Effect of Single Layer Centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen.

ABSTRACT

Additional means are needed for evaluating the quality of stallion spermatozoa in semen doses for AI. Mitochondrial membrane potential (ΔΨm) has been linked to fertility in some species, but is rarely used in the evaluation of cooled stallion semen; metabolic activity may be associated with reactive oxygen species production (ROS). In the present study, ΔΨm and ROS production were measured in doses of cooled stallion semen. The effect of colloid centrifugation on these parameters was also investigated. In this case, colloid centrifugation involves centrifuging a sperm sample through a silane-coated silica colloid formulation to retrieve the most robust spermatozoa. High and low ΔΨm in cooled stallion semen varied between stallions and between ejaculates, but was not affected by single-layer centrifugation (SLC). The SLC-selected spermatozoa produced significantly less hydrogen peroxide than controls (P < 0.001), which could explain the increased longevity and retention of fertilising capacity seen in previous studies. For SLC samples, ΔΨm was positively associated with viable spermatozoa that were not producing reactive oxygen species (r = 0.49; P < 0.001) and negatively associated with ROS production (for superoxide r = -0.4, P < 0.01; for hydrogen peroxide r = -0.39, P < 0.05). There was no clear association between ΔΨm and ROS production in control samples.