Toll-like receptors 7 and 8 can be targeted to enrich Y-chromosome sperm in the domestic dog.

Abstract: An effective method of sperm sex separation for use in the domestic dog is desirable for the breeding of working dogs and for the study of sex-linked diseases in research models. Previous reports identified the X-linked Toll-like receptors 7 and 8 (TLR7/8) as useful targets for immunological sperm separation in bulls, mice and goats using the TLR7/8 ligand resiquimod (R848) to suppress X-sperm motility. This study aimed to localise and assess TLR7/8 as targets for the separation of X- and Y-chromosome spermatozoa in the domestic dog using a simple swim-up assay. Immunocytochemistry revealed that both targets were present in the majority of spermatozoa, with TLR7 found in the sperm tail and base of the sperm head and TLR8 found in the sperm tail and equatorial region of the sperm head. Despite the lack of sex restriction of TLR7/8 in dog sperm, addition of 0.03-0.3 μmol/L R848 in swim-up assays was able to induce a differential response in X- and Y-chromosome sperm. Approximately 88% of sperm contained the Y-chromosome after inclusion of 0.3 μmol/L R848, whilst inclusion of 0.03 μmol/L R848 achieved close to 100%, reducing X-chromosomal content to undetectable levels. In contrast, consistent enrichment was not achieved through swim-up alone. No detrimental impact of R848 treatment on sperm motility or acrosome integrity was noted within these Y-enriched groups after removing R848. Overall, swim-up with 0.03 μmol/L R848 resulted in a highly enriched proportion of Y-chromosome sperm from chilled dog semen without detrimentally impacting sperm health. Lay summary: Males are preferred over females in breeding programs for certain working dogs, such as police dogs, as well as in research programs studying genetic disorders that almost exclusively affect males. This study investigated the presence of two proteins in dog sperm that have been found only in X-chromosome sperm in other species: these X-chromosome sperm fuse with the egg to make XX (female) embryos, with Y-chromosome sperm fusing to make XY (male) embryos. We evaluated whether these proteins could be targeted to increase the Y-chromosome sperm obtained for use in assisted conception in dogs. Overall, we found that whilst these proteins appear to be found in both X- and Y-chromosome dog sperm, their functionality appears to differ. Activating these proteins allowed us to effectively obtain Y-chromosome sperm without damaging them. This is the first successful report of a targeted, yet non-damaging, sperm sexing method in dogs and represents a promising tool for advancing research into currently incurable genetic diseases.