Cryopreservation of bovine embryos cultured under different oxygen tensions with or without addition of antioxidants in differents steps of culture

Abstract

It is known that the oxygen tension and the presence of antioxidants have effects on the development of the embryo produced in vitro. In order to improve the quality, development and success of cryopreservation of bovine embryos, this study has as main objective to investigate some changes in in vitro cultivation system. So, we propose to evaluate the effect of oxygen tension and antioxidant supplementation in the medium of in vitro maturation (IVM) and/or in vitro culture (IVC) of bovine embryos cultured before and after vitrification. In a first step of this work, the oocytes will be matured in vitro in TCM-199 bicarbonate (B-199) at a temperature of 38.5°C, gaseous atmosphere of 5% CO2 and 100% humidity for 22 hours. Following, the oocytes will be fertilized and presumptive zygotes will be cultured for 7 days (168 hpi) in SOF medium supplemented with 0.6 mM cysteine (CIST), or 100 mM ²-mercaptoethanol (²-ME) or 100 IU of catalase (CAT), or without supplementation with antioxidants (SOF - Control). The IVC will be conducted in a incubator with 5% CO2 (20% O2) or with controlled atmosphere (mixture of gases: 7% O2, 5% CO2 and 88% N2). Cleavage (72 hpi) and embryo development to the blastocyst stage will be evaluated after 144 and 168 hpi, when embryos will be vitrificated or evaluated for the presence of ROS. Subsequently, the embryos will thawed and cultured in vitro to evaluate the rate of re-expansion and hatching. The gaseous atmosphere that give better results (in quantity or quality of embryos) will be used in the second stage of the work: the oocytes will be matured in vitro in B-199 (Control) supplemented with CIST, ²-ME or CAT, in atmosphere of 5% CO2, and temperature of 38.5°C for 22 hours. Then, they will be fertilized and presumptive zygotes will be CIV for 7 days (168 hpi) in media supplemented with the same antioxidants used during IVM, in gas atmosphere that will be defined in the first stage of the work (5% CO2 and 7% O2). Cleavage (72 hpi) and embryonic development (144 and 168 hpi) will be evaluated and the embryos will be assessed for quality (presence of ROS or rate of apoptosis) or vitrificated. Subsequently, the embryos will be thawed and cultured in vitro to evaluate the rate of re-expansion and hatching, and embryo quality determined by the total number of cells. (AU)