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Welfare implications for broiler
chickens reared in an insect
larvae-enriched environment:
Focus on bird behaviour,
plumage status, leg health, and
excreta corticosterone
Ilaria Biasato
1
*, Sara Bellezza Oddon
1
, Giulia Chemello
2
,
Marta Gariglio
3
, Edoardo Fiorilla
3
, Sihem Dabbou
4
, Miha Pipan
5
,
Dominik Dekleva
5
, Elisabetta Macchi
3
, Laura Gasco
1
and
Achille Schiavone
3
1
Department of Agricultural, Forest and Food Sciences, University of Turin, Turin, Italy,
2
Department of
Life and Environmental Sciences, Marche Polytechnic University, Ancona, Italy,
3
Department of
Veterinary Sciences, University of Turin, Turin, Italy,
4
Center Agriculture Food Environment, University
of Trento, Trento, Italy,
5
Entomics Biosystems, Cambridge, United Kingdom
The use of insect live larvae as environmental enrichment has recently been
proposed in broiler chickens, but the concomitant administration of black
soldier fly (BSF) and yellow mealworm (YM) has never been tested yet.
Therefore, the present study aims to evaluate the effects of live BSF and YM
larvae as environmental enrichments for broiler chickens by means of plumage
status, behaviour, leg health, and excreta corticosterone metabolites (CM). A
total of 180 4-day old male Ross 308 broiler chickens were randomly
distributed in 3 experimental treatments (6 replicates/treatment, 10 birds/
replicate) and fed for 35 days as follows: 1) control (C, commercial feed), 2)
BSF: C + 5% of the expected daily feed intake [DFI] live BSF larvae and 3) YM: C +
5% of the expected DFI live YM larvae. Feathering, hock burn (HB) and footpad
dermatitis (FPD) scores (end of the trial), as well as behavioural observations
(beginning of the trial [T0] and every 11 days [T1, T2 and T3] during morning,
larvae intake and afternoon) through video recordings, were assessed, and
excreta samples collected to evaluate the CM. Feathering, HB and FPD scores,
and excreta CM were unaffected by insect live larvae administration (p>0.05).
In the morning, the insect-fed birds displayed higher stretching, wing flapping,
ground pecking (at T1 and T3), as well as lower preening (at T1 and T2), than the
C group (p<0.05). During the larvae intake, higher scratching, wing flapping and
ground pecking, as well as lower stretching, preening and laying down, were
observed in the insect-fed (scratching, stretching and laying down) or YM-fed
(wing flapping, ground pecking and preening) groups than the C birds (p<0.05).
In the afternoon, insect live larvae administration increased wing flapping (YM)
and laying down (BSF and YM), as well as decreased ground pecking (YM, p<
0.05). In conclusion, the administration of insect live larvae as environmental
enrichment (especially YM) was capable of positively influencing the bird
OPEN ACCESS
EDITED BY
Vincent M. Cassone,
University of Kentucky, United States
REVIEWED BY
Andrew S. Cooke,
University of Lincoln, United Kingdom
Sabine G. Gebhardt-Henrich,
University of Bern, Switzerland
Xavier Averós,
Neiker Tecnalia, Spain
*CORRESPONDENCE
Ilaria Biasato,
ilaria.biasato@unito.it
SPECIALTY SECTION
This article was submitted
to Avian Physiology,
a section of the journal
Frontiers in Physiology
RECEIVED 27 April 2022
ACCEPTED 19 July 2022
PUBLISHED 25 August 2022
CITATION
Biasato I, Bellezza Oddon S,
Chemello G, Gariglio M, Fiorilla E,
Dabbou S, Pipan M, Dekleva D, Macchi E,
Gasco L and Schiavone A (2022),
Welfare implications for broiler chickens
reared in an insect larvae-enriched
environment: Focus on bird behaviour,
plumage status, leg health, and
excreta corticosterone.
Front. Physiol. 13:930158.
doi: 10.3389/fphys.2022.930158
COPYRIGHT
© 2022 Biasato, Bellezza Oddon,
Chemello, Gariglio, Fiorilla, Dabbou,
Pipan, Dekleva, Macchi, Gasco and
Schiavone. This is an open-access
article distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.
Frontiers in Physiology frontiersin.org01
TYPE Original Research
PUBLISHED 25 August 2022
DOI 10.3389/fphys.2022.930158
welfare through the stimulation of foraging behaviour, increase in activity levels,
and reduction in bird frustration, without affecting the plumage status, leg
health, and excreta CM.
KEYWORDS
black soldier fly, broiler chickens, environmental enrichment, welfare, yellow
mealworm
Introduction
Insects are nowadays recognized as excellent biofactories for
their peculiar ability to valorise a wide spectrum of waste
materials by nutrition upcycling, which allows obtaining
edible high-quality micro- and macro-nutrients that can be
incorporated in the animal feed chain (Gasco et al., 2020).
The so-obtained insect larvae are, indeed, predominantly
fractionated to obtain meals and oils, which can efficiently be
utilized to replace the conventional protein and lipid sources in
monogastric diets (Ravi et al., 2020). However, the scientific
research recently carried on revealed that insect live larvae may
also potentially reach an interesting market share in the form of
environmental enrichments for either poultry (Pichova et al.,
2016;Veldkamp and van Niekerk, 2019;Ipema et al., 2020;Star
et al., 2020;Bellezza Oddon et al., 2021;Tahamtani et al., 2021)or
pigs (Ipema et al., 2021a;Ipema et al., 2021b).
Environmental enrichment can be defined as a modification
of the rearing environment of captive animals aimed at
improving their biological functioning and stimulating their
species-specific behaviours (Newberry, 1995). The enrichment
strategies currently available for broiler chickens can be grouped
in 2 main categories: 1) “point-source objects”, which are
enrichment objects/devices that are generally limited in size
and whose use is often restricted to a single or a few locations
in an animal enclosure; and 2) more complex enriched
environments designed to meet the key behavioural needs of
the animals within them (i.e., outdoor access) (Riber et al., 2018).
Among the “point-source objects”, the provision of food items to
stimulate the bird foraging activity represents one of the most
practical and effective enrichment techniques, as search for
various types of food resources on the litter has been reported
to increase foraging and movement in broiler chickens (Pichova
et al., 2016;Ipema et al., 2020). Such increase in overall activity
levels may have implications for the intensive farming, where the
fast growth rates and the high body weights are the main cause of
leg problems and lameness in broilers, thus, in turn, deeply
limiting their ability to move (Reiter and Bessei, 2009).
Furthermore, as fast-growing broilers spend between 60 and
80% of their time sitting (de Jong and Gunnink, 2018), contact
dermatitis (i.e., hock burns, breast burns and foot pad dermatitis)
may also frequently occur, as a consequence of continuing
contact and pressure of the skin of the breast, hocks and feet
against humid and soiled bedding (Ekstrand et al., 1998). The
limited space and the absence of environmental stimuli of the
commercial conditions can also impair broiler welfare by limiting
the possibility to perform intrinsically motivated behaviours and
diminishing activity levels, thus, in turn, furtherly increasing the
occurrence of leg problems (Vasdal et al., 2019), and the
susceptibility to abdominal dermatitis, plumage soiling and feet
and hock dermatitis (Bruce et al., 1990;Opengart et al., 2018).
Black soldier fly (BSF) and yellow mealworm (YM) live larvae
provision has recently been proposed as promising food
environmental enrichment to promote welfare in broiler
chickens, with increased activity and foraging behaviour (as a
result of the search for larvae on the ground), and reduced
occurrence of hock burns and lameness (as a result of the
increased activity) being observed in the administered birds
(Pichova et al., 2016;Ipema et al., 2020). Welfare assessment
in broiler chickens is usually object of a multiperspective
approach, as heterogeneous parameters (such as plumage
status, hock burns and footpad dermatitis, lameness,
behavioural patterns, and excreta corticosterone) are
commonly evaluated (Weimer et al., 2018;Giersberg et al.,
2021;Iannetti et al., 2021;Lourenço da Silva et al., 2021).
Despite beneficial live insect larvae-related effects on bird
behaviour and feathering scores having recently been
highlighted in either turkeys (Veldkamp and van Niekerk,
2019) or laying hens (Star et al., 2020;Tahamtani et al.,
2021), data about modulation of plumage status and excreta
corticosterone in broiler chickens reared in live insect larvae-
enriched environment are still missing. Furthermore, no studies
assessing the effects of the concomitant administration of BSF
and YM live larvae as environmental enrichments are currently
available in poultry.
Therefore, the present study aims to investigate the effects of
BSF and YM live larvae as environmental enrichments for broiler
chickens, assessing the implications for bird welfare by means of
behaviour, plumage status, leg health, and excreta corticosterone
metabolites (CM).
Materials and methods
Birds and experimental design
The experimental design of the present study is reported in
details by Bellezza Oddon et al. (2021), as the current research is part
of the same project and was performed using the same birds. In order
to provide a brief summary, a total of 180 4-day old male Ross
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Biasato et al. 10.3389/fphys.2022.930158
308 broiler chickens were randomly allotted to 3 experimental
treatments (6 replicate pens/treatment, 10 birds/treatment) as
follows: 1) control (C), where a commercial feed only was
provided (two feeding phases: starter [4–11 days] and grower-
finisher [12–38 days]; ii), BSF, where the C diet was supplemented
with 5% of the expected daily feed intake [DFI] of BSF live larvae
(calculated on dry matter [DM]); and 3) YM, where the C diet was
supplemented with 5% of the expected DFI of YM live larvae
(DM). The starter commercial feed was characterized by
12.5 MJ/kg metabolizable energy (ME) and 224 g/kg crude
protein (CP), while the grower feed contained 13.0 MJ/kg ME
and 220 g/kg CP (Fa.ma.ar.co SPA, Cuneo, Italy). The pens were
1.20 m wide × 2.20 m long (bird density at the end of the growth:
10 kg/m
2
). The daily amount of live larvae was distributed to all the
pens in two plates at the same hour (11.00 a.m.) and 7 days/week
for the wholetrial (35 days). To avoid any potential bias, two plates
with a known amount of control feed inside were also provided to
the C animals to create the same interaction with the operators in
all the treatments, and there was also a visual separation among the
pens (Bellezza Oddon et al., 2021).
Feathering score
At the end of the experimental trial, all the birds were given
feathering scores for back, breast, wing, under-wing and tail
using scores of 1–5 for feather coverage as follows: score 1,
minimal coverage (<25% coverage); score 2, 25%–50% coverage;
score 3, 50%–75% coverage; score 4, >75% coverage; and score 5,
complete coverage (Lai et al., 2010).
Behavioural observations
The behavioural observations were carried out using video
recordings. A total of 3 pens/treatment were filmed for 5 min in
the morning (9.00–9.05 a.m.), 5 min during the larvae intake
(11.00–11.05 a.m.) and 5 min in the afternoon (6.00–6.05 p.m.)
at the beginning of the trial (T0) and every 11 days until the end of
the experiment (T1, T2 and T3). The recorded videos were analysed
by the Behavioural Observation Research Interactive Software
(BORIS, v 7.9.7) (Friard and Gamba, 2016). The considered
behaviours were divided in two categories: the frequency (point
event) and the duration (state event) behaviours (Table 1). The
frequency behaviours were evaluated as the number of times that a
specific behaviour occurred in the pen during the 5 min periods of
observations. The duration behaviours were, instead, assessed as the
percentage of the 5 min periods of observations that 4 identified
subjects in the pen (named as alpha, beta, gamma and delta) spent
performing a specific behaviour.
Feet and hock health assessment
The feet and hocks of the broiler chickens were examined at
the end of the experimental trial in order to assess the incidence
TABLE 1 Description of the broiler ethogram (frequency and duration behaviours) considered in the present study.
Frequency behaviour Definition
Scratching Scraping of the litter with the claws (Ipema et al., 2020)
Preening Grooming of own feathers with beak (Ipema et al., 2020)
Trotting Increasing walking step with head high and breast out (Veldkamp and van Niekerk, 2019)
Pecking pen mate Pecking movements directed at the body or beak of a pen mate (Ipema et al., 2020)
Stretching Stretching one wing together with the leg at the same side or both wings upward and forward (Martin et al., 2005)
Chasing One hen chasing another, with fast running, no vocalisations, no hopping and no wing flapping (Sokołowicz et al., 2020)
Wing flapping Number of wing beats, often while the bird is standing on the toes (Martin et al., 2005)
Shaking Body/wing shake when the plumage is not in order (Martin et al., 2005)
Dust bathing Sitting and performing: vertical wing-shaking, body shaking, litter pecking and/or scratching, bill raking, side and head rubbing
(van Hierden et al., 2002)
Allopreening Social preening (Kenny et al., 2017)
Duration behaviours Definition
Walking Taking one or more step (Webster and Hurnik, 1990)
Preening Grooming of own feathers with beak (Ipema et al., 2020)
Standing still Standing on the feet with extended legs (Webster and Hurnik, 1990)
Ground pecking Pecking at the litter with the head in lower position than the rump (van Hierden et al., 2002)
Lying down Sitting position (Webster and Hurnik, 1990)
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and the severity of the footpad dermatitis (FPD) and the hock
burns (HB). The FPD was scored as follows: 0 = no lesion, slight
discoloration of the skin or healed lesion; 1 = mild lesion,
superficial discoloration of the skin and hyperkeratosis; and
2 = severe lesion, affected epidermis, blood scabs,
haemorrhages and severe swelling of the skin (Ekstrand et al.,
1998). Differently, the HB were scored as follows: 0 = no lesion;
1 = superficial, attached (single) lesion or several single
superficial or deep lesions ≤0.5 cm; 2 = deep lesion >0.5 cm
to ≤1 cm or superficial lesion >0.5 cm; 3 = deep lesion >1.0 cm;
4 = whole hock extensively altered (Louton et al., 2020).
Excreta corticosterone analysis
At the beginning of the trial (T0) and every 11 days (after the
video recordings of the administration of the insect live larvae)
until the end of the experiment (T1, T2, and T3), all the birds
from each pen were housed in wire-mesh cages (100 cm width ×
50 cm length) for 120 min to collect fresh excreta samples. After
collection, the excreta samples were pooled, immediately frozen
at −20°C until corticosterone analysis, and processed according
to Palme et al. (2013) and Costa et al. (2016). In particular, the
excreta were freeze-dried and ground using a cutting mill (MLI
204; Bühler AG, Uzwil, Switzerland). A total of 0.25 g of the
samples were placed into an extraction tube with 3 ml of ether
and stored at −20°C for 1 h. After this time, the aliquots were
mixed for 3 min through multivortex and the supernatant was
recovered and transferred in a new tube. The tubes were then
placed at 50°C for 14 h to obtain a dried extract. Lastly, excreta
CM were analysed with a multi species enzyme immunoassay kit
(Arbor Assay
®
, Ann Arbor, MI, United States) developed for
serum, plasma, saliva, urine, extracted faecal samples, and tissue
culture media. All of the analyses were performed in duplicate.
The inter- and intra-assay coefficients of variation were less than
10% (7% and 9%, respectively). The sensitivity of the assay was
11.2 ng/g of excreta. All of the samples were analysed at multiple
dilutions (1:4, 1:8, 1:16, and 1:32) and all the regression slopes
were parallel to the standard curve (r
2
= 0.979).
Statistical analysis
The statistical analysis was performed using IBM SPSS
Statistics V28.0.0 software (IBM, Armonk, NY, United States).
The pen was considered as the experimental unit for the plumage
status, behaviour, and excreta CM analyses, while the bird was
used for the assessment of the leg health. Shapiro-Wilk’s test
established normality or non-normality of distribution of both
the data and the residuals. The feathering scores were analysed by
fitting a generalized linear mixed model (GLMM) that allowed
them to depend on linear predictors (diet, time, and their
interaction) through a negative binomial response probability
distribution with a nonlinear link function (log). The mean
scores of each body area were included in the statistical
model. A GLMM was also fit to allow the behaviour data to
depend on the same linear predictors through a Poisson loglinear
distribution (frequency behaviours) or a gamma probability
distribution with a nonlinear (log) link function (duration
behaviours). The total number of times that the specific
frequency behaviours occurred in the pen, as well as the mean
percentage of time that the 4 identified subjects of the pen spent
performing the specific duration behaviours, were included in the
corresponding statistical models. Frequency behaviours
occurring less than 0.5 times on average per period of
observation were excluded from the GLMM. The excreta CM
were also analysed by fitting a GLMM that allowed them to
depend on the same linear predictors through a gamma
probability distribution with a nonlinear link function (log).
The mean CM resulting from the duplicate analysis was
included in the statistical model. The replicate was included as
a random effect to account for repeated measurements on the
same pen, and the interactions between the levels of the fixed
factors were evaluated by means of pairwise contrasts. The HB
and FPD scores were analysed by means of Kruskal-Wallis (post-
hoc test: Dunn’s Multiple Comparisons Test). The results were
expressed as least square mean (plumage status, behaviour, and
excreta CM) or mean (leg health) and standard error of the mean
(SEM). pvalues ≤0.05 were considered statistically significant.
Results
Feathering score
The feathering scores of the broiler chickens of the current
research are summarized in Table 2. The administration of both
the BSF and the YM live larvae did not influence the feathering
scores of the birds (p= 0.545). On the contrary, the feathering
scores depended on the body area (p<0.001). In particular, the
back showed better scores when compared to the other body
areas, with breast, under-wing and tail furtherly displaying
greater scores than the wing (p<0.001). No diet × body area
interaction was also identified (p= 0.237).
Behaviour analysis
Frequency behaviours of the broiler chickens of the present
study are summarized in Table 3 and Figures 1–3. In the
morning, stretching and wing flapping were influenced by
both the insect live larvae administration and the time (p<
0.001), but no diet × time interaction was identified (p=
0.686 and p= 0.220, respectively). In details, the insect-fed
broiler chickens performed more stretching and wing flapping
than the C group (p<0.001), and, independently of diet, a
Frontiers in Physiology frontiersin.org04
Biasato et al. 10.3389/fphys.2022.930158
reduction (stretching) and an increase (wing flapping) of such
behaviours was overall observed along the experimental trial (p<
0.001 and p= 0.010, respectively). The wing flapping frequency
also abruptly decreased at T3 when compared to the other
experimental times (p= 0.010). Preening depended on time
only, with an increase being overall identified along the
experimental trial, but an abrupt reduction at T3 (p= 0.001).
On the contrary, no influence of insect live larvae administration
or diet × time interaction were highlighted (p= 0.102 and p=
0.110, respectively). Allopreening, pecking pen mate and shaking
behaviours did not depend on any of the considered variables
(diet: p= 0.549, p= 1.000 and p= 0.001, respectively; time: p=
0.549, p= 0.290 and p= 0.100, respectively; diet × time: p= 0.404,
p= 1.000 and p= 1.000, respectively). During the larvae intake,
scratching and wing flapping behaviours were influenced by
insect live larvae administration only (p= 0.025 and p<
0.001, respectively). In particular, the insect-fed broilers
performed more scratching in comparison with the C birds
(p= 0.025), while increased frequency in wing flapping was
identified in the YM group only (p<0.001). Differently, no
influence of time (p= 0.070 or p= 0.661, respectively) or diet ×
time interaction (p= 0.662 and p= 0.508, respectively) were
identified. Preening and stretching behaviours were influenced
by either the insect live larvae administration or the time (p<
0.001). In particular, the insect-fed birds displayed less preening
and stretching than the C broilers, with the YM group furtherly
showing reduced stretching when compared to the BSF-fed birds
(p<0.001). Furthermore, independently of diet, preening and
stretching frequencies progressively increased in the last 11 days
of the experimental trial (p<0.001). On the contrary, no diet ×
time interaction was highlighted (p= 0.057 and p= 0.104,
respectively). Trotting and shaking behaviours depended on
time only, with trotting frequency progressively decreasing in
the last 11 days of the experimental trial (p<0.001), and shaking
displaying the opposite trend (p<0.001). Differently, no
influence of insect live larvae administration (p= 0.098 or p=
0.687, respectively) or diet × time interaction (p= 1.000 and p=
0.492, respectively) were identified. Allopreening and pecking
pen mate behaviours did not depend on any of the considered
variables (diet: p= 0.624 and p= 0.105, respectively; time: p=
1.000 and p= 0.624, respectively; diet × time: p= 1.000 and p=
1.000, respectively). In the afternoon, a diet × time interaction
was observed for wing flapping only (p<0.001). In details, the YM-
fed broiler chickens performed more wing flapping than the other
groups at T2 and T3 only (p<0.001), while the C birds displayed
higher wing flapping than the HI group at T1 (p<0.05, Figure 3).
On the contrary, preening, stretching and shaking behaviours
depended on time only, with increasing frequencies being
highlighted along the experimental trial (p<0.001). On the
contrary, no influence of insect live larvae administration (p=
0.770, p=0.302orp= 0.378, respectively) or diet × time
interaction (p=0.127,p= 0.106 and p=0.052,respectively)
were highlighted. Allopreening was not influenced by any of the
considered variables (diet: p=1.000;time:p= 0.527; diet × time:
p=0.527).
Duration behaviours of the broiler chickens of the current
research are summarized in Table 4 and Figure 4–6. In the
morning, a diet × time interaction was observed for both the
ground pecking and the preening (p<0.001 and p= 0.006,
respectively). In particular, higher ground pecking was observed
in the insect-fed broilers than the C group at T1 and T3 only (p<
0.001, Figure 4), whereas the C birds spent more time preening in
comparison with the other groups or BSF group alone at T1 and
T2, respectively (p= 0.006, Figure 4). Walking depended on
either the insect live larvae administration or the time (p=
0.001 and p<0.001, respectively). In details, the BSF birds spent
more time walking when compared to the C group (p<0.001),
and, independently of diet, less walking was progressively
observed along the experimental trial (p<0.001). Differently,
no diet × time interaction was identified (p= 0.186). Standing still
and laying down behaviours were influenced by time only (p<
0.001 and p= 0.045, respectively). In particular, broiler chickens
spent less time standing still along the experimental trial (p<
0.001), with an increase in laying down being also observed (p<
0.05). During the larvae intake, ground pecking and laying down
depended on insect live larvae administration only (p<0.001). In
particular, the YM-fed birds displayed higher and lower,
respectively, ground pecking and preening than the other
groups, with either the BSF or the YM broilers spending less
time laying down when compared to the C group (p<0.001). On
the contrary, no influence of time (p= 0.703 and p= 0.190,
respectively) or diet × time interaction (p= 0.118 and p= 0.141,
respectively) were highlighted. Preening was influenced by both
the insect live larvae administration and the time (p<0.001 and
p= 0.001, respectively). In details, the YM-fed birds displayed
lower preening than the other groups (p<0.001), and,
TABLE 2 Feathering score of the broiler chickens depending on diet, body area and their interaction.
Diet (D) Body area (B) SEM p-value Wald test
C BSF YM Back Breast Wing Under-wing Tail D B D B D×B D B D×B
Score, n 1.18 1.16 1.21 3.19
a
1.00
b
0.73
c
1.00
b
0.99
b
0.03 0.05 0.545 <0.001 0.237 1.214 854.780 8.010
C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm live larvae. Means with superscript letters (a, b, c) denote significant differences (p<0.05).
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Biasato et al. 10.3389/fphys.2022.930158
TABLE 3 Frequency behaviours of the broiler chickens depending on diet, time and their interaction.
Diet (D) Time (T) SEM p-value Wald test
C BSF YM T0 T1 T2 T3 D T D T D×T D T D×T
Morning
Scratching, n <0.5 times of occurrence
Preening, n 9.72 8.35 9.96 5.45
a
8.05
b
26.70
c
6.44
a
9.34 0.88 0.102 0.001 0.110 4.980 13.913 4.342
Allopreening, n 0.00 0.00 0.00 0.00 0.00 0.00 0.48 0.00 0.03 0.549 0.549 0.404 1.200 1.200 1.810
Trotting, n <0.5 times of occurrence
Stretching, n 2.07
a
4.08
b
4.74
b
2.92
a
2.89
ab
3.31
ab
4.91
b
0.26 0.71 <0.001 <0.001 0.686 45.794 18.871 0.842
Pecking pen
mate, n
0.00 0.00 0.00 0.00 0.00 0.67 0.00 0.00 0.17 1.000 0.290 1.000 0.000 2.412 0.000
Chasing, n <0.5 times of occurrence
Dust bathing, n <0.5 times of occurrence
Wing
flapping, n
0.00
a
1.67
b
2.77
c
1.88
b
1.44
b
6.38
a
0.00
c
0.21 0.25 <0.001 0.010 0.220 136.671 9.294 3.030
Shaking, n 0.00 0.00 0.00 0.00 0.00 0.87 0.00 0.00 0.03 1.000 0.100 1.000 0.000 4.280 0.000
During larvae intake
Scratching, n 0.33
a
2.28
b
2.52
b
1.20 1.21 1.06 1.49 0.27 0.41 0.025 0.070 0.662 7.416 9.787 0.825
Preening, n 13.05
a
3.59
b
4.74
b
4.00
a
3.85
a
7.32
b
7.89
b
1.16 0.98 <0.001 <0.001 0.057 75.693 206.003 5.716
Allopreening, n 0.00 0.00 0.00 0.40 0.42 0.00 0.00 0.00 0.07 0.624 1.000 1.000 0.240 0.000 0.000
Trotting, n 0.00 0.00 0.00 1.31
a
1.46
a
0.00
b
0.00
b
0.00 0.13 0.098 <0.001 1.000 4.645 39.095 0.000
Stretching, n 4.89
a
2.00
b
1.39
c
1.70
a
1.88
a
2.65
b
2.71
b
0.52 0.29 <0.001 <0.001 0.104 16.280 15.192 4.532
Pecking pen
mate, n
0.00 0.00 0.53 0.00 0.00 0.00 0.00 0.07 0.00 0.105 0.624 1.000 4.950 0.786 0.000
Chasing, n <0.5 times of occurrence
Dust bathing, n <0.5 times of occurrence
Wing
flapping, n
3.15
a
2.63
a
4.73
b
3.45 3.61 3.86 2.81 0.31 0.79 <0.001 0.661 0.508 82.131 0.829 1.356
Shaking, n 0.00 0.00 1.01 0.00
a
0.00
a
0.00
a
2.27
b
0.22 0.03 0.687 <0.001 0.492 0.752 84.592 0.472
Afternoon
Scratching, n <0.5 times of occurrence
Preening, n 7.39 8.12 8.80 4.61
a
6.96
b
8.77
c
15.17
d
1.15 0.90 0.770 <0.001 0.127 0.522 143571.734 4.125
Allopreening, n 0.00 0.00 0.00 0.00 0.53 0.00 0.53 0.00 0.17 1.000 0.527 0.527
Trotting, n <0.5 times of occurrence
Stretching, n 3.73 5.46 4.26 1.59
a
4.61
b
6.31
b
8.33
c
0.66 0.52 0.302 <0.001 0.106 1.891 49.443 5.231
(Continued on following page)
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Biasato et al. 10.3389/fphys.2022.930158
independently of diet, preening duration was reduced in the last
11 days of the experimental trial (p= 0.001). Differently, no
diet×time interaction was identified (p= 0.060). On the contrary,
no influence of insect live larvae administration or diet × time
interaction were observed (p= 0.208 and p= 0.077, respectively).
Standing still did not depend on any of the considered variables
TABLE 3 (Continued) Frequency behaviours of the broiler chickens depending on diet, time and their interaction.
Diet (D) Time (T) SEM p-value Wald test
C BSF YM T0 T1 T2 T3 D T D T D×T D T D×T
Pecking pen
mate, n
<0.5 times of
occurrence
Chasing, n <0.5 times of occurrence
Dust bathing, n <0.5 times of occurrence
Wing
flapping, n
0.00 0.00 1.25 1.30 1.52 0.00 1.19 0.09 0.31 0.309 0.888 0.001 2.346 0.237 14.554
Shaking, n 0.00 0.00 0.00 0.00
a
0.00
a
0.76
b
1.37
c
0.00 0.09 0.378 <0.001 0.052 1.947 20.694 5.975
C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm live larvae. T0 = day 0; T1 = day 11; T2 = day 22; T3 = day 33. Means with superscript letters (a,
b, c, d) denote significant differences (p<0.05).
FIGURE 1
Frequency behaviours of the broiler chickens in the morning (diet*time interaction, p>0.05). (A) Preening. (B) Allopreening. (C) Stretching. (D)
Pecking pen mate. (E) Wing flapping. (F) Shaking. C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm live
larvae. T0 = day 0; T1 = day 11; T2 = day 22; T3 = day 33.
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Biasato et al. 10.3389/fphys.2022.930158
(diet: p= 0.218; time: p= 0.710; diet × time: p= 0.058). In the
afternoon, the insect-fed birds showed higher laying down in
comparison with the C group at T3 only (diet × time interaction,
p<0.001; Figure 6). Ground pecking behaviour depended on
insect live larvae administration, with the YM-fed broiler
chickens spending less time ground pecking than the other
groups (p<0.001). On the contrary, no influence of time or
diet × time interaction were highlighted (p= 0.110 and p= 0.571,
respectively). Finally, walking, standing still and preening
behaviours were influenced by time only (p<0.001), with
broiler chickens spending less time walking and standing still,
as well as more time preening, along the experimental trial (p<
0.001). Differently, no influence of insect live larvae
administration (p= 0.678, p= 0.414 and p= 0.285,
respectively) or diet × time interaction (p= 0.112, p=
0.215 and p= 0.116, respectively) were observed.
Feet and hock health assessment
The administration of BSF and YM live larvae did not influence
either the HB (H = 3.644; C: 0.37 ± 0.09; BSF: 0.73 ± 0.15; YM: 0.77 ±
0.17) or the FPD (H = 2.603; C: 0.60 ± 0.15; BSF: 0.60 ± 0.14; YM:
0.33 ± 0.11) scores (p=0.162andp= 0.272, respectively).
FIGURE 2
Frequency behaviours of the broiler chickens during the larvae intake (diet*time interaction, p>0.05). (A) Scratching. (B) Preening. (C)
Allopreening. (D) Trotting. (E) Stretching. (F) Pecking pen mate. (G) Wing flapping. (H) Shaking. C = control group; BSF = C diet + black soldier fly live
larvae; YM = C diet + yellow mealworm live larvae. T0 = day 0; T1 = day 11; T2 = day 22; T3 = day 33.
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Excreta corticosterone
The excreta CM of the broiler chickens of the present study
are summarized in Table 5 and Figure 7. The administration of
BSF and YM live larvae did not affect the excreta CM of the
broiler chickens of the current research (p= 0.684). Similarly, no
time-related effects or diet × time interactions were identified
(p= 0.288 and p= 0.369, respectively).
Discussion
Feathering score
The administration of neither the BSF nor the YM live larvae
was able to improve the feathering scores of the broiler chickens
of the present study. Previous research highlighted a tendency
towards improvement or a significant improvement in feather
damage of BSF live larvae-fed turkey poults and laying hens,
respectively (Veldkamp and van Niekerk, 2019;Star et al.,
2020). Such improvement has been related to a reduction in
the aggressive pecking directed at the back and tail base, as a
consequence of the re-direction of this behaviour towards the
floor and away from feathers (Veldkamp and van Niekerk,
2019). However, since the aggressive pecking displayed by the
broilers of the current research was not influenced by the
administration of either the BSF or the YM live larvae, it is
reasonable that feather conditions were unaffected as well.
Independently of the utilization of the insect larvae, the back
andthewingofthebirdsshowedthebestandtheworstfeather
coverage, respectively. Little information is currently available
on the feathering scores of the different body parts in broiler
chickens (Lai et al., 2010;Mahmoud et al., 2015;Sevim et al.,
2022), with the totality of the body areas being not always
assessed (Sevim et al., 2022), or the authors reporting a mean
body score only (Mahmoud et al., 2015). Lai et al. (2010)
previously identified similar feathering scores among the
different body regions of broiler chickens, while a clear
FIGURE 3
Frequency behaviours of the broiler chickens in the afternoon (diet*time interaction). (A) Preening. (B) Allopreening. (C) Stretching. (D) Wing
flapping. (E) Shaking. Graph bars (representing least square means) with different superscript letters (a, b) indicate significant differences among the
experimental treatments within the experimental times. C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm
live larvae. T0 = day 0; T1 = day 11; T2 = day 22; T3 = day 33.
Frontiers in Physiology frontiersin.org09
Biasato et al. 10.3389/fphys.2022.930158
separation between the back and the other body areas was
herein outlined. The poor feather coverage of the breast can
reasonably be attributed to the clear predominance of laying
down behaviour in the whole behavioural time budget of the
birds, while wing, under-wing and tail feather damage may be
related to the progressively increase in preening frequency and
duration along the experimental trial. Indeed, wing and
tail—along with breast—represent the plumage areas
receiving preferred attention from the birds during preening
(Duncan and Wood-Gush, 1972). A significant role of the
genetic selection—which aims at growth of meat and not
feathers—cannot be excluded as well.
Behaviour analysis
The variations in the behavioural repertoire of the broiler
chickens of the present study share several similarities between
themorningandthemomentofthelarvaeintake,whilethe
afternoon was characterized by different behavioural patterns.
During the morning and the larvae intake, birds receiving the
insect live larvae spent more time ground pecking (with a
statistical significance being detected at T1 and T3 only, as a
consequence of the higher SEM of T2) and performing
increased scratching behaviour when compared to the non-
supplemented animals. This clear stimulation of a more natural
behaviour such as foraging [characterized by ground pecking
and/or scratching (Ipema et al., 2020)] has already been
observed in turkey poults and broiler chickens administered
with BSF live larvae (Veldkamp and van Niekerk, 2019;Ipema
et al., 2020). Scattering food items on the litter (such insects) or
using different bedding materials (sand, moss-peat, or oat
husks) have previously been reported to stimulate foraging
behaviour in broiler chickens (Arnould et al., 2004;Baxter
and O’Connell, 2016;Pichova et al., 2016). However, similar
environmental enrichments (such as whole wheat, wood
shavings, rice hulls or straw pellets) are not capable of
exerting an analogous effect (Bizeray et al., 2002;Shields
et al., 2005;Toghyani et al., 2010;Jordan et al., 2011;Baxter
and O’Connell, 2016;Pichova et al., 2016), thus suggesting that
birds have a clear preference for certain types of substrates
(Riber et al., 2018). Indeed, the motivational significance behind
each food-based enrichment represents the main driver of the
behavioural changes (Pichova et al., 2016), and the insect
larvae—as alive, moving and part of the natural diet of
birds—seem to be highly interesting for poultry (Bokkers
TABLE 4 Duration behaviours of the broiler chickens depending on diet, time and their interaction.
Diet (D) Time (T) SEM p-value Wald test
C BSF YM T0 T1 T2 T3 D T D T D×T D T D×T
Morning
Ground pecking,
time %
2.59
a
7.12
ab
6.11
b
7.62
a
2.64
c
5.55
b
4.88
b
0.89 0.49 <0.001 <0.001 <0.001 101.932 366.984 235.8011
Walking, time % 4.74
a
5.99
b
3.95
ab
14.43
a
8.00
b
2.76
b
1.66
c
0.45 0.88 0.001 <0.001 0.186 14.706 128.630 3.362
Standing still, time % 23.52 19.91 19.89 41.98
a
27.22
b
8.28
c
20.71
b
2.67 3.21 0.573 <0.001 0.355 1.115 37.646 2.070
Laying down, time % 46.45 51.23 56.03 29.36
a
52.59
ab
73.21
b
60.24
b
3.49 6.56 0.055 0.045 0.107 5.793 6.184 16.710
Preening, time % 7.91
a
4.72
b
7.34
b
2.02
a
5.24
b
12.40
d
6.98
c
0.84 0.88 0.019 0.004 0.006 7.906 11.024 10.203
During larvae intake
Ground pecking,
time %
1.61
ab
1.66
a
2.52
b
2.10 2.06 2.14 2.52 0.85 0.58 <0.001 0.703 0.118 93.006 0.146 5.674
Walking, time % 3.29 4.24 4.78 5.58
a
5.50
a
5.64
a
2.92
b
0.63 0.24 0.208 <0.001 0.077 3.139 38.806 5.132
Standing still, time % 15.32 17.12 20.45 18.58 18.20 17.86 17.15 1.93 1.53 0.218 0.710 0.058 3.050 0.139 6.008
Laying down, time % 75.27
a
33.65
b
44.08
b
43.39 42.78 44.69 51.88 4.55 2.84 <0.001 0.190 0.141 251.827 1.714 3.918
Preening, time % 6.82
a
4.33
a
2.20
b
5.75
a
5.90
a
6.40
a
2.53
b
1.01 0.66 <0.001 0.001 0.060 140.920 12.020 5.640
Afternoon
Ground pecking,
time %
8.12
a
6.13
a
2.87
b
6.26 4.51 6.09 4.34 1.00 0.85 <0.001 0.110 0.571 19.931 4.421 1.120
Walking, time % 5.17 5.25 4.42 23.65
a
6.18
b
2.17
c
1.86
d
0.65 1.03 0.678 <0.001 0.112 0.778 18619.759 4.980
Standing still, time % 16.85 14.79 15.15 45.74
a
16.45
b
7.05
c
11.08
b
1.95 1.64 0.414 <0.001 0.215 1.761 1013.777 3.165
Laying down, time % 36.04
a
52.31
b
64.22
b
17.65
a
60.99
b
75.61
b
73.60
b
5.55 5.09 <0.001 <0.001 <0.001 370.193 44.580 486.225
Preening, time % 2.50 2.96 3.97 1.67
a
2.67
b
2.78
b
7.32
c
0.42 0.47 0.285 <0.001 0.116 2.510 11294.008 5.125
C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm live larvae. T0 = day 0; T1 = day 11; T2 = day 22; T3 = day 33. Means with superscript letters (a,
b, c, d) denote significant differences (p<0.05).
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Biasato et al. 10.3389/fphys.2022.930158
and Koene, 2002;Bruce et al., 2003;Ipema et al., 2020). The
same motivational significance reasonably determined the
increase in the activity levels of the insect-fed broiler
chickens of the current research as well, as demonstrated by
the increased frequency of stretching and wing flapping
behaviours (the latter being mainly detected in the YM-fed
birds), the increased time spent for walking and performing
wing flapping, and the decreased time spent for laying down.
An analogous scenario was also underlined in broilers and
laying hens administered with BSF or YM live larvae as
environmental enrichment (Pichova et al., 2016;Ipema et al.,
2020;Star et al., 2020). It is, however, interesting to notice that
the increase in stretching was observed in the morning only,
while during the larvae intake such behaviour actually
decreased. This may reasonably be related to the parallel
increase in scratching and wing flapping behaviours.
Another peculiar aspect to highlight is the reduced frequency
(independently of time) and duration (mainly with BSF, as a
consequence of the higher SEM of the YM group) preening
displayed by the insect-fed birds of the present study. Preening,
as it keeps plumages well-groomed by distributing lipid-rich oils
from uropygial glands and removing parasites (Delius, 1988),
could take a large time budget (~13%) out of the total
behaviour repertoire of domestic fowl (Dawkins, 1989).
However, overall time spent preening and number of preening
bouts could give useful information about environment
appropriateness for birds (Li et al., 2020). Indeed, absence of
environmental stimuli (i.e., cages) stimulates the birds to spend
more time preening (Delius, 1988) or to perform short-term and
frequent preening (Duncan, 1998), as a sign of boredom and
frustration. Therefore, the administration of insect live larvae may
reduce such negative feelings in broilers. In the afternoon, birds
receiving YM live larvae spent less time ground pecking than the
other groups, whereas either the BSF- or the YM-fed broilers
showed an increased duration of laying down behaviour (with a
statistical significance being detected at T3 only, as a consequence
of the higher SEM of T1 and T2). This may indicate that the need
for foraging was fully rewarded during the morning and the larvae
FIGURE 4
Duration behaviours of the broiler chickens in the morning (diet*time interaction). (A) Preening. (B) Allopreening. (C) Stretching. (D) Wing
flapping. (E) Shaking. Graph bars (representing least square means) with different superscript letters (a, b) indicate significant differences among the
experimental treatments within the experimental times. C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm
live larvae. T0 = day 0; T1 = day 11; T2 = day 22; T3 = day 33.
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Biasato et al. 10.3389/fphys.2022.930158
intake, and that the overall increased activity observed in the
first part of the day predisposed the birds to rest in the
afternoon. However, the wing flapping frequency remained
higher in the YM-fed broiler chickens when compared to the
other groups (with a statistical significance being detected in the
last third of the experimental trial only, as a consequence of the
higher SEM of T1).
Independently of the administration of the insect live
larvae, the broiler chickens of the present study displayed
less active behaviours (i.e., ground pecking, walking and
standing still), as well as more passivity (i.e., laying down),
with increasing age. This is in agreement with previous research
on broilers (Bokkers and Koene, 2003;Castellini et al., 2016;
Ipema et al., 2020;Jacobs et al., 2021), where the rapid increase
in body weights leads to poor mobility and, in turn, inhibits
their ability to express certain behaviours (Bokkers, 2004;
Castellini et al., 2016). The overall increase in preening may
similarly be attributed to frustration related to poor mobility
(Bokkers and Koene, 2003). On the contrary, other active
behaviours such as stretching, shaking and wing flapping
increased with increasing age of birds. It is, however,
important to underline that fast-growing broilers are
motivated to perform the normal behavioural repertoire of
chickens, even after 6 weeks of age and despite being
hampered by the high body weights (Bokkers, 2004).
Furthermore, as behaviours are performed in sitting position
rather than in standing position with increasing age (Bokkers,
2004), it is reasonable to identify an increase in behaviours that
birds can easily perform when laying down.
As a final aspect to consider, the use of YM live larvae yielded
slightly more pronounced effects on bird behaviour (especially in
terms of stimulation of foraging and increase in activity levels)
than the BSF ones. Considering that the broiler chickens of the
current research spent less time consuming the YM live larvae
when compared to BSF (Bellezza Oddon et al., 2021), it is possible
to speculate a bird preference towards the larvae of this insect
FIGURE 5
Duration behaviours of the broiler chickens during the larvae intake (diet*time interaction, p>0.05). (A) Preening. (B) Allopreening. (C)
Stretching. (D) Wing flapping. (E) Shaking. C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm live larvae. T0
= day 0; T1 = day 11; T2 = day 22; T3 = day 33.
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Biasato et al. 10.3389/fphys.2022.930158
species. However, further studies are needed to confirm this
hypothesis.
Feet and hock health assessment
Similarly to what was observed for the feathering scores, the
HB and the FPD scores of the broiler chickens of the current
research were not influenced by the administration of either the
BSF or the YM live larvae. Ipema et al. (2020) highlighted that
FPD occurrence was not affected by insect live larvae provision,
whereas the larvae-administered birds displayed less HB when
compared to the C birds. However, considering that FPD
incidence has been reported to be influenced only in the first
3 weeks of age in turkey poults (Veldkamp and van Niekerk,
2019), it is reasonable that a single evaluation may not be enough
FIGURE 6
Duration behaviours of the broiler chickens in the afternoon (diet*time interaction). (A) Preening. (B) Allopreening. (C) Stretching. (D) Wing
flapping. (E) Shaking. Graph bars (representing least square means) with different superscript letters (a, b) indicate significant differences among the
experimental treatments within the experimental times. C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm
live larvae. T0 = day 0; T1 = day 11; T2 = day 22; T3 = day 33.
TABLE 5 Excreta CM of the broiler chickens depending on diet, time and their interaction.
Diet (D) Time (T) SEM p-value Wald test
C BSF YM T0 T1 T2 T3 D T D T D×T D T D×T
CM, ng/g 2855.8 2955.6 3079.4 3210.3 2978.2 3024.4 2641.4 181.1 209.2 0.684 0.288 0.369 0.382 1.284 1.108
C = control group; BSF = C diet + black soldier fly live larvae; YM = C diet + yellow mealworm live larvae. T0 = day 0; T1 = day 11; T2 = day 22; T3 = day 33.
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Biasato et al. 10.3389/fphys.2022.930158
to observe potential differences in broilers as well. Furthermore,
the identification of very low mean values for both the HB and
the FPD scores of the C birds (less than 1) suggested the presence
of an health status of the legs that was already good
independently of insect live larvae administration, thus, in
turn, making more challenging to improve it.
Excreta corticosterone
The excreta CM of the broiler chickens of the present study
were not affected by the administration of both the BSF and the
YM live larvae as well. The measurement of excreta CM is a well-
recognized, non-invasive method to quantify the stress response
in poultry, which offers a more convenient and less disruptive
alternative to traditional measures that require bird restraint and
blood sampling (Weimer et al., 2018), and does not interrupt the
animal behaviour (Hirschenhauser et al., 2012). However, it is
fundamental to underline that many factors (such as age, sex,
diet, metabolic rate, social status, early life experience, diurnal
and seasonal variations, and differences in the hormone
metabolism of individuals) may influence the excreta CM
(Alm et al., 2014). Therefore, despite the positive, insect-
related modulation in the bird behaviour herein highlighted,
such variability could have probably hidden the potential
differences in the excreta CM.
Conclusion
In conclusion, the administration of BSF and YM live larvae
as environmental enrichment for broiler chickens was capable of
positively influencing the bird welfare through the stimulation of
foraging behaviour, increase in activity levels, and reduction of
behaviours potentially attributable to frustration, without
affecting the plumage status, the leg health, and the excreta
CM. As behavioural outcomes suggested some preference of
the broilers for YM live larvae, further research to confirm
this preference is recommended. Considering that the
administration of insect live larvae in the intensive farming
may potentially lead to different outcomes—as a consequence
of the high rearing densities and competitiveness among
birds—additional research testing such innovative
environmental enrichment in the commercial setup are
strongly recommended.
Data availability statement
The raw data supporting the conclusions of this article will be
made available by the corresponding author upon reasonable
request.
Ethics statement
The animal study was reviewed and approved by the
Bioethical Committee of the University of Turin (Italy).
Author contributions
AS, IB, SBO, and LG designed the study. IB, SBO, MG, EF,
and SD carried out the rearing work. MP, and DD provided the
insect live larvae. IB and SBO gave the feathering scores. GC and
SBO analysed the behavioural video recordings. MG, EF, and SD
collected the excreta samples. SBO, EF, and EM analysed the
excreta corticosterone. IB and SBO performed the statistical
analysis. IB wrote the first draft of the manuscript. All the
authors contributed to the article and approved the submitted
version.
Funding
The research was supported by the European Knowledge and
Innovation Community (KIC), within the EIT Food program
“From waste to farm: insect larvae as tool for welfare
improvement in poultry”(Project ID 19122).
Acknowledgments
The authors are thankful to Entomics Biosystems LDT
(Cambridge, United Kingdom), which provided the live larvae
throughout the experimental trial. The authors are also grateful
to Dario Sola for bird care and technical support.
FIGURE 7
Excreta CM of the broiler chickens (diet*time interaction, p>
0.05). C = control group; BSF = C diet + black soldier fly live larvae;
YM = C diet + yellow mealworm live larvae. T0 = day 0; T1 = day 11;
T2 = day 22; T3 = day 33.
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Biasato et al. 10.3389/fphys.2022.930158
Conflict of interest
Authors MP and DD were employed by the company
Entomics Biosystems.
The remaining authors declare that the research was
conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of
interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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