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ANIMAL NUTRITION |
Agriculture and Agri-Food Canada, Research Centre, Box 3000, Lethbridge, Alberta, Canada
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Abstract |
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Key Words: beef cattle • canola oil • essential oil • fumaric acid • greenhouse gas • methane
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). The Intergovernmental Panel on Climate Change (tier 2, 1996) estimates that cattle lose 6% of GE intake as methane, except for feedlot cattle for which 3.5% of GE intake is lost as methane. Cattle producers are seeking to identify and promote good management practices that reduce production of atmospheric greenhouse gasses from beef operations, particularly those that reduce methane emissions from enteric fermentation. Reducing methane production can be of direct economic benefit because it coincides with greater energy-use efficiency of the feed by the animal.
Methane emissions can be decreased by supplementing the diet with certain additives and ingredients. Adding fats to the diet can reduce methane emissions by lowering ruminal fermentability, and to a lesser degree, through hydrogenation of the unsaturated fats (Johnson and Johnson, 1995). However, added fat can also lower feed intake and fiber digestibility (McGinn et al., 2004), potentially negatively affecting animal performance. Alternative natural feed additives that shift ruminal fermentation may show promise (Wallace, 2004). For example, fumaric acid is a metabolic precursor of propionate and may provide an alternative hydrogen sink within the rumen (Itabashi, 2002). Fumaric acid decreased methane emissions in vitro (Asanuma et al., 1999; López et al., 1999) and in vivo (Bayaru et al., 2001) but not in all studies (McGinn et al., 2004), possibly because of the level of supplementation used. Plant essential oils and spice extracts contain antimicrobial properties (Dean and Ritchie, 1987) that may inhibit methanogenesis by affecting ruminal bacteria.
The purpose of our study was to investigate the impact of several ingredients and feed additives that are currently registered for feeding to cattle on enteric methane production.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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.
Experimental Design and Animals
Sixteen spayed Angus heifers were used in the experiment, which was designed as a repeated Latin square with 2 groups, four 21-d periods, and 4 treatments. Eight heifers were allocated to each group, with 2 cattle in each group fed 1 of 4 treatments. The groups were offset by 1 wk to facilitate measurements.
Before beginning the experiment, the cattle were ear-tagged, vaccinated with a modified live vaccine for infectious bovine rhinotracheitis and parainfluenza 3 viruses (SmithKline Animal Health, Mississauga, ON), dewormed, and spayed. The heifers had been obtained at an early age (3 mo) and conditioned to the environmental chambers used to measure methane before experimentation. This was done to minimize the stress on the cattle while in the chambers. At the beginning of the experiment, the heifers were about 8 mo of age and weighed 260 ± 32 kg (mean ± SD).
From d 1 to 16 of each period, the heifers were housed individually, untethered in pens (4.9 x 1.8 m) equipped with a feeder and water bowl and bedded with straw. The pens were located in a sheltered, unheated barn. Daily feed intake, BW change, and ruminal fermentation were measured in this facility. Before the morning feeding on d 17, the cattle were moved to 4 environmental chambers for measurements of methane and total tract digestibility.
Treatments and Diet
Four treatments were evaluated: control (no additive), fumaric acid (Bartek Ingredients Inc., Stoney Creek, ON, Canada; 175 g/d), essential oil and spice extract (Crina Ruminants; Akzo Nobel Surface Chemistry S.A., Cedex, France; 1 g/d), and canola oil (Canbra Foods Ltd., Lethbridge, AB, Canada; 4.6% of DMI). Sodium bicarbonate (Church & Dwight Co. Inc., Princeton, NJ; 75 g/d) was added to the diet of cattle receiving fumaric acid at approximately 1% of DMI to help neutralize the acidity of the fumaric acid treatment. The essential oil product was a commercial proprietary blend of essential oils and plant extracts and was added to the diet at the manufacturer’s recommended level. However, it was mixed with ground barley before feeding, and the blend was fed at a rate of 10 g/d. The canola oil was poured onto the feed and mixed into the ration manually at the time of feeding.
The basal diet consisted of 75% whole-crop barley silage, 19% steam-rolled barley, and 6% supplement (DM basis), and was the same as used in a previous study by McGinn et al. (2004). Diet ingredient and chemical composition is shown in Table 1. The diet was formulated using the NRC (1996) guidelines to contain 14% CP, and to meet or exceed the mineral and vitamin requirements of cattle gaining 1 kg/d. The supplement contained the supplemental protein sources and provided minerals and vitamins in excess of the NRC (1996) nutrient requirements for animals with an ADG of 1.0 kg/d.
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Measurements of Methane Emissions
Once during each period, the cattle were moved to the environmental chambers to determine methane emissions. Chamber measurements were staggered for group 1 and group 2 because only 4 chambers were available.
Each chamber measured 4.4 m wide x 3.7 m deep x 3.9 m tall (63.5 m3; model C1330, Conviron Inc., Winnipeg, MB, Canada). Chamber air temperature was maintained at 10°C. Within each chamber, the animals were individually restrained in metabolism stalls that measured 2.5 m long x 0.9 m wide, elevated from the floor by 15 cm. Each stall was equipped with a feeder, providing each animal with individual access to feed.
Two animals were housed in each of the 4 chambers. The cattle were paired at the beginning of the experiment such that the total BW of cattle per chamber and per treatment was similar. The pairing of animals was consistent throughout the experiment, such that animals within a chamber received the same treatment. The first day within the chambers was considered an adjustment period, allowing the heifers to adapt before measurements were recorded for 3 consecutive 24-h days. The cattle were returned to their individual stalls on the fifth day. Interruptions occurred daily at 0730, when the chamber floor was cleaned, and at 0900 when cattle were fed. These interruptions had little impact on the daily emissions because fluxes were calculated every 7 to 8 min and then averaged to derive the 24-h emission value. When interruptions occurred, the corresponding fluxes were omitted, allowing for a 15-min reequilibration of the fluxes, where the chamber time constant was 5 min.
Airflow and concentration of methane was measured for the intake and exhaust ducts of each chamber. Details of the procedures used and the precision and accuracy of the technique are given by McGinn et al. (2004). Briefly, air velocity was continuously monitored over the day in each intake and exhaust duct for each chamber (model 8455, TSI Inc., Shoreview, MN). The air stream in each of the 8 ducts (2 per chamber) was sub-sampled, and methane concentration was measured continuously using an analyzer (model Ultramat 5E, Siemens Inc., Karlsruhe, Germany). It took 30 min to sequentially sample the airflow in all intake and exhaust ducts in all chambers.
Emission Calculations
Details of the calculation of methane emissions are reported by McGinn et al. (2004). In summary, the flux (F) of methane for each chamber was calculated for each of the 3 d of measurement during each period from the fresh-air intake (i) and chamber exhaust (e) concentrations (Ci and Ce, respectively; ppm) and mean air velocities (Vi and Ve, respectively; m/s):
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where MW is the molecular weight of gas (g/mole), P is the barometric pressure (Pa), R is the universal gas constant (8.31 J mole–1·deg K–1), T is the stream air temperature (°K), and A is the cross-sectional area of the duct (0.146 m2). The T/P value is a correction for the air velocity meter.
Although all instruments used to measure gas fluxes were calibrated, differences existed between chamber emissions (e.g., due to the range in errors associated with measuring the average air velocity in the ducts, as a result of placement of the sensor inside the ducts). These between-chamber differences were documented by releasing the same amount of gas into each chamber (with no animals) and then determining whole chamber flux when the exhaust concentration reached steady-state (McGinn et al., 2004). The ratio of maximum flux (always chamber 4) to other chamber fluxes was determined. The average correction factors relative to chamber 4 were 1.01, 1.14, 1.13, and 1.00 for chambers 1 to 4, respectively. This procedure decreased the variability in emissions among chambers and improved the sensitivity to treatment differences.
Ruminal Fermentation Measurements
Ruminal pH was measured once per animal at 4 h after feeding on d 14 of each period. A speculum was inserted into the mouth, and a lubricated rubber tube was inserted through the speculum into the rumen via the esophagus. Ruminal contents (200 mL) were removed using an electric pump. Samples were monitored visually to ensure they were not contaminated with saliva. The pH was measured immediately using a pH meter (Accumet model 25, Denver Instrument Company, Arvada, CO). The whole contents were squeezed through 4 layers of cheesecloth. Five milliliters of the filtrate were combined with 1 mL of 25% (wt/vol) meta-phosphoric acid and stored frozen (–30°C) until VFA analysis.
Digestibility
Total tract digestibility of nutrients was determined using an external marker prepared from chromic oxide and ground barley. Ten grams of marker, providing approximately 2 g of Cr, were top dressed once daily onto the feed of individual animals on the last 10 d of each period. In all cases, the entire allotment of marker was consumed. Fecal samples (100 g of wet weight) were collected twice daily at 0900 and 1530 from the rectum of each animal on the last 5 d of each period, while animals were in the chambers (except for the last day when samples were only taken in the morning).
The samples were composited by heifer and period and immediately frozen (–20°C). The pooled samples were later dried at 55°C for 48 h in a forced-air oven, ground through a 1-mm screen, and analyzed for analytical DM, GE, NDF, ADF, and Cr. An additional fecal sample (100 g of wet weight) was also taken from each animal before dosing the marker each period. These samples were analyzed for DM and Cr. The Cr concentration in the feces taken predosing was used to adjust for residual marker excretion. The Cr concentration in fecal samples obtained before dosing was in all cases less than 1% of the Cr concentration in the pooled fecal samples; thus this adjustment was trivial. Chromium was assumed to be completely indigestible, and the digestibility of DM was calculated as follows:
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where DMI represents the average DMI consumed during the 5-d period during which fecal samples were taken. Digestibility of GE, NDF, and ADF was calculated using the same approach.
Chemical Analyses
All chemical analyses were performed on each sample in duplicate, and where the coefficient of variation was >5%, the analysis was repeated.
Ruminal VFA were quantified by gas chromatography (model 5890, Hewlett Parkard, Little Falls, DE) with a capillary column (30 m x 0.25 mm i.d.; 1-µm phase thickness; bonded PEG; Supelco Nukol; Sigma-Aldrich Canada, Oakville, ON, Canada) and flame ionization detection. Crotonic acid was the internal standard. The oven temperature was 100°C for 1 min, which was then increased by 20°C/min to 140°C, and then by 8°C/min to 200°C, and held at this temperature for 5 min. The injector temperature was 200°C, the detector temperature was 250°C, and the carrier gas was helium.
Analytical DM was determined by drying the oven-dried samples at 135°C for 2 h, followed by hot weighing (AOAC, 1995; method 930.05). The OM content was calculated as the difference between 100 and the percentage ash (AOAC, 1995; method 942). Gross energy was determined using an adiabatic calorimeter (model 1241, Parr, Moline, IL). The NDF was determined as described by Van Soest et al. (1991) using heat stable 
-amylase and sodium sulfite, and ADF was determined according to AOAC (1995; method 973.18). For the measurement of CP (N x 6.25), samples were ground using a ball mill (Mixer Mill MM2000, Retsch, Haan, Germany) to a fine powder. Nitrogen was quantified by flash combustion with gas chromatography and thermal conductivity detection (Carlo Erba Instruments, Milan, Italy). Chromium was determined by inductively coupled plasma emission spectrometry (SpectoCi-rosCCD; Specto Analytical Instruments, GmbH & Co., Kleve, KG, Germany) after dry ashing and extraction.
Calculations and Statistical Analysis
Cumulative daily methane emissions from each chamber were calculated for each of the 3 d within each period. The total DMI consumed by the 2 heifers within each chamber on the days that gases were measured was also calculated. The daily methane flux was then expressed per unit of DMI and as a proportion of GE and DE intake of the 2 cattle within the chamber on that same day. Due to a malfunction of one chamber, emission data were treated as missing for periods 1 to 3 for group 1, and periods 1 and 2 for group 2. Thus, data for one period were missing for control, canola oil, and essential oil for group 1, and essential oil and control for group 2.
The data were analyzed using a Mixed model procedure of SAS (SAS Inst. Inc., Cary, NC). The individual animal was the experimental unit for intake, BW, digestibility, and ruminal fermentation variables because these data were obtained from individually penned animals. For these variables, the model included the fixed effect of treatment. Animal nested within group and period nested within group were considered as random effects. The REML method was used for estimating the variance components, and the df were adjusted using the Kenward-Roger’s option in SAS.
Day (1 to 16) was considered a repeated effect for ad libitum DMI with animal x period x group as the subject. The chamber, representing data for 2 animals, was the experimental unit for methane measurements. The model used for methane measurements included the fixed effect of treatment and the random effects of chamber nested within group and period nested within group. Day (1 to 3) was treated as a repeated measure with chamber x period x group as the subject. The variance-covariance error structure that was used was compound symmetry, unstructured, or first order autoregressive, as determined by the fit statistics. Differences among means were tested using a protected (P < 0.05) LSD test. Treatment effects were declared significant at P < 0.05, and trends were discussed at P < 0.10.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Because added oil increased the energy density of the diet by about 6%, total intake of GE was not affected (P = 0.20) by treatment. The ADG was not affected by diet (P = 0.26) in this short study, but the true effect of these dietary treatments on weight gain needs to be evaluated in a longer-term experiment.
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Digestibility
During the period of digestibility measurements, nutrient intakes were similar (P = 0.25 to 0.31) for cattle fed control and essential oil, but intakes were lower than control for cattle fed fumaric acid (P < 0.003) with a further decrease (P < 0.001) for cattle fed canola oil (Table 3). Consequently, during the digestion phase, intakes of GE and fiber were lower (P < 0.05) for animals fed fumaric acid or canola oil compared with the control, despite the fact that adding canola oil to the diet increased its GE content. Digestibility of all nutrients was lowered (P < 0.05) by feeding essential oil, with a further decrease (P < 0.05) for canola oil.
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) compared with their ad libitum intake in the metabolism unit (Table 2
). Once in the chambers, DMI of cattle fed control, fumaric acid, or essential oil was 85% of ad libitum intake, whereas DMI of cattle fed canola oil was 75% of ad libitum intake. Thus, the decrease (P < 0.001) in intake caused by adding oil to the diet was even more pronounced within the chambers than during the period of ad libitum intake.
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). The decrease in methane emission compared with the control tended (P = 0.09) to be maintained when corrected for differences in GE intake; methane emissions decreased from 7.93% of GE for control to 6.30% of GE for cattle fed canola oil. However, feeding canola oil had no effect (P = 0.19) on methane emissions when corrected for differences in DMI, indicating that a large portion of the decline in total methane emissions due to feeding oil was due to the decline in feed intake. Feeding essential oils had no effect on total daily methane emissions (P = 0.83), methane per kilogram of DMI (P = 0.52), or methane as a percentage of GE (P = 0.53), but methane emissions expressed as a percentage of DE intake tended (P = 0.10) to increase compared with the control.
Fumaric acid had no effect on total daily methane emissions compared with the control (P = 0.30 to 0.50), regardless of how the emissions were expressed.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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fed a similar control diet and reported that methane emissions averaged 6.5 to 7.1% of GE intake in 2 experiments. We previously reported that methane emissions from feedlot cattle were greater during the backgrounding phase than during the finishing phase (7.4 vs. 3.4% of GE intake; Beauchemin and McGinn, 2005). Thus, a feed additive or ingredient that reduces methane emissions from cattle fed high-forage diets could have an important impact on reducing the emissions from the feedlot cattle sector in North America.
Adding 4.6% canola oil, a source of unsaturated fat, to a high-forage diet was an effective suppressant of methane, with daily methane emissions decreased by 32% and methane emissions as a percentage of GE intake decreased by 21%. These reductions in methane are important because total methane per animal and methane relative to GE intake are the approaches used by the IPCC (1996) tier 1 and 2, respectively, in calculating methane inventories. Although the methane reduction due to fat feeding was substantial in this study, much of the decrease was due to the reduced intake of DE. Adding canola oil to the diet decreased feed intake by 21% and total tract digestibility of DM by 15% such that DE intake was only 74% of that of the control animals. Consequently, daily methane emissions per animal decreased, which would result in reduced methane inventories using the tier 1 approach of IPCC (1996). However, methane emissions relative to DE intake were only 6% lower for cattle fed canola oil than control cattle.
Others have also reported lower methane emissions for cattle diets supplemented with unsaturated fats. Dohme et al. (2000) observed in vitro that canola oil added to the diet at 5.3% of DM decreased total methane production by 20%. McGinn et al. (2004) reported that adding a similar amount (5% of dietary DM) of sunflower oil to a backgrounding barley-based diet also decreased methane, as a percentage of GE intake, by 21%. Sunflower oil is rich in oleic (45%) acid and linoleic (40%) acid, whereas canola oil contains 54% oleic acid, 22% linoleic acid, and 11% linolenic acid (NRC, 2001). The decreased methane emissions resulting from added canola oil supports a number of other studies in which unsaturated fats were fed, as reviewed by Boadi et al. (2004). Biohydrogenation of mono- and polyunsaturated fats provides an alternative hydrogen sink to reduction of carbon dioxide within the rumen. However, our study indicates that biohydrogenation may play only a minor role in reducing methane emissions as previously suggested by Johnson and Johnson (1995), compared with the effects of added fat on depressing feed intake and ruminal digestion.
Decreased intake due to oil feeding may have resulted from increased ruminal fill due to the decline in fiber digestibility. The reduction in total tract fiber digestibility indicated a possible reduction in ruminal digestion of fiber, which was corroborated by a reduction in ruminal VFA concentration and a decrease in acetate:propionate ratio.
Fat feeding has also been shown to reduce methane through a reduction in protozoal numbers, although protozoal numbers were not measured in this study. Methanogenic bacteria are metabolically associated with ciliate protozoa (Newbold et al., 1995), and feeding oil can cause substantial decreases in protozoal populations (Ivan et al., 2004).
The current study shows that adding high (>4%) levels of supplemental fats to cattle diets can cause substantial reductions in methane emissions (i.e., >20%); however, this strategy may not be commercially viable, given the high cost of most fat sources and the negative impact on intake of DE. As an alternative, we examined the potential use of 2 novel feed additives, fumaric acid and a commercial essential oil product, as a means of reducing methane emissions. Although fumaric acid caused an increase in propionate concentration that is sometimes consistent with decreased methane production, neither additive decreased methane emissions in vivo.
Several in vitro studies have recently fueled interest in the potential use of fumaric acid in ruminant diets to reduce enteric methane emissions. Carro and Ranilla (2003) reported that adding fumarate (from 0 to 10 mM) to concentrate feeds in batch culture increased VFA concentrations, decreased acetate:propionate ratio, and decreased methane production by up to 5%. Similarly, Asanuma et al. (1999) reported that when up to 30 mM of fumarate was added to ruminal inoculum in batch cultures, methane production decreased by about 10%, and propionate concentration increased. López et al. (1999) also observed that adding sodium fumarate (from 0 to 10 mM) to a diet containing 50% hay and 30% barley grain decreased methane production by 5 to 6% in batch culture, which was then confirmed using the rumen simulation technique (Rusitec). In vitro studies indicate that sodium fumarate reduces methane production by diverting hydrogen and stimulates proliferation of cellulolytic bacteria and the digestion of fiber. However, the effects of fumarate in vivo have been less conclusive. McGinn et al. (2004) fed 10.6 g/kg of DMI of fumaric acid to cattle (approximately 15 mM) and reported no effect on total VFA concentration, propionate proportions, or methane emissions. In contrast, Bayaru et al. (2001) added 20 g/kg of DMI of fumaric acid (approximately 18 mM) to a sorghum silage diet fed to cattle and observed a 23% reduction in methane. In that study, only 2 animals were used and methane was measured over a 24-h period using head chambers that measure respired methane and not total methane, unlike the chambers used in the current study. The level of supplementation used in our study (29 g of DMI/ kg) was estimated to be approximately 50 mM (116.07 g/ mol; ruminal volume assumed to be 30 L). The observed decrease in acetate:propionate ratio was consistent with previously reported in vitro observations, but methane emissions were not decreased. The reason for the apparent contradiction between decreased acetate:propionate ratio and the lack of effect on methane emissions is uncertain; however, the ruminal fermentation profiles were only based on one sampling time whereas methane was measured over a 3-d period. Furthermore, VFA concentrations measured in vitro reflect production, whereas in vivo concentrations are the balance between production and absorption. Biochemical calculations indicate that the level of fumaric acid supplementation (175 g/d, MW = 117 g) should have decreased methane production by 0.75 mol/d (assuming 1 mol of fumaric acid uses 2 mol of hydrogen and therefore reduces methane by 0.5 mol). Thus, we expected that the 9.96 mol of methane (159 g/d methane, MW = 16 g) produced by the control cattle would be decreased to 9.2 mol, but instead 10.6 mol were produced (170.6 g/ d). The greater than expected methane production from animals fed fumaric acid could have been due to an increase in ruminal digestion, which is supported by the greater total VFA concentration compared with the control cattle, although this finding was not supported by greater total tract digestibility.
There are numerous essential oils and plant extracts each with varying effects on ruminal fermentation and feed digestion (Kamra et al., 2005). Some essential oils exhibit antimicrobial properties (Helander et al., 1998), and this has motivated recent interest in developing essential oil products as natural feed additives for cattle (Wallace, 2004). Recent studies have shown that some essential oils beneficially affect ruminal fermentation, causing an increase in VFA (Castillejos et al., 2005) and decrease in the rate of AA deamination (McIntosh et al., 2003). The ammonia hyper-producing bacteria within the rumen have been shown to be sensitive to some essential oils (Wallace, 2004), but limited work has been done to determine the effects of essential oils on methanogens. The antimicrobial activity of essential oils and secondary plant metabolites is highly specific, which raises the possibility that these compounds can be used to target methanogens. For example, Kamra et al. (2005) reported that the extract of a number of plants reduced methane production in vitro. Using the same product used in the current study, McIntosh et al. (2003) reported that growth of the methanogen Methanobrevibacter smithii was inhibited but only when the concentration of essential oil product exceeded 1,000 ppm. The maximum concentration used in the present experiment was estimated as 33 ppm (30 L of rumen volume; 1 g/d fed), which is well below this threshold and may account for the lack of effect on methane emissions.
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: beauchemin{at}agr.gc.ca
Received for publication July 14, 2005. Accepted for publication January 17, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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E. M. Ungerfeld, R. A. Kohn, R. J. Wallace, and C. J. Newbold A meta-analysis of fumarate effects on methane production in ruminal batch cultures J Anim Sci, October 1, 2007; 85(10): 2556 - 2563. [Abstract] [Full Text] [PDF]
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