Khatir H, Anouassi A, Tibary A
Veterinary Research Center, Abu Dhabi, United Arab Emirates.
Anim Reprod Sci. 2007 Jun;99(3-4):413-20. doi: 10.1016/j.anireprosci.2006.06.015. Epub 2006 Aug 4.
The aim of this work was to determine the effect of follicle size on camel oocyte quality as measured by developmental competence in vitro and in vivo. Ovaries from a local slaughterhouse were dissected to obtain two classes of follicle size: small (3-6 mm) and large (>6 mm) follicles. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of cumulus oocyte complexes (COCs). All cultures were done in four replicates at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. The COCs were matured for 28 h in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/mL EGF, and 250 microM cysteamine. Nuclear maturation rate for each class of follicle size was determined by contrast phase microscopy in a sample of COCs (n=30) denuded, fixed and stained with aceto-orcein. 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 5% O(2) and 90% N(2). The percentage of COCs reaching metaphase II (MII) after 28 h of maturation was 87% (26/30) and 73% (22/30) for oocytes originating from large and small follicles, respectively (P>0.1). The rate of total cleavage (two cells to blastocyst stage) was greater (P<0.05) for oocytes originating from large follicles (72%; 116/162) than for those derived from small follicles (59%; 140/237). The percentage of fertilized oocytes reaching the blastocyst stage was 35% (57/162) and 20% (48/237) for oocytes collected from large and small follicles, respectively (P<0.05). The viability of in vitro-produced hatched blastocyst from the two groups (15 from 3 to 6mm follicle size and 22 from follicles >6 mm) was assessed by transfer to synchronized recipients. None of the hatched blastocysts from small follicles resulted in a pregnancy whereas 68% (15/22) of the transferred hatched embryos from large follicles developed into a 25-day pregnancy. Of the resulting 15 pregnancies, 53% (n=8) aborted (five between 2 and 4 months and three between 5 and 7 months of pregnancy). The remaining seven pregnant females gave birth to normal healthy offsprings (four females and three males). The present study shows that dromedary oocytes developmental competence is acquired late during the final phase of follicular development and this developmental ability translates into greater pregnancy rates after transfer of in vitro produced hatched blastocysts.
本研究的目的是确定卵泡大小对骆驼卵母细胞质量的影响，卵母细胞质量通过体外和体内发育能力来衡量。从当地屠宰场获取卵巢，解剖以获得两类卵泡大小：小卵泡（3 - 6毫米）和大卵泡（>6毫米）。对卵丘卵母细胞复合体（COCs）进行体外成熟（IVM）、体外受精（IVF）和体外培养（IVC）后，评估卵母细胞的质量。所有培养均在38.5摄氏度、5%二氧化碳和高湿度（>95%）条件下进行四个重复。仅使用带有卵丘且细胞质均匀（深色）的COCs。COCs在补充有10%热处理胎牛血清（FCS）、10纳克/毫升表皮生长因子（EGF）和250微摩尔半胱胺的TCM - 199培养基中成熟28小时。通过相差显微镜在一组去除卵丘、固定并用醋酸洋红染色的COCs样本（n = 30）中确定每类卵泡大小的核成熟率。使用新鲜精液（在改良的TALP溶液中为0.5×10⁶精子/毫升）进行体外受精。受精后的卵母细胞在mKSOMaa中于5%氧气和90%氮气条件下培养。成熟28小时后，来自大卵泡的卵母细胞达到中期II（MII）的COCs百分比为87%（26/30），来自小卵泡的卵母细胞为73%（22/30）（P>0.1）。来自大卵泡的卵母细胞的总分裂率（从二细胞到囊胚阶段）高于（P<0.05）来自小卵泡的卵母细胞（72%；116/162比59%；140/237）。从大卵泡和小卵泡收集的卵母细胞中，分别有35%（57/162）和20%（48/237）的受精卵母细胞达到囊胚阶段（P<0.05）。通过移植到同期受体来评估两组体外产生的孵化囊胚的活力（15个来自3至6毫米卵泡大小，22个来自>6毫米卵泡）。小卵泡的孵化囊胚无一导致妊娠，而来自大卵泡的移植孵化胚胎中有68%（15/22）发育到25天妊娠。在产生的15例妊娠中，53%（n = 8）流产（5例在妊娠2至4个月之间，3例在妊娠5至7个月之间）。其余7只怀孕母羊产下正常健康后代（4只雌性和3只雄性）。本研究表明，单峰驼卵母细胞的发育能力在卵泡发育的最后阶段后期获得，并且这种发育能力在体外产生的孵化囊胚移植后转化为更高的妊娠率。
