南美白对虾精子超低温冷冻保存技术研究

Spermatozoa cryopreservation of Litopenaeus vannamei

【目的】建立一种南美白对虾精子超低温冷冻保存方法,为南美白对虾优良种质的长期保存奠定基础。【方法】利用胰蛋白酶消化法获得游离的南美白对虾精子,分别使用灭菌天然海水、无钙人工海水、3%等渗NaCl溶液和0.9%生理盐水作为基础液,并以不同浓度的二甲基亚砜(DMSO)、甘油、甲醇及其与海藻糖的混合溶液作为抗冻剂,在4种不同降温程序下超低温冷冻保存南美白对虾精子,然后在不同的温度下复苏冷冻精子,以伊红—苯胺黑染色法检测精子存活率。【结果】使用1.00 g/L胰蛋白酶消化精荚5 min能获得大量游离的南美白对虾精子,以灭菌天然海水为基础液,以10%DMSO和0.25 mol/L海藻糖为抗冻剂,在P-1降温程序(4℃平衡30 min,以-5℃/min的速率降至-20℃;-20℃平衡5 min,以-10℃/min的速率降至-80℃;-80℃平衡5 min,然后置于液氮中保存)下超低温冷冻保存南美白对虾精子,经37℃水浴复苏后精子存活率最高。【结论】建立的南美白对虾精子超低温冷冻保存方法具有可行性,为南美白对虾精子冷冻保存库的建立提供了技术支撑。

[Objective ]The technique of cryopreservation in Litopenaeus vannamei spermatozoa was studied to provide a feasible method for long-term storage of the genetic resources of L. vannamei. [Method]Free sperms of L. vannamei were acquired from spermatophore by the trypsinase digestion method. Four extenders （sterilized natural seawater, calcium-free saline, 3% NaCl isotonic solution, and 0.9% physiogieal saline solution） were mixed with different concentrations of DMSO, glycerol, methanol and myeose to form cryoprotectants. Litopenaeus vannamei spermatozoa cryopreservation was performed in four cooling procedures, and then sperms were thawed at different temperatures. The survival rate of sperm was detected using a modified eosin-nigrosin staining method. [Result]A great deal of free sperms were acquired using 1.00 g/ L trypsinase digesting spermatophore for five minutes. A successful cryopreservation of sperms in liquid nitrogen was achieved using sterilized natural seawater containing 10% DMSO and 0.25 mol/L trehalose. Sperms were cryopreserved under the condition of P-1 protocol： suspending for 30 minutes at 4 ℃, 4 ℃ to -20 ℃ cooling rate of -5 ℃/min; suspending for 5 minutes at -20 aC, -20 ℃ to -80 ℃ cooling rate of -10 ℃/min; suspending for 5 minutes at -80 ℃ ,and storing in liquid nitrogen. Frozen sperms were thawed in 37 ℃ water bath, and the survival rate of cryopreserved sperm reached to the highest. [Conclusion ]The established cryopreservation method of L. vannamei spermatozoa was applicable to provide technical support for constructing a cryopreservation storeroom of L. vannmnei.