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To investigate the effects of individual essential amino acids (EAA) on growth and the underlying mechanisms, EAA individually supplemented a low-protein (LP) diet fed to young rats in the present study. Treatments were an LP diet that contained 6% crude protein (CP), a high-protein (HP) diet that contained 18% CP, and 10 LP diets supplemented with individual EAA to achieve an EAA supply equal to that of the HP diet. The CP concentration of the LP diet was ascertained from the results of the first experiment, which examined the effects of dietary CP concentrations on growth rates, with CP ranging from 2% to 26%. Weight gain was increased with the supplementation of His, Ile, Lys, Thr, or Trp as compared to the LP diet (p < 0.05). Feed intake was greater for the His-, Lys-, and Thr-supplemented treatments as compared to the LP group (p < 0.05). Protein utilization efficiency was lower for the HP group than other groups (p < 0.01). The supplementation of Leu, Lys, and Val led to reduced protein utilization efficiency (p < 0.05), but the supplementation of Thr and Trp led to greater efficiency than the LP group (p < 0.05). Compared to the LP group, plasma urea concentrations were elevated with individual EAA supplementation, with the exception of the Thr addition. The added EAA resulted in increased concentrations of the corresponding EAA in plasma, except for Arg and Phe supplementation. The supplementation of Arg, His, Leu, Lys, and Met individually stimulated mTORC1 pathway activity (p < 0.05), and all EAA resulted in the decreased expression of ATF4 (p < 0.05). In summary, the supplementation of His, Ile, Lys, Thr, or Trp to an LP diet improved the growth performance of young rats. Responses to His and Lys additions were related to the activated mTORC1 pathway and feed intake increases. The improved growth performance resulting from the addition of a single EAA is not solely attributed to the inc

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

We hypothesized that the general control nonderepressible 2 (GCN2)/eukaryotic initiation factor 2 (eIF2) signaling pathway and intracellular protein synthesis (PS) are regulated to maintain milk PS in primary bovine mammary epithelial cells (MECs) under essential amino acid (EAA) starvation conditions. We cultured MECs with 0%, 2% (depletion), and 100% (control) EAA for two exposure times (8 and 24 h), followed by three refeeding (RF) times with 100% EAA (0, 8, and 24 h). Subsequently, we measured cell viability, total protein concentration, and proliferation. Western blotting was used to quantify the levels of casein and the expression of total GCN2 and eIF2, as well as phosphorylated GCN2 (GCN2P) and eIF2 (eIF2P). The ISOQuant method was used to assess MEC proteomes, and the resultant data were analyzed using the Kruskal–Wallis test, nonpaired Wilcoxon rank post-hoc test, and ANOVA–Tukey test, as well as principal component analyses and multiple regressions models. Differences in cell viability were observed between the control versus the depleted and repleted MECs, respectively, where 97.2–99.8% viability indicated low cell death rates. Proliferation (range, 1.02–1.55 arbitrary units (AU)) was affected by starvation for 12 and 24 h and repletion for 24 h, but it was not increased compared with the control. Total protein expression was unaffected by both depletion and repletion treatments (median 3158 µg/mL). eIF2P expression was significantly increased (p < 0.05) after treatment with 2% EAA for 8 and 24 h compared with 2% EAA with 8 h + 24 h RF and 2% EAA with 24 h + 8 h RF. GCN2P also showed significantly increased expression (p < 0.05) after treatment with 2% EAA for 24 h compared with the control and 2% EAA with 24 h + 8 h RF. Intracellular casein/α-tubulin expression was unaffected by 2% EAA compared with control (0.073 ± 0.01 AU versus 0.086 ± 0.02 AU, respectively). We studied 30 of the d

To investigate the effects of acetate, propionate, and pH on thermodynamics of volatile fatty acids (VFA) in the rumen, a dual-flow continuous culture study was conducted to quantify production of major VFA, interconversions among the VFA, and H2 and CH4 emissions in a 4 × 4 Latin square design. The 4 treatments were (1) control: pH buffered to an average of 6.75; (2) control plus 20 mmol/d of infused acetate (InfAc); (3) control plus 7 mmol/d of infused propionate (InfPr); and (4) a 0.5-unit decline in pH elicited by adjustment of the buffer (LowpH). All fermentors were fed 40 g of a pelleted diet containing whole alfalfa pellets and concentrate mix pellets (50:50) once daily. After 7 d of treatment, sequential, continuous infusions of [2-13C] sodium acetate (3.5 mmol/d), [U-13C] sodium propionate (2.9 mmol/d), and [1-13C] sodium butyrate (0.22 mmol/d) were carried out from 12 h before feeding for 36 h. Filtered liquid effluent (4 mL) was sampled at 0, 2, 4, 6, 8, 12, 16, and 22 h after feeding, and assessed for VFA concentrations, with another filtered sample (20 mL) used to quantify aqueous concentrations of CH4 and H2. Headspace CH4 and H2 gases were monitored continuously. Ruminal microbes were isolated from the mixed effluent samples, and the microbial community structure was analyzed using the 16S rRNA amplicon sequencing technique. The digestibility of neutral detergent fiber, acid detergent fiber, and starch and microbial C sequestrated from VFA were not affected by treatments. The LowpH treatment increased net propionate production and decreased H2 and CH4 headspace emissions, primarily due to shifts in metabolic pathways of VFA formation, likely due to the observed changes in bacterial community structure. Significant interconversions occurred between acetate and butyrate, whereas interconversions of other VFA with propionate were relatively small. The InfAc and InfPr treatments increased net acetate and propionate production, respectively; however, inter

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

Increased ambient temperature causes heat stress in mammals, which affects physiological and molecular functions. We have recently reported that the dietary administration of a postbiotic from Aspergillus oryzae (AO) improves tolerance to heat stress in fruit flies and cattle. Furthermore, heat-induced gut dysfunction and systemic inflammation have been ameliorated in part by nutritional interventions. The objective of this study was to characterize the phenotypic response of growing calves to heat stress compared to thermoneutral ad libitum fed and thermoneutral feed-restricted counterparts and examining the physiologic alterations associated with the administration of the AO postbiotic to heat-stressed calves with emphasis on intestinal permeability. In this report, we expand previous work by first demonstrating that heat stress reduced partial energetic efficiency of growth in control (45%) but not in AO-fed calves (62%) compared to thermoneutral animals (66%). While heat stress increased 20% the permeability of the intestine, AO postbiotic and thermoneutral treatments did not affect this variable. In addition, AO postbiotic reduced fecal water content relative to thermoneutral and heat stress treatments. Heat stress increased plasma concentrations of serum amyloid A, haptoglobin and lipocalin-2, and administration of AO postbiotic did not ameliorate this effect. In summary, our findings indicated that heat stress led to reduced nutrient-use efficiency and increased systemic inflammation. Results suggest that the AO postbiotic improved energy-use efficiency, water absorption, and the intestinal permeability in heat stress-mediated increase in gut permeability but did not reduce heat stress-mediated rise in markers of systemic inflammation.

Several studies have been conducted to improve grazing management and supplementation in pasture-based systems. However, it is necessary to develop tools that integrate the available information linking the representation of biological processes with animal performance for use in decision making. The objective of this study was to evaluate the precision and accuracy of the Molly cow model predictions of ruminal fermentation, nutrient digestion, and animal performance by cows consuming pasture-based diets to identify model strengths and weaknesses, and to derive new digestive parameters when relevant. Model modifications for adipose tissue, protein synthesis in lean body mass and viscera representation were included. Data used for model evaluations were collected from 25 publications containing 115 treatment means sourced from studies conducted with lactating dairy cattle. The inclusion criteria were that diets contained ≥ 45% perennial ryegrass (Lolium perenne L.), and that dry matter intake, dietary ingredient composition, and nutrient digestion observations were reported. Animal performance and N excretion variables were also included if they were reported. Model performance was assessed before and after model reparameterization of selected digestive parameters, global sensitivity analysis was conducted after reparameterization, and a 5-fold cross evaluation was performed. Although rumen fermentation predictions were not significantly improved, rumen VFA absorption rates were recalculated, which improved the concordance correlation coefficient (CCC) for rumen propionate and ammonia concentration predictions but decreased CCC for acetate predictions. Similar degradation rates of crude protein were observed for grass and TMR diets, but RUP predictions seemed to be affected by the solubility of the protein source as was the intestinal digestibility coefficient. Ruminal fiber degradation was greater after reparameterization, driven primarily by hemicellulose degradati

Mammary epithelial cells (MECs) in culture are a useful model for elucidating mammary gland metabolism and changes that occur under different nutrient disponibility. MECs were exposed to different treatments: 100% EAA for 8 h and 24 h restriction (R); 2% EAA for 8 h and 24 h R; 2% EAA for 8 h and 24 h + 100% EAA for 8 h and 24 h restriction + re-feeding (R + RF). Western blotting and protein quantification was performed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) software identified the amino acids (AAs) and signaling pathways. The chi-squared test, multiple classification analysis, and analysis of variance were used for the purification and identification of data. Intracellular casein levels were not affected. The KEGG analysis revealed that the important pathways of metabolism of AAs, which were involved in processes related to metabolism and biosynthesis of phenylalanine, tyrosine, and tryptophan (fumarate, acetyl-CoA, and tricarboxylic acid (TCA) cycle), were affected by both R and R + RF treatments, mainly through the glutamic-oxaloacetic transaminase-2 enzyme. Additionally, metabolic processes mediated by the mitochondrial malate dehydrogenase, S-adenosylmethionine synthetase, and asparagine synthase proteins positively regulated the carbohydrate pathway, pyruvate, and TCA cycles, as well as the metabolism of alanine, aspartate, and glutamate metabolism (carbohydrate and TCA cycle). We hypothesized that MECs have the capacity to utilize alternative pathways that ensure the availability of substrates for composing milk proteins.

Several studies have been conducted to improve grazing management and supplementation in pasture-based systems. However, it is necessary to develop tools that integrate the available information linking the representation of biological processes with animal performance for use in decision making. The objective of this study was to evaluate the precision and accuracy of the Molly cow model predictions of ruminal fermentation, nutrient digestion, and animal performance by cows consuming pasture-based diets to identify model strengths and weaknesses, and to derive new digestive parameters when relevant. Model modifications for adipose tissue, protein synthesis in lean body mass and viscera representation were included. Data used for model evaluations were collected from 25 publications containing 115 treatment means sourced from studies conducted with lactating dairy cattle. The inclusion criteria were that diets contained ≥ 45% perennial ryegrass (Lolium perenne L.), and that dry matter intake, dietary ingredient composition, and nutrient digestion observations were reported. Animal performance and N excretion variables were also included if they were reported. Model performance was assessed before and after model reparameterization of selected digestive parameters, global sensitivity analysis was conducted after reparameterization, and a 5-fold cross evaluation was performed. Although rumen fermentation predictions were not significantly improved, rumen VFA absorption rates were recalculated, which improved the concordance correlation coefficient (CCC) for rumen propionate and ammonia concentration predictions but decreased CCC for acetate predictions. Similar degradation rates of crude protein were observed for grass and TMR diets, but RUP predictions seemed to be affected by the solubility of the protein source as was the intestinal digestibility coefficient. Ruminal fiber degradation was greater after reparameterization, driven primarily by hemicellulose degradati

Mammary epithelial cells (MECs) in culture are a useful model for elucidating mammary gland metabolism and changes that occur under different nutrient disponibility. MECs were exposed to different treatments: 100% EAA for 8 h and 24 h restriction (R); 2% EAA for 8 h and 24 h R; 2% EAA for 8 h and 24 h + 100% EAA for 8 h and 24 h restriction + re-feeding (R + RF). Western blotting and protein quantification was performed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) software identified the amino acids (AAs) and signaling pathways. The chi-squared test, multiple classification analysis, and analysis of variance were used for the purification and identification of data. Intracellular casein levels were not affected. The KEGG analysis revealed that the important pathways of metabolism of AAs, which were involved in processes related to metabolism and biosynthesis of phenylalanine, tyrosine, and tryptophan (fumarate, acetyl-CoA, and tricarboxylic acid (TCA) cycle), were affected by both R and R + RF treatments, mainly through the glutamic-oxaloacetic transaminase-2 enzyme. Additionally, metabolic processes mediated by the mitochondrial malate dehydrogenase, S-adenosylmethionine synthetase, and asparagine synthase proteins positively regulated the carbohydrate pathway, pyruvate, and TCA cycles, as well as the metabolism of alanine, aspartate, and glutamate metabolism (carbohydrate and TCA cycle). We hypothesized that MECs have the capacity to utilize alternative pathways that ensure the availability of substrates for composing milk proteins.

Background Volatile fatty acids (VFA) generated from ruminal fermentation by microorganisms provide up to 75% of total metabolizable energy in ruminants. Ruminal pH is an important factor affecting the profile and production of VFA by shifting the microbial community. However, how ruminal pH affects the microbial community and its relationship with expression of genes encoding carbohydrate-active enzyme (CAZyme) for fiber degradation and fermentation are not well investigated. To fill in this knowledge gap, six cannulated Holstein heifers were subjected to a continuous 10-day intraruminal infusion of distilled water or a dilute blend of hydrochloric and phosphoric acids to achieve a pH reduction of 0.5 units in a cross-over design. RNA-seq based transcriptome profiling was performed using total RNA extracted from ruminal liquid and solid fractions collected on day 9 of each period, respectively. Results Metatranscriptomic analyses identified 19 bacterial phyla with 156 genera, 3 archaeal genera, 11 protozoal genera, and 97 CAZyme transcripts in sampled ruminal contents. Within these, 4 bacteria phyla (Proteobacteria, Firmicutes, Bacteroidetes, and Spirochaetes), 2 archaeal genera (Candidatus methanomethylophilus and Methanobrevibacter), and 5 protozoal genera (Entodinium, Polyplastron, Isotricha, Eudiplodinium, and Eremoplastron) were considered as the core active microbes, and genes encoding for cellulase, endo-1,4-beta- xylanase, amylase, and alpha-N-arabinofuranosidase were the most abundant CAZyme transcripts distributed in the rumen. Rumen microbiota is not equally distributed throughout the liquid and solid phases of rumen contents, and ruminal pH significantly affect microbial ecosystem, especially for the liquid fraction. In total, 21 bacterial genera, 4 protozoal genera, and 6 genes encoding CAZyme were regulated by ruminal pH. Metabolic pathways participated in glycolysis, pyruvate fermentation to acetate, lactate, and propanoate were downregulated by low

Objective: The objective was to assess if US dairy nutritionists' approaches toward balancing CP in lactating cow diets are influenced by demographic information, feelings toward environmental nitrogen (N) excretion, and dietary CP cost. Materials and Methods: An electronic survey was sent to 886 US certified dairy nutritionists through the database of the American Registry of Professional Animal Scientists (ARPAS) via email. For survey data analysis, a nonparametric statistic was used to check whether the responses to each question were independent. Relationships among demographic data and responses to other questions were performed using Spearman's correlation. Poisson regression and least squares means were used to identify statistically meaningful differences between response counts within each respective question. Results and Discussion: A total of 77 nutritionists representing 1,065 herds from 28 states with a total of 521,000 total lactating dairy cows responded to the questionnaire. Eighty-nine percent of nutritionists balanced diets based on one or more individual AA requirements of dairy cows. Most respondents (72%) reported that they are currently formulating diets with lower dietary CP than they were 3 to 5 yr ago. The primary concern with formulating lower CP diets was the cost per unit of MP, likely due to the high and fluctuating cost of high-RUP feedstuffs. Implications and Applications: Currently, the impetus to reduce dietary CP in lactating cow diets is primarily financially, rather than environmentally, driven. Nutrition models that accurately predict individual AA requirements of dairy cows will allow nutritionists to develop even lower CP diets than is presently possible. Monitoring income over feed cost of low-CP diets supplemented with AA is critical to support the sustainability of the dairy industry.