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Edwards, K. L., Bansiddhi, P., Paris, S., Galloway, M., & Brown, J. L. (2019). The development of an immunoassay to measure immunoglobulin A in Asian elephant feces, saliva, urine and serum as a potential biomarker of well-being. conphys, 7(1).
Abstract: Additional measures of well-being would be beneficial to the management of a variety of species in human care, including elephants. Immunoglobulin A (IgA) is an immune protein associated with pathogen defense, which has been demonstrated to decrease during times of stress, and increase in response to positive stimuli. This paper describes the development and validation of an enzyme immunoassay (EIA) for the quantification of Asian elephant (Elephas maximus) IgA in feces, saliva, urine, and serum. Samples were collected weekly from four females for 6 months to assess IgA and glucocorticoid (GC) concentrations, establish relationships between these two biomarkers, and determine variability in IgA within and between individuals, and across sample types. IgA was quantified in all four sample types, although urinary concentrations were low and sometimes undetectable in individual samples. Concentrations were highly variable within and between individuals, with fecal, salivary and serum IgA, and fecal, salivary and urinary GCs all differing significantly across individuals. Contrary to previous findings, IgA and GC were generally not correlated. Serum IgA was less variable within individuals, with the exception of one female that experienced a brief illness during the study. However, marked inter-individual differences were still apparent. When data from all individuals were combined, fecal IgA was significantly predicted by salivary and urinary IgA; however, this relationship did not hold when individuals were analyzed separately. Analysis of a fifth female that exhibited a more severe systemic illness demonstrated clear increases in fecal IgA and GC, suggesting these may also be useful health biomarkers. Further investigation is needed to determine what sample type is most reflective of biological state in elephants, and how IgA concentrations are associated with health and positive and negative welfare states. Based on observed variability, a longitudinal approach likely will be necessary to use IgA as a measure of well-being.
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Cozzi, B., Povinelli, M., Ballarin, C., & Granato, A. (2014). The Brain of the Horse: Weight and Cephalization Quotients. Brain Behav Evol, 83(1), 9–16.
Abstract: The horse is a common domestic animal whose anatomy has been studied since the XVI century. However, a modern neuroanatomy of this species does not exist and most of the data utilized in textbooks and reviews derive from single specimens or relatively old literature. Here, we report information on the brain of Equus caballus obtained by sampling 131 horses, including brain weight (as a whole and subdivided into its constituents), encephalization quotient (EQ), and cerebellar quotient (CQ), and comparisons with what is known about other relevant species. The mean weight of the fresh brains in our experimental series was 598.63 g (SEM ± 7.65), with a mean body weight of 514.12 kg (SEM ± 15.42). The EQ was 0.78 and the CQ was 0.841. The data we obtained indicate that the horse possesses a large, convoluted brain, with a weight similar to that of other hoofed species of like mass. However, the shape of the brain, the noteworthy folding of the neocortex, and the peculiar longitudinal distribution of the gyri suggest an evolutionary specificity at least partially separate from that of the Cetartiodactyla (even-toed mammals and cetaceans) with whom Perissodactyla (odd-toed mammals) are often grouped.
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Wolter, R. (2018). The behaviour and managementof Przewalski’shorsesin semi-reserves. Ph.D. thesis, University Regensburg, Regensburg.
Abstract: In recent years, Przewalski’s horses have been increasingly kept in semi-reserves. However,there areonly few studies ontheir behaviour and their ability to adaptto management interventions.In the main part of my dissertation, I focus on investigatingthe animals’ behaviour in different semi-reserves with varyinghabitats and living spaces. In addition, I investigate the horses’ behaviour during various management interventionsand analysetheensuing changes instress levels. Another aspect of my dissertation is the studyof social behaviour inPrzewalski’s horses. I investigate theparameters that should be used to demonstrate social bonds between individualsandassess whichdata provide the most meaningful results.In the commentary tochapter 1,several studies investigatingsocial bonds in horsesare discussed. Comparing the various studies, it is strikingthat no homogeneous analyses orevaluation criteria exist. While some authors only considersocial grooming, others include data onthe spatial proximity of the individuals in their evaluations, and various definitionsof proximity can also be found in the literature. Additionally, someauthors use friendly approaches between individuals asa furtherparameter wheninvestigating the social bonds.Continuing with this theme, in chapter 2I investigate the social behaviour of the horses and compare various analysis methods. I show that proactive behaviour, such as friendly approaches, is a good alternative to spatial proximity when investigating social bonds between group members, andis also useful for expanding the often very small data sets of mutual grooming in horses. Comparing Przewalski’s horses with wild living horses, I found no significant differences in the social behavior and the frequency of social interactions, regardlessof group size, group composition, habitat, and individual parameters such as age and gender.Inchapter 3,I investigate the behaviour of a Przewalski’s horse group when exploring a new area of their enclosure. Their behaviour changed, showing less resting and more feeding. Furthermore, the animals maintained greater distances from each other, and the alpha male, instead of herding the group from behind, led the group around the new area and walked in front of the other group members. Moreover, he showed a substantial increase in stress level during the first day.A general comparison of the behaviour of the Przewalski’s horses in different semi-reserves is provided in chapter 4. In it, the habitat choice of the animals and their reactions to various management interventions are investigated. It is shown that Przewalski’s horses prefer open grassland to dense woods, although keeping Przewalski’s horses in a pine forest does not influence the animals’ stress level. In contrast to habitat, food range, and changes in the group composition, which do not appear to change stress levels, individual factors, such as the hierarchy, influence the glucocorticoid level of the animals significantly. The largest increases in stress hormones were demonstrated when the horses were temporarily confined in smaller areas.The importance of the available space is also discussed in chapter 5, where it is shown that horses show less aggressive behaviour when more space is provided. In contrast, the husbandry system does not influence the animals’ aggression, but the way of feeding can additionally reduce agonistic behaviour, for example if food is offered ad libitum.In summary, the results of this study provide indications for the optimization of keeping Przewalski’s horses in semi-reserves. The animals can adapt themselves to the environment and thrive in habitats which do not correspond to their original steppe-like home. Nevertheless, the semi-reserves should provide sufficient grassland, as the horses prefer this type of habitat. General speaking, any types of habitat can only offer a suitable living space if the food range is sufficient for the number of horses. Otherwise, and especially during could winter months, supplementary feeding is necessary according to the body condition of the animals. This is particularly important for older, weakened, or very young animals, which are still adapting to life in the semi-reserve. Without sufficient food, stress hormones can increase and negatively influence the well-being of the horses. The same is true for management interventions: restricting the animals to small enclosures, for example, can adversely affect the horses’ well-being and should be only done if absolutely necessary. Targetedbehaviour observations allow the animals that have a special meaning for the group to be identified, and these should not be taken out of the group unless it is unavoidable, as young and unexperienced horses orientate themselves on those animals. This is especially true for the alpha male in a bachelor group, as these groups are often composed of young horses and the alpha-male provides the necessary stability and experience. Social bonds between individuals can be investigated by observing friendly and proactive behaviour, and social grooming and friendly approaches yield suitable data for such analysis.
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McLean, A. N., & Christensen, J. W. (2017). The application of learning theory in horse training. Appl. Anim. Behav. Sci., 190, 18–27.
Abstract: The millennia-old practices of horse training markedly predate and thus were isolated from the mid-twentieth century revelation of animal learning processes. From this standpoint, the progress made in the application and understanding of learning theory in horse training is reviewed including a discussion of how learning processes are employed or otherwise under-utilised in training. This review describes the process of habituation and the most commonly applied desensitisation techniques (systematic desensitisation, counter-conditioning, overshadowing, response prevention) and propose two additional techniques (approach conditioning and stimulus blending). The salience of different types of cues, the interaction of operant and classical conditioning and the impact of stress are also discussed. This paper also exposes the inflexibility and occasional inadequacy of the terminology of learning theory when translated from the research laboratory situation to the practical setting in horse training. While learning theory provides a rich toolbox for riders and trainers, the training process is subject to the simultaneous use of multiple learning processes. In addition, learning/behavioural outcomes and trained responses are not just the result of simple stimulus-response based interactions but are further shaped by arousal, affective and attachment states. More research is needed in these areas. For the field of equitation science to progress and to improve clarity and use of learning processes, changes in nomenclature are required. In particular, the use of the terms 'positive' and 'negative' as descriptive labels in both reinforcement and punishment modalities are unacceptably misleading for everyday use. These labels inhibit the understanding and recognition of the learning processes that these terms supposedly represent, yet the learning processes they describe are vital for horse riders, handlers and trainers to understand. We therefore propose that these labels should be re-labelled more appropriately as 'addition' or 'subtraction' reinforcement/punishment. This would enlighten trainers on the correct application of learning theory, and safety and welfare benefits for people and horses would follow. Finally it is also proposed that the term 'conflict theory' be taken up in equitation science to facilitate diagnosis of training-related behaviour disorders and thus enable the emergence of improved training practices. The optimal use of learning theory should be established as a fundamental principle in equestrian education.
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Lagos, L., & Blanco, P. (2021). Testing the use of dogs to prevent wolf attackson free ranging ponies in Iberia? CDPnews, 23, 20–27.
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Dwan, K., Altman, D. G., Arnaiz, J. A., Bloom, J., Chan, A. - W., Cronin, E., et al. (2008). Systematic Review of the Empirical Evidence of Study Publication Bias and Outcome Reporting Bias. Plos One, 3(8), e3081.
Abstract: Background The increased use of meta-analysis in systematic reviews of healthcare interventions has highlighted several types of bias that can arise during the completion of a randomised controlled trial. Study publication bias has been recognised as a potential threat to the validity of meta-analysis and can make the readily available evidence unreliable for decision making. Until recently, outcome reporting bias has received less attention. Methodology/Principal Findings We review and summarise the evidence from a series of cohort studies that have assessed study publication bias and outcome reporting bias in randomised controlled trials. Sixteen studies were eligible of which only two followed the cohort all the way through from protocol approval to information regarding publication of outcomes. Eleven of the studies investigated study publication bias and five investigated outcome reporting bias. Three studies have found that statistically significant outcomes had a higher odds of being fully reported compared to non-significant outcomes (range of odds ratios: 2.2 to 4.7). In comparing trial publications to protocols, we found that 40-62% of studies had at least one primary outcome that was changed, introduced, or omitted. We decided not to undertake meta-analysis due to the differences between studies. Conclusions Recent work provides direct empirical evidence for the existence of study publication bias and outcome reporting bias. There is strong evidence of an association between significant results and publication; studies that report positive or significant results are more likely to be published and outcomes that are statistically significant have higher odds of being fully reported. Publications have been found to be inconsistent with their protocols. Researchers need to be aware of the problems of both types of bias and efforts should be concentrated on improving the reporting of trials.
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Jankunis, E. S., & Whishaw, I. Q. (2013). Sucrose Bobs and Quinine Gapes: Horse (Equus caballus) responses to taste support phylogenetic similarity in taste reactivity. Behavioural Brain Research, 256, 284–290.
Abstract: Evidence suggests that behavioural affective reactions to sweet and bitter substances are homologous in humans, nonhuman primates, and rodents. The sweet taste of sucrose elicits facial responses that include rhythmic tongue protrusions whereas the bitter taste of quinine elicits facial responses that include gapes, featuring an opening of the mouth and protrusion of the tongue. The present study using the horse (Equus caballus) was undertaken for three reasons: (1) there is debate about the presence of a sweet receptor gene in the horse, (2) there is a need to expand the examination of facial reactions to taste in lineages other than the closely related lineages of rodents and primates, and (3) the horse provides an opportunity to test the hypothesis that some social signals derive from movements related to taste reaction. The horses were given oral infusions of either sucrose or quinine and their behaviour was examined using frame-by-frame video analysis. Control groups were exposed received water or syringe insertion only. Amongst the many responses made to the infusions, the distinctive response to sucrose was a bob coupled with a slight tongue protrusion and forward movement of the ears; the distinctive response to quinine was a head extension and mouth gape accompanied by a large tongue protrusion and backward movement of the ears. Sucrose Bobs and Quinine Gapes are discussed with respect to: (1) the relevance of facial reactions to both sucrose and quinine to taste receptors in horses, (2) the similarity of features of taste expression in horses to those documented in rodents and primates, and (3) the dissimilarity between facial reactions to taste and other social signals displayed by horses.
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Niederhöfer, S. (2009). Stressbelastung bei Pferden in Abhängigkeit des Haltungssystems. Ph.D. thesis, Tierärztliche Hochschule Hannover, Hannover.
Abstract: Pferdegerechte Haltungssysteme spielen bei der heutigen Nutzung des Freizeit- oder Sportpartners Pferd eine wichtige Rolle, da naturnahe Haltungsbedingungen nur in seltenen Fällen zu realisieren sind. Sämtliche Aufstallungsformen müssen als Kompromiss angesehen werden und bieten somit Vor- und Nachteile. Die vorliegende Untersuchung verfolgte den Zweck, die Stressbelastung von Pferden in verschiedenen Haltungssystemen zu erfassen und vergleichend zu beurteilen. Während der von Mai 2006 bis Juli 2007 durchgeführten Versuche wurden 24 zwei- bis dreijährige Hannoveranerstuten für jeweils 4 Wochen in insgesamt 6 verschiedenen Haltungsformen aufgestallt. Die Haltungsvarianten beinhalteten die Einzelbox (Variante 1), die Einzelbox mit einem frei zugänglichem Paddock (Variante 2), die gemeinsame Haltung von zwei Pferden in zwei Boxen und einem angeschlossenen frei zugänglichem Auslauf (Variante 3) und die Gruppenhaltung von 6 Pferden in einer Mehrraumauslaufhaltung (Variante 4 – 6). Die Haltungsvariante 4 differierte von den Varianten 5 und 6 durch seinen ungegliederten Liegebereich. In den Variante 5 und 6 wurde der Liegebereich der Gruppenhaltung durch eine über die halbe Breite reichende Trennwand in zwei Bereiche unterteilt. Die Trennwand befand sich in Haltungsvariante 5 im rechten Winkel an die Außenwand grenzend, während sie in Variante 6 an der der Stallgasse zugewandten Seite des Liegebereichs aufgestellt wurde. Um eine Vergleichbarkeit mit einem Reitpferd zu schaffen, wurden alle Pferde in allen Haltungssystemen täglich für circa eine Stunde in einer Freilaufanlage bewegt. Die Stressbelastung wurde mittels der Messung der Herzfrequenzvariabilität (Parameter pNN50 und SD1) und der Bestimmung fäkaler Cortisolmetaboliten erfasst. Zusätzlich wurden Videoauswertungen hinsichtlich der Beobachtung von Verhaltensauffälligkeiten in den Haltungsvarianten 1 und 2 durchgeführt. Die durchschnittlich niedrigste Stressbelastung erfuhren die Pferde durch die Haltungsvarianten 4 (pNN50: 46,32 %, SD1:158,58 ms, Cortisolmetaboliten: 21,01nmol/kg Kot) und 6 (pNN50: 47,1 %, SD1: 144,62 ms, Cortisolmetaboliten: 21,01 nmol/kg Kot). Die
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Auswertung der pNN50- und Cortisolmetabolitenwerte ergab die größte Stressbelastung der Pferde in den Varianten 1 (pNN50: 42,81 %, SD1: 134,52 ms, Cortisolmetaboliten: 28,56 nmol/kg Kot) und 3 (pNN50: 42,41 %, SD1: 135,36, Cortisolmetaboliten: 28,60 nmol/kg Kot). Die Auswertung der SD1-Werte zeigte zusätzlich noch eine hohe Stressbelastung der Pferde in der Variante 5 (pNN50: 44,83 %, SD1: 119,24 ms, Cortisolmetaboliten: 27,18 nmol/kg Kot). Die Haltungsvariante 2 (pNN50: 45,77 %, SD1: 144,25 ms, Cortisolmetaboliten: 27,59 nmol/kg Kot) beziehungsweise die Varianten 2 und 5 (bei der Betrachtung der pNN50- und der Cortisolmetabolitenwerte) verursachten im Durchschnitt eine mittlere Stressbelastung. Teilweise waren die Unterschiede jedoch zu gering um die Signifikanzgrenze zu überschreiten. Die Betrachtung der einzelnen Pferde und der Pferdegruppen ergab große interindividuelle Unterschiede und deutliche gruppenspezifische Gemeinsamkeiten. Die Position eines Tieres in der Rangordnung hatte in diesen Untersuchungen bei der Betrachtung der Mittelwerte keinen Einfluss auf die Stressbelastung in Abhängigkeit der Haltungsvariante. Vielmehr zeigten sich auch hier deutliche individuelle Unterschiede in der Stressanfälligkeit und den Vorlieben der einzelnen Pferde bezüglich der verschiedenen Haltungssysteme. Die Analyse der Videoaufzeichnungen ergab, dass mehrere Pferde Kreisbewegungen in der Einzelbox ohne Auslauf (Variante 1) zeigten, während in der Variante 2 (Paddockbox) kein Pferd durch Kreisbewegungen auffiel. Andere Verhaltensauffälligkeiten oder gar Verhaltensstörungen wurden nicht beobachtet. Bei der Auswertung der Aufenthaltshäufigkeit und der Aufenthaltesdauer in den 4 Boxenquadranten zeigten sich einige Pferde sehr aktiv, was ein Hinweis auf eventuelle Unruhe oder Nervosität sein kann, während andere Pferde im Vergleich zum Gruppendurchschnitt sehr ruhig wirkten, da sie sich in der Box kaum bewegten und über lange Zeitabschnitte in einem Quadranten standen. Obwohl die Gruppenhaltung für die Mehrzahl der Pferde eine geringere Stressbelastung bedeutete und auch hinsichtlich des Bewegungs- und Sozialverhaltens am artgerechtesten einzustufen ist, sollte für jedes Pferd individuell unter Berücksichtigung seines Alters, seiner Rasse, seines Nutzungsgrades, aber vor allem seines Charakters und seiner Erfahrungen im Sozialverhalten ein passendes großzügig bemessenes Haltungssystem ermittelt werden, um
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die durch das Haltungssystem ausgeübte Stressbelastung zu minimieren und das Wohlbefinden der Pferde zu steigern.
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Palme, R., Touma, C., Arias, N., Dominchin, M.N., & Lepschy, M. (2013). Steroid extraction: Get the best out of faecal samples. Wien Tierärztl Monat – Vet Med Austria, 100, 238–246.
Abstract: Faecal steroid hormone metabolites are becoming increasingly popular as parameters for reproductive functions and stress. The extraction of the steroids from the faecal matrix represents the initial step before quantification can be performed. The steroid metabolites present in the faecal matrix are of varying polarity and composition, so selection of a proper extraction procedure is essential. There have been some studies to address this complex but often neglected point. Radiolabelled steroids (e.g. cortisol or progesterone) have frequently been added to faecal samples to estimate the efficiency of the extraction procedures used. However, native, unmetabolized steroids are normally not present in the faeces and therefore the results are artifi- cial and do not accurately reflect the actual recoveries of the substances of interest. In this respect, recovery experiments based on faecal samples from radiometabolism studies are more informative. In these samples, the metabolite content accurately reflects the mixture of metabolites present in the given species. As a result, it is possible to evaluate different extraction methods for use with faecal samples. We present studies on sheep, horses, pigs, hares and dogs that utilized samples containing naturally metabolized, 14C-labelled steroids. We recommend extracting faecal steroids by simply suspending the faeces in a high percentage of a primary alcohol (for glucocorticoid metabolites 80% aqueous methanol proved best suited for virtually all mammalian species tested so far). Not only does the procedure significantly increase the total amount of recovered radioactivity, it also increases the percentage of unconjugated metabolites, which are more likely to be recognized by the antibodies used in various immunoassays. The advantages of this extraction procedure are clear: it is very easy to use (no evaporation step is needed), it yields high recoveries and variation based on the extraction procedure is reduced to a minimum.
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Maury, M., Murphy, K., Kumar, S., Mauerer, A., & Lee, G. (2005). Spray-drying of proteins: effects of sorbitol and trehalose on aggregation and FT-IR amide I spectrum of an immunoglobulin G. Eur. J. Pharm. Biopharm., 59(2), 251–261.
Abstract: An immunoglobulin G (IgG) was spray-dried on a Büchi 190 laboratory spray-dryer at inlet and outlet air temperatures of 130 and 190°C, respectively. The IgG solution contains initially 115mg/ml IgG plus 50mg/ml sorbitol. After dialysis, at least 80% of low molecular weight component was removed. After spray-drying the dialyzed IgG and immediate redissolution of the powder, an increase in aggregates from 1 to 17% occurred. A major shift towards increase β-sheet structure was detected in the spray-dried solid, which, however, reverted to native structure on redissolution of the powder. A correlation between aggregation determined by size exclusion chromatography and alterations in secondary structure determined by Fourier transformation infra-red spectroscopy could not therefore be established. On spray-drying a non-dialyzed, sorbitol-containing IgG only some 0.7% aggregates were formed. The sorbitol is therefore evidently able to stabilize partially the IgG during the process of spray-drying. Addition of trehalose to the liquid feed produced quantitatively the same stabilizing action on the IgG during spray-drying as did the sorbitol. This finding again points towards a water replacement stabilization mechanism. The IgG spray-dried powder prepared from the dialyzed liquid feed showed continued substantial aggregation on dry storage at 25°C. This was substantially less in the non-dialyzed, sorbitol-containing spray-dried powder. Addition of trehalose to both dialyzed and non-dialyzed system produced substantial improvement in storage stability and reduction in aggregate formation in storage. The quantitative stabilizing effect of the trehalose was only slightly higher than that of the sorbitol. Taken together, these results indicate that both the sorbitol and trehalose stabilize the IgG primarily by a water replacement mechanism rather than by glassy immobilization. The relevance of this work is its questioning of the importance of the usually considered dominance of glassy stabilization of protein in dried systems of high glass transition temperature, such as trehalose. The low glass transition temperature sorbitol produces almost equal process and storage stability in this case.
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