Efficacy of exogenous xylanases for improving in vitro fermentation of forages

2014 ◽  
Vol 153 (3) ◽  
pp. 538-553 ◽  
Author(s):  
Z. X. HE ◽  
L. Y. YANG ◽  
W. Z. YANG ◽  
K. A. BEAUCHEMIN ◽  
S. X. TANG ◽  
...  
Keyword(s):  
Volatile Fatty Acids ◽  
Xylanase Activity ◽  
Ammonia Nitrogen ◽  
Positive Control ◽  
Linear Increase ◽  
Gas Production ◽  
Pennisetum Purpureum ◽  
Ruminal Fermentation ◽  
Positive Effects

SUMMARYBatch cultures of mixed rumen micro-organisms were used to evaluate varying enzyme products with high xylanase activity (EPX), four of which were recombinant single xylanase activity developmental enzyme products (EPX1–EPX4, products of xylanase genes derived from Trichoderma harzianum, Trichoderma reesei, Orpinomyces and Aspergillus oryzae, respectively), for their potential to improve in vitro ruminal fermentation of three forages [maize (Zea mays) stover (MS), rice (Oryza sativa) straw (RS) and Guimu No. 1 grass (Pennisetum americanum×Pennisetum purpureum, GM)]. The enzyme product EPX5, derived from Trichoderma longibrachiatum, was used as a positive control that could improve in vitro fermentation of forages. Enzymes were supplied at dose rates of 0 (control), 20 (low), 50 (medium) and 80 (high) enzymic units of xylanase/g of dry matter (DM). There were no interactions between EPX and dose for the fermentation characteristics evaluated. Increasing EPX dose linearly increased gas production (GP) kinetic characters [i.e. asymptotic GP (VF), half time when GP is half of the theoretical maximum GP (t0·5), and initial fractional rate of degradation (FRD0)] and methane (CH4) production from RS and GM at 24 h, and increased degradability of DM at 24 h for MS and RS. A linear increase in degradability of neutral detergent fibre (NDF) of the three forages at 24 h was observed with increasing dose of EPX, but at 48 h only NDF degradability of RS was increased. There were differences in the effects of EPX on degradability of DM and NDF from RS at 24 h, with EPX4 having the highest and EPX1 having the lowest. In addition, increasing EPX dose linearly increased acetate proportion at 24 h and total volatile fatty acids (TVFA) at 48 h in MS. Increasing EPX dose linearly increased TVFA at 24 h, and ammonia-nitrogen (NH3-N) concentration at 48 h in RS. For GM, linear or quadratic effects of dose on acetate and butyrate concentration were observed at 24 and 48 h. The present study indicates that applying EPX to low-quality forages has the potential to improve rumen degradability and utilization. Furthermore, EPX from different sources differed in their effects when applied at the same dose rate, with the responses being forage-specific. For RS, the EPX derived from A. oryzae showed the greatest positive effects on forage degradation; whereas for MS and GM, the source of micro-organism where EPX gene was derived did not affect the degradation, with little difference among the EPX evaluated.

scholarly journals Rhodaneses Enzyme Addition Could Reduce Cyanide Concentration and Enhance Fiber Digestibility via In Vitro Fermentation Study

Fermentation ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 207
Author(s):  
Chanadol Supapong ◽  
Anusorn Cherdthong
Keyword(s):  
Volatile Fatty Acids ◽  
Potassium Cyanide ◽  
Insoluble Fraction ◽  
Ammonia Nitrogen ◽  
Gas Production ◽  
Nutrient Digestibility ◽  
Control Group ◽  
In Vitro Digestibility ◽  
Ruminal Fermentation

The use of cyanide-containing feed (HCN) is restricted because it causes prussic acid poisoning in animals. The objective of this study was to see how adding rhodanese enzyme to an HCN-containing diet affected gas dynamics, in vitro ruminal fermentation, HCN concentration reduction, and nutrient digestibility. A 3×4 factorial arrangement in a completely randomized design was used for the experiment. Factor A was the three levels of potassium cyanide (KCN) at 300, 450, and 600 ppm. Factor B was the four doses of rhodanese enzyme at 0, 0.65, 1, and 1.35 mg/104 ppm KCN, respectively. At 96 h of incubation, gas production from an insoluble fraction (b), potential extent (omit gas) (a + b), and cumulative gas were similar between KCN additions of 300 to 450 ppm (p > 0.05), whereas increasing KCN to 600 ppm significantly decreased those kinetics of gas (p < 0.05). Supplementation of rhodanese enzymes at 1.0 to 1.35 mg/104 ppm KCN enhanced cumulative gas when compared to the control group (p < 0.05). Increasing the dose of rhodanese up to 1.0 mg/104 ppm KCN significantly increased the rate of ruminal HCN degradation efficiency (DE) by 70% (p < 0.05). However, no further between the two factors was detected on ruminal fermentation and in vitro digestibility (p > 0.05). The concentration of ammonia-nitrogen (NH3-N) increased with increasing doses of KCN (p < 0.05), but remained unchanged with varying levels of rhodanese enzymes (p > 0.05). The in vitro dry matter digestibility (IVDMD) was suppressed when increasing doses of KCH were administered at 600 ppm, whereas supplementation of rhodanese enzymes at 1.0–1.35 mg/104 ppm KCN enhanced IVDMD (p < 0.05). Increasing doses of KCN affected reduced total volatile fatty acids (TVFA) concentration, which was lowest when 600 ppm was added (p < 0.05). Nevertheless, the concentration of TVFAs increased when rhodanese enzymes were included by 1.0–1.35 mg/104 ppm KCN (p < 0.05). Based on this study, it could be concluded that supplementation of rhodaneses enzyme at 1.0–1.35 mg/104 ppm KCN could enhance cumulative gas, digestibility, and TVAF, as well as lowering ruminal HCN concentration.

scholarly journals Residue of propolis extract in bovine diets with increasing levels of protein on rumen fermentation

2020 ◽  
Vol 55 ◽  
Author(s):  
Roberto Junior Teixeira Nascimento ◽  
Rafael Monteiro Araújo Teixeira ◽  
Thierry Ribeiro Tomich ◽  
Luiz Gustavo Ribeiro Pereira ◽  
Tânia Dayana do Carmo ◽  
...  
Keyword(s):  
Volatile Fatty Acids ◽  
Ammonia Nitrogen ◽  
Rumen Fermentation ◽  
Gas Production ◽  
Ruminal Fermentation ◽  
Propolis Extract ◽  
In Vitro Gas Production ◽  
Dry Matter Degradation ◽  
Kinetics Of

Abstract: The objective of this work was to evaluate the effect of the residue from the extraction of propolis, added to bovine diets with increasing levels of protein, on ruminal fermentation in vitro. For this, the in vitro gas production technique was used. Incubation was carried out with inocula from three fistulated cows, in three periods. In each period, a cow received a daily dose of 100 g propolis residue. Four diets were evaluated: corn silage (control); and 25, 50, and 75% concentrate based on soybean meal. The following were determined: kinetics of rumen fermentation; dry matter degradation; production of gases, volatile fatty acids (acetate, propionate, and butyrate), methane, and ammonia nitrogen; and pH. The inclusion of 14.4, 15.1, and 9.5% propolis residue, respectively, to 25, 50, and 75% concentrate increased the production of gases from the degradation of fibrous carbohydrates, when compared with the control. The propolis residue reduces methane production and the acetate:propionate ratio at all tested concentrate inclusion levels.

scholarly journals Pomegranate seed oil rich in conjugated linolenic acids reduces in vitro methane production

1970 ◽  
Vol 46 (3) ◽  
pp. 325-335
Author(s):  
E. Maleki ◽  
G.Y. Meng ◽  
M. Faseleh Jahromi ◽  
R. Jorfi ◽  
A. Khoddami ◽  
...  
Keyword(s):  
Methane Production ◽  
Volatile Fatty Acids ◽  
Seed Oil ◽  
Control Diet ◽  
Nutrient Utilization ◽  
Gas Production ◽  
Metabolizable Energy ◽  
Ruminal Fermentation ◽  
Ch4 Production

The objective of this study was to determine the effect of pomegranate (Punica granatum L.) seed oil (PSO) on gas and methane (CH4) production, ruminal fermentation and microbial populations under in vitro conditions. Three treatments consisting of a control diet containing 10 mg tallow (CON); the control diet with 5 mg PSO + 5 mg tallow (MPSO) and the control diet containing 10 mg PSO (HPSO) were compared. Ten mg of the experimental fat/oil samples were inserted into a gas-tight 100 mL plastic syringe containing 30 mL of an incubation inoculum and 250 mg of a basic substrate of a hay/concentrate (1/1, w/w) mixture. In vitro gas production was recorded over 0, 2, 4, 6, 8, 10, 12 and 24 h of incubation. After 24 hours, incubation was stopped, and methane production, pH, volatile fatty acids (VFAs) and microbial counts were measured in the inoculant. Gas production at 4, 6, 8, 10, 12 and 24 h incubation, metabolizable energy and in vitro organic matter disappearance increased linearly and quadratically as level of PSO increased. Furthermore, the 10 mg PSO (HPSO) decreased CH4 production by 21.0% compared with the control (CON) group. There were no significant differences in total and individual VFA concentrations between different levels of PSO, except for butyric acid. After 24 h of incubation, methanogenesis decreased in the HPSO compared with the MPSO and CON treatments. In addition, total bacteria and protozoa counts increased with rising PSO levels, while population methanogenesis declined significantly. These results suggested that PSO could reduce methane emissions, which might be beneficial to nutrient utilization and growth in ruminants.

Impact of strain and dose of live yeast and yeast derivatives on in vitro ruminal fermentation of a high-grain diet at two pH levels

2018 ◽  
Vol 98 (3) ◽  
pp. 477-487 ◽  
Author(s):  
P.X. Jiao ◽  
Z.X. He ◽  
S. Ding ◽  
N.D. Walker ◽  
Y.Y. Cong ◽  
...  
Keyword(s):  
Positive Control ◽  
Gas Production ◽  
Additional Treatment ◽  
Ruminal Fermentation ◽  
Selenomonas Ruminantium ◽  
Copy Numbers ◽  
Randomized Design ◽  
Ph Level ◽  
Live Yeast

The objective of this study was to determine the effects of live yeast (LY) or yeast derivatives (YD) on gas production (GP), dry matter (DM) disappearance (DMD), fermentation characteristics, and microbial profiles in batch culture. The study was a completely randomized design with a factorial arrangement: 2 media pH × 5 yeasts × 4 dosages. An additional treatment of monensin (Mon) was added as a positive control for each pH level. Media pH was low (5.8) and high (6.5); the yeasts were three LY (LY1-3) and two YD (YD4-5); and doses were 0, 4 × 106, 8 × 106, and 1.6 × 107 cfu mL−1 for LY and 0, 15, 30, and 60 mg bottle−1 for YD. Substrate consisted of 10% silage and 90% concentrate (DM basis) and samples were incubated for 24 h. Media pH of 6.5 vs. 5.8, increased (P < 0.01) GP, DMD, and volatile fatty acid (VFA) concentrations but decreased (P < 0.01) NH3-N concentration and copy numbers of Ruminococcus albus, Ruminococcus flavefaciens, and Selenomonas ruminantium. Increasing dose of LY2 linearly (P < 0.05) increased DMD. Total VFA concentration was greater with LY2 (P < 0.01) than LY3 and YD5 at pH 6.5. Overall, adding yeast products improved (P < 0.05) DMD at pH 5.8, and increased VFA concentration compared with Mon. These results indicate that in vitro GP and DMD of a high-grain diet varied with source and dose of yeast supplementation. Some yeast products have the potential to improve fermentation of feedlot diets when supplemented at appropriate doses.

scholarly journals Multiple-factor associative effects of peanut shell combined with alfalfa and concentrate determined by in vitro gas production method

2019 ◽  
Vol 64 (No. 8) ◽  
pp. 352-360
Author(s):  
Jiu Yuan ◽  
Xinjie Wan
Keyword(s):  
Fatty Acids ◽  
Volatile Fatty Acids ◽  
Rumen Fluid ◽  
Production Method ◽  
Ammonia Nitrogen ◽  
Gas Production ◽  
Peanut Shell ◽  
Dry Matter Digestibility ◽  
In Vitro Gas Production

The associative effects (AE) between concentrate (C), peanut shell (P) and alfalfa (A) were investigated by means of an automated gas production (GP) system. The C, P and A were incubated alone or as 40 : 60 : 0, 40 : 45 : 15, 40 : 30 : 30, 40 : 15 : 45, 40 : 0 : 60 and 30 : 70 : 0, 30 : 55 : 15, 30 : 40 : 30, 30 : 25 : 45, 30 : 10 : 60, 30 : 0 : 70 mixtures where the C : roughage (R) ratios were 40 : 60 and 30 : 70. Samples (0.2000 ± 0.0010 g) of single feeds or mixtures were incubated for 96 h in individual bottles (100 ml) with 30 ml of buffered rumen fluid. GP parameters were analysed using a single exponential equation. After incubation, the residues were used to determine pH, dry matter digestibility (DMD), organic matter digestibility (OMD), volatile fatty acids (VFA) and ammonia nitrogen (NH<sub>3</sub>-N) of the incubation fluid, and their single factor AE indices (SFAEI) and multiple-factors AE indices (MFAEI) were determined. The results showed that group of 30 peanut shell had higher SFAEI of GP<sub>48 h</sub>, DMD, OMD and total volatile fatty acids (p &lt; 0.05) and MFAEI (p &lt; 0.05) than groups 60, 45 and 0 when C : R was 40 : 60. The group of 10 peanut shell showed higher SFAEI of GP<sub>48 h</sub>, DMD and OMD (p &lt; 0.05) than groups 70, 55 and 40 and MFAEI (p &lt; 0.01) when C : R was 30 : 70. It is concluded that optimal SFAEI and MFAEI were obtained when the C : P : A ratios were 40 : 30 : 30 and 30 : 10 : 60.

scholarly journals Increasing Buffering Capacity Alters Rumen Microbiota Composition and Enhances Rumen Fermentation Characteristics of High-Concentrate Fed Hanwoo Steers

2021 ◽  
Author(s):  
Sonny Ramos ◽  
Seon Ho Kim ◽  
Chang Dae Jeong ◽  
Lovelia L. Mamuad ◽  
A-rang Son ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Volatile Fatty Acids ◽  
Ammonia Nitrogen ◽  
Rumen Fermentation ◽  
Buffering Capacity ◽  
Ruminal Fermentation ◽  
Drastic Decrease ◽  
Degrading Bacteria

Abstract Background: Rumen bacterial community is mainly affected by the type of diet consumed by the host animals. High concentrate diet increases the abundance of lactic acid producers and utilizers due to high level of non-structural carbohydrates thus reducing the number of fiber-degrading bacteria because of drastic decrease in pH. Dietary buffers are essential in regulating rumen pH through the compounds responsible in resisting drastic decrease in pH once cattle were fed with high-concentrate diet. However, no study has evaluated the effects of buffering capacity and efficiency in alleviating chronic acidosis in rumen. Ruminal metataxonomic and fermentation characteristics analyses were conducted to evaluate the effect of different buffering capacities on in vitro and in vivo experiments in high-concentrate fed Hanwoo steers. Results: Results revealed that BC0.9% and BC0.5% had similar and significant effect (P < 0.05) on in vitro ruminal fermentation at 3 to 24 h incubation. Both BC0.9% and BC0.5% had significantly highest (P < 0.05) buffering capacity, pH, and ammonia-nitrogen (NH3-N) than BC0.3% and CON at 24 h of incubation. Individual and total volatile fatty acids (VFA) were significantly lowest in CON. Increasing buffering capacity concentration showed linear effect on pH at 6 to 24 h while total gas and NH3-N at 3 and 12 h. Phylum Bacteroidetes dominated all treatments but a higher abundance of Firmicutes in BC0.5% than others. Ruminoccocus bromii and Succiniclasticum ruminis were dominant in BC0.5% and Bacteroides massiliensis in BC0.3%. The normalized data of relative abundance of observed OTUs’ representative families have grouped the CON with BC0.3% in the same cluster, whereas BC0.5% and BC0.9% were clustered separately which indicates the effect of varying buffering capacity of buffer agents. Principal coordinate analysis (PCoA) on unweighted UniFrac distances revealed close similarity of bacterial community structures within and between treatments and control, in which BC0.9% and BC0.3% groups showed dispersed community distribution. Conclusion: Our findings showed that increasing buffering capacity enhances rumen fermentation parameters and affects rumen microbiome by altering bacterial community through distinct structure between high and low buffering capacity, thus an important factor contributed to the prevention of ruminal acidosis during a high-concentrate diet.

scholarly journals Influence of sulfur on the fermentation characteristics of corn distiller’s dried grains with solubles in in vitro culture

2017 ◽  
Vol 62 (No. 10) ◽  
pp. 417-425
Author(s):  
L. He ◽  
H. Wu ◽  
W. Chen ◽  
Q. Meng ◽  
Z. Zhou
Keyword(s):  
In Vitro Culture ◽  
Production Rate ◽  
Volatile Fatty Acids ◽  
Ammonia Nitrogen ◽  
Gas Production ◽  
Metabolizable Energy ◽  
Composition Analysis ◽  
Sulfur Source ◽  
Net Energy

The effects of sulfur on the fermentation characteristics of corn distiller’s dried grains with solubles (DDGS) in in vitro culture were investigated. Samples (DDGS) were analyzed for nutrient values and then two independent in vitro experiments were conducted to study the effects of various sulfur sources (Na<sub>2</sub>S, Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub>, Na<sub>2</sub>SO<sub>3</sub>, and Na<sub>2</sub>SO<sub>4</sub>) and different sulfur levels (0.346, 0.692, and 1.038%) on the fermentation characteristics of DDGS. Based on sampling and chemical composition analysis, there existed a great variation in the concentrations of sulfur and proximate nutrients of DDGS. In Experiment 1, sulfur source showed a significant (P &lt; 0.01) effect on the gas production parameters (asymptotic gas production (b) and gas production rate (c)) and gas production of DDGS – sulfur from Na<sub>2</sub>SO<sub>4</sub> and Na<sub>2</sub>S produced more (P &lt; 0.01) gas within 48 h with a faster gas production rate as well as higher digestibilities (dry matter degradability and organic matter digestibility) and more energy supplies (metabolizable energy), net energy for maintenance and gain, and net energy for gain than sulfur from Na<sub>2</sub>SO<sub>3</sub> and Na<sub>2</sub>S<sub>2</sub>O<sub>4</sub>. Neither ammonia-nitrogen (NH<sub>3</sub>-N) concentration nor volatile fatty acids (VFA) profile (total VFA and individual VFA proportion) were affected by sulfur source (P &gt; 0.05). In Experiment 2, no significant (P &gt; 0.05) effect on the fermentation characteristics of DDGS with increasing sulfur content was found. The collective findings suggest that regular chemical analyses are necessary to make full use of DDGS, and that the valence state of sulfur in DDGS exerts an effect on its in vitro fermentation characteristics and there appears no dose-related effect of sulfur on the fermentation of DDGS in a short-term in vitro culture.

scholarly journals The effects of compound treatment of Aspergillus oryzae and fibrolytic enzyme on in vitro degradation, gas production and fermentative profile of maize silage and sugarcane silage

2021 ◽  
pp. 1-12
Author(s):  
J. M. Souza ◽  
J. C. S. M. Souza ◽  
D. O. Sousa ◽  
T. A. Del Valle ◽  
L. G. Ghizzi ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Nitrogen Concentration ◽  
Ammonia Nitrogen ◽  
Linear Increase ◽  
Gas Production ◽  
Maize Silage ◽  
Randomized Design ◽  
Fibre Degradation ◽  
Linear Reduction

Abstract The present study was conducted to evaluate the effect of a live culture of Aspergillus oryzae (A; CCT4359) and fibrolytic enzyme (E; Fibrozyme Alltech Inc.) on fibre digestibility by a gas production bioassay and in vitro degradation of maize silage and sugarcane silage. A completely randomized design trial was performed to evaluate: A doses (0, 20, 60 and 100 mg/l), E doses (0, 160, 320 and 480 mg/l) and roughage source (R; maize and sugarcane silage) in a 4 × 4 × 2 factorial arrangement. The inclusion of increasing doses of A and E increased dry matter and neutral detergent fibre in vitro digestibility linearly, but for E this effect occurred only in maize silage. There was a linear increase in the potential for gas production at the highest dose of A only in sugarcane silage, with no effect on lag time (L). Increasing doses of E increased the volume of gases produced linearly, and a trend of linear reduction of L, regardless of the roughage. There was a linear reduction in ammonia-nitrogen concentration in response to increasing doses of A and E, and an increase in acetic acid concentration at the highest dose of A, regardless of roughage. The additives had no synergistic effect on gas production and digestibility, but were efficient in altering the fermentative pattern, demonstrating the potential to increase fibre degradation.

scholarly journals Effects of Thymol Supplementation on Goat Rumen Fermentation and Rumen Microbiota In Vitro

2020 ◽  
Vol 8 (8) ◽  
pp. 1160 ◽  
Author(s):  
Jiangkun Yu ◽  
Liyuan Cai ◽  
Jiacai Zhang ◽  
Ao Yang ◽  
Yanan Wang ◽  
...  
Keyword(s):  
Methane Production ◽  
Volatile Fatty Acids ◽  
Rumen Fluid ◽  
Optimal Dose ◽  
Illumina Miseq ◽  
Ammonia Nitrogen ◽  
Rumen Fermentation ◽  
Gas Production ◽  
Rumen Microbiota

This study was performed to explore the predominant responses of rumen microbiota with thymol supplementation as well as effective dose of thymol on rumen fermentation. Thymol at different concentrations, i.e., 0, 100 mg/L, 200 mg/L, and 400 mg/L (four groups × five replications) was applied for 24 h of fermentation in a rumen fluid incubation system. Illumina MiSeq sequencing was applied to investigate the ruminal microbes in addition to the examination of rumen fermentation. Thymol doses reached 200 mg/L and significantly decreased (p < 0.05) total gas production (TGP) and methane production; the production of total volatile fatty acids (VFA), propionate, and ammonia nitrogen, and the digestibility of dry matter and organic matter were apparently decreased (p < 0.05) when the thymol dose reached 400 mg/L. A thymol dose of 200 mg/L significantly affected (p < 0.05) the relative abundance of 14 genera of bacteria, three species of archaea, and two genera of protozoa. Network analysis showed that bacteria, archaea, and protozoa significantly correlated with methane production and VFA production. This study indicates an optimal dose of thymol at 200 mg/L to facilitate rumen fermentation, the critical roles of bacteria in rumen fermentation, and their interactions with the archaea and protozoa.

scholarly journals Combining Crude Glycerin with Chitosan Can Manipulate In Vitro Ruminal Efficiency and Inhibit Methane Synthesis

Animals ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 37
Author(s):  
Anuthida Seankamsorn ◽  
Anusorn Cherdthong ◽  
Metha Wanapat
Keyword(s):  
Volatile Fatty Acids ◽  
Gas Production ◽  
Nutrient Digestibility ◽  
Crude Glycerin ◽  
Control Group ◽  
In Vitro Digestibility ◽  
Ruminal Fermentation ◽  
Ch4 Production ◽  
In Vitro Gas Production

It was hypothesized that the combination of glycerin and chitosan improves ruminal fermentation efficiency via an enhanced propionate (C3) and reduces in vitro CH4 production. This was explored through in vitro gas production with substrates containing crude glycerin, which replaced cassava chips in the studied ration. The experimental design was organized following a 3 × 3 factorial in completely randomized design and the arrangement of treatments were different levels of crude glycerin supplementations 0, 10.5, and 21% of total mixed ration (TMR) and chitosan levels were added at 0, 1, and 2% dry matter (DM) of substrate. Then, 0.5 g of TMR substrates were added into 40 mL bottles, together with respective doses of chitosan and then incubated at 39 °C. The dietary treatments were performed in three replicates within the incubation, and incubations were repeated on three separate days (runs). No interactions were found between crude glycerin and chitosan doses in terms of theoretical maximum of asymptotic gas production (b), rate of gas production (c), the discrete lag time prior to gas production (L), or the cumulative gas production at 96 h of incubation (p > 0.05). Cumulative gas production at 96 h of incubation was similar among the doses of crude glycerin and levels of chitosan, which ranged from 64.27 to 69.66 mL/g DM basis of substrate (p > 0.05). The concentration of ruminal NH3-N after 2 and 4 h of incubation ranged from 14.61 to 17.10 mg/dL and did not change with the addition of crude glycerin with chitosan (p > 0.05). The concentration of CH4 after 2 h of incubation did not change among treatments (p > 0.05), whereas after 4 h of incubation, CH4 synthesis was significantly reduced by enhancing doses of crude glycerin and chitosan (p < 0.05). The combination of 21% of crude glycerin in TMR with 2% chitosan depressed CH4 production as much as 53.67% when compared to the non-supplemented group. No significant crude glycerin and chitosan interaction effect was detected for in vitro digestibility of nutrients after incubation for 12 and 24 h using the in vitro gas production technique (p > 0.05). In addition, no significant changes (p > 0.05) were observed in total volatile fatty acids, acetate (C2) or butyrate content among treatments and between the main effects of crude glycerin with chitosan. At 4 h of incubation, ruminal C3 content and the C2 to C3 ratio changed significantly when crude glycerin and chitosan was added (p < 0.05). The 21% crude glycerin incorporate into TMR, in combination with 2% additional chitosan, increased C3 content by 26.41%, whereas the ratio of C2 to C3 was reduced by 31% when compared to the control group. Propionate concentration increased by 11.75% when increasing levels of chitosan at 2% of substrate, whereas the C2 to C3 ratio decreased by 13.99% compared to the 0% chitosan group. The inclusion of crude glycerin at 21% in TMR diets with chitosan supplementation at 2% enhanced ruminal propionate concentration and reduced methane production without causing any detrimental effect on the gas kinetics or nutrient digestibility.

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