| This paper takes 2-5 year old Simmental bulls as the research object,and aims to(1)study and compare the changes of physiology,serum biochemical indexes and semen quality of Simmental bulls under non-heat stress and heat stress conditions;(2)Based on the changes of the above indicators,use principal component analysis and membership function analysis to screen heat-sensitive and heat-tolerant Simmental bulls,and use 16 S r DNA and non-targeted metabolomics to discover and regulate the resistance of bulls.The key rumen fluid microbiota and metabolites of thermophilicity,in order to explore the mechanism of interaction regulation between thermotolerance and rumen microbiota in breeding bulls.Experiment 1: In order to study the effect of heat stress on physiology,serum biochemical indexes and semen quality of Simmental bulls,this study was conducted in non-heat stress(THI=66.67±0.91)and heat stress(THI=80.10±1.13))during 18 consecutive days,the physiological values,serum physiological and biochemical indexes and semen quality indexes of 34 Simmental bulls with similar body weight and normal semen collection were measured,and the correlation analysis of each index was carried out.The result is as follows:(1)Heat stress significantly increased the respiratory rate(RR),rectal temperature(RT),serum cortisol(COR)and heat shock protein 70(HSP70)levels of Simmental bulls(P<0.05).The content of triiodothyronine(T3)and thyroxine(T4)in serum,sperm concentration and sperm motility were decreased(P<0.05),but there was no significant change in ejaculate volume(P>0.05).(2)RR,RT,COR,HSP70 were significantly positively correlated with THI(P<0.01),T3,T4 were extremely significantly negatively correlated with THI(P<0.01),THI,RR,RT,COR,HSP70 were significantly correlated with sperm Motility was significantly negatively correlated(P<0.01),T3,T4 were extremely significantly positively correlated with sperm motility(P<0.01),serum testosterone,ejaculate volume,sperm concentration and other indicators were not significantly correlated(P>0.05).Experiment 2: Based on physiology,serum biochemical indexes and semen indexes of 34 Simmental bulls during non-heat stress and heat stress periods,principal component analysis and membership function analysis were used to screen out heatresistant(HT)and heat-sensitive bulls.(HS)group of 6 bulls each.The rumen fluid samples were collected to determine the contents of ammonia nitrogen(NH3-N)and volatile fatty acids(VFAs),and 16 S r DNA and LC-MS techniques were used to explore the rumen flora and metabolites that regulate the heat tolerance of breeding bulls.The result is as follows:(1)The respiratory rate(37.0±1.3 times/min)and rectal temperature(38.7±0.04℃)of bulls in HT group were significantly lower than those in HS group(51.3±2.6times/min),rectal temperature(39.0±0.06 °C)(P < 0.01).(2)COR,T4,HSP70 and sperm motility in the HT group were significantly higher than those in the HS group(P<0.05),and there was no significant difference in T3,HSP90,T,ejaculate volume and sperm concentration between the two groups(P>0.05).The semen quality was higher than that of the HS group.(3)The contents of p H and butyric acid in the rumen fluid of the HT group were significantly higher than those of the HS group(P<0.05),and the content of ammonia nitrogen NH3-N was significantly lower than that of the HS group(P<0.05).There was no significant change in the ratios of acetic acid,propionic acid,and acetic acid and propionic acid(P>0.05).(4)16S r DNA sequencing analysis found that there were differences in the abundance of rumen bacteria between the HT group and the HS group.At the phylum level,Actinobacteriota was significantly decreased(P<0.05)by 1.8%;Desulfobacterota was significantly increased(P<0.05)by 0.28%.At the genus level,Christensenellaceae_R-7_group(Christensenellaceae_R-7_group)and Ruminococcus were significantly decreased(P<0.05)by 1.07% and 1.69%,respectively,and norank_f_F082 was significantly increased(P<0.05),an increase of 0.83%.Differences in relative abundance of KEGG metabolic pathways annotated by bacterial functional genes.Differentially functional bacteria were significantly enriched in 8 metabolic pathways,among which the abundances of transport and catabolism,glycan biosynthesis and metabolism,enzyme family,metabolism,metabolism of cofactors and vitamins,and energy metabolism were in the thermotolerant group(HT)higher,the abundance of carbohydrate metabolism and xenobiotic biodegradation and metabolism was higher in the heat-sensitive group(HS).(5)13 differential metabolites were found by non-targeted metabolomic comparative analysis of rumen fluids from HT and HS bulls.In the HT group,the abundance of 5 metabolites was down-regulated,and the abundance of 8 metabolites was up-regulated.KEGG pathway annotation shows that the metabolite 2’,3’ cyclic CMP is significantly enriched in the metabolism-glutathione metabolism,thyroid hormone synthesis and ferroptosis pathway,and Lyso PC(16:0)is significantly enriched in choline in cancer In the metabolic and glycerophospholipid metabolic pathways,PTolualdehyde was significantly enriched in the xylene degradation pathway.Metabolites 2’,3’ cyclic CMP,Lyso PC(16:0)and P-Tolualdehyde participate in cellular antioxidant,cellular nutrient absorption and enhance the body’s adaptability to improve body homeostasis and production performance.The reason why the test cattle have strong heat resistance.(6)Through metabolomic analysis and correlation analysis of differential bacteria and metabolites in the rumen,it was found that the down-regulation of metabolites 2’,3’ cyclic CMP and Lyso PC(16:0)and the up-regulation of P-Tolualdehyde were closely related to the two phyla.Actinobacteriota and Desulfobacterota at the level,Christensenellaceae_R-7_group,Ruminococcus and norank_f_F082 flora at the three genus level are closely related,In this study,the contents of Actinobacteriota,Christensenellaceae_R-7_group and Ruminococcus in the rumen of the HT group were significantly lower than those of the HS group(P<0.05),and the contents of Desulfobacterota and norank_f_F082 were significantly higher than those of the HS group(P<0.05).Therefore,the rumen fermentation pattern can be regulated by reducing the relative abundance of Actinobacteriota,Christensenellaceae_R-7_group and Ruminococcus in the rumen or increasing the relative abundance of Desulfobacterota and norank_f_F082 to enhance thermotolerance. |
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