How does the microbial load affect the quality of equine cool-stored semen?
Abstract: 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.
Copyright © 2018 Elsevier Inc. All rights reserved.
Publication Date: 2018-03-26 PubMed ID: 29653389DOI: 10.1016/j.theriogenology.2018.03.028Google Scholar: Lookup
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- Journal Article
Summary
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This research article investigates how the presence of bacteria in horse semen affects its quality during storage, and the effectiveness of various techniques to reduce bacterial contamination. The study concludes that a specific process named Single Layer Centrifugation (SLC) is most effective to manage bacteria, maintain sperm quality and enhance overall sperm functionality.
Objective and Methodology
- This research sought to understand the impact of microorganisms on the functionality and quality of cooled-stored horse semen, with the specific goals of evaluating techniques to reduce bacterial presence, examining the connection between bacteriological load and reactive oxygen species (ROS) production, and ascertaining how the bacteria load impacts the semen’s functionality.
- The semen samples were obtained from 11 stallions, divided, and processed in three different ways – via standard extension, standard centrifugation, and Single Layer Centrifugation through a specialized medium called ‘Androcoll-E’.
- The bacteriological load in all samples was measured at different time intervals (immediately, and after 24, 48, and 72 hours of storage). Other parameters assessed included ROS production, sperm viability and motility, and lipid peroxidation.
Findings and Conclusions
- A variety of bacteria were identified in the semen samples, including strains of Corynebacterium, Arcanobacterium, Bacillus, Dermobacter, Staphylococcus, Streptococcus, and Penicilium.
- The bacteria load was found to affect nearly all measured parameters, including sperm survival, ROS production, and motility, suggesting a fairly robust impact on the semen’s overall quality and functionality in storage.
- The SLC process was shown to be the most effective technique, removing over 99% of the bacterial load. Moreover, this process resulted in less production of hydroxyl radicals (HO) and an increased total ROS production, indicating higher sperm functionality.
- The higher ROS production (specifically, superoxide production) demonstrated by samples treated with the Androcoll-E medium suggests that this procedure may be more effective in reducing contamination and maintaining semen viability in cool storage. This could potentially improve the results of artificial insemination protocols.
Cite This Article
APA
Varela E, Rey J, Plaza E, Muñoz de Propios P, Ortiz-Rodríguez JM, Álvarez M, Anel-López L, Anel L, De Paz P, Gil MC, Morrell JM, Ortega-Ferrusola C.
(2018).
How does the microbial load affect the quality of equine cool-stored semen?
Theriogenology, 114, 212-220.
https://doi.org/10.1016/j.theriogenology.2018.03.028 Publication
Researcher Affiliations
- Unit of Infection Diseases, University of Extremadura, Caceres, Spain.
- Unit of Infection Diseases, University of Extremadura, Caceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, University of Extremadura, Caceres, Spain.
- Animal Reproduction and Obstetrics, University of León, León, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, University of Extremadura, Caceres, Spain.
- Animal Reproduction and Obstetrics, University of León, León, Spain.
- Animal Reproduction and Obstetrics, University of León, León, Spain.
- Animal Reproduction and Obstetrics, University of León, León, Spain.
- Animal Reproduction and Obstetrics, University of León, León, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, University of Extremadura, Caceres, Spain.
- Faculty of Veterinary Medicine and Animal Sciences, SLU, Uppsala, Sweden.
- Animal Reproduction and Obstetrics, University of León, León, Spain. Electronic address: cortf@unileon.es.
MeSH Terms
- Animals
- Bacteria / classification
- Bacteria / isolation & purification
- Cold Temperature
- Male
- Semen / microbiology
- Semen Preservation / veterinary
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