DISCIZIA, Faculty of Veterinary Medicine, Federico II University, Napoli, Italy.
Theriogenology. 2011 Oct 15;76(7):1347-55. doi: 10.1016/j.theriogenology.2011.06.004. Epub 2011 Aug 6.
The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.
本研究旨在评估在培养过程中降低葡萄糖浓度或用其他能量底物和/或胚胎营养化合物替代己糖，是否会影响牛胚胎的体外发育、冷冻保存的抗性和性别比例。体外成熟和受精的卵母细胞被随机分配到 4 组进行体外培养，这 4 组在能量底物方面有所不同：A 组，1.5mM 葡萄糖，如标准 SOF 中所示；B 组，0.15mM 葡萄糖；C 组，0.125mM G3P，存在 0.15mM 葡萄糖；D 组，0.34mM 柠檬酸盐，与 2.77mM 肌醇结合。第 7 天评估囊胚，然后用 cryotop 在 16.5% DMSO、16.5%EG 和 0.5M 蔗糖中进行玻璃化冷冻，并在蔗糖浓度逐渐降低（0.25 至 0.15M 蔗糖）的情况下进行解冻。在体外培养 24 小时后评估存活率。最后，通过 PCR 对产生的囊胚进行性别鉴定。与 A 组相比，B、C 和 D 组的囊胚率增加，即降低葡萄糖浓度时（分别为 A、B、C 和 D 组中的 28.2、41.0、35.7 和 35.8；P<0.01）。然而，D 组培养的胚胎发育速度最慢，这表明总囊胚中处于高级阶段的胚胎（扩张和孵化的囊胚）比例最低（分别为 A、B、C 和 D 组中的 56.1、45.8、56.9 和 31.8%；P<0.01）。此外，解冻后 24 小时培养的玻璃化冷冻囊胚的存活率在 D 组也下降（分别为 A、B、C 和 D 组中的 73.3、73.1、71.4 和 58.4%；P<0.01）。有趣的是，与 A 组相比，D 组获得的雌性胚胎比例较高，B 组和 C 组的中间值（分别为 A、B、C 和 D 组中的 45.6、53.4、50.0 和 61.5%；P<0.05）。总之，结果表明，体外培养过程中的能量底物既影响囊胚的产生又影响囊胚的活力。此外，在体外培养过程中改变胚胎的代谢特征可能会对性别比例产生影响。
