| Artificial insemination(AI)technology is an important step in improving the reproductive efficiency of livestocks,therefore,it is very important to screen and obtain high-quality frozen sperm.However,the freezing-thawing process results in reduced sperm quality and fertility,which affects the breeding value of bulls and increases the cost of breeding.Of the established research results showed that cells and organisms can protect cells from external damage by synthesizing heat stress protein HSPs,and the expression level of HSPs is closely related to the fertilization rate of male livestock.At present,there is no report on the relationship between HSP70 and HSP90 and sperm quality in yak.In this study,the same batch of fresh and freezing-thawing sperm were used and purified by a 45% Percoll gradient,various assay techniques were used to analyze the differences in sperm quality and enzymes activity,the expression of HSP70 and HSP90 were detected by qRT-PCR,Western Blot and immunofluorescence techniques,and the correlation between the expression levels of HSP70 and HSP90 and sperm quality were analyzed,which provides a scientific basis for marker-assisted selection of yak sperm quality and fecundity.The results were shown following:1.The RNeasy Kit,hot TRIzol and one-step TRIzol method can obtain total RNA of yak sperm,but the quality of total RNA extracted from the RNeasy kit(concentration,purity and integrity)was superior to the other two methods(P<0.05).2.By comparing sperm motility,malformation rate,acrosome integrity,and plasma membrane integrity of fresh and freezing-thawing sperm,it was found that sperm motility after freezing and thawing decreased by 50.54%(P<0.01),and sperm abnormality rate increased by 56.60%(P<0.01),the spermatozoa acrosome integrity rate decreased by 32.33%(P<0.01),and sperm plasma membrane integrity reduced by 51.28% compared with fresh essence(P<0.01).Scanning electron microscopy showed that the fresh sperm plasma membrane and the acrosome were smooth and intact,while some frozen sperm membranes and acrosome were damaged.The results of enzyme activity test showed that the activities of GSH-PX,SOD,AST,ACP and LDH in sperm after freezing-thawing were significantly lower than those of fresh sperm,and were reduced by 67.65%,67.38%,61.58%,49.53% and 47.06%,respectively.3.In the comparative study on the differences of HSP70 and HSP90 expression in fresh sperm and frozen-thawed sperm in yak,it was found that HSP70 and HSP90 in freezing-thawing sperm were significantly lower than those of fresh sperm at the transcriptional and protein levels(P<0.05).HSP70 protein distributed over the sperm acrosome,acrosome equatorial segment,post-acrosomal and middle piece and tail of sperm in fresh group,while HSP70 protein was mainly located in post-acrosomal and middle piece of sperm,and the average optical density significantly decreased after freezing and thawing(P<0.01).The HSP90 protein in fresh sperm and frozen sperm were expressed in the middle piece and tail of sperm,but the fluorescence signal intensity was extremely lower than that of fresh essence of HSP90 in the middle segment of frozen-thawed sperm(P<0.01),suggesting that protein localization is related to sperm quality.Correlation analysis shows that there was a significant positive correlation between HSP70 expression and sperm motility(r=0.753,P<0.01),plasma membrane integrity(r=0.656,P<0.05)and acrosome integrity(r=0.632,P<0.05).There was a significant positive correlation between the expression of sperm HSP90 and sperm motility(r=0.723,P<0.01),plasma membrane integrity(r=0.586,P<0.05),there was a positive correlation between the expression of HSP90 and acrosome integrity rate(r=0.370,P>0.05).In summary,freezing-thawing resulted in a significant reduction in sperm quality,HSP70,and HSP90 expression levels,and there is a positive correlation between HSP70 and HSP90 mRNA expression levels and sperm quality,which can be used as an index to evaluate the quality of yak sperm. |
PDF Full Text Request