Simple SummaryDairy ewes, just like cows, suffer from the low-fat milk syndrome, also known as milk fat depression (MFD), when marine lipids are added to their diet to modulate milk fatty acid (FA) profile. Alterations in the ruminal metabolism of dietary unsaturated FA seem to be at the core of this MFD but its causative agent has not yet been confirmed. Although some alterations may derive from shifts in oleic acid metabolism, methodological constraints represent a major challenge: the fact that oleic and other dietary FA (e.g., linoleic and linolenic acids) have common intermediates hampers the identification of those metabolites deriving specifically from each one. Thus, we conducted an in vitro trial combining an isotopic tracer approach with the use of rumen inoculum from ewes fed a diet supplemented or not with fish oil (which is known to cause MFD in dairy animals), to provide new insights into shifts in oleic acid metabolism. Fish oil altered the relative contribution of specific pathways, with relevant increments in oxygenated FA, including a major candidate milk fat inhibitor (10-oxo-18:0). Changes in the concentration of trans 18:1 intermediates were also observed. Results are discussed in the context of their potential association with milk fat depression.AbstractShifts in ruminal oleic acid (OA) metabolism have received little research attention but recent studies have suggested their association with marine lipid-induced milk fat depression (MFD) in ewes and cows. Measurement of specific products of OA within the complex mixture of digesta lipids is however challenging. Therefore, this in vitro trial combined the isotopic labelling technique with the use of rumen inoculum from cannulated sheep fed a diet supplemented or not with 2% of fish oil (which has been demonstrated to cause MFD in dairy ruminants) to characterize the metabolism of OA in response to ruminal alterations associated with MFD. The products of 13C-OA after 24 h of incubation were analysed by gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS). Numerous 13C-labeled 18:1 intermediates and oxygenated FA were detected and no elongation or desaturation of 13OA occurred. Diet supplementation with fish oil (i.e., MFD conditions) resulted in no unique metabolites of 13OA but in relevant changes in the relative contribution of specific metabolic pathways. The inhibition of 18:0 saturation caused by this treatment appeared largely attributable to increased oxygenated FA proportion, in particular the candidate milk fat inhibitor 10-oxo-18:0, and warrants further research on the association between MFD and oxygenated FA. Changes in the concentration of 13C-labeled trans 18:1 intermediates but not in cis 18:1, were also observed.

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