Abstract: Objectives To investigate the application of flow cytometric technology in separating X or Y sperm from Labrador retriever, and to define a protocol for producing sex-sorted frozen semen. Methods The semen were collected from 8 Labrador retrievers by manual stimulation and transported in one hour, and then the semen were separated into X- and Y- chromosome by SX_MOFLO flow cytometric technology, a modified high-speed cell sorter, and finally frozen thawed. Sorted X- or Y-sperm and unsorted sperm as control were reanalyzed and quality assessed. Results The purities of flow sorted sex chromosomes in spermatozoa of three dogs ranged from 89% to 91%. The average separation velocity showed significantly differences between sorted X- sperm (4 260/s) and Y- sperm (3 930/s). The sex ratio showed significantly differences between frozen- thawed sorted X- or Y- chromosome bearing sperm (89.11%, 90.67%) and the control. The forward progressive motility was not significantly different between sorted sperm and the control. The survive index on sorted sperm was significantly lower (0.251±0.006\0.054±0.007, P<0.05) than the control (0.048±0.006). But the post thaw acrosome integrity on sorted sperm was significantly higher (84.66±1.22\84.01±1.23, P<0.05) than the control (77.00±0.98). Conclusion It was possible to obtain sorted frozen- thawed Labrador retriever sperm for AI by using flow cytometry techniques.

Key words: Labrador retriever, XY spermatozoa, Flow cytometry, Semen quality