Abstract:
Disclosed are methods for restoring a more nearly normal joint function in an osteoarthritic dog and methods for deceasing the likelihood of a dog developing osteoarthritis. The methods can comprise administering to the dogs a composition comprising an effective concentration of the omega-3 fatty acid, eicosapentaenoic acid.

Description:
CROSS REFERENCE TO RELATED APPLICATION  
       [0001]     This application claims the benefit of U.S. application Ser. No. 10/638,832 filed Aug. 11, 2003, which was converted to a provisional application (Unknown Number), on Aug. 5, 2004. The aforementioned application is incorporated in its entirety by reference. 
     
    
     BACKGROUND  
       [0002]     Osteoarthritis is a degenerative joint disease commonly occurring in humans and in companion animals (Richardson et al.,  Vet. Clin. North Amer. Small Animal Practice  27:883-911, 1997; Curtis et al.,  Drug Disc. Today  9:165-172, 2004). The disease involves progressive deterioration of articular cartilage with minimal inflammation (Schoenherr et al. in  Small Animal Clinical Nutrition  4 th    Ed ., Hand et al. Eds., Walsworth Publishing Company, Marceline, Mo., 2000, 907-921; Hedbom et al.,  Cell Mol. Life Sci  59:45-53, 2002; Pool,  Front Biosci  4:D662-70, 1999). Management of osteoarthritis can include pharmacological treatments, surgery, nutraceutical administration and diet management. Such current management approaches have, however, focused on symptomatic relief and as such, they have not been entirely successful in disease management or in treating the underlying pathologies. Hence, there remains a continuing need for new approaches in managing osteoarthritis in humans and companion animals.  
       SUMMARY  
       [0003]     Accordingly, the inventors herein have succeeded in discovering that administration of an effective amount of omega-3 fatty acids, in particular, Eicosapentaenoic acid (EPA) can provide a new approach for management of osteoarthritis in dogs. The methods are effective in managing osteoarthritis in dogs.  
         [0004]     Thus, in various embodiments, the present invention can include methods for restoring a more nearly normal joint function in an dog having osteoarthritis. The methods can comprise feeding to the dog a composition comprising EPA at a concentration of at least about 0.2% by weight or a concentration of at least about 0.3% by weight.  
         [0005]     The present invention can also include methods for deceasing the likelihood of a dog developing osteoarthritis. The methods can comprise feeding to the dog a composition comprising EPA at a concentration of at least about 0.2% by weight or a concentration of at least about 0.3% by weight.  
         [0006]     The methods of the present invention for restoring a more nearly normal joint function in an osteoarthritic dog can also comprise feeding to the dog a composition on the basis of the composition comprising a fatty acid component comprising a joint-function restoring amount of EPA.  
         [0007]     The methods of the present invention method for deceasing the likelihood of a dog developing osteoarthritis can similarly comprise feeding to the dog a composition on the basis of the composition comprising a fatty acid component comprising a joint-function restoring amount of EPA.  
         [0008]     The present invention can also include methods for deceasing the likelihood of a dog developing osteoarthritis and methods for restoring a more nearly normal joint function in an osteoarthritic dog comprising administering to the dog a composition comprising EPA in amount of about 37.5 mg/kg body weight, about 56.25 mg/kg body weight, about 75 mg/kg body weight or about 93.75 mg/kg body weight. Such compositions can be comprised by an animal food composition, an animal treat or an animal supplement.  
         [0009]     In various embodiments, the methods for restoring a more nearly normal joint function in an osteoarthritic dog can involve treating the osteoarthritic disease or reducing symptoms of the disease in the dog and the methods for decreasing the likelihood of a dog developing osteoarthritis can involve preventing development of the osteoarthritic disease in a dog or preventing or diminishing the appearance of symptoms of the disease in the dog.  
         [0010]     The methods of the present invention can additionally be based upon compositions that further comprise omega-6 fatty acids in a total amount of not more than about 3% by weight and/or a ratio of omega-6 fatty acids to omega-3 fatty acids of about 0.2 to about 1.1 and or a ratio of omega-6 fatty acids to EPA of about 1.0 to about 12.5. 
     
    
     DETAILED DESCRIPTION  
       [0011]     This present invention can involve administration of omega-3 fatty acids, in particular, EPA in managing osteoarthritic diseases and symptoms of such diseases in mammals and in particular, in dogs.  
         [0012]     Omega-3 fatty acids also known as n-3 fatty acids, are a recognized group of polyunsaturated fatty carboxylic acids. In general, the omega-3 fatty acids contain 12-26 carbon atoms with methylene-interrupted double bonds. The physiologically more important omega-3 fatty acids are 18-22 carbons in length and straight chained. The n-3 fatty acids have a double bond between the 3 and 4 carbon atoms as measured from the methyl end of the molecule. Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and alpha-lenolenic acid (ALA) are important n-3 fatty acids for managing osteoarthritis in mammals and EPA is particularly important in managing osteoarthritis in dogs. Derivatives of omega-3 fatty acids can also be used in managing osteoarthritis. Many types of derivatives are well known to one skilled in the art. Examples of suitable derivatives are esters, such as branched or unbranched and/or saturated or unsaturated C 1 -C 30  cycloalkyl esters, in particular C 1 -C 6  alkyl esters of omega-3 fatty acids, particularly EPA.  
         [0013]     The omega-3 fatty acids, and in particular EPA, can be administered to a mammal, and in particular, to a dog, by any of many routes of administration, such as, for example, oral, intranasal, intravenous, subcutaneous and the like. The oral route is particularly suitable and EPA can be administered orally in a wet or dry diet, either incorporated therein or on the surface of any diet component, such as, by spraying, agglomerating, dusting or precipitating on the surface. It can be present in the nutritional diet per se or in a snack, supplement or a treat. It can also be present in the liquid portion of the diet such as water or another fluid. The EPA can be administered as a powder, solid or as a liquid including a gel. If desired the EPA can be orally administered in a nutraceutical or pharmaceutical dosage form such as a capsule, tablet, caplet, syringe, and the like. Within the dosage form the EPA can be present as a powder or a liquid such as a gel. Any of the usual neutraceutical or pharmaceutical carriers can be employed such as water, glucose, sucrose and the like together with the EPA.  
         [0014]     In certain embodiments, the present invention can involve EPA-diet compositions that are essentially free of DHA and/or ALA. Essentially free of DHA or ALA or mixtures thereof is intended to mean that either or both of DHA and ALA are substantially absent or that there are only small insignificant amounts of either or both of DHA or ALA present, for example, less than about 0.1%, less than about 0.03%, less than about 0.01%, less than about 0.03% or less than about 0.001%. In embodiments that are essentially free of DHA and/or ALA, any amount of DHA and/or ALA present is at a concentration sufficiently low so that no substantial effect is produced in an osteoarthritic dog on the disease of osteoarthritis, on the progression of osteoarthritis or on symptoms produced by the osteoarthritis.  
         [0015]     The Omega-3 fatty acids and, in particular, EPA is effective against various forms of osteoarthritis as well as other forms of arthritis including rheumatoid arthritis.  
         [0016]     The omega-3 fatty acid, EPA acts to prevent the development of the degenerative process in joint cartilege or to diminish the degenerative process and thereby improve joint in osteoarthritic dogs or in dogs that might otherwise develop osteoarthritis. This effect is in addition to an anti-inflammatory action of omega-3 fatty acids, which may be of less importance in canine osteoarthritis because a limited involvement of inflammation in the osteoarthritis.  
         [0017]     Use of an in vitro explant procedure involving articular knee cartilage as shown in the examples below, demonstrated that EPA was the only omega-3 fatty acid to significantly decrease induced release of glycosaminoglycan (GAG) from the cartilage. With respect to prevention of joint damage from osteoarthritis a particular target group of pets, especially canines, are those that would be in need of such preventative care as opposed to the general population. For example, pets, particularly large breed canines such as labrador retriever, rottweiler, german shepherd and the like are more susceptible to osteoarthritis as demonstrated by its greater occurrence in these pets. Additionally, pets above the age of six (6) years, particularly dogs, have a significantly greater occurrence of osteoarthritis. EPA can be additionally useful in treating canines and felines with osteoarthritis. Also present with the EPA can be other omega-3 fatty acids such as DHA and ALA as well as omega-6 fatty acids, all of which can be found in sources such as fish oils in relatively large quantities.  
         [0018]     The quantity of EPA which should be employed can vary substantially. As shown in later examples, an actual dose response is observed—the greater the EPA, the greater the anti-arthritic effect. Generally, a minimum of at least about 0.2 wt % based upon the quantity of a nutritious diet satisfying ordinary requirements of a canine or feline on a daily basis. For example, a specific amount can be employed in the usual nutrient food ration on a daily basis or the same daily quantity can be provided to the animal in a treat or supplement on a daily basis. Additionally, a combination of these methods or any other dosing means can be employed as long as the effective quantity of EPA is provided. The range of amounts of EPA includes at least about 0.2%, at least about 0.25%, at least about 0.30%, at least about 0.4%, at least about 0.5%, at least about 0.6% up to about 2%, up to about 2.25%, up to about 2.5%, up to about 3%, up to about 4%, or up to about 5% on a Weight basis. It should be noted that all wt % are on a dry matter basis (DMB). EPA is an omega-3 fatty acid. Generally, the ratios of the EPA or omega-3 to omega-6 fatty acid can vary significantly. In various embodiments, the omega-6:omega-3 ratio can be from about 1.10 to 0.2 omega-6 to 1.0 omega-3 or from about 1.08 to 0.42 omega-6 to 1.0 omega-3 and more particularly, about 0.2, about 0.25, about 0.3, about 0.4, about 0.5, about 0.6, about 0.8, about 1.0, or greater In various embodiments, the omega-6 to EPA ratio can be about 12.5 to about 1.0 omega-6 to 1.0 EPA, or about 12.4 to 1.12 omega-6 to 1.0 EPA and more particularly, about 0.2, about 0.25, about 0.3, about 0.4, about 0.4, about 0.6, about 0.8, about 1.0, about 1.5, about 2.0, about 2.5, about 3, about 4, about 5, about 6, about 7.5, about 10, about 12.5 or greater. The arachidonic acid, AA, (an omega-6) to EPA ratio can be about 0.28 to about 0.01 AA to 1.0 EPA to about 0.28 to 0.08 AA to 1.0 EPA and more particularly about 0.01, about 0.02, about 0.04, about 0.06, about 0.08, about 1.0, about 1.5, about 1.0, about 1.5, about 2.8 or greater.  
         [0019]     The omega-3 fatty acid, and in particular EPA, can be administered in amounts calculated as mg/kg body weight. Thus for example, a 20 kg dog would be expected to consume about 275 g of diet per day. Amounts of EPA in the diet of about 0.2%, about 0.3%, about 4%, about 0.5% or about 0.6% by weight would amount to administering to the dog about 27.5 mg/kg body weight, about 41.25 mg/kg body weight, about 55 mg/kg body weight, about 68.75 mg/kg body weight or about 82.5 mg/kg body weight respectively. More particularly, EPA can be administered in an amount of about 20 mg/kg body weight, about 28 mg/kg body weight, about 30 mg/kg body weight, about 40 mg/kg body weight, about 41 mg/kg body weight, about 50 mg/kg body weight, about 55 mg/kg body weight, about 60 mg/kg body weight, about 69 mg/kg body weight, about 70 mg/kg body weight, about 80 mg/kg body weight, about 82 mg/kg body weight, about 90 mg/kg body weight, about 100 mg/kg body weight, about 120 mg/kg body weight, about 150 mg/kg body weight, or greater.  
         [0020]     As indicated above, the EPA can be in the form of a food provided to the pet. Examples of such foods are regular diets providing all of the animal&#39;s nutrients, treats, supplements and the like. The EPA can be provided in liquids or in pharmaceutical dosage forms such as capsules, tablets, pills, liquids or even parenterally administered such as through syringe. The most important aspect is that the pet be provided an effective amount of EPA to prevent or treat the osteoarthritis. In various embodiments, the route of administration can be oral and the EPA can be incorporated into a food. Foods are generally classified in the pet food industry as “wet” or “dry”. A wet food has a relatively high amount of water and is usually present in a can or a container wherein air is substantially or totally excluded. Examples of such foods are “chunk and gravy”, individual solid particles in the presence of a liquid gravy or a loaf type material which generally takes the shape of the receptacle. The dry food is generally a baked or preferably extruded material, the latter then cut into individual shaped portions, usually known as kibbles. EPA is readily incorporated into a wet food through conventional means. Encapsulation can be employed to protect the EPA from air oxidation in a dry diet. Additionally, use of antioxidants and nitrogen sweeps of packaging can also be employed. This is exemplified by U.S. Pat. No. 4,895,725 which has special emphasis on the micro-encapsulation of specific fish oils. Oils which have high levels of omega-3 fatty acids, are menhaden, salmon, cod and the like.  
         [0021]     The present invention also includes, in various embodiments, methods involving the administration of a composition comprising EPA for reducing the severity and frequency of clinical signs of osteoarthritis and the pain associated with this disease without substantial adverse reactions or side affects. Additionally, in various embodiments, the present invention provides a method of slowing the clinical progression of the osteoarthritic condition of the animal. There is also provided, in various embodiments, a method which substantially improves the overall osteoarthritic condition of the animal so that this benefit can be objectively measured through increased weight bearing in osteoarthritic limbs. The present invention also provides methods involving the administration of EPA in combination with other treatment modalities for osteoarthritis including the administration of various medicaments and/or feeding the animal a weight management diet both of which are known in the art.  
       EXAMPLE 1  
       [0022]     This example illustrates the release of glycosaminoglycan elicited by omega-3 fatty acids in cultured canine cartilage tissue.  
         [0023]     Articular cartilage was obtained from left and right stifles (both femoral condyles and tibial plateau) of dogs. Cartilage explants cultured for 3 days in medium with 10% fetal bovine serum, then washed 3 times in serum free medium. Explants then cultured for 6 days in serum free medium containing 0, 100 or 300 μg/ml n3 fatty acid (EPA, ALA or DHA). After this period in fatty acid medium, all explants were washed 3 times in fatty acid free/serum free medium. Explants were then cultured individually for 4 days in 1 ml of fatty acid and serum free medium containing no additives (C), 10 −6 M retinoic acid (RA) or 50 ng/ml oncostatin M (OSM). Note that not all treatments were possible on all dogs because of cartilage availability. The release of proteoglycan into the medium (μg/mg wet weight) was measured at the termination of culture. In the tables below, the mean and standard deviation of glycosaminoglycan (GAG) release for the triplicate cultures for each of the 4 dogs are given. In addition, the media lactate (μg/mg wt weight) concentrations were given for each treatment.  
                                                                             TABLE 1*                       Dog 1   GAG   GAG       GAG   GAG   Lactate   Lactate       Treatment   Mean   Std Dev   n   Min   Max   Mean   Std Dev                                C   1.363   .497   3   .849   1.84   26.07   33.7       C + carrier   1.630   .306   3   1.31   1.92   21.95   22.6       C + 100   1.590   .291   3   1.29   1.87   23.85   25.4       EPA       C + 300   1.036   .528   3   .57   1.61   NA**       EPA       RA   10.497   1.837   3   8.89   12.5   36   39.3       RA + carrier   7.15   4.527   3   2.   10.5   33.067   45.4       RA + 100   8.677   1.999   3   6.61   10.6   29.367   34.8       EPA       RA + 300   1.593   1.696   3   .436   3.54   26.4   39.1       EPA       OSM   13.6   1.562   3   12.6   15.4   25.367   30.8       OSM+   14.25   6.44   3   7.35   20.1   27.4   33.8       carrier       OSM + 100    6.293   2.301   3   4.34   8.8   33.567   52.5       EPA       OSM + 300    2.167   1.93   3   .93   4.39   20.05   23.8       EPA                 *GAG = glycosaminoglycan; C = serum free medium containing no additives, EPA = eicosapentaenoic acid; RA = retinoic acid; OSM = oncostatin M.            **not analyzed.             
 
         [0024]     As shown in Table 1, significant decrease in GAG release occurred with 100 μg/ml EPA in OSM treated cultures and with 300 μg/ml in RA and OSM treated cultures. There was no significant decrease in media lactate concentrations with any dose of EPA.  
                                                                             TABLE 2*                       Dog 2   GAG   GAG       GAG   GAG   Lactate   Lactate       Treatment   Mean   Std Dev   n   Min   Max   Mean   Std Dev                                C + carrier   .503   .422   3   .127   .96   22.8   NA       C + 100 EPA   .340   .333   3   .1   .72   39.523   24.568       C + 300 EPA   .573   .46   3   .250   1.1   39.2   13.865       OSM + carrier   11.7   5.11   3   7.1   17.2   26.9   4.766       OSM + 100   5.25   3.002   3   2.19   8.19   21.7   9.838       EPA       OSM + 300   2.83   .229   3   2.66   3.09   16.233   3.602       EPA       C + carrier   .973   .222   3   .84   1.23   17.4   NA       C + 100 DHA   .640   .312   3   .45   1   21   6.265       C + 300 DHA   .843   .361   3   .43   1.1   36.2   NA       OSM + carrier   8.73   .777   3   8.1   9.6   25.333   7.106       OSM + 100   8.567   4.219   3   3.7   11.2   28.133   2.715       DHA       OSM + 300   6.073   4.029   3   3.18   10.7   24.8   1.947       DHA       C + carrier   .821   .684   3   .193   1.55   15.567   1.955       C + 100 ALA   1.12   .089   3   1.05   1.22   28.4   13.718       C + 300 ALA   .993   1.104   3   .14   2.24   41.667   14.958       OSM + carrier   7.81   7.471   3   .26   15.2   51.7   28.488       OSM + 100   8.497   4.356   3   4.09   12.8   28.8   4.957       ALA       OSM + 300   6.42   2.730   3   3.44   8.8   55.233   30.305       ALA                 *GAG = glycosaminoglycan; C = serum free medium containing no additives, EPA = eicosapentaenoic acid; RA = retinoic acid; OSM = oncostatin M.             
 
         [0025]     As shown in Table 2, EPA but not ALA or DHA significantly decreased GAG release in OSM treated cultures. There was no significant effect on media lactate concentration by any dose of any of the fatty acids.  
                                                                             TABLE 3*                       Dog 3   GAG   GAG       GAG   GAG   Lactate   Lactate       Treatment   Mean   Std Dev   n   Min   Max   Mean   Std Dev                                C + carrier   2.727   .867   3   2.01   3.69   26.33   4.366       C + 100 ALA   2.117   .428   3   1.81   2.61   24.4   3.995       C + 100 DHA   1.903   .826   3   1.28   2.84   29.35   5.728       C + 100 EPA   1.673   .409   3   1.3   2.11   36.1   NA       C + 300 ALA   2.447   .321   3   2.14   2.18   20.75   7       C + 300 DHA   1.55   .73   3   .73   2.13   28.4   .566       C + 300 EPA   1.567   .387   3   1.3   2.01   10.525   10.854       RA + carrier   20.823   .653   3   20.1   21.37   38.467   4.782       RA + 100 ALA   20.44   .903   3   19.4   21.02   43.233   2.281       RA + 100   21.093   6.881   3   13.38   26.6   45.667   8       DHA       RA + 100 EPA   16.223   6.654   3   8.61   20.93   41.533   2.515       RA + 300 ALA   24.467   2.987   3   21.1   26.8   44.733   4.821       RA + 300   19.457   2.389   3   17.28   22   47.967   9.139       DHA       RA + 300 EPA   1.537   .618   3   1.08   2.24   NA   NA       OSM + carrier   12.773   5.845   3   6.36   17.8   37.867   11.547       OSM-100    22.033   4.596   3   18.4   27.2   32.767   1.815       ALA       OSM-100   11.667   6.007   3   5.5   17.5   32.267   11.467       DHA       OSM-100   17.85   2.051   3   16.4   19.3   39.05   11.526       EPA       OSM-300   23.467   3.102   3   20.3   26.5   34.033   1.38       ALA       OSM-300   11.630   5.069   3   6.79   16.9   30.0   5.963       DHA       OSM-300   8.1   6.767   3   3.79   15.9   21.467   1.93       EPA                 *GAG = glycosaminoglycan; C = serum free medium containing no additives, EPA = eicosapentaenoic acid; RA = retinoic acid; OSM = oncostatin M.             
 
         [0026]     As shown in Table 3, none of the fatty acids significantly altered GAG release from RA- or OSM-stimulated cartilage in this particular animal. There was no change in media lactate associated with any dose of any fatty acid.  
                                                                             TABLE 4*                       Dog 4   GAG   GAG       GAG   GAG   Lactate   Lactate       Treatment   Mean   Std Dev   n   Min   Max   Mean   Std Dev                                C + carrier   1.96   .533   3   1.51   2.55   22.933   4.75       C + 100 ALA   2.103   .107   3   1.98   2.17   20.533   3.478       C + 100 DHA   2.343   .331   3   2   2.66   19.1   2.352       C + 100 EPA   2.687   .996   3   1.72   3.71   23   6.183       C + 300 ALA   1.533   1.244   3   .13   2.5   29.167   22.074       C + 300 DHA   2.307   .361   3   1.93   2.65   24.933   3.4       C + 300 EPA   2.1   .455   3   1.64   2.55   24.767   13.004       RA + carrier   14.113   3.89   3   9.64   16.7   34.533   12.368       RA + 100 ALA   12.547   6.348   3   5.94   18.6   39.933   11.594       RA + 100   11.28   7.123   3   4.79   18.9   25.6   11.766       DHA       RA + 100 EPA   14.393   2.9   3   11.23   16.93   32.967   4.219       RA + 300 ALA   14.093   6.138   3   8.98   20.9   59.367   31.166       RA + 300   11.3   6.815   3   3.5   16.1   25.333   11.684       DHA       RA + 300 EPA   9.093   1.316   3   8.26   10.61   25.1   4.67       OSM + carrier   16.083   3.544   3   12.05   18.7   31.2   5.991       OSM + 100   11.7   2.19   3   9.43   13.8   26.333   9.25       ALA       OSM + 100   24.967   3.262   3   21.2   26.9   36.833   5.066       DHA       OSM + 100   15.883   4.316   3   11.95   20.5   27.237   6.34       EPA       OSM + 300   19.557   3.909   3   15.5   23.3   26.667   6.099       ALA       OSM + 300   16.4   6.27   3   9.4   21.5   36.233   20.342       DHA       OSM + 300   13.493   5.752   3   7.54   19.02   27.8   2.722       EPA                 *GAG = glycosaminoglycan; C = serum free medium containing no additives, EPA = eicosapentaenoic acid; RA = retinoic acid; OSM = oncostatin M.             
 
         [0027]     As shown in Table 4, EPA at 300 μg/ml, but not any other fatty acid at any dose, significantly decreased GAG release from RA treated cultures. There was a significant decrease in media lactate concentration in control. RA- and OSM-treated cultures with the 300 μg/ml OSM pre-treatment.  
       EXAMPLE 2  
       [0028]     This example illustrates the incorporation of n-3 fatty acids into canine chondrocyle membranes.  
         [0029]     The majority of these experiments were performed using monolayer cultures, however, in a single experiment, the incorporation of fatty acids into explant cultures of canine cartilage was analyzed.  
         [heading-0030]     Monolayer Cultures  
         [0031]     Over 24 or 48 hours there was no incorporation of the 18:3 n-3 fatty acid ALA into chondrocyte membranes from two dogs. The % 18:3 n-3 in chondrocytes incubated in medium alone was &lt;1 out of 5 (range=0.3-0.9%) and after 24 or 48 hours of incubation with 100 or 300 μg/ml ALA this percentage had not significantly changed (range=0.3-2.5%).  
         [0032]     Over 48 hours there was significant incorporation of the 20:5 n-3 fatty acid EPA into chondrocyte membranes from one dog. The % 20:5 n-3 increased from &lt;1% (range=0.2-0.6%) to approximately 7% (range=5.6-8%) when cultures were treated with 100 or 300 μg/ml EPA for 48 hours. The incorporation was not different when cultures were performed in the presence or absence of 5% FCS.  
         [0033]     Over 48 hours there was significant incorporation of the 20:5 n-3 fatty acid EPA but not the 18:3 n-3 fatty acid ALA into chondrocyte membranes from one dog (doses of 300 μg/ml for each fatty acid). The % 20:5 n-3 increased from &lt;1% to approximately 15%.  
         [0034]     Over 3 or 6 days there was significant incorporation of the 20:5 n-3 fatty acid EPA into chondrocyte membranes from one dog (dose of 300 μg/ml EPA). The % 20:5 n-3 increased from &lt;1% to 16-18% with no difference between 3 and 6 days incubation.  
         [0035]     Explant Culture  
         [0036]     Over 6 days there was apparent incorporation of the 20:5 n-3 fatty acid EPA, but not the 18:3 n-3 DHA or the n-6 fatty acid AA (arachadonic acid) into cartilage explants from one dog (dose of 300 μg/ml for each fatty acid). The % n-3 20:5 increased from 0% (none detectable) to approximately 2%.  
         [0037]     These data indicated that EPA, but no other n-3 fatty acid was incorporated into canine chondrocyte membranes in either monolayer or explant cultures.  
       EXAMPLE 3  
       [0038]     This example illustrates the effect of n-3 Fatty Acids on Canine Chondrocyte Metabolism.  
         [0039]     To assess the potential effect of n-3 fatty acids on protein and proteoglycan metabolism in canine cartilage, cultures were set up as described in Example 1 except for the final 4 days of culture, no catabolic stimuli were added (i.e., all “control” cultures). During the final 24 hours of culture (1)  35 SO 4 , or (ii)  35 S-methionine and  35 S-cysteine were added to the medium to radiolabel newly synthesized proteoglycans and proteins, respectively. The incorporation of radiolabel into the cartilage matrix was measured at the termination of culture. No attempt was made to quantitate loss of radiolabelled material from the cartilage over the 24-hour labeling period. The mean and standard deviation of the incorporation of  35 SO 4  (“PG”) or  35 S-methionine and  35 S-cysteine (“PROT”) as DPM/mg wet weight are shown in Table 5 below.  
                                                                         TABLE 5*                                       PG   PG       PROT   PROT           Treatment   Mean   Std Dev   N   Mean   Std Dev                                        Carrier   292.667   53.144   3   574.333   198.336           100 ALA   246.333   100.779   3   503.667   184.218           100 DHA   156.0   82.529   3   503.667   81.365           100 EPA   537.333   161.81   3   442.0   72.746           300 ALA   443.0   205.385   3   393.667   34.962           300 DHA   123.333   38.24   3   564.333   220.048           300 EPA   275.667   161.661   3   504.0   44.542                         *PG = incorporation of  35 SO 4  in DPM/mg wet weight; PROT = incorporation of  35 S-methionine and  35 S-cysteine as DPM/mg wet weight; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; ALA = alpha-lenolenic acid.             
 
         [0040]     As shown in Table 5, there was no significant effect of any n-3 fatty acid on protein synthesis and incorporation into the matrix. EPA at 100 μg/ml significantly increased proteoglycan synthesis and incorporation. No other dose or fatty acid significantly altered proteoglycan synthesis and incorporation into the cartilage matrix.  
         [0041]     Reverse transcription-PCR was used to measure the mRNA message expression levels of matrix proteinases (aggrecanases-1 and -2), cyclooxygenases-1 &amp; -2, lipoxygenases-5 and 12, and potential autocrine cytokines and their receptors (e.g. IL-1, IL-6 and TNF).  
         [0042]     The results of this study found that aggrecanase-1 and aggrecanase-2 mRNA messages were expressed in “normal” canine cartilage tissue. In addition, some dogs expressed mRNA message of cyclooxygenase-2 (COX-2) message although there were no signs of joint pathology in these animals. This enabled monitoring the effects of n-3 and n-6 fatty acid supplementation on mRNA expression of aggrecanases and COX-2 in unstimulated canine articular cartilage explants. EPA was the only fatty acid able to reduce the mRNA message for the degradative enzymes, aggrecanase-1 and aggrecanase-2, in canine articular cartilage. This demonstrated the ability of EPA to “turn off” the genes responsible for cartilage degradation.  
       EXAMPLE 4  
       [0043]     This study illustrates the effects of omega-3 fatty acids in canine osteoarthritis clinical studies.  
         [0044]     Three clinical studies were conducted in pet dogs clinically diagnosed with osteoarthritis. Veterinary general practitioners and orthopedic specialists enrolled client owned dogs that met a specific eligibility criteria. All patients were required to: have radiographic evidence of osteoarthritis with measurable clinical manifestations of disease, based on historical accounts by pet owners and physical examinations by veterinarians; be otherwise healthy and free of concurrent diseases based on physical exam, CBC, blood chemistry, and urinalysis; maintain regimen of therapy if receiving medications or supplements prescribed for osteoarthritis during the 30 days prior to enrolling in the study.  
         [0045]     The following measurements were made.  
         [0046]     Serum fatty acid profile: This was determined by a gas chromatography method involving extraction of fatty acids by chloroform and methanol mixture (2:1), methylation using boron trifluride-methanol (BF 3 :MeOH) reagent followed by flame ionization detection (FID). Fatty acid methyl esters were identified by comparison of retention times with those of known standards and quantitated using an internal standard.  
         [0047]     Veterinary clinical evaluation: Veterinarians conducted both a physical exam and a clinical evaluation of the patient&#39;s osteoarthritic condition during the screening phase and at the conclusion of each of the feeding intervals over the course of the clinical trial. Veterinarians assessed the severity of five osteoarthritic parameters: lameness, reluctance to bear weight, reduction in range of motion, reluctance to hold up contra-lateral limb, and pain on palpation of the joint. Changes in severity scores for these individual parameters were measured over the duration of the feeding period. A comprehensive veterinary clinical assessment of the impact of dietary intervention on the osteoarthritic condition of patients was derived by combining the changes in severity scores for all five individual parameters.  
         [0048]     Pet owner subjective evaluation: Pet owners were required to complete an enrollment questionnaire prior to participating in the study and additional questionnaires at the conclusion of each of the feeding intervals over the course of the clinical trial. 
        Enrollment questionnaire—pet owners rated the observed frequency and severity of the most common signs of canine osteoarthritis including difficulty rising from rest, limping, stiffness, soreness when touched, lagging behind during walks, yelping or whimpering in pain, aggressive behaviors, difficulty in running, difficulty in walking, difficulty in climbing steps, difficulty in jumping, difficulty in playing, impaired mobility, and overall activity level. In addition, owners rated the overall osteoarthritic condition of their pet.     Feeding questionnaire—pet owners rated both the frequency and change in severity of the signs of canine osteoarthritis which were benchmarked during enrollment. In addition, the pet owners rated the severity of their animal&#39;s pain associated with osteoarthritis.        
 
         [0051]     Force plate gait analysis: Dogs were evaluated at each respective institution using a computerized biomechanics force plate at day 0, 6 weeks, and 12 weeks. The plate was mounted centrally in and flush with the surface of a 10 m walkway. A handler trotted dogs across the force plate and an observer evaluated each pass across the plate to confirm foot-strikes and gait. A trial was considered valid if there were distinct ipsilateral fore foot and hind foot strikes while the dog was trotted across the force plate at a velocity of 1.7 to 2.0 m/s, with an acceleration variation of −0.5 to 0.5 ms 2 . During each trial, the dog&#39;s forward velocity was measured, using a millisecond timer and two photoelectric switches. Each trial was videotaped for review and confirmation of valid foot-strikes. Care was taken to ensure that the dog triggered the timer and that a consistent speed (as perceived by the handler and observer) was maintained across the plate during each trial.  
         [0052]     Five valid trials for each test period were obtained for each affected limb and each ipsolateral limb of each dog. Orthogonal ground reaction forces of peak vertical force, vertical impulse, braking and propulsive peak forces, and braking and propulsion impulses were measured and recorded by a specialized software program. (Acquire, Sharon Software, DeWitt, Mich.), All forces were normalized with respect to body weight in kilograms. Data from the valid trial for each limb were averaged to obtain a mean value for each force or impulse at each time period.  
         [0053]     Ground reaction force data were compared between treatment and placebo groups as a percentage difference between lame and ipsolateral limbs at each time period. Percentage change of ground force data on the lame limb were compared at the beginning and end of the feeding period.  
       Study #1  
       [0054]     A canine study was conducted to evaluate the dietary effect of feeding high levels of n-3 fatty acids to dogs diagnosed with osteoarthritis. Eighteen veterinary general practitioners were recruited to enroll patients in the study. A total of 131 dogs were randomly assigned to two dietary treatments and fed for 180 days. The test and control foods had similar macronutrient profile, but were significantly different fatty acid composition (Table 6). The test diet contained high levels of ALA, EPA, and DHA, and was formulated with a low n-6/n-3 ratio. The control diet was a leading selling commercially available dog food, with typical levels of n-3 fatty acids and n-6/n-3 ratio characteristic for the industry.  
                                                 TABLE 6*                                       Control Food   Test           Dietary Nutrient   (%)   Food (%)                                        Protein   23.2   19.9           Fat (total)   13.9   13.6           CHO 2  (NFE)   54.7   53.3           C18:3 n-3 (ALA)   0.12   2.8           C20:4 n-6 (AA)   0.03   0.06           C20:5 n-3 (EPA)   &lt;0.01   0.38           C22:6 n-3 (DHA)   &lt;0.01   0.31           Sum n-6   1.99   2.53           Sum n-3   0.09   3.48           n6/n3 ratio   22.8   0.7                         *NFE = Soluble carbohydrate content as Nitrogen Free Extract; ALA = alpha-lenolenic acid; AA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.             
 
         [0055]     Serum fatty acids and pet owner evaluations were recorded at 0, 45, 90 and 180 days. Serum fatty acid profiles were significantly modulated by the test food. The test group had significantly higher concentrations of n-3 fatty acids (P&lt;0.01), specifically EPA, DHA, a-ALA, significantly lower concentrations of AA (P&lt;0.01), and significantly lower n-6:n-3 ratios (P&lt;0.01) as compared to the control group at the conclusion of each feeding interval (Table 7). The test group showed significant improvements for rising from rest, running, and playing at day 45 and walking at days 90 and 180 as compared to the control group based on pet owner observations (P&lt;0.05), even in the presence of a strong placebo effect (Table 8).  
                                                                   TABLE 7*                           Canine Mean Serum Fatty Acid Levels (mg/dl)                Group   Day 0   Day 45   Day 90   Day 180                    C18:3 n-3 (α - ALA)   Control   1.10   0.89   0.52   0.53           Test   1.05   5.61   6.51   7.13       C20:4 n-6 (AA)   Control   71.35   66.34   68.03   68.21           Test   64.32   45.90   46.13   42.65       C20:5 n-3 (EPA)   Control   1.14   0.90   0.67   0.93           Test   1.28   16.28   18.64   19.94       C22:6 n-3 (DHA)   Control   2.67   2.03   1.70   1.98           Test   2.93   11.31   12.24   12.17       Sum n-6   Control   141.08   138.72   137.85   140.28           Test   130.85   118.87   128.71   123.99       Sum n-3   Control   4.95   3.84   2.93   3.51           Test   5.36   33.20   37.39   39.24       n-6:n-3 ratio   Control   33.33   37.95   51.59   51.39           Test   33.90   7.47   8.63   6.92                 *ALA = alpha-lenolenic acid; AA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.*             
 
         [0056]    
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 8* 
               
             
             
               
                   
               
               
                   
               
               
                 Pet Owner Observed Change in Severity of Osteoarthritis* 
               
             
          
           
               
                   
                 Day 0-45 
                   
               
             
          
           
               
                 Osteoarthritic 
                 P 
                 Day 45-90 
                 Day 90-180 
               
             
          
           
               
                 Sign 
                 Group 
                 Mean 
                 Value 
                 Mean 
                 P Value 
                 Mean 
                 P Value 
               
               
                   
               
               
                 Rising 
                 Control 
                 1.77 
                 .041 
                 1.77 
                 nsd** 
                 1.93 
                 nsd** 
               
               
                 from rest 
                 Test 
                 1.56 
                   
                 1.84 
                   
                 1.91 
               
               
                 Running 
                 Control 
                 1.81 
                 .037 
                 1.83 
                 nsd** 
                 1.94 
                 nsd** 
               
               
                   
                 Test 
                 1.56 
                   
                 1.71 
                   
                 1.91 
               
               
                 Walking 
                 Control 
                 1.71 
                 nsd** 
                 2.00 
                 .018 
                 2.19 
                 .002 
               
               
                   
                 Test 
                 1.69 
                   
                 1.71 
                   
                 1.75 
               
               
                 Playing 
                 Control 
                 1.83 
                 .008 
                 1.90 
                 nsd** 
                 2.06 
                 nsd** 
               
               
                   
                 Test 
                 1.50 
                   
                 1.78 
                   
                 1.97 
               
               
                   
               
               
                   *Osteoarthritis severity rating scale: 1 = better, 2 = no change, 3 = worsened.    
               
               
                   **nsd = no significant difference.    
               
             
          
         
       
     
       Study #2  
       [0057]     A canine study was conducted to evaluate the dietary effect of feeding high levels of n-3 fatty acids to dogs diagnosed with osteoarthritis. Two veterinary orthopedic specialists enrolled patients in the study. A total of 38 dogs were randomly assigned to two dietary treatments and feed for 90 days. The test and control diets were manufactured from the same lots of foods as described above (Table 6).  
         [0058]     Serum fatty acids, force plate gait analysis, and veterinary clinical assessments were recorded at 0, 45, and 90 days. Serum fatty acid profiles were significantly modulated by the test food. The test group had significantly higher serum concentrations of n-3 fatty acids (P&lt;0.01), specifically EPA, DHA, ALA, significantly lower concentrations of AA at day 90 (P&lt;0.01), and significantly lower n-6:n-3 ratios (P&lt;0.01) as compared to the control group at the conclusion of each feeding interval (Table 9).  
                                                                                                     TABLE 9*                           Canine Serum Fatty Acid Levels (mg/dl)                Day 0   Day 45   Day 90                        P       P       P       Fatty Acids   Group   Mean   Value   Mean   Value   Mean   Value                    C18:3 n3 (α - ALA)   Control   0.89   0.7764   0.34   &lt;.0001   0.27   &lt;.0001           Test   0.98       4.45       5.04       C20:4 n-6 (AA)   Control   55.55   0.6880   50.78   0.0736   55.95   0.0001           Test   57.13       41.94       38.01       C20:5 n-3 (EPA)   Control   1.19   0.7000   0.34   &lt;.0001   0.20   &lt;.0001           Test   1.54       11.52       11.89       C22:6 n-3 (DHA)   Control   4.30   0.4323   1.82   &lt;.0001   1.32   &lt;.0001           Test   3.37       11.15       11.21       Sum n-6   Control   122.85   0.2508   112.46   0.0148   114.60   0.0036           Test   113.61       91.72       89.85       Sum n-3   Control   6.36   0.8335   2.57   &lt;.0001   1.79   &lt;.0001           Test   5.90       27.14       28.13       n-6:n-3 ratio   Control   32.54   0.2521   66.66   &lt;.0001   75.90   &lt;.0001           Test   45.90       8.48       3.59                 *ALA = alpha-lenolenic acid; AA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.*             
 
         [0059]     A biomechanical assessment of the dogs&#39; most severe osteoarthritic limb was objectively evaluated using force plate gait analysis (Table 10). Vertical peak force is the key parameter measured to determine weight bearing of the affected limb. There was no significant change in mean vertical peak force over the duration of the 90 day feeding for the control group (P=0.91), while there was a significant increase in mean vertical peak force over time for the test group (P=0.01). The percent mean change in vertical peak force was also significantly different between groups (P&lt;0.05), indicating that the test group increased weight bearing in the affected limb, while the control group displayed no change in weight bearing over the course of the study. Weight bearing ability can also be represented by displaying the frequency distribution of percent change in vertical peak for each dietary group. Only 31% of animals in the control group showed improvement in weight bearing after the 90 day feeding, while 82% of the dogs in the test group increased weight bearing over the course of the study.  
                                                                                                             TABLE 10                           Vertical Peak Force                Day 0   Day 90   Change (Day 0-90)                    P       P       Mean = 0   % Mean           Group   Mean   Value   Mean   Value   Mean Change   Pr &gt; 1 t 1   Change   Pr &gt; 1 t 1                    Control   72.80   0.5981   72.63   0.9323   −0.17   0.9144   −0.58   0.0443       Test   69.51       73.21       3.71   0.0103   5.35                  
 
         [0060]     The subjective clinical evaluations performed by the veterinary orthopedic surgeons provided additional support for the efficaciousness of the test diet. Based upon the comprehensive veterinary clinical assessment, a significantly greater percent of dogs were evaluated as improved that consumed the test food as compared to dogs that consumed the control food (P&lt;0.05). The veterinary specialists also observed a greater percent of dogs in the test group displaying a reduction in pain on palpation of the joint as compared to the control group (P=0.05).  
       Study #3  
       [0061]     A canine study was conducted to determine the dose effect of feeding high levels of n-3 fatty acids to dogs diagnosed with osteoarthritis. Twenty-eight veterinary general practitioners enrolled patients in the study. A total of 177 dogs were randomly assigned to three dietary treatments and fed for 90 days. Approximately two-thirds of the dogs participating in the study were receiving medications and/or supplements prescribed for treating osteoarthritis, in addition to consuming the therapeutic diets being evaluated. The three test foods had similar macronutrient profiles, but varied in composition of EPA and DHA, with variable A containing the lowest levels and variable C containing the highest levels (Table 11).  
                                                                   TABLE 11*                                       Test Variable %                Dietary Nutrient   A   B   C                            Protein   19.97   19.51   19.37           Fat (total)   13.78   15.34   19.55           CHO 2  (NFE)   53.92   52.34   47.66           C18:3 n-3 (ALA)   2.65   1.18   1.10           C20:4 n-6 (AA)   0.11   0.18   0.24           C20:5 n-3 (EPA)   0.50   1.18   1.69           C22:6 n-3 (DHA)   0.34   0.80   1.15           Sum n-6   2.70   2.45   2.14           Sum n-3   3.54   3.53   4.52           n6/n3 ratio   0.76   0.7   0.47                         *NFE = Soluble carbohydrate content as Nitrogen Free Extract; ALA = alpha-lenolenic acid; AA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.             
 
         [0062]     Serum fatty acids, pet owner evaluations, and veterinary clinical assessments were recorded at 0, 21, 45, and 90 days. Serum fatty acid profiles were significantly modulated by all dietary variables. The dogs fed test variables B &amp; C had significantly higher serum concentrations of n-3 fatty acids (P&lt;0.01), specifically EPA, DHA, ALA, significantly lower concentrations of n-6 fatty acids, specifically AA (P&lt;0.01), and significantly lower n-6:n-3 ratios (P&lt;0.01) as compared to the dogs feed test variable A at the conclusion of each feeding interval (Table 12).  
                                                                   TABLE 12*                           Canine Serum Fatty Acid Levels (mg/dl)            Fatty       Day 0   Day 21   Day 45   Day 90       Acids   Group   Mean   Mean   Mean   Mean                    C18:3   A   1.34   5.65   5.29   5.63       n-3   B   1.29   3.36   3.99   3.82       (ALA)   C   1.25   2.92   3.32   3.29       C20:4   A   76.37   51.10   47.54   47.77       n-6   B   73.15   41.55   38.94   37.0       (AA)   C   70.05   37.35   36.86   34.73       C20:5   A   1.32   18.74   18.51   19.26       n-3   B   1.54   26.14   29.87   30.03       (EPA)   C   1.85   34.42   35.71   39.04       C22:6   A   3.50   13.75   13.84   13.88       n-3   B   4.72   18.47   19.98   20.16       (DHA)   C   3.91   21.01   21.47   22.49       Sum   A   150.38   114.38   110.12   112.70       n-6   B   143.93   93.83   95.87   92.10           C   139.97   79.71   82.65   80.74       Sum   A   6.16   38.14   37.65   38.77       n-3   B   7.55   47.96   53.84   54.01           C   7.01   58.35   60.50   68.83       n-6:n-3   A   29.99   5.65   3.48   3.75       ratio   B   28.09   3.36   1.92   1.79           C   32.30   2.92   2.02   1.73                 *ALA = alpha-lenolenic acid; AA = arachidonic acid; EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid.             
 
         [0063]     Pet owners reported improvements in 13 of 14 individual osteoarthritic signs for dogs consuming any of the dietary variables for 21 days (Table 13). Additionally, pet owners reported a decrease in severity for 13 of 14 individual osteoarthritic signs for dogs consuming any of the dietary variables for 90 days (Table 14). Pet owners also reported a significant reduction in the frequency of observable osteoarthritic signs after the dogs consumed any of the dietary variables for 90 days (Table 15).  
                                                           TABLE 13                           Pet Owner Observed Improvements in Osteoarthritic Signs (Day 0-21)                Osteoarthritic           Mean = 0           Sign   Diet   Mean   Pr &gt; 1 t 1                            Rising from   A   −0.439   0.0002           rest   B   −0.738   &lt;.0001               C   −0.763   &lt;.0001           Limping   A   −0.720   &lt;.0001               B   −0.731   &lt;.0001               C   −0.837   &lt;.0001           Stiffness   A   −0.537   &lt;.0001               B   −0.783   &lt;.0001               C   −0.627   &lt;.0001           Soreness   A   −0.750   0.0005               B   −0.800   0.0002               C   −0.379   0.0451           Lagging behind   A   −0.564   0.0004           on walks   B   −0.909   &lt;.0001               C   −0.531   0.0022           Pain   A   −0.476   0.0245               B   −0.478   0.0184               C   −0.889   0.0002           Aggression   A   0.000   1.0000               B   −0.313   0.1050               C   −0.429   0.1401           Running   A   −0.524   0.0004               B   −0.682   &lt;.0001               C   −0.674   &lt;.0001           Walking   A   −0.553   0.0007               B   −0.750   &lt;.0001               C   −0.667   &lt;.0001           Stair Climbing   A   −0.449   0.0012               B   −0.667   &lt;.0001               C   −0.723   &lt;.0001           Jumping   A   −0.362   0.0049               B   −0.600   &lt;.0001               C   −0.542   &lt;.0001           Playing   A   −0.622   &lt;.0001               B   −0.763   &lt;.0001               C   −0.487   0.0014           Impaired   A   −0.528   0.0005           Mobility   B   −0.700   &lt;.0001               C   −0.564   0.0001           Activity Level   A   −0.745   &lt;.0001               B   −0.857   &lt;.0001               C   −0.865   &lt;.0001                         The above “p” values refer to the mean change from day 0 to day 21.             
 
         [0064]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                   
               
               
                 Difference in Pet Owners Severity Rating (day 0-90) 
               
             
          
           
               
                   
                 Osteoarthritic Sign 
                 Group 
                 Mean 
                 Pr &gt; t 
               
               
                   
                   
               
             
          
           
               
                   
                 Rising from rest 
                 A 
                 −0.463 
                 &lt;.0001 
               
               
                   
                   
                 B 
                 −0.633 
                 &lt;.0001 
               
               
                   
                   
                 C 
                 −0.518 
                 &lt;.0001 
               
               
                   
                 Limping 
                 A 
                 −0.489 
                 0.0003 
               
               
                   
                   
                 B 
                 −0.588 
                 &lt;.0001 
               
               
                   
                   
                 C 
                 −0.681 
                 &lt;.0001 
               
               
                   
                 Stiffness 
                 A 
                 −0.255 
                 0.0420 
               
               
                   
                   
                 B 
                 −0.483 
                 &lt;.0001 
               
               
                   
                   
                 C 
                 −0.589 
                 &lt;.0001 
               
               
                   
                 Soreness 
                 A 
                 −0.810 
                 &lt;.0001 
               
               
                   
                   
                 B 
                 −0.920 
                 &lt;.0001 
               
               
                   
                   
                 C 
                 −0.926 
                 &lt;.0001 
               
               
                   
                 Lagging behind on 
                 A 
                 −0.657 
                 &lt;.0001 
               
               
                   
                 walks 
                 B 
                 −0.531 
                 0.0014 
               
               
                   
                   
                 C 
                 −0.448 
                 0.0094 
               
               
                   
                 Pain 
                 A 
                 −0.684 
                 0.0002 
               
               
                   
                   
                 B 
                 −0.571 
                 0.0009 
               
               
                   
                   
                 C 
                 −0.667 
                 0.0010 
               
               
                   
                 Aggression 
                 A 
                 −0.750 
                 0.0234 
               
               
                   
                   
                 B 
                 −1.000 
                 0.0025 
               
               
                   
                   
                 C 
                 −1.000 
                 0.0751 
               
               
                   
                 Running 
                 A 
                 −0.579 
                 &lt;.0001 
               
               
                   
                   
                 B 
                 −0.558 
                 &lt;.0001 
               
               
                   
                   
                 C 
                 −0.605 
                 &lt;.0001 
               
               
                   
                 Walking 
                 A 
                 −0.294 
                 0.0358 
               
               
                   
                   
                 B 
                 −0.643 
                 &lt;.0001 
               
               
                   
                   
                 C 
                 −0.595 
                 &lt;.0001 
               
               
                   
                 Stair Climbing 
                 A 
                 −0.419 
                 0.0024 
               
               
                   
                   
                 B 
                 −0.489 
                 0.0002 
               
               
                   
                   
                 C 
                 −0.689 
                 &lt;.0001 
               
               
                   
                 Jumping 
                 A 
                 −0.571 
                 0.0003 
               
               
                   
                   
                 B 
                 −0.479 
                 0.0011 
               
               
                   
                   
                 C 
                 −0.773 
                 &lt;.0001 
               
               
                   
                 Playing 
                 A 
                 −0.606 
                 0.0002 
               
               
                   
                   
                 B 
                 −0.571 
                 0.0003 
               
               
                   
                   
                 C 
                 −0.694 
                 &lt;.0001 
               
               
                   
                 Lameness 
                 A 
                 −0.484 
                 0.0045 
               
               
                   
                   
                 B 
                 −0.778 
                 &lt;.0001 
               
               
                   
                   
                 C 
                 −0.667 
                 &lt;.0001 
               
               
                   
                 Activity Level 
                 A 
                 −0.409 
                 0.0009 
               
               
                   
                   
                 B 
                 −0.704 
                 &lt;.0001 
               
               
                   
                   
                 C 
                 −0.551 
                 &lt;.0001 
               
               
                   
                   
               
               
                   
                   The above “p” values refer to the mean change from day 0 to day 90.    
               
             
          
         
       
     
         [0065]     Dogs consuming higher concentrations of n-3 fatty acids were reported to have more significant improvement in osteoarthritic condition and more significant reduction in the progression of osteoarthritis than those dogs receiving the lowest dosage, based on veterinarians clinical assessments (Table 16). There was no significant difference in improvement in osteoarthritic condition or reduction in the progression of osteoarthritis between the group receiving medications and/or supplements and the non-medicated group (Table 17). This indicates that the therapeutic diets work synergistically with other therapies or at least not withstanding other therapies by providing additional benefit to dogs suffering from osteoarthritis.  
         [0066]     An extremely low incidence of adverse reactions or side effects were reported among dogs participating in this study. Only five dogs out of the 215 animals assigned to food were reported to have diarrhea and vomiting, which could possibly be attributed to consuming one of dietary variables. Similar incidence of adverse reactions or side effects were reported for those dogs consuming the therapeutic diets in the previous two studies discussed ({fraction (1/88)} and {fraction (1/26)} for examples 1 and 2 respectively.  
                                                           TABLE 15                           Difference in Pet Owners Frequency Rating (day 0-90)                Osteoarthritic Sign   Group   Mean   Pr &gt; t                            Rising from rest   A   −0.370   &lt;.0001               B   −0.467   &lt;.0001               C   −0.509   &lt;.0001           Stiffness   A   −0.098   0.2929               B   −0.373   &lt;.0001               C   −0.421   &lt;.0001           Soreness   A   −0.381   0.0146               B   −0.680   &lt;.0001               C   −0.821   &lt;.0001           Running   A   −0.447   0.0004               B   −0.395   0.0009               C   −0.477   &lt;.0001           Jumping   A   −0.357   0.0027               B   −0.354   0.0015               C   −0.467   &lt;.0001           Playing   A   −0.455   0.0013               B   −0.297   0.0238               C   −0.667   0.0010           Limping   A   −0.239   &lt;.0165               B   −0.365   &lt;.0001               C   −0.396   &lt;.0001           Lagging   A   −0.571   &lt;.0001           Behind on   B   −0.643   &lt;.0001           Walks   C   −0.500   0.0004           Aggression   A   −0.417   0.0536               B   −0.467   0.0175               C   −0.167   0.5741           Walking   A   −0.206   0.0911               B   −0.558   &lt;.0001               C   −0.447   0.0002           Stair Climbing   A   −0.302   0.0069               B   −0.348   0.0014               C   −0.457   &lt;.0001           Impaired   A   −0.250   0.0643           Mobility   B   −0.436   0.0005               C   −0.667   &lt;.0001                      
 
         [0067]    
       
         
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                 TABLE 16 
               
               
                   
               
               
                   
               
               
                 Diet 
                 N 
                 Mean 
                 P 
                   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Progression of Osteoarthritic Condition 
               
             
          
           
               
                 A 
                 55 
                 2.327 
                 0.2891 
                 A vs B 
               
               
                 B 
                 62 
                 2.177 
                 0.1619 
                 B vs C 
               
               
                 C 
                 59 
                 1.983 
                 0.0168 
                 A vs C 
               
             
          
           
               
                 Overall Change in Osteoarthritic Condition 
               
             
          
           
               
                 A 
                 54 
                 3.148 
                 0.1675 
                 A vs B 
               
               
                 B 
                 62 
                 2.871 
                 0.0787 
                 B vs C 
               
               
                 C 
                 59 
                 2.525 
                 0.0024 
                 A vs C 
               
               
                   
               
             
          
         
       
     
         [0068]    
       
         
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
             
               
               
               
               
               
             
           
               
                 TABLE 17 
               
               
                   
               
               
                   
               
               
                 Diet 
                 Medicated 
                 N 
                 Mean 
                 P 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Progression of Osteoarthritic Condition 
               
             
          
           
               
                 A 
                 No 
                 22 
                 2.273 
                 0.6665 
               
               
                 A 
                 Yes 
                 33 
                 2.364 
               
               
                 B 
                 No 
                 23 
                 2.130 
                 0.7109 
               
               
                 B 
                 Yes 
                 39 
                 2.205 
               
               
                 C 
                 No 
                 28 
                 2.071 
                 0.4003 
               
               
                 C 
                 Yes 
                 31 
                 1.903 
               
             
          
           
               
                 Overall Change in Osteoarthritic Condition 
               
             
          
           
               
                 A 
                 No 
                 21 
                 3.143 
                 0.9770 
               
               
                 A 
                 Yes 
                 33 
                 3.152 
               
               
                 B 
                 No 
                 23 
                 2.696 
                 0.3247 
               
               
                 B 
                 Yes 
                 39 
                 2.974 
               
               
                 C 
                 No 
                 28 
                 2.750 
                 0.1285 
               
               
                 C 
                 Yes 
                 31 
                 2.323 
               
               
                   
               
             
          
         
       
     
         [0069]     All references cited in this specification are hereby incorporated by reference. Any discussion of references cited herein is intended merely to summarize the assertions made by their authors and no admission is made that any reference or portion thereof constitutes relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.  
         [0070]     The description of the invention is merely exemplary in nature and, thus, variations that do not depart from the gist of the invention are intended to be within the scope of the invention. Such variations are not to be regarded as a departure from the spirit and scope of the invention.