White striping (WS) is a major problem affecting the broiler industry. Fillets affected by this myopathy present pathologies that compromise the quality of the meat, and most importantly, make the fillets more prone to rejection by the consumer. The exact etiology is still unknown, which is why a metabolomics analysis was performed on breast samples of broilers. The overall objective was to identify biological pathways involved in the pathogenesis of WS. The analysis was performed on a total of 51 muscle samples and distinction was made between normal (n = 19), moderately affected (n = 24) and severely affected (n = 8) breast fillets. Samples were analyzed using gas chromatographic mass spectral analysis and liquid chromatography quadrupole time-of-flight mass spectrometry. Data were subsequently standardized, normalized and analyzed using various multivariate statistical procedures. Metabolomics allowed for the identification of several pathways that were altered in white striped breast fillets. The tricarboxylic acid cycle exhibited opposing directionalities. This is described in literature as the backflux and enables the TCA cycle to produce high-energy phosphates through matrix-level phosphorylation and, therefore, produce energy under conditions of hypoxia. Mitochondrial fatty acid oxidation was limited due to disturbances in especially cis-5–14:1 carnitine (log2 FC of 2, P < 0.01). Because of this, accumulation of harmful fatty acids took place, especially long-chain ones, which damages cell structures. Conversion of arginine to citrulline increased presumably to produce nitric oxide, which enhances blood flow under conditions of hypoxia. Nitric oxide however also increases oxidative damage. Increases in taurine (log2 FC of 1.2, P < 0.05) suggests stabilization of the sarcolemma under hypoxic conditions. Lastly, organic osmolytes (sorbitol, taurine, and alanine) increased (P < 0.05) in severely affected birds; likely this disrupts cell volume maintenance. Based on the results of this study, hypoxia was the most likely cause/initiator of WS in broilers. We speculate that birds suffering from WS have a vascular support system in muscle that is borderline adequate to support growth, but triggers like activity results in local hypoxia that damages tissue.

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 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&lt;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 mineral, 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.

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.

Chapter 1 General introduction Chapter 2 Efficacy of L-glutamic acid N,N-diacetic acid to improve the dietary trace mineral bioavailability in broilers Chapter 3 Effect of L-glutamic acid N,N-diacetic acid on the availability of dietary zinc in broiler chickens Chapter 4 L-glutamic acid N,N-diacetic acid has a high tolerance level for broilers and poses no risk for consumer food safety Chapter 5 Dietary L-glutamic acid N,N-diacetic acid affects short term zinc homeostasis in weaned piglets at different levels of dietary zinc

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.