Feeding value of black soldier fly larvae compared to soybean in methionine- and lysine-deficient laying hen diets

Abstract

To increase the sustainability of egg production, alternatives to soybean in poultry nutrition are intensively searched for. Black soldier fly larvae (BSFL) could have a great potential, but the comparative protein value to soybean is not well known. The main objective of this study was to facilitate this comparison by using experimental diets clearly limited in calculated supply of sulphurous amino acids and lysine. Fifty laying hens (Lohmann Brown Classic), aged 40 weeks, were fed one of five diets for 7 weeks (n=10). Two diets were based on soybean cake and oil (SS, SS-) as protein and energy sources, and three diets contained partially defatted BSFL meal and fat from two different origins (AA-, AB-, BB-). Different from SS, all other diets were designed to be deficient in methionine and lysine in relation to requirements by >20%. The realised supply with total sulphurous amino acids and lysine was indeed superior with SS even though this diet was analysed to be more deficient in methionine than the BSFL-based diets. Despite the calculated deficiency in limiting amino acids, laying performance of the hens of all groups was similar and ranged between 93 and 97%. Similarly, egg mass, daily feed intake and feed efficiency were not influenced by the BSFL-based diets. The yolks of group BB- were more intensely coloured compared to the others indicating a difference between BSFL origins. Yolks of SS-, but not of the BSFL-based diets, had lower contents of dry matter and ether extract than those of SS. Including BSFL into the diet did not influence the odour of the eggs tested in scrambled form. The results show that soybean-based feeds for laying hens may be completely replaced by BSFL-based feeds and suggest that the recommendations for amino acid supply of laying hens might need revision.

Full text

ISSN 2352-4588 online, DOI 10.3920/JIFF2021.0178 1
Journal of Insects as Food and Feed, 2022; ##(##): 1-11 Wageningen Academic
Publishers
SPECIAL ISSUE: Insects on the poultry, swine and rabbit menu
1. Introduction
With the increasing demand for animal products, global egg
production has been steadily expanding and amounted to
about 82 million tons in 2019 (FAO, 2021). Therefore, the
demand for feed protein is high, which is currently mainly
covered by soybean. However, the cultivation of this crop
requires substantial land resources and is often associated
with deforestation (Taherzadeh and Caro, 2019). In addition
to ecosystem destabilisation, the cultivation and transport
to consuming countries comes with higher greenhouse gas
emissions from carbon release and expenditure of fossil
fuels (Pendrill et al., 2019). This knowledge triggered an
intensive search for soybean alternatives. Preferable are
those which can be produced with low resource input and
low need for arable land thus associated with low food-feed
competition. Insects as feeds offer a promising solution
in this respect, as they are not well accepted as food by
most people, especially in Europe (Hartmann and Siegrist,
2017), and have certain sustainability benefits, such as
low land demand, possibility of use of residue streams as
feed substrates within circular economy concepts, and
Feeding value of black soldier fly larvae compared to soybean in methionine- and
lysine-deficient laying hen diets
M. Heuel1, M. Kreuzer1, C. Sandrock2, F. Leiber2, A. Mathys3, B. Guggenbühl4,
I.D.M.Gangnat1 and M. Terranova1,5*
1ETH Zurich, Institute of Agricultural Sciences, Animal Nutrition, Eschikon 27, 8315 Lindau, Switzerland; 2Research
Institute of Organic Agriculture (FiBL), Department of Livestock Science, Ackerstrasse 113, 5070 Frick, Switzerland; 3ETH
Zurich, Institute of Food, Nutrition and Health, Laboratory of Sustainable Food Processing, Schmelzbergstrasse 9, 8092
Zurich, Switzerland; 4Agroscope, Microbial Systems of Food, Schwarzenburgstrasse 161, 3003 Bern, Switzerland; 5ETH
Zurich, AgroVet-Strickhof, Eschikon 27, 8315 Lindau, Switzerland; melissa-terranova@ethz.ch
Received: 1 October 2021 / Accepted: 9 March 2022
© 2022 Wageningen Academic Publishers
RESEARCH ARTICLE
Abstract
To increase the sustainability of egg production, alternatives to soybean in poultry nutrition are intensively searched
for. Black soldier fly larvae (BSFL) could have a great potential, but the comparative protein value to soybean is not
well known. The main objective of this study was to facilitate this comparison by using experimental diets clearly
limited in calculated supply of sulphurous amino acids and lysine. Fifty laying hens (Lohmann Brown Classic), aged
40 weeks, were fed one of five diets for 7 weeks (n=10). Two diets were based on soybean cake and oil (SS, SS-)
as protein and energy sources, and three diets contained partially defatted BSFL meal and fat from two different
origins (AA-, AB-, BB-). Different from SS, all other diets were designed to be deficient in methionine and lysine
in relation to requirements by >20%. The realised supply with total sulphurous amino acids and lysine was indeed
superior with SS even though this diet was analysed to be more deficient in methionine than the BSFL-based diets.
Despite the calculated deficiency in limiting amino acids, laying performance of the hens of all groups was similar
and ranged between 93 and 97%. Similarly, egg mass, daily feed intake and feed efficiency were not influenced by
the BSFL-based diets. The yolks of group BB- were more intensely coloured compared to the others indicating a
difference between BSFL origins. Yolks of SS-, but not of the BSFL-based diets, had lower contents of dry matter
and ether extract than those of SS. Including BSFL into the diet did not influence the odour of the eggs tested in
scrambled form. The results show that soybean-based feeds for laying hens may be completely replaced by BSFL-
based feeds and suggest that the recommendations for amino acid supply of laying hens might need revision.
Keywords: Hermetia illucens, soybean, layer diet, larval meal, odour
OPEN ACCESS
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M. Heuel et al.
2 Journal of Insects as Food and Feed ##(##)
beneficial nutrient composition (Smetana et al., 2019;
Van Huis, 2020). In addition to the mealworm (Tenebrio
molitor), various cricket species (Gryllidae), the house
fly (Musca domestica) and other insects, that are also
suitable as feed (Van Huis, 2020), were evaluated. Among
them, from the list of insects recently approved by EU for
feeding to poultry (Regulation (EU) 2021/1372, Annex
IV to EC No 999/2001 (EC, 2021)), the black soldier fly
larvae (Hermetia illucens; BSFL), showed particularly
advantageous characteristics as a substitute for soybean in
hens’ nutrition (Bejaei and Cheng, 2020; Heuel et al., 2021a;
Mwaniki et al., 2020; Patterson et al., 2021). The BSFL are
rich in protein and have a beneficial amino acid composition
for poultry nutrition (Spranghers et al., 2017). The larvae
are a natural source of nutrients for poultry (Bovera et al.,
2016), reproduce fast and efficiently convert almost any
organic material to biomass (Oonincx et al., 2015) which
is characterised by protein of high quality. It has already
been shown that rearing larvae on low-value side streams
or high-impact waste streams can be more sustainable than
using conventional protein sources (Smetana et al., 2019).
Results on the feeding value of BSFL compared to soybean
are contrasting so far, with either higher (Marono et al.,
2017) or lower (Mwaniki et al., 2020) value found for BSFL.
To further clarify the feeding value of BSFL compared to
soybean-based feeds, we had carried out a layer experiment
with diets not supplemented with pure amino acids and
with a calculated small deficiency of methionine (Met) and
total sulphurous amino acids (S-AA; i.e. Met + cysteine
(Cys)) as the first limiting amino acids (Heuel et al., 2021a).
However, this did not trigger any differences to soybean
and between the BSFL sources in the hens’ performance,
which left the question about the comparative protein value
open. Other potential side-effects may also be important
for the implementation of BSFL-feeding to hens in farm
practice. The rearing substrates, depending on the type and
processing, and the BSFL as such may have a strong and
variable odour (Diener et al., 2011; Rana et al., 2015), but the
effects of BSFL on the sensory impression of the eggs from
laying hens are widely unknown. Although taste, texture,
and appearance of boiled eggs were improved, no change
in odour was found by Al-Qazzaz et al. (2016) when adding
either 1 or 5% of BSFL to the diet, a level probably too low
to cause off-odour. Bejaei and Cheng (2020) reported no
changes in sensory texture, taste and odour of boiled eggs
when replacing half or all soybean meal by full-fat dried
BSFL (10 and 18% in the diet), but at the same time various
other feed ingredients were changed in that study.
The present study aimed to evaluate the differences
between soybean protein and BSFL protein by provoking a
pronounced deficiency of limiting amino acids that provides
lysine (Lys) at a borderline level compared to the calculated
requirements of high-performance layer hybrids in their
first third of the laying period. The following hypotheses
were tested: (1) The feeding value of BSFL is superior to
that of soybean, especially in the situation of a deficiency
of limited amino acids. (2) The level of superiority depends
on the source of BSFL. (3) The use of BSFL meal and fat
causes an unfavourable odour of the eggs.
2. Materials and methods
Birds and housing system
The experiment was conducted at the research cooperation
AgroVet-Strickhof (Lindau, Switzerland) and was approved
by the Cantonal Veterinary Office of Zurich, Switzerland
(licence number ZH221/17). For this purpose, 50 Lohmann
Brown Classic layers (Burgmer Geflügelzucht, Weinfelden,
Switzerland) at 40 weeks of age were individually housed
in enriched enclosures (each 80 × 80 × 80 cm). These
contained a meshed floor, a nest, a perch and a scratching
box filled with sawdust. Feed and water were provided ad
libitum with one trough and two nipple drinkers each. The
room climate was kept constantly at 20°C and at about
45% humidity. Throughout the 7-week experiment, the
health status of the hens was monitored daily. No hen
showed signs of illness or died during the experiment.
However, in the beginning of week 3 the water system failed
on one side of the bird house (for half of the hens of each
treatment). Water was provided by an additional trough
instead. No bird was harmed during this period. Still feed
intake declined to some extent which is why no intake and
performance related data from all hens from week 3 were
used for statistical analysis.
Diet composition, experimental design, and sampling
The hens were allocated to one of five experimental diets
according to a randomised design (n=10 hens per diet).
The same hens had been used in the previous experiment
(Heuel et al., 2021a). It was ensured that no hen received
a diet similar to that type already fed in the preceding
experiment. The five diets differed in their main protein
and energy sources (Table 1). There were two control diets
(SS, SS-) based on soybean cake and soybean oil and three
experimental diets (AA-, AB-, BB-) based on different
combinations of partially defatted protein meal and pure fat
from two different BSFL origins (A and B). The difference
between the positive control (SS) and the negative control
(SS-) was intended to be the supply with Met and Lys,
being deficient in SS- but not in SS. Accordingly, diet SS
should meet the breeder’s specifications for requirements
of hybrids of the used type and age, specified as 0.44% Met
and 0.88% Lys/kg diet assuming an average daily feed intake
(ADFI) of 110 g (Lohmann Tierzucht, 2020). Diet SS was
composed of the same ingredients and proportions as the
diet SS used before in Heuel et al. (2021a,b). However, new
batches except for the BSFL materials and soy components
were used. All BSFL-containing diets (AA-, AB-, BB-) were
designed to be similarly deficient in Met and Lys as diet
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Black soldier fly larvae for laying hens
Journal of Insects as Food and Feed ##(##) 3
SS-. Diet AB- served to evaluate whether any effects are
based on the larval protein meal (BB- vs AA- and AB-) or
the larval fat (AA- vs AB- and BB-) or both. Larval meal and
fat A were purchased from a commercial BSFL producer
(InnovaFeed, Paris, France) with the larvae being reared
on >80% wheat bran and solubles from wheat distillation
according the producer. Harvesting of the BSFL took place
before they became prepupae. The BSFL for meal and fat
B were produced in an experimental unit (FiBL, Frick,
Switzerland) and grown on 40% fruit and vegetable raw
waste and 30% each of brewer’s grains and pasta production
waste. Harvesting of batch B took place when ≥50% of
the larvae reached the prepupal stage. A more detailed
description of the production and processing of the two
BSFL batches can be found in Heuel et al. (2021a). Due to
a high residual fat content of BSFL meal B (Table 2), no
additional supplementation of fat B was necessary in diet
BB-. Except for the BSFL meals and fats and the soybean oil,
all components used were certified organic and obtained
from local companies. No synthetic amino acids and no
yolk colour pigments were added. Celite (1.6% of dry matter
(DM)) was added as an indigestible marker to be able to
determine metabolisability according to Vucić-Vranješ et
al. (1994).
Diets were produced on the research station in accordance
with the current Swiss regulations for feed production (SR
916.307; https://www.fedlex.admin.ch/eli/cc/2011/772/de).
At first the individual dry feedstuffs were mixed in a 100
kg single-shaft feed mixer (Gericke, Zurich, Switzerland).
Afterwards, either the soybean oil or the liquefied BSFL
fat was added and mixed again (total mixing time approx.
25 min). The feed was then stored in bags at +4°C.
During the experiment, ADFI and body weight (BW) were
determined weekly and individually. The eggs per hen
were collected and weighed daily. From feeding week 4 on
six eggs per hen were collected to determine egg quality.
Following the determination of the external quality, yolks,
and albumen of four of these eggs per hen were frozen at
-20°C, lyophilised (Beta 1-16 Christ, Osterode am Harz,
Germany) and subsequently homogenised with a kitchen
mortar (Haldenwanger, Berlin, Germany). In week 7, the
hens’ excreta were collected daily for 5 days, weighed and
frozen (-20°C) immediately after collection, lyophilised and
ground to 0.75 mm (centrifugal mill ZM 1, Retsch, Haan,
Germany). Feed samples were taken once before the start of
the experiment and once in week 2. Samples of the soybean
cake and the two BSFL meals were taken once before the
diets were mixed. All feeds were ground to 0.5 mm (same
Table 1. Dietary composition of the control and the four protein reduced diets (% of dry matter).
Diet1SS SS- AA- AB- BB-
Soybean cake 15.0 15.0 - - -
Soybean oil 3.0 3.0 - - -
Defatted larval meal A - - 15.0 15.0
Defatted larval meal B - - - - 15.0
Larval fat A - - 2.0 - -
Larval fat B - - - 2.0 -
Wheat 30.0 16.2 15.8 15.8 15.8
Maize 18.0 34.2 34.1 34.1 37.1
Wheat boll meal 3.16 3.00 2.49 2.49 4.49
Broken rice 2.00 7.00 10.5 10.5 8.50
Wheat bran 8.45 8.49 7.00 7.00 6.00
Sunflower cake 7.28 - - - -
Calcium carbonate 2.7 2.7 2.7 2.7 2.7
Limestone grit 7.0 7.0 7.0 7.0 7.0
Dicalcium phosphate 1.0 1.0 1.0 1.0 1.0
Sodium bicarbonate 0.33 0.33 0.33 0.33 0.33
Sodium chloride 0.20 0.20 0.20 0.20 0.20
Choline chloride 0.08 0.08 0.08 0.08 0.08
Vitamin and trace element premix20.20 0.20 0.20 0.20 0.20
Celite31.6 1.6 1.6 1.6 1.6
1 SS = control, soybean cake and soybean oil; SS- = negative control, soybean cake and soybean oil; AA- = larval meal A and larval fat A; AB- = larval meal A and
larval fat B; BB- = larval meal B rich in larval fat B.
2 Contained per kg: Ca, 86.5 g; P, 0.2 g; Mg, 25 g; Cu, 5 g; Mn, 30 g; J, 400 mg; Zn, 25 g; Fe, 25 g; Se, 100 mg; vitamin A, 5,000,000 IE; vitamin D3, 1,250,000 IU;
vitamin E, 15 g; vitamin K, 1.5 g; vitamin B1, 1 g; biotin, 250 mg; folic acid, 750 mg; niacin, 20 g; pantothenic acid, 8.2 g.
3 No. 545, acid-washed diatomaceous earth (Schneider Dämmtechnik, Winterthur, Switzerland).
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M. Heuel et al.
4 Journal of Insects as Food and Feed ##(##)
mill as for excreta) before being analysed. For the sensory
evaluation, 45 eggs each from groups AA-, AB- and BB- and
90 eggs from group SS (no SS- as only the effect of insect
feeding was targeted) were collected in weeks 4 and 5 and
graded after being stored for 4 days at 4°C.
Laboratory analyses
All analyses were carried out in duplicate. Feed items,
dried yolks and albumens as well as excreta samples were
analysed for their proximate composition according to
standard procedures (AOAC International, 1997) and
methods described in detail in Heuel et al. (2021a).
Table 2. Analysed nutrient contents of the soybean cake, the larval meals A and B and the complete experimental diets (% in dry
matter (DM) unless stated otherwise).1
Item Soybean
cake
Larval meal2Diet3
A B SS SS- AA- AB- BB-
DM (% in original substance) 92.9 95.2 93.7 90.4 90.2 90.3 90.3 90.0
Organic matter 87.1 86.7 88.4 76.9 75.8 74.4 74.5 75.2
Nitrogen 7.08 9.67 7.99 2.61 2.36 2.59 2.64 2.48
Ether extract 9.03 13.3 29.9 7.65 6.76 5.60 5.53 6.79
Chitin - 7.44 6.95 - - 1.06 1.10 0.93
Phosphorus 0.71 1.21 0.68 0.73 0.65 0.69 0.67 0.63
Calcium 0.20 1.64 0.95 4.02 4.26 5.35 5.18 4.68
Magnesium 0.05 0.06 0.06 0.15 0.14 0.14 0.14 0.13
Chloride na40.13 0.14 0.21 0.21 0.24 0.28 0.27
Sodium na 0.34 0.41 0.19 0.19 0.17 0.22 0.19
Amino acids
Methionine 0.56 0.95 0.79 0.27 0.25 0.30 0.31 0.29
Methionine + cysteine 1.09 1.37 1.11 0.56 0.52 0.52 0.53 0.51
Lysine 2.62 3.21 2.36 0.77 0.71 0.76 0.79 0.63
Amino acids (% of total amino acids)
Alanine 0.43 0.74 0.82 0.47 0.51 0.67 0.68 0.68
Arginine 0.75 0.52 0.50 0.69 0.68 0.56 0.57 0.57
Asparagine/aspartic acid 1.13 1.01 0.97 0.91 0.91 0.87 0.88 0.82
Cysteine 0.12 0.08 0.08 0.19 0.19 0.15 0.14 0.16
Glutamine/glutamic acid 1.83 1.19 1.17 2.14 2.02 1.65 1.63 1.75
Glycine 0.42 0.60 0.92 0.47 0.46 0.54 0.54 0.53
Histidine 0.26 0.33 0.30 0.27 0.28 0.30 0.30 0.28
Isoleucine 0.46 0.49 0.50 0.43 0.43 0.43 0.42 0.42
Leucine 0.76 0.74 0.74 0.78 0.84 0.81 0.81 0.82
Lysine 0.61 0.61 0.56 0.50 0.50 0.51 0.51 0.47
Methionine 0.13 0.18 0.19 0.17 0.18 0.20 0.20 0.21
Phenylalanine 0.52 0.45 0.44 0.50 0.51 0.46 0.47 0.47
Proline 0.51 0.61 0.68 0.66 0.65 0.70 0.69 0.75
Serine 0.50 0.45 0.46 0.48 0.48 0.46 0.45 0.45
Threonine 0.39 0.43 0.44 0.37 0.37 0.40 0.40 0.39
Tryptophan 0.13 0.17 0.17 0.14 0.14 0.15 0.15 0.15
Tyrosine 0.34 0.72 0.69 0.33 0.35 0.54 0.53 0.47
Valine 0.48 0.68 0.70 0.51 0.51 0.62 0.62 0.60
Gross energy (MJ/kg dry matter) 21.0 22.5 25.4 17.2 16.8 16.4 16.5 16.9
1 Nutrient composition of the soybean cake and the larval meals according to Heuel et al. (2021a).
2
Insect meal A was produced on wheat bran and dried wheat distillery solubles; insect meal B was produced on 40% fruit and vegetables raw waste (with seasonal
variations); 30% brewers’ grain; 30% pasta production waste.
3 SS = control, soybean cake and soybean oil; SS- = negative control, soybean cake and soybean oil; AA- = larval meal A and larval fat A; AB- = larval meal A and
larval fat B; BB- = larval meal B rich in larval fat B.
4 Not analysed.
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Black soldier fly larvae for laying hens
Journal of Insects as Food and Feed ##(##) 5
DM and total ash were measured with a thermo gravimetric
device (TGS 701, Leco Corporation, St. Josephs, MI, USA;
AOAC index no. 942.05). Organic matter was calculated as
the difference between the two variables. A C/N-analyser
(TruMac CN, Leco Corporation; AOAC index no. 968.06)
was used to determine the N content. The crude protein
content of the lyophilised egg yolks and albumens was
calculated as 6.25 × N. Ether extract (EE) in feeds and yolks
was determined using a Soxhlet extraction system (B-811,
Büchi, Flawil, Switzerland; AOAC index no. 963.15). The
amino acid contents of the diets were analysed using HPLC
(Alliance 2690; Waters Corporation, Milford, MA, USA)
adjusted for amino acid analysis as outlined in Gangnat et al.
(2020). Gross energy (GE) was measured by incineration in a
bomb calorimeter (Calorimeter System C7000 and Cooling
System C7002, IKA-Werke, Staufen, Germany). Chitin in
the BSFL protein meals and diets was determined according
to Black and Schwartz (1950). Shell breaking strength, shell
thickness, yolk colour, yolk and albumen heights as well as
the ratios of the inner egg composition were assessed and
Haugh units (Haugh, 1937) were calculated as described
by Heuel et al. (2021a).
Sensory evaluation
For the sensory evaluation, scrambled egg samples were
prepared by combining the complete egg content from
either three (either from group AA-, AB- or BB-) or six
eggs (group SS) from different hens, respectively, laid 4 days
earlier. These egg materials were fried separately per diet
group in a household pan for 5 min and cooled down to a
temperature of 38 to 40°C. Scrambled egg batches were
then divided into 4-5 portions (groups AA-, AB- and BB-)
and 8-10 portions (group SS), respectively, and filled into
sealable disposable cups (Pacovis, Stetten, Switzerland).
All samples were kept at 38 to 40°C until sensory testing.
As BSFL-fed poultry was not yet allowed to be consumed
by humans at the time of this evaluation, only the odour
of the scrambled egg samples was assessed. As sensory
test method an R-index analysis was chosen. The R-index
is a probability value for discriminating two samples. An
R-index of 100 indicates perfect discrimination, while a
value of 50 means that the two samples are distinguished
just by chance. Each test series consisted of a reference
sample (from SS eggs) and four coded test samples, one of
which was again the reference sample. For each of the coded
samples the panellist had to decide whether it differed in
odour from the reference sample. In addition, a sureness
judgement had to be given to each of the decisions. The
panel evaluated five test series in total. All egg samples
were evaluated by a trained sensory panel (n=12-13) at
Agroscope Liebefeld, Switzerland. No explicit egg related
panel training was conducted since the test set up did
not ask for any product specific odour attributes. All
samples were coded with three-digit random numbers and
presentation order of the samples followed a William Latin
Square design. Tests were conducted at room temperature
under day light conditions.
Calculations and statistical analyses
The coefficients of metabolisability of N and GE were
determined as outlined by Vucić-Vranješ et al. (1994),
considering the intake and excretion of acid-insoluble ash
as an indicator and as described in detail in Heuel et al.
(2021a). Measured data were combined to one value per
hen, feed item or diet and subjected to analysis of variance
using a linear mixed effect model (procedure MIXED of
SAS version 9.4, SAS Institute, Cary, NC, USA), including
the Tukey-Kramer correction for multiple comparisons.
Diet was considered as fixed effect, the individual hen or
egg data as experimental unit. Results are expressed as least
squares means and standard error of the mean. Effects were
assumed to be significant at P<0.05. The sensory data were
collected and statistically analysed with the software FIZZ
(version 2,51 Biosystèmes, Couternon, France). Critical
R-indices (two-sided, 5% significance level) were taken
from Bi and O’Mahony (2007).
3. Results
Nutrient composition of the main protein sources and the
complete diets
The N content as analysed in BSFL meal B was the lowest,
followed by soybean cake and BSFL meal A (Table 2). The
analysed contents of the limiting amino acids and their
proportion of total amino acids differed among the main
protein sources. The BSFL meals contained more Met than
the soybean cake, with meal A having the highest proportion
of Met. The proportion of S-AA was also highest in BSFL
meal A, whereby BSFL meal B and the soybean cake barely
differed. Concerning the content of Lys, the order from
high to low was BSFL meal A, soybean cake and BSFL meal
B. In the complete diets, the BSFL-based diets had higher
levels of Met than the control diets (SS and SS-) following
the differences among the main protein sources, whereas
the content of S-AA did not differ from diet SS-. Diet AB-
contained most Lys and diet BB- least Lys, with the other
diets showing values in between. The EE content in DM
of the defatted BSFL meal A was 16.6% lower than that of
BSFL meal B and 4.3% higher than that of the soybean cake.
The high EE content of BSFL meal B was also reflected in
the diet BB- which was richer in EE than the other BSFL-
based diets, but equal to SS- and lower than that of SS.
Correspondingly, the gross energy content of diet SS was
also the highest but showed only a difference of 0.8 MJ/kg
DM to diet AA-, which had the lowest gross energy content.
With about 7% of DM, the chitin content of the two BSFL
meals was comparable. BSFL meal A was richer in P, Ca
and Mg compared to meal B and the soybean cake. The
two insect meals contained similar amounts of Na and Cl.
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M. Heuel et al.
6 Journal of Insects as Food and Feed ##(##)
Performance
The ADFI was not affected by diet, but groups significantly
differed in the daily amounts of Met and Lys consumed
(Table 3). Accordingly, the Met intake of the SS and SS-
hens was lower by 41 (P=0.003) and 57 mg/day (P<0.001)
than those fed AA-, respectively, with the other groups
being intermediate. The BB- hens had the lowest (P<0.001)
Lys intake, with a consumption being lower by 100 to 190
mg/day compared to the other groups. In all diets, the
realised intake of Met and total S-AA was clearly below the
requirements assumed for laying hens at this performance
stage. For Met this was also true for the positive control diet
(SS), which had been designed to meet the actual demand,
but the deficiency in S-AA was clearly lower in SS than in
the other diets. There was no deficiency in calculated Lys
supply with diets SS, AA- and AB-. Compared to the other
diets, BB- resulted in the poorest (P<0.001) supply of Lys.
The N utilisation was higher (P=0.044) by 5% in the SS-
hens compared to the AA- hens. The N metabolisability
of group SS- was higher (P=0.016) than that of group BB-,
with the other groups ranging in between. The energy
metabolisability was higher (P<0.05) in groups AA- and
AB- than in group SS. The measured dietary contents of
metabolisable energy did not differ significantly between
groups.
Internal and external egg quality as well as sensory
odour perception
Proportions of albumen, yolk and shell of the total egg,
shell breaking strength, shell thickness as well as albumen
composition and the Haugh units did not significantly
differ between the diet groups (Table 4). The BB- hens had
a lower (P=0.019) yolk height than the SS hens. In addition,
the yolks of the SS, AA- and AB- hens had an EE content
higher (P<0.001) by around 3% compared to the SS- hens.
The diet containing BSFL meal B (BB-) intensified yolk
colouration (red and yellow colour space) especially when
compared to diet SS (P<0.001), whereas the yolks of groups
SS-, AA- and AB- were intermediate in colour intensity.
With one exception (test series 3), the calculated R-index
values of the sensory test of the odour of the scrambled
eggs were not significant. Accordingly, the odour perception
did not allow discriminating between the BSFL-based diets
(AA-, AB- and BB-) and diet SS (Table 5).
Table 3. Effect of the insect feeding over 40 d on performance (n=10 per treatment).1,2
SS SS- AA- AB- BB- SEM P-value3
Daily intake
Total (g as fed) 121 119 121 117 115 4.1 ns
Methionine (mg) 290bc 274c331a328ab 299abc 9.6 ***
Methionine + cysteine (mg) 617a559ab 577ab 562ab 529b18.4 *
Lysine (mg) 846a761a847a831a656b25.2 ***
Supply over requirements4 (%)
Methionine -26.7b-29.5b-16.5a-17.8a-21.9ab 1.96 ***
Methionine + cysteine -10.9a-17.9ab -17.1ab -19.7b-21.1b2.09 *
Lysine 6.3a-2.7a6.0a3.5a-14.9b2.52 ***
Bodyweight (kg) 1.99 1.92 2.01 2.02 2.03 0.081 ns
Laying performance (%) 93.5 93.0 97.3 93.5 95.0 2.15 ns
Egg weight (g) 65.9 63.6 65.4 65.9 63.2 1.12 ns
Egg mass (g/day) 61.5 59.3 63.6 61.6 60.1 1.69 ns
Feed efficiency (g feed/g egg) 1.97 1.99 1.90 1.91 1.91 0.044 ns
Nitrogen utilisation5 (%) 42.4ab 45.6a40.6b42.1ab 42.9ab 1.23 ns
Metabolisability6 (%)
Nitrogen 46.6ab 49.2a47.2ab 42.6ab 41.4b1.67 **
Energy 77.2b78.6ab 80.2a80.1a79.5ab 0.67 *
Metabolisable energy (MJ/kg feed dry matter) 13.3 13.2 13.2 13.2 13.4 0.11 ns
1 Least-square means within a row with no common superscript are differ significantly different (P<0.05).
2 SS = positive control, soybean cake and soybean oil; SS- = negative control, soybean cake and soybean oil; AA- = larval meal A and larval fat A; AB- = larval
meal A and larval fat B; BB- = larval meal B rich in larval fat B; SEM = standard error of the mean.
3 ns = not significant; Significant diet effects are indicated as *P<0.05, **P<0.01, ***P<0.001.
4 The requirements were calculated based on performance and recommendations by the National Research Council (1994) and were related to the actual intake
of the hens.
5 Nitrogen excretion via the egg in relation to nitrogen intake.
6 Calculated as outlined by Vucić-Vranješ et al. (1994) for indicator techniques.
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Black soldier fly larvae for laying hens
Journal of Insects as Food and Feed ##(##) 7
4. Discussion
In laying hens, Met and Lys are considered as the first
and second limiting amino acids (Toride, 2004). A Met
deficiency can be counteracted by extra Cys, as this S-AA
can be converted to Met in metabolism. In the present
experiment, the realised supply with these amino acids
turned out to be somewhat different from that planned. This
happened as a result of variations in nutrient composition,
intake and performance. Especially the low Met supply
with the control diets was unexpected, but the higher
Cys supply made the positive control SS still superior in
supply with S-AA. Also, different from expectation, the
Lys supply appeared to be sufficiently high not only in
SS but also in AA- and AB-. The latter shows superiority
of BSFL meal A over B. However, overall, the limiting
amino acids in the diets were far more deficient than in
the previous experiment (Heuel et al., 2021a). Therefore,
and by considering the severe calculated deficiency in the
order of about 20% of requirements, it was unexpected
that again no clear effects on performance of the deficient
diets compared to SS were found and this at a still very
high laying performance (eggs and egg mass per day). Hens
Table 4. Egg quality of the hens receiving the experimental diets (n=10 per treatment).1,2
SS SS- AA- AB- BB- SEM P-value3
Egg composition (g/kg)
Shell 106 106 111 104 105 2.3 ns
Albumen 634 628 617 630 641 6.7 ns
Yolk 260 266 272 266 253 5.5 ns
Shell breaking strength (N) 48.3 49.0 55.5 49.3 47.6 2.28 ns
Shell thickness (mm) 0.43 0.42 0.43 0.42 0.40 0.008 ns
Albumen composition (g/kg wet weight)
Dry matter 118 117 116 118 11 7 1.8 ns
Total ash 6.96 6.69 6.92 6.79 7.32 0.21 ns
Crude protein 93.3 93.6 91.9 93.8 93.3 1.56 ns
Haugh units 86.0 85.4 79.5 84.4 86.7 2.23 ns
Yolk height (mm) 17.9a17.5ab 17.3ab 17.6ab 16.7b0.28 *
Yolk composition (g/kg wet weight)
Dry matter 507a496b505ab 503ab 499ab 2.4 **
Total ash 22.2 21.6 21.3 20.9 21.2 0.56 ns
Crude protein 163 162 159 158 158 1.4 *
Ether extract 262a253b266a265a261ab 2.0 ***
Yolk colour4
Lightness (L*) 67.4 67.1 65.5 65.4 65.8 0.64 ns
Red-green axis (a*) -7.09c-6.13b-5.66b-5.91b-4.72a0.17 ***
Yellow-blue axis (b*) 39.5c47.0ab 44.6b46.2ab 48.5a0.91 ***
1 Least-square means within a row with no common superscript are differ significantly different (P<0.05).
2 SS = positive control, soybean cake and soybean oil; SS- = negative control, soybean cake and soybean oil; AA- = larval meal A and larval fat A; AB- = larval
meal A and larval fat B; BB- = larval meal B rich in larval fat B; SEM = standard error of the mean.
3 ns = not significant; significant diet effect differences are indicated as *P<0.05, **P<0.01, ***P<0.001.
4 L* ranges from black (0) to white (100), a* from red (+) to green (–) and b* from yellow (+) to blue (–).
Table 5. Calculated R-indices
1
for the sensory evaluation of the
odour of the eggs from three experimental feeds compared to
the eggs from the control feed SS (n=13 panellists for series
1, 2 and 3; n=12 panellists for series 4 and 5).
Test series Session Diet2,3
AA- AB- BB-
1 1 66.3 61.8 65.7
2 1 55.9 48.2 61.8
3 2 65.7 64.2 75.4*
4 3 61.1 47.2 63.9
5 3 62.2 63.9 51.4
1
100 indicate perfect discrimination, 50 indicates that samples are distinguished
by chance.
2
AA- = larval meal A and larval fat A; AB- = larval meal A and larval fat B;
BB- = larval meal B rich in larval fat B.
3 Critical R-index values for a significance level of 5% (two-sided) indicated
by * (Bi and O’Mahony, 2007): n=13: 20.24, n=12: 20.93.
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M. Heuel et al.
8 Journal of Insects as Food and Feed ##(##)
tend to compensate for a lack of nutrients by increasing
feed intake (Van Krimpen et al., 2007). Therefore, lack of
performance impairment when feeding a deficient diet
should be accompanied by a higher ADFI, but this did not
happen either. It seems that no indirect compensation for
protein deficiency with a higher ADFI was taking place as
the energy density of the diet was obviously sufficiently
high. Another form of compensation could consist in an
increased digestive or metabolic use efficiency. Marono et
al. (2017) found an improvement in feed efficiency when
Lohmann Brown Classic hens were fed BSFL-based diets
for 21 weeks. Although this did not occur with statistical
significance in the current study, the BSFL-based groups
still tended to have a numerically more favourable feed
efficiency. However, the defatted BSFL meals seem to even
have had a slightly inferior apparent N metabolisability than
the soybean cake as it had been also recorded previously
(Heuel et al., 2021a). Furthermore, it has been observed that
feeding BSFL can lead to morphological changes in the gut,
which can impair digestibility and absorption of nutrients
(Dabbou et al., 2018). Such an impairment of digestibility
could be due to the chitin contained in the insect-based
diets. Chitin is assumed to adversely affect digestion for
instance by binding proteins and amino acids (Longvah et
al., 2011). Accordingly, Bovera et al. (2018) observed decline
in ileal protein digestibility in layers when substituting
half of soybean meal by BSFL meal and explained this by
the chitin supplied with the insect-based diet. Cutrignelli
et al. (2018) found a reduced enzymatic activity in the
ileum in the small intestine and an altered production of
volatile fatty acids in the caecum when feeding a BSFL-
based diet to layers instead of a soybean-based diet which
depressed nutrient digestibility. On the other hand, the
chitin contained in BSFL may help improve the gut milieu
of laying hens by promoting beneficial bacteria producing
short chain fatty acids (Borelli et al., 2017). Since we did
not assess these traits, it is unclear if chitin might explain
the lower N metabolisability found with BSFL material
B. A final possible explanation for the lack of effects on
performance would be that the recommendations are even
more overestimating requirements of Lohmann Brown
Classic hens than speculated earlier (Heuel et al., 2021a).
Applegate and Angel (2014) have already shown that the
recommendations, e.g. of the National Research Council
(NRC, 1994) are probably outdated and need to be adapted
to the current genotypes of the commercial hybrids. This
has yet to be done. Accordingly, a daily supply of 450 mg
Met and 858 mg Lys was considered by Applegate et al.
(2009) to be sufficient to meet the requirements of modern
hybrids. For the Lohmann Brown Classic hens used in the
present study, the requirements seem to be even lower
as the actual supply especially with Met was much lower
than these thresholds in the present experiment. It could
be, though, that requirements are slightly higher under
commercial conditions with group floor housing and access
to outside areas. The lack of clear effects on performance
made it impossible to demonstrate the presence or absence
of BSFL origin differences in the present study.
Another important aspect of the utility of BSFL as feed for
laying hens is its influence on egg quality. Regarding the
egg weight, certain standards have to be met, which should
ensure that as many eggs as possible can be marketed in
the best paid category. No feeding-related influences were
found in the present study, and the average egg weights
of all groups fit into the category of large eggs (63 to 73
g) according to the EU regulation for egg marketing (EC,
2008). Different from this, Mwaniki et al. (2020) found a
decrease in egg weight by 1 g/hen/day and in egg mass by
up to 2 g/day with increasing amounts of BSFL meal (either
10 or 15%) in the diet compared to a control diet with 18%
soybean meal. Similarly, Marono et al. (2017) reported egg
weight and egg mass being lower by 2 g/hen/day and 3 g/
day, respectively, in hens fed a diet containing 17% BSFL
compared to those fed a maize-soybean meal-based diet.
In the present study, shell thickness and stability were also
not significantly influenced by integrating BSFL into the
diets. Mwaniki et al. (2020) reported a numerically higher
shell breaking strength when feeding a diet with 15% BSFL
meal. The authors explained this by the concomitantly
smaller eggs in this group, which have a more favourable
egg surface to egg volume ratio. Different from that, Secci et
al. (2020) found larger eggs as well as lower shell percentage
and shell thickness when replacing half of the soybean
meal with partially defatted BSFL meal in the diet. Apart
from the same argument used by Mwanki et al. (2020),
the authors presumed that hens can mobilise a limited
amount of calcium for shell formation. Considering these
controversial findings concerning the influence of BSFL
meal on egg quality, further research is needed for the
development of feeding recommendations (Heuel et al.,
2021a; Marono et al., 2017; Secci et al., 2020).
Another important point for assessing whether BSFL
are suitable as feed is their influence on the internal egg
quality and composition. Compositional changes of the egg
content determine technological properties, nutritional
value and sensory perception, which are all decisive
for the purchase decision of industry and consumers.
Technological properties are affected by the nutritional
composition of yolk and albumen and the structure of
these two egg fractions. The latter were considered in the
present study via yolk height (a variable related to yolk
membrane stability) and Haugh units (an indicator of
albumen foaming ability). Indeed, there were some diet
effects in some of these variables. Yolk height was low
with diets SS- and BB-, but no reason for that is apparent.
The fat (EE) content of the yolk was increased with diets
AA- and AB- compared to SS- which was opposite to the
fat content of the corresponding diets. One explanation
for this observation could be that diets AA- and AB- had
the highest energy metabolisability and that extra energy
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Black soldier fly larvae for laying hens
Journal of Insects as Food and Feed ##(##) 9
is typically deposited as fat in the yolk (Grashorn, 2016).
Still, the supply with metabolizable energy was not clearly
different among diets. In the previous study (Heuel et al.,
2021a), a diet based on BSFL meal B was superior in yolk
fat content to that with meal A, which is contrary to the
present results. In the investigation of Secci et al. (2018),
BSFL feeding numerically lowered the total lipid content of
the yolk by 15 g/kg compared to the yolks from a soybean-
based diet. This all points towards influencing factors other
than only the exchange of soybean by BSFL.
Sensory perception includes yolk colour quality and absence
of off-flavours. Consumers often equate a darker and more
intensely coloured yolk with free-range or organic farming,
even though it is mainly the feeding that is of influence in
this respect (Beardsworth and Hernandez, 2004). Other
studies have already shown that, depending on the origin
of the larvae, BSFL-based feeding can have a significant
impact on yolk colouration (Mwaniki et al., 2020; Secci et
al., 2018). In the present study, the BSFL-meal B, but not
BSFL fat B, intensified yolk colouration like also found
by Heuel et al. (2021a). This was likely the results of an
enrichment of various colour active compounds in the
BSFL produced on correspondingly different feeding
substrates. An influence of the proportion of maize, rich
in carotenoids, which was low in diet SS, can be excluded
as only meal B had a colouring effect. The effect of the
type of feeding substrate for the BSFL might be responsible
for these findings as they probably were for the sensory
analyses conducted by Bejaei and Cheng (2020) where
hard-boiled eggs from a control group (soybean-based
diet) were rated as more colour-intensive than those from
the group fed a diet with 15% BSFL-based feed. However,
in their study the intensive colouration coincided with a
20% higher proportion of maize in the soybean-based diet.
In addition, Bejaei and Cheng (2020) could not confirm
the sensory finding with corresponding measured egg
colour differences between groups. It still has to be shown
whether the colour intensification noted in yolks of diet
BB- is large enough to be apparent in diets containing
common levels of carotenoids which had been deliberately
omitted in the present study. Consumers are susceptible
to off-flavours, as shown, for example, where feeding a
diet rich in flaxseeds affected the smell and taste of eggs
(Hayat et al., 2010). It could therefore be assumed that
a distinct feed substrate composition and the generally
unpleasant odour of BSFL might even have more adverse
effects. However, except for one deviating test series, the
results of the sensory evaluation indicated that none of the
tested BSFL feeds changed egg odour in a way that it could
be differentiated from the eggs produced without BSFL.
This excludes a general effect of feeding substrate which
differed clearly between BSFL batches A and B but also a
general adverse effect of BSFL feeding. This is consistent
with the study of Al-Qazzaz et al. (2016) using eggs from
laying hens and Dalle Zotte et al. (2019) testing eggs from
quails. In both studies, 10 to 15% of BSFL material was
added to the diet. Al-Qazzaz et al. (2016) were even able
to show that feeding diets based on BSFL may positively
influence sensory palatability and texture of the eggs, traits
which could not be sensorily investigated in the present
study due to the legal reasons outlined above. One aspect,
not specifically looked at in the present study but in the
previous experiment (Heuel et al., 2021b), is the degree to
which the large amounts of lauric, myristic and palmitic
acid present in the BSFL fat are accumulating in the egg
lipids. These fatty acids are considered unfavourable for
human health (Calder, 2015). When larvae material is used
as full-fat meal or, as in the present study, as a combination
of meal and fat, the intake with these fatty acids is high.
Even when feeding only the meals this might be an issue
as these may contain large amounts of residual fats (in the
present study especially BSFL fat B). However, according
to Heuel et al. (2021b) the transfer from BSFL-based diets
to eggs is favourably low.
5. Conclusions
The results of the present study showed that the complete
replacement of soybean in the diets of laying hens with
BSFL meals and fats is possible. In addition, there are no
significant impairments concerning performance and
egg quality, even when the recommendations for the
limiting amino acids are not met. The lack of performance
differences did not allow to prove or disprove hypothesis
1 of a superiority of BSFL over soybean. Also, no clear
difference in performance was found between origins of
BSFL (disproving hypothesis 2), and differences between
origins in egg quality were small as well. Of particular
importance is the finding that the inclusion of BSFL did not
adversely affect the odour of the processed eggs disproving
hypothesis 3. It would be important to assess other sensory
parameters in further studies.
Acknowledgements
This research was financially supported by the Mercator
Research Program of the ETH Zurich World Food System
Center and the Swiss Federal Office for Agriculture (no.
627000824). The authors would like to thank Carmen
Kunz and her team of ETH Zurich for their support in
the lab. Furthermore, we appreciate the supply of BSFL
feed substrate B through Bio Partner Schweiz AG, Seon;
Brauerei Müller AG, Baden, Pastinella AG, Oberentfelden
and Coop-Bananenreiferei, Kaiseraugst and are grateful
to the BSFL production team at FiBL including Markus
Leubin, Jens Wohlfahrt and Uwe Krug.
Conflict of interest
There are no conflicts of interest to declare by the authors.
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M. Heuel et al.
10 Journal of Insects as Food and Feed ##(##)
References
Al-Qazzaz, M.F.A., Ismail, D., Akit, H. and Idris, L.H., 2016. Effect
of using insect larvae meal as a complete protein source on
quality and productivity characteristics of laying hens. Revista
Brasileira de Zootecnia 45: 518-523. https://doi.org/10.1590/S1806-
92902016000900003
Applegate, T.J. and Angel, R., 2014. Nutrient requirements of poultr y
publication: History and need for an update. Journal of Applied
Poultry Research 23: 567-575. https://doi.org/10.3382/japr.2014-
00980
Applegate, T.J., Onyango, E.M., Angel, R. and Powers, W.J., 2009.
Effect of amino acid formulation and dietary direct-fed microbial
supplementation on egg production and egg characteristics in laying
hens. Journal of Applied Poultry Research 18: 552-561. https://doi.
org/10.3382/japr.2009-00010
Association of Official Analytical Chemists International (AOAC),
1997. Official Methods of Analysis. 16th edition. AOAC
International, Gaithersburg, MD, USA.
Beardsworth, P.M. and Hernandez, J.M., 2004. Yolk colour – an
important egg quality attribute. International Poultry Production
12: 17-18.
Bejaei, M. and Cheng, K.M., 2020. The effect of including full-fat dried
black soldier fly larvae in laying hen diet on egg quality and sensory
characteristics. Journal of Insects as Food and Feed 6: 305-314.
https://doi.org/10.3920/JIFF2019.0045
Bi, J. and O’Mahony, M., 2007. Updated and extended table for testing
the significance of the R-index. Journal of Sensory Studies 22: 713-
720. https://doi.org/10.1111/j.1745-459X.2007.00132.x
Black, M.M. and Schwartz, H.M., 1950. The estimation of chitin and
chitin nitrogen in crawfish waste and derived products. Analyst 75:
185-189. https://doi.org/10.1039/an9507500185
Borrelli, L., Coretti, L., Dipineto, L., Bovera, F., Menna, F., Chiariotti,
L., Nizza, A., Lembo, F., and Fioretti, A., 2017. Insect-based diet, a
promising nutritional source, modulates gut microbiota composition
and SCFAs production in laying hens. Scientific Reports 7: 16269.
https://doi.org/10.1038/s41598-017-16560-6
Bovera, F., Loponte, R., Marono, S., Piccolo, G., Parisi, G., Iaconisi, V.,
Gasco, L., and Nizza, A., 2016. Use of Tenebrio molitor larvae meal
as protein source in broiler diet: Effect on growth performance,
nutrient digestibility, and carcass and meat traits. Journal of Animal
Science 94: 639-647. https://doi.org/10.2527/jas.2015-9201
Bovera, F., Loponte, R., Pero, M. E., Cutrignelli, M. I., Calabrò, S.,
Musco, N., Vassalotti, G., Panettieri, V., Lombardi, P., Piccolo, G.,
Di Meo, C., Siddi, G., Fliegerova, K., and Moniello, G., 2018. Laying
performance, blood profiles, nutrient digestibility and inner organs
traits of hens fed an insect meal from Hermetia illucens larvae.
Research in Veterinary Science 120: 86-93. https://doi.org/10.1016/j.
rvsc.2018.09.006
Calder, P.C., 2015. Functional roles of fatty acids and their effects on
human health. Journal of Parenteral and Enteral Nutrition 39: 18-
32. https://doi.org/10.1177/0148607115595980
Cutrignelli, M.I., Messina, M., Tulli, F., Randazzo, B., Olivotto, I., Gasco,
L., Loponte, R., and Bovera, F., 2018. Evaluation of an insect meal
of the black soldier fly (Hermetia illucens) as soybean substitute:
intestinal morphometry, enzymatic and microbial activity in laying
hens. Research in Veterinary Science 117: 209-215. https://doi.
org/10.1016/j.rvsc.2017.12.020
Dabbou, S., Gai, F., Biasato, I., Capucchio, M.T., Biasibetti, E., Dezzutto,
D., Meneguz, M., Plachà, I., Gasco, L. and Schiavone, A., 2018.
Black soldier fly defatted meal as a dietary protein source for
broiler chickens: effects on growth performance, blood traits, gut
morphology and histological features. Journal of Animal Science
and Biotechnology 9: 49. https://doi.org/10.1186/s40104-018-0266-9
Dalle Zotte, A., Singh, Y., Michiels, J. and Cullere, M., 2019. Black
soldier fly (Hermetia illucens) as dietary source for laying quails: live
performance, and egg physico-chemical quality, sensory profile and
storage stability. Animals 9: 115. https://doi.org/10.3390/ani9030115
Diener, S., Zurbrügg, C., Gutiérrez, F.R., Nguyen, D.H., Morel, A.,
Koottatep, T. and Tockner, K., 2011. Black soldier fly larvae for
organic waste treatment – prospects and constraints. In: Proceedings
of the 2nd International Conference on Solid Waste Management
in the Developing Countries, Khulna, Bangladesh, WasteSafe 2:
13-15. Available at: https://www.eawag.ch/fileadmin/Domain1/
Abteilungen/sandec/publikationen/SWM/BSF/Black_soldier_fly_
larvae_for_organic_waste_treatment.pdf.
European Commission (EC), 2008. Commission Regulation (EC)
No 589/2008 of 23 June 2008 laying down detailed rules for
implementing Council Regulation (EC) No 1234/2007 as regards
marketing standards for eggs. Official Journal of the European
Union L 163: 6-23.
European Commission (EC), 2021. Commission Regulation (EU)
2021/1372 of 17 August 2021 amending Annex IV to Regulation
(EC) No 999/2001 of the European Parliament and of the Council
as regards the prohibition to feed non-ruminant farmed animals,
other than fur animals, with protein derived from animals. Official
Journal of the European Union L 295: 1-17.
Food and Agriculture Organization of the United Nations (FAO), 2021.
Global egg production from 1990 to 2019 (in 1,000 metric tons). In
Statista. Available at: https://www.statista.com/statistics/263972/
egg-production-worldwide-since-1990/
Gangnat, I.D.M., Mueller, S., Messikommer, R.E. and Kreuzer, M., 2020.
Performance, egg quality and resilience to nutritional challenges
of Lohmann Dual hens as opposed to layer and traditional dual-
purpose types: a preliminary study. European Poultry Science 84
https://doi.org/10.1399/eps.2020.301
Grashorn, M., 2016. Feed additives for influencing chicken meat and
egg yolk color. In: Carle, R. and Schweigert, R.M. (eds.) Handbook
on natural pigments in food and beverages: industrial applications
for improving food color. Woodhead Publishing, Duxford, UK, pp.
283-302. https://doi.org/10.1016/B978-0-08-100371-8.00014-2
Hartmann, C. and Siegrist, M., 2017. Insects as food: perception and
acceptance. Findings from current research. Ernährungs Umschau
64: 44-50. https://doi.org/10.4455/eu.2017.010
Haugh, R.R., 1937. The Haugh unit for measuring egg quality. United
States Egg and Poultry Magazine 43: 522-555.
Please cite this article as 'in press' Journal of Insects as Food and Feed
https://www.wageningenacademic.com/doi/pdf/10.3920/JIFF2021.0178 - Friday, April 29, 2022 5:59:53 AM - IP Address:217.146.167.9

Black soldier fly larvae for laying hens
Journal of Insects as Food and Feed ##(##) 11
Hayat, Z., Cherian, G., Pasha, T.N., Khattak, F.M. and Jabbar, M.A.,
2010. Sensory evaluation and consumer acceptance of eggs from
hens fed flax seed and 2 different antioxidants. Poultry Science 89:
2293-2298. https://doi.org/10.3382/ps.2009-00575
Heuel, M., Sandrock, C., Leiber, F., Mathys, A., Gold, M., Zurbrügg,
C., Gangnat, I.D.M., Kreuzer, M. and Terranova, M., 2021a. Black
soldier fly larvae meal and fat can completely replace soybean cake
and oil in diets for laying hens. Poultry Science 100: 101034. https://
doi.org/10.1016/j.psj.2021.101034
Heuel, M., Sandrock, C., Leiber, F., Mathys, A., Gold, M., Zurbrügg,
C. Gangnat, I.D.M., Kreuzer, M. and M. Terranova M., 2021b.
Transfer of lauric and myristic acid from black soldier fly larval
lipids to egg yolk lipids of hens is low. Lipids 56: 423-435. https://
doi.org/10.1002/lipd.12304
Lohmann Tierzucht, 2020. Lohmann Brown-Classic Layers
Management Guide. Available at: https://lohmann-breeders.com/
media/strains/cage/management/LOHMANN-Brown-Classic-
Cage.pdf
Longvah, T., Mangthya, K., and Ramulu, P., 2011. Nutrient composition
and protein quality evaluation of eri silkworm (Samia ricinii)
prepupae and pupae. Food Chemistry 128: 400-403. https://doi.
org/10.1016/j.foodchem.2011.03.041
Marono, S., Loponte, R., Lombardi, P., Vassalotti, G., Pero, M.E., Russo,
F., Gasco, L., Parisi, G., Piccolo, G., Nizza, S., Di Meo, C., Attia, Y.A.
and Bovera, F., 2017. Productive performance and blood profiles of
laying hens fed Hermetia illucens larvae meal as total replacement
of soybean meal from 24 to 45 weeks of age. Poultry Science 96:
1783-1790. https://doi.org/10.3382/ps/pew461
Mwaniki, Z., Shoveller, A.K., Huber, L.A. and Kiarie, E.G. 2020.
Complete replacement of soybean meal with defatted black soldier
fly larvae meal in Shaver White hens feeding program (28-43 wks
of age): impact on egg production, egg quality, organ weight, and
apparent retention of components. Poultry Science 99: 959-965.
https://doi.org/10.1016/j.psj.2019.10.032
National Research Council (NRC), 1994. Nutrient requirements of
poultry: 1994. National Academies Press, Washington, DC, USA.
https://doi.org/10.17226/2114
Oonincx, D.G., Van Broekhoven, S., Van Huis, A. and van Loon, J.J.,
2015. Feed conversion, survival and development, and composition
of four insect species on diets composed of food by-products. PLoS
ONE 10: e0144601. https://doi.org/10.1371/journal.pone.0222043
Patterson, P.H., Acar, N., Ferguson, A.D., Trimble, L.D., Sciubba, H.B.
and Koutsos, E.A ., 2021. The impact of dietar y black soldier fly l arvae
oil and meal on laying hen performance and egg quality. Poultry
Science 100: 101272. https://doi.org/10.1016/j.psj.2021.101272
Pendrill, F., Persson, U.M., Godar, J., Kastner, T., Moran, D., Schmidt,
S. and Wood, R., 2019. Agricultural and forestry trade drives large
share of tropical deforestation emissions. Global Environmental
Change 56: 1-10. https://doi.org/10.1016/j.gloenvcha.2019.03.002
Rana, K.S., Salam, M.A ., Hashem, S. and Islam, M.A., 2015.
Development of black soldier fly larvae production technique as
an alternate fish feed. International Journal of Research in Fisheries
and Aquaculture 5: 41-47.
Secci, G., Bovera, F., Nizza, S., Baronti, N., Gasco, L., Conte, G., Serra,
A., Bonelli, A. and Parisi, G., 2018. Quality of eggs from Lohmann
Brown Classic laying hens fed black soldier fly meal as substitute
for soya bean. Animal 12: 2191-2197. https://doi.org/10.1017/
S1751731117003603
Secci, G., Bovera, F., Parisi, G., and Moniello, G., 2020. Quality of
eggs and albumen technological properties as affected by Hermetia
illucens larvae meal in hens’ diet and hen age. Animals 10: 81. https://
doi.org/10.3390/ani10010081
Smetana, S., Schmitt, E. and Mathys, A . 2019. Sustainable use of
Hermetia illucens insect biomass for feed and food: attributional
and consequential life cycle assessment. Resources, Conser vation
and Recycling 144: 285-296. https://doi.org/10.1016/j.
resconrec.2019.01.042
Spranghers, T., Ottoboni, M., Klootwijk, C., Ovyn, A ., Deboosere,
S., De Meulenaer, B., Michiels, J., Eeckjout, M., De Clercq, P. and
De Smet, S., 2017. Nutritional composition of black soldier fly
(Hermetia illucens) prepupae reared on different organic waste
substrates. Journal of the Science of Food and Agriculture 97: 2594-
2600. https://doi.org/10.1002/jsfa.8081
Taherzadeh, O. and Caro, D., 2019. Drivers of water and land use
embodied in international soybean trade. Journal of Cleaner
Production 223: 83-93. https://doi.org/10.1016/j.jclepro.2019.03.068
Toride, Y., 2004. Lysine and other amino acids for feed: production
and contribution to protein utilization in animal feeding. In: Protein
sources for the animal feed industry. FAO, Rome, Italy, pp. 161-166.
Available at: https://www.fao.org/3/y5019e/y5019e0a.htm#bm10
Van Huis, A., 2020. Insects as food and feed, a new emerging
agricultural sector: a review. Journal of Insects as Food and Feed
6: 27-44. https://doi.org/10.3920/JIFF2019.0017
Van Krimpen, M.M., Kwakkel, R.P., André, G., Van der Peet-Schwering,
C.M.C., Den Hartog, L.A. and Verstegen, M.W.A., 2007. Effect of
nutrient dilution on feed intake, eating time and performance of
hens in early lay. British Poultry Science 48: 389-398. https://doi.
org/10.1080/00071660701509619
Vucić-Vranješ, M., Pfirter, H.P. and Wenk, C., 1994. Influence of
processing treatment and type of cereal on the effect of dietary
enzymes in broiler diets. Animal Feed Science and Technology 46:
261-270. https://doi.org/10.1016/0377-8401(94)90144-9
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