A.C. Majumdar

Fertilization and development of Caprine oocytes matured over granulosa cell monolayers

The aim of the present study was to compare the two oocyte maturation systems, i.e. a granulosa cell (GC) monolayer from small (<4mm) or large (>4mm) follicles and a granulosa co-culture for their effects on in vitro maturation (IVM), fertilization and developmental competence of caprine oocytes. A total of 1945 oocytes were used for studies on maturation, fertilization and embryo development. Monolayers were primed with maturation medium, 18-24h before the onset of maturation. Nuclear studies of 263 fertilized oocytes, 18h post-fertilization, revealed that the rate of sperm penetration was not affected by any of the maturation culture systems. Penetration rate was 66.30% versus 69.59% for the control and GC monolayers. On the other hand, progression of fertilization, i.e. sperm head decondensation (32.70% versus 9.78%) and pronucleus formation (8.76% versus 2.17%) were significantly (P<0.05) enhanced in the oocytes matured over GC monolayers, compared to those with GC co-culture respectively. Cleavage rate was significantly (P<0.05) higher in the oocytes matured and cultured over GC monolayers (27.59%) compared to those in oocytes matured and cultured with the GC co-culture (19.28%). Proportionately more embryos derived from oocytes matured and cultured with the GC co-culture blocked (16.53 and 25.92%) at early developmental stages (2-cell and 4-cell, respectively), compared to those derived from oocytes matured and cultured over GC monolayers (7.61% versus 10.56%; 2-cell versus 4-cell). It was concluded that GC monolayers better support cytoplasmic maturation of growing caprine oocytes, which is evident by a better maturation rate, active fertilization, an improved cleavage rate and subsequently a higher rate of morula formation. Granulosa cells from small and large follicles can be used for IVM and IVC with approximately the same efficiency after conditioning them with maturation medium and embryo development medium 18-24h before the onset of culture.

Chronology of maturational events in goat oocytes cultured in vitro

Abstract Follicular oocytes were retrieved from goat ovaries, collected from a local slaughter house. These oocytes were cultured in TCM-199 with 20% heat inactivated goat estrous serum for 6, 12, 18, 24, 30, 31, 32 and 36 h at 39 °C; 5% CO 2 in air and 95% humidity. A total of 1465 good cumulus-enclosed oocytes were matured and fixed at different time intervals to establish the chronology of meiotic events occurring in goat oocytes during in vitro maturation. Results revealed that at 0 h, 78.3% of oocytes were in the GV stage. A total of 57.4% of the oocytes were found in diakinesis stage at 6 h culture. Peak (51.1%) metaphase-I (M-I) was noted at 12 h. M-II was first observed after 18 h of culture, but at 32 h the maximum (71.6%) oocytes were in M-II stage. The percentage of oocytes reaching M-II was comparatively less at 30, 31 and 36 h (50.3, 55.8 and 59.7%), thus indicating that 32 h of culture is the most suitable time for goat oocyte maturation in vitro.