Sexing of Dog Sperm by Fluorescence <i>In Situ</i> Hybridization

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.

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted, frozen–thawed spermatozoa in field conditions

“…The practical use of sexed semen could have a significant effect on canine production, because it allows planned matings for a specific sex of puppy (Garner & Seidel ; Oi et al . ). However, despite the feasibility of the sex‐sorting procedure having been described by several authors (Meyers et al .…”

“…The noticeable deviation of the offspring sex ratio (X 87.5%; Y 86.4%) obtained here is encouraging for the application of sex predetermination using sorted sperm to be practical for obtaining pregnancies with low numbers of frozen-thawed sperm. The practical use of sexed semen could have a significant effect on canine production, because it allows planned matings for a specific sex of puppy (Garner & Seidel 2003;Oi et al 2013). However, despite the feasibility of the sex-sorting procedure having been described by several authors (Meyers et al 2008;Rodenas et al 2014), the technique for AI and the high-dose insemination of the number of sex-sorted sperm preclude the practical application of this procedure at the farm level.…”

“…For dogs, interest in predetermining the sex has been generated from those with pets and has applications for working dogs, such as guide dogs, military and police dogs, guard dogs and tracking dogs (Garner & Seidel ; Oi et al . ). Different traits and temperaments that are sex‐related often underlie the selection of dogs used for various applications by breeders and owners with different demands (Meyers et al .…”

“…The predetermination of sex in livestock offspring is in great demand due to the substantial differences in their sex-related phenotypes, for example the specific production of male calves for beef production and female calves for milk production. For dogs, interest in predetermining the sex has been generated from those with pets and has applications for working dogs, such as guide dogs, military and police dogs, guard dogs and tracking dogs (Garner & Seidel 2003;Oi et al 2013). Different traits and temperaments that are sex-related often underlie the selection of dogs used for various applications by breeders and owners with different demands (Meyers et al 2008).…”

The Effects of Hoechst 33342 Staining and the Male Sample Donor on the Sorting Efficiency of Canine Spermatozoa

“…; Oi et al. ). The application of sex‐sorting technology to the breeding of domestic dogs could be used as a model for the rescue of endangered canids, such as the African wild dog ( Lycaon pictus ) (Johnston et al ) and wolf subspecies.…”