Effect of insect protein and protease on growth performance, blood profiles, fecal microflora and gas emission in growing pig

Abstract

Two experiments were conducted to determine the effect of Hermetia illucens larvae (HIL) as protein and protease on growth performance, blood profiles, fecal microflora, and gas emission in growing pig. In experiment 1, the seventy-two crossbred growing pigs ([Landrace × Yorkshire] × Duroc) with an initial body weight (BW) of 27.98 ± 2.95 kg were randomly allotted to one of four dietary treatments (3 pigs per pen and 6 replicates pen per treatments). The experimental design was a 2 × 2 factorial arrangement of treatments evaluating two diets (Poultry offal diets and HIL diets) without or with supplementing protease. The poultry offal in basal diet has been replaced by HIL. In experiment 2, the four crossbred growing pigs ([Landrace × Yorkshire] × Duroc) with an initial BW of 28.2 ± 0.1 kg were individually accepted in stainless steel metabolism cages. The dietary treatments included: 1) PO- (PO-; poultry offal diet), 2) PO+ (PO- + 0.05% protease), 3) HIL- (3% PO of PO- diet was replacement 3% HIL), 4) HIL+ (HIL- + 0.05% protease). In experiment 1, From weeks 0 to 2, average daily gain (ADG) and feed efficiency (G:F) were significantly increased in the PO diet group compared with the HIL group. From weeks 2 to 4, ADG and G:F were higher for protease group than for non-protease group. At weeks 2 and 4, the PO diet group had lower blood urea nitrogen (BUN) levels than HIL diet group. In experiment 2, crude protein (CP) and nitrogen (N) retention were decreased by HIL diet at weeks 2 and 4. The fecal microflora and gas emission were not affected by HIL and protease. The HIL diet showed lower CP digestibility than PO diet and total essential amino acids digestibility tended to higher in PO diet than HIL diet. In summary, the present study revealed that replacement of the PO protein with the HIL protein and the additive of protease in growing pig diets during the overall experimental period had no negative effect.

INTRODUCTION

Pig industry is the fastest growing livestock industry in the world. Feed costs account for the largest portion when rearing pigs [1]. Protein feed for pigs accounts for 60%–70% of the total feed cost. Fishmeal and soybean meal are mainly used as protein ingredients. However, they are expensive. Furthermore, the use of substances such as fishmeal, fish oil, soybean meal, and grain is growing in both human and animal feed. Food source insecurity has driven up animal feedstock prices in recent years [2,3]

Insects are highly nutritious and good sources of protein. They are considered promising high-quality feed components [4,5]. Many experiments are being conducted by replacing some proteins in feed with insect feed. In addition, replacing 0%–6% of soybean meal with mealworm (Tenebro molitor) can linearly improve average daily gain (ADG) and average daily feed intake (ADFI) [6,7]. Hermetia illucens larvae (HIL) has a fat content of 15% to 49% based on dry matter (DM) [8]. HIL is also high-quality sources of protein as they contain about 40%–44% crude proteins (CP) with an amino acid profile similar or better than soybean meal [9]. Also Yu et al. [9] reported that 4% HIL in the diet of finishing pigs can significantly increase the ADG and decrease the gain:feed (G:F). Although insect diet has advantages, one study showed that the nutrient digestibility of HIL is lower than that of other protein sources because the outer wall of HIL is made of chitin [10]. Sánchez-Muros et al. [11] reported that chitin can negatively affect protein digestibility. Kim et al. [12] reported protease as part of an enzyme cocktail can increase the hydrolysis of proteins in the small intestine, making it profitable for pigs to absorb amino acids and peptides [13]. A diet supplemented with protease can affect the apparent ileal digestibility of gross energy (GE) [14] and improve the apparent ileal digestibility of protein in pigs [15]. We hypothesized that 1) poultry offal (PO) can be completely replaced with HIL. 2) If replacement with HIL is not effective, protease can assist.

A complete replacement of HIL would be possible and that if replacement of HIL does not have a positive effect on the growth performance and nutrient digestibility of growing pigs, it would be assisted using proteases. Therefore, the aim of the present study was to evaluate the effect of HIL as a protein resource added with protease on growth performance, digestibility, blood profiles, fecal microflora, and gas emission in growing pigs.

MATERIALS AND METHODS

Preparation diet and protease

PO diet and Hermetia illucens larvae (HIL) diet were provided by Cherrybro (Okcheon, Korea). The 3% PO of basal diet was replaced with 3% HIL of insect diet. Table 1 shows nutrient compositions of the diets. The chemical compositions and amino acid profiles of HIL are reported in Table 2. The PT125TM (an alkaline serine endopeptidase produced by streptomyces spp.) was provided and used by Eugene-Bio (Suwon, Korea).

Table 1. Nutrient composition of the basal diet (%, as fed basis)

¹⁾Provided per kg of complete diet: copper (as CuSO₄·5H₂O), 12 mg; zinc (as ZnSO₄), 85 mg; manganese (as MnO₂), 8 mg; iodine (as KI), 0.28 mg; and selenium (as Na₂SeO₃·5H₂O), 0.15 mg.

²⁾Provided per kg of complete diet: vitamin A, 11,025 IU; vitamin D₃, 1,103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; riboflavin, 8.3mg; niacin, 50 mg; thiamine, 4 mg; D-pantothenic, 29 mg; choline, 166 mg; and vitamin B₁₂, 33 μg.

PO, poultry offal; HIL, Hermetia illucens larvae; DDGS, Distiller’s dired grains with solubels; MDCP, monodicalcium phophate; Lys, lysine; Met, methionine.

Table 2. Amino acid profile and chemical composition of Hermetia illucens larvae

CP, crude protein; EE, ether extract; CF, crude fiber; Ca, calcium; P, phosphorus.

Animals and experiment design

Experiment 1

Seventy-two crossbred growing pigs ([Landrace × Yorkshire] × Duroc) with an initial body weight (BW) of 27.98 ± 2.95kg were randomly allotted to one of four dietary treatments (3 pigs per pen and 6 replicates pen per treatments) based on BW. The experiment was conducted for 4 weeks. The dietary treatments were arranged in a 2 × 2 factorial design with two types of feedstuffs (3% PO, 3% HIL) and two levels of protease (0%, 0.05%). The dietary treatments included: 1) PO− (PO−; poultry offal diet), 2) PO+ (PO− + 0.05% protease), 3) HIL− (3% PO of PO− diet was replacement 3% HIL), 4) HIL+ (HIL− + 0.05% protease). The pigs had ad libitum access to feed and water. Each pen was equipped with a single-sided stainless steel automatic feeder and nipple drinker. The daily feed allowance was arranged to 2.7 times the requirement to maintain digestible energy (DE, 2.7 × 110 kcal of DE/kg BW^0.75).

Experiment 2

A total of four crossbred growing pigs ([Landrace × Yorkshire] × Duroc) with an initial BW of 28.2 ± 0.1kg were individually accepted in 1.2 m × 0.7 m × 0.96 m stainless steel metabolism cages. Pigs were randomly fed on four diets for four weeks (4 × 4 Latin square design). The dietary treatments included: 1) PO− (PO−; poultry offal diet), 2) PO+ (PO− + 0.05% protease), 3) HIL− (3% PO of PO- diet was replacement 3% HIL), 4) HIL+ (HIL− + 0.05% protease). The diets were provided at 08:00 and 17:00 every day. Water was provided freely for pigs to drink during the experiment. The experimental environment was maintained a relative humidity of 83 ± 2.3%, temperature of 23 ± 1.5°C and a wind speed of 0.25 ± 0.03m/s.

Measurements and sampling

Nutrient digestibility

In experiment 1, fecal samples were collected for each treatment group at 2 and 4 weeks, and then immediately analyzed Chromic oxide (0.2%) as an indigestible marker was added in pigs’ diet to determine the apparent total tract digestibility (ATTD) of DM, CP, and GE. Cr₂O₃ was measured by an acid digestion method using a Spectrophotometer (Model V-550, Jasco, Tokyo, Japan). The ATTD was calculated using the following formula: digestibility (%) = [1] × 100, where Nf = nutrient concentration in feces (% DM), Nd = nutrient concentration in diet (% DM), Cd = chromium concentration in diet (% DM), and Cf = chromium concentration in feces (% DM).

In experiment 2, fecal was collected by total collection method and immediately after collection, fecal was placed in plastic bag and stored in a −21°C freezer. The urine samples were taken daily and placed in buckets with 50 mL of 6 mol/L HCl underneath the metabolic cages. The whole amount of collected urine was weighed and kept at 20°C. Feces and urine collection were performed according to the method described in Kim et al. [12]. Fresh excreta samples were randomly collected every week and stored at 20°C until analysis. The collected samples were combined with chromic oxide three days before sampling. The fecal, urine, and feed samples were thawed and dried at 60°C for 72 hours before being crushed on a 1-mm screen and thoroughly pooled. Subsamples were then collected for chemical analysis. The GE was calculated using a calorimeter bomb to measure the samples’ heat of combustion (Parr 6100, Parr Instrument, Moline, IL, USA). In accordance with the approach outlined in AOAC [16], analyses of DM and CP were performed, and analyses of AAs were performed using High Performance Liquid Chromatography (HPLC) (SHIMADZU Model LC-10AT, Shimadzu, Kyoto, Japan) techniques. A Kjeltec 2300 Analyzer was used to determine the N content (Foss Tecator AB, Hoeganaes, Sweden).

Growth performance

On day 0, week 2, and week 4 growing pigs BW and feed intake were measured, and ADG, ADFI and G:F were calculated.

Fecal microflora and gas emissions

At week 2 and week 4, fresh fecal samples were collected from 2 pigs of each pen (Experiment 1) or each treatment (Experiment 2) by rectal massage. The collected feces were placed in an icebox for analysis and taken to the laboratory. Bacterial colonies were counted by the pour plate method. In order to measure the number of Lactobacillus and Escherichia coli (E. coli), de Man, Rogosa and Sharpe (MRS) agar for Lactobacillus, and MacConkey agar for E. coli were used, and E. coli was cultured at 37°C for 24 h, and Lactobacillus was cultured for 48 h. The feces (300 g) collected per treatment were placed in a plastic box with small holes and the holes were sealed with plaster. The feces in the plastic box were fermented for 24 hours and 48 hours in room temperature (25°C) to ferment. NH₃ and H₂S concentrations were determined in the ranges of 50.0 to 100.0 ppm (No. 3La, detection tube, Gastec, Kanagawa, Japan).

Blood profiles

Blood samples were collected from anterior vena cava of 6 pigs each treatment at 2 and 4 weeks for analyzing concentration of white blood cells (WBCs), red blood cell (RBC), total protein and blood urea nitrogen (BUN) in whole blood and serum. After collection, serum samples were centrifuged (3,000 g) for 15 min at 4°C. The WBC, RBC, total protein and BUN were determined using an automatic blood analyzer (ADVIA 120, Bayer, Leverkusen, Germany).

Statistical analysis

Data for effects of two types of feedstuffs (3% PO, 3% HIL) added with different levels of protease on growth performance, digestibility of DM, CP, GE, blood profiles, gas emissions and fecal microflora of growing pigs were subjected to two-way ANOVA, with different feed, protease addition level, and their interactions as main effects and litter as a covariate. The data were statistically analyzed by the generalized linear model (GLM) procedure in IBM SPSS statistics v.25 (SPSS, Chicago, IL, USA). Each cage was used for each experimental unit. Differences between treatment groups was determined using Tukey’s honest significant difference (HSD) test with a p-value of < 0.05 indicating significance and 0.05 < p-value < 0.10 indicating a tendency.

RESULTE

Experiment 1

Growth performance

From weeks 0 to 2, ADG and G:F were significantly increased (p < 0.05; p < 0.05 respectively) in the PO diet group compared with the HIL diet group (Table 3). From weeks 2 to 4, ADG and G:F were higher (p <0.001; p < 0.006 respectively) for the protease group than for the non-protease group. From weeks 0 to 4, G:F were higher (p < 0.05) for the PO diet group than the HIL diet group of pigs.

Table 3. The effect of replacement dietary of animal protein with insect meal (Hermetia illucens larvae) and additive the protease on the growth performance in growing pigs (Exp1)

PO, poultry offal diet; HIL, Hermetia illucens larvae diet; D, diet; P, protease; BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; G:F, gain:feed.

Nutrient digestibility

The ATTD of DM and GE was not significantly (p > 0.05) affected by diet and protease (Table 4). CP was decreased (p < 0.033) by the HIL diet group at week 2 and 4. The HIL diet group showed lower CP digestibility than the PO diet group.

Table 4. The effect of replacement dietary of animal protein with insect meal (Hermetia illucens larvae) and additive the protease on the nutrient digestibility of in growing pigs (Exp1)

PO, poultry offal diet; HIL, Hermetia illucens larvae diet; D, diet; P, protease; DM, dry matter; CP, crude protein; GE, gross energy.

Blood profiles

From weeks 2 and 4, the PO diet group had lower BUN levels than the HIL diet group (p < 0.004 and p < 0.005, respectively; Table 5). Diet and protease had no effect (p > 0.050) on WBC, RBC, Lymphocyte, and total protein at weeks 2 and 4.

Table 5. The effect of replacement dietary of animal protein with insect meal (Hermetia illucens larvae) and additive the protease on the blood profile in growing pigs (Exp1)

PO, poultry offal diet; HIL, Hermetia illucens larvae diet; D, diet; P, protease; WBC, white blood cell; RBC, red blood cell; BUN, blood urea nitrogen.

Fecal microflora

E. coli and Lactobacillus were not affected (p > 0.05) by diet and protease (Table 6).

Table 6. The effect of replacement dietary of animal protein with insect meal (Hermetia illucens larvae) and additive the protease on the fecal microflora in growing pigs (Exp1)

PO, poultry offal diet; HIL, Hermetia illucens larvae diet; D, diet; P, protease; E. coli, Escherichia coli.

Gas emission

NH₃ and H₂S gas emissions were not affected (p > 0.05) by diet and protease (Table 7).

Table 7. The effect of replacement dietary of animal protein with insect meal (Hermetia illucens larvae) and additive the protease on the fecal gas emission in growing pigs (Exp1)

PO, poultry offal diet; HIL, Hermetia illucens larvae diet; D, diet; P, protease; SE, standard error; NH₃, ammonia; H₂S, hydrogen sulfide.

Experiment 2

Nutrient digestibility

Protease did not affect (p > 0.05) the ATTD of DM, CP, and GE (Table 8). The HIL diet group showed lower (p < 0.05) CP than the PO diet group. The ATTD of arginine, histidine, lysin and tryptophan were higher (p < 0.05) in the PO diet group than the HIL diet group. The ATTD of threonine was higher (p < 0.05) in the protease group than the non-protease group. The ATTD of total essential AAs tended to be higher (p = 0.05) in the PO diet group than the HIL diet group. The ATTD of cysteine was higher (p < 0.05) in the protease group than the non-protease group. The ATTD of glycine was higher (p < 0.05) in the PO diet group than the HIL diet group. There was no interaction between diet and protease in nutrient digestibility.

Table 8. The effect of replacement dietary of animal protein with insect meal (Hermetia illucens larvae) on digestibility of nutrient and amino acids in growing pigs (Exp 2)

PO, poultry offal diet; HIL, Hermetia illucens larvae diet; D, diet; P, protease; DM, dry matter; CP, crude protein; GE, gross energy.

Fecal microflora

E. coli and Lactobacillus were not affected (p > 0.05) by diet and protease (Table 9).

Table 9. The effect of replacement dietary of animal protein with insect meal (Hermetia illucens larvae) and additive the protease on the fecal microflora in growing pigs (Exp2)

PO, poultry offal diet; HIL, Hermetia illucens larvae diet; D, diet; P, protease; E. coli, Escherichia coli.

Nitrogen utilization

The HIL diet group increased (p < 0.05) the fecal N excretion, urine N excretion, total N excretion, and decreased (p < 0.05) N retention (Table 10). The protease group decreased (p < 0.05) the urine N excretion and total N excretion and increased the N retention. There was no interaction between diet and protease in N utilization.

Table 10. The effect of replacement dietary of animal protein with insect meal (Hermetia illucens larvae) and additive the protease on nitrogen utilization in growing pigs (Exp2)

PO, poultry offal diet; HIL, Hermetia illucens larvae diet; D, diet; P, protease; N, nitrogen.

DISCUSSION

Experiment 1

Driemeyer [17] reported that partially replacing fishmeal with HIL (3.5%) does not affect the growth performance of pigs. Biasato et al. [18] showed no significant difference in the effect of replacing 0%, 5%, or 10% soybean meal with HIL on the growth performance of piglets. However, many researchers suggested that replacing protein sources in animal feed with insect protein could improve growth performance and nutrient digestibility [19]. In addition, Ma et al. [20] showed that ADG is increased but feed conversion ratio is decreased when a diet is supplemented with 4% HIL than a diet supplemented with 8% HIL in growing pigs. However, in the present study, replacing PO with HIL decreased growth performance. This reduction was caused by an anti-nutritional factor (i.e., chitin) included in HIL. When the component of chitin in the exoskeleton of HIL is not digested, it can bind to proteins and reduce digestibility [21,22]. Barragan et al. [3] reported that HIL should only be partially replaced because the performance is degraded by factors such as high fat content and ash in insect meal. In the present study, pigs fed diets added with 0.05% protease had better ADG and G:F than pigs fed diets without protease. These results agreed with the results of Zuo et al. [23] and Park et al. [24] showing that supplementation of 200 mg and 0.2% protease in the swine diet, respectively, could improve growth performance. Min et al. [25] reported that the positive effect on growth performance could be explained by increased protein hydrolysis caused by the addition of dietary protease, thereby improving nutrient digestion and utilization.

The BUN concentration is commonly used to determine nitrogen utilization and excretion rates. It is also used to find renal damage in animals and humans [26,27]. BUN is decreased when nitrogen is efficiently used in protein synthesis. It is increased spontaneously as the kidney filtration rate decreases [28,29]. In the current study, the HIL resulted in higher BUN concentrations. Fang et al. [30] reported that BUN levels are negatively correlated with ADG and G:F ratios. Chitin supplementation may decrease ammonia levels, in part by affecting BUN levels [31], consistent with growth performance and BUN results of the current experiment. The BUN level is considered to be increased in this experiment due to low growth performance with the HIL diet compared to the PO diet.

The possibility of improving intestinal health by using insect replacement diet has been suggested based on immune system stimulating properties of chitin, antimicrobial peptides (AMPs), and antimicrobial activity of lauric acid [32-34]. A previous study reported that the antibacterial peptide of HIL shows antibacterial activity against pathogenic bacteria such as E. coli [35]. However, it had no significant effect on fecal microbiota.

Major components of fecal gas that cause air pollution are NH₃ and H₂S [36]. In general, fecal gas emission is related to dietary digestibility. Enhanced digestion, which may reduce microbial fermentation substrates in the large intestine, can lower gas emissions [37]. However, The HIL and protease showed no significant effect on NH₃ or H₂S emission in the overall experiment.

Experiment 2

Many previous studies reported that insect protein sources such as house fly, black soldier fly, HIL, and domestic larvae can be suitable ingredients in the diets of animals [38-41]. They explained that insect ingredients have similar or superior amino acid composition and higher protein content than conventional protein sources. Tan et al. [41] reported that amino acid content in insect or amino acid digestibility of supplement insect meal are similar to soybean meal or fish meal in growing pigs. However, in this experiment, the digestibility of essential AAs including lysine was significantly decreased in the HIL group. CP digestibility of the HIL group also decreased compared to that of the PO group. Bovera et al. [5] reported that digestibility of DM and CP is decreased when Tenebrio molitor is added to broiler diet. The low nutrient digestibility of the HIL group is caused by the mechanisms of chitin in HIL. Endogenous enzymes do not breakdown β1–4 links between N-acetylglucosamine that compose chitin [42]. Therefore, any protein encapsulated by chitin would not be digested or absorbed by the pig’s small intestine [43]. Mammals express acidic mammalian chitinase (AMCase). Pig AMCase is stable in the presence of other digestive proteases. It functions as chitinolytic enzyme under gastrointestinal conditions [44]. Zuo et al. [23] reported that supplement of protease can increase the growth performance when pigs are fed with a low digestible protein source. Protease is a proteolytic active enzyme. It can improve protein digestibility in several experiments [23,45,46]. Therefore, we tried to weaken the antinutrient action of chitin by adding protease. However, the addition of protease showed no significant difference compared to the control without such addition.

CONCLUSION

In summary, the present study revealed that the replacement of PO with HIL and additive of the protease in growing pig diets during the overall experimental period (week 0–4) had no negative effect. The supplementation of protease in the HIL diet increased the ADG and AAs digestibility. But, trends in interactive effects between diet and protease were not observed in this experiment. More research is needed.