Development of bovine eggs collected from ovaries of live animals with ultrasound-guided system

为了建立高效生产活体动物，特别是具有优良遗传性状的牛的系统，我们尝试了通过体外培养系统生产胚胎，包括用带线性探头的超声回声系统对活体动物经阴道抽吸的卵巢进行体外成熟和体外受精。我们首先确定了经阴道抽吸法采集牛卵泡卵母细胞的适宜条件，特别是抽吸压力和针径。结果发现，采用18号针头在80 mm Hg下抽吸卵母细胞，卵母细胞回收率最高。在第二个实验中，为了刺激奶牛卵巢中小卵泡的发育，我们试图利用超声引导的抽吸技术去除对超排刺激反应较低的奶牛的优势卵泡。研究发现，卵泡f-…的吸入较多的优势卵泡液量极大地刺激了小卵泡的发育，而且大多数小卵泡的发育导致了超排处理的排卵。因此，通过卵泡抽吸，低响应性奶牛在超排处理和人工授精后的胚胎回收率得到了极大的提高。此外，我们还发现，给这些奶牛注射针对前列环素抑制素肽的抗抑制素抗体后，对超排治疗的反应与抽吸卵泡的情况相似。在第三个实验中，我们发现从奶牛卵巢的小卵泡中收集的卵母细胞在含有经肝素亲和层析分离的牛卵泡液的培养液中进行体外成熟，促进了体外受精后的囊胚期发育。此外，研究还发现，牛卵泡液中一种新的肝素结合生长/分化因子Midkine具有促进作用。较少

To develop the system for efficient production of live stock animals, especially cattle with superior genetic traits, attempts have been made to produce the embryos by in vitro culture system, including in vitro maturation and in vitro fertilization of oocytes collected from ovaries from live animals with transvaginal aspiration using ultrasound echo system with a linear probe. We first determined a suitable condition, especially aspiration pressure and needle diameter for bovine follicular oocyte collection with transvaginal aspiration. As a result, we found that when the aspiration condition of 18- gauge needle at 80 mmHg was used, the highest oocyte recovery rates were obtained. In the second experiments, to stimulate the development of small sized follicles in cow ovaries, we tried to remove dominant follicles in low responder cows to superovulatory stimulation with follicular aspiration using ultrasound-guided aspiration techniques. It was found that the aspiration of follicular f … More luids of dominant follicles greatly stimulates the development of small-sized follicles, and moreover that most of the small follicles developed result in the ovulation with superovulatory treatments. Thus, with follicular aspiration, recovery rates of embryos after superovulatory treatments followed by artificial insemination were highly improved in low responder cows. Furthermore, we found that injection of anti-inhibin antibody raised against procin inhibin peptide to such cows resulted in similar response to superovulatory treatments to the case of follicular aspiration. In the third experiments, we found that in vitro maturation of oocytes collected from small sized follicles of cow ovaries in medium containing fractions of bovine follicular fluids separated by heparin affinity chromatography promotes subsequent development to the blastocyst stage after in vitro fertilization. Furthermore, it was found that the promoting activities were exerted with midkine, a novel heparin binding growth /differentiation factor in bovine follicular fluids. Less