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 (MII) 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.
Abu Nasar Md. Aminoor Rahman, Ramli Bin Abdullah and Wan Khadijah Wan Embong, 2007. Goat Embryo Development from in vitro Matured Oocytes of Heterogeneous Quality Through Intracytoplasmic Sperm Injection Technique
. Biotechnology, 6: 373-382.