• Korean Journal of Agricultural Science
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• v.38 no.2
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• pp.269-275
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• 2011

The objective of this study was to develop a rapid and reliable PCR method for sexing of morula or blastocyst stage bovine embryo. BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male and bovine specific DNA, respectively. But the unbalanced number of copies of these two repetitive sequences required some modification of PCR method. Karyotyping of blastomeres were carried for the confirmation of sex determination in bovine embryos. The coincidence rate of sex between biopsied-single blastomere and matched blastocyst was 80.0%. When in vivo- and in vitro- derived embryos were compared, 61.8% and 56.7% were male in in vitro- and in vivo-derived embryos, respectively. In vivo-derived embryos showed better hatching rate than in vitro-derived embryos following biopsy of blastomeres. In conclusion, rapid and effective PCR could be applied to sexing of bovine preimplantation embryos using single blastomere. The sensitivity of this assay may eliminate the need for biopsy of more than one nucleated blastomere and reduce trauma to the embryos derived from biopsy procedure.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.283-289
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• 1996

This study was carried out to determine the sex of genomic and embryonic DNA using polymerase chain reaction(PCR). Bovine specific(216bp) and Y chromosome speicific DNA primers(l4lbp) were synthesized and tested for sexing. Bovine embryos used in this study were produced by in vitro fertilization. Few blastomeres for PCR were bisected by nicromanipulator and demi -embryos were cultured in TCM 199 medium containing 0.1% of solcoseryl. The results obtained were as follows; 1. Average optical density of genomic DNA extracted from blood of Hanwoo was 1.79$\pm$ 0.14. 2. 2. The ratio of the demi-embryos developed to blastocyst was 62.1 and 81.9% in morula and blastocyst, respectively. 3. When DNA of 2~4, 5~10 and more than 11 blastomeres was amplified with Y chromosome specific DNA primer by PCR, appreance rate of Y specific DNA band was 16.7, 46.2 and 40.0%, respectively. At least 5 to 10 blastomeres were required to determine the sex of embryos. 4. The rate of demi-embryos developed to blastocyst was 73.3% in TCM 199 medium supplemented with 0.1% solcoceryl. but 55.6% in control.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.443-451
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• 1997

These studies were conducted to determine the sex of preimplantation Hanwoo embryos produced in vitro using polymerase chain reaction(PCR). Y chromosome specific and bovine speicific DNA primers were synthesized and tested for embryo sexing. Bovine IVF embryos were produced in TCM 199 and CR1aa medium, and classified by developmental stages on Day 7 to 9. The effects of developmental rates to bovine IVF blastocysts on sex ratio were also investigated using PCR methods. The results obtained in this study were as follows; 1. Developmental rates to blastocyst from IVM/IVF embryos in TCM 199 and CR1aa medium for 9 days were 23.5 and 30.2%, respectively, and there was significant difference between the media(P<0.05). 2. Male to female ratio of early, mid, expanded and hatching balstocyst produced on Day 7 were 0.7:1, 1.4:1, 2.2:1, and 2.5:1, respectively, and male embryos was significantly higher proportion in expanding and hatching blastocysts(P<0.01). 3. On Day 8, male to female ratio of early, mid, expanded and hatching blastocysts were 0.6:1, 1:1, 2.5:1, and 2.7:1, respectively. Both expanded and hatching blastocysts obtained a significantly higher proportion of males(P<0.01). 4. The male : female ratio of early, mid, expanded and hatching blastocyst produced on Day 9 was 0.6:1, 0.8:1, 1:1, and 2.2:1, respectively. Hatching blastocysts had a significantly higher ratio of males(P<0.01). The developmental rate of IVM/IVF embryos to blastocyst for 9 day culture was higher in CR1aa than that in TCM 199 medium. For the sex ratio by developmental stages of IVF embryos, male ratio was higher in expanded blastocyst but female in early blastocysts.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2007.05a
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• pp.39-50
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• 2007

Sexing from bovine embryos fertilized in vitro implicates a possibility of the sex controlled cattle production. This study was carried out to produce the sex determined cattle through the embryonic sexing by fluorescence in situ hybridization (FISH) technique. FISH was achieved in in vitro fertilized bovine embryos using a bovine Y-specific DNA probe constructed from the btDYZ-1 sequence. Using this probe, a male-specific signal was detected on 100% of Y-chromosome bearing metaphase specimens. The analyzable rate of embryonic sexing by FISH technique was about 93% (365/393) regardless of embryonic stages. As tested single blastomere by FISH and then karyotype with their biopsied embryos, the accuracy of sex determination with FISH was 97.6%. We tried the embryo transfer with sex determined embryos on 15 cattle. Among them, the 5 cattle delivered calf with expected sex last year.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.341-349
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• 1996

초기배의 성판정은 대상가축의 성을 선발하는 수단으로써 뿐만아니라 인간의 유전적 질병의 조기진단법으로서 매우 가치가 크다. 체외수정 소 초기배의 성을 결정하기 위해 PCR과 PRINS를 이용하였으며 성판정에 이용된 8 세포${\sim)$배반포기 초기배는 체외수정후 난관상피세포와의 공배양에 의해 생산되었다. 초기배 의 DNA는 $200{\mu}g/ml$ proteinase K가 함유된 PCR lysis buffer에 하나의 초기배를 부유한 후 $50^{\circ}C$에서 1시간동안 처리하여 준비하였다. 중기 염색체 spreads는 초기배를 nocodazole로 처리한 후 air-drying 방법을 이용하여 준비하였다. 가능한 false positive signals을 배제하기 위해 소특이 및 Y 염색체 특이 primers를 이용하여 PCR을 수행한 결과, 웅성 초기배에서는 두 개의 증폭산물 (소특이 및 Y 염색체 특이)이 합성된 반면 자성 초기배에서는 하나의 증폭산물만 합성되었다. 한편 중기염색체상의 Y 염색체를 동정하기 위해 FISH와 PRINS를 수행한 결과, FISH에서보다 PRINS에서 더 강한 Y 염색체 특이 형광 signals이 탐지되었다. 이러한 결과는 PCR에 의한 체외생산 소 초기배의 신속정확하고 효율적인 성판정이 가능함을 보여주었다. 또한 PRINS를 통해 PCR 에 이용된 Y 특이 probe의 신뢰성이 염색체 수준에서 확인되었다.