title: L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature.
abstract: The spermatozoa of many stallions do not tolerate being cooled, restricting the commercial viability of these animals and necessitating the development of a chemically defined room temperature (RT) storage medium. This study examined the impact of two major modulators of oxidative phosphorylation, pyruvate (Pyr) and L-carnitine (L-C), on the storage of stallion spermatozoa at RT. Optimal concentrations of Pyr (10 mM) and L-C (50 mM) were first identified and these concentrations were then used to investigate the effects of these compounds on sperm functionality and oxidative stress at RT. Mitochondrial and cytosolic reactive oxygen species, along with lipid peroxidation, were all significantly suppressed by the addition of L-C (48 h MitoSOX Red negative: 46.2% vs. 26.1%; 48 and 72 h dihydroethidium negative: 61.6% vs. 43.1% and 64.4% vs. 46.9%, respectively; 48 and 72 h 4-hydroxynonenal negative: 37.1% vs. 23.8% and 41.6% vs. 25.7%, respectively), while the Pyr + L-C combination resulted in significantly higher motility compared to the control at 72 h (total motility: 64.2% vs. 39.4%; progressive motility: 34.2% vs. 15.2%). In addition, supplementation with L-C significantly reduced oxidative DNA damage at 72 h (9.0% vs. 15.6%). To investigate the effects of L-C as an osmolyte, comparisons were made between media that were osmotically balanced with NaCl, choline chloride, or L-C. This analysis demonstrated that spermatozoa stored in the L-C balanced medium had significantly higher total motility (55.0% vs. 39.0%), rapid motility (44.0% vs. 25.7%), and ATP levels (70.9 vs. 12.8 ng/ml) following storage compared with the NaCl treatment, while choline chloride did not significantly improve these parameters compared to the control. Finally, mass spectrometry was used to demonstrate that a combination of Pyr and L-C produced significantly higher acetyl-L-carnitine production than any other treatment (6.7 pg/10(6) spermatozoa vs. control at 4.0 pg/10(6) spermatozoa). These findings suggest that Pyr and L-C could form the basis of a novel, effective RT storage medium for equine spermatozoa.
authors: Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, New South Wales, Australia zamira.gibb@newcastle.edu.au.
authors: Zamira
authors: Z
authors: Gibb
authors: Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, New South Wales, Australia.
authors: Sarah R
authors: SR
authors: Lambourne
authors: Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, New South Wales, Australia.
authors: Julianne
authors: J
authors: Quadrelli
authors: Analytical and Biomolecular Research Facility, Central Scientific Services, Research Services, University of Newcastle, Callaghan, New South Wales, Australia.
authors: Nathan D
authors: ND
authors: Smith
authors: Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, Faculty of Science and IT, University of Newcastle, Callaghan, New South Wales, Australia.
authors: Robert J
authors: RJ
authors: Aitken
mesh_terms: D000177:Acrosome / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002331:Carnitine / Q000494:pharmacology*; D002794:Choline / Q000494:pharmacology; D002843:Chromatin / Q000187:drug effects / Q000648:ultrastructure; D004249:DNA Damage; D006736:Horses; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000648:ultrastructure; D009994:Osmolar Concentration; D019289:Pyruvic Acid / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012662:Semen Preservation; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D013696:Temperature
chemical_list: D002843:Chromatin; D017382:Reactive Oxygen Species; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D002794:Choline; D002331:Carnitine
keywords: ATP; L-carnitine; artificial insemination; assisted reproductive technology; equids (donkeys, horses, zebras); fertility; oxidative stress; pyruvate; reactive oxygen species; sperm; sperm motility and transport; spermatozoa; stallion