Franco M, Hansen P J
Department of Animal Sciences, University of Florida, Gainesville Florida 32611-0910, USA.
In Vitro Cell Dev Biol Anim. 2006 Jan-Feb;42(1-2):40-4. doi: 10.1007/s11626-006-0010-1.
One limitation to the widespread use of in vitro-produced embryos in cattle is their poor survival following cryopreservation. Two approaches for enhancing survival of in vitro-produced bovine embryos following cryopreservation were evaluated: culture in the presence of hyaluronic acid and alterations in the cytoskeleton through cytochalasin B treatment. The experiment was a 2 x 2 factorial design to test main effects of hyaluronic acid added to culture at day 5 after insemination (+ or -) and cryopreservation treatment (control or cytochalasin B). Embryos used for cryopreservation were blastocysts and expanded blastocysts harvested on day 7 after insemination. Cytochalasin B increased the percent of embryos that re-expanded (P < 0.0001) and that hatched following thawing (P < 0.05). The hatching percent was 29.6% for embryos treated with cytochalasin B versus 9.1% for control embryos. There was no significant effect of hyaluronic acid on survival although there was a tendency for embryos cultured with hyaluronic acid to have higher percent hatching if not treated with cytochalasin B (12.7% for hyaluronic acid versus 4.5% for control; hyaluronic acid x cytochalasin B interaction; P = 0.09). In conclusion, cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro. Such a treatment could be incorporated into methods for cryopreservation of bovine embryos provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was of minimal benefit- the increased cryosurvival in the absence of cytochalasin B was not sufficient to allow an adequate number of embryos to survive.
牛体外生产胚胎广泛应用的一个限制因素是其在冷冻保存后的存活率较低。本研究评估了两种提高体外生产牛胚胎冷冻保存后存活率的方法：在透明质酸存在下培养以及通过细胞松弛素B处理改变细胞骨架。实验采用2×2析因设计，以测试在授精后第5天添加到培养液中的透明质酸（+或 -）和冷冻保存处理（对照或细胞松弛素B）的主要效应。用于冷冻保存的胚胎是授精后第7天收获的囊胚和扩张囊胚。细胞松弛素B提高了胚胎解冻后再扩张的百分比（P < 0.0001）和孵化的百分比（P < 0.05）。经细胞松弛素B处理的胚胎孵化率为29.6%，而对照胚胎为9.1%。透明质酸对存活率没有显著影响，尽管未用细胞松弛素B处理时，在透明质酸中培养的胚胎有较高孵化率的趋势（透明质酸组为12.7%，对照组为4.5%；透明质酸×细胞松弛素B交互作用；P = 0.09）。总之，冷冻前用细胞松弛素B处理可提高体外生产牛胚胎的冷冻存活率。如果移植后的存活率足够，这种处理方法可纳入牛胚胎冷冻保存方法中。相比之下，用透明质酸培养益处不大——在没有细胞松弛素B的情况下，冷冻存活率的提高不足以使足够数量的胚胎存活。
