Blackwell Publishing AsiaMelbourne, AustraliaASJAnimal Science Journal1344-39412006 Blackwell Publishing Asia Pty LtdJune 2006773308313Original Article WHITE-ROT FUNGI

TREATMENT OF BAGASSEK. OKANO

et al.

Animal Science Journal (2006) 77, 308313

doi: 10.1111/j.1740-0929.2006.00353.x

ORIGINAL ARTICLE Comparison of in vitro digestibility and chemical composition among sugarcane bagasses treated by four white-rot fungi
Kanji OKANO,1 Yuko IIDA,1 Muhammad SAMSURI,2 Bambang PRASETYA,3 Tomoya USAGAWA4 and Takashi WATANABE5
School of Environmental Science, The University of Shiga Prefecture, Hikone-shi, Japan; 2Ofce of the Ministry of Research and Technology, Jakarta, 3Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong, Indonesia; 4Department of Bioproduction Science, Ishikawa Prefectural University, Nonoichi-machi, Ishikawa-ken, and 5Research Institute for Sustainable Humanosphere, Kyoto University, Uji-shi, Japan
1

ABSTRACT
The capabilities of four white-rot fungi to improve the digestibility of sugarcane bagasse for ruminants were determined. Bagasse was cultured with Lentinula edodes, Pleurotus eryngii, Pleurotus salmoneostramineus, Ceriporiopsis subvermispora (ATCC 90467) or C. subvermispora (CZ-3) at 26C for 8, 12 or 16 weeks. The in vitro organic matter digestibility (IVOMD) and in vitro neutral detergent ber digestibility (IVNDFD) in untreated bagasse were 45.6 and 40.3%, respectively. Meanwhile, the bagasse cultured with L. edodes and two strains of C. subvermispora, respectively, for 12 weeks, were elevated to 68.6 and 59.1%, 60.6 and 49.9% and 59.9 and 49.0%, respectively. In contrast, the IVOMD and the IVNDFD in bagasse cultured with P. eryngii and P. salmoneostramineus were the same or lower than those in untreated bagasse. In vitro gas production (IVGP) in bagasse cultured with L. edodes and two strains of C. subvermispora for 12 weeks was also higher than that of untreated bagasse. These changes in IVOMD, IVNDFD and IVGP demonstrate that L. edodes has a higher capability of improving the digestibility for ruminants than C. subvermispora, P. eryngii or P. salmoneostramineus.

Key words: Ceriporiopsis subvermispora, in vitro digestibility, Lentinula edodes, sugarcane bagasse, white-rot fungi.

INTRODUCTION
Sugarcane is an important crop in the tropics and subtropics. During sugar production, vast amounts of bagasse are discharged as waste product. Around 80 90% of this bagasse is used as a heat source for the sugar production plant, and the remainder is used for papermaking raw material, particle board material or feed for livestock. Since bagasse is discharged at the sugar production plant, it has the advantage of collecting it at a low expense from arable land or forests unlike other biomass resources. The main components of bagasse are cellulose, hemicellulose and lignin. As a result of the high content of lignin, ruminal digestion is inhibited and thus the nutritive value of bagasse is low for ruminants.

Many studies have been conducted that raise the nutritive value of bagasse for ruminants through steaming or chemical treatment. Although the digestibility of bagasse has been improved by these treatments, they are not practical techniques because of the processing costs and residual alkali agent produced. A safe and environment-friendly method for lignin removal using white-rot fungi has also been reported (Kirk & Moore 1972; Zadrazil 1985; Akin et al. 1996).

Correspondence: Kanji Okano, School of Environmental Science, The University of Shiga Prefecture, Hikone-shi, 5228533, Japan. (Email: okano@ses.usp.ac.jp) Received 4 July 2005; accepted for publication 21 December 2005.

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Culturing bagasse with edible basidiomycetes, Pleurotus sajor-caju and Polyporus hirsutus (Kewalramani et al. 1988), Pleurotus abalonus (Suzuki 1995) and Pleurotus eryngii (Zadrazil & Puniya 1995) reportedly improves the digestibility of bagasse or bagasse medium. In contrast, one report showed that the in vitro dry matter digestibility did not improve after culturing with Pleurotus cystidiosus, Pleurotus salmoneostramineus or Auricularia polytricha, although the cellulose digestibility was raised (Yamada et al. 2000). Most edible basidiomycetes cultivated articially are able to degrade lignin and cellulose simultaneously. However, in contrast, the white-rot fungus Ceriporiopsis subvermispora is known to selectively degrade lignin in softwood and hardwood. Owing to its unique wood decay pattern, this fungus has been used in biopulping (Messner & Srebotnik 1994), pretreatment of beech wood for ethanol fermentation (Ito et al. 2003) and conversion of Japanese red cedar (Cryptomeria japonica) into feed for ruminants (Okano et al. 2005). In the present study, we examined the capabilities of four white-rot fungi species, including two strains of the selective white-rot fungus C. subvermispora, for improving the digestibility of bagasse for ruminants.

Scientic Company, Tokyo, Japan). A 15 g sample of bagasse was placed in a 300 mL Erlenmeyer ask with 45 mL distilled water. The ask was closed with a cotton plug and sterilized at 121C for 20 min. After cooling, 4 mL liquid culture medium was inoculated in the ask. All fungi were cultured at 26C. Three samples for each fungus were collected at 8, 12 and 16 weeks after inoculation.

Chemical analysis
All samples were dried at 65C, air-dried then milled through a 1 mm diameter screen. Neutral detergent ber (NDF) was measured according to Chai and Uden (1998) and acid detergent ber (ADF) and acid detergent lignin (ADL) were measured according to Van Soest et al. (1991). Sodium sulte was not used in the determination of NDF, and both NDF and ADF values excluded residual ash. The differences between NDF and ADF, ADF and ADL, and the amount of ADL represent hemicellulose, cellulose and lignin, respectively.

Measurement of in vitro organic matter digestibility, in vitro neutral detergent ber digestibility and in vitro gas production
In vitro organic matter digestibility (IVOMD), in vitro neutral detergent ber digestibility (IVNDFD) and in vitro gas production (IVGP) were determined according to Tilley and Terry (1963) with slight modications. Rumen uid was collected from a stulated wether 2 h after morning feeding. Alfalfa hay cubes (500 g) and unpolished rice (150 g) were fed to the wether twice daily. The uid was then ltered through four folds of gauze. A sample (0.4 g) was placed in a 50 mL centrifuge tube, after which 8 mL ltered rumen uid, 32 mL McDougalls articial saliva (McDougall 1948) and 10.4 mg urea (Horii et al. 1971) were added. The tube was closed with a rubber stopper tted with a 50 mL plastic syringe to measure gas production, which was determined by averaging the readings of the piston at pulled and pushed positions (Suzuki et al. 1995). Simultaneously, rice straw and untreated bagasse were incubated as controls. Each sample was incubated at 39C for 48 h in a water bath and duplicated three times on three different days; that is, each sample was therefore analyzed six times. During incubation, the tubes were stirred every 3 h for 24 h then every 6 h for 48 h after beginning incubation. After incubation, IVOMD and IVNDFD were measured by heating the residue with ND solution at

MATERIALS AND METHODS Preparation of inoculum

A 500 mL Erlenmeyer ask containing 200 mL MYP liquid culture medium (malt extract, 0.7%; yeast extract, 0.05%; soybean peptone, 0.1%; agar, 0.1%) was inoculated with Lentinula edodes (USP no. 456), P. eryngii (USP no. 489), P. salmoneostramineus (USP no. 43), C. subvermispora (ATCC 90467) or C. subvermispora (CZ-3) then homogenized. L. edodes and P. eryngii were previously collected from commercial mushrooms, while P. salmoneostramineus was collected from dead wisteria tree (Suzuki 1992); all were preserved in the School of Environmental Science, The University of Shiga Prefecture, Shiga, Japan. The latter two strains of C. subvermispora were preserved in the Research Institute for Sustainable Humanosphere, Kyoto University, Kyoto, Japan. They were all cultured for 7 days.

Bagasse culture
Fresh sugarcane bagasse obtained from a sugar production plant (Shinko-Sugar, Kagoshima, Japan) was dried at 65C, air-dried then milled through a 2 mm diameter screen using a Wiley hammer mill (Fujiwara

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90C for 16 h (Chai & Uden 1998). Blank corrections were also conducted for the IVOMD, IVNDFD and IVGP measurements. This research was conducted with the approval of the Committee for Animal Experiments of the University of Shiga Prefecture.

Statistical analysis
All IVOMD, IVNDFD, IVGP and chemical composition data were subjected to a one-way analysis of variance and post-hoc multiple comparison test with Tukeys honestly signicant difference test using SPSS statistical software (2002).

RESULTS AND DISCUSSION

IVGP, IVOMD and IVNDFD in bagasse treated with four white-rot fungi species for different cultivation lengths are shown in Table 1. The IVOMD in untreated bagasse was 45.6%; in contrast, the IVOMD in bagasse cultured with L. edodes for 12 weeks was elevated to 68.6%, the greatest improvement among all fungi used in the present study. This value was higher than the 58.2% of rice straw, which was measured simultaneously. The IVOMD also increased with subsequent increases in culture length, reaching 69.1% at 16 weeks, but no signicant difference was observed

between the IVOMD at 8 and 16 weeks. The next greatest improvement in IVOMD was observed in bagasse cultured with the two strains of C. subvermispora. The greatest increases in IVNDFD and IVGP, as with IVOMD, were observed in bagasse cultured with L. edodes, and the next greatest improvement in IVNDFD and IVGP was observed in bagasse cultured with the two strains of C. subvermispora. As with IVOMD, the IVGP in bagasse cultured with L. edodes for 8 weeks, or longer, was higher than that in rice straw. In a previous study examining the conversion of Japanese red cedar into roughage for ruminants by treatment with white-rot basidiomycetes, culturing with C. subvermispora produced a greater improvement in IVOMD and IVGP than culturing with L. edodes (Okano et al. 2005). C. subvermispora is known to be a selective white-rot fungus and, in contrast to non-selective white-rot fungi such as L. edodes, P. salmoneostramineus and P. eryngii, degrades lignin in wood cell walls without penetration of its extracellular enzymes into the cell wall region (Messner & Srebotnik 1994; Watanabe 2003). We therefore expected the improvement in digestibility of bagasse to be greater with C. subvermispora than with L. edodes since the weight loss of cellulose could be lesser with

Table 1 Effects of fungi and cultivation length on in vitro gas production, in vitro organic matter digestibility (IVOMD) and in vitro neutral detergent ber digestibility (IVNDFD) in bagasse

Fungus

Cultivation length (weeks)

Gas production (mL/g organic matter) 12 h 24 h 92 112h 95gh 62bcd 64bcd 70def 32a 50abc 46ab 68cde 94gh 85efg 81defg 93gh 89fg 6 62 77
gh

IVOMD (%)

IVNDFD (%)

48 h 142e 166f 167f 98bc 99bc 108c 67a 85ab 86ab 104bc 137de 140de 118cd 139de 144e 6 104 122 65.5fg 68.6g 69.1g 46.0b 46.9b 47.9b 38.9a 42.5ab 42.7ab 54.6c 60.6def 61.3ef 55.4cd 59.9cde 61.9ef 1.5 45.6 58.2 55.3gh 59.1 h 59.0 h 37.6bc 39.1bcd 39.5bcd 31.0a 34.6ab 35.4ab 43.1cd 49.9fg 51.1fg 44.2de 49.0ef 51.5fg 1.5 40.3 43.3

Lentinula edodes

Pleurotus eryngii

Pleurotus salmoneostramineus

Ceriporiopsis subvermispora (ATCC 90467)

Ceriporiopsis subvermispora (CZ-3)

8 12 16 8 12 16 8 12 16 8 12 16 8 12 16

Standard error of difference Untreated bagasse Rice straw

34 30ef 28def 17abc 20bcd 21bcde 9a 15ab 14ab 18abc 26cdef 22bcde 28def 25cdef 23bcde 3 18 33

Values without untreated bagasse and rice straw represent the means of three media, and those within columns with different superscript letters (ah) denote signicant difference at a 5% level.

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Table 2

Effects of fungi and cultivation length on chemical composition in bagasse

Fungus

Cultivation length (weeks) 8 12 16 8 12 16 8 12 16 8 12 16 8 12 16

Chemical composition of medium (%) Organic matter 95.2(ab2) 94.6a 94.8ab 96.5b 96.6b 96.7b 95.6ab 96.2ab 95.7ab 96.0ab 96.0ab 95.7ab 95.4ab 95.8ab 95.2ab 0.5 96.9 Neutral detergent ber 73.4abc 72.6ab 71.4a 83.6e 84.2e 83.3e 84.7e 84.7e 84.8e 76.6d 75.5bcd 75.6bcd 76.2cd 75.3bcd 74.6bcd 0.9 88.3 Hemicellulose 11.8b 11.2ab 9.5a 22.4de 22.7de 20.9d 23.5e 21.5de 22.8de 15.0c 12.9bc 11.8ab 12.5b 12.7bc 10.9ab 0.6 46.1 Cellulose 54.4cd 54.9d 55.4d 50.9ab 50.9ab 51.4b 48.9a 51.6b 50.4ab 52.4bc 54.8d 56.6d 54.4cd 54.5cd 56.4d 0.6 31.7 Lignin 7.2ab 6.6a 6.5a 10.3de 10.6ef 11.0ef 12.2 g 11.7fg 11.6fg 9.2cd 7.9b 7.3ab 9.3d 8.2bc 7.3ab 0.3 10.5

Lentinula edodes

Pleurotus eryngii

Pleurotus salmoneostramineus

Ceriporiopsis subvermispora (ATCC 90467)

Ceriporiopsis subvermispora (CZ-3)

Standard error of difference Untreated bagasse

Hemicellulose, cellulose and lignin denote neutral detergent ber minus acid detergent ber (ADF), ADF minus acid detergent lignin (ADL) and ADL, respectively. Values without untreated bagasse represent the means of three media, and those within columns with different superscript letters (ag) denote signicant difference at a 5% level.

C. subvermipora than non-selective white-rot fungi; however, the opposite was seen. This nding might have stemmed from differences in cell wall assembly and chemical structures of lignin and lignin carbohydrate complexes. Lignin in grasses is comprised of guaiacylpropanoid, syringylpropanoid and p-hydroxy-benzylpropanoid structures and crosslinked with polysaccharides by cinnmamic acid bridges, while lignin in coniferous trees is primarily comprised of guaiacylpropanoid structures, and directly linked with hemicelluloses by benzyl ether and ester bonds (Watanabe 2003). In a study of basidiomycete treatment to improve digestibility of roughage, it was reported that the effect could differ even within the same fungi species, depending on the strain (Zadrazil 1985; Akin et al. 1996). In our previous study, we cultured Japanese red cedar wood with three strains of C. subvermispora, revealing that the ATCC 90467 strain had a greater effect on improved digestibility than CZ-3 and CBS 347.63 (K. Okano et al., unpubl. data, 2005). In the present study, however, we observed no difference in the effect of improved digestibility between two strains of C. subvermispora. This nding suggests that the effect on improved digestibility is dependent upon a combination of the lignocellulose material and the fungus strain employed.

Chemical composition in bagasse treated with four white-rot fungi species for different cultivation lengths is shown in Table 2. While the lignin content in untreated bagasse was 10.5%, the content in bagasse cultured with L. edodes decreased to 6.5% at 16 weeks. The lignin content in bagasse cultured with both strains of C. subvermispora was 7.3%, respectively, at 16 weeks. This result also indicates that L. edodes has a higher lignin-degrading ability than C. subvermispora, and is proof that L. edodes has a greater effect on improved digestibility of bagasse than C. subvermispora. In the present study, NDF and hemicellulose contents in bagasse cultured with four white-rot fungi were lower than those in untreated bagasse. The greatest decrease of NDF content was observed in bagasse cultured with L. edodes at 16 weeks. During the decay process in wheat straw, rice straw and bagasse medium by white-rot fungi, an increase of soluble substances in neutral detergent has been reported (Agosin et al. 1986; Yamakawa et al. 1992; Suzuki 1995; Suzuki et al. 1995). This increase need not reect the increase of organic matter available as an energy source for ruminants because white-rot fungi metabolize sugar, starch and hemicellulose in preference to cellulose and lignin. Although the decrease in hemicellulose and other carbohydrate causes a loss of nutrients as feedstuff, this disadvantage in fungal

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treatment could be compensated by the increase of digestible NDF content. In this study, the highest values of IVNDFD and IVGP were obtained in bagasse cultured with L. edodes. The digestibility in bagasse was not improved by culturing with P. eryngii or P. salmoneostramineus as shown in Table 1. Kewalramani et al. (1988) reported that culturing bagasse with P. sajor-caju or Polyporus hirsutus raised in vitro dry matter digestibility to 1723%, and Zadrazil and Puniya (1995) reported that culturing bagasse with P. eryngii raised IVOMD to 1020%. In contrast, as seen in the present study, Yamada et al. (2000) reported that culturing bagasse with P. salmoneostramineus, P. cystidiosus, or A. polytricha did not raise the in vitro dry matter digestibility. In the present study, culturing with P. eryngii and P. salmoneostramineus halved the hemicellulose content and increased the cellulose content; nonetheless, the lignin content remained the same or increased. These ndings also suggest that P. eryngii and P. salmoneostramineus have weaker lignin-degrading capabilities than L. edodes and C. subvermispora, and that their effect on improved digestibility was also low. In the present study, the low effect on improved digestibility of P. eryngii might have rst been related to the strain of fungus used. Zadrazil (1985) reported that wheat straws cultured with two different strains of P. eryngii were different from the improvement in IVOMD accordingly. A second reason may be differences in aeration during culturing. Lignin degradation is an oxidative process that requires appropriate oxygen supply. In the case of passive air exchange in culturing with a ask and cotton plug, such as in the present study, insufcient oxygen during culturing decreases the degree of lignin degradation and improvement in digestibility of the culture medium (Kirk et al. 1986; Puniya et al. 1994; Zadrazil & Puniya 1994). Consequently, the amount of air exchange differs depending on the state of the cotton plug insertion. In our study, cotton plugs were inserted very tightly, possibly decreasing lignin degradation by P. eryngii and P. salmoneostramineus and resulting in no improvement in digestibility. Nonetheless, the fact that L. edodes and C. subvermispora demonstrated a large improvement in digestibility under the same conditions is a reliable indication that these fungi have a stronger effect on improved digestibility than do P. eryngii and P. salmoneostramineus. L. edodes has many strains native to Japan, China, and other East Asian countries, and the amount of lignin-degrading enzyme secreted extracellularly dif-

fers depending on the strain (Tokimoto et al. 1988). The recommended temperature for growing L. edodes is 2127C, while the optimum temperature for fruitbody formation is 1618C among low temperature strains and 2127C among high temperature strains. However, there are exceptional instances of strains withstanding temperatures up to 32C (Stamets 2000), indicating that these species are potentially useful in improving bagasse digestibility in the tropics and subtropics.

REFERENCES
Agosin E, Tollier MT, Brillouet JM, Thivend P, Odier E. 1986. Fungal pretreatment of wheat straw: effects on the biodegradability of cell walls, structural polysaccharides, lignin and phenolic acids by rumen microorganisms. Journal of the Science of Food and Agriculture 37, 97106. Akin DE, Morrison WH III, Rigsby LL, Gamble GR, Sethuraman A, Eriksson KEL. 1996. Biological delignication of plant components by the white rot fungi Ceriporiopsis subvermispora and Cyathus stercoreus. Animal Feed Science and Technology 63, 305321. Chai W, Uden P. 1998. An alternative method combined with different detergent strengths in the analysis of neutral detergent bre. Animal Feed Science and Technology 74, 281288. Horii S, Abe A, Kim KS, Kameoka K. 1971. An in vitro technique for estimating digestibility of roughage. Bulletin of National Institute of Animal Industry 24, 99105. (In Japanese) Itoh H, Wada M, Honda Y, Kuwahara M, Watanabe T. 2003. Bioorganosolve pretreatments for simultaneous saccharication and fermentation of beech wood by ethanolysis and white rot fungi. Journal of Biotechnology 103, 273280. Kewalramani N, Kamra DN, Lall D, Pathak NN. 1988. Bioconversion of sugarcane bagasse with white rot fungi. Biotechnology Letters 10, 369372. Kirk TK, Moore WE. 1972. Removing lignin from wood with white-rot fungi and digestibility of resulting wood. Wood Fiber 4, 7279. Kirk TK, Schultz E, Connors WJ, Lorenz LF, Zeikus JG. 1986. Inuence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Archives of Microbiology 117, 277285. McDougall EI. 1948. Studies of ruminant saliva. I. The composition and output of sheeps saliva. Biochemical Journal 43, 99109. Messner K, Srebotnik E. 1994. Biopulping: an overview of developments in an environmentally safe paper-making technology. FEMS Microbiology Reviews 13, 351364. Okano K, Kitagawa M, Sasaki Y, Watanabe T. 2005. Conversion of Japanese red cedar (Cryptomeria japonica) into a feed for ruminants by white-rot basidiomycetes. Animal Feed Science and Technology 120, 235243. Puniya AK, Zadrazil F, Singh K. 1994. Inuence of gaseous phases on lignocellulose degradation by Phanerochaete chrysosporium. Bioresource Technology 47, 181183.

2006 Japanese Society of Animal Science

Animal Science Journal (2006) 77, 308313

WHITE-ROT FUNGI TREATMENT OF BAGASSE 313

SPSS. 2002. SPSS Base 11.5J Users Guide. SPSS Inc, Tokyo. (In Japanese) Stamets P. 2000. Growing Gourmet and Medicinal Mushrooms , 3rd edn. Ten Speed Press, Berkeley, CA. Suzuki Y. 1992. Culture of Pleurotus salmoneostramneus on unsterilized wheat straw. Transactions of Mycological Society of Japan 33, 249253. (In Japanese) Suzuki Y. 1995. Study on nutritional upgrading of underutilized feed resources by basidiomycetes (PhD thesis). Kyoto University, Kyoto. (In Japanese) Suzuki Y, Okano K, Kato S. 1995. Characteristics of whiterotted woody materials obtained from Shiitake mushroom (Lentinus edodes) and nameko mushroom (Pholiota nameko) cultivation with in vitro rumen fermentation. Animal Feed Science and Technology 66, 406411. Tilley JMA, Terry RA. 1963. A two stage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society 18, 104111. Tokimoto K, Fukuda M, Komatsu M. 1988. Variation in removal of wood components of Fagus crenata during decay by monokaryons and dikaryons of Lentinus edodes. Reports of the Tottori Mycological Institute 26, 3745. Van Soest PJ, Robertson JB, Lewis BA. 1991. Methods of dietary ber, neutral detergent ber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74, 35833597.

Watanabe T. 2003. Microbial degradation of lignin-carbohydrate complexes. In: Koshijima T, Watanabe T (eds), Association Between Lignin and Carbohydrates in Wood and Other Plant Tissues, pp. 237287. Springer Verlag, Berlin. Yamada Y, Goto M, Karita S, Takabe K, Fujita M, Suzuki Y, Yurugi Y. 2000. Effect of basidiomycetes (Pleurotus salmoneostramineus, Pleurotus cystidiosus, Auricularia polytricha) on chemical structure and rumen degradability of bagasse. Grassland Science 46, 158166. (In Japanese) Yamakawa M, Abe H, Okamoto M. 1992. Effect of incubation with edible mushroom, Pleurotus ostreatus, on voluntary intake and digestibility of rice straw by sheep. Animal Science and Technology (Japan) 63, 129133. Zadrazil F. 1985. Screening of fungi for lignin decomposition and conversion of straw into feed. Angewandte Botanik 59, 433452. Zadrazil F, Puniya AK. 1994. Inuence of carbon dioxide on lignin degradation and digestibility of lignocellulosics treated with. Pleurotus Sajor-Caju. International Biodeterioration and Biodegradation 33, 237244. Zadrazil F, Puniya AK. 1995. Studies on the effect of particle size on solid-state fermentation of sugarcane bagasse into animal feed using white-rot fungi. Bioresource Technology 54, 8587.

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