162 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE IN RESISTANCE TO EMBRYO CRYOPRESERVATION

Abstract

In vitro embryo production by maturation, fertilization, and culture has become a valuable assisted reproductive technology in cattle breeding systems. However, even with the notable innovations in this system, the greatest obstacles for the complete success of this biotechnology are the low pregnancy rates after transfer and the greater sensitivity to cryopreservation. Thus, this study evaluated the effect of the addition of a meiotic arrest using butyrolactone I and roscovitine on in vitro production of bovine embryos. Nelore oocytes were matured in TCM-199 with Earle's salt + 10% FCS, FSH, and LH, in 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in presence of butyrolactone I (BL-I) 25 μM + roscovitine (ROS) 6.25 μM. Then, the oocytes (Control n = 405; BL-I + ROS n = 386) were cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the conditions above. Semen was selected through Percoll gradient and the concentration adjusted to 2 × 106 sperm mL–1. The presumed zygotes were culture in 90-μL droplets of SOFaa + 0.6% BSA + 2.5% FCS in 5% CO2 until Day 7, when the blastocyst rate was evaluated. We performed 5 replicates. Data were analysed with ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. Statistical differences were observed in blastocyst production rate: Control: 42.64% and BL-I + ROS: 55.96% (P < 0.05). The remaining embryos (Control n = 163; BL-I + ROS n = 182) were vitrified, warmed, and recultured in SOFaa with 10% FCS for 12 h. After this period, re-expansion rate was determined. We found difference in re-expansion rates of groups (Control: 84.66% and BL-I + ROS: 93.40%; P < 0.05). Additionally, differences were found among total number cell groups: Control: 54.33 and BL-I + ROS: 97.5; P < 0.05). The use of BL-I + ROS is able to better a producing rate of in vitro embryos and improve the cryotolerance. The authors acknowledge FAPESP 2010/18994-4 and 2011/08926-4 for financial support.