In vitro embryo development in the pig: impact of oocyte maturation milieu on blastocyst morphology and viability

摘要

In this study, porcine embryos were produced in vitro from slaughterhouse sow or gilt oocytes which were matured and fertilized in vitro and subsequently cultured to the blastocyst stage. In vitro produced blastocysts are of poorer quality than their in vivo counterparts, and suffer from a high incidence of embryo mortality during culture and foetal mortality after (non-) surgical transfer. The causes of such abnormalities can possibly be ascribed to a number of factors such as the high incidence of polyspermy (oocyte penetration by more than one sperm cell), a reduced number of cells in transfer-stage blastocysts, deviant ratio of inner cell mass (cells giving rise to the foetus) to trophectoderm cells (cell layer developing into the placenta) and a higher incidence of programmed cell death (apoptosis). This study demonstrated that the in vitro oocyte maturation milieu, is an important determinant of blastocysts quality. Supplementation of oocyte maturation medium with sow follicular fluid, in contrast with gilt follicular fluid, reduced polyspermy and improved the morphological quality of resultant blastocysts, while modulation of hormone levels and addition of oviductal epithelial cells during in vitro oocyte maturation increased the total blastocyst cell number. Supplementation of the embryo culture medium with growth hormone improved blastocyst quality by reducing DNA fragmentation (albeit that apoptosis in all was not decreased) and enhancing blastocyst expansion. Growth hormone treatment did not improve pregnancy rates up to day 11 following non-surgical transfer, but all pregnancies resulted from batches of blastocysts which had a larger mean batch diameter. In the final part of the study, blastocysts were selected for quality by evaluation of diameter and gross morphology. Blastocysts of Class A quality showed enhanced survival after a novel culture-based test using cytochalasin-B, an actin cytoskeleton depolimerizing agent. In contrast to Class B embryos, the ratio of inner cell mass to trophectoderm cells was normal in Class A embryos and also comparable with in vivo produced embryos. These selection criteria lead to the first reported farrowing of 5 normal and healthy piglets following the non-surgical transfer of in vitro produced blastocysts. The results obtained in this study forms the basis for future research which will focus on the application of embryonic stem cells derived from the inner cell mass of in vitro produced porcine blastocysts.