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.     

Multivariate Cluster Analysis Regression Procedures as Tools to Identify Motile Sperm Subpopulations in Rabbit Semen and to Predict Semen Fertility and Litter Size

Computerized motility analysis (CASA) shows that four separate subpopulations of spermatozoa with different motility characteristics co-exist in rabbit ejaculates. There were significant (p < 0.01) differences in the distribution of these subpopulations among separate genetic lines, total sperm abnormalities and the percentage of altered acrosomes. Furthermore, logistic and linear multivariate regressions among several parameters of rabbit semen quality analysis were tested for use as predictive tools for the fertilizing ability of a specific artificial insemination semen sample. Logistic regression analysis rendered two mathematical, significant (p < 0.01) models: one between sperm viability and conception rate and the other between total sperm abnormalities and conception rate. Multiple linear regression analyses also yielded some significant relationships between both fertility (p < 0.001) and litter size (p < 0.05), with respect to some semen characteristics. Our results support the hypothesis that the predictive in vivo fertility use of the standard rabbit semen quality analysis coupled with a CASA determination could be reasonably achieved by applying linear and logistic regression analyses among several parameters of rabbit semen quality analysis.     

Natriuretic peptide system regulation in granulosa cells during follicle deviation and ovulation in cattle

Natriuretic peptides (NPs) are known to regulate reproductive events in polyovulatory species, but their function and regulation in monovulatory species remain to be fully characterized. Using a well‐established in vivo model, we found that bovine granulosa cells from follicles near the deviation stage express mRNA for the three NP receptors (NPR1, NPR2 and NPR3), but not for NP precursors (NPPA, NPPB and NPPC). The abundance of NPR3 mRNA was higher in dominant compared to subordinate follicles at the expected time of follicular deviation. After deviation, mRNA for all NP receptors was significantly more abundant in the dominant follicle. Intrafollicular inhibition of oestrogen receptors downregulated NPR1 mRNA in dominant follicles. In granulosa cells from preovulatory follicles, NPPC mRNA increased at 3 and 6 h after systemic GnRH treatment, but decreased at 12 and 24 h to similar levels observed in samples collected at 0 h. After GnRH treatment, NPR1 mRNA was upregulated at 24 h, NPR3 mRNA gradually decreased after 3 h, while NPR2 mRNA was not regulated. The mRNA expression of the enzyme FURIN increased at 24 h after GnRH treatment. These findings revealed that the expression of mRNA encoding important components of the NP system is regulated in bovine granulosa cells during follicular deviation and in response to GnRH treatment, which suggests a role of NP system in the modulation of these processes in monovulatory species.