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Journal of assisted reproduction and genetics2021; 38(9); 2465-2480; doi: 10.1007/s10815-021-02134-z

Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the “zombie sperm” dilemma.

Abstract: To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. Results: Using PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results ("zombie sperm"). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0. Conclusions: Flow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.
© 2021. The Author(s).
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Publication Date: 2021-05-15 PubMed ID: 33991296PubMed Central: PMC8490572DOI: 10.1007/s10815-021-02134-zGoogle Scholar: Lookup
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  • Journal Article

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.

This research paper explores the impact of sperm agglutination or clustering, on the accuracy of membrane integrity assessment of sperm cells using flow cytometry, and suggests methods to reduce this effect, including a new staining method. The study asserts that the sperm agglutination can skew the actual data of live sperm cells, causing a mismatch between motility and live acrosome-reacted equine sperm, which may explain the low success rates of A23187 treatment for equine in-vitro fertilization.

Methodology

  • The researchers studied equine or stallion sperm cells and measured their motility using a Computer Assisted Sperm Analysis (CASA).
  • The sperm were stained with either Propidium Iodide-Pisum Sativum Agglutinin (PI-PSA), a commonly used staining method, or a novel approach named Live/Dead-Pisum Sativum Agglutinin (LD-PSA), which uses a fixable live/dead stain and cell dissociation treatment.
  • The sperm cells were then analysed through flow cytometry, a technology that rapidly measures the physical and chemical characteristics of particles in a fluid.
  • Using LD-PSA, acrosome reaction (a process that occurs during fertilization when the sperm enzyme reaches the oocyte or egg) and plasma membrane status were determined in sperm cells treated with A23187, a mobile ion carrier.

Results

  • Membrane Integrity (MI; live sperm) measured with PI-PSA was significantly lower compared to total motility (TMOT), indicating inaccurate results, dubbed as “zombie sperm” in the study.
  • The researchers found out that sperm aggregates or clusters were predominantly of motile sperm. However, sperm agglutination hampers accurate flow cytometry, thus affecting the validity of membrane integrity records.
  • After A23187 treatment, loss of sperm motility was associated with disaggregation and increased MI.
  • The researchers noted that viable sperm become immotile, they individualize; hence, factors that decrease motility, such as A23187, result in an increase in measured MI.
  • Utilizing the LD-PSA method helped to disgregate the sperm cells before assessment, providing a more accurate determination of the sperm membrane’s status.

Conclusion and Implication

  • The research findings highlight the importance of addressing the issue of sperm agglutination or clustering, and its impact on the accuracy of membrane integrity assessment.
  • The study suggests the LD-PSA as a potentially better alternative to PI-PSA method, allowing for more accurate measurements of sperm membrane integrity.
  • These findings provide significant insight about the potential issues associated with using A23187 treatment for equine in-vitro fertilization, hence carrying profound value for future research on sperm capacitation.

Cite This Article

APA
Ortiz I, Felix M, Resende H, Ramírez-Agámez L, Love CC, Hinrichs K. (2021). Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the “zombie sperm” dilemma. J Assist Reprod Genet, 38(9), 2465-2480. https://doi.org/10.1007/s10815-021-02134-z

Publication

Journal of assisted reproduction and genetics
ISSN: 1573-7330
NlmUniqueID: 9206495
Country: Netherlands
Language: English
Volume: 38
Issue: 9
Pages: 2465-2480

Researcher Affiliations

Ortiz, Isabel
  • College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA.
  • Department of Clinical Studies-New Bolton Center, University of Pennsylvania, 382 W. Street Rd., Kennett Square, PA, 19348, USA.
Felix, Matheus
  • College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA.
  • Department of Clinical Studies-New Bolton Center, University of Pennsylvania, 382 W. Street Rd., Kennett Square, PA, 19348, USA.
Resende, Hélène
  • College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Ramírez-Agámez, Luisa
  • College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Love, Charles C
  • College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Hinrichs, Katrin
  • College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, TX, USA. katrinh@vet.upenn.edu.
  • Department of Clinical Studies-New Bolton Center, University of Pennsylvania, 382 W. Street Rd., Kennett Square, PA, 19348, USA. katrinh@vet.upenn.edu.

MeSH Terms

  • Animals
  • Cell Membrane / chemistry
  • Cryopreservation / veterinary
  • Flow Cytometry / veterinary
  • Horses
  • Male
  • Semen Preservation / veterinary
  • Sperm Agglutination
  • Sperm Capacitation
  • Sperm Motility

Conflict of Interest Statement

None.

References

This article includes 44 references
  1. Leemans B, Gadella BM, Stout TA, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A. Why doesn't conventional IVF work in the horse? The equine oviduct as a microenvironment for capacitation/fertilization.. Reproduction 2016 Dec;152(6):R233-R245.
    doi: 10.1530/REP-16-0420pubmed: 27651517google scholar: lookup
  2. Leemans B, Stout TAE, De Schauwer C, Heras S, Nelis H, Hoogewijs M, Van Soom A, Gadella BM. Update on mammalian sperm capacitation: how much does the horse differ from other species?. Reproduction 2019 May;157(5):R181-R197.
    doi: 10.1530/REP-18-0541pubmed: 30721132google scholar: lookup
  3. Palmer E, Bézard J, Magistrini M, Duchamp G. In vitro fertilization in the horse. A retrospective study.. J Reprod Fertil Suppl 1991;44:375-84.
    pubmed: 1795281
  4. Alm H, Torner H, Blottner S, Nürnberg G, Kanitz W. Effect of sperm cryopreservation and treatment with calcium ionophore or heparin on in vitro fertilization of horse oocytes.. Theriogenology 2001 Sep 15;56(5):817-29.
    doi: 10.1016/S0093-691X(01)00610-0pubmed: 11665884google scholar: lookup
  5. Hinrichs K, Love CC, Brinsko SP, Choi YH, Varner DD. In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium, duration of maturation, and sperm calcium ionophore treatment, and comparison with rates of fertilization in vivo after oviductal transfer.. Biol Reprod 2002 Jul;67(1):256-62.
    doi: 10.1095/biolreprod67.1.256pubmed: 12080025google scholar: lookup
  6. Zhang JJ, Muzs LZ, Boyle MS. In vitro fertilization of horse follicular oocytes matured in vitro.. Mol Reprod Dev 1990 Aug;26(4):361-5.
    doi: 10.1002/mrd.1080260411pubmed: 2223085google scholar: lookup
  7. Visconti PE. Understanding the molecular basis of sperm capacitation through kinase design.. Proc Natl Acad Sci U S A 2009 Jan 20;106(3):667-8.
    doi: 10.1073/pnas.0811895106pmc: PMC2630107pubmed: 19144927google scholar: lookup
  8. Visconti PE, Galantino-Homer H, Moore GD, Bailey JL, Ning X, Fornes M, Kopf GS. The molecular basis of sperm capacitation.. J Androl 1998 Mar-Apr;19(2):242-8.
    pubmed: 9570749
  9. Tulsiani DR, Abou-Haila A. Molecular events that regulate mammalian fertilization.. Minerva Ginecol 2011 Apr;63(2):103-18.
    pubmed: 21508901
  10. Boerke A, Brouwers JF, Olkkonen VM, van de Lest CH, Sostaric E, Schoevers EJ, Helms JB, Gadella BM. Involvement of bicarbonate-induced radical signaling in oxysterol formation and sterol depletion of capacitating mammalian sperm during in vitro fertilization.. Biol Reprod 2013 Jan;88(1):21.
    doi: 10.1095/biolreprod.112.101253pubmed: 23115269google scholar: lookup
  11. Flesch FM, Gadella BM. Dynamics of the mammalian sperm plasma membrane in the process of fertilization.. Biochim Biophys Acta 2000 Nov 10;1469(3):197-235.
    doi: 10.1016/S0304-4157(00)00018-6pubmed: 11063883google scholar: lookup
  12. Macías-García B, González-Fernández L, Loux SC, Rocha AM, Guimarães T, Peña FJ, Varner DD, Hinrichs K. Effect of calcium, bicarbonate, and albumin on capacitation-related events in equine sperm.. Reproduction 2015 Jan;149(1):87-99.
    doi: 10.1530/REP-14-0457pubmed: 25349439google scholar: lookup
  13. Bromfield EG, Aitken RJ, Gibb Z, Lambourne SR, Nixon B. Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa.. Reproduction 2014 Feb;147(2):153-66.
    doi: 10.1530/REP-13-0393pubmed: 24194571google scholar: lookup
  14. Leahy T, Rickard JP, Aitken RJ, de Graaf SP. Penicillamine prevents ram sperm agglutination in media that support capacitation.. Reproduction 2016 Feb;151(2):167-77.
    doi: 10.1530/REP-15-0413pubmed: 26705263google scholar: lookup
  15. Leemans B, Gadella BM, Stout TA, Sostaric E, De Schauwer C, Nelis H, Hoogewijs M, Van Soom A. Combined albumin and bicarbonate induces head-to-head sperm agglutination which physically prevents equine sperm-oviduct binding.. Reproduction 2016 Apr;151(4):313-30.
    doi: 10.1530/REP-15-0471pubmed: 26755687google scholar: lookup
  16. Loux SC, Crawford KR, Ing NH, González-Fernández L, Macías-García B, Love CC, Varner DD, Velez IC, Choi YH, Hinrichs K. CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm.. Biol Reprod 2013 Nov;89(5):123.
    doi: 10.1095/biolreprod.113.111708pubmed: 24048572google scholar: lookup
  17. Harayama H, Miyake M, Shidara O, Iwamoto E, Kato S. Effects of calcium and bicarbonate on head-to-head agglutination in ejaculated boar spermatozoa.. Reprod Fertil Dev 1998;10(5):445-50.
    doi: 10.1071/RD98124pubmed: 10461678google scholar: lookup
  18. Harayama H, Miyake M, Kato S. Role of cyclic adenosine 3',5'-monophosphate and serum albumin in head-to-head agglutination of boar spermatozoa.. Reprod Fertil Dev 2000;12(5-6):307-18.
    doi: 10.1071/RD00030pubmed: 11451022google scholar: lookup
  19. Harayama H, Magargee SF, Kunze E, Shidara O, Iwamoto E, Arikawa S, Miyake M, Kato S, Hammerstedt RH. Changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during capacitation in vitro.. Reprod Fertil Dev 1999;11(4-5):193-9.
    doi: 10.1071/RD99056pubmed: 10898283google scholar: lookup
  20. Pavlok A. D-penicillamine and granulosa cells can effectively extend the fertile life span of bovine frozen-thawed spermatozoa in vitro: effect on fertilization and polyspermy.. Theriogenology 2000 Mar 15;53(5):1135-46.
    doi: 10.1016/S0093-691X(00)00258-2pubmed: 10798490google scholar: lookup
  21. Ehrenwald E, Foote RH, Parks JE. Bovine oviductal fluid components and their potential role in sperm cholesterol efflux.. Mol Reprod Dev 1990 Feb;25(2):195-204.
    doi: 10.1002/mrd.1080250213pubmed: 2310569google scholar: lookup
  22. Funahashi H, Day BN. Effects of follicular fluid at fertilization in vitro on sperm penetration in pig oocytes.. J Reprod Fertil 1993 Sep;99(1):97-103.
    doi: 10.1530/jrf.0.0990097pubmed: 8283459google scholar: lookup
  23. Foster ML, Varner DD, Hinrichs K, Teague S, Lacaze K, Blanchard TL, Love CC. Agreement between measures of total motility and membrane integrity in stallion sperm.. Theriogenology 2011 May;75(8):1499-505.
    doi: 10.1016/j.theriogenology.2010.12.011pubmed: 21295826google scholar: lookup
  24. Salazar JL Jr, Teague SR, Love CC, Brinsko SP, Blanchard TL, Varner DD. Effect of cryopreservation protocol on postthaw characteristics of stallion sperm.. Theriogenology 2011 Aug;76(3):409-18.
    doi: 10.1016/j.theriogenology.2011.02.016pubmed: 21496899google scholar: lookup
  25. Loux SC, Macías-Garcia B, González-Fernández L, Canesin HD, Varner DD, Hinrichs K. Regulation of axonemal motility in demembranated equine sperm.. Biol Reprod 2014 Dec;91(6):152.
    pubmed: 25339104doi: 10.1095/biolreprod.114.122804google scholar: lookup
  26. Hidalgo M, Urbano M, Ortiz I, Demyda-Peyras S, Murabito MR, Gálvez MJ, Dorado J. DNA integrity of canine spermatozoa during chill storage assessed by the sperm chromatin dispersion test using bright-field or fluorescence microscopy.. Theriogenology 2015 Aug;84(3):399-406.
    doi: 10.1016/j.theriogenology.2015.03.030pubmed: 25963130google scholar: lookup
  27. Tateno H, Krapf D, Hino T, Sánchez-Cárdenas C, Darszon A, Yanagimachi R, Visconti PE. Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways.. Proc Natl Acad Sci U S A 2013 Nov 12;110(46):18543-8.
    doi: 10.1073/pnas.1317113110pmc: PMC3831971pubmed: 24128762google scholar: lookup
  28. Suarez SS, Vincenti L, Ceglia MW. Hyperactivated motility induced in mouse sperm by calcium ionophore A23187 is reversible.. J Exp Zool 1987 Nov;244(2):331-6.
    doi: 10.1002/jez.1402440218pubmed: 3123593google scholar: lookup
  29. Bedford SJ, Gowdy HL, Hinrichs K. Comparison of the longevity of motility of stallion spermatozoa incubated at 38 degrees C in different capacitating media and containers.. Theriogenology 1999 Feb;51(3):637-46.
    doi: 10.1016/S0093-691X(99)00002-3pubmed: 10729048google scholar: lookup
  30. Darr CR, Varner DD, Teague S, Cortopassi GA, Datta S, Meyers SA. Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production.. Biol Reprod 2016 Aug;95(2):34.
    doi: 10.1095/biolreprod.116.140707pubmed: 27335066google scholar: lookup
  31. Davila MP, Muñoz PM, Bolaños JM, Stout TA, Gadella BM, Tapia JA, da Silva CB, Ferrusola CO, Peña FJ. Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation.. Reproduction 2016 Dec;152(6):683-694.
    doi: 10.1530/REP-16-0409pubmed: 27798283google scholar: lookup
  32. Harayama H, Okada K, Miyake M. Involvement of cytoplasmic free calcium in boar sperm: head-to-head agglutination induced by a cell-permeable cyclic adenosine monophosphate analog.. J Androl 2003 Jan-Feb;24(1):91-9.
    doi: 10.1002/j.1939-4640.2003.tb03134.xpubmed: 12514089google scholar: lookup
  33. Leahy T, Rickard JP, Aitken RJ, de Graaf SP. D-penicillamine prevents ram sperm agglutination by reducing the disulphide bonds of a copper-binding sperm protein.. Reproduction 2016 May;151(5):491-500.
    doi: 10.1530/REP-15-0596pubmed: 26860122google scholar: lookup
  34. Aitken RJ, Gibb Z, Mitchell LA, Lambourne SR, Connaughton HS, De Iuliis GN. Sperm motility is lost in vitro as a consequence of mitochondrial free radical production and the generation of electrophilic aldehydes but can be significantly rescued by the presence of nucleophilic thiols.. Biol Reprod 2012 Nov;87(5):110.
    doi: 10.1095/biolreprod.112.102020pubmed: 22933515google scholar: lookup
  35. WALSHE JM. Penicillamine, a new oral therapy for Wilson's disease.. Am J Med 1956 Oct;21(4):487-95.
    doi: 10.1016/0002-9343(56)90066-3pubmed: 13362281google scholar: lookup
  36. Andrews JC, Bavister BD. Capacitation of hamster spermatozoa with the divalent cation chelators D-penicillamine, L-histidine, and L-cysteine in a protein-free culture medium.. Gamete Res 1989 Jun;23(2):159-70.
    doi: 10.1002/mrd.1120230203pubmed: 2731901google scholar: lookup
  37. Laven R, Smith S. Copper deficiency in sheep: an assessment of relationship between concentrations of copper in serum and plasma.. N Z Vet J 2008 Dec;56(6):334-8.
    doi: 10.1080/00480169.2008.36856pubmed: 19043473google scholar: lookup
  38. Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roederer M. Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry.. J Immunol Methods 2006 Jun 30;313(1-2):199-208.
    doi: 10.1016/j.jim.2006.04.007pubmed: 16756987google scholar: lookup
  39. Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, Roederer M. Amine-reactive dyes for dead cell discrimination in fixed samples.. Curr Protoc Cytom 2010 Jul;Chapter 9:Unit 9.34.
    pmc: PMC2915540pubmed: 20578108doi: 10.1002/0471142956.cy0934s53google scholar: lookup
  40. Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ. L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature.. Biol Reprod 2015 Oct;93(4):104.
    doi: 10.1095/biolreprod.115.131326pubmed: 26316064google scholar: lookup
  41. 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.2011.15pmc: PMC3739346pubmed: 21478895google scholar: lookup
  42. Teague SR, Ligon EM, Serafini R, Varner DD, Love CC. Validation of a fixable stain for assessing the viability of stallion sperm. J Equine Vet Sci 2018;66:42.
    doi: 10.1016/j.jevs.2018.05.018google scholar: lookup
  43. Sampaio B, Ortiz I, Resende H, Felix M, Varner D, Hinrichs K. Factors affecting intracellular calcium influx in response to calcium ionophore A23187 in equine sperm.. Andrology 2021 Sep;9(5):1631-1651.
    pubmed: 33998170doi: 10.1111/andr.13036google scholar: lookup
  44. Gadella BM, Rathi R, Brouwers JF, Stout TA, Colenbrander B. Capacitation and the acrosome reaction in equine sperm.. Anim Reprod Sci 2001 Dec 3;68(3-4):249-65.
    doi: 10.1016/S0378-4320(01)00161-0pubmed: 11744269google scholar: lookup

Citations

This article has been cited 2 times.
  1. Felix MR, Dobbie T, Woodward E, Linardi R, Okada C, Santos R, Hinrichs K. Equine in vitro fertilization with frozen-thawed semen is associated with shortened pre-incubation time and modified capacitation-related changes. Biol Reprod 2025 May 13;112(5):867-879.
    doi: 10.1093/biolre/ioaf043pubmed: 40057974google scholar: lookup
  2. Johannisson A, Morrell JM, Ntallaris T. A combination of biomarkers for predicting stallion sperm fertility. Vet Res Commun 2024 Aug;48(4):2157-2169.
    doi: 10.1007/s11259-024-10372-6pubmed: 38652412google scholar: lookup
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