Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Sex determination of porcine embryos using a new developed duplex polymerase chain reaction procedure based on the amplification of repetitive sequences

Eva Torner A C , Eva Bussalleu A , M. Dolors Briz A , Alfonso Gutiérrez-Adán B and Sergi Bonet A
+ Author Affiliations
- Author Affiliations

A Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, Campus Montilivi, s/n, 17071 Girona, Spain.

B Department of Animal Reproduction, INIA, Carretera De la Coruña Km 5.9, 28040 Madrid, Spain.

C Corresponding author. Email: eva.torner@udg.edu

Reproduction, Fertility and Development 25(2) 417-425 https://doi.org/10.1071/RD12033
Submitted: 11 February 2012  Accepted: 31 March 2012   Published: 14 May 2012

Abstract

Polymerase chain reaction (PCR)-based assays have become increasingly prevalent for sexing embryos. The aim of the present study was to develop a suitable duplex PCR procedure based on the amplification of porcine repetitive sequences for sexing porcine tissues, embryos and single cells. Primers were designed targeting the X12696 Y chromosome-specific repeat sequence (SUSYa and SUSYb; sex-related primer sets), the multicopy porcine-specific mitochondrial 12S rRNA gene (SUS12S; control primer set) and the X51555 1 chromosome repeat sequence (SUS1; control primer set). The specificity of the primer sets was established and the technique was optimised by testing combinations of two specific primer sets (SUSYa/SUS12S; SUSYb/SUS12S), different primer concentrations, two sources of DNA polymerase, different melting temperatures and different numbers of amplification cycles using genomic DNA from porcine ovarian and testicular tissue. The optimised SUSYa/SUS12S- and SUSYb/SUS12S-based duplex PCR procedures were applied to porcine in vitro-produced (IVP) blastocysts, cell-stage embryos and oocytes. The SUSYb/SUS12S primer-based procedure successfully sexed porcine single cells and IVP cell-stage embryos (100% efficiency), as well as blastocysts (96.6% accuracy; 96.7% efficiency). This is the first report to demonstrate the applicability of these repetitive sequences for this purpose. In conclusion, the SUSYb/SUS12S primer-based duplex PCR procedure is highly reliable and sensitive for sexing porcine IVP embryos.

Additional keywords: in vitro-produced embryos, IVF, pig


References

Akane, A., Seki, S., Shiono, H., Nakamura, H., Hasegawa, M., Kagawa, M., Matsubara, K., Nakahori, Y., Nagafuchi, S., and Nakagome, Y. (1992). Sex determination of forensic samples by dual PCR amplification of an X-Y homologous gene. Forensic Sci. Int. 52, 143–148.
Sex determination of forensic samples by dual PCR amplification of an X-Y homologous gene.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK38XhvV2iu7g%3D&md5=81870ff9555f18ab295f5d45e845c764CAS | 1601346PubMed |

Alves, B. C., Hossepian de Lima, V. F., and Moreira-Filho, C. A. (2010). Development of Y-chromosome-specific SCAR markers conserved in taurine, zebu and bubaline cattle. Reprod. Domest. Anim. 45, 1047–1051.
Development of Y-chromosome-specific SCAR markers conserved in taurine, zebu and bubaline cattle.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhs1WrsLjJ&md5=d2276ae6f2eb96edd42c5762b11e20d7CAS | 19602181PubMed |

Appa Rao, K. B., and Totey, S. M. (1999). Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in-vitro developed buffalo embryos using multiplex and nested polymerase chain reaction. Theriogenology 51, 785–797.
Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in-vitro developed buffalo embryos using multiplex and nested polymerase chain reaction.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXjsFegtbc%3D&md5=dae652931a9619c15c8dc9ba2a4c9d0aCAS | 10729003PubMed |

Appa Rao, K. B., Pawshe, C. H., and Totey, S. M. (1993). Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification. Mol. Reprod. Dev. 36, 291–296.
Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2cXlslWms70%3D&md5=62e6419d5c51bee2605f651a3e1b2605CAS |

Bermejo-Álvarez, P., Rizos, D., Rath, D., Lonergan, P., and Gutiérrez-Adán, A. (2008). Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially? Biol. Reprod. 79, 594–597.
Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially?Crossref | GoogleScholarGoogle Scholar | 18579751PubMed |

Bermejo-Álvarez, P., Lonergan, P., Rath, D., Gutiérrez-Adán, A., and Rizos, D. (2010). Developmental kinetics and gene expression in male and female bovine embryos produced in vitro with sex-sorted spermatozoa. Reprod. Fertil. Dev. 22, 426–436.
Developmental kinetics and gene expression in male and female bovine embryos produced in vitro with sex-sorted spermatozoa.Crossref | GoogleScholarGoogle Scholar | 20047728PubMed |

Bodmer, M., Janett, F., Hässig, M., Daas, N., Reichert, P., and Thun, R. (2005). Fertility in heifers and cows after low dose insemination with sex-sorted and non-sorted sperm under field conditions. Theriogenology 64, 1647–1655.
Fertility in heifers and cows after low dose insemination with sex-sorted and non-sorted sperm under field conditions.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD2MrhtFOktA%3D%3D&md5=c99aad2cf37630599b5e27d56b219e3eCAS | 15904953PubMed |

Bredbacka, P. (2001). Progress on methods of gene detection in preimplantation embryos. Theriogenology 55, 23–34.
Progress on methods of gene detection in preimplantation embryos.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXoslOrsg%3D%3D&md5=1f827f0e105929c89367f98a15e68930CAS | 11198085PubMed |

Carneiro, M. C., Takeuchi, P. L., Araújo, A., Lôbo, R. B., Elias, F. P., Vila, R. A., Miranda-Furtado, C. L., and Ramos, E. S. (2011). Sexing single bovine blastomeres using TSPY gene amplification. Genet. Mol. Res. 10, 3937–3941.
Sexing single bovine blastomeres using TSPY gene amplification.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC38zmvV2nsA%3D%3D&md5=e5219359064c8b10e60bd4437822a23aCAS | 22033907PubMed |

Casas, I., Sancho, S., Briz, M., Pinart, E., Bussalleu, E., Yeste, M., and Bonet, S. (2009). Freezability prediction of boar ejaculates assessed by functional sperm parameters and sperm proteins. Theriogenology 72, 930–948.
Freezability prediction of boar ejaculates assessed by functional sperm parameters and sperm proteins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFGlsr3E&md5=cb921d94176a753cb4a0458fc6565cd2CAS | 19651432PubMed |

Chong, S. S., Kristjansson, K., Cota, J., Handyside, A. H., and Hughes, M. R. (1993). Preimplantation prevention of X-linked disease: reliable and rapid sex determination of single human cells by restriction analysis of simultaneously amplified ZFX and ZFY sequences. Hum. Mol. Genet. 2, 1187–1191.
Preimplantation prevention of X-linked disease: reliable and rapid sex determination of single human cells by restriction analysis of simultaneously amplified ZFX and ZFY sequences.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXlvFOisrs%3D&md5=4c70453ceb0ae6ee8c76357978b4a526CAS | 8401500PubMed |

Chowdhary, B. P., and Raudsepp, T. (2006). The horse genome. Genome Dyn. 2, 97–110.
The horse genome.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtVCns7zL&md5=e369419b5e25da64b82716647597ba33CAS | 18753773PubMed |

Fontanesi, L., Scotti, E., and Russo, V. (2008). Differences of the porcine amelogenin X and Y chromosome genes (AMELX and AMELY) and their application for sex determination in pigs. Mol. Reprod. Dev. 75, 1662–1668.
Differences of the porcine amelogenin X and Y chromosome genes (AMELX and AMELY) and their application for sex determination in pigs.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXht1agurbE&md5=e0272a0a876624059c04777df98f2dbeCAS | 18384076PubMed |

Frijters, A. C. J., Mullaart, E., Roelofs, R. M. G., van Hoorne, R. P., Moreno, J. F., Moreno, O., and Merton, J. S. (2009). What affects fertility of sexed bull semen more, low sperm dosage or the sorting process? Theriogenology 71, 64–67.
What affects fertility of sexed bull semen more, low sperm dosage or the sorting process?Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1cjnt1Gmug%3D%3D&md5=a5ff7622275f1ae7685753ac7c29a25cCAS |

Fu, Q., Zhang, M., Qin, W. S., Lu, Y. Q., Zheng, H. Y., Meng, B., Lu, S. S., and Lu, K. H. (2007). Cloning the swamp buffalo SRY gene for embryo sexing with multiplex-nested PCR. Theriogenology 68, 1211–1218.
Cloning the swamp buffalo SRY gene for embryo sexing with multiplex-nested PCR.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXht1Krs7rN&md5=ceea346d8e4589f751a5cc1614e84b05CAS | 17928043PubMed |

Garner, D. L., and Seidel, G. E. (2008). History of commercializing sexed semen for cattle. Theriogenology 69, 886–895.
History of commercializing sexed semen for cattle.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD1c3hsFertw%3D%3D&md5=bf91d0d083060187ac3437ca78901868CAS | 18343491PubMed |

Girish, P. S., Anjaneyulu, A. S., Viswas, K. N., Shivakumar, B. M., Anand, M., Patel, M., and Sharma, B. (2005). Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene. Meat Sci. 70, 107–112.
Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhvVCrtbo%3D&md5=27c2293b21126cc1273125087e04ab31CAS | 22063286PubMed |

Greenlee, A. R., Krisher, R. L., and Plotka, E. D. (1998). Rapid sexing of murine preimplantation embryos using a nested, multiplex polymerase chain reaction (PCR). Mol. Reprod. Dev. 49, 261–267.
Rapid sexing of murine preimplantation embryos using a nested, multiplex polymerase chain reaction (PCR).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXosVOqug%3D%3D&md5=86ac03504248a98a8697ef88eb12fc7dCAS | 9491378PubMed |

Handyside, A. H., Pattinson, J. K., Penketh, R. J., Delhanty, J. D., Winston, R. M., and Tuddenham, E. G. (1989). Biopsy of human preimplantation embryos and sexing by DNA amplification. Lancet 333, 347–349.
Biopsy of human preimplantation embryos and sexing by DNA amplification.Crossref | GoogleScholarGoogle Scholar |

Hirayama, H., Kageyama, S., Takahashi, Y., Moriyasu, S., Sawai, K., Onoe, S., Watanabe, K., Kojiya, S., Notomi, T., and Minamihashi, A. (2006). Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification. Theriogenology 66, 1249–1256.
Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xotleqsbg%3D&md5=56d21363f9785234413c323ab0f3a912CAS | 16672158PubMed |

Jantsch, M., Hamilton, B., Mayr, B., and Schweizer, D. (1990). Meiotic chromosme behaviour reflects levels of sequence divergence in Sus scrofa domestica satellite DNA. Chromosoma 95, 330–335.

Johnson, L. A. (1997). Advances in gender preselection in swine. J. Reprod. Fertil. Suppl. 52, 255–266.
| 1:STN:280:DyaK1c3msFCntg%3D%3D&md5=0c83205f8d7849e1a0b7a85d110a13a5CAS | 9602734PubMed |

Johnson, L. A., and Clarke, R. N. (1988). Flow sorting of X and Y chromosome-bearing mammalian sperm: activation and pronuclear development of sorted bull, boar, and ram sperm microinjected into hamster oocytes. Gamete Res. 21, 335–343.
Flow sorting of X and Y chromosome-bearing mammalian sperm: activation and pronuclear development of sorted bull, boar, and ram sperm microinjected into hamster oocytes.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaL1M7itFOqtA%3D%3D&md5=9ebf1a8c52ee6b029adb462c52b60abeCAS | 3220427PubMed |

Johnson, L. A., Rath, D., Vazquez, J. M., Maxwell, W. M. C., and Dobrinsky, J. R. (2005). Preselection of sex of offspring in swine for production: current status of the process and its application. Theriogenology 63, 615–624.
Preselection of sex of offspring in swine for production: current status of the process and its application.Crossref | GoogleScholarGoogle Scholar | 15626420PubMed |

Leoni, G., Ledda, S., Bogliolo, L., and Naitana, S. (2000). Novel approach to cell sampling from preimplantation ovine embryos and its potential use in embryonic genome analysis. J. Reprod. Fertil. 119, 309–314.
| 1:CAS:528:DC%2BD3cXltlyltrk%3D&md5=a9de1d280232a311b13a7d6ab2cf4007CAS | 10864843PubMed |

Manna, L., Neglia, G., Marino, M., Gasparrini, B., Di Palo, R., and Zicarelli, L. (2003). Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction. Zygote 11, 17–22.
Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXht1WgsrY%3D&md5=2f019e2141344a9dda07aa08c947585bCAS | 12625525PubMed |

Mara, L., Pilichi, S., Sanna, A., Accardo, C., Chessa, B., Chessa, F., Dattena, M., Bomboi, G., and Cappai, P. (2004). Sexing of in vitro produced ovine embryos by duplex PCR. Mol. Reprod. Dev. 69, 35–42.
Sexing of in vitro produced ovine embryos by duplex PCR.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXms1GitLY%3D&md5=e4a6d3881c95d0c3ab68cfefa9b85647CAS | 15278902PubMed |

Martín, I., García, T., Fajardo, V., Rojas, M., Pegels, N., Hernández, P. E., and Martín, I. G. (2009). SYBR-green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs. Meat Sci. 82, 252–259.
SYBR-green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs.Crossref | GoogleScholarGoogle Scholar | 20416744PubMed |

Martínez, E. A., Vazquez, J. M., Roca, J., Cuello, C., Gil, M. A., Parrilla, I., and Vazquez, J. L. (2005). An update on reproductive technologies with potential short-term application in pig production. Reprod. Domest. Anim. 40, 300–309.
An update on reproductive technologies with potential short-term application in pig production.Crossref | GoogleScholarGoogle Scholar | 16008760PubMed |

McGraw, R. A., Jacobson, R. J., and Akamatsu, M. (1988). A male-specific repeated DNA sequence in the domestic pig. Nucleic Acids Res. 16, 10389.
A male-specific repeated DNA sequence in the domestic pig.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL1MXitVyr&md5=c3b0fac8e4db43ae041a9319d32cdfb7CAS | 3194222PubMed |

Naqvi, A. N. (2007). Application of molecular genetic technologies in livestock production: potentials for developing countries. Adv. Biol. Res. 1, 72–84.

Niemann, H., Rath, D., and Wrenzycki, C. (2003). Advances in biotechnology: new tools in future pig production for agriculture and biomedicine. Reprod. Domest. Anim. 38, 82–89.
Advances in biotechnology: new tools in future pig production for agriculture and biomedicine.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3s7ktVentA%3D%3D&md5=165497b904575c2495350d88110ef291CAS | 12654017PubMed |

Paria, N., Raudsepp, T., Pearks Wilkerson, A. J., O’Brien, P. C., Ferguson-Smith, M. A., Love, C. C., Arnold, C., Rakestraw, P., Murphy, W. J., and Chowdhary, B. P. (2011). A gene catalogue of the euchromatic male-specific region of the horse Y chromosome: comparison with human and other mammals. PLoS One 6, e21374.
A gene catalogue of the euchromatic male-specific region of the horse Y chromosome: comparison with human and other mammals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtVKgtLvP&md5=4a17893b84d7bbc2b516c3915d009fcfCAS | 21799735PubMed |

Park, J. H., Lee, J. H., Choi, K. M., Joung, S. Y., Kim, J. Y., Chung, G. M., Jin, D. I., and Im, K. S. (2001). Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere. Theriogenology 55, 1843–1853.
Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXkvFarsbo%3D&md5=8f152981ca0406a81b9bcee7eeb81697CAS | 11414489PubMed |

Petters, R. M., and Wells, K. D. (1993). Culture of pig embryos. J. Reprod. Fertil. Suppl. 48, 61–73.
| 1:STN:280:DyaK2c7psVCktQ%3D%3D&md5=36d4abfea214f4649cde59e3386610e8CAS | 8145215PubMed |

Pierce, K. E., Rice, J. E., Sanchez, J. A., Brenner, C., and Wangh, L. J. (2000). Real-time PCR using molecular beacons for accurate detection of the Y chromosome in single human blastomeres. Mol. Hum. Reprod. 6, 1155–1164.
Real-time PCR using molecular beacons for accurate detection of the Y chromosome in single human blastomeres.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXjsFKntQ%3D%3D&md5=549d391fc997360e011efd91001370cbCAS | 11101699PubMed |

Pomp, D., Good, B. A., Geisert, R. D., Corbin, C. J., and Conley, A. J. (1995). Sex identification in mammals with polymerase chain reaction and its use to examine sex effects on diameter of day-10 or -11 pig embryos. J. Anim. Sci. 73, 1408–1415.
| 1:CAS:528:DyaK2MXlsFSjur4%3D&md5=cfcc629f52cebd3ab087c9f0cad87cccCAS | 7665371PubMed |

Prakash, S., Patole, M. S., Ghumatkar, S. V., Nandode, S. K., Shinde, B. M., and Shouche, Y. S. (2000). Mitochondrial 12S rRNA sequence analysis in wildlife forensics. Curr. Sci. 78, 1239–1241.
| 1:CAS:528:DC%2BD3cXkt1Ckurk%3D&md5=d4bd386a9d1250eda6243647c4bd203bCAS |

Pursel, V. G., and Johnson, L. A. (1975). Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and a new thawing. J. Anim. Sci. 40, 99–102.
| 1:STN:280:DyaE2M%2FnslCjtw%3D%3D&md5=4f504cde4bb8c000eaf1bf861438ac4dCAS | 1110222PubMed |

Rath, D., Long, C. R., Dobrinsky, J. R., Welch, G. R., Schreier, L. L., and Johnson, L. A. (1999). In vitro production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-bearing sperm to produce pigs after embryo transfer. J. Anim. Sci. 77, 3346–3352.
| 1:CAS:528:DC%2BD3cXktFegtQ%3D%3D&md5=0dbd1a7eda107a0e72d078785ba2be24CAS | 10641883PubMed |

Rattanasuk, S., Parnpai, R., and Ketudat-Cairns, M. (2011). Multiplex polymerase chain reaction used for bovine embryo sex determination. J. Reprod. Dev. 57, 539–542.
Multiplex polymerase chain reaction used for bovine embryo sex determination.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXht1GksLnF&md5=296a50c8b3dcac9775b38b35fbfc4047CAS | 21532257PubMed |

Rozen, S., and Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386.
| 1:CAS:528:DyaK1MXmslKqsbo%3D&md5=f49f8151abf9cb9f0cf1067a703936c9CAS | 10547847PubMed |

Sathasivam, K., Kageyama, S., Chikuni, K., and Notarianni, E. (1995). Sex determination in the domestic pig by DNA amplification using the HMG-box sequence. Anim. Reprod. Sci. 38, 321–326.
Sex determination in the domestic pig by DNA amplification using the HMG-box sequence.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXms1ShtLk%3D&md5=4e58c4d06012762b25ebab14b1dc9a8dCAS |

Seidel, G. E. (2010). Brief introduction to whole-genome selection in cattle using single nucleotide polymorphisms. Reprod. Fertil. Dev. 22, 138–144.
Brief introduction to whole-genome selection in cattle using single nucleotide polymorphisms.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXitlagu7g%3D&md5=6bf72311c6b81f984e2dd34236654715CAS | 20003856PubMed |

Sembon, S., Suzuki, S., Fuchimoto, D., Iwamoto, M., Kawarasaki, T., and Onishi, A. (2008). Sex identification of pigs using polymerase chain reaction amplification of the amelogenin gene. Zygote 16, 327–332.
Sex identification of pigs using polymerase chain reaction amplification of the amelogenin gene.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtlKmt7rP&md5=b8a1b0dec5ed92e101ca1512c541d112CAS | 18616845PubMed |

Shea, B. F. (1999). Determining the sex of bovine embryos using polymerase chain reaction results: a six-year retrospective study. Theriogenology 51, 841–854.
Determining the sex of bovine embryos using polymerase chain reaction results: a six-year retrospective study.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3c7ptFemsw%3D%3D&md5=c4ed839ff483a8f957e9aeb2c48bda63CAS | 10729007PubMed |

Tsuchiya, S., Sueoka, K., Matsuda, N., Tanigaki, R., Asada, H., Hashiba, T., Kato, S., and Yoshimura, Y. (2005). The ‘spanning protocol’: a new DNA extraction method for efficient single-cell genetic diagnosis. J. Assist. Reprod. Genet. 22, 407–414.
The ‘spanning protocol’: a new DNA extraction method for efficient single-cell genetic diagnosis.Crossref | GoogleScholarGoogle Scholar | 16331538PubMed |

van Vliet, R. A., Verrinder Gibbins, A. M., and Walton, J. S. (1989). Livestock embryo sexing: a review of current methods, with emphasis on Y-specific DNA probes. Theriogenology 32, 421–438.
Livestock embryo sexing: a review of current methods, with emphasis on Y-specific DNA probes.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD283pvFCmsw%3D%3D&md5=c83820a5e150ee9078053de2dd1a00eeCAS | 16726688PubMed |

Wan, Q. H., Qian, K. X., and Fang, S. G. (2003). A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos taurus. Anim. Reprod. Sci. 77, 1–9.
A simple DNA extraction and rapid specific identification technique for single cells and early embryos of two breeds of Bos taurus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXitlGiu7s%3D&md5=ac8d1246b97f70e90199096e4ff1c677CAS | 12654523PubMed |

Wang, Q., Zhang, X., Zhang, H. Y., Zhang, J., Chen, G. Q., Zhao, D. H., Ma, H. P., and Liao, W. J. (2010). Identification of 12 animal species meat by T-RFLP on the 12S rRNA gene. Meat Sci. 85, 265–269.
Identification of 12 animal species meat by T-RFLP on the 12S rRNA gene.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXjvFeqtbY%3D&md5=e6d11810d769e11dded1201e8307586eCAS | 20374896PubMed |

Westendorf, P., Richter, L., and Treu, H. (1975). Deep freezing of boar sperma. Laboratory and insemination results using the Hülsenberger paillete method. Dtsch Tierarztl. Wochenschr. 82, 261–267.
| 1:STN:280:DyaE28%2FmsFyntg%3D%3D&md5=1f8b0e6f8ee6b25039c7b3beeca3f0f8CAS | 1104331PubMed |