Cryobiology最新文献

The fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a major agricultural pest. Low temperature is an important factor that limits S. frugiperda survival and reproduction. The physiological cold tolerance indexes of S. frugiperda larvae from October 2022 to February 2023 were investigated. The results showed that the cold-resistant substances in S. frugiperda differed significantly among different months. The water content was the lowest in January. Lipids were the highest in December and January, trehalose and protein contents were the highest in December, and glycerol content was the highest in January. The contents of lipid, protein, trehalose, and glycerol were the highest in the 6^th instar larvae, whereas the water content did not differ among the larva stages. The functions of two genes, including endothelial lipase (Eol1) and gastric lipase (Epl1) that were extracted from the transcriptome analysis of S. frugiperda in the cold resistance were assessed. After 24 h of cold stress, the mRNA levels of Eol1 and Epl1 RNA interference were significantly down-regulated by 60.76 % and 82.53 %, and the survival rate of S. frugiperda larvae decreased. Additionally, Eol1 and Epl1 silencing significantly decreased the lipid content and pupal weight of S. frugiperda. The results indicate that S. frugiperda larvae can change the contents in winter to response the cold conditions and the lipids plays an important role in the cold resistance of S. frugiperda.

This study outlines a method for designing an isochoric (constant volume) system to reduce the supercooling preservation temperature without affecting the likelihood of ice nucleation and without the need for cryoprotective additives. The method involves a multiphase system wherein the biological material is separated from a second aqueous solution by a boundary that transfers pressure and heat but not mass. The pressure within the system is passively increased by the confined growth of ice within the secondary solution. This increased pressure in turn lowers the equilibrium freezing temperature of the biological matter, which may be utilized to lower the preservation temperature while maintaining the same degree of supercooling. For example, using this technique, the supercooling preservation temperature may be lowered from -2 °C to -5 °C without increasing the risk of ice nucleation, by ensuring the freezable phase makes up ∼17 % of the total system volume.

Sperm collection and cryopreservation are key technologies for assisted reproduction, with post-mortem sperm collection being the main tool to prevent the decline of endangered species. The present study describes post-mortem sperm collection and two cryopreservation protocols in beauty snakes (Elaphe taeniura), as a model for Colubridae. The vasa deferentia of 18 snakes were collected post-mortem and incubated for 30 min at room temperature to retrieve sperm by float up. Afterwards, fresh sperm was analysed, and 11 samples were diluted into either a single or a double-step TEST-egg yolk-based extender to reach a final concentration of 12 % glycerol. Anatomical and histological analyses of the reproductive organs revealed that those on the right side were larger, heavier, and positioned more cranially than the left ones (P = 0.007, P = 0.003, and P = 0.0002, respectively), but sperm parameters did not differ. Successful sperm retrieval was achieved in 61.1 % of snakes, with positive correlations between seminiferous tubules length and body weight (P = 0.0004) and between seminiferous tubules length and body length (P = 0.001). Sperm-producing animals were heavier and longer compared to azoospermic ones (P = 0.04 and P = 0.04 respectively). Specifically, males longer than 130 cm and heavier than 177g have higher reproductive potential. Both cryopreservation protocols yielded comparable results, preserving sperm viability, motility, and morphology. Therefore, a single-step protocol should be preferred due to its technical simplicity. In conclusion, we described for the first time the reproductive features and characteristics of spermatozoa collected post-mortem in beauty snakes. Although the described protocols are effective, further research is warranted to optimize these techniques.

The valuable anti-cancer and anti-inflammatory secondary metabolite, imperatorin, has been found in the hairy roots (HRs) of Urena lobata. However, an increasing number of problems related to cryo-injury and cryoprotectant toxicity could potentially reduce the quality of root clones, highlighting the need to develop a reliable technique for long-term preservation. Based on the impact of the successive steps of the initial droplet-vitrification procedure employed for cryopreservation of HRs using the histological evaluation of plasmolysis, various selected factors were independently investigated. The maximum plasmolysis was observed after the preculture with 0.5 M sucrose (46.59 %) and the dehydration treatment (48.32 %). In the improved cryopreservation procedure, when prolonged preculture for 72 h in liquid WPM with 0.3 M sucrose and dehydration in the appropriate vitrification solution, which included 30 % (w/v) glycerol and 50 % (w/v) sucrose, for 10 min at 0 °C, the plasmolysis in the two steps was significantly reduced in comparison with the untreated control. The cryopreserved HRs could increase their regeneration to 93.3 % and regenerated root length to 4.65 cm. Their growth and imperatorin production were almost the same as those of untreated controls after three to five subsequent subcultures. Our results have demonstrated that our new approach, which only focuses on the key factors that significantly increase the plasmolysis, modifies their level to fit with the HR cells of U. lobata. The early application of the plasmolysis evaluation method may immediately screen the impact of factors on individual root cells instead of spending time and cost evaluating the recovery of root tips at each step to develop an efficient cryopreservation procedure.

The success of somatic cell cryobanks is dependent on establishing reproducible cryopreservation methodologies. We supposed that associated extracellular cryoprotectants (sucrose and L-proline) with 2.5 or 10 % dimethyl sulfoxide (Me₂SO) could guarantee better northern tiger cat cells quality rates after thawing when compared to Me₂SO alone. Therefore, we evaluated the effects of sucrose or L-proline with 2.5 or 10 % Me₂SO on the cryopreservation of northern tiger cat fibroblasts. Somatic cells were also cryopreserved with 2.5 % or 10 % Me₂SO alone. All cells were analyzed for morphology, membrane integrity, proliferative activity, metabolism, apoptosis classification, reactive oxygen species (ROS) levels, and mitochondrial membrane potential (ΔΨm). Regardless of the cryoprotective solution, cryopreservation did not affect morphology, membrane integrity after culture, proliferative activity, and metabolism (P > 0.05). However, immediately after thawing, 2.5 % Me₂SO with L-proline and 10 % Me₂SO promoted higher rates of membrane integrity when compared to the other cryopreserved groups (P < 0.05). Interestingly, cells cryopreserved with 10 % Me₂SO maintained ROS levels similar to non-cryopreserved cells (P > 0.05). However, the percentage of viable cells evaluated by apoptosis classification was reduced when using 10 % Me₂SO with L-proline compared to non-cryopreserved groups (P < 0.05). Additionally, ΔΨm was altered in all cryopreserved groups (P < 0.05). In summary, sucrose and L-proline were less effective in cryopreservation of northern tiger cat fibroblasts in the presence of 2.5 % or 10 % Me₂SO. Also, 10 % Me₂SO appears to be the most suitable cryoprotectant for the formation of cryobanks of this species.