Cryo letters最新文献

Background: Cryopreservation of sperm cells is a well-established technique. However, cryopreserved sperm cells can have significant cellular damage, primarily due to oxidative stress.

Objective: To investigate the synergistic effects of vitamin C with vitamin E-loaded cyclodextrins on the quality of cryopreserved bull sperm cells in combination with cholesterol-loaded cyclodextrins.

Materials and methods: The ejaculates from nine mature bulls were divided into six equal aliquots for different treatments, including vitamin C (VitC), vitamin E loaded cyclodextrin (CD-VitE), CD-VitE+VitC, cholesterol loaded cyclodextrin (CD-CHL), CD-CHL + CD-VitE + VitC and the control. The sperm motility (CASA), membrane integrity (HOST) and lipid peroxidation (TBARS) were assessed after freezing and thawing.

Results: The combination of CD-CHL + CD-VitE + VitC retained the highest sperm motility and membrane integrity, as well as resulted in the least lipid peroxidation.

Conclusion: The study shows the synergistic effects of vitamin C, vitamin E and cholesterol in improving the cryopreservation outcomes of bull sperm. https://doi.org/10.54680/fr25410110312.

Background: Lipids play a vital role in sperm capacity acquisition, and yet the effect of cryopreservation on lipid profile has received little attention so far.

Objective: To evaluate the change in lipids of ovine sperm upon cryopreservation.

Materials and methods: Ovine semen was aliquoted into two parts: one being the fresh control and the other used for cryopreservation. The targeted lipidomic analysis was performed between fresh and cryopreserved sperm samples.

Results: Cryopreservation resulted in 53 up-regulated and 163 down-regulated lipids. Down-regulation prevails. Most differentiated lipids were glycerophospholipid, fatty acyl, glycerolipid, sphingolipid, prenol lipid and sterol, and they were highly correlated with one another (correlation coefficient r > 0.8). Major pathway enrichments were glycerophospholipid metabolism, glycosylphosphatidylinositol-anchor biosynthesis, glycine, serine and threonine metabolism, biosynthesis of unsaturated fatty acids, actin cytoskeleton regulation, pathogenic infection and autophagy. In glycerophospholipid metabolism, the phosphatidyl-ethanolamine Class II series and phosphatidyl-L-serine Class II series had the largest numbers of differentially-expressed lipids, some significantly down-regulated and others up-regulated. In contrast, all lipids of the 1,2-diacyl-sn-glycerol-3P Class II series were significantly down-regulated.

Conclusion: Cryopreservation altered the sperm lipid profile, and the changes in 1,2-diacyl-sn-glycerol-3P, phosphatidyl-ethanolamine and phosphatidyl-L-serine can be good biomarkers for sperm quality change. https://doi.org/10.54680/fr25410110712.

Background: The inbred C57BL/6 and BALB/c mouse strains are widely recognized and used as foundational models for mutagenesis studies globally. Analyzing molecular damage at cellular and genetic levels from freeze-thaw processes in different mouse strains is crucial for understanding how to preserve sperm function and reproductive efficiency.

Objective: To examine intraspecific variations in fresh and frozen-thawed sperm from outbred (CD-1) and inbred (BALB/c) mouse strains.

Materials and methods: Sperm cryopreservation utilized 3 % (w/v) skim milk powder and 18 % (w/v) raffinose as cryoprotectants.

Results: Post-thaw analysis showed significantly higher progressive sperm motility (p < 0.05), intact plasma membrane integrity (p < 0.01), and viability (p < 0.05) in CD-1 frozen-thawed sperm than in BALB/c. The mRNA expression of XBP1, GRP78, and IRE1 was significantly higher in BALB/c frozen-thawed sperm (p < 0.001). CHOP mRNA levels showed no significant variation (p > 0.05). BAX mRNA was significantly upregulated in both strains after freezing (p < 0.001). While TCP11 mRNA showed no significant differences (p > 0.05), PDIA3 mRNA increased significantly post-thaw (p < 0.001).

Conclusion: Cryopreservation quality was superior in outbred CD-1 sperm compared to inbred BALB/c sperm, evidenced by better post-thaw parameters and elevated endoplasmic reticulum stress-related genes (XBP1, GRP78, and IRE1) and PDIA3. https://doi.org/10.54680/fr25410110512.

Background: Cryopreservation has transformed fertility preservation by enabling the long-term storage of gametes and embryos. Although traditional procedures such as slow freezing and vitrification are routinely used, recent innovations and new cryoprotectants have had a significant impact on reproductive medicine outcomes.

Objective: This review examines advances in cryopreservation techniques, the influence of novel cryoprotectants, and the implications for gamete and embryo survival, viability, and clinical outcomes in fertility preservation.

Materials and methods: We conducted a comprehensive review of the existing literature on classic and novel cryopreservation methods for sperm, oocytes, and embryos. Advances in vitrification methods, the invention of novel cryoprotectants, and comparative effectiveness and toxicity assessments were all evaluated. Clinical data on survival rates, implantation rates, and fertility preservation were thoroughly reviewed.

Results: Improvements in vitrification procedures have drastically enhanced oocyte survival and developmental potential, resolving some of the previously linked cryopreservation issues. Innovative ways to cryopreserve have enhanced sperm survival and motility after thawing. The focus of embryo cryopreservation has switched from traditional slow freezing to precise vitrification, resulting in higher survival rates and better clinical results. Novel cryoprotectants have shown promise in terms of reduced toxicity and improved cryosurvival while retaining biological integrity. Overall, these advances have had a positive impact on fertility preservation techniques and clinical success rates.

Conclusion: Emerging cryopreservation methods, such as breakthroughs in vitrification protocols and the identification of new cryoprotectants, have significantly improved gamete and embryo storage efficiency. Such developments not only increase the longevity and quality of cryopreserved materials, but they also improve therapeutic outcomes in fertility preservation. Additional study and optimization are required to standardize these procedures for optimal use in a variety of patient populations. https://doi.org/10.54680/fr25410110112.

Background: Human semen and epididymal spermatozoa cryopreservation are crucial for men's fertility preservation, particularly for those patients facing neoplastic, autoimmune, urological, and neurological conditions where medical or surgical treatments may pose a risk to fertility or where obstructive or secretory azoospermia is documented. However, there are currently no standardized methods to assure optimal cryosurvival rates.

Objective: To determine the best freezing protocol out of five selected methods based on routine sperm analysis and additional assays including cytofluorimetric analysis, comet assay, and transmission electron microscopy.

Materials and methods: The study is a cross-sectional analysis of 26 fresh semen samples frozen using five different freezing protocols (or methods, M), varying in cooling phase time and temperatures, and utilizing TEST-Yolk Buffer (TYB) as a cryoprotectant. Data on sperm motility, viability, membrane integrity, DNA fragmentation, and ultrastructural shape post-thawing were collected.

Results: Our findings showed that the method 1 (M1) and method 3 (M3) (involving a three-phase cooling process with a phase at +4 degree C, followed by 10 min of exposure to the gas phase of liquid nitrogen before immersion in liquid nitrogen) yielded the best protocols, resulting in minimal deterioration of semen quality.

Conclusion: These results highlight the importance of a pre-freezing phase at +4 degree C when using TYB cryoprotectant on untreated semen, regardless of the duration, despite the less-than-optimal survival rate achieved. It is crucial to use a range of assays to study the effects of cryopreservation procedures, not only assessing sperm motility and viability, but also evaluating membrane integrity, DNA fragmentation, and ultrastructural shape. https://doi.org/10.54680/fr25410110212.

Background: SODs are key enzymes that degrade the superoxide radical, representing a primary defense in the antioxidant system against the toxicity caused by overproduction of ROS under environmental stresses. However, there is scarce data about SOD functions in insects under low temperature.

Objective: In this research, we investigated whether the heterologous superoxide dismutase overexpression in Drosophila melanogaster improves cold tolerance in flies.

Materials and methods: A novel extracellular copper/zinc SOD (MpSOD3) from the desert beetle Microdera punctipennis was transferred to D. melanogaster via P-element-mediated transformation. The protection effect of increased SOD activity on lipid peroxidation and apoptosis were determined by measuring oxidative parameters and TUNEL assay during cold treatment.

Results: Compared to non-transgenic flies exposed to 0 degree C treatment for 12-24 h period, the expression level of MpSOD3 and SOD activity were significantly higher in all transgenic lines with less accumulation of superoxide(O₂^•-). MpSOD3-expressing Drosophila exhibited higher survival rates compared with the control under cold and oxidative exposure. In response to cold, the MDA content and TUNEL assay showed that MpSOD3-expressing adult flies exhibited less lipid peroxidation and apoptotic damage in comparison to control flies.

Conclusion: Collectively, these results indicate that the overexpression of MpSOD3 in transgenic Drosophila lines enhances cold tolerance by eliminating O₂^•-and lessening over-oxidation of the cellular membrane system. https://doi.org/10.54680/fr25410110412.

Background: Sperm cryopreservation is a process that involves preserving sperm cells for future use. Prior to freezing, sperm preparation methods like density gradient centrifugation (DGC) are typically performed to improve sperm quality.

Objective: To compare two sperm freezing methods, with and without seminal fluid, in normal and varicocele patients, focusing on reactive oxygen species (ROS) levels and sperm DNA fragmentation.

Materials and methods: Forty samples from individuals with normal (n=20) and varicocele (n=20) were collected through ejaculation. Each sample was divided into two groups for analysis. One group DGC was performed pre-freezing and another group DGC was done post-freezing. Evaluations were conducted on sperm parameters, DNA fragmentation index, and ROS generation.

Results: In the normal group, progressive motility in Fresh Freeze DGC (19.55 ± 12.90) was lower than Fresh DGC Freeze (32.45 ± 12.86), although viability, morphology, DNA fragmentation and ROS production remained unchanged. Fresh Freeze DGC (44.25 ± 11.38) and Fresh DGC Freeze (53.58 ± 11.41) differed substantially in the varicocele group for viability assessment, whereas other parameters did not change in this group.

Conclusion: It is recommended to prepare sperm before freezing to avoid potential issues with sperm not tolerating the centrifugation process. Removing seminal fluid before freezing can lead to better sperm parameters in both normal and varicocele groups. Doi.org/10.54680/fr25310110712.

Background: Antioxidant is important to prevent ROS attack in boar sperm during the freezing-thawing process.

Objective: To study whether hydrogen (H₂) could improve the survival of boar sperm upon the freeze-thaw process.

Materials and methods: The effects of hydrogen in pre-diluent, freezing extender and thawing solution were investigated. Sperm viability, progressive motility, acrosomal integrity, mitochondrial activity, as well as the levels of malonaldehyde (MDA) and total antioxidant capacity (T-AOC) were measured.

Results: Hydrogen in pre-diluent, freezing extender and thawing solution significantly improved sperm quality parameters (including sperm viability, progressive motility, acrosomal integrity and mitochondrial activity), increased total antioxidant capacity (T-AOC) and decreased malondialdehyde (MDA) of post-thawed boar sperm. The use of hydrogen in all three solutions improved the quality of post-thawed boar sperm.

Conclusion: Hydrogen protects boar sperm against ROS-induced damages during cooling and the freezing-thawing process. Doi.org/10.54680/fr25310110512.

Background: Seed storage globulins have two subunits linked by disulfide bonds. Earlier studies suggested that globulin hydrolysis may enhance the freezing tolerance of hydrated seeds. As a donor for H₂S, NaHS can act as a nucleophile to break the disulfide bond of proteins and convert sulfhydryl group (-SH) of cysteine to the persulfide group (-SSH), which will promote formation of S-persulfidation and de-polymerization of seed globulins.

Objective: To examine the role of the H₂S signal pathway in the freezing tolerance of hydrated brassica seeds.

Materials and methods: Hydrated brassica (Brassica oleracea) seeds were treated with 5 mM NaHS to reduce the disulfide bonds of seed globulins and its effect on seed freezing tolerance was investigated.

Results: NaHS treatment increased embryo supercooling as determined by differential scanning calorimetry (DSC) and seed survival upon slow cooling (control vs NaHS, 30.0% vs 45.3%). NaHS treatment resulted in a significant increase of sulfhydryl groups of storage globulin, indicating the reduction of disulfide bonds. The 2D electrophoresis showed the depolymerization of storage globulins and the accumulation of small polypeptides. In addition, NaHS treatment increased the contents of ascorbate and glutathione for anti-oxidation.

Conclusion: The H₂S signal pathway is likely involved in the freezing tolerance of hydrated brassica seeds via the de-polymerization and hydrolysis of seed storage globulins, as well as the regulation of supercooling. Doi.org/10.54680/fr25310110312.

Background: Cryopreservation of goat sperm is important for goat breeding and population expansion. However, goat spermatozoa are generally less resistant to freezing and the fertilization rate of spermatozoa is lower than that of fresh semen.

Objective: To investigate the effect of different concentrations of Angelica sinensis Polysaccharide (ASP; 0, 150, 300, 600, and 1200 ug/mL) on post-thaw quality, motility characteristics, and oxidative markers of Chongming white goat sperm.

Materials and methods: Fresh semen from Chongming white goat (n=8; mean age, 3-5 years of age) was collected, evaluated, and divided into five treatment groups corresponding to different ASP concentrations. Post-thaw sperm motility kinematics, plasma and acrosomal membrane integrity, and mitochondrial activity were evaluated. Additionally, this study evaluated markers of antioxidant defense, including malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), nitric oxide (NO) content, total antioxidant capacity (T-AOC), as well as 5-methylcytosine (5-MC) level.

Results: The addition of 600 ug/mL ASP enhanced sperm viability and sperm kinematic parameters, improved integrities of acrosome and plasma membrane, and increased mitochondrial activity as compared with the control. Regarding oxidative markers, ASP-added groups had reduced ROS, MDA, and NO levels in semen compared to controls. The ASP 600 µg/mL group had the lowest levels of oxidative markers, and adding 600 ug/mL ASP significantly increased SOD, catalase (CAT), and GSH-Px activities in semen. There were significant increases in T-AOC levels in all ASP-added groups versus the control group. Additionally, the 300 and 600 g/mL ASP-groups showed significantly lower levels of DNA methylation 5-MC than the control groups.

Conclusion: Adding 600 ug/mL ASP to the Chongming white goat low-temperature diluent improves the motility characteristics and antioxidant markers of Chongming white goat semen. Doi.org/10.54680/fr25310110212.