Sexing of Dog Sperm by Fluorescence In Situ Hybridization

Effective preselection of sex has been accomplished in several species of livestock andalso in humans using the flow cytometric sperm sorting method. A guaranteed high sortingaccuracy is a key prerequisite for the widespread use of sperm sexing. The standardvalidation 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 spermseparation, it is not truly independent. Therefore, to be able to specifically produceeither male or female offspring in the dog, we developed a method of direct visualizationof sex chromosomes in a single sperm using fluorescence in situhybridization (FISH) as a validation method. Denaturation of canine spermatozoa byimmersion 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 nosignificant 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 meanpurities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% forthe X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting wasevaluated by using the dual color FISH protocol. Therefore, our results demonstrated thatthe FISH protocol worked reliably for both unsorted and sexed sperm samples.