Saikhun J, Sriussadaporn S, Thongtip N, Pinyopummin A, Kitiyanant Y
Institute of Science and Technology for Research and Development, Mahidol University, Puthamonthon sai 4, Salaya, Nakhonpathom 73170, Thailand.
Theriogenology. 2008 Jun;69(9):1104-10. doi: 10.1016/j.theriogenology.2008.01.024. Epub 2008 Mar 25.
In vitro embryo production in the domestic bitch can provide valuable insights for conservation of endangered canids. In the present study, canine oocytes underwent in vitro maturation (IVM) in simple or complex media, with production of in vitro matured and fertilized (IVM/IVF) canine embryos. Cumulus-oocyte complexes (COCs) were harvested from ovaries by slicing and subjected to IVM in four media (SOF, TCM 199, Ham-F10, and DMEM/F12). After culture for 48h, oocytes were stained and examined for nuclear maturation. There were no significant differences in the mean (+/-S.D.) percentage of nuclear maturation (metaphase II) of oocytes cultured in SOF (18.6+/-7.6%), TCM 199 (18.3+/-4.5%), Ham-F10 (13.9+/-8.2%), or DMEM/F12 (11.9+/-4.2%). For assessment of embryo development, oocytes were matured for 48h in synthetic oviductal fluid (SOF), fertilized with frozen-thawed sperm, and presumptive zygotes were cultured for 7 d, either in SOF or as co-cultures with BRL cells in TCM 199. Percentages of IVM/IVF oocytes that developed to the 2-cell, 3-4-cell, and 5-7-cell stages were higher (P<0.05) following culture in SOF versus BRL cell co-cultures (33.6+/-1.2% vs 13.7+/-1.2%, 24.7+/-0.5% vs 8.7+/-1.1%, and 15.1+/-2.2% vs 4.3+/-1.3%, respectively). However, none of the embryos developed beyond the 8-16-cell stage. In conclusion, simple or complex media successfully induced resumption of meiosis and nuclear maturation of canine oocytes. Furthermore, SOF supported in vitro development of IVM/IVF canine embryos to the 8-16-cell stage.
家犬的体外胚胎生产可为濒危犬科动物的保护提供有价值的见解。在本研究中，犬卵母细胞在简单或复杂培养基中进行体外成熟（IVM），以生产体外成熟并受精（IVM/IVF）的犬胚胎。通过切片从卵巢中采集卵丘-卵母细胞复合体（COC），并在四种培养基（SOF、TCM 199、Ham-F10和DMEM/F12）中进行IVM。培养48小时后，对卵母细胞进行染色并检查核成熟情况。在SOF（18.6±7.6%）、TCM 199（18.3±4.5%）、Ham-F10（13.9±8.2%）或DMEM/F12（11.9±4.2%）中培养的卵母细胞核成熟（中期II）的平均（±标准差）百分比没有显著差异。为了评估胚胎发育，卵母细胞在合成输卵管液（SOF）中成熟48小时，用冻融精子受精，假定的合子在SOF中或与BRL细胞在TCM 199中共培养7天。与BRL细胞共培养相比，在SOF中培养后，发育到2细胞、3-4细胞和5-7细胞阶段的IVM/IVF卵母细胞百分比更高（P<0.05）（分别为33.6±1.2%对13.7±1.2%、24.7±0.5%对8.7±1.1%和15.1±2.2%对4.3±1.3%）。然而，没有一个胚胎发育超过8-16细胞阶段。总之，简单或复杂培养基成功诱导了犬卵母细胞减数分裂的恢复和核成熟。此外，SOF支持IVM/IVF犬胚胎体外发育到8-16细胞阶段。
