Cryoprotectant Solution Research Articles

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster, Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC50), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.

Vitrification tendency and stability of DP6-based vitrification solutions for complex tissue cryopreservation

Vitrification tendency and stability of the amorphous state were analyzed by means of differential scanning calorimetry (DSC) for the vitrification solution DP6, with and without additional solutes to enhance ice suppression. This study is a part of an ongoing research effort to characterize the thermophysical and mechanical properties of DP6 and its derivatives, and their qualities as cryoprotective solutions. DP6 was determined to have a critical cooling rate necessary to ensure vitrification of 2.7 °C/min. The following additional solutions were tested: DP6 + 6% (2R, 3R) 2,3-butanediol, DP6 + 6% 1,3-cyclohexanediol, DP6 + 6% (0.175M) sucrose, DP6 + 12% PEG 400, and DP6 + 17.1% (0.5 M) sucrose. The additives decreased the critical cooling rate of the DP6 solution to rates below 1 °C/min that were not quantifiable by the DSC techniques used. The following critical warming rates necessary to avoid devitrification were identified for DP6 and the modified solutions, respectively: 189 °C/min, 5 °C/min, ≈ 1 °C/min, 15 °C/min, <1 °C/min, and <1 °C/min. Glass transition temperatures and melting temperatures were also measured. Sucrose was the least effective additive on a per mass basis, with 1,3-cyclohexanediol appearing to be the most effective additive for suppressing ice formation in DP6.

Effect of pulsed electric field coupled with vacuum infusion on quality parameters of frozen/thawed strawberries

In this study, pulsed electric field (PEF) was coupled with vacuum infusion (VI) to impregnate strawberries with cryoprotectants. Electroporation of fruits was reached with 5 bi-polar, rectangular pulses of 100 μs width with a nominal electric field strength of 850 V/cm. After PEF treatment, the strawberries were vacuum infused with a cryoprotective solution (12 g/100 g trehalose and 0.2 g/100 g acclimated winter wheat extract containing antifreeze proteins) for 14 min. The strawberries were frozen in liquid nitrogen and thawed in air at 20 °C. Cell survival, texture and color were evaluated before and after freezing and thawing cycle. The fruit pre-treated with PEF prior to VI exhibited higher cell viability in epidermal layer and 30% more red color retention compared to just VI samples. However, no further improvement on strawberry quality in terms of drip loss and texture was observed upon the application of PEF.

Non-Newtonian, shear-thickening behaviour with cryoprotectant solutions

Non-Newtonian, shear-thickening behaviour with cryoprotectant solutions

Cryoprotectant agents and cooling effect on embryos of Macrobrachium amazonicum.

SummaryThere are few reports of cryopreservation and injuries in Macrobrachium amazonicum embryos. Thus, the aim of this study was to analyze the effects of cryoprotectants agents and cooling on stage VIII of this species. Fertilized eggs from ovigerous females were removed from the incubation chamber, then placed in 10 ml Falcon tubes with a cryoprotectant solution and saline-free calcium solution. Thus, the embryos underwent a cooling curve of 1°C per min until reaching 5°C, and then were stored for 2 h. The tubes containing the embryos were washed to remove the cryoprotectant, acclimated for 5 min and then transferred to 50 ml incubators. At the end of the 24-h period, living embryos from each tube were counted and tabulated. A pool of embryos was fixed with 4% formaldehyde and then subjected to histology using 3-mm thick sections and stained with haematoxylin/eosin. Another pool was used for biometric analysis in which length, width and volume were analyzed. The cryoprotectants agents used were: dimethylsulfoxide (DMSO), methyl alcohol, ethylene glycol at 1, 5 and 10% and sucrose (0.5 M). Variance analysis was performed followed by Tukey's honest significant difference (HSD) test at 5% significance level. DMSO cryoprotectant affected embryo survival the least with rates of 71.8, 36.2 and 0% for concentrations of 1, 5 and 10%, respectively. Ethylene glycol caused 100% mortality at all the concentrations used. It was not possible to observe the interference of cooling and cryoprotectants on embryonic structures in this study.

The cryoprotectant system of Cope\u2019s gray treefrog, Dryophytes chrysoscelis: responses to cold acclimation, freezing, and thawing

Cope’s gray treefrog (Dryophytes chrysoscelis) is one of few freeze-tolerant frogs that mobilize glycerol as a cryoprotectant, yet cold and freezing-induced accumulation of this and other osmolytes has received little attention in this species. This study investigated the development of freeze tolerance in D. chrysoscelis, analyzing the response of the cryoprotectant system to cold acclimation, freezing, and thawing. Glycerol production was low and unresponsive to acclimation temperature, or duration of acclimation to 5 °C, except for one cold-acclimated frog that presented elevated glycerol in plasma, liver, and skeletal muscle. Curiously, glycerol concentration in skeletal muscle was higher than that of plasma and liver, in both warm- and cold-acclimated frogs, suggesting glycerol synthesis in muscle. Urea concentration in plasma doubled in response to cold acclimation but did not change during freezing. Freezing induced hepatic glycogen catabolism and an increase in glycerol and glucose in several tissues, although the mobilization dynamics differed between these cryoprotectants, possibly as a result of different transport mechanisms. Although hepatic glucose mobilization was of considerable magnitude, glucose accumulation in peripheral tissues was low and was surpassed by that of glycerol and urea. The muscle production of glycerol and the cold-induced accumulation of urea imply a role for skeletal muscle metabolism in the mobilization of cryoprotective solutes in D. chrysoscelis. The cryoprotectant system of D. chrysoscelis is complex, highly variable, and unique, with glycerol, glucose, and likely urea serving as cryoprotectants.

Sperm vitrification of Litopenaeus vannamei: effect of cryoprotectant solutions on sperm viability and spawning quality after artificial insemination

Vitrification is a fast freezing method with promising results for penaeids sperm cryopreservation. This study evaluated the efficiency of three cryoprotectant solutions for sperm vitrification and artificial insemination with cryopreserved spermatophores of Litopenaeus vannamei. The cryoprotectant solutions tested were 30% methanol, 2% soy lecithin, and 30% methanol + 2% soy lecithin. Fully mature females were artificially inseminated with vitrified and fresh spermatophores as a control group. The vitrification method was efficient in maintaining high rates of sperm survival and membrane integrity. Although the egg fertilization was successfully attained by artificial insemination with cryopreserved spermatophores, low hatching rates suggested that possible DNA fragmentation of sperm cells should be further investigated. This is the first report of artificial insemination using vitrified sperm in penaeids.

Effect of sodium alginate on mouse ovary vitrification

The survival rate of vitrified-thawed ovarian tissues after autotransplantation still needs to be improved. Therefore finding an ideal cryoprectant to reduce the damage to ovaries that caused by vitrification will pave the way for application of ovary cryopreservation on clinics. Experiments were conducted to investigate the effect of sodium alginate in cryoprotectant solution on mouse ovaries during the vitrification process. The ovaries obtained from 6-weeks old CD1 were assigned into six groups from A to F. Group A without treatment was used as the normal control. Group B cryopreserved with the basic cryoprotectant solution containing 15% each Me2SO and EG was used as the experimental control. Groups C, D, E, and F cryopreserved with the basic cryoprotectant solution supplemented with 0.05%, 0.10%, 0.15%, and 0.20% of sodium alginate, respectively, were assigned for the experimental groups. The in vitro analyses showed that the developmental capability of the oocytes isolated from vitrified-thawed ovaries significantly increased with increasing concentration of sodium alginate in the cryoprotectant solution (groups: A = 70 ± 2; B = 43 ± 2; C = 48 ± 3; D = 53 ± 3; E = 60 ± 3; B < C < D < A, P < 0.05), and reached its highest level in group E with 0.15% of sodium alginate (P < 0.05). The lowest developmental capability of all groups was group F (41 ± 1%)(P < 0.05) with 0.20% of sodium alginate. The similar results were obtained by the autotransplantation in vivo. These finding demonstrated that sodium alginate can significantly reduce the damage to ovaries by vitrification.

The impact of preapheresis white blood cell count on autologous peripheral blood stem cell collection efficiency and HSC infusion side effect rate.

Autologous peripheral blood hematopoietic stem cell (PBSC) collection efficiency (CE) is reportedly affected by the patient's blood properties; however, studies to identify factors correlated with CE have shown inconsistent results. Additionally, variables such as stem cell graft granulocyte content and patient age, sex, and underlying disease, may be associated with hematopietic stem cell (HSC) infusion-related adverse reactions. In this study, we evaluated the correlation of preleukapheresis PB granulocyte count and PBSC harvest variables with CD34+ collection yield and efficiency, and thawed HSC infusion side effect occurrence. We evaluated data from 361 patients who had undergone autologous PBSC transplant. Large volume leukapheresis was the method for PBSC collection. Complete Blood Count and CD34+ cell enumeration were performed in the preapheresis PB and the apheresis product sample. The PBSC grafts were submitted to non-controlled rate freezing after addition of 5% DMSO plus 6% hidroxyethylstarch as a cryoprotectant solution. The cryopreserved graft was thawed in a 37°C water bath and then infused without further manipulation. The CD34+ yield was associated with preapheresis PB CD34+ count and immature granulocyte count. The PBSC CE was negatively correlated with preapheresis white blood cell (WBC), immature granulocyte and granulocyte count. The leukapheresis product total nucleated cell (TNC) and granulocyte content was correlated with the thawed graft infusion side effect occurrence. This study has shown that preapheresis PB WBC and granulocyte counts were associated with leukapheresis CE. Additionally, the leukapheresis product TNC and granulocyte content was correlated with thawed graft infusion side effect occurrence.

Strategies for Fast Multiplication and Conservation of Forest Trees by Somatic Embryogenesis and Cryopreservation: a Case Study with Cypress (Cupressus sempervirens L.)

Common cypress (Cupressus sempervirens L.) is one of the most widespread species in the Mediterranean area. It has been traditionally cultivated for its ornamental value, becoming a typical feature of urban and rural landscapes, and high timber quality. In the last 30 years, cypress has been subjected to important breeding programmes, aimed to select clones tolerant to the widespread canker, caused by the pathogenic fungus Seiridium cardinale, leading to various patented varieties today available on the market, as well as for genotypes producing null or low amount of allergenic pollen. Somatic embryogenesis is a suitable in vitro regeneration method for fast cloning of conifer trees, and the cryopreservation of embryogenic callus is a significant tool for the safe long-term conservation of valuable cell lines. Recently, a complete protocol for the production of cypress plants from somatic embryogenesis was developed for the patented clone ‘Mediterraneo’. Here, the coupling of somatic embryogenesis and cryopreservation may offer a superior tool to propagate and maintain selected genotypes of cypress by overcoming repetitive subculturing of selected embryogenic callus lines. For the above, this study aimed to compare different cryopreservation techniques (PVS2-based vitrification and slow cooling) with the ‘Mediterraneo’ embryogenic callus line. Best results were obtained after the optimization of a slow cooling procedure, based on the 30-min treatment of embryogenic masses with a cryoprotective solution containing 180 g l-1 sucrose and 7.5% DMSO, followed by the reduction of the temperature at a rate of -1 °C min-1 up to -40 °C and the subsequent immersion in liquid nitrogen (“two-step freezing”).

Outcome of 51 autologous peripheral blood stem cell transplants after uncontrolled-rate freezing ("dump freezing") using -80°C mechanical freezer.

BACKGROUND AND OBJECTIVE:Controlled-rate freezing is a complicated, expensive, and time-consuming procedure. Therefore, there is a growing interest in uncontrolled-rate freezing (UCF) with −80°C mechanical freezers for cryopreservation of hematopoietic stem cells. This is a retrospective analysis of efficiency of UCF and outcome of autologous peripheral hematopoietic stem cell (PBSC) transplants at our center from December 2011 to June 2016.MATERIALS AND METHODS:Cryoprotectant solutions used included 5% dimethyl sulfoxide and 5% albumin with 2% hydroxyethyl starch and stored at −80°C mechanical freezer till transplant. Evaluation of cryopreservation was studied by analyzing the variation in cellularity, viability, and CD34+ stem cell dose recovery as well as clinical follow-up with engraftment.RESULTS:A total of 51 patients (23 females and 28 males) underwent autologous PBSC transplantations with a median age of 31 years (range: 3–60 years) for both hematological and nonhematological indications. Mean recovery post by UCF at −80°C mechanical was 92.9% ± 15.5% for nucleated cells, 86.6% ± 15.5% for viability, and 80% ± 21.5% in CD34+ dose. The median day to neutrophil engraftment was 10 (range 5–14 days) and platelets engraftment was 15 (range 8–45 days). The cryopreserved products were stored at −80°C for median 7 days (range 2-41 day) before transplant.DISCUSSION/CONCLUSION:Our analysis shows that PBSC can be successfully cryopreserved with mechanical uncontrolled rate freezing. This is a cheap and simple method to freeze the stem cells for a short period in resource-constrained setting.

Wistar rats immature testicular tissue vitrification and heterotopic grafting.

ObjectiveTo evaluate the efficiency of two vitrification protocols for rat immature testicular tissue and heterotopic transplantation.MethodsTwenty-four pre-pubertal Wistar rats were divided into three groups (n=8). After orchiectomy, testicular fragments (3mm) from Groups 1 and 2 were vitrified with different cryoprotectant concentration solutions, using sterile inoculation loops as support. After warming up, the fragments were submitted to cell viability assessment by Trypan blue and histological evaluation. Vitrified (Groups 1 and 2) and fresh (Group 3) fragments were grafted to the animals periauricular region. After 8 weeks of grafting, the implant site was histologically analyzed.ResultsThe viability recovery rate from Group 1 (72.09%) was higher (p=0.02) than that from Group 2 (59.19%). Histological analysis showed similar tubular integrity between fresh fragments from Groups 1 and 3. Group 2 samples presented lower tubular integrity. We ran histological analyses in the grafts from the Groups. In all groups, it was possible to see the implant site, however, no fragment of testicular tissue or signs of inflammation were histologically found in most samples from Groups 1 and 3. In one sample from Group 2, we found degenerated seminiferous tubules with necrosis and signs of an inflammatory process. In another sample from Group 2, we found seminiferous tubules in the implant site.ConclusionThe vitrification of pre-pubertal testicular tissue of rats showed little damage to cell viability through histological analysis when we used cryoprotectants in a lower concentration. Heterotopic transplantation could not preserve the structural organization of the testicular tissue.

Tolerance of jenipapo seeds to cryoprotectants and thawing after immersion in liquid nitrogen

The objective of this study was to evaluate the effects of two cryoprotectants followed by thawing on the physiological potential of Genipa americana L. seeds. Two experiments testing 12 treatments were conducted, one for each cryoprotectant, both in a factorial scheme of 6 × 2 (cryoprotectant concentrations × thawing methods). We tested 0, 5, 10, 15, 20, and 25% for dimethyl sulfoxide, and 0, 0.25, 0.5, 0.75, 1.0, and 1.25 M for sucrose. The thawing methods were fast (38 °C for 30 min) and slow (25 °C for 4 h). The seeds were immersed in the cryoprotectant solution for 3 h, stored for 120 h, and then thawed. The seeds were then sown in substrate (sand and vermiculite, 1:1). Emergence percentage along with speed index, length, fresh and dry matter mass of seedlings were evaluated. Dimethyl sulfoxide and sucrose can be used as cryoprotectants in G. americana seeds. Thawing should be slow when treating seeds with dimethyl sulfoxide.

Post-cooling survival, growth and deformity rates in zebrafish embryos (Danio rerio).

This study investigated and analysed survival, growth and macro- and microscopic damage during the development of zebrafish embryos up to the adult stage after undergoing cooling. The embryos at 50% epiboly stage were selected, submerged in cryoprotectant solution of methanol and sucrose, cooled gradually to 0 ± 2°C temperature, and divided into two groups with different storage times (6 and 18 h). Subsequently, the embryos were reheated, rehydrated and incubated normally. The experiment lasted 5 months and, from hatching onward, the larvae were examined, collected and processed at pre-established time intervals. The hatching rate was significantly higher for the larvae stored for 18 h compared with the 6-h group. However, embryos from this group gave rise to a larger number of malformations, and these were much more severe compared with those in the 6 h group, which led to a higher mortality in the long term. Regarding larval length, the animals of the 6 h group had higher mean total length compared with the 18 h group, but both treatments were inferior to the control. Numerous macro- and microscopic malformations were observed and, in both treatments, only the morphologically normal individuals were able to develop to the adult stage, with organ development similar to the control, except for the gonads that were still undifferentiated in treated animals.

In vitro effect of various cryoprotectants on the semen quality of endangered Oravka chicken.

We aimed to compare the effect of three different permeating cryoprotectants on the post-thaw spermatozoa quality. Pooled semen from Oravka cock line (n = 6) was diluted in Kobidil+ extender and frozen in cryoprotectant solutions containing 8% dimethylsulfoxide (DMSO), 8% ethylene glycol (EG) or 8% glycerol (GL) in liquid nitrogen vapours before being plunged into the liquid nitrogen. Spermatozoa motility parameters were assessed in vitro after freezing-thawing by a computer-assisted semen analysis (CASA) system and viability status was examined using fluorescent probes. The lower percentage (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing were obtained in all experimental groups (DMSO, EG, GL) compared with the control. Significant (P < 0.05) differences in total motility and progressive movement between GL and DMSO, EG groups were observed. However, the higher number (P < 0.05) of acrosome damaged spermatozoa was found in the DMSO and EG groups and no significant differences were observed in the GL group compared with the control. Differences (P < 0.05) between experimental groups and the control in the results of spermatozoa necrosis were observed. No significant differences in the percentage of apoptotic spermatozoa were found between control and experimental groups. However, significant differences (P < 0.05) in number of live and necrotic spermatozoa between GL and DMSO, EG groups were examined. The findings of the present study indicate that glycerol seems to be suitable for semen cryopreservation in the gene banks. In addition, fertility evaluation in vivo is needed in order to evaluate the possible contribution for the bank of animal genetic resources.

Influence of Cryopreservation Solution on the In Vitro Culture of Skin Tissues Derived from Collared Peccary (Pecari tajacu Linnaeus, 1758).

Skin vitrification is a promising and alternative tool for the conservation of biodiversity, especially for wild mammals, such as collared peccaries. Several factors can affect the success of this procedure, such as the cryoprotectant solution used. Therefore, this study was carried out to compare the efficiency of various vitrification solutions for recovery of viable cells after in vitro culture of cryopreserved skin tissues derived from the collared peccary, aiming to study the application in biobanking, where cellular use is not immediately required. Then, Dulbecco's modified Eagle's medium (DMEM) composed of 2.2 g/L sodium bicarbonate and 10% fetal bovine serum (FBS) was supplemented with 3.0 M ethylene glycol (EG) or 3.0 M dimethyl sulfoxide (DMSO) or 1.5 M EG plus 1.5 M DMSO with or without sucrose (SUC; 0.25 M) to produce six solutions for solid-surface vitrification. After warming, skin tissues were cultured in vitro and recovered cells were analyzed for morphology, adhesion, subconfluence, and proliferative activity for developing the growth curve and determining the population doubling time (PDT), and viability by Trypan Blue. The vitrification did not alter the ability of the tissues to adhere to the culture dish, as well as the day of all explants with cell growth, subconfluence samples, subconfluence total time, and PDT (p > 0.05). Moreover, independent of the cryoprotectant solution used, the vitrification altered the day of all attached explants (p < 0.05). Nevertheless, for viability after the first passage, only the EG-SUC (86.9%) and DMSO-SUC (91.4%) groups maintained viable cell recovery similar to the nonvitrified group (96.3%, p > 0.05). Additionally, for viability after the third passage, only the EG-SUC group maintained the cell quality (88.3%), when compared with the nonvitrified (97.8%, p > 0.05). In conclusion, DMEM with 10% FBS, 3.0 M EG, and 0.25 M sucrose was the most efficient solution for vitrifying collared peccary skin tissues, leading to the in vitro culture of viable cells.

Cryopreservation of Human Mesenchymal Stem Cells in an Allogeneic Bioscaffold based on Platelet Rich Plasma and Synovial Fluid

Transplantation of mesenchymal stem cells (MSCs) has emerged as an alternative strategy to treat knee osteoarthritis. In this context, MSCs derived from synovial fluid could provide higher chondrogenic and cartilage regeneration, presenting synovial fluid as an appropriate MSCs source. An allogeneic and biomimetic bioscaffold composed of Platelet Rich Plasma and synovial fluid that preserve and mimics the natural environment of MSCs isolated from knee has also been developed. We have optimized the cryopreservation of knee-isolated MSCs embedded within the aforementioned biomimetic scaffold, in order to create a reserve of young autologous embedded knee MSCs for future clinical applications. We have tested several cryoprotectant solutions combining dimethyl sulfoxide (DMSO), sucrose and human serum and quantifying the viability and functionality of the embedded MSCs after thawing. MSCs embedded in bioscaffolds cryopreserved with DMSO 10% or the combination of DMSO 10% and Sucrose 0,2 M displayed the best cell viabilities maintaining the multilineage differentiation potential of MSCs after thawing. In conclusion, embedded young MSCs within allogeneic biomimetic bioscaffold can be cryopreserved with the cryoprotectant solutions described in this work, allowing their future clinical use in patients with cartilage defects.

Thermal Analyses of a Human Kidney and a Rabbit Kidney During Cryopreservation by Vitrification.

This study focuses on thermal analysis of the problem of scaling up from the vitrification of rabbit kidneys to the vitrification of human kidneys, where vitrification is the preservation of biological material in the glassy state. The basis for this study is a successful cryopreservation protocol for a rabbit kidney model, based on using a proprietary vitrification solution known as M22. Using the finite element analysis (FEA) commercial code ANSYS, heat transfer simulations suggest that indeed the rabbit kidney unquestionably cools rapidly enough to be vitrified based on known intrarenal concentrations of M22. Scaling up 21-fold, computer simulations suggest less favorable conditions for human kidney vitrification. In this case, cooling rates below -100 °C are sometimes slower than 1 °C/min, a rate that provides a clear-cut margin of safety at all temperatures based on the stability of rabbit kidneys in past studies. Nevertheless, it is concluded in this study that vitrifying human kidneys is possible without significant ice damage, assuming that human kidneys can be perfused with M22 as effectively as rabbit kidneys. The thermal analysis suggests that cooling rates can be further increased by a careful design of the cryogenic protocol and by tailoring the container to the shape of the kidney, in contrast to the present cylindrical container. This study demonstrates the critical need for the thermal analysis of experimental cryopreservation and highlights the unmet need for measuring the thermophysical properties of cryoprotective solutions under conditions relevant to realistic thermal histories.

Effect of cryopreservation at -80°C on visco-hyperelastic properties of skeletal muscle tissue.

This paper investigated the effect of cryopreservation at -80°C on mechanical visco-hyperelastic properties of skeletal muscle. For that, both tensile and compression relaxation tests were performed on porcine tissues samples in fibre and cross-fibre directions. Material parameters were identified by using first order Ogden's strain energy function coupled with second order Maxwell's model. The results revealed that the cryopreservation conditions at -80°C with conservative and cryoprotectant solution significantly affected the mechanical properties of the skeletal muscle. Thus, cryopreserved tissue showed a higher instantaneous initial Young's modulus than for the fresh tissue in both tensile and compression deformations, and in the two muscular fibre directions. Furthermore, in compression tests, cryopreserved tissue exhibited a smaller non-linearity and a higher total relaxation ratio in both muscle fibre directions than for the fresh tissue.

Improved solution for vitrification ofProchilodus lineatusembryos based on the reduction in risk factors: Toxicity, osmotic responses and ice-nucleation

The objective of our work was to describe a low toxicity cryoprotectant solution that allowed vitreous solid formation. Embryos of Prochilodus lineatus were submitted to sensitivity evaluations of six internal cryoprotectants (dimethyl sulphoxide – Me2SO, dimethyl acetamide – DMA, dimethyl formamide – DMF, methanol – MET, glycerol – GLY and 1,2-propanediol – PROP) at concentrations of 1–6 M; and two external cryoprotectants (sucrose – SUC and glucose – GLU) at concentrations of 0.1–1 M for 20 min. The capacity of the cryoprotectant solutions to exchange heat with the medium and to form glassy solids was evaluated by immersing 10 μl of cryoprotectant in liquid nitrogen. The PROP had a high survival rate at all concentrations evaluated, and was the only substance that allowed a vitreous solid formation. Thus, it is concluded that the PROP-6 M was the most adequate solution for embryonic vitrification processes, because heat exchange between the system (PROP 6 M/embryos/liquid nitrogen) was faster than for other cryoprotectants and combinations thereof; has low toxicity, promote high rates of dehydration in short periods, and reach the vitreous state, being a good candidate to be used in the tests of embryonic vitrification.