Enhancement of nitric oxide solubility using Fe(II)EDTA and its removal by green algae <Emphasis Type="Italic">Scenedesmus</Emphasis> sp.

A photoautotrophic cultivation of green algae Scenedesmus cells was used for the removal of nitric oxide (NO) from a model flue gas mixture. In an attempt to improve the solubility of NO in the culture broth, the addition of Fe(II)EDTA to the cultivation was investigated. The addition of Fe(II)EDTA greatly enhanced NO-dissolution in the culture broth and subsequently increased the algal-uptake of NO. NO was assimilated as a source of nitrogen for the growth of Scenedesmus cells since there was a steady increase in cell density with no other nitrogen source in the culture except the incoming NO. 40–45% of NO removal was maintained for more than 12 days with the addition of 5 mM Fe(II)EDTA in a 1-L air-lift type photobioreactor system fed with 300 ppm of NO gas at a rate of 0.3 wm. However, the NO-dissolution-enhancing capacity of Fe(II)EDTA did not reach its full potential due to its oxidation to Fe(III)EDTA, possibly induced by molecular oxygen that evolved from algal photosynthesis, and subsequent loss of chelating capabilities.     

Continuous Cr(VI) removal by Scenedesmus incrassatulus in an airlift photobioreactor

Cr(VI) removal by Scenedesmus incrassatulus was characterized in a continuous culture system using a split-cylinder internal-loop airlift photobioreactor fed continuously with a synthetic effluent containing 1.0 mg Cr(VI) l^?1 at dilution rate (D) of 0.3 d^?1. At steady state, there was a small increase (6%) on the dry biomass (DB) concentration of Cr(VI)-treated cultures compared with the control culture. 1.0 mg Cr(VI) l^?1 reduced the photosynthetic pigments content and altered the cellular morphology, the gain in dry weight was not affected. At steady state, Cr(VI) removal efficiency was 43.5 ± 1.0% and Cr(VI) uptake was 1.7 ± 0.1 mg Cr(VI) g^?1 DB. The system reached a specific metal removal rate of 458 μg Cr(VI) g^?1 DB d^?1, and a volumetric removal rate of 132 μg Cr(VI) l^?1 d^?1.     

Bioreactor leaching of uranium from a low grade Indian silicate ore

Bio-leaching studies were carried out in a 2 L bioreactor- BIOSTAT-B^® equipped with a PLC based controller at 20–40% (w/v) pulp density using enriched culture of A.ferrooxidans for Turamdih uranium ore (Jharkhand, India). With the enriched culture of A.ferrooxidans adapted on Fe(II) at pH 2.0, 35 °C and 20% (w/v) pulp density, a 98.3% uranium recovery was recorded in 14 days. The leaching of uranium in the bioreactor improved the dissolution rate by reducing the time from 40 days in shake flask as per our earlier studies to 14 days. While investigating the importance of biogenic Fe(III) in the bio-leaching process a maximum recovery of 84.7% U₃O₈ was observed at pH 2.0 and 20% (w/v) pulp density in 10 h as compared to the uranium leaching of 38.3% in the control experiments. On raising the pulp density to 30%, uranium bio-recovery increased to 87.6% in 10 h at pH 2.0 with <76 μm size material. This showed a distinct advantage because of better mixing of slurry in the bioreactor with auto-controlled conditions that improved the kinetics.     

Arsenic accumulation by aquatic macrophyte coontail (Ceratophyllum demersum L.) exposed to arsenite,and the effect of iron on the uptake of arsenite and arsenate

In this study, the aquatic macrophyte Ceratophyllum demersum L. (coontail or hornwort) was tested for its efficiency of arsenic (As) uptake under laboratory conditions. Our results revealed that the solution pH had a significant effect on As accumulation by C. demersum (p < 0.001). The accumulation was highest at pH 5 and decreased as pH values increased. Plants that were exposed to various concentrations of arsenite (As(III)) for 24 and 48 h, exhibited tolerance and toxic responses, respectively. As accumulation by C. demersum depended on the concentrations of As(III) and the duration of exposure (p < 0.001). At 40 μM after 24 h, plants accumulated 227.5 μg As g⁻¹ dw and showed no visible symptoms of toxicity. However, after 48 h, As level reached 302.4 μg g⁻¹ dw and biomass production decreased significantly. Toxic effects were evident by plant necrosis and negative biomass production, leading to a decrease in the amount of accumulated As. Also, the addition of iron (Fe) into the nutrient solutions (0.18 mM) had contrasting effects on the uptake of 2 As species – the uptake of As(III) was enhanced by the presence of Fe, but the uptake of arsenate (As(V)) was considerably inhibited.     

Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes,antioxidants, and N-acetyl chitooligosaccharides

A chitinase (CHT), a chitosanase (CHS) and a protease (PRO) were purified from the culture supernatant of Serratia sp. TKU020 with squid pen as the sole carbon/nitrogen source. The molecular masses of CHT, CHS and PRO determined by SDS-PAGE were approximately 65 kDa, 55 kDa and 55 kDa, respectively. CHT and CHS were inhibited by Mn²⁺, EDTA and PRO was inhibited by Mg²⁺, EDTA. The antioxidant activity of TKU020 culture supernatant was 78% (DPPH scavenging ability). N-Acetylglucosamine (GlcNAc) and N-acetyl chitobiose (GlcNAc)₂ were also produced from the culture supernatant by using TKU020 strain fermentation. The maximum production of GlcNAc and (GlcNAc)₂ was 1.3 mg/mL and 2.7 mg/mL, respectively, after 4 days of fermentation. With this method, we have shown that squid pen wastes can be utilized and it is effective in the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides, facilitating its potential use in industrial applications and functional foods.     

Effect of partial replacement of concentrates with jackfruit (Artocarpus heterophyllus) leaves on growth performance of kids grazing on native pasture of Tripura,India

Fifteen Black Bengal kids of about 3 months of age and body weight ranging from 3.8 to 4.9 kg were randomly distributed into three groups of five. Kids grazed native pasture 8 h/d. The kids in group I received supplementary concentrate (maize 35%, mustard cake 32%, rice bran 30%, mineral mixture 2% and common salt 1%) at approximately 2% of BW. However, 25 and 50% of the concentrate was replaced with jackfruit leaves for groups II and III, respectively. Total dry matter intake (DMI) was significantly higher in groups II and III than for group I due to greater forage consumption. Digestibility of CP (P < 0.05) decreased and that of NDF increased (P < 0.01) with increasing level of jackfruit leaves in the diet. Digestibility of ADF (P < 0.01), hemi cellulose (P < 0.05) and cellulose (P < 0.01) was higher in groups II and III in comparison to group I. Ruminal pH and TVFA concentration were not significantly different among the groups; however, rumen ammonia-N concentration decreased (P < 0.01) with increased level of jackfruit leaves in the diet. Similarly, plasma urea nitrogen and blood glucose levels were also reduced (P < 0.05) with increasing level of jackfruit leaves in the diet Average daily gain (ADG) was 47.33, 45.11 and 35.56 g/d in groups I, II and III, respectively. ADG and DMI/kg gain were not adversely affected when the level of replacement was restricted to 25%; however, at the 50% of replacement both parameters were adversely affected (P < 0.05). From the results of this experiment, it was concluded that jackfruit leaves might replace 25% of the supplemental concentrate for growing kids grazing in native pasture of northeast India.     

Quercetin is a substrate for the transmembrane oxidoreductase Dcytb

Duodenal cytochrome b (Dcytb) is a transmembrane oxidoreductase protein found in apical membranes of duodenal enterocytes, as well as human erythrocytes, with the capacity to transport electrons donated by cytosolic ascorbate to extracellular electron receptors such as Fe(III), dehydroascorbate, or molecular O₂. We have investigated the capacity of the flavonoid quercetin to act as an electron donor for Dcytb in a manner similar to that of ascorbate by observing the reduction of extracellular Fe(III) to Fe(II) in either Madin–Darby canine kidney (MDCK) cells overexpressing Dcytb (Dcytb⁺) or Dcytb-null MDCK cells. In Dcytb⁺ cells there is a saturable increase in extracellular Fe(III) reduction in response to increasing intracellular quercetin concentrations (K_m = 6.53 ± 1.57 μM), in addition to a small linear response, whereas in Dcytb-null cells there is only a small linear increase in extracellular Fe(III) reduction. No extracellular Fe(III) reduction occurs in Dcytb-null cells when the cells are preloaded with ascorbate. Flavonoids such as quercetin at their physiological concentrations can therefore function as modulators of ferric reductases, enhancing the import of Fe(II) and also providing extracellular reducing potential.     

A new approach for Fe(III)EDTA reduction in NOx scrubber solution using bio-electro reactor

A new process for the removal of NO_x by a combined Fe(II)EDTA absorption and microbial reduction has been demonstrated, in which part of the Fe(II)EDTA will be oxidized by oxygen in the flue gas to form Fe(III)EDTA. In former studies, strain FR-2 has been found to reduce Fe(III)EDTA efficiently. Otherwise, it has been reported that bio-electro reactor could efficiently provide a chance for simultaneous denitrification and metal ion removal. Therefore, a use of bio-electro reactor is suggested to promote the reduction of Fe(III)EDTA by strain FR-2 in this paper. The results showed that the concentration of Fe(III)EDTA decreased rapidly when electric current was applied, and that as the current density rose, the Fe(III)EDTA reduction rate increased while followed by a decrease afterward. The formation of the biofilm on the electrode was observed by ESEM (Environmental Scan Electro-Microscope). In addition, the Fe(III)EDTA reduction rate obviously decreased with the existence of NaNO₂.     

Reduction of Fe(II)EDTA-NO by a newly isolated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain DN-2 in NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> scrubber solution

Biological reduction of nitric oxide (NO) chelated by ferrous ethylenediaminetetraacetate (Fe(II)EDTA) to N₂ is one of the core processes in a chemical absorption–biological reduction integrated technique for nitrogen oxide (NO _x ) removal from flue gases. A new isolate, identified as Pseudomonas sp. DN-2 by 16S rRNA sequence analysis, was able to reduce Fe(II)EDTA-NO. The specific reduction capacity as measured by NO was up to 4.17 mmol g DCW⁻¹ h⁻¹. Strain DN-2 can simultaneously use glucose and Fe(II)EDTA as electron donors for Fe(II)EDTA-NO reduction. Fe(III)EDTA, the oxidation of Fe(II)EDTA by oxygen, can also serve as electron acceptor by strain DN-2. The interdependency between various chemical species, e.g., Fe(II)EDTA-NO, Fe(II)EDTA, or Fe (III)EDTA, was investigated. Though each complex, e.g., Fe(II)EDTA-NO or Fe(III)EDTA, can be reduced by its own dedicated bacterial strain, strain DN-2 capable of reducing Fe(III)EDTA can enhance the regeneration of Fe(II)EDTA, hence can enlarge NO elimination capacity. Additionally, the inhibition of Fe(II)EDTA-NO on the Fe(III)EDTA reduction has been explored previously. Strain DN-2 is probably one of the major contributors for the continual removal of NO _x due to the high Fe(II)EDTA-NO reduction rate and the ability of Fe(III)EDTA reduction.     

Growth and nutrient removal properties of a freshwater microalga Scenedesmus sp. LX1 under different kinds of nitrogen sources

Microalgae have received much attention for the inorganic nutrient removal in tertiary treatment of domestic wastewater. Effect of different kinds of nitrogen sources on the growth and nitrogen/phosphorus removal properties of a newly isolated freshwater microalga, Scenedesmus sp. LX1, from a low-nutrient environment condition was studied and reported in this paper. The order of specific growth rate of the microalga with different nitrogen sources was NH₄-N > urea-N > NO₃-N. With nitrate or urea as nitrogen source, the microalga could grow well and remove both nitrogen and phosphorus efficiently (90% nitrogen and nearly 100% phosphorus were removed). However, with ammonium as the nitrogen source, the maximum algal density was relatively low, and the nitrogen and phosphorus removal efficiencies were as low as 31.1% and 76.4%, respectively. This was caused by the inhibitory effect of algal culture's acid pH due to H⁺ releasing from NH₄⁺ during algal cultivation process.     

Evaluation of in vitro free radical scavenging potential of Streptomyces sp. AM-S1 culture filtrate

The present study was carried out to determine the free radical scavenging potential of culture filtrate of Streptomyces sp. AM-S1. Antioxidant activity of culture filtrate, lyophilized culture filtrate and ethyl acetate extract of Streptomyces sp. AM-S1 was determined by various in vitro assays such as ferric reducing power assay, phosphomolybdenum reduction, DPPH and ABTS radical scavenging activities. The results revealed that the culture filtrate of Streptomyces sp. AM-S1 effectively scavenged DPPH (IC₅₀ 90.2 μl/ml) and ABTS (IC₅₀ 13.2 μl/ml) radicals in a concentration dependent manner. In all the assays, ethyl acetate extract registered higher antioxidant activity when compared with the lyophilized culture filtrate (LCF). In addition, ethyl acetate extract (1123.4 μmole Fe(II)/mg extract) exhibited higher ferric reducing activity than the standard BHA (814.4 μmole Fe(II)/mg extract). Further works are needed on the isolation and identification of antioxidant molecules from the ethyl acetate extract of Streptomyces sp. AM-S1 culture filtrate.     

Earthworm-mediated synthesis of silver nanoparticles: A potent tool against hepatocellular carcinoma,Plasmodium falciparum parasites and malaria mosquitoes

The development of parasites and pathogens resistant to synthetic drugs highlighted the needing of novel, eco-friendly and effective control approaches. Recently, metal nanoparticles have been proposed as highly effective tools towards cancer cells and Plasmodium parasites. In this study, we synthesized silver nanoparticles (EW–AgNP) using Eudrilus eugeniae earthworms as reducing and stabilizing agents. EW–AgNP showed plasmon resonance reduction in UV–vis spectrophotometry, the functional groups involved in the reduction were studied by FTIR spectroscopy, while particle size and shape was analyzed by FESEM. The effect of EW–AgNP on in vitro HepG2 cell proliferation was measured using MTT assays. Apoptosis assessed by flow cytometry showed diminished endurance of HepG2 cells and cytotoxicity in a dose-dependent manner. EW–AgNP were toxic to Anopheles stephensi larvae and pupae, LC₅₀ were 4.8 ppm (I), 5.8 ppm (II), 6.9 ppm (III), 8.5 ppm (IV), and 15.5 ppm (pupae). The antiplasmodial activity of EW–AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. EW–AgNP IC₅₀ were 49.3 μg/ml (CQ-s) and 55.5 μg/ml (CQ-r), while chloroquine IC₅₀ were 81.5 μg/ml (CQ-s) and 86.5 μg/ml (CQ-r). EW–AgNP showed a valuable antibiotic potential against important pathogenic bacteria and fungi. Concerning non-target effects of EW–AgNP against mosquito natural enemies, the predation efficiency of the mosquitofish Gambusia affinis towards the II and II instar larvae of A. stephensi was 68.50% (II) and 47.00% (III), respectively. In EW–AgNP-contaminated environments, predation was boosted to 89.25% (II) and 70.75% (III), respectively. Overall, this research highlighted the EW–AgNP potential against hepatocellular carcinoma, Plasmodium parasites and mosquito vectors, with little detrimental effects on mosquito natural enemies.     

Oxygen regulates the effective diffusion distance of nitric oxide in the aortic wall

Endothelium-derived nitric oxide (NO) is critical in maintaining vascular tone. Accumulating evidence shows that NO bioavailability is regulated by oxygen concentration. However, it is unclear to what extent the oxygen concentration regulates NO bioavailability in the vascular wall. In this study, a recently developed experimental setup was used to measure the NO diffusion flux across the aortic wall at various oxygen concentrations. It was observed that for a constant NO concentration at the endothelial surface, the measured NO diffusion flux out of the adventitial surface at [O₂] = 0 μM is around fivefold greater than at [O₂] = 150 μM, indicating that NO is consumed in the aortic wall in an oxygen-dependent manner. Analysis of experimental data shows that the rate of NO consumption in the aortic wall is first order with respect to [NO] and first order with respect to [O₂], and the rate constant k₁ was determined as (4.0 ± 0.3) × 10³ M^?1 s^?1. Computer simulations demonstrate that NO concentration distribution significantly changes with oxygen concentration and the effective NO diffusion distance at low oxygen level ([O₂] ≤ 25 μM) is significantly longer than that at high oxygen level ([O₂] = 200 μM). These results suggest that oxygen-dependent NO consumption may play an important role in dilating blood vessels during hypoxia by increasing the effective NO diffusion distance.     

Body mass index,iron absorption and iron status in childbearing age women

BackgroundThe prevalence of obesity has increased at an alarming rate worldwide. Some studies have observed an association between iron (Fe) deficiency (ID) and obesity, however more research is needed.ObjectiveTo assess whether body mass index (BMI) is associated with both Fe absorption and Fe status.MethodsA cross sectional sample of 318 Chilean childbearing age women was studied. The women received either a single dose of 0.5 mg of Fe (n = 137, group 1) or 3 mg of Fe plus ascorbic acid (1:2 molar ratio) (n = 181, group 2), both as FeSO₄ with labeled radioisotopes. Fe absorption was assessed through radio Fe erythrocyte incorporation. Fe status was determined by hemoglobin (Hb), mean corpuscular volume, serum Fe, total iron binding capacity, transferrin saturation, erythrocyte Zn protoporphyrin and serum ferritin (SF).Results29%, 47% and 24% of the women were classified as normal, overweight or obese, respectively. Fe absorption was significantly lower in obese women (p < 0.05). In group 1, the geometric mean and range ±1 SD of the percentage of Fe absorption for normal-weight women was 32.9% vs. 19.7% in obese. For group 2, this percentage was 36% vs. 30%, respectively (2-way ANOVA: BMI classification and Fe dose p < 0.05; interaction p = 0.34). Although Fe absorption was lower in obese women, they had higher SF (p < 0.01) and Hb (p < 0.05) concentrations.ConclusionAlthough we did not observe a relationship between BMI and Fe status, obese women displayed lower Fe absorption compared with overweight and normal weight women, possibly due to subclinical inflammation associated with obesity.     

Role of carbon monoxide and nitric oxide in adult rat hepatocytes proliferating in vitro: Effects of CAS 1609

During liver regeneration in vivo carbon monoxide (CO) and nitric oxide (NO) are supposed to play a significant role. We raise the question whether CO and NO are involved in the growth process of cultured hepatocytes. Rat hepatocytes were stimulated into proliferation, growth being estimated by DNA content, mRNA by quantitative RT-PCR, and inducible NO synthase (iNOS) activity by GC–MS. Dexamethasone proved obligatory for fast proliferation. It suppressed the spontaneous rise of iNOS-mRNA in cultures devoid of glucocorticoids, but did not counteract the rise in mRNA in actively dividing cultures. Expression of iNOS-mRNA and cell growth were further enhanced by LiCl (10 mM). NOS activity was completely suppressed by the iNOS-specific inhibitors N-(3-(aminomethyl)benzyl) acetamidine (1400 W,100 μM) and l-N⁶-(1-iminoethyl)lysine (l-NIL, 500 μM), however, without a decrease in hepatocyte growth. Proliferation was attenuated only by very high concentrations (>0.5 mM) of N-nitro-l-arginine methyl ester (l-NAME) and asymmetric dimethylarginine (ADMA). Various NO donors (at 100 μM) did not stimulate cell growth. The furoxan CAS 1609 stimulated growth, decreased iNOS-mRNA expression and transiently increased haem oxygenase-1 (HO-1)-mRNA without releasing considerable amounts of NO. 1H-[1,2,4]Oxadiazolo[4,3,-α]quinoxalin-1-one (ODQ) attenuated the action of CAS 1609. Proliferation was stimulated by Co-protoporphyrin and tricarbonyldichlororuthenium(II) dimer (CORM-2). We conclude that CAS 1609 triggers hepatocyte mitosis most likely via direct, NO-independent induction of HO-1 expression, pointing to CO as a growth-promoting signal in the proliferation cascade in cultured hepatocytes.     

Characterization of microbial communities removing nitrogen oxides from flue gas: the BioDeNOx process

BioDeNOx is an integrated physicochemical and biological process for the removal of nitrogen oxides (NOx) from flue gases. In this process, the flue gas is purged through a scrubber containing a solution of Fe(II)EDTA2-, which binds the NOx to form an Fe(II)EDTA.NO2- complex. Subsequently, this complex is reduced in the bioreactor to dinitrogen by microbial denitrification. Fe(II)EDTA2-, which is oxidized to Fe(III)EDTA- by oxygen in the flue gas, is regenerated by microbial iron reduction. In this study, the microbial communities of both lab- and pilot-scale reactors were studied using culture-dependent and -independent approaches. A pure bacterial strain, KT-1, closely affiliated by 16S rRNA analysis to the gram-positive denitrifying bacterium Bacillus azotoformans, was obtained. DNA-DNA homology of the isolate with the type strain was 89%, indicating that strain KT-1 belongs to the species B. azotoformans. Strain KT-1 reduces Fe(II)EDTA.NO2- complex to N2 using ethanol, acetate, and Fe(II)EDTA2- as electron donors. It does not reduce Fe(III)EDTA-. Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene fragments showed the presence of bacteria closely affiliated with members of the phylum Deferribacteres, an Fe(III)-reducing group of bacteria. Fluorescent in situ hybridization with oligonucleotide probes designed for strain KT-1 and members of the phylum Deferribacteres showed that the latter were more dominant in both reactors.     

Iron containing keratinolytic metallo-protease produced by Chryseobacterium gleum

Chryseobacterium gleum exhibited complete dissolution of whole chicken-feathers (10 g l^?1, pH 8) after 72 h at 30 °C through synthesis of keratinolytic protease when inoculated at 1% (v/v). This enzyme was purified to 67-fold with yield of 2.25% having a specific activity of 1670 U mg^?1 and ～36 kDa Mw. MALDI-TOF MS of this keratinase showed some similarity with the keratinase peptides of Bacillus subtilis (BOFXJ2). The keratinase action was inhibited by EDTA, iodoacetamide and metal ions like mercury, copper and zinc (1 mM each), while it was enhanced by iron and calcium. Keratinase showed presence of 3 mM of Fe M^?1 as tested by atomic absorption spectroscopy and addition of Fe in its apoenzyme retained about 79% of original residual feather degradation activity which portrayed it to be metalloprotease. Purified keratinase revealed significant degradation (85%) of feather concentrate (20 g l^?1) to 3.9 μM ml^?1 of free amino groups in 24 h at an initial pH of 8.0, 30 °C and 120 rpm shaking. This keratinase activity can be controlled precisely by presence of chemical or metal ions which could be of use in biotechnology industry while the culture can be used in poultry waste management.     

Forecasting the effect of feast and famine conditions on biological sulphate reduction in an anaerobic inverse fluidized bed reactor using artificial neural networks

The longevity and robustness of bioreactors used for wastewater treatment is determined by the activity of the microorganisms under steady and transient loading conditions. Two identical continuously operated inverse fluidized bed bioreactors (IFB), IFB R1 and IFB R2, were tested for sulphate removal under the same operating conditions for 140 d (Periods I–IV). Later, IFB R1 was used as the control reactor (Period V), while IFB R2 was operated under feast (Period V-A) and famine (Period V-B) feeding conditions for 66 d. The sulphate removal efficiency was comparable in both IFB, <20% in Period I and ∼70% during Periods II, III and IV. The robustness of the IFB was evident when the sulphate removal efficiency remained comparable during the feast Period (67 ± 15%) applied to IFB R2 compared to continuous feeding Periods (Period IV (71 ± 4%) for IFB R2 and Period V (61 ± 15%) for IFB R1). The IFB performance was modelled using a three-layered artificial neural networks (ANN) model (5-11-3) and a sensitivity analysis, the sulphate removal was found to be dependent on the COD:sulphate ratio. Besides, the robustness, resilience and adaptation time of the IFB were affected by the degree of mixing and the hydraulic retention time.     

Growth and antrum formation of bovine primary follicles in long-term culture in vitro

Successful antral formation in vitro from bovine preantral follicles (145–170 μm) has been described previously, but antrum formation from the primary follicle (50–70 μm) has not yet been achieved in vitro. The aim of the study was to establish an optimal culture system supporting the growth and maturation of bovine primary follicles (50–70 μm) in vitro. Bovine primary follicles were cultured in a three-dimensional culture system for 13 or 21 days in alpha-minimum essential medium. Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β-estradiol (E₂), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450 aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF9; bone morphogenetic protein-15, BMP15; and type III transforming growth factor β receptor, TGFβR3) were analyzed by real-time RT-PCR. After 21 days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH + LH + E₂ + bFGF or FSH + LH + E₂ + bFGF + EGF (p < 0.05). An addition of 50 ng/ml bFGF or bFGF + 25 ng/ml EGF initiated antrum formation by day 19 and day 17 of culture, and the antral cavity formation rate was 16.7% and 33.3% by 21 days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF9, BMP15 and TGFβR3) was significantly altered. We conclude that addition of 50 ng/ml bFGF + 25 ng/ml EGF to media containing FSH + LH + E₂ turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.