Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports

The aim of this study was to improve the freezing protocol of bull sperm, by investigating the influence on sperm viability after freeze/thawing of different freezing medium components, as well as the effect of cooling rates in the different stages of the cooling protocol, in single factor experiments. The experimental variables were: (1) salt-based versus a sugar-based medium (Tris versus sucrose); (2) glycerol concentration; (3) detergent (Equex) concentration; (4) presence of bicarbonate; (5) rate of cooling from 22 degrees C to holding temperature (CR1); (6) holding temperature (HT); (7) rate of cooling from holding temperature to -6 degrees C (CR2); (8) rate of cooling from -10 to -100 degrees C (CR3). All experiments were performed using five bulls per experiment (three ejaculates per bull). Sperm motility after freezing and thawing was assessed by CASA system, and sperm membrane integrity was assessed by flow cytometry. Sucrose-based medium did not offer a clear significant benefit compared to Tris medium. The concentration of Equex that gave the best results in Tris-based media group and sucrose-based media group was in a range between 2-7 and 4-7 g/l, respectively. In both media groups, a glycerol concentration of 800 mM was the best in any post-thaw viability parameters. In the Tris media group, the presence of bicarbonate had a negative effect on sperm viability. CR1 and CR2 had no significant effect on any of the post-thaw sperm viability parameters, but a CR1=0.2 degrees C/min and CR2=4 degrees C/min appeared to give better results in both media. The holding temperature (HT) that gave the best results was found to be in the range of 5-9 degrees C. There was a significant disadvantage of using a low CR3 of 10 degrees C/min, while 150 degrees C/min appeared to be the best cooling rate for either medium.