• Journal of Animal Science and Technology
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• v.61 no.3
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• pp.122-137
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• 2019

Methane, one of the important greenhouse gas, has a higher global warming potential than that of carbon dioxide. Agriculture, especially livestock, is considered as the biggest sector in producing anthropogenic methane. Among livestock, ruminants are the highest emitters of enteric methane. Methanogenesis, a continuous process in the rumen, carried out by archaea either with a hydrogenotrophic pathway that converts hydrogen and carbon dioxide to methane or with methylotrophic pathway, which the substrate for methanogenesis is methyl groups. For accurate estimation of methane from ruminants, three methods have been successfully used in various experiments under different environmental conditions such as respiration chamber, sulfur hexafluoride tracer technique, and the automated head-chamber or GreenFeed system. Methane production and emission from ruminants are increasing day by day with an increase of ruminants which help to meet up the nutrient demands of the increasing human population throughout the world. Several mitigation strategies have been taken separately for methane abatement from ruminant productions such as animal intervention, diet selection, dietary feed additives, probiotics, defaunation, supplementation of fats, oils, organic acids, plant secondary metabolites, etc. However, sustainable mitigation strategies are not established yet. A cumulative approach of accurate enteric methane measurement and existing mitigation strategies with more focusing on the biological reduction of methane emission by direct-fed microbials could be the sustainable methane mitigation approaches.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.10
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• pp.1416-1423
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• 2013

The metabolomic profile of the anaerobic fungus Piromyces sp. F1, isolated from the rumen of goats, and how this is affected by the presence of naturally associated methanogens, was analyzed by nuclear magnetic resonance spectroscopy. The major metabolites in the fungal monoculture were formate, lactate, ethanol, acetate, succinate, sugars/amino acids and ${\alpha}$-ketoglutarate, whereas the co-cultures of anaerobic fungi and associated methanogens produced citrate. This is the first report of citrate as a major metabolite of anaerobic fungi. Univariate analysis showed that the mean values of formate, lactate, ethanol, citrate, succinate and acetate in co-cultures were significantly higher than those in the fungal monoculture, while the mean values of glucose and ${\alpha}$-ketoglutarate were significantly reduced in co-cultures. Unsupervised principal components analysis revealed separation of metabolite profiles of the fungal mono-culture and co-cultures. In conclusion, the novel finding of citrate as one of the major metabolites of anaerobic fungi associated with methanogens may suggest a new yet to be identified pathway exists in co-culture. Anaerobic fungal metabolism was shifted by associated methanogens, indicating that anaerobic fungi are important providers of substrates for methanogens in the rumen and thus play a key role in ruminal methanogenesis.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.4
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• pp.542-549
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• 2009

An in vitro study was conducted to determine the effect of nitrate-nitrogen used as a sole dietary nitrogen source on ruminal fermentation characteristics and microbial nitrogen (MN) synthesis. Three treatment diets were formulated with different nitrogen sources to contain 13% CP and termed i) nitrate-N diet (NND), ii) urea-N diet (UND), used as negative control, and iii) tryptone-N diet (TND), used as positive control. The results of 24-h incubations showed that nitrate-N disappeared to background concentrations and was not detectable in microbial cells. The NND treatment decreased net $CH_4$ production, but also decreased net $CO_2$ production and increased net $H_2$ production. Total VFA concentration was lower (p<0.05) for NND than TND. Suppression of $CO_2$ production and total VFA concentration may be linked to increased concentration of $H_2$. The MN synthesis was greater (p<0.001) for NND than UND or TND (5.74 vs. 3.31 or 3.34 mg/40 ml, respectively). Nitrate addition diminished methane production as expected, but also increased MN synthesis.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.12
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• pp.1746-1751
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• 2005

The objective of this study was to evaluate the effects of sarsaponin on methane production, ruminal fermentation, nutrient digestion and blood metabolites using three Holstein steers in a 3${\times}$3 Latin Square design. The steers were fed Sudangrass hay plus concentrate mixture at a ratio 1.5:1 twice daily, and sarsaponin (0, 0.5 and 1% of DM), which was given at 09:00 and 17:00 h daily by mixing with concentrate. Rumen samples were collected 0, 2, and 5 h after morning dosing. Ruminal pH was numerically decreased and numbers of protozoa were decreased linearly (p<0.01) by treatment. Ruminal ammonia-N was reduced (linear; p<0.05) and total VFA was increased (quadratic; p<0.05) at 2 and 5 h after sarsaponin dosing. The molar proportion of acetate was decreased (quadratic; p<0.05) and propionate was increased (linear; p<0.01) at all sampling times. Blood plasma glucose was increased and urea-N was decreased (linear; p<0.05) at 2 and 5 h after dosing. Methane was decreased by approximately 12.7% (linear; p<0.05). The apparent digestibility of DM and NDF were decreased (quadratic; p<0.05) and that of CP remained unchanged due to the sarsaponin. The numbers of cellulolytic bacteria were decreased (quadratic; p<0.05), while numbers of total viable bacteria remained unchanged due to the sarsaponin. These results show that sarsaponin can partially inhibit rumen methanogenesis in vivo and improve ruminal fermentation, which supports our previous in vitro results.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.1
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• pp.61-68
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• 2020

Objective: The present study explored the effects of grape seed procyanidin extract (GSPE) on rumen fermentation, methane production and archaeal communities in vitro. Methods: A completely randomized experiment was conducted with in vitro incubation in a control group (CON, no GSPE addition; n = 9) and the treatment group (GSPE, 1 mg/bottle GSPE, 2 g/kg dry matter; n = 9). The methane and volatile fatty acid concentrations were determined using gas chromatography. To explore methane inhibition after fermentation and the response of the ruminal microbiota to GSPE, archaeal 16S rRNA genes were sequenced by MiSeq high-throughput sequencing. Results: The results showed that supplementation with GSPE could significantly inhibit gas production and methane production. In addition, GSPE treatment significantly increased the proportion of propionate, while the acetate/propionate ratio was significantly decreased. At the genus level, the relative abundance of Methanomassiliicoccus was significantly increased, while the relative abundance of Methanobrevibacter decreased significantly in the GSPE group. Conclusion: In conclusion, GSPE is a plant extract that can reduce methane production by affecting the structures of archaeal communities, which was achieved by a substitution of Methanobrevibacter with Methanomassiliicoccus.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.6
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• pp.806-811
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• 2012

This study was conducted to evaluate effects of plant extracts on methanogenesis and rumen microbial diversity in in vitro. Plant extracts (Artemisia princeps var. Orientalis; Wormwood, Allium sativum for. Pekinense; Garlic, Allium cepa; Onion, Zingiber officinale; Ginger, Citrus unshiu; Mandarin orange, Lonicera japonica; Honeysuckle) were obtained from the Plant Extract Bank at Korea Research Institute of Bioscience and Biotechnology. The rumen fluid was collected before morning feeding from a fistulated Holstein cow fed timothy and commercial concentrate (TDN; 73.5%, crude protein; 19%, crude fat; 3%, crude fiber; 12%, crude ash; 10%, Ca; 0.8%, P; 1.2%) in the ratio of 3 to 2. The 30 ml of mixture, comprising McDougall buffer and rumen liquor in the ratio of 4 to 1, was dispensed anaerobically into serum bottles containing 0.3 g of timothy substrate and plant extracts (1% of total volume, respectively) filled with $O_2$-free $N_2$ gas and capped with a rubber stopper. The serum bottles were held in a shaking incubator at $39^{\circ}C$ for 24 h. Total gas production in all plant extracts was higher (p<0.05) than that of the control, and total gas production of ginger extract was highest (p<0.05). The methane emission was highest (p<0.05) at control, but lowest (p<0.05) at garlic extract which was reduced to about 20% of methane emission (40.2 vs 32.5 ml/g DM). Other plant extracts also resulted in a decrease in methane emissions (wormwood; 8%, onion; 16%, ginger; 16.7%, mandarin orange; 12%, honeysuckle; 12.2%). Total VFAs concentration and pH were not influenced by the addition of plant extracts. Acetate to propionate ratios from garlic and ginger extracts addition samples were lower (p<0.05, 3.36 and 3.38 vs 3.53) than that of the control. Real-time PCR indicted that the ciliate-associated methanogen population in all added plant extracts decreased more than that of the control, while the fibrolytic bacteria population increased. In particular, the F. succinogens community in added wormwood, garlic, mandarin orange and honeysuckle extracts increased more than that of the others. The addition of onion extract increased R. albus diversity, while other extracts did not influence the R. albus community. The R. flavefaciens population in added wormwood and garlic extracts decreased, while other extracts increased its abundance compared to the control. In conclusion, the results indicated that the plant extracts used in the experiment could be promising feed additives to decrease methane gas emission from ruminant animals while improving ruminal fermentation.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.1
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• pp.71-79
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• 2018

Objective: Gelidium amansii (Lamouroux) is a red alga belonging to the family Gelidaceae and is commonly found in the shallow coasts of many East Asian countries, including Korea, China, and Japan. G. amansii has traditionally been utilized as an edible alga, and has various biological activities. The objective of this study was to determine whether dietary supplementation of G. amansii could be useful for improving ruminal fermentation. Methods: As assessed by in vitro fermentation parameters such as pH, total gas, volatile fatty acid (VFA) production, gas profile (methane, carbon dioxide, hydrogen, and ammonia), and microbial growth rate was compared to a basal diet with timothy hay. Cannulated Holstein cows were used as rumen fluid donors and 15 mL rumen fluid: buffer (1:2) was incubated for up to 72 h with four treatments with three replicates. The treatments were: control (timothy only), basal diet with 1% G. amansii extract, basal diet with 3% G. amansii extract, and basal diet with 5% G. amansii extract. Results: Overall, the results of our study indicate that G. amansii supplementation is potentially useful for improving ruminant growth performance, via increased total gas and VFA production, but does come with some undesirable effects, such as increasing pH, ammonia concentration, and methane production. In particular, real-time polymerase chain reaction indicated that the methanogenic archaea and Fibrobacter succinogenes populations were significantly reduced, while the Ruminococcus flavefaciens populations were significantly increased at 24 h, when supplemented with G. amansii extracts as compared with controls. Conclusion: More research is required to elucidate what G. amansii supplementation can do to improve growth performance, and its effect on methane production in ruminants.

• Journal of Animal Science and Technology
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• v.60 no.11
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• pp.26.1-26.9
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• 2018

Background: This experiment aimed at assessing polyphenol-rich plant biomass to use in complete feed making for the feeding of ruminants. Methods: An in vitro ruminal evaluation of complete blocks (CFB) with (Acacia nilotica, Ziziphus nummularia leaves) and without (Vigna sinensis hay) polyphenol rich plant leaves was conducted by applying Menke's in vitro gas production (IVGP) technique. A total of six substrates, viz. three forages and three CFBs were subjected to in vitro ruminal fermentation in glass syringes to assess gas and methane production, substrate degradability, and rumen fermentation metabolites. Results: Total polyphenol content (g/Kg) was 163 in A. nilotica compared to 52.5 in Z. nummularia with a contrasting difference in tannin fractions, higher hydrolysable tannins (HT) in the former (140.1 vs 2.8) and higher condensed (CT) tannins in the later (28.3 vs 7.9). The potential gas production was lower with a higher lag phase (L) in CT containing Z. nummularia and the component feed block. A. nilotica alone and as a constituent of CFB produced higher total gas but with lower methane while the partitioning factor (PF) was higher in Z. nummularia and its CFB. Substrate digestibility (both DM and OM) was lower (P < 0.001) in Z. nummularia compared to other forages and CFBs. The fermentation metabolites showed a different pattern for forages and their CFBs. The forages showed higher TCA precipitable N and lower acetate: propionate ratio in Z. nummularia while the related trend was found in CFB with V. sinensis. Total volatile fatty acid concentration was higher (P < 0.001) in A. nilotica leaves than V. sinensis hay and Z. nummularia leaves. It has implication on widening the forage resources and providing opportunity to use forage biomass rich in polyphenolic constituents in judicious proportion for reducing methane and enhancing green livestock production. Conclusion: Above all, higher substrate degradability, propionate production, lower methanogenesis in CFB with A. nilotica leaves may be considered useful. Nevertheless, CFB with Z. nummularia also proved its usefulness with higher TCA precipitable N and PF. It has implication on widening the forage resources and providing opportunity to use polyphenol-rich forage biomass for reducing methane and enhancing green livestock production.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.10
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• pp.1389-1394
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• 2012

Sheanut cake (SNC), expeller (SNE) and solvent extractions (SNSE) samples were evaluated to determine their suitability in animal feeding. The CP content was highest in SNSE (16.2%) followed by SNE (14.7%) and SNC (11.6%). However, metabolizable energy (ME, MJ/kg) was maximum in SNC (8.2) followed by SNE (7.9) and SNSE (7.0). The tannin phenol content was about 7.0 per cent and mostly in the form of hydrolyzable tannin (HT), whereas condensed tannin (CT) was less than one per cent. The in vitro gas production profiles indicated similar y max (maximum potential of gas production) among the 3 by-products. However, the rate of degradation (k) was maximum in SNC followed by SNE and SNSE. The $t^{1/2}$ (time taken for reaching half asymptote) was lowest in SNC (14.4 h) followed by SNE (18.7 h) and SNSE (21.9 h). The increment in the in vitro gas volume (ml/200 mg DM) with PEG (polyethylene glycol)-6000 (as a tannin binder) addition was 12.0 in SNC, 9.6 in SNE and 11.0 in SNSE, respectively. The highest ratio of $CH_4$ (ml) reduction per ml of the total gas, an indicator of the potential of tannin, was recorded in SNE (0.482) followed by SNC (0.301) and SNSE (0.261). There was significant (p<0.05) reduction in entodinia population and total protozoa population. Differential protozoa counts revealed that Entodinia populations increased to a greater extent than Holotricha when PEG was added. This is the first report on the antimethanogenic property of sheanut byproducts. It could be concluded that all the three forms of SN byproducts are medium source of protein and energy for ruminants. There is a great potential for SN by-products to be incorporated in ruminant feeding not only as a source of energy and protein, but also to protect the protein from rumen degradation and suppress enteric methanogenesis.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.4
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• pp.515-523
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• 2012

The study investigated the biochemical methane potential (BMP) assay of cellulose supplementing with mixed methanogens and cellulolytic bacteria to improve anaerobic digestion for methane production. For the BMP assay, 7 different microbial supplementation groups were consisted of the cultures of mixed methanogens (M), Fibrobacter succinogenes (FS), Ruminococcus flavefaciensn (RF), R. albus (RA), RA+FS and M+RA+FS including control. The cultures were added in the batch reactors with the increasing dose levels of 1% (0.5 mL), 3% (1.5 mL) and 5% (2.5 mL). Incubation for the BMP assay was carried out for 40 days at $38^{\circ}C$ and anaerobic digestate obtained from an anaerobic digester with pig slurry as inoculum was used. In results, 5% FS increased total biogas and methane production up to 10.4~22.7% and 17.4~27.5%, respectively, compared to other groups (p<0.05). Total solid (TS) digestion efficiency showed a similar trend to the total biogas and methane productions. Generally the TS digestion efficiency of the FS group was higher than that of other groups showing at the highest value of 64.2% in the 5% FS group. Volatile solid (VS) digestion efficiencies of 68.4 and 71.0% in the 5% FS and the 5% RF were higher than other groups. After incubation, pH values in all treatment groups were over 6.4 indicating that methanogensis was not inhibited during the incubation. In conclusion, the results indicated that the hydrolysis stage for methane production in anaerobic batch reactors was the late-limiting stage compared with the methanogenesis stage, and especially, as the supplementation levels of F. succinogenes supplementation increased, the methane production was increased in the BMP assay compared with other microbial culture addition.