Potency of cashew nut shell liquid in rumen modulation under different dietary conditions and indication of its surfactant action against rumen bacteria

( Seongjin Oh ),( Yasuyuki Suzuki ),( Shusuke Hayashi ),( Yutaka Suzuki ),( Satoshi Koike ),( Yasuo Kobayashi ) 한국축산학회 2017 한국축산학회지 Vol.59 No.11

Background: Cashew nut shell liquid (CNSL) is an agricultural byproduct containing alkylphenols that has been shown to favorably change the rumen fermentation pattern only under experimentally fixed feeding conditions. Investigation of CNSL potency in rumen modulation under a variety of feeding regimens, and evidence leading to the understanding of CNSL action are obviously necessary for further CNSL applications. The objective of this study was to evaluate the potency of CNSL for rumen modulation under different dietary conditions, and to visually demonstrate its surfactant action against selected rumen bacteria. Methods: Batch culture studies were carried out using various diets with 5 different forage to concentrate (F:C) ratios (9:1, 7:3, 5:5. 3:7 and 1:9). Strained rumen fluid was diluted with a buffer and incubated with each diet. Gas and short chain fatty acid (SCFA) profiles were characterized after 18 h incubation at 39 °C. Monensin was also evaluated as a reference additive under the same conditions. Four species of rumen bacteria were grown in pure culture and exposed to CNSL to determine their morphological sensitivity to the surfactant action of CNSL. Results: CNSL supplementation decreased total gas production in diets with 5:5 and 3:7 F:C ratios, whereas the F:C ratio alone did not affect any gas production. Methane decrease by CNSL addition was more apparent in diets with 5:5, 3:7, and 1:9 F:C ratios. An interactive effect of CNSL and the F:C ratio was also observed for methane production. CNSL supplementation enhanced propionate production, while total SCFA production was not affected. Monensin decreased methane production but only in a diet with a 1:9 F:C ratio with increased propionate. Studies of pure cultures indicated that CNSL damaged the cell surface of hydrogen- and formate-producing bacteria, but did not change that of propionate-producing bacteria. Conclusion: CNSL can selectively inhibit rumen bacteria through its surfactant action to lead fermentation toward less methane and more propionate production. As CNSL is effective over a wider range of dietary conditions for such modulation of rumen fermentation in comparison with monensin, this new additive candidate might be applied to ruminant animals for various production purposes and at various stages.

Phylogenetic Analysis of 16S rDNA Sequences Manifest Rumen Bacterial Diversity in Gayals (Bos frontalis) Fed Fresh Bamboo Leaves and Twigs (Sinarumdinaria)

Deng, Weidong,Wanapat, Metha,Ma, Songcheng,Chen, Jing,Xi, Dongmei,He, Tianbao,Yang, Zhifang,Mao, Huaming Asian Australasian Association of Animal Productio 2007 Animal Bioscience Vol.20 No.7

Six male Gayal (Bos frontalis), approximately two years of age and with a mean live weight of $203{\pm}17$ kg ($mean{\pm}standard\;deviation$), were housed indoors in metabolism cages and fed bamboo (Sinarundinaria) leaves and twigs. After an adjustment period of 24 days of feeding the diet, samples of rumen liquor were obtained for analyses of bacteria in the liquor. The diversity of rumen bacteria was investigated by constructing a 16S rDNA clone library. A total of 147 clones, comprising nearly full length sequences (with a mean length of 1.5 kb) were sequenced and submitted to an on-line similarity search and phylogenetic analysis. Using the criterion of 97% or greater similarity with the sequences of known bacteria, 17 clones were identified as Ruminococcus albus, Butyrivibrio fibrosolvens, Quinella ovalis, Clostridium symbiosium, Succiniclasticum ruminis, Selenomonas ruminantium and Allisonella histaminiformans, respectively. A further 22 clones shared similarity ranging from 90-97% with known bacteria but the similarity in sequences for the remaining 109 clones was less than 90% of those of known bacteria. Using a phylogenetic analysis it was found that the majority of the clones identified (57.1%) were located in the low G+C subdivision, with most of the remainder (42.2% of clones) located in the Cytophage-Flexibacter-Bacteroides (CFB) phylum and one clone (0.7%) was identified as a Spirochaete. It was apparent that Gayal have a large and diverse range of bacteria in the rumen liquor which differ from those of cattle and other ruminants. This may explain the greater live weights of Gayal, compared to cattle, grazing in the harsh natural environments in which Gayal are located naturally.

Fibrolytic Rumen Bacteria: Their Ecology and Functions

Koike, Satoshi,Kobayashi, Yasuo Asian Australasian Association of Animal Productio 2009 Animal Bioscience Vol.22 No.1

Among rumen microbes, bacteria play important roles in the biological degradation of plant fiber due to their large biomass and high activity. To maximize the utilization of fiber components such as cellulose and hemicellulose by ruminant animals, the ecology and functions of rumen bacteria should be understood in detail. Recent genome sequencing analyses of representative fibrolytic bacterial species revealed that the number and variety of enzymes for plant fiber digestion clearly differ between Fibrobacter succinogenes and Ruminococcus flavefaciens. Therefore, the mechanism of plant fiber digestion is also thought to differ between these two species. Ecology of individual fibrolytic bacterial species has been investigated using pure cultures and electron microscopy. Recent advances in molecular biology techniques complement the disadvantages of conventional techniques and allow accurate evaluation of the ecology of specific bacteria in mixed culture, even in situ and in vivo. Molecular monitoring of fibrolytic bacterial species in the rumen indicated the predominance of F. succinogenes. Nutritive interactions between fibrolytic and non-fibrolytic bacteria are important in maintaining and promoting fibrolytic activity, mainly in terms of crossfeeding of metabolites. Recent 16S rDNA-based analyses suggest that presently recognized fibrolytic species such as F. succinogenes and two Ruminococcus species with fibrolytic activity may represent only a small proportion of the total fibrolytic population and that uncultured bacteria may be responsible for fiber digestion in the rumen. Therefore, characterization of these unidentified bacteria is important to fully understand the physiology and ecology of fiber digestion. To achieve this, a combination of conventional and modern techniques could be useful.

STUDIES ON METHIONINE METABOLISM IN THE RUMEN BACTERIA OF GOATS

Muramatsu, T.,Numa, M.,Ueda, Y.,Furuse, M.,Okumura, J.,Samukawa, K. Asian Australasian Association of Animal Productio 1994 Animal Bioscience Vol.7 No.2

The metabolic fate of methionine in rumen bacteria was studied by intraruminal administration of $^{15}N$ and $1-^{13}C$ labeled methionine in goats. Time course changes in isotopic abundance of amino acids in the rumen bacteria were determined with a computer-controlled gas-chromatograph mass spectrometer. The results from the transition of peak isotopic abundance in amino acids indicated that in rumen bacteria the $^{15}N$ or $^{13}C$ isotope in the methionine molecule was transferred rapidly to into bacteria, methionine administered intraruminally may not be retained as it is, but would be converted quickly to other metabolites in the bacteria.

SOME FACTORS INFLUENCING TRI-L-ALANINE DISAPPEARANCE AND RUMEN BACTERIAL GROWTH YIELD IN VITRO

Ha, J.K.,Kennelly, J.J.,Lee, S.C. Asian Australasian Association of Animal Productio 1991 Animal Bioscience Vol.4 No.4

A series of in vitro incubation studies with washed rumen bacteria were conducted to determine the influence of incubation time and concentrations of peptides, alanine, ammonia nitrogen and carbohydrate on the rate of peptide disappearance and on bacterial growth. Disappearance rate of tri-alanine (ala3) under various conditions was between 30.6 and $58.2mg\;hr^-$ per gram bacterial dry matter. Ala3 was removed from the incubation medium in an almost linear fashion as incubation time and ala3 concentration was increased. Washed rumen bacteria utilized ala3 faster than di-l-alanine (ala2) at all concentrations. Adding 9mM carbohydrate significantly increased ala3 disappearance, but level of ammonia nitrogen had no influence on ala3 disappearance. The presence of alanine in the medium significantly lowered ala3 utilization by rumen bacteria. Bacterial dry matter and nitrogen growth yield were not influenced by alanine and peptides when incubation medium already contained a sufficient level of ammonia nitrogen. Increased ammonia nitrogen in the presence of ala3 did not stimulate bacterial growth. Carbohydrate significantly increased bacterial dry matter and nitrogen growth as expected. Results indicate that the rate of peptide utilization by rumen bacteria may be altered by type and concentration of peptides, and energy supply, and this may be mediated through changes in numbers and type of bacteria.

Recent Advances in Biotechnology of Rumen Bacteria - Review -

Forsberg, C.W.,Egbosimba, E.E.,MacLellan, S. Asian Australasian Association of Animal Productio 1999 Animal Bioscience Vol.12 No.1

Recent advances in the biotechnology of ruminal bacteria have been made in the characterization of enzymes involved in plant cell wall digestion, the exploration of mechanisms of gene transfer in ruminal bacteria, and the development of vectors. These studies have culminated in the introduction and expression of heterologous glucanase and xylanase genes and a fluoroacetate dehalogenase gene in ruminal bacteria. These recent studies show the strategy of gene and vector construction necessary for the production of genetically engineered bacteria for introduction into ruminants. Molecular research on proteolytic turnover of protein in the rumen is in its infancy, but a novel protein high in essential amino acids designed for intracellular expression in ruminal organisms provides an interesting approach for improving the amino acid profile of ruminal organisms.

Isolation and characterization of a novel gossypol-degrading bacteria Bacillus subtilis strain Rumen Bacillus Subtilis

Yunhua Zhang,Zhengyou Zhang,Li Dai,Ying Liu,Maoji Cheng,Lijuan Chen 아세아·태평양축산학회 2018 Animal Bioscience Vol.31 No.1

Objective: The aim of the study was to isolate gossypol-degrading bacteria and to assess its potential for gossypol degradation. Methods: Rumen liquid was collected from fistulated cows grazing the experimental pasture. Approximately 1 mL of the rumen liquid was spread onto basal medium plates containing 2 g/L gossypol as the only source of carbon and was then cultured at 39°C to isolate gossypol-degrading bacteria. The isolated colonies were cultured for 6 h and then their size and shape observed by microscope and scanning electron microscope. The 16S rRNA gene of isolated colonies was sequenced and aligned using National Center for Biotechnology Information-Basic Local Alignment Search Tool. The various fermentation conditions, initial pH, incubation temperature, inoculum level and fermentationperiod were analyzed in cottonseed meal (CSM). The crude protein (CP), total gossypol (TG), and free gossypol (FG) were determined in CSM after fermentation with isolated strain at 39°C for 72 h. Results: Screening results showed that a single bacterial isolate, named Rumen Bacillus Subtilis (RBS), could use gossypol as a carbon source. The bacterium was identified by 16S rDNA sequencing as being 98% homologous to the sequence of Bacillus subtilis strain GH38. The optimum fermentation conditions were found to be 72 h, 39°C, pH 6.5, moisture 50%, inoculum level 107 cell/g. In the optimum fermentation conditions, the FG and TG content in fermented CSM decreased 78.86% and 49% relative to the control. The content of CP and the essential amino acids of the fermented CSM increased respectively, compared with the control. Conclusion: The isolation of a gossypol-degrading bacterium from the cow rumen is of great importance for gossypol biodegradation and may be a valuable potential source for gossypol-degradation of CSM.

INHIBITORY EFFECT OF THE IONOPHORE SALINOMYCIN ON DEAMINATION BY MIXED RUMEN BACTERIA

Kobayashi, Y.,Suda, K.,Wakita, M.,Baran, M.,Hoshino, S. Asian Australasian Association of Animal Productio 1996 Animal Bioscience Vol.9 No.1

A series of in vitro experiments was conducted to investigate response of rumen bacterial deamination to the ionophore salinomycin. Addition of salinomycin to the inoculum, strained rumen fluid, depressed ammonia production from casein, while increased accumulation of ${\alpha}$-amino acids. This suggests an inhibitory effect of salinomycin on ruminal deamination. When the effect in washed bacterial suspension was monitored with individual amino acid, aspartic acid degradation was markedly inhibited by salinomycin. This inhibition was not observed when the mixed rumen bacteria were ultrasonically disrupted and used as the enzyme source. Extent of the inhibition tended to be higher in the bacteria source from sheep on a high roughage diet. From these results it was speculated that the inhibition of deamination with salinomycin is caused by a decreased transport of amino acid into the bacterial cells as well as a decreased proportion of deaminating bacteria in the rumen.

반추위내 서식하는 혼합곰팡이와 박테리아에 의한 Linoleic Acid 가수소화반응과 Stearic Acid 생산에 관한 연구

남인식,Nam, In-Sik 한국미생물학회 2007 미생물학회지 Vol.43 No.2

The objective of this study was to confirm biohydrogenation of linoleic acid and stearic acid production by mixed men fungi and bacteria. In mixed fungal biohydrogenation study, when linoleic acid solution was added to fungal culture (after 24 hr pre-incubation), all linoleic acids were converted to trans-11 vaccenic acid via cis-9, trans-11 conjugated linoleic acid production within 24 hr period of incubation. All linoleic acid solution was hydrogenated to trans-11 vaccenic acid within 24 hr incubation and this was continued until the end of incubation (48 hr). Both treatments (added linoleic acid solution or the same amount of solution without containing linoleic acid into fungal cultures) produced the similar amount of stearic acid. In contrast, 100% of linoleic acid solution was hydrogenated to stearic acid in mixed bacterial culture. It is concluded that the end product of mixed fungal biohydrogenation of linoleic acid is trans-11 vaccenic acid whereas mixed bacteria produced stearic acid as an end product of their biohydrogenation.

Evaluation and Isolation of Phytin Phosphohydrolyzing Bacterial Population in the Rumen

Suzuki, C.,Ushida, K. Asian Australasian Association of Animal Productio 2000 Animal Bioscience Vol.13 No.7

A series of experiments was conducted to evaluate phytin phosphohydrolysis actlVlty in the rumen and to isolate phytase positive rumen bacteria. Endogenous phytase activity of wheat bran was estimated and compared with that of bacterial phytin phosphohydrolysis. Substantial phytase activity was detected in wheat bran during in vitro rumen incubation. Bacterial phytase activity was suggested not to be high. Only two facultative anaerobes, Klebsiella sp. and Corynebacterium sp. were isolated as phytase producing organisms. These belonged to a minor microbial group in the rumen population. Protozoal fraction showed an initial velocity of phytin phosphohydrolysis 7 times higher than the bacterial fraction.