Institut für Nutztierwissenschaften

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Approaches to increase digestibility of Bangladesh ruminant feed resources in order to mitigate enteric methane production
(2024) Bashar, Muhammad Khairul; Rodehutscord, Markus
Multiple analyses have shown that the rising human population, urbanization, and consumer preferences affect the demand for livestock products in developing countries. Concurrently, human population and urbanisation growth are reducing the likelihood of newly cultivated land producing feeds or restoring damaged pastures. Using low-quality roughage more efficiently is an option, but such roughages have drawbacks, including high structural carbohydrate and low nitrogen contents, which lead to poor palatability and nutrient utilisation including emission of methane (CH4) that is harmful to the environment and a loss of energy to the animal. Using commonly accessible legumes and tree foliage could be a possibility to address the need for N and CH4 mitigation due to their high crude protein and secondary compound contents. In order to evaluate the potential of specific combinations of roughages, concentrates, and other feeds, it is necessary to determine the feeding values, in vitro ruminal fermentation, and CH4 production of single feeds, as well as the promising interaction (associative) effects between single feeds when combinded in a ration. This may allow for formulating a better balanced total mixed ration (TMR) for ruminants, which was the main goal of the present doctoral study. Eighteen feedstuffs from Bangladesh were chosen, including roughages such as crop residues (rice straw, urea molasses treated straw and maize stover), silages (Napier silage and Maize silage), common grasses (German grass, Para grass and Napier grass) and leguminous fodder (Ipil-ipil, Glicidia, Alfalfa hay and Moringa tops) and concentrates (crushed wheat, crushed maize, Wheat bran, Kashari bran, and Rice bran). In Study 1, the single feeds were characterized by comprehensive chemical analysis and in vitro production of total gas and methane using the Hohenheim gas test. Based on the results of Study 1, rice straw, German grass, Napier silage, and maize silage were used in Study 2 and incubated in vitro alone or after substitution by Ipil-ipil and Gliricidia at levels of 10%, 20%, 30%, and 40%, respectively. In Study 3, mixtures of roughages and leguminous fodder (rice straw + 40% Ipil-ipil, German grass + 10% Ipil-ipil, Napier silage + 30% Ipil ipil, and maize silage + 30% Ipil-ipil) were combined with single concentrates in order to identify the most promising mixtures in regard to digestibility and methane production. Eventually, 24 TMR were formulated using six for each type of roughage in Study 4. These TMR were comprised of 60% of the same combination of roughage as used in Study 3 and 40% of various combinations of concentrates including TMR-1 (60% rice bran + 10% wheat bran + 30% kashari bran), TMR-2 (30% rice bran + 10% wheat bran + 60% kashari bran), TMR-3 (10% rice bran + 60% wheat bran + 30% kashari bran), TMR-4 (10% rice bran + 30% wheat bran + 60% kashari bran), TMR-5 (60% rice bran + 30% wheat bran + 10 kashari bran), and TMR-6 (30% rice bran + 60% wheat bran + 10% kashari bran) in order to identify suitable TMR for maximising ruminal fermentation and reducing the CH4 production in vitro. In all studies, the total gas production (GP), digestibility of organic matter (dOM), metabolisable energy (ME), and net energy for lactation (NEL) were determined after incubating 200 mg of feed with a rumen fluid-buffer solution for 72 hours. In addition, the CH4 concentration in the produced gas was measured after incubating 120 mg of feed for 24 hours. Measured values of the mixed feed were compared to estimated values, where the estimated values were weighted means of the values of the individual feeds, to determine the associative effects between the feed ingredients (Study 2 and Study 4). In Study 1, the concentration of acid detergent fibre (ADF), neutral detergent fibre (NDF), and acid detergent lignin (ADL) was the lowest in leguminous fodder while the crude protein (CP) concentration was the hightest, followed by the common grasses and the silages. The crop residues showed the lowest CP and the highest cell wall fraction concentrations. The dOM, ME, and NEL of crushed wheat and crushed maize were significantly higher (p<0.05) than those of other feedstuffs. The same differences were found (p<0.05) for CH4 concentration (% of GP) and CH4 production (L CH4/kg dOM). The dOM and ME of German grass and Ipil-ipil were higher (p<0.05), whereas the CH4 concentration and CH4 production were lower compared to that of crop residues and other common grasses. The CH4 production of single feeds decreased with increasing concentrations of CP, ADF, and ADL, whereas it increased with NDF concentration. These findings enabled development of more balanced diets for ruminants with the aim of improved digestibility and reduced CH4 emission while making use of widely available feed resources in Bangladesh. Study 2 aimed to evaluate the in vitro ruminal fermentation, CH4 production, and associative effects between low-quality roughages and CP supplements. The gas production after 24 hours (GP24) and rumen fermentation rate increased (p<0.05) with increasing addition of Ipil-ipil to rice straw. It also resulted in a gradual increase of dOM and ME (p<0.05) of the mixture, reaching maximum values (59.1% and 7.60 MJ ME/kg DM) and maximum reductions in CH4 concentration and CH4/dOM (6.9% and 11.7%) compared to the control with Ipil-ipil addition at 40% of DM. When Ipil-ipil was added at 30% to Napier silage, the same trend was observed for dOM and ME (56.5%, and 7.55 MJ ME/kg DM) and CH4 concentration and CH4/dOM (5.3% and 13.3%). Ipil-ipil did not significantly interact with high quality roughages such as German grass and maize silage. Increasing the addition of Gliricidia to the roughages in Study 2 led to a decrease in GP24, dOM, ME, and CH4 production. The highest levels of the associative effects (p<0.05) were seen when 40% Ipil-ipil was added to rice straw and 30% was added to Napier silage. In conclusion, adding Ipil-ipil to low-quality roughages is superior to Gliricidia and showed promising results, with the ranking as follows: rice straw + 40% Ipil-ipil > Napier silage + 30% Ipil-ipil > maize silage + 30% Ipil-ipil > German gras + 10% Ipil-ipil. Study 3 and Study 4 had the overarching purpose to determine which combination of single concentrates and combinations of concentrates with roughage produced the best mixed feed in terms of rumen fermentation, CH4 reduction potential, and associative effect. The CP and non-stach carbohydrate contents of the Ipil-ipil-based mixed feed was enhanced by adding Wheat bran and Kashari bran as single concentrates, which had a favourable effect on rumen fermentation and gas production but had the opposite effect on CH4 production when compared to the addition of Rice bran. In Study 4, TMR were formulated using roughages and Ipil-ipil and addition of Wheat bran, Kashari bran, and Rice bran in various combinations. The TMR based on German grass and maize silage with specific bran combinations showed significant reductions in in vitro CH4 production, CH4 concentration, and CH4/dOM while maximising GP, dOM, ME, and the associative effects. To conclude, the results of chemical analyses and in vitro fermentation studies showed that specific combinations of roughages, protein feeds, and by-products available in Bangladesh have the potential to formulate rations for cattle that help making livestock production more sustainable. The results may be also relevant for other developing nations. It is suggested to verify the results of the present project by animal trials at local conditions.
Beiträge zur Ermittlung der P-Verwertung bei der Japanischen Wachtel
(2008) Alfoteih, Yassen; Bessei, Werner
Although there are plenty of studies concerning the metabolism and requirement of phosphorous (P) in poultry, the availability of P from organic and inorganic sources has still to be elucidated. Lacking knowledge on the availability of P has not caused problems in the past since commercial diets have been supplemented with P levels far above the requirement. Only when the excretion of P has been recognized as a source of environmental pollution, and poultry producers were forced to reduce P supplementation in the diet according to the requirement, need more of accurate information on the availability of P has stimulated research in this field. A series of experiments on the utilization of P from different organic and inorganic sources has been carried out using Japanese quail as a model for other poultry species. In addition, the role of the Ca: P ratio on the availability of P was investigated. A total of 6 experiments have been carried out. All experiments were carried out using almost the same methodology. Chicks of a commercial fast growing line of Japanese quail of french origin were hatched at the research station of the University of Hoheheheim and reared up to 3 weeks under standard conditions using a starter diet. Starting from week four, the birds were fed the experimental diets and data for the P-balance were collected for 7 consecutive days (fifth week of age). The components to be tested were added to a basal diet containing all nutrients with the exception of Ca and P. The utilization of P was calculated as difference between P intake and P excretion. The basal diet without supplementation of test-components was included in experiments since P utilization of the basal diet was needed to calculate the partial utilization of P from the test-components. In experiment A1 the utilization of P from summer barley and oats was tested. In Experiment A2 experimental diets comprised three different sources of P, each of plant origin (winter wheat, winter barley and maize) and of inorganic sources mono-calcium phosphate (MCP), di-calcium phosphate (DCP) and mono-sodium phosphate (MNP) were used. Mixtures of P sources tested in experiment A3 (MCP+DCP, MCP+MNP, MCP+maize, Wheat+ barley and wheat+ maize) were examined in experiment A3. The P level in the experimental diets was adjusted to a range of 2 to 3 g/kg and the Ca-level to 3 to 5 g/kg. The Ca:P ratio was restricted to a maximum of 2:1. The supplementation of the different types of grain to the diets was 60.0 to 66.0 %. Calcium carbonate (CaCo3) and sodium bicarbonate (NaHCo3) were used to supplement Ca. The result showed that, there was no significant difference among the experimental diets on weight gain of the birds. P utilization was highest in the basal diet (66.6 %) followed by barley and oats (33.3 and 20.8 % respectively), while the standard diet showed the lowest utilization (7.87 %). In experiment A2 the utilization of P of the basal diet (94 %) was significantly higher than of all other P-sources wheat (60 %), maize (66 %), while diet supplemented with barley showed the lowest value (34 %) and the other sources an intermediate position between 47 and 53 %. The partial utilization was somewhat lower than the overall utilization in all diets. In experiment A3 the utilization of P of the basal diet was 69 % and did not significantly differ from the other diets. The combination MCP+MNP showed the highest utilization (76 %), followed by wheat+barley (53 %) and MCP+maize (50 %). The partial utilization showed the similar values as the overall utilization. The three following experiments (B1, B2 und B3) dealt with the effects of different Ca: P ratios on the utilization of P, feed intake, growth rate and selected bone criteria. Four different Ca levels (0.80; 1.20; 1.60 and 2.20 %) were combined with three P levels (0.30; 0.45 and 0.60 %) in experiment B1. During Experiment B2, experimental diets contained three Ca concentrations at levels higher than in experiment B1 (2.60; 3.00 and 3.30 %) and three P concentrations at levels lower than in B1 (0.10; 0.20 and 0.30 %). Experiment B3 was conducted with the same Ca and P concentrations as in experiment B2, but the duration of the balance period was extended from 1 to 3 weeks. In addition of the P utilization and growth rate Ca and P contents of the tibia bones was analyzed and some bone characteristics were measured by computer tomography. In experiments B1 and B2 neither the P nor the Ca concentration showed a consistent effect on feed intake, growth rate, feed conversion or mortality. The P utilization decreased with increasing P concentration and P intake. The effect of the Ca: P interaction on P utilization was only significant in experiment B1. In the extended balance period in experiment B3, the effects of Ca and P concentration as well as their interaction was significant for body weight in the first week of the experiment. From the second week onwards the effects of the sources of variance decreased and fell under the level of significance. The P utilization was only influenced by the P concentration. As in the previous experiments P utilization decreased with increasing P concentration. Bone characteristics showed tendencial response to Ca and P concentration, whereby with the lowest concentrations of Ca and P in the diet, the highest bone weight, ach content, Ca and P content was found. Elevating Ca supply meliorated the total area and cortical area of the tibia. However, the better tibia quality based on evaluating the Strain Strength Index (SSI) as a criterion for the stability of bone was found in groups fed diets with the highest level of P. The results of these experiments showed that there is an obvious variation in the availability of P from the organic and mineral resources. The utilized P value from mineral resources was underestimated, while the utilized P value from organic resources overestimated. The P utilization of the combinations of different P sources which diverged in their P utilization showed intermediate values. The P concentrations of the diets and the P intake were the main influencing factors on P utilization. This result pointed out that the birds were capable to cover their requirements from P through the diets, although the P contents in the diets were low. The P contents of tested diets have met already the requirement of P with quails or even exceeded. The recommendations of P supply in quail diets need to be reduced accordingly. Should quail be used as model for the study of P requirement in broilers, it is essential either to decrease the P content drastically in the diets, or to conduct the tests at earlier age. The lower requirement of P in quail from 3 weeks of age onwards explains the weak reaction of feed intake and growth rate in response to the low P concentration and to extremely high Ca: P ratios. Some bone characteristics responded to low P concentrations and high Ca: P ratios even at higher ages. This confirms previous findings that P requirement for bone building is evidently higher than the P required for growth. The question posed by this result, weather growth rate or bone characteristics should be used to determine the requirement of P in poultry.
Investigations on phytate degradation of rapeseed meal and soybean meal in ruminants
(2023) Chi, Yung-Ping; Rodehutscord, Markus
Oilseed meals are widely used protein feeds in ruminant nutrition. However, aside from the high crude protein (CP) content, oilseed meals also contain high amounts of phosphorus (P), which is predominantly present in organic form as different salts of myo-inositol 1,2,3,4,5,6 hexakis dihydrogen phosphate (InsP6). To become available for intestinal absorption and further utilisation by animals, P must be cleaved from the InsP6 molecule by a specific group of phosphatases, which is known as phytase. Over the decades, ruminants were considered to be capable of utilising nearly all P bound in InsP6 because of the substantial phytase activity exhibited by rumen microbiota. Nevertheless, recent studies have reported variable extents of ruminal InsP6 degradation which seems to be influenced by different factors. In case of an incomplete ruminal InsP6 degradation, post-ruminal InsP6 degradation may be of higher relevance. However, post-ruminal InsP6 degradation has been rarely studied to date. The aim of this thesis was to systematically investigate InsP6 degradation of rapeseed meal (RSM) and soybean meal (SBM) in ruminants, including the possible influencing factors and their combinations. Different study methods (in vivo, in situ, and in vitro) were applied to evaluate the effects of RSM and SBM. The first study (Manuscript 1) was conducted to investigate ruminal and post-ruminal InsP6 degradation in wethers fed a diet containing RSM or SBM, and to link the ruminal disappearance determined in slaughtered wethers with in situ calculated rumen effective degradation of InsP6 (InsP6ED) from cows. Firstly, RSM and SBM was incubated according to a standard in situ procedure in three lactating Jersey cows for 2, 4, 6, 8, 16, 24, 48, and 72 h to obtain InsP6ED for the oilseed meals at rumen passage rates of 0.02 (InsP6ED2) and 0.05 h-1 (InsP6ED5). Secondly, eight wethers were randomly assigned to two treatment groups that were fed a diet containing equal amount of RSM (Diet RSM) or SBM (Diet SBM) for 8 weeks of adaptation. Then, digesta from the reticulo-rumen, omasum, abomasum, jejunum, colon, and rectum were sampled. In consistence with in situ calculated InsP6ED2 (83 and 93% for RSM and SBM, respectively), ruminal InsP6 disappearance was lower in wethers fed Diet RSM (76%) compared to those fed Diet SBM (89%). Post-ruminal InsP6 disappearance did not differ between dietary treatments (6% for Diet RSM vs. 4% for Diet SBM). A higher amount of ruminally degraded InsP6 was observed upon feeding RSM (4.5 g/d for Diet RSM and 3.4 g/d for Diet SBM). Due to the low rumen passage rate in this study, it was suggested that P from InsP6 being available to ruminants is almost entirely from InsP6 degradation in the rumen. As InsP6 is located in a protein-rich structure in seeds and InsP6 degradation has been recently reported to vary in a pattern similar to CP degradation for RSM, the second study (Manuscript 2) was carried out to investigate the variation of in situ ruminal InsP6 degradation of SBM and its relation to CP degradation. In this study, nine commercial solvent-extracted SBM from Europe and South America were incubated in three rumen-fistulated lactating Jersey cows with the same procedure performed in the first study. Rumen effective degradation of CP and InsP6 were calculated for a rumen passage rate of 0.06 h-1 (CPED6 and InsP6ED6). Chemical protein fractions of SBM variants were determined according to Cornell Net Carbohydrate and Protein System (CNCPS). The SBM variants exhibited a considerable variation in CP and InsP6 degradation. Significant correlations were found between InsP6ED6 and CPED6 and between InsP6ED6 and all CNCPS protein fractions, which confirmed the close relationship between CP and InsP6 degradation for SBM. The results suggested that using a general value of InsP6 degradation for diet formulation may not be precise enough, and InsP6ED may be predicted based on CPED or CNCPS protein fractions by using linear regression equations. The third study (Chapter 4.3) aimed to achieve a better understanding of how in vitro InsP6 degradation of RSM and SBM is influenced by different amounts of InsP6 in feed. The same batches of RSM and SBM as used in Manuscript 1 were incubated in a modified rumen simulation technique (RUSITEC) system with different amounts for 3, 6, 12, 24, and 48 h. Degradation of InsP6 from bag residues was calculated and expressed as amount and in percentage using the same equation as applied for in situ calculations. In vitro degradation of InsP6 in response to InsP6 amount differed between RSM and SBM, which may be attributed to the different internal structure and nutrient composition of the oilseed meals. Only when expressing in amounts, the calculated InsP6ED was observed to increase linearly with increasing InsP6 amount in feed. Accordingly, it was recommended to compare InsP6 degradation based on InsP6 amount in the feed and to express degradation as amount instead of using relative value which might not reflect the real degradation kinetics. In conclusion, the results of this thesis showed that the extent of ruminal InsP6 degradation differs when the diet contains either RSM or SBM, while post-ruminal InsP6 degradation is negligibly low given a long rumen retention time. By using linear regression equations, ruminal InsP6 degradation may be predicted from CP degradation due to the close relationship therebetween. Effects of InsP6 amount on InsP6 degradation is dependent on InsP6 source. Based on the high similarity among ruminal InsP6 degradation determined by different methods in this thesis, ruminal InsP6 degradation of oilseed meals measured by in situ or in vitro study may be applicable for in vivo conditions.
Ontogenetic and individual patterns of volatiles in honeybee queens Apis mellifera and its significance for the acceptance of queens in honeybee colonies
(2008) Al Ali Alkattea, Raghdan; Bessei, Werner
Activities of honeybees Apis mellifera L. colony are coordinated by an effective communication network in which the queen plays a central role by controlling behavior and reproduction of workers through pheromones. Most pheromones are produced in the mandibular (QMP) and tergal gland and distributed over the queen?s cuticle. The acquisition of these pheromones from the cuticular body surface of the queen is performed by antennating and licking of the retinue workers. Workers of a colony are able to recognize their own queen. Foreign queens which are introduced without protection are normally killed by the workers. While a lot of work has been performed on the primer and releaser effect of certain queen pheromones, it is still unknown how the workers distinguish their own queen from foreign ones. The fact that queens can be exchanged successfully by protecting the foreign queen for some days demonstrates that workers are able to ?learn? their queen. It is likely that a certain chemical pattern of the cuticle (odor or taste) is finally responsible for the recognition and acceptance as ?own?. In this context, this work has three different objectives: - To better understand the bees? behavior to ?own? and ?foreign? queens and to quantify certain behavioral traits of the queen-workers interaction. - To study the learning ability for own and foreign queens by the use of the Proboscis Extension Reflex (PER) in order to have a tool for future tests of odorous compounds. - To compare the cuticular pattern of queens of different origin. In all three approaches, virgin and mated queens and queens of different kin relation to each other were reared and established in Kirchhainer nuclei colonies. These queens were compared due to the following hypothesis: If the workers perceive their own queen by a distinct smell and if closely related queens have a more similar chemical pattern on the cuticle, then a related foreign queen should be easier ?learned?/ accepted than a non related one. For this purpose, first a specific bioassay had to be developed and established to enable the record of workers behavior to the queen without an inhibition of the complex social interactions between queens and workers. This ?cage bioassay? consists of a small wooden box with a glass front, a wax comb, 30-40 worker bees and a queen. For the tests, the own queen of this mini-colony was removed and a foreign queen was introduced. For a period of about 2 hours certain aggressive and benign actions, respectively, of the workers toward the queen were recorded. In the first set of tests, queens of different kin relations were compared. The results showed, in general, an aggressive reaction against the introduced foreign queens. However, there were clear lower benign and stronger aggression behaviors against unrelated queens compared to the related ones. Some of the unrelated queens were even killed. However, these differences were only significant when virgin queens were exchanged but not when mated queens were used. Concerning the duration of the aggressive action of workers, aggression generally decreased between the beginning and the end of the test; again, this was significant only in the experiments with virgin queens. This indicates, that at least in virgin queens the individual recognition by the worker bees depends on a kin specific odorous pattern of the queen. The same types of queens used in the cage bioassays were used for the learning experiments. A classical olfactory conditioning (PER) of worker honeybees was applied by using a living queen as the source of odor. Hereby the queen was offered in a way that the worker bees could not touch the body surface. The gradual increase in the learning curves was a good indication that the workers are able to learn the queen?s odor and, therefore, can be used as a kind of ?biosensor?. After having learned a queen?s odor, the conditioned workers were tested by offering virgin and mated queens, respectively, with defined kin relation to the queen used for the conditioning before. The results revealed clear differences in the cues used for the ?learning? of individual mated and individual virgin queens, respectively. The workers could significantly discriminate between the learned odor of a mated queen and any other mated queen irrespective of the relatedness. In contrast, worker bees could not discriminate virgin queens from each other. As the worker could only use volatile substances for the associative learning, one can conclude the following: In virgin queens the volatile ?bouquet? is neither individual specific nor kin specific. In mated queens the bouquet has only an individual specificity. Probably, the huge amount of many volatile gland products (including the main component 9-ODA) makes each mated queen ?unique?. But as in the cage bioassays the worker could recognize whether an introduced virgin queen was related to the own queen or not, these recognition must depend on non volatile substances of the virgin queens cuticle which are perceived by licking. If learned and tested queens were of different mating status the worker bees could significantly discriminate between such individuals (except learned odor of mated queens/ tested odor of related virgin queens). This is not surprising because the GC-MS analysis confirmed the huge differences in the odorous pattern (and here mainly the volatile polar gland products) between virgin (= young) and mated (=elder) queens. From the same types of queens used for the cage bioassays and PER, queens? heads and abdomens were extracted in a solvent and the obtained extracts were analyzed using GC-MS. From the extracts of queen abdomens 32 substances (hydrocarbons and polar compounds) were identified and chosen to calculate the ?chemical distance? between queens of different kin relation (sister vs. unrelated) and between sister queens having different ages and mating status. For that purpose, a matrix of Nei-distances was applied as a measure for the similarity of different patterns. The results showed a significantly higher concordance in the chemical pattern within sister queens compared to non related ones. The ?chemical distance? increased from sister queens over half sister to non-related queens. Cluster analyses of the Nei distance and multidimensional scaling clearly confirmed the differentiation between unrelated queens and the similarity of sister queens. Using the same statistical methods, also a clear differentiation between queens of different ages and mating status could be demonstrated. The results presented in this work confirmed with 3 different approaches that workers are able to learn their own queen with different learning cues depending on the mating status of the queen. In virgin queens it could be demonstrated for the first time that the kin relation between different queens can be recognized, presumably by low or non-volatile substances. The chemical analyses confirmed that the cuticular pattern of queens could be used for the differentiation not only according to age but also according to kin.
Parasites, depredators, and limited resources as potential drivers of winter mortality of feral honeybee colonies in German forests
(2023) Kohl, Patrick L.; Rutschmann, Benjamin; Sikora, Luis G.; Wimmer, Norbert; Zahner, Volker; D’Alvise, Paul; Hasselmann, Martin; Steffan-Dewenter, Ingolf; Kohl, Patrick L.; Department of Animal Ecology and Tropical Biology, Biocenter, University of Würzburg, Würzburg, Germany; Rutschmann, Benjamin; Department of Animal Ecology and Tropical Biology, Biocenter, University of Würzburg, Würzburg, Germany; Sikora, Luis G.; NaturKonzept, Pfullingen, Germany; Wimmer, Norbert; Bayerische Landesanstalt Für Wald Und Forstwirtschaft, Freising, Germany; Zahner, Volker; Forest Ecology and Management, University of Applied Sciences Weihenstephan-Triesdorf, Freising, Germany; D’Alvise, Paul; Institute for Medical Microbiology and Hygiene, University Hospital Tübingen, Tübingen, Germany; Hasselmann, Martin; Department of Livestock Population Genomics, Institute of Animal Science, University of Hohenheim, Stuttgart, Germany; Steffan-Dewenter, Ingolf; Department of Animal Ecology and Tropical Biology, Biocenter, University of Würzburg, Würzburg, Germany
Wild honeybees ( Apis mellifera ) are considered extinct in most parts of Europe. The likely causes of their decline include increased parasite burden, lack of high-quality nesting sites and associated depredation pressure, and food scarcity. In Germany, feral honeybees still colonize managed forests, but their survival rate is too low to maintain viable populations. Based on colony observations collected during a monitoring study, data on parasite prevalence, experiments on nest depredation, and analyses of land cover maps, we explored whether parasite pressure, depredation or expected landscape-level food availability explain feral colony winter mortality. Considering the colony-level occurrence of 18 microparasites in the previous summer, colonies that died did not have a higher parasite burden than colonies that survived. Camera traps installed at cavity trees revealed that four woodpecker species, great tits, and pine martens act as nest depredators. In a depredator exclusion experiment, the winter survival rate of colonies in cavities with protected entrances was 50% higher than that of colonies with unmanipulated entrances. Landscapes surrounding surviving colonies contained on average 6.4 percentage points more cropland than landscapes surrounding dying colonies, with cropland being known to disproportionately provide forage for bees in our study system. We conclude that the lack of spacious but well-protected nesting cavities and the shortage of food are currently more important than parasites in limiting populations of wild-living honeybees in German forests. Increasing the density and diversity of large tree cavities and promoting bee forage plants in forests will probably promote wild-living honeybees despite parasite pressure.
Standard Methods for Dissection of Varroa destructor Females
(2021) Piou, Vincent; Vilarem, Caroline; Rein, Carolin; Sprau, Lina; Vétillard, Angélique
Varroa destructor (Anderson and Trueman) is known as a major pest of Apis mellifera L, especially in the Northern Hemisphere where its effects can be deleterious. As an obligate parasite, this mite relies entirely on its host to reproduce and complete its cycle. Studies focusing on isolated organs are needed to better comprehend this organism. To conduct such targeted molecular or physiological studies, the dissection of V. destructor mites is crucial as it allows the extraction of specific organs. Here, we propose a technical article showing detailed steps of females V. destructor dissection, illustrated with pictures and videos. These illustrated guidelines will represent a helpful tool to go further in V. destructor research.
The effect of aging in the murine gut microbiome
(2020) Hernández Arriaga, Angélica; Camarinha-Silva, Amélia
Aging is characterized by several physiological changes. During the lifespan, the biological systems from the body of humans and other animals remain dynamic. Throughout the early stages of life, the microbiome develops into a complex ecosystem with thousands of species. Variations related to diet, environmental changes, medications affect the diversity and composition of the microbiota through the lifespan. Some old individuals with higher incidence of chronic diseases have a loss of the stability of the microbiome and an imbalance occurs between the different colonizers of the gut, also named dysbiosis. One of the most distinctive changes occurring with age is the prevalent low grade inflammation, which is named inflamm-aging. This not only changes the microbial composition of the GIT but also affects the permeability. Murine models are well established and help us to understand the complex dynamics between the host and the microbial communities inhabiting the gastrointestinal tract. These models allow us to analyze microbial communities from tissue and mucosa, from all sections of the gut, which is limited in humans. Methods standardization is an important topic in microbiome research. In chapter 2 it was compared the efficiency of two sample methods, cotton swab and tissue biopsy, in characterizing the mouth microbiota. In recent years, the mouth microbiome is being seen as a diagnostic tool for not only oral diseases but also systemic diseases. As physiological changes occur with aging, the microbiome from the mouth is affected and there is an increase of pathogens present in the oral surfaces. In murine models, cotton swab is a common tool used for sampling the microbiome of the oral cavity. In our study, we observed similar microbial community structure using both methodologies. However, the species Streptococcus danieliae, Moraxella osloensis, and some unclassified members of Streptococcus were affected by the different sampling procedures. In this trial, we included mice at two different ages, 2 months old being considered young and 15 months old considered middle aged mice. We observed changes in the genera Actinobacillus, Neisseria, Staphylococcus, and Streptococcus related to the age of the animal and the sampling type. These results showed the importance of sampling standardization in microbiome research and that age has a strong effect on the microbial ecology of the oral cavity. In chapter 3, it was studied the bacterial communities from duodenum and colon of mice at 2, 15, 24 and 30 months of age in combination with the results of the expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function. Besides, in this chapter were also assessed the indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissues. At 24 and 30 months of age there was an increase in inflammation, they developed fibrosis and the levels of endotoxin in plasma were higher. Regarding changes in the microbiome, the duodenum had more changes than the colon related to age. Allobacullum, Bifidobacterium, Olsenella, Corynebacterium were the genera that differed statistically in the duodenum through the murine lifespan. Fewer changes were observed in the colon, as Allobaculum was the only genus that showed differences between young and old mice. Additionaly, it was analyzed the impact of aging in the active microbial communities of mouth, duodenum and colon at 2, 9, 15, 24 and 30 months of age (chapter 4). Changes were observed at every age and different taxonomical levels, with a greater shift at 15 months of age. This is related to the age of the mice, as at middle age systemic changes related to the aging process start to occur. At old ages, there was an increment of the pathobiontic species Helicobacter hepaticus and Helicobacter ganmani in the duodenum and colon. The oral, duodenal and colonic microbial communities are important pieces of information that can be related to the health status of the host. Research that focuses on assessing the changes in the different niches and not only in the feces, gives a broader overview of the microbial community of the host.
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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