Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillán, Chile.
Department of Animal Science, Faculty of Veterinary Sciences, Universidad de Concepcion, Chillán, Chile.
Theriogenology. 2020 Apr 1;146:94-103. doi: 10.1016/j.theriogenology.2020.02.012. Epub 2020 Feb 9.
The objective of this study was to evaluate, in the domestic cat, the effect of ovarian stimulation with eCG prior to oocyte in vitro maturation (priming) on in vitro and in vivo development after in vitro fertilization (IVF). For this purpose, oocyte donors were either 1) treated with a single dose of 200 IU eCG four days before oocyte recovery (eCG group), or, 2) given no treatment before oocyte recovery (control group). Ovaries of both groups were collected by ovariohysterectomy (OVH) and cumulus-oocyte complexes (COCs) were recovered by slicing. Immature COCs from both groups were matured in vitro (IVM) for 26-28 h. IVF was done with refrigerated epididymal sperm. After 24 h co-incubation, presumptive zygotes were cultured in vitro for eight days. The rates of cleavage, morulae, blastocyst development and hatching were estimated. Some blastocysts were stained for total cell counting and others were used for gene expression analysis of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6). Additionally, to evaluate in vivo development, embryos from the eCG group were transferred at Day 5 and Days 7 or 8 of IVC to synchronized cat recipients. The results showed that, eCG priming increased significantly the rate of blastocyst development as compared to the control group (37.9 and 25.6%, respectively) (P < 0.05). No differences were observed in total cell number of blastocysts and hatching blastocysts (mean ± SD) between the eCG and control groups (420.6 ± 193.6 and 347.0 ± 237.1, respectively) (P > 0.05). In the gene expression analysis, blastocysts generated in the eCG group had higher expression of OCT4 than blastocysts from the control group (P < 0.05). However, no significant differences were observed in the relative expression of SOX2, NANOG, CDX2 and GATA6 (P > 0.05). Additionally, six embryo transfer (ET) procedures were done, three with Day 5 embryos and three with Day 7 or 8 embryos. Recipients from both ET groups delivered live kittens. The total pregnancy rate was 4/6 (67%), meanwhile the live birth rate was 2/6 (33%). In conclusion, eCG priming improved the rate of blastocyst development in vitro and increased relative expression of OCT4. These results demonstrate that eCG priming of oocytes donors before IVM improves oocyte competence, enhance in vitro embryo development and allows live births of healthy offspring after ET.
本研究的目的是评估在猫体内，通过促性腺激素（eCG）对卵母细胞进行体外成熟（预刺激）对体外受精（IVF）后的体外和体内发育的影响。为此，卵母细胞供体 1）接受单次 200IU eCG 治疗，在卵母细胞回收前 4 天（eCG 组），或 2）在卵母细胞回收前不接受任何治疗（对照组）。两组的卵巢均通过卵巢切除术（OVH）收集，通过切片收集卵丘-卵母细胞复合物（COCs）。两组的未成熟 COCs 均在体外进行 26-28 小时的体外成熟（IVM）。使用冷藏的附睾精子进行 IVF。24 小时共孵育后，将推定的受精卵在体外培养 8 天。估计卵裂、桑葚胚、囊胚发育和孵化率。对一些囊胚进行总细胞计数染色，对另一些囊胚进行多能性（OCT4、SOX2 和 NANOG）和分化标记物（CDX2 和 GATA6）的基因表达分析。此外，为了评估体内发育，eCG 组的胚胎在 IVC 的第 5 天和第 7 天或第 8 天转移到同步化的猫受体。结果表明，与对照组相比，eCG 预刺激显著提高了囊胚发育率（分别为 37.9%和 25.6%）（P<0.05）。eCG 和对照组囊胚的总细胞数和孵化囊胚无差异（平均值±标准差）（分别为 420.6±193.6 和 347.0±237.1）（P>0.05）。在基因表达分析中，eCG 组生成的囊胚中 OCT4 的表达高于对照组（P<0.05）。然而，SOX2、NANOG、CDX2 和 GATA6 的相对表达无显著差异（P>0.05）。此外，进行了 6 次胚胎移植（ET）程序，其中 3 次使用第 5 天的胚胎，3 次使用第 7 天或第 8 天的胚胎。来自两个 ET 组的受体均产下活产小猫。总妊娠率为 6/6（67%），活产率为 6/6（33%）。总之，eCG 预刺激提高了体外囊胚发育率，并提高了 OCT4 的相对表达。这些结果表明，在 IVM 之前对卵母细胞供体进行 eCG 预刺激可提高卵母细胞的能力，增强体外胚胎发育，并允许 ET 后健康后代的活产。
