Veterinary Composition Comprising Superoxide Dismutase and at Least One Hydrolysate of Proteins Rich in Bioassimilable Peptides

Abstract

A veterinary composition comprising, in effective amounts, superoxide dismutase and at least one protein hydrolysate rich in bioassimilable peptides.

Claims

1. A veterinary composition, which can preferably be administered orally, comprising, in effective amounts: superoxide dismutase or at least one source of superoxide dismutase, and at least one protein hydrolysate or at least one preparation based on at least one protein hydrolysate, said at least one protein hydrolysate advantageously being devoid of proteins, said at least one protein hydrolysate comprising a peptide fraction in which less than 1% by weight of the peptides have a molecular weight greater than or equal to 10,000 Da, preferably in which 100% by weight of the peptides have a molecular weight less than 10,000 Da, preferably less than 3,000 Da, advantageously less than or equal to 1,800 Da, in a preferred manner less than 1,800 Da.

2. The composition according to claim 1, wherein said peptide fraction represents at least 50% by weight, preferably more than 50% by weight, preferably at least 55% by weight, advantageously at least 60% by weight, in a preferred manner more than 60% by weight, relative to the total weight of said at least one protein hydrolysate.

3. The composition according to claim 1 or 2 comprising at least one source of superoxide dismutase and at least one preparation based on at least one protein hydrolysate in a weight ratio between 0.01:100 and 100:1, preferably between 1:100 and 1:10, preferably between 1:100 and 5:100, advantageously between 1:50 and 2:50.

4. The composition according to any one of the preceding claims, wherein said superoxide dismutase is of plant origin, preferably of fruit origin, advantageously derived from Olea europeae, from Vitis vinifera and/or from at least one Cucurbitaceae such as Cucumis melo; in a preferred manner said superoxide dismutase consisting of the superoxide dismutase of Cucumis melo.

5. The composition according to any one of the preceding claims, wherein said at least one protein hydrolysate is an animal protein hydrolysate or a plant protein hydrolysate.

6. The composition according to any one of the preceding claims, wherein said at least one protein hydrolysate is: a protein hydrolysate of at least one marine animal, preferably a fish protein hydrolysate, advantageously a protein hydrolysate of fish belonging to the Gadidae family, or a plant protein hydrolysate.

7. The composition according to any one of the preceding claims, wherein said at least one protein hydrolysate is a protein hydrolysate of at least one marine animal, preferably a fish protein hydrolysate, advantageously a protein hydrolysate of fish belonging to the Gadidae family.

8. The composition according to any one of claims 1 to 7, said composition comprising: a) from 0.5 to 100 IU, preferably from 20 to 80 IU, preferably from 40 to 65 IU, advantageously from 50 to 60 IU, of superoxide dismutase, and/or b) from 10 to 1,000 mg, preferably from 200 to 800 mg, preferably from 400 to 600 mg, advantageously between 450 and 550 mg, of said at least one preparation based on said at least one protein hydrolysate, preferably said composition comprising a) and b).

9. A nutritional composition, a compound feed for animals such as a complete feed for animals or a supplementary feed for animals, a feed for animals which is aimed at specific nutritional objectives, comprising the composition according to any one of the preceding claims.

10. A veterinary medicine comprising the composition according to any one of claims 1 to 8.

11. The composition according to any one of claims 1 to 8, the nutritional composition, the compound feed for animals, the feed for animals which is aimed at specific nutritional objectives according to claim 9, the veterinary medicine according to claim 10, for its/their use(s) as a veterinary medicine, in particular in domestic animals, preferably in companion animals such as dogs, cats and/or NCAs, advantageously in companion animals such as dogs and/or cats.

12. The composition according to any one of claims 1 to 8, the nutritional composition, the compound feed for animals, the feed for animals which is aimed at specific nutritional objectives according to claim 9, the veterinary medicine according to claim 10, for its/their use(s) to prevent, regulate and/or treat, in animals, in particular in domestic animals, preferably in companion animals such as dogs, cats and/or NCAs, advantageously in companion animals such as dogs and/or cats: fear and/or anxiety, preferably anxiety, in particular in response to chronic mild stress factors and/or at least one behaviour disorder, preferably associated with fear and/or with anxiety, advantageously associated with anxiety.

13. The composition according to the preceding claim, wherein said at least one behaviour disorder is selected from: the act of attacking, aggressiveness, destroying, inappropriate elimination, the act of repeatedly licking a part of its body, the act of scratching itself, astasia, the act of trembling, the act of marking its territory, aberrant motor behaviour, abnormal feeding behaviour such as polyphagia or polydipsia, dysbasia, an abnormal sensory profile, an abnormal posture, an abnormal vocalisation, sleep disorders, loss of expression, loss of sociability and abnormal judgement of situations.

14. The composition according to any one of claims 1 to 8, the nutritional composition, the compound feed for animals, the feed for animals which is aimed at specific nutritional objectives according to claim 9, the veterinary medicine according to claim 10, for its/their use(s) in the improvement of learning processes in animals, in particular in domestic animals, preferably in companion animals such as dogs, cats and/or NCAs, advantageously in companion animals such as dogs and/or cats.

15. The composition according to any one of claims 11 to 14, said composition being administered to the animal in the form of at least one dose, preferably in the form of a plurality of doses, said at least one dose comprising: a) from 0.5 to 100 IU, preferably from 20 to 80 IU, preferably from 40 to 65 IU, advantageously from 50 to 60 IU, of superoxide dismutase per kilogram of body weight of said animal, and/or b) from 10 to 1,000 mg, preferably from 200 to 800 mg, preferably from 400 to 600 mg, advantageously between 450 and 550 mg, of said at least one preparation based on said at least one protein hydrolysate per kilogram of body weight of said animal, preferably said dose comprising a) and b).

Description

DETAILED DESCRIPTION

[0184] The examples below will enable the present invention to be better understood. These examples are given by way of illustration and must under no circumstances be regarded as limiting the scope of said invention in any way.

Example 1Method of Preparing Capsules Comprising the Veterinary Composition According to the Invention

[0185] The preparation method described hereinafter relates to a production of 1,470 pill tubs of 60 capsules each.

A. RAW MATERIALS

[0186] A.1 Active ingredients

[0187] The active ingredients which go into the composition according to the invention are the following: [0188] GABOLYSATPTP 55 (preparation based on fish protein hydrolysate) [0189] SOD B Primo-antioxidant M (5 IU/mg) (melon juice concentrate freeze-dried and coated with palm oil) [0190] A.2 Excipients

[0191] The raw materials used as excipients in the composition according to the invention are the following: [0192] MICROCRYSTALLINE CELLULOSE PDRE460 [0193] MAGNESIUM STEARATEE470b [0194] SILICON DIOXIDEE551 [0195] WHITE GELATIN CAPSULE T100

[0196] These materials are stored at a positive temperature.

B. MIXTURE

[0197] The raw materials presented in point A above are weighed individually then mixed in accordance with any suitable method known to the person skilled in the art. If necessary, a raw material can be milled and/or sieved prior to mixing.

[0198] Prior to mixing, the manufacture of 1,470 pill tubs requires 59.413 kg of mixed powder, according to the following dosage: [0199] Microcrystalline cellulose (powder)E460: 8.0685 kg [0200] Silicon dioxideE551: 0.2475 kg [0201] SOD B Primo-antioxidant M (5 IU/mg): 1.089 kg [0202] GABOLYSATPTP 55: 49.50 kg [0203] Magnesium stearateE470b: 0.495 kg

[0204] The final mixture is bagged, then stored at a positive temperature.

C. MANUFACTURE OF THE CAPSULES

[0205] The capsules used are 720 mg capsules (reference: milgel 4282).

[0206] The dosage per capsule is the following: [0207] WHITE GELATIN CAPSULE TOO: weight 120 mg [0208] Microcrystalline cellulose (powder)E460: 81.50 mg [0209] Silicon dioxideE551: 2.50 mg [0210] SOD B Primo-antioxidant M (5 IU/mg): 11 mg [0211] GABOLYSATPTP 55: 500 mg [0212] Magnesium stearateE470b: 5 mg

[0213] A sampling of capsules is undertaken by the quality department. Analyses are undertaken according to the internal inspection plan.

[0214] Weighings are undertaken throughout the manufacturing process to inspect the weight of the capsules.

[0215] The loose capsules are placed in bags, then into barrels for storage at a positive temperature.

D. PACKAGING/COUNTING

[0216] The loose capsules are packaged in 60-capsule pill tubs in accordance with the work instruction associated with the post. The packaging products are the following: [0217] White 125 ml HDPE pill tub [0218] Tamper-proof cap EP43

[0219] Once the packaging has been completed, the pill tubs are counted, then placed in boxes for storage at a positive temperature.

Example 2Veterinary Clinical Study Performed on Thirty-Nine Dogs

A. Materials and Methods

[0220] A.1 Place

[0221] The study was approved by the committee for the use and care of animals at the cole nationale vtrinaire d'Alfort et Universit Paris-Est [Alfort National Veterinary School and University of Paris-East] (ComERC ENVA; approval number COMERC 2016-01-15). All the dog owners gave their informed consent by way of a written document signed prior to any study procedure. The study took place in the experiment room of the IRCA (Institut de Recherche Clinique Animale [Institute for Animal Clinical Research]) within Alfort National Veterinary School. The 15 m.sup.2 room was adapted for the behavioural tests and the areas used were carefully marked. The floor area was divided into 1 m.sup.2 squares. [0222] A.2 Animals [0223] A.2.1 General inclusion criteria

[0224] Companion dogs of various breeds, aged from 1 to 6 years old, were recruited in the waiting room for vaccinations or via the internet. Prior to their inclusion, the dogs were subjected to a general clinical examination in order to check that they were in good health. Aggressive dogs or dogs which were difficult to control were excluded from the study. A check was performed (clicker test with the Clix multi-clicker) to ensure that all the dogs included had good auditory capacity. Pregnant or lactating dogs and dogs which were on corticosteroids or undergoing psychological treatment were not included. A total of 39 dogs with an average age of 4.01.7 years participated in this study. 26 were female and 13 were male (cf. table 7 below).

TABLE-US-00007 TABLE 7 Individuals tested INCLUSION AGE CBARQ on Placebo/Supplement NUMBER SPECIES (years) GENDER day 0 (S) 160426-ALEFI AUSTRALIAN SHEPHERD 6 f 1 P 160426-ALEIG CHIHUAHUA 3 m 9 P 160426-ALELI LABRADOR 6 f 2 S 160429-CROHU GERMAN SHEPHERD 4 f 1 S 160429-ETHIN CROSS-BREED 1 f 9 S 160429-HGUNI CROSS-BREED 5 f 6 P 160502-MMUEL CROSS-BREED 2 f 7 S 160503-CJAEL CROSS-BREED 3 m 4 P 160503-MRIKA CROSS-BREED 2 m 3 P 160503-MRITI CROSS-BREED 4 m 3 S 160510-EBOGE LABRADOR 5 m 4 S 160510-EBOJO BEAGLE 2 m 9 P 160511-ADEFA YORKSHIRE TERRIER 6 f 3 P 160511-ADEKA YORKSHIRE TERRIER 4 f 2 S 160513-CGAFA PYRENEAN SHEPHERD 6 f 8 P 160516-AGODJ AUSTRALIAN SHEPHERD 6 m 9 P 160516-AGOLI WHIPPET 5 f 1 P 160516-CAPFL AUSTRALIAN SHEPHERD 6 m 3 S 160520-AGOKE AUSTRALIAN SHEPHERD 6 f 2 P 160520-AGOTI BRAZILIAN TERRIER 2 f 2 P 160520-IBALA GOLDEN RETRIEVER 5 f 8 S 160520-MYALE POODLE 2 f 5 P 160520-VPIJA JACK RUSSELL 5 m 8 P 160520-VPIMO JACK RUSSELL 2 f 3 S 160524-SWENI LABRADOR 6 f 3 S 160525-CLEFI GERMAN SHEPHERD 6 m 1 P 160526-CLEIO GOLDEN RETRIEVER 3 f 1 S 160601-HBUNA SHIBA INU 3 f 7 S 160607-IBEIY AKITA 2 f 3 P 160607-NDAFA WEST HIGHLAND TERRIER 5 f 8 S 160607-SJOZA JACK RUSSELL 3 f 6 P 160608-CBEEL BICHON 6 m 8 P 160608-MLELE CROSS-BREED 1 f 7 P 160608-MLEWI CROSS-BREED 5 f 7 S 160613-ECOIZ AUSTRALIAN SHEPHERD 3 f 8 P 160613-ESELY CROSS-BREED 6 f 8 S 160614-ELEHA CANE CORSO 4 f 6 S 160614-ELEJA CANE CORSO 2 m 1 S 160614-LALPA CROSS-BREED 1 m 2 P AVERAGE VALUE SD 4.0 1.7 4.85 2.86 (Standard-Deviation) [0225] A.2.2. Inclusion criteria based on a result of behaviour associated with fear which are adapted from the CBARQ (Serpell & Hsu, 2003)

[0226] Behavioural evaluations of the dogs were obtained by using a simplified version of the CBARQ (Serpell & Hsu, 2003; Bourienne, 2015), a standardised monitoring instrument having established characteristics of reliability and validity. Four questions from the original questionnaire were selected so that the owners gave a mark corresponding to their own assessment of fear in their dog. The marks for the 4 questions (from 0 to 3 points) were added up, and ranged from 0 to 12. This mark was used as the inclusion criterion: the dogs presenting a result ranging from 1 to 9 were selected for this study. The four questions hereinbelow were marked (cf. table 8 below).

TABLE-US-00008 TABLE 8 Simplified CBARQ questionnaire used in order to select the dogs 1. In the presence of loud noises (i.e. cars, fireworks, thunder), does your dog start to tremble, bark or does he/she try to run away? No, never (0 pt) Yes, sometimes (1 pt) Yes, frequently (2 pts) Yes, always (3 pts) 2. When someone unknown to your dog approaches him/her at home, does your dog start to tremble, bark or does he/she try to run away? No, never (0 pt) Yes, sometimes (1 pt) Yes, frequently (2 pts) Yes, always (3 pts) 3. When someone unknown to your dog approaches him/her during a walk, does your dog start to tremble, bark or does he/she try to run away? No, never (0 pt) Yes, sometimes (1 pt) Yes, frequently (2 pts) Yes, always (3 pts) 4. When your dog is left alone at home, does your dog start to bark, destroy furniture, objects or other things? No, never (0 pt) Yes, sometimes (1 pt) Yes, frequently (2 pts) Yes, always (3 pts) [0227] A.3 Experimental protocol [0228] A.3.1. Tested product (S) and placebo (P)

[0229] The tested product is a supplementary feed (abbreviated to supplement) presented in the form of a capsule containing 500 mg of a preparation based on a fish protein hydrolysate (GABOLYSATPTP 55) and 11 mg of SOD B Primo-antioxidant M (5 IU/mg). The principal excipient of this feed supplement is microcrystalline cellulose, said feed supplement containing 81.5 mg thereof. The method for preparing the tested product (S) is that presented in example 1 above.

[0230] The placebo is a capsule of the same size as that containing the supplement, comprising 445 mg per microcrystalline cellulose capsule, and obtained by adapting the preparation method presented in example 1 above.

[0231] The supplement/placebo was given to the dog daily throughout the study (namely for 30 days), from the day after the first test. The supplement was given to 18 dogs and the placebo to 21 dogs. [0232] A.3.2. Protocol

[0233] The present test was adapted from a test developed and applied with the aim of observing the behavioural reactions in dogs when faced with various specific situations (Hoummady et al., 2016).

[0234] Prior to entering the room, the owner of the dog was asked to fill in the simplified CBARQ questionnaire (cf. table 8 above). A saliva sample was collected with the aid of an oral swab (Salimetrics Kit) just prior to entering the test room, and just after leaving the room. The tubes containing the saliva were stored at 4 C., then brought to the laboratory in order to be centrifuged there, frozen to 20 C. and then analysed.

[0235] The dogs were tested 3 times during a period of 30 days (on day 0on day 15on day 30). [0236] A.3.3. Behavioural tests

[0237] The dog enters the room without a lead, without its master and the door is closed by an assistant of the experimenter. The test lasted 6 minutes and 50 seconds in all and was divided into 4 sub-tests. The test was recorded using a CANON (EOS 700D) camera, and the videos were then analysed. The experimenter recorded the duration of each sub-test using a stopwatch.

[0238] Sub-test 1 (ST1). Exploration of a new environment (3 minutes). The experimenter remains seated on a chair within the experiment area (E) without any physical or visual contact. The dog is left free to explore the layout of the room.

[0239] Sub-test 2 (ST2). Interaction with an unknown person (2 minutes). The experimenter stands up from the chair, fetches 2 balls and a string and remains in a low position in the play area (P). He/she calls the dog and invites it to play 3 times.

[0240] Sub-test 3 (ST3). Loud noise (1 minute 20 seconds). The experimenter makes a sudden noise which lasts for 20 seconds. This corresponds to a vacuum-cleaner noise of 85 dB (CD Clix). A conventional CD player was used and a calibration was performed in an acoustic field with the aid of a precision sonometer (type 2235 sonometer with a type 2235 microphone type 1626 +1/3-1/1 Octave Filter Set, Brel &Kjr Sound & Vibration Measurement A/S, Nrum, Denmark). After having made the noise, the experimenter remains seated for one minute.

[0241] Sub-test 4 (ST4). Reaction in relation to an unknown object (30 seconds). From a chair, the experimenter directs a remote-controlled car and places it in area X. The dog is observed throughout this time.

[0242] The dogs were tested on day 0, on day 15 and on day 30. For sub-test 2, the experimenter was changed in each session so as to avoid the dog becoming used to the presence of the experimenter. For sub-test 4, the same unknown object was used all three times (the remote-controlled car), so that the dog could become used to the object.

[0243] Once the test was finished, the door was opened and the dog was invited to leave the room. The camera was switched off. The videos were then analysed with the BORIS v 2.97 software (Behavioural Observation Research Interactive Software). The behaviours listed in table 9 were observed and quantified by two experimenters whose correlation was tested beforehand with the aid of Kendall's tau coefficient (0.9830.012 for 11 videos).

TABLE-US-00009 TABLE 9 Behavioural results during the test Behaviour Event (E)/State (S) Description Sub-test(s) Interaction with the E Bites, claws, touches the door or ST1, ST2, ST3, ST4 door jumps in the direction of the door for at least 2 seconds (one interaction representing 2 seconds) Door area S Time spent in front of the door, ST1, ST2, ST3, ST4 at least half of the body in the door area (no movement for 2 seconds) Whimpering E 1 sound ST1, ST2, ST3, ST4 Yawning E Involuntary opening of the ST1, ST2, ST3, ST4 mouth with extension of the tongue Licking its E Rapid extension of the tongue ST1, ST2, ST3, ST4 chops/snout which passes over the chops and the snout Experimenter area S Time spent in an area less than ST1, ST3, ST4 1 metre from the experimenter Traversings of area E Number of areas (1 m.sup.2) ST1 traversed by at least half of the body Play S Time spent playing with the ST2 toys and/or the experimenter Turning the head in E The dog turns its head in the ST3 the direction of the direction of the source of the noise noise Approaches an E Approaching movement in the ST4 unknown object direction of an unknown object (remote-controlled car) Turns head towards E The dog turns its head in the ST4 an unknown object direction of the unknown object and remains stationary Touches the E The dog touches the unknown ST4 unknown object object (1 contact representing 2 seconds) [0244] A.3.4. Dosage of salival cortisol (prior to the sub-tests)

[0245] A minimum volume of collected saliva was necessary (50 l) in order to be able to perform an analysis. A sample was taken for all the dogs, but only 31 of them produced enough saliva. The cortisol was analysed by a specialist laboratory located in the town of Namur (UNamurUnit de recherche vtrinaire intgre URVI [Integrated Veterinary Research Unit IVRU]). [0246] A.4 Statistical analyses

[0247] Comparisons between the dogs which obtained CBARQs ranging from 1 to 5 and the dogs which obtained CBARQs ranging from 6 to 9 were performed using the Mann-Whitney non-parametric tests.

[0248] The comparisons of the behaviours throughout the 3 sessions were performed with the aid of a non-parametric variance analysis on repeated measurements (when normality had not been ascertained).

[0249] The comparison of the behaviours and of the deltas (day 15day 0; day 30day 0) for the supplement and the placebo was performed using the Mann-Whitney non-parametric tests.

[0250] The averages are expressed with SD.

B. Results

[0251] B.1 Assessment of the behavioural tests and of the simplified CBARQ questionnaire in relation to fear

[0252] The average simplified CBARQ result for the 39 dogs on day 0 was 4.852.86 (cf. table 7 above).

[0253] In order to confirm that the simplified CBARQ questionnaire can be used as a reliable tool making it possible to categorise and include dogs which present different levels of fear and anxiety, the individuals were divided into two groups according to their result: CBARQ ranging from 1 to 5 (less fearful dogs, n=21) and CBARQ ranging from 6 to 9 (more fearful dogs, n=18). The two groups were compared from the point of view of cortisol level prior to the test on day 0, and the behaviours were assessed at the time of the test on day 0. The results showed a significant difference in the cortisol level between the two groups on day 0 (p=0.013; U=56; df=30), the dogs having results between 6 and 9 presenting higher values (6.643.66 nmol/L as opposed to 5.233.24 nmol/L).

[0254] As regards the behaviour, six behavioural units showed significant differences between the two groups of dogs (cf. table 10 below): traversing of area during sub-test 1 (ST1) (p=0.007; U=239; df=38), licking of the chops during sub-test 1 (ST1) (p=0.042; U=267; df=38), time spent in the area of the door during sub-test 2 (ST2) (p=0.019; U=409; df=38), play time during sub-test ST2 (p=0.046; U=264; df=38), licking of the chops during sub-test 3 (ST3) (p=0.025; U=262; df=38) and approaching the unknown object during sub-test 4 (ST4) (p=0.023; U=259; df=38).

[0255] These results confirm the consistency between the simplified CBARQ result attributed to the dogs and the measurements of stress and the behaviours associated with stress which were observed during the tests.

[0256] The most fearful dogs were characterised by a higher cortisol level, lower activity, more time spent in the area of the door, less time spent playing, a lower number of approaches towards the unknown object, a tendency to yawn more, but less licking of the chops.

TABLE-US-00010 TABLE 10 Comparison of the cortisol and behaviours of the dogs with the simplified CBARQ results ranging from 1 to 5 (less fearful dogs) and from 6 to 9 (more fearful dogs) (on day 0) Median CI [25-75] Median CI [25-75] Test p-Value Less fearful dogs More fearful dogs n = 21 n = 18 ST1 Door area 65 26-117 75 47-122 355.5 0.490 ST1 Experimenter 4.5 0-26 0 0-36 315 0.606 area ST1 Interaction with 6 3.5-13.5 8 6-12.5 349.5 0.606 the door ST1 Traversings 34 26.5-43.5 17 5-30 239 0.007 between areas ST1 Whimpering 8 0.5-23.5 0 0-39 290 0.217 ST1 Licking 1 0-4 0 0-1 267 0.042 ST2 Door area 0 0-45 33 4-82 409 0.019 ST2 Play 68 0-117 7 0-27 264 0.046 ST2 Interaction with 0 0-2 1 0-6 379.5 0.139 the door ST2 Whimpering 0 .sup.0-9.5 0 0-7 307.5 0.415 ST2 Yawning 0 0-0 0 0-0 333.5 0.909 ST2 Licking 0 0-3 0 0-1 301.5 0.325 ST3 Door area 14 0-66 47 15-74 397.5 0.053 ST3 Experimenter area 0 0-13 0 0-14 338.5 0.817 ST3 Interaction with 1 .sup.0-3.5 1 .sup.0-3.5 328.5 0.939 the door ST3 Turns head 1 1-2 2 1-3 381 0.125 ST3 Yawning 0 0-0 0 0-9 364 0.483 ST3 Licking 1 0-2 0 .sup.0-0.5 262 0.025 ST4 Door area 0 0-10 9 0-28 382 0.117 ST4 Experimenter area 0 0-0 0 0-6 353 0.362 ST4 Interaction with 0 0-0 0 .sup.0-0.5 336 0.869 the door ST4 Turns head 2 .sup.1-4.5 2 0.5-4.sup. 310 0.528 ST4 Approaches object 1 0.5-1.sup. 0 0-1 259 0.023 ST4 Touches object 0 0-1 0 .sup.0-0.5 310.5 0.463 ST4 Whimpering 0 .sup.0-0.5 0 0-1 342.5 0.662 ST4 Yawning 0 0-0 0 0-0 314 0.211 ST4 Licking 0 .sup.0-0.5 0 0-0 309 0.337 Less fearful dogs More fearful dogs n = 17 n = 14 Cortisol prior to the 3.139 2.655-4.798 5.694 3.972-8.188 287 287 sub-tests in nmol/L

[0257] Once the information on the placebo and the supplement has been revealed, the averages of the simplified CBARQ results on day 0 were calculated. The median results for the placebo were 5.0 (QI ranging from [2.0; 8.0]) and the median results for the supplement were 3.5 (QI ranging from [2.75; 7.25]). There was no significant difference between the two groups of dogs in terms of level of fear (U=176, p=0.722, df=38). The dogs had therefore been affected in an equal way in the group which received the placebo or the group which received the supplement. This means that the two groups of dogs (less fearful dogs and more fearful dogs are homogenous). [0258] B.2 Comparison between the placebo and the supplement: behavioural tests [0259] B.2.1 Comparison on day 0

[0260] On day 0, no difference was detected between the group which received the placebo and the group which received the supplement (cf. table 11 below). However, the dogs of the group which received the placebo showed a tendency to be more active during sub-test 1 (p=0.055) and to remain in the area of the door during sub-test 2 (p=0.09).

TABLE-US-00011 TABLE 11 Comparisons of placebo and supplement on the behaviours during the different sub-tests Day 0 Supplement N = 18 Placebo N = 21 ST Behaviour Median CI [25-75] Median CI [25-75] Test p-Value ST1 Door area 50.9 21.1-132.7 74.9 54.1-118.1 336 0.508 ST1 Experimenter area 2.9 0-29.8 0 0-20.4 382.5 0.494 ST1 Interaction with 6.5 4.5-11.5 8 4-16 338.5 0.553 the door ST1 Traversing 22.5 9.7-33.2 33 24.5-47.5 291.5 0.055 ST1 Whimpering 6 0-22.7 4 0-46 342 0.614 ST1 Yawning 0 0-0 0 0-0 373 0.544 ST1 Licking 1 .sup.0-2.2 0 .sup.0-2.5 386.5 0.424 ST2 Door area 0 0-32 43.2 0-60.8 302 0.09 ST2 Play 27 0-117.3 10.9 0-84.9 401 0.245 ST2 Interaction with 0 .sup.0-2.2 1 .sup.0-6.5 317.5 0.212 the door ST2 Whimpering 0 .sup.0-0.2 0 0-14 319 0.184 ST2 Yawning 0 0-0 0 0-0 342 0.197 ST2 Licking 0 0-2 0 .sup.0-1.5 358.5 0.974 ST3 Door area 17.9 2.5-72.3 28.4 2.1-67 346.5 0.906 ST3 Experimenter area 0 0-21.1 0 .sup.0-5.2 374.5 0.415 ST3 Interaction with 1 .sup.0-4.2 1 .sup.0-2.7 363.5 0.717 the door ST3 Turns head in the 1 1-3 2 1-3 317.5 0.304 direction ST3 Whimpering 2 0-10.7 2 .sup.0-5.5 371 0.552 ST3 Yawning 0 0-0 0 0-0 325 0.203 ST3 Licking 0.5 0-2 0 0-1 375.5 0.439 ST4 Door area 5.1 0-26.7 0 0-17 372.5 0.512 ST4 Experimenter 0 .sup.0-1.2 0 0-0 362 0.650 area ST4 Interaction with 0 0-1 0 0-0 374.5 0.345 the door ST4 Turns head in 1 1-4 2 .sup.1-4.7 332.5 0.59 the direction ST4 Approaches object 1 .sup.0-1.2 0.5 0-1 370.5 0.549 ST4 Touches object 0 0-1 0 0-1 351 1 ST4 Whimpering 0 0-1 0 0-0 373.5 0.406 ST4 Yawning 0 0-0 0 0-0 371 0.141 ST4 Licking 0 0-1 0 0-0 380.5 0.208 [0261] B.2.2. Comparison on day 15

[0262] On day 15, the dogs of the group which received the supplement had a tendency to remain fora longer time in the experimenter area during sub-test 1 (p=0.061; cf. table 12 below).

TABLE-US-00012 TABLE 12 Comparisons of placebo and supplement behaviours during the different sub-tests Day 15 Supplement N = 18 Placebo N = 21 ST Behaviour Median CI [25-75] Median CI [25-75] Test p-Value ST1 Door area 70.9 23-126.6 102.4 53.7-142.6 324 0.438 ST1 Experimenter area 18.5 0-70.6 1.9 0-23.1 413.5 0.061 ST1 Interaction with 5 2.7-8.sup. 5.5 3-14.5 333 0.607 the door ST1 Whimpering 9 1.5-21 9 0-36.7 356 0.894 ST1 Yawning 0 0-0 0 0-0 381 0.246 ST1 Licking 1.5 0-4 1 0-3 382 0.352 ST2 Door area 20 0-71.6 22.7 0-64.7 342.5 0.812 ST2 Play 9.4 0-90.5 2.9 0-80.8 377 0.442 ST2 Interaction with 1 0-4 1 0-5 339 0.726 the door ST2 Whimpering 0 0-8 0 0-20.5 345 0.858 ST2 Yawning 0 0-0 0 0-0 352 0.978 ST2 Licking 2 0-3.250 1 .sup.0-2.7 373 0.517 ST3 Door area 26.7 0-61.5 25.5 12.6-62.6 343 0.825 ST3 Experimenter area 0 0-15 0 0-29.2 357 0.855 ST3 Interaction with 1.5 .sup.0-5.2 0.5 .sup.0-2.7 381.5 0.36 the door ST3 Turns head in the 1 .sup.1-1.2 1 1-2 356 0.881 direction ST3 Whimpering 1.5 .sup.0-6.2 0 0-17 351.5 1 ST3 Yawning 0 .sup.0-0.2 0 0-1 331 0.459 ST3 Licking 1 .sup.0-2.2 0.5 .sup.0-1.7 383 0.331 ST4 Door area 10.9 0-27.9 5.3 0-25.3 372 0.539 ST4 Experimenter area 0 0-2 0 .sup.0-5.2 341 0.72 ST4 Interaction with 0 0-0.25 0 .sup.0-0.7 346 0.86 the door ST4 Turns head in the 1 .sup.1-3.5 2 .sup.1-4.5 336 0.656 direction ST4 Approaches object 0 .sup.0-1.2 0 0-1 370.5 0.519 ST4 Touches object 0 0-25 0 0-0 360.5 0.697 ST4 Whimpering 0 0-1 0 .sup.0-3.5 346 0.873 ST4 Yawning 0 0-0 0 0-0 361 0.317 ST4 Licking 0 0-1 0 0-0 383.5 0.241 [0263] B2.3. Comparison on day 30

[0264] On day 30, the dogs of the group which received the supplement were less prone to yawning during sub-test 2 (p=0.057). No dog from the group which received the supplement yawned (0/18), whereas three dogs from the group which received the placebo (3/21) yawned during sub-test 2 (cf. table 13 below).

TABLE-US-00013 TABLE 13 Placebo and supplement comparisons of the behaviours during the different sub-tests Day 30 Supplement N = 18 Placebo N = 21 ST Behaviour Median CI [25-75] Median CI [25-75] Test p-Value ST1 Door area 79.5 15-143 67 42.7-125.4 357 0.944 ST1 Experimenter area 10 0-50.1 0 0-37.8 394.5 0.321 ST1 Interaction with 6 1-10.5 7 3-10.5 342 0.621 the door ST1 Traversings 18.5 12.7-37.7 25 13.5-40.5 352.5 0.844 ST1 Whimpering 10.5 3-37.5 18 0-34.5 366.5 0.865 ST1 Yawning 0 0-0 0 .sup.0-0.5 324.5 0.116 ST1 Licking 1 0-4 1 0-2 374 0.688 ST2 Door area 4.6 0-100.5 33 2.5-71 344.5 0.665 ST2 Play 23.9 0-96 11 0-46.7 381 0.558 ST2 Interaction with 0.5 .sup.0-3.2 4 .sup.0-8.5 324.5 0.294 the door ST2 Whimpering 0 .sup.0-2.7 0 0-16 312.5 0.131 ST2 Yawning 0 0-0 0-0 0-0 324 0.057 ST2 Licking 0 .sup.0-0.5 0 .sup.0-2.5 322.5 0.213 ST3 Door area 49.8 5.15-66.7 42 9-77.75 345 0.682 ST3 Experimenter area 0 0-11.4 0 0-34.2 329 0.317 ST3 Interaction with 2 .sup.0-4.2 1 0-4 375 0.677 the door ST3 Turns head in the 2 1-3 2 1-3 379.5 0.581 direction ST3 Whimpering 2.5 .sup.0-7.5 3 0-15 365 0.895 ST3 Yawning 0 0-0 0 0-0 358 0.950 ST3 Licking 0.5 .sup.0-2.2 1 0-1 362 0.964 ST4 Door area 6 0-29.2 13 0-30 353 0.848 ST4 Experimenter area 0 0-1 0 .sup.0-7.4 347 0.646 ST4 Interaction with 0 .sup.0-2.2 0 .sup.0-0.5 382 0.445 the door ST4 Turns head in the 1 1-2 2 .sup.1-2.5 321 0.254 direction ST4 Approaches object 0 0-1 0 0-0 375.5 0.565 ST4 Touches object 0 0-0 0 0-0 351 0.680 ST4 Whimpering 0 .sup.0-4.5 0 .sup.0-2.5 376 0.611 ST4 Yawning 0 0-0 0 0-0 351 0.382 ST4 Licking 0 0-1 0 .sup.0-0.5 367.5 0.797 [0265] B.3 Placebo and supplement comparison of the variations between day 0 and day 30: behavioural tests

[0266] Between day 30 and day 0, the dogs from the group which received the supplement saw their activity increase whereas the dogs from the group which received the placebo saw their activity decrease (p=0.036) during sub-test 1 (cf. table 14 below).

[0267] In addition, the dogs from the group which received the supplement were less prone to yawning between day 0 and day 30 (p=0.086) during sub-test 4: two dogs from the group which received the supplement saw their yawning reduce whereas one dog from the group which received the placebo saw its yawning increase (cf. table 14 below).

TABLE-US-00014 TABLE 14 Placebo and supplement comparisons of the variations between day 30 and day 0 for behaviours of different sub-tests Day 30 as opposed to Day 0 Supplement N = 18 Placebo N = 21 ST Behaviour Median CI [25-75] Median CI [25-75] Test p-Value ST1 Door area 5.2 70.1-64.1 5.6 25.1-30.6 362 0.966 ST1 Experimenter area 7.2 10.3-45.7 0 10.4-18.6 386 0.467 ST1 Interaction with 1 6.2-5.sup. 0 5.5-3.5 340 0.582 the door ST1 Traversings 5.5 13-11 15 20-0.5 435 0.036 ST1 Whimpering 5 1.7-11 0 3-10.5 402.5 0.234 ST1 Yawning 0 0-0 0 .sup.0-0.5 317 0.105 ST1 Licking 0 .sup.1-2.2 0 0-2 364 0.919 ST2 Door area 0 4.5-58.7 0 28.1-37.5 376 0.66 ST2 Play 0 26.3-4.6 0 6.2-10.9 346 0.703 ST2 Interaction with 0 1.2-2.5 0 .sup.1-4.5 347 0.72 the door ST2 Whimpering 0 .sup.0-0.5 0 0-10.5 329.5 0.364 ST2 Yawning 0 0-0 0 0-0 342 0.432 ST2 Licking 0 1-0 0 .sup.1-0.5 343 0.612 ST3 Door area 3.2 9.4-24 7.3 6.8-27.8 333 0.609 ST3 Experimenter area 0 2.5-0.sup. 0 5.9-34.5 321 0.362 ST3 Interaction with 0 .sup.1-2.5 0 1-3 355 0.906 the door ST3 Turns head in the 0 0.2-1.2 0 .sup.2-1.7 382.5 0.356 direction ST3 Whimpering 0 4.2-2.5 0 .sup.2-5.5 328 0.505 ST3 Yawning 0 0-0 0 0-0 371 0.423 ST3 Licking 0 .sup.2-2.2 0.5 1-1 331.5 0.573 ST4 Door area 0 0.8-5.3 4.2 0-16.2 305 0.177 ST4 Experimenter area 0 0-1 0 0-0 369 0.545 ST4 Interaction with 0 0.2-1.5 0 0-0 355.5 0.896 the door ST4 Turns head in the 0 2-1 0 2-1 342.5 0.812 direction ST4 Approaches object 0 1-0 0 1-0 323.5 0.39 ST4 Touches object 0 1-0 0 0.7-0.sup. 334.5 0.569 ST4 Whimpering 0 0.2-3.2 0 .sup.0-1.5 364 0.696 ST4 Yawning 0 0-0 0 0-0 323 0.086 ST4 Licking 0 0-0 0 .sup.0-0.7 323 0.329 [0268] B.4. Placebo and supplement variations in the behaviours of the dogs during the tests between days 0, 15 and 30 [0269] B.4.1. Group which received the supplement

[0270] Between day 0, day 15 and day 30, a significant difference was detected between the behaviours of the dogs of the group which received the supplement (cf. table 15 below) as regards the time spent in the experimenter area. Indeed, a significant difference was observed in this regard at day 0, in comparison with day 15 and with day 30; dogs remained for a longer time in the experimenter area on day 15 and on day 30 (p=0.078). This could be explained by a less substantial stress reaction, insofar as the experimenter is an unknown person.

TABLE-US-00015 TABLE 15 Group which received the supplement: variations in the behaviours over the 3 samplings: day 0, day 15, day 30 SUPPLEMENT N = 18 ST Behaviour p-Value ST1 Door area X.sup.2 = 1.000, df = 2 0.607 ST1 Experimenter X.sup.2 = 5.115, df = 2 0.078 area ST1 Interaction with X.sup.2 = 2.030, df = 2 0.362 the door ST1 Traversing X.sup.2 = 1.853, df = 2 0.396 ST1 Whimpering X.sup.2 = 2.000, df = 2 0.368 ST1 Yawning X.sup.2 = 4.455, df = 2 0.108 ST1 Licking X.sup.2 = 0.295, df = 2 0.863 ST2 Door area X.sup.2 = 0.520, df = 2 0.771 ST2 Play X.sup.2 = 1.661, df = 2 0.436 ST2 Interaction with X.sup.2 = 1.814, df = 2 0.404 the door ST2 Whimpering X.sup.2 = 2.971, df = 2 0.226 ST2 Yawning X.sup.2 = 4.000, df = 2 0.135 ST2 Licking X.sup.2 = 9.882, df = 2 0.007 ST3 Door area X.sup.2 = 0.812, df = 2 0.666 ST3 Experimenter X.sup.2 = 1.946, df = 2 0.378 area ST3 Interaction with X.sup.2 = 0.286, df = 2 0.867 the door ST3 Turns head in the X.sup.2 = 4.128, df = 2 0.127 direction ST3 Whimpering X.sup.2 = 1.080, df = 2 0.583 ST3 Yawning X.sup.2 = 2.000, df = 2 0.368 ST3 Licking X.sup.2 = 1.509, df = 2 0.470 ST4 Door area X.sup.2 = 1.368, df = 2 0.504 ST4 Experimenter X.sup.2 = 1.040, df = 2 0.595 area ST4 Interaction with X.sup.2 = 1.389, df = 2 0.499 the door ST4 Turns head in the X.sup.2 = 0.500, df = 2 0.779 direction ST4 Approaches X.sup.2 = 10.759, df = 2 0.005 object ST4 Touches object X.sup.2 = 5.360, df = 2 0.069 ST4 Whimpering X.sup.2 = 0.974, df = 2 0.614 ST4 Yawning X.sup.2 = 3.000, df = 2 0.233 ST4 Licking X.sup.2 = 1.400, df = 2 0.497 [0271] B.4.2. Group which received the placebo

[0272] Between day 0, day 15 and day 30, some significant differences were detected between the behaviours of the dogs of the group which received the placebo (cf. table 16 below).

[0273] The traversing of area was different from one day to another (p =0.032). In particular, the number of traversings of area has reduced on day 30 compared with day 0, indicating that dogs were less active on day 30. This could indicate that the dogs were more stressed on day 30 (less activity).

[0274] The touching of the unknown object (sub-test 4) often presented differences between the three sessions: on day 0, the dogs generally touched the object more than on day 15 and on day 30. This could indicate, in the same way as for the traversing of area, that the dogs were more stressed on day 15 and on day 30.

TABLE-US-00016 TABLE 16 Group which received the placebo: variations in the behaviours over the 3 sampling periods: day 0, day 15, day 30 PLACEBO N = 21 ST Behaviour p-Value ST1 Door area X.sup.2 = 0.1000, df = 2 0.951 ST1 Experimenter area X.sup.2 = 0.275, df = 2 0.872 ST1 Interaction with the X.sup.2 = 0.494, df = 2 0.781 door ST1 Traversing X.sup.2 = 6.861, df = 2 0.032 ST1 Whimpering X.sup.2 = 0.0328, df = 2 0.984 ST1 Yawning X.sup.2 = 2.000, df = 2 0.368 ST1 Licking X.sup.2 = 1.480, df = 2 0.477 ST2 Door area X.sup.2 = 0.375, df = 2 0.829 ST2 Play X.sup.2 = 1.016, df = 2 0.602 ST2 Interaction with the X.sup.2 = 0.679, df = 2 0.712 door ST2 Whimpering X.sup.2 = 1.409, df = 2 0.494 ST2 Yawning X.sup.2 = 1.143, df = 2 0.565 ST2 Licking X.sup.2 = 1.480, df = 2 0.477 ST3 Door area X.sup.2 = 2.676, df = 2 0.262 ST3 Experimenter area X.sup.2 = 1.609, df = 2 0.447 ST3 Interaction with the X.sup.2 = 1.825, df = 2 0.402 door ST3 Turns head in the X.sup.2 = 8.036, df = 2 0.018 direction ST3 Whimpering X.sup.2 = 0.154, df = 2 0.926 ST3 Yawning X.sup.2 = 1.357, df = 2 0.507 ST3 Licking X.sup.2 = 0.931, df = 2 0.628 ST4 Door area X.sup.2 = 8.654, df = 2 0.013 ST4 Experimenter area X.sup.2 = 0.389, df = 2 0.823 ST4 Interaction with the X.sup.2 = 3.130, df = 2 0.209 door ST4 Turns head in the X.sup.2 = 0.406, df = 0.816 direction ST4 Approaches X.sup.2 = 3.368, df = 2 0.186 object ST4 Touches object X.sup.2 = 5.083, df = 2 0.079 ST4 Whimpering X.sup.2 = 0.231, df = 2 0.891 ST4 Yawning X.sup.2 = 2.000, df = 2 0.368 ST4 Licking X.sup.2 = 1.040, df = 2 0.595 df = degree of freedom

C. Conclusions

[0275] C.1. Validation of the behavioural tests

[0276] In line with the CBARQ as the inclusion criterion, it was considered, for the purposes of the present study, that when an enquiry by questionnaire was used as a tool making it possible to determine animal emotions such as fear, joy and aggressiveness, its validity is of the greatest importance. An external validity indicates the effectiveness with which the measurement conceptually predicts pertinent behaviours, results or criteria (John and Soto, 2007). In this case, it was validated successfully by discovering significant differences between the less fearful and more fearful dogs (according to their CBARQ score). As Van den Berg (2010) suggested, this questionnaire can be used in complete confidence as a research tool in order to compare behaviours in different canine populations in this type of study.

[0277] By comparing the dogs which have a score between 6 and 9 (more fearful) for the CBARQ and the dogs which have a score between 1 and 5 (less fearful), the results show that the dogs which were more fearful presented a higher level of cortisol, less activity, more time spent in the area of the door, less time spent playing, and a lower number of approaches towards the unknown object, a tendency to yawn more but more rare licking of the chops. [0278] C.2. Behavioural effects of the supplement on the dogs

[0279] Once the information on the placebo and on the supplement was revealed, the averages of the simplified CBARQ scores on day 0 were calculated. There was no significant difference between the two groups of dogs concerning the level of fear, therefore the dogs were equally affected in the placebo and supplement groups. [0280] C.2.1. Comparisons of the behaviours between the placebo and supplement groups

[0281] On day 15, the dogs of the group which received the supplement had a tendency to remain for a longer time in the experimenter area during sub-test 1 (p=0.061).

[0282] On day 30, the dogs of the group which received the supplement were less prone to yawning during sub-test 2 (p=0.057).

[0283] The supplement thus appears to facilitate dog-human interaction and to reduce emotionality during interactions with humans.

[0284] Between day 0 and day 30, the dogs of the group which received the supplement were more active, whereas the dogs of the group which received the placebo were less active (p =0.036) during sub-test 1. In addition, the dogs of the group which received the supplement had a tendency to yawn less between day 0 and day 30 (p=0.086) during sub-test 4.

[0285] On day 30, the supplement appears to facilitate the learning and familiarisation processes in the dogs, by increasing activity. Indeed, the dogs of the group which received the supplement still explored the environment during the third test session. In addition, the dogs yawned less and consequently showed less emotionality. [0286] C.2.2. Changes in the behaviours over the 30 days for the group which received the placebo and the group which received the supplement

GROUP WHICH RECEIVED THE SUPPLEMENT

[0287] Between day 0, day 15 and day 30, a significant difference was observed on day 0 in comparison with day 15 and day 30; the dogs remained for a longer time in the experimenter area on day 15 and on day 30 (p=0.078). This can presumably be explained by a less substantial stress reaction, insofar as the experimenter is an unknown person. Indeed, during the tests, the person was changed between each session and the dogs therefore could not become familiar with the experimenter.

GROUP WHICH RECEIVED THE PLACEBO

[0288] The traversing of the area during sub-test 1 was different from one day to another (p=0.032). In particular, the number of traversings of area reduced on day 30 in comparison with day 0, which indicates that the dogs were less active on day 30. This could indicate that the dogs were more stressed on day 30 (less activity).

[0289] Touching the unknown object (sub-test 4) had a tendency to vary from one session to another: on day 0, the dogs had a tendency to touch the object more than on day 15 and on day 30. This could indicate, in the same way as for the traversing of area, that the dogs were more stressed on day 15 and on day 30.

[0290] By comparing the changes for the group which received the supplement and for the group which received the placebo, it appears that the dogs of the group which received the supplement seem to explore more and be more curious than the dogs of the group which received the placebo. These dogs therefore could be more motivated to explore and quicker to assimilate new learning.

D. General Conclusion

[0291] The results of the present veterinary clinical study suggest an increase in activity, familiarity with a new person and curiosity, with a reduction in behaviour associated with stress, fear and/or anxiety for dogs which have received the supplement in comparison with those of the group which received the placebo.

[0292] The supplement tested therefore proves to be effective in reducing reactions to stress, fear and/or anxiety (in particular in the context of daily mild stress factors), and promoting learning processes.

[0293] Furthermore, the present study has made it possible to prove the existence of positive effects on dog-human communication and on learning processes caused by chronic stress factors.

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