scholarly journals THE EFFECT OF UREA SUPLEMENTATION IN MATURATION MEDIA OF BOVINE OOCYTE IN VITRO TOWARDS EXPRESSION OF BAX, BCL-2 AND BAX/BCL-2 RATIO

2020 ◽  
Vol 14 (1) ◽  
Author(s):  
Dhesy Kartikasari ◽  
Sri Mulyati ◽  
Suzanita Utama ◽  
Pudji Srianto ◽  
Widjiati Widjiati ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Immunocytochemical Staining ◽  
Control Group ◽  
Bovine Oocyte ◽  
Significant Difference ◽  
In Vitro Oocyte Maturation ◽  
Positive Results

This study aims to evaluate the expression of BAX, BCL-2, and BAX/BCL-2 ratio in maturation media of cow oocytes which supplemented with Urea in vitro because BAX and BCL-2 are the main regulators of apoptosis. A total of 263 oocytes from follicle aspirations originating from ovaries taken from slaughterhouses and were saturated with 3 addition of urea which was divided into three groups. The control group (P0) was control group without the addition of urea, P1 group was added with urea 20 mg/dL, while P2 group was added with urea 40 mg/dL. The results of in vitro oocyte maturation were continued with identification using immunocytochemical staining with the addition of BAX and BCL-2 antibodies. Positive results showed a brownish color on the oocyte and its cumulus. The results of this study indicated that there were significant differences (P0.05) in BAX and BCL-2 expression, although both curves were equally increase. The increase in BCL-2 was more significant than BAX, while the BAX BCL-2 ratio did not show a significant difference (P0.05) in whichthe curve of BAX/BCL-2 ratio was decreased. It can be concluded that the addition of urea does not affect the level of apoptosis.

H1foo is essential for in vitro meiotic maturation of bovine oocytes

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 416-425 ◽  
Author(s):  
Yan Yun ◽  
Peng An ◽  
Jing Ning ◽  
Gui-Ming Zhao ◽  
Wen-Lin Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Linker Histone ◽  
Meiotic Maturation ◽  
Control Group ◽  
Bovine Oocyte ◽  
First Polar Body ◽  
Effective Sirnas ◽  
Bovine Oocytes

SummaryOocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%; P < 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

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.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

176 Effect of epigallocatechin-3-gallate on bovine oocyte in vitro maturation, fertilization, and development

2019 ◽  
Vol 31 (1) ◽  
pp. 212
Author(s):  
Y. Honkawa ◽  
Y. Gen ◽  
S.-H. Hyon ◽  
C. Kubota
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Sperm Concentration ◽  
P Value ◽  
Bovine Oocyte ◽  
Vitro Fertilization ◽  
Significant Difference ◽  
Maturation Rate

Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols, and a strong antioxidant compound. Huang et al. (2018 Asian-australas. J. Anim. Sci.) reported that adding 50μM EGCG can improve the bovine oocyte maturation rate. In this research, we investigated the effect of EGCG supplementation on different periods in bovine IVF. Cumulus-oocyte complex (COC) collected from ovaries of slaughtered cows were cultured in maturation medium (20 to 30 oocytes per 100-µL droplet), which consisted of TCM-199 with Earle’s salts and 25mM HEPES supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1µg mL−1 oestradiol, 0.02mg mL−1 FSH, and antibiotics at 38.5°C in a humidified atmosphere of 5% CO2 in air for 24h (in vitro maturation, IVM). After IVM, COC were fertilized in the fertilization medium (modified Brackett-Oliphant media supplemented with 10 µgmL−1 heparin, 10mM caffeine, and 3mg mL−1 BSA) for 6h using semen of one bull at final sperm concentration of 1×107 mL−1 (IVF). After IVF, COC were denuded and cultured in culture medium [CR1aa supplemented with 10% (vol/vol) FBS and antibiotics] at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90%N2 for 8 days (in vitro culture, IVC). The EGCG was supplemented at 10, 25, 50, and 100M in IVM medium; 25 and 50 µM in IVF medium; and 50 and 100 µM in IVC medium. After 24h in IVM medium, COC were denuded by pipetting, fixed in 3:1 ethanol:acetic acid for 24h and then checked for nuclear and polar body by using aceto-orcein stain. After 18h in IVF, the pronucleus in zygote was fixed in 3:1 ethanol:acetic acid for 24h and checked by aceto-orcein staining. Embryo development was evaluated by counting the total number of embryos that had reached compacted morula by 6 to 8 days after IVF. Significant differences were analysed by chi-squared test and residual analysis. A P-value&lt;0.05 was considered statistically significant. When EGCG was added to IVM, there was no significant difference of oocyte maturation rate between all concentrations (0v. 10v. 25v. 50v. 100 μM: 73.9% v. 56.7% v. 76.7% v. 72.7% v. 63.5%). When EGCG was added to IVF, there was no significant difference of fertilized rate (0v. 25v. 50 μM: 59.4% v. 73.7% v. 64.9%). When EGCG was added to IVC, there was no significant difference in development rate (0v. 50v. 100 μM: 26.2% v. 15.7% v. 22.0%). In this research, EGCG addition did not affect bovine in vitro fertilization.

359 MORPHOLOGIC AND BIOCHEMISTRY ALTERATIONS ON BOVINE OOCYTE MATURATION IN VITRO WITH NITRIC OXIDE AND ITS IMPACT ON EMBRYO DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 336 ◽  
Author(s):  
K. S. Viana ◽  
M. C. C. Bussiere ◽  
C. S. Paes de Carvalho ◽  
B. L. Dias ◽  
M. R. Faes ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Plasma Membrane ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Control Group ◽  
Cortical Granule ◽  
Bovine Oocyte ◽  
Cortical Granules ◽  
Maturation In Vitro

The aim of this study was to evaluate morphologic and biochemistry alterations caused by the addition of sodium nitroprusside (SNP), a NO donor, on bovine oocyte maturation in vitro. Bovine ovaries were collected at a local abattoir. COC were cultured in TCM-199 with 10% FCS, 0.5 μg mL-1 FSH, 5.0 μg mL-1 LH, and antibiotics. Analysis of variance was conducted and the means were compared by t-test at a level of 5%. Experimental design: (1) evaluation of the oocyte plasma membrane viability and integrity using Annexin V/propidium iodide (PI) and Hoechst 33342/PI staining, respectively; (2) microtubule and microfilament organization, and migration of cortical granules by immunofluorescence; (3) oocyte glutathione content and concentration of NO3-/NO2-using the method of Griess (Ricart-Jane D et al. 2002 Nitric Oxide 6, 178-185) and (4) embryo development. In Experiment 1, the addition of 1 mM SNP caused cellular death in the majority of the oocytes [100%, AnnexinV/PI (+) and 80.7% Hoescht/IP (+)] differing from the control group and the 0.01 mM SNP (P < 0.05). In Experiment 2, the microtubule staining was observed in the cytoplasm in both control group and 0.01 mM SNP; however, the group treated with 1 mM of SNP exhibited clear defects in spindle and chromatin arrangements (P < 0.05). No alterations in microfilaments disposition was observed in the control group and 0.01 mM SNP. However, after the addition of 1 mM, the microfilaments arranged into clusters, and not below of the cortex. Oocytes treated with 1 mM SNP (68.2%) showed total cortical granule migration to the periphery of the ooplasm and were similar to the control group (72.2%) (P > 0.05). Nevertheless, in the group treated with 0.01 mM SNP the total cortical granule migration was greater (86.8%, P < 0.05). In Experiment 3, the glutathione content of oocytes cultured in the presence of 1 mM SNP was lower (4.4p mol) when compared to the control group (5.4p mol) and 0.01 mM SNP (5.5 pmol) (P > 0.05). The concentration of NO in the medium were similar to both control group (6.0 ± 3.0 μM) and treated with 0.01 mM SNP (15.8 ± 1.9 μM), however, the treatment with 1 mM SNP increased 10 times (59.9 ± 12.0 μM; P < 0.05) this concentration. In Experiment 4, cleavage rates and embryo development were similar for groups control and 0.01 mM SNP (P > 0.05). Even so, in the group treated with 0.01 mM there was a greater blastocyst cell number when compared to the control group (256.8 ± 52.5 and 196.9 ± 54.0, respectively-P < 0.05). These results indicate that: (1) the addition of 0.01 mM SNP increased the quality of the oocyte maturation, leading to a higher percentage of cortical granules migration and blastocyst cell numbers, in a different pathway from that of glutathione; (2) the addition of 1 mM of SNP caused a cytotoxic effect, leading to cellular death with loss of viability and integrity of plasma membrane, absence of nuclear maturation/organization of cytoskeleton and reduction of the glutathione content, although with no intervention in the migration of cortical granules.

Improvement of the in vitro growth and maturation of isolated mouse preantral follicles in the presence of repaglinide

2019 ◽  
Vol 9 (o3) ◽  
Author(s):  
Sura A. Awadh ◽  
Mehri Azadbakht ◽  
Faris N. A. Alhady
Keyword(s):  
Oocyte Maturation ◽  
Ovarian Follicle ◽  
Control Group ◽  
In Vitro Growth ◽  
Preantral Follicles ◽  
Maturation Rate ◽  
Reproductive Techniques ◽  
In Vitro Oocyte Maturation ◽  
Survival Density

The development of in vitro culture systems that result to preantral follicles growth and increasing of developmental competency of oocytes obtained from follicles has an important role in fertility preservation and assisted reproductive techniques. In this research, we evaluated the effect of repaglinide on in vitro growth and maturation of preantral follicles. Preantral follicles were isolated from 12-14 day-old female NMRI mice ovaries and cultured for 12 days cultured in α-MEM (Control), α-MEM supplemented with 1µM of repaglinide. Follicles examined for development on 1, 3, 6, 9, 12 days of culture. At the end of culture period after HCG administration in vitro oocyte maturation was assessed. Results showed that in vitro follicle growth, survival, density of granulosa cells and steroidogenic activity were higher than the control group (p less than 0.05). In vitro maturation rate in oocytes derived from follicles in the treatment group was higher than control group (p less than 0.05). Therefore the supplementation of the culture medium with repaglinide can improve the ovarian follicle survival, growth and subsequently in vitro oocyte maturation.

scholarly journals Effect of enzymatic pro-oxidant and antioxidant systems on bovine oocyte in vitro maturation

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Sergio Morado ◽  
Stephania Madrid Gaviria ◽  
Gabriel Dalvit ◽  
Pablo Cetica
Keyword(s):  
Xanthine Oxidase ◽  
Oocyte Maturation ◽  
Antioxidant Systems ◽  
Control Group ◽  
Bovine Oocyte ◽  
Cleavage Rate ◽  
Physiological Level ◽  
Chromosome Configuration ◽  
Cell Functions

Abstract The role of reactive oxygen species (ROS) during oocyte in vitro maturation (IVM) is still controversial. Although an increase in ROS production may cause deleterious effects in cells, these reactive species may also act as signaling molecules influencing different cell functions. The aim of this study was to examine the effect of varying endogenous ROS levels during IVM on the process of bovine oocyte maturation. To do so, different enzymatic antioxidant (catalase, or superoxide dismutase + catalase, or diphenyl iodonium) or pro-oxidant systems (xanthine + xanthine oxidase, or xanthine + xanthine oxidase + catalase) were added to the culture medium. ROS levels were determined by 2′,7′-dichlorodihydrofluorescein diacetate stain, nuclear maturation was evaluated by the presence of the metaphase II chromosome configuration at 22h of IVM and cleavage rate was recorded 48hs post- in vitro fertilization. ROS levels were only significantly increased (P<0.05) by the O2 .- generating system (xanthine + xanthine oxidase + catalase), but meiotic maturation rates were significantly lower (P<0.05) in all the evaluated systems compared with the control, except for the diphenyl iodonium group. However, this last group presented a significantly lower (P<0.05) cleavage rate in comparison to the control group. These results indicate that ROS would play an essential role during oocyte maturation, since its increase or decrease beyond a physiological level significantly reduced nuclear or cytoplasmic maturation rates in bovine oocytes.

Regulation of bovine oocyte-specific transcripts during in vitro oocyte maturation and after maternal-embryonic transition analyzed using a transcriptomic approach

2009 ◽  
Vol 76 (8) ◽  
pp. 773-782 ◽  
Author(s):  
Aurore Thelie ◽  
Pascal Papillier ◽  
Christine Perreau ◽  
Svetlana Uzbekova ◽  
Christelle Hennequet-Antier ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Bovine Oocyte ◽  
In Vitro Oocyte Maturation

scholarly journals Toxicity effect of Auxemma oncocalyx fraction and its active principle oncocalyxone A on in vitro culture of caprine secondary follicles and in vitro oocyte maturation

2017 ◽  
Vol 38 (3) ◽  
pp. 1361 ◽  
Author(s):  
Johanna Leiva-Revilla ◽  
Jesús Cadenas ◽  
Luis Alberto Vieira ◽  
Claudio Cabral Campello ◽  
Juliana Jales de Hollanda Celestino ◽  
...  
Keyword(s):  
Growth Rate ◽  
In Vitro Culture ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cellular Proliferation ◽  
Control Group ◽  
Antral Follicles ◽  
Chromatin Configuration ◽  
In Vitro Oocyte Maturation

Crude extract of the heartwood of Auxemma oncocalyx (A. oncocalyx) and its main component i.e., Oncocalyxone A (onco A), have elevated antioxidant and anti-tumoral activity, but studies on the action of these drugs regarding folliculogenesis are lacking. The aim of this study was to evaluate the effect of A. oncocalyx and onco A on the in vitro culture of isolated secondary follicles and on the in vitro maturation of oocytes from caprine antral follicles grown in vivo. Isolated secondary follicles were randomly distributed in six groups; the non-cultured control was immediately fixed upon isolation. The remaining follicles were cultured for 7 days in ?-MEM+ alone (control) or supplemented with DMSO, doxorrubicin, A. oncocalyx or onco A. After culture, follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL) and cellular proliferation (PCNA), as well as gene expression of Bcl2 and Bax. Additionally, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for in vitro maturation: control, cultured only in maturation base medium (TCM 199+); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After in vitro maturation, oocyte chromatin configuration and viability were assessed. After 7 days of culture, there was a reduction (P < 0.05) in the percentage of morphologically intact follicles, antrum formation, growth rate and number of PCNA positive granulosa cells in DXR treatment compared to the other treatments. In the DXR treatment a higher percentage (P < 0.05) of TUNEL positive follicles and higher (P < 0.05) relative BAX:BCL2 mRNA ratio’s were observed. After in vitro maturation of the COCs DXR, A. oncocalyx and onco A treatments had a greater (P < 0.05) percentage of abnormal oocytes and a lower (P < 0.05) percentage of viable oocytes as compared with the control group. However, only DXR and onco A treatments increased (P < 0.05) the percentage of alive oocytes with abnormal chromatin configuration. There were no differences in maturation rates between the control group and DXR, A. oncocalyx and onco A treatments. In conclusion, under our culture conditions, A. oncocalyx and onco A do not exhibit a toxic effect on isolated secondary follicles and on maturation rates of COCs recovered from antral follicles, however, these drugs negatively affected the COCs viability.  Thus, the use of culture biotechnologies as an in vitro secondary follicle culture and in vitro oocyte maturation toxicity testing are appropriated methods to evaluate the possible effects of drugs in folliculogesis.

Comparative Study to Evaluate the Surface Detail Reproduction of Dental Stone after Immersion in Various Different Disinfectant Solutions, Under Stereomicroscope 10 X Magnification –An In-Vitro Study

2017 ◽  
Vol 9 (3) ◽  
Author(s):  
Rathika Rai ◽  
M. A. Easwaran ◽  
K. T. Dhivya
Keyword(s):  
Sodium Hypochlorite ◽  
In Vitro Study ◽  
Control Group ◽  
Immersion Method ◽  
Significant Difference ◽  
Surface Irregularities ◽  
Disinfectant Solution ◽  
Surface Detail ◽  
Group B

Aim: To evaluate the surface detail reproduction of dental stone this is immersed in different disinfectant solution and studied under stereomicroscope. Methodology: Total number of 30 specimens of dental stone (Type III) were made with measurements of 1.5cm diameter and 1cm height .This samples are divided in to 3 groups group A,B,C. were A is immersed in Distilled water which was taken as control group ;B is immersed in 2% Glutaraldehyde and C is immersed in 5%sodium hypochlorite. Each specimen were immersed in the disinfectant solution for 15 minutes and dried under room temperature for 24 hrs. After 24 hrs each specimens are studied under stereomicroscope for surface details. Result: The results showed no significant difference in the surface irregularities and porosities for a group 1 and group 2 except group 3 which showed significant increase in the porosities, surface irregularities and erosions after disinfection with 5% NaHOCl by immersion method. Conclusion: The surface detail reproduction capacity of die stone was adversely affected when 5% Sodium hypochlorite was used as disinfectant solution when compare d to control group and 2% Glutaraldehyde

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