Effect of lipid supplementation on the endogenous synthesis of milk cis-9,trans-11 conjugated linoleic acid in dairy sheep and goats: A tracer assay with 13C-vaccenic acid.

A major proportion of milk rumenic acid (RA; cis-9,trans-11 CLA) is synthesized through mammary Δ9-desaturation of vaccenic acid (VA; trans-11 18:1). Diet composition may determine the relative contribution of this endogenous synthesis to milk RA content, with effects that might differ between ruminant species. However, this hypothesis is mostly based on estimated values, proxies of stearoyl-CoA desaturase (SCD) activity, and indirect comparisons between publications in the literature. With the aim of providing new insights into this issue, in vivo Δ9-desaturation of 13C-labeled VA (measured via milk 13C-VA and -RA secretion) was directly compared in sheep and goats fed a diet without lipid supplementation or including 2% of linseed oil. Four Assaf sheep and 4 Murciano-Granadina goats were used in a replicated 2 × 2 crossover design to test the effects of the 2 dietary treatments during 2 consecutive 25-d periods. On d 22 of each period, 500 mg of 13C-VA were i.v. injected to each animal. Dairy performance, milk fatty acid profile, including isotope analysis, and mammary mRNA abundance of genes coding for SCD were examined on d 21 to 25 of each period. Supplementation with linseed oil improved milk fat concentration and increased the content of milk VA and RA. However, the isotopic tracer assay suggested no variation in the relative proportion of VA desaturated to milk RA, and the percentage of this CLA isomer deriving from SCD activity would remain constant regardless of dietary treatment. These results put into question a major effect of lipid supplementation on the endogenous synthesis of milk RA and support that mammary Δ9-desaturation capacity would not represent a limiting factor when designing feeding strategies to increase milk RA content. The lack of diet-induced effects was common to caprines and ovines, but inherent interspecies differences in mammary lipogenesis were found. Thus, the higher proportions of VA desaturation and endogenous synthesis of milk RA in sheep supported a greater SCD activity compared with goats, a finding that was not associated with the similar mRNA abundance of SCD1 in the 2 species. On the other hand, transfer efficiency of the isotopic tracer to milk was 37% higher in caprine than in ovine, suggesting a greater efficiency in mammary fatty acid uptake from plasma in caprine.

Rumen biohydrogenation and milk fatty acid profile in dairy ewes divergent for feed efficiency.

A sustainable increase in livestock production would require selection for improved feed efficiency, but the mechanisms underlying this trait and explaining its large individual variation in dairy ruminants remain unclear. This study was conducted in lactating ewes to test the hypothesis that rumen biohydrogenation (BH) would differ between high- and low-efficiency animals, and these differences would be reflected in rumen fatty acid (FA) profile and affect milk FA composition. A second aim was to identify differences in FA that may serve as biomarkers of feed efficiency. Data of daily feed intake and milk yield and composition, as well as body weight, were collected individually over a 3-wk period in 40 ewes. The difference between the mean actual and predicted feed intake (estimated through metabolizable energy requirements for maintenance, production, and body weight change) over the period was used as the feed efficiency index (FEI) to select 8 of the highest feed efficiency (H-FE) and 8 of the lowest feed efficiency (L-FE) animals. In addition, residual feed intake (RFI) was estimated as the residual term from the regression of feed intake on various energy sinks. Rumen and milk FA composition were characterized by using gas chromatography, and results were analyzed using a statistical model that included the fixed effect of the group (H-FE vs. L-FE). The FEI averaged -0.29 ± 0.046 and 0.81 ± 0.084 in H-FE and L-FE, respectively, whereas RFI averaged -0.16 ± 0.084 and 0.18 ± 0.082, respectively. The correlation coefficient between both metrics was 0.69. Feed intake was similar in both groups, but H-FE showed greater milk yield, with increases in lactose content and yield, and in milk protein and fat production. Results from rumen FA profiles included a lower proportion of 18:2n-6, cis-9 18:1, and of several of their BH metabolites, and a greater concentration of 18:0, which may indicate that the apparent BH would be more complete in more efficient sheep. Milk FA analysis suggested that the greater fat yield in the H-FE group was mostly explained by increased de novo FA synthesis, whereas their milk would have lower proportions of cis-9 18:1 and C20 to 22n-6 polyunsaturated FA than L-FE. Stepwise multiple linear regression suggested that milk C20 to 22n-6 PUFA might be convenient biomarkers to discriminate more efficient dairy sheep. Further research is needed to validate these findings (e.g., under different dietary conditions).

Effect of dietary lipids and other nutrients on milk odd- and branched-chain fatty acid composition in dairy ewes.

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.

Invited review: Role of rumen biohydrogenation intermediates and rumen microbes in diet-induced milk fat depression: An update.

To meet the energy requirements of high-yielding dairy cows, grains and fats have increasingly been incorporated in ruminant diets. Moreover, lipid supplements have been included in ruminant diets under experimental or practical conditions to increase the concentrations of bioactive n-3 fatty acids and conjugated linoleic acids in milk and meat. Nevertheless, those feeding practices have dramatically increased the incidence of milk fat depression in dairy cattle. Although induction of milk fat depression may be a management tool, most often, diet-induced milk fat depression is unintended and associated with a direct economic loss. In this review, we give an update on the role of fatty acids, particularly originating from rumen biohydrogenation, as well as of rumen microbes in diet-induced milk fat depression. Although this syndrome seems to be multi-etiological, the best-known causal factor remains the shift in rumen biohydrogenation pathway from the formation of mainly trans-11 intermediates toward greater accumulation of trans-10 intermediates, referred to as the trans-11 to trans-10 shift. The microbial etiology of this trans-11 to trans-10 shift is not well understood yet and it seems that unraveling the microbial mechanisms of diet-induced milk fat depression is challenging. Potential strategies to avoid diet-induced milk fat depression are supplementation with rumen stabilizers, selection toward more tolerant animals, tailored management of cows at risk, selection toward more efficient fiber-digesting cows, or feeding less concentrates and grains.

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

The basis for marine lipid-induced milk fat depression (MFD) has not been established yet, but recent reports suggest the putative contribution of shifts in the ruminal metabolism of α-linolenic acid (ALA). To test this hypothesis, an isotopic tracer approach was used in batch cultures of rumen microorganisms with inoculum collected from cannulated ewes fed either a total mixed ration without lipid supplementation (control inoculum) or the same diet supplemented with 2% of fish oil, which is known to cause MFD in lactating sheep (FO-MFD inoculum). The [1-13C]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 biohydrogenation 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-11 to trans-10 shift as an effective marker of diet-induced MFD in sheep, and challenged the involvement of some trans-10 intermediates (e.g., trans-10 18:1 and trans-10,cis-15 18:2) in the low-fat milk syndrome in this species. Conversely, increased abundance of most 18:3 intermediates (including some unidentified isomers) might be representative of ruminal alterations related to fish oil supplementation in ewes. Further research is necessary to examine the potential association between these findings and MFD in lactating animals.

Review: Modulating ruminal lipid metabolism to improve the fatty acid composition of meat and milk. Challenges and opportunities.

Growth in demand for foods with potentially beneficial effects on consumer health has motivated increased interest in developing strategies for improving the nutritional quality of ruminant-derived products. Manipulation of the rumen environment offers the opportunity to modify the lipid composition of milk and meat by changing the availability of fatty acids (FA) for mammary and intramuscular lipid uptake. Dietary supplementation with marine lipids, plant secondary compounds and direct-fed microbials has shown promising results. In this review, we have compiled information about their effects on the concentration of putative desirable FA (e.g. c9t11-CLA and vaccenic, oleic, linoleic and linolenic acids) in ruminal digesta, milk and intramuscular fat. Marine lipids rich in very long-chain n-3 polyunsaturated fatty acids (PUFA) efficiently inhibit the last step of C18 FA biohydrogenation (BH) in the bovine, ovine and caprine, increasing the outflow of t11-18:1 from the rumen and improving the concentration of c9t11-CLA in the final products, but increments in t10-18:1 are also often found due to shifts toward alternative BH pathways. Direct-fed microbials appear to favourably modify rumen lipid metabolism but information is still very limited, whereas a wide variety of plant secondary compounds, including tannins, polyphenol oxidase, essential oils, oxygenated FA and saponins, has been examined with varying success. For example, the effectiveness of tannins and essential oils is as yet controversial, with some studies showing no effects and others a positive impact on inhibiting the first step of BH of PUFA or, less commonly, the final step. Further investigation is required to unravel the causes of inconsistent results, which may be due to the diversity in active components, ruminant species, dosage, basal diet composition and time on treatments. Likewise, research must continue to address ways to mitigate negative side-effects of some supplements on animal performance (particularly, milk fat depression) and product quality (e.g. altered oxidative stability and shelf-life).

Milk fat depression in dairy ewes fed fish oil: Might differences in rumen biohydrogenation, fermentation, or bacterial community explain the individual variation?

Dairy ewes show large individual variation in the extent of diet-induced milk fat depression (MFD) but reasons behind this variability remain uncertain. Previous results offered no convincing support for these differences being related to relevant changes in the milk fatty acid (FA) profile, including potentially antilipogenic FA, or in the transcript abundance of candidate genes involved in mammary lipogenesis. Therefore, we hypothesized that alterations in the processes of rumen biohydrogenation and fermentation, as well as in the bacterial community structure, might account for individual variation in fish oil-induced MFD severity. To test this explanation, 15 ewes received a total mixed ration without lipid supplementation (control; n = 5) or supplemented with 20 g of fish oil/kg of dry matter [10 animals divided into those showing a strong (RESPON+; -25.4%; n = 5) or a mild (RESPON-; -7.7%; n = 5) decrease in milk fat concentration] for 5 wk. Rumen fermentation parameters, biohydrogenation metabolites, and bacterial structure and diversity were analyzed in rumen samples collected before and after treatments. Although the fish oil supplementation increased the concentration of demonstrated or putative antilipogenic FA (e.g., cis-9 16:1, cis-11 18:1, or trans-10,cis-12 CLA), surprisingly, none of them differed significantly in relation to the extent of MFD (i.e., between RESPON- and RESPON+), and this was the case only for a few minor FA (e.g., cis-6+7 16:1 or 17:0 anteiso). Changes in total volatile FA, acetate, and propionate concentrations were associated with MFD severity, with higher decreases in more susceptible animals. Individual responses were not related to shifts in rumen bacterial structure but some terminal restriction fragments compatible with Clostridiales, Ruminococcaceae, Lachnospiraceae, and Succiniclasticum showed greater abundances in RESPON-, whereas some others that may correspond to Prevotella, Mogibacterium, and Quinella-related spp. were more abundant in RESPON+. Overall, the results suggest that individual variation in MFD severity in dairy ewes fed fish oil cannot be fully explained by differences in the processes of rumen biohydrogenation and fermentation or in the bacterial community, and further research would be necessary to elucidate the large variability in the responsiveness to MFD-inducing marine lipids.

In vitro ruminal biohydrogenation of eicosapentaenoic (EPA), docosapentaenoic (DPA), and docosahexaenoic acid (DHA) in cows and ewes: Intermediate metabolites and pathways.

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.

Individual variation of the extent of milk fat depression in dairy ewes fed fish oil: Milk fatty acid profile and mRNA abundance of candidate genes involved in mammary lipogenesis.

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.

Comparison of mammary lipid metabolism in dairy cows and goats fed diets supplemented with starch, plant oil, or fish oil.

A direct comparison of cow and goat performance and milk fatty acid (FA) responses to diets known to induce milk fat depression in the bovine has suggested interspecies differences in rumen and mammary lipid metabolism. Thus, this study was conducted to infer some potential mechanisms responsible for the differences in mammary lipogenesis due to diet and ruminant species. To meet this objective, 12 cows and 15 goats were fed a basal diet (control), a similar diet supplemented with 2.2% fish oil (FO), or a diet containing 5.3% sunflower oil and additional starch (+38%; SOS) according to a 3 × 3 Latin square design with 26-d experimental periods. Milk yield, milk composition, FA profile, and FA secretion were measured. On the last day of each period, the mRNA abundance of 19 key genes in mammary metabolism or the enzyme activity or both were measured in mammary tissue sampled by biopsy or at slaughter or both. The results show significant differences in the response of cows and goats to the dietary treatments. In cows, milk fat content and yield were lowered by FO and SOS (-31%), whereas only FO decreased milk fat content in goats (-21%) compared with the control. In cows and to a lesser extent in goats, FO and SOS decreased the secretion of C16 FA output (mmol/kg of BW). However, SOS increased the secretion of >C16 FA in goats. These changes in milk fat content and FA secretion were not associated with modifications in mammary expression or the activity of 19 proteins involved in the major lipogenic pathways. This absence of variation may be attributable to posttranscriptional regulation for these genes or related to the time of sampling of the mammary tissue relative to the previous meal and milking. Otherwise, the abundances of 15 mRNA among the 19 encoding for genes involved in lipid metabolism in the mammary gland were different among species, with 9 more abundant in cows (FASN, FADS1, SCD1, GPD1, LALBA, SREBF1, LXRA, PPARA, and PPARG1) and 6 more abundant in goats (G6PD, GPAM, SCD5, XDH, CSN2, and SP1). Similarly, a significant effect of the species was observed in the 4 enzyme activities measured; glycerol-3-phosphate dehydrogenase and malic enzyme were higher in cows, and FA synthase and glucose-6-phosphate dehydrogenase activities were higher in goats. In conclusion, the differences between cow and goat performance and milk FA responses to the FO and SOS treatments were not related to changes in the measured mammary lipogenic gene expression. Furthermore, the data provide evidence that the major mammary lipogenic pathways differ between the caprine and the bovine, whose biological significance remains to be unraveled.

In vitro response to EPA, DPA, and DHA: Comparison of effects on ruminal fermentation and biohydrogenation of 18-carbon fatty acids in cows and ewes.

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.

mRNA abundance of genes involved in mammary lipogenesis during fish oil- or trans-10,cis-12 CLA-induced milk fat depression in dairy ewes.

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 composition, and particularly mammary mRNA abundance of candidate lipogenic genes and transcription factors in response to the inclusion of fish oil or trans-10,cis-12 18:2 in the dairy sheep diet. To meet this objective, 12 lactating Assaf ewes (on average, 64 days in milk, producing 1.72 kg of milk/d with 5.17% of fat) were divided into 3 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil (FO treatment) or 1% dry matter of a commercial product rich in trans-10,cis-12 18:2 (CLA treatment) for 39 d. 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 2 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., acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid synthase (FASN), and lipin 1 (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 sterol regulatory element binding transcription factor 1 (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 d in milk on d 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).

Isolation of RNA from milk somatic cells as an alternative to biopsies of mammary tissue for nutrigenomic studies in dairy ewes.

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.

Fish oil-induced milk fat depression and associated downregulation of mammary lipogenic genes in dairy ewes.

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.

Comparison of ruminal lipid metabolism in dairy cows and goats fed diets supplemented with starch, plant oil, or fish oil.

Direct comparison of cow and goat performance and milk fatty acid responses to diets known to induce milk fat depression (MFD) in the bovine reveals relevant species-by-diet interactions in ruminal lipid metabolism. Thus, this study was conducted to infer potential mechanisms responsible for differences in the rumen microbial biohydrogenation (BH) due to diet and ruminant species. To meet this objective, 12 cows and 15 goats were fed a basal diet (control), a similar diet supplemented with 2.2% fish oil (FO), or a diet containing 5.3% sunflower oil and additional starch (+38%; SOS) according to a 3 × 3 Latin square design with 25-d experimental periods. On the last day of each period, fatty acid composition (by gas chromatography) and bacterial community (by terminal-RFLP), as well as fermentation characteristics, were measured in rumen fluid samples. Results showed significant differences in the response of cows and goats to dietary treatments, although variations in some fermentation parameters (e.g., decreases in the acetate-to-propionate ratio due to FO or SOS) were similar in both species. Main alterations in ruminal BH pathways potentially responsible for MFD on the SOS diet (i.e., the shift from trans-11 to trans-10 18:1 and related increases in trans-10,cis-12 18:2) tended to be more pronounced in cows, which is consistent with an associated MFD only in this species. However, changes linked to FO-induced MFD (e.g., decreases in 18:0 and increases in total trans-18:1) were stronger in caprine rumen fluid, which may explain their unexpected susceptibility (although less marked than in bovine) to the negative effect of FO on milk fat content. Altogether, these results suggest that distinct ruminal mechanisms lead to each type of diet-induced MFD and confirm a pronounced interaction with species. With regard to microbiota, differences between cows and goats in the composition of the rumen bacterial community might be behind the disparity in the microorganisms affected by the experimental diets (e.g., Ruminococcaceae, Lachnospiraceae, and Succinivibrionaceae in the bovine, and Pseudobutryrivibrio, Clostridium cluster IV, Prevotella, and Veillonellaceae in the caprine), which hindered the assignation of bacterial populations to particular BH steps or pathways. Furthermore, most relevant variations in microbial groups corresponded to as yet uncultured bacteria and suggest that these microorganisms may play a predominant role in the ruminal lipid metabolism in both cows and goats.

Does supplemental 18:0 alleviate fish oil-induced milk fat depression in dairy ewes?

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 Δ(9)-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.

Comparison of the nutritional regulation of milk fat secretion and composition in cows and goats.

A study with 2 ruminant species (goats and cows) with inherent differences in lipid metabolism was performed to test the hypothesis that milk fat depression (MFD) due to marine lipid supplements or diets containing high amounts of starch and plant oil is caused by different mechanisms and that each ruminant species responds differently. Cows and goats were allocated to 1 of 3 groups (4 cows and 5 goats per group) and fed diets containing no additional oil (control) or supplemented with fish oil (FO) or sunflower oil and wheat starch (SOS) according to a 3 × 3 Latin square design with 26-d experimental periods. In cows, milk fat content was lowered by FO and SOS (-31%), whereas only FO decreased milk fat content in goats (-21%) compared with the control. Furthermore, FO and SOS decreased milk fat yield in cows, but not in goats. In both species, FO and SOS decreased the secretion of C16 FA output. However, SOS increased milk secretion of >C16 FA in goats. Compared with the control, SOS resulted in similar increases in milk trans-10,cis-12 conjugated linoleic acid (CLA) in both species, but caused a 2-fold larger increase in trans-10 18:1 concentration in cows than for goats. Relative to the control, responses to FO in both species were characterized by a marked decrease in milk concentration of 18:0 (-74%) and cis-9 18:1 (-62%), together with a ~5-fold increase in total trans 18:1, but the proportionate changes in trans-10 18:1 were lower for goats. Direct comparison of animal performance and milk FA responses to FO and SOS treatments demonstrated interspecies differences in mammary lipogenesis, suggesting a lower sensitivity to the inhibitory effects of trans-10,cis-12 CLA in goats and that ruminal biohydrogenation pathways are more stable and less prone to diet-induced shifts toward the formation of trans-10-containing intermediates in goats compared with cows. Even though a direct cause and effect could not be established, results suggest that regulation of milk fat synthesis during FO-induced MFD may be related to a shortage of 18:0 for endogenous mammary cis-9 18:1 synthesis, increase in the incorporation of trans FA in milk triacylglycerols, and limitations in the synthesis of FA de novo to maintain milk fat melting point. However, the possible contribution of biohydrogenation intermediates with putative antilipogenic effects in the mammary gland, including trans-9,cis-11 CLA, trans-10 18:1, or cis-11 18:1 to FO-induced MFD cannot be excluded.

Reductions in milk Δ9-desaturation ratios to oral dosing of cobalt-acetate are accompanied by the downregulation of SCD1 in lactating ewes.

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 d. 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.

Dietary sunflower oil modulates milk fatty acid composition without major changes in adipose and mammary tissue fatty acid profile or related gene mRNA abundance in sheep.

There are very few studies in ruminants characterizing mammary and adipose tissue (AT) expression of genes and gene networks for diets causing variations in milk fatty acid (FA) composition without altering milk fat secretion, and even less complementing this information with data on tissue FA profiles. This work was conducted in sheep in order to investigate the response of the mammary gland and the subcutaneous and perirenal AT, in terms of FA profile and mRNA abundance of genes involved in lipid metabolism, to a diet known to modify milk FA composition. Ten lactating Assaf ewes were randomly assigned to two treatments consisting of a total mixed ration based on alfalfa hay and a concentrate (60 : 40) supplemented with 0 (control diet) or 25 (SO diet) g of sunflower oil/kg of diet dry matter for 7 weeks. Milk composition, including FA profile, was analysed after 48 days on treatments. On day 49, the animals were euthanized and tissue samples were collected to analyse FA and mRNA abundance of 16 candidate genes. Feeding SO did not affect animal performance but modified milk FA composition. Major changes included decreases in the concentration of FA derived from de novo synthesis (e.g. 12:0, 14:0 and 16:0) and increases in that of long-chain FA (e.g. 18:0, c9-18:1, trans-18:1 isomers and c9,t11-CLA); however, they were not accompanied by significant variations in the mRNA abundance of the studied lipogenic genes (i.e. ACACA, FASN, LPL, CD36, FABP3, SCD1 and SCD5) and transcription factors (SREBF1 and PPARG), or in the constituent FA of mammary tissue. Regarding the FA composition of AT, the little influence of SO did not appear to be linked to changes in gene mRNA abundance (decreases of GPAM and SREBF1 in both tissues, and of PPARG in the subcutaneous depot). Similarly, the great variation between AT (higher contents of saturated FA and trans-18:1 isomers in the perirenal, and of cis-18:1, c9,t11-CLA and n-3 PUFA in the subcutaneous AT) could not be related to differences in gene mRNA abundance due to tissue site (higher LPL and CD36, and lower SREBF1 in perirenal than in subcutaneous AT). Overall, these results suggest a marginal contribution of gene expression to the nutritional regulation of lipid metabolism in these tissues, at least with the examined diets and after 7 weeks on treatments. It cannot be ruled out, however, that the response to SO is mediated by other genes or post-transcriptional mechanisms.

Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae.

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.