Ruminant Nutrition Platform Session II: Protein and Amino Acid Nutrition

Transcription

1 Ruminant Nutrition Platform Session II: Protein and Amino Acid Nutrition 483 Effects of infused leucine and isoleucine or methionine, lysine, and histidine on cow performance. P. S. Yoder* 1,2, X. Huang 1, and M. D. Hanigan 1, 1 Virginia Tech, Blacksburg, VA, 2 Perdue AgriBusiness, Salisbury, MD. Leucine and to a lesser extent, isoleucine, possess signaling properties promoting cellular anabolic metabolism via mammalian target of rapamycin, but in vivo evidence is limited. In contrast, methionine, lysine, and histidine are well documented to limit milk protein synthesis for some diets. The objective of this study was to evaluate production responses from supplementing 2 groups of amino acids in a 2 2 factorial design. Eight cows (102 ± 19 DIM) were assigned to 4 jugular infusion treatments consisting of saline (SAL), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL in a replicated 4 4 Latin square design. Periods were 18 d in length comprising 8 d of rest followed by 10 d of jugular amino acid infusion. Daily infusion amounts were 21 g of methionine, 38 g of lysine, 20 g of histidine, 50 g of leucine and 22 g of isoleucine. The SAL diet had observed daily deficits of 539 g of metabolizable protein, 20 g of methionine, 48 g of lysine, 7 g of histidine, 89 g of leucine, and 25 g of isoleucine according to CNCPS v6.55 and 498 g of metabolizable protein by NRC (2001). Cows were ad libitum fed a common diet consisting of 15.2% crude protein, and 1.61 Mcal/kg NE L on a dry basis. Dry matter intake increased for MKH by 0.5 kg/d (P = 0.05). Milk yield increased 2.3 kg/d (P = 0.02) by infused IL and no change was observed for MKH (P = 0.39). Milk protein concentration increased by 0.13 percentage units for MKH (P < 0.001) whereas yield increased for both MKH and IL by 88 g/d (P < 0.01) and 57 g/d (P = 0.02) respectively. The milk protein yield increase for MKH + IL was 145 g/d (P < 0.01) versus SAL. Energy corrected milk tended to increase for IL by 1.6 kg/d (P = 0.13). No differences were observed for fat yield or concentrations, milk urea nitrogen, feed efficiency, body weight change, or milk N efficiency, that latter being 38.6%. Increases in milk protein yield were observed from 2 groups of amino acids independently and additively which contradicts the single limiting amino acid theory that a single nutrient will limit milk protein production. Key Words: amino acids, milk protein synthesis 484 Effects of varying extracellular amino acid concentration on amino acid transport in mammary epithelial cells. P. S. Yoder* 1,2, J. J. Castro 3, T. Ruiz-Cortes 4, and M. D. Hanigan 1, 1 Virginia Tech, Blacksburg, VA, 2 Perdue AgriBusiness, Salisbury, MD, 3 Dairy Visions LLC, Chandler, AZ, 4 Universidad de Antioquia, Medellin, Antioquia, Colombia. Understanding mechanisms and quantitative description of cellular responses to changing nutrient supply is critical for predicting milk component yields. Our objective was to evaluate cellular uptake and kinetic behavior of individual amino acids (AA) simultaneously. Bovine mammary epithelial cells were grown to confluency and fed media with an AA profile and concentration similar to dairy cows for 24 h. Treatments were 4 AA concentrations, 0.36, 2.30, 4.28, and 6.24 mm, which represents 16, 100, 186, and 271% of typical plasma AA concentrations in dairy cows. Twenty-four plates of cells ( mm) were assigned to each treatment. Cells were first subjected to treatment media enriched with 15 N labeled AA for 24 h and then incubated with treatment media enriched with 13 C labeled AA for 0, 15, 60, 300, 900, 1800, and 3600 s. Intracellular free AA, intracellular protein-bound AA, and extracellular media AA were analyzed for concentrations and isotopic enrichment using gas chromatography mass spectrometry. Additionally, the total protein, weight, and count of cells were measured. A dynamic 9-pool model was constructed representing extracellular, intracellular, and turnover pools for individual AA and respective isotopes and then solved via numerical integration. Fluxes were extracellular to intracellular entry, intracellular efflux, protein turnover, transamination, oxidation, and synthesis. As an example, cellular influx and efflux ranged from 0.9 to 11.1 and 0 to 9.5 nmol/min for Leu whereas Ala ranged from 1.6 to 50.0 and 0.9 to 56.9 nmol/min for influx and efflux. The average root mean square prediction error as percent of the mean for the 9 pools was 14.6 and 10.6 percent for Leu and Ala respectively. When AA influx was standardized to total pool of the respective AA, Michaelis-Menten kinetics were observed with a K m of 72 and 345 μmol for Leu and Ala respectively. The described model provides insight on individual AA transport kinetics when subjected to varying extracellular concentrations, which might be useful for better understanding and future modeling of AA uptake by the udder. Key Words: amino acids, transport 485 Lactational performance of dairy cows in response to supplementing N-acetyl-l-methionine as a source of rumen-protected methionine. F. X. Amaro* 1, K. G. Arriola 1, Y. Jiang 1, D. Kim 1, A. P. Cervantes 1, V. P. Silva 1, M. C. N. Agarussi 1, J. T. Silva 1, A. T. Adesogan 1, L. F. Ferraretto 1, C. R. Staples 1, J.-S. Eun2,3, J. S. Park 3, J. O. Moon 3, D. Vyas 1, 1 Department of Animal Sciences, University of Florida, Gainesville, FL, 2 Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, UT, 3 Biotechnology Research Institute, CJ CheilJedang, Suwon, South Korea. Adequate supply of essential and limiting amino acids such as methionine is critical for maintaining milk production by high-producing dairy cows. The objective was to assess the effects of supplementing incremental amounts of N-acetyl-l-methionine (NALM), a source of rumenprotected methionine, on DMI, milk production, and dairy efficiency in lactating dairy cows. Sixty multiparous Holstein dairy cows in early lactation (27 ± 4.3 d-in-milk) were fed for 16 wk in a randomized complete block design. Animals were grouped based on actual milk yield, and calving date. Dietary treatments included (1) control (no NALM); (2) 15 g/d NALM; (3) 30 g/d NALM; and (4) 45 g/d NALM. Diets were formulated to meet or exceed nutritional requirements of lactating dairy cows producing at least 42 kg/d of milk and to under supply metabolizable methionine (control) or supply increasing amounts with increasing NALM supplementation. Data were analyzed using the GLIMMIX procedure of SAS, using covariate in the model for all variables tested. Linear and quadratic effects with NALM were tested. Supplementation of NALM did not affect DMI (P = 0.37) regardless of the dose, whereas milk yield tended to be greater (P = 0.07) with intermediate levels of NALM (15 and 30 g/d) compared with the control. Milk true protein concentration was not influenced by supplementing NALM at 15 and 30 g/d, but these inclusion levels increased milk protein yield relative to the control (P = 0.04) possibly due to the increase in milk yield. Dairy efficiency (milk yield/dmi) was greatest at 30 g/d NALM (P = 0.03) and was unaffected by supplementing NALM at 15 or 45 g/d compared with the control. Supplementation of NALM at 30 g/d 407

2 Table 1 (Abstr. 486). Effects of dietary supplementation with RPA on milk production by dairy cows (mean ± SEM; *P = 0.016) Treatment group No. of cows Milk yield (MY) d 0 d 7 Change in MY (kg/cow/7d): d 7 d 0 CV of MY change (%) Non-handling control ± ± ± 3.22 NS 321 Isonitrogenous control (Ala) ± ± ± 3.36 NS 264 Unprotected Arg product ± ± ± 3.59 NS 259 Rumen-protected Arg product ± ± ± 2.70* 132 resulted in greatest milk yield, milk protein yield, and dairy efficiency when fed to early to mid-lactating dairy cows. However, the greatest amount of NALM supplementation (45 g/d) led to no beneficial effects on lactational performance. Overall results in the current study suggest that NALM supplementation can improve productive performance of dairy cows when added at 30 g/d. Key Words: dairy efficiency, milk production, N-acetyl-l-methionine 486 Dietary supplementation with a rumen-protected l-arginine product enhances milk production by dairy cows. A. B. Keith, M. C. Satterfield, F. W. Bazer, and G. Wu*, Texas A&M University, College Station, TX. This proof-of-concept study was conducted on HAW Farms (Belen, NM) to test the hypothesis that l-arginine (Arg) can enhance milk production by lactating cows. A rumen-protected Arg product (RPA, which contained a by-pass matrix) and an unprotected Arg product (UPA) that had the same composition as RPA were manufactured by Biotechnology Services & Consulting Inc. (Coppell, TX). Before parturition, 54 healthy Holstein cows (parities 1 to 4), weighing kg, were assigned randomly to receive either no dietary supplementation (non-handling control) or dietary supplementation of 500 g RPA, 500 g UPA or an isonitrogenous amount of L-alanine (Ala) per day beginning on d 1 to 4 after parturition (the initial day of supplementation = d 0 of the trial). Each cow was fed twice daily a typical silage- and alfalfa hay-based lactation diet containing 17% crude protein (25 kg DM/d) to meet the NRC-recommended requirements of nutrients, and had free access to drinking water. A supplement was administered to cows twice daily (equally divided doses at 8 a.m. and 6 p.m.) by gavaging immediately after consumption of their regular meals. On d 0 and 7 of the trial, milk yields of cows were determined using an auto-milking system. On either day, the composition of nutrients did not differ (P > 0.05) among the 4 groups of cows. Changes in milk yields between d 7 and 0 (analyzed by the paired t-test) were significant (P = 0.016) for the RPA group but not significant (P > 0.05; NS) for the other groups (Table 1). Interestingly, variation in milk yield change was much less for RPA-supplemented cows, compared with the other groups of cows. These results indicate that cows fed RPA had more consistent lactation performances and produced more milk. Key Words: arginine, lactation, milk 487 Efficiency of utilization of amino acid increased with energy supply at low and high metabolizable protein supply in dairy cows. C. Omphalius* 1,2, H. Lapierre 3, L. Bahloul 2, and S. Lemosquet 1, 1 PEGASE, INRA, Agrocampus-Ouest, Rennes, France, 2 Adisseo France S.A.S, Antony, France, 3 Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada. Implementing the concept of a variable efficiency of use of individual AA (AA ef ) in feeding systems requires knowledge on the effect of net energy of lactation (E) in addition to the effect of metabolizable protein supply (P expressed as PDI, INRA). Therefore, AA ef were determined at 2 levels of E (LE: 24.8 vs. HE: 32.6 Mcal/d of NEL) and 2 levels of P (LP: 1,261 vs. HP: 2,227 g/d of PDI) using a 4 4 Latin square with 4 cows and 2-wk periods. Individual AA ef was calculated as the sum of milk protein yield (MPY), endogenous fecal (EFP) and scurf Table 1 (Abstr. 487). Diet P-value In mol/12h of N LELP HELP LEHP HEHP SEM E P E P AA S-EU < MPY <0.01 < EFP < Uptake EAA < NEAA AA eff EAA <0.01 <0.01 <0.01 NEAA <0.01 <0.01 <0.01 MU: milk output EAA Group Group His, Met, Phe, Tyr, and Trp. 2 Lys, Ile, Leu, and Val. 408

3 proteins (INRA, 2018) multiplied by their respective AA profile (Lapierre et al., 2016; CNC: 205) and divided by the AA supply minus the AA endogenous urinary losses (AA S-EU ). Mammary net uptake (MU) of AA (6 samples in 12 h) was determined on d 13 of each period. Data were analyzed by MIXED procedure (SAS) with cow as random effect. Increasing E and P supply increased MPY, essential AA (EAA) MU and only increasing E supply tended to increase non-eaa (NEAA) MU. Increasing E supply was partially achieved by increased DMI (14.5 vs kg/d) increasing EFP. Therefore, the AA ef increased with increasing E supply but at a higher extent in LP diets. The gain in AA ef with increasing E was mainly explained through a change in the partition of AA toward MPY and EFP at whole body level and of MU AA toward MPY as suggested by the lower EAA MU: milk output ratio. The decreased AA ef with increased P supply was related to a decreased NEAA and EAA MU relative to the AA S-EU and to an increased MU: milk output ratio of group 2 AA (Lys, Ile, Leu and Val), both suggesting a higher AA catabolism (Table 1). Key Words: amino acid, efficiency, mammary gland 488 Leucine and lysine alter inflammatory response of immune cells from growing cattle. M. Garcia*, K. A. Pearl, E. C. Titgemeyer, and B. J. Bradford, Kansas State University, Manhattan, KS. Bovine neutrophils alter their inflammatory response upon amino acid supplementation, yet research evaluating effects of specific amino acids is scarce. We examined effects of lysine (Lys) and leucine (Leu) on inflammatory responses of neutrophils (PMN) and mononuclear cells (PBMC) from growing Holstein steers (173 ± 3.7 kg BW). Seven steers were used in a 6 6 Latin square design with a factorial arrangement of 2 levels of Lys (0 and 6 g/d) and 3 levels of Leu (0, 15, and 30 g/d). Steers were fed a basal diet and infused with all required nutrients except Lys. Treatments were delivered by abomasal infusion. Blood was collected on d 7 of each 7-d period. A hematology analyzer was used to measure blood cell composition, and immune cells were isolated using density gradients. To harvest media for cytokine measures, isolated cells were treated with lipopolysaccharide (LPS, 0 or 1 µg/ml) for 2 (PMN) or 24 h (PBMC); PBMC were treated with concanavalin-a (10 µg/ml) to enhance proliferation. PMN were exposed to dihydrorhodamine and labeled E. coli for 50 min to assess oxidative burst and phagocytosis. Period and steer were considered random effects, and data were transformed (square root or log10) to attain normality. Lys reduced lymphocyte numbers (6.40 vs ± /µl, P = 0.04). Leu (30 g/d) almost doubled eosinophil numbers but only for non-lys fed steers (0.36, 0.31, and 0.63 ± /µl, P = 0.02). PMN phagocytic and oxidative burst responses were not impacted by treatment. In the presence of LPS, TNF-α produced by PMN (1.7, 8.5, 17.1 ± 6.9 pg/ ml, P = 0.08) and IFN-γ produced by PBMC (384, 431, 523 ± 87 pg/ ml, P = 0.01) increased or tended to increase linearly as Leu increased. In the absence of LPS, TNF-α produced by PBMC tended to increase quadratically as Leu increased (424, 630, 431 ± 99 pg/ml, P = 0.08). Regardless of LPS, Lys tended to increase production of IFN-γ by PBMC (353 vs. 400 ± 83 pg/ml, P = 0.10). These findings point to a role for dietary Lys and Leu in regulating immune responses. Key Words: amino acid, immunity, steer 489 Alterations in amino acid transporters and the mtor pathway in adipose tissue of Holstein cows during the periparturient period in response to methionine supply. Y. Liang* 1, F. Batistel 1, C. Parys 2, and J. J. Loor 1, 1 Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, Urbana, IL, 2 Evonik Nutrition & Care GmbH, HanauWolfgang, Germany. Recent studies in non-ruminants have revealed that mammalian target of rapamycin (mtor) signaling pathway plays a critical role in the regulation of adipose tissue function. The objective of the current study was to investigate whether increasing the methionine supply would alter mrna expression of amino acid transporters and mtor pathway components in adipose tissue during the periparturient period. Sixty multiparous Holstein cows were used in a block design and assigned to a control diet or the control plus rumen-protected methionine (MET; Mepron, Evonik Nutrition & Care GmbH, Germany). Mepron was fed from 28 to 30 d relative to parturition at 0.09% and 0.10% of the diet dry matter during the prepartum and postpartum period, respectively. Blood sampled from the coccygeal vessel at 14, 7, and 30 d relative to calving date was used for amino acid analysis. Adipose tissue obtained from each group on d 10, 10 and 30 relative to calving date was used for RT-PCR analysis. The statistical model included the random effect of block and fixed effect of treatment, time and its interaction. A treatment time was observed for the plasma concentrations of Asn, Cys, and Gly due to higher responses in MET-supplemented cows (P < 0.05). Compared with control, MET-fed cows had greater concentration of Met, Lys, Thr, Leu, Val, and Phe in plasma (P < 0.05). Among the 7 amino acid transporters measured, MET-supply increased the mrna expression of Gln (SLC38A1), Glu (SLC1A1), L-type amino acid (Met, Leu, Val, Phe; SLC3A2), small zwitterionic α-amino acid (SLC36A1), and neutral amino acid (SLC1A5) transporters in adipose tissue (P < 0.05). Expression of AKT1, RPS6KB1, and EIF4EBP1 were also upregulated in MET-supplemented cows in adipose tissue (P < 0.05), suggesting enhanced insulin signaling and activation of the mtor pathway. Overall, the data indicate that enhanced methionine supply during the periparturient period enhances circulating amino acid concentrations and the availability and uptake of amino acids by adipose tissue. Key Words: amino acid, dairy cow, mtor 490 Methionine supply during the periparturient period alters glutathione metabolism in adipose tissue of Holstein cows. Y. Liang* 1, F. Batistel 1, C. Parys 2, and J. Loor 1, 1 Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, Urbana, IL, 2 Evonik Nutrition & Care GmbH, HanauWolfgang, Germany. The periparturient period is characterized by increased oxidative stress status. Glutathione is one of the major intracellular antioxidants and is important for alleviating oxidative stress. Nuclear factor (erythroidderived 2)-like factor 2 (NFE2L2) is a transcription factor that induces antioxidant-responsive genes. The hypothesis was that enhanced methionine supply has a positive effect on molecular pathways associated with glutathione synthesis and activation of NFE2L2 in adipose tissue. Sixty multiparous Holstein cows were used in a block design and assigned to a control diet or the control plus ethylcellulose rumen-protected methionine (MET; Mepron, Evonik Nutrition & Care GmbH, Germany). Mepron was fed from 28 to 30 d relative to parturition at 0.09% and 0.10% of diet dry matter during the prepartum and postpartum period, respectively. Adipose tissue obtained on d 10, 10, and 30 relative to calving date was used for RT-PCR analysis. Blood was sampled at 14, 7, and 30 d relative to calving date. The statistical model included the random effect of block and fixed effect of treatment, time and its interaction. Compared with control, MET cows had lower plasma concentrations of reactive oxygen metabolites (P = 0.08) and higher 409

4 concentrations of methionine (P = 0.09) and glutathione (P = 0.02). Among enzymes associated with the methionine cycle and transsulfuration, expression of cystathionine-β-synthase in adipose tissue was upregulated in MET cows (P = 0.06). Expression of glutamate-cysteine ligase, glutathione reductase, and transaldolase 1 in adipose tissue also was upregulated in MET cows (P < 0.05), suggesting a positive effect of enhanced methionine supply on intracellular reduced glutathione availability. The upregulation of NFE2L2 in MET cows also is suggestive of a link between adipose tissue methionine metabolism, glutathione synthesis, and the antioxidant response (P = 0.06). No interactions with time were observed for gene expression in adipose tissue. Overall, the data indicate that enhanced methionine supply during the periparturient period can benefit the availability of reduced glutathione and activation of antioxidant-responsive genes in adipose tissue. Key Words: oxidative stress, NFE2L2, transition cow 491 Predictions of rumen outflow of amino acids in dairy cattle. A.J. Myers* 1, H. Lapierre 2, R.R. White 1, H. Tran 3, P. J. Kononoff 3, R. Martineau 2, W. P. Weiss 4, and M. D. Hanigan 1, 1 Virginia Tech, Blacksburg, VA, 2 Agricultural and Agri-Food Canada, Sherbrooke, QC, Canada, 3 Department of Dairy Science, University of Nebraska, Lincoln, NE, 4 Department of Animal Sciences, The Ohio State University, Columbus, OH. The objective of this work was to update and evaluate predictions of essential AA (EAA) outflows from the rumen. The model was constructed based on previously derived equations for ruminally undegraded (RUP), microbial (MiP) and endogenous (EndP) protein rumen outflows, and revised estimates of the EAA composition of each protein fraction. Corrections were included to account for incomplete recovery of EAA during 24-h acid hydrolysis. The predicted protein and EAA rumen outflows were evaluated against a data set of observed values from the literature. Initial evaluations indicated a mean bias for nonammonia, nonmicrobial N flow ([RUP + EndP] / 6.25) of 12 g N/d. Root mean squared errors (RMSE) of EAA predictions ranged from 29.9 to 43.7% of observed mean values. Concordance correlation coefficients (CCC) ranged from 0.29 to Except for Leu, all EAA rumen outflows were overpredicted by 4.0 to 35 g/d. In addition, significant slope bias was present for all EAA except Met and Leu. It is unclear if the observed bias in EAA flows reflects problems in the prediction equations. Under the assumption that the mean and slope biases may be due to problems in the prediction equations, regression equations were derived to adjust predicted EAA flows. The residuals of the EAA flows were regressed on each of the 3 protein flows. Residuals were correlated with each of the 3 protein flows for all EAA. After applying these new linear regressions, RMSE for EAA predictions ranged from 25.0 to 32.9% of observed mean flows. The mean bias was removed for all predicted EAA flows; however, a small slope bias was introduced for Lys, and Thr. The CCC ranged from 0.43 to For the purposes of predicting post-ruminal EAA flows and based on fit statistics, adjusting the mean and slope biases using the adjustments from the protein EAA flows yielded marginally better results than predicting EAA flows without adjustments. Future work should focus on identifying the cause of the observed prediction bias. Key Words: mechanistic model, amino acid, rumen outflow 492 Predicting milk protein production from amino acid supply. M. D. Hanigan* 1, H. Lapierre 2, R. Martineau 2, and A. M. Myers 1, 1 Virginia Tech, Blacksburg, VA, 2 Agriculture and Agri-Food Canada, Lennoxville, QB, Canada. Efficient diet design requires accurate predictions of the relationship between nutrient inputs and milk output. The NRC (2001) model has been shown to be biased in predicting overall milk production and production responses to varying dietary protein intake and exhibits relatively low precision (~25% RMSE). The objective of this work was to test revised representations of the relationship between nutrient supply and milk protein production. Data used were from 237 published studies containing 724 treatment means. Microbial protein outflows were predicted from Roman-Garcia et al. (2016), and RUP flows from White et al. (2017). The AA composition of protein flows was from Sok et al. (2017). Digestibility of the RUP AA was from Paz-Manzano et al. (2014). Two model forms were tested: Milk Protein = DEI + EAA i + EAA 2 i + NEAA + St + FA + Milk Fat % + DIM (1); and Milk Protein = DEI + EAA i + (EAA i /DEI) 2 + NEAA + St + FA + Milk Fat % + DIM (2) where DEI represented digestible energy intake, EAA the digested supply (g/d) of the ith essential AA, NEAA the digested total supply of other AA, and St and FA dietary starch and fatty acid contents (% of DM). Essential AA included all except Trp. All combinations of EAA terms were tested with the presence of squared terms requiring linear term presence. The best submodel for eqn. (1) contained terms for DEI, Arg, Leu, Lys, Met, Thr, NEAA, St, DIM, and milk fat with an AICc of 8191 and RMSE of 12.5%. The best submodel for eqn. (2) contained the same terms plus Phe with an AICc of 8173 and RMSE of 11.9%. Exclusion of His, Ile, and Val from both models indicates they are always provided in excess, or there is inadequate independent variance to define responses to those AA. The results indicate that expressing declining efficiency of AA use for milk protein production as a square of the ratio of AA to DEI is superior to the square of AA. These equations demonstrate that responses to individual EAA and energy are additive and our models must reflect that if improved accuracy and precision are to be achieved in diet formulation. Key Words: essential amino acids, milk protein, model 493 A new model to predict microbial protein synthesis in the rumen. L. E. Moraes* 1, R. R. White 2, and J. L. Firkins 1, 1 The Ohio State University, Columbus, OH, 2 Virginia Tech, Blacksburg, VA. Feeding systems worldwide use metabolizable protein as the unifying unit for computing protein availability in feeds and the protein requirements for various physiological functions. Metabolizable protein is the sum of microbial true protein synthesized in the rumen from RDP and endogenous sources and RUP. In practical feeding situations, these quantities are seldom measured, so mathematical models are needed to predict the amount of ruminally synthesized microbial protein. The objective of this study was to develop a model to predict microbial protein production in the rumen using a more mechanistic and generalizable approach by representing the supply of both RDP and rumen-degraded carbohydrates. A bisubstrate Michaelis-Menten functional form was used to associate the amounts of rumen-degraded NDF (RDNDF) and starch (RDST) with 2 Michaelis-Menten constants and the amount of RDP with the asymptotic response (Vmax). The mechanistic biological reasoning was that, from a mass balance standpoint, the amount of RDP determines the limiting microbial protein produced while the amount of degraded carbohydrates are associated with the affinity (Km) of substrate used for carbon precursors in cellular growth (derivation of cell constituents for dividing cells). The equation is an efficiency derivation with actual microbial N, and actual RDNDF and RDST derived using diet-level prediction factors (White et al., 2017; J. Dairy Sci. 100: ). The model was fitted with 583 treatment means from 154 publications using a Bayesian nonlinear hierarchical modeling approach. The fitted model was Microbial N (g/d) = ( RDP)/[( / 410

5 RDNDF)( /RDST)] where RDP is in g/d and both RNDF and RDST are in kg/d. The Bayesian hierarchical approach allowed successful introduction of between study variability in all model parameters. Model evaluation suggested low slope and mean bias (<2% of MSE). Likewise, a 5-fold cross-validation suggested good predictive ability (Concordance Correlation Coefficient, CCC = 0.52) and relatively low root mean square prediction error (29% of mean) when compared against models from the literature. Key Words: microbial protein, rumen degradable protein, carbohydrate 494 Diets to maximize milk protein secretion: Is the single limiting amino acid model the whole story? L. E. Armentano*, University of Wisconsin, Madison, WI. This talk will challenge the idea of defining the dietary protein requirements for lactating cows based solely on the limiting amino acid concept. One form of the limiting amino acid model is that milk protein yield potential is set, but it can be reduced below this potential by a deficiency in a single essential amino acid. A corollary of this is that the total dietary crude or metabolizable protein (MP) can be minimized to the point that the requirement for this first limiting amino acid is met. This model certainly includes the possibility of sequential second and third limiting amino acids, which become limiting as the original first limiting amino acid reaches its requirement. One problem with this model in the ruminant animal is that a large portion of the MP is derived from rumen microbial reproduction. Unlike animal protein, a community of microbial species has a malleable genetic blueprint and may respond to the amino acid content of the rumen-degraded protein in a complex way. Another limitation of the limiting amino acid model is that potential milk protein yield may be influenced by many dietary (and other) factors. Insulin is known to increase milk protein yield in cases where milk protein yield is limited by MP or where milk protein secretion has been increased due to additional MP. Removing crude protein from the diet and replacing it with other dietary constituents could affect circulating insulin and modulate milk protein secretion independently of the limiting amino acid. One identified mechanism for insulin is through activation of mtor, a regulatory protein in mammary secretory cells. This same mtor is simultaneously regulated by essential amino acids, but the essential amino acids that have the largest effect (e.g., leucine, isoleucine), are different than the those normally suggested as most limiting (methionine, lysine and histidine). Optimizing milk protein secretion and nitrogen efficiency must consider the amino acid composition of MP and animal needs, but careful experimentation and quantitative model development must consider alternatives beyond only the concept of one or a few limiting amino acids. Key Words: insulin, mtor, microbial 411