Effects Of Api-PR19 On Growth Performance And Intestine Health Of Broiler Chickens

In modern poultry production,numerous bacterial diseases are associated with gram-negative bacteria,and representative of them have Salmonella and E.coli.Antibiotic growth promoters?AGPs?plays an important role in reducing the risk of bacterial diseases and promoting the growth of animals,but long-term use of AGPs will cause bacterial resistance,veterinary drug residue and other hazards.With the clarity of the policy that AGPs was withdrawn from the feed by 2020,it's a priority to search for the new safe substitutes of AGPs.Antimicrobial peptides?AMP?are one of the candidates for AGPs substitutes because of the rapid bacteriostasis with multiple targets,broad-spectrum antibacterial activity and difficult to form drug resistance.In this study Apidaecin-derived peptide?Api-PR19?build by genetic engineering was selected to be an alternative to AGPs.Firstly,the antibacterial effect and stress resistance of Api-PR19 was detected in vivo;Secondly,we supplemented Api-PR19 into diet and detected the effects of Api-PR19 for growth performance and intestinal health of broilers,expected to provide a theoretical basis for the application of Api-PR19 in broiler breeding industry.Exp.1 Study on antimicrobial activity and stress resistance of Api-PR19 in vitroThe minimum inhibitory concentration?MIC?and antibacterial effect of Api-PR19 were tested by Escherichia coli ATCC25922,Salmonella typhi ATCC14028,Pasteurella ATCC19427,Helicobacter pylori ATCC43504 and Staphylicoccus aureus ATCC25923.We used Escherichia coli as the indicator,and explored the stress resistance of Api-PR19processed by different temperature,pH and protease.The results showed that:1)the MIC value of Api-PR19 against Escherichia coli ATCC25922,Salmonella typhi ATCC14028,Pasteurella ATCC19427,Helicobacter pylori ATCC43504 and Staphylicoccus aureus ATCC25923 were represented respectively 0.2?0.4?0.1?0.8 and 1.6?g/mL;2)Api-PR19reached the maximum bactericidal efficacy at 4 hour period,and compared with the 1 MIC,the 10 MIC showed no significant increase in bactericidal efficacy;3)With the increase of temperature,the bactericidal activity of Api-PR19 showed a tendency to reduce gradually,and Api-PR19 treated at 80?decreased to about 70%of the primary antimicrobial activity;With the decrease of pH,the germicidal activity of Api-RP19 decreased gradually,and when pH=2,it reduced to about 72%of the pimary antimicrobial activity.Pepsin?3000-3500NFU?,Trypsin?250 NFU?and?-chymotrypsin?1200 U?reduced the antibacterial activity of Api-PR19 by about 52%,63%and 82%,respectively.The results showed in vitro Api-PR19had better antibacterial effect on Escherichia coli,salmonella typhi,Pasteurella and Helicobacter pylori,and had certain antibacterial effect on Staphylicoccus aureus.Api-PR19has the strongest bactericidal effect at 4-hour period.With the increase of concentration of Api-PR19,the bactericidal effect did not increase obviously.Api-PR19 are resistant to high temperature and strong acid,and has good resistance to?-chymotrypsin and trypsin,but poor resistance to pepsin.Exp.2 The effect of Api-PR19 on growth performance of broiler chickensA total of 630 one-day-old male chicks?AA?with similar body weight from a commercial hatchery were randomly divided into 5 groups consisting of 7 replicates with 18birds per replicate.Birds of each group received one of the following dietary treatments:1)control group?CON?fed basal diet;2)AGPs group?AGPs?fed basal diet supplemented with4.96 mg enramycin per kg diet;3)Low-level group?AP1?fed basal diet contaminated with100 mg Api-PR19 per kg diet;4)Middle-level group?AP2?fed basal diet contaminated with200 mg Api-PR19 per kg diet;5)High-level group?AP3?fed basal diet contaminated with300 mg Api-PR19 per kg diet.The results showed that:1)Adding Api-PR19 to the diet significantly reduced feed conversion rate?FCR?in the pre-feeding period?1-21 days?and the whole period of feeding?P<0.05?,and has a tendency to improve the late feeding stage?P<0.10?,and AP2 group had the lowest FCR;2)Adding Api-PR19 to feed had no significant effect on broiler index,spleen index and bursal index of 42 days broilers,but the bursal index of 21 days broilers in AP1 and AP2 groups was significantly lower?P<0.05?;3)Dietary supplementation with Api-PR19 had no significant effect on H₅ antibody levels in influenza subtypes at all stages of broilers,and subtype H₉ antibody levels at 14,21 and 28days did not significantly improve?P>0.05?,but Api-PR19 significantly increased the H₉antibody level at 7 day broiler subfamily;4)It was found that the optimal addition amount of Api-PR19 in the broiler diet was more accurate?related The coefficient is the highest,R²=0.496?,and the recommended value is 200.481 mg/kg.This experiment showed that the addition of Api-PR19 to the diet can improve feed conversion efficiency and improve broiler performance.It could be Api-PR19 improves the intestinal health of broilers;The results of bursal index and antibody titer reveal that Api-PR19 may play a regulatory role in the construction and improvement of early immune defense lines in broilers.Exp.3 The effect of Api-PR19 on intestine health of broiler chickensIn this experiment,duodenal,jejunal and ileal mucosal samples of broilers were collected at 21 and 42 days,as well as cecal chyme of 21 and 42 days,and fixed with paraformaldehyde at 21 and 42 days of age.The fixed intestines were sectioned and stained with hematoxylin-eosin?HE?to detect intestinal morphology.Mucosal samples were assayed for mRNA expression levels of gut glucose transporters and amino acid transporters by RT-qPCR.The expression of sIgA in each intestinal segment was detected by immunohistochemistry.The total number of cecal bacteria and the number of specific microorganisms are detected by absolute quantification of microorganisms.The results showed that:1)Api-PR19 had a significant improvement on the morphology of the ileum;it improved the shape of the duodenum in the broiler at the later feeding period;2)the Api-PR19 significantly up-regulated the relative expression of glucagon passive transporter?GLUT2?and amino acid transporter?rBAT,y+LAT2,CAT1?genes at the 21 day of age?P>0.05?,but had no significant effect on the expression of nutrient transporter genes at the later feeding period;3)The Api-PR19 improved the secretion of sIgA in jejunum and the pre-feeding broiler duodenal sIgA?P>0.05?;4)Api-PR19 significantly reduced the number of cecal Escherichia coli and campylobacter jejuni at the later feeding period?P>0.05?,and maintained the abundance of cecal microbes to some extent.The results of this experiment showed that Api-PR19 can improve the intestinal villus tissue morphology,increase the absorption of nutrients in the small intestine,enhance intestinal immunity,reduce the number of harmful microorganisms in the cecum,maintain intestinal micro-ecological balance,and comprehensively improve intestinal health,and thereby improve feed conversion efficiency and thus,enhanced broiler performance.In all,Api-PR19 has the superior of antimicrobial activity and stress resistance in vivo.Api-PR19 can improve the intestinal villus tissue morphology,increase the absorption of nutrients in the small intestine,reduce the number of harmful microorganisms in the cecum,maintain intestinal micro-ecological balance,and comprehensively improve intestinal health,and thereby improve feed conversion efficiency and broiler performance.