Duque Rodriguez Matteo, Cittadini Camila O, Teplitz Gabriela M, De Stefano Adrian, Lombardo Daniel M, Salamone Daniel F
Facultad de Ciencias Agrarias, Politécnico Colombiano Jaime Isaza Cadavid, Medellin, Colombia.
Laboratorio Biotecnología Animal, Departamento de Producción Animal, Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina.
Front Cell Dev Biol. 2021 Sep 7;9:694889. doi: 10.3389/fcell.2021.694889. eCollection 2021.
Assisted reproductive technologies in canine species are limited due to the low efficiency of maturation (IVM). Unlike other mammals, bitches ovulate oocytes in the germinal vesicle stage and complete metaphase II (MII) after 48-72 h in the oviductal environment and become fertilizable. For this reason, we compared two different IVM media, synthetic oviductal fluid (SOF) supplemented with 8% bovine serum albumin (BSA) or a mixture of 8% BSA-2.5% fetal bovine serum (FBS) and TCM-199 with 10% FBS. Additionally, we evaluated the effect of supplementation with insulin-transferrin-selenium (ITS) and low O tension in oocyte maturation, reactive oxygen species (ROS) levels, membrane integrity, and embryo development following parthenogenetic activation (PA). After 72 h of culture, SOF + BSA, SOF + BSA + FBS, and TCM-199 + FBS show 5, 7, and 4% of MII, respectively, without a statistical difference. However, SOF + BSA produced significantly higher degeneration rates compared to SOF + BSA + FBS (44 and 23%, respectively). Remarkably, supplementation with 1 μl/ml of ITS under high O tension demonstrated a beneficial effect by improving maturation rates up to 20% compared to the other groups. Low O tension increased maturation rates to 36.5%, although there were no statistical differences compared to high O tension in the presence of ITS. Lower ROS levels and higher integrity of the cytoplasmic membrane were found in the presence of ITS despite no differences in maturation rates under low O tension groups. Additionally, after PA, 1% development until the eight-cell stage was obtained after activation of -matured oocytes in the presence of ITS. Taken together, these results indicate that SOF supplemented with 8% BSA and 2.5% FBS is suitable for IVM of canine oocytes and ITS supplementation was beneficial for both high and low O tension. Furthermore, the addition of ITS in the cultured system lowers ROS levels and increases membrane integrity in domestic dog oocytes after IVM.
由于体外成熟（IVM）效率低下，犬类的辅助生殖技术受到限制。与其他哺乳动物不同，母犬在生发泡期排出卵母细胞，并在输卵管环境中48 - 72小时后完成减数分裂中期II（MII），从而具备受精能力。因此，我们比较了两种不同的IVM培养基，即添加8%牛血清白蛋白（BSA）的合成输卵管液（SOF）或8% BSA - 2.5%胎牛血清（FBS）与含10% FBS的TCM - 199的混合物。此外，我们评估了添加胰岛素 - 转铁蛋白 - 硒（ITS）和低氧张力对卵母细胞成熟、活性氧（ROS）水平、膜完整性以及孤雌激活（PA）后胚胎发育的影响。培养72小时后，SOF + BSA、SOF + BSA + FBS和TCM - 199 + FBS的MII分别为5%、7%和4%，无统计学差异。然而，与SOF + BSA + FBS相比，SOF + BSA产生的退化率显著更高（分别为44%和23%）。值得注意的是，在高氧张力下添加1μl/ml ITS显示出有益效果，与其他组相比，成熟率提高了20%。低氧张力使成熟率提高到36.5%，尽管在添加ITS的情况下与高氧张力相比无统计学差异。尽管低氧张力组的成熟率无差异，但在添加ITS的情况下发现ROS水平较低且细胞质膜完整性较高。此外，PA后，在添加ITS的情况下，激活成熟卵母细胞后获得了1%发育至八细胞期的胚胎。综上所述，这些结果表明，添加8% BSA和2.5% FBS的SOF适用于犬卵母细胞的IVM，添加ITS对高氧和低氧张力均有益。此外，在培养系统中添加ITS可降低家犬卵母细胞IVM后的ROS水平并提高膜完整性。
