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Kinetic vitrification: concepts and perspectives in animal sperm cryopreservation

Abstract

Sperm cryopreservation is an important tool for genetic diversity management programs and the conservation of endangered breeds and species. The most widely used method of sperm conservation is slow freezing, however, during the process, sperm cells suffer from cryoinjury, which reduces their viability and fertility rates. One of the alternatives to slow freezing is vitrification, that consist on rapid freezing, in which viable cells undergo glass-like solidification. This technology requires large concentrations of permeable cryoprotectants (P- CPA’s) which increase the viscosity of the medium to prevent intracellular ice formation during cooling and warming, obtaining successful results in vitrification of oocytes and embryos. Unfortunately, this technology failed when applied to vitrification of sperm due to its higher sensitivity to increasing concentrations of P-CPAs. Alternatively, a technique termed ‘kinetic sperm vitrification’ has been used and consists in a technique of permeant cryoprotectant-free cryopreservation by direct plunging of a sperm suspension into liquid nitrogen. Some of the advantages of kinetic vitrification are the speed of execution and no rate-controlled equipment required. This technique has been used successfully and with better results for motility in human (50-70% motility recovery), dog (42%), fish (82%) and donkey (21.7%). However, more studies are required to improve sperm viability after devitrification, especially when it comes to motility recovery. The objective of this review is to present the principles of kinetic vitrification, the main findings in the literature, and the perspectives for the utilization of this technique as a cryopreservation method.

Keywords:
assisted reproduction; cryobank; cryopreservation; cryoprotectant-free; spermatozoa

Introduction

Cryopreservation or biostabilization is a stable and long-term preservation and storage method in which biological materials such as cells, tissues, and embryos are kept in a glassy state (Katkov et al., 2012Katkov II, Bolyukh VF, Chernetsov OA, Dudin PI, Grigoriev AY, Isachenko V, Isachenko E, Lulat AG-M, Moskovtsev SI, Petrushko MP, Pinyaev VI, Sokol KM, Sokol YI, Sushko AB, Yakhnenko I. Kinetic vitrification of spermatozoa of vertebrates: what can we learn from nature? In: Katkov I , editor. Current frontiers in cryobiology. Croatia: IntechOpen; 2012. p. 3-40. http://dx.doi.org/10.5772/34784.
http://dx.doi.org/10.5772/34784...
) and do not undergo biological changes (Akiyama et al., 2019Akiyama Y, Shinose M, Watanabe H, Yamada S, Kanda Y. Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing. Proc Natl Acad Sci USA. 2019;116(16):7738-43. http://dx.doi.org/10.1073/pnas.1808645116. PMid:30936320.
http://dx.doi.org/10.1073/pnas.180864511...
). Although sperm cryopreservation has undergone some changes over the years, conventional freezing (fast or slow) is still the most used method to cryopreserve human and animal sperm samples (Isachenko et al., 2017Isachenko V, Rahimi G, Mallmann P, Sánchez R, Isachenko E. Technologies of cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness. Andrology. 2017;5(6):1055-63. http://dx.doi.org/10.1111/andr.12414. PMid:28992376.
http://dx.doi.org/10.1111/andr.12414...
). This freezing method requires specialized equipment, takes time, and often damages sperm (Ozkavukcu et al., 2008Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J Assist Reprod Genet. 2008;25(8):403-11. http://dx.doi.org/10.1007/s10815-008-9232-3. PMid:18704674.
http://dx.doi.org/10.1007/s10815-008-923...
), mainly owing to the reduced thawing temperature, formation of ice crystals, and stress (physical, chemical, osmotic, and oxidative) that compromise sperm quality and fertilizing capacity (Colás et al., 2009Colás C, Junquera C, Perez-Pe R, Cebrian-Perez JA, Muino-Blanco T. Ultrastructural study of the ability of seminal plasma proteins to protect ram spermatozoa against cold shock. Microsc Res Tech. 2009;72(8):566-72. http://dx.doi.org/10.1002/jemt.20710. PMid:19322897.
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). The main causes of reduced sperm quality in slow freezing are changes in the lipid phase and/or the increase in lipid peroxidation, which results in a reduction in the speed and percentage of mobile sperm and significant losses in the fertilization potential (Celeghini et al., 2008Celeghini ECC, Arruda RP, Andrade AFC, Nascimento J, Raphael CF, Rodrigues PHM. Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin. Anim Reprod Sci. 2008;104(2-4):119-31. http://dx.doi.org/10.1016/j.anireprosci.2007.02.001. PMid:17368970.
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).

To prevent damage, most cryopreservation methods, including slow freezing, use P-CPA that can move across cell membranes and modulate the rate and duration of cell dehydration during membrane phase transitions induced by freezing. P-CPA provide intracellular protection because, among other reasons, they reduce the temperature of ice nucleation and the size of crystals formed (Swain and Smith, 2010Swain JE, Smith GD. Cryoprotectants. In: Chian RC, Quinn P, editors. Fertility Cryopreservation. Cambridge: Cambridge University Press; 2010. p. 24-38. http://dx.doi.org/10.1017/CBO9780511730207.005.
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; Sieme et al., 2016Sieme H, Oldenhof H, Wolkers WF. Mode of action of cryoprotectants for sperm preservation. Anim Reprod Sci. 2016;169:2-5. http://dx.doi.org/10.1016/j.anireprosci.2016.02.004. PMid:26936658.
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).

Equilibrium vitrification, which is used for human (Valojerdi et al., 2009Valojerdi MR, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B. Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet. 2009;26(6):347-54. http://dx.doi.org/10.1007/s10815-009-9318-6. PMid:19513822.
http://dx.doi.org/10.1007/s10815-009-931...
) and animals (Varago et al., 2006Varago FC, Saliba WP, Alvim MTT, Vasconcelos AB, Oliveira CH, Stahlberg R, Lagares MA. Vitrification of in vitro produced Zebu embryos. Anim Reprod. 2006;3(3):353-8.; Gibbons et al., 2019Gibbons A, Bruno-Galarraga M, Fernandez J, Gonzalez-Bulnes A, Cueto M. Vitrified embryo transfer in Merino sheep under extensive conditions. Anim Reprod. 2019;16(2):297-301. http://dx.doi.org/10.21451/1984-3143-AR2018-0108. PMid:33224290.
http://dx.doi.org/10.21451/1984-3143-AR2...
) embryo preservation, requires high concentrations of cryoprotectant; this raises the viscosity of the milieu and prevents ice formation during cooling, as well as during warming. However, high concentrations of cryoprotectant are harmful to the cytoskeletons of oocytes, and especially to those of sperm cells due to harmful cytotoxic (Fahy, 1986Fahy GM. The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology. 1986;23(1):1-13. http://dx.doi.org/10.1016/0011-2240(86)90013-1. PMid:3956226.
http://dx.doi.org/10.1016/0011-2240(86)9...
; Pegg, 2015Pegg DE. Principles of cryopreservation. In: Wolkers WF, Oldenhof H, editors. Cryopreservation and freeze-drying protocols. 3rd ed. New York: Springer; 2015. p. 3-19. (Methods in Molecular Biology; no. 1257). http://dx.doi.org/10.1007/978-1-4939-2193-5_1.
http://dx.doi.org/10.1007/978-1-4939-219...
) and osmotic (Guthrie et al., 2002Guthrie HD, Liu J, Critser JK. Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa. Biol Reprod. 2002;67(6):1811-6. http://dx.doi.org/10.1095/biolreprod67.6.1811. PMid:12444057.
http://dx.doi.org/10.1095/biolreprod67.6...
; Glazar et al., 2009Glazar AI, Mullen SF, Liu J, Benson JD, Critser JK, Squires EL, Graham JK. Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol. Cryobiology. 2009;59(2):201-6. http://dx.doi.org/10.1016/j.cryobiol.2009.07.009. PMid:19646432.
http://dx.doi.org/10.1016/j.cryobiol.200...
) effects. In this context, this technique is not recommended for sperm cells, owing to the use of high concentrations of these cryoprotectants, which can compromise the cells’ biological function (Mphaphathi et al., 2012Mphaphathi ML, Luseba D, Sutherland B, Nedambale TL. Comparison of slow freezing and vitrification methods for Venda cockerel’s spermatozoa. Open J Anim Sci. 2012;3(3):204-10. http://dx.doi.org/10.4236/ojas.2012.23028.
http://dx.doi.org/10.4236/ojas.2012.2302...
). Exceptions include the results obtained by Consuegra et al. (2019)Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Ortiz I, Hidalgo M. Vitrification of stallion sperm using 0.25 ml straws: effect of volume, concentration, and carbohydrates (sucrose/trehalose/raffinose). Anim Reprod. 2019;206:69-77. http://dx.doi.org/10.1016/j.anireprosci.2019.05.009. PMid:31138492.
http://dx.doi.org/10.1016/j.anireprosci....
, who used equilibrium vitrification without applying P-CPA to equine sperm. Thus, cryobiologists are increasingly demanding options to reduce the deleterious effects of cryoprotectants that permeate sperm cells; such measures suggest a reduction (Thananurak et al., 2019Thananurak P, Chuaychu-noo N, Thélie A, Phasuk Y, Vongpralub T, Blesbois E. Sucrose increases the quality and fertilizing ability of cryopreserved chicken sperms in contrast to rafinose. Poult Sci. 2019;98(9):4161-71. http://dx.doi.org/10.3382/ps/pez196. PMid:31065720.
http://dx.doi.org/10.3382/ps/pez196...
) in the frequency of cryoprotectant use or their removal from cryopreservation protocols.

From this perspective, an alternative to the use of P-CPA is kinetic vitrification (Katkov et al., 2012Katkov II, Bolyukh VF, Chernetsov OA, Dudin PI, Grigoriev AY, Isachenko V, Isachenko E, Lulat AG-M, Moskovtsev SI, Petrushko MP, Pinyaev VI, Sokol KM, Sokol YI, Sushko AB, Yakhnenko I. Kinetic vitrification of spermatozoa of vertebrates: what can we learn from nature? In: Katkov I , editor. Current frontiers in cryobiology. Croatia: IntechOpen; 2012. p. 3-40. http://dx.doi.org/10.5772/34784.
http://dx.doi.org/10.5772/34784...
), in which permeable cryoprotectants are not used and the use of N-PCA is optional. Thus, driven by the results of studies using this method in human sperm (Isachenko et al., 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
, bIsachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, Van Der Ven H. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004b;71(4):1167-73. http://dx.doi.org/10.1095/biolreprod.104.028811. PMid:15175233.
http://dx.doi.org/10.1095/biolreprod.104...
, 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
, 2012Isachenko V, Isachenko E, Petrunkina AM, Sánchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod Fertil Dev. 2012;24(2):323-6. http://dx.doi.org/10.1071/RD11061. PMid:22281078.
http://dx.doi.org/10.1071/RD11061...
), researchers have been evaluating and comparing seminal quality in animal models, such as fish (Merino et al., 2011Merino O, Risopatrón J, Sánchez R, Isachenko E, Figueroa E, Valdebenito I, Isachenko V. Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness. Anim Reprod Sci. 2011;124(1-2):125-31. http://dx.doi.org/10.1016/j.anireprosci.2011.02.023. PMid:21392903.
http://dx.doi.org/10.1016/j.anireprosci....
) and rabbits (Rosato and Iaffaldano, 2013Rosato MP, Iaffaldano N. Cryopreservation of rabbit semen: comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. Theriogenology. 2013;79(3):508-16. http://dx.doi.org/10.1016/j.theriogenology.2012.11.008. PMid:23218394.
http://dx.doi.org/10.1016/j.theriogenolo...
). In such scenarios, kinetic vitrification is an alternative to equilibrium or conventional vitrification methods for sperm cells, and stands out for its fast execution (Kim et al., 2012Kim S, Lee Y, Yang H, Kim Y. Rapid freezing without cooling equilibration in canine sperm. Anim Reprod Sci. 2012;130(1-2):111. http://dx.doi.org/10.1016/j.anireprosci.2011.12.014. PMid:22277841.
http://dx.doi.org/10.1016/j.anireprosci....
), not requiring the use of P-CPA, and not requiring programmable refrigeration devices (Ozkavukcu and Erdemli, 2002Ozkavukcu S, Erdemli E. Cryopreservation: basic knowledge and biophysical effects. J Ankara Med Sch. 2002;24:187-96. http://dx.doi.org/10.1501/Jms_0000000030.
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), thereby being a more cost-effective method (Tao et al., 2020Tao Y, Sanger E, Saewu A, Saewu A, Leveille M. Human sperm vitrification: the state of the art. Reprod Biol Endocrinol. 2020;18(1):17. http://dx.doi.org/10.1186/s12958-020-00580-5. PMid:32145746.
http://dx.doi.org/10.1186/s12958-020-005...
). Thus, this review aimed at presenting the principles of kinetic vitrification, its application in sperm samples, the main findings in the literature, and perspectives for the use of this cryopreservation method. Compiling primary studies on this subject is essential to help researchers make decisions about the application of this methodology to different experimental models.

Sperm cell cryopreservation: methods and concepts

Biological and chemical reactions in living cells are dramatically reduced at low subzero temperatures, a phenomenon that can lead to the possible long-term preservation of cells and tissues (Jang et al., 2017Jang TH, Park SC, Yang JH, Kim JY, Seok JH, Park US, Choi CW, Lee SR, Han J. Cryopreservation and its clinical applications. Integr Med Res. 2017;6(1):12-8. http://dx.doi.org/10.1016/j.imr.2016.12.001. PMid:28462139.
http://dx.doi.org/10.1016/j.imr.2016.12....
; Yashaswi and Mona, 2022Yashaswi S, Mona S. Biophysics of Cryopreservation. Int J Thermodyn. 2022;25(1):17-27. http://dx.doi.org/10.5541/ijot.925283.
http://dx.doi.org/10.5541/ijot.925283...
). Biopreservation begins with a reduction in temperature from 37 °C to the 0-10 °C range and cryopreservation at -196 °C is considered an effective method for sperm preservation, which can maintain its structural and functional integrity after thawing (Li et al., 2019Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019;233:84-92. http://dx.doi.org/10.1016/j.ejogrb.2018.11.028. PMid:30580229.
http://dx.doi.org/10.1016/j.ejogrb.2018....
; Nagata et al., 2019Nagata MPB, Egashira J, Katafuchi N, Endo K, Ogata K, Yamanaka K, Yamanouchi T, Matsuda H, Hashiyada Y, Yamashita K. Bovine sperm selection procedure prior to cryopreservation for improvement of postthawed semen quality and fertility. J Anim Sci Biotechnol. 2019;10(1):91. http://dx.doi.org/10.1186/s40104-019-0395-9. PMid:31807306.
http://dx.doi.org/10.1186/s40104-019-039...
; Silva et al., 2019Silva HVR, Silva AR, Silvada LDM, Comizzoli P. Sperm cryopreservation and banking for the conservation of neotropical carnivores. Biopreserv Biobank. 2019;17(2):183-8. http://dx.doi.org/10.1089/bio.2018.0104. PMid:30589562.
http://dx.doi.org/10.1089/bio.2018.0104...
). Water plays a central role in cryobiology. A cell consists of around 60 to 85% water both in free and bounded forms (Yashaswi and Mona, 2022Yashaswi S, Mona S. Biophysics of Cryopreservation. Int J Thermodyn. 2022;25(1):17-27. http://dx.doi.org/10.5541/ijot.925283.
http://dx.doi.org/10.5541/ijot.925283...
) and when refer to sperm cell, different content of water can occur (Hammerstedt et al., 1978Hammerstedt RH, Keith AD, Snipes W, Amann RP, Arruda D, Griel LC Jr. Use of spin labels to evaluate effects of cold shock and osmolality on sperm. Biol Reprod. 1978;18(4):686-96. http://dx.doi.org/10.1095/biolreprod18.4.686. PMid:656535.
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; Kleinhans et al, 1992Kleinhans FW, Travis VS, Du J, Villines PM, Colvin KE, Critser JK. Measurement of human sperm intracellular water volume by electron spin resonance. J Androl. 1992;13(6):498-506. PMid:1338068.; Du et al., 1994Du J, Tao J, Kleinhans FW, Mazur P, Critser JK. Water volume and osmotic behaviour of mouse spermatozoa determined by electron paramagnetic resonance. J Reprod Fertil. 1994;101(1):37-42. http://dx.doi.org/10.1530/jrf.0.1010037. PMid:8064691.
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; Pérez-Sánchez et al., 1994Pérez-Sánchez F, de Monserrat JJ, Soler C. Morphometric analysis of human sperm morphology. Int J Androl. 1994;17(5):248-55. http://dx.doi.org/10.1111/j.1365-2605.1994.tb01250.x. PMid:7698850.
http://dx.doi.org/10.1111/j.1365-2605.19...
; Gravance and Davis, 1995Gravance CG, Davis RO. Automated sperm morphometry analysis (ASMA) in the rabbit. J Androl. 1995;16(1):88-93. PMid:7539416.; Maroto-Morales et al., 2010Maroto-Morales A, Ramón M, García-Alvarez O, Soler AJ, Esteso MC, Martínez-Pastor F, Pérez-Guzmán MD, Garde JJ. Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer. Theriogenology. 2010;73(4):437-48. http://dx.doi.org/10.1016/j.theriogenology.2009.10.003. PMid:20018357.
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; Sánchez et al., 2013Sánchez MV, Bastir M, Roldan ERS. Geometric morphometrics of rodent sperm head shape. PLoS One. 2013;8(11):1-10. http://dx.doi.org/10.1371/journal.pone.0080607. PMid:24312234.
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; Villaverde-Morcillo et al., 2015Villaverde-Morcillo S, Esteso MC, Castaño C, Díaz AT, López-Sebastián A, Campo JL, Santiago-Moreno J. Influence of Staining Method on the Values of Avian Sperm Head Morphometric Variables. Reprod Domest Anim. 2015;50(5):750-5. http://dx.doi.org/10.1111/rda.12574. PMid:26192019.
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; Valverde et al., 2016Valverde A, Arenán H, Sancho M, Contell J, Yániz J, Fernández A, Soler C. Morphometry and subpopulation structure of Holstein bull spermatozoa: variations in ejaculates and cryopreservation straws. Asian J Androl. 2016;18(6):851-7. http://dx.doi.org/10.4103/1008-682X.187579. PMid:27678464.
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; Barquero et al., 2021Barquero V, Roldan ERS, Soler C, Yániz JL, Camacho M, Valverde A. Predictive capacity of boar sperm morphometry and morphometric sub-populations on reproductive success after artificial insemination. Animals. 2021;11(4):920. http://dx.doi.org/10.3390/ani11040920. PMid:33805060.
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) as show in Figure 1. The bound form refers to the water hydrated to complex mixtures of cells like proteins, lipids and glass transition temperature of pure water and no detectable biochemical activity is possible due to lack of sufficient thermal energy. Moreover, the progressive reduction and ultimately the absence of liquid water (once completely frozen) limit all metabolic processes (Pegg, 2007Pegg DE. Principles of cryopreservation. Methods Mol Biol. 2007;368:39-57. http://dx.doi.org/10.1007/978-1-59745-362-2_3. PMid:18080461.
http://dx.doi.org/10.1007/978-1-59745-36...
).

Figure 1
Water proportion graph based on spermatozoa water volume and sperm head morphometry.

This loss of kinetic energy of molecules results in the uncoupling and re-coupling (shunting) of biochemical reactions (Tani and Neely, 1989Tani M, Neely JR. Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+-Na+ and Na+-Ca2+ exchange. Circ Res. 1989;65(4):1045-56. http://dx.doi.org/10.1161/01.RES.65.4.1045. PMid:2551525.
http://dx.doi.org/10.1161/01.RES.65.4.10...
). In addition to metabolic imbalances measurable changes in cell and organelle membrane lipid domains occur. These structural characteristics (transitions) result in a change in membrane fluidity from the liquid-crystalline state to the solid gel state yielding a “leaky” membranous state (Baust et al., 2009Baust JG, Gao D, Baust JM. Cryopreservation: an emerging paradigm change. Organogenesis. 2009;5(3):90-6. http://dx.doi.org/10.4161/org.5.3.10021. PMid:20046670.
http://dx.doi.org/10.4161/org.5.3.10021...
). Freezing is removal of water so that it transforms the liquid water into ice either when within the cell or after it flows out of the cell and freezes externally. The major hurdle for cells to overcome at low temperatures is the water to-ice phase transition (Jang et al 2017Jang TH, Park SC, Yang JH, Kim JY, Seok JH, Park US, Choi CW, Lee SR, Han J. Cryopreservation and its clinical applications. Integr Med Res. 2017;6(1):12-8. http://dx.doi.org/10.1016/j.imr.2016.12.001. PMid:28462139.
http://dx.doi.org/10.1016/j.imr.2016.12....
). Ice crystal formation requires at least one initial nucleation event. The rate of nucleation of intracellular ice crystals is a function of temperature and cytoplasm composition. In classical nucleation theory, a stable ice nucleus is formed by random clustering of water molecules (Karlsson et al., 1993Karlsson JO, Cravalho EG, Borel Rinkes IH, Tompkins RG, Yarmush ML, Toner M. Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide. Biophys J. 1993;65(6):2524-36. http://dx.doi.org/10.1016/S0006-3495(93)81319-5. PMid:8312489.
http://dx.doi.org/10.1016/S0006-3495(93)...
). Hence, the two kinetic processes occurring during the cooling of cells that is growth of ice and the loss of water from the cell, happens at a characteristic rate. This is highly influenced by the cooling rate imposed on the system (Mazur, 2004Mazur P. Principles of cryobiology. In: Fuller BJ, Lane NL, Benson EE, editors. Life in the frozen state. Boca Raton: CRC Press LLC; 2004. p. 3-65. http://dx.doi.org/10.1201/9780203647073.ch1.
http://dx.doi.org/10.1201/9780203647073....
).

In slow freezing, cells are cooled in suspension and ice nucleates first in the extracellular space (Hagiwara et al., 2009Hagiwara M, Choi JH, Devireddy RV, Roberts KP, Wolkers WF, Makhlouf A, Bischof JC. Cellular biophysics during freezing of rat and mouse sperm predicts post-thaw motility. Biol Reprod. 2009;81(4):700-6. http://dx.doi.org/10.1095/biolreprod.109.076075. PMid:19535788.
http://dx.doi.org/10.1095/biolreprod.109...
) leading to the biophysical responses of cellular dehydration. In case a controlled reduction of temperature is maintained, a sufficient osmotic pressure persists that prevents the formation of ice crystals within the cell. However, the cells continually shrink during the process due to the efflux of water (Pegg, 2015Pegg DE. Principles of cryopreservation. In: Wolkers WF, Oldenhof H, editors. Cryopreservation and freeze-drying protocols. 3rd ed. New York: Springer; 2015. p. 3-19. (Methods in Molecular Biology; no. 1257). http://dx.doi.org/10.1007/978-1-4939-2193-5_1.
http://dx.doi.org/10.1007/978-1-4939-219...
). During rapid freezing, there is less time for water to move in the extracellular compartment and gets supercooled very fact leading to intracellular ice formation.

In vitrification the property that viscous liquids allow rapid cooling far below their melting temperature and undergo solidification by avoiding crystallization is used. The supercooled substance with the physical properties of a liquid subsequently acquires solid properties once it reaches below a particular temperature called the glass transition temperature (Tg) (Fahy and Wowk, 2015Fahy GM, Wowk B. Principles of cryopreservation by vitrification. In: Wolkers WF, Oldenhof H, editors. Cryopreservation and freeze-drying protocols. New York: Springer-Verlag; 2015. (Methods in Molecular Biology; no. 1257).). At this point, the molecules of the substance remain in a disordered pattern as in liquids but are locked in place and the consequent “solid-liquid” is called as the glass (Wowk, 2010Wowk B. Thermodynamic aspects of vitrification. Cryobiology. 2010;60(1):11-22. http://dx.doi.org/10.1016/j.cryobiol.2009.05.007. PMid:19538955.
http://dx.doi.org/10.1016/j.cryobiol.200...
). Since there is no crystallization event, vitrification outruns the processes of ice nucleation and growth and thus their potential adverse effects.

Currently, two main approaches for sperm cryopreservation are used on cells, organs, and tissues: conventional freezing (slow and fast) and vitrification (equilibrium and kinetics). However, several methodologies have been used to preserve the fertilizing capacity of sperm cells, differing in terms of the devices used, dilution rates, composition of cryoprotective agents (CPA), freezing rates, and thawing protocols (Li et al., 2019Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019;233:84-92. http://dx.doi.org/10.1016/j.ejogrb.2018.11.028. PMid:30580229.
http://dx.doi.org/10.1016/j.ejogrb.2018....
). Furthermore, slow freezing can be divided into different protocols (Figure 2) such as the One-step that consists of diluting the sample with cryoprotectants and the Two-step in which cryoprotectants are added after cooling (equilibration). And vitrification is divided into equilibrium or traditional and kinetic vitrification (Katkov et al., 2012Katkov II, Bolyukh VF, Chernetsov OA, Dudin PI, Grigoriev AY, Isachenko V, Isachenko E, Lulat AG-M, Moskovtsev SI, Petrushko MP, Pinyaev VI, Sokol KM, Sokol YI, Sushko AB, Yakhnenko I. Kinetic vitrification of spermatozoa of vertebrates: what can we learn from nature? In: Katkov I , editor. Current frontiers in cryobiology. Croatia: IntechOpen; 2012. p. 3-40. http://dx.doi.org/10.5772/34784.
http://dx.doi.org/10.5772/34784...
).

Figure 2
Main sperm cryopreservation protocols. Slow freezing protocol-one step (Khalil et al., 2018Khalil WA, El-Harairy MA, Zeidan AEB, Hassan MAE, Mohey-Elsaeed O. Evaluation of bull spermatozoa during and after cryopreservation: structural and ultrastructural insights. Int J Vet Sci Med. 2018;6(Suppl 1):S549-56. http://dx.doi.org/10.1016/j.ijvsm.2017.11.001. PMid:30761321.
http://dx.doi.org/10.1016/j.ijvsm.2017.1...
); slow freezing protocol-two step (Brito et al., 2017Brito MM, Lúcio CF, Angrimani DS, Losano JD, Dalmazzo A, Nichi M, Vannucchi CI. Comparison of cryopreservation protocols (single and two-steps) and thawing (fast and slow) for canine sperm. Anim Biotechnol. 2017;28(1):67-73. http://dx.doi.org/10.1080/10495398.2016.1203797. PMid:27715465.
http://dx.doi.org/10.1080/10495398.2016....
); equilibrium (Baiee et al., 2020Baiee FH, Al-Wahab A, Almusawi AA, Fitri W, Haron AW, Bustani GS. Effect of vitrification on spermatozoa quality in bull semen. Eurasian J Biosci. 2020;14:3897-904.) and kinetic vitrification protocols (Rosato and Iaffaldano, 2013Rosato MP, Iaffaldano N. Cryopreservation of rabbit semen: comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. Theriogenology. 2013;79(3):508-16. http://dx.doi.org/10.1016/j.theriogenology.2012.11.008. PMid:23218394.
http://dx.doi.org/10.1016/j.theriogenolo...
).

In conventional slow freezing, the sample is gradually cooled from room temperature to 4– 5 °C at a rate of 0.5-1 °C/min. Then, the temperature is further reduced to -80 °C at a rate of 1–10 °C/min and the sample is immersed in liquid nitrogen at -196 °C (Thachil and Jewett, 1981Thachil JV, Jewett MA. Preservation techniques for human semen. Fertil Steril. 1981;35(5):546-8. http://dx.doi.org/10.1016/S0015-0282(16)45498-1. PMid:7227570.
http://dx.doi.org/10.1016/S0015-0282(16)...
). The reduction in temperature is associated with low concentrations of P-CPA and N-CPA (Li et al., 2019Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019;233:84-92. http://dx.doi.org/10.1016/j.ejogrb.2018.11.028. PMid:30580229.
http://dx.doi.org/10.1016/j.ejogrb.2018....
). However, this method takes time, and can last 2–4 h, which varies according to the target species and requires the use of a freezer (Silva et al., 2019Silva HVR, Silva AR, Silvada LDM, Comizzoli P. Sperm cryopreservation and banking for the conservation of neotropical carnivores. Biopreserv Biobank. 2019;17(2):183-8. http://dx.doi.org/10.1089/bio.2018.0104. PMid:30589562.
http://dx.doi.org/10.1089/bio.2018.0104...
; Zainuddin et al., 2020Zainuddin ZZ, Tarmizi MRM, Yap KC, Comizzoli P, Symphorosa S. First evaluations and cryopreservation of semen samples from Sunda Clouded Leopards (Neofelis diardi). Animals. 2020;10(6):1072. http://dx.doi.org/10.3390/ani10061072.
http://dx.doi.org/10.3390/ani10061072...
) or a programmable freezer (Santo et al., 2012Santo MD, Tarozzi N, Nadalini M, Borini A. Human sperm cryopreservation: update on techniques, effect on DNA integrity, and implications for ART. Adv Urol. 2012;2012:854837. http://dx.doi.org/10.1155/2012/854837. PMid:22194740.
http://dx.doi.org/10.1155/2012/854837...
). Conversely, in the conventional rapid freezing protocol, after the addition of a cryoprotectant, the sample comes into direct contact with nitrogen vapors at -80 °C at 15-20 cm for 15 min and only then the sample is immersed in liquid nitrogen (-196 °C) (Sherman, 1990Sherman JK. Cryopreservation of human semen. In: Keel BA, Webster BW, editors. CRC handbook of the laboratory diagnosis and treatment of infertility. Boca Raton: CRC Press; 1990. p. 229-59.). Fast protocols differ from slow protocols in that (i) much of the dehydration and cryoprotective permeation occurs before the start of cooling, and (ii) cooling is usually performed in a single step, in which the sample is directly cooled from a temperature > 0 °C to sub-zero temperatures (< 130 °C) (Shaw and Jones, 2003Shaw JM, Jones GM. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Hum Reprod Update. 2003;9(6):583-605. http://dx.doi.org/10.1093/humupd/dmg041. PMid:14714593.
http://dx.doi.org/10.1093/humupd/dmg041...
). Thus, to optimize time and cost in the sperm cryopreservation process in humans, researchers have focused on the cryopreservation of sperm cells through vitrification, which can be performed with an equilibrium vitrification protocol or with a method that does not use P-CPA, known as kinetic vitrification (Li et al., 2019Li Y, Zhou L, Lv M, Ge P, Liu Y, Zhou D. Vitrification and conventional freezing methods in sperm cryopreservation: a systematic review and meta-analysis. Eur J Obstet Gynecol Reprod Biol. 2019;233:84-92. http://dx.doi.org/10.1016/j.ejogrb.2018.11.028. PMid:30580229.
http://dx.doi.org/10.1016/j.ejogrb.2018....
).

The vitrification process is defined as a method to solidify the liquid at low temperatures, in an amorphous or glassy state, without the formation of extra and intracellular ice crystals in the cryopreserved cells and tissues during the process and during the devitrification of recovered biological material (Kuleshova and Lopata, 2002Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril. 2002;78(3):449-54. http://dx.doi.org/10.1016/S0015-0282(02)03305-8. PMid:12215314.
http://dx.doi.org/10.1016/S0015-0282(02)...
). It is the combination of thermodynamic and kinetic effects that allow ice crystal nucleation and growth to be avoided during cooling of these solutions and is the means by which vitrification of oocytes and embryos are achieved. However, if the system is afforded sufficient time during warming,i.e., if the heating rate is slow, the molecules in the ice crystals may rearrange to form the more favorable hexagonal crystal structure as well as larger crystals (Schiewe and Mullen, 2018Schiewe MC, Mullen SF. Vitrification: fundamental principles and its application for cryopreservation of human reproductive cells. In: Bozkurt Y, editor. Cryopreservation biotechnology in biomedical and biological sciences. London: IntechOpen; 2018. http://dx.doi.org/10.5772/intechopen.79672.
http://dx.doi.org/10.5772/intechopen.796...
). According to MacFarlane (1986)MacFarlane DR. Devitrification in glass-forming aqueous solutions. Cryobiology. 1986;23(3):230-44. http://dx.doi.org/10.1016/0011-2240(86)90049-0.
http://dx.doi.org/10.1016/0011-2240(86)9...
this structure of ice is the most damaging to biological systems. This approach was first proposed in 1937 by Swiss priest Basile J. Luyet, who is considered the founder of cryobiology. Although his theory was tested in a practical application in organic liquids, the cooling rate at the time could not meet the vitrification requirements (amorphous/glassy state). For Luyet, “[...] the essential problem of the vitrification technique is... to obtain a cooling speed sufficient to prevent the formation of crystals” (Luyet, 1937aLuyet B. The vitrification of organic colloids and protoplasm. Biodynamica. 1937a;1:1-14., bLuyet B. Working hypotheses on the nature of life. Biodynamica. 1937b;1:1-7.).

However, the use of P-CPA and/or drying proposed by Luyet et al. was not initially intended to decrease the rate of crystallization or decrease the maximum amount of water crystallization possible, but to dehydrate the biological sample immediately before cooling to minimize the amount of water required in the process (Luyet, 1937bLuyet B. Working hypotheses on the nature of life. Biodynamica. 1937b;1:1-7.). As vitrification requires very high cooling rates, Luyet et al. conducted studies using rapid cooling techniques between 1937 and 1958 (Luyet and Rapatz, 1958Luyet B, Rapatz G. Patterns of ice formation in some aqueous solutions. Biodynamica. 1958;8:1-68.). These years are considered the era of rapid cooling with little or no intracellular cryoprotection, as the use of cryoprotectants to minimize frostbite injuries was not known until 1949 (Fahy, 2015Fahy GM. Overview of biological vitrification. In: Tucker MJ, Liebermann J, editors. Vitrification in assisted reproduction. 2nd ed. Boca Raton: CRC Press; 2015. p. 3-5. http://dx.doi.org/10.1201/b19316-5.
http://dx.doi.org/10.1201/b19316-5...
), when Polge et al. (1949)Polge C, Smith A, Parkes A. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949;164(4172):666. http://dx.doi.org/10.1038/164666a0. PMid:18143360.
http://dx.doi.org/10.1038/164666a0...
made a crucial discovery, that the use of glycerol (a permeable solute) could provide protection to cells at low temperatures.

The first report of cryopreservation by vitrification in sperm consisted of an experiment with frogs by Luyet and Hoddap, in 1938, in their study entitled “Revival of frog's spermatozoa vitrified in liquid air” (Luyet and Hodapp, 1938Luyet BJ, Hodapp EL. Revival of Frog’s Spermatozoa Vitrified in Liquid Air. Exp Biol Med (Maywood). 1938;39(3):433-4. http://dx.doi.org/10.3181/00379727-39-10229P.
http://dx.doi.org/10.3181/00379727-39-10...
). The vitrification method used by these authors became known as kinetic vitrification, which was characterized by its ultra-fast cooling (tens of thousands of °C/min) without P-CPA (Luyet and Hodapp, 1938Luyet BJ, Hodapp EL. Revival of Frog’s Spermatozoa Vitrified in Liquid Air. Exp Biol Med (Maywood). 1938;39(3):433-4. http://dx.doi.org/10.3181/00379727-39-10229P.
http://dx.doi.org/10.3181/00379727-39-10...
; Luyet, 1937aLuyet B. The vitrification of organic colloids and protoplasm. Biodynamica. 1937a;1:1-14., bLuyet B. Working hypotheses on the nature of life. Biodynamica. 1937b;1:1-7.). In later years, studies conducted in rabbit kidneys by Greg Fahy et al. using high pressure and extremely high concentrations of P-CPA, inaugurated another vitrification method that became widely known among cryobiologists as the equilibrium vitrification (Fahy et al., 1984Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrification as an approach to cryopreservation. Cryobiology. 1984;21(4):407-26. http://dx.doi.org/10.1016/0011-2240(84)90079-8. PMid:6467964.
http://dx.doi.org/10.1016/0011-2240(84)9...
). Since then, it was established that vitrification could only be achieved using high concentrations of combinations of P-CPA and N-CPA (Fahy, 1986Fahy GM. The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology. 1986;23(1):1-13. http://dx.doi.org/10.1016/0011-2240(86)90013-1. PMid:3956226.
http://dx.doi.org/10.1016/0011-2240(86)9...
). However, the toxicity of these physicochemical agents related to osmotic damage during saturation, as well as biochemical changes in the sperm cell, have been described as limiting factors for cryobiology by equilibrium vitrification (Mazur et al., 2000Mazur P, Katkov II, Katkova N, Critser JK. The enhancement of the ability of mouse sperm to survive freezing and thawing by the use of high concentrations of glycerol and the presence of an E. coli membrane preparation OxyraseTM to lower the oxygen concentration. Cryobiology. 2000;40(3):187-209. http://dx.doi.org/10.1006/cryo.2000.2238. PMid:10860619.
http://dx.doi.org/10.1006/cryo.2000.2238...
).

Thus, to avoid these toxic effects, some researchers have questioned whether P-CPA are necessary for successful vitrification, proposing the use of very fast heating and cooling rates (50,000 K/min or more) in a very small sample size (Nawroth et al., 2002Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, Mallmann P, Dattena M, Capobianco G, Peters D, Orth I, Isachenko E. Vitrification of human spermatozoa without cryoprotectants. Cryo Lett. 2002;23(2):93-102. PMid:12050777.), without using P-CPA and using only sucrose and other agents such as N-CPA, thus recovering kinetic vitrification, also known as “ultra-fast freezing” (Isachenko et al., 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
, 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
). This methodology is an alternative to the conventional equilibrium sperm vitrification, owing to the low tolerance to sperm osmotic changes from mammals and birds (Donoghue and Wishart, 2000Donoghue AM, Wishart GJ. Storage of poultry semen. Anim Reprod Sci. 2000;62(1-3):201-32. http://dx.doi.org/10.1016/S0378-4320(00)00160-3. PMid:10924826.
http://dx.doi.org/10.1016/S0378-4320(00)...
), which can be caused by high concentrations of P-CPA (Isachenko et al., 2003Isachenko E, Isachenko V, Katkov II, 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(2):191-200. http://dx.doi.org/10.1016/S1472-6483(10)61710-5. PMid:12675999.
http://dx.doi.org/10.1016/S1472-6483(10)...
).

Regarding the differences between cellular damage caused by the cryopreservation process including cooling rates and the use of solutions with or without cryoprotectants, Fig 2 shows that sperm motility, viability, and mitochondrial activity are common cellular damages associated with cryopreservation techniques. Slow freezing can cause morphological damage, reduced acrosomal reaction, and increased chromatin damage. There is also a reduction in the activity of sperm acrosome enzyme (ACE) and hyaluronidase enzyme (HYD) that are positively correlated with motility (Sun et al., 2020Sun W, Jiang S, Su J, Zhang J, Bao X, Ding R, Shi P, Li S, Wu C, Zhao G, Cao G, Sun QY, Yu H, Li X. The effects of cryopreservation on the acrosome structure, enzyme activity, motility, and fertility of bovine, ovine, and goat sperm. Anim Reprod. 2020;17(4):e20200219. http://dx.doi.org/10.1590/1984-3143-ar2020-0219. PMid:34084227.
http://dx.doi.org/10.1590/1984-3143-ar20...
) The cellular damage caused by kinetic vitrification refers mainly to loss of motility and consequently fertility.

Essential factors for vitreous state

Three factors are reported to be essential for obtaining the glassy state: increase cooling rate, increase viscosity and decreasing sample volume (Arav et al., 2002Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H. New trends in gamete’s cryopreservation. Mol Cell Endocrinol. 2002;187(1-2):77-81. http://dx.doi.org/10.1016/S0303-7207(01)00700-6. PMid:11988314.
http://dx.doi.org/10.1016/S0303-7207(01)...
).

Cryopreservation methods vary according to freezing speed (exposure of the sperm solution to low temperatures per minute), cryoprotectant concentration and temperature reduction rates, which includes slow freezing (0.5-0 °C/minute), rapid freezing (50-400°C/minute), ultrarapid freezing (approximately 2500 °C/minute), and vitrification (approximately 20 000 °C/minute) (Schulz et al., 2020Schulz M, Risopatrón J, Uribe P, Isachenko E, Isachenko V, Sánchez R. Human sperm vitrification: a scientific report. Andrology. 2020;8(6):1642-50. http://dx.doi.org/10.1111/andr.12847. PMid:32598551.
http://dx.doi.org/10.1111/andr.12847...
).

In this context, the addition of P-CPA has become essential in conventional cryopreservation methods i.e. slow freezing for cells and equilibrium vitrification for organs and tissues, to protect the sperm cell from damage caused by exposure to low temperatures (Medeiros et al., 2002Medeiros CM, Forell F, Oliveira AT, Rodrigues JL. Current status of sperm cryopreservation: why isn’t it better? Theriogenology. 2002;57(1):327-44. http://dx.doi.org/10.1016/S0093-691X(01)00674-4. PMid:11775978.
http://dx.doi.org/10.1016/S0093-691X(01)...
). According to Fahy and Rall (2007)Fahy GM, Rall WF. Vitrification: an overview. In: Tucker MJ, Liebermann J, editors. Vitrification in assisted reproduction. Boca Raton: CRC Press; 2007. p. 1–20. http://dx.doi.org/10.3109/9780203090022.001.
http://dx.doi.org/10.3109/9780203090022....
the lower the concentration of cryoprotectant, the faster cooling must proceed to avoid ice formation Similarly, if there is an increase in viscosity or or cooling rate or decreasing the volume will increase the chances of obtaining the vitreous state (Arav et al., 2002Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H. New trends in gamete’s cryopreservation. Mol Cell Endocrinol. 2002;187(1-2):77-81. http://dx.doi.org/10.1016/S0303-7207(01)00700-6. PMid:11988314.
http://dx.doi.org/10.1016/S0303-7207(01)...
).

Thus, the addition of cryoprotective agents (CPAs) as carbohydrates and other compounds, to the cryopreservation media increases the viscosity of the solution, thereby facilitating the vitrification process. CPAs are classified as intracellular or permeable (P-CPA) and extracellular or non-permeable (N-CPA). P-CPA are known to protect cells from cryoinjury by increasing the fluidity of the plasma membrane, lipid reordering, and partial dehydration of the cell, reducing the freezing point, thus limiting the formation of intracellular ice crystals, which is one of the main biophysical mechanisms of sperm death (Holt, 2000Holt WV. Basic aspects of frozen storage of semen. Anim Reprod Sci. 2000;62(1-3):3-22. http://dx.doi.org/10.1016/S0378-4320(00)00152-4. PMid:10924818.
http://dx.doi.org/10.1016/S0378-4320(00)...
; Swain and Smith, 2010Swain JE, Smith GD. Cryoprotectants. In: Chian RC, Quinn P, editors. Fertility Cryopreservation. Cambridge: Cambridge University Press; 2010. p. 24-38. http://dx.doi.org/10.1017/CBO9780511730207.005.
http://dx.doi.org/10.1017/CBO97805117302...
).

N-CPA can induce an increase in the osmolarity of the external environment, inducing the passage of water from the interior of the cell to the extracellular environment and preventing the formation of ice crystals during freezing (Meryman, 1971Meryman HT. Cryoprotective agentes. Cryobiology. 1971;8(2):173-83. http://dx.doi.org/10.1016/0011-2240(71)90024-1. PMid:5578883.
http://dx.doi.org/10.1016/0011-2240(71)9...
; Amann and Pickett, 1987Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Equine Vet Sci. 1987;7(3):145-73. http://dx.doi.org/10.1016/S0737-0806(87)80025-4.
http://dx.doi.org/10.1016/S0737-0806(87)...
; Aisen et al., 2002Aisen EG, Medina VH, Venturino A. Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations. Theriogenology. 2002;57(7):1801-8. http://dx.doi.org/10.1016/S0093-691X(02)00653-2. PMid:12041684.
http://dx.doi.org/10.1016/S0093-691X(02)...
). N-CPA are represented by macromolecules with high molecular weight, including complex carbohydrates, such as trehalose, sucrose, and raffinose, in addition to lipoproteins from egg yolk and coconut water (Nunes, 2002Nunes JF. Inseminação artificial em caprinos. In: Gonsalvez PBD, Figueiredo JR, Freitas VJF, editors. Biotécnicas aplicadas à reprodução animal. São Paulo: Varela; 2002. p. 111-25.), milk proteins, and some amino acids (Amann and Pickett, 1987Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Equine Vet Sci. 1987;7(3):145-73. http://dx.doi.org/10.1016/S0737-0806(87)80025-4.
http://dx.doi.org/10.1016/S0737-0806(87)...
). Osmolarity, in turn, can be divided into osmotically active molecules, such as disaccharides (sucrose and trehalose), and osmotically inactive compounds, including polysaccharides, such as maltodextrin, and proteins, such as albumin (Sieme et al., 2016Sieme H, Oldenhof H, Wolkers WF. Mode of action of cryoprotectants for sperm preservation. Anim Reprod Sci. 2016;169:2-5. http://dx.doi.org/10.1016/j.anireprosci.2016.02.004. PMid:26936658.
http://dx.doi.org/10.1016/j.anireprosci....
).

The addition of N-CPA in the equilibrium vitrification can reduce the concentrations of permeable cryoprotectants in the vitrification solution, thereby minimizing damage from their toxicity (Shaw et al., 1997Shaw JM, Kuleshova LL, Macfarlane DR, Trounson AO. Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or Dextran. Cryobiology. 1997;35(3):219-29. http://dx.doi.org/10.1006/cryo.1997.2043. PMid:9367610.
http://dx.doi.org/10.1006/cryo.1997.2043...
; Silva et al., 2015Silva AR, Lima GL, Peixoto G, Souza ALP. Cryopreservation in mammalian conservation biology: current applications and potential utility. Research and Reports in Biodiversity Studies. 2015;4:1-8. http://dx.doi.org/10.2147/RRBS.S54294.
http://dx.doi.org/10.2147/RRBS.S54294...
; Mosca et al., 2016Mosca F, Madeddu M, Sayed AA, Zaniboni L, Iaffaldano N, Cerolini S. Combined effect of permeant and non-permeant cryoprotectants on the quality of frozen/thawed chicken sperm. Cryobiology. 2016;73(3):343-7. http://dx.doi.org/10.1016/j.cryobiol.2016.10.001. PMid:27717656.
http://dx.doi.org/10.1016/j.cryobiol.201...
). A study involving equine spermatozoa evaluated the use of different sugars in equilibrium vitrification; better rates of progressive motility and plasma membrane integrity were obtained with 100 mM trehalose (41.5 and 81.1% respectively) in comparison to 200 mM sucrose (28.7 and 74.4%) and 100 mM (28.6 and 72.9%). The authors attribute these results to the ability of trehalose to preserve the lipid bilayer by stabilizing the water structure around the plasma membrane, thereby protecting sperm against cryoinjuries (Consuegra et al., 2019Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Ortiz I, Hidalgo M. Vitrification of stallion sperm using 0.25 ml straws: effect of volume, concentration, and carbohydrates (sucrose/trehalose/raffinose). Anim Reprod. 2019;206:69-77. http://dx.doi.org/10.1016/j.anireprosci.2019.05.009. PMid:31138492.
http://dx.doi.org/10.1016/j.anireprosci....
).

Sucrose is often used in equilibrium and kinetic vitrification solutions used to increase the cell protective effect (Rall, 1987Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology. 1987;24(5):387-402. http://dx.doi.org/10.1016/0011-2240(87)90042-3. PMid:3652721.
http://dx.doi.org/10.1016/0011-2240(87)9...
); it acts as an osmotic buffer against cellular stress caused during vitrification and devitrification (Pan et al., 2017Pan C, Yu S, Zhang P, Wang B, Zhu Z, Liu Y, Zeng W. Effect of sucrose on cryopreservation of pig spermatogonial stem cells. J Integr Agric. 2017;16(5):1120-9. http://dx.doi.org/10.1016/S2095-3119(16)61489-2.
http://dx.doi.org/10.1016/S2095-3119(16)...
). However, despite these benefits, N-CPA have some disadvantages in kinetic sperm vitrification. When only disaccharides are used as cryoprotectants, there is a marked reduction in motility (Table 1), whose cause remains unknown. Conversely, changes in motility during the vitrification-devitrification process may be due to changes in mitochondrial membrane potential (Isachenko et al., 2019Isachenko V, Sánchez R, Rahimi G, Mallmann P, Isachenko E, Merzenich M. Cryoprotectant‐free vitrification of spermatozoa: fish as a model of human. Andrologia. 2019;51(1):e13166. http://dx.doi.org/10.1111/and.13166. PMid:30298523.
http://dx.doi.org/10.1111/and.13166...
). One way to increase the viscosity of the medium and/or thinner used and to protect the sperm plasma membrane during the kinetic vitrification process, is to include additives, such as bovine serum albumin (BSA). Table 1 shows examples of this inclusion, such as a study on rabbit sperm, in which an increase in DNA motility and integrity was observed when 0.5% BSA was combined with sucrose (0.1 M and 0.25 M) (Rosato and Iaffaldano, 2013Rosato MP, Iaffaldano N. Cryopreservation of rabbit semen: comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. Theriogenology. 2013;79(3):508-16. http://dx.doi.org/10.1016/j.theriogenology.2012.11.008. PMid:23218394.
http://dx.doi.org/10.1016/j.theriogenolo...
).

Table 1
Studies for animal and human spermatozoa kinetic vitrification.

Another factor that has been somewhat neglected is the water content present in the sperm cell and the differences in water permeability. Differences in water permeability account largely for the magnitude of difference in optimal cooling rates for different cell types (Gao and Critser, 2000Gao D, Critser JK. Mechanisms of cryoinjury in living cells. ILAR J. 2000;41(4):187-96. http://dx.doi.org/10.1093/ilar.41.4.187. PMid:11123179.
http://dx.doi.org/10.1093/ilar.41.4.187...
). Cryopreservation protocols must strike an equilibrium between optimal conditions for each cell type, depending on the water content, cell size and morphology, and the water permeability coefficient of the plasma membrane (Curry et al., 1994Curry MR, Millar JD, Watson PF. Calculated optimal cooling rates for ram and human sperm cryopreservation fail to conform with empirical observations. Biol Reprod. 1994;51(5):1014-21. http://dx.doi.org/10.1095/biolreprod51.5.1014. PMid:7849177.
http://dx.doi.org/10.1095/biolreprod51.5...
; Paoli et al., 2014Paoli D, Lombardo F, Lenzi A, Gandini L. Sperm cryopreservation: effects on chromatin structure. In: Baldi E, Muratori M, editors. Genetic damage in human spermatozoa. Switzerland: Springer; 2014. p. 137-50. (Advances in Experimental Medicine and Biology; no. 791). http://dx.doi.org/10.1007/978-1-4614-7783-9_9.
http://dx.doi.org/10.1007/978-1-4614-778...
). In practice, as water volume and sperm morphology are different between specie, cryopreservation protocols must also be different. Katkov et al. (2012)Katkov II, Bolyukh VF, Chernetsov OA, Dudin PI, Grigoriev AY, Isachenko V, Isachenko E, Lulat AG-M, Moskovtsev SI, Petrushko MP, Pinyaev VI, Sokol KM, Sokol YI, Sushko AB, Yakhnenko I. Kinetic vitrification of spermatozoa of vertebrates: what can we learn from nature? In: Katkov I , editor. Current frontiers in cryobiology. Croatia: IntechOpen; 2012. p. 3-40. http://dx.doi.org/10.5772/34784.
http://dx.doi.org/10.5772/34784...
collected semen from three species of birds of prey (gyrfalcon - Falco rusticolus, golden eagle - Aquila chrysaetos, and eastern imperial eagle - Aquila heliaca) and subjected the samples to slow freezing and kinetic vitrification using the same freezing rate used for mammalian sperm. The kinetic vitrification protocol was not successful, and the authors attributed this result to morphological peculiarities and differences in the water content of bird and mammal sperm cells. For these authors, the kinetic vitrification of avian semen would require a faster freezing rate.

Another important point for obtaining the vitreous state is the volume of cryopreserved sample. According to Isachenko et al (2017)Isachenko V, Rahimi G, Mallmann P, Sánchez R, Isachenko E. Technologies of cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness. Andrology. 2017;5(6):1055-63. http://dx.doi.org/10.1111/andr.12414. PMid:28992376.
http://dx.doi.org/10.1111/andr.12414...
there are two methods of cryopreserving samples according to volume and contamination protection: non-aseptic methods, i.e. the one in which the sample is placed directly into liquid nitrogen, and aseptic methods in which devices are used to prevent the samples from being in direct contact with the liquid nitrogen.

Different aseptic vitrification techniques were investigated by Isachenko et al. (2005)Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F, Dessole S, Katkov II, Van der Ven H. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10(3):350-4. http://dx.doi.org/10.1016/S1472-6483(10)61795-6. PMid:15820041.
http://dx.doi.org/10.1016/S1472-6483(10)...
; however, only small volumes, ranging between 1 and 40 µl of sperm suspension could be vitrified in these systems and according to these authors the open-pulled straw method of vitrification is preferable because it allows isolation of the spermatozoa from liquid nitrogen, with a maximum reduction of the potential risk of microbial contamination. In 2011, the Isachenko group reported a novel aseptic cryoprotectant-free vitrification method allowing for the vitrification of larger volumes (up to 500 µl) of spermatozoa. In this study, spermatozoa vitrified with aseptic cryoprotectant-free technology displayed superior functional characteristics. The motility rate, integrity rates of cytoplasmic, and acrosomal membranes were significantly higher after vitrification than after conventional freezing (76% vs 52%, 54% vs 28% and 44% vs 30%, respectively).

Kinetic vitrification: applications and methodologies

Main techniques and protocols used

Kinetic vitrification, in addition to being P-CPA free, differs from other cryopreservation methods in terms of its extremely high rates of cooling (104–106 °C/min) and almost instantaneous devitrification, conditions that prevent the formation of ice inside the cells (Nawroth et al., 2002Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, Mallmann P, Dattena M, Capobianco G, Peters D, Orth I, Isachenko E. Vitrification of human spermatozoa without cryoprotectants. Cryo Lett. 2002;23(2):93-102. PMid:12050777.; Isachenko et al., 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
). Moreover, the entire vitrification and devitrification process takes only a few seconds (Isachenko et al., 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
).

As for the use of non-permeable cryoprotectants only, Isachenko et al. (2004a)Isachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
evaluated different combinations of carbohydrates (sucrose and trehalose) and proteins (human serum albumin) in men and found that slow freezing and kinetic vitrification were similar in terms of sperm motility and DNA integrity. However, in dogs, the results of kinetic vitrification using a thinner and an egg yolk medium, were not considered acceptable for the same parameters (Kim et al., 2012Kim S, Lee Y, Yang H, Kim Y. Rapid freezing without cooling equilibration in canine sperm. Anim Reprod Sci. 2012;130(1-2):111. http://dx.doi.org/10.1016/j.anireprosci.2011.12.014. PMid:22277841.
http://dx.doi.org/10.1016/j.anireprosci....
).

In studies using human (Isachenko et al., 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
, bIsachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, Van Der Ven H. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004b;71(4):1167-73. http://dx.doi.org/10.1095/biolreprod.104.028811. PMid:15175233.
http://dx.doi.org/10.1095/biolreprod.104...
, 2005Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F, Dessole S, Katkov II, Van der Ven H. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10(3):350-4. http://dx.doi.org/10.1016/S1472-6483(10)61795-6. PMid:15820041.
http://dx.doi.org/10.1016/S1472-6483(10)...
, 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
, 2011Isachenko V, Maettner R, Petrunkina AM, Mallmann P, Rahimi G, Sterzik K, Sánchez R, Risopatron J, Damjanoski I, Isachenko E. Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology. Clin Lab. 2011;57(9-10):643-50. PMid:22029178.) and fish sperm (Merino et al., 2011Merino O, Risopatrón J, Sánchez R, Isachenko E, Figueroa E, Valdebenito I, Isachenko V. Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness. Anim Reprod Sci. 2011;124(1-2):125-31. http://dx.doi.org/10.1016/j.anireprosci.2011.02.023. PMid:21392903.
http://dx.doi.org/10.1016/j.anireprosci....
), sperm samples were subjected to swim-up separation and centrifugation (with velocities of 300 g to 400 g for 5-10 min) prior to kinetic vitrification and after devitrification (Nawroth et al., 2002Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, Mallmann P, Dattena M, Capobianco G, Peters D, Orth I, Isachenko E. Vitrification of human spermatozoa without cryoprotectants. Cryo Lett. 2002;23(2):93-102. PMid:12050777.; Isachenko et al., 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
, bIsachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, Van Der Ven H. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004b;71(4):1167-73. http://dx.doi.org/10.1095/biolreprod.104.028811. PMid:15175233.
http://dx.doi.org/10.1095/biolreprod.104...
, 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
). Such procedures allowed the selection of sperm with progressive motility, normal morphology, or with undamaged DNA (Isachenko et al., 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
). This pre-selection resulted in improved sperm quality after devitrification in terms of DNA integrity (Tomlinson et al., 2001Tomlinson MJ, Moffatt O, Manicardi GC, Bizzaro D, Afnan M, Sakkas D. Interrelationships between seminal parameters and sperm nuclear DNA damage before and after density gradient centrifugation: implications for assisted conception. Hum Reprod. 2001;16(10):2160-5. http://dx.doi.org/10.1093/humrep/16.10.2160. PMid:11574509.
http://dx.doi.org/10.1093/humrep/16.10.2...
; O'Connell et al., 2003O’Connell M, McClure N, Powell LA, Steele EK, Lewis SEM. Differences in mitochondrial and nuclear DNA status of high-density and low-density sperm fractions after density centrifugation preparation. Fertil Steril. 2003;79(Suppl 1):754-62. http://dx.doi.org/10.1016/S0015-0282(02)04827-6. PMid:12620488.
http://dx.doi.org/10.1016/S0015-0282(02)...
), morphology (Xue et al., 2014Xue X, Wang W, Shi J, Zhang L, Zhao W, Shi W, Guo B, Qin Z. Efficacy of swim-up versus density gradient centrifugation in improving sperm deformity rate and DNA fragmentation index in semen samples from teratozoospermic patients. J Assist Reprod Genet. 2014;31(9):1161-6. http://dx.doi.org/10.1007/s10815-014-0287-z. PMid:25015033.
http://dx.doi.org/10.1007/s10815-014-028...
), and motility (Fácio et al., 2016Fácio CL, Previato LF, Machado-Paula LA, Matheus PCS, Araújo E Fo. Comparison of two sperm processing techniques for low complexity assisted fertilization: sperm washing followed by swim-up and discontinuous density gradient centrifugation. JBRA Assist Reprod. 2016;20(4):206-11. http://dx.doi.org/10.5935/1518-0557.20160040. PMid:28050954.
http://dx.doi.org/10.5935/1518-0557.2016...
). However, pre-processing the samples is not indicated for all species owing to the sensitivity and peculiarities that the sperm cell can present. In mice, for example, a reduction in progressive motility, mean trajectory velocity, and overall velocity after centrifugation has been observed (Shi et al., 2016Shi X, Wang T, Qiu ZL, Li K, Li L, Chan CPS, Chan SM, Li T, Quan S. Effects of mechanical stresses on sperm function and fertilization rate in mice. Syst Biol Reprod Med. 2016;62(2):152-9. http://dx.doi.org/10.3109/19396368.2015.1115915. PMid:26889695.
http://dx.doi.org/10.3109/19396368.2015....
).

Another peculiarity is the volume used in this technique. The most common procedure is using small sperm volumes ranging from 10 μL to 30 µL (Sanchéz et al. 2011; Slabbert et al., 2015Slabbert M, du Plessis SS, Huyser C. Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa. Andrologia. 2015;47(5):594-9. http://dx.doi.org/10.1111/and.12307. PMid:25040423.
http://dx.doi.org/10.1111/and.12307...
). The great obstacle in using larger volumes was to find an aseptic method that was adequate for this increase. In 2011, the group led by Isachenko reported a new method of aseptic vitrification without the use of a cryoprotectant in humans, allowing the vitrification of larger volumes (up to 500 µL). Later, Slabbert et al. (2015)Slabbert M, du Plessis SS, Huyser C. Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa. Andrologia. 2015;47(5):594-9. http://dx.doi.org/10.1111/and.12307. PMid:25040423.
http://dx.doi.org/10.1111/and.12307...
used 300 µL for human sperm vitrification and observed greater mitochondrial potential and less DNA fragmentation when compared to slow freezing.

The methods previously proposed were described as open and non-aseptic systems because the devices did not prevent the direct contact of the sample with liquid nitrogen (Isachenko et al., 2003Isachenko E, Isachenko V, Katkov II, 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(2):191-200. http://dx.doi.org/10.1016/S1472-6483(10)61710-5. PMid:12675999.
http://dx.doi.org/10.1016/S1472-6483(10)...
, 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
, 2005Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F, Dessole S, Katkov II, Van der Ven H. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10(3):350-4. http://dx.doi.org/10.1016/S1472-6483(10)61795-6. PMid:15820041.
http://dx.doi.org/10.1016/S1472-6483(10)...
). Open systems were used to eliminate ice and create a glassy state in its place, requiring small cool liquid suspensions or just water at ultra-fast freezing speeds (Luyet, 1937aLuyet B. The vitrification of organic colloids and protoplasm. Biodynamica. 1937a;1:1-14.); the technique in which small aliquots or drops of semen (approximately 30 µL) were placed directly in liquid nitrogen aimed at achieving the desired vitrification (Isachenko et al., 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
).

Using sample isolation as an indicator of asepsis (Isachenko et al., 2017Isachenko V, Rahimi G, Mallmann P, Sánchez R, Isachenko E. Technologies of cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness. Andrology. 2017;5(6):1055-63. http://dx.doi.org/10.1111/andr.12414. PMid:28992376.
http://dx.doi.org/10.1111/andr.12414...
) and kinetic vitrification efficacy, different devices such as cryoloop, open pulled straw (OPS), and cut standard straw (CSS) were evaluated (Isachenko et al., 2005Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F, Dessole S, Katkov II, Van der Ven H. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10(3):350-4. http://dx.doi.org/10.1016/S1472-6483(10)61795-6. PMid:15820041.
http://dx.doi.org/10.1016/S1472-6483(10)...
). In a study on human sperm, the OPS device was recommended as it allows the isolation of sperm from liquid nitrogen, with a low risk of microbial contamination during freezing and storage.

Cuevas-Uribe et al. (2011)Cuevas-Uribe R, Leibo SP, Daly J, Tiersch TR. Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling. Cryobiology. 2011;63(3):186-97. http://dx.doi.org/10.1016/j.cryobiol.2011.06.004. PMid:21896271.
http://dx.doi.org/10.1016/j.cryobiol.201...
analyzed the use of eight different devices for the vitrification of fish sperm according to parameters such as: sample filling efficiency, sample storage, sample volume, speed of cooling and heating, visualization of glass formation (characteristic of the glassy state), sample labeling, and cost per sample. According to these authors, the devices that met these parameters were the nichrome loop and the 0.25-mL CSS. Although there is no consensus on the best device, asepsis and isolation of liquid nitrogen are considered crucial for the effectiveness of the technique (Isachenko et al., 2017Isachenko V, Rahimi G, Mallmann P, Sánchez R, Isachenko E. Technologies of cryoprotectant-free vitrification of human spermatozoa: asepticity as criterion of effectiveness. Andrology. 2017;5(6):1055-63. http://dx.doi.org/10.1111/andr.12414. PMid:28992376.
http://dx.doi.org/10.1111/andr.12414...
).

The solution used in kinetic vitrification usually consists of a thinner, an N-CPA, which can be a disaccharide, such as sucrose or trehalose, and proteins, such as human serum albumin (Slabbert et al., 2015Slabbert M, du Plessis SS, Huyser C. Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa. Andrologia. 2015;47(5):594-9. http://dx.doi.org/10.1111/and.12307. PMid:25040423.
http://dx.doi.org/10.1111/and.12307...
), bovine albumin, soy lecithin (Swanson et al., 2017Swanson W, Bateman H, Vansandt L. Urethral catheterization and sperm vitrification for simplified semen banking in felids. Reprod Domest Anim. 2017;52(Suppl 2):255-60. http://dx.doi.org/10.1111/rda.12863. PMid:27807883.
http://dx.doi.org/10.1111/rda.12863...
), fetal bovine serum, and fallopian tube fluid (Isachenko et al., 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
).

The heating of the sample during devitrification depends on the device used with varying time and temperature. Water bath or devitrification solution temperatures range from 37 °C to 42 °C, with 37 °C being reported for human, fish, dog, and domestic cat sperm (Nawroth et al., 2002Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, Mallmann P, Dattena M, Capobianco G, Peters D, Orth I, Isachenko E. Vitrification of human spermatozoa without cryoprotectants. Cryo Lett. 2002;23(2):93-102. PMid:12050777.; Isachenko et al., 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
, 2004bIsachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, Van Der Ven H. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004b;71(4):1167-73. http://dx.doi.org/10.1095/biolreprod.104.028811. PMid:15175233.
http://dx.doi.org/10.1095/biolreprod.104...
, 2005Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F, Dessole S, Katkov II, Van der Ven H. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10(3):350-4. http://dx.doi.org/10.1016/S1472-6483(10)61795-6. PMid:15820041.
http://dx.doi.org/10.1016/S1472-6483(10)...
, 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
, 2012Isachenko V, Isachenko E, Petrunkina AM, Sánchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod Fertil Dev. 2012;24(2):323-6. http://dx.doi.org/10.1071/RD11061. PMid:22281078.
http://dx.doi.org/10.1071/RD11061...
; Merino et al., 2011Merino O, Risopatrón J, Sánchez R, Isachenko E, Figueroa E, Valdebenito I, Isachenko V. Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness. Anim Reprod Sci. 2011;124(1-2):125-31. http://dx.doi.org/10.1016/j.anireprosci.2011.02.023. PMid:21392903.
http://dx.doi.org/10.1016/j.anireprosci....
; Sánchez et al., 2011Sánchez R, Risopatrón J, Schulz M, Villegas J, Isachenko V, Kreinberg R, Isachenko E. Canine sperm vitrification with sucrose: effect on sperm function. Andrologia. 2011;43(4):233-41. http://dx.doi.org/10.1111/j.1439-0272.2010.01054.x. PMid:21486402.
http://dx.doi.org/10.1111/j.1439-0272.20...
; Swanson et al., 2017Swanson W, Bateman H, Vansandt L. Urethral catheterization and sperm vitrification for simplified semen banking in felids. Reprod Domest Anim. 2017;52(Suppl 2):255-60. http://dx.doi.org/10.1111/rda.12863. PMid:27807883.
http://dx.doi.org/10.1111/rda.12863...
), 38 °C used for heating rabbit semen samples (Rosato and Iaffaldano, 2013Rosato MP, Iaffaldano N. Cryopreservation of rabbit semen: comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. Theriogenology. 2013;79(3):508-16. http://dx.doi.org/10.1016/j.theriogenology.2012.11.008. PMid:23218394.
http://dx.doi.org/10.1016/j.theriogenolo...
), and 42 °C also for human semen samples (Slabbert et al., 2015Slabbert M, du Plessis SS, Huyser C. Large volume cryoprotectant-free vitrification: an alternative to conventional cryopreservation for human spermatozoa. Andrologia. 2015;47(5):594-9. http://dx.doi.org/10.1111/and.12307. PMid:25040423.
http://dx.doi.org/10.1111/and.12307...
).

Main results of kinetic vitrification: animal and human models

Kinetic vitrification, as most reproductive techniques, was first tested in animal models. The first time this technique was investigated was in 1938, when Luyet and Hodapp reported the recovery of motility in 20% of sperm from frogs directly immersed in liquid nitrogen using 1 M sucrose. Later, other authors published their experiences with kinetic vitrification. In birds, Shaffner et al. (1941)Shaffner CS, Henderson EW, Card CG. Viability of spermatozoa of the chicken under various environmental conditions. Poult Sci. 1941;20(3):259-65. http://dx.doi.org/10.3382/ps.0200259.
http://dx.doi.org/10.3382/ps.0200259...
subjected rooster semen to what they termed “fast freezing at -76 °C,” using different concentrations of fructose and thawing at 42 °C to 45 °C. Despite the importance of this study, as it is considered as one of the first attempts to apply kinetic vitrification to avian semen, 30% of sperm were mobile after devitrification and there were no fertile eggs after artificial insemination. In a study on rabbits, Hoagland and Pincus (1942)Hoagland H, Pincus G. Revival of mammalian sperm after immersion in liquid nitrogen. J Gen Physiol. 1942;25(3):337-44. http://dx.doi.org/10.1085/jgp.25.3.337. PMid:19873277.
http://dx.doi.org/10.1085/jgp.25.3.337...
tried to recover sperm motility after direct vitrification in liquid nitrogen at -196 °C and obtained a recovery of 0.5% for semen without additives and 0.1% for semen diluted in Ringer’s solution. According to Katkov et al. (2012)Katkov II, Bolyukh VF, Chernetsov OA, Dudin PI, Grigoriev AY, Isachenko V, Isachenko E, Lulat AG-M, Moskovtsev SI, Petrushko MP, Pinyaev VI, Sokol KM, Sokol YI, Sushko AB, Yakhnenko I. Kinetic vitrification of spermatozoa of vertebrates: what can we learn from nature? In: Katkov I , editor. Current frontiers in cryobiology. Croatia: IntechOpen; 2012. p. 3-40. http://dx.doi.org/10.5772/34784.
http://dx.doi.org/10.5772/34784...
these first efforts to perform sperm vitrification did not receive the recognition they deserved, hampered by low repeatability and survival, as well as communication difficulties due to several “iron walls” between scientists in the western allies, Germany, and the USSR, during World War II followed by the Cold War. After the contradictory results obtained by these first studies on sperm kinetic vitrification, the cryoprotective function of glycerol was discovered by Polge et al. (1949)Polge C, Smith A, Parkes A. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949;164(4172):666. http://dx.doi.org/10.1038/164666a0. PMid:18143360.
http://dx.doi.org/10.1038/164666a0...
and Smith and Polge (1950)Smith AU, Polge C. Survival of spermatozoa at low temperatures. Nature. 1950;166(4225):668-9. http://dx.doi.org/10.1038/166668a0. PMid:14780190.
http://dx.doi.org/10.1038/166668a0...
, the egg yolk was discovered by Phillips and Lardy (1940)Phillips PH, Lardy HA. A yolk buffer pabulum for the preservation of bull semen. J Dairy Sci. 1940;23(5):399-404. http://dx.doi.org/10.3168/jds.S0022-0302(40)95541-2.
http://dx.doi.org/10.3168/jds.S0022-0302...
, in addition to the discovery of other CPAs, which moved the focus of the cryopreservation field from kinetic vitrification to slow (or equilibrium) freezing. Slow freezing is still the main sperm cryopreservation method (Katkov et al., 2012Katkov II, Bolyukh VF, Chernetsov OA, Dudin PI, Grigoriev AY, Isachenko V, Isachenko E, Lulat AG-M, Moskovtsev SI, Petrushko MP, Pinyaev VI, Sokol KM, Sokol YI, Sushko AB, Yakhnenko I. Kinetic vitrification of spermatozoa of vertebrates: what can we learn from nature? In: Katkov I , editor. Current frontiers in cryobiology. Croatia: IntechOpen; 2012. p. 3-40. http://dx.doi.org/10.5772/34784.
http://dx.doi.org/10.5772/34784...
) in humans (Mocé et al., 2016Mocé E, Fajardo AJ, Graham JK. Human sperm cryopreservation. Eur Med J. 2016;1:86-91. http://dx.doi.org/10.33590/emj/10313056.
http://dx.doi.org/10.33590/emj/10313056...
) and domestic animals (Thananurak et al., 2019Thananurak P, Chuaychu-noo N, Thélie A, Phasuk Y, Vongpralub T, Blesbois E. Sucrose increases the quality and fertilizing ability of cryopreserved chicken sperms in contrast to rafinose. Poult Sci. 2019;98(9):4161-71. http://dx.doi.org/10.3382/ps/pez196. PMid:31065720.
http://dx.doi.org/10.3382/ps/pez196...
).

In the 2000s, a second “wave” of studies focused on kinetic vitrification gained momentum, pushed by the results of Nawroth et al. (2002)Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu N, Mallmann P, Dattena M, Capobianco G, Peters D, Orth I, Isachenko E. Vitrification of human spermatozoa without cryoprotectants. Cryo Lett. 2002;23(2):93-102. PMid:12050777., who vitrified human sperm without using cryoprotectants and using the swim-up technique for sperm selection, obtaining an increase in motility when compared to slow freezing with permeable cryoprotectants. Subsequently, several studies were conducted comparing vitrification with slow freezing (Isachenko et al., 2004aIsachenko E, Isachenko V, Katkov II, Rahimi G, Schöndorf T, Mallmann P, Dessole S, Nawroth F. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod. 2004a;19(4):932-9. http://dx.doi.org/10.1093/humrep/deh194. PMid:15016773.
http://dx.doi.org/10.1093/humrep/deh194...
, 2004bIsachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, Van Der Ven H. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004b;71(4):1167-73. http://dx.doi.org/10.1095/biolreprod.104.028811. PMid:15175233.
http://dx.doi.org/10.1095/biolreprod.104...
), assessing different techniques and devices (Isachenko et al., 2005Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F, Dessole S, Katkov II, Van der Ven H. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10(3):350-4. http://dx.doi.org/10.1016/S1472-6483(10)61795-6. PMid:15820041.
http://dx.doi.org/10.1016/S1472-6483(10)...
, 2011Isachenko V, Maettner R, Petrunkina AM, Mallmann P, Rahimi G, Sterzik K, Sánchez R, Risopatron J, Damjanoski I, Isachenko E. Cryoprotectant-free vitrification of human spermatozoa in large (up to 0.5 mL) volume: a novel technology. Clin Lab. 2011;57(9-10):643-50. PMid:22029178.), and analyzing parameters beyond sperm motility (Isachenko et al., 2008Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II, Schulz M, Lulat A, Risopatrón MJ, Sánchez R. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136(2):167-73. http://dx.doi.org/10.1530/REP-07-0463. PMid:18483075.
http://dx.doi.org/10.1530/REP-07-0463...
, 2012Isachenko V, Isachenko E, Petrunkina AM, Sánchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod Fertil Dev. 2012;24(2):323-6. http://dx.doi.org/10.1071/RD11061. PMid:22281078.
http://dx.doi.org/10.1071/RD11061...
). According to O’Neill et al. (2019)O’Neill HC, Nikoloska M, Ho H, Doshi A, Maalouf W. Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage. J Assist Reprod Genet. 2019;36(8):1713-20. http://dx.doi.org/10.1007/s10815-019-01505-x. PMid:31273587.
http://dx.doi.org/10.1007/s10815-019-015...
a major limitation to clinical implementation of vitrification for human sperm is the right balance between the volume of spermatozoa suspension cryopreserved and a standardized use of CPAs for survival of spermatozoa.

Following a line of studies similar to that used by the group led by Isachenko, some researchers have evaluated the application of kinetic vitrification to the semen of dogs, fish, and rabbits (Table 1). The results of these studies differ depending on the species, kinetic vitrification protocol and evaluated semen quality parameters. In fish, for example, two studies were conducted in completely different ways: in the first study (Merino et al., 2011Merino O, Risopatrón J, Sánchez R, Isachenko E, Figueroa E, Valdebenito I, Isachenko V. Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: stability of mitochondrion as criterion of effectiveness. Anim Reprod Sci. 2011;124(1-2):125-31. http://dx.doi.org/10.1016/j.anireprosci.2011.02.023. PMid:21392903.
http://dx.doi.org/10.1016/j.anireprosci....
) used sucrose as N-CPA and bovine serum albumin (BSA) and seminal plasma as additives. The sample was previously centrifuged and placed directly into liquid nitrogen; while the second study (Cuevas-Uribe et al., 2011Cuevas-Uribe R, Leibo SP, Daly J, Tiersch TR. Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling. Cryobiology. 2011;63(3):186-97. http://dx.doi.org/10.1016/j.cryobiol.2011.06.004. PMid:21896271.
http://dx.doi.org/10.1016/j.cryobiol.201...
) does not use any type of cryoprotector or additives and compares two devices the cut straw and the nichrome loop. In the first experiment motility was obtained above 80% and plasma membrane integrity for the BSA + seminal plasma combination was 86.7%. In the second, no motility was obtained and the parameter evaluated was fertility which was also low (<2%).

As previously mentioned, the water volume and sperm cell morphology of different species are different, and this may be one of the factors why it is not possible to replicate the same kinetic vitrification protocol in all species. In addition, it is known for example that cryopreservation affects plasma membrane integrity and that plasma membrane composition varies among species (Gautier and Aurich, 2022Gautier C, Aurich C. “Fine feathers make fine birds” – The mammalian sperm plasma membrane lipid composition and effects on assisted reproduction. Anim Reprod Sci. 2022;246:106884. http://dx.doi.org/10.1016/j.anireprosci.2021.106884. PMid:34776291.
http://dx.doi.org/10.1016/j.anireprosci....
) therefore some species are more susceptible to damage during and after cryopreservation. For kinetic vitrification, more studies on these aspects are still needed to make a more precise statement about the interference of the protocol on the quality of the devitrified semen.

This demonstrates that it is necessary that the same protocol and evaluations for the quality of devitrified semen be applied more than once in experiments with similar animal groups or the same species to obtain accuracy of results and repeatability. To date, this is observed in experiments using human spermatozoa, where the number of publications is higher than for animal species.

Final considerations

Kinetic vitrification has great potential as any emerging technology and has recently become an alternative technique showing encouraging results for the use of vitrified sperm in assisted reproductive technologies such as in vitro fertilization (IVF) or intracytoplasmatic sperm injection (ICSI) (Isachenko et al., 2012Isachenko V, Isachenko E, Petrunkina AM, Sánchez R. Human spermatozoa vitrified in the absence of permeable cryoprotectants: birth of two healthy babies. Reprod Fertil Dev. 2012;24(2):323-6. http://dx.doi.org/10.1071/RD11061. PMid:22281078.
http://dx.doi.org/10.1071/RD11061...
; Nagashima et al., 2015Nagashima JB, Sylvester SR, Nelson JL, Cheong SH, Mukai C, Lambo C, Flanders JA, Meyers-Wallen VN, Songsasen N, Travis AJ. Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization. PLoS One. 2015;10(12):1. http://dx.doi.org/10.1371/journal.pone.0143930.
http://dx.doi.org/10.1371/journal.pone.0...
; Cerdeira et al., 2020Cerdeira J, Sánchez-Calabuig MJ, Pérez-Gutiérrez JF, Hijon M, Castaño C, Santiago-Moreno J. Cryopreservation effects on canine sperm morphometric variables and ultrastructure: comparison between vitrification and conventional freezing. Cryobiology. 2020;95:164-70. http://dx.doi.org/10.1016/j.cryobiol.2020.03.007. PMid:32229272.
http://dx.doi.org/10.1016/j.cryobiol.202...
).

The advantages of this methodology include its simplicity, speed, low cost, and the preservation of important physiological parameters, such as mitochondrial membrane potential and DNA integrity (Sánchez et al., 2011Sánchez R, Risopatrón J, Schulz M, Villegas J, Isachenko V, Kreinberg R, Isachenko E. Canine sperm vitrification with sucrose: effect on sperm function. Andrologia. 2011;43(4):233-41. http://dx.doi.org/10.1111/j.1439-0272.2010.01054.x. PMid:21486402.
http://dx.doi.org/10.1111/j.1439-0272.20...
). In addition, cryoprotectant-free vitrification can induce less biological changes in human spermatozoa, in comparison with conventional freezing (Wang et al., 2022Wang M, Todorov P, Wang W, Isachenko E, Rahimi G, Mallmann P, Isachenko V. Cryoprotectants-free vitrification and conventional freezing of human spermatozoa: a comparative transcript profiling. Int J Mol Sci. 2022;23(6):3047. http://dx.doi.org/10.3390/ijms23063047.
http://dx.doi.org/10.3390/ijms23063047...
) and lower acrossomal changes in dog spermatozoa (Caturla-Sánchez et al., 2018Caturla-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. 2018;80:126-9. http://dx.doi.org/10.1016/j.cryobiol.2017.11.001. PMid:29126865.
http://dx.doi.org/10.1016/j.cryobiol.201...
). Another advantage is that the use of high concentrations of permeable cryoprotectants, as used in equilibrium vitrification, are not necessary for kinetic vitrification (O'Neill et al., 2019O’Neill HC, Nikoloska M, Ho H, Doshi A, Maalouf W. Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage. J Assist Reprod Genet. 2019;36(8):1713-20. http://dx.doi.org/10.1007/s10815-019-01505-x. PMid:31273587.
http://dx.doi.org/10.1007/s10815-019-015...
) and it can reduce damage effects to sperm cell.

Also, cryopreservation success is measured by the motility after devitrification, and kinetic vitrification has not yet achieved satisfactory results in this regard in studies for some animals. In dogs, for example, acceptable data were not obtained after devitrification using only sucrose as a non-permeable cryoprotectant requiring the inclusion of proteins, such as bovine serum albumin (Sánchez et al., 2011Sánchez R, Risopatrón J, Schulz M, Villegas J, Isachenko V, Kreinberg R, Isachenko E. Canine sperm vitrification with sucrose: effect on sperm function. Andrologia. 2011;43(4):233-41. http://dx.doi.org/10.1111/j.1439-0272.2010.01054.x. PMid:21486402.
http://dx.doi.org/10.1111/j.1439-0272.20...
) as an additive to stabilize the sperm membrane. Therefore, further studies should investigate which additives can stimulate sperm kinetics after devitrification and what are the heating rates specific for each species. The optimal concentration of non-permeable CPAs is a key factor for sperm vitrification success. It is species-specific but also depends on the methodology (Hidalgo, 2021Hidalgo M. Recent advances in donkey sperm vitrification. Reprod Domest Anim. 2021;56(10):1274-8. http://dx.doi.org/10.1111/rda.13995. PMid:34418185.
http://dx.doi.org/10.1111/rda.13995...
) and need additional attention in terms of research.

Acknowledgements

We would like to thank to the Department of Animal Reproduction of College of Veterinary Medicine and Animal Science of University of São Paulo for the technical support during this research.

  • Financial support: BBB received funding for this research from Fundação Amazônia de Amparo à Estudos e Pesquisas (grant numbers 007/2017).
  • How to cite: Barbosa BB, Evangelista ITA, Soares ARB, Leão DL, Pereira RJG, Domingues SFS. Kinetic vitrification: concepts and perspectives in animal sperm cryopreservation. Anim Reprod. 2023;20(2):e20220096. https://doi.org/10.1590/1984-3143-AR2022-0096

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Publication Dates

  • Publication in this collection
    22 May 2023
  • Date of issue
    2023

History

  • Received
    18 Oct 2022
  • Accepted
    04 Apr 2023
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