• Korean Journal of Veterinary Research
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• v.39 no.1
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• pp.189-203
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• 1999

To determine sex of bovine embryos using hamster histocompatibility Y(H-Y) antibodies, bovine compact morulae were incubated for 6 hours in TCM199 supplemented with 10% hamster H-Y antiserum and the embryos with developmental arrest were diagnosed as male embryos, while the embryos showing development during the incubation as female embryos. This presumptive embryo sexing was confirmed by polymerase chain reaction(PCR)method. 1. In the result of hamster sperm cytotoxicity test to measure H-Y antibody titer, the rate of dead sperm was considerably lower in H-Y antiserum absorbed with hamster male splenocytes than in H-Y antiserum absorbed with hamster female splenocytes or H-Y antiserum unabsorbed with splenocytes(p<0.01). 2. The rate of oocytes fertilized in vitro and the rate of blastocysts of the fertilized oocytes were 58.5% and 32.4%, respectively. The rate of blastocysts on day 8 was 15.9%, denoting the highest rate during whole culture period posterior to in vitro fertilization (IVF). 3. The bovine 16 cell and compact morulae embryos incubated in the medium supplemented with hamster H-Y antibodies showed 37.1% and 48.9% of developmental arrest which were diagnosed as male, respectively, and rates of redeveloped embryos from the arrested were 24.1% in 16 cell and 44.3% in compact morulae embryos, respectively, denoting higher rate of sex determination and rate of redevelopment in compact morulae than 16 cell embryos. 4. Bovine compact morulae of Korean cattle and Holstein were treated with hamster H-Y antibodies for sex determination and the rates of developmental arrest(diagnosed as male) were 48.4% for Korean cattle and 47.9% for Holstein, respectively. The rates of redeveloped embryos to blastocyst after treatment were 42.6% for Korean cattle and 41.8% for Holstein, respectively, showing no significant differences of sex determination and redevelopment between both breed. 5. The sex determination of bovine embryos(Korean cattle and Holstein) using hamster H-Y antibodies was diagnosed by PCR for confirmation, denoting the rates of 86.1% for Korean cattle and 85.9% for Holstein male embryos, respectively, and the rates of 91.9% for Korean cattle and 90.1% for Holstein female embryos, respectively, with no significant differences of sex determination between both breed. These results indicated that hamster H-Y antibodies can be usable for sex determination of bovine embryos of Korean cattle and Holstein, the viability of bovine embryos was sustained while being cultured in the medium supplemented with hamster H-Y antibodies of appropriate titer and sex determination of bovine embryos by PCR can be feasible for confirmation.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.133-137
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• 2004

Sexing from bovine embryos which were fertilized in vitro implicate a possibility of the sex-controlled cattle production. This study was carried out to investigate the possibility of determining of embryo sex by fluorescence in situ hybridization (FISH) technique. FISH was achieved in in vitro fertilized bovine embryos using a bovine Y-specific DNA probe which constructed from the btDYZ-1 sequences. To evaluate Y-chromosome specificity of the FISH probe, metaphase spreads of whole embryos and lymphocytes were prepared and tested. A male-specific signal was detected on 100% of Y chromosome bearing metaphase specimens. Using the FISH technique with a bovine Y-specific probe, 232 whole embryos of 8 cell- to blastocyst-stage were analyzed. Observing the presence of the Y-probe signal on blastomeres, 102 embryos were predicted as male, and 130 embryos as female. The determining rate of embryo sex by FISH technique was about 93% regardless of embryonic stages. In conclusion, the FISH using a bovine Y-specific DNA probe is an accurate, reliable and quick method for determining the sex of bovine embryos.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.169-175
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• 2018

It has been claimed that artificial insemination (AI) of cows with frozen-thawed semen treated with commercially produced kits, Wholemom (in favour of female gender) increases the birth chance of calves with desired sex ratio by approximately 85% without decrease of pregnancy rates. Hence, this study was conducted to investigate the efficacy of wholemom kits as combined with frozen-thawed bovine semen during in vitro fertilization on the in vitro fertilization and developmental efficiency and sex ratios such as some reproductive parameters in bovine. For this, 1,737 oocytes were in vitro fertilized and developed. Agglutination effects on bovine after treatment of Wholemom kit were observed by time passage and dose respectively. To determine sex of embryos, Bovine embryo Y-specific gene primers(ConEY) and Bovine specific universal primer(ConBV) were used as multiple PCR method. Fertilization rate of wholemom-treated group was significantly lower than its of control group[66.9% (1,156/1,737) in Wholemom-treated group; 75.0% (610/813) in control group]. However, developmental rate after fertilization of both wholemom-treated and control groups were not significantly different [26.1% (404/1,156) in Wholemom-treated group; 27.4% (224/610) in control group]. Sex ratio of in vitro fertilized embryo with frozen-thawed semen treated with wholemom kit was determined by multi PCR. Female ratio in wholemom-treated group [85.4% (173/201)] was significantly higher than its of control group [47.2% (66/141)]. In conclusion, wholemom treatments of semen used in the in vitro fertilization and development of bovine oocytes provided increase in female ratio with decrease of fertilization rate.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.92-92
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• 2003

Accurate analysis of nuclear status is needed when biopsied-blastomeres are used for embryo sexing. In this study, the nuclear status of blastomeres derived from 8- to 16-cell stage IVF bovine embryos was analyzed to evaluate the representative of single blastomere for embryo sexing. When 55 embryos were analyzed by PCR following biopsy, the coincident rate of sex determination between biopsied-single blastomere and matched blastocyst by PCR was 80 %. Karyotyping of biastomeres in 8- 16-cell stage bovine embryos was conducted to assess chromosome status of IVF embryos. To establish karyotyping of blastomeres, concentrations of vinblastine sulfate and duration of exposure time for metaphase plate induction with 8- to 16-cell stage bovine embryos were tested. The most effective condition for induction of metaphase plate (>45%) was 1.0 ug/ml vinblastine sulfate treatment for 15 h. In 22 embryos under the condition, only 8 embryos out of ten that had a normal diploid chromosome complement showed a sex-chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four out of the other 11 embryos having a mixoploid chromosomal complement contained haploid blastomere with wrong sex chromosome (18.2%). These results suggested that morphologically normal bovine embryos derived from IVF had considerable proportion of mixoploid and sex-chromosomal mosaicism which could be the cause of discrepancies of the sex between biopsied-single blastomere and matched blastocyst by PCR analysis.

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.343-348
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• 2013

Cell-free fetal RNA has been highlighted as useful tools for the fetal sex determination or other genetic inherent disorder. However, there is no knowledge about the sex determination using cell free fetal RNA in bovine field. Thus, the present study aimed to evaluate the presence of transcripts of DDX3Y, USP9Y and ZRSR2Y genes in maternal plasma of pregnant cows to determine the sex of the fetus using real-time quantitative polymerase chain reaction assay, and verify its accuracy, sensitivity and specificity compared with the molecular testing and the calf sex at birth. Transcripts of USP9Y and DDX3Y genes were expressed in the all plasma of males and females both the control group and the experimental group. However, ZRSR2Y gene was matched up with the molecular testing and the true sex in control group and has an overall accuracy of 82.6%, a sensitivity of 75%, and a specificity of 100% in experimental group. Therefore, these results indicated that real time PCR technique, as a noninvasive and cost-efficient method, is possible to determination fetal sex in the bovine species using circulating cell free RNA in maternal plasma and especially ZRSR2Y gene could be a good candidate for the RNA based sex determination work.

• 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.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.287-287
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• 2004

Sexing from bovine embryos which were fertilized in vitro implicate a possibility of production of the sex controlled cattle. This study was carried out to investigate the possibility of determining of embryo sex by fluorescence in situ hybridization (FISH) technique. FISH was achieved in in vitro fertilized bovine embryos using a bovine Y-specific DNA probe which constructed from the btDYZ-1 sequences. (omitted)

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.299-309
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• 2000

This study was performed 1) to establish the optimal PCR condition of sex determination in Hanwoo IVM/IVF embryos, 2) to examine the sex determination and sex ratio to the developmental stages of Hanwoo IVM/IVF embryos by two-step PCR method. The sexing of bovine IVF embryos were accurately determined by PCR methods using Y chromosome specific DNA primer(BOV 97M, 141bp) and bovine specific DNA primer(216bp). The fregment size were shown at 141 and 216 base pairs(bp) in male, and 216 bp in female. Two-steps PCR method in which the samples were amplified by 15 cycles with Y chromosome specific DNA primer and then amplified by additional 30 cycles with bovine specific DNA primer was effective in the sexing of bovine IVF embryos. The zona-free embryos were more effective than zona-intact embryos in bovine IVF embryo sexing. The appearance of Y chromosome specific band was 45.2% in embryos treated with protease K and 53.3% in embryos treated with freezing and thawing repeatedly. The optimun volume of DNA for sexing of Hanwoo IVF embryos were 2 to 10 $\mu$1 in Zona-free embryos and 12 to 13 $\mu$1 in zona-intact embryos. The sexing rate of bovine IVF embryos by PCR was 96.0% and questionable rate not identified sex was 4.0%, respectively. Among the sexed embryos, the percentage of male and female was 49.7% and 46.3%, respectively, the sex ratio was 1: 1.1. The successful rate of embryo sexing was increased to the developmental stages.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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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.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.10
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• pp.1358-1364
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• 2011

The sex-determining region on the Y (SRY) gene is important in mammalian sex determination and differentiation. We report a study of the abundance of SRY gene products in bovine ejaculate. RT-PCR experiments using RNA extracted from bovine spermatozoa with SRY-specific primers yielded a 456 bp product, but the amount of SRY mRNA in sperm was lower than that in the testes (p<0.01). A protein of approximately 27 KDa was detected by western blotting. The SRY transcript was detected in the midpiece of approximately half the spermatozoa by in situ hybridization, and the SRY protein was detected in the heads of half the spermatozoa by immunofluorescence, indicating that SRY mRNA and protein may only be present in Y-bearing spermatozoa. These results suggest that the SRY transcript and protein are present in bovine ejaculated Y-sperm. The roles of the SRY gene in spermatogenesis, sperm motility, and the sperm-oocyte interaction merit further investigation.