Ratto Marcelo, Gomez Constanza, Berland Marco, Adams Gregg P
Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada S7N 5B4.
Anim Reprod Sci. 2007 Feb;97(3-4):246-56. doi: 10.1016/j.anireprosci.2006.02.002. Epub 2006 Mar 10.
The objective of the present study was to compare the ovarian follicular response, cumulus-oocyte complex (COC) collection rate, and maturational status of COC collected from alpacas subsequent to treatment with two different superstimulatory protocols. Alpacas (n=7 per group) were treated with: (1) 200mg of FSH im divided bid for 3d, plus a single i.v. dose of 1000IU hCG 24h after the last FSH treatment, or (2) 1200IU of eCG as a single i.m. dose, plus a single i.v. dose of 1000IU of hCG on day 3 after eCG treatment (day 0=start of superstimulatory treatment). At 20-24h post-hCG treatment, the ovaries were surgically exposed and COC were collected by needle aspiration of all follicles > or =6mm. The FSH and eCG treatment groups did not differ with respect to the number of follicles > or =6mm at the time of COC collection (20.0+/-7.5 versus 27.0+/-3.3; P=0.5), the number of COC collected (26.2+/-8.4 versus 23.3+/-3.7; P=0.7), or the collection rate per follicle aspirated (89% versus 87%; P=0.7). No differences were detected between FSH- and eCG-treated alpacas in the number of expanded COC collected per alpaca (11.5+/-2.9 versus 8.8+/-2.8; P=0.54), the number of expanded COC in metaphase II (8.5+/-1.9 versus 6.0+/-2.1; P=0.1), or the number of compact COC with > or =3 layers of cumulus cells (12.5+/-4.3 versus 14.3+/-2.6; P=0.72). A greater proportion (P<0.05) of compact COC collected after FSH treatment matured in vitro to the metaphase II stage than after eCG treatment. Eight expanded alpaca COC were fertilized in vitro with llama sperm, three of which were fixed and stained 18h after exposure to sperm and five were cultured in vitro. Two of the three stained oocytes were in the pronuclear stage, and all five of the cultured oocytes developed to the two-cell and morula stages at 2 and 7 days, respectively, after in vitro fertilization. In summary, FSH and eCG treatments were equally effective for ovarian superstimulation and oocyte collection. Cumulus-oocyte complexes were collected from more than 80% of follicles aspirated during laparotomy. Nearly one third of the COC collected after superstimulation were in metaphase II, and more than 70% of the remaining COC progressed to metaphase II after in vitro maturation for 26h, bringing the mean number of oocytes available for in vitro fertilization to 16 per alpaca. Preliminary results support the hypothesis that alpaca oocytes obtained after superstimulation in the absence of progesterone are developmentally competent since morulae developed from all five COC fertilized and cultured in vitro.
本研究的目的是比较用两种不同的超排刺激方案处理后，羊驼的卵泡反应、卵丘-卵母细胞复合体（COC）采集率以及所采集COC的成熟状态。羊驼（每组n = 7）接受以下处理：（1）200mg促卵泡素（FSH）肌肉注射，每日2次，共3天，在最后一次FSH处理后24小时静脉注射1000IU人绒毛膜促性腺激素（hCG）；或（2）1200IU马绒毛膜促性腺激素（eCG）肌肉注射一次，在eCG处理后第3天（第0天 = 超排刺激处理开始）静脉注射1000IU hCG。在hCG处理后20 - 24小时，通过手术暴露卵巢，对所有直径≥6mm的卵泡进行穿刺抽吸来采集COC。在采集COC时，FSH和eCG处理组在直径≥6mm的卵泡数量（20.0±7.5对27.0±3.3；P = 0.5）、采集到的COC数量（26.2±8.4对23.3±3.7；P = 0.7）或每个穿刺卵泡的采集率（89%对87%；P = 0.7）方面没有差异。在每只羊驼采集到的扩张型COC数量（11.5±2.9对8.8±2.8；P = 0.54）、处于中期II的扩张型COC数量（8.5±1.9对6.0±2.1；P = 0.1）或具有≥3层卵丘细胞的紧密型COC数量（12.5±4.3对14.3±2.6；P = 0.72）方面，FSH处理组和eCG处理组之间未检测到差异。与eCG处理后相比，FSH处理后采集到的紧密型COC在体外成熟至中期II阶段的比例更高（P<0.05）。8个扩张型羊驼COC用美洲驼精子进行体外受精，其中3个在与精子接触18小时后固定并染色，5个进行体外培养。3个染色的卵母细胞中有2个处于原核期，所有5个培养的卵母细胞在体外受精后分别在第2天和第7天发育到二细胞期和桑椹胚期。总之，FSH和eCG处理在卵巢超排刺激和卵母细胞采集方面同样有效。剖腹手术期间，超过80%的穿刺卵泡采集到了卵丘-卵母细胞复合体。超排刺激后采集到的COC中近三分之一处于中期II，其余COC中超过70%在体外成熟26小时后发育到中期II，使每只羊驼可用于体外受精的卵母细胞平均数量达到16个。初步结果支持以下假设：在没有孕酮的情况下超排刺激后获得的羊驼卵母细胞具有发育能力，因为所有5个受精并体外培养的COC都发育成了桑椹胚。
