Periparturient dairy cows exhibit intense lipolysis driven by reduced DMI, enhanced energy needs, and the loss of adipose tissue (AT) insulin sensitivity. Extended periods of low insulin sensitivity and negative energy balance induce lipolysis dysregulation, leading to increased disease susceptibility and poor lactation performance. Chromium (Cr) supplementation improves systemic insulin sensitivity, whereas palmitic acid (PA) increases energy availability for milk production. However, the effect of supplementing Cr and PA alone or in combination on insulin sensitivity in AT is unknown. A total of 32 multiparous cows were used in a randomized complete block design experiment and randomly assigned to one of 4 diets fed from 1 to 24 DIM: a control diet with no supplementation (CON, n = 8); the Cr diet (Cr propionate at 0.45 mg/kg Cr/kg DM, n = 8); the PA diet (1.5% DM, n = 8); or Cr+PA (n = 8). Plasma samples were collected at -13 +/- 5.1 d prepartum (PreP), and at 14.4 +/- 1.9 d (PP1) and 21 +/- 1.9 d (PP2) after calving for quantification of albumin, BHB, BUN, calcium, cholesterol, glucose, nonesterified fatty acids (NEFA), total protein, iron, transferrin, triglycerides, and oxylipids. Subcutaneous AT (SCAT) explants were collected at PreP, PP1, and PP2 and incubated in the presence of the lipolytic agent isoproterenol (ISO = 1 mu M, BAS = 0 mu M) for 3 h. The antilipolytic effect of insulin (1 mu L/L) on SCAT explants was evaluated during ISO stimulation (ISO+INS). Lipolysis was quantified by glycerol release in the medium (nmol glycerol/mg AT). Macrophage infiltration and adipocyte size were measured using hematoxylin and eosin-stained AT sections and immunohistochemistry. The Cr diet tended to reduce postpartum NEFA concentrations when compared with CON, PA, and Cr+PA. Likewise, Cr increased the percentage of large adipocytes (>9,000 mu m(2)) postpartum compared with other diets. In line with higher lipid content, Cr-fed cows had higher ex vivo BAS lipolysis at PP2 when compared with PA and Cr+PA. Isoproterenol induced higher lipolysis at PP1 and PP2, but it was not affected by Cr and PA. The ISO+INS treatment reduced lipolysis by 29.91% +/- 11% in Cr compared with ISO. In contrast, ISO+INS did not affect ISO lipolysis in CON, PA, and Cr+PA. Plasma transferrin was reduced by Cr. At PP2, PA cows had 3.3-fold higher macrophage infiltration in SCAT when compared with CON and Cr. Plasma 9-hydroxyoctadecadienoic acid (HODE) and 9-oxo-octadecadienoic acid (oxoODE) were increased by Cr+PA. Palmitic acid increased plasma 13-oxoODE and Cr increased the ratio of 13-HODE to 13-oxoODE. Palmitic acid increased 5-iso prostaglandin F-2 alpha-VI. Our results demonstrate that supplementing Cr during the immediate postpartum enhances SCAT insulin sensitivity and lipid accumulation. Further studies should determine the effects and mechanisms of action of Cr and PA on AT lipogenesis, adipogenesis, and their impact on lactation performance.

Dairy cows with clinical ketosis (CK) exhibit metabolic changes, including intense adipose tissue (AT) lipolysis and systemic insulin resistance, that increase plasma BHB and free fatty acids (FFA). Cows with CK also have systemic inflammation, predisposing them to inflammatory and infectious diseases. This inflammatory process is modulated in part by oxidized fatty acids (oxylipins) that regulate all aspects of inflammation. Oxylipin profiles have been characterized in healthy periparturient cows, but their dynamics during CK are unknown. Clinical ketosis is an acute metabolic disease requiring clinical therapy, commonly including propylene glycol (PG) as a gluconeogenic agent. Recently, we showed that including lipolysis inhibitors such as niacin (NIA) and flunixin meglumine (FM) improved CK recovery. These drugs may modulate oxylipin biosynthesis by regulating the release of PUFA (oxylipin substrates) and cyclooxygenase activity. However, their impact on oxylipin profiles in cows with CK is unknown. The objective of this study was to determine the dynamics of specific linoleic and arachidonic acid-derived oxylipins during CK and following therapy with PG, NIA, and FM. Multiparous Jersey cows (n = 72; 7.1 DIM) with CK from a commercial dairy were sampled. Inclusion criteria were CK symptoms (lethargy, depressed appetite, and reduced rumen fill) and blood BHB >= 1.2 mmol/L. The CK cows (n = 24/treatment) were randomly assigned to one of the 3 treatments: (1) PG: 310 g orally once daily for 5 d, (2) PG + NIA (PGNIA): 24 g orally once daily for 3 d, (3) PG + NIA + FM (PGNIAFM): 1.1 mg/kg i.v. once daily for 3 d. Healthy control cows (HC; n = 24) matched by lactation and DIM (+/- 2 d) were also included. Plasma oxylipins were quantified at enrollment and 7 d later using HPLC-MS/MS. At enrollment, CK had higher concentrations of arachidonic acid (ARA)-derived 5- and 20-HETE, 8,9-, 11,12-, and 14-15-DHET, and lower concentrations of linoleic acid (LA)-derived 12,13-EpOME, 13-oxoODE, 9,10- and 12,13-DiHOME. Integrated analysis of biological pathways and oxylipin profiles using Ingenuity Pathway Analysis revealed ARA metabolism as the top pathway activated during CK. By d 7, treatment with PGNIAFM restored plasma PUFA and oxylipins to profiles similar to HC. Ingenuity Pathway Analysis showed that PGNIAFM activated the zinc transporter SLC30A7, associated with reduced activation of the ARA pathway. Results indicate that higher FAavailability during CK, driven in part by dysregulated lipolysis, increases the pool of substrates for oxylipin biosynthesis. These oxylipins may play a role in both metabolic dysregulation and restoring homeostasis during CK. Inhibiting lipolysis and cyclooxygenase activity with NIA and FM can alter ARA- and LA-derived oxylipin biosynthesis. These findings underscore the potential use of lipolysis inhibitors NIA and FM in CK therapeutics and highlight the importance of understanding oxylipin pathways in the pathogenesis of CK.

Abstract Background As cows transition from pregnancy to lactation, free fatty acids (FFA) are mobilized from adipose tissues (AT) through lipolysis to counter energy deficits. In clinically healthy cows, lipolysis intensity is reduced throughout lactation; however, if FFA release exceeds tissue demands or the liver’s metabolic capacity, lipid byproducts accumulate, increasing cows’ risk of metabolic and infectious disease. Endocannabinoids (eCBs) and their congeners, N-acylethanolamines (NAEs), are lipid-based compounds that modulate metabolism and inflammation. Their synthesis and release depend upon the availability of FFA precursors and the abundance of synthesizing and degrading enzymes and transporters. Therefore, we hypothesized that eCB production and transcription of endocannabinoid system components are modulated by lipolysis pathways in adipocytes. To test this hypothesis, we stimulated canonical (isoproterenol, 1 µmol/L; ISO) and inflammatory (lipopolysaccharide, 1 µg/mL; LPS) lipolysis pathways in adipocytes isolated from the AT of 5 Holstein dairy cows. Following, we assessed lipolysis intensity, adipocytes’ release of eCBs, and transcription of endocannabinoid system components. Results We found that ISO and LPS stimulated lipolysis at comparable intensities. Exposure to either treatment tended to elevate the release of eCBs and NAEs by cultured adipocytes; however, specific eCBs and NAEs and the transcriptional profiles differed by treatment. On one hand, ISO enhanced adipocytes’ release of 2-arachidonoylglycerol (2-AG) but reduced NAE production. Notably, ISO enhanced the cells’ expression of enzymes associated with 2-AG biosynthesis (INPP5F, GDPD5, GPAT4), transport (CD36), and adipogenesis (PPARG). Conversely, LPS enhanced adipocytes’ synthesis and release of N-arachidonoylethanolamide (AEA). This change coincided with enhanced transcription of the NAE-biosynthesizing enzyme, PTPN22, and adipocytes’ transcription of genes related to eCB degradation (PTGS2, MGLL, CYP27B1). Furthermore, LPS enhanced adipocytes’ transcription of eCB and NAE transporters (HSPA1A, SCP2) and the expression of the anti-adipogenic ion channel, TRPV3. Conclusions Our data provide evidence for distinct modulatory roles of canonical and inflammatory lipolysis pathways over eCB release and transcriptional regulation of biosynthesis, degradation, transport, and ECS signaling in cows’ adipocytes. Based on our findings, we conclude that, within adipocytes, eCB production and ECS component expression are, at least in part, mediated by lipolysis in a pathway-dependent manner. These findings contribute to a deeper understanding of the molecular mechanisms underlying metabolic regulation in dairy cows’ AT, with potential implications for prevention and treatment of inflammatory and metabolic disorders. Graphical Abstract

The early period of mammary gland involution is a critical juncture in the lactation cycle that can have significant effects on milk production and mammary gland health. Pegbovigrastim (PEG) administered 1 wk prior and on the day of parturition can enhance immune function and reduce the incidence of mastitis in the early postpartum period. Oxylipids are potent metabolites of polyunsaturated fatty acids (PUFA) and are important mediators of inflammation. The objective of this study was to evaluate effects of PEG given 1 wk before and at the day of dry-off (D0) on concentrations of oxylipids in plasma and milk from 7 d before D0 to 14 after, as well as the effects during the first 14 d of the subsequent lactation. We hypothesized that both pro- and anti-inflammatory oxylipids would vary based on initiation of mammary gland involution and that pegbovigrastim would affect oxylipid concentration, particularly those related to leukocytes. A complete randomized blocked design was used to enroll cows into either a PEG treatment group (n = 10) or control group (n = 10; CON). Blood samples were collected −7, −2, −1, 0, 1, 2, 4, 7, 14 d relative to dry-off and 5, 10, and 14 d post-calving. Samples were analyzed for PUFA and oxylipids in milk and plasma by ultra-performance mass-spectrometry and liquid chromatography tandem quadrupole mass spectrometry, respectively. Overall, 30 lipid mediators were measured both in milk and plasma. Repeated measures analyses revealed a significant interaction of treatment by time for milk 8-iso-keto-15-prostaglandin E2, prostaglandin F2α, plasma 8,12-iso-prostaglandin FαVI, 11-hydroxyeicosatetraenoic acid, and 12-hydroxyheptadecatienoic acid. The majority of milk PUFA and oxylipids differed significantly during early mammary gland involution and into the early postpartum period. This study demonstrated changes in oxylipids in milk secretions and plasma during early involution and further investigation may illuminate multiple complex processes and reveal targets for optimization of mammary gland involution.

Background As cows transition from pregnancy to lactation, free fatty acids (FFA) are mobilized from adipose tissues (AT) through lipolysis to counter energy deficits. In clinically healthy cows, lipolysis intensity is reduced throughout lactation; however, if FFA release exceeds tissue demands or the liver’s metabolic capacity, lipid byproducts accumulate, increasing cows’ risk of metabolic and infectious disease. Endocannabinoids (eCBs) and their congeners, N- acylethanolamines (NAEs), are lipid-based compounds that modulate metabolism and inflammation. Their synthesis and release depend upon the availability of FFA precursors and the abundance of synthesizing and degrading enzymes and transporters. Therefore, we hypothesized that eCB production and transcription of endocannabinoid system components are modulated by lipolysis pathways in adipocytes. To test this hypothesis, we stimulated canonical (isoproterenol, 1 µ M ; ISO) and inflammatory (lipopolysaccharide, 1 µg/mL; LPS) lipolysis pathways in adipocytes isolated from the AT of 5 Holstein dairy cows. Following, we assessed lipolysis intensity, adipocytes’ release of eCBs, and transcription of ECS components. Results ISO and LPS stimulated lipolysis at comparable intensities. Exposure to ISO or LPS tended to elevate the release of eCBs and NAEs from adipocytes. ISO enhanced adipocytes’ release of 2-arachidonoylglycerol (2-AG) but reduced NAE. Conversely, LPS enhanced the synthesis of N- arachidonoylethanolamide (AEA) compared to ISO. Transcriptomic analyses revealed substantial changes in gene expression profiles amongst treatment groups. Notably, ISO enhanced the expression of 2-AG biosynthesizing genes, including INPP5F , GDPD5 , and GPAT4 . LPS augmented adipocytes’ transcription of NAE-biosynthesizing PTPN22 . LPS heightened adipocytes’ transcription of 2-AG degrading COX-2 , MGLL , and CYP27B1 . Furthermore, LPS enhanced the transcription of HSPA1A and SCP2 whereas ISO increased CD36 . ISO promoted the transcription of PPARG , while LPS enhanced expression of TRPV3 and CACNA1C . Conclusions Our data provide evidence for distinct modulatory roles of canonical and inflammatory lipolysis pathways over eCB release and transcriptional regulation of biosynthesis, degradation, transport, and ECS signaling in cows’ adipocytes. Based on our findings, we conclude that, within adipocytes, eCB production and ECS component expression are, at least in part, mediated by lipolysis in a pathway-dependent manner.These findings contribute to a deeper understanding of the molecular mechanisms underlying metabolic regulation in dairy cows’ AT, with potential implications for prevention and treatment of inflammatory and metabolic disorders.

The objective of this experiment was to determine the effect of intramammary calcitriol treatment on indicators of inflammation during an intramammary bacterial infection. Lactating Holstein cows were challenged with intramammary Streptococcus uberis. At the onset of mild or moderate mastitis, cows were randomly assigned to receive 10 µg of intramammary calcitriol (CAL, n = 7) or placebo control (CON; n = 6) after every milking for 5 days. Data were analyzed by ANOVA with mixed models using the MIXED procedure of SAS with significance declared at P ≤ 0.05. Milk somatic cells, mastitis severity scores, rectal temperatures, and milk bacterial counts did not differ between treatments. Calcitriol decreased the percentage of CD11b+CD14- cells in milk compared with CON (CON = 81 vs. CAL = 61 ± 5%). Antioxidant potential and concentrations of 15-F2t- isoprostanes in milk of infected quarters also were lower in CAL compared with CON. Transcripts for the 25-hydroxyvitamin D 24-hydroxylase and inducible nitric oxide synthase were greater in milk somatic cells of CAL compared with CON, but those for β-defensin 7, metallothionein 1 A and 2 A, thioredoxin and thioredoxin reductase did not differ between treatments. Although clinical signs of severity did not differ, CAL influenced the composition of milk somatic cells and redox activity in milk of infected quarters.

OBJECTIVE:The objective of this pilot study was to determine if an alternative dosing schedule of pegbovigrastim (PEG; Imrestor; Elanco Animal Health) affects mammary gland health, rear udder width, or milk production of healthy dairy cows.ANIMALS:20 pregnant late-lactation Holstein cows in November 2019 through April 2020.PROCEDURES:Cows were randomly assigned to receive subcutaneous injections with either 15 mg of PEG (PEG group; n = 10) or a sham injection with saline (0.9% NaCl) solution (control group; 10) administered 7 days before dry-off and at dry-off. Quarter milk samples were collected for bacterial culture and somatic cells before and after dry-off and after calving. Mammary gland width was assessed before and after dry-off. Daily milk yields were evaluated after calving.RESULTS:The incidence of intramammary infection was 5 times greater for quarters of cows in the control group than for quarters of cows that received PEG. The effect of treatment on somatic cell count was not significant, but the effects of period and a treatment-by-period interaction were identified. Treatment did not significantly affect milk production in the subsequent lactation, but the effects of period and an interaction of treatment by period were identified. Rear udder width after dry-off was not significantly affected by treatment, but an effect of period was identified.CLINICAL RELEVANCE:In this pilot study, cows treated with PEG using an alternative dosing schedule had reduced incidence of intramammary infection and an interaction of treatment by sampling period was observed for milk yield. These results suggest that further studies with larger numbers of cows are warranted.

Newborn calves experience altered redox balance upon transition to extrauterine life. In addition to its nutritional value, colostrum is rich in bioactive factors, including pro- and antioxidants. The objective was to investigate differences in pro- and antioxidants as well as oxidative markers in raw and heat-treated (HT) colostrum and in the blood of calves fed either raw or HT colostrum. Eleven colostrum samples (≥8 L) of Holstein cows were each divided into a raw or HT (60°C, 60 min) portion. Both treatments were stored for <24 h at 4°C and tube-fed in a randomized-paired design at 8.5% of body weight to 22 newborn female Holstein calves within 1 h after birth. Colostrum samples were obtained before feeding, and calf blood samples were taken immediately before feeding (0 h) and at 4, 8, and 24 h after feeding. All samples were analyzed for reactive oxygen and nitrogen species (RONS) and antioxidant potential (AOP), from which the oxidant status index (OSi) was calculated. In 0-, 4-, and 8-h plasma samples, targeted fatty acids (FA) were analyzed using liquid chromatography-mass spectrometry, and oxylipids and isoprostanes (IsoP) using liquid chromatography-tandem mass spectrometry. Results for RONS, AOP, and OSi were analyzed by mixed-effects ANOVA or mixed-effects repeated-measures ANOVA, for colostrum and calf blood samples, respectively, whereas FA, oxylipid, and IsoP were analyzed using false discovery rate–adjusted analysis of paired data. Compared with control, HT colostrum showed lower RONS [least squares means (LSM) 189, 95% confidence interval (95% CI): 159–219 vs. 262, 95% CI: 232–292) relative fluorescence units] and OSi (7.2, 95% CI: 6.0–8.3 vs. 10.0, 95% CI: 8.9–11.1), but AOP remained unchanged (26.7, 95% CI: 24.4–29.0 vs. 26.4, 95% CI: 24.1–28.7 Trolox equivalents/µL). Changes in colostrum oxidative markers due to heat treatment were minor. No changes in RONS, AOP, OSi, or oxidative markers were detected in calf plasma. In both groups of calves, plasma RONS activity declined considerably at all postfeeding time points compared with precolostral values, and AOP reached its maximum 8 to 24 h after feeding. Generally, oxylipid and IsoP plasma abundance reached nadirs at 8 h post-colostrum in both groups. Overall, effects due to heat treatment on redox balance of colostrum and newborn calves and on oxidative biomarkers were minimal. In this study, heat treatment of colostrum reduced RONS activity but did not lead to detectable changes in calf oxidative status overall. This indicates that there were only minor changes in colostral bioactive components that could alter newborn redox balance and markers of oxidative damage.

Hepatitis C virus (HCV) vaccine development remains at an early stage. We explored the economic and health consequences of potential HCV vaccines by comparing universal vaccination with a hepatitis C vaccine to no vaccination in two groups: (1) injecting drug users (IDU); (2) all 12 year olds, using a Markov cohort simulation. Among IDUs, vaccination would avert 248 cases of HCV infection and 89 HCV-related deaths per 1000 individuals, and reduce costs. In average risk cohorts, vaccination did not reduce costs but was reasonably cost effective. These results provide encouragement to vaccine developers that a vaccine that is moderately effective and reasonably priced should not face economic barriers to implementation and will be attractive to third party payers.

Horses have one of the highest skeletal muscle oxidative capacities amongst mammals, which, combined with a high glycolytic capacity, could perturb redox status during maximal exercise. We determined the effect of 30 d of oral coenzyme Q10 and N-acetyl-cysteine supplementation (NACQ) on muscle glutathione (GSH), cysteine, ROS, and coenzyme Q10 concentrations, and the muscle proteome, in seven maximally exercising Thoroughbred horses using a placebo and randomized cross-over design. Gluteal muscle biopsies were obtained the day before and 1 h after maximal exercise. Concentrations of GSH, cysteine, coenzyme Q10, and ROS were measured, and citrate synthase, glutathione peroxidase, and superoxide dismutase activities analyzed. GSH increased significantly 1 h post-exercise in the NACQ group (p = 0.022), whereas other antioxidant concentrations/activities were unchanged. TMT proteomic analysis revealed 40 differentially expressed proteins with NACQ out of 387 identified, including upregulation of 13 mitochondrial proteins (TCA cycle and NADPH production), 4 Z-disc proteins, and down regulation of 9 glycolytic proteins. NACQ supplementation significantly impacted muscle redox capacity after intense exercise by enhancing muscle glutathione concentrations and increasing expression of proteins involved in the uptake of glutathione into mitochondria and the NAPDH-associated reduction of oxidized glutathione, without any evident detrimental effects on performance.

Oxidized linoleic acid metabolites (OXLAM) are products of adipocyte lipolysis with the potential to modulate adipose tissue (AT) lipid metabolism and inflammation. In periparturient cows, linoleic acid is preferentially mobilized from AT during lipolysis by hormone-sensitive lipase (HSL) compared with other polyunsaturated fatty acids. Enzymatic and nonenzymatic reactions generate OXLAM from linoleic acid. Among OXLAM, 9-, 10-, and 12-hydroxy-octadecadienoic acids (HODE) are associated with pro-inflammatory responses, whereas 9- and 13-oxo-octadecadienoic acids (oxoODE) and 13-HODE can facilitate inflammation resolution and promote lipogenesis. This study evaluated the effect of HSL activity on OXLAM biosynthesis using subcutaneous AT explants collected from multiparous dairy cows at 10 d before and again at 10 and 24 d after calving. Explants were treated for 3 h without or with the β-adrenergic agonist isoproterenol (ISO; 1 µM; MilliporeSigma, Burlington, MA) to induce HSL activity. The contribution of HSL to OXLAM biosynthesis was determined by inhibiting its activity with CAY10499 (2 µM; Cayman Chemical, Ann Arbor, MI). After treatments, media and explants were collected for lipidomic analysis using HPLC-tandem mass spectroscopy. Results indicated that ISO increased the biosynthesis of 9-, 12-, and 13-HODE and 9-oxoODE, and this effect was reduced at 24 d after calving. Inhibiting HSL activity partially reversed ISO effects on HODE and 9-oxoODE. Our ex vivo model demonstrated for the first time a direct effect of HSL activity on the biosynthesis of OXLAM in AT, especially at 10 d before and 10 d after calving. The biosynthesis of anti-inflammatory OXLAM is limited during the first weeks after parturition and may promote AT inflammation and lipolytic responses to negative energy balance. These results indicate that HSL activity releases linoleic acid for OXLAM biosynthesis in concentrations of a magnitude that may bypass the need for the activation of phospholipases linked with the inflammatory cascade and thus supports, in part, lipolysis-driven inflammation within AT of periparturient cows.

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.

Calves may experience increased oxidative stress at birth through activation of metabolic and respiratory processes. Reducing oxidative stress may enhance calf viability in early life. Our objective was to determine the dose response to fish and flaxseed oil when supplemented in colostrum on concentrations of plasma fatty acid (FA), FA metabolites, and index of oxidative stress during the critical first week of life in calves to understand how supplementing n-3 FA may decrease oxidative stress. We hypothesized that n-3 FA supplemented in colostrum in a linear dose-dependent fashion would associate with increased plasma n-3 FA concentrations and decreased oxidative stress. Twenty-four male and female Holstein calves were randomly assigned to receive 0, 30, 60, or 120 mL of a 1:1 fish to flaxseed oil supplement in colostrum. All calves received 2.8 L of previously frozen colostrum (≥22% Brix) with their respective treatment within 6 h after birth. Blood was sampled before first feeding after birth and on d 1, 2, 4, 7, and 14 d of age to assess oxidant status and plasma free PUFA, phospholipid FA, and oxylipid concentrations. Health indicators were observed daily. Indicators of general health and growth were unaffected by treatment. Supplemented calves exhibited greater concentrations of n-3 FA in plasma as free and phospholipid FA and some n-3 and n-6 FA-derived oxylipids in the first week of life in a linear fashion with increasing supplemental dose. Fish and flaxseed oil treatments did not alter oxidant status but overall decreased isoprostane concentrations in plasma, indicating oxidative stress was decreased. Together, these responses indicate that the fish and flaxseed oil supplement was antiinflammatory. In conclusion, supplementing colostrum with 30, 60, and 120 mL of a 1:1 mixture of fish and flaxseed oil linearly increased plasma concentrations of n-3 FA and metabolites and decreased biomarkers of oxidative stress, but did not alter oxidant status or affect health or growth. Our findings suggest neonatal calves may benefit from n-3 FA supplementation in colostrum to encourage a greater antiinflammatory state.

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-1 beta-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-1 beta stimulation had significant reductions in expression of ADAMTS4, MMP-1, MMP-13, IL-1 beta, 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-1 beta-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 (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-1β 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.

Urinary tract infection (UTI) is an extremely common infectious disease. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. Asymptomatic bacteriuric E. coli (ABEC) strains successfully colonize the urinary tract resulting in asymptomatic bacteriuria (ABU) and do not induce symptoms associated with UTI. Oxylipids are key signaling molecules involved in inflammation. Based on the distinct clinical outcomes of E. coli colonization, we hypothesized that UPEC triggers the production of predominantly proinflammatory oxylipids and ABEC leads to production of primarily anti-inflammatory or proresolving oxylipids in the urinary tract. We performed quantitative detection of 39 oxylipid mediators with proinflammatory, anti-inflammatory, and proresolving properties, during UTI and ABU caused by genetically distinct E. coli strains in the murine urinary bladder. Our results reveal that infection with UPEC causes an increased accumulation of proinflammatory oxylipids as early as 6 h postinoculation, compared to controls. To the contrary, ABEC colonization leads to decreased accumulation of proinflammatory oxylipids at the early time point compared to UPEC infection but does not affect the level of proresolving oxylipids. This report represents the first comprehensive investigation on the oxylipidome during benign ABEC colonization observed in ABU and acute inflammation triggered by UPEC leading to UTI.

The periparturient period of dairy cows is characterized by intense lipolysis in adipose tissues (AT), which induces the release of free fatty acids (FFA) into circulation. Among FFA, polyunsaturated fatty acids are susceptible to oxidation and can modulate inflammatory responses during lipolysis within AT. Linoleic and arachidonic acid oxidized products (oxylipids) such as hydroxy-octadecadienoic acids (HODE) and hydroxy-eicosatetraenoic acids (HETE), were recently identified as products of lipolysis that could modulate AT inflammation during lipolysis. However, the effect of lipolysis intensity during the transition from gestation to lactation on fatty acid substrate availability and subsequent AT oxylipid biosynthesis is currently unknown. We hypothesized that in periparturient dairy cows, alterations in AT and plasma fatty acids and oxylipid profiles coincide with changes in lipolysis intensity and stage of lactation. Blood and subcutaneous AT samples were collected from periparturient cows at -27±7 (G1) and -10±5 (G2) d prepartum and at 8±3 d postpartum (PP). Targeted lipidomic analysis was performed on plasma and AT using HPLC-MS/MS. We report that FFA concentrations increased as parturition approached and were highest at PP. Cows exhibiting high lipolysis rate at PP (FFA>1.0 mEq/L) had higher body condition scores at G1 compared to cows with low lipolysis rate (FFA<1.0 mEq/L). Concentrations of plasma linoleic and arachidonic acids were increased at PP. In AT, 13-HODE, and 5-, 11- and 15-HETE were increased at PP compared to G1 and G2. Concentrations of beta hydroxybutyrate were positively correlated with those of 13-HODE and 15-HETE in AT. Plasma concentrations of 5- and 20-HETE were increased at PP. These data demonstrate that prepartum adiposity predisposes cows to intense lipolysis post-partum and may exacerbate AT inflammation because of increased production of pro-inflammatory oxylipids including 5- and 15-HETE and 13-HODE. These results support a role for certain linoleic and arachidonic acid-derived oxylipids as positive and negative modulators of AT inflammation during periparturient lipolysis.

Gestational diabetes is associated with increased lipolysis and adipose tissue (AT) inflammation, which impair insulin signaling during the second half of pregnancy and in early lactation. Prevalence of gestational diabetes is high in overweight and obese women; similarly, dairy cows with high adiposity exhibit a surge in AT lipolysis and inflammatory responses during the last trimester of gestation that lead to AT insulin resistance. Oxidized FA (oxylipids) can modulate inflammatory responses during lipolysis. Linoleic acid (LNA) and arachidonic acid (AA) derived oxylipids, including hydroxy‐octadecadienoic acids (HODE), oxo‐octadecadienoic acids (oxoODE), and hydroxy‐eicosatetraenoic acids (HETE), were recently identified as potential biomarkers for early detection of insulin resistance. However, the effects of adiposity and lipolysis rate during late gestation and early lactation on oxylipid biosynthesis including FA substrate availability is currently unknown. We hypothesized that periparturient profiles of serum and AT concentrations of PUFA and oxylipids are dependent on body adiposity and lipolytic rate. Blood and subcutaneous AT samples were collected from multiparous Holstein cows with high (HA; n=5) or low (LA; n=4) adiposity scores at −27±7 (FO) and −10±5 (CU) d prepartum and at 8±3 d postpartum (PP). Targeted lipidomic analysis was performed on plasma and AT using HPLC‐MS/MS. We report that circulating free FA (FFA) concentrations increased as parturition approached, and peaked at PP. Across time, HA increased the concentration of circulating FFA compared to LA, reflecting a significant effect of adiposity on lipolytic rate during the periparturient period. Serum concentrations of AA and eicosapentanoic acid (EPA) were decreased at CU, compared to the other timepoints for all cows. Across time, serum AA and docosahexanoic acid (DHA) concentrations were decreased in HA compared to LA. AT content of AA and DHA, as well as LNA content in AT and serum, were unchanged throughout the study and were not influenced by adiposity. There were effects of both adiposity and timepoint on oxylipid concentrations. In AT, 13‐HODE, 5‐HETE, and 11‐HETE were increased at PP compared to FO and CU. Compared to LA, HA had decreased AT concentrations of 9‐HODE,13‐HODE, 5‐HETE, and 15‐HETE. Serum concentration of 9‐oxoODE was increased at PP and was positively correlated with serum FFA concentration (r=0.52. P=0.01), but was unaffected by adiposity. These data demonstrate that prepartum adiposity may limit the availability of serum PUFA, such as DHA, which serve as substrates for anti‐inflammatory oxylipids. Furthermore, lipolysis during late gestation and the onset of lactation may enhance AT biosynthesis and systemic release of HODE and HETE into circulation.Support or Funding InformationUSDA‐NIFA grants 2014‐68004‐21972 and 2015‐67015‐23207Michigan Alliance for Animal Agriculture Effect of adiposity and sampling time point across late gestation and early lactation on fatty add composition and oxylipid profile of subcutaneous adipose tissue and serum (mean + SEM) of dairy cows. Adiposity Sampling Metabolite Low (LA) High (HA) SEM P Far off (FO) Close‐up (CU) Postpartum (PP) SEM P Serum free fatty acids (FFA) mEq/L FFA 0.43 0.63 0.05 <0.01 0.27a 0.34b 0.98c 0.06 <0.01 Serum fatty acids μmol/L Linoleic 341.5 222.6 78.8 NS 205.2 187 454 81.8 NS Arachidonic 42.2 20 10.5 0.02 22.2a 17.4a 53.8b 16.1 0.01 EPA 2.5 0.9 0.8 NS 0.7a 0.5a 2.7b 0.4 0.01 DHA 0.8 0.4 0.1 0.03 0.7 0.4 0.7 0.2 NS Adipose tissue fatty acids μmol/mg Linoleic acid 1340 1110 360 NS 1570 420 1720 500 NS Arachidonic acid 43.5 46.2 17 NS 43.9 9.3 52.4 16 NS EPA 0.83 0.43 0.3 NS 0.9 0.14 1.12 0.4 NS DHA 0.17 0.24 0.1 NS 0.23 0.31 0.07 0.2 NS Adipose tissue oxylipids nM/mg 9‐HODE 1.52 0.63 0.63 0.01 0.59 0.67 1.9 0.5 NS 9‐oxoODE 0.97 0.62 0.35 NS 0.49 1.01 0.87 0.26 NS 13‐HODE 2.42 0.71 0.84 0.05 0.77a 0.95a 2.97b 0.7 0.05 13‐oxoODE 0.62 0.62 0.47 NS 0.32 0.5 1.09 0.38 NS 5‐HETE 0.6 0.33 0.12 0.05 0.24a 0.56b 0.6b 0.12 0.05 11‐HETE 0.16 0.09 0.04 0.03 0.1a 0.08a 0.2b 0.04 0.04 15‐HETE 0.29 0.04 0.08 0.01 0.06a 0.12b 0.42c 0.08 0.01 20‐HETE 0.007 0.007 0.01 NS 0.007 0.007 0.006 0.004 NS Serum oxylipids nM/mL 9‐HODE 140.47 141.5 68.44 NS 210.98 111.45 101.65 60.4 NS 9‐oxoODE 82.3 67.6 36.76 NS 79.64a 87.41a 131b 41.3 0.02 13‐HODE 302 309.67 119.44 NS 364.14 301.73 252.77 98.5 NS 13‐oxoODE 31.97 19.18 2.09 NS 36.47 10.58 23.45 15.66 NS 5‐HETE 13.38 20.59 11.04 NS 12.62 29.75 5.55 10.58 NS 11‐HETE 8.65 7.98 4.69 NS 7.48 9.02 8.43 4.09 NS 15‐HETE 10.31 8.64 5.36 NS 10.72 11.8 5.82 4.87 NS 20‐HETE 26.45 28.45 1.7 NS 5.16 32.55 6.65 16.44 NS EPA=eicosapentaenoic acid; DHA=docosahexaenoic acid; HODE=hydroxyoctadecadienoic acid; oxoODE=oxooctadecadienoic acid; HETE= hydroxyeicosatetraenoic acid. Means in same row with different letter differ between sampling timepoint, P < 0.05. NS= non‐significant effect, P > 0.05.

Uncontrolled inflammation is an underlying etiology for multiple diseases and macrophages orchestrate inflammation largely through the production of oxidized fatty acids known as oxylipids. Previous studies showed that selenium (Se) status altered the expression of oxylipids and magnitude of inflammatory responses. Although selenoproteins are thought to mediate many of the biological effects of Se, the direct effect of selenoproteins on the production of oxylipids is unknown. Therefore, the role of decreased selenoprotein activity in modulating the production of biologically active oxylipids from macrophages was investigated. Thioglycollate-elicited peritoneal macrophages were collected from wild-type and myeloid-cell-specific selenoprotein knockout mice to analyze oxylipid production by liquid chromatography/mass spectrometry as well as oxylipid biosynthetic enzyme and inflammatory marker gene expression by quantitative real-time polymerase chain reaction. Decreased selenoprotein activity resulted in the accumulation of reactive oxygen species, enhanced cyclooxygenase and lipoxygenase expression and decreased oxylipids with known anti-inflammatory properties such as arachidonic acid-derived lipoxin A4 (LXA4) and linoleic acid-derived 9-​oxo-octadecadienoic acid (9-oxoODE). Treating RAW 264.7 macrophages with LXA4 or 9-oxoODE diminished oxidant-induced macrophage inflammatory response as indicated by decreased production of TNFα. The results show for the first time that selenoproteins are important for the balanced biosynthesis of pro- and anti-inflammatory oxylipids during inflammation. A better understanding of the Se-dependent control mechanisms governing oxylipid biosynthesis may uncover nutritional intervention strategies to counteract the harmful effects of uncontrolled inflammation due to oxylipids.

OBJECTIVE:To compare the effects of triamcinolone acetonide (TA) and methylprednisolone acetate (MPA) on expression of selected chondrocyte genes in recombinant equine interleukin-1β (reIL-1β) stimulated articular cartilage explants.DESIGN:In vitro experiment.ANIMALS:Horses (n = 6).PROCEDURES:Articular cartilage explants from 2- to 3- year-old horses were exposed to reIL-1β in the presence and absence of TA and MPA at 10(-7) and 10(-6) M. Resting levels of mRNA of anabolic and catabolic genes of chondrocyte origin were quantified using qPCR after 6- and 12-hour incubations. Genes of interest included aggrecan interglobular domain, aggrecan, and collagen II, matrix metalloproteinases 3 and 13 (MMP3, MMP 13), aggrecanase 1, tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP 1, TIMP 2), BCL 2, vascular endothelial growth factor, and cyclooxygenase 2 (COX 2).RESULTS:IL-1β significantly influenced the expression of most transcripts. MPA and TA inhibited the induction of MMP 13 at 6 and 12 hours; an effect that was significant at 6 hours with MPA at 10(-7) M and TA at 10(-6) M. Similarly, COX 2 was induced by reIL-1β and MPA and TA significantly inhibited its upregulation. TIMP 2 expression was reduced by reIL-1β, an effect that was significantly abrogated by MPA and TA. There were no significant differences observed between glucocorticoids for any gene studied.CONCLUSIONS:No differential effects of MPA or TA on chondrocytic gene expression were identified suggesting that any divergent influences of these glucocorticoids on chondrocyte metabolism are posttranslational.