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Robert John Aitken, Sarah Lambourne, and Ashlee Jade Medica
In brief
A capacity to predict the likelihood of pregnancy following natural matings would be of considerable benefit to the Thoroughbred horse breeding industry. In this article, we describe a strategy for achieving this outcome through the analysis of dismount samples, that achieved an overall accuracy of 94.6%.
Abstract
The purpose of this study was to determine whether the analysis of dismount semen samples from Thoroughbred stallions could be used to predict whether a given mating would result in a pregnancy. The analysis was based on 143 matings of 141 mares by a cohort of 7 Thoroughbred stallions over a 4-week period at an Australian Stud. The criteria of semen quality utilized in this analysis involved a preliminary assessment of the raw dismount sample in terms of semen volume, sperm number, and sperm movement characteristics using an iSperm® Equine portable device. Following this initial assessment, a subpopulation of progressively motile spermatozoa was isolated by virtue of the cells ability to migrate across a 5 µm polycarbonate filter in a Samson™ isolation chamber over a 15-minute period. These isolated cells were again evaluated for their number and quality of movement using the iSperm® system and, in addition, assessed for their ability to reduce WST-1, a membrane impermeant tetrazolium salt. These data were then combined with additional information describing the ages of the animals used in this study, their historical breeding records, and mating frequency during the breeding season. The total data set was then used to predict the occurrence of pregnancy, as confirmed by ultrasound at ~14 days post mating. The criteria used to predict fertility in the ensuing multivariate discriminant analysis were optimized for each individual stallion. Using this strategy, we were able to successfully predict the outcome of a cover with an overall accuracy of 94.6%.
Róisín A Griffin, Mark Baker, Robert John Aitken, Aleona Swegen, and Zamira Gibb
Stallions experience lower per-cycle conception rates compared to other livestock species, largely because they are selected for breeding based on athletic prowess and not reproductive fitness. Mares are seasonal breeders, and pregnancies cannot be detected until 10–14 days post cover via transrectal ultrasonography. This means the detection of stallion fertility fluctuations is delayed by at least 2 weeks, which within the short breeding season employed by the thoroughbred horse breeding industry, can prove quite costly. For these reasons, there is increased demand for robust laboratory assays aimed at the accurate assessment of stallion fertility. This paper reviews our existing knowledge concerning the molecular mechanisms that underpin the functional competence of stallion spermatozoa, highlighting the relative importance of oxidative stress, DNA damage, sperm proteomics and RNA profile. We also consider the way in which fundamental improvements in our understanding of stallion sperm biology are informing the identification and development of possible biomarkers of fertility and thus avenues for the development of specific assays for fertility prediction.
Ashlee Jade Medica, Zamira Gibb, Alecia Sheridan, Natasha Harrison, and Robert John Aitken
MTT is a commonly used cell vitality probe, due to its ability to form insoluble formazan deposits at cellular locations of intense oxidoreductase activity. Although this response is considered a reflection of mitochondrial redox activity, extra-mitochondrial sites of MTT reduction have been recognized within the spermatozoa of several mammalian species. Therefore, the aim of this study was to determine the major sites and causative mechanisms of MTT reduction in stallion spermatozoa. Our results show that stallion spermatozoa displayed substantial mitochondrial formazan deposition, as well as a single extra-mitochondrial formazan deposit in various locations on the sperm head in approximately 20% of cells. The quality and capacitation status of stallion spermatozoa were positively correlated with the presence of an extra-mitochondrial formazan granule. Additionally, extra-mitochondrial formazan deposition was suppressed by the presence of an NADPH oxidase (NOX) inhibitor (VAS2870; active against NOX2, NOX4 and NOX5), MnTMPyP (SOD mimetic) and zinc (NOX5 inhibitor) suggesting that extra-mitochondrial MTT reduction may be facilitated by NOX-mediated ROS generating activity, conceivably NOX5 or NOX2. When comparing MTT to resazurin, another well-known probe used to detect metabolically active cells, MTT reduction had a higher correlation with sperm concentration and motility parameters (R2= 0.91), than resazurin reduction (R2 = 0.76). We conclude that MTT reduction in stallion spermatozoa follows a species-specific pattern due to a high dependence on oxidative phosphorylation and a degree of NOX activity. As such, MTT reduction is a useful diagnostic tool to assess extra-mitochondrial redox activity, and therefore, the functional qualities of stallion spermatozoa.
Robert John Aitken, Diatsendoula Gregoratos, Leslie Kutzera, Emma Towney, Minjie Lin, Alexandra Wilkins, and Zamira Gibb
MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. By contrast, human spermatozoa generally displayed small disorganized midpieces exhibiting moderate MTT reduction activity accompanied by a major extra-mitochondrial formazan deposit on various locations in the sperm head from the neck to the anterior acrosome. Equine spermatozoa presented a combination of these two patterns, with major formazan deposition in the mitochondria accompanied by an extra-mitochondrial formazan deposit in around 20% of cells. The functionality of human spermatozoa was positively associated with the presence of an extra-mitochondrial formazan granule. Subsequent studies indicated that this extra-mitochondrial activity was suppressed by the presence of diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic and NADPH oxidase inhibitors. We conclude that the pattern of MTT reduction to formazan by spermatozoa is species specific and conveys significant information about the relative importance of mitochondrial vs extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.
Róisín A Griffin, Aleona Swegen, Mark Baker, Robert John Aitken, David A Skerrett-Byrne, Antonio Silva Rodriguez, Francisco E Martín-Cano, Brett Nixon, Fernando J Peña, Maryse Delehedde, Nicolas Sergeant, and Zamira Gibb
The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen (n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessments were carried out, and proteomic analyses was conducted using UHPLC-MS/MS. High-quality spermatozoa recorded higher total (95.2 ± 0.52% vs 70.6 ± 4.20%; P ≤ 0.001) and progressive motilities (43.4 ± 3.42% vs 27.3 ± 4.32%; P ≤ 0.05), and a higher proportion of morphologically normal cells (50.2 ± 4.34% vs 38.8 ± 2.72%; P ≤ 0.05). In total, 1069 proteins were quantified by UHPLC-MS/MS, of which 22 proteins were significantly more abundant in the high-quality sperm population (P ≤ 0.05). A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered possible biomarker candidates and their differential expression was confirmed by immunoblot. Protein expression was significantly correlated with total (AKAP4 R 2 = 0.38, P ≤ 0.01; HK1 R 2 = 0.46, P ≤ 0.001) and progressive motilities (AKAP4 R 2 = 0.51, P ≤ 0.001; HK1 R 2 = 0.55, P ≤ 0.01), percentage rapid (AKAP4 R 2 = 0.29, P ≤ 0.05; HK1 R 2 = 0.58, P ≤ 0.001), straight-line velocity (HK1 R 2 = 0.50, P ≤ 0.01) and straightness (HK1 R 2 = 0.40, P ≤ 0.01). Furthermore, AKAP4 was highly susceptible to adduction by 4-hydroxynonenal (4HNE), which resulted in a global reduction in the phosphorylation profiles following capacitation. In conclusion, the proteomic profiles of high- and low-quality stallion spermatozoa differ substantially, and proteins such as AKAP4 and HK1 could serve as biomarkers of ejaculate quality.