Ultra-rapid cooling of ibex sperm by spheres method does not induce a vitreous extracellular state and increases the membrane damages

Autoři: Paula Bóveda aff001;  Adolfo Toledano-Díaz aff001;  Cristina Castaño aff001;  Milagros Cristina Esteso aff001;  Antonio López-Sebastián aff001;  Dimitrios Rizos aff001;  Alejandro Bielli aff002;  Rodolfo Ungerfeld aff003;  Julián Santiago-Moreno aff001
Působiště autorů: Dpto. Reproducción Animal, INIA, Madrid, Spain aff001;  Dpto. Morfología y Desarrollo, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay aff002;  Dpto. Fisiología, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0227946


Sperm cryopreservation by ultra-rapid cooling based on dropping small volumes of sperm suspension directly into liquid nitrogen, has been successful in some wild ruminant species, including the Iberian ibex (Capra pyrenaica). In ultra-rapid cooling, the contents of these droplets are expected to enter a stable, glass-like state, but to the best of our knowledge no information exists regarding the presence or absence of ice formation in the extracellular milieu when using this technique. Different modifications to the extracellular milieu likely inflict different types of damage on the plasmalemma, the acrosome and mitochondrial membranes. The aims of the present work were: 1) to examine the physical state of the extracellular milieu after cryopreservation at slow and ultra-rapid cooling rates—and thus determine whether ultra-rapid cooling vitrifies the extracellular milieu; and 2) to compare, using conventional sperm analysis techniques and scanning and transmission electron microscopy, the damage to sperm caused by these two methods. Sperm samples were obtained by the transrectal ultrasound-guided massage method (TUMASG) from anesthetized Iberian ibexes, and cryopreserved using slow and ultra-rapid cooling techniques. Sperm motility (22.95 ± 3.22% vs 4.42 ± 0.86%), viability (25.64 ± 3.71% vs 12.8 ± 2.50%), acrosome integrity (41.45± 3.73% vs 27.00 ± 1.84%) and mitochondrial membrane integrity (16.52 ± 3.75% vs 4.00 ± 0.65%) were better after slow cooling (P<0.001) than after ultra-rapid technique. Cryo-scanning electron microscopy (Cryo-SEM) suggested that the vitrified state was not achieved by ultra-rapid cooling, and that the ice crystals formed were smaller and had more stretchmarks (P<0.001) than after slow cooling. Scanning electron microscopy revealed no differences in the types of damage caused by the examined techniques, although transmission electron microscopy showed the damage to the plasmalemma and mitochondrial membrane to be worse after ultra-rapid cooling. In conclusion ultra-rapid cooling provoked more membrane damage than slow cooling, perhaps due to the extracellular ice crystals formed.

Klíčová slova:

Acrosomes – Cell membranes – Cryopreservation – Crystals – Mitochondria – Mitochondrial membrane – Sperm – Sperm head


1. Pradiee J, Esteso MC, Castaño C, Toledano-Díaz A, López-Sebastián A, Santiago-Moreno J. Cryopreservation of epididymal sperm from ibexes (Capra pyrenaica) using short equilibration time with glycerol. Theriogenology. 2014; 82(3):525–528. doi: 10.1016/j.theriogenology.2014.05.012 24938799

2. Coloma MA, Toledano-Díaz A, Castaño C, Velázquez R, Gómez-Brunet A, López-Sebastián A, et al. Seasonal variation in reproductive physiological status in the Iberian ibex (Capra pyrenaica) and its relationship with sperm freezability. Theriogenology. 2011; 76:1695–1705. doi: 10.1016/j.theriogenology.2011.07.001 21855981

3. Santiago-Moreno J, Coloma MA, Toledano-Díaz A, Castaño C, Gómez-Brunet A and López-Sebastián A. Assisted reproduction in Mediterranean wild ruminants: lessons from the Spanish ibex (Capra pyrenaica). Soc. Reprod.Fertil. Suppl. 2010; 67: 431–441. 21755689

4. Santiago-Moreno J, Toledano-Díaz A, Pulido-Pastor A, Gómez-Brunet A, López-Sebastián A. Birth of live Spanish ibex (Capra pyrenaica hispanica) derived by artificial insemination with epididymal spermatozoa retrieved after death. Theriogenology. 2006; 66:283–91. doi: 10.1016/j.theriogenology.2005.11.012 16376980

5. Pradiee J, Esteso MC, López-Sebastián A, Toledano-Díaz A, Castaño C, Carrizosa JA, et al. Successful ultra-rapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa. Theriogenology. 2015; 84:1513–1522. doi: 10.1016/j.theriogenology.2015.07.036 26316218

6. Pradiee J, Sánchez-Calabuig MJ, Castaño C, O’Brien E, Esteso MC, Beltran-Breña P, et al. Fertilizing capacity of vitrified epididymal sperm from Iberian ibex (Capra pyrenaica) Theriogenology. 2018; 108:314–320. doi: 10.1016/j.theriogenology.2017.11.021 29288975

7. Mazur P. Freezing of living cells: mechanisms and implications. Am. J. Physiol. Physiol. 1984; 247:125–142.

8. Woelders H, Matthijs A, Engel B. Effects of Trehalose and Sucrose, Osmolality of the Freezing Medium, and Cooling Rate on Viability and Intactness of Bull Sperm after Freezing and Thawing. Cryobiology. 1997; 35:93–105. doi: 10.1006/cryo.1997.2028 9299101

9. Morris GJ, Acton E, Murray B, Fonseca F. Freezing injury: The special case of the sperm cell. Cryobiology. 2012; 64:71–80. doi: 10.1016/j.cryobiol.2011.12.002 22197768

10. González-Fernández L, Morrell JM, Peña FJ and Macías-García B. Osmotic shock induces structural damage on equine spermatozoa plasmalemma and mitochondria. Theriogenology. 2012; 78:415–422. doi: 10.1016/j.theriogenology.2012.02.021 22578615

11. Pegg DE. Principles of cryopreservation. Methods Mol Biol. 2015; 1257:3–19. doi: 10.1007/978-1-4939-2193-5_1 25428001

12. Isachenko E, Isachenko V, Katkov I, Dessole S, Nawroth F. Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success. Reprod Biomed Online. 2003; 6:191–200. doi: 10.1016/s1472-6483(10)61710-5 12675999

13. Isachenko V, Isachenko E, Petrunkina AM, Sanchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod Fertil Dev. 2012; 24:323–26. doi: 10.1071/RD11061 22281078

14. Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N et al. Vitrification of human spermatozoa without cryo-protectants. CryoLetters. 2002; 23:93–102. 12050777

15. Bauchi A, Woods EJ, Critser JK. Cryopreservation and vitrification: recent advances infertility preservation technologies. Expert Rev Med Devices. 2008; 5:359–70. doi: 10.1586/17434440.5.3.359 18452386

16. Katkov I. Kinetic vitrification of spermatozoa of vertebrates: What we can learn from nature? In: Katkov II editor. Current Frontiers in Cryobiology. Rijeka, Croatia. 2012; 3–40.

17. Sánchez R, Risopatrón J, Schulz M, Villegas J, Isachenko V, Kreinberg R. et al. Canine sperm vitrification with sucrose: effect on sperm function. Andrologia. 2011; 43:233–41. doi: 10.1111/j.1439-0272.2010.01054.x 21486402

18. Merino O, Sánchez R, Risopatrón J, Isachenko E, Katkov II, Figueroa E, et al. Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: First report. Andrologia. 2012; 44:390–395. doi: 10.1111/j.1439-0272.2011.01196.x 21806657

19. Pradiee J, Esteso MC, Castaño C, Toledano-Díaz A, López-Sebastián A, Guerra R, et al. Conventional slow freezing cryopreserves mouflon spermatozoa better than vitrification. Andrologia. 2017; 49:1–7.

20. Shaw JM, Jones GM. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Human Reproduction Update. 2003; 9:583–605. doi: 10.1093/humupd/dmg041 14714593

21. Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, et al. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008; 136:167–173. doi: 10.1530/REP-07-0463 18483075

22. Caturla-Sánchez E, Sánchez-Calabuig MJ, Pérez-Gutiérrez JF, Cerdeira J, Castaño C, Santiago-Moreno J. Vitrification of dog spermatozoa: Effects of two cryoprotectants (sucrose or trehalose) and two warming procedures. Cryobiology. 2017; 80:126–129. doi: 10.1016/j.cryobiol.2017.11.001 29126865

23. Said TM, Gaglani A, Agarwal A. Implication of apoptosis in sperm cryoinjury. Reproductive BioMedicine Online. 2010; 21(4):456–462. doi: 10.1016/j.rbmo.2010.05.011 20800544

24. Colás C, Junquera C, Pérez-Pé R, Cebrián-Pérez JA, Muiño-Blanco T. Ultrastructural Study of the Ability of Seminal Plasma Proteins to Protect Ram Spermatozoa Against Cold-shock. Microsc. Res. Tech. 2009; 72:566–572. doi: 10.1002/jemt.20710 19322897

25. Gravance CG, Vishwanath R, Pitt C, Garner DL, Casey PJ. Effects of cryopreservation on bull sperm head morphometry. J Androl. 1998; 19:704–709. 9876021

26. Esteso MC, Fernández-Santos MR, Soler AJ, Garde JJ. Head dimensions of cryopreserved red deer spermatozoa are affected by thawing procedure. Cryo-Letters. 2003; 24:261–268. 12955173

27. Rijsselaere T, Van Soom A, Hoflack G, Maes D, De Kruif A. Automated sperm morphometry and morphology analysis of canine semen by the Hamilton-Thorne analyser. Theriogenology. 2004; 62:1292–306. doi: 10.1016/j.theriogenology.2004.01.005 15325556

28. O’Brien E, Esteso MC, Castaño C, Toledano-Diaz A, Bóveda P, Martínez-Fresneda L, et al. Effectiveness of ultra-rapid cryopreservation of sperm from endangered species, examined by morphometric means. Theriogenology. 2019; 129:160–167. doi: 10.1016/j.theriogenology.2019.02.024 30852388

29. Auger J, Ronot X, Dadoune JP. Human sperm mitochondrial function related to motility: a flow and image cytometric assessment. J Androl. 1989; 10:439–48. doi: 10.1002/j.1939-4640.1989.tb00135.x 2621152

30. Sharafi M, Zhandi M, Sharif AA. Supplementation of soybean lecithin-based semen extender by antioxidants: complementary flowcytometric study on post-thawed ram spermatozoa. Cell Tissue Bank. 2015; 16(2):261–9. doi: 10.1007/s10561-014-9458-5 24907919

31. Emamverdi M, Zhandi M, Shahneh AZ, Sharafi M, Akhlaghi A, Motlagh, et al. Flow cytometric and microscopic evaluation of post-thawed ram semen cryopreserved in chemically defined home-made or commercial extenders. Animal Production Science. 2015; 55:551–558.

32. Pradiee J, O’Brien E, Esteso MC, Castaño C, Toledano-Díaz A, López-Sebastián A, et al. Effect of shortening the prefreezing equilibration time with glycerol on the quality of chamois (Rupicapra pyrenaica), ibex (Capra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia) ejaculates. Anim. Reprod. Sci. 2016; 171:121–128. doi: 10.1016/j.anireprosci.2016.06.007 27346588

33. Santiago-Moreno J, Castaño C, Toledano-Díaz A, Esteso MC, López-Sebastián A, Guerra R, et al. Cryopreservation of aoudad (Ammotragus lervia sahariensis) sperm obtained by transrectal ultrasound-guided massage of the accessory sex glands and electroejaculation. Theriogenology. 2013; 79:383–391. doi: 10.1016/j.theriogenology.2012.10.011 23158213

34. Esteso MC, Rodríguez E, Toledano-Díaz A, Castaño C, Pradiee J, López-Sebastián A. et al. Descriptive analysis of sperm head morphometry in Iberian ibex (Capra pyrenaica): Optimum sampling procedure and. Anim. Reprod. Sci. 2015; 155:1–8. doi: 10.1016/j.anireprosci.2015.01.007

35. Forero-González RA, Celeghini ECC, Raphael CF, Andrade AFC, Bressan FF, Arruda RP. Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes. Andrologia. 2012; 44:154–159. doi: 10.1111/j.1439-0272.2010.01154.x 22506813

36. Chang BS, Randall CS. Use of subambient thermal analysis to optimise protein lyophilisation, Cryobiology. 1992; 29:632–656.

37. Lopera-Vásquez R, Hamdi M, Fernandez-Fuertes B, Maillo V, Beltrán-Breña P, Calle A, et al. Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality. PLoS ONE. 2016; 11(2).

38. Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH. Cold shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as a model. The Journal of Experimental Zoology. 1993; 265:432–437. doi: 10.1002/jez.1402650413 8463792

39. Constantin JG, Schneider M and Corti HR. Glass Transition Temperature of Saccharide Aqueous Solutions Estimated with the Free Volume/Percolation Model. J Phys Chem B. 2016; 120(22):5047–55. doi: 10.1021/acs.jpcb.6b01841 27176640

40. Angell CA, Oguni M and Sichina WJ. Heat capacity of water at extremes of supercooling and superheating. J Phys Chem. 1982; 86:998–1002.

41. Umrath W. Calculation of the freeze-drying time for electron-microscopical preparations. Mikroskopie. 1983; 40:9–34. 6343916

42. Teixeira AS, González-Benito ME and Molina-García AD. Tissue and cytoplasm vitrification in cryopreservation monitored by low temperature scanning electron microscopy (cryo-SEM). In: Méndez-Vilas A (ed) Current microscopy contributions to advances in science and technology. Formatex, Badajoz, 2012; 872–879.

43. Teixeira AS, González-Benito ME and Molina-García AD. Determination of glassy state by cryo-SEM and DSC in cryopreservation of mint shoot tips by encapsulation–dehydration. Plant Cell Tiss Organ Cult. 2014; 119:269–280.

44. Marco-Jiménez F, Casares-Crespo L and Vicente JS. Porcine oocyte vitrification in optimized low toxicity solution with open pulled straws. Zygote. 2012; 204–212. doi: 10.1017/S0967199412000524 23102007

45. Morris GJ. Rapidly cooled human sperm: no evidence of intracellular ice formation. Human Reproduction. 2006; 21:2075–2083. doi: 10.1093/humrep/del116 16613884

46. Morris GJ, Faszer K, Green JE, Draper DE, Grout BWW, Fonseca F. Rapidly cooled horse spermatozoa: Loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation. Theriogenology. 2007; 68:804–812. doi: 10.1016/j.theriogenology.2007.06.009 17645937

47. Ekwall H. Cryo-scanning electron microscopy (Cryo-SEM) of boar semen frozen in medium-straws and MiniFlatPacks. 2007; 67:1463–1472.

48. Isachenko V, Isachenko E, Katkov I, Montag M, Dessole S, Nawroth F, et al. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapour: effect on motility, DNA integrity and fertilization ability. Biol Reprod. 2004; 71:1167–73. doi: 10.1095/biolreprod.104.028811 15175233

49. Cook KLM, Hartel RW. Ice Crystallization in Ice Cream Production. Compr. Rev. food Sci. food Saf. 2010; 9: 213–222.

50. Caldwell KB, Goff HD, Stanley DW. A Low-Temperature Scanning Electron Microscopy Study of Ice Cream. II. Influence of Selected Ingredients and Processes. Food Structure. 1992; 11:11–23.

51. Aboagla EM, Terada T. Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa. Theriogeniology. 2004; 62:1160–1172.

52. Dubochet J. The Physics of Rapid Cooling and Its Implications for Cryoimmobilization of Cells. Methods Cell Biol. 2007; 79:7–21. doi: 10.1016/S0091-679X(06)79001-X 17327150

53. Courtens JL, Paquignon M. Ultrastructure of fresh, frozen and frozen-thawed spermatozoa of the boar in: L.A. Johnson, K. Larsson (Eds.), Proceedings of the First International Conference on Deep Freezing of Boar Semen, Swedish University of Agricultural Sciences. 1985; 61–87.

54. Sherman JK, Liu KC. Ultrastructure before freezing, while frozen, and after thawing in assessing cryoinjury of mouse epididymal spermatozoa. Cryobiology. 1988; 19:503–510.

55. Piomboni P, Focarelli R, Stendardi A, Ferramosca A, Zara V. The role of mitochondria in energy production for human sperm motility. International Journal of Andrology. 2012; 35:109–124. doi: 10.1111/j.1365-2605.2011.01218.x 21950496

56. Somanath PR, Gandhi KK. Isolation and partial characterisation of the plasma and outer acrosomal membranes of goat spermatozoa. Small Ruminant Research. 2004; 53:67–74.

57. Krogenaes A, Berg KA, Hafne AL, Engeland E. Membrane Alterations in Bull Spermatozoa after Freezing and Thawing and after In Vitro Fertilization. Acta vet scand. 1994; 35:17–26. 8209818

58. Hofmo PO, Berg KA. Electron Microscopical Studies of Membrane Injuries in Blue Fox Spermatozoa Subjected to the Process of Freezing and Thawing. Cryobiology. 1989; 26:124–131. doi: 10.1016/0011-2240(89)90042-4 2707028

59. Oliveira LZ, Hossepian de Lima VFM, Levenhagen MA, dos Santos RM, Assumpção TI, Jacomini JO, et al. Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa. J. Vet. Sci. 2011; 12(3): 267–272. doi: 10.4142/jvs.2011.12.3.267 21897100

60. Nath J. Correlative Biochemical and Ultrastructural Studies on the Mechanism of Freezing Damage to Ram Semen. Cryobiology. 1972; 246:240–246.

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