RESEARCH ARTICLE

Effect of black soldier fly larvae as substitutes for fishmeal in broiler diet

Seyeon Chang1,#https://orcid.org/0000-0002-5238-2982, Minho Song2,#https://orcid.org/0000-0002-4515-5212, Jihwan Lee3https://orcid.org/0000-0001-8161-4853, Hanjin Oh1https://orcid.org/0000-0002-3396-483X, Dongcheol Song1https://orcid.org/0000-0002-5704-603X, Jaewoo An1https://orcid.org/0000-0002-5602-5499, Hyunah Cho1https://orcid.org/0000-0003-3469-6715, Sehyun Park1https://orcid.org/0000-0002-6253-9496, Kyeongho Jeon1https://orcid.org/0000-0003-2321-3319, Byoungkon Lee4https://orcid.org/0000-0001-9749-8455, Jeonghun Nam4https://orcid.org/0009-0004-9255-5691, Jiyeon Chun5,*https://orcid.org/0000-0002-4336-3595, Hyeunbum Kim6,*https://orcid.org/0000-0003-1366-6090, Jinho Cho1,*https://orcid.org/0000-0001-7151-0778
Author Information & Copyright
1Department of Animal Science, Chungbuk National University, Cheongju 28644, Korea
2Division of Animal and Dairy Science, Chungnam National University, Daejeon 34134, Korea
3Department of Poultry Science, University of Georgia (UGA), Athens, GA 30602, United States
4Cherrybro Co., Jincheon 27820, Korea
5Department of Food Bioengineering, Jeju National University, Jeju 63243, Korea
6Department of Animal Resources Science, Dankook University, Cheonan 31116, Korea
*Corresponding author: Jiyeon Chun, Department of Food Bioengineering, Jeju National University, Jeju 63243, Korea. Tel: +82-64-754-3615, E-mail: chunjiyeon@jejunu.ac.kr
*Corresponding author: Hyeunbum Kim, Department of Animal Resources Science, Dankook University, Cheonan 31116, Korea. Tel: +82-41-550-3653, E-mail: hbkim@dankook.ac.kr
*Corresponding author: Jinho Cho, Department of Animal Science, Chungbuk National University, Cheongju 28644, Korea. Tel: +82-43-261-2544, E-mail: jinhcho@chungbuk.ac.kr

# These authors contributed equally to this work.

© Copyright 2023 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Jul 11, 2023; Revised: Aug 21, 2023; Accepted: Aug 28, 2023

Published Online: Nov 30, 2023

Abstract

This study investigated the effect of processed forms (defatted or hydrolyzed) of black soldier fly larvae (Hermetia illucens L., BSFL) as a protein substitute on broilers. Experiment 1 was a feeding experiment, and Experiment 2 was a metabolism experiment. In Experiment 1, a total of 120 day-old Arbor Acres broilers (initial body weight 39.52 ± 0.24 g) were used for 28 days. There were 8 replicate pens, and 5 broilers were assigned to each pen. In Experiment 2, a total of 36 day-old broilers (initial body weight 39.49 ± 0.21 g) were used for the metabolism trial. There were 2 broilers in a metabolism cage and six replicate cages per treatment. The dietary treatments were as follows: a basal diet (CON), a basal diet without fishmeal and substitute with defatted BSFL (T1), a basal diet without fishmeal and a substitute with hydrolyzed BSFL (T2). In Experiment 1, during the entire experimental period, the T2 group significantly increased (p < 0.05) body weight gain and feed intake compared to the CON and T1 groups. The feed conversion ratio showed a lower tendency (p = 0.057) in the T2 group than in the CON and T1 groups. At 2 weeks, the CON and T2 groups were significantly higher (p < 0.05) crude protein (CP) digestibility than the T1 group. At 4 weeks, the total protein level significantly increased (p < 0.05) in the CON and T2 groups compared to the T1 group. In Experiment 2, the CP digestibility significantly increased (p < 0.05) in the T2 group compared to the CON and T1 group at weeks 2 and 4. At week 4 amino acid digestibility, the T2 group significantly increased (p < 0.05) lysine, methionine, tryptophan, and glycine digestibility compared to the T1 group. There was no difference in fecal microbiota among the treatment groups. In conclusion, feeding hydrolyzed BSFL as a fishmeal substitute in broiler diets improved growth performance, CP digestibility, and specific amino acid digestibility. Therefore, it is considered that hydrolyzed BSFL in broiler diets can be sufficiently used as a new protein source.

Keywords: Black soldier fly larvae; Broiler; Fishmeal

INTRODUCTION

The environmental trends of global warming, decreasing water availability, and decreasing arable agricultural land are all increasing the importance of finding new feed sources for monogastric animals [1]. Insect meals contain high quality and quantity of protein and also have a high feed-to-protein conversion rate, which has attracted attention to insect meals as a new and promising alternative dietary protein source for monogastric animals [2]. Insects are also easily reared and can promote the reuse of by-products, thus reducing organic waste and waste disposal costs [3,4].

As a specific example, black soldier fly larvae (Hermetia illucens L., BSFL) contain abundant amounts of fat (7%–39% on a dry matter [DM] basis) and protein (37%–63% on a DM basis) [5]. The BSFL has great advantages as a protein source, especially as it contains various essential amino acids (Methionine 1.8%–2.0%; Valine 2.3%–2.8%; Lysine 2.3%–2.6%; Arginine 1.8%–2.0%) [6,7]. Lauric acid, which constitutes up to 64% of the total saturated fatty acid composition of BSFL, has been shown to reduce the number of harmful bacteria in feces and to have antibacterial action against harmful bacteria [810]. Moreover, chitin—which is part of the BSFL exoskeleton—has been reported to have immunomodulatory effects on the innate and adaptive immune systems in mammals [11]. With this advantage, BSFL is already used today as a protein substitute ingredient in the diets of monogastric animals, including poultry, pigs, and dogs [12]. Previous studies have reported that feeding BSFL as a substitute for soybean meal or fishmeal can improve the broiler feed conversion ratio (FCR) [13,14]. Also, insects may be processed in various ways, such as hydrolysis, defatting, and heat processing, and used in animal diet ingredients [15,16]. When insects are defatted, they can be stored for a longer period by preventing the oxidation of lipids occurring during drying and storage [17,18]. In the case of using the hydrolysis processing method using enzymes, enzymes can decompose proteins to promote the absorption of nutrients and increase the digestibility of livestock. Cho et al. [15] reported that processing insects by hydrolysis can reduce anti-nutritional factors in insects, and feeding hydrolyzed Tenebrio molitor larvae in growing pigs improved the apparent ileal digestibility of DM and crude fat compared to feeding defatted T. molitor larvae. Also, the feeding defatted BSFL with a higher protein content at 5% to 19% in a broiler diet, growth performance, carcass quality, and meat quality might be all improved [12,19]. These previous studies show the possibility that insect meals using various processing methods can replace existing protein sources.

However, the results of existing studies examining the effects of BSFL on immunity and the nutrient digestibility of broilers are still inconsistent, and additional research is needed to elucidate the mechanism of these effects. There is also a relative lack of studies comparing the relative efficacies of different processing forms of BSFL. Therefore, this study was conducted to investigate the effect of the processed form of BSFL (defatted or hydrolyzed) as a protein substitute on growth performance, nutrient digestibility, blood profiles, meat quality, and fecal microbiota in broilers.

MATERIALS AND METHODS

Ethics approval and consent to participate

The protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee of Chungbuk National University, Cheongju, Korea (approval no. CBNUA-2049-22-02).

Preparation black soldier fly larvae and diets

The BSFL was supplied after being processed in the form of defatted hydrolyzed at Jeju National University (Jeju, Korea). Table 1 showed the nutritional components of BSFL in the defatted and hydrolyzed forms. The basal diet contained 3% of fishmeal regardless of the feeding phase, and the BSFL diet replaces all 3% of fishmeal in the basal diet with each BSFL form. All diets were fed over 4 phases: pre-starter (days 0–7; Table 2), starter (days 8–14; Table 3), grower (days 15–21; Table 4), and finisher (days 22–28; Table 5). All diets were formulated to meet or exceed the NRC requirement [20].

Table 1. Nutrient components of black soldier fly larvae (BSFL) in the defatted and hydrolyzed form
Items (%) Content
Defatted BSFL Hydrolyzed BSFL
Moisture 6.58 6.59
CP 58.76 38.53
EE 11.51 42.91
CF 9.15 5.61
Ash 10.07 7.68
Aspartic acid 5.15 3.38
Threonine 2.00 1.06
Serine 2.09 1.02
Glutamic acid 6.33 4.37
Glycine 3.01 1.85
Alanine 4.25 2.64
Valine 2.72 1.82
Isoleucine 1.63 1.11
Leucine 3.04 1.94
Tyrosine 3.76 2.19
Phenylalanine 2.89 1.37
Lysine 2.84 1.75
Histidine 2.74 1.67
Arginine 2.06 1.16
Cysteine 0.37 0.22
Methionine 2.58 1.74
Proline 3.33 1.87

CP, crude protein; EE, ether extract; CF, crude fiber.

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Table 2. Ingredient composition of experimental diets (phase 1/days 0–7)
Items Basal diet Defatted BSFL Hydrolyzed BSFL
Ingredients (%) 100.0 100.0 100.0
 Corn 37.6 39.5 38.7
 Wheat fine 15.3 15.3 15.3
 Rice pollards 2.4 2.4 2.4
 Soybean meal 26.9 25.1 25.9
 Cookie wheat flour 1.9 1.9 1.9
 DDGS 5.0 5.0 5.0
 Animal protein 3.3 3.2 3.2
 Fishmeal 3.0 - -
 Defatted BSFL - 3.0 -
 Hydrolyzed BSFL - - 3.0
 Animal fat 1.7 1.7 1.7
 L-Lysine 0.6 0.6 0.6
 L-Methionine 0.4 0.4 0.4
 L-Threonine 0.2 0.2 0.2
 L-Tryptophan 0.1 0.1 0.1
 Salt 0.2 0.2 0.2
 Limestone 0.5 0.5 0.5
 MDCP 0.2 0.2 0.2
 Liquid-Choline 0.1 0.1 0.1
 Vitamin premix1) 0.3 0.3 0.3
 Mineral premix2) 0.3 0.3 0.3
Chemical composition
 AMEn (kcal/kg) 3,000 3,000 3,000
 CP (%) 23.3 23.3 23.3
 Ether extract (%) 5.3 5.3 5.4
 Crude fiber (%) 3.4 3.4 3.4
 Crude ash (%) 5.8 5.9 5.8
 Calcium (%) 0.9 0.9 0.9
 Phosphorus (%) 0.5 0.5 0.5
 Lysine (%) 1.5 1.5 1.5
 SAA (%) 1.1 1.1 1.1

1) Supplied per kg diet: vitamin A, 9,000 IU; vitamin D3, 3,000 IU; vitamin E, 48 mg; vitamin K, 3 mg; thiamin, 1.8 mg; riboflavin, 6 mg; pyridoxine, 3 mg; vitamin B12, 0.012 mg; niacin, 42 mg; folic acid, 1.2 mg; biotin, 0.24 mg; pantothenic acid, 12 mg.

2) Supplied per kg of diet: manganese, 120 mg; zinc, 100 mg; iron, 80 mg; copper, 20 mg; iodine, 2 mg; selenium, 0.3 mg; cobalt, 0.5 mg.

BSFL, black soldier fly larvae; DDGS, dried distiller’s grains with soluble; MDCP, mono-dicalcium phosphate; AMEn, nitrogen-corrected apparent metabolizable energy; CP, crude protein; SAA, sulfur amino acids.

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Table 3. Ingredient composition of experimental diets (phase 2/days 8–14)
Items Basal diet Defatted BSFL Hydrolyzed BSFL
Ingredients (%) 100.0 100.0 100.0
 Corn 42.2 44.2 43.2
 Wheat fine 15.1 15.1 15.1
 Rice pollards 2.5 2.5 2.5
 Soybean meal 21.0 19.2 20.1
 Cookie wheat flour 2.0 2.0 2.0
 DDGS 7.0 7.0 7.0
 Animal protein 2.5 2.3 2.4
 Fishmeal 3.0 - -
 Defatted BSFL - 3.0 -
 Hydrolyzed BSFL - - 3.0
 Animal fat 1.9 1.9 1.9
 L-Lysine 0.6 0.6 0.6
 L-Methionine 0.3 0.3 0.3
 L-Threonine 0.1 0.1 0.1
 L-Tryptophan 0.1 0.1 0.1
 Salt 0.2 0.2 0.2
 Limestone 0.6 0.6 0.6
 MDCP 0.2 0.2 0.2
 Liquid-Choline 0.1 0.1 0.1
 Vitamin premix1) 0.3 0.3 0.3
 Mineral premix2) 0.3 0.3 0.3
Chemical composition
 AMEn (kcal/kg) 3,020 3,020 3,020
 CP (%) 21.3 21.3 21.3
 Ether extract (%) 5.9 5.9 5.9
 Crude fiber (%) 3.4 3.4 3.4
 Crude ash (%) 5.3 5.3 5.3
 Calcium (%) 0.8 0.8 0.8
 Phosphorus (%) 0.6 0.6 0.6
 Lysine (%) 1.3 1.3 1.3
 SAA (%) 1.0 1.0 1.0

1) Supplied per kg diet: vitamin A, 9,000 IU; vitamin D3, 3,000 IU; vitamin E, 48 mg; vitamin K, 3 mg; thiamin, 1.8 mg; riboflavin, 6 mg; pyridoxine, 3 mg; vitamin B12, 0.012 mg; niacin, 42 mg; folic acid, 1.2 mg; biotin, 0.24 mg; pantothenic acid, 12 mg.

2) Supplied per kg of diet: manganese, 120 mg; zinc, 100 mg; iron, 80 mg; copper, 20 mg; iodine, 2 mg; selenium, 0.3 mg; cobalt, 0.5 mg.

BSFL, black soldier fly larvae; DDGS, dried distiller’s grains with soluble; MDCP, mono-dicalcium phosphate; AMEn, nitrogen-corrected apparent metabolizable energy; CP, crude protein; SAA, sulfur amino acids.

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Table 4. Ingredient composition of experimental diets (phase 3/days 15–21)
Items Basal diet Defatted BSFL Hydrolyzed BSFL
Ingredients (%) 100.0 100.0 100.0
 Corn 46.1 47.4 47.1
 Wheat fine 15.6 15.6 15.6
 Rice pollards 2.5 2.5 2.5
 Soybean meal 17.7 16.5 16.8
 Cookie wheat flour 2.0 2.0 2.0
 DDGS 6.0 6.0 6.0
 Animal protein 2.5 2.4 2.4
 Fishmeal 3.0 - -
 Defatted BSFL - 3.0 -
 Hydrolyzed BSFL - - 3.0
 Animal fat 1.9 1.9 1.9
 L-Lysine 0.6 0.6 0.6
 L-Methionine 0.3 0.3 0.3
 L-Threonine 0.1 0.1 0.1
 L-Tryptophan 0.1 0.1 0.1
 Salt 0.2 0.2 0.2
 Limestone 0.5 0.5 0.5
 MDCP 0.2 0.2 0.2
 Liquid-Choline 0.1 0.1 0.1
 Vitamin premix1) 0.3 0.3 0.3
 Mineral premix2) 0.3 0.3 0.3
Chemical composition
 AMEn (kcal/kg) 3070 3070 3070
 CP (%) 20.2 20.2 20.2
 Ether extract (%) 6.0 5.8 5.9
 Crude fiber (%) 3.2 3.2 3.2
 Crude ash (%) 5.1 5.0 5.1
 Calcium (%) 0.8 0.8 0.8
 Phosphorus (%) 0.5 0.5 0.5
 Lysine (%) 1.2 1.2 1.2
 SAA (%) 1.0 1.0 1.0

1) Supplied per kg diet: vitamin A, 9,000 IU; vitamin D3, 3,000 IU; vitamin E, 48 mg; vitamin K, 3 mg; thiamin, 1.8 mg; riboflavin, 6 mg; pyridoxine, 3 mg; vitamin B12, 0.012 mg; niacin, 42 mg; folic acid, 1.2 mg; biotin, 0.24 mg; pantothenic acid, 12 mg.

2) Supplied per kg of diet: manganese, 120 mg; zinc, 100 mg; iron, 80 mg; copper, 20 mg; iodine, 2 mg; selenium, 0.3 mg; cobalt, 0.5 mg.

BSFL, black soldier fly larvae; DDGS, dried distiller’s grains with soluble; MDCP, mono-dicalcium phosphate; AMEn, nitrogen-corrected apparent metabolizable energy; CP, crude protein; SAA, sulfur amino acids.

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Table 5. Ingredient composition of experimental diets (phase 4/days 22–28)
Items Basal diet Defatted BSFL Hydrolyzed BSFL
Ingredients (%) 100.0 100.0 100.0
 Corn 49.7 51.1 50.7
 Wheat fine 15.2 15.2 15.2
 Rice pollards 2.6 2.6 2.6
 Soybean meal 15.5 14.1 14.6
 Cookie wheat flour 2.0 2.0 2.0
 DDGS 5.0 5.0 5.0
 Animal protein 2.4 2.4 2.3
 Fishmeal 3.0 - -
 Defatted BSFL - 3.0 -
 Hydrolyzed BSFL - - 3.0
 Animal fat 1.9 1.9 1.9
 L-Lysine 0.5 0.5 0.5
 L-Methionine 0.4 0.4 0.4
 L-Threonine 0.1 0.1 0.1
 L-Tryptophan 0.1 0.1 0.1
 Salt 0.2 0.2 0.2
 Limestone 0.5 0.5 0.5
 MDCP 0.2 0.2 0.2
 Liquid-Choline 0.1 0.1 0.1
 Vitamin premix1) 0.3 0.3 0.3
 Mineral premix2) 0.3 0.3 0.3
Chemical composition
 AMEn (kcal/kg) 3,100 3,100 3,100
 CP (%) 19.1 19.1 19.1
 Ether extract (%) 5.8 5.7 5.8
 Crude fiber (%) 3.0 3.0 3.0
 Crude ash (%) 4.8 4.8 4.8
 Calcium (%) 0.7 0.7 0.7
 Phosphorus (%) 0.5 0.5 0.5
 Lysine (%) 1.1 1.1 1.1
 SAA (%) 1.0 1.0 1.0

1) Supplied per kg diet: vitamin A, 9,000 IU; vitamin D3, 3,000 IU; vitamin E, 48 mg; vitamin K, 3 mg; thiamin, 1.8 mg; riboflavin, 6 mg; pyridoxine, 3 mg; vitamin B12, 0.012 mg; niacin, 42 mg; folic acid, 1.2 mg; biotin, 0.24 mg; pantothenic acid, 12 mg.

2) Supplied per kg of diet: manganese, 120 mg; zinc, 100 mg; iron, 80 mg; copper, 20 mg; iodine, 2 mg; selenium, 0.3 mg; cobalt, 0.5 mg.

BSFL, black soldier fly larvae; DDGS, dried distiller’s grains with soluble; MDCP, mono-dicalcium phosphate; AMEn, nitrogen-corrected apparent metabolizable energy; CP, crude protein; SAA, sulfur amino acids.

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Experiment 1
Animals and experimental design

A total of 120 one-day-old Arbor Acres broilers (initial body weight [BW] of 39.52 ± 0.24 g) were obtained from a local hatchery (Cherrybro, Eumseong, Korea) and used in this experiment 1 (feeding trial) for 28 days. All broilers were randomly allocated into three dietary treatments in a randomized complete block design. Each treatment had 8 replicate pens, and 5 broilers were assigned to each pen. The dietary treatments were as follows: a basal diet (CON), a basal diet without fishmeal and substitute with defatted BSFL (T1), a basal diet without fishmeal and substitute with hydrolyzed BSFL (T2). The experiment initiation temperature was 33 ± 1°C, after that, the temperature was gradually lowered to maintain 25 ± 1°C. All broilers were given ad libitum access to diet and water throughout the experiments.

Growth performance and Frequency of diarrhea

All broilers were weighed at the beginning of the experiment, at the 2 weeks, and at the end of the experiment (4 weeks) to calculate the body weight gain (BWG). Feed intake (FI) was calculated by subtracting the remaining amount from the diet supply amount until measuring BW. The FCR was calculated by dividing FI by BWG.

To measure the frequency of diarrhea, the same person recorded the diarrhea score at 8:00 and 17:00 for each treatment group during the entire experimental period. The diarrhea scores were as follows: 0, normal feces; 1, soft feces; 2, mild diarrhea; 3, severe diarrhea. The frequency of diarrhea was calculated by counting pen days in which the average diarrhea score of each pen was ≥ 2.

Nutrient digestibility

At 2 and 4 weeks, 0.2% chromium oxide (Cr2O3) was added as an indigestible indicator in all broiler diets for fecal sampling. While collecting feces, the diet was also collected, and immediately stored in a freezer at −20°C. Before analyzing nutrient digestibility, fecal samples were dried at 70°C for 72 h and then crushed on a 1 mm screen. The DM, crude protein (CP), and gross energy (GE) of diet and feces samples were all analyzed according to the method of AOAC [21]. The DM analysis of samples was performed in an oven at 105°C for 16 h. The CP was analyzed according to the Kjeldahl method. An adiabatic oxygen bomb calorimeter (6400 Automatic Isoperibol calorimeter, Parr, Moline, IL, USA) was used to measure GE in diets and feces. Chromium levels were determined via UV absorption spectrophotometry (UV-1201, Shimadzu, Kyoto, Japan) using Williams et al. [22] method. The following equation was used to calculate the apparent total tract digestibility (ATTD).

Digestibility = 1  [ ( Concentration of nutrient in fecal × Concentration of Cr 2 O 3 in the diet ) / ( Concentration of nutrient in diet × Concentration of Cr 2 O 3 in the fecal ) ] × 100.
Blood profile

Blood samples were collected from the brachial wing vein at 2 and 4 weeks (before slaughter), 8 broilers per treatment. Blood samples were collected into vacuum tubes containing K3EDTA for completed blood count analysis and nonheparinized tubes for serum analysis, respectively. After collection, serum samples were centrifuged at 12,500×g at 4°C for 20 min. Red blood cell (RBC), white blood cell (WBC), and lymphocyte were analyzed using an automatic hematology analyzer (XE2100D, Sysmex, Kobe, Japan). Total protein (TP) level was measured using a colorimetric method, and blood urea nitrogen (BUN) level was analyzed using the urease glutamate dehydrogenase method. The TP and BUN in blood were measured using a fully automated chemistry analyzer (Cobas C702, Hofmann-La Roche, Switzerland).

Meat quality

At 4 weeks, all broilers were slaughtered for cervical dislocation and 8 broiler’s breast meat was collected per treatment. General component analysis including moisture, fat, protein, and ash was analyzed according to the AOAC method [21]. The pH was measured with a pH meter (Thermo Orion 535A, Thermo Scientific, Chicago, IL, USA) after adding 100 mL of distilled water to 10 g of breast meat and then homogenizing at 68,400×g for 30 sec using a homogenizer (Bihon seiki, Ace, Osaka, Japan). Water holding capacity (WHC) was analyzed according to the method of Laakkonoen [23]. To analyze the cooking loss (CL), breast meat with a thickness of 3 cm was shaped into a circle, immersed in a 70°C-water bath, and cooled for 30 min. After that, the weight ratio (%) of the initial sample was measured. Drip loss (DL) was calculated as the weight ratio (%) of the initial sample by measuring the amount of loss caused by shaping 2 cm-thick breast meat into a circular shape, vacuum-packing it in a polypropylene bag, and storing it in a refrigerator at 4°C for 24 h. Shear force was analyzed through a shear force cutting test using a rheometer (Compac-100, Sun Scientific, Tokyo, Japan). Color measurement of breast meat was performed using a Minolta colorimeter (CR-410, Konica Minolta, Osaka, Japan). Meat color characteristics were expressed by the CIE L* (lightness), a* (redness), b* (yellowness) system. Two measurements were taken on the surface and cut area of each meat sample.

Experiment 2
Animals and experimental design

A total of 36 one-day-old mixed-sex Arbor Acres broilers (initial BW of 39.49 ± 0.21 g) were used in this experiment 2 (metabolism trial) for 28 days. All broilers were randomly allocated into three dietary treatments based on the initial BW. Dietary treatments were the same as in Experiment 1. There were 2 broilers in a metabolism cage and six replicate cages per treatment. Each cage was 100 cm in width, 40 cm in depth, and 45 cm in height. The experiment was performed in an environmentally controlled room. During the weeks 1 and 3, the diet was fed ad libitum. During the 2nd and 4th weeks (fecal sampling period), the feed supply amount and the remaining amount were recorded every day. All broilers were given ad libitum access to water throughout the experiments.

Nutrient digestibility

The total collection method was used to analyze the ATTD of DM, CP, GE, and amino acid. The diet containing 0.5% Cr2O3 was fed at the 2 and 4 weeks, and feces were collected for 5 days each. The collected feces were stored at -20°C until analysis, dried at 70°C for 72 h at the time of analysis, and then analyzed by crushing with a 1-mm screen. The DM, CP, and GE of diet and feces were analyzed in the same way as in Experiment 1 according to the method of AOAC [21]. Amino acids were analyzed using the high-performance liquid chromatography (HPLC; Shimadzu model LC-10AT, Shimadzu, Kyoto, Japan) method [24]. Cysteine and methionine were oxidized with performic acid for 16 h at 0°C, after that, using cysteic acid and methionine sulfone, respectively, was for analysis.

Fecal microbiota

To analyze fecal microbiota, fresh feces were collected from each cage for each treatment group at the 2 and 4 weeks. Bacterial colonies were counted by the pour plate method. One gram of each fecal sample was diluted with 9 mL of 1× phosphate-buffered saline (PBS) buffer and vortexed for 1 min. Samples were used for measuring the number of viable cells by serial dilution from 10−1 to 10−8. To measure the number of colonies, MacConkey agar was used for Escherichia coli (E. coli), BG sulfa agar was used for Salmonella, and de Man, Rogosa and Sharpe agar (MRS) agar was used for Lactobacillus. All agars were purchased from KisanBio (Seoul, Korea). The MacConkey and BG sulfa agar plates were cultured at 37°C for 24 h. The MRS agar plates were cultured at 37°C for 48 h. After the incubation periods, the agar plates were immediately removed from the incubator, and the number of each colony was counted. The number of microbial colonies was log-transformed before statistical analysis.

Statistical analysis

All data from Experiments 1 and 2 except for Experiment 1’s frequency of diarrhea was analyzed through the general linear model procedure in SAS (SAS Institute, Cary, NC, USA), using each pen as the experimental unit. The frequency of diarrhea was compared with a chi-square test, using the FREQ procedure of SAS. Differences between treatment means were determined using Tukey’s multiple range test. A probability level of p < 0.05 was indicated to be statistically significant, and a level of 0.05 ≤ p < 0.10 was considered to have such a tendency.

RESULTS

Experiment 1
Growth performance and frequency of diarrhea

There was no difference in initial BW among the treatment groups (Table 6). At 2 and 4 weeks, the T2 group had significantly higher (p < 0.05) BW than the T1 group. At weeks 0 to 2, the BWG and FI significantly increased (p < 0.05) in the T2 group compared to the T1 group. At weeks 2 to 4, the T2 group had significantly higher (p < 0.05) BWG and FI than the CON group. For FCR, the T2 group showed a lower tendency (p = 0.063) than the CON and T1 groups. During the entire experimental period, the T2 group significantly increased (p < 0.05) BWG and FI compared to the CON and T1 groups. The FCR showed a lower tendency (p = 0.057) in the T2 group than in the CON and T1 groups. The frequency of diarrhea was no different among the treatment groups.

Table 6. Effect of replacement dietary of fishmeal with black soldier fly larvae (BSFL) on growth performance in broilers (Experiment 1)
Items CON T1 T2 SE p-value
BW (kg)
 Initial 39.52 39.51 39.52 0.415 0.986
 2 weeks 440.50ab 431.00b 465.00a 7.454 0.012
 4 weeks 1,542.00b 1,541.00b 1,669.00a 26.384 0.003
0–2 weeks
 BWG (g) 400.99ab 391.49b 425.48a 7.456 0.012
 FI (g) 479.40b 474.55b 512.85a 3.828 < 0.001
 FCR 1.20 1.21 1.21 0.030 0.828
2–4 weeks
 BWG (g) 1,101.50b 1,110.00b 1,204.00a 26.094 0.020
 FI (g) 1,803.20b 1,838.20ab 1,861.35a 13.085 0.017
 FCR 1.64 1.66 1.55 0.035 0.063
0–4 weeks
 BWG (g) 1,502.49b 1,501.49b 1,629.48a 26.393 0.003
 FI (g) 2,282.60b 2,312.75b 2,374.20a 12.994 < 0.001
 FCR 1.52 1.54 1.46 0.024 0.057
Frequency of diarrhea1)(%) 35.71 30.36 35.72 - 0.670

1) Frequency of diarrhea = (Number of pens with diarrhea / number of pen days) × 100.

a,b Means with different letters are significantly differ (p < 0.05).

CON, basal diet; T1, basal diet without a fishmeal and substitute with defatted BSFL; T2, basal diet without a fishmeal and substitute with hydrolyzed BSFL; BW, body weight; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio.

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Nutrient digestibility

There was no difference in DM digestibility among the treatment groups at weeks 2 and 4 (Table 7). At week 2, the CON and T2 groups were significantly higher (p < 0.05) CP digestibility than the T1 group. The GE digestibility was significantly higher (p < 0.05) in the T2 group than in the T1 group. At week 4, the CON group had significantly higher (p < 0.05) CP digestibility than the T1 group. For GE digestibility, the T2 group showed a similar tendency (p = 0.068) to the CON group.

Table 7. Effect of replacement dietary of fishmeal with black soldier fly larvae (BSFL) on nutrient digestibility in broilers (Experiment 1)
Items (%) CON T1 T2 SE p-value
2 weeks
 DM 78.11 78.22 78.05 0.300 0.920
 CP 70.92a 69.56b 70.62a 0.278 0.006
 GE 78.55ab 78.17b 79.00a 0.159 0.005
4 weeks
 DM 78.54 78.67 78.55 0.278 0.930
 CP 75.12a 73.61b 74.25ab 0.247 0.001
 GE 78.68 77.94 78.47 0.219 0.068

a,b Means with different letters are significantly differ (p < 0.05).

CON, basal diet; T1, basal diet without a fishmeal and substitute with defatted BSFL; T2, basal diet without a fishmeal and substitute with hydrolyzed BSFL; DM, dry matter; CP, crude protein; GE, gross energy.

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Blood profile

At week 2, there was no difference in RBC, WBC, lymphocyte, TP, and BUN levels among the treatment groups (Table 8). At week 4, the TP level significantly increased (p < 0.05) in the CON and T2 groups compared to the T1 group. There was no difference in RBC, WBC, lymphocyte, and BUN levels among the treatment groups at week 4.

Table 8. Effect of replacement dietary of fishmeal with black soldier fly larvae (BSFL) on blood profile in broilers (Experiment 1)
Items CON T1 T2 SE p-value
2 weeks
 RBC (106/µL) 2.31 2.32 2.27 0.181 0.979
 WBC (103/µL) 22.91 23.09 22.66 1.125 0.963
 Lymphocyte (%) 65.15 65.03 67.35 1.630 0.535
 TP (g/dL) 3.23 2.95 2.68 0.296 0.436
 BUN (mg/dL) 3.75 3.50 3.75 0.278 0.767
4 weeks
 RBC (106/µL) 2.26 2.27 2.34 0.142 0.914
 WBC (103/µL) 23.86 24.05 24.06 1.137 0.990
 Lymphocyte (%) 65.05 65.68 66.03 3.026 0.974
 TP (g/dL) 2.93a 2.65b 3.03a 0.068 0.002
 BUN (mg/dL) 2.50 3.00 2.75 0.374 0.646

a,b Means with different letters are significantly differ (p < 0.05).

CON, basal diet; T1, basal diet without a fishmeal and substitute with defatted BSFL; T2, basal diet without a fishmeal and substitute with hydrolyzed BSFL; RBC, red blood cell; WBC, white blood cell; TP, total protein; BUN, blood urea nitrogen.

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Meat quality

The ash content in breast meat had significantly higher (p < 0.05) in the T2 group than in the CON group (Table 9). The pH was significantly higher (p < 0.05) in the T1 and T2 groups than in the CON group. For WHC, the T1 and T2 groups showed a higher tendency (p = 0.097) than the CON group. There was no difference in moisture, fat, protein, CL, DL, shear force, and meat color among the treatment groups.

Table 9. Effect of replacement dietary of fishmeal with black soldier fly larvae (BSFL) on meat quality in broilers (Experiment 1)
Items CON T1 T2 SE p-value
Approximate composition of meat (%)
 Moisture 75.76 75.98 75.81 0.140 0.528
 Ash 1.03b 1.12ab 1.28a 0.047 0.012
 Fat 3.48 2.83 2.59 0.279 0.121
 Protein 19.74 20.07 20.31 0.355 0.537
Meat quality (%)
 pH 5.85b 5.99a 6.03a 0.022 0.001
 WHC 54.41 55.99 55.34 0.454 0.097
 CL 17.54 17.81 17.36 0.622 0.881
 DL 4.73 3.95 3.91 0.299 0.147
Shear force (g) 2,583.75 2,421.25 2,277.50 124.823 0.273
CIE L* 51.95 53.72 55.24 0.983 0.113
CIE a* 5.49 4.31 4.82 0.528 0.329
CIE b* 17.15 16.25 17.18 0.594 0.481

a,b Means with different letters are significantly differ (p < 0.05).

CON, basal diet; T1, basal diet without a fishmeal and substitute with defatted BSFL; T2, basal diet without a fishmeal and substitute with hydrolyzed BSFL; WHC, water holding capacity; CL, cooking loss; DL, drip loss.

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Experiment 2
Nutrient digestibility

At week 2, the DM digestibility was significantly higher (p < 0.05) in the T2 group than in the CON group (Table 10). The CP digestibility significantly increased (p < 0.05) in the T2 group compared to the CON and T1 group at weeks 2 and 4. There was no difference in GE digestibility among the treatment groups at weeks 2 and 4.

Table 10. Effect of replacement dietary of fishmeal with black soldier fly larvae (BSFL) on nutrient digestibility in broilers (Experiment 2)
Items (%) CON T1 T2 SE p-value
2 weeks
 DM 77.88b 79.10ab 79.75a 0.437 0.039
 CP 74.29b 74.21b 76.15a 0.310 0.003
 GE 77.78 78.92 78.02 0.583 0.382
4 weeks
 DM 76.83 75.75 76.20 0.633 0.506
 CP 72.78b 72.73b 73.87a 0.272 0.026
 GE 79.30 79.08 79.63 0.527 0.763

a,b Means with different letters are significantly differ (p < 0.05).

CON, basal diet; T1, basal diet without a fishmeal and substitute with defatted BSFL; T2, basal diet without a fishmeal and substitute with hydrolyzed BSFL; DM, dry matter; CP, crude protein; GE, gross energy.

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At week 2 amino acid digestibility, the T2 group had significantly higher (p < 0.05) valine and leucine digestibility than the CON and T1 groups (Table 11). The glycine digestibility was significantly higher (p < 0.05) in the T2 group than in the CON group. The threonine, phenylalanine, and glutamic acid digestibility showed a higher tendency (p = 0.058, p = 0.072, and p = 0.061, respectively) in the T2 group than in the CON group. At week 4 amino acid digestibility, the T2 group significantly increased (p < 0.05) lysine, methionine, tryptophan, and glycine digestibility compared to the T1 group (Table 12). The glutamic acid digestibility was significantly higher (p < 0.05) in the T2 group than in the CON group. The phenylalanine digestibility showed a higher tendency (p = 0.079) in the T2 group than in the T1 group.

Table 11. Effect of replacement dietary of fishmeal with black soldier fly larvae (BSFL) on amino acid digestibility in broilers at 2 weeks (Experiment 2)
Items (%) CON T1 T2 SE p-value
Indispensable amino acids
 Threonine 85.54 86.63 86.49 0.273 0.058
 Valine 80.26b 79.85b 81.99a 0.369 0.014
 Isoleucine 84.27 83.82 86.08 0.866 0.227
 Leucine 89.00b 89.07b 90.10a 0.132 0.002
 Phenylalanine 88.42 88.42 89.75 0.374 0.072
 Histidine 83.15 83.49 85.47 0.876 0.209
 Lysine 90.65 90.66 91.04 0.277 0.565
 Arginine 92.36 92.17 93.34 0.453 0.226
 Methionine 93.78 94.12 93.47 0.534 0.705
 Tryptophan 84.85 86.98 87.45 2.147 0.678
Dispensable amino acids
 Aspartic acid 85.49 85.65 86.19 0.822 0.824
 Serine 86.12 86.64 86.49 0.890 0.913
 Glutamic acid 89.98 90.22 90.90 0.222 0.061
 Proline 83.14 83.05 83.79 0.586 0.641
 Glycine 81.25b 82.01ab 84.32a 0.578 0.022
 Alanine 88.85 89.21 89.41 0.406 0.633
 Tyrosine 90.73 91.05 91.65 0.471 0.425
 Cysteine 71.47 75.71 75.44 2.475 0.449

a,b Means with different letters are significantly differ (p < 0.05).

CON, basal diet; T1, basal diet without a fishmeal and substitute with defatted BSFL; T2, basal diet without a fishmeal and substitute with hydrolyzed BSFL.

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Table 12. Effect of replacement dietary of fishmeal with black soldier fly larvae (BSFL) on amino acid digestibility in broilers at 4 weeks (Experiment 2)
Items (%) CON T1 T2 SE p-value
Indispensable amino acids
 Threonine 82.23 83.45 84.15 0.579 0.136
 Valine 78.73 79.22 81.01 0.773 0.170
 Isoleucine 85.20 83.69 86.09 0.682 0.116
 Leucine 87.35 87.09 88.20 0.435 0.248
 Phenylalanine 86.39 85.77 87.30 0.385 0.079
 Histidine 81.35 81.31 82.84 1.693 0.775
 Lysine 90.17ab 89.86b 90.66a 0.174 0.045
 Arginine 89.20 90.28 90.33 0.379 0.134
 Methionine 91.37a 89.33b 91.52a 0.467 0.028
 Tryptophan 86.78a 84.01b 86.88a 0.216 < 0.001
Dispensable amino acids
 Aspartic acid 78.14 79.83 80.43 0.684 0.125
 Serine 79.79 80.57 81.79 0.704 0.210
 Glutamic acid 86.84b 87.76ab 88.20a 0.304 0.048
 Proline 77.85 78.35 79.64 1.154 0.559
 Glycine 69.16ab 68.05b 72.81a 1.066 0.045
 Alanine 82.90 84.43 85.15 0.628 0.106
 Tyrosine 87.47 88.14 88.30 1.058 0.847
 Cysteine 66.37 64.48 70.38 3.227 0.466

a,b Means with different letters are significantly differ (p < 0.05).

CON, basal diet; T1, basal diet without a fishmeal and substitute with defatted BSFL; T2, basal diet without a fishmeal and substitute with hydrolyzed BSFL.

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Fecal microbiota

There was no difference in E. coli, Salmonella, and Lactobacillus counts among the treatment groups (Table 13).

Table 13. Effect of replacement dietary of fishmeal with black soldier fly larvae (BSFL) on fecal microbiota in broilers at 4 weeks (Experiment 2)
 Items (Log10CFU/g) CON T1 T2 SE p-value
2 weeks
E. coli 5.97 6.08 6.10 0.082 0.483
Salmonella 2.18 2.28 2.32 0.076 0.427
Lactobacillus 7.53 7.52 7.41 0.078 0.456
4 weeks
E. coli 5.97 6.04 6.08 0.066 0.511
Salmonella 2.29 2.28 2.24 0.064 0.830
Lactobacillus 7.49 7.42 7.52 0.099 0.769

CON, basal diet; T1, basal diet without a fishmeal and substitute with defatted BSFL; T2, basal diet without a fishmeal and substitute with hydrolyzed BSFL; CFU, colony forming unit; E. coli, Escherichia coli.

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DISCUSSION

In Experiment 1, hydrolyzed BSFL showed improvements in both BW and BWG compared to the fishmeal and defatted BSFL throughout the entire experimental period. de Souza Vilela et al. [1] reported significant increases in BW in the grower and finisher phases according to the level of BSFL in broiler diets. Other studies have also reported that feeding BSFL can improve BW and BWG [19,25]. This is consistent with the present study’s findings that feeding hydrolyzed BSFL increased the BW and BWG of broilers. The BSFL is rich in essential nutrients such as protein and fat and is particularly rich in amino acids. Further, chitin, which is a polysaccharide constituting the exoskeleton of insects, can serve as a major energy source for intestinal cells by increasing the production of butyric acid in the cecum [26]. Butyric acid enhances intestinal blood flow, which improves tissue oxygenation and nutrient transport and absorption [27]. Therefore, it is believed that the abundant nutrients and chitin in hydrolyzed BSFL promote the growth of broilers, ultimately resulting in improved BW and BWG. Moreover, in this study, hydrolyzed BSFL showed higher FI than both fishmeal and defatted BSFL. The FI is used as an indicator to evaluate the palatability of a diet [28]. In this study, the increased FI of hydrolyzed BSFL suggests that it is more palatable than fishmeal and defatted BSFL and that it does not adversely affect feed consumption. However, to our knowledge, there has yet to be a study examining hydrolysis among the processing methods of BSFL. We hydrolyzed BSFL using an enzyme called alcalase, which is a serine endopeptidase from Bacillus licheniformis with an alkaline pH optimum and broad substrate specificity, and which has been reported to be helpful in obtaining peptides with antioxidant activity from various protein sources [29,30]. When a protein source is hydrolyzed and used, the enzyme decomposes the protein, thus facilitating the absorption of nutrients and increasing the digestibility of livestock. Therefore, hydrolyzed BSFL—which in this study showed CP digestibility similar to that of fishmeal at weeks 2 and 4—is considered to have improved digestibility and growth performance as protein digestion became easier through the hydrolysis process. Also, in Experiment 2, hydrolyzed BSFL showed higher CP digestibility than both fishmeal and defatted BSFL, while in week 2, DM digestibility was also higher than that of fishmeal. It has been reported that if chitin is included in BSFL that is contained in a large amount in a diet, then monogastric animals cannot easily digest it, which can negatively affect protein digestibility [31,32]. In previous studies, an increase in chitin content when feeding more than 17-29% insect meal has been shown to cause a decrease in protein digestibility [33,34]. The increase in CP digestibility in our study is believed to be due to the fact that the protein is broken down in advance through the hydrolysis process to facilitate the absorption of nutrients. It is also considered to be the case that the digestibility of broilers was not affected because the chitin content was not high, which was achieved by feeding a lower content (3%) of BSFL than has been fed in previous studies. Insect meals have higher amino acid contents than other animal proteins [35]. In our study, the amino acid digestibility of hydrolyzed BSFL was increased in valine and leucine at week 2, and it was increased at lysine and methionine at week 4. The amino acid digestibility obtained in this study was higher than those of other animal proteins (blood meal, feather meal, etc.) reported in previous studies [36,37]. In particular, methionine and lysine—which are the limiting amino acids in broilers—showed higher digestibility than other animal proteins when fed with BSFL in this study. This suggests that BSFL has a rich amino acid profile and can be used as a protein source in broiler diets. However, there have been few studies examining the effect of BSFL on amino acid digestibility to this point, so additional research is needed.

In our study, RBC, WBC, lymphocyte, and BUN did not show significant differences among treatment groups, as the outcomes were all within the physiologically normal range for broilers [38], suggesting that BSFL feeding does not affect broiler health. The TP in serum is positively related to tissue synthesis for growth in broilers, and it may reflect protein synthesis and nutritional status [39,40]. In our study, the TP level at week 4 of hydrolyzed BSFL was significantly similar to that of fishmeal. Therefore, it is believed that hydrolyzed BSFL can play a role similar to fishmeal in tissue synthesis for broiler growth.

In this study, the only general component of broiler breast meat that showed significant differences was ash content. According to Cullere et al. [41], processing insect raw materials can result in higher mineral content than unprocessed insects, particularly when defatted, as the minerals are concentrated and can be even higher. Accordingly, it seems that the ash content of meat was increased by feeding BSFL, which is higher in minerals than fishmeal. Previous studies have shown that the pH of broiler breast meat varies over a wide range of 5.7 to 6.2, with the most cited pH value being 5.8 to 5.9 [4244]. Popova et al. [45] reported that feeding full-fat BSFL showed higher pH than soybean meal and partially defatted BSFL. Therefore, in this study, it is believed that hydrolyzed BSFL, which has a similar fat content to full-fat BSFL (38.53% vs 31.14%), showed a higher pH than fishmeal. Differences in pH values among treatment groups can affect breast meat color and WHC by increasing WHC and decreasing DL, as proteins that are farther from the isoelectric point bind to more water [13]. Meat color is an important quality indicator for consumers [46]. The paleness of meat is indicated by the L* value, where a high L* value indicates poor meat quality [47]. In this study, WHC tended to increase compared to fishmeal when BSFL was fed due to the difference in pH value, but there was no significant difference in DL and meat color. These results indicate that BSFL feeding does not adversely affect broiler meat quality.

The BSFL has a high content of lauric acid, which is known to be a natural antibacterial agent, and which has been reported to be effective in inhibiting the growth of harmful bacteria in intestines by destroying cell membranes [48]. However, there was no significant difference in fecal microbiota among the treatment groups in our study. This is consistent with the results outlined by Cullere et al. [41], and it is considered that all broilers used in this study exhibited optimal health and did not show any difference in fecal microbiota.

CONCLUSION

In conclusion, feeding hydrolyzed BSFL as a fishmeal substitute in broiler diets improved broiler growth performance (increased BW and BWG), improved CP digestibility, and specific amino acid digestibility. Feeding of BSFL did not adversely affect meat quality or blood profiles. Therefore, it is considered that hydrolyzed BSFL in broiler diets can be sufficiently used as a new protein source.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

This experiment was conducted with the support of “Development of production technology for animal substitute materials derived from insect protein hydrolysates” (Project No. 321079-03-2-HD030) of the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET).

Acknowledgements

Not applicable.

Availability of data and material

All data generated or analyzed during this study are included in this published article.

Authors’ contributions

Conceptualization: Chun J, Kim H, Cho J.

Data curation: Chang S, Song M, Lee J.

Formal analysis: Oh H, Song D, An J.

Methodology: Cho H.

Software: Park S, Jeon K.

Validation: Lee B, Nam J.

Investigation: Lee B, Chun J, Cho J.

Writing - original draft: Chang S, Song M, Chun J, Kim H, Cho J.

Writing - review & editing: Chang S, Song M, Lee J, Oh H, Song D, An J, Cho H, Park S, Jeon K, Lee B, Nam J, Chun J, Kim H, Cho J.

Ethics approval and consent to participate

Institutional Animal Care and Use Committee of Chungbuk National University, Cheongju, Korea (approval no. CBNUA-2049-22-02).

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