WO2015162246A1 - Novel use of a nutraceutical composition in animal feed - Google Patents

Abstract

The invention relates to the use of nutraceutical compositions comprising as active ingredients at least one poly-unsaturated fatty acid from microbial source for improving cognitive functions and/or for the treatment or prevention of age-related disorders in pets. The compositions are of primary interest for use in dog and cat food.

Description

Novel use of a nutraceutical composition in animal feed

The present invention relates to a novel use of a nutraceutical composition for animals, especially for pets as dogs and cats, comprising as active ingredients polyunsaturated fatty acids from microbial source.

More specifically, the invention relates to the use of such nutraceutical compositions as feed additives or nutraceuticals in order to improve cognitive functions and/or for the treatment or prevention of age-related disorders in animals, in particular in dogs and cats.

There is an increasing interest in the development of compounds as well as

nutraceuticals compositions that may be used to improve learning memory and alertness for example in elderly pets, and/or that may be used to treat mental disorders or to prevent the development of mental disorders.

In the context of this invention the term "disorder" also encompasses diseases.

The term nutraceutical as used herein denotes usefulness in both the nutritional and pharmaceutical field of application. Thus, the novel nutraceutical compositions can find use as supplement to feed and as pharmaceutical formulations for parenteral application which may be solid formulations such as capsules or tablets, or liquid formulations, such as solutions or suspensions. As will be evident from the foregoing, the term nutraceutical composition also comprises supplement compositions containing the aforesaid active ingredients (Inventive Ingredients) as well as feed and feedstuff including premixes used therefore, especially for pets, which contain the mixture of Inventive Ingredients.

The term "polyunsaturated fatty acids" as used herein (herein also referred as PUFA) denotes a polyunsaturated fatty acid in an esterified (e.g., as triglycerides or ethyl esters) or a free form. A Preferred poly-unsaturated fatty acid is docosahexaenoic acid (4,7,10,13,16,19-docosahexaenoic acid, DHA).

The source of the PUFA is a microorganism, particularly algae. The microorganism can be used in a whole cell form or as a lipid extracted from the microorganism. Preferably, the microorganism is from the order Thraustochytriales, more preferably from the genus Thraustochytrium or Schizochytrium, and in particular Schizochytrium sp. (ATCC 20888 and ATCC 20889). Cognition, broadly defined, refers to mental processes such as perception, awareness, learning, memory, and decision making. Cognition allows an animal to take in information about the environment, process, retain, and make decisions how to act. These mental processes cannot be measured directly. Different cognitive tasks have been developed to evaluate learning and memory in dogs. In order to better understand the behavioural consequences associated with cognitive changes in ageing, a series of behavioural reactivity tests to evaluate stimulus-evoked behaviour were developed. Based on the animal's response to the various stimuli, these tests can distinguish cognitively impaired aged dogs from those who successfully age.

In the following some tasks are summarized:

• The human interaction tests:

The test assesses the reaction of a dog to the presence of a person. A person familiar to the dog sits in the middle of a room while the dog is free to explore. Young dogs spend significantly more time in physical contact with the person than normal aged dogs. Cognitively unimpaired aged dogs spent a lot of time close to the person without actually making physical contact, while impaired aged dogs pay little attention to the person.

• The silhouette and model dog test:

These tests measure social responsiveness to other dogs. The silhouette tests uses a cardboard figure of a dog taped to the wall and the model dog tests uses a life-size plastic replica of a dog sitting in the centre of a room to assess social responsiveness. Young dogs are more responsive to the artificial conspecifics showing significantly more investigative sniffing than both groups of old dogs.

• The curiosity test:

The test measures exploratory behaviour by allowing dogs to examine and play with a variety of toys. Young dogs explore and contact the novel objects significantly more than old dogs. The cognitively impaired aged dogs show almost no interest in toys.

• The mirror test:

It examines an individual animal's reaction to its mirror image. Young dogs and normal aged dogs have mild reactions to the reflection but habituated to the image fairly quickly. The cognitively impaired dogs spent significantly more time reacting to the reflection. Their response can include jumping and barking at the dog in the mirror. • Landmark discrimination learning tasks:

The dogs are presented with two identical objects. To obtain food reward, the animals are required to respond selectively to the object closest to a specific external cue.

• Oddity discrimination learning tasks:

In these task the animal is presented with three objects, two identical and one different. To obtain a reward, the animal is required to respond to the odd object. Difficulty can be increased based on similarity of positive and negative objects.

• Size discrimination learning task and reversal learning tasks:

The test evaluates the animal's ability to learn to distinguish two objects that differ only in size in order to locate a food reward.

Aging involves a progressive deterioration and loss of the cellular processes and physiological functions of an organism that ultimately increase the likelihood of death. The aging process involves a number of molecular pathways such as oxidative stress, cellular stress resistance, neuroendocrine systems, nutrient sensing systems and insulin signaling.

Age related diseases and disorders in general can be grouped as follows:

• Central nervous system disorders: The aging process often causes atrophic changes in the brain. There are substantial age-related declines in brain function, i.e., decrease in norepinephrine and dopamine synthesis.

• Autonomic nervous system disorders: Since the homeostatic mechanisms slow and weaken during advancing age, changes are reflected in the alterations of sympathetic and parasympathetic responsiveness, i.e., decreased sensitivity of baroreceptor and change in thermoregulation.

• Eye and ear disorders: Eye Disorders - Physiological changes of presbyopia and lens opacification subsequently cause decreased accommodation and increased susceptibility to glare. These physiological changes often result in decreased visual acuity as well as blindness. Ear Disorders - For the ear, the physiological change is decreased high frequency acuity, making it difficult to discriminate words if noise is present in the background. Consequently, there is deafness and a decrease in acoustic acuity. • Cardiovascular system disorders (diseases include hypertension, coronary artery disease.

• Congestive heart failure as well as heart block or arrhythmia).

• Respiratory system disorders (Respiratory diseases include emphysema, dyspnea, and hypoxia).

• Gastrointestinal system disorders.

• Endocrine system disorders (include the development of diabetes mellitus, thyroid dysfunction)

• Hematological and immune system disorders.

• Muscular and skeletal system disorders (osteoporosis)

• Cancer.

And specific for pets the following diseases and disorders are known:

• Oxidative damage is, besides plaque and neurofibrillary tangles deposition, another age-dependent type of pathology. Over the course of ageing and normal cellular metabolism, oxidants are produced, that if not reduced by endogenous antioxidants, can damage proteins, lipids and nucleotides. These forms of damage can be measured by biochemical assays for lipid peroxidation

(malondialdehyde), protein carbonyl formation, enzyme dysfunction and the accumulation of DNA/RNA oxidative damage. In canine, these markers of oxidative damage progressively increase with age. Further preliminary evidence suggests that oxidative damage to RNA may precede the accumulation of β- amyloid.

• β-amyloid deposit: One form of neuropathology that was described in dogs over 40 years ago is the development of senile plaques. Plaques are deposits of a toxic peptide called β-amyloid (Αβ) in the space between neurons. Αβ is derived from a longer β-amyloid precursor protein through the activity of two enzymes: β- secretase/BACE or γ-secretase. These enzymes can produce either Αβ that is 40 or 42 amino acids long. The longer species Αβ1 -42 aggregates more rapidly then the shorter Αβ1 -40. The earliest senile plaques contain predominantly Αβ1 -42. Αβ that is associated with blood walls, called Αβ angiopathy, almost entirely consist of the shorter 40 amino acid long peptide. Αβ is deposited in the dog brain with age in a specific pattern: the prefrontal cortex appears to develop Αβ earlier than other regions with dogs over the age of 10 years. This may suggest that prefrontal cortex functions, such as reversal learning, behavioural rigidity and preservative behaviours may be early manifestations of Αβ pathology. As in human brain ageing, the extent of senile plaque formation is higher in dogs with cognitive dysfunction. The extent and location of Αβ-pathology is associated with

performance on specific learning tasks, which are thought to be mediated by these same brain regions (Head, 2002).

• Neurofibrillary tangles. They are formed by the intracellular accumulation of tau protein, which makes up the cytoskeleton of neurons. In Alzheimer's disease, tau protein becomes hyperphosphorylated and forms paired helical filaments, which fill the cytoplasm and lead to neuron dysfunction (Cotman, 2002).

The statement above can be summarized as follows:

• Due to the advances in veterinary medical care and changes in socioeconomic status, dogs in domestic settings are living longer today then in past times.

• An estimated 18 million pet dogs in the United States alone are more than 7 years of age.

• Increased life span appears to be inherently associated with age-related disease process such as (cancer, renal disease, and) cognitive decline.

• As the cognitive aging process is associated with progressive decline in cellular function, it follows that a larger number of older dogs may also be at risk to develop behavioral changes related to cognitive decline.

• These behavioral alterations are often manifested as disorientation (D), altered interactions with the family members (I), disruptions in sleep (S), loss of house training (H), and altered activity levels (A). Collectively, these behavioral attributes may be identified by the acronym "DISHA".

Surprisingly, it has now been found that the present compositions act on different critical signaling pathways involved in aging and hence delay aging and age-related diseases more potently than the individual components. More precisely it has been found that compositions containing as active ingredient PUFA's from microbial source may be useful to improve cognitive function such as perception, awareness, learning, memory, and decision making.

Moreover it has now been found that compositions containing PUFA's have a significant additive and synergistic effect as nutraceutical in preventing and treating of central nervous system disorders, autonomic nervous system disorders, eye and ear disorders, cardiovascular system disorders, congestive heart failure, gastrointestinal system disorders, hematological and immune system disorders, muscular and skeletal system disorders and cancer in pets.

In yet another aspect, the present invention relates to the use of at least one polyunsaturated fatty acid from microbial source to improve cognitive functions and/or for the treatment or prevention of age-related disorders in animals, in particular in dogs and cats.

The present invention further refers to a method to improve cognitive functions and/or for the treatment or prevention of age-related disorders in animals, in particular in dogs and cats, which comprises administering to a pet an effective amount of said composition.

Advantageous embodiments of the invention become evident from the dependent claims.

PUFA may be incorporated into conventional pet food e.g., into dry pet food by spraying a solution, for example an aqueous solution containing the Inventive

Ingredients on the food composition while thoroughly mixing the composition, or by adding the Inventive Ingredients to the dough. Inventive Ingredients may be added simultaneously, e.g. at the same time and even as a premix, or consecutively as single Inventive Ingredient at a time or as a premix. Premixes may also include one or more of the other components of the final composition.

The nutraceutical compositions of the present invention may be in the form of a premix and may contain PUFA's in an amount sufficient to administer to an adult dog (weighing about 20 kg) a dosage from about 1 mg/day to about 5000 mg/day, preferably from about 3 mg/day to about 2500 mg/day.

In the context of this invention "treatment" also encompasses co-treatment as well as prevention. "Prevention" can be the prevention of the first occurrence (primary prevention) or the prevention of a reoccurence (secondary prevention). The following Examples illustrate the invention further. The Examples are for illustrative purposes only and are not intended, nor should they be construed, as limiting the invention in any manner. Those skilled in the art will appreciate that variations and modifications can be made without violating the spirit or scope of the invention.

Said composition may be provided in the form of a concentrate, for example as a simple powdery mixture of its components; or in the form of granules as are obtained for example by spray drying an aqueous slurry of the components or by extruding the mixture; or in the form of tablets as are obtained by compressing the powder into tablets with conventional tableting methods and machinery.

The pet food according to the present invention may be based on any conventional pet food. There is a wide range of pet foods available which may be grouped into (a) complete diets, (b) complementary diets, and (c) snacks and treats. Complete diets may be fed in addition to water for an extended period as the sole source of nutrients and will provide for all the energetic and nutrient needs of the animal and the physiological state for which it is intended. Complementary diets normally are not sufficient to ensure that all nutrient and energy requirements are met unless fed in combination with another foodstuff or diet. Snacks and treats are appetizers or for occasional feeding and are considered as complementary products. There are, however, a number of products available intended to form part of the daily diet or playing a role in animal well-being.

The pet food of the present invention may be in a dry, canned, semi-moist or baked form. Typical components of such compositions, in addition to Inventive Ingredients, are crude protein, crude fat, carbohydrates (NfE), starch, crude fibers, and ash, further on minerals, trace elements, vitamins, fatty acids, protein and amino acids, choline, carnitin, dietary fiber and substances required for balanced diets of the different animal species. Basic ingredients of such food compositions are

• Crude Protein including proteins and N-containing compounds of non- proteinaceous nature, e.g. acid amides, amines, free amino acids, ammonium salts, alkaloids;

• Crude Fat including neutral fats, lipoids (phospho-, sphingolipids, steroids) and other ethersoluble compounds;

• N-free Extractions (NFE) including polysaccharides (starch, glycogen), soluble saccharides (glucose, fructose, saccharose, lactose, maltose and oligosaccharides), and soluble fractions of cellulose, hemicellulose, lignin and pectines;

• Crude Fibers including insoluble fractions of cellulose, hemicellulose, lignin and other components of the cell wall like suberin, cutin etc.;

• Ash including minerals (macrominerals such as calcium, phosphorus, sodium, chloride, potassium, magnesium, and microminerals, i.e., trace elements, such as iron, copper manganese, zinc, iodine, selenium,) and further inorganic substances e.g. silicate.

• Vitamins including vitamins A, B1 , B2, B6, B12, D, pantothenic acid, niacin, folic acid, linolic acid and choline.

Further components may, e.g. L-carnitine, chondroitin sulfate, glucosamine,

glutamine/glutamic acid, arginine, taurine and hydroxyproline.

Typical components which provide the ingredients for a dog food composition, in addition to Inventive Ingredients, comprise, e.g., chicken/beef/turkey, liver, broken pearl barley, ground corn, brute fat, whole dried egg, fowl protein hydrolyzate, vegetable oil, calcium carbonate, choline chloride, potassium chloride, iodinized salt, iron oxide, zinc oxide, copper sulfate, manganese oxide, sodium selenite, calcium iodate, provitamin D, vitamin B1 , niacin, calcium panthothenate, pyridoxin

hydrochloride, riboflavin, folic acid, vitamin B12.

Typical components which provide the ingredients for a cat food composition, in addition to Inventive Ingredients, comprise beef, chicken meat, dried chicken liver, lamb meat, lamb liver, pork, turkey meat, turkey liver, poultry meal, fish meal, fowl protein hydrolysate, animal fats, plant oils, soy bean meal, pea bran, maize gluten, whole dry egg, ground corn, corn flour, rice, rice flour, dry sugar beet molasses, fructooligosaccharides, soluble fibers, plant gums, cellulose powder, clay, bakers yeast, iodized sodium chloride, calcium sulfate, sodium triphosphate, dicalcium phosphate, calcium carbonate, potassium chloride, choline chloride, magnesium oxide, zinc oxide, iron oxide, copper sulfate, iron sulfate, manganese oxide, calcium jodate, sodium selenite, provitamin D, thiamine, niacin, calcium pantothenate, pyridoxine hydrochloride, riboflavin, folic acid, vitamin B12, taurin, L-carnitine, caseine, D- methionine.

Wet pet food contains between about 70 and about 85 % moisture and about 15 and about 25 % dry matter. A typical wet food for adult dogs may, e.g. comprise, in addition to Inventive

Ingredients, at minimum 24 % protein, 15 % fat, 52 % starch, 0.8 % fiber, 3 % linolic acid, 0.6 % calcium, 0.5 % phosphorus, the Ca:P ratio being 1 :1 , 0.2 % potassium, 0.6 % sodium, 0.09 % chloride, 0.09 % magnesium, 170 mg/kg of iron, 15 mg/kg of copper, 70 mg/kg of manganese, 220 mg/kg of zinc, 4 mg/kg of iodine, 0.43 mg/kg of selenium, 74000 lU/kg of vitamin A, 1200 lU/kg of vitamin D, 1 1 mg/kg of vitamin B1 , 6 mg/kg of riboflavin, 30 mg/kg of pantothenic acid, 20 mg/kg of niacin, 4.3 mg/kg of pyridoxine, 0.9 mg/kg of folic acid, 0.2 pg/kg of vitamin B12, 2500 mg/kg of choline, 2500 mg/kg cholin, all percentages being based on dry weight of the total food composition.

A typical wet food for adult cats may, e.g. comprise, in addition to Inventive Ingredients, at minimum 44 % protein, 25 % fat, 20 % starch, 2.5 % fiber, 0.8 % calcium, 0.6 % phosphorus, 0.8 % potassium, 0.3 % sodium, 0.09 % chloride, 0.08 % magnesium, 0.25 % taurin, 170 mg/kg of iron, 15 mg/kg of copper, 70 mg/kg of manganese, 220 mg/kg of zinc, 4 mg/kg of iodine, 0.43 mg/kg of selenium, 74000 lU/kg of vitamin A, 1200 lU/kg of vitamin D, 1 1 mg/kg of vitamin B1 , 6 mg/kg of riboflavin, 30 mg/kg of pantothenic acid, 20 mg/kg of niacin, 4.3 mg/kg of pyridoxine, 0.9 mg/kg of folic acid, 0.2 pg/kg of vitamin B12, 2500 mg/kg of choline, 2500 mg/kg cholin, all percentages being based on dry weight of the total food composition.

Dry pet food contains between about 6 and about 14 % moisture and about 86 % or more dry matter.

A typical dry food for adult dogs may, e.g. comprise, in addition to Inventive

Ingredients, at minimum 25 % protein, 12 % fat, 41 .5 % starch, 2.5 % fiber, 1 % linolic acid, 1 % calcium, 0.8 % phosphorus, the Ca:P ratio being 1 :1 , 0.6 % potassium, 0.35 % sodium, 0.09 % chloride, 0.1 % magnesium, 170 mg/kg of iron, 35 mg/kg of copper, 70 mg/kg of manganese, 220 mg/kg of zinc, 4 mg/kg of iodine, 0.43 mg/kg of selenium, 15000 lU/kg of vitamin A, 1200 lU/kg of vitamin D, 11 mg/kg of vitamin B1 , 6 mg/kg of riboflavin, 30 mg/kg of pantothenic acid, 20 mg/kg of niacin, 4.3 mg/kg of pyridoxine, 0.9 mg/kg of folic acid, 0.2 pg/kg of vitamin B12, 2500 mg/kg of choline, all percentages being based on dry weight of the total food composition.

A typical food for adult cats may, e.g. comprise, in addition to Inventive Ingredients, at minimum 32 % protein, 15 % fat, 27.5 % starch, 11 % dietetic fibers, 4.5 % fiber, 3.4 % linolic acid, 0.08 % arachionic acid, 0.15 % taurin, 50 mg/kg L-carnitin, omega 6/3 = 5, 1 % calcium, 0.8 % phosphorus, the Ca:P ratio being at least 1 :1 , 0.6 % potassium, 0.4 % sodium, 0.6 % chloride, 0.08 % magnesium, 190 mg/kg of iron, 30 mg/kg of copper, 60 mg/kg of manganese, 205 mg/kg of zinc, 2.5 mg/kg of iodine, 0.2 mg/kg of selenium, 25000 lU/kg of vitamin A, 1500 lU/kg of vitamin D, 20 mg/kg of vitamin B1 , 40 mg/kg of riboflavin, 56 mg/kg of pantothenic acid, 153 mg/kg of niacin, 14 mg/kg of pyridoxine, 3.2 mg/kg of folic acid, 0.2 mg/kg of vitamin B12, 3000 mg/kg of choline, all percentages being based on dry weight of the total food composition.

Dry food may be prepared, e.g., by screw extrusion including cooking, shaping and cutting of raw ingredients into a specific kibble shape and size in a very short period of time, while simultaneously destroying detrimental micro-organisms. The ingredients may be mixed into homogenous expandable dough and cooked in an extruder

(steam/pressure) and forced through a plate under pressure and high heat. After cooking, the kibbles are then allowed to cool, before optionally being sprayed with a coating which may include liquid fat or digest including liquid or powdered hydrolyzed forms of an animal tissue such as liver or intestine from, e.g., chicken or rabbit. Hot air drying then reduces the total moisture content to 10 % or less.

Canned (wet) food may be prepared, e.g., by blending the raw ingredients including meats and vegetables, gelling agents, gravies, vitamins, minerals and water. The mix is then fed into cans on a production line, the lids are sealed on and the filled cans are sterilized at a temperature of about 130°C for about 50 to 100 min.

The following Examples illustrate the invention further.

The following examples were designed to assess the effectiveness of dietary supplementation with moderately high level of DHA on cognitive functional and visual processing of aged canines. Twenty eight cognitively experienced beagle dogs between 8.5 and 1 1 years of age were placed on a control diet, Joy's Special Meal, which was selected because if lacked any DHA. After an initial wash-in, subjects were given 10 training sessions on a delayed-non-matching-to-position task, which provides a measure of visuospatial memory. Performance on the DNMP task was used to assign 26 of the dogs to two cognitively equivalent groups. Two animals, which were unreliable responders, were dropped from the study. One of the groups was then maintained on a commercially available diet that lacked any DHA. The second group was maintained on the same diet containing a DHA supplement obtained from dried Schizochytrium sp. The cognitive assessment phase started after two months of treatment and continued for the next four months. All dogs were tested successively on a concurrent discrimination learning protocol (to assess episodic memory), a contrast discrimination learning protocol, a delayed-non-matching-to-position test (to assess visual spatial memory), a variable contrast discrimination protocol (to assess visual processing) and retention of concurrent discrimination learning (to assess long term memory).

The subjects in the DHA supplemented diet were found to perform at higher level that the subjects on the control diet on every one of the cognitive assessment protocols. Furthermore, the differences between the groups achieved statistical significance at the 0.05 level on the contrast discrimination learning protocol (p=0.035; student T-test), the variable contrast discrimination protocol (differences were statistically significant at contrasts of 0.03 (p=0.026) and 0.05 (p=0.01 1 ) on the first test session of test for long- term retention of the concurrent discrimination task (p=0.0488). There was also a significant group by trials interaction on the concurrent discrimination learning task, which revealed superior performance on the DHA supplemented group over the last part of training. The results therefore indicate that 6 months of DHA supplementation can provide significant cognitive benefits in aged beagle dogs, and more generally support the contention that DHA has positive effects on brain health in aged dogs.

In contrast to the other cognitive tasks, the groups on the delayed-non-matching-to- position task did not differ significantly. Because this was a well learned task, the absence of significant effect probably reflects insufficient duration to observe a clear age-dependent decline in the control animals. This result is also consistent with data from human subjects suggesting task differences in sensitivity to DHA in elderly subjects.

The study also assessed several measures of body structure and health, including body weight, body fat, lean mass, clinical chemistry and hematology. With respect to body composition, there were no significant differences between the control and supplemented group. The clinical chemistry, hematology and coagulation data did not reveal any clinically relevant effects.

Introduction and Objectives

Docosahexaenoic acid (DHA) is an omega 3 fatty acid that is found in ^'high

concentrations in the brain and is widely believed to have a critical role in cellular structure function. There has also been considerable speculation that deficits in DHA levels or pathogenic alteration of DHA contribute to cognitive dysfunction and dementia that may develop over the course of aging (e.g., Lukiw & Bazan, 2008). However, randomized control trials of the effects of supplementation with DHA on cognitive function in the elderly and demented have not revealed consistent benefits (e.g., Dacks Shineman @ Fillit, 2013) On the other hand, epidemiological study consistently suggested that high levels of DHA consumption provide protection against age-related cognitive decline or dementia (Huang, 2010) as well as in animal models

The objective of the study is to determine the effects of DHA from dried whole-cell Schizochytrium sp. on cognitive function in aged dogs, as assessed by a battery of tests. Secondary objectives include demonstrating positive effects on structural, biochemical, and vascular measures of brain function, as well as inflammation.

Although this was a non-GLP study, specific GLP measures were employed. These included: (1 ) having the study quality assured by Vivocore's quality assurance committee; (2). Obtaining daily measurements of food intake, (3) The laboratory that performs the clinical chemistry and analysis will be audited by Vivocore's quality assurance committee. (4) Archiving food samples from each bag in a -80°C (±2°C) freezer until the end of the study.

Methods

Study Design Summary

A parallel group design was used to assess the effectiveness of dietary

supplementation with DHA from Dried Schizochytrium sp. on Cognitive Function in Aging Dogs. Twenty six aged and cognitively experienced dogs were initially selected from a larger group of 28 for participation in the study. For the first 42 days of the study, all 28 animals were acclimated to the base diet, which was selected for the study because it did not any DHA. Subject selection was based on performance on a variable-delay delayed-non-matching-to-position (DNMP; see Chan et al., 2002). The two subjects dropped were the animals that showed the most frequency response failures. Baseline DNMP performance was used to rank animals and place them into two cognitively equivalent groups. One of the groups was then maintained on a control diet, which lacked any DHA. The second group was placed on the identical diet, which was supplemented with dried Schizochitrium sp. (S17B) at a concentration of 4.0g/kg

The treatment phase started on the day following group placement. The first 51 days provided the initial wash-in and no other procedures were introduced during this time. Over the next 124 days, the groups were tested on a battery of cognitive tests, including concurrent discrimination learning (days 51 -90), contrast sensitivity learning (days 100-140), retest on DNMP (days 1157-164), performance on variable contrast protocol (days 157-164) and retention of concurrent discrimination learning task (days 167-174). Table 1 shows the schedule of operations. Study Day Procedures Comments

Animal Body Weights

Beginning of Twice Daily

Beginning of In-life phase Observations

-42

Animal Identification

Complete Blood Count

Biochemistry

Begin feeding subjects control

-42 to -1 Baseline Acclimation

diet

To be conducted on one day

-41 to -31 Veterinary Examinations

during this period

Food intake measured daily for

-30 Beginning of food intake monitoring

duration of study

-28 Body weight monitoring Animal Body Weights

Animals maintained on control

-27 to -12 Baseline

diet

Baseline Testing on Visuospatial

-1 1 to -2 DNMP Task (Variable Delay)

Function

Complete Blood Count

Animal Allocation Biochemistry

-1 Randomization to Treatment Fatty Acid Analysis

Groups Blood Collection Animal Body Weights

QMR Imaging

Animals fed as per assigned

O to 50 Beginning of Treatment treatment groups

51 to 90 Episodic Memory Testing Concurrent Discrimination Task

Training on Visual Acuity (Contrast Maximum Contrast & Reduced

100-140

Sensitivity) Contrast

145 to 154 Testing on Visuospatial Function DNMP Task (Variable Delay)

Testing on Visual Acuity (Contrast

157 to 164 Variable Contrast

Sensitivity)

167 to 174 Retention Testing Concurrent Discrimination Task

Complete Blood Count

Biochemistry

Coagulation

175 Blood Collection & QMR Imaging Fatty Acid Analysis

Animal Body Weights

QMR Imaging

Cytokine Analysis

Table 1 : Schedule of Operations

Test diets Joys Special Diet dog food Diet (ME=3510 kcal/kg) served as the control diet and was fed to all of the dogs during the baseline phase, from Days -42 to -1 .

On day 0, the dogs were placed into two treatment groups, one of which was randomly selected to receive the control diet and the other group received control diet

supplemented with Dried Schizochytrium sp. (S17B) at 4 grams per kilogram of chow.

Blood collection

On Days -42, -1 and Day 175 ~ 8ml of whole blood was collected from a suitable vein and placed into K2EDTA tubes and SST tubes for both complete blood count and biochemistry, which include the following : A/G Ratio, Albumin, Globulin, Total Protein, Alkaline Phosphatase, ALT, Bilirubin (Total), Cholesterol, Creatinine, Glucose, Blood Urea Nitrogen, Triglycerides, WBC, RBC, Hemoglobin, Hematocrit, MCH, MCV, MCHC, Platelet Count, Lymphocytes, Monocytes, Eosinophils, Basophils, S. Neutrophils and B. Neutrophils. Tubes were stored at 2-8°C until sent to the Antech Diagnostics for analyses.

On day 175, blood coagulation based upon Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) was also analyzed.

Fatty Acid Analysis

On study Days -1 and 175, -12 ml of whole blood was collected from a suitable vein as per standard operating procedures for the purpose of fatty acid analysis. The animals were fasted for a minimum of 8 hours prior to this collection. The blood samples were sent to the study sponsor for analyses and interpretation.

Plasma Separation

Approximately 12ml of whole blood was placed into K3 EDTA collection tubes and separated as per standard operating procedures. This provided ~6ml of plasma which was separated into 12 aliquots of -500 microlitres. Six aliquots were sent out for analysis to the study sponsor. The remaining six aliquots were retained for archiving purposes in a -80°C freezer.

RBC Separation

After the plasma separation, the remaining RBC in the K3 EDTA tubes was isolated as per standard operating procedures. ~6ml RBC was collected and separated into 12 aliquots of -500 micro litres. Six aliquots were sent out for analysis and the remaining six aliquots were retained for archiving purposes in a -80°C freezer.

Food Consumption and Body Weights

Food consumption was calculated daily for the duration of the study starting at Day -30. On these days, any food remnants will be weighed back and recorded on the Feed Record Form. Feeding of the dogs will be conducted according to standard operating procedures. From Day -42 to -1 all animals were fed using Joy's Special Diet

(ME=3510 kcal/kg), which lacks DHA. The test diet was provided by the study sponsor.

Beginning on Day 0 and continuing until the completion of the study, each animals was fed according to its assigned treatment groups. Those animals fed the control group continued on the feeding regimen above. The dogs assigned to the treatment group were fed the control diet plus the supplement, which consisted of dried Schizochitrium sp. (S17B) at a concentration of 4.0g/kg of diet. All animals were given an equal amount of time, approximately 1 hour, to consume their food. The earliest time that feeding was started on any given day was 16:00.

The finalized protocol stipulated that animals were to be fed an amount equal to 26 g/kg. This was modified by Protocol Amendment 2, signed on May 17, which indicated that the total food provided could be adjusted either up or down if necessary to maintain body condition. Protocol Amendment 3, on June 3, adjusted upwards the food provided to 28.6 grams/kg. Finally, on Aug 2, protocol amendment 4 permitted reduction of total food by 50 grams at a time to maintain body condition scores.

Body weights were assessed every 14 days starting on Day -42 following the facilities standard operating procedures.

Test Apparatus

All cognitive testing utilized a test apparatus (the TGTA) designed for cognitive assessment of dogs. The apparatus consisted of a plastic box (approximately 3'x5'), which was originally described by Milgram et al., 1994.The front contained three- height-adjustable gates through which the dog responds. The experimenter is separated from the dog by a partition containing a one-way mirror and a hinged-door that when opened allows the tray to be presented to the dog. The tray contains one medial and two lateral food wells. Figure 1 is a photograph of the test system, which consisted of the test apparatus and computer. The computer program, Varicog© (CanCog Technologies Inc.), was used for all cognitive testing. The software controls all test sequences and is under administrator control, which assures that the parameters, once set, cannot be modified by the technicians doing the testing. In addition, the same sequence of correct locations was used on each test session for all subjects.

Cognitive Test Protocols

Delayed Non-Matching-To-Position-Task

Animals were tested on the DNMP - Variable Delay task as per standard operating procedures at Baseline over 10 consecutive days (Days -1 1 to -2). There were three delay intervals, set at 5, 55 and 105 S, and there were 18 trials daily, 6 at each delay. The inter-trial interval was set at 30 S. Subjects were allowed to correct their first incorrect response on each session. In scoring, response failures were counted as .5 correct.

Animals were again tested on this task using identical parameters from Days 145 to 154 to examine long-term memory.

Concurrent Discrimination Learning

The dogs were tested on the Concurrent Discrimination task over a maximum of 40 consecutive days, from Days 51 to 90. The task assessed the animals' ability to learn to discriminate four different pairs of objects (A vs B, C vs D, E vs F, and G vs H) simultaneously. The object pairs used were constructed from Lego blocks in such a way that each pair differed on three dimensions, color, size and shape, (see set of object pairs used is shown in Figure 2).

On the day 51 , subjects received a preference test, which consisted of 20

presentations of the object pairs, with each pair presented on 5 occasions. Selecting either object led to food reward; for each pair, the object selected most frequently was designated as the preferred object. On the subsequent training, the dogs were given 24 presentations per session with an inter-trial interval of 15 S and with each object pair presented 6 times. On each trial, the dogs were rewarded only if they choose their initially non-preferred object. For the first incorrect response for each object pair, a correction procedure was used, in which the subject was allowed to reverse its choice. The dogs were tested for either a maximum of 40 consecutive daily sessions following the preference test or until they achieve a score of 85% or higher on two consecutive days.

Contrast Sensitivity

On days 100 to 140, all subjects were trained on a contrast sensitivity protocol, which was designed to assess visual processing abilities. On day 100 the dogs were given a preference test, in which they were given 10 presentations of two objects, a black circle on a white background vs a black triangle on a white background. The object selected most frequently was deemed to be the preferred object. In the case of ties, the preferred object was designed by a coin toss.

Starting on day 101 , subjects were trained to approach their initially non-preferred object to obtain reward. On the initial training, the contrast was at 100% (see Figure 3). Subjects were given 10 trials per day until they either completed 40 test sessions, or successfully completed the following two stage criterion:

Stage 1 Criterion: 90% correct on a given day or 80% or better on 2 consecutive days with no response failures.

Stage 2 Criterion: On 3 consecutive days following completing the Stage 1 criterion, subjects must achieve at least 70% or higher and to respond correctly on at least 30 trials (see Protocol Amendment 6).

After acquiring the Stage 2 criterion, subjects were tested at progressive lower contrasts, in which the contrasts were reduced to 40%, 25%, and 5% by decreasing the luminance of the shapes. At each new level of contrast, the dogs had to complete the two stage criteria defined above. If a dog completed the Stage 2 criterion at 5% luminance, testing was then discontinued.

During both training and testing on Contrast Sensitivity, animals will be allowed to correct their first incorrect response of each session.

On Days 175 to 181 subjects were retested for contrast sensitivity using the variable contrast task. This entailed testing every animal for 8 successive sessions with 2 trials per session at contrasts of 0.01 , 0.03, 0.05, 0.15, 0.20 and 0.40.

Quantitative Magnetic Resonance Imaging Body composition was assessed on study days -1 and -175 using Quantitative

Magnetic Resonance (QMR) Imaging. The imaging used an EchoMRI-D unit and quantified each of the following measures: a. Lean mass

b. Fat mass

c. Free water

d. Total body water

Statistical Analysis

Statistical analyses were completed using both Statistica and Excel. Statistica was used initially for all analyses of variance, and non-parametric statistics. Excel was used for independent sample comparisons using the Student T-Test. All T-tests examining cognitive variables were one-tailed because of the a priori prediction that cognition would either be improved in the group provided with DHA, impaired in the group given food lacking DHA or possibly both. Statistical analysis of all non-cognitive measures used a 2-tail analysis.

The statistical analysis also took into consideration consistency of responding. The general procedure for dealing with response failures was to give the subject a score of .5, which is score that would have been obtained by chance. However, response failures also prolonged training because of the a prior criterion that dogs respond on at least 30 trials before passing the learning criterion. Because response failures depress performance scores, but are not necessarily indicative of cognitive impairment, we used a criterion of 1 .5 failures per session on the average, to justify excluding any given animal the statistical analysis of the data. Generally, all data sets were analyzed twice: the first included the data from all of the subjects and the second analysis was after excluding data from animals that showed frequent response failures.

Results

Group placement:

The subjects originally selected for baseline participation are shown in Appendix 1 , Worksheet 1 . Group placement was based solely on performance on the DNMP during the 10 baseline trials.

To establish that the groups did not differ at baseline, the groups were compared with a repeated measures ANOVA with delay as a within subject variable and grouping as a between subject variable. The results of the analysis revealed a highly significant effect of delay (p=0.000) and no other significant main effects or interactions. Figure 4 shows that the performance of the groups were equivalent and that the delay effect was due to more accurate performance e at 5 S than at 55 or 105 seconds, both of which differed significantly from performance at the 5 S delays (p=0.000 for both). To assure that the groups were still equivalent after removal of the two subjects that did not complete the study, we performed a separate analysis with both Quartz and Almond excluded. The results are shown in Table 3.3 and again indicated a significant effect of delay

(p=0.000) and no other significant main effects or interactions.

Performance on Cognitive Test Protocols

Concurrent Discrimination Learning Data

The raw data from the concurrent discrimination learning task is given in Appendix 5. One animal from the treatment Group (Achilles) was dropped from this phase of the study after the 17th session because of unreliable responding. The initial analyses included the data from all of the subjects, independently of the subjects' consistency in responding. The subjects on the DHA supplemented food required a mean of 27.5 testing sessions and 232 incorrect responses to complete testing.

The next analysis was done after dropping the data from 4 subjects (2 from each group) who averaged more than 1 .5 incorrect responses per trial. The groups were then compared on both errors to criterion and sessions to criterion. The results of the analysis revealed superior performance by the animals receiving the DHA

supplemented diet (for errors, M= 25.1 vs. 30.4 for trials, M=199,05 vs. 235.65), but the differences did not achieve statistical significance.

A third analysis focussed on the probability of a given subject successfully completing the learning criterion. In the control group, 5 of 12 animals were able to successfully complete training. By contrast, 9 of 12 animals in the DHA group passed the learning criterion within 40 test sessions. A chi squared test was used to compare the probability of success between the group. The difference between the groups was marginally significant (p=0.0977) indicating a greater likelihood of success for the animals receiving the DHA supplement.

The final analysis compared groups on every test session using a repeated measures ANOVA. The analysis revealed a highly significant effect of session (p=0.000) and a significant interaction between group and session (p=0.0187). Figure 6 shows that the significant session effect is due to both groups showing progressive improvement with repeated testing. In addition, the group by session interaction reflects the DHA group showing higher levels of performance over the last part of training, but not during initial training.

Performance on the Variable Contrast Object Discrimination Task

Figure 10 indicates that the contrast effect reflected progressively poorer performance with decreasing contrasts beyond .25. Figure 10 also shows that the Group differences were greater at intermediate contrasts (See Figure 10). Accordingly, the groups were compared at each contrast level using Student T- Tests. The results of the analysis are revealed statistically significant differences at contrasts of 0.03 and 0.05 (p=0.026 and 0.01 1 respectively).

We also performed a second analysis in which two animals, one from each group, were dropped because of inconsistent responding. The results again revealed a statistically significant main effect of contrast (p=0.000). There was also a statistically significant effect of Group (p=0.0457, with a one -tailed test). Comparing the groups at each level of contrast again revealed statistically significant effects at contrasts of .03 (p=0.0006) and 0,05 (p=0.006)

Body Composition

The results of the analysis of corrected level of body fat revealed statistically significant effects of group (p=0.0165), Sex (p=0.038) and treatment (p=0.00016). There was also a statistically significant interaction between treatment and sex (p=0.0189). Table 2 shows that Group 1 had a lower level of body fat at both baseline and at the end of the study. Table 2 also shows body fat increased at the end of the study for both groups.

Table 3 shows that the significant effect of Sex reflected greater total fat in males than in females at both the start and the end of the study.

Corrected level of lean mass

Table 4 illustrates that the significant effect of treatment was due to lean mass increasing in both groups. Table 5 shows that the effect of sex reflected showing greater lean mass than females.

Total levels of body fat The final analysis compared the groups with respect to total levels of body fat. The results revealed a marginally significant effect of Group (=0,07819), a statistically significant effect of Treatment (p=0.0000) and a statistically significant interaction between treatment and sex (p=0.0219). Table 8 shows that the marginally significant effect of group and the statistically significant effect of treatment reflected higher total levels of body fat (at baseline and end of treatmentO in the DHA group, and higher levels of body fat at the end of the study than at the beginning.

Food Intake and Body Weight

The results of the analysis revealed a statistically significant effect of day (p=0.0000) and no other statistically significant main effects or interactions.

Figure 12 shows that while there were no group differences, the DHA supplemented group weighed more than the control, on the average.

Blood Chemistry, Clinical Chemistry and Coagulation Data

Blood chemistry (CBC) and clinical chemistry were taken on entry to the study, at baseline and at the end of the study. The coagulation data was only taken once, at the end of the study.

Blood Chemistry

Table 4 summarizes the results of the analyses of the CBC data. There were no statistically significant group by treatment interactions, indicating that two diets did differentially affect any of the measures. There was one statistically significant difference between groups, that of lymphocytes. This result reflected lower levels in the DHA group at both baseline and at the end of the study. Finally, Table 5 shows that on all but five of the measures, there were significant differences at the end of the study for the groups combined compared to the levels at the beginning of the study.

Clinical Chemistry

Table 5 summarizes the results of the clinical chemistry analyses (see also Appendix 3, Worksheet 9). Table 9 shows two significant group by treatment interactions (globulin and biocarbonate) and two marginally significant interactions (total protein and chloride). The globulin data is a reflection of the DHA Group showing a greater decrease than the control group, although at the end of the studies the groups did not differ, and level was still higher in the DHA Group. The bicarbonate result reflects a greater increase in bicarbonate levels in the DHA group than the control. Table 5 also shows that on all but four of the measures, there were significant differences at the end of the study for the groups combined compared to the levels at the beginning of the study. Finally, there was a significant group effect of bicarbonate, reflecting higher levels at both baseline and at the treatment phase in the DHA Group.

Discussion and Conclusions

The primary goal of the present study was to demonstrate that dietary supplementation with DHA has beneficial cognitive effects in aged dogs, but the extent of the benefit appears to vary as a function of task, with significant effects observed in concurrent discrimination learning, acquisition of a contrast sensitivity test, performance on a variable contrast version of the contrast sensitivity test and long-term memory of a concurrent discrimination learning task. By contrast, there was no statistically significant change in performance of a spatial memory task.

The present study was also designed to assess the effect of dietary supplementation with DHA on body composition, measures of blood chemistry, clinical chemistry and coagulation . The results revealed no statistically significant differences attributable to supplementation in body composition, blood chemistry and coagulation. On the clinical chemistry measures, there were statistically significant group differences in levels of globulin and bicarbonate, but these were not deemed to be clinically significant.

Cognitive Assessment measures

Concurrent discrimination learning. .

Although the groups did not differ significantly in errors to criterion or trials to criterion, there was a marginally significant difference in frequency of animals that successfully completed the task, with 9 animals from the supplemented group learning and only 5 from the control group. The design of the concurrent discrimination protocol included testing animals a maximum of 40 sessions, before discontinuing testing. This restriction could reduce the differences between the two groups in both the error and trials to criterion measure by limiting the maximum number of errors and trials possible.

Despite the absence of a significant effect in the total errors measure, there was a statistically significant interaction between group and test session, with the DHA supplemented group performing at a higher overall level than the controls over the last block of training. We interpret this finding to indicate improved learning possibly because of an improved ability by DHA supplementation to recall the outcome of previous trails (in which they were rewarded or not rewarded, depending on which of the object pair that they responded to.

We also compared the groups' retention of the concurrent discrimination learning task in order to assess the likelihood of DHA supplementation affecting long-term memory. The results indicated improved performance over the controls, which was statistically significant on the first and last testing day. The significant difference on the first retention test is consistent with the hypothesis of improved long-term memory.

Contrast sensitivity

The contrast sensitivity tests provided the most compelling indication of positive benefits of DHA supplementation. First, during the initial acquisition phase, the DHA group learned the initial discrimination more rapidly and with fewer errors. Second, during the variable contrast phase, there were highly significant differences between the two groups at contrasts of .03 and .05. By contrast, the groups did not differ significantly at .01 (although on the average, the DHA group performed more accurately). Nor did the groups differ at a contrast of .40; to the contrast, their performance was virtually identical .

We interpret this data as follows. The differences in learning the initial task probably involves the mechanisms that accounted for group differences in learning the concurrent discrimination task, namely improved episodic memory. The results on the performance on the variable contrast test probably reflect differences in visual processing ability. Thus, significant differences were only seen at intermediate level contrasts, which afford high levels of difficulty. These sesults o could represent group differences at the level of the retina, cerebral cortex and possibly both. Performance on the contrast sensitivity protocol depends on both learning and the ability to distinguish objects present at low contrasts to background.

Effect of DHA on performance of DNMP task

Unlike concurrent and contrast sensitivity tasks, there were no significant beneficial effects of DHA supplementation on performance of the DNMP task, although the DHA supplemented group did perform better than the control group.

This may reflect task differences. The DNMP task provides a measure of working memory, Mazereeuw et. al. (2012), for example reported that in human subjects DHA has task specific benefits, with improvement seen in immediate recall, attention, and processing speed and did not extend to composite memory, delayed recall, recognition memory, working memory, or executive function.

Possibly of equal or more importance, all of the dogs used in this study had received extensive training on the DNMP task and were probably performing close to their optimum level at the beginning of the study. If true, this would have provided a possible ceiling effect, making it difficult to see further improvement. On the other hand, although over aging stable performance would be expected to deteriorate, it's likely that the duration of the study was too short to produce sufficient deterioration to allow the detection of group differences.

Body Composition

Measures of body composition did not show selective changes, although there were group differences, the differences were present at baseline and were not linked to the DHA treatment. We also did not find differences in body wt. or food intake.

References

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Dacks, P.A., Shineman, D.W., & Fillit, H.M. (2013). Current evidence for the clinical use of long-chain polyunsaturated N-e fatty acids to prevent age related cognitive decline and Alzheimer's disease. J. Nutr., Health & Aging. 17, 241 -251 .

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Tables

Group TREAMENT DV_1 N

Mean

Contro Corrected Fat 2.45981

1 base 9 12

Contro 3.25991

1 End correct fat 9 12

Corrected Fat

DHA base 3.20515 12

4.27750

DHA End correct fat 6 12

Table 2. Levels of body fat at baseline and at the end of the study. Note that Group 1 showed lower levels than group 2 at baseline as well as the end of the test phase.

Sex TREATMENT DV_1 N

Mean

M Corrected Fat base 2.94607 15

M End correct fat 4.21353 15

F Corrected Fat base 2.643175 9

F End correct fat 3.02735 9

Table 3. Body fat at baseline and at the end of the study as a function of sex.

Group TREATMENT DV_1 N

Mean

Contro 9.37759

I Base Lean Mass 5 12

Contro 10.0564

I End lean Mass 2 12

9.54066

DHA Base Lean Mass 5 12

10.0478

DHA End lean Mass 9 12

Table 4. Lean mass at baseline and at the end of the study as a function of group placement. Sex TREATMENT DV_1 N

Mean

Base Lean 10.034237

M Masss 3 15

M End lean Mass 10.654663 15

Base Lean 8.5006176

F Masss 7 9

9.0479777

F End lean Mass 8 9

Table5. Lean mass at baseline and at the end of the study as a function of sex.

Group TREATMENT DV_1 N

Mean

Contro

I abs water base 6.1775 12

Contro

I end abs water 6.59 12

6.26916

DHA abs water base 7 12

6.59083

DHA end abs water 3 12

Table 6. Absolute level of body water as a function of treatment and Group.

Sex TREATMENT DV_1 N

Mean

M abs water base 6.604 15

6.9793333

M end abs water 3 15

5.5888888

F abs water base 9 9

5.9422222

F end abs water 2 9

Table 7. Absolute level of body water as a function of treatment and Sex. Group TREATMENT DV_1 N

Mean

Contro Total body fat 22.1482

1 base 8 12

Contro end total body 27.1558

1 fat 9 12

Total body fat 26.2302

DHA base 8 12

end total body 32.3747

DHA fat 5 12

Table 8. Total levels of body fat as a function of treatment and Group.

Sex TREATMENT DV_1 N

Mean

Total body fat 23.789998

M base 5 15

31 .103337

M end total body fat 6 15

Total body fat 24.854739

F base 4 9

F end total body fat 27.535286 9

Table 9. Total level of body fat as a function of treatment and sex.

Baseline Means Treatment Means P values

Measure Control DHA Control DHA Group Treatment Group by

Treatment

WBC 8.725 8.717 8.56 8.975 .8466 .912 .6106

RBC 6.1 6.15 6.617 6.367 .6373 0. 0049 .2140

Hemoglobin 141.167 142.583 152.333 145.833 .5520 0.01 13 .1430

Hematocrit 42.583 13.167 47.417 45.75 .6738 0.00027 .2032

MCV 68.917 70.500 71.583 71/917 .6744 0.0000 .6144

MCH 23.333 23.50 23.083 23.00 .9196 0.0097 .3552

MCHC 331.75 330.42 321.25 318.75 .2652 0.0000 .7093

Platelets 343 326.58 335.42 307.167 .4081 .1 195 .4852

Neutrophils 6.1283 5.554 6.09 6.01 12 .7267 .5802 .5135

Lymphocytes 1.8258 2.3991 1.7041 2.1567 0.0359 0.0097 .3449

Monocytes 0.4917 0.4433 0.47 0.4725 .7066 .9041 .4179

Eosinophils .2317 .2667 .2908 .31 17 .5903 0.0231 .7429

Table 4. Group comparisons on each of the blood chemistry measures. The first four columns show mean baseline and treatment data for each of the measures at baseline and at the end of the study. The last columns show the statistical significance of the group comparison, treatment comparison, and group by treatment interaction. Baseline Means Treatment Means P values

Measure Control DHA Control DHA Group Treatment Group by

Treatment

Total Protein 58.917 61.750 58.667 59.417 0.1972 0.0390 0.0906

Albumin 33.167 33.000 34.333 34.250 0.9058 0.0056 0.9167

Globulin 25.750 28.750 24.333 25.167 0.0539 0.0000 0.0399

A/G Ratio 1.2833 1.1583 1.4417 1.3833 0.1842 0.0000 0.3926

Alt 30.017 31.167 32.917 39.00 0.5055 0.0405 0.2100

Alk 36.000 39.167 35.500 40.167 0.6156 0.8855 0.6693

Bilirubin 2.5083 2.4167 2.6083 2.2250 0.9016 0.2166 0.4431

Bun 6.7000 6.5250 6.8167 6.6917 0.8297 0.6443 0.9349

Creatine 59.833 59.167 48.083 45.750 0.651 1 0.0000 0.1345

Glucose 3.5167 3.9750 4.0167 42250 0.1247 0.0007 0.2002

Calcium 2.0658 1.9858 2.1033 2.0358 0.4191 0.0023 0.7726

Sodium 146.75 147.92 147.67 148.33 0.2059 0.1005 0.5269

Potassium 5.0083 4.9750 4.8417 4.7667 0.5598 0.0001 0.6058

Na/K 29.417 30.667 29.833 31.167 0.4369 0.0000 0.8496

Chloride 1 1 1.42 1 11.57 110.75 110.33 0.6059 0.0010 0.0639

Bicarbonate 20.333 20.750 22.250 24.833 0.0424 0.0000 0.0154

Anion 20.000 19.667 19.750 18.083 0.1680 0.0388 0.1571

Cholesterol 4.8433 4.9041 5.1575 5.2517 0.8500 0.0307 0.8794 Baseline Means Treatment Means P values

Measure Control DHA Control DHA Group Treatment Group by

Treatment

Triglyceride 0.7642 0.6141 0.9242 0.8142 0.3332 0.0026 0.7092

Table 5 Clinical chemistry group comparisons. The first four columns show mean baseline and treatment data for each of the measures at baseline and at the end of the study. The last columns show the statistical significance

Claims

1 . A nutraceutical composition, which is a pet food or a supplement composition for a pet food, particularly for dogs or cats, characterized in that said composition comprises at least one poly-unsaturated fatty acid from microbial source.

2. A composition according to claim 1 , characterized in that the at least one component is docosahexaenoic acid.

3. The composition of claim 1 or 2, wherein the microorganism is algae.

4. The composition of claim 3, wherein the microorganism is in a whole cell form.

5. The composition of claim 3 or 4, wherein the microorganism is from the order Thraustochytriales.

6. The composition of claim 5, wherein the microorganism is from the genus

Thraustochytrium or Schizochytrium.

7. The composition of claim 6, wherein the microorganism is Schizochytrium sp. ATCC 20888 or ATCC 20889 and derivatives thereof.

8. The use of at least one poly-unsaturated fatty acid according to claim 1 or 2 for the manufacture of a composition for use as feed additives or nutraceuticals to improve cognitive functions and/or for the treatment or prevention of age-related disorders in animals, in particular in dogs and cats.

9. Use according to claim 3, in order to improve learning memory and alertness.