Sperm Energy-restriction and Recovery (SER) enhances equine sperm function and embryo development after ICSI.

Overview

• This study investigates the effect of sperm energy-restriction and recovery (SER) treatment on the function of frozen-thawed equine sperm and subsequent embryo development following intracytoplasmic sperm injection (ICSI).
• The research demonstrates that SER improves sperm motility and function, leading to a higher rate of blastocyst formation, thereby offering a promising method to enhance assisted reproductive technologies (ART) in horses.

Background and Motivation

• Assisted reproductive technologies (ARTs) are widely used in animal breeding but face challenges such as improving success rates.
• The equine industry struggles particularly with sperm capacitation, which has historically limited conventional in vitro fertilization (IVF) success.
• Due to these limitations, intracytoplasmic sperm injection (ICSI) is the predominant commercial method for producing equine embryos in vitro.
• Despite advancements, ICSI outcomes in horses remain variable, highlighting a need for improved sperm treatments that can boost functional competence.
• The SER strategy has previously shown benefits in mouse and bovine species by transiently restricting sperm energy followed by recovery to enhance fertilization and embryo development.

Experimental Approach

• Frozen-thawed equine sperm were subjected to energy restriction by incubating them in a medium lacking energy substrates such as pyruvate, lactate, and glucose (starvation or ST condition).
• After 20 minutes, when sperm motility was lost, sperm were recovered by transferring them back to a complete medium containing energy substrates (SER phase).
• Sperm functional parameters including motility, velocity, mitochondrial membrane potential, ATP levels, intracellular calcium, and acrosome reaction were measured.
• Phosphorylation markers related to capacitation (PKA substrates and tyrosine phosphorylation) were also assessed to check if SER induced capacitation changes.
• Finally, these sperm were used in ICSI to assess embryo development, specifically blastocyst formation by day 7.

Key Findings

• Motility and Kinetics:
  • Sperm became immotile within 20 minutes under starvation conditions.
  • Sperm motility was fully restored after energy reintroduction via SER.
  • SER-treated sperm showed increased curvilinear velocity (VCL) and lateral head displacement (ALH), indicating enhanced motility characteristics.
  • Linearity (LIN) of sperm movement decreased, reflecting more vigorous but less linear movement patterns compared to controls.
• Energy and Mitochondrial Function:
  • ATP content and mitochondrial membrane potential were reduced during starvation but recovered after SER treatment.
• Calcium and Acrosome Reaction:
  • Intracellular calcium levels increased during starvation and normalized after recovery.
  • Starved sperm exhibited a higher proportion of live acrosome-reacted cells, suggesting a transient physiological activation.
• Capacitation Markers:
  • No significant change in protein kinase A (PKA) substrates or tyrosine phosphorylation levels, indicating no premature or unintended capacitation from SER treatment.
• Embryo Development:
  • ICSI with SER-treated sperm resulted in a significantly higher proportion of blastocyst stage embryos by day 7 (44.2%) compared to controls (18%).

Implications and Significance

• SER is a simple and effective treatment that temporarily restricts sperm energy to improve motility and functional parameters upon recovery.
• The improvement in sperm function translates into better early embryo development outcomes following ICSI in horses.
• This approach potentially addresses a key limitation in equine assisted reproductive technologies by improving sperm quality prior to fertilization.
• SER could optimize commercial and research applications of in vitro embryo production in horses, contributing to more consistent and efficient ART outcomes.
• The study supports broader use of SER treatment across species given its efficacy demonstrated in mice, cattle, and now horses.

Conclusion

• Energy restriction followed by recovery (SER) enhances the functional competence of cryopreserved equine sperm.
• Such treatment results in improved sperm motility, normalized energy metabolism, and better early embryonic development after ICSI.
• SER represents a valuable technique to augment assisted reproduction success in the equine industry and may help overcome current challenges in sperm selection and treatment protocols.