Sansinena M J, Taylor S A, Taylor P J, Schmidt E E, Denniston R S, Godke R A
Embryo Biotechnology Laboratory, Reproductive Biology Center, LSU Agricultural Center, St. Gabriel, LA 70776, USA.
Anim Reprod Sci. 2007 Jun;99(3-4):342-53. doi: 10.1016/j.anireprosci.2006.05.020. Epub 2006 Jul 18.
Assisted reproductive technologies in the llama (Lama glama) are needed to provide alternative methods for the propagation, selection and genetic improvement; however, recovery of adequate quantity and quality of spermatozoa for conventional IVF is problematic. Therefore, an effort was made to adapt the intracytoplasmic sperm injection (ICSI) procedure for the in vitro production of llama embryos. The specific objectives of this study were: (1) to determine in vitro maturation rates of oocytes recovered by transvaginal ultrasound-guided oocyte aspiration (TUGA) or flank laparotomy; (2) to evaluate the effects of activation treatments following ICSI; (3) to evaluate the development of llama ICSI embryos in CR1aa medium or in an oviduct cell co-culture system. Llamas were superstimulated by double dominant follicle reduction followed by oFSH administered in daily descending doses over a 3-day interval. Oocytes were harvested by flank laparotomy or TUGA and matured in vitro for 30 h. Mature oocytes were subjected to ICSI followed by no chemical activation (Treatment A), ionomycin only (Treatment B) or ionomycin/DMAP activation (Treatment C). More oocytes were recovered by flank laparotomy procedure compared with TUGA (94% versus 61%, P<0.05) and a greater number of oocytes harvested by flank laparotomy reached the metaphase-II stage (77% versus 44%, P<0.05). After ICSI, the proportion of cleaved and 4-8-cell stages embryos was significantly greater when injected oocytes were activated with ionomycin/DMAP combination (63% and 38%, respectively, P<0.05). The co-culture of ICSI embryos with llama oviduct epithelial cells resulted in progression to morula (25%) and blastocyst (12%) stages; whereas, all embryos cultured in CR1aa medium arrested at the 8-16-cell developmental stage.
为了提供繁殖、选择和基因改良的替代方法，需要在美洲驼（小羊驼）中应用辅助生殖技术；然而，获取足够数量和质量的精子用于传统体外受精存在问题。因此，人们努力将胞浆内单精子注射（ICSI）程序应用于美洲驼胚胎的体外生产。本研究的具体目标是：（1）确定经阴道超声引导下卵母细胞抽吸术（TUGA）或侧腹剖腹术回收的卵母细胞的体外成熟率；（2）评估ICSI后激活处理的效果；（3）评估美洲驼ICSI胚胎在CR1aa培养基或输卵管细胞共培养系统中的发育情况。通过双优势卵泡减少对美洲驼进行超刺激，随后在3天内每天递减剂量注射促卵泡素（oFSH）。通过侧腹剖腹术或TUGA采集卵母细胞，并在体外成熟30小时。成熟卵母细胞进行ICSI，然后不进行化学激活（处理A）、仅用离子霉素（处理B）或离子霉素/二甲氨基嘌呤激活（处理C）。与TUGA相比，侧腹剖腹术回收的卵母细胞更多（94%对61%，P<0.05），并且通过侧腹剖腹术采集的达到中期II期的卵母细胞数量更多（77%对44%，P<0.05）。ICSI后，当注射的卵母细胞用离子霉素/二甲氨基嘌呤联合激活时，分裂和4-8细胞期胚胎的比例显著更高（分别为63%和38%，P<0.05）。ICSI胚胎与美洲驼输卵管上皮细胞共培养可发育至桑葚胚（25%）和囊胚（12%）阶段；而在CR1aa培养基中培养的所有胚胎均停滞在8-16细胞发育阶段。
