Study On The Physiological Adaptability Mechanisms Of Mongolian Sheep To Cold Environment

Mongolian sheep are characteristically cold-tolerant and thus can survive well and maintain genetic stability in the extremely cold environment of the Mongolian Plateau.However,the physiological adaptive mechanisms,including adaptive thermogenesis and intestinal adaptation,of Mongolian sheep under cold environments remain unknown.Browning of white adipose tissue（WAT）can trigger nonshivering thermogenesis as a potential strategy to promote an animal’s tolerance to cold environments.In addition,gut microbes contribute to regulating nonshivering thermogenesis in cold environments.Therefore,cold tolerance in Mongolian sheep may be due to WAT“browning”,and these adaptive processes are partially modulated by rumen or caecum microbes.Thus,based on WAT and microorganisms,two feeding experiments were conducted in this thesis First,this study aimed to evaluate the adaptive thermogenesis mechanisms of grazing Mongolian sheep in a plateau environment by investigating the changes in WAT morphology at the molecular level.Subsequently,based on gastrointestinal adaptation,the changes in nutrient absorption capacity and microbial composition in the rumen and caecum of Mongolian sheep under cold exposure were investigated.Finally,correlation analysis between rumen microorganisms,fermentation products,and WAT browning in Mongolian sheep under cold exposure was conducted.The thesis includes 7 experiments,which are as follows:Experiment 1:Effects of cold season on the physiology,immune response,antioxidant capacity,and glycolipid metabolism of grazing sheep.A comparative analysis of the biochemical indices,immune response,antioxidant capacity,glycolipid metabolism,lipid metabolism and thermogenesis-related gene expression of the liver in the cold and warm seasons was conducted.A total of twenty-four Mongolian sheep ewes（10 months old,weight=35.34±2.27 kg）raised in different seasons（warm season and cold season）were used in the study.They were allocated into two groups,describing the warm（August）and cold（January）seasons,with 12 ewes assigned to each season.In addition to being allowed to graze in the pasture,each ewe was fed 500 g of concentrate per day per ewe during the cold season.The results showed that（1）compared to those in the warm season,the levels of IL-2,IL-1β,and IL-6 were significantly enhanced in the cold season（P<0.05,P<0.01,and P<0.01,respectively）.（2）The activities of SOD and CAT in serum in the cold season were markedly lower than those in the warm season（all P<0.01）.Additionally,the concentration of MDA in the cold season was significantly higher than that in the warm season（P<0.01）.（3）Compared to those in the warm season,the levels of glucose,glucagon,and IGF-1 in serum were markedly enhanced in the cold season（P<0.05,P<0.01,and P<0.05,respectively）,while the levels of T₃ and T₄ were significantly decreased in the cold season（all P<0.01）.（4）The levels of HDL and glycerol in the cold season were significantly higher than those in the warm season（P<0.05 and P<0.01）.（5）The m RNA expression of CPT1B and UCP2 in the liver was markedly enhanced in the cold season（P<0.01 and P<0.01）.Overall,glycolipid metabolism,thermogenesis,and immune responses were stimulated to meet the requirements of organismal metabolic regulation to enable grazing Mongolian sheep to physiologically adapt to cold climatic conditions.However,the antioxidant defence system was damaged.Experiment 2:Effects of the cold season on morphological and molecular characteristics of WAT in different parts of grazing Mongolian sheep.The present work aimed to examine lipid metabolism,including WAT browning-related mechanisms,in Mongolian sheep during cold acclimation based on changes in WAT adipocyte morphology.The experimental design was identical to that of experiment 1.The results showed that（1）the mean adipocyte diameters of interscapular WAT and subcutaneous WAT were significantly increased in the cold season compared with the warm season（P<0.01 and P<0.05）.The mean adipocyte diameters in retroperitoneal WAT and perirenal WAT in the cold season were smaller than those in the warm season（all P<0.05）.（2）In the cold season,the m RNA expression of peroxisome proliferator-activated receptor gamma（PPAR-γ）and fatty acid-binding protein 4（FABP4）in both interscapular WAT（all P<0.05）and subcutaneous WAT（all P<0.05）was markedly higher than that in the warm season.Additionally,the m RNA expression of lipoprotein lipase（LPL）was reduced in both interscapular WAT and subcutaneous WAT in the cold season（all P<0.01）.（3）The m RNA expression of UCP1-dependent thermogenesis genes,including UCP1,PGC-1α,and PRD1-BF1-RIZ1homologous domain containing 16（PRDM16）（all P<0.05）,and mitochondrial biosynthesis-related genes,including cell death activator（CIDEA）and cytochrome oxidase subunit 4（COX4）（all P<0.01）,in both retroperitoneal WAT and perirenal WAT were significantly elevated in the cold season.Overall,the cold season could trigger an expansion process of both interscapular WAT and subcutaneous WAT.Moreover,the cold season could induce browning of both retroperitoneal WAT and perirenal WAT,resulting in elevated expression of UCP1-dependent browning genes.Experiment 3:Effect of cold exposure on the rumen and caecum short-chain fatty acids（SCFAs）,gastrointestinal morphology,and serum glycolipid metabolism.The present work aimed to examine the rumen and caecum microbial fermentation parameters and nutrient absorptive capacity of Mongolian sheep during cold acclimation.A total of sixteen Mongolian sheep ewes（7 months old,weight=30.94±0.54 kg）were used in the study.They were allocated into two groups,representing the control（temp range is 16-17℃）and cold exposure（temp range is﹣12-﹣15℃）groups,with 8 ewes assigned to each group.The experimental period lasted for 30 days,with 9 days of adaptation to the experimental environment and 21 days for the experimental feeding period.The results showed that（1）the levels of propionate,isobutyrate,butyrate,isovalerate,valerate,and total SCFAs in the rumen were significantly elevated（P<0.05,P<0.01,P<0.05,P<0.05,P<0.05,and P<0.01,respectively）,and the acetate,isobutyrate,and total SCFAs in the caecum were significantly increased（P<0.01,P<0.05,and P<0.05,respectively）in the cold exposure group.（2）In the cold exposure group,the relative weight and the length of the small intestine in Mongolian sheep were significantly increased compared with those in the warm group（P<0.001 and P<0.01）.（3）The height of the papillae of the rumen epithelium,villus height and V/C value of both the duodenum and ileum in the cold exposure group were significantly higher than those in the control group（P<0.001,P<0.001,P<0.001,P<0.01,and P<0.01,respectively）.（4）The levels of glucose,TG,BAs,and leptin in serum in the cold exposure group were significantly higher than those in the control group（P<0.05,P<0.05,P<0.001,and P<0.05,respectively）.Overall,the rumen and caecum fermentation capacity and nutrient absorptive capacity were significantly increased under cold exposure.Experiment 4:Effects of cold exposure on transcriptional regulation of rumen epithelial and the expression levels of absorptive-related genes in the small intestine of Mongolian sheep.The present work aimed to examine the molecular mechanisms of increased nutrient absorption capacity of the rumen epithelium and small intestine in Mongolian sheep under cold exposure based on rumen epithelium transcriptome sequencing and q RT-PCR for the small intestine.The experimental design was identical to that of experiment 3.The results showed that（1）643 differentially expressed genes（DEGs）were identified between the control and cold exposure groups,of which 554 were upregulated and 89 were downregulated in the cold exposure group.（2）The DEGs were mainly enriched in BP terms,including regulation of membrane potential,cell-cell junction organization,morphogenesis of an epithelium,tissue morphogenesis,regulation of transporter activity,transmembrane transport,and tissue morphogenesis.（3）The upregulated DEG-enriched pathways（P<0.05）were mainly involved in butanoate metabolism,propanoate metabolism,gap junctions,the PPAR signalling pathway,bile secretion,etc.H However,the downregulated DEGs were mainly enriched in endocytosis and fatty acid elongation.（4）The m RNA expression of GLUT2,PEPT1,and GLP2R in the duodenum（P<0.05,P<0.01,and P<0.01,respectively）and GLP2R in the ileum（P<0.01）was significantly elevated in the cold exposure group.Overall,the current study further elucidated the molecular mechanisms of increased nutrient absorption capacity of Mongolian sheep under cold exposure.Experiment 5:Effects of cold exposure on the microbial diversity and composition of the rumen and caecum in Mongolian sheep.The present work aimed to examine the changes in microbial composition and function in the rumen and caecum of Mongolian sheep under cold exposure based on 16S r DNA amplicon sequencing technology.The experimental design was identical to that of experiment 3.The results showed that（1）the Shannon index of rumen microbes in the cold exposure group was significantly higher than that in the control group（P<0.05）.（2）At the phylum level,the relative abundance of Firmicutes in the rumen in the cold exposure group was significantly higher than that in the control group（P<0.05）.（3）At the genus level（relative abundance>0.1%）,the relative abundances of Anaerovibrio and Desulfobulbusin in the rumen and Ruminococcus 1 and Oscillibacter in the caecum in the cold exposure group were significantly higher than those in the control group（all P<0.05）.（4）Firmicutes and its members,including class c＿Clostridia,order o＿Clostridiales,and family f＿Lachnospiraceae and Actinobacteria in the rumen and g＿Ruminococcus＿1,g＿Lachnospiraceae＿UCG＿001,and g＿Eubacterium＿ruminantium＿group in the caecum,play a vital role in the cold exposure group.（5）The functional terms,including cell motility,signal transduction mechanisms,and defence mechanisms,play a vital role in the rumen microorganisms of Mongolian sheep in the cold exposure group.Overall,the microbial composition of the rumen and caecum in Mongolian sheep changed significantly under cold exposure.Moreover,the functions of rumen microorganisms also changed significantly.Experiment 6:Effect of cold exposure on the browning of retroperitoneal WAT and perirenal WAT in grazing Mongolian sheep.The present work aimed to examine the m RNA expression of UCP1-dependent thermogenesis genes in retroperitoneal WAT and perirenal WAT in Mongolian sheep under cold exposure.The experimental design was identical to that of experiment 3.The results showed that（1）the mean adipocyte diameters of retroperitoneal WAT and perirenal WAT in the cold exposure group were significantly lower than those in the control group（all P<0.001）.（2）The m RNA and protein expression levels of UCP-1 and PGC-1αin retroperitoneal WAT and perirenal WAT in the cold exposure group were significantly higher than those in the control group（all P<0.05）.Overall,the retroperitoneal WAT and perirenal WAT presented potential browning under cold exposure.Experiment 7:Relationships between rumen microbes,SCFAs,and markers of white adipose tissue browning under cold exposure in Mongolian sheep.Spearman’s correlation test was used to determine the relationships between the rumen microbiota,SCFAs,and markers of WAT browning.The experimental design was identical to that of experiment 3.The results showed the following:（1）At the phylum level,the relative abundance of Actinobacteria,Firmicutes,and Patescibacteria had a positive and statistically significant correlation with PRDM16 in retroperitoneal WAT（P<0.01,P<0.05,and P<0.05,respectively）.（2）The genera g＿＿Selenomonas1,g＿＿Anaerovibrio,g＿＿Ruminococcaceae UCG-014,family f＿＿Lachnospiraceae,order o＿＿Clostridiales,and class c＿＿Clostridia（all within Firmicutes）were significantly positively correlated with markers of browning genes in either retroperitoneal WAT or perirenal WAT（all P<0.05）.（3）The order o＿＿Aeromonadales and class c＿＿Gammaproteobacteria（all within Proteobacteria）were significantly negatively correlated with markers of browning genes in both retroperitoneal WAT and perirenal WAT（all P<0.05）.（4）The relative abundances of Firmicutes,order o＿＿Clostridiales,and class c＿＿Clostridia were significantly positively correlated with the concentration of butyrate（all P<0.05）.（5）The concentration of butyrate was significantly positively correlated with markers of browning genes in either retroperitoneal WAT or perirenal WAT（all P<0.05）.Overall,adaptive thermogenesis in Mongolian sheep is modulated by rumen microbes and SCFAs.In summary,Mongolian sheep depend on improving adaptive thermogenesis to acclimate to cold environments.Concurrently,the nutrient absorption capacity of Mongolian sheep was enhanced to provide the energy substrates for adaptive thermogenesis.More importantly,these adaptive processes are mediated by rumen microbes and their metabolites.