Although stallion spermatozoa produce significant quantities of reactive oxygen species, a lag between 4-hydroxynonenal (4HNE) adduction and the loss of motility in stallion spermatozoa suggests the presence of a robust aldehyde detoxification mechanism. As there is a paucity of literature characterising the role of aldehyde dehydrogenase (ALDH) in sperm functionality, the aim of this study was to ascertain the relationship between 4HNE production, motility and ALDH expression by stallion spermatozoa. PCR analysis revealed the presence of the ALDH1A3, ALDH1B1 and ALDH2 isoforms in these cells. Strong correlations (P < 0.001) were found between ALDH expression and various motility parameters of stallion spermatozoa including the percentage progressive (r = 0.79) and rapidly motile (r = 0.79) spermatozoa, while repeated measurements over 24 h revealed highly significant correlations between progressive motility loss, 4HNE accumulation and ALDH (P ≤ 0.001). ALDH inhibition resulted in a spontaneous increase in 4HNE levels in viable cells (21.1 ± 5.8% vs 42.6 ± 5.2%, P ≤ 0.05) and a corresponding decrease in total motility (41.7 ± 6.2% vs 6.4 ± 2.6%, P ≤ 0.001) and progressive motility (17.0 ± 4.1% vs 0.7 ± 0.4%, P ≤ 0.001) of stallion spermatozoa over 24 h. Similarly, inhibition of ALDH in 4HNE-challenged spermatozoa significantly reduced total motility over 4 h (35.4 ± 9.7% vs 15.3 ± 5.1%, P ≤ 0.05). This study contributes valuable information about the role of the ALDH enzymes in the maintenance of stallion sperm functionality with potential diagnostic and in vitro applications for assisted reproductive technologies.

(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.

This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm^® Equine portable device. After the initial evaluation, a subpopulation of cells was subjected to a migration assay through a 5 µm polycarbonate filter within a Samson™ isolation chamber over a 15 min period. The cells were assessed for their concentration, motility parameters, and ability to reduce the membrane impermeant tetrazolium salt WST-1. The data, combined with the stallion and mare’s ages, were used to predict the likelihood of pregnancy, as confirmed by rectal ultrasound sonography performed 14 days post ovulation. The criteria used to predict pregnancy were optimized for each individual stallion, resulting in an overall accuracy of 87.9% if analyzed pre-chilling and 95% if analyzed post-chilling. This study suggests that an analysis of stallion ejaculate can be used to predict the likelihood of pregnancy resulting from artificial insemination in mares with a high level of accuracy.