Gibbons J R, Krisher R L, Carlin S K, Pearson R E, Gwazdauskas F C
Department of Dairy Science, College of Agriculture and Life Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0315, USA.
Theriogenology. 1995 Apr 15;43(6):1129-39. doi: 10.1016/0093-691x(95)00076-k.
Ultrasound-guided transvaginal follicular aspiration of oocytes from live cows combined with IVM, IVF and in vitro culture (IVC) is a procedure for producing preimplantation-stage bovine embryos and a source of oocytes for pronuclear microinjection of DNA for producing transgenic cattle. This experiment was designed to compare in vitro embryo development rates between oocytes derived from transvaginal follicular aspiration and those obtained from cows at slaughter. Nine cows were subject to a twice-weekly aspiration. Oocytes were aspirated with a 5 MHz ultrasound transducer packaged in a vaginal probe equipped with a dorsal-mounted needle guide (16-ga). All visible follicles (>2 mm) were punctured with a 17-ga, 55-cm needle at each aspiration session and the contents removed under vacuum suction. Oocytes underwent IVM/IVF/IVC. Microinjection of DNA was performed during the pronuclear stage of development, and the zygotes were co-cultured on Buffalo Rat Liver (BRL) cells in modified M199 at 39 degrees C in 5% CO2 and air. After 7 d in culture, embryos were removed and scored for development. A Chi-square analysis was used to compare transvaginal follicular-derived oocytes (microinjected and not) and slaughterhouse-derived, matured in transit oocytes (SHDMT; microinjected and not). Nonmicroinjected embryos resulting from IVF of transvaginal aspiration-derived oocytes developed to blastocysts at a higher rate than SHDMT oocytes (40.0 vs 30.8%; P < 0.05). There was no difference in development rates between the microinjected groups (aspiration = 15.9% vs SHDMT = 12.8%). Higher proportions of the embryos generated from the aspirated oocytes were of excellent or good quality following culture (P < 0.05). In the present experiments the effects of microinjection may overshadow some effects of ova source, but transvaginal follicular aspiration may provide a more consistent, synchronous population of oocytes than those derived from commercial slaughter house sources for use with in vitro systems.
超声引导下从活体母牛经阴道卵泡抽吸卵母细胞，结合体外成熟（IVM）、体外受精（IVF）和体外培养（IVC），是一种生产植入前阶段牛胚胎的方法，也是用于原核显微注射DNA以生产转基因牛的卵母细胞来源。本实验旨在比较经阴道卵泡抽吸获得的卵母细胞与屠宰场获取的卵母细胞的体外胚胎发育率。对9头母牛进行每周两次的抽吸。使用包装在配备背侧针引导器（16号）的阴道探头中的5兆赫超声换能器抽吸卵母细胞。每次抽吸时，用17号、55厘米长的针穿刺所有可见卵泡（>2毫米），并在真空抽吸下吸出内容物。卵母细胞进行IVM/IVF/IVC。在发育的原核阶段进行DNA显微注射，受精卵在改良的M199培养基中，于39℃、5%二氧化碳和空气环境下在水牛大鼠肝（BRL）细胞上共培养。培养7天后，取出胚胎并对发育情况进行评分。采用卡方分析比较经阴道卵泡来源的卵母细胞（显微注射和未显微注射）与屠宰场来源、在运输过程中成熟的卵母细胞（SHDMT；显微注射和未显微注射）。经阴道抽吸来源的卵母细胞IVF产生的未显微注射胚胎发育成囊胚的比率高于SHDMT卵母细胞（40.0%对30.8%；P<0.05）。显微注射组之间的发育率没有差异（抽吸=15.9%对SHDMT=12.8%）。培养后，经抽吸获得的卵母细胞产生的胚胎中，优质或良好质量的比例更高（P<0.05）。在本实验中，显微注射的影响可能掩盖了卵源的一些影响，但经阴道卵泡抽吸可能比商业屠宰场来源的卵母细胞提供更一致、同步的卵母细胞群体，用于体外系统。
