There are numerous publications demonstrating that plant polyphenols can reduce oxidative stress and inflammatory processes in the brain. In the present study we have investigated the neuroprotective effect of plant extract isolated from the roots of L. gmelinii since it contains a rich source of polyphenols and other biologically active compounds. We have applied an oxidative and inflammatory model induced by NMDA, H₂O₂, and TNF-α in human primary neurons and astrocytes, and mouse cerebral endothelial cell (CECs) line in vitro. The levels of ROS generation, NADPH oxidase activation, P-selectin expression, and activity of ERK1/2 were evaluated by quantitative immunofluorescence analysis, confocal microscopy, and MAPK assay. In vivo, sensorimotor functions in rats with middle cerebral artery occlusion (MCAO) were assessed. In neurons NMDA induced overproduction of ROS, in astrocytes TNF-α initiated ROS generation, NADPH oxidase activation, and phosphorylation of ERK1/2. In CECs, the exposure by TNF-α induced oxidative stress and triggered the accumulation of P-selectin on the surface of the cells. In turn, pre-treatment of the cells with the extract of L. gmelinii suppressed oxidative stress in all cell types and pro-inflammatory responses in astrocytes and CECs. In vivo, the treatment with L. gmelinii extract improved motor activity in rats with MCAO.

Modern approaches to reproductive biotechnology include the development of effective methods for long-term storage of gametes and reproductive tissues of animals. One of the promising areas is cryopreservation of ovarian tissue, which allows preserving fertility, especially in rare and valuable species. However, the effectiveness of this technology directly depends on the type of cryoprotectant, freezing mode, cooling rate and storage method. Nonequilibrium cryopreservation is a method in which biological samples are exposed to liquid nitrogen vapor without strict software control of cooling. This approach allows for a simpler procedure but requires careful optimization of conditions to preserve tissue viability. The aim of this work is to evaluate the effect of various cryoprotectants and the altitude of the samples (4, 5 and 6 cm above the surface of liquid nitrogen) on the morphological preservation of follicles in the ovarian tissue of dogs after nonequilibrium cryopreservation. A study was conducted to investigate the effect of nonequilibrium cryopreservation in liquid nitrogen vapor on the morphofunctional state of ovarian tissue in dogs. Four cryoprotectors (dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol) and three temperature regimes (4 cm, 5 cm and 6 cm from the liquid nitrogen level) were used. After thawing, a histological analysis was performed to assess the degree of preservation of different types of follicles. It was found that the best viability indicators were observed when using 1.5 M dimethyl sulfoxide (4 and 6 cm), glycerol (4 cm) and propylene glycol (5 cm). The data obtained are important for the development of effective protocols for cryopreservation of ovarian tissue in dogs in order to preserve the gene pool and reproductive potential.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

The Tobet dog breed, a national heritage of Kazakhstan, is threatened with extinction and conservation measures are urgently needed. This study presents a pioneering approach to preserve the genetic diversity and survival of this endangered breed through cryopreservation and cryobanking. We have optimised methods for cryopreservation of somatic cells (fibroblasts) isolated from skin explants of Tobet dogs and investigated the effects of in vitro cultivation on cell viability. Our results emphasise the importance of selecting appropriate culture media and cryoprotectants. Dulbecco's Modified Eagle Medium (DMEM) and ethylene glycol (EG) were found to be the most effective agents for increasing the growth rate and viability of fibroblasts after thawing. Through careful experimentation, including the evaluation of equilibrium and non-equilibrium cryopreservation techniques and the application of various cryoprotectants, we have established the first Tobet cryobank for somatic cells. The establishment of a cryobank represents a significant step towards the conservation of the Tobet dog breed and provides a model for the conservation of other endangered species. This study not only contributes to the preservation of Kazakhstan's cultural and biological heritage, but also opens up new avenues for the application of biotechnological approaches to wildlife conservation.

One of the important directions in the treatment of ischemic stroke (IS) consequences is revascularization of the damaged brain areas including decreasing acute hypoxia and oxidative stress that occur in the ischemic tissue due to reperfusion syndrome after restoration of blood flow. Plant polyphenols are promising candidates capable of exerting a pronounced antioxidant and neuroprotective effects. There are a number of wild plants growing on the territory of Kazakhstan, and one of these plants containing significant number of polyphenols is Limonium gmelinii (L. gmelinii, genus Limonium Mill). In our study we have applied middle cerebral artery occlusion (MCAO) method to induce focal ischemic cerebral stroke in male Wistar rats. The results of assessment of sensorimotor functions in laboratory animals showed that MCAO resulted in sensorimotor deficiency. At the same time, partial recoveries of sensorimotor functions were observed in animals that were treated with extract of L. gmelinii after stroke compared to untreated animals. Similarly, histological analysis of the damaged brain regions has revealed focal coagulation necrosis with clearly visualized damaged regions in animals with MCAO, whereas brain tissue of animals exposed to L. gmelinii extract after stroke induction was partially restored. Our results demonstrate that extract of L. gmelinii possesses neuroprotective properties that require further investigations.

Both tissue and cell cryopreservation can be applied for biodiversity conservation. The proper preservation of tissues and cells from a wide range of animals of different species is of paramount importance, because these cell samples could be used to reintroduce lost genes back into the breeding pool by somatic cell cloning. The aim of this work was to investigate the effect of different cooling rates on viability of frozen–thawed sheep fibroblasts for conservation of biodiversity so that it might be used in the future to provide nuclear donors (Table 1). Skin samples collected from 10 adult sheep were cut on small pieces (1 × 1 mm), placed into culture Petri dishes containing DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), and covered with coverslips followed by incubation at 5% CO2, 95% relative humidity, and 37°C. During culture, fibroblasts left skin samples and proliferated. Culture medium was changed every 4 days. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10 min in presence of Dulbecco’s phosphate buffered saline + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300g for 10 min. For cryoconservation, fibroblast samples were then diluted at a concentration of 2 × 106 cells mL–1 in DMEM + 20% FBS and 10% dimethyl sulfoxide or 10% ethylene glycol and placed into 0.25-mL plastic straws or 2-mL cryovials. Straws were sealed with modeling clay and maintained at +5°C for 120 min before freezing. Cryopreservation of fibroblasts was carried out by 2 procedures: (1) straws were frozen in programmable freezer Kryo Planer 360-3,5 using the following freezing regimen: +5°C to –40°C at –1°C min–1, –40°C to –85°C at –4°C min–1, and then plunged into liquid nitrogen; (2) cryovials were placed in a Styrofoam box and loaded into a freezer at –70°C for 24 h, and then samples were plunged into liquid nitrogen for storage. Samples were thawed for 1 min in a 37°C water bath. Frozen–thawed samples were diluted with DMEM (1 : 5) and centrifuged at 300g for 7 to 10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 106 cells mL–1. Viability of frozen–thawed fibroblast samples was detected using the Trypan Blue staining method. The values obtained (Table 1) are expressed as mean standard error of the mean (SEM). Statistical analysis was done using Student’s test. Results indicated that there was a significant difference in viability between fresh and cryopreserved fibroblasts. However, there were no differences between the cooling procedures. Importantly, our data suggest that the use of 1.5-M ethylene glycol reduced the toxic elements contained in the cryopreservation solution while maintaining a similar ability to produce viable fibroblasts after cryoconservation. Table 1. Effect of 2 cryoprotectant agent (CPA) on the viability of frozen–thawed ovine fibroblasts