Rapid freezing of the mouse blastocyst: effects of cryoprotectants and of time and temperature of exposure to cryoprotectant before direct plunging into liquid nitrogen

1991 ◽  
Vol 3 (2) ◽  
pp. 175 ◽  
Author(s):  
R Li ◽  
A Trounson
Keyword(s):  
Liquid Nitrogen ◽  
Room Temperature ◽  
Rapid Freezing ◽  
High Concentration ◽  
Freezing And Thawing ◽  
Initial Exposure ◽  
Survival And Development ◽  
High Concentrations

This study investigates the effects of time and temperature of exposure to a high concentration (4.5 M) of dimethyl sulfoxide (DMSO), glycerol, 1,2-propanediol (PROH), or a mixture of DMSO and glycerol (DG) in a solution containing 0.25 M sucrose, on the survival and development of rapidly frozen mouse blastocysts. Embryos had significantly (P less than 0.01) higher rates of survival and development when exposed to cryoprotectant at 0 degree C compared with room temperature. The time of exposure to cryoprotectant at either 0 degree C or room temperature before being plunged into liquid nitrogen significantly (P less than 0.01) affected the survival and development of frozen-thawed embryos. Survival and development of blastocysts in vitro and in vivo was significantly (P less than 0.05) higher when exposed at 0 degree C for 10 min to DG, DMSO and glycerol than to PROH. It is concluded that, unlike early-cleavage stage embryos, blastocysts need to be equilibrated at a low temperature (0 degree C) with high concentrations of cryoprotectant before rapid freezing. Exposure of blastocysts to 4.5 M cryoprotectant and 0.25 M sucrose at room temperature either was toxic or else markedly reduced their viability after freezing and thawing, depending on the duration of the initial exposure.

Survival of rapidly frozen hatched mouse blastocysts

Zygote ◽  
2003 ◽  
Vol 11 (4) ◽  
pp. 361-366 ◽  
Author(s):  
Csaba Pribenszky ◽  
Sándor Cseh ◽  
Zsolt Abonyi-Tóth ◽  
László Solti
Keyword(s):  
Liquid Nitrogen ◽  
Zona Pellucida ◽  
Room Temperature ◽  
Calf Serum ◽  
Significant Loss ◽  
Rapid Freezing ◽  
Foetal Calf Serum ◽  
Freezing Medium

The objective of the present study was to examine the effect of rapid freezing on the in vitro and in vivo survival of zona-pellucida-free hatched mouse blastocysts. Hatched blastocysts were rapidly frozen in a freezing medium containing either ethylene glycol (EG) or glycerol (G) in 1.5 M or 3 M concentration. Prior to freezing, embryos were equilibrated in the freezing medium for 2 min, 10 min, 20 min or 30 min at room temperature. To freeze them, embryos were held in liquid nitrogen vapour [≈1 cm above the surface of the liquid nitrogen (LN2)] for 2 minutes and then immersed into LN2. After thawing, embryos were transferred either to rehydration medium (DPBS + 10% foetal calf serum + 0.5 M sucrose) for 10 minutes or rehydrated directly in DPBS supplemented with foetal calf serum. In vitro survival of embryos frozen with EG was higher than those frozen with G. The highest survival was obtained with 3 M EG and 2 min or 10 min equilibration prior to freezing, combined with direct rehydration after thawing. Frozen blastocysts developed into normal foetuses as well as unfrozen control ones did, with averages of 30% (control), 26% (EG) and 15% (G). The results show that hatching and hatched mouse blastocysts can be cryopreserved by a simple rapid freezing protocol in EG without significant loss of viability. Our data indicate that the mechanical protection of the zona pellucida is not needed during freezing in these stages.

scholarly journals The Swallowing Characteristics of Thickeners, Jellies and Yoghurt Observed Using an In Vitro Model

Dysphagia ◽  
2019 ◽  
Vol 35 (4) ◽  
pp. 685-695 ◽  
Author(s):  
Simmi Patel ◽  
William J. McAuley ◽  
Michael T. Cook ◽  
Yi Sun ◽  
Shaheen Hamdy ◽  
...  
Keyword(s):  
Transit Time ◽  
Mechanical Model ◽  
Xanthan Gum ◽  
In Vitro Model ◽  
High Concentration ◽  
High Concentrations ◽  
Vitro Model ◽  
Thickening Agents

Abstract Drinks and foods may be thickened to improve swallowing safety for dysphagia patients, but the resultant consistencies are not always palatable. Characterising alternative appetising foods is an important task. The study aims to characterise the in vitro swallowing behaviour of specifically formulated thickened dysphagia fluids containing xanthan gum and/or starch with standard jellies and yoghurt using a validated mechanical model, the “Cambridge Throat”. Observing from the side, the model throat can follow an experimental oral transit time (in vitro-OTT) and a bolus length (BL) at the juncture of the pharynx and larynx, to assess the velocity and cohesion of bolus flow. Our results showed that higher thickener concentration produced longer in vitro-OTT and shorter BL. At high concentration (spoon-thick), fluids thickened with starch-based thickener showed significantly longer in vitro-OTT than when xanthan gum-based thickener was used (84.5 s ± 34.5 s and 5.5 s ± 1.6 s, respectively, p < 0.05). In contrast, at low concentration (nectar-like), fluids containing xanthan gum-based thickener demonstrated shorter BL than those of starch-based thickener (6.4 mm ± 0.5 mm and 8.2 mm ± 0.8 mm, respectively, p < 0.05). The jellies and yoghurt had comparable in vitro-OTT and BL to thickeners at high concentrations (honey-like and spoon-thick), indicating similar swallowing characteristics. The in vitro results showed correlation with published in vivo data though the limitations of applying the in vitro swallowing test for dysphagia studies were noted. These findings contribute useful information for designing new thickening agents and selecting alternative and palatable safe-to-swallow foods.

Immunological investigations of oligoethylene glycols functionalized with desaminotyrosine and desaminotyrosyltyrosine

2013 ◽  
Vol 1569 ◽  
pp. 9-14 ◽  
Author(s):  
Konstanze K. Julich-Gruner ◽  
Toralf Roch ◽  
Nan Ma ◽  
Axel T. Neffe ◽  
Andreas Lendlein
Keyword(s):  
Human Blood ◽  
High Concentration ◽  
Inflammatory Conditions ◽  
Tnf Α ◽  
High Concentrations ◽  
Pharmaceutical Agents ◽  
Immunological Evaluation ◽  
Necrosis Factor

ABSTRACTBiomaterials require thorough in vitro testing before being applied in vivo. Unwanted contaminations of biomaterials but also their intrinsic properties can cause uncontrolled immune response leading to severe consequences for the patient. Therefore, immunological evaluation of materials for biomedical applications is mandatory before entering clinical application. In order to introduce physical netpoints, the aromatic compounds desaminotyrosine (DAT) and desaminotyrosyl-tyrosine (DATT) were successfully used to functionalize linear and star-shaped oligoethylene glycol (lOEG and sOEG) as previously described. The materials showed properties of surfactants and have potential to be used for solubilization of lipophilic drugs in water. Furthermore, the materials are susceptible for H2O2 degradation as determined by MALDI-ToF MS analyses. This is important for potential in vivo applications, as macrophages can release reactive oxygen species (ROS) under inflammatory conditions. As it is known that surfactant solutions of high concentration can lead to cell lysis, the effects of OEG-DAT(T) solutions on murine RAW macrophages were investigated. Even at highest OEG-DAT(T) concentration of 1000 µg·mL-1 the viability of the RAW cells was not significantly impaired. Additionally, the polymers were incubated with whole human blood and the production of inflammatory cytokines such as the tumor necrosis factor (TNF)-α and interleukin (IL)-6 was determined. Only at high concentrations, the OEG-DAT(T) solution induced low levels of TNF-α and IL-6, indicating that a mild inflammatory reaction could be expected when such high OEG-DAT(T) concentrations are applied in vivo. Similarly, the OEG-DAT(T) solution did not induce ROS in monocytes and neutrophils after incubation with whole human blood. Conclusively, the data presented here demonstrate that OEG-DAT(T) do not lead to a substantial activation of the innate immune mechanisms and could therefore be investigated for solubilizing pharmaceutical agents.

Comparison of traditional and modified (VitMaster) methods of rabbit embryo vitrification

2009 ◽  
Vol 57 (3) ◽  
pp. 411-416 ◽  
Author(s):  
Krzysztof Papis ◽  
Maciej Korwin-Kossakowski ◽  
Elżbieta Wenta-Muchalska
Keyword(s):  
Liquid Nitrogen ◽  
Supercooled Liquid ◽  
Blastocyst Stage ◽  
Microbiological Contamination ◽  
Cooling Method ◽  
Survival And Development ◽  
Morula Stage ◽  
Rabbit Embryo

In spite of their cryobiological efficacy, minimum-volume vitrification methods suffer from the risk of microbiological contamination and are technically and/or manually demanding. In this study, the effects of a traditional, slightly modified vitrification method and vitrification using supercooled liquid nitrogen (VitMaster) applied for rabbit morula-stage embryos were compared. Embryos were equilibrated in a solution containing 1,2-propanediol (2.72 M) and glycerol (1.36 M) for 7 min and vitrified in 0.25-ml insemination straws after 1-min exposure to a vitrification solution containing additionally 1.0 M sucrose. Cooling was performed in ‘normal’ or supercooled liquid nitrogen. Regardless of the cooling method applied, high in vitro survival and development rates of vitrified embryos were obtained. All embryos were intact after warming, and 61 out of 65 (93.8%) and 23 out of 24 (95.8%) embryos developed to the blastocyst stage after 48-h in vitro culture of embryos vitrified in ‘normal’ or supercooled liquid nitrogen, respectively. The results suggest higher developmental ability of embryos vitrified in supercooled liquid nitrogen (91.7% vs . 83.1% of embryos vitrified traditionally developed to more advanced, expanding and/or hatching blastocyst stages). In vivo survival rate, tested for the traditional vitrification system only, revealed that 36.8% of embryos developed to term. The results show promise for establishing a fully successful method for rabbit embryo vitrification.

scholarly journals High-Dose Benzodiazepines Positively Modulate GABAA Receptors via a Flumazenil-Insensitive Mechanism

2021 ◽  
Vol 23 (1) ◽  
pp. 42
Author(s):  
Na Wang ◽  
Jingjing Lian ◽  
Yanqing Cao ◽  
Alai Muheyati ◽  
Shanshan Yuan ◽  
...  
Keyword(s):  
Gabaa Receptors ◽  
High Dose ◽  
High Concentration ◽  
Concentration Effects ◽  
In Vivo Evaluation ◽  
Chemical Structures ◽  
Loss Of Righting Reflex ◽  
High Concentrations

Benzodiazepines (BZDs) produce versatile pharmacological actions through positive modulation of GABAA receptors (GABAARs). A previous study has demonstrated that high concentrations of diazepam potentiate GABA currents on the α1β2γ2 and α1β2 GABAARs in a flumazenil-insensitive manner. In this study, the high-concentration effects of BZDs and their sensitivity to flumazenil were determined on synaptic (α1β2γ2, α2β2γ2, α5β2γ2) and extra-synaptic (α4β2δ) GABAARs using the voltage-clamp electrophysiology technique. The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice via the loss of righting reflex (LORR) test. Diazepam induced biphasic potentiation on the α1β2γ2, α2β2γ2 and α5β2γ2 GABAARs, but did not affect the α4β2δ receptor. In contrast to the nanomolar component of potentiation, the second potentiation elicited by micromolar diazepam was insensitive to flumazenil. Midazolam, clonazepam, and lorazepam at 200 µM exhibited similar flumazenil-insensitive effects on the α1β2γ2, α2β2γ2 and α5β2γ2 receptors, whereas the potentiation induced by 200 µM zolpidem or triazolam was abolished by flumazenil. Both the GABAAR antagonist pentylenetetrazol and Fa173, a proposed transmembrane site antagonist, abolished the potentiation induced by 200 µM diazepam. Consistent with the in vitro results, flumazenil antagonized the zolpidem-induced LORR, but not that induced by diazepam or midazolam. Pentylenetetrazol and Fa173 antagonized the diazepam-induced LORR. These findings support the existence of non-classical BZD binding sites on certain GABAAR subtypes and indicate that the flumazenil-insensitive effects depend on the chemical structures of BZD ligands.

scholarly journals 93 BLASTOCYST PRODUCTION FROM BOVINE OOCYTES VITRIFIED IN A CLOSED (BIOSECURE) SYSTEM FOLLOWING IN VITRO MATURATION IN THE PRESENCE OR ABSENCE OF VITAMIN E

2005 ◽  
Vol 17 (2) ◽  
pp. 196 ◽  
Author(s):  
V.C. Moreira ◽  
G.J. McCallum ◽  
A. Ainslie ◽  
T.G. McEvoy
Keyword(s):  
Vitamin E ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Assisted Reproductive Technologies ◽  
Calf Serum ◽  
Reproductive Technologies ◽  
Embryo Viability ◽  
Initial Size ◽  
Initial Exposure

The value of assisted reproductive technologies intended for conservation of livestock genetics ultimately will depend on their effectiveness in both sustaining gamete/embryo viability and ensuring stringent biosecurity. This study investigated bovine oocyte survival following vitrification in a sealed system prior to storage in liquid nitrogen. It also tested the effect of supplementary vitamin E on tolerance of oocytes to vitrification procedures. Healthy COCs from abattoir-derived ovaries were matured in TCM-199 supplemented with 10% v/v fetal calf serum (FCS) in the absence (control) or presence (VitE) of 100 μM α-tocopherol (Sigma, Poole, UK) in humidified atmosphere (5% CO2 in air; 38.5°C). Between 22 and 24 h after commencement of IVM, COCs were pipetted to remove excess cumulus cells, and then equilibrated at room temperature in 7.5% DMSO plus 7.5% ethylene glycol (EG) in HEPES-buffered Holding Medium (HM; Irvine Scientific, Santa Ana, CA, USA) for 7 min before transfer to vitrification solution (15% DMSO, 15% EG, and 0.5 M sucrose in HM; Irvine Scientific). Loading of oocytes (n = 78 control and 85 VitE) into CryoTips (Irvine Scientific) and heat-sealing (each end) was achieved within 90 s, with tips then plunged into liquid nitrogen. Subsequent warming and cryoprotectant removal were at room temperature in HM with 1 M sucrose for 2 min, 0.5 M sucrose for 4 min, and HM alone for 6 min. Oocytes were allowed recover for approximately 3 h in TCM-199 with 20% FCS (5% CO2 in air; 38.5°C), and then fertilized in vitro (single sire). After 22 h (Day 1) presumptive zygotes were transferred to SOF containing fatty acid-free BSA (4 mg mL−1) and incubated for up to 8 days (5% O2, 5% CO2, 90% N2; 38.5°C). Cleavage data (Day 2) and blastocyst yields (Days 7 to 9) were analyzed by chi-square test. In addition to those that were vitrified, some oocytes (n = 9 per treatment) were observed via video to permit analysis (ImageJ; NIH, USA) of volume excursions during the 7 min immediately following initial exposure to HM with 7.5% DMSO plus 7.5% EG. Data were compared using ANOVA. Overall incidence of cleavage by Day 2 was 45% (range: 36 to 51%) and 35% (31 to 43%) for control and VitE, respectively, (NS). Day 7 and total control blastocyst yields were 7.4% and 18.5%, respectively; corresponding yields for VitE were 19% and 25% (control vs. VitE, NS). Video evidence indicated that although Control oocytes invariably reached minimal volume later than VitE oocytes (30 vs. 20 s), in each case this was 52% of initial size. By 7 min, both had similar volumes, the respective means being 94% and 92% of initial size. In the present study provision of vitamin E during IVM did not significantly enhance the subsequent resilience and development of oocytes subjected to a vitrification protocol. However, this protocol achieved efficient and biosafe bovine gamete storage. This work was funded by SEERAD; CryoTips and vitrification solutions were donated by Irvine Scientific; VCM was supported by MLC, UK.

scholarly journals ATG5 is instrumental in the transition from autophagy to apoptosis during the degeneration of tick salivary glands

2020 ◽  
Author(s):  
Yanan Wang ◽  
Houshuang Zhang ◽  
Li Luo ◽  
Yongzhi Zhou ◽  
Jie Cao ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Calcium Concentration ◽  
High Concentration ◽  
Normal Feeding ◽  
Feeding Process ◽  
High Concentrations ◽  
Caspase Gene ◽  
Tick Salivary Glands

AbstractFemale tick salivary glands undergo rapid degeneration several days post engorgement. This degeneration may be caused by the increased concentration of ecdysone in the hemolymph during the fast feeding period and both autophagy and apoptosis occur. In this work, we first proved ATG and Caspase gene expression peaks during degeneration of the tick salivary glands. We explored the regulatory role of Rhipicephalus haemaphysaloides autophagy related 5 (RhATG5) in the degeneration of tick salivary glands. During the fast feeding phase, RhATG5 was cleaved and both calcium concentration and the transcription of RhCalpains increased in the salivary glands. Recombinant RhATG5 was cleaved by μ-Calpain only in the presence of calcium; the mutant RhATG5191-199Δ was not cleaved. Treatment with 20-hydroxyecdysone (20E) led to programmed cell death in the salivary glands of unfed ticks in vitro, RhATG8-phosphatidylethanolamine (PE) was upregulated in ticks treated with low concentration of 20E. Conversely, RhATG8-PE decreased and RhCaspase-7 increased in ticks treated with a high concentration of 20E and transformed autophagy to apoptosis. High concentrations of 20E led to the cleavage of RhATG5. Calcium concentration and expression of RhCalpains were also upregulated in the tick salivary glands. RNA interference (RNAi) of RhATG5 in vitro inhibited both autophagy and apoptosis of the tick salivary glands. RNAi of RhATG5 in vivo significantly inhibited the normal feeding process. These results demonstrated that high concentrations of 20E led to the cleavage of RhATG5 by increasing the concentration of calcium and stimulated the transition from autophagy to apoptosis.

scholarly journals ATG5 is instrumental in the transition from autophagy to apoptosis during the degeneration of tick salivary glands

2021 ◽  
Vol 15 (1) ◽  
pp. e0009074
Author(s):  
Yanan Wang ◽  
Houshuang Zhang ◽  
Li Luo ◽  
Yongzhi Zhou ◽  
Jie Cao ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Calcium Concentration ◽  
High Concentration ◽  
Normal Feeding ◽  
Feeding Process ◽  
High Concentrations ◽  
Caspase Gene ◽  
Tick Salivary Glands

Female tick salivary glands undergo rapid degeneration several days post engorgement. This degeneration may be caused by the increased concentration of ecdysone in the hemolymph during the fast feeding period and both autophagy and apoptosis occur. In this work, we first proved autophagy-related gene (ATG) and caspase gene expression peaks during degeneration of the tick salivary glands. We explored the regulatory role of Rhipicephalus haemaphysaloides autophagy-related 5 (RhATG5) in the degeneration of tick salivary glands. During the fast feeding phase, RhATG5 was cleaved and both calcium concentration and the transcription of Rhcalpains increased in the salivary glands. Recombinant RhATG5 was cleaved by μ-calpain only in the presence of calcium; the mutant RhATG5191-199Δ was not cleaved. Treatment with 20-hydroxyecdysone (20E) led to programmed cell death in the salivary glands of unfed ticks in vitro, RhATG8-phosphatidylethanolamine (PE) was upregulated in ticks treated with low concentration of 20E. Conversely, RhATG8-PE decreased and Rhcaspase-7 increased in ticks treated with a high concentration of 20E and transformed autophagy to apoptosis. High concentrations of 20E led to the cleavage of RhATG5. Calcium concentration and expression of Rhcalpains were also upregulated in the tick salivary glands. RNA interference (RNAi) of RhATG5 in vitro inhibited both autophagy and apoptosis of the tick salivary glands. RNAi of RhATG5 in vivo significantly inhibited the normal feeding process. These results demonstrated that high concentrations of 20E led to the cleavage of RhATG5 by increasing the concentration of calcium and stimulated the transition from autophagy to apoptosis.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

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