Donkey sperm activating potential in heterologous ICSI

Abstract

There is a growing interest in the preservation and production of donkeys and mules, both species of significant importance in Argentina. The aim of this study was to evaluate the ability of cryopreserved donkey sperm to activate and induce pronuclear formation when injected into heterologous oocytes, as a potential predictor of future seminal capacity to produce embryos in vitro. Porcine cumulus-oocyte complexes (COCs) were obtained from ovaries of slaughtered gilts, selected, and then incubated, for 44 hours at 39°C, with 5% CO2 and 100% humidity, in 199 medium supplemented with 50 μg/ml gentamicin sulfate, 10% (v/v) porcine follicular fluid, 0.57 mM cysteine, 2 IU/ml equine chorionic gonadotropin, and 10 ng/ml epidermal growth factor. The matured COCs were denuded using serial passages through glass Pasteur pipettes. Donkey and equine sperm, cryopreserved with an egg yolk-free extender using a slow-freezing curve in an automatic freezer, were thawed in a water bath at 37°C for 1 minute and selected using Androcoll-ETM. A small aliquot was placed in a drop of 10% (v/v) polyvinylpyrrolidone in modified Whitten's (MW) injection medium. The oocytes were each placed in a drop of MW and intracytoplasmic sperm injection (ICSI) was performed. Porcine oocytes were injected with either equine sperm (control group) or donkey sperm (experimental group). As a control for the ICSI technique, and to assess parthenogenetic activation, mature oocytes were injected without depositing any sperm into the cytoplasm (Sham). Presumed zygotes were cultured for 16-18 hours in NCSU-23 medium at 39°C, with 5% CO2 and 100% humidity. They were then fixed for 15 minutes in 0.5% (v/v) glutaraldehyde in phosphate-buffered solution and stained for 10 minutes with Hoechst 33342 (5 µl/ml). Finally, they were classified as: 1) Activated: two pronuclei (2-PN) or one pronucleus with the presence of a decondensed sperm and 2) Non-activated: one pronucleus with the presence of a condensed sperm or no evidence of pronuclei. No significant difference was observed (p= 0.73) between activated porcine oocytes injected with donkey sperm: 66.67% (52/78) and those injected with equine sperm: 62.86% (44/70). Parthenogenetic development in the Sham group was 4.6% (3/65). Therefore, similar to equines, heterologous ICSI using donkey sperm could predict the future capacity of donkey semen for in vitro embryo production.