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ESR prepress abstract   -  DOI:

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

Gyöngyi Gazsi, Bence Ivánovics, Izabella Roberta Berta, Tamás Szabó, Zarski Daniel, Balázs Kucska, Béla Urbányi, László Horváth, Ferenc Müller, Tamás Müller*

*Corresponding author:

ABSTRACT: 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 in which in vitro fertilisation (IVF) is not available. The method relies on collection of sperm from several males of ovuliparous fish, and 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 sperm survives the ovary condition and can fertilise ovulated eggs, which are released from the body cavity by 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 reproduction parameters such as egg production or fertilisation rate as compared to controls. In 25 successful spawning experiments, 20 females produced mixed genotype offspring, which contained 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 contributed to 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.