The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Altres ajuts: Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria RZ2009-00008-00-00The Cabra Blanca de Rasquera is a native goat breed from Catalonia (Spain) facing the danger of extinction. To preserve its genetic diversity, ex situ conservation programs for semen cryopreservation and the development of a genetic sperm bank are of major importance. However, the post-thaw sperm quality in this species is still a major concern. Therefore, the aim of this research work was to establish an approach in the thawing protocol of cryopreserved sperm bank doses of this breed in order to achieve a better post-thaw sperm quality and improve reproductive outcomes through artificial insemination. Since seminal plasma has been found to provide a source of nutrients, improve sperm motility, and prevent premature activation during migration of spermatozoa in the female reproductive tract, our study proposed to assess the effect of the addition of 20% seminal plasma (SP) during a 3 h incubation period under in vitro conditions mimicking the in vivo survivability of thawed sperm, in relation to donor age, the season of collection, and melatonin implants of the males in the non-breeding season. However, the strategy of adding seminal plasma to the sperm after thawing failed to improve buck post-thawed sperm quality, thus further investigation is needed. In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant