Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration†

Abstract

Some stallions yield ejaculates which do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through HPLC/MS/MS and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5 °C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine seminal plasma using human orthologs was performed using g: profiler showing enriched Reactome and KEGG pathways related to hexose metabolism, vesicle mediated transport, post translational modification of proteins and immune response. Specific proteins overrepresented in stallions tolerating conservation by refrigeration included a peroxiredoxin-6 like protein, and transcobalamin-2, a primary vitamin B12-binding and transport protein. Also, the protein involved in protein glycosylation, ST3 beta-galactoside alpha-2,3-sialyltransferase 1 was present in good stallions. These proteins were nearly absent in poor stallions. Particularly, annexinA2 appeared as to be the most powerful discriminant variable for identification of stallions needing seminal plasma-removal prior to refrigeration, with a p = 0.002 and a q value = 0.005. Overall this is the first detailed study of the equine SP-proteome, showing the potential value of specific proteins as discriminant bio-markers for clinical classification of stallions for AI.

Dataset of the sperm proteome of stallions with different motility

“…The same protocol as described in [3] , [4] , [5] , [6] , [7] , [8] , [9] was used. Stallion ejaculates were washed in PBS (600gx 10’), then the pellet formed by the spermatozoa was frozen immediately in liquid nitrogen and kept frozen at -80 °C.…”

Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis

“…For sperm morphology analysis, samples of raw semen were xed with buffered-formal saline (BFS; 4.75% formaldehyde) and analyzed using differential interference contrast (DIC) microscopy (1,563x, Olympus BX-60, Olympus Corporation, Melville, NY, USA). Sperm morphological classi cation was done as previously reported 30 . All morphologic abnormalities were counted for each spermatozoon to determine the incidence rate.…”

An integrated overview on the regulation of sperm metabolism (glycolysis-Krebs cycle-oxidative phosphorylation)

“…• − ) and hydrogen peroxide (H 2 O 2 ) are byproducts of these reactions; to maintain these reactions under control, spermatozoa are provided with sophisticated antioxidant systems in both seminal plasma (Gaitskell-Phillips et al, 2020) and the intracellular space (Ozkosem et al, 2016;Lee et al, 2017;O'Flaherty and Matsushita-Fournier, 2017;Fernandez and O'Flaherty, 2018;Fernandez et al, 2019;O'Flaherty et al, 2019;O'Flaherty, 2020).…”