In Vitro Maturation, Fertilization, Embryo Culture And Activation Treatments Of Ovine Oocytes In Different Culture Systems

TLDR: The presence of BRL cell and OOEC even in the absence of heparin significantly improved the fertilization and embryo development suggesting their capacitation and sperm viable ability.

Abstract: Title : IN VITRO MATURATION, FERTILIZATION, EMBRYO CULTURE AND ACTIVATION TREATMENTS OF OVINE OOCYTES IN DIFFERENT CULTURE SYSTEMS Name of the student : AKSHAYA KUMAR KUNDU Degree for which submitted : Ph.D. (Veterinary Physiology) Name of the Chairman : Dr.A.Thangavel, Ph.D., Year and University : 2003 Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 051. There is a rapid development in technologies to produce large number of mammalian embryos, in vitro at low cost for research and commercial purposes, still then in vitro developmental capacity of ovine oocytes is significantly lower than that of in vivo system. Generation of a lot of data may be useful to optimize the requirements for oocyte development, in vitro. This study was aimed to improve ovine oocyte in vitro maturation, fertilization, embryos development and activation of the ovine oocytes to produce parthenogenic embryos. The cumulus oocyte complexes (COCs) were harvested by slicing method from slaughterhouse-derived sheep ovaries. The COCs were matured in different in vitro culture systems, viz., 1. maturation of oocytes in TCM-199, SOF and Ham's F-10 media, 2. supplementation of IGF-I (100 ng/ml), EGF (10 ng/ml) and IGF-I (100 ng/ml) + EGF (10 ng/ml), 3. supplementation of cysteamine @ 50, 100, 200 m and 4. supplementation of s-mercaptoethanol (s-ME) (12.5, 25, 50 M) to the oocyte maturation medium. The cauda epididymal sperm from slaughterhouse-derived testes were utilized for in vitro insemination of the oocytes. The oocytes and sperms were co-incubated over the BRL cell and OOEC monolayers in the fert-TALP medium with and without heparin. The ovine embryos were cultured in different culture systems viz: 1. embryo culture in TCM-199, SOF and Ham's F-10 media, 2. supplementation of IGF-I (100 ng/ml), EGF (10 ng/ml) and IGF-I (100 ng/ml) + EGF (10 ng/ml) to the IVC medium, 3. embryo co-culture with BRL, Vero, BRL/Vero mixed cell and OOEC monolayers and 4. supplementation of Hb (1 g/ml), L-NAME (1 M) and Hb (1 g/ml) + L-NAME (1M) to the IVC medium. The matured ovine oocytes were activated by ethanol, calcium ionophore and thimerosal to produce parthenogenic embryos. The results were observed as follows. TCM-199 and SOF media were found suitable for in vitro ovine oocyte development. IGF-I, EGF and their combination accelerated the oocyte maturation leading to increased fertilization rate and improved embryo development. The presence of cysteamine and s-ME during IVM were found beneficial for ovine oocyte development. The presence of BRL cell and OOEC even in the absence of heparin significantly improved the fertilization and embryo development suggesting their capacitation and sperm viable ability. The SOF medium showed better competence for ovine embryo culture over TCM-199 and Ham's F-10 media. IGF-I, EGF and their combination in IVC medium enhanced the embryo development rate and increased cell number in blastocysts. BRL, Vero, BRL/Vero mixed cell and OOEC monolayers significantly improved the in vitro embryo development. The activated parthenogenic embryos produced were found to be quantitative but not qualitative compared to the IVF oocytes.