Effects Of Glycerol On Apoptotic Signaling Pathways Related Genes During Boar Spermatozoa Cryopreservation

Artificial insemination has been widely used in modern pig industry, AI with frozen-thawed semen was limited to only no more than 1%. However, compared to fresh sperm, AI with post-thawed boar spermatozoa results in low farrowing rates and reduces litter sizes. During cryopreservation, the process of frozen leads to sperm plasma membrane and mitochondrial membrane damaged and changes the membrane permeability, capacitation-like phenomenon even induces apoptosis. Although numerous studies have focused on the constituent of freezing extenders and freezing procedures, antifreeze mechanism of boar spermatozoa remains unclear. In this study, the perspective of cell apoptosis, we treated boar spermatozoa with different concentrations of glycerol to evaluate the sperm quality, the apoptosis-related gene expression and protease activities of Caspases. The results as follows:(1) The viability and motility of spermatozoa in 2% and 3% glycerol were significantly higher than those from 0%,5%,7% and 14% glycerol treatments (P<0.05);(2) The BCL-2/BAX ratio was significantly higher in spermatozoa treated with 2%and 3% glycerol compared to those from 0%,5%,7% and 14% glycerol treatments. The BCL-2/BAX ratio was significantly negatively correlated with Fas expression (r=-0.929, P=0.007).(3) The relative expression levels of Fas in spermatozoa treated with 3% glycerol was significantly lower than those from 0%,2%,5%,7% and 14% glycerol treatments. For FasL, there was significantly lower expression in spermatozoa treated with 2%,3% glycerol than in 0% and 14% glycerol.(4) Caspase-1 expression in spermatozoa treated with 3% glycerol was significantly higher compared to its expression in spermatozoa treated with 0%,2% and 10% glycerol. The expression of Caspase-2 in spermatozoa treated with 3% and 14% glycerol were significantly lower compared to those from other glycerol treatments(0%,2%,3%,7%, 10%), while the Caspase-2 expression in spermatozoa from the 2% glycerol treatment was higher than in spermatozoa from other glycerol treatments(0%,3%,5%,7%,10%, 14%). For Caspase-6 and Caspase-13,5% glycerol treatment induced significantly higher expression levels compared to other treatments(0%,2%,3%,7%,10%,14%), while 10% glycerol treatment induced significantly lower expression levels compared to other treatments(0%,2%,3%,5%,7%,14%). Caspase-8 expression was significantly reduced in spermatozoa from 3% glycerol treatment compared to all other treatments(0%,2%, 5%,7%,14%) except for 10% glycerol. Caspase-8 expression was significantly negatively correlated with the BCL-2/BAX ratio (r=-0.819, P=0.024) other than Fas expression (r=0.786, P=0.036). Spermatozoa exposed to 0% glycerol demonstrated significantly higher Caspase-15 expression than other treatments(2%,3%,5%,7%,10%, 14%). In response to 10% and 14% glycerol, spermatozoa showed significantly lower expression of Caspase-15 compared to other treatments(0%,2%,3%,5%,7%).(5) Glycerol treatments significantly reduced cellular Caspase-3,-6/9,-8 activities compared to the control. No significant differences were found in Caspase-3 protease activity among spermatozoa from all glycerol treatments. Caspase-6/9 activity in 2%x 3% glycerol-treated spermatozoa was significantly lower compared to those treated with 0%glycerol, while that was significantly higher compared to those treated with 5%,7%,10% and 14% glycerol. In spermatozoa from 2%,5% and 14% glycerol treatments, Caspase-8 activity was significantly higher than that in spermatozoa from 3% glycerol. However activity of Caspase-8 in 3% glycerol-treated spermatozoa was significantly higher than those from 7% and 10% glycerol treatments. Caspase-8 protease activity showed a significantly positive correlation with the expression levels of Fas(r=0.754, P=0.049).(6) The percentage of Annexin V+/PI+spermatozoa from 0% glycerol treatment were significantly higher than those from 2%,5%,7% and 10% glycerol treatments. There were no significant differences among spermatozoa from 2%,5%,7% and 10% glycerol treatments. Annexin V+/PI+ spermatozoa demonstrated a significantly positive correlation with Caspase-6/9 protease activity (r=0.840, P=0.037).