Fatty Acid Biohydrogenation, Fermentation, and Digestibility of Ration Containing Napier and King Grass with Different Harvest Ages and Altitudes: In Vitro Study

D. Anzhany, T. Toharmat, Despal, A. Łozicki


Forage is the primary and cheapest source of fatty acids (FA), which includes conjugated linoleic acid (CLA), influencing milk FA. This study aimed to analyze the fermentation, digestibility, biohydrogenation, nutrient composition, and FA content of napier grass (NG) and king grass (KG). Grasses were collected from the Pangalengan (highland) and Dramaga (lowland) districts at three harvest ages (1, 1.5, and 2 months). The feed was then analyzed for nutrients and FA. An in vitro study was performed to analyze the concentrations of NH3, VFA, protozoa populations, and biohydrogenation. No significant differences were observed in protozoa, pH, total VFA, or FA biohydrogenation. NH3 ranged from 5.31 mM to 8.86 mM. Significant differences were found at different altitudes, with an interaction between grass type and harvest age and an interaction between the three factors. The highest NH3 concentration was found in rations containing highland NG at 1.5 months. The DMD value was 58.27%–64.39%, and OMD was 61.07%–67.18%. Different digestibility values were observed at different harvest ages, with an interaction between altitude and harvest age. This aligned with the CF, NDF, and lignin contents in grasses. The highest was at 1.5 months NG. Significant differences were observed in the relative proportions of propionic acids. The highest value was in the ration containing the 1.5-month highland NG. Rations containing KG yielded significantly higher amounts of the C18:0 and C18:1 trans. In conclusion, the 1.5-month highland NG is a potential ration for supporting healthier FA production in milk.


Anzhany, D., T. Toharmat, & Despal. 2022. Ration to produce milk high in conjugated linoleic acid (CLA) at smallholder dairy farm: An in vitro reconstruction. Am. J. Anim. Vet. Sci. 17:130–138. https://doi.org/10.3844/ajavsp.2022.130.138
AOAC. 2005. Official Methods of Analysis of AOAC International. 18th ed. Horwitz W, Latimer GW, editor. Gaithersburg, Maryland: Association of Official Agricultural Chemists.
AOAC. 2000. AOAC Official Method 969.33. AOAC Int. 41:19–61.
Bunzel, M., J. Ralph, & H. Steinhart. 2018. Phenolic compounds as cross-links of plant derived polysaccharides. Czech J. Food Sci. 22:64–67. https://doi.org/10.17221/10613-CJFS
Burgess, P. & B. Huang. 2016. Leaf protein abundance associated with improved drought tolerance by elevated carbon dioxide in creeping bentgrass. J. Am. Soc. Hortic. Sci. 141:85–96. https://doi.org/10.21273/JASHS.141.1.85
Chen, P. B. & Y. Park. 2019. Conjugated linoleic acid in human health: Effects on weight control. Nutrition Prevention Treatment Abdominal Obesity 2:355–382. https://doi.org/10.1016/B978-0-12-816093-0.00025-2
Conte, G., A. Serra, & M. Mele. 2017. Dairy Cow Breeding and Feeding on the Milk Fatty Acid Pattern. p. 19-41. Elsevier Inc. https://doi.org/10.1016/B978-0-12-809762-5.00002-4
Cottyn, B. G. & C. V. Boucque. 1968. Rapid method for the gas-chromatographic determination of volatile fatty acids in rumen fluid. J. Agric. Food Chem. 16:105–107. https://doi.org/10.1021/jf60155a002
Despal, L. A. Sari, I. G. Permana, R. Zahera, & D. Anzhany. 2021. Fibre feeds impact on milk fatty acids profiles produced by smallholder dairy farmers. Int. J. Dairy Sci. 16:98–107. https://doi.org/10.3923/ijds.2021.98.107
Dewhurst, R. J., K. J. Shingfield, M. R. F. Lee, & N. D. Scollan. 2006. Increasing the concentrations of beneficial polyunsaturated fatty acids in milk produced by dairy cows in high-forage systems. Anim. Feed Sci. Technol. 131:168–206. https://doi.org/10.1016/j.anifeedsci.2006.04.016
Duchemin, S., H. Bovenhuis, W. M. Stoop, A. C. Bouwman, J. A. M. van Arendonk, & M. H. P. W. Visker. 2013. Genetic correlation between composition of bovine milk fat in winter and summer, and DGAT1 and SCD1 by season interactions. J. Dairy Sci. 96:592–604. https://doi.org/10.3168/jds.2012-5454
Elgersma, A. 2015. Grazing increases the unsaturated fatty acid concentration of milk from grass-fed cows: A review of the contributing factors, challenges and future perspectives. Eur. J. Lipid Sci. Technol. 117:1345–1369. https://doi.org/10.1002/ejlt.201400469
Enjalbert, F., M. C. Nicot, C. Bayourthe, & R. Moncoulon. 2000. Effects of duodenal infusions of palmitic, stearic, or oleic acids on milk composition and physical properties of butter. J. Dairy Sci. 83:1428–1433. https://doi.org/10.3168/jds.S0022-0302(00)75012-0
Enjalbert, F., S. Combes, A. Zened, & A. Meynadier. 2017. Rumen microbiota and dietary fat: A mutual shaping. J. Appl. Microbiol. 123:782–797. https://doi.org/10.1111/jam.13501
General Laboratory Procedures. 1966. Department of Dairy Science, University of Wisconsin, USA.
Harfoot, C. G. & G. P. Hazlewood. 1997. Lipid Metabolism in the Rumen. In: Hobson PN, Stewart CS, editor. The Rumen Microbial Ecosystem. Springer, Dordrecht. p. 382–426. https://doi.org/10.1007/978-94-009-1453-7_9
Harper, K. J. & D. M. McNeill. 2015. The Role iNDF in the regulation of feed intake and the importance of its assessment in subtropical ruminant systems (the role of iNDF in the regulation of forage intake). Agric. 5:778–790. https://doi.org/10.3390/agriculture5030778
Hayashi, Y., E. Takeya, Y. Ikeno, H. Kumagai, E. M. Cruz, N. P. Garcia, D. L. Aquino, & T. Fujihara. 2021. Periodic changes in chemical composition and in vitro digestibility of locally available Gramineae feed resources in the Philippines. Trop. Anim. Health Prod. 53:123. https://doi.org/10.1007/s11250-021-02572-y
He, D., S. Zheng, J. Xiao, Y. Ye, X. Liu, Z. Yin, & D. Wang. 2022. Effect of lignin on short-chain fatty acids production from anaerobic fermentation of waste activated sludge. Water Res. 212:118082. https://doi.org/10.1016/j.watres.2022.118082
Hendarto, E. & A. Setyaningrum. 2022. Production and king grass nutritional quality number of sources of nitrogen fertilizer. HighTech Innovation Journal 3:252–266. https://doi.org/10.28991/HIJ-2022-03-03-02
Khan, N. A., M. W. Farooq, M. Ali, M. Suleman, N. Ahmad, S. M. Sulaiman, J. W. Cone, & W. H. Hendriks. 2015. Effect of species and harvest maturity on the fatty acids profile of tropical forages. J. Anim. Plant Sci. 25:739–746.
Li, J. C., O. A. Castelán-Ortega, F. A. Galindo-Maldonado, J. Arango, N. Chirinda, R. Jiménez-Ocampo, S. S. Valencia-Salazar, E. J. Flores-Santiago, M. D. Montoya-Flores, I. C. Molina-Botero, A. T. Piñeiro-Vázquez, J. I. Arceo-Castillo, C. F. Aguilar-Pérez, L. Ramírez-Avilés, & F. J. Solorio-Sánchez. 2020. Review: Strategies for enteric methane mitigation in cattle fed tropical forages. Animal 14:453–463. https://doi.org/10.1017/S1751731120001780
Li, Z., Q. Deng, Y. Liu, T. Yan, F. Li, Y. Cao, & J. Yao. 2018. Dynamics of methanogenesis, ruminal fermentation and fiber digestibility in ruminants following elimination of protozoa: A meta-analysis. J. Anim. Sci. Biotechnol. 9:1–9. https://doi.org/10.1186/s40104-018-0305-6
Loften, J. R., J. G. Linn, J. K. Drackley, T. C. Jenkins, C. G. Soderholm, & A. F. Kertz. 2014. Invited review: Palmitic and stearic acid metabolism in lactating dairy cows. J. Dairy Sci. 97:4661–4674. https://doi.org/10.3168/jds.2014-7919
Lourenço, M., B. Vlaeminck, M. Bruinenberg, D. Demeyer, & V. Fievez. 2005. Milk fatty acid composition and associated rumen lipolysis and fatty acid hydrogenation when feeding forages intensively managed or semi-natural grassland. Anim. Res. 54:471–484. https://doi.org/10.1051/animres:2005036
Makmur, M., M. Zain, F. Agustin, R. Sriagtula, & E. M. Putri. 2020. In vitro rumen biohydrogenation of unsaturated fatty acids in tropical grass-legume rations. Vet. World 13:661–668. https://doi.org/10.14202/vetworld.2020.661-668
Makmur, M., M. Zain, Y. Marlida, Khasrad, & A. Jayanegara. 2019. Fatty acids composition and biohydrogenation reduction agents of tropical forages. Biodiversitas 20:1917–1922. https://doi.org/10.13057/biodiv/d200718
Massey, J., J. Antonangelo, & H. Zhang. 2020. Nitrogen fertilization and harvest timing affect switchgrass quality. Resources 9:1–15. https://doi.org/10.3390/resources9060061
McDonald, P., R. A. Edwards, J. F. D. Grennhalgh, C. A. Morgan, L. A. Sinclair, & R. G. Wilkinson. 2020. Animal Nutrition 8th Edition. Harlow, England: Ashford Colour Press. Ltd.
Mertens, D. R. & R. J. Grant. 2020. Digestibility and Intake. In: Moore K. J., M. Collins, C. J. Nelson, D. D. Redfearm, Editor. Forages: The Science of Grassland Agriculture. Vol. II. Seventh Ed. John Wiley & Sons Ltd. p 609–631. https://doi.org/10.1002/9781119436669.ch34
Mwangi, F. W., E. Charmley, O. A. Adegboye, C. P. Gardiner, B. S. Malau-Aduli, R. T. Kinobe, & A. E. O. Malau-Aduli. 2022. Chemical composition and in situ degradability of Desmanthus spp. forage harvested at different maturity stages. Fermentation 8:1–21. https://doi.org/10.3390/fermentation8080377
Nadeau, E., A. Arnesson, & C. Helander. 2015. Effects of grass silage feed value on feed intake and performance of pregnant and lactating ewes and their lambs. Grass Forage Sci. 71:448–457. https://doi.org/10.1111/gfs.12197
Nadeau, E., D. O. de Sousa D, A. Magnusson, S. Hedlund, W. Richardt, & P. Nørgaard. 2019. Digestibility and protein utilization in wethers fed whole-crop barley or grass silages harvested at different maturity stages, with or without protein supplementation. J. Anim. Sci. 97:2188–2201. https://doi.org/10.1093/jas/skz076
Newbold, C. J., G. De la Fuente, A. Belanche, E. Ramos-Morales, & N. R. McEwan. 2015. The role of ciliate protozoa in the rumen. Front. Microbiol. 6:1–14. https://doi.org/10.3389/fmicb.2015.01313
NRC. 2001. Nutrient Requirements of Dairy Cattle: Seventh Revised Edition. Seventh Re. National Academy Press, Washington DC.
Ogimoto, K. & S. Imai. 1981. Atlas of Rumen Microbiology. Japan Scientific Societies Press, Tokyo.
Owens, F. N. & M. Basalan. 2016. Rumenology: Ruminal Fermentation. In: Rumenology. Springer International Publishing Switzerland. p. 63–102. https://doi.org/10.1007/978-3-319-30533-2_3
Putri, E. M., M. Zain, L. Warly, & H. Hermon. 2021. Effects of rumen-degradable-to-undegradable protein ratio in ruminant diet on in vitro digestibility, rumen fermentation, and microbial protein synthesis. Vet. World 14:640–648. https://doi.org/10.14202/vetworld.2021.640-648
Raffrenato, E., R. Fievisohn, K. W. Cotanch, R. J. Grant, L. E. Chase, & M. E. Van Amburgh. 2017. Effect of lignin linkages with other plant cell wall components on in vitro and in vivo neutral detergent fiber digestibility and rate of digestion of grass forages. J. Dairy Sci. 100:8119–8131. https://doi.org/10.3168/jds.2016-12364
Rico, D. E. & K. J. Harvatine. 2013. Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration. J. Dairy Sci. 96:6621–6630. https://doi.org/10.3168/jds.2013-6820
Riestanti, L. U., Despal, & Y. Retnani. 2021. Supplementation of prill fat derived from palm oil on nutrient digestibility and dairy cow performance. Am. J. Anim. Vet. Sci. 16:172–184. https://doi.org/10.3844/ajavsp.2021.172.184
Rira, M., D. P. Morgavi, H. Archimède, C. Marie-Magdeleine, M. Popova, H. Bousseboua, & M. Doreau. 2015. Potential of tannin-rich plants for modulating ruminal microbes and ruminal fermentation in sheep. J. Anim. Sci. 93:334–347. https://doi.org/10.2527/jas.2014-7961
Serafeimidou, A., S. Zlatanos, G. Kritikos, & A. Tourianis. 2013. Change of fatty acid profile, including conjugated linoleic acid (CLA) content, during refrigerated storage of yogurt made of cow and sheep milk. J. Food Compost. Anal. 31:24–30. https://doi.org/10.1016/j.jfca.2013.02.011
Silveira, M. L. & M. M. Kohmann. 2020. Maintaining Soil Fertility and Health for Sustainable Pastures. Elsevier Inc. https://doi.org/10.1016/B978-0-12-814474-9.00003-7
Temple, N. J. 2022. A rational definition for functional foods: A perspective. Front. Nutr. 9. https://doi.org/10.3389/fnut.2022.957516
Tian, X., X. Wang, J. Li, Q. Luo, C. Ban, & Q. Lu. 2022. The effects of selenium on rumen fermentation parameters and microbial metagenome in goats. Fermentation 8:240. https://doi.org/10.3390/fermentation8050240
Tilley, J. M. A. & R. A. Terry. 1963. A two‐stage technique for the in vitro digestion of forage crops. Grass Forage Sci. 18:104–111. https://doi.org/10.1111/j.1365-2494.1963.tb00335.x
Tucak, M., M. Ravlić, D. Horvat, & T. Čupić. 2021. Improvement of forage nutritive quality of alfalfa and red clover through plant breeding. Agronomy 11:1–9. https://doi.org/10.3390/agronomy11112176
Toral, P. G., F. J. Monahan, G. Hervas, P. Frutos, & A. P. Moloney. 2018. Review: Modulating ruminal lipid metabolism to improve the fatty acid composition of meat and milk. challenges and opportunities. Animal 12:S272–S281. https://doi.org/10.1017/S1751731118001994
Vargas, J. A. C. & M. O. Angel. 2021. A protocol for the study of the rumen biohydrogenation of unsaturated fatty acids of lipid supplements mixed with forages using an in vitro approach. 08 July 2021, PROTOCOL (Version 1) available at Protocol Exchange. https://doi.org/10.21203/rs.3.pex-1566/v1
Vidal, A. K. F., T. da C. Barbé, R. F. Daher, J. E. A. Filho, R. S. Nunes de Lima, R. S. Freitas, D. A Rossi, E. da Silva Oliveira, B. R. da Silva Menezes, G. C. Entringer, W. F. S. Peixoto, & S. Cassaro. 2017. Production potential and chemical composition of elephant grass (Pennisetum purpureum Schum.) at different ages for energy purposes. Afr. J. Biotechnol. 16:1428–1433.
Wardeh, M. F. 1981. Models for Estimating Energy and Protein Utilization for Feeds: All Graduate Theses and Dissertations. Utah State University.
Weimer, P. J. 2022. Degradation of cellulose and hemicellulose by ruminal microorganisms. Microorganisms 10:2345. https://doi.org/10.3390/microorganisms10122345
Zhang, Q., S. L. Koser, & S. S. Donkin. 2016. Propionate induces MRNA expression of gluconeogenic genes in bovine calf hepatocytes. J. Dairy Sci. 99:3908–15. https://doi.org/10.3168/jds.2015-10312
Zhang, Y., Y. H. Lee, K. M. Nogoy, C. W. Choi, D. H. Kim, X. Z. Li, & S. H. Choi. 2019. Effect of different harvesting times on the nutritive value and fermentation characteristics of late and early-maturing forage oats by rumen microbes. Korean Journal Agricultural Science 46:125–135.
Zongo, K., S. Krishnamoorthy, J. A. Moses, F. Yazici, A. H. Çon, & C. Anandharamakrishnan. 2021. Total conjugated linoleic acid content of ruminant milk: The world status insights. Food Chem. 334:127555. https://doi.org/10.1016/j.foodchem.2020.127555


D. Anzhany
T. Toharmat
ttoharmat61@gmail.com (Primary Contact)
A. Łozicki
Author Biography

D. Anzhany, IPB University

Study Program Nutrition and Feed Science, Departement of Nutrition and Feed Technology, Faculty of Animal Science, IPB University

AnzhanyD., ToharmatT., Despal, & Łozicki A. (2024). Fatty Acid Biohydrogenation, Fermentation, and Digestibility of Ration Containing Napier and King Grass with Different Harvest Ages and Altitudes: In Vitro Study. Tropical Animal Science Journal, 47(1), 68-78. https://doi.org/10.5398/tasj.2024.47.1.68

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