Roughage to Concentrate Ratio and Saccharomyces cerevisiae Inclusion Could Modulate Feed Digestion and In Vitro Ruminal Fermentation

The objective of this research was to investigate the effect of the roughage-to-concentrate (R:C) ratio and the addition of live yeast (LY) on ruminal fermentation characteristics and methane (CH4) production. The experimental design was randomly allocated according to a completely randomized design in a 4 × 4 factorial arrangement. The first factor was four rations of R:C at 80:20, 60:40, 40:60, and 20:80, and the second factor was an additional four doses of Saccharomyces cerevisiae (live yeast; LY) at 0, 2.0 × 106, 4.0 × 106, and 6.0 × 106 colony-forming unit (cfu), respectively. For the in vitro method, during the incubation, the gas production was noted at 0, 1, 2, 4, 6, 8, 10, 12, 18, 24, 48, 72, and 96 h. The rumen solution mixture was collected at 0, 4, 8, 12, and 24 h of incubating after inoculation. Cumulative gas production at 96 h was highest in the R:C ratio, at 20:80, while the addition of LY improves the kinetics and accumulation of gas (p > 0.05). Maximum in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD) at 24 h after incubation were achieved at the R:C ratio 20:80 and the addition of LY at 6 × 106 cfu, which were greater than the control by 13.7% and 12.4%, respectively. Ruminal pH at 8 h after incubation decreased with an increased proportion of concentrates in the diet, whereas it was lowest when the R:C ratio was at 20:80. Increasing the proportion of a concentrate diet increased total volatile fatty acid (TVFA) and propionic acid (C3), whereas the acetic acid (C2) and C2-to-C3 ratios decreased (p < 0.05). TVFA and C3 increased with the addition of LY at 6 × 106 cfu, which was greater than the control by 11.5% and 17.2%, respectively. No interaction effect was observed between the R:C ratio and LY on the CH4 concentration. The calculated ruminal CH4 production decreased with the increasing proportion of concentrates in the diet, particularly the R:C ratio at 20:80. The CH4 production for LY addition at 6 × 106 cfu was lower than the control treatment by 17.2%. Moreover, the greatest populations of bacteria, protozoa, and fungi at 8 h after incubation were found with the addition of LY at 6 × 106 cfu, which were higher than the control by 19.0%, 20.7%, and 40.4%, respectively. In conclusion, a high ratio of roughage and the concentrate and addition of LY at 6.0 × 106 cfu of the total dietary substrate could improve rumen fermentation, improve feed digestibility, and reduce the CH4 production.

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

South African Journal of Animal Science ◽

10.4314/sajas.v46i3.13 ◽

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.

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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

Canadian Journal of Animal Science ◽

10.1139/cjas-2017-0079 ◽

2018 ◽

Vol 98 (3) ◽

pp. 477-487 ◽

Cited By ~ 1

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.

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In Vitro Ruminal Fermentation and Degradability of Rice Husk on Rice Bran Substitution

ANNALES BOGORIENSES ◽

10.14203/ann.bogor.2020.v24.n1.50-58 ◽

2020 ◽

Vol 24 (1) ◽

pp. 50

Author(s):

Rusli Fidriyanto ◽

Roni Ridwan ◽

Wulansih Dwi Astuti ◽

Rohmatussolihat Rohmatussolihat ◽

Nurul Fitri Sari ◽

...

Keyword(s):

Organic Matter ◽

Rice Husk ◽

Rice Bran ◽

Dry Matter ◽

Acid Level ◽

Gas Production ◽

Ruminal Fermentation ◽

Fatty Acid Production ◽

Dry Matter Digestibility

Rice bran is a by-product of the rice milling process and has been well used as livestock feed. Rice bran is often adulterated with rice husk. The objective of this study was to evaluate the in vitro ruminal fermentation characteristics of rice bran with various compositions of rice husk and assess the relationship between rice husk addition and rice bran quality. The experiment was arranged in a completely randomized design with rice husk addition as a factor and three replications. Data of proximate value, gas production, ruminal degradability, and volatile fatty acid production were analyzed by analysis of variance. Moreover, significant effects of each treatment in the in vitro fermentation were further analyzed by Duncan's multiple range test (P<0.05). It was shown that the addition of rice husk to rice bran could increase acetic acid level, but it reduced potential gas production, gas production rate, organic matter and dry matter digestibility, and propionic acid level. Interestingly, the linear regression of dry matter digestibility, organic matter digestibility, and potential gas production showed the high adjusted R2 values. Moreover, this study also revealed that 10% of rice husk substitution on rice bran could significantly reduce the dry matter digestibility.

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Manipulation of In Vitro Ruminal Fermentation and Feed Digestibility as Influenced by Yeast Waste-Treated Cassava Pulp Substitute Soybean Meal and Different Roughage to Concentrate Ratio

Fermentation ◽

10.3390/fermentation7030196 ◽

2021 ◽

Vol 7 (3) ◽

pp. 196

Author(s):

Gamonmas Dagaew ◽

Anusorn Cherdthong ◽

Sawitree Wongtangtintharn ◽

Metha Wanapat ◽

Chanon Suntara

Keyword(s):

Soybean Meal ◽

Bacterial Population ◽

Gas Production ◽

Neutral Detergent Fiber ◽

Partial Replacement ◽

In Vitro Digestibility ◽

Ruminal Fermentation ◽

Cassava Pulp ◽

Feed Digestibility

Cassava pulp (CS) is high in fiber and low in protein; hence, improving the nutritive value of CS is required to increase its contribution to enhancing ruminant production. The present work hypothesized that CS quality could be enhanced by fermentation with yeast waste (YW), which can be used to replace soybean meal (SBM), as well as lead to improved feed utilization in ruminants. Thus, evaluation of in vitro ruminal fermentation and feed digestibility, as influenced by YW-treated CS and different roughage (R) to concentrate (C) ratios, was elucidated. The design of the experiment was a 5 × 3 factorial arrangement in a completely randomized design. Each treatment contained three replications and three runs. The first factor was replacing SBM with CS fermented with YW (CSYW) in a concentrate ratio at 100:0, 75:25, 50:50, 25:75, and 0:100, respectively. The second factor was R:C ratios at 70:30, 50:50, and 30:70. The level of CSYW showed significantly higher (p < 0.01) gas production from the insoluble fraction (b), potential extent of gas production (a + b), and cumulative gas production at 96 h than the control group (p < 0.05). There were no interactions among the CSYW and R:C ratio on the in vitro digestibility (p > 0.05). Furthermore, increasing the amount of CSYW to replace SBM up to 75% had no negative effect on in vitro neutral detergent fiber degradability (IVNDFD) (p > 0.05) while replacing CSWY at 100% could reduce IVNDFD (p > 0.05). The bacterial population in the rumen was reduced by 25.05% when CSYW completely replaced SBM (p < 0.05); however, 75% of CSWY in the diet did not change the bacterial population (p > 0.05). The concentration of propionate (C3) decreased upon an increase in the CSYW level, which was lowest with the replacement of SBM by CSYW up to 75%. However, various R:C ratios did not influence total volatile fatty acids (VFAs), and the proportion of VFAs (p > 0.05), except the concentration of C3, increased when the proportion of a concentrate diet increased (p < 0.05). In conclusion, CSYW could be utilized as a partial replacement for SBM in concentrate diets up to 75% without affecting gas kinetics, ruminal parameters, or in vitro digestibility.

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

Animals ◽

10.3390/ani10010037 ◽

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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Effect of Cassava Residue Substituting for Crushed Maize on In Vitro Ruminal Fermentation Characteristics of Dairy Cows at Mid-Lactation

Animals ◽

10.3390/ani10050893 ◽

2020 ◽

Vol 10 (5) ◽

pp. 893 ◽

Cited By ~ 1

Author(s):

Yuhui Zheng ◽

Shenglin Xue ◽

Yanyan Zhao ◽

Shengli Li

Keyword(s):

Dairy Cows ◽

Gas Production ◽

The Other ◽

Ruminal Fermentation ◽

Negative Effects ◽

N Content ◽

Dry Matter Digestibility ◽

Substitution Ratio ◽

Cassava Residue

This study was conducted to investigate the effect of using cassava residue to replace crushed maize on in vitro fermentation characteristics of dairy cows at mid-lactation and provide guidance for its utilization. The study included seven treatments with four replicates, which used 0% (control, CON), 5%, 10%, 15%, 20%, 25% and 30% cassava residue to replace crushed maize (air-dried matter basis), respectively. A China-patented automated trace gas recording system was used to perform in vitro gas tests; rumen fluids were collected from three dairy cows at mid-lactation. In vitro dry matter digestibility (IVDMD), gas production (GP), pH, ammonia–N (NH3-N) and microbial protein (MCP) content were analyzed after in vitro incubating for 3, 6, 12, 24 and 48 h, respectively; volatile fatty acid (VFA) content was analyzed after in vitro culturing for 48 h. The results showed that with the increase of substitution ratio of cassava residue, the asymptotic gas production (A) increased quadratically (p < 0.05), cumulative gas production at 48 h (GP48) and the maximum rate of substrate digestion (RmaxS) increased linearly and quadratically (p < 0.05), the time at which the maximum gas production rate is reached (TRmaxG) increases linearly (p < 0.05). In addition, asymptotic gas production in 30% was significantly higher than the other treatments (p < 0.05), RmaxS in 25% and 30% were significantly higher than CON, 5% and 10% (p < 0.05). In addition, with the increase of substitution ratio of cassava residue, when in vitro cultured for 6 h and 12 h, NH3–N content decreased linearly and quadratically (p < 0.05). NH3–N content in 30% was significantly lower than the other treatments except 20% and 25% (p < 0.05) after cultivating for 6 h. Moreover, the content of iso-butyrate, iso-valerate, valerate and total VFA (tVFA) decreased linearly and quadratically (p < 0.05), acetate decreased quadratically (p < 0.05) with the increase of substitution ratio of cassava residue. In conclusion, when the cassava residue substitution ratio for crushed maize was 25% or less, there were no negative effects on in vitro ruminal fermentation characteristics of dairy cows at mid-lactation.

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Effects of Tithonia diversifolia on in vitro methane production and ruminal fermentation characteristics

Animal Production Science ◽

10.1071/an15560 ◽

2016 ◽

Vol 56 (3) ◽

pp. 437 ◽

Cited By ~ 3

Author(s):

S. A. Terry ◽

R. S. Ribeiro ◽

D. S. Freitas ◽

G. D. Delarota ◽

L. G. R. Pereira ◽

...

Keyword(s):

Fatty Acids ◽

Volatile Fatty Acids ◽

Fold Increase ◽

Gas Production ◽

Tithonia Diversifolia ◽

Ruminal Fermentation ◽

Total Volatile ◽

Treatment Groups ◽

Ch4 Production

The present study examined the effects of Tithonia diversifolia on in vitro methane (CH4) production and ruminal fermentation characteristics. The experiment was conducted as a completely randomised design (CRD) using a control (0% T. diversifolia) and three treatment groups with different concentrations (6.9%, 15.2%, 29.2%) of T. diversifolia, which replaced up to 15.2% and 14% dry matter (DM) of fresh sugarcane and concentrates, respectively. Ruminal fluid was obtained from two ruminally cannulated non-lactating Holstein × Zebu heifers maintained on a diet consisting of T. diversifolia, fresh sugarcane and 4 kg of concentrates. The inclusion of T. diversifolia had no effect (P ≥ 0.15) on cumulative gas production (mL, mL/g incubated DM, mL/g digested DM) or in vitro DM disappearance (%). Carbon dioxide (%, mL, mL/g incubated DM) linearly decreased (P ≤ 0.001) and CH4 (%, mL, mL/g incubated DM) quadratically increased (P ≤ 0.01) with increasing concentrations of T. diversifolia replacing fresh sugarcane and concentrates. The total volatile fatty acids (mM) and acetate (A) proportion of total volatile fatty acids (mmol/100 mmol) linearly increased (P < 0.01) with the increasing inclusion of T. diversifolia. Butyrate (mmol/100 mmol) increased quadratically (P ≤ 0.02), while propionate (P; mmol/100 mmol) decreased quadratically (P < 0.02). The A : P ratio increased linearly (P < 0.0001) with increasing amounts of T. diversifolia in the diet. These results indicated that increasing the amount of Tithonia diversifolia in the substrate DM increased the A : P ratio, which resulted in a six-fold increase of CH4 production when fresh sugarcane and concentrates were replaced at up to 15.2% and 14% (DM basis), respectively.

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Methane production and in vitro digestibility of low quality forages treated with a protease or a cellulase

Animal Production Science ◽

10.1071/an14988 ◽

2016 ◽

Vol 56 (10) ◽

pp. 1700

Author(s):

J. M. Cantet ◽

D. Colombatto ◽

G. Jaurena

Keyword(s):

Methane Production ◽

Dry Matter ◽

Gas Production ◽

In Vitro Digestibility ◽

Ruminal Fermentation ◽

Neutral Detergent Fibre ◽

Dry Matter Digestibility ◽

Application Rates ◽

The Impact

The objective was to assess the impact of application of two enzyme mixtures on the in vitro dry matter digestibility, neutral detergent fibre digestibility, net cumulative gas production and methane production after 24 h of incubation of Milium coloratum (formely Panicum coloratum) and a Patagonian meadow grassland. A protease (Protex 6-L) and a fibrolytic enzyme (Rovabio) were assessed at three application rates (30, 60 and 90 mg/100 mL of distiller water) on the substrates. Meadow samples were higher to Milium ones (P < 0.05) for in vitro dry matter digestibility and net cumulative gas production at 24 h. Nevertheless, Milium was ~11% higher than meadow (P < 0.05) for methane when expressed as a proportion of digested dry matter (g/kg). Rovabio did not induce differences in any variable, but the addition of Protex reduced (P < 0.05) in vitro dry matter digestibility in both substrates without bringing about differences in methane production. Collectively, the addition of these enzymes did not benefit in vitro ruminal fermentation of low quality forages.

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44 Effect of Quebracho (Schinopsis balansae) extract inclusion in a high roughage diet upon in vitro gas production

Journal of Animal Science ◽

10.1093/jas/skz397.122 ◽

2020 ◽

Vol 98 (Supplement_2) ◽

pp. 53-54

Author(s):

Luiz Fernando Dias Batista ◽

Madeline E Rivera ◽

Aaron B Norris ◽

Jordan Adams ◽

Roberta Cracco ◽

...

Keyword(s):

Condensed Tannin ◽

Basal Diet ◽

Gas Production ◽

Random Coefficients ◽

Feed Additives ◽

Ruminal Fermentation ◽

Ch4 Production ◽

In Vitro Gas Production ◽

Schinopsis Balansae

Abstract The utilization of natural plant secondary compounds as feed additives in animal nutrition has been extensively studied because of their ability to modify digestive and metabolic functions. Condensed tannin (CT) supplementation can potentially alter ruminal fermentation, and mitigate methane (CH4) emissions. The objective of this study was to determine the effect of quebracho CT extract (QT; Schinopsis balansae) within a roughage-based diet on overall fermentability and CH4 production utilizing the in vitro gas production technique (IVGP). Twenty rumen cannulated steers (227 ± 19 kg) were randomly assigned to four dietary treatments (n=4): QT at 0, 1, 2, and 3% of DM (QT0, QT1, QT2, and QT3). A roughage-based diet containing 88% bermudagrass hay and 12% concentrate was fed daily at 2.1% of shrunk body weight. The animals were adapted to the basal diet for 24-d then introduced to predetermined treatments for 35d. Rumen inoculum was collected weekly from each steer to perform the incubations. Two hundred milligrams of air-dried base diet were incubated for 48-h with a composite rumen inoculum for each treatment over 5 wk. Kinetic analysis of cumulative 48h gas production was performed using Gasfit. Measurements of CH4 were performed via gas chromatography and digested residue was determined post-incubation. Data were analyzed using a random coefficients model. Total gas production was higher for QT0 compared to QT1 and QT3 (P = 0.001), but not different from QT2 (P = 0.554). The fractional rate of gas production was higher for QT2 compared to QT0 (P = 0.011). First and second pool gas production decreased linearly as QT inclusion increased (P = 0.042 and 0.010, respectively). There was no dietary effect in ivNDFD (P = 0.567). However, there was a linear tendency to decrease CH4 production with the addition of QT (P=0.071) likely due to changes in the microbial population.

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Effects of Kikuyu grass (Pennisetum clandestinum) age and different forage: concentrate ratios on methanogenesis

Revista Mvz Córdoba ◽

10.21897/rmvz.43 ◽

2015 ◽

pp. 4726-4738 ◽

Cited By ~ 2

Author(s):

John Ramírez ◽

Sandra Posada O ◽

Ricardo Noguera

Keyword(s):

Dry Matter ◽

Volatile Fatty Acids ◽

Gas Production ◽

Repeated Measure ◽

Pennisetum Clandestinum ◽

Dry Matter Digestibility ◽

Ch4 Production ◽

Kikuyu Grass

ABSTRACT Objective. To evaluate the effect of Kikuyu grass (Pennisetum clandestinum) harvested at two different ages and three forage: concentrate supplement ratios (F/C) on methane (CH4) production, dry matter digestibility (DMD), and fermentation profile using the in vitro gas production technique. Materials and methods. six treatments, resulting from the combination of pasture age (30 or 60 days) and F/C (100/0, 75/25, or 50/50) were evaluated using a 2x3 factorial design. The response variables were measured 6, 12, 24 and 48 hours after incubation. A repeated-measure over time design was used to analyze the data, and differences between means were determined with the LSMEANS procedure of SAS. Results. the youngest grass (30 days) was more digestible, produced less CH4 per gram of digestible dry matter (dDM) and more total volatile fatty acids (VFA) compared to the oldest grass (60 days; p <0.05). Reductions of the F/C ratio increased DMD and CH4 production per gram of dDM (p<0.05) but had no significant effect on VFA concentration (p>0.05). Conclusions. under in vitro conditions and pH close to neutrality, the older grass reduces DMD and increases CH4 production per gram of dDM, while a F/C reduction increases DMD and CH4 production per gram of dDM, which differs with reports conducted in vivo.

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