• Korean Journal of Fisheries and Aquatic Sciences
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• v.51 no.1
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• pp.42-46
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• 2018

Fetal bovine serum (FBS) is essential for cell culture and is used in the determination of infectivity titer and propagation of viruses. To clarify the effects of FBS on the propagation of viral haemorrhagic septicaemia virus (VHSV), which is a causative agent of mass mortalities of olive flounder Paralichthys olivaceus in Korea, VHSV was inoculated into an EPC (epithelioma papulosum cyprinid) cell line supplemented with MEMs (minimal essential medium) with FBS concentrations of 0%, 2%, 5%, and 10% (MEM0, MEM2, MEM5, and MEM10), respectively, and infectivity titers were compared. Cytopathic effects were observed in all experimental groups at 2 days post virus inoculation (dpi) and all cells were detached from cell culture flasks at 7 dpi. Infectivity titers increased to 3 dpi, persisted to 7 dpi, and decreased when cells were detached. The titer of VHSV in EPC cells in MEM0 was the lowest while those in the other experimental groups showed similar levels. In conclusion, 2% (v/v) of FBS was sufficient to propagate VHSV in EPC cells and the withdrawal of VHSV from cell culture flasks should be performed before cell detachment.

• BMB Reports
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• v.28 no.6
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• pp.515-522
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• 1995

The effects of the thyroid hormone ($T_3$) on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were evaluated in a baby hamster kidney cell line, C100. The cells cultured in MEM were supplemented with 10% thyroid hormone-depleted fetal bovine serum (THDS-MEM) and had a 82.5% lower level of HMG-CoA reductase activity than the cells grown in a medium supplemented with fetal bovine serum (FBS-MEM). When $T_3$ was supplemented to THDS-MEM, the reduction of the reductase activity was blocked in a dose-dependent manner. In the cells grown in THDS-MEM containing $T_3$ at a concentration of $10^{-6}$ M, the level of HMG-CoA reductase activity was 91.8% relative to the cells grown in FBS-MEM. These changes in HMG-CoA reductase activity seemed to be at least partly due to the changes of HMG-CoA reductase mRNA levels. The level of HMG-CoA reductase mRNA in cells incubated in THDS-MEM decreased to 76.2% relative to the cells grown in FBS-MEM, while the level of reductase mRNA in cells incubated in THDS-MEM containing $T_3$ at a concentration of $10^{-6}$ M increased to 243.4% relative to the cells grown in FBS-MEM. The increase of HMG-CoA reductase mRNA level after $T_3$ treatment may have been due to the increased stability of reductase mRNA, because the transcriptional rate of the reductase gene did not change significantly in the presence or absence of $T_3$. These results indicate that $T_3$ stabilizes HMG-CoA reductase mRNA at the posttranscriptional level and regulates HMG-CoA reductase activity in a dose-dependent manner.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.7-12
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• 2004

This study was performed to establish the effective culture condition for the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were cultured and passaged two times before use. A single cell was seeded in 96-well plates, cultured in medium supplemented with different concentrations of FBS, catalase or $\beta$-mercaptoethanol ($\beta$ME), and classified by cell size and morphology. Cells were passaged two times into 4-well dish before freezing. The establishment efficiencies were not different among different concentrations of FBS (0.3 to 5.1%). However, population doubling time (PDT) was significantly decreased by increasing the FBS concentration (P＜0.05). The establishment efficiency of $\beta$ME-added group (10.4%) was significantly higher than those of catalase-added and control groups (3.5%, and 3.5%, respectively, p＜0.05), and PDT was significantly decreased (23.6 vs 28.1, and 25.5 h, respectively, p＜0.05). However, catalase did not show a positive effect on the establishment efficiency. Cell size and morphology did not affect the establishment efficiency and PDT of clonal lines. The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 30% FBS and $\beta$ME.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.191-199
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• 2004

This study was conducted to examine in vitro development of porcine embryos constructed by the microinjection of cultured fetal fibroblast cells into porcine oocytes matured in vitro. Single fetal donor cells were deposited into the perivitelline space of enucleated oocytes, followed by electrical fusion and activation. Activated embryos were cultured in NCSU-23 medium supplemented with 5％ FBS, at 38.5$^{\circ}C$ for 6 to 8 days in 5％ $CO_2$ and air. In experiment 1, fusion rates of nuclear transfer embryos did not differ for fetal fibroblast cells incubated in 5％ FBS + NCSU-23 or 5％ FBS + TL Heaps medium, nor did fusion rates of donor cells differ between 1-8 hr incubation durations. Fusion rates for the four treatment subclasses ranged from 72.1％ to 78.0％. In experiment 2, Pre-synchronization in medium containing 0.1 $\mu\textrm{g}$/m Hoechst 33342 an increase from 0 and 8 versus 15 h culture an increased percentage of porcine fibroblast cells in G2/M at the end of the synchronization period (12.4％, 17.5％ and 47.6％). Neither an increase in the concentration of H 33342 (0.2-1.6 $\mu\textrm{g}$/$m\ell$) nor a longer exposure time (12h, 18h and 24h) increased the proportion of porcine G2/M fibroblasts. In experiment 3, fusion rates did not differ significantly far nuclear transfer embryos constructed using donor cells cultured in 5％ FBS + NCSU-23 medium for 1-2, 6-8 or 12-14 days (60.0％, 73.3％ and 62.5％), respectively. The cleavage rate for nuclear transplant embryos using fetal fibroblast cells cultured for 1-2 days was 44.0％, significantly less than 56.7％ and 50.0％. for 6-8 or 12-14 days duration of culture, respectively. In experiment 4, the proportions of nuclear transfer embryos that developed to the $\geq$2 cell and to the blastocyst stage were not affected significantly by culture medium (5％ FBS + NCSU-23 or 5％ FBS + TL-Heaps) or by $O_2$ concentration of the culture (5％ vs 10％). Rates of development to the $\geq$2 cell stage ranged from 65.9％ to 70.1％, and development rates to the blastocyst stage ranged from 9.8％ to 12.5％ for the four treatment subclasses. Developmental rate was highest for embryos cultured in 5％ FBS + NCSU-23 under a gas atmosphere of 5％ $O_2$ in air.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.5
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• pp.615-621
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• 2002

We investigated the effects of the mitogen supplements of 3 types, pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (ConA), to a whole blood culture system on the number of metaphase spreads obtained in perinatal bovine chromosome analysis. In addition, the supplementation of ${\beta}$-mercaptoethanol (${\beta}$-ME) and FBS was examined in such system. Significant differences (p<0.05) were seen in the number of metaphase spreads with PHA stimulation compared to both PWM and ConA stimulation. When examined the effects of ${\beta}$-ME supplementation, the number of metaphase spreads was significantly (p<0.05) increased at $30{\mu}M$ ${\beta}$-ME compared to control. When evaluated FBS supplementation during PWM stimulation, no significant effect of the supplementation was found. Finally, the effects of the cortisol concentration (10-20, 20-30 and >30 ng/ml) of the blood samples were examined. There was no significant effect of cortisol concentration (p>0.05) among these 3 cortisol concentration groups. The mean percentages of normal metaphase plates (2n=60) from each calf 1) with ${\beta}$-ME, 2) without ${\beta}$-ME and 3) with FBS stimulated with PWM were not significantly different (p>0.05). In conclusion, these findings may be useful in cytogenetic screening programs for not only perinatal calves but also for mature cattle.

• Journal of Veterinary Science
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• v.24 no.1
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• pp.4.1-4.16
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• 2023

Background: In vitro culture of preantral follicles is a promising technology for fertility preservation. Objectives: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation. Methods: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E₂) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization. Results: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro. Conclusions: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.6
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• pp.807-812
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• 2007

In order to enhance the flavor and storage stability of meat sauce, the manufacturing process of fermented beef-rib sauce was developed in a two-step process. The fermented sauce base was manufactured with Zygosaccharomyces rouxii Y-80 yeast cultivation in raw sauce ingredients for 3 days at $25^{\circ}C$. The fermented beef-rib sauce (FBS) was produced by mixing fermented sauce base with side ingredients. Comparison of the physicochemical and sensory properties with non-fermented beef-rib sauce (NFBS) revealed that the content of ethanol and volatile flavor compounds were higher in FBS; also, the result of sensory evaluation showed that FBS obtained excellent scores for overall taste. To determine the storage stability, FBS and NFBS were incubated at $25^{\circ}C$ for 7 days. The extent of decrease of pH and increase of titratable acidity in NFBS were faster than FBS. After 7 days, ethanol concentrations in FBS and NFBS were 3.77% and 2.04%, respectively. Therefore, based on these results, it can be suggested that storage stability of FBS is superior.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.532-537
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• 2008

In order to investigate the factors affecting the culture of mouse preantral follicles in vitro, we examined the effect of culture media, protein supplements, and culture period on their growth. The oocyte diameter (initial size: $55.6{\pm}2.5{\mu}m$) was progressively increased during culture, and the maximum size ($72.0{\pm}2.4{\mu}m$) was reached on day 10 of the in vitro culture. The chromatin configuration in the germinal vesicle (GV) oocyte progressively shifted from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN). On day 10 of the culture, most of the oocytes progressed to the SN pattern. The survival and metaphase II rates of the oocytes in alpha-minimal essential medium (alpha-MEM) were significantly higher (p<0.05) than those in Waymouth and tissue culture medium (TCM)-199. As a protein source, fetal bovine serum (FBS) was more suitable for the culture of mouse preantral follicles as compared to human follicular fluid (hFF) and bovine serum albumin (BSA); the optimal concentration of FBS was 5%. These results suggest that in a culture of mouse preantral follicles, alpha-MEM and 5% FBS are an optimal medium and a protein source, respectively; further, the 10 days of culture is required for the complete growth of oocytes in this culture system.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.6
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• pp.862-870
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• 2014

This study was conducted to investigate the effects of brown seaweed (Undaria pinnatifida) by-product and seaweed fusiforme (Hizikia fusiformis) by-product supplementation on growth performance and blood profiles including serum immunoglobulin (Ig) in broilers. Fermentation of seaweeds was conducted by Bacillus subtilis and Aspergillus oryzae. In a 5-wk feeding trial, 750 one-d-old broiler chicks were divided into 5 groups, and were assigned to the control diet or experimental diets including control+0.5% brown seaweed (BS) by-product, control+0.5% seaweed fusiforme (SF) by-product, control+0.5% fermented brown seaweed (FBS) by-product, and control+0.5% fermented seaweed fusiforme (FSF) by-product. As a consequence, body weight gain (BWG) and gain:feed of seaweed by-product groups were clearly higher, when compared to those of control diet group from d 18 to 35 and the entire experimental period (p<0.05). In mortality rate, seaweed by-product groups were significantly lower when compared to control diet group during entire experimental period (p<0.05). However, Feed Intake of experimental diets group was not different from that of the control group during the entire experimental period. Whereas, Feed Intake of fermented seaweed by-product groups was lower than that of non-fermented seaweed groups (p<0.05). Total organ weights, lipids, and glutamic oxalacetic transaminase (GOT) of all treatment groups were not different from those of control group. However, glutamic pyruvate transaminase (GPT) of all treatment groups was higher than that of control group at d 17 (p<0.05). In case of serum Igs concentration, the concentration of IgA antibody in BS, SF, FSF treatment groups was significantly higher than in control group at d 35 (p<0.01). IgA concentration in FBS supplementation groups was negligibly decreased when compared to the control group. IgM concentration in the serums of all treatment groups was significantly higher than in control group (p<0.05) and in fermented seaweed by-product groups were much higher than in non-fermented seaweed groups (p<0.05). On the other hand, IgG concentrations in all treatment groups were lower than in control group (p<0.05). Taken together, our results suggest that by-product dietary supplementation of BS, SF, FBS, and FSF in poultry may provide positive effects of growth performance and immune response.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.