Abstract: Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species.

Abstract: REASONS FOR PERFORMING STUDY: Horses housed in conventional stable environments are exposed to high dust loads. Long-term exposure to inhaled dust may lead to subclinical airway disease. HYPOTHESES: Airway inflammation and mucus accumulation are 1) common in asymptomatic well-performing sport horses, 2) increased in older compared to younger individuals and 3) correlated. METHODS: Mucus quantity and quality (endoscopic scoring) and BALF differential cytology were assessed in 13 younger horses (mean age 5 years) and 13 older horses (mean age 15 years), which had no historical or clinical evidence of lower airway disease, were performing well and were housed permanently in a conventional stable environment. RESULTS: 1) Airway inflammation and/or mucus accumulation were very common. All sport horses showed evidence of subclinical inflammatory airway disease (IAD). 2) Older horses, having been exposed for 10 more years to a conventional stable environment, did not show increased subclinical airway inflammation or mucus accumulation. The only differences between the age groups were increased BALF total and lymphocyte cell counts in the younger horses. 3) Mucus quantity and quality scores were not significantly correlated with each other or with BALF neutrophil percentages and absolute numbers. CONCLUSIONS: Although clinically healthy and performing well, all of the examined horses housed in a conventional stable environment showed evidence of IAD. The degree of IAD was not increased in older horses that had not developed clinical signs. POTENTIAL RELEVANCE: The effect of subclinical degrees of IAD on performance in dressage and show jumping should be investigated further to better understand the clinical significance of IAD in sport horses.

Abstract: Red blood cell hypervolaemia has been used for diagnosis of overtraining in racehorses, and has been suggested as a mechanism of this cause of loss of racing performance. The effects of overload training (OLT) on the plasma, blood and red cell volumes were investigated in a prospective study in 12 Standardbred horses. Measurements of blood volumes were made after eight and 32 weeks of an exercise training study. Horses were randomly allocated to OLT and control groups (n=6) after 16 weeks of training. Training duration and intensity were increased more rapidly for the OLT group from week 16, until overtraining was diagnosed in week 32.There were no significant effects of OLT on plasma, blood or total red cell volumes between weeks eight and 32. These volumes significantly decreased with time. Maximal haematocrit after exercise was lower (P<0.05) in the OT group in week 32 (0.57+/-0.003% L/L) than in week eight (0.59+/-0.004 L/L). It was concluded that red cell hypervolaemia was not a mechanism for the decrease in capacity for exercise that occurs with overtraining.

Abstract: The structure, thermodynamics, and kinetics of heat-induced unfolding of sperm whale apomyoglobin core formation have been studied. The most rudimentary core is formed at pH(*) 3.0 and up to 60 mM NaCl. Steady state for ultraviolet circular dichroism and fluorescence melting studies indicate that the core in this acid-destabilized state consists of a heterogeneous composition of structures of approximately 26 residues, two-thirds of the number involved for horse heart apomyoglobin under these conditions. Fluorescence temperature-jump relaxation studies show that there is only one process involved in Trp burial. This occurs in 20 micro s for a 7 degrees jump to 52 degrees C, which is close to the limits placed by diffusion on folding reactions. However, infrared temperature jump studies monitoring native helix burial are biexponential with times of 5 micro s and 56 micro s for a similar temperature jump. Both fluorescence and infrared fast phases are energetically favorable but the slow infrared absorbance phase is highly temperature-dependent, indicating a substantial enthalpic barrier for this process. The kinetics are best understood by a multiple-pathway kinetics model. The rapid phases likely represent direct burial of one or both of the Trp residues and parts of the G- and H-helices. We attribute the slow phase to burial and subsequent rearrangement of a misformed core or to a collapse having a high energy barrier wherein both Trps are solvent-exposed.

Abstract: This study investigated the effect of stimulus height on the ability of horses to learn a simple visual discrimination task. Eight horses were trained to perform a two-choice, black/white discrimination with stimuli presented at one of two heights: ground level or at a height of 70 cm from the ground. The height at which the stimuli were presented was alternated from one session to the next. All trials within a single session were presented at the same height. The criterion for learning was four consecutive sessions of 70% correct responses. Performance was found to be better when stimuli were presented at ground level with respect to the number of trials taken to reach the criterion (P < 0.05), percentage of correct first choices (P < 0.01), and repeated errors made (P < 0.01). Thus, training horses to carry out tasks of visual discrimination could be enhanced by placing the stimuli on the ground. In addition, the results of the present study suggest that the visual appearance of ground surfaces is an important factor in both horse management and training.