Khatir Hadj, Anouassi Abdelhaq, Tibary Ahmed
Veterinary Research Center, Abu Dhabi, United Arab Emirates.
Theriogenology. 2004 Oct 1;62(7):1175-85. doi: 10.1016/j.theriogenology.2004.01.016.
The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 degrees C, 5% CO2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 microg/ml FSH, and 500 microM cysteamine. Matured COCs (n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 x 10(6)spermatozoa/ml in modified TALP-solution). Inseminated oocytes (n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes (n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively (P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.
本研究的总体目标是利用体外成熟、受精和共培养的卵丘-卵母细胞复合体（COCs）生产单峰驼胚胎。从457个卵巢中总共回收了1598个COCs；其中1308个被认为适合体外成熟，并在补充有10%热处理胎牛血清（FCS）、10 ng/ml表皮生长因子（EGF）、1 μg/ml促卵泡素（FSH）和500 μM半胱胺的TCM-199中，于38.5℃、5%二氧化碳和湿度>95%的条件下培养36小时。将成熟的COCs（n = 88）去除卵丘、固定并染色以确定核状态；63%（56/88）在36小时时达到了中期II（MII）。总体而言，用射出的新鲜精液（在改良的TALP溶液中为0.5×10⁶个精子/ml）对1135个COCs进行授精。检查授精后的卵母细胞（n = 155）是否有受精迹象；68%（106/155）被精子穿透，其中包括52%（55/106）有两个原核和34%（36/106）有多精受精情况。将授精后去除卵丘的卵母细胞（n = 819）与单峰驼输卵管上皮细胞或颗粒细胞在补充有10%热处理FCS的TCM-199中共培养。虽然两种共培养系统的首次分裂率（二至八细胞阶段）相似（分别为32%和33%），但与输卵管细胞共培养的卵母细胞获得了更多的胚胎（二细胞至囊胚阶段）（分别为61%和45%；P < 0.05）。与输卵管或颗粒细胞共培养7天的受精卵母细胞发育至早期桑椹胚阶段的比例分别为19%（80/417）和12%（48/402）（P > 0.05）。然而，只有与输卵管细胞共培养的卵母细胞才能发育至囊胚阶段（占受精卵母细胞的10%）。总之，利用屠宰场来源的卵母细胞、新鲜（射出的）精液和输卵管细胞共培养在体外生产出了单峰驼胚胎。
