• Asian-Australasian Journal of Animal Sciences
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• v.22 no.11
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• pp.1555-1565
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• 2009

Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastix frontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five $\beta$-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.6
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• pp.988-1001
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• 1999

The rumen ecosystem is increasingly being recognized as a promising source of superior polysaccharide-degrading enzymes. They contain a wide array of novel enzymes at the levels of specific activities of 1,184, 1,069, 119, 390, 327 and $946{\mu}mol$ Reducing sugar release/min/mg protein for endoglucanase, xylanase, polygalactouronase, amylase, glucanase and arabinase, respectively. These enzymes are mainly located in the surface of rumen microbes. However, glycoside-degrading enzymes (e.g. glucosidase, fucosidase, xylosidase and arabinofuranosidase, etc.) are mainly located in the rumen fluid, when detected enzyme activities according to the ruminal compartments (e.g. enzymes in whole rumen contents, feed-associated enzymes, microbial cell-associated enzymes, and enzymes in the rumen fluid). Ruminal fungi are the primary contributors to high production of novel enzymes; the bacteria and protozoa also have important functions, but less central roles. The enzyme activities of bacteria, protozoa and fungi were detected 32.26, 19.21 and 47.60 mol glucose release/min/mL mediem for cellulose; 42.56, 14.96 and 64.93 mmol xylose release/min/mL medium after 48h incubation, respectively. The polysachharide-degrading enzyme activity of ruminal anaerobic fungi (e.g. Neocallimastix patriciarum and Piromyces communis, etc.) was much higher approximately 3~6 times than that of aerobic fungi (e.g. Tricoderma reesei, T. viridae and Aspergillus oryzae, etc.) used widely in industrial process. Therefore, the rumen ecosystem could be a growing source of novel enzymes having a tremendous potential for industrial applications.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.5
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• pp.752-757
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• 1999

Studies were made of digestion of timothy (Pheleum pretense) hay, tall fescue (Festuca elatior) hay, and rice (Oryza sativa) straw in pure cultures of rumen anaerobic fungus, Neocallimastix frontails MCH3. The fungus was inoculated on ground forages (1%, w/v) in an anaerobic medium and incubated at $39^{\circ}C$. Incubation was continued for 24, 48, 72 and 96 h. The losses of dry matter, xylose and glucose of forage during incubation were determined at the end of these incubation periods. Xylose and glucose were considered to be released from xylan and cellulose, respectively. The digested xylan to digested cellulose (X/C) ratios of the substrate were calculated. Xylanase and carboxymethyl cellulose (CMCase) of culture supernatant and residual substrate was measured at the same time. The X/C ratios in the cultures on timothy hay and rice straw were greater than 0.5 in the first 24-h incubation period. The values were smaller than 0.3 in tall fesque. The ratio of xylanase activity to that of CMCase in the first 24-h incubation period correlated well with the traits in X/C ratio. However xylanase activity was still superior to CMCase in the following incubation period (48 to 96 h), although the glucose (designated as cellulose) was more intensively digested than xylose (designated as xylan). The production of these polysaccharidases appeared to correlate with substrate cell-wall sugar composition, xylose to glucose ratios, at the beginning of fast growing period.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.8
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• pp.1110-1117
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• 2001

Responses of the rumen fungus, Neocallimastix frontalis RE1, to long chain fatty acid (LCFA) were evaluated by measuring gas production, filter paper (FP) cellulose digestion and polysaccharidase enzyme activities. LCFA (stearic acid, $C_{18:0}$; oleic acid, $C_{18:1}$; linoleic acid, $C_{18:2}$ and linolenic acid, $C_{18:3}$) were emulsitied by ultrasonication under anaerobic condition, and added to the medium. When N frontalis RE1 was grown in culture with stearic, oleic and linoleic acid, the cumulative gas production, gas pool size, FP cellulose digestion and enzymes activities significantly (p<0.05) increased at some incubation times(especially, exponential phases of fungal growth, 48~120 h of incubation) relative to that for control cultures. However, the addition of linolenic acid strongly inhibited all of the investigated parameters up to 120 h incubation, but not after 168 and 216 h of incubation. These results indicated that stearic, oleic and linoleic acids tended to have great stimulatory effects on fungal cellulolysis, whereas linolenic acid caused a significant (p<0.05) inhibitory effects on the cellulolysis by the rumen fungus. These results are the first report of the effect of LCFAs on the ruminal fungi. Further research is needed to identify the mode of action of LCFAs on fungal strains and to verify whether or not ruminal fungi have ability to hydrate unsaturated LCFAs to saturated FAs. There was high correlation between cumulative in vitro gas production and fungal growth (94.78%), FP cellulose degradation (96.34%), CMCase activity(90.86%) or xylanase activity (87.67%). Thus measuring of cumulative gas production could be a useful tool for evaluating fungal growth and/or enzyme production by ruminal fungi.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.5
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• pp.748-763
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• 2003

Anaerobic rumen microorganisms mainly bacteria, protozoa and fungi degrade ligno-cellulosic feeds consumed by the ruminants. The ruminants in developing countries are predominantly maintained on low grade roughage and grazing on degraded range land resulting in their poor nutrient utilization and productivity. Hence, manipulation of rumen fermentation was tried during last two decades to optimize ruminal fermentation for improving nutrient utilization and productivity of the animals. Modification of rumen microbial composition and their activity was attempted by using chemical additives those selectively effect rumen microbes, introduction of naturally occurring or genetically modified foreign microbes into the rumen and genetically manipulation of existing microbes in the rumen ecosystem. Accordingly, rumen protozoa were eliminated by defaunation for reducing ruminal methane production and increasing protein outflow in the intestine, resulting in improve growth and feed conversion efficiency of the animals. Further, Interspecies trans-inoculation of rumen microbes was also successfully used for annulment of dietary toxic factor. Additionally, probiotics of bacterial and yeast origin have been used in animal feeding to stabilize rumen fermentation, reduced incidence of diarrhoea and thus improving growth and feed conversion efficiency of young stalk. It is envisaged that genetic manipulation of rumen microorganisms has enormous research potential in developing countries. In view of feed resource availability more emphasis has to be given for manipulating rumen fermentation to increase cellulolytic activity for efficient utilization of low grade roughage.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.6
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• pp.820-824
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• 2004

The study was to see the effect of administration of ruminal fungi on feed intake, growth rate, rumen fermentation and nutrient digestion of calves (Tharparkar$\times$Holstein-Friesian, average age: 10 months, average body weight: 130 kg). The 6 calves in first group were fed a mixture consisted of 50% wheat straw and 50% concentrate (Maize 62%, Groundnut cake 35%, Mineral mix. 2% and Common salt 1%) along with 1 kg green oats $animal^{-1}$ $day^{-1}$ while second group calves were fed the above-mentioned diet in addition to a dose of 160 ml ($10^{6}$ CFU/ml) fungal culture $calf^{-1}$ $week^{-1}$. The average dry matter intake per day was slightly lowered in fungal fed calves yet feed conversion ratio was higher. The average daily weight gain was significantly higher (15.37%) in fungal administered group as compared to control. The nutrient digestibility was increased for crude fibre, NDF and ADF with fungal administration. Digestible energy value of straw-based diet in terms of percent TDN also increased. The pH and $NH_{3}$-N were lower whereas TVFA, total-N, TCA-N and number of zoospores were higher in rumen liquor in fungal administered group.

• Asian-Australasian Journal of Animal Sciences
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• v.10 no.6
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• pp.599-602
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• 1997

Fluctuation of fungal zoospores on agar strips were observed in the rumen of sheep fed three different levels of dietary concentrate, timothy hay: concentrate = 3:0 (AF diet), timothy hay: concentrate = 2:1 (MC diet), timothy hay : concentrate = 1:2 (HC diet) respectively. The number of zoospores on the strip was drastically decreased after morning feed with AF diet. The number was the highest at 0 h ($1.34{\times}10^2/cm^2$), then declined to $2.0{\times}10^3/cm^2$ at 9 h after feeding. In the rumen of animals fed MC diet, the number of zoospores decreased with time after feeding, although the decrement was slower than that with AF diet. During 0-3 h after feeding, number of zoospores was $1.6{\times}10^4/cm^2$. Although the number slightly decreased at 6 and 9 h, relatively high levels were maintained. It seems that the inducers for zoospore-release were maintained at relatively high concentration throughout incubation period. The fluctuation pattern of number of germinated zoospores was different in the rumen of animals fed HC diet from those of AF and MC diets. The number of zoospores was constantly maintained at lower level ($1.0{\times}10^3/cm^2$) than the other diets. For MC diet, continuous high number of germinated zoospores may be due to the continuous release of zoospores by hemes in timothy hay and concentrate feed, and by unknown mechanisms. Unlike AF diet which promoted relatively rapid decline of zoosporogenesis, supplementation of concentrate feed to the timothy hay did not promote such rapid decline of zoosporogenesis. It was suggested that release of inducers for zoosporogenesis from concentrate feed persisted longer time than from timothy hay. HC diet promoted the lowest zoospore production, suggested the lowest fungal population size in this experiment. These results show that an appropriate amount of concentrate may support fungal growth and stimulate zoosporogenesis in the rumen.

• Journal of Life Science
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• v.28 no.10
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• pp.1244-1253
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• 2018

Rumen microbiome consists of a wide variety of microorganisms, such as bacteria, archaea, protozoa, fungi, and viruses, that are in a symbiotic relationship in a strict anaerobic environment in the rumen. These rumen microbiome, a vital maker, play a significant role in feed fermentation within the rumen and produce different volatile fatty acids (VFAs). VFAs are essential for energy metabolism and protein synthesis of the host animal, even though emission of methane gas after feed fermentation is considered a negative indicator of loss of dietary energy of the host animal. To improve rumen microbial efficiency, a variety of approaches, such as feed formulation, the addition of natural feed additives, dietary feed-microbes, etc., have taken to increase ruminant performance. Recently with the application of high-throughput sequencing or next-generation sequencing technologies, especially for metagenomics and metatranscriptomics of rumen microbiomes, our understanding of rumen microbial diversity and function has significantly increased. The metaproteome and metabolome provide deeper insights into the complicated microbial network of the rumen ecosystem and its response to different ruminant diets to improve efficiency in animal production. This review summarized some recent advances of rumen microbiome techniques, especially "meta-omics," viz. metagenomic, metatranscriptomic, metaproteomic, and metabolomic techniques to increase feed fermentation and utilization in ruminants.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.3
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• pp.379-385
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• 2011

Ferulic acid esterase (FAE) and acetyl esterase (AE) cleave feruloyl groups substituted at the 5'-OH group of arabinosyl residues and acetyl groups substituted at O-2/O-3 of the xylan backbone, respectively, of arabinoxylans in the cell wall of grasses. In this study, the enzyme profiles of FAE, AE and polysaccharide hydrolases of the anaerobic rumen fungus Neocallimastix sp. YQ1 grown on Chinese wildrye grass hay (CW) or alfalfa hay (AH) were investigated by two $2{\times}4$ factorial experiments, each in 10-day pure cultures. The treatments consisted of two glucose levels ($G^+$: glucose at 1.0 g/L, $G^-$: no glucose) and four N sources (N1: 1.0 g/L yeast extract, 1.0 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N2: 2.8 g/L yeast extract and 0.5 g/L $(NH_4)_2SO_4$; N3: 1.6 g/L tryptone and 0.5 g/L $(NH_4)_2SO_4$; N4: 1.4 g/L tryptone and 1.7 g/L yeast extract) in defined media. The optimal combinations of glucose level and N source for the fungus on CW, instead of AH, were $G^-N4$ and $G^-N3$ for maximum production of FAE and AE, respectively. Xylanase activity peaked on day 4 and day 6 for the fungus grown on CW and AH, respectively. The activities of esterases were positively correlated with those of xylanase and carboxymethyl cellulase. The fungus grown on CW exhibited a greater volatile fatty acid production than on AH with a greater release of ferulic acid from plant cell wall.

• Journal of Animal Science and Technology
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• v.46 no.5
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• pp.763-772
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• 2004

The patterns of fungal growth and fiber digestion under the microscope, and tile productions of fibrolytic enzyme were studied in an in vitro culture with Neocallimastix frontalis SA when either filter paper or rice straw was provided as sole energy source. Under the microscopic observation, active zoospores attachment, sporangium development and complex rhizoidal system were founded on the surface and at the edge of filter paper. After 7 days of incubation, a reduced fiber mass, a decreased fiber cohesion and a weakened fiber structure by fungal digestion were clearly observed. Similar fungal development was observed with rice straw, but fungal growth and digestion took place mostly on the damaged and exposed portion of rice straw. Although there were some differences in absolute concentration and pattern, the concentration of both cellulase and xylanase increased with incubation time with the higher activity being obtained with filter paper. Their differences were large especially after 48 and 96hr of incubation(P< 0.05). The filter paper was more good inducer of cellulolytic and xylanolytic enzymes compared with complex substrate, rice straw. These findings suggest that the filter paper is the better energy source for N frontalis than the complex substrate, and structural disintegration by physical process is able to help rumen fungal growth on the lignified roughage although anaerobic rumen fungi have mechanical and enzymatic functions for fiber digestion.