Abstract

Cryopreservation has facilitated advancement of biological research by allowing the storage of cells over prolonged periods of time. While cryopreservation at extremely low temperatures would render cells metabolically inactive, cells suffer insults during the freezing and thawing process. Among such insults, the generation of supra-physiological levels of reactive oxygen species (ROS) could impair cellular functions and survival. Antioxidants are potential additives that were reported to partially or completely reverse freeze-thaw stress-associated impairments. This review aims to discuss the potential sources of cryopreservation-induced ROS and the effectiveness of antioxidant administration when used individually or in combination.

Introduction

The ability to keep an organism alive while frozen and allowing it to survive for a prolonged period of time may sound like a scene lifted directly from a science fiction movie. Although freezing complex multicellular organisms remains challenging and often faced significant obstacles during the revival of the frozen organism [1–4], reviving single-cell organisms after a prolonged period of time has been a reality for several decades. Among many cases, the ability to revive single cell prokaryotic organisms such as Escherichia coli and Treponema pallidum were demonstrated in 1913 and 1954 respectively [5,6]. Such results were also obtained from the unicellular eukaryotic organism Saccharomyces cerevisiae in 1902 [7].

In the field of research involving mammalian cells, significant progress was made when Polge et al. [8] successfully revived frozen fowl spermatozoa in 1949 and Bos taurus spermatozoa cells in 1952 using glycerol as a cryoprotective agent (CPA) [9]. The subsequent use of dimethyl sulfoxide (DMSO) as CPA, which remarkably preserved erythrocytes, was first reported in the 1950s [10] and is now a common component of cryopreservation medium. Although the ability to allow cells to be transported across the world has fostered trans-global scientific collaborations as well as independent verifications of experimental results and clinical advancements, it is frequently taken for granted. One could only imagine the hindrance to scientific advancements if the cryopreservation techniques were absent. For the past few decades, although significant advancements were made in the cryopreservation field on mammalian cells, the technique is far from perfect. Many researchers face challenges such as poor recovery [11,12], loss of functional characteristics of specific cell types [4,13,14] and, in the case of stem cell research, the inability to retain pluripotency [15,16]. In this review, we focus on the role of reactive oxygen species (ROS), a product of cellular metabolism that can be damaging to cells and how ROS contributes to the undesirable results seen after cryopreservation. We further explore current advancements in using antioxidants to negate these undesirable effects observed in cryopreservation.

Cryopreservation and ROS production

Cells have mechanisms to detoxify ROS and once these mechanisms are overwhelmed, ROS can affect various cellular functions and processes by oxidizing proteins, inducing damage to nucleic acids, and peroxidation of lipids [17,18]. Oxidative stress, which is the shift of redox homeostasis toward favoring formation of ROS, dictate the subsequent cellular outcomes such as cellular senescence, apoptosis and altered cellular signaling. Generally, it is known that ROS can modulate cellular survival at low concentrations and death at supraphysiological levels [19]. It is also worth noting that physiological amount of ROS can act as signaling molecules for cellular signaling events [20]. To appreciate the impact of ROS in cryopreservation, it is important to understand the different characteristics of ROS produced in cells, the intracellular sources of these ROS and how cells detoxify these damaging species. Detailed reviews on ROS can be found in published review articles [17,18,21–22] and will not be covered in detail in our current review. A short summary of the sources of ROS and enzymes involved in ROS detexofication is provided in Figure 1 and the section below. Sustained oxidative stress has been believed to be linked to senescence – a response to cellular stress [23], with many lines of evidence supporting this [23–26]. The specific effects of the individual reactive species depend on the relative levels within the cell. The effect of these species at different levels and the biological consequences are summarized in Figure 2.

Metabolism and sources of ROS

Figure 1
Metabolism and sources of ROS

(A) Detoxification and metabolism of reactive oxygen/nitrogen species. (B) Sources of ROS, and localization of enzymes that counteracts ROS in the mitochondria, endoplasmic reticulum (ER), peroxisome, cytosol and the extracellular space. SOD1 is localized in both the mitochondria intermembrane space and cytosol, SOD3 is located extracellularly and SOD2 is found exclusively mostly in the mitochondria matrix. Catalase that reduces hydrogen peroxide (H2O2) into H2O is mostly located in the peroxisomes. Glutathione peroxidase (GPx) is found in the mitochondria and cytosol. Peroxiredoxins (Prx) and thioredoxins (Trx) which constitute the Peroxiredoxin–Thioredoxin (Prx/Trx) system can be found in the nucleus, mitochondria, ER, peroxisome and the extracellular environment. Electron transport chain (ETC), Cytochrome P450 family of enzymes (Cyps), xanthene oxidase (XO) and NADPH oxidases (NOX) are potential sources of O2•−, while ERO1 and acetyl CoA oxidases (AcoX) produce H2O2. Nitric oxide synthase (NOS) is a potential source of NO. Aquaporins (Aqp) facilitate the movement of H2O2 across membranes. Single snowflake indicates ROS detected while two snowflakes indicate an implication with cryopreservation.

Cu2+/Fe3+ (); Cu1+/Fe2+ (); Source of ROS (); Enzyme (); O2•− (); O2 (); H2O (); H2O2 (); •OH (); ONOO (); NO (); NO2 (); H+ (); Detected during cryopreservation (); Implicated during cryopreservation ().

Figure 1
Metabolism and sources of ROS

(A) Detoxification and metabolism of reactive oxygen/nitrogen species. (B) Sources of ROS, and localization of enzymes that counteracts ROS in the mitochondria, endoplasmic reticulum (ER), peroxisome, cytosol and the extracellular space. SOD1 is localized in both the mitochondria intermembrane space and cytosol, SOD3 is located extracellularly and SOD2 is found exclusively mostly in the mitochondria matrix. Catalase that reduces hydrogen peroxide (H2O2) into H2O is mostly located in the peroxisomes. Glutathione peroxidase (GPx) is found in the mitochondria and cytosol. Peroxiredoxins (Prx) and thioredoxins (Trx) which constitute the Peroxiredoxin–Thioredoxin (Prx/Trx) system can be found in the nucleus, mitochondria, ER, peroxisome and the extracellular environment. Electron transport chain (ETC), Cytochrome P450 family of enzymes (Cyps), xanthene oxidase (XO) and NADPH oxidases (NOX) are potential sources of O2•−, while ERO1 and acetyl CoA oxidases (AcoX) produce H2O2. Nitric oxide synthase (NOS) is a potential source of NO. Aquaporins (Aqp) facilitate the movement of H2O2 across membranes. Single snowflake indicates ROS detected while two snowflakes indicate an implication with cryopreservation.

Cu2+/Fe3+ (); Cu1+/Fe2+ (); Source of ROS (); Enzyme (); O2•− (); O2 (); H2O (); H2O2 (); •OH (); ONOO (); NO (); NO2 (); H+ (); Detected during cryopreservation (); Implicated during cryopreservation ().

Effects of different levels of reactive oxygen/nitrogen species on cellular biomolecules

Figure 2
Effects of different levels of reactive oxygen/nitrogen species on cellular biomolecules

Protein can react with ONOO, H2O2, NO, OH and aldehydes such as 4-Hydroxynonenal (4-HNE) can react with protein side chains (e.g. amino acids such as lysine). The formation of oxo-histidine and disulfide bonds are mostly reversible and mediate redox signaling under mild oxidative stress and may not be deleterious. High level of ROS lead to protein aggregation, denaturation and fragmentation. Mitochondrial/nuclear DNA can react with O2•−, ONOO and OH. Mutations and double/single-strand breaks mediated by ROS are minimized by the DNA-Damage Response (DDR). Proteins such as p53, RAD51 and yH2AX are DDR constituents involved in cryopreservation. Severe oxidative stress can overwhelm the DDR, resulting in mutations and double/single strand breaks. Lipids can react with ONOO and OH to cause lipid peroxidation and form lipid peroxides (LPOs). LPOs can decompose into aldehydes (Ald) such as 4-HNE and malondialdehyde (MDA). At low levels of ROS, cells are quiescent. Moderate levels of ROS facilitates beneficial redox signaling to modulate cellular survival, growth and division. Overwhelming levels of ROS can initiate cell death.

Figure 2
Effects of different levels of reactive oxygen/nitrogen species on cellular biomolecules

Protein can react with ONOO, H2O2, NO, OH and aldehydes such as 4-Hydroxynonenal (4-HNE) can react with protein side chains (e.g. amino acids such as lysine). The formation of oxo-histidine and disulfide bonds are mostly reversible and mediate redox signaling under mild oxidative stress and may not be deleterious. High level of ROS lead to protein aggregation, denaturation and fragmentation. Mitochondrial/nuclear DNA can react with O2•−, ONOO and OH. Mutations and double/single-strand breaks mediated by ROS are minimized by the DNA-Damage Response (DDR). Proteins such as p53, RAD51 and yH2AX are DDR constituents involved in cryopreservation. Severe oxidative stress can overwhelm the DDR, resulting in mutations and double/single strand breaks. Lipids can react with ONOO and OH to cause lipid peroxidation and form lipid peroxides (LPOs). LPOs can decompose into aldehydes (Ald) such as 4-HNE and malondialdehyde (MDA). At low levels of ROS, cells are quiescent. Moderate levels of ROS facilitates beneficial redox signaling to modulate cellular survival, growth and division. Overwhelming levels of ROS can initiate cell death.

ROS production has been detected in reproductive and non-reproductive cells. ROS in the form of superoxide (O2•−) which were detected in the cells of various species undergoing cryopreservation can be reduced with the addition of various antioxidants (Tables 1 and 2). O2•− is short-lived and does not cross the mitochondrial or lipid membranes readily due to its charge [27,28]. O2•− cannot react with most biological molecules in the aqueous environment of the cytoplasm [18]. O2•−can be converted into hydrogen peroxide (H2O2) by three known superoxide dismutase (SOD) isoforms; cytosolic-localized SOD1 (Cu, Zn SOD), mitochondrial-localized SOD2 (Mn SOD) and the extracellular SOD3 (Fe SOD). The localization of the SOD isoforms are reviewed in [29]. Significantly elevated levels of O2•− and lipid peroxidation were observed in reviving cryopreserved bull spermatozoa [30]. In alpaca sperm, higher levels of O2•− were detected as compared with other oxidizing intermediates using fluorescent dyes dihydroethidium (DHE) for O2•− and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) for ROS, which were mainly contributed by cells that are propidium iodide negative [31]. This is also seen in human retinal pigment epithelial (hRPE) cells, where apart from an increase in ROS as detected by H2DCFDA only, cellular senescence as well as telomere shortening were reported to increase as a result of cryopreservation [32]. Notably, studies have shown that DMSO treatment of human embryonic stem cells (hES) increases O2•− by two-folds while the same cells that were thawed after cryopreservation in the presence of DMSO lead to a five-fold increase in the O2•−. These data indicate that freeze-thaw stress can promote ROS generation [33]. Other known ice nucleation inhibitors such as anti-freezing protein (AFP) and polyethylene glycol (PEG) have also been known to protect against freeze thaw-induced ROS generation [34–36]. O2•− participates in Fenton and Haber–Weiss (HW) reaction in the presence of a ferrous iron catalyst to generate hydroxyl radical (OH) (Figure 1). OH, unlike O2•−, can function in the aqueous state and is particularly reactive. OH is considered the most damaging member of ROS [37] and was reported to cause oxidation of amino acids [38], and these can result in the fragmentation and disruption of protein conformation [39]. OH can abstract hydrogen and lead to altered nucleic acid bases resulting in DNA damage [40]. Intriguingly, there are no known enzymes to detoxify OH, despite the damage that it can cause to the cell.

Table 1
Antioxidants and their effects on cryopreserved reproductive-associated cells/tissues
CompoundCell typeBeneficial effectsNo effect/adverse effectsCryopreservation method
2,4-dinitrophenol (DNP) Sperm • Motility (↑) [76] • Motility(N/C) [76] 1 cm styrofoam boat on LN at 10 min [76
Ascorbic acid Sperm • ROSa (↓) [149]
• Viability (↑) [149]
• Motility (Weak ↑) [149]
• MMP (↑) [149]
• Apoptotic cells (↓) [149]
• DNA damage (↓) [148,150] [149
• Motility(N/C) [148]
• Viability (N/C) [148]
• DNA fragmentation (N/C or ↑) [148] [150] 
LN vapor phase (6.5-2 cm) at 10–15 min [148]
LN vapor phase (10 cm) at 10 min [149],
−20°C at 10 min + LN vapor phase at 2 h [150
 Mouse embryos • Percentage of intact embryos, blastocyst and number of hatching blastocyst (↑) [183]
• Number of implantation sites (↑) [183
• Fetal development (N/C) [183Vitrification and slow freezing [183
Antifreeze proteins (AFP) Oocytes • ROSa (↓) [35]
• γH2AX+ cells (↓) [35]
• Viability (↑) [35]
• Cleavage rate, blastocyst rate, blastomere count (↑) [35]
• Apoptotic blastomeres (↓) [35]
• Improved chromosomal alignment and spindle organization [35
• Mitochondrial activity (N/C) [35]
• Cells with DNA repair (N/C) [35
Vitrification [35
BHT Sperm • % Motility and viability (↑) [203]
• MDA levelst (↓) [203
• % Sperm with functional intact membrane and GPx activity (N/C) [203LN vapor phase (4 cm) at 15 min [203
Catalase Sperm • ROSa (↓) [149]
• Viability (↑), weak (↑) motility, MMP [149]
• (↓) Apoptotic cells [149]
• (↓) Apoptotic like changes, apoptotic and necrotic cells [244]
• (↓) DNA damage [149]
• Motility (↑) [76]
• Total and progressive motility, viability, % sperm with high MMP(↑) [244
• % cells with intact membrane, H2O2 levelsn, motility, NO levelsp, free iron concentration and functional membrane (N/C) [125]
• Motility (N/C) [76] 
LN vapor phase (10 cm) at 10 min [149]
LN vapor phase at 20 min [125]
1 cm styrofoam boat on liquid nitrogen at 10 min [76
 Oocytes • N.A [124• Oocyte survival and fertility (N/C) [124Controlled rate freezing [124
Coenzyme Q Sperm • (↑) viability, % sperm with functional membrane and active mitochondria [154]
• Weak (↓) number of abnormal cells [154]
• Lipid peroxidation [154]t [218]f (PI staining was done) and DNA fragmentation (↓) [154]
• (↑) total and progressive motility, plasma membrane integrity [218] 
• Slight (↑) or (N/C) ROS k (PI staining was done) [218]∧
• O2•− j (stained in association with Yo-Pro® and MitoStatusRed) (N/C) [218]
• Motility parameters, plasma membrane integrity, MMP and non-capacitated sperm (N/C) [218] 
LN vapor phase (5 cm) at 12 min [154]
LN vapor phase (6 cm) at 20 min [218
Egg yolk Sperm • N.A [116• (↓) NO•d [116LN vapor phase (4 cm) at 10 min [116
Glutathione (GSH) Sperm • Fertilization rate and % cells with ability to undergo acrosome reaction (↑) [41]
• Lipid peroxidationg, mitochondrial ROSq, total ROSs and intracellular ROSr(↓) [41]
• Motility recovery rate (↑) [153]
• % sperm with high MMP, viability, total and Progressive motility(↑) [244]
• Apoptotic like changes, apoptosis and necrosis (↓) [244]
• Sperm DNA damage (↓) [153
• Motility-associated parameters (↓) or (N/C) [41]
• DNA fragmentation (N/C) [244
LN vapor phase (N/I) [41]
LN vapor phase at 10 min [153
 Germ cells enriched with spermatogonial stem cells • N.A [178• ATP (N/C) [178]
• Proliferation (N/C) [178
Slow freeze [178
Hemoglobin (Hb) Oocyte • Survival and fertility (↑) [124N.A [124Controlled freezing [124
Hypotaurine/Taurine Germ cells enriched with spermatogonial stem cells • Proliferation rate and mitochondrial activity (↑) [178• Recovery of cells (N/C) [178Slow-freeze [178
 Sperm • DNA fragmentation (↓) [148• Motility and viability [N/C) [148LN vapor phase (6.5-2 cm) at 10–15 min [148
Iodixanol Sperm • (↑) motility [200,168], protamine, BCL2, protamine2/3 and SPACA3 expression [200]
• (↓) BAX and ROMO1 expression and cellular death [200]
• (↓) MDAt [168]
• (↑) total antioxidant capacity, membrane integrity [168
• Acrosomal integrity(N/C) [168Controlled freezing [168]
LN vapor phase (2 cm) at 15 min [200
L-carnitine Sperm • (↑) viability and motility [147• DNA oxidatione (N/C) [147−20°C at 8 min + LN vapor phase at 2 h [147
L-proline Oocyte • Survival rate (%) (↑) [217]
• MMP (↑) [217]
• (↓) ROS levelsa [217
• Developmental parameters. apoptosis levels, spindle recovery (N/C) [217Vitrification [217
Lactoferrin/apotransferrin Mouse embryos • (↑) percentage of intact embryos and blastocysts [183• Hatching blastocyst (N/C) [183Vitrification or slow freeze [183
 Sperm • (↓) Fe3+, NO2− p (Griess reagent system) [125]
• (↑) percentage of cells with functional plasma membrane [125
• H2O2n, membrane intactness, motility (N/C) [125LN vapor phase at 20 min [125
Melatonin Sperm • (↑) total antioxidant capacity, GSH concentration, functional plasma membrane cells, mitochondrial membrane integrity [199]
• (↑) acrosomal integrity [199,240,241]
• (↑) MMP [199,240]
• (↑) SOD, catalase and GPx activity [199,240,241]
• (↑) BCL-2, SOD2, GSTM1, NRF2, HSP90AA1, catalase and HO-1 gene expression [199]
• (↓) Lipid peroxidation [199]u [240]f [241]u and ROS [199]b levels [240]a [241] u
• (↓) NADPH oxidase activity [199]
• (↓) Nox5 and Bax expression [199]
• (↑) Viability [199,156]
• (↑) motility [199,156,240,241]
• (↑) ATP [156]
• (↓) cleaved caspase 3 and 9 [240]
• (↓) DNA damage [156]
• (↑) 26hpf cleavage rate [156]
• (↑) Bcl2l1 (Bcl-xL) expression and motility [198
• DNA fragmentation and LDH activity (N/C) [199]
• Total blastocyst output (N/C) [156]
• Viability, Bax expression and ROSb (N/C) [198
LN vapor phase (10 cm) at 1 h [199]
Frozen in pellet-form on dry ice
LN vapor phase at 10 min [198]
(N/I) Stored in liquid nitrogen [240]
Pellet freezing in LN [241
 Oocytes (↓) ROS levelsu, DNA fragmentation and apoptotic gene expression [243]
• (↑) expression of telomere maintenance genes [243]
• (↑) embryonic stem cell derivation and implantation rate [243
N.A [243Vitrification [243
MitoTEMPO Sperm • (↑) motility, membrane integrity, sperm vitality, MMP, SOD activity, catalase activity, GPx activity, GPI protein levels [214]
• (↓) MDA levelst [214
• Reversal of some beneficial effects (at 500 μM) [214LN vapor phase (1–5 cm) at 30 min [214
Monothioglycerol (MTG) Sperm • Mitochondrial ROSq and total ROSs (↓) [41]
• Fertility and % cells with ability to undergo acrosome reaction (↑) [41]
• Lipid peroxidationg (↓) [41]
• (↑) motility recovery rate [153]
• (↓) sperm DNA damage [153
• Motility parameters (N/C) [41LN vapor phase (N/I) [41]
LN vapor phase at 10 min [153
NG-nitro-L-arginine methyl ester (l-NAME) Oocyte • (↑) fertility and survival (low concentration) [124• (↓) Fertility and survival (high concentrations) [124Controlled freezing [124
Quercetin Sperm • DNA fragmentation (↓) [153]
• Motility and recovery rate (↑) [153]
• (↓) % high MMP cells [216
• Progressive motility, acrosome and sperm plasma membrane integrity (N/C) [216]
• (↓) sperm motility recovery rate (at 100 μM) [153
Controlled freezing [216]
LN vapor phase at 10 min [153
Resveratrol Sperm • DNA damage [150], MDAt levels [219] and % high MMP cells (↓) [216]
• SOD activity (↑) [219] 
• Progressive motility, acrosome integrity, integrity of sperm plasma membrane (N/C) [216]
• Motility (↓) [219]
• SOD and catalase activity (N/C) [219] 
Slow cool (−20°C) at 10 min followed by LN vapor phase at 2 h [150]
Controlled freezing [216]
Slow cool (−20°C) at 10 min followed by LN vapor phase(N/I) at 2 h [219
SOD Oocyte • (↑) fertility and survival [124• Decrease in fertility [124] (low concentration) Controlled rate freezing [124
 Sperm • (↑) motility [76]
• (↓) Reduced apoptotic like changes [244
• Motility (N/C) [76]∧
• Total and progressive motility, DNA fragmentation, viability, % sperm with high MMP (N/C) [244]
• Increased late apoptotic and necrotic cells [244
1 cm styrofoam boat on LN at 10 min [76
Trehalose Germ cells enriched with spermatogonial stem cells • (↑) proliferation, recovery of colonies after culture and cell viability [207]
• Apoptosis (↓) [207
• Formation of colonies after transplantation (N/C) [207Slow freeze [207
 Testicular tissue • (↑) cell viability, GSH content and T-AOC [204]
• (↓) Lipid peroxidationt [204]
• (↑) SOD and catalase activity [204
• N.A [204−20°C at 2 h, −80°C at 12 h [204
Vitamin E Sperm • (↑) Motility [76]
• (↓) DNA fragmentation [148]
• MDA (↓)t [215
• Viability and motility (N.C) [148]
• Motility (N/C) [215]
• O2•− production in live cellsi (N/C) [215
[Controlled rate freezing] 62.3°C/min [215]
1 cm styrofoam boat on LN at 10 min [76]
LN vapor phase (6.5-2 cm) at 10–15 min [148
Zinc oxide nanoparticles Sperm • DNA damage and lipid peroxidationt (↓) [155• Sperm motility and ability to undergo the acrosome reaction (N/C) [155(N/I) Stored at −196°C [155
Zinc sulfate Sperm • DNA damage (↓) [220]
• (↑) Mitochondria integrity, % sperm with ability to undergo acrosome reaction and capacitation [220]
• (↑) Motility (↑) [220] 
• Motility (N/C) [220] LN vapor phase at 5 min [220
Trolox (Vitamin E analog) Ovarian tissue Viable follicles (↑) [81]
BMP4, BMP15, CTGF, GDF9, KL expression (↑) [81]
• Trolox equivalent antioxidant capacity values (↑). [81
HSP70, ERp60, SOD1 and ERp29, AMH expression (N/C) [812°C/min from 20 to −7°C; cooled at 0.3°C/min to −30°C, into LN (−196°C) [81
CompoundCell typeBeneficial effectsNo effect/adverse effectsCryopreservation method
2,4-dinitrophenol (DNP) Sperm • Motility (↑) [76] • Motility(N/C) [76] 1 cm styrofoam boat on LN at 10 min [76
Ascorbic acid Sperm • ROSa (↓) [149]
• Viability (↑) [149]
• Motility (Weak ↑) [149]
• MMP (↑) [149]
• Apoptotic cells (↓) [149]
• DNA damage (↓) [148,150] [149
• Motility(N/C) [148]
• Viability (N/C) [148]
• DNA fragmentation (N/C or ↑) [148] [150] 
LN vapor phase (6.5-2 cm) at 10–15 min [148]
LN vapor phase (10 cm) at 10 min [149],
−20°C at 10 min + LN vapor phase at 2 h [150
 Mouse embryos • Percentage of intact embryos, blastocyst and number of hatching blastocyst (↑) [183]
• Number of implantation sites (↑) [183
• Fetal development (N/C) [183Vitrification and slow freezing [183
Antifreeze proteins (AFP) Oocytes • ROSa (↓) [35]
• γH2AX+ cells (↓) [35]
• Viability (↑) [35]
• Cleavage rate, blastocyst rate, blastomere count (↑) [35]
• Apoptotic blastomeres (↓) [35]
• Improved chromosomal alignment and spindle organization [35
• Mitochondrial activity (N/C) [35]
• Cells with DNA repair (N/C) [35
Vitrification [35
BHT Sperm • % Motility and viability (↑) [203]
• MDA levelst (↓) [203
• % Sperm with functional intact membrane and GPx activity (N/C) [203LN vapor phase (4 cm) at 15 min [203
Catalase Sperm • ROSa (↓) [149]
• Viability (↑), weak (↑) motility, MMP [149]
• (↓) Apoptotic cells [149]
• (↓) Apoptotic like changes, apoptotic and necrotic cells [244]
• (↓) DNA damage [149]
• Motility (↑) [76]
• Total and progressive motility, viability, % sperm with high MMP(↑) [244
• % cells with intact membrane, H2O2 levelsn, motility, NO levelsp, free iron concentration and functional membrane (N/C) [125]
• Motility (N/C) [76] 
LN vapor phase (10 cm) at 10 min [149]
LN vapor phase at 20 min [125]
1 cm styrofoam boat on liquid nitrogen at 10 min [76
 Oocytes • N.A [124• Oocyte survival and fertility (N/C) [124Controlled rate freezing [124
Coenzyme Q Sperm • (↑) viability, % sperm with functional membrane and active mitochondria [154]
• Weak (↓) number of abnormal cells [154]
• Lipid peroxidation [154]t [218]f (PI staining was done) and DNA fragmentation (↓) [154]
• (↑) total and progressive motility, plasma membrane integrity [218] 
• Slight (↑) or (N/C) ROS k (PI staining was done) [218]∧
• O2•− j (stained in association with Yo-Pro® and MitoStatusRed) (N/C) [218]
• Motility parameters, plasma membrane integrity, MMP and non-capacitated sperm (N/C) [218] 
LN vapor phase (5 cm) at 12 min [154]
LN vapor phase (6 cm) at 20 min [218
Egg yolk Sperm • N.A [116• (↓) NO•d [116LN vapor phase (4 cm) at 10 min [116
Glutathione (GSH) Sperm • Fertilization rate and % cells with ability to undergo acrosome reaction (↑) [41]
• Lipid peroxidationg, mitochondrial ROSq, total ROSs and intracellular ROSr(↓) [41]
• Motility recovery rate (↑) [153]
• % sperm with high MMP, viability, total and Progressive motility(↑) [244]
• Apoptotic like changes, apoptosis and necrosis (↓) [244]
• Sperm DNA damage (↓) [153
• Motility-associated parameters (↓) or (N/C) [41]
• DNA fragmentation (N/C) [244
LN vapor phase (N/I) [41]
LN vapor phase at 10 min [153
 Germ cells enriched with spermatogonial stem cells • N.A [178• ATP (N/C) [178]
• Proliferation (N/C) [178
Slow freeze [178
Hemoglobin (Hb) Oocyte • Survival and fertility (↑) [124N.A [124Controlled freezing [124
Hypotaurine/Taurine Germ cells enriched with spermatogonial stem cells • Proliferation rate and mitochondrial activity (↑) [178• Recovery of cells (N/C) [178Slow-freeze [178
 Sperm • DNA fragmentation (↓) [148• Motility and viability [N/C) [148LN vapor phase (6.5-2 cm) at 10–15 min [148
Iodixanol Sperm • (↑) motility [200,168], protamine, BCL2, protamine2/3 and SPACA3 expression [200]
• (↓) BAX and ROMO1 expression and cellular death [200]
• (↓) MDAt [168]
• (↑) total antioxidant capacity, membrane integrity [168
• Acrosomal integrity(N/C) [168Controlled freezing [168]
LN vapor phase (2 cm) at 15 min [200
L-carnitine Sperm • (↑) viability and motility [147• DNA oxidatione (N/C) [147−20°C at 8 min + LN vapor phase at 2 h [147
L-proline Oocyte • Survival rate (%) (↑) [217]
• MMP (↑) [217]
• (↓) ROS levelsa [217
• Developmental parameters. apoptosis levels, spindle recovery (N/C) [217Vitrification [217
Lactoferrin/apotransferrin Mouse embryos • (↑) percentage of intact embryos and blastocysts [183• Hatching blastocyst (N/C) [183Vitrification or slow freeze [183
 Sperm • (↓) Fe3+, NO2− p (Griess reagent system) [125]
• (↑) percentage of cells with functional plasma membrane [125
• H2O2n, membrane intactness, motility (N/C) [125LN vapor phase at 20 min [125
Melatonin Sperm • (↑) total antioxidant capacity, GSH concentration, functional plasma membrane cells, mitochondrial membrane integrity [199]
• (↑) acrosomal integrity [199,240,241]
• (↑) MMP [199,240]
• (↑) SOD, catalase and GPx activity [199,240,241]
• (↑) BCL-2, SOD2, GSTM1, NRF2, HSP90AA1, catalase and HO-1 gene expression [199]
• (↓) Lipid peroxidation [199]u [240]f [241]u and ROS [199]b levels [240]a [241] u
• (↓) NADPH oxidase activity [199]
• (↓) Nox5 and Bax expression [199]
• (↑) Viability [199,156]
• (↑) motility [199,156,240,241]
• (↑) ATP [156]
• (↓) cleaved caspase 3 and 9 [240]
• (↓) DNA damage [156]
• (↑) 26hpf cleavage rate [156]
• (↑) Bcl2l1 (Bcl-xL) expression and motility [198
• DNA fragmentation and LDH activity (N/C) [199]
• Total blastocyst output (N/C) [156]
• Viability, Bax expression and ROSb (N/C) [198
LN vapor phase (10 cm) at 1 h [199]
Frozen in pellet-form on dry ice
LN vapor phase at 10 min [198]
(N/I) Stored in liquid nitrogen [240]
Pellet freezing in LN [241
 Oocytes (↓) ROS levelsu, DNA fragmentation and apoptotic gene expression [243]
• (↑) expression of telomere maintenance genes [243]
• (↑) embryonic stem cell derivation and implantation rate [243
N.A [243Vitrification [243
MitoTEMPO Sperm • (↑) motility, membrane integrity, sperm vitality, MMP, SOD activity, catalase activity, GPx activity, GPI protein levels [214]
• (↓) MDA levelst [214
• Reversal of some beneficial effects (at 500 μM) [214LN vapor phase (1–5 cm) at 30 min [214
Monothioglycerol (MTG) Sperm • Mitochondrial ROSq and total ROSs (↓) [41]
• Fertility and % cells with ability to undergo acrosome reaction (↑) [41]
• Lipid peroxidationg (↓) [41]
• (↑) motility recovery rate [153]
• (↓) sperm DNA damage [153
• Motility parameters (N/C) [41LN vapor phase (N/I) [41]
LN vapor phase at 10 min [153
NG-nitro-L-arginine methyl ester (l-NAME) Oocyte • (↑) fertility and survival (low concentration) [124• (↓) Fertility and survival (high concentrations) [124Controlled freezing [124
Quercetin Sperm • DNA fragmentation (↓) [153]
• Motility and recovery rate (↑) [153]
• (↓) % high MMP cells [216
• Progressive motility, acrosome and sperm plasma membrane integrity (N/C) [216]
• (↓) sperm motility recovery rate (at 100 μM) [153
Controlled freezing [216]
LN vapor phase at 10 min [153
Resveratrol Sperm • DNA damage [150], MDAt levels [219] and % high MMP cells (↓) [216]
• SOD activity (↑) [219] 
• Progressive motility, acrosome integrity, integrity of sperm plasma membrane (N/C) [216]
• Motility (↓) [219]
• SOD and catalase activity (N/C) [219] 
Slow cool (−20°C) at 10 min followed by LN vapor phase at 2 h [150]
Controlled freezing [216]
Slow cool (−20°C) at 10 min followed by LN vapor phase(N/I) at 2 h [219
SOD Oocyte • (↑) fertility and survival [124• Decrease in fertility [124] (low concentration) Controlled rate freezing [124
 Sperm • (↑) motility [76]
• (↓) Reduced apoptotic like changes [244
• Motility (N/C) [76]∧
• Total and progressive motility, DNA fragmentation, viability, % sperm with high MMP (N/C) [244]
• Increased late apoptotic and necrotic cells [244
1 cm styrofoam boat on LN at 10 min [76
Trehalose Germ cells enriched with spermatogonial stem cells • (↑) proliferation, recovery of colonies after culture and cell viability [207]
• Apoptosis (↓) [207
• Formation of colonies after transplantation (N/C) [207Slow freeze [207
 Testicular tissue • (↑) cell viability, GSH content and T-AOC [204]
• (↓) Lipid peroxidationt [204]
• (↑) SOD and catalase activity [204
• N.A [204−20°C at 2 h, −80°C at 12 h [204
Vitamin E Sperm • (↑) Motility [76]
• (↓) DNA fragmentation [148]
• MDA (↓)t [215
• Viability and motility (N.C) [148]
• Motility (N/C) [215]
• O2•− production in live cellsi (N/C) [215
[Controlled rate freezing] 62.3°C/min [215]
1 cm styrofoam boat on LN at 10 min [76]
LN vapor phase (6.5-2 cm) at 10–15 min [148
Zinc oxide nanoparticles Sperm • DNA damage and lipid peroxidationt (↓) [155• Sperm motility and ability to undergo the acrosome reaction (N/C) [155(N/I) Stored at −196°C [155
Zinc sulfate Sperm • DNA damage (↓) [220]
• (↑) Mitochondria integrity, % sperm with ability to undergo acrosome reaction and capacitation [220]
• (↑) Motility (↑) [220] 
• Motility (N/C) [220] LN vapor phase at 5 min [220
Trolox (Vitamin E analog) Ovarian tissue Viable follicles (↑) [81]
BMP4, BMP15, CTGF, GDF9, KL expression (↑) [81]
• Trolox equivalent antioxidant capacity values (↑). [81
HSP70, ERp60, SOD1 and ERp29, AMH expression (N/C) [812°C/min from 20 to −7°C; cooled at 0.3°C/min to −30°C, into LN (−196°C) [81

Abbreviations: BHT, butylated hydroxytoluene; LN, liquid nitrogen; MDA, malondialdehyde; MMP, mitochondrial membrane potential; N.A, not-applicable; N/C, no changes/no effect. -, no effects have been reported. (↑) and (↓), represent a significant increase or decrease respectively.

, denotes cases where effects are context dependent and due to factors such as cell quality and species.

Method employed for detection of ROS and Oxidative biomarkers are denoted by alphabetical superscripts ‘a’ to ‘u’:

a, H2DCFDA.

b, H2DCFDA/Propidium Iodide (Pi).

c, 4,5-diaminofluorescein diacetate (DAF-2DA).

d, DAF-2DA/Ethidium Homodimer -.

e, 8-OHG.

f, BODIPY 581/591 C11.

g, BODIPY 581/591 C11/Propidium Iodide (Pi)-.

h, Bromopyrogallol Red.

i, DHE/Sytox-.

j, DHE.

k, Dihydrorhodamine(DHR) 123/Propidium Iodide (Pi)-.

l, DHR 123.

m, 2,4-dinitrophenylhydrazine (DNPH) assay.

n, Fox2-modified method.

o, Formamidopyrimidine-DNA glycosylase-sensitive comet assay.

p, Griess reagent system.

q, MitoPY1/SYTOX–.

r, PF6-AM/SYTOX.

s, Peroxy Green 1 (PG1).

t, Thiobarbituric acid reactive substances (TBARS) assay.

u, commercial or obscure ROS detection techniques.

Table 2
Antioxidants and their effects on non-reproductive cell types/tissues
CompoundCell typeBeneficial effectsNo effect/adverse effectsCryopreservation method
Ascorbic acid Bone-marrow mononuclear cells • Clonogenic parameters (↑) [221] (murine model) • Viability and clonogenic parameters (human model) (N/C) [221Controlled rate freezing [221
Astragalosides Pancreatic islets • Restored blood glucose to normal [232]
• Insulin expression after transplantation (↑) [232
• N.A [232Slow-freeze [232
BHT Blood cells • Loss of HUFAs (↓) [184• N.A [184Chromatography paper at −20°C [184
BHT + ascorbic acid Hepatocytes • Post-thaw albumin production (↑) [235• Induced LDH release (↑) [235]
• Urea synthesis, ammonia clearance and cell proliferation (N/C) [235]
• Apoptosis associated DNA fragmentation (N/C) [235
(N/I) stored in −70°C freezer [235
Catalase Mononuclear cells • Clonogenic parameters (↑) (murine model) [221• Viability, clonogenic parameters (human model) (N/C) [221Controlled rate freezing [221
Catalase + Trehalose Hematopoietic cells • DCF fluorescence intensitya (↓) [233]
• Number of DCF+ cellsa (↓) [233]
• (↑) CFU [233,234]
• (↑) pre-CFU [233]
• Better engraftment [233]
• (↑) viability [233]
• Apoptosis (↓) [233]
• (↑) responsiveness to migratory homing associated cytokines, expression of homing-associated receptor and adhesion capacity [234
• N.A [233,234Controlled rate freezing [233,234
Consumption of blueberries by PBMC donors Peripheral blood mononuclear cell • DNA oxidation (↓)o [144• DNA damage induced by H2O2 in cryopreserved cells (N/C) [144Slow freeze [144
Deferoxamine Blood cells • Loss of HUFA (↓) [184• N.A [184Chromatography paper at −20°C [184
Glutathione (GSH) Embryonic stem cells • ROSa (↓) [247]
• Viability (↑) [247
• N.A [247(N/I) Stored in −80°C freezer at 24 h [247
 Embryogenic callus • Post-thaw survival, GSH, ascorbic acid levels, SOD and peroxidase activity (↑) [174]
OHh, H2O2u, O2•−u and MDA levelst (↓) [174
• At high concentrations, survival (↓) or (N/C) [174]
•catalase activity (N/C) [174
Vitrification [174
 Pancreatic islets • MDAt (↓) [222]
• Islet morphometry and glucose clearance rate (↑) [222
• Islet insulin secretion (N/C) [222Slow freeze [222
Peroxiredoxin Murine hepatocytes • Viability (↑) [236]
• Integrin-β1 and β-catenin cell adhesion proteins (↑) [236]
• Urea secretion (↑) [236]
• NO•c(↓) [236]
• ROSa _(↓) [236]
• O2•−j(↓) [236
• E-Cadherin cell adhesion proteins (N/C) [236Slow freeze [236
 Murine insulinoma • Viability (↑) [236]
• Insulin secretion (↑) [236]
• NO•c (↓) [236]
• O2•−j (↓) [236
• ROSa (N/C) [236Slow freeze [236
Polyethylene glycol (PEG) Human embryonic stem cells • (↓) ROSj [36]
• Alleviation of F-actin levels [36
• Cell viability (N/C) [36Slow freeze [36
S-Adenosylmethionine Hepatocytes • (↑) GSH content and cellular viability [177• N.A [177Slow freeze [177
Salidroside Red blood cell • (↓) protein carboxylationm [158]
• (↓) Lipid peroxidationt (when trehalose was used as a CPA) [158
• Lipid peroxidationt (N/C) (when used with glycerol as a CPA) [158N.I [158
SOD Bone-marrow mononuclear cells • N.A [221• Post-thaw recovery (N/C) [221Controlled rate freezing [221
Trehalose Dendritic cells • Preserved cell function and phenotype [205]
• (↑) viability [205]
• Maintained MMP and cytoskeleton integrity [205]
• (↓) apoptosis, BIM-1 and CASP9 expression [205
• N.A [205Controlled rate freezing [205
 Hepatocytes • (↑) albumin secretion, plating efficiency and viability [206]
• (↓) AST activity [206
• EROD and ECOD activity, proliferation, LDH, urea levels (N/C) [206Controlled rate freezing [206
 BM-MNC • (↑) Clonogenic parameters (murine and human models [221• N.A [221Slow controlled rate freezing [221
Wheat proteins or Lipocalins Hepatocytes • (↑) attachment efficiency and viability [227]
• Restoration of cytochrome P450 isoform activity to fresh cells levels [227
• N.A [227Slow freeze [227
CompoundCell typeBeneficial effectsNo effect/adverse effectsCryopreservation method
Ascorbic acid Bone-marrow mononuclear cells • Clonogenic parameters (↑) [221] (murine model) • Viability and clonogenic parameters (human model) (N/C) [221Controlled rate freezing [221
Astragalosides Pancreatic islets • Restored blood glucose to normal [232]
• Insulin expression after transplantation (↑) [232
• N.A [232Slow-freeze [232
BHT Blood cells • Loss of HUFAs (↓) [184• N.A [184Chromatography paper at −20°C [184
BHT + ascorbic acid Hepatocytes • Post-thaw albumin production (↑) [235• Induced LDH release (↑) [235]
• Urea synthesis, ammonia clearance and cell proliferation (N/C) [235]
• Apoptosis associated DNA fragmentation (N/C) [235
(N/I) stored in −70°C freezer [235
Catalase Mononuclear cells • Clonogenic parameters (↑) (murine model) [221• Viability, clonogenic parameters (human model) (N/C) [221Controlled rate freezing [221
Catalase + Trehalose Hematopoietic cells • DCF fluorescence intensitya (↓) [233]
• Number of DCF+ cellsa (↓) [233]
• (↑) CFU [233,234]
• (↑) pre-CFU [233]
• Better engraftment [233]
• (↑) viability [233]
• Apoptosis (↓) [233]
• (↑) responsiveness to migratory homing associated cytokines, expression of homing-associated receptor and adhesion capacity [234
• N.A [233,234Controlled rate freezing [233,234
Consumption of blueberries by PBMC donors Peripheral blood mononuclear cell • DNA oxidation (↓)o [144• DNA damage induced by H2O2 in cryopreserved cells (N/C) [144Slow freeze [144
Deferoxamine Blood cells • Loss of HUFA (↓) [184• N.A [184Chromatography paper at −20°C [184
Glutathione (GSH) Embryonic stem cells • ROSa (↓) [247]
• Viability (↑) [247
• N.A [247(N/I) Stored in −80°C freezer at 24 h [247
 Embryogenic callus • Post-thaw survival, GSH, ascorbic acid levels, SOD and peroxidase activity (↑) [174]
OHh, H2O2u, O2•−u and MDA levelst (↓) [174
• At high concentrations, survival (↓) or (N/C) [174]
•catalase activity (N/C) [174
Vitrification [174
 Pancreatic islets • MDAt (↓) [222]
• Islet morphometry and glucose clearance rate (↑) [222
• Islet insulin secretion (N/C) [222Slow freeze [222
Peroxiredoxin Murine hepatocytes • Viability (↑) [236]
• Integrin-β1 and β-catenin cell adhesion proteins (↑) [236]
• Urea secretion (↑) [236]
• NO•c(↓) [236]
• ROSa _(↓) [236]
• O2•−j(↓) [236
• E-Cadherin cell adhesion proteins (N/C) [236Slow freeze [236
 Murine insulinoma • Viability (↑) [236]
• Insulin secretion (↑) [236]
• NO•c (↓) [236]
• O2•−j (↓) [236
• ROSa (N/C) [236Slow freeze [236
Polyethylene glycol (PEG) Human embryonic stem cells • (↓) ROSj [36]
• Alleviation of F-actin levels [36
• Cell viability (N/C) [36Slow freeze [36
S-Adenosylmethionine Hepatocytes • (↑) GSH content and cellular viability [177• N.A [177Slow freeze [177
Salidroside Red blood cell • (↓) protein carboxylationm [158]
• (↓) Lipid peroxidationt (when trehalose was used as a CPA) [158
• Lipid peroxidationt (N/C) (when used with glycerol as a CPA) [158N.I [158
SOD Bone-marrow mononuclear cells • N.A [221• Post-thaw recovery (N/C) [221Controlled rate freezing [221
Trehalose Dendritic cells • Preserved cell function and phenotype [205]
• (↑) viability [205]
• Maintained MMP and cytoskeleton integrity [205]
• (↓) apoptosis, BIM-1 and CASP9 expression [205
• N.A [205Controlled rate freezing [205
 Hepatocytes • (↑) albumin secretion, plating efficiency and viability [206]
• (↓) AST activity [206
• EROD and ECOD activity, proliferation, LDH, urea levels (N/C) [206Controlled rate freezing [206
 BM-MNC • (↑) Clonogenic parameters (murine and human models [221• N.A [221Slow controlled rate freezing [221
Wheat proteins or Lipocalins Hepatocytes • (↑) attachment efficiency and viability [227]
• Restoration of cytochrome P450 isoform activity to fresh cells levels [227
• N.A [227Slow freeze [227

Abbreviations: AST, aspartate aminotransferase; CFU, colony forming units; DCF, 2′,7′-dichlorofluorescein; BHT, butylated hydroxytoluene; HUFA;highly unsaturated fatty acid; LN, liquid nitrogen; MDA, malondialdehyde; N.A, not-applicable; N/C, no changes/no effect. -, no effects have been reported. (↑) and (↓), represent a significant increase or decrease respectively.

, denotes cases where effects are context dependent and due to factors such as cell quality and species.

Method employed for detection of ROS and Oxidative biomarkers are denoted by alphabetical superscripts ‘a’ to ‘u’:

a, H2DCFDA.

b, H2DCFDA/Propidium Iodide (Pi).

c, 4,5-diaminofluorescein diacetate (DAF-2DA).

d, DAF-2DA/Ethidium Homodimer-.

e, 8-OHG.

f, BODIPY 581/591 C11.

g, BODIPY 581/591 C11/Propidium Iodide (Pi)-.

h, Bromopyrogallol Red.

i, DHE/Sytox-.

j, DHE.

k, Dihydrorhodamine (DHR) 123/Propidium Iodide (Pi)-.

l, DHR 123.

m, 2,4-dinitrophenylhydrazine (DNPH) assay.

n, Fox2 modified method.

o, Formamidopyrimidine-DNA glycosylase-sensitive comet assay.

p, Griess reagent system.

q, MitoPY1/SYTOX–.

r, PF6-AM/SYTOX-.

s, Peroxy Green 1 (PG1).

t, Thiobarbituric acid reactive substances (TBARS) assay.

u, Commercial or obscure ROS detection techniques.

With respect to H2O2 levels in cells during cryopreservation, total H2O2 levels remain largely unchanged, while mitochondrial H2O2 were reported to be increased in spermatozoa [30,41]. H2O2 has highly selective reactivity with only certain biomolecules and can cause the oxidation of thiol groups (SH). H2O2 is toxic at high concentrations because it can be reduced by ferrous Iron, Fe (II), into the more damaging OH via the Fenton reaction (Figure 1) [42–44]. H2O2 has a long half-life which allows it to transduce signals at long ranges [37,45]. When in extracellular space, H2O2 can re-enter the cell through aquaporin-dependent pathways or via direct diffusion [46]. Multiple pathways such as the glutathione peroxidase-glutathione reductase and the peroxiredoxin/thioredoxin-thioredoxin reductase pathways utilize NADPH as reducing equivalent to reduce H2O2 to H2O (Figure 1). The species of ROS and methods used for detection in different cell types used for cryopreservation are summarized in Tables 1 and 2.

Mitochondrial ROS production

Studies from fish [47], sheep [48] and human cells [49] have indicated that cryopreservation induced alterations and/or damages to the mitochondria. Proteins upstream in the electron transport chain (ETC) can generate ROS through the univalent donation of electrons to oxygen in the mitochondria. Sources of ROS in the mitochondria include complex I, complex II and complex III [50]. These enzymes ‘leak’ electrons and as a result, univalently reduce oxygen to O2•−. Through this process, ROS in the form of O2•−, OH and H2O2 are produced (Figure 1).

Factors influencing the production of ROS in the mitochondria include: tissue or cell type, oxygen tension of the extracellular environment, presence of metabolic intermediates and substrates [51], hyperoxia [50,52], the presence of a high proportion of NADH electron donors [51,53] as well as the mitochondrial membrane potential (Δψ) and the pH gradient [54–56], which are constituents of proton-motive force, Δp. The multitude of mitochondria inducers underlie the fact that multiple mechanisms can affect the genesis of mitochondrial ROS in the ETC (reviewed in [50,51,56]).

The MMP or Δψ is a parameter widely used to assess mitochondrial function. Δψ was reported to be altered in thawed cells following cryopreservation [57–59]. Reduction in Δψ in certain cases, such as a mild decrease, is associated with a decline in ROS levels while an increase in Δψ has been noted to promote ROS formation in rat mitochondria isolated from brain [60] and heart muscles [61]. These studies indicated that maintenance of the Δψ is an important aspect to prevent ROS-induced oxidative stress during cryopreservation.

Hyper-polarization of the Δψ can favor ROS generation [62], which is believed to be a result of a reduction in electron transfer [63]. Depolarization of Δψ can be induced by ROS, which impairs oxidative phosphorylation and amplifies ROS generation [64]. Loss of Δψ was reported in cryopreserved human oocytes [57], buffalo sperm [65,58], nucleus pulposus-derived mesenchymal stem cells [66], murine embryos [67], Meleagris gallopavo spermatozoa [68], koala spermatozoa [69] and porcine hepatocytes [59], although a transient elevation in Δψ was reported in murine oocytes after freeze-thawing [70]. Opening of mtochondrial permeability transition pore (mPTP), which involves the formation of a ‘hole’ in the inner mitochondrial membrane (IMM) is known to lead to the dissipation of the Δψ as well as an elevation in ROS levels [64]. Opening of mPTP leads to dissipation of the Δψ, mitochondrial swelling, ATP depletion, relocalization of pro-apoptotic molecules and elevated ROS levels [71,72].

The involvement of mPTP in cryopreservation has been implicated in the study showing that inhibition of mPTP by bongkrekic acid successfully reduced cryopreservation-induced apoptosis in stallion spermatozoa [73]. mPTP opening has been known to enhance H2O2 production through conformational alterations to complex I of ETC [74], depletion of ROS-scavengers as well as intensifying production of ROS from Krebs cycle oxidoreductases [75]. Opening of mPTP is known to be induced during oxidative stress and ROS-mediated alterations to mPTP regulators and components were suggested to be responsible for this. Indeed, it was found that the mild uncoupling agent 2,4-dinitrophenol, which normally reduces ROS, improved motility in sperm with low cryopreservability [76] while the antioxidant, monothioglycerol was found to reduce mitochondria ROS as well as increase fertility and the percentage of cells with the ability to undergo acrosome reaction [41]. ROS-mediated mitochondrial permeabilization involved oxidative attack on the protein thiol groups on the mitochondrial membrane. This may give rise to protein aggregates due to thiol groups cross-linking after being oxidized [77,78].

Protein folding in the endoplasmic reticulum and ROS production

Cryopreservation of cells was known to perturb the homeostasis of the endoplasmic reticulum (ER) [79–82] and ER is a known source of ROS [83,84]. The ER facilitates the proper folding and addition of some post-translational modifications to proteins in the secretory pathway. Accumulation of misfolded proteins in the ER could occur under conditions that perturb ER homeostasis, also known as ER stress. Increased protein synthesis is one such condition. The unfolded protein (UPR) response promotes an adaptive response against ER stress by increasing machineries for protein degradation and protein folding as part of an effort to restore ER homeostasis. The molecular mechanism on how UPR are activated has been comprehensively reviewed by [85,86]. UPR is activated via one of the three membrane-bound transducing receptors (ATF6, PERK, IRE1α), these three sensors thus constitute the three branches of the UPR signaling pathways [85,86]. It was observed that SOD1 and the ER stress marker ERP29 gene expression were significantly up-regulated in response to freeze-thaw stress in primate ovarian tissue [81]. In yeast, genes expression for protein chaperones such as SSA4, HSP26, HSP42 were found to be up-regulated in response to freeze-thaw stress when cells were frozen in the absence of cryoprotectants [87]. In mammals, all three arms of the UPR may be activated during cryopreservation. The XBP-1 protein levels were elevated in vitrified mice oocytes [79] and maturing oocytes exposed to delipidated serum were more susceptible to cryopreservation-induced ER stress [80]. Intriguingly, the handling of oocytes, itself, was sufficient to activate the IRE1α arm [88], highlighting the vulnerability of oocytes to cope with stress during the cryopreservation process.

In addition to its homeostatic role, sustained induction of the UPR in response to severe ER stress caused by a multitude of factors can antagonize cellular survival, resulting in cell death [89]. The activation of the UPR has been implicated in the production of at least two species of ROS: O2•− and H2O2 [90–92] and these ROS were postulated to be an event preceding cellular death. The source of H2O2 may be attributed to oxidative folding via the protein disulfide isomerase (PDI)-ER oxidoreductase (ERO) relay or the cytochrome P450 (CYP) family of enzymes. The PDI-ERO1 pathway has been demonstrated to produce ROS in the form of H2O2 [93–95]. Activation of the PERK-arm of the UPR could lead to downstream ERO1α activation, H2O2 production and mediates the feeding of calcium into the mitochondria which could promote O2•− production and apoptosis [92]. Interestingly, a yeast strain deleted for genes encoding for catalases and glutathione were hypersensitive to exogenous H2O2, but are not sensitive to ERO1 overexpression [96], indicating that there could be other, more potent sources of H2O2 in the ER, or that cytosolic or mitochondria pool of H2O2 are isolated from the ER. Studies have also indicated an ERO1-independent source of H2O2 in the ER [97,98], suggesting the PDI-ERO1 pathway may not be the sole source of H2O2 in the ER. Other possible pathway that could be activated through sustained UPR activation that may lead to mitochondrial ROS generation is through the dimerization of IRE1α, which activates the JNK-SAB axis to initiate cellular death [99].

Nitric oxide synthase and NADPH oxidase

Although not considered ROS, nitric oxide (NO) and peroxynitrite (ONOO) are free radicals. NO can diffuse through the cell membrane. In vivo, NO is not highly reactive to most biomolecules. However, NO can react with metal complexes to form metal nitrosyls. NO reacts with O2•− to form more damaging species, such as ONOO which is thought to occur mostly in the hydrophobic regions of the cell [100]. ONOO can be detoxified by enzymes such as peroxiredoxins and glutathione peroxidase [101] (Figure 1). Unlike NO, ONOO are strong oxidants capable of causing oxidative damage, nitration and S-nitrosylation of proteins [102,103]. In vivo, nitric oxide (NO) is produced by the family of nitric oxide synthase (NOS) which consist of three isoforms: neuronal NOS or NOS1 (‘neuronal’ NOS/nNOS), NOS2 (‘inducible’ NOS/iNOS) and NOS3 (‘endothelial’ NOS/eNOS). NOS typically catalyzes the formation of NO and citrulline from arginine and oxygen. Most NOS isoforms are usually regulated by calmodulin and calcium, and require the cofactors NADPH, FAD, Flavin mononucleotide (FMN) and tetrahydrobiopterin (BH4) [104,105]. Similar to ROS, NO regulates cell death and survival [106–110]. NO is essential for proper cellular physiological function such as vasodilation [111] as well as regulating immunosuppression [112] and tissue repair in mesenchymal stem cells (MSCs) [113]. Conversely, NO, can interfere with hemopoiesis [114]. Moderate levels of NO can initiate capacitation [115] and is essential for motile functions in sperm [116,117].

During cryopreservation, NOS activation or NO production was observed in cryopreserved heart valves [118] and sperm [30,116,119]. While NO itself has not been found to be significantly increased by freeze-thaw stress in RBC, the product of nitric oxide nitrosylation, S-nitrosohemoglobin was found to be increased by freeze-thaw stress [120]. At high levels of NO, sperm functions can be antagonized [116,121–123]. When cryopreserving sperms, the use of low concentrations of NOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME) [124], and anti-nitrosative agents such as hemoglobin [124] and lactoferrin [125] has been found to improve membrane functionality, survival and/or fertilization, indicating that reducing NO may improve assisted reproductive technology outcomes especially since NO was elevated in post-thawed cells. It should however be noted that the use of high concentrations of l-NAME was found to impair sperm function [124].

NADPH oxidases (NOXs) are a family of seven-membered enzymes that are highly regarded due to their role as a major non-mitochondrial ROS generator. NOX enzymes generate ROS, primarily O2•−, by catalyzing the transfer of one electron across the membrane from the electron-donating NADPH to the electron acceptor oxygen, thus reducing oxygen to form O2•−. Exceptions are NOX4, DUOX1 and DUOX2 of the NOX/DUOX family, which was documented to produce mainly H2O2 [126]. While all seven members of the NOX family are found to be located to the plasma membrane, specific NOX isoforms such as NOX4, NOX5 and DUOX2 have also been detected at ER, with NOX4 residing at other subcellular locations including the mitochondria and the nuclear membrane [126,127]. Apart from the mitochondria and ER, the peroxisome is another source of intracellular ROS, which harbors pro-oxidant enzymes such as acyl CoA oxidase (ACOx) and xanthine oxidases (XOs) [128].

Our current understanding of the activation of NOX includes a collection of inducers which can be sorted into three main categories namely: chemical, biological and physical [129]. With respect to cryopreservation, NOX activation induced by chemical and physical inducers are particularly relevant and worth noting. Physical inducers are a broad collection of inducers including temperature [130], osmotic stress [131] and pH changes [132] which are documented NOX-inducers, that are coincidentally generated during cryopreservation [133]. During cryopreservation, the addition and removal of cryoprotectants, as well as freeze-thawing have been proven to subject cells to osmotic stress [134]. Extracellular ice formed during cryopreservation puts the cell through hypertonic conditions as the solute concentration elevates in the unfrozen extracellular portions. As a result, cells shrink as water leaves the cell to re-establish the equilibrium of solute concentration across the cell. The reverse is also true for thawing during cryopreservation, in which this time, cells are put through hypotonic conditions which lead to movement of water into the cell, consequently, cell swelling. Swelling of cells under hypotonic condition, however, was viewed as more pernicious due to the elevation in ROS levels following cryopreservation in the case of stallion sperm [135].

In astrocytes, hypo-osmotic swelling leads to an increase in ROS as well as phosphorylation of p47phox and that, apocynin, an NOX inhibitor abrogated such effects [136]. Moreover, cortical brain slices of mice with p47phox knockout failed to show elevated ROS levels as observed in wild-type mice suggesting hypo-osmotic swelling results in p47phox-NOX-dependent generation of ROS [136]. In agreement with this, supporting evidence from skeletal muscles, in which osmotic stress leads to localized increase in Ca2+ in the cytosol, termed as ‘calcium spark’ and an elevation in ROS levels have further substantiated this viewpoint. In addition to this, treatment of skeletal muscle cells with NOX inhibitors, apocynin and diphenyleneiodonium, reversed this effect. The exclusion of extracellular Ca2+ restrained the increase in levels of ROS as well as calcium spark and the inhibition of Ca2+ release from the sarcoplasmic reticulum by the inhibitors, ryanodine and thapsigargin were able to further reduce ROS levels [131].

Taken together, the above observations suggested that NOX activation via osmotic stress may be dependent on Ca2+ release from the sarcoplasmic reticulum. The Ca2+ could then influx into mitochondria from osmotic stress, leading to induction of NOX activity [131]. Thus, it could be inferred from the above studies that cryopreservation may induce NOX activation. Whether NOX inhibitors can abrogate the ROS generated in post-thawed cells remains to be investigated. Current findings indicate that NOX activation during cryopreservation could be a potential target to reduce ROS-induced damage in cells.

DNA damage, protein oxidation and lipid peroxidation in cryopreservation

Detection of oxidative damage in cryopreserved cells is a valuable measurement to determine the degree of damage. Many consequences of ROS-induced damages can be credited to lipid peroxidation [137], DNA damage [138] and protein oxidation [139,140] (Figure 2). Methods used for measurement of these damages are reviewed by [141].

Cryopreservation significantly increased DNA damage in cells as assessed by the comet assay or DNA fragmentation. Activation of DNA damage repair (DDR) constituents such as p53 [33,142], γH2AX and RAD51 [143] were observed during slow-freeze and/or vitrification. DNA oxidation was increased in cryopreserved human peripheral blood mononuclear cells (PBMCs), indicating oxidative damage has occurred in these cells [144]. In contrast, PBMCs from donors who consumed wild blueberries rich in antioxidants has been reported to have significantly lower DNA oxidation following cryopreservation. Whereas antioxidants may reduce DNA oxidation during freeze-thawing, l-carnitine, an antioxidant [145,146], has however, failed to reduce DNA oxidation in thawed human spermatozoa in vitro [147]. Addition of compounds with known antioxidant properties such as vitamin C [148–150], vitamin E [148,151], resveratrol, [150], β-mercaptoethanol [152], taurine, hypotaurine [148], glutathione (GSH) [153], coenzyme Q [154], quercetin [153], zinc oxide nanoparticles [155], catalase [149] monothioglycerol, glutathione [153] and melatonin [156] have been reported to significantly reduce DNA damage in cryopreserved cells (Table 1).

Protein oxidation, as determined by protein carbonylation were detected in cryopreserved cells. Freezing stress was characterized to lead to the formation of carbonyl groups in intact and homogenized tissue [157]. RBCs cryopreserved with glycerol or trehalose were found to have increased ROS accumulation and protein oxidation. Supplementation of the antioxidant Salidroside ameliorated this effect [158]. Protein oxidation and increased ROS was also detected in cryopreserved sperm cells [159]. Lipid peroxidation can be due to the effect of OH and ONOO [160,161]. Increase in lipid peroxidation was observed in tissue specimens stored at −20°C [162]. Lipid peroxidation as measured by either malondialdehyde (MDA) or 4-Hydroxynonenal (4-HNE) were detected in cryopreserved red blood cells [158], sperm [163–166] and hepatocytes [167]. Notably, the product of lipid peroxidation 4-HNE is extremely reactive, which allows it to react with DNA and proteins. Antioxidants such as iodixanol can reduce lipid peroxidation in cryopreserved buffalo semen [168].

Effectiveness of antioxidants in preventing cryoinjury: lesson learnt so far

Endogenous defense mechanisms and effects of inhibitors on ROS-generating sources in cryopreservation

Transcriptomic studies have shown that many antioxidant genes such as SOD1, cytosolic catalase T (CTT1) and glutaredoxin-1 (GRX1) were induced in the model eukaryote Saccharomyces cerevisiae, also commonly known as the baker’s yeast or brewer’s yeast [169]. These studies indicated the importance of the role of antioxidants in mitigating freeze-thaw stress after cryopreservation [169]. Intriguingly, genetic screening of yeast mutants defective for different antioxidant genes highlighted that not all antioxidants contribute equally in their ability to protect cells from freeze-thaw stress [87,170]. It was found that yeast strains deleted for SOD1 and SOD2 were particularly sensitive to freeze-thaw stress, while single deletion of catalase and glutathione peroxidase were not as sensitive [170]. In mammals, both vitrification and slow freezing were found to up-regulate SOD gene expression and increase proteins levels in murine oocytes [171], embryos [172] and testicular tissue [142]. Furthermore, the addition of O2•− scavenging agent MnCl2 rescued cells deleted for the SOD1 gene [170]. Collectively, these studies highlight the importance of the SOD genes in cryopreservation of various cell types.

Besides SOD, the reduced GSH regeneration system or the pentose-phosphate shunt for NADPH production were up-regulated in S. cerevisiae during freeze-thaw [169]. Given that GSH is the most abundant antioxidant in almost all cell types [173], it is therefore not surprising that the glutathione cycle is required for freeze-thaw tolerance. Studies where spermatozoa were administered with GSH or thiols were demonstrated to modestly reduce ROS [174], increase the motility of spermatozoa [41,175] and the developmental competence of mouse oocytes [176]. In addition, the GSH and cysteine precursor, S-adenosylmethionine, increased the total GSH levels and the viability of cryopreserved cells. While the supplementation of S-adenosylmethionine lead to significantly lower MDA levels in cold-stored rat hepatocytes, it was however not determined in the cryopreserved cell group [177]. The proliferation of spermatogonial stem cells was however noted to be unaffected by administration of glutathione [178].

Interestingly, one of the more oxidizing environment in the cell is the ER, where the reduced GSH to oxidized glutathione (GSSG) ratio is 3:1 as compared with the cytosol where the ratio is 100:1 [179]. Perturbation of ER homeostasis was known to trigger the UPR. The induction of UPR coincides with a reduction in the developmental competence and modest reductions in survival of cryopreserved cells, which can be improved by supplementation of ER stress inhibitor TUDCA [79,80,82]. The use of Trolox, a water-soluble analog of vitamin E, increased antioxidant capacity, prevented ER stress and improved the viability of ovarian tissues. This indicates a role for both oxidative stress and ER stress during cryopreservation [81]. Intriguingly, it was found that an inhibitor that prevents ER stress-induced apoptosis, Salubrinal, did not improve development and viability of bovine blastocyst [180], indicating that preventing ER stress-induced cell death alone may not be sufficient to prevent cryopreservation-induced damage. However, it is worthy to note that in this specific case, the viability of the blastocyst is close to 100% in both control and Salubrinal treated groups [180].

Gene expression encoding for proteins which regulate or sequester the availability of Fenton reaction initiators are up-regulated in transcriptomic studies in freeze-tolerant animals or in yeast undergoing freezing stress [181,182]. As antioxidants, iron chelators such as deferoxamine, lactoferrin and transferrin were found to limit NO production and improve cellular parameters affected by cryopreservation-induced oxidative stress [125,183,184]. Deferoxamine was found to prevent loss of highly unsaturated fatty acids in RBCs stored at −20°C for a shorter period time as compared with the lipophilic free radical scavenger butylated hydroxytoluene (BHT) [184]. Interestingly, supplementation of transferrin, ascorbic acid and a combination of both compounds generally improved the percentage of intact embryos. However only when ascorbic acid was used alone did the number of hatching blastocysts appreciably increase [183]. These studies revealed the complexity of the outcome of cryopreserved cells when using antioxidants as a supplement for cryopreservation.

Apoptosis as an adversity after cryopreservation

The efficiency of the cryopreservation process is still partially compromised due to several factors. Reduced cell viability, increased senescence and impaired cellular functions are among the most widely reported adversities associated with oxidative stress generated during cryopreservation of cells. In spermatozoa, freeze-thawing during cryopreservation greatly reduced cell viability accompanied by a range of structural abnormalities and damages, presumed or found to be a consequence of oxidative stress [185–187]. Supplementation of antioxidants into the cryopreservation media generally yielded good cell viability, indicating that oxidative stress plays a role in inducing cellular death during cryopreservation [147,188,189]. Cryopreservation led to re-localization of phosphatidylserine from the inner to the outer leaflet of plasma membrane, a signal displayed by cells undergoing cell death [166,190]. Caspase activation, which is well-known to be involved in mediating the apoptotic cascade, were also observed in cryopreserved sperm cells [191–193]. The use of caspase inhibitors significantly improved the viability of hepatocytes and human embryonic stem cells after cryopreservation [194–196], indicating that preventing caspase activation can be a plausible approach to improve cell viability.

Some antioxidants may exert their effects through modulation of genes responsible for pro-survival, apoptosis and/or oxidative stress. Melatonin, iodixanol, catalase and vitamin E can up-regulate anti-apoptotic genes such as Bcl2l1 (Bcl-xL) and Bcl-2, while down-regulating pro-apoptotic genes such as BAX/Bax [197–200]. With regard to melatonin, Deng et al. [199] and Chen et al. [198] observed different outcomes on BAX/Bax modulation. This difference could be attributed to the cell type used in each study. Apart from modulating genes responsible for cell survival, the pro-oxidative genes, ROMO1 (in canine) and NOX5 (in humans) have also been reported to be down-regulated by the administration of iodixanol [200] and melatonin, respectively [199]. The use of melatonin in cryopreservation has been noted to increase human antioxidant genes, such as NRF2 and SOD2 among others as shown in Table 1. It remains unclear if the up-regulation of antioxidant genes after antioxidant treatment provides direct benefit, if any, to protect cells against cryopreservation-induced ROS damage. Trehalose [201,202] and BHT [203] were reported to reduce lipid peroxidation [204] in testicular tissue and spermatozoa, respectively, generally enhance total antioxidant capacity, improve cellular viability [204–207] and reduce apoptosis [205,207]. Decrease in mitochondrial membrane potential (Δψ) has been observed in cells stimulated by apoptotic stimulus [208–211], which is also seen in thawed cells after cryopreservation [65]. The reduction in Δψ is, however, prevented through antioxidant administration [212–216]. Administration of amino acid with antioxidant properties such as l-proline is one such case where it reduces ROS levels as well as increases Δψ [217]. These studies indicate that supplementing antioxidants and/or factors that modulate the process of cell death can be a potential solution to reduce cryopreservation-induced cell death.

Context-dependent effects of antioxidants in cryopreservation

The effectiveness of antioxidants in ameliorating functional parameters during cryopreservation is also dependent on the cell type used as well as the integrity of the cells prior to cryopreservation. This could be observed in the cryopreservation of sperm cells from different organisms. In one example, Dong et al. reported that the beneficial effects of SOD administration were only seen in sperm with low post-thaw survivability [76]. This is also observed for other antioxidants namely coenzyme Q [218], resveratrol [219], zinc sulfate [220], ascorbic acid [150], catalase [76] and 2,4-dinitrophenol [76]. These studies concluded that the effectiveness of antioxidants was dependent on sperm quality. While vitamins C and E may generally reduce DNA damage of spermatozoa from human and Gilt-head seabream (Sparus aurata) [148,149,151], these antioxidants can increase DNA damage in cryopreserved sperm from European seabass (Dicentrarchus labrax) [148] suggesting the possibility that antioxidants ameliorate freeze-thaw stress in a species-dependent manner. In cases where trehalose was used to cryopreserve spermatogonial stem cells, while the proliferation capability of such cells was increased in vitro, this did not translate to a real improvement in the number of colonies formed when such cells were subsequently transplanted [207]. Such disagreement between in vitro and in vivo results can also be seen when mouse embryos were incubated with ascorbic acid prior and post-cryopreservation, where the number of normal fetuses was unchanged despite notable improvements such as embryo intactness as well as blastocyst stage [183]. Consistently, there have also been reports in the literature where cellular functionality saw no improvement after antioxidant supplementation for cryopreservation [41,178,219,221,222]. Table 1 is a summary of the effect of antioxidants on cryopreserving reproductive cells and embryos.

Potential practical applications of antioxidants and their effects on cellular function

Hemopoietic stem cells, hepatocytes and islet cells, all possess enormous potential when transplanted. In some studies, it has been reported that the ability to synthesize proteins [223] such as insulin [224,225] or albumin [226], metabolize xenobiotics [226,227], transplantation potential [226–228] and clonogenic potential [229–231] may either be lost or impaired via the process of cryopreservation. Such impairments or undesirable outcomes of cellular functionality have been partly improved through administration of antioxidants. These include ascorbic acid [221], astragalosides [232], taurine [222], hypotaurine [178], vitamin E [76], catalase [221], trehalose [205–207], combination of catalase with trehalose [233,234] as well as combination of BHT with ascorbic acid [235] to cell types such as mononuclear cells, pancreatic islets, germ cells, spermatozoa, dendritic cells, hepatocytes and hemopoietic cells (Table 1). Among these antioxidants, winter wheat lipocalins and peroxiredoxins obtained from wheat are especially notable. They were demonstrated to mollify cryopreservation-associated loss of attachment capacity of hepatocytes, as well as restoring the activity of CYP isoforms to the level similar from fresh, unfrozen murine hepatocytes [227,236]. Table 2 is a summary of the effect of antioxidants on non-reproductive cells.

Collectively, the different studies examined in this review indicated that the effectiveness of antioxidant supplement for cryopreservation very much depends on the cell type, organism as well as the specific antioxidant used. Table 1 provides a summary of the type of the antioxidant used, the cell type and organism as well as the effectiveness of the antioxidant based on the parameters measured.

Moving forward

Oxidative stress is inevitably generated in the cryopreservation process and has been widely cited as the causative factor for some of the cryoinjuries inflicted on the cell [163,165,187,237]. Therefore, administering antioxidants in an effort to counter these deleterious effects on cells during cryopreservation is a plausible solution. Indeed, the use of antioxidants has undoubtedly conferred protection to certain cell type by improving several cellular function parameters and general cryopreservation outcome in specific circumstances as those indicated in the sections above and in Tables 1 and 2. Although effective in some circumstances, antioxidants can be ineffective or even deleterious for some cells. Antioxidants consist of a broad class of substances and molecules with varying physio-chemical properties that dictate their specificity, localization and/or ROS-scavenging roles [238,239]. Mitochondria-targeted antioxidants, MitoTEMPO [214] and melatonin [240,241] are potent antioxidants that prevent oxidative stress-associated damages encountered during cryopreservation. Melatonin in particular, has performed unexpectedly well by exerting its ROS-ameliorating properties through its multi-faceted mechanisms [242]. While the administration of melatonin improved the generation and survival of somatic cell nuclear transferred (SCNT) murine embryos from vitrified oocytes, whether melatonin directly affects ROS or inhibits apoptosis remains to be elucidated [243]. As such, the use of antioxidant in different combinations for the different cell types for cryopreservation may prove to be more effective in countering cryopreservation-induced ROS damage.

There are evidences to indicate that the use of different antioxidants in combination could provide additive protective effect when compared with those administered individually (Tables 1 and 2). In reproductive cells, catalase and low concentration of SOD have been reported to have no effect on oocyte survivability and fertility when used alone. However, when the same dose of SOD was co-administered with catalase, significant improvement in oocyte survivability was observed [124]. For the case of sperm cryopreservation, supplementation of SOD alone has no effect on the general sperm parameters such as motility (total and progressive motility), viability and percentage of sperm with high MMP, while supplementation of catalase alone was beneficial only at high concentrations [244]. Notably, when catalase and SOD were used in combination, sperm parameters such as total motility was greatly improved as compared with individual use of them at the respective dose [244]. In another example, sperm cells frozen in SOD and catalase, or vitamins C and E led to significantly improved parameters such as reduced ROS [215] and increased lateral head displacement of the sperm cells [245] whereas previously such parameters were not improved when the antioxidants were used individually [215,245].

Not all antioxidants used in combination yield additional benefits. For example, single administration of trehalose or catalase improved clonogenic parameters such as burst-forming unit erythroid and colony-forming unit granulocyte-monocyte in fetal liver hematopoietic cells and umbilical cord blood, respectively. However, when trehalose and catalase were administered in combination, no significant improvements in these parameters were observed [246]. Hence, use of different antioxidants in combination does not always imply additional improvement in cellular parameters.

Based on the studies examined in this review, it is notable that antioxidant supplement for cryopreservation can be effective. However, the effectiveness of the specific antioxidants depends on the cell type that undergoes the cryopreservation process. It is therefore important to consider supplementing cryopreservation media with specific antioxidants according to the specific species, cell type, quality and integrity of cells prior to cryopreservation. Additionally, several studies determined the efficacy of the antioxidants on cryopreservation by measuring functional parameters of the cryopreserved cells in an ex vivo setting. Given that many applications for cryopreservation are in the area of reproductive and regenerative medicine, future studies should be attempted to investigate the recovery and efficacy of the cryopreserved cells after transplantation in vivo to better understand the efficacy of the antioxidant used in this process.

Acknowledgments

The authors thank Drs Harmeet Singh, Yiu Wing Kam and Sharon Lim for proofreading the manuscript.

Author Contribution

J.S.L and W.S.D.K. wrote the manuscript, prepared the figures and tables. S.-X.T. supervised and edited the manuscript.

Funding

This work was supported by the Republic Polytechnic Research and Development Grant to S.-X.T.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • ATF6

    activating transcription factor 6

  •  
  • BHT

    butylated hydroxytoluene

  •  
  • CPA

    cryoprotective agent

  •  
  • CYP

    cytochrome P450

  •  
  • DHE

    dihydroethidium

  •  
  • DMSO

    dimethyl sulfoxide

  •  
  • DUOX

    dual oxidase

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERO

    ER oxidoreductase

  •  
  • ETC

    electron transport chain

  •  
  • GSH

    glutathione

  •  
  • H 2DCFDA

    2′,7′-dichlorodihydrofluorescein diacetate

  •  
  • H2O2

    hydrogen peroxide

  •  
  • IRE1α

    inositol-requiring enzyme 1 α

  •  
  • l-NAME

    NG-nitro-l-arginine methyl ester

  •  
  • MDA

    malondialdehyde

  •  
  • MMP

    mitochondrial membrane potential

  •  
  • mPTP

    mitochondria permeability transition pore

  •  
  • NOS

    nitric oxide synthase

  •  
  • NOX

    NADPH oxidase

  •  
  • PBMC

    peripheral blood mononuclear cell

  •  
  • PDI

    protein disulfide isomerase

  •  
  • PERK

    pancreatic eIF-2alpha kinase

  •  
  • ROS

    reactive oxygen species

  •  
  • SAB

    SH3 homology associated BTK binding protein 5

  •  
  • SOD

    superoxide dismutase

  •  
  • TUDCA

    tauroursodeoxycholic acid

  •  
  • UPR

    unfolded protein response

  •  
  • XBP-1

    X-box binding protein 1

  •  
  • 4-HNE

    4-Hydroxynonenal

  •  
  • γH2AX

    H2A histone family member X

References

References
1.
Shu
Z.
,
Heimfeld
S.
and
Gao
D.
(
2014
)
Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion
.
Bone Marrow Transplant.
49
,
469
476
[PubMed]
2.
Lewis
J.K.
,
Bischof
J.C.
,
Braslavsky
I.
,
Brockbank
K.G.M.
,
Fahy
G.M.
,
Fuller
B.J.
et al.
(
2016
)
The grand challenges of organ banking: proceedings from the first global summit on complex tissue cryopreservation
.
Cryobiology
72
,
169
182
[PubMed]
3.
Varghese
A.C.
,
du Plessis
S.S.
,
Falcone
T.
and
Agarwal
A.
(
2008
)
Cryopreservation/transplantation of ovarian tissue and in vitro maturation of follicles and oocytes: challenges for fertility preservation
.
Reprod. Biol. Endocrinol.
6
,
47
4.
Moll
G.
,
Alm
J.J.
,
Davies
L.C.
,
von Bahr
L.
,
Heldring
N.
,
Stenbeck Funke
L.
et al.
(
2014
)
Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?
Stem Cells
32
,
2430
2442
[PubMed]
5.
Keith
S.C.
Jr
(
1913
)
Factors influencing the survival of bacteria at temperatures in the vicinity of the freezing point of water
.
Science
37
,
877
879
[PubMed]
6.
Hollander
D.H.
and
Nell
E.E.
(
1954
)
Improved preservation of Treponema pallidum and other bacteria by freezing with glycerol
.
Appl. Environ. Microbiol.
2
,
164
170
7.
Macfadyen
A.
and
Rowland
S.
(
1902
)
On the suspension of life at low temperatures
.
Ann. Bot. (Lond.)
os-16
,
589
590
8.
Polge
C.
,
Smith
A.U.
and
Parkes
A.S.
(
1949
)
Revival of spermatozoa after vitrification and dehydration at low temperatures
.
Nature
164
,
666
[PubMed]
9.
Polge
C.
and
Rowson
L.E.A.
(
1952
)
Fertilizing capacity of bull spermatozoa after freezing at −79°C
.
Nature
169
,
626
627
[PubMed]
10.
Lovelock
J.E.
and
Bishop
M.W.H.
(
1959
)
Prevention of freezing damage to living cells by dimethyl sulphoxide
.
Nature
183
,
1394
1395
[PubMed]
11.
Ware
C.B.
,
Nelson
A.M.
and
Blau
C.A.
(
2005
)
Controlled-rate freezing of human ES cells
.
BioTechniques
38
,
879
883
[PubMed]
12.
Richards
M.
,
Fong
C.Y.
,
Tan
S.
,
Chan
W.K.
and
Bongso
A.
(
2004
)
An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells
.
Stem Cells
22
,
779
789
[PubMed]
13.
Chinnadurai
R.
,
Garcia
M.A.
,
Sakurai
Y.
,
Lam
W.A.
,
Kirk
A.D.
,
Galipeau
J.
et al.
(
2014
)
Actin cytoskeletal disruption following cryopreservation alters the biodistribution of human mesenchymal stromal cells in vivo
.
Stem Cell Rep.
3
,
60
72
[PubMed]
14.
Hattori
Y.
,
Kato
H.
,
Nitta
M.
and
Takamoto
S.
(
2001
)
Decrease of L-selectin expression on human CD34+ cells on freeze-thawing and rapid recovery with short-term incubation
.
Exp Hematol.
29
,
114
122
[PubMed]
15.
Katkov
I.I.
,
Kim
M.S.
,
Bajpai
R.
,
Altman
Y.S.
,
Mercola
M.
,
Loring
J.F.
et al.
(
2006
)
Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells
.
Cryobiology
53
,
194
205
[PubMed]
16.
Wagh
V.
,
Meganathan
K.
,
Jagtap
S.
,
Gaspar
J.A.
,
Winkler
J.
,
Spitkovsky
D.
et al.
(
2011
)
Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing
.
Stem Cell Rev. Rep.
7
,
506
517
[PubMed]
17.
Temple
M.D.
,
Perrone
G.G.
and
Dawes
I.W.
(
2005
)
Complex cellular responses to reactive oxygen species
.
Trends Cell Biol.
15
,
319
326
[PubMed]
18.
Halliwell
B.
(
2006
)
Reactive species and antioxidants. Redox Biology is a fundamental theme of aerobic life
.
Plant Physiol.
141
,
312
322
[PubMed]
19.
Halliwell
B.
(
2000
)
The antioxidant paradox
.
Lancet
355
,
1179
1180
[PubMed]
20.
Schieber
M.
and
Chandel
N.S.
(
2014
)
ROS function in redox signaling and oxidative stress
.
Curr. Biol.
24
,
R453
R462
[PubMed]
21.
Finkel
T.
and
Holbrook
N.J.
(
2000
)
Oxidants, oxidative stress and the biology of ageing
.
Nature
408
,
239
247
[PubMed]
22.
Bisht
S.
,
Faiq
M.
,
Tolahunase
M.
and
Dada
R.
(
2017
)
Oxidative stress and male infertility
.
Nat. Rev. Urol.
14
,
470
485
23.
von Zglinicki
T.
,
Petrie
J.
and
Kirkwood
T.B.L.
(
2003
)
Telomere-driven replicative senescence is a stress response
.
Nat. Biotechnol.
21
,
229
230
[PubMed]
24.
Rizza
S.
,
Cardaci
S.
,
Montagna
C.
,
Di Giacomo
G.
,
De Zio
D.
,
Bordi
M.
et al.
(
2018
)
S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy
.
Proc. Natl. Acad. Sci. U.S.A.
115
,
E3388
E3397
25.
Passos
J.F.
,
Nelson
G.
,
Wang
C.
,
Richter
T.
,
Simillion
C.
,
Proctor
C.J.
et al.
(
2010
)
Feedback between p21 and reactive oxygen production is necessary for cell senescence
.
Mol. Syst. Biol.
6
,
347
26.
Bigarella
C.L.
,
Liang
R.
and
Ghaffari
S.
(
2014
)
Stem cells and the impact of ROS signaling
.
Development
141
,
4206
4218
[PubMed]
27.
Nozik Grayck
E.
,
Huang
Y.C.T.
,
Carraway
M.S.
and
Piantadosi
C.A.
(
2003
)
Bicarbonate-dependent superoxide release and pulmonary artery tone
.
Am. J. Physiol. Heart Circ. Physiol.
285
,
H2327
H2335
28.
Mumbengegwi
D.R.
,
Li
Q.
,
Li
C.
,
Bear
C.E.
and
Engelhardt
J.F.
(
2008
)
Evidence for a superoxide permeability pathway in endosomal membranes
.
Mol. Cell Biol.
28
,
3700
3712
[PubMed]
29.
Fukai
T.
and
Ushio Fukai
M.
(
2011
)
Superoxide dismutases: role in redox signaling, vascular function, and diseases
.
Antioxid. Redox Signal.
15
,
1583
1606
30.
Chatterjee
S.
and
Gagnon
C.
(
2001
)
Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing
.
Mol. Reprod. Dev.
59
,
451
458
[PubMed]
31.
Evangelista Vargas
S.
and
Santiani
A.
(
2017
)
Detection of intracellular reactive oxygen species (superoxide anion and hydrogen peroxide) and lipid peroxidation during cryopreservation of alpaca spermatozoa
.
Reprod. Domest. Anim.
52
,
819
824
[PubMed]
32.
Honda
S.
,
Weigel
A.
,
Hjelmeland
L.M.
and
Handa
J.T.
(
2001
)
Induction of telomere shortening and replicative senescence by cryopreservation
.
Biochem. Biophys. Res. Commun.
282
,
493
498
[PubMed]
33.
Xu
X.
,
Cowley
S.
,
Flaim
C.J.
,
James
W.
,
Seymour
L.
and
Cui
Z.
(
2010
)
The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells
.
Biotechnol. Prog.
26
,
827
837
[PubMed]
34.
Holt
C.B.
(
2003
)
Substances which inhibit ice nucleation: a review
.
Cryoletters
24
,
269
274
[PubMed]
35.
Lee
H.H.
,
Lee
H.J.
,
Kim
H.J.
,
Lee
J.H.
,
Ko
Y.
,
Kim
S.M.
et al.
(
2015
)
Effects of antifreeze proteins on the vitrification of mouse oocytes: Comparison of three different antifreeze proteins
.
Hum. Reprod.
30
,
2110
2119
[PubMed]
36.
Xu
X.
,
Cowley
S.
,
Flaim
C.J.
,
James
W.
,
Seymour
L.W.
and
Cui
Z.
(
2010
)
Enhancement of cell recovery for dissociated human embryonic stem cells after cryopreservation
.
Biotechnol. Prog.
26
,
781
788
[PubMed]
37.
Dickinson
B.C.
and
Chang
C.J.
(
2011
)
Chemistry and biology of reactive oxygen species in signaling or stress responses
.
Nat. Chem. Biol.
7
,
504
511
[PubMed]
38.
Yoshikawa
T.
,
Takahashi
S.
,
Tanigawa
T.
,
Naito
Y.
,
Ichikawa
H.
,
Takano
H.
et al.
(
1991
)
Investigation into the reactivity between various amino acids and oxygen-derived free radicals by use of the ESR spin trapping method
.
J. Clin. Biochem. Nutr.
11
,
161
169
39.
Liu
F.
,
Lai
S.
,
Tong
H.
,
Lakey
P.S.J.
,
Shiraiwa
M.
,
Weller
M.G.
et al.
(
2017
)
Release of free amino acids upon oxidation of peptides and proteins by hydroxyl radicals
.
Anal. Bioanal. Chem.
409
,
2411
2420
[PubMed]
40.
Cadet
J.
,
Delatour
T.
,
Douki
T.
,
Gasparutto
D.
,
Pouget
J.P.
,
Ravanat
J.L.
et al.
(
1999
)
Hydroxyl radicals and DNA base damage
.
Mutat. Res.
424
,
9
21
41.
Gray
J.E.
,
Starmer
J.
,
Lin
V.S.
,
Dickinson
B.C.
and
Magnuson
T.
(
2013
)
Mitochondrial hydrogen peroxide and defective cholesterol efflux prevent in vitro fertilization by cryopreserved inbred mouse sperm
.
Biol. Reprod.
89
,
1
12
42.
Imlay
J.A.
,
Chin
S.M.
and
Linn
S.
(
1988
)
Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro
.
Science
240
,
640
642
[PubMed]
43.
Fridovich
I.
(
1998
)
Oxygen toxicity: a radical explanation
.
J. Exp. Biol.
201
,
1203
1209
[PubMed]
44.
Thomas
C.
,
Mackey
M.M.
,
Diaz
A.A.
and
Cox
D.P.
(
2009
)
Hydroxyl radical is produced via the Fenton reaction in submitochondrial particles under oxidative stress: implications for diseases associated with iron accumulation
.
Redox Rep.
14
,
102
108
[PubMed]
45.
Winterbourn
C.C.
(
2008
)
Reconciling the chemistry and biology of reactive oxygen species
.
Nat. Chem. Biol.
4
,
278
286
[PubMed]
46.
Holmström
K.M.
and
Finkel
T.
(
2014
)
Cellular mechanisms and physiological consequences of redox-dependent signalling
.
Nat. Rev. Mol. Cell Biol.
15
,
411
421
[PubMed]
47.
Figueroa
E.
,
Valdebenito
I.
,
Zepeda
A.B.
,
Figueroa
C.A.
,
Dumorné
K.
,
Castillo
R.L.
et al.
(
2017
)
Effects of cryopreservation on mitochondria of fish spermatozoa
.
Rev. Aquacult.
9
,
76
87
48.
Dalcin
L.
,
Silva
R.C.
,
Paulini
F.
,
Silva
B.D.M.
,
Neves
J.P.
and
Lucci
C.M.
(
2013
)
Cytoskeleton structure, pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos
.
Cryobiology
67
,
137
145
[PubMed]
49.
O’Connell
M.
,
McClure
N.
and
Lewis
S.E.M.
(
2002
)
The effects of cryopreservation on sperm morphology, motility and mitochondrial function
.
Hum. Reprod.
17
,
704
709
[PubMed]
50.
Turrens
J.F.
(
2003
)
Mitochondrial formation of reactive oxygen species
.
J. Physiol.
552
,
335
344
[PubMed]
51.
Starkov
A.A.
(
2008
)
The role of mitochondria in reactive oxygen species metabolism and signaling
.
Ann. N.Y. Acad. Sci.
1147
,
37
52
[PubMed]
52.
Boveris
A.
and
Chance
B.
(
1973
)
The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen
.
Biochem. J.
134
,
707
716
[PubMed]
53.
Adam Vizi
V.
and
Chinopoulos
C.
(
2006
)
Bioenergetics and the formation of mitochondrial reactive oxygen species
.
Trends Pharmacol. Sci.
27
,
639
645
[PubMed]
54.
Mailloux
R.J.
and
Harper
M.E.
(
2012
)
Mitochondrial proticity and ROS signaling: lessons from the uncoupling proteins
.
Trends Endocrinol. Metab.
23
,
451
458
[PubMed]
55.
Lambert
A.J.
and
Brand
M.D.
(
2004
)
Superoxide production by NADH:ubiquinone oxidoreductase (complex I) depends on the pH gradient across the mitochondrial inner membrane
.
Biochem. J.
382
,
511
517
[PubMed]
56.
Murphy
M.P.
(
2009
)
How mitochondria produce reactive oxygen species
.
Biochem. J.
417
,
1
13
[PubMed]
57.
Jones
A.
,
Van Blerkom
J.
,
Davis
P.
and
Toledo
A.A.
(
2004
)
Cryopreservation of metaphase II human oocytes effects mitochondrial membrane potential: Implications for developmental competence
.
Hum. Reprod.
19
,
1861
1866
[PubMed]
58.
Kadirvel
G.
,
Kumar
S.
and
Kumaresan
A.
(
2009
)
Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen
.
Anim. Reprod. Sci.
114
,
125
134
[PubMed]
59.
Matsushita
T.
,
Yagi
T.
,
Hardin
J.A.
,
Cragun
J.D.
,
Crow
F.W.
,
Bergen
H.R.
III
et al.
(
2003
)
Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver
.
Cell Transplant.
12
,
109
121
[PubMed]
60.
Starkov
A.A.
and
Fiskum
G.
(
2003
)
Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state
.
J. Neurochem.
86
,
1101
1107
[PubMed]
61.
Korshunov
S.S.
,
Skulachev
V.P.
and
Starkov
A.A.
(
1997
)
High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria
.
FEBS Lett.
416
,
15
18
[PubMed]
62.
Votyakova
T.V.
and
Reynolds
I.J.
(
2001
)
ΔΨm‐dependent and ‐independent production of reactive oxygen species by rat brain mitochondria
.
J. Neurochem.
79
,
266
277
[PubMed]
63.
Bolisetty
S.
and
Jaimes
E.A.
(
2013
)
Mitochondria and reactive oxygen species: physiology and pathophysiology
.
Int. J. Mol. Sci.
14
,
6306
6344
[PubMed]
64.
Zorov
D.B.
,
Filburn
C.R.
,
Klotz
L.O.
,
Zweier
J.L.
and
Sollott
S.J.
(
2000
)
Reactive oxygen species (ROS)-induced Ros release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes
.
J. Exp. Med.
192
,
1001
1014
[PubMed]
65.
Castro
L.S.
,
Hamilton
T.R.S.
,
Mendes
C.M.
,
Nichi
M.
,
Barnabe
V.H.
,
Visintin
J.A.
et al.
(
2016
)
Sperm cryodamage occurs after rapid freezing phase: flow cytometry approach and antioxidant enzymes activity at different stages of cryopreservation
.
J. Anim. Sci. Biotechnol.
7
,
17
66.
Chen
S.
,
Deng
X.
,
Ma
K.
,
Zhao
L.
,
Huang
D.
,
Li
Z.
et al.
(
2018
)
Icariin improves the viability and function of cryopreserved human nucleus pulposus-derived mesenchymal stem cells
.
Oxid. Med. Cell. Longev.
2018
,
3459612
67.
Sohn
I.P.
,
Ahn
H.J.
,
Park
D.W.
,
Gye
M.C.
,
Jo
D.H.
,
Kim
S.Y.
et al.
(
2001
)
Amelioration of mitochondrial dysfunction and apoptosis of two-cell mouse embryos after freezing and thawing by the high frequency liquid nitrogen infusion
.
Mol. Cells
13
,
272
280
68.
Słowińska
M.
,
Liszewska
E.
,
Judycka
S.
,
Konopka
M.
and
Ciereszko
A.
(
2018
)
Mitochondrial membrane potential and reactive oxygen species in liquid stored and cryopreserved turkey (Meleagris gallopavo) spermatozoa
.
Poult. Sci.
97
,
3709
3717
[PubMed]
69.
Zee
Y.P.
,
Holt
W.V.
,
Allen
C.D.
,
Nicolson
V.
,
Burridge
M.
,
Lisle
A.
et al.
(
2007
)
Effects of cryopreservation on mitochondrial function and heterogeneity, lipid raft stability and phosphatidylserine translocation in koala (Phascolarctos cinereus) spermatozoa
.
Reprod. Fertil. Dev.
19
,
850
860
70.
Demant
M.
,
Trapphoff
T.
,
Fröhlich
T.
,
Arnold
G.J.
and
Eichenlaub Ritter
U.
(
2012
)
Vitrification at the pre-antral stage transiently alters inner mitochondrial membrane potential but proteome of in vitro grown and matured mouse oocytes appears unaffected
.
Hum. Reprod.
27
,
1096
1111
[PubMed]
71.
Halestrap
A.P.
(
2009
)
What is the mitochondrial permeability transition pore?
J. Mol. Cell Cardiol.
46
,
821
831
[PubMed]
72.
Petronilli
V.
,
Penzo
D.
,
Scorrano
L.
,
Bernardi
P.
and
Di Lisa
F.
(
2001
)
The mitochondrial permeability transition, release of cytochrome c and cell death correlation with the duration of pore openings in situ
.
J. Biol. Chem.
276
,
12030
12034
[PubMed]
73.
Ortega Ferrusola
C.
,
González Fernández
L.
,
Salazar Sandoval
C.
,
Macías García
B.
,
Rodríguez Martínez
H.
,
Tapia
J.A.
et al.
(
2010
)
Inhibition of the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of stallion spermatozoa
.
Theriogenology
74
,
458
465
74.
Batandier
C.
,
Leverve
X.
and
Fontaine
E.
(
2004
)
Opening of the mitochondrial permeability transition pore induces reactive oxygen species production at the level of the respiratory chain complex I
.
J. Biol. Chem.
279
,
17197
17204
[PubMed]
75.
Bonke
E.
,
Siebels
I.
,
Zwicker
K.
and
Dröse
S.
(
2016
)
Manganese ions enhance mitochondrial H2O2 emission from Krebs cycle oxidoreductases by inducing permeability transition
.
Free Radic. Biol. Med.
99
,
43
53
[PubMed]
76.
Dong
Q.
,
Tollner
T.L.
,
Rodenburg
S.E.
,
Hill
D.L.
and
VandeVoort
C.A.
(
2010
)
Antioxidants, oxyrase, and mitochondrial uncoupler 2,4-dinitrophenol improved postthaw survival of rhesus monkey sperm from ejaculates with low cryosurvival
.
Fertil. Steril.
94
,
2359
2361
[PubMed]
77.
Vercesi
A.E.
,
Kowaltowski
A.J.
,
Grijalba
M.T.
,
Meinicke
A.R.
and
Castilho
R.F.
(
1997
)
The role of reactive oxygen species in mitochondrial permeability transition
.
Biosci. Rep.
17
,
43
52
[PubMed]
78.
Valle
V.G.R.
,
Fagian
M.M.
,
Parentoni
L.S.
,
Meinicke
A.R.
and
Vercesi
A.E.
(
1993
)
The participation of reactive oxygen species and protein thiols in the mechanism of mitochondrial inner membrane permeabilization by calcium plus prooxidants
.
Arch. Biochem. Biophys.
307
,
1
7
[PubMed]
79.
Zhao
N.
,
Liu
X.J.
,
Li
J.T.
,
Zhang
L.
,
Fu
Y.
,
Zhang
Y.J.
et al.
(
2015
)
Endoplasmic reticulum stress inhibition is a valid therapeutic strategy in vitrifying oocytes
.
Cryobiology
70
,
48
52
80.
Barrera
N.
,
dos Santos Neto
P.C.
,
Cuadro
F.
,
Bosolasco
D.
,
Mulet
A.P.
,
Crispo
M.
et al.
(
2018
)
Impact of delipidated estrous sheep serum supplementation on in vitro maturation, cryotolerance and endoplasmic reticulum stress gene expression of sheep oocytes
.
PLoS ONE
13
,
e0198742
[PubMed]
81.
Brito
D.C.
,
Brito
A.B.
,
Scalercio
S.R.R.A.
,
Percário
S.
,
Miranda
M.S.
,
Rocha
R.M.
et al.
(
2014
)
Vitamin E-analog Trolox prevents endoplasmic reticulum stress in frozen-thawed ovarian tissue of capuchin monkey (Sapajus apella)
.
Cell Tissue Res.
355
,
471
480
[PubMed]
82.
Lin
T.
,
Lee
J.E.
,
Shin
H.Y.
,
Oqani
R.
,
Kim
S.Y.
and
Jin
D.I.
(
2016
)
Supplement of tauroursodeoxycholic acid in vitrification solution improves the development of mouse embryos
.
Korean J. Agric. Sci.
43
,
575
580
83.
Zeeshan
H.M.A.
,
Lee
G.H.
,
Kim
H.R.
and
Chae
H.J.
(
2016
)
Endoplasmic reticulum stress and associated ROS
.
Int. J. Mol. Sci.
17
,
327
[PubMed]
84.
Tu
B.P.
and
Weissman
J.S.
(
2004
)
Oxidative protein folding in eukaryotes
.
J. Cell Biol.
164
,
341
346
[PubMed]
85.
Walter
P.
and
Ron
D.
(
2011
)
The unfolded protein response: from stress pathway to homeostatic regulation
.
Science
334
,
1081
1086
[PubMed]
86.
Hetz
C.
and
Papa
F.R.
(
2018
)
The unfolded protein response and cell fate control
.
Mol. Cell
69
,
169
181
[PubMed]
87.
Takahashi
S.
,
Ando
A.
,
Takagi
H.
and
Shima
J.
(
2009
)
Insufficiency of copper ion homeostasis causes freeze-thaw injury of yeast cells as revealed by indirect gene expression analysis
.
Appl. Environ. Microbiol.
75
,
6706
6711
[PubMed]
88.
Abraham
T.
,
Pin
C.L.
and
Watson
A.J.
(
2012
)
Embryo collection induces transient activation of XBP1 arm of the ER stress response while embryo vitrification does not
.
Mol. Hum. Reprod.
18
,
229
242
[PubMed]
89.
Tabas
I.
and
Ron
D.
(
2011
)
Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress
.
Nat. Cell. Biol.
13
,
184
190
[PubMed]
90.
Haynes
C.M.
,
Titus
E.A.
and
Cooper
A.A.
(
2004
)
Degradation of misfolded proteins prevents ER-derived oxidative stress and cell death
.
Mol. Cell
15
,
767
776
[PubMed]
91.
Kincaid
M.M.
and
Cooper
A.A.
(
2007
)
ERADicate ER stress or die trying
.
Antioxid. Redox Signal.
9
,
2373
2387
92.
Eletto
D.
,
Chevet
E.
,
Argon
Y.
and
Appenzeller Herzog
C.
(
2014
)
Redox controls UPR to control redox
.
J. Cell Sci.
127
,
3649
3658
[PubMed]
93.
Cuozzo
J.W.
and
Kaiser
C.A.
(
1999
)
Competition between glutathione and protein thiols for disulphide-bond formation
.
Nat. Cell Biol.
1
,
130
135
[PubMed]
94.
Tu
B.P.
and
Weissman
J.S.
(
2002
)
The FAD- and O(2)-dependent reaction cycle of Ero1-mediated oxidative protein folding in the endoplasmic reticulum
.
Mol. Cell
10
,
983
994
[PubMed]
95.
Ramming
T.
,
Okumura
M.
,
Kanemura
S.
,
Baday
S.
,
Birk
J.
,
Moes
S.
et al.
(
2015
)
A PDI-catalyzed thiol–disulfide switch regulates the production of hydrogen peroxide by human Ero1
.
Free Radic. Biol. Med.
83
,
361
372
[PubMed]
96.
Tan
S.X.
,
Teo
M.
,
Lam
Y.T.
,
Dawes
I.W.
and
Perrone
G.G.
(
2009
)
Cu, Zn superoxide dismutase and NADP(H) homeostasis are required for tolerance of endoplasmic reticulum stress in Saccharomyces cerevisiae
.
Mol. Biol. Cell
20
,
1493
1508
[PubMed]
97.
Zito
E.
,
Melo
E.P.
,
Yang
Y.
,
Wahlander
Å.
,
Neubert
T.A.
and
Ron
D.
(
2010
)
Oxidative protein folding by an endoplasmic reticulum-localized peroxiredoxin
.
Mol. Cell
40
,
787
797
[PubMed]
98.
Konno
T.
,
Melo
E.P.
,
Lopes
C.
,
Mehmeti
I.
,
Lenzen
S.
,
Ron
D.
et al.
(
2015
)
ERO1-independent production of H2O2 within the endoplasmic reticulum fuels Prdx4-mediated oxidative protein folding
.
J. Cell Biol.
211
,
253
259
[PubMed]
99.
Win
S.
,
Than
T.
,
Fernandez Checa
J.
and
Kaplowitz
N.
(
2014
)
JNK interaction with Sab mediates ER stress induced inhibition of mitochondrial respiration and cell death
.
Cell Death Dis.
5
,
e989
100.
Habib
S.
and
Ali
A.
(
2011
)
Biochemistry of nitric oxide
.
Indian J. Clin. Biochem.
26
,
3
17
[PubMed]
101.
Trujillo
M.
,
Ferrer Sueta
G.
and
Radi
R.
(
2008
)
Peroxynitrite detoxification and its biologic implications
.
Antioxid. Redox Signal.
10
,
1607
1620
102.
Pacher
P.
,
Beckman
J.S.
and
Liaudet
L.
(
2007
)
Nitric oxide and peroxynitrite in health and disease
.
Physiol. Rev.
87
,
315
424
[PubMed]
103.
Wink
D.A.
,
Vodovotz
Y.
,
Laval
J.
,
Laval
F.
,
Dewhirst
M.W.
and
Mitchell
J.B.
(
1998
)
The multifaceted roles of nitric oxide in cancer
.
Carcinogenesis
19
,
711
721
[PubMed]
104.
Förstermann
U.
and
Sessa
W.C.
(
2012
)
Nitric oxide synthases: regulation and function
.
Eur. Heart J.
33
,
829
837
[PubMed]
105.
Knowles
R.G.
and
Moncada
S.
(
1994
)
Nitric oxide synthases in mammals
.
Biochem. J.
298
,
249
258
[PubMed]
106.
Mitrovic
B.
,
Ignarro
L.J.
,
Vinters
H.V.
,
Akers
M.A.
,
Schmid
I.
,
Uittenbogaart
C.
et al.
(
1995
)
Nitric oxide induces necrotic but not apoptotic cell death in oligodendrocytes
.
Neuroscience
65
,
531
539
[PubMed]
107.
Bal Price
A.
and
Brown
G.C.
(
2000
)
Nitric‐oxide‐induced necrosis and apoptosis in PC12 cells mediated by mitochondria
.
J. Neurochem.
75
,
1455
1464
[PubMed]
108.
Kaneto
H.
,
Fujii
J.
,
Seo
H.G.
,
Suzuki
K.
,
Matsuko
T.A.
,
Masahiro
N.
et al.
(
1995
)
Apoptotic cell death triggered by nitric oxide in pancreatic β-cells
.
Diabetes
44
,
733
738
[PubMed]
109.
Uchiyama
T.
,
Otani
H.
,
Okada
T.
,
Ninomiya
H.
,
Kido
M.
,
Imamura
H.
et al.
(
2002
)
Nitric oxide induces caspase-dependent apoptosis and necrosis in neonatal rat cardiomyocytes
.
J. Mol. Cell Cardiol.
34
,
1049
1061
[PubMed]
110.
Jiang
Z.L.
,
Fletcher
N.M.
,
Diamond
M.P.
,
Abu Soud
H.M.
and
Saed
G.M.
(
2009
)
S‐nitrosylation of caspase‐3 is the mechanism by which adhesion fibroblasts manifest lower apoptosis
.
Wound Repair Regen.
17
,
224
229
[PubMed]
111.
Ignarro
L.J.
,
Cirino
G.
,
Casini
A.
and
Napoli
C.
(
1999
)
Nitric oxide as a signaling molecule in the vascular system: an overview
.
J. Cardiovasc. Pharmacol.
34
,
879
886
[PubMed]
112.
Sato
K.
,
Ozaki
K.
,
Oh
I.
,
Meguro
A.
,
Hatanaka
K.
,
Nagai
T.
et al.
(
2007
)
Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells
.
Blood
109
,
228
234
[PubMed]
113.
Ali
G.
,
Mohsin
S.
,
Khan
M.
,
Nasir
G.A.
,
Shams
S.
,
Khan
S.N.
et al.
(
2012
)
Nitric oxide augments mesenchymal stem cell ability to repair liver fibrosis
.
J. Transl. Med.
10
,
75
114.
Michurina
T.
,
Krasnov
P.
,
Balazs
A.
,
Nakaya
N.
,
Vasilieva
T.
,
Kuzin
B.
et al.
(
2004
)
Nitric oxide is a regulator of hematopoietic stem cell activity
.
Mol. Ther.
10
,
241
248
[PubMed]
115.
Herrero
M.B.
,
Chatterjee
S.
,
Lefièvre
L.
,
de Lamirande
E.
and
Gagnon
C.
(
2000
)
Nitric oxide interacts with the cAMP pathway to modulate capacitation of human spermatozoa
.
Free Radic. Biol. Med.
29
,
522
536
[PubMed]
116.
Ortega Ferrusola
C.
,
González Fernández
L.
,
Macías García
B.
,
Salazar Sandoval
C.
,
Morillo Rodríguez
A.
,
Rodríguez Martinez
H.
et al.
(
2009
)
Effect of cryopreservation on nitric oxide production by stallion spermatozoa
.
Biol. Reprod.
81
,
1106
1111
[PubMed]
117.
Sharafi
M.
,
Zhandi
M.
,
Shahverdi
A.
and
Shakeri
M.
(
2015
)
Beneficial effects of nitric oxide iduced mild oxidative stress on post-thawed bull semen quality
.
Int. J. Fertil. Steril.
9
,
230
237
[PubMed]
118.
Geissler
H.J.
,
Fischer
U.M.
,
Foerster
S.
,
Krahwinkel
A.
,
Antonyan
A.
,
Kroener
A.
et al.
(
2006
)
Effect of thawing on nitric oxide synthase III and apoptotic markers in cryopreserved human allografts
.
Ann. Thorac. Surg.
82
,
1742
1746
[PubMed]
119.
Saeednia
S.
,
Shabani Nashtaei
M.
,
Bahadoran
H.
,
Aleyasin
A.
and
Amidi
F.
(
2016
)
Effect of nerve growth factor on sperm quality in asthenozoosprmic men during cryopreservation
.
Reprod. Biol. Endocrinol.
14
,
29
120.
Rogers
S.C.
,
Dosier
L.B.
,
McMahon
T.J.
,
Zhu
H.
,
Timm
D.
,
Zhang
H.
et al.
(
2018
)
Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes
.
PLoS ONE
13
,
e0209201
[PubMed]
121.
Ramya
T.
,
Misro
M.M.
,
Sinha
D.
,
Nandan
D.
and
Mithal
S.
(
2011
)
Altered levels of seminal nitric oxide, nitric oxide synthase, and enzymatic antioxidants and their association with sperm function in infertile subjects
.
Fertil. Steril.
95
,
135
140
[PubMed]
122.
Werner
C.
,
Cadoná
F.C.
,
da Cruz
I.B.M.
,
Flôres
E.R.D.S.
,
Machado
A.K.
,
Fantinel
M.R.
et al.
(
2017
)
A chemical compound based on methylxanthine–polyphenols lowers nitric oxide levels and increases post-thaw human sperm viability
.
Zygote
25
,
719
730
[PubMed]
123.
O’Bryan
M.K.
,
Zini
A.
,
Cheng
C.Y.
and
Schlegel
P.N.
(
1998
)
Human sperm endothelial nitric oxide synthase expression: correlation with sperm motility
.
Fertil. Steril.
70
,
1143
1147
[PubMed]
124.
Dinara
S.
,
Sengoku
K.
,
Tamate
K.
,
Horikawa
M.
and
Ishikawa
M.
(
2001
)
Effects of supplementation with free radical scavengers on the survival and fertilization rates of mouse cryopreserved oocytes
.
Hum. Reprod.
16
,
1976
1981
[PubMed]
125.
Martins
H.
,
da Silva
G.
,
Cortes
S.
,
Paes
F.
,
Martins Filho
O.
,
Araujo
M.
et al.
(
2018
)
Lactoferrin increases sperm membrane functionality of frozen equine semen
.
Reprod. Domest. Anim.
53
,
617
623
[PubMed]
126.
Meitzler
J.L.
,
Antony
S.
,
Wu
Y.
,
Juhasz
A.
,
Liu
H.
,
Jiang
G.
et al.
(
2014
)
NADPH oxidases: a perspective on reactive oxygen species production in tumor biology
.
Antioxid. Redox Signal.
20
,
2873
2889
127.
Block
K.
and
Gorin
Y.
(
2012
)
Aiding and abetting roles of NOX oxidases in cellular transformation
.
Nat. Rev. Cancer
12
,
627
637
[PubMed]
128.
Bonekamp
N.A.
,
Völkl
A.
,
Dariush Fahimi
H.
and
Schrader
M.
(
2009
)
Reactive oxygen species and peroxisomes: struggling for balance
.
Biofactors
35
,
346
355
[PubMed]
129.
Jiang
F.
,
Zhang
Y.
and
Dusting
G.J.
(
2011
)
NADPH oxidase-mediated redox signaling: roles in cellular stress response, stress tolerance, and tissue repair
.
Pharmacol. Rev.
63
,
218
242
[PubMed]
130.
Shibuya
H.
,
Ohkohchi
N.
,
Seya
K.
and
Satomi
S.
(
1997
)
Kupffer cells generate superoxide anions and modulate reperfusion injury in rat livers after cold preservation
.
Hepatology
25
,
356
360
131.
Martins
A.S.
,
Shkryl
V.M.
,
Nowycky
M.C.
and
Shirokova
N.
(
2008
)
Reactive oxygen species contribute to Ca2+ signals produced by osmotic stress in mouse skeletal muscle fibres
.
J. Physiol.
586
,
197
210
[PubMed]
132.
Abramov
A.Y.
,
Jacobson
J.
,
Wientjes
F.
,
Hothersall
J.
,
Canevari
L.
and
Duchen
M.R.
(
2005
)
Expression and modulation of an NADPH oxidase in mammalian astrocytes
.
J. Neurosci.
25
,
9176
9184
[PubMed]
133.
Watson
P.F.
(
1995
)
Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function
.
Reprod. Fertil. Dev.
7
,
871
891
134.
Oldenhof
H.
,
Gojowsky
M.
,
Wang
S.
,
Henke
S.
,
Yu
C.
,
Rohn
K.
et al.
(
2013
)
Osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents
.
Biol. Reprod.
88
,
1
11
[PubMed]
135.
Ball
B.A.
(
2008
)
Oxidative stress, osmotic stress and apoptosis: Impacts on sperm function and preservation in the horse
.
Anim. Reprod. Sci.
107
,
257
267
[PubMed]
136.
Reinehr
R.
,
Görg
B.
,
Becker
S.
,
Qvartskhava
N.
,
Bidmon
H.J.
,
Selbach
O.
et al.
(
2007
)
Hypoosmotic swelling and ammonia increase oxidative stress by NADPH oxidase in cultured astrocytes and vital brain slices
.
Glia
55
,
758
771
[PubMed]
137.
Flor
A.
,
Doshi
A.
and
Kron
S.
(
2016
)
Modulation of therapy-induced senescence by reactive lipid aldehydes
.
Cell Death Discov.
2
,
16045
[PubMed]
138.
Luo
H.
,
Yang
A.
,
Schulte
B.A.
,
Wargovich
M.J.
and
Wang
G.Y.
(
2013
)
Resveratrol induces premature senescence in lung cancer cells via ROS-mediated DNA damage
.
PLoS ONE
8
,
e60065
[PubMed]
139.
Meng
T.C.
,
Fukada
T.
and
Tonks
N.K.
(
2002
)
Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo
.
Mol. Cell
9
,
387
399
[PubMed]
140.
Schieber
M.
and
Chandel
N.S.
(
2014
)
ROS function in redox signaling and oxidative stress
.
Curr. Biol.
24
,
R453
R462
[PubMed]
141.
Marrocco
I.
,
Altieri
F.
and
Peluso
I.
(
2017
)
Measurement and clinical significance of biomarkers of oxidative stress in humans
.
Oxid. Med. Cell. Longev.
2017
,
6501046
[PubMed]
142.
Devi
L.
,
Makala
H.
,
Pothana
L.
,
Nirmalkar
K.
and
Goel
S.
(
2014
)
Comparative efficacies of six different media for cryopreservation of immature buffalo (Bubalus bubalis) calf testis
.
Reprod. Fertil. Dev.
28
,
872
885
143.
Lee
J.
,
Kim
S.K.
,
Youm
H.W.
,
Kim
H.J.
,
Lee
J.R.
,
Suh
C.S.
et al.
(
2015
)
Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation
.
PLoS ONE
10
,
e0126252
[PubMed]
144.
Del Bo’
C.
,
Fracassetti
D.
,
Lanti
C.
,
Porrini
M.
and
Riso
P.
(
2015
)
Comparison of DNA damage by the comet assay in fresh versus cryopreserved peripheral blood mononuclear cells obtained following dietary intervention
.
Mutagenesis
30
,
29
35
[PubMed]
145.
Cao
Y.
,
Qu
H.J
,
Li
P.
,
Wang
C.B.
,
Wang
L.X.
and
Han
Z.W.
(
2011
)
Single dose administration of L-carnitine improves antioxidant activities in healthy subjects
.
Tohoku J. Exp. Med.
224
,
209
213
[PubMed]
146.
Gülçin
İ.
(
2006
)
Antioxidant and antiradical activities of l-carnitine
.
Life Sci.
78
,
803
811
[PubMed]
147.
Banihani
S.
,
Agarwal
A.
,
Sharma
R.
and
Bayachou
M.
(
2014
)
Cryoprotective effect of l‐carnitine on motility, vitality and DNA oxidation of human spermatozoa
.
Andrologia
46
,
637
641
[PubMed]
148.
Cabrita
E.
,
Ma
S.
,
Diogo
P.
,
Martínez Páramo
S.
,
Sarasquete
C.
and
Dinis
M.T.
(
2011
)
The influence of certain aminoacids and vitamins on post-thaw fish sperm motility, viability and DNA fragmentation
.
Anim. Reprod. Sci.
125
,
189
195
[PubMed]
149.
Li
Z.
,
Lin
Q.
,
Liu
R.
,
Xiao
W.
and
Liu
W.
(
2010
)
Protective effects of ascorbate and catalase on human spermatozoa during cryopreservation
.
J. Androl.
31
,
437
444
[PubMed]
150.
Branco
C.S.
,
Garcez
M.E.
,
Pasqualotto
F.F.
,
Erdtman
B.
and
Salvador
M.
(
2010
)
Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen
.
Cryobiology
60
,
235
237
[PubMed]
151.
Kalthur
G.
,
Raj
S.
,
Thiyagarajan
A.
,
Kumar
S.
,
Kumar
P.
and
Adiga
S.K.
(
2011
)
Vitamin E supplementation in semen-freezing medium improves the motility and protects sperm from freeze-thaw–induced DNA damage
.
Fertil. Steril.
95
,
1149
1151
[PubMed]
152.
Hosseini
S.M.
,
Forouzanfar
M.
,
Hajian
M.
,
Asgari
V.
,
Abedi
P.
,
Hosseini
L.
et al.
(
2009
)
Antioxidant supplementation of culture medium during embryo development and/or after vitrification-warming; which is the most important?
J. Assist. Reprod. Genet.
26
,
355
364
[PubMed]
153.
Shi
X.
,
Hu
H.
,
Ji
G.
,
Zhang
J.
,
Liu
R.
,
Zhang
H.
et al.
(
2018
)
Protective effect of sucrose and antioxidants on cryopreservation of sperm motility and DNA integrity in C57BL/6 mice
.
Biopreserv. Biobank.
16
,
444
450
154.
Yousefian
I.
,
Emamverdi
M.
,
Karamzadeh Dehaghani
A.
,
Sabzian Melei
R.
,
Zhandi
M.
and
Zare Shahneh
A.
(
2018
)
Attenuation of cryopreservation-induced oxidative stress by antioxidant: impact of Coenzyme Q10 on the quality of post-thawed buck spermatozoa
.
Cryobiology
81
,
88
93
[PubMed]
155.
Isaac
A.V.
,
Kumari
S.
,
Nair
R.
,
Urs
D.R.
,
Salian
S.R.
,
Kalthur
G.
et al.
(
2017
)
Supplementing zinc oxide nanoparticles to cryopreservation medium minimizes the freeze-thaw-induced damage to spermatozoa
.
Biochem. Biophys. Res. Commun.
494
,
656
662
[PubMed]
156.
Succu
S.
,
Berlinguer
F.
,
Pasciu
V.
,
Satta
V.
,
Leoni
G.G.
and
Naitana
S.
(
2011
)
Melatonin protects ram spermatozoa from cryopreservation injuries in a dose‐dependent manner
.
J. Pineal Res.
50
,
310
318
[PubMed]
157.
Bortolin
R.C.
,
Gasparotto
J.
,
Vargas
A.R.
,
da Silva Morrone
M.
,
Kunzler
A.
,
Henkin
B.S.
et al.
(
2017
)
Effects of freeze-thaw and storage on enzymatic activities, protein oxidative damage, and immunocontent of the blood, liver, and brain of rats
.
Biopreserv. Biobank.
15
,
182
190
158.
Alotaibi
N.A.S.
,
Slater
N.K.H.
and
Rahmoune
H.
(
2016
)
Salidroside as a novel protective agent to improve red blood cell cryopreservation
.
PLoS ONE
11
,
e0162748
[PubMed]
159.
Mostek
A.
,
Dietrich
M.A.
,
Słowińska
M.
and
Ciereszko
A.
(
2017
)
Cryopreservation of bull semen is associated with carbonylation of sperm proteins
.
Theriogenology
92
,
95
102
[PubMed]
160.
Tai
W.Y.
,
Yang
Y.C.
,
Lin
H.J.
,
Huang
C.P.
,
Cheng
Y.L.
,
Chen
M.F.
et al.
(
2010
)
Interplay between structure and fluidity of model lipid membranes under oxidative attack
.
J. Phys. Chem. B
114
,
15642
15649
[PubMed]
161.
Yusupov
M.
,
Wende
K.
,
Kupsch
S.
,
Neyts
E.C.
,
Reuter
S.
and
Bogaerts
A.
(
2017
)
Effect of head group and lipid tail oxidation in the cell membrane revealed through integrated simulations and experiments
.
Sci. Rep.
7
,
5761
[PubMed]
162.
Whiteley
G.S.W.
,
Fuller
B.J.
and
Hobbs
K.E.F.
(
1992
)
Deterioration of cold-stored tissue specimens due to lipid peroxidation: modulation by antioxidants at high subzero temperatures
.
Cryobiology
29
,
668
673
[PubMed]
163.
Ortega Ferrusola
C.
,
González Fernández
L.
,
Morrell
J.M.
,
Salazar Sandoval
C.
,
Macías García
B.
,
Rodríguez Martinez
H.
et al.
(
2009
)
Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is related to apoptotic-like changes
.
Reproduction
138
,
55
63
[PubMed]
164.
Martin Muñoz
P.
,
Ortega Ferrusola
C.
,
Vizuete
G.
,
Plaza Dávila
M.
,
Rodriguez Martinez
H.
and
Peña
F.J.
(
2015
)
Depletion of intracellular thiols and increased production of 4-hydroxynonenal that occur during cryopreservation of stallion spermatozoa lead to caspase activation, loss of motility, and cell death
.
Biol. Reprod.
93
,
143
[PubMed]
165.
Alvarez
J.G.
and
Storey
B.T.
(
1992
)
Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation
.
J. Androl.
13
,
232
241
[PubMed]
166.
Schuffner
A.
,
Morshedi
M.
and
Oehninger
S.
(
2001
)
Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation
.
Hum. Reprod.
16
,
2148
2153
[PubMed]
167.
Puts
C.F.
,
Berendsen
T.A.
,
Bruinsma
B.G.
,
Ozer
S.
,
Luitje
M.
,
Usta
O.B.
et al.
(
2015
)
Polyethylene glycol protects primary hepatocytes during supercooling preservation
.
Cryobiology
71
,
125
129
[PubMed]
168.
Swami
D.S.
,
Kumar
P.
,
Malik
R.K.
,
Saini
M.
,
Kumar
D.
and
Jan
M.H.
(
2017
)
The cryoprotective effect of iodixanol in buffalo semen cryopreservation
.
Anim. Reprod. Sci.
179
,
20
26
[PubMed]
169.
Odani
M.
,
Komatsu
Y.
,
Oka
S.
and
Iwahashi
H.
(
2003
)
Screening of genes that respond to cryopreservation stress using yeast DNA microarray
.
Cryobiology
47
,
155
164
[PubMed]
170.
Park
J.I.
,
Grant
C.M.
,
Davies
M.J.
and
Dawes
I.W.
(
1998
)
The cytoplasmic Cu,Zn superoxide dismutase of Saccharomyces cerevisiae is required for resistance to freeze-thaw stress. Generation of free radicals during freezing and thawing
.
J. Biol. Chem.
273
,
22921
22928
[PubMed]
171.
Habibi
A.
,
Farrokhi
N.
,
Moreira da Silva
F.
,
Bettencourt
B.F.
,
Bruges Armas
J.
,
Amidi
F.
et al.
(
2010
)
The effects of vitrification on gene expression in mature mouse oocytes by nested quantitative PCR
.
J. Assist. Reprod. Genet.
27
,
599
604
[PubMed]
172.
Boonkusol
D.
,
Gal
A.B.
,
Bodo
S.
,
Gorhony
B.
,
Kitiyanant
Y.
and
Dinnyes
A.
(
2006
)
Gene expression profiles and in vitro development following vitrification of pronuclear and 8‐cell stage mouse embryos
.
Mol. Reprod. Dev.
73
,
700
708
[PubMed]
173.
Coppola
S.
and
Ghibelli
L.
(
2000
)
GSH extrusion and the mitochondrial pathway of apoptotic signalling
.
Biochem. Soc. Trans.
28
,
56
61
[PubMed]
174.
Chen
G.Q.
,
Ren
L.
,
Zhang
D.
and
Shen
X.H.
(
2016
)
Glutathione improves survival of cryopreserved embryogenic calli of Agapanthus praecox subsp. orientalis
.
Acta Physiol. Plant.
38
,
250
175.
Estrada
E.
,
Rivera del Álamo
M.M.
,
Rodríguez Gil
J.E.
and
Yeste
M.
(
2017
)
The addition of reduced glutathione to cryopreservation media induces changes in the structure of motile subpopulations of frozen-thawed boar sperm
.
Cryobiology
78
,
56
64
[PubMed]
176.
Moawad
A.R.
,
Tan
S.L.
and
Taketo
T.
(
2017
)
Beneficial effects of glutathione supplementation during vitrification of mouse oocytes at the germinal vesicle stage on their preimplantation development following maturation and fertilization in vitro
.
Cryobiology
76
,
98
103
[PubMed]
177.
Vara
E.
,
Arias Dı́iaz
J.
,
Villa
N.
,
Hernández
J.
,
Garcı́a
C.
,
Ortiz
P.
et al.
(
1995
)
Beneficial effect of S-Adenosylmethionine during both cold storage and cryopreservation of isolated hepatocytes
.
Cryobiology
32
,
422
427
[PubMed]
178.
Ha
S.J.
,
Kim
B.G.
,
Lee
Y.A.
,
Kim
Y.H.
,
Kim
B.J.
,
Jung
S.E.
et al.
(
2016
)
Effect of antioxidants and apoptosis inhibitors on cryopreservation of murine germ cells enriched for spermatogonial stem cells
.
PLoS ONE
11
,
e0161372
[PubMed]
179.
Hwang
C.
,
Sinskey
A.
and
Lodish
H.
(
1992
)
Oxidized redox state of glutathione in the endoplasmic reticulum
.
Science
257
,
1496
1502
[PubMed]
180.
Do
V.H.
,
Walton
S.
,
Catt
S.
and
Taylor Robinson
A.W.
(
2018
)
Does the addition of salubrinal to in vitro maturation medium enhance bovine blastocyst yields and embryo cryotolerance?
Cryoletters
39
,
219
226
[PubMed]
181.
English
T.E.
and
Storey
K.B.
(
2003
)
Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastropod Littorina littorea
.
J. Exp. Biol.
206
,
2517
2524
[PubMed]
182.
Larade
K.
and
Storey
K.B.
(
2004
)
Accumulation and translation of ferritin heavy chain transcripts following anoxia exposure in a marine invertebrate
.
J. Exp. Biol.
207
,
1353
1360
[PubMed]
183.
Tarín
J.J.
and
Trounson
A.O.
(
1993
)
Effects of stimulation or inhibition of lipid peroxidation on freezing-thawing of mouse embryos
.
Biol. Reprod.
49
,
1362
1368
[PubMed]
184.
Metherel
A.H.
and
Stark
K.D.
(
2015
)
Cryopreservation prevents iron-initiated highly unsaturated fatty acid loss during storage of human blood on chromatography paper at −20°C
.
J. Nutr.
145
,
654
660
[PubMed]
185.
Ozkavukcu
S.
,
Erdemli
E.
,
Isik
A.
,
Oztuna
D.
and
Karahuseyinoglu
S.
(
2008
)
Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa
.
J. Assist. Reprod. Genet.
25
,
403
411
[PubMed]
186.
Paudel
K.
,
Kumar
S.
,
Meur
S.
and
Kumaresan
A.
(
2010
)
Ascorbic acid, catalase and chlorpromazine reduce cryopreservation-induced damages to crossbred bull spermatozoa
.
Reprod. Domest. Anim.
45
,
256
262
[PubMed]
187.
Thomson
L.K.
,
Fleming
S.D.
,
Aitken
R.J.
,
De Iuliis
G.N.
,
Zieschang
J.A.
and
Clark
A.M.
(
2009
)
Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis
.
Hum. Reprod.
24
,
2061
2070
[PubMed]
188.
Beconi
M.T.
,
Francia
C.R.
,
Mora
N.G.
and
Affranchino
M.A.
(
1993
)
Effect of natural antioxidants on frozen bovine semen preservation
.
Theriogenology
40
,
841
851
[PubMed]
189.
Aliakbari
F.
,
Gilani
M.A.S.
,
Amidi
F.
,
Baazm
M.
,
Korouji
M.
,
Izadyar
F.
et al.
(
2016
)
Improving the efficacy of cryopreservation of spermatogonia stem cells by antioxidant supplements
.
Cell. Reprogram.
18
,
87
95
[PubMed]
190.
Duru
N.K.
,
Morshedi
M.
,
Schuffner
A.
and
Oehninger
S.
(
2001
)
Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine
.
Fertil. Steril.
75
,
263
268
[PubMed]
191.
Paasch
U.
,
Sharma
R.K.
,
Gupta
A.K.
,
Grunewald
S.
,
Mascha
E.J.
,
Thomas
A.J.J.
et al.
(
2004
)
Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa
.
Biol. Reprod.
71
,
1828
1837
[PubMed]
192.
Martin
G.
,
Sabido
O.
,
Durand
P.
and
Levy
R.
(
2004
)
Cryopreservation induces an apoptosis-like mechanism in bull sperm
.
Biol. Reprod.
71
,
28
37
[PubMed]
193.
Ortega Ferrusola
C.
,
Sotillo Galán
Y.
,
Varela Fernández
E.
,
Gallardo Bolaños
J.M.
,
Muriel
A.
,
González Fernández
L.
et al.
(
2008
)
Detection of “apoptosis‐like” changes during the cryopreservation process in equine sperm
.
J. Androl.
29
,
213
221
[PubMed]
194.
Stroh
C.
,
Cassens
U.
,
Samraj
A.K.
,
Sibrowski
W.
,
Schulze Osthoff
K.
and
Los
M.
(
2002
)
The role of caspases in cryoinjury: caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells
.
FASEB J.
16
,
1651
1653
[PubMed]
195.
Yagi
T.
,
Hardin
J.A.
,
Valenzuela
Y.M.
,
Miyoshi
H.
,
Gores
G.J.
and
Nyberg
S.L.
(
2001
)
Caspase inhibition reduces apoptotic death of cryopreserved porcine hepatocytes
.
Hepatology
33
,
1432
1440
[PubMed]
196.
Heng
B.C.
,
Clement
M.V.
and
Cao
T.
(
2007
)
Caspase inhibitor Z-VAD-FMK enhances the freeze-thaw survival rate of human embryonic stem cells
.
Biosci. Rep.
27
,
257
264
[PubMed]
197.
Aliakbari
F.
,
Gilani
M.A.S.
,
Yazdekhasti
H.
,
Koruji
M.
,
Asgari
H.R.
,
Baazm
M.
et al.
(
2017
)
Effects of antioxidants, catalase and α-tocopherol on cell viability and oxidative stress variables in frozen-thawed mice spermatogonial stem cells
.
Artif. Cells Nanomed. Biotechnol.
45
,
63
68
198.
Chen
X.J.
,
Zhang
Y.
,
Jia
G.X.
,
Meng
Q.G.
,
Bunch
T.D.
,
Liu
G.S.
et al.
(
2016
)
Effect of melatonin supplementation on cryopreserved sperm quality in mouse
.
Cryoletters
37
,
115
122
[PubMed]
199.
Deng
S.L.
,
Sun
T.C.
,
Yu
K.
,
Wang
Z.P.
,
Zhang
B.L.
,
Zhang
Y.
et al.
(
2017
)
Melatonin reduces oxidative damage and upregulates heat shock protein 90 expression in cryopreserved human semen
.
Free Radic. Biol. Med.
113
,
347
354
[PubMed]
200.
Abdillah
D.A.
,
Setyawan
E.M.N.
,
Oh
H.J.
,
Ra
K.
,
Lee
S.H.
,
Kim
M.J.
et al.
(
2019
)
Iodixanol supplementation during sperm cryopreservation improves protamine level and reduces reactive oxygen species of canine sperm
.
J. Vet. Sci.
20
,
79
86
[PubMed]
201.
Benaroudj
N.
,
Lee
D.H.
and
Goldberg
A.L.
(
2001
)
Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals
.
J. Biol. Chem.
276
,
24261
24267
[PubMed]
202.
Oku
K.
,
Watanabe
H.
,
Kubota
M.
,
Fukuda
S.
,
Kurimoto
M.
,
Tsujisaka
Y.
et al.
(
2003
)
NMR and quantum chemical study on the OH···π and CH···O interactions between trehalose and unsaturated fatty acids:  implication for the mechanism of antioxidant function of trehalose
.
J. Am. Chem. Soc.
125
,
12739
12748
[PubMed]
203.
Naijian
H.R.
,
Kohram
H.
,
Shahneh
A.Z.
,
Sharafi
M.
and
Bucak
M.N.
(
2013
)
Effects of different concentrations of BHT on microscopic and oxidative parameters of Mahabadi goat semen following the freeze–thaw process
.
Cryobiology
66
,
151
155
[PubMed]
204.
Zhang
X.G.
,
Wang
Y.H.
,
Han
C.
,
Hu
S.
,
Wang
L.Q.
and
Hu
J.H.
(
2015
)
Effects of trehalose supplementation on cell viability and oxidative stress variables in frozen-thawed bovine calf testicular tissue
.
Cryobiology
70
,
246
252
[PubMed]
205.
Shinde
P.
,
Khan
N.
,
Melinkeri
S.
,
Kale
V.
and
Limaye
L.
(
2019
)
Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation
.
Transfusion
59
,
686
696
[PubMed]
206.
Katenz
E.
,
Vondran
F.W.R.
,
Schwartlander
R.
,
Pless
G.
,
Gong
X.
,
Cheng
X.
et al.
(
2007
)
Cryopreservation of primary human hepatocytes: the benefit of trehalose as an additional cryoprotective agent
.
Liver Transpl.
13
,
38
45
[PubMed]
207.
Lee
Y.A.
,
Kim
Y.H.
,
Kim
B.J.
,
Kim
B.G.
,
Kim
K.J.
,
Auh
J.H.
et al.
(
2013
)
Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells
.
PLoS ONE
8
,
e54889
[PubMed]
208.
Gottlieb
E.
,
Armour
S.M.
,
Harris
M.H.
and
Thompson
C.B.
(
2003
)
Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis
.
Cell Death Differ.
10
,
709
717
[PubMed]
209.
Scarlett
J.L.
,
Sheard
P.W.
,
Hughes
G.
,
Ledgerwood
E.C.
,
Ku
H.H.
and
Murphy
M.P.
(
2000
)
Changes in mitochondrial membrane potential during staurosporine‐induced apoptosis in Jurkat cells
.
FEBS Lett.
475
,
267
272
[PubMed]
210.
Gottlieb
E.
,
Vander Heiden
M.G.
and
Thompson
C.B.
(
2000
)
Bcl-xL prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis
.
Mol. Cell. Biol.
20
,
5680
5689
[PubMed]
211.
Zamzami
N.
,
Marchetti
P.
,
Castedo
M.
,
Decaudin
D.
,
Macho
A.
,
Hirsch
T.
et al.
(
1995
)
Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death
.
J. Exp. Med.
182
,
367
377
[PubMed]
212.
Lançoni
R.
,
Celeghini
E.C.C.
,
Alves
M.B.R.
,
Lemes
K.M.
,
Gonella Diaza
A.M.
,
Oliveira
L.Z.
et al.
(
2018
)
Melatonin added to cryopreservation extenders improves the mitochondrial membrane potential of postthawed equine sperm
.
J. Equine Vet. Sci.
69
,
78
83
213.
Peña
F.J.
,
Johannisson
A.
,
Wallgren
M.
and
Rodriguez Martinez
H.
(
2003
)
Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate
.
Anim. Reprod. Sci.
78
,
85
98
[PubMed]
214.
Lu
X.
,
Zhang
Y.
,
Bai
H.
,
Liu
J.
,
Li
J.
and
Wu
B.
(
2018
)
Mitochondria-targeted antioxidant MitoTEMPO improves the post-thaw sperm quality
.
Cryobiology
80
,
26
29
[PubMed]
215.
Figueroa
E.
,
Farias
J.G.
,
Lee Estevez
M.
,
Valdebenito
I.
,
Risopatrón
J.
,
Magnotti
C.
et al.
(
2018
)
Sperm cryopreservation with supplementation of α-tocopherol and ascorbic acid in freezing media increase sperm function and fertility rate in Atlantic salmon (Salmo salar)
.
Aquaculture
493
,
1
8
216.
Silva
E.C.B.
,
Cajueiro
J.F.P.
,
Silva
S.V.
,
Soares
P.C.
and
Guerra
M.M.P.
(
2012
)
Effect of antioxidants resveratrol and quercetin on in vitro evaluation of frozen ram sperm
.
Theriogenology
77
,
1722
1726
[PubMed]
217.
Zhang
L.
,
Xue
X.
,
Yan
J.
,
Yan
L.Y.
,
Jin
X.H.
,
Zhu
X.H.
et al.
(
2016
)
L-proline: a highly effective cryoprotectant for mouse oocyte vitrification
.
Sci. Rep.
6
,
26326
[PubMed]
218.
Carneiro
J.A.M.
,
Canisso
I.F.
,
Bandeira
R.S.
,
Scheeren
V.F.C.
,
Freitas Dell’Aqua
C.P.
,
Alvarenga
M.A.
et al.
(
2018
)
Effects of coenzyme Q10 on semen cryopreservation of stallions classified as having good or bad semen freezing ability
.
Anim. Reprod. Sci.
192
,
107
118
[PubMed]
219.
Garcez
M.E.
,
dos Santos Branco
C.
,
Lara
L.V.
,
Pasqualotto
F.F.
and
Salvador
M.
(
2010
)
Effects of resveratrol supplementation on cryopreservation medium of human semen
.
Fertil. Steril.
94
,
2118
2121
[PubMed]
220.
Kotdawala
A.P.
,
Kumar
S.
,
Salian
S.R.
,
Thankachan
P.
,
Govindraj
K.
,
Kumar
P.
et al.
(
2012
)
Addition of zinc to human ejaculate prior to cryopreservation prevents freeze-thaw-induced DNA damage and preserves sperm function
.
J. Assist. Reprod. Genet.
29
,
1447
1453
[PubMed]
221.
Limaye
L.S.
(
1997
)
Bone marrow cryopreservation: improved recovery due to bioantioxidant additives in the freezing solution
.
Stem Cells
15
,
353
358
[PubMed]
222.
Hardikar
A.A.
,
Risbud
M.V.
,
Remacle
C.
,
Reusens
B.
,
Hoet
J.J.
and
Bhonde
R.R.
(
2001
)
Islet cryopreservation: improved recovery following taurine pretreatment
.
Cell Transplant.
10
,
247
253
[PubMed]
223.
De Loecker
P.
,
Fuller
B.J.
and
De Loecker
W.
(
1991
)
The effects of cryopreservation on protein synthesis and membrane transport in isolated rat liver mitochondria
.
Cryobiology
28
,
445
453
[PubMed]
224.
Manning Fox
J.E.
,
Lyon
J.
,
Dai
X.Q.
,
Wright
R.C.
,
Hayward
J.
,
van de Bunt
M.
et al.
(
2015
)
Human islet function following 20 years of cryogenic biobanking
.
Diabetologia
58
,
1503
1512
[PubMed]
225.
Mukherjee
N.
,
Chen
Z.
,
Sambanis
A.
and
Song
Y.
(
2005
)
Effects of cryopreservation on cell viability and insulin secretion in a model tissue-engineered pancreatic substitute (TEPS)
.
Cell Transplant.
14
,
449
456
[PubMed]
226.
Jitraruch
S.
,
Dhawan
A.
,
Hughes
R.D.
,
Filippi
C.
,
Lehec
S.C.
,
Glover
L.
et al.
(
2017
)
Cryopreservation of hepatocyte microbeads for clinical transplantation
.
Cell Transplant.
26
,
1341
1354
[PubMed]
227.
Grondin
M.
,
Hamel
F.
,
Averill Bates
D.
and
Sarhan
F.
(
2009
)
Wheat proteins improve cryopreservation of rat hepatocytes
.
Biotechnol. Bioeng.
103
,
582
591
[PubMed]
228.
Duchez
P.
,
Chevaleyre
J.
,
Brunet de la Grange
P.
,
Vlaski
M.
,
Boiron
J.M.
,
Wouters
G.
et al.
(
2013
)
Cryopreservation of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34+ cells
.
Transfusion
53
,
2012
2019
[PubMed]
229.
Mugishima
H.
,
Harada
K.
,
Chin
M.
,
Suzuki
T.
,
Takagi
K.
,
Hayakawa
S.
et al.
(
1999
)
Effects of long-term cryopreservation on hematopoietic progenitor cells in umbilical cord blood
.
Bone Marrow Transplant.
23
,
395
396
[PubMed]
230.
Othmani
A.E.
,
Rouam
S.
,
Abbad
A.
,
Erraoui
C.
,
Harriba
S.
,
Boukind
H.
et al.
(
2019
)
Cryopreservation impacts cell functionality of long term expanded adipose-derived stem cells
.
J. Stem Cell Res. Ther.
9
,
445
231.
Zhang
X.B.
,
Li
K.
,
Yau
K.H.
,
Tsang
K.S.
,
Fok
T.F.
,
Li
C.K.
et al.
(
2003
)
Trehalose ameliorates the cryopreservation of cord blood in a preclinical system and increases the recovery of CFUs, long‐term culture‐initiating cells, and nonobese diabetic‐SCID repopulating cells
.
Transfusion
43
,
265
272
[PubMed]
232.
Xue
W.J.
,
Luo
X.H.
,
Li
Y.
,
Liu
H.B.
,
Tian
X.H.
,
Feng
X.S.
et al.
(
2011
)
Effects of astragalosides on cultured islets after cryopreservation in rats
.
Transplant. Proc.
43
,
3908
3912
[PubMed]
233.
Sasnoor
L.M.
,
Kale
V.P.
and
Limaye
L.S.
(
2005
)
Prevention of apoptosis as a possible mechanism behind improved cryoprotection of hematopoietic cells by catalase and trehalose
.
Transplantation
80
,
1251
1260
[PubMed]
234.
Sasnoor
L.M.
,
Kale
V.P.
and
Limaye
L.S.
(
2005
)
A combination of catalase and trehalose as additives to conventional freezing medium results in improved cryoprotection of human hematopoietic cells with reference to in vitro migration and adhesion properties
.
Transfusion
45
,
622
633
[PubMed]
235.
Fujita
R.
,
Hui
T.
,
Chelly
M.
and
Demetriou
A.A.
(
2005
)
The effect of antioxidants and a caspase inhibitor on cryopreserved rat hepatocytes
.
Cell Transplant.
14
,
391
396
[PubMed]
236.
Chow-shi-yée
M.
,
Grondin
M.
,
Averill Bates
D.A.
and
Ouellet
F.
(
2016
)
Plant protein 2-Cys peroxiredoxin TaBAS1 alleviates oxidative and nitrosative stresses incurred during cryopreservation of mammalian cells
.
Biotechnol. Bioeng.
113
,
1511
1521
[PubMed]
237.
Tatone
C.
,
Di Emidio
G.
,
Vento
M.
,
Ciriminna
R.
and
Artini
P.G.
(
2010
)
Cryopreservation and oxidative stress in reproductive cells
.
Gynecol. Endocrinol.
26
,
563
567
[PubMed]
238.
Nimse
S.B.
and
Pal
D.
(
2015
)
Free radicals, natural antioxidants, and their reaction mechanisms
.
RSC Adv.
5
,
27986
28006
239.
Halliwell
B.
(
1996
)
Antioxidants: the basics-what they are and how to evaluate them
.
Adv. Pharmacol.
38
,
3
20
240.
Zhu
Z.
,
Li
R.
,
Lv
Y.
and
Zeng
W.
(
2019
)
Melatonin protects rabbit spermatozoa from cryo-damage via decreasing oxidative stress
.
Cryobiology
88
,
1
8
[PubMed]
241.
Appiah
M.O.
,
He
B.
,
Lu
W.
and
Wang
J.
(
2019
)
Antioxidative effect of melatonin on cryopreserved chicken semen
.
Cryobiology
89
,
90
95
[PubMed]
242.
Reiter
R.J.
,
Mayo
J.C.
,
Tan
D.X.
,
Sainz
R.M.
,
Alatorre Jimenez
M.
and
Qin
L.
(
2016
)
Melatonin as an antioxidant: under promises but over delivers
.
J. Pineal Res.
61
,
253
278
[PubMed]
243.
Lee
A.R.
,
Hong
K.
,
Choi
S.H.
,
Park
C.
,
Park
J.K.
,
Lee
J.I.
et al.
(
2019
)
Anti-apoptotic regulation contributes to the successful nuclear reprogramming using cryopreserved oocytes
.
Stem Cell Rep.
12
,
545
556
[PubMed]
244.
Trzcińska
M.
and
Bryła
M.
(
2015
)
Apoptotic-like changes of boar spermatozoa in freezing media supplemented with different antioxidants
.
J. Vet. Sci.
18
,
473
480
245.
Roca
J.
,
Rodríguez
M.J.
,
Gil
M.A.
,
Carvajal
G.
,
Garcia
E.M.
,
Cuello
C.
et al.
(
2005
)
Survival and in vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase
.
J. Androl.
26
,
15
24
[PubMed]
246.
Limaye
L.S.
and
Kale
V.P.
(
2001
)
Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium
.
J. Hematother. Stem Cell Res.
10
,
709
718
247.
Kim
G.A.
,
Lee
S.T.
,
Ahn
J.Y.
,
Park
J.H.
and
Lim
J.M.
(
2010
)
Improved viability of freeze-thawed embryonic stem cells after exposure to glutathione
.
Fertil. Steril.
94
,
2409
2412
[PubMed]

Author notes

*

These authors contributed equally to this work.

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