Abstract
Sperm cryopreservation is a highly efficient method for preserving genetic resources. It extends the reproductive period of males and significantly reduces costs normally associated with maintenance of live animal colonies. However, previous zebrafish (Danio rerio) cryopreservation methods have produced variable outcomes and low post-thaw fertilization rates. To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process. We found that the hypertonic extender E400 preserved motility of sperm held on ice for at least 6 h. We implemented QA by measuring sperm cell densities with a NanoDrop spectrophotometer and sperm motility with computer-assisted sperm analysis (CASA). We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer. Post-thaw motility indicated that the optimal cooling rate in two types of cryogenic vials was between 10 and 15°C/min. Test thaws from this method produced average motility of 20% ± 13% and an average post-thaw fertilization rate of 68% ± 16%.
Highlights
Germplasm cryopreservation is crucial for the storage and availability of genetic resources for biomedical research
To improve post-thaw fertilization rates after cryopreservation, we developed a new extender and cryoprotective medium (CPM), introduced quality assessment (QA), determined the optimal cooling rate, and improved the post-thaw in vitro fertilization process
We developed a CPM, RMMB, which contains raffinose, skim milk, methanol, and bicine buffer
Summary
Germplasm cryopreservation is crucial for the storage and availability of genetic resources for biomedical research. The zebrafish (Danio rerio) research community has generated novel mutations and transgenic lines at an unprecedented rate, producing an enormous resource for studying gene function. Several large-scale mutagenesis programs have produced tens of thousands of novel mutations in the zebrafish genome by exploiting advances in sequencing technologies and the availability of a genome map and sequence.[1,2,3,4] With the recent advent of efficient genome editing technologies,[5,6] even more genetic modifications of the zebrafish genome are being produced. Cryopreservation of sperm is a proven method for long-term storage and reducing maintenance costs of genetic materials
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