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Effects of Cordyceps militaris mycelia on in vitro rumen microbial fermentation.

INTRODUCTION

Cordyceps species are medical fungi well known for their pharmacological actions such as immunomodulatory (Koh et al., 2002; Yu et al., 2003), anti-inflammatory (Yu et al., 2004a, b), antitumor (Nakamura et al., 1999), antifungal (Kneifel et al., 1977) and antibacterial (Ahn et al., 2000) activities, and contain biologically active components such as nucleosides (cordycepin; 3'-deoxyadenosine, and adenosine), polysaccharides and ergosterol (Li et al., 2006). The typical Coryceps used in oriental medicine was Chinese C. sinensis which forms a fruiting body using the larva of a moth as the host. Because natural C. sinensis is rare and expensive, however, techniques for artificial cultivation of Cordyceps species other than the natural Cordyceps have been developed and, thus commercial products of Cordyceps are now widely available. C. militaris is a related species of C. sinensis commonly used as a substitute of the natural Cordyceps (Li et al., 2006).

Although the pharmacological actions of Cordyceps may also affect livestock beneficially, the application of Cordyceps in livestock has received little attention. Recently, however, it has been reported that oral dose of a hot-water extract of mycelia from C. sinensis in an attempt to substitute for antibiotic growth promoter improved body weight gain and immune system in broiler chicks (Koh et al., 2003). The efforts to find biologically active materials that can improve animal health are certainly welcomed to livestock producers since demand for alternative strategies for enhancing livestock production to the same or higher level obtained with antibiotic growth promoters has markedly being increased (Wallace, 2004; Al-Mamun1 et al., 2007; Li et al., 2008). In addition, as long as the biologically active components in Cordyceps can be transferred to the end products of livestock such as milk, meat and eggs, an increase of the market value of livestock products would be expected.

The application of Cordyceps to ruminants is not simple as to mono-gastric animal because of the digestion of rumen microorganisms and the antibacterial and antifungal effects of Cordyceps. Therefore, the objectives of the present study were to examine effects of C. militaris mycelia on rumen microbial fermentation by measuring in vitro gas production, cellulose digestion, and VFA production.

MATERIAL AND METHODS

Sample preparation

Dried C. militaris mycelia cultured on a medium composed of corn gluten, soybean protein, beer yeast and corn steep liquor (culturing method and medium composition are patent pending in Korea) were obtained from EuGene Bio Farm (Hwaseong City, Gyeonggi Province, Korea). The mycelia are composed of 761.6 g CP, 122.4 g crude fat, 9.6 g ether extract, 32.1 g crude ash, 74.3 g nitrogen free extract, and 1.64 g cordycepin per kg of dry matter. The manufacturer reported that C. militaris mycelia used in the present study contained about 2.3 times more cordycepin (1.6 mg/g DM) than C. militaris traditionally cultured on faunal pupae (0.7 mg/g DM).

In vitro batch fermentation

The anaerobic culture techniques of Hungate (1966) were carried out for all incubations with rumen fluid from a 515 kg Korean native steer (Hanwoo) fed a basal diet consisting of rice straw and concentrates mixed at a ratio of 4:6 (fresh weight). The steer was housed in an individual metabolic stall and given the diet at 1.75% of body weight. Rumen fluid was collected via rumen cannulae before the morning feeding into a vacuum flask that was flushed with [O.sub.2]-free C[O.sub.2], and squeezed through four layers of cheese cloths into an Erlenmeyer flask in an anaerobic glove box. The fluid was then mixed with buffer (pH 6.9) (containing 292 mg of [K.sub.2]HP[O.sub.4], 240 mg of K[H.sub.2]P[O.sub.4], 480 mg of [(N[H.sub.4]).sub.2] S[O.sub.4], 480 mg of NaCl, 100 mg of MgS[O.sub.4] x 7[H.sub.2]O, 64 mg of Ca[Cl.sub.2] x 2[H.sub.2]O, 4,000 mg of [Na.sub.2]C[O.sub.3], and 600 mg of cysteine hydrochloride in l,000 ml of [O.sub.2]-free distilled water) at a ratio of 1:2. After mixing, 30 ml of diluted rumen fluid was transferred to 60 ml serum bottles containing 750 mg of Whatman No. 1 cellulose filter paper (FP) as a sole carbon source. Weighed amounts of dried C. militaris mycelia were added to achieve final concentrations of 0.00, 0.10, 0.15, 0.20, 0.25 and 0.30 g/L. The bottles (three replicates per treatment) were sealed with butyl rubber stoppers and aluminum caps, and placed in an incubator at 38[degrees]C for 3, 6, 9, 12, 24, 36, 48 and 72 h without shaking.

Sampling and analysis

At each sampling time, gas production, cellulose filter paper degradation and pH were determined. Gas production (ml/0.1 g substrate) was determined using a water displacement apparatus (Fedorak and Hrudey, 1983) and pH was determined in culture fluid immediately after the vials had been opened. The concentration of VFA was determined only at 24 h incubation, and for analysis of VFA, 1 ml of 25% meta-phosphoric acid was added to 5 ml of fermentation fluid, centrifuged (10,000xg for 10 min at 4[degrees]C) and supernatant was stored at -30[degrees]C until analyzed. VFA analysis was carried out 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[degrees]C, injector and detector temperature = 200[degrees]C). The carrier gas (N2) flow rate was 50 ml/min. Filter paper digestibility was measured by the method of Lee et al. (2004). Residual particulate substrates including adherent microbial biomass were separated from supernatant by centrifugation (3,000 rpm for 20 min) and then treated with 0.15 ml of sodium dodecyl sulphate solution and boiled (100[degrees]C) for 1 h to remove adherent rumen mixed microbial biomass. The pellet was rinsed three times with absolute alcohol at 60[degrees]C and twice with running distilled water, and was dried to a constant weight at 78[degrees]C for 24 h.

Statistical analysis

Data obtained from the experiment were analyzed using the SAS (1996) software package and differences were tested by Duncan's multiple range test, and then P value less than 0.05 was considered significant. Linear, quadratic and cubic responses to C. militaris mycelia level were tested by orthogonal contrast.

RESULTS

The cumulative in vitro gas production at different incubation times for the treatments is shown in Table 1. At all incubation times, the gas production showed a quadratic increase with the supplementation of C. militaris mycelia; maximum responses were seen with 0.25 g/L supplementation from 9 h to 72 h incubation. After incubation for 24 h, the gas production from the 0.25 g/L supplementation was about 50% higher than that from the control treatment. But the gas production was significantly lower for the 0.30 g/L supplementation than for the 0.25 g/L supplementation from 9 h to 72 h incubation.

The effects of the supplementation of C. militaris mycelia on the cellulose FP digestion by mixed rumen microorganisms are shown in Table 2. The cellulose FP digestion showed a quadratic trend, as did the gas production except at 3 h incubation. At all incubation times, the 0.25 g/L supplementation significantly increased cellulose FP digestion compared with the control treatment. After incubation for 24 h, the cellulose FP digestion for the 0.25 g/L supplementation was about 36% higher than that for the control treatment. The cellulose FP digestion was numerically higher for the 0.25 g/L supplementation than for other treatments at all incubation times, although the significant difference was only seen at 72 h incubation.

The effects of the supplementation of C. militaris mycelia on the concentration of VFAs after 24 h incubation are given in Table 3. The concentration of total VFA was significantly increased by the supplementation of C. militaris mycelia compared with the control treatment; the highest response was also seen with 0.25 g/L supplementation. This is true for the responses in the concentration of acetic and propionic acids. But no significant difference was seen in the concentration of butyric and valeric acids among treatments.

As opposed to other responses, the responses of pH to the supplementation of C. militaris mycelia showed a quadratic decrease from 3 h to 36 h incubation (Table 4). The pH was numerically lower for the 0.25 g/L supplementation than for other treatments at all incubation times, although the significant difference was only seen at 36 h incubation.

DISCUSSION

Increasing public concern about antibiotic resistance and residues in animal products has resulted in the search for alternatives, such as prebiotics, probiotics, and other feed additives. Cordyceps species and its constituents have potential as natural products for its various pharmacological actions on human health and still extensive work is being carried out to define the pharmacological efficiency of bioactive materials in Cordyceps (Li et al., 2006). Cordycepin, one of the markers for quality control of Cordyceps was shown to have a selective growth-inhibiting activity towards harmful intestinal bacteria without adverse effects on lactic acid-producing bacteria (Ahn et al., 2000; Yeon et al., 2007) or even with an increase in Lactobacillus sp. (Koh et al., 2003), though the mechanisms underlying the inhibiting or promoting effects are not known. In the present study, the addition of C. militaris mycelia to rumen fluid inoculum caused a marked increase in gas production, cellulose FP degradation and VFA production, suggesting that major changes in the microbial population had occurred in response to the addition of C. militaris. It has been reported that gas production is positively related to microbial protein synthesis (Krishnamoorthy et al., 1991). Therefore, it is tempting to speculate that the increases of cellulose FP digestion and gas production might be due to increased bacteria numbers, the most active organisms for fiber digestion in the rumen (Lee et al., 2004). Protozoa and fungi are also involved in fiber digestion (Lee et al., 2004), but their role in the present study might be excluded or even suppressed since cordycepin was shown to have adverse effects on protozoa (Trigg et al., 1971) and fungi (Kneifel et al., 1977). At 24 h incubation, the production of gas and total VFA and cellulose FP digestion increased linearly with the addition of C. militaris mycelia. However, the addition of 0.3 g/L of C. militaris mycelia reduced the production of gas and total VFA and cellulose FP digestion compared with the 0.25 g/L supplementation. A plateau in the response, rather than a decrease, had been expected. Clearly, this finding needs to be confirmed but, taking the results at face value, it implies that a further increase in the activity of rumen microorganisms could not be expected to supplementing C. militaris mycelia beyond 0.25 g/L.

The production of gas and total VFA measured in the present study was within ranges reported in earlier studies (Doane et al., 1997; Getachew et al., 2004; Lee et al., 2004). In vitro gas methods primarily measure digestion of soluble and insoluble carbohydrates by rumen microorganisms (Menke and Steingass, 1988), and there are linear correlations between the volume of gas and moles of VFA produced and the mass of fiber digested (Calabro et al., 2001). Indeed, the relationship between gas production and cellulose FP digested in the present study was linear ([r.sup.2] = .85, p<0.001; data not shown), and the gas production was also related to VFA production ([r.sup.2] = 0.89, p<0.01; data not shown). The calculated amount of total VFA production based on the culture volume of 30 ml on the 0.25 g/L supplementation at 24 h incubation was 3.66 mmol, which can be converted to 0.013 mmol/mg cellulose FP digested (the amount of cellulose FP digested was 281 mg at 24 h incubation), values that agree well with the estimate (0.01 mmol total VFA/mg NDF digested) reported by Doane et al. (1997). But the value for the control treatment was about half of that for the 0.25 g/L supplementation, implying that rumen fluid inoculum might contain a low level of microbial population. The maximum amount of C. militaris mycelia supplemented (0.30 g/L supplementation) was 9 mg. This degree of nutrients supply over the control treatment implies that the large difference in the production of gas and VFA would be very unlikely to be a result of carbohydrates supplied from C. militaris mycelia.

Alternatives for growth promoting antibiotics for ruminants must ensure that they have no detrimental effect on the basic functions of the fermentation, such as fiber digestion and VFA production (Wallace, 2004). In this connection, C. militaris mycelia used in the present study appears to fulfill these criteria as it increased the fiber digestion and volatile production. But, whether these effects were induced directly by increased bacteria numbers or other mechanisms, such as antiprotozoal activity were involved remains to be investigated.

ACKNOWLEGDMENTS

This experiment was supported by a grant from the Special Grants Research Program of the Ministry of Agriculture and Forestry. S. J. Lee, S. M. Lee and S. H. Shin are supported by scholarships from the BK21 Program, Ministry of Education and Technology, Korea.

REFERENCES

Ahn, Y. J., S. J. Park, S. G. Lee, S. C. Shin and D. H. Choi. 2000. Cordycepin: selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium spp. J. Agric. Food Chem. 48:2744-2748.

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Calabro, S., F. Infascelli, F. Bovera, G. Moniello and V. Piccolo. 2001. In vitro degradability of three forages: fermentation kinetics and gas production of NDF and neutral detergent-soluble fraction of forages. J. Sci. Food Agric. 82:222-229.

Doane, P. H., P. Schofield and A. N. Pell. 1997. Neutral detergent fiber disappearance and gas and volatile fatty acid production during the in vitro fermentation of six forages. J. Anim. Sci. 75:3342-3352.

Fedorak, P. M. and S. E. Hrudey. 1983. A simple apparatus for measuring gas production by methanogenic cultures in serum bottles. Environ. Technol. Lett. 4:425-432.

Getachew, G., P. H. Robinson, E. J. DePeters and S. J. Taylor. 2004. Relationships between chemical compositions, dry matter degradation and in vitro gas production of several ruminant feeds. Anim. Feed Sci. Technol. 111:57-71.

Hungate, R. E. 1966. The rumen bacteria. In: The rumen and its microbes (Ed. R. E. Hungate). Academic Press, New York and London, p. 8.

Kneifel, H., W. A. Konig, W. Loeffler and R. Muller. 1977. Ophiocordin, an antifungal antibiotic of Cordyceps ophioglossoides. Archiv. Microbiol. 113:121-130.

Koh, J. H., H. J. Suh and T. S. Ahn. 2003. Hot-water extract from mycelia of Cordyceps sinensis as a substitute for antibiotic growth promoters. Biotechnol. Lett. 25:585-590.

Koh, J. H., K. W. Yu, H. J. Suh, Y. M. Choi and T. S. Ahn 2002. Activation of macrophages and the intestinal immune system by orally administered decoction from cultured mycelium of Cordyceps sinensis. Biosci. Biotechnol. Biochem. 66:407-411.

Krishnamoorthy, U., H. Steingass and K. H. Menke. 1991. Preliminary observation on the relationship between gas production and microbial protein synthesis in vitro. Arch. Anim. Nutr. 5:521-526.

Li, Zheji, Ganfeng Yi, Jingdong Yin, Peng Sun, Defa Li and Chris Knight. 2008. Effects of organic acids on growth performance, gastrointestinal pH, intestinal microbial populations and immune responses of weaned pigs. Asian-Aust. J. Anim. Sci. 21:252-261.

Lee, S. S., C. K. Choi, B. H. Ahn, Y. H. Moon, C. H. Kim and J. K. Ha. 2004. In vitro stimulation of rumen microbial fermentation by a rumen anaerobic fungal culture. Anim. Feed Sci. Technol. 115:215-226.

Li, S. P., F. Q. Yang and K. W. K. Tsim. 2006. Quality control of Cordyceps sinensis, a valued traditional Chinese medicine. J. Pharm. Biomed. Anal. 41:1571-1584.

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Nakamura, K., Y. Yamaguchi, S. Kagota, Y. M. Kwon, K. Shinozuka and M. Kunitomo. 1999. Inhibitory effect of Cordyceps sinensis on spontaneous liver metastasis of Lewis lung carcinoma and B16 melanoma cells in syngeneic mice. Jpn. J. Pharmacol. 79:335-341.

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Yeon, S. H., J. R. Kim and Y. J. Ahn. 2007. Comparison of growth-inhibiting activities of Cordyceps militaris and Paecilomyces japonica cultured on Bombyx mori pupae towards human gastrointestinal bacteria. J. Sci. Food Agric. 87:54-59.

Yu, K. W., K. M. Kim and H. J. Suh. 2003. Pharmacological activities of stromata of Cordyceps scarabaecola. Phytother Res. 17:244-249.

Yu, R., L. Song, Y. Shao, W. Bin, L. Wang, H. Shang, Y. Wo, W. Ye and X. Yao. 2004a. Isolation and biological properties of polysaccharide CPS-1 from cultured Cordyceps militaris. Fitoterapia. 75:465-472.

Yu, R., L. Wang, H. Zhang, C. Zhou and Y. Zhao. 2004b. Isolation, purification and identification of polysaccharides from cultured Cordyceps militaris. Fitoterapia. 75:662-666.

Joon Mo Yeo (a), Shin Ja Lee (1, a), Sang Min Lee (1), Sung Hwan Shin (1), Sung Hoon Lee (2) Jong K. Ha (3), WanYoung Kim and Sung Sill Lee (1), *

Department of Animal Science, Korea National Agricultural College, RDA, Suwon 445-893, Korea

* Corresponding Author: Sung Sill Lee. Tel: +82-55-751-5411, Fax: +82-55-751-5410, E-mail: lss@nongae.gsnu.ac.kr

(1) Division of Applied Life Science (BK21 Program), Graduate School of Gyeongsang National University, Jinju 660-701, Korea.

(2) Gyeongsangnam-Do Livestock Veterinary Research Institute, Sancheong 666-962, Korea.

(3) Department of Agricultural Biotechnology, Seoul National University, Seoul 151-741, Korea.

(a) Thease authors contributed equally to this work.

Received October 14, 2008; Accepted November 17, 2008
Table 1. Effects of supplement levels (g/L) of Cordyceps
militaris on in vitro cumulative gas production (ml/0.1 g DM
substrate) by mixed rumen microorganisms

 Supplement levels (g/L) of C. militaris
Incubation
times (h) 0.0 0.10 0.15

 3 6.6 (c) 8.5 (ab) 8.0 (b)
 6 7.8 (c) 10.0 (b) 10.2 (ab)
 9 11.4 (c) 13.1 (b) 14.4 (b)
12 13.5 (c) 16.9 (b) 19.2 (b)
24 17.0 (c) 18.7 (bc) 19.9 (b)
36 17.5 (c) 20.8 (b) 22.5 (b)
48 18.7 (d) 21.5(bc) 23.4 (b)
72 19.5 (d) 22.8(bc) 23.6 (b)

 Supplement levels (g/L) of C. militaris
Incubation
times (h) 0.20 0.25 0.30

 3 8.7 (ab) 8.9 (a) 8.0 (ab)
 6 11.5 (a) 10.5 (ab) 9.8 (b)
 9 13.9 (b) 18.6 (a) 13.4 (b)
12 18.2 (b) 24.1 (a) 17.7 (b)
24 20.1 (b) 25.6 (a) 19.8 (b)
36 23.2 (b) 27.0 (a) 21.3 (b)
48 22.2 (b) 28.5 (a) 19.6 (cd)
72 22.8 (bc) 29.4 (a) 20.8 (cd)

 Trend (P)
Incubation
times (h) SEM Linear Quadratic Cubic

 3 0.47 0.006 <0.001 0.814
 6 0.70 0.013 <0.001 0.135
 9 1.21 0.002 0.001 0.023
12 1.37 <0.001 <0.001 0.087
24 1.27 <0.001 <0.001 0.005
36 1.38 <0.001 <0.001 0.123
48 1.19 0.005 <0.001 0.002
72 1.42 0.008 <0.001 0.005

SEM = Standard error of the mean. (a), (b), (c), (d) Means
within a row with different superscripts differ significantly
(p<.05).

Table 2. Effects of supplement levels (g/L) of Cordyceps
militaris on in vitro digestibilities (%) of cellulose filter
paper by mixed rumen microorganisms

 Supplement levels (%) of C. militaris
Incubation
times (h) 0.00 0.10 0.15

 3 10.4 (b) 10.4 (b) 10.4 (b)
 6 13.8 (b) 15.5 (b) 18.2 (a)
 9 16.1 (b) 18.0 (b) 22.1 (a)
12 24.2 (c) 22.3 (c) 28.6 (ab)
24 27.4 (c) 34.8 (ab) 36.2 (a)
36 33.4 (d) 38.3 (c) 39.7 (bc)
48 39.4 (c) 38.9 (c) 45.6 (ab)
72 40.8 (e) 44.7 (d) 47.9 (c)

 Supplement levels (%) of C. militaris
Incubation
times (h) 0.20 0.25 0.30

 3 11.3 (ab) 13.5 (a) 11.7 (ab)
 6 19.1 (a) 19.8 (a) 19.3 (a)
 9 20.9 (a) 21.8 (a) 23.0 (a)
12 29.3 (ab) 30.0 (a) 25.6 (bc)
24 34.8 (ab) 37.5 (a) 32.7 (b)
36 41.4 (ab) 43.2 (a) 37.7 (c)
48 44.2 (b) 47.6 (a) 42.8 (b)
72 51.3 (b) 55.8 (a) 51.5 (b)

 Trend (P)
Incubation
times (h) SEM Linear Quadratic Cubic

 3 1.25 0.015 0.369 0.064
 6 1.43 <0.001 0.007 0.398
 9 1.10 <0.001 0.007 0.004
12 2.31 0.033 0.002 0.417
24 1.77 0.014 <0.001 0.140
36 1.35 <0.001 <0.001 0.634
48 1.73 <0.001 <0.001 0.522
72 1.30 <0.001 <0.001 0.093

SEM = Standard error of the mean. (a), (b), (c), (d) Means
within a row with different superscripts differ significantly
(p<0.05).

Table 3. Effects of supplement levels (g/L) of Cordyceps
militaris on the concentrations (mmol/L) of volatile fatty
acids

 Supplement levels (g/L) of C. militaris

 0.00 0.10 0.15

Total 36.0 (c) 58.5 (b) 62.1 (b)
Acetate 19.1 (c) 33.9 (b) 37.3 (b)
Propionate 6.9 (c) 12.9 (b) 13.9 (b)
Iso-butyrate 0.5 (ab) 0.5 (a) 0.5 (a)
Butyrate 7.4 8.4 7.7
Iso-valerate 1.5 (ab) 1.8 (a) 1.9 (a)
Valerate 0.7 0.8 0.9

 Supplement levels (g/L) of C. militaris

 0.20 0.25 0.30

Total 46.0 (bc) 122.2 (a) 50.5 (bc)
Acetate 26.1 (bc) 84.4 (a) 27.5 (bc)
Propionate 9.3 (bc) 25.3 (a) 11.0 (bc)
Iso-butyrate 0.2 (c) 0.2 (bc) 0.4 (abc)
Butyrate 8.4 10.5 9.1
Iso-valerate 1.3 (ab) 0.8 (a) 1.7 (ab)
Valerate 0.7 1.0 0.8

 Trend (P)

 SEM Linear Quadratic Cubic

Total 11.38 0.001 0.002 <0.001
Acetate 7.39 <0.001 <0.001 <0.001
Propionate 2.81 0.004 0.006 0.009
Iso-butyrate 0.15 0.059 0.055 0.107
Butyrate 1.83 0.110 0.681 0.345
Iso-valerate 0.51 0.274 0.249 0.022
Valerate 0.27 0.778 0.651 0.990

SEM = Standard error of the mean. a, b, c Means within a row
with different superscripts differ significantly (p<0.05).

Table 4. Effects of supplement levels (g/L) of Cordyceps
militaris on pH

 Supplement levels (g/L) of C. militaris
Incubation
times (h) 0.00 0.10 0.15

 3 6.35 (a) 6.38 (a) 6.24 (ab)
 6 6.36 (ab) 6.48 (a) 6.26 (ab)
 9 5.72 (a) 5.44 (ab) 5.15 (bc)
12 5.30 (b) 5.21 (b) 5.08 (b)
24 5.25 (a) 5.16 (abc) 5.06 (abc)
36 5.22 (a) 5.14 (a) 5.06 (a)
48 5.01 4.97 4.95
72 4.96 4.95 5.12

 Supplement levels (g/L) of C. militaris
Incubation
times (h) 0.20 0.25 0.30

 3 6.12 (b) 6.05 (b) 6.20 (ab)
 6 6.16 (b) 6.10 (b) 6.27 (ab)
 9 5.13 (bc) 5.04 (c) 5.41 (ab)
12 5.20 (b) 5.09 (b) 5.61 (a)
24 4.99 (bc) 4.95 (c) 5.20 (ab)
36 5.02 (a) 4.78 (b) 5.05 (a)
48 4.94 4.97 5.01
72 5.12 4.92 5.14

 Trend (P)
Incubation
times (h) SEM Linear Quadratic Cubic

 3 0.12 0.009 0.030 0.262
 6 0.14 0.038 0.052 0.229
 9 0.19 0.035 0.001 0.706
12 0.17 0.068 0.005 0.427
24 0.11 0.189 0.002 0.480
36 0.11 0.005 0.020 0.102
48 0.09 0.877 0.311 0.781
72 0.15 0.369 0.709 0.072

SEM = Standard error of the mean. (a,) (b,) (c) Means within a
row with different superscripts differ significantly (p<0.05).
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Author:Yeo, Joon Mo; Lee, Shin Ja; Lee, Sang Min; Shin, Sung Hwan; Lee, Sung Hoon; Ha, Jong K.; Kim, WanYou
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9SOUT
Date:Feb 1, 2009
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