Intracellular calcium chelating agent (BAPTA-AM) aids stallion semen cooling and freezing-thawing

This study aimed to investigate the effects of different concentrations of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N0N0-tetraacetic acid, tetra-acetoxymethyl ester (BAPTA-AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing-thawing. After collection, semen was extended (1:1 v/v) on a skim milk-based extender, centrifuged and resuspended at 400million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200M BAPTA-AM. Motility parameters were assessed after cooling in Equitainer at 5 degrees C for 12, 24, 48, 72 and 120hr and after freezing-thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved-thawed semen. Cooled stored (48hr) semen containing 50M BAPTA-AM and control extender (0M BAPTA-AM) was used to assess fertility. Inclusion of 50M BAPTA-AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p<0.05). Furthermore, semen cryopreserved in extender containing 50M BAPTA-AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p<0.05). Addition of 50M BAPTA-AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p>0.05). In conclusion, the addition of BAPTA-AM to semen extenders aided stallion semen cryopreservation in a dose-dependent manner. Furthermore, the cooling extender supplemented with 50M BAPTA-AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA-AM.