Content area
Full Text
Address for correspondence: Matej Murin. Institute of Animal Physiology and Genetics CAS, v. v. i., Rumburska 89, 277 21 Libechov, Czech Republic. Tel: +420 315 639 544. E-mail: [email protected]
Introduction
Porcine oocytes are usually retrieved from the ovaries of slaughtered gilts, mixing oocytes of different quality obtained at different stages of the oestrous cycle (Ishizaki et al., 2009). Strict criteria for evaluating oocyte quality before their submission for in vitro culture may increase the number of the oocytes suitable for in vitro embryo production. At present, the most frequent criterion for the selection of oocytes is their morphology, which mainly includes evaluating ooplasm granulation and the number of layers of cumulus cells. Recently, a series of biochemical methods based on biochemical markers has been developed to increase the rate of high quality oocytes retrieval from ovaries.
One of these methods is brilliant cresyl blue (BCB) vital labelling, which is based on the evaluation of glucose-6-phosphate dehydrogenase (G6PDH) activity as the marker of cytoplasmic maturation (Ishizaki et al., 2009). This enzyme is synthesized in BCB-negative (BCB−) oocytes, which are still in the growing phase. BCB− oocytes enzymatically break down BCB and remain colourless. In oocytes that have finished their growth (BCB-positive oocytes, BCB+) this enzyme is inactivated and oocytes stain blue. The quality of BCB+ oocytes is higher than the quality of BCB− oocytes (Kempisty et al., 2011; Antosik et al., 2014). This test has been used in oocytes of different species including pigs, mice, goats, cattle and buffaloes (Opiela and Kątska-Ksiązkiewicz, 2013). Conversely, a negative influence of double BCB staining on fertilization in vitro, cleavage and development to the blastocyst stage has been documented in pigs (Wongsrikeao et al., 2006; Pawlak et al., 2011). However, it is important to state that previously published data showed that single BCB staining is a highly effective method for the evaluation of oocyte quality, as they are still viable after BCB labelling (Kempisty et al., 2011; Antosik et al., 2014).
Evaluations of the germinal vesicle chromatin configuration in porcine oocytes and of the ultrastructure of nucleoli and immunolabelling of their key nucleolar proteins in in vivo-developed, in vitro-produced embryos and parthenotes have already been published (Bjerregaard et al