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Journal of Animal Science - Article

 

 

This article in JAS

  1. Vol. 90 No. 9, p. 3023-3031
     
    Received: June 21, 2011
    Published: January 20, 2015


    2 Corresponding author(s): tanja.hess@colostate.edu
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doi:10.2527/jas.2011-4412

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 horses1

  1. T. M. Hess 2,
  2. J. K. Rexford*,
  3. D. K. Hansen*,
  4. M. Harris,
  5. N. Schauermann*,
  6. T. Ross*,
  7. T. E. Engle*,
  8. K. G. D. Allen and
  9. C. M. Mulligan
  1. Equine Science, Department of Animal Science
    Department of Food Science and Human Nutrition, Colorado State University, Fort Collins 80523

Abstract

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.



INTRODUCTION

Supplementation with omega-3 (n-3) fatty acids has been shown to lead to several benefits in different species. In humans, supplementation with n-3 long chain, highly unsaturated fatty acids (LCHUFA) has been shown to improve inflammatory status and prevent cardiovascular diseases (Calder, 2001), and has been shown to reduce pain and inflammation in human patients with rheumatoid arthritis (MacLean et al., 2004). In arthritic horses, it increased stride length (Woodward et al., 2005) and reduced inflammatory markers (Manhart et al., 2009). In order to ensure incorporation of docosahexaenoic acid (DHA) and eicopentaenoic acid (EPA) to human tissues, these n-3 LCHUFA should be supplied in the diet. There is limited conversion of alpha-linolenic acid (ALA) to DHA in humans (Burdge et al., 2002; Arterburn et al., 2006). Horse studies indicated that supplementation with ALA would not lead to increases in circulating DHA (Hansen et al., 2002; Vineyard et al., 2010), only EPA. Incorporation of n-3 LCHUFA to circulating and muscle tissues could potentially improve chronic inflammatory conditions in horses; however, the optimal type of fatty acid to be supplemented has to be investigated. Horses are herbivores, and adding a marine (fish oil and algae) source of n-3 LCHUFA can lead to palatability problems and refusals. It would be important to know if supplementation of a plant source of n-3 LCHUFA would lead to conversion to n-3 very LCHUFA, and if adding extra amounts of ALA to the corresponding fatty acid existing in forage diets through flaxseed would lead to greater incorporation of n-3 LCHUFA into body tissues.

This study was conducted to investigate the effect of dietary supplementation of marine and plant sources of n-3 LCHUFA on plasma, red blood cell, and equine skeletal muscle fatty acid composition. It was hypothesized that the horses supplemented with the algae and fish source would have greater incorporation of EPA and DHA into the plasma, red blood cell and skeletal muscle over the horses supplemented with flaxseed or the control group.


MATERIALS AND METHODS

Horses were maintained according to the policy of the Equine Sciences program at Colorado State University in Fort Collins, CO, and followed the protocol approved by the Institutional Animal Care and Use Committee.

Horses

Twenty-one mares of mixed stock horse breeding were acclimated to a basal diet of free-choice alfalfa grass hay for 1 mo before dietary intervention. Horses had been dewormed and vaccinated before the start of the study. An initial assessment of BCS was made by 3 trained, unbiased and independent scorers, and averages recorded. Body condition score was based on a scale of 1 to 9 (Henneke et al., 1983). Body weight was measured on an electronic scale. The mares, ranging from 5 to 14 yr of age with an average age of 9, weighed from 525 to 673 kg with an average of 586 kg, and had a BCS ranging from 5 to 8 with an average of 6.7. Body weight and BCS of horses were assessed on a monthly basis during the study. Mares were blocked by age, BW and BCS and were randomly assigned to 1 of 3 dietary treatment groups (7 per group), which were a control group, a group supplemented with a commercial pellet containing algae and fish oil (Magnitude, JBS United, Sheridan, IN), and a group supplemented with a commercial ground flaxseed product (Nutra-Flax, HorseTech, Laurens, IA). Body weight (True-test Inc., Mineral Wells, TX) and BCS were evaluated on a monthly basis 2 to 6 d before sample collection at d 0, 30, 60, and 90. Age, BW and BCS means and ranges by treatment are given in Table 1.


View Full Table | Close Full ViewTable 1.

Treatment group means for age, BCS and BW at baseline1

 
Treatment
Item CON MARINE FLAX SEM
Age, yr 9 9 9 2
BCS, 1 to 9 6.5 6.8 6.9 0.7
BW, kg 588.0 595.5 573.7 42.4
1CON = control diet; MARINE = algae and fish oil supplement added to basal diet; FLAX = flaxseed meal added to basal diet; n = 7 per group.

Diets

Diets included alfalfa-bromegrass mixed hay, rolled barley and a fatty acid supplement that varied with treatment. Diets were formulated to be isocaloric and isonitrogenous and to have correct fatty acid ratios by varying the inclusion of rolled barley and hay (Table 2). The horses underwent a 2-wk adaptation period to the hay fed during the study before baseline samples were taken. Horses were maintained in dry lots within the assigned groups (7 per dry lot) and fed hay once a day. Hay was group-fed to supply 1.6 to 1.7% of the BW of the horses on a DMI per day. Individual hay consumption was not controlled. Horses were fed hay in a bunk feeder, and no hay was left over at the end of the day. Supplements and barley were fed individually in nosebags, and any leftover feed was weighed. All feeds and supplements were analyzed for fatty acid composition using a modified Folch lipid extraction procedure (Folch et al., 1957). In addition, diets were analyzed for nutritional composition (Equi-Analytical Laboratories, Ithaca, NY; Table 2). Treatments and diets formulated (Table 3) were calculated to supply similar fatty acid dosage that has shown to lead to incorporation of EPA and DHA in blood in a previous horse study (King et al., 2008). The first group (MARINE, n = 7) was supplemented daily with an average of 38 g of n-3 LCHUFA provided by a supplement containing algae and fish oil protected from oxidation by vitamin E (Magnitude; JBS United, Sheridan, IN). The 38 g of n-3 LCHUFA consisted of 2 g of ALA, 7.6 g of EPA, 26.6 g of DHA and 1.7 g of docosapentaenoic acid (DPA) supplemented to a basal diet consisting of hay and barley. Barley was fed at 1.1 g/kg BW per day. Although individual hay consumption was not measured, it averaged 1.6% of the BW of the horses on a DM basis, and it was assumed to vary between 1.5 and 2% the BW of the horses on a DMI basis for fatty acid calculation. The n-6:n-3 ratio for the entire diet in the MARINE group was 0.43:1, taking into account the possible variation in individual hay intake. The second treatment group (FLAX, n = 7) consisted of an average of 38 g of n-3 LCHUFA per day provided as ALA by a supplement containing flaxseed meal (Nutra-Flax), which was protected from oxidation by polyphenols in the processed flaxseed (Pizzey, 2002). Barley was fed at 0.51 g/kg BW, and hay was fed at an average of 1.6 to 1.7% of BW. Again, hay intake was assumed to vary between 1.5 and 2% the BW of the horses on a DMI basis for fatty acid calculation. The n-6:n-3 ratio for the entire diet in the FLAX group was 0.44:1, taking into account the possible variation in individual hay intake. The third treatment group (CON, n = 7) did not receive additional n-3 fatty acid supplementation, aside from the n-3 fatty acid ALA that was present in the hay and barley. The third group received an average of 1.6% of BW in hay and 1.8g/kg of BW in supplemental barley daily. The n-6:n-3 ratio for the entire diet in the CON group was 0.79:1, taking into account the possible individual hay intake differences. The CON diet contained a similar amount of n-6 fatty acids as the MARINE and FLAX groups, but approximately one-half the amount of n-3 fatty acids. Fatty acid intake estimates for the 3 treatment groups is shown in Table 3. Supplements were fed for 90 d. Refusals in the nosebags were weighed on a daily basis to estimate a percentage of refusal throughout the study.


View Full Table | Close Full ViewTable 2.

Nutritional analysis and fatty acid content of feedstuffs1

 
Feedstuff
Item Hay Barley Algae and fish oil2 Flaxseed meal3
DM, % 91.20 89.98 95.30 93.20
DE, Mcal/kg 2.14 3.65 3.40 4.90
CP, % 20.60 12.65 12.30 21.80
ADF, % 33.40 7.65 9.00 17.10
NDF, % 42.80 19.27 14.90 25.20
WSC, % 7.70 7.05 5.40 3.80
Simple sugars, % 5.20 2.14 3.80 1.30
Starch, % 4.20 54.62 39.20 4.60
NFC, % 25.40 64.42 45.70 23.50
Fat, % 1.40 2.59 22.50 39.00
Fatty acid, % of fat
    LA 16.98 53.60 5.74 15.69
    ALA 35.92 4.25 2.18 56.52
    ARA - - - -
    EPA - - 7.94 -
    DPA - - 1.72 -
    DHA - - 27.64 -
1WSC = water-soluble carbohydrates; NFC = non-fibrous carbohydrates; LA = linoleic acid; ALA = alpha-linolenic acid; ARA = arachidonic acid; EPA = eicosapentaenoic acid; DPA = docosapentaenoic acid; DHA = docosahexaenoic acid.
2Magnitude (JBS United, Sheridan, IN).
3Nutra-Flax (HorseTech, Scottsdale, AZ).

View Full Table | Close Full ViewTable 3.

Daily estimated average dietary intake for treatment groups on a DM basis1,2

 
Treatments
Item CON MARINE FLAX
DE, Mcal 24.65 24.71 24.64
CP, kg 1.935 1.937 2.075
Starch, kg 0.941 0.891 0.579
Fat, kg 0.256 0.403 0.360
Fatty acids, mg/kg BW
    LA 61.91 61.84 66.08
    ALA 78.15 80.97 149.78
    ARA - - -
    EPA - 13.06 -
    DHA - 45.51 -
1CON = basal control diet; MARINE = algae and fish oil supplement added to basal diet; FLAX = flaxseed meal added to basal diet.
2LA = linoleic acid; ALA = alpha-linolenic acid; ARA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.

Blood and Muscle Collection

Blood samples and muscle middle gluteal biopsies were taken at d 0, 30, 60 and 90 of supplementation 15 h after the last supplementation of FLAX, MARINE and barley for CON. Blood samples (7 mL) were obtained via jugular venipuncture and collected into EDTA evacuated tubes, centrifuged at 2,700 × g for 7 min at 4°C. Plasma was separated from red blood cells by pipetting, and both were immediately transferred to polypropylene tubes, flash-frozen in liquid nitrogen and stored at −80°C until analysis. In preparation for the muscle biopsies, the horses were restrained in stocks, hair was clipped in a 3-cm2 area over the top of the rump in a line between the tuber coxae and tuber ischii, and the area was cleaned with iodine scrub and alcohol. One to 3 mL of lidocaine were administered subcutaneously and a 5- to 10-mm incision was made into the skin. A Bergstrom muscle biopsy needle (Popper and Sons, New York, NY) was used to sample muscle at a depth of 6 cm from the middle gluteal muscle. Approximately 2 to 3 g of wet muscle tissue per horse was extracted, aliquoted and transferred into polypropylene tubes, then flash-frozen in liquid nitrogen and stored at −80°C until analysis.

Fatty Acid Composition

Fatty acid composition of plasma, red blood cells, skeletal muscle and feedstuff were analyzed in duplicate using a modified Folch extraction procedure (Folch et al., 1957). Fatty acid methyl esters were prepared using 14% boron trifluoride (BF3) in methanol (Supelco, Bellefonte, PA). By heating 200 mg lipid with BF3 at 100°C for 30 min, Hexane extracts of fatty acid methyl esters were separated by gas chromatography (Model 6890 GC with autosampler, Agilent, Palo Alto, CA) on a 30 m × 0.25 mm × 0.2 μm film (DBV-225 column, Agilent). This is a standard method for the determination of fatty acid composition in biological samples, and details have been previously published (Reece et al., 1997). Sensitive detection using a flame ionization detector (capable of detecting analyte in the femtomolar range) was used for detection and quantification of fatty acids. Peaks were identified by retention time (RT) compared with known standards using software (Chemstation integration for the GC 6890; Agilent). Accuracy of peak identification by RT was insured using a constant-flow, controlled-temperature ramping program quantitated against known concentrations of standards run at the beginning, middle and end of the run. The program has been previously optimized to prevent co-elution of biologically relevant fatty acids. Inter- and intra-assay CV for feedstuff, plasma, red blood cell and muscle fatty acid composition were 8.1 and 5.2%, respectively.

Statistical Analysis

The effect of fatty acid supplementation (MARINE, FLAX and CON) on plasma, red blood cell and muscle cell fatty acid compositions was analyzed using the PROC MIXED procedure (SAS Inst. Inc., Cary, NC). The between-animal effect was treatment (supplementation), the repeated measures effect was sampling day, and the subject within treatment effect was random. The covariance structure type was autoregressive, which provided the best fit for these analyses according to the Akaike information criterion. Data at sampling d 0 was included as a covariate to adjust for any baseline differences when applicable. For all analyses, a P < 0.05 was accepted as statistically significant. When effects in the analysis of variance were significant, post-hoc t-tests were used for selected comparisons between least square means. A subset of all possible comparisons was made for the interaction means; either treatments were compared at a given time or times compared for a given treatment. Results are reported as mean ± SEM. Residuals from the models were plotted to verify the assumptions of independence and normality.


RESULTS

Horse BW and BCS

Body weights and BCS were evaluated on a monthly basis and are presented in Table 4. Because no differences were observed among treatments, results were pooled and averaged across all treatments. Horses weighed 585.0 ± 41.5 kg at d 0, BW decreased (P < 0.001) progressively from d 0 to 60 (565.0 ± 35.6 kg) and then increased (P < 0.01) from d 60 to 90 (576.1 ± 36.8 kg). Body condition scores ranged from 5 to 8 and decreased (P = 0.02) from d 0 (6.74 ± 0.85) to 90 (6.38 ± 0.82). Horses remained healthy throughout the study with no adverse effects observed because of the different treatments.


View Full Table | Close Full ViewTable 4.

Body condition score and BW across all treatments

 
d BCS (1 to 9) BW, kg
0 6.74 ± 0.85a 585.7 ± 41.5a
30 6.74 ± 0.75a 578.5 ± 36.4b
60 6.57 ± 0.78a 565.0 ± 35.6c
90 6.38 ± 0.82b 576.1 ± 36.8b
a-cValues within a column lacking a common superscript differ, P ≤ 0.05.

Diets

Refusals were not common among horses, except for the MARINE group. Three MARINE horses refused 4% each of the barley and MARINE pellet mixture offered in their feedbags over the 4 mo of the study. The other 4 MARINE horses refused less than 2% each. One FLAX horse refused on multiple occasions, totaling 2% of the flaxseed and barley mixture over the course of the study.

Fatty Acid Composition

Plasma fatty acid composition is shown in Table 5. Linoleic acid (LA; C18:2 n-6) was less in the MARINE horses compared with the FLAX (P < 0.001) and CON (P = 0.05) horses. Overall, LA decreased with time (P < 0.001) from d 0 to 90 of supplementation (data not shown). Linoleic acid decreased (P < 0.001) from d 0 to 60 but did not change to d 90. Additionally, a treatment-by-time interaction was observed (P < 0.001) in which LA was less at d 30, 60 and 90 in the MARINE group compared with the FLAX group. At d 30 and 60, the MARINE group had decreased LA plasma concentrations when compared with the CON group (P ≤ 0.01). At d 90, the MARINE and CON were not different, and both were less (P < 0.05) than the FLAX. Plasma ALA (C18:3 n-3) was different among treatments (P < 0.001). The least ALA values were observed in the MARINE-treated horses compared with CON- (P < 0.001) and FLAX-treated horses (P < 0.001). Plasma ALA overall varied with time (P < 0.001; data not shown). It decreased (P < 0.001) from d 0 to 30, increased (P < 0.001) to d 60 and further increased to d 90 (P < 0.001). There was a treatment-by-time interaction (P < 0.001) in plasma ALA. Plasma ALA was less (P < 0.05) at d 30, 60 and 90 in the MARINE group compared with the FLAX and CON. Plasma ALA was greater in the FLAX group compared with the CON group at d 30 and also at d 90 (P < 0.05), but it was not different from FLAX at d 60. Plasma arachidonic acid (ARA; C20:4) concentrations in the plasma had a treatment effect (P < 0.01), as well as a time effect (P < 0.01), but no interaction. Plasma ARA was greater in the MARINE group compared with the FLAX (P < 0.01) and also the CON (P < 0.01) groups. Plasma ARA increased (P < 0.01) for all treatment groups from d 30 to 60 and decreased from d 60 to 90 (P < 0.01; data not shown). At d 0, EPA was below detectable concentrations in all groups but then was detected in the plasma of the MARINE group from d 30 to 60, when it increased (P < 0.01). It did not change (P = 0.26) from d 60 to 90. In addition to EPA, DHA was below detectable concentrations at d 0 but was detected in only the MARINE group after d 30. Furthermore, DHA increased (P < 0.01) from d 30 to 90.


View Full Table | Close Full ViewTable 5.

Plasma fatty acids composition (% of total fatty acids)1,2

 
Treatment (n = 7)
P-value
Fatty acid CON MARINE FLAX SEM TRT TIME TRT*TIME
LA
    Mean 36.1a 33.4b 36.9a 0.6 < 0.01 < 0.001 < 0.001
    d 0 36.9a 38.1a 37.1a 0.9 - - -
    d 30 40.0a 34.0b 38.5a 0.9 - - -
    d 60 34.5a 30.1b 36.0a 0.9 - - -
    d 90 33.1a 31.5a 35.9b 0.9 - - -
ALA
    Mean 1.53a 0.94b 1.74c 0.55 < 0.001 < 0.001 < 0.001
    d 0 1.53a 1.44a 1.41a 0.08 - - -
    d 30 1.03a 0.55b 1.47c 0.08 - - -
    d 60 1.75a 0.85b 1.85a 0.08 - - -
    d 90 1.82a 0.94b 2.22c 0.08 - - -
ARA
    Mean 1.95a 2.32b 1.87a 0.08 < 0.01 < 0.01 0.66
    d 0 1.54 1.53 2.00 0.07 - - -
    d 30 1.84 2.11 1.91 0.12 - - -
    d 60 2.04 2.52 2.13 0.12 - - -
    d 90 1.89 2.23 1.76 0.12 - - -
EPA
    Mean - 2.69 - 0.19 - < 0.001 -
    d 0 - - - - - - -
    d 30 - 2.18A - 0.14 - - -
    d 60 - 2.80B - 0.14 - - -
    d 90 - 3.07B - 0.14 - - -
DHA
    Mean - 4.00 - 0.19 - < 0.01 -
    d 0 - - - - - - -
    d 30 - 3.68 A - 0.19 - - -
    d 60 - 3.91 A - 0.19 - - -
    d 90 - 4.41 B - 0.19
a-cValues within a row lacking a common superscript differ, P ≤ 0.05.
A-CValues within a column lacking a common superscript differ, by P ≤ 0.05.
1CON = control diet; MARINE = algae and fish oil supplement added to basal diet; FLAX = flaxseed meal added to basal diet; TRT = treatment; TIME = sampling day.
2LA = linoleic acid; ALA = alpha-linolenic acid; ARA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.

Red blood cell (RBC) fatty acid composition is shown in Table 6. Linoleic acid was not different among treatments, and, overall, it varied with time (P < 0.001), decreasing progressively (P < 0.001) from d 30 to 60 and 90 (data not shown). A treatment-by-time interaction effect was observed (P = 0.03), with LA lower in the MARINE group compared with the FLAX (P = 0.01) and CON (P = 0.01) horses at d 90. Alpha-linolenic acid was not different among treatments, but there was a time effect (P < 0.001), with ALA increasing overall from d 30 to 60 and decreasing from d 60 to 90 (P < 0.001; data not shown). There was also no treatment-by-time interaction for ALA. Arachidonic acid concentrations in RBC were different among treatments (P < 0.001); the ARA concentrations in the MARINE group were greater compared with the FLAX (P = 0.001) and CON (P = 0.01) groups. Arachidonic acid concentrations were also different among sampling days (P = 0.01), but no interaction was found (P < 0.06). Overall, ARA concentrations decreased (P = 0.04) from d 30 to 90 (data not shown). Eicosapentaenoic acid was undetected at d 0 in RBC of any treatment and reached detectable concentrations in the MARINE group only after d 30, when it increased (P < 0.001) from d 30 to d 60. In addition, DHA in RBC, which was undetected at d 0, was detected in the RBC of the MARINE group at d 30 and progressively increased (P < 0.001) to d 60 and further increased (P = 0.01) to d 90.


View Full Table | Close Full ViewTable 6.

Red blood cell fatty acid composition (% of total fatty acids)1,2

 
Treatment (n = 7)
P-value
Fatty acid CON MARINE FLAX SEM TRT TIME TRT*TIME
LA
    Mean 29.9 28.6 29.6 0.6 0.30 < 0.001 0.03
    d 0 30.4a 30.0a 29.6a 0.8 - - -
    d 30 31.7a 29.9a 30.6a 0.8 - - -
    d 60 28.8a 28.8a 29.4a 0.8 - - -
    d 90 28.7a 25.8b 28.6a 0.8 - - -
ALA
    Mean 6.14 4.77 5.60 0.38 0.07 < 0.001 0.47
    d 0 3.19 7.48 3.10 0.66 - - -
    d 30 2.71 2.05 2.75 0.66 - - -
    d 60 12.5 9.48 11.30 0.66 - - -
    d 90 3.12 2.28 3.42 0.66 - - -
ARA
    Mean 1.47a 2.15b 1.44a 0.086 < 0.001 < 0.01 0.06
    d 0 3.07 1.92 2.85 0.10 - - -
    d 30 1.78 1.74 1.58 0.10 - - -
    d 60 1.65 1.99 1.67 0.10 - - -
    d 90 1.56 1.78 1.41 0.10 - - -
EPA
    Mean - 2.13 - 0.15 - < 0.001 -
    d 0 - - - - - - -
    d 30 - 1.67A - 0.13 - - -
    d 60 - 2.31B - 0.13 - - -
    d 90 - 2.39B - 0.22 - - -
DHA
    Mean - 2.63 - 0.18 - < 0.001 -
    d 0 - - - - - - -
    d 30 - 2.00 A - 0.18 - - -
    d 60 - 2.76 B - 0.18 - - -
    d 90 - 3.12 C - 0.18 - - -
a-cValues within a row lacking a common superscript differ, P ≤ 0.05.
A-CValues within a column lacking a common superscript differ, by P ≤ 0.05.
1CON = control diet; MARINE = algae and fish oil supplement added to basal diet; FLAX = flaxseed meal added to basal diet; TRT = treatment; and TIME = sampling day.
2LA = linoleic acid; ALA = alpha-linolenic acid; ARA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.

Skeletal muscle fatty acid composition is presented in Table 7. There was a treatment effect for LA (P < 0.001). Overall, LA was lower in the MARINE group compared with the CON (P < 0.001) and FLAX groups (P < 0.001), but FLAX and CON were not different from each other. The values at d 0 were used as a covariate because differences were found among treatments. There was a time effect (P < 0.001) in which LA increased (P < 0.001) from d 30 to 60 but did not change to d 90 (data not shown). A treatment-by-time interaction was observed (P = 0.01); at d 30, the amount of LA was less (P = 0.03) in the MARINE group compared with the CON group but was not different from the FLAX group (P = 0.23). At d 60 and 90, the LA concentration of the MARINE group was less (P < 0.05) than the FLAX and CON groups. Skeletal muscle ALA had a treatment effect (P = 0.01) and was lower in the MARINE compared with the FLAX (P < 0.01) and CON (P < 0.01) groups. However, the skeletal muscle ALA concentration between the FLAX and CON groups did not differ from each other. There was no time effect or treatment-by-time interaction. There was no treatment effect on skeletal muscle ARA or a treatment-by-time effect. Skeletal muscle ARA varied with time, increasing from d 30 to 60 (P < 0.001), but did not vary further to d 90 (data not shown). A treatment effect (P < 0.001) was observed in muscle EPA; overall, it was greater in the MARINE compared with the FLAX and CON (P < 0.001) groups. There was also a treatment-by-time interaction (P < 0.01). Skeletal muscle EPA was greater in the MARINE group at d 60 and 90 than the FLAX and the CON (P < 0.05) groups. There was no time effect in skeletal muscle EPA.


View Full Table | Close Full ViewTable 7.

Skeletal muscle fatty acid composition (% of total fatty acids)1,2

 
Treatment (n = 7)
P-value
Fatty acid CON MARINE FLAX SEM TRT TIME TRT*TIME
LA
    Mean 33.7 a 27.8b 32.7a 0.6 < 0.001 < 0.001 < 0.05
    d 0 32.4a 25.6b 28.4ab 1.0 - - -
    d 30 32.4a 26.8b 28.4ab 1.0 - - -
    d 60 37.2a 27.4b 33.8a 1.0 - - -
    d 90 35.1b 28.6a 34.8b 1.0 - - -
ALA
    Mean 1.47a 1.27b 1.87a 0.12 < 0.01 0.29 0.90
    d 0 1.46 2.30 1.55 0.13 - - -
    d 30 1.79 1.16 1.74 0.14 - - -
    d 60 2.08 1.49 1.93 0.15 - - -
    d 90 1.77 1.18 1.97 0.13 - - -
ARA
    Mean 6.75 6.79 7.11 0.32 0.12 0.001 0.12
    d 0 6.94 6.21 6.66 0.39 - - -
    d 30 6.11 6.94 6.53 0.40 - - -
    d 60 6.90 7.16 7.50 0.41 - - -
    d 90 7.05 6.54 7.73 0.40 - - -
EPA
    Mean 2.00a 2.35b 1.58a 0.15 < 0.001 0.14 < 0.01
    d 0 0 2.23a 2.49a 0.35 - - -
    d 30 0 1.64a 1.58a 0.29 - - -
    d 60 1.33a 2.82b 1.16a 0.21 - - -
    d 90 1.08a 2.72b 1.09a 0.18 - - -
DPA
    Mean 2.49ab 2.18a 2.69b 0.13 < 0.05 0.15 < 0.01
    d 0 2.38a 2.71a 2.34a 0.23 - - -
    d 30 2.50a 2.58a 2.83b 0.18 - - -
    d 60 2.43a 1.90a 2.71ab 0.23 - - -
    d 90 2.66a 1.54b 2.89b 0.18 - - -
DHA
    Mean 4.11a 7.96b 4.55a 0.27 < 0.001 0.02 0.05
    d 0 3.19a 5.44b 4.33ab 0.48 - - -
    d 30 4.00a 6.65 b 4.96a 0.49 - - -
    d 60 2.76a 8.62 b 4.25a 0.51 - - -
    d 90 4.88a 8.96b 4.62a 0.49 - - -
a-cValues within a row lacking a common superscript differ, P ≤ 0.05.
1CON = control diet; MARINE = algae and fish oil supplement added to basal diet; FLAX = flaxseed meal added to basal diet; TRT = treatment; TIME = sampling day.
2LA = linoleic acid; ALA = alpha-linolenic acid; ARA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.

Docosapentaenoic acid (C22:5 n-3), an intermediate fatty acid between EPA and DHA, was detected in skeletal muscle but not in plasma or blood. Docosapentaenoic acid had a treatment effect (P < 0.05) and was lower in MARINE compared with FLAX but was not different from CON. There was no time effect in DPA. There was a treatment-by-time interaction (P < 0.01) in which skeletal muscle DPA was less (P < 0.001) in the MARINE group compared with the FLAX and CON groups at d 90 of treatment. The FLAX and CON groups did not differ at d 90. Skeletal muscle DHA had a treatment effect (P < 0.001) and was greater in the MARINE compared with the FLAX (P < 0.001) and CON (P < 0.001). There was a time effect (P < 0.05) in which overall DHA increased (P = 0.03) from d 0 to 30 and also increased (P < 0.02) from d 60 to 90 (data not shown). Differences among groups were observed at d 0; thus, the time 0 was used as a covariate. There was a treatment-by-time interaction (P = 0.05). Skeletal muscle DHA was greater in the MARINE group compared with the FLAX and CON groups at d 30, 60, and 90.


DISCUSSION

The current study, to the knowledge of the authors, is the first trial to evaluate the effects of supplemental dietary n-3 fatty acids on skeletal muscle fatty acid composition of horses. A salient finding is that horses supplemented with EPA and DHA had higher concentrations of those fatty acids in the skeletal muscles and RBC compared to those supplemented with ALA. In addition, horses supplemented ALA and no EPA and DHA were found to have increases in muscle DPA. In a tracer study (Vermunt at al., 2000), greater dietary ALA led to greater metabolic oxidation of ALA and reduced conversion to EPA and DHA. In this study, we found that horses in the CON group had less ALA compared with FLAX-supplemented horses in plasma, but not in muscle or RBC, indicating a dose effect for dietary ALA on plasma. Previous studies have shown that supplementation with ALA increased the plasma ALA concentration in a dose-dependent manner in humans, hamsters and guinea pigs (Fu and Sinclair, 2000; Morise et al., 2004; Arterburn et al., 2006). Plasma and skeletal muscle ALA were less in the MARINE group compared with the FLAX and CON. The LCHUFA, including ALA, EPA, DHA and ARA, compete for sn-2 position in cell membranes (Arterburn et al., 2006). Therefore, decreased skeletal muscle ALA in the MARINE group in the current study indicates that the n-3 very LCHUFA, EPA and DHA will preferentially be incorporated into muscle cells, and this is indicated when comparing supplementation of ALA of MARINE and CON (Table 3).

In the current study, plasma and RBC LA were less and ARA was greater in the MARINE group, indicating that, perhaps, in the MARINE group, available LA may have been largely converted to ARA in RBC. However, no differences were observed among treatments in skeletal muscle ARA concentrations. The n-6 fatty acid series can be converted from the parent fatty acid, LA, to its long-chain derivative, ARA (Arterburn et al., 2006). The n-3 fatty acid series can be converted from the parent fatty acid, ALA, to its long-chain derivatives, EPA and DHA. These fatty acids compete for conversion enzymes because they use the same elongase and desaturase enzymes (Arterburn et al., 2006). However, other studies have reported a decrease in ARA concentration when evaluating the effects of EPA and DHA supplementation on plasma fatty acid concentrations (Blonk et al., 1990). Previous research in horses supplemented with 6 g of total n-3/100 kg of BW via fish oil led to decreased concentrations of LA and ALA and greater concentrations of ARA in the plasma compared with horses fed 6 g of total n-3/100 kg via ground flaxseed (Vineyard et al., 2010). However, the researchers did supply 1 g/100 g of total fatty acids of ARA in the fish oil group, and the current study did not supply ARA to any treatment group. No other study has shown similar effects, indicating conversion of available LA into ARA. Tracer studies would be necessary to confirm that LA in RBC was converted to ARA.

In the plasma and red blood cells, EPA and DHA were only detected in the MARINE group, indicating that direct supplementation is necessary in order to observe a substantial increase in blood n-3 LCHUFA. The lack of EPA and DHA in the plasma and the red blood cells in the FLAX and CON groups may have been due to EPA or DHA concentration that was below the detection concentration in the current analysis or because horses have a reduced conversion rate to those long-chain fatty acids. Lack of detection of EPA and DHA in horses supplemented with ground flaxseed or a control diet is also supported in previous research conducted by Vineyard et al. (2010), in which both EPA and DHA were only incorporated into the plasma and RBC membrane of the horses supplemented with marine source of EPA and DHA.

Researchers in another study found that horses exhibited an increase in plasma ARA, EPA, and DHA in a dose-dependent manner when supplemented with 0, 10, 20 or 40 g of encapsulated fish oil (King et al., 2008). The 40 g/d dose of fish oil in that study supplied 39.13 g of EPA and DHA. The dose of the MARINE supplement used by the current study was approximately 38 g/d (58.57 mg/kg BW), similar to the greatest dose supplied by King et al (2008).

Previous research has shown that horses receiving flaxseed supplementation (5.6 g/100 kg BW, equivalent to 56 mg/kg) did not result in different plasma ALA concentrations compared with those receiving no supplementation (Siciliano et al., 2003). In the current study, supplementation was done at greater doses and showed a dose effect (CON group less than FLAX group). In contrast, a different study using horses (Hansen et al., 2002) showed that supplementation with 10% flaxseed oil added to the basal pellet did increase plasma ALA concentrations, as well as plasma EPA concentrations. The rate of feeding was much greater than the current study at 442 g of ALA per 500 kg horse from flaxseed oil, which equals to approximately 1.1g/kg BW. Additionally, in humans, it was shown that supplementation with ALA did increase RBC EPA concentrations but not DHA concentrations (Barcelo-Coblijn et al., 2008). Concentrations of DHA only increased in human subjects supplemented with fish oil (Barcelo-Coblijn et al., 2008).

Alpha-linolenic acid can be metabolized by desaturation and elongation to EPA, n-3 DPA and DHA. Docosapentaenoic acid is formed by chain elongation of EPA. The conversion of n-3 DPA to EPA occurs in 2 steps: First, it is elongated to 24:5n-3, desaturated to 24:6n-3 and then oxidated to DHA (chain shortened to 22:6n-3) in the peroxisome (Kaur et al., 2011). Docosapentaenoic acid was only detected in the skeletal muscle, and it was less in MARINE at d 90 of supplementation compared with other treatments. It also increased with time in the FLAX group, indicating conversion of ALA into n-3 DPA. Diets with greater ALA could inhibit the metabolism of 24:5n-3 to 24:6n-3, limiting the conversion to DHA (Pawlosky et al., 2001; Kaur et al., 2011). Muscle EPA decreased with time in FLAX and CON, indicating that ALA was converted to EPA and then n-3 DPA. No EPA was detected in the skeletal muscle of CON horses on d 0 and 30 of supplementation. Concentrations of EPA may have been below detection concentrations in this particular group, or ALA was not being converted to EPA in these horses.

Overall, in this study, the presence of EPA and DHA in the plasma and RBC were both dependent on the presence of those particular long-chain fatty acids in the diet. However, EPA and DHA were evident in all treatment groups in the muscle, indicating that there is conversion from the parent fatty acid, ALA, but it may be limited. The MARINE group had greater amounts of EPA and DHA in all analyzed tissues. This supports previous research that has shown that supplementation with EPA or DHA increased tissue concentrations of these fatty acids, respectively (Arterburn et al., 2006; Barcelo-Coblijn et al., 2008). The results of the current study support previous studies that found supplementation with ALA did not result in greater concentrations of the long-chain derivatives in human plasma (Arterburn et al., 2006), implying that horses may have a limited ability for the conversion, as well.

As far as the authors are aware, this is the first study to report the incorporation of supplemental dietary fatty acids into skeletal muscle in the horse. Increases in n-3 LCHUFA concentrations in muscle membranes may increase in membrane fluidity (Mueller and Talbert, 1988; Simopoulos, 1991). Recently, an n-3 fatty acid receptor was identified and shown to mediate potent anti-inflammatory and insulin sensitizing effects (Oh et al., 2010). Thus, effects on inflammation should be evaluated, because potential beneficial effects have been shown in other species (Calder, 2001).

In summary, direct supplementation of EPA and DHA through a marine source increased the concentrations of those fatty acids in the plasma, RBC and muscle of horses. In the current study, EPA and DHA increased in horses supplemented with a marine source containing these specific fatty acids. Therefore, dietary sources containing EPA and DHA are needed when desiring an increased incorporation of these n-3 LCHUFA to muscle and RBC. Conversion of ALA from flaxseed and forages to EPA and DHA occurs in the skeletal muscle, as indicated by the detection of these fatty acids in horse skeletal muscle in the present study. Supplementation with ALA through flaxseed led to greater incorporation of muscle DPA compared with MARINE supplementation, and the effects of such increases should be evaluated in further studies addressing inflammatory responses and comparing these to EPA and DHA.

 

References

Footnotes