PDF(Pubmed)
Abstract：
The axolotl (Ambystoma mexicanum) draws great attention around the world for its importance as a biomedical research model, but housing and maintaining live animals is increasingly expensive and risky as new transgenic lines are developed. The goal of this work was to develop an initial practical pathway for sperm cryopreservation to support germplasm repository development. The present study assembled a pathway through the investigation of axolotl sperm collection by stripping, refrigerated storage in various osmotic pressures, cryopreservation in various cryoprotectants, and in vitro fertilization using thawed sperm. By the stripping of males, 25-800 µL of sperm fluid was collected at concentrations of 1.6 × 106 to 8.9 × 107 sperm/mL. Sperm remained motile for 5 d in Hanks\' Balanced Salt Solution (HBSS) at osmolalities of 100-600 mOsm/kg. Sperm cryopreserved in 0.25 mL French straws at 20 °C/min in a final concentration of 5% DMFA plus 200 mM trehalose and thawed at 25 °C for 15 s resulted in 52 ± 12% total post-thaw motility. In six in vitro fertilization trials, 20% of eggs tested with thawed sperm continued to develop to stage 7-8 after 24 h, and a third of those embryos (58) hatched. This work is the first report of successful production of axolotl offspring with cryopreserved sperm, providing a general framework for pathway development to establish Ambystoma germplasm repositories for future research and applications.
摘要：
axolotl（Ambystomamexicanum）作为生物医学研究模型的重要性引起了全世界的极大关注，但是，随着新的转基因品系的开发，饲养和维护活体动物变得越来越昂贵和危险。这项工作的目的是开发精子冷冻保存的初始实用途径，以支持种质资源库的开发。本研究通过研究剥离法收集腋窝精子的途径，在各种渗透压下冷藏，在各种冷冻保护剂中冷冻保存，使用解冻的精子进行体外受精。通过男性的剥离，收集25-800μL的精子液，浓度为1.6×106至8.9×107精子/mL。在100-600mOsm/kg的渗透压下，精子在汉克斯平衡盐溶液（HBSS）中保持运动5d。精子以20°C/min的速度冷冻保存在0.25mL法国吸管中，最终浓度为5％DMFA加200mM海藻糖，并在25°C下解冻15s，总解冻后运动性为52±12％。在六项体外受精试验中，使用解冻精子测试的卵子中，有20％在24小时后继续发育到7-8阶段，这些胚胎的三分之一（58）孵化。这项工作是第一个成功生产具有冷冻保存的精子的后代的报告，为建立Ambystoma种质资源库的途径开发提供了一个通用框架，以供将来的研究和应用。
