Browsing by Subject "Phytate"

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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.
Impact of dietary phosphorus and fermentable substrates on the immune system and the intestinal microbiota of the pig
(2016) Heyer, Charlotte Maria Elisabeth; Stefanski, Volker
Phosphorus (P) represents a crucial input for agriculture and food industries as a mineral present in ingredients used for livestock feeding as well as in mineral fertilisers. In the current systems, P is primarily derived from the finite mined phosphate rock resource. Thus, a critical challenge of global P scarcity is directly linked to future food security and sustainable resource management, especially in the European Union which is dependent on raw P from outside Europe. Apart from other future activities in animal nutrition, new dietary formulations of livestock diets emerged as a potential approach to increase the digestibility of plant P, phytate (myo inositol 1,2,3,4,5,6 hexakisphosphate, InsP6), and to reduce the supplementation with mineral phosphate. In non-ruminant animals, such as the pig, InsP6 hydrolysis is incomplete, as the small intestine lacks sufficient enzymes such as endogenous mucosal phytase and phosphatase. As a consequence, there is rising scientific interest to improve the understanding of InsP6 degradation in the digestive tract as well as the effects on nutritional factors and finally animal performance and health. The aim of the present thesis was to investigate the impact of dietary P, InsP6 and InsP6 hydrolysis products in combination with different fermentable substances (protein, carbohydrate) on the porcine immune system, the intestinal microbiota and animal health. First, a comprehensive literature overview describes the impact of P on the immune system and the microbiota along the gastrointestinal tract (GIT), including potential effects on host health with special focus on the pig. Secondly, an in vivo study with growing pigs was conducted to examine the effects of diets with varying mineral calcium-phosphorus (CaP) levels as well as different fermentable substrates on intestinal CaP concentration, InsP6 hydrolysis, the intestinal microbial ecosystem, and the peripheral and gut-associated immune system. In 2 consecutive experiments, 31 growing pigs (55 ± 4 kg) were allotted to a 2 × 2 factorial arrangement with 4 treatment groups, fed either a corn-soybean meal or a corn-pea based diet, each with 2 different CaP levels (low, 66% of the CaP requirement; high, 120% of the CaP requirement) supplemented with monocalcium phosphate and calcium carbonate. After 3 weeks of adaptation to the diets, all pigs were immunized twice with keyhole limpet hemocyanin (KLH). Blood and faeces samples were taken. After slaughtering, immunological tissue (jejunal, ileal mesenteric lymph nodes, spleen) as well as jejunal, ileal, caecal and colonic digesta were taken. Faecal and digesta samples were examined for P, Ca, inositol phosphate (InsP) isomers and for the marker titanium dioxide. The number of different leukocyte subpopulations analysed by flow cytometry, mitogen-induced lymphocytes proliferation in vitro were assessed. In addition, concentrations of plasma anti KLH IgM and plasma anti-KLH IgG analysed by ELISA and haematological parameters analysed by an automated hematology system have been measured in blood and tissue samples. In digesta samples, bacterial 16S rRNA gene copy numbers were determined by quantitative real-time PCR. The concentration of short chain fatty acids (SCFA) and ammonia was assessed. In addition, the use of terminal restriction fragment length polymorphism has been proven to characterize the structure of porcine gut microbiota. Results of the current study demonstrated that CaP and fermentable substrates had a distinct effect on the peripheral and gut-associated immune system, as well as on microbial composition and activity in growing pigs. High dietary CaP concentrations and the corn-pea diets increased P net absorption. Almost no InsP6 degradation could be observed in the GIT, and mainly myo inositol pentakisphosphate (InsP5) isomers were measured in jejunal, caecal digesta and faecal samples. In particular, the high CaP diets showed higher InsP6 and InsP5 concentrations, indicating a reduction of the initial steps of P release from InsP6 and a further breakdown of InsP5 isomers. The low CaP content might cause an impaired first line of defence and activation of the cellular and humoral adaptive immune response. As an example, the high CaP content affected the outcome of the adaptive immune response including a higher number of antigen experienced T-helper cells in the blood as well as higher plasma anti-KLH IgG concentrations. The reactivity of blood and mesenteric lymph node lymphocytes to Concanavalin A in these pigs was impaired, indicating modulating effects of other origin such as migration patterns or activity of antigen-presenting cells. Since results of the present study suggest contradictory effects of CaP level on immune cell numbers and lymphocyte reactivity in vitro and in vivo, further studies are needed to determine effects on cell signalling such as cytokine production profiles. Moreover, the high CaP content and the soybean meal diets increased the number of butyrate-producing bacteria, such as Eubacterium rectale and Roseburia spp. and increased the concentration of various SCFA in the small and large intestine, thereby contributing to improve gut health. Potentially harmful bacteria, such as Enterobacteriaceae and Bacteroides Prevotella Porphyromonas, were increased by the low CaP level and pea diets, indicating a less healthy microbiota. Results demonstrated that both, CaP supply and the amount of fermentable substrates, may beneficially affect gut health due to modulations of the composition and activity of the intestinal microbiota. Further studies should evaluate the impact of CaP on specific pathogenic bacteria known to produce toxic products creating a direct link to the immune system and animal health. Although most parameters of the present study indicate a positive effect of the high CaP diet, not all values showed a consistent effect on animal health, such as immune cell numbers and lymphocyte proliferation in vitro. In conclusion, variations in P availability and the formation of individual InsPs have to be considered when formulating diets in support of a stable intestinal microbial ecosystem and immune functions of the host.
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.
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.
Nutritional evaluation of oilseed press cakes in fish nutrition with emphasis on rainbow trout (Oncorhynchus mykiss, W.)
(2019) Greiling, Alexander Michael; Rodehutscord, Markus
Fishmeal is a valuable, protein rich ingredient for fish feed. It is a source of highly digestible crude protein (CP) with a balanced amino acid (AA) profile, well digestible inorganic phosphorus (P), and a highly digestible energy content. However, its availability is decreasing owing to an increasing demand that is driven by the increased production of fish in feed-based production systems. Research has made great advances in counteracting the limited supply of fishmeal. As a result, the majority of dietary CP in fish feed is made available from oilseeds and their processed by-products. Despite the pre-existing research efforts, the continuous evaluation of feed ingredients in search for alternatives to fishmeal is key to facilitate a sustainable growth of feed-based fish production. Oilseed press cake represents a widely available source of CP. While numerous studies have evaluated the nutritional value of press cake in fish feed, the majority focused on species reared in warmwater production systems. Thus, the objective of this thesis was to add to pre-existing knowledge on press cake and its potential to replace fishmeal in fish feed, with special emphasis on rainbow trout (Oncorhynchus mykiss W.). Initially the nutrient digestibility of various press cakes (linseed, pumpkin seed, rapeseed, soybean, sunflower seed, and walnut kernel cake) was determined in rainbow trout. The press cakes differed greatly in their digestibility of crude nutrients, with CP digestibility ranging from as low as 25% (sunflower seed cake) up to 88% (pumpkin seed cake). Another digestibility experiment was conducted using rapeseed cake and sunflower seed cake whose fibre fractions were reduced using two different processing methods (sieving and dehulling of seeds prior to pressing). The fibre-reduced press cake of rapeseed and sunflower seed cake had a substantially higher CP digestibility than their unprocessed counterpart (Manuscript 1). Three growth experiments were conducted to study the effect of partial replacement of fishmeal with press cake on performance traits of rainbow trout. In all growth experiments groups of rainbow trout were fed with either a basal diet or diets in which fishmeal CP was in part replaced by press cake based on its CP digestibility that was determined in the preceding digestibility experiments. It was found that the performance traits were influenced to a different extent in dependence of the press cake and their inclusion level. Pumpkin seed cake has been shown to have the highest potential to replace substantial amounts of fishmeal of the basal diet without significantly reducing performance traits of rainbow trout. To investigate the potential utilisation of InsP-P and the formation of inositol phosphate isomers in fish two experiments were conducted. The single and interactive effects of a mineral P supplement (monoammonium phosphate; MAP; 1 g P/kg DM of diet) and an InsP6 hydrolysing enzyme (Aspergillus oryzae 6-phytase; 2800 FTU/kg DM diet) were compared between rainbow trout and Atlantic salmon (Salmo salar). For each species a digestibility experiment was conducted under common rearing conditions of each species but using the same four diets (basal diet, basal diet + MAP, basal diet + phytase, and basal diet + MAP + phytase). The faecal disappearance of InsP6 was generally low (approximately 8%) but similar between the species when the diets were devoid of either supplement. The supplementation of phytase significantly increased InsP6 disappearance in both species, but the effect was found to be more pronounced in rainbow trout. The analysis of lower inositol phosphate isomers revealed that their hydrolysis progressed to a greater extent in rainbow trout and it suggested that InsP6 is subject to a different degradation pathway in the two species. While no significant interactive effects on InsP6 disappearance were found between the two supplements for either species, the MAP supplementation slightly decreased InsP6 disappearance in Atlantic salmon but not in rainbow trout. The experiments provide an insight into the breakdown of InsP6 and the faecal appearance of specific lower inositol phosphates and suggest that the use of press cake in feed for rainbow trout seems to be more beneficial than in feed for Atlantic salmon with regards to a more sustainable use of P resources. However, more experiments are recommended to complement these initial findings to gain a better understanding of InsP6 hydrolysis in fish.
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.
Studies on the extent of ruminal degradation of phytate from different feedstuffs
(2017) Haese, Eva; Rodehutscord, Markus
The predominant storage form of phosphorus (P) in plant seeds and grains is phytate (InsP6). To cleave the phosphate group and, thus, make the bound P available for absorption by the animal, the enzyme phytase is required. Rumen microorganisms show substantial phytase activity, however, recent studies have suggested that the extent of InsP6 hydrolysis in ruminants is variable leading to an incomplete hydrolysis of InsP6 in specific conditions followed by the excretion of P from undegraded InsP6. As P is an essential element for the metabolism in animals it is important to ensure that the animals’ requirements are met. Diets for ruminants are often supplemented with mineral P (Pi). However, the global phosphate resources are finite and the excretion of surplus P contributes to eutrophication of surface water when applied to the farmland with manure in excessive amounts. Thus, dietary P supply is of environmental concern. Better knowledge about ruminal InsP6 hydrolysis could help to optimise the utilisation of InsP6 and, thus, reduce the use of Pi as well as unnecessary excretion of P. Hence, the objectives of the present thesis were to examine the InsP6 hydrolysis from different feedstuffs in ruminants and to identify factors that might affect the extent of InsP6 hydrolysis. In the first study, the total digestive tract disappearance of InsP6 from diets differing in amount and source of P was determined in lactating dairy cows. The results confirmed the high potential of rumen microorganisms to hydrolyse InsP6, but the composition of the diet influenced the extent of hydrolysis in vivo. In the second study, two in vitro experiments were conducted in order to determine the InsP6 hydrolysis from maize grain and RSM. In experiment 1, two diets differing in P- and InsP6-P concentration were fed to the donor animals of rumen fluid. In experiment 2, a diet similar to the high P diet of experiment 1 was fed to the donor animals of rumen fluid and the rumen fluid was mixed with artificial saliva containing Pi (PI: 120 mg Pi/l) or no Pi. Maize and RSM were incubated for 3, 6, 12, and 24 h in both experiments and the InsP6 concentration was analysed in fermenter fluids and bag residues. InsP6 disappearance from maize proceeded faster than from RSM. The disappearance of InsP6 was higher when the diet with high P concentration was fed (experiment 1) and lower when the rumen fluid was mixed with Pi containing buffer (experiment 2). In the third study, the in situ disappearance of InsP6 from five different concentrates was examined. Maize, wheat, RSM, heat treated RSM (hRSM), and soybean meal were incubated in the rumen of fistulated dairy cows fed with three diets differing in P- and InsP6-P concentration. Concentrations of InsP6 and isomers of InsP5, InsP4, and InsP3 were determined in the bag residues after 2, 4, 8, 16, and 24 h of incubation. The disappearance of InsP6 from cereals proceeded faster than from oilseed meals, however, averaged over the diets, after 24 h of incubation 95% had disappeared from all concentrates except for hRSM (57%). Feeding the diet with high InsP6 concentrations increased InsP6 disappearance from oilseed meals but not from cereals, while feeding the high Pi diet did not influence ruminal InsP6 hydrolysis from any concentrate. The results derived from analysis of lower InsPs suggested that intrinsic plant phytase activity plays only a minor role in the rumen and that active phytases in the rumen react differently to changes in the ruminal environment. The results of the present thesis suggest that the composition of the diet fed to ruminants affects the extent of ruminal InsP6 hydrolysis. While high InsP6 concentrations have the potential to increase InsP6 hydrolysis, a decrease of InsP6 hydrolysis can occur after addition of Pi to the diet. Differences in the pace of InsP6 hydrolysis between concentrates occurred which could be of importance at high ruminal passage rates when the time available for ruminal hydrolysis decreases.
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.