Wani N A, Nowshari M A
Central Veterinary Research Laboratory, Post Box 597, Dubai, United Arab Emirates.
Theriogenology. 2005 Jul 1;64(1):75-85. doi: 10.1016/j.theriogenology.2004.11.009. Epub 2005 Jan 1.
Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12h without any appreciable effect on the nuclear maturation of the oocytes.
进行实验以研究体外核成熟动力学以及室温下储存卵巢对单峰驼卵母细胞初始染色质构型和体外成熟的影响。从屠宰场的卵巢中收集卵丘卵母细胞复合体（COCs），并在体外成熟4 - 48小时。每隔4小时（从0到48小时），将一组卵母细胞固定、染色并评估核染色质状态。卵母细胞被分类为生发泡（GV）、终变期（DK）、中期I（M-I）、后期I（A-I）、中期II（M-II）阶段，以及那些退化、碎片化、激活或无可见染色质的归为其他类别。培养开始时，74%（66/89）的卵母细胞处于GV期，13%（12/89）处于DK期，12%（11/89）归为其他类别。生发泡破裂在培养中自发开始，培养20小时时97%的卵母细胞已完成此过程。成熟8小时和16小时后，分别有最高比例的卵母细胞（42%，48/114和41%，51/123）处于DK期和M-I期。在培养32小时（42%，50/118）、36小时（45%，47/104）、40小时（49%，57/117）、44小时（52%，103/198）和48小时（46%，55/120）时达到M-II期的卵母细胞比例彼此无差异（P>0.05）。然而，培养40小时后归为其他类别的卵母细胞比例增加，且在48小时时高于其他成熟阶段（P<0.05）。从室温下在生理盐水中储存12小时的卵巢中收集的卵母细胞达到M-II期的比例（43%，64/148）与在37℃温生理盐水中收集并在到达实验室后立即处理的卵母细胞（49%，57/117）之间无差异（P>0.05）。然而，与其他两组相比，从温生理盐水中收集但在室温下储存的卵巢中达到M-II期的卵母细胞数量较少（29%，37/128）（P<0.05）。可以得出结论，单峰驼卵母细胞需要32 - 44小时的体外培养才能使处于M-II期的卵母细胞数量达到最佳。然而，需要进一步研究以找出最合适的成熟时间，这将导致这些卵母细胞在体外受精、卵胞浆内单精子注射、孤雌激活或核移植后进一步发育。卵巢可以在室温下于生理盐水中收集并储存12小时，而对卵母细胞核成熟没有明显影响。
