• 한국동물생명공학회지
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• 제37권2호
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• pp.80-86
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• 2022

The ability to determine the sex of bovine embryos before the transfer is advantageous in livestock management, especially in dairy production, where female calves are preferred in milk industry. The milk production of female and male cattle benefits both the dairy and beef industries. Pre-implantation sexing of embryos also helps with embryo transfer success. There are two approaches for sexing bovine embryos in farm animals: invasive and non-invasive. A non-invasive method of embryo sexing retains the embryo's autonomy and, as a result, is less likely to impair the embryo's ability to move and implant successfully. There are lists of non-invasive embryo sexing such as; Detection of H-Y antigens, X-linked enzymes, and sexing based on embryo cleavage and development. Since it protects the embryo's autonomy, the non-invasive procedure is considered to be the safest. Invasive methods affect an embryo's integrity and are likely to damage the embryo's chances of successful transformation. There are different types of invasive methods such as polymerase chain reaction, detection of male chromatin Y chromosome-specific DNA probes, Loop-mediated isothermal amplification (LAMP), cytological karyotyping, and immunofluorescence (FISH). The PCR approach is highly sensitive, precise, and effective as compared to invasive methods of farm animal embryonic sexing. Invasive procedures, such as cytological karyotyping, have high accuracy but are impractical in the field due to embryonic effectiveness concerns. This technology can be applicable especially in the dairy and beef industry by producing female and male animals respectively. Enhancing selection accuracy and decreasing the multiple ovulation embryo transfer costs.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.183-183
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• 2004

In the bovine embryo transfer industry, sexing preimplantation embryos is an important management tool. Several methods for bovine embryo sexing utilizing polymerase chain reaction (PCR) have been developed. However, they were not popularized because the methods requiretechnical skills and expensive instruments, and are time consuming. PCR also has the risk of false positives due to DNA contamination during the electrophoresis. (omitted)

• Asian-Australasian Journal of Animal Sciences
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• 제20권6호
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• pp.866-871
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• 2007

Testis-specific protein (TSPY) is a Y-specific gene, with up to 200 copy numbers in bulls. In order to make bovine embryo sexing under farm condition more feasible, the possibility of using a non-electrophoretic method to detect the TSPY gene for sexing bovine early embryos was examined. Primers were designed to amplify a portion of the TSPY gene and a common gene as an internal control primer. PCR optimization was carried out using a DNA template from bovine whole blood. Furthermore, embryo samples were diagnosed by this method and the sexing results were contrasted with those of the Loop-Mediated Isothermal Amplification (LAMP) method. The results showed that TSPY was as reliable a sexing method as LAMP. Forty-three morula and blastocyst embryos collected from superovulated donor dairy cattle were sexed by this method, and twenty-one embryos judged to be female embryos were transferred non-surgically to recipients 6 to 8 days after natural estrus. Out of 21 recipients, 9 were pregnant (42.86%) and all delivered female calves. The results showed that the sex predicted by this protocol was 100% accurate. In conclusion, the TSPY gene was a good male specific marker and indicated that a non-electrophoretic method was feasible and accurate to detect the TSPY gene for sexing preimplantation bovine embryos.

• 한국가금학회지
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• 제48권1호
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• pp.41-50
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• 2021

최근 병아리의 성 감별은 닭의 반성유전자를 이용한 깃털 감별법이 산업적으로 널리 이용되고 있으나, 한국토종닭에서는 아직 깃털 감별법이 활용되지 않고 있다. 따라서 본 연구에서는 한국토종닭의 깃털 감별의 산업적 적용을 위하여 닭의 조우성과 만우성 유전자를 이용한 자가성감별 계통을 조성하고자 한 것이다. 기초 계군으로 한국재래닭 적갈색종 557수를 대상으로 하여 조우성과 만우성의 유전자 빈도를 분석한 결과, 각각 0.814와 0.186으로 추정되어 본 집단으로 깃털자가성감별 계통의 구축이 가능한 것으로 판단되었고, 이를 바탕으로 조우성 부계통과 만우성 모계통의 종계 계군을 조성하였다. 모계통 조성에 이용할 만우성 동형접합체(KK) 수컷 선발을 위하여 검정교배를 실시한 결과 40수 중3수가 동형접합개체로 확인되었다. 조우성 종계와 만우성 종계 간의 생산능력을 비교하기 위하여 생존율, 체중, 초산일령, 산란율 및 난중을 분석한 결과, 조우성 닭은 생존율이 우수하였고, 만우성 닭은 산란율이 우수한 것으로 나타났으나, 다른 생산능력에서는 조우성과 만우성 닭 간에 차이가 없는 것으로 나타났다. 더불어 조성된 깃털 자가성감별 계통으로부터 생산된 병아리의 암수 감별의 유효성을 검정한 결과, 98.6%의 감별 일치율을 보였다. 따라서 한국토종닭을 이용한 깃털 자가성감별계통 조성 가능성과 더불어 이의 산업적 활용 가능성을 제시한다.

• 한국수정란이식학회지
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• 제28권3호
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• pp.199-205
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• 2013

The present study was conducted to compare on embryo survival rates by blastomere isolation methods, and establish the optimal PCR procedure for perform the sexing of bovine blastocysts produced by IVF. IVF embryos used in the study was used the Bisected or Sliced methods for blastomere isolation, and the survival rates of blastocyst with rapid way of sexing PCR was assessed. In the present study for survival rates in blastocyst was the total cleavage rate was 75% and a blastocyst development among cleaved embryos was 40%. Survival rate of embryos treated with intact, bisected or sliced method was 100, 63.3 or 81.3%, respectively. Therefore, survival rate of embryos treated with sliced method was higher compared to that of embryos treated with bisected method. The sexing rate of female or male was not significantly different between S4BFBR primer and BSY + BSP primer (1.75 : 1 vs. 1.43 : 1), respectively. Because of the PCR amplification using the S4BFBR primer was simpler method than multiplex PCR amplification method. Furthermore, the accuracy of sexing rate and reduction of PCR work time between 2-step and 3-step of PCR methods was 98.0% / 1.5 hr and 97.0% / 3.5 hr, respectively. Based on these results, it can be suggested that the sliced and PCR methods we developed was very effective method to reduce time consuming and procedure of PCR amplification for sexing with the increase of survival rate on the blastocyst.

• 한국가축번식학회지
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• 제24권3호
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• pp.299-309
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• 2000

본 연구는 한우 체외수정란의 정확한 성판별을 위한 PCR의 적정조건을 검토하였고, Y 염색체 특이적 DNA primer(BOV97M : 141bp)와 소 특이적 DNA primer(216bp)를 이용한 2단계 PCR 방법으로 체외수정란의 발육속도에 따른 성판별율과 성비를 검토하였다. PCR 기법을 이용한 한우 체외수정란의 성판별은 Y 염색체 특이적 DNA primer와 소 특이적 DNA primer로 정확히 성을 판별할 수 있었으며, 웅성 수정란의 경우 141bp와 216bp에서 band가 나타났고, 자성 수정란의 경우 216bp에서 band가 나타났다. 한우 체외수정란의 경우 성판별 정확도를 높이기 위하여 투명대의 제거가 필요하며, 수정란의 DNA 추출방법에 따른 Y 염색체 특이적 band의 출현율은 Proteinase K와 반복 동결융해방법이 각각 45.2 및 53.3%로 나타났다. DNA의 양에 따른 성판별율은 zona free인 경우는 2$\mu$1와 10$\mu$1에서 각각 97.2%와 92.2% 였으며, zona intact의 경우는 12$\mu$1와 13$\mu$1에서는 각각 91.5%와 93.6%로서 zona free 수정란의 경우 2$\mu$1, zona intact의 경우 13$\mu$1의 DNA 양으로 성판별이 가능하였다. 한편, PCR 기법에 의한 한우 체외수정란의 성판별율은 96.0%였으며, 정확히 성판별을 할수 없는 비율은 4.0%였다. 성판별 수정란의 자성과 웅성의 비율은 46.3%와 49.7%로서 성비는 1.1:1이였으며, 발육단계간 자웅성비는 커다란 차이가 인정되지 않았으며, 발육단계가 진행될수록 성판별 정확도가 증가하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권11호
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• pp.1689-1693
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• 2007

The objective of this study was to develop a simplified, efficient, and accurate protocol for sexing goat embryos. Based on the amelogenin gene located on the conservation region of X- and Y- chromosomes, a pair of primers was utilized and the system of PCR was established to amplify a 262 bp fragment from the X- chromosome in female goats, and a 262 bp fragment from X- chromosome and 202 bp fragment from the Y- chromosome in male goats, respectively. The accuracy and specificity of the primers were assessed using DNA template extracted from goat whole blood sample of known sex. 100% (10/10) concordance was obtained by using the PCR assay. Fifty-one biopsied embryos were transferred into 25 recipient goats on the same day that the embryos were collected and sex of the kid was confirmed after parturition. Eighteen kids of predicted sex were born. The biopsied samples from 51 goat embryos were amplified with 100% efficiency and 94.7% accuracy. In conclusion, our results indicated that PCR sexing protocols based on the amelogenin gene is highly reliable and suitable for sex determination of goats.

• Asian-Australasian Journal of Animal Sciences
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• 제16권5호
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• pp.650-654
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• 2003

The accuracy of Polymerase chain reaction (PCR) assay in sexing of sheep embryos was assessed in this study. A total of 174 ovine embryos produced in vitro at different stages of development (2, 4-8 cell stages, morula and blastocyst) were sexed. The universal primers (P1-5EZ and P2-3EZ) used in this assay amplified ZFY/ZFX-specific sequences and yielded a 445 bp fragment in both sexes. Restriction enzyme analysis of ZFY/ZFX-amplified fragments with Sac I exhibited polymorphism between sexes, three and two fragments in males and in females, respectively. For verification of accuracy, blood samples of known sex were utilized as positive controls in each test. The mean percentages of sex identification by this method at 2 cell, 4-8 cell, morula and blastocyst were $73.00{\pm}5.72$, $89.77{\pm}3.79$, $3.33{\pm}8.08$ and $79.6{\pm}9.09$, espectively with the over all male to female ratio of 1:0.87. It is concluded that the ZFY/ZFX based method is highly reliable for the sexing of sheep embryos.

• 한국가축번식학회지
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• 제10권1호
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• pp.27-35
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• 1986

As a preliminary experiment to establish the process on the sexing of mouse embryos by chromosomal analysis, present studies were carried out with inbred (ICR, C57BL) and F1 hybrid [(ICR${\times}$C57BL) = F1 ${\times}$ ICR] mice to investigate the blastomere numbers and mitotic indices (M.I.) to the developmental stage of embryos recovered, the optimum periods of anti-mitotic agent administration, the successful rates of sexing and sex-ratio. The results obtained were summarized as follows: 1. The blastomere numbers (mean${\pm}$S.E.) of the morula and blastocyst were 18${\pm}$0.4 and 54${\pm}$0.7, respectively. 2. Whereas the M.I. of F1 hybrid (16${\pm}$0.2%) was higher than that fo inbred ICR (15${\pm}$0.1%) and C57BL (12${\pm}$0.6%) in the different strains, the morula (7${\pm}$0.6%) was higher than that of blastocyst (6${\pm}$0.4%) in the case of embryo stages. 3. Following to anti-mitotic agents treated, the M.I. of embryos cultured with Colcemid (17${\pm}$1.1%) was superior to that fo embryos cultured with Velban (12${\pm}$0.9%) and the Colcemid injection (7${\pm}$0.4%). 4. The successful rate of sexing in the blastocyst (38.7%; 124/320) was superior to the morula (35.9%; 52/145), and the F1 hybrid (48.1%) was higher than that of inbred ICR (42.4%) and C57 BL (28.2%). 5. In the successful rate of sexing to the methods of administration, the embryos cultured with Colcemid (46.0%) was superior to that of embryos cultured with Velban (39.0%) and the Colcemid injection (38.8%). 6. Of 98 embryos sexed after culture with Colcemid, 89(90.8%) were observed between 2 and 4 hrs. In the case of Velban treatment, 83.1% (74/89) was observed between 2$\frac{1}{2}$ and 4$\frac{1}{2}$ hrs. 7. Out of 761 prepared embryos it was possible to sex 311; 157 were male and 154 were female, i.e.a sex-ratio of 50% a, pp.oximately.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 발생공학 국제심포지움 및 학술대회 발표자료집
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• pp.6-10
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• 2001

No. Sperm can be sexed with 90% accuracy by flow cytometry/cell sorting. No. The current speed of sexing is about 5,000 live sperm of each sex per second, remarkably fast considering that each sperm is individually sexed. No. Although fast, sperm sexing is not fast enough to use standard numbers of sperm per AI dose. No. With well managed heifers, pregnancy rates with low doses of sexed, frozen sperm are 70-80% of those with unsexed sperm with normal sperm numbers. Pregnancy rates are lower in lactating dairy cows. No. Calves from sexed sperm appear to be normal. No. Sexed, frozen semen from a few bulls currently is available commercially in the United Kingdom, and likely will be available in several other countries in 2002, probably at a premium of US ＄30-50 per straw. (omitted)