|Kawarasaki, Tatsuo - SHIZUOKA-KEN, JAPAN|
|Yoshida, Mitsutoshi - SHIZUOKA UNIV, JAPAN|
Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 15, 1998
Publication Date: N/A
Interpretive Summary: Verification of X and Y sorted sperm populations in the laboratory is important to the efficiency of the Beltsvile Sperm Sexing Technology. Although the reanalysis of sorted sperm for DNA has been the basis of validating the technology, it has the drawback of being done on the same instrumentation as the actual sorting was done. This study was designed to odetermine if fluorescence in situ hybridization (FISH) was as effective as DNA reanalysis of flow cytometrically sorted X and Y populations. The results demonstrated that there is no difference between the two methods (FISH and reanalysis) for determining the proportions of X and Y sperm in sorted populations. Each was equally effective, however the reanalysis method was much quicker, in that a determination could be made and results known within 30 minutes after a sort. The FISH method on the other hand took several hours (frequently overnight) to perform and required the counting of the fluorescent chromosome signals under a microscope and thus is much more labor intensive. Scientists will use this comparison to choose the most effective and efficient method for determining the proportions of X and Y sperm within a sorted sperm sample. These results represent yet another validation of the Beltsville Sperm Sexing Technology for sorting sperm of mammalian species.
Technical Abstract: Laboratory verification of flow cytometrically sorted X and Y sperm populations is essential to an effective sperm sexing technology. In this study we have demonstrated that fluorescence in situ hybridization (FISH) is equally as effective as the reanalysis of separate X and Y populations for DNA by flow cytometry. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing Technology. Porcine chromosome - and -1 DNA specific probes were applied to the sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The respective purities of the sorted X bearing sperm was 87.4% for FISH and 87.0% for flow cytometric reanalysis; respective purities for the sorted Y bearing sperm was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm and 4,256 Y sperm were examined by FISH across the 8 ejaculates. For rflow cytometry, 5,000 sorted sperm from X and 5,000 sperm from Y were reanalyzed for DNA content for each ejaculate. These results confirm the validity of the reanalysis for DNA for determining the proportions of X and Y sorted sperm and demonstrate the effectiveness of a rapid FISH procedure using porcine chromosome -Y and -1 DNA specific probe to evaluate the sperm separation from flow cytometric cell sorting. This FISH method may also be useful in studying chromosome abnormalities in sperm such as disomy and aneuploidy.