Clustering of sperm by chromatin integrity features reveals breed and monthly effects, as well as a link between double-strand DNA breaks and litter size in pigs

BACKGROUND: Identifying sperm biomarkers that predict fertility in swine is important for improving animal selection and production. In this context, however, sperm chromatin in swine remains understudied, and whether it varies by breed and season remains unresolved. Additionally, the impact of sperm chromatin protamination, condensation, and DNA integrity on reproductive outcomes (farrowing rate and litter size) is unclear in swine. This study aimed to improve the understanding of sperm chromatin features by evaluating breed- and month-related differences and determining their influence on fertility outcomes. To this end, we evaluated sperm chromatin protamination and condensation, as well as global and double-strand DNA damage, in 91 ejaculates from 41 boars (25 Landrace and 16 Large White) using chromomycin A3, dibromobimane, and alkaline and neutral Comet assays, respectively. RESULTS: Single sperm analysis revealed six clusters associated with chromatin protamination, four clusters related to chromatin condensation, and two clusters corresponding to global DNA damage. Differences between the Landrace and Large White breeds were observed in the percentage of sperm with global DNA damage (P = 0.015), and in chromatin protamination (P = 0.012) and condensation (P < 0.001). Overall, sperm chromatin integrity was better in Large White than in Landrace boars. Monthly analysis revealed that sperm chromatin quality worsened in September due to an increase in global DNA damage (P < 0.01), affecting condensation in one cluster and sperm protamination in two clusters (P < 0.05). Finally, multivariate analysis showed that the percentage of sperm with double-strand DNA breaks was associated with litter size in both breeds. CONCLUSIONS: This study identified sperm subpopulations based on chromatin features in swine. Differences were observed between Large White and Landrace breeds, with the sperm from Large White boars having healthier chromatin quality in terms of DNA damage, protamination, and condensation. Monthly variations were also seen, primarily in relation to global DNA damage. Finally, our study showed an association between the incidence of sperm with double-strand DNA breaks and litter size in both breeds.