Khatir Hadj, Anouassi AbdelHaq
Al Hejen Research Center, PO Box 44857, Abu Dhabi, United Arab Emirates.
Theriogenology. 2006 Jun;65(9):1727-36. doi: 10.1016/j.theriogenology.2005.09.029. Epub 2005 Nov 2.
Dromedary offspring have never been produced fully in vitro. We have previously demonstrated that embryos obtained by culture in semi-defined medium (mKSOMaa) have better in vitro development ability than those cultured with oviductal epithelial cells. The aim of the present experiment was to study the pregnancy rate after embryo transfer of in vitro-produced (IVP) dromedary embryos cultured in semi-defined modified medium (mKSOMaa). IVM/IVF procedures were conducted on six hundred and sixty four (664) cumulus oocytes complexes (COCs) aspirated from ovaries collected at a local slaughterhouse and cultured in vitro (38.5 degrees C; 5% CO2, and maximum humidity >95%). Maturation was completed by incubation in TCM-199 medium supplemented with 10% heat-treated Fetal Calf Serum (FCS), 10 ng/mL EGF, 1 microg/mL FSH, 1 microg/mL E2 and 500 microM cysteamine for 30 h. In vitro fertilization was performed using fresh semen (0.5 x 10(6) spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 38.5 degrees C, 5% CO2 and 90% N2 with maximum humidity (>95%). All IVC steps were done in seven replicates. The cleavage rate (two cells to blastocyst stage) was 64% (425/664) and the percentage of oocytes reaching the blastocyst stage was 23% (155/664). The hatching rate of blastocyst obtained after culture was 46% (71/155). Good quality hatched blastocysts (n = 66) were transferred individually to synchronized recipients. Pregnancy rates, determined by ultrasonography at 15, 60 and 90 days after embryo transfer (ET), were 38%, 32% and 27%, respectively. Out of 18 pregnant females 5 aborted between the fifth and seventh month of pregnancy and 13 females (20%) remained pregnant. After 385 days of pregnancy, the first healthy and normal male-dromedary offspring produced fully in vitro was born at a birth weight of 38 kg. More dromedary calves (n = 4) were born later on. The remaining pregnant females (n = 8) are due to calf within the next months. In conclusion, this is the first reported offspring in camelids obtained by transfer of embryos produced by IVM, IVF and IVC using abattoir-derived oocytes, fresh semen and culture in a semi-defined medium.
单峰驼后代从未完全在体外培育成功。我们之前已经证明,在半限定培养基(mKSOMaa)中培养获得的胚胎比与输卵管上皮细胞共同培养的胚胎具有更好的体外发育能力。本实验的目的是研究在半限定改良培养基(mKSOMaa)中培养的体外生产(IVP)单峰驼胚胎移植后的妊娠率。对从当地屠宰场收集的卵巢中吸出的664个卵丘卵母细胞复合体(COCs)进行体外成熟(IVM)/体外受精(IVF)操作,并在体外培养(38.5摄氏度;5%二氧化碳,相对湿度>95%)。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)、1 μg/mL雌二醇(E2)和500 μM半胱胺的TCM - 199培养基中孵育30小时以完成成熟过程。使用新鲜精液(在改良的输卵管液(TALP)溶液中为0.5×10⁶精子/mL)进行体外受精。受精后的卵母细胞在mKSOMaa中于38.5摄氏度、5%二氧化碳和90%氮气、相对湿度>95%的条件下培养。所有体外培养步骤均进行七次重复。卵裂率(从二细胞期到囊胚期)为64%(425/664),达到囊胚期的卵母细胞百分比为23%(155/664)。培养后获得的囊胚孵化率为46%(71/155)。将质量良好的孵化囊胚(n = 66)分别移植到同期受体中。在胚胎移植(ET)后15天、60天和90天通过超声检查确定的妊娠率分别为38%、32%和27%。在18只怀孕母驼中,有5只在妊娠第五至七个月期间流产,13只母驼(20%)继续妊娠。妊娠385天后,第一只完全在体外培育的健康正常雄性单峰驼后代出生,出生体重为38千克。之后又出生了更多单峰驼幼崽(n = 4)。其余怀孕母驼(n = 8)将在接下来的几个月内产仔。总之,这是首次报道通过使用屠宰场来源的卵母细胞、新鲜精液并在半限定培养基中培养进行IVM、IVF和IVC生产胚胎并移植而获得的骆驼科动物后代。