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.

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.

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.

46 SPINDLE CONFIGURATION OF IN VITRO-MATURED BOVINE OOCYTES EXPOSED TO SODIUM CHLORIDE OR SUCROSE PRIOR TO CRYOTOP VITRIFICATION

2015 ◽  
Vol 27 (1) ◽  
pp. 116 ◽  
Author(s):  
N. Arcarons ◽  
R. Morató ◽  
J. F. W. Spícigo ◽  
M. A. M. M. Ferraz ◽  
T. Mogas
Keyword(s):  
Sucrose Solution ◽  
Statistical Significance ◽  
Normal Chromosome ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Control Group ◽  
Chromosome Configuration ◽  
Chromosome Distribution ◽  
Bovine Oocytes

It has been previously described that a simple treatment with medium containing elevated NaCl or sucrose concentrations increases the cryotolerance and developmental competence of in vitro-matured porcine oocytes after vitrification and parthenogenetic activation (Lin et al. 2009 Reprod. Fertil. Dev. 21, 338–344). This work was designed to study whether the exposure to increased concentrations of NaCl or sucrose before vitrification improves cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, in vitro-matured oocytes were exposed to different NaCl and sucrose concentrations (from 375 to 808 mOsm) for 1 h. In Experiment 2, and according to the results obtained in the first experiment, oocytes were exposed to 375 mOsm NaCl or sucrose solution, vitrified, and warmed. Nontreated oocytes were used as controls. In both experiments, oocytes were fixed after treatment and microtubule, and chromosome distribution was analysed by immunocitochemistry. All statistical analyses were conducted with the IBM SPSS 19 for Windows (IBM corp., Chicago, IL). ANOVA was performed to analyse differences in meiotic spindle. Statistical significance was set at P < 0.05. After exposure to 375 mOsm of NaCl or sucrose, similar percentages of oocytes showing normal chromosome distribution were obtained compared to the control group (83.4, 71.8, and 85.0%, respectively). Groups treated with higher concentrations (443 to 808 mOsm) triggered significantly lower proportions of normal spindles. After vitrification/warming, no significant differences were observed between nonvitrified oocytes (71.3%) and those treated with NaCl before vitrification/warming procedure (41.9%) when normal chromosome organisation was analysed. Significantly higher percentages of normal chromosome configuration were observed when oocytes were exposed to sucrose before vitrification (34.2%) compared with control-vitrified oocytes (23.3%). However, pretreatment with NaCl or sucrose before vitrification did not trigger significant differences in terms of percentages of normal microtubule configuration (41.9 and 32.9%, respectively) compared with control-vitrified oocytes (40.2 and 24.4%, respectively), although both treatments differed significantly from control (79.1 and 81.7%, respectively). In conclusion, this study showed that a 375-mOsm NaCl or sucrose pretreatment of bovine oocytes before vitrification did not have a deleterious effect on the organisation of the meiotic spindle of vitrified/warmed bovine oocytes. Further experiments are required to investigate whether in vitro-matured oocytes subjected to this osmotic treatment could improve their development competence after being vitrified/warmed.

Human oocyte vitrification with corona radiata, in autologous follicular fluid supplemented with ethylene glycol, preserves conventional IVF potential: birth of four healthy babies

2014 ◽  
Vol 26 (7) ◽  
pp. 1001 ◽  
Author(s):  
Xian-Hong Tong ◽  
Li-Min Wu ◽  
Ren-Tao Jin ◽  
Hong-Bing Luan ◽  
Yu-Sheng Liu
Keyword(s):  
Ethylene Glycol ◽  
Embryo Transfer ◽  
Follicular Fluid ◽  
Control Group ◽  
Human Oocyte ◽  
Oocyte Vitrification ◽  
Human Oocytes ◽  
Corona Radiata ◽  
Fertilisation Rate ◽  
Significant Difference

The aim of this study was to examine the effects of vitrification with autologous follicular fluid (AFF) supplemented with ethylene glycol (EG) and sucrose on human oocytes with corona radiata. A total of 182 human oocytes with corona radiata from fifteen infertile patients were vitrified using either equilibration solutions (ES) and vitrification solution (VS) consisting of AFF, EG and sucrose (AFF group, n = 67) or commercial ES and VS (control group, n = 115). All oocytes were thawed in the next cycle, with surviving oocytes being inseminated by conventional IVF. The clinical outcome of vitrified–warmed oocytes by both vitrification methods was analysed retrospectively. In the AFF group, six patients received embryo transfer, with three couples taking four healthy babies home. In the control group, nine patients received embryo transfer, with four couples taking five healthy babies home. There was no significant difference in the survival rate (91.0 vs 92.2%), two pronuclei (2PN) fertilisation rate (73.8 vs 73.6%), cleavage rate (100 vs 100%), top-quality embryo rate (62.2 vs 59.2%), clinical pregnancy rate (50.0 vs 44.4%), implantation rate (33.3 vs 25%) or take-home baby rate (50.0 vs 44.4%) between the AFF group and the control group, respectively. These results show that AFF supplemented with EG and sucrose is an efficient, cost-effective cryoprotectant for human oocyte cryopreservation. A corona radiata on vitrified–warmed oocytes retains the oocytes’ fertilisation capability in conventional IVF.

scholarly journals 81 EFFECT OF SUCROSE CONCENTRATION IN WARMING MEDIUM ON THE DEVELOPMENT POTENTIAL OF VITRIFIED BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 190
Author(s):  
W.C. Chang ◽  
J. Xu ◽  
S. Jiang ◽  
X.C. Tian ◽  
X. Yang ◽  
...  
Keyword(s):  
Sucrose Concentration ◽  
Control Group ◽  
Bovine Oocyte ◽  
Cell Stage ◽  
Significant Difference ◽  
Humidified Air ◽  
Bovine Oocytes ◽  
Experimental Group ◽  
One Way Anova

The aim of this experiment was to determine the effect of the sucrose concentration (0 to 0.33 M) in the dilution medium on the viability, fertilizability, and development of vitrified bovine oocytes. Bovine oocyte-cumulus complexes were collected from slaughterhouse ovaries and in vitro-matured as reported previously. After 24-h maturation in TCM199-based medium under 5% CO2 humidified air at 39°C, these were exposed to hyaluronidase and carefully pipetted to remove all except the 3–5 innermost layers of cumulus. Oocytes were put into the pre-equilibration medium for 3 min and then into vitrification solution containing HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol, and dimethylsulphoxide for 25–30 s; they were then vitrified by modified solid surface vitrification (Dinnyes et al. 2000 Biol. Reprod. 63, 513–518).The oocytes were warmed at 39°C by placing them in holding medium with 0, 0.08, 0.17, 0.25, or 0.33 M sucrose. Non-vitrified oocytes were used as controls. Oocytes were inseminated 30 min after warming, and the presumptive zygotes were cultured in CR1-aa medium supplemented with 6 mg/mL BSA at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for eight days. Data were analyzed by one-way ANOVA. As shown in Table 1, there was no significant difference in survival rate (P > 0.05) of the vitrified oocytes that were placed in dilution solution containing 0.17, 0.25, or 0.33 M sucrose and the non-treated controls. On Day 2 (fertilized on Day 0), cleavage to the 8-cell stage was similar for the 0.17, 0.25, and 0.33 M dilution groups, but the rates for all three were significantly lower (P < 0.05) than for the control group. The blastocyst rate on Day 8 was significantly higher for the 0.25 M group than for any other experimental group but still significantly lower than for the control. In conclusion, this study suggests that with this vitrification/warming procedure the optimum concentration of sucrose in the dilution solution is 0.25 M. Table 1. Oocyte survival after vitrification/warming and subsequent embryo development The authors would like to thank Ms Colleen Shaffer for the preparation of bovine oocytes.

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 Effects of Three Types of Massage on Serum Levels of Malondialdehyde, Superoxide Dismutase and Glutathione Peroxidase After One Session of Exhaustive Exercise in Female Futsal Players

2021 ◽  
Vol 10 (4) ◽  
pp. 328-339
Author(s):  
Bahare Heydari ◽  
◽  
Mohsen Ghofrani ◽  
Mohammad Ebrahim Bahram ◽  
◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Glutathione Peroxidase ◽  
Recovery Period ◽  
Serum Levels ◽  
The Body ◽  
Exhaustive Exercise ◽  
Oxidative Stress Marker ◽  
Control Group ◽  
Significant Difference

Objective: The production of reactive oxygen species in exercise causes oxidative stress which disturbs the balance of oxidants and antioxidants, causing destructive effects on cells. The present study aims to investigate the effect of three types of massage (Swedish, Russian, Thai) on serum levels of Malondialdehyde (MDA), Glutathione Peroxidase (GPX) and Superoxide Dismutase (SOD) following one session of exhaustive exercise. Methods: This quasi-experimental study was conducted on 48 female futsal players aged 17-22 years in Zahedan, Iran who were selected using a purposive sampling method, and randomly divided into four groups of Swedish massage (Long strokes with pressing and tapping using hands), Russian massage (Medium to high pressure), Thai massage (Pressure to certain parts of the body) and Control. The exercise program was based on Bruce protocol. Serum levels of MDA, GPX and SOD were measured by before and immediately after exercise and after massage. Data analysis was performed using repeated measures ANOVA, considering a significance level of P≤0.05. Results: In all three types of massage, there was a significant decrease in serum level of MDA (0.22±0.08), and a significant increase in GPX (1.84±0.46) and SOD (10.02±2.86) levels after exhaustive (P=0.001). No significant difference was observed in the control group. Conclusion: It seems that Russian, Thai, and Swedish types of massage can affect the serum levels of the MDA (as an oxidative stress marker) and the antioxidant enzymes of GPX and SOD during the post-exercise recovery period.

scholarly journals Effect of A-PRF Application on Palatal Wound Healing after Free Gingival Graft Harvesting: A Prospective Randomized Study

2020 ◽  
Vol 14 (01) ◽  
pp. 063-069 ◽  
Author(s):  
Filipa Sousa ◽  
Vanessa Machado ◽  
João Botelho ◽  
Luís Proença ◽  
José João Mendes ◽  
...  
Keyword(s):  
Wound Healing ◽  
Healing Process ◽  
Recovery Period ◽  
Gelatin Sponge ◽  
Control Group ◽  
Total Recovery ◽  
Pain Experience ◽  
Free Gingival Graft ◽  
Significant Difference ◽  
Gingival Graft

Abstract Objective The aim of this study was to investigate the healing effect of advanced platelet-rich fibrin (A-PRF) clot membranes in palatal wounds, resulting from free gingival graft (FGG) harvesting, on the reepithelization rate and on the pain experience after surgery. Materials and Methods Twenty-five patients requiring FGG have participated in this prospective cohort study. After FGG harvesting, the test group (n = 14) received A-PRF clot membranes at the palatal wound and the control group (n = 11) received a gelatin sponge. Epithelialization rate of the palatal wound, wound healing area, correspondent percentage of reduction, and postsurgical pain experience were assessed at 2, 7, 14, 30, and 90 days. Results A-PRF group had higher palatal wound reduction than the control group, at 7, 14, and 30 days of follow-up. The highest difference between the groups was attained at 30 days (91.5% for A-PRF vs. 59.0% control group). At 14 days, a significant difference in the proportion of patients showing total epithelization was found: 64.3% for A-PRF versus 9.1% for the control group. At 90 days, both groups showed total recovery. The control group experienced higher pain level and discomfort until the 14th day, being notably higher on the second day. Conclusion The results suggest that A-PRF membranes haste the healing process, and promote greater reduction along the recovery period and less painful postoperative period.

75 ADDITION OF HYALURONAN TO A VITRIFICATION SOLUTION FOR IMMATURE BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE

2013 ◽  
Vol 25 (1) ◽  
pp. 185
Author(s):  
P. Rodriguez Villamil ◽  
F. Ongaratto ◽  
M. Fernandez Taranco ◽  
G. A. Bó
Keyword(s):  
Ethylene Glycol ◽  
Solid Phase ◽  
Animal Health ◽  
Survival Rates ◽  
Control Group ◽  
Cresyl Blue ◽  
Development Rates ◽  
Vitrification Solution ◽  
Bovine Oocytes

An experiment was designed to evaluate the effect of brilliant cresyl blue (BCB) selection of immature oocytes and the addition of sodium hyaluronate (HA) to the vitrification solution on survival rates of bovine oocytes vitrified using solid-phase vitrification. Bovine cumulus–oocyte complexes (COC; n = 716) obtained from slaughterhouse ovaries were used in 6 replicates. Cumulus–oocyte complexes were washed in tissue culture medium 199 (TCM-199) and randomly allocated to 2 groups to be exposed to BCB stain (Sigma Chemical Company, St. Louis, MO, USA) for 90 min as described by Alm et al. (2005 Theriogenology 63, 2194–2205) or (control) maintained in Vigro holding medium (Bioniche Animal Health, Belleville, Canada) for 90 min (n = 220). Cumulus–oocyte complexes in the BCB group were selected based on their response to BCB as BCB+ (colored, n = 248) or BCB– (colorless, n = 248), whereas those in the control group were selected morphologically as described by Rodríguez-González et al. (2002 Theriogenology 57, 1397–1409). Oocytes from both BCB groups and 100 oocytes in the control group were vitrified by solid-phase vitrification as previously described by Rodriguez et al. (2012 Reprod. Fertil. Dev. 24, 132). The remaining 120 oocytes in the control group were not vitrified and were matured, fertilized, and cultured in vitro (in SOFaa in a controlled atmosphere) for 7 days. Vitrified oocytes were exposed to 10% ethylene glycol for 10 min, and 20% ethylene glycol + 0.2-M trehalose for 30 s, and then were subdivided to be exposed to 30% ethylene glycol + 0.5-M trehalose with or without 0.1 mg mL–1 HA (MAP 5, Bioniche Animal Health). Vitrified oocytes were stored in liquid nitrogen for at least one week and then placed directly into a 0.5-M sucrose solution (in TCM 199) at 37°C for 5 min, 0.25 M of sucrose for another 5 min, and finally TCM-199 and matured, fertilized, and cultured. Development rates (i.e. proportion of blastocysts) were examined on Day 7 after fertilization. Proportional data were first transformed by square root and then analyzed by ANOVA to detect the effect of replicate, type of oocyte (BCB+, BCB–, controls), and vitrified with or without HA or not vitrified as main effects, using the software Infostat (UNC, Argentina, 2010). There was a significant effect of oocyte type on blastocyst rate (P < 0.01) following vitrification (BCB+, 6.4 ± 0.4%. v. BCB–, 1.6 ± 0.6%). Control oocytes (not exposed to BCB) resulted in 3.0 ± 2.0% blastocysts following vitrification, which was lower to that obtained with the BCB+ oocytes. Vitrification also influenced development rates (3.0 ± 2.0 v. 32.0 ± 1.3%) for blastocysts produced from vitrified v. nonvitrified oocytes, respectively (P < 0.01). Furthermore, the use of HA in the vitrification solutions did not have a significant effect on development rates (4.7 ± 0.9 v. 3.3 ± 0.9%, for blastocysts obtained from vitrified oocytes with or without HA, respectively). In conclusion, the selection of oocytes by BCB increased the in vitro development rates of vitrified immature oocytes, whereas the use of HA in the vitrification solution did not improve the survival rates of vitrified oocytes.

32 Vitrification of in vitro-matured bovine oocytes in triacetate cellulose hollow fibres

2019 ◽  
Vol 31 (1) ◽  
pp. 142
Author(s):  
E. V. Kornienko ◽  
A. B. Romanova ◽  
M. A. Ikonopistseva ◽  
G. P. Malenko
Keyword(s):  
Ethylene Glycol ◽  
Fertilization Rate ◽  
Control Group ◽  
Hatching Rate ◽  
High Survival ◽  
Hollow Fibres ◽  
Group 2 ◽  
Bovine Oocytes ◽  
Group 1

A prospective method of vitrification in triacetate cellulose hollow fibres (HF) introduced by Matsunari et al. (2012 J. Reprod. Dev. 58, 599-608) allowed significant simplification and standardization of vitrification/warming procedures and was successfully used for group cryopreservation of various pre-implantation mammalian embryos. The goal of the current work was to evaluate the effectiveness of the HF vitrification method for cryopreservation of in vitro-matured bovine oocytes. The base medium for all the vitrification and rewarming solutions was calcium-free TBP-like protein-HEPES supplied with 20% of fetal bovine serum. Groups of 15 morphologically normal in vitro-matured bovine oocytes were equilibrated with 3% (vol/vol) ethylene glycol for 15min, loaded into HF, and transferred into vitrification solution containing 30% ethylene glycol and either 0.5M (Group 1) or 1.0M (Group 2) sucrose. Hollow fibres were incubated for either 60s (Group 1) or 30s (Group 2) and immediately plunged into LN. Rewarming was conducted at 39°C. Oocytes within HF were placed in decreasing concentrations of sucrose solutions to remove cryoprotectants. Then, oocytes were subjected to IVF. Non-vitrified denuded oocytes were used as a control. Survival rates were evaluated at 21h post-rewarming. Part of the presumptive zygotes were fixed and stained with acetolacmoid for fertilization rate. The remaining zygotes were cultured for 10 days. Developmental rates were evaluated at 44h and 7 and 10 days post-IVF. All results are presented as mean percentage±standard deviation. Data were analysed using Mann-Whitney U test. Significance was set at P&lt;0.05. Survival rate was significantly lower in Group 1 (79.0±8.0%) and Group 2 (75.0±5.0%) compared with the control group (97.0±4.0%). Fertilization rate in Group 1 differed significantly from the control (80.5±18.3% v. 95.5±9.1%). Cleavage rates in Groups 1 and 2 did not differ significantly from the control (42.5±15.7% v. 60.7±11.1% v. 63.0±15.8%, respectively). Blastocyst yields at 7 days post-IVF were 0.9±2.3 (1/116) and 9.6±5.4% (6/65) in Groups 1 and 2, respectively. The former was significantly lower than in the control group (17.0±10.3%, 23/154). It should be noted that hatching in the control group started at 8 days post-IVF and was delayed in Groups 1 and 2 for at least 24h. Day 10 blastocyst yields were 3.0±3.3 (P&lt;0.05), 20.9±13.8, and 30.4±9.6% in Groups 1 and 2 and the control group, respectively. All obtained Day 10 blastocysts (3/116) in Group 1 hatched. Hatching rate in Group 2 was significantly lower than in the control group. Both Groups 1 and 2 showed relatively high survival and fertilization rates, but embryo development rates in both groups had a tendency to be lower than in the control. However, the obtained results indicate that the modifications of the protocol may increase the effectiveness of HF vitrification. The HF vitrification method remains a prospective option for simultaneous cryopreservation of a group of bovine oocytes.

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