Nutritional Composition and In Vitro Digestibility of Spent Mushroom Substrate Supplemented with Urea and Molasses for Ruminants
Abstract
This study aims to evaluate the fermentation characteristics and nutritional potential of spent mushroom substrates (SMS), with or without urea and molasses supplementation, in ruminant diets. SMS derived from the cultivation of four mushroom species—Pleurotus ostreatus (Oyster Mushroom, OYS), Flammulina filiformis (Enoki mushroom, ENK), Hypsizygus marmoreus (Bunashimeji mushroom, SMJ), and Lentinula edodes (Shiitake, STK)—were analyzed for proximate composition, in vitro gas and methane (CH₄) production, dry matter digestibility (DMD), and short-chain fatty acid (SCFA). Statistically significant differences were observed among SMS types across all incubation periods (24, 48, and 72 hours) for gas production, CH₄ production, DMD, and SCFA concentrations (p<0.0001). In addition, CH₄ per gram of digested dry matter (CH₄/DDM) was higher in urea–molasses supplemented SMS compared to non-supplemented substrates (p<0.05). However, no significant differences were observed in total gas production, CH₄ volume, or SCFA concentrations between the two groups, suggesting that fermentation responses were influenced not only by supplementation but also by the inherent differences in substrate composition. The results indicate that different types of SMS vary significantly in their fermentation profiles and digestibility. Supplementation with urea and molasses increased CH₄/DDM but did not significantly affect total gas, CH₄ volume, or SCFA production. These findings demonstrate the potential of SMS as a partial roughage replacement in ruminant diets, as influenced by the type of mushroom substrate, its chemical composition, and the supplementation method.
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References
Akinfemi, A., Adebayo, B. J., & Ogunbosoye, D. O. (2020). Nutritional improvement of rice straw treated with urea-molasses and its effect on in vitro digestibility. Journal of Agricultural Science and Practice, 5(1), 74-79. https://doi.org/10.31248/JASP2019.181
Akinfemi, A., Adebayo, B. J., & Ososanya, T. O. (2018). Effect of urea-molasses treatment on chemical composition and in vitro digestibility of maize cobs. Nigerian Journal of Animal Science, 20(1), 98-105.
Amirault, K., Wright, R., Sujani, S., Dos Reis, B. R., Osorio, J., Fernandes, T., & White, R. R. (2024). Ruminal pH sensing for monitoring volatile fatty acid concentrations in response to short-term dietary disruption. JDS Communications, 5(2), 91-95. https://doi.org/10.3168/jdsc.2023-0409
Andhale, V. T. (2024). Exploring the power of non-conventional feed resources in animal nutrition. Acta Scientific Veterinary Sciences, 6(2), 45-47. https://doi.org/10.31080/ASVS.2024.06.0813
Anotaenwere, C. C., Isikhuemhen, O. S., Dele, P. A., Wuaku, M., Alabi, J. O., Adelusi, O. O., Okedoyin, D. O., Ike, K. A., Gray, D., Kholif, A. E., & Anele, U. Y. (2024). Ensiled Pleurotus ostreatus based spent mushroom substrate from corn: In vitro gas production, greenhouse gas emissions, nutrient degradation, and ruminal fermentation characteristics. AIMS Microbiology, 11(1), 1. https://doi.org/10.3934/microbiol.2025001
Antunes, F., Marçal, S., Taofiq, O., MMB Morais, A., Freitas, A. C., CFR Ferreira, I., & Pintado, M. (2020). Valorization of mushroom by-products as a source of value-added compounds and potential applications. Molecules, 25(11), 2672. https://doi.org/10.3390/molecules25112672.
AOAC International. (2000). Official methods of analysis of AOAC International (Vol. 17, No. 1-2). AOAC International.
Aquino, D., Barrio, A. D., Trach, N. X., Hai, N. T., Khang, D. N., Toan, N. T., & Hung, N. V.. (2020). In Gummert, M., Hung, N., Chivenge, P., & Douthwaite, B. (eds), Sustainable rice straw management (pp. 111–129). Springer. https://doi.org/10.1007/978-3-030-32373-8_7
Aruwayo, A. (2018). Use of urea treated crop residue in ruminant feed. International Journal of Advances in Scientific Research and Engineering, 4(7), 54-64. http://doi.org/10.31695/IJASRE.2018.32794
Bashar, M. K., Haese, E., Sultana, N., & Rodehutscord, M. (2024). In vitro ruminal fermentation, methane emissions, and nutritional value of different tropical feedstuffs for ruminants. Journal of Advanced Veterinary and Animal Research, 11(4), 924. https://doi.org/10.5455/javar.2024.k842
Chen, B. Y., Wu, P. S., & Wang, H. T. (2025). Assessment of alkali-treated spent mushroom substrate supplemented with condensed molasses solubles (CMS) as an alternative forage source for ruminants through in vitro fermentation. Fermentation, 11(2), 92. https://doi.org/10.3390/fermentation11020092
Cheva-Isarakul, B. L., & Kanjanapruthipong, J. (1987). A comparison of urea-treated rice straw with urea-molasses sprayed rice straw as basal diets for growing cattle. Proceedings of the Sixth Annual Workshop of the Australian Asian Fibrous Agricultural Residues Research Network, Los Banos, 1-3 April pp. 191-198.
Crisostomo, J. C. A., Manlapig, J. J. D., Orden, E. A., & Velasco, V. V. (2022). Acacia pods (Samanea saman) as a substitute for concentrate on growing and lactating goats. Philippine Journal of Veterinary & Animal Sciences, 48(2), 65-79.
Etela, I., Ogbeide, A., Ukanwoko, A. I., & Adedokun, O. M. (2018). Spent substrates from three species of mushroom as alternative feed resources for ruminant livestock. The Journal of Solid Waste Technology and Management, 44(1), 15-20. https://doi.org/10.5276/JSWTM.2018.15
Faniyi, T. O., Adegbeye, M. J., Elghandour, M. M. M. Y., Pilego, A. B., Salem, A. Z. M., Olaniyi, T. A., Adediran, T. A., & Adewumi, M. K. (2019). Role of diverse fermentative factors towards microbial community shift in ruminants. Journal of Applied Microbiology, 127(1), 2-11.https://doi.org/10.1111/jam.14212
Getachew, G., Makkar, H. P. S., & Becker, K. (2002). Tropical browses: contents of phenolic compounds, in vitro gas production and stoichiometric relationship between short chain fatty acid and in vitro gas production. The Journal of Agricultural Science, 139(3), 341-352. https://doi.org/10.1017/S0021859602002393
Goyal, M., Kaur, R. P., Pal, R. P., & Singh, D. P. (2023). Effect of mixing paddy straw with molasses and urea on fermentation characteristics and nutritive value of maize silage. The Indian Journal of Animal Sciences, 93(10), 985-992. https://doi.org/10.56093/ijans.v93i10.134389
Grimm, D., & Wösten, H. A. (2018). Mushroom cultivation in the circular economy. Applied Microbiology and Biotechnology, 102, 7795-7803. https://doi.org/10.1007/s00253-018-9226-8.
Jiang, W., Han, G., Via, B. K., Tu, M., Liu, W., & Fasina, O. (2014). Rapid assessment of coniferous biomass lignin–carbohydrates with near-infrared spectroscopy. Wood Science and Technology, 48, 109-122. https://doi.org/10.1371/journal.pone.0256559
Kalvandi, S., Zaboli, K., & Malecky, M. (2018). Effect of spent mushroom compost (Agaricus bisporus) silage processing on its chemical composition, digestibility and ruminal fermentation kinetic in Mehraban sheep. Animal Production Research, 7, 69–82. https://doi.org/10.22124/ar.2018.9256.1270
Kim, S. H., Mamuad, L. L., Jeong, C. D., Choi, Y. J., Lee, S. S., Ko, J. Y., & Lee, S. S. (2013). In vitro evaluation of different feeds for their potential to generate methane and change methanogen diversity. Asian-Australasian Journal of Animal Sciences, 26(12), 1698. https://doi.org/10.5713/ajas.2013.13260
Kim, Y. I., Oh, Y. K., Park, K. K., & Kwak, W. S. (2014). Ensiling characteristics and the in situ nutrient degradability of a by-product feed-based silage. Asian-Australasian Journal of Animal Sciences, 27(2), 201-208. https://doi.org/10.5713/ajas.2013.13448
Kousar, A., Khan, H. A., Farid, S., Zhao, Q., & Zeb, I. (2024). Recent advances on environmentally sustainable valorization of spent mushroom substrate: A review. Biofuels, Bioproducts and Biorefining, 18(2), 639-651. https://doi.org/10.1002/bbb.2559
Kwak, W. S., Kim, Y. I., Seok, J. S., Oh, Y. K., & Lee, S. M. (2009). Molasses and microbial inoculants improve fermentability and silage quality of cotton waste-based spent mushroom substrate. Bioresource Technology, 100(3), 1471-1473. https://doi.org/10.1016/j.biortech.2008.07.066
Leong, Y. K., Ma, T. W., Chang, J. S., & Yang, F. C. (2022). Recent advances and future directions on the valorization of spent mushroom substrate (SMS): A review. Bioresource Technology, 344, 126157. https://doi.org/10.1016/j.biortech.2021.126157
Li, Y., Lv, J., Wang, J., Zhou, S., Zhang, G., Wei, B., Sun, Y., Lan, Y., Dou, X., & Zhang, Y. (2021). Changes in carbohydrate composition in fermented total mixed ration and its effects on in vitro methane production and microbiome. Frontiers in Microbiology, 12, 738334. https://doi.org/10.3389/fmicb.2021.738334
Løvendahl, P., Difford, G. F., Li, B., Chagunda, M. G. G., Huhtanen, P., Lidauer, M. H., Lassen, J., & Lund, P. (2018). Review: Selecting for improved feed efficiency and reduced methane emissions in dairy cattle. Animal, 12(s2), s336-s349. https://doi.org/10.1017/S1751731118002276
Ma, Y., Chen, X., Zahoor Khan, M., Xiao, J., Liu, S., Wang, J., He, Z., Li, C., & Cao, Z. (2020). The impact of ammoniation treatment on the chemical composition and in vitro digestibility of rice straw in Chinese Holsteins. Animals, 10(10), 1854. https://doi.org/10.3390/ani10101854
Mandey, J. S., Kowel, Y. H. S., Regar, M. N., & Leke, J. R. (2017). Effect of different level of energy and crude fiber from sawdust in diets on carcass quality of broiler. https://10.14710/jitaa.42.4.240-246
Manlapig, J. J. D., Ban‐Tokuda, T., & Matsui, H. (2023). Nutritional quality and organic acid profile of rice bran fermented with lactic acid bacteria isolated from horse feces. Animal Science Journal, 94(1), e13860. https://doi.org/10.1111/asj.13860
Manlapig, J. J. D., Kawakami, S., Matamura, M., Kondo, M., Ban-Tokuda, T., & Matsui, H. (2024a). Effect of rice bran extract on in vitro rumen fermentation and methane production. Animal Science Journal, 95(1), e13923. https://doi.org/10.1111/asj.13923
Manlapig, J. J. D., Kondo, M., Ban‐Tokuda, T., & Matsui, H. (2024b). Effect of rice bran fermented with Ligilactobacillus equi on in vitro fermentation profile and microbial population. Animal Science Journal, 95(1), e13955. https://doi.org/10.1111/asj.13955
Matsui, H., Wakabayashi, H., Fukushima, N., Ito, K., Nishikawa, A., Yoshimi, R., Ogawa, Y., Yoneda, S., Ban-Tokuda, T., & Wakita, M. (2013). Effect of raw rice bran supplementation on rumen methanogen population density and in vitro rumen fermentation. Grassland Science, 59(3), 129–134. https://doi.org/10.1111/grs.12023
Mordenti, A. L., Giaretta, E., Campidonico, L., Parazza, P., & Formigoni, A. (2021). A review regarding the use of molasses in animal nutrition. Animals, 11(1), 115. https://doi.org/10.3390/ani11010115
Muqier, X., Eknæs, M., Prestløkken, E., Jensen, R. B., Eikanger, K. S., Karlengen, I. J., troan, G., Vhile, S. G., & Kidane, A. (2023). In vitro rumen fermentation characteristics, estimated utilizable crude protein and metabolizable energy values of grass silages, concentrate feeds and their mixtures. Animals, 13(17), 2695. https://doi.org/10.3390/ani13172695
Oladosu, Y., Rafii, M. Y., Abdullah, N., Magaji, U., Hussin, G., Ramli, A., & Miah, G. (2016). Fermentation quality and additives: a case of rice straw silage. BioMed Research International, 2016(1), 7985167. https://doi.org/10.1155/2016/7985167
Palangi, V., Kaya, A., Kaya, A., & Giannenas, I. (2022). Ecofriendly usability of mushroom cultivation substrate as a ruminant feed: Anaerobic digestion using gas production techniques. Animals, 12(12), 1583. https://doi.org/10.3390/ani12121583
Parchami, M., Rustas, B. O., Taherzadeh, M. J., & Mahboubi, A. (2024). Effect of agro-industrial by products derived from volatile fatty acids on ruminant feed in vitro digestibility. Animals, 14(16), 2330. https://doi.org/10.3390/ani14162330
Ramírez, G. R., Aguilera-Gonzalez, J. C., Garcia-Diaz, G., & Núñez-González, A. M. (2007). Effect of urea treatment on chemical composition and digestion of Cenchrus ciliaris and Cynodon dactylon Hays and Zea mays residues. Journal of Animal and Veterinary Advances, 6(8), 1036-1041.
Rangubhet, K. T., Mangwe, M. C., Mlambo, V., Fan, Y. K., & Chiang, H. I. (2017). Enteric methane emissions and protozoa populations in Holstein steers fed spent mushroom (Flammulina velutipes) substrate silage-based diets. Animal Feed Science and Technology, 234, 78-87. https://doi.org/10.1016/j.anifeedsci.2017.06.005
Rangubhet, T., Kongmun, P., Prasanpanich, S., & Chiang, H. I. (2020). Nutritional evaluation of spent mushroom substrate from Pleurotus ostreatus and P. citrinopileatus as roughage for meat goats. In 58. Kasetsart University Annual Conference, Bangkok (Thailand), 5-7 Feb 2020.
Reddy, P. R. K., & Hyder, I. (2023). Ruminant Digestion. In: Das, P. K., Sejian, V., Mukherjee, J., & Banerjee, D. (Eds), Textbook of Veterinary Physiology. Springer, Singapore. https://doi.org/10.1007/978-981-19-9410-4_14
Ridla, M., Jayanegara, A., & Nahrowi, N. (2025). Evaluation of silage quality, rumen fermentation dynamics, degradability, and methane emissions of total mixed rations formulated from agricultural by-products: an in vitro analysis. Journal of Animal and Feed Sciences. https://doi.org/10.22358/jafs/200863/2025
Rinne, M., Franco, M., Jalava, T., Järvenpää, E., Kahala, M., Blasco, L., Siljander-Rasi, H., & Kuoppala, K. (2019). Carrot by-product fermentation quality and aerobic spoilage could be modified with silage additives. Agricultural and Food Science, 28(2), 59–69. https://doi.org/10.23986/afsci.79829
Royse, D. J., Baars, J., & Tan, Q. (2017). Current overview of mushroom production in the world. In Diego, C. Z., & Pardo-Giménez (Eds.), Edible and medicinal mushrooms: technology and applications, 5-13. https://doi.org/10.1002/9781119149446.ch2
Sallam, S., Rady, A., Attia, M. F., Elazab, M. A., Vargas-Bello-Pérez, E., & Kholif, A. E. (2024). Different maize silage cultivars with or without urea as a feed for ruminant: chemical composition and in vitro fermentation and nutrient degradability. Chilean Journal of Agricultural & Animal Sciences, 40(1), 137-149. https://dx.doi.org/10.29393/chjaas40-14dmsa60014
Shah, A. M., Zhang, H., Shahid, M., Ghazal, H., Shah, A. R., Niaz, M., Naz, T., Ghimire, K., Goswami, N., Shi, W., Xia, D., & Zhao, H. (2025). The vital roles of agricultural crop residues and agro-industrial by-products to support sustainable livestock productivity in subtropical regions. Animals, 15(8), 1184. https://doi.org/10.3390/ani15081184
Shen, J., Zheng, W., Xu, Y., & Yu, Z. (2023). The inhibition of high ammonia to in vitro rumen fermentation is pH dependent. Frontiers in Veterinary Science, 10, 1163021. https://doi.org/10.3389/fvets.2023.1163021
Souza, M. N., Ferrer, M. D., Bezerra, L. R., de Andrade, E. A., Pereira Filho, J. M., Barbosa, A. M., Silva, T. M., Pereira, E. S., da Silva Junior, J. M., Batista, T. S., Mazza, P. H. S. & Oliveira, R. L. (2024). Effect of mushroom crop residue on growth performance, carcass traits, nutrient digestibility, nitrogen balance, ingestive behavior, ruminal and blood parameters of lambs. Animal Feed Science and Technology, 315, 116045. https://doi.org/10.1016/j.anifeedsci.2024.116045
Sundstol, F., & Coxworth, E. M. (1984). Ammonia treatment. In Straw and other fibrous by-products as feed (pp. 196-247). Elsevier Amsterdam.
Van Soest, P. V., Robertson, J. B., & Lewis, B. A. (1991). Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74(10), 3583-3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2
Wahyono, T., Sholikin, M. M., Konca, Y., Obitsu, T., Sadarman, S., & Jayanegara, A. (2022). Effects of urea supplementation on ruminal fermentation characteristics, nutrient intake, digestibility, and performance in sheep: A meta-analysis. Veterinary World, 15(2), 331. https://doi.org/10.14202/vetworld.2022.331-340
Wang, W., Wang, Y., Cui, Z., Yang, Y., An, X., & Qi, J. (2022). Fermented wheat bran polysaccharides intervention alters rumen bacterial community and promotes rumen development and growth performance in lambs. Frontiers in Veterinary Science, 9, 841406. https://doi.org/10.3389/fvets.2022.841406
Widodo, S., Shiddieqy, M. I., Wahyono, T., Widiawati, Y., & Muttaqin, Z. (2023). Analysis of correlation between nutrient content, digestibility, and gas production of forages in Indonesia. Advances in Animal and Veterinary Sciences, 11(11), 1770-1778. https://dx.doi.org/10.17582/journal.aavs/2023/11.11.1770.1778
Yi, Q., Wang, P., Tang, H., Yu, M., Zhao, T., Sheng, Z., & Luo, H. (2023). Fermentation quality, in vitro digestibility, and aerobic stability of ensiling spent mushroom substrate with microbial additives. Animals, 13(5), 920. https://doi.org/10.3390/ani13050920
Zaboli, K., Kalvandi, S., Malecky, M., & Nasrabadi, M. (2023). Nutritional Value of Spent Mushroom (Agaricus bisporus) Compost silage treated with different level of molasses in sheep feeding. Iranian Journal of Applied Animal Science, 13(1), 57-65
Zheng, W., Duan, H., Cao, L., Mao, S., & Shen, J. (2024). Acid-base properties of non-protein nitrogen affect nutrients intake, rumen fermentation and antioxidant capacity of fattening Hu sheep. Frontiers in Veterinary Science, 11, 1381871. https://doi.org/10.3389/fvets.2024.1381871
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