Abstract：
BACKGROUND: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5°C.
OBJECTIVE: To determine if 10 mm pyruvate in a 67 mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C.
METHODS: Stallion ejaculates were extendedin: INRA96 (67 mm glucose, non-pyruvate control), modified Tyrode\'s (67 mm glucose-10 mm pyruvate), supplemented with 0, 10, 50, and 100 μM itaconate. As itaconate was vehiculated in DMSO, a control vehicle was also included. Sperm motility, viability, mitochondrial membrane potential, and production of reactive oxygen species were measured after collection and again after 48 and 96 h of storage at 22°C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3 h in modified Tyrode\'s 67 mm glucose-10 mm pyruvate and modified Tyrode\'s 67 mm glucose, and metabolic analysis conducted.
RESULTS: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6% ± 2.3%, while in the 67 mm glucose-10 mm pyruvate/10 μm itaconate extender, total motility was 34.7% ± 3.8% (p = 0.0066). After 96 h, viability was better in most pyruvate-based media, and the mitochondrial membrane potential in spermatozoa extended in INRA96 was relatively lower (p < 0.0001). Metabolomics revealed that in the spermatozoa incubated in the high pyruvate media, there was an increase in the relative amounts of NAD+, pyruvate, lactate, and ATP.
CONCLUSIONS: Aliquots stored in a 67 mm glucose-10 mm pyruvate-based medium supplemented with 10 μM itaconate, maintained a 35% total motility after 96 h of storage at 22°C, which is considered the minimum acceptable motility for commercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD+.
OBJECTIVE: To determine if 10 mm pyruvate in a 67 mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C.
METHODS: Stallion ejaculates were extendedin: INRA96 (67 mm glucose, non-pyruvate control), modified Tyrode\'s (67 mm glucose-10 mm pyruvate), supplemented with 0, 10, 50, and 100 μM itaconate. As itaconate was vehiculated in DMSO, a control vehicle was also included. Sperm motility, viability, mitochondrial membrane potential, and production of reactive oxygen species were measured after collection and again after 48 and 96 h of storage at 22°C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3 h in modified Tyrode\'s 67 mm glucose-10 mm pyruvate and modified Tyrode\'s 67 mm glucose, and metabolic analysis conducted.
RESULTS: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6% ± 2.3%, while in the 67 mm glucose-10 mm pyruvate/10 μm itaconate extender, total motility was 34.7% ± 3.8% (p = 0.0066). After 96 h, viability was better in most pyruvate-based media, and the mitochondrial membrane potential in spermatozoa extended in INRA96 was relatively lower (p < 0.0001). Metabolomics revealed that in the spermatozoa incubated in the high pyruvate media, there was an increase in the relative amounts of NAD+, pyruvate, lactate, and ATP.
CONCLUSIONS: Aliquots stored in a 67 mm glucose-10 mm pyruvate-based medium supplemented with 10 μM itaconate, maintained a 35% total motility after 96 h of storage at 22°C, which is considered the minimum acceptable motility for commercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD+.
摘要：
背景：大多数马精剂量的商业是在5°C冷藏下使用商业增量剂进行的。
目的：确定在67毫米葡萄糖补充剂中10毫米丙酮酸盐并在22°C下储存是否可以作为冷却至5°C的替代储存方法的基础。
方法：种马射精是延伸素：INRA96（67毫米葡萄糖，非丙酮酸对照)，改良的Tyrode\s（67毫米葡萄糖-10毫米丙酮酸盐），补充0、10、50和100μM衣康酸。当衣康酸酯在DMSO中流动时，还包括控制车辆。精子运动,生存能力，线粒体膜电位，收集后和在22°C下储存48和96小时后再次测量活性氧的产生。为了揭示分子代谢变化，精子在改良的Tyrode67毫米葡萄糖-10毫米丙酮酸和改良的Tyrode67毫米葡萄糖中孵育3小时，并进行代谢分析。
结果：在对照中储存96小时后，INRA96的总运动性非常差，为5.6％±2.3％，而在67毫米葡萄糖-10毫米丙酮酸盐/10μm衣康酸补充剂中，总活动力为34.7%±3.8%(p=0.0066)。96小时后，在大多数基于丙酮酸的培养基中，生存力更好，在INRA96中延伸的精子中线粒体膜电位相对较低(p<0.0001)。代谢组学显示，在高丙酮酸培养基中孵育的精子中，NAD+的相对数量有所增加，丙酮酸,乳酸，和ATP。
结论：等分试样储存在67毫米葡萄糖-10毫米丙酮酸基础培养基中，补充了10μM衣康酸，在22°C下储存96小时后保持35%的总运动性，这被认为是商业化可接受的最低运动性。改善可能与丙酮酸向乳酸的转化和NAD+的再生有关。
目的：确定在67毫米葡萄糖补充剂中10毫米丙酮酸盐并在22°C下储存是否可以作为冷却至5°C的替代储存方法的基础。
方法：种马射精是延伸素：INRA96（67毫米葡萄糖，非丙酮酸对照)，改良的Tyrode\s（67毫米葡萄糖-10毫米丙酮酸盐），补充0、10、50和100μM衣康酸。当衣康酸酯在DMSO中流动时，还包括控制车辆。精子运动,生存能力，线粒体膜电位，收集后和在22°C下储存48和96小时后再次测量活性氧的产生。为了揭示分子代谢变化，精子在改良的Tyrode67毫米葡萄糖-10毫米丙酮酸和改良的Tyrode67毫米葡萄糖中孵育3小时，并进行代谢分析。
结果：在对照中储存96小时后，INRA96的总运动性非常差，为5.6％±2.3％，而在67毫米葡萄糖-10毫米丙酮酸盐/10μm衣康酸补充剂中，总活动力为34.7%±3.8%(p=0.0066)。96小时后，在大多数基于丙酮酸的培养基中，生存力更好，在INRA96中延伸的精子中线粒体膜电位相对较低(p<0.0001)。代谢组学显示，在高丙酮酸培养基中孵育的精子中，NAD+的相对数量有所增加，丙酮酸,乳酸，和ATP。
结论：等分试样储存在67毫米葡萄糖-10毫米丙酮酸基础培养基中，补充了10μM衣康酸，在22°C下储存96小时后保持35%的总运动性，这被认为是商业化可接受的最低运动性。改善可能与丙酮酸向乳酸的转化和NAD+的再生有关。
