During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic cells, which then deposit a mineralized matrix to form calcified cartilage prior to replacement by bone. Previously, we reported that a clonal cell line, ATDC5, undergoes efficient chondrogenic differentiation through a cellular condensation stage. Here we report that the differentiated ATDC5 cells became hypertrophic at the center of cartilage nodules, when the cells ceased to grow. Formation of hypertrophic chondrocytes took place in association with type X collagen gene expression and a dramatic elevation of alkaline phosphate (ALPase) activity. After 5 weeks of culture, mineralization of the culture could be discerned as Alizarin red-positive spots, which spread throughout the nodules even in the absence of ␤-glycerophosphate. Electron microscopy and electron probe microanalysis revealed that calcification was first initiated at matrix vesicles in the territorial matrix and that it advanced progressively along the collagen fibers in a manner similar to that which occurs in vivo. The infrared spectrum of the mineralized nodules indicated two absorption doublets around 1030 cm ؊1 and 600 cm ؊1, which are characteristic of apatitic mineral. Calcifying cultures of ATDC5 cells retained responsiveness to parathyroid hormone (PTH): PTH markedly inhibited elevation of ALPase activity and calcification in the culture in a dose-dependent manner. Thus, we demonstrated that ATDC5 cells keep track of the multistep differentiation process encompassing the stages from mesenchymal condensation to calcification in vitro. ATDC5 cells provide an excellent model to study the molecular mechanism underlying regulation of cartilage differentiation during endochondral bone

Two experiments were conducted to evaluate the effect of amino acid (AA) injections in ovo in Cobb broiler breeder eggs on hatchability and subsequent chick BW. In Experiment 1, moisture, crude fat (CF), and CP were analyzed over time during incubation (Day 0, 7, 14, and 19 of incubation). Moisture, CP, and CF of the embryo increased, and moisture, CP, and CF of eggs decreased, as incubation time increased (P < 0.05). Combined egg and embryo AA contents, except Gly and Pro, decreased (P < 0.05) as incubation time increased. However, the pattern of AA in the egg did not change as the embryo developed. In Experiment 2, AA were injected into the yolk or air cell at Day 0 and 7 of incubation. Hatchability was reduced (P < 0.05) when AA were injected at Day 0 of incubation. However, when the AA solution was injected into the yolk sac at Day 7 of incubation, hatchability was not affected, and BW of chicks increased relative to egg weight prior to incubation. These results suggest that in ovo administration of AA may be an effective method of increasing chick BW at hatch.

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Two experiments were conducted to evaluate the effect of in ovo amino acid (AA) injections in broiler breeder eggs on AA utilization of embryos. All AA used in these experiments were pure crystalline AA in free-base form. Treatments in Experiment 1 comprised 1) control eggs (no injection), 2) 0.5 mL sterile-distilled water injected eggs, and 3) eggs injected with an AA solution suspended in 0.5 mL sterile-distilled water. Injections were administered into the yolk at Day 7 of incubation. At hatch, chicks were killed and bled, and plasma AA concentration was determined. Plasma AA concentration of hatched chicks decreased (P < 0.05) when water was injected. In addition, all AA from eggs injected with AA, except Glu and Lys, were decreased (P < 0.05) at hatch as compared to control eggs. However, AA pattern was not affected by in ovo water injection, but the AA ratio to Lys was reduced by in ovo AA injection. Experiment 2 was conducted to evaluate whole internal egg AA concentrations over incubation time in the presence or absence of in ovo AA administration. Treatments in Experiment 2 comprised 1) control eggs (no injection), and 2) eggs injected with a AA solution at Day 7 of incubation. The AA contents of embryo, yolk, albumen, and allantoic and amnion fluids were analyzed over time during incubation (Days 0, 7, 14, and 19 of incubation). On Day 14 of incubation, there were no differences in AA contents of all tissues between the control group and the group injected with AA on Day 7 of incubation. On Day 19 of incubation, AA contents of embryo, yolk, albumen, and allantoic and amnion fluids were increased (P < 0.05) as mediated by in ovo administration of AA at Day 7 of incubation. These results suggest that in ovo administration of AA may increase AA concentrations in chicken embryos and other egg contents.

Three experiments were conducted to evaluate the effect of differences in in ovo amino acid (AA) injection sites in broiler breeder eggs on subsequent hatchability and BW of chicks. In Experiment 1, an AA solution was injected into eggs with 13-mm or 19-mm, 27-ga needles. Uninjected eggs served as controls. Hatchability was decreased (P < 0.05) in eggs receiving AA injections with the 19-mm needle in comparison to the control and 13-mm-injected groups. However, BW of chicks increased (P < 0.05) relative to pre-incubational egg weight by AA injection with the 13-mm needle. In order to evaluate the in ovo location of AA injections from Experiment 1, India ink was injected into eggs in Experiment 2 with a 13-or 19-mm needle. Immediately after injection, the air cell end of the egg was windowed in order to observe effects of injection site. Windowing of eggs was accomplished by removing a piece of the eggshell over the air cell and the underlying membrane at Day 7 of incubation. The amount of injected India ink was higher in the extra-embryonic coelom in eggs treated by both needles. However, the occurrence of India ink in the extra embryonic coelom was higher (P < 0.05) in the group injected with AA solution using a 13-mm needle as compared to that after injection using a 19-mm needle. The observation of India ink in the amniotic cavity was higher (P < 0.05) in the group injected with AA solution using a 19-mm needle rather than that using a 13-mm needle. In Experiment 3, treatments consisted of control (uninjected eggs) or windowed eggs. Windowed eggs received AA to the chorioallantoic membrane, the yolk, extra-embryonic coelom, or amniotic cavity at Day 7 of incubation. Hatchability was reduced, but chicks hatched when eggs were windowed and when AA were injected into the yolk sac or extra-embryonic coelom. However, chicks did not hatch when AA were administered to the chorioallantoic membrane or into the amniotic cavity. These results suggest that the best AA injection sites in ovo may be the yolk and extra-embryonic coelom.

The production of ‘Bakasang’, an Indonesian fermented fish sauce, was replicated in the laboratory in order to study the physicochemical and microbiological changes associated with the process. Bakasang samples were produced by incubating mixtures of small sardine (Engraulis japonicus) at different concentrations of sodium chloride and glucose at 37°C for 40 days. Changes in pH, total soluble nitrogen, total free amino nitrogen, amino acid composition and total plate counts were observed. The isolation and identification of microflora were also performed. In general, the pH decreased throughout the incubation period, irrespective of NaCl and glucose concentrations. Increases in the amounts of total soluble nitrogen and total free amino nitrogen were noticed during the 40 days of fermentation. The amino acids, glutamic acid, alanine, isoleucine, and lysine were prominent at the end of the process. The total plate count increased during the first 10 days and then decreased gradually for both total microbial population and lactic acid bacteria population.Micrococcus,StreptococcusandPediococcusspp were predominantly present during Bakasang fermentation.

A yeast strain producing high levels of phytase was isolated from soil and identified as Candida krusei. The phytase was located on the yeast cell wall and was a glucanase-extractable protein. The phytase production was controlled by the phosphate concentration in the medium used. The maximum production of phytase occurred in a medium containing 0.5 mg of phosphorus per 100 ml, and most of the cells were ellipsoid-shaped and did not exhibit budding. Increasing the concentration of phosphorus in the medium to more than 5 mg of phosphorus per 100 ml caused inhibition of phytase production and 90% of the cells exhibited budding. On the other hand, transferring cells grown in the high-phosphate medium into a phosphate-free one derepressed the phytase production. For example, transferring cells grown in 2 mg of phosphorus per 100 ml into the phosphate-free medium, enhanced the total phytase activity up to 5.5-fold that in the medium containing 0.5 mg of phosphorus per 100 ml. The phytase showed two optimum pHs of 2.5 and 5.5, an optimum temperature of 40 degrees C and the K(m) value for Na-phytate was 0.03 mM. Using in vitro experiments that simulated the conditions of the digestive tract, 50-80% phosphorus was liberated from different plant samples (wheat bran, rice bran and feeds) by the strain.

Strain CF-66 with strong antifungal activity against Rhizoctonia solani was isolated from compost samples. It is clearly demonstrated that strain CF-66 is belonging to Burkholderia cepacia complex by the morphological and biochemical tests and 16S rDNA sequence. The B. cepacia complex consists of a group of bacteria currently organized into nine genomovars, among them genomovar II and genomovar III, contain the highly epidemic strains. However, it was known that strain CF-66 is not a member of genomovar II or III of the B. cepacia complex by species-specific polymerase chain reaction assay. In this study, the antifungal compound CF66I produced by strain CF-66 was purified and characterized. Based on the nuclear magnetic resonance, GC-MS spectral and infrared spectral data, CF66I was confirmed to have amide bonds, alpha-methyl fatty acid, bromine, and some structural units such as CH(2)CH(2)O. CF66I is stable to high temperature, proteolytic enzymes, and organic solvents. CF66I inhibit the growth of a variety of plant pathogenic fungi and pathogenic yeast, whereas bacterial cells are unaffected. CF66I mainly reduced hyphal extension rates in a dose-dependent manner and induced severe change in cell morphology that resulted in swelled and formed very short hyphae with multiple branches.