Cryo letters最新文献

Background: The skin, the largest organ in the human body, is composed of complex layers that include subcutaneous adipose tissue. Understanding the characteristics of this skin structure is essential to optimize therapeutic interventions, such as cryolipolysis, aiming for more effective and personalized results.

Objective: To evaluate the immunohistochemical effects of skin tissue in adult women undergoing cryolipolysis.

Materials and methods: We carried out an experimental and blind study with immunohistochemical analysis in women with localized abdominal fat, categorized based on the constitution of the skin as flaccid or firm according to the Investigator Assessment Skin Laxity Scoring System scale. Participants were randomized before undergoing the cryolipolysis procedure. Forty-five days after the procedure, they underwent abdominoplasty, with collection of biological material. We evaluated the inflammatory markers EBF-1, TNF-alpha, and CD68, as well as Caspase 3, cleaved Caspase 3, apoptotic BCL2, Ki-67 for fibroblast proliferation, and FIS1 for mitochondrial proliferation.

Results: Six women were included, divided into two groups; three women with loose and three with firm skin. We observed that after cryolipolysis, the group with flaccid skin showed higher expression of the Casp3, TNF-alpha, BCL2, and FIS1 markers compared to those with firm skin.

Conclusion: Cryolipolysis may act differently according to tissue morphology, suggesting that its apoptotic response is more pronounced in the group with flaccid skin. https://doi.org/10.54680/fr25110110212.

Background: Carnitine reduces reactive oxygen species-induced apoptosis and DNA fragmentation through its antioxidant effect.

Objective: To investigate the effect of carnitine on capacitation, mitochondrial activity, acrosomal integrity, reactive oxygen species (ROS), membrane fluidity, and DNA fragmentation during the cryopreservation of Hariana bull spermatozoa.

Materials and methods: Thirty-two semen ejaculates were obtained using artificial vagina (AV) from four seemingly healthy Hariana bulls. Following dilution, the diluted semen samples were split into four aliquots: Group I, the control, included no carnitine; Groups II, III, and IV were the aliquots that contained carnitine supplements of 2.5, 5, and 10 mM, respectively. These four diluted semen samples were then processed immediately for freezing and equilibration.

Results: Regarding post-thaw sperm parameters, such as viability, motility, velocity parameters, capacitation status, mitochondrial activity, acrosomal integrity, reactive oxygen species (ROS), membrane fluidity, and DNA fragmentation, Groups II and III, containing 2.5 mM and 5 mM carnitine respectively, had significantly (P < 0.05) improved parameters compared to the Group I (control). At 5 mM, there was a substantial (P < 0.05) decrease in early apoptotic-like alterations in sperm cells, accompanied by a greater population of sperm cells with high mitochondrial membrane potential.

Conclusion: Carnitine has been shown to have cryoprotective properties in semen extenders. For improved post-thaw sperm quality, carnitine may be added to a Hariana bull semen extender at a dose of 5 mM. https://doi.org/10.54680/fr25110110712.

Background: There is a crucial need to develop appropriate cryopreservation solutions so that somatic resource biobanks of wildlife can be established.

Objective: Here, we propose a cryopreservation protocol to optimize the preservation of skin-derived fibroblasts from six-banded armadillos (Euphractus sexcinctus Linnaeus, 1758) by comparing different concentrations of fetal bovine serum (FBS) in the absence or presence of sucrose as non-permeable cryoprotectants.

Materials and methods: Cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium (DMEM) with 10% dimethyl sulfoxide (DMSO), varying concentrations of FBS (10, 20 and 40%) without or with 0.2 M sucrose, totaling six comparison groups. Cells not subjected to cryopreservation were used as a control. Cells were evaluated for morphological characteristics, viability, metabolism, apoptosis levels, proliferative activity and mitochondrial membrane potential.

Results: Cells maintained similar fusiform morphology and demonstrated high viability (> 90%) before and after cryopreservation in all groups. Cryopreserved cells with 10 and 40% of FBS without sucrose showed lower metabolism, but, when sucrose was added, this parameter was maintained as in the control group. This effect was not observed in the 20% FBS groups in the absence or presence of sucrose, with viability similar to that of the non-cryopreserved group. The addition of sucrose maintained apoptosis levels, while the 20 and 40% FBS without sucrose groups showed alterations in viable, early apoptosis and necrosis stages. Nevertheless, all cryopreserved groups showed lower proliferative activity with a higher population doubling time (16.2-19.9 h) than the non-cryopreserved group (15.2 h). Finally, the 20% FBS groups, in the absence or presence of sucrose, maintained the mitochondrial membrane potential.

Conclusion: We demonstrated that 20% FBS with sucrose was the most suitable cryopreservation solution for six-banded armadillo skin-derived fibroblast lines, promoting high cell survival after thawing. https://doi.org/10.54680/fr25110110412.

Background: Despite advancements in bovine embryos cryopreservation techniques, challenges remain, warranting further investigation into their impact on embryo morphology and viability so that outcomes can be improved.

Objective: To analyze, through transmission electron microscopy (TEM), in vitro-produced bovine embryos vitrified using the Cryotop method.

Materials and methods: Groups of embryos were transferred to a stabilization solution (SS) containing 7.5% EG, 7.5% DMSO in maintenance medium (TCM-199 supplemented with 20% FBS) for 2 min, and then transferred to a vitrification solution (VS) containing 15% EG, 15% DMSO, and 0.5 M sucrose in maintenance medium. Warming was performed in five stages with decreasing concentrations of sucrose. After warming, the blastocysts were cultured for 24 h for subsequent survival analysis and ultrastructural evaluation. In vitro-produced bovine embryos that did not undergo the vitrification process were used as a fresh control.

Results: Blastocoel reestablishment was observed in 52.3% (66/126) of vitrified embryos 24 h after warming, demonstrating the method's effectiveness in post-cryopreservation survival. Ultrastructural analysis of embryos from the fresh control group showed flattened trophoectodermal cells with prominent nuclei, well-preserved mitochondria, and Golgi complexes were also evident. Microvilli were observed in some regions near the zona pellucida. Embryos vitrified using the Cryotop method exhibited lesions consistent with the cryopreservation process, such as intracellular disorganization, mitochondrial injuries, and dispersion of microvilli.

Conclusions: Ultrastructural evaluation of in vitro-produced bovine embryos vitrified using the Cryotop method is an effective tool for increased understanding of the injuries caused to embryonic cells during the cryopreservation process. https://doi.org/10.54680/fr25110110612.

Background: Resveratrol (Res) (3,5,4'-trihydroxystilbene) is a natural polyphenol that exhibits important biological activities.

Objective: To assess the effects of resveratrol (Res) on freeze-thawed survival of semen from Honamli and Hair Bucks.

Materials and methods: Six bucks, aged 2-3 years (three from each breed), were included in the study. Semen was collected from each breed and mixed separately after removing seminal plasma. The mixed semen was diluted with different Res concentrations (0 uM as control, 25 µM, 50 uM, 100 µM, 500 uM, and 1 mM) in Tris diluent and subjected to cryopreservation in liquid nitrogen vapor and frozen. After thawing, the samples were evaluated for motility and some spermatologic quality parameters by flow cytometry.

Results: Data were analyzed separately for Honamli and Hair breeds. The results showed that the Res 1 mM group had the lowest motility in all assessments (P < 0.05). However, no significant differences were observed between the other Res and control groups (P > 0.05). In terms of apoptosis, Hair bucks exhibited a statistically significant difference in late apoptotic parameters, with the control showing the highest values (P. < 0.05). The Res 25 uM group (similar to the control group) showed lower mitochondrial oxidative stress than the Res 1 mM group (P. < 0.05).

Conclusion: Res at a dose of 1 mM did not protect most sperm functional and biochemical parameters except for apoptosis and performed worse than the control group. When all parameters are evaluated collectively, concentrations lower than 1 mM should be used for freezing Honamli and Hair Buck semen with resveratrol. https://doi.org/10.54680/fr25110110312.

Background: Single sperm cryopreservation (SSC) is a method that preserves the limited number of spermatozoa in testicular sperm. But testicular spermatozoa are characterized with low movement, which is not ideal for sperm selection before SSC.

Objective: This study was designed to investigate in vitro incubation (IVC) as a sperm selection technique before SSC on biological factors of testicular spermatozoa.

Materials and methods: Testicular tissue was obtained from 15 azoospermia men. One part of the testicular samples was used as a Control group, which was assessed fresh. One portion was cryopreserved by a vitrification (Vit) method and the two other portions were in vitro cultured for 24 h, with (IVC-Vit) or without (IVC) vitrification. Sperm motility, viability, morphology, DNA fragmentation and mitochondrial membrane potential were evaluated.

Results: Sperm motility and viability were better maintained in the IVC-Vit group compared to the Vit group (P=0.04 and P= 0.003, respectively). Sperm morphology, the fresh, Vit, IVC, and IVC-Vit groups all showed similar results (P > 0.05). Mitochondrial activity was significantly lower in the Vit group compared to the Control fresh group (P = 0.0001). The IVC group had a significantly higher DFI as compared to the Control (P < 0.0001). Compared to the IVC group, the IVC-Vit sperm had a significant increase in DFI (P= 0.0009). There was a statistically significant difference between post warm DFI of the Vit group and IVC-Vit group (P < 0.0001).

Conclusion: IVC as a sperm selection method increased motility and viability of testicular spermatozoa before single sperm vitrification. As DNA fragmentation increased by this technique, this method is not ideal for selecting viable sperm. https://doi.org/10.54680/fr25110110512.

Background: Long-term storage of cowpea pollen is important for the fertilization of spatially or temporally isolated female parents, especially during cowpea crop improvement and wide hybridization programs.

Objective: Experiments were conducted to determine pollen longevity at different storage temperatures and to develop a cryopreservation protocol for pollen of different cowpea accessions.

Materials and methods: The investigation was carried out at the Research Farm of ICAR-NBPGR, New Delhi, India, during the kharif (rainy) season of 2022. Pollen viability was studied after storage for 1, 3, 6, 12 and 24 h, 1 and 2 weeks and 1, 3 and 5 months at three different temperatures (4, -20 and -196 degree C).

Results: Fresh pollen viability ranged from 78 to 91 %. The optimal pollen moisture content was 12-14 % and the optimal air desiccation period under the laminar air flow chamber (22 +/- 1 degree C) was 5 min for subsequent preservation at -196 degree C. Pollen viability was lost completely at 4 and -20 degree C after 1 and 2 weeks of storage, respectively. Pollen stored in liquid nitrogen (-196 degree C) retained vaiblity similar to that of fresh pollen for > 5 months storage. Pollination using cryostored pollen resulted in normal fertilization.

Conclusion: This finding opens a gateway for cowpea haploid germplasm conservation and wide hybridization programs. https://doi.org/10.54680/fr24610110312.

Background: Cryopreservation of spermatozoa is a biotechnology used for fertilization purposes and preservation of genetic material in various domestic species.

Objective: To determine the efficacy of two different commercial semen diluents in the cryopreservation of epididymal semen of domestic cats.

Materials and methods: Twenty male cats aged between 1- 3 years and weighing 2.5- 4.5 kg were used in the study. The testicular tissues removed from the cats were immediately brought to the laboratory in physiological saline and the epididymal parts were trimmed in commercial semen extenders (INRA 96, Group I; OPTIXCELL, Group II). Diluted semen samples were cooled to 4°C and filled into 0.25 mL straws. Semen samples were frozen in a programmable semen freezing device and then placed in a liquid nitrogen container at -196 C. Semen samples were thawed at 38 degree C for 25 s. Thawed semen samples were evaluated in terms of motility and kinematic parameters using CASA.

Results: No statistical difference was found between the groups in terms of total motility, progressive motility, and velocity parameters at 4 degree C. The rate of spermatozoa at slow speeds was found to be lower in group II. In addition, after freezing and thawing process, no statistical difference was observed between the groups in terms of motility, kinematics, and velocity parameters.

Conclusion: Both commercial semen extenders can be used for cryopreservation of cat epididymal semen. https://doi.org/10.54680/fr24610110712.

Background: Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality.

Objective: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion.

Materials and methods: A total of 20 ejaculates from four Marwari stallions (five ejaculates from each) were collected. The gel free semen was extended with primary extender in equal volume and then divided in three equal groups, namely control (C), zinc (ZnO) and gold (Au), and centrifuged to obtain a final concentration of 100-150 x10⁶ mL^-1 and then cryopreserved. Cooled and post-thaw semen evaluations were conducted for various seminal characteristics, viz. progressive sperm motility, sperm plasma membrane integrity, sperm viability, acrosome integrity and mitochondrial membrane potential.

Results: Cooled semen (4 degree C for 2 h) evaluation revealed non-significant differences among the groups (C, ZnO and Au) for all the semen quality parameters. However, at the post-thaw stage, progressive sperm motility, sperm plasma membrane integrity, acrosome integrity and oxidative parameters were significantly (P < 0.01) higher in the ZnO group than Au and C.

Conclusion: The addition of ZnO nanoparticles improves the post thaw quality of stallion spermatozoa by reducing the oxidative stress, but Au nanoparticles had no effect on cooled as well as post-thaw semen quality. https://doi.org/10.54680/fr24610110212.

Cryopreservation is a well-known strategy to conserve genetic resources at ultra-low temperature. However, there is still limited knowledge on the cellular processes and molecular adjustments that allow cells to withstand the multiple stresses to which they are exposed during cryopreservation. To evaluate these processes, transcriptomics, the sub-discipline of omics that simultaneously examines mRNA transcripts formed by transcription from the genome, has been recently used. This article reviews recent scientific studies which use the basic principles of cryopreservation practices together with transcriptomics approaches, within the conceptual framework of cryobiomics. Moreover, the connections between factors that may be useful to optimize and validate approaches for mammalian or plant cell cryopreservation are also assessed. Transcriptomic applications are mainly performed with methods such as reverse transcriptase polymerase chain reaction (RT-PCR), simultaneous polymerase chain reaction (real-time PCR), northern blot, microarray/biochip and gene expression analysis (SAGE). Transcriptomic technologies allow a global view of gene expression profiles of different mammalian or plant cell types to be obtained before and after cryopreservation under multiple stress conditions. For these processes, small amounts of RNA enable efficient transcriptomics analysis. Transcriptomic analysis of cryopreserved mammalian and plant cells provides a conceptual way to identify the genes and their relative alterations in transcriptional abundances together with non-coding RNAs involved in important pathways related to cell viability and proliferation during and after cryopreservation. Moreover, it greatly contributes to understanding of non-fatal cryodamage and related developmental disorders in cryopreserved mammalian oocytes and sperm. In addition, single cell transcriptomics has the potential to non-invasely monitor immune actions and to diagnose the stage of the inflammatory process in kidney. Finally, qRT-PCR and RNA-seq studies have also revealed that some transcription factors are effective at inducing cold tolerance in many plants by elevating the levels of soluble sugars, proline and unsaturated fatty acids in cells. Hence transcriptomics studies may also aid investigations of the main mechanisms behind the so-called 'cryo-recalcitrance' that is observed mostly in plant cells. https://doi.org/10.54680/fr24610110112.