Institut für Nutztierwissenschaften

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Zinc supplementation effects on phytate degradation, mineral digestibility, and bone characteristics in broiler chickens
(2024) Philippi, Hanna; Rodehutscord, Markus
An adequate supply of phosphorus (P) is important in poultry nutrition, as P is essential for numerous metabolic processes. However, oversupply should be avoided to reduce the environmental impact of poultry production. The main source of P in plant feedstuffs commonly used in poultry nutrition is phytate, the salt form of phytic acid (InsP6). For P from InsP6 to be utilized by animals, it needs to be cleaved by phytases or other phosphatases. However, the capacity of endogenous phosphatases of non-ruminant animals does not suffice to release sufficient P to fulfill the animal’s P requirement. Therefore, commercial poultry diets usually are supplemented with P from mineral sources. By using exogenous phytases, the supplementation of mineral P can be reduced, and finite P reserves can be conserved. To feed poultry without mineral P in the future, phytase efficacy must be improved further. Thus, it is important to know and understand all factors influencing phytase efficacy. The results of in vitro studies have indicated that zinc (Zn) may be an influencing factor. The supplementation of Zn could inhibit phytase activity, with the degree of inhibition depending on the exogenous Zn source used. A literature review on the interactions of Zn with phytate and phytase (Manuscript A of this thesis) has identified a lack of in vivo studies investigating the effects of Zn supplementation on phytase with direct measurements, such as intestinal phytate degradation and prececal P digestibility. Therefore, three in vivo studies were conducted as part of this thesis with the main objective to investigate the effect of Zn supplementation and exogenous Zn source on intestinal phytate degradation in broiler chickens. It was hypothesized that due to the formation of insoluble complexes of Zn and phytate, the supplementation of Zn could reduce phytase efficacy with the extent of reduction depending on the exogenous Zn source. Further, other traits that are affected by Zn supply, such as bone mineralization and gene expression, were also investigated in these in vivo studies. The first experiment (Manuscript B) aimed to determine the effect of dietary Zn level and source on intestinal phytate breakdown, mineral digestibility, bone mineralization, and Zn status without and with exogenous phytase in the feed. Ross 308 broiler chickens were fed experimental diets from day 7 to 28. The basal diet contained 33 mg/kg dry matter native Zn and a high phytate-P concentration to challenge interactions in the digestive tract. The experimental diets differed in the level of exogenous phytase (0 or 750 FTU/kg) and in the Zn source (none, 30 mg/kg of Zn-sulfate, or 30 mg/kg of Zn-oxide). Additionally, two experimental diets with a high Zn supplementation level (90 mg/kg) in the form of Zn-sulfate or Zn-oxide, both containing exogenous phytase, were tested. Intestinal phytate breakdown, P digestibility, and bone mineralization were not affected by Zn source or Zn level but only by phytase supplementation. The concentration of ileal myo-inositol was influenced by phytase × Zn source interaction. Birds fed without phytase supplementation had similarly low myo-inositol concentrations whether they received Zn supplementation or not, whereas birds receiving phytase supplementation and Zn supplementation had significantly higher ileal myo-inositol concentrations than birds fed without Zn supplementation but with phytase supplementation. The missing effect of Zn level or Zn source on phytate degradation indicates that no interactions of Zn and phytate relevant for phytase efficacy occurred in the digestive tract of broilers when Zn was supplemented at levels up to 90 mg/kg in the form of Zn-sulfate or Zn-oxide. Based on the results of the first experiment, where Zn alone did not show relevant interactions with phytate, the second experiment (Manuscript C) aimed to investigate whether the combined supplementation of Zn, copper (Cu), and manganese (Mn) from different sources without and with exogenous phytase in the feed affects intestinal phytate breakdown, prececal mineral digestibility, bone mineralization, and mRNA expression of mineral transporters. Cobb 500 broiler chickens received experimental diets from day 0 to 28. Experimental diets differed in the level of phytase supplementation (0 or 750 FTU/kg) and in the trace mineral source (TMS: 100 mg/kg Zn, 100 mg/kg Mn, and 125 mg/kg Cu as sulfates, oxides, or chelates). Prececal InsP6 disappearance and P digestibility were significantly affected by phytase × TMS interaction. Whereas birds receiving exogenous phytase had similar InsP6 disappearance and P digestibility irrespective of TMS, birds fed without exogenous phytase and with chelated trace minerals had a higher InsP6 disappearance and P digestibility than birds receiving no exogenous phytase and oxides or sulfates. These results indicate that the combined supplementation of Zn, Mn, and Cu at high levels may challenge interactions with phytate in non-phytase-supplemented diets with the extent of interaction depending on the TMS. In phytase-supplemented diets however, the choice of TMS was irrelevant for phytate degradation under the conditions of this study. The third experiment (Manuscript D) aimed to determine the impact of Zn level and Zn source on prececal phytate degradation, mineral digestibility, bone mineralization, and mRNA expression of intestinal (trace) mineral transporters. In contrast to the first experiment, an inorganic Zn source and a chelated Zn source were tested. Cobb 500 broiler chickens received experimental diets from day 0 to 21. The experimental diets differed in Zn supplementation level (10, 30, 50 mg/kg Zn) and exogenous Zn source (Zn-oxide or Zn-glycinate). A cornsoybean meal-based diet without Zn supplementation containing 35 mg/kg native Zn was used as a control. All experimental diets were supplemented with 750 FTU/kg phytase. Prececal InsP6 disappearance, P digestibility, and tibia ash quantity and concentration, and Zn concentration in tibia ash were not affected by diet. Bone breaking strength and tibia width did not differ between treatments. Tibia thickness was lower in the treatments with 30 mg Zn as Zn-oxide and 50 mg Zn as Zn-glycinate than in the treatment with 10 mg Zn as Zn-oxide. The expression of intestinal (trace) mineral transporters was not affected by treatment. These results indicate that in phytase-supplemented diets the native Zn concentration of cornsoybean meal-based diets is satisfactory to achieve maximal Zn concentration in tibia ash during the first 3 weeks of age. The missing effect of Zn level or Zn source on phytate degradation confirms the results from Manuscript B, that Zn and phytate do not interact to a level relevant for phytate degradation by exogenous phytase. It is concluded that contrary to the hypothesis that Zn inhibits phytate degradation by complex formation with phytate, the Zn supplementation up to 100 mg/kg does not appear to influence exogenous phytase efficacy. Minor effects were found on the endogenous phytate degradation if Zn, Cu, and Mn were supplemented combined at high levels, where the extent of reduction in endogenous phytate degradation was dependent on the TMS. It remains unclear whether the inhibiting effect on endogenous phytate degradation occurs only due to the combined supplementation or whether an individual high supplementation of a single trace mineral caused the effect. Further experiments are needed to investigate the effect of Zn on endogenous phosphatases, where the activity of endogenous mucosal activity should be determined in broilers fed diets differing in the Zn supplementation level. Moreover, further experiments are needed to test what level of Zn supplementation is needed in phytase-supplemented diets to ensure the birds sufficient supply in all areas. Besides bone development and growth, effects on the immune system, microbiota composition, and the antioxidative system should be considered.
Novel bacterial species from the chicken gastrointestinal tract and their functional diversity
(2023) Rios Galicia, Bibiana; Seifert, Jana
The digestive system of chicken presents different physicochemical conditions along the gastrointestinal tract (GIT), shaping an individual microbial profile along sections with different metabolic capacities and divergence on the adaptations to the environment. Efforts to obtain cultivable bacteria originating from the upper region of chicken GIT enrich the reference genome database and provide information about the site- specific adaptations of bacteria colonizing such GIT sections allowing to understand the metabolic profile and adaptive strategies to the environment. However, the lack of sufficient reference genomes limits the interpretation of sequencing data and restrain the study of complex functions. In this study, 43 strains obtained from crop, jejunum and ileum of chicken were isolated, characterised and genome analysed to observe their metabolic profiles, adaptive strategies and to serve as future references. Eight isolates represent new species that colonise the upper gut intestinal tract and present consistent adaptations that enable us to predict their ecological role, expanding our knowledge on the adaptative functions. Strains of Limosilactobacillus were found to be more abundant in the crop, while Ligilactobacillus dominated the ileal digesta. Isolates from crop encode a high number of glycosidases specialised in complex polysaccharides compared to strains isolated from jejunum and ileum. While isolates from jejunum and ileum encode a higher number of genes that interact with the host such as collagenases and hyaluronidases, indicating preferential persistence and adaptations along the GIT. These results represent the first repository of bacteria obtained from the crop and small intestine of chicken using culturomics, improving the potential handling of chicken microbiome with biotechnological applications
Epidemiological and clinical description of Candidatus Mycoplasma haemosuis, an emerging pathogen in pigs
(2023) Ade, Julia; Hölzle, Ludwig
Candidatus Mycoplasma haemosuis is an emerging pathogen infecting pigs. It belongs to the group of uncultivable hemotrophic mycoplasmas. This group includes other long-known porcine representatives, i. e. Mycoplasma parvum und Mycoplasma suis. M. suis is the causative agent of infectious porcine anemia (IAP), a disease of great economic importance to the pig industry. Previously, Ca. M. haemosuis was only described in China, South Korea and Thailand, with no knowledge of its occurrence outside Asia or of its general clinical and economic importance in general. The present work investigates the occurrence of the novel hemotrophic bacterium and its clinical importance in Germany for the first time. For this, a quantitative real-time PCR was first successfully developed for the detection of Ca. M. haemosuis in pigs. The SYBR® Green-based PCR amplifies a 177-bp fragment of the Ca. M. haemosuis gap, which encodes the NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Using this PCR, Ca. M. haemosuis was detected in a total of seven pigs during an acute clinical disease in May 2017. This represents the first detailed description of a disease induced by Ca. M. haemosuis and the first detection of this novel HM species outside of Asia. In a further study, the newly established PCR was used to comprehensively investigate the occurrence of Ca. M. haemosuis in clinically healthy animals of different age groups in Southern Germany. Ca. M. haemosuis was prevalent in 6.25% of the sows (n=208), in 4.50% of the piglets (n=622), in 17.50% of the pigs (n=200), and in 0.00% of the breeding boars (n=183). By sampling the piglets immediately after birth and prior to the first colostrum uptake, the possibility of a vertical transmission of Ca. M. haemosuis was also determined within this thesis. Since 76.92% of the Ca. M. haemosuis positive sows gave birth to at least one Ca. M. haemosuis positive piglet, a vertical transmission is regarded as very likely. HMs are known to be transmitted blood-dependent and thus, transmitted iatrogenic or via wounds from animal to animal. The detection of M. suis in blood-free excretions such as saliva, urine, nasal, and vaginal secretions from experimentally infected animals has initiated the discussion of additional, blood-independent transmission routes. Saliva (n=148) and urine samples (n=47) were also collected from the sows examined by blood sampling, semen samples (n=183) were also obtained from the examined boars and applied to Ca. M. haemosuis qPCR. The pathogen was not detected in any of the saliva, urine, or semen samples. On the one hand, this demonstrates the lack of suitability of blood-free sample materials for diagnostics; on the other hand, it highlights the blood-dependent transmission pathways known to date and thus strengthens the potential to limit infections through strict hygiene measures during veterinary procedures and through the control of bloodsucking arthropods. In conclusion, based on the newly established qPCR assay for the sensitive and specific detection of Ca. M. haemosuis, the present work provides the first clinical and epidemiological description of the emerging hemotrophic pathogen in pigs. Further, the qPCR assay will be the basis for future studies regarding the epidemiology as well as the clinical relevance and pathogenesis of Ca. M. haemosuis -infections in pigs.
Phytate degradation and phosphorus digestibility in turkeys and broiler chickens fed maize-based diets
(2023) Novotny, Moritz Sebastian Daniel; Rodehutscord, Markus
A growing global human population, stagnation in available land for farming, and an increased interest in sustainable and eco-friendly food production necessitates a highly efficient and environmentally friendly food production. This includes the already very feed-efficient poultry meat production. Currently, using non-renewable mineral phosphate as feed additive is industry standard in poultry nutrition. This can lead to unwanted eutrophication of waterbodies by high faecal concentrations of unutilised plant-based phosphate. Degrading phytate via enzymatic hydrolysation by phytases drastically improves digestibility of plant-based phosphate. With dietary phytase supplementation, a tool is available to reduce necessity of dietary phosphate supplementation. However, predictability of the extent to which phytase supplementation can replace phosphate supplementation is not accurate enough to forego phosphate supplementation entirely. Subject of this doctoral thesis was to study the factors that can influence phytate degradation in the digestive tract of poultry, in order to improve predictability of plant-based phosphate digestibility. The focus was put on maize-based diets, as they are very common worldwide and phytate degradation is challenging due to low intrinsic phytase activity of maize. A literature review on the current state of knowledge on phytate degradation and phosphorus digestibility of chicken fed maize-based diets was conducted. Part of this review was to compare findings for chickens to findings in other poultry species. There is a plethora of studies that investigated the subject in broilers but comparatively little information on turkeys. There were indications of fundamental differences between broilers and turkeys. Consequently, the intention was to identify reasons for these differences and to evaluate to which extent knowledge transfer from chickens to turkeys is possible. Two consecutive trials comparing broilers and turkeys were designed. Factors studied were: supplemented phytase, dietary phosphorus and calcium concentration, age, and endogenous mucosal phosphatase activity. Broilers and turkeys studied were kept simultaneously and under identical conditions, including experimental diets. A total of 480 broiler and 480 turkey hatchlings were obtained at the same day and raised at the experimental facility. Halve of the animals of each species underwent the experiment from day 14 to day 21, the other halve from day 35 to day 42. This set up was chosen to study the influence of physiological development, as species with different maturation rates were compared. In 3-week-old broilers and turkeys, precaecal InsP6 disappearance was the same when no phytase was supplemented and dietary calcium and phosphorus level was low. This coincided with no differences in jejunal mucosal phosphatase activity. Without phytase supplementation, 6-week-old turkeys showed higher precaecal InsP6 disappearance than 6-week-old broilers. This coincided with higher jejunal mucosal phosphatase activity in turkeys than broilers. When phytase was supplemented, precaecal InsP6 disappearance was markedly increased in both species. This increase was always higher in broilers compared to turkeys of the same age. Increased dietary calcium and phosphorus levels led to decreased precaecal InsP6 disappearance in both species. This led to the conclusion that previously reported differences in precaecal InsP6 disappearance between broilers and turkeys were primarily due to the higher dietary calcium and phosphorus concentrations used in turkey diets, and secondly due to more phytate degradation by supplemented phytase in the crop of broilers compared to turkeys. The latter was attributed to more favourable conditions for the supplemented phytase. Although turkeys appeared to have compensated much of that in the more posterior parts of the digestive tract. Jejunal mucosal phosphatase activity was higher in treatments with phytase supplementation than without. As this coincided with high concentrations of lower inositol phosphates in the digesta, these might have triggered increased expression of phosphatases on the brush border membrane. In contrast, an increase in dietary calcium and phosphorus level coincided with a decrease in jejunal mucosal phosphatase activity, numerically in 3-week-old birds, but significantly in 6-week-old birds. This might indicate a downregulation of mucosal phosphatase expression based on phosphate concentration in the small intestine. In conclusion, fundamental mechanisms affecting phytate degradation in the digestive tract of broilers and turkeys seem to be the same. However, there is one big difference in recommended dietary calcium and phosphorus levels and many small differences in important details affecting phytate degradation and phosphate digestibility between the two species. These require dedicated attention to further improve phosphorus efficiency in poultry production.
Screening tools for late drought resistance in tropical potato
(2023) Hölle, Julia; Asch, Folkard
Potato (Solanum tuberosum L.) is a drought sensitive crop, and even short drought spells or infrequent irrigation during stolon formation, tuber initiation, or tuber bulking reduces tuber yields. A number of morphological traits have been described that potentially improve genotypic performance of potato under moisture deficit conditions. In breeding processes, a large set of genotypes are tested at the same time and because the genotypes differ in their phenology, various phenological stages occur simultaneously in the field. Consequently, during a drought spell different varieties will be subjected to soil moisture deficit at different phenological stages. We tested thirteen contrasting genotypes under field conditions in a desert in South Peru in four different irrigation treatments at two different soil types. The irrigation was withheld after 50, 65 and 80 days after planting until final harvest after 120 days. Sequential harvests, remote sensing and phenological evaluation was conducted in five to ten-days intervals. In literature, the belowground and aboveground development of potato has been described as closely and linearly related, meaning that in many studies belowground development is estimated according to aboveground development. The synchrony of the aboveground and belowground development is strongly influenced by both, water deficit and development stage at drought initiation. Under early drought, the aboveground development was accelerated and belowground development slowed. The opposite was found at later development stages. The earlier drought was initiated, the longer the tuber-filling phase, while the bulking phase was shortened. Water deficit also slowed down the aboveground development of flowering by a couple of days. In further drought experiments it is important to evaluated the belowground development separately, as we cannot conclude from the above to the belowground development stage. In conventional breeding experiments often only one final harvest is used to analyze the final tuber yield. This proceeding do not describe under which circumstances like stress intensity the tuber yield was achieved. Genotype evaluation in breeding experiments often relies only on visual evaluation of the aboveground biomass with no harvest of the plant. Besides the phenological stage at drought initiation the stress severity is another important aspect to determinate the drought stress response of potato genotypes. The stress severity depends on the water availability in term of soil water tension and the drought duration. In this study we developed a stress severity index (SSI) which combines all three important parameters, phenology, soil water tension and drought duration. With this SSI the selection processes should be improved and genotypes can be compared independently from environment, seasons and years. The SSI combines the yield response of potato to water deficit based on the soil tension the genotype was subjected to for the duration of the stress modified by the development stage of the genotype and drought duration. SSI allows for comparison of genotypic performance independent of year, location, season, soil type effects, and drought scenario. An SSI value of up to 1000 is able to differentiate between sensitive genotypes from more resistant genotypes. Beyond 1000, yields were generally reduced by more than 60% and a differentiation between genotypes was not possible anymore. SSI allows accumulating stress severity and thus, the higher the yield at a high SSI the stronger are the plants defense and adaptation mechanisms. Therefore, other indices that have looked into stay-green syndrome, rooting depth adaptations, leaf surface temperature, or canopy reflectance indices with only medium success, may benefit from including SSI in their indices to identify the underlying mechanisms of drought tolerance in potato. Remote sensing allows to evaluated many genotype simultaneously at field level. Proven indicators in drought tolerance screening are the normalized vegetation index (NDVI), the photochemical reflectance index (PRI) and thermography which describes the transpirational cooling of the leaves. Therefore, the last objective of this study was to validate the suitability of the SSI in remote-sensing stress diagnosis. The cluster analysis, including SSI, tuber yield reduction, NDVI, PRI and thermography identified three SSI groups with their corresponding physiological reactions under drought. The first group include SSI<1000 with fast decreasing NDVI, PRI and temperature deficit, in the second group matched SSI values from 1000 to 2000 with almost constant NDVI and temperature deficit and in the third group we found SSI beyond 2000 with corresponding small changes of NDVI, PRI and temperature deficit. The combination of these four parameters (tuber yield reduction, NDVI, PRI, thermography) explained 76 % of the variance which indicates this combination as valuable dataset analyzing drought tolerance in potato. Thus, combining these indicators with SSI and tuber yield reduction proved to be a first promising step for a new screening method for drought tolerance in a wider genotypic range. Whereas reflectance data can be recommended for assessing responses under mild to moderate stress severity, thermal imaging should rather be used to screen under mild or early drought stress.
European population genomic differentiation and dispersal pattern of the invasive beetle Anoplophora glabripennis
(2023) Häussermann, Iris Hanna; Hasselmann, Martin
Anthropogenic activities (e.g. homogenized habitats, trade) are the main factors to facilitate the increasing rates of invasive alien species. In this study, the invasion of the Asian long-horned beetle (Anoplophora glabripennis) was examined. Its native distribution is eastern Asia (China, Korean peninsula), but by extensive trade, this beetle was introduced via wood packing materials to North-America (1996) and Europe (2001). ALB attacks healthy broadleaved trees (e.g. Acer spp., Salix spp., Populus spp.), which can become lethal due to larval feeding. This study aims to detect genetic differences and kinship between the European infestation sites in Germany, Switzerland and Italy, from which the introduction and dispersal patterns can be deviated. Therefore, mitochondrial (mt) DNA-markers of the Cytochrome oxidase subunits I and II genes (COI and II) were used (ch. A and B, Sanger sequencing), as well as genome wide single nucleotide polymorphisms (SNPs), which were obtained by a Genotype-by-sequencing (GBS) approach (ch. C, Illumina sequencing). The results of this population genomic study of invasive European ALB populations showed very complex introduction patterns into Europe including multiple independent introductions characterized by the high population structure between the European infestation sites and some cases of human mediated secondary dispersal.
Comprehensive characterization of microbiota in the gastrointestinal tract of quails and two high yielding laying hen breeds
(2023) Roth, Christoph Florian; Camarinha Silva, Amélia
The microbiomes composition in the gastrointestinal tract (GIT) is subject to several changes and influences. In addition to breed, sex, or diet, age affects the GIT microbiome dynamics of laying hens and quails. From the first day, the microbiome develops and increases its bacterial load to thousands of species. Then, depending on the diet fed, the animals microbiome and associated active bacteria vary and directly influence the animals nutrient uptake and efficiency. Omics technologies give insights into changes in microbes in the GIT (crop, gizzard, duodenum, ileum, caeca). In addition, they can reveal how feed supplements such as calcium (Ca) or phosphorus (P) can affect host health and performance through alterations in the microbiome. The Japanese quail has been an established animal model for nutritional and biological studies in poultry for the last 60 years. In particular, its short development time makes it a convenient model for microbiome research. However, compared to broiler microbiome research, the quail microbiome is still poorly understood. Animals of the breed Coturnix japonica were housed under the same conditions, fed a diet with P below recommendation, and the ileum microbiota characterized. Microbiota relations with gender and higher or lower predisposition of the birds for PU, CaU, FI, BWG, and FC were described (Chapter II). In addition, these performance parameters influenced the relative average abundance of bacteria like Candidatus Arthromitus, Bacillus, and Leuconostoc. Gender affects specific bacterial groups of the GIT, such as Lactobacillus, Streptococcus, Escherichia, and Clostridium, which differ in average abundance between male and female quails. Despite the comprehensive microbiota analysis, the interplay between animal genetics, diet, sex, and microbiome functionality is not yet understood. The laying hen breeds Lohmann LSL-Classic and Lohmann Brown-Classic are used worldwide. Little is known about the interaction with microbiome composition, performance, dietary effects, and changes during the productive life that might help develop feeding strategies and microbiome responses on a large scale. Because of the importance of P and Ca in poultry diet, the research in Chapter III was conducted to challenge laying hens with reduced dietary P and Ca and describe the effect on GIT active microbiota. The breed was the primary driver of microbial differences. A core microbiome of active bacteria, present along the complete GIT, was revealed for the first time and consisted of five bacteria detected in 97% of all samples, including digesta and mucosa samples (uncl. Lactobacillus, Megamonas funiformis, Ligilactobacillus salivarius, Lactobacillus helveticus, uncl. Fuscatenibacter). Furthermore, significant microbial differences between the GIT sections and between the breeds were described. Minor dietary effects of the P and Ca reduction on the microbiota showed that a further decrease in Ca and P supplementation might be possible without affecting the gut microbial composition and bird performance. Furthermore, the microbiome of laying hens was characterized at five productive stages (weeks 10, 16, 24, 30, and 60) to analyze the age effect on the GIT microbiome (Chapter IV). Although the two breeds of laying hens were offered the same diet and housed under similar conditions, the active microbiota composition changed between the analyzed productive stages, the breed and the GIT sections. The major shift occurred between weeks 16 and 24 and supported the hypothesis of bacterial fluctuations due to the onset of the laying period. Those changes occurred mainly in the abundance of the genera Lactobacillus and Ligilactobacillus. However, it remains unclear whether the dietary changes, due to the development of the birds, influenced the microbiota shifts or if the anatomical and physiological modifications influenced the GIT microbiota. Furthermore, the shotgun metagenomic analysis revealed differences in regulatory functions and pathways between breeds, sections, and the two production stages. Different relative abundance levels of the microbial composition were observed between the RNA-based targeted sequencing and the DNA-based shotgun metagenomics. In conclusion, the comprehensive characterization of the microbiota in the GIT of quails and two high-yielding breeds of laying hens contributes to a broader knowledge of the microbiome dynamics within the fowl GIT. Age and breed play a more important role than diet in influencing the dynamics of microbial composition in laying hens, and individual performance and sex in quails. Research characterizing the microbiome in poultry and its effect on diet and host genetics will help improve feeding and breeding strategies in the future and reduce excretion of nutrients into the environment while ensuring overall animal health.
Genomic and microbial analyses of quantitative traits in poultry
(2023) Haas, Valentin Peter; Bennewitz, Jörn
Feed and nutrient efficiency will become increasingly important in poultry production in the coming years. In addition to feed efficiency, particular attention is paid to phosphorus (P) in nonruminants. Especially growing animals have a high demand of P but through the low usability of plant-based P sources for nonruminants, mineral P is added to their feeds. Due to worldwide limited mineral P sources, the high environmental impact of P in excretions and high supplementation costs, a better utilization of P from feed components is required. Animals’ P utilization (PU) is known to be influenced by the host genetics and by gastrointestinal microbiota. The overall aim of this thesis was to investigate the relationships between host genetics, gastrointestinal microbiota composition and quantitative traits with the focus on PU and related traits in F2 cross Japanese quail (Coturnix japonica). Japanese quail represent a model species for agriculturally important poultry species. In Chapter one, a genetic linkage map for 4k genome-wide distributed SNPs in the study design was constructed and quantitative trait loci (QTL) linkage mapping for performance as well as bone ash traits using a multi-marker regression approach was conducted. Several genome-wide significant QTL were mapped, and subsequent single marker association analyses were performed to find trait associated marker within the significant QTL regions. The analyses revealed a polygenic nature of the traits with few significant QTL and many undetectable QTL. Some overlapping QTL regions for different traits were found, which agreed with the genetic correlations between the traits. Potential candidate genes within the discovered QTL regions were identified and discussed. Chapter two provided a new perspective on utilization and efficiency traits by incorporating gastrointestinal microbiota and investigated the links between host genetics, gastrointestinal microbiota and quantitative traits. We demonstrated the host genetic influences on parts of the microbial colonization localized in the ileum by estimating heritabilities and mapping QTL regions. From 59 bacterial genera, 24 showed a significant heritability and six genome-wide significant QTL were found. Structural equation models (SEM) were applied to determine causal relationships between the heritable part of the microbiota and efficiency traits. Furthermore, accuracies of different microbial and genomic trait predictions were compared and a hologenomic selection approach was investigated based on the host genome and the heritable part of the ileum microbiota composition. This chapter confirmed the indirect influence of host genetics via the microbiota composition on the quantitative traits. Chapter three further extended the approaches to identify causalities from chapter two. Bayesian learning algorithms were used to discover causal networks. In this approach, microbial diversity was considered as an additional quantitative trait and analyzed jointly with the efficiency traits in order to model and identify their directional relationships. The detected directional relationships were confirmed using SEM and extended to SEM association analyses to separate total SNP effects on a trait into direct or indirect SNP effects mediated by upstream traits. This chapter showed that up to one half of the total SNP effects on a trait are composed of indirect SNP effects via mediating traits. A method for detecting causal relationships between microbial and efficiency traits was established, allowing separation of direct and indirect SNP effects. Chapter four includes an invited review on the major genetic-statistical studies involving the gut microbiota information of nonruminants. The review discussed the analyses conducted in chapter one to three and places the analyses published in these chapters in the context of other statistical approaches. Chapter four completed the microbial genetic approaches published to date and discussed the potential use of microbial information in poultry and pig breeding. The general discussion includes further results not presented in any of the chapters and discusses the general findings across the chapters.
The influence of L-carnitine on hematology and functional blood parameters of dairy cows with special focus on high resolution data around parturition
(2023) Kononov, Susanne Ursula; Huber, Korinna
The transition period, defined as three weeks before to three weeks after parturition, is one of the most critical times in the production cycle of dairy cows. On the one hand, cows have to cope with increased energy demand, while on the other hand, feed intake decreases due to stress and pain during parturition. This results in an negative energy balance and, consequently, at the beginning of body fat tissue mobilization. Lipomobilization increases the blood concentration of NEFA. This is accompanied by an increase in the blood concentration of ketone bodies, such as BHB. In addition to changes in energy metabolism, alterations in the immune function of dairy cows occur during the transition period. Stress and pain during calving lead to elevated blood levels of glucocorticoids, such as cortisol, which affect the immune system. Furthermore, the immune system is affected by increased concentrations of NEFA and BHB. At the same time, oxidative stress occurs due to an imbalance between the production of reactive oxygen species (ROS) and the activity of the antioxidative system. In general, the period around calving and its consequences constitute a very complex process influenced by many interdependent factors. One key factor in energy production is the quaternary amine L-carnitine (LC), which is necessary for the transport of short-chain fatty acids from the cytosol to the mitochondrial matrix. Furthermore, several studies have demonstrated the antioxidant and membrane-stabilizing effects of LC. This study aimed to investigate the effects of dietary LC supplementation on energy metabolism, hematology, and immune functions of dairy cows during the transition period. In addition, the first 72 h after calving were observed at high resolution to show the characteristic courses of the examined parameters, which, to the best of our knowledge, have not yet been analyzed. To attain this aim, 60 pluriparous Holstein Friesian cows were assigned to two groups based on their lactation number, body weight, body condition score, and fat-corrected milk yield from previous lactation. The LC group (CAR) received 25 g of rumen-protected LC. The study started 42 days before excepted calving and ended 110 days after parturition. To evaluate the performance and health of the animals, feed and milk samples were collected regularly, and feed intake, milk yield, body weight, and BCS were documented (Manuscript I). Additionally, NEB was calculated, and NEFA, BHB, and triglyceride concentrations in the blood were determined (Manuscript I). Also, the concentration of LC in the blood as well as that of the precursors γ-butyrobetaine (γBB), Nε-trimethyllysine (TML), and acetylcarnitine (ACA) was examined (Manuscript I). Red blood cell counts and antioxidant enzyme activity were measured to obtain more information on the oxygen supply and antioxidant status of the animals (Manuscript II). To evaluate the immunological status and inflammatory response, white blood cell count, phagocytic activity, ROS production, and lymphocyte populations were analyzed (Manuscript III). Dietary supplementation with LC increases blood LC, γBB, and ACA concentrations. Furthermore, LC supplementation resulted in better utilization of NEFA and TG. This was manifested by an increased blood concentration of triglycerides and a lower concentration of NEFA. Moreover, increased levels of platelets and eosinophils were detected in the CAR group, confirming the membrane-stabilizing effect of LC and the associated longer cell lifespan. Additionally, immunological functions were affected by LC supplementation. The ability of polymorphonuclear cells (PMN) to phagocytose bacteria was analyzed by the mean fluorescence intensity (MFI) of ROS-producing PMN, and the phagocytic capacity decreased compared to the CON group. Simultaneously, the efficiency of ROS production by PMN increased in CAR cows. These results suggest an altered immune function around calving, but not suppression, as is often described in the literature. In addition, this study showed that calving affected almost all analyzed data. The strongest changes in hematology and cell function were found four hours after calving. Furthermore, the influence of LC supplementation on immunological parameters was observed in the first few hours after parturition, indicating that LC supplementation may have an effect at energetically critical times. In conclusion, the present study showed that dietary LC supplementation affected energy metabolism, cell vitality, and cell function during the critical period around calving. However, this study also showed the clear influences of calving, which may be even more pronounced than animal-specific differences. Future studies should record the LC supply of cells to enable a more detailed description of the energetic situation of cells such as blood cells.
Effects of ensiling conditions on the nutritional quality of forage legumes and their impacts on rumen fermentation and nutrient utilization by cattle
(2022) Aloba, Temitope Alex; Uta, Dickhöfer
Silage produced from forage legumes can contribute to the limiting protein supply of ruminants diets in the tropics, and reduced dependence on imported and high-carbon footprint feeds. However, the successes recorded with temperate forage legume silage feeding in ruminants have not been achieved in the tropics. Thus, the effects of silage feeding on ruminants’ performance cannot be isolated from the processes that occur during ensiling. Since controllable and uncontrollable factors govern silage quality, it is imperative to understand the processes that occur during ensiling tropical forage legumes under different conditions to widen knowledge. Therefore, the aim was to determine the effect of ensiling conditions on forage legume nutritional quality, their ruminal and post-ruminal fate, and their potential impact on nutrient utilization by cattle in the tropics. A silage study was conducted to evaluate the effects of ensiling length and storage temperature on the nutritive value and fibre-bound protein of three tropical forage legumes ensiled alone or combined with sorghum. The three forage legumes included soybean (Glycine max), lablab (Lablab purpureus) and jack bean (Cannavalia ensiformis). Silages from each legume were made individually or combined with sorghum (Sorghum bicolor) and stored outdoors or indoors for 30, 75, and 180 days. The results showed that the proportion of soluble nutrients preserved in most silage until 75 d of ensiling declined considerably, thereby increasing dry matter (DM) and crude protein (CP) losses, fibre concentration and reducing digestibility afterwards. Besides, storage temperature affected the fermentation and fibre-bound protein characteristics with higher variation in legume silages’ fibre-bound protein than the sorghum-legume silages. Silages of sorghum and soybean were selected from the first study to compose low and high CP diets with additional ingredients, and the effects of ensiling length, storage temperature, and its interaction with CP levels on in vitro rumen fermentation and post-ruminal digestibility were assessed. Dietary treatments were incubated in duplicate for 8 and 24 h in three runs using the ANKOM RF technique to study rumen fermentation. Post-ruminal digestibility was determined using the pepsin and pancreatic solubility procedure. The results showed that gas production (GP) and ammonia-nitrogen in the rumen inoculum increased quadratically with the ensiling length, with the highest GP and ammonia-nitrogen at 75 d of ensiling, irrespective of incubation times. The GP was higher in diets with low than high CP concentrations, while it was the opposite for ammonia-nitrogen. An interaction between ensiling length and storage temperature effect was found for the apparent CP intestinal digestibility. Overall, ensiling beyond 75 d reduces CP digestibility to the extent that it cannot be recovered by supplying additional CP. In the third study, the effects of CP levels on nutrient intake, digestibility, nitrogen metabolism and performance of growing steers fed corn or corn-soybean silage were investigated. Sixteen growing steers were fed with rations based on corn or corn-soybean silage at high or low CP levels in a 4 × 3 incomplete Latin square design comprising 17 d periods, each with 12 d of adaptation to dietary treatments and 5 d of sampling. While the effect of silages and CP levels were not found for nutrient intake, the apparent total tract digestibility of nutrients was reduced for low than high CP in both silages, with greater differences between the CP levels in corn than corn-soybean silage. The average daily gain and feed efficiency were greater in low than high CP of corn silage, but no differences between CP levels were found in corn-soybean silage. In general, corn silage with low CP concentration but with a high metabolizable energy supply supposedly improved nitrogen use efficiency with a higher yield of microbial protein and average daily gain than other diets. Conclusively, the results of the current thesis showed that ensiling forage legumes individually or in combination with cereal crops beyond 75 d at high temperatures of the tropics leads to a decline in the nutritional quality of legume silage and CP intestinal digestibility even with additional CP sources. Furthermore, prolonged ensiling of combined legume and cereal crops reduces nutrient availability for cattle performance.
Adaptations of Prevotella bryantii B14 to short-chain fatty acids and monensin exposure
(2023) Trautmann, Andrej; Seifert, Jana
The rumen microbiome constitutes a complex ecosystem including a vast diversity of organisms that produce and consume short-chain fatty acids (SCFAs). It is of great interest to analyze these activities as they are of benefit for both, the microbiome and the host. This dissertation aims to display the proteome and metabolome of the predominant ruminal representative Prevotella bryantii B14 in presence of various SCFA and under exposure of the antibiotic monensin in pure and mixed culture (in vitro). Due to the strong contributing abundance of Prevotellaceae in the rumen microbiome, the representative P. bryantii B14 (DSM 11371) was chosen to investigate biochemical factors for the success of withstanding monensin and the impact of SCFA on their growth. The current work is composed of two effective publications. The formatting was aligned to the dissertation. The first publication, studying the supplementation of various SCFAs, showed SCFAs as growth promoting but not essential for P. bryantii B14. Pure cultures of P. bryantii B14 were grown in Hungate tubes under anaerobic conditions. Gas chromatography time of flight mass spectrometry (GC-ToF MS) was used to quantify long-chain fatty acid (LCFA) profiles of P. bryantii B14. Proteins of P. bryantii B14 were identified and quantified by using a mass spectrometry-based, label-free approach. Different growth behavior was observed depending on the supplemented SCFA. An implementation of SCFAs on LCFAs and the composition on membrane proteins became evident. Supplementing P. bryantii B14 with branched-chain fatty acids (BCFAs), in particular isovaleric acid, showed an increase of the 3-IPM pathway, which is part of the branched-chain amino acid (BCAA) metabolism. Findings point out that the structure similarity of isovaleric acid and valine is most likely enhancing the conversion of BCFA into BCAA. The required set of enzymes of the BCAA metabolism supported this perspective. The ionophore monensin has antibiotic properties which are used in cattle fattening but also for treating ketosis and acidosis in ruminants. In the second publication, P. bryantii B14 was exposed to different concentrations of monensin (0, 10, 20 and 50 uM) and to different exposure times (9, 24, 48 and 72 h) with and without monensin. Growth behavior, glucose and intracellular sodium concentration were determined. Proteins were analyzed by label-free quantification method using the same method as in the previous mentioned experiment. Fluorescence microscopy was used to observe extracellular polysaccharides (EPS) of P. bryantii B14. A progressing monensin exposure triggered disconnection between P. bryantii B14 cells to the sacrificial EPS layer by increasing its number and amount of carbohydrate active enzymes (CAZymes). Simultaneously, an increase of extracellular glucose was monitored. Reduction of intracellular sodium was likely to be performed by increasing the abundance of ion-transporters and an increased activity of Na+-translocating NADH:quinone oxidoreductase under monensin supplementation. The role of monensin supplemented Prevotella in a mixed culture of the rumen microbiome was described. Extracted rumen fluid from cows was incubated anaerobically by using the rumen simulation technique (Rusitec). Proteomics of the solid phase was applied by using a similar approach as in the previous related studies. Metabolomics of the liquid phase from the Rusitec content was performed by using 1H-nuclear magnetic resonance (NMR) spectroscopy. Further parameters such as pH, gas and methane production were monitored over time. The experiment was constituted out of three phases starting with an adaptation phase of 7 days. A subsequent treatment phase followed, where monensin was supplemented via the daily introduced total mixed ration (TMR) for further 7 days. The elution phase was the final phase when monensin supplementation was stopped and monitoring was continued for further 3 days. Metabolomics and proteomics showed that members of the genus Prevotella remained most abundant under monensin supplementation. Furthermore, shifting the ruminal metabolism to an increased production of propionate by shifting the metabolism of Prevotella sp. to an enhanced succinate production. The current work shows the impact of SCFAs on various metabolic functions of P. bryantii B14. Diverse defence mechanisms of Prevotella sp., in particular P. bryantii B14, were shown to avoid the antibiotic effects of monensin.
Prediction of ruminal acidosis in dairy cows from milk constituents
(2022) Seyfang, Gero Marc; Rodehutscord, Markus
Subacute rumen acidosis (SARA) is a common, but hardly assumable disease in modern dairy cows’ herds. SARA incidences are prevalent in two circumstances. The first, when the cows have to adapt fast to a ration high in carbohydrates after parturition. Since the feed composition has to be changed fast, to meet the cows’ requirements energy- and nutrients wise, the rumen microbiota climate has to adapt fast, which can cause unbeneficial rumen circumstances. The second, when the lactating cows have, beside high milk yield also a high feed intake in mid-lactation, when feed high in energy but low in structural carbohydrates is fed. This can lead to high density of VFAs in the rumen, if the outflow and absorption through the ruminal wall, as well as the buffer capacity in the rumen is not sufficient for the high production of those acids. Then the ruminal milieu becomes more acid, which can negatively affect the cow’s health. The cows suffering SARA, if at all, show mild symptoms like reduced water and feed intake, depression, diarrhea, reduced rumen motility, laminitis or reduced milk yield and milk fat depression. Since those symptoms can also show up with a delay in time and can be caused by several other factors, monitoring SARA in herds can be difficult. An unambiguous definition of SARA circumstances in the rumen cannot be found in literature, although it is under research for decades. Since SARA can influence the milk yield and can lead to a milkfat depression and a change in composition of milkfat, we focused on milk parameters and milkfat composition in particular with the aim of correlating those with pH conditions in the rumen. Three trials were made with feeding rations that were predictably capable of inducing SARA conditions in mid-lactation. During the trials, besides performance and ruminal parameters, as well as continuous pH measurement, milk samples were taken. The cows used were all rumen cannulated. Therefore, datalogger with integrated pH meter (Large Ruminant Logger M5-T7, Dascor Inc., Oceanside, USA) were placed in the ventral sac of the rumen to measure reliably and continuously. In Trial 1, three feeding rations with constant 20% grass silage were used. One ration consisted of additionally 20% corn silage and 60% concentrate (treatment CS60), the other two rations included 20 respectively 60% pressed sugar beet pulp silage and 60 respectively 20% concentrate (treatments SBPS60 respectively SBPS20). With those rations, low pH values were induced in the rumen, leading to SARA incidences of 89% in the measured days in the CS60, 100% in the SBPS60, and 61% in the SBPS20 treatment. In Trial 2, for all three rations a fix concentration of 52% concentrate was used. The remaining 48% consisted of corn silage (treatment CS), grass silage (treatment GS) or hay (treatment Hay). In the CS treatment, SARA incidence was 23%, while the GS and Hay treatments did not show SARA incidence. While the first two trials were designed as a 3x3 Latin square, in Trial 3 the cows remained in their respective treatment. One group stayed in the barn with a TMR, including 30% concentrate (treatment CG), while the other group was full time grazing and got additional 1.75 kg concentrate per day (treatment PG). SARA incidences were 7% in the CG and 8% in the PG. Additionally, in an intertrial approach, regression models for SARA detection were developed. Therefore, easily accessible performance data from the barn and milk parameters from the official milk control and milk fatty acids were used to estimate the rumen parameters pH mean and the time spent below pH 5.8. One first model was designed to include 63 variables. Besides 11 parameters gained in the barn or from the official milk control, also 52 parameters that were gaschromatographically detected fatty acids and sums of these fatty acids. A second model was designed to be useable if no gaschromatographical milkfat analysis was available. Therefore, only those FAs were included that can be estimated in a good quality with MIR spectroscopy. With those regression models the SARA days from the 185 measurement days were calculated to test the accuracy of the models. From the original 47 SARA days the first model was able to detect 43 days and the second model detected 39 SARA days. Although the accuracy of SARA prediction based on these models might be too inaccurate for a decision if a single day was SARA prevalent or not, an information on herd basis seems assessable. Still the small number of cows and measured days, as well as the fact that two breeds of cows and only cows in the later lactation phase were integrated in the model establishment has to be considered and further developed before it becomes a useful tool in field use for SARA detection.
Entwicklung und ernährungsphysiologische Bewertung mikrobieller Hybrid-Phytasen
(2023) Metten, Alexander; Rodehutscord, Markus
To degrade the organic phosphate storage in the best possible way, it is necessary to increase phytase efficiency in vivo. Both a better understanding of the influencing factors limiting phytate degradation in vivo and a continuous improvement of the biochemical properties of phytases to be best adapted to the conditions in the digestive tract of non-ruminants will help to achieve this. Therefore, the main objective of this work was the generation of a large number of sequentially unique hybrid phytases by directed recombination of known phytase genes with the goal to achieve improved biochemical properties compared to the wild-type phytases used. The focus of this work was the biochemical and nutritional evaluation of the newly generated hybrid phytases with respect to their suitability as feed supplements. All hybrid phytases examined showed more efficient InsP6 degradation at pH 3.0 than at pH 5.5, although the phytase activity supplemented was the same at both pH values. While InsP6 was dephosphorylated to InsP1-2 in many cases at pH 3.0, accumulation of the Ins(1,2,5,6)P4 isomer occurred at pH 5.5. In an in vitro model simulating the digestive tract of broilers, hybrid phytases with high sequential homology to the E. coli and C. braakii phytase showed high accumulation of InsP4 isomers. Interestingly, these phytases preferentially formed the Ins(1,2,5,6)P4 isomer. In contrast, other hybrid phytases were able to degrade all InsP4 isomers and in some cases high InsP2 concentrations were observed. Another in vitro experiment with a complex feed matrix consisting of soybean meal, rapeseed meal, and wheat with a high mineral content, illustrated the negative influence of certain feed-related factors on phytase efficiency. InsP6 present in the feed was significantly less degraded by all phytases used compared to a corn and soy-based feed matrix with a low mineral content. While a hybrid phytase was able to completely dephosphorylate the InsP6 of the corn and soy-based feed matrix down to the InsP3 isomer resulting in high InsP2 concentrations, the InsP6 were still detectable in the in vitro model with the more complex feed matrix and high mineral content, despite identical reaction conditions. In a final feeding trial with broilers, one of the hybrid phytases was supplemented at two doses each (500 and 1500 FTU/kg) to evaluate its suitability as a feed supplement. Also, a commercial phytase was included in the study design at the same doses setting the benchmark for phytase efficiency. A low phosphorus experimental feed based on corn and soybean meal was used. The supplementation of the used hybrid phytase resulted in a dose-dependent increase in broiler performance data such as daily weight gain, feed intake and significantly improved feed efficiency compared to the basal ration without enzyme supplementation. In addition, foot ash content was increased by 21.6% at a dose of 1500 FTU/kg phytase, indicating significantly improved bone mineralization due to the released InsP6 phosphate. By analyzing InsP6 concentration and its degradation products in different segments of the digestive tract, efficient InsP6 degradation was observed. In contrast to the in vitro experiments, no accumulation of InsP3-4 isomers could be detected in crop, gizzard or small intestine. In addition to a high exogenous phytase activity, this result also suggests a high endogenous phytase as well as phosphatase activity in the digestive tract of broilers. It can be assumed that the absence of monocalcium phosphate in the experimental rations may have induced the expression of endogenous phytases and phosphatases. This assumption is confirmed by the high precaecal InsP6 degradation, which was 63.5% in the basal ration without phytase supplementation. Nevertheless, the used hybrid phytase significantly increased the precaecal InsP6 degradation to 76.3%. The high phytase efficiency was also reflected in the measured precaecal phosphorus digestibility, which was increased by 6.8% compared to the basal ration. The commercial phytase used showed comparable improvement in broiler performance data to the non optimized hybrid phytase. This project demonstrated the development of a variety of sequentially unique hybrid phytases by recombination of known phytase genes, which exceeded the biochemical properties of the wild-type phytases in some relevant aspects. Some of the phytases showed very efficient phytate degradation when simulating the digestive tract of broilers in vitro. Also, the suitability of the tested hybrid phytase as feed additives was demonstrated by the increased performance data of broilers. The higher performance data of the broilers could be attributed to efficient phytate degradation. To achieve maximum InsP6 degradation in vivo, the feed-related and animal-related factors on phytase efficiency need to be better understood.
Effects of a reduction of dietary levels of calcium and phosphorus on performance, bone minerals and mineral excretion of turkey breeder hens in the rearing and laying period
(2023) Gickel, Julia Maria; Rodehutscord, Markus
Phosphorus (P) is an essential mineral in feed for livestock and has finite resources all over the world. The aim of this study was to obtain an idea about the reduction potential of P in the diets of turkey breeders. As the metabolism of P is interlinked with the metabolism of calcium (Ca), Ca was also examined. Therefore, the requirements of P and Ca were studied using a factorial approach. As data about the requirements of these minerals in turkey breeders is limited, the present study mostly used data from other poultry species. Thus, this study can be viewed as an approach to building new resilient data for turkey breeder hens. The results of the factorial approach were embedded in a feeding program for turkey breeder hens during rearing and laying with two different treatments. One group was fed a standard feed (practical diet used before the trial), while the other group was fed a Ca/P reduced feed. In total, four trials were conducted: trials I and III focused on the rearing period while trials II and IV focused on the laying period. All trials were observed independently but the hens from trial I were used also in trial II afterwards for studying long-term effects. In each trial, body weight and feed intake were measured throughout the trial period. Bone mineralization was studied in trial I, including analyses of bone ash as well as Ca and P in the bone ash from the tibia of fallen and culled animals. The egg components were studied in eggs from trial II, including analyses of the percentages of albumen, egg yolk, and egg shell; Ca in albumen; P in albumen; Ca in egg yolk; P in egg yolk; water in egg shell; Ca in egg shell; and P in egg shell. In trials II and IV, laying performance, egg weights, number (and causes) of culled eggs, fertility, hatchability, as well as body weight and fitness of the hatchlings were additionally observed. At the end of each trial, the concentrations of dry matter, Ca, and P in manure were analyzed and a nutrient balance was calculated to classify the results. Body weight development exhibited significant differences between the treatments in eight out of 30 weeks in trial I (five weeks had a higher mean weight in the group with standard feed; three weeks had a higher mean weight in the group with Ca/P reduced feed); three out of 28 weeks in trial II (two weeks had a higher mean weight in group with Ca/P reduced feed; one week had a higher mean weight in the group with standard feed); four out of 30 weeks in trial III (higher mean weight in the group with Ca/P reduced feed); and two weeks out of 28 in trial IV (higher mean weight in the group with Ca/P reduced feed). The feed intake data also fluctuated, with three out of 30 weeks exhibiting a significant difference in trial I (higher mean feed intake in the group with Ca/P reduced feed) and one week with a significant difference in trial II (higher mean feed intake in the group with standard feed). The observations throughout all of the trials revealed the tendency for a higher mean feed intake in the groups with Ca/P reduced feed. A significant difference concerning bone mineralization was not observed. Moreover, the results of laying performance, fertility, and hatchability exhibited no differences between the treatments. Regarding the egg weight in trial II, there were eight weeks with a significant difference between the treatments (seven weeks with a higher mean egg weight in the group with standard feed; one week with a higher mean egg weight in the group with Ca/P reduced feed). In trial IV, only one week exhibited a significant difference between the treatments (higher mean egg weight in the group with Ca/P reduced feed). Regarding the weight of hatchlings in trial II, three weeks had a significant difference between the groups (higher mean weight in the group with standard feed). In trial IV, no significant difference was observed for any week all throughout the trial period. The analyses of egg components revealed significant differences between the treatments according to the percentages of albumen and egg yolk and the calculated value of total Ca in the egg (including the shell). In trials I and III, significant differences existed between the treatments in the concentration of Ca in manure, with a lower level in the group with Ca/P reduced feed. This study concluded that a reduction in the dietary levels of Ca from 1.00–1.10% to 0.56–0.80% and of P from 0.48–0.61% to 0.35–0.50% (av. P) or 0.70–0.80% to 0.50–0.60% (total P) in rearing and a reduction of the dietary levels of Ca from 2.90% to 2.80% or 2.60% and of P from 0.36% to 0.30% or 0.24% (av. P) or from 0.65% to 0.50% (total P) in laying in the feed of turkey breeder hens are possible and did not result in disadvantages. As the present study also compared the results with required recommendations and target levels from breeding companies, it was also able to conclude that these levels are obsolete and should be adjusted downwards.
Mitochondrial haplotypes, gene expression and nuclear diversity in two strains of laying hens
(2021) Dreyling, Clara; Hasselmann, Martin
The domesticated chicken (Gallus gallus domesticus) is the most popular and widely spread domestic fowl worldwide, providing human with a stable source of protein in form of meat and eggs for centuries. The ongoing growth of human population increases the need for food and made poultry production one of the fasted growing sectors in the past decades. This need for food has resulted in several different strains which outperform their wild ancestors in terms of meat and egg production. During the past decades not only animal welfare gained importance but also ecological aspects such as global warming and the shortage of resources are becoming more important to society. One important resource for mankind which is becoming shortened is phosphorus (P), whose deposits in form of rock phosphate could be exhausted within the next 50-100 years. 90% of P supply is used in agriculture as fertilizer, whose demand will increase as well with growing population. This thesis focuses on the mitochondrial genetic background and mitochondrial related gene expression in the context of the productive life span and different diets in two contrasting high-yielding strains of laying hens, Lohmann Brown-Classic (LB) and Lohmann LSL-Classic (LSL). Mitochondria, which are commonly known as the powerhouse of the cell due to their role as the main producer of energy, play roles in other processes from cellular homeostasis to the process of ageing. The process of oxidative phosphorylation depends on the availability of P and thus, they become an important part of the complex framework of P utilization. In addition, mitochondrial haplotypes are known to affect physiological traits such as body weight in laying hens or important traits such as e.g. the metabolic capacity in dairy cows. It is known, that single mutations in the mitochondrial genome lead to a better adaptation to height in the Tibetan chicken or play a role in diseases from Alzheimer to obesity or lead to resistance to disease such as Marek’s disease in birds. This work provides insight into the whole mitochondrial genome of 180 laying hens of two commercial strains and links this information to physiological traits and genetic diversity. In addition, the first large-scaled gene expression analyses in the context of the productive life span and different P and Ca contents in laying hens is implemented. The analysis of mitochondrial haplotypes revealed a low level of genetic diversity with only three haplotypes within the LB strain while all LSL hens shared the same mitochondrial genome. Following from this observation, the nuclear genome was analysed based on genotyping data to reveal the whole genetic diversity of both strains. On the nuclear genetic level, both strains appeared as clearly distinct and equally diverse, while some individuals appear as strikingly close related. These individuals are mostly half-siblings sharing the same mitochondrial haplotype, underlining the need for more analyses about the genetic structure about the parental generation, especially the maternal background. Although there were no strong associations were found between the mitochondrial haplotypes and the analysed phenotypic traits (feed intake, body weight, P and Ca utilization), the differences between the strains indicate a potential involvement of the mitochondrial genetic background. The gene expression analyses revealed tissue type and point of the productive life span as the main influencers on gene expression while the influence of the strain is secondary. In addition, the expression of the gene GAPDH, which is frequently used as a reference gene for normalization in gene expression studies, was influenced by tissue and strain, leading to the decision to exclude it as a reference, that should be considered for in further studies. Further, no influence of the changes in dietary P and Ca on gene expression could be observed, suggesting that a reduction of 20% of both minerals is possible without the need to adapt gene expression. However, the results show, that a reduction of both minerals has less effect than a reduction of P alone, leading to an imbalance. In the context of the productive live span, mitochondrial and mitochondrial regulatory genes react contrary, illustrating the complexity of mitochondrial gene expression and regulation. In addition to the higher variance in the analysed phenotypic traits and mitochondrial genome in LB hens, they showed signs of increased oxidative stress compared to LSL hens. In the context of the productive life span, a potential higher demand for energy is suggested, since OXPHOS related gene expression is increasing. As a conclusion this work provides an insight into the mitochondrial genome and provides the first large scaled analysis of mitochondrial linked gene expression in two contrasting laying hen strains.
Impact of age and weaning time on the gut microbiome and the potential host-microbe interactions in calves
(2021) Amin, Nida; Seifert, Jana
The period from birth until the end of weaning is critical for calves as they undergo extreme stress caused by maternal separation, transportation, and weaning related dietary shifts, that can cause long-lasting effects on animal behaviour, health as well as future production parameters. Monitoring the development of microbial ecosystem throughout the gastrointestinal tract of calves and host-microbe interactions during the challenging life periods such as perinatal and weaning is essential for sustainable ruminant production. The present thesis provided new insight on the suitability of buccal swabs as an alternative to complex stomach tubing method for predictive analysis of rumen microbial communities. The changes in oral, rumen and faecal microbial community structure of female German Holstein calves from 8-days to 5-months of age as well as during early- and late-weaning event were identified. The oral microbiota plays a crucial role in animal health. A high dominance of oral pathogens was observed during the first 11-weeks of calves’ life. Similar to the oral microbiota, faeces of 8-day-old calves also showed high abundances of certain opportunistic pathogenic bacteria. Both oral and faecal pathogens showed a decrease in abundance with age and after weaning event in the earlyC group, indicating the age and weaning-dependent maturation of the host immune system. The establishment of dense microbial communities in the faeces of 8-day-old (experimental day 1) pooled herd milk and milk replacer fed Holstein calves was shown and it was dominated by phyla Firmicutes and Actinobacteria and potential lactose- and starch-degrading bacterial species, but as the calves aged and became more mature (5-months of age), their rumen and faecal bacterial communities were dominated by potential fibre-utilizing bacterial genera. The weaning related dietary transitions are critical for calves as their gastrointestinal tract undergoes several modifications, enabling them to digest plant-based diet during the postweaning period. Thus, it was proposed that the age at which animals should be weaned must be carefully considered as it clearly impacted the gastrointestinal tract microbial communities and plasma metabolic profiles of calves in the present study. Early introduction of roughages in the diet of 7-week-old calves increased the abundances of plant fiber degrading bacteria and decreased the abundances of potential lactose- and starch-degrading bacteria in the buccal cavity, rumen and faeces, indicating the weaning-related increase in fiber ingestion and the decrease in milk consumption of the early-weaned group. However, when roughages were introduced in the diet of late-weaned calves at 17-weeks of age, no significant modifications in the structure of gastrointestinal tract microbial communities were observed. Similar to the microbiome, plasma metabolic profiles of early-weaned calves during days 42–112, showed lower concentrations of most of the amino acids, few biogenic amines, and sphingomyelins as compared to the late-weaned calves, suggesting that the liquid diet could provide certain metabolites that can be transported into the bloodstream through gastrointestinal tract. Similarly, the weaning-dependent changes in the quantity of dietary protein, fat and carbohydrates resulted in substantial changes in amino acid metabolism of the early-weaned group. The early-weaning event not only impacted the host microbiome and metabolome but also the host-microbe metabolic interactions as the abundances of potential lactose- and starch degrading bacteria and plasma concentrations of amino acid, biogenic amines and sphingomyelins were strongly positively correlated, both were negatively impacted by the early-weaning event. Thus, it can be concluded that late-weaning was beneficial as it allowed better adaptability of microbes to weaning-related dietary shifts, perhaps due to the greater maturation of their gastrointestinal tract with age as compared to the early-weaning group.
Diagnostics and genome analysis of phloem-limited phytopathogenic bacteria
(2022) Zübert, Christina; Hölzle, Ludwig
This thesis contributes to improving the epidemiological understanding of ‘Candidatus Arsenophonus phytopathogenicus’ and bacteria of the provisional taxon ‘Candidatus Phytoplasma’ by applying genetic markers for identification, differentiation, and phylogenetic reconstruction of this economically relevant plant pathogens.
The intestinal microbiome and metabolome of dairy cows under challenging conditions
(2022) Tröscher-Mußotter, Johanna; Seifert, Jana
The modern dairy cow is confronted with a multitude of stressors throughout live. Especially calving, transition, and microbial infections are strong challenges that can have long-lasting impacts on the cow’s health and performance. Yet, individuals can differ in their response towards these challenges, raising the question which characteristics in the dairy cow contribute to a more or less robust animal. Apart from genetics, the gut microbiome and the entailed metabolome is assumed to play an important role in buffering or promoting host stress. This is also due to the fact that the gut microbiome is strongly involved in the hosts energy metabolism and immune system. As dairy cows often show performance impairments during high energy demanding periods, it could be suggested that improving energy metabolism in these specific phases might reduce the negative phenotypic outcomes. This was tested using dietary L-carnitine, a metabolite inevitably necessary for energy metabolism. However, no supplement effects on the intestinal microbiome or metabolome have been found in the present work. Supplementation was continued throughout the complete trial. Calving functioned as an individual stimulus, and an intra-venous LPS injection induced a standardized inflammatory challenge, as a specific amount of LPS per kg of bodyweight was applied per cow. Supplemented animals were compared to a control group. In total, the animals were studied across 168 days and sampled extensively at several sites. The focus of this thesis was to analyze the bacterial consortia and metabolites of both, host and bacteria, in rumen, duodenum, and feces throughout the given period. This was to elucidate the metabolic reactions and bacterial shifts during the mentioned challenging periods and their response to the L-carnitine supplementation. First, the ruminal and duodenal fluid microbiome of eight double cannulated animals during the two respective challenges was analysed. Before calving and feed change, rumen and duodenal fluid bacterial consortia were significantly different, thereafter very alike. Strong microbial community shifts were observed throughout the complete trial irrespectively of the matrix. Both matrices varied in their metabolite patterns indicating functional variation among sites. Also, a strong increase of Bifidobacterium at three days after calving was observed in almost all animals pointing towards a strong biological purpose. This needs to be investigated in upcoming studies. The study could show increasing ketogenic activities in the animals after calving and proposes a possible protective host-microbial interaction, against a ruminal collapse induced by LPS challenge, here described as "microbial airbag". The second part included fecal samples of the same animals, which were analyzed for their bacterial consortia and targeted metabolites. Different dynamics and diversities of microbial communities amongst the individuals were observed, according to which animals could be grouped into three microbiome clusters. These showed in part fundamentally different metabolic, health, and performance parameters, indicating strong host-microbiome-metabolite interactions. The study demonstrated that microbiome clustering may contribute to identifying different metabo- and production types. Again, the study observed a strong increase of Bifidobacterium at three days after calving and even during the LPS challenge supporting the findings of the former study. This strengthens the hypothesis that also for the cow Bifidobacterium may have protective effects, as this genus is largely involved in health promoting activities. The power of this project lies in the massive sampling of different body sites in dairy cows across a very long period of time and finally, merging of the collected data. This, however, requires high computational efforts as numerous time points, matrices, animals, measurements, treatments, feeding regimen, and challenges resulted into a large bandwidth of parameters and metadata. Yet, it bears the potential to better elucidate and understand actions and reactions of the host, its microbiome and metabolism, as well as organ-axes in dairy cows and thereby gaining a more holistic picture of these complex animals. The aim of analyzing the host, its microbiome and metabolome throughout challenging periods resulted into the following main findings. Time, calving, and feed change remarkably change the microbial communities and to a lesser extent the metabolomes in all three matrices. Rumen and proximal duodenal fluid samples significantly differ in their metabolomes but not in their microbiome. In all matrices, an increase of Bifidobacterium is seen within three days after calving, which has to be further researched. Across the herd, three distinct microbiome clusters are found, which significantly differ in their production and health parameters.
Variation and estimation of nitrogen utilization efficiency in a crossbred pig population
(2022) Berghaus, Daniel; Rodehutscord, Markus
Efficient utilization of dietary nitrogen (N) in pork production is of increasing concern. Previous studies revealed that a genetic basis for N utilization efficiency (NUE) might exist, but to assess the potential of breeding for improved NUE, the between-animal variation of a large number of animals needs to be known. The standard method to determine N retention (NR) in balance trails is laborious and not feasible for the required numbers of animals. However, correlations between protein utilization and blood urea nitrogen (BUN) concentration have been shown to exist and body protein turnover is subject to hormonal control. Hence, the objective of the present thesis was to quantify NR of growing pigs at two different growth stages by N balance and to determine the impact of body protein turnover on NUE. In addition, equations for the estimation of NR were established, using performance data and blood metabolite concentrations, which were applied to evaluate the variation in NUE of a F1 crossbred population. Over a period of 2.5 years, a total of 508 crossbred pigs (German Landrace x Pietrain) from 20 different boars was investigated from the 11th week of life until slaughter. The pigs were housed individually throughout the experimental period and a two-phase fattening was performed. All animals received the same diet for ad libitum intake which was formulated to contain 90% of the recommended lysine concentration so that marginal lysine supply was the limiting factor for protein retention and pigs were allowed to express their full genetic potential of NUE. In both fattening phases, daily feed intake was recorded for each animal in a five-day sampling period (SP), and blood samples were taken from the jugular vein at around 13:00 h on three consecutive days for determination of BUN, cortisol, and insulin-like growth factor 1 (IGF-I) concentration. Additionally, in both SP, N balance was performed in the same experimental barn on a randomly selected subsample of 56 barrows. The barrows were housed in metabolism crates for six days, two days for adaption and four days for quantitative collection of feces and urine. Simultaneously, their body protein turnover was determined using the end-product method after a single oral dose of 15N-labeled glycine. Based on the N balance results, models for estimation of NR were obtained by multiple regression of performance data and blood metabolite concentrations. The significance of the variables was validated using a bootstrapping method to avoid overfitting the models to the observed data. The goodness of fit of the equations was assessed using the coefficient of determination and the root mean square error. The N balance results revealed a high protein retention potential of the animals, which did not differ on average between the two SP. However, large differences in NR were observed between individuals and NR was strongly correlated with N and lysine intake. NUE was also at a high level and varied considerably between individuals. The mean NUE was significantly higher in SP1 than in SP2 and a moderate correlation was observed between NR and NUE. The mean body protein turnover did not differ between the SP and no correlation with NUE was observed. In estimating NR, the model with the best goodness of fit included the variables initial body weight, average daily gain, average daily feed intake, N intake, BUN, cortisol, and IGF-I concentration. This model was used to estimate NR for all animals and subsequently calculate their NUE. Describing NR as a linear function of lysine intake across both SP showed an average marginal efficiency of lysine utilization for protein retention of 67%. Despite a wide variation in NUE within the offspring of the same boars, significant differences were found between the offspring groups of the boars. Under the prevailing circumstances of marginal lysine supply, the NR of fattening pigs could be estimated from performance data and blood metabolite concentrations with satisfying accuracy. This provides a fast and reliable alternative to performing N balance studies, reducing the experimental effort considerably in studies with large numbers of animals. Although lysine supply was the limiting factor for protein retention, only about 70% of the variation in NR could be explained by the level of lysine intake. The remaining part of the variation was likely caused by differences in the intermediary lysine utilization or differences in the lysine content of the retained body protein between individuals. About 50% of the variation in NUE could be explained by differences in the level of NR, implying that pigs with higher protein retention potential utilized dietary N more efficiently. However, this was not accompanied by differences in body protein turnover. Phenotyping of the F1 crossbred population revealed a large variation between individuals and a significant boar effect, indicating the possibility of improving NUE through breeding measures.
Untersuchung der spatio-temporalen Verbreitung von Brucellose-Ausbruchsstämmen in Ägypten mittels cgSNP-Analyse und Multi Lokus VNTR-Analyse
(2022) Holzer, Katharina; Beyer, Wolfgang
Brucellosis is a worldwide zoonosis caused by the genus Brucella, which includes 12 species, and it is of great importance for the public health sector. In animals, the disease can lead to abortions, which entails high economic losses. The disease can be transmitted from animals to humans directly by penetrating through mucous membranes and wounds in the skin, or indirectly by consuming unpasteurized milk, milk products made from it, or meat that has not been sufficiently heated. The bacteria can also be transmitted via aerosols, which among other things categorizes these pathogens in risk class three. Contact with infected animals, especially their reproductive organs, aborted fetuses and discharges therefore represent a risk factor. The disease in humans is serious, an antibiotic treatment lasting several weeks is necessary. Although many countries are considered brucellosis-free, African countries, for example, are still badly affected. This work focuses on Brucella from Egypt. Although brucellosis is endemic in Egypt, there is just one publication with very little data about outbreak analysis of the species Brucella (B.) melitensis and B. abortus based on whole genome sequencing. Some other earlier publications are based on the so-called Multiple Locus VNTR analysis (MLVA), which in this present work turned out to be unsuitable for epidemiological analysis or outbreak analysis. VNTR means variable number of sequence repetitions in the DNA. In order to make concrete statements on outbreak analysis, a so-called single nucleotide polymorphism analysis of the core genome (cgSNP analysis) was carried out based on whole genome sequencing. While the MLVA includes highly mutable DNA regions, in the cgSNP analysis they are excluded. Therefore, the cgSNP analysis enables a more precise classification of the genotypes and thus also the classification of the outbreak strains, while the MLVA can just be used for the differentiation of isolates due to the use of highly mutable sequence regions. This became clear through the direct comparison of both methods including the metadata, so that previously published data that described the outbreak analysis for Brucella using the MLVA cannot be used. Over and above that in contrast to an in silico MLVA based on genome sequencing, a laboratory-based MLVA is prone to errors. In a direct comparison, the in silico MLVA showed reliable results in 100% of the cases and should therefore replace the laboratory-based MLVA. For the cgSNP analysis a total of 185 isolates from animals and humans were available. Out of a total of 185 samples, 137 were classified as B. melitensis and 49 as B. abortus, suggesting that B. melitensis infections are more common than B. abortus infections in Egypt. Furthermore, conserved outbreak strains in Egypt, detected by the cgSNP analysis and genetically differing only minimally, have persisted for several years and have spread or are still spreading, as well as constantly newly introduced outbreak strains or infections. Among the B. abortus isolates, B. abortus RB51 vaccine strains from aborted animals could be detected, confirming that the RB51 vaccine strain also causes abortions. While most B. melitensis strains are assigned to West Mediterranean origin, one isolate each is assigned to American and East Mediterranean origin. Such a classification by a so-called canonical SNP assay (canSNP assay), as was carried out for the B. melitensis isolates, is not possible for B. abortus for methodological reasons. A further cgSNP analysis was used to check existing public database entries from other countries for possible sources of introduction of the detected Egyptian outbreak strains. Italian origin is likely for most B. melitensis isolates, while UK origin is likely for a certain group of B. abortus isolates. The remaining B. abortus isolates could not be assigned unequivocally, but would possibly be close to the isolates from the USA. In order to confirm or refute possible relatives, much more isolates from different regions are necessary for a comparison. Due to the large number of different outbreak strains in Egypt, the origin of these outbreak strains should be outside the country, so that importations can be assumed.

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