Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: Effect of different stages of in vitro maturation

Abstract

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus–oocyte complexes (COCs) were cultured in IVM medium supplemented with 1mM dibutyryl cAMP (dbcAMP) for 22h and then for an additional 22h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0–22, 26, 32, 38 and 44h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44h, but significantly decreased survival rate of oocytes matured for 0 or 22h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P<0.05) than that in the control. At 2h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44h of IVM were similar but were higher (P<0.05) than those of oocytes vitrified at 0 or 22h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38h of IVM did not differ from the control and were higher (P<0.05) than those of oocytes vitrified at 22, 26 or 32h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44h did not differ, but all were lower (P<0.05) than those matured 0h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.