• Donkey
• fertility
• semen cryopreservation
• seminal plasma

Pregnancy rates in donkeys after artificial insemination (AI) with cryopreserved semen are still low, compared to the horse species. The aims of this thesis were to evaluate if the resuspension of frozen-thawed donkey spermatozoa in seminal plasma (SP) could affect semen quality and jennies endometrial response and fertility after AI. In Study 1 it was observed that the proportion of polymorphonucler cells (PMN) in uterine flushings performed 6-10 hours after AI was increased when 70% SP was added to the AI dose, compared to an extender. Moreover, seminal plasma showed a trend toward improvement of pregnancy rates but differences (60% and 20% for addition of SP and extender, respectively) were not statistically significant. In Study 2 it was shown how, compared to control (0% SP), 70% SP significantly decreased post-thaw motility and plasma membrane integrity during 2 hours of in vitro preservation at 37°C. In Study 3, where lower seminal plasma concentrations were tested (5 and 20%) during 4 hours of incubation at 37°C, sperm motility and the proportion of chromatin damaged sperm cells were not affected by the presence of SP immediately post-thaw, compared to control, while plasma membrane integrity was lower in the 20% samples. After 4 hours, however, all main semen characteristics were significantly worse in the 20% seminal plasma samples, compared to control, while the 5% SP samples showed intermediate values. In Study 4 no differences between AIs with 70% SP added or AIs without post-thaw dilution were seen for proportion of PMN evaluated in uterine flushings 24 hours after AI or fertility (1/5 in both groups). Combining the results of Studies 1 and 4, post-thaw addition of 70% seminal plasma to frozen-thawed donkey spermatozoa did not improve pregnancy rates, although there was a trend in this direction. In vitro, both 70% or lower SP concentrations (5% and 20%) did not improve neither motility nor viability or chromatin integrity. The presence of seminal plasma during AI promoted the PMN influx into the uterus 6-10 hours post AI and did not shorten mating induced endometritis compared to the control. In the donkey species, both the interactions of seminal plasma with the sperm cell and the endometrium and their effects on fertility still need to be better explained.