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Enhancing the ability of animals to convert feed into meat or milk by optimizing feed efficiency (FE) has become a priority in livestock research. Although untargeted metabolomics is increasingly used in this field and may improve our understanding of FE, no information in this regard is available in dairy ewes. This study was conducted to (1) discriminate sheep divergent for FE and (2) provide insights into the physiological mechanisms contributing to FE through high-throughput metabolomics. The ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) technique was applied to easily accessible animal fluids (plasma and milk) to assess whether their metabolome differs between high- and low-feed efficient lactating ewes (H-FE and L-FE groups, respectively; 8 animals/group). Blood and milk samples were collected on the last day of the 3-wk period used for FE estimation. A total of 793 features were detected in plasma and 334 in milk, with 100 and 38 of them, respectively, showing differences between H-FE and L-FE. The partial least-squares discriminant analysis separated both groups of animals regardless of the type of sample. Plasma allowed the detection of a greater number of differential features; however, results also supported the usefulness of milk, more easily accessible, to discriminate dairy sheep divergent for FE. Regarding pathway analysis, nitrogen metabolism (either anabolism or catabolism) seemed to play a central role in FE, with plasma and milk consistently indicating a great impact of AA metabolism. A potential influence of pathways related to energy/lipid metabolism on FE was also observed. The variable importance in the projection plot revealed 15 differential features in each matrix that contributed the most for the separation in H-FE and L-FE, such as l -proline and phosphatidylcholine 20:4e in plasma or l -pipecolic acid and phosphatidylethanolamine (18:2) in milk. Overall, untargeted metabolomics provided valuable information into metabolic pathways that may underlie FE in dairy ewes, with a special relevance of AA metabolism in determining this complex phenotype in the ovine. Further research is warranted to validate these findings. [ABSTRACT FROM AUTHOR]

Despite the benefits of adding marine lipids rich in n-3 polyunsaturated fatty acids (PUFA) to ruminant diets to improve milk fatty acid (FA) composition, this strategy induces milk fat depression (MFD), precluding its application under practical conditions. The MFD elicited by marine lipids has tentatively been explained by a shortage of available ruminal 18:0 for mammary uptake and Δ 9 -desaturation to cis -9 18:1, which might increase milk fat melting point and impair fat secretion. This hypothesis was challenged in a recent experiment in dairy ewes, as diet supplementation with 2% DM of 18:0 did not prove useful to alleviate fish oil-induced MFD. However, further research with higher levels of 18:0 inclusion seemed advisable. Therefore, in this study, 16 lactating ewes were allocated to 4 treatments lasting 27 days: a total mixed ration containing no additional lipid (control) or 2% DM of fish oil alone (FO) or in combination with 3% (FOSA3) or 4% (FOSA4) of stearic acid. Fish oil supplementation induced MFD, but addition of 18:0, regardless of the dose, was not able to mitigate it: decreases in milk fat content reached 19% in FO, 20% in FOSA3 and 27% in FOSA4. The reduction in milk 18:0 concentration due to FO (−81%) was completely reverted neither by FOSA3 (−41%) nor by FOSA4 (−25%), and something similar occurred with cis -9 18:1 concentrations (12.53, 5.91, 9.50 and 11.28 g/100 FA, in control, FO, FOSA3 and FOSA4, respectively). Increases in some candidate milk fat inhibitors in FO and FOSA diets (e.g., cis -9 16:1, cis -11 18:1, trans -10 18:1, 10-oxo-18:0, or trans -10 cis -15 18:2) might account for the absence of a positive effect of dietary 18:0. The estimated milk fat melting point was lower in the three supplemented diets (on average, −2.6 °C compared with the control). In conclusion, addition of high doses of stearic acid to the diet (at 3 and 4% DM) was not able to alleviate the MFD caused by the concomitant supplementation with fish oil. This lack of response would further challenge the hypothesis suggesting that fish oil-induced MFD is mainly explained by decreased ruminal production of 18:0 and subsequent problems of milk fat fluidity, but further research would be still needed. [ABSTRACT FROM AUTHOR]

Abstract: In vitro studies have suggested that feeding tannins to ruminants can favourably alter ruminal biohydrogenation of dietary linoleic acid, enhancing accumulation of trans-11 18:1 (VA, vaccenic acid) in the rumen and thereby the content of some human health promoting fatty acids, such as VA and cis-9 trans-11 18:2 (rumenic acid, RA), in dairy or meat products. However, reports on impacts of these phenolic compounds on milk fatty acid (FA) profile are very limited and inconsistent. Therefore, fourteen Assaf ewes in mid lactation were used to examine effects of addition of a mixture (1:1, w/w) of two commercial oenological extracts of quebracho condensed tannins (CT) and chestnut hydrolysable tannins (HT) to a diet containing sunflower oil (SO) on animal performance, milk yield and composition, and ruminal fermentation. All sheep received a total mixed ration based on alfalfa hay and a concentrate (400:600), supplemented with 20g of SO/kg dry matter (DM) plus 0 (Control; n =7) or 10 (TAN; n =7)g of tannins/kg DM. Milk production and composition was analyzed on days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 27 on treatments, and milk FA profile on days 0, 3, 6, 12, 18 and 27. Neither DM intake nor milk, or its component, yield was affected by TAN treatment. Similarly, addition of the extract of tannins to a SO containing ration did not alter concentrations of the major FA classes in milk (i.e., saturates, monounsaturates, and polyunsaturates), had very limited effects on the proportion of particular FA, and was not able to enhance milk VA and RA enrichment above that achieved with SO supplementation. Temporal changes in milk FA composition were characterized by an increase in unsaturated FA with 18 carbons, mainly cis and trans 18:1, and a concomitant reduction in most short and medium chain saturates (6:0 to 12:0 and 16:0; P<0.05) attributable to the presence of SO in the diet. Addition of tannins did not affect ruminal fermentation parameters (i.e., pH, lactate, ammonia, and total volatile fatty acid concentrations) measured after 28days. Reasons for the lack of effects of either type (quebracho CT and chestnut HT) or amount of tannins in the diet are discussed. [Copyright &y& Elsevier]

Small ruminants are susceptible to milk fat depression (MFD) induced by marine lipid supplementation. However, as observed in dairy cows, there is wide individual variation in the response to MFD-inducing diets, which may be due to individual differences in ruminal processes. Therefore, we compared the ruminal 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 concentrations 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 fermentation parameters and fatty acid profiles. In general, 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 microorganisms. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Pomegranate by-products are rich in conjugated linolenic acids (CLnA) and tannins. • Pomegranate tannins protected dietary PUFA and protein from ruminal metabolism. • Biohydrogenation of CLnA yielded trans- 11 18:1 but not cis- 9 trans- 11 CLA. • Pomegranate tannins and CLnA differently affected digesta FA composition. • Pomegranate by-products might detrimentally affect in vitro ruminal fermentation. Increasing demand and production of pomegranate has led to a large amount of by-products that might be used in ruminant feeding. Inclusion of pomegranate by-products in the ovine diet has recently been shown to enrich meat and milk with potentially health-promoting fatty acids (FA). However, it remains unclear whether this effect is due to the action of the bioactive conjugated linolenic acids (CLnA) or of the tannins present in the pomegranate, or perhaps to their interaction. To fill this gap, two in vitro experiments were conducted: the first one tested the effects of pomegranate oil and tannins, alone or in combination, on the biohydrogenation process, and the second one compared the ruminal responses to by-products rich in CLnA (pomegranate seeds, PS), in tannins (pomegranate peels and pulp, PPP) or in both bioactive components (i.e., the whole pomegranate by-product; WPB). Three cannulated ewes were used as donors of inocula for batch cultures of rumen microorganisms. Incubations lasted for 12 and 24 h and were repeated on 3 different days (runs). In both experiments, digesta FA profile was examined by gas chromatography. Results from both trials support that pomegranate tannins and CLnA played different roles in modulating ruminal FA composition. Specifically, tannins would favour the accumulation of potentially health-promoting FA present in dietary lipids (e.g., 18:2n-6 or 18:3n-3) and cis -9 trans -11 conjugated linoleic acid (CLA), whereas the observed increase in trans -11 18:1 would mainly derive from the biohydrogenation of CLnA isomers. Results from the second experiment included evident shifts in some minor FA that would support not only direct saturation steps (e.g., increases in trans- 11 cis- 13 and trans- 11 trans- 13 CLA, and in trans -9, trans -11, cis- 13 and trans -13 18:1) but also a putative isomerisation by rumen bacteria (e.g., increases in trans- 10 cis- 12 CLA and trans- 10, cis- 15, trans- 15 and trans- 16 18:1). Changes in ruminal fermentation parameters (i.e., reductions in ammonia concentration and in the proportions of minor volatile FA) showed that pomegranate tannins protected dietary protein from degradation. Nevertheless, a negative impact on in vitro ruminal fermentation (i.e., reductions in DM disappearance, gas production and total volatile FA concentrations) was observed when 20 % of by-products were included in the diet. Finally, there seem to be no evident synergistic but additive effects between pomegranate bioactive compounds (i.e., tannins and CLnA) on ruminal biohydrogenation or fermentation. [ABSTRACT FROM AUTHOR]

The octadecadienoic conjugated linoleic acid (CLA) isomer with trans-11 and cis-13 double bonds (trans-11, cis-13 CLA) has been described in ruminant milk. For now, this specific CLA is suspected to derive exclusively from ruminal biohydrogenation of dietary α-linolenic acid. However, in rodents, the fatty acid desaturase 3 (FADS3) gene was recently shown to code for an enzyme able to catalyze the unexpected Δ13-desaturation of vaccenic acid, producing a Δ11,13-CLA with all the structural characteristics of the trans-11, cis-13 isomer, although no commercial standard exists for complete conclusive identification. Because the FADS3 gene has already been reported in bovine animals, we hypothesized in the present study that an alternative direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue may therefore coexist with α-linolenic acid biohydrogenation to explain the final ruminant milk trans-11, cis-13 CLA presence. Here, we first confirm that the FADS3 gene is present in ruminant mammal genomic sequence databases. Second, we demonstrate that the Δ11,13-CLA found in milk fat and the highly probable trans-11, cis-13 CLA isomer produced by rodent FADS3 possess exactly the same structural characteristics. Then, we show that bovine mammary MAC-T and BME-UV epithelial cells express both FADS3 and stearoyl-CoA desaturase 1 (SCD1) mRNA and are able to synthesize both the suspected trans-11, cis-13 CLA and cis-9, trans-11CLA (rumenic acid) isomers when incubated with vaccenic acid. Finally, the concomitant presence of the suspected trans-11, cis-13 CLA isomer with FADS3 mRNA was shown in goat mammary tissue, whereas both were conversely very low or even absent in goat liver. Therefore, this study provides several lines of evidence that, by analogy with rumenic acid, trans-11, cis-13 CLA may originate both from ruminal biohydrogenation and from direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue. [ABSTRACT FROM AUTHOR]