Effect of amino acid supplementation and choline chloride for low protein diet on nitrogen efficiency and methane emission of dairy cows

Shahram Shirmohammadi, Akbar Taghizadeh, Ali Hosseinkhani, Hossein Janmohammadi, Rasoul Pirmohammadi, Hadi Valizadeh


Ruminants are one of the largest anthropogenic methane and nitrous oxide emissions. Therefore, the hypothesis was to study the effects of reducing dietary crude protein (CP) level on environmental contaminators when rumen-protected amino acids and choline chloride were supplemented. Sixty Holstein dairy cows were used during the experiment. Test diets were: (1) CD = Control diet with16.2 g of crude protein/ Kg of DM); (2) LM = Low protein diet with 14.2 g of crude protein/ Kg of DM + methionine ; (3) LL = Low protein diet with 14.2 g of crude protein/ Kg of DM + lysine; (4) LML = Low protein diet with 14.2 g of crude protein/ Kg of DM + methionine + lysine; (5) LMLC = Low protein diet with 14.2 g of crude protein/ Kg of DM + methionine + lysine + choline. Dry matter and NDF intake were not different, but the control group received higher CP and ADF compared with other groups (P < 0.05). Fecal CP and ADF of control group were lower (P < 0.05), but no differences were observed for fecal dry matter (DM) and NDF. Milk yield and protein content were higher for LML and LMLC like control group (P < 0.05). Nitrogen intake, urinary N, urinary urea N and total excreta N decreased (P < 0.05) when animals fed low protein. There was no difference in ruminal pH and acetate to propionate ratio, whereas the ruminal ammonia-N decreased with the low protein (P < 0.05). The 120-h gas production test, showed no difference on the kinetics of digestion and in vitro methane emission. However, the inclusion of DMI in the calculations revealed that low protein can reduce (P < 0.05) methane emission. Overall, our findings indicated that low protein can be compensated for by adding rumen-protected amino acids, not only to maintain the animal performance, but also to decrease nitrogen excretion and methane emission.


Crude protein; Environmental; Gas production; Methane; Nitrogen retention; Ruminants.

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Adejoro, F. A., Hassen, A., Akanmu, A. M., & Morgavi, D. P. (2020). Replacing urea with nitrate as a non-protein nitrogen source increases lambs' growth and reduces methane production, whereas acacia tannin has no effect. Animal Feed Science and Technology, 259, 114360. doi: 10.1016/j.anifeedsci.2019.114360

Amanlou, H., Farahani, T. A., & Farsuni, N. E. (2017). Effects of rumen undegradable protein supplementation on productive performance and indicators of protein and energy metabolism in Holstein fresh cows. Journal of Dairy Science, 100(5), 3628-3640. doi: 10.3168/jds.2016-11794

AOAC. (2005). AOAC international guidelines for laboratories performing microbiological and chemical analyses of food and pharmaceuticals: An aid to interpretation of ISO/IEC 17025. Rockville, MD: AOAC International.

Broderick, G., & Kang, J. (1980). Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. Journal of Dairy Science, 63(1), 64-75. doi: 10.3168/jds.S0022-0302 (80)82888-8

Broderick, G., Stevenson, M., Patton, R., Lobos, N., & Colmenero, J. O. (2008). Effect of supplementing rumen-protected methionine on production and nitrogen excretion in lactating dairy cows. Journal of Dairy Science, 91(3), 1092-1102. doi: 10.3168/jds.2007-0769

Buddle, B. M., Denis, M., Attwood, G. T., Altermann, E., Janssen, P. H., Ronimus, R. S.,… Wedlock, D. N. (2011). Strategies to reduce methane emissions from farmed ruminants grazing on pasture. The Veterinary Journal, 188(1), 11-17. doi: 10.1016/j.tvjl.2010.02.019

Dijkstra, J., Bannink, A., Bosma, P. M., Lantinga, E. A., & Reijs, J. W. (2018). Modeling the effect of nutritional strategies for dairy cows on the composition of excreta nitrogen. Frontiers in Sustainable Food Systems, 2, 63. doi: 10.3389/fsufs.2018.00063

Fedorah, P. M., & Hrudey, S. E. (1983). A simple apparatus for measuring gas production by methanogenic cultures in serum bottles. Environmental Technology, 4(10), 425-432. doi: 10.1080/09593338309384228

Geishauser, T., Linhart, N., Neidl, A., & Reimann, A. (2012). Factors associated with ruminal pH at herd level. Journal of Dairy Science, 95(8), 4556-4567. doi: 10.3168/jds.2012-5380

Giallongo, F., Harper, M., Oh, J., Lopes, J., Lapierre, H., Patton, R.,… Hristov, A. N. (2016). Effects of rumen protected methionine, lysine, and histidine on lactation performance of dairy cows. Journal of Dairy Science, 99(6), 4437-4452. doi: 10.3168/jds.2015-10822

Gill, M., Smith, P., & Wilkinson, J. (2010). Mitigating climate change: the role of domestic livestock. Animal, 4(3), 323-333. doi: 10.1017/S1751731109004662

Gomez, A., Mendoza, G. D., Garcìa-Bojalil, C., Barcena, R., Ramos, J. A., Crosby, M. M.,… Lara, A. (2011). Effect of supplementation with urea, blood meal, and rumen-protected methionine on growth performance of Holstein heifers grazing kikuyu pasture. Tropical Animal Health and Production, 43(3), 721-724. doi: 10.1007/s11250-010-9759-z

Greening, C., Geier, R., Wang, C., Woods, L. C., Morales, S. E., McDonald, M. J.,… Leahy, S. C. (2019). Diverse hydrogen production and consumption pathways influence methane production in ruminants. The ISME Journal, 13, 2617-2632. doi: 10.1038/s41396-019-0464-2

Hamamoto, T., Uchida, Y., von Rein, I., & Mukumbuta, I. (2020). Effects of short term freezing on nitrous oxide emissions and enzyme activities in a grazed pasture soil after bovine-urine application. Science of the Total Environment, (740), 140006. doi: 10.1016/j.scitotenv.2020.140006

Henderson, G., Cook, G. M., & Ronimus, R. S. (2018). Enzyme and gene based approaches for developing methanogen specific compounds to control ruminant methane emissions: a review. Animal Production Science, 58(6), 1017-1026. doi: 10.1071/AN15757

Henderson, G., Cox, F., Ganesh, S., Jonker, A., Young, W., Collaborators, G. R. C.,… Arenas, G. N. (2015). Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range. Scientific Reports, 5(1), 14567. doi: 10.1038/srep14567

Hou, Y., Velthof, G. L., & Oenema, O. (2015). Mitigation of ammonia, nitrous oxide and methane emissions from manure management chains: a meta analysis and integrated assessment. Global Change Biology, 21(3), 1293-1312. doi: 10.1111/gcb.12767

Hristov, A. N., & Melgar, A. (2020). Relationship of dry matter intake with enteric methane emission measured with the GreenFeed system in dairy cows receiving a diet without or with 3 nitrooxypropanol. Animal, 14(S3), s484-s490. doi: 10.1017/S1751731120001731

Hristov, A. N., Bannink, A., Crompton, L. A., Huhtanen, P., Kreuzer, M., McGee, M.,… Yanez-Ruiz, D. R. (2019). Invited review: nitrogen in ruminant nutrition: a review of measurement techniques. Journal of Dairy Science, 102(7), 5811-5852. doi: 10.3168/jds.2018-15829

Hristov, A. N., Oh, J., Giallongo, F., Frederick, T. W., Harper, M. T., Weeks, H. L.,… Williams, S. R. O. (2015). An inhibitor persistently decreased enteric methane emission from dairy cows with no negative effect on milk production. Proceedings of the National Academy of Sciences, 112(34), 10663-10668. doi: 10.1073/pnas.1504124112

Huhtanen, P., & Hetta, M. (2012). Comparison of feed intake and milk production responses in continuous and change over design dairy cow experiments. Livestock Science, 143(2-3), 184-194. doi: 10.1016/j. livsci.2011.09.012

Huhtanen, P., & Hristov, A. N. (2009). A meta-analysis of the effects of dietary protein concentration and degradability on milk protein yield and milk N efficiency in dairy cows. Journal of Dairy Science, 92(7), 3222-3232. doi: 10.3168/jds.2008-1352

Hynes, D. N., Stergiadis, S., Gordon, A., & Yan, T. (2016). Effects of concentrate crude protein content on nutrient digestibility, energy utilization, and methane emissions in lactating dairy cows fed fresh cut perennial grass. Journal of Dairy Science, 99(11), 8858-8866. doi: 10.3168/jds.2016-11509.

I.C.O.A.C. (1995). Guide to the care and use of experimental animals. Isfahan, Iran: Isfahan University of Technology Isfahan.

Jarrell, K. F., & Kalmokoff, M. L. (1988). Nutritional requirements of the methanogenic archaebacteria. Canadian Journal of Microbiology, 34(5), 557-576. doi: 10.1139/m88-095

Jenkins, C., Fernando, S., Anderson, C., Aluthge, N., Castillo-Lopez, E., Zanton, G., & Kononoff, P. (2020). The effects of 2 hydroxy 4 methylthio butanoic acid supplementation on the rumen microbial population and duodenal flow of microbial nitrogen. Journal of Dairy Science, 103(11), 10161-10174. doi: 10.3168/ jds.2019-17664

Johnson, K. A., & Johnson, D. E. (1995). Methane emissions from cattle. Journal of Animal Science, 73(8), 2483-2492. doi: 10.2527/1995.7382483x

Kalscheur, K., Vi, R. B., Glenn, B., & Kohn, R. (2006). Milk production of dairy cows fed differing concentrations of rumen degraded protein. Journal of Dairy Science, 89(1), 249-259. doi: 10.3168/jds. S0022-0302(06)72089-6

Kelly, W. J., Leahy, S. C., Li, D., Perry, R., Lambie, S. C., Attwood, G. T., & Altermann, E. (2014). The complete genome sequence of the rumen methanogen Methanobacterium formicicum BRM9. Standards in Genomic Sciences, 9(1), 15. doi: 10.1186/1944-3277-9-15

Kiggundu, M., Nantongo, Z., Kayondo, S. I., & Mugerwa, S. (2019). Enteric methane emissions of grazing short horn zebu weaner bulls vary with estimation method and level of crude protein supplementation. Tropical Animal Health and Production, 52, 1269-1276. doi: 10.1007/s11250-019-02127-2.

Lambie, S. C., Kelly, W. J., Leahy, S. C., Li, D., Reilly, K., McAllister, T. A.,… Altermann, E. (2015). The complete genome sequence of the rumen methanogen Methanosarcina barkeri CM1. Standards in Genomic Sciences, 10(1), 57. doi: 10.1186/s40793-015-0038-5

Larsen, M., Hansen, N. P., Weisbjerg, M. R., & Lund, P. (2020). Evaluation of the ororuminal FLORA sampling device for rumen fluid sampling in intact cattle. Journal of Dairy Science, 103(1), 447-450. doi: 10.3168/jds.2019-16972

Leahy, S. C., Kelly, W. J., Altermann, E., Ronimus, R. S., Yeoman, C. J., Pacheco, D. M.,… Sang, C. (2010). The genome sequence of the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions. PloS one, 5(1), e8926. doi: 10.1371/journal. pone.0008926

Lee, C., Hristov, A. N., Cassidy, T., Heyler, K., Lapierre, H., Varga, G.,… Parys, C. (2012a). Rumen protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet. Journal of Dairy Science, 95(10), 6042-6056. doi: 10.3168/jds.2012-5581

Lee, C., Hristov, A. N., Heyler, K., Cassidy, T., Lapierre, H., Varga, G., & Parys, C. (2012b). Effects of metabolizable protein supply and amino acid supplementation on nitrogen utilization, milk production, and ammonia emissions from manure in dairy cows. Journal of Dairy Science, 95(9), 5253-5268. doi: 10. 3168/jds.2012-5366

Lee, C., Hristov, A. N., Heyler, K., Cassidy, T., Long, M., Corl, B., & Karnati, S. (2011). Effects of dietary protein concentration and coconut oil supplementation on nitrogen utilization and production in dairy cows. Journal of Dairy Science, 94(11), 5544-5557. doi: 10.3168/jds.2010-3889

Li, Y., Leahy, S. C., Jeyanathan, J., Henderson, G., Cox, F., Altermann, E.,… Rakonjac, J. (2016). The complete genome sequence of the methanogenic archaeon ISO4-H5 provides insights into the methylotrophic lifestyle of a ruminal representative of the Methanomassiliicoccales. Standards in Genomic Sciences, 11(1), 59. doi: 10.1186/s40793-016-0183-5

Lutakome, P., Kabi, F., Tibayungwa, F., Laswai, G. H., Kimambo, A., & Ebong, C. (2017). Rumen liquor from slaughtered cattle as inoculum for feed evaluation. Animal Nutrition, 3(3), 300-308. doi: 10.1016/j. aninu.2017.06.010

Martin, C., Morgavi, D., & Doreau, M. (2010). Methane mitigation in ruminants: from microbe to the farm scale. Animal, 4(3), 351-365. doi: 10.1017/S1751731109990620

McDougall, E. (1948). Studies on ruminant saliva. 1. The composition and output of sheep's saliva. Biochemical Journal, 43(1), 99. doi: 10.1042/bj0430099

Moss, A. R., Jouany, J.-P., & Newbold, J. (2000). Methane production by ruminants: its contribution to global warming. Annales de Zootechnie, 49(2000) 231-253. doi: 10.1051/animres:2000119.

National Research Council (2001). Nutrient requirements of dairy cattle (7nd rev. ed.). Washington, DC: National Academy Press.

Noftsger, S., St-Pierre, N., & Sylvester, J. (2005). Determination of rumen degradability and ruminal effects of three sources of methionine in lactating cows. Journal of Dairy Science, 88(1), 223-237. doi: 10.3168/ jds.S0022-0302(05)72680-1

Rehman, A., Arif, M., Saeed, M., Manan, A., Al-Sagheer, A., El-Hack, M. E.,… Alowaimer, A. N. (2020). Nutrient digestibility, nitrogen excretion, and milk production of mid lactation Jersey× Friesian cows fed diets containing different proportions of rumen undegradable protein. Anais da Academia Brasileira de Ciências, 92(Suppl. 1), 1-13. doi: 10.1590/0001-3765202020180787

Russell, J. B., O'connor, J., Fox, D., Van Soest, P., & Sniffen, C. (1992). A net carbohydrate and protein system for evaluating cattle diets: I. Ruminal fermentation. Journal of Animal Science, 70(11), 3551-3561. doi: 10.2527/1992.70113551x

Satter, L., & Slyter, L. (1974). Effect of ammonia concentration on rumen microbial protein production in vitro. British Journal of Nutrition, 32(2), 199-208. doi: 10.1079/BJN19740073

Serrano, R. D. C., Cruz, O. T. B., Coneglian, S. M., & Branco, A. F. (2020). Use of cashew and castor essential oils to improve fibre digestibility in high forage diets: digestibility, ruminal fermentation and microbial protein synthesis. Semina: Ciências Agrárias, 41(6, Suppl. 2), 3429-3440. doi: 10.5433/1679-0359.2020 v41n6Supl2p3429

Seshadri, R., Leahy, S. C., Attwood, G. T., Teh, K. H., Lambie, S. C., Cookson, A. L.,… Varghese, N. J. (2018). Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection. Nature Biotechnology, 36(4), 359. doi: 10.1038/nbt.4110

Shirmohammadi, S., Taghizadeh, A., Hosseinkhani, A., Moghaddam, G. A., Salem, A. Z., & Pliego, A. B. (2020). Ruminal and post ruminal barley grain digestion and starch granule morphology under three heat methods. Annals of Applied Biology, 178(3), 508-518. doi: 10.1111/aab.12662

Solden, L. M., Naas, A. E., Roux, S., Daly, R. A., Collins, W. B., Nicora, C. D.,… Jørgensen, B. (2018). Interspecies cross-feeding orchestrates carbon degradation in the rumen ecosystem. Nature Microbiology, 3(11), 1274. doi: 10.1038/s41564-018-0225-4

Stewart, R. D., Auffret, M. D., Warr, A., Wiser, A. H., Press, M. O., Langford, K. W.,… Walker, A. W. (2018). Assembly of 913 microbial genomes from metagenomic sequencing of the cow rumen. Nature Communications, 9(1), 870. doi: 10.1038/s41467-018-03317-6

Sun, F., Aguerre, M., & Wattiaux, M. (2019). Starch and dextrose at 2 levels of rumen degradable protein in iso nitrogenous diets: Effects on lactation performance, ruminal measurements, methane emission, digestibility, and nitrogen balance of dairy cows. Journal of Dairy Science, 102(2), 1281-1293. doi: 10. 3168/jds.2018-15041

Swanepoel, N., Robinson, P., & Erasmus, L. (2010). Amino acid needs of lactating dairy cows: impact of feeding lysine in a ruminally protected form on productivity of lactating dairy cows. Animal Feed Science and Technology, 157(1-2), 79-94. doi: 10.1016/j.anifeedsci.2010.02.008.

Thauer, R. K. (2012). The wolfe cycle comes full circle. Proceedings of the National Academy of Sciences, 109(38), 15084-15085. doi: 10.1073/pnas.1213193109

Valadares, R., Broderick, G., Valadares, S., Fº., & Clayton, M. (1999). Effect of replacing alfalfa silage with high moisture corn on ruminal protein synthesis estimated from excretion of total purine derivatives. Journal of Dairy Science, 82(12), 2686-2696. doi: 10.3168/jds.S0022-0302(99)75525-6

van Lingen, H. J., Jonker, A., Kebreab, E., & Pacheco, D. (2021). Quantitative joint evaluation of sheep enteric methane emissions and faecal dry matter and nitrogen excretion. Agriculture, Ecosystems & Environment, 305, 107116. doi: 10.1016/j.agee.2020.107116

Van Soest, P. V., Robertson, J., & Lewis, B. (1991). Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74(10), 3583-3597. doi: 10.3168/jds.S0022-0302(91)78551-2

Van Zanten, H., Meerburg, B., Bikker, P., Herrero, M., & De Boer, I. (2016). Opinion paper: the role of livestock in a sustainable diet: a land-use perspective. Animal, 10(4), 547-549. doi: 10.1017/S175173111 5002694

Wedlock, D., Janssen, P., Leahy, S., Shu, D., & Buddle, B. (2013). Progress in the development of vaccines against rumen methanogens. Animal, 7(Suppl. 2), 244-252. doi: 10.1017/S1751731113000682

Weimar, M., Cheung, J., Dey, D., McSweeney, C., Morrison, M., Kobayashi, Y.,… Ronimus, R. (2017). Development of multiwell plate methods using pure cultures of methanogens to identify new inhibitors for suppressing ruminant methane emissions. Applied and Environmental Microbiology, 83(15), e00396-00317. doi: 10.1128/AEM.00396-17

Wu, P., Liu, Z., He, W., Yu, S., Gao, G., & Wang, J. (2018). Intermittent feeding of citrus essential oils as a potential strategy to decrease methane production by reducing microbial adaptation. Journal of Cleaner Production, 194, 704-713. doi: 10.1016/j.jclepro.2018.05.167

Zenobi, M., Gardinal, R., Zuniga, J., Dias, A., Nelson, C., Driver, J.,… Staples, C. (2018). Effects of supplementation with ruminally protected choline on performance of multiparous Holstein cows did not depend upon prepartum caloric intake. Journal of Dairy Science, 101(2), 1088-1110. doi: 10.3168/jds. 2017-13327

DOI: http://dx.doi.org/10.5433/1679-0359.2022v43n1p159

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