Evaluation of Oxidative Profiling in Semen Fractions and Its Relationship With Sperm Function in Kangal Akkaraman Rams

ABSTRACT

The determination of sperm oxidative stress provides significant information related to semen quality. Most studies investigate oxidative stress markers only at the level of seminal plasma, but this approach may provide an incomplete overview of the oxidative status of semen. The aim of the study is to determine the relationship between protein‐normalized oxidative stress markers and spermatological parameters in ram semen fractions. A total of 38 ejaculates obtained from 24 rams were fractionated into seminal plasma, whole semen (non‐centrifuged), and pellet at different concentrations. Total antioxidant status (TAS), total oxidant status (TOS), malondialdehyde (MDA), and oxidative stress index (OSI) were measured and normalized to total protein content using the Lowry method. Fraction‐wise differences in oxidative stress parameters were analysed using linear mixed‐effects models accounting for ram identity, while relationships between spermatological and oxidative parameters were evaluated using Spearman correlation analysis. Principal component analysis (PCA) and hierarchical clustering were performed as exploratory multivariate visualization tools. Motility and viability were positively correlated ( ρ  = 0.856, p  < 0.001), while both parameters were negatively correlated with abnormal sperm rate ( p  < 0.05). In addition, TOS and MDA levels were significantly lower in seminal plasma than in sperm pellet fractions, supporting the presence of fraction‐dependent oxidative profiles ( p  < 0.05). Exploratory PCA suggested that overall multivariate variation was mainly associated with spermatological parameters, whereas oxidative markers had a more limited contribution. These findings provide insight into the relationship between oxidative status and different semen fractions. Additionally, fraction‐specific oxidative profiling may provide complementary information beyond seminal plasma measurements alone.