Department of Soil, Plant, Environment and Animal Production Sciences, University of Naples Federico II, Portici, Italy.
Theriogenology. 2010 Mar 1;73(4):523-9. doi: 10.1016/j.theriogenology.2009.10.007.
The current study was undertaken to investigate the aneuploidy rates in in vitro-matured meiosis II (MII) oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds by using dual-color fluorescent in situ hybridization (FISH). A total of 159 and 144 in vitro-matured MII oocytes of the Italian Friesian and Italian Brown breeds, respectively, were obtained according to the standard methods and analyzed by FISH using "Xcen" and "5" chromosome-specific painting probes, produced by chromosome microdissection and Degenerate Oligonucleotide Primer- Polymerase Chain Reaction (DOP-PCR). Oocytes with unreduced chromosome number were 10.1% and 16.7% in the two breeds, respectively. To avoid bias due to possible artifacts, the aneuploidy rates were determined by analyzing only oocytes with the corresponding polar bodies. In the Italian Friesian, 100 of 143 (69.9%) secondary MII oocytes showed clear MII plates with corresponding first polar bodies and were scored for aneuploidy detection; one oocyte was "nullisomic" for chromosome X (1.0%) and one "disomic" for chromosome 5 (1.0%). In the Italian Brown, 100 of 120 (83.3%) MII oocytes with corresponding first polar bodies were analyzed; one oocyte was nullisomic (1.0%) and one was disomic (1.0%), both for chromosome 5. Totally, 303 oocytes were analyzed, 40 of which showed an unreduced chromosome complement (13.2%); of 200 MII oocytes with the corresponding first polar bodies, the aneuploidy rate (nullisomy+disomy) for the two chromosomes scored was 2%. Assuming that each chromosome is equally involved in aneuploidy, it results that in cattle oocytes matured in vitro, at least 30% of the oocytes (1x30 haploid chromosomes) should be aneuploid. Premature separation of sister chromatids (PSSC) was also observed in 2% of the oocytes in the Italian Friesian breed involving chromosome 5 and in 1% of the Italian Brown breed involving the X chromosome. Estimation of the "baseline" level of aneuploidy in the in vitro-matured oocytes of the various domestic animal species and breeds is, to our opinion, a useful reference for improving the in vitro production of embryos as well as for monitoring future trends of the reproductive health of the species/breeds engaged in zootechnical productions, especially in relation to management errors and environmental hazards.
本研究旨在通过双色荧光原位杂交（FISH）技术，研究两种奶牛（Bos taurus）品种体外成熟减数分裂 II 期（MII）卵母细胞及其相应第一极体的非整倍体率。根据标准方法，分别获得了 159 个和 144 个意大利弗里斯安牛和意大利棕色牛的体外成熟 MII 卵母细胞，并使用染色体微切割和退化寡核苷酸引物-聚合酶链反应（DOP-PCR）产生的“Xcen”和“5”染色体特异性涂染探针进行 FISH 分析。两个品种中未减数的染色体数分别为 10.1%和 16.7%。为了避免因可能的假象而产生偏差，仅分析具有相应极体的卵母细胞来确定非整倍体率。在意大利弗里斯安牛中，143 个次级 MII 卵母细胞中有 100 个（69.9%）显示出清晰的 MII 板和相应的第一极体，并进行非整倍体检测；一个卵母细胞的 X 染色体（1.0%）呈“单体型”，一个卵母细胞的 5 号染色体（1.0%）呈“二体型”。在意大利棕色牛中，120 个具有相应第一极体的 MII 卵母细胞中有 100 个（83.3%）进行了分析；一个卵母细胞为“单体型”（1.0%），一个为“二体型”（1.0%），两者均为 5 号染色体。总共分析了 303 个卵母细胞，其中 40 个显示未减数的染色体组成（13.2%）；在 200 个具有相应第一极体的 MII 卵母细胞中，两条染色体的非整倍体率（单体型+二体型）为 2%。假设每条染色体都同等参与非整倍体形成，则表明在体外成熟的牛卵母细胞中，至少 30%的卵母细胞（30 个单倍体染色体）应为非整倍体。意大利弗里斯安牛品种中涉及 5 号染色体的卵母细胞中观察到姐妹染色单体过早分离（PSSC）的比例为 2%，意大利棕色牛品种中涉及 X 染色体的卵母细胞中也观察到 1%的 PSSC。我们认为，估算各种家畜和品种体外成熟卵母细胞中非整倍体的“基线”水平，是提高胚胎体外生产以及监测参与动物生产的物种/品种生殖健康未来趋势的有用参考，尤其是与管理错误和环境危害有关的趋势。
