Zebrafish sperm cryopreservation with N,N-dimethylacetamide.

High fecundity, rapid generation time, and external development of optically clear embryos make the zebrafish (Danio rerio) a convenient vertebrate model for genetic, developmental, and disease studies. Efficient sperm cryopreservation enhances the zebrafish model system by optimizing productive use of facility space, extending the reproductive lifetime of males, providing an alternative to live stocks for strain recovery, and ensuring the survival of valuable mutant lines. Here we identify a cryoprotective medium, 10% N,N-dimethylacetamide (DMA) (v/v) diluted in buffered sperm motility-inhibiting solution (BSMIS), as well as parameters for zebrafish sperm cryopreservation that enhance cryopreservation efficiency and significantly increase the yield of live embryos from archived stocks. Our experiments emphasize the effect of the ratio of sperm and medium volume and the use of large egg clutches to maximize the recovery of viable embryos.

[1]  R. Plasterk,et al.  Target-Selected Inactivation of the Zebrafish rag1 Gene , 2002, Science.

[2]  James F Amatruda,et al.  Zebrafish as a cancer model system. , 2002, Cancer cell.

[3]  Michael Brand,et al.  Keeping and raising zebrafish , 2002 .

[4]  Ralf Dahm,et al.  Zebrafish: A Practical Approach. Edited by C. NÜSSLEIN-VOLHARD and R. DAHM. Oxford University Press. 2002. 322 pages. ISBN 0 19 963808 X. Price £40.00 (paperback). ISBN 0 19 963809 8. Price £80.00 (hardback). , 2003 .

[5]  J. Briggs.  The zebrafish: a new model organism for integrative physiology. , 2002, American journal of physiology. Regulatory, integrative and comparative physiology.

[6]  D. Kime,et al.  Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus). , 2001, Theriogenology.

[7]  J. Komen,et al.  Sperm cryopreservation of African catfish, Clarias gariepinus: cryoprotectants, freezing rates and sperm:egg dilution ratio. , 2000, Theriogenology.

[8]  Á. Horváth,et al.  Cryopreservation of spermatozoa in cyprinid fishes. , 2000, Theriogenology.

[9]  Kunimasa Suzuki,et al.  Characterization of a testicular 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (a spermiation‐inducing steroid in fish) receptor from a teleost, Japanese eel (Anguilla japonica) 1 , 2000 .

[10]  C. Labbé,et al.  Membrane integrity, mitochondrial activity, ATP content, and motility of the European catfish (Silurus glanis) testicular spermatozoa after freezing with different cryoprotectants. , 1999, Cryobiology.

[11]  I. Babiak,et al.  The effect of egg yolk, low density lipoproteins, methylxanthines and fertilization diluent on cryopreservation efficiency of northern pike (Esox lucius) spermatozoa. , 1999, Theriogenology.

[12]  H. Ohta,et al.  Cryopreservation of sperm from the endangered formosan landlocked salmon (Oncorhynchus masou formosanus). , 1999, Theriogenology.

[13]  A. Johannisson,et al.  Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry. , 1999, Theriogenology.

[14]  J. Postlethwait,et al.  Production of haploid and diploid androgenetic zebrafish (including methodology for delayed in vitro fertilization). , 1999, Methods in Cell Biology.

[15]  F. V. van Eeden,et al.  Developmental mutant screens in the zebrafish. , 1999, Methods in cell biology.

[16]  J. Critser,et al.  Reproductive tissue banking : scientific principles , 1998 .

[17]  D. Ransom,et al.  Appendix 3 Collection, Storage, and Use of Zebrafish Sperm , 1998 .

[18]  P. Mazur,et al.  Fundamental Cryobiology of Mammalian Spermatozoa , 1997 .

[19]  B. Urbányi,et al.  Cryopreservation of common carp (Cyprinus carpio L.) sperm II. Optimal conditions for fertilization , 1996 .

[20]  M. Westerfield.  The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio) , 1995 .

[21]  K. Rana.  Cryopreservation of fish spermatozoa. , 1995, Methods in molecular biology.

[22]  N. Hart,et al.  Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg. , 1987, The Journal of experimental zoology.

[23]  M. Ashwood‐Smith,et al.  Cryopreservation of zebra fish spermatozoa using methanol , 1982 .

[24]  M S Mounib,et al.  Cryogenic preservation of fish and mammalian spermatozoa. , 1978, Journal of reproduction and fertility.

[25]  P. Naccache,et al.  Patterns of Nonelectrolyte Permeability in Human Red Blood Cell Membrane , 1973, The Journal of general physiology.

[26]  A. Karow.  Cryoprotectants—a new class of drugs , 1969, The Journal of pharmacy and pharmacology.