Effect Of Different Antifreeze Agents On The Expression And Distribution Of HSP70 And HSP90 In Cashmere Goat Sperm During Freezing

The purpose of this study was to investigate the effect of different antifreeze agents(glycerin and ethylene glycol)on the expression and distribution of HSP70 and HSP90 in the process of sperm freezing in cashmere goats.The semen of six cashmere goats of the same age and in good condition were collected,mixed and added into the diluent containing different antifreeze for freezing.After thawing,the cryopreservation effect,HSP70 and HSP90 expression and distribution of different cryoprotectants on sperm were detected.The main results are as follows:1.The effects of different concentrations of glycerol and ethylene glycol on semen freezing of Inner Mongolia cashmere goat were compared.When 5%glycerol was added into the semen frozen diluent of Inner Mongolia cashmere goat,the sperm vitality was 61.72%,significantly higher than that of 3%ethylene glycol group and 5%ethylene glycol group(49.71%and 42.6%,P<0.05);the sperm acrosome integrity rate and plasma membrane integrity rate of 5%glycerol group(60.14%and 63.09%)were significantly higher than those of 3%ethylene glycol group(43.26%,41.84%,P<0.05)and 5%ethylene glycol group(36.7%,31.96%,P<0.05)There was no significant difference between 5%glycerol group and 3%ethylene glycol group(95.37%,P>0.05)and 5%ethylene glycol group(93.08%,P>0.05).Therefore,adding glycol to the diluent as sperm antifreeze is not better than glycerin.It is suggested that 5%glycerin has a better protective effect as sperm antifreeze.2.The effects of glycerol and ethylene glycol on the expression and distribution of heat shock protein 70 in Inner Mongolia cashmere goat sperm during cryopreservation were compared.5%glycerol and 3%ethylene glycol were added to the semen frozen diluent of Inner Mongolia cashmere goat as antifreeze during the process of sperm freezing.After sperm freezing-thawing,the expression of HSP70 in 5%glycerol group was 73.98ng/ml,significantly higher than that in 3%ethylene glycol group(72.59ng/ml,P<0.05);HSP70 in 5%glycerol group was distributed in the middle of head and tail,while HSP70 in 3%ethylene glycol group was distributed in the middle of head and tail.The ATP content of 5%glycerol group was significantly higher than that of 3%ethylene glycol group(9.89nmol/ml and 9.49nmol/ml,respectively,P<0.05);after adding rotenone to inhibit the oxidative phosphorylation reaction of sperm,the HSP70 expression of 5%glycerol group and 3%ethylene glycol group(71.7ng/ml and 70.82ng/ml,respectively)was significantly lower than that of 5%glycerol group and 3%ethylene glycol group without rotenone(73.98ng/ml,72.59ng/ml,P<0.05,respectively);HSP70 was only expressed in the head of sperm and not in the tail of sperm in 5%glycerol group after adding rotenone,while HSP70 distribution in sperm in 3%ethylene glycol group did not change;In addition,ATP content of sperm in 5%glycerol group was significantly lower than that in no rotenone group 5%glycerol group(9.36nmol/ml and 9.89nmol/ml,respectively,P<0.05),but the ATP content in sperm of 3%ethylene glycol group did not change significantly after adding rotenone(P>0.05).The above results showed that glycerol and ethylene glycol could affect the metabolism of sperm and than affect the expression and distribution of HSP70 in sperm.3.The effects of glycerol and ethylene glycol on the expression and distribution of heat shock protein 90 in Inner Mongolia cashmere goat sperm during cryopreservation were compared.5%glycerol and 3%ethylene glycol were added to the semen of Inner Mongolia cashmere goat as antifreeze respectively.After sperm freezing-thawing,the HSP90 expression in the sperm of 5%glycerol group was 61.28ng/ml,significantly higher than 3%ethylene glycol group(39.25ng/ml,P<0.05);In 5%glycerol group.HSP90 was distributed in the head and tail,with strong positive signal,while in 3%ethylene glycol group,HSP90 was distributed in the head and tail,and the positive signal was weak.After adding rotenone to inhertit the oxidative phosphorylation reaction of sperm,the expression of HSP90 in the sperm of both 5%glycerol group and 3%ethylene glycol group(37.93ng/ml,36.92ng/ml).All of them were significantly lower than those of 5%glycerol group and 3%ethylene glycol group(61.28ng/ml,39.25ng/ml,respectively,P<0.05);However,the addition of rotenone did not affect the distribution of HSP90 in the two groups.The above results show that although glycerol and ethylene glycol can affect the way of sperm metabolism,they have no effect on the distribution of HSP90 in sperm.In conclusion,glycerol and ethylene glycol as semen antifreeze were added to sperm diluent,which could affect the metabolism of sperm and the expression and distribution of HSP70 in sperm during the freezing-thawing process,but had no effect on distribution of HSP90.The expression and distribution of HSP70 and HSP90 in sperm might be regulated by different mechanisms.