358 MORPHOLOGICAL CHANGES IN VITRIFIED WATER BUFFALO CUMULUS–OOCYTE

COMPLEXES AND THEIR IN VITRO NUCLEAR MATURATION

R. C. S. Yadav, A. Sharma and G. N. Purohit

Abstract

Morphological changes and in vitro nuclear maturation of buffalo cumulus–oocyte

complexes (COCs) was evaluated subsequent to their cryopreservation by vitrification in
solutions containing 4 M, 6 M, 8 M, and 10 M concentrations of glycerol (G) or ethylene
glycol (EG) or their combination. COCs collected from buffalo ovaries by aspiration (n =
1342) were equilibrated in 50% of the vitrification solution and then placed in the
vitrification solution (Dulbecco's phosphate-buffered saline+0.5M sucrose + 0.5% BSA +
cryoprotectant). COCs were transferred to empty semen straws, kept over LN vapor for
2–3 min, and then plunged into LN. After 7–10 days of storage, COCs were warmed and
evaluated for morphological damage. Morphologically normal COCs were cultured in
vitro (9 replicates each with 5–10 oocytes in 50–100-µL culture drops) in TCM-199
medium supplemented with 5µgmL-1 FSH, 5µgmL-1 LH, and 1 ngmL-1 estradiol with
25mM HEPES, 0.25mM pyruvate, and antibiotics. The COCs were incubated for 24 h at
38±1°C and 5% CO2 in humidified air in a CO2 incubator and evaluated for nuclear
maturation at the end of 24 h of culture. Freshly collected COCs were also matured in
vitro and kept as controls (n=142). The proportions of COCs retrieved in morphologically
normal form were compared by chi-square test; the arcsin transformed data of the
proportions of oocytes matured was compared by Duncan's new multiple range test. The
proportions of oocytes recovered in a morphologically normal form were highest in the
6M EG group (95.23%), followed by 8M EG (94.0%) and 6M G (90.6%) groups. At 10M
concentration, a significantly (P <0.05) lower percentage of oocytes was morphologically
normal. The morphological abnormalities recorded were change in shape, rupture of zona
pellucida, and leakage of oocyte contents. A significantly higher (65.62%; P <0.05)
proportion of fresh oocytes reached metaphase-II compared to oocytes vitrified in all
concentrations of G and EG. The proportion of oocytes reaching metaphase-II increased
with increasing concentrations of both G and EG, but at 10M concentration the
proportion of oocytes reaching metaphase-II decreased. The proportions of COCs
reaching metaphase-II in 4M, 6 M, 8M, and 10M glycerol were 6.9%, 21.2%, 25.7%, and
5.5%, respectively. The respective proportions of COCs reaching metaphase-II in 4M, 6
M, 8M, and 10M ethylene glycol were 21.9%, 34.3%, 40.8%, and 7.5%. No significant
benefit of in vitro maturation of oocytes was seen for oocytes vitrified in a combination
of both G and EG. It was concluded that although vitrification brings about some damage
to the oocytes, yet it appears to be a good tool for oocyte cryopreservation, and 8M
concentration of either G or EG appears to be optimum for vitrification of buffalo
oocytes.

Reproduction, Fertility and Development 19(1) 294 - 294