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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.

12 páginas, 4 tablas., Small ruminants are susceptible to milk fat depres-sion (MFD) induced by marine lipid supplementation. However, as observed in dairy cows, there is wide in-dividual variation in the response to MFD-inducing diets, which may be due to individual differences in ruminal processes. Therefore, we compared the rumi-nal responses of goats and sheep with varying degrees of MFD extent to improve our understanding of this complex syndrome. Our specific aims were to attempt to elucidate whether pre-existing variations in ruminal fermentation and biohydrogenation determine a higher tolerance or susceptibility to MFD, and whether the severity of MFD depends exclusively on the response to the diet. The trial was conducted with 25 does and 23 ewes fed a basal diet without lipid supplementation for 3 wk (control period). Then, 2% fish oil (FO) was added to the same diet for 5 additional weeks (MFD period). Based on the extent of the elicited MFD (i.e., the percentage variation between milk fat concentra-tions recorded at the end of the control and MFD periods), the 5 most responsive (RESPON+) and the 5 least responsive (RESPON−) animals were selected within each species. On the last day of each period, ruminal fluid samples were collected to examine fer-mentation parameters and fatty acid profiles. In gen-eral, the individual degree of MFD in sheep and goats did not seem to be predetermined by traits related to ruminal fermentation and biohydrogenation, including fatty acids that may serve as biomarkers of microorgan-isms. Regarding differences in the response to FO, the results suggest no link between MFD susceptibility and concentration of biohydrogenation intermediates such as trans-10-containing C18, C20, and C22 metabolites.The explanation for individual responses based on a shortage of ruminal acetate and 18:0 for mammary uptake also seems to be dismissed, based on the lack of variation in these compounds between RESPON+ and RESPON−. However, the concentration of unsaturated fatty acids provided by FO (e.g., cis-9 16:1, cis-11 18:1, and 20:5n-3) was higher in the rumen of RESPON+ than RESPON− ewes and does. Thus, although further research is needed, the extent of biohydrogenation of these fatty acids might be associated with tolerance or susceptibility to MFD., This work was supported by the Spanish Research State Agency (Agencia Estatal de Investigación) and the European Regional Development Fund (projects AGL2017-87812-R, MINECO/AEI/FEDER, EU and PID2020-113441RB-I00, MCIN/AEI). A. Della Badia benefited from an FPI predoctoral contract, co-funded by the European Social Fund (PRE2018-086174, MCIU/AEI/FSE, EU). The authors thank L. Barrios (Biostatistics Department, CSIC; Madrid, Spain) for her helpful assistance with statistical analysis. The au-thors have not stated any conflicts of interest.

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.

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.

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.

The lactational effects of shearing (CO, control unshorn; SH, shorn) were investigated in 48 dairy ewes of 2 breeds (Lacaune, LC, n = 24; Manchega, MN, n = 24) having a similar stage of lactation (120 ± 6 d in milk) and body frame (65.1 ± 1.5 kg of body weight and 2.4 ± 0.1 body condition score), but differing in fleece and milk production. Ewes were penned indoors, adapted to the diet (alfalfa hay ad libitum and fixed amount of concentrate), and allocated for 30 d in 8 balanced groups to which the experimental treatments were applied. All ewes were sheared on the same day. Feed intake by pen and milk yield by ewe were recorded daily. Individual samples of milk (d -3, 3, 5, 7, and 15) and blood (d -7, 3, 7, and 15) were collected, as well as body weight and body condition score measured (d -15, 0, and 15), related to shearing. Pooled milk samples per pen were also collected before and after shearing for milk fatty acid analysis (d -3 and 15). Average temperatures in the barn before (12.6 ± 0.7°C) and after (13.7 ± 0.4°C) shearing were mild. Fleece was heavier in MN than in LC (1.04 ± 0.10 vs. 0.75 ± 0.09 kg/ewe) and tended to cover more body surface in MN than in LC ewes. Responses to shearing varied according to breed, the rectal temperature after shearing only decreasing significantly in the MN (-0.36 ± 0.09°C). Feed intake increased in the LC-SH (5%), when compared with LC-CO, but did not vary in the MN ewes. Ingestibility of the alfalfa hay, expressed as filling units for sheep and monitored in 2 groups of 6 dry and unshorn ewes of each breed (73.0 ± 2.5 kg of body weight and 3.1 ± 0.2 body condition score), was constant throughout the experiment (0.99 ± 0.03 filling units for sheep/kg of dry matter). Regarding milk production, LC-SH ewes yielded 10% more milk (1.38 ± 0.06 vs. 1.52 ± 0.05 kg/d) than LC-CO ewes, but no differences were detected in MN ewes (0.74 ± 0.03 kg/d, on average). No differences in the concentration of major milk components by effect of the shearing treatment were detected in either breed, but LC-SH ewes yielded 9% more milk protein than did LC-CO ewes. No relevant effects of shearing were detected on milk fatty acid profiles, although MN ewes showed lower C4:0, C6:0, C14:0, trans-11 and trans-12 C18:1 contents than did LC ewes. Moreover, no changes by effect of shearing were detected in plasma glucose, nonesterified fatty acids, cortisol, or insulin values in either breed, or in body weight or body condition score. In conclusion, shearing dairy ewes during lactation under mild winter conditions is a suitable management option that may increase feed intake and milk production, without deleterious effects on milk composition.

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.

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, 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).

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.

11 páginas, 3 tablas, 2 figuras., Previous investigations have shown that cobalt (Co) modifies milk fat composition in cattle, consistent with an inhibition of stearoyl-coenzyme A desaturase (SCD) activity, but it remains unclear whether other ruminant species are also affected. The present study examined the effects of oral administration of Co acetate on intake, rumen function, and milk production and fatty acid (FA) composition in sheep. Twenty lactating Assaf ewes were allocated into 1 of 4 groups and used in a continuous randomized block design that involved a 15-d adaptation, a 6-d treatment, and a 10-d posttreatment period. During the treatment period, animals received an oral drench supplying 0 (control), 3 (Co3), 6 (Co6), and 9 (Co9) mg of Co/kg of BW per day, administered in 3 equal doses at 8-h intervals. Cobalt acetate had no influence on intake or milk fat and protein concentrations, whereas treatments Co6 and Co9 tended to lower milk yield. Results on rumen parameters showed no effects on rumen fermentation, FA composition, or bacterial community structure. Administration of Co acetate decreased milk concentrations of FA containing a cis -9 double bond and SCD product:substrate ratios, consistent with an inhibition of SCD activity in the ovine mammary gland. Temporal changes in milk fat composition indicated that the effects of treatments were evident within 3d of dosing, with further changes being apparent after 6d and reverting to pretreatment values by d 6 after administration. Effect on milk FA composition did not differ substantially in response to incremental doses of Co acetate. On average, Co decreased milk cis-9 10:1/10:0, cis-9 12:1/12:0, cis- 9 14:1/14:0, cis-9 16:1/16:0, cis-9 17:1/17:0, cis-9 18:1/18:0, and cis-9,trans-11 18:2/trans-11 18:1 concentration ratios by 30, 32, 38, 33, 21, 24, and 25%, respectively. Changes in milk fat cis-9 10:1, cis-9 12:1, and cis-9 14:1 concentrations to Co treatment indicated that 51% of cis-9 18:1 and cis-9,trans-11 18:2 secreted in milk originated from δ9-desaturation. In conclusion, results demonstrated the potential of oral Co administration for the estimation of endogenous synthesis of FA containing a cis-9 double bond in the mammary gland of lactating ruminants. Indirect comparisons suggest that the effects of Co differ between sheep and cattle., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Madrid, Spain; AGL2011-23700).