214 EFFECT OF OOCYTE SOURCE ON THE DEVELOPMENTAL CAPACITY OF SOUTH AFRICAN IN VITRO-PRODUCED INDIGENOUS CATTLE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 205
Author(s):  
T. L. Nedambale ◽  
M. B. Raito ◽  
M. L. Mphaphathi
Keyword(s):  
Treatment Group ◽  
South African ◽  
Formation Rate ◽  
Developmental Rate ◽  
Bovine Embryo ◽  
Department Of Agriculture ◽  
Indigenous Cattle ◽  
Developmental Capacity ◽  
Development In Vitro

The present study was undertaken to determine whether the source of oocytes (slaughterhouse ovaries from feedlot cows or naturally grazing indigenous cows) would affect in vitro bovine embryo production. Bovine oocytes (n = 1047), aspirated from slaughterhouse ovaries from feedlot cows and naturally grazing indigenous cows were randomly allocated to Sanyo, Forma, and Thermo 5% CO2 incubators. Oocytes were then in vitro matured in TCM-199 plus 10% fetal bovine serum, 1 μg mL–1 for both FSH and LH at 39°C for 24 h, and fertilized in Brackett and Oliphant (BO) medium per treatment group at 39°C. Presumptive zygotes were cultured in vitro per treatment group. Total cleavage and blastocyst rates were recorded postfertilization. Data were analyzed by ANOVA. Preliminary results demonstrated that there was no effect of incubator or source of oocytes on cleavage and 8-cell embryos. However, the cleavage and embryo developmental rate tended to be lower for the feedlot group, regardless of the incubator used (Table 1). In conclusion, this study suggests that slaughterhouse ovaries obtained from South African indigenous cows from a feedlot resulted in a lower blastocyst formation rate. Further studies are currently underway to count the cell numbers and to conduct embryo transfer. Table 1.Comparison of three different incubators and source of oocytes on embryo development in vitro This work was funded by the South African National Department of Agriculture, DST-PDP, and the National Research Foundation (NRF, Grant. Nos. RT21 and 24000).

scholarly journals 273EFFECT OF EPIDERMAL GROWTH FACTOR (EGF) DURING OOCYTE MATURATION ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 257
Author(s):  
H.J. Hernandez-Fonseca ◽  
R. Palomares-Naveda ◽  
E. Soto-Belloso ◽  
R. Gonzalez-Fernandez ◽  
A.D. De Ondiz-Sanchez ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Medium Components ◽  
Development In Vitro

Medium components during in vitro maturation (IVM) can significantly influence oocyte maturation and subsequent embryo development in vitro (Rose TA and Bavister BD 1992 Mol. Reprod. Dev. 31, 72–77; Harper K and Brackett B 1993 Biol. Reprod. 48, 409–416). The aim of this experiment was to evaluate the effect of EGF during IVM on further development of bovine embryos in vitro. Bovine ovaries were obtained at a slaughterhouse. Cumulus-oocyte complexes (COC) were aspirated from follicles 2–5mm in diameter. COC were incubated for 24h in either of 3 maturation media: T1 (n=72): modified TCM-199; T2 (n=45): modified TCM-199 supplemented with 10ngmL−1 of EGF;; or T3 (n=46): modified TCM-199 supplemented with 10% fetal bovine serum (FBS). After 24h of IVM, COC were inseminated with 2×106 motile spermatozoa/ml. After 18h of gamete coincubation, presumptive zygotes were denuded and placed in culture in SOF rich in glutamine (g-SOf) for 72h, at which time, cleavage rate (%) wass assesed (embryos with >4 cells). Subsequently, cleaved embryos were incubated for an additional 72h in c-SOF (SOF rich in citrate and glucose). Finally, embryos were cultured in modified TCM-199 for 24–48h, at which time blastocyst formation rate (%) was evaluated. Cleavage rates were similar between T2 and T3 but significantly greater than in T1 (P>0.05; see Table 1). Addition of EGF during IVM (T2;; 11/45, 24.4%) did not yield more blastocysts compared to the other two treatments (6/57, 10.5% and 10/29, 34.5%, T1 and T3, respectively). Nonetheless, T3 (with serum) had a greater yield of blastocysts compared to T1 (P>0.01). Results in this study show that the addition of EGF to chemically defined media results in similar cleavage rates and blastocyst yields to those obtained when using serum during IVM. Key words: in vitro maturation, EGF, cleavage, bovine, embryo. Table 1 Effect of EGF and serum during IVM on cleavage rate of bovine oocytes

216 COMPARISON BETWEEN A TRI-GAS THERMO INCUBATOR AND A MODULAR CHAMBER WITH PREMIXED GAS ON THE DEVELOPMENT OF BOVINE EMBRYOS IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 206
Author(s):  
M. B. Raito ◽  
M. L. Mphaphathi ◽  
L. M. Schwalbach ◽  
J. P. C. Greyling ◽  
T. L. Nedambale
Keyword(s):  
South African ◽  
Statistical Difference ◽  
Domestic Animals ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Department Of Agriculture ◽  
Development In Vitro ◽  
Oviductal Fluid

In an attempt to optimize germplasm and reproduction biotechnology IVF laboratory conditions in South Africa, we compared the effects of 2 triple-gas incubation systems, a tri-gas thermo incubator and a modular chamber with premixed gas, on the development of bovine embryos in vitro. After aspirating ovaries collected from a local abattoir, 778 oocytes were matured for 24 h in M-199 supplemented with 10% FBS, and 1 μg mL–1 of FSH and LH at 39°C in 5% CO2. Oocytes were then fertilized in vitro in Brackett and Oliphant (BO) medium at 39°C in 5% CO2. Presumptive zygotes were randomly allocated to the tri-gas thermo incubator or the modular chamber with premixed gas and cultured in synthetic oviductal fluid (SOF) medium at 39°C in 5% CO2, O2, and 90% N2. Total cleavage (Day 2), 8-cell (Day 2), morula (Day 6), and blastocyst (Day 7) rates were recorded postfertilization. Data were analyzed by ANOVA. There was no statistical difference in total cleavage rate between the 2 incubation systems. However, the 8-cell, morula, and blastocyst rates were significantly higher for the modular chamber group compared with the tri-gas incubator group (Table 1). In summary, this study suggests that the modular chamber with premixed gas was a better system for culturing zygotes of South African domestic animals to the blastocyst stage. Table 1.Effect of modular chamber and tri-gas incubator on embryo development in vitro This work was funded by the South African National Department of Agriculture, DST-PDP, and the National Research Foundation (NRF, Grant Nos. RT21 and 24000).

scholarly journals Endoplasmic reticulum stress attenuation promotes bovine oocyte maturation in vitro

Reproduction ◽  
2020 ◽  
Vol 159 (4) ◽  
pp. 361-370
Author(s):  
Hafiza Khatun ◽  
Yasuhiko Wada ◽  
Toshihiro Konno ◽  
Hideki Tatemoto ◽  
Ken-ichi Yamanaka
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Formation Rate ◽  
Critical Factor ◽  
Developmental Rate ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Apoptotic Cells

We have previously reported that regulation of endoplasmic reticulum (ER) stress during in vitro culture acutely increases bovine embryo developmental rate and cryotolerance; these data indicate that ER stress is a critical factor reducing the quality of in vitro-produced embryos. In the current follow-up study, we examined whether ER stress attenuation during in vitro maturation influences meiotic maturation, oocyte quality, and subsequent embryonic development. Bovine cumulus oocyte complexes (COCs) derived from slaughterhouse ovaries were matured with or without tauroursodeoxycholic acid (TUDCA), a selective inhibitor of ER stress (0, 50, 100, and 200 µM) for 22 h followed by in vitro fertilization, and zygotes were cultured for 8 days. Of the different doses of TUDCA, 100 μM TUDCA significantly increased the maturation rate, and decreased reactive oxygen species in denuded oocytes, and appeared lower number of apoptotic cells in matured COCs. Subsequently, treatment of TUDCA (100 µM) decreased the localization and amount of GRP78/BIP protein level as well as ER stress (GRP78/BIP, PERK, IER1, ATF4, and XBP1) and apoptosis (CHOP and BAX)-related gene expression, while it increased the anti-apoptotic gene BCL2 level in matured COCs. Moreover, addition of TUDCA (100 µM) during IVM significantly improved the blastocyst formation rate (43.6 ± 1.8% vs 49.7 ± 1.3%) and decreased the number of apoptotic cells (7.7 ± 1.1% vs 5.03 ± 0.6%) in blastocysts. These findings suggest that the presence of ER stress during maturation impairs the developmental competence of bovine COCs and that this process can be reversed by TUDCA.

scholarly journals 133 INSULIN, TRANSFERRIN AND SELENIUM WITH OR WITHOUT BSA IN A SERUM-FREE CULTURE SYSTEM FOR BOVINE EMBRYO, AND ITS SUITABILITY FOR EMBRYOS CULTURED IN SMALL GROUPS

2005 ◽  
Vol 17 (2) ◽  
pp. 217 ◽  
Author(s):  
C. Daniaux ◽  
B. Verhaeghe ◽  
I. Donnay
Keyword(s):  
Small Groups ◽  
Culture Medium ◽  
Culture Media ◽  
Quality Parameters ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Serum Free ◽  
Abnormal Accumulation ◽  
Development In Vitro

Serum in embryo culture medium may be a potential cause of abnormal accumulation of lipid droplets, which is correlated to a higher sensitivity to cryopreservation. Moreover, serum may introduce pathogens. With the aim of developing a serum-free culture medium, we first (Experiment 1) investigated the effect of adding ITS (5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium) as a serum substitute in SOF medium on embryos cultured in large groups (20 embryos per culture drop of 20 μL) and we then (Experiment 2) analyzed the effect of adding BSA. In this second experiment, our serum-free culture media were also tested on embryos cultured in small numbers (5 embryos per drop of 20 μL) in order to mimic ovum pickup (OPU) conditions. Embryos were obtained from slaughterhouse oocytes, matured in vitro for 24 h in a serum-free enriched 199 medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) containing ITS, and fertilized for 18 h. In experiment 1, embryos were cultured in SOF (Holm et al. 1999 Theriogenology 52, 683–700) supplemented with 0.1 mg/mL polyvinylpyrrolidane (PVP) without (SOF) or with ITS (SOF-ITS), or with 5% FCS (SOF-FCS). Cavitation occurred earlier in presence of serum (Table). Adding ITS to SOF increased blastocyst rates at Day 7 and Day 8 post-insemination (p.i.) and also the hatching rate. In experiment 2, embryos were cultured in SOF-FCS, SOF-ITS, or SOF-ITS supplemented with 4 mg/mL fatty acid free BSA (SOF-ITS-BSA). Within each condition, no differences were observed for blastocyst and hatching rates between embryos cultured in large or in small groups. Adding BSA to SOF-ITS increased blastocyst rate at Day 6 p.i. and also the hatching rate. At Days 7 and 8 p.i., blastocyst rates were higher in SOF-FCS than in SOF-ITS and tended to be higher than in SOF-ITS-BSA, especially for embryos cultured in small groups. Cell numbers of the resulting embryos were unaffected. These results indicate that: (1) ITS as supplement to SOF medium promotes embryo development in vitro. (2) BSA as protein supplement to SOF-ITS medium accelerates blastulation and improves hatching rate. (3) SOF-ITS and SOF-ITS-BSA are two serum-free culture media that can sustain development of embryos, also when cultured in small number, even though SOF-FCS tended to afford better rates of development. Further studies will include evaluation of other quality parameters including resistance to cryopreservation. This work was supported by the Ministery of Agriculture of the Region wallonne de Belgique.

22 EFFECT OF OOCYTE AGE ON THE DEVELOPMENT OF MOUSE PARTHENOGENETIC EMBRYOS AND EFFECT OF OXYGEN CONCENTRATION ON EMBRYO DEVELOPMENT IN VITRO

2006 ◽  
Vol 18 (2) ◽  
pp. 119
Author(s):  
H. Bagis ◽  
S. Arat ◽  
H. Odaman ◽  
A. Tas
Keyword(s):  
Embryo Development ◽  
Formation Rate ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Group 4 ◽  
Mouse Embryo Development ◽  
O2 Concentration ◽  
Group 3 ◽  
Development In Vitro

The objective of this study was to investigate the effects of two parameters on mouse embryo development in vitro. These parameters were the effect of oocyte age on activation and the effect of O2 concentration in culture. In the first experiment, oocytes were recovered from superovutated mice at 15 h (group 1) or 20 h (group 2) after human chorionic gonadotropin (HCG) injection. All oocytes were activated for 6 h with 10 mM Sr2+ in Ca2+ free medium in the presence of 5 �g/mL of cytochalasin B. After activation, embryos were cultured in KSOM.aa medium for 4.5-5.5 days. Zygotes from naturally bred mice were used as control. Differences in blastocyst formation rate and blastocyst cell number among treatments were analyzed by one-way ANOVA after arcsin square transformation. In the first experiment, blastocyst formation rate in the first group was higher than in the second group (62.6% vs. 47.1%; P < 0.05). In addition, blastocyst cell number was also higher in the first group than in the second one (69.4 � 3.2 vs. 52.4 � 2.2; P < 0.05). However, both values were higher in control group (80%, 76.2 � 1.2; P < 0.05) than in the experimental groups. These results showed that young oocytes were activated more effectively than aged oocytes. In the second experiment, mouse zygotes were cultured in a humidified atmosphere of 5% CO2 in air (group 3) or 5% CO2, 5% O2, and 90% N2 (group 4). Blastocyst formation rate and blastocyst cell number of zygotes cultured in low O2 concentration (group 4) for 4.5 days were higher than for group 3 (76.3% vs. 56.4 and 69.0 � 3.4 vs. 52.8 � 2.3; P < 0.05). There was a significant difference in blastocyt formation rate of embryos for 5.5 days between the two groups (25.8% for group 4 vs. 14.4% for group 3; P < 0.05). This suggests that the embryos developed more slowly in high O2 concentration. These results showed that low O2 concentration provided a more suitable environment for mouse embryo development in vitro. The same experiment was repeated with parthenogenetic embryos recently in our laboratory. This study was supported by a grant from TUBITAK, Turkey (VHAG-1022).

100 COMPARISON OF PREGNANCY RATES OF FRESH IN VITRO-PRODUCED BOS INDICUS EMBRYOS PRODUCED IN THE SAME LABORATORY BUT COLLECTED AND TRANSFERRED IN PANAMA OR THE DOMINICAN REPUBLIC

2014 ◽  
Vol 26 (1) ◽  
pp. 164
Author(s):  
L. F. Nasser ◽  
S. C. Feliú ◽  
E. Rodríguez ◽  
K. Mojica ◽  
E. G. Oliveira ◽  
...  
Keyword(s):  
Dominican Republic ◽  
Bos Indicus ◽  
Disease Status ◽  
Pregnancy Rates ◽  
Bovine Embryo ◽  
Department Of Agriculture ◽  
Embryo Production ◽  
Essential Variable ◽  
Laboratory Facility

Because of Panama's stricter sanitary status, a specialised protocol was developed with the Department of Agriculture in the Dominican Republic to legalize the exchange of biological materials (oocytes/embryos). This protocol allows the team of specialised technicians, currently working in Born® Animal Biotechnology's Panamanian facility, to operate using the same in vitro bovine embryo production system (IVP, In vitro Brasil®) to service Dominican producers. Because the donors are not located at a specific centre with controlled sanitary management, a special protocol was developed in which blood tests were done to certify that the entirety of the herd at each client's farm was free of infectious bovine rhinotracheitis, DBVD, leptospirosis, leucosis, brucellosis, and tuberculosis. As timing during IVP is an essential variable that can have detrimental effects on the final results, precautions were taken to ensure that the oocytes arrived at the Panamanian laboratory facility within 24 h of aspiration. A portable incubator was used to transport oocytes and embryos during the import and export portions of the procedure. A comparison of pregnancy rates based on oocyte source and recipient transfers from September 2012 until May 2013 was analysed with ?2 (Table 1). The number of embryos produced in Panama was significantly higher than in the Dominican Republic, which was likely due to the larger number of donors and oocytes from the Panama herd. However, pregnancy rate was higher in the Dominican Republic likely because of the health status of the Dominican recipients, which were free of the diseases mentioned above. Recipients were the same type and breed and under similar management conditions in both countries. The disease status aspect will be examined with greater numbers of animals in the future. The data suggest that the present IVP and recipient management protocols could serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.In vitro embryo production and pregnancy rates of Bos indicus embryos transferred in Panama or the Dominican Republic (September 2012 through May 2013)

208 EFFECT OF GDF8 AND SB-431542 ON PORCINE OOCYTE DURING IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 235
Author(s):  
J. D. Yoon ◽  
E. Lee ◽  
S.-H. Hyun
Keyword(s):  
Treatment Group ◽  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Transforming Growth Factor ◽  
Formation Rate ◽  
Transforming Growth Factor Β ◽  
Nuclear Maturation ◽  
Porcine Oocyte

Growth differentiation factor-8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. SB-431542 (SB) is a specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors such as ALK4, ALK5, and ALK7. The purpose of this study is investigation of the effects of GDF8 and SB on porcine oocytes during in vitro maturation and subsequent embryonic development. We first performed ELISA to detect GDF8 concentrations in follicular fluid for each size of follicle; sizes were as follows: small (<3 mm), medium (>3 mm and <6 mm), and large (>6 mm) follicle. After detection of the GDF8 concentration in follicular fluid, we investigated the effect of GDF8 and SB treatment during in vitro maturation (IVM) on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, and embryonic development after IVF and parthenogenetic activation (PA). Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science, IBM, New York, NY, USA) mean ± SEM. The ELISA result showed different concentrations of GDF8 for each grade of follicular fluid: small, 0.479 ng mL–1; medium, 0.668 ng mL–1; and large, 1.318 ng mL–1. During the IVM process, 1.318 ng mL–1 of GDF8 and 5 ng mL–1 of SB were added to the maturation medium as control, SB, SB+GDF8, and GDF8 treatment groups. After 44 h of IVM, GDF8 group (90.4%) showed a significantly higher nuclear maturation rate than control and SB+GDF8 groups (85.4 and 81.7%). The SB group (78.9%) showed significantly reduced nuclear maturation rate compared with control (P < 0.05). The GDF8 treatment group showed a significant decreased intracellular ROS and increased GSH levels compared with other groups (P < 0.05). The SB+GBF8 treatment group showed a significantly better cytoplasmic maturation than the SB treatment group. In the PA embryonic development analysis, the GDF8 treatment group showed a significantly higher blastocyst formation rate compared with other groups (47.9, 37.2, 46.4, and 58.7% respectively; P < 0.05). In the IVF embryonic development analysis, the GDF8 treatment groups showed significantly higher blastocyst formation rate compared with the SB group (28.2 and 42.2%, respectively; P < 0.05). In conclusion, treatment with GDF8 during porcine oocyte IVM improved the embryonic developmental competence via increased cytoplasmic maturation and led to better oocyte maturation from the ALK receptor inhibition by SB.

Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

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