J. Anim. Sci. 2003. 81:323-328
© 2003 American Society of Animal Science
Effects of 9,10 anthraquinone on ruminal fermentation, total-tract digestion, and blood metabolite concentrations in sheep1
L. Kung, Jr.2,
K. A. Smith,
A. M. Smagala,
K. M. Endres,
C. A. Bessett,
N. K. Ranjit and
J. Yaissle
Department of Animal and Food Sciences, College of Agriculture and Natural Resources, University of Delaware, Newark 19717-1303
2 Correspondence:
Phone: 302-831-2522; fax: 302-831-2822; E-mail:
lkung{at}udel.edu.
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Abstract
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The objective of this study was to evaluate the effects of adding 9,10 anthraquinone, a known inhibitor of methanogenesis and sulfate reduction, on blood metabolites, digestibility, and distribution of gas in sheep. In all experiments, we fed a complete pelleted diet that contained 17.5% crude protein and 24.5% acid detergent fiber. In an 8-wk study, feeding up to 66 ppm (dry matter basis) of 9,10 anthraquinone had no adverse effects on blood metabolites including indicators of normal enzyme function, mineral concentrations, and hematological measurements. Feeding 9,10 anthraquinone had no effect on average daily gain, although sheep fed a diet containing 66 ppm of 9,10 anthraquinone numerically gained the least weight. The ruminal molar proportions of acetic acid were decreased (P < 0.05) and the molar proportions of propionic acid were increased (P < 0.05) in sheep fed 1.5 and 66 ppm 9,10 anthraquinone when compared to those fed an unsupplemented diet. In a digestion trial, 9,10 anthraquinone (33 and 66 ppm) had no effect on the apparent digestion of nutrients in the total gastrointestinal tract. In a metabolism study, ruminal gasses were collected by rumenocentesis and analyzed for methane and hydrogen concentrations. Feeding 500 ppm of 9,10 anthraquinone to sheep resulted in a decrease (P < 0.07) in the concentration of methane, but an increase (P < 0.05) in hydrogen concentration of ruminal gas throughout the 19 d of feeding. There was no indication of ruminal adaptation throughout this time. These results are the first to show that 9,10 anthraquinone can partially inhibit in vivo rumen methanogenesis, which supports previous in vitro findings. In addition, at the concentrations used in this study, 9,10 anthraquinone was not toxic to ruminants.
Key Words: Anthraquinones • Methane Inhibitors • Rumen
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Introduction
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Methane produced in the rumen is a source of lost dietary energy to ruminants. Inhibition of methanogenesis in the rumen may be useful by increasing the efficiency of energy used in the rumen. In recent studies, in vitro evidence has shown that 9,10 anthraquinone (AQ) can partially inhibit methane (Garcia-Lopez et al., 1996) and hydrogen sulfide production (Kung et al., 1998, 2000) in ruminal fermentations. In unadapted ruminal fluid, AQ increased the molar proportions of propionic and butyric acids. It also caused a small decrease in the concentration of total VFA and a decrease in the molar proportion of acetic acid. These changes in ruminal fermentation suggest that addition of AQ to ruminant diets might improve the efficiency of production. However, when methanogenesis was inhibited by the addition of AQ, there was an accumulation of hydrogen in short-term incubations (Garcia-Lopez et al., 1996), partially explaining the decrease in the molar proportion of acetic acid and representing a loss of dietary energy. Many of the changes brought about by AQ on rumen fermentation were similar to findings with other methane inhibitors (Trei et al., 1971). Most of these experimental compounds have not been commercialized for a variety of reasons, including an accumulation of hydrogen, toxicity to the host animal, poor palatability, and ruminal adaptation (Van Nevel and Demeyer, 1996). It is unknown whether feeding AQ to ruminants would also be precluded because of such factors. Our hypothesis was that feeding AQ to sheep would safely decrease the production of ruminal methane.
The objective of this experiment was to determine the effects of feeding AQ to sheep on blood chemistry, nutrient digestion, and in vivo inhibition of methane production.
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Materials and Methods
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Feeding Study
Thirty-five crossbred Dorset x Finn wethers were used in a feeding experiment designed to determine the effects of feeding varying levels of AQ on blood chemistry. Sheep (25 ± 2 kg) were treated to eliminate internal parasites and vaccinated to prevent enterotoxemia 2 wk prior to the start of the study. Animals were fed a complete basal diet once daily (Early Market Lamb Pellet, Agway, Inc., Tully, NY) at 0600 for 2 wk (Table 1
). Sheep (seven per group) were randomly assigned to one of the five pens (10 x 12 m) and fed the basal diet supplemented with: 1) nothing (control), 2) 1.5 ppm (DM basis) of AQ (Akron Life Sciences, Wilmington, DE), 3) 16.5 ppm of AQ, 4) 33 ppm of AQ, or 5) 66 ppm of AQ. The AQ was a 50% soluble suspension that was further diluted with 50 mL of deionized water and sprayed onto 50 kg of feed while mixing in a ribbon mixer. Diets were fed twice daily (0600 and 1800) to obtain ad libitum intake and feed refusals were measured once daily. Fresh water was available at all times. Diets were sampled three times weekly and pooled every 2 wk to obtain four feed samples of each treatment for analysis at the end of the study.
All sheep were weighed at the start and end of the treatment, which lasted 8 wk. On the last day of weeks 2, 4, 6, and 8, sheep were offered their morning allotment of feed at staggered intervals in order to obtain ruminal fluid by stomach tubing 3 h after feeding. Ruminal fluid was strained through four layers of cheesecloth and chilled on ice before processing. Ruminal fluid was analyzed for VFA and ammonia N as described by Sheperd et al. (1995). During the last day of sampling, blood was collected from the jugular vein once, 4 h after feeding, kept on ice, and submitted to the University of Pennsylvania Veterinary School (New Bolton, Pennsylvania) for clinical blood analyses. Blood samples for plasma mineral and biochemical metabolites were collected in tubes containing sodium heparin, and samples for hematology were collected in tubes with sodium fluoride and ethylenediamine tetraacetic acid.
Digestion Study
Nine Dorset x Finn wethers (35 ± 2 kg) were placed in metabolism stalls for separation of feces from urine to determine the effect of varying levels of AQ on nutrient digestion. Sheep were vaccinated as previously described. Sheep were fed the basal diet supplemented with the following: 1) nothing (control), 2) 33 ppm of AQ, or 3) 66 ppm AQ. The experiment was a completely randomized design replicated over time. In the second period, sheep were randomly assigned to treatments with the restriction that no lamb would receive the same treatment as in the first period. Periods were 14 d long. During d 1 to 8 in each period, sheep were given ad libitum access to feed twice daily; thereafter, diets were offered at 90% of ad libitum intake to ensure that there were no feed refusals. From d 9 to 14, total feces were collected and weighed daily. Fresh water was available at all times. A 20% aliquot from each daily sample was composited and frozen at -10°C until analyses for ash, N, ADF, and NDF content as described by Kung et al. (1991).
All animals and procedures were approved under the guidance of the College of Agriculture and Natural Resources Animal Care Committee following approved guidelines (Consortium, 1999).
Rumen Metabolism Study
Ten Dorset x Finn wethers (42 ± 2 kg) were used to evaluate the use of rumenocentesis for collection of ruminal gasses to test the effect of a high level of AQ on ruminal gas composition in sheep. Sheep were housed in metabolism crates. All sheep were vaccinated as previously described. Sheep were fed 1 kg of a basal diet (as described) once daily at 0700 for 7 d and fresh water was available at all times. Two days prior to treatment, during treatment, and post treatment, sheep were limit-fed 700 g per day of the basal diet. During treatment, five sheep received the basal diet and the remaining five sheep were fed the same diet supplemented with 500 ppm (DM basis) AQ. The AQ was sprayed onto the feed and allowed to dry before feeding. Ruminal gas was collected from each lamb starting 3 d before and for 6 d after a 19-d treatment period by rumenocentesis with a 5-mL syringe attached to a sterile 21-gauge needle. The puncture site was shaved, cleaned with betadine, and injected with 0.25 mL of lidocaine. A 2.5-mL sample of gas was withdrawn from the rumen and injected into a 3-mL vaccutainer tube for analyses of methane and hydrogen as described by Garcia-Lopez et al. (1996). Sheep were sampled 4 h after feeding.
Statistics
All data were analyzed by ANOVA using the general linear model procedure of SAS (SAS Inst., Inc., Cary, NC). In all studies, significance was declared at P < 0.05 unless noted otherwise.
Data from the sheep growth study was analyzed as a completely randomized design with degrees of freedom partitioned into treatment, period, and treatment x period interaction. Data from the rumen metabolism study was analyzed as a randomized block design with degrees of freedom partitioned into treatment, period, and treatment x period interaction. The treatment effect was tested by using the treatment x period as the error term. Data from the in vivo gas collection study were analyzed as a study with Repeated Measures Analysis of Variance. Data from the pretreatment period were used as covariates for methane and hydrogen.
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Results and Discussion
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Sheep were judged to be in excellent health throughout the study except for one lamb that received the 1.5-ppm AQ diet, which became lethargic, refused to eat during wk 6 of the study, and was euthanatized during the seventh week on treatment. The specific cause of illness could not be determined. Intake and average daily gains of sheep in the feeding study are shown in Table 2. Assuming a rumen volume of about 4 L, sheep consuming about 1,200 g of feed containing 1.5 and 16.5 ppm of AQ had ruminal fluid containing similar concentrations of AQ to those tested in our previous in vitro studies (about 0.5 to 5 ppm) (Garcia-Lopez et al., 1996). Feed consumption averaged more than 1 kg of feed for all treatments, but because they were group-fed, there was no statistical analysis for intake. Although there is an aversion to seed treated with AQ by birds (Avery et al., 1998), we could not find any data similar to this pertaining to mammalian species. Sheep fed AQ had ADG that were similar to sheep fed the control diet, but the ADG was numerically the lowest in sheep fed the highest level of AQ supplementation.
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Table 2. The effect of feeding various levels of 9,10 anthraquinone (AQ) to sheep for 8 wk on average daily gain and dry matter intake
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The molar proportions of the major VFA from ruminal fluid of sheep in the feeding study are shown in Table 3. The effects of AQ on ruminal VFA were not consistent, nor were they dose dependent. Specifically, sheep fed 1.5 and 66 ppm of AQ had lower (P < 0.05) molar proportions of acetic acid and greater (P < 0.05) molar proportions of propionic acid than did sheep fed the control diet. These findings were similar to previous in vitro data from our laboratory (Garcia-Lopez et al., 1996). For example, in batch fermentations of a 50:50 forage:concentrate diet, the addition of AQ to achieve a final culture concentration of 5 ppm decreased the molar proportion of acetic acid from 53.9% in untreated cultures to 47.5% in treated cultures (Garcia-Lopez et al., 1996). In contrast, the molar proportion of propionic acid was increased from 19.4% in untreated cultures to 25.5% in treated cultures. There were no differences in the molar proportions of the branched-chain fatty isomers (data not shown). Treated cultures also produced more than 15 times less methane than did untreated cultures in the study of Garcia-Lopez et al. (1996).
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Table 3. The molar proportions (mol/100 mol) of volatile fatty acids from sheep fed varying levels of 9,10 anthraquinone (AQ) for 8 wk
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In general, measurements of ions in blood (Table 4), biochemical measurements (Table 5), and hematological measurements (Table 6) after 8 wk on treatment were not different among treatments. Moreover, most measurements were within normal accepted levels for sheep (Hoffman, 1986), with the exception of creatinine phosphokinase (Table 5) concentrations. However, the normal variation in creatinine phosphokinase concentration is always extremely large. Although there were some differences among treatments for blood calcium and creatinine, there was no trend for adverse effects with increasing concentrations of AQ. Digestibility coefficients are shown in Table 7. Feeding 33 and 66 ppm of AQ to sheep had no effects on OM, ADF, NDF, or N digestion in sheep. Overall, the blood and digestibility data suggest that feeding AQ for 8 wk had no adverse effects on the health of sheep.
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Table 5. Plasma metabolite and enzyme concentrations in sheep fed varying levels of 9,10 anthraquinone (AQ) for 8 wk
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Table 7. Nutrient digestion (% dry matter basis) of diets fed to sheep and supplemented with 9,10 anthraquinone (AQ)
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In the in vivo gas collection study, we fed a high dose of AQ because we limited intake to ensure complete consumption of the diet. Assuming a rumen volume of 4 L and an intake of 700 g of diet treated with 500 ppm of AQ (DM basis), this resulted in a liquid concentration of AQ that was equivalent to about 87.5 ppm. The effects of AQ on the concentrations of ruminal hydrogen and methane in sheep are shown in Figures 1 and 2, respectively. Rumenocentesis, withdrawal of fluid from the rumen with a syringe and needle assembly, has been used to collect ruminal fluids for the determination of pH in dairy cattle (Garrett et al., 1999). We used a similar technique to sample ruminal gasses in the current study. As expected, concentrations of hydrogen were low (less than 1 umol/L) prior to feeding of AQ. Within one day of treatment, AQ increased the concentration of hydrogen, which remained elevated (P < 0.05) (about 10 µmol/L) for the duration of the 19-d treatment period. Within 6 d of withdrawal of AQ from the diet, ruminal hydrogen concentration had returned to baseline. Concentrations of methane were more variable than concentrations of hydrogen in ruminal gas, but after treatment with AQ, methane concentrations tended to be lower (P < 0.07) in sheep fed AQ than in sheep fed the control diet. This is the first evidence that shows that AQ can inhibit methanogenesis in in vivo ruminal fermentations. After withdrawing AQ from the diet, the concentration of methane was similar in rumen gas from treated and untreated sheep. A reduction in methane and an increase in hydrogen concentration in ruminal gasses when sheep are fed AQ agree with previous in vitro batch culture and continuous culture data from our laboratory (Garcia-Lopez, 1996). The accumulation of hydrogen suggests that AQ most likely had a direct effect on methanogenic bacteria. In contrast, ionophores like monensin inhibit methane production indirectly by suppressing microorganisms that supply hydrogen to the rumen ecosystem (Chen and Wolin, 1979). Although not well studied, we believe that AQ acts as a redox uncoupler that results in a shortage of ATP for cell growth, which has been the proposed mode of action of AQ on sulfate-reducing bacteria (Cooling et al., 1996). There were no apparent signs of rumen adaptation to AQ because methane remained depressed and hydrogen was elevated for the 19 d of treatment in our study. These findings are significant because ruminal adaptation of many methane inhibitors has been shown to be transient (Immig et al., 1996) due to development of resistant populations or degradation of the inhibitory compounds. A common finding as a result of adding a methane inhibitor to ruminal fermentations is the accumulation of hydrogen. This suggests unwanted alterations in interspecies hydrogen transfer that could continue to lead to an energy loss from the diet. Thus, methods to increase reductive acetogenesis via stimulation or addition of acetogens have been suggested (Lungdhal, 1986). The addition of acetogens into normal ruminal fermentations has not increased acetate production (Le Van et al., 1998), probably because acetogens have much lower affinities for hydrogen than methanogens. Thus, they are unable to compete for substrate when the methanogens are present. However, when combined with methane inhibitors, acetogens have been able to alter ruminal fermentations. Le Van et al. (1998) reported that inhibition of methanogenesis with 2-bromoethanesulfonic acid (BES) and the addition of Acetitomaculum ruminis 1904A to obtain >105 cfu/mL, resulted in reductive acetogenesis. Similar results were obtained by Lopez et al. (1999) with BES and the acetogen Eubacterium limosum ATCC 8486, and by Nollet et al. (1997) with BES and the acetogen Peptostreptococcus productus ATCC 35244.
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Figure 1. The effect of feeding 500 ppm of 9,10 anthraquinone (AQ) to sheep on the concentration of hydrogen in ruminal gas. • = control animals,  = animals fed AQ for 19 d.
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Figure 2. The effect of feeding 500 ppm of 9,10 anthraquinone (AQ) to sheep on the concentration of methane in ruminal gas. • = control animals, = animals fed AQ for 19 d.
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Implications
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The results of this and previous studies from our lab suggest that 9,10 anthraquinone is a potential methane inhibitor for ruminants because 1) it was effective at low concentrations, 2) there were no negative effects on animal health or in vivo digestion, and 3) there was no apparent in vivo adaptation to a reduction in methane production. Long-term studies will be required to further our understanding of how 9,10 AQ may be useful as a rumen modifier.
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Footnotes
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1 Published as paper number 1696 of the DE Agric. Exp. Stn. The authors are grateful to C. Golt for assistance with volatile fatty acid analysis and J. Tabinowski for gas analysis. 
Received for publication April 18, 2002.
Accepted for publication August 20, 2002.
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Abstract
Introduction
