Assessment of the metabolic effects of hydrocortisone on llamas before and after feed restriction

OBJECTIVE: To evaluate the effects of administration of hydrocortisone on plasma concentration of insulin and serum concentrations of glucose, triglyceride, and nonesterified fatty acids (NEFAs) in llamas before and after feed restriction. ANIMALS: 9 adult female llamas. PROCEDURE: Feed was withheld from llamas for 8 hours. Blood samples were collected before (0 minutes) and 120, 180, 240, and 300 minutes after IV injection of hydrocortisone sodium succinate (1 mg/kg) for determination of plasma insulin concentration and serum concentrations of glucose, triglyceride, and NEFAs. The llamas were then fed a limited diet (grass hay, 0.25% of body weight daily) for 21 days, after which the experimental procedures were repeated. RESULTS: Compared with llamas that were not feed-restricted, llamas after feed restriction had significantly higher plasma insulin concentration and serum concentrations of triglycerides and NEFAs. Feed-restricted llamas after hydrocortisone injection had a significantly smaller increase in serum glucose concentration, a decrease (rather than an increase) in serum concentration of NEFAs, and no change in blood concentrations of insulin or triglycerides. CONCLUSIONS AND CLINICAL RELEVANCE: Short-acting glucocorticoid hormones did not appear to increase blood lipid concentrations in healthy llamas, regardless of ongoing fat mobilization. Thus, these hormones appear unlikely to be major direct contributors to diseases such as hepatic lipidosis or hyperlipemia. Although administration of hydrocortisone reduced serum concentration of fatty acids in feed-restricted llamas, its use has not been evaluated in sick camelids and cannot be considered therapeutically useful.     

Extraction methods for quantitation of gentamicin residues from tissues using fluorescence polarization immunoassay

Sodium hydroxide digestion of unhomogenized kidney and skeletal muscle for 20 min at 70 degrees C was a superior method for extracting gentamicin from tissues, compared with simple homogenization, trichloroacetic acid precipitation of homogenized tissue, and sodium hydroxide digestion of homogenized tissue. Fluorescence polarization immunoassay was used to quantitate gentamicin. Sodium hydroxide digestion of unhomogenized tissue allowed for the recovery of 90.0 +/- 5.9% (means +/- SD) from renal cortex and 79.9 +/- 3.5% from skeletal muscle. The limit of sensitivity was 17.4 ng/g kidney tissue, 15.8 ng/g digested muscle, and 39.0 ng/g digested heart. The within-assay coefficient of variation (CV) at 100 ng/g kidney was 9.2%; at 500 ng/g kidney, the CV was 2.5%; and at 2000 ng/g kidney, the CV was 1.5%. The between-assay coefficient of variation was less than 7.5% for all concentrations from kidney, and the 99% confidence interval at 100 ng/g kidney was 71.7-112.4 ng gentamicin/g kidney. The within-assay coefficient of variation (CV) at 100 ng/g muscle was 15%; at 500 ng/g muscle, the CV was 2.6%; and at 2000 ng/g muscle, the CV was 2.3%. The between-assay coefficient of variation was less than 15% for all concentrations from muscle, and the 99% confidence interval at 100 ng/g muscle was 72.5-136.8 ng gentamicin/g muscle. Gentamicin-free milk could be distinguished from milk containing gentamicin concentrations of 10 ng/mL milk with 95% confidence, and from milk containing concentrations of 30 ng gentamicin/mL milk with 99% confidence. Quantitative results at or below the tolerance level can be obtained within 90 min of sample acquisition using these extraction and assay methods.     

Glucose tolerance testing in llamas and alpacas

OBJECTIVE: To determine blood glucose clearance in 2 species of New World camelids after IV challenge and to examine mechanisms of this clearance. ANIMALS: 5 adult female llamas and 5 adult gelded alpacas. PROCEDURE: After food was withheld for 12 hours, camelids received 0.5 g of glucose/kg of body weight by rapid IV infusion. Serum concentrations of glucose, nonesterified fatty acids, cortisol, and insulin, and plasma concentrations of lactate were determined before and 0, 1, 2, 3, 4, 5, 15, 30, 60, 90, 120, 180, and 240 minutes after infusion. Ratios of insulin to glucose and insulin to cortisol were calculated for each time point. RESULTS: Postinfusion glucose concentrations were significantly higher in llamas than alpacas for the first 15 minutes and remained significantly higher than baseline values in both species for 180 minutes. Lactate and cortisol concentrations did not change significantly; nonesterified fatty acid concentrations decreased in both species 30 minutes after infusion. Baseline insulin concentrations were < 6 microU/ml in both species and increased only to 10.1 +/- 0.7 microU/ml in llamas. Insulin concentrations did not change significantly in alpacas. CONCLUSIONS AND CLINICAL RELEVANCE: Llamas and alpacas clear glucose more slowly than other domestic species after challenge, mainly because of a weak insulin response and slow cellular uptake. This response may impair the assimilation of exogenous glucose as well as make llamas and alpacas prone to diabetes-like disorders when an abundance of endogenous or exogenous glucogenic agents are present.     

Simultaneous Flow Cytometric Analysis of Phagocytosis and Oxidative Burst Activity in Equine Leukocytes

This paper describes a method for simultaneously measuring phagocytosis and oxidative burst activity in equine peripheral blood leukocytes by flow cytometry. Opsonized propidium iodide-labelled Staphylococcus aureus (PI-Sa) was used to measure the uptake of bacteria by equine phacocytes and the oxidative burst activity by oxidation of dihydrorhodamine 123. The requirements to achieve optimal activity of phagocytosis and oxidative burst are described. The advantage of the simultaneous technique is that it provides both independent and comparative values for phagocytosis and the oxidative burst, for the detection of impaired mechanisms of microbial destruction. Furthermore, the technique allows evaluation of opsonization activity in this context.     

Effect of dietary iron supplement on growth, haematology and microelements of juvenile grouper, Epinephelus coioides

An experiment was conducted to investigate the effect of dietary iron supplement on growth, haematology and microelements of juvenile grouper, Epinephelus coioides. Casein–gelatine‐based diets supplemented with 0, 50, 100, 150, 200 and 250 mg kg⁻¹ iron from ferrous sulphate were fed to grouper (mean initial weight: 21.0 ± 0.2 g) for 8 weeks. Weight gain was highest in fish fed the diet supplemented with 100 mg kg⁻¹ iron, intermediate in fish fed diets with 50, 150, 200 and 250 mg kg⁻¹ iron and lowest in fish fed the basal diet. Feed efficiency followed a similar trend except that the lowest value was in fish fed the basal diet and the diet supplemented with 250 mg kg⁻¹ iron. Hepatic iron was highest in fish fed diets supplemented with iron ≥100 mg kg⁻¹, followed by fish fed diet with 50 mg kg⁻¹ iron and lowest in fish fed the basal diet. The whole‐body iron was lowest in fish fed the basal diet but not significantly different from other groups, as judged by anova . Iron supplement to the basal diet had no significant effect on haematological parameters (red blood cell count, haematocrit and haemoglobin), hepatic copper concentration or manganese, zinc concentration in liver and whole body. Broken‐line analysis of hepatic iron indicated that iron supplementation of 100 mg kg⁻¹ satisfied the hepatic iron storage and that further supplementation did not expand the iron status.     

Development of an integrated pest management programme for cotton. Part 2: Integration of a lucerne/cotton interplant system,food supplement sprays with biological and synthetic insecticides

Part 1 of the paper showed that the integration of a lucerne/ cotton interplant system with supplementary food sprays retained and increased populations of predatory insects in the cotton crop. The strategic use of biopesticides (Bacillus thuringiensis (Bt), nuclear polyhedrosis virus (NPV)) reduced the use of synthetic insecticides by 50% without sacrificing cotton yield and profitability. In economic terms, the average gross margin for the IPM plot was A$3255 compared with A$3020 and A$3218 for the plots treated with (1) conventional insecticides on transgenic (Ingard®) cotton and (2) conventional insecticides on non-transgenic cotton. The use of transgenic cotton affected a 25% saving on synthetic insecticide usage. The results clearly indicate that a refined IPM approach could have a distinctive advantages in terms of economic and environmental impacts on cotton production. The success of such an approach lies in a strict adherence to its methodology.     

Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.     

Effect of Dietary Nonstructural Carbohydrate Content on Activation of 5′‐Adenosine Monophosphate‐Activated Protein Kinase in Liver,Skeletal Muscle,and Digital Laminae of Lean and Obese Ponies

Background

In EMS‐associated laminitis, laminar failure may occur in response to energy failure related to insulin resistance (IR) or to the effect of hyperinsulinemia on laminar tissue. 5′‐Adenosine‐monophosphate‐activated protein kinase (AMPK) is a marker of tissue energy deprivation, which may occur in IR.

Hypothesis/Objectives

To characterize tissue AMPK regulation in ponies subjected to a dietary carbohydrate (CHO) challenge.

Animals

Twenty‐two mixed‐breed ponies.

Methods

Immunohistochemistry and immunoblotting for total AMPK and phospho(P)‐AMPK and RT‐qPCR for AMPK‐responsive genes were performed on laminar, liver, and skeletal muscle samples collected after a 7‐day feeding protocol in which ponies stratified on body condition score (BCS; obese or lean) were fed either a low‐CHO diet (ESC + starch, approximately 7% DM; n = 5 obese, 5 lean) or a high‐CHO diet (ESC + starch, approximately 42% DM; n = 6 obese, 6 lean).

Results

5′‐Adenosine‐monophosphate‐activated protein kinase was immunolocalized to laminar keratinocytes, dermal constituents, and hepatocytes. A high‐CHO diet resulted in significantly decreased laminar [P‐AMPK] in lean ponies (P = .03), but no changes in skeletal muscle (lean, P = .33; obese, P = .43) or liver (lean, P = .84; obese, P = .13) [P‐AMPK]. An inverse correlation existed between [blood glucose] and laminar [P‐AMPK] in obese ponies on a high‐CHO diet.

Conclusions and Clinical Importance

Laminar tissue exhibited a normal response to a high‐CHO diet (decreased [P‐AMPK]), whereas this response was not observed in liver and skeletal muscle in both lean (skeletal muscle, P = .33; liver, P = .84) and obese (skeletal muscle, P = .43; liver, P = .13) ponies.