Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013;59(1):92-6.
doi: 10.1262/jrd.2012-098. Epub 2012 Oct 11.

Sexing of dog sperm by fluorescence in situ hybridization

Maya Oi  1 , Keisuke Yamada, Hiroyuki Hayakawa, Hiroshi Suzuki
Affiliations

Sexing of dog sperm by fluorescence in situ hybridization

Maya Oi et al. J Reprod Dev. 2013.

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.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Canine spermatozoa decondensed with 1 M NaOH for 4 min. Y chromosomes hybridized with Cy3 labelled probes. Only sperm heads with red a signal were considered to be Y-chromosome-bearing spermatozoa (arrow). Bar=10 µm.
Fig. 2.
Fig. 2.
Detection of X and Y chromosomes on canine spermatozoa by dual color FISH. Sperm heads with green (SpectrumGreen) and red (Cy3) signals were considered to be X- and Y chromosome-bearing spermatozoa, respectively. A sperm head carrying both green and red signals (arrow) was considered to be aneuploid.
See this image and copyright information in PMC

References

    1. Johnson LA, Welch GR. Sex preselection: high-speed flow cytometric sorting of X and Y sperm for maximum efficiency. Theriogenology 1999; 52: 1323–1341 - PubMed
    1. Cran DG, Johnson LA, Polge C. Sex preselection in cattle: a field trial. Vet Rec 1995; 136: 495–496 - PubMed
    1. Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: Live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989; 41: 199–203 - PubMed
    1. Johnson LA. Sex preselection in swine: Altered sex ratios in offspring following surgical insemination of flow sorted X- and Y-bearing sperm. Reprod Dom Anim 1991; 26: 309–314
    1. Cran DG, McKelvey WAC, King ME, Dolman DF, McEvoy TG, Broadbent PJ, Robinson JJ. Production of lambs by low dose intrauterine insemination with flow cytometrically sorted and unsorted semen. Theriogenology 1997; 47: 267