| The Italian Mediterranean buffalo,as a buffalo breed in the world's breeding selection program,has excellent milk performance and high-end quality milk.Guangxi Huaxu Buffalo Biotechnology Co.Ltd.imported a batch of Italian Mediterranean aquatic bulls to improve the local buffalo.When bull semen was produced,it was found that the quality of the semen decreased in July and August of each year,and the rate of sperm deformity increased and the vitality decreased,which directly affected the actual production of semen.In order to understand the effect of ambient temperature on the semen quality of Italian Mediterranean bulls and the reasons,this study examined the THI value,physiological indicators of bulls,semen quality and seminal plasma biochemical indicators of the bullpens in March and July,using UPLC-MS/MS method performed metabolite analysis on the seminal plasma of Italian Mediterranean bulls,and clarified the seminal plasma biochemical reactions and metabolic changes from the seminal plasma metabolism level.The research results provide a theoretical basis for further understanding the mechanism of heat stress on the decline of Italian Mediterranean buffalo semen quality.The experimental results are as follows:1.Analysis of semen quality and determination of physiological and biochemical indexes of bulls in different seasonsFirst,the temperature and humidity of the environment in which 12 Italian Mediterranean bulls located in different seasons and the physiological indexes of the bulls were measured using dry and wet thermometers,and veterinary thermometers.The computer sperm assisted analyzer(CASA)and seminal plasma biochemical indicator kits were used to determine seasonal Italian Mediterranean bull semen quality and triglyceride(TG),testosterone(T),heat shock protein-70(HSP-70),alkaline phosphatase(ALP),acid phosphatase(ACP),fructose(fructose),?-glucosidase(?-Glu),lactate dehydrogenase(LDH),potassium(K)ions,magnesium(Mg)ions,sodium(Na)ions,and the two sets of data Analysis of differences between groups.The results showed:(1)According to the ambient temperature and humidity and using THI=1.8 × t+32-(0.55-0.0055×rh)×(1.8×t-26)formula to calculate the THI value,the THI in March was 66.86±2.98,July the THI was 82.94±3.52.Compared with March,the respiration frequency of Italian Mediterranean bulls increased significantly in July(P<0.01),the rectal temperature increased significantly(P<0.05),the expression of heat shock protein-70(HSP-70)increased significantly(P<0.05)in July.According to the THI threshold of 72 and heat shock protein expression to determine the index of heat stress occurrence,the semen of the Italian Mediterranean bulls in March was set as the non-heat stress group,and July as the heat stress group.(2)Compared with the non-heat stress group,the sperm motility and semen PH value of the Italian Mediterranean bulls in the heat stress group were significantly reduced,and the deformity rate was significantly increased(P<0.05).There was no significant difference in semen collection and semen density(P>0.05).The curvilinear speed,linear motion,average speed,straightness,head amplitude,and whip frequency of sperm were significantly reduced(P<0.05),and the linearity of sperm was significantly reduced(P<0.01).(3)Compared with non-heat stress group,fructose,acid phosphatase(ACP)and ?-glucosidase(?-Glu)in bull seminal plasma of heat stress group were significantly increased(P<0.05),Testosterone(T)is significantly reduced(P<0.05),the difference between alkaline phosphatase(ALP)is not significant(P>0.05),the difference between lactate dehydrogenase(LDH)is not significant(P>0.05),metal ion The middle Mg ion and Na ion decreased significantly(P<0.01),and the K ion difference was not significant(P>0.05).2.Analysis of the influence of heat stress on the metabolic components of seminal plasma of Italian Mediterranean bullsA total of 24 seminal plasma samples of 12 Italian Mediterranean bulls before and after heat stress were analyzed by UPLC-MS/MS,and the identified metabolite data,metabolic pathways were analyzed.The results were as follows:(1)A total of 227 different metabolites were matched using UPLC-MS/MS technology.(2)Most of the seminal plasma metabolites in the heat stress group were related to energy and biofilm repair;most of the seminal plasma metabolites in the non-heat stress group were related to fatty acid oxidation Related metabolites.(3)Six different metabolites,2,5-Furandicarboxylic acid,5,7-Dihydro-1H-purine-2,6,8(3H)-trione,3-mercaptohexyl butyrate,Citric acid and L-alpha-Glycerylphosphorylcholine were screened from the seminal plasma of two groups of Mediterranean bulls before and after heat stress,They were significantly up-regulated in the heat stress group,suggesting that they can be used as a marker of heat stress in Mediterranean bulls.(4)Enrichment analysis of differential metabolites found that they were involved in three metabolic pathways of tricarboxylic acid cycle(TCA cycle),glycerophospholipid metabolism,and glyoxylic acid and dicarboxylic acid metabolism,all of which are significantly up-regulated in the heat stress group Metabolic pathways.The above research shows that the increase in ambient temperature could cause the semen quality of the Italian Mediterranean bulls to decrease,and the change of energy metabolism pathway. |
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