The aim of this diploma thesis was to evaluate the occurrence of ketones and free fatty acids in milk of high-yielding cows. Ketosis is a very common metabolic disorder in dairy cows resulting in reduced milk production, fertility disturbance and more frequent occurrences of other diseases. Ketosis is not only a serious health problem, but also an economic problem, that can be avoided by balancing the feeding dose respecting the physiological needs of energy delivery after birth and at the beginning of lactation of high-yielding cows. The monitoring was carried out in 2016 at Chyšná, which is one of the centers of Agribusiness Košetice a.s. Owerall were 1407 cows monitored during the 6 utility tests (from January to June 2016). The occurrence of subclinical ketoses was defined by the content of ketones in milk (acetone and beta-hydroxybutyric acid). Critical was the period of the first 12 days of lactation, when the occurence of subclinical ketoses was the highest, the occurence of this disease was lower than in other breeds. At Chyšná breeding, the low incidence of subclinical ketoses is mainly influenced by zootechnical work and by working with the feed consultant and the company for the production of compound feeds. As a precautionary process, regular feed analysis and examination of free fatty acids and ketones in milk can be recommended.

La fertilité des vaches laitières s’est beaucoup dégradée au cours des dernières décennies, tant à l’échelle nationale qu’internationale. L’objectif poursuivi est d’apporter un éclairage épidémiologique sur les relations dynamiques entre la conception (réussite à l’insémination artificielle ; IA), les mammites subcliniques et les déséquilibres métabolico-nutritionnels que sont la cétose subclinique et les déséquilibres azotés de la ration. Un premier chapitre bibliographique dresse l’état de l’art des liens entre performances de reproduction et (i) mammites, (ii) cétose subclinique et (iii) excès de protéines dégradables, en identifiant clairement les interactions et associations croisées entre ces différents composants, et en quantifiant autant que possible ces liens. Les trois parties suivantes mobilisent des données exhaustives du contrôle laitier français sur la période 2008-2012, qui ont été confrontées aux données d’IA. La variable d’intérêt retenue est la conception après IA, soit pour la première IA (IA1) ou pour toutes les IA. Les variations des concentrations de cellules somatiques (CCS) autour de l’IA ont été utilisées pour décrire la dynamique des mammites subcliniques autour de l’IA, selon 4 classes (Bas-Bas, Bas-Haut, Haut-Bas et Haut-Haut) et pour différents seuils de CCS. Le statut de cétose subclinique a été évalué grâce aux taux butyreux et protéique du lait. Les concentrations d’urée du lait sont utilisées pour caractériser les déséquilibres azotés. Le second chapitre propose une description succincte des résultats de production et de reproduction des troupeaux bovins laitiers français sur la période d’étude. Le troisième chapitre focalise sur l’interaction entre les CCS et la cétose subclinique dans un modèle de régression de Poisson expliquant la conception. Les chances de conception à l’IA1 sont réduites de 14% (Risque relatif = 0,86 [IC 95%=0,85–0,87]) pour les groupes Bas-Haut et Haut-Haut, comparé au groupe Bas-Bas, et de 3 à 17 %, selon les définitions retenues, lors de cétose subclinique comparé à son absence. Les résultats identifient et quantifient clairement l’interaction entre la cétose subclinique et les mammites subcliniques dans leur association avec la conception : la baisse de la conception est jusqu’à 2 fois supérieure lors de la présence simultanée d’une augmentation des CCS et d’une cétose subclinique par rapport à la situation où il y a seulement augmentation des CCS. Le quatrième chapitre, mobilisant des méthodes proches de celles du chapitre précédent, montre que la baisse de la concentration d’urée dans le lait autour de l’IA (en dessous de 150 mg/kg, 2,6 mmol/L) est associée à une baisse de la conception de 5 à 9% (Risque relatif = 0.91 (IC95%=0,87-0,96)]) par rapport à des concentrations d’urée du lait qui restent stables (250 - 450 mg/kg ou 4,3-7,7 mmol/L). Ceci révèle l’importance de la stabilité du métabolisme azoté autour de la conception, y compris pour des variations d’urée du lait ou du sang à la baisse, alors que seule la hausse de la concentration d’urée était identifiée comme un facteur de risque de dégradation des résultats de reproduction jusqu’alors. La dernière partie permet de mettre en perspective ces éléments originaux. Une des principales limites de ces travaux est la définition imprécise de la cétose subclinique réalisée à partir des taux du lait ; les résultats actuels bénéficieraient d’une actualisation avec un indicateur plus précis de ce trouble. Ces résultats soutiennent qu’une inflammation locale peut affecter la réponse de l’ensemble de l’organisme et altérer les fonctions d'autres organes dans les semaines qui suivent son apparition. Ils illustrent la complexité et les interactions entre les différents troubles chez un même animal. Par ailleurs, maintenir des concentrations basses de l’urée est légèrement pénalisant pour la conception, et ne garantit pas de meilleurs résultats de reproduction. / Reproductive performances of dairy cows are recognized as a key parameter for the profitability of dairy farms, but they are getting worse continuously in many countries during the last decades. Infectious and nutritional disorders are possible contributors to these changes. Mammary infection, nitrogen imbalance and metabolic disorders have been reported to be negatively associated with conception, but their interactions and dynamics are not fully understood. The objective of the present work is to better describe the relationship between fertility, udder health, subclinical ketosis (SCK) and nitrogen imbalance accounting for the temporal variations of these events and their interactions. The first section reviews the link between reproductive performances and (i) mastitis, (ii) metabolic disorders and (iii) nitrogen imbalance in dairy cows in order to clarify the complex interaction among these events. The three other sections are based on exhaustive data from the national French dairy milk improvement system and data on the artificial inseminations (AI) from 2008 to 2012. Fertility was explained as conception at the first (AI1) or at all AI. The udder health status was evaluated through the somatic cells counts (SCC). Several proxies based on the milk fat and protein contents were proposed to define SCK. Milk urea concentration was used to investigate the exposure to nitrogen imbalance. The second section aims to describe the actual situation of milk production and reproduction in French dairy herds. The third section focuses on the interaction between SCC and SCK and their association with conception. On average, the risk of conception at AI1 was 14% lower when the SCC increased or remained high within 40 days before and after AI (Relative risk [and 95% CI] = 0.86 [0.85–0.87]), compared to low SCC before and after AI. The reduction in conception rate associated with SCK (fat and protein contents changes) varied from 3% to 17% depending on the SCK proxy used. Including the interaction term SCC*SCK clearly showed that the association of increased SCC around AI with conception was modified by the presence of SCK. A cow that already has SCK and experiences an increase in SCC around or after AI exhibits up to 2 times further decrease in conception, compared with a cow with a high SCC and no SCK. The fourth section, using similar methods as the previous one, shows that low milk urea concentrations after AI are negatively associated with conception. Cows with a low urea (< 150 mg/kg, 2.6 mM) after AI have a reduced conception (Relative risk RR [and 95% CI] = 0.96 [0.94–0.99]) compared to cows with intermediate urea (250-450 mg/kg, 4.3 - 7.7 mM) after AI. Furthermore, the risk of conception was 5 to 9% lower (relative risk [and 95% CI] = 0.91 [0.87–0.96]) when urea concentrations decrease from intermediate before to low after AI, compared with cows with constant intermediate urea values. This work revealed that a decrease in urea from intermediate (before AI) to low (after AI) is a risk factor for conception failure in addition to the previously known risk factor that is nitrogen excess. The final section aims to highlight the perspectives of these results. The definition of SCK used in this work was identified as the main limitation and the present results would benefit from an update with a better indicator of this disorder. The present work supports that a local inflammation may affect the entire body response and alters the functions of other organs like those of the reproductive tract. Furthermore, maintaining low urea concentrations does not provide any particular advantage and might be negatively associated with conception.

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The aim of this diploma thesis is to evaluate the data about holstein dairy cow body condition at calving and to review its effect on ketones blood level and its relation to incidence of ketosis. The monitoring took place in a selected dairy farm from October 2017 to February 2019. Body condition of 280 cows was evaluated on the day of calving by assessing the body condition score (BCS) and later on 10 days in milk the blood level of B-hydroxybutyrate was measured. Furthermore, parity, body weight in calving day, body weight on 10 days in milk, content of milk fat and protein on 10 days in milk and milk yield on 10 days in milk were monitored. Average BCS of the herd was 3,7, the maximum measured BCS value was 5, the minimum was 2. Total incidence of ketosis on herd level was 21,07 % considering ketosis if BHB 1,0 mmol.l-1. 62,7 % of that was a subclinical form of ketosis while the remaining 37,3 % was the clinical form of ketosis. Correlation analysis of the relation between parity and level of BHB in blood showed positive correlation (r=0,15, p=0,00), when cows on higher lactation had higher level of blood BHB. Positive correlation between fat/protein ratio in milk and level of BHB in blood was evaluated by correlation analysis (r=0,31 , p=0,00). The BCS at calving and BHB level on 10 days in milk showed positive correlation (r=0,14 , p=0,02). As far as negative energy balance is taken into account, the positive correlation between BCS at calving and body weight loss was confirmed (r=0,17 , p=0,00). The fact that heavier cows and cows with higher BCS mobilise more body fat reserves was confirmed by positive correlation between the body weight at calving and the body weight loss (r=0,47, p=0,00) and between the body weight at calving and the fat/protein ratio in milk (r=0,27, p=0,00). Body weight at calving had provable positive impact on the milk yield (r=0,19, p=0,00).

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-10T13:39:50Z No. of bitstreams: 1 Rodolfo Jose Cavalcanti Souto.pdf: 1672049 bytes, checksum: 9d25ef84b343968c6b40907e7241f724 (MD5) / Made available in DSpace on 2017-02-10T13:39:50Z (GMT). No. of bitstreams: 1 Rodolfo Jose Cavalcanti Souto.pdf: 1672049 bytes, checksum: 9d25ef84b343968c6b40907e7241f724 (MD5) Previous issue date: 2013-02-25 / Pregnancy disease (PD) occurs mainly in sheep and goats, supplemented with concentrate, whose expression is due to negative energy balance during the transition period in which the females present in the clinical hyperketonemia, hypoglycemia, ketonuria, anorexia and signs neurological can progress to death. This paper aims to conduct a study of the biochemical and hormonal profile in sheep and goats, and pathology of the liver parenchyma and renal function in sheep with PD diagnosis. We used data (case report forms) and biological materials (serum, plasma and tissue) of 45 sheep and 22 goats that were answered in Bovine Clinic, Campus Garanhuns / UFRPE, diagnosed with PD, from January 2007 to October 2012 . The blood analyzes were: urea, creatinine, albumin, total protein, globulin, β-hydroxybutyrate, non-esterified fatty acids (NEFA), glucose, fructosamine, amylase, aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), vitamin B12 , folic acid, cortisol, insulin, and total ionized Ca, P, Ca: P ratio, Mg, Na, K. Cl. Besides the characterization of macro and histopathological findings of the liver and kidneys of sheep that died. Urine was examined in the presence of ketones employing the reagent strip commercial. Sheep subjected to necropsy were collected fragments of liver and kidneys for histopathological analysis. Marked clinical abnormalities were observed in goats with high values for urea, creatinine, NEFA, β-hydroxybutyrate, amylase and cortisol, while total protein, albumin, total calcium, ionized calcium, phosphorus, the calcium and phosphorus, sodium and insulin were found below the reference values for the species. As for glucose, 77.28% of goats were normoglycemic TP and / or hyperglycemic, while only 22.72% were hypoglycemic. The results of the sheep revealed elevated levels of urea, creatinine, glucose, fructosamine, NEFA's, β-hydroxybutyrate, cortisol, chlorine, AST, GGT amylase and folic acid as the variable insulin, total calcium, ionized calcium and potassium were values below the reference values for the species. As for glucose, 82.90% of the sheep were normoglycemic with PD and / or hyperglycemic, while only 17.10% were hypoglycemic. With regard to the macroscopic group of sheep with lower body score showed an increase of the liver, and yellowing and the cutting surface, while the sheep with higher body score these findings were more intense. Kidney was observed on the surface in some pallor of lean animals and light yellowish in animal fat. Regarding histological alterations, it was found in animals with lower body condition, liver vacuolated ranging from rare discreet. As the renal injury, rare tubular vacuolation of epithelial cells were observed in 28,57%. In sheep with higher body condition scores greater percentage of vacuolation in the liver was observed and ranged from moderate to severe. Regarding renal lesions, 33.33% of the animals showed severe lesion vacuolization of tubular epithelial cells. Marked clinical and metabolic changes were observed in goats and sheep PD, in which the increase of the energy profile certain metabolites and hormones are important diagnostic tools for assessing the magnitude of negative energy balance and the clinical condition of the animals. In sheep PD causes significant changes in renal parenchyma, characterized by vacuolization of cells that are associated tubular body condition score of the animal. / A toxemia da prenhez (TP) ocorre principalmente em ovelhas e cabras, suplementadas com concentrado, cuja manifestação se deve a um balanço energético negativo durante o período de transição, no qual as fêmeas apresentam quadro clínico de hipercetonemia, hipoglicemia, cetonúria, anorexia e sinais neurológicos que pode evoluir para a morte. Este trabalho se propõe a realizar um estudo do perfil bioquímico e hormonal em cabras e ovelhas, e anatomopatológico do parênquima hepático e renal em ovelhas com diagnóstico de TP. Foram utilizados dados (fichas clínicas) e material biológico (soro, plasma e tecido) de 45 ovelhas e 22 cabras que foram atendidas na Clínica de Bovinos, Campus Garanhuns/UFRPE, diagnosticadas com TP, no período de janeiro de 2007 a outubro de 2012. As análises realizadas no sangue foram: uréia, creatinina, albumina, proteína total, globulina, β-hidroxibutirato, ácidos graxos não esterificados (AGNE), glicose, frutosamina, amilase, aspartato aminotransferase (AST), gama glutamiltransferase (GGT), vitamina B12, ácido fólico, cortisol, insulina, Ca total e ionizável, P, relação Ca:P, Mg, Na, K. Cl. Além da caracterização dos achados macro e histopatológicos do fígado e rins das ovelhas que vieram a óbito. Na urina foi examinada a presença de corpos cetônicos empregando-se a fita reagente comercial. Das ovelhas submetidas à necropsia foram coletados fragmentos de fígado e rins para análise histopatológica. Marcadas alterações clínicas foram observadas nas cabras, com valores elevados para ureia, creatinina, AGNE, β-hidroxibutirato, amilase e cortisol, enquanto que a proteína total, albumina, cálcio total, cálcio ionizado, fósforo, a relação cálcio e fósforo, o sódio e a insulina encontraram-se abaixo dos valores de referência para a espécie. Quanto à glicemia, 77,28% das cabras com TP eram normoglicêmicas e/ou hiperglicêmicas, enquanto apenas 22,72% eram hipoglicêmicas. Os resultados obtidos das ovelhas revelaram valores elevados de ureia, creatinina, glicose, frutosamina, AGNE’s, β-hidroxibutirato, cortisol, cloro, AST, GGT amilase e ácido fólico enquanto que a variável insulina, cálcio total, cálcio ionizado e potássio estavam com valores abaixo dos valores de referência para a espécie. Quanto à glicemia, 82,90% das ovelhas com TP eram normoglicêmicas e/ou hiperglicêmicas, enquanto apenas 17,10% eram hipoglicêmicas. Quanto aos achados macroscópicos no grupo de ovelhas com menor escore corporal, foi observado aumento do fígado, além de coloração amarelada na superfície e ao corte, enquanto que nas ovelhas com maior escore corporal estes achados foram mais intensos. Nos rins foi observada palidez na superfície em alguns dos animais magros e leve coloração amarelada em animais gordos. Quanto às alterações histológicas, verificou-se, nos animais com menor escore corporal, fígado com vacuolização que variou de rara a discreta. Quanto às lesões renais, raras vacuolizações em células epiteliais tubulares foram observadas em 28,57%. Nas ovelhas com maior escore corporal maior percentual de vacuolização no fígado foi observada e variou de moderada a acentuada. Quanto às lesões renais, 33,33% dos animais apresentaram lesão acentuada de vacuolizações em células epiteliais tubulares. Marcadas alterações clínicas e metabólicas foram observadas em cabras e ovelhas com TP, em que o aumento de certos metabólitos do perfil energético e hormonal tornam-se importantes ferramentas de diagnóstico para avaliar o balanço energético negativo e a magnitude da condição clínica dos animais. Em ovelhas a TP provoca alterações significativas no parênquima renal, caracterizadas por vacuolização de células tubulares e que estão associados ao escore corporal dos animais.

Alexandra Flocke Untersuchung von Stoffwechselparametern und Lipoproteinen im Blutserum von einlings- und zwillingsträchtigen Merino- und Schwarzköpfigen Fleischschafen im peripartalen Zeitraum Medizinische Tierklinik, Veterinärmedizinische Fakultät, Universität Leipzig Eingereicht im Februar 2012 (94 Seiten, 15 Abbildungen, 8 Tabellen, 199 Literaturangaben, 12 Tabellen im Anhang) Schlüsselwörter: Lipoproteine, Schaf, Stoffwechsel, Endotoxin, Ketose Problemstellung: Der Lipoproteinstatus in der kritischen Phase der Hochträchtigkeit wurde beim Schaf bisher nur wenig untersucht. Die Fähigkeit der Lipoproteine, sowohl Lipide zu transportieren und im Körper umzuverteilen, als auch ihre Rolle in der Endotoxinneutralisation machen sie zu einem wichtigen Parameter in stoffwechselbelasteten Situationen. Das Ziel dieser Arbeit ist es, den Lipoproteinstatus zweier verschiedener Leistungsrassen zu analysieren und zu prüfen, ob und inwiefern peripartal Unterschiede bei Ein- und Zwillingsträchtigkeiten bestehen. Zusätzlich werden die Endotoxine der Tiere bestimmt und mögliche Zusammenhänge mit der Stoffwechselsituation und dem Lipoproteinstatus evaluiert. Tiere, Material und Methoden: Untersucht wurden gesunde einlingsträchtige Merinofleischschafe (MFS1), zwillingsträchtige Merinofleischschafe (MFS2) sowie zwillingsträchtige Schwarzköpfige Fleischschafe (SKF2). Im Abstand von je sieben Tagen wurden den Tieren im Zeitraum von der 5. Woche a.p. bis eine Woche p.p. Blutproben aus der V. jugularis externa entnommen. Aus dem Serum wurden die Konzentrationen von β-Hydroxybutyrat (BHB), Glucose, Freie Fettsäuren (FFS), Triacylglycerol (TG), Cholesterol, Bilirubin, Gesamtprotein (alle Hitachi 912), Insulin (RIA-Kit, Firma IBL, Hamburg), α- und β-Lipoproteine (LIPIDOPHOR All In12®, - ohne prä-β-Lipoproteine), freies Endotoxin (LAL-Test) und Anti-Lipid A Antikörper (ALAAK; ELISA) bestimmt. Ergebnisse: Die α-Lipoproteinkonzentration stieg bei den MFS1 vor dem Ablammen signifikant an und erreichte eine Konzentration von = 208,0 ± 40,1 mg/dl (1. Woche a.p.). Die MFS2 wiesen vor dem Lammen einen konstanten Verlauf zwischen = 180,4 ± 31,1 mg/dl (5. Woche a.p.) und = 196,1 ± 42,1 mg/dl (3. Woche a.p.) auf. Die SKF2 zeigten in der 5. Woche a.p. mit = 206,5 ± 39,0 mg/dl eine größtenteils signifikant höhere Konzentration gegenüber den folgenden Untersuchungszeitpunkten. Bei beiden Rassen ließ sich bei den Zwillingsmuttern nach dem Lammen ein signifikanter Abfall der α-Lipoproteinkonzentration auf = 144,3 ± 46,1 mg/dl (MFS2) bzw. = 170,2 ± 48,8 mg/dl (SKF2) feststellen. Es bestand eine gesicherte Korrelation zu Cholesterol. Die β-Lipoproteinkonzentration lag in der 1. Woche p.p. bei den Zwillingsmuttern (MFS2 und SKF2) signifikant niedriger als vor dem Lammen. Die SKF2 wiesen in der 5. und 4. Woche a.p. signifikant niedrigere Konzentrationen als die MFS1 auf. Gesicherte Korrelationen bestanden zu TG, Cholesterol und ALAAK (MFS1). Für die α-Lipoproteinkonzentration wurde ein Referenzbereich von 144,3 – 208,0 mg/dl und für die β-Lipoproteinkonzentration von 13,6 – 28,0 mg/dl bei Muttertieren der beschriebenen Rassen ermittelt. Ausgenommen die Insulinkonzentration in der 1. Woche a.p. bestanden keine signifikanten Differenzen zwischen MFS1 und MFS2. Im Vergleich der Rassen zeigten sich signifikante Differenzen in der Glucosekonzentration in der 2. Woche a.p. und in der Konzentration von Bilirubin in der 3. und 1. Woche a.p.. Die BHB-Konzentration zeigte einen konstanten Verlauf und stieg bei den MFS1 bis auf = 0,96 ± 0,36 mmol/l eine Woche p.p., bei den MFS2 auf = 0,73 ± 0,35 mmol/l und bei den SKF2 auf = 0,63 ± 0,14 mmol/l eine Woche a.p. signifikant an. Gesicherte Korrelationen bestanden zu FFS (MFS2) und Insulin (MFS2; SKF2). Die Glucosekonzentration zeigte sich bei den Zwillingsmuttern nach zunächst abfallenden Konzentrationen in der Hochträchtigkeit eine Woche p.p. signifikant höher ( = 3,63 ± 0,59 mmol/l; MFS2 und = 3,28 ± 0,42 mmol/l; SKF2) als vor dem Lammen. Gesicherte Korrelationen ließen sich zu FFS (MFS1; MFS2), BHB (MFS2) und dem Zeitpunkt der Untersuchung (MFS2; SKF2) feststellen. Die FFS-Konzentration stieg bei den SKF2 zur 1. Woche a.p. auf = 265 ± 131 μmol/l an, fiel eine Woche p.p. signifikant auf = 128 ± 95 μmol/l ab und korrelierte gesichert mit TG. Die TG-Konzentration fiel von der 1. Woche a.p. bis zur 1. Woche p.p. signifikant auf = 0,19 mmol/l (MFS1; SKF2) bzw. = 0,18 ± 0,05 mmol/l (MFS2) ab und korrelierte mit Gesamtprotein (MFS1), Cholesterol (MFS1; MFS2), β-Lipoproteinen (MFS1; MFS2) und freiem Endotoxin (MFS1). Die Cholesterolkonzentration fiel eine Woche p.p. bei den Zwillingsmuttern signifikant ab, bei den MFS1 war der Verlauf konstant. Die Bilirubinkonzentration stieg eine Woche p.p. signifikant an (MFS1; MFS2) und korrelierte mit der Konzentration von freiem Endotoxin. Das Gesamtprotein sank zum Zeitpunkt des Ablammens hin signifikant ab, erreichte jedoch eine Woche p.p. wieder seine Ausgangskonzentration. Eine gesicherte Korrelation konnte mit ALAAK festgestellt werden (MFS2; SKF2). Freies Endotoxin konnte zu jedem Zeitpunkt der Untersuchungsreihe nachgewiesen werden. Dabei lagen die festgestellten Konzentrationen bei x̃ = 0,06 – 1,30 EU/ml. Eine gesicherte Korrelation fand sich mit der Konzentration der ALAAK (MFS2). ALAAK konnten ebenfalls zu jedem Zeitpunkt in Konzentrationen von x̃ = 22,2 - 56,8 (OD-Wert) nachgewiesen werden. Fazit: Die Lipoproteinkonzentrationen in den einzelnen Gruppen zeigten sich a.p. zunächst regulativ den erhöhten peripartalen Belastungen des Lipidstoffwechsels angepasst, p.p. demonstrierte auch die Abnahme der Lipoproteinkonzentrationen das Ende der lipolytischen Aktivität bei den Schafen. Die vorliegenden Ergebnisse der Stoffwechselparameter lassen auf eine stärkere Belastung der Zwillingsmuttern schließen, welche auch die Lipoproteine mit einbezieht. Freies Endotoxin konnte auch bei gesunden Schafen zwar zu jedem Zeitpunkt nachgewiesen, eine gesicherte Korrelation mit der Lipoproteinkonzentration jedoch nicht festgestellt werden.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 4 2.1 Physiologische Gravidität des Schafes 4 2.2 Stoffwechsel von Mutterschafen in der Hochträchtigkeit 5 2.3 Stoffwechselparameter 6 2.3.1 β-Hydroxybutyrat 6 2.3.2 Glucose 7 2.3.3 Insulin 10 2.3.4 Freie Fettsäuren 12 2.3.5 Triacylglycerol 13 2.3.6 Cholesterol 14 2.3.7 Bilirubin 15 2.3.8 Gesamtprotein 17 2.4 Lipoproteine 18 2.4.1 Definition von Lipoproteinen 18 2.4.2 Stoffwechsel der Lipoproteine 21 2.4.2.1 Chylomikronen 21 2.4.2.2 Very-Low-Density-Lipoproteins 22 2.4.2.3 Low-Density-Lipoproteins 23 2.4.2.4 High-Density-Lipoproteins 24 2.4.3 Besonderheiten der Lipoproteine beim Schaf 24 2.5 Endotoxin 27 2.5.1 Definition von Endotoxin 27 2.5.2 Aufbau von Endotoxin 27 2.5.3 Herkunft und Wirkung des Endotoxins 28 2.5.4 Inaktivierung und Elimination des Endotoxins 29 2.5.5 Beziehung zwischen Endotoxin und Fettstoffwechsel 30 3 Tiere, Material und Methoden 32 3.1 Versuchsgut 32 3.2 Versuchsanordnung 33 3.3 Material 34 3.4 Methodik 34 3.4.1 Gesundheitszustand der Versuchsgruppen 34 3.4.2 Bestimmungsmethoden klinisch-chemischer Parameter 34 3.4.3 Bestimmung der Lipoproteine 35 3.4.4 Bestimmung von freiem Endotoxin 37 3.4.5 Bestimmung von Anti-Lipid A Antikörpern 38 3.5 Statistische Auswertung 39 4 Ergebnisse 41 4.1 Tierbestand 41 4.2 Stoffwechselparameter 41 4.2.1 β-Hydroxybutyrat 41 4.2.2 Glucose 44 4.2.3 Insulin 46 4.2.4 Freie Fettsäuren 48 4.2.5 Triacylglycerol 50 4.2.6 Cholesterol 52 4.2.7 Bilirubin 54 4.2.8 Gesamtprotein 56 4.3 Lipoproteine 58 4.3.1 α-Lipoproteine 58 4.3.2 ß-Lipoproteine 60 4.4 Endotoxin 62 4.5 Anti-Lipid A Antikörper 64 5 Diskussion 66 5.1 Versuchsprinzip 66 5.2 Tiere und Versuchsbedingungen 66 5.3 Methodenkritik 67 5.4 Stoffwechselparameter 68 5.4.1 β-Hydroxybutyrat 68 5.4.2 Glucose 71 5.4.3 Insulin 72 5.4.4 Freie Fettsäuren 74 5.4.5 Triacylglycerol 76 5.4.6 Cholesterol 78 5.4.7 Bilirubin 79 5.4.8 Gesamtprotein 81 5.5 Lipoproteine 83 5.5.1 α-Lipoproteine 83 5.5.2 β-Lipoproteine 86 5.6 Endotoxin 87 5.7 Anti-Lipid A Antikörper 89 6 Zusammenfassung 91 7 Summary 93 8 Literaturverzeichnis 95 9 Anhang 116 9.1 Stoffwechselparameter 116 9.1.1 β-Hydroxybutyrat 116 9.1.2 Glucose 117 9.1.3 Insulin 118 9.1.4 Freie Fettsäuren 119 9.1.5 Triacylglycerol 120 9.1.6 Cholesterol 121 9.1.7 Bilirubin 122 9.1.8 Gesamtprotein 123 9.2 Lipoproteine 124 9.2.1 α-Lipoproteine 124 9.2.2 β-Lipoproteine 125 9.3 Endotoxin 126 9.4 Anti-Lipid A Antikörper 127 / Alexandra Flocke Evaluation of metabolic parameters and lipoproteins in the serum of merino and blackhead sheep for meat production with one or two lambs in the peripartal period Large Animal Clinic for Internal Medicine, Faculty of Veterinary Medicine, University of Leipzig Submitted in February 2012 (94 pages, 15 figures, 8 tables, 199 references, 12 tablets in the appendix) Keywords: lipoproteins, sheep, metabolism, endotoxin, ketosis Objective: The lipoprotein status in the critical phase of the peripartal period of sheep has been few reviewed. The ability of lipoproteins to transport and redistribute lipids in the organism and their capacity for endotoxinneutralisation makes them a considerable parameter in metabolic bonded situations. The objective of this research is to show the lipoprotein status of two competitive breeds and to proof if and to what extend differences between ewes with one and two lambs exist. Additionally, the endotoxic situation is assigned and possible correlations with the metabolic system and the lipoprotein status are evaluated. Animals, materials and methods: The research contains healthy merino sheep with one lamb (MFS1), merino sheep with two lambs (MFS2) and blackhead sheep with two lambs (SKF2). At intervals of seven days, blood samples out of the V. jugularis externa were taken from the animals in a period from the 5th week a.p. to one week p.p.. Concentrations in the serum of the following parameters were defined: β-hydroxybutyrat (BHB), glucose, free fatty acids (FFS), triacylglycerol (TG), cholesterol, bilirubin, total protein (all Hitachi 912), insulin (RIA-kit, IBL, Hamburg), α- and β-lipoprotein (LIPIDOPHOR All In12® - without pre-β-lipoprotein), free endotoxin (LAL-test) and anti-lipid A antibodies (ALAAK). Results: The concentration of α-lipoproteins of the MFS1 increased significantly before parturition and finally reached 208,0 ± 40,1 mg/dl (1st Week a.p.). The group of MFS2 showed a constant deviation between = 180,4 ± 31,1 mg/dl (5th Week a.p.) and = 196,1 ± 42,1 mg/dl (3rd week a.p.) before parturition. The SKF2 evidenced with a concentration of = 206,5 ± 39,0 mg/dl in the 5th week a.p. a predominantly significant higher result as during following points in time. Both groups carrying twins showed a significant decrease of the α-lipoprotein concentration down to = 144,3 ± 46,1 mg/dl (MFS2) or = 170,2 ± 48,8 mg/dl (SKF2) after parturition. There were statistically reliable correlations between the α-lipoprotein concentration and cholesterol. The β-lipoprotein concentration from both twin pregnant groups had been significantly lower one week after parturition than before parturition. The results of the SKF2 in week 5 and 4 a.p. were significantly lower than those of the MFS1. Reliable correlations could be determined with TG, cholesterol and ALAAK (MFS1). An α-lipoprotein reference range for ewes of the introduced breeds could be determined from 144,3 - 208,0 mg/dl and for β-lipoproteins from 13,6 - 28,0 mg/dl. There were no significant differences between MFS1 and MFS2 except the concentration of insulin in the 1st week a.p.. In the comparison of the different breeds, significant differences in the concentration of glucose (2nd week a.p.) and bilirubin (3rd week a.p. and 1st week a.p.) could be shown. The concentration of BHB showed a constant deviation and increased significantly to = 0,96 ± 0,36 mmol/l (MFS1; 1st week p.p.), = 0,73 ± 0,35 mmol/l (MFS2, 1st week a.p.) or = 0,63 ± 0,14 mmol/l (SKF2, 1st week a.p.), respectively. Reliable correlations existed with FFS (MFS2) and insulin (MFS2; SKF2). The concentration of glucose of the twin pregnant ewes decreased initially before parturition and increased significantly again in the 1st week p.p. ( = 3,63 ± 0,59 mmol/l; MFS2 and = 3,28 ± 0,42 mmol/l; SKF2). Reliable correlations had been determined with FFS (MFS1; MFS2), BHB (MFS2) and the time of sampling (MFS2; SKF2). Concentrations of FFS increased in the group of SKF2 to = 265 ± 131 μmol/l in the 1st week a.p., decreased again significantly to = 128 ± 95 μmol/l one week p.p. and showed a correlation with TG. The TG concentration decreased significantly from one week a.p. to one week p.p. down to = 0,19 mmol/l (MFS1; SKF2) and = 0,18 ± 0,05 mmol/l (MFS2), respectively. A reliable correlation existed between TG and total protein (MFS1), cholesterol (MFS1; MFS2), β-lipoproteins (MFS1; MFS2) and free endotoxin (MFS1). Both twin pregnant groups had a significant decrease in the cholesterol concentration one week after parturition, in contrast the group of MFS1 showed a constant deviation. The concentration of bilirubin increased significantly one week p.p. (MFS1; MFS2) and showed a reliable correlation with the concentration of free endotoxin. Total protein decreased significantly towards parturition, but reached its initial concentration again one week p.p.. A statistically reliable correlation could be determined with ALAAK (MFS2; SKF2). Free endotoxin could be detected at any point of the exploration. The observed concentrations ranged from x̃ = 0,06 – 1,30 EU/ml. A reliable correlation could be shown with the concentration of ALAAK (MFS2). ALAAK was detected as well in concentrations from x̃ = 22,2 - 56,8 (OD-value) in any taken sample. Conclusion: The lipoprotein concentrations in each group appeared a.p. initially adapted to the peripartal bonds of the increased lipid metabolism. After parturition, the decrease in lipoprotein concentrations, too, demonstrated the end of the lipolytic activity of the ewes. The results of the metabolic parameters lead to the conclusion of a stronger liability of the ewes with twins. This is also valid for the lipoproteins. Free endotoxin could be detected in healthy sheep at any time, but a reliable correlation with the concentration of lipoproteins was not visible.:Inhaltsverzeichnis 1 Einleitung 1 2 Literaturübersicht 4 2.1 Physiologische Gravidität des Schafes 4 2.2 Stoffwechsel von Mutterschafen in der Hochträchtigkeit 5 2.3 Stoffwechselparameter 6 2.3.1 β-Hydroxybutyrat 6 2.3.2 Glucose 7 2.3.3 Insulin 10 2.3.4 Freie Fettsäuren 12 2.3.5 Triacylglycerol 13 2.3.6 Cholesterol 14 2.3.7 Bilirubin 15 2.3.8 Gesamtprotein 17 2.4 Lipoproteine 18 2.4.1 Definition von Lipoproteinen 18 2.4.2 Stoffwechsel der Lipoproteine 21 2.4.2.1 Chylomikronen 21 2.4.2.2 Very-Low-Density-Lipoproteins 22 2.4.2.3 Low-Density-Lipoproteins 23 2.4.2.4 High-Density-Lipoproteins 24 2.4.3 Besonderheiten der Lipoproteine beim Schaf 24 2.5 Endotoxin 27 2.5.1 Definition von Endotoxin 27 2.5.2 Aufbau von Endotoxin 27 2.5.3 Herkunft und Wirkung des Endotoxins 28 2.5.4 Inaktivierung und Elimination des Endotoxins 29 2.5.5 Beziehung zwischen Endotoxin und Fettstoffwechsel 30 3 Tiere, Material und Methoden 32 3.1 Versuchsgut 32 3.2 Versuchsanordnung 33 3.3 Material 34 3.4 Methodik 34 3.4.1 Gesundheitszustand der Versuchsgruppen 34 3.4.2 Bestimmungsmethoden klinisch-chemischer Parameter 34 3.4.3 Bestimmung der Lipoproteine 35 3.4.4 Bestimmung von freiem Endotoxin 37 3.4.5 Bestimmung von Anti-Lipid A Antikörpern 38 3.5 Statistische Auswertung 39 4 Ergebnisse 41 4.1 Tierbestand 41 4.2 Stoffwechselparameter 41 4.2.1 β-Hydroxybutyrat 41 4.2.2 Glucose 44 4.2.3 Insulin 46 4.2.4 Freie Fettsäuren 48 4.2.5 Triacylglycerol 50 4.2.6 Cholesterol 52 4.2.7 Bilirubin 54 4.2.8 Gesamtprotein 56 4.3 Lipoproteine 58 4.3.1 α-Lipoproteine 58 4.3.2 ß-Lipoproteine 60 4.4 Endotoxin 62 4.5 Anti-Lipid A Antikörper 64 5 Diskussion 66 5.1 Versuchsprinzip 66 5.2 Tiere und Versuchsbedingungen 66 5.3 Methodenkritik 67 5.4 Stoffwechselparameter 68 5.4.1 β-Hydroxybutyrat 68 5.4.2 Glucose 71 5.4.3 Insulin 72 5.4.4 Freie Fettsäuren 74 5.4.5 Triacylglycerol 76 5.4.6 Cholesterol 78 5.4.7 Bilirubin 79 5.4.8 Gesamtprotein 81 5.5 Lipoproteine 83 5.5.1 α-Lipoproteine 83 5.5.2 β-Lipoproteine 86 5.6 Endotoxin 87 5.7 Anti-Lipid A Antikörper 89 6 Zusammenfassung 91 7 Summary 93 8 Literaturverzeichnis 95 9 Anhang 116 9.1 Stoffwechselparameter 116 9.1.1 β-Hydroxybutyrat 116 9.1.2 Glucose 117 9.1.3 Insulin 118 9.1.4 Freie Fettsäuren 119 9.1.5 Triacylglycerol 120 9.1.6 Cholesterol 121 9.1.7 Bilirubin 122 9.1.8 Gesamtprotein 123 9.2 Lipoproteine 124 9.2.1 α-Lipoproteine 124 9.2.2 β-Lipoproteine 125 9.3 Endotoxin 126 9.4 Anti-Lipid A Antikörper 127

Acetoacetato (AA) e 2-metilacetoacetato (MAA) são compostos &#946;-cetoácidos acumulados em diversas desordens metabólicas como no diabetes e na isoleucinemia, respectivamente. Examinamos o mecanismo de oxidação aeróbica de AA e MAA iniciada por intermediários reativos de mioglobina de coração de cavalo (Mb) gerados pela adição de H2O2. Uma rota quimioluminescente que envolve um intermediário dioxetânico cuja termólise gera espécies &#945;-dicarbonílicas (metilglioxal e biacetilo) foi proposta e estudada. Emissão de luz ultra fraca acompanha a reação, e sua intensidade aumenta linearmente pelo aumento da concentração tanto de Mb (10-500 &#181;M) quando AA (10-100 mM). Estudos de consumo de oxigênio mostraram que MAA é, como esperado, quase uma ordem de grandeza mais reativo que AA. Estudos de EPR com captação de spin, utilizando MNP, possibilitaram detectar adutos de MAA atribuíveis a um radical centrado no C&#945; (aN = 1.55 mT) e ao radical acetila (aN = 0.83 mT). O sinal do radical acetila é totalmente suprimido por sorbato, um conhecido e eficiente supressor de espécies tripletes, o que é consistente com uma rota reacional envolvendo um intermediário dioxetânico. Clivagem-&#945; da ligação carbonila-carbonila do produto biacetilo triplete produziria, de fato, radicais acetila. Além disso, utilizando AA como substrato para Mb/H2O2, um sinal de EPR atribuível ao aduto MNP-AA&#8226; (aN = 1.46 mT e aH = 0.34 mT) foi observado e confirmado por efeito isotópico. O consumo de oxigênio e o rendimento de compostos &#945;-dicarbonílicos foram dose-dependentes à concentração de AA ou MAA (1-50 mM) bem como à concentração de H2O2 adicionado às misturas de reação contendo Mb (até 1:10 quando medido o consumo de oxigênio, e até 1:25 quando medido o rendimento de compostos &#945;-dicarbonílicos) e tert-butilhidroperóxido (até 1:200). Os perfis de pH (5,8-7,8) para consumo de oxigênio e rendimento de compostos &#945;-dicarbonílicos mostraram maiores rendimentos para baixos valores de pH, indicativo de ferrilMb formada no ciclo peroxidático da proteína. Avaliando os níveis de lesão de Mb, os &#946;-cetoácidos diminuíram o nível de desorganização protéica na estrutura secundária e terciária elicitada por H2O2. Ainda, houve maior preservação da estrutura primária da proteína, sendo que MAA protegeu mais em comparação a AA, embora quando utilizado este último composto, foi mostrado que há acetilação dose-dependente de Mb. Acetoacetato aumentou a velocidade de descoramento da hemeproteína, provavelmente por ataque de espécies tripletes geradas no sistema. Músculos de rato, plantar e sóleo, expostos ex vivo a concentrações citotóxicas de glicose oxidase (GOX, gera H2O2 em fluxo), foram protegidas pelos ésteres etílicos AAE e MAAE. Foi detectado biacetilo no meio intracelular em músculos expostos a MAAE e GOX. A concentração deste composto &#945;-dicarbonílico é claramente relacionada à abundância de Mb em cada um dos tipos de músculos estudados. Em resumo, Mb tratada com metabólitos &#946;-cetoácidos (AA e MAA) gera radicais centrados em carbono e produtos &#945;-dicarbonílicos altamente reativos no estado triplete. Experimentos realizados com tecido muscular ex vivo sugerem que esta reação possivelmente ocorra in vivo. Levantamos a hipótese de que a geração de espécies carbonílicas reativas e seus adutos em condições de desbalanço metabólico possam contribuir para a compreensão das bases moleculares de desordens cetogênicas. / Acetoacetate (AA) and 2-methylacetoacetate (MAA) are &#946;-ketoacids accumulated in several metabolic disorders such as diabetes and isoleucinemia, respectively. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by the reactive enzyme intermediates formed by the reaction of muscle horse myoglobin (Mb) with H2O2. A chemiluminescent route involving a dioxetane intermediate whose thermolysis yields triplet &#945;-dicarbonyl species (methylglyoxal and diacetyl) is envisaged. Accordingly, the ultraweak light emission that accompanies the reaction increases linearly by raising the concentration of both Mb (10-500 &#181;M) and AA (10- 100 mM). Oxygen uptake studies revealed that MAA is, expectedly, almost one order of magnitude more reactive than AA. EPR spin-trapping studies with MNP detected spin adducts from MAA attributable to an &#945;-carbon-centered radical (aN = 1.55 mT) and to an acetyl radical (aN = 0.83 mT). As the acetyl radical signal is totally suppressed by sorbate, a well-known efficient triplet species quencher, the dioxetane hypothesis seems to be reliable. The &#945;-cleavage of the carbonyl-carbonyl bond of a putative excited triplet diacetyl product would, in fact, leads to an acetyl radical. Furthermore, using AA as substrate for Mb/H2O2, an EPR signal assignable to a MNP-AA&#8226; adduct (aN = 1.46 mT and aH = 0.34 mT) was observed and confirmed by isotope effect. Oxygen consumption and &#945;-dicarbonyl yield were also dependent on AA or MAA concentrations (1-50 mM) as well as on the concentration of peroxide added to the Mb-containing reaction mixtures: H2O2 (up to 1:10 when measuring oxygen uptake and up to 1:25 when measuring the &#945;-dicarbonyl yield) and t-butOOH (up to 1:200). The pH profiles (5.8-7.8) of oxygen consumption and &#945;-dicarbonyl yield show higher reaction rates at lower pHs, indicative of a ferrylMb intermediate. Evaluating Mb lesion, both &#946;-ketoacids reduced disorganization of the secondary and tertiary protein structure elicited by H2O2. Therefore, Mb primary structure was more preserved, and MAA was more protective than AA. Moreover using the later compound, it was shown that Mb acetylation is dose-dependent. Acetoacetate increased the rate of the hemeprotein bleaching, probably due to the attack of triplet products generated in the system. Plantaris and soleous rat muscles exposed to damaging concentrations of glucose oxidase (GOX, generates H2O2 in flux), was cytoprotected by AAE and MAAE. Intracellular diacetyl was detected in muscle samples exposed to MAAE and GOX. The &#945;-dicarbonyl concentration is clearly related to the Mb abundance in the muscle types. In summary, Mb treated with peroxides reacts with &#946;-ketoacid metabolites (AA and MAA), yielding carbon-centered radicals and highly reactive &#945;-dicarbonyl products in the triplet state. Experiments carried out ex vivo with muscle tissue showed that this reaction possibly occurs in vivo. A new route for generation and accumulation of carbonyl reactive species and adducts is here proposed to occur in unbalanced metabolic situations, such as is the case of ketogenic disorders.

O presente estudo teve como objetivo relacionar os níveis de indicadores sanguíneos energéticos e de cálcio na ocorrência de afecções uterinas, mastite clínica e mastite subclínica em vacas de raça Holandesa. A presença de mastite foi monitorada através do California Mastitis Test (CMT) e da contagem de células somáticas, para a identificação de mastite subclínica, e do Teste da Caneca de Fundo Preta (TCFP), para identificação de mastite clínica. A presença das afecções uterinas foi identificada mediante exame clínico. Foram feitas coletas de sangue para a determinação dos níveis de cálcio (Ca), glicose e β-hidroxi-butirato (BHB). O CMT foi realizado nas duas primeiras semanas após o parto, o TCFP foi realizado conforme a rotina de ordenha, estabelecida pela propriedade, e as coletas de sangue foram realizadas na semana anterior ao parto e duas coletas até os 21 dias posteriores ao parto. A ocorrência de hipocalcemia subclínica foi determinada quando o Ca no soro atingiu valores ≤ 8,00 mg/dL, hipoglicemia com valores de glicose ≤ 50 mg/dL e hipercetonemia com valores de BHB ≥ 1,2 mmol/L. Nas vacas com mastite clínica foi realizado tratamento conforme estabelecido pelo protocolo de ordenha e tratamentos da fazenda. Os dados de mastite subclínica obtidos através do teste de CMT, por quarto mamário, foram validados através de cultura microbiológica realizada em laboratório de referência e em laboratório na fazenda para identificar crescimento bacteriano, bem como através da contagem de células somáticas (CCS) e da contagem bacteriana total (CBT). Não foi encontrada relação significativa entre a hipocalcemia subclínica, a hipoglicemia e a hipercetonemia com afecções uterinas e da glandula mamária nas vacas após o parto. / This study aimed to relate the levels of energy and calcium in the occurence of both uterine diseases, clinic and subclinic mastitis in Holstein cows. The presence of mastitis was monitored through California Mastitis Test (CMT) and the counting of somatic cells (CSC), for the identification of subclinic mastitis, and strip cup test, for the identification of clinic mastitis. The presence of uterine disorders was identified by clinical examination. Blood collections were made for determining calcium, glicose and β-hydroxy-butyrate (BHB). CMT was realized in the first two weeks after calving, BBMT was realized according to the routine of milking settled by the farm and the blood collections were made in the week before the calving and also two collections were made after it. The occurrence of subclinic hypocalcemia was determined when serum Ca reached ≤ 8,00 mg/dL, hypoglycemia with glucose values ≤ 50 mg/dL and hyperketonemia values with BHB BHB ≥ 1,2 mmol/L. Data of clinic mastitis were obtained through the identification of altered milk in BBMT and subsequently realized treatment as settled by the milking protocol and treatments of the farm. Data of subclinic mastitis were obtained through CMT test, for mammary quarter, and validated by microbiological culture in reference laboratory, culture in the farm for identifying the bacterial growth, counting of somatic cells (CSC) and total bacterial counting (TBC). It was not identified in the study any relationship between subclinical hypocalcemia and uterine and mammary gland diseases in cows after calving.

A multiple trait, random regression, test day (TD) model was used to determine the heritability and genetic correlations of milk, fat, protein, and SCS with metritis, cystic ovary disease, mastitis, lameness, ketosis, milk fever, and displaced abomasum for four periods in lactations 1, 2, and 3-5 using two disease definitions. Definition 1 assumed that the first disease occurrence affects all TD thereafter equally and definition 2 assumed that disease affects only the next TD. Results were similar between definitions. The models were modified to allow the estimation of differences in disease occurrence by level of production, and to estimate production differences between diseased and non-diseased cows. Few significant associations were found between production levels and disease occurrence, the largest was between MAST and SCS. Diseased animals had lower levels of production than healthy animals for all diseases except COD. Animals with MAST had increased SCS. / Natural Sciences and Engineering Research Council (NSERC). Dairy Cattle Genetics Research and Development (DairyGen) Council.