Subjects participating in magnetic resonance imaging (MRI) examinations regularly report anxiety and stress related reactions. This may result in impaired data quality and premature termination of scans. Moreover, cognitive functions and neural substrates can be altered by stress. While prior studies investigated pre–post scan differences in stress reactions only, the present study provides an in-depth analysis of mood changes and hormonal fluctuations during the time course of a typical fMRI session. Thirty-nine subjects participated in the study. Subjective mood, salivary alpha-amylase (sAA) and cortisol were assessed at six time points during the lab visit. Associations between hormonal data and neural correlates of a visual detection task were observed using a region of interest approach applied to the thalamic region. Mood and hormonal levels changed significantly during the experiment. Subjects were most nervous immediately after entering the scanner. SAA was significantly elevated after MRI preparation. A subgroup of n = 5 (12.8%) subjects showed pronounced cortisol responses exceeding 2.5 nmol/l. Preliminary fMRI data revealed an association between sAA levels and left thalamic activity during the first half of the experiment that disappeared during the second half. No significant correlation between cortisol and thalamic activity was observed. Results indicate that an fMRI experiment may elicit subjective and neuroendocrine stress reactions that can influence functional activation patterns.

Summary: Salivary alpha-amylase: More than an enzyme - Investigating confounders of stress-induced and basal amylase activity (Dipl.-Psych. Jana Strahler) The hypothalamus-pituitary-adrenal (HPA) axis and the autonomic nervous system (ANS) are two of the major systems playing a role in the adaptation of organisms to developmental changes that threaten homeostasis. The HPA system involves the secretion of glucocorticoids, including cortisol, into the circulatory system. Numerous studies have been published that introduced salivary cortisol to assess HPA axis activity and therefore strengthens its role as an easy obtainable biomarker in stress research that can be monitored easily and frequently. Recent findings suggest a possible surrogate marker of autonomic activity due to autonomic innervation of salivary glands: salivary alpha-amylase (sAA). Up to date, additional methodological research is needed for a better understanding of the advantages and disadvantages of sAA activity in comparison to already established markers of ANS activity. The aim of the present thesis is to further our knowledge of confounders of sAA activity under basal and acute stress conditions and to strengthen the validity of this enzyme as an easy obtainable alternative for ANS testing. After introducing classical and modern stress concepts and stress system physiology (chapter 2), the reader is acquainted with anatomical basics of salivary gland innervation and secretion of salivary proteins, including sAA, due to autonomic innervation (chapter 3 and 4). Afterwards, a more nuanced review of methodological considerations of sAA determination shows gaps of knowledge concerning its usefulness as a marker of ANS activity (chapter 5). Given the fact that the integration of sAA into developmental and aging research is a relative recent phenomenon, several issues have to be addressed before a final conclusion could be drawn. Therefore, we conducted a series of studies incorporating these considerations regarding behavioral correlates of inter- and intraindividual differences in sAA activity with a special emphasis on older adults. Chapter 7 deals with sAA activity under psychological stress conditions in different age groups. Since vulnerability to disease and disease prevalence patterns change with age, it is important to investigate stress reactivity of people in different age groups. We therefore investigated children between 6 and 10 years, because childhood is a sensitive period of growth and development, and thus plays an important role for later life health. Young adults were included to represent the most studied human age group as a reference. Older adults between 59 and 61 years were investigated, because at this age the course is set for the further development of a person’s health in later life, and because autonomic stress responses in older age might be important determinants of cardiovascular and inflammatory aging. Our goal is to test for associations of sAA with more established stress system markers, i.e., salivary cortisol as outcome measurement of HPA reactivity, heart rate (HR) and heart rate variability (HRV) as markers for autonomic reactivity, and to directly compare these responses between different age groups across the life span. Secretion of sAA and cortisol was repeatedly assessed in 62 children, 78 young adults, and 74 older adults after exposure to a standardized psychosocial stressor, the Trier Social Stress Test. In addition, cardiovascular activity was measured in both adult groups. Older adults showed attenuated sAA, HR, and HRV responses. Furthermore, we found higher sAA but lower cortisol at baseline as well as lower sAA and cortisol responses in children. Age by sex interactions were observed only for cortisol with higher responses in older male participants. No associations between the parameters were found. Results in children and young adults confirm previous results. Overall, findings implicate sAA as an alternative or additional autonomic stress marker throughout the life span, with marked and rapid responsiveness to stress in three relevant age groups. The impact of age and chronic stress on basal sAA activity is the center of interest in chapter 8. We therefore assessed diurnal profiles of sAA and salivary cortisol in 27 younger and 31 older competitive ballroom dancers as well as 26 younger and 33 older age- and sex-matched controls. According to the Allostatic Load concept, repeated, non-habituating responses to social-evaluative conditions, which characterize the lives of competitive ballroom dancers, should be associated with stress system dysregulations. Furthermore, we expect to see an increased sympathetic drive associated higher overall alpha-amylase activity in older adults. Analyses revealed an elevated daily overall output of sAA in older adults while there was no effect of age on mean cortisol levels. Alterations of diurnal rhythms were only seen in younger male dancers showing a flattened diurnal profile of sAA and younger dancers and female older dancers showing a blunted diurnal rhythmicity of cortisol. Furthermore, we found a negative correlation between summary indices of basal sAA and the amount of physical activity. In conclusion, higher overall output of sAA in older adults was in line with the phenomenon of a “sympathetic overdrive” with increasing age. Furthermore, a lower output of sAA in people who are more physical active was in line with the hypothesis of an exercise-induced decrease of sympathetic activity. Taken together, results of chapter 7 and 8 show a clear impact of age on sAA activity, either under acute stress or basal conditions. One problem when integrating sAA into developmental and aging research is the use of adrenergic agonists and antagonists what is very common in older adults, i.e. antihypertensive drugs (AD). As well, the previously shown sympathetic overactivity that occurs with normal aging is associated with higher blood pressure (BP). Therefore, chapter 9 deals with a possible impact of high BP and AD on diurnal sAA activity in 79 older adults (33 normotensive adults, 16 medicated vs. 45 hypertensive adults, 34 medicated). Results showed a pronounced rhythm of sAA in all groups. Diurnal profiles differed significantly between men and women with men lacking the typical decrease of sAA in the morning and showing more pronounced alterations throughout the day. An effect of AD on sAA profiles and area under the curve values indicates that subjects not using AD´s show a heightened diurnal profile and a higher total output of sAA. Descriptively, this was also true for hypertensive older adults. Hypertensive subjects and those not using AD showed the highest diurnal output of sAA and the steepest slope. In sum, our results show an impact of antihypertensive medication and a difference between normotensive and hypertensive subjects on characteristics of diurnal sAA activity. Hence, findings are of particular interest in research using sAA as a prognostic indicator of pathological states and processes. Given the fact that hypertension was also shown to be associated with substantial changes of transmitters within the suprachiasmatic nucleus (SCN) - the “biological clock” that receives photic input from retinal glands via the retinohypothalamic pathway - and an altered output from the SCN to the sympathetic nervous system, we broaden the idea of a possible effect of different lighting conditions on morning sAA profiles in chapter 10. In a counterbalanced within-subjects design six men and 16 women of different ages collected sAA morning profiles on two consecutive days with leaving their shutters closed on the one day (= dark) and open their shutters on the other day (= bright). We were able to replicate earlier findings of light-induced changes of salivary cortisol with higher responses during the bright condition. On either day, women showed larger cortisol increases than men. Despite multisynaptic autonomic connections arising from the SCN projecting to multiple organs of the body, we could not find an effect of sunlight on sAA morning profiles. Evidence for circadian clock gene expression in human oral mucosa might account for this result and indicates that peripheral oscillators may act more like integrators of multiple different time cues, e.g. light, food intake, instead of a “master” oscillator (SCN). Results of chapter 7 to 10 provide clear evidence that sAA is heightened in states of autonomic arousal, i.e. stress, aging and hypertension, and that its circadian rhythmicity seems to be regulated rather integrative than directly via efferent input from hypothalamic SCN neurons. In chapter 11 this thesis tries to approach one central question: What is the biological meaning of the findings made? According to this enzyme´s anti-bacterial and digestive action short term changes might not have a biological meaning itself but rather reflect just a small part of multiple coordinated body responses to stressful stimuli. While the sympathetic branch of the ANS mainly stimulates protein secretion, the parasympathetic branch stimulates saliva flow. Acute stress responses might therefore be interpreted as reflecting predominant sympathetic activity together with parasympathetic withdrawal. The same mechanism could also be suitable for the finding of higher diurnal levels of sAA in older adults or hypertensive subjects reflecting a higher peripheral sympathetic tone in these groups. Diurnal profiles of sAA itself may reflect circadian changes in autonomic balance. Circadian rhythms are of great advantage since they enable individuals to anticipate. This pre-adaptation enables the individual to cope with upcoming demands and challenges. Our finding of a relationship between sAA and salivary cortisol what strengthens the relevance of glucocorticoids that were previously shown to be able to phase shift circadian rhythms in cells and tissue. Within a food-related context there is evidence that decreasing levels of sAA in the morning could reflect increases of feeling hungry since sAA systematically increases during food consumption and with the subjective state of satiety. So far, much more research is needed to identify underlying physiological mechanisms of circadian sAA rhythmicity. Taking the next step, future studies will have to focus on the integration of sAA assessment into longitudinal studies and different disease states to prove its applicability as a marker of sympathetic neural functioning in the genesis and prognosis of disease.

Die vorliegende Arbeit befasst sich mit der Evaluierung einer neuen klinischen Handlungsempfehlung zur Rapid Sequence Induction (RSI) des nichtnüchternen Säuglings sowie eines neuen Modells zur simulatorbasierten Evaluation akutmedizinischer, klinischer Leitlinien.  Bei der klassischen RSI kommt es aufgrund der fehlenden Zwischenbeatmung während der Narkoseeinleitung zu hohem Zeitdruck. Sie ist technikbedingt assoziiert mit Hypoxämie beim Patienten und einem hohen Stresslevel beim Anwender (Eich et al. 2009a). Stress wiederum ist ein potentieller Trigger bei der Entstehung unsicherer Handlungen und kritischer Ereignisse. Aus diesem Grund gab der wissenschaftliche Arbeitskreis Kinderanästhesie der Deutschen Gesellschaft für Anästhesiologie und Intensivmedizin (DGAI) im Jahre 2007 eine neue Handlungsempfehlung zur Narkoseeinleitung des nichtnüchternen Säuglings (kontrollierte RSI) heraus (Schmidt et al. 2007; Schreiber 2007).  Wesentliche Änderungen sind die geplante Durchführung einer sanften Zwischenbeatmung via Maske, der Verzicht auf Succinylcholin als Muskelrelaxans und das Weglassen des Krikoiddrucks. Dadurch sollten Zeitdruck und Stress vermindert sowie die Inzidenz der damit zusammenhängenden unsicheren Handlungen und kritischen Ereignisse reduziert werden. Für unsere Studie entwickelten wir ein simulatorbasiertes Modell, um die kontrollierte (neue) RSI-Technik mit der klassischen (bisherigen) RSI-Technik vergleichend zu evaluieren. Dabei kombinierten wir Methoden der Observation, der Stressmessung und der der Selbsteinschätzung. An der kontrollierten, randomisierten Studie nahmen 30 männliche Anästhesisten (Fachärzte und Weiterbildungsassistenten) teil. Sie absolvierten am Säuglingssimulator (SimBaby®) eine standardisierte Narkoseeinleitung bei einem vier Wochen alten Säugling mit obstruktiver Pylorusstenose. Jeweils 15 Probanden absolvierten die klassische und die kontrollierte RSI (Eich et al 2009a; Eich et al. 2009b). Das Szenario mit den Vitalzeichen und deren Trends wurden zuvor programmiert. Während des Versuchs beobachteten wir unsichere Handlungen und kritische Ereignisse wie Hypoxämie (SpO2 < 90%), forcierte Maskenbeatmung, Intubationsschwierigkeiten und -Komplikationen. Zusätzlich maßen wir den Stress der Probanden mithilfe der Ergospirometrie (Herz-, Atemfrequenz und Atemminutenvolumen sowie O2-Verbrauch und CO2-Produktion), der Analyse von Kortisol und α-Amylase im Speichel und eines Selbsteinschätzungsfragebogens (subjektives Stress- und Sicherheitsempfinden)  (Eich et al. 2009a; Eich et al. 2009b). Bei allen Probanden konnte im Verlauf des Szenarios ein deutlicher, reproduzierbarer Anstieg aller Stressparameter gemessen werden. Bei der kontrollierten RSI waren objektiv gemessene Stressparameter sowie das subjektive Stressempfinden niedriger als bei der klassischen RSI. Bei der klassischen RSI entwickelte sich, im Gegensatz zur kontrollierten RSI, immer eine Hypoxämie. Die Inzidenz der übrigen unsicheren Handlungen und kritischen Ereignisse war bei beiden Methoden relativ gering.  Unsere Daten zeigen, dass die kontrollierte RSI - im Vergleich zur klassischen RSI - den Stress bei den Anwendern und damit das Entstehen unsicherer Handlungen und kritischer Ereignisse zu reduzieren vermag (Eich et al 2009a). Das neuentwickelte simulatorbasierte Modell hat sich dabei als praktikabel und valide erweisen zur Evaluation akutmedizinischer, klinischer Leitlinien (Eich et al 2009b).

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2007. / Sugarcane is cultivated because of the high levels of sucrose it stores in its internodes. Starch metabolism has been a neglected aspect of sugarcane research despite the problems caused by it during sugarcane processing. Currently there is no information available on the starch content in different South African commercial sugarcane varieties. This project had two main aims of which the first was to determine the starch content in the internodal tissues of six commercial sugarcane varieties. The activities of ADP-Glucose Pyrophosphorylase (AGPase) and β- amylase were also determined. The second aim of the project was to manipulate starch metabolism in sugarcane using transgenesis. To achieve this, transformation vectors for the down-regulation of AGPase activity and over-expression of β-amylase activity were designed. These vectors were then used to transform sugarcane calli and the results were analysed in suspension cultures. Starch levels in sugarcane internodal tissue increased more than 4 times from young to mature internodes. There were also large differences between varieties. When mature tissues of different varieties were compared, their starch concentration varied between 0.18 and 0.51 mg g-1 FW, with the majority of the varieties having a starch concentration between 0.26 and 0.32 mg g-1 FW. NCo376’s starch concentration was much lower than the rest at 0.18 mg g-1 FW and N19’s was much higher at 0.51 mg. g-1 FW. There was also a very strong correlation between starch and sucrose concentration (R2 = 0.53, p ≤ 0.01) which could be due to the fact that these metabolites are synthesized from the same hexose-phosphate pool. No correlation was evident between starch concentration and AGPase activity. This was true for correlations based on either tissue maturity or variety. β-amylase activity expressed on a protein basis was almost 5 times higher in the young internodes compared to mature internodes, suggesting that carbon might be cycled through starch in these internodes. AGPase activity in the transgenic suspension cultures was reduced by between 0.14 and 0.54 of the activity of the wild type control. This reduction led to a reduction in starch concentration of between 0.38 and 0.47 times that of the wild type control. There was a significant correlation between the reduction in AGPase activity and the reduction in starch (R2 = 0.58, p ≤ 0.05). β-amylase activity in the transgenic suspension cultures was increased to 1.5-2 times that of the wild type control. This led to a reduction in starch concentration of between 0.1 and 0.4 times that of the wild type control. Once again the increase in β-amylase activity could be correlated to the reduction in starch concentration of the transgenic suspension cultures (R2 = 0.68, p ≤ 0.01). In both experiments there was no significant effect on sucrose concentration.

Le suivi et la compréhension des états de stress et de récupération des sportifs est un enjeu majeur, notamment dans le cadre de périodisations complètes de l’entraînement. Un déséquilibre mal maîtrisé entre le stress et la récupération peut induire une fatigue voire un épuisement psychologique et physiologique du sportif. Il en découlera des conséquences néfastes sur le bien-être et la santé de la personne mais également sur la performance individuelle et collective. Ces conséquences sont complexes et impliquent des dimensions environnementales, sociales, psychologiques et physiologiques non-spécifiques. Enfin, ces symptômes sont soumis à des variations intra et interindividuelles importantes rendant le diagnostic d’autant plus complexe à établir. En s’appuyant sur plusieurs suivis de sportifs en pôle « Espoir », ce travail de recherche montre l’évolution de variables psychologiques et physiologiques intervenant dans la réponse au stress chez le sportif. Ces travaux permettent également de mettre en évidence des variables prédictives des états de stress et de récupération. Enfin, nous démontrons que les états de stress et de récupération des sportifs impactent les états émotionnels au niveau intra et interindividuel. / Monitoring stress and recovery states is a key issue, especially in high performance sport. An imbalance between stress and recovery can induce fatigue, psychological and physiological exhaustion. Thus, negative consequences on well-being, health and individual and collective performance can be experienced. These consequences are complex as they stem from non-specific environmental, social, psychological and physiological dimensions. These phenomena are subject to important intra-individual and inter-individual variations, which make a diagnosis hard to establish. Based on a several followings of athletes from high performance structures, this research shows the evolution of psychological and physiological variables intervening as a response to stress. From these studies, some predictive variables also showed up in predicting stress and recovery states. Finally, we made evidence of the impact of athletes’ stress and recovery states on emotional states, on the intra and inter-individual levels.

L’objectif de cette étude est d’étudier le rôle de la salive dans la libération des molécules odorantes, par deux approches, in vitro et in vivo. L’effet des protéines salivaires sur la libération de 10 molécules odorantes (5 esters et 5 cétones, de longueur de chaîne hydrophobe variable) a été étudié in vitro dans des systèmes modèles composés de salives artificielles et humaine. Les salives artificielles contiennent les protéines majoritairement présentes dans la salive (mucine et alpha-amylase), seules et en mélange. Les quantités de chaque molécule odorante présentes dans la phase gazeuse à l’équilibre thermodynamique ont été mesurées par une analyse headspace en mode statique couplée à la chromatographie en phase gazeuse (SH-GC). Les coefficients de partage entre l’air et chacun des systèmes modèles ont été calculés pour chacune des molécules. Cette approche in vitro nous a permis de démontrer une diminution des coefficients de partage air/salive artificielle en présence de mucine et d’alpha-amylase, par un effet hydrophobe. Aucun effet cumulatif n’est observé lorsque les deux protéines sont mises ensemble en solution. En présence de salive humaine, une diminution des coefficients de partage est également observée, les esters étant plus affectés par la présence de salive humaine que les cétones. Cette observation est due à une activité des estérases de la salive, qui augmente avec l’hydrophobicité des esters. La libération in vivo du propanoate d’éthyle et de l’hexanoate d’éthyle a été suivie sur 10 sujets par spectrométrie de masse à ionisation chimique à pression atmosphérique (APCI-MS) dans des conditions physiologiques différentes : au repos, après stimulation et après élimination du film salivaire résiduel. La salive de chaque sujet a été caractérisée dans les différentes conditions physiologiques testées. De grandes variations de flux, viscosité et de composition salivaire ont été mises en évidence entre les sujets, ainsi qu’entre les conditions physiologiques pour un même sujet. Les différences observées sur les paramètres de libération in vivo des molécules odorantes sont discutées en regard de ces paramètres physiologiques. Nous avons ainsi observé qu’une viscosité salivaire élevée diminue la quantité de molécules odorantes libérées sur un temps donné. Dans le même temps, la présence d’une quantité importante d’alpha amylase dans la salive augmente de façon significative le temps de libération de la molécule la plus hydrophobe, l’hexanoate d’éthyle. Nous avons ainsi mis en évidence que la rétention des molécules hydrophobes par les protéines salivaires peut induire une modification de leur cinétique de libération en conditions réelles de consommation et pourrait intervenir dans la persistance aromatique. / The aim of this work is to give a deeper understanding of the impact of the salivary composition on aroma release, by two approaches, an in vitro and an in vivo approach. The impact of salivary proteins on the release of 10 aroma compounds (5 esters and 5 ketones, varying in their hydrophobic chain length) was first investigated by in vitro model systems composed of artificial and human saliva. Artificial salivas were composed of the main salivary proteins, mucins and alpha amylase, alone and in mixture.The amount of aroma released in the vapor phase at equilibrium was analyzed by Static Headspace Gas Chromatography analysis. Air/system partition coefficients have been calculated. This in vitro approach allowed us to demonstrate the ability of both mucin and alpha-amylase to decrease the release of aroma compounds by hydrophobic effect (increase of retention with aroma hydrophobicity). Interestingly, no cumulative effect was observed when both proteins were mixed together in solution. The release of ketones in presence of human saliva is lower than in water and slightly higher than in the presence of artificial saliva. Esters are more affected by the presence of human saliva than ketones. This observation is due to an esterase activity of saliva, which increases with the hydrophobicity of esters. The in vivo release of ethyl propanoate and ethyl hexanoate was followed on ten subjects by Atmospheric Pressure Chemical Ionization mass spectrometry (APCI-MS) under different physiological conditions: at rest, after stimulation and after removing the superficial salivary coat. The saliva was characterized for each subject and each physiological condition. Great variations were observed between the subjects on the salivary flow, viscosity, composition and for each subject between the physiological conditions. The differences observed on in vivo release parameters are discussed as a function of physiological parameters. We observed that subjects with a more viscous saliva present a lower amount of aroma released. The presence of higher amounts of alpha-amylase increased the time needed to release the more hydrophobic compound, ethyl hexanoate. Our results suggest that the retention of hydrophobic aroma compounds by salivary proteins induces a modification of the kinetics of aroma release in real consumption conditions, and could be responsible for aroma persistence.

Aim: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. Methods: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4 ′ ,6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. Results: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. Conclusion: The enzyme activities as well as the pellicle’s ultrastructure are of high similarity in cariesactive and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3–120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.

Subjects participating in magnetic resonance imaging (MRI) examinations regularly report anxiety and stress related reactions. This may result in impaired data quality and premature termination of scans. Moreover, cognitive functions and neural substrates can be altered by stress. While prior studies investigated pre–post scan differences in stress reactions only, the present study provides an in-depth analysis of mood changes and hormonal fluctuations during the time course of a typical fMRI session. Thirty-nine subjects participated in the study. Subjective mood, salivary alpha-amylase (sAA) and cortisol were assessed at six time points during the lab visit. Associations between hormonal data and neural correlates of a visual detection task were observed using a region of interest approach applied to the thalamic region. Mood and hormonal levels changed significantly during the experiment. Subjects were most nervous immediately after entering the scanner. SAA was significantly elevated after MRI preparation. A subgroup of n = 5 (12.8%) subjects showed pronounced cortisol responses exceeding 2.5 nmol/l. Preliminary fMRI data revealed an association between sAA levels and left thalamic activity during the first half of the experiment that disappeared during the second half. No significant correlation between cortisol and thalamic activity was observed. Results indicate that an fMRI experiment may elicit subjective and neuroendocrine stress reactions that can influence functional activation patterns.

Despite the abundance of research conducted into the effects of exercise on mucosal immunity the results remain controversial. Much of the inconsistencies arise from the exercise protocols, the participants studied and their nutritional status, as well as methodological and analytical differences. The purpose of this thesis was to examine the influence of some of these factors, and to investigate potential means of enhancing the mucosal immune response to exercise. In study 1 (Chapter 3) it was shown that a fed or fasted state 2 h prior to exercise had no effect on the s-IgA concentration or secretion rate during prolonged exercise. However, when participants were fed during exercise (Chapter 4), the secretion rate of salivary antimicrobial proteins lysozyme and a-amylase increased, but sIgA remained unchanged. These changes were likely due to the activation of mechanical and gustatory receptors leading to a reflex stimulation of protein secretion via the autonomic nerves, rather than changes in stress hOnliones, since cortisol did not change significantly during exercise. Study 3 (Chapter 5) extended these findings where it was demonstrated that chewing flavoured gum during exercise enhanced lysozyme and a-amylase secretion but resulted in a small reduction in s-IgA secretion rate. Salivary antimicrobial proteins are affected by the exercise intensity since both s-IgA and lysozyme secretion rate increased post -exercise following an incremental test to exhaustion, but not after exercise at 50% Y02max. Moreover, lysozyme secretion rate was also elevated following exercise at 75% Y02mru<, whereas s-IgA remained unchanged. These effects are thought to be mediated by increased sympathetic nervous system activity reflected by the concomitant increases in (lamylase and chromogranin A, rather than the hypothalamic-pituitary-adrenal axis. Resting mucosal immunity exhibits significant gender differences. In study 1 (Chapter 3) s-IgA concentration, secretion rate and osmolality were found to be lower in females than in males at rest. In addition, saliva flow rate was found to be lower in females compared with males in study 5 (Chapter 7). However, these differences did not appear to influence the salivary responses to acute exercise or exercise training. Chronic exercise training in elite male and female swimmers resulted in lower levels of s-IgA secretion rate following periods of intense training prior to competition compared with post-competition (Chapter 7), but these levels were not directly associated with reported episodes of respiratory illness.

Foram realizados dois experimentos para comparar os efeitos da Monensina (MON, DSM Nutritional Products Ltd.), uma mistura de óleos funcionais (OF; Crina&reg; Ruminants, DSM Nutritional Products Ltd.) isoladamente ou associado com enzima amilolítica (OFA; Crina- Ronozyme Rumistar&trade;) em dietas de vacas em lactação. No experimento 1, oito vacas holandesas multíparas (576 &#177; 100 Kg peso corporal, e 146,5 &#177; 35,1 dias em lactação e 35.1 &#177; 4,0 kg/d de produção de leite no início do experimento) canuladas no rúmen foram distribuídas em quadrado latino replicado com períodos experimentais de 21 dias para determinar os efeitos da MON, OF e OFA sobre a digestibilidade dos nutrientes, fermentação ruminal, síntese de proteína microbiana, metabolitos sanguíneos, produção e composição do leite, utilização de energia e nitrogênio de vacas em lactação. Os tratamentos nos experimentos foram: controle (CON), sem aditivo; monensina (MON), adição de 13mg/Kg MS de monensina; óleos funcionais (OF), fornecimento de 44 mg Crina/Kg MS e óleos funcionais associado a enzima amilolítica (OFA), adição de 44 mg Crina/Kg e 330 KNU de enzima com atividade amilolítica por Kg de MS. Os dados foram analisados através de contrastes ortogonais para avaliar o efeito de aditivos (C1), comparar ionóforo com óleos funcionais (C2) e óleos funcionais isolados ou combinados com enzima amilolítica (C3). Embora não tenha sido encontrado efeito sobre a ingestão de nutrientes, os aditivos tenderam a aumentar (P=0,058) a digestibilidade da proteína bruta. No entanto, MON tendeu a aumentar (P=0,085) a digestibilidade da fibra em detergente neutro em comparação a OF e OFA. Os aditivos diminuíram (P=0,026) a concentração de nitrogênio amoniacal (NH3-N) no rúmen, notadamente a MON e OFA. Os tratamentos contendo OF apresentaram menor pH (P=0,017) e maior NH3-N no rúmen (P=0,004). Não foram encontradas diferenças na produção e composição do leite, mas o fornecimento de aditivos melhorou a eficiência produtiva (P= 0,004). No experimento 2, trinta vacas multíparas da raça holandesa (574&#177;68 Kg peso corporal, 30.9&#177;4,1 Kg/dia de produção de leite e 152,2 &#177; 54,1 dias em lactação no início do experimento) foram distribuídas em um delineamento em blocos ao acaso em três tratamentos: MON, OF e OFA, previamente descrito no experimento 1. Os tratamentos foram proporcionados ao longo de 9 semanas. Contrates ortogonais foram utilizados para comparar MON com óleos funcionais isolados ou com amilase. Não foram observadas diferenças na ingestão de nutrientes e digestibilidade quando comparado MON com os tratamentos com OF. No entanto, tratamentos contendo óleos funcionais tendem a aumentar (P=0,062) a ingestão de MS das vacas. Não foi observado efeito de interação tempo tratamento. Os tratamentos contendo óleos funcionais apresentaram menor teor de lactose e proteína no leite (P&le;0.039) em comparação a MON. Concluindo, todos os aditivos tiveram pequeno efeito sobre a digestibilidade e ingestão de nutrientes, sem alterar a produção de leite das vacas. Contudo, os aditivos diminuíram a concentração de amônia ruminal, em especial a MON. Não foi encontrado efeito sinérgico quando se combinou óleos funcionais com enzima amilolítica. / Two experiments were carried out to compare the effects of monensin (MON, DSM Nutritional Products Ltd.,), a blend of functional oils (FO; Crina&reg; Ruminants, DSM Nutritional Products Ltd.) alone or associated with amylolytic enzyme (FOA; Crina- Ronozyme Rumistar&trade;) in a dairy cow diet. In experiment 1, eight multiparous Holstein cows (576&#177;100 kg LW, 146&#177;35 DIM, and 35.1&#177;4.0 kg/d milk yield at the start of experiment, cannulated in the rumen were assigned into a replicated Latin square experiment design with 21-d periods in order to determine the effects of MON, FO or FOA on nutrient digestibility, ruminal fermentation, microbial protein synthesis, blood metabolites, milk yield and composition, energy and nitrogen utilization of dairy cows. Treatments in experiments were: control (CON), with no additive; MON, addition of 13 mg monensin/kg diet DM; FO, provision of 44 mg Crina/kg diet DM and FOA, addition of 44 mg Crina and 330 KNU of enzyme with amylolytic activity per kg diet DM. Data were analyzed through orthogonal contrasts to evaluate the effect of additives (C1), compare ionophore with functional oils (C2), and contrast functional oils alone or with amylolytic enzyme (C3). Although no effects were found on nutrient intake, additives tended to increase (P=0.058) crude protein digestibility. Yet, MON tended to increase (P=0.085) neutral detergent fiber digestibility in comparison with FO and FOA. Additives decreased (P=0.026) ammonia nitrogen concentration (NH3-N) in rumen, notably due to MON and FOA. Treatments containing FO showed lower (P=0.017) pH and higher (P=0.004) NH3-N in rumen. No differences were found on milk yield and composition, but the provision of additives improved (P=0.04) productive efficiency (kg milk ÷ kg DM intake) of cows. In experiment 2, thirty multiparous Holstein cows (574&#177;68 kg LW, 152,2&#177;54,1 DIM and 30.9&#177;4.1 kg/d milk yield at the start of experiment) were assigned to a randomized block design experiment and distributed to three treatments: MON, FO or FOA, as previously described in Experiment 1. Treatments were provided throughout 9 weeks. Orthogonal contrasts were also used to compare MON with functional oils (C1) and essential oils alone or with amylase(C2). No differences were detected on nutrient intake and digestibility when comparing MON with FO-treatments. However, treatments containing functional oils tended to increase (P=0,062) DM intake of cows. No treatment by time interaction effect was observed. Treatments containing functional oils had lower (P&le;0.039) lactose and protein content in milk than MON. In conclusion, all additives had small effect on nutrient intake and digestibility, without altering milk production of cows. Nonetheless, feed additives decreased ammonia concentration in rumen notably because of MON. No synergetic effect was found when combining FO with amylolytic enzyme.