Goat Embryo Development from in vitro Matured Oocytes of Heterogeneous Quality Through Intracytoplasmic Sperm Injection Technique

Abstract

Experiments were conducted to develop an intracytoplasmic sperm injection (ICSI) protocol to produce goat embryos from in vitro matured heterogeneous oocytes. Two different protocols ('Protocol 1' or P-1 and 'Protocol 2′ or P-2) were formulated and tested to develop a suitable one. Cumulus-oocyte complexes (COCs) were obtained either by LOPU (P-1) or by ovariectomy and slicing (P-2). Recovered COCs were cultured in a microdrop (50 μL) of HEPES-buffered in vitro maturation (IVM) medium with 10% oestrus goat serum (OGS) (P-1) or IVM medium without HEPES with 15% OGS (P-2) in presence of CO2(5%) at 38.5°C either for 24 h (P-1) or 27 h (P-2). After IVM, COCs were denuded to select a mature oocyte (Mil) and held in the incubator in presence of CO2 (5%) until ICSI. Motile sperm were selected from frozen-thawed buck semen by swim-up either in mDM (P-1) or mSOF (P-2) medium. A motile sperm was immobilized and injected, head-first, into the ooplasm of a MII oocyte. Sham injections were also performed as control. Injected oocytes were cultured in mSOF medium either with 10% (P-1) or 20% (P-2) goat serum in presence of CO2 (5%) at 38.5°C A significantly higher (p<0.01) number of COCs was recovered with P-2 (17.5 COCs/donor) than P-1 (6.7 COCs/donor). However, the quality of the COCs was better with P-1 than P-2. A higher proportion of COCs were matured with P-2 (69.3%) than P-1 (55.5%), which was not statistically significant. A fertilization rate of 28.6% was obtained in P-1 without any cleavage. Fertilization rate (54.1 %) was significantly higher (p<0.05) in P-2 with a cleavage rate of 18.2% and few morula-stage embryos were obtained (2.9%). In conclusion, better fertilization and embryo development rates were obtained with P-2 than P-1. Morula-stage embryos were obtained using ICSI technique from in vitro matured heterogeneous goat oocytes without any artificial activation and first reported in Malaysia. © 2007 Asian Network for Scientific Information.