The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilising 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). 1,069 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.

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.