Effects of Dietary Supplementation of Antimicrobial Peptide on Growth Performance, Serum Biochemistry and Intestinal Health in Broilers

Antibiotics have been widely used for the growth promotion and prevention of diseases in livestock and poultry. However, the misuse of antibiotics disrupts the micro-ecological balance of the intestine and also leads to bacterial antibiotic resistance and antibiotic residues in meat and eggs (Marazuela and Bogialli, 2009). The emergence and rapid spread of antibiotic-resistant microbes poses risk for human health. Hence, there is an urgent need to search for novel antibiotic substitutes. Probiotics, prebiotics, organic acids, phytobiotics, enzymes and antimicrobial peptides (AMPs) are a few potential alternatives.
               
AMPs are small biological molecules (<10 kDa) containing less than 100 amino acids, found in all species, ranging from plants and insects to animals and having broad-spectrum antimicrobial activity against bacteria, some viruses and fungi (Meyerholz and Ackermann, 2005). Cathelicidins and β-defensins are the two major families of AMPs in avian species. Defensins are cysteine-rich peptides that play an important role in the innate immune system and are capable of killing a broad spectrum of pathogens. About 14 types of β-defensins have been discovered in chicken from different tissues such as bone marrow, tongue, trachea, bursa of Fabricius, brain, kidney, testicle, ovary, male and female reproductive tract, liver and the urogenital tract, gastrointestinal tract and lung so far (Lynn et al., 2004). Among these, the expression of avian β-defensin 9 is weak, 2 is moderate and 13 is strong in the small intestine of birds. Natural compounds such as AMPs are attractive possibilities to replace antibiotics because of their natural antimicrobial, immunomodulatory properties and a low tendency for the development of bacterial resistance. Consequently, the present study was undertaken to evaluate the effect of chicken intestinal AMPs on growth performance and intestinal health in broiler chicks.

Chicken intestinal AMP
 
The AMPs were extracted from chicken small intestines, as described by Ma et al., (2004). Size exclusion column chromatography was used to purify the AMP rich supernatant (Bio Rad, USA). The SDS-PAGE analysis of active fraction with potential antimicrobial activity revealed band size with a molecular mass of less than 5 kDa.  The fraction was then outsourced to the Sophisticated Analytical Instruments Facility, Indian Institute of Technology, Madras for confirmation of molecular mass by LC-MS (Ashraf and Azad, 2017). The antimicrobial peptide had a molecular mass of 3.76 kDa, which closely corresponded to the molecular weight of avian β-defensin 2 (3.9 kDa) with the NCBI Accession Number: AAB30585. The peptide-rich fractions were lyophilized and kept at 0°C until further use.
 
Animals, diet and experimental design
 
A total of 90 one-day-old healthy broiler chicks (Cobb 400) were randomly allotted to three dietary treatments based on body weight. There were 3 replicates in each treatment with 10 birds per replicate. The dietary treatments included, negative control (NC; basal diet), positive control (PC; basal diet supplemented with 335 mg chlortetracycline/kg) and antimicrobial peptide groups (AMP; basal diet supplemented with 100 mg AMP/kg). The experimental diets were formulated based on three phases: pre-starter (day 1 to 7), starter (day 8 to 21) and finisher (day 22 to 35). Birds had adlibitum access to feed and water during the trial. The standards of care used in the study were approved by Institutional Animal Ethics Committee (Approval Lr. No. 370/DFBS/IAEC/2021). All the diets were formulated to meet BIS (2007) broiler nutrient recommendations. The feed ingredients and nutrient levels of the basal diet in three phases were presented in Table 1.
 

 
Sample preparation and measurements
 
Body weights of individual birds were measured on days 1, 7, 14, 21, 28 and 35 and the weight gain was calculated from that data. The FCR was calculated by dividing the respective feed intake of the chicks by weight gain during the respective period. On day 35th the intestinal tissues were collected from the midpoint of the duodenum, jejunum and ileum for the assessment of intestinal morphology and a part of intestinal tissue was used for measurement of antioxidant activity. And remaining intestinal tissue was immediately processed for RNA isolation.
 
Serum biochemical indicators
 
The serum samples collected on day 21^st were analyzed for Alanine transaminase (ALT), Albumin, Aspartate aminotransferase (AST), Glucose, Total protein, Uric acid and Alkaline Phosphatase (ALP) using A15 Biosystem auto analyzer.
 
Intestinal morphology
 
The intestinal tissues were processed and stained with hematoxylin and eosin (Uni et al., 1998) and histological indices measured by an image analyzer software (Alpha imager hp version 5.0) to assess the intestinal length to crypt depth (V/C) ratio.
 
Intestinal antioxidant capacity
 
Intestinal tissue was crushed and 10% (w/v) homogenate was prepared with 0.05M PBS (pH 7.4). The homogenate was centrifuged and used for the estimation of malondialdehyde (MDA), superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC). Malondialdehyde was determined by the thiobarbituric acid reactive substances method as described by Ohkawa et al., (1979). The GSH-Px activity was determined by the method of Rotruck et al., (1973). SOD activity was determined by the method described by Marklund and Marklund (1974). Catalase activity was assayed according to the method of Caliborne and Greenwald (1985). The DPPH radical scavenging assay was performed to determine the T-AOC as described by Xing et al., (2015). 
Gene expression analysis of cytokines and tight junction proteins
 
A piece (25 mg) of the intestine was ground and treated with 750 µl Trizol Reagent (Sigma) to extract the RNA as described by Kaiser et al., (2006). The High-Capacity cDNA Reverse Transcription Kit (Takara Kit) was used for cDNA synthesis as per the manufacturer’s instructions. The expression of cytokine and tight junction protein genes (IL-17A, IFN-α, IFN-γ, TGF-β, IL-10, MUC-2, ZO-1 and Claudin-3) was measured using real-time PCR with the 16s rRNA gene as the housekeeping gene. Primers used for real-time PCR were given in Table 2. The data were analyzed by the 2^-ΔΔCt method and normalized using the expression level of the housekeeping gene. The thermal cyclic conditions used for Real-Time PCR were as follows: 95°C for 3 minutes, 40 cycles of 95°C for 30 seconds, 60°C for 15 seconds and 72°C for 35 seconds.  
 

                                                 
Statistical analysis
 
Data were analyzed by one-way ANOVA using SPSS v.20.0 statistical package. When dietary treatment was significant (P<0.05), means were compared using the Duncan test. The results were expressed as mean±standard deviation.

The effect of supplementation of AMP on growth performance is presented in Table 3. A significantly (P<0.05) higher body weight gain was observed in the AMP supplemented group during finisher and overall periods when compared to NC. FCR did not significantly (P>0.05) vary among the treatments during pre-starter, starter and overall period. These results were in agreement with Bao et al., (2009) who reported greater body weight gain in broilers supplemented with swine gut antimicrobial peptides. The improvement in weight gain may be because of an increase in nutrient absorption and utilization as it has been reported that AMP could improve the apparent digestibility of dry matter, crude protein, crude fat and gross energy in broilers (Wen and He, 2012). In contrast to the present findings, no effect of supplementation of AMP on growth performance (Shan et al., 2007), while significantly lower FCR was reported by Bai et al., (2019). This inconsistency in results might be due to variation in the types of AMPs supplemented and the form of feed (Landy et al., 2020). The present study revealed no difference in the overall weight gain and FCR between PC and AMP groups which makes it evident that AMP could safely replace antibiotic growth promoters in the broiler diet.
 

       
AMP revealed no significant (P>0.05) difference in serum biochemical indicators (glucose, total protein, albumin, ALT, AST, ALP and uric acid) on day 21 is presented in Fig 1. These findings were in agreement with Xie et al., (2020) who reported that supplementation of AMPs (100mg plectasin and cecropin/kg) with and without cinnamaldehyde had no effect on serum ALT, AST, albumin, total protein, glucose and uric acid.
       

 
AMP revealed no significant (P>0.05) difference in the villus length of the duodenum of 21 days old broiler chicken is presented in Fig 2. However, there was a significant (P<0.05) increase in the villus length in the AMP group compared to NC in jejunum and ileum. A significant increase (P<0.05) in villus length to crypt depth ratio was noticed in AMP group compared to the NC in the ileum. In line with the present study, Bao et al., (2009) reported a significant increase in jejunal villus length of AMP supplemented birds. Increased villus length suggests an increased surface area for greater absorption of the available nutrients (Caspary, 1992).
       

 
The dietary supplementation of AMP significantly (P<0.01) improved the glutathione peroxidase activity when compared to PC and NC. The SOD activity was significantly (P<0.05) higher in the AMP group compared to the NC. There was no significant (P>0.05) difference in the total antioxidant capacity, malondialdehyde and catalase activity among all the treatment groups (Fig 3). These findings were in agreement with Xie et al., (2020) who reported that supplementation of AMPs (100 mg plectasin and cecropin/kg) with and without cinnamaldehyde had no effect on malondialdehyde, catalase and total antioxidant capacity.
 

               
AMP significantly (P<0.01) lowered the gene expression of pro-inflammatory cytokines viz., IL-17 A, IFN-α, IFN-γ, anti-inflammatory cytokine IL-10 and tight junction proteins viz., ZO-1 and Claudin-3 compared to the NC group (Fig 4). The expression of interferons and the extent of inflammation in the body have a positive correlation. As a result, there is reduced intestinal inflammation and the function of digestion and absorption of nutrients is retained. The above findings were in agreement with Xie et al., (2020) who reported a reduction in the expression of IL-17 A, IFN-α and Zhang et al., (2021) who reported reduced expression of IL-17A, IFN-α and IFN-γ in the intestine on AMP supplementation. AMP significantly increased the expression of anti-inflammatory cytokine TGF-γ and intestinal tight junction protein MUC-2. TGF-γ can increase tight junction protein expression in intestinal epithelial cells, maintain transmembrane potential balance and strengthen the intestinal mucosal barrier (Howe et al., 2005). The upregulation of MUC-2 gene by AMP indicated that MUC-2 provided a protective barrier between the epithelial surfaces and the gut lumen (Allen et al., 1998).

Supplementation of AMP improved the weight gain of broiler chicks by 7.32%. There was a 5.81% rise in FCR in the AMP group, which was however not statistically significant. AMP supplementation increased the villus length of jejunum, ileum and V/C ratio of ileum and intestinal Superoxide dismutase and Glutathione peroxidase in the AMP group. AMP supplementation modulates the expression of cytokines and intestinal tight protein genes. Hence, this study concludes that antimicrobial peptide improved the growth performance and intestinal health in broilers and has the potential for application in broiler diets as an alternative to antibiotic growth promoters.

The authors thank Tamil Nadu Veterinary and Animal Sciences University for providing the necessary facilities to carry out the research work.

No potential conflict of interest was reported by the authors.