Effect of controlled freezing and open-pulled straw (ops) vitrification on atp content and reactive oxygen species (ros) level of bovine in vitro produced blastocysts | 不同冷冻方法对牛体外胚胎ATP含量与ROS水平的影响

Chinese. 【目的】探讨冷冻保存对牛体外生产胚胎能量代谢、有氧代谢造成的影响。【方法】采用常规法或玻璃化法（open pulled straw，OPS法）冷冻保存牛体外受精（in vitro fertilization，IVF）囊胚和体细胞核移植（somatic cell nuclear transfer，SCNT）囊胚，利用ATP Bioluminescence Assay Kit HS II 和 GENMED ROS Assay Kit检测了冷冻-解冻后囊胚ATP含量和ROS水平。【结果】（1）IVF和SCNT囊胚OPS法冷冻-解冻后存活率（（93.25±5.17）%和（77.56±3.52）%）均显著高于常规冷冻法（（81.25±4.98）%和（49.41±2.24）%）（P＜0.05）；（2）IVF囊胚和SCNT囊胚OPS法冷冻-解冻后ATP含量（（0.63±0.05）和（0.33±0.02）pmol）均显著高于常规冷冻法（（0.45±0.03）和（0.22±0.01）pmol）（P＜0.05）；但是均显著低于相应的IVF或SCNT新鲜囊胚（（0.76±0.04）和（0.40±0.02）pmol）（P＜0.05）；（3）玻璃化冷冻IVF囊胚、SCNT囊胚ROS水平（（74.34±4.24）和（43.21±3.35）cps）高于新鲜IVF囊胚（（48.52±2.65）cps）、SCNT囊胚（（27.36±2.23）cps）（P＜0.05），常规冷冻组则与此相反（（35.61±4.32）和（16.56±2.52）cps）（P＜0.05）。【结论】玻璃化冷冻较适用于牛IVF、SCNT囊胚冷冻保存；玻璃化冷冻和常规冷冻均显著影响牛IVF囊胚、SCNT囊胚中ATP含量和ROS水平。

English. 【Objective】 The experiment was designed to investigate the effect of cryopreservation on ATP content and ROS level of bovine blastocysts produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). 【Method】 Bovine in vitro produced blastocysts were cryopreserved by controlled freezing and OPS vitrification. ATP Bioluminescence Assay Kit HS II and GENMED ROS Assay Kit were used to analyze ATP content and ROS level in cryopreserved blastocysts. 【Result】 (1) For each kind of blastocysts (IVF or SCNT), significant difference was observed between survival rates of controlled freezing group ((81.25±4.98)% or (49.41±2.24)%) and OPS vitrification group ((93.25±5.17)% or (77.56±3.52)%) (P＜0.05). (2) For IVF blastocysts, ATP content was significantly decreased from (0.76±0.04) pmol (fresh group) to (0.45±0.03) pmol (controlled freezing group) or (0.63±0.05) pmol (vitrification group) (P＜0.05), and the same was found for SCNT blastocysts ((0.40±0.02) pmol to (0.22±0.01) pmol or (0.33±0.02) pmol) (P＜0.05). ATP content of each kind of blastocysts (IVF or SCNT) in controlled freezing group ((0.45±0.03) pmol or (0.22±0.01) pmol) was significantly lower than that in OPS vitrification group ((0.63±0.05) pmol or (0.33±0.02) pmol) (P＜0.05). (3) ROS level of fresh IVF blastocysts ((48.52±2.65) cps) or SCNT blastocysts ((27.36±2.23) cps) was significantly lower than that of OPS vitrification group ((74.34±4.24) cps or (43.21±3.35) cps) (P＜0.05), but higher than that of controlled freezing group ((35.61±4.32) cps or (16.56±2.52) cps) (P＜0.05). 【Conclusion】 The present work indicated that vitrification was more efficient in the cryopreservation of bovine blastocysts derived from IVF or SCNT with comparison of controlled freezing. Meanwhile, both vitrification and controlled freezing significantly altered ATP content and ROS level in those blastocysts.