Effects Of Heat Stress On Semen Quality And Seminal Plasma Metabolites In Male New Zealand White Rabbits

It is of great significance to understand the influence and reasons of environmental temperature on the semen quality of male rabbits,in order to alleviate the "summer infertility" of domestic rabbits.The THI value,physiological indexes,semen quality and seminal plasma biochemical indexes of New Zealand white rabbits in May and August were detected.To explore the effects of heat stress on rabbits,provide a research direction for the technical research on the adaptation of rabbit heat stress,and promote the sustainable and healthy development of rabbit industry.The main experimental results are as follows:1.Twenty male rabbits were selected and the ambient temperature and relative humidity were recorded by thermohygrometer 3 times a day（8:00,14:00 and 20:00）.Semen of male rabbits was collected in May and August for analysis.The results showed that the average temperature and humidity of rabbit house in May were 22.04℃and 61.82%respectively.The average temperature and humidity of rabbit house in August were 30.15℃ and 78.62%respectively.THI may value of 20.94;The THI value in August was 29.10.The male rabbits in May were defined as the non-heat-stressed group,and the male rabbits in August were defined as the heat-stressed group.Compared with the non-heat-stressed group,the heat-stressed group had significantly lower sperm motility and semen pH（P<0.01）,significantly higher abnormality rate（P<0.05）,and significantly lower semen volume（P<0.05）,the sperm density decreased significantly（P<0.05）.In the heat stress group,the number of grade A sperm was significantly decreased（P<0.01）,the number of grade B and C sperm was significantly increased（P<0.01）,and there was no significant difference in the number of grade D sperm between the two groups（P=0.112）.The three indexes of TSO,TMS and TFSF in the heat stress group were significantly decreased（P<0.01）.Heat stress protein 70（HSP70）,acid phosphatase（ACP）in seminal plasma of heat stress group were significantly increased（P<0.01）,seminal plasma testosterone（T）,α-glucosidase（αGlu）,Fructose（Fructose）was significantly decreased（P<0.01）;Mg2+,Na+,K+ in metal ions were significantly decreased（P<0.01）;alkaline phosphatase（P=0.148）,lactate dehydrogenase isoenzyme（P=0.775）There was no significant difference between the two groups.Heat stress can affect the semen quality of male rabbits,resulting in semen quality decline.2.UPLC-MS/MS technique was used to analyze seminal plasma samples of heat stress group and non-heat stress group.346 metabolites were identified.VIP>1.0,FC>1.5 or FC<0.667 and P<0.05;A total of 71 metabolites were matched,including stearic acid,betaine,arachidonic acid,L-malic acid,indole and so on.KEGG analysis of differential metabolites showed that the metabolic pathways of differential metabolites were energy metabolism and amino acid metabolism,including niacin and nicotinamide metabolism,citric acid cycle and tryptophan metabolism.3.RNA-Seq technology was used to analyze the semen of the heat-stressed group and the non-heat-stressed group.The results showed that there were 1676 differential genes in the heat-stressed group and the non-heat-stressed group,including 894 upregulated genes and 782 down-regulated genes.Analysis results of differentially expressed genes GO and KEGG showed that the differentially expressed genes GO were enriched in metabolic process,defense response,cell response to cytokines stimulation,organic nitrogen compound metabolism and response to biological stimulation,including SIRT1,NT5C3A,MSMO1 and PHGDH,etc.KEGG analysis showed that the main enrichment factors were cytokine-cytokine receptor interaction,folic acid biosynthesis,citric acid cycle,etc.Combined analysis of differential genes and metabolites revealed 14 metabolites and 27 genes,including palmitic acid,arachidonic acid and L-malic acid,and genes including ACSBG1,PPT1,THEM5,ME2,MDH2 and ME1.KEGG analysis of differential metabolites and differential genes showed that the enriched metabolic processes included glyoxylic acid and dicarboxylic acid metabolism,arachidonic acid metabolism,citric acid cycle and pyruvate metabolism.The results provide reference for further understanding the mechanism of heat stress on semen quality decline of male rabbits.