| In China,the development of swine industry is seriously restricted by the excess emission of nitrogen and the shortage of feed protein resources.The use of low-protein diets is an effective solution.Furthermore,previous studies showed that a moderate reduction(?4%reduction vs NRC)of dietary crude protein(CP)level,supplemented with essential amino acids(AAs),can improve the intestinal micro-ecology and barrier function without damaging the growth performance of pigs.However,greatly reducing the dietary CP level(>4%reduction),even supplemented with essential and nonessential AAs,detrimentally affects the growth performance,metabolism and intestinal health of pigs,which may be due to the excess lack of protein-derived peptides,as the role of protein is not only to provide AAs(the theory of ideal protein has been questioned),peptides released from the protein can play some other bioactive roles.Therefore,we hypothesize that protein-derived peptides can improve the metabolism and intestinal environment under extremely low CP(LP)level.To test this hypothesis,using the casein hydrolysate as the peptide source,the first animal feeding test(first three chapter)was carried out:twenty-one pigs(initial BW 19.90 ± 1.00 kg,63±1 days of age),housed individually,were assigned to 3 groups and fed with control diet(16%CP,CON),LP diets(13%CP)supplemented with free AAs(LPA)or casein hydrolysate(LPC)for 28 days.For the growing pigs,16%CP level in control diet was a moderately LP level for 20-50 kg pigs according to the NRC(2012)(the recommended CP level is 18%);13%CP level in both LPA and LPC diets was extremely LP level.The aim of this animal feeding test was to evaluate the effect of supplementation of casein hydrolysate in extremely LP diets,in comparison with AAs supplementation,on metabolism and intestinal health in pigs.The specific research contents and results are as follows:1 Effects of low-protein diets supplemented with AAs or casein hydrolysate on metabolism in pigsThrough the animal test,we found that LPC diet increased the average daily gain(ADG)of pigs as compared with CON and LPA diets.Metabolism is closely related to growth performance.Therefore,in this chapter,we firstly investigated the effects of two LP diets on metabolism.Pigs were slaughtered on day 29.Serum samples were collected before the slaughter;and liver and back fat were collected after the slaughter.Results of blood metabolic network analysis showed that ketone body metabolic flux,representing the lipid catabolism,was decreased(P<0.05)in LPC group as compared with CON and LPA groups;and the pentose phosphate pathway(PPP)metabolic flux,representing anabolism,was increased(P<0.05)in LPC group as compared with LPA group.Compared with CON and LPA diets,LPC diet increased(P<0.05)the content of glucose-6-phosphate dehydrogenase(G6PDH),and decreased(P<0.05)the content of pyruvate dehydrogenase complex(PDHC)in serum.In the liver and back fat,compared with CON group,the gene expression of carnitine palmitoyl transferase-1?(CPT-1?)was up-regulated(P<0.05)in LPA group,but it was down-regulated(P<0.05)in LPC group.Moreover,the gene expression of hormone sensitive lipase(HSL)in the back fat was down-regulated(P<0.05)in LPC group as compared with CON and LPA groups.Collectively,compared with LPA diet,LPC diet decreased lipid catabolism,which may promote the growth performance of pigs.2 Effects of low-protein diets supplemented with AAs or casein hydrolysate on the small intestinal barrier function,bacterial community and mucosal immunity in pigsWe found that LPC diet can increase the growth performance.Therefore,whether the LPC diet can also positively affect the intestinal health?In this chapter,we aim to investigate the effects of LPC diet,in comparison with LPA diet,on the small intestinal barrier function,bacterial community and immunity in pigs.Small intestinal tissue and digesta were collected for measuring the barrier and immune factors and the bacterial community,respectively.Results showed that compared with CON diet,LPA diet decreased(P<0.05)the gene expression level of zonula occludens-1(ZO-1)and claudin-1 and the protein expression level of intestinal stem-cell proliferation factor Leucine-rich repeat-containing G-protein-coupled receptor-5(Lgr-5)in the jejunum;whereas LPC diet increased(P<0.05)the gene expression level of occludin and ZO-1 and the protein expression level of Lgr-5 in the jejunum,and the protein expression level of mucin-2(MUC-2)in the ileum.LPA diet reduced(P<0.05)Lactobacillus counts,whereas LPC diet increased(P<0.05)Lactobacillus counts and reduced(P<0.05)Escherichia coli counts in the ileum.LPA diet increased protein expression levels of pro-inflammatory cytokine IL-6 and IL-22(P<0.05),whereas LPC diet decreased protein expression levels of pro-inflammatory IL-1?,IL-17A and TNF-?(P<0.05)in the ileum.Collectively,compared with LPA diet,LPC diet showed a beneficial effect on the small intestinal health in pigs.3 Effects of low-protein diets supplemented with AAs or casein hydrolysate on the colonic microbiota and mucosal immunity in pigsIn the previous chapter,we found that LPC diet positively affected the small intestinal health in pigs.Relative to the small intestine,the colon has the higher activity of protein fermentation by microbes.The colonic mucosa represents the main interacting surface of the gut microbiota and the immune system.Therefore,in this chapter,we aim to study the effect of LPC diet,in comparison with LPA diet,on the colonic microbiota and mucosal immunity in pig.The colonic digesta were collected and used for the bacterial high-through sequencing and the detection of microbial metabolites;colonic tissues were collected and used for the detection of intestinal barrier and immune factors.Results showed that compared with CON diet,LPA and LPC diets decreased(P<0.05)the abundance of Streptococcus and Escherichia coli,and LPC diet further decreased the abundance of Proteobacteria and Escherichia.Further,LPC diet increased(P<0.05)the abundance of Lactobacillus reuteri.LPA and LPC diets decreased(P<0.05)concentrations of ammonia and cadaverine,and LPC diet further decreased(P<0.05)concentrations of putrescine and phenol.Moreover,LPC diet increased(P<0.05)total short-chain fatty acid(SCFA)concentration.Compared with CON diet,LPA and LPC diets decreased(P<0.05)the protein expression levels of toll-like receptor-4(TLR4),nuclear factor-?B(NF-?B),IL-1?and TNF-?,and LPC diet further decreased(P<0.05)the protein expression level of IFN-?.Moreover,LPC diet increased(P<0.05)the protein expression levels of G-protein coupled receptor-43(GPR43),IL-4,transforming growth factor-?(TGF-?)and immunoglobulin A(IgA).Collectively,compared with LPA diet,LPC diet showed a beneficial effect on the colonic health in pigs.Combining the results of the first animal feeding test,we found that the supplementation of casein hydrolysate to the extremely LP diets improved growth performance,metabolism and intestinal health in growing pigs.However,others have reported that compared with CP level 17%and 15%diets,CP level 13%diet supplemented with intact casein detrimentally affected the growth performance,metabolism,immunity and intestinal health in growing pigs.In order to compare the different effects of the supplementation of intact casein and casein hydrolysate to extremely LP diets on the physiological functions of pigs and understand the mechanics behind these,we conducted the second animal feeding test:sixteen pigs(initial BW 19.09±0.61 kg,63±2 days of age),housed individually,were randomly assigned to two groups and fed with CP level 13%diet supplemented with intact casein(LIC)or casein hydrolysate(LCH)for 28 days.The research contents and results are as follows:4 The differences in growth performance,metabolism and immunity in pigs fed low-protein diets supplemented with intact casein or casein hydrolysateIn this chapter,we compared the differences in growth performance,metabolism and immunity in pigs fed LIC and LCH diets.Pigs were weighed at the beginning and end of the experiment for calculating the ADG.Pigs were slaughtered on day 29.Serum samples,collected before the slaughter,were used for measuring the metabolic and immune factors;the liver tissues,collected after the slaughter,were used for measuring the liver metabolic factors.Results showed that LCH diet increased(P<0.05)ADG as compared with LIC diet.Blood metabolic network analysis showed that lactate metabolic flux,representing the catabolism,was decreased(P<0.05),but PPP metabolic flux,representing the anabolism,was increased(P<0.05)by LCH diet relative to LIC diet.Compared with LIC diet,LCH diet decreased(P<0.05)the content of pyruvate kinase(PK),and increased(P<0.05)the content of G6PDH in serum.Liver metabolomics analysis showed that metabolites differences between two groups were mainly concentrated in carbohydrate metabolism,with the content of glucose,galactose and xylitol was increased and of pyruvate was decreased in LCH group as compared with LIC group.Moreover,compared with LIC group,the liver gene expressions of PK and citrate synthase(CS),catalyzing glycolysis pathway,were down-regulated(P<0.05),and gene expressions of phosphoenol pyruvate carboxykinase(PEPCK)and pyruvic carboxylase(PC),catalyzing gluconeogenesis,were up-regulated(P<0.05)in LCH group.For the immune response,compared with LIC diet,LCH diet decreased(P<0.05)the concentrations of IgE and pro-inflammatory IL-6,IL-22 and IL-17A in serum.Collectively,compared with LIC diet,LCH diet increased systemic anabolism,decrease catabolism,and decrease systemic immune response in pigs.5 The differences in intestinal digestion and absorption and barrier development in pigs fed low-protein diets supplemented with intact casein or casein hydrolysateIn the previous chapter,we found that LIC and LCH diets differently affected glycometabolism and immunity in pigs.Intestinal carbohydrate digestion and absorption and barrier function development respectively directly affect the animal glycometabolism and immune response.Therefore,in this chapter,we aim to compare the difference in intestinal carbohydrate digestion and absorption and barrier function development in pigs fed LIC and LCH diets.The intestinal tissues were collected for measuring the carbohydrate digestion and absorption factors and the barrier function factors.Ileal digesta were collected for the metabonomic analysis.Results showed that compared with LIC diet,LCH diet up-regulated(P<0.05)the gene expressions of sucrase,maltose and sodium-glucose transport proteins 1(SGLT-1)in the ileum.Metabolomic analysis showed that the differences of metabolites in the ileal digesta were mainly concentrated in carbohydrate,with the contents of glucose,sucrose and maltose were increased(P<0.05)in LCH group as compared with LIC group.Compared with LIC diet,LCH diet up-regulated(P<0.05)the gene expressions of jejunal occludin and ZO-1,ileal MUC-2 and colonic MUC-2 and MUC-4.Collectively,compared with LIC diet,LCH diet promoted the intestinal carbohydrate digestion and absorption and the intestinal barrier function development in pigs.These changes in intestinal function may mediate the differential effects of LIC and LCH diets on metabolism and immunity.6 The differences in gut microbiota in pigs fed low-protein diets supplemented with intact casein or casein hydrolysateThe important role of intestinal microbes in regulating the animal growth and health has been widely recognized.In this chapter,we aim to compare the difference in gut microbiota in pigs fed LIC and LCH diets,and to explore the mechanism of the effect of these two diets on host physiological function at the microbial level.Total bacterial genomic DNA isolated form the intestinal(duodenal,jejunal,ileal and colonic)digesta was used for 16S rRNA sequencing.Bioinformatics analysis indicated that there was a higher bacterial diversity in the ileum and colon in LCH group than that in LIC group.The difference of microbial composition between two groups mainly occurred in the jejunum and ileum,characterized by that the higher abundance of Firmicutes and Actinobacteria and the low abundance of Proteobacteria in the jejunum,and the higher abundance of Bacteroidetes in the ileum were found in LCH group compared with LIC group.Interestingly,Lactobacillus were enriched throughout the entire intestine in LCH group.Functional prediction by PICRUSt suggested that metabolic pathways associated with carbohydrates and AAs were enriched and reduced,respectively,in LCH group.Collectively,compared with LIC diet,LCH diet positively regulated the microbial diversity and composition.Jejunum and ileum are the key response sites,and Lactobacillus are the key taxa.These changes in microbial composition and metabolic function may mediate the differential effects of LIC and LCH diets on intestinal carbohydrate digestion and absorption and barrier function development.Taken together,these findings suggest that a certain quantity of protein-derived peptides rather than only AAs or intact protein are required to maintain or improve growth performance,metabolism,immunity and intestinal health of growing pigs fed with extremely LP diets. |
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