Study The Effects Of Stress On Blood And Intestinal Metabolism Of Growing Meat Rabbits Based On Multi-omics

Stress response is a change in the physiological(psychological)state of livestock and poultry when they are stimulated by the outside world.Stress is a common and key problem in the production of meat rabbits.However,at present,there are few studies on the effects of stress on meat rabbits by using the methods of system biology.As an important terminal regulatory factor on the hypothalamic-pituitary-adrenal(HPA)axis,glucocorticoid can regulate the dynamic balance of the body and cause the body's response to stress.In this paper,the changes of protein and small molecular metabolites in meat rabbits under stress were studied by subcutaneous injection of dexamethasone(a synthetic glucocorticoid)and high-throughput analysis techniques of proteomics and metabolomics.To reveal the differential proteins and their biological functions in the intestinal tract of stressed meat rabbits,the changes of serum metabolites and their overall metabolic pathway,and to analyze the effects of stress on the intestinal function of meat rabbits based on the changes of stress intestinal flora.Furthermore,through the study of serum metabolomics and cecal flora of meat rabbits,the effect of heat stress on meat rabbits was analyzed,and the molecular mechanism of stress response of meat rabbits was explored.The main contents are as follows:1.Sixty 42-day-old Hyla rabbits were randomly divided into 3 groups with 20 rabbits in each group.DEX group:subcutaneous injection of dexamethasone sodium phosphate injection(1.3mg/kg BW);Control group:subcutaneous injection of the same dose of normal saline;Pair-fed group:subcutaneous injection of the same dose of normal saline and feeding DEX group feed intake matching group.During the experimental period,the rabits were injected continuously for 7 days.The effect of dexamethasone on growing meat rabbits was detected by measuring the production performance,slaughtering performance and blood biochemical indexes of meat rabbits.The results showed that compared with the control group,subcutaneous injection of dexamethasone could significantly reduce the average daily gain of meat rabbits(P<0.05),increase the ratio of feed to meat(P<0.05),and affect the production performance of meat rabbits.The contents of serum alkaline phosphatase(ALP),Alanine aminotransferase(ALT),Aspartate aminotransferase(AST),total cholesterol(TC),TNF-?and IL-6 in DEX group were significantly higher than those in Control group,while the contents of total protein(TP)and glucose(GLU)in serum were significantly lower in Dexamethasone group than those in Control group.Therefore,subcutaneous injection of dexamethasone could induce stress response in meat rabbits.2.The effects of dexamethasone-induced stress on meat rabbits were studied based on TMT labeled quantitative proteomics and UPLC-MS/MS metabonomics.Intestinal differential proteins and serum differential metabolites were screened in DEX group and Control group.Serum metabonomics analysis of meat rabbits showed that serum differential metabolites were significantly enriched with pyrimidine metabolism,amino acid biosynthesis and aminoacyl-t RNA biosynthesis pathway.Compared with Control group,the serum contents of phosphatidylcholine and?-aminobutyric acid in DEX group were significantly decreased(P<0.05),and?-aminobutyric acid could participate in tricarboxylic acid cycle.The levels of glycine and shikimate in serum of meat rabbits in DEX group were significantly down-regulated(P<0.05).Dexamethasone-induced stress may affect protein synthesis by inhibiting the biosynthesis of aminoacyl t RNA.366 differential proteins were successfully identified in the jejunum of meat rabbits.GO annotation and enrichment analysis showed that the differential proteins were mainly involved in ion binding and oxidation-reduction process.KEGG enrichment analysis showed that the differential proteins were significantly enriched in carbohydrate digestion and absorption,fat digestion and absorption,mineral absorption,conversion of pentose and gluconate,metabolism of arginine and proline,protein digestion and absorption,DNA replication and PPAR signal pathway.Seven differential proteins in PPAR signal pathway were significantly down-regulated.ACOX1 is the key rate-limiting enzyme of fatty acid?oxidation in the pathway.The decrease of its expression can activate PPAR?to regulate fatty acid oxidation metabolism.Among the differential proteins,PepT1,B0AT,FATP4and SGLT1 were significantly down-regulated(P<0.05).RT-PCR analysis results showed that the expression levels of PepT1,B~0AT and SGLT1 mRNA were significantly down-regulated in all segments of the small intestine(P<0.05),while the transcriptional level of FATP4 was significantly different only in the ileum.Thus,dexamethasone-induced stress may affect the digestion and absorption of intestinal nutrients by inhibiting the expression of intestinal nutrient transporters PepT1,B~0AT and SGLT1 at the transcriptional and translational levels,and down-regulate the expression of FATP4 at the translation level through PPAR?signal pathway,affecting fatty acid transport and fat deposition.3.The study on the diversity of intestinal flora of meat rabbits in dexamethasone-induced stress was analyzed based on 16S r RNA gene amplicon sequencing and q PCR.The results showed that dexamethasone induced stress affected the microflora and microbial abundance in jejunum and cecum of meat rabbits,significantly decreased the abundance of thick-walled bacteria in jejunum of meat rabbits(P<0.05),and significantly decreased the abundance of Bacteroides in cecum(P<0.05).Dexamethasone induced stress significantly increased the abundance of deformans in jejunum and cecum(P<0.05).It can be seen that stress induced by dexamethasone increase the number of harmful bacteria in the intestinal tract.4.Eighty 35-day-old Hyla meat rabbits with similar body weight were pre-fed at 24±1?for 7 days,and then 60 42-day-old meat rabbits were randomly divided into two groups:moderate temperature group(24±1?)and continuous heat stress group(34±2?).A The experimental period was 14 days.The production performance and blood biochemical indexes of meat rabbits were measured.UPLC-MS/MS metabolites and 16s amplifiers were used to study the effects of heat stress on cecal flora of meat rabbits.The results showed that after continuous high temperature exposure,the average daily gain,average daily feed intake and thymus index were significantly decreased,the contents of HSP70,ALP and Cortisol in blood were significantly increased,while TP,GLU,T3 and T4 were significantly decreased(P<0.05),indicating that the establishment of heat stress model of meat rabbit was basically successful.Heat stress significantly increased the expression of HSP70 at mRNA level and decreased the expression of PepT1,B~0AT and SGLT1mRNA in small intestine of meat rabbits(P<0.05).A total of 39 differential metabolites were screened by serum metabolomics,including 12 up-regulated differential metabolites and 27 down-regulated differential metabolites,which can effectively distinguish between heat stress group and moderate temperature group,and can be used as biomarkers of heat stress in meat rabbits.The selected differential metabolites were analyzed by KEGG annotation and enrichment analysis.The results showed that 14 pathways affected by heat stress were identified by KEGG pathway enrichment analysis,including Sphingolipid metabolism,Glyoxylate and dicarboxylate metabolism,Pyrimidine metabolism,pyruvate metabolism,Alanine,aspartate and glutamate metabolism,Lysine biosynthesis,Citrate cycle(TCA cycle)),aminoacyl-t RNA biosynthesis and so on.The analysis of the effect of heat stress on the cecal microflora of meat rabbits showed that the abundance of cecal Proteus in the heat stress group was significantly higher than that in the moderate temperature group.The number of Candidatus-saccharimonas in the cecum microflora was significantly higher than that in the moderate temperature group(P<0.05)which may be related to inflammatory diseases in the heat stress group.To sum up,meat rabbits may respond to dexamethasone-induced stress and heat stress by:1)affecting pyrimidine metabolism and aminoacyl-t RNA biosynthesis pathway in blood,reducing the synthesis of amino acids and proteins;2)inhibiting the expression of intestinal nutrient transporters such as PepT1,B0AT and SGLT1,thus affecting intestinal digestion and absorption of sugars and proteins;2)affecting lipid metabolism and deposition through PPAR signal pathway.3)the thick-walled bacteria and Bacteroides in the jejunum and cecum of meat rabbits are absolutely dominant bacteria,and stress will change the ratio of thick-walled bacteria and Bacteroides in the jejunum and cecum of meat rabbits.Stress increases the number of amoeba in the intestinal tract,affects the balance between beneficial and harmful bacteria in the intestinal tract,and affects the health of the intestinal tract.