Ability of three dairy feed evaluation systems to predict postruminal outflows of amino acids in dairy cows: A meta-analysis.

Adequate prediction of postruminal outflows of essential AA (EAA) is the starting point of balancing rations for EAA in dairy cows. The objective of this meta-analysis was to compare the performance of 3 dairy feed evaluation systems (National Research Council [NRC], Cornell Net Protein and Carbohydrate System version 6.5.5 [CNCPS], and National Academies of Sciences, Engineering and Medicine [NASEM]) to predict EAA outflows (Trp was not tested). The data set included a total of 354 treatment means from 70 duodenal and 24 omasal studies. To avoid Type I error, mean and linear biases were considered of concern if statistically significant and representing > 5.0% of the observed mean. Analyses were conducted on raw observed values and on observations adjusted for the random effect of study. The analysis on raw data indicates the ability of the feed evaluation system to predict absolute values whereas the analysis on adjusted values indicates its ability to predict responses of EAA outflows to dietary changes. For the prediction of absolute values (based on raw data), NRC underpredicted outflows of all EAA, from 5.3 to 8.6% of the observed mean (%obs.mean) except for Leu, Lys, and Val; NASEM overpredicted Lys (10.8%obs.mean); and CNCPS overpredicted Arg, His, Lys, Met, and Val (5.2 to 26.0%obs.mean). No EAA had a linear bias of concern with NASEM, followed by NRC for His (6.8%obs.mean), and CNCPS for all EAA (5.6 to 12.2%obs.mean) except Leu, Phe, and Thr. On the other hand, for the prediction of responses to dietary changes (based on adjusted data), NRC had 2 EAA presenting a linear bias of concern, followed by NASEM and CNCPS with 4 and 6 EAA, respectively. Predictions of His showed a linear bias of concern (5.3 to 9.6%obs.mean) with the 3 feed evaluation systems. Measured chemistry of crude protein and EAA were reported for 1 or more feed ingredients of the ration in 36% of the studies, and resulted in decreased linear biases in the 3 feed evaluation systems. The difference in mean biases of Met outflows was systematically positive when comparing omasal versus duodenal studies. Predictions of Met outflows with NRC had a higher concordance correlation coefficient in duodenal (used to develop NRC equations) versus omasal studies, whereas the opposite was observed with CNCPS, the latter showing the lowest mean bias for Met in omasal sampling studies. The 30% difference in Met mean biases between sampling sites appeared related to a similar difference found for observed Met versus NAN outflows between duodenal and omasal studies, which is independent of predictions. In conclusion, NRC and NASEM yielded accurate predictions of EAA outflows, with a small superiority of NASEM to predict absolute values, and slight superiority of NRC to predict the responses to dietary changes. In comparison, CNCPS may present mean and linear biases of concern for many EAA. Moreover, it remains to determine which sampling site is more representative of the true supply of EAA to the cows.

Ability of three dairy feed evaluation systems to predict postruminal outflows of nitrogenous compounds in dairy cows: A meta-analysis.

Adequate prediction of postruminal outflow of protein fractions is the starting point for the determination of metabolizable protein supply in dairy cows. The objective of this meta-analysis was to compare the performance of 3 dairy feed evaluation systems (National Research Council [NRC], Cornell Net Protein and Carbohydrate System [CNCPS], and National Academies of Sciences, Engineering and Medicine [NASEM]) to predict outflows (g/d) of nonammonia nitrogren (NAN), microbial N (MiN), and nonammonia nonmicrobial N (NANMN). Predictions of rumen degradabilities (% of nutrient) of protein (RDP), NDF, and starch were also evaluated. The data set included 1,294 treatment means from 312 digesta flow studies. The 3 feed evaluation systems were compared using the concordance correlation coefficient (CCC), the ratio of root mean square prediction error (RMSPE) on standard deviation of observed values (RSR), and the slope between observed and predicted values. Mean and linear biases were deemed biologically relevant and are discussed if higher than a threshold of 5% of the mean of observed values. The comparisons were done on observed values adjusted or not for the study effect; the adjustment had a small effect on the mean bias but the linear bias reflected a response to a dietary change rather than absolute predictions. For the absolute predictions of NAN and MiN, CNCPS had the best-fit statistics (8% greater CCC; 6% lower RMSPE) without any bias; NRC and NASEM underpredicted NAN and MiN, and NASEM had an additional linear bias indicating that the underprediction of MiN increased at increased predictions. For NANMN, fit statistics were similar among the 3 feed evaluation systems with no mean bias; however, the linear bias with NRC and CNCPS indicated underprediction at low predictions and overprediction at elevated predictions. On average, the CCC were smaller and RSR ratios were greater for MiN versus NAN indicating increased prediction errors for MiN. For NAN responses to a dietary change, CNCPS also had the best predictions, although the mean bias with NASEM was not biologically relevant and the 3 feed evaluation systems did not present a linear bias. However, CNCPS, but not the 2 other feed evaluation systems, presented a linear bias for MiN, with responses being overpredicted at increased predictions. For NANMN, responses were overpredicted at increased predictions for the 3 feed evaluation systems, but to a lesser extent with NASEM. The site of sampling had an effect on the mean bias of MiN and NANMN in the 3 feed evaluation systems. The mean bias of MiN was higher in omasal than duodenal studies in the 3 feed evaluation systems (from 55 to 61 g/d) and this mean bias was twice as large when 15N labeling was used as a microbial marker compared with purines. Such a difference was not observed for duodenal studies. The reasons underlying these systematic differences are not clear as the type of measurements used in the current meta-analysis does not allow to delineate if one site or one microbial marker is yielding the "true" postruminal N outflows. Rumen degradabilities of protein was underpredicted with CNCPS, and RDP responses to a dietary change was underpredicted by the 3 feed evaluation systems with increased RDP predictions. Rumen degradability of NDF was underpredicted and had poor fit statistics for NASEM compared with CNCPS. Fit statistics were similar between CNCPS and NASEM for rumen degradability of starch, but with an underprediction of the response with NASEM and absolute values being overpredicted with CNCPS. Multivariate regression analyses showed that diet characteristics were correlated with prediction errors of N outflows in each feed evaluation system. Globally, compared with NAN and NANMN, residuals of MiN were correlated with several moderators in the 3 feed evaluation systems reflecting the complexity to measure and model this outflow. In addition, residuals of NANMN were correlated positively with RDP suggesting an overestimation of this parameter. In conclusion, although progress is still to be made to improve equations predicting postruminal N outflows, the current feed evaluation systems provide sufficient precision and accuracy to predict postruminal outflows of N fractions.

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.

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.

Milk production and efficiency of utilization of nitrogen, metabolizable protein, and amino acids are affected by protein and energy supplies in dairy cows fed alfalfa-based diets.

Alfalfa has a lower fiber digestibility and a greater concentration of degradable protein than grasses. Dairy cows could benefit from an increased digestibility of alfalfa fibers, or from a better match between nitrogen and energy supplies in the rumen. Alfalfa cultivars with improved fiber digestibility represent an opportunity to increase milk production, but no independent studies have tested these cultivars under the agroclimatic conditions of Canada. Moreover, decreasing metabolizable protein (MP) supply could increase N use efficiency while decreasing environmental impact, but it is often associated with a decrease in milk protein yield, possibly caused by a reduced supply of essential AA. This study evaluated the performance of dairy cows fed diets based on a regular or a reduced-lignin alfalfa cultivar and measured the effect of energy levels at low MP supply when digestible His (dHis), Lys (dLys), and Met (dMet) requirements were met. Eight Holstein cows were used in a double 4 × 4 Latin square design, each square representing an alfalfa cultivar. Within each square, 4 diets were tested: the control diet was formulated for an adequate supply of MP and energy (AMP_AE), whereas the 3 other diets were formulated to be deficient in MP (DMP; formulated to meet 90% of the MP requirement) with deficient (94% of requirement: DMP_DE), adequate (99% of requirement: DMP_AE), or excess energy supply (104% of requirement; DMP_EE). Alfalfa cultivars had no significant effect on all measured parameters. As compared with cows receiving AMP_AE, the dry matter intake of cows fed DMP_AE and DMP_EE was not significantly different but decreased for cows fed DMP_DE. The AMP_AE diet provided 103% of MP and 108% of NEL requirements whereas DMP_DE, DMP_AE, and DMP_EE diets provided 84, 87, and 87% of MP and 94, 101, and 107% of NEL requirements, respectively. In contrast to design, feeding DMP_EE resulted in a similar energy supply compared with AMP_AE, although MP supply has been effectively reduced. This resulted in a maintained milk and milk component yields and improved the efficiency of utilization of N, MP, and essential AA. The DMP diets decreased total N excretion, whereas DMP_AE and DMP_EE diets also decreased milk urea-N concentration. Reducing MP supply without negative effects on dairy cow performance is possible when energy, dHis, dLys, and dMet requirements are met. This could reduce N excretion and decrease the environmental impact of milk production.

Diet supplementation with canola meal improves milk production, reduces enteric methane emissions, and shifts nitrogen excretion from urine to feces in dairy cows.

The objective of this study was to examine the effect of isonitrogenous substitution of solvent-extracted soybean meal (SBM) with solvent-extracted canola meal (CM) on enteric CH4 production, ruminal fermentation characteristics (including protozoa), digestion (in situ and apparent total-tract digestibility), N excretion, and milk production of dairy cows. For this purpose, 16 lactating Holstein cows, of which 12 were ruminally cannulated, were used in a replicated 4 × 4 Latin square (35-d periods; 14-d adaptation). The cows averaged (mean ± SD) 116 ± 23 d in milk, 692 ± 60 kg of body weight, and 47.5 ± 4.9 kg/d of milk production. The experimental treatments were control diet (no CM; 0%CM) and diets supplemented [dry matter (DM) basis] with 7.9% CM (8%CM), 15.8% CM (16%CM), or 23.7% CM (24%CM) on a DM basis. The forage:concentrate ratio was 52:48 (DM basis) and was similar among the experimental diets. Canola meal was included in the diet at the expense of SBM and soybean hulls, whereas the percentages of the other diet ingredients were the same. Intake of DM increased linearly, whereas apparent total-tract digestibility of DM, crude protein, neutral detergent fiber, and gross energy (GE) declined linearly as CM inclusion in the diet increased. Total volatile fatty acids concentration and butyrate molar proportion decreased linearly, whereas molar proportion of propionate increased linearly, and that of acetate was unaffected by CM inclusion in the diet. Ruminal ammonia concentration was not affected by inclusion of CM in the diet. Energy-corrected milk (ECM) yield increased linearly (up to 2.2 kg/d) with increasing CM percentage in the diet, whereas milk production efficiency averaged 1.63 kg of ECM/kg of DM intake and was unaffected by CM inclusion in the diet. Daily CH4 production decreased linearly with increasing CM percentage in the diet (489, 475, 463, and 461 g/d for 0%CM, 8%CM, 16%CM and 24%CM diets, respectively). As a consequence, CH4 emission intensity (g of CH4/kg of ECM) also declined linearly by up to 10% as the amount of CM increased in the diet. Methane production also decreased linearly when expressed relative to GE intake (5.7, 5.2, 5.1, and 4.9% for 0%CM, 8%CM, 16%CM and 24%CM diet, respectively). Quantity of manure N excretion was not affected by replacing SBM with CM; however, N excretion shifted from urine to feces as dietary percentage of CM increased, suggesting reduced potential for N volatilization. Results from this study show that replacing SBM with CM as a protein source in dairy cow diets reduced enteric CH4 emissions (g/d, % of GE intake, and adjusted for milk production) and increased milk production. The study indicates that CM can successfully, partially or fully, replace SBM in lactating dairy cow diets, with positive effects on animal productivity and the environment (i.e., less enteric CH4 emission and urinary N excreted). We conclude that compared with SBM, inclusion of CM meal in dairy cow diets can play a key role in reducing the environmental footprint of milk production.

Economic and environmental effects of revised metabolizable protein and amino acid recommendations on Canadian dairy farms.

The objective of this research was to evaluate the potential economic and environmental effects of the formulation model used to balance dairy rations for metabolizable protein (MP) or 3 essential AA (EAA: His, Lys, and Met) in 3 regions of Canada with different farming systems. The Maritimes, Central Canada, and the Prairies reference dairy farms averaged 63, 71, 144 mature cows per herd and 135, 95, 255 ha of land, respectively. Using N-CyCLES, a whole-farm linear program model, dairy rations were balanced for (1) MP, based on National Research Council (NRC) requirements (MP_2001); (2) MP plus Lys and Met, based on NRC (AA_2001); (3) MP (MP_Rev); or (4) for His, Lys, and Met (AA_Rev), both based on a revised factorial approach revisiting both supply and requirements of MP and EAA. Energy was balanced to meet requirements based on NRC (2001). Assuming the requirements were met within each approach, it was considered that milk yield and composition were not affected by the type of formulation. Given the assumptions of the study, when compared with MP_2001 formulation, balancing dairy rations using the AA_Rev approach reduced calculated farm N balance by 3.8%, on average from 12.71 to 12.24 g/kg of fat- and protein-corrected milk; it also enhanced farm net income by 4.5%, from 19.00 to 19.70 $CAN/100 kg of fat- and protein-corrected milk, by reducing inclusion of protein concentrate in dairy rations. Calculated animal N efficiency was on average 4.3% higher with AA_Rev than with MP_2001 for mid-lactation cows. This gain in N efficiency would result in a reduction in N2O emission by manure, contributing to a partial decrease of total greenhouse gas emission by 1.7%, through a reduction of N excreted in manure. With the AA_2001 formulation, farm N balance was 1% higher than with MP_2001 formulation while reducing farm net income by 6.4%, due to the need to purchase rumen-protected AA, with no effect on total greenhouse gas emission. Both MP formulations lead to fairly similar outputs. The AA_Rev formulation also indicated that His might be a co-limiting AA with Met in dairy rations balanced with ingredients usually included in Canadian dairy rations. Given the assumptions of the study, balancing dairy rations for 3 EAA (His, Lys, and Met) rather than MP, has some potential positive effects on Canadian dairy farms by increasing net incomes through a reduction of crude protein supply, leading to a decreased environmental effect.

Histidine optimal supply in dairy cows through determination of a threshold efficiency.

Two His deletion studies were conducted to examine the mechanisms used by dairy cows to support milk true protein yield (MTPY) when His supply is altered. The potential mechanisms involved in how the efficiency of utilization of His varied included reduced catabolism, more efficient mammary usage, and use of His labile pools. For the first study, 5 multicatheterized cows were used in a 4 × 4 Latin square plus 1 cow with 14-d periods. Treatments were abomasal infusion of increasing doses of His (0, 7.6, 15.2, and 20.8 g/d) in addition to a mixture of AA (595 g/d; casein profile excluding His). Cows were fed the same protein-deficient diet throughout the study. The MTPY increased linearly with a quadratic tendency with increasing doses of His. Muscle concentrations of carnosine, a His-based dipeptide, tended to increase in a quadratic manner with increasing His supply, suggesting that the 0- and 7.6-g doses were insufficient to cover His requirement. Liver catabolism of His decreased as His supply decreased. Mammary fractional removal of His was considerably greater at low His supply, but the ratio of His mammary net uptake to milk output was not affected by the rate of His infusion, averaging 1.02. The mechanisms to face a reduced His supply included reduced His hepatic catabolism, more efficient His mammary use of lowered arterial supply, and, to a lesser extent, use of His labile pools. Two independent estimates of His efficiency were calculated, one based on the sum of exported proteins (measured MTPY plus estimated metabolic fecal protein and scurf; i.e., the anabolic component, EffMTPY) and the other based on liver removal (i.e., the catabolic component). These 2 estimates followed the same pattern of response to His supply, decreasing with increasing His supply. The EffMTPY at which MTPY peaked was 0.785. For the second study, 6 cows were used in a 6 × 6 Latin square with 7-d periods. Two greater doses of His (30.4 and 38.0 g/d) were added; otherwise, the nutritional design was similar to the first study. In this second study, the indicator AA oxidation technique was used instead of the multiorgan approach, with labeled Leu as the indicator of His utilization. The MTPY peaked and Leu oxidation reached the nadir at an average EffMTPY of 0.763. Combined across both studies, the data indicate that optimal usage of His would occur at a threshold EffMTPY of 0.77. The agreement between experimental approaches across both studies indicates that the biological optimal supply of His expressed in grams per day could be calculated as the sum of exported proteins divided by this EffMTPY plus estimated endogenous urinary excretion.

Review: Impact of protein and energy supply on the fate of amino acids from absorption to milk protein in dairy cows.

Making dairy farming more cost-effective and reducing nitrogen environmental pollution could be reached through a reduced input of dietary protein, provided productivity is not compromised. This could be achieved through balancing dairy rations for essential amino acids (EAA) rather than their aggregate, the metabolizable protein (MP). This review revisits the estimations of the major true protein secretions in dairy cows, milk protein yield (MPY), metabolic fecal protein (MFP), endogenous urinary loss and scurf and associated AA composition. The combined efficiency with which MP (EffMP) or EAA (EffAA) is used to support protein secretions is calculated as the sum of true protein secretions (MPY + MFP + scurf) divided by the net supply (adjusted to remove the endogenous urinary excretion: MPadj and AAadj). Using the proposed protein and AA secretions, EffMP and EffAA were predicted through meta-analyses (807 treatment means) and validated using an independent database (129 treatment means). The effects of MPadj or AAadj, plus digestible energy intake (DEI), days in milk (DIM) and parity (primiparous v. multiparous), were significant in all models. Models using (MPadj, MPadj × MPadj, DEI and DEI × DEI) or (MPadj/DEI and MPadj/DEI × MPadj/DEI) had similar corrected Akaike's information criterion, but the model using MPadj/DEI performed better in the validation database. A model that also included this ratio was, therefore, used to fitting equations to predict EffAA. These equations predicted well EffAA in the validation database except for Arg which had a strong slope bias. Predictions of MPY from predicted EffMP based on MPadj/DEI, MPadj/DEI × MPadj/DEI, DIM and parity yielded a better fit than direct predictions of MPY based on MPadj, MPadj × MPadj, DEI, DIM and parity. Predictions of MPY based on each EffAA yielded fairly similar results among AA. It is proposed to ponder the mean of MPY predictions obtained from each EffAA by the lowest prediction to retain the potential limitation from AA with the shortest supply. Overall, the revisited estimations of endogenous urinary excretion and MFP, revised AA composition of protein secretions and inclusion of a variable combined EffAA (based on AAadj/DEI, AAadj/DEI × Aadj/DEI, DIM and parity) offer the potential to improve predictions of MPY, identify which AA are potentially in short supply and, therefore, improve the AA balance of dairy rations.

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.

Does blending canola meal with other protein sources improve production responses in lactating dairy cows? A multilevel mixed-effects meta-analysis.

The objective of this meta-analysis was to evaluate the effect of blending canola meal (CM) with other protein sources on production responses in lactating dairy cows. To evaluate this effect, a data set was assembled containing 22 studies reporting at least 3 isonitrogenous dietary treatments (total of 74 treatment means). Each study needed to report 1 diet with CM <0.3 kg/d, 1 or more diets consisting of CM blended with another protein source, and 1 diet with CM as the main protein source in the protein supplement (>85%). The crude protein (CP) concentration of CM averaged 37.4 ± 3.09% (dry matter basis), and the predictor of interest was the intake of CP from CM, which averaged 0.46 ± 0.413 kg/d among studies. The maximal CP from CM ranged from 0.47 to 1.55 kg/d among studies. The quadratic relationship between CP from CM and responses in milk true protein concentration was significant, the maximum response (3.19%) being reached at 0.79 kg of CP from CM; the quadratic relationships were not significant for the other dependent variables. Responses in dry matter intake; yields of milk, energy-corrected milk, and milk true protein; and apparent N efficiency were related positively to CP from CM and negatively for responses in milk fat and milk urea N concentrations. Remembering that diets were isonitrogenous within studies, this indicates no nutritional benefit of blending CM with another protein source. Taken together, the results indicate that the whole-body N utilization efficiency by the dairy cow improved and that more dietary protein was used to synthesize milk protein when CM was used as the sole protein source in the protein supplement up to 1.55 kg/d.

Plasma essential amino acid concentrations in response to casein infusion or ration change in dairy cows: A multilevel, mixed-effects meta-analysis.

The objectives of the current meta-analysis were to review the relationships between plasma individual essential AA concentrations and their respective AA digestible flows (AADI) in 2 independent sets of studies. The first set of studies included 36 casein infusion studies (CN; 83 treatment means) and was regarded as the best comparison standard available, because differences in AADI achieved by casein infusion (up to 40% of total metabolizable protein supply) did not rely on any model assumptions and were directly estimated from casein infusions. The second set of studies included 42 feeding trials (FT; 94 treatment means) in which AADI were predicted using the 2001 National Research Council model. The 2 sets of studies were not balanced for dry matter intake and the supplies of metabolizable protein and net energy for lactation; therefore, a subset of 17 CN studies (35 treatment means) and 19 FT trials (49 treatment means) balanced for these variables was assembled to allow the comparison of linear terms from CN and FT studies. In the subset of data set, the linear terms of individual AA did not differ between CN and FT studies except for Met and Thr, with a slope lower by 23 and 62%, respectively, in CN versus FT studies. The agreement in linear slopes between CN and FT studies indicates, indirectly, that AADI were predicted accurately by the National Research Council model. In the large data set, the relationships between plasma concentrations of Ile, Leu, Val, and their sum (branched-chain AA; BCAA) and their respective AADI shared common characteristics that distinguished them from the other AA. For the plasma concentration of BCAA, the linear terms were significant in CN and FT studies, but the quadratic terms were significant only in FT studies. This decline in the response of plasma concentration of BCAA to increased BCAA digestible flow in FT studies was associated with diets rich in energy, diets with a high concentrate level, or diets based on corn silage. These dietary conditions can stimulate insulin secretion and decrease plasma concentration of BCAA. For the non-BCAA, a quadratic term was significant for plasma His, Lys, Met, and Thr in each set of studies, indicating an increased removal of these AA by the liver as AADI increased.

Estimation of correction factors to determine the true amino acid concentration of protein after a 24-hour hydrolysis.

Although it has been acknowledged for a long time that a single period of hydrolysis, normally 21 to 24 h, is not the optimal time for most of the AA, a single period is routinely used due to time and cost constraints. As models to balance dairy rations for proteins are evolving toward balancing for AA, it becomes critical to improve the predictions of AA supply from digested proteins. Our objective was to develop correction factors that could systematically be applied to AA concentrations obtained after a 24-h hydrolysis of proteins to account for incomplete recovery and therefore determine their true AA composition. Thirteen substrates were selected to represent different types of proteins commonly used to estimate the supply of AA in ration formulation models: feed ingredients (grass silage, corn silage, soybean meal, canola meal, high-protein corn dried distillers grains, and wheat dried distillers grains plus solubles), 16-h rumen residues (soybean meal and canola meal), digesta (duodenal digesta and feces), and rumen microorganisms (fluid-associated bacteria, particle-associated bacteria and protozoa). Each protein was hydrolyzed in 6 N HCl for multiple hydrolysis times: 13 (2, 4, 8, 12, 18, 21, 24, 30, 48, 72, 96, 120, and 168 h) for feed ingredients, rumen residues, and digesta, and 9 (2, 4, 8, 18, 24, 30, 48, 96, and 168 h) for rumen microorganisms; all analyses were conducted in triplicate. Using nonlinear regression, the AA composition in the protein before the hydrolysis (A0) was derived for each AA in each protein. Two ratios were calculated as potential correction factors: A0/24-h concentration (A0/24h) and the maximal concentration/24-h concentration (max/24h). Both ratios were tested to determine if the type of proteins was affecting them. The ratios A0/24h were not affected by the type of proteins, whereas the ratios max/24h were also not affected by the type of proteins except for 3 nonessential AA (Ala, Glu, and Gly). In an attempt to propose correction factors, our results were combined with results from the literature reporting ratios A0/24h, ratios max/24h, or the ratio of the AA composition calculated from gene structure/24 h. The correction factors proposed for individual AA varied from 1.02 (Asp) to 1.12 (Thr). For the essential AA, the highest ratios were obtained, as expected, for the branched-chain AA and Thr. Formulation programs balancing dairy rations for essential AA would need to acknowledge the incomplete recovery of AA when obtained from 24-h hydrolysis and include correction factors, specific for each AA, but the same across different types of proteins, to correctly estimate the true AA supply to dairy cows.

Relationships between postruminal casein infusion and milk production, and concentrations of plasma amino acids and blood urea in dairy cows: A multilevel mixed-effects meta-analysis.

The relationships between postruminal casein infusion and production variables and concentrations of plasma AA and blood urea were evaluated using multilevel mixed-effects models derived from literature data collected in dairy cows. The data set contained 147 treatment means [i.e., 66 controls (CTL) and 81 casein-infused (CAS) means]. Each CAS mean was paired with its corresponding CTL mean to create 81 mean differences (CAS minus CTL), which were analyzed as absolute and percentage-based units (i.e., percentage increase or decrease in CAS relative to CTL). The primary variable of interest was the difference in estimated metabolizable protein (MP) supply (ΔMP) between CAS and CTL. The other explanatory variables were based on levels in CTL: MP supply, MP balance, the ratio of duodenal microbial protein (MCP) to MP supply (MCPMP), the stage of lactation (early or mid/late) and the type of forage (grass/legume- or corn silage-based). The MP supply and MP balance influenced negatively the relationship between ΔMP and the response of true protein yield. Responses of milk urea, blood urea, and plasma urea cycle AA concentrations were associated positively with ΔMP, indicating that a large amount of infused AA was catabolized to urea. Responses of plasma essential AA concentrations were related positively to ΔMP. The relative effect of ΔMP was highest for responses of plasma His concentration in cows fed grass/legume-based diets and at high MCPMP ratios. This relationship suggests that positive responses of plasma His concentrations are associated with diets relying heavily on microbial protein synthesis (high MCP), low in crude protein (low estimated MP supply), or both. The relationship between ΔMP and responses of plasma group 2 AA (Ile, Leu, Lys, and Val) concentrations was approximately 2 times greater than that for group 1 AA (His, Met, and Phe+Tyr) at mean MCPMP and MP supply. This could reflect the low hepatic removal group 2 AA compared with group 1 AA in dairy cows. Collectively, these results provide novel information on how dietary and cow conditions may alter responses to protein supplementation.

Amino acid composition of rumen bacteria and protozoa in cattle.

Because microbial crude protein (MCP) constitutes more than 50% of the protein digested in cattle, its AA composition is needed to adequately estimate AA supply. Our objective was to update the AA contributions of the rumen microbial AA flowing to the duodenum using only studies from cattle, differentiating between fluid-associated bacteria (FAB), particle-associated bacteria (PAB), and protozoa, based on published literature (53, 16, and 18 treatment means were used for each type of microorganism, respectively). In addition, Cys and Met reported concentrations were retained only when an adequate protection of the sulfur groups was performed before the acid hydrolysis. The total AA (or true protein) fraction represented 82.4% of CP in bacteria. For 10 AA, including 4 essential AA, the AA composition differed between protozoa and bacteria. The most noticeable differences were a 45% lower Lys concentration and 40% higher Ala concentration in bacteria than in protozoa. Differences between FAB and PAB were less pronounced than differences between bacteria and protozoa. Assuming 33% FAB, 50% PAB, and 17% of protozoa in MCP duodenal flow, the updated concentrations of AA would decrease supply estimates of Met, Thr, and Val originating from MCP and increase those of Lys and Phe by 5 to 10% compared with those calculated using the FAB composition reported previously. Therefore, inclusion of the contribution of PAB and protozoa to the duodenal MCP flow is needed to adequately estimate AA supply from microbial origin when a factorial method is used to estimate duodenal AA flow. Furthermore, acknowledging the fact that hydrolysis of 1 kg of true microbial protein yields 1.16 kg of free AA substantially increases the estimates of AA supply from MCP.

Deletion of the GAA repeats from the human frataxin gene using the CRISPR-Cas9 system in YG8R-derived cells and mouse models of Friedreich ataxia.

The Friedreich ataxia is a monogenic disease due to a hyperexpanded GAA triplet located within the first intron of the frataxin gene that causes transcriptional issues. The resulting frataxin protein deficiency leads to a Fe-S cluster biosynthesis dysfunction in the mitochondria and to oxidative stress and cell death. Here we use the CRISPR-Cas9 system to remove the mutated GAA expansion and restore the frataxin gene transcriptional activity and protein level. Both YG8R and YG8sR mouse models and cell lines derived from these mice were used to CRISPR-edited successfully the GAA expansion in vitro and in vivo. Nevertheless, our results suggest the YG8sR as a better and more suitable model for the study of the CRISPR-Cas9 edition of the mutated frataxin gene.

Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice.

Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases.

Casein infusion rate influences feed intake differently depending on metabolizable protein balance in dairy cows: A multilevel meta-analysis.

The effects of casein infusion have been investigated extensively in ruminant species. Its effect on responses in dry matter intake (DMI) has been reviewed and indicated no significant effect. The literature reviewed in the current meta-analysis is more extensive and limited to dairy cows fed ad libitum. A total of 51 studies were included in the meta-analysis and data were fitted to a multilevel model adjusting for the correlated nature of some studies. The effect size was the mean difference calculated by subtracting the means for the control from the casein-infused group. Overall, casein infusion [average of 333 g of dry matter (DM)/d; range: 91 to 1,092 g of DM/d] tended to increase responses in DMI by 0.18 kg/d (n=48 studies; 3 outliers). However, an interaction was observed between the casein infusion rate (IR) and the initial metabolizable protein (MP) balance [i.e., supply minus requirements (NRC, 2001)]. When control cows were in negative MP balance (n=27 studies), responses in DMI averaged 0.28 kg/d at mean MP balance (-264 g/d) and casein IR (336 g/d), and a 100g/d increment in the casein IR from its mean increased further responses by 0.14 kg/d (MP balance being constant), compared with cows not infused with casein. In contrast, when control cows were in positive MP balance (n=22 studies; 2 outliers), responses in DMI averaged -0.20 kg/d at mean casein IR (339 g/d), and a 100g/d increment in the casein IR from its mean further decreased responses by 0.33 kg/d, compared with cows not infused with casein. Responses in milk true protein yield at mean casein IR were greater (109 vs. 65 g/d) for cows in negative vs. positive MP balance, respectively, and the influence of the casein IR on responses was significant only for cows in negative MP balance. A 100g/d increment in the casein IR from its mean increased further responses in milk true protein yield by 25 g/d, compared with cows not infused with casein. Responses in blood urea concentration increased in casein studies (+0.59 mM) and the influence of the casein IR was greatest for cows in positive MP balance (0.26 vs. 0.11 mM per 100g/d increment). Responses in DMI were also correlated negatively with responses in blood urea concentration only for cows in positive MP balance. Together, these results suggest an association between satiety and deamination and oxidation of AA supplied in excess of requirements for cows in positive MP balance. Therefore, casein stimulated appetite in cows fed MP-deficient diets possibly via the supply of orexigenic AA or through a pull effect in response to an increased metabolic demand. Conversely, casein induced satiety in cows fed diets supplying MP in excess of requirements. Not precluding other factors involved in satiety (e.g., insulin, gut peptides), casein could have increased the supply of AA (e.g., Ser, Thr, Tyr), which might depress appetite at the brain level or increase the deamination and the oxidation of AA in oversupply in agreement with the hepatic oxidation theory.

Short communication: Casein hydrolysate and whey proteins as excipients for cyanocobalamin to increase intestinal absorption in the lactating dairy cow.

Bioavailability of vitamin B12 is low in humans and animals. Improving vitamin B12 absorption is important for optimal performance in dairy cows and for increasing vitamin B12 concentrations in milk for human consumption. However, when supplemented in the diet, 80% of synthetic vitamin B12, cyanocobalamin (CN-CBL), is degraded in the rumen of dairy cows and only 25% of the amount escaping destruction in the rumen disappears from the small intestine between the duodenal and ileal cannulas. In pigs, vitamin B12 from milk is more efficiently absorbed than synthetic CN-CBL. The objective of this study was to determine the efficacy of casein hydrolysate and whey proteins as excipients for CN-CBL to increase portal-drained viscera (PDV) flux of the vitamin in lactating dairy cows. Four multiparous lactating Holstein cows (237 ± 17 DIM) equipped with a rumen cannula and catheters in the portal vein and a mesenteric artery were used in a randomized Youden square design. They were fed every 2 h to maintain steady digesta flow. On experimental days, they received a postruminal bolus of (1) CN-CBL alone (0.1 g), (2) CN-CBL (0.1 g) + casein hydrolysate (10 g), or (3) CN-CBL (0.1 g) + whey proteins (10 g). Starting 30 min after the bolus, blood samples were taken simultaneously from the 2 catheters every 15 min during the first 2 h and then every 2 h until 24 h postbolus. Milk yield, DMI, and vitamin B12 portal-arterial difference and PDV flux were analyzed using the MIXED procedure of SAS. Milk yield and DMI were not affected by treatments. The portal-arterial difference of vitamin B12 during the 24-h period following the bolus of vitamin was greater when the vitamin was given in solution with casein hydrolysate (2.9 ± 4.6 pg/mL) than alone (-17.5 ± 5.2 pg/mL) or with whey protein (-13.4 ± 4.2 pg/mL). The treatment effects were similar for the PDV flux. The present results suggest that CN-CBL given with casein hydrolysate increases vitamin B12 absorption as compared with CN-CBL given alone.