Effect And Mechanism Of Maternal Dietary Crude Protein Level On Jejunum Development In Huzhu Bamei Suckling Piglets

The growth and development of gastrointestinal tract in suckling piglet directly affect the survival rate of piglets,while piglets survival rate is one of the main factors on pig production efficiency.The purpose of this study was to investigate the effect of maternal dietary crude protein(CP)level on growth performance,jejunum development of Huzhu Bamei suckling piglets,and the interaction between jejunum microbiota as well as metabolites of piglets under maternal nutrition.A total of 60 healthy and multiparous(4～5 fetuses)purebred Huzhu Bamei sows with similar body weight(88.33±2.26 kg)were randomly divided into 3 groups: normal protein group(NP,14% CP),low protein group(LP,12% CP)and very low protein group(VLP,10%CP).Each group had 5 replicates and each replicate had 4 sows.The piglets from sows in each treatments were then used for the further experiment.The piglets were weighed at birth,7,14 and 21 days of age.The average daily gain(ADG)was calculated and the daily diarrhea of piglets was recorded.On the 21 st day after birth,9 piglets in each group were randomly selected for slaughter and sampling.16 S r RNA gene sequencing,metabonomics,q PCR,molecular biology and bioinformatics technique were used to explore the effect and mechanism of maternal dietary CP level on jejunum development of suckling piglets,and perform correlation analysis on microbiome and metabolome data.The main results were presented as follows:1.Reducing maternal dietary CP level(from 14% to 12% or to 10%)had no significant effect on body weight of piglets(p>0.05),but the diarrhea rate of suckling piglets decreased significantly(p<0.05).Jejunal weight and villus height in LP group were significantly higher than NP and VLP group(p<0.05),and there was no significant difference between VLP and NP group(p>0.05).Villus width and muscle thickness in LP group were significantly higher than NP and VLP group(p<0.05).The crypt depth of VLP and NP group was significantly deeper than LP group(p<0.05),and there was no significant difference between VLP and NP group(p>0.05).The ratio of villus height to crypt depth(V/C)and goblet cells in NP and LP group were significantly higher than VLP group(p<0.05),and there was no significant difference between LP and NP group(p>0.05).Trypsin activity and Ig G concentration in NP and LP group were significantly higher than VLP group(p<0.05),and there was no significant difference between LP and NP group(p>0.05).Lipase activity in LP group was significantly higher than NP and VLP group(p<0.05),and NP group was significantly higher than VLP group(p<0.05).Lactase activity in LP group was significantly lower than VLP group(p<0.05),but there was no significant difference among other groups(p>0.05).The relative expression of occludin m RNA in LP group was significantly higher than NP and VLP group(p<0.05),and there was no significant difference between VLP and NP group(p>0.05).The relative m RNA expressions of TLR4 and TNF-α in NP group were significantly higher than LP and VLP group(p<0.05).The relative expression of TGF-β and IL-10 m RNA in LP group was significantly higher than NP group(p<0.05),and there was no significant difference between VLP and NP group(p>0.05).The results showed that moderate reduction of maternal dietary CP level(12%)had no effect on growth of suckling piglets,but could reduce diarrhea rate,improve the jejunum tissue morphology and mucosal barrier function of suckling piglets.2.Based on the result of high-throughput sequencing of 16 S r RNA gene,Firmicutes and Proteobacteria were the two dominant bacteria in jejunum of 21 days old piglets.OTUs,ACE and Chao1 index in VLP group were significantly higher than NP groups(p<0.05),and there was no significant difference between LP and NP group(p>0.05).Compared with NP group,Acidaminococcus,Megasphaera and Romboutsia in Firmicutes,Sutterella in Proteobacteria and Prevotella＿7 in Bacteroidetes were significantly increased in LP group(q<0.05),Buchnera in Proteobacteria was significantly increased in VLP group(q<0.05),and Enterobacter in Proteobacteria and Prevotella-1 in Bacteroidetes were significantly decreased in VLP group(q<0.05).KEGG pathway analysis showed that compared with NP group,Aminoacyl t RNA biosynthesis pathway was significantly down-regulated in LP and VLP groups(q<0.05).Phosphotransferase system was significantly up-regulated in LP group and downregulated in VLP group(q<0.05).3.Using liquid chromatography-mass spectrometry(UHPLC-Q-TOF-MS/MS)technology,according to the orthogonal partial least squares discrimination analysis(OPLS-DA),the variable importance for the projection(VIP)of metabolites was calculated.With VIP>1,and q<0.05 as the screening criteria,59 different metabolites were screened out(43 in positive ion mode,16 in negative ion mode).Furthermore,14 potential biomarkers such as bilirubin,indole,indole acrylic acid and L-tryptophan were screened by AUC>0.90 and p<0.05.KEGG pathway analysis showed that different metabolites were mainly enriched in Cysteine and methionine metabolism,Amino sugar and nucleotide sugar metabolism,Glycolysis / Gluconeogenesis,Tryptophan metabolism and other pathways.These biomarkers played an important role in maternal nutrition regulation of jejunum development in piglets through metabolic pathways.4.By calculating the Spearman correlation coefficients of bacteria at the jejunal differential genus level with jejunum development and differential metabolites of 21 days old piglets,Acidaminococcus,Enterobacter and Prevotella＿7 were positively correlated with jejunum development(p<0.05),and Wolbachia was significantly negative correlated with jejunum development(p<0.05).Enterobacter was positively correlated with 3-indoleacetic acid,DL-O-leucine,Indole,Indoleacrylic acid and Lmethionine(p<0.05),while Megasphaera was negatively correlated with L-leucine,Lphenylalanine,L-tryptophan and Phenyllactic acid(p<0.05).These results indicated that these differential microorganisms and metabolites were closely related to the development of jejunum.In conclusion,moderate reduction of CP level in maternal diet(12%)under the condition of balanced 5 amino acids cannot affect the growth of Huzhu Bamei suckling piglets,but can reduce the diarrhea rate and promote the development of jejunum.This study provided a theoretical basis for revealing the relationship and internal mechanism between jejunal microbial function and metabolites of piglets regulated by maternal nutrition.It also reflected the biological characteristics of Bamei pig’s roughage tolerance from the perspective of adapting to low protein diet,and provided a scientific basis for the formulation of feed formula and breeding of Huzhu Bamei pig in production practice.