Comparison of bull sperm morphology evaluation methods under field conditions
Keywords:
Bull, sperm morphology, eosin-nigrosin, triple stain, microscope, phase contrast
Abstract
Sperm morphological assessment is a critical component in bull breeding soundness evaluations. Although sperm morphology is an important parameter to identify subfertile from fertile bulls, the evaluation can be biased because sperm staining methods used under field conditions may not be exact due to various factors, including artifacts. Objective was to compare 2 sperm morphological evaluation methods. Prebreeding season ejaculates of 1,216 Angus cross bulls collected via electroejaculation were evaluated. For each bull, an unstained (UNS) and an eosin-nigrosin stained (ENS) semen smear were viewed under a phase contrast microscope and a brightfield microscope with an oil immersion lens, both at 1,000 × magnification. Normal and percentage of abnormal sperm were identified by counting 200 sperm. Inter-rater agreements between 2 clinicians for the percentage of abnormal sperm determination and its categories were very good (ENS method, r = 0.84 – 0.96; UNS method, r = 0.76 – 0.96; p < 0.01). No differences (p > 0.1) were observed for abnormal sperm percentage determination and its categories between 2 methods. Correlation was very good between 2 methods for total abnormal sperm percentage determination (r = 0.91; p < 0.01) and its categories (r = 0.84 – 0.96; p < 0.05). Additionally, 60 ejaculates were evaluated by triple stain (TS), ENS, and UNS methods. Agreements between TS (percentage of sperm with damaged membrane) and ENS (percentage of abnormal sperm) and between TS and UNS methods were moderate (r = 0.58; p < 0.05) and fair (r = 0.43; p < 0.05), respectively. Based on our findings, either technique can be used for bull sperm morphological evaluation under field conditions. Considering the ease of semen smear preparation, the UNS method can be a viable alternative to the ENS method.
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References
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12. Nagy S, Johannisson A, Wahlsten T, et al: Sperm chromatin structure and sperm morphology: their association with fertility in AI-dairy Ayrshire sires. Theriogenology 2013;79:1153–1161. doi: 10.1016/j.theriogenology.2013.02.011
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14. Auger J: Assessing human sperm morphology: top models, underdogs or biometrics? Asian J Androl 2010;12:36–46. doi: 10.1038/aja.2009.8
15. Gatimel N, Mansoux L, Moreau J, et al: Continued existence of significant disparities in the technical practices of sperm morphology assessment and the clinical implications: results of a French questionnaire. Fertil Steril 2017;107:365.e3–372.e3. doi: 10.1016/j.fertnstert.2016.10.038
16. Brito LFC: A multilaboratory study on the variability of bovine semen analysis. Theriogenology 2016;85:254–266. doi: 10.1016/j.theriogenology.2015.05.027
17. Freneau GE, Chenoweth PJ, Ellis R, et al: Sperm morphology of beef bulls evaluated by two different methods. Anim Reprod Sci 2010;118:176–181. doi: 10.1016/j.anireprosci.2009.08.015
18. Brito LF, Greene LM, Kelleman A, et al: Effect of method and clinician on stallion sperm morphology evaluation. Theriogenology 2011;76:745–750. doi: 10.1016/j.theriogenology.2011.04.007
19. Hook KA, Fisher HS: Methodological considerations for examining the relationship between sperm morphology and motility. Mol Reprod Dev 2020;87:633–649. doi: 10.1002/mrd.23346
20. Celeghini ECC, Arruda RP, Albuquerque R, et al: Utilization of fluorescent probe association for simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes of rooster spermatozoa. Braz J Poultry Sci 2007;9:143–149. doi: 10.1590/S1516-635X2007000300001
21. Angrimani DSR, Bicudo LC, Luceno NL, et al: A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs. Cryobiology 2022;105:56–62. doi: 10.1016/j.cryobiol.2021.12.001
22. Oliveira BM, Arruda RP, Thomé HE, et al: Fertility and uterine hemodynamic in cows after artificial insemination with semen assessed by fluorescent probes. Theriogenology 2014;82:767–772. doi: 10.1016/j.theriogenology.2014.06.007
23. Lin LI: A concordance correlation coefficient to evaluate reproducibility. Biometrics 1989;45:255–268.
24. Altman DG, Altman E: Practical statistics for medical research. 1st ed. Boca Raton, FL, USA: Chapman & Hall/CRC; 1999.
25. Hatamoto-Zervoudakis LK, Duarte Júnior MF, Zervoudakis JT, et al: Free gossypol supplementation frequency and reproductive toxicity in young bulls. Theriogenology 2018;110:153–157. doi: 10.1016/j.theriogenology.2018.01.003
26. Komsky-Elbaz A, Kalo D, Roth Z: Effect of aflatoxin B1 on bovine spermatozoa’s proteome and embryo’s transcriptome. Reproduction 2020;160:709–723. doi: 10.1530/REP-20-0286
27. Silva EFSJD, Missio D, Martinez CS, et al: Mercury at environmental relevant levels affects spermatozoa function and fertility capacity in bovine sperm. J Toxicol Environ Health A 2019;82:268–278. doi: 10.1080/15287394.2019.1589608
28. van den Hoven L, Hendriks JC, Verbeet JG, et al: Status of sperm morphology assessment: an evaluation of methodology and clinical value. Fertil Steril 2015;103:53–58. doi: 10.1016/j.fertnstert.2014.09.036.
29. Natali I, Muratori M, Sarli V, et al: Scoring human sperm morphology using Testsimplets and Diff-Quik slides. Fertil Steril 2013;99:1227.e2–1232.e2. doi: 10.1016/j.fertnstert.2012.11.047.
30. Henkel R, Schreiber G, Sturmhoefel A, et al: Comparison of three staining methods for the morphological evaluation of human spermatozoa. Fertil Steril 2008;89:449–455. doi: 10.1016/j.fertnstert.2007.03.027
31. Davis RO, Gravance CG: Standardization of specimen preparation, staining, and sampling methods improves automated sperm-head morphometry analysis. Fertil Steril 1993;59:412–417. doi: 10.1016/s0015-0282(16)55686-6.
32. Mallidis C, Cooper TG, Hellenkemper B, et al: Ten years’ experience with an external quality control program for semen analysis. Fertil Steril 2012;98:611.e4–616.e4. doi: 10.1016/j.fertnstert.2012.05.006
33. Menkveld R: Sperm morphology assessment using strict (tygerberg) criteria. Methods Mol Biol 2013;927:39–50. doi: 10.1007/978-1-62703-038-0_5
34. Eustache F, Auger J: Inter-individual variability in the morphological assessment of human sperm: effect of the level of experience and the use of standard methods. Hum Reprod 2003;18:1018–1022. doi: 10.1093/humrep/deg197
35. Barthelemy C, Royere D, Hammahah S, et al: Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation. Arch Androl 1990;25:29–40. doi: 10.3109/01485019008987591
36. Aalseth EP, Saacke RG: Morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees C. J Reprod Fertil 1985;74:473–478. doi: 10.1530/jrf.0.0740473
37. Kasimanickam R, Kasimanickam V, Pelzer KD, et al: Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees C. Anim Reprod Sci 2007;101:60–73. doi: 10.1016/j.anireprosci.2006.09.001
38. Graham JK, Kunze E, Hammerstedt RH: Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry. Biol Reprod 1990;43:55–64. doi: 10.1095/biolreprod43.1.55.
39. Peña AI, Quintela LA, Herradon PG: Flow cytometric assessment of acrosomal status and viability of dog spermatozoa. Reprod Dom Anim 1999;34:495–502.
40. Pintado B, de la Fuente J, Roldan ER: Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability. J Reprod Fertil 2000;118:145–152.
41. Cummins JM, Fleming AD, Crozet N, et al: Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status. J Exp Zool 1986;237:375–382. doi: 10.1002/jez.1402370310
42. Tesarik J, Mendoza C: Alleviation of acrosome reaction prematurity by sperm treatment with egg yolk. Fertil Steril 1995;63:153–157. doi: 10.1016/s0015-0282(16)57311-7
43. Esteves SC, Sharma RK, Thomas AJ Jr, et al: Effect of in vitro incubation on spontaneous acrosome reaction in fresh and cryopreserved human spermatozoa. Int J Fertil Womens Med 1998;43:235–242.
44. Valle RR, Valle CM, Nichi M, et al: Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm. Theriogenology 2008;70:115–120. doi: 10.1016/j.theriogenology.2008.03.011
45. Bruinjé TC, Ponce-Barajas P, A. Dourey A, et al: Morphology, membrane integrity, and mitochondrial function in sperm of crossbred beef bulls selected for residual feed intake. Can J Anim Sci 2019;99:456–464. doi: 10.1139/cjas-2018-0103
46. Veeramachaneni R: Spermatozoal morphology. In: Mckinnon AO, Squires EL, Vaala WE, et al: editors. Equine reproduction. 2nd edition, Oxford; Wiley: 2011:1297–1307.
47. Koppers AJ, De Iuliis GN, Finnie JM, et al: Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. J Clin Endocrinol Metab 2008;93:3199–3207. doi: 10.1210/jc.2007-2616
48. Amaral A, Lourenço B, Marques M, et al: Mitochondria functionality and sperm quality. Reproduction 2013;146:R163–R174. doi: 10.1530/REP-13-0178
2. Gatimel N, Moreau J, Parinaud J, et al: Sperm morphology: assessment, pathophysiology, clinical relevance, and state of the art in 2017. Andrology 2017;5:845–862. doi: 10.1111/andr.12389
3. Kasimanickam R, Tibary A, Kastelic J: Fundamentals of bull selection. Clin Theriogenol 2013;5:97–107.
4. Butler ML, Bormann JM, Weaber RL, et al: Selection for bull fertility: a review. Transl Anim Sci 2019;4:423–441. doi: 10.1093/tas/txz174
5. Koziol JH, Armstrong CL: Manual for breeding soundness examination of bulls. 2nd edition, Montgomery, AL, USA; Society for Theriogenology: 2018.
6. Barth AD, Oko RJ: Abnormal morphology of bovine spermatozoa. 1st edition, Ames, Iowa, USA; Iowa State University Press: 1989:285.
7. Vishwanath R, Moreno JF: Review: semen sexing – current state of the art with emphasis on bovine species. Animal 2018;12:s85–s96. doi: 10.1017/S1751731118000496.
8. Morgentaler A, Fung MY, Harris DH, et al: Sperm morphology and in vitro fertilization outcome: a direct comparison of World Health Organization and strict criteria methodologies. Fertil Steril 1995;64:1177–1182. doi: 10.1016/s0015-0282(16)57981-3
9. Kandel ME, Rubessa M, He YR: Reproductive outcomes predicted by phase imaging with computational specificity of spermatozoon ultrastructure. Proc Natl Acad Sci USA 2020;117:18302–18309. doi: 10.1073/pnas.2001754117
10. Eliasson R: Semen analysis with regard to sperm number, sperm morphology and functional aspects. Asian J Androl 2010;12:26–32. doi: 10.1038/aja.2008.58
11. Al-Makhzoomi A, Lundeheim N, Håård M, et al: Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden. Theriogenology 2008;70:682–691. doi: 10.1016/j.theriogenology.2008.04.049
12. Nagy S, Johannisson A, Wahlsten T, et al: Sperm chromatin structure and sperm morphology: their association with fertility in AI-dairy Ayrshire sires. Theriogenology 2013;79:1153–1161. doi: 10.1016/j.theriogenology.2013.02.011
13. Holroyd RG, Doogan W, De Faveri J, et al: Bull selection and use in northern Australia. 4. Calf output and predictors of fertility of bulls in multiple-sire herds. Anim Reprod Sci 2002;71:67–79. doi: 10.1016/S0378-4320(02)00026-X
14. Auger J: Assessing human sperm morphology: top models, underdogs or biometrics? Asian J Androl 2010;12:36–46. doi: 10.1038/aja.2009.8
15. Gatimel N, Mansoux L, Moreau J, et al: Continued existence of significant disparities in the technical practices of sperm morphology assessment and the clinical implications: results of a French questionnaire. Fertil Steril 2017;107:365.e3–372.e3. doi: 10.1016/j.fertnstert.2016.10.038
16. Brito LFC: A multilaboratory study on the variability of bovine semen analysis. Theriogenology 2016;85:254–266. doi: 10.1016/j.theriogenology.2015.05.027
17. Freneau GE, Chenoweth PJ, Ellis R, et al: Sperm morphology of beef bulls evaluated by two different methods. Anim Reprod Sci 2010;118:176–181. doi: 10.1016/j.anireprosci.2009.08.015
18. Brito LF, Greene LM, Kelleman A, et al: Effect of method and clinician on stallion sperm morphology evaluation. Theriogenology 2011;76:745–750. doi: 10.1016/j.theriogenology.2011.04.007
19. Hook KA, Fisher HS: Methodological considerations for examining the relationship between sperm morphology and motility. Mol Reprod Dev 2020;87:633–649. doi: 10.1002/mrd.23346
20. Celeghini ECC, Arruda RP, Albuquerque R, et al: Utilization of fluorescent probe association for simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes of rooster spermatozoa. Braz J Poultry Sci 2007;9:143–149. doi: 10.1590/S1516-635X2007000300001
21. Angrimani DSR, Bicudo LC, Luceno NL, et al: A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs. Cryobiology 2022;105:56–62. doi: 10.1016/j.cryobiol.2021.12.001
22. Oliveira BM, Arruda RP, Thomé HE, et al: Fertility and uterine hemodynamic in cows after artificial insemination with semen assessed by fluorescent probes. Theriogenology 2014;82:767–772. doi: 10.1016/j.theriogenology.2014.06.007
23. Lin LI: A concordance correlation coefficient to evaluate reproducibility. Biometrics 1989;45:255–268.
24. Altman DG, Altman E: Practical statistics for medical research. 1st ed. Boca Raton, FL, USA: Chapman & Hall/CRC; 1999.
25. Hatamoto-Zervoudakis LK, Duarte Júnior MF, Zervoudakis JT, et al: Free gossypol supplementation frequency and reproductive toxicity in young bulls. Theriogenology 2018;110:153–157. doi: 10.1016/j.theriogenology.2018.01.003
26. Komsky-Elbaz A, Kalo D, Roth Z: Effect of aflatoxin B1 on bovine spermatozoa’s proteome and embryo’s transcriptome. Reproduction 2020;160:709–723. doi: 10.1530/REP-20-0286
27. Silva EFSJD, Missio D, Martinez CS, et al: Mercury at environmental relevant levels affects spermatozoa function and fertility capacity in bovine sperm. J Toxicol Environ Health A 2019;82:268–278. doi: 10.1080/15287394.2019.1589608
28. van den Hoven L, Hendriks JC, Verbeet JG, et al: Status of sperm morphology assessment: an evaluation of methodology and clinical value. Fertil Steril 2015;103:53–58. doi: 10.1016/j.fertnstert.2014.09.036.
29. Natali I, Muratori M, Sarli V, et al: Scoring human sperm morphology using Testsimplets and Diff-Quik slides. Fertil Steril 2013;99:1227.e2–1232.e2. doi: 10.1016/j.fertnstert.2012.11.047.
30. Henkel R, Schreiber G, Sturmhoefel A, et al: Comparison of three staining methods for the morphological evaluation of human spermatozoa. Fertil Steril 2008;89:449–455. doi: 10.1016/j.fertnstert.2007.03.027
31. Davis RO, Gravance CG: Standardization of specimen preparation, staining, and sampling methods improves automated sperm-head morphometry analysis. Fertil Steril 1993;59:412–417. doi: 10.1016/s0015-0282(16)55686-6.
32. Mallidis C, Cooper TG, Hellenkemper B, et al: Ten years’ experience with an external quality control program for semen analysis. Fertil Steril 2012;98:611.e4–616.e4. doi: 10.1016/j.fertnstert.2012.05.006
33. Menkveld R: Sperm morphology assessment using strict (tygerberg) criteria. Methods Mol Biol 2013;927:39–50. doi: 10.1007/978-1-62703-038-0_5
34. Eustache F, Auger J: Inter-individual variability in the morphological assessment of human sperm: effect of the level of experience and the use of standard methods. Hum Reprod 2003;18:1018–1022. doi: 10.1093/humrep/deg197
35. Barthelemy C, Royere D, Hammahah S, et al: Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation. Arch Androl 1990;25:29–40. doi: 10.3109/01485019008987591
36. Aalseth EP, Saacke RG: Morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees C. J Reprod Fertil 1985;74:473–478. doi: 10.1530/jrf.0.0740473
37. Kasimanickam R, Kasimanickam V, Pelzer KD, et al: Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees C. Anim Reprod Sci 2007;101:60–73. doi: 10.1016/j.anireprosci.2006.09.001
38. Graham JK, Kunze E, Hammerstedt RH: Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry. Biol Reprod 1990;43:55–64. doi: 10.1095/biolreprod43.1.55.
39. Peña AI, Quintela LA, Herradon PG: Flow cytometric assessment of acrosomal status and viability of dog spermatozoa. Reprod Dom Anim 1999;34:495–502.
40. Pintado B, de la Fuente J, Roldan ER: Permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or Hoechst 33258, or to eosin: accuracy in the assessment of cell viability. J Reprod Fertil 2000;118:145–152.
41. Cummins JM, Fleming AD, Crozet N, et al: Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status. J Exp Zool 1986;237:375–382. doi: 10.1002/jez.1402370310
42. Tesarik J, Mendoza C: Alleviation of acrosome reaction prematurity by sperm treatment with egg yolk. Fertil Steril 1995;63:153–157. doi: 10.1016/s0015-0282(16)57311-7
43. Esteves SC, Sharma RK, Thomas AJ Jr, et al: Effect of in vitro incubation on spontaneous acrosome reaction in fresh and cryopreserved human spermatozoa. Int J Fertil Womens Med 1998;43:235–242.
44. Valle RR, Valle CM, Nichi M, et al: Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm. Theriogenology 2008;70:115–120. doi: 10.1016/j.theriogenology.2008.03.011
45. Bruinjé TC, Ponce-Barajas P, A. Dourey A, et al: Morphology, membrane integrity, and mitochondrial function in sperm of crossbred beef bulls selected for residual feed intake. Can J Anim Sci 2019;99:456–464. doi: 10.1139/cjas-2018-0103
46. Veeramachaneni R: Spermatozoal morphology. In: Mckinnon AO, Squires EL, Vaala WE, et al: editors. Equine reproduction. 2nd edition, Oxford; Wiley: 2011:1297–1307.
47. Koppers AJ, De Iuliis GN, Finnie JM, et al: Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. J Clin Endocrinol Metab 2008;93:3199–3207. doi: 10.1210/jc.2007-2616
48. Amaral A, Lourenço B, Marques M, et al: Mitochondria functionality and sperm quality. Reproduction 2013;146:R163–R174. doi: 10.1530/REP-13-0178
Published
2023-08-11
How to Cite
Hanson R., Reddick S., Thuerauf S., Webb K., Kasimanickam V., & Kasimanickam R. (2023). Comparison of bull sperm morphology evaluation methods under field conditions. Clinical Theriogenology, 15. https://doi.org/10.58292/ct.v15.9425
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Research Reports
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