Utilizing gas flux from automated head chamber systems to estimate dietary energy values for beef cattle fed a finishing diet.

Dietary net energy for maintenance (NEm) and gain (NEg) can be estimated using calculations based on live performance or adjusted-final body weight, which is calculated based on carcass characteristics. These values are commonly referred to as performance-adjusted (pa) NEm (paNEm) and NEg (paNEg). The NEm and NEg of a diet can also be estimated by adding recovered energy (RE) with heat production (HP) derived from an automated head chamber system (AHCS), which we will term gas-adjusted (ga) NEm (gaNEm) and NEg (gaNEg). Furthermore, HP from the Brouwer equation requires an estimate of urinary nitrogen (UN) excretion, which can be calculated based on N intake, blood urea N, UN concentration, and urine creatinine, or it could be zeroed. Alternatively, HP can be calculated using an alternative equation based on the respiratory quotient. Demonstrating agreement between pa and ga derived dietary energy values provides an opportunity to validate using the AHCS for energetic experiments and this comparison has not been conducted previously. Accordingly, the objective of this experiment was to assess the agreement between live and carcass paNEm and paNEg with gaNEm and gaNEg, where HP was calculated using 4 different approaches. Estimates of HP were not different (P = 0.99) between the 4 approaches employed, indicating that all options investigated are appropriate. Live paNEm and paNEg had a higher agreement (Lin's concordance correlation coefficient [CCC] = 0.91) with gaNEm and gaNEg than carcass values (CCC ≤ 0.84). These results suggest that researchers can implement the AHCS to provide good estimates of dietary energy values in finishing beef cattle that are unrestrained.

Automated head chamber systems (AHCS) implemented into beef cattle research allow estimation of gas flux, heat production (HP), and calculated gas-adjusted dietary net energy for maintenance (gaNEm) and gain (gaNEg) values when paired with recovered energy. However, a comparison between AHCS-derived values and performance-adjusted NEm (paNEm) and NEg (paNEg) from either live performance (live paNEm and paNEg) or carcass data (carcass paNEm and paNEg) has not been conducted. Accordingly, the objectives of this experiment were to evaluate the agreement between gaNEm and gaNEg, estimated using different approaches for calculating HP, with live paNEm and paNEg or carcass paNEm and paNEg. Accounting for urinary nitrogen or methane when calculating HP does not appreciably influence HP estimates or subsequent calculations to estimate dietary NEm and NEg. There was excellent agreement between live paNEm and gaNEm, and between paNEg and gaNEg. Measures of precision, accuracy, and agreement were lower for carcass than for live-derived values when compared to gaNEm and gaNEg but were still acceptable. These results suggest that researchers can implement the AHCS to provide estimates of HP, gas flux, and estimates of dietary energy values in unrestrained finishing beef cattle-fed diets ranging in crude protein content (10.8% to 12.5%). Additional research is warranted on the use of the AHCS to conduct energetic studies across varying diets and production systems, particularly grazing systems.

Recommendations on visit duration and sample number requirements for an automated head chamber system.

Automated head chamber systems (AHCS; GreenFeed, C-Lock Inc., Rapid City, SD) increasingly are being used for measuring the gas flux of unrestrained cattle. There are a wide range of recommendations for what constitutes a "good" visit (i.e., duration) to an AHCS and how many visits are required for the AHCS to quantify gas fluxes accurately and precisely. Accordingly, the purpose of this experiment was to investigate the effects of visit duration thresholds and the subsequent effects of these thresholds on the number of visits needed to provide adequate estimates of carbon dioxide (CO2) and methane (CH4) emissions, and oxygen (O2) consumption by beef cattle. This analysis utilized data from three previously published experiments with grazing beef steers and one experiment with finishing beef steers, with 103 steers total. When comparing all available visits, there was excellent agreement [Lin's concordance correlation coefficient (CCC) ≥ 0.96] between visits ≥ 3 min in duration and those ≥ 2 min for the three gases in all four experiments. When data from all four experiments were pooled, there was excellent agreement between visits ≥ 3 min and those ≥ 2 min and ≥ 1 min for all gases (CCC ≥ 0.96). These results suggest that estimates from visits ≥ 2 min are like those from visits ≥ 3 min. Next, we investigated if including visits ≥ 2 min or ≥ 1 min would increase the minimal number of visits required to provide excellent agreement with the "gold-standard" (mean of all visits ≥ 3 min). For this, we used only one of the experiments and randomly selected visits per animal ranging from n = 5 to 60, in increments of 5. The sole experiment was used because all animals had more than 60 visits. We then assessed the agreement between the "gold-standard" (mean of all visits ≥ 3 min [144 ±â€…55.01 visits per steer]) estimates of CO2, O2, and CH4. The minimum number of visits required to achieve excellent agreement (CCC ≥ 0.90) to the "gold-standard" estimate for all gases was 30 visits ≥ 3 min in duration, or 40 visits ≥ 2 min in duration. Visits ≥ 1 min in duration did not achieve excellent agreement, even when 60 were used. Based on these results, we recommend excluding visits < 3 min in duration with 30 minimum visit records per animal. However, if researchers choose to implement a 2-min visit duration threshold then 40 visit records are needed per animal.

GreenFeed systems (C-Lock Inc., Rapid City, SD) are being increasingly used for measuring carbon dioxide (CO2) and methane (CH4) emissions and oxygen consumption (O2) of free-roaming cattle. These systems utilize averages of multiple visits to provide estimates of daily gas flux. There currently exists a range of recommendations for what constitutes a "good" visit to GreenFeed. Additionally, the number of recommended visits required to achieve adequate estimates of these gas fluxes appears to be dependent on the minimum visit duration that is used. To date, there has been only one experiment that has investigated visit duration and the recommended number of visits for CO2 and CH4 emissions and to our knowledge this has not been assessed for O2 consumption. Based on the results of this experiment, we recommend using a 3-min minimum visit duration threshold with 30 visit records per animal. If researchers choose to use a 2-min visit duration threshold, then 40 visit records per animal are recommended.

An Experimental Field Trial Investigating the Use of Bacteriophage and Manure Slurry Applications in Beef Cattle Feedlot Pens for Salmonella Mitigation.

Post-harvest Salmonella mitigation techniques are insufficient at addressing Salmonella harbored in cattle lymph nodes, necessitating the exploration of pre-harvest alternatives that reduce Salmonella prior to dissemination to the lymph nodes. A 2 × 2, unbalanced experiment was conducted to determine the effectiveness of pre-harvest treatments applied to the pen surface for Salmonella mitigation in cattle. Treatments included manure slurry intended to mimic pen run-off water (n = 4 pens), a bacteriophage cocktail (n = 4), a combination of both treatments (n = 5), and a control group (n = 5) that received no treatment. Environment samples from 18 feedlot pens and fecal grabs, hide swabs, and subiliac lymph nodes from 178 cattle were collected and selectively enriched for Salmonella, and Salmonella isolates were sequenced. The combination treatment was most effective at reducing Salmonella, and the prevalence was significantly lower compared with the control group for rump swabs on Days 14 and 21. The treatment impact on Salmonella in the lymph nodes could not be determined due to low prevalence. The reduction on cattle hides suggests that bacteriophage or water treatments applied to the feedlot pen surface may reduce Salmonella populations in cattle during the pre-harvest period, resulting in reduced contamination during slaughter and processing.

Transcription factor gene Pea3 regulates erectile function during copulation in mice.

Male mice with homozygous loss of function mutations of the transcription factor gene Pea3 (Pea3 null) are infertile due to their inability to inseminate females, however the specific deficits in male sexual behaviors that drive this phenotype are unknown. Here, the copulatory behavior of male mice (Pea3 null and control) with hormonally primed ovariectomized females was monitored via high-speed and high-resolution digital videography to assess for differences in female-directed social behaviors, gross sexual behaviors (mounting, thrusting), and erectile and ejaculatory function. Pea3 null male mice exhibit greatly reduced erectile function, with 44% of males displaying no visible erections during copulation, and 0% achieving sustained erections. As such, Pea3 null males are incapable of intromission and copulatory plug deposition, despite displaying largely normal female-directed social behaviors, mounting behaviors, and ejaculatory grasping behavior. Additionally, the organization and timing of thrusting behaviors is impaired in Pea3 null males. Our results show that the transcription factor gene Pea3 regulates the ability to achieve and maintain erections during copulation in mice.

Pain and itch processing by subpopulations of molecularly diverse spinal and trigeminal projection neurons.

A remarkable molecular and functional heterogeneity of the primary sensory neurons and dorsal horn interneurons transmits pain- and or itch-relevant information, but the molecular signature of the projection neurons that convey the messages to the brain is unclear. Here, using retro-TRAP (translating ribosome affinity purification) and RNA sequencing, we reveal extensive molecular diversity of spino- and trigeminoparabrachial projection neurons. Among the many genes identified, we highlight distinct subsets of Cck+ -, Nptx2+ -, Nmb+ -, and Crh+ -expressing projection neurons. By combining in situ hybridization of retrogradely labeled neurons with Fos-based assays, we also demonstrate significant functional heterogeneity, including both convergence and segregation of pain- and itch-provoking inputs into molecularly diverse subsets of NK1R- and non-NK1R-expressing projection neurons.

In situ antibody phage display yields optimal inhibitors of integrin α11/ß1.

Integrins are transmembrane multi-conformation receptors that mediate interactions with the extracellular matrix. In cancer, integrins influence metastasis, proliferation, and survival. Collagen-binding integrin-α11/ß1, a marker of aggressive tumors that is involved in stroma-tumor crosstalk, may be an attractive target for anti-cancer therapeutic antibodies. We performed selections with phage-displayed synthetic antibody libraries for binding to either purified integrin-α11/ß1 or in situ on live cells. The in-situ strategy yielded many diverse antibodies, and strikingly, most of these antibodies did not recognize purified integrin-α11/ß1. Conversely, none of the antibodies selected for binding to purified integrin-α11/ß1 were able to efficiently recognize native cell-surface antigen. Most importantly, only the in-situ selection yielded functional antibodies that were able to compete with collagen-I for binding to cell-surface integrin-α11/ß1, and thus inhibited cell adhesion. In-depth characterization of a subset of in situ-derived clones as full-length immunoglobulins revealed high affinity cellular binding and inhibitory activities in the single-digit nanomolar range. Moreover, the antibodies showed high selectivity for integrin-α11/ß1 with minimal cross-reactivity for close homologs. Taken together, our findings highlight the advantages of in-situ selections for generation of anti-integrin antibodies optimized for recognition and inhibition of native cell-surface proteins, and our work establishes general methods that could be extended to many other membrane proteins.

In utero heat stress causes reduced testicular area at puberty, reduced total sperm production, and increased sperm abnormalities in boars.

In utero stress has been shown to negatively affect intact male rats and mice, though very little research has been conducted in boars. The objectives of the present studies were to determine the effects of in utero heat stress (IUHS) on postnatal development and the response to postnatal heat stress of boars. Ten boars were selected at weaning from litters subjected to IUHS or in utero thermoneutral (IUTN) during 30-60 days of gestation. The boars were evaluated for reproductive performance from birth through 57 weeks of age (WOA). Testicular area tended to be smaller for IUHS boars compared to IUTN boars at 24 WOA (P = 0.080). Libido did not differ for IUHS or IUTN (P = 0.818). Total sperm production was reduced in IUHS boars compared to IUTN boars (P ≤ 0.038). Semen volume and semen concentration did not differ (P ≥ 0.469 and P ≥ 0.664, respectively). Total motility and progressive motility did not differ for IUHS and IUTN boars (P ≥ 0.430 and P ≥ 0.652, respectively). In utero heat stressed boars had a greater incidence of sperm with tail abnormalities than IUTN (P ≤ 0.042). In utero heat stressed boars had a lower incidence of sperm with proximal droplets following mild, postnatal heat stress compared with IUTN (P = 0.005). In utero heat stress resulted in significant reductions in sperm production and increased sperm abnormalities in boars. The IUHS boars may be slightly more tolerant to postnatal heat stress, though more research is needed.

A Role for Dystonia-Associated Genes in Spinal GABAergic Interneuron Circuitry.

Spinal interneurons are critical modulators of motor circuit function. In the dorsal spinal cord, a set of interneurons called GABApre presynaptically inhibits proprioceptive sensory afferent terminals, thus negatively regulating sensory-motor signaling. Although deficits in presynaptic inhibition have been inferred in human motor diseases, including dystonia, it remains unclear whether GABApre circuit components are altered in these conditions. Here, we use developmental timing to show that GABApre neurons are a late Ptf1a-expressing subclass and localize to the intermediate spinal cord. Using a microarray screen to identify genes expressed in this intermediate population, we find the kelch-like family member Klhl14, implicated in dystonia through its direct binding with torsion-dystonia-related protein Tor1a. Furthermore, in Tor1a mutant mice in which Klhl14 and Tor1a binding is disrupted, formation of GABApre sensory afferent synapses is impaired. Our findings suggest a potential contribution of GABApre neurons to the deficits in presynaptic inhibition observed in dystonia.

Sensory-Derived Glutamate Regulates Presynaptic Inhibitory Terminals in Mouse Spinal Cord.

Circuit function in the CNS relies on the balanced interplay of excitatory and inhibitory synaptic signaling. How neuronal activity influences synaptic differentiation to maintain such balance remains unclear. In the mouse spinal cord, a population of GABAergic interneurons, GABApre, forms synapses with the terminals of proprioceptive sensory neurons and controls information transfer at sensory-motor connections through presynaptic inhibition. We show that reducing sensory glutamate release results in decreased expression of GABA-synthesizing enzymes GAD65 and GAD67 in GABApre terminals and decreased presynaptic inhibition. Glutamate directs GAD67 expression via the metabotropic glutamate receptor mGluR1ß on GABApre terminals and regulates GAD65 expression via autocrine influence on sensory terminal BDNF. We demonstrate that dual retrograde signals from sensory terminals operate hierarchically to direct the molecular differentiation of GABApre terminals and the efficacy of presynaptic inhibition. These retrograde signals comprise a feedback mechanism by which excitatory sensory activity drives GABAergic inhibition to maintain circuit homeostasis.

Wafer-scale fabrication of patterned carbon nanofiber nanoelectrode arrays: a route for development of multiplexed, ultrasensitive disposable biosensors.

One of the major limitations in the development of ultrasensitive electrochemical biosensors based on one-dimensional nanostructures is the difficulty involved with reliably fabricating nanoelectrode arrays (NEAs). In this work, we describe a simple, robust and scalable wafer-scale fabrication method to produce multiplexed biosensors. Each sensor chip consists of nine individually addressable arrays that uses electron beam patterned vertically aligned carbon nanofibers (VACNFs) as the sensing element. To ensure nanoelectrode behavior with higher sensitivity, VACNFs were precisely grown on 100 nm Ni dots with 1 microm spacing on each micro pad. Pretreatments by the combination of soaking in 1.0 M HNO(3) and electrochemical etching in 1.0M NaOH dramatically improved the electrode performance, indicated by the decrease of redox peak separation in cyclic voltammogram (DeltaE(p)) to approximately 100 mV and an approximately 200% increase in steady-state currents. The electrochemical detection of the hybridization of DNA targets from E. coli O157:H7 onto oligonucleotide probes were successfully demonstrated. The 9 arrays within the chip were divided into three groups with triplicate sensors for positive control, negative control and specific hybridization. The proposed method has the potential to be scaled up to NxN arrays with N up to 10, which is ideal for detecting a myriad of organisms. In addition, such sensors can be used as a generic platform for many electroanalysis applications.

Systemic low-grade inflammation does not decrease skeletal muscle mass and protein synthesis in old rats.

Age-associated low-grade systemic inflammation may contribute to sarcopenia. We hypothesized that skeletal muscle mass and protein synthesis rate would be reduced in old rats exhibiting persistent low-grade inflammation compared to age-matched controls. Male 24-month-old Wistar rats exhibiting a low-grade systemic inflammation for at least one month (LGI group) were compared to non-inflamed rats (C group). Tissue protein synthesis rates were quantified using the L-[1-(13)C]-valine flooding dose method. Body weight, gastrocnemius muscle and spleen weights were not significantly different between groups, but liver and small intestine weights were 13 and 14% higher in LGI than in C. Fractional and absolute protein synthesis rates were not significantly different between groups for gastrocnemius, spleen and small intestine, but higher for liver in LGI than in C. Despite an increase in liver protein synthesis, low-grade inflammation did not reduce skeletal muscle mass, suggesting that age-associated low-grade systemic inflammation occurs independently of sarcopenia.

Free and protein-bound glutamine have identical splanchnic extraction in healthy human volunteers.

The objectives of the present study were to determine the splanchnic extraction of glutamine after ingestion of glutamine-rich protein ((15)N-labeled oat proteins) and to compare it with that of free glutamine and to determine de novo glutamine synthesis before and after glutamine consumption. Eight healthy adults were infused intravenously in the postabsorptive state with L-[1-(13)C]glutamine (3 micromol x kg(-1) x h(-1)) and L-[1-(13)C]lysine (1.5 micromol x kg(-1) x h(-1)) for 8 h. Four hours after the beginning of the infusion, subjects consumed (every 20 min) a liquid formula providing either 2.5 g of protein from (15)N-labeled oat proteins or a mixture of free amino acids that mimicked the oat-amino acid profile and contained L-[2,5-(15)N(2)]glutamine and L-[2-(15)N]lysine. Splanchnic extraction of glutamine reached 62.5 +/- 5.0% and 66.7 +/- 3.9% after administration of (15)N-labeled oat proteins and the mixture of free amino acids, respectively. Lysine splanchnic extraction was also not different (40.9 +/- 11.9% and 34.9 +/- 10.6% for (15)N-labeled oat proteins and free amino acids, respectively). The main conclusion of the present study is that glutamine is equally bioavailable when given enterally as a free amino acid and when protein bound. Therefore, and taking into consideration the drawbacks of free glutamine supplementation of ready-to-use formulas for enteral nutrition, protein sources naturally rich in this amino acid are the best option for providing stable glutamine.

Neither glutamine nor arginine supplementation of diets increase glutamine body stores in healthy growing rats.

The aim of the work was to resolve whether glutamine and arginine supplemented diets affect plasma and tissue (muscle, liver and intestinal mucosa) glutamine concentrations, as well as glutaminase and glutamine synthetase specific activities. The trial was performed in growing rats fed 10% protein diets for 3 weeks. Protein sources were: whey proteins (W); whey proteins+free glutamine (WG); whey proteins+arginine (WA); and casein+wheat protein hydrolysate+acid whey (39:39:22), as source containing protein-bound glutamine (CGW). Rats fed the control diet (6.4% glutamine) (W) showed comparable glutamine body stores to those of rats fed the WG diet. In fact, glutamine sup- plementation down-regulated the hepatic glutamine synthetic capacity of growing rats (W/WG: 6.8+/-0.3 vs 6.0+/-0.2 nmol/min/mg protein). Arginine supplementation of the diet (up to 9% of the protein content) resulted in a decrease in plasma and tissue glutamine concentrations (W/WA: plasma, 1218+/-51 vs 1031+/-48 micromol/L; liver 7.5+/-0.4 vs 6.5+/-0.2 micromol/g; muscle: 5.7+/-0.2 vs 4.0+/-0.2 micromol/g). These data suggest that glutamine supplementation of the diet does not increase plasma and tissue glutamine concentrations in healthy growing rats, while the addition of arginine to the diet decreases glutamine body stores.

Protein hydrolysate vs free amino acid-based diets on the nutritional recovery of the starved rat.

BACKGROUND AND AIMS: To test the hypothesis that a peptide-based enteral product was equivalent to a low-fat, free amino acid-based formula in the nutritional and functional recovery of the starved rat. METHODS: Sixteen male Wistar rats were starved for 3 days. Then, rats were randomised to a whey protein hydrolysate-based diet or a free amino acid-based diet and refed for 3 days. The experiment was designed to provide the same energy intake in both groups. The parameters studied included body weight gain, nitrogen retention, plasma free amino acid concentrations, muscle glutamine concentrations and glutathione levels in gut mucosa and liver. RESULTS: Weight gain was statistically higher on the peptide-based diet than on the elemental diet after the refeeding period. This difference in weight gain was associated with a statistically higher nitrogen retention. Plasma and muscle free glutamine concentrations were higher in rats fed the whey protein hydrolysate-based diet than those in rats refed the free amino acid-based diet, even though the glutamine intake was higher in the latter group. Glutathione concentrations in liver and gut mucosa were similar in the groups. CONCLUSION: We conclude that enteral diets containing peptides were more effective than a diet containing free amino acids in the nutritional recovery of the starved rat.

Food deprivation and refeeding influence growth, nutrient retention and functional recovery of rats.

The objective of this work was to determine the effects of starvation and refeeding on growth, nutritional recovery and intestinal repair in starved rats. Male Wistar rats, weighing 200 g, were starved for 3 d, then refed a soy-based diet for another 3 d. Normally fed rats were given the same diet and used as controls. The variables assessed were as follows: body weight gain and nitrogen retention during recovery after starvation; muscle glutamine concentration; tissue protein content; gut mucosa and liver glutathione levels; intestinal permeability to ovalbumin, lactulose and mannitol; and intestinal tissue apoptosis. Starvation was associated with lower muscle glutamine levels and intestinal mucosa impairment, including a lower content of mucosal protein, a higher level of oxidized glutathione, enhanced permeability to macromolecules and greater numbers of apoptotic cells. Refeeding for 3 d resulted in rapid repair of gut atrophy and normalization of not only intestinal permeability but also of the majority of metabolic markers assessed in other tissues. In conclusion, with the use of severely starved rats, we have established a reversible experimental animal model of malnutrition that might prove useful in comparing the effectiveness of different enteral diets.