157 EFFECTS OF FETAL CALF SERUM OR PHENAZINE ETHOSULFATE AND FRUCTOSE OR GLUCOSE ON EMBRYONIC DEVELOPMENT AND LIPID ACCUMULATION OF BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 186
Author(s):  
M. Barcelo-Fimbres ◽  
G. Seidel Jr
Keyword(s):  
Amino Acids ◽  
Lipid Accumulation ◽  
Lipid Droplets ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Bovine Embryos ◽  
Cell Embryo ◽  
Phenazine Ethosulfate

Slaughterhouse oocytes (n = 6222) were maturated in a chemically defined medium (CDM) similar to SOF plus 0.5% fatty acid-free BSA (FAF-BSA) and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Oocytes and frozen-thawed sperm, centrifuged through a Percoll gradient, were co-cultured for 18 h in F-CDM (CDM + heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 (CDM + nonessential amino acids, 10 μM EDTA, 0.5% FAF-BSA, and 0.5 mM fructose or glucose in Expt 1 and glucose in Expt 2). In both experiments, after 48 h, 8-cell embryos were cultured 135 h in CDM-1 (CDM-1 + essential amino acids, no EDTA, and 2 mM fructose or glucose). A factorial design with two hexoses and three additives in CDM-2 (control; 10% fetal calf serum (FCS); and 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH) was used for both experiments, each replicated eight times. For Expt 1, Day 7.5 blastocysts were fixed and stained with Sudan Black B to quantify cytoplasmic lipid droplets. A digital photo at 600× of the equatorial part of the embryo was evaluated by classifying lipophilic droplet diameters as small (S, <2 μm), medium (M, 2 to 6 μm), or large (L, >6 μm), reported as number of lipid droplets (LD) per 1000 μm2. Data were analyzed by ANOVA. For Expt 1, 8-cell embryo production per oocyte matured was not affected by fructose or glucose (P > 0.1) (70 vs. 68%, respectively); however, blastocyst rates per oocyte matured (B/O) and per 8-cell embryo (B/E) were higher (P < 0.01) for fructose than glucose (Table 1). There were no differences between control, PES, and FCS (P > 0.1) for B/O, or B/E. For Expt 2, B/O and B/E were higher (P < 0.01) for fructose than for glucose. No differences were found for additives (P > 0.1) control, FCS, or PES for B/O or B/E. There was an interaction (P < 0.05) between additives and hexoses for blastocyst production, because the benefit of fructose compared to glucose was greater for controls than for FCS or PES (means not presented). Accumulations of each size of LD were less for PES (P < 0.05) than for control and FCS. Control and PES were lower than FCS (P < 0.05) for S, M, and L droplets. There was no effect of fructose or glucose (P > 0.1) on numbers of S, M, or L droplets (Table 1). In conclusion, fructose produced more blastocysts than glucose after 8-cell development, but there was no hexose effect either before this stage or in lipid accumulation. PES reduced and FCS increased lipid accumulation relative to controls. Table 1. Main effects of additives and hexoses on development of bovine embryos (±SE)

89 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECT IN THE ULTRASTRUCTURE AND DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 157 ◽  
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
T. S. Rascado ◽  
L. F. Crocomo ◽  
M. D. Guastali ◽  
...  
Keyword(s):  
Lipid Content ◽  
Lipid Droplets ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Culture Methods ◽  
Mitochondrial Number ◽  
Phenazine Ethosulfate

Over the past decades, there have been great advances in in vitro production (IVP) systems, with improved culture methods and new knowledge regarding embryo physiology, ultrastructure and morphology. Currently, the major obstacle associated with the extensive use of this technology is the great sensitivity of IVP embryos to cryopreservation. According to the literature, the reduced cryotolerance of IVP embryos is frequently associated with their high lipid content. Although is not clear until now how the lipid accumulation occurs, it may be influenced by the use of undefined culture media, supplemented with fetal calf serum (FCS); or as a result of embryo energy metabolism abnormalities that affect mitochondrial function, leading to the decrease in both the embryo quality and survival after cryopreservation. In this context, phenazine ethosulfate (PES), a reducer of NADPH electrons, which favours pentose–phosphate pathways and also inhibits the fatty acids synthesis, has been used to increase IVP embryo cryotolerance (Sudano et al. 2011 Theriogenology 75, 1211–1220). The aim of the present study was to evaluate the phenazine ethosulfate and FCS effect in the ultrastructure of IVP bovine embryos. A 2 × 2 factorial experiment design was used to test 2 FCS concentrations (0 or 10%) and the addition of PES (without or with PES) in the culture media. Slaughterhouse ovaries were used to obtain oocytes which were matured and fertilized in vitro (Day 0). Presumptive zygotes (n = 1440) were divided in 4 culture media: SOFaa without FCS; SOFaa without FCS + 0.3 μM PES (started on Day 4); SOFaa + 10% FCS; SOFaa + 10% FCS + 0.3 μM PES (started on Day 4). Embryo development was evaluated after 7 days under standard culture conditions (at 38.5°C in atmosphere of 5% O2, 5% CO2 and 90% N2). Transmission electron microscopy (TEM) was performed on Day-7 blastocysts from each group (n = 5) through standard protocol. For the statistical analysis, the arcsine transformation was applied to blastocyst percentage data and submitted to the ANOVA, followed by Tukeys' test through PROC GLM (SAS Institute Inc., Cary, NC, USA). In the absence of significant interactions, only main effect means are presented. The blastocyst production was not affected (P = 0.47) by the use of PES (42.7 ± 3.2 vs 39.3 ± 3.2, respectively for control and PES Day 4). The addition of 10% of FCS increased (P < 0.0001) the percentage of blastocysts (48.9 ± 3.2 vs 33.0 ± 3.2, respectively, for 10% and 0% of FCS). The ultrastructure analysis showed similar features in embryos from all studied groups. However, embryos cultured in the absence of FCS presented fewer and smaller lipid droplets. Moreover, embryos cultured without FCS presented more cellular debris in the perivitelinic space and in the blastocoele, indicating loss of blastomeres. The use of PES was able to reduce lipid droplets and increase the mitochondrial number in serum-produced embryos. Therefore, the PES decreased lipid content and increased mitochondrial number without affecting the development and ultrastructure of IVP bovine embryos. FAPESP 09/54513-3, 10/09922-0.

137 PHENAZINE ETHOSULFATE AND FETAL CALF SERUM EFFECTS ON THE DEVELOPMENT AND APOPTOSIS OF IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 173
Author(s):  
M. J. Sudano ◽  
D. M. Paschoal ◽  
T. S. Rascado ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Bos Indicus ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Quality ◽  
Phenazine Ethosulfate

Phenazine ethosulfate (PES) is a metabolic regulator that inhibits fatty acid synthesis and favours the pentose-phosphate pathway. Supplementation of fetal calf serum (FCS) during culture has been correlated with the reduction of quality of in vitro produced bovine embryos (IVPE). The aim of the present study was to evaluate embryo development and apoptosis in blastocysts after the supplementation of PES and FCS in culture medium of IVPE. Oocytes (N = 4320) were matured and fertilized in vitro (Day 0). The zygotes (Bos indicus) were cultured in SOFaa medium with 4 concentrations of FCS (0, 2.5, 5, and 10%) and with the use or not of 0.3 μM PES from Day 4 (after 96 h of embryo culture). Embryo development was evaluated after 7 days of culture. Apoptosis in blastocysts (N = 60–80) was accessed through TUNEL reaction. Embryos (Bos indicus) recovered from superstimulated cows were used as in vivo control (n = 15). Data were analysed by ANOVA followed by LSD using PROC GLIMMIX (SAS; SAS Institute Inc., Cary, NC, USA) means ± SEM. Increasing FCS concentration in the culture media did not change cleavage (86.7 ± 1.7, 82.3 ± 1.6, 86.3 ± 1.4, 87.0 ± 1.5, P > 0.05) and augmented blastocyst production (30.5 ± 2.5a, 41.8 ± 2.4b, 40.5 ± 2.6b, 47.2 ± 2.8b, P < 0.05), respectively, for 0, 2.5, 5, and 10%. Additionally, increasing FCS concentration increased apoptosis in blastocysts (13.8 ± 1.2b, 19.1 ± 1.8b, 20.7 ± 1.9bc, 28.4 ± 2.3c, P < 0.05, respectively, for 0, 2.5, 5, and 10%). The addition of PES from Day 4 in the culture medium did not affect (P > 0.05) cleavage (87.0 ± 1.3 and 84.4 ± 1.3), blastocyst production (42.0 ± 2.8 and 43.0 ± 2.0), and apoptosis in blastocysts (20.7 ± 2.0b and 18.9 ± 2.1b), respectively, for control and PES Day 4 groups. Independent of FCS withdrawal or PES addition to culture medium, the in vivo control group presented the lowest apoptosis rate (6.3 ± 1.1a). Therefore, increasing FCS concentration augmented embryo development and reduced blastocyst quality. However, the addition of 2.5% of FCS in the culture medium increased the embryo development without the reduction of blastocyst quality. Moreover, the PES supplementation from Day 4 did not affect embryo development and blastocyst quality. São Paulo Research Foundation – FAPESP.

The quantitative requirements of human diploid cells (strain MRC-5) for amino acids, vitamins and serum

1975 ◽  
Vol 17 (3) ◽  
pp. 397-411
Author(s):  
K. Lambert ◽  
S.J. Pirt
Keyword(s):  
Amino Acids ◽  
Growth Factor ◽  
Inorganic Compounds ◽  
Calf Serum ◽  
Essential Amino Acids ◽  
Defined Medium ◽  
Cell Populations ◽  
Maximum Cell ◽  
Human Diploid Cells ◽  
Diploid Cells

The uptakes of all essential amino acids, vitamins (except riboflavin), glucose and serum during growth of human diploid cells (MRC-5) were determined. The amino acid uptakes varied considerably with the conditions of culture. The glucose requirement is several times greater than that for mouse LS or human HeLa cells. These analytical results were used to modify the medium so as to ensure that an excess of all defined medium constituents was present and pH was not limiting during study of the serum requirements. It was then found that maximum cell populations were directly proportional to the serum concentration. Hence the growth was limited by the supply of an unknown growth factor in serum. The serum growth factor was not replaced by a mixture of over 60 vitamins, co-enzymes, hormones and other organic and inorganic compounds considered to be possible growth factors, although this mixture did not lower the growth rate and somewhat (22%) increased the yield from the serum growth factor. The unit of serum growth factor is precisely defined in terms of the amount in a standard batch of calf serum. This standard contains 10 units/ml whereas the other batch of serum used contained only 5 units/ml.

Growth of human diploid cells (strain MRC-5) in defined medium; replacement of serum by a fraction of serum ultrafiltrate

1979 ◽  
Vol 35 (1) ◽  
pp. 381-392
Author(s):  
K. Lambert ◽  
S.J. Pirt
Keyword(s):  
Amino Acids ◽  
Active Material ◽  
Maximum Yield ◽  
Calf Serum ◽  
Defined Medium ◽  
Cell Yield ◽  
Maximum Cell ◽  
Macromolecular Component ◽  
Human Diploid Cells ◽  
Diploid Cells

A calf serum ultrafiltrate fraction permitted growth for at least 3.5 generations, including one subculture, of MRC-5 cells in defined medium in the absence of whole serum. The active material has a molecular weight of 10 000 Daltons or less. This suggests that there may be no requirement for a large macromolecular component of serum. The ultrafiltrate was assayed by maximum cell yield from a serum-limited inoculum in a defined medium containing non-limiting amounts of vitamins, amino acids, glucose, a 68-component supplement, iron and methylcellulose. The levels of vitamins, amino acids and glucose were based on quantitative measurements of uptake and the levels of the other components by minimum amount required for maximum yield in defined medium without ultrafiltrate or serum. With excess ultrafiltrate maximum cell yield was limited by the defined part of the medium, probably the supplement. The cell doubling time in defined medium with ultrafiltrate fractions was 70 h compared with 27 h in the medium with serum. Excess ultrafiltrate did not inhibit growth. The lowered growth rate is attributed to a nutritional deficiency in the supplement.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

294 USE OF FORSKOLIN TO PRODUCE IN VITRO BOVINE EMBRYOS UNDER TWO CONCENTRATIONS OF FETAL CALF SERUM

2010 ◽  
Vol 22 (1) ◽  
pp. 303
Author(s):  
D. M. Paschoal ◽  
M. J. Sudano ◽  
L. C. O. Magalhães ◽  
L. F. Crocomo ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Fetal Calf Serum ◽  
Formation Rate ◽  
Calf Serum ◽  
Basic Medium ◽  
Intracellular Camp ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Viability ◽  
High Concentration

The increased storage of lipid granules in in vitro-produced (IVP) bovine embryos seems to be related to the presence and concentration of fetal calf serum (FCS) during culture. The presence of high concentration of lipids on embryos reduces their viability after cryopreservation, which has been one of the main obstacles for the success of vitrification of IVP bovine embryos (Moore et al. 2007 Theriogenology 68, 1316-1325). The present experiment aimed to induce cytoplasmic lipolysis in IVP bovine embryos using forskolin (Sigma-Aldrich, St. Louis, MO, USA), which raises the levels of intracellular cAMP (Seamon et al. 1981 Proc. Natl. Acad. Sci. USA, 78, 3363-3367). Nelore oocytes were matured in TCM-199 + 10% FCS, FSH, and LH in 5% CO2 in air atmosphere, at 38.5°C. After 24 h of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions. Presumptive zygotes were cultured in 2 concentrations of FCS: Control 0% (SOFaa + 5 mg mL-1 BSA; basic medium, BM), and Control 2.5% (BM supplemented with 2.5% FCS). On Day 6 of culture embryos were divided into 2 additional treatments: Forskolin 0% (BM + 10 μM forskolin; and Forskolin 2.5% (BM supplemented with 2.5% FCS and 10 μM forskolin). All embryos were cultured in a 5% CO2, 5%O2, and 90% N2 atmosphere at 38.5°C for 7 days, when blastocyst formation rate was evaluated. Embryo viability was also checked by staining the embryos with Hoechst 33342 and propidium iodide. Data were analyzed by ANOVA followed by Tukey’s test, using a 5% significance level. No statistical differences were observed among treatments on cleavage rates, evaluated on Day 3 of culture, or on blastocyst formation rates. Although no statistical differences was observed between treatments on percentage of viable cells, embryos cultured with 0% FCS, independently of the presence of forskolin, presented significantly more damaged cells than embryos cultured with 2.5% FCS (P < 0.05). The results indicate that the presence of FCS is important to reduce degeneration of blastomeres during culture. Moreover, the presence of forskolin on Day 6 of culture did not influence embryo development, indicating that this drug could be a good alternative to reduce embryo lipid content in bovine IVP embryos produced in presence of FCS. Table 1.Effect of fetal calf serum and forskolin on embryo culture Acknowledgments: FAPESP 07/53505-1.

109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 154 ◽  
Author(s):  
M. Barcelo-Fimbres ◽  
G. E. Seidel
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Embryonic Development ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Nile Red ◽  
Essential Amino Acids

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P > 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P < 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P < 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P < 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P < 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos. Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Daniela Martins Paschoal ◽  
Mateus José Sudano ◽  
Midyan Daroz Guastali ◽  
Rosiára Rosária Dias Maziero ◽  
Letícia Ferrari Crocomo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Similar Rate ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Vitrification Solution ◽  
Oviductal Fluid

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

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