Colostrum supplementation with n-3 fatty acids does not alter calf outcome on a healthy commercial farm.

Our objective was to supplement colostrum with n-3 fatty acids (FA) to provide anti-inflammatory mediators that may improve the immune response of neonatal calves. Elevated markers of inflammation have been associated with increased occurrence of calf disease in early life, thus decreasing animal productivity. We hypothesized that a colostrum supplement containing 60-mL of a 1:1 ratio fish:flaxseed oil blend with or without 200 mg of α-tocopherol might provide an advantageous start to early life by decreasing oxidative stress and regulating the inflammatory response. Calves were blocked by birth order and randomly assigned to 1 of 3 treatments: no supplement added to colostrum (control), 60 mL of 1:1 fish:flaxseed oil blend, and 60 mL of 1:1 fish:flaxseed oil blend with 200 mg of α-tocopherol. In total, 180 heifer calves (n = 60 per treatment) were enrolled on a commercial farm. After colostrum feeding, all calves were housed in individual hutches and fed milk replacer 3 times per day. Health was scored 3 times per week until weaning at 8 wk of age. Weight, wither height, and heart girth were measured after birth, 3 wk, and 8 wk of age to assess preweaning growth. A subgroup of 54 calves (18 blocks or 18 calves per treatment) were sampled 2 d (± 8 h) after birth to evaluate oxidant status, serum total protein, and inflammatory gene and cytokine protein expression in blood after an in vitro lipopolysaccharide (LPS) challenge as indicators of health and immunity. At 9 wk, calves were transported 18 h to another farm, and medical records were kept as an indicator of disease incidence up to 13 wk of age. Calf mortality was 1.8%, which is below industry average, and exceptional health scores were observed throughout the study. Health scores and growth were similar throughout the preweaning period regardless of treatment. Serum total protein indicated successful passive transfer in all calves, and oxidant status index was not affected by treatments on d 2 of age. Concentrations of tumor necrosis factor α increased with LPS stimulation, but the increase was not altered by treatment. Likewise, leukocyte gene expression of tumor necrosis factor α, IL-8 and IL-10, and cyclooxygenase-2 increased upon LPS stimulation, but the fold change did not differ with treatment. In conclusion, 60 mL of 1:1 ratio fish:flaxseed oil colostrum supplement did not enhance preweaning calf performance. Supplementing n-3 FA in a one-time meal may not provide the anti-inflammatory benefits observed with continuous feeding.

Omega-3 fatty acids and docosahexaenoic acid oxymetabolites modulate the inflammatory response of equine recombinant interleukin1ß-stimulated equine synoviocytes.

Omega-3 fatty acid (n-3 PUFA) supplementation may have beneficial effects in certain chronic diseases, potentially including osteoarthritis. Favorable effects are attributed, in part, to downstream pro-resolving oxylipid metabolites. We investigated the role of n-3 PUFA and docosahexaenoic acid (DHA)-derived oxylipids (docosanoids) on equine synoviocyte metabolism. We hypothesized that n-3 PUFA and selected docosanoids would modulate inflammatory mediator gene expression by recombinant equine (re)IL-1ß-stimulated synovial fibroblasts. Synoviocyte monolayer cultures were prepared from grossly normal equine carpal synovium. Cellular incorporation of eicosapentaenoic acid (EPA) and DHA was determined using LC-MS and docosanoid biosynthesis by LC-MS-MS. The influence of n-3 PUFA and docosanoids on osteoarthritis marker gene expression was determined by quantitative real time polymerase chain reaction (qPCR). Synoviocytes incorporated EPA and DHA in significant amounts and DHA treatment augmented the synthesis of several docosanoids. Synoviocyte cultures pre-treated with EPA or DHA followed by reIL-1ß stimulation had significant reductions in expression of ADAMTS4, MMP-1, MMP-13, IL-1ß, IL-6 and COX-2. The docosanoids resolvin D1 and D2, maresin 1 and protectin DX, alone and in combination, abrogated ADAMTS4, MMP-1, MMP-13, and IL-6 gene expression in reIL-1ß-stimulated synoviocytes. Similarly, both resolvins and maresin 1 stifled COX-2 expression. Our results demonstrate that synoviocytes readily incorporate n-3 PUFA. DHA incorporation was sufficient for biosynthesis of significant concentrations of several docosanoids which modulated the synovial inflammatory response in vitro. These data indicate n-3 PUFA supplementation may prove useful in the prevention or treatment of osteoarthritis.

Docosahexaenoic acid-derived oxidized lipid metabolites modulate the inflammatory response of lipolysaccharide-stimulated macrophages.

Docosahexaenoic acid (DHA) supplementation has demonstrated beneficial effects in a number of inflammatory diseases. Increasingly, important contributions to its favorable effects are attributed downstream metabolites called docosanoids. Herein, we investigated the role of DHA-derived oxidized lipid metabolites on inflammatory mediator expression by RAW 264.7 murine macrophages. Specifically, macrophage incorporation of DHA, and the resultant biosynthesis of selected pro-resolving docosanoids was quantified. Docosanoid effects on the expression of selected pro-inflammatory cytokines in LPS-stimulated cultures was determined. Macrophages incorporated DHA in significant amounts. In the presence of DHA macrophages produced statistically significant amounts of several putative pro-resolving docosanoids compared to untreated controls. Among them, resolvins D1 and D2 and maresin 1 abrogated COX-2 and IL-1ß gene expression in LPS-stimulated macrophages. In addition to these mediators, protectin DX inhibited LPS-stimulated macrophage expression of IL-6. Our results demonstrate that macrophages incorporate DHA in quantities sufficient for the biosynthesis of biologically-relevant concentrations of a number of pro-resolving docosanoids, certain of which modulate the inflammatory response of macrophages under conditions mimicking acute inflammation. These data provide further information on the mechanism(s) by which DHA exerts salutary effects on the inflammatory response of macrophages.

Ethyl pyruvate decreases proinflammatory gene expression in lipopolysaccharide-stimulated equine monocytes.

Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation protocol and enriched to 76% purity by adhesion to tissue culture dishes. Isolated monocytes were incubated with 0, 1, 5, 10 and 50 mM ethyl pyruvate. Cell viability, production of caspase 3/7, and caspase-3 gene expression were determined. In a separate experiment, monocytes were stimulated with LPS (0.1 ng/ml for 1h) followed by incubation with 0, 1, 5, or 10 mM ethyl pyruvate for 1 h. Proinflammatory gene expression was determined by real-time PCR. Ethyl pyruvate at 50 mM adversely affected monocyte viability. Ethyl pyruvate at 10mM or less had no significant effect on monocyte viability, and did not increase activity of caspase 3/7 nor caspase-3 gene expression. Incubation with LPS alone induced a significant upregulation in proinflammatory gene expression. Subsequent treatment of monocytes with ethyl pyruvate significantly reduced IL-8 expression in LPS stimulated monocytes at 5 mM, and IL-8, TNF-α and COX-2 at 10 mM. No beneficial effect on expression of IL-1ß or IL-6 was detected. Overall, 10 mM ethyl pyruvate did not adversely affect monocyte viability and suppressed LPS-induced proinflammatory gene expression. Ethyl pyruvate may be a beneficial anti-inflammatory therapy in equine endotoxemia.