Stallion spermatozoa primarily utilise oxidative phosphorylation (OXPHOS), rather than glycolysis, to produce the energy required to reach the site of fertilisation [2]. In contrast, bull and ram spermatozoa utilise both OXPHOS and glycolysis to fulfil their energy requirements. Intense mitochondrial activity due to OXPHOS makes these cells susceptible to oxidative stress (due to the production of reactive oxygen species; ROS). ROS are important signalling molecules in many cellular processes such as sperm capacitation. However, in order to maintain homeostasis, ROS must be rapidly neutralised by cellular antioxidants, and removed from the cell. The continued, unchecked production of ROS will disrupt vital cellular functions, leading to a state of oxidative stress. Increased ROS generation is associated with decreased sperm quality, fertilizing potential, and fertility [1, 3, 4]. The role of ROS in male infertility is discussed by O’Flaherty [5], and in greater detail by Wagner [6].

The extent of mitochondrial oxidative stress is assessed using flow cytometry.  Spermatozoa are incubated with a fluorogenic dye (MitoSOX™ Red; MSR) and a nuclear stain (Sytox® Green; SyG). MSR rapidly and selectively targets sperm mitochondria, where it fluoresces red when oxidized by ROS, while MSR oxidation is prevented by the potent antioxidant, superoxide dismutase. Meanwhile, the nucleic acid stain SyG cannot penetrate live cells, and is therefore used to identify dead cells within the population. As dead cells cannot produce ROS, we only report on live cells. The reported populations are:

• MSR neg / SyG neg: % of live cells not suffering mitochondrial oxidative stress
• MSR pos / SyG neg: % of live cells with high mitochondrial oxidative stress