Effect of milrinone on the developmental competence of growing lamb oocytes identified with brilliant cresyl blue

Abstract

Juvenile in vitro embryo transfer is a novel technique that can be used to increase the rate of genetic gain in a population and presents an alternative to embryo technologies on the basis of adult animals. However, oocytes from prepubertal animals have a lower viability than those obtained from adult ewe oocyte donors. In this research, we aimed to determine the optimum concentration and time of treatment of oocytes from prepubertal lambs with brilliant cresyl blue (BCB) stain and milrinone during IVM. This would improve the developmental rate of lamb oocytes and embryos after IVF. First, lamb cumulus–oocyte complexes were cultured under different concentrations (13 or 26 μM) of BCB staining. Treated lamb oocytes were then divided into BCB− (colorless cytoplasm) and BCB+ (colored cytoplasm) groups on the basis of their glucose-6-phosphate dehydrogenase activity. The blastocyst efficiency rate of BCB+ oocytes treated with 13 μM BCB (37.03%) was significantly higher than that of BCB+ oocytes treated with 26 μM BCB (23.25%) and that of nontreated BCB control oocytes (15.37%), as well as that of BCB− oocytes (6.28%). Both control oocytes and BCB+ oocytes exhibited significantly higher cleavage rates (60.15% and 73.44%, respectively) than that of BCB− oocytes (36.19%). Moreover, the diameter and glutathione content of BCB+ oocytes were found to be significantly greater than those of BCB− oocytes (163.37 vs. 159.25 μm and 6.39 vs. 0.26 pM, respectively). After culturing BCB− oocytes in different concentrations of milrinone (0, 50, 75, and 100 μM) for 3, 6, or 9 hours, results reported that supplementation of IVM medium with 75 μM milrinone for 6 hours yielded a significantly higher proportion of blastocysts than the other treatments. These results show that the staining of lamb cumulus–oocyte complexes with 13 μM BCB before IVM may be used to select developmentally competent lamb oocytes. Furthermore, they suggest that milrinone can be used to promote lamb developmental competence of lamb embryos produced during IVF.