title: Sex determination of porcine embryos using a new developed duplex polymerase chain reaction procedure based on the amplification of repetitive sequences.
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.
authors: 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. eva.torner@udg.edu
authors: Eva
authors: E
authors: Torner
authors: Eva
authors: E
authors: Bussalleu
authors: M Dolors
authors: MD
authors: Briz
authors: Alfonso
authors: A
authors: Gutiérrez-Adán
authors: Sergi
authors: S
authors: Bonet
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004622:Embryo, Mammalian / Q000737:chemistry*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000379:methods*; D012743:Sex Preselection / Q000379:methods / Q000662:veterinary*; D013552:Swine / Q000235:genetics*
chemical_list: D017931:DNA Primers