Fisheries and Aquatic Sciences, School of Forest, Fisheries, and Geomatics Sciences, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, 32653, USA.
Primo Broodstock USA LLC, 3901 County Line Ditch Rd, Mims, Florida, 32754, USA.
Cryobiology. 2022 Feb;104:70-78. doi: 10.1016/j.cryobiol.2021.10.003. Epub 2021 Oct 30.
The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration for: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA or methanol, membrane integrity was significantly decreased (70-75%) compared to controls. With catalase, sperm cells maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.
本研究旨在从高通量遗传资源库操作的角度，探讨在冷冻保护液中添加抗氧化酶过氧化氢酶（CAT）是否能有效用于斑马鱼精子的冷冻保存。研究使用了三种冷冻保护剂（10%，v/v）：二甲基乙酰胺（DMA）、二甲基甲酰胺（DMF）和甲醇。研究目的是评估 CAT 对精子活力、质膜完整性和浓度的影响，包括：1）新鲜精子在平衡期至 60 分钟内；2）冷冻后在 10、20 和 40°C/min 下冷却的精子；3）冷冻后受精和胚胎存活率。无论添加何种冷冻保护剂，CAT 的添加都不能提高精子活力。DMA 或甲醇暴露 10 分钟后，与对照组相比，质膜完整性显著降低（70-75%）。用 CAT 处理后，精子细胞保持质膜完整性，与仅用冷冻保护剂的试验组相比，在 50 分钟平衡期后细胞浓度得以维持。然而，在冷冻保存和解冻后，CAT 对精子活力、质膜完整性、细胞浓度、受精或胚胎存活率的影响没有影响。冷却速率的分析也表明，CAT 不影响 3 小时pf 的受精率或 24 小时 pf 的存活率。总的来说，CAT 的添加可以在冷冻前为精子提供一些抗氧化应激的保护，但解冻后则无效。我们提出，关于 CAT 用于资源库的常规使用的决策，特别是那些每年处理数万份冷冻样本的资源库，将取决于是否高效的高通量操作，或者特定的研究问题是否是项目目标。
