scholarly journals RESPON PENGGUNAAN BERBAGAI BAHAN AKTIVATOR PADA AKTIVASI PARTENOGENESIS OOSIT KAMBING HASIL IVM

SAINSTIS ◽  
2012 ◽  
Author(s):  
Kholifah Holil, Eva Ari Wahyuni, Hari Soepriandono Gatot Ciptadi
Keyword(s):  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Reproductive Technologies ◽  
Cleavage Rate ◽  
Parthenogenetic Activation ◽  
Sperm Extract ◽  
Fetal Bovine ◽  
Humidified Air

Parthenogenetic activation is one method that can be used to determine the quality of IVM oocytes results before further use to other reproductive technologies (IVF and transfer core). In parthenogenetic activation can be used various activators such as ethanol, Ca Ionophore, and Crude Sperm Extract (CSE). Therefore, the aim of this experiment is to know the response use a variety of materials activator of parthenogenetic activation of goat oocytes IVM.<br />The sample used in this experiment was oocytes aspirated from goat ovarian follicles taken from RPH Sukun of Malang. Oocytes were matured for 24 hr in TCM-199 supplemented with fetal bovine serum (FBS), follicle-stimulating hormone (FSH) and lutheinizing hormone (LH) at a temperature of 38,5oC and 5% CO2 in humidified air. After another 30 hours of in vitro maturation, they were then activated by various treatments. The treatment of experiment are treatment 1, activation using ethanol 7% for 7 minute, treament 2, activation using Ca Ionophore 20 µM for 7 minute. Treatment 3, activation using CSE 2,5 µg/ml for 2 hr.<br />Based on the result of research, it is showed that activation by using 7% ethanol for 7 minutes is able to produce cleavage rate of 70.40%. Activation by Ca Ionophore 20 μM for 7 minutes is able to produce cleavage rate of 52.75%. While the use of CSE activation with 2.5 ug / ml for 2 hours produces cleavage rate of 36.33%. Thus it can be concluded that the goat oocyte IVM able to respond to a variety of materials activators on parthenogenetic activation performed. The highest response given by the successive results goat IVM oocytes activated using 7% ethanol for 7 minutes, 20 μM Ca Ionophore for 7 minutes, and the CSE 2.5 microg / ml for 2 hours.<br /><br />Keywords: Parthenogenetic activation, goat oocytes IVM, etanol, calsium ionophore, Crude Sperm Extract (CSE). <br /><br />

43 MASS VITRIFICATION OF GERMINAL-VESICLE STAGE EQUINE OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 151
Author(s):  
H. S. Canesin ◽  
I. Ortiz ◽  
J. G. Brom-de-Luna ◽  
Y. H. Choi ◽  
K. Hinrichs
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Control Group ◽  
Cleavage Rate ◽  
Fetal Bovine ◽  
Vitrification Solution

Oocyte cryopreservation has the potential to preserve female genetics. In addition, equine oocytes are not readily available in some areas, and vitrification could be used to accumulate oocytes at remote locations to provide material for research. To preserve large numbers of oocytes, a method for rapid vitrification of multiple oocytes is needed. First, we determined whether immature equine oocytes could be held overnight before vitrification, and we tested the use of a mesh+capillary-action media-removal vitrification platform. Oocytes were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to either immediate vitrification or overnight holding (24 to 27 h in 40% M199-Earle’s salts, 40% M199-Hanks’ salts, 20% fetal bovine serum, and 0.3 mM pyruvate) then vitrification. Oocytes were vitrified using different times (1 or 4 min) in vitrification solution and first warming solution: 1v1w, 1v4w, 4v1w, and 4v4w. The base solution was MH (80% M199-Hanks’ salts and 20% fetal bovine serum). Cryoprotectant concentration (vol/vol) was increased in 3 steps until reaching 7.5% dimethyl sulfoxide and 7.5% ethylene glycol. The oocytes were then held in vitrification solution (MH with 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose) for either 1 or 4 min, according to treatment, and 3 to 10 oocytes were transferred to a 75-μm sterile stainless steel mesh. The mesh was placed on sterile paper to absorb excess medium, then plunged in LN. The oocytes were warmed in MH solution with 1.25 M sucrose for either 1 or 4 min, then placed in 0.62 M and 0.31 M sucrose solutions for 5 min each and undetermined time in MH. After warming, oocytes were cultured for maturation (in vitro maturation) in M199-Earle’s salts, 5 mU mL–1 FSH, and 10% fetal bovine serum. After 30 to 36 h, the oocytes were denuded and stained with Hoechst 33258. Data were analysed by Fisher’s exact test. There were no significant differences (P > 0.05) in rates of meiotic resumption among timing treatments (35, 24, 26, and 39% for 1v1w, 1v4w, 4v1w, and 4v4w, respectively), nor between immediately vitrified (17/55, 31%) and overnight held-vitrified groups (18/56, 32%). In the second experiment, all oocytes were held overnight. They were vitrified and warmed using only the 1v1w and 4v4w schedules, then subjected to in vitro maturation, intracytoplasmic sperm injection, and embryo culture. The MII rate of the control group (27/37, 73%) was higher (P < 0.05) than that for 1v1w (12/33, 36%) or 4v4w treatments (10/35, 29%). The cleavage rate for control (25/27, 93%) was higher than that for 1v1w (5/9, 56%) but not than that for 4v4w (6/9, 67%). Blastocyst rates were 19% (5/27), 11% (1/9), and 0% (0/9) for control, 1v1w, and 4v4w, respectively (P > 0.05). These results indicate that blastocysts may be produced from equine immature oocytes vitrified en masse; however, both the maturation and blastocyst production rates were relatively low. Additional studies are required to improve the efficiency of this technique. This work was supported by the Clinical Equine ICSI Program, Texas A&M University.

181 RESVERATROL DURING IN VITRO MATURATION IMPROVES THE QUALITY OF BOVINE OOCYTE AND ENHANCES EMBRYONIC DEVELOPMENT IN VITRO

2017 ◽  
Vol 29 (1) ◽  
pp. 199 ◽  
Author(s):  
V. Torres ◽  
L. Muñoz ◽  
R. Urrego ◽  
J. J. Echeverry ◽  
A. Lopez
Keyword(s):  
Fatty Acid ◽  
Embryonic Development ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Fetal Bovine ◽  
Development In Vitro ◽  
And Control

It is known that reactive oxygen species (ROS) are accumulated within the oocyte during in vitro maturation (IVM) and have been related to poor quality and decreased embryo development in vitro. The use of antioxidants in culture media is an alternative to overcome oxidative stress damage in the oocyte. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a phenol produced naturally by several plants and has shown protection against oxidative damage in numerous cell types. Two different experiments were performed to evaluate the effect of resveratrol on the quality of bovine oocytes matured in vitro assessed by levels of ROS and intracellular glutathione (GSH) as well as in vitro embryo development rates. Experiment 1 used different concentrations of resveratrol [0 (Control), 1 (R1), 10 (R10), 20 (R20), and 40 (R40) μM] were used to supplement IVM media. Ovaries were collected from Bos indicus cows at a local abattoir and cumulus-oocyte complexes were matured in vitro for 24 h in TCM199 with 6 mg mL−1 of fatty acid-free BSA, 5% fetal bovine serum, 0.2 mM Na-pyruvate, 50 μg mL−1 of gentamicin, 0.5 μg mL−1 of FSH, and 0.5 µg mL−1 of LH at 38°C in 5% CO2 and 90% humidity. The ROS were evaluated by 2′,7′-dichlorodihydrofluorescein diacetate staining (n = 301) and intracellular GSH levels were determined by Cell Tracker Blue fluorescent stain (n = 310). Denuded oocytes were observed under an epifluorescence microscope. Fluorescence intensities of oocytes were analysed by ImageJ software (Version 1.49v, National Institutes of Health, Bethesda, MD, USA) and normalized to control oocytes. Experiment 2 used cumulus-oocyte complexes (n = 674) collected and matured in vitro under the same conditions described for Exp. 1. In vitro fertilization was performed for 18 h at 38°C in 5% CO2 in Tyrode’s medium with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, and 6 mg mL−1 of fatty acid-free BSA. Additionally 10 µg mL−1 of heparin and 20 μM d-penicillamine, 10 μM hypotaurine, and 1 μM epinephrine were added. The presumptive zygotes were cultured in vitro in SOFaa medium with 5% fetal bovine serum, at 38°C, in 5% CO2, 5% O2, and 90% humidity until Day 7, when embryonic development was assessed. Data were analysed by ANOVA followed by Fisher´s multiple range test using Statgraphics Centurion XVI (Version 16.2.04, Statpoint Technologies Inc., Warrentown, VA). Data are presented as percentage mean ± standard error of the mean (P < 0.05). All concentrations of resveratrol in treated oocytes showed reduced intracellular levels of ROS compared to control (R1: 0.66 ± 0.04, R10: 0.55 ± 0.04, R20: 0.62 ± 0.04, R40: 0.64 ± 0.04, and Control: 1 ± 0.04 pixel/oocyte; P < 0.01). Intracellular levels of GSH were significantly higher for R1 (1.4 ± 0.06; P < 0.01) and R10 (1.3 ± 0.06; P < 0.01) compared with the control. On the other hand, R10 showed a significantly higher blastocyst rate (51% ± 3) compared with R1 (39% ± 4), R20 (39% ± 3), R40 (33% ± 3), and control (38% ± 4). Treatments R1, R20, and R40 showed no significant differences compared to control. These results indicate that resveratrol at 10 μM during IVM improves maturation conditions by decreasing ROS level, increasing intracellular GSH, and improving embryonic developmental competence.

160 In vitro maturation of pre-pubertal goat oocytes and their development after chemical activation

2019 ◽  
Vol 31 (1) ◽  
pp. 205
Author(s):  
N. A. Wani ◽  
S.-B. Hong
Keyword(s):  
In Vitro Maturation ◽  
Culture Media ◽  
Chemical Activation ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Epidermal Growth ◽  
Humidified Air ◽  
Activation Status

Experiments were conducted to study in vitro maturation of pre-pubertal goat oocytes and their developmental potential after chemical activation. Cumulus-oocyte complexes (n=1170) collected from the ovaries of pre-pubertal goats slaughtered at a local abattoir were matured in TCM-199 supplemented with 0.15mg mL−1 l-glutamine, 0.25mM sodium pyruvate, 0.1mM l-cysteine, 20ng mL−1 epidermal growth factor, 10mg mL−1 FSH, 10mg mL−1 LH, 1μg mL−1 oestradiol and 10% FCS for 24h at 39°C under 5% CO2 in humidified air. In Experiment 1, matured oocytes were activated (r=6) with either 5mM ionomycin (n=85) or 7% ethanol (n=91) followed by culture in 6-DMAP for 4h. All the activated oocytes were then cultured in KSOM supplemented with 3mg mL−1 BSA and were fixed and stained with Hoechst 33342 after 18h of culture to evaluate their activation status. In Experiment 2, oocytes activated with 5mM ionomycin and 6-DMAP were cultured for 7 days (r=6) in 1 of the 4 different culture media [Charles Rosenkrans medium (CR-1), modified TCM-199, KSOM and SOF] to study their developmental potential. All media were supplemented with 3.0mg mL−1 BSA for the first 3 days and 10% FCS for the subsequent 4 days. Of these pre-pubertal oocytes, 59% reached metaphase II stage, and 83% of these oocytes were classified as activated in the group using ionomycin in comparison with 69% in the group using ethanol as an activating agent (P&lt;0.05). No difference was observed in the cleavage rate of activated oocytes cultured in any of the 4 culture media (65.7v. 55.0v. 61.0v. 56.2%, respectively). However, the development to blastocyst stage was observed in only KSOM (16%) and SOF (5%) media. In conclusion, the present study demonstrates that pre-pubertal goat oocytes can mature in vitro and can be activated with 5mM ionomycin, and KSOM, and to a lesser extent SOF, supports development to the blastocyst stage. We plan to use these oocytes as a cytoplast source for interspecies somatic cell NT; however, before that, more studies are needed to evaluate their requirements in culture media to enhance their development to the blastocyst stage.

75 Improvement of Developmental Competence of Bovine In Vitro-Produced Embryos by Using Charcoal:Dextran-Stripped Fetal Bovine Serum on In Vitro Culture Media

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
A. Mesalam ◽  
R. Kong ◽  
B.-H. Choi ◽  
K.-L. Lee ◽  
B.-Y. Park ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Mrna Levels ◽  
Fetal Bovine

Serum has widely been used as a main supplement to embryo in vitro culture media as it contains embryotrophic factors. Charcoal:dextran treatment of fetal bovine serum (FBS) removes lipophilic chemicals and certain steroid hormones and growth factors. The objective of this study was to investigate the effects of charcoal:dextran-stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium (SOF-BE1 medium supplemented with 10% of serum) on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The experiment was conducted in 6 replicates (350 oocytes per group). The differences in embryo development, integrated optical intensity, and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P < 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% v. 36.85 ± 0.89%, respectively). The total number of cells per Day 8 blastocyst was not significantly different (P > 0.05) between the CDS FBS group (208.40 ± 14.77) and the HI FBS group (195.11 ± 19.15). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly increased mitochondrial activity, as identified by MitoTracker Green, and reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P < 0.05) higher after 24 h in the CDS FBS than in the HI FBS group (85.33 ± 4.84% v. 68.67 ± 1.20%). Quantitative reverse transcription PCR showed that the mRNA levels of lipid metabolism-related genes, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, and the cholesterol metabolism related gene hydroxymethylglutaryl-CoA reductase were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of survival gene sirtuin 1, antioxidant gene superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen–thawed blastocysts were significantly (P < 0.05) higher in the CDS FBS group than in the HI FBS group; however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. In conclusion, these data suggest that supplementation of in vitro culture medium with CDS FBS improves in vitro bovine embryo developmental competence and the quality of blastocysts in terms of their crytolerance and gene expression. This research was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets), BK21plus, and KGSP.

scholarly journals Effect of fetal bovine serum on in vitro maturation and fertilization of bovine oocytes

2020 ◽  
Vol 8 (5) ◽  
pp. 2187-2191
Author(s):  
Dipannita Baishya ◽  
Arundhati Bora ◽  
J Goswami ◽  
Aunbha Baruah ◽  
DJ Dutta ◽  
...  
Keyword(s):  
Bovine Serum ◽  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Fetal Bovine ◽  
Bovine Oocytes

185 PARTHENOGENETIC ACTIVATION OF SIKA DEER (CERVUS NIPPON TEMMINCK) OOCYTES WITH CHEMICALS

2012 ◽  
Vol 24 (1) ◽  
pp. 204
Author(s):  
Y. P. Yin ◽  
L. N. Tang ◽  
A. R. Fan ◽  
S. Zhang ◽  
X. Ma ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Sika Deer ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Fetal Bovine

Parthenogenetic activation of the oocyte represents an important step in the somatic cell nuclear transfer. The aim of the present study was to establish optimizing conditions for parthenogenetic activation of Sika deer oocytes necessary for cloning Sika deer. Sika deer ovaries were collected from a slaughter house during oestrus season (October and November), placed into saline (25°C) supplemented with 1% (v/v) penicillin and streptomycin and transported into the laboratory within 4 h. The small vesicular follicles (diameter, 2–5 mm) on the ovarian surface were incised with a scalpel in a Petri dish containing PBS to release the cumulus–oocyte complexes (COC). Only COC with uniform cytoplasm and at least 3 layers of compact cumulus cells were cultured in vitro for 24 h. The media of in vitro maturation (IVM) was TCM-199 supplemented with 10% fetal bovine serum, 10 μg mL–1 FSH, 1 μg mL–1 LH, 0.2 mM cysteamine and 50 ng mL–1 epidermal growth factor. After IVM, the cumulus cells were denuded with 0.2% hyaluronidase in TCM-199 at 38.5°C by pipetting. The cumulus-free Sika deer oocytes were stimulated by 1 of the following treatments: 1) ethanol + 6-DMAP, treated with 7% ethanol for 7 min and 2 mM 6-dimethylaminopurine (6-DMAP) in DSOF for 4 h; or 2) ionomycin + 6-DMAP, treated with 5 μM ionomycin for 5 min and 2 mM 6-DMAP in DSOF for 4 h. Then, oocytes were transferred into culture media for 7 days [Day 0 (D0) = activation]. On D3, embryos were transferred into fresh DSOF drops supplemented with 10% (v/v) fetal bovine serum. All cultures were overlaid with mineral oil and kept in a humidified modular incubation chamber gassed with 5% CO2. Effects of these chemicals on oocyte activation were then examined and compared with the controls, in which oocytes were cultured in TCM-199 for 4 h without chemical supplement. Our results showed that rates of cleavage, morula and blastocyst were 72.7, 43.9 and 32.4% (n = 139), respectively, by treatment with ionomycin + 6-DMAP. And rates of cleavage, morula and blastocyst were 61.1, 29.7 and 17.8% (n = 134), respectively, by treatment with ethanol + 6-DMAP. However, the rates of cleavage, morula and blastocyst were 5, 0 and 0% (n = 101) in the control group. Meanwhile, the rates of oocyte cleavage (72.7% vs 61.1%), morula (43.9% vs 29.7%) and blastocyst (32.4% vs 17.8%) between 2 treatments of ionomycin + 6-DMAP and ethanol + 6-DMAP were significantly different (P < 0.05). In conclusion, parthenogenetic activation of Sika deer oocytes with ionomycin + 6-DMAP is more effective than that with ethanol + 6-DMAP. These results have begun to elucidate parameters important for animal modeling and cloning with the Sika deer and should facilitate the development of genetically defined animal models in this species. This work was supported by the grant from the China Postdoctoral Science Foundation (No. 20090451135).

Replacement of fetal bovine serum and FSH with buffalo follicular fluid in in vitro maturation of buffalo oocytes

1996 ◽  
Vol 45 (1) ◽  
pp. 245 ◽  
Author(s):  
S.K. Das ◽  
M.S. Chauhan ◽  
P. Palta ◽  
P.K. Katiyar ◽  
M.L. Madan
Keyword(s):  
Follicular Fluid ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Fetal Bovine

scholarly journals Influence of bovine serum albumin and fetal bovine serum supplementation during in vitro maturation on lipid and mitochondrial behaviour in oocytes and lipid accumulation in bovine embryos

2016 ◽  
Vol 28 (11) ◽  
pp. 1721 ◽  
Author(s):  
Maite del Collado ◽  
Naiara Z. Saraiva ◽  
Flavia L. Lopes ◽  
Roberta C. Gaspar ◽  
Luciana C. Padilha ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Bovine Embryos ◽  
Fetal Bovine

Proper oocyte maturation is crucial for subsequent embryo development; however, oocyte mitochondrial and lipid-droplet behaviour are still poorly understood. Although excessive lipid accumulation during in vitro production (IVP) of bovine embryos has been linked with impaired cryotolerance, lipid oxidation is essential for adequate energy supply. Fetal bovine serum (FBS) and bovine serum albumin (BSA) are supplements used during IVP, containing high and low lipid content, respectively. This study aimed to understand how these supplements influence oocyte mitochondrial and lipid behaviour during in vitro maturation (IVM) in comparison to in vivo maturation, as well as their influence on development rates and embryo lipid accumulation during IVP. We demonstrate that only in vivo-matured oocytes maintained correlation between lipid content and active mitochondria. IVM media containing FBS increased total lipid content 18-fold and resulted in higher lipid accumulation in oocytes when compared with media with BSA. IVM using a lower FBS concentration combined with BSA resulted in satisfactory maturation and embryo development and also reduced lipid accumulation in blastocysts. In conclusion, IVM causes changes in mitochondrial and lipid dynamics, which may have negative effects on oocyte development rates and embryo lipid accumulation. Moreover, decreasing FBS concentrations during IVM may reduce embryo lipid accumulation without affecting production rates.

Sign in / Sign up

Export Citation Format

Share Document