• Asian-Australasian Journal of Animal Sciences
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• v.22 no.8
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• pp.1107-1112
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• 2009

The separation of X and Y chromosome-bearing sperm is of use in many aspects of livestock maintenance. In this study, we sought to determine the difference in DNA content between X- and Y-bearing sperm, separate sperm into X- and Y-enriched pools, and assess the efficacy of sorting. Sperm collected from Duroc and miniature pigs were stained with 20.8 $\mu{M}$ Hoechst 33342 and analyzed using a high-speed cell sorter. Measurement of the fluorescence intensity of stained sperm nuclei revealed that the X-bearing sperm of Duroc and miniature pigs respectively contain 2.75% and 2.88% more DNA than Y-bearing sperm. In total, 50.18% of the sperm were assigned to the X-sorted sample and 49.82% was assigned to the Y-sorted sample for Duroc pigs. For miniature pigs, the Xsorted sample represented 50.19% of the population and the Y-sorted represented 49.81% of the population. Duplex PCR was used to evaluate accuracy of sorting. A fast and reliable method for porcine sexing was developed through amplification of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed to amplify the conserved porcine SRY high motility group (HMG) box sequence motif. We found that the primer pair designed in this study was 1.46 times more specific than previously reported primers. Thus, this study shows that the present method can be applied in porcine breeding programs to facilitate manipulation of the sex ratio of offspring and to achieve precise sexing of porcine offspring by amplification of the HMG box of the SRY gene.

• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1819-1826
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• 2015

Poly(ε-ʟ-lysine) (ε-PL) is a novel bioactive polymer secreted by filamentous bacteria. Owing to lack of a genetic system for most ε-PL-producing strains, very little research on enhancing ε-PL biosynthesis by genetic manipulation has been reported. In this study, an effective genetic system was established via intergeneric conjugal transfer for Streptomyces albulus PD-1, a famous ε-PL-producing strain. Using the established genetic system, the Vitreoscilla hemoglobin (VHb) gene was integrated into the chromosome of S. albulus PD-1 to alleviate oxygen limitation and to enhance the biosynthesis of ε-PL in submerged fermentation. Ultimately, the production of ε-PL increased from 22.7 g/l to 34.2 g/l after fed-batch culture in a 5 L bioreactor. Determination of the oxygen uptake rate, transcriptional level of ε-PL synthetase gene, and ATP level unveiled that the expression of VHb in S. albulus PD-1 enhanced ε-PL biosynthesis by improving respiration and ATP supply. To the best of our knowledge, this is the first report on enhancing ε-PL production by chromosomal integration of the VHb gene in an ε-PL-producing strain, and it will open a new avenue for ε-PL production.

• The Plant Pathology Journal
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• v.25 no.2
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• pp.179-183
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• 2009

To facilitate the genetic manipulation of Cladosporium phlei, a causal agent of leaf spot disease in timothy (Phleum pretense), protoplast-mediated transformation of C. phlei has been developed and the resulting transformants were characterized in this study. Hygromycin B resistance was applied as a dominant selection marker due to the sensitivity of C. phlei to this antibiotic. The transformation efficiency ranged from approximately 20-100 transformants per experiment. Southern blot analysis of stable transformants revealed that transformation occurred by way of stable integration of the vector DNA into the fungal chromosome. PCR analysis and plasmid rescuing of randomly selected transformants suggested that integration of tandem repeat copies of vector DNA was common. In addition, multiple integrations of the transforming vector at different chromosomal sites were also observed. The establishment of a transformation method for C. phlei facilitates strain improvement of this fungus and can be applied as an initial step in the molecular analysis of pigment production in this fungus.

• Development and Reproduction
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• v.22 no.1
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• pp.111-117
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• 2018

The objective of this study was to determine the mitotic intervals (${\tau}_0$) of two consecutive cell divisions and synchronous embryonic cleavage in grass puffer, Takifugu niphobles at different water temperatures (18, 20, 22, and $24^{\circ}C$). The color of the fertilized egg was light yellowish. The egg type was demersal and unadhesive. Egg weight was $0.09{\pm}0.002mg$. The sizes of unfertilized eggs were smaller than fertilized eggs in major axis and minor axis at $20^{\circ}C$ (p<0.05). The size of the fertilized egg of $18^{\circ}C$ water temperature group at the blastodisc stage was the smallest (p<0.05), but no significant differences were observed in the other water temperatures group except $18^{\circ}C$ water temperature group (p>0.05). The first cleavage stages at 18, 20, 22, and $24^{\circ}C$ were at 75, 90, 105, and 120 mins, respectively. As water temperature was increased, embryonic development and formation time of the first cleavage furrow were accelerated. There were negative correlation between ${\tau}_0$ and water temperature for grass puffer (Y=-1.225X+70.05, $R^2=0.988$, n=10, where Y was ${\tau}_0$ and X was temperature). This study confirmed that successful hatching of grass puffer was related to water temperature. Chromosome manipulation will be helpful for this species using cleavage frequency and ${\tau}_0$.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.29-29
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• 2003

In order to establish effective procedures for chromosome manipulation in Haliotis gigantea and H. discus, which are of enormous aquacultural potential, temperature-dependent measures of mitotic intervals ($\tau$$_{0}$) were determined. Mitotic intervals ($\tau$$_{0}$) in these abalone were determined by averaging the duration of the first and third embryonic divisions over a range of temperatures from 8 to 26$^{\circ}C$. The relationships of each mitotic interval at two cell ($\tau$$_{I}$), four cell ($\tau$$_{II}$ ), eight cell ($\tau$$_{III}$), sixteen cell ($\tau$$_{IV}$ ) and $\tau$$_{0}$, to temperature (T in $^{\circ}C$) in H. gigantea were log $\tau$$_{I}$ : 176.1-28.3T, log $\tau$$_{II}$ : 199.5-12.4T, log $\tau$$_{III}$ = 236.2-12.2T, log $\tau$$_{IV}$ = 269.3-14.lT and log $\tau$$_{0}$ : 83.1-32.8, respectively. The relationships of each mitotic interval at $\tau$$_{I}$, $\tau$$_{II}$ , $\tau$$_{III}$, $\tau$$_{IV}$ and $\tau$$_{0}$, to temperature in H. discus were log $\tau$$_{I}$ = 104.9-13.8T, log $\tau$$_{II}$ : 138.3-10.5T, $\tau$$_{III}$ : 172.4-10.2T, log $\tau$$_{IV}$ : 211.3-12.2T and log $\tau$$_{0}$=85.6-33.3T, respectively. There were strong, negative correlations between mitotic interval and water temperatures for all ten temperatures in these two species (H. gigantea: Y = -138.75 logX + 341.25, $R^2$ = 0.97; H. discus: Y = -112.33 logX + 255.22, $R^2$ = 0.98, where Y is mitotic interval and X is temperature).d X is temperature).rature).

• Journal of Aquaculture
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• v.8 no.4
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• pp.295-306
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• 1995

The present study was performed to produce supermales and superfemales in Oreochromis niloticus by combination of induced sex reversal and diploid gynogenesis. More than $95\%$ of female was obtained by oral administration of $17\beta-estradiol$ (480 mg/kg diet) to the eutheroembryonic larvae of this species. The result of progeny tests with XY pseudofemales showed that incidences of male progeny were ranged from $71.4\%\;to\;73.7\%$. p.opo.lion of male from XY pseudofemale was not significantly different (P>0.05) from that of $\chi^2$ (1:3), but it was significantly different (P<0.01) from that of $\chi^2$(1:1) Gynogenetic diploids were produced by applying cold shocks 3 min after insemination to the eggs of XY pseudofemale sperms were genetically inactivated by ultraviolet rays of 4,050 $erg/mm^2$. Fertilization rates of gynogenetic diploids were not different from that of their controls, however, hatching rates and early survival rates were silghtly lower than those of controls. In the cytogenetic studies, there was no difference in the size of cell and nucleus between the gynogenetic diploids and the controls. At 80 days after hatching, frequencies of gynogenetic male were higher than those of gynogenetic female (P<0.01) in their population.

• 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.

• Journal of Aquaculture
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• v.13 no.4
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• pp.331-338
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• 2000

To establish an alternate technology for all male seed production of Nile tilapia, Oreochromis niloticus at hatchery scale, the performance of progenies produced by crossing superfemale with sex reversed male was evaluated. Free crossings between 21 superfemales and 7 sex reversed males stocked in an aquarium for 60 days resulted in the production of 93.5 % male progenies, compared to 53.2 % males produced by an equal number of normal females and normal males. Spawning frequency was lower (19 times) in the experimental cross including superfemales than that (24 times) in the control including normal females. Seed production in the experimental aquarium was (3,085) significantly lower than that (3,797) of the control. Both in terms of seeds per female per day or seeds per g female per day, seed productivity did not significantly differ between these groups. The alternate technology involving crossing between superfemale and sex reversed male has proven to be an efficient alternate technology for mass production of all male seeds at the hatchery scale.