Abstract
ABSTRACT: INTRODUCTION: Despite advances in Assisted Reproductive Technologies (ART), obtaining developmentally competent embryos remains a major challenge. We previously demonstrated that modulating sperm metabolism through Sperm Energy Restriction and Recovery (SER) enhances sperm function and improves fertilization and embryo development rates following in vitro fertilization in mice. OBJECTIVE: To optimize SER in bovine and equine species. METHODS: Cryopreserved sperm were incubated in either complete medium (control) or nutrient-deficient medium (ST), followed by recovery with metabolic substrates (SER). Sperm function was assessed by evaluating motility and capacitation-associated markers, including phosphorylation of PKA substrates (pPKAs), tyrosine phosphorylation (pY), and intracellular Ca(2+) levels. Control and SER bovine sperm were then used for intracytoplasmic sperm injection (ICSI). RESULTS: SER improved sperm motility in both species, while no differences were observed in pPKAs or pY levels in either species. ST equine sperm showed higher intracellular Ca(2+) levels and a greater percentage of live, acrosome-reacted cells compared to controls (14.4% vs. 6.6%, p < 0.05). In bovines, ICSI using SER sperm yielded higher 2-cell embryo rates than controls (50% vs. 16%, p < 0.05), and 17% of these embryos developed to the blastocyst stage, whereas none did in the control. SER also enhanced egg activation post-ICSI, with more oocytes displaying greater than 3 Ca(2+) peaks (40% vs. 10%, p < 0.05). CONCLUSION: SER improves sperm function in both bovine and equine species and enhances egg activation after bovine ICSI. These findings suggest that targeting sperm metabolism may increase the efficiency of in vitro embryo production across species. (Supported by USDA 2022-67016-41939)