title: Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm.
abstract: Repeatable methods for IVF have not been established in the horse, reflecting the failure of standard capacitating media to induce changes required for fertilization capacity in equine sperm. One important step in capacitation is membrane cholesterol efflux, which in other species is triggered by cholesterol oxidation and is typically enhanced using albumin as a sterol acceptor. We incubated equine sperm in the presence of calcium, BSA, and bicarbonate, alone or in combination. Bicarbonate induced an increase in reactive oxygen species (ROS) that was abolished by the addition of calcium or BSA. Bicarbonate induced protein tyrosine phosphorylation (PY), even in the presence of calcium or BSA. Incubation at high pH enhanced PY but did not increase ROS production. Notably, no combination of these factors was associated with significant cholesterol efflux, as assessed by fluorescent quantitative cholesterol assay and confirmed by filipin staining. By contrast, sperm treated with methyl-β-cyclodextrin showed a significant reduction in cholesterol levels, but no significant increase in PY or ROS. Presence of BSA increased sperm binding to bovine zonae pellucidae in all three stallions. These results show that presence of serum albumin is not associated with a reduction in membrane cholesterol levels in equine sperm, highlighting the failure of equine sperm to exhibit core capacitation-related changes in a standard capacitating medium. These data indicate an atypical relationship among cholesterol efflux, ROS production, and PY in equine sperm. Our findings may help to elucidate factors affecting failure of equine IVF under standard conditions.
authors.affiliation: CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain bea_macias@hotmail.com.
authors.forename: B
authors.initials: B
authors.lastname: Macías-García
authors.affiliation: CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: L
authors.initials: L
authors.lastname: González-Fernández
authors.affiliation: CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: S C
authors.initials: SC
authors.lastname: Loux
authors.affiliation: CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: A M
authors.initials: AM
authors.lastname: Rocha
authors.affiliation: CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: T
authors.initials: T
authors.lastname: Guimarães
authors.affiliation: CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: F J
authors.initials: FJ
authors.lastname: Peña
authors.affiliation: CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: D D
authors.initials: DD
authors.lastname: Varner
authors.affiliation: CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain CECA/ICETA - Animal Sciences CentreICBAS-University of Porto, Campus Agrario de Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, PortugalDepartments of Veterinary Physiology and PharmacologyLarge Animal Clinical SciencesCollege of Veterinary Medicine and Biomedical Science, Texas A&M University, College Station, Texas, USALaboratory of Equine ReproductionFaculty of Veterinary Medicine, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: K
authors.initials: K
authors.lastname: Hinrichs
mesh_terms: D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology*; D002021:Buffers; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D005260:Female; D006736:Horses; D007124:Immunoenzyme Techniques; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D013075:Sperm Capacitation / Q000187:drug effects*; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001639:Bicarbonates; D002021:Buffers; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D002784:Cholesterol; D002118:Calcium