- Email: [email protected]
Mode of action of cryoprotectants for sperm preservation
Accepted Manuscript Title: Mode of action of cryoprotectants for sperm preservation Author: Harald Sieme Harri¨ette Oldenhof Willem F. Wolkers PII: DOI: Reference:
S0378-4320(16)30034-3 http://dx.doi.org/doi:10.1016/j.anireprosci.2016.02.004 ANIREP 5359
To appear in:
Animal Reproduction Science
Received date: Revised date: Accepted date:
30-11-2015 28-1-2016 1-2-2016
Please cite this article as: Sieme, Harald, Oldenhof, Harri¨ette, Wolkers, Willem F., Mode of action of cryoprotectants for sperm preservation.Animal Reproduction Science http://dx.doi.org/10.1016/j.anireprosci.2016.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MODE OF ACTION OF CRYOPROTECTANTS FOR SPERM PRESERVATION
Harald Sieme1,*, Harriëtte Oldenhof1, Willem F. Wolkers2
1
Clinic for Horses - Unit for Reproductive Medicine, University of Veterinary Medicine
Hannover, Germany, 2Institute of Multiphase Processes, Leibniz Universität Hannover, Germany
*corresponding
author: Harald Sieme, Clinic for Horses - Unit for Reproductive Medicine,
University of Veterinary Medicine Hannover, Bünteweg 15, 30559 Hannover, Germany; phone: +49 511 953 8530, fax: +49 511 953 82 8530, e-mail address: [email protected]
1
ABSTRACT Sperm cryopreservation facilitates storage and transport for use in artificial reproduction technologies. Cryopreservation processing, however, exposes cells to stress resulting in cellular damage compromising sperm function. Cryoprotective agents are needed to minimize cryopreservation injury, but at higher concentration they are toxic to cells. In this review, we describe cryoinjury mechanisms, and modes of action of different types of cryoprotective agents. Furthermore, measures are discussed how to minimize toxic effects caused by adding and removing cryoprotective agents. Cryoprotective agents can be divided into permeating and non-permeating agents. Permeating agents such as glycerol can move across cellular membranes and modulate the rate and extent of cellular dehydration during freezing-induced membrane phase transitions. Permeating protectants provide intracellular protection because they are preferentially excluded from the surface of biomolecules thereby stabilizing the native state. Non-permeating agents can be divided into osmotically active smaller molecules and osmotically inactive macromolecules. Both, permeating and non-permeating protectants form a protective glassy state during freezing preserving biomolecular and cellular structures. Freezing extenders for sperm contain salts, buffer compounds, sugars, proteins and lipids, and typically contain glycerol as the main permeating cryoprotective agent providing intracellular protection. Non-permeating protectants including sugars and proteins are used as bulking agents and to increase the glass transition temperature of the freezing extender. Ultra-heattreated milk and egg yolk are frequently added as membrane modifying agents to enhance the inherent sperm cryostability. The protocol how to use and add cryoprotectants is a compromise between their beneficial and potentially detrimental effects.
keywords: cryopreservation, cryoprotective agents, equine spermatozoa, cytotoxicity, membrane phase behavior, osmotic tolerance
2
CRYOPRESERVATION
Since the introduction of glycerol as a permeating cryoprotective agent (Polge et al., 1949), and the subsequent discovery of dimethyl sulfoxide (Lovelock and Bishop, 1959), many different types of cells have been cryopreserved. Despite these successes, not all cell types can be cryopreserved using standard cryopreservation methods. Cryopreservation requires use of cryoprotective agents that have minimal cytotoxic effects (Davidson et al., 2014). In addition, a specific cooling rate is needed for maximum survival (Mazur, 1963). This cooling rate varies greatly depending upon the cell type and is related to cell-type specific membrane properties (Benson et al., 2012). Cryopreservation damage is particularly manifested as a loss in membrane integrity after thawing (Meryman, 2007). Insights in the impact of cooling, freezing and protective agents on membrane properties may help to rationally design cryopreservation protocols.
CELLULAR DAMAGE INDUCED BY FREEZING
The process of cryopreservation exposes cells to stress induced by low-temperature and osmotic imbalances. Exposing biomolecules to decreasing temperatures may lead to (irreversible)
conformational
changes.
Osmotic
stress
during
cryopreservation
is
predominantly the result of extracellular ice formation. Upon extracellular ice formation, the solute concentration in the extracellular unfrozen fraction increases causing cells to dehydrate (Mazur, 2004; Meryman, 2007). Cellular dehydration during freezing is the result of water transport out of the cell in order to retain equilibrium between the intra- and extracellular solute concentration. Dehydration especially occurs when low cooling rates are used (Mazur, 1963). When cells shrink or swell beyond their osmotic tolerance limits this can be lethal
3
(Woods et al., 2004; Benson et al., 2012; Yeste, 2016). At fast cooling rates, cells do not have enough time to loose water and as a result intracellular water contents remain relatively high leading to intracellular ice formation (Mazur, 1963). Moreover, addition and removal of cryoprotective agents, also exposes cells to osmotic stress (Mazur, 2004; Kashuba et al., 2014; Meryman, 2007). Freezing also results in accumulation of damaging reactive oxygen species. Cryotolerance of stallion spermatozoa is related to the extent ROS production in mitochondria (Yeste, 2015). The ‘two-factor hypothesis’ postulates that cells display a specific optimal cooling rate, where damage due to dehydration (solute effects injury) and intracellular ice formation is minimal and cellular survival after thawing is maximal (Mazur, 1963). The capacity of cells to change their volume in response to freezing-induced osmotic stress is determined by the rate of water transport into and out of the cells defined by the membrane hydraulic permeability and activation energy for water transport (Mazur, 1963). The membrane permeability to water is determined by the membrane phospholipid composition, the presence of water and ion channel proteins, cytoskeletal elements (Elmoazzen et al., 2009), and is also altered by cryoprotective agents (Devireddy et al., 2002; Akhoondi et al., 2011, Oldenhof at al., 2010). Membrane hydraulic permeability decreases with decreasing temperature. The cell-specific membrane hydraulic permeability can be described by an empirical Arrhenius relationship, which predicts that the rate of water transport across the membrane exponentially slows down with decreasing temperature (Mazur, 1963). A plot of the natural logarithm of the membrane hydraulic permeability versus the reciprocal of the absolute temperature can be fitted with a linear regression line. Ice formation, however, changes the effect of temperature on membrane hydraulic permeability resulting in a different Arrhenius relationship compared to that in the absence of ice (Akhoondi et al., 2011). Cryoprotective agents increase membrane permeability to water,
4
and thereby facilitate cellular dehydration during freezing even at low subzero temperatures (Devireddy et al., 2002; Oldenhof et al., 2010; Akhoondi et al., 2011; Xu et al., 2014). Moreover, cryoprotective agents typically shift the optimal cooling rate to lower temperatures and broaden the range of cooling rates resulting in optimal survival.
DIFFERENT TYPES OF CRYOPROTECTIVE AGENTS
Cryopreservation requires protection of intracellular structures and biomolecules, and hence requires protective agents that are able to pass the cellular membrane. Permeating cryoprotective agents are generally small non-ionic molecules. The most commonly used membrane permeable cryoprotective agents are dimethyl sulfoxide (DMSO) and glycerol. Alternatively, in cases where the above mentioned agents are toxic to the cells, ethylene glycol, methyl-formamide, or dimethyl-formamide may be used (Squires et al., 2004). Permeating cryoprotective agents are osmotically inactive, because they are equally distributed in the intracellular and extracellular spaces. However, addition itself poses osmotic stress to cells because water moves faster across the cellular membrane compared to the permeating cryoprotective agents. This results in an initial osmotic unbalance and shrinkage of the cells followed by an influx of water and cryoprotective agents until an equal distribution of cryoprotective agent inside and outside the cell is reached. Cryopreservation solutions may also include non-permeable cryoprotective agents. These can be divided into osmotically active molecules such as disaccharides (sucrose, trehalose), and osmotically inactive compounds including polysaccharides (hydroxyl ethylene starch, maltodextrin), and proteins (albumin, polyvinylpyrrolidon). Compounds such as sucrose are believed not to pass cellular membranes, and will cause cellular dehydration since they increase the osmolality of the cryopreservation medium. Addition of large
5
macromolecules contributes little to the medium osmolality, and this does not cause cellular dehydration (Oldenhof et al., 2013b). Protective effects of various proteins that are synthesized in response to stress have been investigated. Examples include antifreeze proteins, heat shock proteins (HSP), and socalled late embryo abundant (LEA) proteins (Park et al., 2013; Popova et al., 2011). Mixed results have been found in terms of the effects of these compounds on cryosurvival. Stress proteins are generally not supplemented in cryopreservation solutions for cells. In addition to cryoprotective compounds as described above, properties of cellular membranes can be modified by supplementing or extracting lipids and/or cholesterol. This can be achieved using cholesterol-loaded cyclodextrins or liposomes, which change the cholesterol/phospholipid ratio or the phospholipid composition thereby increasing osmotic tolerance limits, susceptibility to lipid peroxidation and cryostability (Glazar et al., 2009). It has been shown that the use a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing extender of equine sperm (Blommaert et al 2015).
MODELS EXPLAINING ACTION OF CRYOPROTECTIVE AGENTS
Permeating cryoprotective agents have been attributed a variety of different cryoprotective properties. First of all, they decrease the ice nucleation temperature, and ice crystal size. The ‘preferential exclusion theory’ explains the stabilizing effects of small cosolutes on biomolecules by preferential interaction of biomolecules with water rather than with the added co-solute (Arakawa and Timasheff, 1985). This means that cryoprotective agents such as glycerol and sucrose are being excluded from the surface of biomolecules thereby stabilizing the native conformation. This theory is developed to explain the stabilizing effects of compatible solutes on proteins at suprazero temperatures (Arakawa and
6
Timasheff, 1985; Arakawa et al., 1990; Crowe et al., 1992), but it is assumed that this mechanism also explains stabilizing effects during freezing. In different temperature regimes cryoprotective agents may have different functions/modes of action. At low subzero temperatures, they modulate the rate of cellular dehydration upon ice formation and facilitate dehydration (Oldenhof et al., 2013a). Upon further decreasing the temperature, cryoprotective agents enter into a so-called glassy (vitrified) state. Molecular reactions are slowed in the highly viscous glassy state, which stabilizes cells during long-term storage. The glassy state functions as a matrix in which biomolecular and cellular structures are embedded and preserved (Saragusty et al., 2009). The glass transition temperature of sugars and polysaccharides is relatively high as compared to glycerol (Slade and Levine, 1991). Their use in freezing formulations increases the glass transition temperature, which allows storage at higher subzero temperatures (Stoll et al., 2012; Oldenhof et al., 2013b). The water replacement hypothesis postulates that protectants can replace water and interact with phospholipids and other biomolecules through hydrogen bonding (Crowe et al., 1992), whereas the water entrapment hypothesis suggests that protectants trap water around biomolecules, therewith preventing dehydration-induced conformational changes (Belton and Gil, 1994). Both these hypotheses have been proposed for dry preservation of biomolecules and cells. We have recently shown that water surrounding membrane phospholipids is being removed during freezing-induced dehydration, and that this is not prevented by cryoprotective agents, suggesting that neither replacement nor entrapment play a role in protection of membranes during freezing (Oldenhof et al., 2010; Akhoondi et al., 2011; Oldenhof et al., 2013b).
7
CRYOPRESERVATION OF SPERM
Sperm cryopreservation facilitates transport and storage, for use in artificial reproduction technologies. Cryopreserved sperm, however, displays a high degree of variation in survival after thawing among species and individuals. Such differences could be genetic in origin, causing differences in the inherent cryostability. Also the presence of damaging compounds in an ejaculate may decrease sperm cryostability. Centrifugation processing and clean-up approaches can be employed to remove cell debris and damaging compounds in an ejaculate and to enrich samples with good quality sperm (Sieme and Oldenhof, 2015b). Freezing extenders for sperm contain nutrients (salts, sugars, proteins and lipids), and typically contain glycerol as the main permeating cryoprotective agent. However, other permeating cryoprotective agents such as ethylene glycol and (di)methyl-formamide, or combinations of various permeating agents may yield similar or even higher cryosurvival rates (Squires et al., 2004, Álvarez et al., 2014, Wu et al., 2015). In addition to ultra-heat-treated milk, egg yolk is frequently added as membrane modifying agent in sperm freezing extenders (Sieme, 2011, Varisli et al., 2013). Because of its undefined composition and animal origin, a lot of efforts have been made to find alternatives for egg yolk. It has been shown that liposomes, particularly composed of unsaturated lipids, can be used instead of egg yolk, resulting in similar cryosurvival rates (Röpke et al., 2011, Pillet et al., 2012). Also cyclodextrin-cholesterol complexes can be used to replace egg yolk in sperm freezing extenders (Blommaert et al 2015, Moraes et al., 2015), Nowadays, sperm is generally cryopreserved in 0.25- or 0.5-mL plastic straws, which have a large surface area to volume ratio. Conventional (equiaxial) freezing of semen packaged in straws is typically done using different cooling velocities in different
8
temperature regimes. Typically, sperm diluted in freezing extender is slowly cooled from room temperature to 5°C at a rate of about 0.1°C min−1, followed by freezing at a rate of 10– 60 °C min−1 down to temperatures as low as −80 °C, after which samples are plunged into liquid nitrogen for storage (Sieme and Oldenhof, 2015a). Most cryoprotectants show some degree of toxicity to sperm. This toxicity can be minimized by taking several measures. The cryoprotectant concentration should be reduced as much as possible (Hoffmann et al., 2011). The temperature at which cryoprotective agents are added is also critical. It has been reported that cryoprotectants have to be added at room temperature (Vidament et al., 2000), whereas others suggest adding pre-cooled cryoprotectants at low temperatures, which is referred to as the so called cold cryoprotectant procedure (Clarke et al., 2003). It is beneficial to add cryoprotectants in a stepwise manner to semen to reduce osmotic stress (Sieme and Oldenhof, 2015a). Measures after thawing include stepwise dilution of the sample to slowly return to isotonic conditions. The protocol how to use and add cryoprotectants is a compromise between their beneficial and potentially detrimental effects. In all likelihood, the composition of the freezing extender may need to be custom-designed and adapted for individual donors in order to assure maximal cryosurvival.
ACKNOWLEDGMENTS The work in our laboratories is supported by the ‘Mehl-Mülhens Stiftung’, as well as the German Research Foundation (DFG: Deutsche Forschungsgemeinschaft) via the Cluster of Excellence ‘From regenerative biology to reconstructive therapy’ (REBIRTH) and grants WO1735/6-1 and SI1462/4-1.
9
REFERENCES
Akhoondi, M., Oldenhof, H., Stoll, C., Sieme, H., Wolkers, W.F., 2011. Membrane hydraulic permeability changes during cooling of mammalian cells. Biochim. Biophys. Acta 1808, 642–648. Álvarez, C., Gil, L., González, N., Olaciregui, M., Luño, V., 2014. Equine sperm post-thaw evaluation after the addition of different cryoprotectants added to INRA 96 extender. Cryobiology 69, 144–148. Arakawa, T., Bhat, R., Timasheff, S.N., 1990. Why preferential hydration does not always stabilize the native structure of globular proteins. Biochemistry 29, 1924–1931. Arakawa, T., Timasheff, S.N., 1985. The stabilization of proteins by osmolytes. Biophys. J. 47, 411–414. Belton, P.S., Gil, A.H., 1994. Raman IR spectroscopic studies of the interaction of trehalose with hen egg lysozyme. Biopolymers 34, 957–961. Benson, J.D., Woods, E.J., Walters, E.M., Critser, J.K., 2012. The cryobiology of spermatozoa. Theriogenology 78, 1682–1699. Blommaert, D., Franck, T., Donnay, I., Lejeune, J-P., Detilleux, J., Serteyn, D., 2015. Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm, Cryobiology, (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.008 Clarke, G.N., Liu, D.Y., Baker, H.W.G., 2003. Improved sperm cryopreservation using cold cryoprotectant. Reprod. Fert. Dev. 15, 377–381. Crowe, J.H., Hoekstra, F.A., Crowe, L.M., 1992. Anhydrobiosis, Annual Review of Physiology 54, 570–599.
10
Davidson, A.F., Benson, J.D., Higgins, A.Z., 2014. Mathematically optimized cryoprotectant equilibration procedures for cryopreservation of human oocytes. Theor. Biol. Med. Model. 11, 13. Devireddy, R.V., Swanlund, D.J., Olin, T., Vincente, W., Troedsson, M.H.T., Bischof, J.C., Roberts, K.P., 2002. Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry. Biol. Reprod. 66, 222–231. Elmoazzen, H.Y., Elliott, J.A., McGann, L.E., 2009. Osmotic transport across cell membranes in nondilute solutions: a new nondilute solute transport equation. Biophys. J. 96, 2559-2571. Glazar, A.I., Mullen, S.F., Liu, J., Benson, J.D., Critser, J.K., Squires, E.L., Graham, J.K., 2009. Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol. Cryobiology 59, 201–206. Hoffmann, N., Oldenhof, H., Morandini, C., Rohn, K., Sieme, H., (2011). Optimal concentrations of cryoprotective agents for semen from stallions that are classified ‘good’ or ‘poor’ for freezing. Anim. Reprod. Sci. 125, 112–118. Kashuba, C.M., Benson, J.D., Critser, J.K., 2014. Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I--Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols. Cryobiology 68, 166–175. Lovelock, J.E., Bishop, M.W.H., 1959 Prevention of Freezing Damage to Living Cells by Dimethyl Sulphoxide. Nature 183, 1394-1395. Mazur, P., 1963. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. Gen. Physiol. 47, 347–369.
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Mazur, P., 2004. Principles of cryobiology. in Fuller, B.J., Lane, N.L., Benson, E.E. (Eds.), Life in the frozen state. CRC Press LLC, Boca Raton, FL, pp 3–65. Meryman, H.T., 2007. Cryopreservation of living cells: principles and practice. Transfusion. 47, 935–945. Moraes, E.A., Matos, W.C., Graham, J.K., Ferrari, W.D., 2015. Cholestanol-loadedcyclodextrin improves the quality of stallion spermatozoa after cryopreservation. Anim. Reprod. Sci. 158, 19–24. Oldenhof, H., Friedel, K., Sieme, H., Glasmacher, B., Wolkers, W.F., 2010. Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy. Cryobiology 61, 115–122. Oldenhof, H., Akhoondi, M., Sieme, H., Wolkers, W.F., 2013a. Use of Fourier transform infrared spectroscopy to determine optimal cooling rates for cryopreservation of cells. Biomedical Spectroscopy and Imaging 2. 83–90. Oldenhof, H., Gojowsky, M., Wang, S., Henke, S., Yu, C., Rohn, K., Wolkers, W.F., Sieme, H., 2013b. Osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents. Biol. Reprod. 88, 68. Park, S.J., Choi, H.R., Nam, K.M., Na, J.I., Huh, C.H., Park, K.C., 2013. Immediate induction of heat shock proteins is not protective against cryopreservation in normal human fibroblasts. Cryo Letters 34, 239-247. Pegg, D.E., 2015. Principles of cryopreservation. in: Wolkers, W.F., Oldenhof, H. (Eds.), Cryopreservation and freeze-drying protocols, third edition, Methods in molecular biology 1257, Springer, New York, pp. 3–19. Pillet, E., Labbe, C., Batellier, F., Duchamp, G., Beaumal, V., Anton, M., Desherces, S., Schmitt, E., Magistrini, M., 2012. Liposomes as an alternative to egg yolk in stallion freezing extender. Theriogenology 77, 268–279.
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Polge, C., Smith, A.U., Parkes, A.S., 1949. Revival of spermatozoa after vitrification and dehydration at law temperatures. Nature 164, 666–676. Popova, A.V., Hundertmark, M., Seckler, R., Hincha, D.K. 2011. Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes. Biochim. Biophys. Acta 1808, 1879-1887. Röpke, T., Oldenhof, H., Leiding, C., Sieme, H., Bollwein, H., Wolkers, W.F., 2011. Liposomes for cryopreservation of bovine sperm. Theriogenology 76, 1465–1472. Saragusty, J., Gacitua, H., Rozenboim, I., Arav, A., 2009. Do physical forces contribute to cryodamage? Biotechnol. Bioeng. 104, 719–728. Sieme, H., 2011. Semen extenders for frozen semen. in: McKinnon, A.O., Squires, E.L., Vaala, W.E., Varner, D.D. (Eds.), Equine Reproduction, second edition, Blackwell Publishing Ltd., Chichester, UK, pp. 2964–2971. Sieme, H., Oldenhof, H., 2015a. Cryopreservation of domestic livestock semen. in: Wolkers, W.F., Oldenhof, H. (Eds.), Cryopreservation and freeze-drying protocols, third edition, Methods in molecular biology 1257, Springer, New York, pp. 277–287. Sieme, H., Oldenhof, H., 2015b. Sperm cleanup and centrifugation processing for cryopreservation. in: Wolkers, W.F., Oldenhof, H. (Eds.), Cryopreservation and freeze-drying protocols, third edition, Methods in molecular biology 1257, Springer, New York, pp. 343–352. Slade, L., Levine, H., 1991. Beyond water activity: recent advances based on an alternative approach to the assessment of food quality and safety. Crit. Rev. Food Sci. Nutr. 30, 115–136. Squires, E.L., Keith, S.L., Graham, J.K., 2004. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology 62, 1056–1065.
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Stoll, C., Holovati, J.L., Acker, J.P., Wolkers, W.F., 2012. Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes. Biotechnol. Prog. 28, 364–171. Varisli, O., Scott, H., Agca, C., Agca, Y., 2013. The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm. Cryobiology 67, 109–116. Vidament, M., Ecot, P., Noue, P., Bourgeois, C., Magistrini, M., Palmer, E., 2000. Centrifugation and addition of glycerol at 22C instead of 4C improve post-thaw motility and fertility of stallion spermatozoa. Theriogenology 54, 907–919. Woods, E.J., Benson, J.D., Agca, Y., Critser, J.K., 2004. Fundamental cryobiology of reproductive cells and tissues. Cryobiology 48, 146–156. Wu, Z., Zheng, X., Luo, Y., Huo, F., Dong, H., Zhang, G., Yu, W., Tian, F., He, L., Chena, J., Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants. Anim. Reprod. Sci. 163, 75–81. Xu, Y., Zhao, G., Zhou, X., Ding, W., Shu, Z., Gao, D., 2014. Biotransport and intracellular ice formation phenomena in freezing human embryonic kidney cells (HEK293T). Cryobiology 68, 294–302. Yeste M., 2016. Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology. 85, 47–64. Yeste, M., Estrada, E., Rocha, L.G., Marın, H., Rodrıguez-Gil, J.E., Miro, J., 2015. Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus. Andrology 3, 395– 407.
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S0378-4320(16)30034-3 http://dx.doi.org/doi:10.1016/j.anireprosci.2016.02.004 ANIREP 5359
To appear in:
Animal Reproduction Science
Received date: Revised date: Accepted date:
30-11-2015 28-1-2016 1-2-2016
Please cite this article as: Sieme, Harald, Oldenhof, Harri¨ette, Wolkers, Willem F., Mode of action of cryoprotectants for sperm preservation.Animal Reproduction Science http://dx.doi.org/10.1016/j.anireprosci.2016.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MODE OF ACTION OF CRYOPROTECTANTS FOR SPERM PRESERVATION
Harald Sieme1,*, Harriëtte Oldenhof1, Willem F. Wolkers2
1
Clinic for Horses - Unit for Reproductive Medicine, University of Veterinary Medicine
Hannover, Germany, 2Institute of Multiphase Processes, Leibniz Universität Hannover, Germany
*corresponding
author: Harald Sieme, Clinic for Horses - Unit for Reproductive Medicine,
University of Veterinary Medicine Hannover, Bünteweg 15, 30559 Hannover, Germany; phone: +49 511 953 8530, fax: +49 511 953 82 8530, e-mail address: [email protected]
1
ABSTRACT Sperm cryopreservation facilitates storage and transport for use in artificial reproduction technologies. Cryopreservation processing, however, exposes cells to stress resulting in cellular damage compromising sperm function. Cryoprotective agents are needed to minimize cryopreservation injury, but at higher concentration they are toxic to cells. In this review, we describe cryoinjury mechanisms, and modes of action of different types of cryoprotective agents. Furthermore, measures are discussed how to minimize toxic effects caused by adding and removing cryoprotective agents. Cryoprotective agents can be divided into permeating and non-permeating agents. Permeating agents such as glycerol can move across cellular membranes and modulate the rate and extent of cellular dehydration during freezing-induced membrane phase transitions. Permeating protectants provide intracellular protection because they are preferentially excluded from the surface of biomolecules thereby stabilizing the native state. Non-permeating agents can be divided into osmotically active smaller molecules and osmotically inactive macromolecules. Both, permeating and non-permeating protectants form a protective glassy state during freezing preserving biomolecular and cellular structures. Freezing extenders for sperm contain salts, buffer compounds, sugars, proteins and lipids, and typically contain glycerol as the main permeating cryoprotective agent providing intracellular protection. Non-permeating protectants including sugars and proteins are used as bulking agents and to increase the glass transition temperature of the freezing extender. Ultra-heattreated milk and egg yolk are frequently added as membrane modifying agents to enhance the inherent sperm cryostability. The protocol how to use and add cryoprotectants is a compromise between their beneficial and potentially detrimental effects.
keywords: cryopreservation, cryoprotective agents, equine spermatozoa, cytotoxicity, membrane phase behavior, osmotic tolerance
2
CRYOPRESERVATION
Since the introduction of glycerol as a permeating cryoprotective agent (Polge et al., 1949), and the subsequent discovery of dimethyl sulfoxide (Lovelock and Bishop, 1959), many different types of cells have been cryopreserved. Despite these successes, not all cell types can be cryopreserved using standard cryopreservation methods. Cryopreservation requires use of cryoprotective agents that have minimal cytotoxic effects (Davidson et al., 2014). In addition, a specific cooling rate is needed for maximum survival (Mazur, 1963). This cooling rate varies greatly depending upon the cell type and is related to cell-type specific membrane properties (Benson et al., 2012). Cryopreservation damage is particularly manifested as a loss in membrane integrity after thawing (Meryman, 2007). Insights in the impact of cooling, freezing and protective agents on membrane properties may help to rationally design cryopreservation protocols.
CELLULAR DAMAGE INDUCED BY FREEZING
The process of cryopreservation exposes cells to stress induced by low-temperature and osmotic imbalances. Exposing biomolecules to decreasing temperatures may lead to (irreversible)
conformational
changes.
Osmotic
stress
during
cryopreservation
is
predominantly the result of extracellular ice formation. Upon extracellular ice formation, the solute concentration in the extracellular unfrozen fraction increases causing cells to dehydrate (Mazur, 2004; Meryman, 2007). Cellular dehydration during freezing is the result of water transport out of the cell in order to retain equilibrium between the intra- and extracellular solute concentration. Dehydration especially occurs when low cooling rates are used (Mazur, 1963). When cells shrink or swell beyond their osmotic tolerance limits this can be lethal
3
(Woods et al., 2004; Benson et al., 2012; Yeste, 2016). At fast cooling rates, cells do not have enough time to loose water and as a result intracellular water contents remain relatively high leading to intracellular ice formation (Mazur, 1963). Moreover, addition and removal of cryoprotective agents, also exposes cells to osmotic stress (Mazur, 2004; Kashuba et al., 2014; Meryman, 2007). Freezing also results in accumulation of damaging reactive oxygen species. Cryotolerance of stallion spermatozoa is related to the extent ROS production in mitochondria (Yeste, 2015). The ‘two-factor hypothesis’ postulates that cells display a specific optimal cooling rate, where damage due to dehydration (solute effects injury) and intracellular ice formation is minimal and cellular survival after thawing is maximal (Mazur, 1963). The capacity of cells to change their volume in response to freezing-induced osmotic stress is determined by the rate of water transport into and out of the cells defined by the membrane hydraulic permeability and activation energy for water transport (Mazur, 1963). The membrane permeability to water is determined by the membrane phospholipid composition, the presence of water and ion channel proteins, cytoskeletal elements (Elmoazzen et al., 2009), and is also altered by cryoprotective agents (Devireddy et al., 2002; Akhoondi et al., 2011, Oldenhof at al., 2010). Membrane hydraulic permeability decreases with decreasing temperature. The cell-specific membrane hydraulic permeability can be described by an empirical Arrhenius relationship, which predicts that the rate of water transport across the membrane exponentially slows down with decreasing temperature (Mazur, 1963). A plot of the natural logarithm of the membrane hydraulic permeability versus the reciprocal of the absolute temperature can be fitted with a linear regression line. Ice formation, however, changes the effect of temperature on membrane hydraulic permeability resulting in a different Arrhenius relationship compared to that in the absence of ice (Akhoondi et al., 2011). Cryoprotective agents increase membrane permeability to water,
4
and thereby facilitate cellular dehydration during freezing even at low subzero temperatures (Devireddy et al., 2002; Oldenhof et al., 2010; Akhoondi et al., 2011; Xu et al., 2014). Moreover, cryoprotective agents typically shift the optimal cooling rate to lower temperatures and broaden the range of cooling rates resulting in optimal survival.
DIFFERENT TYPES OF CRYOPROTECTIVE AGENTS
Cryopreservation requires protection of intracellular structures and biomolecules, and hence requires protective agents that are able to pass the cellular membrane. Permeating cryoprotective agents are generally small non-ionic molecules. The most commonly used membrane permeable cryoprotective agents are dimethyl sulfoxide (DMSO) and glycerol. Alternatively, in cases where the above mentioned agents are toxic to the cells, ethylene glycol, methyl-formamide, or dimethyl-formamide may be used (Squires et al., 2004). Permeating cryoprotective agents are osmotically inactive, because they are equally distributed in the intracellular and extracellular spaces. However, addition itself poses osmotic stress to cells because water moves faster across the cellular membrane compared to the permeating cryoprotective agents. This results in an initial osmotic unbalance and shrinkage of the cells followed by an influx of water and cryoprotective agents until an equal distribution of cryoprotective agent inside and outside the cell is reached. Cryopreservation solutions may also include non-permeable cryoprotective agents. These can be divided into osmotically active molecules such as disaccharides (sucrose, trehalose), and osmotically inactive compounds including polysaccharides (hydroxyl ethylene starch, maltodextrin), and proteins (albumin, polyvinylpyrrolidon). Compounds such as sucrose are believed not to pass cellular membranes, and will cause cellular dehydration since they increase the osmolality of the cryopreservation medium. Addition of large
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macromolecules contributes little to the medium osmolality, and this does not cause cellular dehydration (Oldenhof et al., 2013b). Protective effects of various proteins that are synthesized in response to stress have been investigated. Examples include antifreeze proteins, heat shock proteins (HSP), and socalled late embryo abundant (LEA) proteins (Park et al., 2013; Popova et al., 2011). Mixed results have been found in terms of the effects of these compounds on cryosurvival. Stress proteins are generally not supplemented in cryopreservation solutions for cells. In addition to cryoprotective compounds as described above, properties of cellular membranes can be modified by supplementing or extracting lipids and/or cholesterol. This can be achieved using cholesterol-loaded cyclodextrins or liposomes, which change the cholesterol/phospholipid ratio or the phospholipid composition thereby increasing osmotic tolerance limits, susceptibility to lipid peroxidation and cryostability (Glazar et al., 2009). It has been shown that the use a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing extender of equine sperm (Blommaert et al 2015).
MODELS EXPLAINING ACTION OF CRYOPROTECTIVE AGENTS
Permeating cryoprotective agents have been attributed a variety of different cryoprotective properties. First of all, they decrease the ice nucleation temperature, and ice crystal size. The ‘preferential exclusion theory’ explains the stabilizing effects of small cosolutes on biomolecules by preferential interaction of biomolecules with water rather than with the added co-solute (Arakawa and Timasheff, 1985). This means that cryoprotective agents such as glycerol and sucrose are being excluded from the surface of biomolecules thereby stabilizing the native conformation. This theory is developed to explain the stabilizing effects of compatible solutes on proteins at suprazero temperatures (Arakawa and
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Timasheff, 1985; Arakawa et al., 1990; Crowe et al., 1992), but it is assumed that this mechanism also explains stabilizing effects during freezing. In different temperature regimes cryoprotective agents may have different functions/modes of action. At low subzero temperatures, they modulate the rate of cellular dehydration upon ice formation and facilitate dehydration (Oldenhof et al., 2013a). Upon further decreasing the temperature, cryoprotective agents enter into a so-called glassy (vitrified) state. Molecular reactions are slowed in the highly viscous glassy state, which stabilizes cells during long-term storage. The glassy state functions as a matrix in which biomolecular and cellular structures are embedded and preserved (Saragusty et al., 2009). The glass transition temperature of sugars and polysaccharides is relatively high as compared to glycerol (Slade and Levine, 1991). Their use in freezing formulations increases the glass transition temperature, which allows storage at higher subzero temperatures (Stoll et al., 2012; Oldenhof et al., 2013b). The water replacement hypothesis postulates that protectants can replace water and interact with phospholipids and other biomolecules through hydrogen bonding (Crowe et al., 1992), whereas the water entrapment hypothesis suggests that protectants trap water around biomolecules, therewith preventing dehydration-induced conformational changes (Belton and Gil, 1994). Both these hypotheses have been proposed for dry preservation of biomolecules and cells. We have recently shown that water surrounding membrane phospholipids is being removed during freezing-induced dehydration, and that this is not prevented by cryoprotective agents, suggesting that neither replacement nor entrapment play a role in protection of membranes during freezing (Oldenhof et al., 2010; Akhoondi et al., 2011; Oldenhof et al., 2013b).
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CRYOPRESERVATION OF SPERM
Sperm cryopreservation facilitates transport and storage, for use in artificial reproduction technologies. Cryopreserved sperm, however, displays a high degree of variation in survival after thawing among species and individuals. Such differences could be genetic in origin, causing differences in the inherent cryostability. Also the presence of damaging compounds in an ejaculate may decrease sperm cryostability. Centrifugation processing and clean-up approaches can be employed to remove cell debris and damaging compounds in an ejaculate and to enrich samples with good quality sperm (Sieme and Oldenhof, 2015b). Freezing extenders for sperm contain nutrients (salts, sugars, proteins and lipids), and typically contain glycerol as the main permeating cryoprotective agent. However, other permeating cryoprotective agents such as ethylene glycol and (di)methyl-formamide, or combinations of various permeating agents may yield similar or even higher cryosurvival rates (Squires et al., 2004, Álvarez et al., 2014, Wu et al., 2015). In addition to ultra-heat-treated milk, egg yolk is frequently added as membrane modifying agent in sperm freezing extenders (Sieme, 2011, Varisli et al., 2013). Because of its undefined composition and animal origin, a lot of efforts have been made to find alternatives for egg yolk. It has been shown that liposomes, particularly composed of unsaturated lipids, can be used instead of egg yolk, resulting in similar cryosurvival rates (Röpke et al., 2011, Pillet et al., 2012). Also cyclodextrin-cholesterol complexes can be used to replace egg yolk in sperm freezing extenders (Blommaert et al 2015, Moraes et al., 2015), Nowadays, sperm is generally cryopreserved in 0.25- or 0.5-mL plastic straws, which have a large surface area to volume ratio. Conventional (equiaxial) freezing of semen packaged in straws is typically done using different cooling velocities in different
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temperature regimes. Typically, sperm diluted in freezing extender is slowly cooled from room temperature to 5°C at a rate of about 0.1°C min−1, followed by freezing at a rate of 10– 60 °C min−1 down to temperatures as low as −80 °C, after which samples are plunged into liquid nitrogen for storage (Sieme and Oldenhof, 2015a). Most cryoprotectants show some degree of toxicity to sperm. This toxicity can be minimized by taking several measures. The cryoprotectant concentration should be reduced as much as possible (Hoffmann et al., 2011). The temperature at which cryoprotective agents are added is also critical. It has been reported that cryoprotectants have to be added at room temperature (Vidament et al., 2000), whereas others suggest adding pre-cooled cryoprotectants at low temperatures, which is referred to as the so called cold cryoprotectant procedure (Clarke et al., 2003). It is beneficial to add cryoprotectants in a stepwise manner to semen to reduce osmotic stress (Sieme and Oldenhof, 2015a). Measures after thawing include stepwise dilution of the sample to slowly return to isotonic conditions. The protocol how to use and add cryoprotectants is a compromise between their beneficial and potentially detrimental effects. In all likelihood, the composition of the freezing extender may need to be custom-designed and adapted for individual donors in order to assure maximal cryosurvival.
ACKNOWLEDGMENTS The work in our laboratories is supported by the ‘Mehl-Mülhens Stiftung’, as well as the German Research Foundation (DFG: Deutsche Forschungsgemeinschaft) via the Cluster of Excellence ‘From regenerative biology to reconstructive therapy’ (REBIRTH) and grants WO1735/6-1 and SI1462/4-1.
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REFERENCES
Akhoondi, M., Oldenhof, H., Stoll, C., Sieme, H., Wolkers, W.F., 2011. Membrane hydraulic permeability changes during cooling of mammalian cells. Biochim. Biophys. Acta 1808, 642–648. Álvarez, C., Gil, L., González, N., Olaciregui, M., Luño, V., 2014. Equine sperm post-thaw evaluation after the addition of different cryoprotectants added to INRA 96 extender. Cryobiology 69, 144–148. Arakawa, T., Bhat, R., Timasheff, S.N., 1990. Why preferential hydration does not always stabilize the native structure of globular proteins. Biochemistry 29, 1924–1931. Arakawa, T., Timasheff, S.N., 1985. The stabilization of proteins by osmolytes. Biophys. J. 47, 411–414. Belton, P.S., Gil, A.H., 1994. Raman IR spectroscopic studies of the interaction of trehalose with hen egg lysozyme. Biopolymers 34, 957–961. Benson, J.D., Woods, E.J., Walters, E.M., Critser, J.K., 2012. The cryobiology of spermatozoa. Theriogenology 78, 1682–1699. Blommaert, D., Franck, T., Donnay, I., Lejeune, J-P., Detilleux, J., Serteyn, D., 2015. Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm, Cryobiology, (2015), http://dx.doi.org/10.1016/j.cryobiol.2015.11.008 Clarke, G.N., Liu, D.Y., Baker, H.W.G., 2003. Improved sperm cryopreservation using cold cryoprotectant. Reprod. Fert. Dev. 15, 377–381. Crowe, J.H., Hoekstra, F.A., Crowe, L.M., 1992. Anhydrobiosis, Annual Review of Physiology 54, 570–599.
10
Davidson, A.F., Benson, J.D., Higgins, A.Z., 2014. Mathematically optimized cryoprotectant equilibration procedures for cryopreservation of human oocytes. Theor. Biol. Med. Model. 11, 13. Devireddy, R.V., Swanlund, D.J., Olin, T., Vincente, W., Troedsson, M.H.T., Bischof, J.C., Roberts, K.P., 2002. Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry. Biol. Reprod. 66, 222–231. Elmoazzen, H.Y., Elliott, J.A., McGann, L.E., 2009. Osmotic transport across cell membranes in nondilute solutions: a new nondilute solute transport equation. Biophys. J. 96, 2559-2571. Glazar, A.I., Mullen, S.F., Liu, J., Benson, J.D., Critser, J.K., Squires, E.L., Graham, J.K., 2009. Osmotic tolerance limits and membrane permeability characteristics of stallion spermatozoa treated with cholesterol. Cryobiology 59, 201–206. Hoffmann, N., Oldenhof, H., Morandini, C., Rohn, K., Sieme, H., (2011). Optimal concentrations of cryoprotective agents for semen from stallions that are classified ‘good’ or ‘poor’ for freezing. Anim. Reprod. Sci. 125, 112–118. Kashuba, C.M., Benson, J.D., Critser, J.K., 2014. Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I--Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols. Cryobiology 68, 166–175. Lovelock, J.E., Bishop, M.W.H., 1959 Prevention of Freezing Damage to Living Cells by Dimethyl Sulphoxide. Nature 183, 1394-1395. Mazur, P., 1963. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. Gen. Physiol. 47, 347–369.
11
Mazur, P., 2004. Principles of cryobiology. in Fuller, B.J., Lane, N.L., Benson, E.E. (Eds.), Life in the frozen state. CRC Press LLC, Boca Raton, FL, pp 3–65. Meryman, H.T., 2007. Cryopreservation of living cells: principles and practice. Transfusion. 47, 935–945. Moraes, E.A., Matos, W.C., Graham, J.K., Ferrari, W.D., 2015. Cholestanol-loadedcyclodextrin improves the quality of stallion spermatozoa after cryopreservation. Anim. Reprod. Sci. 158, 19–24. Oldenhof, H., Friedel, K., Sieme, H., Glasmacher, B., Wolkers, W.F., 2010. Membrane permeability parameters for freezing of stallion sperm as determined by Fourier transform infrared spectroscopy. Cryobiology 61, 115–122. Oldenhof, H., Akhoondi, M., Sieme, H., Wolkers, W.F., 2013a. Use of Fourier transform infrared spectroscopy to determine optimal cooling rates for cryopreservation of cells. Biomedical Spectroscopy and Imaging 2. 83–90. Oldenhof, H., Gojowsky, M., Wang, S., Henke, S., Yu, C., Rohn, K., Wolkers, W.F., Sieme, H., 2013b. Osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents. Biol. Reprod. 88, 68. Park, S.J., Choi, H.R., Nam, K.M., Na, J.I., Huh, C.H., Park, K.C., 2013. Immediate induction of heat shock proteins is not protective against cryopreservation in normal human fibroblasts. Cryo Letters 34, 239-247. Pegg, D.E., 2015. Principles of cryopreservation. in: Wolkers, W.F., Oldenhof, H. (Eds.), Cryopreservation and freeze-drying protocols, third edition, Methods in molecular biology 1257, Springer, New York, pp. 3–19. Pillet, E., Labbe, C., Batellier, F., Duchamp, G., Beaumal, V., Anton, M., Desherces, S., Schmitt, E., Magistrini, M., 2012. Liposomes as an alternative to egg yolk in stallion freezing extender. Theriogenology 77, 268–279.
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Polge, C., Smith, A.U., Parkes, A.S., 1949. Revival of spermatozoa after vitrification and dehydration at law temperatures. Nature 164, 666–676. Popova, A.V., Hundertmark, M., Seckler, R., Hincha, D.K. 2011. Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes. Biochim. Biophys. Acta 1808, 1879-1887. Röpke, T., Oldenhof, H., Leiding, C., Sieme, H., Bollwein, H., Wolkers, W.F., 2011. Liposomes for cryopreservation of bovine sperm. Theriogenology 76, 1465–1472. Saragusty, J., Gacitua, H., Rozenboim, I., Arav, A., 2009. Do physical forces contribute to cryodamage? Biotechnol. Bioeng. 104, 719–728. Sieme, H., 2011. Semen extenders for frozen semen. in: McKinnon, A.O., Squires, E.L., Vaala, W.E., Varner, D.D. (Eds.), Equine Reproduction, second edition, Blackwell Publishing Ltd., Chichester, UK, pp. 2964–2971. Sieme, H., Oldenhof, H., 2015a. Cryopreservation of domestic livestock semen. in: Wolkers, W.F., Oldenhof, H. (Eds.), Cryopreservation and freeze-drying protocols, third edition, Methods in molecular biology 1257, Springer, New York, pp. 277–287. Sieme, H., Oldenhof, H., 2015b. Sperm cleanup and centrifugation processing for cryopreservation. in: Wolkers, W.F., Oldenhof, H. (Eds.), Cryopreservation and freeze-drying protocols, third edition, Methods in molecular biology 1257, Springer, New York, pp. 343–352. Slade, L., Levine, H., 1991. Beyond water activity: recent advances based on an alternative approach to the assessment of food quality and safety. Crit. Rev. Food Sci. Nutr. 30, 115–136. Squires, E.L., Keith, S.L., Graham, J.K., 2004. Evaluation of alternative cryoprotectants for preserving stallion spermatozoa. Theriogenology 62, 1056–1065.
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Stoll, C., Holovati, J.L., Acker, J.P., Wolkers, W.F., 2012. Synergistic effects of liposomes, trehalose, and hydroxyethyl starch for cryopreservation of human erythrocytes. Biotechnol. Prog. 28, 364–171. Varisli, O., Scott, H., Agca, C., Agca, Y., 2013. The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm. Cryobiology 67, 109–116. Vidament, M., Ecot, P., Noue, P., Bourgeois, C., Magistrini, M., Palmer, E., 2000. Centrifugation and addition of glycerol at 22C instead of 4C improve post-thaw motility and fertility of stallion spermatozoa. Theriogenology 54, 907–919. Woods, E.J., Benson, J.D., Agca, Y., Critser, J.K., 2004. Fundamental cryobiology of reproductive cells and tissues. Cryobiology 48, 146–156. Wu, Z., Zheng, X., Luo, Y., Huo, F., Dong, H., Zhang, G., Yu, W., Tian, F., He, L., Chena, J., Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants. Anim. Reprod. Sci. 163, 75–81. Xu, Y., Zhao, G., Zhou, X., Ding, W., Shu, Z., Gao, D., 2014. Biotransport and intracellular ice formation phenomena in freezing human embryonic kidney cells (HEK293T). Cryobiology 68, 294–302. Yeste M., 2016. Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology. 85, 47–64. Yeste, M., Estrada, E., Rocha, L.G., Marın, H., Rodrıguez-Gil, J.E., Miro, J., 2015. Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus. Andrology 3, 395– 407.
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