| The feed cost accounts for 60%-70% of the total cost of animal breeding.The intestine tract not only works as an vital organ to digest and absorb nutrients but also serves as an important mucosal barrier.The intestinal epithelial structure is the basis of various intestinal physiological functions.Intestinal health is the basis for animal healthy development and rapid growth,and plays an vital role for obtaining reasonable profit.Early weaning stress often causes intestinal barrier dysfunction in piglets.In the past,antibiotic growth promoters were added to feed to alleviate weaning stress.However,the use of such substances has been banned considering antibiotics residues.Now,the safe,efficient and natural products are urgently needed as alternatives of antibiotics.Unlike conventional antibiotics,it is difficult for bacteria to develop resistance to these antimicrobial peptides(Amp)because they act as an important component of innate immune defenses.However,synthetic Amp are extremely complex,and the application and mechanism of many Amp are still kept unknown.These studies were conducted to investigate the effects of different antimicrobial peptides products on growth performance,intestinal gene transcription,intestinal digestion and immune function,intestinal microbiota and its metabolites in weaned piglets,reveal the molecular mechanisms of Amp effects on intestinal health of weaned piglets,and provide solutions to alleviate weaning stress and reference for development of new alternatives of antibiotics.Study 1: Regulation and mechanism of composite antimicrobial peptides derived from insects and fungi on intestinal function of weaned pigletsA total of 42 piglets(Duroc × Landrace × Large Yorkshire)of 21 days old,with average body weight of 8.01 ± 0.64 kg,were selected and randomly assigned to three groups(14 replicates per group): 1)Con group,basal diet;2)Ant group:basal diet + guitaramycin 500 mg/kg,chlortetracycline 500 mg/kg,polymyxin200 mg/kg);3)Amp group: basal diet + cecropin AD300 mg/kg,plectasin 500mg/kg).The feeding experiment lasted for 25 days.The results of the present study showed that: compared with Ant group,the villus height,villus height/crypt depth of jejunum and the maltase activity of jejunal mucosa in Amp group were significantly increased(P < 0.05),Amp obtained better improvements in the small intestine than that of antibiotic.Jejunal RNA-Seq results showed that there were 690 DEGs between Ant group and Amp group.And these gens were mainly involved in pathways related to nutrient metabolism,digestive enzyme secretion and immunity(P < 0.05).Further analysis demonstrated that compared with Ant group,the content of pro-inflammatory factor tumor necrosis factor alpha in jejunal mucosa in Amp group was significantly decreased,and the mRNA expression of tumor necrosis factor alpha and interleukin-1? in jejunum mucosa was also significantly decreased(P < 0.05).The analysis of microflora in hindgut showed that compared with Ant group,the content of total short chain fatty acids(P = 0.080)and acetic acid(P = 0.095)in cecum feces of Amp group tended to increase;at the genus level,the abundance of Prevotella?9 in Amp group was significantly increased(P < 0.05),the abundance of Succinivibrio tended to increase(P = 0.070),the abundance of Prevotellaceae?NK3B31?group and Lactobacillus were significantly decreased(P < 0.05).Spearman correlation analysis showed that the maltase content of jejunal mucosa was positively correlated with abundances of Lactobacillus and Prevotella-UCG-003 in cecum(P < 0.05);the tumor necrosis factor alpha content of jejunal mucosa was positively correlated with abundance of Prevotella?NK3B31?group,and negatively correlated with abundance of Prevotella-9(P < 0.05)in cecum.In summary,composite Amp supplementation improved the digestion and immune function of small intestine,altered the micoflora structure and increased the content of short chain fatty acids in hindgut,thus Amp improved the barrier function of whole intestine tract.Study 2: Regulation and mechanism of Natucin P derived from the intestines of semi-wild pigs on intestinal function of weaned pigletsA total of 204 piglets(Duroc × Landrace × Large Yorkshire)of 28 days old with average body weight of 7.97 ± 1.04 kg,were selected and randomly assigned to four groups(three replicates per group,17 piglets per replicate): 1)Con group:basal diet;2)Ant group: basal diet + chlortetracycline(d 1-24 added 1000 mg/kg,d 25-37 added 500 mg/kg);3)Npl group: basal diet + low dosage Natucin P(d1-14 added 400 mg/kg,d15-37 added 300 mg/kg);4)Nph group: high dosage Natucin P(d1-14 added 600 mg/kg,d15-37 added 500 mg/kg).The feeding experiment lasted for 37 days.The results showed that: compared with Ant group,the average feed intake in Npl group during d1-24 tended to increase(P = 0.075);the jejunal crypt depth(P = 0.0001)in Npl group was significantly decreased,the ratio of villus height/crypt depth in the jejunum and ileum were significantly increased(P < 0.05);the number of Ki-67 positive cells in the jejunum of Npl group tended to increase(P = 0.070).RNA-Seq results showed that there were867 DEGs between Npl group and Ant group,their gens terms were mainly involved in pathways of nutrient metabolism,and barrier function(P < 0.05).Further analysis demonstrated that the values of maltase,lactase,sucrase,intestinal alkaline phosphatase and SIgA in the jejunal mucosa of the Npl group were the highest,and were significantly higher than that in Con group and Nph group(P < 0.05).In terms of the serum intestinal barrier function markers,compared with Con group,the concentration of D-lactate in Npl group was significantly decreased(P < 0.05),and the concentration of diamine oxidase(P =0.074)and endotoxin(P = 0.068)also had a decreasing trend,indicating that the intestinal barrier function of Npl group was significantly improved.Natucin P elicited similar improvements as an antibiotic.16 S rDNA sequencing results showed that: compared with Ant group,the alpha diversity index was significantly increased in Npl group,including ACE,chao1,observed species(P< 0.05);and beta diversity index was also tended to increase in Npl group(P =0.054).There were significant differences in the composition of microbial flora among the groups at the species level: Nph group had higher abundance of beneficial Alloprevotella?sp?feline?oral?taxon?309,and lower abundance of Clostridium?disporicum,one kind of conditional pathogenic bacteria than those in Ant group(P < 0.05).Compared with Npl group,the abundance of Clostridium?disporicumwas significantly decreased in Nph group(P < 0.05).Therefore,it is recommended to use high dose Natucin P in pig farms with poor feeding environment.Spearman correlation analysis showed that the abundance of Lactobacillus reuteri in colon was positively correlated with the contents of maltase,lactase,sucrase and SIgA in jejunual muscoa(P < 0.05).Therefore,Natucin P regulated the digestion and immune function in jejunum mucosa by shaping intestinal microbiota,thus improving the barrier function of whole intestinal tract.In conclusion,dietary antimicrobial peptides improved the digestion and immune function of the small intestine by increased the intestinal morphology and the content of jejunal digestive enzymes,and inhibited the secretion and expression of inflammatory cytokines,promoted the secretion of SIgA and the proliferation of crypt cells.The action mechanism was that Amp altered the gene transcription related to nutritional digestion and absorption and immune function.Moreover,the supplementation of antimicrobial peptides affected the diversity and composition of the hindgut microbiota and increased the concentration of short chain fatty acids and affected the secretion of intestinal digestive enzymes,inflammatory factors and SIgA by shaping the intestinal microbiota,thereby antimicrobial peptides improving the intestinal barrier function of the whole intestinal tract.These studies provided an important reference to looking for new antibiotic alternatives and nutritional regulation of piglet intestinal health. |
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