1. Introduction
Ž Induced gynogenesis is a unique tool for inbreeding purposes in teleost fish Chour-
. rout, 1988; Mair, 1993 . It involves an artificial reproduction, using UV-irradiated sperm
to activate the eggs and the application of a physical or chemical shock to restore the diploid status of the embryo. The shocks destroy the aster formation or the microtubules
Ž .
of the spindle figure and inhibit nuclear division Diter et al., 1993 . The result is a Ž
. diploid embryo containing maternal genetic material only Fig. 1 . Meiogynogenesis is
achieved by inhibiting the extrusion of the second polar body. The resulting offspring are homozygous at a locus only if no recombination occurred. By determining the
percentage of heterozygous offspring, one can calculate the recombination frequency.
Ž . Inbreeding F is lower for those traits showing much residual heterozygosity. For some
Ž species, like rainbow trout, this residual heterozygosity is so high up to 100 at some
. loci
that meiogynogenesis cannot be considered as an efficient inbreeding tool Ž
. Thorgaard et al., 1983; Guyomard, 1984 .
Ž .
Mitogynogenesis or endomitosis results in fully homozygous offspring F s 1 since Ž
. it is achieved by inhibiting the first mitotic cleavage after duplication of the haploid
Ž .
Ž .
genome Fig. 1 . Homozygous inbred strains of genetically identical fish clonal lines may be obtained after two generations using this reproduction method. It has been
Ž .
achieved in zebrafish Danio rerio Streisinger et al., 1981 , medaka Oryzia latipes Ž
. Ž
. Naruse et al., 1985 , common carp Cyprinus carpio
Komen et al., 1991 , ayu Ž
. Ž
Plecoglossus altiÕelis Han et al., 1992 , rainbow trout Oncorhynchus mykiss Quillet et .
Ž .
al., 1991; Scheerer et al., 1991 , hirame Yamamoto, 1999 and Nile tilapia Ore- Ž
. ochromis niloticus Hussain et al., 1993 . The importance of inbred strains in research
Ž .
and aquaculture has been stressed by Bongers 1997 . Inbreeding represents an impor- tant tool to improve production characteristics with increased chances to directly select
specific genotypes. The genome, purged of deleterious genes, may be reconstituted by Ž
. crossing several inbred lines Yamamoto, 1999 . Also, crossing clonal lines may induce
heterotic effects. Finally, as a result of reduced genetic variation in inbred lines, less animals are necessary to obtain statistically significant experimental designs.
The African catfish Clarias gariepinus is an economically important fish species which is cultivated on many continents. In Africa and Asia, an extensive as well as
Ž .
intensive culture exist Huisman and Richter, 1987 , whereas in Europe, they are mostly intensively produced. The specific conditions for the production of meiogynogens
through the retention of the second polar body have already been optimised in C. Ž
. Ž
. gariepinus Volckaert et al., 1994, 1997 . High survival rates 46 were obtained when
Ž .
a shock of 418C at 3 min after activation m.a.a. was applied for 2 min. Cold and Ž
pressure shocks applied 4 m.a.a., generated even higher survival rates 80 and 68, .
respectively . The optimal conditions to induce endomitosis remained to be determined. Preliminary experiments showed a higher survival in mitogynogenic embryos using heat
Ž .
shocks, as predicted by Hussain et al. 1993 . Furthermore, androgenesis in C. gariepi- Ž
. Ž
nus has been successfully induced up to 10.5 survival by heat shocking Bongers et .
al., 1995 . The genetic background of the presumed gynogenic progeny requires verification.
Haploid progeny are not viable but incomplete UV-irradiation, possibly through differ-
Fig. 1. Induced and spontaneous gynogenesis in fish. When the oogonium of a female heterozygous at loci T and Q is activated by a UV-irradiated sperm cell, it completes meiosis-II and develops into a haploid
Ž .
Ž .
Ž non-viable embryo. Viable diploid 2n offspring can be obtained by inhibiting first mitosis A: mitogyno-
. Ž
gens 2n; complete homozygous offspring or second meiosis B: meio-II-gynogens 2n; heterozygous offspring .
depending on the recombination frequency, r, of the locus; 0 heterozygosity, H, if r s 0 . Spontaneous Ž
gynogenesis can occur via the latter mechanism but also by inhibition of the first meiosis C: meio-I-gynogens .
Ž 2n . In this case, recombination leads to 50 heterozygous offspring 100 heterozygous clonal offspring if
. r s 0 .
Ž .
ences in the spermatocrit Mair, 1993 , could generate progeny with paternal genetic Ž
. Ž
material ‘false’ gynogens . This may lead to misinterpretation of the results Carter et .
al., 1991 . Furthermore, one should verify the homozygous nature of endomitotic Ž
. gynogens Bongers, 1997 and determine the degree of residual heterozygosity, through
recombination, in meiogynogenic progeny. It is possible to assess the level of recom- Ž
bination and hence the inbreeding rate by using heterozygous females Chourrout, .
1988 . Genetic markers are required to detect paternal contamination and to determine the
degree of heterozygosity in gynogenic offspring. Morphological markers can be used if Ž
. the trait is based on a recessive allele; such phenotypic markers e.g., albinism were not
available in our broodstock. Multilocus DNA fingerprinting has been used for detecting Ž
paternal contamination but it is not efficient to verify heterozygosity levels Carter et al., .
Ž 1991; Volckaert et al., 1994; Bongers, 1997 . Allozyme markers are useful Linhart et
. al., 1987 but they cannot compete with the level of polymorphism and the information
Ž .
content of single locus microsatellite markers Wright, 1992 . Furthermore, these latter markers allow biopsies.
We show the low feasibility of applying heat shocks to induce endomitosis and the presence of a few ‘false’ and relatively many spontaneous gynogens. We provide
alternative hypotheses for the high percentage of residual heterozygosity at one of the DNA markers and suggest an alternative estimation of the recombination rate. Finally,
we show evidence for male heterogametic sex chromosomes in C. gariepinus.
2. Materials and methods
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