Amino acid metabolism in periparturient dairy cattle

International Dairy Nutrition Symposium October 2017 H. Lapierre, D.R. Ouellet, M. Larsen and L. Doepel Agriculture and Agri-Food Canada Aarhus University Foulum, Denmark Trouw Nutrition, Canada

MP: metabolizable protein Postpartum protein deficiency Fairly little focus despite acknowledged (Bell et al. 2000)

1. AA metabolism post-calving 2. From pre- to post-calving 3. Can we reduce the deficit? AA supplementation 4. Other roles of AA glutamine supplementation 5. Conclusions

AA metabolism

artery portal hepatic liver PDV LIVER POST-LIVER =SPLANCHNIC AA across tissues MPY

1. AA metabolism post-calving 10 cows prepared with splanchnic catheters before parturition 4 Control (post-rumen water infusion) Blood samples collected on DIM 5, 15 and 29

Postpartum protein deficiency (Larsen et al. 2015; NRC 2001)

Postpartum protein deficiency -571-291 -64 (Larsen et al. 2015; NRC 2001)

Lysine net flux (mmol/h) (Larsen et al. 2015)

Lysine net flux (mmol/h) Group 2 AA Ile, Leu, Val (Larsen et al. 2015)

Methionine net flux (mmol/h) (Larsen et al. 2015)

Methionine net flux (mmol/h) Group 1 AA: His, Phe+Tyr, Trp (Larsen et al., 2015)

Methionine net flux (mmol/h) Uptake:output U:O (Larsen et al., 2015)

Mammary uptake : output (Larsen et al. 2014 & 2015)

? Excess uptake is used for what? Leucine: 13 C from 13 C-Leucine was recovered in 13 CO 2 ->oxidation = energy (Raggio et al. 2006) Lysine: 15 N from 15 N-Lysine was recovered into milk non EAA (Asx, Glx,Ser and Ala; Lapierre et al. 2008) EAA: in vitro, labelled AA into milk lactose (Bequette et al. 2006)

NEAA net flux (mmol/h) (Larsen et al. 2015)

1. AA metabolism post-calving DIM 5: post-liver supply of AA clearly insufficient to cover MPY utilization of body proteins DIM 29: post-liver supply of AA MPY «Pattern» similar to established lactation for Groups 1 and 2-AA

2. Pre- vs. post-calving 6 dairy cows Pre-calving: 18 days before calving 1326 g MP/d Post-calving: 21 or 42 DIM 2136 g MP/d 40.2 kg/d milk

Lysine net flux (mmol/h) (Doepel et al. 2009)

Methionine net flux (mmol/h) (Doepel et al. 2009)

Methionine net flux (mmol/h) Established lactation (Raggio et al. 2004)

Methionine net flux (mmol/h) (Doepel et al. 2009)

Methionine net flux (mmol/h) Liver/Portal 0.62 Liver/Portal 0.32 (Doepel et al. 2009)

Liver inflow of AA arterial input + PDV liver

Liver inflow of AA pre-calving PDV + arterial inflow liver

Liver inflow of AA post-calving PDV + arterial inflow liver

Methionine net flux (mmol/h) Liver/Inflow 0.11 Liver/Inflow 0.08 (Doepel et al. 2009)

Non-essential AA net flux (mmol/h) (Doepel et al. 2009)

2. Pre- vs. post-calving The comparison helped to delineate that the liver is not «THE» key control but acts in response to both the supply and utilization of AA by other tissues At the initiation of lactation, the liver «spares» AA: no increment of AA removal AA to support gluconeogenesis

3. Can we reduce the deficit? 2 studies with post-rumen infusion 1 field study

2 studies with post-rumen infusion: «Close-the-gap» strategy Study 1: casein (CN: 720 to 194 g/d) Study 2: free AA, CN profile (791 to 226 g/d) Net release - milk secretion, g/d 300 PDV release TSP release 200 100 0-100 -200-300 n = 18 Larsen & Kristensen, 2012 Raun & Kristensen, 2011 Dalbach et al., 2011 Larsen & Kristensen, 2009a,b 4 15 29 Days relative to parturition Amino acids, g/d 900 800 700 600 500 400 300 200 100 0 Sampling Sampling 0 5 10 15 20 25 30 Days relative to calving Sampling

Milk protein yield (MPY) in response to CN infusion (study 1) Efficiency > 70% (Larsen et al. 2014)

Milk yield in response to AA-CN infusion (study 2) 60 P trt < 0.01, P DIM < 0.01, P trt x DIM = 0.29 kg/d 50 40 30 20 10 0 Efficiency = 45% -15-10 -5 0 5 10 15 20 25 30 Days relative to calving 46.0 ± 0.8 38.2 ± 0.9 7.8 ± 1.3 kg greater with AA-CN Similar to +7.2 kg infusing CN

MP balance in response to AA-CN infusion (study 2) (Larsen et al. 2015)

MP balance in response to AA-CN infusion (study 2) P trt = 0.30 (Larsen et al. 2015)

Group 1-AA net flux (mmol/h) His, Met, Phe+Tyr, Trp (Larsen et al. 2015)

Group 1-AA net flux (mmol/h) * 95% recovery (Larsen et al. 2015)

Group 1-AA net flux (mmol/h) * * * * (Larsen et al. 2015)

Group 2-AA net flux (mmol/h) Ile, Leu, Lys,Val (Larsen et al. 2015)

Group 2-AA net flux (mmol/h) (Larsen et al., 2015)

Group 2-AA net flux (mmol/h) * * * * (Larsen et al., 2015)

NEAA net flux (mmol/h) (Larsen et al. 2015)

NEAA net flux (mmol/h)? * (Larsen et al. 2015)

Liver glucose total flux (mmol/h) = (Galindo et al. 2015)

Glucose total flux (mmol/h) (Galindo et al. 2015)

Glucose total flux (mmol/h) * Established lactation (Galindo et al. 2011)

NE L balance in response to AA-CN infusion (study 2) P trt DIM = 0.10 * (Galindo et al. 2015)

[NEFA], mm * P trt DIM = 0.05 -> increased fat mobilisation at DIM 5 (Galindo et al. 2015)

[BHBA], mm * P trt DIM = 0.03 (Galindo et al. 2015)

Field trial (preliminary results): 91 Holsteins randomised block design Control ~15.5% CP Protein ~20.5% CP Energy ~15.0% CP PMR 3 kg conc./d PMR with extra protein 3 kg conc./d PMR 3 kg conc. + 2 kg barley/d Same feed Calv. 14 d 29 d

Milk yield 50 P diet x parity < 0.01 40 kg/d 30 20 CONTROL PROTEIN ENERGY 10 0 Primi Multi Wk 1 to 4: +5.5 kg/d for older cows (Larsen et al. EAAP, 2017)

Milk protein and fat yield followed milk yield Milk protein yield P diet x parity < 0.05 Milk fat yield P diet x parity < 0.05 2.0 2.0 kg/d 1.5 1.0 kg/d 1.5 1.0 CONTROL PROTEIN ENERGY 0.5 0.5 0.0 Primi Multi 0.0 Primi Multi Concentrations did not differ among diets (Larsen et al. EAAP, 72 2017)

Greater fat mobilisation with high protein allocation Plasma NEFA 1000 P diet x parity x week < 0.01 M 800 600 C multi P multi E multi 400 200 0 1 2 3 4 5 8 Week after calving (Larsen et al. EAAP, 2017)

BHBA tended to be greatest with control Plasma BHB 1.4 P diet = 0.07; P parity < 0.01 mm 1.2 1.0 0.8 C multi P multi E multi 0.6 0.4 0.2 1 2 3 4 5 8 Week after calving (Larsen et al. EAAP, 74 2017)

3. Can we reduce the deficit? Increased protein supply increased MPY -> failed to decrease protein deficiency! BUT: increased AA concentrations!!!

Although the deficit was not reduced: CN increased fractional synthesis rate of albumin at DIM 4 CN increased rumen papillae proliferation CN stabilized inflammatory responsiveness of leukocytes (Larsen et al. 2017)

4. Other effects of AA Glutamine: conditionally essential NEAA Immune system (lymphocyte, cytokine) Precursor or purine and pyrimidine synthesis Major energy source Glu + Gln 20 % AA in milk Plasma concentrations still low at 29 DIM -> post-rumen infusion of 300 g/d Gln 21 days post-calving (Doepel et al. 2006)

Effect of Gln infusion non significant increment of milk yield 83% recovery of infused Gln in the portal vein Milk, kg/d 44 40 36 32 28 24 20 4 7 11 14 18 21 Day from calving Ctl Gln no effect on immune parameters (Doepel et al. 2006 & 2007)

5. Conclusions: AA metabolism AA deficiency mirrors estimated MP balance: post-liver vs. mammary uptake AA metabolism post-calving follows the same pattern as in established lactation: Group 1: liver catabolism Mammary U:O of EAA 1 Group 2: Very little liver catabolism Mammary U:O of EAA > 1; but decreases with low supply

5. Conclusions: pre- vs. post-calving The liver is NOT the key regulator Responds to both absorption and tissue utilization AA priority is to make protein Initiation of lactation does NOT increase AA liver removal

5. Conclusions: reducing the deficit Increased AA supply: Very efficient use of extra AA into MPY Does not reduce AA deficiency No increment of liver gluconeogenesis Increased energy deficiency But limited effect on [NEFA & BHBA] Protein appears more limiting than energy in the month post-calving Beneficial to reach the biological potential of a larger production?

Thanks to: Dairy Farmers of Canada, Dairy Farmers of Québec (Novalait) Aarhus University, Denmark Agriculture and Agri-Food Canada Ajinomoto Heartland, Inc. Danish Agri-Fish Agency Danish Council for Independent Research Danish Council for Technology and Innovation Danish Milk Levy Fund Evonik Industries AG Ministry of Food, Agriculture and Fisheries, Denmark Natural Science and Engineering Research Council of Canada (NSERC)

Questions?

Thank you!