zebrafish reproduction revisiting in vitro fertilization to increase sperm cryopreservation success斑马鱼繁殖回顾体外受精增加精子冷冻保存成功.pdf

Zebrafish Reproduction: Revisiting In Vitro Fertilization to Increase Sperm Cryopreservation Success Mary Hagedorn1,2*, Virginia L. Carter1,2 1 Smithsonian Conservation Biology Institute, Smithsonian National Zoological Park, Washington, D.C. United States of America, 2 Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, Hawaii, United States of America Abstract 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 pool