obvious typos or mistakes, and non- to increase it by 10.2% with the CTS concentrate (P = 0.066). A sim-
physiological values. The DM intake of two cows in week 8 of the ilar response was observed on CH4 as a proportion of ingested
study was unusually low (6.75 kg/d) or high (30.3 kg/d), and those gross energy (Ym; interaction P = 0.011). For treatment interac-
observations were discarded for the DM intake, digestibility, and tions on CH4 yield and Ym, additional contrasts, conducted between
CH4 variables analyses. Because the reason for the unusually high CTS and STA within the PBO treatment, showed no effects of CTS
or low DM intake was clearly related to DM intake estimation supplementation (P = 0.23; contrast not shown). Supplementing
but not to those two animals themselves, which had a DM intake 3-NOP had no effects on CH4 intensity (P = 0.20) or CH4 over
within the main range in week 4, those observations were retained ECM yield (P = 0. 56). Supplementing CTS had no effects on CH4
for the analyses of all other response variables. For all other intensity (P = 0.10) or CH4 expressed over ECM yield (P = 0.40).
response variables where outliers with physiological values were
identified, each analysis was run again excluding them. As the con-
clusions of the analysis did not change substantially when outliers Rumen fermentation
within physiological values were removed, the results are pre-
sented including those observations. All data were analysed using Rumen pH tended to be lower with 3-NOP than with PBO only
JMP 17.2.1 (SAS Institute Inc., Cary, NC) software. in the afternoon sampling and with the STA supplement (interac-
tion 3-NOP
CTS
Time, P = 0.028; Table 5). There were no other
significant 3-way interactions between treatments and time on
Results rumen fermentation. There tended to be an interaction between
3-NOP and CTS on isobutyrate molar percentage with 3-NOP
Diet chemical composition numerically increasing isobutyrate with the CTS supplement, but
not with the STA supplement (P = 0.097). There tended to be an
The chemical composition of pasture and concentrates are pre- interaction between 3-NOP and CTS on valerate molar percentage
sented in Table 1. As the trial progressed from week 4 to week 8, with 3-NOP numerically decreasing valerate with the STA supple-
the pasture had a similar composition of CP and NDF, increased ment, and numerically increasing it with the CTS supplement
DM and ADF, and decreased ether extract and IVD. Compared to (P = 0.088). Supplementing 3-NOP decreased acetate (P < 0.001)
STA, the CTS concentrate had higher proportions of CP, ether and increased propionate (P = 0.002) molar percentage, resulting
extract and NDF, and lower starch and IVD. The concentrates con- in a decreased acetate to propionate ratio (P < 0.001). Molar per-
taining PBO or 3-NOP were very similar to each other. The calcu- centage of butyrate (P = 0.008) and 2- and 3-methylbutyrate
lated chemical composition of the experimental diets is (P = 0.005) were increased by 3-NOP, and 3-NOP had no effects
presented in Table 2. In weeks 4 and 8 of the study, ingested diets on total VFA concentration (P = 0.21) or caproate (P = 0.34) molar
had numerically similar CP contents and CTS diets had higher ether percentage. Supplementing CTS decreased total VFA concentration
extract content. The NDF content of the CTS diet was numerically (P < 0.001), increased acetate (P < 0.001), and decreased propionate
somewhat higher than the STA diet. (P = 0.015), thus increasing the acetate to propionate ratio
(P = 0.002). The CTS supplement had no effects on molar percent-
Feed intake and diet DM digestibility ages of butyrate (P = 0.11), 2- and 3-methylbutyrate (P = 0.30) or
caproate (P = 0.14).
Supplementing 3-NOP decreased pasture intake (P = 0.003) and
total DM intake (P = 0.003), and increased diet concentrate propor- Milk production, milk composition and performance
tion (P = 0.005; Table 3). Supplementing CTS had no effects on pas-
ture intake, total DM intake or concentrate proportion (all The 3-NOP and CTS treatments tended to interact on milk yield
P  0.39). There was an interaction between 3-NOP and CTS on diet (P = 0.078) with 3-NOP numerically decreasing milk yield with the

Table 2
Chemical composition of the experimental diets ingested by dairy cows offered grazed grass supplemented with concentrates without cottonseed (STA) or with whole cottonseed
with lint (CTS) on weeks 4 and 8 of the study1,2.

Week 4 Week 8
Items STA CTS STA CTS
DM, % 35.3 38.3 37.8 39.2
CP, % DM 19.1 19.5 19.0 19.5
Ether extract, % DM 2.73 6.35 2.50 6.35
NDF, % DM 33.8 35.8 33.3 35.8
Ash, % DM 10.3 9.39 9.76 9.39
1
Calculated as ingestion of nutrients in dietary components divided by DM intake of components.
2
Placebo and 3-Nitrooxypropanol additives were assumed not to alter chemical composition of diets.

5
 C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

Table 3
Diet intake and digestibility of grazing dairy cows supplemented concentrates without cottonseed (STA), with whole cottonseed with lint (CTS), and with a placebo (PBO) or the
methanogenesis inhibitor 3-nitrooxypropanol (3-NOP).

STA CTS P-value1,2

Items PBO 3-NOP PBO 3-NOP SEM INH CNT INH
CNT
Pasture intake, kg DM/d 11.1 10.3 11.5 9.87 0.37 0.003 0.95 0.26
Concentrate offered, kg DM/d 4.44 4.45 4.54 4.54 – – – –
Total estimated intake, kg DM/d 15.5 14.8 16.0 14.4 0.37 0.003 0.84 0.25
Concentrate proportion, kg DM/kg DM 0.290 0.303 0.288 0.318 0.007 0.005 0.39 0.27
Diet DM digestibility 0.684a 0.715a 0.722y 0.654x 0.013 0.16 0.38 <0.001

Abbreviations: INH = CH4 inhibitor effect; CNT = concentrate type effect; INH
CNT = CH4 inhibitor and concentrate type interaction.
1
Main effects of week for all variables: P < 0.001. CNT
week interaction for all variables: P  0.45, except diet digestibility (P < 0.001). INH
week interaction for all
variables: P  0.026, except diet digestibility (P = 0.41). CNT
INH
week interaction for all variables: P  0.22, except diet digestibility (P = 0.004).
2
Diet DM digestibility contrast between PBO and 3-NOP within the STA concentrate (P = 0.11), and within the CTS concentrate (P < 0.001).
a
Within the STA concentrate, values within a row with different superscripts differ (P < 0.05) due to INH
CNT interaction.
x,y
Within the CTS concentrate, values within a row with different superscripts differ (P < 0.05) due to INH
CNT interaction.

Table 4
Methane emissions of grazing dairy cows supplemented concentrates without cottonseed (STA), with whole cottonseed with lint (CTS), and with a placebo (PBO) or the
methanogenesis inhibitor 3-nitrooxypropanol (3-NOP).

STA CTS P-value1,2

Items PBO 3-NOP PBO 3-NOP SEM INH CNT INH
CNT
CH4, g/d 319 276 307 307 10.7 0.054 0.39 0.050
CH4 yield, g/kg DM intake 20.8a 18.8a 19.5x 21.5x 0.75 0.99 0.35 0.011
CH4 intensity, g/kg milk yield 13.6 13.0 14.7 13.8 0.57 0.20 0.10 0.83
CH4, g/kg energy-corrected milk 13.7 13.4 14.4 13.9 0.68 0.56 0.40 0.90
YM, MJ CH4/100 MJ GE intake3 6.68a 6.04a 6.27x 6.92x 0.24 0.99 0.35 0.011

Abbreviations: INH = CH4 inhibitor effect; CNT = concentrate type effect; INH
CNT = CH4 inhibitor and concentrate type interaction; Ym = CH4 conversion factor.
1
Main effects of week for all variables: P  0.011, except absolute CH4 (P = 0.43). CNT
week interaction for all variables: P  0.63, except for CH4 intensity (P = 0.074) and
CH4 intensity of energycorrected milk yield (P = 0.074). INH
week interaction for all variables: P  0.22. CNT
INH
week interaction for all variables: P  0.27.
2
CH4 yield contrast between PBO and 3-NOP within the STA concentrate (P = 0.067), and within the CTS concentrate (P = 0.066). YM contrast between PBO and 3-NOP
within the STA concentrate (P = 0.070), and within the CTS concentrate (P = 0.064).
3
Ym calculated as [CH4 production (g/d)
1.396 (g/L)
0.03954 (MJ/L)  gross energy intake (MJ/d)
100)].
a
Within the STA concentrate, values within a row with different superscripts differ (P < 0.05) due to INH
CNT interaction.
x
Within the CTS concentrate, values within a row with different superscripts differ (P < 0.05) due to INH
CNT interaction.

Table 5
Rumen fermentation characteristics of grazing dairy cows supplemented concentrates without cottonseed (STA), with whole cottonseed with lint (CTS), and with a placebo (PBO)
or the methanogenesis inhibitor 3-nitrooxypropanol (3-NOP).

STA CTS P-value1,2,3

Items PBO 3-NOP PBO 3-NOP SEM INH CNT INH
CNT INH
CNT
time
Rumen pH
a.m. 7.30m 7.27m 7.51n 7.29n 0.91 0.32 0.99 0.74 0.028
p.m. 7.12o 6.77o 6.85p 6.80p
Total volatile fatty acids, mM 118 109 90.4 87.5 4.52 0.21 <0.001 0.52 0.20
Acetate, mol/100 mol 64.5 60.8 66.7 63.5 0.61 <0.001 <0.001 0.68 0.21
Propionate, mol/100 mol 15.2 17.0 13.7 15.7 0.53 0.002 0.015 0.82 0.59
Butyrate, mol/100 mol 16.9 19.0 16.8 17.5 0.47 0.008 0.11 0.15 0.10
Isobutyrate, mol/100 mol 0.89 0.87 0.85 0.95 0.034 0.25 0.52 0.097 0.92
2- and 3-methylbutyrate, mol/100 mol 0.97 1.09 0.96 1.23 0.062 0.005 0.30 0.25 0.52
Valerate, mol/100 mol 1.33 1.04 0.79 0.84 0.093 0.22 <0.001 0.088 0.13
Caproate, mol/100 mol 0.23 0.23 0.17 0.22 0.026 0.34 0.14 0.33 0.71
Acetate/Propionate, mM/mM 4.29 3.66 5.02 4.20 0.18 <0.001 0.002 0.60 0.58

Abbreviations: INH = CH4 inhibitor effect; CNT = concentrate type effect; INH
CNT = CH4 inhibitor and concentrate type interaction.
1
Main effects of time (a.m. or p.m. sampling) for all variables: P < 0.001, except for pH (P = 0.80) and butyrate (P = 0.55). INH
time (a.m. or p.m. sampling) interaction for
all variables: P  0.10, except for 2- and 3-methylbutyrate (P = 0.050). CNT
time (a.m. or p.m. sampling) interaction for all variables: P  0.12, except for pH (P = 0.037), total
volatile fatty acids (P = 0.076), acetate (P = 0.018), caproate (P = 0.014) and acetate/propionate (P = 0.016).
2
Main effects of parity (primiparous or multiparous) for all variables P  0.16, except for butyrate (P = 0.065). INH
parity interaction for all variables: P  0.23, except
rumen pH (P = 0.057). CNT
parity interaction for all variables: P  0.10, except for total VFA (P = 0.003). CNT
INH
parity interaction for all variables: P  0.24, except for
total VFA (P = 0.078).
3
pH contrast between PBO and 3-NOP within the STA concentrate in a.m. sampling (P = 0.90), and in p.m. sampling (P = 0.066), and within the CTS concentrate in both a.m.
and p.m. sampling (P  0.22).
m,n,o,p
Within concentrates at a set sampling time (a.m. or p.m.), values within a row with different superscripts differ (P  0.10) due to INH
CNT
time interaction.

STA concentrate, but not with the CTS concentrate (Table 6). Sup- the concentration of milk fat (P = 0.40) or protein (P = 0.46). Sup-
plementing 3-NOP had no effects on the yield of milk fat plementing 3-NOP increased milk urea by 6.6% (P = 0.046). BW
(P = 0.30), protein (P = 0.70) or ECM (P = 0.36), nor did it affect change (P = 0.35) and milk somatic cell count (P = 0.68) were not

6
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

Table 6
Milk production and composition of grazing dairy cows supplemented concentrates without cottonseed (STA), with whole cottonseed with lint (CTS), and with a placebo (PBO) or
the methanogenesis inhibitor 3-nitrooxypropanol (3-NOP).

STA CTS P value1,2

Items PBO 3-NOP PBO 3-NOP SEM INH CNT INH
CNT
Milk and component yield, kg/d
Whole milk 23.3 21.9 21.7 22.1 0.52 0.37 0.18 0.078
Fat 0.93 0.89 0.92 0.88 0.040 0.30 0.80 0.87
Protein 0.84 0.80 0.79 0.81 0.026 0.70 0.35 0.27
Energy-corrected milk 24.2 23.1 23.4 23.0 0.82 0.36 0.58 0.62
Milk composition
Fat, % 3.81 3.88 4.02 3.75 0.12 0.40 0.77 0.17
Protein, % 3.43 3.50 3.45 3.45 0.047 0.46 0.67 0.48
Milk urea, mg/L 341 357 320 350 11.3 0.046 0.22 0.54
BW change, kg/d 0.45 0.44 0.21 0.42 0.10 0.35 0.22 0.31
Milk Log SCC5 1.61 1.74 1.80 1.77 0.11 0.68 0.30 0.45

Abbreviations: INH = CH4 inhibitor effect; CNT = concentrate type effect; INH
CNT = CH4 inhibitor and concentrate type interaction; SCC = somatic cell count.
1
Main effects of week for all variables: P < 0.001, except milk log SCC (P = 0.025). CNT
week interaction for all variables: P  0.053, except milk log SCC (P = 0.11).
INH
week interaction for all variables: P  0.27, except milk protein %, milk urea and milk log SCC (all P  0.094). CNT
INH
week interaction for all variables: P  0.095,
except milk fat, milk Log SCC, and milk protein yield (all P  0.17).
2
Milk yield contrast between PBO and 3-NOP within the STA concentrate (P = 0.065), and within the CTS concentrate (P = 0.52).

affected by 3-NOP. Supplementing CTS had no effects on the yield Discussion

of milk fat (P = 0.80), protein (P = 0.35) or ECM (P = 0.58), nor did it
affect the concentration of milk fat (P = 0.77), protein (P = 0.67) or Effects on rumen fermentation and CH4 emissions
urea (P = 0.22). The CTS concentrate had no effects on BW change
(P = 0.22) or milk somatic cell count (P = 0.30). In the present study, the interactions found between the 3-NOP
and CTS treatments on CH4 emissions disproved our hypothesis.
Pasture characteristics That is, there were no additive effects on CH4 mitigation between
the two strategies evaluated when supplemented twice daily to
There was an interaction between 3-NOP and CTS on pasture grazing dairy cows. A previous study evaluated the combined
allowance (P = 0.002) with 3-NOP increasing pasture allowance effects of 3-NOP and lipids on CH4 emission. The authors reported
with the STA concentrate (P = 0.019) and decreasing it with the effective methanogenesis inhibition by both 3-NOP (32% decrease)
CTS concentrate (P = 0.035; Table 7). Supplementing 3-NOP had and canola oil (25% decrease) on their own, and their combination
no effects on pasture pregrazing mass (P = 0.19), pregrazing height resulted in additive (52% decrease) CH4 emission mitigation (Zhang
(P = 0.68), density (P = 0.60) or the grazing area offered to adjust for et al., 2021). In that study, there were no interactions between the
equal pasture availability (P = 0.45). Pasture postgrazing height strategies evaluated and both strategies were independently effec-
was lower with 3-NOP (P = 0.009). Supplementing CTS had no tive in decreasing CH4. This contrasts with our study where the
effects on pasture pregrazing mass (P = 0.55), pregrazing height individual main effects of each strategy on CH4 yield were not sig-
(P = 0.19), density (P = 0.51), grazing area offered (P = 0.38), and nificant, and the mitigation strategies did not act independently.
postgrazing height (P = 0.43). Recently, (Maigaard et al., 2024) reported decreases in CH4 yield

Table 7
Pasture characteristics and botanical composition as affected by grazing dairy cows supplemented concentrates without cottonseed (STA), with whole cottonseed with lint (CTS),
and with a placebo (PBO) or the methanogenesis inhibitor 3-nitrooxypropanol (3-NOP).

Items3 STA CTS P value1,2

PBO 3-NOP PBO 3-NOP SEM INH CNT INH
CNT
Pregrazing mass, kg of DM/ha 1 994 2 316 1 995 2 119 158.7 0.19 0.55 0.54
Pregrazing height, cm 11.7 11.9 11.6 11.1 0.32 0.68 0.19 0.28
Density, kg DM/ha per cm 282 288 292 318 29.1 0.60 0.51 0.74
Area offered, m2/cow per day 75.6 80.3 74.3 75.1 3.69 0.45 0.38 0.60
Pasture allowance4, kg DM/cow per day 17.2b 19.6a 18.3x 16.2y 0.71 0.87 0.11 0.002
Postgrazing height, cm 6.3 5.6 6.3 6.0 0.20 0.009 0.43 0.30
Botanical composition4
Lolium perenne 0.69 0.56 0.64 0.64 – – – –
Trifolium repens 0.11 0.15 0.13 0.14 – – – –
Other species 0.02 0.05 0.03 0.03 – – – –
Dead material 0.09 0.10 0.11 0.13 – – – –

Abbreviations: INH = CH4 inhibitor effect; CNT = concentrate type effect; INH
CNT = CH4 inhibitor and concentrate type interaction.
1
Main effects of week for all variables: P < 0.001, except pregrazing pasture mass (P = 0.68) and density (P = 0.086). CNT
week interaction for all variables: P  0.18.
INH
week interaction for all variables: P  0.59. CNT
INH
week interaction for all variables: P  0.12, except pregrazing mass (P = 0.007).
2
Pasture allowance contrast between PBO and 3-NOP within the STA concentrate (P = 0.019), and within the CTS concentrate (P = 0.035).
3
Measured > 3 cm.
4
Mean values from 2 composite samples per treatment in CH4 measurement weeks.
a,b
Within the STA concentrate, values within a row with different superscripts differ (P < 0.05) due to INH
CNT interaction.
x,y
Within the CTS concentrate, values within a row with different superscripts differ (P < 0.05) due to INH
CNT interaction.

7
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

of 6–7% with supplementation of cracked rapeseeds, 12–13% with Jayanegara et al., 2018; Kim et al., 2020; Almeida et al., 2021), 3-
nitrate and 18–23% with 3-NOP supplementation. Similarly to our NOP was supplied mixed in either partial mixed rations or TMR
study, their combinations did not enhance their individual effects leading to a continuous supply of the additive. In our study, the
(absence of additivity between strategies). 3-NOP supply pattern restricts the delivery of the compound into
In our study, 3-NOP significantly increased propionate concen- the rumen to twice daily when pulse-dosed in the milking parlour
tration with both supplements with no interaction and decreased with concentrate supplementation. Thus, synchrony between the
the acetatetopropionate molar ratio from 4.72 to 3.90 mM/ CH4-inhibiting activity of 3-NOP and the presence of grass from
mM. So, despite the mild inhibition caused by 3-NOP on CH4 pro- main grazing bouts was limited, causing a mismatch between peak
duction, 3-NOP did act on ruminal VFA concentrations shifting fer- 3-NOP inhibitory activity and CH4 production. A limitation of the
mentation from acetate towards propionate. This shift is consistent SF6 technique for CH4 measurement, while suitable for grazing ani-
with the beef and all ruminant meta-regressions reported by Kim mals, is that it collects a 24-h gas sample, not allowing for analysis
et al. (2020), but not entirely with the dairy cattle meta- of intraday CH4 emissions. There have been previous reports that
regressions, where 3-NOP decreased acetate, but did not affect pro- CH4 production inhibition by 3-NOP can be limited when not sup-
pionate concentrations. Mild CH4 inhibition (10%), yet with notice- plemented continuously. When 3-NOP was dosed in a supplement
able reductions in the acetatetopropionate ratio (6.5%), have during milking but access to non-grazed pasture rations was with-
been previously reported when 3-NOP was dosed twice daily held for 1 h (to simulate the delay in time between milking and
through the rumen cannula (Reynolds et al., 2014). The authors grazing), no significant effects on CH4 production were detected
speculated that greater inhibition was not observed due to the 3- (Muetzel et al., 2019). Reynolds et al. (2014) reported transitory
NOP compound rapidly washing out of the rumen with liquid CH4 inhibitory effects of 3-NOP lasting for only 2–3 h, and smaller
outflow. residual effects were maintained throughout the day. In addition to
The level of methanogenesis inhibition by 3-NOP observed with passage out of the rumen (Reynolds et al., 2014), 3-NOP is rapidly
the STA supplement in our study is more modest than previously metabolised in the rumen and disappears through its own mode of
reported results (13.4% absolute CH4 and 9.7% CH4 yield). Based action (Yu et al., 2021). In vitro, Duin et al., 2016 reported almost all
on a meta-analysis of 14 experiments with a range of 3-NOP doses, 3-NOP to be broken down after 12 h. Thus, because 3-NOP inhibi-
supplemental methods, diet compositions, and animal species, 3- tory activity on methanogenesis is relatively short-lived in the
NOP consistently reduced CH4 emissions (all three metrics) rumen, in systems where 3-NOP cannot be supplied continuously
by > 30% (Kebreab et al., 2023). Also using meta-analyses, others with feed, shortening the interval between additive delivery and
(Dijkstra et al., 2018; Jayanegara et al., 2018; Kim et al., 2020; digestion and fermentation of grass, which is the largest portion
Almeida et al., 2021) have reported similar efficacy evidence to of daily ingested feed, is critical. Slowrelease formulations of 3-
Kebreab et al (2023). NOP especially intended for pasturefed cows are underway
The lower effectiveness of 3-NOP to decrease CH4 obtained in (Muetzel et al., 2019).
the present study cannot be explained by the dose rate used. In addition to the 3-NOP delivery restricted to twice daily in our
Methane inhibition by 3-NOP has been reported to be dose depen- study, on CH4 measurement weeks, the time gap between 3-NOP
dant i.e., increasing with increased 3-NOP dose (Kim et al., 2020; dosing in the milking parlour and pasture grazing in the paddocks
Kebreab et al., 2023). A range of 3-NOP doses have been reported was augmented because of the experimental handling of the ani-
in the literature (40–340 mg/kg DM), but a practical dose of mals. We estimate this time gap to be around 3.5 h in the morning
60 mg 3-NOP/kg DM for dairy cows has been recommended and 1.5 h in the afternoon. In this time frame, the cows were
(Hegarty et al., 2021). The 3-NOP dose rate of our study (130 mg/ moved from the milking parlour to the management chute where
kg DM intake) was higher than the practical dose recommended experimental procedures associated with the SF6 and n-alkane
and that of other experiments with confined dairy cows (Haisan techniques were carried out, and then back to the grazing pad-
et al., 2014; Hristov et al., 2015; Lopes et al., 2016; Haisan et al., docks; a greater morning gap was due to the SF6 canister replace-
2017). ment being done in the mornings. This may help explain why our
Another possibility to consider is that the 3-NOP dose targeted rumen fermentation results showed shifts consistent with studies
in this study could have induced some level of supplement rejec- where more substantial decreases in CH4 production were
tions or palatability issues of the 3-NOP supplements. Palatability obtained (Kim et al., 2020). That is, because rumen fermentation
studies with doses of 3-NOP of up to 120 mg/kg DM intake in dairy (assessed in week 6 and not on CH4 measurement weeks) was
cows fed TMR diets reported no observed palatability issues measured closer in time to 3-NOP supplementation. It has been
(Melgar et al., 2018), and beef cattle fed 3-NOP at 100 mg/kg diet reported that the diurnal pattern of CH4 production follows closely
DM rapidly adjusted to 3-NOP showing no eating preferences the diurnal pattern of rumen fermentation (Brask et al., 2015). Out-
within 7 days (Lee et al., 2020). Also, studies with dairy (Melgar side CH4 measurement weeks, the time spent between 3-NOP dos-
et al., 2020b) and beef (Vyas et al., 2016) cattle using 3-NOP doses ing and feeding in paddocks was estimated to be around 60 min. It
of up to 200 mg 3-NOP/kg DM did not notice effects on DM intake. is not uncommon for dairy cows to daily spend varying amounts of
That said, the daily 3-NOP dose supplied in two portions of concen- time away from grazing paddocks due to prolonged milking times
trate in our study still had a considerably higher concentration in and/or long walking distances towards the milking parlour, espe-
feed (380 mg 3-NOP/kg concentrate) compared to the studies by cially in large pasture-based dairy herds. For example, a survey
Vyas et al. (2016), Melgar et al. (2018), and Lee et al. (2020). The of Australian dairy farmers reported that herds of over 300 cows
animals did not seem to reject the supplements. Although, unfortu- spent between 1.18 and 3.45 h away from the paddock between
nately, and due to a lack of appropriate infrastructure, supplement the end of milking and the beginning of grazing (Beggs et al., 2015).
refusals in the milking parlour, if they took place, were not mea- The possible impact of the basal diet (of higher roughage levels
sured. It is recommended that further studies evaluate whether than with TMR) on the observed lack of efficacy of 3-NOP in this
supplying 3-NOP in the concentrate at concentrations like the study is unknown. The increased 3-NOP dose compared to most
one we used, as required by pulse-dose supplementation, may studies with dairy cows might have helped improving 3-NOP effi-
impair concentrate palatability. cacy with diets that were also somewhat high in NDF, especially
One major factor that could have compromised the effective- with CTS supplementation (Dijkstra et al., 2018; Kebreab et al.,
ness of 3-NOP in the present study is its mode of supply. In all 2023). The tendency towards a lack of effect of 3-NOP with the
studies used in meta-regressions (Dijkstra et al., 2018; CTS supplement on absolute CH4 emissions obtained in the present
8
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

study can be partly explained by the higher NDF, and also ether would also increase pasture postgrazing height indicating that less
extract concentrations of the CTS supplement. Diet nutrient com- forage was removed by the cows, but the opposite was observed.
position has been reported to be influential over 3-NOP efficacy, How this result was affected by the interaction found between 3-
with lower additive efficacy when diet NDF and crude fat concen- NOP and CTS on pasture allowance is difficult to interpret. This
trations increase (Kebreab et al., 2023). Lower 3-NOP efficacy with illustrates that the relationship between 3-NOP supply and grass
high forage diets of higher NDF concentration has been speculated diets (as affected by composition and grazing management) and
to be a consequence of greater CH4 formation with forage than its effects on DM intake are unknown and merit further study.
grain diets (Vyas et al., 2018). Greater CH4 production with higher The present study found that in the afternoon rumen sampling,
methanogen abundance and/or higher concentrations of methyl 3-NOP supplementation tended to decrease rumen pH with the
coenzyme M (3-NOP’s structural analogue) imply that there is STA concentrate. This contrasts with previous results of meta-
more competition for 3-NOP to bind with methyl coenzyme M analyses that have shown increased rumen pH with 3-NOP supple-
reductase and inhibit CH4 production. In addition, the CH4 mitigat- mentation (Jayanegara et al., 2018; Kim et al., 2020). Also, in our
ing efficacy of 3-NOP at a given dose is enhanced by 3.08% for each study, supplementing 3-NOP decreased DM digestibility with the
1% DM decrease in dietary crude fat concentration for TMR diets CTS supplement. Yet, meta-analyses have reported no effects of
between 3 and 6% crude fat levels (Kebreab et al., 2023). Our 3-NOP on nutrient digestibility (Jayanegara et al., 2018; Kim
results are consistent with this meta-analysis. It is noted that the et al., 2020). This was most likely related to lipid content, as CTS
tendency to increase CH4 yield with 3-NOP and CTS has a mathe- can decrease DM digestibility, particularly fibre digestibility
matical explanation driven by a numerical decrease in DM intake. (Muñoz et al., 2019). The discrepancies between our findings and
Additional factors to consider for evaluating 3-NOP effectiveness that of others are difficult to explain and may be related to differ-
with grass diets are pasture’s varying composition with season ences in basal diet composition (Kebreab et al., 2023), and/or the
progression and grazing management, and its digestibility, NDF, mode of supply of the additive and its higher 3-NOP concentration
and water contents compared to TMR diets. in feed.
The lack of a consistent effect of CTS on decreasing CH4 emis- In the present study, 3-NOP tended to decrease milk yield with
sion in our study was unexpected, and disagrees with what others the STA supplement, but not with the CTS supplement. Impor-
have reported. Total mixed rations with whole CTS (3.28 kg/d) tantly, ECM yield was unaffected by treatments and this result
were more effective in decreasing CH4 emission (14% CH4 yield) was mainly driven by the lack of effect of treatments on milk com-
than TMR diets with whole linseed or rapeseed (Muñoz et al., position. Meta-analyses based on TMR diets have reported that 3-
2019), and the authors attributed the results to a higher oil concen- NOP does not compromise milk yield (Jayanegara et al., 2018;
tration and a lower intake of digestible fibre with CTS. A concen- Arndt et al., 2022). However, a tendency to decrease milk yield
trate supplement including whole CTS (2.61 kg DM/d) decreased with increasing 3-NOP supplementation has been reported by
CH4 yield up to 15% in lactating dairy cows fed forage-based diets another meta-analysis (Kim et al., 2020). In our study, the tendency
(Grainger et al., 2010). Muñoz et al. (2021) offered grazing dairy towards decreased milk yield agreed with the negative effects of 3-
cows 2.3 kg DM/d of whole CTS as part of a concentrate supple- NOP on DM intake. The higher milk urea concentration as a result
ment and found a 14% decrease in CH4 yield, although the effects of 3-NOP supplementation found in our study agrees with a meta-
were transient. These discrepancies may be influenced by the analysis of 5 studies by Hristov et al., 2022, but not with Jayanegara
lower level of CTS used in our study compared to others. et al., 2018 meta-analysis. The MUN increase with 3-NOP has been
In our study, there were no interactions of 3-NOP or CTS with explained as a consequence of increased butyrate absorption from
parity for CH4 variables. A recent study by Maigaard et al. (2024) the rumen, which could stimulate blood flow and NH3 absorption
evaluated the effects of combined supplementation of primiparous from the rumen (Hristov et al., 2022). In agreement, 3-NOP
and multiparous cows with 3-NOP, rapeseed, and nitrate. The increased rumen butyrate concentration in our study, which may
authors found 2-way interactions between treatments and parity be an indication of greater butyrate absorption.
on DM intake, with treatments exerting a more pronounced In our study, 3-NOP supplementation had no effect on BW
decrease in DM intake in multiparous than in primiparous cows. change. Results of 3-NOP on BW of dairy cows have been inconsis-
The authors attributed the 2-way interactions of parity with 3- tent with some studies reporting increased BW (Haisan et al.,
NOP and rapeseed on DM intake as responsible for the 3-way inter- 2014; Hristov et al., 2015) and others not (Reynolds et al., 2014;
action they observed between fat, 3-NOP, and parity on CH4 yield, Melgar et al., 2020a). Similarly, CTS supplementation also did not
with primiparous but not multiparous cows supplemented with affect BW change or milk SCC. Few studies have evaluated the
rapeseed responding to 3-NOP. Based on the differential responses effects of whole CTS on variables associated with lactation cow’s
of DM intake and CH4 yield of primiparous and multiparous cows health. Our results agree with Sun et al. (2022) that reported no
to treatments, the authors deemed that the efficiency of additives effects of CTS on MUN or milk SCC, and Muñoz et al. (2021) who
was in fact similar across parities, which agrees with our study. reported no effects of CTS supplementation on health or reproduc-
tion performance.
Effects on diet intake, milk production and performance We are unaware of any published study reporting 3-NOP sup-
plementation twice daily to dairy cows at milking under grazing
In the present study, 3-NOP supplementation decreased pasture condition. The forage quality of the present study was representa-
intake by 10.5% across both concentrate supplements. Reports on tive of temperate grasslands in spring, typical of pasture-based
the effect of 3-NOP on DM intake are not consistent. A meta- dairy systems in the south of Chile (Muñoz et al., 2016), and the
analysis reported up to 4.5% decrease in DM intake with 3-NOP diet concentrate proportion supplemented (30%) is on the high
inclusion (Almeida et al., 2021); another meta-analysis reported end of concentrate spring supplementation levels (Muñoz et al.,
a tendency towards decreased DM intake in beef, but not in dairy 2015).
cattle (Kim et al., 2020), and others have reported no effect of 3-
NOP on DM intake (Jayanegara et al., 2018; Arndt et al., 2022).
The mechanism involved in lower intake with 3-NOP may be Conclusion
related to increased portal concentrations of propionate affecting
satiety (Allen, 2000), which would be consistent with our findings. Twice daily supplementation of grazing dairy cows with supple-
Furthermore, we expected that the lower DM intake with 3-NOP ments that provided a combination of 3-NOP and CTS did not
9
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

increase the effectiveness of each CH4 mitigation strategy supple- Unit at INIA Remehue for assistance with the care of experimental
mented separately, disproving our hypothesis. The 3-NOP additive animals.
modestly decreased or tended to decrease CH4 emissions (absolute
and yield) only with the STA concentrate, but not with the CTS con- Financial support statement
centrate. The lesser CH4 inhibitory effects of 3-NOP than previously
reported with TMR were most likely related to the mode of 3-NOP This work was supported by the Agencia Nacional de Investi-
supply (twice daily dosing at milking) being uncoupled with the gación y Desarrollo (ANID), projects Fondecyt Regular 1191476
pasture grazing bouts. Extended time gaps between additive deliv- and 1240525. The 3-NOP was provided by DSM Nutritional Prod-
ery and access to pasture due to experimental handling likely fur- ucts AG (Kaiseraugst, Switzerland).
ther impaired 3-NOP effectiveness. The 3-NOP additive decreased
grass intake; however, ECM yield and milk composition were lar-
gely unaffected. Supplementing grazing dairy cows with CTS did References
not decrease CH4 production and this may be related to insufficient
Allen, M.S., 2000. Effects of diet on short-term regulation of feed intake by lactating
levels of supplementation. There is a need for effective and pro- dairy cattle. Journal of Dairy Science 83, 1598–1624. https://doi.org/10.3168/
nounced CH4 mitigation strategies that can be applicable to grazing jds.S0022-0302(00)75030-2.
dairy systems by pulse-dosing at milking. Alternatives for continu- Almeida, A.K., Hegarty, R.S., Cowie, A., 2021. Meta-analysis quantifying the potential
of dietary additives and rumen modifiers for methane mitigation in ruminant
ous delivery of 3-NOP into the rumen are required for boosting its production systems. Animal Nutrition 7, 1219–1230. https://doi.org/10.1016/j.
CH4 mitigation effectiveness in grazing systems. aninu.2021.09.005.
AOAC International, 2016. Official Methods of Analysis of AOAC International, 20th.
Washington, DC, USA.
Ethics approval Arndt, C., Hristov, A.N., Price, W.J., McClelland, S.C., Pelaez, A.M., Cueva, S.F., Oh, J.,
Dijkstra, J., Bannink, A., Bayat, A.R., Crompton, L.A., Eugène, M.A., Enahoro, D.,
Kebreab, E., Kreuzer, M., McGee, M., Martin, C., Newbold, C.J., Reynolds, C.K.,
Animal care and experimental procedures were performed in Schwarm, A., Shingfield, K.J., Veneman, J.B., Yáñez-Ruiz, D.R., Yu, Z., 2022. Full
accordance with the requirements of the Chilean Law 20.380 on adoption of the most effective strategies to mitigate methane emissions by
Animal Protection and with the approval of INIA’s Institutional ruminants can help meet the 1.5 °C target by 2030 but not 2050. Proceedings of
the National Academy of Sciences 119, e2111294119. https://doi.org/10.1073/
Animal Care and Use Committee (Certificate 01/2019). pnas.2111294119.
Nutrición animal: Manual de métodos analíticos. Herrero Hermanos Sucesores S. A.,
Mexico City, Mexico.
Data and model availability statement Beauchemin, K.A., McGinn, S.M., Benchaar, C., Holtshausen, L., 2009. Crushed
sunflower, flax, or canola seeds in lactating dairy cow diets: effects on methane
None of the data were deposited in an official repository. The production, rumen fermentation, and milk production. Journal of Dairy Science
92, 2118–2127. https://doi.org/10.3168/jds.2008-1903.
data that support the study findings are available from the authors Beauchemin, K.A., Ungerfeld, E.M., Eckard, R.J., Wang, M., 2020. Review: fifty years
upon request. of research on rumen methanogenesis: lessons learned and future challenges
for mitigation. Animal 14, 2–16. https://doi.org/10.1017/S1751731119003100.
Beauchemin, K.A., Ungerfeld, E.M., Abdalla, A.L., Alvarez, C., Arndt, C., Becquet, P.,
Declaration of Generative AI and AI-assisted technologies in the Benchaar, C., Berndt, A., Mauricio, R.M., McAllister, T.A., Oyhantçabal, W.,
writing process Salami, S.A., Shalloo, L., Sun, Y., Tricarico, J., Uwizeye, A., De Camillis, C., Bernoux,
M., Robinson, T., Kebreab, E., 2022. Invited review: current enteric methane
mitigation options. Journal of Dairy Science 105, 9297–9326. https://doi.org/
During the preparation of this work the author(s) did not use 10.3168/jds.2022-22091.
any AI and AI-assisted technologies. Beggs, D.S., Fisher, A.D., Jongman, E.C., Hemsworth, P.H., 2015. A survey of
Australian dairy farmers to investigate animal welfare risks associated with
increasing scale of production. Journal of Dairy Science 98, 5330–5338. https://
Author ORCIDs doi.org/10.3168/jds.2014-9239.
Brask, M., Weisbjerg, M.R., Hellwing, A.L.F., Bannink, A., Lund, P., 2015. Methane
production and diurnal variation measured in dairy cows and predicted from
Camila Muñoz: https://orcid.org/0000-0003-4080-9325. fermentation pattern and nutrient or carbon flow. Animal 9, 1795–1806.
I.A. Muñoz: N/A. https://doi.org/10.1017/S1751731115001184.
Dijkstra, J., Bannink, A., France, J., Kebreab, E., van Gastelen, S., 2018. Short
R. Rodríguez: https://orcid.org/0000-0001-6702-4689.
communication: antimethanogenic effects of 3-nitrooxypropanol depend on
N.L. Urrutia: https://orcid.org/0000-0002-6196-0465. supplementation dose, dietary fiber content, and cattle type. Journal of Dairy
E.M. Ungerfeld: https://orcid.org/0000-0002-5422-5462. Science 101, 9041–9047. https://doi.org/10.3168/jds.2018-14456.
Dillon, P.G., 1993. The use of n-alkanes as markers to determine herbage intake,
botanical composition of available or consumed herbage and in studies of
CRediT authorship contribution statement digesta kinetics with dairy cows. The National University of Ireland, Dublin,
Ireland.
Duin, E.C., Wagner, T., Shima, S., Prakash, D., Cronin, B., Yáñez-Ruiz, D.R., Duval, S.,
C. Muñoz: Writing – original draft, Visualization, Resources, Rümbeli, R., Stemmler, R.T., Thauer, R.K., Kindermann, M., 2016. Mode of action
Project administration, Methodology, Funding acquisition, Data uncovered for the specific reduction of methane emissions from ruminants by
curation, Conceptualization. I.A. Muñoz: Investigation, Data cura- the small molecule 3-nitrooxypropanol. Proceedings of the National Academy
of Sciences 113, 6172–6177. https://doi.org/10.1073/pnas.1600298113.
tion. R. Rodríguez: Writing – review & editing, Investigation. N.L. FAO, 2003. Calculation of the energy content of foods - Energy conversion factors. In
Urrutia: Writing – review & editing, Resources, Investigation. E. Food energy - methods of analysis and conversion factors (ed. sine nomine).
M. Ungerfeld: Writing – review & editing, Formal analysis, Food and Agriculture Organization of the United Nations, Rome, Italy, pp. 18–
37.
Conceptualization. Goering, H.K., Van Soest, P.J., 1970. Forage Fiber Analyses (Apparatus, Reagents,
Procedures, and Some Applications). ARS-USDA, Washington, DC, USA.
Grainger, C., Clarke, T., Beauchemin, K.A., McGinn, S.M., Eckard, R.J., 2008a.
Declaration of interest Supplementation with whole cottonseed reduces methane emissions and can
profitably increase milk production of dairy cows offered a forage and cereal
The authors declare no conflicts of interest. grain diet. Australian Journal of Experimental Agriculture 48, 73–76. https://doi.
org/10.1071/ea07224.
Grainger, C., Clarke, T., Eckard, R.J., 2008b. Effect of whole cottonseed
Acknowledgments supplementation on energy and nitrogen partitioning and rumen function in
dairy cattle on a forage and cereal grain diet. Australian Journal of Experimental
Agriculture 48, 860–865. https://doi.org/10.1071/ea07400.
We gratefully acknowledge S. Hube, R. Saldaña, and C. Uribe for Grainger, C., Williams, R., Clarke, T., Wright, A.D.G., Eckard, R.J., 2010.
technical assistance during the study, and the staff of the Dairy Supplementation with whole cottonseed causes long-term reduction of

10
C. Muñoz, I.A. Muñoz, R. Rodríguez et al. Animal 18 (2024) 101203

methane emissions from lactating dairy cows offered a forage and cereal grain response effect of 3-nitrooxypropanol on enteric methane emissions in dairy
diet. Journal of Dairy Science 93, 2612–2619. cows. Journal of Dairy Science 103, 6145–6156. https://doi.org/10.3168/
Haisan, J., Sun, Y., Guan, L.L., Beauchemin, K.A., Iwaasa, A., Duval, S., Barreda, D.R., jds.2019-17840.
Oba, M., 2014. The effects of feeding 3-nitrooxypropanol on methane emissions Miller, G., Eory, V., Duthie, C., Newbold, J., 2023. Existing and near-to-market
and productivity of Holstein cows in mid lactation. Journal of Dairy Science 97, methane reducing feed additives and technologies: Evidence of Efficacy,
3110–3119. https://doi.org/10.3168/jds.2013-7834. Regulatory Pathways to Market and Mechanisms to Incentivise Adoption.
Haisan, J., Sun, Y., Guan, L., Beauchemin, K.A., Iwaasa, A., Duval, S., Kindermann, M., Scotland’s Rural College, Aberdeen, UK.
Barreda, D.R., Oba, M., 2017. The effects of feeding 3-nitrooxypropanol at two Muetzel, S., Lowe, K., Janssen, P., Pacheco, D., Bird, N., Walker, N., Vidoni, O.,
doses on milk production, rumen fermentation, plasma metabolites, nutrient Schweikert, L., Clasadonte, L., Kindermann, M., 2019. Towards the application of
digestibility, and methane emissions in lactating Holstein cows. Animal 3-nitrooxypropanol in pastoral farming systems. In: Proceedings of the 7th
Production Science 57, 282–289. https://doi.org/10.1071/an15219. Greenhouse Gas and Animal Agriculture Conference, 4-8 August 2019, Iguassu
Hegarty, R.S., Cortez Passetti, R.A., Dittmer, K.M., Wang, Y., Shelton, S.W., Emmet- Falls, Brazil, p. 81.
Booth, J., Wollenberg, E.K., McAllister, T., Leahy, S., Beauchemin, K., Gurwick, N., Muñoz, C., Hube, S., Morales, J.M., Yan, T., Ungerfeld, E.M., 2015. Effects of
2021. An evaluation of emerging feed additives to reduce methane emissions concentrate supplementation on enteric methane emissions and milk
from livestock. Edition 1. A report coordinated by Climate Change, Agriculture production of grazing dairy cows. Livestock Science 175, 37–46. https://doi.
and Food Security (CCAFS) and the New Zealand Agricultural Greenhouse Gas org/10.1016/j.livsci.2015.02.001.
Research Centre (NZAGRC) initiative of the Global Research Alliance (GRA). Muñoz, C., Letelier, P.A., Ungerfeld, E.M., Morales, J.M., Hube, S., Pérez-Prieto, L.A.,
Retrieved on 2 December 2021 from https://globalresearchalliance.org/wp- 2016. Effects of pregrazing herbage mass in late spring on enteric methane
content/uploads/2021/12/An-evaluation-of-evidence-for-efficacy-and- emissions, dry matter intake, and milk production of dairy cows. Journal of
applicability-of-methane-inhibiting-feed-additives-for-livestock-FINAL.pdf. Dairy Science 99, 7945–7955. https://doi.org/10.3168/jds.2016-10919.
Hristov, A.N., Oh, J., Giallongo, F., Frederick, T.W., Harper, M.T., Weeks, H.L., Branco, Muñoz, C., Sánchez, R., Peralta, A.M.T., Espíndola, S., Yan, T., Morales, R., Ungerfeld,
A.F., Moate, P.J., Deighton, M.H., Williams, S.R.O., Kindermann, M., Duval, S., E.M., 2019. Effects of feeding unprocessed oilseeds on methane emission,
2015. An inhibitor persistently decreased enteric methane emission from dairy nitrogen utilization efficiency and milk fatty acid profile of lactating dairy cows.
cows with no negative effect on milk production. Proceedings of the National Animal Feed Science and Technology 249, 18–30. https://doi.org/10.1016/j.
Academy of Sciences of the United States of America 112, 10663–10668. anifeedsci.2019.01.015.
https://doi.org/10.1073/pnas.1504124112. Muñoz, C., Villalobos, R., Peralta, A.M.T., Morales, R., Urrutia, N.L., Ungerfeld, E.M.,
Hristov, A.N., Melgar, A., Wasson, D., Arndt, C., 2022. Symposium review: effective 2021. Long-term and carryover effects of supplementation with whole oilseeds
nutritional strategies to mitigate enteric methane in dairy cattle. Journal of on methane emission, milk production and milk fatty acid profile of grazing
Dairy Science 105, 8543–8557. https://doi.org/10.3168/jds.2021-21398. dairy cows. Animals 11, 2978.
Jayanegara, A., Sarwono, K.A., Kondo, M., Matsui, H., Ridla, M., Laconi, E.B., Nahrowi, Pérez-Prieto, L.A., Peyraud, J.L., Delagarde, R., 2012. Does pre-grazing herbage mass
2018. Use of 3-nitrooxypropanol as feed additive for mitigating enteric methane really affect herbage intake and milk production of strip-grazing dairy cows?
emissions from ruminants: a meta-analysis. Italian Journal of Animal Science Grass and Forage Science 68, 93–109.
17, 650–656. https://doi.org/10.1080/1828051X.2017.1404945. Reynolds, C.K., Humphries, D.J., Kirton, P., Kindermann, M., Duval, S., Steinberg, W.,
Kebreab, E., Bannink, A., Pressman, E.M., Walker, N., Karagiannis, A., van Gastelen, S., 2014. Effects of 3-nitrooxypropanol on methane emission, digestion, and
Dijkstra, J., 2023. A meta-analysis of effects of 3-nitrooxypropanol on methane energy and nitrogen balance of lactating dairy cows. Journal of Dairy Science 97,
production, yield, and intensity in dairy cattle. Journal of Dairy Science 106, 3777–3789. https://doi.org/10.3168/jds.2013-7397.
927–936. https://doi.org/10.3168/jds.2022-22211. Sun, X., Su, Y., Hao, Y., Zhang, J., Yue, X., Wang, W., Ma, Z., Chu, K., Wang, S., Wang, Y.,
Kim, H., Le, H.G., Beek, Y.C., Lee, S., Seo, J., 2020. The effects of dietary Li, S., 2022. Novel process methods for the whole cottonseed: effect on the
supplementation with 3-nitrooxypropanol on enteric methane emissions, digestibility, productivity, fat profile, and milk gossypol levels in lactating dairy
rumen fermentation, and production performance in ruminants: a meta- cows. Frontiers in Nutrition 9, 801712. https://doi.org/10.3389/
analysis. Journal of Animal Science and Technology 62, 31–42. https://doi.org/ fnut.2022.801712.
10.5187/jast.2020.62.1.31. Tyrrell, H.F., Reid, J.T., 1965. Prediction of the energy value of cow’s milk. Journal of
Lee, C., Kim, S.-H., Beauchemin, K., Celi, P., Duval, S., 2020. Short-term eating Dairy Science 48, 1215–1223.
preference of beef cattle fed high forage or high grain diets supplemented with Ungerfeld, E.M., Aedo, M.F., Martínez, E.D., Saldivia, M., 2019. Inhibiting
3-Nitrooxypropanol. Animals 10, 64. methanogenesis in Rumen batch cultures did not increase the recovery of
Lopes, J.C., de Matos, L.F., Harper, M.T., Giallongo, F., Oh, J., Gruen, D., Ono, S., metabolic hydrogen in microbial amino acids. Microorganisms 7, 115.
Kindermann, M., Duval, S., Hristov, A.N., 2016. Effect of 3-nitrooxypropanol on Van Soest, P.v., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral
methane and hydrogen emissions, methane isotopic signature, and ruminal detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.
fermentation in dairy cows. Journal of Dairy Science 99, 5335–5344. https://doi. Journal of Dairy Science 74, 3583–3597.
org/10.3168/jds.2015-10832. Vargas, J., Ungerfeld, E., Muñoz, C., DiLorenzo, N., 2022. Feeding strategies to
Maigaard, M., Weisbjerg, M.R., Johansen, M., Walker, N., Ohlsson, C., Lund, P., 2024. mitigate enteric methane emission from ruminants in grassland systems.
Effects of dietary fat, nitrate, and 3-nitrooxypropanol and their combinations on Animals 12, 1132. https://doi.org/10.3390/ani12091132.
methane emission, feed intake, and milk production in dairy cows. Journal of Vyas, D., McGinn, S.M., Duval, S.M., Kindermann, M., Beauchemin, K.A., 2016. Effects
Dairy Science 107, 220–241. https://doi.org/10.3168/jds.2023-23420. of sustained reduction of enteric methane emissions with dietary
Martínez, M.E., Benavente, A., Morales, R., 2017. Potential of n-alkanes as supplementation of 3-nitrooxypropanol on growth performance of growing
biomarkers in grass-feeding steers. Ciencia e Investigación Agraria: Revista and finishing beef cattle. Journal of Animal Science 94, 2024–2034. https://doi.
Latinoamericana De Ciencias De La Agricultura 44, 239–251. org/10.2527/jas2015-0268.
Mayes, R.W., Lamb, C.S., Colgrove, P.M., 1986. The use of dosed and herbage n- Vyas, D., McGinn, S.M., Duval, S.M., Kindermann, M.K., Beauchemin, K.A., 2018.
alkanes as markers for the determination of herbage intake. The Journal of Optimal dose of 3-nitrooxypropanol for decreasing enteric methane emissions
Agricultural Science 107, 161–170. https://doi.org/10.1017/ from beef cattle fed high-forage and high-grain diets. Animal Production
S0021859600066910. Science 58, 1049–1055. https://doi.org/10.1071/an15705.
Melgar, A., Nedelkov, K., Martins, C., Welter, K., Chen, X., Harper, M., Duval, S., Yu, G.H., Beauchemin, K.A., Dong, R.L., 2021. A Review of 3-nitrooxypropanol for
Hristov, A., 2018. Palatability of total mixed rations containing 3-nitro- enteric methane mitigation from ruminant livestock. Animals 11, 3540. https://
oxypropanol for lactating dairy cows. Journal of Dairy Science 101, 92. doi.org/10.3390/ani11123540.
Melgar, A., Harper, M., Oh, J., Giallongo, F., Young, M., Ott, T., Duval, S., Hristov, A., Zhang, X.M., Smith, M.L., Gruninger, R.J., Kung Jr, L., Vyas, D., McGinn, S.M.,
2020a. Effects of 3-nitrooxypropanol on rumen fermentation, lactational Kindermann, M., Wang, M., Tan, Z.L., Beauchemin, K.A., 2021. Combined effects
performance, and resumption of ovarian cyclicity in dairy cows. Journal of of 3-nitrooxypropanol and canola oil supplementation on methane emissions,
Dairy Science 103, 410–432. rumen fermentation and biohydrogenation, and total tract digestibility in beef
Melgar, A., Welter, K.C., Nedelkov, K., Martins, C.M.M.R., Harper, M.T., Oh, J., cattle. Journal of Animal Science 99, skab081. https://doi.org/10.1093/jas/
Räisänen, S.E., Chen, X., Cueva, S.F., Duval, S., Hristov, A.N., 2020b. Dose- skab081.

11