Cows in first (n = 163) and second (n = 101) lactation were studied to determine if reproduction, progesterone (P4), IGF-I, insulin, NEFA, and milk production differed between genetic groups. Thirty-five cows were Holstein-Jersey (HJ) crosses, 48 were Jersey-Holstein (JH) crosses, 51 were Holsteins (HH), and 29 were Jerseys (JJ). Days open (DO) was affected by genetic group. HH had 168.6 ± 9.6 DO which was different from HJ (142.9 ± 11.3 d), JJ (132.1 ± 12.1 d), and JH (127.2 ± 9.3 d). HH had 2.4 ± 0.1 services per conception which was different from JH (1.9 ± 0.1), but not different from HJ (2.1 ± 0.2) or JJ (2.1 ± 0.2). In HH P4 concentrations (1.6 ± 0.11 ng/mL) were not different from HJ (1.5 ± 0.12 ng/mL), but lower than JH (1.8 ± 0.10 ng/mL) and JJ (1.8 ± 0.13 ng/mL). NEFA concentrations were greater in lactation 2 (0.52 ± 0.02 mEq/L) than in lactation 1 (0.45 ± 0.02 mEq/L). Insulin in HH (0.83 ± 0.03 ng/mL) was not different from HJ (0.87 ± 0.04 ng/mL) or JH (0.76 ± 0.03 ng/mL), but was greater than JJ (0.66 ± 0.04 ng/mL). IGF-I gradually increased over the 10 wk period. The HH produced 10,348 ± 208 kg of milk, which was greater than the HJ (9,129 ± 230 kg), the JH (9,384 ± 192 kg), and the JJ (7,080.9 ± 261 kg). Reproductive measures, milk yield, hormones and metabolites were affected by genetic groups. / Master of Science

Orientador: Roberto de Oliveira Roça / Resumo: RESUMO O objetivo do presente estudo foi investigar o período de jejum alimentar pré-abate mais adequado para Nelores terminados em confinamento e submetidos à curta jornada de transporte, considerando aspectos de bem-estar animal e qualidade da carne. O experimento foi realizado com 60 animais, machos não castrados, selecionados de mesmo lote do confinamento e divididos em 3 grupos: 6, 12 e 24 horas de jejum. As amostras de sangue foram colhidas antes do embarque (pré-jejum) e na calha de sangria (pós-jejum), assim como, foram avaliados os parâmetros relacionados a desidratação e função renal (proteínas totais, sódio, potássio, cloreto, cálcio ionizado, intervalo aniônico – “ânion GAP” e Nitrogênio ureico sanguíneo ou “Blood Ureia Nitrogen” - BUN, hematócrito percentual, hemoglobina), mobilização energética (ácidos graxos não esterificados – NEFA, β-hidroxibutirato – BHB e creatinina), resposta das proteínas de fase aguda (haptoglobina - Hp e proteína amiloide A - SAA) e avaliação do estresse (cortisol, glicose, lactato e dióxido de carbono total). Durante o resfriamento, o pH foi mensurado no Músculo longissimus thoracis, e colhidas amostras para análise do potencial glicolítico (glicogênio, glicose, glicose-6-fosfato e lactato). Após o resfriamento, amostras do M. longissimus thoracis foram colhidas e avaliadas para perda de peso por descongelamento, cor objetiva (L*, a* e b*, H* e C*), umidade, perda por cozimento e força de cisalhamento. Os resultados para os parâmetros ... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

No description available.

Die Methan (CH4) Produktion der Milchkühe wird durch eine Vielzahl von umwelt- und wirtsspezifischen Faktoren beeinflusst, wobei Trockensubstanzaufnahme und Rationszusammensetzung die größte Auswirkung haben. Der größte Teil des CH4 wird von Archaeen im Pansen produziert. Auch die kurzkettige Fettsäure (SCFA) Acetat wird im Pansen durch mikrobielle Fermentation gebildet und kann vom Wirtstier zur Milchfettsynthese im Euter verwendet werden. Die Acetatbildung im Pansen korreliert mit der CH4 Produktion. Allerdings kann Milchfett auch aus nicht veresterten Fettsäuren (NEFA) und Triacylgylcerolen endogenen Ursprungs synthetisiert werden, insbesondere aus mobilisiertem Körperfett. In dieser Studie wurde die Hypothese überprüft, dass eine Verdrängung des zur Milchfettbildung genutzten Acetats durch eine höhere Körperfettmobilisation in der Frühlaktation die ruminale Acetatproduktion senkt und damit die Bildung von CH4 verringert. Ein weiteres Ziel war zu untersuchen, ob der Anstieg der CH4 Produktion im Laktationsverlauf mit einer Veränderung des Mikrobioms assoziiert ist, und ob sich Kühe mit hoher oder niedriger CH4 Emission in ihrer Bakterien- und Archaeen-Zusammensetzung unterscheiden. 20 Holstein Kühe wurden in ihrer ersten Laktation untersucht; ihre Futteraufnahme und Rationszusammensetzung wurde analysiert. Im Verlauf des Versuchs wurden mehrfach Blut- und Pansensaftproben gewonnen. Die Plasma-NEFA-Konzentrationen wurden photometrisch, die Pansen-SCFA-Konzentrationen mittels Gaschromatographie analysiert. Während des Beobachtungszeitraums wurde an 4 Zeitpunkten die individuelle CH4 Produktion in Respirationskammern erfasst. In einer Untergruppe von 9 Kühen wurden Pansensaftproben von 3 Zeitpunkten während der Laktation einer DNA-Extraktion unterzogen und bakterielle und archaeale 16S rRNA Amplicons wurden sequenziert. Die Bakterien- und Archaeenpopulation im Pansensaft wurden beschrieben und Pansenmikrobiom der CH4 Ausbeute gegenübergestellt. Statistische Auswertungen wurden mit repeated measurements ANOVA und Tukey Tests, sowie mit der Pearsons‘ Korrelation für ausgewählte Parameter durchgeführt. Mikrobielle Daten wurden mit multivariaten Analysen (PERMANOVA) weiterverarbeitet und Bray-Curtis-Unähnlichkeiten ermittelt. Die gesamte CH4 Produktion stieg signifikant von durchschnittlich 208 l/Tag in der Trockenperiode auf 516 l/Tag in der Spätlaktation an. Der Grad der Körperfettmobilisation, ausgedrückt als Plasma NEFA Konzentration, und die CH4 Ausbeute waren in der Frühlaktation negativ korreliert (p = 0,002). Kühe mit hoher Fettmobilisation (NEFA > 580 μmol/l) neigten nur vor der Geburt, aber nicht während der Laktation zu höheren Pansenacetat Konzentrationen als Tiere mit niedriger Mobilisation (NEFA < 580 μmol/l). Trotz einer möglichst gleichbleibenden Rationszusammensetzung während der Laktation änderte sich das Mikrobiom mit der Zeit signifikant, was sich in einer Abnahme des Artenreichtums und der Biodiversität zeigte. In der Spätlaktation, als die CH4 Ausbeute am höchsten war, gab es keinen Unterschied in der bakteriellen oder archealen Populationsstruktur zwischen den drei Kühen mit der schwächsten und den dreien mit der stärksten CH4 Ausbeute. Parallel zum Anstieg der CH4 Produktion von 434,3 l/Tag auf 540,5 l/Tag veränderte sich das Verhältnis von (Acetat + Butyrat) / Propionat im Pansensaft mit dem Fortschreiten der Laktation von 3,5 auf 4,4. Dennoch war kein Zusammenhang zwischen der Konzentration der ruminalen SCFA und der CH4 Ausbeute festzustellen. Der Stoffwechselzustand des Tieres, insbesondere der Grad der Körperfettmobilisierung bei negativer Energiebilanz, nahm Einfluss auf die CH4-Ausbeute. Die Zusammensetzung des Mikrobioms im Pansen und dessen Stoffwechselnetzwerk veränderte sich mit der Zeit. Es war jedoch in dieser Studie nicht möglich, einzelne Mikroorganismen als Prädiktor für die CH4-Emission von Milchkühen zu identifizieren. Vielmehr scheinen Verschiebungen der mikrobiellen Gemeinschaften insgesamt für die Veränderung der CH4 Ausbeute verantwortlich zu sein.:1 Introduction 1 2 Background 2 2.1 Greenhouse Gases 2 2.2 Dairy cows and their importance to food production 3 2.3 Rumen functions 5 2.3.1 Anatomy and Physiology 5 2.3.2 Rumen microbes 7 2.3.2.1 Bacteria 8 2.3.2.2 Archaea 11 2.3.3 Short-chain fatty acids 12 2.3.4 Methane formation 15 2.4 Interrelationship between methane and host animal physiology 15 2.4.1 Physiologic aspects affecting methane formation 15 2.5 Effects of feed composition and feed contents on methane production 16 2.5.1 Relationship of ruminal short-chain fatty acids and methane production 17 2.5.2 Milk fatty acids to estimate methane emission 19 2.6 Description of methods 20 2.6.1 Methane Measurement 20 2.6.2 Sampling of rumen contents 21 2.6.3 Methods to identify microbes 22 2.7 Objective and realization of the studies 23 3 Publications 26 3.1 First Publication 26 3.1.1 Supplement first Publication 40 3.2 Second Publication 42 3.2.1 Supplement second Publication 56 4 Discussion 60 4.1 Assessment of experimental design 60 4.1.1 Animals 60 4.1.2 Feed 61 4.1.3 Rumen fluid 61 4.1.4 Blood and milk metabolites 62 4.2 Assessment of results 62 4.2.1 Variance of methane emissions 62 4.2.2 Rumen short-chain fatty acids and methane 65 4.2.3 Acetate in the cows’ metabolism and methane production 66 4.2.4 Fat mobilization in early lactation 67 4.2.5 NEFA in the context of metabolism 68 4.2.6 Rumen microbes 69 4.2.6.1 Microbial community change over time 70 4.2.6.2 Community differences between individuals 71 4.2.6.3 Relationship between microbes and methane production levels 72 4.2.7 Further considerations 74 5 Conclusions 75 6 Summary 77 7 Zusammenfassung 79 8 References 81 / Methane (CH4) production in dairy cows is influenced by a variety of environmental and host-specific factors, among which dry matter intake and ration composition have the greatest impact. The major part of CH4 is produced in the rumen by Archaea. The short-chain fatty acid (SCFA) acetate is also produced in the rumen by microbial fermentation and can be used by the host to synthesize milk fat in the mammary gland. The production of acetate is correlated with ruminal CH4 production. Milk fat can also be synthesized from non-esterified fatty acids (NEFA) and triacylglycerol that originate from endogenous fat stores of dairy cows, especially during times of fat mobilization. This study checked the hypothesis that a higher fat mobilization during early lactation decreases ruminal acetate production by replacing acetate for milk fat synthesis and, thus, decreases synthesis of CH4. Another aim of this study was to test the hypothesis that increases in CH4 yield over the course of lactation are associated with changes in rumen microbial community profile, and that high and low CH4 emitting cows differ in their bacterial and archaeal community structure. A herd of 20 Holstein cows was studied during the course of their first lactation; feed intake and diet composition was monitored. Blood and rumen fluid were repeatedly sampled throughout the trial. Plasma NEFA concentrations were analyzed by photometrical analysis, and rumen SCFA concentrations by gas chromatography. Individual CH4 production was measured in respiration chambers at four times during the observation period. In a subgroup of 9 cows, rumen fluid samples from 3 timepoints during lactation were subjected to DNA extraction and bacterial and archaeal 16S rRNA amplicons were sequenced. The bacterial and archaeal community structures in the rumen fluid were described, and the rumen microbiome composition linked to CH4 yield. Statistical analysis was conducted using repeated measurement ANOVA and Tukey tests, as well as Pearsons’ correlation for selected parameters. Microbial data was further treated with multivariate analyses (PERMANOVA) and Bray-Curtis dissimilarities were determined. Total CH4 production increased significantly over time from an average 208 L/day during the dry period to 516 L/day in late lactation. The level of fat mobilization, expressed as blood plasma NEFA concentrations, and CH4 yield showed an inverse relationship in early lactation (p = 0.002). High mobilizing cows (NEFA > 580 μmol/L) tended to show higher ruminal acetate concentrations than low mobilizing cows (NEFA < 580 μmol/L) only before parturition and not during lactation. Despite a diet composition that was kept as constant as possible throughout the lactation, the microbial community changed significantly over time as indicated by a decrease in species richness and species evenness. However, in late lactation when CH4 yield was highest, no difference in bacterial or archaeal community structure could be detected between the three highest CH4 yielding cows and the three lowest CH4 yielding cows. The ratio of (acetate + butyrate) / propionate in rumen fluid changed significantly with progressing lactation from 3.5 to 4.4, accompanied by an increase in CH4 production from 434.3 L/d to 540.5 L/d. However, no correlation between the concentration of ruminal SCFA and CH4 yield was found. The metabolic state of the animal, especially the degree of fat mobilization during times of negative energy balance, had an impact on CH4 yield. Also, the microbial community composition in the rumen and its metabolic network is adaptable and changes over time. However, in this study individual microorganisms could not be identified to serve as predictor for CH4 emission from dairy cows at the moment. Rather, shifts in the microbial communities as a whole appear to be responsible for the changes in CH4 yield.:1 Introduction 1 2 Background 2 2.1 Greenhouse Gases 2 2.2 Dairy cows and their importance to food production 3 2.3 Rumen functions 5 2.3.1 Anatomy and Physiology 5 2.3.2 Rumen microbes 7 2.3.2.1 Bacteria 8 2.3.2.2 Archaea 11 2.3.3 Short-chain fatty acids 12 2.3.4 Methane formation 15 2.4 Interrelationship between methane and host animal physiology 15 2.4.1 Physiologic aspects affecting methane formation 15 2.5 Effects of feed composition and feed contents on methane production 16 2.5.1 Relationship of ruminal short-chain fatty acids and methane production 17 2.5.2 Milk fatty acids to estimate methane emission 19 2.6 Description of methods 20 2.6.1 Methane Measurement 20 2.6.2 Sampling of rumen contents 21 2.6.3 Methods to identify microbes 22 2.7 Objective and realization of the studies 23 3 Publications 26 3.1 First Publication 26 3.1.1 Supplement first Publication 40 3.2 Second Publication 42 3.2.1 Supplement second Publication 56 4 Discussion 60 4.1 Assessment of experimental design 60 4.1.1 Animals 60 4.1.2 Feed 61 4.1.3 Rumen fluid 61 4.1.4 Blood and milk metabolites 62 4.2 Assessment of results 62 4.2.1 Variance of methane emissions 62 4.2.2 Rumen short-chain fatty acids and methane 65 4.2.3 Acetate in the cows’ metabolism and methane production 66 4.2.4 Fat mobilization in early lactation 67 4.2.5 NEFA in the context of metabolism 68 4.2.6 Rumen microbes 69 4.2.6.1 Microbial community change over time 70 4.2.6.2 Community differences between individuals 71 4.2.6.3 Relationship between microbes and methane production levels 72 4.2.7 Further considerations 74 5 Conclusions 75 6 Summary 77 7 Zusammenfassung 79 8 References 81

Le protéasome est responsable de la dégradation régulée d'une majeure partie des protéines intracellulaires. Cette machinerie multimoléculaire est composée d'un cœur catalytique, le protéasome 20S, qui peut être activé par plusieurs types de protéines régulatrices, en particulier la particule régulatrice 19S ou PA700, les complexes heptamériques formés par les membres de la famille 11S (ou PA28) et PA200. Au cours de ce travail, nous nous sommes focalisés sur PA28gamma, un régulateur nucléaire du protéasome, qui active la dégradation de plusieurs substrats par le protéasome 20S de façon indépendante de l'ubiquitine et de l'ATP. Malgré de multiples études montrant l'implication de PA28gamma dans de nombreux processus cellulaires essentiels tels que le cycle cellulaire, la prolifération, l'apoptose, l'architecture nucléaire, la dynamique de la chromatine, les infections virales et la réponse au stress, ses fonctions exactes ne sont pas encore comprises. De plus, les mécanismes impliqués dans la régulation de l'activité de PA28gamma et de son association avec le protéasome 20S restent mystérieux. Une analyse SILAC des partenaires d'interaction de PA28gamma endogène a révélé l'existence d'un nouveau facteur, non caractérisé, que nous avons appelé PIP30 (PA28gamma Interacting Protein 30 kDa). Le gène PIP30 contient un domaine très conservé chez les Eucaryotes. Nous avons produit et purifié la protéine PIP30 recombinante et montré qu'elle est faiblement structurée, malgré le fait qu'elle puisse se dimériser. Nous avons confirmé, aussi bien in vitro qu'in cellulo, que PIP30 interagit directement et spécifiquement avec PA28gamma. En analysant la co-immunoprécipitation de PA28gamma avec différents mutants tronqués de GFP-PIP30, nous avons pu identifier la séquence de PIP30 responsable de l'interaction avec PA28gamma dans sa partie C-terminale. Nous essayons maintenant d'identifier la séquence de PA28gamma impliquée dans la liaison de PIP30 et de cristalliser le complexe PA28gamma/PIP30. L'élaboration d'un anticorps anti-PIP30 « maison » nous a permis de montrer que PIP30 est une protéine nucléaire stable. Son niveau d'expression diminue en réponse à la déplétion de PA28gamma, ce qui suggère que PIP30 est stabilisée par son interaction avec PA28gamma in cellulo. Nous avons démontré in vitro que PIP30 inhibe partiellement l'activation médiée par PA28gamma des activités de type chymotrypsine et caspase, mais pas trypsine, du protéasome. Cependant, nous avons montré, par une approche ELISA, que PIP30 n'affecte pas la liaison de PA28gamma au protéasome 20S. Par ailleurs, nous avons testé l'effet de PIP30 sur la dégradation de p21 par le complexe PA28gamma/protéasome 20S et observé que PIP30 augmente la vitesse de dégradation de p21 dans ce test. Nos tentatives pour élucider la fonction exacte de PIP30 in cellulo n'ont jusqu'ici pas abouti à une conclusion convaincante. L'ensemble de ces résultats suggère que PIP30 pourrait être impliqué dans le recrutement sélectif des substrats de PA28gamma et/ou dans la modulation de l'activation du protéasome par PA28gamma. / The proteasome is responsible for the regulated degradation of most intracellular proteins. This multi-subunit machinery is composed of a common catalytic core, the 20S proteasome, which can be activated by various types of regulators, notably the 19S regulatory particle or PA700, the heptameric complexes formed by the members of the 11S (or PA28) family and PA200. This work has been focused on PA28gamma, a nuclear regulator of the proteasome, which has been shown to activate degradation of several proteasomal substrates in an ATP- and ubiquitin- independent manner. Despite many evidences revealing the involvement of PA28gamma in many essential cellular processes, such as cell cycle progression, proliferation, apoptosis, nuclear architecture, chromatin dynamics, viral infection and stress response, its exact function(s) remain to be understood. In addition, how PA28gamma activity and association to the 20S proteasome are regulated is completely unclear. A SILAC-based analysis of endogenous PA28gamma interaction partners revealed the existence of a novel, completely uncharacterized protein, which we called PIP30 (PA28gamma Interacting Protein 30 kDa). Evolutionary analysis indicates that PIP30 gene contains a domain highly conserved in Eukaryotes, without any alternative splicing or gene duplication evidences. We produced and purified the recombinant PIP30 protein and showed that it is poorly structured, although it is able to make dimers. We confirmed both in vitro and in cellulo that PIP30 directly and specifically interacts with PA28gamma. By analyzing the co-immunoprecipitation of PA28gamma with various GFP-PIP30 truncation mutants, we identified the sequence of PIP30 responsible for PA28gamma binding in its C-terminal part. Ongoing analyses now focus on the identification of PIP30 binding motif on PA28gamma sequence and the crystallization of the PA28gamma-PIP30 complex. Using homemade anti-PIP30 antibodies, we showed that PIP30 is a stable nuclear protein. Its expression level is decreased in response to PA28gamma depletion, suggesting that it is stabilized by its interaction with PA28gamma in cellulo. We demonstrated in vitro that PIP30 partially inhibits PA28gamma-mediated activation of the chymotrypsin- and caspase-, but not the trypsin-like, activities of the proteasome. However, we showed by an ELISA-based approach that PIP30 does not affect PA28gamma binding to 20S. Considering the limitations of probing proteasome activity with small fluorogenic substrates, we tested the effect of PIP30 on the PA28gamma-dependent proteasomal degradation of in vitro translated p21, a known protein substrate of PA28gamma. We unexpectedly found that PIP30 enhanced the rate of p21 degradation. Our attempts to elucidate the exact functions of PIP30 in cellulo were unsuccessful so far. Altogether, our results suggest that PIP30 could be involved in the selective recruitment of PA28gamma protein substrates and/or modulate PA28gamma-mediated proteasome activation.

Made available in DSpace on 2014-08-20T14:37:50Z (GMT). No. of bitstreams: 1 tese_elizabeth_schwegler.pdf: 1198143 bytes, checksum: 306f08a6253b951c8751a8eba9c7194c (MD5) Previous issue date: 2012-08-15 / The peripartum in dairy cows (three weeks before and three weeks after calving) is characterized by large changes in physiological demands in the animal where management practices, particularly nutrition, strongly influences the incidence of peripartum disorders and subsequent milk production. Most of the studies in that period in dairy cows are focused on confined systems where milk production is high. Therefore, the aim of this study was to assess metabolic markers associated with the occurrence of clinical and subclinical mastitis and insulin resistance in dairy cows in semi extensive system. Our study was divided into two major experiments with the following hypothesis: 1) medium milk production primiparous cows in semi extensive system have predictive metabolic markers of clinical and subclinical mastitis in the prepartum, 2) medium milk production pluriparous dairy cows with low rate of glucose metabolism during the prepartum, which is an indicative of insulin resistance, have higher minerals excretion in the postpartum period. In experiment 1, blood concentrations of NEFA in the prepartum period were higher, in contrast, phosphorus and glucose were lower (P <0.05) in animals with clinical mastitis postpartum. In experiment 2, pluriparous dairy cows with low rate of glucose metabolism in the prepartum had higher urinary calcium excretion in both prepartum and postpartum periods, and also the highest NEFA concentration in the prepartum period (P <0.05). The blood concentrations of calcium in dairy cows with high rate of glucose metabolism in the postpartum was elevated from the prepartum period and remained in the postpartum (P <0.05). Dairy cows kept in semi extensive system with moderate production level had predictive markers of mastitis in the prepartum, as previously demonstrated by other authors in more intensified systems of higher requirements for the animal. The highlighted marker was NEFA, however, in the second study it was shown in higher concentrations in the pluriparous dairy cows with low rate of glucose metabolism at prepartum, emphasizing the importance of this marker also in the insulin resistance. / O periparto nas vacas leiteiras (três semanas anteriores e as três posteriores ao parto) é caracterizado por grandes mudanças nas demandas fisiológicas do animal, sendo que, as práticas de manejo, principalmente nutricionais, influenciam intensamente a incidência de desordens no periparto e a subsequente produção de leite. A maioria dos estudos neste período na vaca leiteira é em sistemas confinados de alta produção leiteira, por essa razão o objetivo desta tese foi identificar marcadores metabólicos preditivos com a ocorrência de mastite clínica e subclínica e com a excreção de minerais em vacas leiteiras em sistema semi-extensivo. Nosso estudo foi estratificado em dois trabalhos com as seguintes hipóteses: 1) vacas leiteiras primíparas de média produção em sistema semi-extensivo possuem marcadores metabólicos preditivos de mastite clínica e subclínica no pré-parto; 2) vacas leiteiras pluríparas de média produção com menor taxa de metabolização de glicose, que é um indicativo de resistência da insulina, no pré-parto possuem maior excreção de minerais no pós-parto. No experimento 1 as concentrações sanguíneas de NEFA no pré-parto foram maiores e de fósforo e glicose menores (P<0,05), em animais com mastite clínica no pós-parto. No experimento 2 as vacas leiteiras pluríparas com menor taxa de metabolização de glicose no pré-parto apresentaram maior excreção urinária do cálcio no pré e pós-parto, e ainda, o NEFA mais alto no pré-parto (P<0,05). As concentrações sanguíneas de cálcio em vacas leiteiras com maior taxa de metabolização de glicose no pós-parto foi elevada desde o pré parto, mantendo-se no pós (P<0,05). Nas condições estudadas, em vacas leiteiras mantidas em sistema semi-extensivo de mediana produção foi observado que há marcadores preditivos de mastite já no pré-parto, como previamente comprovado por outros autores em sistemas mais intensificados de maiores exigências para o animal. O marcador em destaque foi o NEFA, sendo que o mesmo no segundo estudo se apresentou de forma mais elevada nas vacas leiteiras pluríparas com menor taxa de metabolização de glicose no pré-parto, enfatizando a importância deste marcador também na resistência à insulina.

Made available in DSpace on 2014-08-20T14:38:48Z (GMT). No. of bitstreams: 1 dissertacao_diego_andres_velasco_acosta.pdf: 554045 bytes, checksum: 4c51ec170f50997dc62e7caef5c6f9d2 (MD5) Previous issue date: 2012-02-28 / Negative energy balance (NEB) has been associated with changes in hormones that support follicular development in early lactation. Prepartum recombinant bovine somatotropin (rbST) injection led to reduced non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) in the early postpartum period. Therefore, prepartum administration of somatotropin may result in metabolic changes that benefit the energetic balance of the postpartum dairy cows. The aim of this study was to determine the effect of prepartum injections of somatotropin in dairy heifers with high BCS on the markers of NEB, resumption of ovarian activity and milk production. Holstein heifers (n=20) with high body condition score (scale 1-5; BCS>3.5) were randomly divided into two groups: somatotropin group (n=10) that received two doses of 500 mg of rbST at -28 and -14 days from calving; and control group (n=10) that received applications of placebo in a similar manner to somatotropin group. Blood samples were collected weekly from 28 to 42 d after calving to evaluate BHBA and NEFA concentrations. Follicular development was also monitored via ultrasound examinations starting at days 10 postpartum and every 3 days until ovulation. Prepartum somatotropin administration did not affect (P =0.28) the moment of the first postpartum ovulation. The proportion of cows ovulating the first follicular wave postpartum was not different between groups (P=0.49). There was no effect (P>0.05) of prepartum treatment on the diameter of the first postpartum dominant follicle. There was a interaction days by group for plasma NEFA concentrations during the prepartum period (P= 0.03) and a tendency for the day (P=0.07) but no effect on the group (P=0.35). There was only an effect of the week (P<0.001) for postpartum NEFA concentrations. Postpartum NEFA concentration was higher at days 0, 7 and 14 than at 21, 28, 35 and 42 (P<0.05). For pre and postpartum BHBA there was not effect of days (P>0.05), group (P>0.05) or days by group (P=0.05). In conclusion, prepartum somatotropin treatment in dairy heifers with high body condition did not improve parameters that indicate that these heifers had a less severe NEB during the early postpartum period,had not affect development of the first follicular wave in the postpartum period and milk production. / O balanço energético negativo (BEN) tem sido associado com alterações nos hormônios que auxiliam o desenvolvimento folicular no início da lactação. A aplicação pré-parto de somatotropina recombinante bovina (rbST) pode levar a uma redução sérica dos ácidos graxos não esterificados (AGNE) e β-hidroxibutirato (BHBA) no período pós-parto. Portanto, a administração pré-parto de somatotropina pode resultar em alterações metabólicas que beneficiam o equilíbrio energético da vaca leiteira após o parto. O objetivo deste estudo foi determinar o efeito da administração pré-parto de somatotropina em novilhas leiteiras com elevado escore de condição corporal (ECC) sobre marcadores BEN, a retomada da atividade ovariana e produção de leite. Foram utilizadas novilhas da raça Holandês (n = 20) com ECC elevada (Escala 1-5; ECC > 3,5), foram divididos aleatoriamente em dois grupos: grupo somatotropina (n = 10) que recebeu duas aplicações de 500 mg de rbST nos dias -28 e -14 referente ao parto e grupo controle (n = 10) que recebeu aplicações de placebo no mesmo local e forma do grupo somatotropina. Amostras de sangue foram coletadas entre os dias -28 a 42 relativos ao parto para a avaliação das concentrações de BHBA e AGNE. O desenvolvimento folicular também foi monitorado através de exames de ultrassom a partir do dia 10 pós-parto, e cada 3 dias até o momento da primeira ovulação. A administração pré-parto de somatotropina não afetou (P = 0,28) o momento da primeira ovulação pós-parto. A proporção de vacas que ovularam na primeira onda folicular pós-parto não foi diferente entre os grupos (P = 0,49). Não houve efeito (P> 0,05) do tratamento pré-parto de somatotropina sobre o diâmetro do folículo dominante da primeira onda folicular pós-parto. Houve interação entre o dia e grupo para as concentrações plasmáticas de AGNE durante o período pré-parto (P = 0,03) e uma tendência para o dia (P = 0,07), mas nenhum efeito sobre o grupo (P = 0,35). Houve apenas um efeito da semana (P <0,001) para as concentrações de AGNE no pós-parto. As concentrações de AGNE pós-parto foram maiores nos dias 0, 7 e 14 do que em 21, 28, 35 e 42 (P <0,05). Para BHBA pré e pós-parto não houve efeito de dias (P> 0,05), grupo (P > 0,05) ou dias por grupo (P>0,05). Em conclusão, o tratamento pré-parto de somatotropina em novilhas leiteiras com condição corporal elevada não melhorou os parâmetros que indicam que estas novilhas tiveram um BEN menos grave durante o período pós-parto, não teve efeito sobre o desenvolvimento da primeira onda folicular pós-parto e produção leiteira.

Current methods of energy restriction are not successful for achieving long-term weight loss and maintenance for the majority of individuals. As a result, the prevalence of obesity and obesity related diseases continue to increase. This calls for the development of novel lifestyle interventions to combat the obesity epidemic. Hunger has been highlighted as a major factor influencing the long-term success of weight management methods and therefore how a given dietary intervention affects the appetite regulatory system may dictate the success of the diet by augmenting long-term adherence. In addition, the effect of a given dietary intervention on exercise may determine its suitability for exercising individuals and may influence the energy deficit that can be achieved by the diet. This thesis investigated the acute effects of two novel methods of dietary restriction; breakfast omission and severe energy restriction. The main aims for this thesis were to determine the effect of these methods of energy restriction on ad-libitum energy intake, subjective appetite sensations, and peripheral concentrations of hormones involved in appetite regulation. In addition, this thesis also investigated the effects of these methods of energy restriction on metabolism and glycaemic control at rest, and performance and perceived exertion during exercise. This work found that moderate and severe energy deficits induced by breakfast omission and 24 h of severe energy restriction, respectively, resulted in either no (Chapter VIII) or partial (Chapters IV and VII) energy intake compensation over the subsequent 24-48 h. Subjective appetite was increased during (Chapters IV, V, VII and VIII) and shortly after (Chapter VII) energy restriction, but this effect was transient and was offset after an ad-libitum (Chapters IV and VII) or standardised (Chapters V and VIII) meal. In addition, none of the work presented in this thesis demonstrated an appetite hormone response to energy restriction that was indicative of compensatory eating behaviour. Compared to breakfast omission, breakfast consumption resulted in an increased in resting energy expenditure and carbohydrate oxidation, with a concurrent reduction in fat oxidation during the morning. However, there were no differences after lunch (Chapter V). In response to a standardised breakfast, resting energy expenditure was suppressed (Chapter VII) or not different (Chapter VIII) the following morning, after 24 h severe energy restriction compared to energy balance. Plasma NEFA and fat oxidation was greater, carbohydrate oxidation was reduced, and postprandial insulin sensitivity was impaired in the after 24 h severe energy restriction (Chapter VI, VII and VIII). In Chapter IV, omission of breakfast in the morning was shown to reduce exercise performance in evening, even after provision of an ad-libitum lunch 4 h before. However, there was no difference in perception of effort during steady state exercise, independent of breakfast consumption or omission in the morning (Chapters IV and V). Collectively, breakfast omission and 24 h severe energy restriction reduce energy intake and promote an appetite regulatory response conducive to maintenance of a negative energy balance. Chronic intervention studies are now required to confirm whether these effects persist after long-term practice of these dietary interventions.

O objetivo deste trabalho consistiu em esclarecer os mecanismos bioquímicos e hormonais da ação da somatotropina recombinante bovina (r-bST), recomendada no tratamento de toxemia da prenhez (TP). Para tal, foram realizados dois experimentos nos quais induziram-se quadros de hipercetonemia ou hiperlipidemia em 39 ovelhas com elevada condição corporal tratadas ou não previamente com 160 mg de um produto comercial de r-bST, pela via subcutânea. A hipercetonemia foi provocada pela infusão IV de 5 mM/kg P.V. de beta hidroxibutirato (BHB) no decorrer de duas horas e a hiperlipidemia com injeção IV de solução 20% de óleo de peixe, perfazendo 0,54g de triacilglicerol/kg P.V. no decorrer de quatro dias. Essas quantidades infundidas lograram provocar os quadros esperados. No primeiro experimento, o tratamento com r-bST promoveu aumento significativo da gliconeogênese por meio da conversão de BHB em glicose, maiores teores de insulina, com consequente aumento da resistência insulínica, incremento na concentração de IGF1 que levou a mobilização de nitrogênio ureico para os depósitos e maior atividade de GGT. No segundo experimento, o rbST gerou destacado aumento da gliconeogênese, pela metabolização dos AGNEs e de BHB em glicose, com maior destaque aos AGNEs, elevação dos teores de insulina e do percentual de resistência insulínica, e maior atividade da AST e GGT e bilirrubinal direta e total. Assim, conclui-se que a atuação farmacológica do r-bST se baseia na vigorosa capacidade do aumento da gliconeogênese, diminuindo concomitantemente os nefastos catabólitos (AGNEs e BHB) no processo da TP. Foi realizado ainda um experimento subsidiário para comparar a capacidade diagnóstica da detecção de BHB, em sangue total, entre o kit diagnóstico enzimático (Randox®), padrão ouro, e tiras reativas comerciais (Precision Xceed®) lidas em dosímetro portátil. Foram obtidas 247 amostras de sangue das ovelhas nas quais tinham sido infundida solução de BHB, no experimento um. Duas faixas de acetonemia foram consideradas: de 0,7 a 1,5 mM caracterizada como cetose inaparente e acima deste valor como cetose clínica. A correlação entre os métodos foi alta (r = 0,98 ; P < 0,001). Na cetose inaparente as tiras reativas apresentaram sensibilidade de 0,97 e especificidade de 0,89 e na cetose aparente sensibilidade de 0,99 e especificidade de 0,75, respectivamente. As tiras reativas podem ser empregadas eficientemente no diagnóstico precoce de casos de TP, com custo baixo e rápida leitura ao pé dos animais. / The aim of this study was to clarify the biochemical and hormonal mechanisms of action of recombinant bovine somatotropin (r-bST), recommended in the treatment of pregnancy toxemia (PT). Two experiments were conducted inducing hyperlipidemia or hyperketonemia in 39 high body condition sheep pretreated or no with 160 mg of a commercial product of rbST subcutaneously. The hyperketonemia was caused by IV infusion of 5 mM / kg BW of beta-hydroxybutyrate (BHB) during two hours and hyperlipidemia with IV injection of a 20% fish oil solution corresponding to 0.54 g of triglyceride / kg BW in four days course. These infusions successfully caused the expected effects. In the first experiment, treatment with rbST caused a significant increase of gluconeogenesis by converting BHB into glucose, higher levels of insulin, with consequent increased insulin resistance, increase in the concentration of IGF1 leading to mobilization of urea nitrogen for deposits and increased activity of GGT. In the second experiment, the r-bST greatly increased gluconeogenesis generated by metabolism of non esterified fatty acids (NEFA), BHB and glucose, with emphasis on NEFA, elevated levels of insulin and the percentage of insulin resistance, and increased activity of AST and GGT and bilirrubin direct and total. In conclusion, the pharmacological action of r-bST is based on vigorous increase capacity on gluconeogenesis, concomitantly reducing the adverse catabolites (NEFA and BHB) in the PT process. An additional experiment was performed to compare the diagnostic capability of detecting BHB in whole blood, between the enzymatic diagnostic kit (Randox®), considered gold standard, and commercial test strips (Precision Xceed®) read in hand-held meter. 247 sheep blood samples were obtained in the first experiment in which BHB solution had been infused. Two ketonemic tracks were considered: 0.7 to 1.5 mM characterized as unapparent ketosis and above this value as a clinical ketosis. The correlation between the methods was high (r = 0.98, P <0.001). In test strips method, ketosis unapparent showed sensitivity of 0.97 and specificity of 0.89 and in ketosis apparent sensitivity of 0.99 and specificity of 0.75, respectively. The test strips can be used efficiently in the early diagnosis of cases of TP, with fast reading and low cost.

O objetivo deste trabalho consistiu em esclarecer os mecanismos bioquímicos e hormonais da ação da somatotropina recombinante bovina (r-bST), recomendada no tratamento de toxemia da prenhez (TP). Para tal, foram realizados dois experimentos nos quais induziram-se quadros de hipercetonemia ou hiperlipidemia em 39 ovelhas com elevada condição corporal tratadas ou não previamente com 160 mg de um produto comercial de r-bST, pela via subcutânea. A hipercetonemia foi provocada pela infusão IV de 5 mM/kg P.V. de beta hidroxibutirato (BHB) no decorrer de duas horas e a hiperlipidemia com injeção IV de solução 20% de óleo de peixe, perfazendo 0,54g de triacilglicerol/kg P.V. no decorrer de quatro dias. Essas quantidades infundidas lograram provocar os quadros esperados. No primeiro experimento, o tratamento com r-bST promoveu aumento significativo da gliconeogênese por meio da conversão de BHB em glicose, maiores teores de insulina, com consequente aumento da resistência insulínica, incremento na concentração de IGF1 que levou a mobilização de nitrogênio ureico para os depósitos e maior atividade de GGT. No segundo experimento, o rbST gerou destacado aumento da gliconeogênese, pela metabolização dos AGNEs e de BHB em glicose, com maior destaque aos AGNEs, elevação dos teores de insulina e do percentual de resistência insulínica, e maior atividade da AST e GGT e bilirrubinal direta e total. Assim, conclui-se que a atuação farmacológica do r-bST se baseia na vigorosa capacidade do aumento da gliconeogênese, diminuindo concomitantemente os nefastos catabólitos (AGNEs e BHB) no processo da TP. Foi realizado ainda um experimento subsidiário para comparar a capacidade diagnóstica da detecção de BHB, em sangue total, entre o kit diagnóstico enzimático (Randox®), padrão ouro, e tiras reativas comerciais (Precision Xceed®) lidas em dosímetro portátil. Foram obtidas 247 amostras de sangue das ovelhas nas quais tinham sido infundida solução de BHB, no experimento um. Duas faixas de acetonemia foram consideradas: de 0,7 a 1,5 mM caracterizada como cetose inaparente e acima deste valor como cetose clínica. A correlação entre os métodos foi alta (r = 0,98 ; P < 0,001). Na cetose inaparente as tiras reativas apresentaram sensibilidade de 0,97 e especificidade de 0,89 e na cetose aparente sensibilidade de 0,99 e especificidade de 0,75, respectivamente. As tiras reativas podem ser empregadas eficientemente no diagnóstico precoce de casos de TP, com custo baixo e rápida leitura ao pé dos animais. / The aim of this study was to clarify the biochemical and hormonal mechanisms of action of recombinant bovine somatotropin (r-bST), recommended in the treatment of pregnancy toxemia (PT). Two experiments were conducted inducing hyperlipidemia or hyperketonemia in 39 high body condition sheep pretreated or no with 160 mg of a commercial product of rbST subcutaneously. The hyperketonemia was caused by IV infusion of 5 mM / kg BW of beta-hydroxybutyrate (BHB) during two hours and hyperlipidemia with IV injection of a 20% fish oil solution corresponding to 0.54 g of triglyceride / kg BW in four days course. These infusions successfully caused the expected effects. In the first experiment, treatment with rbST caused a significant increase of gluconeogenesis by converting BHB into glucose, higher levels of insulin, with consequent increased insulin resistance, increase in the concentration of IGF1 leading to mobilization of urea nitrogen for deposits and increased activity of GGT. In the second experiment, the r-bST greatly increased gluconeogenesis generated by metabolism of non esterified fatty acids (NEFA), BHB and glucose, with emphasis on NEFA, elevated levels of insulin and the percentage of insulin resistance, and increased activity of AST and GGT and bilirrubin direct and total. In conclusion, the pharmacological action of r-bST is based on vigorous increase capacity on gluconeogenesis, concomitantly reducing the adverse catabolites (NEFA and BHB) in the PT process. An additional experiment was performed to compare the diagnostic capability of detecting BHB in whole blood, between the enzymatic diagnostic kit (Randox®), considered gold standard, and commercial test strips (Precision Xceed®) read in hand-held meter. 247 sheep blood samples were obtained in the first experiment in which BHB solution had been infused. Two ketonemic tracks were considered: 0.7 to 1.5 mM characterized as unapparent ketosis and above this value as a clinical ketosis. The correlation between the methods was high (r = 0.98, P <0.001). In test strips method, ketosis unapparent showed sensitivity of 0.97 and specificity of 0.89 and in ketosis apparent sensitivity of 0.99 and specificity of 0.75, respectively. The test strips can be used efficiently in the early diagnosis of cases of TP, with fast reading and low cost.