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.

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Abstract. Eleven individual additives were incubated with either perennial ryegrass or with grass silage+barley grain (50 : 50) and the in vitro methane output was assessed using the gas production technique (GPT). Additives were: fatty acids (lauric, oleic, linoleic and linolenic acids), halogenated methane analogues (bromoethanesulfonate and bromochloromethane), pyromellitic diimide, statins (mevastatin and lovastatin), a probiotic (Saccharomyces cerevisiae) and an unsaturated dicarboxylic acid (fumaric acid). Each additive was included at a range of concentrations. Effects on methane output per gram of feed dry matter (DM) incubated (CH 4 /DMi) and disappeared (CH 4 /DMd), as well as other fermentation variables, were evaluated after 24 h of incubation. The addition of increased concentrations of individual fatty acids, bromoethanesulfonate and pyromellitic diimide caused a dose-dependent decline in methane output (CH 4 /DMi, CH 4 / DMd), when incubated with either perennial ryegrass or grass silage+barley grain. No methane output was detected for either feed with the addition of !5 mM bromochloromethane. The statins were ineffective inhibitors of methane output regardless of feed type. For perennial ryegrass, S. cerevisiae caused a dose-dependent decline in CH 4 /DMd and fumaric acid a dose-dependent decline in CH 4 /DMi and CH 4 /DMd. The effectiveness of lauric, oleic, linoleic and linolenic acids and bromoethanesulfonate to reduce methane output was more pronounced when incubated with grass silage+barley grain than with perennial ryegrass, and therefore the type of feed is an important component for any future in vitro and in vivo studies to be undertaken with these additives. Thus, incorporating different feed types in the initial in vitro screening protocols of all new additives is recommended.Additional keywords: in vitro total gas production technique, rumen fermentation modifiers, ruminant diets.