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.

No abstract

In the present work we report a non-aqueous electrophoresis method (NACE) for the separation and determination of eight tricyclic antidepressants (TCAs) in human serum. Separation is carried out in 1 M acetic acid, 50 mM ammonium acetate and 10 mM SDS in ACN. Standards and samples were prepared in 0.8 M acetic acid in ACN and introduced by electrokinetic injection. The interactions between TCAs and acetate ions were studied and deduced from comparison of both the actual experimental and theoretical mobility. The establishment of calibration curves in the presence or absence of serum matrix, between 20 and 200 ng/mL, together with the determination of most important analytical parameters and a study of robustness were performed. Validation of the CE method was carried out with serum samples spiked with the analytes. The NACE determination method offers better resolution and higher sensitivity than the determination of TCAs in aqueous media.

No abstract

No abstract