237 INCREASE OF BLASTOCYST DEVELOPMENTAL RATE IN VITRO BY SELECTION OF DEVELOPMENTALLY COMPETENT COW OOCYTES BEFORE IVM USING A STAINING TEST

Abstract

A large proportion of bovine oocytes fail to develop to blastocyst stage following maturation, fertilization, and culture in vitro. While suboptimal culture conditions undoubtedly contribute to this poor development, it is recognized that immature oocytes, especially from cows with reduced reproductive performance or which are slaughtered on the end of their use, are heterogeneous in quality and developmental competence (Gordon 2003). The aim of the present study was to increase the efficiency of blastocyst production from cows after IVM/IVF by oocyte selection before maturation. Immature oocytes are known to synthesize a variety of proteins (Wassarman PM 1988, Annu. Rev. Biochem. 57, 415–442), among them, glucose-6-phosphate dehydrogenase (G6PDH). This enzyme is active in the growing oocyte, but has decreased activity in oocytes that have finished their growth phase. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colorless (Erisson et al. 1993 Theriogenology 39, 214). The ovaries were obtained from a slaughterhouse and transported to the laboratory; cumulus-oocyte complexes (COCs) were recovered by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control – placed immediately into culture; (2) holding control – COCs kept in PBS containing 0.4% BSA for 90 min at 38.5°C before placement into culture; and (3) treatment – incubation with brilliant cresyl blue for 90 min at 38.5°C before culture. Treated oocytes were then divided into BCB− (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction in control, BCB−, and BCB+ groups; activity was significantly increased in BCB− COCs in comparison to the control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to Day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes (77.1 and 72.5%, respectively) than for BCB− oocytes (58.1%). The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than either control group (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB− oocytes (3.9%). The number of nuclei in the blastocysts was comparable in BCB+ and both control groups (105.5 ± 5.8 and 117.5 ± 8.5, 101.8 ± 6.2, respectively). Blastocysts in the BCB− group had a significantly lower cell number (61.0 ± 2.6) than did controls. The results show that the staining of COCs from cows before IVM may be useful in increasing the efficiency of blastocyst production during standard IVF procedures. In addition, classification of G6PDH activity on the basis of BCB staining may be used to effectively select cow oocytes with further developmental competence. To our knowledge, this is the first study to evaluate the association between G6PDH activity in oocytes and further blastocyst development in cows.