ORIGINAL

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

Efecto de la edad del pasto kikuyo (Pennisetum clandestinum) y la relación forraje: concentrado sobre la metanogénesis

John Ramírez,¹ M.Sc, Sandra Posada O,^1* Ph.D, Ricardo Noguera,¹ Ph.D.

¹Universidad de Antioquia, Facultad de Ciencias Agrarias, Grupo de Investigación en Ciencias Agrarias-GRICA, AA 1226, Medellín, Colombia.

*Correspondence: slposada@gmail.com

Received: September 2014; Accepted: February 2015.

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.

Key words: Carbohydrates, in vitro digestibility, grass maturity, methane production (Sources: CAB, USDA).

RESUMEN

Objetivo. Evaluar el efecto de la edad del pasto kikuyo (Pennisetum clandestinum) y de la relación forraje/suplemento (F/S) sobre la producción de metano (CH₄), la digestibilidad de la materia seca (DMS) y el perfil de fermentación mediante la técnica in vitro de producción de gas. Materiales y métodos. Seis tratamientos, resultantes de la combinación de los factores edad del pasto (30 y 60 días) y relación F/S (100/0, 75/25 y 50/50), fueron evaluados bajo un diseño factorial 2x3. Las variables respuesta se midieron a las 6, 12, 24 y 48 horas de incubación. Para el análisis de los datos se empleó un diseño de medidas repetidas en el tiempo y las diferencias entre medias se determinaron con el procedimiento LSMEANS de SAS. Resultados. El pasto de menor edad (30 días) fue más digestible, produjo menos CH₄ por gramo de materia seca digestible (MS_d) y más ácidos grasos volátiles totales (AGV) que el pasto de mayor edad (60 días, p<0.05). La reducción en la relación F/S aumentó la DMS y la producción de CH₄ por gramo de MS_d (p<0.05), pero no tuvo efecto estadístico sobre la concentración de AGV (p>0.05). Conclusiones. Bajo condiciones in vitro, con pH próximo a la neutralidad, la mayor edad del pasto reduce la DMS y aumenta la producción de CH₄ por gramo de MS_d, mientras que la reducción en la relación F/S aumenta la DMS y la producción de CH₄ por gramo de MS_d, último hallazgo que contrasta con los reportes in vivo.

Palabras clave: carbohidratos, digestibilidad in vitro, estado de madurez, producción de metano (Fuentes: CAB, USDA).

INTRODUCTION

Methane (CH₄) is one of the main products of ruminal fermentation. It is considered a greenhouse gas (GHG) and represents between 2 and 15% of the energy consumed by ruminants (1). CH₄ is produced in the rumen by a group of microorganisms of the Archaea domain, which use hydrogen (H₂) to reduce carbon dioxide (CO2) to CH₄, maintaining a low H₂ pressure that favors fermentation processes (2).

Most dairy farms in Colombia are based on Kikuyu grass (Pennisetum clandestinum) as the main grazing grass for the animals. Average levels of neutral detergent fiber (NDF), nonstructural carbohydrates (NSC) and crude protein (CP) in Kikuyu are 58.1, 13.4, and 20.5%, respectively (on a dry matter bases (DM)) and fluctuate depending on the agronomic management (3). Because of its composition, Kikuyu grass presents nutritional constraints affecting milk yield and quality. Thus, supplements are commonly offered to improve the availability of NSC and energy in the rumen.

Feeding concentrate supplements to grazing ruminants is associated with a reduction of CH₄ emissions. A change in the relationship feed: concentrate (DM bases) from 47: 53 to 68: 32 increased CH₄ emissions from 538 to 648 g/cow/day (4). Different carbohydrates change the volatile fatty acids (VFA) profile and the production of CH₄. Cellulose and starch degradation leads to glucose formation, which can be fermented in different pathways resulting in varying amounts of H₂. The production of CH₄ per unit of food digested depends on the amount of H₂ formed, according to the stoichiometric ratio 4H₂+CO₂→CH₄+2H₂O. The formation of acetate and butyrate results in 1 and 0.5 moles of CH₄ per mole of fermented glucose, respectively. On the other hand, propionate formation requires a net input of reduced equivalents, decreasing CH₄ production. Fiber fermentation results in the preferential production of acetate, while feeding NSC increases the proportion of propionate (2). The objective of this study was to determine 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

Location. The study was conducted at the Nutrition and Animal Feeding Laboratory facilities of Universidad de Antioquia, Medellin, Colombia.

Substrates and chemical analysis. The substrates tested were Kikuyu grass (Pennisetum clandestinum) and a commercial concentrate supplement formulated for dairy cows. The grass was harvested at Betania farm, owned by Solla SA Company, during vegetative stage by cutting it 10 cm above the ground at 30 and 60 days of age. This farm is located in Santa Rosa de Osos municipality (Ant., Colombia) at 2460 m.a.s.l., with 18°C average temperature (range: 4 to 27°C), 70% relative humidity and 2400 mm average annual precipitation, corresponding to a lower montane wet forest (LMWF). The pasture was managed under a rotational grazing system with chemical fertilization (190, 54, 16, and 9 kg N, P, K, and S/ha/year).

Grass samples were dried in forced air oven at 65°C for 48 hours. Grass and supplement samples were ground to 1 mm for chemical analysis including DM, CP, ether extract (EE), ash (5), NDF (6), acid detergent fiber (ADF), acid detergent lignin (ADL) (7) and gross energy (GE). The GE was assessed in an adiabatic bomb calorimeter (IKA C5000, Rhys International Ltd., United Kingdom). NSC values were obtained by the equation, NSC=100-(%CP+%EE+%Ash %NDF) (8). The chemical description of substrates is presented in table 1.

Experimental treatments. Six treatments, resulting from the combination of pasture age (30 to 60 days) and inclusion level of concentrate supplement (0, 25 and 50%) were evaluated in a 2x3 factorial design. The treatments were: K30-100/0, K30-75/25, K30-50/50, K60-100/0, K60-75/25, and K60-50/50. Descriptors K30 and K60 correspond to Kikuyu age, while 100/0, 75/25 and 50/50 to the F/C ratio (DM basis). Treatments were replicated three times, corresponding to three sources of ruminal inoculum, each obtained from a different animal.

Gas production technique. This technique was conducted following protocols by Posada et al (9). Grass and concentrate supplement were ground to 1 mm and added (0.5 g) to each flask (100 ml) according to the percentages set for each treatment.

The inocula were obtained from three Holstein Friesian adult cows from La Montaña farm, owned by Universidad de Antioquia (San Pedro de los Milagros, Ant., Colombia). The cows were grazing on 42 days old Kikuyu and were provided mineralized salt and water ad libitum. The inocula were collected in the morning (6:00 AM) and transported in thermos preheated with 40°C water to the laboratory, where they were filtered through a cotton mesh, gassed with CO2 and kept in a warm water bath during inoculation.

Preparation of the buffer solution followed recommendations by McDougall (10), using a 9/1 (45/5 ml/ml) buffer/inoculum ratio and (1M) citric acid was added to the mixture to obtain pH 6.5 (11). The flasks were sealed with rubber stoppers and stored in a forced-air oven at 39 °C throughout the fermentation process.

Total gas and CH₄. Total gas and CH₄ production was determined at 6, 12, 24 and 48 h of incubation. Gas pressure generated in the incubation flasks was measured with a digital transducer (Ashcroft 2089QG - Precision Digital Test Gauges, Stratford, CT, USA) and the PSI readings were converted to volume (ml) using the equation

Y = -0.1833 + 0.0598 X² (9).

After reading the pressure, the generated gas was removed from the flasks using a plastic syringe and then stored in plastic bags for later CH₄ analysis. For this purpose, gas samples (100 µl) were taken with a gas-tight syringe and manually injected into a gas chromatograph (Thermo TRACE GC Ultra^TM, Thermo Scientific, USA) equipped with a flame ionization detector (FID). The chromatographic conditions corresponded to a 30 m nonpolar column, 0.32 mm and 0.25 µm, 200°C injection port temperature, 50: 1 split injection mode, 250°C detector temperature, 30°C (5 min) up to 200°C (30°C/min) oven temperature, and helium as a carrier gas (1.0 ml/min). CH₄ data were processed according to Lopez and Newbold (12). CH₄ production (ml) was calculated from total gas volume (ml) and CH₄ concentration. Gas and CH₄ production (ml) was expressed per gram of incubated dry matter (_iDM) and per gram of digestible dry matter (_dDM). CH₄ production was also expressed as a percentage of the incubated GE (_iGE) and incubated digestible energy (_iDE), giving CH₄ a caloric value of 9.45 kcal/L (13).

Dry matter digestibility. DMD was determined gravimetrically at the times of gas pressure readings by filtering the incubation flasks contents using glass crucibles (porosity 1, 100-160 µm) and a vacuum pump. The residues were dried in the crucibles at 60°C for 48 hours in a forced-air oven and weighed.

Fermentation profile and pH. The pH and concentration of acetic, propionic and butyric acids were determined in the liquid after filtration. This liquid was preserved by adding sulfuric acid (98% v/v) until pH 2 was reached. The pH was determined with digital pH meter (Handylab pH 1, Schott® Instruments, SI Analytics GmbH, Germany) before acidification.

To determine VFA, liquid samples (1 µl) were injected into a gas chromatograph (Thermo TRACE GC Ultra^TM, Thermo Scientific, USA) equipped with a 30 m, 0.32 mm and 0.25 microns column. Temperatures were 300°C (FID), 210°C (inlet), and 68°C (1 min), ramp 10°C/min until reaching 140°C (1 min), ramp 40°C/min until reaching 200°C (1 min) (oven). The mode of injection was split and helium was used as a carrier gas (2.5 ml/min).

Statistical analysis. A repeated measures analysis was performed using PROC MIXED of SAS (14). The fixed effects of the model were grass, F/C, and incubation time; the random effect was the ruminal inoculum. Treatment means were compared using PROC LSMEANS of SAS (14).

RESULTS

Table 2 shows the chemical composition of the treatments. The 30 days-old grass had higher CP and NSC content, but lower NDF, ADF and ADL compared to 60 days. The NSC/NDF ratio increased as concentrate increased in the mix.

Tables 3 and 4 show that grass age significantly (p<0.05) affected DMD, cumulative production of gas and CH₄ per gram of _dDM, and the percentage of _iDE transformed into CH₄. Following 48 hours of incubation, the 30 days old grass showed greater DMD (68.97 vs. 65.30%), decreased production of gas (463.56 vs. 499.27 ml) and CH₄ (101.37 ml vs. 109.97) per gram of _dDM, and lower production of CH₄ as a percentage of _iDE (22.83 vs. 24.70%) compared with the 60 days grass.

The F/C ratio significantly affected all variables (p<0.05). The F/C presented a growing trend as grass was reduced and concentrate increased between 0 and 48 hours of incubation. The average CH₄ production in this time frame, regardless of grass age, was 57.40, 73.70, and 81.85 ml (per gram of _iDM) and 96.15, 107.15 and 113.70 ml (per gram of _dDM) for 100/0, 75/25, and 50/50 F/C ratios, respectively. For the same ratios, the average CH₄ production corresponded to 12.50, 16.50 and 18.80% of the _iGE, and 21.30, 23.80 and 26.20% of the _iDE, respectively. After incubation, DMD was 60.55, 68.85 and 72.00% for 100/0, 75/25 and 50/50 F/C ratios, respectively. Results behaved in a similar way when the means of different F/C rates were compared inside each grass age.

Table 5 shows that grass age significantly affected (p<0.05) acetic, propionic and butyric acid concentration, with the highest values for the young (30 days) compared with the 60 days old grass (117.87, 36.83 and 23.60 vs. 104.67, 33.40 and 22.47 mmol/L, respectively). The F/C ratio had no statistical effect on VFA concentration or the acetate/propionate ratio (A/P) (p>0.05), but it significantly affected pH (p<0.05). Treatments including more concentrate had lower pH values (6.50 and 6.52 for 50/50 and 75/25 ratios, respectively, compared with 6.58 for the 100/0 ratio). The grass age*F/C interaction was not significant (p>0.05) for the variables presented in tables 3, 4 and 5.

DISCUSSION

Effect of grass age on production of gas, CH₄, and DMD. Caro and Correa (15) reported a 7.8% increase in NDF and 2.7% reduction in NSC for Kikuyu grass harvested at 32 vs. 58 days of regrowth. In the present study, NDF increase and NSC reduction exceeded the figures reported by those researchers (Table 1). It is expected that forage quality decrease with maturity, which was verified by Caro and Correa (15), who found that changes in the chemical composition affect in situ digestibility at 24h incubation. They reported a 6.7% increase in grass digestibility after 32 days of regrowth compared to 58 days. This is in agreement with the present study, where DMD at 24 hours of incubation was higher for the younger grass (Table 3). Within species, increased harvest age is associated with higher NDF, ADF and ADL levels, and lower CP content (Table 1) as well as DMD reduction.

Chaves et al (16) concluded that CH₄ produced by grazing heifers was significantly affected by the type of grass, reflecting differences in maturity and chemical composition and confirming that CH₄ production is primarily a function of the amount of cell wall digested in the rumen. In the present study, pasture age significantly affected gas and CH₄ production (per gram of _dDM) and CH₄ (as percent of _iDE) (Tables 3 and 4). According to Sun et al (17), CH₄ production should be expressed in relation to _dDM, instead of _iDM. They reported that Cichorium intybus and Lolium perenne fodder had similar CH₄ production on _iDM basis despite their differences in NDF (28.1 and 49.9%, respectively) and the higher digestibility of Cichorium intybus. In the present study, treatment K30-100/0 resulted in lower NDF, ADF and ADL (Table 2), higher accumulated digestibility at 48 hours (p<0.05), and similar gas and CH₄ production (per gram of _iDM) compared to treatment K60-100/0. However, when gas production and CH₄ were expressed per gram of _dDM (Tables 3 and 4) the values were statistically higher for the older grass (60 days) (p<0.05). Accordingly, highly digestible forage can produce the same gas and CH₄ per gram of _iDM; which switches when production of both is expressed as per gram of _dDM. The higher digestibility of the substrate mathematically reduces gas and CH₄ production and biologically increases Y_ATP. Van Soest et al (18) defined microbial efficiency as the proportion of the substrate energy fixed by the cells and, thus, it is inversely related to VFA and CH₄ production.

Effect of F/C ratio on DMD, CH₄, and gas production. The increase in gas production and CH₄ per gram of _iDM observed with decreasing F/C ratios (Tables 3 and 4) can be attributed to higher NSC vs. NDF in the incubated substrate (Table 2). The high NSC proportion in the concentrate supplement (Table 1) explains its upward trend as F/C decreased, resulting in a higher NSC/NDF ratio (Table 2). Pellikaan et al (19) also found increased in vitro CH₄ and gas production from substrates with low NDF and high starch content. They obtained 399.9 and 66.8 ml for gas and CH₄, respectively, from potato starch (per gram of incubated organic matter); while values were 273.3 and 47.6 ml, respectively, from grass silage. Navarro-Villa et al (11) also reported increased production of gas and CH₄ for incubated barley grain compared with barley straw and grass silage. In the present study, the increased gas and CH₄ production from substrates with concentrate supplement can be explained by their higher DMD (Table 3). However, it is important to note that this increased digestibility was not sufficient to generate a downward trend in gas and CH₄ output (at 48 hours of incubation) as F/C decreased when they were expressed as per gram of _dDM. The only exception to this was gas production per gram of _dDM from grass harvested at 60 days (Tables 3 and 4). Lower levels of incubated protein as F/C decreased -especially for 30 days grass- may be associated with reductions in CH₄. According Inthapanya and Preston (20), less protein degradation in the incubated substrate decreases CH₄ production. However, this effect was smaller in the present study compared to that generated by a higher NSC/NDF ratio, as previously stated.

Increased in vitro CH₄ production per gram of _iDM and _dDM in treatments with low F/C (Table 4) is not in agreement with in vivo results (21), where it is accepted that rapidly fermenting diets decrease CH₄ production. The effect of these diets on in vivo CH₄ production is explained by a decrease in ruminal pH, affecting the growth of methanogens, protozoa (22) and cellulolytic bacteria (23), and increasing passage rate, which reduces the number of protozoa and thus interspecies transfer of H₂ and methanogenesis (24). The in vitro gas technique does not allow replacing the liquid and/or solid fractions, and a buffer solution is added to guarantee pH stability between 6.5 to 7.0 (Table 5). Both things may explain the divergence between in vitro and in vivo results regarding CH₄ for increasing NSC levels. This was reported by Navarro-Villa et al (11), who measured 70% higher CH₄ for grains compared with forages incubated in vitro, while McGeough et al (25) reported lower CH₄ production from ruminants fed cereal based diets.

The CH₄ increase, as a percentage of the _iGE and _iDE, is consistent with CH₄ emissions per gram of _iDM and _dDM (Table 4). Values ranged from 8.8 (K60-100/0) and 15.1% (K60-50/50) of _iGE after 24 hours of incubation, surpassing the results by Bhatta et al (26) who evaluated mixed diets also at 24 hours of incubation and obtained between 4.4 and 7.8% of GE.

Effect of grass age on fermentation profile. Glucose is a common intermediary among carbohydrates degraded in the rumen and their final products (VFA, CO2 and CH₄). The mechanism that controls glucose partition between different VFA depends on food composition (2). Tropical grasses usually grow and mature faster, rapidly turning into fibrous and less digestible. The lower NDF content (Table 2) and the larger DMD (Table 3) of 30-day grass treatments is consistent with the higher VFA concentration, as reported in Table 5, explaining the significant effect of age on acetate, propionate and butyrate production (p<0.05).

Effect of F/C ratio on fermentation profile. When fiber-rich diets change to diets based on concentrates, a decrease can be expected in ruminal pH (27), number of fibrolytic bacteria (23) and the A/P ratio (28). Although in the present study F/C significantly affected the pH, it tended to remain high and stable due to the HCO3-/CO2 balance of the buffer solution, which could explain the lack of significant effect of the F/C ratio on the A/P ratio (Table 5). Citric acid has been suggested to overcome the buffer effect and perform in vitro evaluations at pH lower than 6 (11). However, that strategy did not work in the present study; pH readings at 6 hours of incubation (Table 5) were higher than those recorded at the beginning (pH 6.5). This could be explained by citric acid being rapidly fermented by rumen microorganisms to CO2 and acetic acid (29).

The in vitro gas technique does not remove VFA via absorption, so its concentration depends on the fermentation rate. Since DMD increases with incubation time, a higher VFA concentration is expected as fermentation continues. However, this was not observed in this study (Table 5). It can be assumed that microorganisms degraded VFA to obtain energy, as observed by Wang et al (30), which would explain the stability of its concentration over the incubation time.

The results of this study confirm that grass age affects DMD, CH₄, gas production, and fermentation profile due to a higher proportion of cell wall. A reduction of F/C ratio simultaneous with a high NSC/NDF ratio improves substrate digestibility and increases CH₄ and gas production, contradicting the in vivo reports. The pH, which is controlled by the buffer solution, hinders the study of the effect of F/C ratio on methanogenesis in in vitro systems. Therefore, for future studies we suggest to change the formulation of the buffer solution to enable the assessments of mixed diets at a pH close to in vivo.

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