• Asian-Australasian Journal of Animal Sciences
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• v.20 no.11
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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.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.2
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• pp.111-118
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• 2003

Objectives: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. Materials and Methods: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. Results: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were $1.0{\mu}M$ of vinblastine (20.3%), $5.0{\mu}M$ of nocodazole (28.1%) and $1.0{\mu}M$ colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine ($1.0{\mu}M$) and nocodazole ($1.0{\mu}M$). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. Conclusions: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.97-108
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• 2003

In-vitro fertilized Hanwoo embryos were biopsied for sex determination by PCR. Biopsied embryos were incubated for 1∼2 hours for the recovery. Those sexed Hanwoo embryos were transferred to 49 Hanwoo and 16 Holstein recipients from February 2000 to February 2001. Of 65 recipients, 14 cows(12 Hanwoo and 2 Holstein) delivered the same offspring as sex-determined by PCR, therefore the conception rate was 21.5%. 1. Total 65 embryos(male 35, female 30) were transferred to recipients, and 14 calves (male 6, female 8) were delivered. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex determination was 100.0%. 2. The conception rate after transfer with biopsied embryo between Hanwoo and Holstein was 24.5% and 12.5% 3. The conception rate after transfer with biopsied embryo between fresh and frozen-thawed embryos was 23.5% and 14.3%. 4. The conception rate according to the season when embryo was transferred was 11.8, 29.4, 23.5 and 20.0% for spring, summer, autumn and winter, respectively. 5. The conception rate according to embryo quality after biopsy was 41.7, 30.0 and 0.0% for excellent, good and fair quality. 6. The conception rate according to thickness of uterine horn was 71.4, 18.9, 11.8 and 0.0% for 0, +, ++ and +++ thickness. 7. The conception rate according to the site in the uterine hem where embryo was put was 30.0, 20.0 and 10.0% for cranial, mid, and caudal part of uterine horn. 8. The conception rate according to the quality of corpus luteum ipsilateral to the uterine horn where embryos was transferred was 41.2, 14.3 and 15.4% for excellent, good and fair quality. 9. The conception rate according to the time required for embryo transfer was 18.2, 30.0, 30.0, 0.0 and 25.0% for 10, 15, 20, 25 and 30 minutes. 10. The conception rate according to parity of recipients was 26.5, 19.1, 14.3 and 0.0% for the primiparous, the 2nd parous, the 3rd parous and the 4th parous recipients. These results indicated that fresh embryos are more demanded than frozen-thawed embryos for good conception rate in embryo transfer with biopsied-sexed embryo. Also, it was indicated that we should consider embryo-recovering condition, recipient's uterine thickness, transfer site in uterine horn, quality of corpus luteum, time required for transfer and parity of recipient to achieve good conception rate in ET with biopsied-sexed embryos.

• Journal of Animal Science and Technology
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• v.49 no.2
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• pp.287-294
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• 2007

The present study was to conducted the sexing efficiency and accuracy of bovine embryo by LAMP (Loop-mediated isothermal amplification) method, the development of the biopsied embryos into re- reformation and the freezability of these blastocysts by slow-freezing and vitrification. In vivo embryos were superovaluted with gonadotropin(Antorin R-10) for 4 days combined with progestrone releasing intravaginal(CIDR) insertion in Hanwoo donors, and in vitro embryos were used blastocyst embryos at Day 7 or Day 8 after post-insemination in vitro. The biopsy of bovine embryo was carried out in a 80μl drop with Ca2+-Mg2+ free D-PBS and the viability of biopsied embryos were evaluated in IVMD medium at over 12 h culture time in 5% CO2 incubator.For embryo sexing, about five or seven blastomeres were isolated from in vitro and in vivo embryos at blastocysts with microblade. and were then subjected to LAMP. The survivability of biopsied embryos were no difference in the development rate to re-formation of blastocysts between in vivo and in vitro embryos(100% and 90% respectively). The rates of sexed embryos were compared according to two groups, the female rate was lower than that the male in the in vivo and in vitro embryos(46% vs, 54% and 40% vs, 60%, respectively). However, there were no difference in the overall sexing ratio between the two groups. The survivability of frozen-thawed sexed embryos were lower in the in vitro than in vivo embryos in the slow-freezing(Group 1) and vitrification method(Group 2), (41.7% vs. 58.8% and 57.1% vs, 77.8. respectively).

• 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.15 no.2
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• pp.175-180
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• 2000

The possible use of micromanipulative biopsy and PCR of the biopsied embryonic cells was tested to produce sexed bovine embryos in practical terms. By micromanipulation and PCR techniques, higher survival rate and accurate sexing of demi-embryos were btained. Bovine oocytes matured and fertilized in vitro were co-cultured with bovine oviductal epithelial cell (BOEC) monolayer in USU-6 medium supplemented with 15% FBS, and the embryos of 37% (327/885) were developed to blastocysts. Among 111 blastocysts produced by invitro, only 7 (6.3%) embryos were found unable to determine their sex, probably due to the loss of cells, since no PCR product was found from those cells. All the remaining 104 (93.7%) demi-embryos survived micromanipulation and demonstrated male-specific product or bovine-specific product alone suggesting that correct sexing of the sample. Forty-three point one percent(25/58) of manipulated and cryopreserved demi-embryos after thawing were survived. Final verification of the sexed embryos is necessary to make sure the same sex in fetus and newborn calf upon embryo transfer. The established sexing method on a large number of bovine embryos from previous and this study suggests that this a could be used practically in the field.

• Reproductive and Developmental Biology
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• v.36 no.4
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• pp.269-273
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• 2012

The present study was to assess the in vitro viability and sexing rate of bovine embryos. Blastocysts were harvested on day 7~9 day after insemination(in vitro and in vivo), and the sex of the embryos was examined using the LAMP method. Embryo cell biopsy was carried out in a $80{\mu}l$ drop $Ca^{2+}$, $Mg^{2+}$ free D-PBS and, biopsied embryos viability were evaluated after more 12 h culture in IVMD culture medium. The formation of recovered embryo to expanded and hatching stages had ensued in higher of sexed embryo in vivo than in vitro (100% vs. 89%, p<0.05), and in vitro, the rates of degeneration after sexing were significantly (p<0.05) higher in vitro than in vivo(11% vs. 0.0%). The rates of the predicted sex were female 61% vs. 56%, and male 39% vs. 44% in vivo and in vitro, respectively. The rates of survival following different biopsy methods were seen between punching and bisection method in vivo and in vitro (100% vs. 89% and 100% vs, 78% respectively). Biopsy method by punching was significantly (p<0.05) higher than bisection between produced embryos in vivo and in vitro. The present data indicate that with microblade after punching for embryo sexing results in high incidence of survivability on development after embryo biopsy. It is also suggested that LAMP-based embryo sexing suitable for field applications.

• 대한생식의학회:학술대회논문집
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• 2003.12a
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• pp.137.1-137.1
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• 2003

• Clinical and Experimental Reproductive Medicine
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• v.47 no.4
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• pp.293-299
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• 2020

Objective: The goal of the present study was to investigate the rate of chromosomal aneuploidies in surplus embryos after sex determination at the cleavage stage. Then, the same chromosomal aneuploidies were evaluated in blastocysts after extended culture. Methods: Sixty-eight surplus embryos were biopsied at the cleavage stage and incubated for an additional 3 days to allow them to reach the blastocyst stage. The embryos were reanalyzed via fluorescence in situ hybridization (FISH) to examine eight chromosomes (13, 15, 16, 18, 21, 22, X, and Y) in both cleavage-stage embryos and blastocysts. Results: Although the total abnormality rate was lower in blastocysts (32.35%) than in cleavage-stage embryos (45.58%), the difference was not significant (p=0.113). However, when we restricted the analysis to autosomal abnormalities, we observed a significant difference in the abnormality rate between the cleavage-stage embryos (44.11%) and the blastocysts (17.64%, p=0.008). A higher rate of sex chromosomal abnormalities was also observed in cleavage-stage embryos (29.4%) than in blastocysts (14.70%, p=0.038). Conclusion: The data indicated that embryo biopsy should be conducted at the blastocyst stage rather than the cleavage stage. The results also emphasized that examination of common chromosomal aneuploidies apart from sex selection cycles can be conducted in the blastocyst stage with the FISH method.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.43-49
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• 2008

This study was carried out to examine the efficiency of biopsy methods, and the pregnancy rate, calving and abortion rates, gestation length and birth weight of Hanwoo calves following transfer of fresh, frozen and sexed Hanwoo embryos produced in vitro. The survivability of biopsied embryos was 80.0 and 90.0% using aspiration and punching methods at 24 h after culturing, respectively. The ratios of male and female embryos were 42.1 and 52.6%, respectively, and the percentage of sex unidentified was 5.3%. Pregnancy rates was not significantly different between hCG and control group (46.4 vs. 38.5%), fresh and frozen embryos (41.3 vs. 35.0%), and sexed and IVP embryos (27.5 vs. 41.2%) (p>0.05). Calving and abortion rates of IVP and sexed embryos were not significantly different in calving (85.0 vs. 87.0%) and in abortion (15.2 vs. 13.3%) (p<0.05). Gestation length of IVP and sexed calves were 281.3 and 288.2 days in female and 283.0 and 282.3 days in male, and the birth weight of IVP and sexed calves were 23.6 and 25.0 kg in female and 24.6 and 23.8 kg in male, respectively. There were no difference in gestation length and birth weight between IVP embryos and sexed embryos (p>0.05). Administration of hCG to recipients did not improve the pregnancy rate following transfer of Hanwoo embryos produced in vitro and sexed embryos. Although the production of calves derived from sexed Hanweoo embryos cultured in vitro can be obtained, the efficiency of sexed calves production need to be improved in biopsy methods and pregnancy rate. Further study should be focused on the improvement of pregnancy rates for commercial application of embryo transfer.