102 EFFECT OF DEMECOLCINE PRE-TREATMENT ON VIABILITY, TIMING OF FIRST POLAR BODY EXTRUSION, SPINDLE CONFIGURATION, AND SUBSEQUENT DEVELOPMENT OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE

Abstract

Oocyte cryopreservation is a potentially valuable way of preserving female germ cells. However, to date the reported developmental competence of cryopreserved oocytes is low. The objectives of this study were to investigate the effects of demecolcine pretreatment on viability, timing of the first polar body extrusion (PBI), spindle, chromatin organization, and in vitro embryo development of ovine vitrified germinal vesicle (GV) oocytes after in vitro fertilization (IVF) and parthenogenetic activation. Cumulus-oocyte complexes (COC) aspirated from ovine ovaries collected at slaughter were selected and randomly divided into 3 groups: (1) untreated (in vitro matured, IVM) as a control, (2) vitrified (Moawad AR et al. 2009 Reprod. Fertil. Dev. 21, 135 abst), and (3) deme + vitrified (oocytes were incubated with 0.1 μg mL-1 demecolcine for 20 min before vitrification). After vitrification COC were thawed and matured in vitro for 24 h. Following IVM, oocytes from 3 groups were subsequently subjected to (1) immunostaining, (2) IVF, or (3) activation. Presumptive zygotes were cultured in vitro in SOF media for 7 days. Data were analyzed using chisquare and t-test. No significant differences (P > 0.05) were observed in survival rates between deme + vitrified (90.8%, 324/357) and vitrified (87.2%, 211/242). However, the numbers of oocytes with PBI in two vitrified groups at 18 h (20.4 and 8.5 v. 47.1%) and 24 h post IVM (51 and 43.2 v. 88.5%) were significantly lower (P < 0.01) than those in the control. Percentage of normal spindle and chromatin configuration in the two vitrified groups also significantly decreased (P < 0.05) compared with those in the control (42.5 and 41.8 v. 76.5%), whereas missing spindle in the 2 vitrified groups significantly increased (P < 0.001) compared with the controls (47.5 and 32.7 v. 3.9%). Following IVF (pi), cleavage rates at 24.48 hpi and morula development (5 days pi) were significantly lower (P < 0.001) in deme + vitrified (6.1, 43.1, and 28.5%) and vitrified groups (3.3, 30.1, and 22.9%) than control (50.4, 82.4, and 46.4%). Blastocyst development in deme + vitrified (9.8%) and control (33.6%) was significantly higher (P < 0.01) than in vitrified group (1.3%). Hatched blastocysts were observed only in deme + vitrified and control groups (4.9 v. 12.8%). In addition, post activation (pa) cleavage rates in deme + vitrified (10.3 v. 40.7%) and control (52.5 v. 76.7%) at 24 and 48 hpa were significantly higher (P < 0.05) than those in the vitrified group. Blastocyst development in deme + vitrified (4.8%) was higher than that in the vitrified group (1.8%), but not significant (P > 0.05); however, these values were still significantly lower (P < 0.001) than those in the control (24.2%). No significant differences were observed in total cell numbers per blastocyst between all the groups. Taken together, these results suggest that pretreatment of oocytes with demecolcine before vitrification could improve the developmental competence of ovine vitrified-thawed GV-stage oocytes. A. R. Moawad was supported by the Egyptian government.