Cryo letters最新文献

Background: Cryopreservation of spermatozoa is a biotechnology used for fertilization purposes and preservation of genetic material in various domestic species.

Objective: To determine the efficacy of two different commercial semen diluents in the cryopreservation of epididymal semen of domestic cats.

Materials and methods: Twenty male cats aged between 1- 3 years and weighing 2.5- 4.5 kg were used in the study. The testicular tissues removed from the cats were immediately brought to the laboratory in physiological saline and the epididymal parts were trimmed in commercial semen extenders (INRA 96, Group I; OPTIXCELL, Group II). Diluted semen samples were cooled to 4°C and filled into 0.25 mL straws. Semen samples were frozen in a programmable semen freezing device and then placed in a liquid nitrogen container at -196 C. Semen samples were thawed at 38 degree C for 25 s. Thawed semen samples were evaluated in terms of motility and kinematic parameters using CASA.

Results: No statistical difference was found between the groups in terms of total motility, progressive motility, and velocity parameters at 4 degree C. The rate of spermatozoa at slow speeds was found to be lower in group II. In addition, after freezing and thawing process, no statistical difference was observed between the groups in terms of motility, kinematics, and velocity parameters.

Conclusion: Both commercial semen extenders can be used for cryopreservation of cat epididymal semen. https://doi.org/10.54680/fr24610110712.

Background: Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality.

Objective: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion.

Materials and methods: A total of 20 ejaculates from four Marwari stallions (five ejaculates from each) were collected. The gel free semen was extended with primary extender in equal volume and then divided in three equal groups, namely control (C), zinc (ZnO) and gold (Au), and centrifuged to obtain a final concentration of 100-150 x10⁶ mL^-1 and then cryopreserved. Cooled and post-thaw semen evaluations were conducted for various seminal characteristics, viz. progressive sperm motility, sperm plasma membrane integrity, sperm viability, acrosome integrity and mitochondrial membrane potential.

Results: Cooled semen (4 degree C for 2 h) evaluation revealed non-significant differences among the groups (C, ZnO and Au) for all the semen quality parameters. However, at the post-thaw stage, progressive sperm motility, sperm plasma membrane integrity, acrosome integrity and oxidative parameters were significantly (P < 0.01) higher in the ZnO group than Au and C.

Conclusion: The addition of ZnO nanoparticles improves the post thaw quality of stallion spermatozoa by reducing the oxidative stress, but Au nanoparticles had no effect on cooled as well as post-thaw semen quality. https://doi.org/10.54680/fr24610110212.

Background: The low-temperature cholesterol esterase is primarily used in industries such as papermaking and healthcare.

Objective: To discover a microorganism with high cholesterol esterase activity and tolerance to low temperatures, leading to the promotion of the sustainable utilization of marine cold-adapted microbial resources and fostering industrial development.

Materials and methods: This study isolated a strain producing low-temperature cholesterol esterase from marine samples in the China Bohai Sea. The strain was identified through 16S rDNA sequencing and named Panthenia agglutinosa Y03. The cholesterol esterase gene (PaChe) from P. agglutinosa Y03 was cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme PaChe was purified and characterized. The structure of PaChe was predicted using AlphaFold2, and molecular docking was performed with cholesterol linoleate as the ligand.

Results: The enzyme protein has a molecular weight of 56.35 KDa, a theoretical pI of 7.24, lacks a signal peptide, and exhibits structural features of the α/β hydrolase superfamily protein. The concentration of the purified PaChe is 0.5 mg/mL, with a specific activity of 42.7 U/mg. The optimal working temperature is 30 degree C, and the enzyme retains activity at degree C, demonstrating weaker thermal stability. The optimal pH is 7, and the enzyme maintains over 70 % activity at pH 9. Na⁺, Ca²⁺ and Mg²⁺ are the primary activators, while Ba²⁺, Fe²⁺, Mn²⁺, Cu²⁺ and chemical agents such as SDS as inhibitors, with Cu²⁺ exhibiting particularly significant inhibitory effects.

Conclusion: This study establishes the theoretical groundwork for the development and utilization of a novel low-temperature cholesterol esterase. https://doi.org/10.54680/fr24610110412.

Background: Most plant species produce seeds that can be dried to c. 3-7% moisture content, i.e., orthodox, and successfully stored in liquid nitrogen. However, tea seeds are often described as recalcitrant, i.e., they die when to lower moisture levels safe for cryopreservation.

Objective: In this study, we investigated the tolerance to desiccation and freezing of Longjing tea (Camellia sinensis) seeds and analyzed the reason behind seed mortality during desiccation and cryopreservation.

Materials and methods: We analyzed the desiccation capacity of tea seeds from Hangzhou, China and subjected the desiccated seeds to programmed cooling and thermal analysis using differential scanning calorimeter (DSC). Subsequently, we used the exotherms to determine the frozen and unfrozen water. We analyzed seed germination and seedling establishment at different water content after storing seeds in liquid nitrogen and correlated the survival with the bound and unbound water.

Results: Results showed that the seeds could tolerate drying to 7.9 % moisture content without significant viability loss. Moreover, the germination percentage declined sharply after cryopreservation, and the lowest moisture content at which a normal seedling could develop was 11.3+/-1 %, although seeds dried to lower MC did germinate but did not grow into the seedling phase.

Conclusion: Longjing tea seeds used in this study were neither orthodox nor recalcitrant. We show that the interaction between lipids and freezable water in seeds may lead to the cryoinjury. https://doi.org/10.54680/fr24610110612.

Cryopreservation is a well-known strategy to conserve genetic resources at ultra-low temperature. However, there is still limited knowledge on the cellular processes and molecular adjustments that allow cells to withstand the multiple stresses to which they are exposed during cryopreservation. To evaluate these processes, transcriptomics, the sub-discipline of omics that simultaneously examines mRNA transcripts formed by transcription from the genome, has been recently used. This article reviews recent scientific studies which use the basic principles of cryopreservation practices together with transcriptomics approaches, within the conceptual framework of cryobiomics. Moreover, the connections between factors that may be useful to optimize and validate approaches for mammalian or plant cell cryopreservation are also assessed. Transcriptomic applications are mainly performed with methods such as reverse transcriptase polymerase chain reaction (RT-PCR), simultaneous polymerase chain reaction (real-time PCR), northern blot, microarray/biochip and gene expression analysis (SAGE). Transcriptomic technologies allow a global view of gene expression profiles of different mammalian or plant cell types to be obtained before and after cryopreservation under multiple stress conditions. For these processes, small amounts of RNA enable efficient transcriptomics analysis. Transcriptomic analysis of cryopreserved mammalian and plant cells provides a conceptual way to identify the genes and their relative alterations in transcriptional abundances together with non-coding RNAs involved in important pathways related to cell viability and proliferation during and after cryopreservation. Moreover, it greatly contributes to understanding of non-fatal cryodamage and related developmental disorders in cryopreserved mammalian oocytes and sperm. In addition, single cell transcriptomics has the potential to non-invasely monitor immune actions and to diagnose the stage of the inflammatory process in kidney. Finally, qRT-PCR and RNA-seq studies have also revealed that some transcription factors are effective at inducing cold tolerance in many plants by elevating the levels of soluble sugars, proline and unsaturated fatty acids in cells. Hence transcriptomics studies may also aid investigations of the main mechanisms behind the so-called 'cryo-recalcitrance' that is observed mostly in plant cells. https://doi.org/10.54680/fr24610110112.

Background: The development of assisted reproduction techniques for birds is useful for ex situ conservation but is limited. For the golden eagle (Aquila chrysaetos), only artificial insemination procedures using extenders developed over 50 years ago have been described.

Objective: To evaluate the effect of the viscosity of the cryopreservation medium on the acrosomal reaction ability of A. chrysaetos sperm.

Materials and methods: Viscosity was determined in 45 ejaculates. A design using the Lake medium, 6 % dimethylacetamide (DMA), and Ficoll (10 %, 35 %, and 45 %) was developed to create conditions of viscosity that was lower, similar, and higher than those determined in fresh semen.

Results: The viscosity of fresh semen was 3.2320 mPa/s. In aliquots of the Lake medium supplemented with 6 % DMA and 10 %, 35 %, and 45 % Ficoll, the viscosities were 2.1698 mPa/s, 3.5393 mPa/s, and 6.1123 mPa/s, respectively. Post-thaw, in the aliquot with 10 % Ficoll, 74 % of sperm were alive, with 28 % mobility, and 21 % exhibited an acrosomal reaction with percentages that were significantly lower (P < 0.05) than those observed in sperm frozen with 30 % and 45 % Ficoll.

Conclusion: Viscosity has a positive influence on the viability of cryopreserved semen. https://doi.org/10.54680/fr24610110512.

Background: Characterization of intracellular ice formation (IIF) in oocytes during the freezing and thawing processes will contribute to optimizing their cryopreservation. However, the observation of the ice formation process in oocytes is limited by the spatiotemporal resolution of the cryomicroscope systems.

Objective: To observe the intracellular icing of oocytes during cooling and rewarming, and to study the mechanism of formation and growth of intracellular ice in oocytes.

Materials and methods: Mouse oocytes were frozen at different cooling rates to induce intracellular ice formation using a cryomicroscopy system consisting of a microscope equipped with a cryogenic cold stage, an automatic cooling system, a temperature control system, and a high-speed camera. The growth patterns of intracellular ice in oocytes were analyzed from the images recorded. Finally, the growth rate of intracellular ice formation in oocytes was calculated using an automatic intracellular ice tracking method.

Results: The IIF temperature decreased gradually with the increase in cooling rate. Initiation sites of IIF could be classified into three categories: marginal type, internal type and coexisting type. There was a strong predominance for ice crystal initiation site in the oocytes, with up to 80% of the initiation sites located in the marginal region. The intracellular ice growth modes of darkening and twitching cells were characterized by "spreading" and "clustering", respectively. In addition, twitching cells started to recrystallize during rewarming, while darkening cells did not. The instantaneous maximal growth rate of ice crystals in twitching cells was about 10 times higher than that in darkening cells.

Conclusion: By visualising the growth of ice crystals in mouse oocytes during cooling and rewarming, we obtained valuable information on the kinetics of ice formation and melting in these cells. This information can help us understand how ice formation and melting affect the viability and quality of oocytes after cryopreservation. Doi.org/10.54680/fr24310110412.

Background: Ovarian tissue cryopreservation for fertility preservation carries a risk of malignant cell re-seeding. Artificial ovary is a promising method to solve such a problem. However, ovary decellularization protocols are limited. Hence, further studies are necessary to get better ovarian decellularization techniques for the construction of artificial ovary scaffolds.

Objective: To establish an innovative decellularization technique for whole porcine ovaries by integrating liquid nitrogen with chemical agents to reduce the contact time between the scaffolds and chemical reagents.

Materials and methods: Porcine ovaries were randomly assigned to three groups: novel decellularized group, conventional decellularized group and fresh group. The ovaries in the novel decellularized group underwent three cycles of freezing by liquid nitrogen and thawing at temperatures around 37 degree C before decellularization. The efficiency of the decellularization procedure was assessed through histological staining and DNA content analysis. The maintenance of ovarian decellularized extracellular matrix(ODECM) constituents was determined by analyzing the content of matrix proteins. Additionally, we evaluated the biocompatibility of the decellularized extracellular matrix(dECM) by observing the growth of granulosa cells on the ODECM scaffold in vitro.

Results: Hematoxylin and eosin staining, DAPI staining and DNA quantification techniques collectively confirm the success of the novel decellularization methods in removing cellular and nuclear components from ovarian tissue. Moreover, quantitative assessments of ODECM contents revealed that the novel decellularization technique preserved more collagen and glycosaminoglycan compared to the conventional decellularized group (P<0.05). Additionally, the novel decellularized scaffold exhibited a significantly higher number of granulosa cells than the conventional scaffold during in vitro co-culture (P<0.05).

Conclusion: The novel decellularized method demonstrated high efficacy in eliminating DNA and cellular structures while effectively preserving the extracellular matrix. As a result, the novel decellularized method holds significant promise as a viable technique for ovarian decellularization in forthcoming studies. Doi.org/10.54680/fr24310110212.

Background: Aquaporins (AQPs) are essential proteins that facilitate the rapid movement of water and cryoprotective agents (CPAs) during the cryopreservation process, and ensure the cryo-tolerance of sperm cells.

Objective: This study evaluated the preservation of aquaporin levels in human sperm after undergoing freezing using natural deep eutectic solvents (NADES) as CPAs for cryoprotection.

Materials and methods: From June 2021 to October 2022, 35 semen samples with normal sperm parameters were acquired from the Mehr Infertility Treatment Institute in Rasht, Iran. The samples were divided into several groups for analysis: control group (not frozen), group frozen with SpermFreeze Solution, and groups frozen with different NADESs, including ChS, ChX, ChU, ChG, GlyP, and EtP. After thawing, various aspects for each group were assessed, including the integrity and condensation of sperm chromatin, viability, motility, integrity of acrosome, and the expression of AQP1, AQP3, AQP7, AQP8, and AQP9 genes.

Results: The analysis of gene expression revealed that freezing with ChS and GlyP preserved the expression of the AQP1 and AQP3 genes compared to the control group. Regarding AQP7 and AQP8, significant differences were not observed in expression levels between certain NADES groups (e.g., ChS, ChU, and GlyP) and the control group. Additionally, samples frozen with specific NADESs, such as ChS, ChG, EtP, and GlyP, exhibited preserved levels of AQP9 expression when compared to the control group.

Conclusion: These findings emphasize the importance of NADES in preserving the expression of aquaporins in cryopreserved human sperm and their important fertility parameters. Doi.org/10.54680/fr24310110512.

Background: The industrial scale cryo-storage of raw tissue materials requires a robust, low-cost and easy-to-operate method that can facilitate the down-stream process.

Objective: The study was aimed to develop the multifunctional protective solutions (MPS) for transportation at ambient conditions and also subsequent cryo-storage below -20 degree C of raw porcine hides for tissue engineering and regenerative medicine.

Materials and methods: Protective solutions with antimicrobial activity and proteinase-inhibiting activity were developed and tested for its efficacy in preserving the extracellular matrix of porcine dermis from microbial spoilage, proteolytic degradation, freeze damage and excessive dehydration during shipping and cryo-storage. The MPSs contained phosphate-buffered saline with ethylene diamine tetra acetic acid (EDTA) added as chelator and proteinase inhibitor, as well as glycerol or maltodextrin (M180) as cryoprotectants.

Results: MPSs prepared with EDTA and glycerol or M180 had significant antimicrobial activity and proteinase-inhibiting activity during the period of shipping and handling. Glycerol and M180 prevented eutectic salt precipitation and excessive freeze dehydration upon cryo-storage of porcine hides. Without glycerol or M180, hides could be freeze-dehydrated to the low hydration at ~0.4 g/g dw, and formed irreversible plications after freezing. A critical hydration (0.8~0.9 g/g dw) was observed for the extracellular matrix of porcine dermis, and dehydration to a lower level could impose enormous stress and potential damage. The soaking of porcine hides in MPSs decreased water content as glycerol and M180 entered into dermis. Upon equilibration, the glycerol content in the tissue was about 94% of the incubating glycerol solution, but the M180 content in the tissue was only about 50% of the incubating M180 solution, indicating that M180 did not get into the entire aqueous domain within dermis. MPSs reduced ice formation and increased the unfrozen water content of porcine raw hides upon cryo-storage.

Conclusion: MPSs prepared with EDTA and glycerol or M180 have antimicrobial activity and proteinase-inhibiting activity, which can be used for transportation and cryo-storage of raw hides at the industrial scale. Glycerol at 7.5% w/v and M180 at 20% w/v were sufficient to prevent freeze damage and excessive freeze dehydration. Doi.org/10.54680/fr24310110312.