Sperm energy-restriction and recovery enhances equine sperm function and embryo development after ICSI

Assisted reproductive technologies (ARTs) are extensively used in domestic animal reproduction; however, improving their efficiency and consistency remains a major challenge for achieving reliable reproductive outcomes. The equine industry faces unique challenges related to sperm capacitation, which limited the success of conventional in vitro fertilization (IVF) for many years. Consequently, intracytoplasmic sperm injection (ICSI) became the most widely used technique for commercial in vitro embryo production (IVP). Even though a successful IVF protocol was recently developed, substantial opportunities remain to improve its efficiency and consistency in the equine species. In this context, strategies that enhance sperm functional competence represent a key avenue to improve equine IVP. Sperm energy-restriction and recovery (SER) treatment has been shown to improve fertilization and embryo development rates in mice and bovine species following IVF and ICSI, respectively. Here, we evaluated the effect of SER on cryopreserved equine spermatozoa by assessing sperm functional parameters and embryo development after ICSI. Frozen-thawed sperm were incubated without pyruvate, lactate, and glucose (starvation; ST) and subsequently recovered using a complete medium (SER). Under ST conditions, sperm became immotile within 20 min, and motility was restored by SER to control levels. Compared with controls, SER-treated sperm exhibited increased curvilinear velocity (VCL; 246.4 vs. 201.6 µm/s) and amplitude of lateral head displacement (ALH; 9.8 vs. 7.3 µm), accompanied by reduced linearity (LIN; 34.4% vs. 43.4%; P < 0.05). ST reduced ATP content and mitochondrial membrane potential and elevated intracellular Ca2+ levels; all were restored following SER. Consistent with this transient Ca2+ rise, ST sperm displayed a higher percentage of live acrosome-reacted cells than controls (P < 0.05). No differences were detected in protein kinase A substrates or tyrosine phosphorylation, two markers of capacitation. Finally, ICSI using SER-treated sperm resulted in a greater proportion of day-7 blastocysts compared with controls (44.2% vs. 18%; P < 0.05). Collectively, these findings indicate that SER enhances equine sperm function and improves early embryo development, highlighting its potential to advance reproductive biotechnologies in this species.