Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen

The aim of the present study was to evaluate three thawing rates for bull semen frozen in 0.25-ml straws: placing the straws in a water bath at 37 °C for 40 s, at 50 °C for 15 s or at 70 °C for 5 s. In a first experiment, the three thawing rates were compared in relation to post-thaw sperm motility, determined subjectively, and sperm plasma and acrosomal membrane integrity, examined by flow cytometry, after 0 and 5 h of incubation at 37 °C. In a second experiment, the three thawing rates were evaluated based on post-thaw sperm motility, determined using a CASA system, after 0 and 2 h of incubation at 37 °C. In addition, for the motile spermatozoa, the individual motility descriptors were analysed using a multivariate clustering procedure to test the presence of separate sperm subpopulations with specific motility characteristics in the thawed bull semen samples. Finally, it was investigated if the thawing rate had any influence on the relative frequency distribution of spermatozoa within the different subpopulations. In terms of overall post-thaw motility or plasma and acrosomal sperm membrane integrity there were no significant differences between the three thawing methods evaluated. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patterns of movement: (1) moderately slow and progressive sperm (27%); (2) “hyperactivated-like” sperm (15.4%); (3) poorly motile non-progressive sperm (34.3%); (4) fast and progressive sperm (23.3%). The thawing rate had no significant influence on the frequency distribution of spermatozoa within the four subpopulations, but there was a significant effect (P < 0.05) of the interaction between thawing rate and incubation time. Higher proportions of spermatozoa with fast and progressive movement were observed after 2 h of post-thaw incubation when the thawing was at the faster rates (35 °C/40 s: 8.3%, 50 °C/15 s: 18.1% and 70 °C/5 s: 16.5%). Whether this subtle difference might affect to the in vivo fertility of the thawed bovine semen is not known.