Addition Of Cryoprotectants Research Articles

Enhanced probiotic viability and aromatic profile of yogurts produced using wheat bran (Triticum aestivum) as cell immobilization carrier

Abstract The effect of wheat bran ( Triticum aestivum ) as a cell immobilization carrier for probiotic yogurt production on cell viability, composition of volatile compounds and sensory characteristics, was studied. Wheat bran was delignified and separately used as a carrier for the immobilization of Lactobacillus casei ATCC393 and Lactobacillus bulgaricus DSM20081. Both biocatalysts were freeze-dried without the addition of cryoprotectants and were used for yogurt fermentation at 40 °C. Their operational stability was evaluated during successive yogurt fermentation batches until they were inactivated. The yogurts fermented using the immobilized biocatalysts were compared with those fermented with free Lactobacillus cells and with conventional yogurt culture ( Streptococcus thermophilus and L. bulgaricus ). The novel yogurts showed significantly higher cell viabilities during storage at 4 °C. In addition, the immobilized biocatalysts showed higher survival rates during exposure to simulated gastric juice (pH 3.0). The immobilized biocatalysts significantly affected the production of volatile compounds, indicating, in combination with the sensory evaluations, potential for good-quality probiotic yogurt production by freeze-dried ready-to-use immobilized starters.

Optimization of cryoprotectants for Streptococcus thermophilus during freeze-drying using Box-Behnken experimental design

The strain Streptococcus thermophilus are widely used in the ways of probiotic formulations form or starter culture powder depending on the function of the strain and viability during production process. The pros and cons of freeze-drying on the viability and storage of starter lactic acid bacteria are well documented. In the present study, we focused on the optimization of freeze-drying medium with addition of cryoprotectants for Streptococcus thermophilus. The interactive effects of sucrose, soluble starch and ascorbic acid on the survival rate as well as the viable counts of S. thermophilus during freeze-drying were investigated by Box-Behnken experimental design. The results showed that, with the mixture of 17 g/100mL sucrose, 17 g/100mL soluble starch and 0.4 g/mL ascorbic acid, the viability could be well preserved when freeze-drying was employed. Moreover, the survival rate and viable counts of S. thermophilus were observed to (69.93±0.07) % and (2.79±0.03) ×1011 cfu/g during freeze-drying using the optimal compound cryprotectants, respectively, which verified the adequacy of predicted value 70.19 % and 2.81×1011 cfu/g. Our results demonstrated that the mixture of the cells in the presence of above three cryprotectants before freeze-drying protected S. thermophilus cells from the adverse freeze-drying environment efficiently.

Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes

Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All fresh and vitrified oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group). Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos.

113 DEVELOPING A CRYOPRESERVATION PROTOCOL FOR DESERT TORTOISE SPERM (GOPHERUS AGASSIZZII)

The desert tortoise (Gopherus agassizzii) is listed as threatened by the USA Fish and Wildlife Service and population declines continue to occur throughout most of their range. This species’ low reproductive rate, combined with the advanced age at which they reach sexual maturity, makes them vulnerable to multiple threats. Although assisted reproductive technologies can enhance breeding of many species, they are not widely used in tortoises. The objective of this study was to identify effective sperm cryopreservation protocols for the desert tortoise and possibly to other members of Testudinidae. We compared the effects of various concentrations of the cryoprotectants dimethyl sulfoxide (DMSO) and glycerol (6–20%) using 3 freezing devices at 4 freeze rates (CryoCooler, Ops Diagnostics, 2.3°C/m, 6.4°C/m; CryoMed, Thermo Scientific, 0.3°C/m or 1.0°C/m; and CoolCell, Biocision, 1.0°C/m) on several sperm parameters. Sperm was collected postmortem from the vas deferens of 9 individuals and tested either individually (n = 2), combined into 1 pool of 3 individuals, or 2 pools of 2 individuals. Sperm was extended in TEST-yolk buffer. Initial motility score (IMS; % motile × speed of progression2), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded before cryoprotectant addition and freezing. For each treatment group, triplicate vials were thawed at 37°C for 60 s. Cryoprotectant was removed by centrifugation and the sperm pellet was resuspended in M199 + HEPES. Sperm were evaluated immediately following resuspension (T0), as well as 30 (T30) and 60 (T60) minutes postincubation at 22°C. All data were expressed as a percentage of initial (%IMS, %IPL, and %IAC). A sperm quality index (SQI) was calculated as (%IMS × %IPL × %IAC)/1,000, giving equal weight to each indicator of cryosurvival. The effects of freeze method on %IMS, %IPL, %IAC, and SQI were analysed by ANOVA and Tukey’s test. The effect of freeze method was significant at T0 and T60, with the 16% DMSO, 6.4°C/m method resulting in the highest %IMS at T0 (46.1%) and T30 (33.8%) and the 12% glyercol at 0.3°C/m highest at T60 (48.7%). Sperm frozen in 16% glycerol at 0.3°C/m had the highest %IPL at T0, T30, and T60 (91.9, 90.4, and 85.4%, respectively). Acrosome integrity was best maintained when sperm were frozen in 16% DMSO at 6.4°C/min (91.9%). The SQI was highest at T0 when sperm was frozen at 1.0°C/min in the CryoMed with highest post-thaw sperm parameters in 16% (T0) or 12% glycerol (T30 and T60). Interestingly, there were significant differences in SQI between the two 1.0°C/min freeze methods at each period, indicating that freezing device affected sperm cryosurvival, perhaps due to different freezing curves. This study indicates that mid-range (12 and 16%) cryoprotectant concentrations and slow freeze rates (0.3°C/m and 1.0°C/m) are optimal for desert tortoise sperm frozen in TEST-yolk buffer. Future studies will determine fertilizing capability of these sperm. These results may serve as a starting point for the study of sperm cryopreservation in other Testudinidae species.

Substitution of egg yolk with different concentrations of soybean lecithin in tris-based extender during bulls' semen preservability

ObjectiveTo investigate the effect of various concentrations of soybean lecithin as an alternative for egg yolk in bull semen extender on post-chilling and post-thaw sperm quality characteristics. MethodsSemen ejaculates were collected from three mature bulls' once/week for 5 weeks. After initial evaluation the approved ejaculates were pooled and extended gradually 1:7 with tris-citrate-fructose egg yolk extender (control) and tris-citrate-fructose (TCF)+different concentration of soybean lecithin (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% and 4.0%) to ensure 60 million motile spermatozoa/mL, and then proceeded an adopted international cryopreservation protocol. Frozen straws were thawed at 37 °C for 30 s. The parameters studied were sperm motility, viability, abnormality, membrane integrity and normal intact acrosome percentages in chilled and frozen-thawed semen. ResultsThe substitution of egg yolk into TCF with 1% soya lecithin significantly (P < 0.0001) ameliorated the maintenance of semen characters motility (86.67% ± 0.80%), life (87.60% ± 0.88%), and membrane integrity (82.47% ± 0.94%), meanwhile it had significantly (P < 0.0001) reduced the abnormality (13.20% ± 0.60%) of spermatozoa to its least value compared to some other concentrations in use. Moreover, the addition of 1.5% of soya lecithin to TCF had maintained the semen characteristics compared to the control tris-citrate-fructose egg yolk. The significantly higher mean values of post-thawing sperm motility % were observed in 1% soybean lecithin (56.00% ± 1.00%) as compared to the control (41.00% ± 1.00%). Conclusion1–1.5% soya lecithin can effectively alternate egg yolk as cryoprotective additives for cryopreservation extender, without any detrimental effects on post-chilling and post-thaw semen quality in cattle bull.

Mince from Tilapia-Backbone: Effects of Washing and Cryoprotectant Addition during Frozen Storage

Mince obtained from tilapia (Oreochromis niloticus) (backbone) was evaluated; the effect of washing and the addition of a commercial cryoprotectant on the quality of the mince obtained were also assessed. Physicochemical, microbiological and sensorial analyses were carried out at 0, 15, 30, 45, 60, 90, 120 and 180 days of frozen storage (-20 ºC). During washing treatment 91% of lipids was removed from the mince (p&lt;0.05). The proximal composition was stable during the storage time (p&gt;0.05). Parameters such as L* and “θ” increased while a*, b*, chroma and TBARS decreased due to the washing treatment (p&lt;0.05). The cryoprotectant effect resulted in a decrease of L*, “θ” (Hue) and TBARS (p&lt;0.05). The addition of the cryoprotectant caused a significant decrease of L*, “θ” and TBARS. During the storage period, the proximate composition was stable and the microbial load remained below the official limits. The panelists detected changes on the odor, color and texture in the mince evaluated. Results suggest that the washing treatment improved the stability of the mince compared to the addition of cryoprotectant.

Impact of Sample Aging on Freeze-Thaw Stability of Oil-in-Water Emulsions Prepared with Soy Protein Isolates

The freeze-thaw stability of oil-in-water emulsions prepared with unheated and heated aqueous dispersions of fresh and stored soy protein isolates was evaluated in the absence and presence of glucose or sorbitol (0.75–15.0% w/w). Sample aging had a negative impact of freeze-thaw stability. The cryoprotectant addition enhanced the freeze-thaw stability, but at low concentrations emulsions prepared with unheated soy protein isolates showed better response to freeze-thawing. Nevertheless, at the highest cryoprotectant concentration, a total stabilization was evidenced for all emulsions. The results of this article indicated that the cryoprotectants act on proteins at interfacial level.

The nematode Caenorhabditis elegans survives subfreezing temperatures in an isochoric system

This study is the first experimental evidence showing that a living multicellular organism, the nematode Caenorhabditis elegans, can survive subfreezing temperatures in an isochoric (constant volume) thermodynamic system, while immersed in a simple isotonic solution, without the addition of cryoprotectants. Some of the test conditions were more extreme than those found at the ice/water interface of the Antarctic subglacial Vostok lake. On earth, life takes place in an isobaric (constant pressure) environment. In isobaric systems, subfreezing temperature survival of organisms in nature and subfreezing temperature preservation of living material for biotechnology and medicine, is made possible by use of cryoprotective chemicals additives. Our theoretical thermodynamic studies suggested that in an isochoric system, living biological material could survive subfreezing temperatures, without any cryoprotective chemicals. By confirming the theoretical predictions, this paper suggests a new technology for subfreezing preservation of cells, organs and organisms of possible value for biotechnology and medicine as well as new possible mechanisms of living organism survival in nature.

Suppression of protein inactivation during freezing by minimizing pH changes using ionic cryoprotectants

Freezing and lyophilization are often used for stabilization of biomolecules; however, this sometimes results in partial degradation and loss of biological function in these molecules. In this study we examined the effect of freezing-induced acidity changes on denaturation of the model enzyme haloalkane dehalogenase under various experimental conditions. The effective local pH of frozen solutions is shown to be the key causal factor in protein stability. To preserve the activity of frozen-thawed enzymes, acidity changes were prevented by the addition of an ionic cryoprotectant, a compound which counteracts pH changes during freezing due to selective incorporation of its ions into the ice. This approach resulted in complete recovery of enzyme activity after multiple freeze-thaw cycles. We propose the utilization of ionic cryoprotectants as a new and effective cryopreservation method in research laboratories as well as in industrial processes.

Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome.

Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation.

Toxicity Minimized Cryoprotectant Addition and Removal Procedures for Adherent Endothelial Cells.

Ice-free cryopreservation, known as vitrification, is an appealing approach for banking of adherent cells and tissues because it prevents dissociation and morphological damage that may result from ice crystal formation. However, current vitrification methods are often limited by the cytotoxicity of the concentrated cryoprotective agent (CPA) solutions that are required to suppress ice formation. Recently, we described a mathematical strategy for identifying minimally toxic CPA equilibration procedures based on the minimization of a toxicity cost function. Here we provide direct experimental support for the feasibility of these methods when applied to adherent endothelial cells. We first developed a concentration- and temperature-dependent toxicity cost function by exposing the cells to a range of glycerol concentrations at 21°C and 37°C, and fitting the resulting viability data to a first order cell death model. This cost function was then numerically minimized in our state constrained optimization routine to determine addition and removal procedures for 17 molal (mol/kg water) glycerol solutions. Using these predicted optimal procedures, we obtained 81% recovery after exposure to vitrification solutions, as well as successful vitrification with the relatively slow cooling and warming rates of 50°C/min and 130°C/min. In comparison, conventional multistep CPA equilibration procedures resulted in much lower cell yields of about 10%. Our results demonstrate the potential for rational design of minimally toxic vitrification procedures and pave the way for extension of our optimization approach to other adherent cell types as well as more complex systems such as tissues and organs.

Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life

Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze–thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in β-sheet content (band maximum detected at a frequency of 1634cm−1 in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life.

21. A lesson from Nature for controlling ice during organ cryopreservation by vitrification

Banking of organs using current tissue banking practices employing conventional cryopreservation by freezing is not feasible due to the well known damage caused by intra and extra-cellular ice formation. An alternative cryopreservation approach is known as vitrification. There has been considerable success in ice-free vitrification of small tissue samples but better ice control is required for samples > 10 mL in volume. Development of novel cryoprotectants and methods that will facilitate clinically effective banking by vitrification of large vascularized tissues and organs is needed. There are lessons to be learned for organ vitrification from Nature. Freeze avoiding insects such as Cujucus clavipes beetle larvae from the interior of Alaska vitrify and survive temperatures that routinely reach −60 °C with some individuals surviving exposure to −100 °C. At the onset of winter these insects combine changes in behavior with increases in cryoprotectant content (such as glycerol, proline, trehalose), dehydration, production of antifreeze compounds (proteins and glycolipids), and diapause. The cryoprotectant concentration can achieve 4.2 M, approximately half the concentration required for ice-free cryopreservation of tissue and organ therapy products. Recombinant copies of insect-derived antifreeze proteins are available, so they can be manufactured, and they are up to 10 times more effective in ice modulation (thermal hysteresis) than the well known fish-derived antifreeze proteins that have proven ineffective or detrimental for cell cryopreservation. Recently we have discovered that certain insect-derived antifreeze proteins significantly increase cell viability during cryopreservation by freezing. Similarly, in unpublished studies we have found that an insect-derived antifreeze glycolipid, with thermal hysteresis activity similar to that of hyperactive insect antifreeze proteins, can significantly increase cell viability post-cryopreservation using freezing methods. These glycolipids have been found in several species including freeze tolerant and avoiding insects, freeze tolerant frogs, and freeze tolerant plants. Unlike the antifreeze proteins, which are diverse in structure across species, these glycolipids are nearly identical. While we anticipate that eventually it will be possible to synthesize the saccharide component (s1-4 linked xylose-mannose), at present the antifreeze glycolipid is harvested from natural sources, especially plants. Translation of this lesson from Nature requires a better understanding of how animals prepare themselves for diapause and hibernation so that we can reproduce these phenomena in organ donors in vivo, under physiological conditions post-mortem prior to organ procurement, and/or organs ex-vivo before cryopreservation in cryoprotectant formulations that incorporate antifreeze compounds. It is anticipated that utilization of antifreeze proteins and glycolipids will enable utilization of less cytotoxic cryoprotectant formulations with enhanced ice control during cooling and warming of larger biological structures than currently possible. In the big picture it is likely that they will be combined with other strategies presented in this session on controlling ice formation including improved cryoprotectant addition and removal methods, pressure assisted cooling, stress relief by annealing, synthetic antifreeze compounds, and nanoheater assisted rewarming. Once we have ice formation under control we must be concerned with ischemic reperfusion injury, the final hurdle upon in vivo transplantation in patients.

Cryoprotective ability of betaine-type metabolite analogs during freezing denaturation of enzymes.

To evaluate an analog library of betaine-type cellular metabolites, which are naturally found in polar fish for survival in subzero temperatures, for preventing denaturation of enzymes during freezing. Comparison of the cryoprotective ability of reported cryoprotectants, such as dimethylsulfoxide, glycerol, ectoine, hydroxyectoine, and trehalose, with betaine-type analogs using α-glucosidase revealed that analogs introducing C3-C6 alkyl chains into an ammonium cation retained 20 % higher activity than the control cryoprotectants at the same concentration. In particular, the analog possessing triplicate n-butyl chains showed a profound effect. It allowed retention of enzyme activity to 95 % even after 100 freeze-thaw cycles, while addition of the control cryoprotectants decreased the activity to 10-20 %. The cryoprotective ability of betaine-type analogs can be applied not only to α-glucosidase but also other enzymes such as β-glucosidase, alkaline phosphatase, lactose dehydrogenase, sulfatase, and horseradish peroxidase. Synthetic betaine-type metabolite analogs possess practicable cryoprotective ability for various enzymes, and are considerably superior to previously reported cryoprotectants.

Cell blebbing upon addition of cryoprotectants: a self-protection mechanism.

In this work, the mechanism of cell bleb formation upon the addition of cryoprotectants (CPAs) was investigated, and the role of cell blebs in protecting cells was determined. The results show that after adding CPAs, the hyperosmotic stress results in the breakage of the cortical cytoskeleton and the detachment of the cell membrane from the cortical cytoskeleton, causing the formation of cell blebs. Multiple blebs decrease the intracellular hydrostatic pressure induced by the extracellular hyperosmotic shock and alleviate the osmotic damage to cells, which reduces the cell mortality rate. In the presence of a low concentration of CPAs, cell blebs can effectively protect cells. In contrast, in the presence of a high concentration of CPAs, the protective effect is limited because of severe disruption in the cortical cytoskeleton. To determine the relationship between blebs and the mortality rate of cells, we defined a bleb index and found that the bleb index of 0.065 can be regarded as a reference value for the safe addition of DMSO to HeLa cells. The bleb index can also explain why the stepwise addition of CPAs is better than the single-step addition of CPAs. Moreover, the mechanism of the autophagy of cells induced by the hyperosmotic stress was studied, and the protective effect associated with the autophagy was compared with the effect of the blebbing. The findings reported here elucidate a self-protection mechanism of cells experiencing the hyperosmotic stress in the presence of CPAs, and they provide significant evidence for cell tolerance in the field of cryopreservation.

C-2026: Observations of damage to stage V zebrafish oocytes in contact with extracellular ice

The successful banking of zebrafish germplasm by embryo or oocyte cryopreservation remains elusive, due to the rapid onset of cryoinjury following extracellular ice formation. The present study used high-speed imaging to investigate the causes of damage to Stage V zebrafish oocytes following contact with extracellular ice and subsequent cooling. During cryomicroscopy experiments in the absence of cryoprotectant additives, extracellular ice formation was initiated approximately 8 mm away from the oocyte; the resulting crystals were then allowed to envelop the oocyte during an isothermal hold at −0.9 °C. When oocytes were exposed to the external ice for up to 20 min at this temperature, only 13% exhibited any evidence of injury (rupture or darkening). In contrast, our previous studies have shown that when zebrafish embryos are maintained in contact with external ice for 20 min at −0.4 °C, mechanical damage occurred in as many as 47% of embryos. Controlled-cooling experiments with oocytes were performed at rates of 0.5, 1, and 2 °C/min, all of which were preceded by a 5-min isothermal hold at −0.9 °C, to allow equilibration with the extracellular ice. In all 68 oocytes observed, cryoinjury manifested as a sudden increase in oocyte opacity (darkening) during the cooling ramp. The average darkening temperature decreased slightly with increasing cooling rate, but this effect was not statistically significant (ANOVA, p > 0.25). However, we found a statistically significant effect of oocyte age (time since collection) on the darkening temperature (ANOVA, p p

Principles of cryopreservation by vitrification.

Vitrification is an alternative approach to cryopreservation that enables hydrated living cells to be cooled to cryogenic temperatures in the absence of ice. Vitrification simplifies and frequently improves cryopreservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed cell type populations, eliminates the need to find a frequently imperfect compromise between solution effects injury and intracellular ice formation, and enables cooling to be rapid enough to "outrun" chilling injury, but it complicates the osmotic effects of adding and removing cryoprotective agents and introduces a greater risk of cryoprotectant toxicity during the addition and removal of cryoprotectants. Fortunately, a large number of remedies for the latter problem have been discovered over the past 30+ years, and the former problem can in most cases be eliminated or adequately controlled by careful attention to technique. Vitrification is therefore beginning to realize its potential for enabling the superior and convenient cryopreservation of most types of biological systems (including molecules, cells, tissues, organs, and even some whole organisms), and vitrification is even beginning to be recognized as a successful strategy of nature for surviving harsh environmental conditions. However, many investigators who employ vitrification or what they incorrectly imagine to be vitrification have only a rudimentary understanding of the basic principles of this relatively new and emerging approach to cryopreservation, and this often limits the practical results that can be achieved. A better understanding may therefore help to improve present results while pointing the way to new strategies that may be yet more successful in the future. To assist this understanding, this chapter describes the basic principles of vitrification and indicates the broad potential biological relevance of vitrification.

Sperm quality of the Amazon catfish Leiarius marmoratus (Gill, 1870) after cold storage.

This study aimed at assessing the sperm quality of the Amazon catfish, Leiarius marmoratus ¸ after refrigeration without extenders. After capturing the animals and stripping of semen, the following parameters were analyzed: progressive motility, motility quality score, duration of motility and sperm morphology. An aliquot of fresh semen from each male was kept at room temperature (28 ± 2°C) as a control, for further comparison with cooled semen. The semen from each animal was stored in extenders-free individual syringes. The syringes were kept in ice within polystyrene boxes at 13 ± 2°C. For both fresh and cooled semen, seminal parameters were evaluated every one-hour interval, reaching seven hours of analysis. Fresh semen showed a significant decrease in motility, motility quality score and duration of motility remaining viable only for three hours. Progressive motility of the cooled semen displayed a negative linear pattern (P<0.05). The duration of motility increased (P<0.05), reaching its peak after three hours of storage. The motility quality score showed a quadratic pattern. No statistical differences were observed when sperm morphology was assessed (P>0.05), even though the mean values of total abnormalities have increased over the storage time. Further studies focusing on the application of this technique should be performed, including the addition of extenders and cryoprotectants for preservation of the sperm over longer periods.

Preservation of spotted buffalo epididymal spermatozoa in andromed with raffinose addition

Raffinose is an external cryoprotectant that would protect cell membrane from cold shock effect during preservation process. This study was perform to observe the effect of raffinose on the quality of spotted buffalo epididymal spermatozoa during storage at 4 °C for 24 h. Three different extenders, i.e. commercial tris-egg yolk free extender (Andromed) as control (AM), Andromed Andromed plus 0.2% w/v raffinose (AMR0.2), and Andromed plus 0.4% w/v raffinose (AMR0.4), were used in this study. The results showed that the progressive motility and viability of spermatozoa reduced significantly during storage period. At 12 h of storage, the progressive motility was 60.33% in AM versus 60% in both AMR0.2 and AMR0.4 (P>0.05). Meanwhile, after 24 h of storage, the progressive motility in both AM and AMR0.4 extenders was 55% versus 55.33% in AMR0.2 (P>0.05). Spermatozoa viability in AM, AMR0.2, and AMR0.4 were 70.30%, 73%, 72.33% after 12 h of storage and 66.33%, 68.67% and 69.67% after 24 h of storage, respectively (P>0.05). This results suggested that Andromed without an external cryoprotectant addition could maintain the quality of epididymal spermatozoa as good as the one with 0.2% and 0.4% raffinose addition.

Effect of cryopreservation on the enzyme activity of Russian sturgeon (Acipenser gueldenstaedtiiBrandt &amp; Ratzeburg, 1833) semen

The hypothesis was tested that cryodamage occurs during the storage of semen at −196°C and that cryopreservation has a negative effect on the enzyme activity and motility of semen in the Russian sturgeon, Acipenser gueldenstaedtii. In addition, the aim of the study was to determine the activity of metabolic enzymes, including adenosintriphosphatase (total ATPase), creatine kinase (CK), succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), plus antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR). After cryopreservation (storage at −196°C), the motility and duration of semen fell and the total ATPase, SDH, LDH and CK activity in the spermatozoa was reduced. In the seminal plasma, however, total ATPase, SDH, LDH and CK activity increased markedly. Activity also notably increased in the group with no diluents or cryoprotectants. SOD, CAT and GSH-Px activities also increased in the seminal plasma but dropped with regard to the spermatozoa, whereas the GR activity varied. The antioxidant enzyme activity trend between the groups with and without the addition of cryoprotectants was the same as in the metabolic enzyme activity. Cryopreservation had a great effect on the enzyme activity and motility of A. gueldenstaedtii semen; however, the cryoprotectant protected the semen during cryopreservation.