(5) Effect of Canine Sperm Concentration Using a Milk-Based Extender on Sperm Quality and Proteostasis After Cooling for 24 hours

Michal Zigo¹; Margaret Caldwell²; Edgar Diaz-Miranda³; Peter Sutovsky^1,3; Julia Baldrighi²

¹Division of Animal Sciences, University of Missouri, Columbia, MO, USA 65211

² Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA 65211

³Department of Obstetrics, Gynecology and Women’s Health, University of Missouri School of Medicine, Columbia, MO, USA 65201

Abstract Text:

Shipping cooled canine semen for artificial insemination (AI) offers numerous benefits, including increased convenience, decreased costs and lower risk of venereal diseases, while also allowing access to the best genetic pools. Studies have shown higher pregnancy rates and larger litter sizes for cooled-shipped semen, compared to frozen semen in dog AI. However, there is currently no standardized protocol for canine semen extension during cooled shipment. Conventional semen parameters that depend heavily on sperm motility and morphology, are still predominantly used to evaluate sperm quality and fertility. Our ongoing work on the sperm ubiquitin-proteasome system confirms that imbalanced proteostasis, reflected by the formation of stress induced protein aggregates called aggresomes, is associated with abnormal sperm morphology and function in mammals. We hypothesize that dog semen cooling conditions can be optimized to boost semen quality. This study included 11 healthy, mature adult dogs of medium to giant size breeds, of which ten were proven studs, and one was maiden/unproven (no family history of infertility). The sperm-rich fractions were collected by manual stimulation. After initial light-microscopic evaluation, the raw semen was diluted to 1:1 (v/v) with INRA96 at 37 °C, centrifuged, and the pellets were resuspended with INRA96 to 200 mil/mL concentration (C200), followed by serial dilutions to 100 mil/mL (C100) and 50 mil/mL (C50). Aliquots of 1 mL per concentration were kept in canine semen cooling transport boxes for 24 h. We evaluated the semen parameters of all three concentration groups after a 24 hour incubation (t24) and the control group (Ctrl) at the time of resuspension (t0) by using computer assisted semen analysis (CASA), morphology analysis, acrosomal status (peanut agglutinin labeling), and the presence of aggresomes (ProteoStat kit, cat # ENZ51035K100, Enzo LifeSciences). No statistical differences in total and progressive motilities, sperm velocities and sperm beat cross frequencies were observed between C50-t24, C100-t24, C200-t24 and Ctrl-t0 groups. However, C50-t24 showed a higher amplitude of lateral head displacement than C200-t24 and Ctlr-t0 . When examining sperm movement patterns, C50-t24 had lower linearity and straightness than C200-t24, while C200-t24 had higher straightness than Ctrl-t0. We observed a substantial decrease in the total aggresome mean fluorescence intensity (MFI) in the C50-t24 when compared to Ctrl-t0, as well as the aggresome MFI decrease in both midpiece (mitochondrial sheath and cytoplasmic droplet) and head when compared to Ctrl-t0. No differences in acrosomal integrity were observed between the treatment groups. These results indicate that the extended canine sperm concentration of 50 million per milliliter affected the evaluated fertility parameters. Evaluation of aggresome status could be used as an additional fertility marker for dog semen evaluation. Further studies are underway comparing aggresome patterns in dogs with their wild relative wolves, adding information on species conservation to our domesticated species data. Correlations between aggresome patterns and sperm morphology can be used for machine learning thus putting Artificial Intelligence into Artificial Insemination (AI²).

Keywords: canine spermatozoa, cooled semen, concentration, male fertility, aggresome, fertility diagnostics

 
Supported by USDA-NIFA grant 2021-67015-33404 (PS), MU Research Council grant (PS), MU CAFNR Joy of Discovery Seed Grant program (PS), MU-CVM Office of Research (JB), and MU-CVM Start-Up Fund (JB).