Cold-shock androgenesis in common carp (Cyprinus carpio)

Abstract

Methods of induction of uniparental inheritance have been developed for model organisms as well as for species of commercial interest to fix sex-specific traits in breeding programs or to restore breeds or lines from cryopreserved sperm in aquaculture important species. Androgenesis in common carp (Cyprinus carpio) has been successfully induced by egg nucleus inactivation with γ-, X- or UV-ray irradiation but these techniques are not widely applicable for laboratory or commercial use on practical, financial and safety grounds. In the past decade a promising low-cost, low-tech cold-shock approach for successfully inducing androgenesis has been demonstrated for loach, Japanese flounder and zebrafish. The aim of the current study was to develop a cold shock methodology for application in common carp, a freshwater species of high commercial interest and a widely used model species. Gametes were collected from wild-type females (dominant green phenotype) and Koi males (recessive blonde phenotype) to enable easy identification of successful induction of androgenetic haploid progeny. A combination of different temperature treatments (0, 2, 4, 6, 8 °C) and different cold-shock durations (15, 30, 45, 60, 75 min) applied shortly after gamete activation (3 s after fertilization) were initially trialed. Optimal condition for egg nucleus elimination was a cold-shock at 2 °C for 60 min duration where hatching rate of haploid progeny reached 35.6% and 26.26 ± 10.19% across two attempts. Double haploid induction was then attempted with three replicates (300 g egg) with following parameters 2 °C cold-shock for 60 min, then transferred into 20 °C and subsequently treated by a heat shock arresting first mitotic cleavage applied 40 min at 40 °C for 2 min. These combined treatments resulted in reduced fertilization and hatching rates for all replicates and low yield of progeny (1.09–1.28% in experimental incubation, <1% in hatchery incubation). A genome wide SNP analysis of a subset of progeny confirmed that they were double haploids. Cold-shock androgenesis was found to be effective in common carp, providing a new possibility of uniparental inheritance induction for isogenic line production.