Sharma G Taru, Loganathasamy K
Reproductive Physiology Laboratory, Physiology and Climatology Division, Indian Veterinary Research Institute, Izatnagar-243122, UP, India.
Vet Res Commun. 2007 Oct;31(7):881-93. doi: 10.1007/s11259-007-0059-7.
The present study was conducted to investigate the effect of meiotic stages during in vitro maturation (IVM) on the survival of vitrified-warmed buffalo oocytes, vitrified at different stages of IVM. Cumulus oocyte complexes obtained from slaughterhouse ovaries were randomly divided into 6 groups: control (non-vitrified, matured for 24 h at 38 +/- 1 degrees C, 5% CO2 in humidified air), and those matured for 0 h (vitrified before IVM) or 6, 12, 18 and 24 h before vitrification. Cumulus oocyte complexes were vitrified in solution consisting of 40% w/v propylene glycol and 0.25 mol/L trehalose in phosphate-buffered saline supplemented with 4% w/v bovine serum albumin. Vitrified cumulus oocyte complexes were stored at -196 degrees C (liquid nitrogen) for at least 7 days and then thawed at 37 degrees C; cryoprotectant was removed with 1 mol/L sucrose solution. Cumulus oocyte complexes in the 0, 6, 12, 18 and 24 h groups were then matured for an additional 24, 18, 12, 6 and 0 h, respectively, to complete 24 h of IVM. Among the five vitrification groups, 89-92% of cumulus oocyte complexes were recovered, after warming, of which 84-91% were morphologically normal. Overall survivability of vitrified cumulus oocyte complexes was lower (p < 0.05) than that of non-vitrified cumulus oocyte complexes (94.5%). Survival rates of cumulus oocyte complexes matured 24 h prior to vitrification (61.3%) were higher (p < 0.05) than those matured for 12 h (46.7%), 6 h (40.6%) and 0 h (37.6%). Nuclear status following 24 h IVM was assessed. A higher proportion of non-vitrified (control) oocytes (72.7%) reached metaphase II (M-II) stage in control than oocytes vitrified for 24 h (60.0%), 18 h (54.4), 12 h (42.3%), 6 h (33.3%) and 0 h (31.6%) (p < 0.05). The results suggest that length of time in maturation medium prior to vitrification influences post-thaw survivability of buffalo oocytes; longer intervals resulted in higher survival rates.
本研究旨在调查体外成熟（IVM）过程中的减数分裂阶段对不同IVM阶段玻璃化冷冻-解冻后的水牛卵母细胞存活的影响。从屠宰场卵巢获取的卵丘-卵母细胞复合体随机分为6组：对照组（未玻璃化，在38±1℃、5%二氧化碳的湿润空气中成熟24小时），以及在玻璃化前成熟0小时（IVM前玻璃化）或6、12、18和24小时的组。卵丘-卵母细胞复合体在由40%（w/v）丙二醇和0.25mol/L海藻糖组成的溶液中玻璃化，该溶液存在于补充有4%（w/v）牛血清白蛋白的磷酸盐缓冲盐水中。玻璃化的卵丘-卵母细胞复合体在-196℃（液氮）中储存至少7天，然后在37℃解冻；用1mol/L蔗糖溶液去除冷冻保护剂。然后，0、6、12、18和24小时组的卵丘-卵母细胞复合体分别再成熟24、18、12、6和0小时，以完成24小时的IVM。在五个玻璃化组中，解冻后89%-9%的卵丘-卵母细胞复合体得以恢复，其中84%-91%形态正常。玻璃化的卵丘-卵母细胞复合体的总体存活率低于未玻璃化的卵丘-卵母细胞复合体（94.5%）（p<0.05）。玻璃化前成熟24小时的卵丘-卵母细胞复合体的存活率（61.3%）高于成熟12小时（46.7%）、6小时（40.6%）和0小时（37.6%）的（p<0.05）。评估24小时IVM后的核状态。对照组中未玻璃化（对照）的卵母细胞达到中期II（M-II）期的比例（72.7%）高于玻璃化24小时（60.0%）、18小时（54.4%）、12小时（42.3%）、6小时（33.3%）和0小时（31.6%）的卵母细胞（p<0.05）。结果表明，玻璃化前在成熟培养基中的时间长度会影响水牛卵母细胞解冻后的存活率；较长的间隔时间会导致更高的存活率。
