FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Theriogenology2000; 53(9); 1691-1703; doi: 10.1016/s0093-691x(00)00308-3

Assessment of equine sperm mitochondrial function using JC-1.

Abstract: The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged through a Percoll gradient (PERC). Spermatozoa were resuspended to 25 x 10(6) cells/mL, samples were split, and one sample was repeatedly flash frozen (FF) in LN2 and thawed. The following gradients of PERC:FF were prepared: 100:0 (100), 75:25(75), 50:50 (50), 25:75 (25) and 0:100 (0). Samples were stained with 2.0 microM JC-1 and assessed for staining by flow cytometry and by a fluorescence microplate reader. A total of 10,000 gated events was analyzed per sample with flow cytometry. The mean percentage of cells staining orange for the 100, 75, 50, 25 and 0 treatments was 92.5, 72.8, 53.4, 27.3 and 7.3, respectively. The expected percentage of spermatozoa forming JC-1 aggregates was correlated with the actual percentage of orange labeled sperm cells determined by flow cytometry (r2=0.98). Conversely, JC-1 monomer formation was negatively correlated with expected mitochondrial membrane potential (r2=-0.98). The blank corrected orange fluorescence, assessed by microplate assay, was significantly (P<0.0001) correlated with the expected (r2=0.49) and with the flow cytometric (r2=0.50) determination of percentage of spermatozoa with mitochondria of high membrane potential. Total orange and orange:green fluorescence was also correlated with mitochondrial function. These results indicate that JC-1 staining can accurately detect changes in mitochondrial membrane potential of equine spermatozoa. The relative fluorescence of JC-1 labeling patterns of equine spermatozoa can be accurately and objectively determined by flow cytometry and by a fluorescence microplate reader assay.
Get Full Text
Publication Date: 2000-09-01 PubMed ID: 10968415DOI: 10.1016/s0093-691x(00)00308-3Google 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.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study examines and validates the use of a specific dye called JC-1 in identifying the presence of high or low mitochondrial function in equine sperm cells. The application of this fluorescent dye has been used in other cell types but not yet on horse sperm cells.

Objective of the Study

  • The central aim of this research was to apply JC-1 staining, a method not previously used with equine spermatozoa, and assess it by flow cytometry or a fluorescence microplate reader. The goal was to evaluate the mitochondrial function of equine spermatozoa.

Methodology

  • Six ejaculates from four stallions were collected and centrifuged through a Percoll gradient (PERC).
  • The sperm cells were then resuspended to 25 x 10(6) cells/mL and split into samples. Among them, one sample was repeatedly flash frozen in LN2 and thawed.
  • These prepared samples were stained with 2.0 microM JC-1 and then evaluated for staining by flow cytometry and by a fluorescence microplate reader.
  • For each sample, 10,000 events were analysed.

Findings

  • The results showed that the percentage of cells staining orange, an indication of high mitochondrial function, decreased as the ratio of freeze-thawed cells increased in the sample mix.
  • The mean percentage of cells staining orange for the 100, 75, 50, 25 and 0 treatments was 92.5, 72.8, 53.4, 27.3 and 7.3, respectively.
  • The expected percentage of sperm cells forming JC-1 aggregates correlated strongly with the actual percentage of orange labeled sperm cells, as determined by flow cytometry (r2=0.98).
  • A negative correlation (r2=-0.98) was identified between JC-1 monomer formation and the expected mitochondrial membrane potential.
  • The microplate reader assessment of the blank-corrected orange fluorescence, or high mitochondrial membrane potential, strongly correlated with both the expected (r2=0.49) and the flow cytometric (r2=0.50) determinations.

Conclusion

  • Overall, the researchers concluded that JC-1 staining can accurately detect changes in mitochondrial membrane potential of equine spermatozoa and the relative fluorescence of JC-1 labelling patterns of equine spermatozoa.
  • This can be accurately and objectively determined by flow cytometry and by a fluorescence microplate reader assay, suggesting possibilities for its practical application in equine reproduction and genetics.

Cite This Article

APA
Gravance CG, Garner DL, Baumber J, Ball BA. (2000). Assessment of equine sperm mitochondrial function using JC-1. Theriogenology, 53(9), 1691-1703. https://doi.org/10.1016/s0093-691x(00)00308-3

Publication

Theriogenology
ISSN: 0093-691X
NlmUniqueID: 0421510
Country: United States
Language: English
Volume: 53
Issue: 9
Pages: 1691-1703

Researcher Affiliations

Gravance, C G
  • Department of Population, Health and Reproduction, University of California, Davis, USA.
Garner, D L
    Baumber, J
      Ball, B A

        MeSH Terms

        • Animals
        • Benzimidazoles / chemistry
        • Carbocyanines / chemistry
        • Flow Cytometry / veterinary
        • Fluorescent Dyes / chemistry
        • Horses / physiology
        • Male
        • Microscopy, Fluorescence / veterinary
        • Mitochondria / chemistry
        • Mitochondria / physiology
        • Propidium / chemistry
        • Regression Analysis
        • Semen / chemistry
        • Spermatozoa / chemistry
        • Spermatozoa / physiology

        Citations

        This article has been cited 35 times.
        1. Medica AJ, Lambourne S, Aitken RJ. Predicting the Outcome of Equine Artificial Inseminations Using Chilled Semen. Animals (Basel) 2023 Mar 30;13(7).
          doi: 10.3390/ani13071203pubmed: 37048459google scholar: lookup
        2. Dziekońska A, Lecewicz M, Partyka A, Niżański W. Fluorescence Microscopy and Flow-Cytometry Assessment of Substructures in European Red Deer Epididymal Spermatozoa after Cryopreservation. Animals (Basel) 2023 Mar 8;13(6).
          doi: 10.3390/ani13060990pubmed: 36978531google scholar: lookup
        3. Dziekońska A, Neuman NM, Burdal KK, Wiszniewska-Łaszczych A, Bogdaszewski M. The Effect of Different Extenders on the Quality Characteristics of European Red Deer Epididymal Sperm Stored at 5 °C. Animals (Basel) 2022 Oct 4;12(19).
          doi: 10.3390/ani12192669pubmed: 36230410google scholar: lookup
        4. Silva AM, Ribeiro CT, Bernardino RL, Jarak I, Carvalho RA, Pereira-Sampaio MA, de Souza DB, Alves MG, Oliveira PF. Stress Hormone Corticosterone Controls Metabolic Mitochondrial Performance and Inflammatory Signaling of In Vitro Cultured Sertoli Cells. Biomedicines 2022 Sep 19;10(9).
          doi: 10.3390/biomedicines10092331pubmed: 36140432google scholar: lookup
        5. Eberhardt M, Prochowska S, Duszewska AM, Van Soom A, Olech W, Niżański W. The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa. BMC Vet Res 2022 Aug 10;18(1):305.
          doi: 10.1186/s12917-022-03408-zpubmed: 35945588google scholar: lookup
        6. Su J, Wang C, Song Y, Yang Y, Cao G. Effect of lactoferrin on ram sperm motility after cryopreservation. Anim Biosci 2022 Sep;35(9):1351-1359.
          doi: 10.5713/ab.21.0561pubmed: 35240020google scholar: lookup
        7. Orsolini MF, Meyers SA, Dini P. An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section I. Animals (Basel) 2021 Nov 13;11(11).
          doi: 10.3390/ani11113248pubmed: 34827983google scholar: lookup
        8. Usuga A, Tejera I, Gómez J, Restrepo O, Rojano B, Restrepo G. Cryoprotective Effects of Ergothioneine and Isoespintanol on Canine Semen. Animals (Basel) 2021 Sep 22;11(10).
          doi: 10.3390/ani11102757pubmed: 34679779google scholar: lookup
        9. Gallo A, Esposito MC, Tosti E, Boni R. Sperm Motility, Oxidative Status, and Mitochondrial Activity: Exploring Correlation in Different Species. Antioxidants (Basel) 2021 Jul 16;10(7).
          doi: 10.3390/antiox10071131pubmed: 34356364google scholar: lookup
        10. Gao X, Jiang Z, Yan X, Liu J, Li F, Liu P, Li J, Wei Y, Sun YE, Zhang Y, Wang C. ATF5, a putative therapeutic target for the mitochondrial DNA 3243A > G mutation-related disease. Cell Death Dis 2021 Jul 14;12(7):701.
          doi: 10.1038/s41419-021-03993-1pubmed: 34262025google scholar: lookup
        11. Bucci D, Spinaci M, Galeati G, Tamanini C. Different approaches for assessing sperm function. Anim Reprod 2020 May 22;16(1):72-80.
          doi: 10.21451/1984-3143-AR2018-122pubmed: 33299480google scholar: lookup
        12. Habib MR, Ghoname SI, Ali RE, El-Karim RMG, Youssef AA, Croll RP, Miller MW. Biochemical and apoptotic changes in the nervous and ovotestis tissues of Biomphalaria alexandrina following infection with Schistosoma mansoni. Exp Parasitol 2020 Jun;213:107887.
          doi: 10.1016/j.exppara.2020.107887pubmed: 32224062google scholar: lookup
        13. Barbagallo F, La Vignera S, Cannarella R, Aversa A, Calogero AE, Condorelli RA. Evaluation of Sperm Mitochondrial Function: A Key Organelle for Sperm Motility. J Clin Med 2020 Jan 29;9(2).
          doi: 10.3390/jcm9020363pubmed: 32013061google scholar: lookup
        14. Marzano G, Moscatelli N, Di Giacomo M, Martino NA, Lacalandra GM, Dell'Aquila ME, Maruccio G, Primiceri E, Chiriacò MS, Zara V, Ferramosca A. Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm. Biology (Basel) 2020 Jan 27;9(2).
          doi: 10.3390/biology9020022pubmed: 32012799google scholar: lookup
        15. Ugur MR, Saber Abdelrahman A, Evans HC, Gilmore AA, Hitit M, Arifiantini RI, Purwantara B, Kaya A, Memili E. Advances in Cryopreservation of Bull Sperm. Front Vet Sci 2019;6:268.
          doi: 10.3389/fvets.2019.00268pubmed: 31552277google scholar: lookup
        16. Daniele JR, Heydari K, Dillin A. Mitochondrial Subtype Identification and Characterization. Curr Protoc Cytom 2018 Jul;85(1):e41.
          doi: 10.1002/cpcy.41pubmed: 29944197google scholar: lookup
        17. Li CY, Zhao YH, Hao HS, Wang HY, Huang JM, Yan CL, Du WH, Pang YW, Zhang PP, Liu Y, Zhu HB, Zhao XM. Resveratrol significantly improves the fertilisation capacity of bovine sex-sorted semen by inhibiting apoptosis and lipid peroxidation. Sci Rep 2018 May 15;8(1):7603.
          doi: 10.1038/s41598-018-25687-zpubmed: 29765076google scholar: lookup
        18. Carreira JT, Trevizan JT, Carvalho IR, Kipper B, Rodrigues LH, Silva C, Perri SHV, Drevet JR, Koivisto MB. Does sperm quality and DNA integrity differ in cryopreserved semen samples from young, adult, and aged Nellore bulls?. Basic Clin Androl 2017;27:12.
          doi: 10.1186/s12610-017-0056-9pubmed: 28649382google scholar: lookup
        19. Zou P, Liu L, Zheng LD, Payne KK, Manjili MH, Idowu MO, Zhang J, Schmelz EM, Cheng Z. Coordinated Upregulation of Mitochondrial Biogenesis and Autophagy in Breast Cancer Cells: The Role of Dynamin Related Protein-1 and Implication for Breast Cancer Treatment. Oxid Med Cell Longev 2016;2016:4085727.
          doi: 10.1155/2016/4085727pubmed: 27746856google scholar: lookup
        20. Daniele JR, Heydari K, Arriaga EA, Dillin A. Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry. Anal Chem 2016 Jun 21;88(12):6309-16.
          doi: 10.1021/acs.analchem.6b00542pubmed: 27210103google scholar: lookup
        21. Yin Y, Liu L, Yang C, Lin C, Veith GM, Wang C, Sutovsky P, Zhou P, Ma L. Cell Autonomous and Nonautonomous Function of CUL4B in Mouse Spermatogenesis. J Biol Chem 2016 Mar 25;291(13):6923-35.
          doi: 10.1074/jbc.M115.699660pubmed: 26846852google scholar: lookup
        22. Plaza Davila M, Martin Muñoz P, Tapia JA, Ortega Ferrusola C, Balao da Silva C C, Peña FJ. Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility. PLoS One 2015;10(9):e0138777.
          doi: 10.1371/journal.pone.0138777pubmed: 26407142google scholar: lookup
        23. Sellami H, Znazen A, Sellami A, Mnif H, Louati N, Ben Zarrouk S, Keskes L, Rebai T, Gdoura R, Hammami A. Molecular detection of Chlamydia trachomatis and other sexually transmitted bacteria in semen of male partners of infertile couples in Tunisia: the effect on semen parameters and spermatozoa apoptosis markers. PLoS One 2014;9(7):e98903.
          doi: 10.1371/journal.pone.0098903pubmed: 25019616google scholar: lookup
        24. Chis R, Sharma P, Bousette N, Miyake T, Wilson A, Backx PH, Gramolini AO. α-Crystallin B prevents apoptosis after H2O2 exposure in mouse neonatal cardiomyocytes. Am J Physiol Heart Circ Physiol 2012 Oct 15;303(8):H967-78.
          doi: 10.1152/ajpheart.00040.2012pubmed: 22904156google scholar: lookup
        25. Bhogal RH, Weston CJ, Curbishley SM, Adams DH, Afford SC. Autophagy: a cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress. Autophagy 2012 Apr;8(4):545-58.
          doi: 10.4161/auto.19012pubmed: 22302008google scholar: lookup
        26. Hossain MS, Johannisson A, Wallgren M, Nagy S, Siqueira AP, Rodriguez-Martinez H. Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art. Asian J Androl 2011 May;13(3):406-19.
          doi: 10.1038/aja.2011.15pubmed: 21478895google scholar: lookup
        27. Li J, Qin Z, Liang Z. The prosurvival role of autophagy in Resveratrol-induced cytotoxicity in human U251 glioma cells. BMC Cancer 2009 Jun 30;9:215.
          doi: 10.1186/1471-2407-9-215pubmed: 19566920google scholar: lookup
        28. Katila T. In vitro evaluation of frozen-thawed stallion semen: a review. Acta Vet Scand 2001;42(2):199-217.
          doi: 10.1186/1751-0147-42-199pubmed: 11503365google scholar: lookup
        29. Strassner FM, Demattio L, Siuda M, Malama E, Muffels G, Bollwein H. Relationships Between Metabolism of Cryopreserved Equine Sperm Determined by the Seahorse Analyzer and Sperm Characteristics Measured by Flow Cytometry and Computer-Assisted Analysis of Motility. Vet Sci 2025 Nov 21;12(12).
          doi: 10.3390/vetsci12121109pubmed: 41472089google scholar: lookup
        30. Aiwale BS, Maurya R, Naqvi S. Green synthesized selenium nanoparticles mitigate cyclophosphamide-induced reproductive toxicity in male Wistar rats. Naunyn Schmiedebergs Arch Pharmacol 2025 Dec 26;.
          doi: 10.1007/s00210-025-04929-8pubmed: 41452359google scholar: lookup
        31. Pasciu V, Nassif C, Dattena M, Succu S, Sotgiu FD, Cannas A, Cossu I, Baralla E, Chessa F, Berlinguer F, Mara L. Effect of Different Extenders on the Oxidative Status and Fertility of Sarda Ram Liquid Semen Stored at 15 °C. Antioxidants (Basel) 2025 Jul 30;14(8).
          doi: 10.3390/antiox14080932pubmed: 40867829google scholar: lookup
        32. Feng Q, Yang Y, Zhang B, Shi W, Fang Y, Xu C, Deng Z, Feng W, Shi D. The Effects of Limonin, Myo-Inositol, and L-Proline on the Cryopreservation of Debao Boar Semen. Animals (Basel) 2025 Jul 27;15(15).
          doi: 10.3390/ani15152204pubmed: 40804994google scholar: lookup
        33. Solís JM, Sevilla F, Silvestre MA, Araya-Zúñiga I, Roldan ERS, Saborío-Montero A, Valverde A. Effect of Thawing Procedure and Thermo-Resistance Test on Sperm Motility and Kinematics Patterns in Two Bovine Breeds. Animals (Basel) 2024 Sep 25;14(19).
          doi: 10.3390/ani14192768pubmed: 39409716google scholar: lookup
        34. Hu M, You Y, Li Y, Ma S, Li J, Miao M, Quan Y, Yu W. Deacetylation of ACO2 Is Essential for Inhibiting Bombyx mori Nucleopolyhedrovirus Propagation. Viruses 2023 Oct 12;15(10).
          doi: 10.3390/v15102084pubmed: 37896861google scholar: lookup
        35. Diak N, Śliwińska MA, Student S, Świątek P. The three-dimensional conformation and activity of mitochondria in syncytial male germ line-cysts of medicinal leeches. Cell Tissue Res 2023 Nov;394(2):325-342.
          doi: 10.1007/s00441-023-03825-ypubmed: 37642734google scholar: lookup
        Subscribe to our newsletter
        Join 106,698 horse owners like you who receive our email updates on horse health and nutrition.