No abstract

Summary: Reproductive biotechnologies such as cooling and freezing semen are employed in order to obtain genetic enhancement in domestic animal breeds. However, cryopreservation of donkey semen, as in several other domestic mammalian species, has not yet reached a standard procedure that provides satisfactory and repeatable results, as in bulls. Despite there are over 43 million donkeys worldwide, there are only few studies about donkey semen preservation. The aim of this study was to evaluate the predictive potential of hypo-osmotic test and the supra vital staining in raw semen to be used as indicators for donkey semen freezability. Ejaculates were collected twice per week from 10 fertile donkey stallions. Volume, appearance (color and density), concentration, total motility, morphology, sperm viability (supra vital staining -eosin-nigrosin-EN) and plasma membrane integrity (hypo-osmotic swelling test -HOST) were evaluated in raw semen. After dilution the samples were centrifugated and sperm pellets were resuspended in a freezing extender to a concentration of 50 ×10 6 cells/mL. Aliquots were packed into 0.5 mL straws and placed in the refrigerator (5°C) for 20 min. Subsequently these straws were kept above liquid nitrogen for 20 min, then plunged into nitrogen and stored in a holding tank. Post-thaw analyzes consisted in computerized analysis of sperm movement characteristics, sperm viability (EN), plasma membrane integrity (HOST and fluorescent probe) and acrosome membrane integrity using fluorescent probe. High correlation was found between EN (0.93) and HOST (0.69) in raw semen when compared with total motility after frozen-thawed semen. Low correlation was found between EN and HOST when compared with plasma and acrosomal membrane integrity in post-thawed with fluorescent probes. Since sperm motility is one of the key parameters to evaluate the quality of frozen semen, hypo-osmotic test and supra vital staining can be considered good predictive indicators in raw semen for donkey semen freezability.Keywords: Equus Asinus, cryopreservation, eosin-nigrosin, plasma membrane integrity, semen, reproduction Citation: Arruda de Oliveira R., Batista Silva Teixeira A., Almeida Pignataro T., Freitas M. L., Castro Alves Teixeira H., de Oliveira Carvalho J., Alves Nune Dode M., Pivato I., Budik S. (2017) Evaluation of Hypo-osmotic swelling test and supra vital staining technique as indicators for donkey semen freezability. Pferdeheilkunde 33,[159][160][161][162][163][164]

The objective of this study was to evaluate hormone and energy profiles in the postpartum period and to correlate these profiles with the resumption of ovarian cyclicity, as well as characterizing the postpartum short estrous cycle of Curraleiro Pé-Duro cows. Twelve Curraleiro Pé-Duro cows were examined via rectal palpation and ultrasound at 10 days postpartum, and subsequently examined daily to evaluate the resumption of ovarian cyclicity as well as every five days in order to evaluate uterine involution. Upon analysis of the data, it was possible to observe the formation of two distinct groups, one of which was comprised of those animals which returned to cyclicity within 60 days postpartum and another comprised of those animals which returned to cyclicity more than 105 days postpartum. Therefore, animals were divided into two groups; precocious, designated Ov Group, and delayed, designated NOv Group, wherein the cut-off time for all tests was 60 days postpartum. Statistically significant differences (P < 0.01) between the groups occurred only regarding the day of 1st ovulation, which in the Ov Group averaged 51.4 ± 9.3 days and in the NOv Group averaged 138.3 ± 19.8 days postpartum. The other postpartum short estrous cycle variables assessed did not show statistically significant differences (P > 0.05) between the groups. NEFA, BHBA and thyroxine concentration levels did not differ (P > 0.05) between the groups in any of the statistical analyses. However, in the analysis comparing growth curves, triglycerides levels were higher for the Ov Group (P = 0.04) and cholesterol levels were higher for the NOv Group (P = 0.02). In this experiment, a small influence of a negative energy balance between the groups was observed, suggesting that these animals can present significant genetic variability due to natural selection, as evidenced by the formation of groups of animals with precocious and delayed reproductive characteristics.

This study aimed to compare the effects a commercial milk-based extender and a self-made egg yolk extender had on the quality of canine semen stored at two different temperatures, 5ºC or 15ºC. The ejaculate obtained was split into two aliquots of equal volume and diluted with the milk or egg yolk extender. The final concentration was 100×106 spermatozoa/mL. Diluted semen was placed in transport containers and maintained at final storage temperatures of 5ºC and 15ºC. The quality of the chilled semen was assessed 12, 24, and 36 hours after storage. Semen diluted with the milk extender had higher motility, vigour, and plasma membrane integrity (p<0.05) of the spermatozoa than that diluted with the egg yolk extender. No difference in the semen quality was observed between the stored temperatures in both the groups. The difference observed between the extenders could be due to the standard formulation of the commercial milk extender and the presence of glucose in the mixture. In conclusion, the milk extender was better than the egg yolk extender at preserving the motility, viability, and membrane integrity of chilled canine semen for up to 36 hours. The storage temperature did not seem to affect the semen quality, suggesting that canine semen can be refrigerated at 15ºC.