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

为了开发高效生产活畜，特别是具有优良遗传性状的牛的系统，已经尝试通过体外培养系统生产胚胎，包括使用带线性探头的超声回波系统经阴道抽吸从活畜卵巢收集的卵母细胞进行体外成熟和体外受精。我们首先确定了一个合适的条件，特别是抽吸压力和针直径的牛卵泡卵母细胞收集经阴道抽吸。结果发现，采用80 mmHg 18号针吸出条件时，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，我们尝试使用超声引导下的卵泡抽吸技术去除对超排卵刺激反应低的奶牛的优势卵泡。结果表明，抽吸优势卵泡的液体较多，可极大地促进小卵泡的发育，而且多数小卵泡发育后，经超排卵治疗可导致排卵。因此，在低反应奶牛中，通过卵泡抽吸，超排卵治疗后人工授精的胚胎恢复率得到了极大的提高。此外，我们发现，对这些奶牛注射针对procin抑制素肽的抗抑制素抗体，对超排卵治疗的反应与滤泡吸入病例相似。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肝素亲和层析分离的牛卵泡液部分的培养基中体外成熟，促进了体外受精后囊胚期的后续发育。此外，还发现了一种新的肝素结合生长/分化因子midkine在牛卵泡液中发挥促进作用。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