Changes in functional parameters of cryopreserved boar spermatozoa capacitated in vitro

The aim of this study was to assess functional status of spermatozoa after cryopreservation, separation and capacitation to characterize the influence of these procedures on spermatozoa functional characteristics. The ejaculates of 7 boars were frozen-thawed, motile spermatozoa were separated on Percoll-gradient and capacitated in Tris-buffered medium with 1 mM caffeine at 39 deg C in atmosphere of 5 % CO2 for 3 hours. Motility, viability and chromatine integrity of spermatozoa were evaluated by phase contrast, Annexin V-FICT apoptosis detection kit and Sperm chromatin structure assay (SCSA), respectively. Data were analysed for high cryotolerant- (HCT-) boars (n = 4) and low cryotolerant- (LCT-) boars (n = 3). There were no differences in motility, viability or chromatin integrity between spermatozoa of both boar groups before semen freezing, but significantly higher motility and viability rates were found for spermatozoa of HCT-boars compared with those of LCT-boars after thawing. In HCT-boars, mean proportions of motile and viable spermatozoa decreased gradually during cryopreservation, separation and capacitation. The decrease in motility and viability of spermatozoa after thawing (from 78.8+/-2.1 to 46.3+/-7.8 % and from 85.3+/-2.1 to 43.0+/-7.3 %, respectively) and capacitation (from 51.8+/-5.5 to 36.8+/-7.4 % and from 35.0+/-3.7 to 29.9+/-4.6 %, resp.) was significant. In LCT-boars, mean proportions of motile and viable spermatozoa decreased after thawing (from 81.7+/-5.7 to 16.7+/-6.8 % and from 82.9+/-2.1 to 39.1+/-2.9 %, resp.), increased after separation (to 50.0+/-6.3 % and to 51.8+/-5.5 %, resp.) and decreased again after capacitation (to 44.2+/-3.7 % and 37.4+/-9.0 %, resp.). All differences were significant. In both boar groups mean percentages of spermatozoa with intact DNA did not changed during the whole evaluated period. In conclusion, the cryotolerance of spermatozoa in boars cannot be predicted before cryopreservation of their semen. Separation of motile spermatozoa on a Percoll- gradient was more effective for the low-cryotolerant than for the high-cryotolerant boars. The DNA of spermatozoa was highly stable during cryopreservation, separation and capacitation by caffeine.