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Fish are widely used in numerous fields of basic and applied research. Currently, they are the third laboratory animal group in numbers, and will become increasingly important. Common carp is a major species in both aquaculture and research. Inbred strains of carp by gynogenetic (only female inheritance) and androgenetic (only male inheritance) reproduction techniques were developed at our university. With these methods, homozygous animals are produced in one generation and we present the production of homozygous inbred and F1 hybrid strains of common carp. As in mammalian research, using genetically well defined fish is a methodological necessity since in outbred stocks: (1) repeatability between experiments is low, (2) high levels of inbreeding may have accumulated and (3) high intrastrain variability might obscure treatment effects. Within inbred strains, the variation is reduced and as a result, less animals (compared to outbreds) are necessary to obtain statistically significant results. We illustrate this with experimental data from an F1 hybrid and partly outbred strain of common carp, both subjected to an antibiotic treatment resulting in reduced gonadal growth. Results obtained from a single inbred strain should be generalized with the use of a panel of inbred strains. We show that optimal allocation of animals between and within inbred strains depends on the ratio (variation between strains): (variation within strains). When selecting a panel of inbred strains, attention has to be paid to genetic relations between strains to avoid testing within a limited genetic range. It should be considered that in inbred strains, (genic) dominance and interaction effects are absent, due to the absence of heterozygous genotypes. In general, variation within inbred strains will be reduced for traits with a high degree of genetic determination. However, in inbred strains of carp produced by gynogenesis or androgenesis, the chromosome manipulation treatment induces considerable (environmental) variation. By using F1 hybrids of carp, derived from crossing homozygous clonal siblings this source of variation can be avoided. Still, variation in F1 hybrids of carp is relatively large and varies greatly between strains when compared to inbred strains of laboratory rodents. It is assumed that their poikilothermic nature makes them more susceptible to environmental variation. Using inbred fish lines will increase experimental quality and leads to a more efficient use of experimental animals.

Growth rate and phenotypic variation in length and body weight of two homozygous gynogenetic clones E4-gyn and E20-gyn, and four F 1 hybrids E4×E5, E4×E6, E20×E5 and E20×E6, was compared with two partially outbred groups E4×WT and E20×WT. There was a significant effect of sire on growth rate and food conversion ratio (FCR) in both the E4 and E20 groups, caused by differences between WT groups and homozygous clones. There was no significant difference in growth rate or FCR between individual E4 groups. Within the E20 offspring, both E20×WT and E20×E5 had a significantly higher growth rate than the E20-gyn group. All E4-derived groups differed significantly in length and body weight at 24 weeks post hatching, but there were no significant differences in length and body weight between E20-derived groups. The variation in length and body weight increased in homozygous clones when compared with the other groups, while the E4×E5 and E20×E6 fish exhibited the lowest variation, (5% for length and 15–16% for body weight). Replicates of the homozygous clones and F 1 hybrids, treated with 50 ppm 17 α -methyltestosterone (MT) to induce sex reversal, showed a significantly depressed growth rate and FCR when compared to the untreated groups. Mean length and body weight were also significantly depressed in all E4-derived groups but not the E20 groups. The phenotypic variation increased in all groups except E20×E6. Sex reversal was in most cases incomplete. The percentage of sex-reversed males varied from 17 to 23% in the homozygous clones and from 3% to 28% in the F 1 hybrids. Only the E20×E5 group contained 69% males.

Pubertal female Clarias gariepinus (recipients) were exposed for 62 days to holding water from intact, completely castrated, seminal vesicles-extirpated or testes-extirpated adult male or from intact adult female conspecifics (donors). In operated male donor groups, levels of six androgens (measured by RIA) were lowest in testes-extirpaied and completely castrated males. However, even complete absence of the testes (GSI 0%) did not eliminate circulating androgens. Male holding water exerted primer pheromonal action in enhancing ovarian development of recipient females, irrespective of the male donor group. Holding water from female donors was ineffective, comparable to controls (fresh water). Thus, although the effect of holding water is linked to the male gender, the gonadal origin of ovarian growth-stimulating compounds in the holding water could not be demonstrated.

Stimulation and inhibition of gonadal development by intersexual contact was studied in pubertal African catfish, Clarias gariepinus. The effect of a possible interaction was studied by evaluation after a 98-day experimental period of gonadal development in combinations of intact and anosmic males and females. In addition, separate groups of males and females, respectively, were exposed to holding water from these combinations. A tentative model of stimulation of gonadal development by intersexual contact in pubertal fish was developed. Males stimulate ovarian development of females by both olfactory and tactile cues. In addition, males seem to enhance gonadal development of other males through olfactory stimulation via holding water. In contrast, females tended to inhibit male gonadal development, especially through tactile cues. It seems that although males are hampered by female tactile stimuli in their gonadal development and ability to stimulate male gonadal development, their ability to stimulate female gonadal development is not affected.

Homozygous gynogenetic fry of common carp (Cyprinus carpio L.) were produced by heat-shocking eggs, activated with UV-irradiated sperm (1 : 3 diluted, 10 ml, at 2200 J m−2 min−1) during metaphase of the first mitosis. Consistent yields of 5–15% viable, gynogenetic fry were obtained when eggs were shocked at 40°C for 2 min, 28–30 min after fertilization. The homozygous nature of the gynogenetic fry was demonstrated by the mendelian segregation patterns of three recessive mutant pigment genes. Homozygous inbred strains were produced by heterozygous gynogenetic reproduction (second polar body retention) of homozygous gynogenetic females, while F1 hybrids were produced by crossing these females with homozygous gynogenetic male siblings. The clonal nature of these strains was unequivocally demonstrated by the acceptance of reciprocally exchanged skin allografts.

Through gynogenetic and androgenetic reproduction, homozygous individuals can be produced in one generation. Selection of homozygous gynogenetic or androgenetic individuals facilitates the production of genetically defined clones. However, to obtain a selection response additive genetic variance is required. The additive genetic variance in homozygous populations doubles as compared to the additive genetic variance in the base population. We examined the possibility of using gynogenesis and androgenesis in estimating additive genetic variance. Therefore, additive genetic relations and the distribution of additive genetic variance between and within homozygous gynogenetic and androgenetic families were derived. Within gynogenetic families variance equals V A + V E and between-family variation equals V A . This theory is used to analyze experimental data on gonad development and fertility in homozygous gynogenetic common carp, Cyprinus carpio . Five gynogenetic families were produced from full sib, outbred female parents. Regular sampling for maturation stage between the age of 13 and 25 months revealed large between-and within-family variance for the gonado somatic index (GSI) and egg quality parameters. Heritabilities were estimated using Gibbs sampling. For the traits GSI at 13 months and the percentage of normal larvae, obtained after fertilization of egg samples at 19 months, heritability estimates were 0.71 and 0.72, respectively. This suggests that these traits are influenced by additive genetic effects; and that homozygous individuals can be successfully selected from our carp families to produce early or late maturing homozygous gynogenetic inbred strains with high egg quality.

Genetics of testis development was examined in 12 all-male F1 hybrid strains of common carp, Cyprinus carpio, produced after crossing four homozygous androgenetic males with three homozygous gynogenetic females. These parents were selected for fast or slow gonad development. In the F1 hybrids, variance components for general and specific combining abilities were examined by regular sampling between 100 and 240 days of age. The variation between F1 crosses for the onset of spermatogenesis (ranging from 100–130 days after hatching) was mainly due to general combining ability and thus additive genetic differences between the inbred parent lines. Testis somatic index (TSI) ranged from 2.0–5.5% at the end of the experiment. Early onset of spermatogenesis coincided with high TSI and late onset of spermatogenesis with low TSI. In the experiment, the female parent affected TSI to a greater extent than the male parent. For this parameter, general combining ability also accounted for most of the variance observed. These data fit to a great extent with the established genetic model for gonadal development in the platyfish, Xiphophorus maculatus.

After androgenetic or gynogenetic reproduction, a large expansion of phenotypic variance is generally observed. Within one homozygous family, this expansion is the result of increased environmental variance, since genetic variance does not increase. We consider three types of environmental variance (VE) within homozygous offspring: (1) “true” VE (inter-individual variance), (2) VE, due to developmental instability (DI, intra-individual variance) and (3) VE originating from embryonic damage (ED) caused by the chromosome manipulation treatment. We examined the importance of these three types of VE. It is thought that homozygous individuals show high levels of true VE and DI. Therefore, in the first experiment we compared three F1-isogenic and one partly outbred strain of common carp, Cyprinus carpio, in true VE of length, body weight, and number of dorsal fin rays. The isogenic strains varied in degree of homozygosity (coefficient of inbreeding F: 0 to 0.99). DI was determined by measuring fluctuating asymmetry (FA) of five bilateral symmetric characteristics. We found the strain with the highest F to display the lowest true VE. FA was equal in all isogenic strains but highest in the partly outbred strain. In a second experiment, similar observations were performed on gynogenetic and androgenetic offspring from parents with identical genotypes. Homozygous (endomitosis, EM: F = 1) and partly heterozygous (2pb-gynogenesis: F = 0.74) gynogenetic groups were produced. Normal fertilizations (F = 0.75) served as controls. The androgenetic groups showed highest FA and variations caused by ED, followed by 2pb- and EM-gynogenetic groups, respectively. We conclude that increased variation within gynogenetic or androgenetic of offspring is the result of ED, caused by the chromosome manipulation treatment. J. Exp. Zool. 277:72–79, 1997. © 1997 Wiley-Liss, Inc.

Summary Antibody production to dinitrophenyl-keyhole limpet haemocyanin (DNP-KLH) served as the immune parameter to divergently select carp (Cyprinus carpio L.) to produce high- and low-responder F, hybrid lines. Antibody production to trinitrophenyl-lipopolysaccharide [TNP-LPS) and to DNP-KLH were similar in magnitude. By contrast, some high-responder lines were low responders to DNP-human serum albumin, and vice versa. Low-responder carp were relatively susceptible to infection with the parasite Trypanoplasma borreli. This suggested that at least one gene with a major influence on resistance differed between the two homozygous parents (69, 85) used to generate the high- and low-responder homozygous families, respectively. The isogenic lines showed no within-line variation in DNA fingerprints, but differed with respect to their MhcCyca-DAB genes.

To test for a residual maternal influence in the performance of androgenetic offspring in common carp, Cyprinus carpio, an experiment was conducted where three females and three isogenic males were used to produce nine androgenetic families. Eggs were irradiated with UV (175 mJ cm−2) and diploidy was induced using a heat shock (40 °C, 2 min). Yields of androgenetic diploids depended mainly on the egg donor. After raising the larvae for a period of 10 weeks, significant differences were observed regarding the frequency of normal and deformed androgenetic animals, also depending on the egg donor. It is hypothesized that the UV-irradiation causes damage in maternal RNA's, resulting in disturbed embryonic development.

Haploid androgenetic offspring (max. 53.9%) were obtained after UV-irradiating eggs using dosages of 150–300 mJ/cm 2 (duration: 135 to 270 s at an intensity of 1.1 mW/cm 2 ). During irradiation, eggs were stirred in a synthetic ovarian fluid to assure a homogenous irradiation of all pronuclei, without activating the eggs. Androgenetic origin was checked using a recessive colour marker (“blond”). Absence of black fry (dominant trait) in the offspring confirmed inactivation of the female genome. Besides androgenetic haploids, few biparental diploids hatched at the optimal dose (250 mJ/cm 2 ). Diploidy could be restored by heat shocks (40.0°C, 2 min) applied at different times after fertilization (26, 28 and 30 min). Yields of putative androgenetic diploids ranged from 7.2 to 18.3% at the time of hatching. Also, survival of the larvae 24 days after hatching was high (78–89% of the initial number). After testing the homozygous nature of the fish, they will be used to generate isogenic strains.

Vitellogenesis was monitored in Clarias gariepinus females during puberty and after artificially induced ovarian recrudescence. In two experiments (with pubertal and adult females, respectively), the effect of male stimulation of vitellogenesis was assessed by weekly sampling of females kept with males of exposed to male+female holding water. The parameters examined were the gonadosomatic index (GSI), the proportion and diameter of exogenous vitellogenic oocytes, and plasma levels of estradiol (E2) and vitellogenin (VTG). In pubertal females the pattern and sequence of appearance of these parameters are in general agreement with data from other teleosts: a peak in E2 values precedes a peak in VTG, resulting in an increase of GSI and in a concomitant change in yolk deposition in the oocytes. Stimulation causes both a shift in timing and an increase in levels of plasma E2 and VTG, resulting in faster ovarian growth. In adult females, stimulation of vitellogenesis, although resulting in distinct differences in GSI and ovarian development, is not accompanied by a shift in timing of plasma E2 and VTG. E2 values tend to be higher in stimulated adult females. These differences between first and subsequent cycles are thought to be due to the extremely short recrudescence period of C. gariepinus in captivity.

Adult female African catfish, Clarias gariepinus , were manipulated to enter the phase of ovarian recrudescence by induced ovulation and stripping. These females were exposed to various conspecific stimuli for 33 days. At the end of the experimental period, gonadosomatic indexes (GSIs) were higher in females which had received holding water from a mixed-sex population than in comparable controls. The effect was abolished when recipient females had been rendered anosmic, indicating the presence of chemical substances perceptible through olfaction. Fecundity estimates and histological analysis suggest that the increase in GSI due to pheromonal stimulation is caused by both an increased recruitment of oocytes into the stage of exogenous vitellogenesis and by an enhanced deposition of yolk material in the oocytes. It is concluded that male C. gariepinus release pheromones with a stimulatory effect on vitellogenesis. Neither anosmia itself nor metabolites in holding water affected ovarian recrudescence or body weight increase. The biological significance of such pheromones is discussed in the context of observations on reproduction of C. gariepinus made in the natural environment.