Application of embryo biopsy and sex determination via polymerase chain reaction in a commercial equine embryo transfer program in Argentina
F. L. Riera A B , J. E. Roldán A B , J. M. Espinosa A B , J. E. Fernandez A , I. Ortiz C and K. Hinrichs
C D
+ Author Affiliations
- Author Affiliations
A Centro de Reproducción Equina Doña Pilar, Ruta 188, Km 200, (6070) Lincoln (B), Argentina.
B Laboratorio de Biotecnologia Reproductiva Prof. Robert M. Kenney, Sargento Cabral 748, (1669) La Lonja (B), Argentina.
C Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine & Biomedical Sciences, 4466 TAMU, Texas A&M University, College Station, TX 77843-4466, USA.
D Corresponding author. Email: khinrichs@cvm.tamu.edu
Reproduction, Fertility and Development 31(12) 1917-1925 https://doi.org/10.1071/RD19228
Submitted: 25 June 2019 Accepted: 17 September 2019 Published: 28 October 2019
Abstract
Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20 h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller embryos (70–85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive embryos were confirmed as male and 78% of Y-DNA-negative embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%.
Additional keywords: genetic diagnosis, pregnancy, sexing.
References
Carnevale, E. M., Squires, E. L., and McKinnon, A. O. (1987). Comparison of Ham’s F10 with CO2 or HEPES buffer for storage of equine embryos at 5C for 24 h. J. Anim. Sci. 65, 1775–1781.| Comparison of Ham’s F10 with CO2 or HEPES buffer for storage of equine embryos at 5C for 24 h.Crossref | GoogleScholarGoogle Scholar | 3443591PubMed |
Carney, N. J., Squires, E. L., Cook, V. M., Seidel, G. E., and Jasko, D. J. (1991). Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos. Theriogenology 36, 23–32.
| Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos.Crossref | GoogleScholarGoogle Scholar | 16726974PubMed |
Chang, H. F., Tsai, Y. L., Tsai, C. F., Lin, C. K., Lee, P. Y., Teng, P. H., Su, C., and Jeng, C. C. (2012). A thermally baffled device for highly stabilized convective PCR. Biotechnol. J. 7, 662–666.
| A thermally baffled device for highly stabilized convective PCR.Crossref | GoogleScholarGoogle Scholar | 22241586PubMed |
Choi, Y. H., Gustafson-Seabury, A., Velez, I. C., Hartman, D. L., Bliss, S., Riera, F. L., Roldan, J. E., Chowdhary, B., and Hinrichs, K. (2010). Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. Reproduction 140, 893–902.
| Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis.Crossref | GoogleScholarGoogle Scholar | 20843896PubMed |
Choi, Y. H., Penedo, M. C. T., Daftari, P., Velez, I. C., and Hinrichs, K. (2015). Accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts. Reprod. Fertil. Dev. 28, 1382–1389.
| Accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts.Crossref | GoogleScholarGoogle Scholar |
Chua, K. H., Lee, P. C., and Chai, H. C. (2016). Development of insulated isothermal PCR for rapid on-site malaria detection. Malar. J. 15, 134.
| Development of insulated isothermal PCR for rapid on-site malaria detection.Crossref | GoogleScholarGoogle Scholar | 26931146PubMed |
Clark, K. E., Squires, E. L., McKinnon, A. O., and Seidel, G. E. (1987). Viability of stored equine embryos. J. Anim. Sci. 65, 534–542.
| Viability of stored equine embryos.Crossref | GoogleScholarGoogle Scholar | 3624099PubMed |
Cuervo-Arango, J., Aguilar, J. J., Vettorazzi, M. L., and Martínez-Boví, R. (2015). eCG concentrations, luteal structures, return to cyclicity, and postabortion fertility in embryo transfer recipient mares. Theriogenology 84, 1003–1013.
| eCG concentrations, luteal structures, return to cyclicity, and postabortion fertility in embryo transfer recipient mares.Crossref | GoogleScholarGoogle Scholar | 26143362PubMed |
Cuervo-Arango, J., Claes, A. N., and Stout, T. A. (2018). Effect of embryo transfer technique on the likelihood of pregnancy in the mare: a comparison of conventional and Wilsher’s forceps-assisted transfer. Vet. Rec. 183, 323.
| Effect of embryo transfer technique on the likelihood of pregnancy in the mare: a comparison of conventional and Wilsher’s forceps-assisted transfer.Crossref | GoogleScholarGoogle Scholar | 29798842PubMed |
Curran, S., and Ginther, O. J. (1991). Ultrasonic determination of fetal gender in horses and cattle under farm conditions. Theriogenology 36, 809–814.
| Ultrasonic determination of fetal gender in horses and cattle under farm conditions.Crossref | GoogleScholarGoogle Scholar | 16727049PubMed |
El-Gayar, M., and Holtz, W. (2005). Transfer of sexed caprine blastocysts freshly collected or derived from cutured morulae. Small Rumin. Res. 57, 151–156.
| Transfer of sexed caprine blastocysts freshly collected or derived from cutured morulae.Crossref | GoogleScholarGoogle Scholar |
Flood, P. F., Betteridge, K. J., and Diocee, M. S. (1982). Transmission electron microscopy of horse embryos 3–16 days after ovulation. J. Reprod. Fertil. Suppl. 32, 319–327.
| 6962867PubMed |
Garcia, J. F. (2001). Practical considerations of embryo manipulation: preimplantation genetic typing. Theriogenology 56, 1393–1399.
| Practical considerations of embryo manipulation: preimplantation genetic typing.Crossref | GoogleScholarGoogle Scholar | 11768806PubMed |
Hasler, J. F., Cardey, E., Stokes, J. E., and Bredbacka, P. (2002). Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment. Theriogenology 58, 1457–1469.
| Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment.Crossref | GoogleScholarGoogle Scholar | 12374117PubMed |
Herrera, C., Morikawa, M. I., Bello, M. B., von Meyeren, M., Centeno, J. E., Dufourq, P., Martinez, M. M., and Llorente, J. (2014). Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program. Theriogenology 81, 758–763.
| Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program.Crossref | GoogleScholarGoogle Scholar | 24439164PubMed |
Hirayama, H., Kageyama, S., Moriyasu, S., Sawai, K., Onoe, S., Takahashi, Y., Katagiri, S., Toen, K., Watanabe, K., Notomi, T., Yamashina, H., Matsuzaki, S., and Minamihashi, A. (2004). Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification. Theriogenology 62, 887–896.
| Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification.Crossref | GoogleScholarGoogle Scholar | 15251240PubMed |
Hirayama, H., Kageyama, S., Moriyasu, S., Sawai, K., and Minamihashi, A. (2013). Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes. J. Reprod. Dev. 59, 321–326.
| Embryo sexing and sex chromosomal chimerism analysis by loop-mediated isothermal amplification in cattle and water buffaloes.Crossref | GoogleScholarGoogle Scholar | 23965599PubMed |
Huhtinen, M., Peippo, J., and Bredbacka, P. (1997). Successful transfer of biopsied equine embryos. Theriogenology 48, 361–367.
| Successful transfer of biopsied equine embryos.Crossref | GoogleScholarGoogle Scholar | 16728134PubMed |
Ideta, A., Aoyagi, Y., Tsuchiya, K., Kamijima, T., Nishimiya, Y., and Tsuda, S. (2013). A simple medium enables bovine embryos to be held for seven days at 4°C. Sci. Rep. 3, 1173.
| A simple medium enables bovine embryos to be held for seven days at 4°C.Crossref | GoogleScholarGoogle Scholar | 23378907PubMed |
Kuo, H. C., Lo, D. Y., Chen, C. L., Tsai, Y. L., Ping, J. F., Lee, C. H., Lee, P. A., and Chang, H. G. (2017). Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay on a field-deployable device. Poult. Sci. 96, 35–41.
| Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay on a field-deployable device.Crossref | GoogleScholarGoogle Scholar | 27389062PubMed |
Lin, Y. H., Lin, Y. J., Chang, T. D., Hong, L. L., Chen, T. Y., and Chang, P. F. (2016). Development of a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay for detection of Fusarium oxysporum f. sp. cubense Race 4. PLoS One 11, e0159681.
| Development of a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay for detection of Fusarium oxysporum f. sp. cubense Race 4.Crossref | GoogleScholarGoogle Scholar | 28030597PubMed |
Lopes, R. F. F., Forell, F., Oliveira, A. T. D., and Rodrigues, J. L. (2001). Splitting and biopsy for bovine embryo sexing under field conditions. Theriogenology 56, 1383–1392.
| Splitting and biopsy for bovine embryo sexing under field conditions.Crossref | GoogleScholarGoogle Scholar |
Martinez, C. A., Nohalez, A., Parrilla, I., Lucas, X., Sanchez-Osorio, J., Roca, J., Cuello, C., Rodriguez-Martinez, H., Martinez, E. A., and Gil, M. A. (2018). Simple storage (CO2-free) of porcine morulae for up to three days maintains the in vitro viability and developmental competence. Theriogenology 108, 229–238.
| Simple storage (CO2-free) of porcine morulae for up to three days maintains the in vitro viability and developmental competence.Crossref | GoogleScholarGoogle Scholar | 29253666PubMed |
Martinez, C. A., Cambra, J. M., Nohalez, A., Parrilla, I., Roca, J., Vazquez, J. L., Rodriguez-Martinez, H., Gil, M. A., Martinez, E. A., and Cuello, C. (2019). Prevention of hatching of porcine morulae and blastocysts by liquid storage at 20 degrees C. Sci. Rep. 9, 6219.
| Prevention of hatching of porcine morulae and blastocysts by liquid storage at 20 degrees C.Crossref | GoogleScholarGoogle Scholar | 30996298PubMed |
Moussa, M., Duchamp, G., Mahla, R., Bruyas, J. F., and Daels, P. F. (2003). In vitro and in vivo comparison of Ham’s F-10, Emcare holding solution and ViGro holding plus for the cooled storage of equine embryos. Theriogenology 59, 1615–1625.
| In vitro and in vivo comparison of Ham’s F-10, Emcare holding solution and ViGro holding plus for the cooled storage of equine embryos.Crossref | GoogleScholarGoogle Scholar | 12559466PubMed |
Pashen, R. L. (1987). Short-term storage and survival of horse embryos after refrigeration at 4°C. J. Reprod. Fertil. 32, 697–698.
Pietrani, M., Losinno, L., and Cuervo Arango, J. (2019). Effect of the interval from prostaglandin F2alpha treatment to ovulation on reproductive efficiency rates in a commercial equine embryo transfer program. J. Equine Vet. Sci. 78, 123–126.
| Effect of the interval from prostaglandin F2alpha treatment to ovulation on reproductive efficiency rates in a commercial equine embryo transfer program.Crossref | GoogleScholarGoogle Scholar | 31203975PubMed |
Pomar, F. J., Ducro-Steverink, D. W., Hazeleger, W., Teerds, K. J., Colenbrander, B., and Bevers, M. M. (2004). Development, DNA fragmentation and cell death in porcine embryos after 24 h storage under different conditions. Theriogenology 61, 147–158.
| Development, DNA fragmentation and cell death in porcine embryos after 24 h storage under different conditions.Crossref | GoogleScholarGoogle Scholar | 14643869PubMed |
Riera, F. L. (2009). Equine embryo transfer. In ‘Equine Breeding Management and Artificial Insemination’. (Ed Juan C. Samper.) pp. 185–199. (Saunders : Philadelphia, PA USA.)
Seidel, G. E., Cullingford, E. L., Stokes, J. E., Carnevale, E. M., and McCue, P. M. (2010). Pregnancy rates following transfer of biopsied and/or vitrified equine embryos: evaluation of two biopsy techniques. Anim. Reprod. Sci. 121S, 297–298.
Squires, E. L., McCue, P. M., and Vanderwall, D. (1999). The current status of equine embryo transfer. Theriogenology 51, 91–104.
| The current status of equine embryo transfer.Crossref | GoogleScholarGoogle Scholar | 10729065PubMed |
Stout, T. A., Meadows, S., and Allen, W. R. (2005). Stage-specific formation of the equine blastocyst capsule is instrumental to hatching and to embryonic survival in vivo. Anim. Reprod. Sci. 87, 269–281.
| Stage-specific formation of the equine blastocyst capsule is instrumental to hatching and to embryonic survival in vivo.Crossref | GoogleScholarGoogle Scholar | 15911176PubMed |
Vogelsang, S. G., Bondioli, K. R., and Massey, J. M. (1985). Commercial application of equine embryo transfer. Equine Vet. J. 17, 89–91.
Wilkes, R. P., Tsai, Y. L., Lee, P. Y., Lee, F. C., Chang, H. F., and Wang, H. T. (2014). Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction. BMC Vet. Res. 10, 213.
| Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.Crossref | GoogleScholarGoogle Scholar | 25200113PubMed |
Wilsher, S., and Allen, W. R. (2004). An improved method for nonsurgical embryo transfer in the mare. Equine Vet. Educ. 16, 39–44.
| An improved method for nonsurgical embryo transfer in the mare.Crossref | GoogleScholarGoogle Scholar |
Zoheir, K. M., and Allam, A. A. (2011). A rapid improved method for sexing embryo of water buffalo. Theriogenology 76, 83–87.
| A rapid improved method for sexing embryo of water buffalo.Crossref | GoogleScholarGoogle Scholar | 21396688PubMed |
