Clinical applications of flow cytometric sperm analyses
Keywords:
Semen analysis, stallion, flow cytometry, sperm membrane, livestock
Abstract
Animal andrology has benefitted by flow cytometry use (to analyze various sperm components). Examples include its use to explore DNA intactness, to sort sperm for chromosomal sex, or to explore via suitable fluorophores the functional intactness of essential attributes (e.g. plasma membrane). These explorations can provide the clinician with evidence of the extent of cell death, a major variable in using semen for breeding. Such gains have evolved over the past decade(s) through the production of versatile markers, including biomarkers, the design of simpler protocols, and the availability of more user-friendly and less expensive bench-flow cytometers. The present review summarizes the current state of flow cytometry use within animal andrology. It attempts to be critical of research gains, separating them for clinical relevance and focusing on its use to examine the early events that occur in the labile sperm plasma membrane (leading to cell death) – a most relevant marker for clinical decisions during diagnosis and prognosis of fertility when using ejaculated sperm (either unprocessed or processed) for cooling, freezing, or sperm sorting for in vitro fertilization or artificial insemination.
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References
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3. Rodriguez-Martinez H: State of the art in farm animal sperm evaluation. Reprod Fertil Dev 2007;19:91–101. doi: 10.1071/RD06104
4. Rodriguez-Martinez H, Barth AD: In vitro evaluation of sperm quality related to in vivo function and fertility. Soc Reprod Fertil Suppl 2007;64:39–54. doi: 10.5661/RDR-VI-39
5. Al-Makhzoomi A, Lundeheim N, Haard 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
6. Rodrígue Martínez H: Semen evaluation:can we forecast fertility? Vet Stanica 2019;50:293–305.
7. DeJarnette JM, Harstine BR, McDonald K, et al: Commercial application of flow cytometry for evaluating bull sperm. Anim Reprod Sci 2022;246:106838. doi: 10.1016/j.anireprosci.2021.106838
8. Rodriguez-Martinez H, Kvist U, Ernerudh J, et al: Seminal plasma proteins: what role do they play? Am J Reprod Immunol 2011;66 Suppl 1:11–22. doi: 10.1111/j.1600-0897.2011.01033.x
9. Perez-Patino C, Parrilla I, Barranco I, et al: New in-depth analytical approach of the porcine seminal plasma proteome reveals potential fertility biomarkers. J Proteome Res 2018;17:1065–1076. doi: 10.1021/acs.jproteome.7b00728
10. Rodriguez-Martinez H, Martinez EA, Calvete JJ, et al: Seminal plasma: relevant for fertility? Int J Mol Sci 2021;22:4368. doi: 10.3390/ijms22094368
11. Rodriguez-Martinez H, Roca J: Extracellular vesicles in seminal plasma: a safe and relevant tool to improve fertility in livestock? Anim Reprod Sci 2022;244:107051. doi: 10.1016/j.anireprosci.2022.107051
12. Pena FJ, Saravia F, Johannisson A, et al: Detection of early changes in sperm membrane integrity pre-freezing can estimate post-thaw quality of boar spermatozoa. Anim Reprod Sci 2007;97:74–83. doi: 10.1016/j.anireprosci.2005.12.014
13. da Silva BCM, Ortega-Ferrusola C, Morrell JM, et al: Flow cytometric chromosomal sex sorting of stallion spermatozoa induces oxidative stress on mitochondria and genomic DNA. Reprod Domest Anim 2016;51:18–25. doi: 10.1111/rda.12640
14. Gardela J, Ruiz-Conca M, Wright D, et al; Semen modulates cell proliferation and differentiation-related transcripts in the pig peri-ovulatory endometrium. Biology (Basel) 2022;11:616. doi: 10.3390/biology11040616
15. Alvarez-Rodriguez M, Martinez C, Wright D, et al: The transcriptome of pig spermatozoa, and its role in fertility. Int J Mol Sci 2020;21:1572. doi: 10.3390/ijms21051572
16. Martinez CA, Roca J, Alvarez-Rodriguez M, et al: miRNA-profiling in ejaculated and epididymal pig spermatozoa and their relation to fertility after artificial insemination. Biology (Basel) 2022;11:236. doi: 10.3390/biology11020236
17. Pertille F, Alvarez-Rodriguez M, da Silva AN, et al: Sperm methylome profiling can discern fertility levels in the porcine biomedical model. Int J Mol Sci 2021;22:2679. doi: 10.3390/ijms22052679
18. Kerns K, Zigo M, Drobnis EZ, et al: Zinc ion flux during mammalian sperm capacitation. Nat Commun 2018;9:2061. doi: 10.1038/s41467-018-04523-y
19. Zigo M, Kerns K, Sen S, et al: Zinc is a master-regulator of sperm function associated with binding, motility, and metabolic modulation during porcine sperm capacitation. Commun Biol 2022;5:538. doi: 10.1038/s42003-022-03485-8
20. Hossain MS, Johannisson A, Wallgren M, et al: Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art. Asian J Androl 2011;13:406–419. doi: 10.1038/aja.2011.15
21. Ortega-Ferrusola C, Gil MC, Rodriguez-Martinez H, et al: Flow cytometry in spermatology: a bright future ahead. Reprod Domest Anim 2017;52:921–931. doi: 10.1111/rda.13043
22. Roca J, Parrilla I, Gil MA, et al: Non-viable sperm in the ejaculate: lethal escorts for contemporary viable sperm. Anim Reprod Sci 2016;169:24–31. doi: 10.1016/j.anireprosci.2016.02.028
23. Pena FJ, Gibb Z: Oxidative stress and reproductive function: oxidative stress and the long-term storage of horse spermatozoa. Reproduction 2022;164:F135–F144. doi: 10.1530/REP-22-0264
24. Pena FJ, O’Flaherty C, Ortiz Rodriguez JM, et al: The stallion spermatozoa: a valuable model to help understand the interplay between metabolism and redox (De)regulation in sperm cells. Antioxid Redox Signal 2022;37:521–537. doi: 10.1089/ars.2021.0092
25. Gonzalez-Castro RA, Pena FJ, Herickhoff LA: Validation of a new multiparametric protocol to assess viability, acrosome integrity and mitochondrial activity in cooled and frozen thawed boar spermatozoa. Cytometry B Clin Cytom 2022;102:400–408. doi: 10.1002/cyto.b.22058
26. Ortiz-Rodriguez JM, Martin-Cano FE, Gaitskell-Phillips G, et al: The SLC7A11: sperm mitochondrial function and non-canonical glutamate metabolism. Reproduction 2020;160:803–818. doi: 10.1530/REP-20-0181
27. Ortega-Ferrusola C, Martin Munoz P, Ortiz-Rodriguez JM, et al: Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoadagger. Biol Reprod 2019;100: 1090–1107. doi: 10.1093/biolre/ioy241
28. Aitken RJ, Findlay JK, Hutt KJ, et al: Apoptosis in the germ line. Reproduction 2011;141:139–150. doi: 10.1530/REP-10-0232
29. Boerke A, Brouwers JF, Olkkonen VM, et al: Involvement of bicarbonate-induced radical signaling in oxysterol formation and sterol depletion of capacitating mammalian sperm during in vitro fertilization. Biol Reprod 2013;88:21. doi: 10.1095/biolreprod.112.101253
30. Aitken RJ: The capacitation-apoptosis highway: oxysterols and mammalian sperm function. Biol Reprod 2011;85:9–12. doi: 10.1095/biolreprod.111.092528
31. Zerbinati C, Caponecchia L, Puca R, et al: Mass spectrometry profiling of oxysterols in human sperm identifies 25-hydroxycholesterol as a marker of sperm function. Redox Biol 2017;11:111–117. doi: 10.1016/j.redox.2016.11.008
32. Luque GM, Dalotto-Moreno T, Martin-Hidalgo D, et al. Only a subpopulation of mouse sperm displays a rapid increase in intracellular calcium during capacitation. J Cell Physiol 2018;233: 9685–9700. doi: 10.1002/jcp.26883
33. Escoffier J, Navarrete F, Haddad D, et al: Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation. Biol Reprod 2015;92:121. doi: 10.1095/biolreprod.114.127266
34. Sui X, Zhang R, Liu S, et al: RSL3 drives ferroptosis through GPX4 inactivation and ROS production in colorectal cancer. Front Pharmacol 2018;9:1371. doi: 10.3389/fphar.2018.01371
35. Ortiz-Rodriguez JM, Martin-Cano FE, Ortega-Ferrusola C, et al: The incorporation of cystine by the soluble carrier family 7 member 11 (SLC7A11) is a component of the redox regulatory mechanism in stallion spermatozoadagger. Biol Reprod 2019;101:208–222. doi: 10.1093/biolre/ioz069
36. Ortiz-Rodriguez JM, da Silva BCM, Masot J, et al: Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3. PLoS One 2019;14:e0211994. doi: 10.1371/journal.pone.0211994
37. Aitken RJ, Baker MA, Nixon B: Are sperm capacitation and apoptosis the opposite ends of a continuum driven by oxidative stress? Asian J Androl 2015;17:633–639. doi: 10.4103/1008-682X.153850
38. Pena FJ, Johannisson A, Wallgren M, et al: Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture. Zygote 2004;12:117–124. doi: 10.1017/S096719940400262X
39. Pena FJ, Johannisson A, Wallgren M, et al: Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation. Theriogenology 2004;61:63–70. doi: 10.1016/S0093-691X(03)00181-X
40. Tienthai P, Johannisson A, Rodriguez-Martinez H: Sperm capacitation in the porcine oviduct. Anim Reprod Sci 2004;80:131–146. doi: 10.1016/S0378-4320(03)00134-9
41. Gibbons SJ, Washburn KB, Talamo BR: P2X(7) receptors in rat parotid acinar cells: formation of large pores. J Auton Pharmacol 2001;21:181–190. doi: 10.1046/j.1365-2680.2001.00224.x
42. Xiao F, Waldrop SL, Khimji AK, et al: Pannexin1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated free fatty acids in liver cells. Am J Physiol Cell Physiol 2012;303:C1034–C1044. doi: 10.1152/ajpcell.00175.2012
43. Gallardo BJM, da Silva BCM, Martin MP, et al: Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7. Reproduction 2014;148:221–235. doi: 10.1530/REP-13-0191
44. da Silva BCM, Ortega FC, Morillo RA, et al: Sex sorting increases the permeability of the membrane of stallion spermatozoa. Anim Reprod Sci 2013;138:241–251. doi: 10.1016/j.anireprosci.2013.02.021
45. Van der Paal J, Neyts EC, Verlackt CCW, et al: Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress. Chem Sci 2016;7:489–498. doi: 10.1039/C5SC02311D
46. Ribeiro SC, Sartorius G, Pletscher F, et al: Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting. Fertil Steril 2013;100:686–694. doi: 10.1016/j.fertnstert.2013.05.030
47. Ortega FC, Anel-Lopez L, Ortiz-Rodriguez JM, et al: Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na. Andrology 2017;5:1174–1182. doi: 10.1111/andr.12419
48. Dasheiff RM: A new method of monitoring membrane potential in rat hippocampal slices using cyanine voltage-sensitive dyes. J Neurosci Methods 1985;13:199–212. doi: 10.1016/0165-0270(85)90068-8
49. Ortega-Ferrusola C, Anel-Lopez L, Martin-Munoz P, et al: Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes. Reproduction 2017;153: 293–304. doi: 10.1530/REP-16-0539
50. Aitken RJ, Gibb Z, Baker MA, et al: Causes and consequences of oxidative stress in spermatozoa. Reprod Fertil Dev 2016;28:1–10. doi: 10.1071/RD15325
51. Koppers AJ, Mitchell LA, Wang P, et al: Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. Biochem J 2011;436:687–698. doi: 10.1042/BJ20110114
52. Munoz PM, Ferrusola CO, Lopez LA, et al: Caspase 3 activity and lipoperoxidative status in raw semen predict the outcome of cryopreservation of stallion spermatozoa. Biol Reprod 2016;95:53. doi: 10.1095/biolreprod.116.139444
53. Estaras M, Pena FJ, Tapia JA, et al: Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression. J Physiol Biochem 2020;76:345–355. doi: 10.1007/s13105-020-00740-6
54. Martin Munoz P, Ortega Ferrusola C, Vizuete G, et al: Depletion of intracellular thiols and increased production of 4-hydroxynonenal that occur during cryopreservation of stallion spermatozoa lead to caspase activation, loss of motility, and cell death. Biol Reprod 2015;93:143. doi: 10.1095/biolreprod.115.132878
55. Gallardo BJM, da Silva BCM, Martin MP, et al: Caspase activation, hydrogen peroxide production and Akt dephosphorylation occur during stallion sperm senescence. Reprod Domest Anim 2014;49:657–664. doi: 10.1111/rda.12343
56. Caselles AB, Miro-Moran A, Morillo Rodriguez A, et al: Identification of apoptotic bodies in equine semen. Reprod Domest Anim 2014;49:254–262. doi: 10.1111/rda.12264
57. Ortega FC, Gonzalez FL, Salazar SC, et al: Inhibition of the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of stallion sperm. Theriogenology 2010;74:458–465. doi: 10.1016/j.theriogenology.2010.02.029
2. Rodriguez-Martinez H: Semen evaluation and handling In: PJCaSL, editor. Animal andrology:theory and applications. Oxfordshire, UK; Cabi: 2014:509–594.
3. Rodriguez-Martinez H: State of the art in farm animal sperm evaluation. Reprod Fertil Dev 2007;19:91–101. doi: 10.1071/RD06104
4. Rodriguez-Martinez H, Barth AD: In vitro evaluation of sperm quality related to in vivo function and fertility. Soc Reprod Fertil Suppl 2007;64:39–54. doi: 10.5661/RDR-VI-39
5. Al-Makhzoomi A, Lundeheim N, Haard 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
6. Rodrígue Martínez H: Semen evaluation:can we forecast fertility? Vet Stanica 2019;50:293–305.
7. DeJarnette JM, Harstine BR, McDonald K, et al: Commercial application of flow cytometry for evaluating bull sperm. Anim Reprod Sci 2022;246:106838. doi: 10.1016/j.anireprosci.2021.106838
8. Rodriguez-Martinez H, Kvist U, Ernerudh J, et al: Seminal plasma proteins: what role do they play? Am J Reprod Immunol 2011;66 Suppl 1:11–22. doi: 10.1111/j.1600-0897.2011.01033.x
9. Perez-Patino C, Parrilla I, Barranco I, et al: New in-depth analytical approach of the porcine seminal plasma proteome reveals potential fertility biomarkers. J Proteome Res 2018;17:1065–1076. doi: 10.1021/acs.jproteome.7b00728
10. Rodriguez-Martinez H, Martinez EA, Calvete JJ, et al: Seminal plasma: relevant for fertility? Int J Mol Sci 2021;22:4368. doi: 10.3390/ijms22094368
11. Rodriguez-Martinez H, Roca J: Extracellular vesicles in seminal plasma: a safe and relevant tool to improve fertility in livestock? Anim Reprod Sci 2022;244:107051. doi: 10.1016/j.anireprosci.2022.107051
12. Pena FJ, Saravia F, Johannisson A, et al: Detection of early changes in sperm membrane integrity pre-freezing can estimate post-thaw quality of boar spermatozoa. Anim Reprod Sci 2007;97:74–83. doi: 10.1016/j.anireprosci.2005.12.014
13. da Silva BCM, Ortega-Ferrusola C, Morrell JM, et al: Flow cytometric chromosomal sex sorting of stallion spermatozoa induces oxidative stress on mitochondria and genomic DNA. Reprod Domest Anim 2016;51:18–25. doi: 10.1111/rda.12640
14. Gardela J, Ruiz-Conca M, Wright D, et al; Semen modulates cell proliferation and differentiation-related transcripts in the pig peri-ovulatory endometrium. Biology (Basel) 2022;11:616. doi: 10.3390/biology11040616
15. Alvarez-Rodriguez M, Martinez C, Wright D, et al: The transcriptome of pig spermatozoa, and its role in fertility. Int J Mol Sci 2020;21:1572. doi: 10.3390/ijms21051572
16. Martinez CA, Roca J, Alvarez-Rodriguez M, et al: miRNA-profiling in ejaculated and epididymal pig spermatozoa and their relation to fertility after artificial insemination. Biology (Basel) 2022;11:236. doi: 10.3390/biology11020236
17. Pertille F, Alvarez-Rodriguez M, da Silva AN, et al: Sperm methylome profiling can discern fertility levels in the porcine biomedical model. Int J Mol Sci 2021;22:2679. doi: 10.3390/ijms22052679
18. Kerns K, Zigo M, Drobnis EZ, et al: Zinc ion flux during mammalian sperm capacitation. Nat Commun 2018;9:2061. doi: 10.1038/s41467-018-04523-y
19. Zigo M, Kerns K, Sen S, et al: Zinc is a master-regulator of sperm function associated with binding, motility, and metabolic modulation during porcine sperm capacitation. Commun Biol 2022;5:538. doi: 10.1038/s42003-022-03485-8
20. Hossain MS, Johannisson A, Wallgren M, et al: Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art. Asian J Androl 2011;13:406–419. doi: 10.1038/aja.2011.15
21. Ortega-Ferrusola C, Gil MC, Rodriguez-Martinez H, et al: Flow cytometry in spermatology: a bright future ahead. Reprod Domest Anim 2017;52:921–931. doi: 10.1111/rda.13043
22. Roca J, Parrilla I, Gil MA, et al: Non-viable sperm in the ejaculate: lethal escorts for contemporary viable sperm. Anim Reprod Sci 2016;169:24–31. doi: 10.1016/j.anireprosci.2016.02.028
23. Pena FJ, Gibb Z: Oxidative stress and reproductive function: oxidative stress and the long-term storage of horse spermatozoa. Reproduction 2022;164:F135–F144. doi: 10.1530/REP-22-0264
24. Pena FJ, O’Flaherty C, Ortiz Rodriguez JM, et al: The stallion spermatozoa: a valuable model to help understand the interplay between metabolism and redox (De)regulation in sperm cells. Antioxid Redox Signal 2022;37:521–537. doi: 10.1089/ars.2021.0092
25. Gonzalez-Castro RA, Pena FJ, Herickhoff LA: Validation of a new multiparametric protocol to assess viability, acrosome integrity and mitochondrial activity in cooled and frozen thawed boar spermatozoa. Cytometry B Clin Cytom 2022;102:400–408. doi: 10.1002/cyto.b.22058
26. Ortiz-Rodriguez JM, Martin-Cano FE, Gaitskell-Phillips G, et al: The SLC7A11: sperm mitochondrial function and non-canonical glutamate metabolism. Reproduction 2020;160:803–818. doi: 10.1530/REP-20-0181
27. Ortega-Ferrusola C, Martin Munoz P, Ortiz-Rodriguez JM, et al: Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoadagger. Biol Reprod 2019;100: 1090–1107. doi: 10.1093/biolre/ioy241
28. Aitken RJ, Findlay JK, Hutt KJ, et al: Apoptosis in the germ line. Reproduction 2011;141:139–150. doi: 10.1530/REP-10-0232
29. Boerke A, Brouwers JF, Olkkonen VM, et al: Involvement of bicarbonate-induced radical signaling in oxysterol formation and sterol depletion of capacitating mammalian sperm during in vitro fertilization. Biol Reprod 2013;88:21. doi: 10.1095/biolreprod.112.101253
30. Aitken RJ: The capacitation-apoptosis highway: oxysterols and mammalian sperm function. Biol Reprod 2011;85:9–12. doi: 10.1095/biolreprod.111.092528
31. Zerbinati C, Caponecchia L, Puca R, et al: Mass spectrometry profiling of oxysterols in human sperm identifies 25-hydroxycholesterol as a marker of sperm function. Redox Biol 2017;11:111–117. doi: 10.1016/j.redox.2016.11.008
32. Luque GM, Dalotto-Moreno T, Martin-Hidalgo D, et al. Only a subpopulation of mouse sperm displays a rapid increase in intracellular calcium during capacitation. J Cell Physiol 2018;233: 9685–9700. doi: 10.1002/jcp.26883
33. Escoffier J, Navarrete F, Haddad D, et al: Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation. Biol Reprod 2015;92:121. doi: 10.1095/biolreprod.114.127266
34. Sui X, Zhang R, Liu S, et al: RSL3 drives ferroptosis through GPX4 inactivation and ROS production in colorectal cancer. Front Pharmacol 2018;9:1371. doi: 10.3389/fphar.2018.01371
35. Ortiz-Rodriguez JM, Martin-Cano FE, Ortega-Ferrusola C, et al: The incorporation of cystine by the soluble carrier family 7 member 11 (SLC7A11) is a component of the redox regulatory mechanism in stallion spermatozoadagger. Biol Reprod 2019;101:208–222. doi: 10.1093/biolre/ioz069
36. Ortiz-Rodriguez JM, da Silva BCM, Masot J, et al: Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3. PLoS One 2019;14:e0211994. doi: 10.1371/journal.pone.0211994
37. Aitken RJ, Baker MA, Nixon B: Are sperm capacitation and apoptosis the opposite ends of a continuum driven by oxidative stress? Asian J Androl 2015;17:633–639. doi: 10.4103/1008-682X.153850
38. Pena FJ, Johannisson A, Wallgren M, et al: Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture. Zygote 2004;12:117–124. doi: 10.1017/S096719940400262X
39. Pena FJ, Johannisson A, Wallgren M, et al: Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation. Theriogenology 2004;61:63–70. doi: 10.1016/S0093-691X(03)00181-X
40. Tienthai P, Johannisson A, Rodriguez-Martinez H: Sperm capacitation in the porcine oviduct. Anim Reprod Sci 2004;80:131–146. doi: 10.1016/S0378-4320(03)00134-9
41. Gibbons SJ, Washburn KB, Talamo BR: P2X(7) receptors in rat parotid acinar cells: formation of large pores. J Auton Pharmacol 2001;21:181–190. doi: 10.1046/j.1365-2680.2001.00224.x
42. Xiao F, Waldrop SL, Khimji AK, et al: Pannexin1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated free fatty acids in liver cells. Am J Physiol Cell Physiol 2012;303:C1034–C1044. doi: 10.1152/ajpcell.00175.2012
43. Gallardo BJM, da Silva BCM, Martin MP, et al: Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7. Reproduction 2014;148:221–235. doi: 10.1530/REP-13-0191
44. da Silva BCM, Ortega FC, Morillo RA, et al: Sex sorting increases the permeability of the membrane of stallion spermatozoa. Anim Reprod Sci 2013;138:241–251. doi: 10.1016/j.anireprosci.2013.02.021
45. Van der Paal J, Neyts EC, Verlackt CCW, et al: Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress. Chem Sci 2016;7:489–498. doi: 10.1039/C5SC02311D
46. Ribeiro SC, Sartorius G, Pletscher F, et al: Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting. Fertil Steril 2013;100:686–694. doi: 10.1016/j.fertnstert.2013.05.030
47. Ortega FC, Anel-Lopez L, Ortiz-Rodriguez JM, et al: Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na. Andrology 2017;5:1174–1182. doi: 10.1111/andr.12419
48. Dasheiff RM: A new method of monitoring membrane potential in rat hippocampal slices using cyanine voltage-sensitive dyes. J Neurosci Methods 1985;13:199–212. doi: 10.1016/0165-0270(85)90068-8
49. Ortega-Ferrusola C, Anel-Lopez L, Martin-Munoz P, et al: Computational flow cytometry reveals that cryopreservation induces spermptosis but subpopulations of spermatozoa may experience capacitation-like changes. Reproduction 2017;153: 293–304. doi: 10.1530/REP-16-0539
50. Aitken RJ, Gibb Z, Baker MA, et al: Causes and consequences of oxidative stress in spermatozoa. Reprod Fertil Dev 2016;28:1–10. doi: 10.1071/RD15325
51. Koppers AJ, Mitchell LA, Wang P, et al: Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. Biochem J 2011;436:687–698. doi: 10.1042/BJ20110114
52. Munoz PM, Ferrusola CO, Lopez LA, et al: Caspase 3 activity and lipoperoxidative status in raw semen predict the outcome of cryopreservation of stallion spermatozoa. Biol Reprod 2016;95:53. doi: 10.1095/biolreprod.116.139444
53. Estaras M, Pena FJ, Tapia JA, et al: Melatonin modulates proliferation of pancreatic stellate cells through caspase-3 activation and changes in cyclin A and D expression. J Physiol Biochem 2020;76:345–355. doi: 10.1007/s13105-020-00740-6
54. Martin Munoz P, Ortega Ferrusola C, Vizuete G, et al: Depletion of intracellular thiols and increased production of 4-hydroxynonenal that occur during cryopreservation of stallion spermatozoa lead to caspase activation, loss of motility, and cell death. Biol Reprod 2015;93:143. doi: 10.1095/biolreprod.115.132878
55. Gallardo BJM, da Silva BCM, Martin MP, et al: Caspase activation, hydrogen peroxide production and Akt dephosphorylation occur during stallion sperm senescence. Reprod Domest Anim 2014;49:657–664. doi: 10.1111/rda.12343
56. Caselles AB, Miro-Moran A, Morillo Rodriguez A, et al: Identification of apoptotic bodies in equine semen. Reprod Domest Anim 2014;49:254–262. doi: 10.1111/rda.12264
57. Ortega FC, Gonzalez FL, Salazar SC, et al: Inhibition of the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of stallion sperm. Theriogenology 2010;74:458–465. doi: 10.1016/j.theriogenology.2010.02.029
Published
2023-09-25
How to Cite
Peña F., & Rodriguez-Martinez H. (2023). Clinical applications of flow cytometric sperm analyses. Clinical Theriogenology, 15. https://doi.org/10.58292/ct.v15.9406
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Review Reports
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