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SUPPLEMENTARY MATERIAL - Please download it using the following link: http://dx.doi.org/10.20350/digitalCSIC/13779 (OPEN ACCESS), This work was supported by the Junta de Castilla y León (JCyL, Spain; project CSI276P18). P. G. Toral benefited from a Ramón y Cajal research contract from the Spanish Ministry of Economy and Competitiveness (MINECO; RYC-2015-17230) and C. Fernández-Díez from a postdoctoral research contract from JCyL. Co-funding by the European Regional Development Fund (ERDF/FEDER) and the European Social Fund (ESF) is also acknowledged.

11 páginas, 3 tablas, 3 figuras., The basis for marine lipid-induced milk fat depression (MFD) has not been stablished 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 [1-13C]ALA was added at a dose of 1% of incubated dry matter and the proportions of 13C-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 BH of 13ALA was extensive and yielded multiple metabolites, with a total of 48 chromatographic peaks showing 13C 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 13C-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-10 to trans-11 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., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2014- 54587-R and AGL2017-87812). P. G. Toral benefits from a Ramón y Cajal research contract from the MINECO (RYC-2015-17230). Co-funding by the European Regional Development Fund is also acknowledged. The authors thank V. Peiró, R. Sedano, and R. Redondo (Autonomous University of Madrid, Spain) for their assistance in gas chromatography-combustion isotope ratio mass spectrometry analysis.

13 páginas, 1 figura, 6 tablas., Both sheep and goats can display very different individual degrees of milk fat depression (MFD), which might explain some apparent contradictions in the literature. Because the antilipogenic effect of certain fatty acids (FA) is the most likely origin of MFD, characterizing the milk FA profile of animals showing different degrees of MFD seems a helpful step to understand the physiological basis of the tolerance or susceptibility to the syndrome. Analyzing whether specific traits may predetermine a particular responsiveness would also be of relevance to meet this aim. However, information about these aspects is scant, not only in goats and sheep but in ruminants in general. This study was conducted with 25 Murciano-Granadina does and 23 Assaf ewes that were fed a total mixed ration without lipid supplementation for 3 wk (control period). Then, all animals received the same basal diet supplemented with 2% of fish oil (FO) for 5 additional weeks (MFD period). At the end of this second period, and on the basis of the extent of FO-induced decreases in milk fat concentration, the 5 most responsive (RESPON+) and the 5 least responsive (RESPON−) animals were selected within each species, 20 in total. Milk yield and composition, including a comprehensive FA profile, were examined at the end of each period. By design, between-group variation in milk fat concentration and yield was substantial, but no significant interaction with the effect of species was detected. Reductions in these 2 performance traits averaged 6% in RESPON− and 26% in RESPON+. Results do not allow suggesting that responsiveness to MFD would be clearly predetermined neither by the studied performance traits nor by milk FA profile, although a certain relationship with energy balance might exist. Furthermore, variations in ewes and does displaying different individual degrees of MFD may be associated with changes in certain candidate milk fat inhibitors, such as trans-10 18:1 and cis-9 16:1, whereas trans-10,cis-12 conjugated linoleic acid would only have a minor role in determining MFD severity. Alterations in the molar yield of de novo and preformed FA suggest relevant differences in the mechanisms underlying MFD in RESPON+ and RESPON−, with interspecies effects being observed only in more tolerant animals. Further research is still required to elucidate key determinants of responsiveness to MFD., This work was supported by the Spanish Research State Agency (Agencia Estatal de Investigación) and the European Regional Development Fund (project AGL2017-87812-R, AEI/FEDER, EU). A. Della Badia benefited from an FPI predoctoral contract (PRE2018-086174) and P. G. Toral from a Ramón y Cajal research contract (RYC-2015-17230), from the Spanish Ministries Science and Innovation (MICINN) and of Economy and Competitiveness (MINECO), respectively. Co-funding by the European Social Fund is also acknowledged. The authors have not stated any conflicts of interest.

13 páginas, 4 figuras, 2 tablas., A great deal of uncertainty still exists about intermediate metabolites and pathways explaining the biohydrogenation (BH) of 20- and 22-carbon polyunsaturated fatty acids (PUFA). Therefore, this study was conducted to provide further insight into the ruminal metabolism of 20:5 n-3 (EPA), 22:5 n-3 (DPA), and 22:6 n-3 (DHA), the main n-3 PUFA present in the marine lipids used in dairy ruminant feeding, and to examine potential differences between bovine and ovine. To meet this aim, we investigated the 20- and 22-carbon metabolites accumulated during in vitro incubation of EPA, DPA, and DHA with rumen inocula from cows and ewes. The PUFA were added at a dose of 2% incubated dry matter and digesta samples were analyzed after 24 h of incubation using complementary gas-liquid chromatography of fatty acid methyl esters and gas chromatography-mass spectrometry of 4,4-dimethyloxazoline derivatives. Results suggested that the main BH pathway of EPA and DPA would proceed via the reduction of the double bond closest to the carboxyl group (cis-5 in EPA and cis-7 in DPA); curiously, this mechanism seemed of much lower importance for DHA. Thus, DPA would not be a major intermediate product of DHA and their BH might actually follow separate pathways, with the accumulation of numerous unique metabolites in each case. A principal component analysis supported this hypothesis, with a clear separation between PUFA treatments in the score and loading plots. Within EPA and DPA groups, cow and ewe samples loaded separately from each other but not distant. No conjugated 20:5, 22:5, or 22:6 isomer compatible with the initial product of EPA, DPA, or DHA metabolism, respectively, was identified in the ruminal digesta, although this would not unequivocally exclude their transient formation. In this regard, results from DPA incubations provided the first indication that the metabolism of this very long chain PUFA may involve the formation of conjugated double bond structures. The BH of EPA, DPA, and DHA resulted in the appearance of several tentative trans-10-containing metabolites, showing a general trend to be more abundant in the digesta of ewes than in that of cows. This finding was speculated to have some relationship with the susceptibility of dairy sheep to marine lipid-induced milk fat depression. Differences in the relative proportion of intermediate products would also suggest an influence of ruminant species on BH kinetics, with a process that would likely be slower and less complete in cows than in ewes. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2014- 54587-R). P. G. Toral benefited from a Ramón y Cajal research contract from the MINECO. Co-funding by the European Regional Development Fund is also acknowledged.