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ANIMAL NUTRITION |
,2,3,4
* University of Western Australia, Faculty of Natural and Agricultural Sciences, Crawley, Western Australia 6009, Australia; and
Commonwealth Scientific and Industrial Research Organisation Livestock Industries, Centre for Environment and Life Sciences, Private Bag 5, Wembley, Western Australia 6913, Australia
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: acetate:propionate ratio • methane • methanogen • pH • sheep • volatile fatty acid
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The microbes producing methane, methanogenic archaea (i.e., methanogens), compete with bacteria in the rumen for substrates such as hydrogen (Zinder, 1993). Furthermore, during fermentation, hydrogen is produced, and the removal of hydrogen is important for the efficiency of rumen fermentation (Stewart et al., 1997). Propionate is an end-product of fermentation that requires hydrogen for production, and the acetate:propionate ratio in the rumen has an inverse relationship with methanogenesis (Lana et al., 1998; Russell, 1998).
The addition of grain to the diet increases the amount of starch in the rumen and changes rumen fermentation. Methane production is generally reduced per unit of feed intake when grain content in the diet is increased. This reduction is indicated by a lower acetate:propionate ratio and pH (Moss et al., 1995; Lana et al., 1998). Rumen fermentation is also affected by decreasing retention time. The type of diet can reduce retention time (Evans, 1981a,b) with high-grain diets having shorter retention times compared with roughage diets. Retention time has also been decreased in the past using nylon mesh balls, commonly known as pot scrubbers, in which the physical contact and scratching against the ruminal wall is thought to increase ruminal turnover (Matsuyama et al., 2000). Matsuyama et al. (2000) found that decreasing ruminal retention time decreased methanogenesis.
Our experiment was conducted to examine the interaction of pot scrubbers and feeding a concentrate-based diet on methane production in vitro, acetate:propionate ratio, and pH. It was hypothesized that the interaction of increasing the grain content in the diet and reducing retention time in the rumen would decrease methane production and acetate:propionate ratios more than the individual effect of each treatment.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experimental Design
During the experiment 24, five-year-old, Merino rams, surgically fitted with rumen cannulas, were individually penned indoors at the Large Animal Facility at the University of Western Australia. The sheep were fed once daily in the morning and had free access to water at all times.
Before and during the experiment, all sheep were weighed and fed at maintenance according to their individual BW. All sheep ate 95% or more of their rations during the whole experiment. The 24 sheep were divided into 4 equal groups based on live BW [average 67.4 ± 0.27 kg (SEM)], making up a complete factorial design. All sheep were fed an oaten chaff diet [98% oaten chaff (8.7 MJ of ME/kg of DM and 86% DM)], supplemented with 2% vitamin and mineral stock mix (Siromin, Narrogin Mineral Stockmix, Narrogin, Western Australia, Australia; White et al., 1992) during the 5-wk acclimatization period. Two weeks after beginning the acclimatization period, 2 of the 4-sheep groups had 6 pot scrubbers (5-cm diam.) inserted into their rumen as a way of decreasing the retention time of digesta without changing the diet composition (Matsuyama et al., 2000) and to examine the interaction of retention time and grain content in the diet. One group with pot scrubbers and 1 group without pot scrubbers were assigned to the oaten chaff diet throughout the experiment. The sheep in the other 2 groups, 1 with and 1 without pot scrubbers, were assigned to an oaten grain diet. In the first diet phase (3-wk duration after acclimatization), the sheep assigned to the grain diet were offered an oaten chaff diet supplemented with oaten grain accounting for 35% of energy intake (low grain) [35% oaten grain (10.7 MJ of ME/kg and 86% DM), 63% oaten chaff, and 2% Siromin]. In the second diet phase (an additional 3-wk duration after the first diet phase), the oaten chaff diet was supplemented with oaten grain accounting for 70% of energy intake (high grain) [70% oaten grain (10.7 MJ of ME/kg and 86% DM), 28% oaten chaff, and 2% Siromin].
Estimation of Mean Retention Time of Liquid and Particulate Matter
Chromium and Yb were used to measure the retention time of digesta in the rumen for each sheep using the double-marker dose technique (Faichney, 1992a). Ytterbium, as YbCl3, was used to associate with the particulate matter in the rumen (Faichney, 1992b), and Cr-EDTA was used to associate with the liquid fraction (Faichney, 1992a). A single dose of 50 mL of Cr-Yb solution was added to the rumen 1 h after feeding. The disappearance of these markers per unit of time was used to calculate the mean retention time of the particulate and liquid fractions from the rumen according to Faichney (1975).
Preparation of Cr and Yb Solution. The Cr-EDTA and YbCl3 stock solutions were prepared in the following manner. A total of 36.5 g of Na2-EDTA was boiled in 1 L of deionized H2O for 1 h, and 33.3 g of CrCl3-6H2O (Sigma-Aldrich Pty. Ltd., Castle Hill, New South Wales, Australia) was dissolved in 200 mL of deionized H2O and slowly added to the boiling Na2-EDTA solution. The mixture was boiled for 4 h and allowed to cool to room temperature before 125 mL of concentrated NH4OH was added and mixed. The solution was left overnight. The solution was then filtered through Whatman No. 1 filter paper, and pH was adjusted to pH 7 with 10 N HCl. Finally, 36.25 g of YbCl3 (Sigma-Aldrich Pty. Ltd.) was added to the solution and made up to 2 L with deionized H2O.
Rumen Sample Collection and Processing. Samples for measuring retention time of fluid and particulate matter in the rumen were collected on d 17 to 19 in diet phase 1 and 2. The sheep were fed 1 h before 50 mL of the Cr-Yb solution was administered. The solution was delivered to the rumen through the cannulas using a syringe and flexible tubing situated approximately in the middle of the rumen. To detect the disappearance of Cr and Yb from the rumen, samples were collected at 2, 4, 6, 8, 14, 26, 36, 48, 58, and 72 h after administration. At every sampling, 120 mL were collected at different locations of the rumen and mixed. After mixing, approximately 20 mL were stored at –20°C. The remaining 100 mL were put back into the rumen through the cannulas.
Rumen samples were prepared for Cr and Yb analysis according to the following protocol. Rumen samples were thawed, and weights were recorded. The samples were then lyophilized for 96 h using a freeze dryer (Heto, FD4.0, Thermo Fisher Scientific, Waltham, MA). When dry, the weights of the containers and the dried samples were recorded. The dried samples were then transferred to a conical flask, and concentrated nitric acid was used to remove all carbon. The slurry of dried salts was then dissolved in 20 mL of deionized H2O.
Cr and Yb Assay.
The Cr standards were prepared from K2Cr2O7 (Sigma-Aldrich Pty. Ltd.), and the Yb standards were prepared from Yb2O3 (Sigma-Aldrich Pty. Ltd.). The concentrations used were equivalent to 0 to 12 µg/mL for both trace elements. The diluted digested samples were aspirated into an atomic absorption spectrophotometer (AA300, Varian, Palo Alto, CA), and concentrations of Cr were measured using the following parameters: lamp current 7 mA, slit width 0.2 nm, wavelength 357.9 nm, and an air-acetylene oxidizing flame; concentrations of 1.0 ppm yielded absorbance of 0.100. The concentrations of Yb were measured via atomic absorption spectrophotometery. The parameters used were as follows: lamp current 5 mA, slit width 0.5 nm, wavelength 398.8 nm, and a nitrous oxide-acetylene reducing flame. All calculations were done according to the protocols published by Faichney (1975) and Faichney et al. (1999).
VFA, pH, and In Vitro Methane Production
Rumen Sampling. On the last day of each of the 3-wk diet phases (d 21 and 42), samples were collected 1 h after feeding for analyzing VFA and methane production in vitro. The sample for VFA measurement was strained through 2 layers of muslin cloth, and a 9-mL sample was added to 1 mL of 1 N NaOH and stored at –20°C until used. Approximately 100 mL of crude rumen fluid was collected for measuring pH before the sample was prepared for measuring methanogenesis in vitro.
VFA. Frozen samples were allowed to reach room temperature before being vortexed for 30 s, and the particulate matter was allowed to settle before 0.5 mL of the supernatant was centrifuged (5415C, Eppendorf, Hamburg, Germany) at maximum speed for 20 min. The concentrations of acetic and propionic acids and the total amount of VFA were determined by GC [Agilent 6890 series GC (Agilent Technologies, Palo Alto, CA) with an HP 6890 injector and using HP chemstation software (Agilent Technologies)] using the standard procedure for separation of VFA (Supelco Bulletin No. 749D, Supelco Inc., Bellefonte, PA). The GC was fitted with a HP-FFAP capillary column 30 m x 0.53 mm x 1 µm (Agilent Technologies). All measurement and calculations were performed by the Department of Agriculture Western Australia, Perth, Australia.
In Vitro Methanogenesis.
Methane production from sampled digesta was performed in 100-mL serum bottles, which were purged with N2 before 30 mL of whole rumen fluid was added to each bottle. Afterwards, N2 was bubbled through each bottle for 1 min to ensure anaerobic conditions. The experiment was performed in triplicate for each sheep. The bottles were then incubated for 24 h at 39°C in an Innova 4080 shaking incubator (New Brunswick Scientific, Edison, NJ) at 100 rpm. The incubation was terminated with injection of 3 mL of 17.5% formalin. Gas pressures in the serum bottles were measured with a pressure transducer, and the composition of the headspace in the bottles and the method to calculate the amount of methane produced was done according to the methods of Klein and Wright (2006) with the following changes: the column temperature was 190°C and the injector and detector temperatures were 250°C.
Statistical Analyses
All statistical analyses were conducted using the multivariate statistical package PRIMER v6 (Clarke and Gorley, 2006). Differences between diet phases were based on changes within the same group of sheep but on different diets. Changes within a diet phase were based on comparison between 2 or more sheep groups within the same diet phase. The data was transformed according to draftsman plots where necessary. Multivariate data analysis was normalized and reassembled in a Euclidean distance matrix before using analysis of similarities, a multivariate analogue to ANOVA. Non-metric multidimensional scaling (MDS; MDS ordination) was used to investigate the effect of grain and pot scrubbers on methane production, pH, retention time of digesta, and VFA; on MDS plots, most similar communities are grouped closer together (Clarke, 1993; Clarke and Warwick, 2001).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Grain was found to affect rumen fermentation, but it did not change rumen retention time for either the liquid or the particulate phase. High grain-fed sheep without pot scrubbers produced significantly less methane in vitro than both groups fed oaten chaff diets (P < 0.01). Similarly, the increase from low to high grain resulted in a 16% decrease in in vitro methane production (P < 0.01) in the sheep without pot scrubbers.
High grain-fed sheep without pot scrubbers showed a significantly lower acetate:propionate ratio than oaten chaff-fed sheep (P < 0.01). Additionally, there was a significant decrease in acetate:propionate ratio for grain-fed sheep without pot scrubbers (P < 0.05) when grain content was increased from low to high (Figure 1). High grain-fed sheep without pot scrubbers had significantly greater propionate concentrations than oaten chaff-fed sheep (P < 0.01). Furthermore, going from a low- to a high-grain diet significantly increased ruminal propionate concentration (P < 0.05; Table 1).
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). The analysis also showed an overall change when increasing the grain content in the diet from low to high as grain-fed sheep without pot scrubbers changed significantly (P < 0.05; Table 1). Effect of Pot Scrubbers
There was no effect of pot scrubbers on liquid or particulate retention time in the rumen (Table 1). However, oaten chaff-fed sheep with pot scrubbers had a significantly lower acetate:propionate ratio than sheep fed the same diet but without pot scrubbers (P < 0.05; Figure 1). The same was observed for high grain-fed sheep with and without pot scrubbers (Figure 1).
Interaction of Diet and Pot Scrubbers
The interaction between diet and pot scrubbers was found to alter rumen fermentation, but no effect was recorded on liquid or particulate retention time (Table 1). There was a significantly lower level of methane produced in vitro when a low-grain diet was combined with pot scrubbers than when the oaten chaff was fed to sheep without pot scrubbers (P < 0.05; Table 1). The significantly lower in vitro methane production between these 2 groups also remained after a high grain was fed (P < 0.05; Table 1).
When a high-grain diet was fed to sheep with pot scrubbers, they showed a significantly lower acetate:propionate ratio than grain-fed sheep without pot scrubbers (Figure 1; P < 0.05). The acetate:propionate ratio was also significantly lower than in the 2 groups offered an oaten chaff diet (P < 0.01; Figure 1). Furthermore, there was a significant decrease in acetate:propionate ratio (P < 0.05) when grain content was increased from low to high (Figure 1), with a larger and more significant (P < 0.01) decrease for sheep with pot scrubbers. Propionate concentrations showed similar responses as did the acetate:propionate ratio when a high-grain diet was fed to sheep with pot scrubbers. The change from low to high grain also resulted in a significant increase in propionate concentrations, with a larger increase for sheep with pot scrubbers (Table 1). The high-grain diet combined with pot scrubbers also increased total VFA concentration significantly compared with sheep given oaten chaff without pot scrubbers (P < 0.05; Table 1). Furthermore, when a high-grain diet was offered, rumen pH was significantly less in sheep fed grain with pot scrubbers compared with oaten chaff-fed sheep with or without pot scrubbers (Table 1).
When differences were tested using a multivariate statistical analysis including all data, sheep fed high grain with pot scrubbers were significantly different from grain-fed sheep without pot scrubbers (P < 0.05), as well as from both groups given oaten chaff (P < 0.01). Furthermore, when diet was shifted from a low- to a high-grain diet, the sheep with pot scrubbers fed the high-grain diet were found to change more than the grain-fed sheep without pot scrubbers (Table 1).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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It was hypothesized that the interaction of increasing the grain content in the diet and decreasing retention time of digesta from the rumen would decrease methane production and acetate:propionate ratios more than either one of these strategies individually. Our data partly support this hypothesis. When a high-grain diet was combined with pot scrubbers, the acetate:propionate ratio was found to be significantly lower than for all other sheep groups (Figure 1). However, high grain-fed sheep without pot scrubbers exhibited slightly, but not significantly, lower in vitro methane productions (Table 1). In vitro methane production was less in low grain-fed sheep with pot scrubbers compared with oaten chaff-fed sheep without pot scrubbers. This reduction was not accompanied by a significant alteration in acetate:propionate ratio or pH, which means that other factors may be responsible for the reduction in methane production during this phase. However, we expected a further reduction in in vitro methane production in high grain-fed sheep with pot scrubbers compared with when they were fed a low-grain diet. A possible explanation for why this did not occur could be because the balance between methanogens and bacteria competing for hydrogen had already been manipulated and established by the interaction of pot scrubbers and low grain. To fully support this argument, further investigations to enumerate methanogens and other hydrogen-utilizing bacteria under different dietary conditions would be necessary.
Reasons for the lower acetate:propionate ratio observed in high grain-fed sheep with pot scrubbers are unknown, but the lower methane production in vitro in low grain-fed sheep with pot scrubbers could explain why more propionate was produced when a high level of grain was offered. In this situation, the microorganisms competing with methanogens for hydrogen to produce propionate in the rumen of sheep with pot scrubbers would have had more time to adjust to the increasing amount of hydrogen available.
The lower pH measured in the rumen of high grain-fed sheep with pot scrubbers may also have had an effect on both methane production and the acetate:propionate ratio. Rumen methanogens are sensitive to even modest decreases in pH (Van Kessel and Russell, 1996), and changes in pH can explain up to 25% of the changes in the acetate:propionate ratio, although the effect of pH is more subtle than the effect of diet (Russell, 1998). Lana et al. (1998) concluded that lower pH in grain-fed sheep is due to greater VFA production, less motility, and slower dilution rate of the rumen. Our results indicate that the total concentration of VFA influences ruminal pH more than dilution rate.
The multivariate statistical analysis, which can be illustrated by the nonmetric MDS plot, supports the above findings and shows clearly that the 4 groups were different (Figure 2). The MDS plot (Figure 2) illustrates the differences found between the groups when a high-grain diet was offered, showing the oaten chaff-fed sheep further from grain-fed sheep with pot scrubbers than from grain-fed sheep, which we also found in our analysis (Table 1). Further analyses carried out by overlaying the MDS plot with the methane production, acetate:propionate ratio, and pH of individual sheep confirmed that these were the main factors separating the sheep groups (data not shown). Overall, the MDS plot in Figure 2 illustrates that pot scrubbers and grain influenced the rumen fermentation and that there was an interaction of these 2 variables.
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The decline in in vitro methanogenesis when sheep were fed a high-grain diet (Table 1) is consistent with previous observations (Van Kessel and Russell, 1996; Baker, 1997; Russell, 1998). As well, the significantly lower acetate:propionate ratio for high grain-fed sheep is consistent with the literature (Hodgson and Thomas, 1975; Lana et al., 1998; Hristov et al., 2001).
Interestingly, in the current study, methane levels were reduced in vitro for both groups of high grain-fed sheep with or without pot scrubbers, but it is possible that a similar decrease was a result of a different balance of microorganisms competing for hydrogen, because we found in vitro methane production from low grain-fed sheep with pot scrubbers was already significantly less than that of oaten chaff-fed sheep (Table 1). The differences found using multivariate statistical analysis confirm the above-mentioned effects of grain.
Effect of Pot Scrubbers
The possible explanations for the observed effect of pot scrubbers on acetate:propionate ratio when a high-grain diet was offered would be similar to the reasons described above. Surprisingly, pot scrubbers did not affect rumen retention time as hypothesized and observed by Matsuyama et al. (2000). However, this supports the findings of Loerch (1991), who observed that there was no effect of pot scrubbers on ruminal pH, VFA concentrations, dilution rate, and rumen volume for cattle on a high-grain diet (100%). Instead, he concluded that the beneficial effects observed were due to stimulation of the rumen wall. Based upon our findings, it is reasonable to conclude that the main benefit of pot scrubbers may be stimulation of the rumen wall, but there is no doubt that adding pot scrubbers affected microbial fermentation in the rumen.
In conclusion, in this experiment we changed the fermentation characteristics in the rumen of sheep by using combinations of pot scrubbers and grain and examined the effect this has on in vitro methanogenesis. We found that pot scrubbers, in combination with low grain in the diet, significantly decreased the amount of methane produced (P < 0.05), and we observed a similar effect in sheep consuming a high-grain diet without pot scrubbers. The acetate:propionate ratio was lower in high grain-fed sheep with pot scrubbers compared with sheep fed the same diet but without pot scrubbers in their rumen, indicating that we altered the balance of hydrogen-utilizing bacteria. These findings were supported by our multivariant statistical analysis including all data, which showed that sheep given the combined treatment of high grain and pot scrubbers were different from all other sheep groups in this experiment (Table 1). Overall, this indicates that the interaction was important and that further effort should be made to fully clarify the relationship between increasing grain supplement in the diet and pot scrubbers and the microbes inhabiting the rumen, with a focus especially on methanogens and their role. In conclusion, introducing pot scrubbers into the rumens of livestock consuming low levels of grain may be a way to lower methane emissions.
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Footnotes |
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2 Present address: CSIRO Human Nutrition, Kintore Ave., Adelaide, SA. 5000 Australia.
4 Present address: CSIRO Livestock Industries, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia, Qld. 4067 Australia.
3 Corresponding author: c.christophersen{at}csiro.au
Received for publication June 20, 2007. Accepted for publication November 8, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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