Abstract
In this paper, we show a protocol of a refined and useful application of rapid fluorescence in situ hybridization (FISH) for sexing bovine blastocysts. About ten blastomeres per bovine blastocyst are fixed in an equal volume of PBS(-) and fixative freshly made of acetic acid and methanol (1:3, v/v), and mounted onto a clean glass slide, on which the fixative is dropped. The PCR primer pair is designed from a bovine male- specific DNA sequence termed as BC1.2. The following sequences of the oligonucleotide primers are used: upstream 5'-ATCAGTGCAGGGACCGAGATG-3' and downstream 5'AAGCAGCCGATAAACACTCCTT-3'. A Dig-labeled probe produced by PCR is added to the hybridization mixture (as a final concentration, 50% formamide, 10% dextran sulfate, 30 ng/μl BSA and 2 × SSC, pH 7.0). The hybridization mixture adjusted is dropped on each glass slide, and denatured at 72°C for 8 min on an aluminum block. Immediately after denaturation, they are hybridized at 38.5°C for 5 min, washed in 0.5 × SSPE at 72°C for 5 min and followed by washing in PN buffer (0.1% sodium phosphate, pH 8.0, 0.1% NP-40) for 2 min. The Dig is detected by incubation with anti-Dig-fluorescein (1.0 μg/ml) in PN buffer plus 1% Block Ace at 38.5°C for 5 min. After washing with PN buffer, the blasomeres are counterstained with propidium iodide (0.3 μg/ml) in an anti-fade solution. The observation is carried out under an epifluorescence microscope. The whole process of FISH can be carried out within almost 1 hr. This protocol with a male-specific DNA probe is a powerful tool for sexing bovine embryos.
