Paternal determinants of Offspring Sex Ratio in horses: A retrospective analysis of stallion breed, age, and breeding method.

Offspring Sex Ratio (OSR) at birth in mammals is generally assumed to follow a 1:1 male-to-female distribution, yet deviations in OSR have been observed and may be influenced by maternal and paternal factors. In this study we have focused on the effect of paternal factors (stallion age and breed) and breeding methods on the OSR. Using a retrospective dataset of 251,783 foal births recorded in France between 2015 and 2024, we applied generalized linear mixed models to evaluate the effects of these variables on the likelihood of producing a female foal. Stallion identity was included as a random effect. Our analysis revealed that stallion breed and breeding methods significantly influenced the sex ratio, while stallion age did not. The Anglo-Arab, Comtois, and French Sport Horse stallions were more likely to sire female foals compared to the French Trotter. Pasture breeding was associated with a higher proportion of female offspring, whereas artificial insemination using frozen or refrigerated semen reduced this likelihood. Overall, deviations in OSR were toward female offspring and were associated with stallion breed and breeding method. These patterns suggest possible contributions from paternal factors, including breed-related differences in semen characteristics and post-meiotic or peri-fertilization processes - such as semen handling, preservation, and insemination timing- that may differentially affect X- and Y-chromosome-bearing spermatozoa. Maternal factors previously reported to influence Offspring Sex Ratio cannot be excluded as contributors to the observed patterns.