Effects of strain, lifespan and dietary myo-inositol sources on poultry metabolism

Poultry production has shown a significant increase during the last decade. Meat and egg industry rapid growth implicates accelerating metabolic rate and general performance of birds. To maintain a high level of production, several strategies to achieve optimal raising and feeding have been implemented. Previous studies demonstrated the importance of MI metabolism on animal physiology; however, at present there is a substantial lack of information about the roles of MI and its metabolism in poultry. For instance, no information is available about MI concentration in organs of poultry. Moreover, it remains no elucidated, which are the effects of dietary sources of MI such as dietary phytase or pure MI supplementation. This thesis focused on gaining a comprehensive understanding of the potential roles of strain, productive period, and dietary sources of MI on poultry metabolism. To obtain the state of the art research on MI metabolism and its dietary sources in poultry, a comprehensive review of dietary MI was written (manuscript 1, chapter 3). This review revised information about MI in poultry such as feed sources, transport and cell metabolism, physiological meaning, and the influence of dietary MI in poultry. The revision indicated that MI appears to play critical roles in several different metabolic pathways so that understanding them could be an essential approach for future research in poultry. The second study was performed to study the effects of phytase and pure MI supplementation on the metabolite profile of broilers (manuscript 2, chapter 3). It was observed that phytase supplementation affected differently the metabolite profile than the supplementation of pure MI. Metabolites affected by phytase comprised several groups of metabolites such as acylcarnitines, phosphatidylcholines, sphingomyelins, lysophosphatidylcholines, and biogenic amines, whereas pure MI supplementation increased plasma concentrations of dopamine and serotonin. The third study was performed to get preliminary information about the effects caused by dietary phytase on systemic MI on the gastrointestinal tract, blood, and organs MI of broiler chickens (manuscript 3, chapter 3). Supplementation of 1500 FTU phytase/kg feed increased plasma and kidney MI concentrations. Plasma MI correlated negatively with InsP6 and positively with intestinal MI concentrations. A fourth study gave a general description of MI concentrations and general metabolite profile during the productive life of Lohmann Classic Brown and Lohmann LSL Classic laying hens. It was found that productive period affected MI and MI key enzymes expression. Moreover, the analyses showed differences in metabolite profiles being the onset of egg production, a determinant point. Differences were attributed to different groups of metabolites such as amino acids, biogenic amines, phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins. The above mentioned, indicated each strain could express different MI concentrations and metabolite profiles during distinct productive periods what should be considered to future interventions. To conclude, findings from these investigations suggested intrinsic traits such as breed and stage of production and diet could affect MI and MI key enzymes expression as well as metabolite profiles. Future studies are needed to establish the roles of MI on poultry metabolism.Die Geflügelproduktion hat im letzten Jahrzehnt erheblich zugenommen. Das schnelle Wachstum der Fleisch- und Eierindustrie impliziert eine Beschleunigung des Stoffwechsels und der allgemeinen Leistung von Geflügel. Um ein hohes Produktionsniveau aufrechtzuerhalten, wurden verschiedene Strategien zur Erzielung einer optimalen Aufzucht und Fütterung umgesetzt. Frühere Studien haben die Bedeutung des MI-Metabolismus für die Tierphysiologie aufgezeigt. Derzeit fehlen jedoch wesentliche Informationen über die Rolle des MI und seinem Metabolismus bei Geflügel. Beispielsweise sind keine Informationen über die MI-Konzentration in Geflügelorganen verfügbar. Darüber hinaus ist es nicht geklärt, welche Auswirkungen diätetische MI-Quellen wie z.B. durch Phytase-basierte Freisetzung aus der Nahrung oder reine MI-Supplementierung haben. Diese Dissertation fokussierte sich auf ein umfassendes Verständnis der möglichen Rolle von Zuchtlinie, Lebensdauer und diätetischem MI für den Stoffwechsel des Geflügels. Mit dem Ziel, den neuesten Stand der Forschung zum MI-Metabolismus und seinen Nahrungsquellen bei Geflügel zu erhalten, wurde eine umfassende Übersicht über das MI in der Nahrung verfasst (Manuskript 1, Kapitel 3). Diese Übersichtsarbeit fasst Informationen über MI bei Geflügel zusammen, wie etwa Futterquellen, Transport und Zellstoffwechsel, physiologische Bedeutung und den Einfluss von MI in der Nahrung bei Geflügel. Es wurde erfasst, dass MI eine Schlüsselrolle in einer Reihe verschiedener Stoffwechselwege zu spielen scheint, so dass das Verständnis dieser Stoffwechselwege ein wichtiger Ansatz für die künftige Geflügelforschung sein könnte. Die zweite Studie wurde durchgeführt, um die Auswirkungen von Phytase und reiner MI-Supplementierung auf das Metabolitenprofil von Broilern zu untersuchen (Manuskript 2, Kapitel 3). Es wurde beobachtet, dass eine Phytase-Supplementierung das Metabolitenprofil anders beeinflusste als die Supplementierung von reinem MI. Unter Phytasefütterung betroffene Metaboliten umfassten mehrere Gruppen von Metaboliten wie Acylcarnitines, Phosphatidylcholines, Sphingomyelines, Lysophosphatidycholines und biogene Amine, während eine reine MI-Supplementierung die Plasmakonzentrationen von Dopamin und Serotonin erhöhte. Die dritte Studie wurde durchgeführt, um vorläufige Informationen über die Auswirkungen der diätetisch supplementierten Phytase auf den systemischen MI auf den Magen-Darm-Trakt, den Blut- und Organ-MI von Masthühnern zu erhalten (Manuskript 3, Kapitel 3). Die Ergänzung von 1500 FTU Phytase / kg Futter erhöhte die Plasma- und Nieren-MI-Konzentrationen. Die Plasma-MI-Konzentration korrelierte negativ mit InsP6 und positiv mit den intestinalen MI-Konzentrationen. Eine vierte Studie lieferte eine allgemeine Beschreibung der MI-Konzentrationen und der Metabolitenprofile während des produktiven Lebens von Legehennen der Linien Lohmann Classic Brown und Lohmann LSL Classic. Es wurde festgestellt, dass die Produktionsperiode die Expression von MI- und MI-Schlüsselenzymen beeinflusste. Darüber hinaus zeigten die Analysen Unterschiede in den Metabolitenprofilen, wobei der Beginn der Eiproduktion ein bestimmender Punkt war. Unterschiede wurden verschiedenen Gruppen von Metaboliten wie Aminosäuren, biogenen Aminen, Phosphatidylcholinen, Lysophosphatidycholinen und Sphingomyelinen gefunden. Dies zeigte deutlich, dass jede Linie unterschiedliche MI-Konzentrationen und Metabolitenprofile entlang der Lebensdauerzeigte, was bei zukünftigen Interventionen berücksichtigt werden sollte. Zusammenfassend lässt sich sagen, dass die Ergebnisse dieser Untersuchungen darauf hindeuten, dass intrinsische Merkmale wie Zuchtlinie, Produktionsstadium und Ernährung die Expression von MI- und MI-Schlüsselenzymen sowie die Metabolitenprofile beeinflussen könnten. Zukünftige Studien sind erforderlich, um die Rolle des MI für den Geflügelstoffwechsel zu bestimmen

Clicker Training Mice for Improved Compliance in the Catwalk Test

The CatWalk test relies on the run of mice across the platform to measure a constant speed with low variation. Mice usually require a stimulus to walk to the end of the catwalk. However, such stimuli are usually aversive and can impair welfare. Positive reinforcement training of laboratory animals is a thriving tool for refinement and contributes to meeting the demands instituted by Directive 2010/63/EU. We have already demonstrated the positive effects of clicker training. In this study, we trained male and female mice to complete the CatWalk protocol while assessing the effects of training on their well-being (Open Filed and Elevated Plus Maze). In the CatWalk test, we observed that clicker training improved the running speed of the mice. In addition, clicker training reduced the number of runs required by mice, which was more pronounced in males. Clicker training lowered anxiety-like behaviors in our mice, especially in females, where a significant difference was observed between trained and untrained ones. Based on our findings, we hypothesize that clicker training is an effective tool to motivate mice and increase performance on the CatWalk test without potentially impairing their welfare (e.g., by puffing them)

Development of a 3D simulator for training the mouse in utero electroporation.

In utero electroporation (IUE) requires high-level training in microinjection through the mouse uterine wall into the lateral ventricle of the mouse brain. Training for IUE is currently being performed in live mice as no artificial models allow simulations yet. This study aimed to develop an anatomically realistic 3D printed simulator to train IUE in mice. To this end, we created embryo models containing lateral ventricles. We coupled them to uterus models in six steps: (1) computed tomography imaging, (2) 3D model segmentation, (3) 3D model refinement, (4) mold creation to cast the actual model, (5) 3D mold printing, and (6) mold casting the molds with a mix of soft silicones to ensure the hardness and consistency of the uterus and embryo. The results showed that the simulator assembly successfully recreated the IUE. The compression test did not differ in the mechanical properties of the real embryo or in the required load for uterus displacement. Furthermore, more than 90% of the users approved the simulator as an introduction to IUE and considered that the simulator could help reduce the number of animals for training. Despite current limitations, our 3D simulator enabled a realistic experience for initial approximations to the IUE and is a real alternative for implementing the 3Rs. We are currently working on refining the model

Simulation of <i>in utero</i> electroporation (IUE).

(A) Opening of the mouse abdominal cavity. (B) Handling of embryos outside of the abdominal cavity. (C) Injection of the DNA-containing solution in the lateral ventricle. (D) Application of voltage through forceps-type platinum electrodes.</p

Questionnaire for the <i>in utero</i> electroporation simulation model.

Questionnaire for the in utero electroporation simulation model.</p

Comparison of the mechanical properties from biosamples and their models.

(A) Statistical comparison between actual embryos and the simulator to evaluate the load [N] per displacement [mm] as a measure of hardness. The two-way ANOVA with Sidak multiple comparison was used to compare the statistics (Embryo’s (E15): n = 19, Embryo model: n = 3). The bars in the graph represent the mean and the SD. For displacement of 1 mm: mean difference = -0.1981, 95% CI -0.3992 to 0.002982. For 1.5 mm displacement: mean difference = -0.3966, 95% CI: -0.5977 to -0.1956, P-valueex vivo uteri as well (uterus(mean = 0.02574) vs. simulator Sh00-20(mean = 0.09674): P-value = 0.0079; uterus versus simulator Sh00-30(mean = 0.1349): P-value<0.0001). Significance was set as P-value less than 0.05. Bars in the graph represent the SD.</p

Steps of mold creation from the digital uterus model.

The mold creation for the uterus model followed the same steps as for the embryo model, but used a three-part design to accommodate the anatomical tube shape of a single uterus horn. (A) and (B) Import of the uterus model into Netfabb and Meshmixer to clean up the model. (C) An additional uterus shape was created within the original, with an offset of 0.5 mm. (D)The offset uterus shape was equipped with cylinders at both ends and a cube at the lower end. (E) The original uterus model and the cube were subtracted from a surrounding box. The printed model consists of the two outer parts that outline the original uterus shape and the merged offset uterus model combined with the cylinders and the box to sit inside the two outer parts.</p

Evaluation of the IUE simulator.

(A-E) The user responses to each of our questions to evaluate the IUE simulator. The X-axis indicates percentage points, while the Y-axis indicates the level of user experience. Statistical comparisons were made according to the ’ ’user’s level of expertise (Expert in white and beginner/intermediate in black). Student’s t-test was used for statistical comparison (n = 3). Significance was set as P-value less than 0.05. Bars in the graph represent SD. (F) indicates the statistical comparison between the questions. The X axes indicate percentage points, whereas the Y axis indicates the respective question. The one-way ANOVA followed by Tukey’s HSD (honestly significant difference) test was used for statistical comparison. Significance was set as P-value less than 0.05. Different letters indicate significant differences. Data are depicted in percentage units.</p

Steps of mold creation from the digital embryo model.

(A) Importation of embryo model into Netfabb. (B) Importation of the posterior ventricle into Netfabb. (C) Creation of a box from the part library; it was scaled to cover the embryo model. (D) The plane cut through the box, the embryo model, and the lateral ventricle model. (E) Merging of the upper and lower parts of the lateral ventricle after the plane cut. (F) Subtraction of the head part of the embryo after the plane was cut from the surrounding box with the "Boolean difference" function. (G) Subtraction of the embryo body model after the plane is cut from w the surrounding box with the "Boolean difference" function. (H) Separation of the embryo’s body mold into two halves to create the final silicon embryo.</p

Assembly of the silicone embryo model.

(A) The upper part of the head of the embryo model. The silicone was poured into the mold and hardened overnight. Due to the partial shape of the lateral ventricle on the mold cover on the right, the silicone contains two cavities representing the respective part of the lateral ventricle. (B) The model of the embryo body as seen above. Using a cover, the remaining shape of the lateral ventricle is represented as two cavities in the embryo model. (C) The upper and lower parts of the embryo model are assembled so that the two parts of the lateral ventricle meet each other. (D) The silicone embryo model was assembled with the upper and lower parts glued. The two parts of the lateral ventricle meet each other due to automatic registration in the design process, forming the complete shape of the lateral ventricle.</p