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ANIMAL PRODUCTION |
Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: cattle • enteric methane • ionophore • lasalocid • monensin • protozoa
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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) and identified as one avenue through which reduction of global CH4 emissions can be achieved. Considerable effort is being devoted to strategies that will reduce CH4 production by ruminal microorganisms. These strategies are at various stages of development, with relatively few inhibitors commercially available or economically feasible to the producer. Use of ionophores as a strategy to decrease CH4 emission from ruminants is feasible because this group of feed additives is commonly used in beef and dairy production.
Ionophores are highly lipophilic compounds and toxic to many bacteria, protozoa, and fungi (Russell, 1996). Two ionophores, monensin and lasalocid, are extensively used to manipulate ruminal fermentation and, thereby, improve feed efficiency (Russell and Strobel, 1989). Studies reporting decreased ruminal methanogenesis with ionophore supplementation have varied with respect to extent of the decrease and persistence of the response (Carmean and Johnson, 1990; Mbanzamihigo et al., 1996). Although there is no evidence that ionophores affect ruminal methanogens directly, there may be an indirect effect if protozoa act as a symbiotic host for methanogens (Ushida and Jouany, 1996).
The objectives were to 1) evaluate impact of ionophore administration on enteric CH4 emissions, 2) discern duration of ionophore-mediated suppression of CH4 production and alterations in ruminal fermentation, 3) investigate effect of the ionophore rotation (monensin and lasalocid) on CH4 emission and ruminal fermentation, and 4) assess effects of ionophores on concentrations of ruminal ciliate protozoa in cattle fed low- or high-concentrate diets.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Thirty-six Angus yearling steers averaging 328 ± 24.9 kg were used to assess the effects of ionophore supplementation on enteric CH4 production, ruminal fermentation characteristics, and ruminal fluid ciliate populations. Before the beginning of the experiment, steers were divided into 2 groups to adapt, over a 6-wk period, to a low- or high-concentrate diet. Composition (DM basis) of the low-concentrate diet was 10.8% alfalfa silage, 75.2% corn silage, 12.9% canola meal, and 1.1% mineral mix; and that of the high-concentrate diet was 22.7% alfalfa silage, 8.3% corn silage, 67.9% barley grain, and 1.1% mineral mix (Table 1). Steers were fed daily at 0900. All steers received 500,000 IU of vitamin A and 75,000 IU of vitamin D3 by injection during the adaptation phase and again 3 mo later.
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Experimental Plan
The experiment, which lasted 16 wk, consisted of 4 experimental periods. The first period, wk 1 and 2, served to establish baseline values. During this period, steers assigned to LC-C, LC-M, and LC-M/L were fed the low-concentrate diet without ionophore, and all other steers were fed the high-concentrate diet without ionophore. The second period, from wk 3 to 6, served to measure any short-term effects of ionophore supplementation on ruminal CH4 production, fermentation characteristics, and ciliate protozoal populations; the third period, from wk 7 to 14, served to measure any long-term effects of ionophore supplementation. All ionophores were removed from the low- and high-concentrate diets in the fourth period, wk 15 and 16, to examine the effects of ionophore withdrawal on ruminal CH4 production, fermentation characteristics, and ciliate protozoal populations.
Feed Sampling and Analyses
Feed and ort samples were collected weekly and dried in a forced-draught oven at 60°C for at least 48 h to determine DM concentrations. Dried feed samples were ground using a Wiley Mill (Thomas Scientific, Swedenboro, NJ) fitted with a 1-mm screen. Feed samples were analyzed for CP (N x 6.25) using a Leco CNS 2000 analyzer (Leco Corp., St. Joseph, MI), as described by Bilous (1999), and GE using a Parr 1241 adiabatic bomb calorimeter. In vitro OM digestibility was determined by the method of Tilley and Terry (1963) using bovine inoculum. Neutral detergent fiber, ADF, ash, Ca, P, K, Mg, and Na were analyzed using methodology outlined by Undersander et al. (1993), method No. 973.18 (AOAC, 1997), method No. 942.05 (AOAC, 1997), and method No. 985.01 (AOAC, 1997), respectively. Ionophore concentrations were analyzed by Animal Health Laboratory, Guelph, Ontario, Canada. Monensin was determined using the method Fd-DRUGS-ION04 (CFIA, 1997a), and lasalocid was determined using the method OMAF Toxi-010 (CFIA, 1997b).
Methane Gas Sampling and Analyses
One week before the first CH4 gas collection, stainless steel permeation tubes, as described by Boadi et al. (2002), containing 0.27 to 0.34 mg of SF6 and with known release rates (292 to 659 ng of SF6/min), were placed in the rumen (through the throat) using a speculum. This allowed enough time for the tracer gas to equilibrate in the rumen. Animals were trained to wear the gas collection apparatus during the equilibration period. Exhaled gas from the nose and mouth was drawn into preevacuated (30 mmHg) stainless steel collection canisters (130-mm diam.) through 900-mm capillary tubing (128 µm i.d.) with an in-line 15-µm filter and flexible nosepiece fitted to a halter (Boadi et al. 2002).
Over the course of the 16-wk feeding experiment, 24-h gas samples were collected in wk 1, 3, 4, 5, 6, 8, 10, 12, 14, and 16. Background air samples were collected at each time using a similar collection apparatus. After a 24-h period of collection, canisters were removed, and the pressure was tested to identify blocked or leaking capillary systems. Thereafter, canisters were pressurized to 110 kPa with pure N2 to prevent sample contamination before analyses and to allow injection of gas samples into the sample loop of a gas chromatograph.
A gas chromatograph (Star 3600, Varian, Mississauga, Ontario, Canada), fitted with an electron capture detector and a flame ionization detector, was used for determining SF6 and CH4, respectively, in the collected samples (Boadi et al., 2002). A Molecular Sieve with bead diameter 0.5 nm (1,800 mm) and a Poropak QS (1,800 mm) column were used for SF6 and CH4, respectively (Varian, Mississauga, Ontario, Canada). The column and injector temperatures were 35 and 350°C, respectively, and N2 was used as the carrier gas with a flow rate of 30 mL/min. Before sample analysis, prepared standards were used to standardize the gas chromatograph for SF6 (20.73 ppt; Scott-Marrin Inc., Riverside, CA) and CH4 (99.7 ppm; Supelco, Mississauga, Ontario, Canada).
Ruminal Fluid Sample Collection, Preparation and Analyses
Ruminal fluid samples were collected 2 h postfeeding on the day after gas collection. Approximately 300 mL of fluid was aspirated using a Geishauser oral probe (Geishauser, 1993), with the first 100 mL of fluid discarded to avoid saliva contamination, and the remainder subsampled: 30 mL for VFA and ammonia-N concentrations and 50 mL for enumeration of ciliate protozoa. The pH of the ruminal fluid samples was immediately measured using an Accumet Basic 15 pH meter and an Accumet gel-filled polymer-body combination pH electrode (Fisher Scientific, Fairlawn, NJ), calibrated with pH 4.0 and pH 7.0 buffer solutions (Fisher Scientific). Ruminal fluid used for VFA and ammonia-N analyses was centrifuged, and the supernatant was frozen and stored at –20°C. Ruminal fluid samples for determination of ciliate protozoal populations was mixed gently by inversion. Three 500-µL subsamples were taken from each sample, and combined with 500-µL of 20% formalin. Subsamples were stored at 4°C until ready for counting of ciliate protozoa.
Ammonia-N concentration of ruminal fluid samples was determined using the method described by Novozamsky et al. (1974). A 50-µL sample of the thawed supernatant was combined with 1.5 mL of reagent I (100 mL of alkaline phenolate + 200 mL of 0.05% sodium nitroprusside + 10 mL of 4% Na EDTA). The mixture was vortexed, 2.5 mL of reagent II (400 mL of phosphate buffer + 100 mL of 10% NaOH) was added, and the contents were vortexed again. Test tubes were covered and put in darkness for 30 min. Thereafter, absorbance was determined at 630 nm on a spectrophotometer, and ammonia-N concentrations were calculated from regression equations of the standard curve.
Volatile fatty acid concentrations in ruminal fluid were measured using the method described by Erwin et al. (1961). Frozen ruminal fluid samples were thawed at room temperature and 25% metaphosphoric acid solution was added in the ratio of 1:5, ruminal fluid:metaphosphoric acid. Tubes were vortexed and placed in a –20°C freezer overnight. Thawed samples were centrifuged at 2,000 x g for 20 min. Approximately 2 mL of supernatant was transferred into a clean, dry vial and capped. Concentrations of VFA were determined by gas chromatography (Model 3400 Star, Varian, Walnut Creek, CA), with injector and detector temperatures at 170 and 195°C, respectively, and initial and final column temperatures at 120 and 165°C, respectively.
Ciliate protozoa were counted as described by Sambrook and Russell (2001) using a standard hemocytometer chamber, and 4 major genera were identified as outlined by Dehority (1993).
Statistical Analyses
Statistical analyses were performed using a repeated measures model, assuming autoregressive covariance structure for DMI data and spatial power model covariance structure for the remaining data sets. The statistical design was a split-plot with a 2 x 3 factorial arrangement of the treatments in the main plot and time and treatment x time interactions in the subplot.
All statistical analyses were conducted with PROC MIXED (SAS Inst. Inc., Cary, NC), with least squares means and associated SE reported. Statistical analysis of total ciliate protozoa and individual species was performed on log10-transformed data, with one observation added onto actual counts to meet the requirement of SAS analysis for observations of zero. Treatment, week, and treatment x week were included in the model, and effects of treatment were tested using animal within treatment as the error term. Although a factorial design was used for treatments, main effect interactions required data presentation as 6 separate treatments. PROC CORR was used to define the correlation between CH4 emissions and total ciliate protozoa. Means were separated at the 5% level of significance using the predicted differences option.
One steer, assigned to HC-M/L, was removed from the test in wk 6 because of an unrelated illness, and all of that animal’s data were removed. Also, improper handling of collection spheres in wk 14 required all CH4 emission data collected that week to be removed. Numbers of observations are reported in all cases.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Cold exposure can decrease beef animal performance as a result of increased maintenance needs and faster gastrointestinal passage rate resulting in lowered digestibility of feed (Kennedy and Milligan, 1978). Decreased ruminal retention time or reduced feed degradation in the rumen could reduce enteric CH4 emissions.
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). The in vitro OM digestibility of the 2 diets was 70.3 and 57.4%, respectively.
Steers assigned the high-concentrate diet without ionophores consumed an average of 8.21 ± 0.21 kg of DM/d, which was greater (P < 0.05) than DMI for all other diets over the course of the 16-wk experiment (Table 2); gross energy intake followed the same pattern as DMI. Use of the 5% orts as a criterion for adjustment of daily feed offered disadvantaged animals assigned to the low-concentrate diets because animals were not consuming the larger pieces of corncob (>5 cm) once frozen. Animals consuming the low-concentrate diet also had a lower F:G (P < 0.05) than those fed the high-concentrate diet because the OM digestibility was lower (Table 1). Digestible OM intake was greater (P < 0.05) for all high-concentrate diets than for the 3 low-concentrate diets, with animals consuming the high-concentrate diet without ionophore supplementation having the greatest digestible OM intake (Table 2).
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; Zinn et al., 1994). However, when animals were fed the high-concentrate diet, both monensin and the rotation of monensin and lasalocid decreased (P < 0.05) DMI (kg/d) and improved (P < 0.05) F:G, whereas ADG was not influenced (P > 0.05). As previously reported (Russell and Strobel, 1989), improved feed efficiency with ionophore supplementation of high-concentrate diets was related to the reduction in DMI and not a direct effect on growth rate. Goodrich et al. (1984) observed that cattle consuming diets with a high ME had a greater response to monensin than did cattle consuming diets with lower ME concentrations, with the optimum concentration for monensin supplementation at 12.13 kJ of ME/kg. Metabolizable energy, calculated using standard NRC (1996) values for diet ingredients, for the low- and high-concentrate diets used in the present experiment were 9.3 and 11.2 kJ/kg, respectively.
Enteric CH4 Production
Week 1 enteric CH4 production by animals consuming the high- and low-concentrate diets was 173.8 ± 17.2 and 193.1 ± 13.1 L/d, representing energy losses of 5.9 ± 0.38 and 6.7 ± 0.36% of GE intake, respectively (Figures 2 and 3). Although volume of CH4 emitted was not affected by diet, the greater digestible OM concentrations of the high-concentrate diets resulted in lower (P < 0.05) fractional CH4 losses.
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and 3; Table 3). A rotation of monensin and lasalocid every 2 wk did not influence the extent or duration of the response compared with supplementation of monensin only. Neither monensin supplementation nor rotation of monensin and lasalocid supplementation affected enteric CH4 production, expressed as liters per day or liters per kilogram of BW, for the high-or low-concentrate diets.
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These data support previous in vivo experiments, which demonstrated that the inhibitory effects of ionophores on ruminal methanogenesis do not persist. Control values were restored on d 12 (Rumpler et al., 1986) and on d 9 (Carmean and Johnson, 1990) of ionophore feeding, in studies where the experimental animals were fed a high (70 to 90% DM basis) concentrate diet. Johnson et al. (1997) reported an adaptive response, as measured by enteric CH4 emissions, within 21 d of ionophore supplementation when cattle consumed high-concentrate diets, whereas there was no evidence of adaptation in the initial 44 d when cattle consumed forage diets that included pasture. Results of Johnson et al. (1997) and the current study suggest that an adaptation response for methanogenesis may be related to the composition of the diet fed because available energy was greater for the high-concentrate diets. It should be noted that the extent of ionophore-mediated suppression of CH4 production in the current study was similar for the high- and low-concentrate diets but that the duration of suppression was longer when animals were fed the low-concentrate diets than when animals were fed the high-concentrate diets.
Individual measurements for 24-h enteric CH4 emissions ranged from 54.7 to 369.3 L. This represents CH4 energy losses that ranged from 1.1 to 9.2% GE intake on the low-concentrate diet and from 0.9 to 8.4% on the high-concentrate diet daily. The range in CH4 emissions was relatively large and is probably a function of the wide range of weather conditions experienced during the course of the 16-wk study. For example, average 24-h ambient temperatures on day of sampling ranged from –36.4 to 6.4°C. The week-to-week variation (P < 0.01) in CH4 emission, expressed as liters per day, liters per kilogram of DMI or percentage of GE intake, therefore, reflects increased DMI over time typical of growing animals, the impact of ionophore supplementation, and fluctuating intake in response to weather. Boadi and Wittenberg (2002) demonstrated a strong correlation between CH4 production and DMI for cattle provided ad libitum access to feed, accounting for 64% of the variation. Therefore, it is expected that the short-term reduction in enteric CH4 emissions observed in this study should be achievable in a commercial setting.
Ruminal Fluid Characteristics
Mean ruminal fluid pH was greater (P < 0.001) when animals were fed the low-concentrate diet (6.56 ± 0.03) compared with the high-concentrate diet (6.38 ± 0.03, Figures 4 and 5). Animals fed the high-concentrate diet had a decrease (P < 0.05) in ruminal fluid pH in the first week of ionophore supplementation compared with animals fed the same diet without supplementation (Table 4). Ruminal fluid pH levels were similar for animals receiving diets with and without ionophores for the remainder of the experiment. This initial decline was not observed for animals fed the low-concentrate diet.
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) but did not influence ruminal fluid pH in cattle consuming a low-concentrate diet (Branine and Galyean, 1990). In contrast, Lana and Russell (1997) reported a reduced pH with ionophore supplementation. The effect of ionophores on ruminal pH is related to the inhibition of gram-positive bacteria, which are responsible for much of the ruminal lactate production (Russell and Strobel, 1989). Reduced lactate production could increase ruminal pH due to the low pKa (3.9) of lactate compared with the pKa of VFA (4.8) normally associated with ruminal fermentation (Russell and Hino, 1985). In the present experiment, with the exception of the first week of monensin supplementation in the high-concentrate diet, neither monensin supplementation nor rotation of monensin and lasalocid caused a change (P > 0.05) in ruminal pH.
Total VFA concentrations were not changed (P > 0.05) as a result of ionophore addition to either the high-concentrate (115.6 ± 1.8 mM) or low-concentrate (111.1 ± 1.8 mM) diets. However, the acetate:propionate ratio in the ruminal fluid was decreased (P < 0.001) from the time ionophores were introduced until they were removed from either diet (Figures 4 and 5; Table 4). Original acetate:propionate ratios were restored within 2 wk of ionophore withdrawal in either diet. Likewise, the ionophore-induced decrease (P < 0.001) in ruminal ammonia-N concentration persisted through the entire ionophore feeding period in both the high- and low-concentrate diets. This depression in ammonia-N disappeared within 2 wk after ionophore removal from the diets.
Fuller and Johnson (1981) observed that both monensin and lasalocid decreased the acetate:propionate ratio and ammonia-N production without affecting total VFA production in vitro when they were added to either high- or low-concentrate substrates. Similar results were obtained by Russell and Strobel (1989). These observations were confirmed in vivo for animals fed low-(Lana and Russell, 1997) and high-concentrate (Hristov et al., 2001) diets. As previously reported by Rogers et al. (1997), ruminal fermentation characteristics returned to presupplementation levels relatively quickly after ionophores are withdrawn from the diet.
Changes in ruminal fermentation characteristics can be explained by varying sensitivities of microbial species to ionophores in the rumen. Gram-positive bacteria are sensitive to ionophores, which influences the fermentation processes producing acetate, butyrate, lactate, and ammonia. On the other hand, gram-negative bacteria, generally engaged in fermentation pathways associated with the production of propionate, are resistant to ionophores (Russell and Strobel, 1989; Russell, 1996). Proteolytic and obligate amino acid fermenting bacteria are also sensitive to ionophores (Russell, 1996). Consequently, the acetate:propionate ratio and ammonia-N concentration in ruminal fluid decrease.
Ruminal Protozoa
The ruminal probe used to collect ruminal fluid does not retrieve a representative sample of all regions of the rumen (Duffield et al., 2004), and as such organisms associated with the epithelium and ventral region of the rumen are likely underrepresented. Because ruminal fluid samples included particulate matter, it is assumed that there was a mixture of microorganisms, both free floating and those sequestered by feed particles.
Ruminal fluid protozoa counts averaged 2.60 x 105 cfu/mL in the initial 2 wk cattle were fed the high-concentrate diet without ionophore supplementation (Figure 6 and 7). Protozoal populations for cattle fed the low-concentrate diets without ionophore supplementation during the same period were 1 log lower (P < 0.05, 5.50 x 104 cfu/mL). Several studies (Franzolin and Dehority, 1996; Hristov et al., 2001) have reported ruminal protozoa counts in a similar range when cattle were fed diets containing 62 to 75% concentrate. The high-concentrate diet contained approximately 70% concentrate.
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). Total ciliate protozoal populations decreased (P < 0.001) to 1.21 x 104 cfu/mL when ionophores were added to the low-concentrate diet, the response for monensin only and monensin and lasalocid in rotation being similar. Protozoal counts were restored within 3 and 6 wk of ionophore supplementation for the high- and low-concentrate diets, respectively.
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and 7, Table 5).
It is recognized that ruminal ciliate protozoa are reduced by initial ionophore exposure and that they may subsequently develop resistance. Dennis and Nagaraja (1986) conducted short-term in vitro and in vivo trials in which a reduction of total protozoa numbers was observed when monensin or lasalocid were added to ruminal fluid. A study by Rogers et al. (1997) showed that extent of reductions in total ruminal protozoa numbers as a result of monensin supplementation decreased with time for animals fed a high-concentrate diet. They also reported that the effect of monensin on the protozoa population was species-dependent; Entodinium was inhibited, whereas Isotricha numbers increased as a result of monensin supplementation. Many methanogen spp. are found in association with ciliate protozoa in the rumen (Chagan and Ushida, 2004), living on the exterior surface or as endosymbionts within the protozoa. Ushida and Jouany (1996) reported apparent daily CH4 emission per protozoan cell to range from a trace amount to 2 nmol for in vitro systems.
In summary, the decrease in CH4 production resulting from ionophore supplementation appears to be short-lived when cattle are fed either low- or high-concentrate diets. Methanogens do not appear to be directly affected by the presence of ionophores (Chen and Wolin, 1979), suggesting an indirect response. Although total VFA concentration in ruminal fluid was not changed, the decrease in the acetate:propionate ratio, and ammonia-N concentration persisted as long as the ionophores were supplemented in the low- or high- concentrate diet.
The data suggest that the effect of ionophores on enteric CH4 production may be related to ciliate protozoal populations. Total protozoal numbers declined for both high- and low-concentrate diets when ionophores were introduced, with the transitory effect lasting no more than 3 and 6 wk, respectively. The transitory suppression of CH4 emissions when ionophores are introduced to the ruminant diet was related to the transitory decline in ruminal ciliate protozoal populations. As these populations recover, CH4 emissions returned to presupplementation levels. Further, the data indicate that ruminal ciliate protozoal populations can adapt to the ionophores presented in either low- or high-concentrate cattle diets, with a more rapid adaptation when digestible OM is greater. Rotation of monensin and lasalocid does not prevent or retard ciliate protozoal adaptation compared with continual use of monensin.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: km_wittenberg{at}umanitoba.ca
Received for publication November 8, 2005. Accepted for publication February 10, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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