Effects of cryopreservation on the motile sperm subpopulations in semen from Asturiana de los Valles bulls.

ABSTRACT

The aim of this study was to identify different motile sperm subpopulations in ejaculates from an autochthonous bull breed (Bos taurus) and to determine possible modifications in these subpopulations resulting from cryopreservation. Ejaculates were collected and cryopreserved following a conventional protocol. The overall sperm motility and the kinematic parameters of individual spermatozoa were evaluated in fresh ejaculates, after 4h at 5 degrees C, and at 0 and 2h postthaw. A multivariate clustering procedure separated 23,585 motile spermatozoa into four subpopulations Subpopulation 1 showed medium velocity (VCL 99.4+/-17.8 microm/sec) and high progressiveness (LIN 65.1+/-14.0%); Subpopulation 2 included spermatozoa with high velocity (VCL 148.7+/-25.6 microm/sec) but a nonprogressive trajectory (LIN 33.1+/-10.5%); Subpopulation 3 represented slowly motile (VCL 58.3+/-24.3 microm/sec) and nonprogressive sperm (LIN 39.6+/-18.3%); and Subpopulation 4 included very rapid (VCL 152.8+/-25.7 microm/sec) and highly progressive sperm (LIN 70.9+/-13.7%). Subpopulation 4 was present in the greatest quantity in fresh ejaculates (36%), but after cooling, it significantly decreased (21%) concomitantly with an increase (P<0.001) in Subpopulation 2 (from 21% in fresh to 34% in postcooled semen). After freezing and thawing, the overall sperm motility was reduced, mainly due to Subpopulation 2 decreasing from 34% after cooling to 14% after thawing. Differences among bulls in the frequency distribution of spermatozoa within subpopulations were evidenced after thawing by different proportions of spermatozoa in Subpopulations 2 and 4. The current results indicate that a structure of four sperm subpopulations may be a common characteristic of bovine ejaculates and that the cooling phase of cryopreservation seems to be the determinant of postthaw semen quality.