Wani Nisar A, Hong Seungbum
Reproductive Biotechnology Center, Post Box 299003, Dubai, United Arab Emirates.
Reproductive Biotechnology Center, Post Box 299003, Dubai, United Arab Emirates.
Theriogenology. 2018 Jun;113:44-49. doi: 10.1016/j.theriogenology.2018.02.004. Epub 2018 Feb 8.
Experiments were conducted to investigate the development of in vitro matured camel oocytes after their intra-cytoplasmic sperm injection (ICSI) with epididymal sperm collected from slaughtered male camels. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 °C and on ice (0-1 °C), respectively. Cumulus-oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25 COCs/well) containing 500 μL of maturation medium and cultured at 38.5 °C in an atmosphere of 5% CO in air for about 30 h. Spermatozoa were collected from the cauda epididymites in syringes containing 2-3 mL of tris-tes egg yolk extender. They were cooled down slowly and stored at refrigeration (4 °C) temperature. On the day of use, spermatozoa were prepared by the swim up technique before use in ICSI. The injected oocytes were either activated by ionomycin and roscovitine or put into the culture without any chemical activation. In Experiment 1, presumptive zygotes were fixed and stained with Hoechst 33342 for evaluation of fertilization after 18 h of culture, while, in Experiment 2, they were cultured in 500 μL of the culture medium at 38.5 °C in an atmosphere of 5% CO 5% O and 90% N in air for 7 days to evaluate their development. The proportion of oocytes activated when ICSI was followed by chemical activation was significantly higher (P < 0.05) when compared with non-activated ones. In experiment 2, a higher number of oocytes cleaved (59 vs. 35%) and developed to blastocysts (20 vs. 7%) in the group with post-ICSI activation when compared with the group without chemical activation, respectively. In conclusion, to the best of our knowledge, this is the first report where embryos were produced by ICSI in camels. Chemical activation of oocytes by ionomycin and roscovitine, post -ICSI, enhanced their cleavage and development to blastocyst stage.
进行了实验，以研究体外成熟的骆驼卵母细胞在进行胞浆内精子注射（ICSI）后，与从屠宰的雄性骆驼附睾采集的精子的发育情况。卵巢和睾丸分别从当地屠宰场收集，卵巢置于37°C的生理盐水（NSS）中，睾丸置于冰（0 - 1°C）上。从卵泡中吸出的卵丘-卵母细胞复合体（COCs）随机分配到含有500μL成熟培养基的4孔培养板（每孔20 - 25个COCs）中，并在38.5°C、含5% CO₂的空气环境中培养约30小时。精子从附睾尾部收集到含有2 - 3 mL三羟甲基氨基甲烷-卵黄稀释液的注射器中。它们缓慢冷却并储存在冷藏（4°C）温度下。在使用当天，精子在用于ICSI之前通过上浮技术制备。注射后的卵母细胞要么用离子霉素和罗曲伏丁激活，要么不经过任何化学激活直接放入培养液中。在实验1中，假定的受精卵在培养18小时后固定并用Hoechst 33342染色以评估受精情况，而在实验2中，它们在38.5°C、含5% CO₂、5% O₂和90% N₂的空气环境中，在500μL培养液中培养7天以评估其发育情况。与未激活的卵母细胞相比，ICSI后进行化学激活时激活的卵母细胞比例显著更高（P < 0.05）。在实验2中，与未进行化学激活的组相比，ICSI后激活组中有更多的卵母细胞发生分裂（分别为59%对35%）并发育成囊胚（分别为20%对7%）。总之，据我们所知，这是首次关于在骆驼中通过ICSI产生胚胎的报道。ICSI后用离子霉素和罗曲伏丁对卵母细胞进行化学激活，可增强其分裂并发育到囊胚阶段。
