Cooled stallion semen transported without quality certification: the first Lithuanian study on semen quality biomarkers and fertility after 24-hour shipment

In Lithuania, the growing equine breeding industry has increased the use of artificial insemination with cooled stallion semen imported from abroad and delivered within 24 hours of collection. Upon arrival, motility is routinely assessed, but pH is rarely measured despite its potential as an early biomarker of semen quality deterioration before motility visibly declines. This study aimed to evaluate correlations between semen pH and key morphological and functional parameters. Twenty semen samples were collected from Warmblood stallions aged 4–22 years at breeding centers in Europe, cooled and sent to Lithuania. Following 24 hours of transport, semen concentration, pH, total sperm motility, sperm viability (eosin/nigrosin staining) and morphology were determined in specialized Animal Reproduction Laboratory according to the methodologies used there. Then, the semen was used to artificially inseminate the mares. Conception rates were observed after 14 days. Despite all sperm samples quality meets international limits, some tendencies were noted. Semen motility (ρ = +0.40, p = 0.040) and concentration in sperm dose (ρ = +0.68; p = 0.000039) statistically significantly correlated with conception rates. Tail and sperm midpiece pathologies also correlated statistically significant (ρ = +0.67, p = 0.001). Semen pH values in the analyzed samples ranged from 7.61 to 9.63. A statistically significant positive correlation was observed between pH and detached pathological sperm heads (ρ = 0.54, p = 0.036) also pH and sperm heads with a narrow base (ρ = 0.68, p = 0.0057). The analysis revealed positive statistically significant associations between semen pH and conception rates (ρ = 0.65, p = 0.0158). Our study results suggest that although all semen samples met international standards, higher motility, sperm concentration, and pH were positively correlated with conception rates. In stallions, increased numbers of detached heads and narrow-base heads may be associated with handling stress, pH changes, or osmotic imbalance. Monitoring pH alongside morphological assessment, total sperm motility, and viability could improve the evaluation of stallion semen quality and serve as an early biomarker of alterations in the seminal environment. Further investigation with a larger sample size is warranted.