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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.

Isotopic tracers are used to directly quantify the effect of mammary Δ9-desaturation on milk fatty acid (FA) composition, but very few studies have applied this method to measuring the endogenous synthesis of rumenic acid (RA; cis-9,trans-11 conjugated linoleic acid) in cows and goats, and no publications exist in ewes. In sheep, knowledge about the contribution of stearoyl-coenzyme A desaturase (SCD) to milk FA secretion is derived mostly from indirect estimates based on inhibition of the enzyme by oral administration of cobalt, a cost-effective method that has not been validated to date. To fill both gaps, we conducted an isotopic tracer assay in sheep to quantify the proportion of endogenous RA in milk for the first time in this species. We then compared the results with estimates derived from a Co administration assay performed on the same animals. First, 5 lactating ewes received an intravenous injection of 200 mg of [1-13C]trans-11 18:1 (vaccenic acid, VA), the precursor for RA production by SCD activity. At -24, -15, 0, 9, 24, 33, 48, 57, 72, 81, and 96 h post-injection, we recorded milk yield and collected milk samples to examine fat concentration and FA profile. We conducted compound-specific isotope analysis of VA and RA by gas chromatography-combustion isotope ratio mass spectrometry. Afterward, in the Co administration assay, ewes received a daily dose of 7 mg of Co/kg of body weight for 5 d. We analyzed milk samples for composition before and on the last days of cobalt dosing. On average, 17% of the injected amount of [1-13C]VA was transferred to milk within 96 h post-injection, and up to 29% of the VA taken up by the mammary gland was desaturated to milk RA. Under our conditions, the mean proportion of this conjugated linoleic acid isomer deriving from Δ9-desaturation represented 82 to 90% of the amount secreted in milk. However, the proportion estimated in the Co assay with calculations based on individual FA concentrations was lower (on average, 46%). When we calculated the same estimates based on changes in Δ9-desaturation ratios after Co dosing, the higher values of endogenous RA (75%) did not differ from the results of the isotopic tracer assay. Nevertheless, correlation analysis between the results obtained through [1-13C]VA or Co administration revealed no significant relationship, which would prevent acceptance of the latter as a reliable alternative to isotopic labeling to examine mammary Δ9-desaturation in dairy ewes.

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.

11 páginas, 4 tablas, 1 figura., Milk odd- and branched-chain fatty acids (OBCFA) are largely derived from bacteria leaving the rumen, which has encouraged research on their use as biomarkers of rumen function. Targeted research has examined relationships between these fatty acids (FA) and dietary components, but interactions between the effects of lipids and other nutrients on milk OBCFA are not well characterized yet. Furthermore, factors controlling milk OBCFA in sheep are largely unknown. Thus, the present meta-analysis examined relationships between diet composition and milk OBCFA using a database compiled with lot observations from 14 trials in dairy ewes fed lipid supplements. A total of 47 lots received lipid supplements, whereas their respective controls (27 lots) were fed the same basal diets without lipid supplementation. Relationships between milk OBCFA and dietary components were first assessed through a principal component analysis (PCA) and a correlation analysis. Then, responses of milk OBCFA to variations in specific dietary components (selected on the basis of the PCA) were examined in more detail by regression analysis. According to the loading plot, dietary unsaturated C18 FA loaded opposite to major milk OBCFA (e.g., 15:0, 15:0 anteiso, and 17:0) and were strongly correlated with principal component 1, which described 46% of variability. Overall, regression equations supported this negative, and generally linear, relationship between unsaturated C18 FA levels and milk OBCFA. However, the influence of C20–22 n-3 polyunsaturated FA and saturated FA was more limited. The PCA also suggested that dietary crude protein is not a determinant of milk OBCFA profile in dairy ewes, but significant relationships were observed between some OBCFA and dietary fiber or starch, consistent with a potential role of these FA as biomarkers of rumen cellulolytic and amylolytic bacteria. In this regard, regression equations indicated that iso FA would show opposite responses to increasing levels of acid detergent fiber (positive linear coefficients) and starch (negative linear coefficients). Lipid supplementation would not largely affect these associations, supporting the potential of OBCFA as noninvasive markers of rumen function under different feeding conditions (i.e., with or without lipid supplementation). Because consumption of these FA may have nutritional benefits for humans, the use of high-fiber/low-starch rations might be recommended to maintain the highest possible content of milk OBCFA in dairy sheep., 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, UE). P. G. Toral benefited from a Ramón y Cajal research contract (RYC-2015-17230) and A. Della Badia from a FPI predoctoral contract (PRE2018-086174), from the Spanish Ministries of Economy and Competitiveness (MINECO) and Science and Innovation (MICINN), respectively. Co-funding by the European Social Fund is also acknowledged. The authors thank M.-P. Létour-neau-Montminy (Université Laval, Québec, Canada) for helpful assistance with statistical analysis. The authors have not stated any conflicts of interest.

12 páginas, 5 tablas., Dairy ewes are less prone than cows to milk fat depression (MFD) but suffer from this syndrome when marine lipids are added to their diet to modulate milk fatty acid (FA) profile. However, there are large individual differences in MFD extent, and the reasons behind this variability are uncertain. On this basis, a study was conducted in lactating sheep to test the hypotheses that individual susceptibility to the low-fat milk condition may be explained by differences in (1) the milk concentration of some FA, particularly antilipogenic FA, or (2) the transcriptional regulation of mammary lipogenesis. For 5 wk, 15 ewes received a total mixed ration supplemented with 0 (control; n = 5) or 20 g of fish oil/kg of dry matter [10 animals selected out of 22 and divided into those showing marked (RESPON+; n = 5) or mild (RESPON-; n = 5) MFD]. Milk production and composition, including a comprehensive FA profile, were examined on 3 consecutive days before and after treatments. Candidate gene expression was also analyzed before the start of the trial and at its end using RNA isolated from milk somatic cells. According to the experimental design, the fish-oil-induced decrease in milk fat concentration was much stronger in RESPON+ (-25.4%) than in RESPON- (-7.7%). Milk from all ewes fed the supplemented diet showed rather uniform changes in the proportion of potentially healthy FA (such as cis-9,trans-11 18:2, trans-11 18:1, or 20:5n-3) and of those with confirmed or putative antilipogenic effects (e.g., cis-9 16:1, trans-10 and cis-11 18:1, trans-9,cis-11 18:2, and 10-oxo-18:0), without significant variation between RESPON+ and RESPON-. It was not possible to relate the very few exceptions to this general trend (e.g., in cis-7 16:1 and 22:6n-3) to responsiveness. Major mechanisms involved in mammary lipogenesis, specifically the uptake and de novo synthesis of FA, appeared to be unequally inhibited in ewes displaying different degrees of MFD, with molar yields of >16C FA being unaffected in RESPON-. However, this was not reflected in candidate gene expression. Supplementation with fish oil showed a tendency to lower the mRNA abundance of lipogenic genes such as ACSS2, FASN, LPIN1, FADS2, and INSIG1, but only SCD and GPAT4 tended to differ between RESPON- and RESPON+. Overall, these results offer no convincing support for the initial hypotheses, so further research must be pursued to explain the individual variation in MFD severity. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved., This study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2014- 54587-R). The authors thank L. Rodríguez (FPI predoctoral grant, MINECO) and A. G. Mendoza for their help with experimental animals and fatty acid analysis. P. G. Toral benefits from a Ramón y Cajal research contract from the MINECO. Co-funding by the European Regional Development Fund is also acknowledged.

11 páginas, 4 tablas, 2 figuras., Milk fat depression (MFD) caused by trans-10 cis-12 18:2 is known to be mediated in cows and ewes by downregulation of mammary lipogenic genes. However, transcriptional mechanisms underlying marine lipid-induced MFD have not been well defined yet and the few available studies in ovine are not consistent. This trial was conducted to directly compare changes in animal performance, milk fatty acid (FA) composition and particularly mammary mRNA abundance of candidate lipogenic genes and transcription factors (14 genes in total) in response to the inclusion of fish oil or trans-10 cis-12 18:2 in dairy sheep diet. To meet this objective, 12 lactating Assaf ewes (days in milk (DIM) = 64 ± 1.9; milk yield = 1.72 ± 0.135 kg/d; milk fat concentration = 5.17 ± 0.228%) were divided into 3 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% DM of fish oil (FO) or 1% DM of a commercial product rich in trans-10 cis-12 18:2 (Lutrell Pure; CLA) for 39 days. Measurements and samplings were conducted before starting the treatments and at the end of the trial. Milk samples were used for RNA extraction from somatic cells. Feed intake was not affected by lipid supplements and, as designed, reductions in milk fat concentration (‒31%) were similar in the two treatments, although the unpredicted increase in milk production with FO counteracted the anticipated reduction in milk fat yield. Nevertheless, this did not preclude the detection of FO-induced decreases in the mRNA abundance of candidate lipogenic genes (e.g., ACSS2, FASN and LPIN1), thus supporting the hypothesis that transcriptional regulation would be a relevant component of this type of MFD in sheep. Expected CLA-induced downregulation of some genes, such as FASN or SREBF1, could not be detected in our samples, which might be related, at least in part, to high inter-individual variation and relatively advanced lactation stage (on average 102-103 DIM on days 38 and 39). Overall, direct comparison of the effects of dietary FO and CLA on transcript abundance of candidate lipogenic genes and transcription factors suggest that there might be relevant differences in the transcriptional control mechanisms underlying the MFD induced by each kind of supplement (i.e., fish oil or CLA)., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2014-54587, co-funded by the European Regional Development Fund). P. G. Toral gratefully acknowledges receipt of a postdoctoral research contract from the MINECO (Juan de la Cierva program). The authors thank Y. Chilliard, L. Bernard, C. Leroux (INRA, Clermont-Ferrand, France), and J. J. Arranz and A. Suárez-Vega (University of León, Spain), for helpful assistance with nutrigenomic techniques.

12 páginas, 6 tablas., Supplementation of dairy ewe diet with marine lipids may be an effective strategy for modulating milk fatty acid composition but induces milk fat depression (MFD). This syndrome has been associated with a shortage of 18:0 for uptake and Δ9-desaturation that may impair the capacity of the mammary gland to achieve an adequate fluidity for milk fat secretion. On this basis, it was suggested that supplemental 18:0 may contribute to alleviate marine lipid-induced MFD in sheep. To test this hypothesis, 12 lactating ewes were allocated to 1 of 3 lots and used in a 3 × 3 Latin square design with 3 periods of 28 d each and 3 experimental treatments: a total mixed ration without lipid supplementation (control) or supplemented with 20 g/kg of DM of fish oil alone (FO) or in combination with 20 g/kg of DM of 18:0 (FOSA). Diets were offered ad libitum, and animal performance and rumen and milk fatty acid composition were studied at the end of each period. After completing the Latin square trial and following a change-over design, the in vivo digestibility of supplemental 18:0 was estimated using 6 lactating sheep. As expected, diet supplementation with fish oil increased the milk content of some potentially health-promoting fatty acids (e.g., cis-9,trans-11 18:2, trans-11 18:1, 20:5n-3, 22:5n-3, and 22:6n-3), but reduced milk fat concentration and yield (−20% in both FO and FOSA treatments). Thus, although reductions in milk 18:0 and cis-9 18:1 output caused by FO (−81 and −51%, respectively) were partially reversed with FOSA diet (−49 and −27%, respectively), the addition of 18:0 to the diet did not prove useful to alleviate MFD. This response, which could not be fully accounted for by the low digestibility coefficient of supplemental 18:0, may challenge the theory of a shortage of this fatty acid as a mechanism to explain fish oil-induced MFD in sheep. Effects of FO and FOSA on rumen and milk fatty acid composition would support that increases in the concentration of some candidate milk fat inhibitors (e.g., cis-9 16:1 or 10-oxo-18:0) might play a relevant role in this type of MFD., This work was supported by the Council of Castile and Leon (CSI023U13) and the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2011- 23700). P. G. Toral and D. Carreño benefit from a Juan de la Cierva research contract and an FPI predoctoral grant, respectively, from the MINECO.

12 páginas, 3 tablas, 1 figura., The modulation of milk fat nutritional quality through fish oil supplementation seems to be largely explained by the action of n-3 very long chain polyunsaturated fatty acids (PUFA) on ruminal biohydrogenation (BH) of C18 fatty acids (FA). However, relationships among this action, disappearance of those PUFA in the rumen, and potential detrimental consequences on ruminal fermentation remain uncertain. This study compared the effect of 20:5n-3 (eicosapentaenoic acid; EPA), 22:5n-3 (docosapentaenoic acid; DPA), and 22:6n-3 (docosahexaenoic acid; DHA) on rumen fermentation and BH of C18 FA and was conducted simultaneously in cows and sheep to provide novel insights into interspecies differences. The trial was performed in vitro using batch cultures of rumen microorganisms with inocula collected from cannulated cows and ewes. The PUFA were added at a dose of 2% incubated dry matter, and treatment effects on ruminal C18 FA concentrations, PUFA disappearances, and fermentation parameters (gas production, ammonia and volatile FA concentrations, and dry matter and neutral detergent fiber disappearances) were examined after 24 h of incubation. A principal component analysis suggested that responses to PUFA treatments explained most of the variability; those of ruminant species were of lower relevance. Overall, EPA and DHA were equally effective for inhibiting the saturation of trans-11 18:1 to 18:0 and had a similar influence on ruminal fermentation in cows and sheep (e.g., reductions in gas production and acetate:propionate ratio). Nevertheless, DHA further promoted alternative BH pathways that lead to trans-10 18:1 accumulation, and EPA seemed to have specific effects on 18:3n-3 metabolism. Only minor variations attributable to DPA were observed in the studied parameters, suggesting a low contribution of this FA to the action of marine lipids. Although most changes due to the added PUFA were comparable in bovine and ovine, there were also relevant specificities, such as a stronger inhibition of 18:0 formation in cows and a greater increase in 18:3n-3 metabolites in sheep. No direct relationship between in vitro disappearance of the incubated PUFA and effect on BH (in particular, inhibition of the last step) was found in either cows or ewes, calling into question a putative link between extent of disappearance and toxicity for microbiota. Conversely, an association between the influence of these PUFA on ruminal lipid metabolism and fermentation may exist in both species. In vivo verification of these findings would be advisable. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved., We dedicate this article to the memory of Kevin J. Shingfield, a renowned expert in ruminant lipid metabolism and, above all, a good friend of ours. This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2014-54587-R) and the Spanish National Research Council (CSIC; PIE201540E104). P. G. Toral benefits from a Ramón y Cajal research contract and D. Carreño benefits from an FPI predoctoral grant, both from MINECO. Cofunding by the European Regional Development Fund is also acknowledged.

8 páginas, 4 tablas, 1 figura., The use of stable isotopes is a reliable and risk-free alternative to radioactive tracers to directly examine in vivo fatty acid (FA) metabolism. However, very limited information is available in ruminants, and there is none in sheep. Therefore, we conducted an experiment in dairy ewes to determine, for the first time in this species, the uptake, Δ9-desaturation and secretion of 13C-labeled stearic acid (SA) into milk, with the aim of measuring in vivo endogenous synthesis of milk oleic acid (OA) and stearoyl-CoA desaturase (SCD) activity. Six lactating Assaf ewes fed a total mixed ration (forage:concentrate ratio 30:70) received an intravenous injection of 2 g of 13C-labeled SA. At −24, −15, 0, 4, 8, 12, 16, 20, 24, 36, 48, 60 and 72 h postinjection (p.i.), milk yield was recorded and milk samples were collected to examine fat concentration and FA composition, including compound-specific isotope analysis of SA and OA by gas chromatography-combustion isotope ratio mass spectrometry. Over the p.i. period, the SA proportion ranged from 7.6 to 8.3% of total FA, with a maximum 13C enrichment of 1.9%, whereas OA was more abundant (14.3-15.4% of total FA) and had lower 13C enrichments (up to 0.69%). On average, 15% of the isotopic tracer was transferred to milk within 72 h p.i., and 47-50% of the SA taken up by the mammary gland would have been desaturated to OA. The proportion of oleic acid being synthesized endogenously was estimated to represent between 48% and 57% of the amount secreted in milk. Further research under different dietary conditions would be now recommended., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2011-23700). P. G. Toral and D. Carreño benefit from a Juan de la Cierva research contract and an FPI predoctoral grant, respectively, from the MINECO. The authors thank Drs. L. Bernard and Y. Chilliard (INRA, Clermont-Ferrand, France) for helpful training in stable carbon isotope techniques during Toral’s postdoctoral stay, and Drs. R. Redondo and V. Peiró (Autonomous University of Madrid, Spain) for their contribution to GC-C-IRMS analysis.

9 páginas, 3 tablas, 1 figura., Addition of marine algae (MA) to the diet of dairy ruminants has proven to be an effective strategy to enhance the milk content of some bioactive lipids, but it has also been associated with the syndrome of milk fat depression. Little is known, however, about the persistency of the response to dietary MA in sheep. Based on previous experiments with dairy ewes fed sunflower oil plus MA, it was hypothesized that the response might be mediated by time-dependent adaptations of the rumen microbiota, which could be evaluated indirectly through milk fatty acid (FA) profiles. Animal performance and milk FA composition in response to MA in the diet were studied using 36 Assaf ewes distributed in 6 lots and allocated to 2 treatments (3 lots/ treatment) consisting of a total mixed ration (40:60 forage:concentrate ratio) supplemented with 25 g of sunflower oil (SO)/kg of dry matter plus 0 (SO; control diet) or 8 g of MA/kg of dry matter (SOMA diet). Milk production and composition, including FA profile, were analyzed on d 0, 6, 12, 18, 24, 34, 44, and 54 of treatment. Diet supplementation with MA did not affect milk yield but did decrease milk fat content. Differences in the latter were detected from d 18 onward and reached −17% at the end of the experiment (i.e., on d 54). Compared with the control diet, the SOMA diet caused a reduction in milk 18:0 and its desaturation product (cis-9 18:1) that lasted for the whole experimental period. This decrease, together with the progressive increase in some putative fat synthesis inhibitors, especially trans-10 18:1, was related to the persistency of milk fat depression in lactating ewes fed MA. Additionally, inclusion of MA in the diet enhanced the milk content of trans-11 18:1, cis-9,trans-11 18:2, and C20–22 n-3 polyunsaturated FA, mainly 22:6 n-3. Overall, the persistency of the responses observed suggests that the ruminal microbiota did not adapt to the dietary supply of very long chain n-3 polyunsaturated fatty acids., This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2011- 23700). E. Bichi gratefully acknowledges receipt of a predoctoral grant from the Spanish National Research Council (CSIC; JAE Programme) supported by European Social Fund. The authors thank K. J. Shingfield and V. Toivonen (MTT Agrifood Research Finland, Jokioinen, Finland) for invaluable help with FA identification. We also thank the research farm staff from the Instituto de Ganadería de Montaña (León, Spain) for their help in the fieldwork.

11 páginas, 5 tablas., Several studies in dairy cows have shown a relationship between milk fat depression (MFD) and alterations caused in lipogenic gene expression by dietary nutrients. However, information on small ruminants is not only scarce but also inconsistent. Therefore, this experiment was conducted in dairy ewes to study the effect of a diet known to induce MFD on milk fatty acid (FA) composition and mRNA abundance of key candidate genes involved in mammary lipogenesis. Twelve lactating Assaf ewes (on average 63d in milk) were randomly assigned to 2 treatments consisting of a total mixed ration based on alfalfa hay and concentrates (50:50), supplemented with 0 (control) or 17g of fish oil/kg of diet dry matter (FO). Profiles of milk FA and mRNA abundance of candidate genes in biopsied mammary tissue were examined before starting the treatments and after 1 and 4.5wk on the diets. As expected, FO induced MFD and modified milk FA composition. Compared with the control, reductions in milk fat concentration and yield were not detected on d 7, but reached up to 25 and 22%, respectively, on d 30. However, increases in confirmed or putative antilipogenic FA (trans-10,cis-12 and trans-9,cis-11 18:2, cis-9 16:1, cis-11 18:1, and oxo-FA) were already established on the early stage of the treatment and lasted until the end of the feeding period. These changes were accompanied by decreases in the mRNA abundance of genes encoding lipogenic enzymes. The coordinated nature of the downregulation, which tended to affect most studied metabolic pathways, including FA activation (ACSS1), de novo synthesis (ACACA and FASN), uptake and transport (LPL and FABP3), desaturation (SCD1), and esterification (AGAPT6), supports the involvement of a central regulator of milk fat synthesis. In this regard, without ruling out the potential contribution of PPARG, our results suggest that SREBF1 would have a relevant role in the MFD syndrome in sheep fed FO. Among the other studied transcription factors, the tendency to a downregulation of INSIG1 was associated with that of SREBF1, whereas no variation was detected for SCAP or THRSP. Fish oil had no significant effects on the transcript abundance of CD36, GPAM, DGAT1, LPIN1, and XDH. Overall, changes in potential antilipogenic FA and mRNA abundance of candidate lipogenic genes support a relationship between them and suggest that FO-induced MFD in dairy ewes would be mediated by transcriptional mechanisms., This work was supported by the Council of Castile and Leon (CSI023U13) and the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2014-54587, co-funded by the European Regional Development Fund). D. Carreño and P. G. Toral benefitted from an FPI predoctoral grant and a Juan de la Cierva research contract, respectively, from the MINECO. The authors thank A. González-Mendoza from the Instituto de Ganadería de Montaña (Spain) for his help in the field work and laboratory analysis. Special gratitude is extended to Y. Chilliard, L. Bernard, C. Leroux (INRA, Clermont-Ferrand, France), and J. J. Arranz (University of León, Spain), and also to L. Barrios (Biostatistics Department, CSIC; Madrid, Spain) for helpful assistance with nutrigenomic techniques and statistical analysis, respectively.

11 páginas, 4 tablas, 1 figura., Nutrigenomic studies of mammary lipogenesis in ruminants often rely on the use of mammary tissue (MT) collected either by biopsy or at slaughter. However, isolating RNA from milk would be a useful and cost-effective technique that may avoid distress to the animal and facilitate the collection of samples in time series experiments. This assay was therefore conducted to test the hypothesis that RNA extracted from milk somatic cells (MSC) in dairy sheep would be a feasible alternative to the performance of MT biopsies for nutrigenomic analyses. To meet this objective, 8 lactating Assaf ewes were divided in 2 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil, which was known not only to elicit milk fat depression but also to downregulate the expression of some candidate genes involved in mammary lipogenesis. Total RNA was extracted from MSC and biopsied MT to examine whether the potential changes in the abundance of transcripts was similarly detected with both RNA sources. Milk fatty acid profile was also analyzed by gas chromatography, and variations in mRNA abundance were determined by reverse transcription quantitative PCR. Values of RNA integrity number were always ≥7.7. The expected and designed decrease of milk fat concentration with fish oil (−29%), was associated with a lower transcript abundance of genes coding for enzymes involved in fatty acid activation (ACSS1), de novo synthesis (ACACA and FASN), uptake from plasma lipids (LPL), and esterification of fatty acids to glycerol (LPIN1), as well as of a transcription factor that may regulate their expression (INSIG1). Stable mRNA levels were showed in other candidate genes, such as FABP3, GPAT4, or SCD. Changes due to the dietary treatment were similarly detected with both RNA sources (MSC and MT biopsies), which supports the initial hypothesis and would validate the use of milk as an alternative RNA source for nutrigenomic analyses in dairy sheep., This work was supported by the Ministry of Economy and Competitiveness (MINECO, Spain; AGL2014-54587, co-funded by the European Regional Development Fund, European Union). P. G. Toral gratefully acknowledges receipt of a postdoctoral research contract from the MINECO (Juan de la Cierva program).

11 páginas, 2 figuras, 4 tablas., This study was conducted in lactating ewes to examine the involvement of Δ9-desaturase in mammary lipogenesis, especially in the endogenous synthesis of cis-9, trans-11 18:2 and cis-9 18:1, because no information on this matter was available for dairy sheep. With this aim, 6 Assaf ewes were monitored in a 15-d experiment, which included a 5-d pretreatment period, a 5-d treatment period, and a 5-d posttreatment period. During the treatment period, ewes received 0.5 g/d of sterculic acid (a cyclopropene fatty acid that inhibits Δ9-desaturase), delivered intravenously in 4 equal doses at 6-h intervals. Animals were fed pasture to supply mainly α-linolenic acid and minimize the amount of milk cis-9, trans-11 18:2 of ruminal origin. Sterculic acid administration was calculated to inhibit Δ9-desaturase by 70% based on the milk content of cis-9 14:1. This inhibition resulted in decreases in the milk content of the enzyme products (e.g., cis-9 10:1, cis-9 14:1, cis-9 16:1, cis-9 18:1, and cis-9, trans-11 18:2) and increases in its substrates (e.g., 14:0, 18:0, and trans-11 18:1), as well as in reductions in the desaturase indexes. Some other milk fatty acids, further to previously reported products or substrates of Δ9-desaturase (e.g., cis-15 18:1 and cis-9, cis-15 18:2, or trans-11, trans-15 18:2, and cis-9, trans-11, trans-15 18:3), were also affected by sterculic acid administration. Endogenous synthesis was the major source of cis-9 18:1 and cis-9, trans-11 18:2, accounting for 63 and 74% of its content in milk fat, respectively. To our knowledge, the present study provides the first estimates of endogenous synthesis of these 2 bioactive fatty acids in ovine milk fat., This work was supported by the Spanish Ministry of Science and Innovation (MICINN, Madrid; AGL2008- 04805). The authors from Instituto de Investigación en Ciencias de la Alimentación wish to thank the Comunidad Autónoma de Madrid (S2009-AGR-1469) and the MICINN (Consolider Ingenio 2010 Programme; FUN-C-FOOD CSD2007-063) for their financial support. E. Bichi gratefully acknowledges receipt of a predoctoral grant from the Spanish National Research Council [Consejo Superior de Investigaciones Científicas (CSIC), Madrid; JAE Programme] supported by the European Social Fund.

35 pages, 1 figure, 5 tables., Rumen microbial biohydrogenation of dietary unsaturated fatty acids has a major effect on the process of developing healthier dairy products. This study aimed to investigate in vivo the effect of diet supplementation with sunflower (SO) and fish (FO) oils on the rumen bacterial community in dairy sheep. First, 32 lactating ewes, divided in 8 lots of 4 animals each (2 lots per treatment), were fed a high-concentrate total mixed ration supplemented with 0, 2% SO, 1% FO, or 2% SO plus 1% FO. After 21 d, rumen fluid samples were taken from each lot for DNA extraction and fluorescence in situ hybridization (FISH) analysis. In a second experiment, 5 cannulated ewes were first fed the same TMR, with the exception of a higher forage level, and then changed to the same diet supplemented with 2% SO plus 1% FO. After 0, 3, and 10 d, rumen content samples were taken for DNA extraction and FISH analysis (fluid). Total bacteria and the Butyrivibrio group were studied in microbial DNA by terminal RFLP analysis (T-RFLP), and real-time PCR was used to quantify Butyrivibrio bacteria that produce vaccenic acid or stearic acid. In rumen fluid samples, total bacteria and clostridial clusters IX and XIV were analyzed by FISH. Dietary supplementation with SO plus FO seemed to induce important changes in the total bacteria and Butyrivibrio populations, and a high interindividual variation was observed, and the speed of the effect of the lipid supplementation depended on the individual microbial composition. Analysis by T-RFLP and FISH showed increases in cluster IX bacteria with SO plus FO supplementation, presumably Quinella-like microorganisms. The abundances of vaccenic acid- and stearic acid-producing Butyrivibrio relative to total bacteria, estimated by real time PCR, were low (0.28 and 0.18%, respectively, in rumen fluid, and 0.86 and 0.81% in rumen contents) and only that of SA-producing bacteria seemed to be reduced by diets containing FO, although differences were only significant in lactating ewes. The T-RFLP analysis showed a variable effect of lipid supplementation on different bacteria of the family Lachnospiraceae, which includes the cultured bacteria known to be actively involved in rumen biohydrogenation. These results suggest that the latter bacteria do not play a dominant role in this process, and therefore other as-yet-uncultivated microorganisms might be more relevant., A. Belenguer and P. G. Toral gratefully acknowledge receipt of a research contract and a predoctoral grant, respectively, from the Spanish National Research Council (CSIC, I3P Programme). This work was supported by the Spanish Ministry of Science and Innovation (Project AGL2008-04805-C02-02), the Council of Castile and Leon (Projects CSI01B08) and the Spanish National Research Council (Project 200940I034).

9 pages, 5 tables, 1 figure.-- ISI Article Identifier: 000268136400045., Previously published as proceeding at the Options Méditerranéennes A, 85: 439-444 (2009). https://digital.csic.es/handle/10261/15655, This work was conducted to investigate the effect of supplementing grazing ewes on pasture with a cereal concentrate on the milk fatty acid (FA) profile. Ninety Assaf ewes in mid lactation were distributed in 9 lots of 10 animals each and allocated to 3 feeding regimens: 1) pasture—ewes were only allowed to graze pasture (an irrigated sward of Lolium perenne, Trifolium pratense, and Dactylis glomerata); 2) PS—grazing ewes were supplemented with oat grain (700 g/animal and day); and 3) TMR—ewes were fed ad libitum a total mixed ration (TMR; 80:20 concentrate/forage ratio). Milk yield and composition were recorded for 5 wk. The highest milk yield was observed in ewes receiving the TMR and the lowest in grazing ewes supplemented with oat grain. Productions of milk fat, protein, and total solids showed the lowest values in treatment PS. The atherogenicity index, which comprises C12:0, C14:0, and C16:0, in PS milk fat was no different from that observed in milk from animals on pasture (1.53 for pasture, 1.54 for PS, and 3.22 for TMR). Oat grain supplementation generated higher amounts of C18:0 and cis-9 C18:1 in milk fat than the pasture-only diet, but significantly decreased the levels of α-linolenic acid and most of intermediates of the process of biohydrogenation of this FA. Cis-9 trans-11 C18:2 and trans-11 C18:1, its precursor for endogenous synthesis in the mammary gland, were lower in PS (0.58 and 1.59 g/100 g of total FA) than in TMR (0.72 and 1.92 g/100 g of total FA) and very different from the results observed in grazing ewes receiving no supplement (1.21 and 3.88 g/100 g of total FA). Furthermore, the lowest levels of trans-10 C18:1 and trans-10 cis-12 C18:2 were detected in the milk fat of ewes fed pasture. It is concluded that, when pasture quality and availability do not limit dairy production, supplementation of grazing ewes with oat grain compromised the milk FA profile without any significant positive effect on milk production., This work was supported by the Ministerio de Educación y Ciencia (AGL2005–04760). The authors from Instituto del Frío (Consejo Superior de Investigaciones Científicas, CSIC) thank Comunidad Autónoma de Madrid (S-505/AGR-0153) and the Ministerio de Educación y Ciencia (Consolider CSD2007–063) for the financial support. The authors from Instituto de Ganadería de Montaña (CSIC-Universidad de León) thank CSIC (200640I212) for the financial support.

9 pages, 1 figure, 4 tables., The effects of ruminant diet supplementation with linoleic or different polyunsaturated fatty acids (FA) have been well documented. Less abundant information, however, exists on the effects of incorporating monounsaturated FA, such as oleic acid, on lipid metabolism or animal performance. The purpose of this work was to assess the effects of feeding dairy ewes a diet supplemented with high levels of olive oil (OO) on milk yield and composition, paying particular attention to the FA profile. Twenty-four Assaf ewes were fed ad libitum with 2 diets, control or supplemented with 6% OO (2 lots of 6 animals per diet) for 4 wk. Milk yield and composition and dry matter intake were recorded weekly. Milk FA composition was determined by gas chromatography and conjugated linoleic acid profile by silver ion HPLC. Milk yield increased in ewes receiving OO, with no differences in dry matter intake. The OO diet decreased the milk protein percentage but increased the milk fat, protein, and total solids yield. Medium-chain saturated FA (C10:0 to C16:0) content was reduced with OO supplementation, whereas C18:0 and cis-9 C18:1 content increased. Leaving aside trans- 11, most trans C18:1 isomers, mainly trans-10, increased in supplemented ewes. The main conjugated linoleic acid isomer (cis-9, trans-11 C18:2) decreased with OO supplementation, whereas trans-7, cis-9 and trans-9, cis-11 C18:2 exhibited a remarkable increase. These results support the argument that the supplementation of ewe diets with high levels of OO does not have any detrimental effects on animal performance but substantially modifies the FA profile. Key words: oleic acid, olive oil, conjugated linoleic acid, monounsaturated fatty acid, This work was supported by the Ministerio de Educación y Ciencia (Spain; Research Projects AGL2005-04760 and Consolider CSD2007-063) and the Comunidad Autónoma de Madrid (Project S-0505/AGR/000153).

10 pages, 5 tables, 1 figure.-- PMID: 18349249 [PubMed].-- © 2008 American Dairy Science Association., Previously published as proceeding at the XXXVIII Jornadas de Estudio AIDA (XII Jornadas sobre Producción Animal) (Zaragoza, Spain, May 16-17, 2007). https://digital.csic.es/handle/10261/4374, Feeding vegetable oils rich in linoleic acid has been demonstrated to be an effective strategy to enrich milk with conjugated linoleic acid (CLA). However, high amounts of vegetable oil in the diet in free form could adversely affect animal performance, mainly in sheep. The aim of this work was to improve the ewe milk fatty acid profile by increasing potentially healthy acids such as CLA without any detrimental effects on milk production and ruminal fermentation with soybean oil (SBO) diet supplementation. Twenty-four ewes were assigned to 2 treatments and fed 2 diets (control or supplemented with 6% of SBO; 2 lots of 6 animals per treatment) and fed ad libitum for 4 wk. The forage:concentrate ratio was 20:80. Batch cultures of rumen microorganisms were used to study in vitro rumen fermentation. Changes in fatty acid profile were characterized as a reduction in C6:0 to C16:0 at the expense of an increase in C18:0, C18:1 isomers, and CLA concentrations. Proportions of milk CLA and trans-11 C18:1 (vaccenic acid) went from 1.04 to 3.44 and 2.08 to 6.20 g/100 g of total fatty acids, respectively. However, the SBO diet also increased trans-10 C18:1 and other trans C18:1 content. No significant decreases were found in the treatments for dry matter intake and milk production. The notable increases in trans-10, cis-12 and trans-9, cis-11 were not accompanied by fat level decreases in ewe milk. Concerning in vitro ruminal fermentation, no significant differences were found in the extent and rate of gas production, effective degradability, in vitro true digestibility, and volatile fatty acid production. The results demonstrate that dairy sheep milk CLA content can be substantially increased (more than 3-fold) by adding high levels of SBO in the diet as free oil, without any negative effects on animal performance., This work was supported by the Ministerio de Educación y Ciencia (Spain; Research Projects AGL2005-04760) and the Comunidad Autónoma de Madrid (Project S-0505/AGR/000153).

11 páginas, 3 tablas, 3 figuras., Oral administration of cobalt has been proven to alter milk fatty acid (FA) composition consistent with an inhibition of mammary stearoyl-coenzyme A desaturase (SCD) activity in ruminants, but the mechanisms explaining its mode of action remain uncertain. In this study, Co (as Co-acetate) was dosed to lactating ewes with the aims of examining mammary gene expression during Co-induced changes in milk FA composition, and estimating the endogenous synthesis of SCD products in milk of sheep fed an 18:3n-3-enriched diet. Twelve Assaf ewes fed a diet supplemented with 2% linseed oil were allocated to 2 experimental groups and received an oral drench supplying either 0 (Control) or 9 mg of Co/kg of body weight per day. Treatments were administered in 3 equal doses at 8-h intervals for 6 days. No effects of Co administration on animal performance were observed. The changes in milk FA (namely, reductions in most cis-9-containing FA) were consistent with an inhibition of SCD in the absence of detectable effects on the relative importance of mammary de novo synthesis and FA uptake. The high proportion of endogenous cis-9 trans-11 18:2 observed in this study (89%) would agree with a greater supply of trans-11 18:1 of ruminal origin in ewes fed linseed oil, compared with previous estimates in sheep fed a diet without lipid supplementation. Differences between studies could also be related to diet-induced changes in SCD activity. Altogether, both mechanisms would support that basal diet composition is a major determinant of the relative contribution of Δ9-desaturation to milk FA profile. Similarly, the consumption of a diet rich in 18:3n-3 might also explain the low proportion of milk cis-9 18:1 estimated to derive from Δ9-desaturation (29%). The administration of Co to ewes fed linseed oil allowed to discriminate minor 18:3 isomers in milk, such as cis-9 trans-12 cis-15 18:3, as SCD products. Finally, Co dosing lowered the mRNA abundance of SCD1 in the mammary secretory tissue (33%), whereas no changes were detected in the SCD5 isoform or in the studied transcription factors (SREBF1, PPARG, SP1, and EGR2). These results suggest that the mode of action of Co in dairy ewes would be at least partly mediated by the downregulation of SCD1., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; AGL2011- 23700). P. G. Toral gratefully acknowledges receipt of a postdoctoral research contract from the MINECO (Juan de la Cierva program).

PMID: 16428640 [PubMed], Previously published as proceeding at the XXXVII Jornadas de Estudio AIDA (XI Jornadas sobre Producción Animal) (Zaragoza, Spain, 2005). https://digital.csic.es/handle/10261/5104, Based on the potential benefits of cis-9, trans-11 conjugated linoleic acid (CLA) for human health, there is a need to develop effective strategies for enhancing milk fat CLA concentrations. Levels of cis-9, trans-11 CLA in milk can be increased by supplements of fish oil (FO) and sunflower oil (SO), but there is considerable variation in the response. Part of this variance may reflect time-dependent ruminal adaptations to high levels of lipid in the diet, which lead to alterations in the formation of specific biohydrogenation intermediates. To test this hypothesis, 16 late lactation Holstein-British Friesian cows were used in a repeated measures randomized block design to examine milk fatty acid composition responses to FO and SO in the diet over a 28-d period. Cows were allocated at random to corn silage-based rations (8 per treatment) containing 0 (control) or 45 g of oil supplement/kg of dry matter consisting (1:2; wt/wt) of FO and SO (FSO), and milk composition was determined on alternate days from d 1. Compared with the control, the FSO diet decreased mean dry matter intake (21.1 vs. 17.9 kg/d), milk fat (47.7 vs. 32.6 g/kg), and protein content (36.1 vs. 33.3 g/kg), but had no effect on milk yield (27.1 vs. 26.4 kg/d). Reductions in milk fat content relative to the FSO diet were associated with increases in milk trans-10 18:1, trans-10, cis-12 CLA, and trans-9, cis-11 CLA concentrations (r2 = 0.74, 0.57, and 0.80, respectively). Compared with the control, the FSO diet reduced milk 4:0 to 18:0 and cis 18:1 content and increased trans 18:1, trans 18:2, cis-9, trans-11 CLA, 20:5 n-3, and 22:6 n-3 concentrations. The FSO diet caused a rapid elevation in milk cis-9, trans-11 CLA content, reaching a maximum of 5.37 g/100 g of fatty acids on d 5, but these increases were transient, declining to 2.35 g/100 g of fatty acids by d 15. They remained relatively constant thereafter. Even though concentrations of trans-11 18:1 followed the same pattern of temporal changes as cis-9, trans-11 CLA, the total trans 18:1 content of FSO milk was unchanged because of the concomitant increases in the concentration of other isomers (4–10 and 12–15), predominantely trans-10 18:1. In conclusion, supplementing diets with FSO enhances milk fat cis-9, trans-11 CLA content, but the high level of enrichment declines because of changes in ruminal biohydrogenation that result in trans-10 replacing trans-11 as the major 18:1 biohydrogenation intermediate formed in the rumen., Financial support from the Biotechnology and Biological Sciences Research Council, Department for Environment, Food, and Rural Affairs, The Scottish Executive Environment and Rural Affairs Department and the Milk Development Council under the Eating, Food and Health LINK scheme is gratefully acknowledged. The authors thank the staff of the Center for Dairy Research Animal Production Research Unit for diligent care of experimental animals and sample collection and Vesa Toivonen for assistance with the interpretation of mass-spectral data.

9 páginas, 3 figuras, 3 tablas., Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment length polymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition., This study was supported by the Spanish Ministry of Economy and Competitiveness (Madrid, Spain; AGL2011-23700). T. Castro-Carrera was granted a predoctoral fellowship from the Spanish National Research Council (CSIC, Madrid, Spain; JAE Programme) supported by the European Social Fund (European Commission, Brussels, Belgium).

13 páginas, 7 tablas,1 figura., Supplementation of ruminant diets with plant oils and marine lipids is an effective strategy for lowering saturated fatty acid (FA) content and increasing the concentration of cis-9,trans-11 conjugated linoleic acid and long-chain n-3 FA in ruminant milk. However, changes in populations of ruminal microorganisms associated with altered biohydrogenation of dietary unsaturated FA are not well characterized. Twenty-five lactating Assaf ewes were allocated at random to 1 of 5 treatments composed of dehydrated alfalfa hay and concentrates containing no additional lipid (control), or supplemented with 25 g of sunflower oil and 0 (SO), 8 (SOMA1), 16 (SOMA2), or 24 (SOMA3) g of marine algae/kg of diet dry matter. On d 28 on diet, samples of rumen fluid were collected for lipid analysis and microbial DNA extraction. Appearance and identification of biohydrogenation intermediates was determined based on complementary gas chromatography and Ag+-HPLC analysis of FA methyl esters. Total bacteria and the Butyrivibrio group were studied in microbial DNA by terminal RFLP analysis, and real-time PCR was used to quantify the known Butyrivibrio bacteria that produce trans-11 18:1 or 18:0. Dietary supplements of sunflower oil alone or in combination with marine algae altered the FA profile of rumen fluid, which was associated with changes in populations of specific bacteria. Inclusion of marine algae in diets containing sunflower oil resulted in the accumulation of trans 18:1 and 10-O-18:0 and a marked decrease in 18:0 concentrations in rumen fluid. At the highest levels of supplementation (SOMA2 and SOMA3), marine algae also promoted a shift in ruminal biohydrogenation pathways toward the formation of trans-10 18:1 at the expense of trans-11 18:1. Changes in the concentration of biohydrogenation intermediates were not accompanied by significant variations in the abundance of known cultivated ruminal bacteria capable of hydrogenating unsaturated FA. However, certain bacterial groups detected by terminal RFLP (such as potentially uncultured Lachnospiraceae strains or Quinella-related bacteria) exhibited variations in their relative frequency consistent with a potential role in one or more metabolic pathways of biohydrogenation in the rumen., This work was supported by the Spanish Ministry of Science and Innovation (MICINN; AGL2008- 04805-C02-02) and the Spanish National Research Council (CSIC; 200940I034). P. G. Toral was granted a fellowship from the CSIC (I3P Program). The authors thank R. John Wallace and Nest McKain from the Rowett Institute of Nutrition and Health (Aberdeen, UK), and C. Jamie Newbold from the Institute of Biological, Environmental and Rural Sciences (Aberystwyth, UK) for kindly providing the bacterial strains.

12 páginas, 4 figuras, 4 tablas., Ruminant diet supplementation with sunflower oil (SO) and fish oil (FO) has been reported as a good strategy for enhancing some milk fat compounds, such as conjugated linoleic acid (CLA) and omega-3 polyunsaturated fatty acids (PUFA) in dairy cows, but no information is available regarding dairy sheep. In this work, ewe diet was supplemented with FO, alone or in combination with SO, with the aim of improving milk nutritional value and evaluating its effect on animal performance. Sixty-four Assaf ewes in mid-lactation, fed a high-concentrate diet, were distributed in 8 lots of 8 animals each and assigned to 4 treatments (2 lots per treatment): no-lipid supplementation (Control), or supplementation with 20 g SO/kg (SO), 10 g FO/kg (FO), or 20 g SO plus 10 g FO/kg (SOFO). Milk production and composition, including a complete fatty acid profile, were analysed on days 0, 3, 7, 14, 21, and 28 on treatments. FO supplementation tended to reduce DM intake in comparison with the Control treatment (-15%), and its use in combination with SO (SOFO) resulted in a significant decrease in milk yield as well (-13%). All lipid supplements reduced milk protein content, and FO also reduced milk fat content, by up to 21% alone (FO) and 27% in combination with SO (SOFO). Although the mechanisms involved in FO-induced milk fat depression are not wellestablished yet, the observed increase in some milk trans-FA that are putative inhibitors of milk fat synthesis, such as trans-9 cis-11 CLA, and the almost 3-fold decrease in C18:0 (consistent with the theory of reduced milk fat fluidity) may be involved. When compared with the Control, lipid supplementation remarkably improved the milk content of rumenic acid (cis-9 trans-11 CLA; up to 4-fold increases with SO and SOFO diets), whereas FO-containing diets also increased milk omega-3 PUFA, mainly docosahexanoic acid (DHA, with mean contents of 0.29 and 0.38% of total fatty acids for SOFO and FO, respectively), and reduced the omega-6/omega-3 FA ratio to approximately half the control value. All lipid supplements resulted in high levels of some trans-FA, mainly trans-11 C18:1 (vaccenic acid) but also trans-10 C18:1., This work was supported by the Spanish Ministry of Science and Innovation (MICINN; AGL2008-04805). The authors from the Instituto de Ganadería de Montaña would like to thank the Council of Castile and Leon for complementary financial support (GR158) and the farm staff for their help in the field work. P.G. Toral gratefully acknowledges receipt of a predoctoral grant from the CSIC (I3P Programme). The authors from the Instituto del Frío (CSIC) wish to thank the Comunidad Autónoma de Madrid (S- 505/AGR-0153) and the Ministerio de Ciencia e Innovación (Consolider Ingenio 2010 Programme; FUN-C-FOOD CSD2007-063) for their financial support. They also wish to thank M.V. Rodríguez-Pino for her technical assistance in the chromatographic analysis.

14 pages, 9 tables, 2 figures.-- Available online 19 September 2010, Studies in ruminants have shown that supplementing the diet with a mixture of fish oil (FO) and sunflower oil (SO) enhances the concentration of cis-9, trans-11 conjugated linoleic acid (CLA), 20:5 n-3, and 22:6 n-3 in milk due to alterations in ruminal biohydrogenation but the intermediates formed under these conditions are not well characterised. Five ewes fitted with rumen cannula and fed a high concentrate diet were used to examine the effect of a mixture (30 g/kg DM) of FO and SO (1:2, wt/wt) on temporal changes in rumen fermentation characteristics and the relative abundance of biohydrogenation intermediates in ruminal digesta collected after 0, 3, and 10 d on diet. Appearance and identification of biohydrogenation intermediates was determined based on complementary gas-liquid chromatography and Ag+-HPLC analysis of fatty acid methyl esters and gas chromatography-mass spectrometry analysis of corresponding 4,4-dimethyloxazoline derivatives. Inclusion of FO and SO in the diet had no effect on rumen pH, VFA concentrations or nutrient digestion, but altered the fatty acid composition of ruminal digesta, changes that were characterised by time-dependent decreases in 18:0 and 18:2 n-6 and the accumulation of trans 16:1, trans 18:1, 10-O- 18:0, and trans 18:2. Lipid supplements enhanced the proportion of 20:5 n-3 and 22:6 n-3 in digesta and resulted in numerical increases in cis-9, trans-11 CLA concentrations, but decreased the relative abundance of trans-10, cis-12 CLA. Furthermore, detailed analysis revealed the appearance of several unique 20:1, 20:2, 22:1, 22:3, and 22:4 products in ruminal digesta that accumulated over time, providing the first indications of 20 and 22 carbon fatty acid intermediates formed during the biohydrogenation of long-chain unsaturated fatty acids in sheep. In conclusion, FO and SO in a high concentrate diet caused a time-dependent inhibition of the complete biohydrogenation of 18 carbon unsaturated fatty acids resulting in the accumulation of trans 16:1, trans 18:1, trans 18:2, 20, and 22 carbon metabolites in ruminal digesta of sheep, with no evidence of a shift in ruminal biohydrogenation pathways towards trans-10 18:1 formation., P. G. Toral gratefully acknowledges receipt of a predoctoral research grant from the Spanish National Research Council (CSIC, I3P Programme). This work was supported by the Spanish Ministry of Science and Innovation (MICINN; AGL2008-04805-C02- 02) and the Council of Castile and Leon (Project GR158).

13 pages, 3 figures, 4 tables., In an attempt to develop strategies for enhancing the nutritional value of sheep milk fat, dairy ewe diet was supplemented with three incremental levels of marine algae (MA), in combination with sunflower oil, to evaluate the effects of these marine lipids on milk fatty acid (FA) profile and animal performance. Fifty Assaf ewes in mid-lactation were distributed in 10 lots of 5 animals each, and allocated to 5 treatments (2 lots per treatment): non-lipid supplementation (Control), or supplementation with 25 g of sunflower oil/kg DM plus 0 (SO), 8 (SOMA1), 16 (SOMA2), or 24 (SOMA3) g of MA (56.7 % ether extract)/kg DM. Milk production and composition, including FA profile, were analysed on days 0, 3, 7, 14, 21 and 28 on treatments. Neither intake nor milk yield were significantly affected by lipid addition but all MA supplements decreased milk fat content from d 14 onward, reaching a 30% reduction after 28 d on SOMA3. This milk fat depression might be related not only to the joint action of some putative fat synthesis inhibitors, such as trans-9 cis-11 C18:2 and probably trans-10 C18:1, but also to the limited ability of the mammary gland to maintain a desirable milk fat fluidity, that would have been caused by the noticeable rise in trans-C18:1 together with the lowered availability of stearic acid for oleic acid synthesis through the delta-9 desaturase. Furthermore, all lipid supplements, and mainly MA, reduced the secretion of de novo FA (C6:0-C14:0) without increasing the yield of pre-formed FA (> C16). SOMA supplementation resulted in larger increases in RA (cis-9 trans-11 C18:2) than those observed with SO alone, achieving a mean content as high as 3.22% of total FA, and representing more than a 7-fold increase compared to the control. Vaccenic acid (trans-11 C18:1) was also significantly enhanced (on average +794% in SOMA treatments), as was C22:6 ω-3 (DHA) content, although the transfer efficiency of the latter, from the diets to the milk, was very low (5%). All the same, the highest levels of MA inclusion (SOMA2 and SOMA3) reduced the milk ω-6/ω-3 ratio. On the other hand, MA supplements caused an important increase in trans-10 C18:1, which would disallow from considering that this milk has a healthier fat profile before unravelling the specific role of each individual FA and ensuring that this trans-FA is at least innocuous in relation to the cardiovascular disease risk., This work was supported by the Ministerio de Ciencia e Innovación (MICINN; AGL2008-04805). The authors from the Instituto de Ganadería de Montaña would like to thank the research farm staff for their help in the field work. P.G. Toral gratefully acknowledges receipt of a predoctoral grant from the CSIC (I3P Programme). The authors from the Instituto del Frío (CSIC) wish to thank the Comunidad Autónoma de Madrid (S-505/AGR-0153) and the Ministerio de Ciencia e Innovación (Consolider Ingenio 2010 Programme; FUN-C-FOOD CSD2007-063) for their financial support. They also wish to thank M.V. Rodríguez-Pino for her technical assistance in the chromatographic analysis.