How does the microbial load affect the quality of equine cool-stored semen?

Contaminating bacteria present in stallion ejaculates may compromise sperm quality during storage. Different procedures have been used to reduce the load of microorganisms in semen and avoid bacterial growth during storage. The aims of this study were: 1) to evaluate different techniques to eliminate bacteria in semen 2) to study the relationship between total microflora load (TML) and ROS production; and 3) to determine if TML affects the functionality of cool-stored sperm. Ejaculates from 11 stallions were split and processed in 3 ways: A. extended semen; B. conventional centrifuged semen, and C. Single layer centrifugation through Androcoll-E (SLC). All samples were preserved in INRA 96 at 5 °C for 72 h. Aliquots from native semen and from different treatments were taken for bacteriological analysis at T0, T24, T48 and T72h of storage and Total microbial load (TML: CFU (colony-forming units/ml) was calculated. The ROS production (dichlorodihydrofluorescein diacetate for H2O2, dihydroethidium for superoxide anion and CellROX deep red for total ROS), viability (YO-PRO-1-Ethidium) and lipid peroxidation (BODIPY-C11) were assessed by flow cytometry, and motility by CASA. The bacteria isolated were Corynebacterium spp, Arcanobacterium spp, Bacillus spp, Dermobacter, Staphylococcus spp, Streptococcus spp, Penicilium spp. TML of semen showed correlations with live sperm (r: -0.771), dead sperm (r: 0.580), H2O2 production (r: 0.740), and total ROS production (CellROX (+)) (r: -0.607), Total motility (r: 0.587), Progressive motility (r: -0.566), VCL (r: -0.664), VSL (r: -0,569), VAP (r: -0.534) (p ≤ 0.05). SLC removed 99.34% of the microbial load, which was assicated with a significanlty reduced H2O2 production (p ≤ 0.05). However, only samples treated with Androcoll-E had a higher total ROS production (CellROX +) (p ≤ 0.05). These results suggest that CellROX stain probably identifies superoxide production rather than H2O2 and this higher superoxide production may reflect an intense sperm functionality. The bacterial load increased the production of H2O2 in cool-stored semen which was associated with lower tolerance to refrigeration. SLC was the sperm processing technique that was most efficient at removing bacteria, reducing H2O2 production and selecting the most functional sperm.