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ANIMAL NUTRITION |
,2
* Bioresearch Corporation of Yokohama, Yokohama 230-0004, Japan and
and
Laboratory of Agricultural Production Technology, Tokyo University of Agricultureand Technology, Tokyo 183-8509, Japan
Abstract
The effects of 
-cyclodextrin-horseradish oil complex (CD-HR) on methane production and ruminal fermentation were studied in vitro and in steers. In the in vitro study, diluted ruminal fluid (30 mL) was incubated anaerobically at 38°C for 6 h with or without CD-HR, using cornstarch as substrate. The CD-HR was added at various concentrations (0, 0.17, 0.85 and 1.7 g/L). Treatment affected neither the pH of the medium nor the number of protozoa. Total VFA increased in a linear manner (P = 0.02), and NH3-N decreased quadratically (P = 0.04) as the concentration of CD-HR increased from 0.17 g/L to 1.7 g/L. Molar proportions of acetate decreased in a linear manner (P = 0.03), and propionate increased linearly (P = 0.008) with increasing concentrations of CD-HR. Production of methane was inhibited up to 90%, whereas accumulation of dihydrogen was increased 36-fold by 1.7 g/L of CD-HR supplementation relative to controls. The effect of CD-HR on methane production, ruminal fermentation and microbes, and digestibility was further investigated in vivo using four Holstein steers in a crossover design. The CD-HR supplement was mixed into the concentrate portion of a (1.5:1) Sudangrass hay plus concentrate mixture that was fed twice daily to the steers. Ruminal samples were collected 0, 2, and 5 h after the morning feeding. No effects of CD-HR supplementation on ruminal pH (P = 0.63) or protozoal numbers (P = 0.44) were observed. Molar proportion of acetate was decreased (P = 0.04) and propionate was increased (P = 0.005) by CD-HR treatment. Molar proportion of butyrate was increased (P = 0.05) in CD-HR-supplemented steers. Ruminal NH3-N was decreased (P = 0.05) by treatment. Blood plasma glucose concentration was increased (P = 0.02) and urea-N was decreased (P = 0.04) with CD-HR supplementation. Daily DMI was decreased (P = 0.04), and apparent digestibility of DM (P = 0.13), NDF (P = 0.14), and CP tended (P = 0.14) to be increased by treatment. Methane production was decreased (P = 0.03) by 19%, and the number of methanogens was also decreased (P = 0.03). Although N retention (P = 0.11), total viable bacteria (P = 0.15), and sulfate-reducing bacteria (P = 0.17) were not significantly altered by treatment, tendencies for increases were noted with CD-HR supplementation. The number of cellulolytic (P = 0.38) and acetogenic bacteria (P = 0.32) remained unchanged by treatment. These results indicate that CD-HR supplementation can be used to decrease methane production in steers.
Key Words: -Cyclodextrin-Horseradish Oil Complex • Methane • Ruminal Fermentation
Introduction
Methane production in the rumen may account for as much as 6 to 10% of the GE intake of ruminant animals (Blaxter and Clapperton, 1965
), and has also received attention as a potential contributor to global warming (Leng, 1991; Moss, 1993). Many chemical compounds, such as halogenated methane analogs, diaryliodonium derivatives, coenzyme-M analogs, and uncouplers of proton motive force, have been found to inhibit methane production in vitro and in vivo (Bauchop, 1967; Czerkawaski and Breckenridge, 1972; Martin and Macy, 1985). However, ruminal microbial populations have been found to adapt in vivo (Clapperton, 1977) or degrade in vitro (Martin and Macy, 1985) many of these compounds.
Sinigrin (allyl glucosinolate) is converted to allyl isothiocyanate (AIT) by the enzyme myrosinase (Simon et al., 1984) in mustard seeds and horseradish root. Tyagi and Singhal (1998) reported that mustard oil and glucosinolate reduced ruminal methane production in vitro. They speculated that biohydrogenation of mustard oil fatty acids would withdraw dihydrogen from methane formation. However, they ignored AIT, which is the volatile oil of mustard. In a subsequent study, Lila et al. (2003) reported that synthetic AIT also inhibited ruminal methanogenic bacteria and reduced methane production in vitro. Japanese horseradish (HR) oil contains similar active compounds to mustard oil (Simon et al., 1984). Our hypothesis was that supplementation with HR oil may inhibit methane emission from ruminants. However, using HR as a supplement for ruminant feed is difficult due to its pungent odor, and the substance may also decrease the appetite of animals (our unpublished observations). To overcome this problem, we used cyclodextrin (CD) as an encapsulated material to allow for a gradual release of the coated compound. This study was conducted to evaluate the effects of -cyclodextrin-horseradish oil complex (CD-HR) on methane production and ruminal fermentation in vitro and in steers.
Materials and Methods
The dairy cows and steers were cared in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Tokyo University of Agriculture and Technology, No. 15-21.
In Vitro Experiment
Cyclodextrin was the product of Bio Research Corporation of Yokohama (Yokohama, Japan), and HR oil was purchased from Nisshin Koryo Co. Ltd. (Tokyo, Japan). The CD-HR complex was prepared according to Ajisaka et al. (2002). Briefly, 10% of CD was dissolved in distilled water at 60°C, and a weighed amount of HR oil was added to achieve equal amounts of CD and HR. The mixture was homogenized at 8,000 rpm for 10 min. After 3 d of sitting at room temperature, the precipitate of the CD-HR complex was filtered (qualitative filter paper No. 1, ashless; Toyo Roshi Kaisha Ltd., Tokyo, Japan) and dried under reduced pressure. The CD-HR was found to contain 3.5% (wt/wt) HR (GC-2010, Shimadzu Co. Ltd., Kyoto, Japan).
A 600-kg lactating Holstein dairy cow, fed 10 kg of forage (Sudangrass hay) and 10 kg of concentrate mixture (Table 1) per day (DM basis), was used as the donor animal for ruminal fluid. About 600 mL of ruminal fluid was drawn prior to the morning feeding through a stainless steel stomach tube into a warm Thermos flask with O2-free CO2 and immediately transported into the laboratory. The ruminal fluid was strained through four layers of surgical gauze into an Erlenmeyer flask and passed O2-free CO2. The fluid was then mixed with buffer in a 1:2 ratio (Russell and Martin, 1984). After mixing, 30 mL of diluted ruminal fluid were anaerobically transferred to 60-mL serum bottles containing 200 mg of cornstarch (Wako Pure Chemical Industries, Ltd., Tokyo, Japan). Weighed amounts of CD-HR were added to achieve final concentrations of 0, 0.17, 0.85, and 1.7 g/L. Serum bottles were anaerobically sealed under O2-free CO2 atmosphere and placed in an incubator at 38°C for 6 h.
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). A 0.5-mL sample of gas was removed from each of the bottles with a gas-tight syringe, and methane and hydrogen were measured with a gas chromatograph (model GC-8A, Shimadzu Co. Ltd., Kyoto, Japan) using a molecular sieve 5A column (1.6 m x 3.2 mm i.d., 60 to 80 mesh, Shinwakako, Kyoto, Japan) and a thermal conductivity detector (column temperature = 60°C, injector and detector temperature = 80°C). The carrier gas (Ar) flow rate was 50 mL/min. The pH was measured with a portable pH meter (ATC pH meter Piccolo2, Arvore-Vila do Conde, Portugal). About 5 mL of fermentation fluid was taken for analysis of NH3-N and VFA, and stored immediately at –30°C until analysis. A 1-mL sample of the incubated fluid was diluted with 4 mL of methylgreen-formalin-saline to count ruminal ciliate protozoa. One milliliter of thawed fermentation fluid was mixed with 0.2 mL of deproteinizing agent (12% meta-phosphoric acid in 3 N H2SO4), centrifuged (10,000 x g for 10 min at 4°C), and VFA was analyzed by a gas chromatograph (model GC-14B, Shimadzu Co. Ltd.) using a Thermon-3000 5% Shincarbon A column (1.6 m x 3.2 mm i.d., 60 to 80 mesh, Shinwakako) and flame ionization detector (column temperature = 130°C, injector and detector temperature = 200°C). The carrier gas (N2) flow rate was 50 mL/min. The micro diffusion method was used to determine NH3-N (Conway, 1962). Total protozoa and its composition were counted using a Fuchs-Rosenthal counting chamber (Hausser Scientific Partnership, Horsham, PA) as described previously (Ogimoto and Imai, 1981).
In the in vitro experiment, each group contained three bottles; consequently, the fermentation was realized in 12 fermentation bottles. The in vitro fermentation experiment was conducted in three runs on different days within a 1-wk interval, using three replicates per run. Data were analyzed (Table 2) using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC) with four levels of CD-HR, and run was included in the model as a random blocking factor (n = 3). Treatment effects were tested using day x treatment interaction, and the overall mean CD-HR random effect of run interaction residual was reported. Orthogonal contrasts were performed to determine linear and quadratic trends.
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Four 6- to 7-mo-old (mean BW = 141 ± 27 kg) castrated Holstein steers (half-sib) were housed in digestion stalls in a lighted and ventilated room. The steers were adapted with Sudangrass hay:concentrate mixture (1.5:1; Table 1
) twice daily at 0900 and 1700 at the maintenance level. Water and trace mineral salt were continuously available. During the first period, two steers received the control diet and the other two steers received the same diet supplemented with CD-HR. The CD-HR was supplemented at 2% of feed DM by mixing it into the concentrate mixture twice daily before feeding. The diets were switched during the second period. Each period consisted of 14 d of adaptation and 12 d of measurement.
From d 15 to 18 of each period, methane production was determined by head hood collection chamber. A 4-d total collection digestibility trial was carried out during d 19 to 23 of each period. During the digestion trial, feces were collected daily, weighed, and 10% aliquots were dried in a forced-air oven at 60°C, ground to pass a 1-mm screen, and then assayed for CP and NDF. Urine was collected in a 20-L container containing 300 mL of 3 N H2SO4, and a 50- to 100-mL sample was frozen until analyzed. On the final day of each period, samples of ruminal fluid and jugular venous blood were collected before feeding and at 2 and 5 h after feeding. Approximately 300 mL of ruminal digesta was collected using a flexible polyvinyl chloride stomach tube and a subsample (100 mL) of the 2-h postfeeding sample was taken in a sterilized container for microbiology assay. The ruminal fluid was strained through four layers of surgical gauze. The pH was measured and samples were taken and stored as described above. Blood samples were collected into heparinized tubes, immediately placed on ice, and centrifuged at 11,000 x g for 15 min at 4°C. Plasma was removed and stored at –30°C.
Hungate’s anaerobic technique (1969) was used to prepare media and to cultivate microorganisms. Total viable counts were determined in roll tubes (triplicate) with the complete medium described by Leedle and Hespell (1980). Other counts were estimated by the most probable number method according to Alexander (1982). Cellulolytic bacteria were cultivated in Halliwell and Bryant (1953) medium. Media for hydrogenotrophs were the same as previously described (Morvan et al., 1994).
Samples were collected in a sterile 100-mL container and processed within 15 min. Serial 10-fold dilutions in an anaerobic mineral solution (Bryant and Burkey, 1953) were prepared. Dilutions 102 to 105 were used to cultivate acetogens; 104 to 108 for cellulolytic and sulfate-reducing bacteria and methanogens; and 106 to 109 for total viable bacteria. Afterward, 1 mL of each tube was used to inoculate 5 mL of culture media in Hungate tubes. Five tubes were used for each diluted sample. Hydrogenotrophic cultures were pressurized to 202 kPa with O2-free H2/CO2 (80:20), and all incubations were carried out at 38°C. For total viable bacteria, cultures were incubated for 4 d; cellulolytic and sulfate-reducing bacteria and methanogenic archaea were incubated for 2 wk; and acetogenic bacteria were incubated for 3 wk. Cellulolytic tubes were considered positive when the filter paper in them was degraded (Morvan et al., 1996). A black FeS precipitate developed in tubes associated with a gas consumption counted as positive for sulfate reduction (Morvan et al., 1996). Tubes with methane concentrations above 100 ppm were counted for the determination of methanogens (Butine and Leedle, 1989). Tubes associated with gas consumption in which the increase in acetate concentration was above 10 mM were counted as positive for the estimation of H2/CO2 acetogenic bacteria (Morvan et al., 1996).
A head hood collection chamber was used for the measurement of methane, which was measured from 0900 to 1700 h after feeding. Methane concentration was determined every minute throughout the measurement period with an infrared gas analyzer detector (Fuji Electric Co., Ltd., Tokyo, Japan). During the measurement of methane production, the chambers were maintained at 20°C and 50% relative humidity. The nitrogen content of the feed, feces, and urine samples were determined by the Kjeldahl method (AOAC, 1965). The NDF was determined by the procedure of Goering and Van Soest (1970). The VFA, protozoa, and NH3-N were determined as described before. Plasma glucose was determined by the o-toluidine boric acid method using a kit (Wako Pure Chemical Industries, Ltd.) and urea-N was measured by the diacetylmonooxime method using a kit (Wako Pure Chemical Industries, Ltd.).
Data were analyzed using the GLM procedure of SAS. The statistical model used was as follows:
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where yijk = dependent variable for cow on treatment i during period j; m = overall mean; Ti = treatment effect (i = 1,2); Pj = period effect (j = 1,2); Ck = cow effect (k = 1 to 4); and eijk = error. Ruminal fermentation measurements were tested using split-plot ANOVA. The model was as follows:
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where Hl = time effect (l = 1 to 3) with respective interactions, and m, T, P, and C are as defined above. Orthogonal contrasts were also examined for ruminal fermentation parameters due to the time of three observations within a day. Data are presented as least squares means and were considered statistically different if P < 0.05. Differences of P < 0.20 to P < 0.05 are discussed as trends.
Results
In Vitro Experiment
Table 2 shows the in vitro effects of CD-HR on microbial fermentation with cornstarch after 6 h of incubation. Methane production decreased in a linear manner (P = 0.001) on CD-HR supplementation at 0.17, 0.85, and 1.70 g/L by 19, 41, and 90% relative to controls, respectively. The increase (P = 0.001) in dihydrogen accumulation was 4-, 21- and 36-fold by CD-HR supplementation relative to controls at 0.17, 0.85, and 1.70 g/L, respectively. The pH of the medium was unaffected by CD-HR supplementation. A quadratic effect (P = 0.04) was observed on NH3-N with increasing levels of CD-HR. Increasing the concentration of CD-HR resulted in a linear increase (P = 0.02) in total VFA concentration. Compared with untreated cultures, increasing levels of CD-HR caused a linear decrease in molar proportion of acetate (P = 0.03), and a linear increase in propionate (P = 0.008) and butyrate (P = 0.03). Different treatments did not significantly change numbers of protozoa. The CD-HR supplementation exerted little effect (P = 0.09; quadratic effect) on minor VFA. Increasing levels of CD-HR caused linear increases (P = 0.006) in total gas production.
In Vivo Experiment
Table 3 shows the effect of CD-HR on ruminal gasses, feed intake, and digestibility. Methane production was decreased (P = 0.03) by 19%, and carbon dioxide production was unchanged (P = 0.83) with CD-HR. Feed intake decreased (P = 0.04) by 10.3% in CD-HR-supplemented steers. Although the results were not significant, the apparent digestibility of DM (P = 0.13), NDF (P = 0.14), and CP (P = 0.14) tended to be increased by treatment.
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. The pH value of the fluid was unaffected by CD-HR supplementation. Ruminal NH3-N concentrations fluctuated with time after feeding (quadratic effect; P = 0.008). On average, supplementation with CD-HR decreased NH3-N by 14.3%. Total concentrations of VFA increased (linear effect; P = 0.05) after feeding, but were unaffected by treatment (P = 0.68). Ruminal feeding with CD-HR significantly altered the patterns of ruminal fermentation. Concentrations of acetate decreased (P = 0.04), but average ruminal propionate concentration was 3.7 mM higher (P = 0.005) in CD-HR-supplemented steers than in controls. Acetate:propionate ratio decreased (P = 0.03) by 22.9% on CD-HR supplementation. Ruminal average butyrate concentration increased (P = 0.03) by 11.3% in CD-HR-supplemented steers. Minor VFA were unaffected (isobutyrate, P = 0.54; isovalerate, P = 0.49; and valerate, P = 0.66) by CD-HR supplementation.
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shows the effect of CD-HR on rumen microbiota in steers. The number of methanogenic bacteria was decreased (P = 0.03) by CD-HR supplementation, whereas the number of total viable bacteria (P = 0.15) and sulfate-reducing bacteria (P = 0.17) tended to be increased. The number of cellulolytic (P = 0.38) and acetogenic bacteria (P = 0.32) was unchanged by treatment. Concentrations of protozoa in the rumen fluctuated with time (linear effect; P = 0.03). Ruminal ciliate protozoa comprised Entodinium spp., Dasytricha sp., and Isotricha sp. The total number and composition of protozoa were unaffected (P = 0.44) by CD-HR supplementation.
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). Postfeeding glucose concentration increased (P = 0.02) by 9.2% after treatment, whereas urea-N decreased (P = 0.04) by 8.9%. Nitrogen content in feces was unaffected (P = 0.42) by CD-HR supplementation, but was decreased in urine (P = 0.02) and feces plus urine (P = 0.05). However, N-retention tended to be increased (P = 0.11) by CD-HR supplementation.
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Cyclodextrin was used as an encapsulating material and contained the pungent odor of HR. There are two advantages to coating HR with CD. First, the volatile nature of the active ingredient is reduced, and second, the CD-matrix delays the release of HR to provide sustained release in the rumen when CD-HR is fed to steers on a daily basis. Total gas was increased with CD-HR in the in vitro study, indicating improved ruminal fermentation. Methane production was decreased for both in vitro and in vivo studies, and dihydrogen accumulation was increased in the in vitro study by CD-HR. Tyagi and Singhal (1998) reported that mustard oil cake reduced ruminal microbial methane in vitro, and Lila et al. (2003) reported that AIT, an active compound of mustard oil, reduced ruminal microbial methane and increased dihydrogen, supporting the present results. Accumulation of dihydrogen by CD-HR in our in vitro study resembled the effect of direct methane inhibitors (Bauchop, 1967; Trei et al., 1971).
Molar proportion of acetate was decreased and propionate was increased with corresponding decreases in acetate:propionate ratio with CD-HR supplementation in both in vitro and in vivo studies. These results confirm similar responses in fermentation patterns of ruminants where methane production is inhibited by various halogenated analogs of methane (Bauchop, 1967; Trei et al., 1971) and other antimethanogenic compounds, such as ionophores (Goodrich et al., 1984). The decreased acetate:propionate ratio reflects both the reduced production of methane and there direction of hydrogen from methane to propionate (Demeyer and Van Nevel, 1975).
However, inhibiting one chemical reaction in a system as complex as that in the rumen may result in numerous other interrelated effects, one of which is the inhibition of fiber digestion following changes to the microbial population (Van Nevel and Demeyer, 1995). The decreased fiber digestibility observed in previous reports has been attributed to problems with the disposal of reducing equivalents. Accumulation of dihydrogen indicates that methanogens were probably directly inhibited in this study. Decreased methane formation was thus probably not caused by competition for electrons due to fatty acid biohydrogenation. Due to the decrease in methane production and number of methanogens, problems with reoxidation of cofactors and disposal of metabolic-H2 would presumably also have occurred in the present experiment. However, supplementation with CD-HR did not affect the apparent digestibility of DM and NDF, which may be due to the moderate inhibition of methane. Similar effects with moderate inhibitions of methane affecting fiber digestibility, and numbers of cellulolytic bacteria and methanogens have been reported before (Dong et al., 1997). Supplementation with CD-HR decreased feed intake, as seen with other inhibitors of methane production (Horton, 1980; Van Nevel and Demeyer, 1996; Nagaraja et al., 1997). Apparent digestibility of DM, NDF, and CP was numerically increased, in contrast with other methane inhibitors (Van Nevel and Demeyer, 1995). This is attributed to lower intakes causing lower passage rates and higher digestibilities (Paladines et al., 1964; Horton, 1980). Fermentative digestion is also reportedly improved with monensin in animals on high-roughage diets due to a reduction in rumen turnover rate (Nagaraja et al., 1997). Measuring the effects of CD-HR supplementation on the true digestibility of DM, NDF, and CP would therefore be important.
Ruminal NH3-N was decreased by CD-HR. This is supported by the results observed with isobutyrate, valerate, and isovalerate because these VFA result from deamination of AA. The decrease in ruminal NH3-N could not have been caused by decreased protozoal numbers since the number of protozoa was unchanged by CD-HR supplementation. The CD-HR supplementation might therefore reduce the rate of deamination, similar to the results reported for other methane inhibitors (Johnson et al., 1972; Horton, 1980), as disposal of metabolic-H2 occurs by deamination releasing one pair of reducing equivalents (Russell and Martin, 1984; Hino and Russell, 1985).
Higher blood glucose and lower urea-N concentrations were found in CD-HR-supplemented steers. Increased blood glucose could potentially improve performance in energy-limiting animals. Also, a higher glucose supply could mean that glucogenic AA are spared from catabolism, resulting in lower urea-N in blood plasma. Nitrogen content in both urine and feces plus urine was decreased significantly in CD-HR-supplemented steers, so supplementation with CD-HR may reduce N voided into the environment.
In the present study, inhibition of methane production was found to be much greater in vitro than in vivo. In the in vitro experiment, CD-HR at 0.17, 0.85, and 1.70 g/L represented 2.5, 11, and 20% of the total substrate plus additive. Comparable low proportions of CD-HR in total feed yielded similar decreases in methane production (approximately 19%) both in vitro (2.5% of feed DM) and in vivo (2.0% of feed DM). In contrast, CD-HR at 11 and 20% of total CD-HR plus substrate in vitro resulted in substantially greater inhibitions of methane production than in vivo. Passage of CD-HR out of the rumen in vivo therefore seems likely to exert some influence.
Implications
The present study has shown that an -cyclodextrin-horseradish oil complex offers the potential to inhibit ruminal methane production both in vitro and in vivo. Addition of -cyclodextrin-horseradish oil complex decreased methanogenesis and nitrogen content of feces plus urine, increased propionate concentration, and numerically increased the apparent digestibility of dry matter, neutral detergent fiber, and crude protein. The additive caused changes in fermentation end products that were similar to findings for other classical methane inhibitors. However, further research is needed to evaluate the longer-term efficacy of -cyclodextrin-horseradish oil complex for inhibiting methanogenesis and to examine animal performance trials.
Footnotes
1 The present research was financially supported by the research grant from the Institute of the Society for Techno-inovation of Agriculture, Forestry, and Fisheries (STAFF), Japan. 
2 Correspondence: Fuchu-shi, Saiwai-cho 3-5-8 (phone: +81-042-367-5805; fax: +81-042-367-5801; e-mail: hita{at}cc.tuat.ac.jp).
Received for publication July 11, 2003. Accepted for publication February 18, 2004.
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