(1) Background: This study set out to develop a series of simple, novel, rapid methods for assessing different forms of antioxidant activity. (2) Methods: An ABTS platform was used to engineer: (i) an electrochemical post-activation assay to assess free radical scavenging activity; (ii) an electrochemical pre-activation strategy to assesses the suppression of free radical formation; (iii) a horseradish peroxidase-mediated oxidation system to monitor hydrogen peroxide scavenging activity and (iv) a cumene peroxide-hematin system to determine the ability of samples to scavenge the mixture of organic peroxides and peroxyl and alkoxyl radicals generated in the presence of these reagents. Each assay was assessed against a panel of candidate antioxidant compounds to determine their relative activities and specificities. In addition, human semen samples were analyzed to determine how the results of these antioxidant assays correlated with semen quality. (3) Results: All 4 assays revealed dose-dependent antioxidant activity on the part of vitamin C, N-acetyl cysteine, hypotaurine, BSA, melatonin, glutathione, resveratrol and epigallocatechin gallate. The other compounds tested either completely lacked antioxidant activity or were only active in one of the assays. Using unfractionated human semen as an exemplar of biological fluids rich in antioxidants, the outputs from the individual assays were found to reflect different aspects of semen quality. When the data from all 4 assays were combined, accurate predictions were generated reflecting the importance of oxidative stress in defining semen quality as reflected by sperm count, seminal lipid aldehyde content, sperm DNA damage and free radical generation by the sperm mitochondria. (4) Conclusions: The methodologies described in this paper constitute the basis for rapid, point-of-care assessments of oxidative stress.

(1) Background: The RoXsta^TM system has been developed as a rapid, effective means of profiling different types of antioxidant activity. The purpose of this study was to examine its performance utilizing a diverse array of biological fluids including semen, blood plasma, serum, urine, saliva, follicular fluid and plant extracts. (2) Methods: The RoXsta^TM system was used to assess the ability of different fluids to suppress free radical formation as well as scavenge a variety of toxic oxygen metabolites including free radicals and both hydrogen and organic peroxides. (3) Results: Human semen was shown to have significantly (p < 0.001) more peroxide scavenging power than any other fluid tested (10–14 mM vitamin C equivalent compared with 1–2 mM for blood serum or plasma), while urine was particularly effective in scavenging free radicals and preventing free radical formation (p < 0.001). The powerful antioxidant properties of human semen were shown to reside within the seminal plasma (SP) fraction, rather than the spermatozoa, and to be resistant to snap freezing in liquid nitrogen. Moreover, comparative studies demonstrated that human SP exhibited significantly (p < 0.001) higher levels of antioxidant potential than any other species examined (stallion, bull, dog) and that this intense activity reflected the relative vulnerability of human spermatozoa to peroxide attack. (4) Conclusions: The RoXsta^TM system provides valuable information on the antioxidant profile of complex biological fluids, supporting its diagnostic role in conditions associated with oxidative stress. Based on the results secured in this study, human semen is identified as a particularly rich source of antioxidants capable of scavenging both hydrogen and organic peroxides, in keeping with the high susceptibility of human spermatozoa to peroxide-mediated damage.

Purpose: To create a novel medium that retained human sperm quality following cryopreservation at a higher level than that seen with currently available commercial cryoprotectants. Methods: Cryopreservation was achieved via 1:1 dilution with cryoprotectant followed by slow-programmed freezing. A NaCl-free cryopreservation carrier medium based on the use of histidine as the major osmolyte was designed that was capable of sustaining human sperm motility over 6 days at ambient temperature. This medium was supplemented with ethylene glycol, glycerol, and DMSO to create the basis for a novel cryopreservation medium. Dose-dependent studies with various supplements were then conducted to optimize the effectiveness of this formulation including assessments of vitamin C, EDTA, crocin, zinc, ergothioneine, and myo-inositol, as well as the potential replacement of DMSO by Cyrene™. Post-thaw samples were assessed for motility, vitality, and DNA integrity and then reassessed following sperm isolation with the Felix™ System. Results: The completed cryopreservation formulation comprised 4.5% ethylene glycol, 4.5% glycerol, 1% DMSO in a carrier medium supplemented with 0.4 mM vitamin C, 1 mM EDTA, and 22 mM myo-inositol. Spermatozoa frozen in this medium and isolated using the Felix™ System had significantly greater total motility, progressive motility, vitality, and DNA integrity than spermatozoa frozen in a commercially available product that is widely used in infertility clinics. Conclusion: A novel cryopreservation medium has been developed in this study that represents a significant improvement over existing technologies.

(1) Background: The RoXstaTM system has been developed as a potential diagnostic tool to profile the antioxidant activity of complex biological fluids. (2) Methods: This methodology was used to assess the ability of different biofluids (semen, blood plasma, serum, urine, saliva, follicular fluid and plant extracts) to suppress free radical formation, as well as scavenge a variety of toxic oxygen metabolites including free radicals and peroxides. (3) Results: Human semen was shown to have significantly (p < 0.001) more peroxide scavenging power than any other fluid tested, while urine was particularly effective in scavenging free radicals and preventing free radical formation. The powerful antioxidant properties of human semen were shown to reside within the seminal plasma (SP) fraction, rather than the spermatozoa, and to be resistant to snap freezing in liquid nitrogen. Moreover, comparative studies demonstrated that human SP exhibited significantly (p < 0.001) higher levels of antioxidant potential than any other species examined (stallion, bull, dog) and that this intense activity reflected the relative vulnerability of human spermatozoa to peroxide attack. (4) Conclusions: the RoXstaTM system provides valuable information on the antioxidant profile of complex biological fluids, supporting its diagnostic role in conditions associated with oxidative stress. Based on the results secured in this study, human semen is identified as a particularly rich source of antioxidants capable of scavenging both hydrogen-, and organic- peroxides, in keeping with the high susceptibility of human spermatozoa to peroxide-mediated damage.