Browsing by Subject "Inositol phosphate"

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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.
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 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.