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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.
© 2015 Society for Reproduction and Fertility.
Publication Date: 2014-10-27 PubMed ID: 25349439DOI: 10.1530/REP-14-0457Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research investigates the influence of calcium, bicarbonate, and albumin on the capacitation process of horse sperm, an essential step for successful in vitro fertilisation (IVF)—the method of achieving pregnancy by combining egg and sperm outside the body. The study highlights that horse sperm react atypically to the traditional methods leading to capacitation, indicating a potential reason for the inconsistent success of equine IVF procedures.
Research Context and Objective
- IVF in horses has failed to establish repeatable methods due to the lack of necessary changes in equine sperm when subjected to standard capacitating media. A crucial step of capacitation is the efflux or loss of membrane cholesterol, typically enhanced using albumin— a protein that can accept sterols and assist in cholesterol removal.
- The goal of this research was to investigate the effect of calcium, BSA (an albumin type), and bicarbonate on equine sperm capacitation processes, and to determine their collective or individual impacts.
Methods and Findings
- The team incubated horse sperm with calcium, BSA, and bicarbonate, used separately or together. They found that bicarbonate alone led to an increase in reactive oxygen species (ROS), which was counteracted by the addition of calcium or BSA.
- In spite of the presence of calcium or BSA, bicarbonate still induced protein tyrosine phosphorylation (PY) — a process that is typically associated with sperm capacitation.
- Although a high pH environment intensified PY, it didn’t increase ROS production. Importantly, applying these factors did not result in a noticeable cholesterol efflux, this was assessed by a fluorescent cholesterol assay and confirmed by filipin staining – a technique used to visualize cholesterol.
- When sperm was treated with methyl-β-cyclodextrin, a significant reduction in cholesterol levels was observed, but in the absence of PY or ROS increase. Adding BSA led to an increase in sperm binding to bovine zonae pellucidae — a thick membrane surrounding the egg — across all three stallions studied.
Conclusion and Significance
- The research demonstrates that the presence of serum albumin does not decrease membrane cholesterol levels in horse sperm, pointing to the fact that equine sperm does not show core capacitation changes even in a standard capacitating medium.
- The findings indicate an unusual interplay among cholesterol efflux, ROS production, and PY in horse sperm, which might help explain the frequent failure of equine IVF under standard conditions. These insights could be valuable in refining IVF procedures for horses, potentially leading to more consistent success rates.
Cite This Article
APA
Macías-García B, González-Fernández L, Loux SC, Rocha AM, Guimarães T, Peña FJ, Varner DD, Hinrichs K.
(2014).
Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm.
Reproduction, 149(1), 87-99.
https://doi.org/10.1530/REP-14-0457 Publication
Researcher Affiliations
- 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.
- 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.
- 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.
- 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.
- 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 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.
MeSH Terms
- Animals
- Bicarbonates / pharmacology
- Buffers
- Calcium / pharmacology
- Cattle
- Cholesterol / metabolism
- Female
- Horses
- Immunoenzyme Techniques
- Male
- Oocytes / cytology
- Oocytes / drug effects
- Oocytes / metabolism
- Phosphorylation / drug effects
- Reactive Oxygen Species / metabolism
- Serum Albumin, Bovine / pharmacology
- Signal Transduction / drug effects
- Sperm Capacitation / drug effects
- Spermatozoa / cytology
- Spermatozoa / drug effects
- Spermatozoa / metabolism
- Tyrosine / metabolism
Citations
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