Aging of stallion spermatozoa stored in vitro is de-layed at 22ºC using a 67mM glucose–10mM py-ruvate-based media

Abstract

Background: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5◦C. Objectives: 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. Material and methods: Stallion ejaculates were extendedin: INRA96 (67 mM glucose, non-pyruvate control), modified Tyrode’s (67 mM glu-cose–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 modi-fied Tyrode’s 67mM glucose–10mM pyruvate and modified Tyrode’s 67mM glucose, andmetabolic analysis conducted. Results: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motil-ity 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. Discussion and conclusions: Aliquots stored in a 67 mM glucose–10 mM pyruvatebased 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 com-mercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD+.