Submitted to: Pig Reproduction National Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: March 31, 2005
Publication Date: June 12, 2005
Citation: Guthrie, H.D., Welch, G.R. 2005. New assays for reactive oxygen species (ros) formation and mitochondrial membrane potential in fresh and cryopreserved boar spermatozoa [abstract]. 7th International Conference on Pig Reproduction National Conference. p. 141. Technical Abstract: Cryopreservation of boar spermatozoa has not been adopted by the swine industry because, even corrected for viable sperm number, cryopreserved sperm fail to perform as well as liquid semen in terms of fertilization and pregnancy rate. Part of the loss in sperm functionality and fertility may be due to oxidative damage from formation of ROS. Evidence for this is that the addition of antioxidants or ROS scavengers to semen extenders can improve sperm viability and in vitro embryo production after in vitro fertilization. In addition, sperm lipid peroxidation increases during liquid semen storage and cryopreservation and can be partially eliminated by presence of antioxidants. Seminal plasma contains numerous natural antioxidants and ROS scavengers. However this natural protection is lost during sperm washing in preparation for cryopreservation. Many questions remain; it is not known what proportion of the sperm population is producing ROS, which ROS are being produced, or whether viable, nonviable or damaged sperm are producing ROS. Experiments were conducted to determine if cryopreserved spermatozoa produced more ROS than fresh spermatozoa and to determine the effects of cryopreservation and ROS on mitochondrial function and motility. Fresh and cryopreserved sperm were incubated in a Tyrodes noncapacitating medium for 30 min at 1) 25C, 2) 38C, and 3) 38C with xanthine (XA)/xanthine oxidase (XO) as an extracellular ROS source or menadione as an intracellular ROS source. Sperm were collected for computer-assisted motion analysis. Novel dual staining flow cytometric procedures were developed for superoxide anion using the oxidation of hydroethidine (HE) to ethidium bromide (EB), for hydrogen peroxide using the oxidation of 2,7-dichorodihydrofluorescein to dichlorofluorescein (DCF), and for measurement of high mitochondrial membrane potential (MMP) using 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) aggregate red fluorescence. The impermeant nuclear stains Yo Pro-1 and propidium iodide were used to distinguish between viable and nonviable sperm. Both superoxide anion and hydrogen peroxide were capable of oxidizing HE to HB, but DCF was only generated by hydrogen peroxide. Surprisingly the incidence ROS formation was low and did not differ between fresh and cryopreserved viable spermatozoa incubated at 25C or 38C. However, compared to 25C, incubation at 38C increased the incidence of EB positive viable sperm from 1.5 to 3.2%. In samples containing high levels of ROS, most ROS containing cells were found in the nonviable portion of the sperm population. Viable sperm were very responsive to XA (1 uM)/XO (0.1 U/ml) with 94.6 and 87.3% of fresh and cryopreserved sperm, respectively, being positive for EB along with a 4-fold increase in mean EB fluorescence intensity without a decrease in viability. Following XA/XO treatment > 90% of viable sperm had intact mitochondrial membranes. However, motion parameters were decreased by 34-62%. Treatment of sperm with menadione increased ROS formation and caused a progressive decrease MMP (mean JC-1 red fluorescence intensity). When MMP had decreased by 60% of control levels motion parameters were decreased by 25-60%. We conclude that the mechanism of inhibition of motility by ROS formation may be related a decrease in mitochondrial charge potential below a critical threshold.