Compound bioengineering protein supplementation improves intestinal health and growth performance of broilers

Abstract

Currently, antimicrobial peptides (AMPs) are of growing interest as potential substitutes for antibiotic growth promoters in animal production. The present study was conducted to evaluate the effects of dietary supplementation of bioengineering artificial Parasin I protein (API) and artificial plectasin protein (APL) (named as compound bioengineering protein, CBP) on growth performance and intestinal health of broilers. A total of 450 one-day-old Arbor Acres male healthy broilers were randomly allotted to 5 dietary groups with 10 replicates of 9 individuals in each replicate and supplemented with 0, 250, 500, 750, and 1,000 mg/kg CBP for 6 wk. Dietary CBP supplementation increased (P < 0.01) body weight (6 wk), average daily gain (0–6 wk), and average daily feed intake (3–6 wk and 0–6 wk). CBP addition enhanced antioxidant capacity, which was accompanied by the higher (P < 0.05) activity of serum total antioxidant capacity (T-AOC) (750 mg/kg), jejunal glutathione peroxidase (750 mg/kg), and T-AOC (500 and 1,000 mg/kg). Dietary CBP addition improved intestinal health, reflecting by the increased (P < 0.05) villus height to crypt depth ratio in the duodenum, the upregulated (P < 0.01) mRNA levels of claudin-1 (500 and 750 mg/kg) in the ileum, the downregulated (P < 0.01) mRNA expression of occludin (500 mg/kg) in the duodenum and claudin-1 (500 mg/kg) and occludin (500 and 750 mg/kg) in the jejunum, and the upregulated mRNA expression of (P < 0.01) mucin2 (MUC2) (1,000 mg/kg) in the duodenum. In addition, CBP upregulated (P < 0.01) IL-10 (1,000 mg/kg) in duodenum and ileum, and downregulated (P < 0.05) the mRNA expression of IL-6 (750 and 1,000 mg/kg), interferon-γ (1,000 mg/kg) in the jejunum and TNF-α (250 mg/kg) in the ileum. Furthermore, dietary CBP increased (P < 0.01) the abundance of total bacteria and Lactobacillus (500 and 750 mg/kg), and reduced (P < 0.05) the abundance of Escherichia coli (750 mg/kg) in the cecum. In conclusion, CBP supplementation enhances the antioxidant capacity, intestinal health, immune function, and ameliorates the gut microflora population, thus improving the growth performance of broilers. Dietary supplementation of 750 mg/kg CBP exhibits a better beneficial effect.

INTRODUCTION

Antibiotics are successfully used in animals for growth promotion, preventing diarrhea, and enhancing the immune function of the body for a long period (Kumar et al., 2005). However, the abuse of antibiotics has caused drug-resistant strains, which may cause negative effects on animal and human health (Neu, 1992; Spellberg et al., 2008; Ong et al., 2014). Thus, there is an urgent need to explore safe, effective, and natural alternatives to replace antibiotics. Antimicrobial peptides (AMPs) are intrinsic part of the innate immune system (Andersson et al., 2016). They are small molecules substances produced by organisms and play important roles in defending against bacterial, fungi, and protists invasion (Haines et al., 2009; Roudi et al., 2017). AMPs possess the advantages of efficient broad-spectrum antimicrobial activity and are less prone to develop bacterial resistance, making them promising candidates for drug development (Yeaman and Yount, 2003; Hancock and Sahl, 2006).

Parasin I (PI), a 19-residue antimicrobial peptide isolated from the skin mucus of wounded catfish, shows potent antimicrobial activity against gram-positive, gram-negative bacteria, and fungi, such as Salmonella, Escherichia coli, and Crytococcus neoformans, without any hemolytic activity (Park et al., 1998). PI is generated from unacetylated histone proteins 2 (H2A). When catfish got wounded or injured, cathepsin D was secreted to the mucosal surface as an inactive proenzyme and activated to the mature enzyme by matrix metalloproteinase 2 (MMP2), and then induced cleavage of the peptide bond of Ser₁₉-Arg₂₀ occurs, to release the active substance PI (Cho et al., 2002; Koo et al., 2008). PI was a membrane-bound peptide, it causes osmotic rupture of the cell by binding to the cell surface, thus killing microorganisms (Park et al., 1998; Koo et al., 2008). Therefore, resistant strains were not easily produced against antimicrobial peptides. The antimicrobial activity of PI was mainly through the α-helixes and N-terminal basic residues. Studies demonstrated that both α-helixes and N-terminal basic residues alterations cause the activity of PI decreases sharply or even be lost (Brogden, 2005).

Plectasin (PL), a 40-amino acid produced by the fungus Pseudoplectania nigrella, was reported by Mygind et al. (2005). It was the first defensin derived from fungus that showed therapeutic potential and strong activity against gram-positive bacteria, including Staphylococcus, Streptococcus, and S. pneumoniae. While its efficacy was comparable to that of vancomycin and penicillin, even against various strains resistant to conventional antibiotics (Mygind et al., 2005; Brinch et al., 2009; Mandal et al., 2009; Ong et al., 2014). Differing from other defensins, PL was active only toward gram-positive bacteria for its particular antimicrobial mechanism, this small peptide could bind the bacterial cell-wall precursor lipid II directly to inhibit the synthesis of bacterial cell walls, thus leading to the death of bacteria (Hara et al., 2008; Schneider et al., 2010). Studies have shown that PL does not affect cell viability or IL-8 production, and it could be used as an inoffensive antibiotic alternative for clinical application (Hancock and Sahl, 2006; Hara et al., 2008).

However, the high cost and low-expression levels limit their practical application of AMPs, and genetic recombination of AMPs is considered the ideal solution. The bioengineering artificial PI protein (API, a fusion protein contains PI functional segments) (Zhao et al., 2015) and the bioengineering artificial PL protein (APL, a fusion protein contains PL functional segments) were constructed and expressed in the methylotrophic yeast Pichia pastoris, and get high yields using a high cell density fermentation strategy in our laboratory. Previous studies show that levels of dietary API supplementation exhibits beneficial effects in rabbits (Zhang et al., 2022) and broilers (Peng et al., 2022). However, whether the combination supplementation of these 2 bioengineering proteins could improve the growth performance and gut health of broilers remains unknown. Therefore, the present study was conducted to evaluate the effects of dietary supplementation of API and APL (named as compound bioengineering protein, CBP) on the growth performance, antioxidant capacity, immune function, and intestinal health of broilers, and the potential dosages were explored.

MATERIALS AND METHODS

The Animal Care and Use Committee of Sichuan Agricultural University (Chengdu, Sichuan, China) approved the procedures used in this experiment (approval No. SCAUAC202009-01).

Compound Bioengineering Protein

The 2 high-expression strains containing API (Zhao et al., 2015) and APL used in this study were obtained from our laboratory. The API and APL were prepared and supplied from a 50 L fermenter (Shanghai Baoxing Bio-engineering Equipment Co. Ltd., Shanghai, China) containing basal salt medium through high-density fermentation, respectively. The stirring speed was adjusted to maintain a relative percentage of dissolved oxygen at 20 to 30%. Following methanol induction at 29°C for 3 d. The yeast fermentation broth was adsorbed with adsorbent and dried. The prepared API and APL were then mixed according to their expressions to get the CBP. Each kilogram of CBP provided approximately 4 g API and 2 g APL.

Animals, Diets, and Experimental Design

A total of 450 one-day-old healthy male Arbor Acres broilers were randomly classified into 5 dietary groups, each group consisting of 10 replicates with 9 broilers per pen based on body weight (BW). Broilers in different groups were fed on the corn-soybean meal basal diet (Control), or basal diet supplemented with 250, 500, 750, and 1,000 mg/kg CBP. The basal diet was formulated based on 2 phases, including the starter phase (d 1–21) and the grower phase (d 22–42), to meet the nutrient requirements of white-feathered broilers according to NRC1994 and NY/T33-2004, as shown in Table 1. The feed is in mash form. Broilers were housed in wire-mesh cages and the temperature was maintained at 32°C, 28°C, and 25°C for the first, second, and subsequent weeks, respectively. The trial lasted for 6 wk. All chickens were free to access feed and water during the experiment period. Daily observations were made to record general health and mortality.

Table 1.

Ingredient and nutrient levels of basal diets at different phases (as-fed basis).

Ingredients (%)	0–3 wk	3–6 wk
Corn	56.90	61.50
Soybean meal	33.25	28.60
Soybean oil	2.50	2.70
Wheat bran	2.70	3.03
CaCO3	1.45	1.40
CaHPO4	1.80	1.53
L-Threonine	0.07	0.05
L-Lysine HCl	0.15	0.11
D, L-Methionine	0.31	0.21
NaCl	0.37	0.37
Mineral and vitamin premix1	0.50	0.50
Total	100	100
Calculated nutrient levels2
Poultry metabolizable energy (Mcal/kg)	2.889	2.949
Crude protein (%)	20.00	18.20
Calcium (%)	1.03	0.94
Available phosphorus (%)	0.45	0.40
Digestible lysine (%)	1.15	1.01
Digestible methionine+cysteine	0.90	0.76
Digestible methionine (%)	0.59	0.47
Digestible threonine	0.81	0.73
Digestible valine	0.91	0.83
Digestible tryptophan	0.23	0.21

Mineral and vitamin premix provided the following per kilogram of diet:

¹0–3 w: vitamin A, 8,000 IU; vitamin B_1, 2 mg; vitamin B₂, 8 mg; vitamin B₆, 3.5 mg; vitamin B₁₂, 0.01 mg; vitamin D₃, 1,000 IU; vitamin E, 20 IU; vitamin K, 0.5 mg; pantothenic acid, 10 mg; niacin, 35 mg; folic acid, 0.55 mg; choline, 1,300 mg; Fe (FeSO₄·7H₂O), 100 mg; Cu (CuSO₄·5H₂O), 8 mg; Mn (MnSO₄·H₂O), 120 mg; Zn (ZnSO₄·H₂O), 100 mg; I (KI), 0.7 mg; Se 0.3 mg.

3–6 w: vitamin A, 6,000 IU; vitamin B_1, 2 mg; vitamin B₂, 8 mg; vitamin B₆, 3.0 mg; vitamin B₁₂, 0.01 mg; vitamin D₃, 750 IU; vitamin E, 20 IU; vitamin K, 0.5 mg; pantothenic acid, 10 mg; niacin, 30 mg; folic acid, 0.55 mg; choline, 1,000 mg; Fe (FeSO₄·7H₂O), 80 mg; Cu (CuSO₄·5H₂O), 8 mg; Mn (MnSO₄·H₂O), 100 mg; Zn (ZnSO₄·H₂O), 80 mg; I (KI), 0.7 mg; Se 0.3 mg.

²These values were calculated from data provided by NRC1994 and NY/T33-2004.

Sample Collection and Preparation

Birds were weighed at d 1, 21, and 42 and recorded as BW, and the feed consumption per pen was measured weekly to determine average daily gain (ADG), average daily feed intake (ADFI), and feed to gain (F/G). At the end of the 6 wk, birds fasted for 8 h overnight, then 6 birds from each group were selected according to the average BW of the corresponding pen and slaughtered to collect blood, duodenum, jejunum, and ileum. The blood samples were centrifuged at 3,000 rpm for 10 min and the serum was collected and stored immediately at −20°C. The middle of the duodenum, jejunum, and ileum segment was fixed in 4% formaldehyde in phosphate buffer and kept at room temperature for the intestinal morphology analyses. The duodenal, jejunal, and ileal mucosa was gently scraped from the intestinal tissues after being flushed with physiological saline, the collected mucosa was frozen immediately in liquid nitrogen and stored at −80°C until further analysis. The chyme was collected from the middle of the cecum and frozen immediately in liquid nitrogen and stored at −80°C for later analysis.

Intestinal Morphology Assays

Morphology measurements of the duodenum, jejunum, and ileum tissue samples were using paraffin embedding techniques. Three samples (n = 3) were randomly selected from 6 groups for tissue morphology measurements, and 10 visual fields were observed for each sample. Measurements included villus height (VH), crypt depth (CD), and villus height to crypt depth ratio (VH/CD). The VH was measured from the crypt-villus junction to the villus tip. The CD was measured from the crypt-villus junction to the crypt base, and the VH/CD was calculated by dividing VH by CD.

Antioxidant Capacity of Serum and Jejunum

Serum samples are directly used for determination after appropriate dilution, and jejunum mucosa samples were homogenized with physiological saline and centrifuged at 3,000 × g for 15 min at 4°C, and then the supernatants were collected for further analysis. Glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) were measured by colorimetric assay according to the commercial kits (no. 005, A015-1, A001-1-1, A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and the details of these components followed the kit instructions.

Serum Immune Factor

Serum samples are directly used for determination. Interleukin-10 (IL-10) and tumor necrosis factor α (TNF-α) were measured by enzyme-linked immunosorbent assay according to the commercial kits (No. MM-114501, MM-093801, Jiangsu Meimian Industrial Co., Ltd., China) and the details of these components followed the kit instructions.

Enzyme Activities Assays

Jejunal enzyme activities were determined using an ultraviolet spectrophotometer. The pretreatment of jejunal mucosa samples was the same as above. The activities of lipase, amylase, chymotrypsin, and trypsin were measured using commercial kits (No. A054-1-1, C016-1-14, A080-3-1, A080-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

Gene Expression Assays

The total RNA of intestinal mucosa samples was extracted using RNAiso Plus, according to the manufacturer's protocol (No. 9109, Takara, Dalian, China). RNA concentration and quality were detected by spectrophotometer (NanoDrop 2000, Thermo, Waltham, MA), and then the RNA was reverse-transcribed using the HIScript III RT SuperMix for qPCR (No. R323-01, Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). Real-time PCR was performed with SYBR Green Premix Ex Taq II reagents (No. RR820A, Takara, Dalian, China) on a QuantStudio 5 Flex system (Applied Biosystems, Foster City, CA). The relative mRNA abundances of 5 tight junction-related genes, 1 mucin-related gene, and 4 inflammation-related genes were normalized to β-actin and calculated by the 2^−ΔΔCt method. Primers were designed with Primer Express 3.0 (Applied Biosystems, Foster City, CA) as shown in Table S1.

The abundance of microbiota in cecum chyme was determined using TaqMan methods. DNA was extracted from the cecum chyme using the Stool DNA Kit (No. D4015-01, Omega Bio-Tek, Doraville, CA) according to the manufacturer's protocol. Primers and probes for gut total bacteria, Lactobacillus, and Escherichia coli were designed with Primer Express 3.0 (Applied Biosystems, Foster City, CA) as shown in Table S2. TaqMan real-time PCR amplification was performed on a Bio-Rad CFX96 Touch real-time PCR instrument (BioRad Laboratories, Richmond, CA). The standard curve and the abundance of microbiota were calculated by the method according to previous studies (Whelan et al., 2003; Yi et al., 2022).

Statistical Analysis

Statistical analysis was performed by SAS Ver. 9.4 (SAS Institute Inc., Cary, NC). Values were analyzed using analysis of variance (ANOVA). When ANOVA P values were less than 0.05, means were compared by using Tukey's multiple comparison procedure. Furthermore, these data were analyzed by multiple linear regression analysis to assess optimal dietary compound bioengineering protein levels. The data were expressed as the mean and standard error (SEM). Statistical significance was considered as P < 0.05, and a trend was expressed when 0.05 < P < 0.10.

RESULTS

Growth Performance

Dietary CBP supplementation improved the growth performance of broilers (Table 2). Compared with the control group, dietary CBP supplementation increased (P < 0.05) BW at 6 wk, ADG during 0 to 6 wk, ADFI during 3 to 6 wk, and 0 to 6 wk of broilers, and the ADFI changed linearly (P < 0.05) with the increase of CBP during the 3 to 6 wk and 0 to 6 wk. Compared with the control group, 250 mg/kg CBP increased the ADFI of broilers during 3 to 6 wk and 0 to 6 wk (P < 0.05); 750 mg/kg CBP increased the BW at 6 wk, the ADG, and ADFI during 0 to 6 wk (P < 0.05. Levels of CBP exhibited a limited effect on the F/G of broilers during 0 to 3 wk, 3 to 6 wk, and 0 to 6 wk (P > 0.05).

Table 2.

Effects of dietary supplementation of compound bioengineering protein (CBP) on growth performance in broilers.

	Dietary CBP level, mg/kg						P value
Items	0	250	500	750	1,000	SEM	ANOVA	Linear	Quadratic
BW, g
D 0	41.97	42.00	42.22	42.18	42.10	0.045	0.317	0.143	0.655
3 wk	670.06	680.22	684.72	698.44	668.67	4.254	0.162	0.186	0.752
6 wk	2252.71b	2333.49ab	2298.34ab	2362.19a	2250.51b	12.437	0.009	0.056	0.514
ADG, g/d
0–3 wk	29.91	30.39	30.60	31.25	29.84	0.202	0.166	0.191	0.756
3–6 wk	75.37	77.27	76.84	79.23	75.33	0.488	0.062	0.175	0.851
0–6 wk	52.64b	54.56ab	53.72ab	55.24a	52.58b	0.296	0.009	0.057	0.512
ADFI, g/d
0–3 wk	42.48	43.65	44.08	45.00	43.22	0.311	0.113	0.087	0.821
3–6 wk	127.13b	133.39a	129.99ab	131.82ab	127.88b	0.631	0.001	0.015	0.103
0–6 wk	84.80b	88.52a	87.04ab	88.41a	85.55ab	0.398	0.004	0.008	0.162
F/G, g: g
0–3 wk	1.42	1.44	1.44	1.45	1.45	0.004	0.143	0.269	0.128
3–6 wk	1.71	1.69	1.69	1.66	1.70	0.010	0.325	0.459	0.712
0–6 wk	1.63	1.62	1.62	1.60	1.63	0.005	0.618	0.704	0.903

Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; BW, body weight; F/G, feed to gain ratio. Data are shown as means and SEM, n = 10 in each group.

^a,bMeans within the same row not bearing the common superscript are significantly different (P < 0.05).

Intestinal Morphology

The effects of dietary supplementation of CBP on intestinal morphology in broilers are shown in Table 3. Compared with the control group, dietary CBP supplementation improved (P < 0.05) the VH/CD of the duodenum, which changed linearly (P < 0.05) with the increase of CBP. Dietary CBP supplementation tended to increase (0.05 < P < 0.10) the VH of the duodenum. Dietary CBP supplementation did not affect the intestinal morphology of the jejunum and ileum.

Table 3.

Effects of dietary supplementation of compound bioengineering protein (CBP) on intestinal morphology in broilers.

Items	Dietary CBP level, mg/kg					SEM	P value
	0	250	500	750	1,000		ANOVA	Linear	Quadratic
Duodenum
VH (μm)	1351.04	1645.67	1766.61	1670.85	1561.80	51.559	0.085	0.010	0.642
CD (μm)	140.69	124.49	122.94	120.48	109.05	5.083	0.454	0.475	0.215
VH/CD (μm/μm)	9.82b	13.61a	14.58a	14.05a	14.45a	0.616	0.045	0.016	0.109
Jejunum
VH (μm)	1561.46	1325.84	1437.95	1327.97	1411.77	50.061	0.622	0.405	0.291
CD (μm)	118.05	125.91	110.51	127.34	114.99	7.351	0.960	0.884	0.745
VH/CD (μm/μm)	13.99	11.60	13.30	10.57	13.12	0.755	0.669	0.663	0.491
Ileum
VH (μm)	1009.41	1019.40	922.24	912.52	1044.22	46.231	0.893	0.594	0.583
CD (μm)	114.44	110.50	114.90	91.44	115.41	4.125	0.321	0.930	0.849
VH/CD (μm/μm)	8.94	9.32	8.22	10.03	9.06	0.354	0.663	0.624	0.483

Abbreviations: CD, crypt depth; VH, villus height; VH/CD, villus height to crypt depth ratio. Data are shown as means and SEM, n = 3 in each group.

^a,bMeans within the same row not bearing the common superscript are significantly different (P < 0.05).

Antioxidant Capacity of Serum and Jejunum

We explored the antioxidant capacity of the serum and jejunum (Figure 1). Compared with the control group, dietary 750 mg/kg CBP supplementation increased (P < 0.05) the activity of T-AOC (Figure 1C), while CBP showed limited effects (P > 0.05) on activities of GSH-Px, T-SOD, and MDA levels in the serum. Dietary 750 mg/kg CBP supplementation increased (P < 0.05) the activity of GSH-Px in the jejunum (Figure 1E). Dietary 500 and 1,000 mg/kg CBP supplementation increased (P < 0.05) the activity of T-AOC (Figure 1G), while dietary CBP did not affect (P > 0.05) the activity of T-SOD and MDA level in the jejunum.

Figure 1.

Effects of dietary supplementation of compound bioengineering protein on serum and jejunal antioxidant capacity in broilers. (A–D) serum, (E–H) jejunum. GSH-Px, glutathione peroxidase; MDA, malondialdehyde; T-AOC, total antioxidant capability; T-SOD, total superoxide dismutase. Data are shown as means and SEM, n = 6 in each group. ^a–cMean values with different letters were significantly different among diets with 0, 250, 500, 750, and 1,000 mg/kg of compound bioengineering protein (1-way ANOVA, P < 0.05, Tukey's post hoc test).

Digestive Enzyme Activity in the Mucosa of the Jejunum

We explored digestive enzyme activity in the mucosa of the jejunum (Table 4). Compared with the control group, dietary CBP supplementation exhibited limited effects (P > 0.05) on the activities of lipase, amylase, chymotrypsin, and trypsin in the mucosa of the jejunum.

Table 4.

Effects of dietary supplementation of compound bioengineering protein (CBP) on jejunal enzyme activities in broilers.

Items	Dietary CBP level, mg/kg					SEM	P value
	0	250	500	750	1,000		ANOVA	Linear	Quadratic
Jejunal enzyme activity
Lipase U/g prot	13.12	9.77	7.63	8.26	11.51	0.745	0.109	0.014	0.864
Amylase U/mg prot	1.15	0.82	1.14	0.96	0.86	0.085	0.657	0.926	0.137
Chymotrypsin U/mg prot	0.66	0.32	0.36	0.34	0.40	0.092	0.762	0.300	0.499
Trypsin U/mg prot	248.67	112.29	104.60	112.44	153.45	17.584	0.061	0.008	0.225

Data are shown as means and SEM, n = 6 in each group.

Tight Junction-Related Genes and MUC2 Abundance

We further explored the mRNA abundance of tight junction-related genes and MUC2 in the duodenum, jejunum, and ileum (Figure 2). Compared with the control group, dietary 500 mg/kg CBP supplementation downregulated (P < 0.05) the mRNA levels of OCLN in the duodenum (Figure 2A), CLDN-1, and OCLN in the jejunum (Figure 2B), while upregulated (P < 0.05) the mRNA levels of CLDN-1 in the ileum (Figure 2C). A total of 750 mg/kg CBP downregulated (P < 0.05) the mRNA expression of OCLN in the jejunum (Figure 2B) and upregulated (P < 0.05) the mRNA expression of CLDN-1 in the ileum (Figure 2C). The other levels of CBP exhibited limited effect (P > 0.05) on the expression of small intestinal tight junction-protein-related genes.

Figure 2.

Effects of dietary supplementation of compound bioengineering protein on the mRNA expression of tight junction-related genes and MUC2 in the intestinal mucosa of broilers. (A) Duodenum, (B) jejunum, (C) ileum, (D) MUC2. ZO-1, zonula occluden-1; ZO-2, zonula occluden-1; CLDN-1, claudin-1; CLDN-2, claudin-2; OCLN, occludin, MUC2, mucoprotein-2. Data are shown as means and SEM, n = 6 in each group. ^a–cMean values with different letters were significantly different among diets with 0, 250, 500, 750, and 1,000 mg/kg of compound bioengineering protein (1-way ANOVA, P < 0.05, Tukey's post hoc test).

Compared with the control group, dietary 1,000 mg/kg CBP supplementation upregulated (P < 0.05) the mRNA level of MUC2 in the duodenum (Figure 2A). Dietary CBP supplementation had a limited effect (P > 0.05) on the expression of MUC2 in the jejunum or ileum.

Serum Immune Factor Content and Inflammation-Related Genes Abundance

Dietary CBP supplementation also enhanced intestinal immune function, reflecting by regulating the serum cytokines and the mRNA abundance of inflammation-related genes in the duodenum, jejunum, and ileum (Figure 3). Dietary 250 mg/kg CBP supplementation downregulated (P < 0.05) the mRNA levels of TNF-α in the ileum (Figure 3D), 750 mg/kg CBP downregulated (P < 0.05) the mRNA expression of IL-6 in the jejunum (Figure 3C), and 1,000 mg/kg CBP upregulated (P < 0.05) the mRNA expression of IL-10 in the duodenum (Figure 3B) and ileum (Figure 3D), and downregulated (P < 0.05) the mRNA levels of IL-6 and IFN-γ in the jejunum (Figure 3C). However, dietary CBP inclusion had a limited effect (P > 0.05) on IL-10 and TNF-α content in the serum.

Figure 3.

Effects of dietary supplementation of compound bioengineering protein on serum immune factor content and mRNA abundance of intestinal mucosa inflammation-related genes in broilers. (A) The content of serum immune factor, (B) inflammation-related genes abundance in the duodenum, (C) inflammation-related genes abundance in the jejunum, and (D) inflammation-related genes abundance in the ileum. IL-10, interleukin-10; IL-6, interleukin-6; TNF-α, tumor necrosis factor α; IFN-γ, interferon-γ. Data are shown as means and SEM, n = 6 in each group. ^{a, b}Mean values with different letters were significantly different among diets with 0, 250, 500, 750, and 1,000 mg/kg of compound bioengineering protein (1-way ANOVA, P < 0.05, Tukey's post hoc test).

Cecal Microbial Population

Dietary CBP supplementation improved the abundance of cecal microbial (Table 5). Compared with the control group, dietary 500 and 750 mg/kg CBP supplementation increased the abundance of total bacteria and Lactobacillus (P < 0.05), and 750 mg/kg CBP reduced the abundance of Escherichia coli (P < 0.05) in cecal chyme.

Table 5.

Effects of dietary supplementation of compound bioengineering protein (CBP) on cecal microflora (copies/mg) in broilers.

Items	Dietary CBP level, mg/kg					SEM	P value
	0	250	500	750	1,000		ANOVA	Linear	Quadratic
Total bacteria	11.08b	11.05b	11.23a	11.23a	11.11b	0.022	0.007	0.003	0.745
Escherichia coli	10.66a	10.27ab	10.24ab	9.98b	10.33ab	0.071	0.045	0.003	0.673
Lactobacillus	5.48b	5.69ab	6.22a	6.24a	5.97ab	0.075	0.006	<0.001	0.533

Data are presented as means and SEM (n = 6).

^a,bMean values with different letters were significantly different (P < 0.05).

DISCUSSION

In the present study, dietary CBP supplementation, especially 750 mg/kg, improved the growth performances of broiler chickens, which increased the BW, ADG, and ADFI during the late and whole growth periods. Similar results are observed in previous studies that levels of antibacterial peptides (cecropin antimicrobial peptides, porcine antibacterial peptide or composite antibacterial peptide containing lactoferrin antibacterial peptide, plant defensin, and active yeast) supplementation in diets or water improved the growth performance of piglets and broilers (Bao et al., 2009; Wu et al., 2012; Xiao et al., 2013). CBP contains API and APL, these 2 bioengineering proteins were origin from antimicrobial peptides Parasin I and plectasin, respectively. Previous studies show dietary API supplementation promotes intestinal health in rabbits and improves the growth performance of broilers (Peng et al., 2022; Zhang et al., 2022), and dietary supplementation 200 mg/kg recombinant plectasin increases ADG and decreases F/G by enhancing intestinal health and innate immunity of broilers (Ma et al., 2020; Zhang et al., 2021). Our previous study indicated that the API could be activated by enterokinase to release Parasin I in vitro (Zhao et al., 2015). In consequence, the improved growth performances of broilers in the present study may be attributed to the beneficial effect of Parasin I and plectasin released from corresponding fusion proteins.

The improved body antioxidant capacity contributes to the health and growth of animals. GSH-Px, T-SOD, and T-AOC are important indicators to reflect the body's antioxidant capacity of animals. GSH-Px and T-SOD can specifically inhibit the formation of hydroxyl radicals and play important roles in scavenging reactive oxygen species, thus protecting the animal body from peroxidative damage (Colakoglu et al., 2017). T-AOC reflects the overall antioxidant capacity of animals. MDA is an end product of lipid peroxidation and can serve as a marker of oxidative stress (Castillo et al., 2005). In this study, dietary CBP supplementation enhanced T-AOC in the serum and jejunum, and the activity of GSH-Px in the jejunum of broilers, suggesting that CBP enhanced the antioxidant capacity of the gut and whole body in broilers.

The intestinal tract is the most important part for the digestion and absorption of nutrients in animals, and maintaining normal intestinal morphology is crucial for broilers. The VH reflects the mitotic activity of crypt intestinal cells and the replacement rate of epithelial cells (Cera et al., 1988; Fan et al., 1997). The VH to CD ratio reflects the intestinal development and functional status. The increased VH/CD value indicates that the function of the intestinal mucosa is improved, and the ability of digestion and absorption is enhanced (Caspary, 1992). Previous studies show that antimicrobial peptides contribute to improving the intestinal morphology of broilers. Supplementation of antimicrobial peptides increased VH in the duodenum and maintained the integrity of the duodenal morphology and structure of broilers (Bao et al., 2009; Wen and He, 2012). Similarly, in the present study, dietary CBP supplementation increased VH and VH/CD in the duodenum of broilers, indicating the beneficial effects of dietary CBP on the intestinal morphology of broilers.

We further explored the mRNA expression of tight junction proteins and found that dietary CBP supplementation exhibited different regulations on these genes in different intestinal segments. CBP significantly upregulated the mRNA levels of CLDN-1 in the ileum. CLDN-1 can prevent the increase of intestinal epithelial permeability and inhibit the adhesion of bacteria to the intestinal mucosa (Weber et al., 2008). OCLN has the function of regulating macromolecular flux (Turner, 2009). Our results are consistent with the previous study in that orally administering the antimicrobial peptide buforin II upregulated the mRNA levels of CLDN-1 in the ileum in weaned piglets challenged by enterotoxigenic Escherichia coli (Tang et al., 2013). CBP downregulated the mRNA levels of CLDN-1 and OCLN in the duodenum and jejunum may be associated with better absorption and utilization of nutrients in front of the intestinal tract (duodenum and jejunum), but further study is needed. The mRNA expression of intestinal tight junction proteins in animals exhibits different responses to AMPs. Studies have shown that antimicrobial peptides WK3 have no impact on the expression of tight junction proteins in piglets (Cao et al., 2021), and antimicrobial peptide composition (containing antimicrobial peptides ABPs and plant essential oils) downregulate the mRNA levels of CLDN-3 (Xie et al., 2020). While, OCLN deficiency exhibited no impact on the intestinal barrier function of mice (Saitou et al., 2000). Therefore, those disparate results may be due to different characterizations of antimicrobial peptides, species, and the physical status of the animal. Dietary CBP supplementation also increased the mRNA levels of MUC2 in the small intestine in the current study. MUC2 is a large glycoprotein produced by intestinal goblet cells, which is the key part of the intestinal mucus layer (Hansson, 2020). MUC2 plays an important role in eliminating bacteria (Wenzel et al., 2014). MUC2 deficiency cannot prevent the invasion of pathogens, thus causing spontaneous colitis in mice (Tai et al., 2008). In the present study, dietary CBP supplementation upregulated the mRNA levels of MUC2, indicating the protective effects of CBP on the intestinal health of broilers.

Cytokines play crucial roles in inflammation and immune response. When the body suffered from foreign microorganisms or toxic substances, the immune system will immediately secrete inflammatory factors such as IL-6, IL-10, TNF-α, and IFN-γ, thus promoting immune response and restoring immune homeostasis (Brandtzaeg, 2017). Studies have shown that AMPs can enhance adaptive immunity by regulating proinflammatory and anti-inflammatory factors and inducing chemotactic activity (Wang et al., 2016). Dietary recombinant plectasin supplementation downregulates the mRNA levels of IL-6 and IFN-γ in the ileum of broilers (Zhang et al., 2021). Antibacterial peptide ABPs upregulate the mRNA levels of IL-10 in the ileum (Xie et al., 2020). In this study, dietary CBP addition upregulated the mRNA levels of IL-10 in the duodenum and ileum, and downregulated IL-6, IFN-γ in the jejunum, and TNF-α in the ileum. Based on these results, dietary CBP supplementation enhances intestinal immunity and promoted intestinal health.

Supplementation of CBP in the diets also effectively meliorated the microflora component in cecal chyme, which increased the abundance of Lactobacillus and reduced the abundance of E. coli in the cecum of broilers. The intestinal microbiota homeostasis of broilers plays an important role in nutrition, immunity, and metabolism, and healthy intestinal flora contributes more to promoting the digestion and absorption of nutrients (Cao et al., 2013). Our previous studies indicated that activated API exhibited an inhibitory impact on pathogenic microorganisms such as gram-negative bacteria in vitro, Escherichia coli (Zhao et al., 2015). Besides, previous study shows that antimicrobial peptide P5 decreases the number of E. coli in the ileum and cecum of broilers on d 21 and 35 (Choi et al., 2013). Our present results were consistent with the above studies, indicating the inhibition effect of CBP on harmful bacteria in the cecum of broilers.

In summary, dietary combination supplementation of API and APL (CBP) improves the growth performance of broilers. The improvement effects of CBP are associated with the enhanced antioxidant status of serum and jejunum, improved intestinal morphology and health, enhanced intestinal immunity, and ameliorated gut microflora. In general, 750 mg/kg CBP supplementation is more effective. Our results suggested that bioengineering protein (API and APL) could be potentially used as an alternative to antibiotics in poultry.