Effects of Some Respiratory and Glycolytic Inhibitors on Mitochondrial Functionality in Bovine Semen

Fertility potential of spermatozoa depends on maintenance of the mitochondrial membrane potential ( m) that provides energy for sperm hyperactivation immediately prior to successful fertilization. Mitochondrial structure and integrity are associated with sperm motility and reduced fertility, and measurement of m may be suitable for determining bull semen quality. Mitochondrial membrane potential in four commercial AI bulls was assessed using JC-1 and propidium iodide in the presence of the glycolytic inhibitors 2-deoxy-D-glucose (DOG) and iodoacetamide (IAM) and the respiratory inhibitor valinomycin (VAL) to determine the maximum m at minimum incubation. Flow cytometry recorded m within a unified population for all treatments that represented sperm with low and high m respectively. Maximum m was seen at 40 min incubation. Mean high fluorescence intensity (MFI) (orange) was significantly greater for untreated compared to the treated sperm, at 40 and 80 min incubation, in both fresh and frozenthawed semen. In sperm treated with VAL and IAM, m was lowered significantly, and the proportion of sperm with high: low m ratio was higher in control and DOGtreated samples representing more active mitochondria. In samples treated with VAL and IAM, the ratio was reversed, representing loss in activity. Cryopreservation increased the high:low m ratio only in bull 1 by 30% and lowered it in bulls 2, 3 and 4 by 20% 70% compared to fresh semen. The rise in bull 1 may relate to be the product of sperm demonstrating a capacitationlike effect of the freeze-thaw process which stimulated sperm hyperactivity. We conclude that mitochondrial function was affected adversely on freeze-thaw process. The 40 min incubation is satisfactory for future studies. Furthermore, IAM, a known glycolytic inhibitor, has a similar effect to VAL, a recognized respiratory inhibitor, which should provide a basis for further research.