Cryo letters最新文献

Background: Mico-volume semen freezing is essential and used popularly for fertility preservation of patients suffering cancer or undergoing male reproductive system related surgeries, and for other reasons that may risk fertility potential in ART cycles. However, clinicians and embryologists still face some unresolved technical and theoretical issues about the frozen-thawed efficiency.

Objective: To choose the appropriate freezing method for different volumes of normal semen samples.

Materials and methods: We investigated the frozen-thawed outcomes of semen with different volumes (20 uL, 50 uL, 100 uL, 200 uL, 500 uL and 1 mL) using two freezing methods (FLNV, static liquid nitrogen vapour cooling followed by liquid nitrogen preservation; RFLN, direct rapid freezing in liquid nitrogen) and analyzed the vitality, progressive motility and DNA fragmentation index of thawed sperm.

Results: We found that semen freezing with volumes more than 100 uL had better outcomes than volumes less than or equal to 50 uL after thawing. FLNV presented a higher efficiency for cryopreservation of semen with volumes less than 50 uL.

Conclusion: For smaller (micro) volumes, the FLNV technique is better than the RFLN method. https://doi.org/10.54680/fr24110110412.

Background: When sperm are cryopreserved, reactive oxygen species (ROS) are formed that are detrimental to the sperm.

Objective: To evaluate the effects of oleic acid and trehalose added to ram semen extender on sperm parameters, lipid peroxidation (MDA), and superoxide dismutase (SOD) enzyme levels of spermatozoa following the freeze/thawing processes.

Materials and methods: Ejaculates were collected from four rams and pooled at 35 degree C. Pooled ejaculates were diluted with oleic acid at 0 mM and trehalose at 0 mM (O0 T0) as the control. The Tris-based extender was supplemented with either 0.5 (O0.5) or 1 (O1) mM of oleic acid or 25 (T25) or 50 (T50) mM of trehalose alone, and in combination [0.5 mM oleic acid + 25 mM trehalose (O0.5 T25), 0.5 mM oleic acid + 50 mM trehalose (O0.5 T50), 1 mM oleic acid + 25 mM trehalose (O1 T25) and 1 mM oleic acid + 50 mM trehalose (O1 T50)]. The semen was frozen by the traditional liquid nitrogen vapour method and stored at -196C in the liquid nitrogen tank.

Results: Semen extender containing O1T25 significantly improved the total motility, when compared with other treatment groups (P<0.05), except for O1 T50. O1 T50 had a higher viability rate than any other treatment. The addition of O1 T25 and O1 T50 increased DNA and membrane integrity of spermatozoa post-thawing compared to other treatments (P<0.05). The level of MDA was significantly (P<0.05) lower in extenders supplemented with O1, O0.5 T25, O0.5 T50, O1 T25 and O1T50 compared to the other treatment groups. In addition, SOD levels were higher in groups treated with O1 T25 and O1 T50 than the other treatment groups (P<0.05).

Conclusion: The addition of a combination of oleic acid and trehalose concentrations to Tris-based extender improved the quality of ram semen post-thawing. Doi.org/10.54680/fr23610110712.

Background: Antioxidant present in sperm cells protects them from oxidative damage. However, sperm are more susceptible to peroxidative damages due to the loss of these enzymes during cryopreservation and their survival and fertility may be compromised. Insulin like growth factor-1 (IGF-1) has an antioxidant effect and could maintain sperm motility.

Objective: To improve seminal parameters, mitochondrial membrane potential (MMP), oxidative status and DNA integrity of buck semen after freeze-thawing by fortification of goat semen diluent with various concentrations of IGF-1.

Materials and methods: Fifty ejaculates were collected and were extended with tris- citric acid- fructose diluent with 10% egg yolk and 6% glycerol with sperm concentrations of 1×10⁸ mL^-1. Post-cryopreserved sperm were assessed for motility and a range of other functional parameters.

Results: In post-thaw semen sperm motility, live sperm count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, malondialdehyde (MDA), protein carbonyl content (PCC), TUNEL positive sperm differed significantly (P<0.05) with the various concentrations of IGF-1 used. Sperm functional parameters post-thawing were significantly (P<0.05) better in 250 ng/mL IGF-1. IGF-1 protects against lipid peroxidation by lowering MDA and PCC production, thus reducing the harmful effect of reactive oxygen species. The kidding percentage using the artificial insemination technique was significantly higher ( i.e., 40%) in the group supplemented with 250 ng/mL of IGF-1 than in the non-supplemented group (i.e., 30%).

Conclusion: IGF-1 may be used to improve post-thaw semen quality and fertility as measured by actual kidding rate. Doi.org/10.54680/fr23610110312.

Background: Cicer microphyllum Benth. is a crop wild relative (CWR) of chickpea (C. arietinum L.), that possess useful genes for cold and drought tolerance. The species is being conserved in the In Vitro Active Genebank for short- to medium-term conservation. Cryopreservation would be a useful complementary approach for its long-term conservation.

Objective: The current work aimed to develop an efficient cryoconservation protocol for cryobanking of C. microphyllum shoot tips.

Materials and methods: In vitro shoot tips excised from 4-month old shoot cultures grown on B5 + 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO₃ medium were cryoconserved using a droplet-vitrification technique. Post-thaw regrowth was evaluated after: (i) preculture medium (B5 basal, B5 + 3, 4, 6 and 10% sucrose), (ii) preculture incubation temperature (25 ± 2, 10, 8 and 22/5 degree C), (iii) PVS2 duration (10, 20, 30. 40, 50 and 60 min) and (iv) regrowth medium (B5) supplemented with 0.5 mg/L KIN + 0.1 NAA mg/L; 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO₃; 0.2 mg/L BAP + 10 mg/L AgNO₃; 0.2 mg/L BAP + 20 mg/L AgNO₃ and 0.2 mg/L BAP + 30 mg/L AgNO₃.

Results: In vitro shoot tips grown on B5 + 0.5 mg/L KIN + 0.1 mg/L NAA + 10 mg/L AgNO₃, precultured on B5 + 6% sucrose at 10 degree C for 3 days, followed by PVS2 treatment for 20 min, unloading solution for 60 min and regrowth on B5 + 0.2 mg/L BAP + 20 mg/L AgNO₃ resulted in highest survival (57%) and regrowth (40%) after cryoconservation.

Conclusion: The standardized protocol was successfully used for cryobanking of in vitro shoot tips of C. microphyllum in the In Vitro Base Genebank of ICAR-NBPGR, New Delhi. Doi.org/10.54680/fr23610110412.

Background: The search for compounds that can prevent cold stress-attributed apoptosis is of immediate interest. In this regard, the use of neuropeptides, in particular synthetic leu-enkephalin, as protectors is promising, due to their ability to prevent the development of apoptosis under some stresses.

Objective: To study apoptotic phenomena after cold stress and to evaluate the protective effect of dalargin on these processes.

Materials and methods: The study was performed on a L929 fibroblast line. The impact of cold stress and the protective effect of dalargin on apoptosis against cold stress were evaluated using morphological parameters, distortion of cell membrane asymmetry and release of cytochrome C into the cell cytoplasm. To assess the proliferative potential of fibroblasts, mechanical damage to the monolayer was modeled as a scratch wound.

Results: The study showed that cold stress induced apoptosis in L929 fibroblasts and reduced proliferation in the fibroblast monolayers. Conspicuous apoptotic changes were found to develop only after a certain time after cold exposure, when the cells were returned to normothermia. Dalargin was demonstrated to exert a protective effect on proliferation and against apoptosis during cold stress. Using the opioid receptor antagonist naloxone, we revealed that the protective mechanism of dalargin appeared to be due to activation of delta-opioid receptors of L929 fibroblasts, which affected the development of apoptosis.

Conclusion: In addition to their fundamental value, these findings are of practical importance since neuropeptides, in particular dalargin, added to perfusion solutions and media for hypothermic preservation of organs and cells, can improve their efficiency. Doi.org/10.54680/fr23610110212.

Background: Cryopreservation currently represents the most suitable strategy for the long-term conservation of plant germplasm. While much effort has focused on the development of protocols to enable successful cryostorage, there are few, if any reports, that consider the effect of cryogenic temperatures on the phytohormone status of the seed and developing seedlings.

Objective: To investigate the effect of cryopreservation on external seed coat features as well as levels of indole-3-acetic acid (IAA), abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in maize.

Materials and methods: Two groups of seeds at 6% moisture content were compared: one was maintained at 4 degree C (control) while the other was exposed to LN within cryo-vials.

Results: Seeds exposed to cryogenic temperatures were characterized by the presence of large cracks in the seed coat compared with control seeds. Cryogenic exposure also resulted in a reduction in biomass and plant height. Results from the phytohormone analysis showed an initial reduction in the levels of IAA, ABA and ACC after 7 days of growth followed by sharp increase in levels relative to the control by 14 days. Whilst the roles of ABA and ethylene (and by extension, its precursor ACC) are well studied as stress response molecules, much less is known about the potentially vital role of auxins in regulating plant growth under conditions of low temperature stress.

Conclusion: It is postulated that the interaction of all three hormones modulate crosstalk between various stress responses and recovery pathways to ameliorate the damage caused by freezing stress and enable plant survival. Given the dearth of information on phytohormones in cryobiology, more studies are needed to fully elucidate these relationships in the context of freezing stress caused by liquid nitrogen. Doi.org/10.54680/fr23610110612.

Background: Amides are low molecular weight cryoprotectants. N-methylacetamide (MA) is one of the cryoprotectant agents in this group.

Objective: To investigate the cryoprotective effect of MA in rabbit semen.

Materials and methods: For this purpose, six ejaculates from six New Zealand rabbits were collected and pooled using an artificial vagina. Pooled semen was divided into four equal parts and diluted with TCG+ egg yolk. CPA was added to form the following groups: Control with 6% DMSO; Group 1 with 1% MA; Group 2 with 2% MA; and Group 3 with 3% MA. After the addition of CPA, the semen eqilibration procedure was started. Sperm were then drawn into 0.25 mL straws, frozen by automatic semen freezing and stored in a liquid nitrogen container. Pipettes were thawed after 24 h and analyses were performed.

Results: Total, progressive and rapid motility values of the Control group were higher than those of the MA groups (p<0.05). However, there was no statistical difference between the Control and Group 2 in terms of these parameters. While there was no statistical difference between the groups in terms of acrosome damage and mitochondrial membrane potential, the best results were observed in Control, Group 2, Group 1 and Group 3, respectively. When we compared all groups, no difference was found in terms of MDA, CAT and GSH-Px. There was a statistical difference between Group 3 and the Control in terms of GSH level (p<0.05).

Conclusion: DMSO appeared to be more useful for the cryopreservation of rabbit semen compared to MA. Doi.org/10.54680/fr23610110812.

Background: Tarenaya spinosa is a medicinal species used for treating respiratory and inflammatory diseases. Various biotechnological studies have been developed for in vitro establishment of plants and long-term conservation of this species.

Objective: This study aimed to establish a new cryopreservation protocol using the D cryo-plate technique for in vitro-grown shoot tips of T. spinosa.

Materials and methods: Different steps of the cryopreservation procedure were evaluated in this work: preculture; sucrose concentration of calcium alginate gel; concentration and time of exposure to osmoprotective loading solution; time of exposure to silica gel; and regrowth on recovery medium.

Results: The optimal procedure included preculture with increasing sucrose concentration (from 0.25 to 0.50 M), encapsulation and dehydration over silica-gel for 60 min. Increasing sucrose concentration in the loading solution or exposure duration had a negative effect on recovery of cryopreserved shoot tips. However, the association of calcium alginate gel enriched with 0.6 M sucrose with post-rewarming culture with BAP for 2 weeks resulted in the most efficient cryopreservation protocol (76% survival and 70% shoot recovery).

Conclusion: The plants developed after cryopreservation maintained their in vitro multiplication capacity and demonstrated the efficiency of long-term conservation by D cryo-plate technique for T. spinosa. Doi.org/10.54680/fr23610110512.

The process of freezing biological material at extremely low temperatures is known as cryopreservation. To ensure the preservation of cells and tissues over an extended period of time, low temperatures are applied since biological processes, including the biochemical ones, come to a halt under cryogenic conditions and thus it is possible to maintain their structural and functional integrity. The field of cryopreservation gained more prominence in the 20th century and emerged as an unavoidable technology for different applications such as cell therapy, tissue engineering, or assisted fertilization. In this work we provide an overview of various technologies in the field of cryotechnology with regard to the freezing, storage and thawing of living cells. The first part covers the freezing process, starting with cryoprotective agents regarding their protection mechanisms and compositions, passing by cryo-imaging, micro-fluidic systems, and the currently available freezing and biobanking equipment. The second part focusses on the thawing process as well as the hypothermic preservation for the short-term storage of biological materials and constructs. Doi.org/10.54680/fr23610110112.

Background: Chitin is the second largest carbon source on the earth, and chitosan oligosaccharides produced by its degradation have good application prospects in medicine, cosmetics, and agricultural production.

Objective: The discovery of a chitinase with high efficiency, high stability and clear degradation mechanism is of great help to promote the research of chitin derivatives and the development of the industrial chain.

Materials and methods: In this experiment, a low-temperature chitinase-producing strain Photobacterium sp. LG-29 was isolated from deep-sea mud in the Bohai Sea, and studied by means of molecular biology, biochemistry and bioinformatics.

Results: Purification of chitinase yielded an enzyme solution with a concentration of 0.918 mg per mL and a specific activity of 21.036 U per mg. The optimum action temperature is 35 degree C, and it is still active at 4 degree C, showing low-temperature enzymatic activity, and also has certain thermal stability. The optimum pH is 8.0, and it maintains more than 70% of the enzyme activity at pH 11, which is very stable in an alkaline environment. Mn²⁺, Ca²⁺, and Mg²⁺ are the main activators of enzymes, while Fe²⁺, Zn²⁺, etc. have extremely significant inhibitory effects on enzymes. The Km and Kcat of chitinase were determined to be 269.05 μmol/L and 0.49 min^-1, respectively. Chitinase PbCHI5 has both endonuclease and exonuclease activity. The theoretical pI of the enzyme is 4.16, which is a stable hydrophilic protein.

Conclusion: This experiment laid a theoretical foundation for the development and utilization of new low-temperature chitinases. Doi.org/10.54680/fr23510110212.