Further provided, in some embodiments of the present-disclosed subject matter, are methods of modulating fatty acid profiles in a horse or, in other words, modulating the balance between the amounts and types of omega-3 and omega-6 fatty acids present in the horse. In some embodiments, a method of modulating fatty acid profiles in a horse is provided that comprises administering to the horse an effective amount of a composition of the presently-disclosed subject matter that includes a combination of an amount of gamma linolenic acid (GLA), an amount of docosahexaenoic acid (DHA), and an amount of eicosapentaenoic acid (EPA).

The term “modulate” as used herein in relation to the modulation of fatty acid profiles in a target subject is used to refer to varying or changing the fatty acid profiles of a subject, including both increasing and/or decreasing the amounts or balance of omega-3 and omega-6 fatty acids in a subject. In this regard, the skilled artisan will understand that the terms “modulate,” “increase,” “decrease,” and the like do not necessarily refer to an entire alteration of a fatty acid profile or other factor in a subject, but can instead be determined relative to a control, wherein the control can be representative of a subject or environment in which a composition of the presently-disclosed subject matter is not administered or is administered at a particular level. For example, in some embodiments, a modulation in a fatty acid profile relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% increase or decrease in a level of a particular omega-3 or omega-6 fatty acid, or in a level of longer-chain fatty acid produced from the administered shorter-chain fatty acids (e.g., a change in DGLA from GLA). In some embodiments, the increases and/or decreases described herein can be in reference to a control subject that has not been treated with one of the presently-disclosed compositions. In other embodiments, the increases and/or decreases described herein can be in reference to a baseline obtained in a subject that is in need of treatment, but has not yet begun a particular therapeutic regimen with the compositions described herein.

For administration of a therapeutic composition as disclosed herein, in some embodiments, the amounts administered can be expressed as grams administered per day or g/d. In some embodiments, the amounts of the fatty acids included in the compositions described herein are administered in the range of about 1 g/d to about 15 g/d to achieve the desired response or modulation. For example, in some embodiments, the amount of a mixture of EPA and DHA administered is in the range of about 1 g/d to about 20 g/d, while the amount of GLA administered is in the range of about 1 g/d to about 15 g/d. In some embodiments, the GLA is administered at about 1 g/d to about 10 g/d, while the mixture of DHA and EPA is administered at about 5 g/d to about 15 g/d. In some embodiments, the GLA is administered at about 3 g/d to about 7 g/d, while the mixture of DHA and EPA is administered at about 7 g/d to about 12 g/d.

The compositions of the presently-disclosed subject matter are typically administered as an oral supplement and are thus generally administered to horses orally as a top-dressing or dietary treatment on a desired food source (e.g., grain). It is contemplated, however, that suitable methods for administering a composition in accordance with the methods of the presently-disclosed subject matter can also include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and/or intraarterial administration), buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, intratracheal installation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hyper-velocity injection/bombardment. In some embodiments, the administration of the composition is via oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intraaural administration, rectal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravitreous administration, subconjunctival administration, intracameral administration, intraocular administration or combinations thereof.

Regardless of the route of administration, the compositions of the presently-disclosed subject matter are typically administered in an amount effective to achieve the desired response. As such, the term “effective amount” is used herein to refer to an amount of the therapeutic composition (e.g., a composition comprising an amount of DHA, EPA, and GLA) sufficient to produce a measurable biological response (e.g., a change in a fatty acid profile). Actual dosage levels of active ingredients in a composition of the presently-disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular horse and/or application. Of course, the effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the horse being treated. Preferably, a minimal dose is administered, and the dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art.

For additional guidance regarding formulation and dose, see U.S. Pat. Nos. 5,326,902; 5,234,933; PCT International Publication No. WO 93/25521; Berkow et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, New Jersey; Goodman et al., (1996) Goodman & Gilman's: The Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Health Professions Division, New York; Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca Raton, Florida; Katzung, (2001) Basic & Clinical Pharmacology, 8th ed. Lange Medical Books/McGraw-Hill Medical Pub. Division, New York; Remington et al., (1975) Remington's Pharmaceutical Sciences, 15th ed. Mack Pub. Co., Easton, Pennsylvania; and Speight et al., (1997) Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland/Philadelphia; Duch et al., (1998) Toxicol. Lett. 100-101:255-263.

In some embodiments, and as noted above, administering the composition increases an amount of EPA, DHA, GLA, and/or dihomo-gamma-linolenic acid (DGLA) in a horse, including in a biological sample obtained from the horse. In this regard, the term “biological sample” as used herein thus refers to any body fluid or tissue potentially comprising a fatty acid. In some embodiments, for example, the biological sample can be a blood sample, a serum sample, a plasma sample, or subfractions thereof. In some embodiments, the biological sample comprises plasma or red blood cells. In some embodiments, the biological sample comprises red blood cells, and the EPA, DHA, GLA, and/or DGLA is incorporated into the cell membranes of the red blood cells.

Turning now to the step of measuring the fatty acid profile in a horse, including identifying an amount of omega-3 and/or omega-6 fatty acids present in a horse, various methods known to those skilled in the art can be used to identify the fatty acids in the provided biological sample. For instance, in some embodiments, the amount of DHA, EPA, GLA, and/or DGLA can be measured in a biological sample obtained from a subject using standard gas chromatography techniques.

In some further embodiments, by virtue of modulating the fatty acid profiles in a subject (e.g., a horse), the fatty acid profiles in a subject can be improved such that the administration of the composition can be used to alleviate or treat one or more inflammatory conditions in the subject, such as reducing an amount of or incidence of gastric ulcers, airway inflammation, and/or joint inflammation in the horse. As such, further provided in some embodiments of the presently-disclosed subject matter are methods of treating an inflammatory condition in a horse that includes administering an effective amount of a composition of the presently-disclosed subject matter (e.g., a composition including an effective amount of a combination of DHA, EPA, and GLA).

The terms “treatment” or “treating,” as used herein, refer to the medical management of a subject with the intent to modulate, cure, ameliorate, stabilize, or reduce the occurrence of a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In some embodiments, the subject is known to have or currently be suffering from a particular condition (e.g., gastric ulcers), while in other embodiments, the subject may be at a risk of suffering from such a condition.

As used herein, the term “inflammatory condition” includes diseases or disorders which are caused, at least in part, or exacerbated, by inflammation, which is generally characterized by increased blood flow, edema, activation of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis), heat, redness, swelling, pain and/or loss of function in the affected tissue or organ. The cause of inflammation can be due to physical damage, chemical substances, micro-organisms, tissue necrosis, cancer, or other agents or conditions. Such inflammatory conditions include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders. Acute inflammatory disorders are generally of relatively short duration, and last for from about a few minutes to about one to two days, although they can last several weeks. Characteristics of acute inflammatory disorders include increased blood flow, exudation of fluid and plasma proteins (edema) and emigration of leukocytes, such as neutrophils. Chronic inflammatory disorders, generally, are of longer duration, e.g., weeks to months to years or longer, and are associated histologically with the presence of lymphocytes and macrophages and with proliferation of blood vessels and connective tissue. Recurrent inflammatory disorders include disorders which recur after a period of time or which have periodic episodes. Some inflammatory disorders fall within one or more categories. Exemplary inflammatory disorders include capable of treatment with the presently-disclosed compositions include, in some embodiments, gastric ulcers, airway inflammation, and/or joint inflammation, and may be diagnosed or assessed by standard methods known to those skilled in the art of medicine, including veterinary medicine.

With further respect to the therapeutic methods described herein, including the above-described methods of treating an inflammatory conditions, in some embodiments of the therapeutic methods, administering a composition of the presently-disclosed subject matter reduces an amount of an inflammatory cytokine and/or increases an amount of an anti-inflammatory cytokine in a subject. In some embodiments, administering the composition reduces an amount of IL-8 (inflammatory cytokine) in a subject and/or increases an amount of IL-10 (anti-inflammatory cytokine) in the subject.

Various methods known to those skilled in the art can also be used to determine a reduction in the amount of an inflammatory cytokine in a subject such as a horse. For example, in certain embodiments, the amounts of expression of an inflammatory cytokine in a subject can be determined by probing for mRNA of the gene encoding the inflammatory cytokine in a biological sample obtained from the subject (e.g., a tissue sample, a urine sample, a saliva sample, a blood sample, a serum sample, a plasma sample, or sub-fractions thereof) using any RNA identification assay known to those skilled in the art. Briefly, RNA can be extracted from the sample, amplified, converted to cDNA, labeled, and allowed to hybridize with probes of a known sequence, such as known RNA hybridization probes immobilized on a substrate, e.g., array, or microarray, or quantitated by real time PCR (e.g., quantitative real-time PCR, such as available from Bio-Rad Laboratories, Hercules, CA). Because the probes to which the nucleic acid molecules of the sample are bound are known, the molecules in the sample can be identified. In this regard, DNA probes for one or more of the mRNAs encoded by the inflammatory genes can be immobilized on a substrate and provided for use in practicing a method in accordance with the presently-disclosed subject matter.

With further regard to determining levels of an inflammatory cytokine in samples, mass spectrometry and/or immunoassay devices and methods can also be used to measure the inflammatory cytokine in samples, although other methods can also be used and are well known to those skilled in the art. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is hereby incorporated by reference in its entirety. Immunoassay devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, can be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety.

Any suitable immunoassay can be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like. Specific immunological binding of the antibody to the inflammatory molecule can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionucleotides, and the like, attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.

The use of immobilized antibodies or fragments thereof specific for the inflammatory molecules is also contemplated by the present invention. The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as microtiter wells), pieces of a solid substrate material (such as plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test biological sample and then processed quickly through washes and detection steps to generate a measurable signal, such as for example a colored spot.

Mass spectrometry (MS) analysis can be used, either alone or in combination with other methods (e.g., immunoassays), to determine the presence and/or quantity of an inflammatory molecule in a subject. Exemplary MS analyses that can be used in accordance with the present invention include, but are not limited to: liquid chromatography-mass spectrometry (LC-MS); matrix-assisted laser desorption/ionization time-of-flight MS analysis (MALDI-TOF-MS), such as for example direct-spot MALDI-TOF or liquid chromatography MALDI-TOF mass spectrometry analysis; electrospray ionization MS (ESI-MS), such as for example liquid chromatography (LC) ESI-MS; and surface enhanced laser desorption/ionization time-of-flight mass spectrometry analysis (SELDI-TOF-MS). Each of these types of MS analysis can be accomplished using commercially-available spectrometers, such as, for example, triple quadropole mass spectrometers. Methods for utilizing MS analysis to detect the presence and quantity of peptides, such as inflammatory cytokines, in biological samples are known in the art. See, e.g., U.S. Pat. No. 6,925,389; 6,989,100; and 6,890,763 for further guidance, each of which are incorporated herein by this reference.

With still further regard to the various methods described herein, although certain embodiments of the methods disclosed herein only call for a qualitative assessment (e.g., the presence or absence of the expression of an inflammatory cytokine in a subject), other embodiments of the methods call for a quantitative assessment (e.g., an amount of a reduction in the level of an inflammatory cytokine in a subject). Such quantitative assessments can be made, for example, using one of the above mentioned methods, as will be understood by those skilled in the art.

The skilled artisan will also understand that measuring a reduction in the amount of a certain feature (e.g., cytokine levels) or an improvement in a certain feature (e.g., inflammation) in a subject is a statistical analysis. For example, a reduction in an amount of an inflammatory cytokine in a subject can be compared to control level of an inflammatory cytokine, and an amount of an inflammatory cytokine of less than or equal to the control level can be indicative of a reduction in the amount of inflammatory cytokines, as evidenced by a level of statistical significance. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and/or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, incorporated herein by reference in its entirety. Preferred confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

Finally, while the compositions and methods described herein have been described with particular reference to horses, it is contemplated that the present compositions and methods can be performed on a wide variety of subjects. Indeed, the term “subject” as used herein is not particularly limited. The term “subject” is inclusive of vertebrates, such as mammals, and the term “subject” can include human and veterinary subjects. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, camel, cat, guinea pig, rodent, or the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.

The presently-disclosed subject matter is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the present invention.

EXAMPLES
Example 1—Analysis of the Effect of Varied Levels of Omega-3 and Omega-6 Oil Supplementation on Plasma and RBC Fatty Acid Content
Materials & Methods

Twenty-three Thoroughbred horses (aged 4 to 19 years) were used in a longitudinal design to study how supplementation of varied levels of EPA, DHA, and GLA impact the equine blood fatty acid profile. Dietary treatments included no supplementation (control) or oil supplementation at different levels of EPA, DHA, and GLA top-dressed on grain concentrate twice daily for three months. Horses were assigned one of five treatments (4 horses per treatment; Table 1) for three months and were followed an additional three months after cessation of supplementation. Fatty acid composition of plasma and red blood cell (RBC) was measured monthly from July to December.

Horses were subjected to varied management practices from full-time pasture turnout with no exercise to horses stalled and in consistent exercise work six days per week. Horses received varied amounts and types of grain concentrate and hay. They also had widely variable access to fresh grass pasture (4-24 h/d).

TABLE 1

Treatment compositions.

TRT

Fatty Acid
GLA
EPA +

No.
n
Supply
(g/d)
DHA (g/d)

1
4
22.5% GLA
9.690
.001

2
4
20% GLA
7.251
.109

3
4
10% GLA with
3.732
4.315

EPA + DHA

(0.86:1 GLA:

EPA + DHA)

4
4
5% GLA with
1.946
6.434

EPA + DHA

(0.30:1 GLA:

EPA + DHA)

5
4
None
—
—

Results

Direct supplementation with GLA, EPA, and/or DHA modified the fatty acid profile of equine plasma and red blood cells (RBCs). There was a consistent dose response (FIGS. 1-2) to supplementation of all three fatty acids. Varied management styles and intakes of grain, hay, and fresh grass did not affect the ability for supplemental EPA, DHA, and GLA to alter the equine blood fatty acid profile. Additionally, fresh grass access high in ALA and LA did not seem to impact fatty acid changes in any of the longer-chain fatty acids EPA, DHA, and GLA. Horses were not able to effectively elongate ALA or LA into these longer-chain fatty acids. These results supported the need for direct supplementation in order to increase levels of these fatty acids in plasma and RBCs.

There was a strong positive linear relationship (R²=0.87) between GLA intake and RBC DGLA incorporation after 90 days of supplementation (FIG. 1). As GLA intake increased, more DGLA was incorporated into RBCs. This illustrated horses can effectively convert GLA to DGLA and indicated an important dose response. Intake levels of EPA and DHA responded similarly against incorporation of EPA and DHA into RBCs (FIG. 2; R²=0.83). Significantly, when not supplemented with EPA and DHA (data not shown, intake of 0), there was a loss of EPA and DHA incorporated into RBCs.

The highest level of GLA supplementation (22.5% GLA) was able to incorporate the most DGLA into RBCs by three months of supplementation before potentially reaching maximal change (FIG. 3). This level also took longer to reduce after cessation of supplementation. Results further indicated that higher levels of supplementation take longer to elicit maximal changes and more time to washout.

In summary, the above-described studies showcased clear and distinct manipulation of equine plasma and RBC fatty acid composition depending on levels of direct supplementation with GLA, EPA, or DHA. Supplying longer-chain fatty acids directly allowed the horse to avoid minimally effective and competitive elongation steps. A preferred equine fatty acid profile, however, was not determined in these initial studies and thus further research was undertaken to determine what beneficial physiological changes can result from an altered fatty acid profile supported by a specific proprietary ratio of GLA, EPA, and DHA.

Example 2—Assessment of Physiological Response to GLA, EPA, and DHA Supplementation in Intensely-Exercised Thoroughbreds
Materials & Methods

Thirteen physically fit Thoroughbred horses of racing age (aged 2 to 8 years) were used in a crossover design to study the effects of supplementing long-chain anti-inflammatory omega-3 fatty acids EPA and DHA and a specific SC-PUFA omega-6 fatty acid GLA on physiological responses in intensely exercised racehorses. Horses underwent baseline testing prior to supplementation and were split into two groups balanced for age, gender, bodyweight, breeze speed, ulcer score, gastric pH, and RBC count. Groups were supplemented with a blend of GLA, EPA and DHA (GLA-EPA-DHA; n=13) or a blend of LA and ALA (LA-ALA; n=13). These treatments provided similar daily intakes of omega-3 and omega-6 fatty acids but at different fatty acid lengths. GLA-EPA-DHA provided longer-chain fatty acids while the LA-ALA blend provided the shorter-chain parent fatty acids. Horses were supplemented twice daily for three months on one treatment before switching treatments for another three months. Horses were in intense exercise training throughout the trial consisting of galloping three days per week on a dirt racetrack and walking 30 min per day on a mechanical exerciser three days per week. Fatty acid composition of plasma and red blood cells (RBC) were measured monthly. Prior to supplementation and at the end of each treatment period, horses underwent a series of collection procedures. Blood, bronchoalveolar lavage (BAL) fluid, gastric fluid, muscle biopsies, and radiographs were collected around or after breezing (14-16 m/s) for three furlongs on a dirt racetrack. At minimum, measurements were collected for assessment of exercise-induced pulmonary hemorrhage (EIPH), ulcer score, gastric pH, mitochondrial respiration, and inflammatory cytokines.

The daily intake of 60 ml/d of GLA-EPA-DHA oil supplied 9.0 g of omega-3 fatty acids consisting mainly of eicosapentaenoic acid (EPA) (C20:5n3) and docosahexaenoic acid (DHA) (C22:6n3), while 35 ml/d of LA-ALA oil supplied 10.2 g of omega-3 fatty acids as alpha-linolenic acid (ALA) (C18:3n3).

The GLA-EPA-DHA and LA-ALA oil blends provided 8.2 g and 8.0 g of omega-6 fatty acids, respectively. All of the omega-6 fatty acid in the LA-ALA oil blend was linoleic acid (LA) (C18:2n6), while 5.4 g of the GLA-EPA-DHA oil blend was supplied as gamma linolenic acid (GLA) (C18:3n6) and 2.5 g was linoleic acid (LA) (C18:2n6).

TABLE 2

Daily Intake of Fatty Acids with Supplementation.

LA-ALA
GLA-EPA-DHA

Fatty Acid
supplement (g/d)
supplement (g/d)

LA
8.0
2.5

ALA
10.2
0

GLA
0
5.4

AA
0
0.3

EPA
0
4.4

DPA
0
0.8

DHA
0
3.8

EPA + DHA
0
8.2

OMEGA 3
10.2
9.0

OMEGA 6
8.0
8.2

Results

Red blood cell EPA (FIG. 4) and DHA (FIG. 5) were significantly increased in the GLA-EPA-DHA supplemented horses, but not in the LA-ALA oil group. Also, novel eicosanoids generated from long-chain omega-3 PUFAs, termed resolvins and protectins, have anti-inflammatory properties. The most potent anti-inflammatory omega-3 PUFAs are EPA and DHA. Studies in humans have shown that these long-chain omega-3 PUFAs are nine times as potent as ALA. The present study clearly demonstrated that daily intake of EPA+DHA from the GLA-EPA-DHA composition described herein significantly increased RBC membrane EPA+DHA in horses, but that a similar amount of omega-3 in the form of alpha-linolenic acid (ALA) from LA-ALA oil does not. Horses did not appear to be able to efficiently convert ALA into EPA and DHA. Therefore, a dietary source of EPA and DHA like the presently-described GLA-EPA-DHA oil was believed to be needed to affect long-chain omega-3 PUFA cellular membrane composition.

Red blood cell DGLA (FIG. 6) was significantly increased in the GLA-EPA-DHA supplemented horses, but not in the LA-ALA oil group. Daily intake of 5.4 g GLA from the GLA-DHA-EPA oil blend resulted in a significant increase in both gamma linolenic acid (GLA) (C18:3n6) and dihomo-gamma-linolenic acid (DGLA) (C20:3n6). DGLA is made in the horse's body by the elongation of GLA using an efficient enzyme that does not appear to suffer any form of (dietary) inhibition. DGLA is a precursor of series-1 thromboxane and prostaglandin and a 15-hydroxyl derivative that blocks the transformation of arachidonic acid to leukotrienes. All of these effects are anti-inflammatory.

The GLA-EPA-DHA oil blend was further believed to reduce the incidence of gastric ulcers, airway inflammation, and joint inflammation, and can be of benefit in reducing the inflammatory response after exercise. An experiment to examine ulcer score showed positive results for reducing ulcer score (FIG. 7). When horses started supplementation with a high ulcer score (>2), the scores and/or ulcers were significantly reduced after 3 months of supplementation with the GLA-EPA-DHA composition, but not with the LA-ALA oil, compared to the control. This was supported by higher gastric pH when supplemented with the GLA-EPA-DHA oil (FIG. 8). Additionally, GLA-EPA-DHA supplemented horses showed lower heart rates during breezing (FIG. 9), fewer RBCs and eosinophils in bronchoalveolar lavage fluid post-breeze (FIG. 10-11), lower levels of the inflammatory cytokine IL-8 (FIG. 12A), and higher levels of the anti-inflammatory cytokine IL-10 (FIG. 12B) following an exercise stressor. Cytokine levels of IL-8 and IL-10 were altered during exercise recovery when horses were supplemented with GLA-EPA-DHA. IL-8, considered to be an inflammatory cytokine, was decreased at 24 h post-exercise (FIG. 12A). Alternatively, IL-10 is considered to be an anti-inflammatory cytokine and was increased at 4 h post-exercise (FIG. 12B). These results indicate that supplementation with GLA-EPA-DHA can attenuate the inflammatory environment after exercise stress.

Results described herein show positive physiological benefits to supplementing a proprietary blend of GLA, EPA, and DHA. This presently-described blend supplies potent anti-inflammatory long-chain fatty acids directly to the horse.

Example 3—Effect of Polyunsaturated Fatty Acid (PUFA) Supplementation on Red Blood Cell and Plasma Fatty Acid Composition and Its Relationship to the Incidence and Severity of Squamous Gastric Ulcers in Exercised Thoroughbreds

As described herein above, omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are essential nutrients for horses. Omega-3 PUFAs include alpha-linolenic acid (ALA, 18:3n3), eicosapentaenoic acid (EPA, 20:5n3), docosapentaenoic acid (DPA, 22:5n3), and docosahexaenoic acid (DHA, 22:6n3). Omega-6 PUFAs include linoleic acid (LA, 18:2n6), gamma-linolenic acid (GLA, 18:3n6), dihomo-gamma-linolenic acid (DGLA, 20:3n6), and arachidonic acid (AA, 20:4n6). PUFAs with 18 or less carbons are considered short chain (SC), while those with 20 or more carbons are long chain (LC). LC-PUFAs serve as precursors for numerous lipid mediators, which are together referred to as eicosanoids and include prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs). Eicosanoids produced from AA tend to be pro-inflammatory, while DGLA, EPA, and DHA give rise to lipid mediators, which are anti-inflammatory and inflammation-resolving. In other species, both omega-3 and omega-6 fatty acids play a role in the prevention and resolution of gastric ulcers.

With that in mind, additional studies were conducted to further evaluate how supplementation with short-chain (e.g., LA, ALA) or longer-chain (e.g., GLA, EPA, DHA) PUFAs affects plasma and red blood cell (RBC) PUFA composition, and how these changes relate to the incidence and severity of squamous gastric ulcers.

Materials and Methods

Horses and feeding management. Thirteen fit Thoroughbred horses (age 4.1±1.9 y, BW 496.0 kg±36.5 kg; mean±SD) housed at the Kentucky Equine Research Performance Center in Ocala, Florida, were used in a 3-period study. During each 88-d period, the horses were fed 4.9±1.4 kg/d (mean±SD) of a textured horse feed fed in 3 meals/d along with 1.0-1.5% BW/d of timothy hay and 60 g loose salt. The horses were stalled for 8 h/d and were turned out in bahiagrass-pasture paddocks for 16 h each night. The proximate analysis and PUFA composition of the feed, hay, and pasture is shown in Table 3 along with the PUFA supplements. The feed, hay, and pasture provided 2.3, 4.9, and 2.9 g/kg (as fed) of ALA and 24.9, 1.5, and 0.8 g/kg (as fed) of LA, respectively. These feedstuffs did not supply measurable quantities of other PUFAs. The study protocol was reviewed and approved by an internal company Institutional Animal Care and Use Committee.

TABLE 3

Proximate analysis and fatty acid composition of

feed, hay, pasture, and PUFA supplements.

Textured
Timothy
Pasture

feed
hay
(100%

GLA-

(as-fed
(as-fed
DM
LA-
EPA-

basis)
basis)
basis)
ALA
DHA

Digestible energy
2.97
1.90
1.98

(DE, Mcal/kg)

Crude protein
12.7
10.3
17.3

(CP, %)

Crude fat (%)
4.9
2.3
2.8
100
100

Acid detergent
7.9
32.5
35.9

fiber (ADF, %)

Neutral detergent
14.6
53.9
62.2

fiber (NDF, %)

Starch (%)
36.2
0.7
1.8

Water-soluble
8.4
11.3
3.9

carbohydrate

(WSC, %)

Nonstructural
44.6
12.0
5.7

carbohydrate

(NSC, %)

Omega-6
g/100 g total identified fatty acids

Linoleic acid
48.4
16
14.6
28.9
5.6

(LA, C18:2n6)

Gamma-linolenic
0.1
0.3
0.2
0.2
12.1

acid

(GLA, C18:3n6)

Dihomo-g-
0
0
0
0
0.2

linolenic

(DGLA,

C20:3n6)

Arachidonic acid
0
0
0
0
0.8

(AA, C20:4n6)

Omega-3

Alpha-linolenic
4.5
51.1
51.3
37
1.4

acid

(ALA, C18:3n3)

Eicosapentaenoic
0
0
0
0
11.1

acid

(EPA, C20:5n3)

Docosapentaenoic
0
0
0
0
2

acid

(DPA, C22:5n3)

Docosahexaenoic
0
0
0
0
9.5

acid

(DHA, C22:6n3)

PUFA supplementation. During period 1, the horses received no PUFA supplementation other than what was contained in the feed, hay, and pasture (CON). Following period 1, horses were split into two groups balanced for age, concentrate intake, bodyweight, and baseline values for exercise speed, gastric pH, and ulcer score. During periods 2 and 3, the horses received either 35 ml/d of a corn oil-flax oil supplement (LA-ALA) or 60 ml/d of a high gamma-linolenic acid (GLA) safflower oil-fish oil supplement (GLA-EPA-DHA) in a switchback design. Seven horses received GLA-EPA-DHA first while the other six horses received LA-ALA first before switching to the other treatment. During a 10-day washout period between periods 2 and 3, the horses did not receive PUFA supplementation. Each supplement was divided into two daily feedings. The LA-ALA supplement provided 8.0 g LA and 10.2 g ALA per day. The GLA-EPA-DHA supplement provided 2.5 g LA, 5.4 g GLA, 0.3 g AA, 4.4 g EPA, 0.8 g DPA, and 3.8 g DHA per day. Each supplement provided a similar amount of total omega-6 (8.0 g LA-ALA vs 8.2 g GLA-EPA-DHA) and omega-3 (10.2 g LA-ALA vs 9.6 g GLA-EPA-DHA) per day.

Exercise schedule. During the first 77 days of each period, horses were exercised 3 times per week on a 1,600-m dirt racetrack. Across all periods, each session averaged 3,640±590 m and lasted 12.3±0.5 min. During each session, horses walked 275±67 m (1-3 m/s), trotted 1,115±285 m (3-5 m/s), cantered 1,520±470 m (5-10 m/s), and galloped 730±325 (>10 m/s). The horses cantered or galloped 4.0±1.1 min of each session with heart rates≥154 bpm. The horses also walked 30 min/session 3 days per week on a 20-m diameter mechanical walker.

On day 81 of each period, the horses performed a 4,600-m standardized exercise test (SET) on the racetrack consisting of 1,800-m trot (4.5±0.4 m/s), 1,200-m canter (8.2±1.0 m/s), 600-m fast gallop (15.3±0.6 m/s), and 1,000-m canter (8.7±1.7 m/s). Horses did not perform any forced exercise for 2 weeks following the SET.

Gastroscopy and gastric fluid collection. Five days after the SET (d 86), the gastric mucosa of the horses was examined using a flexible 3-m videoendoscope (Olympus OSF-V60) while sedated with detomidine (0.01 mg/kg) (Dormosedan, Zoetis). Prior to gastroscopy, feed and hay were withheld 15-18 h and water withheld 2-3 h. The severity of squamous gastric ulceration was scored on a scale of 0-4 in accordance with an equine gastric lesion scoring system by a researcher who was unaware of the diet each horse was being fed. A score of 0 was considered free of ulceration, scores of 1-2 were considered mild to moderate ulceration, and scores of 3-4 were considered severe ulceration. Gastroscopic evaluations followed the collection of 200-300 mL gastric fluid through the biopsy port of the endoscope for pH analysis. Gastric fluid samples were immediately evaluated for pH after collection using a benchtop pH meter (Oakton pH 700 Benchtop Meter, model 35419-12).

Blood collection and fatty acid analysis. Blood samples were collected monthly during each period to evaluate changes in plasma and RBC fatty acid composition. The final sample from each period was collected two days after the gastroscopy (d 88). Prior to morning feeding (0600-0700 h), samples were collected via venipuncture of the jugular vein directly into K₂EDTA vacutainers. Vacutainers were placed at 4° C. until they could be centrifuged at 1,500×g for 10 minutes as soon as possible (≤10 minutes). After centrifugation, plasma was collected and placed into separate aliquots. The buffy coat was carefully discarded via aspiration to access RBCs. RBCs were then collected and placed into separate aliquots. Samples were either immediately sent overnight at ambient temperature to a commercial laboratory (OmegaQuant Analytics, Sioux Falls, SD) where they were frozen at −80° C. until analysis, or they were frozen at −80° C. after collection and sent overnight on dry ice to the same laboratory. Plasma and RBC samples were processed and analyzed for fatty acid composition using gas chromatography with flame ionization. A total of 24 fatty acids were identified for every sample.

Statistical Analysis. Data was analyzed using Prism (GraphPad) statistical software. Values are mean±SD unless specified otherwise and levels of significance were set at α≤0.05. Significant differences in RBC and plasma fatty acid levels between treatments were determined using a one-way ANOVA for repeated measures followed by a Tukey post-hoc test. Significant differences in RBC fatty acid composition between ulcer groups were determined using a two-tailed, unpaired T test. Odds ratios were used to quantify the relationship between PUFA supplementation and severity of squamous ulcer score (MedCalc3).

Results

Supplementation with GLA-EPA-DHA resulted in a decrease in RBC and plasma LA and ALA (p<0.05) and an increase in RBC and plasma GLA, DGLA, EPA, and DHA (p<0.05) compared to CON or LA-ALA-supplemented horses. RBC AA was also higher in GLA-EPA-DHA-supplemented horses (p<0.05). RBC GLA, DGLA, EPA, and DHA were elevated in LA-ALA horses compared to CON horses during period 3 due to a carryover effect following GLA-EPA-DHA supplementation in period 2 (Table 4 and FIG. 13).

TABLE 4

RBC and plasma PUFA composition (d 88) when horses were unsupplemented (CON) or fed

LA-ALA or GLA-EPA-DHA supplements (g/100 g total identified fatty acids).

CON
LA-ALA
GLA-EPA-DHA

Period 1
Period 2
Period 3
TOTAL
Period 2
period 3
TOTAL

(n = 13)
(n = 6)
(n = 7)
(n = 13)
(n = 7)
(n = 6)
(n = 13)

RBC fatty acids

LA C18:2n6
39.64 ± 0.93^a
39.93 ± 0.97
38.33 ± 0.82
39.07 ± 1.19^a*
37.98 ± 1.13
37.12 ± 0.70
37.59 ± 1.02^b

ALA C18:3n3
1.27 ± 0.33^a
1.34 ± 0.26
1.09 ± 0.18
1.21 ± 0.25^a
1.04 ± 0.24
1.10 ± 0.24
1.07 ± 0.23^b

GLA C18:3n6
0.10 ± 0.04^a
0.12 ± 0.01
0.17 ± 0.03
0.15 ± 0.03^b*
0.38 ± 0.04
0.37 ± 0.09
0.37 ± 0.07^c

DGLA C20:3n6
0.27 ± 0.05^a
0.25 ± 0.02
0.38 ± 0.05
0.32 ± 0.07^a*
0.57 ± 0.13
0.51 ± 0.08
0.54 ± 0.11^b

AA C20:4n6
1.48 ± 0.26^a
1.31 ± 0.21
1.39 ± 0.24
1.36 ± 0.22^b
1.68 ± 0.23
1.53 ± 0.22
1.61 ± 0.23^c

EPA C20:5n3
0.09 ± 0.05^a
0.09 ± 0.02
0.2 ± 0.04
0.15 ± 0.06^b*
0.65 ± 0.11
0.61 ± 0.13
0.63 ± 0.12^c

DHA C22:6n3
0.09 ± 0.07^a
0.12 ± 0.07
0.39 ± 0.10
0.27 ± 0.17^b*
0.83 ± 0.14
0.69 ± 0.21
0.77 ± 0.18^c

(EPA + DHA)
0.18 ± 0.11^a
0.16 ± 0.07
0.60 ± 0.11
0.39 ± 0.24^b*
1.47 ± 0.20
1.30 ± 0.34
1.39 ± 0.28^c

(DGLA + AA)
1.76 ± 0.30^a
1.57 ± 0.23
1.77 ± 0.26
1.67 ± 0.26^a
2.25 ± 0.32
2.04 ± 0.29
2.15 ± 0.31^b

(DGLA + AA + EPA + DHA)
1.94 ± 0.37^a
1.72 ± 0.27
2.36 ± 0.24
2.07 ± 0.41^a*
3.72 ± 0.47
3.34 ± 0.59
3.55 ± 0.54^b

Plasma fatty Acids

LA C18:2n6
54.04 ± 1.46^a
55.12 ± 1.24
54.80 ± 0.45
54.94 ± 0.88^a
53.52 ± 1.19
52.34 ± 1.01
52.98 ± 1.23^b

ALA C18:3n3
2.87 ± 1.04^a
2.63 ± 0.38
2.10 ± 0.23
2.34 ± 0.40^a
1.81 ± 0.46
1.89 ± 0.48
1.85 ± 45^b

GLA C18:3n6
0.08 ± 0.04^a
0.13 ± 0.01
0.17 ± 0.06
0.15 ± 0.05^b*
0.83 ± 0.12
0.77 ± 0.22
0.80 ± 0.16^c

DGLA C20:3n6
0.33 ± 0.08^a
0.30 ± 0.03
0.51 ± 0.05
0.41 ± 0.12^a*
0.69 ± 0.15
0.81 ± 0.09
0.75 ± 0.13^b

AA C20:4n6
1.39 ± 0.21^a
1.14 ± 0.14
1.25 ± 0.13
1.20 ± 0.14^b
1.45 ± 0.19
1.47 ± 0.14
1.46 ± 0.16^a

EPA C20:5n3
0.09 ± 0.04^a
0.07 ± 0.02
0.18 ± 0.06
0.13 ± 0.07^a
0.57 ± 0.09
0.65 ± 0.06
0.61 ± 0.08^b

DHA C22:6n3
0.07 ± 0.05^a
0.12 ± 0.06
0.35 ± 0.12
0.24 ± 0.15^b*
0.86 ± 0.12
0.85 ± 0.12
0.85 ± 0.12^c

EPA + DHA
0.16 ± 0.06^a
0.18 ± 0.08
0.53 ± 0.14
0.36 ± 0.21^b*
1.43 ± 0.17
1.5 ± 0.17
1.46 ± 0.17^c

DGLA + AA
1.73 ± 0.26^a
1.44 ± 0.15
1.76 ± 0.13
1.61 ± 0.21^a
2.14 ± 0.27
2.28 ± 0.12
2.20 ± 0.22^b

(DGLA + AA + EPA + DHA)
1.89 ± 0.29^a
1.62 ± 0.18
2.29 ± 0.10
1.98 ± 0.37^a*
3.57 ± 0.36
3.77 ± 0.26
3.66 ± 0.32^b

Unlike superscripts in a row are significantly different.

*Period effect seen in LA-ALA due to carryover effect of GLA-EPA-DHA supplementation in period 2

Gastric ulcers. The overall prevalence of gastric ulcers (grade 1-4) was 54%, 46%, and 31% in the CON, LA-ALA, and GLA-EPA-DHA groups, respectively (Table 5). Five of the 13 horses (38%) had severe ulcers (grade 3-4) at the end of the CON period. None of the other 8 horses developed severe ulcers during the rest of the study. When supplemented with GLA-EPA-DHA, significantly fewer horses (1 of 13, 8%) had a severe ulcer (OR 0.13; 95% CI 0.013-1.36; p<0.05; z score 1.70). LA-ALA supplemented horses had a 23% (3 of 13) incidence of severe ulceration, which was not different from CON or GLA-EPA-DHA (p>0.05).

TABLE 5

Number of horses with each ulcer score per treatment.

ULCER SCORE

0
1-2
3-4

TREATMENT
number
%
number
%
number
%

CON
6
46%
2
15%
5
38%

LA-ALA
7
54%
3
23%
3
23%

GLA-EPA-DHA
9
69%
3
23%
1
8%

All observations (n=39) were grouped to compare RBC fatty acid composition to ulcer score. The PUFA composition of all horses with no or mild to moderate ulcers (grade 0-2; n=30) is shown in Table 6 compared to horses with severe (grade 3-4) ulcer scores (n=9). RBC GLA, DGLA, EPA and AA were all higher in horses with grade 0-2 ulcers compared to horses with severe ulcers (grade 3-4) (p<0.05). Gastric fluid pH was also higher (1.69±0.20) in the horses with no or mild to moderate ulcers compared to the horses with severe ulcers (1.51±0.22) (p<0.05).

TABLE 6

RBC PUFA composition of horses with either grade

0-2 ulcers (n = 30) or grade 3-4 ulcers (n = 9).

Ulcer Score

0-2
3-4

RBC Fatty
n = 30
n = 9

acid
g/100 g total fatty acids
p value

ALA
1.23 ± 0.26
1.03 ± 0.32
0.06

LA
38.58 ± 1.36
39.4 ± 1.18
0.11

GLA
0.23 ± 0.14
0.13 ± 0.05
<0.05

DGLA
0.40 ± 0.15
0.30 ± 0.09
<0.05

EPA
0.33 ± 0.27
0.14 ± 0.13
<0.05

DHA
0.42 ± 0.34
0.21 ± 0.21
0.09

AA
1.54 ± 0.24
1.29 ± 0.22
<0.01

DGLA + AA
1.94 ± 0.35
1.59 ± 0.24
<0.01

EPA + DHA
0.75 ± 0.61
0.35 ± 0.32
0.06

GLA-EPA-
2.69 ± 0.88
1.94 ± 0.40
<0.05

DHA

The five horses that had severe ulceration initially (period 1, CON) were subgrouped to evaluate supplementation. In these horses, supplementation with GLA-EPA-DHA resulted in higher RBC DGLA, EPA, and DHA (p<0.05) (FIG. 14), and four of the five had no ulcers (grade 0) after 3 months of GLA-EPA-DHA supplementation, regardless of period. Two of these horses redeveloped ulcers when subsequently placed on the LA-ALA supplement and one horse was resistant to any changes.

Discussion

There are two families of essential fatty acids (EFAs), omega-6 polyunsaturated fatty acids (PUFAs) derived from linolenic acid (LA), and the omega-3 PUFAs derived from alpha-linolenic acid (ALA). Essential fatty acids cannot be synthesized by the body, but the body can desaturate and elongate EFAs provided by the diet. These pathways are responsible for not only producing PUFAs found in the membranes for virtually every cell in the body, but also generating prostaglandins (PGs) and other eicosanoids involved in the pathophysiology, prevention, and treatment of gastric ulcers. Generally, these pathways work in balance, producing pro-inflammatory and anti-inflammatory compounds. Linolenic acid, the parent of the omega-6 pathway, is desaturated to form gamma-linolenic acid (GLA), which in turn is elongated to form dihomo-GLA (DGLA), the precursor of PG1 series. DGLA can also be desaturated by delta-5-desaturase to arachidonic acid (AA), the precursor of PG2 series. Alpha-linolenic acid, the parent of the omega-3 pathway, is desaturated and elongated by the same set of enzymes to give rise to eicosapentaenoic acid (EPA), the precursor of PG3 series, and further to docosahexaenoic acid (DHA), a precursor to resolvins and protectins involved in inflammation.

These pathways compete for the same enzymes in their elongation steps. Although the ideal omega-6: omega-3 ratio has not been established for horses, it is clear that the current feeding practices of performance horses has drastically skewed the ratio from a natural forage-based diet. An all-forage diet would provide a 0.3:1 (omega-6: omega-3) ratio. In this study, the basal diet provided about 140 g/d LA and 62 g/d ALA for a ratio of 2.2:1. When horses were supplemented in the current study, treatments were matched to provide similar omega-6: omega-3 ratios (0.8:1) so as to only evaluate the type of FA (SC vs LC) fed rather than an impact on the ratio. Studies in other species have ratios from 5.3-665:1, and studies in horses have ratios of 0.8-7.5:1, if they are listed at all.

Traditionally, PUFAs in equine diets have been provided as SC-PUFAs from vegetable oils and forage. The predominant form of omega-6 SC-PUFA (LA) comes from vegetable oils, such as corn and soy oil, while the major SC-PUFA omega-3 (ALA) is supplied from forage or vegetable oils such as flaxseed oil. In the present study, horses fed supplemental LA and ALA from corn oil and flax oil did not increase LC-PUFAs in plasma or RBC. Horses fed GLA-EPA-DHA had reduced RBC and plasma LA and ALA, and increased EPA, DHA, and AA. This agrees with previous studies in which horses fed fish oil had greater (p<0.05) proportions of EPA, DHA, and AA in plasma and RBC, and plasma LA and ALA were less (p<0.05) compared with those supplemented with flax oil. Additionally, horses not supplemented directly with EPA and DHA did not increase their plasma or RBC levels. This reflects an inability to adequately elongate dietary SC-PUFAs like ALA and LA to LC-PUFAs like DGLA, EPA, and DHAs and highlights the benefits of supplementing LC-PUFAs directly. This allows the horse to avoid minimally effective and competitive elongation steps. Interestingly, GLA is considered a SC-PUFA, but has been shown both here and in other unpublished research from the present inventors research to be efficiently elongated to the LC-PUFA DGLA by the horse. It is possible that concurrently supplying LC-PUFAs EPA and DHA allow for GLA to be efficiently converted to DGLA and AA and their respective PGs. Some research has shown a reduction in AA when fed fish oil, but the present study has demonstrated an increase in DGLA and AA that is likely attributable to supplying GLA. The GLA-EPA-DHA treatment with GLA increased RBC levels of AA compared to CON, and the LA-ALA treatment showed a decrease in RBC AA. Although plasma levels of AA were not different between GLA-EPA-DHA and CON, LA-ALA had significantly lower AA levels than CON.

RBCs are longer-lived than platelets and lipoproteins, so their fatty acid composition is more stable and reflective of chronic omega-3 status. Previous studies in horses demonstrated augmented levels of omega-3 PUFAs in plasma, RBCs, leukocytes, synovial fluid, and skeletal muscle following EPA and DHA supplementation. Data from mice demonstrated that the gastrointestinal tissues are highly responsive to dietary LC-PUFA supplementation and that the sum of EPA+DHA in RBCs, expressed as a percentage of total fatty acids (the omega-3 index), can serve as a valid biomarker for assessing dietary EPA+DHA incorporation into gastrointestinal tissues. Although gastrointestinal tissue was not measured in this study, levels of fatty acids in RBCs, including EPA+DHA, were altered by dietary supplementation and may be reflective of changes to local gastric tissue.

Prostaglandins produced from fatty acids can influence acid, mucus, and bicarbonate secretion in addition to accelerated healing of ulcers. AA from the omega-6 pathway produces PGE2 while PGE1 and PGE3 are produced from DGLA and EPA through the omega-6 and omega-3 pathway, respectively. NSAIDS block the production of all three PGEs through disruption of the cyclooxygenase (COX) pathway and have been implicated in increased risk for gastric ulcers. When it comes to gastric protection, PGE2 from AA is thought to play an important role during stress. Studies have shown that PGE2 deficiency can increase mucosa susceptibility to ulceration and that increased erosion has been seen with decreased levels of AA, largely due to a decrease in PGE2 production. When supplied with LA as a precursor to AA, the horse has been able to increase gastric PGE2 production and lower gastric acid output. However, GLA supplementation was preferred to LA in rats subjected to aspirin-induced gastric hemorrhage. GLA supplementation produced higher levels of AA and no gastric hemorrhage compared to hemorrhage in 3 of 8 rats on the LA diet. It was believed GLA was able to bypass depressed levels of delta-6-desaturation in the LA group and protect gastric mucosa through improved synthesis of AA and PGEs. Similarly, supplementation of LA in the present study's LA-ALA treatment did not increase AA levels but reduced levels. Although gastric PGE2 was not measured in this study, it may be inferred that an increase in AA in the GLA-EPA-DHA treatment likely resulted in an increase in PGE2 and could be attributed to the reduced pH and ulcer prevalence and severity in the GLA-EPA-DHA group. Horses with severe ulceration (grade 3-4) had lower RBC AA levels as well as lower GLA, DGLA, and EPA compared to those with no or mild to moderate ulcers (grade 0-2).