B. Kovács1*, M. Bercsényi2, Zs.Csenki1, K. Bakos1, R. Kovács1, A. Csepeli1, S. Németh2, Cs. Uri1, G. Bernáth1, Á. Horváth1, Z. Bokor1, B. Urbányi1, L. Orbán3
1Department of Aquaculture, Szent István University, Páter Károly u. 1., H-2100 Gödöllő, Hungary
2Department of Animal Sciences and Breeding, Georgikon Faculty, University of Pannonia, Deák Ferenc u. 16, H-8230 Keszthely, Hungary
3 Reproductive Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link National University of Singapore, Singapore 117604, Singapore
Introduction
Contrary to other model animals, highly homogeneous/inbred strains of zebrafish (Danio rerio) are not available to the scientific community. Only two gynogenetic lines (C32 strain by Streisinger et al. (1981) and another line by Shinya and Sakai2011 were generated) and some partially inbred strains (SJD, SJC, SJA derived from DAR, C32, and AB, respectively and the IM from India strain) were established but genetic analysis of them, revealed that 7% (C32), 11% (SJD) 15% (SJA) and 5% (IM) of tested loci were polymorphic in them, respectively (Guryev et al. 2006; Bradford et al. 2011; Shinya and Sakai 2011.). However, it is not clear whether these types of strains have such a "high" genetic diversity in zebrafish or will become extinct.
Gynogenesis is one of the fastest ways to produce highly inbred strains. However, the production of gynogenetic zebrafish lines has some disadvantages (i.e., logistical difficulties related to sperm, high mortality levels of embryos and larvae, biased sex ratio, low quantity and variable quality sperm, etc.).
In this study, we have initiated the production of a highly inbred line with gynogenesis using interspecific cryopreserved sperm to avoid disadvantages of the process.
Material and methods
An outbred AB-type strain was used to generate genetically homogeneous strains of zebrafish. Eggs were obtained by stripping anesthetized spawners. Gamma irradiated carp (Cyprinus carpio) or goldfish (Carassius auratus auratus) sperm were cryopreserved (Horváth et al. 2007) and later used for in vitro egg activation. To obtain homozygous fish, the activated eggs were subjected to a heat shock according to the protocol described by of Streisinger et al. (1981). The heat shock converts haploid eggs into homozygous diploids (di-haploid) by blocking the first cell cleavage. The survivors were reared in a ZebTec Zebrafish housing system (Tecniplast) according the recommendations of the Zebrafish Book (Westerfield 2000). Upon sexual maturity of gynogenotes, they produced the later generations by induction of natural spawning as described in the Zebrafish Book.
Results
Gynogenetic zebrafish individuals were produced successfully with interspecific cryopreserved sperm. Only 184 larvae hatched from more than 18,000 eggs. Most of them died of different developmental disturbances at different developmental stages. Finally, 14 individuals reached adulthood. Five of them (4 males and one putative female) have not produced offspring, while 7 males and 2 females were fertile. These individuals were used to produce the F1 generation. Until now in the F1 generation consists of 46 individuals produced by 1 couple and only one individual displays the male phenotype.
Discussion and conclusion
Our results show that irradiated and cryopreserved sperm of carp and goldfish can be used for gynogenesis in zebrafish. However, adequate control groups were not available to check the sperm and egg quality before the treatments because the unfertilized, haploid and interspecific hybrid individuals (from carp/goldfish X zebrafish crosses) have a similar phenotype and are not viable. Nevertheless, the survival rate was lower than 1% as it was expected. We observed a sex-biased nature of the highly homogenous lines both in the gynogenetic (male biased) and the F1 generation (female biased; ~98%). Presumably this is a result of the multi chromosomal sex determination of Danio rerio (Liew et al. 2012).
To the best of our knowledge, this is the first experiment that produced gynogenetic zebrafish individuals with interspecific, cryopreserved sperm.
Acknowledgements
This work was supported by OTKA (105393) and KTIA-AIK-12-1-2012-0010 projects
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