Pavlok A, Cech S, Kubelka M, Lopatárová M, Holý L, Jindra M
Czech Academy of Sciences, Institute of Animal Physiology and Genetics, 277 21 Libechov, Czech Republic.
Anim Reprod Sci. 2006 Nov;96(1-2):186-95. doi: 10.1016/j.anireprosci.2005.12.008. Epub 2006 Jan 31.
The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.
研究了储存在分离卵泡中的牛卵母细胞的活力。这项工作的目的是延长卵母细胞在成熟前的体外操作时间，并开发一种新的卵母细胞“获能”方法，以提高体外生产胚胎的质量。从屠宰母牛的卵巢中解剖出卵泡；随后，根据卵泡直径将其分为三类（2-3毫米、3-4毫米和4-6毫米），并在pH值降低的改良组织培养液-199（TCM-199）中于17-18摄氏度下保存24或48小时。此后，分离出卵丘-卵母细胞复合体（COCs），使其成熟、受精，并将胚胎体外培养总共9天。在3-6毫米大小类别的卵泡中，最初在17-18摄氏度下保存24小时（“获能”）的卵母细胞发育而成的总囊胚和孵化囊胚的百分比，显著高于对照组[对照卵母细胞]（分别为44.9%和30.3%，而对照组为36.2%和20.4%）。在17-18摄氏度下保存48小时的卵泡中的卵母细胞，其发育能力已经下降。当将3-4毫米和4-6毫米类别的COCs根据其推测的发育史（源自假定生长中的“健康”卵泡，与假定退化中的“不健康”卵泡相对）进一步分为两个亚组时，获得了有趣的数据。仅在源自“健康”COCs的亚组中，观察到24小时保存的卵母细胞孵化囊胚率有更高的提高（在3-4毫米和4-6毫米类别中，分别为15.3%对25.0%，以及20.0%对34.4%）。将26个囊胚（由在17-18摄氏度下保存24小时的卵泡发育而成）移植到26头受体小母牛体内，结果有18头怀孕。卵泡在17-18摄氏度下体外保存，不仅能获得数量更多、质量更好的囊胚，还便于卵泡的长途安全运输。在适当的卵母细胞成熟之前延长卵泡保存时间，还能根据分离卵泡的数量使适当数量的受体动物同步。
