Diploid Gynogens Research Articles

Two generations of meiotic gynogenesis significantly elevate homogeneity and confirm genetic mode of sex determination in bighead carp (Hypophthalmichthys nobilis)

Gynogenesis is an effective technique for establishing pure line and validating possible chromosomal mode of sex determination in fish. As one of the major aquaculture fish historically cultured in China, bighead carp (Hypophthalmichthys nobilis) is famous for its market value of the head, but slow in its genetic improvement due to a long period of sexual maturation. In this study, females of bighead carp (F0 dams) with fast growth and bigger head were chose to induce first and second generation of meiotic gynogenesis (GF1 and GF2 for short) by using UV-irradiated heterologous sperm of common carp (Cyprinus carpio). On average, diploid gynogens were obtained by cold shock with a survival rate of 7.36% for GF1 and 6.24% for GF2, respectively. Results of morphology comparison and parentage assignment showed that all survival gynogens were genetically derived from the dams. Compared with their F0 parents, heterozygosities of GF1 and GF2 gynogens decreased by 42% and 60% when analyzed using 50 SSRs, and by 35% and 53% when using 1505 SNP markers, respectively. Sexing of the GF1 and GF2 gynogens showed all-female genotypes and phenotypes when examined by male-specific DNA markers, histology section and morphology of gonads. Overall, large number of meiotic gynogens have been successfully induced in two consecutive generations, providing valuable resources for fast domestication and genetic improvement in bighead carp. Our evidence of sex ratios in both GF1 and GF2 gynogens also strongly supports a proposed chromosomal mechanism of sex determination (XX/XY) for this large-size cyprinid fish.

Ploidy level and performance in meiotic gynogenetic offsprings of grass carp using UV-irradiated blunt snout bream sperm

Diploid (2n) gynogens, triploid (3n) hybrids and 2n hybrids fish were produced in meiotic gynogenetic offsprings of grass carp (Ctenopharyngodon idella) eggs using UV-irradiated blunt snout bream (Megalobrama amblycephala) sperm. Flow cytometry revealed that a total of 306 (54.2%) 2n gynogens, 157 (27.7%) 2n hybrids and 102 (18.1%) 3n hybrids were identified at one year old stage examined. Chromosome analysis showed that 2n gynogens and 2n hybrid individuals had the same chromosome number as their parents (2n = 48), while the chromosome number of 3n hybrids was 3n = 72. The morphological characters of 2n hybrids and 3n hybrids were intermediate between their parents, and the 2n gynogens were similar to grass carp. Microsatellite genetic analysis showed that the expected heterozygosity (He) of 2n gynogens, 2n hybrids, 3n hybrids and grass carp control were 0.3775, 0.6518, 0.6601, 0.6502, respectively, and the polymorphism information content (PIC) was 0.3791, 0.5749, 0.5907, 0.5738, respectively. Compared with 2n and 3n hybrids, the He and PIC of 2n gynogens were significantly decreased, indicating that high homozygous could be obtained by the gynogenetic method. The 3n hybrids grew faster than both 2n gynogens and 2n hybrid individuals, suggesting that 3n hybrids had an obvious growth advantage.

Massive production of all-female diploids and triploids in the crucian carp.

In many species of aquaculture importance, all-female and sterile populations possess superior productivity due to faster growth and a relatively homogenous size of individuals. However, the production of all-female and sterile fish in a large scale for aquaculture is a challenge in practice, because treatments necessary for gynogenesis induction usually cause massive embryonic and larval mortality, and the number of induced gynogens is too small for their direct use in aquaculture. Here we report the massive production of all-female triploid crucian carp by combining artificial gynogenesis, sex reversal and diploid-tetraploid hybridization. Previously, we have obtained an allotetraploid carp population (4n = 200) by hybridization between red crucian carp (Carassius auratus red var; ♀) and common carp (Cyprinus carpio; ♂). We induced all-female diploid gynogens of the Japanese crucian carp (Carassius cuvieri; 2n = 100). We also generated male diploid gynogens of the same species treated gynogenetic fry with 17-α-methyltestosterone, leading to the production of sex-revered gynogenetic males. Finally, these males were used to cross with the female diploid Japanese crucian carp gynogens and the allotetraploid females, resulting in the production of fertile all-female diploid Japanese crucian carp (2n=100) and sterile all-female triploid hybrids (3n = 150), respectively. Therefore, diploid crucian carp gynogenetic females and sex-reversed male together with an allotetraploid line provide an opportunity to produce all-female triploid populations in a large scale to meet demands in aquaculture industry.

Induction of meiotic gynogenesis in the stinging catfish Heteropneustes fossilis (Bloch) and evidence for female homogamety

This study reports the results on induced meiotic diploid gynogenesis and female homogametic nature in the Indian catfish, Heteropneustes fossilis. The eggs of H. fossilis were inseminated with conspecific sperm. The sperm suspension was diluted to 1 × 107 sperm mL−1 in Hanks balanced salt solution. Sperm were irradiated under UV light, with the exposure time ranging from 15 to 360 s (7500 ergs mm−2 for 60 s). The genetic inactivation of paternal chromosomes was confirmed by chromosome counting from the larval cells and the larvae also had a characteristic haploid syndrome. A typical ‘Hertwig effect’ in the yield of hatched larvae was observed with doses of UV exposure >75 s (9375 ergs mm2). Larvae resulting from sperm UV irradiated above 120 s (15 000 ergs mm2) were 100% haploids. Application of heat shock to the activated eggs was effective in suppressing the release of the second polar body (meiotic gynogenesis) and resulted in diploid gynogenetic larvae morphologically identical to those of the control. The best yield of diploid gynogens (49.3% with respect to the control) was found to be at 6 min after egg activation and the heat shock at 41 °C for a 1-min duration, at an ambient water temperature of 27 °C. A total of 113 diploid gynogenetic fry from seven different female fish were reared and subjected to sexing. All gynogenetic fish were female in contrast to the control, which had a mean sex ratio of 56.7% females (which was not significantly different from 50% female). From these results, the sex determination mechanism in H. fossilis was presumed to be female homogamety.

Induction of gynogenesis in stellate sturgeon (Acipenser stellatusPallas, 1771) and its verification using microsatellite markers

Normal egg fertilization was carried out in stellate sturgeon Acipenser stellatus sperm by 60-, 90- and 120-s ultraviolet irradiation, and a cold shock was carried out afterwards. The treatments included haploids, diploid gynogens, triploid and control groups. Fertilization, hatching and survival rate at the 60-s irradiation time were significantly greater than those in the 90-s and 120-s irradiation and control groups. Two pairs of microsatellite markers were used to find gynogenesis in comparison with the parental and offspring genome in one locus. The results showed all maternal genomes among gynogen offspring with no paternal genome. The markers also showed the maximum gynogenesis induction in the Gy60 treatment.

Induction of diploid gynogenesis in turbot Scophthalmus maximus with left-eyed flounder Paralichthys olivaceus sperm

Turbot Scophthalmus maximus exhibits sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. In this paper, gynogenetic diploids of turbot were induced by activating egg development with ultraviolet (UV)-irradiated left-eyed flounder Paralichthys olivaceus sperm combined with cold shock to prevent extrusion of the second polar body. The results of UV irradiation experiments showed that survival, motility, and duration of activity of P. olivaceus sperm generally decreased with increase in UV dose. The typical Hertwig's effect was observed after fertilized turbot eggs with UV-irradiated P. olivaceus sperm and the optimal UV dose for gynogenetic haploid production was 36,000 erg mm(-2). At 15 degrees C, appropriate timing of cold shock for retention of the second polar body in turbot eggs was at 6 min after fertilization. Results of different combinations of two shock temperatures (1 or 3 degrees C) and four shock durations (15, 25, 35 or 45 min) at 6 min after fertilization demonstrated that shock of 25 min at 1 degrees C gave the highest production of diploid gynogens (39.58% relative to its diploid control). The results of this study reveal that the use of UV-irradiated P. olivaceus sperm for activation of turbot eggs and cold shock for polar body retention is an effective method to produce gynogenetic offspring.

A Genetic Linkage Map of Atlantic Halibut (Hippoglossus hippoglossus L.)

A genetic linkage map has been constructed for Atlantic halibut on the basis of 258 microsatellites and 346 AFLPs. Twenty-four linkage groups were identified, consistent with the 24 chromosomes seen in chromosome spreads. The total map distance is 1562.2 cM in the female and 1459.6 cM in the male with an average resolution of 4.3 and 3.5 cM, respectively. Using diploid gynogens, we estimated centromere locations in 19 of 24 linkage groups. Overall recombination in the female was approximately twice that of the male; however, this trend was not consistent along the linkage groups. In the centromeric regions, females had 11-17.5 times the recombination of the males, whereas this trend reversed toward the distal end with males having three times the recombination of the females. Correspondingly, in the male, markers clustered toward the centromeric region with 50% of markers within 20 cM of the putative centromere, whereas 35% of markers in the female were found between 60 and 80 cM from the putative centromere. Limited interspecies comparisons within Japanese flounder and Tetraodon nigroviridis revealed blocks of conservation in sequence and marker order, although regions of chromosomal rearrangement were also apparent.

Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (Pseudosciaena crocea) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1: 100 with Ringer's solution and UV irradiated with doses ranging from 0-150 J cm (-2). The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm(-2), after fertilization, motility was < 10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm -2. A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 208 degrees C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures ( 28 degrees C, 38 degrees C or 48 degrees C) and five shock durations ( 4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 +/- 2.99%) were obtained at 38 degrees C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.

Induction of gynogenesis in endangered sarpunti, Puntius sarana (Hamilton) and evidence for female homogamety

The experiment was conducted to optimize a procedure of gynogenesis induction in Puntius sarana by suppressing meiotic cell division in fertilized eggs. Gynogenesis was induced by fertilizing normal eggs with genetically inactivated sperm followed by heat shocking. The sperm were inactivated by exposing them to a UV dose of 250 μW cm − 2 for 2 min at a concentration of 1 × 10 8 sperm/ml. Seven different heat shock temperatures viz., 32, 34, 36, 38, 39, 40 and 41 °C were tested for different lengths of time to find out the best combination of heat shock temperature and shock duration. Exposure of inseminated eggs to heat shock at 39 °C for 2–4 min after fertilization increased the yield of diploid gynogenetic offspring. Early developmental stages of P. sarana were studied to know the life history of the female homogamety. Histological examination of the gonads of the diploid gynogens revealed that all the fishes were female, indicating female homogamety (XX) in P. sarana.

RAPD and microsatellite analysis of diploid gynogens from allotetraploid hybrids of red crucian carp ( Carassius auratus)×common carp ( Cyprinus carpio)

Genetic variation was comparatively analyzed between the artificially induced diploid gynogen population from F 10 allotetraploid hybrids of red crucian carp (♀) ( Carassius auratus red var., 2n=100)×common carp (♂) ( Cyprinus carpio L., 2n=100) and the normal F 10 allotetraploid hybrid population used as the control, using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. The specific 600-bp fragment for diploid gynogen population was detected by S45 and the specific 900-bp fragment for allotetraploid F 10 hybrid population was detected by S134. The results from RAPD assay and microsatellite analysis were in agreement with each other, that is to say, the diploid gynogens presented lower level of polymorphism than allotetraploid F 10 hybrids. Furthermore, as expected, microsatellite analysis revealed more detailed information on genetic diversity than RAPD assay. The mean percentage of polymorphic loci (12.71%) and Shannon's index of phenotypic diversity (0.25) from RAPD data for diploid gynogen population were significantly lower than those (30.69% and 0.63, respectively) for F 10 allotetraploid hybrid population. The mean number of alleles per microsatellite locus (1.73) in diploid gynogen population was considerably lower than that (2.55) in F 10 allotetraploid hybrid population. The average observed (0.36) and expected heterozygosity (0.26) in diploid gynogen population were lower than those (0.58 and 0.40, respectively) in F 10 allotetraploid hybrid population, indicating that the diploid gynogens presented lower genetic diversity than the allotetraploids. In addition, the mean effective number of alleles at 11 microsatellite loci (1.60) in diploid gynogen population was lower than that (1.88) in F 10 allotetraploid hybrid population. The significant differences between two populations in the average observed and expected heterozygosity, mean number of alleles and effective number of alleles, suggested that the effect of gynogenesis resulted in rather higher genetic homogeneity in diploid gynogens. The comparative RAPD analysis of diploid gynogens and their parents was performed with 34 primers. The identical RAPD pattern was detected between diploid gynogens and their female parent, however, some clear specific RAPD bands were detected between diploid gynogens and their male parents, but not detected in their female parent. The result indicated that heterologous genetic material had incorporated into diploid gynogenetic fish (G 1).

Production of gynogenetic progeny from allotetraploid hybrids red crucian carp×common carp

Following activation by UV-irradiated sperm, diploid eggs of the allotetraploid hybrids (red crucian carp ♀×common carp ♂) developed into normal live gynogens. Cold-shocking these activated diploid eggs failed to induce the expected tetraploidy in them. All these gynogenetic progenies, generated with or without the cold shock, were female diploids with 100 chromosomes, indicating the XXXX genotype of the tetraploid females. The gynogenetic females matured at the age of 2+ suggesting delayed maturity, in comparison to the diploid females of the red crucian carp. Thirty percent of these gynogens were fertile with normal ovary and mature eggs. These gynogens were not morphologically distinguishable from the F 2 hybrids. Like the diploid F 2 hybrids, these diploid gynogens produced diploid eggs, for these eggs developed into triploids with 150 chromosomes, when crossed with red crucian carp. Without the treatment for doubling the chromosome number, the diploid eggs, produced by the F 1 gynogens, developed into the F 2 gynogens following activation by UV-irradiated sperm. Production of these diploid eggs from the diploid gynogens may have resulted from the pre-meiotic endoreduplication of chromosomes. For aquaculture, these diploid eggs remain an important gamete source for the production of tetraploids and triploids, and establishment of diploid gynogenetic hybrid line.

Cold-Shock Induction of Diploid Gynogenesis in Stinging Catfish, Heteropneustes fossilis

The work was conducted to optimize a procedure of gynogenesis induction in the stinging catfish, Heteropneustes fossilis by suppressing meiotic cell division in fertilized eggs. Gynogenesis was induced by fertilizing normal eggs with genetically inactivated sperm followed by cold-shocking. The sperm were inactivated by exposing them to aUV dose of 250 μw/cm2for2.5 minutes at a concentration of 1 × 108sperm/mL. The optimum cold-shock condition to produce maximum percentage of gynogens was found to be at 2°C for 10 or 15 minutes initiated within 3-7 minutes of fertilization. Karyological examination of one day-old larvae and comparison of the erythrocyte area and volume of 3 month-old putative gynogen fry with those of normal fry were used to confirm the success of gynogenesis induction. The findings of the present work are expected to open up avenues for production of diploid gynogens and creation of monosex female populations in stinging catfish.

Meiotic Gynogenesis Induction in Muskellunge Esox masquinongy by Thermal and Pressure Shocks

AbstractMass production of fast‐growing, all‐female muskellunge Esox rnasquinongy by gynogenesis requires optimized techniques of preventing second polar body extrusion. Heat, cold, and pressure shocks were evaluated for their efficiency of doubling the maternal genome. Muskellunge eggs (20–40 g) were activated with 1 mL ultraviolet (UV)‐irradiated (1,248 J/ m2) heterologous sperm of yellow perch Perca flavescens. Survival and ploidy (by flow cytometry) were determined during the eyed‐stage. Cold shocks of 1.3 × 1 C were applied at 5 or 20 min after gamete activation with water (time of initiation, TI) for a duration of 150 min and pressure shocks of 48,263 or 55,158 kPa (7,000 or 8,000 psi, respectively) at a TI of 4 rnin for 12 min. These shock treatments resulted in 43.7–95.0% diploid gynogens with corresponding yield of diploid gynogen (percent diploid gynogens × total percent survival) of 2.6–11.I%. Cold shocks applied at TI of 5 or 20 min after activation resulted in statistically similar percent survival, percent diploid gynogens, and yield of diploid gynogens. Heat shocks of 31 × 0.1C applied at a TI of 5 to 15 min for a duration of 5 min resulted in 4.8–21.1% diploid gynogens with yields of 0.1–0.4%. Cold and pressure shocks have better potential than heat shock for preventing the second polar body extrusion. Muskellunge eggs activated with UV‐irradiated yellow perch sperm, but not exposed to shock, resulted in 100% haploids with survival of 2.3–5.8%. No viable embryos were produced from the hybrid cross between muskellunge and yellow perch, thus, all diploids produced after the shock treatments were unambiguous meiotic gynogens. Muskellunge eggs fertilized with fresh muskellunge sperm (controls) showed 60.4–64.0% survival to the eyedstage and 100% diploidy. Considering that the sex‐determining mechanism in muskellunge follows the WZ female, ZZ male system, future efforts should be directed to test the efficiency of cold and pressure shocks for mass‐producing gynogenetic super female (WW) muskellunge.

Meiotic Gynogenesis Induction in Muskellunge Esox masquinongy by Thermal and Pressure Shocks

Mass production of fast-growing, all-female muskellunge Esox rnasquinongy by gynogenesis requires optimized techniques of preventing second polar body extrusion. Heat, cold, and pressure shocks were evaluated for their efficiency of doubling the maternal genome. Muskellunge eggs (20–40 g) were activated with 1 mL ultraviolet (UV)-irradiated (1,248 J/ m2) heterologous sperm of yellow perch Perca flavescens. Survival and ploidy (by flow cytometry) were determined during the eyed-stage. Cold shocks of 1.3 × 1 C were applied at 5 or 20 min after gamete activation with water (time of initiation, TI) for a duration of 150 min and pressure shocks of 48,263 or 55,158 kPa (7,000 or 8,000 psi, respectively) at a TI of 4 rnin for 12 min. These shock treatments resulted in 43.7–95.0% diploid gynogens with corresponding yield of diploid gynogen (percent diploid gynogens × total percent survival) of 2.6–11.I%. Cold shocks applied at TI of 5 or 20 min after activation resulted in statistically similar percent survival, percent diploid gynogens, and yield of diploid gynogens. Heat shocks of 31 × 0.1C applied at a TI of 5 to 15 min for a duration of 5 min resulted in 4.8–21.1% diploid gynogens with yields of 0.1–0.4%. Cold and pressure shocks have better potential than heat shock for preventing the second polar body extrusion. Muskellunge eggs activated with UV-irradiated yellow perch sperm, but not exposed to shock, resulted in 100% haploids with survival of 2.3–5.8%. No viable embryos were produced from the hybrid cross between muskellunge and yellow perch, thus, all diploids produced after the shock treatments were unambiguous meiotic gynogens. Muskellunge eggs fertilized with fresh muskellunge sperm (controls) showed 60.4–64.0% survival to the eyedstage and 100% diploidy. Considering that the sex-determining mechanism in muskellunge follows the WZ female, ZZ male system, future efforts should be directed to test the efficiency of cold and pressure shocks for mass-producing gynogenetic super female (WW) muskellunge.

Histocompatibility analysis of clonal diversity in unisexual hybrids of the killifishesFundulus heteroclitusandFundulus diaphanus

Hybrids of the killifishes Fundulus diaphanus and Fundulus heteroclitus, found at two sites on the Atlantic coast of Nova Scotia, are unisexual diploid gynogens. Because there seemed to be many opportunities for the sexual progenitor species to hybridize and form new clones at these sites, we initially had expected that clonal diversity might be high among the hybrids. However, examination of their histocompatibility genomes, via inter-individual scale grafting, provides evidence, albeit not conclusive, that most of the hybrids at the two sites, which are separated by 125 km, comprise a single clone. Grafts made among F. diaphanus were rejected within an average of 16-20 days, establishing that sufficient diversity exists within the histocompatibility genomes of these fish to permit their immune systems to reject foreign melanophores. Grafts made from F. diaphanus to the hybrids were also rejected, within an average of 17-26 days, demonstrating that the hybrids possessed competent immune systems. Grafts made between hybrids were nearly always accepted. Those few that appeared to be rejected were usually accepted when regrafted. Melanophores carried on the graft remained visible for the duration of the study (90-180 days) or, if they slowly disappeared, they did so at a rate comparable with that of autograft scales.

Histocompatibility analysis of clonal diversity in unisexual hybrids of the killifishes &lt;i&gt;Fundulus heteroclitus&lt;/i&gt; and &lt;i&gt;Fundulus diaphanus&lt;/i&gt;

Hybrids of the killifishes Fundulus diaphanus and Fundulus heteroclitus, found at two sites on the Atlantic coast of Nova Scotia, are unisexual diploid gynogens. Because there seemed to be many opportunities for the sexual progenitor species to hybridize and form new clones at these sites, we initially had expected that clonal diversity might be high among the hybrids. However, examination of their histocompatibility genomes, via inter-individual scale grafting, provides evidence, albeit not conclusive, that most of the hybrids at the two sites, which are separated by 125 km, comprise a single clone. Grafts made among F. diaphanus were rejected within an average of 16-20 days, establishing that sufficient diversity exists within the histocompatibility genomes of these fish to permit their immune systems to reject foreign melanophores. Grafts made from F. diaphanus to the hybrids were also rejected, within an average of 17-26 days, demonstrating that the hybrids possessed competent immune systems. Grafts m...

Distribution and reproductive capacity of natural triploid individuals and occurrence of unreduced eggs as a cause of polyploidization in the loach,Misgurnus anguillicaudatus

Loaches (Misgurnus anguillicaudatus) were collected from 35 localities in Japan and assayed by flow cytometry to determine ploidy status. No tetraploids were found, with samples from 33 localities having no or few (1.2–3.2%) triploids. Samples collected from Ichinomiya Town, Aichi Prefecture, showed a relatively high rate of triploidy (7.7%). Samples collected from a fish farm in Hirokami Village, Niigata Prefecture, also showed high proportions of triploids (2.0–15.8%), these triploid males being sterile, but the females producing both large-sized triploid and small-sized haploid eggs. Such eggs developed bisexually rather than gynogenetically, giving rise to viable tetraploid and diploid offspring after normal fertilization. Of eight diploid females obtained from the same locality, one produced a high incidence of viable diploid gynogens (55%) after gynogenetic induction by fertilization with UV-irradiated spermatozoa. These observations indicated the presence of diploid fish which produced both diploid and haploid eggs. Thus, triploid and diploid individuals were also produced after fertilization with haploid spermatozoa. These results suggested that the occurrence of such unreduced eggs may be a cause of natural polyploidization in this species.

Induced Diploid Gynogenesis in White Bass

Abstract Optimal conditions were determined for induction of meiotic diploid gynogenesis in white bass Morone chrysops. In two series of experiments, ultraviolet light (UV) irradiation doses to inactivate sperm genome and optimal temperature shock to restore diploidy were established. Eggs of white bass were inseminated with sperm from striped bass M. saxatilis diluted (1:30) in saline and irradiated with UV at doses ranging from 50 to 1,200 J/m2. A typical “Hertwig effect” was observed with increasing UV dose. Larvae obtained at doses of 400 J/m2 and greater were abnormally developed (haploid syndrome) and had a haploid chromosome number (n = 24). Heat shocks to cause retention of the second polar body were applied. Heat shocks of 2-min duration at 36°C, 38°C, or 40°C were applied at 2 or 3 min after insemination to white bass eggs inseminated with irradiated (800 J/m2) striped bass sperm. The best results were obtained after application of 36°C heat shock at 3 min after insemination, which induced a significant increase in yield of diploid gynogens (24–39%, from initial numbers of gynogenetic embryos) and provided relatively high postshock embryo survival. The gynogenetic origin of diploids obtained was confirmed by the lack of melanophores in putative gynogens (contrary to pigmented control hybrid larvae).

Suitable conditions for induction of gynogenesis in the catfish, Clarias macrocephalus, using sperm of Pangasius sutchi

Gynogenesis was induced in Clarias macrocephalus using UV-irradiated sperm of Pangasius sutchi. Sperm was diluted 1:100 in Ringer's solution and irradiated for 2 min with a 30-W UV-lamp placed 30 cm over the sperm. Optimum shock duration was determined by applying cold shock (10°C) to eggs activated with the irradiated sperm, 4.5 min after activation, with durations of 3–12 and 8–17 min (at 1-min intervals). The highest percentage of diploid gynogens was obtained from the groups cold-shocked for 12, 13 or 14 min. To determine the optimum time for starting cold shock, a cold shock at 10°C with duration of 14 min was applied to groups of eggs 2.5–8 min after activation (at 0.5-min intervals). Results revealed that the optimum time to start cold shock was 3.5, 4 or 4.5 min after activation. Cold shock at 6–11°C applied for 14 min, 4 min after activation, was equally effective in inducing diploidization of gynogenetic fry. However, the highest mean percentage of diploid gynogens was observed in the 7°C-treated group.

Hybridization and gynogenesis in two species of the genus Brachydanio

Reciprocal crosses between Brachydanio frankei and B. rerio produced hybrids phenotypically intermediate between the parents. Eggs activated by UV-irradiated sperm yielded haploid progenies, which succumbed within 3 days after hatching. When B. frankei eggs were inseminated with UV-irradiated (4 min) B. rerio sperm and heat shocked at 39 °C for 3 min, 57% of the embryos hatched; all the survivors were diploids. Heat shock (41 °C) applied for 4 min to 2.5-min-old eggs of B. rerio fertilized with 5 min UV-irradiated B. frankei sperm induced gynogenesis. Adult diploid gynogens in B. frankei and B. rerio were identified by the spots or stripes, a maternal colour marker.