Cryobiology最新文献

Ex situ conservation of plant genetic resources (PGR) plays a crucial role in sustainable growth and development, as highlighted by the Global Strategy for Plant Conservation (GSPC). Seed genebanks, a key component of ex situ conservation, have been instrumental in preserving plant diversity. However, challenges arise with the conservation of non-orthodox (recalcitrant and intermediate) seeds and vegetative tissues, which are not amenable to storage in traditional genebanks at temperatures of -20°C. Cryopreservation, the storage of biological materials at ultra-low temperatures in liquid nitrogen, has emerged as a viable solution for conserving such non-orthodox seeds, pollen, and dormant buds. This review presents insights into the National Cryogenebank Facility at ICAR-NBPGR, India, a pioneer in developing cryopreservation techniques and cryobanking of PGR. Established in 1987, the facility focuses on conserving difficult-to-conserve species of various agri-horticultural crops, including recalcitrant and intermediate species. With a capacity to hold a quarter of a million samples, the facility employs species-specific protocols to conserve rare, threatened, and endangered plant species, wild and weedy crop relatives, and genetic stocks. Over the past 3 decades, cryopreservation protocols have been developed at this facility using a diverse range of explants, including seeds, excised embryos, embryonic axes, pollen grains, and dormant buds. Successful cryopreservation protocols have been developed for temperate and tropical plant species important for horticultural, plantation, agro-forestry, and industrial use. Priority is given to conserving indigenous crop species and capturing the genetic diversity of indigenous tropical and temperate major and minor fruits. Additionally, the facility has successfully conserved pollen grains and dormant buds of tropical and temperate fruit crops, ensuring their viability and survival over extended periods of cryostorage. Furthermore, the cryobank regularly retests cryostored germplasm to assess viability and regrowth, with promising results indicating retention of seed viability even after 25-30 years of cryostorage. This highlights the potential of cryobanking as a long-term solution for conserving plant genetic resources. The National Cryogenebank Facility at ICAR-NBPGR exemplifies advancements in cryopreservation techniques applicable to plant genetic resource conservation, contributing significantly to national, regional and global efforts towards ex situ conservation of difficult-to-store plant species and overall sustainable agricultural development.

The application of spermatogonial stem cells (SSC) will be more effective and feasible following the successful cryopreservation and transfer of SSCs in livestock. Like other cells, SSCs are also sensitive to cryoinjury; hence composition of the cryomedia and freezing protocols need to be optimized. The present study aims to optimising the best freezing rates by minimising the ice crystallization and dehydration effect in order to maximize the post-thaw SSCs survivability and stemness characteristics. Three different freezing protocols with varied cooling profiles, cooling profile 1 (isopropanol based freezing): 1°C/min from 0°C to -10°C, 0.5°C/min upto -40°C, further reduced to 0.25°C/min upto -50°C and 0.1°C/min to -60°C, cooling profile 2 (using programmable freezer): 1°C/min up to 4°C, 0.3°C/min up to -8°C, and cooled at 0.5°C/min to -50°C, further decrease to -90°C (8°C/min) and cooling profile 3 (uncontrolled rapid freezing): 3.3°C/min from 0°C to -10°C, 5°C/min upto -40°C, 2°C/min to -50°C and 1.2°C/min upto -60°C, were compared for cryopreservation efficiency. The overall viability (91.41 ± 2.00% Vs 74.59 ± 2.34%), stemness activity (1.34 ± 0.095 OD units Vs 0.356 ± 0.026 OD units), and proliferation rate (0.849 ± 0.019 OD units Vs 0.749 ± 0.015 OD units) of post-thaw SSC culture irrespective of the freezing regimes was significantly decreased when compared to pre-freeze SSC culture characteristics. The post-thaw viability was significantly greater in cooling profile 1 (79.64 ± 4.1%) when compared to cooling profile 2 (69.72 ± 2.4%) and cooling profile 3 (75.43 ± 4.8%). Also, cooling profile 1 yielded greater (p<0.05) post-thaw stemness activity (0.456±0.044 OD units) when compared to other methods. The study suggests that the cooling profile 1 using isopropanol based freezing can be recommended for preservation of viability and stemness characteristics of SSCs.

This study evaluated the effectiveness of Percoll® density gradient centrifugation (Percoll-DGC) for selecting bull epididymal sperm prior to conventional slow (CS) or ultra-rapid (UR) freezing and its effects on sperm quality. Fifteen pooled samples from 30 epididymides (2 different samples/pool) of 15 bulls were split into two aliquots assigned to either CS or UR freezing. Samples were either selected using Percoll-DGC (40/80 %) or left non-selected (control), resulting in four pre-freezing treatments: Percoll-CS, Control-CS, Percoll-UR, and Control-UR. The CS freezing used 5 % glycerol, exposing sperm straws to liquid nitrogen (LN₂) vapors, while UR freezing used 100 mM sucrose with direct submersion of 30 μL samples into LN₂. Overall, sperm quality was higher in CS treatments than in UR treatments. Pre-freezing, Percoll-CS improved straight-line velocity (VSL), linearity (LIN), and beat-cross frequency (BCF) compared to Control-CS (P < 0.05). Similarly, Percoll-UR treatment enhanced progressive motility (PSM), velocities, straightness (STR), amplitude of lateral head displacement (ALH), and BCF compared to Control-UR (P < 0.05). Post-thaw, Percoll-CS demonstrated higher kinematic parameters, viability, and acrosome integrity compared to Control-CS (P < 0.05). Meanwhile, Percoll-UR improved viability and acrosome integrity relative to Control-UR (P < 0.05). Notably, both Percoll-UR and Control-UR resulted in lower DNA fragmentation compared to Percoll-CS. In conclusion, Percoll-DGC selection prior to CS freezing significantly improved post-thaw sperm quality, including kinematics, viability, and acrosome integrity. For UR freezing, Percoll-DGC primarily enhanced post-thaw viability and acrosome integrity.

Resveratrol is a polyphenol compound showing strong antioxidant properties. It is believed that semen cryopreservation causes significant sperm losses which eventually affects sperm quality. Improving antioxidant status of semen may reduce this damage and enhance sperm fertilizing potential. This study investigated the resveratrol treated freeze-thawing sperm motion, kinetics, structural parameters and antioxidant/oxidant status of Kamori buck spermatozoa. Thirty-two ejaculates from 4 fertile Kamori bucks were processed in tris-citric-glyercol-egg yolk (TCG-EY) based with varying concentrations of resveratrol (0, 10, 20, 40 and 50 μM). Over 75% sperm motility were pooled and frozen in liquid nitrogen. The results unveiled that adding resveratrol at 40 and 50 μM concentration significantly enhanced (P < 0.05) total motility (progressive motility, rapid velocity, and average path velocity, straight line velocity, curvilinear velocity, amplitude of lateral head displacement, beat cross frequency, straightness and linearity in contrast with control and other groups. Supplementing resveratrol at 40 and 50 μM concentration significantly improved (P < 0.05) functional plasma membrane integrity, acrosome integrity, whereas, all resveratrol groups had same significant (P < 0.05) effect on DNA integrity in response to control group. The 40 and 50 μM resveratrol significantly promoted (P < 0.05) superoxide dismutase (SOD), catalase (CAT), peroxidases (POD), and total antioxidant capacity (TAC) levels while significantly reducing (P < 0.05) the total oxidant status (TOS) and malondialdehyde (MDA) contents as compared to control and other groups, respectively. The principal component analysis (PCA) exhibited samples were present in different clusters except two groups which had partially overlapped. Using hierarchical clustering analysis, two clusters were constructed showing the relationship within the treated groups. The results of spearman correlation coefficient showed that yellow color showed highly positive correlation while turquoise color exhibited highly negative association between sperm variables. Overall, the results concluded that resveratrol at 50 μM performed slightly better results than 40 μM in terms of improving sperm quality parameters.

Fewer studies investigate the effects of underlying genetic factors related to semen characteristics, significantly affecting sheep farm profitability. This study aimed to identify single nucleotide polymorphisms (SNP) and genomic regions associated with fresh and frozen-thawed semen traits in rams with low (Hasak) and high (Hasmer) cryotolerance. Semen collected from 11 (5 Hasak with low and 6 Hasmer with high cryotolerance) rams cryopreserved in 0.25 ml straws in the breeding season. Quality characteristics were determined in fresh, equilibrated, and frozen-thawed semen. A Genome-Wide Association Study (GWAS) was conducted to unveil the genetic structure that might be attributed to cryotolerance in low and high cryotoleranced rams. Fresh (regarding total and progressive motility) and equilibrated semen quality were similar in Hasak and Hasmer rams (p > 0.6). However, the freeze-thawing process had a more pronounced negative effect on ram semen traits in Hasak than in Hasmer (p < 0.05). GWAS revealed 27 SNPs correlated with post-thaw semen parameters. Moreover, network analyses revealed pathways related to sperm ion channels and their activities, providing insights into the intricate molecular mechanisms underlying sperm physiology and emphasizing their role in potentially impacting sperm cryotolerance. The functional significance of detected SNPs and the associated pathways require further exploration.

Osmotic stresses during cryoprotectant loading induce changes in cellular volume, leading to membrane damage or even cell death. Appropriate model-guided mitigation of these osmotic gradients during cryoprotectant loading is currently lacking, but would be highly beneficial in reducing viability loss during the loading process. To address this need, we reformulate the two-parameter formalism described by Jacobs and Stewart for cryoprotectant loading under the constraint of constant cell volume. We then derive simple, concise, analytic solutions to these equations, showing the transient extracellular permeating and nonpermeating cryoprotectant concentrations required to load a cell at constant volume, thus eliminating osmotic stresses during cryoprotectant loading. Additionally, we show analytic approximations of both ramp (linear) as well as step-wise loading and how one can use the hydraulic conductivity L_p, membrane permeability P_s, cell volume V_o, and osmotically inactive fraction to derive cryoprotectant loading protocols that minimize osmotic stress. We also present timescales for water and cryoprotectant transport which can be used to estimate loading times as well as L_p and P_s. We discuss how previous optimized loading strategies are inherently sensitive to parameter uncertainties and biological variability, increasing the likelihood of exceeding critical osmotic limits. By contrast, the proposed protocol provides a larger buffer against deviations, offering a safer and more robust solution to CPA loading. Importantly, we demonstrate that the volume-loss-free CPA loading protocols outlined in this paper occur on the same timescale as conventional and step-loading methods, suggesting that these protocols could be a safer, more efficient alternative for CPA loading.

Graphene oxide (GO) has been extensively studied for its diverse biomedical applications, including drug delivery, imaging, and tissue engineering. Silymarin, as a flavonoid complex derived from the milk thistle plant, has recently shown potential health benefits, particularly concerning reproductive health. This study aims to evaluate the effects of GO and silymarin supplementation, both individually and in combination, on the characteristics of frozen-thawed ram sperm. Semen samples were collected using standard artificial insemination (AI) techniques with an artificial vagina. The collected semen was evaluated and cryopreserved in a tris-based extender containing varying concentrations of silymarin and GO (0, 10, or 20 μg/mL) or their combination. Post-thaw assessments evaluated sperm motility, viability, morphological abnormalities, DNA integrity, membrane integrity, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and total antioxidant capacity (TAC). Our findings revealed that the combination of 20 μg/mL silymarin and 20 μg/mL GO significantly enhanced total motility, viability, membrane integrity, and DNA integrity of sperm. Additionally, this treatment effectively reduced morphological abnormalities and MDA levels post-thawing. Notably, SOD and TAC activities were improved following the freeze-thaw compared to other treatment groups. In conclusion, the combination of silymarin and GO significantly improves the quality of frozen-thawed ram sperm by enhancing sperm parameters while reducing oxidative stress markers. The results suggest their potential as effective additives in cryopreservation protocols, providing a promising avenue for improving reproductive outcomes in rams and potentially other livestock species.

Skin banks are valuable tools for the maintenance of biodiversity. The red-rumped agouti is a wild rodent of ecological importance in South America because it acts as a seed disperser, and skin banks could serve as alternatives to conserve genetic variability. Nevertheless, the most suitable skin region for forming these banks must still be determined to guarantee tissue quality after cryopreservation. We harvested skin tissues from the ear, abdominal, and inguinal regions of red-rumped agouti and evaluated the effects of cryopreservation on the quantitative and qualitative histological parameters of these somatic tissues. All tissues were evaluated for ultrastructure, skin thickness, cell count, number of perinuclear halos, collagen density, proliferative activity, and cellular ability during culture. Cryopreservation altered the thicknesses of the abdominal and inguinal dermises. Cryopreservation did not alter the number of fibroblasts and perinuclear halos in any region. The abdominal region had a higher number of fibroblasts than the other regions. All groups showed increased collagen fibers after cryopreservation, while maintaining a similar proliferative potential. During culture, all regions presented with cell viability above 90 % before and after cryopreservation, and the doubling time was maintained. However, cells from the inguinal and abdominal regions had lower metabolic rates than those from the ear. In summary, the ear skin was found to be the best region for cell recovery. However, abdominal skin has shown positive results and may be an alternative source of somatic cells.

Vitrification is a conventional and mature method for embryo cryo-preservation, but ice crystals formed during the vitrification process can damage embryos. HPC has the property of forming a high-viscosity gel under low-temperature conditions, so it can be added to vitrification solutions to investigate whether it improves the negative impact of vitrification on embryos. The results showed that the addition of HPC (50 μg/ml) to the vitrification solution significantly increased the post-warming survival rate of sheep morula embryos. Compared to the vitrification group, the addition of HPC significantly reduced the level of ROS (reactive oxygen species, ROS) and increased the level of GSH (glutathione, GSH) in embryos after warming, mitigating the damage to mitochondria caused by vitrification. It also had clear positive effects on the developmental potential of vitrified-warmed sheep embryos. Overall, the addition of 50 μg/ml HPC to the vitrification solution can significantly improve the quality of sheep morula embryos after vitrification and warming.