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

Overview

• This research investigates the ability of donkey sperm to activate oocytes and support the development of mule embryos through Intracytoplasmic Sperm Injection (ICSI), focusing on comparing its effectiveness in different species and understanding molecular mechanisms involved.

Background and Significance

• The Equus genus includes species capable of hybridizing to create healthy offspring, with mules (offspring of a mare and a jack donkey) being the most common hybrid.
• While assisted reproductive technologies like ICSI have rapidly advanced in domestic horses, there is limited knowledge and application of in vitro embryo production in other related equids such as donkeys.
• Studying donkey sperm’s capability to fertilize horse oocytes in vitro can help address this gap and improve reproductive techniques for donkeys.

Objectives of the Study

• Characterize the localization of Phospholipase C zeta (PLCζ), a key sperm protein involved in oocyte activation, in donkey sperm cells.
• Evaluate donkey sperm’s ability to activate oocytes and support embryo development through ICSI in two models: pig oocytes (heterologous system) and horse oocytes (homologous system for hybrids).
• Assess the development of embryos up to blastocyst stage after injecting donkey sperm, including comparisons between frozen and refrigerated sperm samples from different donkey males (jacks).

Key Experimental Approaches

• PLCζ Localization: Examined binding patterns of PLCζ in donkey sperm and compared them to horse sperm, to identify any differences in the fertilization-associated protein distribution.
• ICSI in Pig Oocytes: Donkey sperm was injected into pig oocytes to test the sperm’s capacity to induce pronuclear formation and recruit maternal SMARCA4, a chromatin remodeling factor important during embryo development.
• ICSI in Horse Oocytes: Donkey sperm was injected into horse oocytes, with subsequent monitoring of embryo cleavage and development up to the blastocyst stage to evaluate in vitro mule production.
• Sperm Sample Variability: Sperm from different jacks and handling methods (frozen vs. refrigerated) were tested to determine consistency in activation ability and embryo development outcomes.

Main Findings

• Donkey sperm showed distinct patterns of PLCζ localization compared to horse sperm, implying potential species-specific differences in how sperm initiate oocyte activation.
• Donkey sperm has a reduced capacity to activate pig oocytes, indicating that the molecular signals or mechanisms triggered by donkey sperm may not be efficiently recognized by pig oocytes.
• When donkey sperm was injected into horse oocytes, oocyte activation was effectively induced as evidenced by comparable cleavage and blastocyst rates between mule embryos and horse embryos produced via ICSI.
• The recruitment of the maternal factor SMARCA4 did not show noticeable differences after donkey sperm injection, suggesting that this aspect of embryo development was similarly triggered despite species differences.
• Frozen and refrigerated donkey sperm samples from different individuals similarly supported the in vitro production of mules, highlighting practical implications for assisted reproduction.

Implications and Conclusions

• This study provides novel insight into the molecular and functional capabilities of donkey sperm during fertilization, particularly through the lens of PLCζ localization and activation potential.
• The reduced activation observed in pig oocytes demonstrates how species-specific interactions play a role in fertilization efficacy during ICSI, which could inform species-tailored reproductive technologies.
• Successful activation and embryo development using donkey sperm and horse oocytes confirm the feasibility of producing mule embryos in vitro, potentially advancing conservation and breeding programs.
• Understanding sperm factors like PLCζ and maternal responses such as SMARCA4 recruitment offers a molecular foundation for improving assisted reproduction techniques across equid species.
• Overall, donkey sperm is capable of effectively activating horse oocytes and supporting embryo development despite some molecular differences from horse sperm, enabling efficient in vitro mule production.