Browsing by Person "Seifert, Jana"

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The active core microbiota of two high-yielding laying hen breeds fed with different levels of calcium and phosphorus
(2022) Roth, Christoph; Sims, Tanja; Rodehutscord, Markus; Seifert, Jana; Camarinha-Silva, Amélia
The nutrient availability and supplementation of dietary phosphorus (P) and calcium (Ca) in avian feed, especially in laying hens, plays a vital role in phytase degradation and mineral utilization during the laying phase. The required concentration of P and Ca peaks during the laying phase, and the direct interaction between Ca and P concentration shrinks the availability of both supplements in the feed. Our goal was to characterize the active microbiota of the entire gastrointestinal tract (GIT) (crop, gizzard, duodenum, ileum, caeca), including digesta- and mucosa-associated communities of two contrasting high-yielding breeds of laying hens (Lohmann Brown Classic, LB; Lohmann LSL-Classic, LSL) under different P and Ca supplementation levels. Statistical significances were observed for breed, GIT section, Ca, and the interaction of GIT section x breed, P x Ca, Ca x breed and P x Ca x breed (p < 0.05). A core microbiota of five species was detected in more than 97% of all samples. They were represented by an uncl. Lactobacillus (average relative abundance (av. abu.) 12.1%), Lactobacillus helveticus (av. abu. 10.8%), Megamonas funiformis (av. abu. 6.8%), Ligilactobacillus salivarius (av. abu. 4.5%), and an uncl. Fusicatenibacter (av. abu. 1.1%). Our findings indicated that Ca and P supplementation levels 20% below the recommendation have a minor effect on the microbiota compared to the strong impact of the bird’s genetic background. Moreover, a core active microbiota across the GIT of two high-yielding laying hen breeds was revealed for the first time.
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.
Antiviral defense systems in the rumen microbiome
(2025) Sáenz, Johan S.; Rios-Galicia, Bibiana; Seifert, Jana
The continuous interaction between phages and their respective hosts has resulted in the evolution of multiple bacterial immune mechanisms. However, the diversity and prevalence of antiviral defense systems in complex communities are still unknown. We therefore investigated the diversity and abundance of viral defense systems in 3,038 high-quality bacterial and archaeal genomes from the rumen. In total, 14,241 defense systems and 31,948 antiviral-related genes were identified. Those genes represented 114 unique system types grouped into 49 families. We observed a high prevalence of defense systems in the genomes. However, the number of defense systems, defense system families, and system density varied widely from genome to genome. Additionally, the number of defense system per genome correlated positively with the number of defense system families and the genome size. Restriction modification, Abi, and cas system families were the most common, but many rare systems were present in only 1% of the genomes. Antiviral defense systems are prevalent and diverse in the rumen, but only a few are dominant, indicating that most systems are rarely present. However, the collection of systems throughout the rumen may represent a pool of mechanisms that can be shared by different members of the community and modulate the phage–host interaction.
Central carbon metabolism, sodium-motive electron ransfer, and ammonium formation by the vaginal pathogen Prevotella bivia
(2021) Schleicher, Lena; Herdan, Sebastian; Fritz, Günter; Trautmann, Andrej; Seifert, Jana; Steuber, Julia
Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.
Characterization of host and bacterial proteins in crossbred grower pigs at marginal lysine concentration
(2025) Kurz, Alina; Seifert, Jana
The current pork production strategies still result in high nitrogen emissions and thus harmful effects on the environment. The animals excrete about two-thirds of the protein in the diet unused, which increases nitrogen emissions and makes feeding the animals according to their needs expensive and inefficient. To minimize the negative aspects of pork production, it is important to reduce the use of proteins in feed while improving the protein efficiency of pigs. Improving protein efficiency requires a comprehensive understanding of physiological processes, particularly enzymatic activities, amino acid transporter capacities, the proteome, the metabolome and the microbiome. The aim of this work was therefore to identify animal- specific and quantitatively expressed proteins, enzymes, amino acid transporters and metabolites and to correlate these findings with protein turnover data. The study also examined whether the efficiency of protein utilization is influenced by these variables. A total of 48 male crossbred animals (German Landrace x Pietrain) were slaughtered in the 21st week of life for the present experiment. The animals were kept individually under the same conditions from the 75th day of life until the day of slaughter. A two-phase ad libitum feeding was carried out, with the change from phase 1 to phase 2 taking place in the 14th week of life. Since the genetic potential of protein utilization efficiency was to be estimated, the content of the first limiting amino acid lysine was 90 %, just below the GfE (2006) requirement limit in order to ensure a limiting factor for the protein approach. Thus, lysine was the limiting factor for protein retention and allowed animals to exploit their full genetic potential for protein utilization. After euthanizing, the animals were opened, and the stomach, small intestine and large intestine removed. Mucosal samples were taken from the sections of the pars nonglandularis, cardiac gland zone and pars pylorica, as well as digesta samples from the stomach. In addition, digesta and mucosal samples were collected from the duodenum, jejunum, ileum, colon, and cecum. The proteome in all samples was analyzed. The metabolome and enzyme activities were determined in all digesta samples. In addition, amino acid transporter expression was examined in all small intestinal mucosal samples. The activity of the enzymes trypsin, chymotrypsin, carboxypeptidase A and carboxypeptidase B were examined in the digesta samples from all sections. The highest activities were found in the small intestine, the lowest activities in the stomach and large intestine. The trypsin and chymotrypsin activity was highest in the jejunum, whereas the activities of the carboxypeptidases were highest in the duodenum. In addition, individual animal differences in enzyme activities could be identified. In addition, the expression rate of various amino acid transporters was examined in the present work. In the duodenum the transporter SLC1A5 showed the highest expression rate, in the jejunum the transporters SLC5A1 and SLC6A19. In the ileum, the rates of the transporters SLC1A1, SLC7A1 and SLC7A9 were at the highest level. The identification of the host proteome revealed different protein patterns in the examined sections of the gastrointestinal tract. In particular, host proteins were identified in the mucosa samples, with the exception of those from the ileum and cecum. The high number of host proteins in the mucosa samples, which are associated with cellular processes and metabolism, underlines its role in physiological digestive processes. In addition, the examined host proteins could be assigned to numerous KEGG pathways, thus creating a deeper understanding of physiological and metabolic pathways. The study of the bacterial proteome allowed not only categorization into different metabolic pathways, but also identification of the active microbiome in the different sections. For example, it was shown that Firmicutes dominate in the stomach and small intestine, whereas Bacteroidetes are mainly found in the large intestine. This highlights the different roles of the sections examined, with Firmicutes being primarily responsible for breaking down proteins and carbohydrates and Bacteroidetes playing a central role in the fermentation of undigested proteins and carbohydrates in the large intestine. Additionally, the increased presence of Tenericutes in the mucosal samples indicated a possible specialization of this family to the conditions in the mucosal environment, where they interact with host cells and contribute to metabolic processes. The identification of numerous unclassified bacterial groups also shows how complex the composition of the microbiome is and that further research must be carried out in order to be able to fully identify connections. The examination of the various metabolites in the digesta samples from the gastrointestinal tract showed clear differences in the detection rate. While the fatty acids acetate, butyrate, valerate, isobutyrate and propionate were found primarily in the sections of the large intestine, lactate, isovalerate and the amino acids showed a higher occurrence in the sections of the small intestine. This is in line with the scientific knowledge that fatty acids are primarily formed during microbial fermentation in the large intestine and should therefore occur in higher quantities here. Overall, the results of the present work showed that, in addition to physiological differences between the sections of the gastrointestinal tract, there also appear to be significant animal- specific differences in all parameters examined. These differences can have an influence on the efficiency of the animals, but it is still important to find out which factors cause these differences. Since the feeding, husbandry concept and experimental design were chosen in such a way that there is as little variation and discrepancy between the animals as possible, a genetic influence on the efficiency of the animals cannot be ruled out. If the animals' genetic potential to use nitrogen efficiently becomes apparent, a further foundation stone has been laid for making pork production tailored to needs and conserving resources and the environment.
Cow’s microbiome from antepartum to postpartum: a long-term study covering two physiological challenges
(2022) Tröscher-Mußotter, Johanna; Deusch, Simon; Borda-Molina, Daniel; Frahm, Jana; Dänicke, Sven; Camarinha-Silva, Amélia; Huber, Korinna; Seifert, Jana
Little is known about the interplay between the ruminant microbiome and the host during challenging events. This long-term study investigated the ruminal and duodenal microbiome and metabolites during calving as an individual challenge and a lipopolysaccharide-induced systemic inflammation as a standardized challenge. Strong inter- and intra-individual microbiome changes were noted during the entire trial period of 168 days and between the 12 sampling time points. Bifidobacterium increased significantly at 3 days after calving. Both challenges increased the intestinal abundance of fiber-associated taxa, e.g., Butyrivibrio and unclassified Ruminococcaceae. NMR analyses of rumen and duodenum samples identified up to 60 metabolites out of which fatty and amino acids, amines, and urea varied in concentrations triggered by the two challenges. Correlation analyses between these parameters indicated a close connection and dependency of the microbiome with its host. It turns out that the combination of phylogenetic with metabolite information supports the understanding of the true scenario in the forestomach system. The individual stages of the production cycle in dairy cows reveal specific criteria for the interaction pattern between microbial functions and host responses.
Dynamic development of viral and bacterial diversity during grass silage preservation
(2023) Sáenz, Johan S.; Rios-Galicia, Bibiana; Rehkugler, Bianca; Seifert, Jana
Ensilaging is one of the most common feed preservation processes using lactic acid bacteria to stabilize feed and save feed quality. The silage bacterial community is well known but the role of the virome and its relationship with the bacterial community is scarce. In the present study, metagenomics and amplicon sequencing were used to describe the composition of the bacterial and viral community during a 40-day grass silage preservation. During the first two days, we observed a rapid decrease in the pH and a shift in the bacterial and viral composition. The diversity of the dominant virus operational taxonomic units (vOTUs) decreased throughout the preservation. The changes in the bacterial community resembled the predicted putative host of the recovered vOTUs during each sampling time. Only 10% of the total recovered vOTUs clustered with a reference genome. Different antiviral defense mechanisms were found across the recovered metagenome-assembled genomes (MAGs); however, only a history of bacteriophage infection with Lentilactobacillus and Levilactobacillus was observed. In addition, vOTUs harbored potential auxiliary metabolic genes related to carbohydrate metabolism, organic nitrogen, stress tolerance, and transport. Our data suggest that vOTUs are enriched during grass silage preservation, and they could have a role in the establishment of the bacterial community.
Effects of diets with different phosporus availability on the intestinal microbiota of chickens and pigs
(2019) Tilocca, Bruno; Seifert, Jana
In the research works of the present thesis, 16S rRNA gene sequencing and metaproteomics were employed to investigate the gut microbiota of chickens and pigs kept at experimental diets with varying amount of calcium-phosphorus (CaP) and supplemented MP. This represents a valuable approach to investigate the bacterial specimens involved in the P absorption, allowing for a comprehensive understanding of how the intestinal bacteria adapt to a new diet and which metabolic routes are affected by changing levels of supplemented P and/or MP. Two major experimental trials were performed during the investigation. The first one was conducted on chickens operating a modulation in the dietary levels of Ca, P and MP. This trial highlighted a shift in the composition of the crop and ceca-associated microbial community depending on the composition of the diet fed. Also, investigated protein inventory revealed that the stress condition due to the reduced P availability is mirrored in the gastrointestinal tract (GIT)-associated microbiota. Marked differences were observed in the functions of the bacterial community in the case of P-available diets versus P-deficient ones. Protein repertoire of the first case draws a thriving microbial community focused on complex and anabolic functions. Contrariwise, the bacterial community in the case of P-lacking diets appears to deal with catabolic functions and stress response. The second trial was conducted on pigs and attempts to define the dynamics featuring the microbiota adaptation to a new challenging diet composed of different protein sources and varying levels of Ca and P. Statistical evidences reveal a stepwise adaptation of the fecal microbiota to the experimental diets fed. Both DNA-based approach and metaproteomics independently reveal three main adaptation phases: -before the feeding of the experimental trial (i.e. Zero), -the response of the microbial community to the challenging factor (i.e. MA) and, finally, - the newly achieved homeostatic balance (i.e. EQ). As observed in the first trial, feeding of the experimental diets impairs the overall fecal microbiota composition, stimulating the presence of phase-specific bacterial specimens and a characteristic relative abundance of the shared ones. Bacterial families responsible for the phase-specific architecture of the fecal microbiota are also active in the biochemical pathways driving the functional peculiarities of each adaptation phase. A deeper investigation of the identified protein repertoire revealed that the observed statistical differences among the adaptation phases are uniquely due to the Ca and P composition of the diets fed. None of the observed effects can be attributed to the diverse protein sources supplemented with the diets. Functional categorization of the identified protein inventory depicts three diverse functional assets of the microbial community. Specifically, prior the feeding of the experimental diets, bacteria are hypothesized to live under homeostatic condition, since they appear to be involved in complex and highly-specialized functions. Following the administration of the experimental diets microbial community changes its functional priority and reduce the expression of highly specialized functions to focus on more essential ones. Proteins involved in complex functions such as widening the substrates array and facing complex sugars tend to increase in abundance while the new homeostatic balance is achieved. Altogether, data from both trials provide useful information for future studies aimed to design effective breeding strategies finalized to reduce the P supplementation in the routinely breeding of livestock and maintain a balanced microbial activity in the animal GIT. Investigation of the dynamics of the porcine microbiota provides instructions on the minimal exposure time required from the intestinal microbiota to adapt to a new dietary composition. This is of fundamental importance for the design of future studies aimed to confirm and/or continue our results. Moreover, the anatomical and physiological similarities occurring between humans and pigs, make our findings of interest for future human nutritional studies, where the mechanisms and lasts of the microbiota adaptation process is still object of discussion.
Effects of feeding diets without mineral P supplement on intestinal phytate degradation, blood concentrations of Ca and P, and excretion of Ca and P in two laying hen strains before and after onset of laying activity
(2024) Sommerfeld, Vera; Bennewitz, Jörn; Camarinha-Silva, Amélia; Feger, Martina; Föller, Michael; Huber, Korinna; Oster, Michael; Ponsuksili, Siriluck; Schmucker, Sonja; Seifert, Jana; Stefanski, Volker; Wimmers, Klaus; Rodehutscord, Markus
The objective of this study was to characterize intestinal phytate degradation and mineral utilization by 2 laying hen strains before and after the onset of egg laying using diets without or with a mineral phosphorus (P) supplement. One offspring of 10 roosters per strain (Lohmann Brown-classic [LB] and Lohmann LSL-classic [LSL]) was sacrificed before (wk 19) and after (wk 24) the onset of egg-laying activity and following 4 wk placement in a metabolic unit. Diets were corn-soybean meal-based and without supplemented P (P-) or with 1 g/kg supplemented P (P+) from monocalcium phosphate. In wk 19 and 24, the blood plasma and digesta of duodenum+jejunum and distal ileum were collected. The concentration of P in blood plasma was higher in hens fed P+ than P- (P < 0.001). In duodenum + jejunum and ileum content, the concentrations of InsP6, Ins(1,2,4,5,6)P5 and Ins(1,2,3,4,5)P5 were lower in P- than in P+ (P ≤ 0.009). In duodenum+jejunum, the concentrations of InsP6, Ins(1,2,4,5,6)P5 and Ins(1,2,3,4,5)P5 were lower in wk 24 than 19 and lower in LSL than LB hens (P < 0.001). The concentration of myo-inositol (MI) in duodenum + jejunum content was lower in wk 19 than 24 (P < 0.001). Following a 4-d total excreta collection, the retained amount of P was higher in P+ than P- (P < 0.001). Phosphorus retention was lower in LB hens fed P- than in other treatments (P × strain: P = 0.039). In the jejunal tissue, some genes related to intracellular InsP metabolism were higher expressed in LB than LSL hens. The renunciation of mineral P increased endogenous phytate degradation, but more P was retained with supplemented P. Differences in endogenous phytate degradation between the periods before and after the onset of egg laying might be attributed to different Ca concentrations in intestinal digesta caused by different Ca needs in both periods.
Effects of myo-inositol supplementation in the diet on myo-inositol concentrations in the intestine, blood, eggs, and excreta of laying hens
(2025) Sommerfeld, Vera; Hanauska, Anna; Huber, Korinna; Bennewitz, Jörn; Camarinha-Silva, Amélia; Feger, Martina; Föller, Michael; Oster, Michael; Ponsuksili, Siriluck; Schmucker, Sonja; Seifert, Jana; Stefanski, Volker; Wimmers, Klaus; Rodehutscord, Markus
The objectives of this study were to investigate whether an increased dietary myo-inositol (MI) supply translates into changes in MI concentrations and endogenous mucosal phosphatase activities in the intestine of laying hens and whether different laying hen strains respond differently to MI supplementation. The diets were corn–soybean meal-based and supplemented without (MI0) or with 1 (MI1), 2 (MI2), or 3 (MI3) g MI/kg feed. Ten hens per strain (Lohmann Brown-classic (LB) and Lohmann LSL-classic (LSL)) and diet were sacrificed at the age of 30 wk following a 4-wk stay in a metabolic unit. The blood plasma, digesta of the duodenum+jejunum and distal ileum, mucosa of the duodenum, and eggs were collected at wk 30. The concentration of MI in the blood plasma was increased by MI supplementation (P < 0.001); however, that of MI3 did not further increase compared with MI2. The concentration of MI in the duodenum+jejunum and ileum increased steadily (P < 0.001). The MI concentration in the duodenum+jejunum was higher in LB than in LSL hens (P = 0.017). The MI concentration in egg yolk was increased by MI supplementation (P < 0.001) and was higher in LB than in LSL hens (P = 0.015). Strain or diet did not affect mucosal phosphatase activity. Myo-inositol flow at the terminal ileum and postileal disappearance increased with each increment in MI supplementation (P < 0.001) and was higher in LB than in LSL hens (P ≤ 0.041). Regression analysis indicated that, on average, 84% of supplemented MI was retained in the body or metabolized and excreted in a different form. Based on the measured MI concentrations in the blood and eggs, dietary MI was not completely absorbed in the small intestine and, to a different extent, in the two laying hen strains. A higher dietary MI supply was followed by higher intestinal absorption or metabolism by microorganisms. The fate of supplemented MI and its relevance to birds warrant further research.
Evolution of rumen and oral microbiota in calves is influenced by age and time of weaning
(2021) Amin, Nida; Schwarzkopf, Sarah; Kinoshita, Asako; Tröscher-Mußotter, Johanna; Dänicke, Sven; Camarinha-Silva, Amélia; Huber, Korinna; Frahm, Jana; Seifert, Jana
Background: The rumen bacterial communities are changing dynamically throughout the first year of calf’s life including the weaning period as a critical event. Rumen microbiome analysis is often limited to invasive rumen sampling procedures but the oral cavity of ruminants is expected to harbour rumen microbes due to regurgitation activity. The present study used buccal swab samples to define the rumen core microbiome and characterize the shifts in rumen and oral microbial communities occurring as result of calf’s age as well as time of weaning. Results: Buccal swab samples of 59 calves were collected along the first 140 days of life and compared to stomach tubing sample of the rumen at day 140. Animals were randomly divided into two weaning groups. Microbiota of saliva and rumen content was analysed by 16S rRNA gene amplicon sequencing. Our study showed that most rumen-specific bacterial taxa were equally observed in rumen samples as well as in the buccal swabs, though relative abundance varied. The occurrence of rumen-specific OTUs in buccal swab samples increased approximately 1.7 times from day 70 to day 140, indicating the gradual development of rumen as calf aged. The rumen-specific bacterial taxa diversity increased, and inter-animal variations decreased with age. Early weaning (7 weeks of age) rapidly increased the rumen microbial diversity from pre- to post-weaned state. Rumen microbiota of early-weaned calves seemed to have a suppressed growth of starch- and carbohydrate-utilizing bacteria and increased fibre degraders. Whereas, in late-weaned calves (17 weeks of age) no impact of dietary modifications on rumen microbiota composition was observed after weaning. Oral-specific bacterial community composition was significantly affected by calf’s age and time of weaning. Conclusions: The present study showed the significant impact of calf’s age and weaning on the establishment of rumen- and oral-specific bacterial communities utilizing buccal swab samples. The results emphasize the possibility of using buccal swab samples as a replacement of complex stomach tube method for large-scale predictive studies on ruminants. For in-depth rumen microbiome studies, the time of sampling should be carefully considered using an active phase of regurgitation.
Factors influencing proteolysis and protein utilization in the intestine of pigs: A review
(2021) Kurz, Alina; Seifert, Jana
Pigs are among the most important farm animals for meat production worldwide. In order to meet the amino acid requirements of the animals, pigs rely on the regular intake of proteins and amino acids with their feed. Unfortunately, pigs excrete about two thirds of the used protein, and production of pork is currently associated with a high emission of nitrogen compounds resulting in negative impacts on the environment. Thus, improving protein efficiency in pigs is a central aim to decrease the usage of protein carriers in feed and to lower nitrogen emissions. This is necessary as the supply of plant protein sources is limited by the yield and the cultivable acreage for protein plants. Strategies to increase protein efficiency that go beyond the known feeding options have to be investigated considering the characteristics of the individual animals. This requires a deep understanding of the intestinal processes including enzymatic activities, capacities of amino acid transporters and the microbiome. This review provides an overview of these physiological factors and the respective analyses methods.
Food fermentation: an essential unit operation towards secure, sustainable, safe, and sustaining food systems
(2025) Gänzle, Michael G.; Seifert, Jana; Weiss, Jochen; Zijlstra, Ruurd T.
Functionality of the Na+-translocating NADH:quinone oxidoreductase and quinol:fumarate reductase from Prevotella bryantii inferred from homology modeling
(2024) Hau, Jann-Louis; Schleicher, Lena; Herdan, Sebastian; Simon, Jörg; Seifert, Jana; Fritz, Günter; Steuber, Julia
Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiota. In Prevotella bryantii , the Na + -translocating NADH:quinone oxidoreductase (NQR) and quinol:fumarate reductase (QFR) interact using menaquinone as electron carrier, catalyzing NADH:fumarate oxidoreduction. P. bryantii NQR establishes a sodium-motive force, whereas P. bryantii QFR does not contribute to membrane energization. To elucidate the possible mode of function, we present 3D structural models of NQR and QFR from P. bryantii to predict cofactor-binding sites, electron transfer routes and interaction with substrates. Molecular docking reveals the proposed mode of menaquinone binding to the quinone site of subunit NqrB of P. bryantii NQR. A comparison of the 3D model of P. bryantii QFR with experimentally determined structures suggests alternative pathways for transmembrane proton transport in this type of QFR . Our findings are relevant for NADH-dependent succinate formation in anaerobic bacteria which operate both NQR and QFR.
Host metabolome and faecal microbiome shows potential interactions impacted by age and weaning times in calves
(2023) Amin, Nida; Schwarzkopf, Sarah; Tröscher-Mußotter, Johanna; Camarinha-Silva, Amélia; Dänicke, Sven; Huber, Korinna; Frahm, Jana; Seifert, Jana
Background: Calves undergo nutritional, metabolic, and behavioural changes from birth to the entire weaning period. An appropriate selection of weaning age is essential to reduce the negative effects caused by weaning-related dietary transitions. This study monitored the faecal microbiome and plasma metabolome of 59 female Holstein calves during different developmental stages and weaning times (early vs. late) and identified the potential associations of the measured parameters over an experimental period of 140 days. Results: A progressive development of the microbiome and metabolome was observed with significant differences according to the weaning groups (weaned at 7 or 17 weeks of age). Faecal samples of young calves were dominated by bifidobacterial and lactobacilli species, while their respective plasma samples showed high concentrations of amino acids (AAs) and biogenic amines (BAs). However, as the calves matured, the abundances of potential fiber-degrading bacteria and the plasma concentrations of sphingomyelins (SMs), few BAs and acylcarnitines (ACs) were increased. Early-weaning at 7 weeks significantly restructured the microbiome towards potential fiber-degrading bacteria and decreased plasma concentrations of most of the AAs and SMs, few BAs and ACs compared to the late-weaning event. Strong associations between faecal microbes, plasma metabolites and calf growth parameters were observed during days 42–98, where the abundances of Bacteroides, Parabacteroides , and Blautia were positively correlated with the plasma concentrations of AAs, BAs and SMs as well as the live weight gain or average daily gain in calves. Conclusion: The present study reported that weaning at 17 weeks of age was beneficial due to higher growth rate of late-weaned calves during days 42–98 and a quick adaptability of microbiota to weaning-related dietary changes during day 112, suggesting an age-dependent maturation of the gastrointestinal tract. However, the respective plasma samples of late-weaned calves contained several metabolites with differential concentrations to the early-weaned group, suggesting a less abrupt but more-persistent effect of dietary changes on host metabolome compared to the microbiome.
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.
Metaomic studies of the dietary impact on the structural and functional diversity of the rumen microbiome
(2018) Deusch, Simon; Seifert, Jana
Ruminant production efficiency and related emission of greenhouse gases are mainly determined by the rumen microbiome. The structure and activity of the microbial communities in turn are mostly influenced by the animal’s feed intake. The most widely used forage sources for ruminant production in Europe are corn silage, grass silage and grass hay. Progress in animal production requires optimized feeding strategies which presuppose an improved understanding of the dietary impact on the complex bionetwork residing in the rumen. A broad range of different methods are applicable to investigate archaea and bacteria which represent the most active members of the rumen microbiome. Most rumen studies available are restricted to nucleic acid-based approaches with limited functional insights. To improve knowledge about the prokaryotic communities and their adaptation responses to different animal feeds, it is essential to focus on the actual functions out of numerous possibilities that are encoded by the genomes of the rumen microbiome. Therefore proteins are best suited since representing the actual function of investigated cells combined with phylogenetic information. The major aim of this project was the feasible, first-time establishment of a metaproteomics-based characterization of the ruminal prokaryotic communities to further investigate the dietary impact on the prokaryotic rumen metaproteome. The first part was providing an overview about research that used state of the art technologies to investigate the microbiome of the gastrointestinal tract of farm animals. Yet, Omics-technologies and their combination are rarely employed in livestock science. The considered studies relied mainly on stand-alone, DNA-based molecular methods which clearly emphasized the importance of introducing contemporary methods such as shotgun metaproteomics to study the rumen microbiome and to gain deeper, more complete insights into the actual functions carried out by the specific members of the prokaryotic communities. The second part of the current project focused on a suitable, mass spectrometry-based analysis of the prokaryotic communities in the rumen ecosystem. Metaproteomic studies are challenged by the heterogeneity of the rumen sample matter that contains, besides archaeal and bacterial cells, also eukaryotic cells of rumen fungi and protozoa as well as enormous amounts of plant cells from ingested feed and epithelial cells of the animals. Shotgun metaproteomic studies require the extraction of proteins preferably of the desired target organisms to increase the coverage of the respective metaproteome and the reliability of subsequent protein identifications. This entails the avoidance of undesired proteins present in the rumen samples. In contrast to nucleic acids, proteins cannot be enriched or amplified by PCR thus, optimized sample preparation protocols are necessary in order to retrieve enhanced amounts of prokaryotic instead of plant-derived or other eukaryotic cells before protein extraction and subsequent LC-MS/MS analysis. The final step and the major aim of this project was the in depth analysis of the metaproteome of archaea and bacteria and their adaptive response to the most common forages, corn silage, grass silage and grass hay accessing as well host-related influences and variations between different ecological niches within the rumen. Improved mass-spectrometric measurements and the construction of a customized, sample-specific in-house database for enhanced bioinformatic quantification of proteins yielded comprehensive datasets comprising 8,163 bacterial and 358 archaeal proteins that were identified across 27 samples from three different rumen fractions of three Jersey cows, fed rotationally with three different diets. The functional and structural data of the metaproteomic analysis was further flanked by 16S rRNA gene-based analyses of the archaeal and bacterial community structures and the metabolomes of the rumen fluid fractions were quantified by nuclear magnetic resonance. So far, to the best of our knowledge, there are no studies investigating the metaproteome expressed by the entirety of archaeal and bacterial communities in the different phases of the rumen ecosystem under varying dietary influence. Dietary treatments revealed significant variations in the metaproteome composition and community structures of ruminal bacteria. Host-related effects were not significant. In conclusion, within this project the application of shotgun metaproteomics to characterize the prokaryotic rumen metaproteome was successfully implemented and the obtained results clearly emphasized the benefits of using complementary, state of the art methods to study the microbiome of complex ecosystems like the rumen. Considering the specific functional niches of the rumen microbiome have been shown to be of great importance.
Microbiota and nutrient portraits of European roe deer (Capreolus capreolus) rumen contents in characteristic Southern German habitats
(2023) Dahl, Sarah-Alica; Seifert, Jana; Camarinha-Silva, Amélia; Cheng, Yu-Chieh; Hernández-Arriaga, Angélica; Hudler, Martina; Windisch, Wilhelm; König, Andreas
Roe deer ( Capreolus capreolus ) are found in various habitats, from pure forest cultures to agricultural areas and mountains. In adapting to the geographically and seasonally differentiating food supply, they depend, above all, on an adapted microbiome. However, knowledge about the microbiome of wild ruminants still needs to be improved. There are only a few publications for individual species with a low number of samples. This study aims to identify a core microbiota for Bavarian roe deer and present nutrient and microbiota portraits of the individual habitat types. This study investigated the roe deer’s rumen (reticulorumen) content from seven different characteristic Bavarian habitat types. The focus was on the composition of nutrients, fermentation products, and the rumen bacterial community. A total of 311 roe deer samples were analysed, with the most even possible distribution per habitat, season, age class, and gender. Significant differences in nutrient concentrations and microbial composition were identified for the factors habitat, season, and age class. The highest crude protein content (plant protein and microbial) in the rumen was determined in the purely agricultural habitat (AG), the highest value of non-fibre carbohydrates in the alpine mountain forest, and the highest fibre content (neutral detergent fibre, NDF) in the pine forest habitat. Maximum values for fibre content go up to 70% NDF. The proportion of metabolites (ammonia, lactate, total volatile fatty acids) was highest in the Agriculture-Beech-Forest habitat (ABF). Correlations can be identified between adaptations in the microbiota and specific nutrient concentrations, as well as in strong fluctuations in ingested forage. In addition, a core bacterial community comprising five genera could be identified across all habitats, up to 44% of total relative abundance. As with all wild ruminants, many microbial genera remain largely unclassified at various taxonomic levels. This study provides a more in-depth insight into the diversity and complexity of the roe deer rumen microbiota. It highlights the key microorganisms responsible for converting naturally available nutrients of different botanical origins.
Na+-coupled respiration and reshaping of extracellular polysaccharide layer counteract monensin-induced cation permeability in Prevotella bryantii B14
(2021) Trautmann, Andrej; Schleicher, Lena; Pfirrmann, Jana; Boldt, Christin; Steuber, Julia; Seifert, Jana
Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.
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