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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.

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 of furthering 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 β-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 β-oxidation, reduced motility parameters, indicating that β-oxidation contributes to maintenance of motility in stallion spermatozoa.

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.

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.