Effect of Sambucus peruviana (extracts and forage) and oils on the kinetics of degradation and in vitro fermentation of diets for dairy cattle

N Arias-Ortiz, R Barahona-Rosales and DM Bolívar-Vergara*

Department of Animal Production, Faculty of Agricultural Sciences, Universidad Nacional de Colombia, Medellín Campus, Colombia
* dmboliva@unal.edu.co

Summary

The objective of this experiment was to evaluate the effect of the inclusion of different raw materials in diets for dairy cattle on the fermentation kinetics and the degradation of dry matter (DM) through the in vitro gas production technique. The control treatment was Cenchrus clandestinus plus commercial concentrate (70:30 w/w). Other treatments were fish (FO) and linseed (LO) oils added at 5% of DM, Sambucus. peruviana forage added at 10 (Sam10%) and 20% (Sam20%) of diet DM, aqueous S. peruviana extract added at 2 (EH2%) and 4% (EH4%) of the diet and ethanolic S. peruviana extract added at 2% (ET2%) and 4% (ET4%) of the diet. There were differences (P<0.05) for DM disappearance at 48 hours, going from 61 to 69% for ET4% and Sam20%, respectively and for total gas production going from 151.9 to 203 ml for ET4% and LO, respectively. There were no differences (P>0.05) for lag time with values between 3.9 (Sam10%) and 4.7 (ET2%). Methane emission differed (P<0.05), both in total production at 48 hours (mg) and per gram of incubated DM and ranged from 7.72 to 10.47 for Sam 20% and EH4% and from 12.44 to 16.08 for Sam20% and LO, respectively. The ET4% treatment had lower emissions (P<0.05) than FO, LO, EH2% and control treatments. With Sam20%, lower emissions (P<0.05) were obtained than FO, LO, EH2% and EH4%. The addition of ethanolic extracts of S. peruviana reduces the production of CH ₄ per gram of degraded DM without affecting DM degradability.

Keywords: additives, methane, gas production, ruminants

Introduction

Livestock production is one of the main activities in Colombia, which contributes the most to the social and economic development and occupies more than 70% of the area with an agricultural vocation (Zuluaga 2013). Unlike meat farming, specialized milk systems make better use of the grazing area. However, the feeding of grazing ruminants in the Colombian high tropics is limited to the use of few plant species such as Kikuyu grass (Cenchrus clandestinus) or Ryegrass (Lolium multiflorum ), the quality and production, with environmental conditions and or management practices, seldom allowing for the optimal performance of the animal or with the need for the use of high dosages of external inputs such as balanced feed, veterinary drugs and synthetic fertilizers (Blanco et al 2005). For this reason, it is necessary to search for forage alternatives such as non-traditional plant species, such as shrubs, typically used in livestock farms as part of living fences or to provide shade and which could contribute to reduce the environmental footprint of cattle farms, such as methane (CH₄) emissions, one of the main greenhouse gases (GHG) resulting from enteric fermentation. Mitigating the production of this gas represents a challenge for the agricultural activity around the world, and a lot of effort has been made to identify strategies to reduce the emissions of this gas. Among the alternatives for ruminal methane mitigation are plant secondary metabolites (Bodas et al 2012; Santra et al 2012) and there is evidence that certain natural compounds have antimicrobial properties, which could work against rumen methanogenic groups (Santra et al 2012). Compounds such as saponins, tannins and essential oils can reduce ruminal methane production in vitro (Goel et al 2008; Busquet et al 2006; Tana et al 2011, Molina et al 2013) and the level of reduction varies depending on the concentration and type of metabolite present. The objective of this work was to evaluate the inclusion of oils, S. peruviana forage or its extracts on in vitro degradation of dry matter (DM) with emphasis on gas production, dry matter degradation and methane production.

Materials and Methods

Experimental diets

Forages were collected at "La Montaña" farm of the Universidad de Antioquia, located in the municipality of San Pedro de los Milagros, department of Antioquia, Colombia with an ecological classification of Low Montane Humid Forest (bh-MB), an average temperature of 15°C, a precipitation of 1575 mm and located 2425 m above sea level. The forages collected from a silvopastoral system were 35-day-old Kikuyu grass (Cenchrus clandestinus), and elderberry (Sambucus peruviana) collected with a regrowth age of 90 days. The concentrate sample was collected from feed manufactured for high-producing Holstein breed cows, composed mainly of corn, soybean meal, cottonseed and mineralized salt. Extracts obtained from S. peruviana was made from dried material placed in contact with ethanol for 10 minutes or with water for two hours depending on the treatment. The extracts were subsequently filtered and the filtrate was subjected to roto evaporation and percolation to eliminate the solvent.

A basal diet was formulated for lactating cows that were grazing on Cenchrus clandestinus pastures and supplemented with a commercial concentrate (14% PC) in a ratio of 70% grass and 30% concentrate. Starting from this basal (control) diet, eight additional treatments were generated by the addition of different feeds or aqueous or ethanol S. peruviana extracts. The final treatments were fish (FO) and linseed (LO) oils added at 5% of DM, S. peruviana forage added at 10 (Sam10%) and 20% (Sam20%) of diet DM, aqueous S. peruviana extract added at 2 (EH2%) and 4% (EH4%) of the diet and ethanolic S. peruviana extract added at 2% (ET2%) and 4% (ET4%) of the diet.

Laboratory analyses

Bromatological analyzes were carried out in the Bromatology Laboratory of the Universidad Nacional de Colombia, Medellín campus. Samples were dried in a forced ventilation oven at a temperature of 65°C for 72 h and then ground to pass a 1 mm sieve, using a Romer RAS mill (Romer Labs, Mexico). In each sample the following analyses were carried out: dry matter content (DM, in a forced air oven at 105°C until reaching constant weight, based on ISO 6496), crude protein (CP, by the Kjeldahl method, according to NTC 4657), fiber insoluble in neutral and acid detergent (NDF and ADF) respectively, according to the sequential technique described by Van Soest et al (1991), lignin (Van Soest et al 1963) and ether extract (EE, by Soxhelet extraction by immersion NTC 668). The ash content (CEN) was determined by direct incineration in a muffle at 500°C, according to AOAC 942.05. Content of calcium (Ca) and phosphorus (P) was determined by Spectrophotometry AA and UV-VIS (based on NTC 5151 and 4981), respectively and protein insoluble in neutral detergent by the gravimetric and Kjeldahl method. Finally, the gross energy content was determined using the energy content of the nutrients.

Tannin content was determined in the Animal Nutrition and Feeding Research Laboratory (NUTRILAB) at the Sede de Investigación Universitaria (SIU) of the Universidad de Antioquia, Medellín, Colombia, following the butanol-HCl method (Terrill et al 1992), modified by Barahona (1999). For thequantification of soluble tannins, samples averaging 21.3 mg were weighed in triplicate and placed in 15-ml Falcon tubes. then, 4 ml of 70% acetone was added and the Falcon tube was vortexed for 10 seconds. Then, 3 to 4 ml of diethyl ether were added, and samples were vortexed for 10 seconds and then centrifuged at 1000 RPM for 5 minutes. The upper layer (acetone + ether) was removed with a pasteur micropipette and four ml of distilled water was added to each tube, to obtain a final volume of 5 ml. Next, samples were vortexed again for 10 seconds and centrifuged at 1800 RPM for 5 minutes. The supernatant was recovered, and the solid residue was preserved for subsequent determination of insoluble tannins. For determination of soluble tannins, 0.5 ml of each sample was mixed with 4.5 ml of a 95:5 butanol-HCl solution. The resulting solutions were then heated in a water bath at 95ºC for 1 h, and once cooled, read in a spectrophotometer at 550 nm using a blank sample as control.

For thequantification of insoluble (bound) tannins, the tubes containing the solid residues were further centrifuged for 6 minutes at 3500 RPM. After complete elimination of the liquid, five ml of butanol were added, shaking to break up the pellet and tubes were placed on a water bath for 45 minutes at a temperature of 95ºC, centrifuging again for 6 minutes at 3500 RPM. Samples were read in a spectrophotometer against a blank sample at 550 nm. An extra sample was included to be used as a blank, both for the soluble and insoluble tannins. The treatment of this was the same as that of the samples for analysis, but instead of butanol HCl, butanol-H₂O was used as a blank reagent.

Gas production kinetics

The in vitro gas production technique was carried out in the animal nutrition laboratory of the Universidad Nacional de Colombia, Medellín, following the procedure of Theodorou et al (1994). To do this, six 0.7g samples of each diet were weighed and packed in previously weighed ANKON SKU F57 fiber bags with a porosity of 25 microns, which were incubated for a period of 48 hours.

The ruminal fluid was obtained from two fistulated Holstein cows, which were receiving a diet based on Kikuyo grass (Cenchrus clandestinus) with 35 days of regrowth and concentrate for lactating cows between 100 and 200 days in milk. The collection of ruminal fluid was carried out at the La Montaña farm located in San Pedro de los Milagros, Antioquia. Once removed from the rumen, the digesta was quickly squeezed to obtain the ruminal fluid which was immediately stored in thermoses previously heated at 39°C for transport to the laboratory. Once there, the ruminal fluid was re-filtered through a cotton cloth and transferred to an Erlenmeyer flask immersed in a water bath at 39°C, with constant CO ₂ gassing.

To carry out the in vitro gas production experiment, 110 ml glass bottles were used that previously received the corresponding substrate. With the aid of a graduated syringe, 45 ml of the culture medium and 5 ml of rumen fluid were added and bottles were sealed with 14-mm rubber lids. For every 10 bottles with substrate, medium and inoculum, there was one bottle that only contained culture medium and inoculum, or "blanks", included with the purpose of correcting the pressure generated by gassing with CO ₂ and the pressure produced by the presence of microorganisms from the rumen fluid.

Pressure (mm Hg) readings were taken at 10 times ( i.e 2, 4, 6, 9, 12, 18, 24, 30, 36 and 48 h post-inoculation), with the help of a pressure transducer (Ashcroft Inc, USA), connected to a digital reader and a three-way valve. The first outlet was connected to a 10 mm hypodermic needle that was inserted into the bottles, the second to the pressure transducer and the third to a plastic syringe that was used for volume measurement. The volume of gas collected up to 48 hours was accumulated in airtight and sterilized 250 ml bags. Once the readings were carried out, the bottles were shaken manually and placed back in the water bath. To correct the volume of gas production at different times, the equation: Y=-0.009X2+3.6932X-0.0338 was used, where x is the pressure at each measurement point.

To estimate dry matter degradation, the contents of bottles removed from fermentation at different times ( i.e 12, 24, and 48 h) were recovered, dried in a forced air oven at 65°C for 48 hours and then weighed using an analytical balance with four digits of precision (OHAUS, Mexico). Then by relating the initial and final DM weights, the percentage dry matter disappearance (DMD) was calculated.

In vitro methane accumulation was measured using a F10 photoacoustic Multigas Analyzer®, a portable device that allows the measurement of up to nine gases in just a 30 ml sample. A three-way valve was used, the first outlet was connected to a 20 mL plastic syringe that captured air, and the second outlet was attached to a hypodermic needle and a 250 mL IV bag without prior use. The gas samples collected in the bags were analyzed immediately after collection. The total liters of methane produced were calculated using the ideal gas law (López and Newbold 2007), based on the concentration measured (ppm).

Statistical analysis

The gas production data were fitted to the non-linear Gompertz (Lavrencic et al 1997) mathematical model, which has the following equation:

Where, y is equal to the accumulated gas production at a time x, a > 0 is the maximum gas production, the parameter b > 0 is the difference between the initial gas and the final gas at a time x and the parameter c > 0 describes the specific rate of gas accumulation. The practical application of this model requires the conversion of parameters a, b, c into parameters with biological meaning. For the purposes of this study, these are: time to inflection point (TIP, h), gas to inflection point (GIP, ml), maximum gas production rate (MGPR, ml h^-1) and Lag phase (LP or microbial establishment, h). For its estimation, the following formulas were used: TIP = b/c; GIP = a/e; MGPR = (a*c)/e; LP= ((b/c)-(1/c)); where "e" is the Euler number, equivalent to ˜ 2.718281828459.

The results were analyzed using a Randomized Block Design, where 9 diets were evaluated (control, 5% fish oil (FO), 5% linseed oil (LO), 10% and 20% of S. peruviana forage , 2% and 4% S. peruviana aqueous extract and 2% and 4% S. peruviana ethanolic extract, three (12, 24 and 48 h) destructive sampling schedules, and three different inoculums, which were used as a blocking factor. The model used was the following:

Yij = µ +i +ßj +ij

Where,

Yij: Observations of the jth subject assigned to treatment i

µ: General mean of the population

i: Effect of the ith treatment

ßj: Effect of the jth block

ij: Experimental error

The variables analyzed were maximum gas production (a, ml), TIP (h), GIP (ml), MGPR (ml*h^-1), LP (h), disappearance of DM (%) and methane expressed as total methane (,mg per g of incubated DM (g/Kg IDM) and per g of degraded DM (g/Kg DMD) using the PROC GLM procedure of SAS® (SAS Institute Inc., Cary, NC, USA, 2001). Other variables analyzed were the percentage of gross energy and of digestible energy transformed into methane. Comparison of the means was carried out using the Tukey test.

Results and discussion

Nutritional Quality

Both forages used in this experiment have a high protein content. Kikuyu grass, for example, contained 22.9% CP, which is similar to the values between 20.84% and 21.83% reported by Marin et al. (2013) for pastures in this same region. In the case of S. peruviana the CP content was even greater (28.6%), somehow higher than the 25.7% reported by Rivera et al (2013). In turn, the concentrate used was of low protein (15.9%) and high energy (4.31 Mcal/kg) content. It is thought that this combination of feeds, should improve diet degradability parameters as energy sources of immediate availability such as concentrate are combined with those of slow availability such as forages. In terms of protein, fertilized pastures should be a source of easily available protein, subsequently converted into microbial protein (Naranjo et al 2016). The presence of a feed of slow protein availability such as soybean cake should contribute to a synergism in the ruminal metabolism of protein and energy.

In terms of nutritional requirements, the amount of effective fiber in the diet is a critical characteristic of dairy ration formulation. Thus, a ration with a NDF content lower than 26% to 28% or that contains fiber of very small particle size, can lead to metabolic and production problems. The amount of fiber that should be included in the diet of dairy cattle depends on the body condition and weight of the cow, the particle size of the feed, the buffer capacity of the diet, the frequency of intake and economic aspects. The NRC advises that rations for dairy cattle should contain between 19% ADF in high-producing animals to 27% in dry cows. According to the NRC, the NDF should range between 25% and 35%, values lower than those observed in the diets evaluated, which ranged from 40.5% to 52.2% for the Sam20% and ET4% diets, respectively (Table 1). Given the above, the diets evaluated are more adequate for animals of medium production or on the second third of lactation. Lignin contents are high in shrubs such as S. peruviana compared to Kikuyu grass (8.3% and 4% respectively), which indicates that the use of this shrub in high quantities can reduce digestibility. The gross energy contents (Mcal Kg^-1) ranged from 4.23 in the 10% and 20% of S. peruviana forage diets and 4.9 in the ET4% diet. These values exceed the energy contents in diets with S. peruviana and Tithonia diversifolia reported by Rivera et al (2013).

Gas Production

Figure 1 shows that the diets evaluated had very similar fermentative behavior, showing an LP between 3.9 and 4.7 h, suggesting that all diets are highly degradable. The gas production technique, unlike other in vitro techniques, offers the possibility of determining the importance of the soluble fraction of feed in the fermentation process (Posada and Noguera 2005). Early on the fermentation, soluble sugars, are fermented immediately. However, soluble sugars generally only constitute a small part of the potentially digestible material (Stefanon et al 1996). As the fermentation process progresses, the material is hydrated and colonized by rumen microorganisms, which causes different degradation rates depending on the concentration of structural carbohydrates. According to Van Soest (1994), gas production is a reference point to measure the efficiency of the metabolic activity of microorganisms. This explains why in the first 24 h, almost 70% of the total gas was produced in all treatments. At 24 h, there was greater gas accumulation for LO and FO (146 and 141 ml g ^-1 DM, respectively) and less gas accumulation for ET2% with 119.8 ml g ^-1 DM (P< 0.001).

Figure 1. Cumulative gas production per gram of incubated organic matter. AP: Fish oil, AL: Linseed oil, ET2%: 2% ethanolic extract, ET4%: 4% ethanolic extract, EH2%: 2% water extract, EH4%: 4% water extract, Sam10%: Sambucus forage at 10%, Sam20%: Sambucus forage at 20%, Control: Only grass and concentrate

Table 2 shows the parameters obtained by the Gompertz model. For the maximum gas production "a", highly significant differences were observed between treatments (P<0.01). The treatments with the highest production were those with the addition of linseed and fish oil, with accumulations at 48 hours of 203 and 196 ml g ^-1 DM, respectively and that of the lowest gas production was ET4% with 151 ml g ^-1 DM. In work carried out by Molina et al (2013), the gas accumulation for Guinea grass, Leucaena and 70:30 Guinea+Leucaena at 24 h was only 34.4, 35.4 and 24.5 ml g ^-1 OM, respectively, P=0.045), much lower values than those found in this work, indicating that the diets evaluated in the present study are rapidly degradable.

Figure 2 shows the behavior of the gas production rate, showing a similar curve for all treatments. After 4 h, there is an increase in the gas production rate until reaching the inflection point is reached between 12.8 (FO) and 14.5 (control) h, at which time the gas production rate begins to decrease. Diets with oil (FO and LO) reached the inflection point faster than the control (p=0.0066). There were no significant differences among the other treatments (P>0.05; Table 3). In all treatments, the greatest fermentation was obtained in the first 15 h of incubation, suggesting that these diets, because of their high degradability, are suitable for medium-production animals, as they should favor the rumen passage rate and lead to increased intakes.

Figure 2. Gas production rate at inflection point ml/h. AP: Fish oil, AL: Linseed oil, ET2%: 2% ethanolic extract, ET4%: 4% ethanolic extract, EH2%: 2% water extract, EH4%: 4% water extract, Sam10%: Sambucus forage at 10%, Sam20%: Sambucus forage at 20%, Control: Only grass and concentrate

Donney's (2016) evaluating diets with 91% C. clandestinus + 9% supplement, and 63% C. clandestinus+ 30% T. diversifolia+ 7% supplement, reported that the inflection points in gas accumulation occurred at 16.5 and 16.3 h, respectively, similar to what was found in this work, suggesting that these diets are of rapid degradation. Gas production at the inflection point (Table 3) was lower in the ET4% treatment (55.9 ml) compared to the other treatments (P<0.0001). Likewise, the maximum gas production (a) was lower inET4% than the other treatments (P<0.01), suggesting that the content of secondary metabolites affected the fermentation process. The addition of these extracts and their content of secondary metabolites suggests an effect on microorganism populations by inhibiting the growth of bacteria (McSweeney et al 2001). The antibacterial activity of these compounds is linked to the formation of complexes with the cell wall, which causes morphological changes and induces nutritional deficiencies (Posada et al 2005).

Addition of the S. peruviana extracts (ET2%, ET4%, EH2%) led to lower observed in the treatments containing oil and the control (P<0.0001), explained by the increase in dietary energy coming from lipids and due to its low inclusion, there was no negative effect on the degradation of DM. For the Lag Phase, no significant differences were observed between the treatments (P>0.05), it is important to highlight that the colonization time was between 3.9 and 4.7 hours for all treatments, which again confirms that the diets evaluated are of high fermentability. Rivera et al (2013) reported gas volumes at 96 hours for S. peruviana of 63.4 ml with a face lag of 3.28 h. By relating the amount of degraded substrate (mg) and the volume of gas produced (ml) during the fermentation process, they reported significant differences when comparing a traditional (high concentrate plus kikuyu) diet with one containing S. peruviana, with values of 3.96 and 11.3 ml/mg, respectively. The gas production data from this experiment agree with what was found by Rivera et al (2013), when comparing the traditional diet with the 20% Sam and 10% Sam treatment. However, the treatments with extracts (ET2%, ET4%, EH2%) presented a significant difference (P<0.05) which could be attributed to the contents of extracted tannins (Table 4), which were higher in relation to what was reported by Lezcano et al (2014) and Cardenas et al (2016).

In vitro dry matter degradability

Table 5 shows the disappearance of dry matter (DMD) at 12, 24 and 48 h of incubation. A DM disappearance of more than 50% was observed in all treatments after 24 h, which indicates a diet composed of rapidly fermenting nutrients. The greatest disappearance of MS at 12 h occurred in the ET4% treatment, being greater (P<0.05) with respect to the other treatments. At 24 h, the extracts and Sam10% diets had greater DMD than the control. After 48 h, the same treatments maintained their difference with the control diet, except for EH2%.

The Sam20% treatment showed a low DMD in relation to the other treatments with S. peruviana, which is probably related to its high concentration of secondary metabolites, (1.23±0.1% of soluble tannins and 5.66±0.9% of bound tannins) Levels of tannins in the extracts, were not as high (Table 4). Tannins can bind to proteins and structural polysaccharides (cellulose, hemicellulose and pectin), delaying their digestion rate and interfering with the activity of microbial enzymes (McSweeney et al 2001), and this can reduce the degradability of DM. Waghorn (2008) and Patra (2010) stated that reduced digestibility in diets containing condensed tannins is almost universal and inevitable. This is an important factor that must be considered when supplemental tannins or tanniferous plants are added to diets, since the relationships with digestion are affected by the type of tannin and the composition of the diet. In the work of Rivera et al (2013), they reported digestibilities at 48 hours of less than 58% for the diet composed of C. clandestinusplus commercial concentrate and S. peruviana in a 60:30:10 proportion.

The results found in thus study in response to the addition of oils differ from previous reports (Machmüller et al 1998; Dohme et al 2000; Yabuuchi et al 2006; Patra and Yu 2013), as the addition of oils in the current study did not decrease the in vitro degradation of DM, total gas production, or CH ₄ produced per g of DMD with respect to the control. On the contrary, addition of FO increased DMD at 12, 24 and 48 hours, compared to the control (P<0.05; Table 5).

Previous studies have suggested that lipids have toxic effects on the rumen populations of fibrolytic microorganisms. However, other works suggest that the toxic effects are not only confined to microbial population (Machmüller et al 2006; Patra and Yu 2013). For treatments with polyunsaturated oils LO and FO, a decrease in the availability of H ₂ could be explained by a partial use of it in the biohydrogenation process of fatty acids. Van Soest (1994) suggested that the use of H ₂ in biohydrogenation is minor and that H ₂ could go to other fermentative routes, including the synthesis of propionate. Additionally, H ₂ could be used for the reduction of nitrate (NO₃), the synthesis of microbial protein, the reduction of sulfates or be expelled in gaseous form (Van Soest, 1994). It is very likely that under the conditions of the present experiment, microbial protein synthesis will increase, since the degradation of DM and fiber was not reduced, not compromising the energy available for its synthesis. Therefore, it can be speculated that the production of H ₂ gas most likely increased.

In vitro methane production

With regard to total methane production at 48 h, the lowest (7.72 mg) methane accumulation occurred with Sam20%, which was lower than that of the treatments FO, LO, EH2% and EH4%. However, its DM disappearance after 48 h (61%) was lower than that observed with the FO, ET2%, ET4%, EH4% and Sam 10% diets (Table 5). Tannins and saponins that have been extensively shown to possess promising mitigation potential. Tanniferous plants have often, although not always, been shown to have a potential to reduce enteric CH ₄ emissions up to 20%. Apráez et al (2012) indicated that forage resources such as Acacia decurrens, Sambucus nigra, Ambrosia arborescensy , or Otholobium munyense, show promise to be included in the diet of ruminants since, in addition to their nutrient contribution, they also contribute to reduce CH ₄ production due to their tannin content.

When methane production is expressed in grams per kg of DMD, the ET4% treatment had the lower values and was different (P<0.05) to the oil, control and EH2% treatments. One of the reasons for this reduction is attributed to the inhibitory effect of tannins on ruminal methanogenesis, which is directly related to its effect on methanogenic Archaea and protozoa, and indirectly by their depression in ruminal fiber degradation (Patra and Saxena 2011). When observing the production of CH ₄ (g/kg DMD), the difference observed with ET4% over other treatments should be highlighted because it did not negatively affect DM disappearance. The structure, molecular weight and concentration of condensed tannins affect the nutritional value of the diet; therefore, it is important that the benefits of CH ₄ reduction are not overshadowed by any detrimental effects on diet digestion and animal production (Grainger et al 2009).

Different mechanisms that explain observed reductions in enteric CH₄ in the presence of lipids have been suggested, such as: physical limitations due to the coating of lipids on food particles that prevent enzymatic attack by microorganisms (Palmquist 1984; Dohme et al 2000) , toxic effects of fatty acids on some populations of microorganisms (Machmüller et al 2006; Yang et al 2009; Patra and Yu 2013) and modifications in fermentation patterns due to changes in microbial populations (Yabuuchi et al 2006; Martin, et al. to 2010). In this study, when CH ₄ production was expressed in different units, there were no differences between the control and with the treatments with oil addition, suggesting that there was no toxic effects on microbial populations.

There are not many reports of fractional gas production rates from tropical forage mixtures. However, the results of this study coincide with reports for tropical legumes and pastures incubated alone, in which legumes show greater values than grasses. Fractional rates of gas production may be associated with yields in the production of protein of microbial origin. There is an inverse relationship between gas production and microbial biomass production.

Conclusions

The inclusion of non-traditional forage species in diets under grazing conditions may possibly allow a decrease in gas production during fermentation. The addition of ethanolic extracts of S. peruviana managed to achieve 5% less gas than the control diet respectively, without affecting its degradability. Hydrolyzable and condensed tannins are bioactive plant components that may offer an opportunity to reduce enteric CH ₄ production, although addition in high quantities could compromise dry matter digestibility. The addition of ethanolic extracts of S. peruviana in low quantities allows the production of CH ₄ per gram of degraded MS to be reduced without affecting the degradability of the diet.

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