Effects of Individual Essential Amino Acids on Growth Rates of Young Rats Fed a Low-Protein Diet.

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 increased plasma availability of EAA. Rather, it may be the consequence of a confluence of factors encompassing signaling pathways, the availability of amino acids, and other associated elements. The additivity of these factors results in independent responses to several EAA with no order of limitation, as is universally encoded in growth models for all production animal species.

Responses in splanchnic and mammary amino acid metabolism to short-term graded removal of methionine in lactating goats.

Four multi-catheterized lactating goats were used in a 4 × 4 Latin square experiment to investigate the responses of amino acid metabolism in portal-drained viscera (PDV), liver, and mammary glands to short-term varying supplies of methionine (Met). During the last 45 h in each experimental period, goats were fasted for 12 h and then abomasally infused with an amino acid (AA) mixture plus glucose for 33 h. Treatments consisted of graded removal of Met from an infused AA mixture to achieve Met content in the infusate of 100% (complete), 60%, 30%, or 0% that in casein. Graded Met removal decreased the production of milk, milk protein, lactose, and fat linearly whilst also decreasing arterial Met concentration linearly (P < 0.05). Meanwhile, net PDV uptake and liver removal of Met decreased linearly (P < 0.05) due to decreased Met affinity of PDV and liver (P < 0.05). Net mammary uptake of Met (P > 0.1) was maintained as Met supply declined. This was achieved through increased mammary affinity (P < 0.05) and increased mammary blood flow (P < 0.05) totally offsetting the negative effect of decreased circulating Met concentration. Graded removal of Met from the infusate linearly decreased mammary uptake-to-milk output ratios of Met (P < 0.05) and tended to decrease essential amino acid (EAA) linearly (0.05 < P < 0.1). Treatments also linearly decreased circulating concentration of prolactin and linearly increased insulin concentration (P < 0.05). In conclusion, results of the present study indicated there were several mechanisms used to mitigate a Met deficiency, including reduced catabolism of Met in PDV, liver, and peripheral tissue (including mammary glands) and a linear increase in mammary blood flow. The observed decreases in milk protein production as Met supply decreased appear to be a result of regulatory events which may have been driven by decreased circulating prolactin, rather than as a result of decreased mammary Met uptake.

Trichostrongylus colubriformis infection damages intestine brush board cells and could negatively impact postabsorptive parameters of Santa Ines lambs.

This study aimed to evaluate histological, digestive and postabsorptive physiological parameters in Santa Ines lambs infected with Trichostrongylus colubriformis and fed different levels of phosphorus. Therefore, eighteen Santa Ines, castrated male, six-month old, healthy lambs (initial body weight 22.4 ± 2.7 kg) were distributed in one of four treatments arranged in a 2 × 2 split-plot arrangement: Sufficient dietary P level and uninfected (SPui; n = 4), Sufficient dietary P level and infected (SPi; n = 5), Deficient dietary P level and uninfected (DPui; n = 4), Deficient dietary P level and infected (DPi; n = 5). Infected lambs received, orally, a single pulse dose of 40.000 T. colubriformis infective larval stage (L3). Animals were fed Tifton 85 hay (Cynodon ssp.; 60%), and cassava meal and maize gluten meal (40%). Measurement of nutrient apparent digestibility and nitrogen metabolism were performed in individual metabolic stalls. To achieve the trial results, it was measured methane emissions in respiratory chambers, urine purine derivatives, ruminal short-chain fatty acids (SCFA), histological cuts of duodenal mucosal tissues and passage rates fluxes, analyzed by external (Yb, Cr, and Co) and internal (iNDF) markers. Statistical procedures were performed in R studio. The fixed main effects of treatment and the interactions were tested by ANOVA, and means compared by Duncan's test at 5% significance. Apparent digestibility was not affected by treatments, however, nitrogen retained decreased (P < 0.01) and urinary nitrogen losses increased (P < 0.01) in infected animals. Small intestine digesta content, empty segment weight, and length were higher in infected animals (P < 0.05). Passage rate was not majorly affected by infection or dietary P levels. Methane emissions, SCFA concentrations, and purine derivative excretion were also not affected by treatments. Regarding the histology, the vilosity weight (P < 0.05), and crypt depth (P < 0.01) decreased in infected animals. In conclusion, T. colubriformis infection can damage intestinal mucosa and affect nitrogen metabolism, but did not affect the digesta transit, and nutrient digestibility. The P dietary levels did not promote any modification in GIT physiological parameters tested in this study.

Effects of Essential Amino Acid Deficiency on General Control Nonderepressible 2/Eukaryotic Initiation Factor 2 Signaling and Proteomic Changes in Primary Bovine Mammary Epithelial Cells.

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 detected 1180 proteins, 16 of which were differentially expressed in starved and refed MECs. Cells faced with EAA deficiency activated the GCN2P/eIF2P pathway, and the lack of change in the levels of casein and other milk proteins suggested that the EAA deficit was mitigated by metabolic flexibility to maintain homeostasis.

An evaluation of the validity of an in vitro and an in situ/in vitro procedure for assessing protein digestibility of blood meal, feather meal and a rumen-protected lysine prototype.

In vitro procedures are commonly used to estimate rumen protein degradability and protein digestibility of feed ingredients. However, it is unclear how well these assays correlate to in vivo data. The objectives of this work were to compare postruminal protein availability estimates from one in vitro procedure and one in situ/in vitro procedure with in vivo observations for blood meal (BM), feather meal (FM), and a rumen-protected lysine prototype (RP-Lys). The FM and BM used for this experiment were subsamples of material assessed in vivo by an isotope-based method and the RP-Lys subsamples were of a prototype tested in two in vivo trials: a lactation trial and by plasma appearance. Subsamples of the BM (n = 14) and the FM (n = 22) were sent to each of three different laboratories for in vitro or in situ/in vitro analysis of crude protein (CP) and determination of rumen undegraded protein (RUP) and digested RUP (dRUP). Subsamples of the RP-Lys (n = 5) were sent to one laboratory for in vitro analysis of CP, RUP, and dRUP. Two diets containing BM or FM were assessed using the Cornell Net Carbohydrate and Protein System (CNCPS) v6.55 with ingredient inputs derived from either the CNCPS feed library, the isotope dilution method, or an average of the in vitro results from the three laboratories to determine how much the differences among estimates affected ingredient values. In vitro dRUP estimates for BM from one laboratory closely matched those determined in vivo (66.7% vs. 61.2%, respectively), but no in vitro estimates for FM matched the in vivo values. Surprisingly, there were significant differences in protein digestibility estimates from the modified three-step procedure across the two laboratories for BM (P < 0.0001) and for FM (P < 0.0001) indicating significant variation among laboratories in application of the method. Within all laboratories, BM estimates were reported in a narrow range (CV values of 2.6 or less). However, when testing multiple samples of FM or the RP-Lys prototype, CV values within a laboratory ranged up to 11 and 34, respectively. For the RP-Lys, dRUP estimates from the in vitro method were roughly half of that determined by the in vivo methods suggesting poor concordance between the in vitro and in vivo procedures for this ingredient. The inconsistencies within and among laboratories accompanied with dissimilarities to in vivo data is problematic for application in nutrition models. Additional refinement to the in vitro techniques is warranted.

Supplementing Ruminally Protected Lysine, Methionine, or Combination Improved Milk Production in Transition Dairy Cows.

The objectives of this study were to evaluate the effects of dietary supplementation of ruminally protected lysine (RPL), or methionine (RPM), and their combination (RPML) on the production efficiency of transition cows. A total of 120 pre-partum multiparous Holstein cows were assigned to four treatments based on previous lactation milk production, days (d) of pregnancy, lactation, and body condition score (BCS). Cows were fed a basal diet [pre-calving: 1.53 Mcal/kg dry matter (DM) and post-calving: 1.70 Mcal/kg DM] with or without supplemental ruminally protected amino acids (RPAA). Treatments were the basal diets without supplemental amino acids (CONTROL, n = 30), with supplemental methionine (RPM, pre-calving at 0.16% of DM and post-calving at 0.12% of DM, n = 30), with supplemental lysine (RPL, pre-calving at 0.33% of DM and post-calving at 0.24% DM, n = 30), and the combination (RPML, pre-calving at 0.16% RPM + 0.33% RPL of DM and post-calving at 0.12% RPM + 0.24 % RPL DM, n = 30). The dietary content of lysine was balanced to be within 6.157.2% metabolizable protein (MP)-lysine and that of methionine was balanced within 2.1-2.35% MP-methionine. Dry matter intake (DMI) was measured daily. Milk samples were taken on d 7, 14, and 21 days relative to calving (DRC), and milk yields were measured daily. Blood samples were taken on d -21, -14, -7 before expected calving and d 0, 7, 14, and 21 DRC. Data were analyzed using SAS software. There were significant Trt × time interactions (P < 0.01) for DMI pre- and post-calving period. The CON cows had lower DMI than RPM, RPL, and RPML, both pre-calving (P < 0.01) and post-calving periods (P < 0.01). Energy-corrected milk (P < 0.01), milk fat (P < 0.01), protein (P = 0.02), and lactose (P < 0.01) percentage levels were greater for RPM, RPL, and RPML cows compared to CON. Supplementing RPAA assisted in maintaining BCS post-calving than CON (P < 0.01). Blood concentrations of ß-hydroxybutyrate decreased with RPM or RPL or the combination pre-calving (P < 0.01) and tended to decrease post-calving (P = 0.10). These results demonstrated that feeding RPL and RPM improved DMI and milk production efficiency, maintained BCS, and reduced ß-hydroxybutyrate concentrations of transition cows.

Automated acquisition of top-view dairy cow depth image data using an RGB-D sensor camera.

Animal dimensions are essential indicators for monitoring their growth rate, diet efficiency, and health status. A computer vision system is a recently emerging precision livestock farming technology that overcomes the previously unresolved challenges pertaining to labor and cost. Depth sensor cameras can be used to estimate the depth or height of an animal, in addition to two-dimensional information. Collecting top-view depth images is common in evaluating body mass or conformational traits in livestock species. However, in the depth image data acquisition process, manual interventions are involved in controlling a camera from a laptop or where detailed steps for automated data collection are not documented. Furthermore, open-source image data acquisition implementations are rarely available. The objective of this study was to 1) investigate the utility of automated top-view dairy cow depth data collection methods using picture- and video-based methods, 2) evaluate the performance of an infrared cut lens, 3) and make the source code available. Both methods can automatically perform animal detection, trigger recording, capture depth data, and terminate recording for individual animals. The picture-based method takes only a predetermined number of images whereas the video-based method uses a sequence of frames as a video. For the picture-based method, we evaluated 3- and 10-picture approaches. The depth sensor camera was mounted 2.75 m above-the-ground over a walk-through scale between the milking parlor and the free-stall barn. A total of 150 Holstein and 100 Jersey cows were evaluated. A pixel location where the depth was monitored was set up as a point of interest. More than 89% of cows were successfully captured using both picture- and video-based methods. The success rates of the picture- and video-based methods further improved to 92% and 98%, respectively, when combined with an infrared cut lens. Although both the picture-based method with 10 pictures and the video-based method yielded accurate results for collecting depth data on cows, the former was more efficient in terms of data storage. The current study demonstrates automated depth data collection frameworks and a Python implementation available to the community, which can help facilitate the deployment of computer vision systems for dairy cows.

Amino Acid Metabolomic Profiles in Bovine Mammary Epithelial Cells under Essential Amino Acid Restriction.

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.

Metatranscriptomic analyses reveal ruminal pH regulates fiber degradation and fermentation by shifting the microbial community and gene expression of carbohydrate-active enzymes.

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 pH in the liquid fraction. CONCLUSIONS: The ruminal microbiome changed the expression of transcripts for biochemical pathways of fiber degradation and VFA production in response to reduced pH, and at least a portion of the shifts in transcripts was associated with altered microbial community structure.

Interactions of amino acids and hormones regulate the balance between growth and milk protein synthesis in lactating rats fed diets differing in protein content.

Insulin-like growth factor-I (IGF-I), growth hormone (GH), and prolactin (PRL) play important roles in milk protein synthesis, and their plasma concentrations were reported to be affected by dietary protein intake. To investigate the relationship between circulating amino acid (AA) and concentrations of these hormones, 18 Wistar rats aged 14 wk were assigned to a low (LP; 9% protein), standard (SP; 21% protein), or high-protein (HP; 35% protein) diet from parturition through day 15 of lactation. Plasma, liver, pituitary gland, skeletal muscle, and mammary gland samples were collected at the end of treatment. Circulating and hepatic IGF-I concentrations increased linearly with elevated dietary protein concentrations (P < 0.0001). Rats receiving the HP diet had higher circulating GH (P < 0.01) and pituitary PRL concentrations (P < 0.0001) but lower pituitary GH concentration (P < 0.0001) relative to those in rats receiving the LP and SP diets. Pearson correlation test performed on composed data across treatments showed that several circulating AAs were correlated with circulating and tissue concentrations of IGF-I, GH, and PRL. Multiple linear regression analyses identified Leu, Gln, Ala, Gly, and Arg as the main AAs associated with hormone responses (R2 = 0.37 ~ 0.80; P < 0.05). Rats fed the LP and HP diets had greater Igf1 and Ghr gene expression in skeletal muscle than those fed the SP diets (P < 0.01). However, LP treatment decreased Prlr mRNA abundance in mammary glands as compared with the SP and HP treatments (P < 0.05). The HP diets increased AA transporter expression (P < 0.01) but decreased mammalian target of rapamycin (P < 0.05) and 70 kDa ribosomal protein S6 kinase 1 (P < 0.01) phosphorylation in mammary glands as compared with the LP and SP diets. The results of the present study suggested that several circulating AAs mediated the effects of dietary protein supply on concentrations of IGF-I, GH, and PRL, which in turn altered the metabolism status in peripheral tissues including the lactating mammary glands.

A revised representation of ruminal pH and digestive reparameterization of the Molly cow model.

Ruminal pH is a critical factor to regulate nutrient degradation and fermentation. However, it has been poorly predicted in the Molly cow model, and recent improvements in the representation of nitrogen cycling across the rumen wall altered some of the modeled responses to feed nutrients, resulting in some model bias. The objectives of this study were to further improve the representation of pH and to refit parameters related to ruminal metabolism and nutrient digestion in the model to resolve this bias, and to use the improved model to estimate nitrogen and energy fluxes with varying rumen-degradable protein (RDP; 40 vs. 60%) and ruminally degraded starch (RDSt; 50 vs. 75%). A meta data set containing 284 peer reviewed studies with 1,223 treatment means was used to derive parameter estimates for ruminal metabolism and nutrient digestions. Refitting the parameters significantly improved the accuracy and precision of the model predictions for ruminal nutrient outflow [acid detergent fiber (ADF), neutral detergent fiber (NDF), total N, microbial N, nonammonia N, and nonammonia nonmicrobial N], ammonia and blood urea concentrations, and fecal nutrient outflow (protein, ADF, and NDF). The prediction error for body weight was decreased from 19.3 to 6.2% with decreased mean bias (from 76.0 to 11.5%) and slope bias (from 17.2 to 7.7%), primarily due to improved representations of ruminal dry matter and liquid pool size. Adding ammonia concentration as a driver to the pH equation increased the precision of predicted ruminal pH and, thereby, the precision of predicted volatile fatty acid (VFA) concentrations, due to improved representation of pH regulation of VFA production rates. Although minor mean and slope bias were observed for ruminal pH and VFA concentrations, the concordance correlation coefficients indicated that much of the observed variation in these variables remains unexplained. Overall, the biological functions of nutrient degradation and digestion appear to be represented without bias. Simulated results indicated that decreasing RDP and RDSt proportions in an isonitrogenous and isocaloric diet can slightly improve N efficiency, and increasing RDSt proportions can increase energy efficiency.

Ruminal volatile fatty acid absorption is affected by elevated ambient temperature.

The objective of this study was to investigate the effect of short-term elevated ambient temperature on ruminal volatile fatty acid (VFA) dynamics and rumen epithelium gene expression associated with the transport and metabolism of VFA. Eight ruminally cannulated Holstein heifers (200 kg) were used in a factorial, repeated measures experiment with two treatments and two periods. During the first period, animals were provided with feed ad libitum and housed at 20 °C. During the second period, one group (HS) was housed at 30 °C and fed ad libitum. The other group (PF) was housed at 20 °C and pair-fed to match the intake of the HS group. During each period, animals were kept on treatment for 10 day, with sample collection on the final day. In the second period, indicators of heat stress were significantly different between PF and HS animals (P < 0.05). There was a thermal environment effect on butyrate production (P < 0.01) that was not associated with feed intake (P = 0.43). Butyrate absorption decreased in HS animals (P < 0.05) but increased in PF animals (P < 0.05) from period 1 to period 2. There was a feed intake effect on BHD1 expression (P = 0.04) and a tendency for a thermal environment effect (P = 0.08), with expression increasing in both cases. Expression of MCT4 was affected by feed intake (P = 0.003) as were all NHE genes (NHE1, NHE2, and NHE3; P < 0.05). These results indicate that with low feed intake and heat stress, there are shifts in rumen VFA dynamics and in the capacity of the rumen epithelium to absorb and transport VFA.

Use of Mechanistic Nutrition Models to Identify Sustainable Food Animal Production.

To feed people in the coming decades, an increase in sustainable animal food production is required. The efficiency of the global food production system is dependent on the knowledge and improvement of its submodels, such as food animal production. Scientists use statistical models to interpret their data, but models are also used to understand systems and to integrate their components. However, empirical models cannot explain systems. Mechanistic models yield insight into the mechanism and provide guidance regarding the exploration of the system. This review offers an overview of models, from simple empirical to more mechanistic models. We demonstrate their applications to amino acid transport, mass balance, whole-tissue metabolism, digestion and absorption, growth curves, lactation, and nutrient excretion. These mechanistic models need to be integrated into a full model using big data from sensors, which represents a new challenge. Soon, training in quantitative and computer science skills will be required to develop, test, and maintain advanced food system models.

Using artificial neural networks to predict pH, ammonia, and volatile fatty acid concentrations in the rumen.

The objectives of this study were (1) to predict ruminal pH and ruminal ammonia and volatile fatty acid (VFA) concentrations by developing artificial neural networks (ANN) using dietary nutrient compositions, dry matter intake, and body weight as input variables; and (2) to compare accuracy and precision of ANN model predictions with that of a multiple linear regression model (MLR). Data were collected from 229 published papers with 938 treatment means. The data set was randomly split into a training data set containing 70% of the observations and a test data set with the remaining observations. A series of ANN with a range of 1 to 9 artificial neurons in 1 hidden layer were examined, and the best one was selected to compare with the best-fitted MLR model. The performance of model predictions was evaluated by root mean square errors (RMSE) and concordance correlation coefficients (CCC) using cross-evaluations with 100 iterations. When using the ANN to predict ruminal pH and concentrations of ammonia, total VFA, acetate, propionate, and butyrate, the RMSE were 4.2, 41.4, 20.9, 22.3, 32.9, and 29.7% of observed means, respectively. The RMSE for the MLR were 4.2, 37.8, 18.3, 19.9, 29.8, and 26.6% of the observed means. The CCC for ruminal pH, ruminal concentrations of ammonia, total VFA, acetate, propionate, and butyrate were 0.57, 0.49, 0.45, 0.40, 0.52, and 0.40, using the ANN, and 0.37, 0.48, 0.40, 0.29, 0.43, and 0.35, using the MLR. Evaluations of the MLR and the ANN indicated that these 2 model forms exhibited similar prediction errors, with 4.2, 39.6, 19.6, 21.1, 31.3, and 28.1% of observed means for pH, ammonia, total VFA, acetate, propionate, and butyrate. Although the ANN increased the precision of predictions related to ruminal metabolism, it failed to improve the accuracy compared with the linear regression model.

A revised representation of urea and ammonia nitrogen recycling and use in the Molly cow model.

Accurately predicting nitrogen (N) digestion, absorption, and metabolism will allow formulation of diets that more closely match true animal needs from a broad range of feeds, thereby allowing efficiency of N utilization and profit to be maximized. The objectives of this study were to advance representations of N recycling between blood and the gut and urinary N excretion in the Molly cow model. The current work includes enhancements (1) representing ammonia passage to the small intestine; (2) deriving parameters defining urea synthesis and ruminal urea entry rates; (3) adding representations of intestinal urea entry, microbial protein synthesis in the hindgut, and fecal urea-N excretion; and (4) altering existing urinary N excretion equations to scale with body weight and adding purine derivatives as a component of urinary N excretion. After the modifications, prediction errors for ruminal outflows of total N, microbial N, and nonammonia, nonmicrobial N were 29.8, 32.3, and 26.2% of the respective observed mean values. Prediction errors of each were approximately 7 percentage units lower than the corresponding values before model modifications and fitting due primarily to decreased slope bias. The revised model predicted ruminal ammonia and blood urea concentrations with substantially decreased overall error and reductions in slope and mean bias. Prediction errors for gut urea-N entry were decreased from 70.5 to 26.7%, which was also a substantial improvement. Adding purine derivatives to urinary N predictions improved the accuracy of predictions of urinary N output. However, urinary urea-N excretion remains poorly predicted with 69.0% prediction errors, due mostly to overestimated urea-N entry rates. Adding representations of undigested microbial nucleic acids, microbial protein synthesized in the hindgut, and urea-N excretion in feces decreased prediction errors for fecal N excretion from 21.1 to 17.1%. The revised model predicts that urea-N entry into blood accounts for approximately 64% of dietary N intake, of which 64% is recycled to the gut lumen. Between 48 and 67% of the urea recycled to the gut flows into the rumen largely depending on diet, which accounts for 29 to 54% of total ruminal ammonia production, and 65 to 76% of this ammonia-N is captured in microbial protein, which represents 17% of N intake. Based on model simulations, feeding a diet with moderately low crude protein and high rumen-undegradable protein could increase apparent ruminal N efficiency by 20%.

An evaluation of Molly cow model predictions of ruminal metabolism and nutrient digestion for dairy and beef diets.

Model evaluation, as a critical process of model advancement, is necessary to identify adequacy and consistency of model predictions. The objectives of this study were (1) to evaluate the accuracy of Molly cow model predictions of ruminal metabolism and nutrient digestion when simulating dairy and beef cattle diets; and (2) to identify deficiencies in representations of the biology that could be used to direct further model improvements. A total of 229 studies (n = 938 treatments) including dairy and beef cattle data, published from 1972 through 2016, were collected from the literature. Root mean squared errors (RMSE) and concordance correlation coefficients (CCC) were calculated to assess model accuracy and precision. Ruminal pH was very poorly represented in the model with a RMSE of 4.6% and a CCC of 0.0. Although volatile fatty acid concentrations had negligible mean (2.5% of mean squared error) and slope (6.8% of mean squared error) bias, the CCC was 0.28, implying that further modifications with respect to volatile fatty acid production and absorption are required to improve model precision. The RMSE was greater than 50% for ruminal ammonia and blood urea-N concentrations with high proportions of error as slope bias, indicating that mechanisms driving ruminal urea N recycling are not properly simulated in the model. Only slight mean and slope bias were exhibited for ruminal outflow of neutral detergent fiber, starch, lipid, total N, and nonammonia N, and for fecal output of protein, neutral detergent fiber, lipid, and starch, indicating the mechanisms encoded in the model relative to ruminal and total-tract nutrient digestion are properly represented. All variables related to ruminal metabolism and nutrient digestion were more precisely predicted for dairy cattle than for beef cattle. This difference in precision was mostly related to the model's inability to simulate low forage diets included in the beef studies. Overall, ruminal pH was poorly simulated and contributed to problems in ruminal nutrient degradation and volatile fatty acid production predictions. Residual analyses suggested ruminal ammonia concentrations need to be considered in the ruminal pH equation, and therefore the inaccuracies in predicting ruminal urea N recycling must also be addressed. These modifications to model structure will likely improve model performance across a wider array of dietary inputs and cattle type.

Plasma and Pancreas Islet Hormone Concentrations in Lactating Rats Are Associated with Dietary Protein Amounts.

Background: Circulating amino acid (AA) and nitric oxide (NO) concentrations and hepatic gluconeogenesis are affected by previous protein intake. However, information about their relations and islet hormone responses is limited. Objective: This study investigated the associations between islet hormone concentrations with circulating AA and NO concentrations as well as with hepatic gluconeogenesis in lactating rats. Methods: At delivery, 18 Wistar rats aged 14 wk were assigned either to low-protein (LP; 9% protein), standard-protein (SP; 21% protein), or high-protein (HP; 35% protein) diets for 15 d in groups of 6 pups/dam. Circulating AA and NO concentrations, circulating and pancreas islet hormone concentrations, and the activities and gene expressions of hepatic phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were measured at the end of treatment. Results: Circulating insulin and glucagon concentrations were greater in the HP than in the LP (25% and 17%, respectively) and SP (37% and 31%) diet groups, whereas compared with the SP group, pancreatic concentrations were lower in the LP (32% and 49%) and HP (34% and 46%) groups (P < 0.01). Hepatic PEPCK and G6Pase activities in the HP group were greater than those in the SP (15% and 15%) and LP (8% and 19%) groups (P < 0.05). In all groups, plasma NO concentrations were correlated negatively to circulating insulin (r = -0.77, P = 0.0003) and positively to pancreas insulin and glucagon concentrations and the insulin-to-glucagon ratio (r = 0.50-0.63; P < 0.05). Some circulating AAs correlated positively to circulating insulin and pancreas insulin and glucagon (r = 0.50-0.82, P < 0.05) but negatively to circulating glucagon (r = -0.53-0.68, P < 0.05). Conclusion: Variations in circulating AA and NO concentrations and hepatic gluconeogenic enzyme activities are likely intermediary responses involved in the effects of dietary protein amounts on the synthesis and secretion of islet hormones in lactating rats.

SESN2 negatively regulates cell proliferation and casein synthesis by inhibition the amino acid-mediated mTORC1 pathway in cow mammary epithelial cells.

Amino acids (AA) are one of the key nutrients that regulate cell proliferation and casein synthesis in cow mammary epithelial cells (CMEC), but the mechanism of this regulation is not yet clear. In this study, the effect of SESN2 on AA-mediated cell proliferation and casein synthesis in CMEC was assessed. After 12 h of AA starvation, CMECs were cultured in the absence of all AA (AA-), in the presences of only essential AA (EAA+), or of all AA (AA+). Cell proliferation, casein expression, and activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway were increased; but SESN2 expression was decreased in response to increased EAA or AA supply. Overexpressing or inhibiting SESN2 demonstrated that cell proliferation, casein expression, and activation of the mTORC1 pathway were all controlled by SESN2 expression. Furthermore, the increase in cell proliferation, casein expression, and activation of the mTORC1 pathway in response to AA supply was inhibited by overexpressing SESN2, and those effects were reversed by inhibiting SESN2. These results indicate that SESN2 is an important inhibitor of mTORC1 in CMEC blocking AA-mediated cell proliferation and casein synthesis.

Effects of heat stress during porcine reproductive and respiratory syndrome virus infection on metabolic responses in growing pigs.

Heat stress (HS) and immune challenges negatively impact nutrient allocation and metabolism in swine, especially due to elevated heat load. In order to assess the effects of HS during Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection on metabolism, 9-wk old crossbred barrows were individually housed, fed ad libitum, divided into four treatments: thermo-neutral (TN), thermo-neutral PRRSV infected (TP), HS, and HS PRRSV infected (HP), and subjected to two experimental phases. Phase 1 occurred in TN conditions (22 °C) where half the animals were infected with PRRS virus (n = 12), while the other half (n = 11) remained uninfected. Phase 2 began, after 10 d with half of the uninfected (n = 6) and infected groups (n = 6) transported to heated rooms (35 °C) for 3 d of continuous heat, while the rest remained in TN conditions. Blood samples were collected prior to each phase and at trial completion before sacrifice. PPRS viral load indicated only infected animals were infected. Individual rectal temperature (Tr), respiration rates (RR), and feed intakes (FI) were determined daily. Pigs exposed to either challenge had an increased Tr, (P < 0.0001) whereas RR increased (P < 0.0001) with HS, compared to TN. ADG and BW decreased with challenges compared to TN, with the greatest loss to HP pigs. Markers of muscle degradation such as creatine kinase, creatinine, and urea nitrogen were elevated during challenges. Blood glucose levels tended to decrease in HS pigs. HS tended to decrease white blood cell (WBC) and lymphocytes and increase monocytes and eosinophils during HS. However, neutrophils were significantly increased (P < 0.01) during HP. Metabolic flexibility tended to decrease in PRRS infected pigs as well as HS pigs. Fatty acid oxidation measured by CO2 production decreased in HP pigs. Taken together, these data demonstrate the additive effects of the HP challenge compared to either PRRSV or HS alone.

Bovine Mammary Gland Biopsy Techniques.

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.