Benign prostatic hyperplasia (BPH) is a common condition in intact male dogs, often progressing from subclinical to symptomatic stages with increasing clinical and structural impact. This study evaluated canine prostate-specific esterase (CPSE) as a biomarker for BPH progression, focusing on clinical severity, complexity, and ultrasonographic features. Seventy-one dogs were included: subclinical-BPH (n = 14), clinical-BPH (n = 26), BPH-prostatitis (n = 9), and controls (n = 22). CPSE levels, measured using a canine-specific enzyme-linked immunosorbent assay, were significantly correlated with clinical severity (ρ = 0.800, p ≤ 0.001) and complexity (ρ = 0.818, p ≤ 0.001). CPSE predicted mild (OR = 1.260, B = 0.231, p ≤ 0.001) and moderate severity (OR = 1.300, B = 0.262, p ≤ 0.001), as well as low (OR = 1.225, B = 0.203, p ≤ 0.05), moderate (OR = 1.235, B = 0.211, p ≤ 0.01), and high (OR = 1.346, B = 0.297, p ≤ 0.001) clinical complexity. CPSE showed a trend toward predicting structural alterations (OR = 1.227, B = 0.204, p = 0.069) and was associated with larger stippled areas, increased prostatic volume, and larger cysts/abscesses (all p ≤ 0.001). CPSE appears to be a promising marker for BPH progression.

Standardized methods for investigating the canine vaginal microbiome are not available yet. Data using next-generation sequencing (NGS) are still limited, and methodologies lack consistency. In theory, microbiome results can be significantly affected by factors like sampling technique, storage, DNA extraction methods, and reference databases, which can all introduce bias. To address these concerns, we compared two storage methods for the sample (no medium vs. medium), examined the effect of host DNA depletion, and tested two reference databases (Emu vs. SILVA) using samples from six bitches, totaling 26 samples. Host depletion showed no significant impact on bacterial composition, nor did the storage conditions. However, when comparing reference databases, we found significant differences in beta diversity, emphasizing the importance of database choice when comparing studies. The proposed protocol paves the way for future studies on the canine vaginal microbiome, setting the basis for more precise and comprehensive microbiome profiling in this field. IMPORTANCE: Understanding the vaginal microbiome in dogs could lead to new insights into reproductive health and fertility, but progress is limited by the lack of clear guidelines on how samples should be collected, stored, and analyzed. This study helps clarify which steps in the process truly matter and which have little impact, offering practical guidance for researchers entering this field. By highlighting where inconsistencies can influence outcomes and which methodological choices affect results, we take an important step toward more reliable and comparable research. These findings support future scientific studies and hold potential to improve veterinary care over time.

Despite the widespread use of cryopreserved dog semen, reliable predictors of individual semen freezability are still not available. This study investigated the relationship between post-ejaculatory blood plasma concentrations of canine prostate-specific esterase (CPSE) and semen quality following cryopreservation. The CPSE levels were quantified using a canine-specific sandwich-type immunoassay, and sperm quality was evaluated in fresh and frozen–thawed samples using motility, kinematic, and morphological sperm parameters. Cryopreservation resulted in a significant decline in sperm motility and morphology, with marked variability in the magnitude of post-thaw changes among individuals. CPSE showed significant correlations with several morpho-functional parameters in both fresh and frozen–thawed sperm (p ≤ 0.05). However, its strongest predictive value for freezability was observed for total motility (p = 0.004), with a minimum threshold of 53 ng/mL identifying dogs showing a ≥20% post-thaw motility decline (AUC = 0.785, p = 0.010). These findings suggest that post-ejaculatory CPSE levels in blood plasma may reflect sperm cryoresilience and serve as a potential biomarker of canine sperm freezability.

Gonadectomy is performed in bitches to prevent unwanted reproduction and reduce the risk of sex hormone-related conditions. However, growing evidence suggests that the timing of spaying may influence long-term susceptibility to non-reproductive diseases. This retrospective case–control study (2013–2023) evaluated the association between timing of spaying and the development of orthopedic and neoplastic disorders in a Belgian referral-hospital population. Cases included bitches diagnosed with cranial cruciate ligament rupture, hip dysplasia, elbow dysplasia, lymphoma, mast cell tumor, osteosarcoma, or hemangiosarcoma, while disease-free bitches served as controls. Associations between disease occurrence and spaying status were assessed using multivariable logistic regression adjusted for age, weight category, and body condition score. Age at gonadectomy (<12 vs. ≥12 months) and timing relative to the first estrus were evaluated in separate models. Spaying <12 months of age was associated with increased odds of all conditions compared with intact females. Spaying ≥12 months of age was associated with lower odds of several orthopedic and neoplastic outcomes compared with early spaying, although odds were not always comparable to those in intact females. Large body size and higher body condition score were independently associated with increased odds of orthopedic outcomes. These findings support individualized spaying strategies rather than a universal age threshold.

Semen cryopreservation is widely used in canine breeding, but individual variation in post-thaw semen quality limits its efficiency. This study investigated whether testicular artery Doppler-derived blood-flow parameters and circulating vascular endothelial growth factor (VEGF) concentrations are associated with semen quality and sperm freezability in clinically healthy dogs. Sixty-four privately owned male dogs underwent reproductive screening, semen collection, testicular Doppler ultrasonography, blood sampling, fresh and post-thaw semen analysis, and plasma VEGF quantification. Testicular artery Doppler parameters were not significantly associated with fresh or post-thaw semen quality or with most freezability outcomes. Plasma VEGF concentration was positively correlated with fresh linearity (LIN) (p = 0.034), straightness (STR) (p = 0.031), and beat-cross frequency (BCF) (p = 0.043). Higher VEGF concentrations were associated with greater post-thaw declines in progressive motility (PM) (p = 0.036), straight-line velocity (VSL) (p = 0.032), LIN (p = 0.001), STR (p = 0.005), and wobble (WOB) (p = 0.010). In multivariable analysis, VEGF was associated with linearity decline (B = 0.595, 95% CI: 0.254–0.937; p = 0.001). Circulating VEGF may therefore be associated with individual variation in canine sperm cryotolerance, whereas testicular artery Doppler assessment showed limited value.