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.

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.

Dousing the flame: reviewing the mechanisms of inflammatory programming during stress-induced intrauterine growth restriction and the potential for ω-3 polyunsaturated fatty acid intervention.

Intrauterine growth restriction (IUGR) arises when maternal stressors coincide with peak placental development, leading to placental insufficiency. When the expanding nutrient demands of the growing fetus subsequently exceed the capacity of the stunted placenta, fetal hypoxemia and hypoglycemia result. Poor fetal nutrient status stimulates greater release of inflammatory cytokines and catecholamines, which in turn lead to thrifty growth and metabolic programming that benefits fetal survival but is maladaptive after birth. Specifically, some IUGR fetal tissues develop enriched expression of inflammatory cytokine receptors and other signaling cascade components, which increases inflammatory sensitivity even when circulating inflammatory cytokines are no longer elevated after birth. Recent evidence indicates that greater inflammatory tone contributes to deficits in skeletal muscle growth and metabolism that are characteristic of IUGR offspring. These deficits underlie the metabolic dysfunction that markedly increases risk for metabolic diseases in IUGR-born individuals. The same programming mechanisms yield reduced metabolic efficiency, poor body composition, and inferior carcass quality in IUGR-born livestock. The ω-3 polyunsaturated fatty acids (PUFA) are diet-derived nutraceuticals with anti-inflammatory effects that have been used to improve conditions of chronic systemic inflammation, including intrauterine stress. In this review, we highlight the role of sustained systemic inflammation in the development of IUGR pathologies. We then discuss the potential for ω-3 PUFA supplementation to improve inflammation-mediated growth and metabolic deficits in IUGR offspring, along with potential barriers that must be considered when developing a supplementation strategy.

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.

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.

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.

Intermittent maternofetal oxygenation during late gestation improved birthweight, neonatal growth, body symmetry, and muscle metabolism in intrauterine growth-restricted lambs.

In humans and animals, intrauterine growth restriction (IUGR) results from fetal programming responses to poor intrauterine conditions. Chronic fetal hypoxemia elevates circulating catecholamines, which reduces skeletal muscle ß2 adrenoceptor content and contributes to growth and metabolic pathologies in IUGR-born offspring. Our objective was to determine whether intermittent maternofetal oxygenation during late gestation would improve neonatal growth and glucose metabolism in IUGR-born lambs. Pregnant ewes were housed at 40 °C from the 40th to 95th day of gestational age (dGA) to produce IUGR-born lambs (n = 9). A second group of IUGR-born lambs received prenatal O2 supplementation via maternal O2 insufflation (100% humidified O2, 10 L/min) for 8 h/d from dGA 130 to parturition (IUGR+O2, n = 10). Control lambs (n = 15) were from pair-fed thermoneutral ewes. All lambs were weaned at birth, hand-reared, and fitted with hindlimb catheters at day 25. Glucose-stimulated insulin secretion (GSIS) and hindlimb hyperinsulinemic-euglycemic clamp (HEC) studies were performed at days 28 and 29, respectively. At day 30, lambs were euthanized and ex vivo HEC studies were performed on isolated muscle. Without maternofetal oxygenation, IUGR lambs were 40% lighter (P < 0.05) at birth and maintained slower (P < 0.05) growth rates throughout the neonatal period compared with controls. At 30 d of age, IUGR lambs had lighter (P < 0.05) hindlimbs and flexor digitorum superficialis (FDS) muscles. IUGR+O2 lambs exhibited improved (P < 0.05) birthweight, neonatal growth, hindlimb mass, and FDS mass compared with IUGR lambs. Hindlimb insulin-stimulated glucose utilization and oxidation rates were reduced (P < 0.05) in IUGR but not IUGR+O2 lambs. Ex vivo glucose oxidation rates were less (P < 0.05) in muscle from IUGR but not IUGR+O2 lambs. Surprisingly, ß2 adrenoceptor content and insulin responsiveness were reduced (P < 0.05) in muscle from IUGR and IUGR+O2 lambs compared with controls. In addition, GSIS was reduced (P < 0.05) in IUGR lambs and only modestly improved (P < 0.05) in IUGR+O2. Insufflation of O2 also increased (P < 0.05) acidosis and hypercapnia in dams, perhaps due to the use of 100% O2 rather than a gas mixture with a lesser O2 percentage. Nevertheless, these findings show that intermittent maternofetal oxygenation during late gestation improved postnatal growth and metabolic outcomes in IUGR lambs without improving muscle ß2 adrenoceptor content.

Inflammatory Mediation of Heat Stress-Induced Growth Deficits in Livestock and Its Potential Role as a Target for Nutritional Interventions: A Review.

Heat stress is detrimental to well-being and growth performance in livestock, and systemic inflammation arising during chronic heat stress contributes to these poor outcomes. Sustained exposure of muscle and other tissues to inflammation can impair the cellular processes that facilitate muscle growth and intramuscular fat deposition, thus reducing carcass quality and yield. Climate change is expected to produce more frequent extreme heat events, increasing the potential impact of heat stress on sustainable livestock production. Feedlot animals are at particularly high risk for heat stress, as confinement limits their ability to seek cooling from the shade, water, or breeze. Economically practical options to circumvent heat stress in feedlot animals are limited, but understanding the mechanistic role of inflammation in heat stress outcomes may provide the basis for treatment strategies to improve well-being and performance. Feedlot animals receive formulated diets daily, which provides an opportunity to administer oral nutraceuticals and other bioactive products to mitigate heat stress-induced inflammation. In this review, we examine the complex associations between heat stress, systemic inflammation, and dysregulated muscle growth in meat animals. We also present evidence for potential nutraceutical and dietary moderators of inflammation and how they might improve the unique pathophysiology of heat stress.

The Price of Surviving on Adrenaline: Developmental Programming Responses to Chronic Fetal Hypercatecholaminemia Contribute to Poor Muscle Growth Capacity and Metabolic Dysfunction in IUGR-Born Offspring.

Maternofetal stress induces fetal programming that restricts skeletal muscle growth capacity and metabolic function, resulting in intrauterine growth restriction (IUGR) of the fetus. This thrifty phenotype aids fetal survival but also yields reduced muscle mass and metabolic dysfunction after birth. Consequently, IUGR-born individuals are at greater lifelong risk for metabolic disorders that reduce quality of life. In livestock, IUGR-born animals exhibit poor growth efficiency and body composition, making these animals more costly and less valuable. Specifically, IUGR-associated programming causes a greater propensity for fat deposition and a reduced capacity for muscle accretion. This, combined with metabolic inefficiency, means that these animals produce less lean meat from greater feed input, require more time on feed to reach market weight, and produce carcasses that are of less quality. Despite the health and economic implications of IUGR pathologies in humans and food animals, knowledge regarding their specific underlying mechanisms is lacking. However, recent data indicate that adaptive programing of adrenergic sensitivity in multiple tissues is a contributing factor in a number of IUGR pathologies including reduced muscle mass, peripheral insulin resistance, and impaired glucose metabolism. This review highlights the findings that support the role for adrenergic programming and how it relates to the lifelong consequences of IUGR, as well as how dysfunctional adrenergic signaling pathways might be effective targets for improving outcomes in IUGR-born offspring.

Going Up Inflame: Reviewing the Underexplored Role of Inflammatory Programming in Stress-Induced Intrauterine Growth Restricted Livestock.

The impact of intrauterine growth restriction (IUGR) on health in humans is well-recognized. It is the second leading cause of perinatal mortality worldwide, and it is associated with deficits in metabolism and muscle growth that increase lifelong risk for hypertension, obesity, hyperlipidemia, and type 2 diabetes. Comparatively, the barrier that IUGR imposes on livestock production is less recognized by the industry. Meat animals born with low birthweight due to IUGR are beset with greater early death loss, inefficient growth, and reduced carcass merit. These animals exhibit poor feed-to-gain ratios, less lean mass, and greater fat deposition, which increase production costs and decrease value. Ultimately, this reduces the amount of meat produced by each animal and threatens the economic sustainability of livestock industries. Intrauterine growth restriction is most commonly the result of fetal programming responses to placental insufficiency, but the exact mechanisms by which this occurs are not well-understood. In uncompromised pregnancies, inflammatory cytokines are produced at modest rates by placental and fetal tissues and play an important role in fetal development. However, unfavorable intrauterine conditions can cause cytokine activity to be excessive during critical windows of fetal development. Our recent evidence indicates that this impacts developmental programming of muscle growth and metabolism and contributes to the IUGR phenotype. In this review, we outline the role of inflammatory cytokine activity in the development of normal and IUGR phenotypes. We also highlight the contributions of sheep and other animal models in identifying mechanisms for IUGR pathologies.

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.

Homework questions designed to require higher-order cognitive skills in an undergraduate animal physiology course did not produce desirable difficulties, testing effects, or improvements in information retention.

Studies show that retrieval practices such as homework assignments that are completed during the encoding phase of learning benefit knowledge acquisition and retention. In addition, desirable difficulties, which are strategies that intentionally create a greater challenge during initial learning to enhance encoding and retrieval pathways, also benefit learning long term. Our objective was to determine whether weekly homework questions intended to create desirable difficulties by requiring higher-order cognitive skills (HOCS) benefited students' long-term retention of physiology concepts compared to questions designed to require lower-order cognitive skills (LOCS). Undergraduate students in a junior-level animal physiology course were presented information during weekly laboratory periods, and then required to complete retrieval practices in the form of online homework assignments 5 d after each lab. Homework questions were formatted per Bloom's Taxonomy to require HOCS (i.e., level 4 or 5) or LOCS (i.e., level 1 or 2). Information retention was assessed the next week via performance on an in-class quiz and again at semesters' end via performance on a final practical exam. We observed no differences in performance on the in-class quiz or final practical exam between students randomly assigned to complete homework with HOCS questions compared to LOCS questions. However, students that received homework with HOCS questions had decreased (P < 0.05) performance scores on 9 out of the 11 homework assignments compared to those receiving homework with LOCS questions. These findings indicate that desirable difficulties were not created by our HOCS homework questions because students receiving these more difficult retrieval practices did not achieve equal success on them. As a result, this attempt to create variations in cognitive demand did not enhance retention of knowledge in this study.

Lipopolysaccharide endotoxin injections elevated salivary TNFα and corneal temperatures and induced dynamic changes in circulating leukocytes, inflammatory cytokines, and metabolic indicators in wether lambs.

Pathogenic infections increase morbidity and reduce performance in livestock, and thus understanding the comprehensive physiological changes associated with infections can benefit production sustainability. In this study, we sought to investigate such physiological responses to an acute immune challenge in lambs. Polypay wethers received single IV injections of 1.5 µg/kg lipopolysaccharide endotoxin (LPS-injected; n = 6) or saline (controls; n = 6). Corneal temperatures (via infrared thermography), rectal temperatures, blood, plasma, and saliva were assessed every 2 hr for 10 hr after injections. Blood was also assessed at 24 hr. LPS-injected lambs exhibited elevated (P < 0.05) corneal and rectal temperatures that peaked at 4 hr but were still slightly greater (P < 0.05) than controls at 10 hr. Circulating total white blood cells, monocytes, and granulocytes were reduced (P < 0.05) in LPS-injected lambs within the first 4 hr but were subsequently greater (P < 0.05) than in controls. Lymphocytes were reduced (P < 0.05) in LPS-injected lambs over the first 8 hr and did not differ from controls thereafter. Red blood cells, hematocrit, and hemoglobin were increased (P < 0.05) in LPS-injected lambs over the first 6 hr, indicating mild dehydration. Blood glucose briefly increased (P < 0.05) in LPS-injected lambs at 2 hr but was less (P < 0.05) than in controls thereafter. Blood lactate was greater (P < 0.05) in LPS-injected lambs between 6 and 10 hr after injections, which together with reduced (P < 0.05) CO2 partial pressure indicated a metabolic shift toward glycolysis. LPS-injected lambs exhibited a transient increase (P < 0.05) in plasma TNFα at 2 and 4 hr only and sustained increases (P < 0.05) in CXCL9 and CXCL10 beginning at 6 and 4 hr, respectively. They also exhibited a mild, paradoxical increase (P < 0.05) in the anti-inflammatory sFRP3. Salivary TNFα was increased (P < 0.05) in LPS-injected lambs at 2 hr only. Regression analyses indicated that rectal temperatures were a generally poor predictor of the other inflammatory components in this study, with the exception of circulating leukocyte populations. Likewise, correlations among the 10 cytokines measured in this study were generally weak, with notable exceptions between CXCL9 and CXCL10 and between IL-21 and IFNγ. These findings demonstrate that physiological changes to even short-lived immune challenges are dynamic in nature and persist beyond the time frame of febrile responses and other common assessments.

Maternofetal inflammation induced for 2 wk in late gestation reduced birth weight and impaired neonatal growth and skeletal muscle glucose metabolism in lambs.

Intrauterine stress impairs growth and metabolism in the fetus and offspring. We recently found that sustained maternofetal inflammation resulted in intrauterine growth-restricted (MI-IUGR) fetuses with asymmetric body composition, impaired muscle glucose metabolism, and ß-cell dysfunction near term. These fetuses also exhibited heightened inflammatory tone, which we postulated was a fetal programming mechanism for the IUGR phenotype. Thus, the objective of this study was to determine whether poor growth and metabolism persisted in MI-IUGR lambs after birth. Polypay ewes received serial lipopolysaccharide or saline injections in the first 2 wk of the third trimester of pregnancy to produce MI-IUGR (n = 13) and control (n = 12) lambs, respectively. Lambs were catheterized at 25 d of age. ß-Cell function was assessed at 29 d, hindlimb glucose metabolism at 30 d, and daily blood parameters from day 26 to 31. Glucose metabolism was also assessed in flexor digitorum superficialis (FDS) muscle isolated at necropsy on day 31. Asymmetric body composition persisted in MI-IUGR neonates, as these lambs were lighter (P < 0.05) than controls at birth and 31 d, but body and cannon bone lengths did not differ at either age. FDS muscles from MI-IUGR lambs were smaller (P < 0.05) and exhibited reduced (P < 0.05) glucose oxidation and Akt phosphorylation but similar glucose uptake compared with controls when incubated in basal or insulin-spiked media. Similarly, hindlimb glucose oxidation was reduced (P < 0.05) in MI-IUGR lambs under basal and hyperinsulinemic conditions, but hindlimb glucose utilization did not differ from controls. Circulating urea nitrogen and cholesterol were reduced (P < 0.05), and triglycerides, high-density lipoprotein cholesterol, and glucose-to-insulin ratios were increased (P < 0.05) in MI-IUGR lambs. Glucose and insulin concentrations did not differ between groups during basal or hyperglycemic conditions. Although circulating monocyte and granulocyte concentrations were greater (P < 0.05) in MI-IUGR lambs, plasma tumor necrosis factor α (TNFα) was reduced (P < 0.05). FDS muscle contained greater (P < 0.05) TNF receptor 1 (TNFR1) and IκBα protein content. These findings indicate that maternofetal inflammation in late pregnancy results in fetal programming that impairs growth capacity, muscle glucose oxidation, and lipid homeostasis in offspring. Inflammatory indicators measured in this study appear to reflect heightened cytokine sensitivity in muscle and compensatory systemic responses to it.

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.