Effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations.

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.

Assessment of ram sperm membrane integrity following different thawing procedures.

Semen from five 2.5-yr-old rams selected for use in an AI program was collected over 3 consecutive days using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in French mini-straws (approx. 100 mill/straw), and frozen in a programmable freezer. Three freezing operations were carried out per ram. Three straws per freezing operation were subjected to the following thawing procedures: 1) 70 degrees C, 5 sec; 2) 50 degrees C, 9 sec and 3) 35 degrees C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluorochromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), the hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NAR's) were used to determine the degree of sperm membrane integrity. Significant differences between thawing treatments were found for post-thaw motility (P < .05) and membrane integrity (P < 0.01), and variation among rams was statistically significant. Post-thaw sperm motility as well as the percentage of spermatozoa showing intact membranes were significantly higher (P < 0.01) for straws thawed at 70 degrees C than for those thawed at 35 degrees C (67.0 +/- 1.1 and 63.0 +/- 1.1%, and 50.5 +/- 1.5 and 41.7 +/- 1.5%, respectively). However, no corresponding statistically significant difference could be found for these parameters when 70 degrees C and 50 degrees C thawing were compared. It was concluded that sperm can be thawed at 50 degrees C for 9 sec instead of 70 degrees C for 5 sec without further reducing sperm motility or membrane integrity. This lower thawing temperature would facilitate the widespread use of frozen/thawed ram semen under farm conditions in Sweden.