Effect of charcoal:dextran stripped fetal bovine serum on in vitro development of bovine embryos

The use of fetal bovine serum (FBS) as protein supplementation in IVP of bovine embryos has presented difficulties because it can introduce a number of pathogenic components in culture systems, can be related to the birth of calf with abnormal growth and development, and precludes the establishment of the actual nutritional needs of the embryo, because it contains an unlimited variety of substances. This study evaluated the replacement of the FBS in the medium of in vitro culture (IVC) of bovine embryos, using the knockout serum replacer (KSR) as protein supplementation and culture medium conditioned with stem cells. Therefore, bovine oocytes from ovaries of slaughterhouse were selected and matured in vitro in TCM-199 medium supplemented with 10% FBS (Crypion), 1.0 μg mL-1 FSH (Pluset®, Calier, Barcelona, Spain), 50 μg mL-1 hCG (Profasi®, Serono, Geneva, Switzerland), 1.0 μg mL-1 estradiol (Sigma E-2758, Sigma Chemical, St. Louis, MO, USA), 0.2 mM sodium pyruvate, and 83.4 μg mL-1 amikacin for 24 h. After that, 1144 oocytes were fertilized in IVF-TALP medium containing 6 mg mL-1 of BSA. After 18 to 22 h, the zygotes were cultured in SOF + 5% FBS (group 2); SOF + 5% KSR (group 3); SOF (5% FBS) + 10% SOF (5% FBS) conditioned by stem cells (group 4); or SOF (5% KSR) + 10% SOF (5% KSR) conditioned by stem cells (group 5), in an atmosphere of 5% O2 at 38.5°C for 8 days. A control group outside the controlled atmosphere was added, supplemented with 5% FBS (group 1). The SOF medium supplemented with 5% FBS or KSR was conditioned by stem cells and added to SOF medium for the culture of embryo at a concentration of 10%. The rates of cleavage and production of blastocysts were assessed 48 hours and 7 days after IVF, respectively, and analyzed by chi-square test, with a significance level of 5% in the statistical program Minitab® (release 14.1, Minitab, State College, PA, USA). On the eighth day, the TUNEL test for determination of the percentage of apoptosis and the differential staining technique for determination of inner cell mass (ICM) and trophoblast (TF) were performed. The results were submitted to ANOVA, followed by comparing the means by Tukey’s test using the program GraphPad Prism (GraphPad, San Diego, CA, USA). The treatments did not differ in the production of embryos, being similar to the control group: G1 = 31.75% (74/233), G2 = 35.26% (79/224), G3 = 32.70% (74/226), G4 = 28.76% (63/219), and G5 = 26.85% (65/242). With regard to the assessment of embryonic quality, the treatments showed similar results to the control groups. No differences were observed among groups both in color and ICM/TF ratio (G1 = 0.60, G2 = 0.62, G3 =0.65, G4 = 0.60, and G5 = 0.60). Furthermore, the TUNEL showed no significant difference in the percentage of apoptosis among groups (G1 = 7.10%, G2 = 3.76%, G3 = 5.58%, G4 = 4.50%, and G5 = 4.11%). The data obtained so far indicate that it is possible to produce embryos in vitro by replacing the FBS in the culture, achieving results similar to those obtained with serum. Financial support: FAPESP 2007/58506-6.

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In vitro development of bovine embryos as affected by different lots of bovine serum albumin and citrate

Theriogenology ◽

10.1016/0093-691x(94)90678-c ◽

1994 ◽

Vol 42 (3) ◽

pp. 397-403 ◽

Cited By ~ 11

Author(s):

R.W. Rorie ◽

G.F. Miller ◽

K.B. Nasti ◽

R.W. McNew

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Bovine Serum ◽

Bovine Embryos ◽

In Vitro Development ◽

Vitro Development

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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

Reproduction Fertility and Development ◽

10.1071/rd15067 ◽

2016 ◽

Vol 28 (11) ◽

pp. 1721 ◽

Cited By ~ 33

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.

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76 Tetrahydrofuran does not have Embryo Toxic Effects on In Vitro-Produced Bovine Embryos

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab76 ◽

2018 ◽

Vol 30 (1) ◽

pp. 176

Author(s):

M. M. R. Chowdhury ◽

I. Khan ◽

A. Mesalam ◽

K.-L. Lee ◽

J.-Y. Hwang ◽

...

Keyword(s):

In Vitro Culture ◽

Bovine Serum ◽

Culture Media ◽

Fetal Bovine Serum ◽

Control Group ◽

Bovine Embryos ◽

Sodium Pyruvate ◽

Fetal Bovine ◽

The Impact

In vitro embryo developmental potentials are still suboptimal compared with in vivo potential due to the challenge of various unknown stressors that must be overcome by in vitro-cultured oocytes. To improve existing embryo developmental potentials, many chemicals have been treated in maturation media by dissolving in toxic substances such as dimethyl sulfoxide (DMSO) or other carrier molecule. The foremost effort of this study was to investigate the impact of the solvent tetrahydrofuran (THF) on the cytotoxicity of in vitro embryo production (IVP). The experiment was completed within 8 replicates. Statistical analyses were performed using SPSS version 22.0 (IBM/SPSS, Armonk, NY, USA), a one-way ANOVA followed by multiple pairwise comparisons (Tukey’s test), and Duncan’s multiple range post hoc test. The level of statistical significance was considered P < 0.05. Oocytes were cultured in vitro maturation media (IVM) followed by in vitro fertilization (IVF), in vitro culture media 1 (IVC1), and in vitro culture media 2 (IVC2). Composition of the media was as follows: IVM medium was TCM-199 supplemented with 10% (v/v) fetal bovine serum, 1 µg mL−1 oestradiol-17β, 10 µg mL−1 FSH, 0.6 mM cysteine, and 0.2 mM sodium pyruvate. The IVC1 medium consisted of CR1-aa supplemented with 44 µg mL−1 sodium pyruvate, 14.6 µg mL−1 glutamine, 10 IU mL−1 penicillin, 0.1 mg mL−1 streptomycin, 3 mg mL−1 BSA, and 310 µg mL−1 glutathione. The IVC2 medium was the same composition as IVC1 except that BSA was replaced with 10% (v/v) fetal bovine serum. The final concentration of the optimized (0.5 µM) THF in culture medium was 0.4%. When coculturing with 0.5 µM THF in the IVM stage, the cleavage rate (58.65 ± 1.90% v. 56.87 ± 1.68%) was not significantly different, but the blastocyst rate (35.21 ± 1.44% v. 28.34 ± 2.11%) was significantly higher compared with the control group. The TUNEL assay confirmed that apoptotic nuclei in THF group were significantly reduced compared with the control group (2.32 ± 0.14 v. 5.65 ± 0.12). The total cell number of trophectoderm (TE) in control and THF groups was 115.34 ± 0.98 and 132.13 ± 1.55, and that of the inner cell mass (ICM) was 29.67 ± 0.40 and 39.94 ± 0.44, respectively. However, the ICM:TE ratio in control and treated blastocysts was 1:3.34 and 1:3.9, which was not statistically significant. Immunocytochemistry analysis (using antibodies to IKBKB, NFkB, COX2, CASP9, and CASP3) demonstrated that THF supplementation significantly attenuated expression of these proteins. The quantitative recerse transcription PCR data established that relative mRNA expression level of the anti-apoptotic gene BCL2 was up-regulated, whereas that of COX2, iNOS, BAX, IKBKB, NFkB, CASP9, and CASP3 were significantly down-regulated in the THF treated group compared with the control. In conclusion, 0.5 µM THF supplement in the IVM media did not have injurious effects on in vitro-cultured bovine embryos. This work was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets) and BK21plus.

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163 EFFECTS OF SERUM AND L-CARNITINE ON DEVELOPMENT AND CRYOTOLERANCE OF BOVINE EMBRYOS PRODUCED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab163 ◽

2016 ◽

Vol 28 (2) ◽

pp. 211

Author(s):

A. Zolini ◽

E. L. Carrascal-Triana ◽

A. Ruiz ◽

J. M. Penitente-Filho ◽

P. J. Hansen ◽

...

Keyword(s):

Bovine Serum ◽

Fetal Bovine Serum ◽

Bovine Embryos ◽

Blastocyst Development ◽

Beneficial Effects ◽

Serum Supplementation ◽

Fetal Bovine ◽

Concentration Table ◽

Hatching Rates

Cryotolerance of bovine embryos produced in vitro (PIV) can be improved by l-carnitine. The objective of the present study was to determine whether the optimal concentration of l-carnitine is dependent on serum. Bovine embryos were produced in vitro with abattoir-derived oocytes. After fertilization (Day 0), oocytes (n = 2768) were randomly assigned in a 2 × 4 factorial design to culture in SOF-BE1 medium supplemented with or without 5% fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5, and 3.03 mM at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. The proportion of oocytes that cleaved was assessed on Day 3, and the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, and hatched) stages was determined on Day 7. Blastocysts and expanded blastocysts (n = 466) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. After thaw, embryos were cultured for 72 h in SOF-BE1 supplemented with 10% (v/v) fetal bovine serum and 50 mM dithiothreitol at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 9 times, and data were analysed by logistic regression. There was no interaction between serum and l-carnitine, at any of the concentrations tested, on embryo development or cryotolerance. Cleavage rates were not affected by serum or l-carnitine. Addition of serum during culture increased (P < 0.05) development to the blastocyst (19.7 ± 1.1% v. 25.3 ± 1.4%) and advanced blastocyst (9.1 ± 0.8% v. 12.4 ± 1.2%) stages. While l-carnitine did not affect blastocyst development, advanced blastocyst development was reduced (P < 0.05) for l-carnitine at 3.03 mM (0 mM: 10.9 ± 1.2%, 0.75 mM: 12.2 ± 1.4%, 1.5 mM: 13.5 ± 1.5%, 3.03 mM: 7.0 ± 1.0%). Serum reduced (P < 0.01) re-expansion (78.1 ± 3.4% v. 65.5 ± 3.1%, 81.0 ± 3.0% v. 68.4 ± 2.7%, 78.4 ± 3.4% v. 65.8 ± 3.1%, for 24, 48, and 72 h, respectively) and hatching (52.0 ± 4.0% v. 39.8 ± 3.6%, 61.2 ± 4.1% v. 45.4 ± 3.8%, 61.2 ± 4.1% v. 45.4 ± 3.8%, for 24, 48, and 72 h, respectively) rates at all time points. In contrast, treatment of embryos with l-carnitine during culture increased (P < 0.05) post-thaw re-expansion rates at 24 and 48 h, regardless of concentration (Table 1). In conclusion, post-thaw viability of bovine embryos PIV can be improved by the addition of l-carnitine during culture. Moreover, the beneficial effects of l-carnitine on cryosurvival are not dependent on serum supplementation. Table 1.Effect of addition of l-carnitine during culture on post-thaw re-expansion and hatching rates

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Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2015000700002 ◽

2015 ◽

Vol 35 (7) ◽

pp. 605-612

Author(s):

Daniel R. Arnold ◽

Carolina A.P. Corrêa ◽

Laura L.G. Lorena ◽

Roberta C. Gaspar ◽

Guilherme F. Rossi ◽

...

Keyword(s):

Histone Modification ◽

Bovine Serum ◽

Fetal Bovine Serum ◽

Bovine Embryos ◽

In Vitro Production ◽

Cell Stage ◽

Morula Stage ◽

Fetal Bovine

Abstract In vitro production (IVP) of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS). Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst). Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05). Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05), 16-cell (P<0.05) and morula (P<0.05) stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01). Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

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