Sex ratio determination in bovine semen: a new approach by quantitative real time PCR.
Theriogenology
; 66(9): 2202-9, 2006 Dec.
Article
em En
| MEDLINE
| ID: mdl-16956656
ABSTRACT
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 11) 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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pré-Seleção do Sexo
/
Espermatozoides
/
Cromossomo X
/
Cromossomo Y
/
Bovinos
/
Reação em Cadeia da Polimerase
/
Processos de Determinação Sexual
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Theriogenology
Ano de publicação:
2006
Tipo de documento:
Article
País de afiliação:
Itália