| Post-weaning diarrhoea,a leading cause of death in piglets,is a common problem in animal husbandry.It has become increasingly clear that nutritional factors are associated with post-weaning diarrhoea and play an important role in the gastrointestinal health of animals.Intestinal microbial metabolites from undigested dietary protein fermentation include ammonia,indole and various phenols.These metabolites can provoke post-weaning diarrhoea.A low-protein diet,as a vital nutritional strategy,may reduce protein fermentation indices(e.g.the concentration of ammonia in the intestine of piglets)and post-weaning diarrhoea.However,it has also been reported that piglets which were fed a low-protein diet had reduced growth performance compared to piglets on a high-protein diet.Thus,the concept of compensatory growth can be applied to this situation.Compensatory growth is a phenomenon in which animals exhibit a faster growth rate after their nutritional levels return to normal due to prior malnutrition or artificial restriction.Although the compensatory growth induced by protein restriction may reduce diarrhoea level without degrading the ultimate growth performance,the effects of low protein and succedent realimentation on gut microbiota in weaned piglets are still unclear.In the present study,we investigated the effects of low protein and realimentation on ileal morphology,ileal microbial composition and metabolites in weaned piglets.This paper is divided into three parts.1.Effects of low protein and succedent realimentation on the growth performance,diarrhoea index and ileal digestive and absorptive function of weaned pigletsThis part aims to investigate the effects of low protein and succedent realimentation on the growth performance,diarrhoea index and ileal digestive and absorptive function of weaned piglets.Thirty-six 28-day-old weaned piglets with an average body weight of 6.47±0.04 kg were randomly divided into a control group and a treatment group with six pens per group and three piglets housed in the same pen.The experiment was divided into two stages.During the protein restriction stage(1-14 days),piglets were fed diets containing 18.83%(normal)of crude protein in the control group and 13.05%(low)of crude protein in the treatment group.During the protein realimentation stage(15-28 days),piglets were fed diets containing 18.83%of crude protein in both groups.Six piglets from each group were euthanized for sampling on day 14 and 28.The daily feed intake of the piglets in each pen was accurately recorded;the piglets were weighed every other day,and the index of diarrhoea was calculated.Results showed that the average daily gain(ADG)of piglets in the treatment group declined significantly(P<0.01),but average daily feed intake(ADFI)and feed:gain ratio(F:G ratio)increased significantly(P<0.05)during the 14-day protein restriction.Compared to the control group,the piglets in the treatment group has increased ADG(P=0.07)and decreased F:G ratio(P<0.05)after realimentation for 14 days.The treatment group had a decreased diarrhoea index during the entire experimental period when compared to the control group(P<0.05).In addition,compared with the control group,piglets in the treatment group had higher maltase activity and expression of APN,SGLT-1 and PEPT-1 of ileal mucosa on day 14(P<0.05).The villus height and villus height:crypt depth and expression of SGLT-1 and PEPT-1 in the ileal mucosa were higher in the treatment group than those in the control group on day 28(P<0.05).Moreover,piglets in the treatment group had higher cecal mucosa thickness on day 14(P<0.01).In conclusion,low protein and realimentation could induce compensatory growth and reduce the post-weaning diarrhoea of piglets.In addition,the ileal digestive and absorptive function was improved during the protein realimentation stage.2.Effects of low protein and succedent realimentation on ileal barrier function,bacterial community and metabolites of weaned pigletsThis part aims to investigate the effects of low protein and succedent realimentation on ileal barrier function,bacterial community and metabolites of weaned piglets.The experiment design was same as that of chapter 1.Results showed that DAO activity and expression of ZO-1,MUC-2 and β-Defensin of ileal mucosa in the treatment group declined significantly on day 14(P<0.05).Immunohistochemistry analysis showed that the treatment group had an increased H-score of Claudin-1 on day 14(P<0.05)and increased H-score of Occludin on day 28(P<0.05)when compared to the control group.Pyrosequencing showed that none of bacterial diversity(Shannon and Simpson)and richness(Chao and OTU)were significantly affected by the protein restriction and succedent realimentation.PCoA analysis showed that ileal digesta samples were apparently clustered separately in the 2 groups on day 14.Compared with the control group,the treatment group had an increased abundance of Actinobacteria,Clostridiaceae 1,Halomonadaceae,Clostridium sensu stricto 1,Streptococcus,Halomonas and Pseudomonas on day 14(P<0.05)and a decreased abundance of Lactobacillaceae,Lactobacillus and Salmonella on day 14(P<0.01).The abundance of Firmicutes,Lactobacillaceae,Clostridiaceae 1,Lactobacillus and Clostridium sensu stricto 1 were significantly increased and the abundance of Proteobacteria,Pseudomonadaceae,Enterobacteriaceae,Moraxellaceae,Halomonadaceae,Salmonella,Halomonas and Pseudomonas were significantly decreased in the treatment group during the protein realimentation stage(P<0.05).Moreover,the concentrations of lactic acid,acetic acid,isovaleric acid,total SCFAs and total BCFAs in the ileal content of piglets in the treatment group significantly declined on day 14(P<0.05),and the concentrations of lactic acid,acetic acid and total SCFAs increased significantly on day 28(P<0.05)compared to the control group.Specifically,the ammonia concentration in the ileal content in the treatment group decreased on both day 14 and 28(P<0.05)compared to the control group.In conclusion,low protein may damage the barrier of ileal mucosa,but it was repaired after succedent realimentation.In addition,low protein and realimentation changed the ileal microbial composition and metabolites,reduced the abundance of pathogenic bacteria and increased the concentration of SCFAs.These changes maintained the intestinal health and reduced post-weaning diarrhoea.3.Effects of low protein and succedent realimentation on cecal barrier function,bacterial community and metabolites of weaned pigletsThis part aims to investigate the effects of low protein and succedent realimentation on cecal barrier function,bacterial community and metabolites of weaned piglets.The experiment design was same as that of chapter 1.Results showed that the treatment group had higher relative mRNA expression of cecal barrier-related genes on day 14 and 28(P<0.05),and higher protein expression level of Occludin on day 28(P<0.05)than the control group.Immunohistochemistry analysis showed that the treatment group had an increased H-score of ZO-1,Claudin-1 and Occludin on day 14(P<0.01)when compared to the control group.16S rDNA high throughput sequencing showed that the treatment group increased Shannon index,Chao and OTU,and decreased Simpson index(P<0.05)relative to the control group on day 14.Compared with the control group,the treatment group had a decreased abundance of Firmicutes,Lactobacillaceae and Lactobacillus on day 14(P<0.05)and an increased abundance of Proteobacteria,Ruminococcaceae,Lachnospiraceae,Erysipelotrichaceae,Peptostreptococcaceae,Neisseriaceae,Ruminococcus 2,Faecalibacterium,Lachnospiraceae_uncultured,Peptococcus,Terrisporobacter and Leeia on day 14(P<0.05).The abundance of Peptococcaceae and Peptococcus were significantly decreased and the abundance of Ruminococcaceae UCG-014 were significantly increased in the treatment group on day 28(P<0.05).RT-PCR analysis showed that the gene copies of Lactobacillus were significantly decreased and the gene copies of Ruminococcus were increased in the treatment group compared with the control group on day 14(P<0.05).Moreover,the concentrations of lactic acid,propionic acid,isovaleric acid and total BCFAs in the treatment group significantly decreased on day 14(P<0.05).The concentrations of acetic acid and total SCFAs increased in the treatment group relative to the control group on day 28(P<0.05).In addition,the ammonia concentration reduced in the treatment group during the entire experimental stage(P<0.01).In conclusion,low protein and realimentation improved cecal barrier function,changed the cecal microbial composition and metabolites,reduced protein fermentation and increased the concentrations of total SCFAs. |
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