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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.

Summary 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-1β). 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-1β), 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-1β 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.

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 (P0.01), 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% (P0.001) from d 30 to 60, and DHA 19% (P0.01) 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% (P0.01) from d 30 to 60, and DHA increased 56% (P0.001) between d 30 and 90. Skeletal muscle LA was at least 17% less (P0.001) 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 (P0.001) in MARINE group compared with other treatments and increased (P0.001) by 71% from d 30 to 60. Skeletal muscle DHA was at least 57% greater (P0.001) in the MARINE group compared with other groups and increased (P0.001) 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.

Summary Six mature mares of Quarter Horse and Paint breeding (average body weight 560 kg) were used in a replicated, completely randomized experiment to evaluate the acceptance and digestibility of diets containing untreated or ammoniated wheat straw. Horses were fed one of three dietary treatments: (1) 50% chopped, ammoniated wheat straw/50% chopped alfalfa hay (AMWS), (2) 100% chopped alfalfa hay (ALF), or (3) 50% chopped wheat straw/50% chopped alfalfa hay (WS). Horses were maintained in dry lots during the two experimental periods with ad libitum access to water, and were fed diets in two equal feedings daily totaling 1.6% of body weight. Dietary adjustment periods were 14 days, followed by fecal collection periods of 4 days. Total fecal output was determined using chromic oxide as an external indicator. Feed and fecal samples were analyzed for dry matter, nitrogen, acid detergent fiber, neutral detergent fiber and energy content. Dietary intake (dry-matter basis) was similar between treatments with means of 8.7, 9.1 and 8.8 kg/d for AMWS, ALF, and WS, respectively. Energy and protein digestibilities were greater for ALF than WS. Dry matter digestibility was greater for both ALF and AMWS than WS. Digestibility of acid detergent fiber was not different among treatments, however a trend (P

What accounts for the wide spectrum of folate-responsive dysmorphogeneses? Both embryonic and fetal cells are entirely dependent on maternal folate to support their requirement for precisely timed proliferative bursts during gestation. Folate receptors (FRs) mediate transport into cells and are central to transplacental maternal-to-fetal folate transport. FRs are also critical for neural tube and neural crest development because recent murine "knock-out" and "knock-down" of FRs results in a high percentage of folate-responsive neural tube defects (NTDs) and neurocristopathies.Central to our hypothesis is the fact that folate deficiency is accompanied by a reduction in the proliferative capacity of highly mitotic neural tube or neural crest cells. Therefore, depending on when in pregnancy various cohorts of highly proliferative cells are deprived of folate, and the origin of the affected cells will determine the type of developmental dysmorphogenesis. Thus, selective folate deficiency in early pregnancy of only highly proliferative neural tube or neural crest cells predisposes to NTDs or gross dysmorphogenesis, respectively. Folate deficiency that compromises placental development will predispose to small-for-date babies due to an overall nutrient deficiency, and the development of folate insufficiency later in pregnancy could predispose to more subtle midline birth defects involving atresia of neural crest cell-derived structures. Finally, a congenital folate transport defect would only be corrected by suprapharmacological doses of folate, which ensures passive diffusion.This hypothesis can explain the results of several earlier and more recent clinical trials on folate supplementation in pregnancy, but it also raises the possibility that there may be several as yet undiscovered neurocristopathies that are folate responsive. Teratology 62:42-50, 2000. Published 2000 Wiley-Liss, Inc.

In the United States, the Food and Drug Administration (FDA) is the agency responsible for ensuring that the direct food additives and color additives used in food are safe for all consumers. In order to determine the safety of these additives for consumption, appropriate information and results from a series of tests must be made available to the agency. In 1982, in an effort to provide guidance to the food industry concerning the appropriate tests for the determination of safety, the FDA issued the Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Foods, commonly referred to as the Redbook. In 1993, based on the expansion of technology and the use of food additives, as well as the refinement of the scientific criteria for establishing safety, the FDA updated its guidelines and issued the draft Redbook II. Since Redbook II was issued, additional refinements have been made in the procedures for the multigeneration reproduction study and for the assessment of effects on male reproduction. The latest proposed guidelines for multigeneration studies are provided here.

The Food and Drug Administration (FDA) is the agency responsible for ensuring that the direct food additives and color additives used in food in the United States are safe for all consumers. In 1982, in an effort to provide guidance concerning appropriate tests, the FDA issued Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food, commonly known as the Redbook. The Redbook included detailed guidelines for testing the effects of direct and indirect food and color additives on mothers and their developing fetuses. Based on refinements in safety assessment and risk evaluation as well as expansion of knowledge concerning the metabolism and pharmacokinetics of food and color additives, the need to revise and update the 1982 document became apparent. In 1993, Redbook II in draft form was made available for public comment. Since then, test end points and developmental landmarks have been refined. The latest proposed guidelines for developmental toxicity studies are provided here.

Interest in folic acid has increased recently as a result of reports of a protective effect of dietary supplementation on the incidence of neural tube defects (NTD) in humans. This chapter will describe folic acid, assays for this vitamin, and characteristics of the deficient state. Biochemical reactions involving folic acid will be reviewed, as will studies which report a protective effect with respect to NTD. A number of compounds which adversely affect folic acid concentrations will be reviewed along with any evidence of a role for folic acid in the developmental toxicity of these compounds.

Carbamazepine (Tegretol, CBZ) is an anticonvulsant drug that is very effective in the treatment of tonic-clonic seizures and is gaining acceptance as a treatment for various psychiatric disorders. The drug is embryotoxic in rodents and has been reported to produce neural tube defects in approximated 1% of prenatally exposed human offspring. It is metabolized by the cytochrome P-450 system to a stable, pharmacologically active epoxide intermediate, carbamazepine-10, 11-epoxide. It is currently unknown whether the parent compound, the epoxide intermediate or some other metabolite is the embryotoxic agent. The present study was designed to determine the embryotoxicity of CBZ and its epoxide intermediate (CBZ-E) in a rodent whole embryo culture system. Rat embryos were cultured beginning on day 9 of gestation (GD 9), and mouse embryos were cultured beginning in GD 8. All embryos were cultured for 48 hr in medium containing various concentrations of either CBZ or CBZ-E. Mice were more sensitive to the effects of CBZ than were rats. The parent compound was embryotoxic to mouse embryos at concentrations as low as 12 micrograms, but it was only embryotoxic at 60 micrograms/ml to rat embryos. CBZ-E was not embryotoxic to either species at concentrations as high as 48 micrograms/ml. These results suggest that the parent compound is the embryotoxic agent and that the epoxide intermediate plays no role in the drug's embryotoxic mechanism.

s / Journal of Equine Veterinary Science 31 (2011) 230-356 270 Nutrition: Oral Presentations I Synovial Prostaglandin E2 Concentrations in Horses Fed Long Chain Polyunsaturated Fatty Acids: An Inquiry Study T. Ross, T.Hess,D.Hansen, T. Engle,N. Schauermann, and J. Rexford Colorado State University, Fort Collins, CO USA Introduction: Elevating n-3 fatty acids in mammalian diets may decrease inflammatory processes in the joint.1 One hypothesized mechanism is the potential of n-3 long chain polyunsaturated fatty acids (LCPUFA) to reduce the production of potent inflammatory eicosanoids, primarily prostaglandin E2 (PGE2). 2 Little to no information exists regarding oral n-3 supplementation and effects on inflammatory markers common in the equine joint. Therefore, mature horses were fed dietary n-3 LCPUFA for ninety (90) days to determine if oral supplementation alters synovial concentrations of PGE2. Materials and Methods: Twenty-onematuremixed breedmares with no history of joint disease and free of lameness were blocked by age, weight and body condition score and randomly assigned into one of three dietary treatment groups. Basal diet consisted of alfalfa/bromegrass mixed hay with the addition of rolled barley grain. Amount of barley varied with treatment groups to keep diets isocaloric and isonitrogenous. Group 1 (FISH) received 69 mg/kg BW of the n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) via a commercial fish oil supplement (Magnitude ; JBS United, Sheridan, IN) daily; Group 2 (FLAX) received 68.6 mg/kg BW of the n-3 polyunsaturated fatty acid alpha linolenic acid (ALA) via a flaxseed supplement (Nutra-Flax) daily and Group 3 (CONT) did not receive an oral n-3 supplement. Total diet composition of n-3 LCPUFA were approximately 143.5 mg/kg BW (FISH), 142.5 mg/kg BW (FLAX) and 78.7 mg/kg BW (CONT), respectively. On day 90, approximately 3 ml of synovial fluid was extracted from a random carpal joint of each horse and immediately placed into a 5 ml EDTA tube. Samples were processed and stored at -20 C until laboratory analysis was conducted. Prostaglandin E2 levels were determined using a two step process. In short, synovial fluid samples were first subjected to solid-phase extraction procedures using C2 ethyl minicolums(SampliQ ; Agilent Technologies, Wilmington, DE). Samples were then evaluated in triplicate using a commercially available PGE2 ELISA kit (Assay Designs; Ann Arbor, MI). Prostaglandin E2 levels were expressed in pg/ml. Data were analyzed using statistical software (SAS; Cary, NC) by use of one-way analysis of variance with significant differences (P < .05) being further evaluated using Fisher's LSD test. Results and Discussion: Synovial PGE2 levels at day 90 were within concentration ranges reported previously for values obtained from healthy equine joints.3 Though not significant, therewas a trend (P1⁄4 .075) for PGE2 concentrations to be lower in FISH group (21.261 +/4.01 pg/ml) compared to CONT group (32.2302 +/4.01 pg/ml). A ninety (90) day supplementation of the n-3 fatty acid ALA resulted in no difference in PGE2 concentrations (28.202 +/4.9 pg/ml) when compared to FISH (P1⁄4 .2932) or CONT (P 1⁄4 .5360) horses. In a companion inquiry, synovial fluid fatty acid levels for EPA and DHA were significantly higher in the FISH group compared to either CONT or FLAX groups.4 Though levels did not differ between treatments, the FISH group expressed the lowest levels of PGE2 in the current study; likely due to having higher concentrations of EPA and DHA in the joint. Prostaglandin E2 concentrations have been reported to be higher in joints of horses with existing osteoarthritis compared to levels in normal, healthy joints.3 Additionally, synovial fluid PGE2 levels have been positively correlated to degree of lameness and joint effusion in dogs,5 indicating usefulness as an indicator of joint inflammation and as a therapeutic target. Osteoarthritic horses receiving oral n-3 fatty acid supplementation for 90 days had lower plasma PGE2 and synovial white blood cell concentrations when compared to arthritic horses receiving no n-3 fatty acid supplementation,6 demonstrating a potential benefit of oral n-3 fatty acids for reducing severity of pain and inflammation. The lack of difference in synovial fluid PGE2 concentrations in the current study may be due to all horses being healthy and free of existing joint inflammation or disease. Conclusion: Data presented here indicate a possible inhibition of inflammatory eicosanoid prostaglandin E2 production by oral supplementation of the polyunsaturated fatty acids EPA and DHA. Further research is needed to determine if oral n-3 LCPUFA supplementation can be therapeutically advantageous in horses experiencing joint inflammation. Total and Water-soluble Phosphorus Excretion in Horses Consuming All-Forage Diets L.K. Warren, S. Dilling, J. Kivipelto, J. Bobel, and J. Eckert University of Florida, Gainesville, FL USA Introduction: Disposal of animal manure is under increasing scrutiny because of growing public concern over the long-term effects on phosphorus (P) accumulation in soils and its contribution to water quality degradation and eutrophication. When manure is applied to pastures or deposited by the grazing animal, soils will retain most of the P applied in excess of plant uptake. Despite the generally high adsorption capacity of most soils, P leaching can occur and P losses to runoff or surface erosion can be high. Potential P loss is not only related to the amount of P excreted in manure, but also how easily the manure P is dissolved in storm water and subject to runoff loss. Specifically, the chemical forms of P and their relative proportions inmanure influence runoff risk. Sharpley and Moyer [1] found a strong correlation betweenwater soluble P in livestock and poultry manures and the amount of P leached from soil following simulated rainfall events. Thus the soluble P fraction in manure is thought to pose the greatest environmental risk. Water soluble P has been shown to be highly correlated with daily P intake in livestock and poultry.[2] Although total P excretion has been positively correlated with P intake in horses,[3] the soluble P content of horse manure, as well as the effect of diet on fractional P excretion in horses is unknown. This study was undertaken to evaluate total and soluble P excretion in horses consuming an allforage diet. Materials and Methods: Six mature horses (mean SE, 530 14 kg) were fed 3 different hays in a 3 X 3 Latin square design. In

It is unknown whether orofacial clefting, one consequence of teratogenic exposure, results from a direct interaction between the teratogen and the embryonic palate, or indirectly from maternal alterations caused by the teratogen. In the current study pregnant A/J mice were exposed to one of three cleft-inducing agents in order to examine the relationship between drug-induced clefting and the response of maternal plasma corticosterone to drug administration. The agents used, haloperidol (HAL), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or phenytoin (PHT), were administered in teratogenic doses between 0800 and 0930 on gestational day 10 (GD 10). For corticosterone determinations, mice were dosed on GD 10, and blood was collected at 1, 4, 24, or 48 hr after dosing. For fetal evaluation of cleft lip and/or cleft palate, mice were dosed on GD 10 and killed on GD 18. Phenytoin was the most potent inducer of cleft lip and palate and induced a sustained elevation of plasma corticosterone in maternal animals. The other treatments, in order of decreasing potency to induce clefting and/or cause an elevation of corticosterone in plasma were 2,4,5-THALcontrols. Correlations between maternal corticosterone levels and clefting incidence were very high at all time points examined; total exposure (area under the curve) was also highly correlated. A linear relationship between drug-induced increases in maternal corticosterone levels and the incidence of clefting in A/J mice was evident. Based on these findings, we believe that increased maternal corticosterone levels may play a role in orofacial clefting in A/J mice.

Inbred strains of mice differ in their response to the embryopathic effects of phenytoin (PHT). A/J animals, the most susceptible strain, were mated to C57BL/6J mice, the most resistant strain. The susceptibility of the F1 hybrid offspring was determined by the susceptibility of the mother. B6AF1 animals were as resistant as C57BL/6J parental mice, and AB6F1 hybrids were as susceptible as A/J mice. This was especially evident when orofacial anomalies were tallied. (B6A)F2 hybrid offspring were as resistant as their C57BL/6J grandparents.

Exposure of pregnant rats to the anesthetic gas, nitrous oxide (N2O), has been reported to be teratogenic. This gas decreases activity of the enzyme 5-methyltetrahydrofolate homocysteine methyltransferase (methionine synthetase) and alters the level and distribution of folic acid derivatives in embryonic tissue. Because of their role in purine, thymidine and amino acid metabolism, alterations in the levels of various folate forms could affect macromolecular synthesis. The effect of N2O exposure on the number of somites, the content of DNA, RNA and protein as well as the incorporation of thymidine, deoxyuridine or serine into DNA were determined. Pregnant rats were exposed for 24 hr to 50% N2O-50% O2 beginning on day 10 of gestation. Control animals were exposed to 50% N2-50% O2. Embryos were removed and cultured for 4 hr using a rodent whole embryo culture system in medium containing radiolabeled precursors for DNA. Treatment with N2O had no effect on somite number, RNA or protein content. However, there was a significant decrease in DNA content. There was an increase in the incorporation of thymidine into DNA, but the incorporation of deoxyuridine and serine into DNA was decreased. These data demonstrate that treatment of pregnant rats with N2O results in decreased DNA synthesis and content in exposed embryos.

Acute administration of the anticonvulsant drug, phenytoin (PHT) has been shown to result in embryotoxicity and teratogenicity in several strains of mice. The A/J strain is reported to be most susceptible to the effects of the drug including an increased incidence of resorptions and orofacial clefts in treated animals. When administered chronically, the drug has been shown to be teratogenic in the absence of maternal toxicity and embryolethality in Swiss Webster mice [Hansen and Billings, 1985]. In this paper, we have compared the embryopathic effects of chronic and acute administrations of PHT to A/J mice. PHT was administered to pregnant females by intraperitoneal (i.p.) injection on day 10 of gestation at a dose of either 60 or 75 mg/kg body weight. Alternatively, PHT was added to ground chow and fed to animals prior to and throughout gestation; animals received a daily dose of either 60 or 75 mg/kg body weight. Pregnant animals were sacrificed on day 18 or 19 of gestation, and fetuses were examined for the presence of orofacial clefts and other anomalies. There was a significant increase in the frequency of cleft lip and palate in animals receiving the drug by i.p. administration, but there was no increase in the incidence of clefts if the drug were added to the diet. The results of this study reiterate the importance of the route of administration of a drug in determining its embryopathic effect.

Phenytoin (PHT) is teratogenic in A/J but not in C57BL/6J mice. Teratogenesis in F1 hybrid offspring of these two strains is dependent on the susceptibility of the maternal parent. Hepatic microsomes from untreated pregnant A/J females produced more of both the phenolic and diol metabolites than did microsomes from pregnant C57BL/6J females. This difference disappeared when the animals were pretreated with phenobarbital or phenytoin. It seems unlikely that the genetic difference in susceptibility to PHT-induced teratogenesis can be explained on the basis of maternal metabolism of the drug.

Exposure of pregnant rats to the anesthetic gas, nitrous oxide (N2O), has been reported to be teratogenic. This gas decreases activity of the enzyme 5-methyltetrahydrofolate homocysteine methyltransferase (methionine synthetase) and alters the level and distribution of folic acid derivatives in embryonic tissue. Because of their role in purine, thymidine and amino acid metabolism, alterations in the levels of various folate forms could affect macromolecular synthesis. The effect of N2O exposure on the number of somites, the content of DNA, RNA and protein as well as the incorporation of thymidine, deoxyuridine or serine into DNA were determined. Pregnant rats were exposed for 24 hr to 50% N2O-50% O2 beginning on day 10 of gestation. Control animals were exposed to 50% N2-50% O2. Embryos were removed and cultured for 4 hr using a rodent whole embryo culture system in medium containing radiolabeled precursors for DNA. Treatment with N2O had no effect on somite number, RNA or protein content. However, there was a significant decrease in DNA content. There was an increase in the incorporation of thymidine into DNA, but the incorporation of deoxyuridine and serine into DNA was decreased. These data demonstrate that treatment of pregnant rats with N2O results in decreased DNA synthesis and content in exposed embryos.