Sexing of in vitro produced ovine embryos by duplex PCR

The aim of this article was to
develop a fast and easy duplex polymerase chain
reaction (PCR) method, for sex determination of ovine
in vitro produced embryos prior to implantation. We
tested the approach with 107 samples of autosomal
cells (oviductal sheep cells and male lamb fibroblasts),
divided into three groups for each sex according to the
number of cells employed (30, 5, 2, respectively). We
then used the test on 21 embryos at blastocyst stage.
On the same day the embryos were transferred in pairs
into 11 recipient synchronized ewes. The PCR utilized
two different sets of primers: the first pair recognized a
bovine Y-chromosome-specific sequence (SRY), that
showed 100% homology with the corresponding
sequence of the ovine Y-chromosome and is amplified
in males only. The second pair recognized the bovine
1.715 satellite DNA (SAT) which was amplified in all
ovine samples but, when submitted to the GenBank
database did not show homology with any of the reported
ovine sequences. However, after sequencing,
ovine amplification product showed 98% homology
with the bovine specific satellite sequence. The autosomal
samples were amplified with 85.0% efficiency
and 91.2% accuracy, while amplification was successful
with all 21 embryos (100% efficiency). Eight
lambs were born and the sex as determined by PCR
corresponded to the anatomical sex in seven (87.5%
accuracy). These results confirm that this method can
be applied in ovine breeding programs to manipulate
sex ratio of offspring.