Influence of an n-3 long-chain polyunsaturated fatty acid-enriched diet on experimentally induced synovitis in horses.

Dietary n-3 long-chain polyunsaturated fatty acid (LCPUFA) supplementation has previously been shown to modify joint-related inflammation in several species, although information in the horse is lacking. We investigated whether dietary supplementation with n-3 LCPUFA would modify experimentally induced synovitis in horses. Twelve, skeletally mature, non-pregnant mares were randomly assigned to either a control diet (CONT) or an n-3 long-chain fatty acid-enriched treatment diet (N3FA) containing 40 g/day of n-3 LCPUFA for 91 days. Blood samples taken on days 0, 30, 60 and 90, and synovial fluid collected on days 0 and 90 were processed for lipid composition. On day 91, joint inflammation was stimulated using an intra-articular (IA) injection of 100 ng of recombinant equine IL-1beta (reIL-1ß). Synovial fluid samples taken at post-injection hours (PIH) 0, 4, 8 and 24 were analysed for prostaglandin E2 (PGE2 ), matrix metalloproteinase (MMP) activity and routine cytology. Synovium and articular cartilage samples collected at PIH 8 were analysed for gene expression of MMP 1 and MMP 13, interleukin-1beta (IL-1ß), cyclooxygenase 2 (COX-2), tumour necrosis factor-alpha and the aggrecanases, a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5. A 90-day feeding period of n-3 LCPUFA increased serum phospholipid and synovial fluid lipid compositions of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) compared to CONT horses. The reIL-1ß injection caused an inflammatory response; however, there was no effect of dietary treatment on synovial fluid PGE2 content and MMP activity. Synovial tissue collected from N3FA horses exhibited lower expression of ADAMTS-4 compared to CONT horses. Despite the presence of EPA and DHA in the synovial fluid of N3FA horses, dietary n-3 LCPUFA supplementation did not modify synovial fluid biomarkers compared to CONT horses; however, the lower ADAMTS-4 mRNA expression in N3FA synovium warrants further investigation of n-3 LCPUFA as a joint therapy.

Changes in gait associated with acute stage II posterior tibial tendon dysfunction.

The purpose of this study was to examine differences in gait mechanics between patients with acute stage II PTTD and healthy volunteers. Hindfoot and midfoot kinematics, plantar foot pressures and electromyographic (EMG) activity of the posterior tibialis, gastrocnemius, anterior tibialis and the peroneals were measured in five patients with acute stage II PTTD. Kinematics and kinetics were compared to a database of 20 healthy volunteers. EMG and plantar pressure data were obtained from five healthy volunteers. Hindfoot moments and powers were also calculated. The center of pressure excursion index (CPEI) was calculated from the plantar pressures. Significant differences were observed between the two groups, which confirmed clinical observations. Limited hindfoot eversion and increased midfoot external rotation occurred during the first and third rockers. The EMG data suggested that tendon dysfunction in the posterior tibialis is associated with compensatory activity, not only in its antagonists (the peroneals), but also in the anterior tibialis and the gastrocnemius. These data suggest that non-operative treatment of patients with PTTD should consider minimizing the activity of the posterior tibialis as well as the peroneals, the anterior tibialis and the gastrocnemius.

An overview of rodent toxicities: liver and kidney effects of fumonisins and Fusarium moniliforme.

Fumonisins are produced by Fusarium moniliforme F. verticillioides) and other Fusarium that grow on corn worldwide. They cause fatal toxicoses of horses and swine. Their effects in humans are unclear, but epidemiologic evidence suggests that consumption of fumonisin-contaminated corn contributes to human esophageal cancer in southern Africa and China. Much has been learned from rodent studies about fumonisin B1(FB1), the most common homologue. FB1 is poorly absorbed and rapidly eliminated in feces. Minor amounts are retained in liver and kidneys. Unlike other mycotoxins, fumonisins cause the same liver cancer promotion and subchronic (studies (3/4) 90 days) liver and kidney effects as (italic)F. moniliforme. FB 1 induces apoptosis of hepatocytes and of proximal tubule epithelial cells. More advanced lesions in both organs are characterized by simultaneous cell loss (apoptosis and necrosis) and proliferation (mitosis). Microscopic and other findings suggest that an imbalance between cell loss and replacement develops, a condition favorable for carcinogenesis. On the molecular level, fumonisins inhibit ceramide synthase, and disrupt sphingolipid metabolism and, theoretically, sphingolipid-mediated regulatory processes that influence apoptosis and mitosis. Liver sphingolipid effects and toxicity are correlated, and ceramide synthase inhibition occurs in liver and kidney at doses below their respective no-observed-effect levels. FB1 does not cross the placenta and is not teratogenic in vivoin rats, mice, or rabbits, but is embryotoxic at high, maternally toxic doses. These data have contributed to preliminary risk evaluation and to protocol development for carcinogenicity and chronic toxicity studies of FB1 in rats and mice.

Pharmacokinetic considerations of dexamethasone-induced developmental toxicity in rats.

Dexamethasone (DEX) has been shown to elicit growth stunting and cleft palate in rat fetuses. This investigation characterized DEX dosimetry as various pharmacokinetic parameters and evaluated their impact on developmental toxicity endpoints. DEX pharmacokinetics was evaluated as a single dose on either gestation day (GD) 9 or 14, as well as on GD 14 after multiple daily dosing from GD 9 to GD 14. An additional set of pregnant rats was dosed with DEX on GD 9 through GD 14, pharmacokinetic evaluation was conducted on GD 14 through GD 16, and teratological evaluation was conducted following sacrifice on GD 20. For all pharmacokinetic evaluations, a subcutaneous (sc) injection of 0.8 mg DEX/kg body weight together with 50 microCi 3H-DEX was administered to Sprague-Dawley rats. Blood, urine, and feces were collected for 24 or 48 h. At GD 20 sacrifice, maternal tissues as well as fetal brain and liver samples were collected as part of the laparotomy. All samples were assayed using scintillation spectrometry. DEX pharmacokinetic parameters remained similar whether dosing occurred early (GD 9) or late (GD 14) in organogenesis, or dosing occurred on multiple sequential days (GD 9-14). DEX produced maternal and fetal weight loss, fetal lethality, and cleft palate. DEX a-half-life was positively correlated with the percentage of implants affected [(number of non-live + number with cleft palate)/number of implants]/litter. Neither the area under the concentration-time curve (AUC), the maximum maternal plasma concentration (Cmax), nor the terminal phase beta-half-life correlated with any fetal outcome parameters. The correlation between the percentage of the litter that was affected and half-life was improved if AUC was added in a stepwise multiple regression. These data suggest that the length of time that DEX is present in the maternal plasma at a sufficiently high concentration (i.e., slower tissue distribution of DEX) appears to be important in determining the risk of an adverse outcome in the offspring.

Evaluation of the BBMEC model for screening the CNS permeability of drugs.

Combinatorial synthesis and high-throughput pharmacology screening have greatly increased compound throughput in modern drug-discovery programs. For CNS drugs, it is also important to determine permeability to the blood--brain barrier. Yet, given the increased pace of discovery, it difficult to conduct this screen in a timely fashion. In this presentation, we describe several improvements to an existing CNS permeability screen, the bovine brain microvessel endothelial cell (BBMEC) model. By implementation of these incremental process improvements, we have achieved a robust, facile screen for determination of CNS permeability of multiple compounds.

Effect of nitrous oxide exposure on maternal and embryonic S-adenosylmethionine levels and ornithine decarboxylase activity.

Nitrous oxide is suspected to be a developmental toxicant in humans. The anesthetic does produce increases in the resorption and malformation frequencies in rodents. The mechanism for the drug's developmental toxicant effects is unknown. Embryonic DNA synthesis is decreased; however, this decrease does not appear to be due to depressed levels of adenine or guanine. In this investigation, we examined the effect of N2O on maternal and embryonic S-adenosylmethionine (AdoMet) levels and ornithine decarboxylase (ODC) activity, and the effect of exogenous methionine (Met) on these parameters was also examined. AdoMet and ODC are involved in polyamine synthesis, and polyamines are involved in regulation of macromolecular synthesis. Pregnant rats were treated with N2O for 24 hours beginning on the morning of day 10 of gestation. There was no effect of N2O on maternal hepatic AdoMet or S-adenosylhomocysteine (AdoHcy) levels; there was also no effect on embryonic AdoMet. Embryonic AdoHcy could not be detected in many of the samples; however, N2O treatment did significantly increase the number of embryonic samples in which AdoHcy was detectable. ODC activity was not affected by either treatment in dams but was increased by N2O in embryos. It is possible that the embryotoxic effect of this anesthetic is mediated by alterations in the AdoMet to AdoHcy ratio or to changes in ODC activity and polyamine synthesis.

Retinoic acid-induced stress protein synthesis in the mouse.

We have previously demonstrated that stress proteins (SPs) are synthesized in tissues in which malformations are later observed following treatment with the developmental toxicant, retinoic acid (RA), on day 11 of gestation (GD 11). These proteins were not synthesized in tissues which did not present with malformations near partuition. The purpose of the present investigation was to determine if this correlation between early SP synthesis and later malformation was present at other times during gestation. CD-1 strain mice were dosed orally with corn oil or 100 mg/kg body weight RA on GD 10 or 13. Some of the mice in each group were given an intraperitoneal injection of 3H-leucine to label embryonic protein synthesis one hour after dosing with RA. These animals were sacrificed 1.5 hour later, and embryonic protein synthesis was determined by two-dimensional gel electrophoresis followed by autoradiography. Other animals in each group were sacrificed on day 17 of gestation, and fetuses were examined for the presence of malformations. Following treatment with RA on day 10 of gestation, malformations were observed in the forelimbs, the hindlimbs and the tail; heart defects were not observed. SPs of 20-25,000 and 90,000 relative molecular mass (Mr) were synthesized in the forelimb bud and tail; in addition, a second low molecular weight (20-25,000) and a 84,000 Mr SPs were synthesized in forelimb buds. No SPs were synthesized in the hindlimb bud or the heart. Following RA treatment on GD 13, cleft palate was observed in 58% of fetuses; no other malformations were found. Proteins of 34,000, 84,000 and 90,000 Mr were synthesized in craniofacial tissue; SPs were not observed in forelimb bud, hindlimb bud, heart or tail tissues at this time. Therefore, it appears that there may be a correlation between tissue-specific SP synthesis early in organogenesis and the presence of a malformation later in gestation.

Determination of kynurenic acid by capillary electrophoresis with laser-induced fluorescence detection.

Kynurenic acid (KA) is an excitatory amino acid receptor antagonist that is believed to play an important role in a host of diseases of the neuropsychiatric and central nervous system. A method for the determination of KA in microdialysate samples using capillary electrophoresis (CE) separation with laser-induced fluorescence (LIF) detection is described. CE is advantageous for the analysis of microdialysis samples due to its short analysis times and small sample volume requirements. Three complexation approaches were evaluated in an attempt to achieve the best limit of detection. The best approach was found to be pre-column complexation with inclusion of Zn(II) in the background electrolyte. After optimization of the zinc acetate concentration and pH, a limit of detection of 1 nM KA was achieved. However, when KA was present in the dialysate, the limit of detection increased 50-fold. Even though the endogenous levels of KA in rat brain are below this limit of detection, this methodology could be used to monitor the increase of KA levels in rat brain following dosing with its precursors, tryptophan and kynurenine.

Lack of embryotoxicity of homocysteine thiolactone in mouse embryos in vitro.

Recent work from humans and chick embryos has suggested that homocysteine may play a role in producing neural tube defects (NTDs). In an effort to determine if homocysteine is able to produce NTDs in mammalian embryos, mouse embryos were explanted on GD 8 and cultured for 44 h. When either homocysteine or homocysteine thiolactone was added to the culture medium, treated embryos developed as well as controls and had closed neural tubes. Homocysteine thiolactone was also microinjected into the amniotic sac of mouse embryos. Again, development proceeded normally with no significant increase in the number of embryos with open neural tubes at the end of the culture period. HPLC analysis of embryonic thiols 24 h after microinjection revealed a significant increase in embryonic cystathionine levels. These data suggest that homocysteine does not produce NTDs in mouse embryos cultured in vitro and that early organogenesis-stage embryos are able to metabolize homocysteine.

Antisense modulation of 5,10-methylenetetrahydrofolate reductase expression produces neural tube defects in mouse embryos.

The role of folate metabolism in producing neural tube defects (NTDs) in humans is unknown. In the current study, antisense oligodeoxyribonucleotide technology was utilized to disrupt normal expression of the gene for 5,10-methylenetetrahydrofolate reductase (MTHFR) in organogenesis-stage mouse embryos. Two different antisense probes were microinjected into the amniotic sac of gestation day (GD) 8 mouse embryos with PBS or scrambled sense oligodeoxyribonucleotides injected into control embryos. Concentration-dependent increases in the frequencies of embryos with NTDs were observed for both antisense sequences. The level of mRNA for MTHFR was decreased in embryos treated with the higher concentration of one antisense sequence, indicating that the sequence is able to decrease gene expression. 5-methyltetrahydrofolate, the product of the MTHFR reaction, was able to decrease the incidence of antisense-induced NTDs, but co-injection with L-methionine did not. These results suggest that reduced expression of MTHFR may play a role in producing NTDs.

Dose-response of retinoic acid induced stress protein synthesis and teratogenesis in mice.

Stress proteins are synthesized in response to a variety of stressors, including several teratogenic agents. However, their role, if any, in the teratogenic process is unknown. We have previously demonstrated that all-trans-retinoic acid administered to pregnant CD-1 mice on gestational day 11 or 13 produced limb defects and cleft palate near term in a dose-responsive manner. This chemical also induced the synthesis of several nuclear stress proteins in embryonic tissues within several hours of dosing. The stress proteins were only observed in tissues that eventually became malformed and not in tissues that appeared normal at term. In the current work, we examined the stress response in embryonic target tissues after several different doses of retinoic acid. The nuclear stress proteins were synthesized in a dose-related manner and at a lower retinoic acid dose than doses producing malformations in the corresponding tissue at birth. Each individual stress protein and the total stress protein response were highly correlated, across dose, with the respective malformations observed at term.

Behavioral teratology and dominant lethal evaluation of nitrous oxide exposure in rats.

Epidemiological studies have suggested that spontaneous abortion may be increased in medical personnel following the sort of chronic low-level exposure to the anesthetic gas nitrous oxide (N2O) seen in surgical or dental operatories. These results are supported by some, but not all, animal studies, and results are less well established at low exposure levels. Behavioral effects in exposed animal offspring have also been observed, but again not in all studies. To further examine this problem, we conducted the present experiments. Adult male or female rats were exposed to trace concentrations of N2O (0%, 0.1%, 0.5%, or 1.0% in air) for 6 h daily either throughout gestation (females) or for 9 weeks (males). Offspring from treated adults were subjected to an extensive behavioral test battery. There were no clear dose-response effects on any of eight behavioural tests for any offspring. Maternal and offspring weights were normal from conception through adulthood. Additionally, we studied effects of N2O on male fertility by mating treated males with untreated females and examining uterine contents. There was no evidence for a substantial decline in fertility of exposed males, although there was a small dose-related trend for resorptions to increase and live births to decrease with increasing paternal N2O exposure. There results suggest that there is little alteration in male or female fertility following chronic exposure to low levels of N2O. There are also no significant long-term behavioral alterations in offspring exposed gestationally to trace levels of N2O via dam or sire.

In vitro effects of folate derivatives on valproate-induced neural tube defects in mouse and rat embryos.

The anticonvulsant drug valproic acid (VPA), produces neural tube defects in mouse and rat embryos treated in vivo or in vitro. The mechanism for the drug's embryotoxic effect is unknown, but 5-formyltetrahydrofolate has been reported to decrease the incidence of VPA-induced neural tube defects in mice treated in vivo. In the present study we have examined the ability of 5-formyltetrahydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate and folic acid to protect against VPA-induced neural tube defects in CD-1 mouse or CD rat embryos grown in a whole embryo culture system. Mouse embryos with 2-5 somite pairs were cultured for 48 hr beginning on gestation day 8; presomite stage rat embryos were cultured beginning on gestation day 9 (for both species gestation day 0 was taken as the day a vaginal sperm plug was found). VPA at 1.2 mm (rats) or 1.8 mm (mice) produced a high incidence of open neural tubes. None of the folate derivatives in concentrations up to 100 mug/ml was able to decrease the incidence of VPA-induced defects in either species. These data suggest that folate is not involved in the mechanism of VPA-induced neural tube defects.

Lack of attenuation of valproic acid-induced embryotoxicity by compounds involved in one-carbon transfer reactions.

Previous reports have indicated that valproic acid (VPA) is a developmental toxicant in vitro as well as in vivo, producing primarily neural tube defects. The mechanism for the drug's embryotoxic effects is unknown; however, work from our laboratory over the last few years has indicated that addition of various folate derivatives did not significantly decrease the incidence of VPA-induced neural tube defects. It is possible that some compound involved in one-carbon transfer reactions other than folate could protect against VPA embryotoxicity. We have examined the ability of l- or d-serine, sodium formate and l-methionine to decrease VPA-induced embryotoxicity. CD strain rat embryos were cultured for 48 hr beginning on day 9 of gestation. Each compound was examined for embryotoxicity, and a non-embryotoxic concentration was added to the culture medium 3 hr before the addition of 150 mug VPA/ml. Neither l-serine (300 mug/ml), d-serine (300 mug/ml), sodium formate (600 mug/ml) or l-methionine (1.12 mg/ml) were able to decrease VPA-induced developmental toxicity in vitro. The morphological scores of the embryos were not increased, nor was the frequency of embryos with open neural tubes decreased by treatment with any of these compounds. These data suggest that the mechanism for VPA-induced embryotoxicity does not involve compounds involved in one-carbon transfer reactions.

Partial attenuation of hydroxyurea-induced embryotoxicity by deoxyribonucleotides in mouse and rat embryos treated in vitro.

Hydroxyurea (HU) is a well known developmental toxicant in all animal species tested. It inhibits DNA synthesis, and addition of deoxycytidine monophosphate (dCMP) has been shown previously to attenuate the developmental toxicant effects of HU in vivo. The purpose of the present investigation was to determine whether addition of deoxyadenosine monophosphate (dAMP) or dCMP would attenuate HU-induced embryotoxicity using a rodent whole embryo culture system. Rat embryos were removed on the morning of day 10 of gestation, and mouse embryos were removed on day 8 of gestation (day 0 = the day a vaginal plug was found). Embryos were treated with various concentrations of HU (up to 500 mug/ml) for 1 hr at 37 degrees C before being washed and cultured for 43 hr in rat serum containing dAMP or dCMP. At the end of the culture period, six endpoints were evaluated for each viable embryo: morphological score; number of somite pairs; crown-rump and head lengths, DNA and protein contents. HU (300 mug/ml) significantly decreased values for all endpoints in embryos from both mice and rats; however, mouse embryos appeared to be more sensitive to the effects of the drug. dAMP and dCMP alone produced some embryotoxicity at high concentrations. The combination of HU + dAMP had no consistent beneficial effect in rat embryos and no effect on mouse embryos. The combination of HU + dCMP improved growth and development slightly. As determined by HPLC analysis, HU treatment (300 mug/ml for 1 hr) decreased all nucleotide pools. Subsequent treatment with dAMP increased all pool levels, although these levels remained below those of control embryos. These results suggest that the developmental toxicant effects of HU are not due solely to alterations in deoxyribonucleotide pool levels.

Effects of fumonisin B1 in pregnant rats.

Fumonisin B1 (FB1), the major mycotoxin from Fusarium moniliforme, has been implicated as a causative agent in several animal and human diseases. Despite animal toxicity studies and human epidemiological studies of FB1, knowledge of its reproductive effects is scarce. In this study, one of a series of proposed studies that will allow extrapolation to humans, pregnant rats were given oral doses of 0, 1.875, 3.75, 7.5 or 15 mg FB1/kg on gestation days 3 16. Caesarean sections were performed on day 17 or 20, and maternal condition, implantation efficiency, foetal viability and foetal development were measured. Dose-related decreases in overall feed consumption and body weight gain were seen, but only the feed consumption decrease at 15 mg/kg, and the decreased body weight gain at 15 mg/kg on days 0-17 were statistically significant. Foetal body weights at day 17 were similar in control and treated groups; but in day-20 foetuses, female weight and crown-rump length were significantly decreased at 15 mg/kg. FB1 was not teratogenic at the doses tested, and no dose-related effects were seen in either skeletal or soft-tissue development. In day-17 animals, maternal and foetal brain, liver and kidney tissues, and maternal serum were preserved to study the levels of sphinganine (Sa), sphingosine (So), and the Sa/So ratios. Dose-related increases were seen in Sa/So ratios in maternal livers, kidneys and serum. Sa/So ratios of maternal brains were not affected, nor were those of foetal kidneys, livers or brains.

Effects of fumonisin B1 in pregnant rats. Part 2.

The developmental toxicity of purified fumonisin B1 (FB1), a mycotoxin from the common corn fungus Fusarium moniliforme, was examined in Charles River rats. Pregnant rats were dosed orally on gestation days 3-16 at 0, 6.25, 12.5, 25 or 50 mg FB1/kg body weight/day. FB1 was not teratogenic at the doses tested. At 50 mg/kg, maternal toxicity (inappetence, emaciation, lethargy, death, resorption of entire litters) and foetal toxicity (increased number of late deaths, decreased foetal body weight, decreased crown rump length, increased incidence of hydrocephalus, increased incidence of skeletal anomalies) were seen. The foetal toxicity observed at 50 mg/kg may be related to maternal toxicity. Histopathological evaluation of tissues from dams of control and all treated groups revealed dose-related toxic changes in kidney and liver tissues. Acute toxic tubular nephrosis was seen in kidneys from all treated groups. Hepatocellular cytoplasmic alteration and individual cellular necrosis of the liver was seen in the two high-dose groups. Sphinganine (Sa) and sphingosine (So) were measured in day-17 adult and foetal tissues. Dose related increases in Sa/So ratios were seen in maternal liver, kidney, serum and brain, but there was no effect on foetal liver, kidney and brain. These data suggest that FB1 does not cross the placenta and further suggest that the observed foetal toxicity is a secondary response to maternal toxicity.

Comparison of in vitro BBMEC permeability and in vivo CNS uptake by microdialysis sampling.

The studies presented in this report were designed to assess the correlation of the bovine brain microvessel endothelial cell (BBMEC) apparent permeability coefficient (P(app)) and in vivo BBB penetration using microdialysis sampling. A mathematical model was developed to describe the relationship of brain extracellular fluid (ECF) concentration to free drug in plasma. The compounds studied have a broad range of physico-chemical characteristics and have widely varying in vitro and in vivo permeability across the blood-brain barrier (BBB). BBMEC permeability coefficients vary in magnitude from a low of 0.9 x 10(-5) cm/s to a high value of 7.5 x 10(-5) cm/s. Corresponding in vivo measurements of BBB permeability are represented by clearance (CL(in)) into the brain ECF and range from a low of 0.023 microl/min/g to a high of 12.9 microl/min/g. While it is apparent that in vitro data from the BBMEC model can be predictive of the in vivo permeability of a compound across the BBB, there are numerous factors both prior to and following entry into the brain which impact the ultimate uptake of a compound. Even in the presence of high BBB permeability, factors such as high plasma protein binding, active efflux across the BBB, and metabolism within the CNS can greatly limit the ultimate concentrations achieved. In addition, concentrations in the intracellular space may not be the same as concentrations in the extracellular space. While these data show that the BBMEC permeability is predictive of the in vivo BBB permeability, the complexity of the living system makes prediction of brain concentrations difficult, based solely on the in vitro measurement.

Effects of two different dietary sources of long chain omega-3, highly unsaturated fatty acids on incorporation into the plasma, red blood cell, and skeletal muscle in horses.

The objective of this study was to examine the effects of different sources of dietary omega-3 (n-3) fatty acid supplementation on plasma, red blood cell, and skeletal muscle fatty acid compositions in horses. Twenty-one mares were blocked by age, BW, and BCS and assigned to 1 of 3 dietary treatments with 7 mares per treatment. Dietary treatments were: 1) control or no fatty acid supplement (CON), 2) 38 g of n-3 long chain, highly unsaturated fatty acid (LCHUFA) supplement/d provided by algae and fish oil (MARINE) containing alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA), and 3) 38 g of n-3 LCHUFA supplement/d provided by a flaxseed meal (FLAX) containing ALA. Each supplement was added to a basal diet consisting of hay and barley and was fed for 90 d. Blood samples and muscle middle gluteal biopsies were taken at d 0, 30, 60 and 90 of supplementation. Plasma, red blood cell and skeletal muscle fatty acid profiles were determined via gas chromatography. Plasma linoleic acid (LA) and ALA were at least 10 and 60% less (P < 0.01), respectively, in the MARINE compared with the FLAX and CON groups. Plasma EPA and DHA were only detected in the MARINE group, and EPA increased 40% (P < 0.001) from d 30 to 60, and DHA 19% (P < 0.01) from d 30 to 90. Red blood cell LA and ALA were not different among treatments. Red blood cell EPA and DHA were only detected in the MARINE group, where EPA increased 38% (P < 0.01) from d 30 to 60, and DHA increased 56% (P < 0.001) between d 30 and 90. Skeletal muscle LA was at least 17% less (P < 0.001) in the MARINE group compared with the other treatments. Skeletal muscle ALA was 15% less (P = 0.03) in the MARINE group compared with FLAX and CON groups. Skeletal muscle EPA was at least 25% greater (P < 0.001) in MARINE group compared with other treatments and increased (P < 0.001) by 71% from d 30 to 60. Skeletal muscle DHA was at least 57% greater (P < 0.001) in the MARINE group compared with other groups and increased (P < 0.001) by 40% between d 30 and 90. As far as the authors are aware, this is the first study to demonstrate that dietary fatty acid supplementation will affect muscle fatty acid composition in horses. Incorporation of n-3 LCHUFA into blood and muscle depends directly on dietary supply of specific fatty acids.