Sexing of Dog Sperm by Fluorescence In Situ
Hybridization |
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| Authors: | Maya OI Keisuke YAMADA Hiroyuki HAYAKAWA Hiroshi SUZUKI |
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| Institution: | 1)Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan;2)Genetics Hokkaido Association, Hokkaido 089-0103, Japan;3)The United Gradate School of Veterinary Sciences, Gifu University, Gifu 501-1193, Japan |
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| Abstract: | Effective preselection of sex has been accomplished in several species of livestock and
also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting
accuracy is a key prerequisite for the widespread use of sperm sexing. The standard
validation method is flow cytometric remeasurement of the DNA content of the sexed sperm.
Since this method relies on the same instrument that produced the original sperm
separation, it is not truly independent. Therefore, to be able to specifically produce
either male or female offspring in the dog, we developed a method of direct visualization
of sex chromosomes in a single sperm using fluorescence in situ
hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by
immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97%
hybridization efficiency and a good preservation of sperm morphology. There was no
significant difference between the theoretical ratio (50:50) and the observed ratio of X-
and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean
purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for
the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was
evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that
the FISH protocol worked reliably for both unsorted and sexed sperm samples. |
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| Keywords: | Dog Fluorescence in situ hybridization (FISH) Sexing Sperm |
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