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Cryopreservation is a pivotal technique in safeguarding genetic material across diverse species, despite its inherent challenges linked to induced spermatozoa damage, notably apoptosis and lipid peroxidation (LPO). Given the insufficient antioxidant defense of spermatozoa against LPO, there is a rising interest in integrating additional additives into extenders to ameliorate mammalian semen quality. Among these additives, flavonoids have garnered considerable attention due to their potent antioxidative properties. Hence, our study aimed to assess the efficacy of flavone (FL) and 3-hydroxyflavone (3-OH = ) supplementation in the cryopreservation medium to protect canine sperm against the damaging impacts of freezing and ensure the preservation of their reproductive potential. Semen was collected from five Beagle stud dogs and then pooled. Then, the sample was divided into 7 groups, each treated with 1) 0 mM, 2) 0.1 mM FL, 3) 0.2 mM FL, 4) 0.4 mM FL, 5) 0.1 mM 3-OH = , 6) 0.2 mM 3-OH = , 7) 0.4 mM 3-OH = . Semen samples were subjected to cryopreservation in French straws and glycerol as a cryoprotectant. In the frozen thawed semen, sperm motility parameters by CASA system and sperm membrane integrity, acrosome status, mitochondrial activity, DNA fragmentation, early apoptosis with capacitation, and LPO were assessed using flow cytometry just after thawing (0 h) and 4 h post thaw. Results reveal significant increase in the proportion of live spermatozoa with undamaged acrosomes in the FL 0.1 and 3-OH = 0.2 groups at 0 h post thaw. At this time point, 3-OH = 0.1 significantly reduced the DNA fragmentation index (DFI) compared to the FL 0.1 and 0.2 groups. However, after the next 4 h, 3-OH = 0.4 exhibited the lowest (P < 0.05) DFI compared to FL 0.2 and 3-OH = 0.1. Additionally, 3-OH = 0.4 showed the highest (P < 0.05) proportion of non apoptotic and non capacitated spermatozoa compared to FL 0.1 0 h post-thaw. Simultaneously, the same group demonstrated significant reduction in apoptotic and capacitated sperm cells, at 0 h and 4 h post-thaw. Moreover, 3-OH = at 0.1 (0 h and 4 h) and 0.2 mM (4 h) significantly enhances the proportion of live sperm without LPO post thaw. Whitin the FL groups, only 0.4 FL significantly increased the percentage of live sperm without LPO. No significant effect of the tested substances was observed on sperm motility, cell membrane integrity, or mitochondrial activity. These findings highlight the promising role of flavone and 3-hydroxyflavone in enhancing sperm resilience during cryopreservation, suggesting their protective function against acrosome damages, capacitation, apoptosis and lipid peroxidation., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The effects of Mn 2+ -, Zn 2+ - or Cu 2+ -nanosuccinate added to freezing extender on select post-thaw semen characteristics were determined in six Texel rams (aged 2-4 years) during seasonal anestrus (April-May). Ejaculates (n = 6 per ram) collected into an artificial vagina were divided into ten isovolumetric fractions each. Semen was diluted in lactose-yolk-tris-citrate-glycerin medium and nanosuccinates (Mn 2+ - and Zn 2+ -nanosuccinate: 0.0 (control), 2.5, 5.0 and 7.5 μg/l; Cu 2+ -nanosuccinate: 0.0 (control), 1.25, 2.5 and 3.75 μg/l) were added to semen extender. Extended semen was loaded into 0.25-ml straws and frozen in liquid nitrogen. After thawing, sperm motility parameters were determined with computer assisted semen analysis (CASA), and the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) was measured with a spectrophotometric technique. The addition of 5.0 μg/l of Mn 2+ - and Zn 2+ -nanosuccinate significantly increased the sperm progressive motility and both 2.5 and 5.0 μg/l improved sperm motion kinetics. Further, both nanosuccinates at a dose of 5.0 μg/l significantly decreased SOD activity and stimulated an increase in GPx and CAT activity in semen samples. Alternatively, the addition of Cu 2+ -nanosuccinate (highest dose) significantly reduced the progressive motility and velocity of ram spermatozoa, increased the percentage of sperm with acrosomal/head defects and seminal SOD activity, and depressed CAT (highest dose) and GPx (all doses) activity. In summary, the addition of Mn 2+ - and Zn 2+ -nanosuccinate to semen extender had beneficial effects on sperm motility/motion kinetics and structural integrity, whereas Cu 2+ -nanosuccinate generally had debilitating effects on the post-thaw semen characteristics in rams., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose. All co-authors have read and approved the contents of this manuscript, and there is no financial or other interest to report. We certify that this submission is an original work and is not under review at any other journal or with another publisher., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

In recent decades, there has been a growing interest in optimizing the protocols intended to sperm cryopreservation in domestic animals. These protocols include initial cooling, freezing, and thawing. While different attempts have been devised to improve sperm cryopreservation, the efficiency of this reproductive biotechnology is still far from being optimal. Furthermore, while much attention in improving cooling/freezing, less emphasis has been made in how thawing can be ameliorated. Despite this, the conditions through which, upon thawing, sperm return to physiological temperatures are much relevant, given that these cells must travel throughout the female genital tract until they reach the utero-tubal junction. Moreover, the composition of the media used for artificial insemination (AI) may also affect sperm survival, which is again something that one should bear because of the long journey that sperm must make. Furthermore, sperm quality and functionality decrease dramatically during post-thawing incubation time. Added to that, the deposition of the thawed sperm suspension devoid of seminal plasma in some species during an AI is accompanied by a leukocyte migration to the uterine lumen and with it the activation of immune mechanisms. Because few reviews have focused on the evidence gathered after sperm thawing, the present one aims to compile and discuss the available information concerning ruminants, pigs and horses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Vitrification is commonly used for in vitro fertilization and has significant impact on gametes., Objective: To investigate changes in ultrastructure, membrane potential and distribution of mitochondria in mouse oocytes after vitrification., Materials and Methods: Mouse oocytes were divided into three groups: one group as fresh control, one group for the toxicity test (treated with cryoprotectant but without vitrification), and the other for vitrification., Results: Most mitochondria in oocytes were damaged after cooling and warming, being rough and fuzzy in appearance, even swollen and broken. The membrane potential of the toxicity test group and the vitrification group was 0.320 +/-0.030 and 0.244 +/- 0.038, respectively, in comparison to the fresh group (0.398 +/- 0.043). The membrane potential of the vitrified oocytes was significantly lower than fresh oocytes and the toxicity test oocytes (P % 0.05), but there was no significant difference between fresh oocytes and the toxicity test oocytes (P > 0.05). Mitochondria in fresh oocytes were denser and strained stronger, with 59.5> distributed homogeneously and 36.4> polarized. The majority of mitochondria in the toxicity-tested oocytes were clustered (69.3>) and only a small portion were distributed homogeneously (19.6>), while mitochondria in vitrified oocytes were clustered (56.3>) and deficient (24.4>), and their fluorescent staining was weak and blurred. There was a significant disruption in mitochondrial function after vitrification., Conclusion: Vitrification alters the ultrastructure, membrane potential and distribution of mitochondria in oocytes, most likely caused by toxicity and mechanical injury. Doi.org/10.54680/fr24510110212.

Background: Walking catfish, Clarias batrachus is one of the native and most popular freshwater catfish species in Indonesia. However, cultivation faces challenges, particularly due to the scarcity of larvae resulting from underdeveloped breeding technologies. Cryopreservation is a method of storing sperm to maintain viability for a long period and support the breeding technology of the fish. Cryoprotectant, in this context, plays an important role in determining the success of sperm cryopreservation., Objective: To determine the best type and concentration of cryoprotectant for cryopreservation of walking catfish sperm., Materials and Methods: A total of five different types of cryoprotectants, namely DMSO, glycerol, ethyl glycol, ethanol, and methanol, were tested at four concentration levels namely 0%, 5%, 10%, 15%, and 20%, each with four replications., Results: The type and concentration of cryoprotectant had a significant effect on sperm motility and viability (P < 0.05). The best outcomes were obtained with 5% DMSO and ethyl glycol, 10% glycerol and methanol, as well as 15% ethanol., Conclusion: The highest motility and viability values were obtained with 5% DMSO, resulting in its recommendation for cryopreservation of walking catfish sperm. Doi.org/10.54680/fr24510110612.

Background: Various antioxidant substances are added to sperm extenders to protect spermatozoa against oxidative stress and cryodamage., Objective: To investigate the effects of the flavonoid diosmin (DIO) and a flavanone glycoside naringin (NAR) on the freezability of ram semen., Materials and Methods: In this study, six Merino rams were used during the breeding season. The ejaculates were pooled after collection from the rams. Pooled ejaculates were divided into six groups: control, NAR 1 mM, NAR 2 mM, NAR 4 mM, DIO 2 mM, and DIO 4 mM, and then diluted with a TRIS-based diluent. The pooled semen was equilibrated, placed in 0.25 mL pipettes with 10 × 10 7 sperm cells in each pipette, and frozen in liquid nitrogen vapor. After 24 h, the pipettes were thawed at 37 degree C for 25 s and analyzed in terms of spermatological parameters., Results: The highest plasma membrane integrity ratio was found in the DIO 4 mM group, whereas a statistically significant difference was found between the NAR 1 mM and NAR 2 mM groups (p < 0.05). While the DIO 4 mM group had the highest acrosome integrity rate, a statistically significant difference was found between the other groups (p < 0.05). Mitochondrial activity was the highest in the NAR 4 mM, DIO 4 mM and DIO 2 mM groups (p < 0.05). In the analysis of the sperm membrane lipid profile, it was observed that the DIO group had the highest lipid-phospholipid ratio. In sperm membrane protein profile analysis, it was found that both additives exerted protective effects at different levels. The highest total protein content was seen in the DIO 4 mM and NAR 4 mM groups. 8-hydroxydeoxyguanosine (8-OhDG) positivity was more common in the control group than in the DIO and NAR groups. Cu-Zn superoxide dismutase (SOD) expression was lower in the control group and more intense in all other groups. Positive results were especially observed in the acrosome of the sperm cells., Conclusion: The addition of NAR and DIO to the ram semen extender increased the quality of sperm parameters after the freeze-thaw process. Doi.org/10.54680/fr24510110412.

Background: In reproductive biotechnology, sperm cryopreservation has a vital role to play. Cryopreservation of sperm produces reactive oxygen species (ROS), which disrupt sperm function and structural competence. Numerous protective chemicals, including fructans, have been used during sperm cryopreservation., Objectives: To evaluate the effect of different concentrations of the fructosan inulin on ram sperm quality parameters, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) production after freezing and thawing., Materials and Methods: The pooled samples from four healthy rams were divided into seven equal aliquots and diluted in a Tris-base extender supplemented with 1, 2, 4, 8, 16, and 28 mM of inulin or without inulin supplementation (control). By using liquid nitrogen vapor, the semen was frozen and stored at 196 degree C., Results: The total motility, viability, and DNA integrity were significantly improved after freeze-thawing with 28 mM inulin, compared to other treatment groups (P < 0.05). A Tris-based extender containing 16 and 28 mM of inulin displayed the highest levels of ram sperm membrane integrity when compared with the control (p <0.05). The abnormality of ram sperm was increased during freeze-thawing at control and 1 mM of inulin, compared to 16 and 28 mM of inulin (P < 0.05). Additionally, 28 mM of inulin decreased MDA and increased SOD activity in ram sperm in comparison with the other treatments (P < 0.05)., Conclusion: As a result, 28 mM of inulin could be beneficial for the cryopreservation industry and reduce the harmful effects of freeze-thawing on ram sperm. Doi.org/10.54680/fr24510110512.

This study aimed at scrutinizing efficiency of incorporating L-carnitine or M. oleifera leaves extract into semen diluent on improving cryopreservation capacity and in vitro fertilization ability of buck spermatozoa. Ejaculates (n=48) were collected by an artificial vagina from six adult Damascus bucks twice weekly during the breeding season (September-October). Following initial evaluation, ejaculates of each collection session from the same bucks were pooled, diluted (1:10) with glycerolized (3 % glycerol, v/v) tris-citric acid egg yolk diluent and were split into three aliquots. The first aliquot served as control, whereas the second and third aliquots were supplemented with 4 μL/mL L-carnitine and 400 μL/mL moringa leaves extract (v/v), respectively. Thereafter, all specimens were processed for cryopreservation and were stored in liquid nitrogen (-196 °C) for 12 months before post-thaw sperm criteria were analyzed by a computer-assisted sperm analysis (CASA) system. Integrity of sperm DNA post thawing was visualized in all semen groups by fluorescence imaging, and in vitro fertilization ability of spermatozoa was also determined. Inclusion of L-carnitine or moringa leaves extract into the diluent improved (P<0.05) post-thaw sperm physical, morphofunctional and kinematic attributes, whilst maintaining (P<0.05) integrity of sperm DNA throughout the freezing and thawing cycle. Consequently, both supplemented groups yielded higher (P<0.05) in vitro fertilization rates compared to control. These results accentuate the protective roles of these antioxidants on buck sperm against consequences of cryopreservation-induced oxidative stress, hence ameliorating post-thaw sperm quality and fertilization competence. This is crucial for successful application of AI and IVF in goat selective breeding programs., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests, financial or personal, that could have appeared to influence conducting and reporting this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The freezability of chicken spermatozoa is low, therefore, effective cryoprotectants is desiderated. Antifreeze proteins (AFPs) are widely found in cold-tolerant species and help them to survive in freezing environments. This study was the first to evaluate the effects of different concentrations of plant-originated antifreeze glycoprotein (AFGP) (0, 0.1, 1, and 5 μg/mL) on post-thawed sperm motion characteristics, morphology, mitochondrial function, antioxidant activity, and fertilizing potential in chickens. Results showed that the total motility of 0.1 to 1 μg/mL AFGP groups were significantly higher than those of the 5 μg/mL AFGP group (P < 0.05). The post-thawed sperm viability of 0.1 μg/mL AFGP group was significantly higher than any of test groups (P < 0.05). Higher abnormal morphology rate of post-thawed sperm was observed in the control group (0 μg/mL AFGP) than in the 0.1, 1, and 5 μg/mL AFGP groups (P < 0.05). The concentrations of malondialdehyde (MDA) decreased gradually with the increase of AFGP concentration. ATP was significantly higher in the 0.1 and 1 μg/mL AFGP groups than those of control and any of test groups (P < 0.05). The 0.1 to 1 μg/mL AFGP groups had increased mitochondrial membrane potential (MMP) level (P > 0.05). The 0.1 μg/mL AFGP group had the highest average fertility (61.36%) compared with control group (57.02%) and any of test groups of chickens at 31 wk of age, and the 1 μg/mL AFGP group had the highest average fertility (37.72%) compared with control group (21.73%) and any of test groups of chickens at 65 wk of age. In conclusion, the results from this study suggest lower concentration of AFGP (0.1-1 μg/mL) showed positive effect for sperm function. This study inspires the continuous evaluation and seeking right way of adopting different kinds of AFPs in rooster semen cryopreservation., Competing Interests: DISCLOSURES The authors declare that they have no conflicts of interest to this work., (Copyright © 2024. Published by Elsevier Inc.)

The aim of this study was to assess the effect of lyophilized freezing extenders, which can be stored at room temperature, on stallion post-thaw sperm total motility (TM). Ejaculates of 28 stallions were frozen with four different extenders: two commercial freezing extenders offered worldwide and two novel lyophilized extenders (STAR and MX3), and two different cryopreservation protocols (CP1 with an equilibration period of 20 min. and CP2 with an equilibration period of 60 min.). The TM was assessed after thaw. Mean TM did not show significant differences between cryopreservation protocols within each extender. Mean TM was greater in samples diluted with STAR than in samples diluted with Botucrio (P ˂ 0.05), but no significant differences were observed for this variable between the other studied extenders. From all evaluated samples, twenty ejaculates showed the greatest TM when using the lyophilized extenders and the CP1. Thus, lyophilized extenders are a promising option for stallion sperm cryopreservation and have the advantage of storage and distribution at room temperature for at least one year., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cryopreservation is highly desired for long-term maintenance of the viability of living biosamples, while effective cell cryopreservation still relies heavily on the addition of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS). However, the intrinsic toxicity of DMSO is still a bottleneck, which could not only cause the clinical side effect but also induce cell genetic variants. In the meantime, the addition of FBS may bring potentially the risk of pathogenic microorganism contamination. The liquid marbles (LMs), a novel biotechnology tool for cell cryopreservation, which not only have a small volume system that facilitated recovery, but the hydrophobic shell also resisted the harm to cells caused by adverse environments. Previous LM-based cell cryopreservation relied heavily on the addition of FBS. In this work, we introduced acidic polyaspartic acid and polyglutamic acid as cryoprotectants to construct LM systems. LMs could burst in an instant to facilitate and achieve ultrarapid recovery process, and the hydrophilic carboxyl groups of the cryoprotectants could form hydrogen bonds with water molecules and further inhibit ice growth/formation to protect cells from cryoinjuries. The L929 cells could be well cryopreserved by acidic polyamino acid-based LMs. This new biotechnology platform is expected to be widely used for cell cryopreservation, which has the potential to propel LMs for the preservation of various functional cells in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sperm cryopreservation can lead to subfertility due to potential damage to sperm DNA, membranes, and overall motility caused by the freeze-thaw process. Interleukin-6 (IL-6) is a versatile cytokine with various roles in reproductive processes. However, the impacts of IL-6 supplementation on cryopreserved ram sperm have not been thoroughly investigated. Therefore, this study aims to assess the influence of IL-6 on the sperm quality of cryopreserved ram sperm. Ram semen was collected, pooled, and extended with tris-citrate soybean lecithin extender supplemented with 0, 50, 100, and 200 ng/mL of IL-6. The samples experienced a standard freezing protocol, and sperm quality, kinematic parameters, ultrastructure, and molecular docking of cryopreserved ram spermatozoa were evaluated. The results showed that sperm kinematics, viability, progressive motility, and membrane integrity were significantly enhanced by the addition of 100 or 200 ng of IL-6/mL (p < 0.05). Semen supplemented with 100 or 200 ng/mL of IL-6 also exhibited higher percentages of sperm kinematics, including DAP, DCL, DSL, VSL, VAP, VCL, and ALH, compared to other groups (p < 0.05). IL-6 supplementation enhanced acrosome integrity, and reduced caspase-3 activity in post-thawed ram spermatozoa (p < 0.05) compared to untreated group. Supplementation with IL-6 (200 ng/mL) significantly decreased oxidative biomarkers (NO, MDA, and H 2 O 2 ) (p < 0.001) and improved total antioxidant capacity (p < 0.05). The percentage of sperm damage (tail, head, and midpiece) was significantly reduced by IL-6 supplementation (p < 0.05). Electron micrographs showed that supplementation with 100 or 200 ng/mL IL-6 protected acrosome stability, plasma membrane integrity, and sustained the ultrastructure integrity of cryopreserved ram spermatozoa. The docking exploration indicates a higher binding affinity with sperm function biomarkers, including caspase 3, BCL2, and PSMA6, with binding energies of - 52.30 kcal/mol, - 56.04 kcal/mol, and - 57.06 kcal/mol, respectively. In conclusion, the addition of IL-6 to the freezing extender can enhance the post-thaw quality of cryopreserved ram spermatozoa., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

The shortage of suitable donor meniscus grafts from the knee and temporomandibular joint (TMJ) impedes treatments for millions of patients. Vitrification offers a promising solution by transitioning these tissues into a vitreous state at cryogenic temperatures, protecting them from ice crystal damage using high concentrations of cryoprotectant agents (CPAs). However, vitrification's success is hindered for larger tissues (>3 mL) due to challenges in CPA penetration. Dense avascular meniscus tissues require extended CPA exposure for adequate penetration; however, prolonged exposure becomes cytotoxic. Balancing penetration and reducing cell toxicity is required. To overcome this hurdle, a simulation-based optimization approach is developed by combining computational modeling with microcomputed tomography (µCT) imaging to predict 3D CPA distributions within tissues over time accurately. This approach minimizes CPA exposure time, resulting in 85% viability in 4-mL meniscal specimens, 70% in 10-mL whole knee menisci, and 85% in 15-mL whole TMJ menisci (i.e., TMJ disc) post-vitrification, outperforming slow-freezing methods (20%-40%), in a pig model. The extracellular matrix (ECM) structure and biomechanical strength of vitreous tissues remain largely intact. Vitreous meniscus grafts demonstrate clinical-level viability (≥70%), closely resembling the material properties of native tissues, with long-term availability for transplantation. The enhanced vitrification technology opens new possibilities for other avascular grafts., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Background: The present study aimed to investigate the cryoprotective effect of epigallocatechin gallate (EGCG) replacing sucrose on surimi during frozen storage. Substitution or partial substitution of 0.1% EGCG for sucrose (1.5%) was added to surimi, and the surimi samples without and with commercial cryoprotectants (4% sucrose and 4% sorbitol) were used as the control group., Results: The results obtained suggest that, with the increase in frozen storage time, the structural performance of surimi protein gradually weakened (e.g. the decrease in the surface hydrophobicity, the increase in the total sulfhydryl and solubility, and the protein myosin heavy chain bands became shallow) and surimi gel quality gradually deteriorated (e.g. the decrease in water-holding capacity, gel strength and all texture profile attributes). However, compared with the other three group surimi samples during the frozen period, the surimi proteins with partial replacement of sucrose by EGCG had a higher total sulfhydryl group content and solubility of proteins, as well as lower surface hydrophobicity of protein, suggesting that the addition of EGCG as a partial substitute for sucrose can enhance the antifreeze ability of surimi. Meanwhile, the surimi gel with the partial replacement of sucrose by EGCG had a higher water retention capacity, gel strength and texture attributes (e.g. hardness, springiness, cohesiveness, chewiness, and resilience), indicating that the addition of EGCG as a partial substitute for sucrose can inhibit the deterioration of surimi gel quality., Conclusion: Overall, EGCG partially replacing sucrose can play an alternative cryoprotectant with a lower sweetness to prevent the quality of surimi from deteriorating. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Ultrasound-assisted freezing (UAF) is a clean technique for meat cryoprotections; however, its effectiveness is still limited compared to conventional cryoprotectants, e.g., sugars, polyols, especially at high dosages. To resolve this problem, a synergistic cryoprotection strategy was developed in this study. Adenosine monophosphate (AMP), an adenosine-type food additive, was introduced into frozen surimi at a considerably reduced content (0.08%), yet substantially enhanced the efficiency of UAF to comparable levels of commercial cryoprotectant (4% sucrose with 4% sorbitol). Specifically, UAF/AMP treatment retarded denaturation of surimi myofibrillar protein (MP) during 60-day frozen storage, as evidenced by its increased solubility, Ca 2+ -ATPase activity, sulfhydryl content, declined surface hydrophobicity, particle size, and stabilized protein conformation. Gels of UAF/AMP-treated surimi also demonstrated more stabilized microstructures, uniform water distributions, enhanced mechanical properties and water-holding capacities. This study provided a feasible approach to boost the cryoprotective performance of UAF, thus expanding its potential applications in frozen food industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Conventional cryoprotectant mixtures (sucrose and sorbitol) impart excessive sweetness and calories to surimi. Therefore, there is a need to explore alternative cryoprotectants with low sweetness and low-calorie content. The cryoprotective effects and possible mechanisms of soybean oligosaccharides (SBOS) on the frozen stability of grass carp (Ctenopharyngodon idellus) surimi were investigated during 120 days of frozen storage in a comparison with commercial cryoprotectants (4% sucrose and 4% sorbitol, w/w)., Results: SBOS at 6-8% (w/w) and commercial cryoprotectants could restrain water mobility and reduce thawing loss of frozen surimi by increasing non-freezable water content. SBOS could maintain the structural stability of proteins by preventing sulfhydryl groups from being rapidly oxidized to disulfide bonds, retarding the reduction of the solubility, Ca 2+ -ATPase activity and α-helix content of myofibrillar proteins (MP), as well as hindering the increasing surface hydrophobicity of MP of surimi during 120 days of frozen storage. The introduction of SBOS increased the gel strength and water-holding capacity of frozen-stored surimi. Compared with commercial cryoprotectants, 8% SBOS was more effective in stabilizing protein structure, whereas it was slightly less effective with respect to ice-forming inhibition., Conclusion: The results obtained in the present study suggest that 8% SBOS could be potentially developed as a new cryoprotectant for surimi as a result of its ice-forming inhibition abilities and protein structure stability. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions-consistent with crystallization of most free water-during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied., (© 2024. The Author(s).)

Plaque assays quantify the amount of active, replicating virus to study and detect infectious diseases by application of samples to monolayers of cultured cells. Due to the time taken in thawing, propagating, plating, counting, and then conducting the assay, the process can take over a week to gather data. Here, we introduce assay-ready cryopreserved Vero monolayers in multiwell plates, which can be used directly from the freezer with no cell culture to accelerate the process of plaque determination. Standard dimethyl sulfoxide cryopreservation resulted in just 25% recovery, but addition of polyampholytes (macromolecular cryoprotectants) increased post-thaw recovery and viability in 12- and 24-well plate formats. Variability between individual wells was reduced by chemically induced ice nucleation to prevent supercooling. Cryopreserved cells were used to determine influenza viral plaques in just 24 h, matching results from nonfrozen controls. This innovation may accelerate viral detection and quantification and facilitate automation by eliminating extensive cell culturing.

This study investigated the impact of various Ge132 (Bis-carboxyethyl germanium sesquioxide) concentrations on frozen bovine semen. Ejaculates from three bulls were pooled and divided into six groups, each one with different Ge132 concentrations (0, 500, and 1000 μg/mL) and each group was incubated in different conditions (33°C for 30 min (D: D0, D500, and D1000), and the other was immediately cooled to 4°C (R: R0-control; R500 and R1000)). Thawed semen was evaluated for sperm characteristics by CASA and flow cytometer. Results showed better motility in the immediate cooling group without Ge132 compared with high Ge132 concentrations. Values for total motility dropped after 5 and 60 min in groups with high Ge132 levels and some control groups. Linearity increased with 1000 μg/mL Ge132, while straightness differed between moments in multiple groups. Membrane integrity was higher in a control group and certain Ge132 groups. Lower O 2 - generation occurred without Ge132. After oxidative stress induction, lipid peroxidation intensity increased with arachidonic acid, but D1000 had lower peroxidation than R0. Overall, Ge132 appears to have provided protection against PLM when subjected to oxidative stress, since even at high concentrations it maintained sperm metabolism., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

We researched the ability of tanacetan pectin from inflorescences of common tansy Tanacetum vulgare L. to change the osmolarity and freezing point of water in solutions of cryoprotectants: glycerol-3.5%, dimethyl sulfoxide (DMSO)-10%, dimethylacetamide-10% (DMAC), and 1.2-propanediol (1.2-PD)-10%, as well as the effect of solutions of tanacetan (0.2%, 0.4%) on the kinetics of crystallization processes and the nature of crystal formation. We used a combination of protector and pectin that we tested earlier, which provided effective protection for human leukocytes and platelets, as well as bovine spermatozoa, at temperatures below freezing (-20°C and -80°C). It has been established that tanacetan slows down the process of water freezing in glycerol, but not in DMSO, DMAC, and 1.2-PD, promotes deeper supercooling of the medium, and affects the morphological structure of ice. The addition of pectin to the cryosolution increases the activity of the main cryoprotectant glycerol even at its low concentrations. The combination of glycerol and tanacetan can be effective in freezing biological materials, which is confirmed by the preservation of leukocytes at -20°C and -80°C for 7 days, platelets at -80°C for 30 days, and spermatozoa at -80°C within 1 day. A comprehensive analysis of the chemical, physicochemical, and cryoprotective properties of tanacetan indicates the prospect of using pectin in the cryopreservation of biological objects at temperatures of electric freezers.

Aim: The aim of this study is the evaluation effect of nanoliposome-loaded Mito-Tempo on sperm parameters during human sperm cryopreservation., Methods: Semen samples of 50 Asthenoteratozoospermia men (random) were collected. Sperm parameters were analyzed based on World Health Organization (WHO, 2010) criteria (2021) and each sample was divided into 5 groups (E1-E5). E1 (control group): the sperm was cryopreserved without nanoliposome, and Mito-Tempo. E2: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.1 mM) + freezing medium. E3: sperm cryopreservation with Mito-Tempo-loaded nanoliposome (Mito-Tempo 0.2 mM) + freezing medium. E4: in this group, the cryopreservation sperm with Mito-Tempo 0.3 mM + freezing medium. E5: the cryopreservation sperm with Mito-Tempo 0.2 mM + freezing medium., Results: The result of this study indicated that sperm parameters and total antioxidant capacity (TAC) significantly increase in E3 and E4 groups, compared to E1, E2, and E5 groups respectively (P < 0.05). The percentage of abnormal morphology, DNA fragmentation index (DFI), malondialdehyde (MDA), and the levels of ROS significantly decrease in E3 and E4 groups, compared to E1, E2, and E5 groups (P < 0.05). In addition, the sperm parameters and stress oxidative factors significantly improve in E3 group compared to other groups (P < 0.05)., Conclusions: In conclusion, the combination of Mito-Tempo with nanoliposome due to its ability to cooperate with lipid layers may lead to significant performance in reducing oxidative stress damage and increasing the quality of sperm parameters., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Recently, we proposed a low-glycerol cryoprotectant formulation (consisting of 0.4 M trehalose and 5% glycerol) for cryopreservation of human red blood cells (RBCs), which greatly reduced the concentration of glycerol, minimized intracellular ice damage, and achieved high recovery. Although this study was successful in cellular experiments, the nonequilibrium phase transition behaviors of the cryoprotective agent solution have not been systematically analyzed. Therefore, it is essential to provide reliable thermodynamic data to substantiate the viability of this cryopreservation technique. In this study, the phase change behaviors and thermal properties of typical trehalose and/or glycerol solutions quenched in liquid nitrogen were investigated using differential scanning calorimetry and cryomicroscopy. It was found that the glass transition temperatures of both the trehalose aqueous solution (<1.0 M) and glycerol aqueous solution (<40% w/v) did not vary apparently with the concentration at low concentrations, while they increased significantly with increasing concentration at high concentrations. Moreover, it was revealed that the inhibitory effect of trehalose on ice growth was affected by glycerol. We further found that the addition of low concentrations of glycerol facilitates the partial glass transition of trehalose solutions at low concentrations. The results of this work provide reliable thermodynamic data to support the cryopreservation of human RBCs with unusually low concentrations of glycerol.

During cryopreservation, the growth of ice crystals can cause mechanical damage to samples, which is one of the important factors limiting the quality of preserved samples. To enhance the preservation quality of biological samples, scholars have tried various engineering methods. Among them, an electric field is an essential factor affecting solution freezing. Dimethyl sulfoxide, as a commonly used cryoprotectant, can cause mechanical damage to cells due to ice crystals even when freezing at the optimal freezing rate. Water is a strongly polar dielectric material, and the applied alternating current (AC) electric field will affect the water freezing performance. Therefore, a quantitative study of ice crystal nucleation and growth during freezing of dimethyl sulfoxide solutions under different AC electric field conditions is needed to try to reduce ice crystal damage. We created a liquid-film device to approximate the ice crystal growth process as a two-dimensional image. The frequency of the AC voltage was set from 0 to 50 kHz. We measured the supercooling of the dimethyl sulfoxide solution under AC electric field conditions. As an objective and accurate quantitative analysis of the ice crystal growth process, we propose a Dilated Convolutional Segmentation Transformer for semantic segmentation of ice crystal images. It is concluded that the average area and the growth rate of single ice crystals decrease with increasing electric field frequency at a certain concentration of dimethyl sulfoxide solution. Lower concentrations of dimethyl sulfoxide solution in combination with an AC electric field can achieve similar ice suppression effects as when higher concentrations of dimethyl sulfoxide solution act alone. We believe that AC electric fields are expected to be an aid to cryopreservation and provide some theoretical basis and experimental foundation for its development.

Freeze drying has been well applied in the preparation of high-efficiency viability probiotic powders. However, the process is generally accompanied by probiotic viability deficiency, which poses a problem for further application. In this study, various kinds of cryoprotectant formulations (skim milk, maltodextrin, and sucrose) were tested to enhance the survival of Lactiplantibacillus plantarum NBC99 after freezing and freeze-drying. An I-optimal experimental design-oriented optimization model was presented to optimize the cryoprotective medium, and the highest cell survival was observed with 25% skim milk, 8.71% maltodextrin, and 1.13% sucrose cryoprotectant as the optimum condition. L. plantarum NBC99 has been a good potential strain for the manufacture of an industrial probiotic, and this research has aimed to investigate the long-term protective effects of optimum cryoprotectant formulations on the viability of bacteria. The results showed the potential value of freeze-dried probiotic L. plantarum NBC99 culture for commercialization.

The present study was conducted to evaluate the effects of trehalose supplementation in egg-yolk (EY)-free tris extender on dog spermatozoa. Pooled spermatozoa were diluted with extender 1 (EY-free tris extender supplemented with 0, 10, 15, 20, or 30 mM trehalose) and cooled (2 × 10 8 sperm/mL) for 1 hour at 4°C. After that, extender 2 (extender 1 containing 1 M glycerol) was added (v:v) to the diluted sperm, loaded in 0.5-mL straws (1 × 10 8 sperm/mL), and incubated at 4°C for 30 minutes. The sperm straws were frozen over liquid nitrogen (LN 2 ) vapor for 20 minutes and then plunged directly into LN 2 . After thawing at 37°C for 25 seconds, sperm progressive motility (CASA), viability (SYBR-14/PI), apoptosis (Annexin V/PI), and reactive oxygen species (ROS; H 2 DCFDA/PI) were evaluated. Thereafter, the optimal concentrations of trehalose were selected, and the gene expression of BAX , BCL2 , NOX5 , SMOX , OGG1 , and ROMO1 was evaluated after freeze-thawing. Supplementation with 20 and 30 mM trehalose significantly increased sperm progressive motility and viability compared to the control. However, trehalose had no significant effect on sperm ROS or phosphatidylserine translocation index. There were minor numerical increases and decreases in gene expression when the selected optimal concentrations of trehalose (20 and 30 mM) were compared to the control. However, there were no significant differences. We conclude that the addition of trehalose (20 and 30 mM) in EY-free extender could improve sperm motility and viability without significant effects on ROS, apoptosis, or gene expression.

Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 μg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 μg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) ( p  < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.

Cryopreservation is the most effective technology for the long-term preservation of biological materials, including cells, tissues, and even organs in the future. The process of cooling and rewarming is essential to the successful preservation of biological materials. One of the critical problems in the development of cryopreservation is the optimization of effective rewarming technologies. This article reviewed rewarming methods, including traditional boundary rewarming commonly used for small-volume biological materials and other advanced techniques that could be potentially feasible for organ preservation in the future. The review focused on various rewarming technique principles, typical applications, and their possible limitations for cryopreservation of biological materials. This article introduced nanowarming methods in the progressing optimization and the possible difficulties. The trends of novel rewarming methods were discussed, and suggestions were given for future development.

Oocyte vitrification has become a widely adopted method in clinical practice. However, the solidification behavior and its impact on oocytes during the ultrarapid cooling process remain poorly understood. In this study, we established a system and methodology to observe crystallization behavior in oocytes during quench cooling and warming. Subsequently, the threshold concentration of cryoprotective agents (CPAs) required for oocyte vitrification was determined through a visualization method. The results demonstrated that the ice front could not be observed in the image sequence when using 16.5% DMSO +16.5% EG during high-speed quench cooling (2821.58°C/min). Finally, oocytes were encapsulated with an antifreezing hydrogel (7.5% EG +7.5% DMSO +0.5% alginate) and subjected to high-speed quench cooling. No ice crystals appeared in the antifreezing hydrogel-encapsulated oocytes at a low concentration of osmotic CPA (2.4 M). This research opens up new possibilities for oocyte vitrification with a reduced concentration of CPA.

Oocyte cryopreservation is increasingly being used in reproductive technologies for conservation and breeding purposes. Further development of oocyte cryopreservation techniques requires interdisciplinary insights in the underlying principles of cryopreservation. This review aims to serve this purpose by: (1) highlighting that preservation strategies can be rationally designed, (2) presenting mechanistic insights in volume and osmotic stress responses associated with CPA loading strategies and cooling, and (3) giving a comprehensive listing of oocyte specific biophysical membrane characteristics and commonly used permeation model equations. It is shown how transport models can be used to simulate the behavior of oocytes during cryopreservation processing steps, i.e., during loading of cryoprotective agents (CPAs), cooling with freezing as well as vitrification, warming and CPA unloading. More specifically, using defined cellular and membrane characteristics, the responses of oocytes during CPA (un)loading were simulated in terms of temperature- and CPA type-and-concentration-dependent changes in cell volume and intracellular solute concentration. In addition, in order to determine the optimal cooling rate for slow programmable cooling cryopreservation, the freezing-induced cell volume response was simulated at various cooling rates to estimate rates with tolerable limits. For vitrification, special emphasis was on prediction of the timing of reaching osmotic tolerance limits during CPA exposure, and the need to use step-wise CPA addition/removal protocols. In conclusion, we present simulations and schematic illustrations that explain the timing of events during slow cooling cryopreservation as well as vitrification, important for rationally designing protocols taking into account how different CPA types, concentrations and temperatures affect the oocyte., Competing Interests: Declaration of Competing Interest We have no conflicts of interest to report., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods-liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability., (© 2024. The Author(s).)

Extensive studies have been conducted on deicing nanomaterials to improve the cryoprotective effects on cells, tissues, and organs. The nanomaterials with different composition, sizes, and shapes can inhibit the formation and growth of ice crystals, thereby reducing the damage to the cryopreserved samples. In this study, the carbon composite particles (CCPs) with different sizes and shapes were prepared by the hydrothermal method. The results demonstrated that the cryoprotective effect of CCPs enhanced with the decrease in particle size. Compared with spherical CCPs, Janus nanoparticles and WSP nanoflower with special shapes demonstrated improved protective effects on cryopreserved cells. In addition, the combination of deicing micro/nanomaterials at appropriate concentrations with commercial cryoprotectants exerted improved cryoprotective effects on cells. The prepared deicing micro/nanomaterials can improve cell cryopreservation, demonstrating great application potential in biomedical research and cryopreservation.

Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1's efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research., (© 2024. The Author(s).)

Cryopreservation of human corneal stroma-derived mesenchymal stromal cells (hCS-MSCs) with dimethylsulfoxide (DMSO) as a cryoprotective agent (CPA) has not been previously compared to that with glycerol under standard conditions. The hCS-MSCs were hereby cryopreserved with both compounds using a freezing rate of 1 °C/minute. The CPAs were tested by different concentrations in complete Minimum Essential Medium (MEM) approved for good manufacturing practice, and a medium frequently used in cell laboratory culturing-Dulbecco's modified eagle serum. The hCS-MSCs were isolated from cadaveric human corneas obtained from the Norwegian Eye Bank, and immunophenotypically characterized by flow cytometry before and after cryopreservation. The survival rate, the cellular adhesion, proliferation and cell surface coverage after cryopreservation of hCS-MSCs has been studied. The hCS-MSCs were immunofluorescent stained and examined for their morphology microscopically. The results showed that cryopreservation of hCS-MSCs in MEM with 10% glycerol gives a higher proliferation rate compared to other cryopreserving media tested. Based on the results, hCS-MSCs can safely be cryopreserved using glycerol instead of the traditional use of DMSO., (© 2024. The Author(s).)

Allograft transplantation is an important method for tendon reconstruction after injury, and its clinical success highly relies on the storage and transportation of the grafts. Cryopreservation is a promising strategy for tendon storage. In this study, we report a novel cryopreservation agent (CPA) formulation with a high biocompatibility for tendon cryopreservation. Mainly composed of natural zwitterionic betaine and the biocompatible polymer poly(vinylpyrrolidone) (PVP), it exhibited ideal abilities to depress the freezing point and inhibit ice growth and recrystallization. Notably, after cryopreservation via plunge-freezing for 1 month, Young's modulus (144 MPa, 98% of fresh tendons) and ultimate stress (46.7 MPa, 99% of fresh tendons) remained stable, and the cross-linking of collagen microfibers, protein structures, and glycosaminoglycan (GAG) contents changed slightly. These results indicate that the formulation (5 wt % betaine and 5 wt % PVP in phosphate-buffered saline, PBS solution) effectively maintains the biomechanical properties and tissue structure. This work offers a novel cryopreservation method for tendons and may also provide insights into the long-term preservation of various other tissues.

Cryopreservation of red blood cells (RBCs) plays an indispensable role in modern clinical transfusion therapy. Researchers are dedicated to finding cryoprotectants (CPAs) with high efficiency and low toxicity to prevent RBCs from cryopreservation injury. This study presents, for the first time, the feasibility and underlying mechanisms of a novel CPA called tris(hydroxymethyl)aminomethane-3-propanesulfonic acid (TAPS) in RBCs cryopreservation. The results demonstrated that the addition of TAPS achieved a post-thaw recovery of RBCs at 79.12 ± 0.67%, accompanied by excellent biocompatibility (above 97%). Subsequently, the mechanism for preventing RBCs from cryopreservation injury was elucidated. On one hand, TAPS exhibits a significant amount of bound water and effectively inhibits ice recrystallization, thereby reducing mechanical damage. On the other hand, TAPS demonstrates high capacity to scavenge reactive oxygen species and strong endogenous antioxidant enzyme activity, providing effective protection against oxidative damage. Above all, TAPS can be readily removed through direct washing, and the RBCs after washing showed no significant differences in various physiological parameters (SEM, RBC hemolysis, ESR, ATPase activity, and Hb content) compared to fresh RBCs. Finally, the presented mathematical modeling analysis indicates the good benefits of TAPS. In summary, TAPS holds potential for both research and practical in the field of cryobiology, offering innovative insights for the improvement of RBCs cryopreservation in transfusion medicine.

To investigate the mechanisms underlying the differences in the freezability of boar semen, Yorkshire boars with freezing-tolerant semen (YT, n = 3), Yorkshire boars with freezing-sensitive semen (YS, n = 3), Landrace boars with freezing-tolerant semen (LT, n = 3), and Landrace boars with freezing-sensitive semen (LS, n = 3) were selected for this study. Their sperm was subjected to protein extraction, followed by data-independent acquisition proteomics and functional bioinformatics analysis. A total of 3042 proteins were identified, of which 2810 were quantified. Some key KEGG pathways were enriched, such as starch and sucrose metabolism, carbohydrate digestion and absorption, mineral absorption, the HIF-1 signaling pathway, and the necroptosis pathways. Through PRM verification, we found that several proteins, such as α-amylase and epididymal sperm-binding protein 1, can be used as molecular markers of the freezing resistance of boar semen. Furthermore, we found that the addition of α-amylase to cryoprotective extender could significantly improve the post-thaw motility and quality of boar semen. In summary, this study revealed some molecular markers and potential molecular pathways contributing to the high or low freezability of boar sperm, identifying α-amylase as a key protein. This study is valuable for optimizing boar semen cryopreservation technology.

Objectives: This study aimed to examine the effects of supplementation of vitamin D to the egg-yolk extender on characteristics of frozen-thawed ram semen., Methods: Semen samples obtained from adult rams were pooled and divided into five equal volumes. It was reconstituted with extenders containing different concentrations of vitamin D: 0 (control), 12.5 (VITD 12.5), 25 (VITD 25), 50 (VITD 50), and 100 ng/mL (VITD 100), and then they were frozen. Sperm motility parameters, plasma membrane functional integrity, acrosomal integrity, DNA fragmentation, and mitochondrial membrane potential of the groups were evaluated after sperm thawing., Results: Total motility and progressive motility were higher in VITD 50 than in all other groups (p < 0.05). Higher sperm straightness, linearity, and wooble were higher in VITD 50 than in the control group (p < 0.05). A similar pattern of VITD 50 was observed for plasma membrane integrity and mitochondrial membrane potential (p > 0.05)., Conclusions: In the study, it was observed that adding vitamin D to the extender had a beneficial effect on ram spermatological parameters. In addition, it was concluded that the use of the 50 ng/mL vitamin D in the extender provided more effective protection than the other doses., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

This study aimed to evaluate the effects of replacing egg yolk extender with sesame oil on the quality of sperm in goats following incubation at 37°C, chilling at 4°C, and freezing. Semen samples were collected from four intact male goats. The individual semen sample was divided into six groups consisting of a control group and five treatment groups with different egg yolk-to-sesame oil ratios. Seminal plasma was removed, and the sperm pellet was diluted with experimental semen extenders. The control group contained an extender of 10 % egg yolk (SO0), and the experimental extenders were composed of 8.75 % egg yolk and 1.25 % sesame oil (SO1.25); 7.5 % egg yolk and 2.5 % sesame oil (SO2.5); 5 % egg yolk and 5 % sesame oil (SO5); 2.5 % egg yolk and 7.5 % sesame oil (SO7.5); and 10 % sesame oil (SO10). Each group of semen was divided into three groups, incubated at 37°C for 1 h, chilled at 4°C for 4 h, or frozen for 24 h. Five replicates were performed. Sperm quality was evaluated, including motility, viability, and functional membrane integrity. The SO1.25 group achieved the highest sperm quality rate among the treatment groups, and the extender did not have a negative effect compared to the control. However, the total replacement of egg yolk with sesame oil in an extender resulted in the lowest sperm quality. In conclusion, the ratios of egg yolk and sesame oil that were acceptable for goat semen cryopreservation were 8.75 % and 1.25 %, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Today, synthetic chemicals are used in vitrification solutions for cryopreservation studies to mimic natural cryoprotectants that supply tolerance to organisms in nature against freezing stress. In the case of plants, PVS2, containing glycerol, dimethyl sulfoxide (Me2SO), ethylene glycol and sucrose, is considered as the golden standard for successful cryopreservation. However, Me2SO can generally cause toxicity to certain plant cells, adversely affecting viability after freezing and/or thawing. Hence, the replacement (or substantial reduction) of Me2SO by cheap, non-toxic and natural cryoprotectants became a matter of high priority to vitrification solutions or reducing their content gained escalating importance for the cryopreservation of plants. Fructans, sucrose derivatives mainly consisting of fructose residues, are candidate cryoprotectants., Objective: Inspired by their protective role in nature, we here explored, for the first time, the potential of an array of 8 structurally different fructans as cryoprotectants in plant cryopreservation., Materials and Methods: Arabidopsis thaliana L. seedlings were used as a model system with a one-step vitrification method. PVS2 solutions with different Me2SO and fructan contents were evaluated., Results: It was found that branched low DP graminan, extracted from milky stage wheat kernels, led to the highest recovery (85%) among tested fructans with 12.5% Me2SO after cryopreservation, which was remarkably close to the viability (90%) observed with the original PVS2 containing 15% Me2SO. Moreover, its protective efficacy could be further optimized by addition of vitamin C acting as an antioxidant., Conclusion: Such novel formulations offer great perspectives for cryopreservation of various crop species. Doi.org/10.54680/fr24410110512.

Background: The natural population of Colchicum figlalii (Varol) Parolly and Eren grows in a narrow area of serpentine rock clearings at an altitude of 1900-2100 m in Southwestern Anatolia (Sandras Mountain, Mugla, Turkey). The species is regarded as endangered according to the IUCN Red List Categories., Objective: To develop an optimum procedure for in vitro propagation and cryopreservation of germplasm of this rare endemic., Materials and Methods: A total of 281 bulbs were used as in vitro culture starting material and after surface sterilization, clean material was obtained from 157 of them. Woody Plant Medium (WPM), Olive Medium (OM), and Murashige and Skoog medium (MS) were used for in vitro culture establishment., Results: The maximum regeneration rate (~67.3%) was obtained after four weeks of incubation on OM. The calli were successfully induced by using OM supplemented with 10.7 uM NAA from leaves of in vitro grown C. figlalii bulbs. A PVS2-vitrification procedure was used for cryopreservation of C. figlalii callus tissue. After cryo-storage, the best result for regeneration (66.7%) was obtained from calli treated with PVS2 for 75 min before plunging into liquid nitrogen. All rooted seedlings derived from cryopreserved calli were successfully acclimatized to greenhouse conditions., Conclusion: This study is an effective reference for future long-term conservation of similar species that are difficult to cryopreserve. Doi.org/10.54680/fr24410110412.

This work investigated the cryoprotective effect of trehalose (TH) and sodium pyrophosphate (SPP) alone and in combination on myofibrillar protein (MP) oxidation and structural changes in silver carp surimi during 90 days of frozen storage (-20 °C). TH combined with SPP was significantly more effective than single TH or SPP in preventing MP oxidation (P < 0.05), showing a higher SH content (6.05 nmol/mg protein), and a lower carbonyl (4.24 nmol/mg protein) and dityrosine content (1280 A.U.). SDS-PAGE results indicated that TH combined with SPP did not differ significantly from TH and SPP in inhibiting protein degradation but was more effective in inhibiting protein crosslinking. Moreover, all cryoprotectants could stabilise the secondary and tertiary structures and inhibit unfolded and aggregation of MP, with the combination of TH and SPP being the best. It's worth noting that TH combined with SPP had a synergistic effect on inhibiting the decrease in α-helix content and gel-forming ability, and the increase in surface hydrophobicity. Overall, TH combined with SPP could significantly inhibited MP oxidation and structural changes in surimi during frozen storage and improve the gel-forming ability, which was significantly better than single TH or SPP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This comprehensive review delves into the evolving landscape of assisted reproductive technologies (ARTs) in bovine species, particularly focusing on the pivotal roles of semen additives in the cryopreservation of buffalo and cattle semen. In developing nations, where ARTs are still emerging, these techniques significantly influence bovine reproductive strategies. In contrast, developed regions have embraced them as primary approaches for dairy buffalo and cattle breeding. Semen cryopreservation, while offering advantages like extended storage and genetic propagation, also presents challenges. These include diminished sperm quality due to reactive oxygen species (ROS) production, alterations in sperm structure, and temperature fluctuations. Further, the effect of cryopreservation differs between cattle and buffaloes, with the latter exhibiting poorer semen viability and fertility due to inherent lipid composition susceptibilities. The generation and implications of ROS, especially hydrogen peroxide, contribute significantly to sperm DNA damage and functional impairments. To counteract these challenges, research has intensified on semen additives, aiming to bolster semen quality and protect against oxidative stress-induced damage. As the field advances, the review emphasizes the need for optimized cryopreservation techniques and tailored antioxidant strategies to harness the full potential of ARTs in bovine breeding programs. Doi.org/10.54680/fr24410110112.

Sperm cryopreservation plays an important role in the artificial insemination (AI) industry of small ruminants. It, however the use of frozen-thawed goat semen is limited due to the insufficient number of sperm with good biological functions. Mitochondria are the most sensitive organelles to cryopreservation damage in sperm. This study was conducted to determine the effects of MitoQ, the mitochondrial-targeted antioxidant, in a plant-based extender on the quality parameters of Markhoz goat sperm after the freezing and thawing process. Semen samples were collected and diluted in the extender, divided into five equal aliquots and supplemented with 0, 1, 10, 100 and 1000 nM MitoQ and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, lipid peroxidation (LPO), DNA fragmentation, reactive oxygen species (ROS) concentration, viability and apoptotic-like changes were measured. The use of 10 and 100 nM MitoQ resulted in higher (P≤0.05) total motility (TM), progressive motility (PM), viability, membrane functionality, mitochondrial activity, and acrosome integrity compared to the other groups. On the other hand, LPO, apoptotic-like changes, DNA fragmentation and ROS concentration were lower (P≤0.05) in MQ10 and MQ100 groups compared to the other groups. MitoQ has no effect (P>0.05) on sperm abnormal morphology and velocity parameters. In conclusion, MitoQ can reduce oxidative stress by regulating mitochondrial function during the cryopreservation process of buck sperm and could be an effective additive in the cryopreservation media to protect sperm quality., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Equine embryos >300 μm require puncture before vitrification. Protocols that do not require pre-puncture would make vitrification easier and allow for its widespread use., Objectives: To design a successful vitrification protocol for embryos >300 μm without puncture as a pre-treatment., Study Design: Experimental in vivo study., Methods: Thirty-eight embryos were divided into 3 groups (G1: ≤300 μm, n = 11; G2: >300-500 μm, n = 20; G3: >500 μm, n = 7). Embryos were vitrified using a human vitrification kit. Following a 15 min exposure to equilibration solution (ES; 7.5% DMSO +7.5% ethylene glycol [EG] in a base medium [BM] of M199 HEPES-buffered medium [H199] + hydroxypropyl cellulose + gentamycin), embryos were exposed for ≤90 s to a vitrification solution (15% DMSO +15% EG + 0.5 M trelahose in BM), loaded onto a Cryolock and plunged into LN2. Warming was undertaken by plunging the Cryolock tip into 1 mL of H199 + 20% FBS + pen/strep +1 M sucrose at 42°C for 1 min. The embryos were then moved to a 0.5 M sucrose solution for 4 min, then placed in Vigro Hold for 4 min prior to transfer to a recipient., Results: Pregnancy rates were 81.8% (9/11) for G1, 80% (16/20) for G2, and 0% (0/7) for G3. The largest embryo to survive was 480 μm., Main Limitations: Limited numbers and only one pregnancy was followed to term., Conclusions: Equine embryos ≤480 μm can be successfully vitrified using a protocol with a longer exposure time to the ES. This does not appear to have a negative effect on early embryonic development., (© 2024 EVJ Ltd.)

The study investigated the impact of resveratrol (RES) on bull sperm cryopreservation employing conventional slow (CS) and ultra-rapid (UR) freezing methods on sperm quality and in vitro fertility. Twenty-four ejaculates from four bulls were divided into four groups based on the cryopreservation method and RES addition: CS-RES (n = 80), CS-Co (n = 80), UR-RES (n = 24), and UR-Co (n = 24). The CS freezing involved exposing sperm straws with 5% glycerol to liquid nitrogen (LN 2 ) vapors, while UR freezing submerged sperm drops with 100 mM sucrose directly into LN 2 . Overall, sperm kinematic parameters and integrity of plasma and acrosome membranes significantly decreased (P < 0.001) after cryopreservation. Post-thaw values of motilities (total [TM] and progressive [PSM]), velocities (curvilinear and straight-line), beat cross frequency (BCF), and sperm with intact plasma membrane/intact acrosome (PI-/PNA-) were higher (P < 0.05) with CS-RES and CS-Co treatments compared to UR-RES and UR-Co treatments. CS-RES treatment resulted in greater percentages (P < 0.05) of TM, PSM, PI-/PNA-, and fertility (blastocyst rate) than their control, CS-Co; while UR-RES showed higher BCF values (P < 0.05) than its control, UR-Co. Additionally, UR-RES treatment exhibited lower oxidative stress percentages than UR-Co (P < 0.05). This study presents the following conclusions: (1) the CS freezing resulted in better cryosurvival of bull sperm than UR freezing; (2) the RES supplementation to CS freezing medium improved sperm motility, membrane integrity, and fertility; and (3) despite low cryosurvival sperm and fertility, the RES addition to ultra-rapid freezing medium reduced oxidative stress., Competing Interests: Declaration of Competing Interest None of the authors have any conflict of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Vitrification is a technique of cryopreservation that has been proposed as a promising alternative method for the preservation of oocytes, embryos and gonadal tissue., Objective: To determine the effect of different antioxidants on post-thaw viability, morphology of retrieved oocytes and histology of vitrified ovarian tissue., Materials and Methods: Four different antioxidants [i.e., resveratrol (20 uM), ZnSO 4 (500 uM), curcumin (25 uM) and quercetin (1 uM)] were evaluated after their addition to the vitrification and warming media for their effects on the viability and morphology of retrieved oocytes and the histology of vitrified ovarian tissue., Results: The number of oocytes retrieved from ovarian tissue from the above mentioned antioxidants and vitrified control were 34, 41, 26, 31 and 46 respectively. Among these the number of viable oocytes were found to be 24 (70.6%), 30 (73.1 %), 20 (76.9%), 26 (83.9%) and 33 (71.7%) and the number of oocytes found morphologically normal were 24 (70.6%), 26 (63.4%), 18 (69.2%), 21 (67.7%) and 34 (73.9%) for the above mentioned different antioxidants and vitrified control, respectively. Non-significant (P. > 0.05) differences were found between different treatment groups. Histomorphological evaluation of the ovarian cortical tissue showed that the percentage of intact follicles was significantly (P < 0.05) higher in the fresh control (84.19±3.9) than in other groups. Non-significant differences were found between resveratrol (50.2±5.5), curcumin (48.7±5.7), quercetin (51.6±4.8) and the vitrified control (42.7±6.1) groups; however, the ZnSO4 supplemented group (23.1±8.54) differed significantly (P < 0.05) from other antioxidant groups but was non-significant (P > 0.05) with the vitrified control group (42.7±6.1)., Conclusion: The addition of antioxidants resveratrol, curcumin and quercetin at these concentrations tended to non-significantly improve the follicular integrity after vitrification. Doi.org/10.54680/fr24410110212.

For sperm cryopreservation, the conventional method, which requires glycerol, has been used for a long time. In addition, the permeable cryoprotectant-free vitrification method has been continuously studied. Although the differences of cryopreservation effects between the two methods have being studied, differences in microRNA (miRNA) profiles between them remain unclear. In this study, we investigated the differences in miRNA expression profiles among conventional freezing sperm, droplet vitrification freezing sperm and fresh human sperm. We also analyzed the differences between these methods in terms of differentially expressed miRNAs (DEmiRs) related to early embryonic development and paternal epigenetics. Our results showed no significant differences between the cryopreservation methods in terms of sperm motility ratio, plasma membrane integrity, DNA integrity, mitochondrial membrane potential, acrosome integrity, and ultrastructural damage. However, sperm miRNA-sequencing showed differences between the two methods in terms of the numbers of DEmiRs (28 and 19 with vitrification using a nonpermeable cryoprotectant and the conventional method, respectively) in postthaw and fresh sperm specimens. DEmiRs related to early embryonic development and paternal epigenetics mainly included common DEmiRs between the groups. Our results showed that the differences between conventional freezing and droplet vitrification were minimal in terms of miRNA expression related to embryonic development and epigenetics. Changes in sperm miRNA expression due to freezing are not always detrimental to embryonic development. This study compared differences in miRNA expression profiles before and after cryopreservation between cryopreservation by conventional and vitrification methods. It offers a new perspective to evaluate various methods of sperm cryopreservation., (Copyright © 2024 Copyright: ©The Author(s)(2024).)

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC 3 C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DS ion ) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC 3 C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC 3 C)&#95;DS ion(100%) /1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect., (© 2024 Wiley‐VCH GmbH.)

One of the factors responsible for less pregnancy rates is the use of frozen semen in sheep due to the oxidative stress created by the process. The aim of this experiment was to test the effects of adding coenzyme Q-10 (CoQ10) to the seminal extender on sperm quality and the pregnancy rate of sheep. In this study, ejaculates from eight Dorper rams of reproductive age were used and tested in four treatments: Control (pure BotuBov®), C1 (175 µM of CoQ10), C3 (350 µM of CoQ10), and C7 (700 µM of CoQ10). Samples were collected in triplicate from each animal, and sperm analysis was performed by CASA after thawing at 0 h and 2 h. The samples were also analyzed by flow cytometry for plasma and acrosomal membrane integrity, stability, lipid peroxidation, mitochondrial potential, and superoxide anion production. In total, 198 ewes were inseminated by laparoscopy and divided into two groups: control (n=98) and C7 (n=100). Pregnancy diagnosis was performed at 30 days. Coenzyme Q10 proved to be safe for semen cryopreservation, not altering sperm kinetic values between the groups post-thawing. In flow cytometry, the C1 and C7 groups achieved a better index of plasma membrane integrity and membrane stability (P<0.05). A increased pregnancy rate was observed in C7 (52 %) compared to the control (38 %). In conclusion, coenzyme Q10 assists in the cryopreservation process, protecting the sperm cell and improving pregnancy rates in ewes., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The effective combination of semen cryopreservation and artificial insemination has a positive effect on the conservation of germplasm resources, production and breeding, etc. However, during the process of semen cryopreservation, the sperm cells are very susceptible to different degrees of physical, chemical, and oxidative stress damage. Oxidative damage is the most important factor that reduces semen quality, which is affected by factors such as dilution equilibrium, change of osmotic pressure, cold shock, and enzyme action during the freezing-thawing process, which results in the aggregation of a large amount of reactive oxygen species (ROS) in sperm cells and affects the quality of semen after thawing. Therefore, the method of adding antioxidants to semen cryoprotective diluent is usually used to improve the effect of semen cryopreservation. The aim of this experiment was to investigate the effects of adding five antioxidants (GLP, Mito Q, NAC, SLS, and SDS) to semen cryoprotection diluent on the cryopreservation effect of semen from Saanen dairy goats. The optimal preservation concentrations were screened by detecting sperm viability, plasma membrane integrity, antioxidant capacity, and acrosomal enzyme activities after thawing, and the experimental results were as follows: the optimal concentrations of GLP, Mito Q, NAC, SLS, and SDS added to semen cryopreservation diluent at different concentrations were 0.8 mg/mL, 150 nmol/L, 0.6 mg/mL, 0.15 mg/ mL, 0.6 mg/mL, and 0.15 mg/mL. The optimal concentrations of the five antioxidants were added to the diluent and analyzed after 1 week of cryopreservation, and it was found that sperm viability, plasma membrane integrity, and mitochondrial activity were significantly enhanced after thawing compared with the control group (P < 0.05), and their antioxidant capacity was significantly enhanced (P < 0.05). Therefore, the addition of the above five antioxidants to goat sperm cryodilution solution had a better enhancement of sperm cryopreservation. This study provides a useful reference for exploring the improvement of goat semen cryoprotection effect., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)