Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Regional Campus of International Excellence, University of Murcia, Murcia, Spain.
Cryobiology. 2013 Apr;66(2):144-50. doi: 10.1016/j.cryobiol.2012.12.009. Epub 2013 Jan 9.
This study was aimed to determine the effect of forskolin on the viability of in vivo-derived porcine embryos vitrified by the superfine open pulled straw (SOPS) or solid surface vitrification (SSV) methods at the 2-cell, 4-cell, and blastocyst stages. Zygotes, 2- to 4-cell embryos, and morulae were obtained from superovulated sows. After collection, embryos were cultured for 24h with 0 or 10 μM forskolin and then vitrified using the SOPS and SSV method, or not vitrified (fresh controls). Fresh and vitrified-warmed 2-cells, 4-cells, and blastocysts were cultured for additional 96 h, 72 h and 24 h, respectively. At the end of the culture, embryos were evaluated for progression to the blastocyst stage and total cell number. The vitrification method did not affect any of the parameters evaluated for any embryo stage. Forskolin increased (P<0.01) the blastocyst formation and the final developmental stage of vitrified 2- and 4-cell embryos. However, these embryos exhibited lower (P<0.003) blastocyst formation rates than their fresh counterparts. The total cell number and hatching rate were similar in both groups (vitrified and fresh) of 2- and 4-cell embryos. Vitrified blastocysts exhibited viabilities, final developmental stages, hatching rates, and total cell numbers that were similar to those of their fresh counterparts, regardless of the addition of forskolin. In conclusion, the SOPS and SSV methods are suitable for the cryopreservation of in vivo-derived 2- to 4-cell porcine embryos. Pre-treatment with forskolin for 24h before vitrification improves the cryotolerance of 2- and 4-cell porcine embryos.
本研究旨在确定毛喉素（forskolin）对采用超细开口拉制吸管（SOPS）或固体表面玻璃化（SSV）方法在 2-细胞、4-细胞和囊胚阶段对体内来源的猪胚胎进行玻璃化冷冻的活力的影响。卵裂期胚胎、2-细胞胚胎和 4-细胞胚胎以及桑葚胚均从超排卵母猪中获得。收集后，胚胎在 0 或 10μM 毛喉素存在下培养 24 小时，然后使用 SOPS 和 SSV 方法进行玻璃化冷冻，或不进行玻璃化冷冻（新鲜对照）。新鲜和玻璃化冷冻-解冻的 2-细胞、4-细胞和囊胚分别培养 96 小时、72 小时和 24 小时。培养结束时，评估胚胎向囊胚阶段的进展和总细胞数。玻璃化方法对任何胚胎阶段的任何评估参数均无影响。毛喉素增加（P<0.01）了玻璃化冷冻的 2-细胞和 4-细胞胚胎的囊胚形成率和最终发育阶段。然而，这些胚胎的囊胚形成率低于新鲜对照组（P<0.003）。2-细胞和 4-细胞胚胎的总细胞数和孵化率在两组（玻璃化和新鲜）之间相似。玻璃化囊胚的活力、最终发育阶段、孵化率和总细胞数与新鲜囊胚相似，无论是否添加毛喉素。总之，SOPS 和 SSV 方法适用于体内来源的 2-至 4-细胞猪胚胎的冷冻保存。玻璃化冷冻前用毛喉素预处理 24 小时可提高 2-和 4-细胞猪胚胎的抗冻能力。
