Estrategias de mejora de la inseminación artificial ovinauna perspectiva seminal

Abstract

The optimization of artificial insemination (AI) in the ovine species is crucial to increase its implementation, maximizing production performance and genetic gain in this farming. Different strategies are analyzed in this sense in the following Doctoral Thesis, compiled into a total of five scientific articles. The first two papers focused on defining the ideal conditions for centrifuging ram semen, considering that sperm damage caused by this procedure is species-specific. Publication I evaluated the performance of three centrifugal forces (600, 3,000 and 6,000 x g for 10 minutes at room temperature) and their effects on sperm motility and functionality. Centrifugation forces equal to or greater than 3,000 x g induced a deleterious effect in sperm quality at both levels, and 600 x g not provide a successful cell packaging. Therefore, the first trial of Publication II assessed the effect of two cooling methods at 15 °C (fast or slow) and two diluents (INRA 96® y Tyrode’s) on sperm recovery rate, pellet weight and sperm quality after centrifugation of semen at 600 x g for 10 minutes. INRA 96® combined with slow refrigeration and Tyrode’s at room temperature registered the best results. In a second step, the influence of three centrifugal forces (600, 1,200 and 6,000 x g for 10 minutes) on the same parameters was evaluated with INRA 96® and Tyrode’s under the previously selected conditions. The highest quantitative and qualitative efficiency was achieved at 1,200 x g in both extenders, since 6,000 x g impaired sperm motility. Finally, to test the in vivo effects of the centrifugation protocol, a fertility trial was performed using semen centrifuged and stored at 15 °C for 6 hours. The damage produced by 6,000 x g on sperm motility was maintained until 6 hours, and a decrease in fertility after cervical AI was also observed. This confirmed the suitability of the centrifugation protocol at 1,200 x g for 10 minutes (in INRA 96® and at 15 °C). In Publication III, because of the scarce and conflicting information on seminal plasma (SP) withdrawal as part of ram semen refrigeration protocols, the role of SP in medium-term semen preservation in liquid form (up to 48 hours at 5 °C) was investigated. To this end, the effect of SP addition to ram epididymal sperm at the beginning or at the end of the preservation protocol was assessed in a first step, and sperm motility and functionality were analyzed after 6 hours at 15 °C, and 24 and 48 hours at 5 °C. Sperm showed higher quality at 48 hours when stored in the absence of SP, while a final SP supplementation resulted in improved sperm motility and functionality. In addition, the effect of SP removal from freshly ejaculated ram seminal samples using the previously optimized centrifugation protocol was evaluated under the same storage conditions in a second experience. The removal of SP decreased sperm functionality after 24 and 48 hours at 5 °C, while the final supplementation had positive effects on quality and fertility in cervical AI. Although SP absence proved to be beneficial in a medium-term liquid preservation protocol in a sperm model without previous contact with this substance (epididymal sperm), the lack of this finding in ejaculated seminal samples under the same preservation protocol indicates the possible failure of the SP removal method. The last two articles aimed to optimize sperm quality evaluation in rams, as the selection of males and ejaculates with good fertilizing potential is essential for use in ovine AI. In this regard, Publication IV focused on determining the best time to conduct this assessment within a preservation protocol at 15 °C for 6 hours. Sperm motility and functionality were determined at 0 hours (30 °C), 3 and 6 hours (15 °C), and 24 hours (5 °C) as a positive damage control, and both were found to improve at 6 hours. Additionally, the responsible factor of the sperm instability during the first hours of liquid storage was investigated testing the interactions of epididymal sperm with SP and extender (independently and in combination) under the same storage conditions used in the previous experience. Sperm from groups with diluent showed altered motility and functionality at 0 hours, indicating that extender addition initially acts as a destabilizing factor, requiring 3 to 6 hours of adaptation to the extender before an accurate assessment of sperm quality. Finally, in Publication V, when trying to identify some male factors involved in the different fertility rates obtained after cervical AI at two times of the breeding season (early and late), no differences were found in the ultrasonographic evaluation of ram testicular complex nor in the sperm quality after 6 hours at 15 °C. In contrast, a sperm proteome analysis detected a lower expression of proteins related to energy metabolism, sperm-oocyte interactions, and flagellum structure at the time of the reproductive season with lower fertility. Overall, the results of this Doctoral Thesis demonstrated the importance of optimizing protocols for ram semen handling, preservation and quality analysis in order to design really effective strategies to improve sheep AI.