Artificial sperm insemination in externally fertilised fish as a novel tool for ex situ and in situ conservation of valuable populations

Loss of genetic diversity and accumulation of deleterious mutations may lead to inbreeding depression in captive breeding. To address the problem of maintaining genetic diversity, we developed a new fish spawning method which offers flexibility in crossing diverse species when in vitro fertilisation (IVF) is not available. This method involves the collection of sperm from several males of ovuliparous fish; the sperm mix is then injected by catheter into the ovarian cavity of a female through the oviduct. We demonstrate, using zebrafish as a model for externally fertilised fish, that the sperm survives the ovarian conditions and can fertilise ovulated eggs, which are released from the body cavity during natural spawning. Wild type females were injected with reporter transgenic sperm from homozygous transgenic males before intended spawning with wild type males. The sperm injection method did not have an impact on reproductive parameters such as egg production or fertilisation rate compared to controls. In 25 successful spawning experiments, 20 females produced mixed genotype offspring comprising both transgenic and wild type larvae in varying ratios, indicating that the injected transgenic sperm efficiently competed with sperm released by non-transgenic wild type mating males, and both sperm types contributed to the fertilisation of the released eggs. This experiment provides proof of principle for increasing the genetic base of offspring of fish species, including that of many endangered fish species for which IVF is not available due to lack of timed induction of ovulation or when gametic release cannot be synchronised