Received: 20 November 2020 | Accepted: 7 April 2021

DOI: 10.1111/jpn.13567

SUPPLEMENT ARTICLE

Black soldier fly and yellow mealworm live larvae for broiler
chickens: Effects on bird performance and health status

Sara Bellezza Oddon1 | Ilaria Biasato1 | Arianna Imarisio2 | Miha Pipan3 |

Dominik Dekleva3 | Elena Colombino2 | Maria Teresa Capucchio 2
|
Marco Meneguz1 | Bergagna Stefania4 | Raffaella Barbero4 | Marta Gariglio2 |
Sihem Dabbou5,6 | Edoardo Fiorilla2 | Laura Gasco1 | Achille Schiavone2

1
Department of Agricultural, Forest
and Food Sciences, University of Turin, Abstract
Grugliasco, Italy
The commercial broiler chicken strains are the result of successful selection pro-
2
Department of Veterinary Sciences,
University of Turin, Grugliasco, Italy
grammes. Most of the problems related to welfare arise from the high growth rate
3
Entomics Biosystems, Cambridge, UK and body weight. The use of environmental enrichments in intensive farming could
4
Veterinary Medical Research Institute have a positive effect on birds by increasing animal welfare. The aim of the study was
for Piemonte, Liguria and Valle d'Aosta,
to evaluate the effects of Hermetia illucens (HI) and Tenebrio molitor (TM) live larvae in
Turin, Italy
5
Center Agriculture Food Environment, the diets of broiler chickens on growth performance, carcass yield and health status.
University of Trento, Trento, Italy A total of 180 four-­day-­old male broiler chickens (Ross 308) were randomly allotted
6
Research and Innovation Centre,
to 18 pens. Each pen was assigned to one of the three dietary treatments (6 repli-
Fondazione Edmund Mach, San Michele
all’Adige, Italy cates/treatment, 10 birds/replicate) as follows: (i) control diet (C): commercial feed
(two feeding phases: starter [4–­11 days] and grower [12–­38 days]), (ii) HI: C + 5% of
Correspondence
Ilaria Biasato, Department of Agricultural, the expected daily feed intake (DFI) HI live larvae (calculated on dry matter [DM]) and
Forest and Food Sciences, University of
(iii) TM: C + 5% of DFI TM live larvae (DM). At 39 days of age, birds were slaughtered.
Turin, Grugliasco, Italy.
Email: [email protected] Growth performance parameters were overall not affected by dietary treatments,
except for the grower phase feed conversion ratio (FCR) and the overall FCR being
Funding information
The research was supported by the better in the TM broilers than the others (p < 0.01). No differences were observed
European Knowledge and Innovation
for slaughtering performance and haematological and serum parameters, except for
Community (KIC), within the EIT Food
program ‘FROM WASTE TO FARM: insect the spleen relative weight being higher (p < 0.01) in the birds administered with lar-
larvae as tool for welfare improvement in
vae when compared to the C group. Gut morphometric indexes and histopathologi-
poultry’ (Project ID 19122).
cal alterations were not influenced by insect larvae administration. In conclusion, the
administration in limited quantities of HI and TM live larvae as environmental enrich-
ment has no negative effects on broiler chicken growth performance and health sta-
tus. A behavioural study could confirm that live insect larvae represent a novel natural
environmental enrichment in broiler farming.

KEYWORDS
black soldier fly, environmental enrichment, growth performance, gut health, insect larvae,
poultry, yellow mealworm

This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial-­NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made.
© 2021 The Authors. Journal of Animal Physiology and Animal Nutrition published by Wiley-­VCH GmbH.

J Anim Physiol Anim Nutr. 2021;00:1–9.wileyonlinelibrary.com/journal/jpn | 1

2 | BELLEZZA ODDON et al.

1 | I NTRO D U C TI O N numerically higher and lasted for a longer time in broiler chickens
fed 10% of larvae and 4 times per day when compared to the control
In Europe, poultry industry has progressively moved on towards an birds (Ipema et al., 2020). According to EU legislation, in particular
industrialized and vertically integrated broiler production, with sub- the Commission Regulation No. 999/2001, the use of live insects
stantial increase in per capita poultry consumption and significant de- in poultry feeding is allowed as the feed ban regarding the use of
cline of market prices for broilers (Magdelaine et al., 2019). Currently, insect-­derived proteins does not apply to live insects (IPIFF, 2020).
the commercial broiler chicken strains are the result of successful Nowadays, the standard evaluation of the bird performance has
selection programmes for two important productive characteristics: progressively been accompanied by the concomitant assessment
the rapid growth and the body conformation (Scheuermann et al., of the gut health dynamics, as a healthy, well-­functioning intestine
2003). The maximization of the productive efficiency has progres- is considered a synonymous of animal health and it represents the
sively led to the adoption of intensive indoor systems to carry out main determinant of the animal growth (Kogut & Arsenault, 2016).
the broiler chicken production (Robins & Phillips, 2011). However, In particular, the morphometric evaluation of selected gut segments
the intensive rearing may frequently cause the parallel degradation and/or mucosal elements (such as the crypts and the villi)—­together
of the welfare status of the birds, thus being furtherly exacerbated with the blood profile and organ histopathological analyses—­has
by barren environment (Riber et al., 2018). Therefore, environmen- previously been resulted to be an effective, easy-­to-­use approach to
tal enrichments have recently been proposed as effective tools to simultaneously characterize the gut health and overall health status
create a more diverse rearing habitat for the animals. The environ- of broilers (Biasato et al., 2018). However, such analyses have not
mental enrichment is generally defined as an improvement in the been performed yet on birds administered with live insect larvae as
biological functioning of captive animals resulting from modification environmental enrichments.
of their environment (Newberry, 1995). Since in nature birds eat Based on the above-­mentioned background, the goal of this
insects in larval, pupal and adult forms, they can be used as envi- study is to evaluate the effects of the administration of TM and HI
ronmental and nutritional enrichments in broiler chicken farming live larvae on performance, blood profile, gut morphology and organ
(Zuidhof et al., 2003). The recovery of a natural behaviour could histopathology in broiler chickens.
therefore have positive effects on the animal welfare and, conse-
quently, on the breeding and final product images that the consumer
creates. Consumers are increasingly interested in the issue of animal 2 | M ATE R I A L S A N D M E TH O DS
welfare, as they seem to prefer food products which are perceived
to be more ‘animal friendly’ (Harper & Makatouni, 2002). 2.1 | Birds and husbandry
The introduction of a natural element, such as the live insect
larvae, into a fully artificial breeding could expand the range of be- The present trial was performed in the poultry facility of the
havioural expression and increase the motility of the animals. For University of Turin (Italy). The experimental protocol (ID: 814715)
this reason, the use of live larvae can be considered a novel, prom- was approved by the Bioethical Committee of the University of Turin
ising environmental enrichment. Indeed, the addition of variable (Italy). A total of 180 four-­day-­old male broiler chickens (Ross 308;
food items (such as the insects) may be attractive for birds and could average initial live weight: 88.6 ± 9.8 g) were randomly allotted to 18
potentially increase the amount of time spent for the foraging be- pens. Each pen was 1.20 m wide × 2.20 m long and was covered with
haviour and, consequently, make animals more active (Koene, 1999; rice hulls as litter. The poultry house was equipped with a waterproof
Bizeray et al., 2002). floor and walls, completely covered by tiles and provided with an au-
Currently, the use of insects in animal feed is a topic of growing tomatic ventilation system. The lighting schedule was 16-­h light:8-­h
interest (Gasco et al., 2019). So far, many researchers have focused darkness for the whole trial. Environmental conditions in the house,
their attention on the introduction of insect meal in poultry diets. In temperature and relative humidity, were set according to the Ross
particular, the available studies investigated the effects of Tenebrio guidelines (Aviagen, 2019). The animals and the environmental pa-
molitor (TM) and Hermetia illucens (HI) larva meal on broiler chicken rameters were daily checked during the whole experimental period.
growth performance and animal health (Bovera et al., 2016; Biasato
et al., 2017; Dabbou et al., 2018; Biasato et al., 2019). In the recent
years, the interest has also been directed to the use of live insect 2.2 | Diets
larvae as feed ingredients or enrichments in poultry nutrition. For
this reason, there are few scientific papers on this issue; trials have According to birds’ requirements (Aviagen 2019), the basal diet was a
been carried out on turkeys (Veldkamp & van Niekerk, 2019) and lay- commercial feed having two feeding phases: starter (1–­11 days; me-
ing hens (Star et al., 2020) fed HI live larvae, with improved growth tabolizable energy [ME]: 12.5 MJ/kg and crude protein [CP]: 224 g/
performance (in terms of better feed efficiency) having overall been kg) and grower (12–­38 days; ME: 13.0 MJ/kg and CP: 220 g/kg) (Fa.
observed. In broiler chickens, on the other hand, the effects of HI ma.ar.co SPA). Each pen was assigned to the dietary treatments (6
live larvae on animal activity and leg health were recently evaluated replicates/treatment, 10 birds/replicate) as follows: (i) control (C), (ii),
(Ipema et al., 2020). The authors observed that the activity was HI: C + 5% of the expected daily feed intake [DFI] HI live larvae and
BELLEZZA ODDON et al. | 3

TA B L E 1 Proximate composition of HI
Chemical compositiona HI early instar HI late instar TM early TM late
and TM larvae
(as fed basis, %) larvae larvae instar larvae instar larvae

DM 25.32 25.32 27.54 27.54

CP 12.01 8.07 16.78 10.82
Ash 3.05 2.00 1.69 0.90
EE 0.42 1.93 0.59 5.50
GE (MJ/kg) 5.03 6.76 5.90 7.65

Abbreviations: CP, crude protein; DM, dry matter; EE, ether extract; GE, gross energy.
a
Values are reported as mean of duplicate analyses.

(iii) TM: C + 5% of the expected of DFI TM live larvae. The amount of As viable larva was needed for the chickens to be attracted to
the live larvae was calculated on the dry matter (DM) of the expected the environmental enrichment, prior to administration to birds, the
DFI reported in the Ross guidelines (Aviagen, 2019). To prevent the larvae underwent a revitalization phase at a temperature of 28°C for
chicks from not being able to feed due to the too large size of the 10 min. In this period of time, they reactivated their metabolism and,
larvae, different larvae dimensions were provided in the two growth consequently, their motility.
periods and precisely: early or late instar (length: 0.80 cm ± 0.05 and
1.5 ± 0.05 cm, respectively) for starter and grower period respec-
tively. The commercial diet and the water were distributed ad libitum 2.4 | Chemical analysis
in all the treatments. For each pen, the daily larvae quantity was
placed in two plates and distributed at 11.00 a.m. To avoid the bias, At each arrival, a sample of larvae was stored (−20°C) for subsequent
two plates with a known amount of control feed inside were also analytical evaluation. The larvae samples were subsequently freeze-­
provided to the C animals to create the same interaction with the dried and ground using cutting mill (MLI 204; Bühler AG). Samples
operators in all the treatments. When the larvae intake was ended in were analysed for: DM (method number 943.01), ash (method num-
all the boxes, the leftover C feed was returned in the feeders. Every ber 924.05), crude protein (CP, method number 984.13) and ether
day, the time spent by birds eating the larvae was recorded in the extract (EE, method number 2003.05) according to International
larvae treatments only by stopwatch, starting from the moment the AOAC (DM, ash, CP: AOAC, 2000; EE; AOAC, 2003). The gross en-
plates with the larvae were located in the box until the plates were ergy (GE) content was determined using an adiabatic calorimetric
empty. Samples of larvae were periodically collected, killed by freez- bomb (C7000; IKA). The HI and TM larvae proximate composition
ing (−20°C) and stored to carry out the chemical analyses. is shown in Table 1.

2.3 | Larvae preservation 2.5 | Growth performance

The quantity of HI and TM larvae needed to satisfy the 5% of the At the beginning of the experimental trial, birds were individually
DFI was weekly sent by Entomics Biosystems (Cambridge, UK) and labelled with a wing mark. The live weight (LW) of the animals was
by Italian Cricket Farm (Turin, Italy) respectively. recorded at an individual level (4, 11 and 38 days of age) using elec-
Since the HI larvae underwent one day of transport, they were tronic scales (KERN PLE-­N v. 2.2; KERN & Sohn GmbH; d: 0.1). The
shipped along with the substrate to secure feed. Upon arrival, the average daily gain (ADG) and the daily feed intake (DFI) were cal-
larvae were separated from the substrate. According to Holmes et al. culated on pen basis at the end of each growth period (4–­11 days;
(2016), the HI larvae were kept in a climatic chamber at 16°C to trig- 12–­38 days), while the feed conversion ratio (FCR) was calculated
ger the diapause mechanism. The diapause is a dynamic process con- on pen basis at the end of each growth period and for the overall
sisting of several successive phase, and it is generally defined as an experimental trial (4–­11 days; 12–­38 days; 4–­38 days). The FCR was
endogenously arrest of the direct development that proceeds with calculated by including in the formula the amount (g) of larvae (based
an alternative programme of physiological events (Koštál, 2006). As on DM: 25.3% for HI larvae and 27.5% for TM larvae) intake by the
far as TM larvae are concerned, the study of Qin & Walker (2005) birds.
was taken as reference. In particular, as these authors reported that
at 5.5°C, the activity of thermal hysteresis is still found in the hae-
molymph of TM, a pre-­trial (data not reported) to induce diapause 2.6 | Slaughtering procedure and recording
was held placing TM larvae at different refrigeration temperatures
(6°C, 8°C and 10°C). Once returned at room temperature in all cases, At 39 days of age, after 12 h of feed withdrawal, final LW was re-
the diapause ended. For this trial, it was then decided to store larvae corded. On the basis of the average final LW, 3 birds/pen (18 broil-
in a climatic chamber at 6°C. ers/diet) were electrically stunned and slaughtered at a commercial
4 | BELLEZZA ODDON et al.

TA B L E 2 Effects of the dietary treatments on the growth

System), while the electrophoretic pattern of the serum was ob-
performance of the broiler chickens (n = 6)
tained using a semi-­a utomated agarose gel electrophoresis sys-
Dietary treatmentsa tem (Sebia Hydrasys®). The albumin, alaninoaminotransferase
Age p
Items (days) C HI TM SEM Valueb (ALT), aspartate aminotransferase (AST), triglycerides, choles-
terol, uric acid and HDL serum concentrations were measured
LW, g 4 87 87 88 0.38 0.796
by means of enzymatic methods in a clinical chemistry analyser
11 220 216 225 3.60 0.603
(Screen Master Touch, Hospitex diagnostics Srl.), while the LDL
38 2488 2527 2452 22.28 0.619
serum concentration was calculated according to the Friedewald
ADG, 4–­11 19 18 20 0.49 0.610
formula as follows: total cholesterol—­H DL—­(triglycerides/5)
g/d 12–­38 76 72 80 1.98 0.348 (Friedewald et al., 1972).
DFI, g/d 4–­11 24 22 22 0.67 0.679
12–­38 110 108 103 3.45 0.753
FCR, 4–­11 1.25 1.23 1.16 0.02 0.223 2.8 | Histomorphological investigations
g/G 12–­38 1.36 ab
1.39 a
1.32 b
0.01 **
4–­38 1.37a 1.38a 1.31b 0.01 ** The slaughtered birds were also submitted to anatomo-­pathological
investigations. Samples of intestine (duodenum, jejunum and ileum),
Abbreviations: ADG, average daily gain; DFI, daily feed intake; FCR,
feed conversion ratio; LW, live weight; SEM, standard error of the mean. liver, spleen, thymus and bursa of Fabricius were collected and pro-
a
Three dietary treatments: C = control; HI: C + 5% of DFI HI larvae; and cessed according to Biasato et al. (2017). The processed samples
TM: C + 5% of DFI TM larvae. were submitted to morphometric (gut segments) and histopatho-
b
**p value < 0.01. logical (organs) investigations, following the procedures previously
described (Biasato et al., 2017). In addition, gut histopathological
abattoir. The plucked and eviscerated carcasses were obtained, and findings were separately assessed for mucosa (inflammatory infil-
the head, neck, feet and abdominal fat were removed to obtain the trates) and submucosa (inflammatory infiltrates and gut-­associated
chilled carcass. Then, the weight of heart, spleen, bursa of Fabricius, lymphoid tissue [GALT] activation) for each segment. The total
liver, gut, gizzard, glandular stomach, visceral fat, thigh and breast score of each gut segment was obtained by adding up the mucosa
were immediately recorded and the data were expressed as percent- and submucosa scores, while the total score of each bird was rep-
age of LW. The caeca from the slaughtered animals were also iso- resented by the mean value of the duodenum, jejunum and ileum
lated and photographed orthogonally with a metric scale (mm). The scores.
images were subsequently analysed with the software Image®-­Pro
Plus software (6.0 version, Media Cybernetics) in order to record the
caecal length. 2.9 | Statistical analysis

The statistical analysis was performed using IBM SPSS Statistics

2.7 | Haematological and serum parameters V26.0.0 software (IBM). Each pen was considered as the experimen-
tal unit for the growth performance and larvae intake times (n = 6
At slaughter, blood samples were collected from the slaughtered per treatment), while the individual bird was used as the experimen-
birds. A total of 2.5 ml was placed in an EDTA tube and 2.5 ml in a tal unit to analyse the slaughtering performance, the blood param-
serum-­separating tube. From a drop of blood without anticoagulant, eters, the gut histomorphology and the histopathological features
a blood smear was prepared and the May–­Grünwald and Giemsa (n = 18 per treatment).
were used as stain (Campbell, 1995). The blood samples previously Shapiro–­Wilk’s test established normality or non-­normality of
treated with a 1:200 Natt–­Herrick solution were employed for the distribution. Growth performance and blood parameters were an-
total red and white blood cell counts through an improved Neubauer alysed by means of one-­way ANOVA (post hoc test: Bonferroni’s
haemocytometer. On every slide, one hundred leucocytes, including multiple comparison test). Both the larvae intake times and the gut
granular (heterophils, eosinophils and basophils) and non-­granular histomorphological findings were analysed by fitting a general lin-
(lymphocytes and monocytes) leucocytes, were counted, and the ear mixed model (GLMM). In the first case, the GLMM allowed the
heterophils-­to-­lymphocytes (H/L) ratio was calculated (Salamano larvae intake minutes to depend on three fixed factors (diet, time
et al., 2010). and the interaction between the diet and the time). The replicate
The serum was obtained by keeping the tubes without an- was included as a random effect to account for repeated measure-
ticoagulant in a standing position at room temperature for ments on the same pen. In the second case, the GLMM allowed the
approximately two hours. Later, the serum was separated by cen- morphometric indices (Vh, Cd and Vh/Cd, separately) to depend on
trifugation (700 g for 15 min) and frozen at −80°C until analy- three fixed factors (diet, intestinal segment and the interaction be-
sis. The total proteins were quantified by means of the ‘Biuret tween the diet and the intestinal segment). Animal was included as a
method’ (Bio Group Medical System kit; Bio Group Medical random effect to account for repeated measurements on the same
BELLEZZA ODDON et al. | 5

TA B L E 3 Effects of the dietary treatments on the slaughtering

bird. The interactions between the levels of the fixed factors were
performance of the broiler chickens (n = 18)
evaluated by means of pairwise comparisons. The histopathological
scores were analysed by means of the Kruskal–­Wallis test (post hoc Dietary treatmentsa

test: Dunn’s multiple comparison test). Items C HI TM SEM p Valueb

The results were expressed as the mean (growth and slaughter-
LW (g) 2477 2517 2442 20.13 0.741
ing performance, blood parameters and histopathological features)
Hot carcass 76.48 76.01 76.07 0.153 0.402
or least square mean (larvae intake times and gut histomorphological yield (%LW)
findings) and standard error of the mean (SEM). p values ≤0.05 were
CCW (g) 1853 1841 1879 12.15 0.429
considered statistically significant.
Cold carcass 74.42 73.94 73.99 0.159 0.402
yield (%LW)
Breast yield 32.42 32.08 31.83 0.227 0.574
3 | R E S U LT S (%CCW)
Thigh yield 29.76 29.47 29.02 0.168 0.205
3.1 | Growth performance (%CCW)
Spleen (%LW) 0.78a 0.94b 0.88b 0.00 **
The data relating to the growth performance parameters are Liver (%LW) 1.61 1.68 1.67 0.03 0.602
shown in Table 2. The final LW (39 days of age), the ADG and the Bursa of 0.18 0.20 0.19 0.01 0.562
DFI taking into account the larvae intake did not differ among the Fabricius
groups. The FCR in the starter period was also not affected by the (%LW)
dietary treatment. Differently, the grower period FCR significantly Heart (%LW) 0.42 0.45 0.45 0.01 0.078
differed among the experimental groups, with the TM birds dis- Intestine (%LW) 3.25 3.48 3.34 0.05 0.111
playing a better feed efficiency than the HI (p < 0.01). Significant Glandular 0.30 0.31 0.30 0.00 0.474
differences were also identified in the overall FCR calculation, as stomach
the TM broilers showed a better FCR than the other dietary treat- (%LW)

ments (p < 0.01). Gizzard (%LW) 1.19 1.23 1.28 0.08 0.894
Abdominal fat 1.33 1.24 1.29 0.03 0.598
(%LW)

3.2 | Larva ingestion time Caecal length 16.92 17.25 17.14 0.03 0.771
(cm)

The broilers fed TM larvae showed lower intake times of larvae than Abbreviations: CCW, cold carcass weight; LW, live weight; SEM,
standard error of the mean.
the HI birds (p < 0.05; Figure 1), with the fastest larva consumption
a
Three dietary treatments: C = control; HI: C + 5% of DFI HI larvae; and
being also recorded during the second week of the trial for both the
TM: C + 5% of DFI TM larvae.
groups (p < 0.05). b
**p value < 0.01.

HI TM

7.00

6.00

5.00

4.00
MINUTES

3.00

2.00

1.00

0.00
W1 W2 W3 W4 W5
WEEKS

F I G U R E 1 Time spent by the broiler chickens on eating the insect larvae

6 | BELLEZZA ODDON et al.

TA B L E 4 Effects of dietary treatments

Dietary treatmentsa
p on the haematochemical parameters of
Items C HI TM SEM Value the broiler chickens (n = 18)
6
Erythrocytes (10 cell/µl) 2.85 3.13 3.43 1.23 0.165
Leucocytes (106 cell/µl) 12.49 14.15 13.13 11.26 0.836
H/L 1.09 1.13 0.91 0.07 0.361
Albumin (g/dl) 1.41 1.46 1.57 0.05 0.396
ALT (U/L) 3.50 2.89 6.00 0.60 0.076
AST (U/L) 372.67 360.33 345.06 15.70 0.779
Cholesterol (mg/dl) 165.22 165.56 162.50 2.43 0.859
Total protein (g/dl) 8.24 8.53 9.36 0.31 0.325
Triglycerides (mg/dl) 39.94 45.61 48.44 1.75 0.131
Uric acid (mg/dl) 3.86 3.47 3.13 0.24 0.468
HDL (mg/dl) 115.50 114.67 111.67 1.45 0.534
LDL (mg/dl) 41.73 41.7 41.14 1.34 0.978

Abbreviations: ALT, alaninoaminotransferase; AST, aspartate aminotransferase; HDL, high-­density

lipoproteins; H/L, heterophils/lymphocytes; LDL, low-­density lipoproteins; SEM, standard error of
the mean.
a
Three dietary treatments: C = control; HI: C + 5% of DFI HI larvae; and TM: C + 5% of DFI TM
larvae.

TA B L E 5 Intestinal morphometric indices in the broiler chickens in relation to diet and intestinal segment (n = 18)

Diet (D) Intestinal segment (IS) p Value†

Index C HI TM Duodenum Jejunum Ileum SEM D IS D × IS

a b c
Vh, mm 1.20 1.27 1.20 1.69 1.17 0.82 0.03 0.129 *** 0.866
a b c
Cd, mm 0.09 0.10 0.09 0.11 0.09 0.08 0.00 0.057 *** 0.576
Vh/Cd 14.01 13.55 12.80 16.61a 15.21a 9.46c 0.47 0.159 *** 0.949

Note: The means with different superscript letters (a, b, c) within the same row per fixed effect (i.e. diet, intestinal segment) differ significantly
(p < 0.05).
Abbreviations: C control, HI C + 5% of DFI HI larvae, TM C + 5% of DFI TM larvae, SEM standard error of the mean, Vh villus height, Cd crypt depth
and Vh/Cd villus height-­to-­crypt depth ratio.
†
***p value < 0.01.

3.3 | Slaughtering performance morphometric indices. On the contrary, the intestinal segment sig-
nificantly affected the gut histomorphology (p < 0.001). In particular,
No significant differences were overall observed for the slaughter- the duodenum showed greater Vh, Cd and Vh/Cd than the other gut
ing performance among the experimental treatments, except for the segments (p < 0.001), with morphometric indices being also greater
spleen relative weight being higher (p < 0.01) in the birds adminis- in the jejunum when compared to the ileum (p < 0.001).
tered with insect larvae when compared to the C group (Table 3). Absent or mild to moderate histopathological alterations were
observed in all the organs from all the experimental treatments, as
shown in Table 6. Independently of the insect larvae administration,
3.4 | Haematological and serum parameters the spleen showed mild, multifocal white pulp hyperplasia, while mild
to moderate, multifocal to diffuse follicular depletion was detected in
As reported in Table 4, the haematological and the serum param- the bursa of Fabricius. Furthermore, the liver showed mild to moder-
eters were not influenced by the dietary treatments. ate, multifocal to diffuse vacuolar degeneration of the hepatocytes,
as well as mild to moderate, multifocal lymphoplasmacytic infiltrates,
whereas in gut mild to moderate, multifocal to diffuse, mucosal or
3.5 | Histomorphological investigations submucosal lymphoplasmacytic infiltrates with or without GALT ac-
tivation were observed. No alterations were detected in the thymus.
As summarized in Table 5, there was no significant influence of diet However, the insect larvae administration did not affect the histo-
and interaction between diet and intestinal segment on the gut pathological scores.
BELLEZZA ODDON et al. | 7

TA B L E 6 Histopathological alterations in the broiler chickens

uncovered that the consumption of larvae up to 5% of the dietary
(n = 18)
DM has a neutral effect on the growth rate (Ipema et al., 2020). The
Dietary treatmentsa reduction of FCR in the TM broilers may result from the proximate
p
Items C HI TM SEM Value composition of the larvae. Indeed, in the late instar, the TM larvae
of the present study were characterized by a greater quantity of
Spleen 0.05 0.32 0.38 0.06 0.062
DM, CP and EE than the HI larvae (27.54, 10.82, 5.50; 25.32, 8.07
Thymus Absence of alterations
and 1.93 respectively). Therefore, due to its composition, the TM
Bursa of 0.25 0.17 0.41 0.06 0.308
larvae appear to be more nutritious than the HI larva and its effect
Fabricius
appeared in the second period of growth when, proportionally, the
Liver 1.47 1.55 1.13 0.10 0.197
quantity of larvae administered became more conspicuous.
Gut 1.88 1.22 1.94 0.14 0.069

Note: The data are expressed as the mean of the scores (0 = absence of
alterations; 1 = mild alterations; 2 = moderate alterations; 3 = severe
4.2 | Larva ingestion time
alterations). The gut scores resulted from the mean of the three gut
segment scores (in turn obtained from the adding of the mucosa and
submucosa scores). The assessment of the larva ingestion time revealed that broiler
Abbreviation: SEM, standard error of the mean. chickens were very eager to eat live larvae, especially the TM larvae.
a
Three dietary treatments: C = control; HI: C + 5% of DFI HI larvae; and The TM broilers showed lower intake times of larvae than the HI birds
TM: C + 5% of DFI TM larvae.
(3.45 and 4.27 min, respectively), with the fastest larva consumption
being also recorded during the second week of the trial for both the
4 | DISCUSSION groups. Since in the first week the animals needed to adapt to the
new feed provided, the larva ingestion time was longer than the sec-
4.1 | Growth performance ond week. In the last three weeks, the intake time slowed down in
relation to the increase in body mass of the birds and, consequently,
During the experimental trial, no mortality was recorded. Since in the increased difficulty of movement. The daily assessment of the
nature birds eat insects, especially in the first weeks of their life larvae intake times represents an innovative parameter. In the study
(Bruns, 1960), they can be used as environmental and nutritional conducted with turkeys, in which HI larvae were provided as 10% of
enrichments in order to improve animal welfare. Chickens that have the DFI, the authors observed that the larvae were consumed within
access to outdoor zone pick up insects at all life stages and eat two minutes after provision (Veldkamp & van Niekerk, 2019). This
them on a voluntary basis, thus meaning that they are evolutionar- result is in contrast to the broilers’ intake times of the first, third and
ily adapted to insects as a natural part of their diet (Bovera et al., fourth week, but is similar to the second week (2.17 min). Apart from
2016). This is the reason why the birds of the trial were very eager the different bird species, the discrepancies between the results
to eat TM and HI larvae. The consumption of the larvae did not may be due to dissimilar methods of measuring the intake time—­as
affect the final LW, the ADG, the DFI and the starter period FCR. Veldkamp & van Niekerk (2019) did not indicate the exact registra-
Despite the absence of a statistical significance, in both the feeding tion start moment—­and the type of larvae administration.
phases the DFI displayed by the C birds was found to be numeri-
cally greater than that of the larvae-­administered group. Therefore,
it is conceivable that the nutrient supply derived from dietary in- 4.3 | Slaughtering performance
sect larvae supplementation may have partially influenced this
growth parameter. In the grower phase, the HI birds showed a simi- The slaughtering performance of the broiler chickens was overall not
lar FCR to the C group, but a worse feed efficiency when compared influenced by the dietary treatments. On the contrary, the spleen
to the TM broiler chickens. Furthermore, the birds administered weight was found to be significantly higher in the larvae-­fed animals
with TM larvae showed a better overall FCR when compared to than in the C group. Indeed, the study showed a reactivity of the
the other groups. The result derived from the comparison of the C spleen white pulp in all the groups, with numerically heavier spleen
group in respect of the HI animals contrasts with those reported in being identified in the treated animals in comparison with the control
literature. Indeed, in either the turkey or the laying hens trials, in birds. This outcome can be related to the chitin, a polysaccharide ex-
which the quantity of supplied HI larvae was equal to 10% of the isting in insect that exerts an immunostimulant effect (Gasco et al.,
DFI, an increase in the feed efficiency was observed (Veldkamp & 2018). Bovera et al. (2016) have reached the same conclusion, since
van Niekerk, 2019; Star et al., 2020). The turkeys fed HI larvae also the authors observed spleen weight gain in animals fed TM meal and
showed a higher body weight gain and final body weight than the associated the results with the antimicrobial and antifungal chitin
C treatment (Veldkamp & van Niekerk, 2019). On the contrary, the properties. Furthermore, more stressed animals have a lower spleen
productive parameters in the laying hens (laying rate, egg weight, weight due to the corticosterone effect, which depresses lymphoid
egg mass and mortality rate) were not affected by the larvae ad- organs growth (Bovera et al., 2016). Consequently, the birds fed in-
ministration (Star et al., 2020). Moreover, previous studies have sect larvae could be in a situation of greater welfare than the C group.
8 | BELLEZZA ODDON et al.

4.4 | Haematological and serum parameters observed. Moreover, it has been shown that different insect spe-
cies in larval stage can differently affect the production parameters
Prior to this study, no research has focused its attention on evaluat- of the birds. The live larvae, thanks to its chitin component, could
ing the effects of live larvae on broilers’ blood parameters. From a have immunostimulant properties and, therefore, can positively in-
general point of view, blood tests represent useful indicators of the fluence the broiler chicken health. Future behavioural and welfare
health and metabolic status of the birds. In particular, the assessment studies will be required to confirm that live insect larvae can be used
of blood biochemical parameters, such as total protein, albumin, AST, as natural environmental enrichment in intensive poultry farming.
ALT, cholesterol, triglycerides, uric acid and others, can serve as diag-
nostic tool for identifying specific metabolic disturbances occurring AC K N OW L E D G M E N T S
in the main organs (Nunes et al., 2018). Considering that the haema- The research was supported by the European Knowledge and
tological and serological analyses led to similar results among the ex- Innovation Community (KIC), within the EIT Food programme ‘From
perimental treatments, it is reasonable to hypothesize that the insect waste to farm: insect larvae as tool for welfare improvement in
larvae administration has no negative effects on bird metabolic status. poultry’ (Project ID 19122). The authors are thankful to Entomics
Furthermore, the blood values that have been taken into considera- Biosystems (Cambridge, UK), which provided the live larvae through-
tion in the present study are within the broiler chickens physiological out the experimental trial. The authors are also grateful to Mr. Dario
ranges (Lumej, 2008). The results obtained are also in agreement with Sola for bird care and technical support.
those of Biasato et al. (2018) and Dabbou et al. (2018), in which no ef-
fect on blood traits was observed in broiler chickens fed with increas- A N I M A L W E L FA R E S TAT E M E N T
ing level of insect (HI or TM) meal inclusion (inclusion range 5%–­15%). The authors confirm that the ethical policies of the journal, as noted
on the journal’s author guidelines page, have been adhered to and
the appropriate ethical review committee approval has been re-
4.5 | Histomorphological investigations ceived. The authors confirm that they have followed EU standards
for the protection of animals used for scientific purposes and feed
So far, no histomorphological investigations have been performed in legislation.
poultry reared with live insect larvae as environmental enrichments.
The broiler chickens administered with insects of the present study ORCID
showed similar gut morphology when compared to the C birds, thus Sara Bellezza Oddon https://orcid.org/0000-0001-8942-0682
probably revealing no significant effects of the live larvae on the Ilaria Biasato https://orcid.org/0000-0002-8855-4248
intestinal digestion and absorption properties. Differently, increas- Arianna Imarisio https://orcid.org/0000-0002-6137-0377
ing inclusion levels of TM (Biasato et al., 2018) and HI (Dabbou et al., Miha Pipan https://orcid.org/0000-0001-8391-5924
2018) meals in diets for broiler chickens have previously been re- Dominik Dekleva https://orcid.org/0000-0003-1685-541X
ported to deeply worsen the gut morphology of the birds (in terms Elena Colombino https://orcid.org/0000-0002-6371-2000
of shorter villi, deeper crypts and reduced Vh/Cd), also determining Maria Teresa Capucchio https://orcid.org/0000-0002-1068-0551
a significant impairment in their growth performance (in terms of Marco Meneguz https://orcid.org/0000-0001-8007-2044
increased FCR). Since the intestinal morphometric indices resulted Bergagna Stefania https://orcid.org/0000-0001-5390-4108
herein unaffected, the improvement in the feed efficiency observed Raffaella Barbero https://orcid.org/0000-0003-4497-6382
in the TM broilers was probably related to the better nutritional pro- Marta Gariglio https://orcid.org/0000-0001-5224-8604
file of the TM larvae in comparison with the HI species. Sihem Dabbou https://orcid.org/0000-0002-3525-1614
The insect larvae administration in the broiler chickens of the cur- Edoardo Fiorilla https://orcid.org/0000-0002-0173-1118
rent trial did not lead to the development of significant histopatho- Laura Gasco https://orcid.org/0000-0002-1829-7936
logical alterations, thus confirming the safety of including insects Achille Schiavone https://orcid.org/0000-0002-8011-6999
and insect-­based products in poultry nutrition. Indeed, dietary insect
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