Effect of holding at room temperature on initial chromatin configuration and in vitro maturation rate of equine oocytes

2002 ◽  
Vol 57 (8) ◽  
pp. 1973-1979 ◽  
Author(s):  
C.C. Love ◽  
L.B. Love ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
In Vitro Maturation ◽  
Room Temperature ◽  
Maturation Rate ◽  
Chromatin Configuration

209 MATURATION KINETICS AFTER HOLDING EQUINE OOCYTES IN EMBRYO HOLDING MEDIUM

2016 ◽  
Vol 28 (2) ◽  
pp. 235
Author(s):  
P. Dini ◽  
O. Bogado ◽  
K. Smits ◽  
A. VanSoom ◽  
P. Daels
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Developmental Competence ◽  
Hoechst 33342 ◽  
Immature Oocytes ◽  
Maturation Rate ◽  
Chromatin Configuration ◽  
One Way Anova ◽  
Selection Of

It has been reported that immature, equine oocytes can be maintained in meiotic arrest at 24°C. To evaluate a commercial equine embryo holding medium for storage of equine oocyte at 24°C and to determine the effect of holding on maturation kinetics, cumulus‐oocyte complexes (COC) were recovered from slaughtered mares and placed in Syngro® Embryo Holding Solution at 22–25°C for 18–20 h (OH Group) or placed directly in DMEM-F12-based in vitro maturation (IVM) conditions (D-Mat Group) at 5% CO2 in air at 38.5°C. Maturation rate (metaphase II percentage; MII%) was assessed (presence of polar body under stereomicroscope) after denudation at 22, 24, and 28 h. After assessment, the denuded oocytes that were considered immature were placed back in IVM, reassessed at 24 and 28 h, and MII% was compared with that of oocytes remaining uninterrupted in IVM for 24 and 28 h. One-way ANOVA was used to compare dependent variable in different groups using PROC ANOVA (SAS, version 9.2, SAS Institute, Cary, NC, USA). A random selection of mature oocytes from both groups were fertilised using intracytoplasmic sperm injection (ICSI). A total of 250 injected oocytes were cultured in DMEM-F12 with 10% FCS. Blastocyst rates in OH and D-mat groups were similar (7.1% v. 6.3%). At 22 h, significantly more oocytes reached the MII stage in the OH group than in the D-Mat group, but MII% was similar in both groups at 24 and 28 h (Table 1). Denuded, immature oocytes reached similar maturation rate as the undenuded oocytes in the same group. Our data suggest that oocytes can be held in Syngro® Embryo Holding Solution at 22–25°C for 18–20 h without compromising oocyte developmental competence. Overnight holding of oocytes accelerates maturation with similar maturation rate at 22, 24, and 28 h of IVM in the OH group. Denudation of immature oocytes after 22 h of IVM and returning the denuded oocytes to IVM does not affect the progression of maturation. In subsequent experiment, overnight held oocytes were fixed and stained (Hoechst 33342) and MII% was evaluated after 20, 22, and 28 h of IVM. Chromatin configuration confirmed that stored oocytes reach the MII stage at 22 h. Maturation rates were significantly lower at 20 h, suggesting that 22 h of IVM is required for stored oocytes. Table 1.Maturation rates (% in MII stage) at 22, 24, and 28 h of IVM for equine oocytes held in Syngro® Embryo Holding Medium before IVM (OH) and oocytes placed directly in IVM (D-Mat) Thanks to I. Lemahieu and P. Van Damme. Study was supported by the Special Research Fund at UGent.

225 IN VITRO MATURATION OF LION OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 220 ◽  
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
G. Mari ◽  
D. Zambelli
Keyword(s):  
In Vitro Maturation ◽  
Room Temperature ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
Corpora Lutea ◽  
Culture Methods ◽  
Maturation Rate ◽  
Simple Medium

The potential for rescuing immature oocytes from the ovaries of females of rare species of felids that die or undergo ovariohysterectomy has been reported in only two studies (Johnston et al. 1991 Biol. Reprod. 45, 898-906; Jewgenow et al. 1997 J. Reprod. Fertil. Suppl. 51, 33-39), in which oocytes were maturated in complex media (Eagle's MEM, M199, respectively). The domestic cat is used as a research model for endangered species but there may be some differences that perturb the adaptation of in vitro maturation (IVM), fertilization, or culture methods of lion oocytes compared to cat oocytes. Therefore, in the present study we evaluated the in vitro response of lion oocytes to our system for in vitro maturation of domestic cat oocytes in a simple medium (Merlo et al. 2005 Theriogenology 63, 2032-2039). A 14-year-old lioness, referred to our clinic because of pyometra, underwent ovariohysterectomy. After premedication with acepromazine 0.1 mg/kg i.m. (Prequillan; ATI, Bologne, Italy) and ketamine 5 mg/kg i.m. (Ketavet 100; Intervet, Boxmeer, The Netherlands), anesthesia was induced with ketamine 0.05 mg/kg i.v. and diazepam 0.02 mg/kg i.v. (Diazepam; Intervet) and maintained with isoflurane (Forane; Abbott, Rome, Italy) after intubation. Ovaries were removed and stored in saline solution at room temperature until collection of oocytes (within 1 h). A total of 53 small follicles (2-4 mm), five corpora lutea and a 15-mm follicle were present on the ovaries. Oocytes were collected by aspirating visible follicles of each ovary with a 21-ga needle connected to a vacuum pump (K-MAR-5100; Cook Australia, Brisbane, Australia) at -75 mmHg; then CL were removed and the ovaries were minced with a scalpel blade in a 60-mm petri dish containing HEPES-SOF (H-SOF) for recovery of additional oocytes. A total of 45 oocytes were recovered, of which 19 were degenerate (42.2%); of the remaining 26, 12 were fully surrounded by cumulus cells, 9 had only corona radiata, and 5 were denuded. Degenerate oocytes were discarded and all other oocytes were washed twice in H-SOF and matured in a 35 mm petri dish containing SOFaaBSA 5 mg/mL plus 0.1 IU of porcine FSH-LH (Pluset; Laboratorios Calier, Barcelona, Spain), 25 �L/mL insulin-transferrin-selenium (ITS) (Sigma, Madrid, Spain), 1.2 mm l-cysteine (Sigma), and 25 ng/mL epidermal growth factor (EGF) (Sigma) for 24 h in a humidified atmosphere of 5% CO2 in air at 38.5�C. After maturation, cumulus cells were removed by pipetting oocytes into a 0.25% trypsin solution for 2 min. Then, denuded oocytes were washed once in H-SOF plus 10% FCS to inactive trypsin and twice in H-SOF before being stained with Hoechst 33342 (10 �g in 10 mL PBS) for 30 min at room temperature. After washing in PBS, oocytes were observed using fluorescence microscopy to determine maturation rate. Oocytes in telophase I or metaphase II were considered mature. Of 26 oocytes, 19 (73.1%) were mature and 7 (26.9%) were at the GV stage. These results demonstrated that lion oocytes can undergo successful IVM at a frequency that is similar to that of cat oocytes cultured in the same system (76.9%, P > 0.05). Furthermore, the maturation rate obtained in a simple medium was higher or similar to those previously reported (mean: 22.9% and 69.7% respectively, by Johnston et al. 1991 and Jewgenow et al. 1997).

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P >0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

293 EFFECT OF DIFFERENT CONCENTRATIONS OF LH, FSH, AND E2 ON THE MATURATIONAL RATE OF INDIGENOUS SOUTH AFRICAN CATTLE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
K. P. M. Lekola ◽  
J. W. Ng'ambi ◽  
N. Nkadimeng ◽  
M. L. Mphaphathi ◽  
T. L. Nedambale
Keyword(s):  
South African ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
African Cattle ◽  
Maturation Medium ◽  
Maturation Rate

In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26 µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB– (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB–) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199) + 10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 µg mL–1 of FSH, 5 mg mL–1 of LH, and 2 µg mL–1 of E2; the T2 group was matured in the presence of 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2; and the T3 group was matured in the presence of 1.5 µg mL–1 of FSH, 7 mg mL–1 of LH, and 4.5 µg mL–1 of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24 h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB–, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB–, and not exposed to BCB respectively). However, BCB– oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

scholarly journals In Vitro Maturation of Horse Oocytes: Characterization of Chromatin Configuration Using Fluorescence Microscopy1

1993 ◽  
Vol 48 (2) ◽  
pp. 363-370 ◽  
Author(s):  
K. Hinrichs ◽  
A. L. Schmidt ◽  
P. P. Friedman ◽  
J. P. Selgrath ◽  
M. G. Martin
Keyword(s):  
In Vitro Maturation ◽  
Chromatin Configuration

scholarly journals Effect of follicle-stimulating hormone on Bligon goat oocyte maturation and embryonic development post in vitro fertilization

2020 ◽  
Vol 13 (11) ◽  
pp. 2443-2446
Author(s):  
Diah Tri Widayati ◽  
Mulyoto Pangestu
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Follicle Stimulating Hormone ◽  
Fertilization In Vitro ◽  
Vitro Fertilization ◽  
Maturation Rate ◽  
Stimulating Hormone

Background and Aim: Bligon goat is a crossbreed between Etawah and Kacang goat. This crossbreed goat is mostly reared by small farmers. In vitro maturation allows female goat (does) contributes toward reproduction despite the fact that the animal has been slaughtered. The aim of this study was to determine the in vitro maturation rate of Bligon goat oocytes supplemented with follicle-stimulating hormone (FSH), and their ability for further embryonic development after in vitro fertilization. Materials and Methods: Experiment was conducted at the Laboratory of Animal Physiology and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, using Bligon goat ovaries obtained from local slaughterhouse around Yogyakarta. One thousand five hundred cumulus-oocyte complexes were matured for 24 h in tissue culture medium 199 supplemented with 50 IU/L FSH or without FSH (control). First, matured oocytes were evaluated its morphology based on the expansion of cumulus cells and PB1 extrusion. Next, 600 oocytes were then stained with 1% aceto-orcein to examine maturation based on changes in the configuration of chromosomes and nuclear membrane breakdown. Oocytes were considered mature when they reached metaphase II. To prove the ability of mature oocytes to develop into embryos, 900 oocytes were processed for fertilization in vitro. The data were analyzed using analysis of variance. Results: The results indicated that FSH supplementation significantly increased oocyte maturation rate (65.21±7.26 vs. 43.25±6.23%) as indicated by extrusion of PB1 and homologous chromosome pairing and lined in the equator. The rate of degeneration was lower in the FSH-supplemented medium (3.21±0.25 vs. 10.17±3.15%). The blastocyst stage of oocyte developed embryos was reached by 12.43±2.15% and 22.28±4.86% of the control and treatment groups, respectively. Conclusion: FSH supplementation significantly improves oocyte maturation and yields mature oocytes for future embryo development in vitro.

Sign in / Sign up

Export Citation Format

Share Document