Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined bovine embryo culture medium (BECM) or polyvinylalcohol (PVA)-free BECM supplemented with 10% heat-treated or unheated fetal calf serum (FCS). When PVA in BECM was replaced with FCS 8 h postinsemination, the proportion of >/=-cell-stage embryos 48 h postinsemination was significantly lower in heat-treated FCS (74%) than in PVA (91%) medium, but the value (84%) in unheated FCS was not different from the values in PVA and heat-treated FCS. However, the addition of either heat-treated FCS or unheated FCS to PVA-free BECM did not depress or increase the proportions (63 to 70%) of >/=-cell-stage embryos 96 h postinsemination and stimulated blastocyst formation 144 (21 to 28%) and 192 (47 to 54%) h postinsemination. Blastocyst formation was also stimulated when heattreated FCS or unheated FCS was added to PVA-free BECM 120 h postinsemination. The mean cell number of blastocysts developed in PVA-free BECM with heat-treated FCS (150 cells) or unheated FCS (152 cells) significantly increased compared with that (119 cells) developed without FCS. These results indicate that, although BECM can support bovine embryo development, a high proportion (about 50%) of morphologically normal blastocysts can be produced from oocytes matured and fertilized in vitro when they are cultured in PVA-free BECM containing FCS.     

Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer

Supplementation of synthetic oviduct fluid (SOF) medium plus amino acids and bovine serum albumin (BSA) with either fetal calf serum (FCS) or charcoal-treated FCS (CT-FCS) from Day 5 of development was investigated to determine if either in vitro or post-transfer development was altered. Development to the compact morula stage or beyond was similar for all 3 treatments. However, blastocyst development at Day 7 was accelerated when serum was added to the medium (21.6, 40.1 and 39.4% blastocysts from cleaved embryos for BSA, FCS and CT-FCS, respectively; P < 0.01), but cell number of the resulting embryos was unaffected. Furthermore, addition of CT-FCS decreased the between replicate variation in embryo development and produced more Grade 1 and 2 quality embryos (25.8%) than BSA supplementation (18.1%; P < 0.05). The transfer of Grade 1 and 2 embryos at Day 7 following culture resulted in similar pregnancy and embryo survival rates for the 3 treatments, with a tendency for lower embryo survival of embryos cultured in FCS (embryo survival at Day 50 = 37.7% vs 53.3% and 57.6% for FCS, BSA and CT-FCS, respectively; P = 0.1). Significant fetal loss from Day 50 to term occurred within all 3 treatments. There were no birth weight differences for calves amongst the 3 culture treatments; however, one of the sires produced calves that were significantly heavier than expected, suggesting a possible sire-by-embryo interaction. These results demonstrate that addition of FCS may promote blastocyst development; however, there was also a tendency for lower embryo survival. Thus charcoal treatment of FCS is recommended, because it decreases variability in embryo development between runs and results in embryo survival rates to term similar to that BSA-supplemented media.     

The ability of fetal calf serum, new-born calf serum and normal steer serum to promote the in vitro development of bovine morulae

The objective of this study was to compare fetal calf serum, new-born calf serum and normal steer serum as medium supplements in the development of bovine morulae in vitro . Bovine morulae were cultured in Hams F-10 tissue culture medium (HF-10) supplemented with 5% or 10% (v/v) fetal calf serum (FCS), new-born calf serum (NBCS) or normal steer serum (NSS). Embryos were recovered at slaughter from mixed bred donor cows of mixed breeding following estrus synchronization with prostaglandin and superovulation with follicle stimulating hormone. A total of 88 morulae were recovered, washed in HF-10 + 1% Bovine Serum Albumin and randomly assigned to treatments. Embryos were cultured in microdrops of medium under paraffin oil at 37 degrees C in a 5% CO(2) humidified atmosphere. Observations for stage of development were made every 24 hours. In vitro development was analyzed by assigning to each embryo a value of 0-5 based on the most advanced stage reached (0= no development, 5= development to a hatched blastocyst). Analysis of variance of these data revealed a significant treatment effect (P<.001) while no level effect or treatment x level interaction was apparent. Comparison of treatment means by Duncans new mulitple range test showed that NSS was superior to NBCS (P<.05) which was in turn superior to FCS (P<.05) as supplements of HF-10 in promoting the in vitro development of bovine morulae.     

Culture of chondrocytes in medium supplemented with fetal calf serum or a serum substitute: Ultroser G

Fetal calf serum and a serum substitute, Ultroser G, were compared for their effects on the growth curves, clonal growth and cell cycle progression of rabbit chondrocytes in primary culture and during at least three cell passages and included a screen for the maintenance of cartilage-like differentiation i.e. the presence of type II collagen. Proliferation was also compared with another serum substitute, Nu-Serum. Ultroser G is shown to be equivalent to fetal calf serum as far as chondrocyte proliferation is concerned, clonal growth is improved and biosynthesis of type II collagen is maintained in primary culture.     

Asparaginase and glutaminase activities in culture media containing dialyzed fetal calf serum

Summary Degradation of asparagine in media containing dialyzed fetal calf serum has been shown to be the result of asparaginase, which is active even at 4°C. The asparaginase activity of undialyzed fetal calf serum is only one-tenth of that found in the dialyzed serum. Glutaminase activity was also demonstrated in the medium containing dialyzed fetal calf serum. Minor changes in some other amino acids were also observed. This work was supported in part by Grant CA02568, National Cancer Institute, National Institutes of Health.     

Growth and development of rabbit oocytes in vitro: Effect of fetal bovine serum concentration on culture medium

The objective was to develop a culture system that produced blastocyst stage embryos from rabbit oocytes grown in vitro. Two experiments were performed. First, various concentrations of fetal bovine serum (FBS, 0, 0.05, 0.5 and 5%) were used in the culture medium for in vitro growth (IVG) of oocytes recovered from follicles 200 to 299 μm in diameter. Intracytoplasmic sperm injection (ICSI) was performed on mature oocytes obtained after IVG for 8 days and in vitro maturation for 14 to 16 h. Rates of survival and pronuclear formation after ICSI were higher for oocytes grown in a medium with 0.05% FBS compared to oocytes grown in a medium lacking FBS (97.6 vs. 76.9%, 97.5 vs. 70%, P < 0.1). The rate of development to the blastocyst stage was also higher in the medium containing 0.05% FBS than in the medium lacking FBS (9.5 vs. 17.9%, P < 0.05). Next, using oocytes recovered from follicles 200 to 399 μm in diameter which were cultured in 0.05% FBS, oxygen consumption and the number of cells were analyzed. Blastocysts from oocytes grown in vitro with 0.05% FBS had reduced oxygen consumption and number of cells compared with those from ovulated oocytes (21.66 ± 4.54 × 10¹⁴ vs. 50.19 ± 4.61 × 10¹⁴ mol/sec, 244 ± 25 vs. 398 ± 24, P < 0.05). Rabbit oocytes grown in vitro with 0.05% FBS achieved pregnancy, but pregnancies were not maintained to term. In conclusion, the addition of 0.05% FBS to the culture medium for IVG improved developmental competence of rabbit oocytes grown in vitro.     

A simple and effective method for the removal of trace metal cations from a mammalian culture medium supplemented with 10% fetal calf serum

Direct batch addition of sterile Chelex ion-exchange resin to Dubecco's modified Eagle's medium supplemented with 10% fetal calf serum with gentle stirring removed a very wide variety of trace metal ions from the medium to varying extents dependent upon Chelex content (between 0.01 and 4% w/v), exposure time (between 5 min and 10 days) and temperature 4, 25 and 37 °C). Prolonged treatment (10 days) with 4% w/v Chelex at 4°C reduced the concentration of zinc, strontium, aluminum, copper, manganese, nickel and chromium from 100 to 2.7, 12.1, 7.7, 22.6, 13.0, 14.7 and 53.3% of their original concentrations, respectively. Re-supplementation of the metal depleted medium with a defined cocktail of metals restored the growth potential of the medium which was then capable of supporting growth over at least three subcultures without a decrease in fibroblast cell yield, demonstrating its suitability in cell culture studies on trace metal ions.     

Utilization of whey fractions as a substitute for fetal calf serum in culture media

Summary A dairy by-product was used in cell line cultures as a replacement for fetal calf serum (FCS). Various fractions were prepared from whey. All fractions have the ability to stimulate DNA synthesis in human and mouse 3T3 fibroblast and hybridoma cultures. They also allow hybridoma proliferation in the long term using only 0.5% FCS. In these latter experiments, heated and ultrafiltered fractions were more effective than 10% fetal calf serum.     

Mutual dependence of growth modifying effects of 4-hydroxynonenal and fetal calf serum in vitro

Recently, the hypothesis has been put forward that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, contributes to the mechanism of oxygen toxicity and to the selective pressure exerted by exposure to hyperoxia. Here it has been studied whether HNE itself is involved in mechanisms that convey increased resistance of the cells to the toxicity of HNE. The following four cell lines, different in their basic biological features, were used: nonmalignant Chinese hamster lung fibroblasts V79 (established cell line), human carcinoma HeLa (established cell line), pigmented murine melanoma B16f10 (primary culture), and amelanotic murine melanoma B16BL6 (primary culture). The cells were pretreated in vitro with a toxic dose of HNE (50 μM), and afterwards the effects of a second exposure to the same dose of HNE on ³H-thymidine incorporation was examined. Cells were cultured in the absence and in the presence of fetal calf serum (FCS), because it had been shown that a growth modifying effect of HNE depends on an unknown serum factor. The results showed that, regardless of the type of cells, preculturing them with 50 μM HNE in the presence of serum changed the reactivity of the cells to added serum as well as to additional HNE treatment. Thus, HNE precultured cells incorporated less ³H-thymidine in the presence of serum than if cultured under serum-free conditions. On the other hand, HNE precultured cells became less sensitive to further HNE treatment, but only if cultured in the presence of serum. It was concluded that a toxic dose of HNE renders surviving cells more resistent to oxidative stress, possibly by forming a bioactive conjugate with a serum peptide/protein. It is supposed that the same humoral factor might be responsible for the growth modifying effects of high doses of HNE as well as for the growth inhibition in the presence of serum observed for HNE precultured cells.     

In vitro lead-induced cell toxicity and cytoprotective activity of fetal calf serum in human fibroblasts

The underlying mechanisms by which lead ions produce their deleterious effects prior to the onset of clinical symptoms are incompletely understood. This study aimed to assess lead-induced cell toxicity mechanisms by focusing on the effects of the metal on cell growth, DNA synthesis, cellular ATP, intracellular hexosaminidase activity and lysosomal function, and examine the possible cytoprotective role of fetal calf serum (FCS). Several human dermal cultured fibroblast lines were exposed to Pb (400 M) for 1–6 days with 2, 5, and 10% FCS. The earliest toxic effect of Pb was significant inhibition of DNA synthesis after 24 h direct exposure; this harmful effect was not progressive during the first 3 days, but worsened clearly on the 4th day regardless of the FCS concentration. A time-dependent depletion of intracellular ATP content was also caused by ionic lead, thereby compromising the cell energy charge which precedes cell death. Fibroblast growth was progressively and significantly inhibited from day 2 onwards; the greatest noxious effect was observed in the presence of 2% FCS: 49% reduction in cell proliferation after 5 days. Lead salts produced loss of cell adhesion to the culture dish which worsened from the 2nd day and was more pronounced when FCS in growth medium was decreased. Toxic actions on lysosomal membrane integrity provoked a decrease in neutral red uptake (NRU) which was exposure time-dependent and more marked with 2% FCS. In contrast, increased relative NRU (to 20% at 4 days), suggestive of endocytosis-induced lysosome enlargement, was observed in Pb-exposed cells. Intracellular hexosaminidase activity was not negatively affected until 5 days after exposure to Pb salts. FCS had a significant cytoprotective effect on Pb-induced toxicity.     

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.     

Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes

Bovine serum albumin (BSA) and fetal calf serum (FCS) were evaluated as protein supplements for in vitro maturation and fertilization of oocytes from cows and hamsters. BSA and low doses of FCS (0.1 or 1.0%) did not support viability or maturation of the cumulus-oocyte complex as well as higher doses of FCS (5, 10, or 20%) for either species. BSA failed to support cumulus expansion for bovine or hamster cumulus-oocyte complexes. All doses of FCS examined supported cumulus expansion in bovine cumulus-oocyte complexes, whereas the hamster complexes required at least 1.0% FCS to induce cumulus expansion. The addition of a serum filtrate, Solcoseryl, with BSA improved viability of the cumulus in the bovine but did not support cumulus expansion or completion of Meiosis I in bovine complexes. In vitro fertilization could be accomplished in media containing FCS by increasing the heparin concentration in the bovine system or reducing FCS for the hamster system. Polyspermy was increased when FCS was the protein supplement. It is not known whether this is an interaction of FCS with the sperm or oocyte. In conclusion, FCS was found necessary for follicle-stimulating-hormone (FSH)-induced cumulus expansion. It also improved cumulus cell viability and completion of the first meiotic division in complexes of both species compared with BSA.     

Assessment of culture medium without commercial ammonium nitrate for in vitro culture of industrially important plant species

Plant Cell, Tissue and Organ Culture (PCTOC) - Ammonium nitrate (AN) is one of the major nitrogen sources of Murashige and Skoog (MS) medium. It is prohibited in various countries, including India...     

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture

We studied the effect of fetal calf serum and serum protein fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine β-globulins reduced this transfer, although to a lesser extent than whole serum. α-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A γ-globulin fraction failed to exhibit any effect on the uptake of [¹⁴C]phosphatidylcholine by the cells.All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles preincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine to parenchymal cells.These observations were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of the liposomal surface membrane as a result of the binding of certain serum proteins. On the other     

Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation

This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.     

Insect cell culture medium supplementation with fetal bovine serum and bovine serum albumin: effects on baculovirus adsorption and infection kinetics

The effects of insect cell culture medium supplementation with FBS were investigated. BSA was found to be the factor responsible for the increased baculovirus infection rate of FBS-supplemented cultures in a concentration-dependent form up to 25 g L(-)(1). Lower rates of baculovirus binding to cells were observed with FBS- and BSA-supplemented cultures compared with infections carried out in serum-free media. Virus attachment constants were found to depend on medium matrix composition. An efficiency factor dependent on the medium matrix composition was introduced to account for these effects, and a mathematical model was developed to describe the virus-cell interactions. It was shown that BSA acts by minimizing the nonspecific virus binding leading to an increased cell infection rate. Cell specific Porcine parvovirusvirus-like particles (PPV-VLPs) expression was unaffected by medium supplementation pointing out that BSA and/or FBS affects mainly the initial phase of the baculovirus infection cycle. Implications for process definition are discussed.