The crystallization properties of antifreeze GelMA hydrogel and its application in cryopreservation of tissue-engineered skin constructs.

Gelatin methacrylate (GelMA) hydrogels are expected to be ideal skin tissue engineering dressings for a wide range of clinical treatments. Herein, we report the preparation of GelMA or antifreeze GelMA hydrogel sheets with different GelMA concentrations, crosslinking times, and cryoprotectant (CPA) concentrations. The crystallization properties of GelMA or antifreeze GelMA hydrogel sheets were studied by cryomicroscopy and differential scanning calorimetry (DSC). It was found that the growth of ice crystals was slower when GelMA hydrogel concentration was more than 7%. The 10% DMSO-7% GelMA hydrogel sheets crosslinked for 60 min showed no ice crystal formation and growth during cooling and warming. The DSC results showed that the vitrification temperature of the 10% DMSO-7% GelMA hydrogel sheet was -111°C. Furthermore, slow freezing and rapid freezing of fibroblast-laden GelMA or antifreeze GelMA hydrogel sheets, and tissue-engineered skin constructs were studied. The results showed no significant difference in cell survival between slow (88.8% ± 1.51) and rapid (89.2% ± 3.00) freezing of fibroblast-loaded 10% DMSO-7% GelMA hydrogel sheets, and significantly higher than that of 7% GelMA hydrogel sheets (33.4% ± 5.46). The cell viability was higher in tissue-engineered skin constructs after slow freezing (86.34% ± 1.45) than rapid freezing (72.74% ± 1.34). We believe that the combination of antifreeze hydrogels and tissue engineering will facilitate the cryopreservation of tissue engineering constructs.

Use of Antifreeze Protein from Tenebrio molitor (TmAFP) in Vitrification of In Vitro-Produced Bovine Embryos: An Ultrastructural Study.

The objective of this study was to evaluate the effects of different concentrations of antifreeze protein (AFP) extracted from the larva of the beetle, Tenebrio molitor (TmAFP), on vitrification of in vitro-produced bovine embryos. In vitro-produced blastocysts were divided into three experimental groups and vitrified using a cryotop. TmAFP was added to the equilibrium solution (ES) and vitrification solution (VS) at a concentration of 0 ng/mL (control), 500 ng/mL (500TmAFP), or 1000 ng/mL (1000TmAFP). Vitrification was carried out by first placing the blastocysts in ES for 2 minutes (7.5% ethylene glycol [EG] and 7.5% dimethyl sulfoxide [DMSO]). The blastocysts were then transferred to VS (15% EG and 15% DMSO) and promptly deposited on a cryotop stem and submerged in liquid nitrogen. Warming was carried out in three steps with decreasing sucrose concentrations. After warming, the blast cells were cultured for 24 hours for subsequent survival analysis and ultrastructural evaluation. There was a significant difference in the survival rate and expansion in the 500TmAFP group compared with the other groups. The ultrastructural analysis revealed intracellular lesions in all vitrified embryos; however, the embryos of the 500TmAFP and 1000TmAFP groups showed fewer cytoplasmic lesions compared with the control group. Taken together, addition of TmAFP can mitigate cellular changes that involve organelles and cellular components essential for proper functioning and improve the viability of warmed and vitrified in vitro-produced bovine embryos.

Observation on the ice crystal formation process of large yellow croaker (Pseudosciaena crocea) and the effect of multiple cryoprotectants pre-soaking treatments on frozen quality.

By observing the formation behavior of ice crystals, the quality of food products under different freezing conditions can be intuitively judged. In this paper, large yellow croaker was taken as the research object, and a novel cryomicroscopic system was developed to directly observe the structure of ice crystals during the freezing process. The cryoprotective effects of 4% sucrose +4% sorbitol (SU + SO), 4% xylo-oligosaccharide (XO), 4% xylo-oligosaccharide + 0.3% tetrasodium pyrophosphate (XO + TSPP) and 0.2% antifreeze protein (AFP) at different freezing temperatures were investigated. And the evaluation indicators, such as cell deformation degree, equivalent diameters, roundness, elongation and fractal dimension were introduced to quantify the damage of ice crystals to muscle tissues and fibers. The results indicate that reducing the freezing temperature and adding cryoprotectants can improve the quality of large yellow croaker. AFP has the best cryoprotective effect, with a reduction in cell deformation degree of 54.78% and 67.83% compared to the Control group at -5 °C and -20 °C, respectively. SU + SO and XO have the equivalent antifreeze effect, which is slightly inferior to XO + TSPP. In addition, physical parameters of large yellow croaker samples were measured to verify the influence of ice crystal structure on product quality. Therefore, direct observation of the ice crystal formation process and evaluation of ice crystal structure can accurately reflect the quality of frozen products, which is of great significance for the development of refrigeration and preservation technology.

Antifreeze protein type III addition to freezing extender comprehensively improves post-thaw sperm properties in Okinawan native Agu pig.

Sperm cryopreservation often leads to physical cell damage through ice crystal formation. This study evaluates the improvements to freezing extender cryoprotective activity due to antifreeze protein (AFP) addition, which primarily acts on ice crystal formation, through investigating the post-thaw sperm properties of Okinawan native Agu pig. Six individual boar sperm samples were diluted with the freezing extender supplemented with 1 µg/mL of AFP I or AFP III and then subjected to cryopreservation. Treatment with AFP I during the freezing procedure had no improvement for any characteristics after thawing compared to untreated sperm. In contrast, the addition of AFP III to the freezing extender strongly increased sperm motility, mitochondria and cell membrane integrity, and the acrosomal proteolytic activity of frozen-thawed sperm in 5 of 6 individuals (P < 0.05). Furthermore, cryoinjury prevention by AFP III significantly enhanced sperm viability (by ATP content), and maintained DNA quality and in vitro sperm penetrability compared with AFP I treatment (P < 0.05). These findings demonstrate that AFP III addition to the freezing extender of boar sperm is more effective in maintaining sperm characteristics than the extender without AFP III or AFP I, despite individual differences in response.

Effect of the addition of antifreeze protein type I on the quality of post-thawed domestic cat epididymal sperm.

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen-thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 µg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

Improving the cryoprotective effect of antifreeze proteins from Daucus carota on plant-based meat by eliminating N-glycosylation.

As a novel animal meat alternative, plant-based meat (PBM) frequently suffers from quality problems as a result of freeze-thaw cycles in commercial transportation and household storage. There is a need to reduce the deterioration of PBM attributes, such as water holding capacity, as a result of these freeze-thaw cycles. In this study, Daucus carota antifreeze protein (DcAFP) and its deglycosylated mutant DcAFP-N294G were heterologously expressed in Komagataella phaffii X33. The effects of pretreatment with recombinant AFPs (rAFPs) on the microstructure, rheological properties, water mobility, and water distribution of PBM were assessed. The rDcAFP-N294G-treated PBM samples had superior viscoelasticity and water distribution features compared to the rDcAFP-treated group because the complex N-linked oligosaccharides did not interfere with the binding of rAFPs to ice molecules. In addition, rAFP pretreatment resulted in a smoother and flatter surface of the high-moisture protein extrudate matrix compared to the commercial cryoprotectant trehalose. Deglycosylated DcAFP has potential applications as a new effective cryoprotectant in meat alternatives.

Effect of antifreeze protein type III on frozen/thawed of spermatozoa recover from goat epididymis.

The objective of this study was to evaluate the effect of antifreeze protein type III (AFP III) on the freezing of epididymal spermatozoa of goats. A total of 16 pairs of testicles were collected in a slaughterhouse and transported at approximately 5 °C in a thermal box. Epididymal spermatozoa were recovered by retrograde lavage and evaluated using a phase contrast microscope. Then, they were cryopreserved in extender based on Tris-egg yolk, supplemented with AFP III (0, 1, 10, 100 µg/mL), using an automated system. After thawing (37 °C/30 s), the spermatozoa kinetics were evaluated using the CASA automated system; and plasma and acrosome membrane integrity, mitochondrial membrane potential, and intracellular ROS production, by flow cytometry. There was no difference (P ≥ 0.05) between the experimental groups for the parameters of spermatozoa kinetics, mitochondrial membrane potential, and ROS production. However, the integrity of plasma and acrosome membranes of frozen spermatozoa with 100 µg/mL of AFP III was lower (P < 0.05) than the control group. It was concluded that the addition of AFP III to the Tris-egg yolk extender, used in the freezing of sperm obtained from the epididymis of goats, did not improve the preservation of these cells.

New insight into the mechanism by which antifreeze peptides regulate the physiological function of Streptococcus thermophilus subjected to freezing stress.

INTRODUCTION: Antifreeze peptides regulate the physiological functions of frozen cells and even their apoptosis; however, the mechanisms by which antifreeze peptides regulate these processes remain unclear, although the interactions between cell membranes and ice are well known to be important in this process. OBJECTIVES: Our study aims to investigate how antifreeze peptides regulate cell physiological functions during the freezing process. METHODS: We investigated the cryoprotective effect of rsfAFP on the physiological functions of S. thermophilus under freezing stress by measuring cellular metabolism activity, intracellular enzyme activity, cell membrane characterization, and cell apoptosis. The mechanism by which rsfAFP impacts S. thermophilus physiological functions under freezing stress was investigated using multispectral techniques and cryo-TEM. RESULTS: We show that a recombinant antifreeze peptide (rsfAFP) interacts with the extracellular capsular polysaccharides and peptidoglycan of Streptococcus thermophilus and ice to cover the outer layer of the membrane, forming a dense protective layer that regulates the molecular structure of extracellular ice crystals, which results in reduced extracellular membrane damage, depressed apoptosis and increased intracellular metabolic activity. This interaction mechanism was indicated by the fact that S. thermophilus better maintained its permeability barrier, membrane fluidity, membrane structural integrity, and cytoplasmic membrane potential during freezing stress with rsfAFP treatment. CONCLUSION: These results provide new insights into the mechanism by which rsfAFP regulates frozen cellphysiological functionsand apoptosis under freezing stress.

Effects of Antifreeze Protein Type I and Glycerol in Diluents on Cryopreserved Goat Epididymal Sperm.

The effect of antifreeze protein (AFP) as a cryoprotectant used in different concentrations of glycerol on post-thaw quality of epididymal sperm was investigated. Sperm were isolated from 50 testicles, obtained from 25 healthy mature goat bucks, with progressive motility >80%, and total morphological abnormalities <10% were pooled in each replication. The semen samples were diluted with Tris-citrate-fructose-soybean lecithin extender containing different concentration of AFP [0 µg/mL (A0), 5 µg/mL (A5), 10 µg/mL (A10)]. Each concentration of AFP was added in an extender containing either 7% (G7) or 5% (G5) glycerol. Post-thaw total and progressive motility were found to be higher (p < 0.05) in groups A5G5 and A5G7. Plasma membrane integrity, sperm acrosome integrity, DNA integrity, acrosome intact sperm, and mitochondrial membrane potential were found to be higher (p < 0.05) in groups A5G5 and A10G5. Sperm viability was found to be higher (p < 0.05) in group A5G5, while lipid peroxidation was recorded lower (p < 0.05) in groups A5G5 and A5G7. Regarding the apoptosis occurrence, the results demonstrate higher (p < 0.05) live post-thawed spermatozoa for groups containing 5 µg/mL AFP with 5% and 7% glycerol in addition to the lowest (p < 0.05) value for groups containing 0 µg/mL AFP with 5% and 7% glycerol. Based on these results, the present study concludes that the addition of 5 µg/mL AFP in combination with 5% glycerol in freezing extender improves the post-thaw quality, structure, and function parameters for buck spermatozoa.

Intracellular and Extracellular Antifreeze Protein Significantly Improves Mammalian Cell Cryopreservation.

Cell cryopreservation is an essential part of the biotechnology, food, and health care industries. There is a need to develop more effective, less toxic cryoprotective agents (CPAs) and methods, especially for mammalian cells. We investigated the impact of an insect antifreeze protein from Anatolica polita (ApAFP752) on mammalian cell cryopreservation using the human embryonic kidney cell line HEK 293T. An enhanced green fluorescent protein (EGFP)-tagged antifreeze protein, EGFP-ApAFP752, was transfected into the cells and the GFP was used to determine the efficiency of transfection. AFP was assessed for its cryoprotective effects intra- and extracellularly and both simultaneously at different concentrations with and without dimethyl sulfoxide (DMSO) at different concentrations. Comparisons were made to DMSO or medium alone. Cells were cryopreserved at -196 °C for ≥4 weeks. Upon thawing, cellular viability was determined using trypan blue, cellular damage was assessed by lactate dehydrogenase (LDH) assay, and cellular metabolism was measured using a metabolic activity assay (MTS). The use of this AFP significantly improved cryopreserved cell survival when used with DMSO intracellularly. Extracellular AFP also significantly improved cell survival when included in the DMSO freezing medium. Intra- and extracellular AFP used together demonstrated the most significantly increased cryoprotection compared to DMSO alone. These findings present a potential method to improve the viability of cryopreserved mammalian cells.

Freezing-enhanced oxidation of iodide by hydrogen peroxide in the presence of antifreeze proteins from the Arctic yeast Leucosporidium sp.AY30.

Ice-binding proteins (IBPs), originating from Arctic or Antarctic microorganisms, have freeze-inhibiting characteristics, allowing these organisms to survive in polar regions. Despite their significance in polar environments, the mechanism through which IBPs affect the chemical reactions in ice by controlling ice crystal formation has not yet been reported. In this study, a new mechanism for iodide (I-) activation into triiodide (I3-), which is the abundant iodine species in seawater, by using hydrogen peroxide (H2O2) in a frozen solution with IBPs was developed. A significant enhancement of I- activation into I3- was observed in the presence of Arctic-yeast-originating extracellular ice-binding glycoprotein (LeIBP) isolated from Leucosporidium sp. AY30, and a further increase in the I3- concentration was observed with the introduction of H2O2 to the frozen solution (25 times higher than in the aqueous solution after 24 h of reaction). The reaction in the ice increased with an increase in LeIBP concentration. The in-situ pH measurement in ice using cresol red (CR) revealed protons accumulated in the ice grain boundaries by LeIBP. However, the presence of LeIBP did not influence the acidity of the ice. The enhanced freeze concentration effect of H2O2 by LeIBP indicated that larger ice granules were formed in the presence of LeIBP. The results suggest that LeIBP affects the formation and morphology of ice granules, which reduces the total volume of ice boundaries throughout the ice. This leads to an increased local concentration of I- and H2O2 within the ice grain boundaries. IBP-assisted production of gaseous iodine in a frozen environment provides a previously unrecognized formation mechanism of active iodine species in the polar regions.

Ice crystal recrystallization inhibition of type I antifreeze protein, type III antifreeze protein, and antifreeze glycoprotein: effects of AF(G)Ps concentration and heat treatment.

This study compared ice recrystallization behaviors of frozen dessert model systems containing type I antifreeze protein (AFP I), type III antifreeze protein (AFP III), and antifreeze glycoprotein (AFGP) at -10 °C. Specifically, effects of AF(G)P concentration and heat treatment (95 °C for 10 min) were examined. The concentration dependence of the ice recrystallization rate constant reasonably well fit a sigmoidal function: the fitting procedure was proposed, along with cooperative coefficient α, and a new index of AF(G)P ice recrystallization inhibition (IRI) activity (C50). After 95 °C heat treatment for 10 min, AFP III lost its ice crystal recrystallization inhibitory activity the most: AFP I was less affected; AFGP was almost entirely unaffected. These different thermal treatment effects might reflect a lower degree of protein aggregation because of hydrophobic interaction after heat treatment or might reflect the simplicity and flexibility of the higher order structures of AFP I and AFGP.

An insect antifreeze protein from Anatolica polita enhances the cryoprotection of Xenopus laevis eggs and embryos.

Cryoprotection is of interest in many fields of research, necessitating a greater understanding of different cryoprotective agents. Antifreeze proteins have been identified that have the ability to confer cryoprotection in certain organisms. Antifreeze proteins are an evolutionary adaptation that contributes to the freeze resistance of certain fish, insects, bacteria and plants. These proteins adsorb to an ice crystal's surface and restrict its growth within a certain temperature range. We investigated the ability of an antifreeze protein from the desert beetle Anatolica polita, ApAFP752, to confer cryoprotection in the frog Xenopus laevis. Xenopus laevis eggs and embryos microinjected with ApAFP752 exhibited reduced damage and increased survival after a freeze-thaw cycle in a concentration-dependent manner. We also demonstrate that ApAFP752 localizes to the plasma membrane in eggs and embryonic blastomeres and is not toxic for early development. These studies show the potential of an insect antifreeze protein to confer cryoprotection in amphibian eggs and embryos.

Low-temperature adaptive conductive hydrogel based on ice structuring proteins/CaCl2 anti-freeze system as wearable strain and temperature sensor.

Conductive hydrogels as wearable devices meet the basic demands of mechanical flexibility and smart sensing. However, achieving anti-freeze property in conductive hydrogels is still challengeable. Here, a novel anti-freezing system based on ice structuring proteins and CaCl2 was introduced to enable a conductive hydrogel with low-temperature adaptability. Both formation of ice nuclei and ice growth of the hydrogel at sub-zero temperature could be inhibited. Supported by the anti-freeze system, the hydrogel revealed good flexibility (890% at -20 °C), recovery and conductivity (0.50 S/m at -20 °C) at both room temperature and sub-zero temperature. The low-temperature adaptability enabled the hydrogel to be used as strain and temperature sensors at both room temperature and sub-zero temperature. The anti-freeze system in this work is expected to open up a new avenue to promote the conductive hydrogel with low-temperature adaptability.

Effects of Antifreeze Protein III on Sperm Cryopreservation of Pacific Abalone, Haliotis discus hannai.

Pacific abalone (Haliotis discus hannai) is a highly commercial seafood in Southeast Asia. The aim of the present study was to improve the sperm cryopreservation technique for this valuable species using an antifreeze protein III (AFPIII). Post-thaw sperm quality parameters including motility, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, fertility, hatchability, and mRNA abundance level of heat shock protein 90 (HSP90) were determined to ensure improvement of the cryopreservation technique. Post-thaw motility of sperm cryopreserved with AFPIII at 10 µg/mL combined with 8% dimethyl sulfoxide (DMSO) (61.3 ± 2.7%), 8% ethylene glycol (EG) (54.3 ± 3.3%), 6% propylene glycol (PG) (36.6 ± 2.6%), or 2% glycerol (GLY) (51.7 ± 3.0%) was significantly improved than that of sperm cryopreserved without AFPIII. Post-thaw motility of sperm cryopreserved with 2% MeOH and 1 µg/mL of AFPIII was also improved than that of sperm cryopreserved without AFPIII. A combination of 10 µg/mL AFPIII with 8% DMSO resulted in the highest post-thaw motility, showing AI of 60.1 ± 3.9%, PMI of 67.2 ± 4.0%, and MMP of 59.1 ± 4.3%. DNA integrity of sperm cryopreserved using 10 µg/mL AFPIII combined with 8% DMSO was not significantly (p > 0.05) different from that of fresh sperm. Cryopreservation using a combination of AFPIII with 8% DMSO improved fertilization and hatching rates of sperm compared to that of cryopreservation without supplementation of 10 µg/mL AFPIII. Sperm cryopreserved using AFPIII showed higher mRNA abundance levels of HSP90 than those cryopreserved without AFPIII. Results of the present study suggest that 10 µg/mL AFPIII combined with 8% DMSO can be used for large scale cryopreservation of Pacific abalone sperm and for hatchery production.

Hypothermic preservation of rat hearts using antifreeze glycoprotein.

Antifreeze proteins are an effective additive for low-temperature preservation of solid organs. Here, we compared static hypothermic preservation with and without antifreeze glycoprotein (AFGP), followed by nonfreezing cryopreservation of rat hearts. The heart was surgically extracted and immersed in one of the cardioplegia solutions after cardiac arrest. Control rat hearts (n=6) were immersed in University of Wisconsin (UW) solution whereas AFGP-treated hearts (AFGP group) (n=6) were immersed in UW solution containing 500 ?g/ml AFGP. After static hypothermic preservation, a Langendorff apparatus was used to reperfuse the coronary arteries with oxygenated Krebs-Henseleit solution. After 30, 60, 90, and 120 min, the heart rate (HR), coronary flow (CF), cardiac contractile force (max dP/dt), and cardiac diastolic force (min dP/dt) were measured. Tissue water content (TWC) and tissue adenosine triphosphate (ATP) levels in the reperfused preserved hearts were also assessed. All the parameters were compared between the control and AFGP groups. Compared with the control group, the AFGP group had significantly (p<0.05) higher values of the following parameters: HR at 60, 90, and 120 min; CF at all four time points; max dP/dt at 90 min; min dP/dt at 90 and 120 min; and tissue ATP levels at 120 min. TWC did not differ significantly between the groups. The higher HR, CF, max dP/dt, min dP/dt, and tissue ATP levels in the AFGP compared with those in control hearts suggested that AFGP conferred superior hemodynamic and metabolic functions. Thus, AFGP might be a useful additive for the static/nonfreezing hypothermic preservation of hearts.

Bioinspired l-Proline Oligomers for the Cryopreservation of Oocytes via Controlling Ice Growth.

Various types of cells are routinely cryopreserved in modern regenerative and cell-based medicines. For instance, the oocyte is one of the most demanding cells to be cryopreserved in genetic engineering and human-assisted reproductive technology (ART). However, the usage of cryopreserved oocytes in ART clinics is still limited mainly because of the unstable survival rate. This is due to the fact that oocytes are more prone to be damaged by ice crystals in comparison to other cells, as oocytes are larger in size and surface area. Meanwhile, oocytes contain more water, and thus, ice crystals are easier to form inside the cells. Currently, to avoid injury by the formed ice crystals, cryopreservation (CP) of oocytes has to use large amounts of small molecules as cryoprotectants such as dimethyl sulfoxide (DMSO) and ethylene glycol (EG), which can permeate into the cell and prevent ice formation inside. However, these molecules are chemically and epigenetically toxic to cells. Therefore, great efforts have been focused on reducing the amount of DMSO and EG used for oocyte CP. In nature, the antifreeze (glyco)proteins (AFGPs) locate extracellularly with the ability to protect living organisms from freezing damage via controlling ice growth. Inspired by this, biocompatible and nontoxic L-proline oligomers (L-Pron), which have the same polyproline II helix structure as that of AFGPs, are first employed for the CP of oocytes. The experimental results reveal that L-Pro8 has a profound activity in inhibiting ice growth as that of AFGP8. Also, by the addition of 50 mM L-Pro8, the amount of DMSO and EG can be greatly reduced by ca. 1.8 M for oocyte CP; moreover, the survival rate of the cryopreserved oocytes is increased up to 99.11%, and the coefficient of variance of the survival rate is decreased from 7.47 to 2.15%. These results mean that almost all oocytes can survive after CP with our method; importantly, the mitochondrial function as a critical criterion for the quality of the frozen-thawed oocytes is also improved. It is proposed that with the addition of L-Pro8, the extracellular ice growth is slowed down, which prevents the direct injuries of cells by large ice crystals and the accompanying osmotic pressure increase. As such, this work is not only significant for meeting the ever-increasing demand by the ART clinics but also gives guidance for designing materials in controlling ice growth during CP of other cells and tissues.

Antifreeze protein from Anatolia polita (ApAFP914) improved outcome of vitrified in vitro sheep embryos.

Embryo cryopreservation is an important tool to preserve endangered species. As a cryoprotectant for mouse oocytes, antifreeze protein from Anatolica polita (ApAFP914) has demonstrated utility. In the present study, the effects of controlled slow freezing and vitrification methods on the survival rate of sheep oocytes fertilized in vitro after freezing-thawing were compared. Different ApAFP914 concentrations were added to the vitrification liquid for exploring the effect of antifreeze protein on the warmed embryos. The results showed that the survival and hatching rates of in vitro derived embryos were significantly higher than that of the slow freezing method. Furthermore, among the cryopreserved embryos at different developmental stages, the survival and hatching rates of the expanded blastocyst were significantly higher than those of the blastocysts, early blastocysts and morula. The survival and the hatching rates of the fast-growing embryos were both significantly higher than that of the slow-growing embryos. Additionally, treatment of ApAFP914 (5-30 µg/mL) did not increase the freezing efficiency of the 6-6.5 d embryos. However, addition of 10 µg/mL of ApAFP914 significantly increased the hatching rate of slow-growing embryos. In conclusion, our study suggests that the vitrification is better than the slow freezing method for the conservation of in vitro sheep embryos, and supplementation of ApAFP914 (10 µg/mL) significantly increased the hatching rate of slow-growing embryos after cryopreservation.

Pre-grafting histological studies of skin grafts cryopreserved in α helix antarctic yeast oriented antifreeze peptide (Afp1m).

A number of living creatures in the Antarctic region have developed characteristic adaptation of cold weather by producing antifreeze proteins (AFP). Antifreeze peptide (Afp1m) fragment have been designed in the sequence of strings from native proteins. The objectives of this study were to assess the properties of Afp1m to cryopreserve skin graft at the temperature of -10 °C and -20 °C and to assess sub-zero injuries in Afp1m cryopreserved skin graft using light microscopic techniques. In the present study, a process was developed to cryopreserve Sprague-Dawley (SD) rat skin grafts with antifreeze peptide, Afp1m, α-helix peptide fragment derived from Glaciozyma antractica yeast. Its viability assessed by different microscopic techniques. This study also described the damages caused by subzero temperatures (-10 and -20 °C) on tissue cryopreserved in different concentrations of Afp1m (0.5, 1, 2, 5 and 10 mg/mL) for 72 h. Histological scores of epidermis, dermis and hypodermis of cryopreserved skin grafts showed highly significant difference (p < 0.01) among the different concentrations at -10 and -20 °C. In conclusion, the integrity of cryopreserved skin grafts with lower concentrations of Afp1m (0.5, 1 and 2 mg/mL) or at -20 °C was not maintained. The present study attested that Afp1m is a good cryoprotective agent for the cryopreservation of skin graft. Higher Afp1m concentrations (5 and 10 mg/mL) at -10 °C found to be suitable for the future in vivo study using (SD) rat skin grafts.

Effects of gelatin-based antifreeze peptides on cell viability and oxidant stress of Streptococcus thermophilus during cold stage.

Cold stage adversely affects cell proliferation and cell viability of probiotics such as Streptococcus thermophilus in food industry, new type of cryoprotectants continues to be needed. Gelatin-based antifreeze peptide becomes a popular topic because of its cryoprotective effects on cold-stressed probiotics. In this study the effects of tilapia scales antifreeze peptides (TSAPP) on cell viability and oxidant stress of S. thermophilus during cold stage were investigated. The results showed that the percentage of viable cells was increased 10.85 folds compared with control groups. Addition of TSAPP activated the activities of ATPases, relieved the hyperpolarization of cell membrane potential and regulated the intracellular Ca2+ concentration. Furthermore, TSAPP significantly inhibited reactive oxygen species level and malonaldehyde content in cells. Under cryopreservation with TSAPP, cells of S. thermophilus maintained higher activities of antioxidant enzymes including catalase, peroxidase and total antioxidant capacity. These findings indicate that TSAPP likely offered its cellular protection by maintaining membrane integrity and alleviation of oxidative stress.