Animal (2018), 12:10, pp 2032–2039 © The Animal Consortium 2018

doi:10.1017/S1751731117003743
animal

Black soldier fly larva fat inclusion in finisher broiler chicken diet

as an alternative fat source
A. Schiavone1,5, S. Dabbou1, M. De Marco1, M. Cullere2, I. Biasato1, E. Biasibetti1,
M. T. Capucchio1, S. Bergagna3, D. Dezzutto3, M. Meneguz4, F. Gai5, A. Dalle Zotte2† and
L. Gasco4,5
1
Department of Veterinary Sciences, University of Turin, Largo Paolo Braccini 2, 10095 Grugliasco (TO), Italy; 2Department of Animal Medicine, Production and
Health, University of Padova, Agripolis, Viale dell’Università 16, 35020 Legnaro, Padova, Italy; 3Veterinary Medical Research Institute for Piemonte, Liguria and Valle
d’Aosta, Via Bologna 148, 10154 Turin, Italy; 4Department of Agricultural, Forest and Food Sciences, University of Turin, Largo Paolo Braccini 2, 10095 Grugliasco
(TO), Italy; 5Institute of Science of Food Production, National Research Council, Largo Paolo Braccini 2, 10095 Grugliasco (TO), Italy

(Received 17 May 2017; Accepted 13 December 2017; First published online 18 January 2018)

The objective of the present study was to evaluate the effects of partial or total replacement of finisher diet soybean oil with black
soldier fly (Hermethia illucens L.; HI) larva fat on the growth performance, carcass traits, blood parameters, intestinal morphology
and histological features of broiler chickens. At 21 days of age, a total of 120 male broiler chickens (Ross 308) were randomly
allocated to three experimental groups (five replicates and eight birds/pen). To a basal control diet (C; 68.7 g/kg as fed of soybean
oil), either 50% or 100% of the soybean oil was replaced with HI larva fat (HI50 and HI100 group, respectively). Growth
performance was evaluated throughout the trial. At day 48, 15 birds (three birds/pen) per group were slaughtered at a commercial
abattoir. Carcass yield and proportions of carcass elements were recorded. Blood samples were taken from each slaughtered
chicken for haematochemical index determination. Morphometric analyses were performed on the duodenum, jejunum and ileum.
Samples of liver, spleen, thymus, bursa of fabricius, kidney and heart were submitted to histological investigations. Growth
performance, carcass traits, haematochemical parameters and gut morphometric indexes were not influenced by the dietary
inclusion of HI larva fat. Histopathological alterations developed in the spleen, thymus, bursa of fabricius and liver and were
identified in all of the experimental groups, but HI larva fat inclusion did not significantly affect ( P > 0.05) the severity of the
histopathological findings. The present study suggests that 50% or 100% replacement of soybean oil with HI larva fat in broiler
chickens diets has no adverse effects on growth performance or blood parameters and had no beneficial effect on gut health.

Keywords: Hermetia illucens, broiler chickens, dietary fat source, growth performance, gut morphometry

Implication Introduction
The black soldier fly (Hermetia illucens L.; HI) has been the The majority of the lipid sources used in poultry diets consists
subject of recent attention in poultry nutrition as an alter- mainly of rendered fat, coming from the part of the slaugh-
native ingredient. The lipid content of whole non-defatted tered animal which is not used for human consumption, and
black soldier fly larvae is notable (26% to 35%). The process crude vegetable oils. Among vegetable oils, soybean oil is
of larva defatting results in two potential feed ingredients: utilised as an ingredient in poultry diets due to its high
fat and protein-concentrate meal. Recently, the fat derived energy value, digestibility and metabolizable energy content
from larvae of HI has been shown to be an acceptable lipid compared with other vegetable oils (Fascina et al., 2009).
source for broiler chickens. This study demonstrated that fat However, currently, the limited supply of soybeans and their
from HI can totally and partly substitute for conventional high price has caused increasing interest in the search for
lipid sources in finisher broiler diet without impairing per- new alternative lipid sources for poultry feed.
formance, carcass characteristics, intestinal morphology or Insects represent an opportunity for partial or total replace-
histological features. ment of the conventional lipid feed sources. Furthermore,
insects have a potential as a feed ingredient due to their
†
E-mail: [email protected] appropriate nutritional quality (Veldkamp and Bosch, 2015)

2032
 Black soldier fly larva fat in broiler diet

and greater acceptance by poultry (Leiber et al., 2017). Indeed, care and use of experimental animals (European directive 86/
insects (adult, larval and pupal form) are naturally consumed 609/EEC, put into law in Italy with D.L.116/92) and approved
by wild birds and free-range poultry (Rumpold et al., 2016). In by the Ethical Committee of the Department of Veterinary
recent years, most of the attention on insects as a feed source Sciences of the University of Turin (Italy).
has been focussed on their protein content (Belforti et al.,
2015; Gasco et al., 2016; Renna et al., 2017). Certain insect Experimental design and feed preparation
species have a high fat content with higher levels in larval The study was carried out at the experimental poultry farm of
stages than in adults (Ramos-Elorduy, 1997; Barroso et al., the Department of Agricultural, Forest and Food Sciences
2014). In fact, lipids are also a main component of insects and (DISAFA) of the University of Turin, located in Carmagnola (TO),
are produced during protein isolation. Surendra et al. (2016) Italy. The poultry house was 7 m wide × 50 m long × 7 m high,
pointed out the importance of defatting insect meal and then equipped with waterproof floor and walls, covered completely
using insect protein concentrate as an animal feed ingredient by tiles and with an automatic ventilation system. The impact
and the lipids for both animal nutrition and the production of of partial or total replacement of soybean oil with HI larva fat in
biodiesel. Such authors showed that fat removal is necessary to broiler chickens’ finisher diet (from 21 to 48 days of age) was
improve the storability of the feed and to increase the protein evaluated by replacement in a basal control diet (C: 100%
digestibility of the insect-derived feed. soybean oil; 68.7 g/kg as fed of soybean oil), either at 50% or
Black soldier fly (Hermetia illucens L.; HI) is a very pro- 100% HI larva fat (HI50 and HI100 groups, respectively). All
mising species because of its amino acid profile, lipid content diets were formulated according to Aviagen (2014) broiler
and calcium content (Cullere et al., 2016; Schiavone et al., nutrition specifications (Table 1). Chickens had free access to
2017b). The larva of the black soldier fly contains up to 45% water and feed throughout the trial.
of lipids (Li et al., 2016). In the fatty acid (FA) profile of HI
larvae, linoleic acid content is greater than α-linolenic acid Birds and husbandry
content, similar to many plant oils (such as soybean oil and Day-old male broiler chickens (Ross 308) were farmed in a
sunflower oil). The FA profile is also rich in medium-chain floor pen until 21 days of age and fed a commercial broiler
fatty acids (MCFAs) such as lauric acid (C12:0) and its esters, starter diet (217 g/kg of CP; 12.9 MJ/kg metabolizable
which represent 21.4% to 49.3% of the total profile (Tran energy). At hatching, all the birds were vaccinated against
et al., 2015; Li et al., 2016; Ushakova et al., 2016). The use of Newcastle disease, Marek disease, infectious bronchitis and
dietary fats rich in lauric acid and myristic acid (C14:0) in coccidiosis. At day 21, after equalizing for mean initial BW,
broilers could be advantageous in terms of gut health and 120 birds were chosen and homogeneously distributed over
growth performance. The underlying mechanism involves three dietary treatments, each one consisting of five pens as
antimicrobial effects on gut bacteria and changes in gut replicates with eight chicks per pen. The animals were reared
morphology (Zeitz et al., 2015). to the slaughter age set at 48 days. Each pen was 1.0 m
Chicken growth directly depends on the morphological wide × 1.5 m long. Each pen was equipped with a feeder, an
and functional integrity of the digestive tract. Microscopic automatic drinker and rice hulls as litter. Up to 21 days of age
structure parameters such as villus height and crypt depth infrared lamps were used to keep a suitable body tempera-
have been reported to be good indicators of intestinal ture according to standard breeding practices (Aviagen,
development, health and functionality, influencing nutrient 2014). The lighting schedule was 18 h light : 6 h dark for
digestion and absorption (Wang and Peng, 2008). Despite the whole experimental period. Clinical signs of illness and
the relationship between dietary modifications and gut mortality were monitored daily throughout the experimental
morphology being widely investigated in poultry, limited period.
data about the utilization of feed fats are currently available
(Ozdogan et al., 2014; Zeitz et al., 2015). Chemical composition and fatty acid profile of the
Currently, knowledge about the suitability of HI larvae fat as experimental diets
a poultry feed ingredient is still scarce, and to the best of our Chemical analyses were carried out on three replicates of each
knowledge, no studies have yet been carried out to determine feed sample. Diet samples were ground through a 0.5-mm
the effects of soybean oil replacement by HI larva fat on gut sieve and stored in airtight plastic containers. They were ana-
morphology and histological features. For this reason, the lysed for DM (#930.15), ash (#924.05), and CP (#984.13)
objective of the present work was to evaluate the effects of according to the Association of Official Analytical Chemists
finisher diet HI larva fat on the growth performance, carcass (2005) methods. The gross energy content was determined
traits, blood parameters, intestinal morphology and histo- using an adiabatic calorimetric bomb (IKA C7000; IKA, Staufen,
logical features of broiler chickens. Germany). The lipid extraction and FA profiling of the experi-
mental diets were carried out at the laboratory of the Depart-
ment of Animal Medicine, Production and Health, University of
Material and methods
Padova, Legnaro, Italy. The lipid extraction was performed by
Ethical approval Accelerated Solvent Extraction (M-ASE) using petroleum ether
The experimental protocol was designed according to the as a solvent. The FA profile was determined as described by
guidelines of the current European and Italian laws on the Mattioli et al. (2016) and Schiavone et al. (2007). Samples were

2033
Schiavone, Dabbou, De Marco, Cullere, Biasato, Biasibetti, Capucchio, Bergagna, Dezzutto, Meneguz, Gai, Dalle Zotte and Gasco

transmethylated using a methanolic solution of H2SO4 (4%) in GA, USA). The aspartate-aminotransferase (AST), alanine-
order to determine fatty acid methyl esters (FAME). A biphasic aminotransferase (ALT), triglycerides, cholesterol, phosphorus,
separation was obtained by adding 0.5 ml of distilled magnesium, iron, uric acid and creatinine serum concentrations
water and 1.5 ml of N-heptane to each sample. Fatty acid were measured by means of enzymatic methods in a clinical
methyl esters were quantified by gas chromatography chemistry analyser (Screen Master Touch; Hospitex diagnostics
(Shimadzu GC17A), equipped with an Omegawax 250 column Srl., Florence, Italy).
(30 m × 0.25 µm × 0.25 µm) and flame ionization detector.
Helium was used as the carrier gas at a constant flow of 0.8 ml/ Histomorphological investigations
min. The injector and detector temperatures were 260 °C. The slaughtered animals were submitted to anatomopatho-
Peaks were identified based on commercially available FAME logical investigations. Intestinal segment samples (~5 cm in
mixtures (37-Component FAME Mix; Supelco Inc., Bellefonte, length) of duodenum, jejunum and ileum were excised and
PA, USA). The results are expressed as % of total detected flushed with 0.9% saline to remove all the contents. The
FAME. The proximate and FA composition of the feeds are collected segments of intestine were the loop of the duode-
reported in Table 1. num, the tract before Meckel’s diverticulum (jejunum) and
the tract before the ileocolic junction (ileum). Samples of
Growth performance liver, spleen, thymus, bursa of fabricius, kidney and heart
During the whole experimental period (21 to 48 days), the were also collected. Gut segments were fixed in Carnoy’s
BW and feed consumption (FC) of all birds were measured solutions for morphometric analysis, while the other organ
weekly to calculate daily feed intake, daily weight gain samples were fixed in 10% buffered formalin solution for
(DWG) and the feed conversion ratio (FCR). Final BW (FBW) histological examination. Tissues were routinely embedded
was recorded on day 48. All measurements were carried out in paraffin wax blocks, sectioned at 5 µm thickness, mounted
on the pen basis using a high precision electronic scale on glass slides and stained with haematoxylin and eosin
(Sartorius – Signum®). (HE). The evaluated morphometric indexes were the villus
height (Vh, from the tip of the villus to the crypt), crypt depth
Slaughtering procedures (Cd, from the base of the villus to the submucosa) and the
At day 48, 15 birds (three birds/pen) from each feeding group Vh/Cd ratio (Laudadio et al., 2012). Morphometric analyses
(chosen on the basis of pen average FBW) were individually were performed on 10 well-oriented and intact villi and 10
identified with a shank ring and weighed. The chickens were crypts chosen from the three collected intestinal segments
electrically stunned and slaughtered at a commercial abat- (Qaisrani et al., 2014). Histopathological alterations were
toir. The plucked and eviscerated carcasses were obtained, evaluated using a semi-quantitative scoring system as pre-
and the head, neck, feet and abdominal fat were removed to viously assessed by Biasato et al. (2016): absent/minimal
obtain the chilled carcass. Then, the weights of the liver, (score = 0), mild (score = 1) and severe (score = 2).
heart, spleen, bursa of fabricius, abdominal fat, and breast
and thighs were immediately recorded. The breast and thigh Statistical analysis
weights were expressed as percentage of live weight. The statistical analysis was performed using the SPSS software
package (version 17 for Windows; SPSS Inc., Chicago, IL, USA).
Haematological and serum parameters The experimental unit was the pen for growth performance,
At slaughtering (day 48), blood samples were collected from while it was the individual bird for all of the other parameters.
three birds per pen. A total of 2.5 ml was placed in an Data were tested by one-way ANOVA, followed by Tukey’s
ethylenediaminetetraacetic acid tube and 2.5 ml in a serum- post hoc test for growth performance, carcass traits and blood
separating tube. Then, a blood smear was prepared, using parameters. One-way ANOVA (Duncan’s multiple range test) or
one glass slide for each bird, from a drop of blood without the Kruskal–Wallis test (post hoc test: Dunn’s Multiple Com-
anticoagulant. The smears were stained using May- parison test) was used to compare (1) the morphometric
Grünwald and Giemsa stains (Salamano et al., 2010). The indexes among the dietary treatments within each intestinal
total red (erythrocytes) and white (leucocytes) blood cell segment and (2) the morphometric indexes among the intest-
counts were determined in an improved Neubauer haemo- inal segments within each dietary treatment. Histopathological
cytometer on blood samples previously treated with 1 : 200 scores were analysed by the Kruskal–Wallis test (post hoc test:
Natt-Herrick solution. A total of 100 leukocytes, including Dunn’s Multiple Comparison test). Significance was declared at
granular (heterophils, eosinophils and basophils) and non- P < 0.05. A statistical trend was considered for P < 0.10. The
granular (lymphocytes and monocytes) leucocytes, were results are expressed as the mean and pooled standard error of
counted on the slide, and the H/L ratio was calculated. The the mean (SEM).
tubes without anticoagulant were left to clot in a standing
position at room temperature for ~2 h to obtain serum. The
Results
serum was separated by means of centrifugation at 700 × g for
15 min and frozen at −80°C until analysis. The electrophoretic Diet composition and fatty acid profile
pattern of the serum was obtained using a semi-automated The ingredients and the chemical and FA composition of
agarose gel electrophoresis system (Sebia Hydrasys®, Norcross, the three experimental diets are summarized in Table 1.

2034
 Black soldier fly larva fat in broiler diet

Table 1 Ingredients (g/kg as fed), chemical (g/kg DM) and fatty acid Table 2 Effects of dietary Hermetia illucens larvae fat (HI) inclusion on
composition (% of total FAME) of the experimental diets the growth performance of broiler chickens (n = 5 pens/treatment)
C HI50 HI100 C HI50 HI100 SEM P-value

Ingredients Initial BW (g; day 21) 823.1 823.1 817.8 3.12 0.762
Maize meal 547.4 547.4 547.4 Final BW (g; day 48) 3621.6 3576.9 3751.3 34.46 0.087
Soybean meal 294 294 294 Daily weight gain (g) 107.6 105.9 112.8 1.36 0.084
Soybean oil 68.7 34.3 – Daily feed intake (g) 208.3 207.3 212.4 1.16 0.175
Hermetia illucens larvae fat – 34.3 68.7 Feed conversion ratio 2.01 2.03 1.95 0.01 0.172
Gluten meal 50 50 50
C = control; HI50 = Hermetia illucens 50%; HI100 = Hermetia illucens 100%.
Dicalcium phosphate 12.4 12.4 12.4
Calcium carbonate 11.2 11.2 11.2
Sodium chloride 2.2 2.2 2.2
Table 3 Effects of dietary Hermetia illucens (HI) larvae fat inclusion on
Sodium bicarbonate 1.5 1.5 1.5
carcass traits and internal organs weight (n = 15/treatment)
DL-methionine 1.6 1.6 1.6
L-Lysine 3.6 3.6 3.6 C HI50 HI100 SEM P-value
Threonine 1 1 1
Mineral and vitamin finisher premix1 5 5 5 LW (g) 3661 3681 3756 22.5 0.189
Choline chloride 0.4 0.4 0.4 Chilled carcass (g) 2594 2624 2651 19.1 0.483
3-phytase (E-300; natuphos bio/G500) 1 1 1 Chilled carcass (% of LW) 70.8 71.3 70. 6 0.25 0.494
Analysed composition Breast (g) 743.3 756.6 778.1 9.92 0.360
Gross energy (MJ/kg) 19.78 19.65 19.70 Breast (% of LW) 20.3 20.6 20.7 0.24 0.745
Chemical composition Thighs (g) 800 821 838 7.45 0.113
Dry matter (g/kg) 898.7 892.9 898.2 Thighs (% of LW) 21.9 22.3 22.3 0.15 0.373
CP 219.5 210.6 210.4 Abdominal fat (g) 32.0 39.9 41.6 2.03 0.120
Crude fat 90.3 85.7 90.1 Liver (g) 69.1 66.9 64.0 1.29 0.289
Ash 57.5 55.3 57.0 Heart (g) 18.6 18.8 17.5 0.39 0.317
Fatty acid composition (% of total FAME) Spleen (g) 5.0 4.2 5.1 0.19 0.111
SFA 16.0 36.2 56.9 Bursa of fabricius (g) 8.1 7.8 7.6 0.39 0.846
C12:0 0.0 17.3 34.7
C14:0 0.0 3.4 6.5 C = control; HI50 = Hermetia illucens 50%; HI100 = Hermetia illucens 100%;
LW = live weight.
MUFA 23.8 19.9 15.2
PUFA 59.8 43.3 27.9
UFA/SFA 5.22 1.75 0.76 Growth performance and slaughtering traits
∑n-6 54.9 40.1 26.2 During the whole experimental period, the birds remained
∑n-3 4.88 3.22 1.66
healthy, no signs of illness were observed, and the mortality
∑n-6/∑n-3 11.2 12.5 15.8
rate was zero in all groups. The growth performance of the
C = control; HI50 = Hermetia illucens 50%; HI100 = Hermetia illucens 100%, broiler chickens is reported in Table 2. The initial BW of
DM = dry matter; AME = apparent metabolizable energy; FAME = fatty acids chicks did not differ (P > 0.05) among the three dietary
methyl esters; FA = fatty acids; SFA = saturated fatty acids; MUFA =
monounsaturated fatty acids; PUFA = polyunsaturated fatty acids; UFA = treatments. Growth performance was not influenced by the
unsaturated fatty acids. partial or total replacement of soybean oil with HI larva fat
1
Mineral-vitamin finisher premix (Final B Prisma, IZA SRL, Forlì, Italy), given
values are supplied per kg of diet: 2.500.000 IU of vitamin A; 1.000.000 IU of
(P > 0.05). However, a positive numerical trend was
vitamin D3; 7.000 IU of vitamin E; 700 mg of vitamin K; 400 mg of vitamin B1; observed in the HI100 group for FBW (P = 0.087) and DWG
800 mg of vitamin B2; 400 mg of vitamin B6; 4 mg of vitamin B12; 30 mg of (P = 0.084). No statistical differences were observed for
biotin; 3.111 mg of Ca pantothenate acid; 100 mg of folic acid; 15.000 mg of
vitamin C; 5.600 mg of vitamin B3; 10.500 mg of Zn, 10.920 mg of Fe; 9.960 mg
FCR; nevertheless, the HI100 group displayed the lowest
of Mn; 3.850 mg of Cu; 137 mg of I; 70 mg of Se. numerical value. Dietary HI larva fat inclusion did not
significantly affect (P > 0.05) the slaughtering traits of the
chickens (Table 3).
The three diets were isonitrogenous and had similar gross
energy, total lipid, protein and ash contents but differed Haematological and serum parameters
greatly in their FA composition. In the experimental diets, The dietary HI larva fat inclusion level did not significantly
C12:0 and C14:0 FA and total saturated fatty acids (SFAs) influence (P > 0.05) the haematological or serum biochemi-
increased with increasing inclusion of HI larva fat. Total cal traits of the birds (Table 4).
monounsaturated FAs (MUFAs), polyunsaturated FAs
(PUFAs), total n-3 PUFAs and total n-6 PUFAs decreased in Histomorphological investigations
the experimental diets as the HI larva fat inclusion level The intestinal morphology of the broiler chickens is
increased, which determined an increase of the ∑n-6/∑n-3 summarized in Table 5. Dietary HI larva fat inclusion did
ratio in the experimental diets (from 11.2% to 15.8% of total not significantly affect (P > 0.05) gut morphometric indexes.
FAME for HI0 and HI100, respectively). In all dietary treatments, the duodenum showed greater Vh

2035
Schiavone, Dabbou, De Marco, Cullere, Biasato, Biasibetti, Capucchio, Bergagna, Dezzutto, Meneguz, Gai, Dalle Zotte and Gasco

Table 4 Effects of dietary Hermetia illucens (HI) larvae fat inclusion on Table 6 Effects of dietary Hermetia illucens (HI) larvae fat inclusion on
blood parameters of broiler chickens (n = 15/treatment) histopathological scores of broiler chickens (n = 15/treatment).
C HI50 HI100 SEM P-value C HI50 HI100 SEM P-value

Erythrocytes (106cell/μl) 4.19 3.90 3.88 0.08 0.208 Spleen 0.84 1.06 0.94 0.14 0.488
Leucocytes (103 cell/μl) 12.3 12.5 12.4 0.17 0.861 Thymus 0.16 0.27 0.13 0.10 0.637
H/L ratio 0.64 0.57 0.63 0.03 0.679 Bursa of fabricius 1.06 0.88 1.19 0.13 0.275
Total proteins 4.22 4.15 4.03 0.14 0.864 Liver 0.40 0.50 0.69 0.16 0.428
Uric acid (mg/dl) 3.47 3.85 3.61 0.23 0.808 Heart No alterations
Creatinine (mg/dl) 0.30 0.31 0.32 0.01 0.302 Kidney No alterations
AST (UI/l) 279 285 294 9.31 0.817
C = control; HI50 = Hermetia illucens 50%; HI100 = Hermetia illucens 100%.
ALT (UI/l) 22.7 25.7 23.7 0.88 0.363
GGT (UI/l) 29.8 25.3 26.4 1.46 0.407
Triglycerides (mg/dl) 32.3 32.7 33.0 1.30 0.980
Cholesterol (mg/dl) 59.8 66.7 65.8 1.58 0.160 of the dietary treatments. The histopathological scores of the
Phosphorus (mg/dl) 6.51 6.84 5.72 0.24 0.151 broiler chickens are shown in Table 6. Dietary HI larva fat
Magnesium (mEq/l) 2.17 1.55 1.82 0.16 0.263 inclusion did not significantly affect (P > 0.05) the severity of
Iron (μg/dl) 72.95 73.86 73.27 3.47 0.994 the histopathological findings. Spleen showed mild (69% of
the broilers in all the dietary treatments) to severe (C = 6%;
AST = aspartate-aminotransferase; ALT = alanine-aminotransferase; GGT =
gamma-glutamyl transferase; C = control; HI50 = Hermetia illucens 50%; HI50 = 19%; HI100 = 13%) white pulp depletion or hyper-
HI100 = Hermetia illucens 100%. plasia. Of the animals, 25% (C), 13% (HI50) and 19%
(HI100) had a normal spleen. In the thymus, mild cortical
Table 5 Effects of dietary Hermetia illucens (HI) larvae fat inclusion on depletion was found in all the groups (C = 13% of the
intestinal morphometric indexes of broiler chickens (n = 15/treatment) broilers; HI50 = 25%; HI100 = 13%). A normal thymus was
C HI50 HI100 SEM P-value
observed in 88% (C), 75% (HI50) and 88% (HI100) of the
animals. The bursa of fabricius showed mild (C = 69% of the
Duodenum broilers; HI50 = 63%; HI100 = 81%) to severe (C = 19%;
Vh (mm) 2.53a 2.52a 2.38a 0.10 0.579 HI50 = 13%; HI100 = 19%) follicular depletion. Of the
Cd (mm) 0.15 0.17 0.15 0.01 0.415 animals, 13% (C), 25% (HI50) and 0% (HI100) had a normal
Vh/Cd 17.62× 16.53 16.89× 1.42 0.859 bursa of fabricius. The liver showed mild (C = 25% of the
Jejunum broilers; HI50 = 50%; HI100 = 44%) to severe (C = 6%;
Vh (mm) 2.02b 2.01b 2.04b 0.13 0.987 HI50 = 0%; HI100 = 13%) perivascular lymphoid tissue
Cd (mm) 0.16 0.17 0.15 0.01 0.390 activation. A normal liver was observed in 63% (C), 50%
Vh/Cd 13.26y 13.07 14.40xy 1.06 0.469
(HI50) and 44% (HI100) of the animals.
Ileum
Vh (mm) 1.74b 1.71b 1.56b 0.09 0.352
Cd (mm) 0.16 0.15 0.13 0.01 0.159 Discussion
Vh/Cd 11.71y 11.60 11.98y 0.78 0.942
P Vh 0.000 0.000 0.000 0.05 Growth performance and slaughtering traits
P Vh/Cd 0.002 0.430 0.004 0.43 The inclusion of HI larva fat substituting 50 or 100% of the
soybean oil in finisher broiler chickens’ diet did not lead to
Vh = villus height; Cd = crypt depth; Vh/Cd = villus height to crypt depth ratio;
C = control; HI50 = Hermetia illucens 50%; HI100 = Hermetia illucens 100%. any adverse effects on growth performance or carcass traits.
Different superscript letters in the same column mean significant differences Feed intake was not affected by the partial or total replace-
(P < 0.05) among the intestinal segments (duodenum, jejunum and ileum) ment of soybean oil with HI larva fat, indicating that 50%
for Vh (a, b) and Vh/Cd (x, y) within each dietary treatment. P-values of the
significant differences are indicated as P Vh and P Vh/Cd. and 100% HI inclusion were both acceptable to broiler
chickens. This finding suggests that it is possible to replace
up to 100% of the soybean oil with HI larva fat in diets for
(C = 2.53 mm; HI50 = 2.52 mm; HI100 = 2.38 mm) (P < 0.001) finisher chickens without any negative effects on feed utili-
than the jejunum (C = 2.02 mm; HI50 = 2.01 mm; HI100 = zation or productive performance. Comparing HI larva fat
2.04 mm) and ileum (C = 1.74 mm; HI50 = 1.71 mm; HI100 = with other vegetable oils used in poultry diets, it seems to be
1.56 mm). In the C group, the Vh/Cd ratio was higher similar to coconut oil rich in saturated oils (about 90%) with
(P < 0.01) in the duodenum (17.62) compared with the jeju- 60% of its total FA composition being MCFAs (Kappally
num (13.26) and ileum (11.71). In the HI100 dietary treatment, et al., 2015). Furthermore, lauric acid content in coconut oil
the Vh/Cd ratio was also higher (P < 0.01) in the duodenum accounts for ~36.95 g/100 g FA (Wang et al., 2015). A study
(16.89) than the ileum (11.98). In contrast, no significant dif- on male broiler finisher chickens (days 22 to 42) found that
ferences (P > 0.05) regarding Cd were observed within any dietary replacement (25%, 50%, 75% and 100%) of soybean
intestinal segment in any of the groups. oil with coconut oil did not affect growth performance (Wang
Histopathological alterations developed for spleen, et al., 2015). The results of the present study agree with the
thymus, bursa of fabricius and liver and were observed in all findings of Schiavone et al. (2017a), which reported that the

2036
 Black soldier fly larva fat in broiler diet

partial or total replacement of soybean oil with HI larva fat in treatments of the present experiment in which important dif-
growing broiler diets did not affect the growth performance, ferences in the dietary contents of C12:0 and C14:0 FA were
the feed choice or the carcass traits. In the same context, observed with increasing HI fat inclusion levels. Such finding
Cullere et al. (2016) showed that defatted HI larva meal could be due to the already optimal health status of the
could be introduced into the diet for growing broiler quails at animals which was expressed by satisfactory productive per-
10% and 15% inclusion levels, partially replacing conven- formance and normal intestinal morphology. It is hypothesized
tional soybean meal and soybean oil, with no negative that a sub-optimal farming condition and/or health status are
effects on productive performance, mortality or carcass traits. necessary to test the real potential of dietary MCFA in exerting
Regarding the use of HI larva fat in fish feed, Li et al. (2016) a possible positive effect on chicken gut health and growth
did not report any significant effects on growth performance performance. This confirmed the previous findings of Biasato
in juvenile Jian carp (Cyprinus carpio var. Jian) fed with diets et al. (2016), who observed no differences related to insect
where soybean oil was substituted with 25%, 50%, 75% or meal utilization in free-range chickens. According to previous
100% HI larva fat. studies (de Verdal et al., 2010; Biasato et al., 2016), the
duodenum shows greater morphometric indexes among the
Haematological and serum parameters intestinal segments. This finding represents physiological gut
The physiological and biochemical status of animals can be development because the duodenum is the segment with the
expressed through haematology and blood chemistry. All the fastest cell renewal and is also the first one to be stimulated by
blood parameters obtained in the present trial suggested the presence of diet in the lumen (de Verdal et al., 2010).
that HI larva fat did not affect the health status of the ani- Furthermore, the severity of the histopathological altera-
mals. The H/L ratio has recently been used for measurement tions in the broilers was not affected regardless of diet.
of distress conditions in chickens in which an increased H/L However, the lymphoid system activation represented an
ratio may indicate that the animals suffered from infections, interesting finding. Some stressful situations, in particular
inflammation or stress (Salamano et al., 2010; De Marco overcrowding, can frequently occur in modern poultry rear-
et al., 2013; Pozzo et al., 2013). In the present study, the H/L ing operations. Measures of immunity that have been
ratio of chickens fed the experimental diets did not present reported to be affected under stress conditions in poultry are
any significant differences compared with the control group. lymphoid organ weight (Heckert et al., 2002), the H/L ratio
Their leukocytes fell within the normal range, thus suggest- (De Marco et al., 2013) and lymphocyte blastogenesis
ing that the feeding treatments did not affect the immune (Cunnick et al., 1994). Despite no histopathological studies
system of the broiler chickens. It should be highlighted that being currently available in relation to stress evaluation in
the present study was performed in a standard environment broilers, the lymphoid system activation detected in the
without infections, stress or other factors that could have present study may reflect this aspect. A potential role of HI in
influenced the haematological parameters. The activities of the development of these alterations seems unlikely as they
serum AST and ALT are generally related to liver damage, were also found in the animals fed with the control diet, their
acting as indicators of liver necrosis when they increase severity was unaffected, and the percentage of their occur-
(Hyder et al., 2013). The identification of no influence on ALT rence was heterogeneous and quite similar among the diet-
or AST activity suggests that HI larva fat may not cause ary treatments. The greater percentage of normal and
negative effects on the hepatopancreas or liver health. No moderately affected organs in all the groups may suggest the
significant differences (P > 0.05) were found in the mean presence of tolerable stress levels. This hypothesis could also
values of creatinine, thus implying that HI larva fat has be supported by the identification of the overall positive
similar effects on the kidney functionality of the birds. The health and welfare status of the broilers.
results of the present research agree with those of Schiavone In conclusion, the important differences in the dietary
et al. (2017a) and Li et al. (2016), who showed that the use contents of lauric and myristic FAs with increasing HI fat
of HI larva fat in substitution for soybean oil in broilers and inclusion had no beneficial effect on chicken gut health.
juvenile Jian carp diets, respectively, had no negative effects Despite this, the findings of this study suggest that HI
on the blood traits of the animals and confirmed the nutri- larva fat could totally replace soybean oil in finisher chickens’
tional adequacy of these diets. diet without any adverse effects on growth performance,
haematological parameters, serum biochemical indices,
Histomorphological investigations intestinal morphology or histological features. These findings
An initial hypothesis was that lauric and myristic MCFA would suggest that HI larva fat can be a suitable ingredient for
exert a certain positive effect on chicken growth performance poultry diets. Further research efforts are necessary to deeply
through two main mechanisms: an antimicrobial effect on gut investigate the impact of HI larva fat on the meat quality
pathogens that would improve gut health together with redu- traits and FA profile of broiler chickens.
cing the competition for nutrients, and modifications to the gut
morphology, mainly increasing villi length and the villi : crypt
ratio in the duodenum and jejunum, which could improve Acknowledgements
nutrient absorption (Zeitz et al., 2015). However, the intestinal The research was supported by a University of Turin (ex 60%)
morphology of broiler chickens was not affected by the dietary grant (Es. fin. 2014 and 2015), by University of Padova funds

2037
Schiavone, Dabbou, De Marco, Cullere, Biasato, Biasibetti, Capucchio, Bergagna, Dezzutto, Meneguz, Gai, Dalle Zotte and Gasco

(ex 60%, 2016 – DOR1603318) and by a University of Padova Kappally S, Shirwaikar A and Shirwaikar A 2015. Coconut oil: a review of
Senior Researcher Scholarship (Prot. n. 1098, 2015). The potential applications. Hygeia Journal of Drugs and Medicine 7, 34–41.
authors are grateful to Dr Paolo Montersino, Mr Dario Sola and Laudadio V, Passantino L, Perillo A, Lopresti G, Passantino A, Khan RU and
Tufarelli V 2012. Productive performance and histological features of intestinal
Mr Mario Colombano for bird care and technical support. mucosa of broiler chickens fed different dietary protein levels. Poultry Science
91, 265–270.
Declaration of interest Leiber F, Gelencsér T, Stamer A, Amsler Z, Wohlfahrt J, Früh B and Maurer V
2017. Insect and legume-based protein sources to replace soybean cake in an
The authors declare that they have no competing interests. organic broiler diet: Effects on growth performance and physical meat quality.
Renewable Agriculture and Food Systems 32, 21–27.
Ethics Statement Li L, Ji H, Zhang B, Tian J, Zhou J and Yu H 2016. Influence of black soldier fly
(Hermetia illucens) larvae oil on growth performance, body composition, tissue
The experimental protocol was approved by the Ethical Com- fatty acid composition and lipid deposition in juvenile Jian carp (Cyprinus carpio
mittee of the Department of Veterinary Sciences of the Uni- var. Jian). Aquaculture 465, 43–52.
versity of Turin (Italy) (protocol number 1/2016). Mattioli S, Dal Bosco A, Szendrő ZS, Cullere M, Gerencsér ZS, Matics ZS,
Castellini C and Dalle Zotte A 2016. The effect of dietary Digestarom® herbal
supplementation on rabbit meat fatty acid profile, lipid oxidation and anti-
Software and data repository resources oxidant content. Meat Science 121, 238–242.
The datasets analyzed in the current study are available from Ozdogan M, Topal E, Paksuz EP and Kirkan S 2014. Effect of different levels of
crude glycerol on the morphology and some pathogenic bacteria of the small
the corresponding author on reasonable request. intestine in male broilers. Animal 8, 36–42.
Pozzo L, Salamano G, Mellia E, Gennero MS, Doglione L, Cavallarin L, Tarantola
M, Forneris G and Schiavone A 2013. Feeding a diet contaminated with
References ochratoxin A for broiler chickens at the maximum level recommended by the EU
Association of Official Analytical Chemists 2005. Official methods of analysis, for poultry feeds (0.1 mg/kg). 1. Effects on growth and slaughter performance,
18th edition. AOAC, Washington, DC, USA. haematological and serum traits. Journal of Animal Physiology and Animal
Aviagen 2014. Ross 708 broiler. Broiler performance objectives. Retrieved on 10 Nutrition 97, 13–22.
May 2017 from http://en.avi-agen.com/ross-708/. Qaisrani SN, Moquet PC, van Krimpen MM, Kwakkel RP, Verstegen MW and
Barroso FG, de Haro C, Sánchez-Muros MJ, Venegas E, Martínez-Sánchez A and Hendriks WH 2014. Protein source and dietary structure influence growth per-
Pérez-Bañón C 2014. The potential of various insect species for use as food formance, gut morphology, and hind gut fermentation characteristics in broilers.
for fish. Aquaculture 422, 193–201. Poultry Science 93, 3053–3064.
Belforti M, Gai F, Lussiana C, Renna M, Marfatto V, Rotolo L, De Marco M, Ramos-Elorduy J 1997. Insects: a sustainable source of food? Ecology of Food
Dabbou S, Schiavone A, Zoccarato I and Gasco L 2015. Tenebrio molitor meal in and Nutrition 36, 247–276.
rainbow trout (Oncorhynchus mykiss) diets: effects on animal performance, Renna M, Schiavone A, Gai F, Dabbou S, Lussiana C, Malfatto V, Prearo M,
nutrient digestibility and chemical composition of fillets. Italian Journal of Capucchio MT, Biasato I, Biasibetti E, De Marco M, Brugiapaglia A, Zoccarato I
Animal Science 14, 670–676. and Gasco L 2017. Evaluation of the suitability of a partially defatted black
Biasato I, De Marco M, Rotolo L, Renna M, Dabbou S, Capucchio MT, Biasibetti E, soldier fly (Hermetia illucens L.) larvae meal as ingredient for rainbow
Tarantola M, Costa P, Gai F, Pozzo L, Dezzutto D, Bergagna S, Gasco L and trout (Oncorhynchus mykiss Walbaum) diets. Journal of Animal Science and
Schiavone A 2016. Effects of dietary Tenebrio molitor meal inclusion in free-range Biotechnology 8, 57.
chickens. Journal of Animal Physiology and Animal Nutrition 100, 1104–1112. Rumpold BA, Klocke M and Schlüter O 2016. Insect biodiversity: underutilized
Cullere M, Tasoniero G, Giaccone V, Miotti-Scapin R, Claeys E, De Smet S and bioresource for sustainable applications in life sciences. Regional Environmental
Dalle Zotte A 2016. Black soldier fly as dietary protein source for broiler Change 17, 1445-1454.
quails: apparent digestibility, excreata microbial load, feed choice, performance, Salamano G, Mellia E, Tarantola M, Gennero MS, Doglione L and Schiavone A
carcass and meat traits. Animal 10, 1923–1930. 2010. Acute phase proteins and heterophil:lymphocyte ratio in laying hens in
Cunnick JE, Kojic LD and Hughes RA 1994. Stress induced changes in immune different housing systems. Veterinary Record 167, 749–751.
function are associated with increased production of an interleukin-1-like factor Schiavone A, Chiarini R, Marzoni M, Castillo A, Tassone S and Romboli I 2007.
in young domestic fowl. Brain, Behavior and Immunity 8, 123–136. Breast meat traits of Muscovy ducks fed on a microalga (Crypthecodinium
De Marco M, Martinez S, Tarantola M, Bergagna S, Mellia E, Gennero MS and cohnii) meal supplemented diet. British Poultry Science 48, 573–579.
Schiavone A 2013. Effect of genotype and transport on tonic immobility and Schiavone A, Cullere M, De Marco M, Meneguz M, Biasato I, Bergagna S,
heterophil/lymphocyte ratio in two local Italian breeds and Isa Brown hens kept Dezzutto D, Gai F, Dabbou S, Gasco L and Dalle Zotte A 2017a. Partial or
under free-range conditions. Italian Journal of Animal Science 12, 481–485. total replacement of soybean oil by black soldier larvae (Hermetia illucens L.)
de Verdal H, Mignon-Grasteau S, Jeulin C, Le Bihan-Duval E, Leconte M, Mallet fat in broiler diets: effect on growth performances, feed-choice, blood traits,
S, Martin C and Narcy A 2010. Digestive tract measurements and histological carcass characteristics and meat quality. Italian Journal of Animal Science 16,
adaptation in broiler lines divergently selected for digestive efficiency. Poultry 93–100.
Science 89, 1955–1961. Schiavone A, De Marco M, Martínez S, Dabbou S, Renna M, Madrid J, Hernandez
Fascina VB, Carrijo AS, Souza KMR, Garcia AML, Kiefer C and Sartori JR 2009. F, Rotolo L, Costa P, Gai F and Gasco L 2017b. Nutritional value of a partially
Soybean oil and beef tallow in starter broiler diets. Brazilian Journal of Poultry defatted and a highly defatted black soldier fly larvae (Hermetia illucens L.) meal
Science 11, 249–256. for broiler chickens: apparent nutrient digestibility, apparent metabolizable
Gasco L, Henry M, Piccolo G, Marono S, Gai F, Renna M, Lussiana C, Antono- energy and apparent ileal amino acid digestibility. Journal of Animal Science and
poulou F, Mola P and Chatzifotis S 2016. Tenebrio molitor meal in diets for Biotechnology 8, 51.
European sea bass (Dicentrarchus labrax, L.) juveniles: growth performance, Surendra KC, Olivier R, Tomberlin JK, Jha R and Khanal SK 2016. Bioconversion
whole body composition and in vivo apparent digestibility. Animal Feed Science of organic wastes into biodiesel and animal feed via insect farming. Renewable
and Technology 220, 34–45. Energy 98, 197-202.
Heckert RA, Estevez I, Russek-Cohen E and Pettit-Riley R 2002. Effects of Tran G, Heuzé V and Makkar HPS 2015. Insects in fish diets. Animal Frontiers 5,
density and perch availability on the immune status of broilers. Poultry Science 37–44.
81, 451–457. Ushakova NA, Brodskii ES, Kovalenko AA, Bastrakov AI, Kozlova AA
Hyder MA, Hasan M and Mohieldein AH 2013. Comparative levels of ALT, AST, and Pavlov ADS 2016. Characteristics of lipid fractions of larvae of the
ALP and GGT in liver associated diseases. European Journal of Experimental black soldier fly Hermetia illucens. Doklady Biochemistry and Biophysics 468,
Biology 3, 280–284. 209–212.

2038
 Black soldier fly larva fat in broiler diet

Veldkamp T and Bosch G 2015. Insects: a protein-rich feed ingredient in pig and Wang JX and Peng KM 2008. Developmental morphology of the small intestine
poultry diets. Animal Frontiers 5, 45–50. of African ostrich chicks. Poultry Science 87, 2629–2635.
Wang J, Wang X, Li J, Chen Y, Yang W and Zhang L 2015. Effects of dietary Zeitz J, Fennhoff OJ, Kluge H, Stangl GI and Eder K 2015. Effects of
coconut oil as a medium-chain fatty acid source on performance, carcass com- dietary fats rich in lauric and myristic acid on performance, intestinal
position and serum lipids in male broilers. Asian-Australasian Journal of Animal morphology, gut microbes, and meat quality in broilers. Poultry Science 94,
Sciences 28, 223–230. 2404–2413.

2039