| Artificial insemination has been widely used in modern pig industry, but only about 1% was used with frozen semen. The main reason was that the structure and function of sperm has been severely damaged during cryopreservation, resulting in a severely decline of frozen-thawed sperm motility farrowing rate, and litter size. Although lots of works had been done about the components of frozen extender and freezing procedures, the effect was still not satisfied. Studies have showed that cryopreservation was involved in sperm Caspase-mediated apoptosis processes. However, the effect of types and concentrations of cryoprotectants (CPAs) on apoptosis-related gene expression of Caspases is not well known. Therefore, the viability, motility of frozen-thawed boar spermatozoa with different cryoprotectants, sugars, antioxidants were evaluated by qRT-PCR, Annexin V-FITC. Furthermore, the mRNA expression of caspase genes, caspase protease activities and phosphatidylserine (PS) externalization level of frozen-thawed boar spermatozoa were detected. The results were as follows:(1) The GeNorm results revealed that the expression stability of PGK1, ACTB and RPL4 in all of the samples were high, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order.(2) The sperm motility and viability in LEY plus 6% EG、5% DMSO and in LEY +3% glycerol plus 0.035g/mL sucrose、250mmol/L trehalose.0.045g/mL lactose、200 IU/mL CAT、4 mmol/L GSH and 200 IU/mL SOD was the highest in the respective groups, but significant lower than LEY+3% glycerol treatment (50.81±1.33%,43.22±2.99%).(3) qRT-PCR analysis showed that, except for Caspase-1 and-13, the expression levels of Caspase-2,-6,-8, and -15 in 3% glycerol were significant lower than the control, which suggested that 3% glycerol could effectively inhibit apoptosis initiators. EG could not effectively inhibit the expression of Caspase-1,-6,-13 rather than Caspase-2,-8 and-15. The inhibitory effeciency of EG was less than 3% glycerol. The expression levels of Caspase-1,-2,-6 and -15 in 14% DMSO were significant lower or no significant difference compared to 3% glycerol. In addition, the expression levels of Caspase-1,-8 and-15 in 5% DMSO treatment had no significant difference with 3% glycerol. These resuts indicated that DMSO was a potential cryoprotectant for boar sperm cryopreservation. 0.035g/mL sucrose could effectively inhibit the expression of Caspase-1 and-15, but could not inhibit the expression of apoptosis initiators Caspase-2, Caspase-8 and inflammatory mediators Caspase-13.0.015g/mL lactose can effectively inhibit the expression of Caspase-2,-8,-13,-15.400IU/mL CAT could effectively inhibit the expression of Caspase-1, while 300IU/mL CAT could effectively inhibit the expression of Caspase-2, Caspase-8 and Caspase-13.4 mmol/L GSH and 300 lU/mL SOD could effectively inhibit the expression of Caspases.(4) The protease activity of caspase-3 In EG, DMSO, sucrose, trehalose and lactose treatments were very low. Except for 6% EG, Caspase-3 protease activities in each groups were significantly lower than the direct freezing group, but there was no significant difference between 3% glycerol and the control. Except for 2% EG and 5% DMSO, the Caspase-6/9 protease activity levels in the other treatments were significant lower than the control, but no significant difference was found compared to 3% glycerol. Caspase-6/9 protease activities in different concentrations of sucrose were significantlower than control and direct freezing; There was no significant difference of caspase-6/9 protease activities between 0.015g/mL and 0.025g/mL sucrose, but significant lower than 3% glycerol. The Caspase-6/9 protease activities in all concentrations of trehalose, lactose treatments were significant lower than control and direct freezing, and the Caspase-6/9 protease activities in 250mmol/L trehalose and 0.035g/mL lactose were the lowest., Caspase-8 protease activity in glycerol, EG, DMSO, sucrose and lactose treatments were significant lower than control and direct freezing. Caspase-8 protease activities were the lowest in 2% EG,5% DMSO and 0.035g/mL lactose, and all significantly lower than 3% glycerol. In addition, Caspase-8 activities in different concentrations of sucrose were significantly lower than 3% glycerol, and lowest in 0.035g/mL sucrose. In the trehalose treatment group, except for 375mmol/L trehalose, the remaining treatments were significantly higher than control, direct freezing and 3% glycerol.(5) Although the. percentage of Annexin-V+/PI+sperm in EG and DMSO treatments were significantly lower than the control, direct freezing and 3% glycerol, no significant differences between the control and 3% glycerol. Additionally, the percentage of Annexin-V+/PI+sperm in 0.015g/mL,0.045g/mL sucrose and lactose were significantly lower than the control, direct freezing and 3% glycerol. The percentage of Annexin-V+/PI+ sperm in 250 mmol/L and 425 mmol/L trehalose were significantly lower than direct freezing, but no significant differences compared with the control and 3% glycerol. The percentage of Annexin-V+/PI+ sperm in 200IU/mL and 300IU/mL CAT,3 mmol/L and 4 mmol/L GSH and 200IU/mL SOD were significantly lower than direct freezing, there were no significant differences compared to control and 3% glycerol. In addition, the percentage of Annexin-V+/PI+ sperm in EG treatment group was lowest, were high in enzymes and trehalose.In conculusion, adding appropriate concentrations of CPAs had protective effects on frozen-thawed boar spermatozoa, improving post-thawed sperm motility and viability, inhibiting caspases expression and protease activities of Caspase-3,-6/9 and -8,reducing PS externalization during boar spermatozoa cryopreservation. Our results play an important role for further choosing the appropriate CPAs and optimizing and improving the freezing extenders for boar sperm. |
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