Unit of Infection Diseases, University of Extremadura, Caceres, Spain.
Laboratory of Equine Reproduction and Equine Spermatology, University of Extremadura, Caceres, Spain.
Theriogenology. 2018 Jul 1;114:212-220. doi: 10.1016/j.theriogenology.2018.03.028. Epub 2018 Mar 26.
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 HO, 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), HO 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 HO 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 HO and this higher superoxide production may reflect an intense sperm functionality. The bacterial load increased the production of HO 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 HO production and selecting the most functional sperm.
精液中存在的污染细菌可能会在储存过程中降低精子质量。已经采用了不同的方法来减少精液中的微生物负荷并避免储存过程中的细菌生长。本研究的目的是：1）评估消除精液中细菌的不同技术；2）研究总微生物负荷（TML）与 ROS 产生之间的关系；3）确定 TML 是否会影响冷藏精子的功能。从 11 头种马的精液中分离并通过 3 种方式处理：A. 延长精液；B. 常规离心精液，和 C. 通过 Androcoll-E（SLC）进行单层离心。所有样本均在 5°C 的 INRA 96 中保存 72 小时。在储存的 T0、T24、T48 和 T72 小时，从原始精液和不同处理的样本中取出等分试样进行细菌分析，并计算总微生物负荷（TML：CFU（菌落形成单位/ml）。通过流式细胞术评估 ROS 产生（HO 的二氯二氢荧光素二乙酸酯、超氧化物阴离子的二氢乙啶和总 ROS 的 CellROX 深红色）、活力（YO-PRO-1-碘化丙啶）和脂质过氧化（BODIPY-C11），并通过 CASA 评估运动性。分离出的细菌为棒状杆菌、放线杆菌、芽孢杆菌、皮肤杆菌、葡萄球菌、链球菌、青霉。精液的 TML 与活精子（r：-0.771）、死精子（r：0.580）、HO 产生（r：0.740）和总 ROS 产生（CellROX（+））（r：-0.607）、总运动性（r：0.587）、进行性运动性（r：-0.566）、VCL（r：-0.664）、VSL（r：-0.569）、VAP（r：-0.534）（p≤0.05）呈负相关。SLC 去除了 99.34%的微生物负荷，这与 HO 产生显著降低（p≤0.05）有关。然而，只有经过 Androcoll-E 处理的样本才有更高的总 ROS 产生（CellROX+）（p≤0.05）。这些结果表明，CellROX 染色可能识别超氧化物的产生而不是 HO，并且这种更高的超氧化物产生可能反映出精子功能的增强。细菌负荷增加了冷藏精液中 HO 的产生，这与对冷藏的耐受性降低有关。SLC 是去除细菌、降低 HO 产生和选择最具功能精子的最有效的精子处理技术。
