Cryo letters最新文献

Background: One of the most essential ingredients of artificial insemination (AI) programs is semen processing which needs extender with additives to protect spermatozoa against cold stress during cryopreservation. The selection of cryoprotectant and composition of extenders are of great importance for sperm survival during and after cryopreservation. The addition of small quantities of iodixanol in the freezing extender can protect spermatozoa by altering the glass transition temperature of the mixture and changing the structure of the growing ice crystals.

Objective: To reveal the effect of iodixanol fortification in semen extender on post thawed sperm qualities, DNA intactness and antioxidant status.

Materials and methods: The ejaculates were collected from Barbari goats using artificial vagina method and samples with > 80% initial progressive sperm motility were extended with tris-citric acid-fructose diluent. Iodixanol was added to the sperm preparation medium at different concentration (Control, 0 uM; 100 uM; 200 uM; 300 uM and 400 uM). Sperm concentrations were adjusted 400 million per mL and diluted semen was kept for equilibration at 5 degree C for 4 h before being cryopreserved in liquid nitrogen. Semen samples were evaluated for sperm quality at the pre-freeze and post-thaw stages.

Results: Semen samples fortified with 400 uM iodixanol had improved cryopreservation, with significantly higher motiliy, live sperm count, acrosome integrity and hypo osmotic swelling (HOS) positive spermatozoa compared to the control. In addition, fortified semen had significantly lower sperm MDA and protein carbonyl contents after cryopreservation and greater sperm DNA integrity (fewer TUNEL+ve sperm) and lower mitochondrial membrane potential. Finally, semen fortification resulted in a small improvement in post-cryopreservation kidding rate.

Conclusion: Iodixanol at 400 uM significantly improves post-thaw sperm qualities and fertility in Barbari goat and its value as a freezing extender seems to relate to an enhanced antioxidant capacity. Doi.org/10.54680/fr25310110612.

Brazil is a megadiverse country with continental dimensions. It is long acknowledged as the richest country in plant diversity, encompassing approximately 20% of the world's flora, with more than 50,000 species of plants, algae and fungi distributed in six major biomes, including two biodiversity hotspots. However, significant environmental challenges, primarily driven by climate changes and intensive, non-sustainable land use practices, have led to widespread deforestation, habitat reduction and, consequently, shifts in species distribution, genetic erosion and increased vulnerability. Considering the high rates of endemism and the global economic value of numerous Brazilian native species as crops and wild relatives, ornamentals and medicinal plants, cryopreservation emerges as a fundamental ex situ complementary strategy to safeguard its plant genetic resources. This article aims to provide a comprehensive overview of cryopreservation of native plants in Brazil during the past decade, which shows that more than 85 species from 23 families have been cryopreserved. Methods for assessing cryoinjury at the morphophysiological, biochemical, molecular and metabolic levels are reviewed. The main challenges, as well as future perspectives for the cryopreservation of Brazilian floristic diversity, are also discussed. Doi.org/10.54680/fr25310110112.

Background: Adipose tissue grafting is one of the reconstruction methods for damaged tissue repair.

Objective: To develop a convenient procedure for human adipose tissue cryopreservation without any special equipment.

Materials and methods: Adipose tissues were frozen using different combinations of permeating and non-permeating cryoprotectants at a cooling rate of -1 degree C per min and stored at -20 degree C for 1, 3, 6 and 9 months. Histo-morphological characteristics, mitochondrial activity, oil ratio (OR) index, survival and differentiation potential of mesenchymal stem cells of thawed adipose tissue were evaluated.

Results: The most damage or degeneration and OR indices of adipose tissues were detected in phosphate-buffered saline without any cryoprotectant at 1, 3, 6, and 9 months after cryopreservation (P≤0.05). The best protection of adipose tissue against freezing damage was observed when using a solution of 0.5 M DMSO + 9% FBS + 0.2 M trehalose (P < 0.05). Similarly, mitochondrial activities of thawed adipose tissues were the highest in the 0.5 M DMSO + 9% FBS + 0.2 M trehalose, but lowest in the phosphate-buffered saline. There was no difference in the stemness and differentiation potential of adipose tissue-derived mesenchymal stem cells among different cryopreservation treatments.

Conclusion: The combination of 0.5 M DMSO, 9% FBS and 0.2 M trehalose has the best protection for human adipose tissue during cryopreservation. Doi.org/10.54680/fr25310110412.

Background: The field of sperm cryopreservation requires the search for and development of new effective cryopreservatives with low toxicity.

Objective: The purpose of this study was to evaluate the characteristics of bull spermatozoa subjected to freezing and storage at -80 degree C for 28 days in a diluent with the addition of Hericium erinaceus polysaccharides.

Materials and methods: The sperm was frozen under the protection of a complex cryoprotectant and stored in an electric freezer at -80 degree C for 28 days. Before cooling and after defrosting, the biological parameters of the sperm were analyzed using the Argus-CASA system.

Results: It was established that two fractions isolated from H. erinaceus (PF1, PF2) are heteropolysaccharide peptides with a high content of glucose, xylose, mannose, galactose and arabinose. It was shown that before cooling, when a fraction obtained from dry fruiting bodies of H. erinaceus (PF2) was added to the sperm, the number of progressively motile sperm with intact DNA decreased, but their kinematic parameters did not change. After thawing, after 28 days of storage at -80 degree C, an increase in the number of progressively motile sperm with intact DNA and their kinematic parameters was observed.

Conclusion: Thus, fungal polysaccharides are obviously underestimated macromolecular substances with enormous cryostabilization potential, and polysaccharide fractions from dried fruiting bodies of H. erinaceus may in the future find wide application in the composition of new cryopreservation media. https://doi.org/10.54680/fr25210110912.

Cryopreservation is a fundamental technique for preserving the structural and functional integrity of biological material, particularly for the conservation of genetic resources in avian species. Since its development in the 1940s, this technology has advanced significantly, although challenges persist, primarily due to the unique morphology of avian sperm, which complicates cryoprotectant penetration and increases the risk of structural damage. Overcoming these challenges is crucial for improving semen preservation, supporting the sustainability of avian species, and contributing to conservation efforts. In domestic production birds, cryopreservation is essential for maintaining genetic diversity. However, these species often exhibit low tolerance to the freezing process, primarily due to the high concentration of polyunsaturated fatty acids in their sperm membranes, making them susceptible to oxidative damage. This has driven research aimed at developing more effective cryoprotectants and techniques to enhance semen quality post-thaw. Wild birds, particularly endangered species, face additional challenges in cryopreservation. These species are often managed in captivity to prevent extinction, with artificial insemination serving as a valuable tool. However, artificial insemination is constrained by low post-thaw motility rates, even when advanced cryoprotectants are employed. Research indicates that certain cryopreservation media can improve sperm motility and fertility rates, although further optimization of these methods is required. The future of avian semen cryopreservation will concentrate on customizing extenders and cryoprotectants, optimizing freezing techniques, and improving post-thaw semen quality. These advancements are essential for enhancing commercial poultry production and for the conservation of endangered species. Research in this area is expected to evolve over the next decade, developing effective solutions to address both commercial and conservation needs. https://doi.org/10.54680/fr25210110312.

Background: Allium ramosum is an important member of the genus Allium, which is commonly known as Chinese chive or fragrant-flowered garlic. Conserving the genetic diversity of different species of Allium is crucial, and cryopreservation has emerged as an important strategy for long-term conservation of alliums.

Objective: To develop a reliable protocol for the cryoconservation of A. ramosum shoot bases.

Materials and methods: Different parameters, viz. (a) cold-hardening (5 degree C for 16/8 h photoperiod), (b) PVS2 dehydration (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min), (c) pregrowth medium (MM3: MS + 0.1 mg/L NAA + 0.5 mg/L 2iP + 10 mg/L spermidine + 3% sucrose; MM10: MS + 0.1 mg/L NAA + 0.5 mg/l 2iP + 10 mg/L spermidine + 10% sucrose) and (d) preculture duration (1, 2, 3, 4 and 5 days) were tested using a vitrification technique.

Results: Shoot bases excised from 4-wk old in vitro cultures that had been cold-hardened at 5 degree C (16/8 h photoperiod) and precultured on MM10 with 10% sucrose at 5 degree C for 3 days resulted in highest post-thaw regrowth of 43% after conventional vitrification. However, when droplet-vitrification was used, post-thaw regrowth was increased to 77%. Retesting of shoot bases after 10 years of cryobanking, revealed no significant difference in the post-thaw regrowth of A. ramosum.

Conclusion: This is the first report of the long-term cryopreservation of A. ramosum shoot bases using vitrification and droplet-vitrification techniques. https://doi.org/10.54680/fr25210110512.

Background: The long-term preservation of plant germplasm is critical to ensure a pool of genetic diversity for future breeding efforts and for conservation management.Although it is important to assess whether the biochemical properties and vigour of the germplasm remain unchanged.

Objective: To investigate the potential effects of cryopreservation in liquid nitrogen (LN) as a strategy for conservation of carrot.

Results: Seeds were exposed to cryogenic temperatures and growth and development was monitored in Petri dishes (for up to 14 days) and in a pot trial (90 days). By day 7, 53% control seeds had germinated compared with 70% seeds exposed to LN. Biochemical attributes were also assessed. Initial differences were observed in the growth rate of plants, where plants originating from seeds exposed to LN emerged faster than those originating from control seeds. However, at the end of the pot trial, differences were only observed in belowground biomass (i.e., mass of carrots). Biochemical evaluations showed that carotenoids and cell wall-linked phenolics were elevated in carrots produced from seeds exposed to LN.

Conclusion: Overall, the results show that cryopreservation can be used as a viable strategy for long-term preservation of carrot germplasm. https://doi.org/10.54680/fr25210110812.

Background: The polycystic ovary syndrome (PCOS) is a substantial obstacle to female fertility due to ovulation inhibition. Oocyte cryopreservation is crucial for preserving fertility in women with fertility-compromising disorders such as PCOS.

Objective: In this study, the ultrastructural damages of oocytes were evaluated following freezing from PCOS mouse model.

Materials and methods: This experimental study was conducted on 30 adult NMRI mouse. The study comprised three groups: 1) unfrozen PCOS oocytes; 2) vitrified-thawed control oocytes; and 3) vitrified-thawed PCOS oocytes. Transmission electron microscopy was employed for ultrastructure examination across all groups. Moreover, the expression of apoptotic genes, including BAX and Bcl2, was assessed using real time-quantitative polymerase chain reaction (RT-PCR).

Results: The oocyte cryopreservation process had a high impact on the destruction of ooplasm cortical granules, Golgi complexes and mitochondria in vitrified-thawed PCOS oocytes compared to the other groups. In PCOS oocytes, particularly those that were vitrified-thawed, there was a notable increase in vacuolation, with a higher abundance of larger and more numerous vacuoles observed compared to the control group. The vitrified-thawed PCOS group also exhibited a notable increase in the expression of the apoptotic gene compared to the other groups (p < 0.05).

Conclusion: A precise evaluation of oocyte cryopreservation is imperative for improving this technique and for producing high-quality oocytes with enhanced fertility potential. This study contributes valuable insights into understanding the intricate relationship between PCOS, cryopreservation and oocyte quality. https://doi.org/10.54680/fr25210110412.

Background: Progesterone, which is present in the semen extender as a component of egg yolk is a potential inducer of capacitation in spermatozoa during cryopreservation. An anti-progesterone component in the extender may protect the spermatozoa from being capacitated and pre-acrosome reacted during cryopreservation. It may lead to better quality of post-thaw sperm population for improved conception.

Objective: To investigate the effect of mifepristone on the cryo-survivability of cattle spermatozoa.

Materials and methods: Thirty two semen ejaculates were collected from four Sahiwal bulls and divided into three fractions. These fractions were extended with egg yolk-based TRIS extender supplemented with different concentrations of mifepristone (0, 10 and 20 uM) and subjected to cryopreservation. Cryopreserved semen samples were thawed and evaluated for spermatozoa motion parameters (CASA), viability (flow cytometer), hypo-osmotic swelling test (HOST) responsiveness, capacitation status (CTC), acrosome reaction (flow cytometer) and intracellular calcium ion concentrations (flow cytometer).

Results: There was no definitive effect of mifepristone on sperm motility and kinematics. However, the semen samples which were treated with mifepristone showed significantly higher spermatozoa viability and HOST responsiveness. Mifepristone also protected spermatozoa from being cryo-capacitated during the preservation process. Higher percentages of uncapacitated and acrosome intact spermatozoa were found at the post-thaw stage in comparison to the untreated group. Mifepristone-treated groups showed fewer spermatozoa with high intracellular calcium levels.

Conclusion: A 10 uM concentration of mifepristone has better potential to protect the spermatozoa from progesterone-induced cryo-capacitation and premature acrosome reaction during cryopreservation. https://doi.org/10.54680/fr25210110612.

We briefly examine how cold-hardiness in general, including freeze-tolerance, freeze-avoidance and dehydration strategies allow survival in cold climates, through the eyes of some specific insects and fish. Strategies do vary with geography and latitude, even between two types of insects living in the same area. We look at ice nucleation proteins to enhance freezing and antifreeze proteins to help avoid ice formation or, in some cases, to hinder what is known as recrystallization, as a frozen organism thaws. https://doi.org/10.54680/fr25210110112.