Ice Crystals Research Articles

Structural features of rice starch-protein system: Influence of retrogradation time and quick-freezing temperature

This work investigated the effect of retrogradation time (0 h, 2 h, 4 h, 6 h, 8 h) and freezing temperature (−20 °C, −32 °C, −80 °C) on the muti-scale structures of the rice starch-protein system of quick-frozen wet rice noodles. The Relative crystallinity and porosity of the rice starch-protein system increased with increasing retrogradation time. However, while longer retrogradation does lead to an improvement in relative crystallinity, it also results in significant damage to the microstructure. When the retrogradation time was 6 h, the microstructure of the rice starch-protein system was less damaged and the quality was better. The mass fractal dimension and relative crystallinity of the rice starch-protein system exhibited an increase as the freezing temperature was decreased from −20 to −80 °C. Additionally, the retrogradation degree of starch decreased, the size of ice crystals decreased, and the disruption of microforms was reduced. The muti-scale structures of the rice starch-protein systems were similar when quick-frozen at temperatures of −32 and −80 °C. Therefore, the optimal treatment method for practical production is to quick-freeze at −32 °C and age for 6 h to obtain high-quality quick-frozen wet rice noodles.

The structure of ice under confinement in periodic mesoporous organosilicas (PMOs).

We investigated the structure of ice under nanoporous confinement in periodic mesoporous organosilicas (PMOs) with different organic functionalities and pore diameters between 3.4 and 4.9nm. X-ray scattering measurements of the system were performed at temperatures between 290 and 150K. We report the emergence of ice I with both hexagonal and cubic characteristics in different porous materials, as well as an alteration of the lattice parameters when compared to bulk ice. This effect is dependent on the pore diameter and the surface chemistry of the respective PMO. Investigations regarding the orientation of hexagonal ice crystals relative to the pore wall using x-ray cross correlation analysis reveal one or more discrete preferred orientation in most of the samples. For a pore diameter of around 3.8nm, stronger correlation peaks are present in more hydrophilically functionalized pores and seem to be connected to stronger shifts in the lattice parameters.

Freeze-thaw stability of high-internal-phase emulsion stabilized by chickpea protein microgel particles and its application in surimi.

Future applications of high-internal-phase emulsions (HIPEs) are highly regarded, but poor freeze-thaw stability limits their utilization in frozen products. This study aimed to characterize the structure of chickpea protein microgel particles (HCPI) induced by NaCl and to assess its impact on the freeze-thaw stability of HIPEs. The results showed that NaCl induction (0-400 mmol L-1) increased the surface hydrophobicity (175.9-278.9) and interfacial adsorbed protein content (84.9%-91.3%) of HCPI. HIPEs prepared with HCPI induced by high concentration of NaCl exhibited superior flocculation index and centrifugal stability, and their freeze-thaw stability was better than that of natural chickpea protein. The increase in NaCl concentration reduced the droplet aggregation and coalescence index of the freeze-thaw emulsions, diminishing the precipitation of oil from the emulsion. Linear and nonlinear rheology showed that the strengthened gel structure (higher G' values) restricted water flow and counteracted the damage to the interfacial film by ice crystals at 100-400 mmol L-1 NaCl, thus improving the viscoelasticity of the freeze-thaw emulsions. Finally, the thawing loss of surimi gel with HCPI-200 HIPE was reduced by 2.04% compared to directly adding oil. This study provided a promising strategy to improve the freeze-thaw stability of HIPEs and reduce the thawing loss of frozen products. © 2024 Society of Chemical Industry.

Poor Sperm Chromatin Condensation Is Associated with Cryopreservation-Induced DNA Fragmentation and Cell Death in Human Spermatozoa.

Background/Objectives: Semen cryopreservation is routinely performed in fertility clinics for a variety of reasons, including fertility preservation and storage of donor sperm, yet the freeze-thaw process leads to cellular damage via ice crystal formation, osmotic shock, and supraphysiological levels of oxidative stress. Sperm resistance to damage during the freeze-thaw process varies widely, yet the intrinsic factors associated with sperm cryotolerance are largely unknown. The study aimed to investigate whether poor chromatin condensation renders sperm vulnerable to DNA fragmentation and cell death induced by the freeze-thaw process. Methods: Participants (n = 51) from the general community who met the inclusion criteria collected a semen sample after 3-8 days of abstinence. Neat semen samples underwent traditional semen analysis, aniline blue (AB)-eosin staining for chromatin condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay for DNA fragmentation, and the Annexin V assay for apoptosis/necrosis, prior to being cryopreserved using the liquid nitrogen vapour method and stored at -196 °C. Stored samples were later thawed at room temperature and processed using density gradient centrifugation. Motile sperm concentration, DNA fragmentation and apoptosis/necrosis were analysed in post-thaw samples. Results: As indicated by a significant interaction effect in linear mixed models, an increased proportion of AB-positive sperm in the pre-freeze sample exacerbated the adverse effect of freezing on sperm DNA fragmentation (p = 0.004), late apoptosis (p = 0.007), and necrosis (p = 0.007). AB-staining was positively correlated with all three parameters in the post-thaw sample (all rs ≥ 0.424, all p < 0.01) and remained significant after adjusting for neat sperm concentration (all partial rs ≥ 0.493, all p < 0.01). Similarly, AB-staining was significantly correlated with the percentage point change in sperm DNA fragmentation (rs = 0.366, p = 0.014) and necrosis (rs = 0.403, p = 0.009), both of which remained significant after adjusting for neat sperm concentration (both partial rs ≥ 0.404, both p < 0.01), and borderline significantly correlated with percentage point change in late apoptosis (rs = 0.307, p = 0.051). Conclusions: Sperm with poorly condensed chromatin may be more susceptible to cellular damage during the freeze-thaw process, independent of pre-freeze sperm concentration. These findings may help to explain the intrinsic variation in sperm resistance to cryodamage within and between individuals that is poorly understood.

Analysis of cryopreservation media thermophysical characteristics after ultra-rapid cooling through differential scanning calorimetry

Cryoprotective agents play a critical role in minimizing cell damage caused by ice formation during cryopreservation. However, high concentrations of CPAs are toxic to cells and tissues. Required concentrations of CPAs can be reduced by utilizing higher cooling and warming rates, but insight into the thermophysical properties of biological solutions in the vitrification method is necessary for the development of cryopreservation protocols. Most studies on thermophysical properties under ultra-rapid cooling conditions have been qualitatively based on visualization. Differential scanning calorimetry methods are ideal for studying the behavior of biomaterials in various freezing conditions quantitatively and accurately, though previous studies have been predominantly restricted to slower cooling rates. Here, we developed an ultra-rapid cooling method for DSC that can achieve minimal cooling rates exceeding 2000 °C/min. We investigated the thermophysical vitrification behavior of ternary solutions of phosphate buffer saline (1X), dimethyl sulfoxide or glycerol and ice blocking polymers (X-1000 or Z-1000). We quantified the impact of solute concentration on ice crystal formation during rapid cooling. Our findings support the expectation that increasing the solute concentration reduces the amount of ice formation, including devitrification. Devitrification increases from 0 % to 40 % (v/v) Me2SO and then reduces significantly. The relative amounts of devitrification to the total ice formation are 0 %, 60 %, 0 % in 20 %, 40 %, 60 % (v/v) Me2SO, and 2 %, 48 %, 49 % in 20 %, 40 %, 60 % (v/v) glycerol, respectively. The results suggest that at low concentrations, such as below 20 % (v/v) for Me2SO or glycerol, increasing the warming rate after ultra-rapid freezing is not essential to eliminate devitrification. Furthermore, ice blocking polymers do not reduce ice formation substantially and cannot eliminate devitrification under ultra-rapid cooling conditions. In conclusion, our results provide insights into the impact of solute concentration on ice formation and devitrification during rapid cooling, which can be practical for optimizing cryopreservation protocols.

The use of xanthan gum in a milk-containing ice cream with the whey protein microparticulate

Hydrocolloids of stabilization systems are necessary components in ice cream production. They influence viscosity, stabilization of structural elements and melting rate. Their role is especially important in production of ice cream with the low content of fat and nonfat milk solids. Today, specialized stabilization systems for production of such ice cream are absent. Moreover, when choosing stabilization systems, there are problems of economic character that are linked with an increase in prices on the effective polysaccharide — locust bean gum. The aim of the research was substantiation of the composition of the effective formulation of hydrocolloids using their available variety, xanthan gum, to use in production of milk-containing ice cream (with the reduced content of fat and dry nonfat milk substances). To achieve the best quality indicators, a whey protein microparticulate was introduced into milk-containing ice cream. Based on the synergetic properties of hydrocolloids in terms of dynamic viscosity, the composition of four formulations was determined with the content of xanthan gum of 8.6% (in samples 1 and 2), 16% (in sample 3) and 3% (in sample 4). Ice cream with the complex stabilization system of the trademark Cremodan 334 was produced as a control sample. The following indicators were determined in all samples: dynamic viscosity, viscoelastic characteristics (hardness, adhesion strength, gumminess), melting rate, condition and dispersity of the air phase and ice crystals. All developed formulations were superior to the control sample in terms of viscosity by 1.2–2 times. It has been found that replacement of the kappa-carrageenan fraction with iota-carrageenan in combination with guar gum and xanthan gum in an amount of 50% leads to a decrease in viscosity by 1.3 times. A reduction of visco-elastic characteristics was noted in the samples of hydrocolloid formulations under study. When using iota-carrageenan (samples 2 and 4), a notable reduction of thermal stability of ice cream was revealed in sample 4. Furthermore, a decrease in dispersity of the air phase was observed; the content of air bubbles with a size of 50 µm reduced by almost 30%. Based on the results of the investigations, it has been established that the formulation of hydrocolloids of ice cream sample 1, which consists of mono- and di-glycerides of fatty acids, guar gum, xanthan gum and kappa-carrageenan, allows obtaining a product with technologically necessary quality indicators and the most cream-like consistency.

Technologically important quality indicators of low-lactose ice cream with milk and whey protein concentrates

Protein concentrates have a high biological value and contain significantly less lactose in comparison with dry skimmed milk. The use of protein concentrates in ice cream without the content of non-fat milk solids allows not only making up for protein but also developing the component composition of a product with the low content of lactose. In this regard, the study of technologically significant quality indicators of ice cream with the low content of lactose without non-fat milk solids when using milk and whey protein concentrates is an urgent task. The rheological, microstructural, color and thermostatic characteristics, as well as the oxidative stability of fat of the test samples were determined in the research. It has been found that viscosity decreased by 1.8-2.6 times when using whey proteins. The use of protein concentrates led to an increase in hardness and adhesiveness by 2.2-4.1 and 1.6-5.0 times, respectively, but did not significantly affect the thermal stability. The mass fraction of fat had the greatest influence on these characteristics. The study of color characteristics of ice cream made it possible to reveal a decrease in the yellowness index when using milk protein concentrate. It has been established that color differences in most of the samples were not recognizable to the human eye. The induction period was characterized by the lowest value in the samples with whey proteins, thus indicating a probability of a faster oxidation process in them. The use of protein concentrates did not have a significant effect on ice crystals. The stability of the air phase was significantly reduced when using the whey proteins. A multivariate analysis of the studied samples divided them into the control and the samples containing protein concentrates. Based on the technologically significant quality indicators, the use of milk protein concentrates in the production of low-lactose ice cream is more preferable than the use of whey protein concentrates.

Supercooled preservation of cultured primary rat hepatocyte monolayers.

Supercooled preservation (SCP) is a technology that involves cooling a substance below its freezing point without initiating ice crystal formation. It is a promising alternative to prolong the preservation time of cells, tissues, engineered tissue products, and organs compared to the current practices of hypothermic storage. Two-dimensional (2D) engineered tissues are extensively used in in vitro research for drug screening and development and investigation of disease progression. Despite their widespread application, there is a lack of research on the SCP of 2D-engineered tissues. In this study, we presented the effects of SCP at -2 and -6°C on primary rat hepatocyte (PRH) monolayers for the first time and compared cell viability and functionality with cold storage (CS, + 4°C). We preserved PRH monolayers in two different commercially available solutions: Hypothermosol-FRS (HTS-FRS) and the University of Wisconsin (UW) with and without supplements (i.e., polyethylene glycol (PEG) and 3-O-Methyl-Α-D-Glucopyranose (3-OMG)). Our findings revealed that UW with and without supplements were inadequate for the short-term preservation of PRH monolayers for both SCP and CS with high viability, functionality, and monolayer integrity. The combination of supplements (PEG and 3-OMG) in the HTS-FRS solution outperformed the other groups and yielded the highest viability and functional capacity. Notably, PRH monolayers exhibited superior viability and functionality when stored at -2°C through SCP for up to 3days compared to CS. Overall, our results demonstrated that SCP is a feasible approach to improving the short-term preservation of PRH monolayers and enables readily available 2D-engineered tissues to advance in vitro research. Furthermore, our findings provide insights into preservation outcomes across various biological levels, from cells to tissues and organs, contributing to the advancement of bioengineering and biotechnology.

The Valence-Dependent Activity of Colloidal Molecules as Ice Recrystallization Inhibitors.

Inspired by advances in cryopreservation techniques, which are essential for modern biomedical applications, there is a special interest in the ice recrystallization inhibition (IRI) of the antifreeze protein (AFPs) mimics. There are in-depth studies on synthetic materials mimicking AFPs, from simple molecular structure levels to complex self-assemblies. Herein, we report the valence-dependent IRI activity of colloidal organic molecules (CMs). The CMs were prepared through polymerization-induced particle-assembly (PIPA) of the ABC-type triblock terpolymer of poly(acryloxyethyl trimethylammonium chloride)-b-poly(benzyl acrylate)-b-poly(diacetone acrylamide) (PATAC-b-PBzA-b-PDAAM) at high monomer conversions. Stabilized by the cationic block of PATAC, the strong intermolecular H-bonding and incompatibility of the PDAAM block with PBzA contributed to the in situ formation of Janus particles (AX1) beyond the initial spherical seed particles (AX0), as well as the high valency clusters of linear AX2 and trigonal AX3. Their distribution was controlled mainly by the polymerization degrees (DPs) of PATAC and PDAAM blocks. IRI activity results of the CMs suggest that the higher fraction of AX1 results in the better IRI activity. Increasing the fraction of AX1 from 27% to 65% led to a decrease of the mean grain size from 39.8% to 10.9% and a depressed growth rate of ice crystals by 58%. Moreover, by replacing the PDAAM block with the temperature-responsive one of poly(N-isopropylacrylamide) (PNIPAM), temperature-adjustable IRI activity was observed, which is well related to the reversible transition of AX0 to AX1, providing a new idea for the molecular design of amphiphilic polymer nanoparticle-based IRI activity materials.

Efficient all-electron hybrid density functionals for atomistic simulations beyond 10 000 atoms.

Hybrid density functional approximations (DFAs) offer compelling accuracy for abinitio electronic-structure simulations of molecules, nanosystems, and bulk materials, addressing some deficiencies of computationally cheaper, frequently used semilocal DFAs. However, the computational bottleneck of hybrid DFAs is the evaluation of the non-local exact exchange contribution, which is the limiting factor for the application of the method for large-scale simulations. In this work, we present a drastically optimized resolution-of-identity-based real-space implementation of the exact exchange evaluation for both non-periodic and periodic boundary conditions in the all-electron code FHI-aims, targeting high-performance central processing unit (CPU) compute clusters. The introduction of several new refined message passing interface (MPI) parallelization layers and shared memory arrays according to the MPI-3 standard were the key components of the optimization. We demonstrate significant improvements of memory and performance efficiency, scalability, and workload distribution, extending the reach of hybrid DFAs to simulation sizes beyond ten thousand atoms. In addition, we also compare the runtime performance of the PBE, HSE06, and PBE0 functionals. As a necessary byproduct of this work, other code parts in FHI-aims have been optimized as well, e.g., the computation of the Hartree potential and the evaluation of the force and stress components. We benchmark the performance and scaling of the hybrid DFA-based simulations for a broad range of chemical systems, including hybrid organic-inorganic perovskites, organic crystals, and ice crystals with up to 30 576 atoms (101 920 electrons described by 244 608 basis functions).

Characterization of gelatin-oxidized riclin cryogels and their applications as reusable ice cubes in shrimp preservation

Traditional ice is usually employed to preserve food freshness and extend shelf life. However, ice cannot bear repeated freeze − thaw cycles during the transportation and retailing process, resulting in microbial cross-contamination and spoilage of foods. Herein, succinoglycan riclin was oxidated (RO) and crosslinked with gelatin (Ge), the Ge-RO cryogels were prepared via Schiff base reaction and three freeze − thaw cycles. The Ge-RO cryogels showed improved storage modulus (G′) and thermal stability compared with pure gelatin hydrogel. The polymer framework of Ge-RO gels exhibited stable properties against ice crystals destructions during nine freeze − thaw treatments. During the storage and repeated freeze − thaw treatments of shrimps, Ge-RO cryogels exhibited a remarkable preservation effect on shrimps, and their freshness was evaluated using an electronic nose technique equipped with ten sensors. The results demonstrated that the shrimp muscle preserved in ice generated off-odors and resulted in high sensor responses. The sensor responses were reduced sharply of shrimps preserved in cryogels. Moreover, 1H NMR-based metabolomics analysis revealed that shrimps in Ge-RO cryogels group reversed the metabolic perturbations compared with the traditional ice group, the metabolic pathways were related to energy metabolism, nucleotide metabolism, and amino acid metabolism, which provide new clues to the freshness of shrimps. Furthermore, RO exhibited superior antimicrobial activity against E. coli and S. aureus microorganisms. Thus, the crosslinked cryogels are potentially applicable to food preservation, offering sustainable and reusable solutions against traditional ice.

Pathways of ice multiplication in nimbostratus clouds during the Indian summer monsoon

The present study illustrates the microphysical parameters of nimbostratus clouds during the Indian summer monsoon using airborne and radar observations conducted as part of the Cloud Aerosol Interaction and Precipitation Enhancement Experiment (CAIPEEX), and simulations with the Weather Research and Forecasting (WRF) model. The study also analyzes the impact of secondary ice production (emphasizing the microphysical pathways of Hallett-Mossop (HM) as represented in the models) on cloud processes using model sensitivity simulations. The effect of possible pathways of riming during the HM process is the focus of the sensitivity simulations. Aircraft observations showed higher ice particle concentrations in the Hallett–Mossop zone (−3 °C to - 8 °C) along with the existence of smaller and larger cloud droplets, rimed needles, columns, and snow particles. Observations strongly suggested the active presence of the HM process in this cloud. The observed mean values of microphysical parameters including liquid water content, ice number concentrations, and maximum vertical velocity, agreed well with model simulations. The number concentration and water content of ice crystals and snow decreased in the HM zone during the HM inactive case. A reduction in rainfall is also observed in the absence of HM. HM increases convection as it causes a sudden increase in the number concentration of small ice particles, which causes rainwater to freeze quickly and water vapour to be consumed by diffusional growth. Latent heating is produced by both effects, which energizes the convection.

Effect of low-frequency electric field assisted freezing on ice crystals of tilapia fish protein

The mechanism by which low-frequency alternating electric fields affect ice crystals and quality of aquatic products remain unclear. To study the effect of low-frequency electric field freezing (LF-EFAF) on product ice crystals, differences between LF-EFAF treated samples and air freezing (AF) were compared. The results showed that LF-EFAF increased the subcooling of fish protein by 0.3 °C and doubled the freezing time compared to AF. The equivalent diameter and cross-sectional area of LF-EFAF samples were reduced by up to 20% and 35% respectively, compared to AF samples. Moisture distribution of results showed that LF-EFAF restrained the increase in free water content of fish proteins. The LF-EFAF had a positive effect on the size and morphology. This resulted in less mechanical damage to the muscle fibres, which led to better water retention and inhibited moisture migration. Therefore, LF-EFAF can be an effective means of fish proteins preservation.

Disaccharide-assisted inkjet freezing for improved cell viability

Non-permeable disaccharides are widely used as cryoprotectant agents due to their low cytotoxicity, but their protective effect is insufficient when the disaccharides are present only extracellularly. On the other hand, cryoprotectant agent (CPA)-free cryopreservation has been recently achieved by instantaneously inkjet-freezing cells as tiny droplets. However, CPA-free cryopreservation requires skilled handling operations due to instability of the vitreous water without the CPA. In this study, the effectiveness of separately adding two types of disaccharides in inkjet freezing of 3T3 cells was evaluated and the following results were obtained. First, trehalose showed the highest effect at 0.57 M, twice the plasma osmolarity, with a maximum cell viability of over 90 % when freezing 70 pL droplets. However, higher concentrations of trehalose decreased cell viability due to damage caused by dehydration. Similarly, sucrose gave cell viability close to 90 % at 0.57 M with 70 pL droplets, and higher concentrations decreased cell viability. Next, the relationship between minimum trehalose concentrations to prevent intracellular and extracellular ice crystal formation and droplet size was analyzed. The results indicated that trehalose of less than 0.57 M was able to inhibit intracellular ice crystal formation even in the largest droplet used in this study, 450 pL, while trehalose of nearly 0.57 M was required to inhibit extracellular ice crystal formation in the smallest droplet, 70 pL. In other words, the suppression of extracellular ice crystals by the addition of CPA was shown to be crucial in improving the viability of inkjet superflash freezing.

Effect of different freezing conditions on ice crystal formation behavior and ice-growth inhibition by cryoprotectants.

The formation of ice crystals will have adverse effects on aquatic products, and the key to ensure the long-term preservation and better quality preservations of the product is to evaluate the intercellular ice crystal formation to find suitable refrigeration conditions and cryoprotectants. The ice crystal formation was successfully captured by using an inverted microscope cryomicroscopic system equipped with a low-temperature stage, the ice crystals formed under different freezing methods between tuna muscle cells were observed directly, the deformation degree of muscle tissue pores during crystallization was evaluated, and the effect of freeze-thaw times on tuna samples was analyzed. The effects of the use of cryoprotectant such as cellobiose and carboxylated cellulose nanofibers on ice-growth inhibition were investigated, and the reliability of the ice crystal observation results was further verified by the determination of physical properties. The results showed that carboxylated cellulose nanofibers had the best ice-growth inhibition effect, they prevented about 50% cell deformation compared with the control group, and also reduced the minimum size of ice crystal formation. In addition, the addition of cellobiose and sodium tripolyphosphate gave the ice crystals a more uniform size and roundness. The experiment proposed a stable and clear observation method for the process of intercellular ice crystal formation, and the accuracy of the observation method was further verified by some physical indicators. This may help in the selection of suitable measurement methods to directly observe ice crystal formation behavior and screen cryoprotectants. © 2024 Society of Chemical Industry.

Effect of Plasma Electric Field Assistance on the Freezing Process and Ice Crystal Formation of Bactrian Camel Meat

Background: The longissimus dorsi muscle of the camel was chosen as the experimental material in order to investigate the impact of plasma electric field assisted freezing on the quality and freezing process of Bactrian camel meat. Methods: The effect of the auxiliary plasma electric field of different field strengths (1 kV/m, 3 kV/m and 5 kV/m) on the freezing velocity of the camel meat and the distribution of ice crystals as well as the diameter and roundness of ice crystal in the frozen meat were studied and analyzing them with the help of the thermodynamic theory. Result: The findings demonstrated that the meat samples in the auxiliary 5 kV/m plasma field environment took 60 min and 30 min, respectively, at -20°C and -35°C, to pass the maximal ice crystal production zone. The tissue sections revealed that at -20°C and -35°C assisted 5 kV/m plasma field freezing, the smallest ice crystal area and grain size were formed, the roundness of the ice crystals was highest. Using Boltzmann’s entropic equation to calculate the system’s entropy, the results revealed that the meat samples frozen by the aided 5 kV/m plasma electric field had the lowest entropy at -20°C and -35°C. The electric field assisted freezing requires less energy to improve the subcooling degree and accelerate the freezing rate of meat samples, proving that the auxiliary 5 kV/m plasma electric field can significantly improve the system frozen meat moisture vitrification degree and can make the crystallization process more controllable.

P-152 The impact of one-step fast warming protocol on vitrified-warmed blastocysts on reproductive outcome

Abstract Study question Does the implementation of one-step fast warming protocol affect the post warming quality of embryos and/or the reproductive outcome in frozen embryo replacement cycles? Summary answer The implementation of a one-step fast warming protocol provides similar survival, pregnancy, and clinical pregnancy rates with standard warming protocols. What is known already Cryopreservation process presents a significant challenge for embryos, as the formation of ice crystals can be lethal to their cells. Therefore, effective cryopreservation protocols are essential to mitigate this risk. Vitrification method protects the cells during cryopreservation by transforming water from a liquid state to a glass-like phase, thereby preventing the ice crystals formation. Moreover, the warming procedure can itself contribute to ice crystal formation, particularly through recrystallization. Studies have shown that embryo survival is related to warming rate and a one-step fasting warming protocol can be equally or more effective than the conventional protocol. Study design, size, duration This prospective randomized study was performed at Embryolab Fertility Clinic, in Thessaloniki, Greece between July and December 2023 and included 118 frozen embryo transfers. Both study and control group included only expanded blastocysts of good quality (AA or AB according to Gardner’s criteria). Control group was matched to the study group according to treatment, time period, woman age, quality of embryos and number of transferred embryos. The embryos were vitrified using open system. Participants/materials, setting, methods Fast warming group included embryos that were warmed using the one-step warming protocol: the embryos were exposed to 1M sucrose solution for 1’ at 37 °C and then immediately placed into culture media where laser-assisted hatching was performed. Control group included embryos that were warmed using conventional protocol: 1’ in 1M, 2’ in 0.5M, 2’ in 0.25M and 3’ in washing solution at room temperature and then placed into culture media for laser-assisted hatching. Main results and the role of chance The analyzed cases were 118 in total, 59 in the fast-warming group and 59 in the control group. Survival rate was 100% in both groups. Pregnancy rate (positive b-HCG) was not significantly different between the two groups: 72.41% in the fast-warming group and 67.79% in the control group (p = 0.687). Clinical pregnancy rate was also not significant between the two groups: 78.26% in the fast-warming group and 81.81% in the control group (p = 0.753). Limitations, reasons for caution The study included a limited number of cases, and information on the live birth rate was still pending. To establish the efficacy of fast warming conclusively, it is recommended that additional prospective randomized studies be conducted to validate its potential benefits. Wider implications of the findings One-step fast warming protocol resulted in similar survival rates post warm compared to the conventional warming protocol, while pregnancy and clinical pregnancy rates were not significantly different. Furthermore, it is worth noting that by minimizing the duration of the warming protocol, the workflow efficiency in the IVF laboratory is enhanced. Trial registration number N/A

Mitigation of mechanical damage and protein deterioration in giant river prawn (Macrobrachium rosenbergii) by multi-frequency ultrasound-assisted immersion freezing

In order to investigate the effects of multi-frequency ultrasound-assisted immersion freezing (MUIF) on the meat quality of Macrobrachium rosenbergii, tail meat was subjected to different MUIF treatments respectively, namely 20 + 40 kHz (MUIF-20 + 40), 20 + 60 kHz (MUIF-20 + 60), 40 + 60 kHz (MUIF-40 + 60) and 20 + 40 + 60 kHz (MUIF-20 + 40 + 60), and the immersion freezing (IF) as control. Results showed that average diameter of ice crystals was 28 μm in IF, and that was only 8 μm in MUIF-20 + 40 + 60. When compared to IF, MUIF alleviated oxidative deterioration of lipids and proteins, but only at higher ultrasound frequency (MUIF-40 + 60; MUIF-20 + 40 + 60). Carbonyl content of MUIF-20 + 40 + 60 was only 40% of that in IF. Similarly, protein denaturation was inhibited in MUIF (except for MUIF-20 + 40). Transmission electron microscopy showed greater distortion of the ultrastructural components in IF, MUIF-40 + 60, and MUIF-20 + 40 + 60, suggested by bended Z-line. In conclusion, MUIF can be an effective strategy to mitigate mechanical damage and protein deterioration in the meat of Macrobrachium rosenbergii.

P-325 Comparing slow-freezing and vitrification endometrial biopsy cryopreservation protocols for tissue cryodamage and functional organoid formation

Abstract Study question Are slow-freezing and vitrification suitable methods for cryopreserving endometrial biopsies intended for downstream research applications and organoid formation? Summary answer Both slow-frozen and vitrified endometrial biopsies resulted in successful organoid formation thus can be considered similar to fresh endometrial biopsies in preserving the cellular viability. What is known already Passive slow freezing (PSF) protocols have demonstrated efficacy for cryopreserving endometrial biopsies with subsequently viable isolated epithelial and stromal cells. Additionally, single-cell transcriptomic analyses and successful generation of endometrial organoids suggest a little impact of cryopreservation. By avoiding ice crystal formation, vitrification (VT) could potentially prevent mechanical injury and improve tissue integrity compared to slow freezing. Study design, size, duration Experimental study using endometrial biopsies collected using a Pipelle from eight healthy volunteers. Samples were retrieved at three cycle phases: proliferative (n = 2), mid-secretory (7 days post-LH surge, LH + 7, n = 2) and late-secretory phase (LH + 11, n = 4). Each biopsy was divided into fresh, slow-freezing and vitrification sub-samples for further comparison of outcomes according to microscopic histological analysis, transmission electron microscopy (TEM), gene expression profiling evaluation and generation of endometrial epithelial organoids (from 2 slow-frozen and 2 vitrified samples). Participants/materials, setting, methods PSF was performed using Mr Frosty with samples embedded in 1x DMEM, 30% FBS and 7.5% DMSO at -80ºC. Vitrification was performed using 40% ethylene glycol (EG); 30% Ficoll; 0.5 M Sucrose and 10 mg/ml HSA, and preserved in liquid nitrogen. Tissue morphology was evaluated by traditional histology, TEM and gene expression profiling. The viability of endometrial cells and the ability to form epithelial organoids were evaluated before and after two different freezing protocols. Main results and the role of chance Our analysis showed that both PSF and vitrification techniques are appropriate for cryopreservation and storage of endometrial biopsies aimed at organoid generation, as no significant differences were found between fresh and cryopreserved tissue samples. However, ultrastructural changes were observed by TEM in PSF samples indicating an impact on mitochondria of epithelial and stromal cells with increased swelling. Changes were also observed in epithelial cells in vitrified samples, with decreased nuclear granularity, clarity, and open karyoplasm. On TEM analyses changes in the chromatin status were more evident in vitrified samples compared to fresh and PSF samples. PSF treated samples showed similarly high quality of epithelial and stromal nuclear staining when compared to the fresh samples in haematoxylin and eosin-stained slides, while VT of samples resulted in a significant decrease of both epithelial and stromal nuclear clarity. However, despite these changes observed in the tissue morphology, all three groups displayed similar transcriptional profiles and endometrial organoids were generated from both slow-frozen and vitrified samples, indicating very little or no impact of the freezing method used for post-thaw tissue functionality. Limitations, reasons for caution Although our study demonstrated relatively mild damages in both freezing protocols, we cannot exclude the absence of additional changes not evaluated in this study, such as those concerning the epigenetic constitution of the cells. Therefore, more extended research with larger sample size is needed for further validation of the results. Wider implications of the findings Our findings indicate that both slow-freezing and vitrification methods applied to endometrial biopsies are effective in maintaining tissue morphology and functionality. Further optimization of cryopreservation methods of endometrium could contribute to future experimental and clinical use of cryopreserved endometrial tissue. Trial registration number not applicable

O-294 Human oocyte survival, early embryo development, metabolic fingerprinting, and pregnancy outcomes following ultra-rapid or standard vitrification and thawing

Abstract Study question Is ultra-rapid vitrification and thawing superior to standard vitrification in terms of preserving oocyte viability, embryo competence and enhancing pregnancy outcomes? Summary answer Ultra-rapid vitrification and thawing preserves oocyte viability and embryo quality as confirmed by their euploidy, non-invasive biomarkers in the culture media, and favorable pregnancy outcomes. What is known already Cryopreservation and vitrification protocols for oocytes and embryos have varying cooling rates, cryoprotectant chemical composition and concentration. While vitrification avoids intracellular ice crystal formation, the elevated concentrations of cryoprotectants may be cytotoxic. Over the past 20 years, parameters for vitrification protocols were optimized to improve efficiency and viability of human oocytes and embryos, however few studies have evaluated the metabolic and secretome profiles in addition to embryology and pregnancy outcomes, particularly following ultra-rapid vitrification and thawing. Mid-infrared spectroscopy is a great option for characterizing environmental components and metabolic profiles using the more spectrally complex fingerprint region from 1450-500 cm-1. Study design, size, duration This prospective study included 1,214 donor oocytes collected between January 2022 and November 2023. Sibling oocytes were assigned to standard or ultra-rapid vitrification and thawing. Corresponding blastocysts underwent the same type of vitrification following trophectoderm biopsies for PGT-A. Spent embryo culture fluid was collected on day 1, 3, and 5 for spectroscopy and PCR analysis, as well as on day 6 for metabolic fingerprinting. Clinical pregnancy was assessed 5 days following single embryo transfer. Participants/materials, setting, methods Vitrified oocyte survival was assessed one hour after thawing. Secretome profiling of day 1, 3, and 5 embryos was conducted by Attenuated Total Reflection Fournier Transform Infrared (ATR-FTIR) spectroscopy. Metabolic profiling of blastocysts (day 6) was conducted by Fournier Transform Infrared (FTIR) spectroscopy. Embryology and pregnancy data together with lipid, protein, and nucleic acid expression profiles were evaluated using the Shapiro-Wilk test followed by the student t-test (parametric variables) or the Mann-Whitney U-Test (non-parametric variables). Main results and the role of chance There was 100% survival in oocytes subject to ultra-rapid vitrification-thawing (n = 531) compared to 94.5% in oocytes that underwent standard vitrification (n = 634). There were no significant differences in the fertilization rates following intracytoplasmic sperm injection (81.3% with ultra-rapid and 83.2% with standard vitrification) or potential to reach day 3 of embryo development[RL1]. Ultra-rapid vitrification-thawing produced significantly more blastocysts than standard vitrification-thawing (67.3% vs. 53.0%, respectively) but both techniques had similar euploidy rates (61.8% vs. 61.4%, respectively; p &amp;gt; 0.05). Spectra in the 4000-450 cm-1 region revealed there were no significant differences in lipid and protein expression profiles of day 1, 3, 5 and 6 embryos. The similar result obtained for the culture media for day 1,3,5 and 6 day embryo development with no significant difference. Day 3 blastomere biopsy samples and Day5, Day 6 trophectoderm biopsy sample showed no significant differences for total protein concentration, lipid and nucleic acid experssion profiles in both group.This finding was corroborated by PCR analysis showing no significant difference in nucleic acid profiles. Finally, clinical pregnancy rates were 65.22% with ultra-rapid and 56.5% [RL2] with standard vitrification. (p &amp;lt; 0.05) Limitations, reasons for caution While both our ultra-rapid and standard vitrification-thawing protocols appear to preserve embryo competence to the blastocyst stage and euploidy, future studies should evaluate whether RNA expression levels are altered with these approaches. Wider implications of the findings This study shows competent and euploid embryos can be generated following ultra-rapid vitrification and provides embryo metabolites that can serve as non-invasive biomarkers for the identification of healthy embryos before embryo transfer. Trial registration number NA