Dr Aleona Swegen

The oxidation of leucomethylene blue (LMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) are colorigenic reactions, generating methylene blue (M+) and the ABTS¿+ cation radical, respectively. In this study, we have analyzed the conditions under which these probes become oxidized and explored their application in the assessment of antioxidant activity. Using hematin as a catalyst, LMB responded to hydrogen peroxide and cumene hydroperoxide, whereas ABTS could only respond to the latter. However, in the presence of horse radish peroxidase, both probes responded specifically and dose-dependently to hydrogen peroxide. ABTS and LMB could also be oxidized in the anodic chamber of an electrochemical cell, permitting both pre-activation assays of free radical formation and post-activation assessments of free radical scavenging activity. In the pre-activation mode, both ABTS and LMB successfully revealed DMSO's capacity to inhibit free radical formation, in contrast to the 'green' solvent Cyrene¿, which was virtually devoid of such activity. In the post-activation mode, the LMB oxidation product, methylene blue, was shown to be particularly sensitive to the 2-electron reducing properties of vitamin C. In contrast, ABTS responded more sensitively to compounds, like resveratrol, that used hydrogen atom transfer and one electron reduction to achieve their antioxidant action. Only LMB-based antioxidant assessments correlated with sperm motility (P < 0.001), suggesting this probe's sensitivity to 2-electron reduction may find particular application in diagnostic andrology. Evidently, not all redox sensors are created equal, so in future assessments of antioxidant activity, it will be important to match the chemistry of the probe with its analytical application.

Lipids are dynamic biological molecules that play key roles in metabolism, inflammation, cell signalling and structure. They are biologically significant in the physiology of conception and reproduction. Many of the mechanisms surrounding equine conception and the early feto-maternal dialogue are yet to be understood at a biochemical level. Recently, lipidomic technologies have advanced considerably and analytical strategies have been enhanced and diversified. Consequently, in-depth lipidomic exploration now has the potential to reveal new lipid biomarkers and biochemical relationships that improve our understanding of the processes leading to efficient and successful reproduction. This review considers the role of lipids in conception and establishment of pregnancy, providing new insights into the enigmatic pathways governing early reproductive physiology of the mare.

The ongoing mass extinction of animal species at an unprecedented rate is largely caused by human activities. Progressive habitat destruction and fragmentation is resulting in accelerated loss of biodiversity on a global scale. Over decades, captive breeding programs of non-domestic species were characterized by efforts to optimize species-specific husbandry, to increase studbook-based animal exchange, and to improve enclosure designs. To counter the ongoing dramatic loss of biodiversity, new approaches are warranted. Recently, new ideas, particularly the application of assisted reproduction technologies (ART), have been incorporated into classical zoo breeding programs. These technologies include semen and oocyte collection, artificial insemination, and in-vitro embryo generation. More futuristic ideas of advanced ART (aART) implement recent advances in biotechnology and stem-cell related approaches such as cloning, inner cell mass transfer (ICM), and the stem-cell-associated techniques (SCAT) for the generation of gametes and ultimately embryos of highly endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni) of which only two female individuals are left. Both, ART and aART greatly depend on and benefit from the rapidly evolving cryopreservation techniques and biobanking not only of genetic, but also of viable cellular materials suitable for the generation of induced pluripotent stem cells (iPSC). The availability of cryopreserved materials bridges gaps in time and space, thereby optimizing the available genetic variability and enhancing the chance to restore viable populations.

In vitro maturation (IVM) is an important reproductive technology used to produce embryos in vitro. However, the developmental potential of oocytes sourced for IVM is markedly lower than those matured in vivo. Previously, NAD+-elevating treatments have improved oocyte quality and embryo development in cattle and mice, suggesting that NAD+ is important during oocyte maturation. The aim of this study was to examine the effects of nicotinic acid (NA), nicotinamide (NAM) and nicotinamide mononucleotide (NMN) on oocyte maturation and subsequent embryo development. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation medium supplemented with NA, NAM and resveratrol or NMN. Mature oocytes were artificially activated and presumptive zygotes cultured for 7 days. Additionally, oocytes were matured without treatment then cultured for 7 days with NMN. Supplementing the IVM medium with NA improved maturation and blastocyst formation while NAM supplementation improved cleavage rates compared with untreated controls. Supplementing the IVM or embryo culture media with NMN had no effect on maturation or embryo development. The results show that supplementing the maturation medium with NA and NAM improved maturation and developmental potential of porcine oocytes.

Maternal recognition of pregnancy (MRP) is a process by which an early conceptus signals its presence to the maternal system and prevents the lysis of the corpus luteum, thus ensuring a maternal milieu supportive of pregnancy continuation. It is a fundamental aspect of reproductive biology, yet in the horse, the mechanism underlying MRP remains unknown. This review seeks to address some of the controversies surrounding the evidence and theories of MRP in the equine species, such as the idea that the horse does not conform to the MRP paradigm established in other species or that equine MRP involves a mechanical, rather than chemical, signal. The review examines the challenges of studying this particularly clandestine phenomenon along with the new tools in scientific research that will drive this quest forward in coming years, and discusses the value of knowledge gleaned along this path in the context of clinical applications for improving breeding outcomes in the horse industry.

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 R2 = 0.38, P = 0.01; HK1 R2 = 0.46, P = 0.001) and progressive motilities (AKAP4 R2 = 0.51, P = 0.001; HK1 R2 = 0.55, P = 0.01), percentage rapid (AKAP4 R2 = 0.29, P = 0.05; HK1 R2 = 0.58, P = 0.001), straight-line velocity (HK1 R2 = 0.50, P = 0.01) and straightness (HK1 R2 = 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.

Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell-cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC-MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim offurthering our understanding of this cell's complex biological machinery. We were able to identify 9883 peptides comprising 1030 proteins, which were subsequently attributed to 975 gene products. Gene ontology analysis for molecular and cellular processes revealed new information about the metabolism, antioxidant defences and receptors of stallion spermatozoa. Mitochondrial proteins and those involved in catabolic processes constituted dominant categories. Several enzymes specific to ß-oxidation of fatty acids were identified, and further experiments were carried out to ascertain their functional significance. Inhibition of carnitine palmitoyl transferase 1, a rate-limiting enzyme of ß-oxidation, reduced motility parameters, indicating that b-oxidation contributes to maintenance of motility in stallion spermatozoa.