Aggregation and activity of mitochondria during in vitro maturation of cattle oocytes

Deckblatt-Impressum persönlicher Dank Inhaltsverzeichnis Abkürzungsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Einleitung Literatur Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Anhang Literaturverzeichnis Danksagung SelbständigkeitserklärungNur ca. 30% aller in vitro gereiften bovinen Oozyten entwickeln sich nach In- vitro-Fertilisation (IVF) zu Blastozysten. Dabei ist die heterogene Entwicklungskompetenz der verwendeten Oozyten ein entscheidender Einflussfaktor. Das Ziel der vorliegenden Arbeit bestand in der parallelen Charakterisierung der Aktivität und der Aggregation von Mitochondrien im Ooplasma und der Chromatinkonfiguration boviner Oozyten verschiedener Cumulus- Oozyten-Komplex (COK)-Klassen während der In-vitro-Maturation (IVM) sowie die Beurteilung der weiteren Entwicklungsfähigkeit nach IVF. Zudem wurde die Apoptose in Oozyten und umgebenden Cumuluszellen definierter COK untersucht werden. Die verwendeten COK wurden nach der Gewinnung wie folgt klassifiziert: kompakter Cumulus oophorus Klasse I, aufgelockerter Cumulus oophorus Klasse II, expandierter Cumulus oophorus Klasse III, denudierte Oozyte Klasse IV. Bei den Oozyten der Klasse II konnte eine frühere Wiederaufnahme der Meiose sowie ein früheres Auftreten reifer Oozyten während der IVM im Vergleich zu den Oozyten der Klasse I beobachtet werden. Die Oozyten der Klasse III und IV befanden sich zu Beginn der Reifung häufig bereits in fortgeschrittenen Stadien der Meiose und zeigten vermehrt Degenerationserscheinungen. Im Verlauf der IVM kam es bei den Oozyten der COK- Klassen I und II zu einem Wechsel von einer feinkörnigen zu einer kristallinen Aggregationsstruktur der Mitochondrien. Die Zunahme der kristallinen Aggregationsform war vor allem bei besonders energieverbrauchenden Prozessen der nukleären Reifung (MI-Spindelaufbau, Polkörperausstoß) zu beobachten. Die Oozyten der COK-Klasse II zeigten einen signifikanten Anstieg der kristallinen Aggregationsform bereits nach 20 h. In der frühen Phase der IVM (0 - 12 h) konnte bei Oozyten der Klasse I und besonders deutlich bei den Oozyten der Klasse II, ein Aktivitätsanstieg in den Mitochondrien beobachtet werden. Die höchste Aktivität konnte bei Oozyten im Diplotän, in der Diakinese und in der Metaphase II verzeichnet werden. Ein Anstieg der Apoptoserate im Verlauf der IVM wurde vor allem in den Cumuluszellen der COK-Klasse III beobachtet. Während der IVM wurde in den Cumuluszellen aller COK-Klassen aktivierte Caspase-3 nachgewiesen (höchste Aktivität nach 12 h IVM). In den Oozyten der verschiedenen COK-Klassen konnte jedoch nur die inaktive Form der Caspase-3 festgestellt werden. Es wurden hohe Teilungs- und Blastozystenraten unter Verwendung von Oozyten der COK-Klassen I und II beobachtet. Eine tendenzielle Überlegenheit bezüglich der Entwicklungskompetenz von Oozyten der Klasse II war festzustellen. Der Status von Nukleus und Mitochondrien bei Oozyten verschiedener COK-Klassen korrelierte mit deren Entwicklungskompetenz. Das Auftreten von MII-Stadien und einer kristallinen mitochondrialen Aggregation bereits nach 20 h IVM sowie die stärkere Dynamik bei der mitochondrialen Aktivität in der frühen Phase der IVM in den Oozyten der Klasse II waren mit einer tendenziell höheren Entwicklungskompetenz gegenüber den Oozyten der Klasse I verbunden. Die Ergebnisse der vorliegenden Arbeit lassen den Schluss zu, dass Aussagen über mitochondriale Parameter zu definierten Zeitpunkten der IVM geeignete Kriterien zur Feststellung der Oozytenqualität sind.The activity and the status of aggregation of mitochondria during in vitro maturation of cattle oocytes were examined in relation to cumulus-oocyte morphology and chromatin configuration of oocytes. In addition the rate of apoptosis was determined in oocytes and cumulus cells. The results were correlated with the developmental capacity of oocytes after IVM and IVF. For the experiments the cumulus-oocyte-complexes (COCs) were evaluated morphologically and classified into 4 groups: Compact cumulus oophorus (class I), slightly expanded cumulus oophorus (class II), expanded cumulus cells (class III) and denuded oocytes (class IV). There was a clear relationship between chromatin configuration and morphology of freshly recovered COCs. More class II oocytes showed diakinesis after 4 hrs of IVM when compared to class I oocytes. After 20 hrs of IVM, the percentage of oocytes in metaphase II was 16.2%, 43% and 70% in class I, II and III oocytes, respectively. Signs of degeneration and activation could be observed in oocytes with an expanded cumulus oophorus (class III) at the end of the IVM period. Meiosis was mostly irregular in denuded oocytes (class IV). Mitochondrial aggregation and activity was correlated with time of IVM, morphology of COCs and chromatin configuration. Oocytes from class I and II had evenly finely dispersed mitochondria wich turned into a crystalline appearance during IVM, mostly notably at time when energy consuming events like MI-spindle formation or extrusion of the first polar body occurred. Only in class II oocytes these changes could be detected as early as 20 hrs after start of IVM. An increase of mitochondrial activity could be observed in the early phase of IVM in class I and II oocytes, the latter having the highest increase of activity. Diplotan, diakinesis and metaphase II were the stages of meiosis with the highest mitochondrial activity of oocytes. Apoptosis of cumulus cells increased during IVM, especially in class III oocytes. The active form of caspase-3 could be observed in cumulus cells of COCs from class I-IV. In oocytes from all classes the inactive form of caspase-3, but no active caspase-3, could be observed. Oocytes of class I and II provided the highest rates of cleavage and blastocyst formation. The best results, though not statistically significant were obtained with class II oocytes. Morphology of COCs was clearly correlated with chromatin configuration, mitochondrial status and developmental competence. Oocytes from class II showed strong mitochondrial activity early during IVM and developed a crystalline appearance of mitochondria already after 20 hrs of IVM wich was accompanied by the extrusion of the first polar body (Metaphase II). These events might be correlated with the slightly better developmental competence of class II oocytes when compared to class I oocytes. The mitochondrial parameters investigated in this study may be helpful to assess the quality of oocytes in cattle

Equine Arteritis Virus (EAV) Outbreak in a Show Stallion Population

(1) Background: Equine arteritis virus (EAV) infection causes reproductive losses and systemic vasculitis in susceptible equidae. The intact male becomes the virus’ reservoir upon EAV infection, as it causes a chronic-persistent infection of the accessory sex glands. Infected semen is the main source of virus transmission. (2) Here, we describe acute EAV infection and spread in a stallion population after introduction of new members to the group. (3) Conclusions: acute clinical signs, acute phase detection of antigen via (PCR) nasal swabs or (EDTA) blood, and seroconversion support the idea of transmission via seminal fluids into the respiratory tract(s) of others. This outbreak highlights EAV’s horizontal transmission via the respiratory tract. This route should be considered in a chronic-persistently infected herd, when seronegative animals are added to the group

Equine Arteritis Virus (EAV) Outbreak in a Show Stallion Population

(1) Background: Equine arteritis virus (EAV) infection causes reproductive losses and systemic vasculitis in susceptible equidae. The intact male becomes the virus’ reservoir upon EAV infection, as it causes a chronic-persistent infection of the accessory sex glands. Infected semen is the main source of virus transmission. (2) Here, we describe acute EAV infection and spread in a stallion population after introduction of new members to the group. (3) Conclusions: acute clinical signs, acute phase detection of antigen via (PCR) nasal swabs or (EDTA) blood, and seroconversion support the idea of transmission via seminal fluids into the respiratory tract(s) of others. This outbreak highlights EAV’s horizontal transmission via the respiratory tract. This route should be considered in a chronic-persistently infected herd, when seronegative animals are added to the group

Toward evidence-based severity assessment in rat models with repeated seizures: I. Electrical kindling

Objective: Rodent epilepsy models can significantly contribute to our understanding of pathophysiological mechanisms and to validation of biomarker and target candidates. Evidence-based severity assessment is a presupposition for the ethical evaluation of animal experimentation allowances as well as for the development of efficacious refinement concepts. Methods: Aiming to improve our understanding of the impact of experimental procedures and repeated seizures, we have completed a comprehensive behavioral and biochemical analysis assessing various parameters that can inform about the influence of an electrical kindling paradigm on well-being in rats. Thereby, we have focused on the immediate effects of phases with focal and generalized seizures with behavioral testing during kindling acquisition. Results: Electrode implantation exerted mild effects on anxiety-associated behavior and reduced serum corticosterone at 3 weeks, but not 7 weeks, following surgery. Analysis in kindled rats excluded any relevant impact of focal seizures on behavioral and biochemical parameters. Assessment in rats with generalized seizures revealed an impact on nest complexity scores, nest soiling, and selected parameters in paradigms evaluating anxiety-associated behavior. Moreover, serum corticosterone levels, but neither hair corticosterone nor fecal corticosterone metabolite concentrations were lowered as a consequence of repeated generalized seizures. The assessment of various other behavioral and biochemical parameters did not reveal any other relevant effects of generalized seizures. Cross-correlation analysis suggested that assessment of nest building and maintenance can provide information comparable to that from more elaborate behavioral assays. This finding provides first evidence that nest scoring might serve as a simple and valid approach to evaluate rat well-being during routine assessment schemes. Significance: The findings argue against a persistent level of pronounced distress and suggest a classification of the kindling paradigm as a model with moderate severity based on a longer-lasting mild impact on animal behavioral patterns. This suggestion provides a basis for a prospective and retrospective case-by-case severity assessment