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Methods in molecular biology (Clifton, N.J.)2025; 2954; 241-259; doi: 10.1007/978-1-0716-4698-4_14

Analysis of Motion Characteristics and Plasma Membrane Intactness (Viability) in Sperm from Domestic Animals.

Abstract: Sperm quality analysis using computer-assisted sperm analysis (CASA) systems and fluorescence-based techniques has become common in the animal reproduction industry, particularly for large animals (i.e., bovine, porcine, equine). In this chapter, the methods commonly utilized in the author's laboratory to examine sperm motion characteristics via CASA and plasma membrane intactness by flow cytometry will be described. These include methods to properly dilute fresh (stallions, bulls, boars), cool-stored (stallions, boars), or frozen/thawed (stallions, bulls, boars) sperm for assessment of sperm motion characteristics using CASA, preparation of fluorochromes (SYBR-14/propidium iodide) utilized to distinguish between intact (i.e., viable or "live") versus non-intact (i.e., nonviable or "dead") sperm, and the analysis and interpretation of plasma membrane intactness using flow cytometry.
© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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Publication Date: 2025-07-02 PubMed ID: 40601280DOI: 10.1007/978-1-0716-4698-4_14Google Scholar: Lookup
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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 article illustrates various methods for analysing the quality of animal sperm, specifically focusing on techniques used in assessing motion characteristics of sperm and their plasma membrane intactness (viability).

Article Explanation

The featured article’s overarching focus is an analysis of sperm quality in large domestic animals, including bovine (cows), porcine (pigs), and equine (horses). The techniques outlined are designed to evaluate the sperm’s motion characteristics and the intactness of the plasma membrane, which is a critical measure of sperm viability.

  • Methods Used: The primary methods used in the author’s laboratory are discussed in the paper. They include the Computer-Assisted Sperm Analysis (CASA) systems and fluorescence-based flow cytometry. CASA helps in the examination of the sperm’s motion characteristics, whilst flow cytometry is employed to evaluate the sperm’s plasma membrane intactness. The plasma membrane of a sperm must be intact for the sperm to be considered viable.
  • Sperm Processing: To prepare the sperm for analysis, it is first diluted. The procedures for dilution could alter depending on whether the sperm under study is fresh (from stallions, bulls, and boars), cool-stored (from stallions and boars), or has been frozen and thawed (from stallions, bulls and boars), and such specifics are explained in detail in the research piece.
  • Fluorochromes Preparation: Fluorochromes, specifically SYBR-14 and propidium iodide, are prepared for use in distinguishing between ‘live’ (intact) sperm and ‘dead’ (non-intact) sperm. The article goes into the preparation of these vital distinguishing agents.
  • Analysis and Interpretation: After the process of sperm dilution and fluorochromes preparation, analysis and interpretation are conducted using flow cytometry. This analysis process determines the intactness of the plasma membrane, thus providing vital information about the sperm’s viability. The article describes in depth how this analysis and interpretation is carried out.

Overall, this study provides an essential guide to studying sperm quality in large domestic animals, which is particularly significant in the animal reproduction industry, presenting techniques that offer in-depth analysis of sperm viability.

Cite This Article

APA
Hernández-Avilés C. (2025). Analysis of Motion Characteristics and Plasma Membrane Intactness (Viability) in Sperm from Domestic Animals. Methods Mol Biol, 2954, 241-259. https://doi.org/10.1007/978-1-0716-4698-4_14

Publication

Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
NlmUniqueID: 9214969
Country: United States
Language: English
Volume: 2954
Pages: 241-259

Researcher Affiliations

Hernández-Avilés, Camilo
  • Equine Fertility Laboratory, Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA. chernandez@cvm.tamu.edu.

MeSH Terms

  • Animals
  • Male
  • Cell Membrane / metabolism
  • Cell Membrane / physiology
  • Spermatozoa / cytology
  • Spermatozoa / physiology
  • Flow Cytometry / methods
  • Sperm Motility / physiology
  • Semen Analysis / methods
  • Animals, Domestic
  • Horses
  • Cell Survival
  • Cryopreservation
  • Swine
  • Cattle

References

This article includes 49 references
  1. Kenney RM, Hurtgen J, Pierson R, Witherspoon D, Simons J. Society for Theriogenology manual for clinical fertility evaluation of the stallion. .
  2. Koziol JH, Armstrong C. Society for Theriogenology manual for breeding soundness examination of bulls, 2nd edn. .
  3. Hurtgen JP. Society for Theriogenology manual for the reproductive examination of the boar. .
  4. Barth AD. Review: the use of bull breeding soundness evaluation to identify subfertile and infertile bulls. Animal 12(s1):s158–s164.
    pubmed: 29560847doi: 10.1017/S1751731118000538google scholar: lookup
  5. Hernández-Avilés C, Love CC. Prebreeding season/prepurchase examination of the stallion: testicular ultrasonography, semen evaluation, and considerations. Clin Theriogenol 13:289–298.
  6. Jørgensen N, Auger J, Giwercman A, Irvine DS, Jensen TK, Jouannet P. Semen analysis performed by different laboratory teams: an intervariation study. Int J Androl 20:201–208.
    pubmed: 9401822doi: 10.1046/j.1365-2605.1997.00052.xgoogle scholar: lookup
  7. Cooper TG, Björndahl L, Vreeburg J, Nieschlag E. Semen analysis and external quality control schemes for semen analysis need global standardization. Int J Androl 25:306–311.
    pubmed: 12270029doi: 10.1046/j.1365-2605.2002.00370.xgoogle scholar: lookup
  8. Holt WV. Is quality assurance in semen analysis still necessary? A spermatologist’s viewpoint. Hum Reprod 20:2983–2986.
    pubmed: 16006459doi: 10.1093/humrep/dei189google scholar: lookup
  9. De Jonge C. Semen analysis: looking for an upgrade in class. Fertil Steril 97:260–266.
    pubmed: 22289285doi: 10.1016/j.fertnstert.2011.12.045google scholar: lookup
  10. Christensen P, Brockhoff PB, Lehn-Jensen H. The relationship between semen quality and the nonreturn rate of bulls. Reprod Domest Anim 34:503–507.
    doi: 10.1111/j.1439-0531.1999.tb01410.xgoogle scholar: lookup
  11. Broekhuijse MLWJ, Šoštarić E, Feitsma H, Gadella BM. Additional value of computed assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination. Theriogenology 76:1473–1486.
    pubmed: 21872316doi: 10.1016/j.theriogenology.2011.05.040google scholar: lookup
  12. Brito LFC. A multilaboratory study on the variability of bovine semen analysis. Theriogenology 85:254–266.
    pubmed: 26671653doi: 10.1016/j.theriogenology.2015.05.027google scholar: lookup
  13. Whitesell K, Stefanovski D, McDonnell SM, Turner R. Evaluation of the effect of laboratory methods on semen analysis and breeding soundness examination (BSE) classification in stallions. Theriogenology 142:67–76.
    pubmed: 31581045doi: 10.1016/j.theriogenology.2019.09.035google scholar: lookup
  14. Hernández-Avilés C, Ramírez-Agámez L, Pearson M, Beckham AMN, Varner DD, Love CC. A matter of agreement: the effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm. Theriogenology 202:74–83.
    pubmed: 36924698doi: 10.1016/j.theriogenology.2023.02.025google scholar: lookup
  15. Amann RP, Hammerstedt RH. Validation of a system for computerized measurements of spermatozoal velocity and percentage of motile sperm. Biol Reprod 23:647–656.
    pubmed: 7448267doi: 10.1095/biolreprod23.3.647google scholar: lookup
  16. Katz DF, Davis RO, Delandmeter BA, Overstreet JW. Real-time analysis of sperm motion using automatic video image digitization. Comput Methods Prog Biomed 21:173–182.
    doi: 10.1016/0169-2607(85)90002-1google scholar: lookup
  17. Amann RP. Computerized evaluation of stallion semen. Proc Am Assoc Equine Pract 32:453–473.
  18. Garner DL, Pinkel D, Johnson LA, Pace MM. Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses. Biol Reprod 34:127–138.
    pubmed: 3955132doi: 10.1095/biolreprod34.1.127google scholar: lookup
  19. Harrison RA, Vickers SE. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J Reprod Fertil 88:343–352.
    pubmed: 1690300doi: 10.1530/jrf.0.0880343google scholar: lookup
  20. Graham JK, Kunze E, Hammerstedt RH. Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry. Biol Reprod 43:55–64.
    pubmed: 2393693doi: 10.1095/biolreprod43.1.55google scholar: lookup
  21. Amann RP, Waberski D. Computer-assisted sperm analysis (CASA): capabilities and potential developments. Theriogenology 81:5–17.
    pubmed: 24274405doi: 10.1016/j.theriogenology.2013.09.004google scholar: lookup
  22. Yesté M, Bonet S, Rodríguez-Gil JM, Rivera Del Álamo MM. Evaluation of sperm motility with CASA-Mot: which factors may influence our measurements?. Reprod Fertil Dev 30:789–798.
    pubmed: 29533761doi: 10.1071/RD17475google scholar: lookup
  23. Hernández-Avilés C, Ramírez-Agámez L, Varner DD, Love CC. Factors affecting the analysis and interpretation of sperm quality in frozen/thawed stallion semen. Theriogenology 218:35–44.
    pubmed: 38295678doi: 10.1016/j.theriogenology.2024.01.039google scholar: lookup
  24. Boyers SP, Davis RO, Katz DF. Automated semen analysis. Curr Probl Obstet Gynecol Fertil 12:167–200.
  25. Budworth PR, Amann RP, Chapman PL. Relationships between computerized measurements of motion of frozen-thawed bull spermatozoa and fertility. J Androl 9:41–54.
    pubmed: 3346179doi: 10.1002/j.1939-4640.1988.tb01007.xgoogle scholar: lookup
  26. Farrell PB, Presicce GA, Brockett CC, Foote RH. Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology 49:871–879.
    pubmed: 10732095doi: 10.1016/S0093-691X(98)00036-3google scholar: lookup
  27. Gliozzi TM, Turri F, Manes S, Cassinelli C, Pizzi F. The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull sperm. Animal 11:1975–1982.
    pubmed: 28397643doi: 10.1017/S1751731117000684google scholar: lookup
  28. Holt C, Holt WV, Moore HDM, Reed HCB, Curnock RM. Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials. J Androl 18:312–323.
    pubmed: 9203061doi: 10.1002/j.1939-4640.1997.tb01925.xgoogle scholar: lookup
  29. Broekhuijse MLWJ, Šoštarić E, Feitsma H, Gadella BM. Application of computer-assisted semen analysis to explain variations in pig fertility. J Anim Sci 90:779–789.
    pubmed: 22064743doi: 10.2527/jas.2011-4311google scholar: lookup
  30. Jasko DJ, Little TV, Lein DH, Foote RH. Comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987–1988). J Am Vet Med Assoc 200:979–985.
    pubmed: 1577655doi: 10.2460/javma.1992.200.07.979google scholar: lookup
  31. Love CC. Relationship between sperm motility, morphology and the fertility of stallions. Theriogenology 76:547–557.
    pubmed: 21497893doi: 10.1016/j.theriogenology.2011.03.007google scholar: lookup
  32. Barrier Battut I, Kempfer A, Becker J, Lebailly L, Camugli S, Chevrier L. Development of a new fertility prediction model stallion semen, including flow cytometry. Theriogenology 86:1111–1131.
    pubmed: 27207472doi: 10.1016/j.theriogenology.2016.04.001google scholar: lookup
  33. Waite JA, Love CC, Brinsko SP, Teague SR, Salazar JL Jr, Mancill SS, Varner DD. Factors impacting equine sperm recovery rate and quality following cushioned centrifugation. Theriogenology 70:704–714.
    pubmed: 18573520doi: 10.1016/j.theriogenology.2008.04.047google scholar: lookup
  34. Hoogewijs ML, Govaere JL, Rijsselaere T, de Schauwer C, Vanhaesebrouck EM, de Kruif A, de Vliegher SP. Influence of technical settings on CASA motility parameters of frozen-thawed stallion semen. Proc Am Assoc Equine Pract 55:336–337.
  35. Althouse GC, Hopkins SM. Assessment of boar sperm viability using a combination of two fluorophores. Theriogenology 43:595–603.
    pubmed: 16727651doi: 10.1016/0093-691X(94)00065-3google scholar: lookup
  36. Cross NL, Morales P, Overstreet JW, Hanson FW. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 15:213–226.
    doi: 10.1002/mrd.1120150303google scholar: lookup
  37. Garner DL, Johnson LA, Yue ST, Roth BL, Haughland RP. Dual DNA staining assessment of bovine sperm viability using SYBR-14 and propidium iodide. J Androl 15:620–629.
    pubmed: 7721666doi: 10.1002/j.1939-4640.1994.tb00510.xgoogle scholar: lookup
  38. Nagy S, Jansen J, Topper EK, Gadella BM. A triple-stain flow cytometric method to assess plasma- and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in the presence of egg-yolk particles. Biol Reprod 68:1828–1835.
    pubmed: 12606354doi: 10.1095/biolreprod.102.011445google scholar: lookup
  39. Garner DL. Hoechst 33342: the dye that enabled differentiation of living X- and Y-chromosome bearing mammalian sperm. Theriogenology 71:11–21.
    pubmed: 18952273doi: 10.1016/j.theriogenology.2008.09.023google scholar: lookup
  40. Merkies K, Chenier T, Plante C, Buhr MM. Assessment of stallion sperm viability by flow cytometry and light microscope analysis. Theriogenology 54:1215–1224.
    pubmed: 11192180doi: 10.1016/S0093-691X(00)00428-3google scholar: lookup
  41. Foote RH. Fertility of bull semen at high extension rates in Tris-buffered extenders. J Dairy Sci 53:1475–1477.
    doi: 10.3168/jds.S0022-0302(70)86417-7google scholar: lookup
  42. Pursel VG, Johnson LA. Freezing of boar spermatozoa: fertilizing capacity with concentrated semen and a new thawing procedure. J Anim Sci 40:99–102.
    pubmed: 1110222doi: 10.2527/jas1975.40199xgoogle scholar: lookup
  43. Blanchard TL, Varner DD, Schumacher J. Semen preservation. In: Blanchard TL, Varner DD, Schumacher J (eds) Manual of equine reproduction. Mosby, St. Louis, pp 143–154.
  44. Rigby SL, Varner DD, Thompson JA, Love CC, Brinsko SP, Blanchard TL. Measurement of sperm concentration in stallion ejaculates using photometric or direct sperm enumeration techniques. Proc Am Assoc Equine Pract 47:236–238.
  45. Hernández-Avilés C, Serafini R, Love CC, Teague SR, LaCaze KA, Lawhon SD, Wu J. The effects of antibiotic type and extender storage method on sperm quality and antibacterial effectiveness in fresh and cooled stored stallion semen. Theriogenology 122:23–29.
    pubmed: 30219312doi: 10.1016/j.theriogenology.2018.08.022google scholar: lookup
  46. Palmer CW. Welfare aspects of theriogenology: investigating alternatives to electroejaculation in bulls. Theriogenology 64:469–479.
    pubmed: 15955553doi: 10.1016/j.theriogenology.2005.05.032google scholar: lookup
  47. King GJ, Macpherson JW. A comparison of two methods for boar semen collection. J Anim Sci 36:563–565.
    pubmed: 4693863doi: 10.2527/jas1973.363563xgoogle scholar: lookup
  48. Love CC. Semen collection techniques. Vet Clin North Am Equine Pract 8:111–128.
    pubmed: 1576545doi: 10.1016/S0749-0739(17)30470-4google scholar: lookup
  49. Hernández-Avilés C, Ramírez-Agámez L, Varner DD, Love CC. Effects of egg yolk level, penetrating cryoprotectant, and pre-freeze cooling rate, on the post-thaw quality of stallion sperm. Anim Reprod Sci 248:107162.
    pubmed: 36469980doi: 10.1016/j.anireprosci.2022.107162google scholar: lookup

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