Effects of L-ascorbic acid or insulin-transferrin-selenium on in vitro maturation and energy metabolism on the developmental competence of porcine parthenogenetic embryos

Introduction: Porcine parthenogenetic embryos have emerged as promising tools in biomedical science. However, in vitro maturation and in vitro development often lead to elevated levels of reactive oxygen species and reduced developmental competence of the embryo. This study focused on advancing our understanding of how to increase the developmental competence of embryos. In addition, the embryos of large animals have specific metabolic pathways, and their energy substrate requirements for preimplantation are also more flexible than those of rodents. Little is known about the metabolism of porcine embryos from the 4-cell stage until morula. This study examines the effects of adding insulin-transferrin-selenium and/or L-ascorbic acid to the medium for maturing prepubertal porcine oocytes and establishes a new strategy of using glucose and pyruvate/lactate in porcine embryos cultured for standard protocols. Methods: The effects of insulin-transferrin-selenium and/or L-ascorbic acid during in vitro maturation were examined in experiment 1. In experiment 2, porcine parthenogenetic embryos were cultured in energy-free medium (days 0--5, 1--5, and 2--5, respectively) for the remaining duration in medium containing lactate-pyruvate or glucose for days 0--5 to investigate the effects of energy substrates in early developmental stages. Finally, the strategies of using glucose and lactate-pyruvate for in vitro development in Experiment 3 were determined, and the blastocyst quality and quantity were evaluated by culturing in different combinations of energy substrate and culture durations: (1) glucose (7 days); (2) lactate-pyruvate/energy-free/glucose (2 days/3 days/2 days, respectively); (3) lactate-pyruvate (7 days); and (4) lactate-pyruvate/glucose (2 days/5 days, respectively). Results: Supplementation with a combination of insulin-transferrin-selenium and L-ascorbic acid during in vitro maturation significantly improved the quantity and quality of blastocysts, whereas insulin-transferrin-selenium alone did not have a significant effect. Moreover, the embryos were capable of self-growth without an energy substrate throughout the preimplantation stages, resulting in late cleavage and 2-cell block. The exposure of the embryo to energy substrates for at least the first 48 hrs tended to have greater beneficial effects on embryo developmental competence. In addition, compared with glucose, exposure to lactate and pyruvate significantly increased both embryo quantity and quality. Conclusion: Adding a combination of insulin-transferrin-selenium and L-ascorbic acid to in vitro maturation medium could increase the developmental competence of porcine parthenogenetic diploid embryos. Survival and development at the 1-cell stage are energy dependent, but those from the 4-cell stage to the early blastocyst stage are energy independent. The morula-early blastocyst formation with lactate-pyruvate had a better result than those exposed to glucose only.