• Journal of Aquaculture
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• v.6 no.4
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• pp.285-293
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• 1993

Gynogenetic diploid of olive flounder, Paralichthys olivaceus were induced by cold shock to fertilized eggs with red sea bream, Paragus major sperm that had been genetically inactivated with 4,800 ergs/$mm^2$ ultraviolet (UV) rays. Cold shock to the eggs at $2^{\circ}C$ for 45 minutes proved to be optimum condition to retain the second polar body. At this treatment, hatching rates of normal fry obtained were more than $33.8\%$. No different growth rates were observed up to 200 days after hatching between control and gynogenetic diploid offsprings. However, body weights of gynogenetic diploids were significantly heavier than that of control 300 days after hatching (p> 0.05). A proportion of female in gynogenetic diploid was significantly higher than that in the control (p< 0.01).

• Journal of Life Science
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• v.20 no.12
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• pp.1902-1905
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• 2010

This study was conducted to increase the efficiency of farming practice in rainbow trout, Oncorhynchus mykiss, by sex reversal and chromosome-set manipulation techniques. Induction of sex-reversed gynogenetic diploid rainbow trout males and mass production of all-female rainbow trout by genetic sex reversal was performed. Phenotypic males in the gynogenetic diploid group were induced successfully by dietary administration of 5 mg of 17 alpha-methyltestosterone per kg diet for 82 days. All females were produced by crossing between normal female and sex-reversed gynogenetic diploid male rainbow trout.

• Fisheries and Aquatic Sciences
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• v.4 no.1
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• pp.39-46
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• 2001

To examine the effect of copy number-dependent transgenic genotype on the expression of foreign gene, stable hemizygous and homozygous transgenic breeding line was established using artificial parthenogenesis. For this purpose, induced diploid gynogenetic transgenesis was optimized in mud loach (Misgurnus mizolepis) using UV-irradiated cyprinid loach (M. anguillicaudatus) sperm and thermal shocks. Optimum UV range for inactivation of cyprinid loach sperm was between 3,150 to $4,050\;ergs/mm^2$ The UV-irradiated sperm were inseminated into eggs from recessive color strain (yellow) or heterozygous transgenic mud loach containing CAT gene. Cold shock at $2^{\circ}C$ for 60 min, 5 min post fertilization successfully restored the diploidy of eggs inseminated with UV-irradiated sperm. Restoration to diploidy was confirmed by flow cytometry and gynogenetic status was verified by examining maternal exclusive inheritance of multi-locus DNA fingerprints, body color and transgenic marker. Putative isogenic transgenic fish clearly showed homozygous status at trans gene locus based on Southern blot hybridization and progeny testing. Further, such homozygous gynogenetic diploids revealed the increased levels of transgene expression, when compared to those of heterozygous (hemizygous) transgenic fish.

• Journal of Aquaculture
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• v.9 no.4
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• pp.429-435
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• 1996

We investigated the effects of rearing water temperatures during sex differentiation period on sex ratios in olive flounder, Paralichthys olivaceus. Control and gynogenetic diploid juvenile flounder reared at water temperature of 18, 21, 24 and $27^{\circ}C$ for 65 days from 35 to 100 days after hatching. Fish were sampled to examine sex ratios at 195 or 260 days after hatching. Female ratios of control and gynogenetic diploid flounder declined rapidly as water temperature increased. Sex ratio of gynogenetic diploid reared at $27^{\circ}C$ was very close to 1 : 1 ratio (P<0.01), The survival rates of control and gynogenetic diploid reared at $27^{\circ}C$ were different from other water temperature groups. The growth of body weight of control and gynogenetic diploid reared at $18^{\circ}C$ were different from other water temperature groups.

• Development and Reproduction
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• v.4 no.1
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• pp.79-85
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• 2000

This study was made to optimize the conditions needed to produce two types of gynogenetic diploids in the sweet fish, Plecoglossus altivelis. Firstly, ultraviolet (UV) ray doses between 3,000 erg to 14,000 erg/$\textrm{mm}^2$ were tested to inactivate sperm genetically. Based on the appearance of the haploid syndromes in the embryo, a dose of UV ray 6000~7000 erg was required to inactivate sperm genetically. Then, cold shock treatment at 1~2$^{\circ}C$ for 15~30 min were conducted to retain the 2nd polar body in inseminated egg. The best elapsed time before the start of the cold shock was examined between 5~8 min. The experiments in which began 5 min after insemination at 1~2$^{\circ}C$ during 17.5 min gave 21.2％ survival rate and 89.7% normal eyed embryo rate. The gynogenetic diploid produced by suppression of the first cleavage, a considerably high number of heteroploids appeared and high mortality was observed at the metamorphosis stage, so further investigation is needed. The production of gynogenetic diploids were confirmed by GPI isozyme marker. The heterozygous type in Gpi-1 locus was observed in the meiotic-G2N as a result of gene-centromere recombination during meiosis. The heterozygous type was never observed in mitotic-G2N and showed segregation into two homozygous types at Gpi-1 locus.

• Development and Reproduction
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• v.4 no.2
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• pp.153-160
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• 2000

In this study, genetic variation in the 5 strains of 2N-cont, meiotic-G2N, mitotic-G2N and two types of clones with different genetic backgrounds was investigated by developing their tolerance to water temperature and salinity, which is a physiological trait, into a quantitative trait. The temperature was set at 19$^{\circ}C$, 22.5$^{\circ}C$, 25$^{\circ}C$ and 30$^{\circ}C$, each of which was combined with 0$\textperthousand$, 15$\textperthousand$ and 30$\textperthousand$ of salinity respectively, making 12 groups in all. In the mean survival time (MST), samples with 15$\textperthousand$ of salinity showed the longest survival time at all temperatures. The 2N-cont had the longest 126.16 h followed by clone-11 and clone-15 surviving for 113.22 h and 91.05 h respectively. Gynogenetic diploids showed the shortest 87,32 h and 36.56 h. At 22.5 and 25$^{\circ}C$, MST of each strain was significantly short, showing similar results to those of the groups at 19$^{\circ}C$. The 2N-cont had the longest MST while clones had a longer MST than gynogenetic diploids. This could be due to gynogenesis which causes homozygosis among malignant harmful genes, leading to its appearance in populations and resulting in early death in individuals with such genes. On the other hand, MST of clones was longer than that of gynogenetic fish. This could be because the 1st gynogenetic generation, which is a parental population, has already had its malignant genes removed, while the clones of the 2nd gynogenetic generation have had their superior genes fixed as well as their tolerance and survival improved. When temperature was raised to 22.5$^{\circ}C$ and 25$^{\circ}C$, increase in variation was observed in gynogenetic diploids and decrease in clones in 15$\textperthousand$ of salinity. This shows that such a trait is genetic to a certain extent. Consequently, if this character is developed into a quantitative trait and applied to selective breeding, it could be a useful character to secure superior strains and individuals, and also it would be possible to improve populations genetically through selection.

• Journal of Life Science
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• v.20 no.11
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• pp.1634-1639
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• 2010

This study was conducted to increase the efficiency of farming practice in rainbow trout, Oncorhynchus mykiss, by sex reversal and chromosome-set manipulation techniques. To obtain phenotypic males, hormonal sex reversal was carried out using an exogenous hormone treatment method. 5 mg of 17 alpha-methyltestosterone per kg diet was supplied for 82 days after first feeding at $10^{\circ}C$ and $13^{\circ}C$. More than 93% of the male population was produced by this method and growth of hormone-treated fish at $13^{\circ}C$ was faster than that of untreated bi-sexual groups. Induced diploid gynogenesis was carried out using artificial insemination of UV-irradiated sperm into haploid eggs. Based on the appearance of the rate of haploid syndrome and survival of embryo, a UV ray dose of at least $3,600\;erg/cm^2$ was required to inactivate rainbow trout sperm genetically. Haploid embryos were restored to diploid by blocking the extrusion of the second polar body using heat shock treatment at $28^{\circ}C$ for 20 min, 10 min post insemination. Gynogenetic diploid sex ratios were confirmed after maturation of the fish erythrocyte measurements and chromosome counts.

• Development and Reproduction
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• v.6 no.2
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• pp.71-76
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• 2002

Present study was aimed to summary the recent reports of chromosomal technology such like a polyploidv, sex differentiation, gynogenesis, transgenic fish and gene manipulation. Triploid cells for rainbow trout and channel catfish were induced through thermal shocks of varying temperature levels and produced as a industrial use. A monosex fish with homogametic females of 15 species of high valued fish were produced by exposing to irradiation. It seemed that different irradiation was suitable to inactivate the sperm and block the formation in producing the gynogenetic diploids. Since 1985, transgenic fish have been successfully produced by microinjecting or electroporating desired foreign DNA into unfertilized or newly fertilized eggs using about 40 fish species. More recently, transgenic fish have also been produced by infecting newly fertilized eggs with pantropic, defective retroviral vectors carrying desired foreign DNA. These transgenic fish can serve as excellent experimental models for basic scientific investigations as well as in marine biotechnological applications.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.38-38
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• 2003

Blocking the first mitotic cleavage of the zygote is a key tool for chromosome-set manipulations in fish. We developed an improved method for inducing tetraploidy by blocking the mitosis with a combination of heat shock at 40.5$^{\circ}C$ for 1, 2 or 3 min followed by cold shock at $1.5^{\circ}C$ for 30, 45 or 60 min. When applied during the first cleavage metaphase of mud loach (Misgurnus mizolepis) zygotes, the optimal combination was heat for 2 min followed by cold for 45 min. At 1 month, the frequency of 4N survivors and the yield from total eggs fertilized was 55.7% and 14.4%, respectively, compared to heat shock alone with 20.0% efficiency and 3.6% yield. The effectiveness of the procedure was confirmed by diploid mitotic gynogenesis using transgenic markers. The overall yield of homozygous diploids, 34.0%, was better than that for single heat shock, 17.3%. The tetraploids and homozygous diploids had higher early mortality than normal diploid controls. However at 1 month, the viability of the tetraploids was the same as normal diploids. For gynogenetic diploids, the survival was similar to normal diploids after 3 months. The high efficiency of this new protocol extends the opportunity to study polyploidy in basic and applied research.

• Journal of Aquaculture
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• v.19 no.2
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• pp.99-108
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• 2006

Sex of the Nile tilapia Oreochromis niloticus is mainly determined by an XX/XY system. However, accumulating evidences suggest the existence of additional sex modifying factors including environmental, autosomal and parental influences. In order to investigate the possibility of parental effects on sex ratios of tilapia progenies, in this study, a series of crosses was carried out using gynogenetic clonal fish, neomales, normal males and females, and YY fish. Crosses between clonal XX male and clonal female have yielded only female progenies and no parental influences were observed. However, in the crosses between clonal males and normal females, female parents were significantly associated with the progeny sex ratios ($X^2$=20.046, 7 d.f., p<0.01). Progeny sex ratios from the crosses between neomales and normal females ($X^2$=60.491, 5 d.f and $X^2$=28.072, 2 d.f.) also showed significant association with female parents (P<0.001). The stability of progeny sex ratios from repeated spawns were confirmed by using 6 different parental pairs. In 16 crosses between normal males and normal females, sex ratios of progenies showed clear maternal influences, and further analysis of the results revealed a negative correlation ($r^2$=0.7718, p<0.05) between the sex ratios of progenies from two different males, indicating a strong paternal influence. No statistically significant relationship between survival rates and sex ratios of progenies was observed in any genotypic groups. Taken together, the influence of parental pairs on progeny sex ratios in this species is evident although the cause of this influence is not clear.