Search Results (24)

Sperm cryopreservation is a powerful tool for the conservation of endangered species, but its application requires adapting protocols to particular species, due to differences in sperm structure, function, and cryosensitivity. Research on the biology of endangered felids primarily relies on the domestic cat as an experimental model. Semen from live animals can be collected using several methods. However, in animals that die due to roadkill or in the field, spermatozoa must be retrieved from the epididymis. Differences may exist in the cryosensitivity of epididymal and ejaculated sperm due to the influence of secretions from accessory genital glands. We analyzed the effects of several factors on the motility and acrosomal integrity of cryopreserved cat epididymal spermatozoa, including cooling rate, storage system, time and temperature of straw loading, and the freezing method in nitrogen vapors. There were no significant differences in motility or acrosomal integrity at thawing between fast (−0.5 °C/min) or slow (−0.125 °C/min) cooling rates or between loading straws at room temperature versus 5 °C. Post-thaw motility was significantly higher when using straws compared to pellets and when freezing in nitrogen vapors at two levels rather than at a single level. Additionally, interactions between the loading temperature of straws and both motility and acrosomal integrity were not significant. These results are important for standardizing protocols to cryopreserve feline epididymal sperm, facilitating the rescue of genetic material from endangered species in the field. Full article

In this work, five different dextran-based cryogels for controlled drug release are investigated. Vitamin B12 was used as a model drug for in vitro release tests. Two different drug-loading procedures were adopted, leading to very different drug release curves. Indeed, a fast Fickian release was observed when freeze-dried samples of DEX₄₀PEG₃₆₀MA and DEX₄₀PEG₅₀₀MA were infused with the drug after cryogel formation. On the contrary, a slowed highly non-Fickian behavior arises when the drug is loaded before the low-temperature crosslinking step, leading to the cryogel formation. The non-Fickian drug release, observed for all the five different dextran-based cryogels investigated, is actually due to the cryoconcentration phenomenon, modeled with a two-step release process. The proposed transport model accurately predicts experimental release curves characterized by a long lag time, confirming that dextran-based cryogels are suitable for controlled release. Full article

Pancreatic surgery is extremely challenging and demands an extended learning curve to be executed with a low incidence of post-operative complications. The soft consistency of the human pancreas poses a primary challenge for pancreatic surgeons. This study aimed to analyze the preliminary mechanical characteristics of the human pancreas to develop a realistic synthetic phantom for surgical simulations in the near future. Pancreatic specimens, comprehensive of the pancreatic parenchyma and main pancreatic duct, were collected during pancreatic resections and analyzed through nano-bio-indentation (Bioindenter^TM UNHT³ Bio, Anton Paar GmbH, Graz, Austria) to measure the elastic modulus. Comparisons were made between slow and fast loading rates, immediate and post-freezing analyses, and multipoint indentations. The results demonstrated that a slow loading rate (30 μN/min), immediate analysis, and multipoint measurements are crucial for obtaining accurate values of the elastic modulus of the human pancreas (1.40 ± 0.47 kPa). In particular, the study revealed that analysis after freezing could impact the outcomes of the indentation. Moreover, the study suggested that both the pancreatic parenchyma and the main pancreatic duct should be analyzed to achieve a more precise and comprehensive definition of the. mechanical features of the pancreas. These preliminary findings represent the initial steps toward defining the consistency and mechanical characteristics of human pancreatic tissue with the goal of creating a realistic synthetic phantom. Full article

The wound healing process is often slowed down as a result of complications from bacterial infections and inflammatory reactions. Therefore, it is necessary to develop dressings with fast antibacterial and anti-inflammatory activity that shorten the wound healing period by promoting cell migration and proliferation. Chitosan (CS)-based hydrogels have been widely studied for their antibacterial and wound healing capabilities. Herein, we developed a composite hydrogel based on CS and PVA embedding silver nanoparticles (AgNPs) with antibacterial properties and ibuprofen (Ib) as an anti-inflammatory agent. The hydrogel prepared by double physical cross-linking, with oxalic acid and by freeze–thawing, loaded with 0.225 wt.% AgNPs and 0.264 wt.% Ib, displayed good mechanical properties (compressive modulus = 132 kPa), a high swelling degree and sustained drug delivery (in simulated skin conditions). Moreover, the hydrogel showed strong antibacterial activity against S. aureus and K. pneumoniae due to the embedded AgNPs. In vivo, this hydrogel accelerated the wound regeneration process through the enhanced expression of TNF alpha IP8, by activating downstream cascades and supporting the healing process of inflammation; Cox2, which enhances the migration and proliferation of cells involved in re-epithelization and angiogenesis; MHCII, which promotes immune cooperation between local cells, eliminating dead tissue and controlling infection; the intense expression of Col I as a major marker in the tissue granulation process; and αSMA, which marks the presence of myofibroblasts involved in wound closure and indicates ongoing re-epithelization. The results reveal the potential healing effect of CS/PVA/AgNPs/Ib hydrogels and suggest their potential use as wound dressings. Full article

This study involves an investigation of the effects of various cooking temperatures, freeze–thaw processes, and food preservatives on the quality and shelf-life of sous vide Mediterranean mussels. Cooking temperatures of 80 °C or above significantly improved the microbiological quality, with bacterial counts remaining within the acceptability range for human consumption even after 21 days of refrigerated storage. Fast freezing followed by slow thawing preserved the highest moisture content, potentially improving texture. Sensory analysis revealed that refrigerated sous vide mussels maintained a comparable taste to freshly cooked samples. Frozen samples reheated via microwaving exhibited more intense flavour than pan-reheated or fresh mussels. Food additives, including citric acid, potassium benzoate, and potassium sorbate, alone or in combination with grape seed oil, significantly reduced total volatile basic nitrogen and thiobarbituric acid-reactive substances during 28 days of storage, indicating decreased spoilage and lipid oxidation. Mussels with a combination of these additives registered a nitrogen content as low as 22 mg of N/100g after 28 days, well below the limit of acceptability (<35 mg of N/100g). Food additives also inhibited bacterial growth, with mesophilic bacteria count below 3.35 Log CFU/g after 28 days, compared with 5.37 Log CFU/g in control samples. This study provides valuable insights for developing optimal cooking and preservation methods for sous vide cooked seafood, underscoring the need for further research on optimal cooking and freeze–thaw protocols for various seafood types. Full article

This article presents data from experiments to determine the cryoresistance of Charollais sheep embryos, depending on the stage of embryo development and the method of freezing, as well as the results of embryo transfer. The study design consisted of a study on the cryopreservation of ewe embryos at different developmental stages (early, 2–8 blastomeric and late, at the morula/blastocyst stage), two cryopreservation protocols (slow freezing and ultra-fast vitrification), and embryo transfer of cryo- and fresh embryos. Embryos from Charollais sheep donors (n = 12) were recovered after induction of superovulation. The embryos were recovered surgically (laparotomy) on days 2 and 6 after insemination. Before there was transfer to recipients, part of embryos was cryopreserved using standard slow freezing and ultra-fast vitrification methods. The average ovarian response was 7.54 ovulations per donor, and 5.83 embryos per donor were collected. No effect of the cryopreservation method and embryo development stage on the preservation of the morphological structure of embryos was found. There were no significant differences in the survival rate of cryoembryos at different development stages, frozen using different techniques, and after transfer to recipients. Differences in cryoresistance between embryonic developmental stages in favor of the morula/blastocyst stage were found (survival after thawing 86.4% vs. 75.0% in early embryos). At different stages of development, the survival rate of fresh embryos (45.8%) compared to cryopreserved ones (30.2%) was significantly higher (p < 0.05), while among fresh ones, the best survival rate (50.0%) was observed after the transfer of morules and blastocysts. Full article

Unfiltered olive oils (UO) have gained popularity in the global market, but they lose their quality characteristics faster than filtered oils (FO). In this work, refrigeration and freezing temperatures were explored to maintain UO quality features during storage. A full factorial design was applied to an UO and to the same oil after filtration to evaluate the effect of storage temperature (room temperature, 4 °C and –20 °C) and freezing speed (slow-freezing, in the freezer at −20 °C and fast-freezing, in a bath of liquid nitrogen). Official quality parameters, polar and nonpolar phenolic compounds, oxidative stability index, volatile compounds and descriptive sensory profile were measured periodically over 24 months of storage in the dark. Storage temperature influenced the quality of both UO and FO, but in different ways. At non-freezing temperature, UO experienced a severe decrease in its sensory quality compared to FO, mainly due to the hydrolysis of secoiridoids and degradation of the volatile fraction, but storage at −20 °C allowed to effectively preserve UO quality traits, thus resulting as a suitable strategy to increase the shelf-life of UO to satisfy the demand of consumers for this particular product. The results showed that slow-freezing was the most appropriate method for freezing. Full article

Sodium alginate (SA)-based hydrogels are often employed as bioink for three-dimensional (3D) scaffold bioprinting. They offer a suitable environment for cell proliferation and differentiation during tissue regeneration and also control the release of growth factors and mesenchymal stem cell secretome, which is useful for scaffold biointegration. However, such hydrogels show poor mechanical properties, fast-release kinetics, and low biological performance, hampering their successful clinical application. In this work, silk fibroin (SF), a protein with excellent biomechanical properties frequently used for controlled drug release, was blended with SA to obtain improved bioink and scaffold properties. Firstly, we produced a printable SA solution containing SF capable of the conformational change from Silk I (random coil) to Silk II (β-sheet): this transition is a fundamental condition to improve the scaffold’s mechanical properties. Then, the SA-SF blends’ printability and shape fidelity were demonstrated, and mechanical characterization of the printed hydrogels was performed: SF significantly increased compressive elastic modulus, while no influence on tensile response was detected. Finally, the release profile of Lyosecretome—a freeze-dried formulation of MSC-secretome containing extracellular vesicles (EV)—from scaffolds was determined: SF not only dramatically slowed the EV release rate, but also modified the kinetics and mechanism release with respect to the baseline of SA hydrogel. Overall, these results lay the foundation for the development of SA-SF bioinks with modulable mechanical and EV-release properties, and their application in 3D scaffold printing. Full article

A two-step freezing cryoprotocol preceded by desiccation to 15 to 25% moisture content was developed and successfully applied to winter dormant buds of mulberry (different Morus spp.) of a core set comprising 238 accessions studies in our laboratory. The survival and recovery percentage of diverse accessions cryobanked for various periods were tested under in vitro conditions, and several factors were analyzed to determine their role in optimizing the recovery of low-viability accessions. The effect of rates of freezing and thawing (both fast and slow), were tested and recovery compared. Recovery conditions such as dark incubation and rehydration in sterile moist moss grass for different durations after cryopreservation led to a higher survival percentage compared to controls. Two different recovery culture media were compared for their efficiency in survival. On average, the survival under in vitro culture conditions using optimized conditions was high: above 60% in majority of the accessions. Dormant buds showed viability in the range of 25 to 100% with an average of 50.4%. The recovery percentage of winter dormant buds after cryopreservation via slow freezing and slow thawing with rehydration by moist moss grass for 2 h was recorded in the range from 63.3 to 90.9% with an average of 81.05%. Without rehydration, it ranged from 50 to 75% with an average of 60.4%. Regeneration of cryopreserved mulberry germplasm after 6 years of storage indicated no survival loss over different years of storage, and 33–40% of the accessions showed viability above 40%, up to a maximum of 100%. Maximum shoot formation (100%) was obtained from Morus alba. The majority of the accessions were rooted in vitro within 20–25 days of subculture in the auxin rich rooting media, except in wild species M. latifolia and M. laevigata, which took longer (45 to 60 days) for root development. All the rooted plantlets were then transferred to the field and successfully established in a glasshouse. Full article

Premium extra virgin olive oils (PEVOO) are oils of exceptional quality and retail at high prices. The green color of recently extracted olive oils is lost during storage at room temperature, mainly because of the pheophytinization of chlorophylls. Since a green color is perceived as a mark of high-quality oils by consumers, it is especially important for PEVOO to maintain their initial green color. This study assessed the effect of applying low temperatures (refrigeration and freezing) and modified atmospheres on the color of four PEVOO for 24 months. Also, the effect of two freezing methods (slow freezing by placing the oil at −20 °C and fast freezing by immersing the oil in a bath of liquid nitrogen) was studied. Results showed that the green color was better preserved in oils frozen and stored at −20 °C whereas in oils frozen with liquid nitrogen the green color was lost much faster during frozen storage. An in-depth study of this unexpected phenomenon showed that this loss of green color was mainly due to a pheophytinization of chlorophylls. This phenomenon did not happen at the moment of freezing with liquid nitrogen, but over the first 100 days of storage at −20 °C. In addition, correlations between single chlorophyll and pheophytin contents and chromatic coordinates were established. Full article

The aim of this study was to investigate the effect of application of pulsed electric field (PEF) and different freezing methods (fast, slow and vacuum freezing) on the drying kinetics as well as selected physical properties of freeze-dried apple. The apples were subjected to PEF treatment with range of pulses from 0 to 160 and the intake energy from 0 to 1327 kJ·g⁻¹. Apples with and without PEF treatment were frozen with different rates and the freeze-dried. The water content, water activity and colour attributes of freeze-dried apples were investigated. Regression analysis and fitting procedures showed that among six different models, the Midilli et al. model the best described the drying curves of all dried samples. The highest value of the parameter L* = 71.54 was obtained for freeze-dried sample prepared without PEF pre-treatment and fast frozen. Application of PEF pre-treatment resulted in increase in browning index of freeze-dried apples (BI). The studies confirmed the positive effect of PEF on the freeze drying rate only in the case of the slow or fast freezing of the material after the application of low-energy PEF treatment. However, the increase in drying rate was also observed after application of slow and vacuum freezing of the material without PEF pre-treatment. These technologies can be recommended for optimization of the freeze drying process of apples. The statement that the freeze drying process with application of appropriately selected PEF processing parameters causing only partial destruction of cell membranes can be considered as an innovative contribution to the development of science about the possibilities of PEF application. Full article

Thermal and non-thermal technologies used in food processing should be not only effective in terms of decontamination and preservation but also minimize undesirable losses of natural bioactive compounds. Arabica (Coffea arabica) is the most cultivated variety of coffee, making it a valuable source of phytonutrients, including antioxidants. In the present study, green and roasted Arabica coffee beans were treated with slow freezing (SF), fast freezing (FF), microwave radiation (MWR) and cold atmospheric plasma (CAP). Moisture content (MC) of coffee beans and antioxidant activity (AOA) of aqueous extracts were measured. Green coffee showed a decrease in MC after MWR treatment, and roasted coffee showed an increase in MC after freezing. After SF and FF at −19 °C for 24 h, all extract samples showed an increase in AOA by 4.1–17.2%. MWR treatment at 800 W for 60 s was accompanied by an increase in the AOA of green coffee extracts by 5.7%, while the changes in the AOA of roasted coffee extracts were insignificant. Sequential combined treatments of SF + MWR and FF + MWR resulted in an additive/synergistic increase in the AOA of green/roasted coffee extracts, up to +23.0%. After CAP treatment with dielectric barrier discharge (DBD) parameters of 1 μs, 15 kV and 200 Hz for 5 and 15 min, green coffee showed a decrease in the extract AOA by 3.8% and 9.7%, respectively, while the changes in the AOA of roasted coffee extracts were insignificant. A high positive correlation (r = 0.89, p < 0.001) between AOA and MC was revealed. The results obtained indicate that SF, FF, MWR and combined treatments may be applied at the pre-extraction stage of coffee bean preparation in order to increase the yield of antioxidant extractives. Full article

Gelatine is a well-known and extensively studied biopolymer, widely used in recent decades to create biomaterials in many different ways, exploiting its molecular resemblance with collagen, the main constituent of the extra-cellular matrix, from which it is derived. Many have employed this biopolymer in tissue engineering and chemically modified (e.g., gelatin methacryloyl) or blended it with other polymers (e.g., alginate) to modulate or increase its performances and printability. Nevertheless, little is reported about its use as a stand-alone material. Moreover, despite the fact that multiple works have been reported on the realization of mould-casted and three-dimensional printed scaffolds in tissue engineering, a clear comparison among these two shaping processes, towards a comparable workflow starting from the same material, has never been published. Herein, we report the use of gelatine as stand-alone material, not modified, blended, or admixed to be processed or crosslinked, for the realization of suitable scaffolds for tissue engineering, towards the two previously mentioned shaping processes. To make the comparison reliable, the same pre-process (e.g., the gelatin solution preparation) and post-process (e.g., freeze-drying and crosslinking) steps were applied. In this study, gelatine solution was firstly rheologically characterized to find a formulation suitable for being processed with both the shaping processes selected. The realized scaffolds were then morphologically, phisico-chemically, mechanically, and biologically characterized to determine and compare their performances. Despite the fact that the same starting material was employed, as well as the same pre- and post-process steps, the two groups resulted, for most aspects, in diametrically opposed characteristics. The mould-casted scaffolds that resulted were characterized by small, little-interconnected, and random porosity, high resistance to compression and slow cell colonization, while the three-dimensional printed scaffolds displayed big, well-interconnected, and geometrically defined porosity, high elasticity and recover ability after compression, as well as fast and deep cell colonization. Full article

The increased interest in breeding dogs and cats and their use as models for other canids and felids demand research to improve reproductive techniques. Among them, testicular cryopreservation stands out. Testicular cryopreservation enables the maintenance of reproductive capacity and allows the establishment of germplasm banks for several species of commercial value or at risk of extinction. Furthermore, it enables the transport of genetic material among different regions. It is noteworthy that this biotechnology represents the only possibility of preserving the fertility of prepubertal animals that have died, so it has great importance in the propagation of the genetic material of animals. The spermatogonia present in the testes can be cultivated in vitro and the sperm obtained can be used in artificial reproduction programs. Although advances have been achieved with the use of testicular fragments to obtain viable and functional germ cells, the establishment of protocols that can be used in clinical routine have not been concluded yet. The testicular cryopreservation process can be carried out through techniques such as slow freezing, fast freezing and vitrification. However, the protocols used for the canine and feline species are still in the experimental phase. Given the importance of the topic, the aim of this review is to draw a profile of the subject approaching the main works on testicular cryopreservation in dogs and cats. Full article

Spin freeze-drying, as a part of a continuous freeze-drying technology, is associated with a much higher drying rate and a higher level of process control in comparison with batch freeze-drying. However, the impact of the spin freezing rate on the dried product layer characteristics is not well understood at present. This research focuses on the relation between spin-freezing and pore size, pore shape, dried product mass transfer resistance and solid state of the dried product layer. This was thoroughly investigated via high-resolution X-ray micro-computed tomography (µCT), scanning electron microscopy (SEM), thermal imaging and solid state X-ray diffraction (XRD). It was concluded that slow spin-freezing rates resulted in the formation of highly tortuous structures with a high dried-product mass-transfer resistance, while fast spin-freezing rates resulted in lamellar structures with a low tortuosity and low dried-product mass-transfer resistance. Full article