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PubMed2004

Effect of different cryoprotectants on the structural preservation of follicles in frozen zebu bovine (Bos indicus) ovarian tissue.

Cryopreservation of ovarian tissue is a new and promising technique for germ-line storage. The objective of this study was to evaluate the effect of four cryoprotectants (at two concentrations each) on the preservation of zebu bovine preantral follicles after ovarian cryostorage. Strips of ovarian cortex were cryopreserved using glycerol (GLY; 10 or 20%), ethylene glycol (EG), propanediol (PROH) or dimethylsulphoxide (DMSO; 1.5 or 3M). In addition, a toxicity test was performed for each cryoprotectant by exposing the ovarian tissue to them without freezing. Tissues were analyzed by histology and transmission electron microscopy. Ovarian tissue frozen in either concentration of DMSO or PROH or in 10% GLY retained a higher percentage of morphologically normal follicles (73-88%) than tissue frozen in 20% GLY or in either concentration of EG (16-52%). In the toxicity test, exposure of tissues to DMSO, PROH or GLY resulted in higher percentages of normal follicles (80-97%) than exposure to EG (49%). Electron microscopy revealed damage to the ultrastructure of follicles frozen in 10% GLY, while follicles cryopreserved in DMSO and PROH at either concentration exhibited normal ultrastructure. In conclusion, DMSO and PROH were the most effective cryoprotectants for zebu ovarian tissue, preserving the structural integrity of somatic and reproductive cells within the ovary.

Animals↗
PubMed2004

Evaluation of alternative cryoprotectants for preserving stallion spermatozoa.

Although use of cryopreserved stallion spermatozoa is currently accepted by many breed registries, utilization of this technique remains limited due to poor fertility for some stallions. One reason for these results is osmotic stress that spermatozoa experiences when the cryoprotectant (glycerol) is added to the cells prior to freezing and removal from the cells after thawing. In an effort to minimize osmotic damage, alternative cryoprotectants, having lower molecular weights and greater membrane permeability than glycerol, were evaluated to determine their effectiveness for cryopreserving stallion spermatozoa. In the first experiment, equal molar concentrations of several amides were compared to determine if they could preserve the motility of sperm as well as glycerol. At 0.55 M concentration, addition of glycerol to a skim milk-egg yolk (SMEY) diluent resulted in higher percentages of motile sperm (61%) than methyl formamide (40%) or dimethyl formamide (38%, P<0.05), while formamide, acetamide, and methyl acetamide resulted in recovery of less than 20% motile cells (P<0.05). When methyl formamide or dimethyl formamide were increased to 0.6 or 0.9 M they resulted in percentages of motile cells (48-54%) similar to that achieved with glycerol (52%). Similarly, 0.9 M ethylene glycol also resulted in similar percentages of motile cells (43%). Replacing the glucose and fructose in the SMEY diluent with either raffinose or trehalose did not result in higher percentages of motile sperm (65 and 66%, respectively) than the control SMEY (63%). Similarly, addition of methyl cellulose also did not increase the percentages of motile spermatozoa in the samples, after cryopreservation (P>0.05). In conclusion, both methyl formamide and dimethyl formamide protected stallion spermatozoa from cryodamage as effectively as glycerol. Since these compounds permeate the plasma membrane more effectively than glycerol, they should cause less osmotic damage to stallion spermatozoa than glycerol. Therefore, these compounds may prove very effective in the cryopreservation of stallion spermatozoa, and may be particularly useful for spermatozoa from stallions that produce spermatozoa that have poor post-thaw characteristics when glycerol is used as the cryoprotectant.

Acetamides↗
PubMed2004

Methanol as a cryoprotectant for equine embryos.

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.

Animals↗
PubMed2003

Thermal expansion measurements of cryoprotective agents. Part I: a new experimental apparatus.

As part of an ongoing effort to characterize the mechanical behavior of biological tissues in the cryogenic temperature range, the current study focuses on the thermal expansion measurements of cryoprotective agents. This study focuses on the upper part of the cryogenic temperature range, where the cryoprotectant behaves like low viscous liquid at all practical cooling rates. For the purpose of this study, a new apparatus for thermal expansion measurements has been designed and constructed. Part I of this study (the current report) includes: a description of the new experimental apparatus, the techniques of operation, calibration, system validation, and a detailed uncertainty analysis. Part II of this study (the report following) includes thermal expansion measurements of the cryoprotectant mixtures DP6 and VS55, and comparison with data from the literature on DMSO solutions.

Cryopreservation↗
PubMed2001

Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm.

The process of freezing and thawing induces certain cellular damage in rainbow trout (Oncorhynchus mykiss) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Oncorhynchus mykiss) spermatozoa, is related to sublethal damage to the plasma membrane. External cryoprotectants are known to stabilize the sperm cell membrane against such damage. In the current study, we used a basic freezing extender containing #6 Erdahl and Graham and 7% DMSO and added egg yolk, BSA, and a soybean-protein complex (DanPro S760) singly and in various combinations. To assess the effect of these cryoprotectants we evaluated the percentage of cells with progressive motility, permeability of cells to propidium iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hypo- and isoosmotic solutions of 10 and 300 mOsm, and the in vitro fertility rate. Fertility trials were performed using 1.87 x 10(7) spermatozoa/egg. Some of the tested stabilizers increased motility, increased viability, or reduced cell fragility after freezing and thawing. Nevertheless these quality improvements demonstrated by the "in vitro" tests do not always correlate with high fertility. The best membrane protection in terms of resistance to hypoosmotic shock was achieved when BSA and egg yolk were added to the extender. The highest fertility rates were obtained with DanPro S760 alone or in combination with BSA; the use of BSA with egg yolk did not improve this parameter. Our results demonstrated that some external cryoprotectants effectively increased membrane resistance during freezing and thawing, but some of the tested mixtures interfered with fertilization. Soybean protein concentrate provided good protection and increased fertility rates in cryopreserved trout spermatozoa.

Animals↗
PubMed2002

Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen.

Hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders in order to protect the spermatozoa against cold shock. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). In recent years, arguments concerning the presence of cryoprotective antagonists in egg yolk, have reinforced interest in the use of only the LDL extracted from egg yolk in the extenders. However, current methods of LDL purification do not support the use of LDL in commercial extenders because they offer a poor recovery rate. Consequently, we have developed an easy method to extract LDL from egg yolk. Several concentrations of purified LDL (between 2.5 and 20%, w/v) were tested in freezing extenders for bull semen, and compared with commercial extenders. Our extraction method reached 97% purity and about 67% yield, and is easily reproducible on an industrial scale. Analysis of sperm motility showed that the motility and characteristics of spermatozoa movement were improved with LDL in the extender, as compared to a commercial extender containing egg yolk. The optimum LDL concentration in the extender was 8%. In conclusion, we propose that an extender containing LDL extracted from egg yolk could be used as cryoprotective media with a better efficiency than present commercial extenders.

Animals↗
PubMed2003

Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success.

The use of cryoprotective agents for the conventional cryopreservation of human spermatozoa, oocytes, zygotes, early cleavage stage embryos and blastocysts is an integral part of almost every human IVF programme. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen usually requires high cryoprotectant concentrations with consequent cytotoxic effects. This review covers the history of this problem, and in this light offers an explanation, through physico-chemical concepts, for one of the most recent developments in this area: the recovery of motile and potent spermatozoa after cryoprotectant-free vitrification.

Cryopreservation↗
PubMed2001

Comparison of cryoprotectants in the preservation of Theileria parva sporozoites using an in vitro infectivity assay.

An in vitro infectivity assay was used to examine five cryoprotectants for their suitability for preserving Theileria parva sporozoites. All five were capable of preserving T. parva sporozoites through freezing, the optimal concentrations being 7.5% for glycerol, 5% for dimethyl sulphoxide (DMSO), poly (vinylpyrrolidone) (PVP) and poly(ethylene glycol) (PEG), and 2.5% for hydroxyethyl starch (HES). When the five cryoprotectants were compared at their optimal concentrations, using a modification of the standard method of stabilate preparation, glycerol was significantly better than the others (p < 0.05). Measurement of the effects of each cryoprotectant on the osmolality of the media revealed that glycerol and DMSO elevated the osmolality significantly (p < 0.05). Resuscitation of glycerol-preserved sporozoites required the presence of glycerol in the diluent to maintain infectivity. Studies on the effects of equilibration time in glycerol on the infectivity of sporozoites showed that those frozen immediately after mixing (2 min) were as infective as those frozen after 60 min of equilibration.

Animals↗
PubMed2018

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants.

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

Cell Survival↗
PubMed2000

Osmoprotectants and cryoprotectants for Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen that can grow in high osmotic strength environments and at refrigeration temperatures. Glycine betaine, proline betaine, acetylcarnitine, carnitine, gamma-butyrobetaine and 3-dimethylsulphoniopropionate all acted as osmoprotectants, as evidenced by an increase in growth rate of L. monocytogenes 10403S and Scott A when provided with these compounds, while being stressed in defined medium containing 0.7 M NaCl. These same compounds exhibited cryoprotective activity, as evidenced by increasing the growth rate of L. monocytogenes at 5 degrees C. Ectoine, hydroxy ectoine, pipecolic acid and proline were ineffective as osmoprotectants or cryoprotectants under these conditions. The presence of osmoprotectants and cryoprotectants in foods may provide compounds assisting L. monocytogenes to overcome the barriers of high osmotic strength and low temperature that otherwise control microbial growth.

Acetylcarnitine↗
PubMed1999

Permeability characteristics of human oocytes in the presence of the cryoprotectant dimethylsulphoxide.

Equilibration of oocytes with cryoprotectants is a prerequisite of low temperature storage. However, cryoprotectant exposure may induce damage via osmotic stress. Knowledge of cell membrane permeability characteristics and their temperature dependence would facilitate the design of cryopreservation protocols in which osmotic stress is minimized and the incidence of intracellular freezing is reduced. To obtain such data, the volume change of donated human oocytes following exposure to cryoprotectant was measured at a variety of temperatures. After removal of cumulus cells, each oocyte was placed in a 5 microl droplet of phosphate-buffered medium. The oocyte was held in position by suction generated using a fine pipette and perfused with 1 ml 1.5 mol/l dimethylsulphoxide (DMSO) at 30, 24 or 10 degrees C. The volume of the oocyte before, during and after perfusion was recorded by videomicroscopy. Oocyte volume was calculated from radius measurements and the Kedem-Katchalsky (K-K) passive coupled transport coefficients, namely L(p) (hydraulic permeability), P(DMSO) (permeability to DMSO) and sigma (reflection coefficient) were derived. The resulting coefficients were L(p) = 1. 65 +/- 0.15, 0.70 +/- 0.06 and 0.28 +/- 0.04 microm/min.atm; P(DMSO) = 0.79 +/- 0.10, 0.25 +/- 0.04 and 0.06 +/- 0.01 microm/s and sigma = 0.97 +/- 0.01, 0.94 +/- 0.03 and 0.96 +/- 0.01 at 30, 24 and 10 degrees C respectively. The activation energy for L(p) was 14.70 and for P(DMSO) was 20.82 kcal/mol. The permeability parameters of human oocytes are higher than those of murine oocytes, suggesting that they require a shorter period of exposure to DMSO with concomitantly reduced toxic effects.

Cell Membrane Permeability↗
PubMed2005

Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants.

BACKGROUND: Cryopreservation of testicular tissue is an option in fertility preservation for pre-pubertal boys who will lose spermatogenic cells as a result of chemotherapy. We compared three different protocols and cryoprotectants in cryopreservation of testicular tissue. METHODS: Testicular tissue obtained from 16 infertile men was evaluated by light microscopy(LM), immunostaining against MAGE-A4, transmission electron microscopy (TEM) and organ culture. Seminiferous tubules (1312) from non-frozen (n = 16) and frozen-thawed samples (n = 34) were studied following cryopreservation using protocols with either 1,2-propanediol (PrOH), glycerol or dimethylsulphoxide (DMSO) as cryoprotectants. RESULTS: Normal structure was seen in 86 +/- 6% (mean +/- SD) of the fresh tissue. After freezing with DMSO, 70 +/- 6% and after PrOH, 37+/-3% of the tubules were judged to be good. When glycerol was used, the structure of the basal compartment of the tubules was severely damaged. The ultrastructure of the cryopreserved samples as revealed by TEM and MAGE-positive spermatogonia confirmed the findings. Cryopreserved Leydig cells maintained their morphology and ability to release testosterone in culture. CONCLUSION: DMSO as a cryoprotectant (at a 0.7 mol/l concentration) proved to maintain the structure of testicular tissue, especially spermatogonia, after cryopreservation better than PrOH or glycerol.

Adult↗
PubMed1995

Pentoxifylline-supplemented cryoprotectant improves human sperm motility after cryopreservation.

In this study, human spermatozoa obtained from donors (n = 15) with normal semen characteristics were cryopreserved in human sperm preservation medium, supplemented with the phosphodiesterase inhibitor pentoxifylline at concentrations of 0, 1, 3 and 10 mM. The effect of pentoxifylline on cryopreserved spermatozoa was determined by monitoring changes in sperm motility and acrosome morphology by labelling the spermatozoa with fluorescein-conjugated concanavalin A lectin. Cryoprotectant supplemented with 1 mM pentoxifylline was found to improve post-thaw progressive motility from 15.3 +/- 2.4 (control) to 23.1 +/- 3.8% (P < 0.01), and total motility from 27.4 +/- 3.3 (control) to 38.2 +/- 3.9% (P < 0.05) without reducing the percentage of spermatozoa with normal acrosomal regions, and so appears useful for cryopreservation purposes. The beneficial effects of 1 mM pentoxifylline on sperm motility were shown to be maintained post-thaw over a 6 h time course. Cryoprotectant supplemented with 3 mM pentoxifylline was found to improve only post-thaw progressive motility, from 15.3 +/- 2.4 (control) to 20.7 +/- 3.0% (P < 0.05). However, cryopreservation in the presence of 10 mM pentoxifylline was found to have a significantly (P < 0.01) detrimental effect on acrosome morphology post-thaw, reducing it from 29.0 +/- 2.0 (control) to 21.0 +/- 2.4% without affecting sperm motility. This suggests that assessment of the acrosomal region may indicate subtle deleterious effects of cryoprotectant supplements that cannot be determined from post-thaw motility assessments alone. These findings differ from previous studies in that a lower concentration of pentoxifylline (1 mM) was found to be optimal for cryopreservation purposes.

Acrosome↗
PubMed1999

Comparison of cryoprotectants and methods of cryopreservation of fowl spermatozoa.

The deleterious effects of three cryoprotectants, glycerol, dimethylsulfoxide (DMSO), and dimethylacetamide (DMA), were compared on fowl spermatozoa. The viability and integrity of spermatozoa were measured with eosin-nigrosin smears. Glycerol was the least deleterious cryoprotectant, followed by DMA, and DMSO was the most toxic. Methods employing either glycerol or DMA were then compared for the cryopreservation of semen in either straws or pellets. Fertility was measured following artificial insemination. The highest fertility rates were obtained with semen frozen with DMA in pellets directly plunged in liquid nitrogen, DMA being added at -6 C (92.7%) or 5 C (84.7%). When semen was frozen in straws, glycerol equilibrated for 1 or 30 min gave the highest fertility results, but the fertility rates were lower (53.7 and 63.9%) than with DMA in pellets. The lowest results (26.7%) were obtained when semen was frozen in straws with DMA. When semen was frozen in pellets at very high cooling rates, DMA was superior to glycerol as a cryoprotectant, as evidenced by fertility. In contrast, when straws and low freezing rates were used, glycerol gave better results; however these results were never as high as those obtained with DMA and pellets. In conclusion, under our experimental conditions, the highest fertility rates were achieved with DMA and pellets. However, for gene banking, which requires high levels of safety and clear identification, glycerol and straws are more convenient.

Acetamides↗
PubMed2000

Washing and cryoprotectant effects on frozen storage of spent hen surimi.

Mince of 98-wk-old spent hens was washed two times with 0.1% NaCl. Portions of unwashed and washed mince were mixed with a cryoprotectant (CP) composed of 4% sucrose, 4% sorbitol, and 0.2% Na-tripolyphosphate and were immediately frozen and stored at -20 C. Mince without CP was run as control. Textural properties of the stored mince and surimi were measured at 1-mo intervals for 6 mo, after being thawed at 4 C overnight, ground with 3% NaCl, and heated at 90 C for 15 min. For freeze-thaw stability study, minces were subjected to six freeze-thaw cycles. Each freeze-thaw cycle was carried out at 1 mo of storage. Textural quality parameters (gel strength, breaking strength, deformation, protein solubility, expressible moisture, cooking yield, folding test, drip-loss, and sensory scores) were decreased in both unwashed and washed mince, mostly during the early stages of storage. Washed mince showed significantly better textural properties than unwashed mince. Washing protected the gel quality of the hen mince from degradation during frozen storage. Cryoprotectants could not protect the gel strength or breaking strength, but deformation was slightly improved. Water-retention properties were protected, and folding test and sensory scores were well preserved in the mince with added CP. Cryoprotectants had a beneficial effect on frozen, stored spent hen surimi to protect the elasticity and cohesiveness of the gel.

Animals↗
PubMed1999

Effects of Percoll separation, cryoprotective agents, and temperature on plasma membrane permeability characteristics of murine spermatozoa and their relevance to cryopreservation.

Cryopreservation of murine spermatozoa would provide an efficient method for preserving important genotypes. However, to date such methods have resulted in low survivals with significant variability. To address this issue, a series of five experiments was performed to determine the cryobiological characteristics of murine spermatozoa. Experiments 1 and 2 investigated the effect of Percoll separation on the hydraulic conductivity (L(p)) of murine spermatozoa. Both Percoll separation and cryoprotective agents (CPAs) decreased the L(p). However, these effects were not additive. Experiment 3 was performed to determine the effect of temperature on L(p) in the presence of cryoprotectants (L(p)(CPA)), cryoprotectant permeability (P(CPA)), and the reflection coefficient (sigma) in spermatozoa from both ICR and B6C3F1 mice. Permeability parameters decreased as temperature decreased, and permeability characteristics differed between strains. In experiments 4 and 5, theoretical simulations for CPA addition and removal were developed and empirically tested. Strain-specific methods for CPA addition and removal based upon the fundamental cryobiological characteristics of murine spermatozoa resulted in higher survivals than current methods or procedures, which were used as controls.

Animals↗
PubMed2003

Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals.

Collection of quality data on high-energy synchrotron sources requires the selection of a cryoprotectant that allows protein crystals to be cryocooled without damage to the crystal and with suppression of ice formation. The use of sodium malonate as a versatile cryoprotectant for salt-grown protein crystals is presented here. In addition to its useful cryoprotectant properties, sodium malonate can also function as a versatile stabilizing solution that allows the manipulation, derivatization and ligand soaking of crystals grown from salt that may not be possible in the crystal mother liquor.

Bacterial Proteins↗
PubMed1993

Purification and characterization of a protein cryoprotective for Vibrio cholerae extracted from the prawn shell surface.

A substance cryoprotective for Vibrio cholerae on the prawn shell surface was purified by ammonium sulfate precipitation and gel filtration. It was a protein of 81 kDa and called cryoprotective protein (CPP). The cryoprotective activity of this protein for V. cholerae was sensitive to heat at 100 C and trypsin treatment. In the presence of Mg ion the protein can bind to the bacterial cell surface. V. cholerae can adhere to the shell surface of the prawn. The number of adhered bacteria was reduced by treating the shell with anti-CPP serum, heat or by trypsin. The presence of Mg ion promoted the adherence. These results suggest that the CPP could serve as an adherence site for V. cholerae on the shell surface.

Amino Acid Sequence↗