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.

中文翻译：

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质谱法揭示了高质量和低质量种马精子中不同的蛋白质组学特征

马匹繁育业依赖于最佳种马繁殖力。传统的精子评估提供的关于射精质量的信息有限，不能单独预测受精潜力。本研究的目的是利用质谱法比较高质量和低质量种马精子的蛋白质组学特征，最终目标是确定生育生物标志物候选者。使用 Percoll 密度梯度对延长的种马精液 (n = 12) 进行分馏，以分离低质量和高质量的精子种群。进行了运动性和形态学评估，并使用 UHPLC-MS/MS 进行了蛋白质组学分析。高质量精子的总数较高（95.2±0.52% vs 70.6±4.20%；P≤0.001）和进行性运动（43.4±3.42% vs 27.3±4.32%；P≤0.05），形态正常细胞比例更高（50.2±4.34% vs 38.8±2.72%；P≤0.05）。UHPLC-MS/MS 对 1,069 种蛋白质进行了定量，其中 22 种蛋白质在高质量精子群体中的含量明显更高（P≤0.05）。A-激酶锚定蛋白 4 (AKAP4) 和己糖激酶 1 (HK1) 被认为是可能的生物标志物候选物，它们的差异表达通过免疫印迹得到证实。蛋白表达与总（AKAP4 R2=0.38，P≤0.01；HK1 R2=0.46，P≤0.001）和进行性运动（AKAP4 R2=0.51，P≤0.001；HK1 R2=0.55，P≤0.01）、百分比显着相关快速（AKAP4 R2=0.29，P≤0.05；HK1 R2=0.58，P≤0.001），直线速度（HK1 R2=0.50，P≤0.01）和直线度（HK1 R2=0.40，P≤0.01）。此外，AKAP4 极易被 4-羟基壬烯醛 (4HNE) 加成，这导致获能后磷酸化谱的整体减少。总之，高质量和低质量种马精子的蛋白质组学特征存在很大差异，AKAP4 和 HK1 等蛋白质可作为射精质量的生物标志物。