• Journal of Animal Science and Technology
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• v.45 no.4
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• pp.617-626
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• 2003

This experiment employed a rumen simulated continuous culture system to examine the possibility of improving the rumen bypass of polyunsaturated fatty acids (PUFA) by using a high proportion of concentrate in the feed, and compared soya and linseed in terms of conjugated linoleic acid (CLA) production. No effect of type of fat source was observed on ruminal fermentation. A high proportion of concentrate (80%) in the feed decreased (P<0.001) vessel pH but increased (P<0.01) ammonia nitrogen, total VFA, acetate, butyrate and valerate concentrations compared with a low proportion (40%). Fat sources (soya vs. linseed) and concentrate ratio in the feed did not affect digestibilities of organic matter (OM), total nitrogen, neutral detergent fiber (NDF) and acid detergent fiber (ADF). Soya increased the flows of trans C18:1, C18:2 n-6 and C18:3 n-3 compared with linseed. The difference in fat source alone did not affect the flow of CLA but this was increased when high levels of soya and linseed were associated with a high proportion of concentrate in the feed. There was no effect of fat source on biohydrogenation of C18:1 n-9 and C18:2 n-6, but biohydrogenation of C18:3 n-3 and total C18 PUFA was higher with the linseed than with the soya treatment. A high proportion of concentrate decreased biohydrogenation of C18:2 n-6, C18:3 n-3 and total C18 PUFA compared with a low proportion.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.10
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• pp.1425-1429
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• 2005

Megaspahera elsdenii YJ-4, which was previously isolated as a producer of trans-10, cis-12 CLA, was studied for its carbon source on the CLA production. M. elsdenii YJ-4, was incubated with glucose and lactose, and cultured in batch and continuous culture systems with linoleic acid at various pHs to investigate CLA production. Batch cultures of the ruminal bacterium, M. elsdenii YJ-4, were resistant to stearic acid and linoleic acid, and little growth inhibition was observed even when the fatty acid concentration in the culture was as much as 4 mg $ml^{-1}$. Stationary phase batch cultures (0.25 mg bacterial protein $ml^{-1}$) that had been grown on lactate and incubated with linoleic acid (0.20 mg $ml^{-1}$) produced approximately 12 ${\mu}g$ trans-10, cis-12 CLA mg $protein^{-1}$ and little cis-9, trans-11 CLA was detected. Some linoleic acid was converted to hydrogenated products (chiefly stearic acid), but these fatty acids were less than 5 ${\mu}g$ mg bacterial $protein^{-1}$. Stationary phase batch cultures that had been grown on glucose produced at least 3-fold less trans-10, cis-12 CLA than ones grown on lactate. Cells from lactate-limited continuous cultures produced less trans-10, cis-12 CLA than those from batch culture, but only if the pH was greater than 6.4. When the pH of the lactate-limited continuous cultures was lower than 6.4, trans-10, cis-12 CLA and hydrogenated products declined. Cells from glucose-limited continuous cultures produced less trans-10, cis-12 CLA and hydrogenated products than the cells that had been limited by lactate, but pH had little impact on this production. These results support the idea that M. elsdenii YJ-4 could be one of the major producers of trans-10, cis-12 CLA which causes cows to produce milk with a low fat content.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.12
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• pp.1743-1748
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• 2003

The effect of pH on the fermentation characteristics and the formation of cis-9, trans-11 conjugated linoleic acid (CLA) and trans-11 octadecenoic acid by mixed ruminal bacteria was examined in vitro when incubated with linseed or rapeseed. Concentrate (1%, w/v) with ground linseed (0.6%, w/v) or rapeseed (0.5%, w/v) was added to 600 ml mixed solution of strained rumen fluid with artificial saliva (1:1, v/v), and was incubated anaerobically for 12 h at $39^{\circ}C$. The pH of culture solution was maintained at level close to 4.5, 5.3, 6.1 and 6.9 with 30% $H_2SO_4$ or 30% NaOH solution. pH increment resulted in increases of ammonia and total volatile fatty acid (VFA) concentration in culture solutions containing both oilseeds. Fermentation did not proceeded at pH 4.5. Molar proportion of acetate decreased but that of propionate increased as pH increased when incubated with oilseeds. While the hydrogenating process was very slow at the pH range of 4.5 to 5.3, rapid hydrogenation was found from the culture solutions of pH 6.1 and 6.9 when incubated with linseed or rapeseed. As pH in culture solution of linseed or rapeseed increases proportions of oleic acid (cis-9 $C_{18:1}$) and trans-11 octadecenoic acid increased but those of linoleic acid and linolenic acid decreased. The CLA proportion increased with pH in culture solution containing rapeseed but CLA was mostly not detected from the incubation of linseed.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.6
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• pp.819-826
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• 2009

An in vitro study was conducted to investigate the effect of malate or fumarate on fermentation characteristics, and production of conjugated linoleic acid (CLA) and methane ($CH_4$) by rumen microbes when incubated with linolenic acid (${\alpha}-C_{18:3}$). Sixty milligrams of ${\alpha}-C_{18:3}$ alone (LNA), or ${\alpha}-C_{18:3}$ with 24 mM malic acid (M-LNA) or ${\alpha}-C_{18:3}$ with 24 mM fumaric acid (F-LNA) were added to the 150 ml culture solution consisting of 75 ml strained rumen fluid and 75ml McDougall's artificial saliva. Culture solution for incubation was also made without malate, fumarate and ${\alpha}-C_{18:3}$ (Control). Two grams of feed consisting of 70% concentrate and 30% ground alfalfa (DM basis) were also added to the culture solution of each treatment. In vitro incubation was made anaerobically in a shaking incubator up to 12 h at $39^{\circ}C$. Supplementation of malate (M-LNA) or fumarate (F-LNA) increased pH at 6 h (p<0.01) and 12 h (p<0.001) incubation times compared to control and linolenic acid (LNA) treatments. Both malate and fumarate did not influence the ammonia-N concentration. Concentration of total VFA in culture solution was higher for M-LNA and F-LNA supplementation than for control and LNA treatments from 6 h (p<0.040) to 12 h (p<0.027) incubation times, but was not different between malate and fumarate for all incubation times. Molar proportion of $C_3$ was increased by F-LNA and M-LNA supplementation from 6 h (p<0.0001) to 12 h (p<0.004) incubation times compared to control and LNA treatments. No differences in $C_{3}$ proportion, however, were observed between M-LNA and F-LNA treatments. Accumulated total gas production for 12h incubation was increased (p<0.0002) by M-LNA or F-LNA compared to control or LNA treatment. Accumulated $CH_4$ production for 12 h incubation, however, was greatly reduced (p<0.0002) by supplementing malate or fumarate compared to the control, and its production from M-LNA or F-LNA treatment was smaller than that from LNA treatment. Methane production from LNA, M-LNA or F-LNA treatment was steadily lower (p<0.01 - p<0.001) from 3 h incubation time than that from the control, and was also lower for M-LNA or F-LNA treatment at incubation times of 6 h (p<0.01) and 9 h (p<0.001) than for LNA treatment. Methane production from LNA, however, was reduced (p<0.01 - p<0.001) from 3 h to 9 h incubation times compared to the control. Both malate and fumarate increased concentration of trans11-$C_{18:1}$ from 3 h to 12 h incubation (p<0.01), cis9,trans11-CLA up to 6 h incubation (p<0.01 - p<0.01), trans10,cis12-CLA at 3 h (p<0.05) and 12 h (p<0.01), and total CLA for all incubation times (p<0.05) compared to corresponding values for the ${\alpha}-C_{18:3}$ supplemented treatment (LNA). In conclusion, malate and fumarate rechanneled the metabolic $H_2 pathway to production of propionate and CLA, and depressed the process of biohydrogenation and methane generation. Linolenic acid alone would also be one of the optimistic alternatives to suppress the $CH_4$ generation.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.2
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• pp.200-207
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• 2007

In vitro rumen incubation studies were conducted to determine effects of initial pH on bacterial attachment and fiber digestion. Ruminal fluid pH was adjusted to 5.7, 6.2 and 6.7, and three major fibrolytic bacteria attached to rice straw in the mixed culture were quantified with real-time PCR. The numbers of attached and unattached Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminocococcus albus were lower (p<0.05) at initial pH of 5.7 without significant difference between those at higher initial pH. Lowering incubation media pH to 5.7 also increased bacterial numbers detached from substrate regardless of bacterial species. Dry matter digestibility, gas accumulation and total VFA production were pH-dependent. Unlike bacterial attachment, maintaining an initial pH of 6.7 increased digestion over initial pH of 6.2. After 48 h in vitro rumen fermentation, average increases in DM digestion, gas accumulation, and total VFA production at initial pH of 6.2 and 6.7 were 2.8 and 4.4, 2.0 and 3.0, and 1.2 and 1.6 times those at initial pH of 5.7, respectively. The lag time to reach above 2% DM digestibility at low initial pH was taken more times (8 h) than at high and middle initial pH (4 h). Current data clearly indicate that ruminal pH is one of the important determinants of fiber digestion, which is modulated via the effect on bacterial attachment to fiber substrates.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.1
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• pp.86-91
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• 2012

An in vitro rumen batch culture study was completed to compare effects of common grasses, leguminous shrubs and non-leguminous shrubs used for livestock grazing in Australia and Ghana on $CH_4$ production and fermentation characteristics. Grass species included Andropodon gayanus, Brachiaria ruziziensis and Pennisetum purpureum. Leguminous shrub species included Cajanus cajan, Cratylia argentea, Gliricidia sepium, Leucaena leucocephala and Stylosanthes guianensis and non-leguminous shrub species included Annona senegalensis, Moringa oleifera, Securinega virosa and Vitellaria paradoxa. Leaves were harvested, dried at $55^{\circ}C$ and ground through a 1 mm screen. Serum bottles containing 500 mg of forage, modified McDougall's buffer and rumen fluid were incubated under anaerobic conditions at $39^{\circ}C$ for 24 h. Samples of each forage type were removed after 0, 2, 6, 12 and 24 h of incubation for determination of cumulative gas production. Methane production, ammonia concentration and proportions of VFA were measured at 24 h. Concentration of aNDF (g/kg DM) ranged from 671 to 713 (grasses), 377 to 590 (leguminous shrubs) and 288 to 517 (non-leguminous shrubs). After 24 h of in vitro incubation, cumulative gas, $CH_4$ production, ammonia concentration, proportion of propionate in VFA and IVDMD differed (p<0.05) within each forage type. B. ruziziensis and G. sepium produced the highest cumulative gas, IVDMD, total VFA, proportion of propionate in VFA and the lowest A:P ratios within their forage types. Consequently, these two species produced moderate $CH_4$ emissions without compromising digestion. Grazing of these two species may be a strategy to reduce $CH_4$ emissions however further assessment in in vivo trials and at different stages of maturity is recommended.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.10
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• pp.1399-1406
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• 2000

A continuous culture study was conducted to determine the impact of ruminal degradable soy protein (S-RDP) level and dilution rate (D) on growth of ruminal non-structural carbohydrate-fermenting microbes. Corn starch, urea and isolated soy protein (ISP) were used to formulate three diets with S-RDP levels of 0, 35 and 70% of total dietary CP. Two Ds were 0.03 and $0.06h^{-1}$ of the fermenter volume in a single-effluent continuous culture system. As S-RDP levels increased, digestibilities of dietary dry matter (DM), organic matter (OM) and crude protein (CP) linearly (p=0.001) decreased, whereas digestion of dietary starch linearly (p=0.001) increased. Increasing D from 0.03 to $0.06h^{-1}$ resulted in decreased digestibilities of dietary DM and OM, but had no effect on digestibilities of dietary starch (p=0.77) and CP (p=0.103). Fermenter pH, the concentration of volatile fatty acids (VFA) and daily VFA production were unaffected (p=0.159-0.517) by S-RDP levels. Molar percentages of acetate, propionate and butyrate were greatly affected by S-RDP levels (p=0.016-0.091), but unaffected by D (p=0.331-0.442). With increasing S-RDP levels and D, daily bacterial counts, daily microbial N production (DMNP) and microbial efficiency (MOEFF; grams of microbial N produced per kilogram of OM truly digested) were enhanced (p=0.001). The increased microbial efficiency with increasing S-RDP levels is probably the result of peptides or amino acids that served as a stimulus for optimal protein synthesis. The quantity of ruminal degradable protein from soy proteins required for optimum protein synthesis of non-structural carbohydrate-fermenting microbes appears to be equivalent to 9.5% of dietary fermented OM.

• Journal of the Korean Society of Food Culture
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• v.4 no.3
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• pp.245-251
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• 1989

To estimate the nutritional values and the optimum cooking method of the Cow's rumen-reticulum (tripe) which is a Korean traditional food, the nutrients of raw tissue of tripes, the changes in total N, ${\alpha}-amino$ N, calcium and phosphorus contents and nucleotides and their related compounds contents in soup stock prepared of tripe tissues according to various boiling time period with saucepot or pressure cooker and various ratios of the water to the tripe (wt/wt) were measured. No significant difference was observed in nutrients contents between rumen (1st stomach) and reticulum (2nd stomach) which contained 83% moisture, 0.4-0.5% ash, 3% fat, 13% protein, 50-56 mg% calcium, 75-76 mg% phosphorus. 75-77 mg% ${\alpha}-amino$ N on fresh weight basis. The results obtained show that the significant loss of nutrients observed after removing epitherial cell layer from tripe, and in aspect of the nutrients contents, the nucletides contents, and the sensory evaluation score in soup stock, the optimum cooking time period of tripe was 8 hours in boiling in saucepot and 1 or 2 hours in cooking in pressure cooker, and the ratio of the water to the tripe (wt/wt) was above ten.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.880-884
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• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.1
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• pp.66-74
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• 2012

The effects of exogenous fibrolytic enzymes (EFE; a mixture of two preparations from Trichoderma spp., with predominant xylanase and ${\beta}$-glucanase activities, respectively) on colonization and digestion of ground barley straw and alfalfa hay by Fibrobacter succinogenes S85 and Ruminococcus flavefaciens FD1 were studied in vitro. The two levels (28 and 280 ${\mu}g$/ml) of EFE tested and both bacteria were effective at digesting NDF of hay and straw. With both substrates, more NDF hydrolysis (p<0.01) was achieved with EFE alone at 280 than at 28 ${\mu}g$/ml. A synergistic effect (p<0.01) of F. succinogenes S85 and EFE on straw digestion was observed at 28 but not 280 ${\mu}g$/ml of EFE. Strain R. flavefaciens FD1 digested more (p<0.01) hay and straw with higher EFE than with lower or no EFE, but the effect was additive rather than synergistic. Included in the incubation medium, EFE showed potential to improve fibre digestion by cellulolytic ruminal bacteria. In a second batch culture experiment using mixed rumen microbes, DM disappearance (DMD), gas production and incorporation of $^{15}N$ into particle-associated microbial N ($^{15}N$-PAMN) were higher (p<0.001) with ammoniated (5% w/w; AS) than with native (S) ground barley straw. Application of EFE to the straws increased (p<0.001) DMD and gas production at 4 and 12 h, but not at 48 h of the incubation. EFE applied onto S increased (p<0.01) $^{15}N$-PAMN at 4 h only, but EFE on AS increased (p<0.001) $^{15}N$-PAMN at all time points. Prehydrolysis increased (p<0.01) DMD from both S and AS at 4 and 12 h, but reduced (p<0.01) $^{15}N$-PAMN in the early stage (4 h) of the incubation, as compared to non-prehydrolyzed samples. Application of EFE to barley straw increased rumen bacterial colonization of the substrate, but excessive hydrolytic action of EFE prior to incubation decreased it.