Browsing by Subject "Phytase"

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Bone ash data in the context of phosphorus and phytase evaluation in poultry
(2021) Künzel, Susanne; Rodehutscord, Markus
Phosphorus (P) is an essential element that is crucial for various metabolic processes in the body of animals and humans. To keep the animals healthy and to obtain food products rich in nutrients, an adequate P supply is indispensable. Plant feedstuffs, the main components of poultry diets, contain P in a form that is only partially available to poultry. For this reason, poultry diets are often supplemented with mineral P. However, global rock phosphate reserves, where mineral P is mined from, are limited. Additionally, excessive P supply should also be avoided because of the environmental impact of P accumulation in the soil. Consequently, P supply not exceeding the requirements of poultry is essential to ensure animal wellbeing and to protect the environment. In order to feed diets with adequate concentrations of P, it is necessary to have suitable approaches for the determination of available P in the animal. The availability of P varies widely between feed components and it is also influenced by feed supplements and other factors. Bone ash analysis is an often-used tool to evaluate the relative bioavailability of P since a high amount of P is stored in the bones. A standard assay for bone ash analyses has never been agreed on. Therefore, many different approaches are described in the literature with an unknown impact on the results of P bioavailability studies. The main objective of the present thesis was to examine the suitability of bone ash data for the evaluation of available P in poultry with emphasis on methodological aspects. Therefore, different studies with broiler chickens and Japanese quail were conducted. The experiments comprised various aspects related to P availability in poultry. The effect of feed supplements in the form of phytase products, myo-inositol and a coccidiostat were evaluated. Furthermore, quantitative genetic analyses were performed. All experiments had in common that tibiotarsus (tibia) or foot ash data or both were used for the examination of the relative bioavailability of P. Based on the data that accrued during the studies described in the four manuscripts of this thesis, comprehensive methodological analyses were performed. The tibia and foot were compared regarding their appropriateness as a trait for the evaluation of the relative bioavailability of P by using bone ash data. The relationship between the two traits was investigated, as well as the relationship between foot or tibia ash and quantitative P measurements. Additionally, P concentration in the ash of both bone fractions was analysed and compared. Results indicated only minor differences between tibia and foot ash data. No clear preference for one of them could be deduced from the data. The left and right feet of broiler chickens were compared in terms of both ash concentration and total ash amount. Significant differences between the two feet of the same animal were detected for both traits. Consequently, not only the choice of the bone fraction but also of the body side should be considered when sampling for bone ash data. Ash data are mostly expressed as a concentration of the dry matter content of the bone. Also possible is the use of the absolute ash amount. The relationship of both ways of expression with traits of quantitative P measurements was analysed by using correlation coefficients and regression analyses. Results showed that the absolute ash amount was at least as suitable as ash concentration but has the advantage that it is easier to determine. Possible selection procedures for animals for bone ash analyses were simulated with data from two of the experiments. Often it is not possible to use all animals involved in an experiment for bone ash analyses. Therefore, the influence of sampling frequency and selection method on the outcome of P availability studies was evaluated. Results indicated that the number and selection method of animals for bone ash data might influence the results. However, it was not possible to recommend a specific selection method based on the obtained results. Estimates of heritability and genetic correlations showed the suitability of bone ash data as a proxy trait for P efficiency breeding of poultry. The absolute amount of bone ash data appeared to be most promising for this purpose. Bone ash data are a very useful and easy to determine trait to estimate the relative bioavailability of P. However, investigations performed in this thesis showed the importance of a careful selection of methods. A standardised assay would be helpful to obtain meaningful and more comparable estimates of relative P bioavailability.
Entwicklung und ernährungsphysiologische Bewertung mikrobieller Hybrid-Phytasen
(2023) Metten, Alexander; Rodehutscord, Markus
To degrade the organic phosphate storage in the best possible way, it is necessary to increase phytase efficiency in vivo. Both a better understanding of the influencing factors limiting phytate degradation in vivo and a continuous improvement of the biochemical properties of phytases to be best adapted to the conditions in the digestive tract of non-ruminants will help to achieve this. Therefore, the main objective of this work was the generation of a large number of sequentially unique hybrid phytases by directed recombination of known phytase genes with the goal to achieve improved biochemical properties compared to the wild-type phytases used. The focus of this work was the biochemical and nutritional evaluation of the newly generated hybrid phytases with respect to their suitability as feed supplements. All hybrid phytases examined showed more efficient InsP6 degradation at pH 3.0 than at pH 5.5, although the phytase activity supplemented was the same at both pH values. While InsP6 was dephosphorylated to InsP1-2 in many cases at pH 3.0, accumulation of the Ins(1,2,5,6)P4 isomer occurred at pH 5.5. In an in vitro model simulating the digestive tract of broilers, hybrid phytases with high sequential homology to the E. coli and C. braakii phytase showed high accumulation of InsP4 isomers. Interestingly, these phytases preferentially formed the Ins(1,2,5,6)P4 isomer. In contrast, other hybrid phytases were able to degrade all InsP4 isomers and in some cases high InsP2 concentrations were observed. Another in vitro experiment with a complex feed matrix consisting of soybean meal, rapeseed meal, and wheat with a high mineral content, illustrated the negative influence of certain feed-related factors on phytase efficiency. InsP6 present in the feed was significantly less degraded by all phytases used compared to a corn and soy-based feed matrix with a low mineral content. While a hybrid phytase was able to completely dephosphorylate the InsP6 of the corn and soy-based feed matrix down to the InsP3 isomer resulting in high InsP2 concentrations, the InsP6 were still detectable in the in vitro model with the more complex feed matrix and high mineral content, despite identical reaction conditions. In a final feeding trial with broilers, one of the hybrid phytases was supplemented at two doses each (500 and 1500 FTU/kg) to evaluate its suitability as a feed supplement. Also, a commercial phytase was included in the study design at the same doses setting the benchmark for phytase efficiency. A low phosphorus experimental feed based on corn and soybean meal was used. The supplementation of the used hybrid phytase resulted in a dose-dependent increase in broiler performance data such as daily weight gain, feed intake and significantly improved feed efficiency compared to the basal ration without enzyme supplementation. In addition, foot ash content was increased by 21.6% at a dose of 1500 FTU/kg phytase, indicating significantly improved bone mineralization due to the released InsP6 phosphate. By analyzing InsP6 concentration and its degradation products in different segments of the digestive tract, efficient InsP6 degradation was observed. In contrast to the in vitro experiments, no accumulation of InsP3-4 isomers could be detected in crop, gizzard or small intestine. In addition to a high exogenous phytase activity, this result also suggests a high endogenous phytase as well as phosphatase activity in the digestive tract of broilers. It can be assumed that the absence of monocalcium phosphate in the experimental rations may have induced the expression of endogenous phytases and phosphatases. This assumption is confirmed by the high precaecal InsP6 degradation, which was 63.5% in the basal ration without phytase supplementation. Nevertheless, the used hybrid phytase significantly increased the precaecal InsP6 degradation to 76.3%. The high phytase efficiency was also reflected in the measured precaecal phosphorus digestibility, which was increased by 6.8% compared to the basal ration. The commercial phytase used showed comparable improvement in broiler performance data to the non optimized hybrid phytase. This project demonstrated the development of a variety of sequentially unique hybrid phytases by recombination of known phytase genes, which exceeded the biochemical properties of the wild-type phytases in some relevant aspects. Some of the phytases showed very efficient phytate degradation when simulating the digestive tract of broilers in vitro. Also, the suitability of the tested hybrid phytase as feed additives was demonstrated by the increased performance data of broilers. The higher performance data of the broilers could be attributed to efficient phytate degradation. To achieve maximum InsP6 degradation in vivo, the feed-related and animal-related factors on phytase efficiency need to be better understood.
High-throughput sequencing techniques to analyze microbial communities in the gastrointestinal tract of broiler chickens
(2018) Borda Molina, Daniel Enrique; Camarinha-Silva, Amélia
Broiler chicken represents an excellent case-study to elucidate the inter-communication between the host and its microbial communities. The general aim of this thesis was to describe the changes in bacterial community structure that occurred in chickens, in response to different experimental diets. An update of the state of the art of the chicken gastrointestinal microbiota was done in chapter 2. The composition and functionality are described through the most recent technologies that provide taxonomic information at DNA level using 16S rRNA genes. Gene catalogs and their abundance are deciphered through shotgun metagenome sequencing, which is still at its infancy and only eight publications have been published so far. At the protein level, only two studies were found that contribute metaproteomic information. Thanks to these technologies many studies were able to focus on answering how feed supplementations altered the microbes in GIT sections. The second part presented in chapter 3 comprises an extensive investigation of the broiler chicken microbiota composition in digesta and mucosa of individual samples under varying supplementation of calcium, phosphorus, and phytase. The dietary impact on the distribution of the microbial communities was studied in the crop, ileum, and caecum through illumina sequencing of the 16S rRNA gene. One important outcome was the high variability in the microbial composition between individual samples. Significant differences were observed between the digesta and mucosa samples, supporting the hypothesis that being close to the host, mucosa-associated communities show a different composition. A calcium effect on the performance was observed, where values for body weight gain and feed conversion were lower in comparison to the other treatments. Microbial communities in the crop mucosa revealed a dietary effect, while in the digesta samples no significant changes were seen. Regarding the ileum mucosa, there was an effect of P addition on the microbial distribution. As expected, caeca-derived samples showed an increase in the diversity indexes when compared to the ileum and crop and butyrate producers were detected in higher abundance. A lower microbial diversity in the crop was linked to lower growth performance regarding the supplementation of Ca. Hence, each dietary treatment affected the microbial communities; nevertheless, none of the dietary treatments displayed a consistent effect across the studied gut sections. Additionally, the effects of supplementing different proteases and one phytase on the microbial community of the ileum of broiler chickens was assessed. Thus, the specific aim of chapter 4 was to determine how enzyme supplementation affects the microbiota composition in the ileum of broilers and whether these effects were related to differences in pre-caecal AA digestibility. Three different protease sources at a low and high level were included. The microbial taxonomy was assessed through 16S rRNA gene Illumina amplicon sequencing. Performance results revealed a significant increase in growth and feed efficiency in broilers fed with phytase only and the high dosage of protease C, in comparison to the control. Most of the AA showed a significant difference between the control diet and protease C at high dosage and phytase diets. Effects on microbiota composition were observed at the genus level for some protease and phytase supplementations. The genera Streptococcus, Lactobacillus, and uncultured Clostridiaceae were responsible for these differences. This study demonstrates that effects of enzyme supplementation were evident in the terminal small intestine microbiota composition, and, to a lesser extent, in pc AA digestibility. However, the changes in microbiota composition and pc AA digestibility could not be correlated which may indicate the absence of a causal relationship. Finally, an outlook with metagenome sequencing is presented in chapter 5, to further characterize the result of feeding strategies. Metabolism information, essential to microbial activities registered 50% of abundant genes in the supplemented diets while being reduced to 40% in the control samples Phosphatases pathways and butyrate production increased in the supplemented diets while calcium signaling pathway was higher in the control. In conclusion, within this project a method of standardization to study the microbiota along the gastrointestinal tract of broiler chickens was successfully established. The obtained results revealed a significant impact of both, enzyme and mineral supplementation in the individual sections of the GIT. Also, it was proved that even if the GIT works as an interconnected system, its compartmentalization creates different environmental conditions which influence the microbiota. This study provides insights into the responses of the bacteria and their functionality which were stimulated by the feed supplementations.
In vivo and in vitro studies of degradation of inositol phosphates in the digestive tract of broiler chickens
(2017) Sommerfeld, Vera; Rodehutscord, Markus
Phosphorus (P) is an important element in poultry nutrition, which must be adequately supplied in the diet. However, for non-ruminant animals, it is only partially available from plant seeds—the major components of poultry diets—where P is predominantly bound as phytic acid (myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate); InsP6) and its salts, called phytate. InsP6-P can be utilized after the stepwise cleavage of the P from the phytate molecule by phytases and other phosphatases. After the theoretical complete dephosphorylation of InsP6, six phosphate groups and myo-inositol (MI) are potentially available for absorption. Recent studies assume an effect of MI on growth performance when it is added in its free form to the diet or released as a result of InsP6 breakdown. Because P is of specific economic and environmental relevance, the improvement of the digestibility of plant P in poultry is of great interest. The overarching aim of this thesis was therefore to gain a deeper insight into the degradation of InsPs in the digestive tract of broiler chickens, with a focus on the intermediate and end-products as influenced by the diet composition. An in vitro assay was established to study the disappearance of InsP6 and the formation of lower inositol phosphate (InsP) isomers in the poultry digestive tract. The assay simulates the conditions (pH, temperature, proteolytic enzymes, water content, and retention time) of the crop, stomach, and small intestine, using a poultry diet as matrix. The assay yielded highly reproducible results and was sensitive to the factors that varied in the three experiments. A diminishing effect on InsP degradation was found by the supplementation of P and Ca. The described assay is a suitable tool that can be used to screen feed enzymes and to investigate the effects of supplements in the absence of endogenous phytases. The first in vivo experiment aimed to distinguish between the single and interactive effects of P, calcium (Ca), and phytase. Effects on lower InsP esters and their isomers and MI in different segments of the digestive tract, and on the prececal digestibility of P, Ca, and amino acids (AAs) in broiler chickens were studied. Moreover, a complete picture was drawn of all dephosphorylation steps from InsP6 to MI in the digesta of the terminal ileum. The dietary treatments included diets without (P-) or with (P+) monosodium phosphate supplementation, without (Ca-) or with (Ca+) additional limestone supplementation, and without or with 1500 FTU phytase/kg feed. Up to the terminal ileum, InsP6 disappearance was found to decrease in P+Ca-, and even more so in P+Ca+, when no phytase was added. Adding phytase removed all effects of P and Ca. However, P+Ca+ increased the concentrations of lower InsP esters and reduced free MI in the ileum, even in the presence of phytase. Supplementation with phytase increased the concentration of MI in all segments of the digestive tract and in blood plasma, demonstrating the ability of broilers to fully degrade phytate and absorb the released MI. While the prececal AA digestibility was not affected by P and Ca or an interaction between P, Ca, and phytase, it increased with the addition of phytase. The objective of the second in vivo experiment was to investigate the effects of supplementation with free MI or graded levels of phytase on InsP degradation, concentrations of MI in the digestive tract and blood, bone mineralization, and prececal digestibility of AA. Birds were fed a control diet with adequate levels of all nutrients without or with MI supplementation, or one of three experimental diets that differed in phytase level, with reduced P and Ca levels. These outcomes indicate that MI might have been a relevant cause for the increase in gain:feed. Therefore, it is likely that the release of MI after complete dephosphorylation of phytate is one of the beneficial effects of phytase, along with the release of P and improvement in digestibility of other nutrients. It can be concluded that the established in vitro assay is a suitable tool to investigate effects on feed enzymes or differences between different feed enzymes in a feed matrix under standardized conditions without the interference of endogenous phytases, or depending on animal-specific variations. Based on the outcome of the in vitro and in vivo experiments of the present project, the combined supplementation of P and Ca—rather than supplementation of P or Ca solely—seems to be crucial for InsP degradation. There is now some evidence that MI can affect the growth and feed efficiency of broiler chickens without affecting the metabolism of InsPs or AAs. As the results regarding the effects of P and Ca on InsP6 degradation or phytase effects on AA digestibility are not consistent across studies, and studies investigating the effects of MI are scarce and not consistent, further systematic research is needed.
Mineral and phytic acid content as well as phytase activity in flours and breads made from different wheat species
(2023) Longin, C. Friedrich. H.; Afzal, Muhammad; Pfannstiel, Jens; Bertsche, Ute; Melzer, Tanja; Ruf, Andrea; Heger, Christoph; Pfaff, Tobias; Schollenberger, Margit; Rodehutscord, Markus
Wheat is of high importance for a healthy and sustainable diet for the growing world population, partly due to its high mineral content. However, several minerals are bound in a phytate complex in the grain and unavailable to humans. We performed a series of trials to compare the contents of minerals and phytic acid as well as phytase activity in several varieties from alternative wheat species spelt, emmer and einkorn with common wheat. Additionally, we investigated the potential of recent popular bread making recipes in German bakeries to reduce phytic acid content, and thus increase mineral bioavailability in bread. For all studied ingredients, we found considerable variance both between varieties within a species and across wheat species. For example, whole grain flours, particularly from emmer and einkorn, appear to have higher mineral content than common wheat, but also a higher phytic acid content with similar phytase activity. Bread making recipes had a greater effect on phytic acid content in the final bread than the choice of species for whole grain flour production. Recipes with long yeast proofing or sourdough and the use of whole grain rye flour in a mixed wheat bread minimized the phytic acid content in the bread. Consequently, optimizing food to better nourish a growing world requires close collaboration between research organizations and practical stakeholders ensuring a streamlined sustainable process from farm to fork.
Phytate degradation and phosphorus digestibility in turkeys and broiler chickens fed maize-based diets
(2023) Novotny, Moritz Sebastian Daniel; Rodehutscord, Markus
A growing global human population, stagnation in available land for farming, and an increased interest in sustainable and eco-friendly food production necessitates a highly efficient and environmentally friendly food production. This includes the already very feed-efficient poultry meat production. Currently, using non-renewable mineral phosphate as feed additive is industry standard in poultry nutrition. This can lead to unwanted eutrophication of waterbodies by high faecal concentrations of unutilised plant-based phosphate. Degrading phytate via enzymatic hydrolysation by phytases drastically improves digestibility of plant-based phosphate. With dietary phytase supplementation, a tool is available to reduce necessity of dietary phosphate supplementation. However, predictability of the extent to which phytase supplementation can replace phosphate supplementation is not accurate enough to forego phosphate supplementation entirely. Subject of this doctoral thesis was to study the factors that can influence phytate degradation in the digestive tract of poultry, in order to improve predictability of plant-based phosphate digestibility. The focus was put on maize-based diets, as they are very common worldwide and phytate degradation is challenging due to low intrinsic phytase activity of maize. A literature review on the current state of knowledge on phytate degradation and phosphorus digestibility of chicken fed maize-based diets was conducted. Part of this review was to compare findings for chickens to findings in other poultry species. There is a plethora of studies that investigated the subject in broilers but comparatively little information on turkeys. There were indications of fundamental differences between broilers and turkeys. Consequently, the intention was to identify reasons for these differences and to evaluate to which extent knowledge transfer from chickens to turkeys is possible. Two consecutive trials comparing broilers and turkeys were designed. Factors studied were: supplemented phytase, dietary phosphorus and calcium concentration, age, and endogenous mucosal phosphatase activity. Broilers and turkeys studied were kept simultaneously and under identical conditions, including experimental diets. A total of 480 broiler and 480 turkey hatchlings were obtained at the same day and raised at the experimental facility. Halve of the animals of each species underwent the experiment from day 14 to day 21, the other halve from day 35 to day 42. This set up was chosen to study the influence of physiological development, as species with different maturation rates were compared. In 3-week-old broilers and turkeys, precaecal InsP6 disappearance was the same when no phytase was supplemented and dietary calcium and phosphorus level was low. This coincided with no differences in jejunal mucosal phosphatase activity. Without phytase supplementation, 6-week-old turkeys showed higher precaecal InsP6 disappearance than 6-week-old broilers. This coincided with higher jejunal mucosal phosphatase activity in turkeys than broilers. When phytase was supplemented, precaecal InsP6 disappearance was markedly increased in both species. This increase was always higher in broilers compared to turkeys of the same age. Increased dietary calcium and phosphorus levels led to decreased precaecal InsP6 disappearance in both species. This led to the conclusion that previously reported differences in precaecal InsP6 disappearance between broilers and turkeys were primarily due to the higher dietary calcium and phosphorus concentrations used in turkey diets, and secondly due to more phytate degradation by supplemented phytase in the crop of broilers compared to turkeys. The latter was attributed to more favourable conditions for the supplemented phytase. Although turkeys appeared to have compensated much of that in the more posterior parts of the digestive tract. Jejunal mucosal phosphatase activity was higher in treatments with phytase supplementation than without. As this coincided with high concentrations of lower inositol phosphates in the digesta, these might have triggered increased expression of phosphatases on the brush border membrane. In contrast, an increase in dietary calcium and phosphorus level coincided with a decrease in jejunal mucosal phosphatase activity, numerically in 3-week-old birds, but significantly in 6-week-old birds. This might indicate a downregulation of mucosal phosphatase expression based on phosphate concentration in the small intestine. In conclusion, fundamental mechanisms affecting phytate degradation in the digestive tract of broilers and turkeys seem to be the same. However, there is one big difference in recommended dietary calcium and phosphorus levels and many small differences in important details affecting phytate degradation and phosphate digestibility between the two species. These require dedicated attention to further improve phosphorus efficiency in poultry production.
Phytate hydrolysis and formation of inositol phosphates in the digestive tract of broilers
(2015) Zeller, Ellen; Rodehutscord, Markus
Phytate (any salt of myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) or InsP6) represents the major binding form of phosphorus (P) in plant seeds. In the digestive tract, availability of P from plant seeds and feedstuffs obtained thereof largely depends on the enzymatic hydrolysis of InsP6 and less phosphorylated inositol phosphate isomers (InsPs). High prices of mineral P supplements and environmental burden linked with excessive P excretion of animals as well as exhaustion of the global rock phosphate stores demand for maximization of phytate-P utilization in animal feeding. The major objective of this thesis was to understand better InsP6 hydrolysis and formation of lower InsPs in different segments of the digestive tract of broilers and how they can be influenced by different dietary factors. In the first study (Manuscript 1), broilers (n=10 pens per dietary treatment) were fed low-P (5.2 g/kg DM) corn-soybean meal-based diets without (basal diet) or with one of three different phytase supplements (an Aspergillus and two E. coli derived phytases) from days 16 to 25 of age. InsP6 hydrolysis until the lower ileum (74%) of birds fed the basal diet indicated a high potential of broilers and their gut microbiota to hydrolyse InsP6 in low-P diets. Different InsP pattern in different gut segments suggested the involvement of phosphatases of mucosal or microbial origin. Supplemented phytases significantly increased InsP6 hydrolysis in the crop but not in the lower ileum. Measurements in the crop and proventriculus/gizzard confirmed published in vitro degradation pathways of 3- and 6-phytases for the first time in broilers. Presence of InsP4 and InsP5 isomers specifically formed by different supplemented phytases indicated activity of these enzymes still in the small intestine. InsP4 accumulation differed between the 6- and 3-phytases in the anterior segments of the gut. In the second study (Manuscript 2), effects of supplemental mineral P were studied using different basal diets. Semi-synthetic and corn-soybean meal-based basal diets (experiment 1), or corn-based and wheat-based basal diets were used (experiment 2). Anhydrous monosodium phosphate (MSPa) or monocalcium phosphate monohydrate (MCPh) was supplemented to increment the P concentration by 0.05, 0.10, and 0.15% or by 0.075 and 0.150% in experiment 1 and 2, respectively. In experiment 1, total excreta were collected from day 20 to 24 of age (7 replicated birds per diet). In experiment 2, digesta from the terminal ileum was collected when broilers were 22 days old (5 replicated pens per diet, 19 birds per pen). No differences were found in InsP6 hydrolysis between the maize- and wheat-based diets (experiment 2). Mineral P supplements significantly decreased InsP6 hydrolysis from the InsP-containing diets in both experiments. The choice of the basal diet did not affect the evaluation of the supplemented mineral P sources. This lead to the conclusion that calculated availability values for mineral P sources need to be adjusted for the decline in hydrolysis of InsP contained in the basal diet resulting from the P supplement. In the third study (Manuscript 3), broilers (20 birds per pen; n=8 pens per treatment) were fed two low-P corn-soybean meal-based diets without (BD-; 4.4 g P/kg DM) or with monocalcium phosphate (MCP) (BD+; 5.2 g P/kg DM) and without or with added phytase at 500 or 12,500 FTU/kg from days 15 to 24 of age. Digesta samples were taken from the duodenum/jejunum and lower ileum. Another 180 broilers (n=6 pens per treatment, 10 birds each) were fed the three BD+ diets from day 1 to 21 of age to assess the influence of supplemented phytase on tibia mineralization and strength. Interactions between MCP and phytase affected InsP6 hydrolysis and the concentrations of specific lower InsPs. Supplementation with 12,500 FTU/kg phytase resulted in 92% prececal InsP6 hydrolysis and strong degradation of InsP5. This resulted in higher P net absorption, affirmed by higher body weight gain, tibia strength, and mineralization compared to treatments without or with 500 FTU/kg of phytase. MCP supplementation reduced InsP6 hydrolysis and the degradation of specific lower InsPs in birds fed diets without phytase or with 500 FTU/kg of phytase, but did not reduce InsP6 hydrolysis or degradation of InsP5 at the high phytase dose. Hence effects of added MCP on phytase efficacy depend on the dose of supplemented phytase. In the fourth study (Manuscript 4), broilers (15 birds per pen, n=8 pens per treatment) were fed a wheat-soybean meal diet low in P (4.8 g/kg DM) and containing either microwave-treated (BDTW; 121 U/kg of phytase) or non-microwave treated (BDUTW; 623 U/kg of phytase) wheat meal from d 16 to 23 of age. Diets were used without or with supplementation of a phytase, alone or in combination with a xylanase. Interactions between microwave treatment and enzyme supplementation were found for InsP6 hydrolysis in the ileum and P net absorption in the duodenum/jejunum and ileum. In the ileum, P net absorption was similar, but InsP6 hydrolysis was significantly higher for BDTW (78%) than for BDUTW (69%) in the absence of supplemental phytase. Microwaving may have disrupted wheat aleurone structures in ways that increased the accessibility of the phytate and may have encouraged higher levels of activity among specific phytases of microbial or endogenous mucosal origin in the lower small intestine. In both segments, InsP6 hydrolysis and P net absorption were significantly increased by supplementation of phytase, but no further by additional supplementation of xylanase. In birds that were fed the phytase-supplemented diets, microwave treatment of wheat had no effect on InsP6 hydrolysis, but it significantly reduced P net absorption in both segments. The fifth study compromised two experiments (Manuscript 5) in which the influence of different dietary factors on InsP6 degradation in the crop was investigated. The experimental designs was as mentioned for Manuscript 3 (experiment 2) and 4 (experiment 1) since the samples were taken in the same trials. In experiment 1, InsP6 hydrolysis in the crop was significantly increased by supplementation of phytase, but not further by the additional supplementation of xylanase. Microwave treatment of wheat reduced InsP6 hydrolysis and degradation of InsP5, due to reduction in intrinsic enzyme activity. The effect of 500 FTU/kg of supplemental phytase on InsP6 hydrolysis was much higher in broilers fed the maize- compared to those fed the wheat-based diets (experiment 2 and 1). Thus, for supplemental phytase the accessibility of phytate in wheat seems to be lower than in maize, perhaps due to different storage sites. Supplementation of 12,500 FTU/kg of phytase caused high InsP6 hydrolysis (up to 80%) and stronger degradation of InsP3-5 than supplementation of 500 FTU/kg (experiment 2). In both experiments, degradation of Ins(1,2,5,6)P4 was a limiting step in the breakdown process of InsP6 by the supplemented phytase. However, upon phytase supplementation Ins(1,2,5,6)P4 accumulated in BDTW diets whereas InsP4 degradation proceeded in untreated wheat diets (experiment 1). Ins(1,2,5,6)P4 seemed to be degraded synergistically by intrinsic wheat phosphatases and the supplemented phytase. Taking all studies together, it can be concluded that broilers and their gut microbiota have a very high potential to hydrolyze InsP6 in the digestive tract when diets low in P and Ca are fed. Differences in the concentrations of lower InsPs showed that the initial step of InsP6 hydrolysis is not the only catabolic step influenced by different dietary factors. To optimize efficacy of phytases and achieve a maximal InsP degradation and minimal P excretions the separate and interactive effects of different dietary influencing factors on InsP hydrolysis need to be better understood and considered in future diet formulations.
Studies of phytate degradation and associated mineral utilization in growing pigs
(2024) Klein, Nicolas; Rodehutscord, Markus
Phosphorus (P) is primarily stored in plant seeds as phytate, which is any salt of phytic acid (myo-inositol hexakisphosphate; InsP6). The enzymatic capacity of pigs and the microorganisms present in their digestive tract allows for only partial and incomplete prececal degradation of InsP6. To address this limitation, the addition of exogenous phytases to feed has been state-of-the-art for more than three decades. Phytases, found in various organisms like bacteria, fungi, plants, and animals, initiate the breakdown of InsP6 and inorganic phosphate, thereby making the liberated phosphate available for digestion. Substantial scientific research and practical field applications have shown that adding exogenous phytases to the diet of non-ruminants leads to an improvement in P digestibility and a reduction in P excretion. Over time, phytases with enhanced thermal profiles and higher activity at physiological pH milieus have been engineered. Additionally, advancements in plant breeding have increased intrinsic phytase activity in feed ingredients relevant to animal nutrition. Thus, this thesis aimed to deepen the understanding of inositol phosphate (InsP) degradation in ileal-cannulated pigs, focusing on how dietary variables can influence this process along the digestive tract. In the first study (Manuscript 1), diets based on wheat with variable intrinsic phytase activity and the addition of an exogenous phytase on InsP degradation in pigs were investigated. Although wheat intrinsic phytase contributed to increased InsP degradation and P digestibility in pigs, variation in intrinsic phytase activity by crossbreeding wheat, as achieved in this study, was not reflected in InsP degradation and P digestibility data. This discrepancy may be attributed to an inadequate proteolytic resistance of wheat phytase in the gastric compartment of the pig. A marked increase in prececal InsP degradation and P digestibility was achieved by adding a microbial phytase to the wheat-based feed. While exogenous phytase increases the digestibility of InsP-P, other dietary components may impair its hydrolytic activity. In the second study (Manuscript 2), the single and interactive effects of dietary Ca concentration and exogenous phytase in pigs were investigated. The study assessed the effects of dietary Ca and exogenous phytase on InsP degradation and nutrient digestibility in growing pigs. Additional dietary Ca decreased prececal InsP6 disappearance, but only with exogenous phytase. Concentrations of lower InsP isomers and myo-inositol in the ileal digesta and prececal P digestibility were greater with exogenous phytase, but not affected by dietary Ca concentration. In contrast, fecal InsP6 disappearance was lower and the concentration of InsP4-5 isomers in feces was greater with additional dietary Ca. The study revealed that dietary Ca concentration is relevant for InsP disappearance in the hindgut, but not in the ileum. However, when exogenous phytase is used, the dietary Ca concentration is important because prececal InsP degradation is affected by the Ca level in the diet. The third study (Manuscript 3) aimed to investigate the effects of dietary Ca sources, exogenous phytase, and formic acid on InsP degradation and nutrient digestibility in growing pigs. Replacing limestone with Ca formate reduced prececal InsP6 disappearance and prececal P digestibility. However, adding formic acid to a diet containing limestone and phytase enhanced prececal InsP6 disappearance and increased InsP2 and myo-inositol concentrations in the distal ileal digesta. Accordingly, prececal P digestibility was increased when formic acid was added to the diet containing limestone and exogenous phytase. This suggested that Ca formate is not a suitable alternative Ca source to limestone for optimizing prececal P release from InsP in growing pigs. The release of P from InsP by exogenous phytase in limestone diets can still be further increased by adding formic acid. In conclusion, the degradation of InsP in growing pigs is a complex process affected by dietary Ca concentration, dietary Ca source, and the presence of phytase. However, investigated dietary traits mainly affected InsP6, with lesser effects on lower InsP isomers, highlighting the importance of prececal hydrolytic breakdown of InsP6 to InsP5. This directly affected P digestibility of growing pigs. Intrinsic phytase activity in feed ingredients, such as wheat, can contribute to prececal InsP degradation but may be neglected when exogenous phytase is present in the feed. Exogenous phytase addition notably increased prececal InsP degradation and P digestibility in plant-based diets, and complete dephosphorylation of InsP in the prececal tract was implied by the increase of the final degradation product myo-inositol in the ileum content, urine, and blood. However, some P remained bound to InsP at the end of the ileum of growing pigs and was therefore not available for digestion. Nevertheless, some diets with exogenous phytase tested in this thesis met the recommended digestible P concentration. This suggests that diets formulated with adequate InsP-containing ingredients and exogenous phytase may obviate the need for mineral P addition, reducing the reliance on finite P sources in growing pig nutrition.
Zinc supplementation effects on phytate degradation, mineral digestibility, and bone characteristics in broiler chickens
(2024) Philippi, Hanna; Rodehutscord, Markus
An adequate supply of phosphorus (P) is important in poultry nutrition, as P is essential for numerous metabolic processes. However, oversupply should be avoided to reduce the environmental impact of poultry production. The main source of P in plant feedstuffs commonly used in poultry nutrition is phytate, the salt form of phytic acid (InsP6). For P from InsP6 to be utilized by animals, it needs to be cleaved by phytases or other phosphatases. However, the capacity of endogenous phosphatases of non-ruminant animals does not suffice to release sufficient P to fulfill the animal’s P requirement. Therefore, commercial poultry diets usually are supplemented with P from mineral sources. By using exogenous phytases, the supplementation of mineral P can be reduced, and finite P reserves can be conserved. To feed poultry without mineral P in the future, phytase efficacy must be improved further. Thus, it is important to know and understand all factors influencing phytase efficacy. The results of in vitro studies have indicated that zinc (Zn) may be an influencing factor. The supplementation of Zn could inhibit phytase activity, with the degree of inhibition depending on the exogenous Zn source used. A literature review on the interactions of Zn with phytate and phytase (Manuscript A of this thesis) has identified a lack of in vivo studies investigating the effects of Zn supplementation on phytase with direct measurements, such as intestinal phytate degradation and prececal P digestibility. Therefore, three in vivo studies were conducted as part of this thesis with the main objective to investigate the effect of Zn supplementation and exogenous Zn source on intestinal phytate degradation in broiler chickens. It was hypothesized that due to the formation of insoluble complexes of Zn and phytate, the supplementation of Zn could reduce phytase efficacy with the extent of reduction depending on the exogenous Zn source. Further, other traits that are affected by Zn supply, such as bone mineralization and gene expression, were also investigated in these in vivo studies. The first experiment (Manuscript B) aimed to determine the effect of dietary Zn level and source on intestinal phytate breakdown, mineral digestibility, bone mineralization, and Zn status without and with exogenous phytase in the feed. Ross 308 broiler chickens were fed experimental diets from day 7 to 28. The basal diet contained 33 mg/kg dry matter native Zn and a high phytate-P concentration to challenge interactions in the digestive tract. The experimental diets differed in the level of exogenous phytase (0 or 750 FTU/kg) and in the Zn source (none, 30 mg/kg of Zn-sulfate, or 30 mg/kg of Zn-oxide). Additionally, two experimental diets with a high Zn supplementation level (90 mg/kg) in the form of Zn-sulfate or Zn-oxide, both containing exogenous phytase, were tested. Intestinal phytate breakdown, P digestibility, and bone mineralization were not affected by Zn source or Zn level but only by phytase supplementation. The concentration of ileal myo-inositol was influenced by phytase × Zn source interaction. Birds fed without phytase supplementation had similarly low myo-inositol concentrations whether they received Zn supplementation or not, whereas birds receiving phytase supplementation and Zn supplementation had significantly higher ileal myo-inositol concentrations than birds fed without Zn supplementation but with phytase supplementation. The missing effect of Zn level or Zn source on phytate degradation indicates that no interactions of Zn and phytate relevant for phytase efficacy occurred in the digestive tract of broilers when Zn was supplemented at levels up to 90 mg/kg in the form of Zn-sulfate or Zn-oxide. Based on the results of the first experiment, where Zn alone did not show relevant interactions with phytate, the second experiment (Manuscript C) aimed to investigate whether the combined supplementation of Zn, copper (Cu), and manganese (Mn) from different sources without and with exogenous phytase in the feed affects intestinal phytate breakdown, prececal mineral digestibility, bone mineralization, and mRNA expression of mineral transporters. Cobb 500 broiler chickens received experimental diets from day 0 to 28. Experimental diets differed in the level of phytase supplementation (0 or 750 FTU/kg) and in the trace mineral source (TMS: 100 mg/kg Zn, 100 mg/kg Mn, and 125 mg/kg Cu as sulfates, oxides, or chelates). Prececal InsP6 disappearance and P digestibility were significantly affected by phytase × TMS interaction. Whereas birds receiving exogenous phytase had similar InsP6 disappearance and P digestibility irrespective of TMS, birds fed without exogenous phytase and with chelated trace minerals had a higher InsP6 disappearance and P digestibility than birds receiving no exogenous phytase and oxides or sulfates. These results indicate that the combined supplementation of Zn, Mn, and Cu at high levels may challenge interactions with phytate in non-phytase-supplemented diets with the extent of interaction depending on the TMS. In phytase-supplemented diets however, the choice of TMS was irrelevant for phytate degradation under the conditions of this study. The third experiment (Manuscript D) aimed to determine the impact of Zn level and Zn source on prececal phytate degradation, mineral digestibility, bone mineralization, and mRNA expression of intestinal (trace) mineral transporters. In contrast to the first experiment, an inorganic Zn source and a chelated Zn source were tested. Cobb 500 broiler chickens received experimental diets from day 0 to 21. The experimental diets differed in Zn supplementation level (10, 30, 50 mg/kg Zn) and exogenous Zn source (Zn-oxide or Zn-glycinate). A cornsoybean meal-based diet without Zn supplementation containing 35 mg/kg native Zn was used as a control. All experimental diets were supplemented with 750 FTU/kg phytase. Prececal InsP6 disappearance, P digestibility, and tibia ash quantity and concentration, and Zn concentration in tibia ash were not affected by diet. Bone breaking strength and tibia width did not differ between treatments. Tibia thickness was lower in the treatments with 30 mg Zn as Zn-oxide and 50 mg Zn as Zn-glycinate than in the treatment with 10 mg Zn as Zn-oxide. The expression of intestinal (trace) mineral transporters was not affected by treatment. These results indicate that in phytase-supplemented diets the native Zn concentration of cornsoybean meal-based diets is satisfactory to achieve maximal Zn concentration in tibia ash during the first 3 weeks of age. The missing effect of Zn level or Zn source on phytate degradation confirms the results from Manuscript B, that Zn and phytate do not interact to a level relevant for phytate degradation by exogenous phytase. It is concluded that contrary to the hypothesis that Zn inhibits phytate degradation by complex formation with phytate, the Zn supplementation up to 100 mg/kg does not appear to influence exogenous phytase efficacy. Minor effects were found on the endogenous phytate degradation if Zn, Cu, and Mn were supplemented combined at high levels, where the extent of reduction in endogenous phytate degradation was dependent on the TMS. It remains unclear whether the inhibiting effect on endogenous phytate degradation occurs only due to the combined supplementation or whether an individual high supplementation of a single trace mineral caused the effect. Further experiments are needed to investigate the effect of Zn on endogenous phosphatases, where the activity of endogenous mucosal activity should be determined in broilers fed diets differing in the Zn supplementation level. Moreover, further experiments are needed to test what level of Zn supplementation is needed in phytase-supplemented diets to ensure the birds sufficient supply in all areas. Besides bone development and growth, effects on the immune system, microbiota composition, and the antioxidative system should be considered.
Zum Einfluß von mikrobieller Phytase und Calcium auf die Blei-, Cadmium und Zinkretention beim wachsenden Schwein
(1997) Zacharias, Bernhard; Drochner, Winfried
In the present study the influence of microbial phytase and/or calcium supplementation in rations of growing pigs (15 to 30 kg resp. 50 kg) on the retention of lead, cadmium and zinc in kidneys, liver, muscles and bones was investigated. The rations consisted of a barley-soy mixture supplemented with lead, cadmium and zinc either in the form of Pb-, Cd- and Zn-polluted barley or of CdCl2, Pb(CH3COO)2, and ZnSO4. The rations contained an average of 1,45 mg Pb, 0,78 mg Cd, and 55,7 mg Zn per kg dry matter and were given either with or without an addition of 800 U microbial phytase per kg. Compared to the reference group the addition of microbial phytase resulted in diets with a nomal calcium supply (6 g/kg) to a significant increase in the concentration of lead in the phalanx 1 and cadmium in kidneys and liver. The zinc concentration, however, only was increased in tendency in bones and liver. By augmenting the calcium concentration to 12 g/kg it was possible to avoid phytase-induced increase in the retention of lead in phalanx 1 and in the deposition of cadmium in kidneys and liver. For zinc, however, this effect of calcium could not be detected. The higher lead concentration in liver, kidneys and bones after addition of Pb(CH3COO)2 may be due to the 51 higher dietary Pb level as compared to the normally polluted barley rations. The reduced Cd-accumulation in livers and kidneys after feeding CdCl2 supplemented diets with a high calcium level may be explained by an increased formation of insoluble cadmium-calcium-phytate that cannot be hydrolysed by phytase. For the heavy metals lead, cadmium, and zinc the addition of phytase might probably result in an increased metal availability due to phytase-induced hydrolysis of the phytate complex. The effect of a calcium supply exceeding the recommended level may be explained by a reduction in the solubility of phytate which results in a decreased ability of phytate to be splitted by phytase and therefore leads to a reduced