Decreased lactose percentage in milk associated with quarter health disorder and hyperketolactia, a proxy for negative energy balance, in dairy cows.

Several studies have described variations in lactose content (LC) in dairy cows during udder quarter health disorder or negative energy balance (NEB). However, their joint effects on LC have never been described. This was the aim of a longitudinal observational study performed on 5 Quebec dairy farms using automatic milking systems. Quarter milk samples were collected every 14 d from 5 to 300 DIM. Quarter health status was described by combining SCC level (SCC- or SCC+: < or ≥100,000 cells/mL, respectively) and infectious status (Patho- or Patho+: absence or presence of pathogens on a milk culture, respectively). Cows with NEB in early lactation (DIM <70) were identified using milk BHB content: <0.15 mM = BHB-; 0.15 to 0.19 mM = BHB+; >0.19 mM = BHB++. A total of 14,505 quarter cisternal milk samples were collected from 380 lactating cows. The quarter LC was analyzed using a mixed linear regression model with the following fixed effects: quarter health status, parity, time interval between last milking and sampling, quarter milk yield (in kg/d), DIM, and herd. A random quarter intercept with a repeated measures correlation structure and a cow random intercept were also specified. The LC of SCC+ quarters was lower (-0.17 ± 0.013 percentage points) compared with LC of SCC- quarters for both primiparous and multiparous cows. Of the 162 bacterial species identified, only 8 species had a prevalence greater than 4.0%, and just 5 of them were associated with a reduction in LC: Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus epidermidis, and Staphylococcus simulans. Cows identified as BHB+ and BHB++ in early lactation had a lower LC (-0.05 ± 0.019 and -0.13 ± 0.020 percentage points, respectively) compared with BHB- cows. For BHB++ cows, in both parity groups the decrease in LC (-0.20 ± 0.025 percentage points) was higher in SCC+ quarters compared with SCC- quarters. Moreover, the additive effect of the quarter health status and NEB on milk LC was greater with larger increases in BHB. Our findings highlight the necessity to jointly take into consideration both quarter health status and milk BHB concentration when using LC as a biomarker for NEB.

Synthesis of milk components involves different mammary metabolism adaptations in response to net energy and protein supplies in dairy cows.

Net energy for lactation (NEL) and metabolizable protein (MP) are the 2 main nutritional forces that drive synthesis of milk components. This study investigated mammary-gland metabolism in dairy cows in response to variations in the supply of NEL and MP. Four Holstein dairy cows were randomly assigned to a 4 × 4 Latin square design, in which each experimental period consisted of 14 d of dietary treatment. The diets provided 2 levels of NEL (low energy, 25.0 Mcal/d vs. high energy, 32.5 Mcal/d) and 2 levels of MP (low protein, 1,266 g/d vs. high protein, 2,254 g/d of protein digestible in the intestine) in a 2 × 2 factorial arrangement. Performance and dry matter intake (DMI) were measured during the last 5 d of each period, and the mammary net balance was measured on d 13 by collecting 6 sets of blood samples from the left carotid artery and left mammary vein. Mammary plasma flow was measured according to the Fick principle for Phe and Tyr. The mammary net balance of carbon equaled the uptake of nutrients expressed as carbon minus the output of lactose, fatty acids (FA) synthesized in the mammary gland, AA of milk protein, and glycerol-3P from triglyceride on d 13. Milk, lactose, fat, and protein yields increased when NEL and MP supplies increased. However, increasing the NEL supply increased FA synthesis more than increasing the protein supply did. In addition, FA secretion increased more than lactose secretion when the NEL supply increased. Increasing the NEL supply increased the left half-udder uptake of all major energy-yielding nutrients by increasing mammary plasma flow. However, nutrient uptake increased more than milk output did, which in turn increased carbon dioxide output. This increase in nutrient oxidation by the mammary gland decreased the mammary efficiency of nutrients utilization when the NEL supply increased. Increasing MP supply tended to increase glucose uptake through mammary clearance and increased mammary AA uptake with no change in mammary plasma flow. In addition, the protein supply did not change the mammary uptake of acetate or ß-hydroxybutyrate. The increase in milk-component secretions in response to either NEL or MP supplies occurred through different metabolic adaptations (increase in mammary plasma flow vs. clearances, respectively). These results suggest that the nutrient use by the mammary gland is highly flexible, which helps in maintaining milk and milk-component yields even with limiting nutrient supplies.

Extrusion of lupines with or without addition of reducing sugars: Effects on the formation of Maillard reaction compounds, partition of nitrogen and Nε-carboxymethyl-lysine, and performance of dairy cows.

The extrusion of leguminous seeds induces the formation of Maillard reaction compounds (MRC) as a product of protein advanced glycation and oxidation, which lowers protein degradability in the rumen. However, the quantitative relationship between the parameters of pretreatment (i.e., addition of reducing sugars) and extrusion, and the formation of MRC has not been established yet. Moreover, the fate of the main stable MRC, Nε-carboxymethyl-lysine (CML), in the excretory routes has never been investigated in ruminants. We aimed to test the effects of the temperature of extrusion of white lupines with or without addition of reducing sugars on the formation of MRC, crude protein (CP) degradability in the rumen, N use efficiency for milk production (milk N/N intake), and performance of dairy cows. Two experiments with a replicated 4 × 4 Latin square design were conducted simultaneously with 16 (3 rumen-cannulated) multiparous Holstein cows to measure indicators of ruminal CP degradability (ruminal NH3 concentration, branched-chain volatile fatty acids), metabolizable protein supply (plasma essential AA concentration), N use efficiency (N isotopic discrimination), and dairy performance. In parallel, apparent total-tract digestibility of dry matter, organic matter, neutral detergent fibers, N, total Lys and CML, and partition of N and CML were measured with 4 cows in both experiments. The diets consisted on a DM basis of 20% raw or extruded lupines and 80% basal mixed ration of corn silage, silage and hay from permanent grasslands, pelleted concentrate, and a vitaminized mineral mix. Expected output temperatures of lupine extrusion were 115°C, 135°C, and 150°C, without and with the addition of reducing sugars before extrusion. The extrusion numerically reduced the in vitro ruminal CP degradability of the lupines, and consequently increased the predicted supply of CP to the small intestine. Nitrogen balance and urinary N excretion did not differ among dietary treatments in either experiment. Milk yield and N use efficiency for milk production increased with extrusion of lupines at 150°C without addition of reducing sugars compared with raw lupines. Nitrogen isotopic discrimination between dietary and animal proteins (the difference between δ15N in plasma and δ15N in the diet) were lower with lupines extruded at 150°C without and with addition of reducing sugars. Regardless of sugar addition, milk true protein yield was not affected, but milk urea concentration and fat:protein ratio were lower with lupines extruded at 150°C than with raw lupines. In the CML partition study, we observed that on average 26% of the apparently digested CML was excreted in urine, and a much lower proportion (0.63% on average) of the apparently digested CML was secreted in milk, with no differences among dietary treatments. In conclusion, we showed that the extrusion of white lupines without or with addition of reducing sugars numerically reduced enzymatic CP degradability, with limited effects on N partition, but increased milk yield and N use efficiency at the highest temperature of extrusion without addition of reducing sugars.

Essential amino acid profile of supplemental metabolizable protein affects mammary gland metabolism and whole-body glucose kinetics in dairy cattle.

This study investigated mammary gland metabolism and whole-body (WB) rate of appearance (Ra) of glucose in dairy cattle in response to a constant supplemental level of metabolizable protein (MP) composed of different essential AA (EAA) profiles. Five multiparous rumen-fistulated Holstein-Friesian dairy cows (2.8 ± 0.4 lactations; 81 ± 11 d in milk; mean ± standard deviation) were abomasally infused according to a 5 × 5 Latin square design with saline (SAL) or 562 g/d of EAA delivered in different profiles where individual AA content corresponded to their relative content in casein. The profiles consisted of (1) a complete EAA mixture (EAAC), (2) Ile, Leu, and Val (ILV), (3) His, Ile, Leu, Met, Phe, Trp, Val (GR1+ILV), and (4) Arg, His, Lys, Met, Phe, Thr, Trp (GR1+ALT). A total mixed ration (58% corn silage, 16% alfalfa hay, and 26% concentrate on a dry matter basis) was formulated to meet 100 and 83% of net energy and MP requirements, respectively, and was fed at 90% of ad libitum intake on an individual cow basis. Each experimental period consisted of 5 d of continuous abomasal infusion followed by 2 d of no infusion. Arterial and venous blood samples were collected on d 4 of each period for determination of mammary gland AA and glucose metabolism. On d 5 of each period, D-[U-13C]glucose (13 mmol priming dose; continuous 3.5 mmol/h for 520 min) was infused into a jugular vein and arterial blood samples were collected before and during infusion to determine WB Ra of glucose. Milk protein yield did not differ between EAAC, GR1+ILV, and GR1+ALT, or between SAL and ILV, and increased over SAL and ILV with EAAC and GR1+ILV. Mammary plasma flow increased with ILV infusion compared with EAAC and GR1+ILV. Infusion of EAAC tended to increase mammary gland net uptake of total EAA and decreased the mammary uptake to milk protein output ratio (U:O) of non-EAA compared with SAL. Infusion of ILV increased mammary net uptake and U:O of Ile, Leu, and Val markedly over all treatments. The U:O of total Ile, Leu, and Val increased numerically (25%) with GR1+ILV infusion compared with EAAC, and the U:O of total Arg, Lys, and Thr tended to decrease, primarily from decreased U:O of Lys. During GR1+ALT infusion, U:O of total Arg, Lys, and Thr was greater than that during EAAC infusion, whereas U:O of Ile, Leu, and Val did not differ from EAAC. Glucose WB Ra increased 16% with GR1+ALT over SAL, and increased numerically 8 and 12% over SAL with EAAC and GR1+ILV, respectively. The average proportion of lactose yield relative to glucose WB Ra did not differ across treatments and averaged 0.53. On average, 28% of milk galactose arose from nonglucose precursors, regardless of treatment. In conclusion, intramammary catabolism of group 2 AA increased to support milk component synthesis when the EAA profile of MP was incomplete with respect to casein. Further, WB and mammary gland glucose metabolism was flexible in support of milk component synthesis, regardless of absorptive EAA profile.

Comparison of feed evaluation models on predictions of milk protein yield on Québec commercial dairy farms.

Feed evaluation models (FEM) are a core part in dairy cow feeding. As these models are developed using different biological and mathematical approaches mainly tested in a research context, their abilities to predict production in commercial farms need to be validated, even more so when they are used outside the context of their development. Four FEM-National Research Council, 2001 (NRC_2001); Cornell Net Carbohydrate and Protein System, 2015 (CNCPS); NorFor, 2011; and INRA, 2018 (INRA_2018)-were evaluated on their abilities to predict daily milk protein yield (MPY) of 541 cows from 23 dairy herds in the province of Québec, Canada. The effects of cow and diet characteristics were tested on the residuals of MPY. Sensitivity and uncertainty analyses were then performed to evaluate the influence of the uncertainty of the main characteristics of cows and feed ingredients measured on the farm and used in the 4 FEM on the predictions of metabolizable protein (MP) supply and MPY. The 4 models had acceptable predictions of MPY, with concordance correlation coefficients (CCC) ranging from 0.75 to 0.82 and total bias ranging from 12.8% to 19.3% of the observed mean. The Scandinavian model NorFor had the best predictions with a CCC of 0.82, whereas the 3 other models had similar CCC at 0.75 to 0.76. The INRA_2018 and NRC_2001 models presented strong central tendency biases. Removing herd effect put the 4 FEM at the same level of performance, with 11.9 to 12.4% error. Analyzing model behavior within a herd seems to partly negate the effect of using predicted dry matter intake (DMI) in the comparison of models. Diet energy density, days in milk, and MPY estimated breeding value were related to the residual in the 4 models, and Lys and Met (as percent of MP) only in NRC_2001 and NorFor. This suggests that inclusion of these factors in these models would improve MPY predictions. From the sensitivity analysis, for the 4 FEM, DMI and factors affecting its prediction had the greatest influence on the predictions of MP supply and MPY. Of the feed ingredients, forage composition had the greatest effect on these predictions, including a strong effect of legume proportion with NorFor. Diet acid detergent fiber concentration had a very strong effect on MP supply and MPY predictions only in INRA_2018, because of its effect on organic matter digestibility estimation. The range of predictions of MP supply and MPY when combining all these potential uncertainties varied depending on the models. The INRA_2018 model presented the lowest standard deviation (SD) and NorFor the highest SD for the predictions of both MP supply and MPY. Overall, despite the fact that FEM were developed in a research context, their use in a commercial context yields acceptable predictions, with NorFor yielding the best predictions overall, although within-herd responses varied similarly for the 4 tested models.

A multiparametric approach to assessing residual pain experienced by dairy cows undergoing digestive tract surgery under multimodal analgesia.

This study assessed residual pain responses of dairy cows undergoing fistulation surgery under multimodal analgesia using a multiparametric method combining behavioural and physiological indicators. A longitudinal study was conducted on five dairy cows, each acting as her own control. The surgery consisted of implanting a ruminal and a duodenal cannula in each cow. The multimodal drug protocol consisted of a combination of N-Methyl-D aspartic Acid antagonists, α2-agonists, and local anaesthetic during surgery, and non-steroidal anti-inflammatory drugs (NSAIDs) and opioid treatment postsurgery. Cow responses to surgery were monitored by direct behavioural observation, physiological assay indicators, and milk production from day (D) -6 days before surgery (D-6) to D13 postsurgery. From the data recorded, the variables that contributed most to the discrimination of days pre- and postsurgery were identified using factorial discriminant analysis. Components 1 and 2 of the factorial discriminant analysis explained 68.2% and 17.9%, respectively, of the total variance. Component 1 was mainly explained by haptoglobin (contribution to axis: 0.885), oxidative stress (ratio of oxidized gluthatione to reduced glutathione (GSH/GSSG), -0.746; vitamin E, -0.683; vitamin A, -0.597; malondialdehyde (MDA), 0.416), and behavioural indicators (general attitude, 0.594). On this axis, the higher the score, the higher were the apathy and haptoglobin and MDA concentrations, and the lower were the GSH/GSSG ratio and concentrations of vitamins A and E. This axis opposed cows on D-6 to cows on D3 and D5; cows on D1 and D13 were intermediate. Component 2 was mainly explained by the Hypothalamic-pituitary-adrenal axis (non-esterified fatty acid (NEFA), 0.686; cortisol, 0.669), milk yield (-0.725), oxidative stress (MDA, -0.584; nitric oxide (NO), 0.454), and behavioural indicators of pain (ear position, 0.467; leg postures, 0.431). On this axis, the higher the score, the higher the NEFA, cortisol, and nitric oxide concentrations; the more the ear and leg pain postures; and the lower the milk production and MDA concentrations. This axis opposed cows on D13 to cows on D1. These results suggest that cows may experience some pain only on D1, whereas on subsequent days, the inflammatory response and oxidative stress did not seem to be associated with pain. Our results should be considered for different surgeries to improve analgesia immediately after surgery, and to provide antioxidants along with NSAIDs to promote recovery.

Postruminal infusions of amino acids or glucose affect metabolisms of splanchnic, mammary, and other peripheral tissues and drive amino acid use in dairy cows.

Effects of AA and glucose infusions on efficiency of use of essential AA (EAA) were studied according to a 2 × 2 factorial using 5 multicatheterized cows in a 4 × 4 Latin square plus one cow, with 2-wk periods. The diet provided 87% of energy and 70% of metabolizable protein requirements, and the 4 treatments were abomasal infusions of (1) water, (2) an AA mixture with a casein profile (695 g/d), (3) glucose (1,454 g/d), or (4) a combination of AA and glucose infusions. Milk samples were collected on the last 6 milkings. On d 14, 6 blood samples were collected from arterial, and portal, hepatic, and mammary venous vessels. Splanchnic plasma flow was calculated by dilution of p-aminohippurate and mammary flow by the Fick principle using Phe + Tyr. The net flux of AA across tissues [splanchnic, i.e., portal-drained viscera (PDV) + liver, and mammary gland] was calculated as the efflux minus the influx across that tissue. The efficiency of EAA was calculated as the sum of exported true proteins [milk protein yield (MPY), scurf, and metabolic fecal protein] multiplied by their respective AA profile and divided by the predicted AA supply minus AA endogenous urinary loss. In addition, catabolism was estimated for each tissue: AA supply - (portal net flux + metabolic fecal protein) for the PDV; -hepatic net flux for the liver; splanchnic net flux - (-mammary net flux + scurf) for the other peripheral tissues; and -mammary net flux - milk for the mammary gland. The MIXED procedure (SAS Institute Inc., Cary, NC) was used with cow as a random effect. No AA × glucose interaction existed for most of the measured parameters. With infusions of AA and glucose, MPY increased by 17 and 14%, respectively. The decreased efficiency of EAA-N with AA infusion resulted from increased EAA-N in MPY smaller than the increased EAA-N supply and was accompanied by increased liver catabolism of His + Met + Phe (representing group 1 AA) and increased mammary and PDV catabolisms of group 2 AA-N (Ile, Leu, Lys, and Val). In contrast, the increased efficiency of EAA-N with glucose infusion, resulting from increased EAA-N in MPY with no change in EAA-N supply, was accompanied by decreased mammary catabolism of group 2 AA-N and hepatic catabolism of His + Met + Phe. No mammary catabolism of His, Met, and Phe existed in all treatments, as indicated by the mammary uptake to milk output ratio close to one for these EAA. Therefore, the mammary gland contributes significantly to variations of efficiency of group 2 AA-N through variations of AA catabolism, in response to both AA and glucose supplies, whereas additional PDV catabolism was observed with increased AA supply. Partition of AA use between tissues allows to delineate their anabolic or catabolic fate across tissues and better understand changes of efficiency of EAA in response to protein and energy supplies.

Amino acid efficiencies of utilization vary by different mechanisms in response to energy and protein supplies in dairy cows: Study at mammary-gland and whole-body levels.

Variations of mammary gland (MG) metabolism were studied in dairy cows in response to diets containing 2 levels of net energy of lactation [NEL; 25.0 and 32.5 Mcal/d for low (LE) and high energy (HE), respectively], combined with 2 levels of metabolizable protein [MP, 1,266 and 2,254 g/d of protein digestible in the intestine for low (LP) and high protein (HP), respectively] in a 2 × 2 factorial arrangement. Four cows received 4 diets (LELP, HELP, LEHP, and HEHP) in a 4 × 4 Latin square design with 2-wk experimental periods. Milk production and feed intake were measured on the last 5 d of each period, whereas MG net uptake of AA was determined on d 13. Efficiencies were estimated as the sum of measured milk true protein yield (MPY) and of estimations of metabolic fecal and scurf proteins multiplied by their respective AA profile and divided by the estimated AA supply minus the AA endogenous urinary loss. The increased MPY in the HE compared with the LE diets (higher by 123 g/d) was accompanied by increased mammary plasma flow and MG uptake of the nonessential AA (NEAA) and the essential AA (EAA), except for branched-chain AA. In contrast, the increase in MPY (higher by 104 g/d) observed in the HP compared with the LP diets was linked to increased MG uptake of EAA without a change in mammary plasma flow and a decreased NEAA uptake. Because MG uptake of total AA-N was almost equal to cows' milk output on a nitrogen basis, these different mechanisms involve a large MG flexibility, with variable synthesis of NEAA. In addition, MP efficiency did not increase only through increased MPY in the HE compared with the LE diets but also through metabolic fecal protein, estimated to increase (by 65 g/d) with dry matter intake. The MPY increased in the HP compared with the LP diets, but the increase was smaller than the calculated increase (greater by 993 g/d) in MP supply. The highest MG clearance rates of individual EAA could suggest that Met, His, and Lys were limiting in LP, and Met was the most limiting AA in HP. Interestingly, a similar hypothesis could be stated by analyzing estimated AA efficiencies. The highest efficiencies among EAA, observed for His in HELP and for Met with the other diets, could indicate that they were the most limiting AA in these respective diets, whereas other EAA (including Lys) efficiencies varied with MP efficiency. The MG metabolic flexibility with regard to individual AA utilization partially contributes to the anabolic fate of AA through MPY; however, other export proteins also contribute to variations in MP and AA efficiencies.

Effects of soybean meal or canola meal on milk production and methane emissions in lactating dairy cows fed grass silage-based diets.

This study evaluated the effects of soybean meal (SBM) and heat-moisture-treated canola meal (TCM) on milk production and methane emissions in dairy cows fed grass silage-based diets. Twenty-eight Swedish Red cows were used in a cyclic change-over experiment with 4 periods of 21 d and with treatments in 2 × 4 factorial arrangement (however, the control diet without supplementary protein was not fed in replicate). The diets were fed ad libitum as a total mixed ration containing 600 g/kg of grass silage and 400 g/kg of concentrates on a dry matter (DM) basis. The concentrate without supplementary protein consisted of crimped barley and premix (312 and 88 g/kg of DM), providing 130 g of dietary crude protein (CP)/kg of DM. The other 6 concentrates were formulated to provide 170, 210, or 250 g of CP/kg of DM by replacing crimped barley with incremental amounts of SBM (50, 100, or 150 g/kg of diet DM) or TCM (70, 140, or 210 g/kg of diet DM). Feed intake was not influenced by dietary CP concentration, but tended to be greater in cows fed TCM diets compared with SBM diets. Milk and milk protein yield increased linearly with dietary CP concentration, with greater responses in cows fed TCM diets compared with SBM diets. Apparent N efficiency (milk N/N intake) decreased linearly with increasing dietary CP concentration and was lower for cows fed SBM diets than cows fed TCM diets. Milk urea concentration increased linearly with increased dietary CP concentration, with greater effects in cows fed SBM diets than in cows fed TCM diets. Plasma concentrations of total AA and essential AA increased with increasing dietary CP concentration, but no differences were observed between the 2 protein sources. Plasma concentrations of Lys, Met, and His were similar for both dietary protein sources. Total methane emissions were not influenced by diet, but emissions per kilogram of DM intake decreased quadratically, with the lowest value observed in cows fed intermediate levels of protein supplementation. Methane emissions per kilogram of energy-corrected milk decreased more when dietary CP concentration increased in TCM diets compared with SBM diets. Overall, replacing SBM with TCM in total mixed rations based on grass silage had beneficial effects on milk production, N efficiency, and methane emissions across a wide range of dietary CP concentrations.

Diets rich in starch improve the efficiency of amino acids use by the mammary gland in lactating Jersey cows.

The objective of this study was to test whether the greater milk N yield usually observed when feeding diets based on starch versus fiber was the consequence of a higher efficiency of AA use across the mammary gland and whether this effect depended on dietary crude protein (CP) content. Five midlactation multicatheterized Jersey cows were fed 4 isoenergetic diets to provide 2 different carbohydrate compositions (CHO; rich in starch vs. rich in fiber) crossed by 2 different protein levels (12.0 vs. 16.5% CP) and according to a 4 × 4 Latin square design. Blood samples were collected at the end of each treatment period from the mesenteric artery and mammary vein to determine mammary net nutrient fluxes. The nature of nutrients taken up by the mammary gland differed between starch and fiber diets: mammary net uptake of acetate increased with fiber versus starch diets, whereas mammary net uptake and clearance rate of glucose increased with starch versus fiber diets but only at a normal CP level. In addition, the mammary net uptake of total, essential, and branched-chain AA (BCAA) was significantly enhanced (12, 11, and 26% on average, respectively) when feeding starch versus fiber diets, in line with a greater milk protein yield (7% on average) and regardless of the CP level. The conversion efficiency of plasma essential AA into milk protein was improved with starch diets (33.7% on average) compared with fiber diets (27.5% on average). This higher mammary efficiency use of AA with starch diets was accompanied by a greater fractional extraction and clearance rate of AA belonging to group 2 (BCAA, Lys, Thr) by the mammary gland in absence of effects of CHO on either the mammary blood flow or the mammary AA metabolism. The positive effect of starch diets on mammary clearance rate and uptake of BCAA observed in this study was further improved when increasing dietary CP from 12.0 to 16.5%. Concerning the individual AA, Leu was the only whose mammary uptake accounted for a higher proportion of total essential AA in diets based on starch versus fiber and whose mammary uptake to milk output ratio was modified (together with Pro). Diets rich in starch versus fiber improved the mammary AA utilization; however, some CHO × CP interactions on mammary metabolism support the concept of different metabolic pathways by which starch diets improve milk protein yield at the 2 studied CP levels. Results from this study suggest that mammary Leu and glucose metabolism can be modulated by the supply of glucogenic nutrients to the mammary gland.

Changes in mammary metabolism in response to the provision of an ideal amino acid profile at 2 levels of metabolizable protein supply in dairy cows: Consequences on efficiency.

The aim of this study was to compare the modifications in mammary gland metabolism by supplying an ideal versus an imbalanced essential AA (EAA) profile at low and high metabolizable protein (or PDIE, its equivalent in the INRA feeding system). Four lactating, multiparous Holstein cows received 4 treatments composed of 2 basal diets containing 2 levels of PDIE (LP or HP) and 2 different infusions of AA mixtures (AA- or AA+) in the duodenum. The AA+ mixture contained Lys, Met, Leu, His, Ile, Val, Phe, Arg, Trp, and Glu, whereas the AA- mixture contained Glu, Pro, and Ser. The infusion mixtures were iso-PDIE. The diet plus infusions provided 13.9 versus 15.8% of crude protein that corresponded to 102 versus 118g/kg of dry matter of PDIE in LP and HP treatments, respectively. The treatments were designed as a 2×2 crossover design of 2 levels of PDIE supply (LP vs. HP) with 28-d periods. Infusions of AA in the duodenum (AA- vs. AA+) were superimposed to diet within each 28-d period according to 2×2 crossover designs with 14-d subperiods. Increasing the PDIE supply tended to increase milk protein yield; however, the efficiency of PDIE utilization decreased and the plasma urea concentration increased, indicating a higher catabolism of AA. The AA+ treatments increased milk protein yield and content similarly at both levels of protein supply. This was explained by an increase in the mammary uptake of all EAA except His and Trp. The mammary uptake of non-EAA (NEAA) was altered to the increase in EAA uptake so that the total AA uptake was almost equal to milk protein output on a nitrogen basis. The ratio between NEAA to total AA uptake decreased from 46% in LPAA- to 40% in LPAA+, HPAA-, and HPAA+ treatments. The PDIE efficiency tended to increase in the AA+ versus the AA- treatments because the NEAA supply and the amount of NEAA not used by the mammary both decreased. Nevertheless, our AA+ treatments seemed not to be the ideal profile: the mammary uptake-to-output ratio for Thr was higher than 1 in LPAA-, but it decreased to 1 in all the other treatments, suggesting that Thr was deficient in these treatments. Conversely, an excess of His was indicated because its uptake was similar in AA+ and AA- treatments. In conclusion, balancing the EAA profile increased milk protein yield and metabolizable protein efficiency at both levels of protein supply by increasing the mammary uptake of EAA and altering the NEAA uptake, leading to less AA available for catabolism.

Diets rich in starch increase the posthepatic availability of amino acids in dairy cows fed diets at low and normal protein levels.

Five mid-lactation multicatheterized Jersey cows were used in a 4×4 Latin square design to investigate whether the increase in milk N yield associated with diets rich in starch versus fiber could originate from changes in the splanchnic AA metabolism and if these changes depended upon the dietary crude protein (CP) content. Four isoenergetic diets were formulated to provide 2 different carbohydrate compositions [diets rich in starch (350g of starch and 310g of neutral detergent fiber/kg of dry matter) versus rich in fiber (45g of starch and 460g of neutral detergent fiber/kg of dry matter)] crossed by 2 different CP contents (12.0 vs. 16.5% CP). At the end of each treatment period, 6 hourly blood samples were collected from the portal and hepatic veins as well as the mesenteric artery to determine net nutrient fluxes across the portal-drained viscera (PDV), liver, and total splanchnic tissues. Dry matter and calculated energy intake as well as total absorbed energy were similar across treatments. However, the net portal appearance (NPA) of acetate, total volatile fatty acids, and ß-hydroxybutyrate were higher with diets rich in fiber versus starch, whereas that of oxygen, glucose, butyrate, and insulin were lower. Concomitant to these changes, the percentage of N intake recovered as total AA (TAA) in the portal vein was lower for diets rich in fiber versus starch (42.3 vs. 51.4%, respectively), without, however, any difference observed in the NPA of the main AA used as energy fuels by the PDV (Glu, Gln, and Asp). Despite a higher NPA of TAA with starch versus fiber diets, no differences in the net hepatic flux of TAA, essential and nonessential AA were observed, resulting in a higher (+22%) net splanchnic release of AA and, hence, a greater (+7%) milk N yield. The net hepatic flux and hepatic fractional removal of none of the individual AA was affected as the main carbohydrate changed from fiber to starch, except for Gly and Lys, which were higher for the latter. After correcting for differences in NPA of TAA, the net hepatic uptake of TAA tended to be lower with starch versus fiber diets. The higher transfer of N from feed to milk with diets rich in starch is not the consequence of a direct sparing AA effect of glucogenic diets but rather the result of lower energy requirements by the PDV along with a higher microbial N flow to the duodenum. A better AA use by peripheral tissues with starch versus fiber diets was also hypothesized but more studies are warranted to clarify this issue.

Dietary carbohydrate composition modifies the milk N efficiency in late lactation cows fed low crude protein diets.

Nitrogen emissions from dairy cows can be readily decreased by lowering the dietary CP concentration. The main objective of this work was to test whether the milk protein yield reduction associated with low N intakes could be partially compensated for by modifying the dietary carbohydrate composition (CHO). The effects of CHO on digestion, milk N efficiency (milk N/N intake; MNE) and animal performance were studied in four Jersey cows fed 100% or 80% of the recommended protein requirements using a 4×4 Latin square design. Four iso-energetic diets were formulated to two different CHO sources (starch diets with starch content of 34.3% and NDF at 32.5%, and fiber diets with starch content of 5.5% and NDF at 49.1%) and two CP levels (Low=12.0% and Normal=16.5%). The apparent digestible organic matter intake (DOMI) and the protein supply (protein digestible in the small intestine; PDIE) were similar between starch and fiber diets. As planned, microbial N flow (MNF) to the duodenum, estimated from the urinary purine derivatives (PD) excretion, was similar between Low and Normal CP diets. However, the MNF and the efficiency of microbial synthesis (g of microbial N/kg apparently DOMI) were higher for starch v. fiber diets. Milk and milk N fractions (CP, true protein, non-protein N (NPN)) yield were higher for starch compared with fiber diets and for Normal v. Low CP diets. Fecal N excretion was similar across dietary treatments. Despite a higher milk N ouput with starch v. fiber diets, the CHO modified neither the urinary N excretion nor the milk urea-N (MUN) concentration. The milk protein yield relative to both N and PDIE intakes was improved with starch compared with fiber diets. Concentrations of ß-hydroxybutyrate, urea and Glu increased and those of glucose and Ala decreased in plasma of cows fed starch v. fiber diets. On the other hand, plasma concentration of albumin, urea, insulin and His increased in cows fed Normal compared with Low CP diets. This study showed that decreasing the dietary CP proportion from 16.5% to 12.0% increases and decreases considerably the MNE and the urinary N excretion, respectively. Moreover, present results show that at similar digestible OM and PDIE intakes, diets rich in starch improves the MNE and could partially compensate for the negative effects of Low CP diets on milk protein yield.

Milk protein responses in dairy cows to changes in postruminal supplies of arginine, isoleucine, and valine.

An ideal profile of essential AA (EAA) can improve the efficiency of metabolizable protein (or PDIE, the equivalent in the INRA feeding system) utilization in dairy cows. Compared with other EAA, existing recommendations for the requirements of Arg, Ile, and Val are few and inconsistent. Four multiparous Holstein dairy cows at 22±6 wk of lactation received 4 treatments (duodenal infusions of 445±22.4 g/d of an EAA mixture complementing a low-protein diet in a 4×4 Latin square design with a period length of 1 wk). The control treatment provided a balanced supply (in % of PDIE) of 5.1% Arg, 5.2% Ile, and 5.9% Val, whereas in the 3 subsequent treatments of -Arg, -Ile, and -Val, the concentrations of these 3 EAA were reduced to 3.5, 4.1, and 4.5%, respectively. All treatments were made isonitrogenous and were balanced to provide 7 other EAA (Lys, Met, His, Leu, Phe, Thr, and Trp), according to the recommendations described in the literature. Combined, the diet and the infusions provided 14.3±0.1% crude protein on a dry matter basis, and 66.0±1.2 g of PDIE/Mcal of net energy for lactation. Neither dry matter intake (19.2 kg/d) nor milk yield (30.4±0.4 kg/d) was affected by treatments. The -Arg and -Ile treatments did not modify milk protein synthesis or the efficiency of N utilization. However, the -Val treatment decreased milk protein content by 4.9% and milk crude protein content by 4.3%, and tended to decrease the efficiency of N use for milk protein yield by 3.7% (compared with the control). These effects of Val were related to a decrease in the plasma concentration of Val as well as a trend toward decreasing plasma concentrations of Met, His, and the sum of all EAA and nonessential AA in the -Val treatment, which indicates a different utilization of all AA in response to the Val deficit. The deletion of Ile, compared with the deletion of Val, tended to decrease the milk protein-to-fat ratio by 3.8%. In conclusion, the supply of Arg at 3.5% of PDIE was not limiting for milk protein synthesis. The slight effect on the milk protein-to-fat ratio caused by decreasing the supply of Ile suggests a need to reevaluate the Ile requirement more precisely. A low Val supply could be limiting for milk protein synthesis, provided that the requirements of Lys, Met, and His are met.

Milk protein synthesis in response to the provision of an "ideal" amino acid profile at 2 levels of metabolizable protein supply in dairy cows.

Providing a well-balanced supply of essential AA (EAA) can serve as an opportunity to reduce the protein intake for dairy cows by increasing the efficiency of metabolizable protein (or PDIE, its equivalent in the INRA feeding system) utilization for milk protein yield. Our objectives were to compare the effect of supplying an "ideal" EAA profile (EAA+) with an imbalanced AA profile (control) at 2 levels of PDIE/NE(L) (net energy for lactation) supplies to study the interaction between PDIE and AA profiles, and to compare this ideal profile with a simple mixture of the 4 most deficient EAA (4 EAA) in the diets of dairy cows. Six lactating multiparous Holstein cows received 6 treatments with 2 different levels of PDIE supplied by diets and AA infusions in the duodenum according to a changeover design with 3-wk periods. Within each PDIE supply level, the cows received 3 different AA infusions in the duodenum according to a 3×3 Latin square design with 1-wk subperiods, which corresponded to the following treatment groups: control (Glu), 4EAA (Lys, Met, His, Leu), and EAA+ (4 EAA plus Ile, Val, Phe, Trp, and Tyr). In the low and high PDIE treatments, diets and infusions provided 54.7 and 64.0 g/Mcal of PDIE/NE(L), respectively, which corresponded to crude protein levels of 13.6 and 15.2%, respectively. High-PDIE supplies increased the milk protein yield by 163 g/d, the milk protein content by 1.4 g/kg, the milk yield by 4.1 kg/d, and the lactose yield by 178 g/d and decreased the PDIE efficiency of utilization by 12.4%, whereas the N efficiency of utilization remained unaffected. Supplying the 2 EAA profiles (4EAA and EAA+) increased the milk protein yield by 67 g/d, the milk protein content by 1.3g/kg, and the milk yield by 0.9 kg/d, whereas the milk fat and milk lactose contents were decreased by 2.4 and 1.6g/kg, respectively. The responses regarding milk yield and its composition were similar whether the cows received the 4 EAA or the EAA+ treatment. The responses were similar for the milk yield and composition whether the EAA were supplied by low- or high-PDIE supplies. In conclusion, the efficiency of PDIE utilization was improved by 6.6% and the N efficiency was improved by 7.0% by correcting the EAA profiles, independent of the level of PDIE supplied. In addition, the increased efficiency observed, associated with provision of the 4 EAA, was similar to the provision of all EAA (EAA+) in this experiment.

Triennial Lactation Symposium: Mammary metabolism of amino acids in dairy cows.

Although in dairy cows the mammary gland (MG) is the major net user of essential AA (EAA) supply, milk protein synthesis from absorbed EAA is not a straightforward process. Early studies identified 2 groups of EAA based on different pattern of mammary utilization: group 1 [Met, Phe (+Tyr), Trp], where MG uptake was similar to secretion in milk protein, and group 2 (Arg, Ile, Leu, Lys, Thr, and Val), where uptake exceeded milk protein output. This review examines the validity of this classification under variable protein supply through a meta-analysis, with the outcomes then explained with studies in which the fates of individual EAA were monitored using isotope approaches. For the meta-analysis, the Fick principle, based on stoichiometric transfer of Phe+Tyr uptake to milk protein, was used to estimate mammary plasma flow across all studies. This approach was judged acceptable because doubling Phe supply did not result in mammary oxidation of Phe+Tyr and either limited or no contribution of peptides to Phe and Tyr mammary supply could be detected. The AA content of proteins synthesized by the MG was estimated from milk protein composition, and the uptake-to-output ratio (U:O) for individual AA was re-calculated based on these assumptions. Analysis of individual samples by isotopic dilution resulted in reduced variance compared with analysis on pooled samples performed with an AA analyzer. Globally, the U:O of His and Met is maintained close to unity under variable protein supply. The group 2 AA could be subdivided. First, the U:O for group "2v" AA (Ile, Leu, Val, and Lys) is greater than 1 and varied with protein supply. Accordingly, the increased U:O of Leu, induced by duodenal casein infusion, led to extra-mammary Leu oxidation. Decreasing Lys supply decreased Lys U:O and the associated transfer of N to non-EAA, mainly to Glx, Asx, Ser, and Ala. Second, the U:O of group "2nv" AA, Arg and Thr, does not vary with protein supply. The Arg U:O averages 2.5, whereas the Thr U:O, albeit averaging 1.2, does not differ from unity. Excess of both these AA is probably directed toward the synthesis of non-EAA rather than energy supply. Overall, the ability of the MG to use excess EAA-N supply offers alternative sources of N and C for energy provision, lactose synthesis and non-EAA synthesis. The latter function spares dietary non-EAA for other necessary processes, such as gluconeogenesis and energy supply, in other tissues to support lactation.

Alteration of the nutrient uptake by the udder over an extended milking interval in dairy cows.

Little is known about modifications of the mammary utilization of nutrients circulating in blood plasma when milk yield is strongly decreased by once-daily milking. A trial was carried out to describe the mammary nutritional adjustments linked to the downregulation of milk synthesis as milk accumulated over an extended milking interval in the bovine udder. Three Holstein dairy cows yielding 34.0 kg/d of milk were fitted with an ultrasound flow probe around the left external pudic artery and with catheters inserted into the left carotid and milk vein to estimate mammary blood flow (MBF) and mammary uptake of acetate, ß-hydroxybutyrate, nonesterified fatty acids, glycerol, glucose, O(2), and CO(2) release. The trial was carried out over 2 consecutive weeks, with wk 2 repeating wk 1. Cows were milked twice daily at 12-h milking intervals. On d 3, cows were milked at 0630 h and were not milked for 36 h until d 4 at 1830 h. Over the following days, twice-daily milking was resumed using 12-h milking intervals. Each half-udder was milked separately. Secretion rates of milk and milk proteins decreased 67% during the 12-to-36-h interval of milk accumulation, whereas that of milk fat fell 30%. Timing of changes in MBF and lactose levels in blood plasma was concomitant and significant after 19.5 and 21.5h of milk accumulation in the udder, respectively. The MBF decreased, most likely because the usual increases in MBF no longer occurred when the udder was full of milk. After 24h of milk accumulation, MBF did not increase further when cows lay down, and did not increase as usual 3h after a meal, suggesting a possible physical effect of milk accumulated in the udder on MBF, complementing metabolic regulation. Mammary uptake or release of nutrients was lowered before 24h for glucose, acetate, and ß-hydroxybutyrate and after 24h for total glycerol, O(2), and CO(2), mostly associated with the impaired MBF. However, these decreases ranged from 12 to 17%, and cannot entirely explain the -45 and -20% decreases in milk secretion rates observed during the entire 36 h of milk accumulation, thus confirming the primary role of intramammary metabolic regulation in the downregulation of milk secretion. The larger amount of nutrients taken up by the udder could explain the enhanced milk fat levels, involving a strongly modified metabolic fate of nutrients.

Effect of amino acid or casein supply on whole-body, splanchnic, and mammary glucose kinetics in lactating dairy cows.

This study was conducted to establish how AA supplied in a free form or as protein (casein, CN) affect the whole-body rate of appearance (WB Ra) of glucose, splanchnic and mammary glucose kinetics, and milk lactose secretion in lactating dairy cows. Five Holstein cows fitted with a rumen cannula and permanent indwelling catheters in the abomasum, portal, hepatic, and mesenteric veins, and one mesenteric artery, were used in a Youden square with 4 periods of 14 d each. Cows were fed a hay-based diet providing 100 and 70% of their net energy and metabolizable protein (MP) requirements, respectively. Treatments consisted of abomasal infusions of water (70% of MP requirements: control, Con), free AA (95% of MP requirements: AA1; and 120% of MP requirements: AA2), or CN (95% of MP requirements: CN1). The free AA mixture had the same profile as CN. On d 14 of each period, [6,6-(2)H(2)]glucose (25.8 mmol/h) was infused into a jugular vein, and blood samples (n=8) were taken over 4h from arterial, portal, hepatic, and mammary sources to measure glucose enrichment and concentration. Splanchnic and mammary plasma flows were determined by downstream dilution of para-aminohippurate and with the Fick principle, respectively. The last 6 milkings of each period were weighed and sampled to measure the yields of milk and components. The AA1 and CN1 treatments were not different for any of the measured parameters. Supplying AA linearly increased glucose WB Ra (AA2 vs. Con: +151 mmol/h) and liver net flux (+149 mmol/h). Utilization of glucose from the plasma compartment by the portal-drained viscera and liver and true portal absorption were not affected by AA supply. From these observations, we suggest that the increased WB Ra was due to increased net hepatic production. The AA from the infusion, in excess of that used to cover the increase in milk protein, were converted to glucose with an apparent efficiency close to 100% of maximum theoretical efficiency. Milk and lactose yields increased linearly with infusions of AA, by 14 and 16% with AA2 treatment, respectively. However, mammary glucose uptake was not significantly altered by AA infusions; this suggests that the mammary gland exerts active control on the uptake and utilization of glucose. For all treatments, the sum of true portal glucose absorption and true hepatic glucose production contributed more than 99% of WB Ra in the lactating cow; this would suggest that renal glucose synthesis makes only a small contribution to WB Ra under these conditions.

Whole-body glucose metabolism and mammary energetic nutrient metabolism in lactating dairy cows receiving digestive infusions of casein and propionic acid.

This study analyzed the effect of propionate (C3) and casein (CN) on whole-body and mammary metabolism of energetic nutrients. Three multiparous Holstein cows fitted with both duodenal and ruminal cannulas were used in 2 replicated Youden squares with 14-d periods. Effects of CN (743 g/d in the duodenum) and C3 (1,042 g/d in the rumen) infusions, either separately or in combination as supplements to a grass silage diet, were tested in a factorial arrangement. The control diet provided 97% of energy and protein requirements. Within each period, blood samples were taken (d 11) from the carotid artery and the right mammary vein to determine net uptake of energetic nutrients. Plasma blood flow was calculated using the Fick principle (based on Phe and Tyr). On d 13, [6,6-(2)H(2)]glucose was infused in the jugular vein to determine whole-body glucose rate of appearance (Ra) based on enrichments in arterial plasma. Both C3 and CN treatments increased whole-body Ra (17% and 13%, respectively) but only CN increased milk (18%) and lactose (14%) yields, suggesting no direct link between whole-body Ra and milk yield. When CN was infused alone, the apparent ratio of conversion of CN carbon into glucose carbon was 0.31 but, when allowance was made for the CN required to support the extra milk protein output, the ratio increased to 0.40, closer to the theoretical ratio (0.48). This may relate to the observed increases in arterial glucagon concentrations for CN alone. Conversely, the apparent conversion of infused C3 carbon alone to glucose was low (0.31). With C3, mammary plasma flow increased as did uptakes of lactate, Ala, and Glu whereas the uptake for beta-hydroxybutyrate (BHBA) decreased. Mammary net carbon balance suggested an increase with C3 treatment in glucose, lactate, Ala, and Glu oxidation within the mammary gland. Mammary glucose uptake did not increase with CN treatment, despite an increase in glucose arteriovenous difference and extraction rate, because plasma flow decreased (-17%). Whereas CN, alone or in combination with C3, increased both lactose and protein yields, only mammary AA (and BHBA in CN alone) uptake increased because plasma flow decreased (-17%). These data suggest that the observed variations of milk lactose yield (and other milk components) are linked to metabolic interchanges between several energetic nutrients at both the whole-body and mammary levels and are not explained by increases in whole-body glucose availability.

The production of intrinsically labeled milk protein provides a functional tool for human nutrition research.

Oral or intravenous administration of labeled, free amino acids does not allow the direct assessment of protein digestion and absorption kinetics following dietary protein intake. Consequently, dietary protein sources with labeled amino acids incorporated within the protein are required. The aim of this study was to produce milk proteins intrinsically labeled with l-[1-(13)C]phenylalanine that would allow the assessment of protein digestion and absorption kinetics and the subsequent muscle protein synthetic response to dietary protein intake in vivo in humans. Two Holstein cows (body weight of 726 +/- 38 kg) were continuously infused with l-[1-(13)C]phenylalanine at 402 micromol/min for 44 to 48 h, during and after which plasma samples and milk were collected. After milk protein separation, casein was used in a subsequent human proof-of-principle experiment. Two healthy males (aged 61 +/- 1 yr; body mass index of 22.4 +/- 0.1 kg/m(2)) ingested 35 g of casein highly enriched with [1-(13)C] phenylalanine. Plasma samples were collected at regular intervals, and skeletal muscle biopsies were collected before and 6 h after casein ingestion. In the initial experiment, a total of 5.83 kg of l-[1-(13)C]phenylalanine-enriched milk protein (casein enrichment was 29.4 molar percent excess) was collected during stable isotope infusion in the cows. In the proof-of-principle study, ingestion of 35 g of intrinsically labeled casein resulted in peak plasma l-[1-(13)C]phenylalanine enrichments within 90 min after protein ingestion (9.75 +/- 1.47 molar percent excess). Skeletal muscle protein synthesis rates calculated over the entire 6-h period averaged 0.058 +/- 0.012%/h. The production of intrinsically labeled milk protein is feasible and provides dietary protein that can be used to investigate protein digestion and absorption and the subsequent muscle protein synthetic response in vivo in humans.