Combinations Of Cryoprotectants Research Articles

Effect of melatonin on developmental competence, mitochondrial distribution, and intensity of fresh and vitrified/thawed in vitro matured buffalo oocytes

Background: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10−9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10−9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

Synergistic combination of cryoprotectants improved freeze‐dried survival rate and viable counts of Lactiplantibacillus plantarum

Well‐designed cryoprotectants are crucial for successful probiotic application. In this study, based on literature analysis, characteristics and economic analysis of cryoprotectants, the sucrose (8.2%)–skim milk (8.0%)–inulin (8.2%) combination was proposed and optimised to enhance freeze‐drying resistance of probiotics. Survival, viable cell counts and inactivation rate during storage at 4°C of Lactiplantibacillus plantarum grx16 protected by the combination was 94.9%, 1.89 × 1011 cfu/g and 0.0116. The optimised sucrose–skim milk–polymer prebiotics combination could also effectively protect several other probiotics, with the survival rate being 89.2–95.6%. These results will aid in the rational development of synergistic cryoprotectant formulations for probiotics.

EFFICIENCY OF AMIDES AS CRYOPROTECTORS IN THE COMPOSITION OF PROTECTIVE ENVIRONMENTS FOR BULLS SPERM CRYOCONSERVATION

The article highlights the results of research on the effectiveness of the introduction of cryoprotectants of the amide group – dimethylformamide (DMF), dimethylacetamide (DMAC) into the cryoprotective media for bull sperm and to determine their effect on the main physiological characteristics of germ cells. Materials and methods. Immediately after obtaining sperm, it was diluted with medium No. 1 containing 5.6% glycerol and 1.4% DMF (experimental group 1). Experimental group 2 was also diluted and treated with medium No. 1, but with the addition of 5.6% glycerol and 1.4% DMAC. After saturation of the sperm with the protective substances of medium № 1, it was diluted with medium No 2 containing 4% glycerol and 1% DMF (experimental group 1) and 4% glycerol and 1% DMAC (experimental group 2). Control samples were diluted according to a standard two-moment protocol using 7% glycerol in medium No. 1 and 5% glycerol in medium No. 2. Cryopreservation of spermatozoid was carried out by direct immersion of standard flat metal cassettes with coated granules in a liquid nitrogen medium. At the same time, cooling was carried out in the following mode: 4°C to minus 10°C at minus 3°C/min and from minus 10°C to minus 80°C at minus 40°C/min. Deconservation of spermatozoa was carried out at a temperature of 38–39°C. Cows were inseminated by the recto-cervical method. The results. The use of penetrating cryoprotectants from the amide group - dimethylformamide and dimethylacetamide - in media for sperm dilution and cryopreservation ensures a certain increase in the motility of germcells by 5.9% and 9.0%, respectively. The positive effect of the composition of cryoprotectants was also noted regarding the survivat of sperm after deconservation. Thus, the survival rate in the second experimental group was 0.67 hours or 10.3% higher than in the control group. The environments supplemented with cryoprotective substances of the amide group ensured sufficiently high sperm survival at body temperature (38ºС), which significantly exceeded the established minimum physiological norm (5 hours). In percentage terms, this excess was 34.6% and 42.6%. Conclusions. The feasibility of using combinations of endocellular cryoprotectants made from glycerol and dimethylformamide (DMF) or glycerol and dimethylacetamide (DMAC) in cryoprotective media (diluents) for bull sperm has been proven, which increases the quality characteristics of the biomaterial.

Effect of freeze-dried protectants on the survival rate and fermentation performance of fermented milk's directed vat set starters

A directed vat set (DVS) starter was proposed to improve the drawbacks of liquid starters in fermented production and enhance the survival rates of B. animalis subsp. lactis BZ11, S. thermophilus Q-1, and Lactiplantibacillus plantarum LB12. The protective agent formula was optimized using the response surface method (RSM), with the survival rate as the benchmark. The best combination of cryoprotectants was determined to be BZ11: 10 % skimmed milk powder, 3 % sodium glutamate, and 15 % trehalose; LB12: 10 % skim milk powder, 5 % glutamate sodium, and 10 % trehalose; Q-1: 10 % skimmed milk powder, 3 % sodium glutamate, and 10 % trehalose. The survival rate of BZ11 significantly increased to 92.87 ± 1.25 %. The DVS fermented milk did not differ significantly from the control group regarding cholesterol removal, live cell counts and pH (p > 0.05). All DVS can be stored for at least 2500 d at −20 °C—this DVS starter for fermented milk benefits from its large-scale and automated commercial production.

Influence of cryopreservation techniques and low concentrations of permeating cryoprotectants on the conservation of ear cartilage and skin derived from six-banded armadillos (Euphractus sexcinctus Linnaeus, 1758)

Considering the importance of efficiently obtaining somatic resource banks as an ex-situ conservation strategy for wild mammals, we evaluated two techniques (slow freezing - SF and solid-surface vitrification - SSV) for the cryopreservation of ear cartilage and skin from six-banded armadillos. Additionally, we analyzed the effects of two combinations of intracellular cryoprotectants (3.0 M or 6.0 M ethylene glycol - EG and dimethyl sulfoxide - Me2SO) on SSV. Tissues not subjected to cryopreservation were used as controls. All samples were evaluated for morphological analysis and cell ability during culture. The thickness of the basal layer was similar to the control only for tissues derived from SF, while SSV ensured the preservation of the cartilage thickness. Moreover, fragments derived from SF and SSV, especially in the 3.0 M EG-Me2SO group, resulted in dermis thickness and total skin similar to the control. All cryopreserved techniques maintained normal patterns of the fibroblasts, epidermal cells, and melanocytes. While only SF-derived fragments maintained the number of degenerated chondrocytes similar to the control, no difference was observed between groups for normal chondrocytes and lacunae. Moreover, SSV maintained the collagen fibers percentage of the tissues even after warming. After culture, SF and SSV techniques were efficient for the recovery of the somatic cells in all parameters evaluated, except the day of subconfluence, which was greater for the SSV group with 6.0 M EG-Me2SO. In summary, SSV, especially with 3 M EG-Me2SO, was as efficient as SF in preserving ear skin and cartilage derived from six-banded armadillos.

Cryoprotectant Effects on Duck Surimi During Frozen Storage

The surimi-like material made from duck (DSLM) was investigated on the freeze-thaw stability during 4 months of frozen storage using different low-sweetness cryoprotectants such as palatinose (PAL), trehalose (TRE), polydextrose (PLD), maltodextrin (MAL), sucrose and sorbitol (SS), and lactitol (LAC) at a 6% concentration. As compared to the CON, the inclusion of cryoprotectants significantly improved the water-holding capacity of all treated samples. Particularly, as compared to all of the cryoprotectants evaluated, the PLD and PAL samples displayed greater stability concerning Ca2+-ATPase activity, sulfhydryl concentration, and protein solubility. All samples' endothermic peaks declined over time, showing changes in thermal characteristics during storage. A scanning electron microscope (SEM) observation of the CON and LAC-treated samples after 4 months of frozen storage revealed an increase in the number of pores. These findings imply that low-sweetness PLD and PAL outperformed the usual cryoprotectant combination of sucrose and sorbitol (SS) and that they could be used in frozen DSLM. They provide greater water-holding capacity, anti-freezing characteristics, lowered sulfhydryl oxidation, increased protein solubility, and structural integrity, which renders them useful components for innovation in the frozen surimi-like material industry.

Soybean lecithin and cholesterol-loaded cyclodextrin in combination to enhances the cryosurvival of dairy goat semen

The objective of the study was to examine the effect of soy lecithin (SL) and cholesterol loaded cryclodestrin (CLC) on cryo-survival of sperm cryopreserved in the presence or absence of seminal plasma in Saanen dairy goats. Tris-based dilutions containing various concentrations of SL (0, 0.5%, 1.0% or 2.0%) and CLC (0, 2.0 g/L, 4.0 g/L or 6.0 g/L CLC) were used to cryopreserve Saanen dairy goat sperm. The quality of frozen-thawed sperm, including progressive motility, viability, acrosome and plasma membrane integrity, as well as fertility were detected. Results found that the optimal combination of the two cryoprotectants was 1.0% SL+4.0 g/L CLC, which significantly increased progressive motility, viability, acrosome and plasma membrane integrity of frozen thawed sperm. The impact of the two cryoprotectants in combination was not affected by the presence of seminal plasma. The conception rates obtained after artificial insemination using sperm cryopreserved with and without seminal plasma were 88.89% and 91.67% (P > 0.05), respectively. The respective values for average number of litter sizes were 1.55 ± 0.17 and 1.56 ± 0.21 (P > 0.05). Therefore, this study improved the cryopreservation efficiency of goat semen, enhanced the sperm cryosurvival, and layed a foundation for the wide application of frozen goat semen.

P-190 Spontaneous blastocyst collapse during pre-vitrification equilibration is related to a lower live birth rate: a prospective cohort study

Abstract Study question This report provides updated data from a study investigating the association between the spontaneous collapse patterns of blastocysts during pre-vitrification equilibration and pregnancy success. Summary answer Live birth rates were lower for completely collapsed blastocysts, suggesting the possibility for including blastocyst collapse pattern as a criterion for selecting embryos for transfer. What is known already Previous time-lapse studies have found that blastocysts exhibiting strong contraction during culture have a low probability of hatching in animals and implanting in humans, suggesting blastocyst contraction has a negative impact on reproductive outcomes. Conversely, artificial shrinkage of human blastocysts before vitrification is considered to improve embryo survival and/or pregnancy rates by promoting cryoprotectant permeation inside the blastocoel. However, shrinkage is not induced in some vitrification protocols, including ours, as the viability is high; this demonstrates the potential of existing protocols to achieve sufficient cryoprotectant permeation. Moreover, the Alpha consensus meeting on cryopreservation did not issue recommendations regarding artificial shrinkage. Study design, size, duration This study included 798 patients who were undergoing their first autologous IVF/ICSI cycle followed by a freeze-all strategy, including blastocyst vitrification, at our clinic between June 2018 and November 2021. For patients with multiple vitrified blastocysts, embryos for transfer were selected hierarchically based only on morphological scoring during culture. To reduce bias, only data from a single blastocyst (day 5, Gardner score 4, excluding CC) transfer from each patient’s first warmed cycle was analyzed. Participants/materials, setting, methods Blastocysts were vitrified-warmed in in-house-prepared solutions using Rapid-i carriers. Prior to vitrification, intact blastocysts were equilibrated in 10% ethylene glycol (15 min, 37 °C). Their spontaneous collapse patterns were assessed under an inverted microscope before they were vitrified in 15% ethylene glycol + 15% dimethylsulfoxide + 0.5 M sucrose. Blastocyst collapse was defined as the separation of the trophectoderm cells from the zona pellucida. Collapsed blastocysts with/without a blastocoel cavity were defined as partially/completely collapsed blastocysts. Main results and the role of chance Non-collapsed (Nc), partially collapsed (Pc), and completely collapsed (Cc) blastocyst transfers (embryo survival rate, 99.6%) constituted 27.1%, 54.6%, and 18.3% of the 798 cycles, respectively. Live birth rates differed significantly between groups (Nc, 54.2%; Pc, 46.6%; Cc, 34.2%; P < 0.001). Regarding perinatal outcomes of 366 singletons, the female rate tended to differ between groups (Nc, 35.3%; Pc, 44.3%; Cc, 53.1%; P = 0.084). Significant differences in maternal age (years: Nc, 34.6 ± 4.0; Pc, 35.4 ± 3.7; Cc, 36.4 ± 4.0; P < 0.001), MII oocyte number (Nc, 13.0 ± 6.2; Pc, 12.0 ± 6.6; Cc, 10.8 ± 6.9; P = 0.009), blastocyst diameter (μM: Nc, 184 ± 18; Pc, 181 ± 20; Cc, 173 ± 23; P < 0.001), and good-quality (4AA/4AB/4BA) blastocyst percentage (Nc, 70.4%; Pc, 61.2%; Cc, 47.9%; P < 0.001) were found between groups. Upon logistic regression analysis including these potential confounders, only maternal age (odds ratio (OR), 0.89; 95% confidence interval (CI), 0.86–0.93; P < 0.001) and collapse pattern (Cc/Nc: OR, 0.59; 95% CI, 0.37–0.93; P = 0.023) significantly impacted the live birth rate, whereas blastocyst morphology (good/not: OR, 1.37; 95% CI, 0.97–1.94; P = 0.069) tended to impact it. Limitations, reasons for caution The cryoprotectant concentration of our pre-vitrification equilibration solution is relatively low. Thus, caution is required when assessing the collapse pattern, as the solution osmolarity can influence blastocyst shrinkage. Follow-up studies with more participants are warranted to confirm these results and the health of the children born after vitrified-warmed embryo transfers. Wider implications of the findings A negative relationship was found between spontaneous blastocyst collapse during pre-vitrification equilibration and live birth rate. Studying blastocyst collapse pattern may assist selection of the most viable blastocysts after vitrification in ethylene glycol and dimethylsulfoxide—a widely used permeable cryoprotectant combination—thus, increasing IVF success rates. Trial registration number Not applicable

Exploring the protective effects of freeze-dried Lactobacillus rhamnosus under optimized cryoprotectants formulation

To prolong the preservation time of Lactobacillus rhamnosus and improve the cell survival rate, the cryoprotectants formulation was optimized by response surface experiments with the survival rate as the index. The optimal combination of cryoprotectants is 11.1% trehalose, 9.1% glycerin, 3.5% sodium glutamate, and 15.7% skimmed milk powder. The freeze-dried powder could be stored stably at −20 °C for 60 days and the survival rate was enhanced to 97.8% compared to the 22% survival rate of the control group. Further study of the freeze-dried powder revealed that the cell membrane was intact and could be maintained for at least 60 days, and the cell surface morphology was smooth and fully covered by the cryoprotectants, and the Na+/K+-ATPase activity was only decreased by 4.7%. These results demonstrated a mechanism whereby the composite cryoprotectants could reduce the damage to cells by ice crystal formation through enhanced protection of cell membranes, thus obtaining the effect of extended preservation time and improved survival rate, which provided a theoretical basis for the freeze-drying of Lactobacillus rhamnosus.

Synergistic combination of cryoprotectants for high freeze-dried survival rate and viable cell counts of Streptococcus thermophilus

Vacuum freeze-drying is one of the common methods to prepare probiotic powder. However, the low temperature and low moisture content could lead to the death of probiotics. The method of adding cryoprotectants is efficient to improve the survival rate of probiotics during vacuum freeze-drying. The synergistic combination of different cryoprotectants could play a better protective effect. However, the principle of the synergistic combination of different cryoprotectants during vacuum freeze-drying is not clear. In this study, literature review and analysis were firstly used to reveal commonly used cryoprotectants for coccus. Taking the price of cryoprotectants into account, the combination is preliminarily determined as sucrose/skim milk/sodium glutamate. Then, taking Streptococcus thermophilus 937 as the research target strain, the effect of this cryoprotectant combination was verified by a single-factor screening experiment and concentration optimization of cryoprotectants. It was found that the preferred cryoprotectant combination for Streptococcus thermophilus 937 is 6% sucrose/8% skim milk/4% sodium glutamate, with the freeze-dried survival rate and viable counts being 90.59% and 1.78 × 1011CFU/g, respectively. Meanwhile, this cryoprotectant combination also had a good protective effect on the other two cocci, Streptococcus thermophilus Grx02 and Enterococcus faecium 218. This result might have guiding and reference significance on the vacuum freeze-drying of coccus.

The Effect of Cryoprotectants Concentration on Ice Crystal Propagation Velocity.

Background: Two physicochemical effects occur during vitrification: nucleation and crystallization. Nucleation is a statistical occurrence by its nature. Thus, the more water molecules that are present the higher are the chances for nucleation to occur. Crystallization is a first-order transition where a water molecule is incorporated into ice crystal. Intracellular viscosity, which is the combination of water, salts, and cryoprotectants (CPs), affects both the nucleation and crystal growth rates. Ice velocity is inversely correlated with the viscosity and directly proportional to the function of the system's supercooling. However, little is known about the speed of ice crystal propagation in vitrification solutions containing different concentrations of CPs. Methods: This article describes the ice crystal propagation velocity while referring to vitrification. Ice crystal propagation velocity was measured in solutions containing different CP (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG], and glycerol) concentrations at a supercooled temperature. The different CPs solutions were inserted into 0.25 mL straws and placed in different temperatures of an alcohol bath (Tc) at supercooling temperatures of -8°C to -10°C. Results: We found that ice crystal propagation is inversely correlated to CP concentrations. Interestingly, PG showed, with statistically significant results, lower ice crystal growth velocities up to concentrations of 30% (v/v), compared with DMSO, and EG at the same concentrations. The combination of EG with PG showed better results (0.25 mm/s) than EG with DMSO (0.39 mm/s) in terms of decreasing the ice crystal growth velocity. When the concentration was increased to 40% (v/v), EG showed the lowest ice crystal propagation velocity (0.09 mm/s), although not significantly different than PG and glycerol but significantly lower than DMSO (0.13 mm/s). Conclusion: These results suggest that current vitrification solutions are not optimized. Based on our results, we suggest that combining PG with EG has advantages over the combination of DMSO and EG, which might promote successful cell and tissue vitrification.

The Role of Cryoprotective Agents in Liposome Stabilization and Preservation.

To improve liposomes’ usage as drug delivery vehicles, cryoprotectants can be utilized to prevent constituent leakage and liposome instability. Cryoprotective agents (CPAs) or cryoprotectants can protect liposomes from the mechanical stress of ice by vitrifying at a specific temperature, which forms a glassy matrix. The majority of studies on cryoprotectants demonstrate that as the concentration of the cryoprotectant is increased, the liposomal stability improves, resulting in decreased aggregation. The effectiveness of CPAs in maintaining liposome stability in the aqueous state essentially depends on a complex interaction between protectants and bilayer composition. Furthermore, different types of CPAs have distinct effective mechanisms of action; therefore, the combination of several cryoprotectants may be beneficial and novel attributed to the synergistic actions of the CPAs. In this review, we discuss the use of liposomes as drug delivery vehicles, phospholipid–CPA interactions, their thermotropic behavior during freezing, types of CPA and their mechanism for preventing leakage of drugs from liposomes.

Development of Milt Cryopreservation Protocol for Economically Important Minor Carp Cirrhinus reba (Hamilton, 1822)

Background: Reba carp is one of the promising candidates for species diversification in fresh-water aquaculture. It is available in all the major rivers of India. Cryopreserved milt will be useful in genetic exchange between remotely located farms. For using cryomilt as a tool for genetic exchange, species specific milt cryopreservation protocol is required. Methods: Milt was cryopreserved using three extenders (7,7B and 9C) and two cryoprotectants (DMSO and methanol) and evaluated with fresh milt as control. Milt was cryopreserved in 1:6 dilution in French medium straws of 0.5 ml. The feasibility of selected extender and cryoprotectant combinations was assessed by the ability of cryopreserved sperm to fertilize the C. reba eggs and hatching percentage. Fertilized eggs were incubated in 0.5 L bowl and a flow-through incubation system. Analysis was done for both bowl and flow-through incubation separately and combinedly as well. Result: Better hatching percentage was obtained in flow-through incubation system for all the treatments. In combined analysis extender 7 with 10% DMSO as cryoprotectant gave significantly higher hatching rate (44.5%) and (75%) in comparison to control. Better performance was noticed in flow-through incubation system (50.09%) as compared to bowl (39.1%) in the same treatment. Thus, it can be concluded that extender 7 with 10% DMSO (V/V) is suitable to retain sperm quality in reba carp having optimal sperm motility, high fertility and hatching close to the values obtained with fresh sperm in flow-through incubation system.

P-220 Ice crystals propagation velocity is related to the type and concentration of cryoprotectants and impacts cells and tissue vitrification success

Abstract Study question How different cryoprotectants at various concentrations affect ice crystals formation and propagation during vitrification? Summary answer The velocity of ice crystal propagation is inversely related to concentration and type of cryoprotectants and is responsible for downstream cell damage during vitrification. What is known already Vitrification has replaced slow freezing for oocyte and embryo cryopreservation and is now being considered also for gonadal tissue. However, the results in terms of both vitrification and rewarming survival rates are still not uniform. Understanding the complex physical events controlling ice crystals formation, propagation and expansion are key for improving cryoprotectant solutions and overcoming challenges. Study design, size, duration Basic science research. Ice crystal propagation velocity was measured in solutions of four cryoprotectants: dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG) and glycerol (Gly), at various concentrations in supercooled temperatures. Each experiment used five 0.25ml and five 2ml straws (volume 0.1 ml and 1 ml) containing different cryoprotectant combinations (DMSO:EG, EG:PG, DSMO:PG, DMSO:Gly, EG:Gly, PG:Gly and EG+PG vs EG+DMSO), immersed in alcohol baths prepared at 8-10 °C below the freezing points of each solution. Participants/materials, setting, methods Alcohol baths were set as follows: (1) between -10 °C and -12 °C for straws with 0% and 10% (v/v) DMSO, EG, PG and Gly; (2) -15 °C to -19 °C for the 20%; (3) -19.5 to -24 °C for the 30%, and for the mixture of 15% EG and 15% PG and 15% EG and 15% DMSO (most used vitrification solutions); and (4) -25 to -36 °C for the 40%. A 50µm thermocouple measured supercooling. Student’s T-test analyzed differences. Main results and the role of chance Seeding was done by touching the straw end with LN- precooled forceps until visualizing a small ice crystal. Following 10 to 60s the distance to which the ice crystals grew inside the straws was evaluated using a fine caliber. Ice crystal propagation was inversely correlated to type and cryoprotectant concentrations. Propylene glycol showed statistically significant lower ice crystals growth velocity up to concentrations of 30% (v/v), compared to DMSO and EG at the same concentrations (p < 0.05). Moreover, the ice crystal velocity of PG was also lower than Gly at these same concentrations. The combination of EG with PG decreased ice crystal propagation better than EG with DMSO. At concentrations of 40%, there were no differences. These results suggest that current cryopreservation vitrification solutions are not optimized and combining PG with other CPs has advantages. Using a vitrification solution of 40% DMSO or EG, the velocity of crystal growth was in the range of 0.1mm/sec (6mm/min). This means that to vitrify large tissues, a cooling rate of over 1000 °C/min from RT to a temperature of -196 °C (ΔT of 220 °C in 13 seconds) is necessary to avoid a single ice nucleus to grow to a size of 1.3mm during cooling or warming. Limitations, reasons for caution Assessing the velocity of ice propagation is a novel concept to perfect vitrification/warming solutions. Wider implications of the findings These results pave the way to develop protocols and platforms able to minimize the deleterious impact of cryoprotectant concentrations on both cells and tissues survival after vitrification. Trial registration number not applicable

Cryopreservation of sperm in cobia, Rachycentron canadum (Linnaeus, 1766)

This study evaluates the quality and optimization of cryopreservation protocol for sperm of Rachycentron canadum, (Cobia) by screening various cryoprotectants, extenders, freezing protocols and equilibration times. Earlier studies on toxicity assay confirmed the suitability of the extender marine fish ringer and cryoprotectants such as DMSO, EG and DMA for long-term storage. Experiments were performed with milt along with extender, cryoprotectants (DMSO, DMA and DMSO + EG) at dilution ratio of 1:40 and equilibration time of 15 s with single step freezing protocol (−6 °C/min from 4 °C to −150 °C and in LN2 at −196 °C). The outcome of these experiments revealed that the sperm quality (motility, viability, functional integrity and DNA damage) was high with DMSO (5%) + EG (5%) compared to other individual and combination of cryoprotectants after 120 days storage. Maximum sperm motility of 62.8 ± 2.7% and viability of 72.4 ± 1.9% registered with DMSO (5%) + EG (5%). With reference to morphology, the post-thaw cryopreserved sperm of DMSO (5%) + EG (5%) showed intact sperm without any distortion. Under TEM also showed normal architecture of cellular organelles and plasma membrane. Metabolic enzymes activities (LDH, Asp and ALP), were high in post-thaw cryopreserved sperm with DMSO (5%) + EG (5%) compared to other cryoprotectants, but lesser than control. Similarly, antioxidant enzymes (SOD, GPx and LPO) registered high with DMSO (5%) + EG (5%), but low LPO compared to other cryoprotectants. Comet assay of post-thaw cryopreserved sperm of 120 days storage showed minimum DNA damage (5.84 ± 1.8%) with DMSO (5%) + EG (5%). Functional integrity of post-thaw cryopreserved sperm registered maximum of 68.7 ± 3.6% with DMSO (5%) + EG (5%). This protocol thus established remarkable cryoresistance of cobia sperm; imply to be amenable for long-term storage.

Efficacy of the incorporation between self-encapsulation and cryoprotectants on improving the freeze-dried survival of probiotic bacteria.

This study aimed to improve the viability of probiotic bacteria during freeze-drying by the combination of self-encapsulation and cryoprotectants. Lactiplantibacillus plantarum VAL6 and Lactobacillus acidophilus VAR1 were exposed to environmental stresses including temperature, pH and increased CO2 concentration before performing freeze-drying with the addition of cryoprotectants. The results proved that tested stresses can stimulate the bacteria to synthesize more extracellular polymeric substances to form self-encapsulation that increases their freeze-dried viability. In combination with cryoprotectants to form double-layered microencapsulation, L. plantarum VAL6 stressed at pH 3.5 in combination with whey protein isolate could achieve the highest Improving Cell Viability of 4361-fold, while L. acidophilus VAR1 stressed at 25o C in combination with alginate gave a maximum Improving Cell Viability of 73.33-fold. The combination of self-encapsulation and cryoprotectants significantly improves the freeze-dried viability of probiotics. This is the first report that uses environmental stress to stimulate extracellular polymeric substance synthesis for self-encapsulation formation combined with the addition of cryoprotectants to enhance the freeze-dried survival of probiotics. This could be a novel approach in improving the viability of probiotic strains for various applications.

Cryopreservation of Testicular Tissue from Adult Red-Rumped Agoutis (Dasyprocta leporina Linnaeus, 1758).

Simple SummaryTesticular tissues are composed of many types of germ cells, including early stages which can be grown up to fully formed spermatozoa following grafting or in vitro culture. The systematic banking of testicular tissues at freezing temperatures is useful for future use in assisted reproduction and to improve the reproductive management of rare mammalian species. The present study explored testicular tissue cryopreservation in the red-rumped agouti by slow freezing or vitrification methods, using different combinations of cryoprotectants. Solid-surface vitrification using the combination of dimethyl sulfoxide and ethylene glycol was the most effective protocol to preserve testicular cell morphology and proliferative potential.This study measured the effects of different freezing techniques and permeating cryoprotectants on the preservation of testicular tissues from adult red-rumped agoutis. Tissue biopsies (3.0 mm3) from five individuals were allocated to different experimental groups: control (non-cryopreserved); slow freezing (SF), solid-surface vitrification (SSV), and conventional vitrification (CV). Each method used dimethyl sulfoxide (DMSO), ethylene glycol (EG), or a DMSO + EG combination. Morphology, viability, mitochondrial activity, and proliferative potential were assessed in fresh and frozen tissue samples. Testicular morphology was better using SSV with a combination of DMSO and EG. Across the different cryopreservation approaches, as well as cryoprotectant combinations, cell viability was comparable. Regarding mitochondrial activity, DMSO + EG/SSV or CV, and DMSO + EG/CV were similar to the EG/SF group, which was the best group that provided values similar to fresh control groups. Adequate preservation of the proliferative potential of spermatogonia, Leydig cells, and Sertoli cells was obtained using SSV with DMSO + EG. Overall, the use of SSV with DMSO + EG was the best protocol for the preservation of testicular tissues from adult red-rumped agoutis.

A successful vitrification technique for goat morulae conservation

The aim of this study was to evaluate the effect of different vitrification and warming processes on the in vitro embryo survival of caprine morulae, considering the day of recovery. A total of 136 morulae of Criolla-Neuquina goats recovered on Days 7 or 8 after sponge removal, were exposed to three different vitrification processes; V1 (n = 48): glycerol (G) + ethylene glycol (EG); V2 (n = 44): EG + 0.5 M sucrose and V3 (n = 44): G + EG + 0.5 M sucrose. The morulae of each vitrification process were randomly assigned to three warming processes; W1 (n = 45): 0.5 M sucrose at 25 °C; W2 (n = 44): 0.5 M sucrose at 39 °C; and W3 (n = 47): solution containing half the concentration of the cryoprotectants + 0.5 M sucrose at 25 °C. After, embryos were cultured in 100 μL TCM 199 drops under mineral oil, at 39 °C and a 6.5% CO2 atmosphere for 72 h according to the different treatments. There were no viable embryos in V1 and V2 in none of their three respective warmings. Only V3 showed an embryo survival rate to hatched blastocyst stage of 59.1%. When considering embryo survival according to the warming processes, the survival rate was higher in V3W2 (76.9%) and V3W3 (66.7%) groups compared to the V3W1 group (37.5%; P < 0.05). The embryo survival of V3 for Day 8 after sponge removal (81.3%) was higher compared to Day 7 (46.4%; P < 0.05). In conclusion, a successful embryo survival is obtained by using a combination of cryoprotectants (G + EG) with addition of sucrose in the vitrification process for conservation of caprine morulae in embryo transfer programs. The survival rates in vitro of vitrified-warmed morulae in goats were influenced by their recovery day. Further studies should be conducted to determine if these results are reproducible in vivo embryo transfer on field situations.

Effect of two Different Permitting and Combination of Cryoprotectants on Cattle Oocytes Maturation Rate Following Brilliant Cresyl Blue Exposure

Effect of two Different Permitting and Combination of Cryoprotectants on Cattle Oocytes Maturation Rate Following Brilliant Cresyl Blue Exposure

Freezing Regimens and Gel Carrier Composition Influence Safety of Saccharomyces boulardii Immobilized Yeast Cells

The effect of DMSO and sucrose added to alginate gel on the viability of immobilized Saccharomyces boulardii yeast cells after rapid and slow cooling to –196 °C has been studied. Thermomechanical analysis revealed the inflection of phase transitions corresponding to each component of the solutions when cooling those of 1% sodium alginate contained 20% sucrose or 5% DMSO down to –196 °C. Phase transitions caused by the presence of DMSO and sucrose in sodium alginate solutions were observed at lower temperatures than in impurity-free sodium alginate solution. In experiments to study the viability of free and immobilized yeast cells after freezing, it was found that the highest viability of free cells suspended in solutions of sodium alginate, DMSO, sucrose, and the cells immobilized in gel granules, was provided by a cooling rate of 1 deg/min followed by immersion in liquid nitrogen. After adding DMSO and sucrose separately to the alginate gel, as well as their combinations, the viability of immobilized cells increased. The maximum viability of immobilized cells was found in gel granules, which contained the following combinations of cryoprotectants: 5% DMSO and 10% sucrose, 5% DMSO and 20% sucrose, 10% DMSO and 10% sucrose and 10% DMSO and 20% sucrose.