VERIFICATION OF FLOW CYTOMETRICALLY-SORTED X-BEARING AND Y-BEARING PORCINE SPERMATOZOA AND REANALYSIS OF SPERMATOZOA FOR DNA CONTENT USING THE FLUORESCENCE IN-SITU HYBRIDIZATION (FISH) TECHNIQUE

Flow cytometric sperm sorting based on X and Y sperm DNA difference ha s been established as the only effective method for sexing the spermat ozoa of mammals. The standard method for verifying the purity of sorte d X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and acc uracy of DNA reanalysis by fluorescence in situ hybridization (FISH) u sing chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromo some Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm sa mples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flo w cytometric reanalysis; purity for the sorted Y-bearing spermatozoa w as 85.9% for FISH arid 84.8% for flow cytometric reanalysis. A total o f 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH acr oss the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa a nd 5,000 Y spermatozoa were reanalyzed for DNA content for each ejacul ate. These results confirm the high purity of flow sorted porcine X an d Y sperm cells and the validity of reanalysis of DNA in determining t he proportions of X- and Y-sorted spermatozoa from viewing thousands o f individual sperm chromosomes directly using FISH. (C) 1998 by Elsevi er Science Inc.