Aging of stallion spermatozoa stored in vitro is delayed at 22°C using a 67mm glucose-10mm pyruvate-based media.

Abstract

Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5°C. To determine if 10mm pyruvate in a 67mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C. Stallion ejaculates were extendedin: INRA96 (67mm glucose, non-pyruvate control), modified Tyrode's (67mm glucose-10mm pyruvate), supplemented with 0, 10, 50, and 100μM 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 96h of storage at 22°C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3h in modified Tyrode's 67mm glucose-10mm pyruvate and modified Tyrode's 67mm glucose, and metabolic analysis conducted. After 96h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6%±2.3%, while in the 67mm glucose-10mm pyruvate/10μm itaconate extender, total motility was 34.7%±3.8% (p=0.0066). After 96h, 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. Aliquots stored in a 67mm glucose-10mm pyruvate-based medium supplemented with 10μM itaconate, maintained a 35% total motility after 96h 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+.