The delta 6 desaturase knock out mouse reveals that immunomodulatory effects of essential n-6 and n-3 polyunsaturated fatty acids are both independent of and dependent upon conversion

Typically fatty acids (FA) exert differential immunomodulatory effects with n-3 [α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and n-6 [linoleic acid (LA) and arachidonic acid (AA)] exerting anti- and pro-inflammatory effects, respectively. This over-simplified interpretation is confounded by a failure to account for conversion of the parent FA (LA and ALA) to longer-chain bioactive products (AA and EPA/DHA, respectively), thereby precluding discernment of the immunomodulatory potential of specific FA. Therefore, we utilized the Δ6-desaturase model, wherein knockout mice (D6KO) lack the Fads2 gene encoding for the rate-limiting enzyme that initiates FA metabolism, thereby providing a model to determine specific FA immunomodulatory effects. Wild-type (WT) and D6KO mice were fed one of four isocaloric diets differing in FA source (9 weeks): corn oil (LA-enriched), arachidonic acid single cell oil (AA-enriched), flaxseed oil (ALA-enriched) or menhaden fish oil (EPA/DHA-enriched). Splenic mononuclear cell cytokine production in response to lipopolysaccharide (LPS), T-cell receptor (TCR) and anti-CD40 stimulation was determined. Following LPS stimulation, AA was more bioactive compared to LA, by increasing inflammatory cytokine production of IL-6 (1.2-fold) and TNFα (1.3-fold). Further, LPS-stimulated IFNγ production in LA-fed D6KO mice was reduced 5-fold compared to LA-fed WT mice, indicating that conversion of LA to AA was necessary for cytokine production. Conversely, ALA exerted an independent immunomodulatory effect from EPA/DHA and all n-3 FA increased LPS-stimulated IL-10 production versus LA and AA. These data definitively identify specific immunomodulatory effects of individual FA and challenge the simplified view of the immunomodulatory effects of n-3 and n-6 FA.     

Reduced protein oxidation in Wistar rats supplemented with marine ω3 PUFAs

The potential effects of various dietary eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) ratios (1:1, 2:1, and 1:2, respectively) on protein redox states from plasma, kidney, skeletal muscle, and liver were investigated in Wistar rats. Dietary fish oil groups were compared with animals fed soybean and linseed oils, vegetable oils enriched in ω6 linoleic acid (LA; 18:2) and ω3 α-linolenic acid (ALA; 18:3), respectively. Fish oil treatments were effective at reducing the level of total fatty acids in plasma and enriching the plasmatic free fatty acid fraction and erythrocyte membranes in EPA and DHA. A proteomic approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labeling of protein carbonyls, FTSC intensity visualization on 1-DE or 2-DE gels, and protein identification by MS/MS was used for the protein oxidation assessment. Albumin was found to be the most carbonylated protein in plasma for all dietary groups, and its oxidation level was significantly modulated by dietary interventions. Supplementation with an equal EPA:DHA ratio (1:1) showed the lowest oxidation score for plasma albumin, followed in increasing order of carbonylation by 1:2 <2:1 ≈ linseed < soybean. Oxidation patterns of myofibrillar skeletal muscle proteins and cytosolic proteins from kidney and liver also indicated a protective effect on proteins for the fish oil treatments, the 1:1 ratio exhibiting the lowest protein oxidation scores. The effect of fish oil treatments at reducing carbonylation on specific proteins from plasma (albumin), skeletal muscle (actin), and liver (albumin, argininosuccinate synthetase, 3-α-hydroxysteroid dehydrogenase) was remarkable. This investigation highlights the efficiency of dietary fish oil at reducing in vivo oxidative damage of proteins compared to oils enriched in the 18-carbon polyunsaturated fatty acids ω3 ALA and ω6 LA, and such antioxidant activity may differ among different fish oil sources because of variations in EPA/DHA content.     

Expression of “brown-in-white” adipocyte biomarkers shows gender differences and the influence of early dietary exposure

Fish oil supplementation provides an inconsistent degree of protection from cardiovascular disease (CVD), which may be attributed to genetic variation. Single nucleotide polymorphisms (SNPs) in the elongation-of-very-long-chain-fatty-acids-2 (ELOVL2) gene have been strongly associated with plasma proportions of n-3 long-chain polyunsaturated fatty acids (LC-PUFA). We investigated the effect of genotype interaction with fish oil dosage on plasma n-3 LC-PUFA proportions in a parallel double-blind controlled trial, involving 367 subjects randomised to treatment with 0.45, 0.9 and 1.8 g/day eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (1.51:1) or olive oil placebo for 6 months. We genotyped 310 subjects for ELOVL2 gene SNPs rs3734398, rs2236212 and rs953413. At baseline, carriers of all minor alleles had lower proportions of plasma DHA than non-carriers (P = 0.021–0.030). Interaction between genotype and treatment was a significant determinant of plasma EPA (P < 0.0001) and DHA (P = 0.004–0.032). After the 1.8 g/day dose, carriers of ELOVL2 SNP minor alleles had approximately 30 % higher proportions of EPA (P = 0.002–0.004) and 9 % higher DHA (P = 0.013–0.017) than non-carriers. Minor allele carriers could therefore particularly benefit from a high intake of EPA and DHA in maintaining high levels of plasma n-3 PUFA conducive to protection from CVD.     

Regulation of human lymphocyte proliferation by fatty acids

In the present study, the effect of increasing concentrations of palmitic (PA, C16:0), stearic (SA, C18:0), oleic (OA, C18:1, n-9), linoleic (LA, C18:2n-6), docosahexaenoic (DHA, C22:6 n-3) and eicosapentaenoic (EPA, C20:5 n-3) acids on lymphocyte proliferation was investigated. The maximal non-toxic concentrations of these fatty acids for human lymphocytes in vitro were determined. It was also evaluated whether these fatty acids at non-toxic concentrations affect IL-2 induced lymphocyte proliferation and cell cycle progression. OA and LA at 25 microM increased lymphocyte proliferation and at higher concentrations (75 microM and 100 microM) inhibited it. Both fatty acids promoted cell death at 200 microM concentration. PA and SA decreased lymphocyte proliferation at 50 microM and promoted cell death at concentrations of 100 microM and above. EPA and DHA decreased lymphocyte proliferation at 25 and 50 microM being toxic at 50 and 100 microM, respectively. PA, SA, DHA and EPA decreased the stimulatory effect of IL-2 on lymphocyte proliferation, increasing the percentage of cells in G1 phase and decreasing the proportion of cells in S and G2/M phases. OA and LA caused an even greater pronounced effect. The treatment with all fatty acids increased neutral lipid accumulation in the cells but the effect was more pronounced with PA and DHA. In conclusion, PA, SA, DHA and EPA decreased lymphocyte proliferation, whereas OA and LA stimulated it at non-toxic concentrations.     

Long-Chain Omega-3 Polyunsaturated Fatty Acids Have Developmental Effects on the Crop Pest,the Cabbage White Butterfly Pieris rapae

Nutritional enhancement of crops using genetic engineering can potentially affect herbivorous pests. Recently, oilseed crops have been genetically engineered to produce the long-chain omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at levels similar to that found in fish oil; to provide a more sustainable source of these compounds than is currently available from wild fish capture. We examined some of the growth and development impacts of adding EPA and DHA to an artificial diet of Pieris rapae, a common pest of Brassicaceae plants. We replaced 1% canola oil with EPA: DHA (11:7 ratio) in larval diets, and examined morphological traits and growth of larvae and ensuing adults across 5 dietary treatments. Diets containing increasing amounts of EPA and DHA did not affect developmental phenology, larval or pupal weight, food consumption, nor larval mortality. However, the addition of EPA and DHA in larval diets resulted in progressively heavier adults (F _{4, 108} = 6.78; p = 0.011), with smaller wings (p < 0.05) and a higher frequency of wing deformities (R = 0.988; p = 0.001). We conclude that the presence of EPA and DHA in diets of larval P. rapae may alter adult mass and wing morphology; therefore, further research on the environmental impacts of EPA and DHA production on terrestrial biota is advisable.     

Enzymatic preparation of [1-13C]d-fructose-6-phosphate from [13C]formaldehyde and d-ribose-5-phosphate using the formaldehyde-fixing system of Methylomonas aminofaciens 77a

The intracellular concentration of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-cis-docosahexaenoic acid (DHA) was carried out by Mortierella alpina 1S-4 in a medium containing fish oil as the main carbon source. The EPA and DHA contents reached 29.2% and 20.0% of total fatty acids, respectively, when the fungus was grown in a medium containing salmon oil (EPA and DHA contents, 14.0% and 17.3%, respectively) as the main carbon source in a 5-1 bench-scale fermentor. EPA and DHA in the added fish oil were incorporated into both the mycelial polar lipids and triglycerides.On leave from Suntory Ltd. Correspondence to: S. Shimizu     

Improvement of nutritional quality of cultured sobaity sea bream,Sparidentex hasta (Valenciennes) muscle by preharvest feeding of finisher feeds

A 6‐month long study was conducted to improve the nutritional quality of the cultured sobaity bream, Sparidentex hasta by feeding them finisher feeds containing high docosahexaenoic acid (DHA) at the last two months of the grow‐out stage so that the muscle DHA level be increased at par to the wild. A grow‐out feed used from the beginning until the end of the trial was considered as the control (Diet 1). Experimental diets 2 and 3 were formulated to contain 9.0% DHA (e.g. 1.68 g DHA/100 g feed) and 10.5% DHA (2.20 g DHA/100 g feed), by incorporating high DHA tuna oil into a sea bream grow‐out diet. For comparison, a commercial finisher feed (Diet 4) from Skretting, Italy was also used. The results of this study demonstrated that fish fed DHA enriched finisher diets resulted in significantly (p < .05) better growth, feed utilization and higher muscle eicosapentaenoic acid (EPA) and DHA content compared to those fed grow‐out diet. The muscle DHA and EPA of fish fed finisher diets were also higher than those of the whole year average DHA and EPA content of wild sobaity. An organoleptic evaluation showed no significant (p > .05) differences between sensory attributes of muscle from cultured and wild sea bream. The results of the study demonstrated that feeding finisher feed enriched with DHA at the later part of the grow‐out operation, the n‐3 PUFA levels of cultured sobaity can cost‐effectively be increased at par to the wild.     

Cold storage effects on flesh quality of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) fed diets containing different vegetable oils

The intention of this experiment was to assess the effects of different sources of dietary lipid on the fatty acid composition of the fillet and liver and the flesh quality traits of rainbow trout (Oncorhynchus mykiss) after a 70‐day feeding period. Four iso‐nitrogenous (approx. 51% crude protein) and iso‐lipidic (approx. 14% crude lipid) experimental diets were formulated. The control diet contained only fish oil (FO) as the primary lipid source. In the other three dietary treatments, fish oil was replaced by 100% (LO30/SO35/SFO35) and 70% (FO30/LO35/SO35 or FO30/SO35/SFO35) sesame oil (SO), linseed oil (LO), or sunflower oil (SFO). Triplicate groups of 40 rainbow trout (~46 g) held under similar culture conditions were hand‐fed daily to apparent satiation for 70 days. At the end of the feeding trials, no difference in growth performance among experimental groups was noted (P > 0.05). There were some differences in the proximate composition of fish fillets (P < 0.05): the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels were highest in fish fed the control diet (P < 0.05); and EPA and DHA levels in fish fed the FO30/LO35/SO35 diet were closest to the control diet (P < 0.05). In contrast, fish fed the diet containing 100% plant oils (LO30/SO35/SFO35) had the highest level of total n‐6 fatty acids in the fillet and liver. In a 12‐day refrigerated storage at 1°C the thiobarbituric acid (TBA), trimethylamin nitrogen (TMA‐N) and pH values gradually increased in all dietary groups (P < 0.05). The chemical indicators of spoilage, TBA, TMA‐N, and pH values were within the limit of acceptability for human consumption.     

Micellar oleic and eicosapentaenoic acid but not linoleic acid influences the β-carotene uptake and its cleavage into retinol in rats

Improving the bioavailability of β-carotene is vital to manage vitamin A deficiency. The influence of micellar oleic (OA), linoleic (LA) and eicosapentaenoic (EPA) acids on plasma β-carotene response and its conversion to retinol has been studied in rats employing single (9 h time course) and repeated (10 days) dose administrations. After a single dose, the levels (area under the curve) of plasma β-carotene and retinyl palmitate in OA and EPA groups were higher (p < 0.05) by 13, 7 and 11, 6 folds than LA group. The liver β-carotene level in OA and EPA groups were higher (p < 0.05) by 3 and 1.2 folds than LA group. After repeated dose, the plasma β-carotene and retinyl palmitate levels in OA (6.2%, 51.7%) and EPA (25.4%, 17.23%) groups were higher (p < 0.05) than LA group. The liver β-carotene level in OA (21.2%) and EPA (17.6%) groups were higher (p < 0.05) than LA group. In both the experiments, the activity of β-carotene 15,15′-dioxygenase in the intestinal mucosa and plasma triglyceride levels were also higher in OA and EPA groups than LA group. β-Carotene excreted through urine and feces of OA and EPA groups was lower than the LA group. These results demonstrate an improved absorption and metabolism of β-carotene when fed mixed micelles with OA or EPA compared with LA. Although the mechanism involved in selective absorption of fatty acids needs further studies, intestinal β-carotene uptake and its conversion to vitamin A can be modulated using specific fatty acids.     

Eicosapentaenoic Acid and Rosiglitazone Increase Adiponectin in an Additive and PPARγ‐Dependent Manner in Human Adipocytes

Adiponectin, an anti‐inflammatory and insulin‐sensitizing protein secreted from adipose tissue, may be modulated by dietary fatty acids, although the mechanism is not fully known. Our objective was to investigate the effect of long‐chain n‐3 polyunsaturated fatty acids (PUFAs) on adiponectin in cultured human adipocytes, and to elucidate the role of peroxisome proliferator‐activated receptor‐γ (PPARγ) in this regulation. Isolated human adipocytes were cultured for 48 h with 100 µmol/l eicosapentaenoic acid (C20:5n‐3, EPA), docosahexaenoic acid (C22:6n‐3, DHA), palmitic acid (C16:0), 100 µmol/l EPA plus 100 µmol/l DHA, or bovine serum albumin (control). Additionally, adipocytes were treated for 48 h with a PPARγ antagonist (BADGE) or agonist (rosiglitazone) in isolation or in conjunction with either EPA or DHA. At 48 h, EPA and DHA increased (P < 0.05) adiponectin secretion by 88 and 47%, respectively, while EPA, but not DHA, also increased (136%, P < 0.001) cellular adiponectin protein. Interestingly, PPARγ antagonism completely abolished the DHA‐mediated increase in secreted adiponectin, but only partially attenuated the EPA‐mediated response. Thus, EPA's effects on adiponectin do not appear to be entirely PPARγ mediated. Rosiglitazone increased (P < 0.001) the secreted and cellular adiponectin protein (90 and 582%, respectively). Finally, the effects of EPA and rosiglitazone on adiponectin secretion were additive (+230% at 48 h combined, compared to 121 and 124% by EPA or rosiglitazone alone, respectively). Overall, our findings emphasize the therapeutic importance of long‐chain n‐3 PUFA alone, or in combination with a PPARγ agonist, as a stimulator of adiponectin, a key adipokine involved in obesity and related diseases.     

Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human THP-1 macrophages more effectively than eicosapentaenoic acid

A number of studies have investigated the effects of fish oil on the production of pro-inflammatory cytokines using peripheral blood mononuclear cell models. The majority of these studies have employed heterogeneous blends of long-chain n-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which preclude examination of the individual effects of LC n-3 PUFA. This study investigated the differential effects of pure EPA and DHA on cytokine expression and nuclear factor kappaB (NF-kappaB) activation in human THP-1 monocyte-derived macrophages. Pretreatment with 100 microM EPA and DHA significantly decreased lipopolysaccharide (LPS)-stimulated THP-1 macrophage tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and IL-6 production (P<.02), compared to control cells. Both EPA and DHA reduced TNF-alpha, IL-1beta and IL-6 mRNA expression. In all cases, the effect of DHA was significantly more potent than that of EPA (P<.01). Furthermore, a low dose (25 microM) of DHA had a greater inhibitory effect than that of EPA on macrophage IL-1beta (P<.01 and P<.04, respectively) and IL-6 (P<.003 and P<.003, respectively) production following 0.01 and 0.1 microg/ml LPS stimulation. Both EPA and DHA down-regulated LPS-induced NF-kappaB/DNA binding in THP-1 macrophages by approximately 13% (P< or =.03). DHA significantly decreased macrophage nuclear p65 expression (P< or =.05) and increased cytoplasmic IkappaBalpha expression (P< or =.05). Although similar trends were observed with EPA, they were not significant. Our findings suggest that DHA may be more effective than EPA in alleviating LPS-induced pro-inflammatory cytokine production in macrophages - an effect that may be partly mediated by NF-kappaB. Further work is required to elucidate additional divergent mechanisms to account for apparent differences between EPA and DHA.     

Dietary linoleic acid has no effect on arachidonic acid,but increases n-6 eicosadienoic acid,and lowers dihomo-γ-linolenic and eicosapentaenoic acid in plasma of adult men

High intakes of linoleic acid (LA,18:2n-6) have raised concern due to possible increase in arachidonic acid (ARA, 20:4n-6) synthesis, and inhibition of alpha linolenic acid (ALA, 18:3n-3) desaturation to eicosapentaenoic (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In healthy men, 10.5% energy compared to 3.8% energy LA with 1% energy ALA increased plasma phospholipid LA and 20:2n-6, the elongation product of LA, and decreased EPA, with no change in ARA. However, LA was inversely related to ARA at both 10.5% energy and 3.8% energy LA, (r=?0.761, r=?0.817, p<0.001, respectively). A two-fold variability in ARA among individuals was not explained by the dietary LA, ARA, ALA, or fish intake. Our results confirm LA requirements for ARA synthesis is low, <3.8% energy, and they suggest current LA intakes saturate Δ-6 desaturation and adversely affect n-3 fatty acid metabolism. Factors other than n-6 fatty acid intake are important modifiers of plasma ARA.     

Omega-6 and omega-3 fatty acids metabolism pathways in the body of pigs fed diets with different sources of fatty acids

This study was carried out on 24 gilts (♀ Polish Large White × ♂ Danish Landrace) grown with body weight (BW) of 60 to 105 kg. The pigs were fed diets designed on the basis of a standard diet (appropriate for age and BW of pigs) where a part of the energy content was replaced by different fat supplements: linseed oil in Diet L, rapeseed oil in Diet R and fish oil in Diet F (6 gilts per dietary treatment). The fat supplements were sources of specific fatty acids (FA): in Diet L α-linolenic acid (C18:3 n?3, ALA); in Diet R linoleic acid (C18:2 n?6, LA) and in Diet F eicosapentaenoic acid (C20:5 n?3, EPA), docosapentaenoic acid (C22:5 n?3, DPA) and docosahexaenoic acid (C22:6 n?3, DHA). The protein, fat and total FA contents in the body did not differ among groups of pigs. The enhanced total intake of LA and ALA by pigs caused an increased deposition of these FA in the body (p < 0.01) and an increased potential body pool of these acids for further metabolism/conversions. The conversion efficiency of LA and ALA from the feed to the pig’s body differed among groups (p < 0.01) and ranged from 64.4% to 67.2% and from 69.4% to 81.7%, respectively. In Groups L and R, the level of de novo synthesis of long-chain polyunsaturated FA was higher than in Group F. From the results, it can be concluded that the efficiency of deposition is greater for omega-3 FA than for omega-6 FA and depends on their dietary amount. The level of LA and ALA intake influences not only their deposition in the body but also the end products of the omega-3 and omega-6 pathways.     

T helper cells subtypes and their cytokine gene expression affected by carvacrol in sensitized mice administered during sensitization period

Th1, Th2, Th17, and Treg cells and their cytokine gene expressions in splenocytes of control mice, ovalbumin sensitized (S), and S treated with dexamethasone and carvacrol during a sensitization period were examined. Th2 and Th17 population as well as the gene expression of IL-4, IL-17, and TGF-β were increased, but Th1, Th1/Th2 ratio, the gene expression of IFN-γ and FOXP3 as well as the IFN-γ/IL-4 ratio were decreased in S compared with control group ( P < 0.001 for all cases). Carvacrol treatment caused significant reduction of Th2 and Th17 population as well as gene expression of IL-4, IL-17, and TGF-β but increase in Treg cells, Th1/Th2 ratio, gene expressions of FOXP3, IFN-γ, and IFN-γ/IL-4 ratio ( P < 0.05 to P < 0.001). The population of Th1, Th2, Th17 cells as well as the gene expression of IL-4, IL-17, and TGF-β were significantly decreased, but only Treg was increased in the dexamethasone treatment group ( P < 0.05 to P < 0.001). Carvacrol treatment during the sensitization period showed a more specific effect on Th1/Th2 imbalance in sensitized mice than dexamethasone, which may indicate the therapeutic potentials of carvacrol in disorders associated with Th1/Th2 imbalance such as asthma.     

Long-chain conversion of [13C]linoleic acid and alpha-linolenic acid in response to marked changes in their dietary intake in men

We studied the long-chain conversion of [U-13C]alpha-linolenic acid (ALA) and linoleic acid (LA) and responses of erythrocyte phospholipid composition to variation in the dietary ratios of 18:3n-3 (ALA) and 18:2n-6 (LA) for 12 weeks in 38 moderately hyperlipidemic men. Diets were enriched with either flaxseed oil (FXO; 17 g/day ALA, n=21) or sunflower oil (SO; 17 g/day LA, n=17). The FXO diet induced increases in phospholipid ALA (>3-fold), 20:5n-3 [eicosapentaenoic acid (EPA), >2-fold], and 22:5n-3 [docosapentaenoic acid (DPA), 50%] but no change in 22:6n-3 [docosahexanoic acid (DHA)], LA, or 20:4n-6 [arachidonic acid (AA)]. The increases in EPA and DPA but not DHA were similar to those in subjects given the SO diet enriched with 3 g of EPA plus DHA from fish oil (n=19). The SO diet induced a small increase in LA but no change in AA. Long-chain conversion of [U-13C]ALA and [U-13C]LA, calculated from peak plasma 13C concentrations after simple modeling for tracer dilution in subsets from the FXO (n=6) and SO (n=5) diets, was similar but low for the two tracers (i.e., AA, 0.2%; EPA, 0.3%; and DPA, 0.02%) and varied directly with precursor concentrations and inversely with concentrations of fatty acids of the alternative series. [13C]DHA formation was very low (<0.01%) with no dietary influences.     

Heat stress upregulation of Toll-like receptors 2/4 and acute inflammatory cytokines in peripheral blood mononuclear cell (PBMC) of Bama miniature pigs: an in vivo and in vitro study

Global warming is a challenge to animal health, because of increased heat stress, with subsequent induction of immunosuppression and increased susceptibility to disease. Toll-like receptors (TLR) are pattern recognition receptors that act as sentinels of pathogen invasion and tissue damage. Ligation of TLRs results in a signaling cascade and production of inflammatory cytokines, which eradicate pathogens and maintain the health of the host. We hypothesized that the TLR signaling pathway plays a role in immunosuppression in heat-stressed pigs. We explored the changes in the expression of TLR2, TLR4 and the concentration of acute inflammatory cytokines, such as IL-2, IL-8, IL-12 and IFN-γ in Bama miniature pigs subjected to 21 consecutive days of heat stress, both in vitro and in vivo models. The results showed that heat stress induced the upregulation of cortisol in the plasma of pigs (P<0.05); TLR4 mRNA was elevated, but IL-2 was reduced in peripheral blood mononuclear cells (PBMC, P<0.05). The white blood cell count and the percentage of granulocytes (eosinophilic+basophilic) decreased significantly in heat-stressed pigs (P<0.05). In the in vitro model (PBMC heat shocked for 1 h followed by a 9 h recovery period), TLR2 and TLR4 mRNA expression also increased, as did the concentration of IL-12 in supernatants. However, IFN-γ was significantly reduced in PBMC culture supernatants (P<0.05). We concluded that a consecutive heat stress period elevated the expression of TLR2 and TLR4 in PBMC and increased the plasma levels of inflammatory cytokines. These data indicate that TLR activation and dysregulation of cytokine expression in response to prolonged heat stress may be associated with immunosuppression and increased susceptibility to antigenic challenge in Bama miniature pigs.     

Eicosapentaenoic acid- and docosahexaenoic acid-rich fish oil in sow and piglet diets modifies blood oxylipins and immune indicators in both,sows and suckling piglets

Over the last decades, genetic selection has increased sows’ litter size. Consequently, there is a high proportion of piglets born with low weight which are vulnerable. Their viability may potentially be enhanced through early nutrition. The aim of the current study was to evaluate whether including a fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the diets of the sow and piglets was able to increase concentrations of anti-inflammatory molecules in their blood. Thirty-six sows, in four consecutive batches, were randomly assigned to either a control diet with animal fat (15 g/kg in gestation and 30 g/kg in lactation) or an n-3 long-chain fatty acid (n-3 LCFA) diet from insemination until the end of lactation. From day 11 of lactation, piglets were also offered a diet containing 30 g/kg of animal fat or n-3 LCFA. To prepare the n-3 LCFA diet, 15 g/kg or 30 g/kg of animal fat in the control diet were replaced by an equivalent amount of solid fish oil for sows and piglets, respectively. All the sows were sampled for serum and plasma at day 108 of gestation and at weaning. Additionally, only for the first batch of sows, blood samples were also obtained at weaning from the two lightest (>800 g) and the two heaviest birth weight piglets in each litter. Serum fatty acids (FAs) were quantified by gas chromatography, plasma oxylipins by ultra-HPLC-MS and plasma immunoglobulins (Ig) and cytokines by ELISA. The n-3 LCFA diet increased the concentrations of n-3 FAs in gestating and lactating sows and in piglets (P < 0.001, P < 0.001 and P = 0.011, respectively), particularly EPA (P < 0.001, P < 0.001 and P < 0.001, respectively) and DHA (P < 0.001, P < 0.001 and P < 0.001, respectively), and also their oxygenated derivatives. In addition, fish oil increased plasma IgM in gestating and lactating sows (P = 0.014 and P = 0.008, respectively), interleukin (IL) 6 in sows at weaning (P = 0.012), and IL1β in piglets (P = 0.018). Birth BW of piglets, regardless of diet, slightly influenced some of the n-6-derived oxylipins. In conclusion, fish oil addition in diets increased the blood concentrations of n-3 FAs and their oxygenated derivatives, some of which have anti-inflammatory activity, in gestating and lactating sows and piglets, IgM in gestating and lactating sows, IL6 in lactating sows and IL1β in piglets.     

Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen

Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12–18:2) substantially. The most common product was vaccenic acid (trans-11–18:1), produced by species related to Butyrivibrio fibrisolvens. α-Linolenic acid (LNA; cis-9,cis-12,cis-15–18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n − 3)) and docosahexaenoic acid (DHA; 22:6(n − 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 μg ml⁻¹, nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11–18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.     

Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo

Previous studies suggest that the n-3 polyunsaturated fatty acids (PUFAs) eicosapenteinoic acid (EPA) and docosahexaenoic acid (DHA), constituents of fish oil, exert chemopreventive activity in colon cancer. One of the mechanisms involved is the facilitation of apoptosis. While a pro-apoptotic potential of n-3 PUFAs has been suggested, it is still unclear whether additional consumption of fish will also lead to comparable results. The aim of this study was to assess EPA- and DHA-mediated effects on endpoints of apoptosis and to use a novel biomarker-approach to measure modulation of apoptosis by consumption of fish. LT97 human colon adenoma and HT29 human colon adenocarcinoma cells were used to investigate modulation of apoptosis by EPA, DHA or linoleic acid (LA) using a set of endpoints, namely phosphatidylserine staining with Annexin-V (flow cytometry), Bcl-2 expression (Real-time RT–PCR), and Bid, caspase 3, 8 and 9 expression as well as PARP cleavage (Western Blot). Furthermore, faecal water (FW) of volunteers (n = 89) from a human trial intervening with fish was used to investigate changes in apoptosis by flow cytometry. DHA was more effective at inducing apoptosis than EPA. LT97 cells were more prone to DHA and EPA induced apoptosis than HT29 cells. Treatment of LT97 cells with FW from volunteers consuming fish did not result in any changes in apoptosis. Taken together, our results show that adenoma cells are highly susceptible to n-3 PUFA-induced apoptosis. By using a biomarker-approach (FW) to measure apoptosis-induction ex vivo no change in apoptosis after additional fish consumption was detectable.