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
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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
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