The ability of donkey sperm to induce oocyte activation and mule embryo development after ICSI.

Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey sperm's fertilization capability in vitro to further improve donkey ICSI. In this work, we initially studied the localization of donkey sperm Phospholipase C zeta (PLCζ) and assessed the sperm's capacity to induce pronuclear formation and maternal SMARCA4 recruitment upon injection into pig oocytes through ICSI. Subsequently, we investigated the injection of donkey sperm into horse oocytes, evaluating in vitro production up to the blastocyst stage using sperm from different jacks, including frozen and refrigerated samples. Distinct patterns of PLCζ localization were observed for donkey sperm cells compared to their horse counterparts. Additionally, donkey sperm exhibits a reduced ability to induce porcine oocyte activation. However, when injected into horse oocytes, donkey sperm demonstrated sufficient capability to induce oocyte activation as no discernible differences in cleavage or blastocyst rates are observed between in vitro produced mules and horse ICSI embryos. Our study not only delineates PLCζ localization in donkey sperm but also suggests potential differences in the ability to induce oocyte activation in pigs compared to horses while observing no distinctions in pronuclear recruitment of SMARCA4. Interestingly, donkey sperm remains sufficiently capable of inducing horse oocyte activation for in vitro mule blastocyst production.

Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation.

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

Cryopreservation of Prochilodus lineatus semen: effect of cryoprotectants combination / Criopreservação do sêmen do Prochilodus lineatus: efeito da combinação de crioprotetores

The study was conducted with the objective of comparing the toxicity and the effect of the combination of intra and extracellular cryoprotectants in curimba Prochilodus lineatus sperm cells subjected to cryopreservation. Semen from 19 males were analyzed and diluted in four solutions comprise of intra and extracellular cryoprotectants in the following combinations: methanol+lactose, methanol+egg yolk, DMSO+lactose and DMSO+egg yolk. A portion of the diluted semen was frozen while the remaining fraction was kept in repose and evaluated after 10 min. For freezing, the diluted samples were packaged in 0.50 mL straws and placed into liquid nitrogen vapor for 24 h and, after this time, submerged into liquid nitrogen for 10 days. The combination of DMSO+lactose was less toxic to the diluted semen, resulting in higher motility rates and durations when compared to the other treatments. After thawing, the highest motility rate and duration were obtained using lactose as extracellular cryoprotectant, regardless of its combination. There was no significant difference between treatments when analyzing sperm morphology after thawing. Considering the effects of the tested treatments, the use of lactose as an extracellularcryoprotectant added with DMSO or methanol is the most suitable, since these combinations presented the highest motility rates and durations and low morphological change rate after thawing.(AU)

O estudo foi realizado com o objetivo de comparar a toxicidade e o efeito da combinação de crioprotetoresintra e extracelulares em espermatozóides de curimba Prochilodus lineatus submetidos à criopreservação. O sêmen de 19 machos foi analisado e diluído em quatro soluções com crioprotetores intra e extracelulares nas seguintes combinações: metanol+lactose, metanol+gema de ovo, DMSO+lactose e DMSO+gema de ovo. Uma porção do sêmen diluído foi congelada enquanto que a fração remanescente foi mantida em repouso e avaliada após 10 min. Para o congelamento, as amostras diluídas foram envasadas em palhetas de 0,50 mL e colocadas em vapor de nitrogênio líquido durante 24 h e, após este tempo, submersas em nitrogênio líquido durante 10 dias. A combinação de DMSO+lactose se mostrou menos tóxica para o sêmen diluído, resultando em taxas mais elevadas de motilidade e duração espermática quando comparadas com os outros tratamentos. Após descongelamento, as maiores taxas de motilidade e duração foram obtidas usando lactose como crioprotector extracelular, independentemente da sua combinação. Não houve diferença significativa entre os tratamentos ao analisar a morfologia espermática após a descongemento. Considerando-se os efeitos dos tratamentos utilizados, o uso de lactose como crioprotector extracelular, adicionada ao DMSO ou metanol, é o mais adequado, uma vez que estascombinações apresentaram maiores taxas de motilidade e duração espermática, e baixa taxa de alteração morfológica após o descongelamento.(AU)

Cryopreservation of Prochilodus lineatus semen: effect of cryoprotectants combination / Criopreservação do sêmen do Prochilodus lineatus: efeito da combinação de crioprotetores

The study was conducted with the objective of comparing the toxicity and the effect of the combination of intra and extracellular cryoprotectants in curimba Prochilodus lineatus sperm cells subjected to cryopreservation. Semen from 19 males were analyzed and diluted in four solutions comprise of intra and extracellular cryoprotectants in the following combinations: methanol+lactose, methanol+egg yolk, DMSO+lactose and DMSO+egg yolk. A portion of the diluted semen was frozen while the remaining fraction was kept in repose and evaluated after 10 min. For freezing, the diluted samples were packaged in 0.50 mL straws and placed into liquid nitrogen vapor for 24 h and, after this time, submerged into liquid nitrogen for 10 days. The combination of DMSO+lactose was less toxic to the diluted semen, resulting in higher motility rates and durations when compared to the other treatments. After thawing, the highest motility rate and duration were obtained using lactose as extracellular cryoprotectant, regardless of its combination. There was no significant difference between treatments when analyzing sperm morphology after thawing. Considering the effects of the tested treatments, the use of lactose as an extracellularcryoprotectant added with DMSO or methanol is the most suitable, since these combinations presented the highest motility rates and durations and low morphological change rate after thawing.

O estudo foi realizado com o objetivo de comparar a toxicidade e o efeito da combinação de crioprotetoresintra e extracelulares em espermatozóides de curimba Prochilodus lineatus submetidos à criopreservação. O sêmen de 19 machos foi analisado e diluído em quatro soluções com crioprotetores intra e extracelulares nas seguintes combinações: metanol+lactose, metanol+gema de ovo, DMSO+lactose e DMSO+gema de ovo. Uma porção do sêmen diluído foi congelada enquanto que a fração remanescente foi mantida em repouso e avaliada após 10 min. Para o congelamento, as amostras diluídas foram envasadas em palhetas de 0,50 mL e colocadas em vapor de nitrogênio líquido durante 24 h e, após este tempo, submersas em nitrogênio líquido durante 10 dias. A combinação de DMSO+lactose se mostrou menos tóxica para o sêmen diluído, resultando em taxas mais elevadas de motilidade e duração espermática quando comparadas com os outros tratamentos. Após descongelamento, as maiores taxas de motilidade e duração foram obtidas usando lactose como crioprotector extracelular, independentemente da sua combinação. Não houve diferença significativa entre os tratamentos ao analisar a morfologia espermática após a descongemento. Considerando-se os efeitos dos tratamentos utilizados, o uso de lactose como crioprotector extracelular, adicionada ao DMSO ou metanol, é o mais adequado, uma vez que estascombinações apresentaram maiores taxas de motilidade e duração espermática, e baixa taxa de alteração morfológica após o descongelamento.