Use of focused ultrasonication in activity-based profiling of deubiquitinating enzymes in tissue.

To develop a reproducible tissue lysis method that retains enzyme function for activity-based protein profiling, we compared four different methods to obtain protein extracts from bovine lung tissue: focused ultrasonication, standard sonication, mortar & pestle method, and homogenization combined with standard sonication. Focused ultrasonication and mortar & pestle methods were sufficiently effective for activity-based profiling of deubiquitinases in tissue, and focused ultrasonication also had the fastest processing time. We used focused-ultrasonicator for subsequent activity-based proteomic analysis of deubiquitinases to test the compatibility of this method in sample preparation for activity-based chemical proteomics.

Daily Eicosapentaenoic Acid Infusion in IUGR Fetal Lambs Reduced Systemic Inflammation, Increased Muscle ADRß2 Content, and Improved Myoblast Function and Muscle Growth.

Intrauterine growth-restricted (IUGR) fetuses exhibit systemic inflammation that contributes to programmed deficits in myoblast function and muscle growth. Thus, we sought to determine if targeting fetal inflammation improves muscle growth outcomes. Heat stress-induced IUGR fetal lambs were infused with eicosapentaenoic acid (IUGR+EPA; n = 9) or saline (IUGR; n = 8) for 5 days during late gestation and compared to saline-infused controls (n = 11). Circulating eicosapentaenoic acid was 42% less (p < 0.05) for IUGR fetuses but was recovered in IUGR+EPA fetuses. The infusion did not improve placental function or fetal O2 but resolved the 67% greater (p < 0.05) circulating TNFα observed in IUGR fetuses. This improved myoblast function and muscle growth, as the 23% reduction (p < 0.05) in the ex vivo differentiation of IUGR myoblasts was resolved in IUGR+EPA myoblasts. Semitendinosus, longissimus dorsi, and flexor digitorum superficialis muscles were 24-39% lighter (p < 0.05) for IUGR but not for IUGR+EPA fetuses. Elevated (p < 0.05) IL6R and reduced (p < 0.05) ß2 adrenoceptor content in IUGR muscle indicated enhanced inflammatory sensitivity and diminished ß2 adrenergic sensitivity. Although IL6R remained elevated, ß2 adrenoceptor deficits were resolved in IUGR+EPA muscle, demonstrating a unique underlying mechanism for muscle dysregulation. These findings show that fetal inflammation contributes to IUGR muscle growth deficits and thus may be an effective target for intervention.

Daily Injection of the ß2 Adrenergic Agonist Clenbuterol Improved Muscle Glucose Metabolism, Glucose-Stimulated Insulin Secretion, and Hyperlipidemia in Juvenile Lambs Following Heat-Stress-Induced Intrauterine Growth Restriction.

Stress-induced fetal programming diminishes ß2 adrenergic tone, which coincides with intrauterine growth restriction (IUGR) and lifelong metabolic dysfunction. We determined if stimulating ß2 adrenergic activity in IUGR-born lambs would improve metabolic outcomes. IUGR lambs that received daily injections of saline or the ß2 agonist clenbuterol from birth to 60 days were compared with controls from pair-fed thermoneutral pregnancies. As juveniles, IUGR lambs exhibited systemic inflammation and robust metabolic dysfunction, including greater (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, increased (p < 0.05) intramuscular glycogen, reduced (p < 0.05) circulating IGF-1, hindlimb blood flow, glucose-stimulated insulin secretion, and muscle glucose oxidation. Daily clenbuterol fully recovered (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, hindlimb blood flow, muscle glucose oxidation, and intramuscular glycogen. Glucose-stimulated insulin secretion was partially recovered (p < 0.05) in clenbuterol-treated IUGR lambs, but circulating IGF-1 was not improved. Circulating triglycerides and HDL cholesterol were elevated (p < 0.05) in clenbuterol-treated IUGR lambs, despite being normal in untreated IUGR lambs. We conclude that deficient ß2 adrenergic regulation is a primary mechanism for several components of metabolic dysfunction in IUGR-born offspring and thus represents a potential therapeutic target for improving metabolic outcomes. Moreover, benefits from the ß2 agonist were likely complemented by its suppression of IUGR-associated inflammation.

Use of bioluminescent Escherichia coli to determine retention during the life cycle of the housefly, Musca domestica (Diptera: Muscidae, L).

Researchers have documented that the housefly (Musca domestica) can serve as a vector for the spread of foodborne pathogens to livestock, food, and humans. Most studies have investigated Musca domestica as a vector only after the fly comes into contact or consumes the pathogen as an adult. The objective of this study was to determine whether the larvae of Musca domestica could ingest Escherichia coli from bovine manure and whether the E. coli could survive the metamorphosis process and be transmitted. Larvae (n=960) were incubated in sterilized bovine manure inoculated with 0, 3, 5, and 8 log10 colony-forming units (CFU)/mL of bioluminescent E. coli for 24 (larvae stage), 48 (larvae stage), 120 (pupae stage), and 192 h (adult stage). Larvae incubated for 24 h in bovine manure possessed 0.0, 2.7, 2.9, and 3.5 log(10) CFU/mL of E. coli, from inoculated with 0, 3, 5, and 8 log(10) CFU/mL of E. coli, respectively. Concentrations of E. coli within the pupae were 0.0, 1.7, 1.9, and 2.2 log(10) CFU/mL for each inoculation concentration, respectively. Flies that emerged from the pupae stage contained 0.0, 1.3, 2.2, and 1.7 log(10) CFU/mL of E. coli from larvae incubated in manure inoculated with concentrations of E. coli, respectively. These results suggest the housefly can emerge with quantities of E. coli. While this was an enteropathogenic E. coli (EPEC), these data may suggest that if the fly is capable of retaining similar concentrations of an enterohemorrhagic E. coli (EHEC), these concentrations may be capable of initiating illness in humans. Furthermore, the E. coli concentration within and on adult flies is related to environmental exposure. It must be noted that larvae were incubated in sterilized bovine manure, and there was no other bacterial competition for the E. coli. Thus, the rate of positive flies and concentrations present when flies emerged may vary under more realistic conditions.

Survival of Escherichia coli O157:H7 transformed with either the pAK1-lux or pXEN-13 plasmids in in vitro bovine ruminal and fecal microbial fermentations.

The use of luminescent plasmids in bacteria may serve as a viable model for the real-time validation of various pre-harvest interventions on the colonization or shedding patterns of Escherichia coli O157:H7 within cattle. The objective of this study was to determine if the growth characteristics of E. coli O157:H7 in mixed ruminal and fecal microbial fluid cultures would be altered when transformed with one of the two luminescent plasmids: pAK1-lux (PAK) or pXEN-13 (XEN). Transformants harboring the luminescent plasmids were compared to the non-transformed parental strain (wild type [WT]) after incubating in mixed ruminal or fecal microbial fluid media for 6 h in triplicate (n=3). The transformants and WT exhibited similar growth rates. Within mixed ruminal microbial fluid fermentations and mixed fecal microbial fluid, all transformants grew similarly (p=0.28) through the 6-h study. The reflective light unit (RLU; photons/pixel per second) photonic emissions of each plasmid within ruminal fluid differed at 0 h (p=0.002) and 2 h (p=0.02) and within fecal fluid at 0 h (p=0.009) and 2 h (p=0.04). The RLU remained the same within rumen fluid at 4 h (p=0.22) and 6 h (p=0.80) and within fecal fluid at 4 h (p=0.06) and 6 h (p=0.29). Growth of E. coli O157:H7 transformed with the bioluminescent plasmids was not altered in comparison to the WT, suggesting that both plasmids may serve as useful models for in vivo studies.

Evaluation of Precision Livestock Technology and Human Scoring of Nursery Pigs in a Controlled Immune Challenge Experiment.

The objectives were to determine the sensitivity, specificity, and cutoff values of a visual-based precision livestock technology (NUtrack), and determine the sensitivity and specificity of sickness score data collected with the live observation by trained human observers. At weaning, pigs (n = 192; gilts and barrows) were randomly assigned to one of twelve pens (16/pen) and treatments were randomly assigned to pens. Sham-pen pigs all received subcutaneous saline (3 mL). For LPS-pen pigs, all pigs received subcutaneous lipopolysaccharide (LPS; 300 µg/kg BW; E. coli O111:B4; in 3 mL of saline). For the last treatment, eight pigs were randomly assigned to receive LPS, and the other eight were sham (same methods as above; half-and-half pens). Human data from the day of the challenge presented high true positive and low false positive rates (88.5% sensitivity; 85.4% specificity; 0.871 Area Under Curve, AUC), however, these values declined when half-and-half pigs were scored (75% sensitivity; 65.5% specificity; 0.703 AUC). Precision technology measures had excellent AUC, sensitivity, and specificity for the first 72 h after treatment and AUC values were >0.970, regardless of pen treatment. These results indicate that precision technology has a greater potential for identifying pigs during a natural infectious disease event than trained professionals using timepoint sampling.

Daily injection of the ß2 adrenergic agonist clenbuterol improved poor muscle growth and body composition in lambs following heat stress-induced intrauterine growth restriction.

Background: Intrauterine growth restriction (IUGR) is associated with reduced ß2 adrenergic sensitivity, which contributes to poor postnatal muscle growth. The objective of this study was to determine if stimulating ß2 adrenergic activity postnatal would rescue deficits in muscle growth, body composition, and indicators of metabolic homeostasis in IUGR offspring. Methods: Time-mated ewes were housed at 40°C from day 40 to 95 of gestation to produce IUGR lambs. From birth, IUGR lambs received daily IM injections of 0.8 µg/kg clenbuterol HCl (IUGR+CLEN; n = 11) or saline placebo (IUGR; n = 12). Placebo-injected controls (n = 13) were born to pair-fed thermoneutral ewes. Biometrics were assessed weekly and body composition was estimated by ultrasound and bioelectrical impedance analysis (BIA). Lambs were necropsied at 60 days of age. Results: Bodyweights were lighter (p ≤ 0.05) for IUGR and IUGR+CLEN lambs than for controls at birth, day 30, and day 60. Average daily gain was less (p ≤ 0.05) for IUGR lambs than controls and was intermediate for IUGR+CLEN lambs. At day 58, BIA-estimated whole-body fat-free mass and ultrasound-estimated loin eye area were less (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. At necropsy, loin eye area and flexor digitorum superficialis muscles were smaller (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Longissimus dorsi protein content was less (p ≤ 0.05) and fat-to-protein ratio was greater (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Semitendinosus from IUGR lambs had less (p ≤ 0.05) ß2 adrenoreceptor content, fewer (p ≤ 0.05) proliferating myoblasts, tended to have fewer (p = 0.08) differentiated myoblasts, and had smaller (p ≤ 0.05) muscle fibers than controls. Proliferating myoblasts and fiber size were recovered (p ≤ 0.05) in IUGR+CLEN lambs compared to IUGR lambs, but ß2 adrenoreceptor content and differentiated myoblasts were not recovered. Semitendinosus lipid droplets were smaller (p ≤ 0.05) in size for IUGR lambs than for controls and were further reduced (p ≤ 0.05) in size for IUGR+CLEN lambs. Conclusion: These findings show that clenbuterol improved IUGR deficits in muscle growth and some metabolic parameters even without recovering the deficit in ß2 adrenoreceptor content. We conclude that IUGR muscle remained responsive to ß2 adrenergic stimulation postnatal, which may be a strategic target for improving muscle growth and body composition in IUGR-born offspring.

Survival of O157:H7 and non-O157 serogroups of Escherichia coli in bovine rumen fluid and bile salts.

While Shiga toxin-producing Escherichia coli (STEC) reside asymptomatically within ruminants, particularly cattle, these strains pose a serious health risk to humans. Research related to STEC has historically focused upon O157:H7. However, with an increase in foodborne outbreaks of non-O157 origin and recent changes in testing for non-O157 by the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS), there is now a critical need to understand the biological activity of non-O157 serogroups. The focus of this study was to determine whether variations exist in the ability of different serotypes of STEC to survive within bovine rumen fluid medium and bile salts. The results of this study demonstrated through viable plate count analysis that the five serotypes tested (O157:H7, O111:H8, O103:K.:H8, O145:H28, and O26:H11) were capable of growing in rumen fluid medium. However, the concentrations of the serotypes O103:K.:H8 and O26:H11 after 24 h were significantly less (p < 0.05) than that observed for the other serotypes tested. A significant decrease (p = 0.03) in the survival of O103:K.:H8 in 50 mg/mL of bovine bile salts in comparison to the other STEC strains tested was also observed. Collectively, these data suggest that non-O157 serogroups of E. coli respond differently to the environment of the bovine gastrointestinal tract. Further research is needed to elucidate how these differential physiological variations correlate with alterations in colonization success within ruminants and how they may impact human illnesses.

Evaluation of a novel computer vision-based livestock monitoring system to identify and track specific behaviors of individual nursery pigs within a group-housed environment.

Animal behavior is indicative of health status and changes in behavior can indicate health issues (i.e., illness, stress, or injury). Currently, human observation (HO) is the only method for detecting behavior changes that may indicate problems in group-housed pigs. While HO is effective, limitations exist. Limitations include HO being time consuming, HO obfuscates natural behaviors, and it is not possible to maintain continuous HO. To address these limitations, a computer vision platform (NUtrack) was developed to identify (ID) and continuously monitor specific behaviors of group-housed pigs on an individual basis. The objectives of this study were to evaluate the capabilities of the NUtrack system and evaluate changes in behavior patterns over time of group-housed nursery pigs. The NUtrack system was installed above four nursery pens to monitor the behavior of 28 newly weaned pigs during a 42-d nursery period. Pigs were stratified by sex, litter, and randomly assigned to one of two pens (14 pigs/pen) for the first 22 d. On day 23, pigs were split into four pens (7 pigs/pen). To evaluate the NUtrack system's capabilities, 800 video frames containing 11,200 individual observations were randomly selected across the nursery period. Each frame was visually evaluated to verify the NUtrack system's accuracy for ID and classification of behavior. The NUtrack system achieved an overall accuracy for ID of 95.6%. This accuracy for ID was 93.5% during the first 22 d and increased (P < 0.001) to 98.2% for the final 20 d. Of the ID errors, 72.2% were due to mislabeled ID and 27.8% were due to loss of ID. The NUtrack system classified lying, standing, walking, at the feeder (ATF), and at the waterer (ATW) behaviors accurately at a rate of 98.7%, 89.7%, 88.5%, 95.6%, and 79.9%, respectively. Behavior data indicated that the time budget for lying, standing, and walking in nursery pigs was 77.7% ± 1.6%, 8.5% ± 1.1%, and 2.9% ± 0.4%, respectively. In addition, behavior data indicated that nursery pigs spent 9.9% ± 1.7% and 1.0% ± 0.3% time ATF and ATW, respectively. Results suggest that the NUtrack system can detect, identify, maintain ID, and classify specific behavior of group-housed nursery pigs for the duration of the 42-d nursery period. Overall, results suggest that, with continued research, the NUtrack system may provide a viable real-time precision livestock tool with the ability to assist producers in monitoring behaviors and potential changes in the behavior of group-housed pigs.

Short Communication: Beta-adrenergic agonists alter oxidative phosphorylation in primary myoblasts.

Beta-adrenergic agonists (ß-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which ß-AAs achieve this outcome. Our objective was to identify the influence of ractopamine HCl and zilpaterol HCl on mitochondrial respiratory activity in muscle satellite cells isolated from crossbred beef steers (N = 5), crossbred barrows (N = 2), Yorkshire-cross gilts (N = 3), and commercial weather lambs (N = 5). Real-time measurements of oxygen consumption rates (OCRs) were recorded using extracellular flux analyses with a Seahorse XFe24 analyzer. After basal OCR measurements were recorded, zilpaterol HCl, ractopamine HCl, or no ß-AA was injected into the assay plate in three technical replicates for each cell isolate. Then, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, and rotenone were injected into the assay plate sequentially, each inducing a different cellular state. This allowed for the measurement of OCR at these states and for the calculation of the following measures of mitochondrial function: basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, adenosine triphosphate (ATP)-linked respiration, and spare respiratory capacity. Incubation of bovine cells with either zilpaterol HCl or ractopamine HCl increased maximal respiration (P = 0.046) and spare respiratory capacity (P = 0.035) compared with non-supplemented counterparts. No difference (P > 0.05) was observed between zilpaterol HCl and ractopamine HCl for maximal respiration and spare respiratory capacity in bovine cell isolates. No measures of mitochondrial function (basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, ATP-linked respiration, and spare respiratory capacity) were altered by ß-AA treatment in ovine or porcine cells. These findings indicate that ß-AAs in cattle may improve the efficiency of oxidative metabolism in muscle satellite cells by modifying mitochondrial respiratory activity. The lack of response by ovine and porcine cells to ß-AA incubation also demonstrates differing physiological responses to ß-AA across species, which helps to explain the variation in its effectiveness as a growth supplement.

Beta-adrenergic agonists (ß-AAs) are supplemented to pigs and cattle to improve growth performance, carcass weight, and loin muscle area. Little is known about the mechanism taking place within individual cells by which ß-AAs achieve this outcome. Previous work reported that ß-AA supplementation improves the efficiency in which cells use glucose as an energy source and alters the expression of genes related to mitochondrial function, a key component of cellular energy production. To further our understanding of the impact of ß-AA supplementation on these cellular functions, our objective was to identify the influence of two ß-AAs used in livestock production, ractopamine HCl and zilpaterol HCl, on the mitochondrial respiratory activity of cells collected from the loin muscle and grown in culture. We isolated cells from cattle, pig, and sheep muscle and measured the oxygen consumption of the cells after treatment with ractopamine HCl, zilpaterol HCl, or with no supplement. We found that both ractopamine HCl and zilpaterol HCl enhance the efficiency of cellular energy production during a state of cellular stress in bovine muscle cells. There was no appreciable effect of the supplement on the energy production of pig or sheep cells. These data indicate that ß-AA supplementation in cattle may increase the muscle cell energy production capacity compared with non-supplemented cells. This study also demonstrates that the efficiency of cell energy production is one plausible mechanism underlying species differences in the response to ß-AA supplementation.

Transcriptome analyses indicate that heat stress-induced inflammation in white adipose tissue and oxidative stress in skeletal muscle is partially moderated by zilpaterol supplementation in beef cattle.

Heat stress (HS) triggers oxidative stress, systemic inflammation, and disrupts growth efficiency of livestock. ß-adrenergic agonists supplemented to ruminant livestock improve growth performance, increase skeletal muscle mass, and decrease carcass fat. The objective of this study was to understand the independent and interacting effects of HS and zilpaterol hydrochloride (ZH) supplementation on the transcriptome of subcutaneous white adipose tissue and the longissimus dorsi muscle in steers. Twenty-four Red Angus-based steers were assigned to thermoneutral (TN; Temperature Humidity Index [THI] = 68) or HS (THI = 73-85) conditions and were not supplemented or supplemented with ZH (8.33 mg/kg/d) for 21 d in a 2 × 2 factorial. Steers in the TN condition were pair-fed to the average daily feed intake of HS steers. RNA was isolated from adipose tissue and skeletal muscle samples collected via biopsy on 3, 10, and 21 d and sequenced using 3' Tag-Seq to an achieved average depth of 3.6 million reads/sample. Transcripts, mapped to ARS-UCD1.2, were quantified. Differential expression (DE) analyses were performed in DESeq2 with a significance threshold for false discovery rate of 0.05. In adipose, 4 loci (MISP3, APOL6, SLC25A4, and S100A12) were DE due to ZH on day 3, and 2 (RRAD, ALB) were DE due to the interaction of HS and ZH on day 10 (Padj < 0.05). In muscle, 40 loci (including TENM4 and OAZ1) were DE due to ZH on day 10, and 6 loci (HIF1A, LOC101903734, PDZD9, HNRNPU, MTUS1, and TMCO6) were DE due to environment on day 21 (Padj < 0.05). To explore biological pathways altered by environment, supplement, and their interaction, loci with DE (Praw < 0.05) were evaluated in Ingenuity Pathway Analysis. In adipose, 509 pathways were predicted to be altered (P < 0.01): 202 due to HS, 126 due to ZH, and 181 due to the interaction; these included inflammatory pathways predicted to be upregulated due to HS but downregulated due to the interaction of HS and ZH. In muscle, 113 pathways were predicted to be altered (P < 0.01): 23 due to HS, 66 due to ZH, and 24 due to the interaction of HS and ZH. Loci and pathway data in muscle suggest HS induced oxidative stress and that the stress response was moderated by ZH. Metabolic pathways were predicted to be altered due to HS, ZH, and their interaction in both tissues. These data provide evidence that HS and ZH interact to alter expression of genes in metabolic and immune function pathways and that ZH moderates some adverse effects of HS.

Heat stress (HS) negatively impacts livestock health and carcass quality. Supplementation of livestock with ß-adrenergic agonists (ß-AA) increases muscle mass and decreases fat deposition. The purpose of this study was to understand how HS and zilpaterol hydrochloride (ZH), a ß-AA, alter gene expression in muscle and in adipose of cattle. Twenty-four steers were assigned to thermoneutral (TN) or HS conditions and were not supplemented (NS) or supplemented with ZH for 21 d. RNA was isolated from muscle and adipose collected on days 3, 10, and 21 to identify changes in gene expression. Several individual loci were differentially expressed (DE) due to HS or ZH in both tissues while the interaction of HS and ZH altered expression in adipose. A less stringent definition of DE used to explore biological pathways predicted that both treatments alter metabolism. Pathway analyses also supported that HS increased inflammation in adipose, but that these inflammatory pathways were downregulated by ZH. HS also was predicted to induce oxidative stress in muscle although ZH moderated this response. This study provides information on how HS and ß-AA act independently and interact to alter physiology, lending insight useful for the development of management and mitigation strategies for stress.

Primary myoblasts from intrauterine growth-restricted fetal sheep exhibit intrinsic dysfunction of proliferation and differentiation that coincides with enrichment of inflammatory cytokine signaling pathways.

Intrauterine growth restriction (IUGR) is linked to lifelong reductions in muscle mass due to intrinsic functional deficits in myoblasts, but the mechanisms underlying these deficits are not known. Our objective was to determine if the deficits were associated with changes in inflammatory and adrenergic regulation of IUGR myoblasts, as was previously observed in IUGR muscle. Primary myoblasts were isolated from IUGR fetal sheep produced by hyperthermia-induced placental insufficiency (PI-IUGR; n = 9) and their controls (n = 9) and from IUGR fetal sheep produced by maternofetal inflammation (MI-IUGR; n = 6) and their controls (n = 7). Proliferation rates were less (P < 0.05) for PI-IUGR myoblasts than their controls and were not affected by incubation with IL-6, TNF-α, norepinephrine, or insulin. IκB kinase inhibition reduced (P < 0.05) proliferation of control myoblasts modestly in basal media but substantially in TNF-α-added media and reduced (P < 0.05) PI-IUGR myoblast proliferation substantially in basal and TNF-α-added media. Proliferation was greater (P < 0.05) for MI-IUGR myoblasts than their controls and was not affected by incubation with TNF-α. Insulin increased (P < 0.05) proliferation in both MI-IUGR and control myoblasts. After 72-h differentiation, fewer (P < 0.05) PI-IUGR myoblasts were myogenin+ than controls in basal and IL-6 added media but not TNF-α-added media. Fewer (P < 0.05) PI-IUGR myoblasts were desmin+ than controls in basal media only. Incubation with norepinephrine did not affect myogenin+ or desmin+ percentages, but insulin increased (P < 0.05) both markers in control and PI-IUGR myoblasts. After 96-h differentiation, fewer (P < 0.05) MI-IUGR myoblasts were myogenin+ and desmin+ than controls regardless of media, although TNF-α reduced (P < 0.05) desmin+ myoblasts for both groups. Differentiated PI-IUGR myoblasts had greater (P < 0.05) TNFR1, ULK2, and TNF-α-stimulated TLR4 gene expression, and PI-IUGR semitendinosus muscle had greater (P < 0.05) TNFR1 and IL6 gene expression, greater (P < 0.05) c-Fos protein, and less (P < 0.05) IκBα protein. Differentiated MI-IUGR myoblasts had greater (P < 0.05) TNFR1 and IL6R gene expression, tended to have greater (P = 0.07) ULK2 gene expression, and had greater (P < 0.05) ß-catenin protein and TNF-α-stimulated phosphorylation of NFκB. We conclude that these enriched components of TNF-α/TNFR1/NFκB and other inflammatory pathways in IUGR myoblasts contribute to their dysfunction and help explain impaired muscle growth in the IUGR fetus.

Myoblasts are stems cells whose functional capacity can limit muscle growth. However, stressful intrauterine conditions cause these cells to be intrinsically dysfunctional. This restricts muscle growth capacity, leading to intrauterine growth restriction (IUGR) of the fetus, low birth weight, and less muscle mass after birth. Consequently, meat yield is reduced in IUGR-born food animals and glucose homeostasis is impaired in IUGR-born humans, which contributes to metabolic dysfunction. Intrinsic dysfunction of IUGR myoblasts has been previously observed, but the fetal programming changes (i.e., permanent changes in the development of cellular mechanisms that explains different functional outcomes) have not been identified. This study shows that one mechanism is the enhancement of signaling pathways for TNF-α and other inflammatory cytokines. These cytokines have roles in stress responses and regulation of muscle growth. Programmed enhancement of these pathways means that IUGR myoblasts are more responsive to even normal amounts of circulating cytokines. Unfortunately, the primary response of myoblasts to cytokines is slower differentiation (i.e., cellular transformation necessary for muscle growth). Programmed enhancement of this response directly impedes myoblast-dependent muscle growth, and the deficit is lifelong. However, identifying this mechanism is a fundamental step for developing strategies to improve muscle growth in low birth weight offspring.

Sustained heat stress elevated corneal and body surface temperatures and altered circulating leukocytes and metabolic indicators in wether lambs supplemented with ractopamine or zilpaterol.

Understanding how ß adrenergic agonists influence the physiology of heat stress could lead to mitigation options. We sought to investigate body surface temperatures in feedlot wethers supplemented with ractopamine or zilpaterol and exposed to heat stress for 18 d. Corneal and skin temperatures were assessed via infrared thermography at 1- and 2-m distances. Rectal temperatures and circulating leukocytes, metabolites, and electrolytes were also measured. Heat stress increased (P < 0.05) rectal temperatures in unsupplemented and zilpaterol-supplemented lambs but not in ractopamine-supplemented lambs. Heat stress also increased (P < 0.05) surface temperatures of the cornea, nose, ear, and back, regardless of supplement. Observations were comparable between thermography performed at 1 and 2 m, and higher emissivity settings generally produced less variation. Heat stress tended to increase (P = 0.08) blood monocytes in unsupplemented but not ractopamine- or zilpaterol-supplemented lambs. Granulocytes were increased (P < 0.05) by heat stress in ractopamine-supplemented lambs but decreased (P < 0.05) in zilpaterol-supplemented lambs. Blood glucose, triglycerides, and cholesterol did not differ among groups, and blood lactate was reduced (P < 0.05) by heat stress in zilpaterol-supplemented lambs only. Blood Na+ was reduced (P < 0.05) and Ca2+ increased (P < 0.05) by heat stress, regardless of supplement. These findings indicate that ß1- and ß2-adrenergic agonists differentially relieve some but not all heat stress-induced changes in stress indicators. Moreover, corneal and skin surface temperatures measured by infrared thermography reasonably identified body temperature changes at a distance of 2 m.

Heat stress-induced deficits in growth, metabolic efficiency, and cardiovascular function coincided with chronic systemic inflammation and hypercatecholaminemia in ractopamine-supplemented feedlot lambs.

Heat stress hinders growth and well-being in livestock, an effect that is perhaps exacerbated by the ß1 agonist ractopamine. Heat stress deficits are mediated in part by reduced feed intake, but other mechanisms involved are less understood. Our objective was to determine the direct impact of heat stress on growth and well-being in ractopamine-supplemented feedlot lambs. Commercial wethers were fed under heat stress (40 °C) for 30 d, and controls (18 °C) were pair-fed. In a 2 × 2 factorial, lambs were also given a daily gavage of 0 or 60 mg ractopamine. Growth, metabolic, cardiovascular, and stress indicators were assessed throughout the study. At necropsy, 9th to 12th rib sections (four-rib), internal organs, and feet were assessed, and sartorius muscles were collected for ex vivo glucose metabolic studies. Heat stress increased (P < 0.05) rectal temperatures and respiration rates throughout the study and reduced (P < 0.05) weight gain and feed efficiency over the first week, ultrasonic loin-eye area and loin depth near the end of the study, and four-rib weight at necropsy. Fat content of the four-rib and loin were also reduced (P < 0.05) by heat stress. Ractopamine increased (P < 0.05) loin weight and fat content and partially moderated the impact of heat stress on rectal temperature and four-rib weight. Heat stress reduced (P < 0.05) spleen weight, increased (P < 0.05) adrenal and lung weights, and was associated with hoof wall overgrowth but not organ lesions. Ractopamine did not affect any measured indicators of well-being. Heat stress reduced (P < 0.05) blood urea nitrogen and increased (P < 0.05) circulating monocytes, granulocytes, and total white blood cells as well as epinephrine, TNFα, cholesterol, and triglycerides. Cortisol and insulin were not affected. Heat stress reduced (P < 0.05) blood pressure and heart rates in all lambs and increased (P < 0.05) left ventricular wall thickness in unsupplemented but not ractopamine-supplemented lambs. No cardiac arrhythmias were observed. Muscle glucose uptake did not differ among groups, but insulin-stimulated glucose oxidation was reduced (P < 0.05) in muscle from heat-stressed lambs. These findings demonstrate that heat stress impairs growth, metabolism, and well-being even when the impact of feed intake is eliminated by pair-feeding and that systemic inflammation and hypercatecholaminemia likely contribute to these deficits. Moreover, ractopamine improved muscle growth indicators without worsening the effects of heat stress.

Whole blood transcriptome analysis in feedlot cattle after 35 days of supplementation with a ß1-adrenergic agonist.

Ractopamine HCl (RHC) is supplemented to feedlot cattle to improve feed efficiency and increase carcass weight. Supplementation of RHC clearly benefits livestock production, but it is of note that the adrenergic system through which it acts is typically associated with stress. The purpose of this study was to identify changes in the transcriptome of whole blood in RHC-supplemented feedlot cattle. We hypothesized that transcripts related to inflammatory processes would be upregulated after 35 days of dietary RHC supplementation. To test this hypothesis, RNA from whole blood collected from 16 cattle before and after supplementation with 300 mg/day of RHC was sequenced using 3' tag-seq. Eight transcripts were differentially expressed (Adjp < 0.10) between pre- and post-supplementation blood samples. Although several of these transcripts including IFI35, TYROBP, and TP53INP1 are associated with inflammation, a systemic dysregulation of inflammatory pathways was not evident. These data provide insight into the response of cattle to RHC supplementation that will direct future studies examining how the transcriptome of whole blood and other tissues responds during acute exposure to RHC and how this supplement mechanistically improves growth performance.

Hypertrophic muscle growth and metabolic efficiency were impaired by chronic heat stress, improved by zilpaterol supplementation, and not affected by ractopamine supplementation in feedlot lambs1.

Feedlot performance is reduced by heat stress and improved by ß adrenergic agonists (ßAA). However, the physiological mechanisms underlying these outcomes are not well characterized, and anecdotal reports suggest that ßAA may confound the effects of heat stress on wellbeing. Thus, we sought to determine how heat stress and ßAA affect growth, metabolic efficiency, and health indicators in lambs on a feedlot diet. Wethers (38.6 ± 1.9 kg) were housed under thermoneutral (controls; n = 25) or heat stress (n = 24) conditions for 21 d. In a 2 × 3 factorial, their diets contained no supplement (unsupplemented), ractopamine (ß1AA), or zilpaterol (ß2AA). Blood was collected on days -3, 3, 9, and 21. On day 22, lambs were harvested and ex vivo skeletal muscle glucose oxidation was determined to gauge metabolic efficiency. Feet and organ tissue damage was assessed by veterinary pathologists. Heat stress reduced (P < 0.05) feed intake by 21%, final bodyweight (BW) by 2.6 kg, and flexor digitorum superficialis (FDS) muscle mass by 5%. ß2AA increased (P < 0.05) FDS mass/BW by 9% and average muscle fiber area by 13% compared with unsupplemented lambs. Blood lymphocytes and monocytes were greater (P < 0.05) in heat-stressed lambs, consistent with systemic inflammation. Plasma insulin was 22% greater (P < 0.05) and glucose/insulin was 16% less (P < 0.05) in heat-stressed lambs than controls. Blood plasma urea nitrogen was increased (P < 0.05) by heat stress on day 3 but reduced (P < 0.05) on days 9 and 21. Plasma lipase and lactate dehydrogenase were reduced (P < 0.05) by heat stress. Glucose oxidation was 17% less (P < 0.05) in muscle from heat-stressed lambs compared with controls and 15% greater (P < 0.05) for ß2AA-supplemented compared with unsupplemented lambs. Environment and supplement interacted (P < 0.05) for rectal temperature, which was increased (P < 0.05) by heat stress on all days but more so (P < 0.05) in ß2AA-supplemented lambs on days 4, 9, and 16. Heat stress increased (P < 0.05) the frequency of hoof wall overgrowth, but ßAA did not produce any pathologies. We conclude that reduced performance in heat-stressed lambs was mediated by reduced feed intake, muscle growth, and metabolic efficiency. ß2AA increased muscle growth and improved metabolic efficiency by increasing muscle glucose oxidation, but no such effects were observed with ractopamine. Finally, ßAA supplementation was not detrimental to health indicators in this study, nor did it worsen the effects of heat stress.

ASAS-SSR Triennnial Reproduction Symposium: Looking Back and Moving Forward-How Reproductive Physiology has Evolved: Fetal origins of impaired muscle growth and metabolic dysfunction: Lessons from the heat-stressed pregnant ewe.

Intrauterine growth restriction (IUGR) is the second leading cause of perinatal mortality and predisposes offspring to metabolic disorders at all stages of life. Muscle-centric fetal adaptations reduce growth and yield metabolic parsimony, beneficial for IUGR fetal survival but detrimental to metabolic health after birth. Epidemiological studies have reported that IUGR-born children experience greater prevalence of insulin resistance and obesity, which progresses to diabetes, hypertension, and other metabolic disorders in adulthood that reduce quality of life. Similar adaptive programming in livestock results in decreased birth weights, reduced and inefficient growth, decreased carcass merit, and substantially greater mortality rates prior to maturation. High rates of glucose consumption and metabolic plasticity make skeletal muscle a primary target for nutrient-sparing adaptations in the IUGR fetus, but at the cost of its contribution to proper glucose homeostasis after birth. Identifying the mechanisms underlying IUGR pathophysiology is a fundamental step in developing treatments and interventions to improve outcomes in IUGR-born humans and livestock. In this review, we outline the current knowledge regarding the adaptive restriction of muscle growth and alteration of glucose metabolism that develops in response to progressively exacerbating intrauterine conditions. In addition, we discuss the evidence implicating developmental changes in ß adrenergic and inflammatory systems as key mechanisms for dysregulation of these processes. Lastly, we highlight the utility and importance of sheep models in developing this knowledge.

National Beef Quality Audit-2016: assessment of cattle hide characteristics, offal condemnations, and carcass traits to determine the quality status of the market cow and bull beef industry.

To continue the series that began in 1994, the National Beef Quality Audit (NBQA) - 2016 was conducted to quantify the quality status of the market cow and bull beef sector, as well as determine improvements made in the beef and dairy industry since 2007. The NBQA-2016 was conducted from March through December of 2016, and assessed hide-on carcasses (n = 5,278), chilled carcasses (n = 4,285), heads (n = 5,720), and offal items (n = 4,800) in 18 commercial processing facilities throughout the United States. Beef cattle were predominantly black-hided; 68.0% of beef cows and 67.2% of beef bulls possessed a black hide. Holstein was the predominant type of dairy animal observed. Just over half (56.0%) of the cattle surveyed had no mud contamination on the hide, and when mud was present, 34.1% of cattle only had small amounts. Harvest floor assessments found 44.6% of livers, 23.1% of lungs, 22.3% of hearts, 20.0% of viscera, 8.2% of heads, and 5.9% of tongues were condemned. Liver condemnations were most frequently due to abscess presence. In contrast, contamination was the primary reason for condemnation of all other offal items. Of the cow carcasses surveyed, 17.4% carried a fetus at the time of harvest. As expected, mean carcass weight and loin muscle area values observed for bulls were heavier and larger than cows. The marbling scores represented by cull animal carcasses were most frequently slight and traces amounts. Cow carcasses manifested a greater amount of marbling on average than bull carcasses. The predominant fat color score showed all carcasses surveyed had some level of yellow fat. Only 1.3% of carcasses exhibited signs of arthritic joints. Results of the NBQA-2016 indicate there are areas in which the beef and dairy industries have improved and areas that still need attention to prevent value loss in market cows and bulls.

The effects of nutritional management on carcass merit of beef cattle and on sensory properties of beef.

The ultimate goal of feeding beef cattle is to develop a meat product that satisfies consumer needs. Nutritional management plays a significant role in determining carcass merit of beef cattle. Certain management procedures (eg, metabolic modifiers, early weaning), general animal health (eg, history of respiratory disease), and certain feed ingredients (eg, trace minerals, antioxidants) can have positive and negative effects on the overall quality of beef that is supplied to the consuming public.

Effect of dietary lipoic acid on metabolic hormones and acute-phase proteins during challenge with infectious bovine rhinotracheitis virus in cattle.

OBJECTIVE: To determine the effect of dietary supplemental lipoic acid (LA) on serum concentrations of metabolic hormones and acute-phase proteins of steers challenged with infectious bovine rhinotracheitis virus (IBRV). ANIMALS: 32 steers. PROCEDURES: Steers were randomly assigned to 4 treatments: negative control (NC; no LA, no IBRV challenge), control (CON; no LA, IBRV challenge), 16 mg of LA/kg of body weight (BW)/d plus IBRV challenge (LA16), and 32 mg of LA/kg of BW/d plus IBRV challenge (LA32). Following a 21-day adaptation period, CON, LA16, and LA32 steers received IBRV (2 mL/nostril [day 0]); NC steers received saline (0.9% NaCl) solution. Blood samples, nasal swab specimens, BW, and rectal temperatures were obtained 0, 1, 3, 5, 7, 14, and 21 days after challenge. Serum was analyzed for concentrations of haptoglobin, amyloid-A, leptin, and anti-IBRV antibodies. RESULTS: Steers fed LA32 began gaining BW by day 7, whereas BW of CON and LA16 steers declined. Serum haptoglobin concentration of LA32 steers was lower than that of CON and LA16 steers on day 7. Serum neutralization titers for 30 of 32 steers were negative for anti-IBRV antibodies before challenge; however, all steers (including NCs) had antibodies on day 21. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that LA supplementation augmented certain aspects of the immune response; LA32 steers had a more rapid recovery from IBRV viral challenge than did others.