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Sex determination of buffalo embryos (Bubalus bubalis) by polymerase chain reaction

Published online by Cambridge University Press:  14 February 2003

Laura Manna
Affiliation:
Dipartimento di Scienze Cliniche Veterinaria, Facoltà de Medicina Veterinaria, Università di Napoli ‘Federico II’, Naples, Italy
Gianluca Neglia
Affiliation:
DISCIZIA, Facoltà di Medicina Veterinaria, Università di Napoli ‘Federico II’, Naples, Italy
Marcella Marino
Affiliation:
DISCIZIA, Facoltà di Medicina Veterinaria, Università di Napoli ‘Federico II’, Naples, Italy
Bianca Gasparrini
Affiliation:
DISCIZIA, Facoltà di Medicina Veterinaria, Università di Napoli ‘Federico II’, Naples, Italy
Rossella Di Palo
Affiliation:
DISCIZIA, Facoltà di Medicina Veterinaria, Università di Napoli ‘Federico II’, Naples, Italy
Luigi Zicarelli
Affiliation:
DISCIZIA, Facoltà di Medicina Veterinaria, Università di Napoli ‘Federico II’, Naples, Italy

Abstract

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at −20 °C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).

Type
Research Article
Copyright
2003 Cambridge University Press

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