Sex ratio determination in bovine semen: a new approach by quantitative real time PCR.
Sex preselection of livestock offspring in cattle represents, nowadays, a big potential for genetic improvement and market demand satisfaction. Sperm sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos but, an accurate verification of the yield of sperm separation remains essential for a field application of this technique or for improvement and validation of other related semen sexing technologies. In this work a new method for the determination of the proportion of X- and Y-bearing spermatozoa in bovine semen sample was developed by real time PCR. Two sets of primers and internal TaqMan probes were designed on specific X- and Y-chromosome genes. To allow a direct quantification, a standard reference was established using two plasmid cDNA clones (ratio 1:1) for the specific gene targets. The method was validated by a series of accuracy, repeatability and reproducibility assays and by testing two sets of sorted and unsorted semen samples. A high degree of accuracy (98.9%), repeatability (CV=2.58%) and reproducibility (CV=2.57%) was shown. The results of X- and Y-sorted semen samples analysed by real time PCR and by flow cytometric reanalysis showed no significant difference (P>0.05). The evaluation of X-chromosome bearing sperms content in unsorted samples showed an average of 51.11+/-0.56% for ejaculates and 50.17+/-0.58% for the commercial semen. This new method for quantification of the sexual chromosome content in spermatozoa demonstrated to be rapid and reliable, providing a valid support to the sperm sexing technologies.
Parati
K
K
Istituto Sperimentale Italiano Lazzaro Spallanzani, V.le Forlanini 23, 20134 Milan, Italy. isils@tin.it
Bongioni
G
G
Aleandri
R
R
Galli
A
A
D000818:Animals; D002417:Cattle / Q000235:genetics*; D002469:Cell Separation / Q000662:veterinary; D005260:Female; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000662:veterinary; D019849:Sex Determination Processes; D012743:Sex Preselection / Q000379:methods / Q000662:veterinary*; D012744:Sex Ratio; D013094:Spermatozoa / Q000166:cytology* / Q000502:physiology; D014960:X Chromosome; D014998:Y Chromosome