Reproductive ability of triploid ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids and characteristics of their offspring

The purpose of this study was to investigate reproductive ability of backcross triploid koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. These triploids have been obtained by crossing of F₁ hybrid females producing diploid eggs with males of parental species. Triploid hybrid females, when crossed with goldfish or koi males, produced mostly aneuploid fish with ploidy range from approximately 2.2n–3.2n with a mean value 2.5n; some fish in crosses of triploid females with koi males were tetraploid (4.0n). Since analysed fish had in their genomes one haploid set from parental males, the data indicate that triploid hybrid females mostly produced aneuploid eggs with ploidy range from approximately 1.2n–2.2n and a mean ploidy around 1.5n while some eggs were triploid. Triploid hybrid males were completely sterile and have not released any sperm after hormonal injection. Despite their low viability, some aneuploid fish obtained from triploid hybrid females were raised in indoor recirculating systems until the age of 2 years and their reproductive ability has been evaluated. One aneuploid female with ploidy 2.1n produced larvae with ploidy range from 2.9n to 3.4n with a mean ploidy of 3.1n when crossed with a koi male; about 60% of obtained larvae had ploidy from 3.0n to 3.2n. These data indicate that this female produced mostly eggs with unreduced ploidy level.     

Studies on sperm of diploid and triploid tench, Tinca tinca (L.)

The tench Tinca tinca is an interesting fish from the viewpoint of polyploidy and related atypical reproduction aspects. Triploid tench were produced artificially. Studies of spermiation as well as of sperm motility and structure were performed on several triploid and diploid males simultaneously with individual experimental crosses with diploid females to define their reproductive capacities. The testes of triploids visually looked less developed in the most of cases with lower sperm production (0.05 cm³ sperm per male), GSI and weight of testes compared to diploids (0.58 cm³ sperm per male). Analysis of variance showed significant influence of ploidy level on the percentage of motile spermatozoa. Triploidy did not change percentage of live spermatozoa and velocity of spermatozoa at the first time of sperm movement. The study of sperm structure by scanning electron microscopy revealed that most sperm cells were of normal structure with some anomalies. Sperm heads of triploid and diploid males were mostly round-shaped, 1.86±0.2 and 1.6±0.18 μm in diameter. The midpiece of triploid spermatozoa was slightly narrower than that of diploid ones with typical cylindrical shape. Flow cytometry revealed sperm cells of triploids to be largely aneuploid (1.47 n) with high mosaic DNA, oscillating from haploid DNA content (1.0 n) to diploid DNA content (1.9 n). Experimental crosses between triploid males and diploid females revealed that these males were capable to stimulate effective development with relatively high level of fertilization and hatching rates from 0 to 70%. In conclusion, triploidization does not seem to guarantee sterility of tench.     

Reproductive performance and mature gonad morphology of triploid and diploid Black Tiger shrimp (Penaeus monodon) siblings

Sibling harvest age Black Tiger shrimp triploids and diploids of both sexes were reared to reproductive maturity, crossed with wild caught females and males, conditioned for spawning and a comprehensive reproductive performance trial was undertaken. Ovarian development, spawning frequency, fecundity, hatch rate, gonad morphology, male reproductive tracts and thelycum impregnation rates of the wild female × triploid male cross were assessed. After ablation, ovarian development and cycling between wild G₀ diploid and G₁ diploids was not significantly different, whereas G₁ triploids failed to show any signs of ovarian development and cycling, thus resulting in no G₁ triploid female spawnings. There were 10 G₀ diploid female × G₀ diploid male first‐spawnings and 9 G₀ diploid female × G₁ diploid male first‐spawnings, all of which produced viable nauplii. In comparison, there were 7 G₀ diploid female × G₁ triploid male first‐spawnings, none of which produced viable nauplii. The 26 wild G₀ diploid female spawnings had more eggs than the 1 G₁ diploid female spawning. Gonad morphology and male reproductive tract assessments showed impaired reproductive development in triploid gonadal tissues of both sexes (compared with sibling diploids and wild shrimp) to a point where complete maturation had not occurred. The thelycum of 16 wild G₀ diploid females crossed with G₁ triploid males had no visible spermatophore present, suggesting that G₁ triploid males are incapable of developing viable spermatophores and mating with females. This study demonstrates that the triploid females and males are incapable of producing viable gametes and are thus reproductively sterile.     

Variability in microsatellite DNA markers in gynogenetic and backcross progenies obtained from ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrid females

Inheritance and segregation at five microsatellite loci were studied in diploid gynogenetic and triploid backcross progenies obtained from koi × goldfish hybrid females, which produce diploid eggs. Gynogenetic and backcross progenies were obtained from three individual hybrid females by inseminating eggs with genetically inactivated and intact sperm of parental species respectively; no shock treatments were applied to the early embryos. Complete absence of paternally specific alleles at all investigated microsatellite loci has proven successful genetic inactivation of spermatozoa by irradiation and confirmed gynogenetic origin of progenies. Genotypic segregations at microsatellite loci showed almost complete homogeneity of gynogenetic progenies and their identity to female parents. These results correspond with previous cytogenetic data on the occurrence of premeiotic endomitosis in hybrid females producing diploid eggs. Fish from triploid backcross progenies had genotypes resulting from combination of entire diploid female genome and haploid genome from male.     

Ploidy polymorphism and morphological variation among reciprocal hybrids of Pseudosciaena crocea (♀) × Miichthys miiuy (♂)

Diploid and triploid hybrids of large yellow croaker (Pseudosciaena crocea, Pc) and Miiuy croaker (Miichthys miiuy, Mm) were produced by interspecific hybridization. The result showed that Pc (♀) and Mm (♂) can be successfully crossed and hybrid offspring of normal hatch rate were produced. In contrast, a lower fertilize and hatch rate were produced for hybrids of Pc (♂) and Mm (♀), and the offspring died day 19 after hatching. To confirm the ploidy of hybrid offspring, we examined the karyotypes of offspring. The result of DNA relative content investigation and genetic relationship analysis demonstrated that the major hybrid offspring were hybrid diploid. In addition, a small number of hybrid were triploid and gynogenesis diploid. Morphological analysis based on the measurable traits data indicated the success of the interspecific crosses. The natural gynogenetic diploid large yellow croaker (PP), diploid hybrids (PM) and triploid hybrids (PPM) were closer to maternal parent (Pc) than paternal parent (Mm). This study provided genetic evidences of the chromosome, DNA content and morphological levels to support the successful establishment of the polyploidy hybrids of Pc (♀) and Mm (♂).     

All-male Tilapia Hybrids of Two Strains of Oreochromis niloticus

Hybridization between Nile tilapia Oreochromis niloticus (Local and Stirling University strains) and blue tilapia O. aureus was found to be a tool to produce monosex populations. In order to select a purebred that produces all- or nearly all-male hybrids with high productivity, O. niloticus females and their diploid gynogenetics (meiogynes and mitogynes) were hybridized with O. aureus males. The sex ratio of progenies was evaluated from inter- and intraspecific crosses of two strains of Nile tilapia. Single-pair matings and group spawns under hatchery conditions showed no deviation ( P > 0.01) from the expected sex ratio of intraspecific crosses among two strains of Nile tilapia. However, a higher proportion of male progenies in blue tilapia was observed in group spawns in pond ( P < 0.004) and hatchery conditions ( P < 0.01). Only the Stirling strain and mitogynes produced all-male progenies under laboratory conditions. There were significant differences ( P < 0.05) in growth among hybrid progeny groups, when gynogens and their regular O. niloticus (Local strain) females were crossed with O. aureus males. Six-month hybrid offspring from mitogyne female parents grew better than those from regular and meiogyne female broods.     

Gynogenesis and sex determination in large-scale loach Paramisgurnus dabryanus (Sauvage)

Gynogenesis was induced using heterologous sperms in large-scale loach, Paramisgurnus dabryanus (Sauvage), in which a ZW/ZZ sex determination was previously proposed. Three microsatellite loci were used to monitor exclusive maternal inheritance of gynogenetic progenies. The results showed that high percentages of meiogynogens were produced at 4 min post-fertilization and mitogynogens were produced at 18 min post-fertilization by heat shocks, while meiotic gynogenesis was induced by cold shocks within a wide period and high heterozygosity was even observed in gynogens produced at 24 min post-fertilization. The sex ratios of the F₁ progenies in three gynogenetic families were significantly deviated from 1:1 expectation with a female bias in two families and a male bias in one family (P < 0.05), and the other four gynogenetic families showed approximate 1:1 sex ratios. Moreover, the self-mating between gynogenetic F₁ progenies and mating between gynogenetic F₁ progenies and normal individuals produced all-female progenies or identical proportions of females and males. The data of sex ratios generally confirmed that the sex determination in large-scale loach was determined by the putative ZW/ZZ system, and the possible reasons causing the biased sex ratios are discussed.     

Induced diploid gynogenesis and polyploidy in crucian carp, Carassius auratus gibelio (Bloch), x common carp, Cyprinus carpio L., hybrids.

Abstract. Results of a 16 year (1977–1992) study of hybrids obtained from crossing two cyprinid fish, crucian carp, Carassius auratus gibelio (Bloch) (bisexual form), and common carp, Cyprinus carpio L., are presented. The ability to produce diploid eggs in F₁ females (resulting from chromosome endoreduplication in early oogenesis) was responsible for the high yield of spontaneous diploids in gynogenetic progenies of F₁ females and for triploidy in hybrids from back-crosses of F₁ females with males of the parent species. Different types of hybrids, including diploid and triploid gynogenetic, tetraploid (amphidiploid) and androgenetic progenies, were obtained. Diploid and some triploid hybrids (females only) were reproduced inter se by induced gynogenesis in several subsequent gynogenetic generations. Amphidiploid hybrids were obtained by crossing triploid F₈ females with diploid common carp males or by crossing diploid gynogenetic females with diploid inverted hybrid males producing diploid sperm. Results of cytogenetic investigations and fish culture properties in hybrids are presented. The scientific and practical significance of the data obtained are discussed.     

Plasma levels of Gonadotropin-II and gonadal sex steroids in triploid catfish, Heteropneustes fossilis (Bloch)

Triploid fish have under-developed gonads due to altered reproductive endocrinology. Triploids of Indian catfish (H. fossilis) showed significantly reduced plasma levels of gonadotropin (GtH-II), testosterone (T) and estradiol-17 (E₂) than that of diploids throughout the year, except for the resting phase, irrespective of sex. Plasma levels of GtH-II were significantly different (p<0.001) between diploid and triploid fish during preparatory, prespawning and spawning phase. The plasma testosterone contents in triploids were significantly less (p<0.001) than that of diploids, except during the resting phase. Triploid females showed very low titres of estradiol-17 (<1 ng ml^–1) throughout the annual reproductive cycle in contrast to highly fluctuating levels in diploid females. Thus, this study for the first time provides information on reduced levels of GtH-II and sex steroids in plasma of male triploid fish and additional information on species-specific alteration of sex hormones in female triploids.     

The obvious heterosis and genetic characters of intergeneric cross and backcross juveniles between blunt snout bream (Megalobrama amblycephala) and topmouth culter (Culter alburnus)

In this study, we used artificial insemination to generate hybrid groups of fish [MC‐F₁(MA♀×CA♂) and MC‐F₂(MC‐F₁♀×♂)] by intergeneric crosses of Megalobrama amblycephala (MA) and Culter alburnus (CA); sequential backcrosses [CAM‐B₁ (CA♀×MC‐F₁♂) and MCC‐B₁ (MC‐F₁♀×CA♂)] were also performed. All these hybrids showed high rates of fertilization, hatching and survival (p > 0.05). For genetic traits, compared with those of the M. amblycephala and C. alburnus parental lines (Table 1), the fertilization rate, hatching rate and 7‐day survival rate of MC‐F1(MA♀×CA♂), MC‐F2(MC‐F1♀×♂), CAM‐B1 (CA♀×MC‐F1♂) and MCC‐B1 (MC‐F1♀×CA♂) by artificial insemination exhibited similar high rates (p > 0.05). The morphology of the four hybrids MC‐F₁/F_2, CAM‐B₁ and MCC‐B₁ were intermediate between those of their parents. Compared with their parents of MA and CA, weight gain rate (WG), specific growth rate (SGR) and protein efficiency ratio (PER) of hybrids MC‐F₁/F₂, CAM‐B₁ and MCC‐B₁ were significantly (p < 0.05) increased and feed conversion ratio (FCR) was significantly (p < 0.05) decreased after 3 months feeding. Moreover, protein content of muscle for MC‐F₁/F₂, CAM‐B₁ and MCC‐B₁ was significantly (p < 0.05) higher and carbohydrate content of muscle was significantly (p < 0.05) lower than their parents. The females and males of the four hybrids had normal gonadal development. In this study, we successfully generated intergeneric and backcross hybridization lines with fertile potential among fish of the Cultrinae subfamily and these hybrids had obvious heterosis in terms of growth performance, feed utilization and muscle quality.     

Hormonal profile of growing male and female diploids and triploids of the blue tilapia,Oreochromis aureus,reared in intensive culture

Triploidy as a result of thermal shock exposure of fertilized eggs decreases the growth rate ofOreochromis aureus as compared to their diploid controls, but this is due to the higher female ratio present in triploids (86%) and the lower growth rate of females. When females and males are considered separately, the growth rate is not significantly different in diploids and triploids. Since triploidy results in a malfunctioning steroidogenesis in females (mainly testosterone (T) and 17β-estradiol (E₂)), but does not affect the growth rate, it is concluded that female gonadal steroids do not influence growth unless in pharmacological concentrations. These low levels of gonadal steroids are generally accompanied by higher levels of gonadotropin (GtH), but the difference is not always significant. Despite their lower growth rate diploid females have higher plasma concentrations of growth hormone (GH) during several months compared to the triploid females and diploid males. 3,5,3′-triiodo-L-thyronine (T₃) levels, however, are comparable between diploid and triploid females (except for 1 month), but higher in diploid males in 4 of the 5 months studied. 11-ketotestosterone (11kT) is always higher in males. These results indicate that the higher growth rate of males may be related to the high circulating levels of T₃ and 11kT.     

Allelic segregation of sex‐linked microsatellite markers in Nile tilapia (Oreochromis niloticus) and validation of inheritance in YY population

Stocking of all‐male fingerling produced by direct administration of male hormone 17‐α‐methyltestosterone is the most preferred method for present‐day aquaculture of the Nile tilapia Oreochromis niloticus. However, due to the growing concern of negative impact of steroid hormone in food fish, production of ‘genetically male’ tilapia, which depends on the concrete and thorough understanding of sex determination, has long been a scientific curiosity. The objective of the present study was to identify reliable sex‐linked markers and to evaluate the applicability of those markers in terms of monosex production approach. ‘XY’ neofemales were produced by using synthetic oestrogen and identified through selective breeding and progeny testing. Three females with progeny not deviating from 3:1 sex ratio (male:female) were designated as ‘XY’ neofemales and were used subsequently to produce putative YY progeny. Among the fifteen microsatellite markers tested, marker ARO172 was most informative in differentiating male and female genotypes. Twenty‐seven F₂ fish from three families were identified as putative YY males based on marker genotyping, and four of them were crossed to produce F₃ to validate marker association by progeny testing. The YY males produced 86%–100% male progeny indicating ARO172 a unique sex‐linked marker applicable in marker‐assisted selection.     

Phenotypic trait of Crassostrea hongkongensis♀× C. angulata♂ hybrids in southern China

The Hong Kong oyster (Crassostrea hongkongensis) and the Portuguese oyster (Crassostrea angulata) are important aquaculture species in southern China. To understand the potential feasibility of hybridization between the two species, two‐by‐two factorial cross‐experiments were conducted in Shenzhen (Guangdong province). An asymmetry in fertilization was revealed: C. hongkongensis eggs could be fertilized by C. angulata sperm, but the reciprocal cross was not possible. The fertilization and hatching success in the C. hongkongensis females × C. angulata males (HA) cross was lower than the two intraspecific crosses. Moreover, hybrid larvae had a slower growth rate than those from parental progenies due to genome incompatibility. However, the size of the spat and adult growth were greater than those of the two parental progeny, with obvious heterosis during the grow‐out stage. The survival advantage of hybrids was examined at all times. The surprising finding was that all hybrids were completely fertile, meaning they could produce normal and functional gametes. Genetic analysis by molecular markers was applied to confirm that the HA spat were true hybrids. Our results revealed that the interspecific hybridization between C. hongkongensis and C. angulata is completely successful in one direction. Furthermore, hybrids are viable, fast growing and completely fertile, which provides a promising method for the genetic improvement of oysters as a new aquaculture stock in southern China.     

Assessment of homozygosity and fertility in meiotic gynogens of the European sea bass (Dicentrarchus labrax L.)

Analysis of 5–6 microsatellite loci was used to measure the increment of homozygosity in two meiogynogenetic progenies (A and B) of sea bass with respect to their mother. In progeny A and B, 20% and 12% of the meiogynogens retained heterozygosity for all investigated maternal loci, respectively, while complete homozygosity was observed only in 6% of B and in none of A, indicating the occurrence of significant allelic recombination during meiosis. The overall increment of homozygosity for the investigated loci was 27% for A and 47% for B. Although survival at hatching of meiogynogens was about half that of controls, they subsequently grew as controls and displayed the same onset of puberty and reproductive potential at adulthood during three consecutive years when crossed between themselves (11 crosses) or with control fish (13 crosses). In particular, meiogynogenetic females (n=24) underwent vitellogenesis and yielded eggs of good quality upon stimulation with LH–RH analogue similarly to normal fish. Sperm released by meiogynogenetic males (n=23) was equivalent to that of controls in terms of volume, quality and fertilization capability.Second generation meiogynogens were obtained by chromosome set manipulation from meiogynogenetic females and found to be morphologically normal at 3 years of age. Interestingly, under our culture conditions, the percentage of meiogynogenetic males in the second generation was 7% as opposed to 39% in the first generation.     

Sex differentiation of summer flounder (Paralichthys dentates L.) raised at practical hatchery temperatures

Female summer flounder grow considerably faster than males, and rearing temperature has been shown to influence sex differentiation in related species. The present studies were conducted to examine sex differentiation of post‐metamorphic juveniles reared at temperatures conducive for commercial production. In experiments 1 and 2, offspring produced from crosses with meiogynogenetic, sex‐reversed males and normal females were raised at 14, 16 or 18°C for ≥300 days or 12°C for 30, 60 or 120 days and then transferred to a male‐determining temperature (21°C). In all cases, the fish developed predominantly as phenotypic males (≥74%). In a third experiment, offspring produced from crosses with normal males and females were reared at 15, 17 and 19°C for 111, 227 and 278 days and then transferred to 21°C. Most fish (≥92.1%) developed as phenotypic males irrespective of rearing temperature or length of exposure. Several hypotheses are proposed for these findings and further research is necessary to understand the sex‐determining mechanisms in this species before the economic advantages associated with sexually dimorphic growth can be realized.     

Ploidy level,morphological characteristics and performance of interploid hybrids by Megalobrama amblycephala♀× allo‐tetraploids ♂

Interploid hybrids were produced by mating allo‐tetraploid males, an interspecific tetraploidy induced by blunt snout bream (Megalobrama amblycephala) ♀× black bream (Megalobrama terminalis) ♂, with blunt snout bream females. The fertilization rate was high in the interploid hybrids. Although significant (P<0.05) mortalities were observed at hatching, swim‐up and 30‐day stages, respectively, the survival percentage of the interploid hybrids became stabilized after the 60‐day stage. In different interploid hybridization batches, 64–97% of juveniles were identified as 3n and 1–36% as 2n. Less than 2% of tetraploids were obtained in these interploid hybridization batches. The daily growth rate of the interploid 3n increased 7.5%, compared with that of diploid blunt snout breams during the 180‐day culture. Moreover, a method using seven morphological parameter ratios was set up with a relatively high classification accuracy of 93.8% to harmlessly discriminate interploid 3n from allo‐tetraploids, black bream and blunt snout bream.     

The growth and reproductive endocrinology of adult triploid Pacific salmonids

This paper describes the effect of triploidy on growth and reproductive endocrinology in the months leading up to and including spawning in rainbow trout,Salmo gairdneri, and pink salmon,Oncorhynchus gorbuscha. Growth rates were the same for diploid and triploid rainbow trout, but triploid female pink salmon were smaller than maturing diploid females and diploid and triploid males of the same age. Triploid males of both species developed typical secondary sexual characteristics and had normal endocrine profiles, although their cycle appeared to be delayed by about one month. Triploid females remained silvery in appearance and showed no endocrine signs of maturation, even at the level of the pituitary. Thus, although triploids of both sexes are genetically sterile, only the females do not undergo physiological maturation.Reported in part at the Third International Symposium on Reproductive Physiology of Fish, St. John's, Newfoundland, August 2–7, 1987.     

Survival of Diploid and Triploid Rhamdia quelen Juveniles Under Different Oxygen Concentrations

The objective of this study was to determinate the lethal concentration of dissolved oxygen (DO) over 96 hours of exposure (LC50–96h) for diploid and triploid jundia Rhamdia quelen juveniles. Diploid and triploid fish weighing approximately 4 g were subjected to DO concentrations varying between 0.4 and 1.3 mg O₂ L^?1; water temperature was maintained at 27?°C and pH at 6.3. The LC50–96h for diploids of Rhamdia quelen was 0.535 mg O₂ L^?1, while the value obtained for triploids was 6% greater. These results demonstrated that triploids of Rhamdia quelen juveniles have greater sensitivity to hypoxia compared to diploids.     

Verification of mitotic gynogenesis in ornamental (koi) carp (Cyprinus carpio L.) using microsatellite DNA markers

The Japanese ornamental (koi) carp is a popular decorative fish all over the world. In koi, clones have not yet been obtained, although production of fish with identical colour patterns could be of commercial interest. Mitotic gynogenetic progenies are essential for subsequent production of clones in fish. However, resulting late‐shocked progenies may be contaminated with meiotic gynogens from spontaneous suppression of the second meiotic division in eggs. In this study, microsatellite DNA markers were used to confirm mitotic gynogenetic origin of obtained late‐shocked progenies. Recombination rate (y) and mapping distance relative to centromere (M‐C) of 10 microsatellite loci were determined based on percentage of heterozygotes in meiotic gynogenetic progenies. The range of y varied from 0.01 to 0.96 and the M‐C map ranged from 0.5 to 48 cM. The mean value of y over the 10 loci was 0.481. Six loci, which had y 0.47 and higher, were used as markers in two late‐shocked gynogenetic progenies. Complete homozygosity was revealed at all six microsatellite loci indicating mitotic gynogenetic origin of analysed progenies.     

Saltwater performance of triploid Atlantic salmon Salmo salar L. brown trout Salmo trutta L. hybrids

Survival and growth in a saltwater net-pen of sexually immature triploid Atlantic salmon Salmo salar L. brown trout Salmo trutta L. hybrids was comparable to that of immature diploid Atlantic salmon. Following 17 months of freshwater rearing, the experimental fish were individually tagged and transferred to a saltwater net-pen where they were raised communally for 376 days. Initial and final average weights were 158 and 760 g per fish for the diploid Atlantic salmon and 209 and 1010 g per fish for the triploid hybrids; weights for the hybrids were significantly larger in both cases (α= 0.05). Survival from transfer to harvest was 43% for the Atlantic salmon and 48% for the hybrids. Average specific growth rate of fish which survived to harvest was 0.42% day^-1 for Atlantic salmon and 0.41% day^-1 for hybrids; these values were not significantly different. No significant differences were observed in average condition factor and dress-out percentage between crosses. Average gonadal weights and gonadosomatic indices were not significantly different for male diploid Atlantic salmon, female triploid hybrids and male triploid hybrids, but were significantly greater for female Atlantic salmon.