Effects of energy supplementation on energy losses and nitrogen balance of steers fed green-chopped wheat pasture I: Calorimetry.

Cattle grazing wheat pasture in the southern Great Plains are sometimes fed an energy supplement; however, the benefits of supplementation on nutrient balance, energy metabolism, and greenhouse gas emissions have not been elucidated. Therefore, we used 10 British crossbred steers (206 ± 10.7 kg initial BW) in a respiration calorimetry study to evaluate the effects of energy supplementation on energy losses, N balance, and nutrient digestibility of steers fed green-chopped wheat forage. The study design was an incomplete replicated 4 × 4 Latin square with treatments in a 2 × 2 factorial arrangement. Steers ( = 8) were assigned to 1 of 2 BW blocks (4 steers per block) with dietary factors consisting of 1) no supplementation (CON) or supplemented with a steam-flaked corn-based energy supplement (that also contained monensin sodium) at 0.5% of BW daily (SUP) and 2) NEm intakes of 1 times (1x) or 1.5 times (1.5x) maintenance. Wheat forage was harvested daily and continuously fed as green-chop to steers during the 56-d study. There were no differences ( ≥ 0.32) between CON and SUP for OM (78.3 vs. 80.7%, respectively) or NDF (68.3 vs. 64.8%, respectively) digestibility. At the 1.5x level of intake, there was no difference ( ≥ 0.16) in energy lost in feces (4.27 vs. 3.92 Mcal/d) or urine (0.58 vs. 0.55 Mcal/d), heat production (8.69 vs. 8.44 Mcal/d), or retained energy (3.10 vs. 3.46 Mcal/d) between supplementation treatments. Oxygen consumption (1,777 vs. 1,731 L/d; = 0.67) and CO production (1,704 vs. 1,627 L/d; = 0.56) of CON and SUP steers, respectively, were not different; however, SUP steers tended to have ( = 0.06) lower CH production (115 vs 130 L/d) than CON steers. Methane, as a proportion of GE intake, was similar for CON (6.87%) and SUP (6.07%; = 0.18), as was the ME:DE ratio ( = 0.24; 86.3% for CON and 87.9% for SUP). Fractional N excretion in urine and feces, as a proportion of total N excreted ( ≥ 0.84) or N intake ( ≥ 0.63), was not different between treatments. Calculated NEm and NEg values for CON were 1.76 and 1.37 Mcal/kg DM, respectively, whereas the NEm and NEg values for the SUP treatment were 2.32 and 1.61 Mcal/kg DM, respectively. Calculated NE values for steers fed additional energy were approximately 17.5% greater than the expected difference in energy content. This was probably the result of the inconsistent response at the 1x DMI level. Under these circumstances, energy supplementation did appear to enhance NEm and NEg value of the supplemented wheat forage diet.

Effect of condensed tannin extract supplementation on growth performance, nitrogen balance, gas emissions, and energetic losses of beef steers.

Condensed tannins (CT) may decrease greenhouse gas emissions and alter the site of N excreted by ruminants. We evaluated the effect of top-dressing a steam-flaked corn-based finishing diet (14.4% CP and NEg 1.47 Mcal/kg) for beef cattle with a commercially available CT extract at 3 levels (0, 0.5, and 1.0% of diet, DM basis). Angus-crossbred steers ( = 27; 350 ± 32 kg initial BW) were individually fed via Calan gates for 126 d. Diet digestibility and N balance were estimated after 34 and 95 d on feed (Phase 1 and Phase 2, respectively) using titanium dioxide as a marker of fecal output and the creatinine:BW ratio as a marker for urine output. Ruminal CH and metabolic CO fluxes were measured using a GreenFeed system (C-Lock Inc., Rapid City, SD) for 2 sampling periods that coincided with fecal and urine sampling. Urine energy loss was estimated from urine N excretion, assuming all excreted N was urea. Oxygen consumption was estimated from CO production assuming a respiratory quotient of 1.05. Average daily gain (2.08, 2.14, and 2.08 kg/d for 0, 0.5, and 1.0% CT, respectively) and G:F did not differ ( = 0.88) among treatments. Starch intake and OM intake did not differ ( ≥ 0.42) among treatments during each phase. Apparent total tract starch digestibility during Phase 1 linearly decreased ( = 0.04) with inclusion of CT. Apparent total tract digestibility of OM and starch were not different among treatments ( ≥ 0.13) during Phase 2. Nitrogen intake did not differ ( ≥ 0.16) among treatments during each phase, but fecal N excretion linearly increased ( = 0.05) with inclusion of CT during Phase 1. Urinary N excretion was not different ( ≥ 0.39) among treatments during both phases, but urinary N as a proportion of total N excretion linearly decreased ( = 0.01) when CT was included in the diet during Phase 1. Retained N was not different ( ≥ 0.27) among treatments during each phase. Fluxes of CO were similar ( ≥ 0.37) among treatments during both phases. No differences ( ≥ 0.23) were observed for percentage of GE intake lost as CH (2.99, 3.12, and 3.09% in Phase 1 and 3.54, 3.55, and 4.35% in Phase 2) for 0, 0.5, and 1.0% CT, respectively. No difference ( ≥ 0.42) was observed for heat production lost as a percent of GE intake during both phases. Growth performance, gas emissions, and energetic losses were not affected by the inclusion CT in a steam-flaked corn-based finishing diet.

Novel animal models for studying complex brain disorders: BAC-driven miRNA-mediated in vivo silencing of gene expression.

In schizophrenia, glutamic acid decarboxylase 1 (GAD1) disturbances are robust, consistently observed, cell-type specific and represent a core feature of the disease. In addition, neuropeptide Y (NPY), which is a phenotypic marker of a sub-population of GAD1-containing interneurons, has shown reduced expression in the prefrontal cortex in subjects with schizophrenia, suggesting that dysfunction of the NPY+ cortical interneuronal sub-population might be a core feature of this devastating disorder. However, modeling gene expression disturbances in schizophrenia in a cell type-specific manner has been extremely challenging. To more closely mimic these molecular and cellular human post-mortem findings, we generated a transgenic mouse in which we downregulated GAD1 mRNA expression specifically in NPY+ neurons. This novel, cell type-specific in vivo system for reducing gene expression uses a bacterial artificial chromosome (BAC) containing the NPY promoter-enhancer elements, the reporter molecule (eGFP) and a modified intron containing a synthetic microRNA (miRNA) targeted to GAD1. The animals of isogenic strains are generated rapidly, providing a new tool for better understanding the molecular disturbances in the GABAergic system observed in complex neuropsychiatric disorders such as schizophrenia. In the future, because of the small size of the silencing miRNAs combined with our BAC strategy, this method may be modified to allow generation of mice with simultaneous silencing of multiple genes in the same cells with a single construct, and production of splice-variant-specific knockdown animals.

Bacterial artificial chromosome transgenic analysis of dynamic expression patterns of regulator of G-protein signaling 4 during development. II. Subcortical regions.

A large family of regulator of G protein signaling (RGS) proteins modulates signaling through G-protein-coupled receptors. Previous studies have implicated RGS4 as a vulnerability gene in schizophrenia. To begin to understand structure-function relationships, we have utilized bacterial artificial chromosome (BAC) methods to create transgenic mice that express green fluorescent protein (GFP) under the control of endogenous RGS4 enhancer elements, circumventing the lack of suitable antibodies for analysis of dynamic patterns of expression. This report follows from the accompanying mapping paper in cerebral cortex, with a focus on developmental and mature expression patterns in subcortical telencephalic, diencephalic and brainstem areas. Based on reporter distribution, the data suggest that alterations in RGS4 function will engender a complex phenotype of increased and decreased neuronal output, with developmental, regional, and cellular specificity.

Bacterial artificial chromosome transgenic analysis of dynamic expression patterns of regulator of G-protein signaling 4 during development. I. Cerebral cortex.

Signaling through G-protein-coupled receptors is modulated by a family of regulator of G protein signaling (RGS) proteins that have been implicated in several neurological and psychiatric disorders. Defining the detailed expression patterns and developmental regulation of RGS proteins has been hampered by an absence of antibodies useful for mapping. We have utilized bacterial artificial chromosome (BAC) methods to create transgenic mice that express GFP under the control of endogenous regulator of G-protein signaling 4 (RGS4) enhancer elements. This report focuses on expression patterns in the developing and mature cerebral cortex. Based on reporter distribution, RGS4 is expressed by birth in neurons across all cortical domains, but in different patterns that suggest region- and layer-specific regulation. Peak expression typically occurs before puberty, with complex down-regulation by adulthood. Deep and superficial neurons, in particular, vary in their patterns across developmental age and region and, in primary sensory cortices, layer IV neurons exhibit low or no expression of the GFP reporter. These data suggest that altering RGS4 function will produce a complex neuronal phenotype with cell- and subdomain-specificity in the cerebral cortex.

Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons.

Distinct classes of neurons are generated from progenitor cells distributed in characteristic dorsoventral patterns in the developing spinal neural tube. We define restricted neural progenitor populations by the discrete, nonoverlapping expression of Ngn1, Math1, and Mash1. Crossinhibition between these bHLH factors is demonstrated and provides a mechanism for the generation of discrete bHLH expression domains. This precise control of bHLH factor expression is essential for proper neural development since as demonstrated in both loss- and gain-of-function experiments, expression of Math1 or Ngn1 in dorsal progenitor cells determines whether LH2A/B- or dorsal Lim1/2-expressing interneurons will develop. Together, the data suggest that although Math1 and Ngn1 appear to be redundant with respect to neurogenesis, they have distinct functions in specifying neuronal subtype in the dorsal neural tube.

Immature CD4+CD8+ thymocytes do not polarize lipid rafts in response to TCR-mediated signals.

TCR-mediated stimulation induces activation and proliferation of mature T cells. When accompanied by signals through the costimulatory receptor CD28, TCR signals also result in the recruitment of cholesterol- and glycosphingolipid-rich membrane microdomains (lipid rafts), which are known to contain several molecules important for T cell signaling. Interestingly, immature CD4(+)CD8(+) thymocytes respond to TCR/CD28 costimulation not by proliferating, but by dying. In this study, we report that, although CD4(+)CD8(+) thymocytes polarize their actin cytoskeleton, they fail to recruit lipid rafts to the site of TCR/CD28 costimulation. We show that coupling of lipid raft mobilization to cytoskeletal reorganization can be mediated by phosphoinositide 3-kinase, and discuss the relevance of these findings to the interpretation of TCR signals by immature vs mature T cells.

Cloning the partial cDNAs of mu-calpain and m-calpain from porcine skeletal muscle.

Calpains are non-lysosomal proteases involved in myofibrillar protein degradation. To facilitate studying the expression of the porcine calpain genes and their influence on protein accretion, we have cloned partial cDNAs for mu- and m-calpain from porcine skeletal muscle via PCR amplification. A 289 bp fragment for mu-calpain and a 629 bp fragment for m-calpain were cloned into the EcoRV site of pBluescript II KS+ vector. The nucleotide sequence for porcine mu-calpain and m-calpain were 92% and 90% identical to corresponding regions of rabbit mu- and m-calpain, respectively. The deduced amino acid sequences for both mu- and m-calpain share 94% identity with respective rabbit mu- and m-calpains. Isoform specificity was validated by Southern hybridization of mu- and m-calpain probes with cloned mu- and m-calpain fragments and Northern hybridization with pig muscle mRNA. These clones will be used to evaluate the role of calpain expression in muscle hypertrophy.