The absence of well defined inbred lines is an important problem associated with scientific research on fish. Inbred lines can be produced by conventional full-sib mating, but at least 10-15 generations are needed to produce homozygous inbred lines. Using common carp, which reach maturity at 1.5 years, this would last some 15-30 years. Nowadays experimental fishes are usually obtained from commercial fish farms, or bred in the laboratory using a limited number of broodstock fish. In both cases the genetic background and the degree of inbreeding of the experimental animal is unknown.
In consequence the results from various laboratories are difficult to compare. Bioassays often show a large variation in the experimental results and a relative low reproducability. Moreover, large numbers of fish are needed to obtain statistically significant results. In order to solve these problems this research project was started with the aim to develop homozygous inbred lines of fish by gynogenetic breeding. Furthermore, in our university there was a high need for inbred lines with specific (mutant) genotypes, which could be used in the ongoing research on the immune system and sex determination of common carp.
In gynogenesis, eggs are fertilized with genetically inactivated sperm. The resulting haploid embryo can be made diploid by inhibition of the second meiotic division (retention of the second polar body or 2PB method), or by inhibition of the first mitotic division (endomitosis or EM method). In the first case the gynogenetic offspring will be partly heterozygous due to recombination during the preceeding meiotic prophase. In the second case the haploid genome of the embryo is duplicated while the first cell division is prevented. The resulting diploid offspring will be fully homozygous.
In a first series of experiments (chapter 3) the optimal conditions for irradiation and dilution of milt, and for administration of a temperature shock to inhibit the second meiotic division, were investigated. Milt was irradiated with U.V. light (235.7 nm). Dilution (in physiological saline) and irradiation duration were important parameters for the survival of spermatozoa. Sperm, diluted 1:3, could be irradiated for 60 minutes (2200 J/m2,min) without loss of fertilization capacity. This fertilization capacity was considerably reduced when higher dilutions were used, while a shorter irradiation period failed to inactivate all spermatozoa.
The effectiveness of genetic inactivation was checked by using sperm from scaled males (a dominant trait) and eggs from scattered females (recessive trait). Gynogenetic offspring turned out to be all scattered. Inhibition of the second meiotic division was achieved by administering eggs, fertilized with genetically inactivated sperm, a temperature shock at various moments after fertilization. Consistent yields of 25-50 % viable fry were obtained when eggs were cold shocked (0°C) for 45 minutes, 1-2 or 7-9 minutes after fertilization (at 24 °C). This bimodal response was typical for common carp, but essentially different from other investigations on common carp gynogenesis, where lower incubation temperatures and degumming of egg was practised.
In a second series of experiments (chapter 4) the optimal conditions for inhibition of the first mitotic division were investigated. The occurrence of metaphase of the first mitotic division was histologically determined. Consistent yields of 5 - 15 % viable fry were obtained when eggs were heat shocked at 40 °C). for 2 minutes, 28-30 minutes after fertilization (i.e. at metaphase). Accurate timing of the heat shock, as well as the heat shock temperature and duration, were critical in obtaining an optimal yield of diploid fry. The homozygous nature of the gynogenetic fry was demonstrated by the Mendelian segregation patterns of two recessive colour mutations (chapter 4).
An important aspect of the described gynogenetic breeding techniques is the effect of the expected homozygosity in a first generation of gynogenetic offspring. In order to investigate this effect, we compared homozygous carps (EM method) with heterozyous gynogenetic carps (2PB method) and a group obtained by full-sib mating (chapter 5). The three groups were all obtained from the same mother, and allowed a comparison of the effects of increasing levels of homozygosity. Skin grafts were exchanged between animals of the same group and between animals of different groups. Skin allografts exchanged among heterozygous gynogenetic carp exhibited prolonged survival. Furthermore a strong histocompatibility (H) locus was seen to segregate in this group. In contrast skin allografts exchanged among homozygous gynogenetic siblings or among normal full-sibs were all rejected in an acute manner, with homozygous fish showing the most vigorous allograft reactions. These findings were explained by assuming that acute allograft reactions were the result of a single strong H-locus disparity, or of a multiple minor H-loci barrier which mimics a strong H-locus effect (chapter 5).
In a follow-up experiment (chapter 6) the effects of increasing levels of homozygosity on sex, gonad development and fertility of carps from these three groups were compared. Surprisingly nearly 50 % males and fishes with intersex gonads were found in the EM group while males were absent in the 2PB group. This excluded a possible contamination with non-irradiated (non-inactivated) sperm. Inbreeding significantly increased the mean gonad weight as well as the variation in gonad weights. Full sib (FS) and heterozygous gynogenetic offspring (2PB) were normal in gonad development, but gonads from homozygous gynogenetic (EM) carp were often retarded in vitellogenesis. The ovulation response was significantly reduced with increasing levels of inbreeding. Eggs from ovulated females of the FS, 2PB and EM groups were fertilized with milt from males of the FS and EM groups. Yields of normal fry were reduced in crosses involving FS and 2PB eggs when compared to crosses with EM eggs or milt. This indicated that homozygous fish were essentially free of recessive lethal genes affecting embryo survival (chapter 6).
New inbred lines were produced using a combination of both gynogenetic techniques. Homozygous inbred strains were produced by gynogenetic reproduction (2PB method) of homozygous gynogenctic (EM) females. F 1 hybrid strains were produced by crossing homozygous females with homozygous gynogenetic male siblings. The clonal nature of these strains was unequivocally demonstrated by reciprocally exchanged skin allografts. All grafts exchanged among members of the same strain were permanently accepted. Likewise grafts from homozygous strain members were accepted by fish from the related half-sib F 1 hybrid strains, while the reverse grafts were rejected. These results provided evidence for the idea that in carp, as in other vertebrates studied so far, histocompatibility genes exist as major and minor loci which are codominantly expressed (chapter 5).
The inbred strains and F 1 hybrids were comparable in body weight and gonad development (chapter 6), but the F 1 hybrids showed a much lower variation in body weight and gonad development. In contrast the phenotypic variation was considerably enlarged in the homozygous inbred strains. This phenomenon is well known in inbred strains of mice and rats, and are generally attributed to developmental instability. The F 1 hybrids are therefore more suited for use in bioassay's, especially since they might possess an increased viability.
One of the advantages of the described gynogenetic inbreeding system is that selection of the most interesting and viable genotypes is required only in the first generation. The selected females can be propagated to produce inbred strains are identical to their parents in overall performance. However, in order to obtain males within a gynogenetic inbred line, some females should be sex-inversed by hormonal treatment. Therefore juvenile, non-inbred carps were treated with various doses of orally administrated 17αmethyltestosterone during different periods after hatching. The treatment periods were 3-8 weeks, 6-11 weeks and 10-15 weeks after hatching. The tested hormone concentrations in the food were 50 and 100 ppm, while a dose of 150 ppm was also applied during 6-11 weeks after hatching. The gonads were inspected at 6 months after hatching. Administration of 50 ppm 17α-MT in the food between 6 and 11 weeks after hatching resulted in 92,7% males. Earlier treatments with 17α-MT in concentrations of 50 and 100 ppm of hormone in the food resulted in high percentages of sterile fish while later treatments produced a high percentage of intersex gonads (chapter 7). Surprisingly a similar experiment using 178 estradiol failed to induce female gonads in any of the periods tested and irrespective of the concentrations of hormone used.
The optimal treatment with methyltestosterone was used to induce sex-inversion in the produced homozygous inbred strains and F1 hybrids (chapter 8). The untreated groups contained females and a single fish with intersex gonads. In the treated groups however, mainly intersex gonads were observed. Only one F 1 hybrid group contained significantly more males (60 %) than animals with intersex gonads. These results can only be explained by assuming that the success of hormone induced sex inversion is genetically determined.
Maleness in common carp is thought to be determined by dominant sex determining genes, since heterozygous gynogenetic offspring were all female. However, in some homozygous gynogenetic offspring nearly 50 % males and intersexes were found. It was therefore suggested that maleness in these groups might he caused by recessive mutations in sex determining genes. The mother of one offspring group, probably heterozygous for a putative mutation, was crossed with an unrelated gynogenetic male from another experimental group. The offspring of this cross was exclusively female, but crosses of these females with gynogenetic males contained again 50 % males and intersexes. It was concluded that these males and intersexes were homozygous for a recessive mutant sex determining gene termed mas-1. To our knowledge such mutations have not been described in fish before (chapter 8).
In conclusion, it can be stated that gynogenesis is a very successful and rapid method for the production of homozygous inbred lines of the common carp, Cyprinus carpio. Such inbred lines have until now only been produced in two small aquarium fish species, zebrafish ( Brachydanio rerio ), and medaka ( Oryzias latipes ). Our new inbred lines of common carp will be very important for future scientific research. The use of F 1 hybrids in endocrinological and immunological bioassays will result in an increased standardisation and thus in a reduction of the number of experimental animals needed. Perhaps the inbred lines can also provide an alternative for the use of other experimental vertebrate animals. The present study also demonstrated the possibilities of gynogenetic breeding in unravelling complex biological processes as graft rejection and sex determination. Moreover, the rapid isolation of specific mutants with an abnormal development may offer important possibilities for future research.