Trace mineral chelation for sustainable animal nutrition : enhancing zinc availability with L-glutamic acid N,N-diacetic acid

In current farm animal practice, the uncertainty in the availability of zinc (Zn), as affected by dietary and digestive factors, is compensated by calculating gross requirements from net requirements using a worse-case availability factor in the conversion. Consequently, the higher levels of Zn inclusion lead to a reduction in relative efficiency of uptake, as levels fed are higher than Zn requirements. Ultimately, the result of this is an increase in Zn manure which can result in high Zn levels in soil when this manure is used, increasing the environmental impact of farm animals. A novel chelator, L-glutamic acid N,N-diacetic acid (GLDA), is a chelating agent, capable of binding di- and trivalent metal ions. By binding to these metal ions, it potentially provides stability of the complex in the upper gastrointestinal tract, which minimizes the formation of insoluble complexes, thereby improving nutritional bioavailability. This thesis aims to improve our understanding on trace mineral nutrition and determine the potential of using GLDA to increase the availability of minerals in livestock production.In Chapter 2 the impact of GLDA was compared to the well-established chelating agent ethylenediaminetetraacetic acid (EDTA). Previous work in literature showed effects of EDTA on trace mineral retention, but EDTA suffers from low biodegradability and its high chelation strength can be considered to be too high compared to metal transporters in the body. In experiment 1 broilers were fed Zn sulphate with GLDA or EDTA in molar amounts equivalent to chelate the level of Zn added. In experiment 2 the effect of GLDA on a basal diet containing no additional minerals was established. Serum and tibia Zn clearly responded to the increasing doses of dietary zinc with a significant response to the presence of EDTA and GLDA. These results are also indicative of the equivalent nutritional properties between GLDA and EDTA. In experiment 2, zinc levels in serum and tibia were also increased with the addition of GLDA to a basal diet lacking supplemental trace mineral, where serum zinc levels were 60% higher at the 216 mg/kg inclusion level. The study demonstrated that dietary GLDA enhanced availability of Zn.The aim of Chapter 3 was to quantify the reduction of dietary Zn that could be achieved in broilers to obtain the same Zn status. Broiler were fed Zn in a dose response manner with and without GLDA. The results indicated that when GLDA was included in the diet, based on tibia Zn, the same Zn status was achieved with a 19 mg/kg smaller Zn dose while based on serum Zn this was 27 mg/kg less Zn.Chelators are known to have negative side-effects when fed at high levels, for example due to chelation of minerals within the cell walls, leading to cell wall disruption. In order to determine the effects of high GLDA inclusion a dose response with GLDA up to 10000 mg/kg was performed in Chapter 4. The results of this study indicated that there are no negative side effects of GLDA inclusion up to 3000 mg/kg of GLDA/kg feed. The GLDA residue levels in breast tissue indicated that 0.01% of total GLDA absorption is stored in breast tissue. Higher values are found in kidney and liver for the highest inclusion level, indicating that the fraction of GLDA that is absorbed is actively excreted by the animal. The limited absorption of GLDA indicates that the role of GLDA affecting Zn availability takes place within the gastrointestinal tract of the animal, by sustaining solubility during digestive processes.As the Zn load in manure of pigs is greater than that of broilers, the effect of GLDA in piglets was determined in Chapter 5. On top, pigs are a better model for potential human implications than broilers, which is important considering the high prevalence of Zn deficiency in the developing world. GLDA appeared to protect a significant fraction of soluble luminal Zn from being captured by phytic acid and mediated it towards the Zn transport mechanisms in the gut mucosa, thereby promoting higher Zn retention in GLDA-supplemented animals compared to control animals. This led to lower necessity for mobilization of body Zn stores to compensate for endogenous losses in the presence of GLDA, lowering the chance of subclinical or clinical Zn deficiency.In Chapter 6 we discussed the impact of GLDA on Zn availability and retention in farm animals by combining the results of previous chapters with existing literature. The importance of study design when assessing trace mineral availability is discussed. The impact of GLDA on trace mineral availability is discussed by showing the increased retention of Zn when using adding GLDA to diets of animals. The potential impact on the environment by implementing GLDA is discussed and put into context regarding sustainable farming, emphasizing the key role GLDA can play

Effect of L-glutamic acid N,N-diacetic acid on the availability of dietary zinc in broiler chickens

Chelating agents can be used to improve the nutritional availability of trace minerals within the gastrointestinal tract. This study was conducted to determine the effect of a novel chelating agents, L-glutamic acid N,N-diacetic acid (GLDA), a biodegradable alternative to ethylenediaminetetraacetic acid on the nutritional bioavailability of zinc in broilers. Twelve dietary treatments were allocated to 96 pens in a randomized block design. Pens contained 10 Ross 308 male broilers in a factorial design with 6 incremental zinc levels (40, 45, 50, 60, 80, and 120 ppm of total Zn), with and without inclusion of GLDA (0 and 100 ppm) as respective factors. Experimental diets were supplied from day 7 to 21/22 and serum, liver and tibia Zn content were determined in 3 birds per pen. Growth performance and liver characteristics were not affected by dietary treatments, but both supplemental Zn and GLDA enhanced tibia and serum zinc concentration. The positive effect of GLDA was observed at all levels of the dietary Zn addition. The amount of zinc needed to reach 95% of the asymptotic Zn response was determined using nonlinear regression. When GLDA was included in the diet, based on tibia Zn, the same Zn status was achieved with a 19 ppm smaller Zn dose while based on serum Zn this was 27 ppm less Zn. Dietary GLDA reduces supplemental Zn needs to fulfill nutritional demands as defined by tibia Zn and serum Zn response. Considering the positive effect on the nutritional availability of Zn in broilers, GLDA presents an opportunity as biodegradable additive, to reduce Zn supplementation to livestock and thereby reducing Zn excretion into the environment, while fulfilling the nutrition Zn needs of farmed animals.</p

Metabolomic Analysis of Wooden Breast Myopathy Shows a Disturbed Lipid Metabolism

Myopathies have risen strongly in recent years, likely linked to selection for appetite. For white striping (WS), causes have been identified; but for wooden breast (WB), the cause remains speculative. We used metabolomics to study the breast muscle of 51 birds that were scored for both at 35 days of age to better understand potential causes. A partial least square discriminant analysis revealed that WS and WB had distinct metabolic profiles, implying different etiologies. Arginine and proline metabolism were affected in both, although differently: WB increased arginine in breast muscle implying that the birds did not use this pathway to increase tissue blood flow. Antioxidant defenses were impeded as shown by low anserine and beta-alanine. In contrast, GSH and selenium concentrations were increased. Serine, linked to anti-inflammatory properties, was increased. Taurine, which can stabilize the cell’s sarcolemma as well as modulate potassium channels and cellular calcium homeostasis, was also increased. Mineral data and depressed phosphatidylethanolamine, cAMP, and creatine-phosphate suggested compromised energy metabolism. WB also had drastically lower diet-derived lipids, suggesting compromised lipid digestion. In conclusion, WB may be caused by impaired lipid digestion triggered by a very high appetite: the ensuing deficiencies may well impair blood flow into muscle resulting in irreparable damage

Efficacy of l-glutamic acid, N,N-diacetic acid to improve the dietary trace mineral bioavailability in broilers

Trace minerals are commonly supplemented in the diets of farmed animals in levels exceeding biological requirements, resulting in extensive fecal excretion and environmental losses. Chelation of trace metal supplements with ethylenediaminetetraacetic acid (EDTA) can mitigate the effects of dietary antagonists by preserving the solubility of trace minerals. Lack of EDTA biodegradability, however, is of environmental concern. l-Glutamic acid, N,N-diacetic acid (GLDA) is a readily biodegradable chelating agent that could be used as a suitable alternative to EDTA. The latter was tested in sequential dose-response experiments in broiler chickens. Study 1 compared the effect of EDTA and GLDA in broilers on supplemental zinc availability at three levels of added zinc (5, 10, and 20 ppm) fed alone or in combination with molar amounts of GLDA or EDTA equivalent to chelate the added zinc, including negative (no supplemental zinc) and positive (80 ppm added zinc) control treatments. Study 2 quantified the effect of GLDA on the availability of native trace mineral feed content in a basal diet containing no supplemental minerals and supplemented with three levels of GLDA (54, 108, and 216 ppm). In study 1, serum and tibia Zn clearly responded to the increasing doses of dietary zinc with a significant response to the presence of EDTA and GLDA (P < 0.05). These results are also indicative of the equivalent nutritional properties between GLDA and EDTA. In study 2, zinc levels in serum and tibia were also increased with the addition of GLDA to a basal diet lacking supplemental trace minerals, where serum zinc levels were 60% higher at the 216 ppm inclusion level. Similar to the reported effects of EDTA, these studies demonstrate that dietary GLDA may have enhanced zinc solubility in the gastrointestinal tract and subsequently enhanced availability for absorption, resulting in improved nutritional zinc status in zinc-deficient diets. As such, GLDA can be an effective nutritional tool to reduce supplemental zinc levels in broiler diets, thereby maintaining health and performance while reducing the environmental footprint of food-producing animals.</p

Dietary l-glutamic acid N, N-diacetic acid improves short-term maintenance of zinc homoeostasis in a model of subclinical zinc deficiency in weaned piglets

This study compared the Zn response in selected tissues of weaned piglets fed L-glutamic acid, N,N-diacetic acid (GLDA), while challenged with short-term subclinical Zn deficiency (SZD). During a total experimental period of eight days, 96 piglets were fed restrictively (450 g/d) a high phytate (9 g/kg) diet containing added Zn at 0, 5, 10, 15, 20, 25, 45 and 75 mg/kg with and without 200 mg/kg of GLDA. No animals showed signs of clinical Zn deficiency and no phenotypical differences were observed. Broken line analysis of Zn status parameters such as liver Zn and apparently absorbed Zn indicated that the gross Zn requirement threshold was around 55 mg/kg diet. Supplementation of Zn above this threshold led to a saturation of the response in apparently absorbed Zn and linear increase in liver Zn. Bone and serum Zn responded to the dose in a linear fashion, likely due to the time-frame of Zn homoeostatic adaptation. Inclusion of GLDA into the diets yielded a higher intercept for bone Zn (P < 0·05). Liver Zn accumulation and MT1A gene expression was higher for piglets receiving GLDA (P < 0·05), indicating higher Zn influx. This study indicates that a strong chelator such as GLDA mitigates negative effects of phytate in plant-based diets, by sustaining Zn solubility, thereby improving nutritional Zn availability