Challenges in Development of Sperm Repositories for Biomedical Fishes: Quality Control in Small-Bodied Species.
Challenges in Development of Sperm Repositories for Biomedical Fishes: Quality Control in Small-Bodied Species.
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生物医学鱼类精子库开发的挑战：小型物种的质量控制。
DOI：
10.1089/zeb.2017.1426
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发表时间：
2017
期刊：
影响因子：
2
通讯作者：
Tiersch,TerrenceR
中科院分区：
文献类型：
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作者：
Torres,Leticia;Liu,Yue;Guitreau,Amy;Yang,Huiping;Tiersch,TerrenceR
质量控制（QC）是种质资源库可重复性和有效运作的关键。然而，许多生物医学鱼类模型由于体型小（<5 cm）和精子体积小（<5 μL）而存在显著的QC挑战。使用最小体积的精子，我们使用斑马鱼来评估常见的QC终点，作为冷冻保存沿着顺序步骤中受精成功的替代物。首先，通过使用不同的样品体积和混合方法，用Makler®计数室评估校准珠悬浮液的浓度。对于精子分析，首先用Hanks平衡盐溶液（HBSS）以1：30的比例稀释样本。通过使用不同比例的精子和活化培养基来评价运动性，并在不同浓度下用流式细胞术分析膜完整性。使用小至1 μL的体积可以可靠地估计浓度和精子活力，而膜完整性需要至少2 μL（1 × 106个细胞/mL）。因此，需要<5 μL的精子悬液（用HBSS稀释至30-150 μL后），以使用三个终点评价精子质量。使用互补终点的组合进行精子质量评估可增强冷冻保存期间的QC工作，提高可靠性和重现性，并减少时间和资源的浪费。
Quality control (QC) is essential for reproducible and efficient functioning of germplasm repositories. However, many biomedical fish models present significant QC challenges due to small body sizes (<5 cm) and miniscule sperm volumes (<5 μL). Using minimal volumes of sperm, we used Zebrafish to evaluate common QC endpoints as surrogates for fertilization success along sequential steps of cryopreservation. First, concentrations of calibration bead suspensions were evaluated with a Makler®counting chamber by using different sample volumes and mixing methods. For sperm analysis, samples were initially diluted at a 1:30 ratio with Hanks' balanced salt solution (HBSS). Motility was evaluated by using different ratios of sperm and activation medium, and membrane integrity was analyzed with flow cytometry at different concentrations. Concentration and sperm motility could be confidently estimated by using volumes as small as 1 μL, whereas membrane integrity required a minimum of 2 μL (at 1 × 106cells/mL). Thus, <5 μL of sperm suspension (after dilution to 30–150 μL with HBSS) was required to evaluate sperm quality by using three endpoints. Sperm quality assessment using a combination of complementary endpoints enhances QC efforts during cryopreservation, increasing reliability and reproducibility, and reducing waste of time and resources.