Equilibration Solution Research Articles

Synthesis and Sorption Performance of Novel Sorbents for Selective Solid-Phase Extraction of Eu(III) Ions from Aqueous Solutions

Magnesium-cadmium hydroxyapatite [(Mg-Cd)HAP] and novel multi-wall carbon nanotubes Mg-Cd hydroxyapatite (CNTs/(Mg-Cd)HAP) composites were synthesized by a co-precipitation method. The synthesized sorbents were characterized using BET surface area measurements, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). They have possessed specific surface area of 34.85 and 60.41 m2 g–1 for (Mg-Cd)HAP and CNTs/(Mg-Cd)HAP, respectively. Furthermore, the synthesized sorbents were applied in removal of Eu(III) ions from aqueous solutions. Sorption performance of the synthesized sorbents towards Eu(III) ions was studied under different sorption parameters such as equilibration time, sample mass, solution pH and ionic strength. Sorption of europium reached equilibrium within 6 h at pH 2.5. The maximum sorption capacity (Qmax) was 137.31 and 151.47 mg g–1 for sorption of Eu(III) onto (Mg-Cd)HAP and CNTs/(Mg-Cd) HAP, respectively. Kinetically, the sorption process followed the pseudo-second-order model and both film and pore diffusions participated in ruling the diffusion of Eu(III) ions. Equilibrium sorption data of Eu(III) were analyzed using Freundlich and Langmuir isotherm models. The thermodynamic parameters (ΔG°, ΔH°, and ΔS°) for sorption of Eu(III) onto the synthesized sorbents were determined and the reaction is found to be feasible, spontaneous and endothermic process.

Порівняльний аналіз різних еквілібраційних розчинів при кріоконсервуванні ооцит-кумулюсних комплексів свинок та подальший розвиток ембріонів in vitro

Порівняльний аналіз різних еквілібраційних розчинів при кріоконсервуванні ооцит-кумулюсних комплексів свинок та подальший розвиток ембріонів in vitro

Vitrification of blastocysts at various degrees of blastocoele expansion using different exposure times to the equilibration solution

Vitrification of blastocysts at various degrees of blastocoele expansion using different exposure times to the equilibration solution

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

Impact of cryopreservation method on dromedary camel ovary structure, viability, and development of antral follicular oocytes

The objectives of this study were, a) to compare two different vitrification techniques, the solid surface vitrification (SSV) and direct vitrification (DV) method, b) to evaluate the effect of four cryoprotectant agents and their toxicity on the morphological appearance and ultrastructural of camel ovarian cortex, c) to examine the development of oocytes recovered from the vitrified ovarian cortex. Fragments of ovarian cortex were exposed to equilibration solution consisting of TCM- 199 with 10% fetal camel serum (FCS); 0.10M sucrose and including one of the following cryoprotectants; 20% glycerol (GLY); 3.5M ethylene glycol (EG); 3.5M propanediol (PROH) or 3M dimethylsulphoxide (DMSO). After vitrification of ovarian fragments, they were warmed and evaluated by histological and transmission electron microscope. The oocytes isolated from vitrified ovarian cortex were cultured in TCM-199 at 38.5°C under 5% CO2 for 44h. Maturation was indicated through cumulus expansion and calculated by oocytes reaching first telophase and second metaphase (TI+MII). The percentage of morphologically normal and viable follicles of SSV was significantly higher P<0.05 than DV group (52.9 vs. 38.1, respectively). In conclusion, viability, histological and ultrastructural observations revealed that SSV method and ethylene glycol-based freezing solution were able to remain morphology better follicle and oocyte. Additionally, most organelles of oocytes are able to recover their normal morphology in camel ovarian cortex following cryopreservation and thawing processes, and oocytes isolated from vitrified ovarian cortex can exhibit maturation and reaches to (TI+MII).

Vitrification of bovine ovarian tissue: effect of perforated antral follicles on the structural preservation of follicles

Despite all scientific studies, researches are continuing to increase outcomes of freezing ovarian tissue. The objective was to evaluate whether follicular perforation could improve the survival of antral follicles after vitrification. The present study was conducted on 20 bovine ovaries collected at slaughterhouse and a total of 344 antral follicles (less than 4 mm in diameter). For each ovary, three slices (approximately 1 × 1 cm) were separated from the cortex of each ovary and randomly were assigned in three groups, control (without freezing, fixed in 10% buffered formalin), non­perforated vitrified and perforated vitrified (after exposure to equilibration solution, antral follicles were immediately perforated) groups. After thawing, the tissue slices were evaluated histopathologically. Our histological evaluation showed that vitrification in group V1 did not significantly influence the degree of follicle damage compared to the follicles in fresh group as 69.8% of vitrified follicles were appeared as normal. Increasing follicular size had a significant effect on the vulnerability of follicles using different methods (p < 0.05). Therefore, perforation of follicles of bovine ovarian cortical tissue did not improve freezability of antral follicles by vitrification.

Effect of different moisture equilibration process on the quality of apple chips dried by instant controlled pressure drop (dic)-assisted hot air drying

Moisture equilibration process (MEP) is an important process before controlled pressure drop (DIC) drying to obtain greater products quality and avoid producing bubbles. The aim of this work was to study the effect of MEP on the quality of apple chips dried by instant controlled pressure drop-assisted hot air drying (DIC-AD). After predrying by AD, four types of moisture equilibration solutions were applied to equilibrate the moisture of apple chips before DIC. The quality of apple chips equilibrated in water for 10 min was superior to those for 30 and 60 min with better color, brittleness, hardness, puffing degree (PD), and microstructure. Sugar solutions showed positive effect on brittleness and hardness, PD, and soluble solids of apple chips. In addition, calcium chloride was beneficial to the color of apple chips, and the product showed good rehydration and PD possibly due to the reaction between pectin and calcium ions. Practical applications Instant controlled pressure drop (DIC) was a new sort of united drying method that can combine with other drying technology, such as hot air drying and freezing drying. In practical production, apple slices are usually dried to low moisture content for long time storage and regained appropriate moisture content before drying. After predrying, moisture equilibration process (MEP) is an important process before DIC drying which made water homogenous distributed in the sample to obtain greater products quality and avoid producing bubbles during DIC drying. In this study, MEP with different solutions including water, fructose syrup, maltitol, and calcium chloride were performed before DIC drying, and the changes of physiochemical properties of dried products were studied. Based on the analysis of experimental results, predried apple slice equilibrated in water for 10 min, 10% fructose syrup and 30% maltitol solution might produce apple chips with better quality.

First successful vitrification of salmonid ovarian tissue

Due to a lack of cryopreservation protocols for fish eggs and embryos, alternative techniques which will enable storage of female genetic resources are crucial for future development of reproduction management in conservation biology and aquaculture. Experiments were conducted to develop an optimal vitrification protocol for cryopreservation of brown trout Salmo trutta juvenile ovarian tissue. Needle immersed vitrification (NIV) method was used where ovaries were pinned on an acupuncture needle, passaged through equilibration and vitrification solutions containing different combinations and concentrations of methanol (MeOH), propylene glycol (PG) and dimethyl sulfoxide (Me2SO) and subsequently plunged into liquid nitrogen. Vitrification solutions containing equal cryoprotectant concentrations (3M Me2SO and 3M PG) yielded the highest oogonia survival rates (up to 40%) and qualitatively and quantitatively unaltered perinucleolar follicles. The method developed for brown trout could be applied to the conservation of female genetic resources of other salmonid species, including endangered and endemic species or populations.

PEDOT(PSS) as Solid Contact for Ion-Selective Electrodes: The Influence of the PEDOT(PSS) Film Thickness on the Equilibration Times.

To understand the rate determining processes during the equilibration of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate-based (PEDOT(PSS)-based) solid contact (SC) ion-selective electrodes (ISEs), the surfaces of Pt, Au, and GC electrodes were coated with 0.1, 1.0, 2.0, and 4.0 μm thick galvanostatically deposited PEDOT(PSS) films. Next, potential vs time transients were recorded with these electrodes, with and without an additional potassium ion-selective membrane (ISM) coating, following their first contact with 0.1 M KCl solutions. The transients were significantly different when the multilayered sensor structures were assembled on Au or GC compared to Pt. The differences in the rate of equilibration were interpreted as a consequence of differences in the hydrophilicity of PEDOT(PSS) in contact with the substrate electrode surfaces based on X-ray photoelectron spectroscopy (XPS) and synchrotron radiation-XPS (SR-XPS) analysis of 10-100 nm thick PEDOT(PSS) films. The influence of the layer thickness of the electrochemically deposited PEDOT(PSS)-films on the hydrophilicity of these films has been documented by contact angle measurements over PEDOT(PSS)-coated Au, GC, and Pt electrode surfaces. This study demonstrates that it is possible to minimize the equilibration (conditioning) time of SC ISEs with aqueous solutions before usage by optimizing the thickness of the SC layer with a controlled ISM thickness. PEDOT(PSS)-coated Au and GC electrodes exhibit a significant negative potential drift during their equilibration in an aqueous solution. By coating the PEDOT(PSS) surface with an ISM, the negative potential drift is compensated by a positive potential drift related to the hydration of the ISM and activity changes at the PEDOT(PSS)|ISM interface. The potential drifts related to activity changes in the ISM have been determined by a novel adaptation of the "sandwich membrane" method.

Effect of Trace Elements on Carbonate Thermodynamic Constants

This study is a preliminary work on the description and the modeling of mechanisms potentially causing discrepancies between experimental data and thermodynamic calculation performed on carbonate equilibrium at high temperature. A better understanding of dolomite dissolution/precipitation processes that occur during its equilibration is important for investigations of high-level radioactive waste disposal or Carbon and Capture Storage (CCS). The first results demonstrated the fast dolomite equilibration in solution (within 12 days) and the good agreement between experimental and thermodynamic calculations at 25°C for dolomite and calcite-dolomite systems whereas discrepancies are observed at higher temperature (80°C). As shown from cathodoluminescence photomicrographs, one of the expected mechanisms causing those differences is the incorporation of trace elements such as Mg, Mn or Ba found in natural systems.

Effect of vitrification on meiotic maturation, mitochondrial distribution and glutathione synthesis in immature silver fox cumulus oocyte complexes

The present study was designed to investigate the effects of vitrifying oocytes obtained from silver foxes on nuclear maturation, mitochondrial distribution and glutathione (GSH) synthesis after in vitro culture for 72 h. Immature oocytes were randomly divided into three groups: (1) fresh GV (germinal vesicle) oocytes (Control group), (2) exposure to the equilibration and vitrification solution but without being plunged into liquid nitrogen (exposed group), and (3) vitrification by the cryoloop method (vitrified-warmed group). The number of survival oocytes was not decreased by either being exposed to the cryoprotectant or being vitrified-warmed compared with the control group (P > 0.05). After IVM, the percentage of resumption of meiosis for vitrified-warmed oocytes (41.9%) was significantly lower than in the control (81.2%) and exposed (79.1%) groups (P < 0.05). However, the proportion of oocytes reaching the metaphase II (MII) stage was similar among the different groups (11.4%, 9.3% and 5.2%, respectively, P > 0.05). The translocation of active mitochondria during fox oocyte maturation was revealed using MitoTracker Red staining and confocal laser microscopy. For fresh oocytes at the GV stage, active mitochondria were distributed around the entire cortex with small granulations and various-sized cavities (no MitoTracker signals). After IVM, the mitochondria formed large granulations and clumps throughout the cytoplasm. Vitrification significantly decreased the proportion of MII oocytes with normal mitochondrial distribution compared with the control and exposed groups (35.4%, 71.9% and 59.2%, respectively, P < 0.05). Similarly, the GSH content was significantly lower in vitrified-warmed oocytes compared with the control and exposed oocytes after IVM (3.4, 5.7 and 4.7 pM/oocyte, respectively, P < 0.05). However, no significant difference was observed between the cryoprotectant exposed and control groups with regard to the normal mitochondrial distribution or GSH content (P > 0.05). These results indicate that vitrification of fox immature oocytes using a cryoloop allows them to resume meiosis and develop to the MII stage. The damage to mitochondria and the GSH synthesis deficiency may be associated with the reduced developmental competence of cryopreserved oocytes.

48 EFFECT OF DIMETHYL SULFOXIDE- OR GLYCEROL-BASED VITRIFICATION PROTOCOLS ON THE DNA METHYLATION OF BOVINE CUMULUS-OOCYTE COMPLEXES

Although vitrification is becoming increasingly common for oocyte preservation, there has been recent evidence that some cryoprotectants may alter DNA methylation and so result in decreased oocyte developmental competence and abnormal embryonic development post-warming. The objective of this study was to determine the effect of dimethyl sulfoxide (DMSO)- or glycerol-based vitrification protocols on DNA methylation in bovine cumulus-oocyte complexes (COC). The vitrification protocols evaluated included a combination of ethylene glycol (EG) with either DMSO or glycerol (GLY). Cumulus-oocyte complexes were first exposed to equilibration solution (ES) consisting of 7.5% DMSO or GLY and 7.5% EG for 9 min at room temperature (RT) before being transferred to vitrification solution (VS) containing 15% DMSO or GLY, 15% EG, and 0.5 M sucrose. While in VS, 3 to 4 COC were loaded into an open system vitrification device (Cryolock®, BioTech Inc.) and plunged into LN within 1 min. For warming, COC were exposed to dilution solution 1 consisting of 0.5 M sucrose (37°C) for 2.5 min and to dilution solution 2 consisting of 0.25 M sucrose for 2.5 min (RT). Base media for all solution was PBS supplemented with 20% FBS. Cumulus-oocyte complexes (n = 137) were collected from crossbred cows by ovum pick-up on 3 separate days (repetitions), with half of the oocytes each day being matured to metaphase II (MII) stage before vitrification and the other half vitrified as germinal vesicle (GV) COC. The resulting treatments were DMSO GV v. Glycerol GV and DMSO MII v. Glycerol MII. Fresh COC (GV and MII stage, respectively) were utilised as controls. DNA methylation analysis of oocytes was performed through fluorescent staining using a primary antibody anti-5 mC (1:1000) and secondary antibody Alexa Fluor 488 (1:500). Fluorescence intensity of samples was measured using ImageJ software (NIH, Bethesda, MD, USA). Data were analysed through ANOVA with post hoc Tukey’s test. No differences between the vitrification groups or controls were found when analysing DNA methylation of GV stage COC (P = 0.1825). Metaphase II stage COC showed statistical difference between groups (P &lt; 0.001) where glycerol-treated MII oocytes exhibited higher DNA methylation compared with DMSO MII and fresh MII COC. Results of the experiment showed that the glycerol-based vitrification protocol causes alteration of DNA methylation of bovine metaphase II cumulus-oocyte complexes, and as such may not be an optimal choice in mature oocyte vitrification protocols.

40 THE EFFECT OF EXPOSURE TIME ON TOXICITY OF VITRIFICATION SOLUTION ON PORCINE CUMULUS–OOCYTE COMPLEXES BEFORE IN VITRO MATURATION

Although offspring have been produced from porcine cumulus-oocyte complexes (COC) vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731), embryo development rates have remained low. Numerous vitrification protocols are reported with a wide variation in the applied exposure time to the vitrification solution. Because cryoprotectants in the vitrification solution can be detrimental to the oocytes and their subsequent development, it is important to verify the effect of their exposure time to the COC. In this study, we compared the development of a control group with 3 toxicity control (TC) groups in which COC were exposed to the vitrification solution for 30 s, 1 min, or 1.5 min (TC1, TC2, and TC3, respectively) at 38.5°C. Before exposure, the COC were rinsed and equilibrated in 7 µg mL−1 cytochalasin B. The equilibration solution consisted of 2% (vol/vol) ethylene glycol + 2% (vol/vol) propylene glycol and the vitrification solution contained 17.5% (vol/vol) ethylene glycol + 17.5% (vol/vol) propylene glycol, 50 mg mL−1 polyvinylpyrrolidone and 0.3 M sucrose. The COC were not exposed to liquid nitrogen. After washing in a warming solution of 0.4 M sucrose at 42°C, COC were washed in a sucrose gradient from 0.2 to 0.0 M. Subsequently, the COC were subjected to in vitro maturation in porcine oocyte medium. During the first 20 h of in vitro maturation, the porcine oocyte medium was supplemented with 10 IU mL−1 eCG, 10 IU mL−1 hCG, 1 mM dibutyryl cAMP, and 10 ng mL−1 epidermal growth factor. Then, the medium was replaced with dibutyryl cAMP-free porcine oocyte medium for an additional 28 h. After in vitro maturation, oocytes were parthenogenetically activated (Day 0) and cultured for 7 days in porcine zygote medium. Survival, nuclear maturation, cleavage, and blastocysts rates (Days 6 and 7) were assessed. All parameters were statistically analysed by binary logistic regression. Only the survival rate of TC3 was significantly lower than that of the control group (89.2 v. 95.6%). Exposure to cryoprotectants significantly decreased maturation rates in TC1, TC2, and TC3 compared with the control (72.6%, 75.2%, 76.3% v. 86.1%). Cleavage rates were significantly lower in TC2 and TC3 than that in the control (82.8% and 81.7% v. 92.9%). Concerning blastocyst rates on Day 6 and Day 7 of in vitro culture, only TC1 could reach the same level as the control, expressed on the total number of activated oocytes (54.6% v. 67.7%, and 64.0% v. 72.9%, respectively) as well as expressed on the cleaved oocytes (61.4% v. 72.4% and 72.0% v. 78.0%, respectively). Consequently, despite the reduced maturation rate, TC1 provides the same quantity of blastocysts from matured oocytes as the control. In conclusion, exposure to the vitrification solution for longer than 30 s has toxic effects on COC and therefore is not recommended for vitrification. R. Appeltant is an International Research Fellow of the JSPS Japan (P15402).

Novel substituted Hydroxyapatite nanoparticles as a solid phase for removal of Co(II) and Eu(III) ions from aqueous solutions

Ionic substitution is a tool to improve the sorptive properties of hydroxyapatite (HAP). Based on this concept, strontium and magnesium/cadmium substituted HAP nanoparticles were synthesized, by a co-precipitation method, and applied in removal of Co(II) and Eu(III) ions from aqueous solutions. The influence of different sorption parameters such as equilibration time and solution pH on sorbed amount of Co(II) and Eu(III) was studied and discussed. The sorption process followed the pseudo-second-order kinetic with necessary time of 24h to reach equilibrium. The maximum sorption capacity of Co(II) was 33.31mgg−1 for Sr-HAP at pH 4.5 and 28.93mgg−1 for (Mg-Cd)HAP at pH 1.1, while it was 115.74 and 137.31mgg−1 for sorption of Eu(III) onto Sr-HAP and (Mg-Cd)HAP at pH 2.5, respectively. Desorption of Co(II) and Eu(III) from loaded samples was studied using various eluents and maximum recovery was obtained using FeCl3 and HCl solutions.

Presence of sucrose in the vitrification solution and exposure for longer periods of time improve post-warming follicle integrity in cat ovarian tissues.

Ovarian tissue cryopreservation followed by tissue culture is a promising approach to preserving the fertility of biomedical models and endangered species. The objective of this study was to investigate the impact of exposure time to vitrification solution and presence of sucrose using different exposure temperatures and base media on intra-ovarian follicle integrity. Peripubertal ovarian cortical pieces were obtained by isolating the cortex and dissecting it into 1×1×0.2mm3 pieces. The cortical pieces were then exposed to equilibration solution and then vitrification solutions (VS) in one of the conditions mentioned above, plunged directly into liquid nitrogen and stored for ≥24hr in liquid nitrogen. After thawing, the cortical pieces were cultured in vitro for 0, 1 or 7days to determine the follicle integrity (through histological assessment) and the ability of the tissue to recover from cryoinjury. Fresh controls maintained a constant level of normal morphology (>60% of the total follicles) throughout the culture period. Cortical pieces exposed to VS with sucrose for 10min had the highest percentage of normal follicles (approximately 20% after 7days of culture) throughout the culture period. Other conditions using different base medium, lower exposure temperatures or different thawing methods did not improve the follicle integrity. This protocol provides a solid foundation on which to optimize ovarian tissue cryopreservation in the domestic cat and to investigate the molecular effects of vitrification.

Successful vitrification of human embryos using equilibration solution

Successful vitrification of human embryos using equilibration solution

Automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry for the determination of ten antipsychotics in human plasma.

An automatic on-line solid-phase extraction with ultra-high performance liquid chromatography and tandem mass spectrometry method was developed for the simultaneous determination of ten antipsychotics in human plasma. The plasma sample after filtration was injected directly into the system without any pretreatment. A Shim-pack MAYI-C8 (G) column was used as a solid-phase extraction column, and all the analytes were separated on a Shim-pack XR-ODS III column with a mobile phase consisting of 0.1% v/v formic acid in water with 5mM ammonium acetate and acetonitrile. The method features were systematically investigated, including extraction conditions, desorption conditions, the equilibration solution, the valve switching time, and the dilution for column-head stacking. Under the optimized conditions, the whole analysis procedure took only 10 min. The limits of quantitation were in the range of 0.00321-2.75μg/L and the recoveries ranged from 75.9 to 122%. Compared with the off-line ultra-high performance liquid chromatography and the reported methods, this validated on-line method showed significant advantages such as minimal pretreatment, shortest analysis time, and highest sensitivity. The results indicated that this automatic on-line method was rapid, sensitive, and reliable for the determination of antipsychotics in plasma and could be extended to other target analytes in biological samples.

Shortened equilibration time can compromise clinical outcomes in human embryo vitrification

Vitrification is an important way to cryopreserve human embryos and the recommended time of embryo exposure to the vitrification solution is 1 min. However, practically speaking, the duration of embryos exposure to equilibration solution can vary from 5 to 15 min. The purpose of this study was to investigate the effect of different equilibration times on the outcomes of frozen-thawed embryo transfer cycles. The data were collected from our medical records from January 2012 to June 2013 and a total of 517 cycles were included. These cycles were divided into four groups according to the equilibration time: (i) 5–6 min; (ii) 7–8 min; (iii) 9–10 min and (iv) 11–12 min. The results show that there were no differences in terms of survival rate and fully intact embryo rate among the four groups. However, lower clinical pregnancy, embryo implantation and live birth rates were observed in the 5–6 min exposure group (54.6%, 31.9% and 48.2%, respectively) compared with the three other groups. The corresponding rates in the 9–10 min group (73.5%, 47.6% and 64.7%) were the highest. This study indicated that different equilibration times influenced the clinical outcomes of human embryo vitrification and vitrification with shortened equilibration time compromised the clinical outcomes. Appropriate prolongation of the equilibrium time would probably improve the clinical outcomes.

Comparison of the cryo-tolerance of vitrified gorgonian oocytes

Coral reefs have been declining considerably in recent years because of changes to the environment and climate. The cryopreservation of coral gametes is an essential alternative method that yields immense success in preserving corals. This study focuses on developing vitrification techniques for Junceella fragilis and Ellisella robusta oocytes, and presents a comparison on the cryotolerance of their vitrified oocytes. The results revealed that these coral oocytes could be preserved for a longer period in equilibration solution 2 and vitrification solution (VS) 2 at 5 °C than at 26 °C. Oocyte viability decreased significantly when VS2 was used for >4 min at 26 °C compared with the control. Cryoprotectant tolerance was higher in E. robusta oocytes than in J. fragilis oocytes. However, E. robusta was determined to be more cryo-tolerant, with differences attributed to their habitats, thus making E. robusta is likely a superior candidate species for further study. The results of this study on the effects of coral cryopreservation provide a foundation for developing protocols further for the cryopreservation of the oocytes of gorgonian corals.

Oocyte maturation and expression pattern of follicular genes during in-vitro culture of vitrified mouse pre-antral follicles

Our aim was to evaluate the oocyte maturation rate and follicular genes expression pattern during in-vitro culture of vitrified mouse pre-antral follicles. Middle sized pre-antral follicles were isolated mechanically from the ovaries of pre-pubertal mice and distributed in vitrification and control groups. In the vitrification group, follicles were washed in equilibration and vitrification solutions and then were immersed in liquid nitrogen after loading on cryotop tips. After warming in descending concentrations of sucrose solutions, fresh and vitrified-warmed follicles were cultured for 13 days. Follicles survival rate and follicular genes expression were assessed during in vitro culture. Finally, at the end of the culture period oocytes maturation rate were compared in both groups. In the vitrification group, follicles survival rate was lower significantly comparing to the control group (P < 0.05), whereas oocytes maturation rate were similar. Although at the beginning of the culture period, expression of some genes such as Gdf9, Bmp15, Tgfβ1 and BmprII were higher in the vitrification group (P < 0.05), during the rest of the culture period expression pattern of all follicular genes were similar in both groups. In conclusion, survival rate of cryotop vitrified pre-antral follicles reduced during culture period while oocytes maturation and follicular genes expression did not show any noticeable alteration.