Impact of dietary protein and fiber on the nutritional physiology of poultry

The present thesis is based on ten peer-reviewed publications that were published between 2013 and 2021 and focus on i) the establishment and further development of analytical methods characterizing physiological features and processes in the intestinal tract and ii) the significance of dietary protein and iii) dietary insoluble fiber on the nutritional physiology of poultry. Chapter I comprises three studies aimed at developing and refining laboratory techniques that will provide better insight into the physiological properties and functions of the intestinal tract. Analytical methods will shed light on the intestinal nutrient transport in chickens, the intestinal mucus layer formation in pigs and the distribution and frequency of intraepithelial immune cells in the gut of chickens. Chapter II includes three publications that examined the effect of qualitative or quantitative differences in dietary protein on the nutritional physiology of chickens. In particular, the impact of feeding differently processed grain legumes on immunological, morphological and functional alterations in the gastrointestinal tract of broilers are investigated. Furthermore, studies regarding protein requirements of male dual purpose chickens were performed investigating the effect of dietary protein reduction on digestive physiology and growth performance. Chapter III is based on a series of four publications evaluating the impact of dietary lignocellulose as an insoluble fiber source in poultry nutrition. Feeding experiments with laying hens and broilers are presented investigating the effect of varying dietary lignocellulose concentrations on chicken’s productive performance, digestive physiology and intestinal microbiota. Finally, a review article summarizes and evaluates studies on the effects of lignocellulose in poultry nutrition and compares the results with those observed in feeding trials using traditional insoluble fiber sources. In the general discussion the most important results from Chapters I-III are highlighted and intensively discussed in comparison with previous literature findings. Furthermore, open research questions are identified and future research perspectives addressed

Einfluss der Futterstruktur auf die Leistung, die gastrointestinale Morphologie, das darmassoziierte lymphatische Gewebe und auf den jejunalen Glukosetransport von Legehennen

New developments in the production and processing of feed for the use in the poultry sector are associated with changes in the feed structure, which is characterized by the particle size and the particle size distribution as well as the physical form of the diet, i.e. mash, pellets or expandate. Different grinding methods, grinding intensities as well as thermal treatment processes are used in order to achieve more energy-efficient feed production and to increase the quality and safety of feed. The aim of this study was to investigate whether gastrointestinal function and consequently animal performance and health were affected by the feeding of differently structured diets. For this purpose, investigations were focused on morphological, histological and immunological parameters and on the jejunal glucose transport of the digestive tract in laying hens. The eight experimental diets were produced using two different mills, the roller mill and the hammermill. By changing the grinding intensities, coarsely and finely ground feed was produced either as a mash or it was processed further to expandate. In total, the following eight diets were produced, which differed in their feed structure, but showed an identical botanical and chemical composition (30 % corn, 29 % wheat and 22 % extracted soybean mash): Coarsely and finely ground mash diets, coarsely and finely ground expanded diets, each of them produced by one of the two mills. In eight consecutive trial runs, a total of 384 laying hens (Lohmann Brown), aged 20 weeks were randomly allocated to eight different experimental groups (in total 48 hens per feeding group) with six animals per floor pen each representing a single experimental unit. The hens had ad libitum access to feed and water and were kept in barn systems. Each experimental group received differently structured diets over a period of 21 days. During the experimental trial, the laying performance, i.e. the egg production and the egg weight of each feeding group was recorded daily, and the feed intake and body weight recorded weekly. At the end of each trial run, the hens, aged 23 weeks, were slaughtered. In order to examine the effect of different feed structures on the development of the digestive organs, the pancreas, proventriculus, gizzard and the three segments of the small intestine of hens of each feeding group were extracted and subsequently weighed. For morphometric analysis, segments from the duodenum, jejunum and ileum were removed, processed histologically and finally the villi lengths and crypt depths were determined. In order to investigate the effect of the different diets on the active glucose transport in the jejunum, Ussing chamber experiments were conducted. On the basis of flow cytometric investigations, the intraepithelial lymphocytes (IELs) of the duodenum and jejunum of laying hens were characterized, and the effect of different feed structures on their distribution and relative frequency examined. All collected data were analyzed by using a three factorial analysis of variance in order to control for interaction and single factor effects with respect to the factors milling method, physical form and particle size of the diet. Concerning the collected animal performance data, the physical form of the diet had an impact on the overall feed intake. Hens fed with mash diets showed significantly higher feed intakes than those given the expandate. Regarding the total experimental period, neither the particle size of the feed nor the milling method used affected the overall animal performance. However, the results showed that both the physical form as well as the particle size of the diet influenced the development of the digestive organs. Laying hens that received mash instead of expandate had significantly higher gizzard and proventriculus weights as well as increased pancreas weights. Hens fed the coarsely ground diets showed significantly higher relative gizzard weights than those fed the finely ground diets. Regarding the microscopic structure of the small intestine, the results demonstrated that only the physical form of the diet had an impact on the villus lengths and crypt depths. Laying hens fed with mash diets showed significantly longer duodenal and shorter ileal villi as well as increased duodenal villus height to crypt depth ratios than those given the expandate. Moreover, the results of the Ussing chamber experiment showed that the active glucose transport in the jejunum, mediated by the SGLT-1, was influenced by the factor physical form of the diet. Mash-fed hens had a higher glucose transport rate than expandate-fed hens. The protocol for the flow cytometric analysis, which was established in order to determine the different lymphocyte subsets from the gut-associated lymphoid tissue of laying hens, led to reliable and reproducible results. With regard to the effect of the feed structure, the results showed no difference in the distribution and relative frequency of the IELs. To conclude, the results of this study illustrated that in general the feed structure had no impact on the animal performance, with the exception that the physical form of the diet (mash vs. expandate) influenced the FI. The feeding of mash diets compared with the feeding of expanded diets showed positive effects regarding the development of gastrointestinal organs and the intestinal microstructure of epithelium of laying hens, which may have led to the observed heightened glucose absorption of the jejunum. Furthermore, the feeding of coarsely ground diets also resulted in increased gizzard weights, but did not influence the intestinal microstructure of the epithelium and the jejunal glucose absorption. This study provides a characterization of different lymphocyte subpopulations from the gut-associated lymphoid tissue of Lohmann Brown hens. However, the feed structure did not seem to influence the distribution and relative frequency of IELs in healthy hens of this age. The milling method used for the feed production had no effect on the investigated parameters. Finally, it should be noted that based on the results of the present study, neither intensive grinding nor the use of thermal treatment processes led to improved animal performance. In contrast, the feeding of coarsely ground mash diets seemed to have positive effects on the gastrointestinal function and thus may lead to improved animal health and welfare. From an environmental and economical point of view, it should be emphasized that the production of coarsely ground mash diets using energy-saving milling methods is resource-efficient and therefore contributes to climate protection.Neue Entwicklungen in der Herstellung und Bearbeitung von Futter im Geflügelsektor sind mit Veränderungen in der Futterstruktur verbunden. Der Begriff der Futterstruktur umfasst dabei einerseits die Partikelgröße und Partikelgrößenverteilung, andererseits die Form des Angebots, das heißt, ob beispielsweise schrotförmiges, pelletiertes oder expandiertes Futter verfüttert wird. Dabei werden unterschiedliche Vermahlungsarten und -intensitäten sowie thermische Behandlungsverfahren genutzt, um eine energieeffizientere Futterproduktion zu erzielen und die Qualität und Sicherheit der Futtermittel zu erhöhen. Ziel der Studie war es daher zu überprüfen, ob die Fütterung unterschiedlich strukturierten Futters die Magen- Darm-Funktion sowie die damit verbundene Leistung und Gesundheit von Legehennen beeinflusst. Zu diesem Zweck wurden morphologische, histologische, transportphysiologische und immunologische Untersuchungen des Gastrointestinaltrakts bei Legehennen durchgeführt. Die Futtervarianten wurden mittels zwei verschiedener Mühlen, der Hammermühle und dem Weizenstuhl hergestellt, grob und fein vermahlen und entweder ohne Behandlung als Schrot verfüttert oder noch zusätzlich einer thermisch-mechanischen Druckkonditionierung unterzogen und als Expandat verfüttert. Es entstanden die folgenden acht Futtervarianten, die sich in ihrer Futterstruktur unterschieden, jedoch aus botanisch und chemisch identischen Ausgangskomponenten bestanden (30 % Mais, 29 % Weizen und 22 % Sojaextraktionsschrot): grob und fein vermahlenes Schrot, grob und fein vermahlenes Expandat, jeweils produziert durch eine der beiden Mühlen. In acht konsekutiv angelegten Versuchsreihen wurden insgesamt 384 Legehennen (Lohmann Brown; Alter: 20 Wochen) acht Fütterungsgruppen (48 pro Gruppe) zugeordnet, die jeweils über einen Zeitraum von 21 Tagen das entsprechende Futter erhielten. Dabei wurden sechs Tiere zusammen in einer Gruppe in Bodenhaltung gehalten (experimentelle Einheit), denen ad libitum-Fütterungs- und Tränkeeinrichtungen zur Verfügung standen. Während des gesamten Versuchszeitraumes wurden verschiedene Leistungsparameter der Legehennen erhoben, d.h. pro Fütterungsgruppe wurde die Legeleistung und die Eigewichte täglich, die Futteraufnahme sowie das Gewicht der Tiere wöchentlich bestimmt. Im Alter von 23 Wochen wurden die Versuchstiere nach vorheriger Betäubung durch Blutentzug getötet. Um den Einfluss der Futterstruktur auf die Entwicklung der Verdauungsorgane der Legehennen in den verschiedenen Versuchsgruppen zu untersuchen, wurden Pankreas, Drüsen- und Muskelmagen sowie die drei Dünndarmabschnitte entnommen und anschließend gewogen. Für die histologischen Untersuchungen wurden Gewebeproben vom Duodenum, Jejunum und Ileum entnommen, histologisch bearbeitet und die Länge der Darmzotten und Kryptentiefe morphometrisch beurteilt. Mit Hilfe von Ussing Kammer Versuchen wurde der aktive Glukosetransport im Jejunum der Tiere gemessen und überprüft, ob dieser durch die Fütterung der verschieden Diäten beeinflusst wurde. Mittels durchflusszytometrischer Messungen wurden intraepitheliale Lymphozyten (IEL) im Duodenum und Jejunum zum einen charakterisiert und zum anderen mögliche Effekte der Futterstruktur auf deren Verteilung und relative Häufigkeit untersucht. Die statistische Analyse der Ergebnisse erfolgte durch eine dreifaktorielle Varianzanalyse, so dass der Einfluss der einzelnen Faktoren (Mühle, Form und Partikelgröße) sowie deren Interaktionseffekte überprüft werden konnte. Die Auswertung der Leistungsdaten ergab, dass die Form des Futters einen Einfluss auf die Futteraufnahme der Tiere hatte. Legehennen, die mit Schrot gefüttert wurden, hatten signifikant höhere Futteraufnahmen als jene, die das Expandat erhielten. Bezogen auf den gesamten Versuchszeitraum beeinflusste dagegen weder die Partikelgröße des Futters noch die verwendete Mühle die Leistung der Tiere. Allerdings hatte die Form sowie die Partikelgröße des Futters einen Einfluss auf die Entwicklung einzelner Organgewichte des Gastrointestinaltraktes. Legehennen, die mit Schrot anstelle von Expandat gefüttert wurden, hatten signifikant schwerere Drüsen- und Muskelmägen sowie höhere Pankreasgewichte. Außerdem wiesen Hennen, die mit grob vermahlenen Diäten gefüttert wurden im Vergleich zu denen, die fein vermahlenes Futter erhielten, signifikant höhere Muskelmagengewichte auf. Die Ergebnisse der histologischen Untersuchungen des Dünndarmgewebes veranschaulichten, dass einzig die Form des Futters Einfluss auf die Länge der Darmzotten und Kryptentiefe hatte. Die mit Schrot gefütterten Legehennen hatten signifikant längere duodenale und kürzere ileale Darmzotten als Hennen, die Expandat als Futter erhielten. Außerdem führte die Fütterung von Schrot zu einem größeren Verhältnis von Villuslänge zur Kryptentiefe im Duodenum. Die Resultate der Ussing Kammer Versuche ergaben, dass der durch den SGLT-1 vermittelte aktive Glukosetransport im Jejunum durch den Faktor Form des Futters beeinflusst wird. Somit wiesen die mit Schrot gefütterten Hennen höhere Glukosetransportraten auf als Hennen, die mit Expandat gefüttert wurden. Das eigens etablierte Protokoll für die durchflusszytometrische Bestimmung unterschiedlicher Lymphozytensubpopulationen aus dem darmassoziierten lymphatischen Gewebe führte zu aussagekräftigen und reproduzierbaren Ergebnissen. Allerdings konnten keine durch Futter bedingten Effekte auf die Verteilung und relative Häufigkeit der IEL festgestellt werden. Die Ergebnisse der vorliegenden Studie deuten darauf hin, dass die Futterstruktur im Allgemeinen keinen Einfluss auf die Leistung der Legehennen hat, mit Ausnahme der Futteraufnahme, die durch die Form des Futters (Schrot vs. Expandat) beeinflusst wurde. Die Fütterung von Schrot im Vergleich zu Expandat hatte positive Effekte auf die Entwicklung der Organe des Magen-Darm- Traktes und auf die Mikroarchitektur der intestinalen Schleimhaut, wodurch möglicherweise auch die nachgewiesenen erhöhten Glukoseaufnahmeraten im Jejunum zu erklären sind. Zudem führte die Verfütterung von grob vermahlenen Diäten zu erhöhten Muskelmagengewichten, während die Mikroarchitektur des Darmepithels sowie der intestinale Glukosetransport davon nicht beeinflusst wurde. Durch die vorliegende Arbeit konnten verschiedene Lymphozytensubpopulationen im darmassoziierten lymphatischen Gewebe von Legehennen der Rasse Lohmann Brown charakterisiert werden. Allerdings deuten die Ergebnisse daraufhin, dass die Futterstruktur keinen Einfluss auf die Verteilung und relative Häufigkeit der IELs in gesunden Legehennen dieses Alters hat. Die zur Futterproduktion genutzten Mühlen hatten insgesamt keinen Einfluss auf die untersuchten Parameter. Auf Grundlage der Ergebnisse dieser Studie ist abschließend festzuhalten, dass weder die intensive Vermahlung noch die Nutzung thermischer Behandlungsverfahren von Futter zu verbesserten Leistungen der Legehennen führten. Vielmehr deuten die Untersuchungsergebnisse darauf hin, dass insbesondere durch die Fütterung von grob vermahlenem Schrot die Funktion des Magen-Darm-Traktes positiv beeinflusst wird und dies auch positive Auswirkungen auf die Gesundheit und das Wohlbefinden der Tiere haben könnte. Zudem ist aus ökologischer und ökonomischer Sicht zu betonen, dass eine ressourcenschonende Futtermittelproduktion durch die Herstellung von grob vermahlenem Schrot und die Verwendung energiesparender Mühlen gewährleistet wird und somit zum Klimaschutz beitragen könnte

Lignocellulose as an insoluble fiber source in poultry nutrition: a review

Extensive research in recent years into the use of various fiber sources in poultry nutrition has led to the perception that dietary fiber is more than a simple diet diluent. Several studies showed that the feeding of insoluble fiber sources such as oat hulls, sunflower hulls or wood shavings may affect digestive physiology and function improving chickens health and growth performance. In this context, the effect of lignocellulose as an insoluble dietary fiber source is increasingly being investigated. Lignocellulose is a component of plant cell walls and consists mainly of the insoluble carbohydrate polymers cellulose and hemicelluloses as well as the phenolic polymer lignin. Lignocellulose is chemically and physicochemically different from other insoluble fiber sources and thus possibly has different effects on poultry compared to traditional fiber sources. Several studies investigated the effect of dietary lignocellulose on growth performance, nutrient digestibility, gastrointestinal tract development and intestinal microbiota in broilers and laying hens. Studies differed in terms of feed formulation and lignocellulose inclusion level as well as products of different suppliers were used. The results obtained are inconsistent; beneficial, indifferent or detrimental effects of feeding lignocellulose were observed, so that a final assessment of lignocellulose as a “novel” insoluble fiber source is difficult. This review article summarizes the results of studies in connection with the feeding of lignocellulose to poultry, compares them with those that have used other insoluble fiber sources and illuminates the possible mechanisms of action

Comparison of different histological protocols for the preservation and quantification of the intestinal mucus layer in pigs

The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production. The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness. Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution. The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution. After dehydration, methacarn fixed tissues were embedded in paraffin wax. Both sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness. Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared. In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area. Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements. Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness. The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness. Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon. Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples. In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs. Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness. The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs

The effects of particle size, milling method, and thermal treatment of feed on performance, apparent ileal digestibility, and pH of the digesta in laying hens

Various milling methods result in different particle size distributions and, in combination with mash and thermal treatment (expandate) of the feed, may have an impact on nutrient digestibility, pH of the digesta and subsequently the performance of an animal. Since this aspect has not been widely considered in laying hens, the objective of the present study was to investigate the effects of milling method, expansion, and particle size of feed on performance, apparent ileal nutrient digestibility, and pH of digesta in laying hens. Twelve variants of the same diet were produced. Four different milling techniques (hammer mill, roller mill, disc mill, and wedge-shaped disc mill) were used to grind the feed cereals. Coarse feed was obtained from all four mills. Additionally, fine feed was obtained from the hammer mill and the roller mill. Each of the six feed variants was offered as mash or expandate, resulting in a total of 12 treatments. The duration of the experimental period was 21 days. A total of 576 layers, each 19 weeks of age, were used in eight replicates. The statistical analysis for the four milling methods and two thermal treatments was performed using a 4 × 2 factorial arrangement. The effect of particle size was investigated using a 2 × 2 × 2 factorial arrangement including the coarse and fine particle sizes that were produced with the hammer mill and the roller mill as well as the mash and expandate. The animal performance and the pH of the digesta were not affected by the treatments. Ileal digestibility of starch was significantly improved by feeding mash compared to expandate (P = 0.013) and by feeding coarse compared to fine feed (P = 0.028). Based on this study, the tested milling methods can be used for the production of feed for laying hens without affecting performance and digestibility of nutrients