
%0 Journal Article
%A Yadav, R. C. S. 
%A Sharma, A. 
%A Purohit, G. N. 
%T 358 MORPHOLOGICAL CHANGES IN VITRIFIED WATER BUFFALO CUMULUS&#8211;OOCYTE COMPLEXES AND THEIR <i>IN VITRO</i> NUCLEAR MATURATION
%J Reproduction, Fertility and Development
%D 2006/12/12
%V 19
%N 1
%P 294-294
%K 
%R https://doi.org/10.1071/RDV19N1AB358
%U https://www.publish.csiro.au/paper/RDV19N1AB358 
%X Morphological changes and in vitro nuclear maturation of buffalo cumulus&#8211;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&#39;s phosphate-buffered saline+0.5M sucrose + 0.5% BSA + cryoprotectant). COCs were transferred to empty semen straws, kept over LN vapor for 2&#8211;3 min, and then plunged into LN. After 7&#8211;10 days of storage, COCs were warmed and evaluated for morphological damage. Morphologically normal COCs were cultured in vitro (9 replicates each with 5&#8211;10 oocytes in 50&#8211;100-&#181;L culture drops) in TCM-199 medium supplemented with 5&#181;gmL-1 FSH, 5&#181;gmL-1 LH, and 1 ngmL-1 estradiol with 25mM HEPES, 0.25mM pyruvate, and antibiotics. The COCs were incubated for 24 h at 38&#177;1&#176;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&#39;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 &#60;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 &#60;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. 
