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Over the past decades, cryobiology has become increasingly integrated across a variety of domains. With broad intersection, this discipline has recently also emerged as a cornerstone for advancing and clinically translating tissue-engineered constructs (TECs). This review provides a comprehensive overview of recent advances at the intersection of cryobiology and tissue engineering. Vitrification of both scaffold-based and scaffold-free TECs as well as approaches of its upscaling including benefits of polymers and technical devices are comprehensibly discussed. The development of biologically inspired nanoscale materials is outlined as an integral part of this research. Success in vitrification of organoids is also discussed. Developments in controlled-rate slow-cooling/freezing protocols are then examined, with particular attention to xeno-free and Me2SO-free cryoprotective systems that enhance cell viability and biocompatibility. Preclinical studies utilizing cryopreserved TECs in animal models are further outlined as key milestones toward clinical translation. Furthermore, this review introduces emerging synergistic approaches that make TECs more adaptable to cryopreservation by incorporating cryoprotective agents (CPAs), nanoparticles, cold-responsive polymers and ice recrystallization/devitrification inhibitors at the scaffold design stage. Finally, cryobioprinting as an emerging approach that unites cryobiology and tissue engineering, offering new opportunities for the fabrication, storage, and on-demand deployment of viable tissue constructs is reviewed. Overall, the overviewed experimental evidence underscores the transformative role of cryobiology in driving recent advances in the field of tissue engineering and fostering innovative and forward-looking strategies in this field. Ultimately, a closer convergence of cryobiology, tissue engineering, and transplantation science will be essential to advance TECs toward scalable, off-the-shelf availability.
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Cryopreservation of marine species is a key tool for biodiversity conservation, population management, and the advance of aquaculture and biological research. Currently exist significant progress in the cryopreservation of gametes, embryos, larvae and even juveniles of several genus of marine invertebrates. However, cnidarians, and particularly jellyfish, remain largely underexplored. In this study, we report on the first successful cryopreservation of Aurelia aurita ephyrae, the first larval stage of this jellyfish species. This species is characterized by extremely high-water content (96.3 %), which represents a unique challenge in the method of cryopreservation. In this study we done toxicity tests using different concentrations ranging from 0.5 to 6 M of dimethyl sulfoxide (Me2SO). On the other hand, we designed two cocktails of cryoprotectants and evaluated the efficiency in a protocol of cryopreservation. The first one is based on 1 M Me2SO + 0.5 M dimethylformamide (DMF) and the second used 1.5 M Me2SO with 0.04 M trehalose (TRE) and 1 % BSA as a post-thaw supplement. The first protocol achieved 100 % survival immediately after thawing and 33 % survival at 48 h, while the second maintained a consistent 33 % survival across both time points. The damage and viability of the larvae was assessed using live/dead fluorescence dye and allowed confirming the integrity of surviving individuals. These results mark the first demonstration of successful cryopreservation protocol for jellyfish larvae and represent a significant advance for the ex-situ conservation of gelatinous zooplankton. This work lays the foundation for further research into cryopreservation protocols for marine species, understand most better water flow for organisms with high water content and opens new possibilities for marine biodiversity conservation and cnidarian model systems.
Understanding cell volume changes in response to osmotic challenges is important both to fundamental cell physiology and to cryobiology where large cell volume excursions are caused by the formation of extracellular ice and the addition of osmotically active cryoprotectants. In this work, we report experimental measurements of human umbilical vein endothelial cell (HUVEC) volume response to hypertonic solutions of non-permeating solutes (either phosphate-buffered saline (PBS) or sucrose) using a Coulter Multisizer 4e Analyzer. Fitting to both ideal-solution, and non-ideal-solution thermodynamic models, we obtain the osmotic properties of HUVECs. We show that the osmotic properties are consistent when obtained from fitting experimental volume data for exposure to hypertonic solutions of 3.3× PBS, 5× PBS, or 0.6 M sucrose. However, the obtained osmotic parameters differ for 1.1 M sucrose with statistically higher membrane permeabilities than for other hypertonic solutions. We demonstrate that HUVECs that have been re-exposed to isotonic solutions after exposure to hypertonic solutions of PBS or sucrose recover their expected volume, confirming that HUVEC volume regulation is predominantly governed by osmotic mechanisms. We also demonstrate that in order to reach such a conclusion it is important to account for the effects of mixings of volumes of experimental and cell-carrying solutions as a confounding variable in interpreting cell volume responses.
Earth is undergoing accelerated loss of biodiversity. As wild animal populations decline, the resulting erosion of genetic diversity threatens species' adaptive potential and long-term survival. Wildlife biobanking, defined as the systematic collection, preservation, and use of biological materials and their associated metadata, has emerged as a critical conservation strategy. Importantly, building on decades of progress in cryobiology and reproductive biotechnologies, gonadal and somatic tissues and cells from thousands of wild species have been preserved worldwide. These biobanks serve as genetic insurance policies, supporting animal care and management, research, assisted reproduction, and eventual population recovery. This review discusses current strategies, challenges, and future opportunities in wildlife biobanking. Cryopreservation approaches for different sample types and taxa also are reviewed, emphasizing the challenges of sample availability and species-specific cryotolerance. The objective is also to integrate increasingly large amount of data associated with biological samples (digital biobanking) into conservation efforts. New frontiers in biobanking include innovative ambient-temperature preservation, precision biobanking informed by omics technologies, data interpretation using artificial intelligence, and establishment of global networks. These promising developments provide more opportunities to enhance and expand wildlife biobanking more quickly and broadly.
Haematopoietic stem cell transplantation (HSCT) offers curative potential for several malignant and non-malignant hematologic disorders. Despite its proven efficacy, access to HSCT in sub-Saharan Africa remains limited, especially in francophone countries, due to the lack of infrastructure, cryobiology facilities and trained personnel. Senegal recently launched a national initiative to establish its first HSCT program. We report the first autologous HSCT performed in Senegal in February 2025 at Dalal Jamm University Hospital, in a 51-year-old man diagnosed with high-risk IgA-lambda multiple myeloma (ISS stage III, del17p). Mobilisation was achieved using filgrastim (10 µg/kg/day for 7 days). A total of 2.8 × 10⁶ CD34⁺ cells/kg were collected by apheresis and stored at 4°C for 24 hours without cryopreservation. Conditioning consisted of high-dose intravenous melphalan (200 mg/m2) followed by reinfusion of the graft on day 0. Hematologic recovery occurred by day +10, with transient grade 3 anemia and grade 4 thrombocytopenia requiring transfusion support. The main complications were manageable febrile neutropenia and mild gastrointestinal and renal toxicities. The patient was discharged on day +17, remained infection-free and achieved complete hematologic and biochemical remission at five months post-transplant. Consolidation therapy with bortezomib-thalidomide-dexamethasone and lenalidomide maintenance was subsequently administered. This first non-cryopreserved autologous HSCT in Senegal demonstrates the feasibility, safety and cost-effectiveness of transplantation under resource-limited conditions. Establishing local cryopreservation and molecular diagnostic capabilities will be essential to enable tandem and allogeneic HSCT, ensuring sustainability and regional self-sufficiency in advanced hematologic care.
Hibernation is a specialized adaptive energy-saving survival strategy evolved by animals to withstand winter cold stress and food scarcity. Its core feature lies in profound metabolic suppression, characterized by a drastic reduction in metabolic rate during hibernation, accompanied by the coordinated downregulation of multiple physiological functions such as body temperature, heart rate, and respiratory rate. The establishment and maintenance of this deep metabolic suppression state essentially rely on the systemic reprogramming of energy metabolism, which serves as the core driving force of hibernation adaptation. During this reprogramming process, lipid metabolism acts as a key executive link: fats stored in adipose tissue not only function as the primary energy reserve pool during hibernation but also undergo precise regulatory remodeling in terms of their compositional characteristics, mobilization efficiency, and catabolic processes, thereby synchronously adapting to the demands of energy supply and environmental adaptation goals. Importantly, metabolic suppression often precedes cooling and can exceed Q10 predictions, indicating active regulatory control rather than passive thermal effects. Reliance on lipid oxidation and cyclic torpor-arousal transitions should heighten oxidative stress risk: electron leakage from mitochondrial complexes I/III during deep torpor, relative hypoxia from reduced perfusion, and rapid "metabolic restart" upon arousal may resemble ischemia-reperfusion. Yet hibernators show minimal oxidative damage, implying robust antioxidant and repair programs. This review summarizes recent advances in the metabolic remodeling of lipids, substrate conversion, and oxidative stress adaptation in hibernating animals. It reveals the evolutionary mechanisms underlying energy metabolism adaptation and provides potential insights for applications in metabolic diseases, cryobiology, and related fields.
Microfluidic devices offer precise control over solution mixing and gradient generation, essential for cell-based assays in cryobiology and biomedical research. However, traditional fabrication methods are time-consuming, costly, and require specialized expertise, which limits accessibility. To address these challenges, we developed a cost-effective, reliable, and fully 3D-printed microfluidic device workflow to facilitate rapid and inexpensive prototyping using a consumer-grade printer and biocompatible plastic resins. Here, we demonstrate this workflow with a fluidic mixing device capable of generating programmable concentration gradients and solution combinations. Commercial mixing devices cost more than $300 each and cannot be customized. By utilizing affordable resin materials and an innovative open-channel design sealed with transparent adhesive tape, we overcame common fabrication issues such as channel clogging, enabling rapid and reproducible fabrication of complex microfluidic architectures, all at a materials cost of less than $5. Here we demonstrate this workflow, integrating dual-syringe pumps to create linear osmotic gradients, ranging from iso-osmotic (~300 mOsm/kg) to hyperosmotic (~9,000 mOsm/kg) conditions, followed by a return to isotonicity over defined intervals. To ensure automation and reproducibility, we developed an open-source Python-based software tool that precisely regulates syringe pump activation, flow rates, and gradient timing. The device's performance was validated through continuous osmometric measurements, which confirmed both the linearity and accuracy of gradient generation, and colorimetric measurements to confirm mixing efficacy. This accessible and cost-effective microfluidic platform significantly improves the reproducibility of osmotic exposure studies and shows potential for various biomedical applications, including drug screening and precise chemical modulation.
Vitrification is enabling successful cryopreservation of progressively larger organs. Heating the whole volume of an organ simultaneously instead of only the surface during recovery from vitrification has been vital to this success. Volumetric warming enables faster and more uniform warming. Faster warming reduces ice crystal growth, ice recrystallization, and toxic effects of cryoprotectants by reducing ice growth time and cryoprotectant exposure time during warming. Nanowarming of intravascular magnetic nanoparticles by alternating magnetic fields, and direct dielectric warming by alternating electric fields, have both been used successfully for volumetric warming of vitrified organs. Dielectric warming predates vitrification, having been studied intermittently in cryobiology since the 1950s. Most early research was empirical, using microwaves at 915 MHz or 2.45 GHz because of wide availability of magnetron generators and microwave ovens. With greater theoretical understanding, interest later grew in frequencies near 400 MHz and lower. Larger energy absorption and smaller wavelengths inside tissue warming from vitrification instead of thawing from freezing made optimum frequencies for vitrification even lower, below 100 MHz for organs more than 10 cm in diameter. Recent dielectric warming research has used 27, 40, and 55 MHz, achieving heating rates of 200 - 700 °C/min. The advantages of dielectric warming not being dependent upon exogenous particles or vascular volume, high energy efficiency, and ability to monitor temperature by impedance behavior during warming, are counterbalanced by the need for organ immersion before vitrification and pre-warming to a uniform starting temperature above the glass transition temperature. With measurements of two electrical properties of a vitrification solution, permittivity and loss factor as function of temperature, detailed theoretical analysis and modeling of dielectric warming is possible. Comparatively little research has been done on dielectric warming at frequencies optimal for vitrification. This review covers the history, theory, equipment types, practical aspects, and future directions of dielectric warming of cryopreserved tissue.
Ice growth inhibition is crucial in cryotechnology, as uncontrolled recrystallization during the frozen state and freeze-thaw cycles causes irreversible damage to biological samples. Nanoscale materials that mimic antifreeze proteins and exhibit ice recrystallization inhibition (IRI) activity have been explored as cryoprotectants; however, the structural features that govern potent IRI activity remain unclear. This study investigated the effects of nanoparticle size and functionality on the IRI activity. Polystyrene nanoparticles (PSNPs, 30-1000 nm) were used as inert nanoscale models, and amino acid derivatives with phenyl groups with or without hydroxyl functionality, including L-phenylalanine monomers, pentamers of L-phenylalanine (Phe-5), L-tyrosine, and 3,4-dihydroxy-L-phenylalanine, were examined. Among these, we found that tyrosine monomer nanocrystals (TMNs) display exceptionally potent IRI activity under both extracellular and intracellular conditions, which is attributed to nanoscale structure formation, hydroxyl functionality, and high colloidal stability. TMNs enhance cell survival during cryopreservation, even at low dimethyl sulfoxide concentrations, whereas Phe-5 and other analogs show limited activity owing to aggregation or lack of hydroxyl groups. These results elucidate the key factors influencing IRI activity, including nanoscale assembly with high colloidal stability and the presence of a hydroxy functional group. Therefore, considering the biocompatibility of L-tyrosine, our study shows that TMNs are promising supplementary materials for cryobiology.
Sperm cryopreservation is pivotal for conserving fish germplasm, yet cryodamage-induced quality decline limits its application. This study focused on Sichuan bream (Sinibrama taeniatus), an endemic and economically important fish species in the upper Yangtze River. Based on an established cryopreservation protocol, we evaluated sperm quality using computer-assisted sperm analysis (CASA) and fertility assays, followed by a systematic assessment of structural and functional damage via flow cytometry (membrane integrity, mitochondrial potential, reactive oxygen species, and DNA fragmentation), enzymatic assays (energy metabolism and antioxidant enzymes), Western blotting, and ultrastructural observation. Finally, integrated proteomic and metabolomic analyses were employed to elucidate the underlying physiological mechanisms. The results demonstrated that freeze-thawing significantly impaired sperm motility, fertility, and ultrastructure, concurrently disrupting energy metabolism and the antioxidant system. Crucially, multi-omics revealed that these functional declines were linked to dysregulation in key pathways involving cytoskeleton organization, lipid metabolism, energy homeostasis, and oxidative stress, forming a coherent network from initial molecular perturbation to phenotypic dysfunction. This study provides a comprehensive characterization of sperm cryodamage in Sichuan bream, advancing the understanding of fish sperm cryobiology and informing targeted cryoprotection strategy development.
Partial freezing has been proposed in various forms since the 1990s as a high sub-0°C technique for prolonging the viable preservation window of organs relative to conventional hypothermic storage. However, despite the fundamental dependence of this technique on equilibrium thermodynamic behaviors, solution and protocol design have thus far remained largely empirical. The multisolute osmotic virial equation (MSOVE) has previously been used for quantitative protocol design in cryobiology, including the optimization of cryoprotectant addition and removal procedures and the prediction of osmotic responses in multi-solute systems. Here, we apply the MSOVE to model the freezing process of partial freezing solutions utilized in recent rat liver preservation protocols, computing melting points, liquidus curves, equilibrium ice fractions, and solute concentration trajectories down to -20 °C. The calculations provide simple mechanistic insights into observed differences in preservation quality between different pairings of composition and storage temperature, and suggest practical routes to optimize electrolyte content, polymer concentration, and cryoprotectant choice when rationally designing future partial freezing protocols.
An in-silico and experimental dual-screening lyophilization strategy was developed for long-term ambient storage of two model restriction endonucleases (REase) of XbaI (oxidation-resistant) and XhoI (oxidation-sensitive). After molecular docking and experimental screening of lyoprotectants and their combinations, a trehalose-povidone-arginine ternary lyoprotectant "combo" was finally developed. The lyophilized formulations were primarily evaluated by four critical quality attributes (CQAs), including lyocake appearance, reconstitution rate, relative enzymatic activity (REA) and short-term storage stability. Four ternary lyophilized formulation candidates were further evaluated by thermogravimetric analysis (TGA) for the residual moisture contents (<2%). Based on the results of four CQAs and TGA, the optimized formulation was finally determined, including trehalose (15%), arginine (5%) and povidone (5%). Differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) results indicated that REase lyocakes formed a co-amorphous glassy matrix. Scanning electron microscopy (SEM) images showed REase lyocakes with intact, loose and porous structure. Following six months of long-term stability testing under controlled conditions (25 °C, 60% RH), the lyophilized REase products still maintained comparable DNA cleavage efficiency to that of freshly prepared samples. This study presents a unique in-silico and experimental dual-screening strategy for developing cold-chain-free solid-state enzyme reagents.
This study assessed, in sheep, the effects of antifreeze protein (AFP) type I on redox balance at different embryonic stages by measuring mitochondrial activity (MITO), reactive oxygen species (ROS), and glutathione (GSH) levels in the same embryos immediately before cryopreservation and after warming. A total of 45 GI-GII embryos were divided into morulae (MO; n = 18) and blastocysts (BL; n = 27), and then, each group was subjected to slow freezing in a solution containing 0.1 μg/mL AFP I or not. In the MO-CONT group, cryopreservation reduced (P < 0.05) GSH levels and MITO, whereas ROS levels remained unchanged. In contrast, no differences (P > 0.05) were observed between pre- and post-cryopreservation in the MO-AFP group. Interestingly, the MO-AFP exhibited higher (P < 0.05) MITO than the MO-CONT group. In blastocysts, cryopreservation led to reductions (P < 0.05) in ROS, GSH levels, and MITO. In conclusion, AFP I supplementation preserved MITO in sheep morula embryos following cryopreservation, suggesting a stage-dependent protective effect.
Using rapid nanopore whole-genome sequencing, we assembled the genome of Bacillus anthracis strain ter21 (5,229,480 bp), a Tsiankovskii-I group isolate cultured from a fatal case in 2021 of a pony from a zoo in Ternopil, Ukraine, identifying virulence plasmid pXO1 that encodes anthrax toxin, and pXO2.
The efficacy of cryopreservation is fundamentally limited by the difficulty of achieving high intracellular concentrations of cryoprotective agents (CPAs) without inducing osmotic injury or chemical toxicity during loading. This Short Communication advances a thermodynamic hypothesis proposing that intracellular cryoprotection could be achieved through the in situ generation of cryoprotective solutes via pressure-activated disassembly of supramolecular complexes composed of cryoprotectant monomers or oligomers. The physical trigger for this disassembly is the hydrostatic pressure that arises intrinsically during isochoric (constant-volume) freezing: as ice forms, the fixed-volume constraint produces a substantial pressure increase. We propose that the stability of these assemblies is governed by the Helmholtz free energy, and that isochoric pressure shifts the free-energy landscape to favor the dissociated state for assemblies with a negative reaction molar volume. Because the pressures generated during isochoric freezing reach levels known to destabilize supramolecular complexes, this mechanism offers a plausible route for generating CPAs precisely during the freezing process. This approach would decouple cryoprotectant availability from membrane transport and synchronize protection with the onset of freezing. The purpose of this contribution is to articulate the thermodynamic basis and physical plausibility of this mechanism and to motivate future investigation of pressure-mediated preservation strategies.
Vacuum freeze-drying is a critical technology for extending the shelf life of probiotics while preserving their functional viability. However, during processing, water migration and ice crystal formation can lead to substantial cellular damage and reduced viability. This review aims to address the following key scientific questions: (i) how water migration evolves across different freeze-drying stages, (ii) how these mechanisms affect probiotic stability, and (iii) how these processes can be controlled through formulation and process design. We systematically examine water migration mechanisms during freezing, primary drying (sublimation), and secondary drying (desorption), and their effects on probiotic survival. Each stage involves distinct physical transformations of water, which influence ice crystal growth, membrane integrity, and the development of a glassy matrix. These processes are governed by interactions between intrinsic factors (e.g., enzymes, proteins, lipids, and exopolysaccharides) and extrinsic factors (e.g., medium composition, cryoprotectants, and process parameters), which collectively regulate water behavior and phase transitions. Recent advances in characterization techniques-including differential scanning calorimetry, nuclear magnetic resonance, Raman spectroscopy, and process analytical technologies-now enable multi-scale monitoring of water mobility and state changes. The review highlights that optimizing process control, developing hybrid freeze-drying strategies, innovating cryoprotectant formulations, and implementing multiscale modeling of water kinetics are essential for precise moisture management during probiotic freeze-drying. These strategies are key to improving probiotic survival and maintaining biological activity. In conclusion, this mechanistic understanding provides a scientific basis for the rational design of freeze-drying protocols and the development of next-generation probiotic products with enhanced stability and functionality.
The cryopreservation of primary human brain vascular pericytes (HBVPs) has facilitated their commercial availability as an essential component of the blood-brain barrier. Among the commercial suppliers of HBVPs in suspension, only one provides information regarding the cryoprotectant and vehicle used, i.e., 5% dimethyl sulfoxide (Me2SO) in complete medium, with no cryopreservation procedure given. Published protocols for the cryopreservation of brain vascular pericytes derived from human-induced pluripotent stem cells use traditional 10% Me2SO. In this study, we characterized the cryobiological response of HBVPs using graded freezing, both in the absence of cryoprotectants, which identifies their susceptibility to the main types of cryoinjury, and in the presence of several different cryoprotectants with the aim of quantifying post-thaw outcome. We first validated our graded freezing technique using human cerebral microvascular endothelial cell/D3 clone (hCMEC/D3) by comparing the membrane integrity results in this study in the absence and presence of 5% Me2SO plus 6% hydroxyethyl starch (HES) in hCMEC/D3 complete medium, with those previously published by our group. We then assessed the cryobiological response of HBVPs to controlled slow cooling at 1 °C/min in the absence and presence of various cryoprotectants, namely 5% Me2SO, 10% Me2SO, 5% Me2SO plus 6% HES, or 10% glycerol, all in HBVP complete medium. We found that all cryoprotectants tested yielded immediate post-thaw membrane integrity >93%. Cryopreservation with 10% Me2SO resulted in 95.3 ± 0.7% membrane integrity; using a lower concentration of Me2SO (5%) alone, or in the presence of 6% HES, resulted in membrane integrities of 93.3 ± 0.8% and 94.2 ± 0.8%, respectively. For applications where avoiding Me2SO is preferred, we showed that HBVPs can be cryopreserved in 10% glycerol (post-thaw membrane integrity of 95.7 ± 0.5%). The AlamarBlue reduction assay with 3 h incubation time indicated that HBVPs cryopreserved with either 5% Me2SO, 10% Me2SO, 5% Me2SO plus 6% HES, or 10% glycerol exhibited post-thaw metabolic activities which were not statistically different from those of unfrozen controls. Our study presents various application-specific options for cryopreservation of primary HBVPs with validated post-thaw membrane integrity and metabolic activity.
Obesity is a global public health problem, and new non-surgical or pharmacological treatments are being explored. Among these, cryostimulation has emerged as a new beneficial non-invasive method. Several studies have reported a greater decrease in skin temperature following cryostimulation in individuals with higher BMI or body fat percentage. The aim of this study was to evaluate the effect of a single 3-min whole-body cryostimulation (WBC) session on skin temperature and cold perception in male and female obese subjects. Skin temperature changes before and after the 3-min cryostimulation exposure were measured in twenty-one participants. Our results showed that the average skin temperature decreased by 12.9 °C without reaching dangerous levels. Individual characteristics such as sex and age should nevertheless be carefully considered to ensure the safety and efficacy of the treatment.
Sperm cryopreservation is a common method of preserving male fertility; however, the cryopreservation cycle of spermatozoa and the thawing process cause irreparable changes in the structural stability and biologic activity of the sperms. The current study was made to mitigate the cryodamage with the help of liposomes that entrap gallic acid (GA-loaded LIPO). Liposomes (LIPOs) were prepared from phosphatidylcholine, cholesterol, and Tween-80, and gallic acid (GA) was encapsulated within the vesicles. Their physicochemical properties were characterized by dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). GA-loaded LIPOs were incubated with spermatozoa for 1 h, after which semen samples were mixed with CryoSperm medium, loaded into straws, exposed to nitrogen vapor, and stored in liquid nitrogen. After one month, samples were thawed and evaluated for motility, viability, membrane integrity, mitochondrial activity, acrosome integrity, and DNA fragmentation (DFI). The ready LIPOs were also homogenous in morphology, with a mean particle size of 58.27 nm and a zeta potential of -15.5 mV. GA was successfully encapsulated into the liposomal matrix, and the encapsulation was 85.7 percent. The sperm motility, viability, plasma membrane integrity, mitochondrial activity and acrosome integrity, were significantly enhanced in parallel with lowering the DFI (p < 0.05) by treatment of spermatozoa with GA-loaded LIPOs compared to the control group (p < 0.05). The use of LIPOs has significant opportunities in reducing the effects of cryodamage to spermatozoa and at the same time increasing the biopotential of antioxidant substances.