Karja Ni Wayan Kurniani, Wongsrikeao Pimprapar, Murakami Masako, Agung Budiyanto, Fahrudin Mokhamad, Nagai Takashi, Otoi Takeshige
Laboratory of Animal Reproduction and Biotechnology, The United Graduate School of Veterinary Science, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.
Theriogenology. 2004 Dec;62(9):1585-95. doi: 10.1016/j.theriogenology.2004.03.012.
The present study was conducted to examine the effect of oxygen tension during in vitro culture (IVC) of porcine oocytes/embryos on their development and quality using two different culture systems. Porcine cumulus oocyte complexes (COCs) were matured (IVM) and fertilized (IVF) in vitro, and subsequently cultured for 6 days in a simple and economical portable incubator or a standard CO(2) incubator. While the same temperature (38.5 degrees C) and CO(2) concentration (5%) were used in the both systems, the portable incubator was operated in a negative air pressure (- 300 mmHg) to create an O(2) level at 8-10% (low O(2) concentration), or in a positive air pressure (high O(2) concentration). To compare the two culture systems, IVM and IVF of COCs and subsequent IVC of in vitro produced (IVP) embryos were carried out in the portable incubator with a low O(2) concentration (Group I) or in the standard incubator with a high O(2) concentration (Group II). To assess the effect of O(2) concentration on IVC of IVP embryos, some oocytes that had been cultured in the standard incubator for IVM and IVF were subsequently cultured in the portable incubator with a low O(2) concentration (Group III) or a high O(2) concentration (Group IV). The occurrence of DNA fragmentation in the blastocysts produced under different culture conditions was examined by TUNEL staining to assess embryo quality. The rates of oocytes that reached MII and were penetrated by spermatozoa following IVF did not differ between the two incubation systems. In contrast, the proportions of development to blastocysts and the mean cell number of blastocysts in Group I were higher than those in Group II and Group IV. The index of DNA-fragmented nucleus in the blastocysts of Group I was significantly lower than that in the blastocysts of Group II. Therefore, low oxygen tension during IVM, IVF and IVC enhanced the subsequent development of IVP embryos to the blastocyst stage and improved their quality.
本研究旨在使用两种不同的培养系统，检测猪卵母细胞/胚胎体外培养（IVC）期间的氧张力对其发育和质量的影响。猪卵丘卵母细胞复合体（COCs）在体外成熟（IVM）并受精（IVF），随后在一个简单且经济的便携式培养箱或标准二氧化碳培养箱中培养6天。虽然两个系统都采用相同的温度（38.5摄氏度）和二氧化碳浓度（5%），但便携式培养箱在负压（-300 mmHg）下运行以产生8-10%的氧水平（低氧浓度），或在正压下运行（高氧浓度）。为比较这两种培养系统，COCs的IVM和IVF以及体外产生（IVP）胚胎随后的IVC在低氧浓度的便携式培养箱中进行（I组），或在高氧浓度的标准培养箱中进行（II组）。为评估氧浓度对IVP胚胎IVC的影响，一些在标准培养箱中进行IVM和IVF培养的卵母细胞随后在低氧浓度（III组）或高氧浓度（IV组）的便携式培养箱中培养。通过TUNEL染色检测在不同培养条件下产生的囊胚中DNA片段化的发生情况，以评估胚胎质量。两种培养系统中，IVF后达到MII期并被精子穿透的卵母细胞比率没有差异。相反，I组中发育到囊胚的比例和囊胚的平均细胞数高于II组和IV组。I组囊胚中DNA片段化细胞核指数显著低于II组囊胚。因此，IVM、IVF和IVC期间的低氧张力促进了IVP胚胎随后发育到囊胚阶段并提高了其质量。
