Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success.
Zebrafish reproduction: revisiting in vitro fertilization to increase sperm cryopreservation success.
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DOI：
10.1371/journal.pone.0021059
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Carter VL
中科院分区：
文献类型：
--
作者：
Hagedorn M;Carter VL
虽然传统的超低温保存是一种经过验证的长期、安全地储存遗传物质的方法，但斑马鱼群落使用的程序没有标准化，结果不一致，从而将个别实验室和储存中心的许多基因类型的安全置于危险之中。成功的斑马鱼精子冷冻保存计划的一个重要挑战是解冻后体外受精成功率(0-80%)的巨大变异性。但是，这种变异在多大程度上是由于体外受精过程的生殖特征，而不是由于冷冻保存过程？这些实验只评估了新鲜精子的体外过程，但也得出了使用冷冻精子成功进行体外受精所需的基本指标。我们分析了具有严格身体条件范围的雄性斑马鱼的生殖特征。它与精子体积或活动率无关(P>；0.05)，但与精子密度有关。年轻男性的精子密度更高(P<；0.05)。为了最大限度地减少体外受精过程中的精子损耗，106个/毫升是实现≥70%的体外受精成功率所需的最低精子浓度。在体外过程中，汇集精子不会降低受精成功率(P>；0.05)，但汇集卵子会使受精率降低约30%至50%(P<；0.05)。这种受精成功率的下降不是由于雌性卵子的汇集，而是因为处理卵子的工具类型。加强斑马鱼体外培养过程的建议包括：1)使用身体状况接近1.5的雄性斑马鱼，以获得最大的精子密度；2)使用106个活动细胞/毫升的工作精子密度进行体外受精，以尽量减少精子浪费；以及3)在受精之前，不要使用金属或锋利的工具处理卵子。
Although conventional cryopreservation is a proven method for long-term, safe storage of genetic material, protocols used by the zebrafish community are not standardized and yield inconsistent results, thereby putting the security of many genotypes in individual laboratories and stock centers at risk. An important challenge for a successful zebrafish sperm cryopreservation program is the large variability in the post-thaw in vitro fertilization success (0 to 80%). But how much of this variability was due to the reproductive traits of the in vitro fertilization process, and not due to the cryopreservation process? These experiments only assessed the in vitro process with fresh sperm, but yielded the basic metrics needed for successful in vitro fertilization using cryopreserved sperm, as well. We analyzed the reproductive traits for zebrafish males with a strict body condition range. It did not correlate with sperm volume, or motility (P>0.05), but it did correlate with sperm concentration. Younger males produced more concentrated sperm (P<0.05). To minimize the wastage of sperm during the in vitro fertilization process, 106 cells/ml was the minimum sperm concentration needed to achieve an in vitro fertilization success of ≥ 70%. During the in vitro process, pooling sperm did not reduce fertilization success (P>0.05), but pooling eggs reduced it by approximately 30 to 50% (P<0.05). This reduction in fertilization success was due not to the pooling of the females' eggs, but to the type of tools used to handle the eggs. Recommendations to enhance the in vitro process for zebrafish include: 1) using males of a body condition closer to 1.5 for maximal sperm concentration; 2) minimizing sperm wastage by using a working sperm concentration of 106 motile cells/ml for in vitro fertilization; and 3) never using metal or sharp-edged tools to handle eggs prior to fertilization.
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