Aquaculture Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, USA.
Zebrafish. 2011 Dec;8(4):167-79. doi: 10.1089/zeb.2011.0704. Epub 2011 Sep 1.
This study reports the first production of offspring with vitrified sperm from a live-bearing fish Xiphophorus hellerii. The overall goal of this study was to develop streamlined protocols for integration into a standardized approach for vitrification of aquatic species germplasm. The objectives were to (1) estimate acute toxicity of cryoprotectants, (2) evaluate vitrification solutions, (3) compare different thawing methods, (4) evaluate membrane integrity of post-thaw sperm vitrified in different cryoprotectants, and (5) evaluate the fertility of vitrified sperm. Nine cryoprotectants and two commercial vitrification additives were tested for acute toxicity and glass forming ability, alone and in combination. Two vitrification solutions, 40% glycerol (Gly) and 20% Gly+20% ethylene glycol (EG) in 500 mOsmol/kg Hanks' balanced salt solution (HBSS), were selected for vitrification of 10 μL sperm samples using inoculating loops plunged into liquid nitrogen. Samples were thawed at 24°C (one loop in 5 μL of HBSS or three loops in 500 μL of HBSS). Samples thawed in 500 μL were concentrated by centrifugation (1000 g for 5 min at 4°C) into 5 μL for artificial insemination. Offspring were produced from virgin females inseminated with sperm vitrified with 20% Gly+20% EG and concentrated by centrifugation.
本研究报告了首例通过玻璃化保存的活产鱼类剑尾鱼精子生产后代。本研究的总体目标是开发简化的方案，将其整合到水生物种种质的玻璃化标准化方法中。具体目标是：(1) 评估冷冻保护剂的急性毒性，(2) 评估玻璃化溶液，(3) 比较不同的解冻方法，(4) 评估不同冷冻保护剂中解冻后精子的膜完整性，以及 (5) 评估玻璃化精子的生育能力。单独和组合使用了九种冷冻保护剂和两种商业玻璃化添加剂进行急性毒性和玻璃形成能力测试。选择 40%甘油 (Gly) 和 20% Gly+20%乙二醇 (EG) 的两种玻璃化溶液，在 500 mOsmol/kg Hank's 平衡盐溶液 (HBSS) 中，用接种环浸入液氮中对 10 μL 精子样品进行玻璃化处理。在 24°C 下解冻样品(在 5 μL HBSS 中一个环或在 500 μL HBSS 中三个环)。在 500 μL 中解冻的样品在 4°C 下以 1000 g 离心 5 分钟浓缩至 5 μL 进行人工授精。用经 20% Gly+20% EG 玻璃化并离心浓缩的精子对处女雌鱼进行人工授精，产生后代。
