Sperm cryopreservation of a live-bearing fish, Xiphophorus couchianus: Male-to-malevariation in post-thaw motility and production of F_1hybrid offspring

摘要

Fishes of the genus Xiphophorus are well-studied biomedical research models, and some species, such asX. couchianus, are currently listed as endangered in the wild. Sperm cryopreservation in these live-bearingfishes has begun recently. Thus far, live young have been produced with cryopreserved sperm only in onespecies (Xiphophorus helleri). In this study, the goal was to develop a practical protocol for spermcryopreservation of Xiphophorus couchianus, and to produce live young with cryopreserved sperm. Spermwere collected by crushing of testis in Hanks' balanced salt solution at an osmolality of 500 mOsmol/kg(HBSS500), and were cryopreserved with 14% glycerol (v/v) as cryoprotectant at a cooling rate of 20 °C/minfrom 5 to -80 °C in 250-4 French straws. For artificial insemination, samples were thawed at 40 °C for 5 s ina water bath, washed once using fresh HBSS500 by centrifuging at 1000 g for 5 min at 4 °C, concentrated into-5μL,and injected into virgin females ofXiphophorus maculatus.The inseminated were monitoredfor 90 days for subsequent discharge of live young. Results from 2006 and 2007 showed considerable male-to-male variation in post-thaw motility (from 1 to 70%). Offspring were produced by cryopreserved sperm intwo tanks (of three) at 36 and 66 days after insemination in 2007. Paternity was confirmed via phenotypes(body color) and genotypes (microsatellite genetic marker) of the hybrid offspring. Overall, a practicalprotocol for sperm cryopreservation and artificial insemination is provided to preserve X. couchianus, whichis an important biomedical research model, and also currently listed as an endangered species in theInternational Union for Conservation of Nature (IUCN) red list.