Mucosubstances in the porcine gastrointestinal tract: Fixation, staining and quantification

Mucins are of great interest in intestinal research and histochemical methods are often employed to identify them. Since it is in the nature of mucins that they are “hard to hold onto” once they come into contact with water, a frequently used medium in histochemistry, there are a number of challenges that may decrease diagnostic accuracy. As the outcome of methods published for microscopic detection of mucosubstances proved to be unsatisfactory in our hands, the aim was the establishment of a reliable and reproducible protocol. Tissue samples were available from pig feeding experiments. In the present study, we focus on a fixation / staining procedure without making comparisons between differently fed pigs. Several fixation and staining procedures were evaluated for their use in semiautomatic quantification and quality assessment of different mucus fractions simultaneous on one tissue section. Cryostat sectioning, subsequent fixation steps with heat, ethanol and modified Bouin’s solution, followed by triple staining with high iron diamine, alcian blue and periodic acid-Schiff turned out to be the best method to identify sulfomucin, sialomucin and neutral mucin simultaneous on one tissue section. This methodology resulted in very good morphology of goblet cells with intact mucin containing vesicles within the cells, which was comparable to ultrastructural electron microscopical observations. Semiautomatic quantification of different mucins was possible. In conclusion, reliable mucus quantification and assessment of mucus quality requires strictly tested procedures. According to our experience, the most important aim after cryosectioning is fast fixation of the mucosubstances, which requires a combination of different fixation steps

evaluation of different fixatives for histochemical staining techniques considering tissue shrinkage

Staining of mast cells (MCs), including porcine ones, is critically dependent upon the fixation and staining technique. In the pig, mucosal and submucosal MCs do not stain or stain only faintly after formalin fixation. Some fixation methods are particularly recommended for MC staining, for example the fixation with Carnoy or lead salts. Zinc salt fixation (ZSF) has been reported to work excellently for the preservation of fixation-sensitive antigens. The aim of this study was to establish a reliable histological method for counting of MCs in the porcine intestinum. For this purpose, different tissue fixation and staining methods that also allow potential subsequent immunohistochemical investigations were evaluated in the porcine mucosa, as well as submucosa of small and large intestine. Tissues were fixed in Carnoy, lead acetate, lead nitrate, Zamboni and ZSF and stained subsequently with either polychromatic methylene blue, alcian blue or toluidine blue. For the first time our study reveals that ZSF, a heavy metal fixative, preserves metachromatic staining of porcine MCs. Zamboni fixation was not suitable for histochemical visualization of MCs in the pig intestine. All other tested fixatives were suitable. Alcian blue and toluidine blue co-stained intestinal goblet cells which made a prima facie identification of MCs difficult. The polychromatic methylene blue proved to be the optimal staining. In order to compare MC counting results of the different fixation methods, tissue shrinkage was taken into account. As even the same fixation caused shrinkage-differences between tissue from small and large intestine, different factors for each single fixation and intestinal localization had to be calculated. Tissue shrinkage varied between 19% and 57%, the highest tissue shrinkage was found after fixation with ZSF in the large intestine, the lowest one in the small intestine after lead acetate fixation. Our study emphasizes that MC counting results from data using different fixation techniques can only be compared if the respective study- immanent shrinkage factor has been determined and quantification results are adjusted accordingly

Preliminary Studies on the Intrahepatic Anatomy of the Venous Vasculature in Cats.

Hepatic surgeries are often performed in cats to obtain a disease diagnosis, for the removal of masses, or for the treatment of shunts. Whereas the vascular anatomy of the liver has been studied in dogs, such evidence is lacking in cats. The current study used corrosion casts of portal and hepatic veins and computed tomography (CT) analysis of the casts to identify and describe the intrahepatic anatomy in healthy cat livers (n = 7). The results showed that feline livers had a consistent intrahepatic portal and venous anatomy, with only minor disparities in the numbers of secondary and tertiary branches. The feline portal vein consistently divided into two major branches and not three, as previously described in the literature for cats. The finding of a portal vein originating from the right medial lobe branch leading to the quadrate lobe in 4/7 specimens is a novelty of the feline anatomy that was not previously described in dogs. Partial to complete fusion of the caudate process of the caudate and the right lateral lobe, with a lack of clear venous separation between the lobes, was present in two specimens. These findings allowed a detailed description of the most common intrahepatic venous patterns in cats. Further anatomical studies should be encouraged to confirm the present findings and to investigate the utility of this information in surgical settings

Dietary Enterococcus faecium NCIMB 10415 and Zinc Oxide Stimulate Immune Reactions to Trivalent Influenza Vaccination in Pigs but Do Not Affect Virological Response upon Challenge Infection

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy- two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Zn(high); 50 ppm, Zn(low)). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn(high) and E. faecium groups gained weight after infection while those in the control group (Zn(low)) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn(high)+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Zn(high) and E. faecium groups at single time points after infection compared to the Zn(low) control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology

Effect of Dietary Zinc Oxide on Morphological Characteristics, Mucin Composition and Gene Expression in the Colon of Weaned Piglets

The trace element zinc is often used in the diet of weaned piglets, as high doses have resulted in positive effects on intestinal health. However, the majority of previous studies evaluated zinc supplementations for a short period only and focused on the small intestine. The hypothesis of the present study was that low, medium and high levels of dietary zinc (57, 164 and 2,425 mg Zn/kg from zinc oxide) would affect colonic morphology and innate host defense mechanisms across 4 weeks post-weaning. Histological examinations were conducted regarding the colonic morphology and neutral, acidic, sialylated and sulphated mucins. The mRNA expression levels of mucin (MUC) 1, 2, 13, 20, toll-like receptor (TLR) 2, 4, interleukin (IL)-1β, 8, 10, interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β) were also measured. The colonic crypt area increased in an age-depending manner, and the greatest area was found with medium concentration of dietary zinc. With the high concentration of dietary zinc, the number of goblet cells containing mixed neutral-acidic mucins and total mucins increased. Sialomucin containing goblet cells increased age-dependently. The expression of MUC2 increased with age and reached the highest level at 47 days of age. The expression levels of TLR2 and 4 decreased with age. The mRNA expression of TLR4 and the pro-inflammatory cytokine IL-8 were down-regulated with high dietary zinc treatment, while piglets fed with medium dietary zinc had the highest expression. It is concluded that dietary zinc level had a clear impact on colonic morphology, mucin profiles and immunological traits in piglets after weaning. Those changes might support local defense mechanisms and affect colonic physiology and contribute to the reported reduction of post-weaning diarrhea

Das schleimhautassoziierte Netzwerk im Darm des Schweins: Eine histologische Untersuchung von Ernährung-Mikrobiota-Pathogen-Wirts-Interaktionen

List of tables VIII List of figures IX List of abbreviations X 1\. General introduction 1 2\. Literature review 4 2.1. The pig as a model organism in biomedical sciences 4 2.2. Porcine intestinal morphology, physiology and immunology 4 2.2.1. Macroscopic and microscopic anatomy of the porcine intestinal tract 4 2.2.2. Physiology of the porcine intestinal tract 6 2.2.3. Immunology of the porcine intestinal tract 7 2.3. The intestinal mucosal network 8 2.3.1. Gut associated lymphoid tissue - GALT 12 2.3.2. Mucosal homeostasis and barrier function 13 2.4. Nutritional influences on microbiota, pathogens and host 17 2.4.1. Probiotics as feed additives 17 2.5. Histological techniques applied to investigate the gastrointestinal tract 18 2.6. Nutrition-microbiota-pathogen-host-interactions: A graphic abstract 19 3\. Aims and objectives of the thesis 20 4\. Intraepithelial lymphocyte numbers and histomorphological parameters in the porcine gut after Enterococcus faecium NCIMB 10415 feeding in a Salmonella Typhimurium challenge 22 4.1. Introduction and aim 25 4.2. Materials and methods 26 4.2.1. Study design and probiotic feeding in the Salmonella challenge trial 26 4.2.2. Sample collection and histochemical staining 27 4.2.3. Morphometric parameters assessed by light microscopy 27 4.2.4. Counting of IEL by light microscopy 29 4.2.5. Statistical analysis 29 4.3. Results 30 4.3.1. Pathological findings 30 4.3.2. Morphometric parameters and IEL counts assessed by light microscopy 32 4.4. Discussion 37 4.4.1. Pathological findings 37 4.4.2. Morphometric parameters were influenced by the time post infection (age) and probiotic feeding 37 4.4.3. IEL quantities and distribution were altered by time post infection and probiotic feeding 39 4.5. Conclusion 40 5\. Enhancement of immunohistochemical detection of Salmonella in tissues of experimentally infected pigs 48 5.1. Introduction 51 5.2. Materials and Methods 51 5.2.1. Sample collection 51 5.2.2. Immunohistochemistry 52 5.2.3. Pigment differentiation / iron demonstration 52 5.3. Results 54 5.3.1. Immunohistochemical detection of Salmonella 54 5.3.2. Pigment differentiation / iron demonstration 55 5.4. Discussion 58 6\. Porcine intestinal mast cells. Evaluation of different fixatives for histochemical staining techniques considering tissue shrinkage 64 6.1. Introduction 67 6.2. Materials and Methods 68 6.2.1. Sampling and tissues 68 6.2.2. Tissue processing 69 6.2.3. Histochemical staining 70 6.2.4. Tissue shrinkage 71 6.2.5. Average shrinkage factor 71 6.2.6. Quantification of mast cells 71 6.2.7. Statistics 71 6.3. Results 72 6.3.1. Fixation of mast cells, histochemistry and morphology 72 6.3.2. Tissue shrinkage and resulting shrinkage factor 75 6.3.3. Quantification and statistics 76 6.4. Discussion 78 6.4.1. Fixations 78 6.4.2. Histochemistry and morphology 79 6.4.3. Shrinkage 80 6.4.4. Mast cell quantification results of porcine intestinal tissue 81 6.5. Conclusions 82 7\. Discussion 87 7.1. The intestinal mucosal network: Developmental changes and the influence of weaning 87 7.2. The intestinal mucosal network: Nutrition related changes 89 7.2.1. Effects of Enterococcus faecium 89 7.3. The intestinal mucosal network: Methodological aspects 92 7.3.1. Histological methodologies applied to improve characterization of microorganisms and immune cells 92 7.3.1.1. Microorganisms 92 7.3.1.2. Lymphocytes 92 7.3.1.3. Mast cells 93 7.3.2. Comparability of results 94 8\. Summary / Zusammenfassung 95 8.1. Summary of the PhD-Thesis 95 8.2. Zusammenfassung der Dissertation 98 9\. References 101 10\. List of publications 109 11\. Danksagung (Acknowledgment) 114 12\. Declaration of academic honesty 115This thesis was designed under the umbrella of the collaborative research centre (German: Sonderforschungsbereich) “SFB 852 Nutrition and intestinal microbiota - host interactions in the pig”, where different feeding strategies for pigs were examined in vivo, including probiotic treatment. There are many literature reports on the efficacy of the frequently-used probiotic Enterococcus faecium (E. faecium). The described effects range from no effect at all to a positive influence on performance (e.g. weight gain or feed conversion ratio) or protection from pathogens or reduced resistance to pathogens. Since the intestine is the interface where nutrition, microbiota, pathogens and host meet, the objective of the thesis was to further our knowledge on the effect of E. faecium in a Salmonella challenge situation, with particular focus on critically involved components of the intestinal mucosal network. Chapter 1 gives a general introduction on the pig as one of the most important farm animals for meat production as well as for biomedical research. For a long time, antibiotic growth promotors (in-feed-antibiotics) were used to compensate for the negative effects of intensive pig husbandry. It is known that the overly intensive and uncontrolled use of antibiotics affects human as well as animal health due to a rise in multiple resistances of bacterial infections. Consequently, research efforts of the SFB were focused on feed additives, particularly on mechanistic studies on the effects of probiotics. The field of work for this thesis was the “Histology Platform”, whose purpose was to apply and refine a broad spectrum of histological methodologies to the porcine organism in the course of several feeding trials. The effects of different nutritional strategies on gastrointestinal histological parameters under either normal housing conditions or in challenge situations were to be examined. Chapter 2 gives a literature overview on the pig as a model organism in biomedical sciences; porcine intestinal morphology, physiology and immunology; the intestinal mucosal network; nutritional influences on microbiota, pathogens and host and histological techniques applied to investigate the gastrointestinal tract. The objective of this thesis as well as the hypotheses are summarized in chapter 3 and are also found in the following descriptions. Chapters 4, 5 and 6 cover the three main publications for this thesis. Chapter 4 reports on intraepithelial lymphocyte numbers and histomorphological parameters in the porcine gut after Enterococcus faecium NCIMB 10415 feeding in a Salmonella Typhimurium challenge. Morphological parameters and the number of intraepithelial lymphocytes (IEL) were evaluated for the effect of the factors “time post infection/age” and “probiotic treatment” by light microscopy. The time post- infection had significant effects (P < 0.05) on the treated animals as well as in the control animals. Older animals showed longer and wider villi, deeper and wider crypts, a higher villus enlargement factor, a higher ratio between villus and crypt enlargement factors as well as more intraepithelial lymphocytes. Probiotic treatment resulted in a non-significant tendency for longer villi (P = 0.037), a slightly, but non-significant, higher ratio of villus surface/crypt circumference enlargement factors (P = 0.046) and significantly more IEL (P < 0.025). As the most important result, the population of intraepithelial lymphocytes situated at the nuclear level of the epithelium was identified to be strongly influenced by probiotic treatment (P = 0.004 at villus tip and P < 0.001 at villus base). It was concluded that the probiotic may have an immune modulatory effect by increasing the number of intraepithelial lymphocytes. These results confirmed the hypothesis that favourable effects of E. faecium treatment under a Salmonella challenge would involve beneficial changes in performance and immunological related parameters of the intestinal mucosal network. As an indicator for enhanced performance, the mucosal surface available for nutrient absorption was slightly enlarged (1.14-1.31-fold increase in absorptive surface related parameters “villus length”, “enlargement factor villi” and “ratio of villus surface/crypt circumference enlargement factors”). The epithelial barrier defending or regenerating intraepithelial lymphocytes were increased (1.09-2.32-fold), wich is a sign for improved immune protection. As an additional indicator for improved immune protection, the number of immunohistochemically detectable bacteria (Salmonella) invading the mucosa was expected to be significantly lower. Due to technical issues (see chapter 5), this hypothesis could not be tested. Chapter 5 reports on the enhancement of immunohistochemical detection of Salmonella in tissues of experimentally infected pigs. Samples were obtained from a challenge trial in which piglets were infected with Salmonella enterica serovar Typhimurium DT104. Tissue samples were fixed in Zamboni’s fixative and paraffin-embedded. Different immunohistochemical staining protocols were evaluated. Salmonella was detected in varying amounts in the tissues, and detergents like Triton X-100 or Saponin were found to enhance the sensitivity of the detection method. Additionally, a detection limit for Salmonella in immunohistochemical preparations was estimated (102-103 CFU per g tissue). It was concluded that the use of detergents could result in a higher sensitivity in the immunohistochemical detection of salmonellae. To the best of my knowledge, this is the first report on this issue. The results confirmed the hypothesis that (species-) specific histological protocols improve the detection of pathogens within porcine tissues. Since mast cells were identified as a central cell population with a multitude of physiological and pathological functions, including regulation of intestinal barrier function and host defence within the intestinal mucosal network, it was hypothesised that these cells are influenced by probiotic and other treatments in the SFB 852 trials. A species-specific protocol was to be established to reliably quantify mast cells in porcine tissues and find a basal cell number for further investigations in other SFB 852 trials. This aim was accomplished and in Chapter 6 the evaluation of different fixatives for histochemical staining techniques for porcine intestinal mast cells is described, including the effect of tissue shrinkage during fixation and embedding. Different tissue fixation and staining methods were evaluated in the porcine intestine. Metachromatic staining of mast cells was found to be critically dependent on the fixation and staining technique. The study revealed that zinc salt fixation preserved metachromatic staining in mast cells, which is the first report on this topic. Polychromatic methylene blue was deemed the optimal staining and in order to compare mast cell counting results between different fixation methods, tissue shrinkage has to be taken into account. These results also confirmed the hypothesis that (species-) specific histological protocols improve the detection and identification of immune cells within porcine tissues. In a general discussion (chapter 7), 3 topics concerning the intestinal mucosal network are discussed that turned out to be important issues in histological studies on nutrition-microbiota-pathogen-host- interactions: developmental changes, nutrition-related changes and methodological aspects.Die vorliegende Doktorarbeit wurde im Rahmen des Sonderforschungsbereiches „SFB 852“ konzipiert, in dem verschiedene Fütterungskonzepte, einschließlich Probiotikagabe, für Schweine in vivo untersucht wurden. In der Literatur finden sich verschiedene Berichte über die Effektivität des Probiotikums Enterococcus faecium (E. faecium). Sie reichen von gar keinem Effekt, über eine positive Wirkung auf die Leistung (z.B. Gewichtzunahme oder Futterumsatzrate) und einem Schutz vor Pathogenen, bis hin zu einer reduzierten Resistenz gegenüber Pathogenen. Da der Darm die Schnittstelle ist an der Ernährung, Mikrobiota, Krankheitserreger und Wirt aufeinandertreffen, war das Ziel der Arbeit die Vermehrung unseres Wissens über die Wirkung von E. faecium in einem Salmonellen-Infektionsversuch. Besonderer Fokus lag dabei auf beteiligten Komponenten mit kritischen Funktionen innerhalb des schleimhautassoziierten Netzwerkes im Darm. Kapitel 1 gibt einen Überblick über das Schwein, eines der wichtigsten Nutztiere in der Fleischproduktion sowie in der biomedizinischen Forschung. Für eine lange Zeit wurden Fütterungsantibiotika in der intensiven Nutztierhaltung eingesetzt, um negative Effekte auszugleichen, die dieses Haltungssystem mit sich bringt. Es ist bekannt, dass ein übermäßig intensiver und unkontrollierter Einsatz von Antibiotika die Human-, wie auch die Tiergesundheit beeinflusst und zu einem Anstieg von Infektionen mit multiresistenten Keimen führt. Die Forschungstätigkeit des SFB 852 war daher auf Fütterungszusätze ausgerichtet, im speziellen auf mechanistische Studien zum Effekt von Probiotika. Das Arbeitsfeld dieser Dissertation war die „Histologie Plattform“, deren Aufgabe darin bestand in Fütterungsversuchen mit Schweinen ein breites Spektrum an histologischen Methoden am porzinen Organismus anzuwenden und zu verbessern. Der Effekt verschiedener Fütterungsstrategien auf histologische Parameter sollte unter normalen Haltungsbedingungen und auch in Infektionsversuchen untersucht werden. Kapitel 2 gibt einen Einblick in die Literatur über das Schwein als Modellorganismus in der biomedizinischen Forschung; porzine Darmmorphologie, -physiologie und -immunologie; das schleimhautassoziierte Netzwerk im Darm; ernährungsbedingte Einflüsse auf Mikrobiota, Pathogene und den Wirt sowie histologische Techniken, die zur Untersuchung des Magendarmtraktes eingesetzt werden können. Das Ziel und die Hypothesen dieser Arbeit werden in Kapitel 3 zusammengefasst. Sie sind ebenfalls in der folgenden Beschreibung enthalten. Kapitel 4, 5 und 6 enthalten die drei Hauptpublikationen für diese Arbeit. Kapitel 4 berichtet über intraepitheliale Lymphozyten-Zahlen und histomorphologische Parameter im Schweinedarm nach Fütterung von Enterococcus faecium NCIMB 10415 innerhalb eines Infektionsversuchs mit Salmonella Typhimurium. Lichtmikroskopisch wurde die Wirkung der Faktoren "Zeit nach der Infektion / Alter" und "probiotische Behandlung" auf morphologische Parameter und die Anzahl der intraepithelialen Lymphozyten (IEL) untersucht. Die vergangene Zeit seit der Infektion zeigte signifikante Auswirkungen (P < 0.05), sowohl in der Behandlungs- als auch in der Kontrollgruppe. Ältere Tiere hatten längere und breitere Zotten, tiefere und breitere Krypten, einen höheren Zotten-Vergrößerungsfaktor, ein höheres Verhältnis zwischen Zotten- und Krypten-Vergrößerungsfaktoren sowie mehr intraepitheliale Lymphozyten. Die probiotische Behandlung führte tendenziell, aber nicht signifikant, zu längeren Zotten (P = 0.037) sowie einem leicht erhöhten Verhältnis von Zotten-Oberfläche/Krypten-Umfang-Vergrößerungsfaktoren (P = 0.046) und signifikant mehr intraepithelialen Lymphozyten (P < 0.025). Als wichtigstes Ergebnis wurde festgestellt, dass die auf Kernebene des Epithels gelegene Population der intraepithelialen Lymphozyten stark durch die probiotische Behandlung beeinflusst war (P = 0.004 an der Zottenspitze und P < 0.001 an der Zottenbasis). Daraus wurde geschlossen, dass das Probiotikum durch Erhöhung der Anzahl der intraepithelialen Lymphozyten eine immun- modulierende Wirkung haben könnte. Diese Ergebnisse bestätigen die Hypothese, dass positive Auswirkungen einer E. faecium Behandlung während einer Salmonellen-Infektion mit positiven Veränderungen in den Leistungs- und immunologischen Parametern des schleimhautassoziierten Netzwerkes verbunden sein müssten. Als Indikator für eine verbesserte Leistung war die für die Nährstoffaufnahme zur Verfügung stehende Schleimhautoberfläche leicht vergrößert (1.14-1.31-fache Zunahme der die absorptive Oberfläche betreffenden Parameter “Zottenlänge”, “Vergrößerungsfaktor Zotten” und “Verhältnis der Vergrößerungsfaktoren Zottenoberfläche/Kryptenumfang”). Die intraepithelialen Lymphozyten zur Verteidigung oder Regeneration der epithelialen Barriere waren vermehrt (1.09-2.32-fach). Dies ist ein Hinweis auf einen verbesserten Immunschutz. Als zusätzlicher Indikator für einen verbesserten Immunschutz sollte die Zahl der immunohistochemisch nachweisbaren Bakterien (Salmonella), die in die Schleimhaut eindringen, signifikant niedriger sein. Aufgrund technischer Aspekte (siehe Kapitel 5), konnte diese Hypothese nicht getestet werden. Kapitel 5 berichtet über die Verbesserung des immunhistochemischen Nachweises von Salmonella in Geweben experimentell infizierter Schweine. Zur Untersuchung kamen Proben eines Infektionsversuches, in denen Ferkel mit Salmonella enterica Serovar Typhimurium DT104 infiziert waren. Gewebeproben wurden in Zambonis Fixiermittel fixiert und in Paraffin eingebettet. Anschließend erfolgte die Erprobung und Auswertung verschiedener immunhistochemischer Färbeprotokolle. Salmonella konnte in unterschiedlichen Mengen in den Geweben nachgewiesen werden und Detergentien, wie Triton X-100 oder Saponin, erhöhten die Empfindlichkeit des Nachweisverfahrens. Zusätzlich wurde eine Nachweisgrenze für Salmonella in immunohistochemischen Verfahren geschätzt (102-103 CFU pro g Gewebe). Daraus ergab sich die Schlussfolgerung, dass die Verwendung von Detergentien zu einer höheren Empfindlichkeit im immunhistochemischen Nachweis von Salmonellen führen kann. Nach bestem Wissen ist dies die erste Abhandlung zu diesem Thema. Die Ergebnisse bestätigten die Hypothese, dass (Spezies-) spezifische histologische Protokolle notwendig sind, um die Erkennung von Krankheitserregern in Schweinegewebe zu verbessern. Da Mastzellen eine zentrale Zellpopulation mit einer Vielzahl von physiologischen und pathologischen Funktionen darstellen, einschließlich der Regulierung der Darmbarrierefunktion und Wirtsverteidigung im schleimhautassoziierten Netzwerk, wurde die Hypothese aufgestellt, dass diese Zellen durch probiotische und anderen Behandlungen in den SFB 852 Studien beeinflusst sein müssten. Ein Spezies-spezifisches Protokoll sollte etabliert werden, um Mastzellen in Schweinegeweben zuverlässig zu identifizieren und quantifizieren und eine basale Zellzahl für weitere Untersuchungen in anderen SFB 852 Studien zu finden. Dieses Ziel wurde erreicht und somit wird in Kapitel 6 über die Evaluierung verschiedener Fixiermittel für histochemische Färbetechniken von Mastzellen im Schweinedarm berichtet. Besondere Berücksichtigung findet dabei die Gewebeschrumpfung, die während der Fixierung und Einbettung auftritt. Verschiedene Gewebefixierungs- und Färbemethoden wurden am Schweinedarm untersucht. Die metachromatische Färbung von Mastzellen erwies sich als kritisch abhängig von der Fixierungs- und Färbungstechnik. Desweiteren ergab die Studie, dass eine Zinksalz-Fixierung die metachromatische Färbung von Mastzellen erhält. Dies ist der erste Bericht zu diesem Thema. Polychromatisches Methylenblau hat sich als optimale Färbung zum Mastzellnachweis herauskristallisiert. Um Mastzellen-Zählergebnisse zwischen den verschiedenen Fixierungsmethoden zu vergleichen, musste die Gewebeschrumpfung berücksichtigt werden. Diese Ergebnisse bestätigten auch die Hypothese, dass (Spezies-) spezifische histologische Protokolle die Detektion und Identifizierung von Immunzellen in porzinen Geweben verbessern. In einer allgemeinen Diskussion (Kapitel 7) werden 3 Themen aufgegriffen, die das schleimhautassoziierte Netzwerk im Darm betreffen. Diese haben sich als wichtig in den histologischen Studien zu Ernährungs-Mikrobiota-Pathogen-Wirt- Interaktionen erwiesen: entwicklungsbedingte Veränderungen, ernährungsbedingte Veränderungen und methodische Aspekte

Influence of Age and Breed on Bovine Ovarian Capillary Blood Supply, Ovarian Mitochondria and Telomere Length

Worldwide, dairy cows of the type of high-producing cattle (HPC) suffer from health and fertility problems at a young age and therefore lose productivity after an average of only three lactations. It is still contentious whether these problems are primarily due to genetics, management, feeding or other factors. Vascularization plays a fundamental role in the cyclic processes of reproductive organs, as well as in the regeneration of tissues. In a previous study, HPC were shown to have a greater ovarian corpus luteum vascularization compared to dual-purpose breeds. We hypothesize that this activated angiogenesis could likely lead to an early exhaustion of HPC′s regenerative capacity and thus to premature reproductive senescence. The objective of this study was to investigate if a HPC breed (Holstein-Friesian, HF) exhibits higher ovarian angiogenesis than a dual-purpose breed (Polish Red cow, PR) and if this is related to early ovarian aging and finally reproductive failure. For this purpose, we assessed the degree of vascularization by means of ovarian blood vessel characterization using light microscopy. As indicators for aging, we measured ovarian mitochondrial size and telomere length in peripheral leukocytes. We report in this study that in both breeds the distance between capillaries became smaller with increasing age and that the mean telomere length decreased with increasing age. The only difference between the two breeds was that PR developed larger capillaries than HF. Neither a relationship between telomere length, nor the morphology of the mitochondrial apparatus and nor angiogenesis in HF was proven. Although the data trends indicated that the proportion of shortened telomeres in HF was higher than in the PR, no significant difference between the two breeds was detected