Genomic and functional evaluation of exopolysaccharide produced by Liquorilactobacillus mali t6-52: technological implications.

BACKGROUND: This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype. RESULTS: The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain's adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage. CONCLUSIONS: The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain's safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals.

Optimization of Culture Medium for the Production of an Exopolysaccharide (p-CY02) with Cryoprotective Activity by Pseudoalteromonas sp. RosPo-2 from the Antarctic Sea.

When cells are exposed to freezing temperatures, high concentrations of cryoprotective agents (CPA) prevent ice crystal formation, thus enhancing cell survival. However, high concentrations of CPAs can also cause cell toxicity. Exopolysaccharides (EPSs) from polar marine environments exhibit lower toxicity and display effects similar to traditional CPA. In this study, we sought to address these issues by i) selecting strains that produce EPS with novel cryoprotective activity, and ii) optimizing culture conditions for EPS production. Sixty-six bacteria producing mucous substances were isolated from the Ross Sea (Antarctic Ocean) using solid marine agar plates. Among them, Pseudoalteromonas sp. RosPo-2 was ultimately selected based on the rheological properties of the produced EPS (p-CY02). Cryoprotective activity experiments demonstrated that p-CY02 exhibited significantly cryoprotective activity at a concentration of 0.8% (w/v) on mammalian cells (HaCaT). This activity was further improved when combined with various concentrations of dimethyl sulfoxide (DMSO) compared to using DMSO alone. Moreover, the survival rate of HaCaT cells treated with 5% (v/v) DMSO and 0.8% (w/v) p-CY02 was measured at 87.9 ± 2.8% after freezing treatment. This suggests that p-CY02 may be developed as a more effective, less toxic, and novel non-permeating CPA. To enhance the production of EPS with cryoprotective activity, Response Surface Methodology (RSM) was implemented, resulting in a 1.64-fold increase in production of EPS with cryoprotective activity.

The effect of different concentrations of laminarin on the quality of cryopreserved ram semen.

BACKGROUND: Increasingly, sheep breeders are using artificial insemination to produce lambs, so finding methods that preserve ram sperm can be useful. OBJECTIVE: To determine the protective effects of different concentrations of laminarin on ram sperm motility, viability, abnormalities, membrane, and DNA integrity, superoxide dismutase enzyme (SOD) activity, and malondialdehyde (MDA) production after freeze-thawing. MATERIALS AND METHODS: The ejaculates of four rams were collected and stored at 35 degree C. Semen samples were diluted with a tris-base extender containing 100, 200, 400, and 800 ug/mL of laminarin and a control extender containing no laminarin, then frozen in liquid nitrogen after 4 h in the refrigerator. RESULTS: In the treatment of frozen-thawed spermatozoa with 800 ug/mL laminarin, motility, viability, membrane integrity, and DNA integrity were significantly higher than in the control. In spermatozoa that were exposed to 800 ug/mL laminarin after thawing, MDA production was significantly lower than in the control group. The percentage of abnormal spermatozoa in 800 ug/mL laminarin was significantly lower than that in the control. CONCLUSION: The addition of 800 ug/mL laminarin to the freezing extender increases motility, viability, SOD activity, and plasma membrane integrity, while reducing abnormality and MDA production in freeze-thawed ram semen. https://doi.org/10.54680/fr24110110812.

Metabolites assay offers potential solution to improve the rooster semen cryopreservation.

Semen cryopreservation represents a promising technology utilized for preserving high-quality chicken varieties in husbandry practices. However, the efficacy of this methodology is significantly impeded by the diminished quality of sperm. Metabolites, as the end products of metabolic reactions, serve as indicators of biological processes and offer insights into physiological conditions. In this study, we investigaged the sperm quality and alteration in metabolic profiles during the cryopreservation of Longyou Partridge Chicken semen. Following artificial semen collection, four groups of semen samples were established based on four points of the cryopreservation process (Ⅰ, fresh semen; Ⅱ, semen added extender and chilled at 4 °C for 30 min; Ⅲ, semen added cryoprotectants; Ⅳ, semen gradient freezed and stored in liquid nitrogen). Semen cryopreservation has a negative effect on the percentage of sperm in a straight-line trajectory (LIN), has no significant effect on total motile sperms (TM) or the proportion of sperm with typical morphology (NM). Metabolites were identified using LC-MS technique and analyses including Principal Component Analysis (PCA), Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA), Univariate statistical analysis, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were employed to identify metabolites. A total of 2471 metabolites had been identified, with the majority of the list being made up of amino acids and their metabolites as well as benzene and substituted derivatives. Group II exhibits 882 metabolites with significantly elevated abundance relative to Group I, alongside 37 metabolites displaying decreased abundance. In Group III, 836 metabolites demonstrate notably augmented abundance compared to Group II, while 87 metabolites exhibit reduced abundance. Furthermore, Group IV showcases 513 metabolites with markedly heightened abundance in comparison to Group III, and 396 metabolites with decreased abundance. Specific metabolites such as 5-Hydroxylysine, Phosphocholine, and alpha-d-glucose-6-phosphate exhibited a progressive decline during the cryopreservation process, correlating with either dilution and chilling, cryoprotectant addition, or freezing. In conclusion, our investigation systematically examined the changes of seminal metabolome and sperm quality throughout the cryopreservation process of rooster semen.

Exploring the relationship between pollen viability and inclusion in Paeonia lactiflora after cryopreservation.

Pollen, as the male gametophyte, carries half of plant genetic information and is an important source of germplasm. The cryopreservation of pollen can not only preserve germplasm, but also solve the problem of time and space barrier in crossbreeding. So it is of great significance to explore the mechanism of pollen viability maintenance after cryopreservation. In this paper, 10 cultivars of Paeonia lactiflora with different fresh pollen viability that did not change after cryopreservation were taken as objects and the effects of pollen inclusions such as soluble sugar, starch, soluble protein, free amino acids, and proline were explored. The results showed that: (1) The contents of pollen inclusions in the fresh pollen of 10 cultivars were different. After cryopreservation, the contents of starch and free amino acids significantly decreased in 10 cultivars, and the soluble sugar, soluble protein, and proline varied with cultivars. (2) Correlation analysis showed that fresh pollen viability was significantly positively correlated with the soluble sugar (R-values of 0.630) and starch content (R-values of 0.694) in fresh pollen. But after cryopreservation pollen viability was only significantly positively correlated with the starch content (R-values of 0.725). These results suggest that the effects of pollen inclusions on pollen vitality are different before and after cryopreservation. The fresh pollen with higher soluble sugar and starch is more vital. But after cryopreservation, the pollen with high starch content has higher viability. The maintenance of stable pollen viability after cryopreservation appears to be related to starch content or starch metabolism, which requires further to study for a final determination.

Engineered ice-binding protein (FfIBP) shows increased stability and resistance to thermal and chemical denaturation compared to the wildtype.

Many polar organisms produce antifreeze proteins (AFPs) and ice-binding proteins (IBPs) to protect themselves from ice formation. As IBPs protect cells and organisms, the potential of IBPs as natural or biological cryoprotective agents (CPAs) for the cryopreservation of animal cells, such as oocytes and sperm, has been explored to increase the recovery rate after freezing-thawing. However, only a few IBPs have shown success in cryopreservation, possibly because of the presence of protein denaturants, such as dimethyl sulfoxide, alcohols, or ethylene glycol, in freezing buffer conditions, rendering the IBPs inactive. Therefore, we investigated the thermal and chemical stability of FfIBP isolated from Antarctic bacteria to assess its suitability as a protein-based impermeable cryoprotectant. A molecular dynamics (MD) simulation identified and generated stability-enhanced mutants (FfIBP_CC1). The results indicated that FfIBP_CC1 displayed enhanced resistance to denaturation at elevated temperatures and chemical concentrations, compared to wildtype FfIBP, and was functional in known CPAs while retaining ice-binding properties. Given that FfIBP shares an overall structure similar to DUF3494 IBPs, which are recognized as the most widespread IBP family, these findings provide important structural information on thermal and chemical stability, which could potentially be applied to other DUF3494 IBPs for future protein engineering.

Cryopreservation with DMSO affects the DNA integrity, apoptosis, cell cycle and function of human bone mesenchymal stem cells.

Cryopreservation (CP) enables pooling and long-term banking of various types of cells, which is indispensable for the cell therapeutics. Dimethyl sulfoxide (DMSO) is universally used as a cryoprotectant in basic and clinical research. Although, the use of DMSO has been under serious debate due to significant clinical side effects correlated with infusions of cellular therapy products containing DMSO, the effect of CP with DMSO on the cell properties and functions remains unknown. Here, we experimentally found that the CP of human bone mesenchymal stem cells (hBMSCs) with 10 % DMSO results 10-15 % of cells apoptosis upon immediate freeze-thaw, ca. 3.8 times of DNA damage/repair relative to the fresh ones after post-thaw cultured in 48 h, and cell cycle arrests at G0/G1 after post-thaw cultured in 24 h. Moreover, CP with 10 % DMSO significantly increases the reactive oxygen species (ROS) level of the frozen-thawed MSCs which may be one of the causes impair cellular properties and functions. Indeed, we found that the differentiation and migration ability of post-thaw cultured hBMSCs decrease as the expression of adipogenic, osteogenic genes and F-actin reduces in the comparison with those of the fresh cells.

Inulin Can Improve Red Blood Cell Cryopreservation by Promoting Vitrification, Stabilizing Cell Membranes, and Inhibiting Ice Recrystallization.

In transfusion medicine, the cryopreservation of red blood cells (RBCs) is of major importance. The organic solvent glycerol (Gly) is considered the current gold-standard cryoprotectant (CPA) for RBC cryopreservation, but the deglycerolization procedure is complex and time-consuming, resulting in severe hemolysis. Therefore, it remains a research hotspot to find biocompatible and effective novel CPAs. Herein, the natural and biocompatible inulin, a polysaccharide, was first employed as a CPA for RBC cryopreservation. The presence of inulin could improve the thawed RBC recovery from 11.83 ± 1.40 to 81.86 ± 0.37%. It was found that inulin could promote vitrification because of its relatively high viscosity and glass transition temperature (Tg'), thus reducing the damage during cryopreservation. Inulin possessed membrane stability, which also had beneficial effects on RBC recovery. Moreover, inulin could inhibit the mechanical damage induced by ice recrystallization during thawing. After cryopreservation, the RBC properties were maintained normally. Mathematical modeling analysis was adopted to compare the performance of inulin, Gly, and hydroxyethyl starch (HES) in cryopreservation, and inulin presented the best efficiency. This work provides a promising CPA for RBC cryopreservation and may be beneficial for transfusion therapy in the clinic.

Development of a Me2SO-free cryopreservation medium and its long-term cryoprotection on the CAR-NK cells.

Cryopreservation is a crucial step in the supply process of off-the-shelf chimeric antigen receptor engineered natural killer (CAR-NK) cell products. Concerns have been raised over the clinical application of dimethyl sulfoxide (Me2SO) due to the potential for adverse reactions following infusion and limited cell-specific cytotoxic effects if misapplied. In this study, we developed a Me2SO-free cryopreservation medium specifically tailored for CAR-NK cells to address this limitation. The cryopreservation medium was formulated using human serum albumin (HSA) and glycerol as the base components. Following initial screening of seven clinically-compatible solutions, four with cryoprotective properties were identified. These were combined and optimized into a single formulation: IF-M. The viability, phenotype, and function of CAR-NK cells were evaluated after short-term and long-term cryopreservation to assess the effectiveness of IF-M, with Me2SO serving as the control group. The viability and recovery of CAR-NK cells in the IF-M group were significantly higher than those in the Me2SO group within 90 days of cryopreservation. Moreover, after 1 year of cryopreservation the cytotoxic capacity of CAR-NK cells cryopreserved with IF-M was comparable to that of fresh CAR-NK cells and significantly superior to that of CAR-NK cells cryopreserved in Me2SO. The CD107a expression intensity of CAR-NK cells in IF-M group was significantly higher than that of Me2SO group. No statistical differences were observed in other indicators under different cryopreservation times. These results underscore the robustness of IF-M as a suitable replacement for traditional Me2SO-based cryopreservation medium for the long-term cryopreservation and clinical application of off-the-shelf CAR-NK cells.

Effect of melatonin on cryopreservation of Beijing you chicken (gallus gallus) spermatozoa.

Beijing You Chicken, a valuable local chicken breed from Beijing, China, was once listed as an endangered breed. From the point of view of conservation, the preservation of this breed is an important task for the local researchers. Semen cryopreservation is a popular method to maintain valuable species. However, during cryopreservation, semen is susceptible to oxidative damage. Melatonin is a potent antioxidant and free radical scavenger, so it has been selected to improve the efficiency of sperm cryopreservation. In this study, the chicken semen was treated with different concentrations of melatonin in the cryopreservation solution. The results showed that melatonin at concentrations of 10-3 M and 10-5 M significantly improved sperm progressive motility and total motility, respectively, compared to the control (P < 0.05). Melatonin at 10-3 M also significantly improved the plasma membrane and acrosome integrity of spermatozoa compared to the control. The mechanisms are that melatonin significantly reduces the level of ROS and preserves sperm mitochondrial membrane potential. Most importantly, the melatonin-treated cryopreserved chicken sperm after artificial insemination significantly increased the hatching rate of chicks compared to the control (p < 0.05). The results show that melatonin has a positive effect on the quality of the cryopreserved spermatozoa. These results provide the theoretical and practical basis for using melatonin to improve Beijing You Chicken conservation, and they may also be applicable to poultry as a whole.

Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes.

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL-1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 µm s-1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 µm s-1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Determination of cell membrane permeability coefficients: Comparison of models in the case of oocytes.

Values of cell membranes permeability coefficients for water and molecules of cryoprotective agents (CPAs) are the necessary characteristics for developing physical-mathematical models describing mass transfer processes through cell membranes in order to predict optimal cell cooling rates. We carried out a comparative analysis of the permeability coefficients of mouse oocyte membranes for molecules of water, ethylene glycol (EG), propane-1,2-diol (1,2-PD) and dimethyl sulfoxide (Me2SO), determined by applying the classical Kedem-Katchalsky model, which considers only the penetration of non-electrolyte molecules (water and CPA) through the membrane, and the model developed by us, which takes into account the transmembrane transfer of ions and the associated changes in the transmembrane electric potential. We shown that calculations based on the developed modified model provide lower values of the permeability coefficients of the oocyte membrane for water and CPA molecules. What is important that the obtained by our modified model permeability coefficients for water molecules do not depend on the type of cryoprotectant, while the application of the classical model both in our studies and works of other authors always gave different values of these coefficients in solutions with different cryoprotectants. Our modified model also makes it possible to determine the dynamics of the transmembrane electric potential of the cell under the conditions of transmembrane mass transfer and the duration of the membrane being influenced by the changes in electric potential, that is a parameter that can directly affect the viability of cells.

Osmotic properties of T cells determined by flow imaging microscopy in comparison to electrical sensing zone analysis.

To develop cryopreservation methods for cell-based medicinal products it is important to understand osmotic responses of cells upon immersion into solutions with cryoprotective agents (CPAs) and during freezing. The aim of this study was to assess the osmotic response of T cells by using flow imaging microscopy (FIM) as a novel cell-sizing technique, and to corroborate the findings with electrical impedance measurements conducted on a Coulter counter. Jurkat cells were used as a potential model cell line for primary T cells. Cell volume responses were used to derive important cell parameters for cryopreservation such as the osmotically inactive cell volume Vb and the membrane permeability towards water and various CPAs. Unlike Coulter counter measurement, FIM, combined with Trypan blue staining can differentiate between viable and dead cells, which yields a more accurate estimation of Vb. Membrane permeabilities to water, dimethyl sulfoxide (Me2SO) and glycerol were measured for Jurkat cells at different temperatures. The permeation of Me2SO into the cells was faster in comparison to glycerol. CPA permeation decreased with decreasing temperature following Arrhenius behavior. Moreover, membrane permeability to water decreased in the presence of CPAs. Vb of Jurkat cells was found to be 49% of the isotonic volume and comparable to that of primary T cells. FIM proved to be a valuable tool to determine the membrane permeability parameters of mammalian cells to water and cryoprotective agents, which in turn can be used to rationally design CPA loading procedures for cryopreservation.

Engineered Test Tissues: A Model for Quantifying the Effects of Cryopreservation Parameters.

Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.

Is glycerol a good cryoprotectant for sperm cells? New exploration of its toxicity using avian model.

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation.

Brassica juncea leaf cuticle contains xylose and mannose (xylomannan) which inhibit ice recrystallization on the leaf surface.

MAIN CONCLUSION: Conjugated sugars showed antifreeze activity in the cuticle by ice recrystallization inhibition rather than thermal hysteresis, enhancing freezing capacity at the surface of B. juncea leaves. Antifreeze biomolecules play a crucial role in mitigating the physical damage from frost by controlling extracellular ice crystal growth in plants. Antifreeze proteins (AFPs) are reported from the apoplast of different plants. Interestingly, there is no report about antifreeze properties of the cuticle. Here, we report the potential antifreeze activity in the Brassica juncea (BJ) leaf cuticle. Nano LC-MS/MS analysis of a cuticle protein enriched fraction (CPE) predicted over 30 putative AFPs using CryoProtect server and literature survey. Ice crystal morphology (ICM) and ice recrystallization inhibition (IRI) analysis of ABC supernatant showed heat and pronase-resistant, non-protein antifreeze activities as well as hexagonal ice crystals with TH of 0.17 °C and IRI 46%. The ZipTip processed ABC supernatant (without peptides) had no effect on TH activity, confirming a non-protein antifreeze molecule contributing to activity. To understand the origin and to confirm the source of antifreeze activity, cuticular membranes were isolated by pectinase and cellulase hydrolysis. FTIR analysis of the intact cuticle showed xylose, mannose, cellulose, and glucose. Xylanase and cellulase treatments of the ZipTip processed ABC supernatant led to an increase in sugar content and 50% loss in antifreeze activity. UV spectroscopy and NMR analysis supported the finding of FTIR and enzyme hydrolysis suggesting the contribution of xylose and mannose to antifreeze activity. By TLC analysis, conjugated sugars were found in the cuticle. This work has opened up a new research area where the antifreeze capacity needs to be established with regard to complete characterization and mechanism of action of the antifreeze carbohydrates (conjugated sugars) on the leaf surface.

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.

Histone H3 and H4 Modifications Point to Transcriptional Suppression as a Component of Winter Freeze Tolerance in the Gall Fly Eurosta solidaginis.

The goldenrod gall fly (Eurosta solidaginis) is a well-studied model of insect freeze tolerance. In situations of prolonged winter subzero temperatures, larvae of E. solidaginis accept ice penetration throughout extracellular spaces while protecting the intracellular environment by producing extreme amounts of glycerol and sorbitol as cryoprotectants. Hypometabolism (diapause) is implemented, and energy use is reprioritized to essential pathways. Gene transcription is one energy-expensive process likely suppressed over the winter, in part, due to epigenetic controls. The present study profiled the prevalence of 24 histone H3/H4 modifications of E. solidaginis larvae after 3-week acclimations to decreasing environmental temperatures (5 °C, -5 °C and -15 °C). Using immunoblotting, the data show freeze-mediated reductions (p < 0.05) in seven permissive histone modifications (H3K27me1, H4K20me1, H3K9ac, H3K14ac, H3K27ac, H4K8ac, H3R26me2a). Along with the maintenance of various repressive marks, the data are indicative of a suppressed transcriptional state at subzero temperatures. Elevated nuclear levels of histone H4, but not histone H3, were also observed in response to both cold and freeze acclimation. Together, the present study provides evidence for epigenetic-mediated transcriptional suppression in support of the winter diapause state and freeze tolerance of E. solidaginis.

Cryopreservation of human cerebral microvascular endothelial cells with glycerol.

The cryopreservation of human cerebral microvascular endothelial cells (hCMEC) has facilitated their commercial availability for research studying the blood-brain barrier. The currently employed cryopreservation protocol uses 10% dimethyl sulfoxide (Me2SO) in cell medium, or 5% Me2SO in 95% fetal bovine serum (FBS) as cryoprotective agents (CPAs). However, Me2SO is toxic to cells and FBS is animal-derived and not chemically defined, so reducing the concentrations of these components is desirable. Recently, we showed that cryopreserving hCMEC in cell medium with 5% Me2SO and 6% hydroxyethyl starch (HES) results in over 90% post-thaw cell viability. This previous work was performed using an interrupted slow cooling (graded freezing) approach followed by SYTO13/GelRed staining to assay for membrane integrity. In this paper, we repeated graded freezing of hCMEC in cell medium containing 5% Me2SO and 6% HES, but this time using Calcein AM/propidium iodide staining to ensure that the stain is an equivalent alternative to SYTO13/GelRed for assessment of cell viability, and that results are comparable to those previously published. Next, using graded freezing experiments and Calcein AM/propidium iodide staining, we examined the effectiveness of non-toxic glycerol as a CPA at different concentrations, loading times, and cooling rates. The cryobiological response of hCMEC was used to develop a protocol that optimizes both the permeating and non-permeating capabilities of glycerol. HCMEC in cell medium loaded with 10% glycerol for 1 h at room temperature, ice nucleated at -5 °C and held for 3 min, and then cooled at -1 °C/min to -30 °C before plunging into liquid nitrogen had post-thaw viability of 87.7% ± 1.8%. Matrigel tube formation assay and immunocytochemical staining of junction protein ZO-1 were carried out on post-thaw hCMEC to ensure that the cryopreserved cells were viable and functional, in addition to being membrane-intact.

Effects of cryopreservation in the presence of Natural Deep Eutectic Solvents (NADESs) on sperm parameters.

Natural Deep Eutectic Solvents (NADESs) are being considered as a potential alternative to traditional cryoprotective agents (CPAs) in sperm freezing. The study aimed to assess the effects of NADESs as a CPA on human sperm parameters. A total of 32 normozoospermic semen samples were collected from the Alzahra infertility treatment center (Iran) between July 2021 and September 2022. The samples were categorized into eight different groups: 1) a control (nonfrozen), and groups frozen with 2) SpermFreeze Solution, 3) ChX (Choline chloride and Xylitol), 4) ChS (Choline chloride and D-sorbitol), 5) ChG (Choline chloride and Glucose), 6) ChU (Choline chloride and Urea), 7) EtP (Ethylene glycol and l-proline), and 8) GlyP (Glycerol and l-proline). The study also analyzed the quality of sperm parameters, such as chromatin condensation and integrity, acrosome integrity, and survival, along with the expression of some genes that affect sperm fertility (TRPV1, TRPV4, SPACA3, and OGG1). The study found there were notable variations in sperm parameters (such as viability, chromatin condensation and integrity, and acrosome integrity) among frozen groups with some NADESs compared to the SpermFreeze Solution and control groups (P < 0.05). Analysis of gene expression demonstrated that the levels of TRPV1, TRPV4, SPACA3, and OGG1 genes were superior in the GlyP group compared to the other groups (P < 0.05). Additionally, the ChS and ChU groups exhibited preserved expression of these genes compared with the SpermFreeze Solution group. The use of NADESs led to the discovery of a more appropriate CPA that has low toxicity and is highly effective in maintaining the fertility potential of sperm.