Slides
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Exploring flexibilities in protein nutrition for a sustainable dairy industry Kelly Nichols, PhD August 5th, 2025 Balchem – Real Science Lecture Series
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The Nichols Lab Acknowledgements
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Protein nutrition intersects with sustainability concerns NH3 N2O N runoff NO3 - FEED PROTEIN MILK PROTEIN MANURE N Feces Urine Imported feed is the largest N input to whole-farm N balance • Approximately 2x the volume of fertilizer N Dutch dairies: Jarvis et al. 2011; The European Nitrogen Assessment USA dairies: Ros et al. 2023; J. Dairy Sci. 106:3268-3286
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The “Nitrogen Crisis”: Not just a European problem
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The “Nitrogen Crisis”: Not just a European problem Roy et al. 2021; Environ. Res. Lett. 16:035004 Probability that groundwater systems violate nitrate regulations Top 20 US regions with the greatest cropland N surplus Pennino et al. 2020; Sci. Total Environ. 722:137661
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Growing emphasis on N discharge regulations and N-source air pollution is likely to continue in agriculture-intensive areas of the US Resource availability cost of protein-rich ingredients, variable global markets, public perception (e.g., feed vs. food debate, GMO) Understanding metabolic flexibility in ruminants determine strategies for improved protein efficiency that align with goals and requirements of producers Protein nutrition intersects with sustainability concerns Ruminants deliver efficiencies and inefficiencies with their transfer of dietary N into human-edible protein • How to deal with manure N surplus? reduce manure N content
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Exploring flexibilities in protein nutrition RUMEN POST- ABSORPTIVE MANURE N FEED PROTEIN MILK PROTEIN POST-RUMEN
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Exploring flexibilities in protein nutrition 1) Impact of energy source 2) Rumen N balance 3) Mammary gland amino acid metabolism
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Exploring flexibilities in protein nutrition 1) Impact of energy source 2) Rumen N balance 3) Mammary gland amino acid metabolism
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Metabolizable protein efficiency is related to energy supply n = 825 -20 MJ/d -10 MJ/d 0 MJ/d 10 MJ/d 20 MJ/d Energy-expensive protein synthesis Glucogenic potential of amino acids Energy-induced endocrine signaling regulates protein synthesis What about the source of this energy? Daniel et al. 2016; Animal 10:1975-1985 Metabolizable protein supply (g/d)
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Energy source affects the transfer of dietary N into milk N LIPOGENIC • Rumen-inert fat supplements (Ca-salts, saturated long-chain fatty acids) • Fiber-rich feedstuffs acetate, butyrate GLUCOGENIC • Post-ruminal glucose • Ruminal propionate • Starch-rich feedstuffs • Post-ruminal infusion of fat or fatty acids
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Energy source affects the transfer of dietary N into milk N 32.3% 30.5% 28.6% 24.3% Cantalapiedra-Hijar et al. 2014; Animal 8:275-285 GLUCOGENIC LIPOGENIC 12% CP - starch 12% CP - fiber 16.5% CP - fiber 16.5% CP - starch
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0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34 LP-LF LP-HF HP-LF HP-HF Milk N/N Intake 0.28 0.29 0.30 0.31 0.32 0.33 0.34 0.35 0.36 L-C L-GG L-LG H-C H-GG H-LG Milk N/N Intake LF HF HFLFC GG LG C GG LG PGG < 0.01 ↑ milk N efficiency with glucogenic energy Low MP High MP Low protein High protein PPT x FT = 0.07 Nichols et al. 2018; J. Dairy Sci. 101:7857-7870 Nichols et al. 2019; J. Dairy Sci. 102:395-412 Energy source affects the transfer of dietary N into milk N
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0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34 LP-LF LP-HF HP-LF HP-HF Milk N/N Intake 0.28 0.29 0.30 0.31 0.32 0.33 0.34 0.35 0.36 L-C L-GG L-LG H-C H-GG H-LG Milk N/N Intake LF HF HFLFC GG LG C GG LG Milk N efficiency tends to ↑ with fat supplementation at low protein levels Low MP High MP Low protein High protein PPT x FT = 0.07 Nichols et al. 2018; J. Dairy Sci. 101:7857-7870 Nichols et al. 2019; J. Dairy Sci. 102:395-412 Energy source affects the transfer of dietary N into milk N 0.324 0.342 0.307 0.307
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Glucogenic energy stimulates partitioning of branched-chain AA (BCAA) towards extra-mammary peripheral tissues Lipogenic energy (saturated LCFA) does not affect AA partitioning ↑ [glucose] extra-mammary lipogenesis acetate, BHB insulin+ + BCAA muscle+ ↓ [BCAA] ↓ [acetate, BHB] ↑ mammary blood flow Maintained milk protein + fat output Energy type affects amino acid partitioning Nichols et al. 2019; J. Dairy Sci. 102:1160-1175 Nichols et al. 2019; J. Dairy Sci. 102:7150-7167 Lemosquet et al. 2009; J. Dairy Sci. 92:6068-6082 Nichols et al. 2016; J. Dairy Sci. 99:1145-1160 Curtis et al. 2018; J. Dairy Sci. 101:4542-4553
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Summary: Impact of energy source on protein efficiency • Stimulates insulin cascade re-partitioning of BCAA and energy metabolites towards extra-mammary peripheral tissues (muscle and adipose) = ↑ mammary blood flow • Positive responses in milk N efficiency at low protein levels more research needed • No insulin response no AA re-partitioning • Stimulates milk protein yield and ↑ N efficiency Consideration for dietary ingredient inclusion, especially novel ingredients or byproducts does this offer more glucogenic or lipogenic energy, and what might the implication be on protein metabolism? GLUCOGENIC LIPOGENIC
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Exploring flexibilities in protein nutrition 1) Impact of energy source 2) Rumen protein balance 3) Mammary gland amino acid metabolism
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Refining rumen protein balance Urinary N, g/kg BW Rumen protein balance, g N/kg BW Y = 0,22 + 0,79 X (NTREAT = 251, NEXP = 62, RMSE = 0,03) Sauvant et al. 2015; INRA Productions Animales: 347-368 79% of additional rumen available N = excreted in urine Everything >0 is excess Rumen protein balance: difference between maximum possible microbial protein synthesis from rumen-available N and energy
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Refining rumen protein balance Diet formulation parameters are dimensioned to achieve ruminal synchrony between protein and energy supply In practice, may lead to excessive safety margins for rumen N supply • Assumes N metabolism, N recycling, and N utilization are constant • Ignores contribution from endogenous N Nichols et al. 2022; Animal. 16:100537
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Refining rumen protein balance Base + Met Base + Met + Urea P-value Rumen N requirement* 0.83 0.96 - CP , g/kg DM 136 146 - Calculated based on bacterial growth depression % from CNCPS v7 estimate based on ruminal nitrogen requirements given the number of bacteria that can be produced from the fermentable carbohydrate Higgs et al. 2023; J. Dairy Sci. 106:1826-1836 • Both diets deficient in MP relative to estimated requirements • CNCPS v.7 does account for endogenous urea-N recycling *
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Refining rumen protein balance Base + Met Base + Met + Urea P-value Rumen N requirement* 0.83 0.96 - CP , g/kg DM 136 146 - DM intake, kg/d 24.1 24.7 0.05 ≤ P < 0.10 ECM yield, kg/d 38.7 39.4 > 0.05 Milk true protein, g/kg 29.5 29.7 > 0.05 Milk true protein, kg/d 1.14 1.14 > 0.05 Higgs et al. 2023; J. Dairy Sci. 106:1826-1836
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Refining rumen protein balance Base + Met Base + Met + Urea P-value Rumen N requirement* 0.83 0.96 - CP , g/kg DM 136 146 - DM intake, kg/d 24.1 24.7 0.05 ≤ P < 0.10 ECM yield, kg/d 38.7 39.4 > 0.05 Milk true protein, g/kg 29.5 29.7 > 0.05 Milk true protein, kg/d 1.14 1.14 > 0.05 Milk urea N, mg/dL 7.1 8.6 < 0.05 Milk N efficiency, % 35.4 32.3 - Plasma urea N, mg/dL 5.7 8.5 < 0.05 Predicted urine N, g/d 130 170 < 0.05 NDF digestibility, % 40.1 42.4 > 0.05 Higgs et al. 2023; J. Dairy Sci. 106:1826-1836
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Refining rumen protein balance Base + Met Base + Met + Urea P-value Rumen N requirement* 0.83 0.96 - CP , g/kg DM 136 146 - DM intake, kg/d 24.1 24.7 0.05 ≤ P < 0.10 ECM yield, kg/d 38.7 39.4 > 0.05 Milk true protein, g/kg 29.5 29.7 > 0.05 Milk true protein, kg/d 1.14 1.14 > 0.05 Milk urea N, mg/dL 7.1 8.6 < 0.05 Milk N efficiency, % 35.4 32.3 - Plasma urea N, mg/dL 5.7 8.5 < 0.05 Predicted urine N, g/d 130 170 < 0.05 NDF digestibility, % 40.1 42.4 > 0.05 If trying to reduce N excretion, do we need to supplement extra RDP? Balancing for estimated rumen N requirements by adding urea (likely) ↑ N excretion with no positive impact on ECM yield, milk protein content, or milk protein yield Higgs et al. 2023; J. Dairy Sci. 106:1826-1836
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Source of protein supply between RUP and RDP and the balance between them matters more as dietary CP content ↓ Can we rely on endogenous urea to compensate? Mutsvangwa et al. 2016; J. Dairy Sci. 99:6298-6310 Chibisa and Mutsvangwa, 2013; J. Dairy Sci. 96:6550-6563 What are the underlying mechanisms that may be inhibiting greater return to the GI tract of endogenous urea? RUMEN LIVER KIDNEY Urea recycled to gastrointestinal tract Urea synthesis URINE FECES INTESTINE Absorbed ammonia and amino acids from gastrointestinal tract Proportion of hepatic urea output that recycles to the GIT ↑ as dietary CP content ↓
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Summary: Reconsidering rumen protein balance • In practice, assess the need for excess rumen N supply (e.g., highly positive rumen protein balance) for a given diet Cattle can use endogenous N to support short-term periods of ruminal N insufficiency may be able to ↓ safety margin on rumen-available N Lowering ruminal N supply is a route to reducing dietary CP content and N excretion Deeper understanding of urea recycling mechanism in lactating dairy cattle will enhance opportunities to rely on this mechanism to improve N efficiency
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Exploring flexibilities in protein nutrition 1) Impact of energy source 2) Rumen protein balance 3) Mammary gland amino acid metabolism
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Digestible amino acid profile RUMEN • rumen undegradable (RUP) • microbial SMALL INTESTINE But what is the complementary profile? • RUP this can be changed based on feed ingredients Optimizing N efficiency = aiming for RUP AA profile that complements microbial AA flow • Microbial CP assume a fixed AA profile
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Absorbed amino acid profile affects marginal efficiency Post-ruminal profile (infused or rumen-protected) AA dose (g/d) Marginal efficiency Reference Complete essential AA (EAA) 359 0.31 Doepel and Lapierre, 2010 J. Dairy Sci. 93:3264-3274Complete non-EAA (NEAA) 356 0.03 EAA+NEAA 715 0.25
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Absorbed amino acid profile affects marginal efficiency Post-ruminal profile (infused or rumen-protected) AA dose (g/d) Marginal efficiency Reference Complete essential AA (EAA) 359 0.31 Doepel and Lapierre, 2010 J. Dairy Sci. 93:3264-3274Complete non-EAA (NEAA) 356 0.03 EAA+NEAA 715 0.25 Complete EAA 562 0.35 Nichols et al., 2019 J. Dairy Sci. 102:8963-8976Group 1 AA+Ile+Leu+Val 562 0.28 Group 1 AA+Arg+Lys+Thr 562 0.18 Ile+Leu+Val 562 0.12 *Group 1 = His, Met, Phe, Trp
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Absorbed amino acid profile affects marginal efficiency Post-ruminal profile (infused or rumen-protected) AA dose (g/d) Marginal efficiency Reference Complete essential AA (EAA) 359 0.31 Doepel and Lapierre, 2010 J. Dairy Sci. 93:3264-3274Complete non-EAA (NEAA) 356 0.03 EAA+NEAA 715 0.25 Complete EAA 562 0.35 Nichols et al., 2019 J. Dairy Sci. 102:8963-8976Group 1 AA+Ile+Leu+Val 562 0.28 Group 1 AA+Arg+Lys+Thr 562 0.18 Ile+Leu+Val 562 0.12 *Group 1 = His, Met, Phe, Trp
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Absorbed amino acid profile affects marginal efficiency Post-ruminal profile (infused or rumen-protected) AA dose (g/d) Marginal efficiency Reference Complete essential AA (EAA) 359 0.31 Doepel and Lapierre, 2010 J. Dairy Sci. 93:3264-3274Complete non-EAA (NEAA) 356 0.03 EAA+NEAA 715 0.25 Complete EAA 562 0.35 Nichols et al., 2019 J. Dairy Sci. 102:8963-8976Group 1 AA+Ile+Leu+Val 562 0.28 Group 1 AA+Arg+Lys+Thr 562 0.18 Ile+Leu+Val 562 0.12 His+Met+Lys 101 0.67 Nichols et al., 2024 J. Dairy Sci. 107:6797-6816soybean meal + rapeseed meal (EAA+NEAA) 101 0.17
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Absorbed amino acid profile affects marginal efficiency Post-ruminal profile (infused or rumen-protected) AA dose (g/d) Marginal efficiency Reference Complete essential AA (EAA) 359 0.31 Doepel and Lapierre, 2010 J. Dairy Sci. 93:3264-3274Complete NEAA 356 0.03 EAA+NEAA 715 0.25 Complete EAA 562 0.35 Nichols et al., 2019 J. Dairy Sci. 102:8963-8976Group 1 AA+Ile+Leu+Val 562 0.28 Group 1 AA+Arg+Lys+Thr 562 0.18 Ile+Leu+Val 562 0.12 His+Met+Lys 101 0.67 Nichols et al., 2024 J. Dairy Sci. 107:6797-6816soybean meal + rapeseed meal (EAA+NEAA) 101 0.17 Marginal efficiency ↑ as digestible AA profile more closely resembles casein Greater efficiency if EAA supply is prioritized within this profile
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Flexibility in mammary gland amino acid metabolism 400 600 800 1000 1200 SAL EAAC ILV Gr1+ILV Gr1+ALT g/d a b a b abP < 0.01 Group 1 ILV ALT His, Met, Phe, Trp Ile, Leu, Val Arg, Lys, Thr ↑ milk protein yield with incomplete EAA profiles when group 1 AA are present Milk Protein Yield Nichols et al. 2019; J. Dairy Sci. 102:8963-8976
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Flexibility in mammary gland amino acid metabolism ARTERYVEIN AA UPTAKE = sequestration (irreversible loss) of AA MILK PROTEIN ~90% structural proteinTRANSFORMATION oxidation galactose fatty acids
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Mammary gland adaptation to essential AA profiles Blood flow altered by changes in vasodilatory systems and intramammary metabolite and energy balance • AA deficiency and imbalance ↑ mammary blood flow 0 200 400 600 800 1000 1200 SAL EAAC −HIS GLC EAAC+GLC −HIS+GLC Plasma flow, L/h His deletion ↑ mammary plasma flow P-HIS = 0.05 Doelman et al., in preparation
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Mammary gland adaptation to essential AA profiles 400 500 600 700 800 900 1000 1100 SAL EAAC ILV Gr1+ILV Gr1+ALT Plasma flow, L/h ↑ plasma flow with greater imbalance in EAA profile P = 0.04 ab ab b a b Blood flow altered by changes in vasodilatory systems and intramammary metabolite and energy balance • AA deficiency and imbalance ↑ mammary blood flow Nichols et al. 2022; J. Dairy Sci. 105:7354-7372
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Intramammary flexibility Group 1 PROTEIN Ile, Leu, Val Arg, Lys, Thr Group 1 1:1 >1:1 >1:1 MAMMARY CELL NEAAILV ALT Nichols et al. 2022; J. Dairy Sci. 105:7354-7372
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Intramammary flexibility Group 1 PROTEIN Arg, Lys, Thr Group 1 1:1 >1:1 >1:1 MAMMARY CELL NEAAILV ALT N and carbon skeletons from Ile, Leu, Val Nichols et al. 2022; J. Dairy Sci. 105:7354-7372 Ile, Leu, Val
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Group 1 PROTEIN Arg, Lys, Thr Group 1 1:1 >1:1 >1:1 MAMMARY CELL NEAAILV ALT N and carbon skeletons from Arg, Lys, Thr Intramammary flexibility Nichols et al. 2022; J. Dairy Sci. 105:7354-7372 Ile, Leu, Val
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Summary: Mammary amino acid metabolism Consider the profile of EAA groups, not necessarily individuals, when supplementing rumen-undegradable protein in dairy cattle rations The profile of digestible AA will impact the efficiency of use of dietary protein sources • Look for ingredient profiles that enrich digestible protein with EAA that more closely match the AA profile of casein Intramammary compensation for N and carbon between non-group 1 EAA supports milk protein synthesis when absorbed EAA profile is incomplete • What is the scope of this flexibility?
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The mammary gland is flexible in its use of AA Harnessing flexibility: Take-home messages Minimize rumen degradable protein balance Energy source matters with respect to improving protein efficiency • To a degree, recycling of endogenous urea can compensate for periods of low rumen N supply utility is overlooked, and the dynamics are not well understood • Glucogenic and lipogenic energy elicit different effects on postabsorptive AA metabolism but both can improve N efficiency • Can achieve moderate increases in milk protein efficiency, and substantial decreases in urinary N excretion • Digestible profile of AA groups should become a focus alongside balancing for individual AA