• Development and Reproduction
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• v.5 no.1
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• pp.47-52
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• 2001

Predetermination of sex in livestock of offpring is in great demand and is of critical importance to providing for the most efficient production of the animal ariculture. Such a sexing techlology would also enhance the economy of conventional artificial insemination(AI) and aid the porcine industry. The purpose of this study was to evaluate the efficiency of enriching X-bearing porcine sperm using discontinuous percoll gradients and PCR mefhod. Semen was collected from mature boars of proven fertility center (AI center KimHae). Sperm was leaded on the isotonic discontinuous percoll gradient and then it was centrifuged at 120 ${\times}$ g for 20 minutes. After centrifugation, sperm included in each fraction were recovered (7${\times}$10$^6$ sperms/ml) and then sperm genomic DNA was extractedfor the PCR. SRY gene was used to evaluate the ratio between X and Y sperm in the separated fractions. Ju viro ffrtilization wascarried out by adding the unseparated sperm (control) or separated (experimental poop) to the matured oocytes in TCM-199. Embryos for sex determination were obtained at 2 cell stage and then was used for SRY gene amplification. After centrifugation of discontinuous percoll gradient, the most motile sperm was obtained at 95% fiaction (94.4% ${\pm}$ 5.1%, p < 0.01). The PCR analysis evaluated that 30%, 50% and 65% fractions were Y sperm rich, whereas 80% and 95% fractions were X sperm rich. PCR analysis with each porcineembryo showed that 33.3% of control and 66.7% of experimental group were determined as female embryos. In conclusion, in vitro matured oocytes inseminated with sperms (95% fraction) prepared by percoll gradient centrifugation showed high fertilization rates and female embryos than control sperms.

• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.113-120
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• 1989

In order to determine the sex of preimplantation embryos prior to transfer in cattle, a series of experiments were carried out using 45 Holstein donor cows to examine the ovarian response on the gonadotropin and PGF2${\alpha}$, and the morphology of fresh embryos or frozen/thawed embryos after deep freezing at -196$^{\circ}C$. The sexing of embryos treated with the medium containing H-Y antiserum(10%, v/v) and FITC anti-mouse IgG(10%, v/v) were analysed by chromosomal analysis, and the sex of the embryos which survived were ascertain after delivering the pups. The results obtained were summarized as follows ; 1. The average number of developed follicle and corpus luteum per cow were 13.5 and 8.1, and the ovalation rate was 60.1%. 2. Of 220-ova recovered, 75(34.1%) were morula and 91(41.4%) were blastocyst, and the morphological normal and abnormal rate of ova recovered were 75.5% and 24.5%, respectively. 3. Of 39 frozen/thawed embryos, the scores of normal morula and blastocyst, after thawing were 79.2%(19/24) and 73.3%(11/15). The average rate of frozen/thawed embryos which appeared morphologically normal post thawing was 76.9%(30/39). 4. The sex ratio was measured using the embryos treated with immunofluorescence assay to examine the relationship between embryo developmental stage, sex ratio of morula stage embryo was 42.2%(19/45) fluorescing and 57.8%(26/45) non-fluorescing, on the other hand, the ratio switched to 46.8%(29/62) fluorescing and 53.2%(33/62) non-fluorescing embryo in blastocyst stage. The sex ratio was also measured between fresh and frozen/thawed embryos, fresh and frozen/thawed treated embryos were indicated 45.8%(38/83) fluorescing, 54.2%(45/83) non-fluorescing and 41.7%(10/24) fluorescing, 58.3%(14/24) non-fluorescing. This trend indicated the approximal sex ratio was 1 : 1. 5. The result of karyotype test showed the successful rate of sexing embryo is fluorescing and non-fluorescing was 21.2%(7/33) and 29.6%(8/27). The female to male ratio within 33 fluorescing was 28.6 : 71.4, and the ratio of 27 non-fluorescing embryos was 87.7 : 12.5. 6. Of the embryo transferred after assignment of H-Y phenotype, five of the fluorescing embryos survived to term, all was males. Whereas six non-fluorescing embryos also survived to term and the sexes of the calves were 1 male 5 female.

• Journal of Animal Science and Technology
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• v.49 no.1
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• pp.1-8
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• 2007

This study was focused on discriminating the molecular sexes of red deer and elk by duplex polymerase chain reaction(PCR) using two primer sets. Sex differentiation of mammals is primarily dependent on the presence or absence of sex determining region Y(SRY) gene encoded on Y chromosome which plays a key role for male development. Zinc finger X-Y(ZFX-ZFY) gene, one of X-Y homology gene group was found on X- and Y- chromosomes, respectively. At first, the nucleotide sequences were characterized for the intron 9 flanking region of ZFX-ZFY genes. The intron 9 of ZFX and ZFY is 529-bp and 665-bp in length, respectively. A transposable element sequence similar to bovine SINE element Bov-tA was detected only in ZFY gene of Cervidae. Sexing analysis was conducted by duplex PCR assay for amplification of SRY and ZFX-ZFY genes. Two differentially amplified patterns were found: one for females has a common band amplified only from ZFX as a template, and another for males had three bands(a common ZFX and two male-specific ZFY and SRY). On the separate tests using each gene, the results was identical to those from duplex PCR assay. Moreover, the results from PCR assays provide also identical information to phenotypic investigation of individuals of red deer, elk as well as their hybridized progenies collected from two isolated farms. These results suggest that it may be a rapid and precise method for determining the sexes by duplex PCR amplification using Y-chromosome specific SRY and X- and Y- homologous ZFX-ZFY genes showing sexual dimorphism in red deer and elk without any other controls.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.53-61
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• 1996

This study was carried out to determine effectively the sex of rabbit embryos using H-Y antiserum. H-Y antiserum was obtained from inbred SD strain female rat which was immunized by injection of testis cell of inbred SD strain male rate into its spleen. The titer of antiserum was identified by sperm cytotoxicity test and culture of rabbit embryos with antiserum. The developed or undeveloped embryos were separated by exposure the embryos to the antiserum with H-Y antibody. Developed embryo were transferred to the recipients and sex of offspring were examined. 1. In the sperm cytotoxicity test, the rate of dead sperm showed no difference between two antisera from spleen and testis cell as antigens. It is confirmed that H-Y antibody in antiserum was absorbed by H-Y antigen in male rat spleen cells. 2. When rabbit morulae were exposed to antiserum and complement, the rate of embryos developed or arrested was 51 and 49% respectively and the rate was closely same as natural sex ratio of 50:50%. 3. When rabbit morulae were cultured for 12h in the medium containing antiserum produced by antigen of testis cell, the rate of embryos developed or arrested was 48 and 52% respectively and the rate was closely same as natural sex ratio of 50:50%. 4. Eighty rabbit embryos which were not affected by H-Y antiserum were transferred to four recipients. Two recipients were pregnant and born 13 pups among which 2 (14%) were male and 11 (86%) were female. In conclusion, existence of H-Y antibody in the serum from female rat immunized by injecting testis cell from newborn male rat to the spleen of the female rat was confirmed. When rabbitmorulae were exposed to H-Y antiserum and complement, about a half of embryos were developed to blastocysts. When the rabbit embryos not affected by H-Y antiserum were transferred, the rate of female offspring was 86%. Therefore, it was identified that most of embryos which were not affected by H-Y antiserum were female.

• 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.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.323-331
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• 1996

Predetermination of sex in mammalian species has many aspects of application including the prenatal diagnoses of genetic disorders in humans and sex-selected breeding programs in the animal industry. Embryos sexing can be carried out using the polymerase chain reaction (PCR) to amplify specific sequences present in the sex chromosomes, or by fluorescent in situ hybridization (FISH) of specific probes to the X and Y chromosomes. A 3.3 kb porcine male-specific DNA fragment (pEM39) was cloned previously in our laboratory. In this study, FISH and PCR methods were employed to examine if the pEM39 can be used a sex-specific DNA probes Porcine ovaries were obtained from a local slaughter house and oocytes collected. All oocytes were subjected to in vitro maturation followed by 1n vitro fertilization. Parthenogenetically activated embryos were served as a negative control. Embryonic samples were collected at the 2-cell stages and PCR was performed to analyze DNA. Among 10 embryos examined, four embryos were identified as males and six were females. The cloned male-specific DNA fragment showed male-specificity for the cells in the liver tissue and the porcine early embryos by FISH. It was also demonstrated that the cloned male-specific DNA is localized on the hetero chromatic region of the long arm in the Y chrom-osome (Yq) as shown by the FISH and karyotyping. The results suggest that the cloned male-specific DNA fragment may be useful for predetermination of sex with a few embryonic cells. The porcine male-specific sequence can be a reliable index for embryo sexing by PCR.

• Korean Journal of Poultry Science
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• v.20 no.4
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• pp.177-188
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• 1993

This study was performed to find out the reasonable sexing methods In the chicken, obtain the basic information for the mechanisms related to chicken sexual differentiation and identify the genes which known to involved in chicken sex differentiation. The chromosome analysis of chicken embryonic fibroblast was a simple method to determine sex of chicken by means of Z and W chromosome identification. The bands of female chicken genomic DNA digested with Xho Ⅰ and Eco RI restriction endonuclease showed to be useful in direct sex determination and these repetitive sequences of Xho Ⅰ and Eco RI families were proposed to be very homologous in their sequences by colony hybridization analysis. Seven of 150 random primers were selected to amplify the W chromosome-specific band by using arbitrary primed PCR and three of them were useful to identify the sex of chicken. To identify the sex differentiation genes in the chicken, PCR for the amplification of ZFY and SRY sequences was performed. ZFY and SRY sequences were amplified successfully in the chicken genome, implying that chicken genome might have the sex-related conserved sequences similar to mammalian ones. The PCR products of ZFY amplification were the same in both sexes, suggesting that these sequences may be located on autosome or Z chromosome. The profile of PCR amplification for SRY sequences showed variation between sexes, but this result was not enough to specify whether the SRY gene in chicken is on the autosome or sex chromosome.

• Korean journal of applied entomology
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• v.13 no.3 s.20
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• pp.141-144
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• 1974

The vinyl-shielded rearing chamber was controlled by automatic electric heater to maintain relatively constant temperature and under this condition, it took 13 to 14 days from egg to adult and 8 days for their preoviposition. The pupal weight was not varied with the advance of generation, whereas was greatly effected by temperature changes. Since only $60\%$ of the pupae was able to classify the sex by pupal weight, a mechanical device will be needed for the further sexing.

• Korean Journal of Animal Reproduction
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• v.7 no.2
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• pp.41-52
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• 1983

Animal in dustry in Korea urgently needs the domestic introduction and the industrial a, pp.ication of embryo transfer technique. Namely, this technique can be utilized effectively, as means of the improvement of livestocks, as means of the increase of meat production, as means of substitute for the livestock import, and dissemination of new breed. However, as this technique avaliable in our country is remaining initial stage, we can not make use of the technique industrially unless we make much improvement as follows; induction of superovulation, non-surgical recovery of embryos, synchronization between the estrus such cycles of donor and recipient, non-surgical transfer of embryos, etc. Simultaneously, the basic studies such as harvesting oocytes from ovary, in vitro culture of oocytes, in vitro capacitation of spermatozoa, cloning by culture of blastomeres and transfer of nuclei, sexing embryo, etc. should not be neglected in order to make the technique of embryo transfer more simple and convenient. For the success of these studies, universities, national and public institutes, large scale cattle farms, and small scale cattle farms should cooperate each other. For instance, universities undertake basic researches, and the national and public institutes a, pp.y the results of the researches to animal industry along with cooperation by large scale cattle farms. By the help of the cooperative organizations, the technique relevant to our environment and farm condition may be able to be finalized, and to be a, pp.ied to samll scale cattle farm. Consequently, being served to stimulate animal productivity, this technique can be contributed to the development of livestock industry in Korea.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.106-110
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• 2019

Sexed sperm can contribute to increase the profitability of the cow industry through the production of offspring of the craved sex, such as males for meat or females for dairy production. Therefore, the utilization of sexed sperms plays a very important role in the production of offspring of superior cattle. In this study, we examined the pregnancy rates and calves sexing proportion of male and female calves produced using AI, both performed using sexed and conventional sperm. In the result, the conception rates after ET were 73.3% (33/45) sexed semen and 52% (55/104) conventional semen. Thus, the sex ratio for sexed-semen inseminations was 70% (21/30) females for singleton births within a 272 to 292 day gestation interval. The sex ratio for conventional semen was 61% (34/56) females for births. As a result, it is suggested that the use of sex classification sperm will play a very important role in the offspring production of Korean bovine.