Ozawa Manabu, Nagai Takashi, Kaneko Hiroyuki, Noguchi Junko, Ohnuma Katsuhiko, Kikuchi Kazuhiro
Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan.
Theriogenology. 2006 Mar 1;65(4):860-9. doi: 10.1016/j.theriogenology.2005.06.012. Epub 2005 Aug 9.
We investigated the effects of HEPES in the medium (to maintain pH) and paraffin oil covering the medium (to maintain osmolality) on the developmental ability of porcine embryos produced in vitro using tightly closed glass tubes in the absence of a CO2 gas-regulated incubator. Putative porcine zygotes obtained by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes (day of IVF=Day 0) were cultured in 5% CO2 gas-equilibrated NCSU-37 media containing pyruvate and lactate during Days 0-2, and glucose during Days 2-6, in open glass tubes in a CO2 incubator or tightly closed glass tubes without a CO2 incubator at 38.5 degrees C. The following four media were used: (1) medium covered with paraffin oil and supplemented with HEPES; (2) medium covered with paraffin oil but with no HEPES supplementation; (3) medium not covered with paraffin oil but supplemented with HEPES; (4) medium not covered with paraffin oil and with no HEPES supplementation. As a control group, zygotes were cultured in medium with neither paraffin oil coverage nor HEPES supplementation using a four-well dish in a CO2 gas-regulated incubator. After culture, the osmolality in each of the four closed conditions was maintained at approximately 285-286 mOsm, lower (P<0.05) than that in the control (291 mOsm). In the two HEPES-supplemented media groups in the closed-tube system, the pH was maintained at 7.5-7.7, and the blastocyst development rates (15.5% in non-oil covered and 18.5% in oil covered group) did not differ significantly from that of the control (20.2%), although the mean cell numbers in the blastocysts in the two closed-tube condition groups (28.2 and 33.0) were lower (P<0.05) than in the control (43.5). In contrast, the pH was higher in the two groups without HEPES supplementation (approximately 8.0) than the control (7.4; P<0.05), and the blastocyst development rates (10.9% in non-oil covered and 7.5% in oil covered group) or total cell numbers in the blastocyst (24.8 and 28.7) in the two non-HEPES groups were drastically decreased (P<0.05) compared to those in the control (20.2% and 43.5). These results suggested that maintenance of pH is important for successful in vitro porcine embryo culture under closed-air conditions, whereas the range of osmolality that suits embryo development is not limited to a small range. Furthermore, blastocyst production was possible in a glass tube without a CO2 incubator, although blastocyst quality was lower compared to those produced in an incubator.
我们研究了培养基中的HEPES（用于维持pH值）和覆盖在培养基上的石蜡油（用于维持渗透压）对在无CO₂气体调节培养箱的情况下使用密闭玻璃管体外生产的猪胚胎发育能力的影响。通过体外成熟（IVM）卵母细胞的体外受精（IVF）获得的假定猪受精卵（IVF日=第0天），在第0 - 2天培养于含有丙酮酸和乳酸的5% CO₂气体平衡的NCSU - 37培养基中，第2 - 6天培养于含有葡萄糖的该培养基中，培养条件为在38.5℃下，于CO₂培养箱中的开放玻璃管或无CO₂培养箱的密闭玻璃管中。使用了以下四种培养基：（1）覆盖有石蜡油并添加了HEPES的培养基；（2）覆盖有石蜡油但未添加HEPES的培养基；（3）未覆盖石蜡油但添加了HEPES的培养基；（4）未覆盖石蜡油且未添加HEPES的培养基。作为对照组，受精卵在有CO₂气体调节的培养箱中使用四孔培养皿培养于既无石蜡油覆盖也无HEPES添加的培养基中。培养后，四种密闭条件下的渗透压均维持在约285 - 286 mOsm，低于对照组（291 mOsm，P<0.05）。在密闭管系统中添加了HEPES的两种培养基组中，pH维持在7.5 - 7.7，囊胚发育率（未覆盖油组为15.5%，覆盖油组为18.5%）与对照组（20.2%）相比无显著差异，尽管两种密闭管条件组囊胚中的平均细胞数（28.2和33.0）低于对照组（43.5，P<0.05）。相比之下，未添加HEPES的两组pH高于对照组（约8.0 vs 7.4；P<0.05），且未添加HEPES的两组囊胚发育率（未覆盖油组为10.9%，覆盖油组为7.5%）或囊胚中的总细胞数（24.8和28.7）与对照组（20.2%和43.5）相比大幅降低（P<0.05）。这些结果表明，在密闭空气条件下，维持pH值对猪胚胎体外培养成功很重要，而适合胚胎发育的渗透压范围并不局限于一个小范围。此外，在无CO₂培养箱的玻璃管中也可以产生囊胚，尽管与在培养箱中产生的囊胚相比质量较低。
