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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.

The results of a socio-economical survey of farming systems practising rice culture with introduced fish, rice culture with indigenous fish and rice monoculture in the semi-deep waters of the Mekong Delta, Vietnam, are presented. Rice and fish yields, inputs, and cost-benefits were computed to evaluate the agricultural effects of rice with fish culture. Household size, labour availability and educational level were not significantly different among the three farming systems. Rice-fish farming systems differed mainly from rice monoculture by a higher fertilizer/ water requirement and less pesticide use. The total farm cash and net return did not differ among the farming systems. The main beneficial effects of rice-fish culture are thought to be related to environmental sustainability, system biodiversity, farm diversification and household nutrition.

Fish husbandry and rice culture management factors influencing the yield of introduced fish in ricefields of the Vietnamese Mekong Delta were studied by multiple regression analysis. A significant (P < 0.001) regression model was computed in which feed input and duration of culture period positively and ricefield area, rice seeding rate and the year of the survey negatively affected the yield of introduced, as well as indigenous, fish. The negative impact of larger ricefields is probably the result of the escape of fish. This is also probably the reason for the year of survey since the average yield of introduced fish was 92.5 kg ha−1 in 1995 (because of an extreme flood) as compared to 164.8 kg ha−1 in 1994. A high seeding rate of rice results in a dense stand which suppresses the growth of fish. Opportunities for improvement of fish production are proper ricefield construction, reduced seeding rates, stocking fingerlings early in the dry season and more intensive feeding.

Migrating European eel ( Anguilla anguilla L.) glass eels were collected annually in the Garonne Basin (France) during 1987–1989 and subsequently raised under freshwater conditions. In 1990 gonadal development was determined and after about 1 year it was re-examined to check if sex ratios had changed. Undifferentiated gonads were seldom found, and were mainly present in the youngest year sample 89-1 + (numbers refer to year of catch followed by freshwater age). This gonadal stage can develop directly into an ovary. Differentiation of male sex proceeded via an intersexual stage with male and female sex cells. Phenotypic expression of female gonadal sex was considered as being gonochoristic and differentiated. The female gonad gradually developed towards a fourth stage ovary, characterised by the presence of follicles containing oocytes arrested in the late prophase of meiosis. Yolk formation in oocytes was not observed. The intersexual progression to males was termed pre-maturational sex change. The testes of full grown males contained numerous tubules with spermatogonia in mitotic arrest. The percentage of intersexual eels in 1990 of the youngest year sample (89-1+) was very high (91%). Most of these animals differentiated into males in 1991 (89-2+). The percentages of undifferentiated and intersexual eels in the older year samples of 1988 and 1987 examined in 1990 were low, showing that sex differentiation in most animals had occurred after 2–3 years. The sex ratios expressed as males/(males + females) were 64% (88-2+) and 76% (88-3+); 84% (87-3+) and 83% (87-4+). Comparison of these sex ratios suggests that sex reversal did not occur. Selective mortality and migration, population density, feeding conditions and temperature are reviewed as possible factors controlling sex differentiation in European eel.

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.

The gonad development and fertility were compared for three offspring groups produced by full-sib mating (FS group: F = 0.25), gynogenesis by retention of the second polar body (2PB group; F unknown) and gynogenesis by endomitosis (EM group: F = 1), using the same common carp female. 2PB offspring were all female, but the homozygous EM offspring consisted of 50% males and intersexes which were homozygous for a recessive mutant sex-determining gene. Another mutant, affecting pigmentation in eggs, was likewise detected in the EM offspring but not in the 2PB offspring. The variation in body weight, gonad weight and egg size in each group increased with increasing degree of inbreeding. FS and 2PB offspring were comparable in mean gonadal development, but gonads from homozygous gynogenetic carp were often retarded. The ovulation response significantly decreased with increasing levels of inbreeding. Yields of normal fry were reduced in crosses involving milt from FS males and eggs from FS and 2PB females as compared with crosses with eggs or milt from EM animals. FS eggs fertilized with EM milt gave significantly better yields of normal fry than any other group. Taken together these results suggest that 2PB gynogenetic offspring are largely heterozygous for many deleterious genes affecting reproduction. In contrast, homozygous EM offspring display considerable inbreeding depression in gonad development but recessive lethal genes are excluded from the genepool.

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.

1. 1. Pubertal vitellogenic and previtellogenic female Clarias gariepinus were exposed to holding water from a mixed-sex population of adult conspecifics. 2. 2. Exposure to holding water caused an accelerated ovarian growth in both cases. 3. 3. Analysis of holding water by gas chromatography and mass spectrometry (GCMS) yielded the presence of several steroids and steroid glucuronides known to be produced in adult, prespawning C. gariepinus . 4. 4. The possible role of these compounds in stimulating ovarian growth is discussed in relation to earlier findings on their pheromonal properties.

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.

Migrating European eel (Anguilla anguilla L.) glass eels were collected annually in the Garonne Basin (France) during 1987–1989 and subsequently raised under freshwater conditions. In 1990 and 1991 sex differentiation, gonadal development, length/weight and eye size were studied in relation to metamorphosis from yellow to silver eel stage. Differentiation from intersexual gonads to testes took place after 2 years and was synchronized with metamorphosis. Differentiated ovaries, however, were already present in 2 + yellow eels indicating that expression of female gonadal sex was not synchronized with metamorphosis. Body length and eye size were used to assess the ontogenetic stage of individual fish. The majority of female yellow eel and male and female silver eel of the experimental fish could be identified using these external morphological characteristics. Intersexual yellow eel could not be identified with this method. Intersexual and female yellow eel reached the silver stage at ages of 2–3 and 3–4 years and at body weight ranges of 78–410 and 309–830 g, respectively, implying that metamorphosis in captivity took place at earlier ages and at heavier body weights than in nature. Metamorphosis with respect to enlargement of eyes up to the size of mature silver eels occurred in the hatchery population without hormonal intervention. The testes of silver eels contained spermatogonia in mitotic arrest and the ovaries had oocytes, which were blocked in late prophase of meiosis. Body length and eye size were not suitable characteristics to assess sexual maturity in hatchery raised silver eel. Males and females continued to eat and grow after they became silvery whereas gonadal development gradually stopped. The relationship between age, length/weight, feed intake and metamorphosis (including integumental colour change, enlargement of eyes, degeneration of the alimentary tract, gonadal growth/maturation) as well as the role of gonadotropins are discussed.

Thc African catfish, Cluricts gariepinus, is a highly appreciated species for aquaculture, becausc of its favourable food conversion, its resistance to discascs, its relatively low rcquirements for watcr quality, the posnbility for high stocking density and the excellent meat quality. For those reasons cven in the Netherlands, there is a modest, but Europc's largest and still expanding, African catlish farming activity. Although this spccics grows and maturcs in captivity, therc is no spontaneous reproduction. We could demonstratc that the failure to rcproduce resides in the brain-pituiary-gonad axis. Hormones required for oogcncsis and spermatogencsis are being produced in sufficient quantitics. However, final oocyte maturation, ovulation, spermiation and spawning behaviour do not occur, duc to the lack of a gonadotropin surgc. In nature, the prespawning gonadotropin surge is induced by environmental factors such as the water level in the spawning area. Under farming conditions the environmental cues are hard to identify andlor to mimic. In conihination with unavoidable stress this causes a blockadc of the release of gonadotropin releasing hormone (GnRH). Consequently, gonadotropin surge relcasc fails to occur, which is enforccd by an effective hypothalamic dopaminergic inhibition. The gonadrotropin surge induccs the conversion of 17 tuOH-progcsterone into 17 ruhydroxy-20 (1-dihydroprogcsterone, the final maturation inducing substance. Based on these data, several protocols for artificial propagation could be developed. Thcy include either a treatment with a GnRH analogue in combination with a dopamine receptor antagonist, a treatrnent with homologous gonadotropin or HCG, or a treatment with 17 aOH-progesterone. Since a number of years WC have used the African catfish as a mode1 for fundamental research on fish rcproductive endocrinology. Till now one gonadotropic hormone (GTH) could be demonstrated. Its amino acid composition and sequence was analysed and appeared to bc homologous with known forms of the maturational GTH (GTH-II). Specific radioimmuno assays for the complete hormonc and its a- and /?-subunit respectively, have been developed. cDNAS encoding the subunits have been cloned. They are applied now for Northern blotting and in situ hybridization. GnRHs were fully characteriscd (a specific catfish-GnRH and chicken-GnRH-II). Specific antibodies against these peptides were raised and the cDNAS encoding the hormone precursor molecules were cloncd and used for respectively immunocytochemical localisation and radioimmunoassays, and in situ hybridisation. The importance of the two GnRH forms for gonadotropin release was studicd. ChickenGnRH-II appears to be 10 to 100 times morc potent than catfish GnRH, probably due to its higher receptor affinity. Catfish GnRH, however, is present in the brain and pituitary about 100 times more than chicken GnRH-II. Steroid hormone synthesis by "varies, testis and seminal vescicles was analyscd. The sex sieroids that play a rolc in the negative feedback control of gonadotopin release were identified (1 1-keto-testosterone and tcstosterone) and their interaction with hypothalamic dopamine metabolism was demonstratcd as one of the possible mechanisms of action. Several stcroid conjugates from the seminal vesicles were shown to have pheromonal activities, involved in reproductive behaviour. They induce under certain physiological conditions attraction between conspecifics and synchronization of ovulation.

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.

1. 1. In teleost fish, sex pheromones are involved in advanced phases of reproduction, priming actions influencing oocyte maturation and ovulation in females, and spermiation in males; releasing actions involving attraction of prospective mates and triggering spawning behavior and release of gametes. 2. 2. In the African catfish Clarias gariepinus, sex pheromones were recently demonstrated to also influence earlier phases of the gonadal cycle, i.e. ovarian development in females. 3. 3. Aspects of interaction between sexes in emission of the pheromones, their source and identity are reviewed. 4. 4. The possible function of pheromonal stimulation in the natural environment and implications for fundamental and applied research methodologies are discussed.

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.

The effects of genetic manipulations of eggs and sperm and incubation conditions of eggs on the yields of gynogenetic fry were investigated. Ten ml of sperm (1:3 diluted) was inactivated using UV-irradiation at a dose of 2200 J m −2 min −1 for 1 h. Gynogenesis was achieved by cold-shocking eggs, fertilized with irradiated sperm, at different times after fertilization. Consistent yields of 25–50% viable, gynogenetic fry were obtained when eggs were incubated at 24°C and cold-shocked (0°C, 45 min) 1–2 or 7–9 minutes after fertilization. This bimodal response of eggs to cold shocks was essentially different from the responses found by other authors who researched gynogenesis in carp using NaCl/urea solutions and temperatures below 24°C for incubation. Although the latter conditions proved to delay the first sensitive period, this could not fully account for the observed differences.

Gonad development in Clarias gariepinus was studied from day 12 to day 114 after hatching. Ovaries were histologically recognizable at day 28 and characterized by the formation of an ovarian cavity and the presence of germ cells with nuclei in meiotic prophase. Testes were manifest at day 42 by their crenated appearance and the presence of spermatogonia and Leydig cells distributed between the germ cells. Exogenous androgens clearly affected the process of gonad differentiation. Treatment with 17α-methyltestosterone (30 μg/l) and 11β-hydroxyandrostenedione (300 μg/l) for 4 weeks from day 28 influenced the sex ratio in favour of males, and had no effect on the gonadosomatic index (GSI). In females, GSIs decreased when high dosages of methyltestosterone were administered. Methyltestosterone (100 μg/l) from day 14 had a feminizing effect. It is concluded that this effect is pharmacological rather than physiological.

Reproduction of the African catfish under hatchery conditions depends on the artificial induction of egg maturation and ovulation. In this study the combination of pimozide/LHRHa as ovulation-inducing substance has been evaluated. At a dosage, given as one single injection, of 5 mg pimozide + 0.05 mg LHRHa per kilogram body weight, 100% ovulation was achieved. Most of the eggs, collected after stripping, could be fertilized and hatched normally.

Administration of 11-desoxycorticosterone-acetate (DOCA) or carp pituitary suspension (CPS) to broodfish of the African catfish Clarias lazera leads to maturation of postvitellogenic oocytes. CPS also causes ovulation, but not oviposition. After DOCA injection, ovulation and oviposition of mature oocytes can be evoked mechanically by stripping. Only postvitellogenic oocytes measuring more than 1 mm in diameter undergo maturation, and only eggs that have passed through all stages of maturation develop into normal fry upon fertilization. The time required for postvitellogenic oocytes to give the highest hatching percentages and the lowest fractions of deformed fry is 11–14 h for CPS and about 14 h for DOCA at 25°C. By producing an eosinophilic material, the granulosa cells of atretic follicles and postovulatory follicles contribute to the disintegration and clearance of degenerating oocytes and over-ripe eggs.

Ovarian growth in pubertal African catfish was followed during a 100-day period. Different combinations of male sensory stimuli resulted in three distinct levels of ovarian development. A basic level (ovarian weight 5.3–7.5 g; GSI 1.9–2.7%) was reached in controls (no contact with males) and in females having visual and auditory contact with males. An intermediate level (ovarian weight 11 g; GSI 4%) was reached if olfactory contact with males was possible. A maximum level (ovarian weight nearly 20 g; GSI 7.3%) resulted from unlimited contact between the two sexes. The question is discussed whether the difference between maximum and intermediate levels of ovarian growth should be ascribed to a better water quality due to experimental conditions rather than to the presence of male tactile stimuli. The possible existence of male primer pheromones responsible for the observed stimulation of ovarian growth in pubertal African catfish by enhancing vitellogenesis is also discussed.

At Schiermonnikoog the population structure of Calathus melanocephalus was analysed during four consecutive years (1962–1965), using the structure of the ovaries (in one year also the testes) as a key to the ages of individual adults. The following conclusions were drawn: 1. Part of the population which emerges June–July, and reproduces in August, hibernates. The ovaries of these individuals are spent with conspicuous corpora lutea. 2. Specimens belonging to this hibernating group resume activity in April and continue to the end of July. The females reproduce in July for a second time. 3. The hibernated specimens may be distinguished from newly emerged females by their clear corpora lutea, and lower mean egg-number. The newly emerged females are immature, or have only inconspicuous corpora lutea. 4. It seems that about one third of the adult population at Schiermonnikoog is of the second year category

The effects of carp pituitary suspension (CPS) and 11-desoxycorticosterone-acetate (DOCA) on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and glucose-6-phosphate dehydrogenase (G6PD) activity in the ovary of Clarias lazera are described. Strong 3 beta-HSD and G6PD activities are localized in the stroma, of both control and treated fish. A single CPS injection stimulates 3 beta-HSD activity in the granulosa of postvitellogenic, maturing and post-ovulatory follicles, but DOCA has no such effect on the postvitellogenic and maturing follicles, and only stimulates a weak response in the postovulatory ones.

The effects of oral administration of 17α-methyltestosterone (17α-MT) and 17β-estradiol (17β-E2) on the gonadal development of common carp were investigated during periods of 5 weeks between 3 and 15 weeks after hatching. Administration of 50 ppm 17α-MT in the food between 6 and 11 weeks after hatching resulted in 92.7% males. Earlier or later treatments with 17α-MT at concentrations of 50 and 100 ppm of hormone in the food resulted in high percentages of sterile fish. Administration if 17β-E2 failed to induce female gonads in any of the periods tested and irrespective of the concentrations of hormone used. Gonad weight and gonadosomatic index of both males and females decreased with increasing doses of 17β-E2, indicating that the hormone had been absorbed by the fish. For hormonal sex control in other fish species it is recommended to apply hormone dosages which are related to body weight and not to body weight gain.

A comparison was made between the fecundity of female African catfish, Clarias gariepinus, transferred from their natural habitat in Northern Israel to nearby fish ponds and an indoor hatchery respectively, and conspecifics reared and kept in an indoor hatchery in The Netherlands. The results indicate that an inherent endogenous rhythm rather than the natural light periodicity determines the cyclical changes in ovarian activity, and that this internal rhythm is determined by environmental factors at an early stage of development. High water temperatures and the presence of males seem to enhance ovarian activity and to shorten its resting period. The significance of gonadotropin secretion by the pituitary for the ovarian cycle is discussed. It is concluded that for propagation of C. gariepinus throughout the year, independent of the seasons, broodfish should be reared and kept in hatchery tanks at a water temperature of 25°C, with proper feeding, in the presence of males, and at any local light periodicity.

The paper summarizes recent advances in the culture of the African catfish, Clarias gariepinus (Burchell). Rhythms in egg production, artificial spawning, the role of pheromones, induction of triploidy, nutrition, feeding and growth of larvae and fingerlings, as well as health control are treated. The economics of artisanal pond farming and the aquacultural potential of the fish species in intensive flow-through husbandry systems are discussed.

Glycoprotein gonadotropic hormone (GTH) was purified from 2000 pituitaries of male and female African catfish, Clarias gariepinus . The first step was chromatography on concanavalin A-Sepharose followed by filtration on Ultrogel Aca 54, chromatography on DEAE-cellulose, and filtration on Ultrogel Aca 54, respectively. Finally, the purified fractions were analyzed by polyacrylamide gel electrophoresis. The gonadotropic activity in the different fractions was characterized using two tests: the radioimmunoassay for carp gonadotropin-β subunit was used to quantify the immunoreactive GTH and a cAMP accumulation test was applied to measure the GTH biological activity. The purified glycoprotein GTH was used to raise antibodies and to develop a radioimmunoassay. This resulted in an assay with a variation between assays of approximately 4%, a precision of 4–8%, and an accuracy of 4–8%. GTH levels can be measured over a range of 0.8 to 12.5 ng/ml.

Female catfish ( Clarias gariepinus ), raised from eggs to maturity under hatchery conditions and kept at 25°C under a natural photoperiod, were injected with 17α-hydroxy-progesterone in order to induce oocyte maturation and ovulation. This was achieved using dosages of 8 μg/g body weight, administered in two successive injections of 3 and 5 μg/g, with a time interval of 4 h. Eggs could be stripped within 12.5 h after administration of the first injection. Most of the eggs could be fertilized and hatched normally.

The effect was studied of a preovulatory gonadotropic hormone (GTH) surge, induced by pimozide/LHRHa treatment, and of 17α-hydroxyprogesterone on oocyte maturation and ovulation and on the plasma levels of 17α-hydroxyprogesterone, 17α-hydroxy-20β-dihydroprogesterone, testosterone and estradiol. GTH and 17α-hydroxyprogesterone initially caused an increase in plasma testosterone levels, followed by an increase in 17α-hydroxy-20β-dihydroprogesterone. Neither treatment affected estradiol levels. All fish treated with pimozide/LHRHa or 17α-hydroxyprogesterone showed oocyte maturation and ovulation.

In this thesis, the development of genetically uniform strains of common carp, Cyprinus carpio L. is described. As in research on mammals, the use of genetically uniform fish could increase the quality (replicability, reproducability and repeatability) of experiments. Inbreeding was done by gynogenetic and androgenetic reproduction. With these methods, fully homozygous. offspring are produced in only one generation. The principle of these reproduction techniques is to eliminate the genetic contribution of one of the parents by (UV) irradiating the sexual products, followed by artificially inducing diploidy by physical shocks in the developing zygotes. Inbred strains, congenic strains and F1 hybrids were produced. Congenic strains were used to study sex determination in carp. Inbred strains and F1 hybrids were used to study the genetic control of gonad development after a quantitative genetic model had been developed to facilitate estimation of genetic variance. We show that genetically uniform carp strains are suitable as experimental animal model in genetic research and in bioassays (toxicological research). It is obvious that other research areas could also profit from genetically uniform carp strains.

The aim of the work described in this thesis is to investigate the possibility to select for immune responsiveness, and subsequently produce isogenic carp ( Cyprinus carpio L.) lines, via gynogenesis, that express the trait under selection. If possible, this would allow the repeated production of numerous isogenic fish lines, all selected for different immune parameters, that can be used for immunogenetic studies on disease resistance. As a model, we chose a defined antigenic determinant (dinitrophenyl: DNP), coupled to a carrier molecule (keyhole limpet haemocyanin: KLH) in combination with a reliable read-out system (enzymelinked immunosorbent assay: ELISA), to divergently select carp for the magnitude of their primary antibody response. The possibility to reproduce both homozygous gynogenetic females and functional males with a high or a low antibody response, resulted in the establishment of a number of F 1 hybrid crosses with high or low immune responsiveness to DNP-KLH. Typically, these isogenic lines showed no within-line genetic variation. Between-line genetic variation in susceptibility to infection with Trypanoplasma borreli, a haemoflagellate parasite of carp, was dependent upon the immune response type of the carp lines. Major histocompatibility complex (Mhc) class II β-chain polymorphism could be associated with the immune response types described and, in retrospect, may have contributed to the observed differences in magnitude of immune responsiveness to DNP-KLH, and possibly T.borreli.

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.