Artificial insemination of African catfish (Clarias gariepinus) using cryopreserved sperm

In this study, we aimed to develop a practical protocol for using cryopreserved sperm for induced/wild/tank spawning of fish species with external fertilization. Experiments were carried out on African catfish (Clarias gariepinus) as a model species. Sperm was collected for cryopreservation and diluted with the cryomedium (266 mM fructose, 20% methanol) at a ratio of 1:1 with a final methanol concentration of 2.47 M pH7.73. Diluted sperm was loaded into 0.5-ml straws and cryopreserved by conventional protocol. Samples were prepared for insemination 24 h later, by thawing for 13 s in a 40 °C water bath, and centrifuged at 500 × g for 10 min at 20 °C. The seminal plasma, extender and external cryoprotectant were removed from the concentrated spermatozoa. The pellet was then resuspended in common carp (Cyprinus carpio) seminal plasma to reconstitute the lost volume. Sperm samples were then injected by a catheter into the ovarian cavity through the oviduct of the experimental females by the so-called ovarian lavage method in parallel with the intramuscular hormonal administration (5 mg carp pituitary/kg bw). Inseminated females (n = 9) were monitored for 10 h and ovulated eggs and spermatozoa stored in in the ovary were stripped. Stripped gamete samples were divided into two batches: (1) the first batch contained only the previously injected spermatozoa and was activated by aerated water (WA) immediately after stripping; (2) in case of the second batch additional, freshly stripped sperm was added as positive control to the stripped eggs before water activation (PC). Furthermore, five females were propagated by using the dry fertilization method (in vitro fertilization) as negative control (NC). All sperm and hormone injected females produced fertilised eggs with a hatching rate of 17.7 ± 13.2%, 12.5 ± 9.3%, and 61 ± 11.5% for WA, PC and NC respectively. These results indicate that artificial insemination based on using cryopreserved sperm with ovarian lavage can be a viable alternative to in vitro fertilization in a catfish species. Thus, we describe a proof of principle for a practical protocol for the induced/wild/tank spawning of an externally fertilising fish species with economical importance and propose that the protocol could be also applied to endangered marine or fresh fish species. •Cryopreserved sperm was injected post-thaw by catheter into the ovarian lobes of C. gariepinus before induced spawning.•Gametes were stripped and fertilised with water, a