Mechanism Of Glutamate On Improving Growth Performance And Gastrointestinal Tract Function Of Heat-stressed Hu Sheep

Heat stress has become a major factor that restricted the development of sheep industry.Glutamate is a functional amino acid,which was reported to alleviated heat stress of pigs and broilers in summer.However,the application and mechanisms regarding the anti-heat stress effects of glutamate in ruminants have not been elucidated yet.Therefore,this study aimed to investigate the effects of dietary glutamate supplementation on the production performance,rumen fermentation characteristics,gastrointestinal structure and function in heat-stressed Hu sheep by using metagenomic and metabolomic methods,aiming to provide theoretical basis for glutamate application in ruminant production.Exp.1 Effects of glutamate on rumen fermentation characteristics of Hu sheep in vitroThe rumen fluid was obtained from six Hu sheep.The diet for growing-Hu sheep was serve as basic substrate（CON group）,and the experiment groups were supplemented with0.5,1,2 mmol/L L-glutamate,respectively.The diet in each group was cultured with rumen fluid for 24 h in vitro.The results showed that glutamate supplementation increased the rumen microbial crude protein（MCP）（P<0.05）when compared with CON.In addition,the contents of NH₃-N and TVFA were positively correlated with the dosage of glutamate,the effect was significant when glutamate supplementation reached 2 mmol/L.Dietary 2 mmol/L glutamate supplementation also increased the rumen p H values,the contents and kinetic parameters of gas production,and the concentrations of acetic acid,butyric acid,isobutyric acid,valeric acid,and isovaleric acid（P<0.05）,whereas no effect on the ratio of acetic acid to propionic acid was observed.Collectively,dietary glutamate supplementation promoted rumen degradation efficiency,improved rumen fermentation characteristics,increased TVFA and MCP production.Moreover,the best promotion effect was observed when supplemented with 2 mmol/L glutamate.Exp.2 Effects of glutamate on production performance,slaughter performance,serum biochemical indexes and hormones in Hu SheepThis experiment was to determine the effects of glutamate on the growth performance,slaughter performance,nutrient apparent digestibility and serum metabolites of Hu sheep.A total of forty-eight male Hu sheep with an average initial body weight of 17.74±0.17 kg were randomly divided into two groups.Each group had six replications,with four sheep in each replication.The control group（CON）was fed with basal diet,while the treatment group（GLU）was fed with basal diet+3 g/head/day glutamate.The pre-test lasted for 15days,followed by a formal test for 90 days.The results showed that there were no differences in growth performance,serum glutamate contents,serum biochemical indexes and serum hormones between CON and GLU during 1-30 days and 31-60 days,but a higher average daily gain（ADG）,a lower average dairy feed intake（ADFI）and a lower ratio of ADFI/ADG were observed during 61-90 days（P<0.05）.Glutamate supplementation notably increased the levels of serum glutamate,globulin,immunoglobulin A,immunoglobulin G,immunoglobulin M,growth hormone and testosterone during 61-90 days（P<0.05）,and significantly reduced the levels of serum heat shock protein 70（HSP70）,adrenocorticotrophic hormone,cortisol,corticosterone,triiodothyronine and thyronine during 61-90 days（P<0.05）.The results of nutritional digestion test indicated that glutamate markedly improved the apparent digestibility of dry matter,organic matter and crude protein（P<0.05）.The slaughter test results showed that glutamate had no significant effect on carcass traits and organ indexes（P>0.05）.In conclusion,glutamate supplementation alleviated heat stress and improved growth performance by increasing the level of serum glutamate to promote growth hormone level,decrease heat stress-related hormones and HSP70 levels,and enhance immune function in Hu sheep.These improvements might be related to feeding time of glutamate supplementation.Exp.3 Effects of glutamate on rumen fermentation and gastrointestinal VFA contents in Hu SheepThis experiment was conducted to investigate the effects of glutamate on rumen fermentation characteristics and gastrointestinal volatile fatty acids（VFA）contents of Hu sheep.The results showed that glutamate supplementation increased the rumen p H values,the contents of NH₃-N,MCP and isovaleric acid,the proportions of isobutyric acid,valeric acid and isovaleric acid in heat-stressed Hu sheep,when compared with CON（P<0.05）.Besides,the concentrations of isobutyric acid in abomasum and the contents of NH₃-N in jejunum and cecum were higher in GLU than those in CON（P<0.05）.However,no differences were observed in the VFAs in jejunum,cecum and colon between GLU and CON（P>0.05）.The results suggested that glutamate supplementation improved rumen fermentation characteristics of heat stressed Hu sheep,but had no effect on VFAs in intestine.Exp.4 Effects of glutamate on the morphological structure and barrier function of gastrointestinal tract in Hu SheepThe effects of glutamate supplementation on the morphological structure and barrier function in rumen epithelium and jejunal mucosa of heat-stressed Hu sheep were evaluated.The results showed that the height of rumen papilla and jejunum villi,and the thickness of rumen epithelial stratum corneum were higher in GLU than those in CON（P<0.05）.Lower level of serum diamine oxidase（DAO）,malondialdehyde,interleukin-6 and tumor necrosis factor-α,and higher level of serum total antioxidant capacity,superoxide dismutase,glutathione reductase and interleukin-4（IL-4）were observed in GLU than those in CON during 61-90 days（P<0.05）,whereas these indices showed no differences during1-30 days and 31-60 days（P>0.05）.Glutamate supplementation increased the m RNA expression levels of adenylate activated protein kinase（AMPK）,heat stock protein 70（HSP70）and tight junction protein（claudin-1,occludin,and ZO-1）in rumen epithelium（P<0.05）,decreased the m RNA expression level of DAO in rumen epithelium,when compared with CON（P<0.05）.Moreover,glutamate supplementation decreased the m RNA expression levels of DAO,toll like receptor 9（TLR₉）,myeloid differentiation factor88（My D88）,nuclear factor kappa-B（NF-κB）and apoptotic gene（Bax）in jejunal mucosa as compared to CON（P<0.05）.Collectively,these results suggested that glutamate supplementation enhanced the antioxidant capacity and reduced inflammation of body,improved morphological structure of rumen and jejunal epithelium,enhanced tight junction function of rumen epithelial cells by increasing the m RNA expression of AMPK/HSP70 pathway,and reduced inflammation and apoptosis of jejunum mucosa cells by decreasing the m RNA expression of TLR9/My D88/NF-κB pathway.Exp.5 Regulatory mechanism of glutamate on gastrointestinal function of heat-stressed Hu sheep based on metagenomics and metabolomicsThe metagenomics and metabolomics were empolyed to investigate the effects of glutamate on microbial structure in rumen and metabolites in rumen and jejunum.The results showed that glutamate supplementation improved the metabolism pathway abundance of cell growth and death by increasing the activities of DNA polymeraseⅢ,DNA mismatch endonuclease,lysozyme,DNA repair protein Rad D and HSP20 family proteins.Furthermore,the structure of rumen microflora was changed,as evidenced by the increased relative abundance of Saccharibacteria,Planctomycetes,Elusmicrobia,Ignavibacteria,Nitrospirae and Poribacteria at phylum level（P<0.05）.Glutamate supplementation increased the relative abundance of Bacteroides＿sp＿CAG:709、Bacteroides＿ovatus、Bacteroides＿thetaiotaomicron、Aistipes＿sp.、Phocaeicola＿vulgatus、Methanobrevibacter＿sp.＿Ab M4 and Methanobrevibacter＿boviskoreani at the species level（P<0.05）,and decreased the relative abundance of Selenomonas＿bovis、Selenomonas＿sp.＿AE3005 and unclassfied＿g＿Butyrivibrio（P<0.05）.The correlation analysis results indicated these rumen microorganisms had a positive correlation effect on improving rumen fermentation,nutrition apparent digestibility,immune ability,inflammation and gastrointestinal barrier function in heat-stressed Hu sheep.Additionally,Metabolomic analysis indicated that glutamate supplementation significantly affected the enrichment degree of purine metabolism,thyroid synthesis,junction function,olfactory conduction,salivary secretion and bile secretion in rumen（P<0.05）.In addition,these altered metabolisms were closely correlated with the microorganisms changed due to glutamate supplementation,and closely associated the decrease of heat stress related hormones secretion,the improvement of rumen epithelium tight function,and the increase of nutrient apparent digestibility.Meanwhile,the phenylalanine metabolism,glycerophospholipid metabolism,choline metabolism,and endocannabinoid signaling were significantly altered,indicating that glutamate changed amino acid metabolism,improved the environment and decreased the inflammation through neuromodulation in jejunum.In conclusion,dietary glutamate supplementation at a dosage of 3 g/head/day improved the metabolism pathway abundance of cell growth and death by increasing the activities of DNA polymeraseⅢ,DNA mismatch endonuclease,lysozyme,DNA repair protein Rad D and HSP20 family proteins,which led to the rumen microbial structure changed.Besides,the contents of serum glutamate were increased during 61-90 days.All of which contributed to improve the rumen fermentation function,nutrition apparent digestibility,immune ability,antioxidant ability,inflammation and increase growth performance of heat-stressed Hu sheep during 61 to 90 days.Moreover,glutamate supplementation improved the morphological structure of rumen epithelium and jejunum mucosa,enhanced tight junction function of rumen epithelial cells by increasing the m RNA expression of AMPK/HSP70,and reduced the inflammation and apoptosis of jejunum mucosa cells by decreasing the m RNA expression of TLR₉/My D88/NF-κB pathway.These results indicated that a longer period of glutamate supplementation（not less than 60 d）at a level of 3 g/head/day improved the growth performance and gastrointestinal function of heat-stressed Hu sheep,which was beneficial to their production performance in summer.