In vitro biohydrogenation of 13C-labeled α-linolenic acid in response to ruminal alterations associated with diet-induced milk fat depression in ewes

Abstract

The basis for marine lipid-induced milk fat depression (MFD) has not been established yet, but recent reports suggest the putative contribution of shifts in the ruminal metabolism of α-linolenic acid (ALA). To test this hypothesis, an isotopic tracer approach was used in batch cultures of rumen microorganisms with inoculum collected from cannulated ewes fed either a total mixed ration without lipid supplementation (control inoculum) or the same diet supplemented with 2% of fish oil, which is known to cause MFD in lactating sheep (FO-MFD inoculum). The [1-13 C]ALA was added at a dose of 1% of incubated dry matter and the proportions of 13 C-labeled fatty acids (FA) were examined after 24 h of incubation, using complementary gas chromatography and gas chromatography-combustion isotope ratio mass spectrometry (GC-C-IRMS) analyses. Expected differences in FA profiles were confirmed between control and FO-MFD inocula (e.g., large decreases in 18:0 and increases in most 18:1 and 18:2 intermediates, particularly trans isomers, to fish oil supply). The biohydrogenation of 13 ALA was extensive and yielded multiple metabolites, with a total of 48 chromatographic peaks showing 13 C enrichment, regardless of the inoculum type. However, although ALA was biohydrogenated through common pathways under standard or MFD conditions, large changes in the accumulation of 13 C-labeled FA suggest important differences in the relative contribution of each specific route. First, increased accumulation of trans-11-containing FA in FO-MFD incubations was accompanied by a general repression of the trans-13/14 pathway (supported by lower trans-13+14 18:1 or trans-11,trans-13 18:2 proportions), together with a lower production of cis FA (e.g., cis-9, -12, and -15 18:1 and some cis,cis 18:2). Results also downplayed the relevance of the trans-11 to trans-10 shift as an effective marker of diet-induced MFD in sheep, and challenged the involvement of some trans-10 intermediates (e.g., trans-10 18:1 and trans-10,cis-15 18:2) in the low-fat milk syndrome in this species. Conversely, increased abundance of most 18:3 intermediates (including some unidentified isomers) might be representative of ruminal alterations related to fish oil supplementation in ewes. Further research is necessary to examine the potential association between these findings and MFD in lactating animals.

Conditions Associated with Marine Lipid-Induced Milk Fat Depression in Sheep Cause Shifts in the In Vitro Ruminal Metabolism of 1-13C Oleic Acid

“…This experiment is an integral part of a larger study using stable isotopes (namely 13 C-labeled FA) to provide new insights into ruminal BH. A first paper on the in vitro BH of 13 C α-linolenic acid has recently been published [ 11 ] and contains detailed information about diets and rumen inocula composition that will therefore be only mentioned briefly in this new article.…”

“…Five rumen-cannulated dry ewes (BW = 65.5 ± 2.28 kg) were used as rumen inocula donors for the batch cultures of microorganisms, which were conducted as described previously [ 11 , 21 ]. Sheep were randomly divided into two groups and fed a total mixed ration (TMR) with no lipid supplementation (to obtain the control inoculum; 3 ewes) or supplemented with 2% dry matter (DM) of fish oil (to obtain the FO-MFD inoculum; 2 ewes).…”

“…The FA composition of both diets (% of total FA, data for control and supplemented TMR, respectively) included mainly 16:0 (22.6 vs. 21.7), cis -9 18:1 (14.8 vs. 14.5), 18:2n-6 (40.1 vs. 22.8) and 18:3n-3 (13.2 vs. 7.2), while EPA (3.3), DPA (0.7) and DHA (9.3) were only detected in the fish oil diet. Please see Toral et al [ 11 ] for details.…”

“…The percentage of FA originating from the incubated 13 OA was calculated as explained by Toral et al [ 11 ]. First, the 13 C% in the methanol was used to correct for the isotopic shift due to the extra methyl group in 13 C-labeled FAME that were detected during GC-C-IRMS analysis.…”

“…The most prominent among them would be those occurring in recently proposed candidate milk fat inhibitors, such as cis -11 18:1, trans -10 cis -15 18:2 and 10-oxo-18:0 [ 6 , 9 , 10 ]. Some of these changes would be associated with shifts in the BH of α-linolenic acid, as reported in a recent in vitro study [ 11 ] but some others, particularly variations in 18:1 and oxygenated FA, may derive from alterations in the metabolism of oleic acid (OA) in the rumen [ 12 , 13 ]. In this regard, supplementation with OA-rich lipids has been reported to induce MFD in dairy cows [ 14 , 15 , 16 ] and, combined with fish oil, to decrease milk fat concentration and yield to a greater extent than a blend of this marine lipid and a source of α-linolenic acid [ 17 ].…”