160 EFFECT OF cAMP MODULATORS ON IN VITRO MATURATION OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 193 ◽  
Author(s):  
S. E. Farmer ◽  
T. L. Adams ◽  
J. A. Sarmiento-Guzmán ◽  
C. L. Bailey ◽  
K. R. Bondioli
Keyword(s):  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Inhibitory Effect ◽  
Maturation Process ◽  
In Vitro Production ◽  
Angus Cattle ◽  
Significant Difference ◽  
Chi Squared ◽  
Bovine Oocytes

The efficiency of in vitro production (IVP) has remained low due to an inadequate in vitro maturation (IVM) system. Thus far, the most promising methods of improving IVM utilise cAMP modulators to slow the maturation process by keeping cAMP high. This experiment compares standard IVM to an extended IVM as previously described by Albuz (2010 Hum. Reprod. 25, 2999–3011). The extended IVM consists of a 2-h pre-IVM phase with FSK (an adenylate cyclase activator) and 3-isobutyl-1-methylxanthine [IBMX, a nonspecific phosphodiesterase (PDE) inhibitor], followed by a 31-h IVM phase with cilostamide (a PDE3 inhibitor). 3-Isobutyl-1-methylxanthine inhibits all PDE in both the oocyte and CC, whereas cilostamide inhibits PDE3 only in the oocyte, allowing a gradual reversal of inhibition. Bovine oocytes (n = 363) were obtained by transvaginal ultrasound-guided aspiration of both Brahman and Angus cattle over 4 collection days. Oocytes from each cow were divided into 2 groups. The first group was placed in standard 23-h IVM medium composed of TCM-199 supplemented with 10% fetal bovine serum, sodium pyruvate, penicillin and streptomycin, glutamine, and FSH, and cultured in 5% CO2 at 39°C. A subset of oocytes was removed from maturation at 8, 13, 18, and 23 h. The second group was placed into a pre-IVM medium of HEPES-TALP supplemented with 100 μM FSK and 500 μM IBMX for 2 h at 39°C, and then moved into standard maturation medium supplemented with 20 μM cilostamide for 31 h (5% CO2, 39°C). Oocytes were sampled at 8, 13, 18, 23, 28, and 33 h. Oocytes were stained with 1% orcein and nuclear status was examined for each sample time (Table 1). Data were analysed using a chi-squared test. At 8 h, there was a significant difference (P < 0.001) between GV stage of IVM and extended IVM (7.1 v. 73.2%), and between metaphase I (MI) stage IVM and extended IVM (76.2 v. 4.9%). At the 23-h time sample, there was a significant difference between metaphase II (MII) IVM and MII extended IVM (78.9 v. 30.6%; P < 0.001). There was also a significant difference between MII oocytes at 23 h IVM and MII oocytes at 33 h extended IVM (78.9 v. 33.3%; P < 0.001). These results are consistent with the hypothesis that cAMP modulators slow the nuclear maturation process. However, results also suggest that the inhibitory effect of the cAMP modulators is not completely reversible. Oocytes appear to arrest at MI by 23-h extended IVM and do not progress to MII at the same rate as standard IVM. Table 1.Nuclear status of bovine oocytes after standard IVM and extended IVM with cAMP modulators

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).

31 PROTOCOL OPTIMIZATION AND EVALUATION OF MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITIES IN BOVINE CYTOPLASTS OBTAINED BY CHEMICAL ENUCLEATION TECHNIQUES

2014 ◽  
Vol 26 (1) ◽  
pp. 130
Author(s):  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
M. del Collado ◽  
M. R. de Lima ◽  
R. Vantini ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activity ◽  
Significant Difference ◽  
Mitogen Activated Protein ◽  
Chi Squared ◽  
High Concentrations ◽  
Bovine Oocytes ◽  
Maturation Promoting Factor

Chemical enucleation using microtubule-depolymerizing drugs is an attractive procedure to simplify the enucleation process in nuclear transfer. The aim of this study was to optimize chemically assisted (CA) and chemically induced (CI) enucleation protocols using metaphase II (MII) and pre-activated bovine oocytes, respectively, and to evaluate the activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in cytoplasts generated by these techniques. Initially, we determined the shortest effective treatment of MII and activated oocytes with 0.05 μg mL–1 demecolcine. Bovine oocytes in vitro matured (IVM) for 19 h (MII) or activated artificially with 5 μM ionomycin (5 min) and 10 μg mL–1 cycloheximide (5 h) after 26 h IVM were treated with demecolcine and samples were collected at 0, 0.25, 0.5, 1.0, 1.5, and 2.0 h of treatment. Oocytes were then stained with 10 μg mL–1 Hoechst 33342 and the protrusion or enucleation rates were determined. Next, we evaluated histone H1 and myelin basic protein (MBP) kinases, reflecting MPF and MAPK activities, respectively, in oocytes obtained from these treatments, and for that we used the method described by Kubelka et al. (2000 Biol. Reprod. 62, 292–302). Protrusion and enucleation rates were evaluated by the chi-squared (χ2) test, and MPF and MAPK activities were submitted to ANOVA and Tukey's test at 5% significance. For MII oocytes, effects of demecolcine were observed as early as 15 min, with a significant difference (P < 0.05) between control (12/112, 10.7%) and treated (33/114, 28.9%) groups in relation to protrusion rates. The largest number of protrusions was observed after 1.0 h of treatment (control: 15/113, 13.3%a; treated: 45/111, 40.5%b). In pre-activated oocytes, effects of demecolcine were also observed after 15 min, and in both techniques there were no significant differences between groups treated with demecolcine for 1.0, 1.5, or 2.0 h (CA: 40.5 to 52.5% of protrusion; CI: 35.2 to 46.7% of enucleation). In contrast to previous reports in which high concentrations of demecolcine for CA enucleation increased MPF activity, we observed no alterations in the activity of this factor at a demecolcine concentration of 0.05 μg mL–1. Activity of MAPK also did not differ significantly between the control and treated groups throughout evaluation. In the CI technique, a significant difference in MPF activity was observed after 0.5 h (70.3%) and 2.0 h of activation (39.1%), considering that the activity was 100% at the beginning of the evaluation. However, we observed no significant difference between the control and treated groups at any of the time points studied, as verified for MAPK activity. The exact effect of MPF on the nucleus in mammals is not well established. We believe that the use of low concentrations of demecolcine for short periods is less damaging to embryonic development and, until we have a better understanding of the effect of these kinases on the transferred nucleus, we recommend its use for chemical enucleation protocols in bovine. Financial support: FAPESP 2010/20744-6 and 2011/12983-3.

158 EFFECTS OF APOTRANSFERRIN ON IN VITRO MATURATION OF CUMULUS - OOCYTES COMPLEXES (COCs) IN HANWOO, KOREAN NATIVE COWS

2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
S.-H. Choi ◽  
S.-R. Cho ◽  
M.-H. Han ◽  
H.-J. Kim ◽  
C.-Y. Choe ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Basic Fuchsin ◽  
In Vitro Production ◽  
Control Groups ◽  
Significant Difference ◽  
Native Cattle ◽  
Vitro Development ◽  
Vitro Production

For in vitro production of embryos, animal sera have been used as energy sources, maturation promoters, vitamins, growth factors, and antioxidative compounds. However, the sera had risk of virus and mycoplasma infections which could result in too big offspring and cause dystocia in ovine and bovine. Apotransferrin (apo-Tf) is a component of mammalian sera and has played a role as an antioxidant in media. A study was conducted to investigate the effects of apo-Tf on in vitro maturation of cumulus-oocytes complexes (COCs) in Hanwoo, Korean native cows. Ovaries were collected from a slaughterhouse and COCs were taken from 2-6-mm antral follicles. The collected COCs were washed three times with 0.1M polyvinyl alcohol (PVA)-TCM199 and matured in 0, 1, 10, or 100 �g/mL apo-Tf with TCM-199 at 39�C, 5% CO2, 95% air for 6, 12, or 24 h. Mature COCs were fertilized with frozen-thawed Korean native cattle semen treated with BO medium (Brackett and Oliphants 1975 Biol. Reprod. 12, 260-274) containing 5 mM caffeine and 1 �g/mL heparin for 8 h and developed to the blastocyst stage in 5% FBS and 0.3% BSA in TCM199-IVMD (IFP, Japan). To evaluate the morphology of nuclear types, the matured COCs were fixed in 1:3 acetic acid-ethanol for 30 s and stained with 3% basic Fuchsin. IVM and IVF were replicated three times. All of the results were analyzed by ANOVA using the STATVIEW program. The maturation rates of control were 34.2%, 37.3%, and 45.8% for 6, 12, and 24 h, respectively. There were no differences among the concentrations of apo-Tf, and nuclear types at 78.3-87.0% GVBD for 6 h, 82.8-91.3% MI for 12 h, and 88.9-100.0% MII for 24 h, with 1, 10, and 100 �g/mL apo-Tf, respectively. Conversely, there was significant difference between 1 �g/mL and 10 �g/mL in terms of cleavage rates, although the others did not vary significantly (P < 0.05). There were significant differences among the concentrations of apo-Tf for blastocyst formation (P < 0.05). Blastocysts matured with 1, 10, and 100 �g/mL apo-TF and developed in 5% FBS and 0.3% BSA in TCM199-IVMD showed rates of 8.8-21.6%, 9.4-35.3%, and 9.1-19.1%, respectively. The control groups developed to the blastocyst stage showed rates of 8.6%, 10.8%, and 10.5% in 5% FBS and 0.3% BSA in TCM199-IVMD, respectively. These results suggest that apo-Tf is an important factor for the in vitro maturation and in vitro development of bovine COCs.

271 EFFECT OF BULL ON IN VITRO FERTILIZATION OF BOVINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 292
Author(s):  
O. S. Garcia ◽  
R. S. Ferro ◽  
J. M. D. Bezerra ◽  
M. S. Sales
Keyword(s):  
Culture Medium ◽  
Sperm Concentration ◽  
Maturation Process ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Cumulus Oophorus ◽  
Significant Difference ◽  
Moist Environment ◽  
Bovine Oocytes

The main objective of this work was to evaluate the effect of the bull on IVF of bovine oocytes. These oocytes were obtained from ovaries collected in the slaughterhouse and selected for their maturation process in TCM-199 culture medium with 20% estrus cow serum, 10 IU of eCG, and 1 mg mL-1 gentamicin. The oocytes were cultivated for 20 to 24 hours in 39.0°C culture incubator and moist environment with 5% CO2. For the oocyte insemination, frozen/thawed semen of 8 bulls was used by means of swim-up capacity procedure according to Parrhit (1989) and then motility, sperm concentration, and strengh were evaluated. In all cases, 25 to 30 oocytes, previously selected according to cumulus oophorus density were artificially inseminated with 1 × 106 spermatozoids mL-1. The total of selected oocytes by bull for this work was: 5028 (1), 1247 (2), 2888 (3), 650 (4), 1529 (5), 699 (6), 984 (7), and 1250 (8). The results were evaluated according to the average of oocytes matured in vitro and to embryonic division 48 hours after AI. In order to evaluate the effects by bull, the statistical examination of chi-square proportion comparison was used. The average values and standard deviation (SD) motility were 57.0% ± 13.7; 53.2% ± 11.3; 51.2% ± 8.20; 40.9% ± 5.0; 48.1% ± 4.92; 33.5% ± 4.42; 53.5% ± 11.1 and 40.0% ± 8.9 for bulls 1 to 8, respectively. There was a highly significant difference (P < 0.001) in relation to the percentage of division: 46, 42, 34, 18, 30, 31, 30, and 17% among bulls 1 to 8, respectively, in addition to variability among these bulls. The results obtained confirm the effect of the bull on conception rate. The conclusion is that frozen/thawed semen with 50% motility can increase the percentage of embryonic division.

304 SHORT-PERIOD SPERM - OOCYTE INCUBATION AND FERTILIZATION MEDIA EFFECTS ON THE IN VITRO PRODUCTION OF SWINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 267
Author(s):  
M. G. Marques ◽  
A. B. Nascimento ◽  
L. F. Martins ◽  
F. R. O. Barros ◽  
M. D. Goissis ◽  
...  
Keyword(s):  
Incubation Period ◽  
In Vitro Production ◽  
Standard Medium ◽  
Sodium Pyruvate ◽  
Short Period ◽  
Significant Difference ◽  
Incubation Periods ◽  
Chi Squared ◽  
Vitro Production

The aim of this study was to evaluate the effect of 2 sperm–oocyte incubation periods (30 min and 6 h) and 3 IVF media (TCM-199, TCM-199 with Ca-lactate, or mTBM). Cumulus–oocyte complexes (COCs) from abattoir-derived swine ovaries were matured in TCM-199 supplemented with 3.05 mM glucose, 50µg mL−1 gentamycin, 0.91 mM sodium pyruvate, 10% swine follicular fluid, 0.57 mM cysteine, 10 ng mL−1 of epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 of hCG for 22 h, followed by a 22-h incubation without hormones. After in vitro maturation, oocyteswere allocated into 3 IVF media: TCM-199 (supplemented with 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 50µg mL−1 gentamycin, 1 mg mL−1 BSA, and 3.06 mM mL−1 caffeine-standard medium);TCM-199 Ca (standard medium with 2.92 mM Ca lactate); or mTBM (supplemented with 5 mM mL−1 sodium pyruvate, 0.57 mM cysteine, 50µg mL−1 entamycin, 1 mg mL−1 BSA, and 2 mM mL−1 caffeine). The refrigerated semen at 15°C was centrifuged and capacitated for 2 h at 38.5°C in an atmosphere of 5% CO2 in air and under high humidity conditions in the respective IVF media. Oocytes were denuded, washed with the respective IVF media, and inseminated with the capacitated spermatozoa at a concentration of 6 × 105 sperm mL−1 in 90-µL microdroplets. After 30 min, a group of oocytes from each fertilization medium was gently washed to remove nonbound spermatozoa and returned to a new medium droplet for another 5.5 h (30-min group). The other group of oocytes remained in the same droplet with spermatozoa for 6 h (6-h group). After IVF, oocytes were cultured in porcine zygote medium-3 for 7 days. Data were analyzed by chi-squared test (P &lt; 0.05).With regard to cleavage rates on the third day of embryo development (Day 3), no significant difference was observed among sperm–oocyte incubation periods; however, there was a difference in the TCM-199 group, compared with the mTBM and TCM-199 Ca groups, at the 30-min incubation period. With 6 h of incubation, no differences were observed among groups. When blastocyst (Day 7) rates were evaluated, no significant differences were observed between the 2 TCM-199 media groups; however, the mTBM groups showed higher blastocyst rates. The sperm–oocyte incubation period of 30 min or 6 h of IVF did not interfere with the blastocyst rate. In conclusion, the IVF medium mTBM was more efficient than the TCM-199 media when considering embryo production. Moreover, 30 min of IVF was sufficient for the spermatozoa responsible for oocyte fertilization to bind to the zona pellucida. Table 1.Cleavage (Day 3) and blastocyst (Day 7) rates of embryos incubated in two sperm–oocyte periods (30 min or 6 h) and cultivated in three IVF media This work was supported by the FAPESP 05/01420-7.

94 USE OF β-MERCAPTOETHANOL AND CYSTEINE FOR IN VITRO MATURATION AND CULTURE OF SHEEP EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 159
Author(s):  
J. Pradiee ◽  
L. L. Viana ◽  
E. C. S. Santos ◽  
A. Gonçalves ◽  
R. G. Mondadori ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Maturation ◽  
In Vitro Production ◽  
Embryo Viability ◽  
Negative Effects ◽  
Cleavage Rate ◽  
Chi Squared ◽  
Antibiotic Solution ◽  
The Media

During in vitro production (IVP), embryos are sensitive to suffering negative effects from catabolites, such as reactive oxygen species (ROS). Under physiological conditions, the action of the ROS is blocked by antioxidants such as glutathione, but glutathione's concentration is reduced during the main steps of the IVP process. The objective of the present study is to evaluate the effect of the supplementation of the media for in vitro maturation (IVM) and in vitro culture (IVC) with β-mercaptoethanol and cysteine on the rates of embryo development and viability after vitrification in open pulled straws (OPS). Ten IVP routines were conducted for IVP, using ovaries form pubertal sheep collected in a slaughterhouse. The ovaries were kept in a saline/antibiotic solution at 30°C during transport to the laboratory. The cumulus oophurus–oocytes complexes (COC) selected for IVM were allocated to 2 treatments: T1 (control), including no antioxidants in the IVM and IVC media (n = 676); and T2, including 50 μM β-mercaptoethanol and 600 μM cysteine, in the IVM and IVC media (n = 729). The IVM was conducted using the TCM 199 medium including oestradiol, FSH, LH, pyruvate, heat inactivated sheep serum and antibiotics, for 22 to 24 h. Sperm selection was conducted by swim-up in medium with tris-glucose-citric acid with fresh semen. For IVF, conducted for 18 to 22 h, 1 × 106 spermatozoa per mL were used in SOF medium including 2% heat-inactivated sheep serum. Both IVM and IVF were conducted with incubation with 5% CO2 at 39°C with saturated humidity. After IVF, the probable zygotes were denuded and cultured for 8 days in SOF medium with 0.4% BSA, at 39°C, in bags with 3 gases (5% CO2, 90% N2 and 5% O2). The criteria considered for embryo viability were: cleavage rate at Day 2 (cleaved/inseminated), embryo development at Day 7 (blastocysts/cleaved) and the reexpansion rate 24 h post-vitrification. Such frequencies were compared between treatments by the chi-squared test. The cleavage rate did not differ (P > 0.05) for T1 (60.3%) and T2 (64.3%). The rate of embryro development at Day 7 was also similar (P > 0.05) for T1 (33.6%) and T2 (36.6%). The reexpansion rate for T1 (76.9%) and T2 (54.1%) were also similar (P > 0.05). Thus, supplementation of IVM and IVC media with β-mercaptoethanol and cysteine presented no effect in the development and viability of vitrified sheep embryos. CAPES, MARFRIG Group.

Structural modifications induced by an in vitro maturation process in zona pellucida glycoproteins of bovine oocytes. A Raman microspectroscopy analysis

RSC Advances ◽  
2016 ◽  
Vol 6 (86) ◽  
pp. 83429-83437 ◽  
Author(s):  
G. Rizo ◽  
M. Roldán-Olarte ◽  
D. C. Miceli ◽  
L. E. Jiménez ◽  
R. M. S. Álvarez
Keyword(s):  
Zona Pellucida ◽  
In Vitro Maturation ◽  
Raman Microspectroscopy ◽  
Maturation Process ◽  
Structural Modifications ◽  
Bovine Oocytes

Raman microspectroscopy is useful for discrimination between immature and in vitro matured bovine oocytes. Modifications in the glycoproteins of the zona pellucida exerted by the maturation methods might influence the process of in vitro production.

scholarly journals Meiotic maturation and in vitro maturation of bovine oocytes

2006 ◽  
Vol 22 (1-2) ◽  
pp. 29-34 ◽  
Author(s):  
Tatjana Smiljakovic ◽  
Wolfgang Tomek
Keyword(s):  
Meiotic Division ◽  
In Vitro Maturation ◽  
Meiotic Maturation ◽  
In Vitro Production ◽  
Vitro Production ◽  
Bovine Oocytes

In vitro production of embryos, and, as part of this method, in vitro maturation of oocytes, have received great attention in last ten-fifteen years. It is well established in bovine. Here, in this review is presented importance of this method, usual meiotic division is described, as well, as importance of biochemical investigations of several protein factors and enzymes, which control these processes.

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