42 Effect of vitamin D (cholecalciferol) on the cryosurvival of canine epididymal spermatozoa

Vitamin D deficiency is associated with poor sperm quality and motility (Adamczewska et al. 2023 Biomedicines 11, 90). Dietary supplementation of vitamin D in women also improves the outcome of infertility treatments (Baldini et al. 2024 Arch. Gynecol. Obstet. 309, 2881–2890). Addition of vitamin D in semen extenders improved cryosurvival of bovine spermatozoa (Asadpour et al. 2021 Basic Clin. Androl. 31, 20); however, the role of nutrients such as vitamin D3 in canine reproductive health and sperm function is not well known. This study aimed to determine (1) the effect of vitamin D3 on the kinematics and cryosurvival of spermatozoa before freezing and post-thaw, and (2) the presence of vitamin D receptor (VDR) within the epididymis of dogs. In Experiment 1 (Table 1), Tris-citrate-sucrose (TCS) + 20% egg yolk without 8% glycerol (cooling medium) and with 8% glycerol (freezing medium) was supplemented with vitamin D in four concentrations (treatments): (1) 0 nM, (2) 0.01 nM, (3) 0.1 nM, and (4) 1 nM. Spermatozoa were recovered by slicing the cauda of the epididymis in TCS-based medium. Samples (n = 5 males) with >70% initial motility were processed for cooling and slow/standard freezing. Computer-aided sperm analysis (CASA) and staining for membrane integrity were used to determine sperm motility, kinematics, and viability immediately after recovery (precool), after cooling/before freezing, and post-thaw. In Experiment 2, immunofluorescence staining was applied using rabbit monoclonal antibody to determine the presence and localization of VDR within the canine epididymis (n = 3 males). Data on sperm parameters were analyzed using R and R studio. The average initial sperm motility was 90.0% (range: 85.9%–95.3%). After cooling, sperm motility declined by up to 31% regardless of vitamin D treatment. Vitamin D supplementation did not affect (P < 0.05) sperm kinematics, the proportion of post-thaw motility, or membrane integrity; however, the regression line plot of vitamin D concentration versus mean sperm parameters indicated a strong positive correlation for progressive motility of cooled (R² = 0.92) and thawed (R² = 0.81) spermatozoa. Immunofluorescence assessments of tissue sections revealed localization of VDR within the caput, corpus, and cauda lumina but not within tubular tissue. This study demonstrates distinct localization of VDR in the epididymis and that vitamin D3 in extenders has cryoprotective effects.