Graduate Student Literature Review: System, plant, and animal factors controlling dietary pasture inclusion and their impact on ration formulation for dairy cows.

Diet formulation in a pasture-based dairy system is a challenge as the quality and quantity of available pasture, which generally constitutes the base diet, is constantly changing. The objective of this paper is to cover a more in-depth review of the nutritional characteristics of pasture-based diets, identifying potential system, plant, and animal factors that condition pasture dietary inclusion in dairy cows. In practice, there is a wide diversity of pasture-based systems with predominant to minimal use of pasture requiring a more specific classification that potentially considers the amount and time of access to pasture, access to housing, length of grazing season, seasonality of calving, and level and method of supplementation. There are important differences in the nutritional quality between pasture species and even cultivars. However, under management practices that promote maintenance of pasture in a vegetative state as well as controlling the availability of pasture, it is possible to achieve high dry matter intakes (∼2.9%-3.4% of live weight) of pasture with moderate to high diet energy density, protein supply, and digestibility. The amount of pasture to include in the diet will depend on several factors, such as the type of production system, the cost of supplementary feeds, and the farmer's objectives, but inclusions of ∼40% to 50% of the diet seem to potentially reduce costs while apparently not limiting voluntary feed intake. Considering that there seems to be a continuum of intermediate management systems, a better understanding of the factors inherent to the feed ingredients used, as well as the use of nutrients by cows, and potential interactions between animal × system should be addressed in greater depth. This requires a meta-analysis approach, but given the diversity of the pasture-based system in practice, the existing information is highly fragmented. A clear definition of "subsystems" is necessary to direct the future research and development of mechanistic models.

Effects of rumen-encapsulated methionine and lysine supplementation and low dietary protein on nitrogen efficiency and lactation performance of dairy cows.

Low crude protein (CP) diets might be fed to dairy cows without affecting productivity if the balance of absorbed AA were improved, which would decrease the environmental effect of dairy farms. The aim of this study was to investigate the effects of supplementing ruminally protected Lys (RPL) and Met (RPM) at 2 levels of dietary CP on nutrient intake, milk production, milk composition, milk N efficiency (MNE), and plasma concentrations of AA in lactating Holstein cows and to evaluate these effects against the predictions of the new NASEM (2021) model. Fifteen multiparous cows were used in a replicated 3 × 3 Latin square design with 21-d periods. The 3 treatments were (1) a high-protein (HP) basal diet containing 16.4% CP (metabolizable protein [MP] balance of -130 g/d; 95% of target values), (2) a medium-protein diet containing 15% CP plus RPL (60 g/cow per day) and RPM (25 g/cow per day; MPLM; MP balance of -314 g/d; 87% of target values), and (3) a low-protein diet containing 13.6% CP plus RPL (60 g/cow per day) and RPM (25 g/cow per day; LPLM; MP balance of -479 g/d; 80% of target values). Dry matter intake was less for cows fed MPLM and LPLM diets compared with those fed the HP diet. Compared with the HP diet, the intake of CP, neutral detergent fiber, acid detergent fiber, and organic matter, but not starch, was lower for cows fed MPLM and LPLM diets. Milk production and composition were not affected by MPLM or LPLM diets relative to the HP diet. Milk urea N concentrations were reduced for the MPLM and LPLM diets compared with the HP diet, indicating that providing a low-protein diet supplemented with rumen-protected AA led to greater N efficiency. There was no significant effect of treatment on plasma AA concentrations except for proline, which significantly increased for the MPLM treatment compared with the other 2 treatments. Overall, the results supported the concept that milk performance might be maintained when feeding lactating dairy cows with low CP diets if the absorbed AA balance is maintained through RPL and RPM feeding. Further investigations are needed to evaluate responses over a longer time period with consideration of all AA rather than on the more aggregated MP and the ratio between Lys and Met.

Review: How the efficiency of utilization of essential amino acids can be applied in dairy cow nutrition.

How the efficiency of utilization of essential amino acids (EffUEAA) can be applied in dairy cow nutrition is presented in this review. The concept of EffUEAA proposed by the National Academies of Sciences, Engineering and Medicine (NASEM, 2021) is first detailed. It represents the proportion of the metabolisable essential amino acids (mEAA) supply used to support protein secretions and accretions (scurf, metabolic fecal, milk and growth). For these processes, the efficiency of each individual EAA is variable, and considered to vary similarly for all the protein secretions and accretions. The anabolic process of gestation is ascribed to a constant efficiency (33%), whereas the efficiency of endogenous urinary loss (EndoUri) is set at 100%. Therefore, the NASEM model EffUEAA was calculated as the sum of EAA in the true protein of secretions and accretions divided by the available EAA (mEAA - EndoUri - gestation net true protein/0.33). In this paper, the reliability of this mathematical calculation was tested through an example where the experimental efficiency of His was calculated assuming that liver removal represents catabolism. The NASEM model and experimental efficiencies were in the same range and varied in similar manner. Assuming that the NASEM model EffUEAA reflects EAA metabolism in the dairy cow, its different applications were examined. In NASEM, target efficiencies were determined for each EAA: 75, 71, 73, 72, 73, 60, 64, 86 and 74% for His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val, respectively. From these, recommendations for mEAA supply can be calculated as: [(secretions + accretions)/(target EffUEAA × 0.01) + EndoUri + gestation/0.33], assuming energy supply is adequate. In addition to NASEM propositions, equations to predict EffUEAA with precision and accuracy are detailed, using the ratio of (mEAA-EndoUri) to digestible energy intake, in a quadratic model that includes days in milk. Moreover, milk true protein yield predictions from predicted EffUEAA or efficiency of utilization of metabolisable protein are better than those from the multivariate equation of NASEM (2021) and superior to those predicted with a fixed efficiency. Finally, either the NASEM model or the predicted EffUEAA can be used to assess the responsiveness of a ration to supplementation with a single EAA. If the EffUEAA of the EAA to supplement is higher than the target EffUEAA, while the EffUEAA of the other EAA are lower than the target value, this suggests a potential improvement in milk true protein yield to supplementation with this EAA.

Models to predict milk fat concentration and yield of lactating dairy cows: A meta-analysis.

Few models have attempted to predict total milk fat because of its high variation among and within herds. The objective of this meta-analysis was to develop models to predict milk fat concentration and yield of lactating dairy cows. Data from 158 studies consisting of 658 treatments from 2,843 animals were used. Data from several feed databases were used to calculate dietary nutrients when dietary nutrient composition was not reported. Digested intake (DI, g/d) of each fatty acid (FA; C12:0, C14:0, C16:0, C16:1, C18:0, C18:1 cis, C18:1 trans C18:2, C18:3) and absorbed amounts (g/d) of each AA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val) were calculated and used as candidate variables in the models. A multi-model inference method was used to fit a large set of mixed models with study as the random effect, and the best models were selected based on Akaike's information criterion corrected for sample size and evaluated further. Observed milk fat concentration (MFC) ranged from 2.26 to 4.78%, and milk fat yield (MFY) ranged from 0.488 to 1.787 kg/d among studies. Dietary levels of forage, starch, and total FA (dry matter basis) averaged 50.8 ± 10.3% (mean ± standard deviation), 27.5 ± 7.0%, and 3.4 ± 1.3%, respectively. The MFC was positively correlated with dietary forage (0.294) and negatively associated with dietary starch (-0.286). The DI of C18:2 (g/d) was more negatively correlated with MFC (-0.313) than that of the other FA. The best variables for predicting MFC were days in milk, FA-free dry matter intake, forage, starch, DI of C18:2, DI of C18:3, and absorbed Met, His, and Trp. The best predictor variables for MFY were FA-free dry matter intake, days in milk, absorbed Met and Ile, and intakes of digested C16:0 and C18:3. This model had a root mean square error of 14.1% and concordance correlation coefficient of 0.81. Surprisingly, DI of C18:3 was positively related to milk fat, and this relationship was consistently observed among models. The models developed can be used as a practical tool for predicting milk fat of dairy cows, while recognizing that additional factors are likely to also affect fat yield.

Assessing amino acid uptake and metabolism in mammary glands of lactating dairy cows intravenously infused with methionine, lysine, and histidine or with leucine and isoleucine.

The objective of this study was to evaluate the effect of jugular infusions of 2 groups of AA on essential AA (EAA) transport and metabolism by mammary glands. Four Holstein cows in second lactation (66 ± 10 d in milk) were used in 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Treatments were jugular infusions of saline; Met, Lys, and His (MKH); Ile and Leu (IL); or MKH plus IL (MKH+IL). Each period consisted of 8 d of no infusion followed by 8 d of jugular vein infusion of the treatment solutions. Amino acids were infused at rates of 21 g of Met, 38 g of Lys, 20 g of His, 50 g of Leu, and 22 g of Ile per day. Cows were fed a basal diet consisting of 15.2% crude protein with adequate rumen degradable protein but 15% deficient in MP based on estimates by Cornell Net Carbohydrate and Protein System (v6.5). On the last day of each period, 13C-AA derived from algae was infused into the jugular vein over 6 h, and blood and milk samples were collected before, during, and after infusion. Plasma and milk samples were analyzed for AA isotopic enrichment, and a mammary compartmental model was fitted to the data to derive bidirectional transport and metabolism rates for individual EAA. Influx of Leu increased with IL, whereas influx of other EAA was not different among treatments. Cellular efflux of Met and Lys to venous plasma represented 12 to 34% of influx, whereas cellular efflux of Phe and BCAA represented 29 to 59% of influx. Increased efflux/influx ratios of Ile and Leu with IL but not Met and Lys with MKH demonstrated that increased Ile and Leu influx was mostly returned to plasma resulting in no change in net uptake or efficiency. The isotope results showed that mammary net uptake of Lys and Ile increased during MKH infusion. Net uptake of Met increased with MKH but only in the absence of IL. Catabolism of Lys and Met only increased with MKH alone, resulting in decreased efficiency for milk protein, which demonstrated that Ile and Leu infusion can spare Lys and Met for milk protein synthesis. Total AA uptake to milk output was not different from 1, implying the catabolized Met and Lys contributed nitrogen to nonessential AA. Overall, EAA uptake and metabolism in mammary glands of dairy cows varied across individual EAA and responded differently to respective AA supplements. In addition, uptake, retention, and end use of AA by mammary tissue is variable and dependent on the mix of AA provided. This variability, depending on the mix of AA absorbed, will change the efficiency of utilization of individual AA at the mammary gland level and consequently the whole-body level. Thus, it is inaccurate to use a fixed, constant efficiency within and across AA to represent tissue activity.

Modeling fatty acids for dairy cattle: Models to predict total fatty acid concentration and fatty acid digestion of feedstuffs.

Development of predictive models of fatty acid (FA) use by dairy cattle still faces challenges due to high variation in FA composition among feedstuffs and fat supplements. Two meta-analytical studies were carried out to develop empirical models for estimating (1) the total FA concentration of feedstuffs, and (2) the apparent total-tract digestibility of total FA (DCFATTa) in dairy cows fed different fat types. In study 1, individual feedstuff data for total crude fat (EE) and FA were taken from commercial laboratories (total of 203 feeds, 1,170,937 samples analyzed for total FA, 1,510,750 samples analyzed for total EE), and data for FA composition were collected from the Cornell Net Carbohydrate and Protein System feed library. All feedstuffs were grouped into 7 classes based on their nutritional components. To predict total FA concentration (% of dry matter) for groups of feeds, the total EE (% of dry matter) was used as an independent variable in the model, and all models were linear. For forages, data were weighted using the inverse of the standard error (SE). Regression coefficients for predicting total FA from EE (% of dry matter) were 0.73 (SE, 0.04), 0.98 (0.02), 0.80 (0.02), 0.61 (0.04), 0.92 (0.03), and 0.93 (0.03), for animal protein, plant protein, energy sources, grain crop forage, by-product feeds, and oilseeds, respectively. The intercepts for plant protein and by-product groups were different from zero and included in the models. As expected, forages had the lowest total FA concentration (slope = 0.57, SE = 0.02). In study 2, data from 30 studies (130 treatment means) that reported DCFATTa in dairy cows were used. Data for animal description, diet composition, intakes of total FA, and DCFATTa, were collected. Dietary sources of fat were grouped into 11 categories based on their fat characteristic and FA profile. A mixed model including the random effect of study was used to regress digested FA on FA intake with studies weighted according to the inverse of their variance (SE). Dietary intake of extensively saturated triglycerides resulted in markedly lower total FA digestion (DCFATTa = 44%) compared with animals consuming unsaturated FA, such as Ca-salts of palm (DCFATTa = 76%) and oilseeds (DCFATTa = 73%). Cows fed saturated fats had lower total FA digestion among groups, but it was dependent on the FA profile of each fat source. The derived models provide additional insight into FA digestion in ruminants. Predictions of total FA supply and its digestion can be used to adjust fat supplementation programs for dairy cows.

Effects of jugular infused methionine, lysine, and histidine as a group or leucine and isoleucine as a group on production and metabolism in lactating dairy cows.

Essential AA (EAA), particularly leucine, isoleucine, methionine, and histidine, possess signaling properties for promoting cellular anabolic metabolism, whereas methionine, lysine, and histidine are considered also to be substrate limiting AA. The objective of this study was to evaluate production responses to supplementation of 2 AA groups in a 2 × 2 factorial design. Eight cows (99 ± 18 days in milk) were assigned to 4 jugular infusion treatments consisting of saline (CON), 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 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. Cows were ad libitum fed a common diet consisting of 15.2% crude protein and 1.61 Mcal/kg NEL on a dry matter basis that was predicted to meet rumen degradable protein requirements but was 15% deficient in metabolizable protein. Milk and energy-corrected milk yields increased by 2.3 kg/d and 1.9 kg/d, respectively, with infused IL, and no change was observed for MKH. Milk protein concentration increased by 0.13 percentage units for MKH, whereas milk protein yield increased for both MKH and IL by 84 g/d and 64 g/d, respectively. The milk protein yield increase for MKH+IL was 145 g/d versus CON. Gross feed efficiency tended to increase with IL infusion, and N efficiency tended to increase with MKH infusion. Aggregate arterial EAA concentrations less Met, Lys, and His declined by 7.2% in response to MKH infusion. Arterial EAA less Ile and Leu also declined by 6.2% in response to IL infusion. Net total AA (TAA) and EAA uptake by the udder tended to increase in response to MKH infusion, whereas mammary blood flow increased in response to IL infusion, but TAA and EAA net uptakes were unaffected. Apparent udder affinity increased for TAA and EAA less Met, Lys, and His in response to MKH infusion, whereas affinity for EAA less Ile and Leu increased for IL infusion. Venous Met and Leu concentrations increased by 192% and 35% from the MKH and IL infusions, respectively, compared with CON, which indicates that intracellular concentration of these EAA changed substantially. 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 EAA will limit milk protein yield.

Effects of jugular infusions of isoleucine, leucine, methionine, threonine, and other amino acids on insulin and glucagon concentrations, mammalian target of rapamycin (mTOR) signaling, and lactational performance in goats.

The supply and profile of absorbed AA may affect milk protein synthesis through hormonal changes and mammalian target of rapamycin (mTOR) signaling pathways; and Ile, Leu, Met, and Thr (ILMT) are the 4 AA that have been reported to have the greatest effect on mammary mTOR signaling. The extent to which ILMT and the other remaining AA (RAA) differ in their effects on milk protein synthesis needs to be systematically investigated. In this study, 5 lactating goats, averaging 120 ± 10 d in milk, fitted with jugular vein and carotid artery catheters, were fasted for 24 h, followed by intravenous infusions of a mixture containing AA and glucose for 8 h in a 5 × 5 Latin square design. The AA mixtures were formulated according to the profile of casein. The amounts of AA infused were calculated based on supplies of AA when metabolizable protein (MP) was at requirement (MR). Treatments were an infusate containing glucose without AA (NTAA); an infusate containing 3 × the MR of Ile, Leu, Met and Thr (3F0R); and infusates containing 3F0R plus 1, 2, or 3 × MR of RAA (3F1R, 3F2R, and 3F3R, respectively) according to amounts provided when fed to meet MP requirements for maintenance and lactation for each goat. Milk, arterial blood, and mammary tissue samples were collected immediately after halting the infusion. Relative to NTAA, supplementation of ILMT tended to increase milk protein production and plasma glucose concentrations, and increased milk and lactose production, but had no effects on production or content of milk fat. Graded supplementation of RAA tended to quadratically affect production of milk and lactose. Arterial glucose and glucagon concentrations decreased linearly, and plasma insulin concentrations decreased quadratically with increased RAA. Mammary p70-S6K1 phosphorylation was decreased by addition of ILMT compared with NTAA but increased linearly with increased RAA infusion. Furthermore, EIF4EBP1 gene expression was much lower for 3F-treated goats than for the NTAA treatment. Both MTOR and RPS6KB1 gene expressions were decreased quadratically with increased RAA supply. These results suggested that short-term milk protein yield tended to be increased by elevated ILMT availability, and this trend was not explained by variations in mammary mTOR signaling or pancreatic hormone secretions, whereas graded increase of RAA in combination with ILMT appeared to regulate the efficiency of conversion of glucose to lactose in a manner not involving milk protein production.

Assessing bioavailability of ruminally protected methionine and lysine prototypes.

Met and Lys are essential AA that can limit lactational performance in dairy cattle fed protein-sufficient diets. Thus, there is industry demand for ruminally protected (RP) sources of Met and Lys. One method of providing ruminal protection for Met and Lys is lipid encapsulation. The objective of this work was to assess 3 lipid-encapsulated Met prototypes (P1, P2, and P3) and 1 Lys prototype (P4) to determine ruminal protection, small intestine absorption (experiment 1), and animal production responses (experiment 2). Ruminal protection was estimated from 8-h in situ retention during ruminal incubation and intestinal absorption from plasma appearance after an abomasal bolus of the in situ retentate. Blood samples were collected over time to determine plasma Met and Lys concentration responses compared with unprotected Lys and Met infused abomasally. The prototypes were not exposed to the total diet or subjected to typical feed handling methods before evaluation. The bioavailability of P1, P2, and P3 Met prototypes was found to be 14, 21, and 18% of the initial AA material, respectively. The RP-Lys prototype had a bioavailability of 45%. To evaluate production responses, 20 Holstein cows were randomly assigned to 2 trials (n = 10 each) in a replicated Latin square design with 14-d periods. The base diet was predicted to be deficient in metabolizable Met (-14.8 g/d) and Lys (-16.1 g/d) per the Cornell Net Carbohydrate and Protein System (version 6.55). In the Met trial, the base diet was supplemented with RP-Lys to meet Lys requirements, and treatments were as follows: no added RP-Met (NCM), NCM plus Smartamine M (SM; Adisseo, Alpharetta, GA), and NCM plus P1, P2, or P3 at 148% of the Met content of SM. In the Lys trial, the base diet was supplemented with RP-Met to meet the Met requirement, and treatments were as follows: no added Lys (NCL), NCL plus AjiProL (AL; Ajinomoto Heartland Inc., Chicago, IL), and NCL plus P4 at 55, 78, or 102% of the reported absorbed Lys in AL. All products were top dressed on the diet without prior mixing or extended exposure to the rest of the diet. Milk protein concentration significantly increased when diets were supplemented with P2, P3, or SM (3.12, 3.12, and 3.11%, respectively) compared with NCM (3.02%). Only P1 (3.04%) was significantly lower than SM. Prototype P2 had the greatest numerical milk protein output response among the 3 RP-Met prototypes, suggesting that it may have had the greatest efficacy when supplemented into these rations. There was a numerical milk protein concentration response to AL and a linear increase in milk protein concentration for P4. The P4 and AL treatments resulted in comparable milk protein production regardless of P4 dose.

Meta-analysis of 2-hydroxy-4-methylthio-butanoic acid supplementation on ruminal fermentation, milk production, and nutrient digestibility.

Methionine is considered one of the most important essential AA for milk protein synthesis in dairy cows. Supplementation of unprotected, free Met is nearly 100% degraded by ruminal microorganisms, which complicates supplementation. 2-Hydroxy-4-methylthio-butanoic acid (HMTBa) can be converted to Met in the body and is used as a Met source in dairy production. However, results of published studies assessing the effects of supplementing Met sources, including HMTBa, on performance variables are inconsistent. A meta-analysis was performed to quantitatively summarize the accumulated results of HMTBa supplementation on animal performance and nutrient digestibility. Data pertaining to HMTBa dose, dietary composition, and major performance variables (rumen volatile fatty acid composition, milk production, nutrient digestibility) were collected from 39 articles containing 169 treatment means. Publications were from scientific journals published from 1970 to 2018; 1 internal report from Novus International Inc. (St. Charles, MO) was also included. The HMTBa effects on response variables were analyzed using linear mixed models with random study effects. Other explanatory variables tested included neutral detergent fiber and crude protein percent as well as days in milk. Results showed that HMTBa supplementation increased blood Met concentration and milk fat yield but had no effect on nutrient digestibility.

Effect of dietary phosphorus on intestinal phosphorus absorption in growing Holstein steers.

The effect of dietary P intake on intestinal P absorption was evaluated in growing Holstein steers. Diets varying in P content (0.15, 0.27, 0.36, and 0.45%, DM basis) were fed to 8 steers (174±10kg of BW) fitted with permanent duodenal and ileal cannulas in a replicated 4×4 Latin square with 14-d periods. Ytterbium-labeled corn silage and cobalt-EDTA were used as particulate and liquid phase markers, respectively, to measure digesta flow. Duodenal and ileal samples and spot urine samples were collected every 9 h from d 11 to 14. Total fecal collection was conducted on d 11 to 14 with fecal bags. Blood samples were collected from the coccygeal vessel on d 14. Feed, digesta, and fecal samples were analyzed for total P and inorganic P. Data were analyzed using PROC GLIMMIX in SAS with a model including treatment, square, period, and interaction of treatment and square. Preplanned contrasts were used to evaluate linear and quadratic treatment effects. Results were reported as least squares means. Dry matter intake (mean=4.90kg/d, 2.8% of BW) and apparent DM digestibility (mean=78.1%) were unaffected by treatment. Duodenal and ileal flow of total P increased linearly with increasing P intake (13.4, 18.5, 23.0, and 27.4g/d; 6.80, 7.87, 8.42, and 10.4g/d). Increasing P intake increased the quantity of P absorbed from the small intestine linearly (6.96, 11.1, 14.6, and 17.2g/d), but absorption efficiency was unchanged (mean=59.6%). Phosphorus was absorbed on a net basis from the large intestine, but this was not affected by treatment and was a small proportion of total P absorption. Blood inorganic P increased linearly with increased dietary P (4.36, 6.31, 7.68, and 8.5mg/dL) and salivary P secretion was unchanged (mean=5.79g/d), suggesting that rumen function was prioritized during short-term P deficiency. These data showing an absence of change in absorption efficiency and salivary P secretion in the face of short-term P deficiency may be used to improve published models of P digestion, absorption, and metabolism.

Effects of reduced dietary protein and supplemental rumen-protected essential amino acids on the nitrogen efficiency of dairy cows.

When fed to meet the metabolizable protein requirements of the National Research Council, dairy cows consume an excess of N, resulting in approximately 75% of dietary N being lost to the environment as urine and feces. Reductions in environmental N release could be attained through an improvement in N efficiency. The objective of this study was to determine if the predicted reduction in milk yield associated with feeding a low-protein diet to lactating dairy cows could be avoided by dietary supplementation with 1 or more ruminally protected (RP) AA. Fourteen multiparous and 10 primiparous Holstein cows, and 24 multiparous Holstein × Jersey crossbred cows were used in a Youden square design consisting of 8 treatments and 3 periods. The 8 dietary treatments were (1) a standard diet containing 17% crude protein [CP; positive control (PC)], (2) a 15% CP diet [negative control (NC)], (3) NC plus RP Met (+M), (4) NC plus RP Lys (+K), (5) NC plus RP Leu (+L), (6) NC plus RP Met and Lys (+MK), (7) NC plus RP Met and Leu (+ML), and (8) NC plus RP Met, Lys, and Leu (+MKL). Dry matter intake was not affected by treatment. Crude protein intake was lower for NC and RP AA treatments compared with the PC treatment. No detrimental effect was detected of the low-CP diet alone or in combination with AA supplementation on milk and fat yield. However, milk protein yield decreased for NC and +MKL diets, and lactose yield decreased for the +MKL compared with the PC diet. Milk urea N concentrations were lower for all diets, suggesting that greater N efficiency was achieved by feeding the low-protein diet. Minimal effects of treatments on arterial plasma essential AA concentrations were detected, with only Ile and Val being significantly lower in the NC than in the PC diet. Phosphorylation ratios of signaling proteins known to regulate mRNA translation were not affected by treatments. This study highlights the limitations of requirement models aggregated at the protein level and the use of fixed postabsorptive efficiency to calculate milk protein requirements. Milk protein synthesis regulation by signaling pathways in vivo is still poorly understood.

Casein synthesis is independently and additively related to individual essential amino acid supply.

Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P<0.1 and were removed from the model. Increasing concentrations of Ile, Leu, Met, and Thr simultaneously increased CFSR curvilinearly with a predicted maximum response of 4.32 ± 0.84%/h at 63% of DMEM concentrations. The maximum response to each of the 4 AA was at 71, 49, 60, and 32% of the concentration in DMEM, for Ile, Leu, Met, and Thr, respectively. These values correspond to 270, 120, 440, and 140% the plasma concentrations of Ile, Leu, Met, and Thr observed in lactating cows fed to meet National Research Council requirements, respectively. The CFSR estimated at those maxima were similar among AA (3.6 ± 0.6%/h). Individual AA effects on CFSR did not correlate with mammalian target of rapamycin (mTOR) signaling. Independent responses of CFSR to individual essential AA observed in this study contradict the single-limiting AA theory assumed in current requirement systems. The saturable responses in CFSR to these 4 AA also highlight the inadequacy of using a fixed postabsorptive AA efficiency approach for determining AA requirements for milk protein synthesis.

Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells.

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 × 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h(-1), whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h(-1). Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.

Effects of jugular-infused lysine, methionine, and branched-chain amino acids on milk protein synthesis in high-producing dairy cows.

In addition to lysine and methionine, current ration-balancing programs suggest that branched-chain amino acid (BCAA) supply may also be limiting in dairy cows. The objective of this study was to investigate whether BCAA, leucine, isoleucine, and valine become limiting for milk protein synthesis when methionine and lysine supply were not limiting. Nine multiparous Holstein cows with an average milk production of 53.5±7.1 kg/d were randomly assigned to 7-d continuous jugular infusions of saline (CTL), methionine and lysine (ML; 12 g and 21 g/d, respectively), or ML plus leucine, isoleucine, and valine (ML+BCAA; 35 g, 15 g, and 15 g/d, respectively) in a 3×3 Latin square design with 3 infusion periods separated by 7-d noninfusion periods. The basal diet consisted of 40% corn silage, 14% alfalfa hay, and a concentrate mix, and respectively supplied lysine, methionine, isoleucine, leucine, and valine as 6.1, 1.8, 4.7, 8.9, and 5.3% of metabolizable protein. Dry matter intake (23.9 kg/d), milk yield (52.8 kg/d), fat content (2.55%), fat yield (1.33 kg/d), lactose content (4.77%), lactose yield (2.51 kg/d), and milk protein efficiency (0.38) were similar across treatments. Protein yield and protein content were not significantly different between ML (1.52 kg/d and 2.88%, respectively) and ML+BCAA (1.51 kg/d and 2.83%, respectively), but they were significantly greater than that of CTL (1.39 kg/d and 2.71%). Cows that received ML+BCAA had less milk urea nitrogen content (10.9 mg/dL) compared with milk of CTL cows (12.4 mg/dL) and ML cows (11.8 mg/dL). Whereas high-producing cows responded positively to methionine and lysine supplementation, no apparent benefits of BCAA supplementation in milk protein synthesis were found. Infusion of BCAA may have stimulated synthesis of other body proteins, probably muscle proteins, as evidenced by decreased milk urea nitrogen.

Effects of lipid supplementation on the yield and composition of milk from cows with different beta-lactoglobulin phenotypes.

Responses to lipid supplementation differ between dairy breeds and genetic lines suggesting nutrition by genotype interactions. beta-Lactoglobulin phenotype is associated with changes in yield and composition of milk. The response of cows with different beta-lactoglobulin phenotypes to lipid supplementation has not been examined. Furthermore, we examined whether lipid supplementation alters milk protein composition. By using a randomized block design, we fed Holstein cows for 3 wk either a control diet containing 2.8% crude fat (n = 19) or an experimental diet that was supplemented with 4.2% tallow (n = 20). Before randomization, all cows were fed the supplemental tallow diet for at least 2 wk. Dry matter intake, body weight, milk yield, and milk composition were measured in the last week before and during the experimental period. Feeding supplemental tallow increased dry matter intake and yields of milk and milk components, including casein content, without decreasing milk component content or altering milk protein composition. On the low-fat control diet, cows with the beta-lactoglobulin allele B had a greater milk and milk component yield than cows with the A allele, whereas no differences by beta-lactoglobulin phenotype were observed in cows on the tallow supplement diet. Our results suggest that cows that differ in beta-lactoglobulin phenotype respond differently to a low-fat diet and that feeding cows 4.2% of additional tallow increases milk yield without affecting milk component content and milk protein composition.

Dietary calcium has little effect on mineral balance and bone mineral metabolism through twenty weeks of lactation in Holstein cows.

Calcium and P balance and mobilization from bone were evaluated through 20 wk of lactation to determine the timing and extent of net resorption of bone mineral and mineral balance in lactating dairy cows. Eighteen Holstein cows were blocked by parity and calving date and randomly assigned to 1 of 3 dietary treatments: high (1.03%, HI), medium (0.78%, MED), or low (0.52%, LOW) dietary Ca. Dietary P was 0.34% in all diets. Cows consumed treatment diets from calving to 140 DIM. Total collection of milk, urine, and feces was conducted 2 wk before expected calving and in wk 2, 5, 8, 11, and 20 of lactation. Blood samples were collected at 14 and 10 d before expected calving and 0, 1, 3, 5, 10, 14, 21, 28, 35, 56, 70, 84, 98, and 140 d after calving. Blood samples were analyzed for Ca, P, and parathyroid hormone concentration. Serum concentrations of osteocalcin (OC), a marker of bone formation, and deoxypyridinoline (DPD), a marker of bone resorption, were measured to assess bone mobilization. Rib bone biopsies were conducted within 10 d postcalving and during wk 11 and 20 of lactation. Dietary Ca concentration affected Ca balance, with cows consuming the HI Ca diet in positive Ca balance for all weeks with the exception of wk 11. Interestingly, all cows across all treatments had a negative Ca balance at wk 11, possibly the result of timed estrous synchronization that occurred during wk 11. At wk 20, Ca balances were 61.2, 29.9, and 8.1 g/d for the HI, MED, and LOW diets, respectively. Phosphorus balances across all treatments and weeks were negative. Bone Ca content on a fat-free ash weight basis was least in cows consuming the MED diet, but bone P was not different. Serum Ca and P were not affected by treatment. Dietary Ca concentration did not affect P balance in the weeks examined, but there was a clear effect of parity on balance, markers of bone metabolism, and bone P. Primiparous cows had greater serum OC and DPD concentrations than multiparous cows. Regardless of dietary treatment, serum OC concentration peaked around d 35 of lactation. Simultaneously, DPD concentration began to decrease, which may indicate a switch from net bone resorption to formation after d 35. However, this was not reflected in balance measures. This information may help refine dietary mineral recommendations for lactating dairy cows and suggests that dietary P requirements are independent of dietary Ca.

A model of phosphorus digestion and metabolism in the lactating dairy cow.

A dynamic, mechanistic, compartmental model of phosphorus (P) digestion and metabolism was constructed in the Advanced Continuous Simulation Language using conservation of mass principles and mass action kinetics. Phosphorus was assumed to exist in 3 forms: inorganic (Pi), phytic acid (Pp), and organic (excluding phytic acid; Po). All 3 forms were assumed to be present in the digestive tract with absorption of Pi into blood. Inputs to the model were total P intake; Pp, Po, and Pi as proportions of total P; milk yield; rate of salivation (fixed at 239 L/d); and rate of liquid passage from the rumen (fixed at 198 L/d). The model was fitted to 2 experiments from the literature. Derived parameters were well defined by the data. With a mean observed P intake of 75 g/d, total tract P digestibility was 38%. Phytic acid P digestibility in the rumen was 74%, with no additional Pp digestion in the lower tract. Inorganic P and Po digestibility in the lower tract were 48 and 89%, respectively. Flows of Po and Pi from the rumen were 2.4 and 3.0 times greater than intake, respectively. The increase in Po was apparently due to microbial growth. The increase in Pi arose primarily from secretion of Pi into the rumen via salivation where 65% of absorbed P was recycled to the rumen. Milk synthesis used 30% of absorbed Pi, and 1% was excreted in urine. This research suggested that the primary regulation points for maintaining blood P were bone deposition and resorption and absorption from the intestine. However, because bone P balance was related to both dietary P intake and ruminal phytase activity, it is critical to achieve a better understanding of phytate digestibility across several feeds if dietary P is to be reduced below current requirements.

Evaluation of solids, nitrogen, and phosphorus excretion models for lactating dairy cows.

Monitoring or accurately predicting manure quantities and nutrient concentrations is important for dairy farms facing strict environmental regulations. The objectives of this project were to determine the daily out-flow of manure nutrients from a free-stall barn using mass balance and to compare results with published excretion models. The project was conducted at the free-stall facility housing the lactating cow herd of the Virginia Tech Dairy Center in 2005. The herd consisted of 142 (+/-8.9) Holstein and Jersey cows with a mean body weight of 568 (+/-6.2) kg and average milk yield of 29.8 (+/-1.7) kg/d with 3.18% (+/-0.07) true protein and 3.81% (+/-0.13) milk fat on 18 sampling days. The intakes of dry matter (DM), N, and P were estimated from the formulated ration. Daily consumption averaged 21.7 (+/-0.27) kg of DM with 17.7% (+/-0.26) crude protein and 0.46% (+/-0.03) P. Approximately 110 (+/- 27.9) kg/d of sawdust was used as bedding; its contribution to manure flow was subtracted. The alleys in the free-stall barn were flushed every 6 h with recycled wastewater, and the slurry was collected. On 18 sampling days the volumes and constituents of the flushwater and the flushed manure were determined for a 6-h flush cycle and extrapolated to daily values. Net daily flow of solids and nutrients in manure were calculated as the differences between masses in flushed slurry and flushwater. Nitrogen and P excretion were also calculated from dietary inputs and milk output. The flow was compared with the American Society of Agricultural Engineers' (ASAE) standards. Each cow produced 5.80 kg/d of total solids (remainder after drying at 105 degrees C). The ASAE standard predicted DM (remainder after drying at 60 degrees C) excretion of 8.02 to 8.53 kg/d per cow. Recovery of P amounted to 74.8 g/d per cow. Overall, 102% of intake P was recovered; 75.1% in the manure outflow and 26.9% in milk. About 285 g/d and 148 g/d of N per cow were recaptured in manure and milk, respectively; 182 g/d was presumably volatilized. All models of N excretion appeared to underestimate N excretion. Volatilization rate of N amounted to 18.1%/h for the 6-h flush interval. Measured outflow of manure-P from the facility was similar to excretion predictions. Presentation of excreted solids as both total solids and DM is warranted. We conclude that using excretion prediction equations is useful for predicting excretion and outflow of P in a lactating cow facility, but N excretion predictions exhibited bias and have to be used prudently for predicting N outflow and N volatilization.

Milk protein synthesis as a function of amino acid supply.

Most prediction schemes of milk protein secretion overestimate milk protein yield from dairy cows at high protein intakes, thereby overestimating milk protein yield response to protein supplementation. This study was conducted to determine factors contributing to such an overestimation. Using published studies, a database was constructed that was limited to amino acid (AA) infusion studies, as then only the digestible amino acid of dietary origin needed to be estimated, whereas the amount infused was known exactly, thereby reducing the dependence on estimated values. Although milk protein yield was positively related with total energy supply, and both digestible duodenal supply and infused AA, in this database there was no relationship between milk protein yield response above control treatments and the nutrient status of the cows (energy or protein). Total milk protein yield was defined as a function of individual AA supply, using a segmented-linear and a logistic model to obtain estimates of the efficiency of conversion of AA into milk protein. Except for Lys and Met supply, the segmented-linear model yielded lower root mean square error and better correlation, but both models were similar in their reliability. For both models, the estimated efficiency of conversion of AA to milk differed among AA. Estimations of the ideal profile of AA for lactating dairy cows were similar between models, with requirements for Lys and Met in line with 2001 National Research Council recommendations. The major difference is that the segmented-linear model yields a constant efficiency of conversion of an AA until requirements are met, with zero efficiency beyond this point. The logistic model allows for an estimation of the decreasing marginal efficiency of conversion of AA as the supply approaches the requirements. The use of variable efficiency factors should improve our ability to predict protein yield in response to supplemental protein.