Effect of BAPTA-AM on Thawed Stallion Spermatozoa Extended in INRA 96 or Tyrodeʹs Medium

We studied the effect of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) on the outcome of cryopreservation of stallion spermatozoa and whether reextension of thawed sperm in a more physiological and Ca2+-containing medium might improve the characteristics of thawed stallion spermatozoa. Individual ejaculates from six stallions were collected and split into three subsamples. The first two samples were supplemented with the membrane-permeable Ca2+ chelator BAPTA-AM at final concentrations of 5 and 10 μM, respectively, while the third subsample served as control. After 4 weeks of storage, samples were thawed in a water bath at 37°C and evaluated using flow cytometry and computer-assisted sperm analysis (CASA). In a second experiment, in order to determine whether restoring Ca2+ could improve sperm quality after cryopreservation, thawed semen was washed by centrifugation and resuspended in Tyrodeʹs complete medium. BAPTA-AM supplementation did not modify the outcome of cryopreservation; however, changing the spermatozoa from INRA 96 to Tyrodeʹs complete medium resulted in significant improvements in the percentages of live sperm and total motility post thaw.