Impact of ergot alkaloid and steroidal implant on whole-body protein turnover and expression of mTOR pathway proteins in muscle of cattle.

Introduction: Holstein steers (n = 32) were used to determine if the ergot analog, bromocriptine decreases muscle protein synthesis through inhibitory action on the mTOR pathway via a direct effect on signal proteins, and if these negative effects can be alleviated with anabolic agents. Methods: Steers were treated with intramuscular administration of bromocriptine (vehicle or 0.1 mg/kg BW) and a subdermal commercial steroidal implant containing trenbolone acetate (TBA) and estradiol 17ß (with or without), in a 2×2 factorial design. During the 35 day experiment, intake was restricted to 1.5 times maintenance energy requirement. On days 27 through 32, steers were moved to metabolism stalls for urine collection, and whole-body protein turnover was determined using a single pulse dose of [15N] glycine into the jugular vein on day 28. On day 35, skeletal muscle samples were collected before (basal state) and 60 min after (stimulated state) an i.v. glucose challenge (0.25 g glucose/kg). Blood samples were collected at regular intervals before and after glucose infusion for determination of circulating concentrations of glucose and insulin. Results: Bromocriptine reduced insulin and glucose clearance following the glucose challenge, indicating decreased insulin sensitivity and possible disruption of glucose uptake and metabolism in the skeletal muscle. Conversely, analysis of whole-body protein turnover demonstrated that bromocriptine does not appear to affect protein synthesis or urea excretion. Western immunoblot analysis of skeletal muscle showed that it did not affect abundance of S6K1 or 4E-BP1, so bromocriptine does not appear to inhibit activation of the mTOR pathway or protein synthesis. Estradiol/TBA implant decreased urea excretion and protein turnover but had no effect on protein synthesis, suggesting that steroidal implants promote protein accretion through unchanged rates of synthesis and decreased degradation, even in the presence of bromocriptine, resulting in improved daily gains. Implanted steers likely experienced increased IGF-1 signaling, but downstream activation of mTOR, S6K and 4E-BP1, and thus increased protein synthesis did not occur as expected. Conclusions: Overall, this data suggests that bromocriptine does not have a negative impact on muscle protein synthetic pathways independent of DMI.

Differential effect of two dietary protein sources on time course response of muscle anabolic signaling pathways in normal and insulin dysregulated horses.

The objective of the study was to characterize the temporal changes of phosphorylation patterns of mTOR signaling proteins in response to two dietary protein sources in insulin dysregulated (ID, n = 8) and non-ID (n = 8) horses. Horses were individually housed and fed timothy grass hay and 2 daily concentrate meals so that protein was the first limiting nutrient and the total diet provided 120% of daily DE requirements for maintenance. On sample days, horses randomly received 0.25 g CP/kg BW of a pelleted alfalfa (AP) or commercial protein supplement (PS). Blood samples were collected before and 30, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420, and 480 min post feeding and analyzed for plasma glucose, insulin and amino acid (AA) concentrations. Gluteus Medius muscle samples were obtained before and 90, 180, and 300 min after feeding and analyzed for relative abundance of phosphorylated mTOR pathway components using western immunoblot analysis. There was no effect of protein source on postprandial glucose and insulin responses (P ≥ 0.14) but consumption of PS elicited a 2 times larger AUC for essential AA (EAA), greater peak concentrations of EAA and a shorter time to reach peak EAA concentrations compared to AP. Abundance of phosphorylated mTOR (P = 0.08) and rpS6 (P = 0.10) tended to be ~1.5-fold greater after consumption of PS at 90 min compared to AP. Dephosphorylation patterns differed between protein sources and was slower for AP compared to PS. ID horses had a 2 times greater (P = 0.009) AUC and 3 times higher postprandial peak concentrations (P < 0.0001) for insulin compared to non-ID horses after consumption of both treatment pellets, but EAA responses were similar between groups (P = 0.53). Insulin status did not affect rpS6 or mTOR phosphorylation after consumption of either protein source (P ≥ 0.35), but phosphorylated rpS6 abundance was twice as high in ID compared to non-ID horses (P = 0.007). These results suggest that the consumption of higher quality protein sources may result in greater postprandial activation of the mTOR pathway compared to equal amounts of a forage-based protein source. Moreover, ID does not impair postprandial activation of mTOR and rpS6 proteins in horses following a protein-rich meal.

Effects of Bromocriptine on Glucose and Insulin Dynamics in Normal and Insulin Dysregulated Horses.

The objectives of the study were to study the effects of the synthetic ergot alkaloid (EA), bromocriptine, on glucose and lipid metabolism in insulin dysregulated (ID, n = 7) and non-ID (n = 8) mares. Horses were individually housed and fed timothy grass hay and two daily concentrate meals so that the total diet provided 120% of daily DE requirements for maintenance. All horses were given intramuscular bromocriptine injections (0.1 mg/kg BW) every 3 days for 14 days. Before and after 14 days of treatment horses underwent a combined glucose-insulin tolerance test (CGIT) to assess insulin sensitivity and a feed challenge (1 g starch/kg BW from whole oats) to evaluate postprandial glycemic and insulinemic responses. ID horses had higher basal plasma concentrations of insulin (P = 0.01) and triglycerides (P = 0.02), and lower concentrations of adiponectin (P = 0.05) compared with non-ID horses. The CGIT response curve showed that ID horses had slower glucose clearance rates (P = 0.02) resulting in a longer time in positive phase (P = 0.03) and had higher insulin concentrations at 75 min (P = 0.0002) compared with non-ID horses. Glucose (P = 0.02) and insulin (P = 0.04) responses to the feeding challenge were lower in non-ID compared to ID horses. Regardless of insulin status, bromocriptine administration increased hay intake (P = 0.03) and decreased grain (P < 0.0001) and total DE (P = 0.0002) intake. Bromocriptine treatment decreased plasma prolactin (P = 0.0002) and cholesterol (P = 0.10) and increased (P = 0.02) adiponectin concentrations in all horses. Moreover, in both groups of horses, bromocriptine decreased glucose clearance rates (P = 0.02), increased time in positive phase (P = 0.04) of the CGIT and increased insulin concentrations at 75 min (P = 0.001). The postprandial glycemic (P = 0.01) and insulinemic (P = 0.001) response following the oats meal was lower after bromocriptine treatment in all horses. In conclusion, in contrast to data in humans and rodents, bromocriptine treatment reduced insulin sensitivity in all horses, regardless of their insulin status. These results indicate that the physiological effects of EA might be different in horses compared to other species. Moreover, because bromocriptine shares a high degree of homology with natural EA, further investigation is warranted in horses grazing endophyte-infected grasses.

Correction to "Synthesis, Evaluation, and Characterization of an Ergotamine Imprinted Styrene-Based Polymer for Potential Use as an Ergot Alkaloid Selective Adsorbent".

[This corrects the article DOI: 10.1021/acsomega.1c02158.].

Synthesis, Evaluation, and Characterization of an Ergotamine Imprinted Styrene-Based Polymer for Potential Use as an Ergot Alkaloid Selective Adsorbent.

Alkaloid toxicities negatively impact livestock health and production. To assess alkaloid occurrences, adsorbent technologies may offer effective means to their extraction and isolation from a complex feed matrix. In this study, molecularly imprinted polymers (MIPs) were synthesized and evaluated for their specificity of binding to various ergot alkaloids. Co-polymers of styrene and hydroxyethyl methacrylate were synthesized in the absence or presence of an ergotamine (ETA) template, yielding non-imprinted polymer (NIP) and molecularly imprinted polymer (MIP), respectively. The influence of parameters such as pH, temperature, and initial concentration on the adsorption of ergot alkaloids was evaluated along with their application as solid phase extraction materials. Chemical and morphological properties were characterized. Adsorption was generally greater for MIP compared to NIP. Cross-reactivity with related alkaloids existed due to similarities in structure and functional groups and was dependent on the type and concentration of alkaloid and polymer type (alkaloid type × concentration × product; P < 0.05). The pH of the medium had no influence on the binding properties of polymers toward ETA within a pH range of 2-10. Binding was independent of temperature between 36 and 42 °C. When kinetics of adsorption were evaluated, the Langmuir isotherm had a better fit (R 2 > 0.95) to adsorption equilibrium data than the Freundlich equation. The maximum amounts adsorbed (Q o) from the Langmuir model were 8.68 and 7.55 µM/g for MIP and NIP, respectively. Fourier transform infrared, scanning and tandem electron microscopy, and Brunauer-Emmett-Teller analysis confirmed a highly porous MIP structure with a greater surface area compared to NIP. Binding characteristics evaluated with computational strategy using molecular docking experiments and in vitro in a complex media (rumen fluid) indicated a stronger ETA adsorption by the tested composition selected among other polymeric materials and affinity of MIP compared with NIP. This study suggested the possible utility of MIP as a solid phase extraction sorbent for applications in analytical chemistry or sensing devices tailored to track ergot alkaloid incidence and the fate of those alkaloids in complex ruminal digestive samples.

Feeding Cannabidiol (CBD)-Containing Treats Did Not Affect Canine Daily Voluntary Activity.

Growing public interest in the use of cannabidiol (CBD) for companion animals has amplified the need to elucidate potential impacts. The purpose of this investigation was to determine the influence of CBD on the daily activity of adult dogs. Twenty-four dogs (18.0 ± 3.4 kg, 9 months-4 years old) of various mixed breeds were utilized in a randomized complete block design with treatments targeted at 0 and 2.5 mg (LOW) and at 5.0 mg (HIGH) CBD/kg body weight (BW) per day split between two treats administered after twice-daily exercise (0700-0900 and 1,700-1,900 h). Four hours each day [1,000-1,200 h (a.m.) and 1,330-1,530 h (p.m.)] were designated as times when no people entered the kennels, with 2 h designated as Quiet time and the other 2 h as Music time, when calming music played over speakers. Quiet and Music sessions were randomly allotted to daily a.m. or p.m. times. Activity monitors were fitted to dogs' collars for continuous collection of activity data. Data were collected over a 14-day baseline period to establish the activity patterns and block dogs by activity level (high or low) before randomly assigning dogs within each block to treatments. After 7 days of treatment acclimation, activity data were collected for 14 days. Data were examined for differences using the MIXED procedure in SAS including effects of treatment, day, session (Quiet or Music), time of day (a.m. or p.m.), and accompanying interactions. CBD (LOW and HIGH) did not alter the total daily activity points (P = 0.985) or activity duration (P = 0.882). CBD tended (P = 0.071) to reduce total daily scratching compared with the control. Dogs were more active in p.m. sessions than in a.m. sessions (P < 0.001). During the p.m. session, dogs receiving HIGH tended (P = 0.091) to be less active than the control (CON). During the a.m. and p.m. sessions, CBD reduced scratching compared with CON (P = 0.030). CBD did not affect the activity duration during exercise periods (P = 0.143). These results indicate that, when supplemented with up to 4.5 mg CBD/kg BW/day, CBD does not impact the daily activity of adult dogs, but may exert an antipruritic effect.

Endophyte Infected Tall Fescue: Plant Symbiosis to Animal Toxicosis.

Endophyte-infected fescue is a major cool season forage used for livestock production in the United States and through other areas of the world. A unique aspect of this forage resource is the symbiotic relationship with an endophytic fungus (Epichloë coenophiala) that has detrimental impact on herbivores due to toxic ergot alkaloids. Research over the past 50 years has unveiled details regarding this symbiotic relationship. This review focuses on the origin of tall fescue in the United States and the consequences of its wide-spread utilization as a livestock forage, along with the discovery and toxicodynamics of ergot alkaloids produced by E. coenophiala. The majority of past ergot alkaloid research has focused on observing phenotypic changes that occur in livestock affected by ergot alkaloids, but recent investigation of the metabolome, transcriptome, and proteome have shown that fescue toxicity-related illnesses are much more complex than previous research suggests.

Pathways regulating equine skeletal muscle protein synthesis respond in a dose-dependent manner to graded levels of protein intake.

Activation of the mechanistic target of rapamycin (mTOR)-controlled anabolic signaling pathways in skeletal muscle of rodents and humans is responsive to the level of dietary protein supply, with maximal activation and rates of protein synthesis achieved with 0.2 to 0.4 g protein/kg body weight (BW). In horses, few data are available on the required level of dietary protein to maximize protein synthesis for maintenance and growth of skeletal muscle. To evaluate the effect of dietary protein level on muscle mTOR pathway activation, five mares received different amounts of a protein supplement that provided 0, 0.06, 0.125, 0.25, or 0.5 g of crude protein (CP)/kg BW per meal in a 5 × 5 Latin square design. On each sample day, horses were fasted overnight and were fed only their protein meal the following morning. A preprandial (0 min) and postprandial (90 min) blood sample was collected and a gluteus medius muscle sample was obtained 90 min after feeding the protein meal. Blood samples were analyzed for glucose, insulin, and amino acid concentrations. Activation of mTOR pathway components (mTOR and ribosomal protein S6 [rpS6]) in the muscle samples was measured by Western immunoblot analysis. Postprandial plasma glucose (P = 0.007) and insulin (P = 0.09) showed a quadratic increase, while total essential amino acid (P < 0.0001) concentrations increased linearly with the graded intake of the protein supplement. Activation of mTOR (P = 0.02) and its downstream target, rpS6 (P = 0.0008), increased quadratically and linearly in relation to the level of protein intake, respectively. Comparisons of individual doses showed no differences (P > 0.05) between the 0.25 and 0.5 g of protein intake for either mTOR or rpS6 activation, indicating that protein synthesis may have reached near maximal capacity around 0.25 g CP/kg BW. This is the first study to show that the activation of muscle protein synthetic pathways in horses is dose-dependent on the level of protein intake. Consumption of a moderate dose of high-quality protein resulted in near maximal muscle mTOR pathway activation in mature, sedentary horses.

Contractile Response of Bovine Lateral Saphenous Vein to Ergotamine Tartrate Exposed to Different Concentrations of Molecularly Imprinted Polymer.

Ergot alkaloids, in their active isomeric form, affect animal health and performance, and adsorbents are used to mitigate toxicities by reducing bioavailability. Adsorbents with high specificity (molecularly imprinted polymers: MIP) adsorb ergot alkaloids in vitro, but require evaluation for biological implications. Using ex vivo myography, synthetic polymers were evaluated for effects on the bioactivity of ergotamine tartrate (ETA). Polymers were first evaluated using isotherms. Lateral saphenous veins were collected from 17 steers for four independent studies: dose response of ETA, adsorbent dose response, validation of pre-myograph incubation conditions and MIP/ non-molecularly imprinted polymer (NIP) comparison. Norepinephrine normalized percent contractile response to increasing ETA exhibited a sigmoidal dose response (max: 88.47 and log of the effective molar concentration (EC50) (-log [ETA]) of 6.66 ± 0.17 M). Although sample preparation time affected contractile response (p < 0.001), pre-myograph incubation temperature (39 vs. 21 °C, 1 h) had no effect (p > 0.05). Isothermal adsorption showed a maximum adsorption of 3.27E-008 moles·mg-1 and affinity between 0.51 and 0.57 mg (R²: 0.83-0.92) for both polymers, with no significant difference between polymers (p > 0.05). No significant differences in maximum inhibitory (p = 0.96) and IC50 responses (p = 0.163) between MIP and NIP were noticed. Normalized percent contraction could be predicted from the in vitro adsorption data (R² = 0.87, p < 0.01), for both polymers. These studies indicate that synthetic polymers are potentially effective adsorbents to mitigate ergot toxicity caused by ergot alkaloids, with little evidence of significant differences between MIP and NIP in aqueous media.

Consumption of endophyte-infected fescue seed during the dry period does not decrease milk production in the following lactation.

Ergot alkaloids in endophyte-infected grasses inhibit prolactin (PRL) secretion and may reduce milk production of cows consuming these grasses. We investigated the effects of consuming endophyte-infected fescue seed during late lactation and the dry period on mammary growth, differentiation, and milk production. Twenty-four multiparous Holstein cows were randomly assigned to 3 treatment groups. Starting at 90±4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3×/wk subcutaneous injections of bromocriptine (0.1mg/kg of body weight, positive control; BROMO), or endophyte-infected fescue seed (INF) as 10% of the diet on an as fed basis. Although milk yield of groups did not differ before treatment, at dry off (-60 d prepartum) INF and BROMO cows produced less milk than CON. Throughout the treatment period, basal concentrations of PRL and the prepartum increase in plasma PRL were reduced in INF and BROMO cows compared with CON cows. Three weeks after the end of treatment, circulating concentrations of PRL were equivalent across groups. In the subsequent lactation milk yield was not decreased; in fact, BROMO cows exhibited a 9% increase in milk yield relative to CON. Evaluation of mammary tissue during the dry period and the subsequent lactation, by quantitative histology and immunohistochemical analysis of proliferation markers and putative mammary stem or progenitor cell markers, indicated that feeding endophyte-infected fescue seed did not significantly affect mammary growth and development. Feeding endophyte-infected grasses during the dry period may permit effective utilization of feed resources without compromising milk production in the next lactation.

Discrimination of healthy versus sick steers by means of continuous remote monitoring of animal activity.

OBJECTIVE: To test a unique electronic ear tag designed to collect movement data to determine whether physical activity of sick steers differed from that of healthy steers. ANIMALS: 206 steers. PROCEDURES: Physical activity in 2 groups of steers during November and December of 2010 (101 steers; the tag of 1 steer failed, and thus that steer was removed from the study, which resulted in data for 100 steers) and 2011 (105 steers) was monitored with an electronic ear tag device with an on-board triple-axis accelerometer. The accelerometer recorded motion in all 3 axes in the form of counts per minute. A radio-frequency transmitter on the ear tag delivered serial packets of motion data to a local server. An algorithm was developed to analyze the activity data to determine whether this technique could be used to assess health status with high accuracy. RESULTS: Steers that became sick had significantly fewer activity counts (approx 25% fewer), compared with the activity counts of steers that remained healthy the entire time. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, automated detection of health status in growing cattle was feasible through remote monitoring of animal activity. Early identification of sick animals should lead to improved health outcomes, increased marketability, and improved animal well-being and help to minimize the use of antimicrobials that could contribute to resistant bacteria.

In vitro culture of Parascaris equorum larvae and initial investigation of parasite excretory-secretory products.

Currently, diagnosis of Parascaris equorum infection in equids is limited to patent infections. The goals of this study were to culture P. equorum larvae in vitro and identify excretory-secretory (ES) products for prepatent diagnostic testing. Parascaris equorum L2/L3 larvae were hatched and cultured for up to 3 weeks for ES product collection. Fifth stage (L5) P. equorum were also cultured for ES product collection. Examination of ES fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver stain revealed L2/L3 products ranging from 12-94 kDa and L5 products ranging from 12-189 kDa. Western blot analyses were conducted using polyclonal antibodies produced against P. equorum or Baylisascaris procyonis L2/L3 ES products, sera from rabbits inoculated with B. procyonis or Toxocara canis eggs, and sera from animals naturally infected with P. equorum or T. canis. Western blot results indicated parasite antigens migrating at 19 and 34 kDa may be useful for specifically detecting P. equorum infections.

The effects of crude protein concentration and slow release urea on nitrogen metabolism in Holstein steers.

This experiment was conducted to determine the effects of slow release urea (SRU) and its interaction with crude protein (CP) level in the diet on N metabolism in Holstein steers. Eight rumen-cannulated Holstein steers (body weight 265 ± 18 kg) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial treatment structure. Treatment factors were the CP level in the diet, 10.9% versus 12.1% CP, and the non-protein nitrogen source used, urea versus SRU. Total collection of urine and faeces for 7 days allowed the estimation of N retention and diet digestibility. In addition, blood and rumen sampling allowed estimation of rumen fermentation and blood N profiles. Decreasing CP intake from 12.1% to 10.9% reduced urinary N output, but also reduced diet digestibility and N retention. When compared to urea, SRU did not alter N retention, but reduced ruminal ammonia and plasma urea concentrations. Although SRU did not improve N retention at either CP level, rumen ammonia and plasma urea concentrations were reduced, which may indicate that SRU may carry a lower risk for toxicity when compared to urea when fed at higher dietary concentrations.