Bernal-Ulloa Sandra Milena, Lucas-Hahn Andrea, Herrmann Doris, Hadeler Klaus-Gerd, Aldag Patrick, Baulain Ulrich, Niemann Heiner
Institute of Farm Animal Genetics, Biotechnology, Friedrich-Loeffler-Institute, Mariensee, Germany; Facultad de Ciencias Agropecuarias, Universidad de Ciencias Aplicadas y Ambientales, Bogotá, Colombia.
Institute of Farm Animal Genetics, Biotechnology, Friedrich-Loeffler-Institute, Mariensee, Germany.
Theriogenology. 2016 Sep 15;86(5):1222-30. doi: 10.1016/j.theriogenology.2016.04.013. Epub 2016 Apr 20.
Cryopreservation of in vitro produced bovine embryos is associated with significantly reduced survival rates, mainly due to insufficient quality of the embryos. Caffeine supplementation during IVM has been used to delay meiotic resumption and concomitantly also increased embryo quality. Here, we investigated the influence of pre-IVM with caffeine on oocyte maturation, intraoocyte cAMP concentration, developmental competence after IVF, and blastocyst cryotolerance. Oocytes were obtained by slicing of ovaries and were submitted to either 2 hours culture before IVM with or without caffeine (0, 1, 5, 10, 20, 30 mM), or standard IVM (no pre-IVM). Oocytes were in vitro matured and fertilized and zygotes were cultured under standard in vitro conditions until Day 8. Expanded blastocysts derived from either standard control or the 10-mM caffeine treatments were submitted to vitrification. Caffeine delayed meiotic resumption after 9-hour IVM in a concentration-dependent manner. The cAMP levels were similar before and after IVM. Matured oocytes, cleavage, and blastocyst rates were reduced in the 30-mM caffeine concentration and were similar among the other treatment groups. Number and proportion of inner cell mass and trophectoderm cells in blastocysts did not differ among treatments. Forty-eight hours after thawing, hatching rates were higher in the 10-mM caffeine group (73.8%) compared with the standard control (59.7%). Reexpansion rates and total number of cells after 48 hours were similar in both treatments. The ratio of live/total cells was higher in the caffeine treatment. These results suggest that caffeine supplementation before IVM delayed meiotic resumption and improved blastocyst quality shown in higher cryotolerance.
体外生产的牛胚胎冷冻保存与存活率显著降低有关，主要原因是胚胎质量不足。体外成熟（IVM）过程中添加咖啡因已被用于延迟减数分裂恢复，同时也提高了胚胎质量。在此，我们研究了IVM前用咖啡因处理对卵母细胞成熟、卵母细胞内cAMP浓度、体外受精（IVF）后的发育能力以及囊胚冷冻耐受性的影响。通过切割卵巢获得卵母细胞，在有或无咖啡因（0、1、5、10、20、30 mM）的IVM前进行2小时培养，或进行标准IVM（无IVM前培养）。卵母细胞进行体外成熟和受精，受精卵在标准体外条件下培养至第8天。来自标准对照或10 mM咖啡因处理的扩张囊胚进行玻璃化冷冻。咖啡因在9小时IVM后以浓度依赖的方式延迟减数分裂恢复。IVM前后cAMP水平相似。30 mM咖啡因浓度组的成熟卵母细胞、分裂率和囊胚率降低，其他处理组相似。各处理组囊胚内细胞团和滋养外胚层细胞的数量和比例无差异。解冻后48小时，10 mM咖啡因组的孵化率（73.8%）高于标准对照组（59.7%）。两种处理48小时后的再扩张率和细胞总数相似。咖啡因处理组活细胞/总细胞的比例更高。这些结果表明，IVM前添加咖啡因可延迟减数分裂恢复，并提高囊胚质量，表现为更高的冷冻耐受性。
