Ding Li-Jun, Tian Hai-Bin, Wang Jing-Jun, Chen Juan, Sha Hong-Ying, Chen Jian-Quan, Cheng Guo-Xiang
Rujin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
Mol Reprod Dev. 2008 Dec;75(12):1710-5. doi: 10.1002/mrd.20922.
The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.
本研究的目的是确定不同频率经阴道采卵（OPU）对回收的卵丘卵母细胞复合体（COC）数量的影响，以及随后成熟卵母细胞支持克隆牛胚胎植入前发育的能力。通过经阴道卵泡抽吸术，从6头中国荷斯坦奶牛的卵巢中采集COC，每周两次（每3或4天一次）（第一组）、每5天一次（第二组）、每周一次（每7天一次）（第三组）、每10天一次（第四组）、每2周一次（每14天一次）（第五组）。通过优势卵泡（DF）直径和采集的COC确定卵泡的发育阶段，并阐明卵泡波的动态变化。此外，在体外成熟（IVM）开始后的不同时间间隔观察卵母细胞第一极体（PB I）的排出情况，以确定体细胞克隆核移植的合适培养时间窗口。将成熟卵母细胞用于生产克隆牛胚胎，随后在山羊输卵管中培养。7天后，冲洗出胚胎，比较五组的囊胚发育率。结果表明，所有直径≥3 mm（D1）卵泡的抽吸诱导并同步了卵泡波的动态变化，次级卵泡在4天后（D5）开始闭锁。另一个卵泡波在D7至D10之间开始，第二个卵泡波中的次级卵泡闭锁在D14开始。通过OPU获得的卵母细胞减数分裂进程（从IVM开始到PB I排出）的时间晚于从屠宰场获得的卵母细胞。在IVM开始后20至24小时之间，20%的卵母细胞排出了PB I。此外，在IVM开始22小时时，80%（520/650）采集的COC停滞在中期II（MII），并用作细胞质供体。重构胚胎发育到囊胚阶段的比率分别为23.1%（第一组）、15.0%（第二组）、10.9%（第三组）、4.9%（第四组）和29.0%（第五组）。结果表明，采用不同的OPU间隔时，来自相同活体供体的卵泡发育潜力不同，且第二个卵泡波中的早期闭锁卵泡支持克隆牛胚胎早期发育的能力更高。
