冷冻保护剂对犊牛睾丸组织标志性酶活性的保护作用

Protective Effects of Cryoprotectants on the Activities of Calf Testicular Tissue Iconic Enzymes

为了探讨犊牛睾丸组织适宜的冷冻保存体系,设计以甘油、DMSO、乙二醇、海藻糖、BSA和睾丸液作为主要成分的6种冷冻保护液,以碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、乳酸脱氢酶(LDH)和β-D葡萄糖苷酶(β-D-Glucosidase)的酶活性作为测定指标,以新鲜犊牛睾丸组织的4种酶活性为空白对照,使用6种冷冻保护液将睾丸组织于液氮中冷冻保存7d,然后37℃水浴解冻并无菌培养24h,再制备成φ=10%的睾丸组织匀浆液并测定4种酶活性,最后分析6种冷冻保护液对犊牛睾丸组织标志性酶活性的冷冻保护效果。结果表明,含ρ=0.015g/mL牛血清白蛋白(BSA)的冷冻保护液冷冻7d后,睾丸组织AKP、ACP、LDH和β-D葡萄糖苷酶的活性分别为599.99、129.11、1 891.53和489.98U/g,均显著优于其他组(P<0.05);含ρ=0.04g/mL海藻糖的冷冻保护液冷冻7d后,AKP、ACP、LDH和β-D葡萄糖苷酶活性分别为454.88、114.63、1 862.79和457.8U/g,其保护效果次于0.015g/mL BSA组,但均显著优于其他组(P<0.05);经DMSO、乙二醇和睾丸液冷冻7d后,4种标志性酶的活性均低于0.015g/mL BSA组和0.04g/mL海藻糖组,甘油组冷冻保护液的效果最差。因此,含ρ=0.015g/mL BSA和ρ=0.04g/mL海藻糖的冷冻保护液能够较好地保护犊牛睾丸组织的酶活性,是犊牛睾丸组织适宜的冷冻保护剂。

In order to research and develop feasible cryopreservation system of calf testicular tissue, 6 kinds of cryoprotectants that used Glycerin, Glycol, DMSO, Trehalose, BSA and Testicular Liquid as main ingredients were designed. The activities of alkaline phosphatase （AKP）, acid phosphatase （ACP）, lactic dehydrogenase （LDH） and β-glucosaccharase （β-D-Glueosidase） were used as determined indexes, and the activities of above 4 enzymes of fresh calf testicular tissue were blank controls. Testicular tissue was eryopreserved for 7 d in liquid nitrogen by 6 kinds of cryoprotectants, then thawed in 37 ℃ water bathe and sterile culture for 24 h, after that, the volume fraction of 10% testicular tissue homogenate was prepared and it was used to determine activities of 4 kinds of enzymes, finally, freeze protection effects of the activities of calf testicular tissue iconic enzymes by 6 kinds of cryoprotectants were analyzed. The results showed as follows：after cryopreservation of calf testicular tissue for 7d with cryoprotectant containing 0. 015 g/mL bovine serum albumin （BSA）, the activities of AKP, ACP, LDH and β-D-Glucosidase of calf testicular tissue were 599.99,129.11,1 891.53 and 489.98 U/g respectively, which all were significantly higher than other groups （P〈0.05） ;after cryo- preservation for 7d with cryoprotectant containing 0.04 g/mL trehalose, the activities of AKP, ACP, LDH and β-D-Glucosidase of calf testicular tissue were 454.88, 114.63, 1 862.79 and 457.8 U/g respectively,which were less than 0. 015 g/mL BSA group but higher than the other groups, significantly（P〈0.05） ; after cryopreservation for 7d with cryoprotectants containing DMSO, testicular liquid and glycol, the activities of iconic enzymes were all less than 0. 015 g/mL BSA group and 0.04 g/mL trehalose group. The effect of glycerin group was the worst. In conclusion, cryoprotectant that containing 0. 015 g/mL BSA and 0.04 g/mL trehalose can well protect the activity of calf testicular tissue enzymes, and suitable served as cryoprotectants for calf testicular tissue.