Peripheral Nerve Injury (PNI) has a multifactorial etiology and results in functional impairment in both motor and sensory behavior, which can be reversed by appropriate treatment strategies. As PNI results in an inflammatory response, conservative modalities with antioxidant potential, such as cryotherapy, are promising tools to be studied. The aim of this study was to analyze the effects of cold water immersion on functional, oxidative, and morphological changes after a PNI model in rats. PNI was surgically induced by compression of the sciatic nerve, and cryotherapy started the day after the injury and was applied daily for 15 days. PNI caused a significant impairment in motor behavior that was accompanied by microscopic damage to the structure of the sciatic nerve. The observed functional and morphological changes were accompanied by a significant oxidative damage in the skeletal muscle innervated by the injured sciatic nerve. The effects of cryotherapy in reducing motor impairment were corroborated by the modulation of morphological damage to the sciatic nerve and also by the reduction of oxidative damage in innervated areas. Taken together, the results presented suggest that cryotherapy may be a potential treatment resource after a PNI; however, more studies are needed to clarify the mechanisms underlying the origin of its therapeutic effects.

Glycerol is commonly used as a cryoprotectant in bovine sperm cryopreservation; however, its cytotoxic effects can negatively impact sperm quality. This study aimed to reduce glycerol concentration in semen extenders by supplementing with natural antioxidants (taxifolin and fetuin) and the disaccharide trehalose. Twelve ejaculates were collected from three bulls and initially evaluated, then diluted in a tris-egg yolk (TEY) extender containing either 7 % or 4 % glycerol. The control group received 7 % glycerol (TEY), while experimental groups included G7F (TEY + fetuin), G7T (TEY + taxifolin), G7M (TEY + fetuin + taxifolin), and 4 % glycerol groups supplemented with 50 mM trehalose: G4 (TEY + trehalose), G4F (TEY + fetuin), G4T (TEY + taxifolin), and G4M (TEY + fetuin + taxifolin). Post-thaw evaluations included assessments of sperm motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), DNA fragmentation, and oxidative stress markers. The G4F and G4T groups exhibited significantly higher total and progressive motility, viability, plasma membrane integrity, and MMP compared to other groups (p < 0.05). Furthermore, these groups showed significantly lower malondialdehyde (MDA) levels, indicating reduced lipid peroxidation and oxidative stress (p < 0.05). In conclusion, co-supplementation of 50 mM trehalose with 50 μM taxifolin and 5 mg/mL fetuin in a cryopreservation extender containing 4 % glycerol may enhance the post-thaw quality of bull spermatozoa.

The Daurian ground squirrel (Spermophilus dauricus) is a mammalian hibernator that undergoes prolonged torpor bouts in winter, periodically interrupted by spontaneous arousals. However, whether the function of the Harderian gland (HG)-an orbital endocrine organ-and its associated glycogen and lipid metabolic activity vary across different physiological states of hibernation remains unclear. In this study, we conducted quantitative analyses of HG morphology and assessed glycogen and lipid metabolism in Daurian ground squirrels across distinct hibernation phases. Results showed that: (1) Compared with the pre-hibernation (PRE) period, HG glycogen content declined during torpor, while elevated glycogen synthase (GS) activity during inter-bout arousal (IBA) restored glycogen levels. (2) Triacylglycerols (TAG) and free fatty acid (FA) concentrations in the HG remained stable and higher than PRE levels throughout hibernation. Notably, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) activities peaked during torpor, while lipase (LPS) activity was highest during IBA. These findings suggest that lipid accumulation in the HG is regulated through a dynamic balance of synthesis and degradation, with lipid metabolism predominating as the main energy strategy during hibernation. In contrast, glycogen metabolism remains suppressed during torpor, primarily regulated by GS activity.

Due to the lack of specificity in diagnosing death from hypothermia through anatomical and pathological examinations, it is often necessary to rule out other causes of death, such as injury or disease. Thus, finding a method that can distinguish fatal hypothermia from deaths due to alternative causes poses a significant challenge in forensic practice. Cold-inducible RNA-binding protein (CIRBP), a cold shock protein, increases when the body temperature drops below 32 °C. Recently, an increasing number of researchers have focused on the role of CIRBP in response to cold stress. However, few studies have evaluated the value of CIRBP in the forensic identification of fatal hypothermia. To address this, we established a rat model of fatal hypothermia and examined the expression of CIRBP in the brain, which is considered a key organ for thermoregulation. Additionally, we employed catRAPID omics v2.1 and RNA sequencing to predict the potential targets of CIRBP. Finally, we modeled scenarios commonly observed in cases of death from hypothermia-diabetes, alcoholism, and starvation-to assess the limitations of CIRBP and its potential target ONECUT2 in the identification of fatal hypothermia. The results indicated that CIRBP and ONECUT2 in the prefrontal cortex (PFc) may be potential biomarkers for identifying fatal hypothermia. Moreover, ONECUT2 was found to be less influenced by individual conditions such as diabetes and alcoholism compared to CIRBP.

Cryopreservation is a technique used to store cells or tissues for extended periods of time. It plays a significant role in assisted reproductive technologies (ART). A primary challenge associated with cryopreservation is cellular cryodamage, which is caused by oxidative stress from the excessive production of reactive oxygen species (ROS). Astaxanthin (AST), a carotenoid with robust antioxidative properties, has been shown to have a positive function on cryopreserved sperm and oocytes when added to cryo-extender. In sperm, AST improves quality by increasing motility and viability. In oocytes, it enhances post-thaw survival rates and developmental competence. This study aims to review the effect of AST in the cryopreservation of reproductive cells and tissues, with an emphasis on its molecular mechanisms.

Our objectives were to investigate how permeating cryoprotectant agents (CPAs; dimethyl sulfoxide, methanol), non-permeating CPAs (trehalose at 100 and 300 mmol/L; lactose at 5 and 10 %), and freezing rates (-1.0, -5.0, -10, -20 °C/min) impact post-thaw sperm motility and velocity (VCL) in blue catfish (Ictalurus furcatus) using 2.5 mL straws. Sperm frozen with methanol at -20 °C/min had the highest kinematics (VCL = 141.8 ± 18.5 μm/s, motility = 30.5 ± 12.2 %), and showed similar results to fresh sperm (VCL = 162.7 ± 5.4 μm/s, motility = 50.2 ± 15.1 %). Adding non-permeating CPAs did not improve VCL, and sperm motility was inhibited by lactose 5-10 % and trehalose 300 mmol/L. However, trehalose 100 mmol/L increased motility by 41.2 %, relative to straws without the addition of non-permeating CPAs. Development of this new freezing technology has merit for selective breeding, large research projects, and commercial-scale production for an economically important farm animal.

In this Anthropocene era, we are witnessing a rapid loss of biodiversity. To date, nearly 25% of the approximately 6,000 vertebrate species assessed by the International Union for Conservation of Nature are threatened with extinction. While the preservation of natural habitats remains crucial, the management of wildlife populations under human care has also played a critical role in the recovery of several threatened and endangered species. Because ex situ wildlife populations are often small and geographically isolated, careful and strategic genetic management is of utmost importance. The ability to store reproductive cells and tissues long-term-either in liquid nitrogen or as dry material at room temperature-is extremely valuable. It enables the infusion of genetic material into living populations, extends generation intervals, and helps preserve genetic diversity. Furthermore, these banked materials serve as a form of insurance for future species restoration programs. Consequently, efforts have been made to develop cryopreservation protocols for sperm, eggs, embryos, and gonadal tissues in wildlife species. Despite several achievements and milestones-particularly in sperm cryopreservation-significant challenges remain. In this review, we examine the status of cryopreservation protocols for gametes, embryos, and gonadal tissues across various wildlife taxa, highlighting recent successes and ongoing obstacles to advancing these efforts. Additionally, we explore emerging technologies that enhance cell survival and facilitate the integration of genome resource banking into species conservation programs.

Sperm ultra-rapid freezing techniques cryopreserve selected spermatozoa without seminal plasma using non-permeable cryoprotectants. Existing methods are limited to cryopreserving low sperm volumes. This study aims to enhance the volume of ultra-rapid frozen sperm by developing a new carrier, the Sperm Freezing Catheter (SFC), and by concentrating sperm through pellet cryopreservation. Twenty-five normal semen samples, prepared using the swim-up method, were divided into five groups:1. Fresh 2. Sperm suspension cryopreserved with the Cryotop Vial Device (CVD(S)) 3. Sperm suspension cryopreserved with the SFC (SFC(S)) 4. Sperm pellet cryopreserved with the CVD (CVD(P)) 5. Sperm pellet cryopreserved with the SFC (SFC(P)) with ultra-rapid freezing method. Sperm parameters, DNA fragmentation index (DFI), acrosome reaction (AR), and mitochondrial membrane potential (MMP) were evaluated across all groups. Progressive motility (83.96 ± 5.012), viability (88.92 ± 4.84), and normal morphology (7.000 ± 2.363) were significantly reduced in cryopreserved groups compared to fresh samples (P < 0.0001). Between carriers, progressive motility was higher in CVD(S) compared to SFC(S) (67.52 ± 3.44 vs. 60.00 ± 6.564, P = 0.001), but no significant differences were observed between CVD(P) and SFC(P) (62.68 ± 7.052 vs. 57.72 ± 9.026, P = 0.055). Viability, morphology, DFI, AR, and MMP were similar between CVD and SFC in both suspension and pellet groups. In conclusion, ultra-rapid freezing of concentrated spermatozoa using the SFC allows for cryopreservation of a larger sperm count in a limited volume, presenting a practical option for fertility preservation with outcomes comparable to conventional methods.

This study examined morphological changes in preantral follicles, stromal cells, and the extracellular matrix of cattle ovarian tissue vitrified in medium supplemented with Croton argyrophyllus Kunth essential oil (CaEO). Ovarian cortical slices were cryopreserved using solid surface vitrification in α-MEM alone (control) or supplemented with 0.01, 0.1, 1.0, or 10.0 μg/mL CaEO. After thawing, the ovarian fragments were cultured for 24 h. Then, follicular morphology, stromal cells, collagen distribution, thiol levels, and the activity of enzymatic antioxidants catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) were analyzed. Histological analysis revealed an increased rate of healthy follicles in the ovarian tissues vitrified in medium supplemented with CaEO in comparison to the control group. However, no significant differences were found in the rate of primordial follicle activation (P < 0.05). Collagen fibers were more abundant in fragments vitrified with 0.1, 1, or 10 μg/mL CaEO than in those stored with 0.01 μg/mL CaEO or in the control medium (P < 0.05). A higher thiol content was observed in tissues cryopreserved with 1 or 10 μg/mL CaEO than in the control group (P < 0.05). Catalase activity was greater in samples stored in the presence of 0.01 or 0.1 μg/mL CaEO than in tissue cryopreserved with 10 μg/mL CaEO (P < 0.05). In conclusion, CaEO enhances the efficiency of cryopreservation by preserving follicular health, increasing collagen fibers, and regulating antioxidant enzyme activity.

Interrupted cooling protocols are effective techniques used in cryobiology to study how cells respond to the freeze/thaw processes. By interrupting either rapid or slow cooling processes within specific sub-zero temperature ranges, researchers can improve the viability of cells after cryopreservation. These methods also provide insights into the differences between slow and rapid cooling injuries. Interrupted cooling protocols help researchers optimize critical factors affecting post-thaw viability and function, such as cooling profiles, cryoprotectants, and plunge temperatures. Over more than 60 years, numerous studies have used interrupted cooling protocols to understand the nature of damage during cooling and warming, identify the sub-zero temperature ranges where most damage occurs, and select appropriate types and concentrations of cryoprotectants for specific cell types. This paper begins with a review of crucial cryobiology concepts that are important to understand interrupted cooling, such as slow cooling and rapid cooling injuries, different types of cryoprotectants and how each type protects cells during cooling and warming, and the damage caused by cryoprotectants. This paper continues with providing a comprehensive review of studies that used different interrupted cooling protocols and to highlight their important findings for a broad range of cell types.

Cryopreservation is essential for long-term plant germplasm preservation, yet the mechanisms underlying cellular responses during the critical dehydration phase remain poorly understood. In this study, callus tissues of 'Light Yellow' Petunia × Calibrachoa were used to compare the cytological, physiological, biochemical, and molecular effects of one-step (T1) and two-step (T2-1, T2-2) osmo-protection. The one-step method (T1) directly exposed tissues to MS medium with 2 M glycerol and 0.35 M sucrose for 30 min, while the two-step strategy involved 15 min in glycerol alone (T2-1) followed by 15 min in glycerol-sucrose (T2-2). The two-step method markedly improved cell viability, reaching 90.15 % after 30 min, compared with 12.32 % in T1. LF-NMR revealed that two-step treatment reduced total water content (71.35 %) and free water proportion (52.6 %), while increasing bound water (7.26 %), accompanied by upregulation of aquaporin genes (TIP, PIP), indicating enhanced water regulation. After 30 min, cell shrinkage reached 69.65 % in T1 but only 49.59 % in the two-step treatment, suggesting a more moderate dehydration rate. Transmission electron microscope (TEM) confirmed that two-step treatment could protect the integrity of cell membranes, vacuoles, and organelles, while T1 caused plasmolysis, membrane collapse, and mitochondrial damage. Biochemical assays showed reduced malondialdehyde (MDA) and conductivity in two-step treatments, alleviating lipid peroxidation and permeability change. Analysis of cell wall by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS) revealed that T2-2 treatments not only promoted accumulation of hemicellulose (54.52 mg/g) and pectin (303.82 μmol/g) for enhancing wall strength, but also facilitated the accumulation of osmoprotective sugars, such as glucose (171.42 μg/mL), fructose (25 μg/mL), and xylose (13.76 μg/mL). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results further elucidated alongside upregulation expression of sugar metabolism-related the genes (INV, HK). Antioxidant enzymes (CAT, POD) and wall remodeling genes (XTH, PMEI) were also regulated during two-step treatment, suggesting multiple pathways mediate osmotic stress responses. In conclusion, the two-step osmo-protection effectively mitigates dehydration stress, preserves viability and structural integrity, and activates molecular defense mechanisms, demonstrating clear advantages for cryopreservation of high-moisture, freeze-sensitive plant materials.

This meta-analysis evaluated and compared the efficacy and safety of cryoablation and thermal ablation in the treatment of lung cancer. A comprehensive search of relevant literature was conducted in databases including PubMed, Web of Science, Embase, The Cochrane Library, CNKI, Wanfang, and VIP, covering the period from the establishment of each database up to April 1, 2025. Studies were selected based on predefined inclusion and exclusion criteria. The meta-analysis was performed using Review Manager 5.3 software. Ultimately, 12 retrospective studies involving a total of 907 patients were included, with 422 patients undergoing cryoablation and 485 receiving thermal ablation. The results indicated that cryoablation was associated with a longer operative duration (p < 0.00001), but demonstrated superior intraoperative pain control (p < 0.00001) and a lower postoperative fever rate (p = 0.01). With regard to short-term and long-term therapeutic outcomes, as well as certain complications, no statistically significant differences were observed between the two modalities. In summary, both ablation techniques exhibited comparable efficacy in the treatment of lung cancer, with cryoablation offering improved intraoperative comfort and a reduced incidence of postoperative fever compared to thermal ablation.

Cryopreservation is a common assisted reproductive technology in goat breeding, but it often impairs sperm quality and fertilizing ability. This study aimed to explore the effects of cryopreservation on sperm quality and protein abundance profiles in Nubian goat spermatozoa using TMT-based quantitative proteomics. Semen from six healthy adult bucks was divided into fresh and frozen-thawed groups. Key sperm quality parameters, including total motility, membrane integrity, acrosomal integrity, and abnormality rates, were evaluated. TMT-labeled proteomic analysis was performed, followed by GO, KEGG, and PPI network analyses. Results showed that cryopreservation significantly reduced total motility, membrane and acrosomal integrity, while increasing abnormality rates (P < 0.0001). A total of 204 differentially abundant proteins (DAPs) were identified, including 108 upregulated and 96 downregulated proteins. Functional enrichment revealed that these DAPs were mainly involved in glycolysis, cytoskeleton organization, lysosomal function, and oxidative stress. PPI analysis identified GAPDHS, ODF2, and ALB as key proteins potentially regulating sperm cryoinjury. This study provides new insights into the molecular mechanisms of sperm cryodamage in goats and suggests candidate biomarkers for improving cryopreservation strategies.

Many terrestrial hibernating amphibians accumulate low-molecular cryoprotectants to endure winter cold, while some overwinter in warmer water. However, high-altitude Rana kukunoris overwintering in water face near-zero, ice-covered conditions, with their physiological adaptations-particularly cold tolerance and osmotic/ion regulation-remaining unclear. We simulated this state in the lab, comparing underwater-hibernating (WH) and non-hibernating (NH) frogs. After 8 weeks of hibernation, WH frogs showed elevated plasma urea (2.67 × ), glycerol (7.01 × ), and glucose (1.23 × ), with upregulated freeze-responsive protein genes (Fr10, Li16) in liver, muscle, heart, and brain. WH frogs survived air-freezing better than NH frogs but perished when frozen in water. They also had higher body water content (86.38 % vs. 78.82 %) and increased aquaporin (AQP1, AQP3) expression in kidneys and skin, promoting water retention. Kidney histology revealed enlarged renal tubules and reduced urine concentration efficiency. To counter osmotic imbalance, WH frogs maintained higher plasma osmolality (239.5 vs. 163.17 mOsm·kg) and excreted more dilute urine (20.75 vs. 39.83 mOsm·kg). These results reveal a trade-off: low temperatures drive R. kukunoris to accumulate cryoprotectants to enhance cold tolerance, but this disrupts osmotic balance in hypotonic conditions. This study provides the first integrative evidence that R. kukunoris adopts a trade-off strategy between cryoprotection and osmotic regulation during underwater hibernation, a scenario distinct from terrestrial freeze-tolerant species. With stable inorganic ions, osmotic shifts likely stem from cryoprotectant accumulation, making water regulation (e.g., hypotonic urine excretion) vital. Elevated body water suggests that prolonged stress may overwhelm osmotic regulation, while stark survival differences between air- and water-freezing underscore hibernation site selection's role in overwintering success.

Oxidative stress plays a crucial role in cellular damage during nerve graft storage, impairing optimal regeneration after transplantation. This study evaluated the effects of the association of vitamins C (Vit C) and polyethylene glycol encapsulated vitamin E (PEG-Vit E) on histological changes and oxidative stress markers during nerve preservation under hypothermic conditions (4 °C) in vitro. Sciatic nerve segments extracted from male red chickens (Gallus domesticus) were stored in either a saline solution (0.9 % NaCl) or a saline solution supplemented with combined doses of Vit C (0.1 mg/mL) and PEG-Vit E (2.5 mg/mL). The segments were divided into subgroups based on preservation duration: 0 days, 1 day, 2 days, 3 days, 5 days, and 7 days. Malondialdehyde (MDA) levels, as an indicator of lipid peroxidation, were measured in stored tissues using the TBARS assay. Total antioxidant status (TAS), which reflects total effect of all antioxidants, was assessed in tissue samples and vitamin-supplemented storage solutions using the ABTS assay. Structural changes were evaluated by histological analysis, and the percentage of degenerated axons was determined by counting morphologically altered fibers on stained transverse nerve sections. The results indicated that the Vit C/PEG-Vit E complex offered the most protective effect on days 2 and 3. On day 3, MDA levels decreased significantly in the treated group (0.29 ± 0.02 vs. 1.42 ± 0.15 nmol/mL; p < 0.001), and axonal degeneration was reduced to (24.97 ± 0.42 % vs. 66.40 ± 2.26 %; p < 0.001). On day 2, TAS was higher in the treated tissues (66.59 % ± 0.32 % vs. 52.65 % ± 4.91 %; p < 0.01). From day 5, the TAS of the solution declined by 26.53 % (p < 0.0001), along with its protective effects. In conclusion, supplementing the preservation medium with Vit C and PEG-Vit E may improve the conservation of nerve segments and prevent axonal degeneration.

The purpose of this study was to investigate the freeze-drying protective effect of cold stress and arginine on Enterococcus faecalis L118 and to develop a new freeze-drying protection system in combination with glycerol and skim milk. The freeze-drying protection effect of cold stress and arginine on E. faecalis L118 was evaluated using one-way ANOVA and response surface methodology (RSM), and the freeze-drying protection system was optimized. Subsequently, the protective mechanism and efficacy of the optimized system were further assessed via cell membrane integrity analysis, storage stability tests, probiotic property assays, and RT-qPCR. In this study, cold stress on E. faecalis L118 before freeze-drying and arginine as a cryoprotectant significantly enhanced its survival rate. The optimal protection system was optimized via RSM to consist of cold stress at 8 °C for 8 h, 1.5 % arginine, 10.4 % skim milk, and 9.8 % glycerol, and the survival rate of E. faecalis L118 achieved under this system was 87.90 ± 1.28 %. The E. faecalis L118 treated with the optimal protection system had relatively high storage stability at -20 °C and could maintain the cell membrane integrity and probiotic properties of E. faecalis L118. RT-qPCR results indicated that cold stress significantly upregulated (P < 0.05) the expression of atpA, plsX, luxS, and SOD genes, while arginine upregulated (P < 0.05) atpA, cspC, LDH1, FabH, FabF, plsX, and mprF2 genes. All genes in the optimal protection system were upregulated (P < 0.05). Our study demonstrated the protective effects of cold stress and arginine on E. faecalis L118 during freeze-drying and established a novel freeze-drying protective system, providing a theoretical basis for the preservation and industrial-scale production of E. faecalis L118.

The marine annelid Platynereis dumerilii is a functional molecular model organism for developmental, evolutionary and chronobiological studies. Research on Platynereis is rapidly growing, and with it, the number of genetic variants that laboratories isolate or generate and must subsequently maintain and propagate. Therefore, there is an urgent need to alleviate the burden of live culture maintenance by developing cryopreservation techniques for this species. We report the first cryopreservation protocol for P. dumerilii larvae, which combined with a careful post-thawing culturing regime, allowed us to obtain animals that survived to adulthood and successfully reproduced. Our experiments show highest survival rate in 6-8 dayold larvae (dpf). Equilibration with cryoprotecting agents takes 1h in 5 % (v/v) Me2SO + 0.1 %(v/w) sucrose, followed by transfer to 0.25 ml straws. The protocol cools larvae at 2.5 °C/min from 20 °C to -35 °C using a programmable freezer, followed by a rapid transfer to liquid N. Larvae are thawed in a water bath at 18 °C. The post-thaw larvae feeding regime consisted of 50 % Tetraselmis + 50 % diatom strains mixture (Grammatophora marina and Nitzschia laevis). The maximum survival obtained with this protocol so far produced 34 % survival after ∼5 months, the average is 8.69 ± 13.08 % (140 days) with a large variability inter-individual batches.

Inkjet-based superflash freezing (SFF) enables vitrification without cryoprotective agents (CPAs) by miniaturizing droplets. However, the insufficient cooling and warming rates of large droplets have restricted the applicable volume of SFF to 70 pL or less. The large thermal mass of the substrate in SFF has been a major factor limiting the thawing process. In this study, an impact-induced droplet release (IIDR) thawing method was developed, in which frozen droplets are mechanically separated from the substrate and directly thawed in prewarmed medium. This approach also improves cooling efficiency by direct freezing on an aluminum transporter with high thermal conductivity. Numerical analyses demonstrated that the IIDR thawing method improved both cooling and warming rates by approximately threefold for 200 pL droplets compared with the substrate-based method. Consequently, high cell viability of over 80 % was achieved in 200 pL droplets using NIH 3T3 fibroblasts (approximately 1600 droplets, corresponding to about 320 cells), extending the applicability of CPA-free SFF cryopreservation from the previous limit of 70 pL-200 pL. By contrast, in 70 pL droplets, viability decreased because the droplets remained at the medium interface, preventing sufficient warming, thereby indicating the need for further optimization of the thawing process. From the relationship with viability, the critical cooling and warming rates for cells were estimated to be approximately 1.5 × 10 °C/s and 3 × 10 °C/s, respectively. Overall, the IIDR thawing method provides a reproducible platform that overcomes key limitations of SFF and represents a promising basis for CPA-free inkjet-based cryopreservation.

Current hypothermic preservation solutions are insufficient to prevent donor organ injury during hypothermic storage, which negatively impacts graft functional recovery and survival after transplantation. This study was to evaluate whether supplementation of the preservation solutions with antioxidant propyl gallates (PG) reduced cell injury during hypothermic storage and subsequently rewarming-reoxygenation or transplantation. Human cells and rat aorta were stored in University of Wisconsin (UW) or hyperbranched polyglycerol (HPG) solution supplemented with PG at 4 °C, followed by rewarming-reoxygenation with atmospheric O at 37 °C and isotransplantation, respectively. Cell or tissue injury was measured by lactate dehydrogenase release, flow cytometric and histologic analyses. Here, we showed that PG supplementation significantly increased antioxidative activities of both UW and HPG solutions, and its concentrations for maximum protection of different human cells from hypothermic storage-induced cell death were estimated between 25 and 50 μM, which was also indicated by increased cell survival or decreased cell apoptosis during rewarming-reoxygenation. The PG cytoprotection was correlated with its inhibition of lipid peroxidation, prevention of mitochondrial dysfunction or an increase in ATP content. The enhanced cytoprotection of preservation solutions by PG supplementation was confirmed in hypothermic storage of rat aortas, indicated by less tissue damage and higher levels of tissue ATP during the hypothermic storage and less chronic injury after transplantation. In conclusion, cell injury during hypothermic storage and subsequently rewarming-reoxygenation was substantially prevented by the hypothermic preservation with PG, suggesting that PG is a promising antioxidant for hypothermic preservation of donor cells or organs in transplantation.

The aim of this study was to assess the effects of incorporating either free rosmarinic acid (RA) or RA-loaded chitosan nanoparticles (RA-CHNPs) into a buffalo semen extender on various parameters, including semen quality, antioxidant capacity, expression of apoptotic genes, semen microbiota, ultra-structural alterations, and fertility outcomes of cryopreserved buffalo sperm. Semen samples were collected from five healthy, fertile Egyptian buffalo bulls (Bubalus bubalis), aged 5-6 years, using the artificial vagina technique. The ejaculates were cryopreserved in a Tris-based extender supplemented with 100 μg/mL of either RA or RA-CHNPs, alongside a control group without antioxidant supplementation. Supplementing the cryopreservation extender with 100 μg/mL RA-CHNPs significantly improved sperm viability, progressive motility, membrane integrity, and kinematic parameters. Additionally, it significantly enhanced the activity of major antioxidant enzymes and markedly reduced levels of hydrogen peroxide, malondialdehyde, nitric oxide, and semen microbiota counts compared to the control group (p < 0.05). Furthermore, RA-CHNPs treatment significantly modulated the expression of apoptosis-involved genes, with reduced expression of caspase-3 and Bax and increased Bcl-2 expression. Transmission electron microscopy confirmed that RA-CHNPs helped preserve the ultra-structural integrity of spermatozoa, particularly the acrosome and plasma membrane. Notably, pregnancy rates increased by 19 % in buffalo cows inseminated with RA-CHNPs-treated semen compared to those inseminated with supplemented semen (p = 0.0157). In conclusion, loading rosmarinic acid onto chitosan nanoparticles enhanced its stability, water solubility, and bioavailability, ultimately improving its effectiveness in the semen freezing extender. The addition of 100 μg of RA-CHNPs significantly improved post-thaw sperm quality.