title: Bovine embryo sex determination by multiplex loop-mediated isothermal amplification.
abstract: In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing.
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand; Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand.
authors.forename: Trisadee
authors.initials: T
authors.lastname: Khamlor
authors.affiliation: Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand.
authors.forename: Petai
authors.initials: P
authors.lastname: Pongpiachan
authors.affiliation: Embryo Technology and Stem Cell Research Center and School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
authors.forename: Rangsun
authors.initials: R
authors.lastname: Parnpai
authors.affiliation: Embryo Technology and Stem Cell Research Center and School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
authors.forename: Kanchana
authors.initials: K
authors.lastname: Punyawai
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand.
authors.forename: Siwat
authors.initials: S
authors.lastname: Sangsritavong
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand. Electronic address: nipa.cho@biotec.or.th.
authors.forename: Nipa
authors.initials: N
authors.lastname: Chokesajjawatee
mesh_terms: D000818:Animals; D002417:Cattle / Q000196:embryology* / Q000502:physiology; D004622:Embryo, Mammalian / Q000378:metabolism*; D005260:Female; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000662:veterinary*; D011094:Polyethyleneimine; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000662:veterinary*
chemical_list: D011094:Polyethyleneimine
keywords: Bovine; Embryo sexing; Multiplex LAMP; Sex determination