36 Extended culture after vitrification-warming helps in spindle recovery of bovine oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 144
Author(s):  
E. Gutierrez ◽  
Z. Jiang ◽  
K. Bondioli
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Incubation Period ◽  
The Other ◽  
Meiotic Spindle ◽  
Chromosome Arrangement ◽  
Extended Culture ◽  
Base Medium ◽  
Maturation Medium ◽  
Bovine Oocytes

The meiotic spindle is one of the most vulnerable cytoplasmic organelles when performing oocyte vitrification. It has been proposed that submitting oocytes to a post-warming incubation period in maturation medium helps in the reorganization of microtubules and chromosomes. Our previous experiments found no differences in spindle morphology after submitting vitrified oocytes to a 2-h incubation period. The aim of this experiment was to determine the effect of extended culture on the reorganization of the meiotic spindle of vitrified-warmed bovine oocytes. Oocytes were purchased from a commercial vendor (n=86) and matured during shipment. In this experiment, three treatments were evaluated: fresh oocytes (F) (n=30), vitrified-warmed (VW; n=26), and extended culture (EC; n=30). Cumulus-oocyte complexes were removed at 18h of maturation. Fresh oocytes were denuded by vortexing in hyaluronidase (1.5mgmL−1) and immediately fixed using 4% paraformaldehyde. Oocytes undergoing vitrification were partially denuded by pipetting in hyaluronidase (1.5mgmL−1). The vitrification protocol consisted of incubation in equilibration solution (7.5% dimethyl sulfoxide + 7.5% ethylene glycol) for 9min and then in vitrification solution (15% dimethyl sulfoxide + 15% ethylene glycol + 0.5M sucrose). While in vitrification solution, oocytes were mounted onto a Cryolock and plunged into liquid nitrogen in less than 1min. Warming was performed by placing a Cryolock into 0.5M sucrose for 3min and then into 0.25M sucrose for 3min. Finally, oocytes were washed in base medium. The base medium used for cryoprotectant and warming solutions was Dulbecco's phosphate-buffered saline supplemented with 20% fetal bovine serum. Both, vitrification and warming, were performed at 38.5°C. After warming, half of the oocytes were completely denuded and fixed and the other half underwent a 6-h incubation period in maturation medium (IVF-Bioscience). To examine microtubule distribution and chromosome arrangement, fixed oocytes were submitted to an immunostaining protocol using α tubulin antibody (1:100) and anti IgG-Alexa Fluor 488 (1:1000; Thermo Fisher Scientific) and counterstained with Hoechst. The effect of extended culture on the incidence of abnormal microtubule distribution and chromosome arrangement was analysed using logistic regression with a binomial response variable (normal/abnormal). There was no difference in maturation rates among groups (F=73.3%, VW=77%, EC=86.6%; P=0.43). For microtubule distribution, oocytes fixed immediately after warming had a higher incidence of abnormal spindles (57.7%) when compared with oocytes submitted to extended culture (26.6%; P=0.02). The most common abnormality seen in oocytes fixed after warming was small and faintly stained spindles. Microtubule distribution in fresh oocytes did not differ from oocytes in the other groups. There were no differences in chromosome arrangement among groups (P=0.11). Future research will focus on evaluating the benefits that this technique offers to improve development following IVF using vitrified-warmed oocytes.

25 Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 120
Author(s):  
E. Girka ◽  
K. R. Bondioli
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Recovery Period ◽  
Normal Chromosome ◽  
Meiotic Spindle ◽  
Control Group ◽  
Chromosome Arrangement ◽  
Epothilone B ◽  
Significant Difference ◽  
Bovine Oocytes

Vitrification has the potential to be a valuable technique for preservation of bovine oocytes; however, this method often results in abnormal microtubule and chromosome arrangement. The aim of this experiment was to evaluate taxol and epothilone B as meiotic spindle stabilising pretreatments in a vitrification protocol. Bovine oocytes were purchased and matured invitro during shipment. At 18h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in invitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45s. For warming, a Cryolock was placed directly into a 0.5M sucrose solution and incubated for 3min. Oocytes were transferred to a 0.25M solution for 3min and washed in the basal solution used for vitrification and warming media (Dulbecco’s phosphate-buffered saline, 20% fetal bovine serum). Once warmed, oocytes were transferred to IVM medium for a 4-h recovery period and completely denuded before staining. Staining to evaluate spindle morphology was performed with anti α-tubulin primary antibody and secondary antibody Alexa Fluor 488. Oocytes were also stained with Hoechst to evaluate chromosome arrangement. Both spindle morphology and chromosome arrangement data were analysed using a logistic regression with a binomial response variable (normal/abnormal). Both 0.5μM and 1.0μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P>0.05). The 2.0μM taxol treatment improved chromosome configuration (P<0.05) with no effect on microtubule distribution compared with the control group (P>0.05). All epothilone B treatments resulted in disruption of microtubule distribution and chromosome arrangement compared with control (P<0.001) and resulted in a consistent abnormality hypothesised to be tubulin polymerization. These results indicate that taxol is capable of increasing the occurrence of normal chromosome arrangement in vitrified bovine oocytes and that epothilone B may cause additional harm to the oocyte that is not associated with the metaphase plate. Table 1. Effect of stabilisation agents on meiotic spindle of invitro-matured bovine oocytes Treatment n Normal microtubule distribution (%) Normal chromosome arrangement (%) Control 100 44 47 0.5μM Taxol 104 44 37 1.0μM Taxol 98 43 56 2.0μM Taxol 102 49 62a 0.5μM Epothilone B 103 11b 11b 1.0μM Epothilone B 97 6b 8b 2.0μM Epothilone B 100 2b 1b aP<0.05;. bP<0.001: Different superscripts within a column indicate a significant difference.

48 Effect of Dimethyl Sulfoxide- or Glycerol-Based Vitrification Protocols on Meiotic Spindle of In Vitro-Matured Bovine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 163
Author(s):  
E. J. Gutierrez ◽  
F. A. Diaz ◽  
B. A. Foster ◽  
K. R. Bondioli
Keyword(s):  
Dimethyl Sulfoxide ◽  
Incubation Period ◽  
Meiotic Spindle ◽  
Chromosome Arrangement ◽  
Significant Difference ◽  
Fetal Bovine ◽  
High Concentrations ◽  
Binomial Response ◽  
Bovine Oocytes

There is evidence suggesting that high concentrations of cryoprotectants (CPA) and very low temperatures during vitrification cause disruption of the meiotic spindle, resulting in poor post-warming meiotic resumption and other abnormalities at fertilization. This study sought to determine the damage caused by CPA and freezing upon the meiotic spindle of bovine oocytes vitrified at the metaphase II stage, and whether a subsequent incubation could promote recovery from this damage. Bovine cumulus–oocyte complexes were purchased from a commercial vendor (n = 154). Oocytes were removed from in vitro maturation media at 22 h, denuded by vortexing in hyaluronidase, and divided into 4 groups according to CPA exposure and whether they were incubated or not. The resulting groups were DMSO I (n = 36), DMSO NI (n = 41), GLY I (n = 39), GLY NI (n = 38). Two repetitions were carried out for each protocol evaluated, which included a combination of ethylene glycol (EG) with either dimethyl sulfoxide (DMSO) or glycerol (GLY). Oocytes were exposed to equilibration solution consisting of 7.5% EG and 7.5% DMSO or GLY for 9 min at room temperature (RT) and then placed into vitrification solution (VS) that contained 15% EG, 15% DMSO or GLY, and 0.5 M sucrose. While in VS, 3 to 4 denuded oocytes were loaded onto a Cryolock® (Biotech Inc., Alpharetta, GA, USA) and plunged into liquid nitrogen within 1 min. For warming, oocytes were exposed to previously warmed (37°C) dilution solution 1 (DS1) consisting of 0.5 M sucrose for 1 and 2 min at RT, for a total of 3 min in DS1, and then placed in dilution solution 2 containing 0.25 M sucrose for 3 min. Finally, oocytes were washed in base media. Base media for all solutions was PBS supplemented with 20% fetal bovine serum. After warming, half of the oocytes were fixed and the rest were submitted to a 2-h incubation period in maturation media at 37°C and 5.5% CO2, and then fixed. To examine microtubule distribution and chromosome arrangement, fixed oocytes were submitted to an immunofluorescence protocol using α-tubulin antibody (1:100) as primary antibody, Alexa Fluor 488 (1:1000) as secondary antibody, and counterstained with propidium iodide (10 mg mL−1). Oocytes were observed under a fluorescence microscope. The effects of CPA and incubation on the incidence of abnormal spindles measured with both microtubules distribution and chromosome arrangement were evaluated using logistic regression with a binomial response variable (normal/abnormal). For microtubule distribution, results showed that oocytes treated with DMSO presented significantly lower normality (31.17%) than those treated with glycerol (54.55%; P < 0.003). The most common abnormality observed in oocytes treated with DMSO was that the spindle was smaller and more faintly stained than those treated with glycerol. For chromosome arrangement, there was no significant difference between treatments (P = 0.7093). Additionally, there was no sign of improvement when submitting the oocytes to an incubation period for any of the components examined.

28 Effect of deuterium oxide on bovine oocyte cryotolerance

2019 ◽  
Vol 31 (1) ◽  
pp. 140
Author(s):  
F. Salerno ◽  
M. Rubessa ◽  
B. Gasparrini ◽  
M. Wheeler
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Deuterium Oxide ◽  
Control Group ◽  
Crystal Formation ◽  
Cleavage Rate ◽  
Maturation Medium

It is known that cryopreservation triggers spindle disassembly, increased aneuploidy risk, decreased post-thaw survival, fertilization, and embryo development. We hypothesised that a treatment with D2O before vitrification would slow down oocyte metabolism and reduce ice crystal formation by replacing water inside the cells. The aim of the study was to evaluate the effect of a 4-h treatment with different D2O concentrations (0, 3, 15, and 30%) on cryotolerance of bovine in vitro-matured oocytes. Abattoir-derived bovine oocytes were matured in vitro for 20h in TCM-199 medium with 15% of bovine serum (BS), 0.5µg mL−1 of FSH, 5µg mL−1 of LH, 0.8mM l-glutamine, and 50µg mL−1 of gentamicin at 39°C with 5% of CO2 and randomly divided into 5 experimental groups. A group of non-vitrified oocytes was used as the fresh oocyte control group, whereas the remaining oocytes were incubated for 4h in in vitro maturation medium with 0% (vitrified control; n=205), 3% (n=205), 15% (n=205), and 30% D2O (n=205) before vitrification. The experiment was repeated 4 times. Oocytes were denuded in HEPES-buffered TCM-199 (H199)+5% BS and vitrified using a cryotop freezing straw. The oocytes were incubated in 200μL of H199+20% BS with 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 3min. After that, oocytes were collected in 50μL of H199+20% fetal bovine serum with 15% ethylene glycol+15% dimethyl sulfoxide and 0.5M sucrose for 20s and plunged into LN2. One month later, oocytes were warmed in thawing media with decreasing concentrations of sucrose (1.35M to 0.31M) and then placed into in vitro maturation medium for 2h before IVF. Matured oocytes were IVF and cultured according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). Cleavage and blastocyst rates were evaluated after 7 days of culture. Data were analysed using the GLM procedure of SPSS (SPSS Inc., Chicago, IL, USA). The least statistical difference post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. As expected, both cleavage [60.5±4.6 (fresh control); 36.9±2.6 (0% D2O); 46.3±3.7 (3% D2O); 31.6±2.4 (15% D2O); and 24.4±2.6 (30% D2O)] and blastocyst rates [25.7±0.8 (fresh control); 9.0±0.8 (0% D2O); 9.0±0.7 (3% D2O); 3.6±0.2 (15% D2O); and 4.3±0.8 (30% D2O)] decreased in all vitrified groups compared with the fresh control group. Within vitrified oocytes, cleavage rate increased (P&lt;0.05) with 3% D2O treatment compared with the other groups. However, pretreatment with higher (15-30%) D2O concentrations decreased (P&lt;0.05) blastocyst rates of vitrified-warmed oocytes. In conclusion, a pretreatment with low concentrations (3%) of D2O improved the cleavage rate of bovine vitrified-warmed oocytes, suggesting a potential beneficial effect, whereas deleterious effects were observed using the higher concentrations. Therefore, further studies are required to assess a potential use of D2O to improve oocyte cryotolerance, likely testing different incubation times.

scholarly journals 46 VITRIFICATION OF IMMATURE AND MATURE BOVINE OOCYTES

2017 ◽  
Vol 29 (1) ◽  
pp. 130
Author(s):  
P. T. Hardin ◽  
F. A. Diaz ◽  
B. A. Foster ◽  
E. J. Gutierrez ◽  
K. R. Bondioli
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Maturation Stage ◽  
Crystal Formation ◽  
Ice Crystal ◽  
Exact Test ◽  
Immature Oocytes ◽  
Commercial Source ◽  
Bovine Oocytes

While vitrification has become a valuable system used in oocyte and embryo preservation, there is still much to be learned in optimizing this protocol. Both mature and immature oocytes can be vitrified but each presents challenging aspects. Mature oocytes have microfilaments that are not yet developed in immature oocytes, which are fragile and may be disrupted by ice crystal formation during freezing. Further, currently many different cryoprotectants are used in different concentrations, most being combinations of dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol. This study aimed to determine if vitrification solutions composed of ethylene glycol and either dimethyl sulfoxide or glycerol resulted in more-competent post-thaw oocytes, and to determine if maturation stage affected optimal vitrification solution. As validation of the IVF protocol, fresh mature oocytes from a commercial source were fertilized and proportion, with pronuclei formation 48 h post-IVF was recorded. Two experiments evaluated 2 cryoprotectant solutions by analysing post-vitrification and thaw competence of in vitro-fertilized oocytes to form pronuclei. Oocytes in both studies were exposed to 2 sequential vitrification solutions containing 10% DMSO or glycerol, 10% ethylene glycol and 0.5 M sucrose, and then 20% DMSO/glycerol and ethylene glycol and 0.5 M sucrose, before vitrification on cryolocks. In the first study, immature bovine oocytes (n = 200) were vitrified. Following thawing and IVM, they were analysed for pronuclei formation, with 8.49% and 0% fertilization following vitrification in DMSO and glycerol, respectively (P < 0.01). In the second study, mature oocytes were vitrified (n = 200), thawed, and fertilized using the same methods as in study 1. In total, 12.62% and 3.4% of the mature oocytes were successfully fertilized following vitrification in DMSO and glycerol, respectively (P < 0.05). Fisher’s exact test was used for all statistics in both studies. These results suggest that DMSO in combination with ethylene glycol may be superior to glycerol for vitrification of both immature and mature bovine oocytes.

Effect of type of cryoprotectant on morphology and developmental competence of in vitro-matured buffalo (Bubalus bubalis) oocytes subjected to slow freezing or vitrification

2008 ◽  
Vol 20 (4) ◽  
pp. 490 ◽  
Author(s):  
S. K. Gautam ◽  
V. Verma ◽  
P. Palta ◽  
M. S. Chauhan ◽  
R. S. Manik
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bubalus Bubalis ◽  
The Other ◽  
Developmental Competence ◽  
Slow Freezing ◽  
Cleavage Rate ◽  
Cryopreservation Method

The present study examined the effects of different cryoprotectants on morphology and developmental competence of in vitro-matured buffalo oocytes after slow freezing or vitrification. After slow freezing in dimethyl sulfoxide (DMSO), ethylene glycol (EG) or 1,2-propanediol (PROH), at 1.0 or 1.5 m each, the proportion of morphologically normal oocytes recovered was significantly higher (P < 0.05) with 1.5 than 1.0 m for all cryoprotectants and was highest (P < 0.05) for 1.5 m DMSO. Following vitrification, the percentage of morphologically normal oocytes recovered was lower (P < 0.01) for 40% EG than for 40% DMSO, 20% EG + 20% DMSO or 20% EG + 20% PROH. The most common damage, irrespective of the cryopreservation method, was loss of cumulus mass. The cleavage rate and the proportion of vitrified–warmed oocytes that developed to morulae/blastocysts were significantly higher (P < 0.01) for 20% EG + 20% DMSO than for the other groups. A higher proportion of oocytes developed to morulae (11.5% v. 4.3%) or blastocysts (5.4% v. 0.6%) after vitrification in 20% EG + 20% DMSO than after slow freezing in 1.5 m DMSO. In conclusion, vitrification was more effective than slow freezing for the cryopreservation of in vitro-matured buffalo oocytes.

scholarly journals Clicked and long spaced galactosyl- and lactosylcalix[4]arenes: new multivalent galectin-3 ligands

2014 ◽  
Vol 10 ◽  
pp. 1672-1680 ◽  
Author(s):  
Silvia Bernardi ◽  
Paola Fezzardi ◽  
Gabriele Rispoli ◽  
Stefania E Sestito ◽  
Francesco Peri ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Ethylene Glycol ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Lectin Binding ◽  
The Other ◽  
Isomeric Form ◽  
Synthetic Pathway ◽  
Cuaac Reaction ◽  
Galectin 3

Four novel calix[4]arene-based glycoclusters were synthesized by conjugating the saccharide units to the macrocyclic scaffold using the CuAAC reaction and using long and hydrophilic ethylene glycol spacers. Initially, two galactosylcalix[4]arenes were prepared starting from saccharide units and calixarene cores which differ in the relative dispositions of the alkyne and azido groups. Once the most convenient synthetic pathway was selected, two further lactosylcalix[4]arenes were obtained, one in the cone, the other one in the 1,3-alternate structure. Preliminary studies of the interactions of these novel glycocalixarenes with galectin-3 were carried out by using a lectin-functionalized chip and surface plasmon resonance. These studies indicate a higher affinity of lactosyl- over galactosylcalixarenes. Furthermore, we confirmed that in case of this specific lectin binding the presentation of lactose units on a cone calixarene is highly preferred with respect to its isomeric form in the 1,3-alternate structure.

scholarly journals Comparative Study of Ag Nanostructures: Molecular Simulations, Electrochemical Behavior, and Antibacterial Effect

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Álvaro de Jesús Ruíz-Baltazar ◽  
Simón Yobanny Reyes-López ◽  
D. Larrañaga ◽  
R. Pérez
Keyword(s):  
Cyclic Voltammetry ◽  
Ethylene Glycol ◽  
Comparative Study ◽  
Sodium Borohydride ◽  
Electrochemical Behavior ◽  
Antibacterial Effect ◽  
Chemical Reduction ◽  
Molecular Simulations ◽  
The Other ◽  
E Coli

Nanoparticles of Ag with different sizes and structures were obtained and studied. Two methods for reductions of Ag ions were employed, chemical reduction by sodium borohydride and ethylene glycol. Cuboctahedral and icosahedral structures were obtained. Molecular simulations were carried out in order to evaluate the reactivity of both structures. On the other hand, the electrochemical activity and antibacterial effect (E. coli) of the cuboctahedral and icosahedral structures were measured experimentally. The results obtained by molecular simulation, cyclic voltammetry, and antibacterial effect were compared and discussed in this work.

cis-5-Cyclooctene-1,2-dione and its Ethylene Ketals

1972 ◽  
Vol 50 (10) ◽  
pp. 1548-1556 ◽  
Author(s):  
Peter Yates ◽  
E. G. Lewars ◽  
P. H. McCabe
Keyword(s):  
Hydrogen Peroxide ◽  
Acetic Anhydride ◽  
Ethylene Glycol ◽  
Dimethyl Sulfoxide

Oxidation of cis-cis-1,5-cyclooctadiene with hydrogen peroxide gives cis-5-cyclooctene-trans-1,2-diol (3) which is converted to cis-5-cyclooctene-1,2-dione (6) on treatment with dimethyl sulfoxide and acetic anhydride. Bromination of 6 is accompanied by transannular bonding to give a dibromo keto ether 9a or b. Ketalization of 6 with ethylene glycol gives a monoketal 11 and two diketals 12 and 13 with 1,3-dioxolane and 1,4-dioxane rings, respectively. Bromination of 12 with bromine or pyridinium perbromide is accompanied by transannular bonding and fission of one of the 1,3-dioxolane rings to give a dibromo monoketal ether 15a (or b). Bromination of 12 with N-bromosuccinimide followed by dehydrobromination gives a cyclooctadiene-1,2-dione diketal 20a (or b).

On the incubation period in neurolues

1927 ◽  
Vol 23 (10) ◽  
pp. 1046-1050
Author(s):  
E. V. Sukhova
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Incubation Period ◽  
The Other ◽  
Other Hand ◽  
General Number ◽  
The Central Nervous System

Speaking about syphilis lesions of the central nervous system, it is impossible not to note that these lesions are among the most severe diseases of the latter. But, on the other hand, their severity is redeemed to some extent by the specific means of combating them which we have in our hands. In this case, the fight against neurolues is reduced not so much to its treatment as to its prevention. Hence the interest with which the question of the influence of various conditions on the occurrence of syphilitic lesions of the central nervous system has recently begun to be comprehensively discussed and the exact causes which, from the general number of syphilitics, distinguish the group subsequently condemned to neurolues have been sought to be elucidated.

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