COMPARISON OF THE IMPACT OF NUTRITION AND ENVIRONMENTAL FACTORS ON THE WELFARE OF SHELTER DOGS IN LITHUANIA AND

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Faculty of veterinary medicine

Anastasia Stylianidou

COMPARISON OF THE IMPACT OF NUTRITION AND ENVIRONMENTAL FACTORS ON THE WELFARE OF SHELTER DOGS IN LITHUANIA AND

CYPRUS

MITYBOS IR APLINKOS VEIKSNIŲ ĮTAKOS

PRIEGLAUDŲ ŠUNŲ GEROVEI LIETUVOJE IR KIPRE PALYGINIMAS

MASTER THESIS

Of Integrated Studies of Veterinary Medicine

Supervisor: Dr. Vaida Andrulevičiūtė

KAUNAS 2019

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2 THE WORK WAS DONE IN THE DEPARTMENT OF BIOCHEMISTRY

CONFIRMATION OF THE INDEPENDENCE OF DONE WORK

I confirm that the presented Master Thesis “COMPARISON OF THE IMPACT OF NUTRITION AND ENVIRONMENTAL FACTORS ON THE WELFARE OF SHELTER DOGS IN

LITHUANIA AND CYPRUS”.

1. Has been done by me;

2. Has not been used in any other Lithuanian or foreign university;

3. I have not used any other sources not indicated in the work and I present the complete list of the used literature.

Anastasia Stylianidou

(date) (author’s name, surname) (signature)

CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE ENGLISH LANGUAGE IN THE DONE WORK

I confirm the correctness of the English language in the done work.

Anastasia Stylianidou

CONCLUSION OF THE SUPERVISOR REGARDING DEFENSE OF THE MASTER THESIS

THE MASTER THESIS HAVE BEEN APPROVED IN THE DEPARTMENT OF BIOCHEMISTRY

(Date of approbation) (Name, surname of the manager of department/clinic) (Signature) Reviewers of the Master Theses

1) 2)

(Name, surname) (Signatures) Evaluation of defense commission of the Master Theses:

(Date) (Name, surname of the secretary of the defense commission) (Signature)

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SUMMARY

The objective of this study was to evaluate the influence of nutrition, and environmental factors to a dog’s wellness by comparing the health of shelter dogs in Lithuania and Cyprus.

The main parameters of the dogs’ welfare reflecting the influence of accommodation facilities and environment factors, considering living conditions (from shelters in Lithuania and Cyprus), were analysed and compared. Biochemical blood profiling for each dog was conducted, reflecting wellness of dogs and how does the dogs feed quality affects their health. All the animals were physically examined before the blood sampling, and information about their overall health condition and status was collected. A total of 23 blood samples was collected, 9 in Cyprus and 14 in Lithuania (all samples are from the same shelter of each country), during March and May of 2019. The samples were analyzed in the LHSU, Biochemistry Department, in Lithuania. A small questionnaire was prepared in order to collect information about feeding management (type and amount feed, snacks, supplements etc.), as well as, questions about their facilities and environment.

The research revealed that, the shelter dogs participating from the Lithuanian shelter, and the Cypriot shelter had normal acid base balance, with no indication of renal or hepatic dysfunction according to their biochemical profiles. Abnormally high values of Urea (P<0.05), creatinine (P<0.05), total protein (P<0.05) were obtained from both populations of the shelters. These are associated with dehydration in dogs. The value of Triglycerides was elevated in both countries, possibly due to a surplus of calories intake, which were not burned off by the dogs, and instead were stored in fat cells due to insufficient physical activity in addition to the possibility of overfeeding.

Female dogs from Lithuanian and Cyprus did not differ widely, but it is assumed that the female shelter dogs from Cyprus were more dehydrated than those in Lithuania. No significant differences in the biochemical parameters of male blood were identified and were not associated with health problems.

A set of recommendation was designed for the owner of shelters, following the findings of this research, which consider feed quality, accommodation facilities and environment (physical/mental) influences.

Keywords: Canine, Nutrition, Dry feed, Biochemical blood parameters, Welfare, Environment.

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5 MITYBOS IR APLINKOS VEIKSNIŲ ĮTAKOS PRIEGLAUDŲ ŠUNŲ GEROVEI

LIETUVOJE IR KIPRE PALYGINIMAS

Anastasia Stylianidou Magistro baigiamasis darbas

SANTRAUKA

Šio tyrimo tikslas buvo ištirti mitybos ir aplinkos veiksnių įtaką šuns gerovei, lyginant prieglaudos šunų sveikatą Lietuvoje ir Kipre.

Išanalizuoti ir palyginti šunų gerovės parametrai, atspindintys apgyvendinimo patalpų ir aplinkos veiksnių įtaką. Visi tyrime naudoti šunys, prieš imant kraujo mėginius, buvo ištirti fiziškai ir surinkta informacija apie jų bendrą sveikatos būklę. Buvo atlikta kraujo biocheminių rodiklių analizė, atspindinti šunų savijautą ir pašaro kokybės daromą įtaką šunų sveikatai. Tyrimui 2019 m.

kovo ir gegužės mėn. buvo paimti 23 kraujo mėginiai, 9 – Kipre ir 14 – Lietuvoje (visi šunys yra iš tos pačios prieglaudos). Mėginiai buvo analizuojami LSMU Biochemijos katedroje, Lietuvoje.

Parengta anketa žmonėms, dirbantiems prieglaudose, kurios atsakymai buvo naudoti surenkant informaciją apie šėrimą(pašaro rūšis ir kiekis, užkandžiai, papildai ir kt.), aplinką ir esamas sąlygas.

Tyrimas atskleidė, kad ir Lietuvos, ir Kipro prieglaudoje gyvenantys šunys, turėjo normalią rūgščių ir šarmų pusiausvyrą, ir nenustatyti inkstų ar kepenų funkcijų sutrikimai. Abiejose prieglaudų populiacijose buvo gautos neįprastai aukštos karbamido (P <0,05), kreatinino (P <0,05) ir bendro baltymo (P <0,05) vertės. Tai susiję su šunų dehidratacija. Trigliceridų padidėjusi vertė nustatyta abiejose šalyse, galimai dėl nepakankamo fizinio aktyvumo ir dėl galimybės per daug maitintis.

Vertinant lyčių sveikatingumą, nustatyta jog Lietuvos ir Kipro prieglaudų kalių biocheminiai parametrai nesiskyrė, tačiau nustatyta, kad Kipro prieglaudos šunys buvo labiau dehidratuoti. Patinų kraujo biocheminių parametrų reikšmingi skirtumai nenustatyti ir nesusieti su sveikatos problemomis. Remiantis šio tyrimo išvadomis, atsižvelgiant į pašarų kokybę, apgyvendinimo galimybes ir aplinkos (fizinę / psichinę) įtaką, paruoštos rekomendacijos prieglaudų savininkams.

Raktiniai žodžiai: šuo, mityba, sausas pašaras, biocheminiai kraujo rodikliai, gerovė, aplinka.

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ABBREVIATIONS

ALKP Alkaline phosphatase GGT Gamma-glutamyltransferase AST Aspartate aminotransferase ALT Alanine aminotransferase BCS Body condition score

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INTRODUCTION

The dog is considered by some, to be a member of their family, and nowadays human has the role of the feed provider (1). There are several types of commercial pet food available in the market, but each dog has its own daily energy requirements, according to its weight, age, activity, breed, reproduction condition/state, and current medical conditions. Therefore, to determine which nutritional diet would be optimal, that could provide a dog all the necessary nutrients, and energy ,to maintain a proper health status, we need to evaluate it as an individual (1). By incorporating nutritional assessment, for each dog, we can notably improve a pet’s quality and duration of life.

Several guidelines for nutrition schemes ,have as their goal the perfection of a dog’s health and response to control diet- linked diseases, or even the management of other diseases ,such as chronic renal conditions (2).

The environment in which the dog lives in ,should be considered specifically, and must not be neglected when we are evaluating the total energy and nutrient requirements needed by a dog as an individual (3).

The objective of this study was to evaluate the influence of nutrition and environmental factors to dog’s wellness by comparison of the wellness of sheltered dogs in Lithuania and Cyprus.

Tasks of the work:

1. To analyse the main dogs welfare parameters considering accommodation facilities and environment factors reflecting living conditions in shelters of Lithuania and Cyprus.

2. To compare the main blood parameters reflecting wellness of dogs and how they are affected by their feed quality in Lithuania and Cyprus.

3. To set recommendations for owners of shelters considering feed quality, accommodation facilities and environment (physical/mental) influences.

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1 LITERATURE REVIEW

1.1 The types of commercial pet food

There are several types of commercial pet food available, which can be categorized according to their moisture levels, processing and preservation methods. Dry, semi moist and canned foods are the three major types of commercial dog food found today. Dry pet food having moisture content of 14 % or less. Canned pet food or otherwise called “Wet food”, with a moisture content of 60 % or more, and last but not least Semi moist food which consists with moisture level content of 14 % or more or less than 60 % (1,4).

Dry pet food occupies the biggest share on the market of commercial pet food, as there are several advantages which are advertised to the pet owners (5). In comparison to semi moist and canned foods, dry pet food is more economical, and has an extended period of storage, due to its moisture content. Another unconfirmed, but plausible factor why pet owners choose dry feed over the other types, is the dental hygiene advantages. Manufacturing companies and veterinarians ,promote the use of dry food, as the chewing and grinding of the feed by the dog, is considered a prevention method of accumulation of plaque and calculus on dog teeth (4,5).

Dry food can be classified in three world wide spread categories: economic, premium, and super premium.We could also include “Extra Premium” as a fourth category, however, these feeds can only be found in some regions around the globe, which is why they are excluded from this research.

This classification is according to the quality of ingredients used, quantity and type. Manufactures created this classification by their long experience in feed production. In order to be used on food labels, their commercial and advertising information. There isn’t any governmental authority on a global basis which has recognized or set criteria that would validate this classification official. While this classification is technically unofficial, manufacturers, shouldn’t and aren’t allowed to write any false information regarding the classification of their product(4–6) .The composition of economic food, has cereals and corns as the most prevalent ingredients, followed by animal origin by-products (meat of 10% or less) with inadequate nutritional value. These foods are not categorized by the age, weight or different life stages, activity and reproduction stage. Usually they can be found in small markets, supermarkets or kiosks. In contrast, premium food have ingredients of higher biological value, as meat or meat by- products are listed as the first ingredient in the food label provided by the manufacturer. The meat or animal byproduct constitutes from a range of 10% -30 % of the feed. It is also formulated to support different stages of life, activity and reproduction. Super premium is consisted by great or even excellent quality of ingredients. There are no animal byproducts, only meat, concentrated not less than 50%. There are less cereal and corn concentrations, and instead, the use of better digestible carbohydrates such as rice, potatoes, etc. Generally, super premium pet food

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9 digestibility is heightened by the inclusion of good-quality ingredients, increased levels of fat, and the use of proper processing techniques during preparation. There are different categories of feeds available for several life stages, breeds, weight and veterinary diets (4–6).

1.2 Regulation of pet food

In Europe, there are several governmental committees that issue laws, regulations, standards and implementation policies, which establish nutrient profiles for dogs. In addition, they set standards to evaluate the health condition of each animal individually, taking into consideration the feeding management system that the owner establishes, as well as the environmental factors. These, nutritional assessment guidelines, which have been created by the appropriate committees, have as their goal, the enhancement of a pet’s quality, and duration of life. In addition, by applying optimal animal care, they aid the maintenance of health, and promote the appropriate response to any illness or injury. Proper feeding management for the duration of all life stages of the dog, can support and help avoid diet linked diseases. Additionally, it can also aid in the management of other conditions (1,2).

For example, age and obesity are linked with the predisposition of several medical conditions.

These include but are not limited to: cardiovascular and respiratory disorders, hypertension, and diabetes mellitus in addition to several more chronic health conditions. Other than these hazardous disorders, obesity has an adverse effect on the duration of the dog’s lifespan. Therefore, nutritional assessment with the use of proper nutritional guidelines, is essential to control or even prevent the conditions associated with morbidity (7,8).

Taking everything into consideration, nutritional profiles differ considerably between individuals, even in different dogs living under the same conditions. Several factors, such as: age, weight, body size and condition, health condition and activity, but also environmental factors such as: housing, ambient temperature, humidity e.t.c might greatly affect the health status of a dog (1,2,4).

1.3 Food labelling

EU Regulation 767/2009 (9), issues rules for proper labelling, declarations and other types of marketing advertisements, to ensure that the information provided on the package, is accurate and genuine. The label must incorporate the product name, the estimated weight of the product, excluding the weight of its container or packaging, name and address of the manufacturer, a guaranteed analysis for crude protein, fiber and fat concentration, as well as moisture levels. A list of ingredients must also be included, in descending order, according to the prevalence of weight, in addition to any feed additives that might be included. The label should also include the type of nutrition the product provides, that is to say, whether it is “Complete” and thus sufficient for daily rations, or if it is

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10 considered “Complementary” feed, which has increased content of specific substances but should be used in combination with additional feed to accomplish sufficient nourishment on a daily ration. The time interval from the date and time of production to the date of expiration or a “Best Used By” date, must be included. Last but not least, the species or category of animal the feed is intended to be used for, as well as the caloric content and feeding instructions , should be clearly indicated (9,10).

1.4 Energy utilization

1.4.1 Metabolism of Energy

All metabolic actions require energy, as it involves a transfer and/or expenditure to complete a metabolic processes (11). Dogs, like all living organisms, cannot absorb all of the energy which is incorporated within their food, as some of the energy vanishes in the form of solid, fluid and gaseous excretions and as radiant heat produced by the body. The potential energy, which a food can produce, after its complete oxidation of the organic matter, can be defined as gross energy (GE). Gross energy can be estimated by the absolute combustion of food in a bomb calorimeter (1). Digestible energy (DE) is defined as the energy, which an animal is physically able to digest and absorb from its food, and is calculated by the total GE minus the energy lost with the excreted feces (1).

Metabolizable Energy (ME), is the remaining amount of energy, after fecal and urinary energy loss. But it also represents the energy available for growth, reproduction and the energy which facilitates metabolic processes such as, locomotion thermoregulation and etc. (12). The continuous production of heat resulted by the basal metabolism and locomotion, and ingestion of a meal can be called as the heat increment (HI). Last but not least, procedure which the body of a dog metabolizes energy, is the net energy (NE) ,which is the energy available for the maintenance of body tissues, and production needs (3).

Figure 1 Scheme plan depicting how total gross energy of food is broken down (3)

1.5 Energy requirements in a healthy adult dog

Each dog has its own a daily energy requirement, which are needed to support its resting metabolic rate. Resting metabolic rate can be defined as, the energy consumed by the cells and tissues of the animal, to control homeostasis amid resting periods (4). Additionally, energy is needed for the

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11 voluntary muscular activity, and overall thermoregulation of the body, which includes the production of meal induced thermogenesis during digestion, absorption and utilization of nutrients. This sum of energy can be otherwise defined as, Maintenance energy requirements (1).

1.6 Assessment guideline for energy requirements

In order to optimize the diet of a dog, incorporating nutritional assessment can notably improve a pet’s quality and duration of life. Several guidelines have as their goal the perfection of a dog’s health and response to control diet-linked diseases, or even the management of other diseases ,such as chronic renal conditions (2).

1.6.1.1 History of feeding management

To be able to make an “educated” determination of the amount of Metabolizable Energy needed by a dog for the maintenance of constant healthy body weight. A diet history should provide the following data (1,2):

1. Type and composition of feed provided to the animal (i.e., dry, semi moist, canned foods of commercial dog food. Category: economic, premium, and super premium. extra premium.

Ingredients of home cooked diets. Energy density, etc.).

2. Quantity provided to the animal (i.e. Amount described in weight or volume.).

3. Feeding and providing water schedule

4. Form, kind and amount of treats or table leftovers provided to animals (i.e., commercial treats product nutritional data and quantity. Owners foods which are used as treats, quantity and how frequently given).

Figure 2 Scheme plan of energy ﬂow in mammals (15)

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12 5. Kind, quantity and frequency of nutritional supplements provided to the animal.

6. Who provides the feed and water every day?

7. Do the animals have access to other species or pet(s) food? Or do other pet have access to the animal’s diet, such as other dogs, pests, etc.

1.6.1.2 Physical inspection

Evaluating the current physical condition of a dog, and monitoring the changes throughout its life, by assessing body weight, oral condition, body condition score, last but not least, the appearance and state of both the coat and skin. Can be considered as the main indicating parameters of nutritional adequacy and provide us information about its overall health status (3).

1.6.1.2.1 Body Condition Score

Body condition score, is a non-objective, semi-quantitative method of estimating the percentage of body fat, as well as, the scale of how over- or underweight an animal is. There are several types of scales for both cats and dogs, but the most used and recognized are the scale of 1 to 9 and 1 to 5 (2,13). On a scale of 1 to 9 the score of 4 to 5 is considered to be the optimal, and in the scale of 1 to 5 the optimal is 2.5 to 3, which corresponds to body fat percentage of 12 to 20% (1,2). The ideal body configuration of a dog during palpation and visual examination, is to have easily palpable ribs and bony prominences such as the tail-base, covered with a thin layer of fat. Their abdominal area ,should have a slight abdominal tuck when viewed from the side ,and a distinct lumbar waist ,giving the shape of an hourglass figure when seen from above (1,3). When dogs are considered to be underweight, their ribs and rest of bony prominences can be visibly and easily palpated, with barely or nonexistent overlying fat. Their abdominal tuck is very prominent and when seen from the side and above their hourglass figure is exaggerated. In case of being overweight or obese, is more difficult to palpate the ribs, and a thick fat layer can be seen covering the bony prominences. The abdomen is obviously more extended, which makes the abdominal tuck less prominent when seen from the side, and the hourglass shape is less pronounce or nonexistent is some cases (1,2,14).

1.6.1.3 Animal-Individual Factor

Each organisms has its own needs, and in order to provide a dog with the necessary nutrients and energy to maintain a proper health status, we need to evaluate it as an individual. Weight, age, activity, breed, sexual reproduction condition/state, current medical conditions and medications are the most important parameters, needed to assess an animal on an individual level. Therefore to determine which would be its optimal nutritional diet (1).

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13 1.6.1.4 Environment

The environment in which the dog lives in ,should be highly considered, and must not be neglected when we are evaluating the total energy and nutrient requirements needed by a dog as an individual (3). Temperature , atmospheric and indoor moisture ,ventilation, type of housing(indoors or outdoors) , environmental stressors and the degree of acclimatization , must be regulated according to the breed, body composition, activity, behavior ,and the life stage nutrient requirements of a dog (1,3,15). The normal ambient temperature range, in which dogs, do not need to spend any energy, to maintain their body core temperature, is defined as Thermo-neutral zone. It has been estimated to range from 15 to 20 degrees of Celsius for long-haired dogs , and 20 to 25 degrees of Celsius for short-haired dogs (1). In case the ambient temperature is lower than the body temperature, a dog may need 10 to 90% more calories than during the summer time. Therefore, it’s estimated that dogs which are kept outdoors need more energy and nutrients in comparison to the dogs which live indoors.

Hence, is highly advisable to provide the animal with high-fat content feeds ,for better cold- acclimatization in temperatures lower than the Thermo-neutral zone (1,15). Compared to lower ambient temperatures, when the temperatures are higher than the estimated Thermo-neutral zone, the amount of energy expended is relatively smaller. However, the amount of water requirements increase, due to the vaporization of water via insensible perspiration, respiration, panting and sweating. It is estimated that the metabolic rate increased only 10% in adult dogs, when the ambient temperature was higher or equal to 35 degrees of Celsius (3,15). The number of dogs within a household or shelter also alter the adequacy of the facility. As the more concentrated an enclosement is, the temperature in the local environment increases. Animals try to minimize the heat loss , by huddling together, especial neonates (1).

Dogs which are individually housed, and have limited opportunity to move, might have daily energy requirements as low as 70kcal ME/Kg^0.75. But in multi-dog households, where there is a lot of mutual interaction, that triggers the animals to become more active it may rise to 144kcal ME/Kg^0.75(1).

Stress can be another factor which influences the amount of energy and nutrients required by a dog. Dogs which are influenced by environmental stressors such as, conflict over resources, change of location due to boarding or hospitalization etc., require a slight increase in energy requirements (3).

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1.7 Energy intake

The main nutrients, which are considered to be the vital source of energy in an animal’s diet , are proteins, carbohydrates, and fats (4). There are more nutrients which are also majorly important for the body’s functions, but they do not supply the body with energy .Such nutrients are minerals(micronutrients) and vitamins (1). A complete- diet must be formulated to serve the optimal quantities of all vital nutrients, when the dog consumes the appropriate amount of feed, which meets its daily energy requirements (4). Hence for the selection of a proper dog food, one should choose on the basis of the caloric distribution, which refers to the percentage of calories from fat, protein, and carbohydrate, and also the micronutrient levels compared to patient’s requirements, and ingredient source (16). Age, activity level and stage of life should always be considered when selecting a dog feed (4).

1.7.1 Carbohydrates

Carbohydrates are composed of carbon, hydrogen and oxygen. According to their structural form they can be classified into monosaccharaides, disaccharides and polysaccharides (4). They are the primary energy containing constituents of plants. Monosaccharaides are otherwise named as simple sugars, and are composed of single units, which consists of three and seven carbon atoms.

Glucose, fructose and galactose are the most significant, metabolically and nutrition- wise. Glucose is one of the major carbohydrates used by cells of the body to generate energy (4). It circulates within the blood and is the end product of glycogen hydrolysis and the digestion of starch within the body.

Disaccharides consist of two monosaccharaides combined together (4). Lactose and galactose form Figure 3 Recommended caloric distribution of dog food for different life stages (5)

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15 lactose which is the major disaccharide found in the milk of mammals, hence the puppy’s source of energy (17). Polysaccharides on the other hand, are composed of by many single units of monosaccharaides, creating long and complex chains, such as, starch and dietary fiber (4,18). Cereal grains of corn ,rice ,oats, barley ,wheat, beet pulp , soybeans ,potatoes, carrots ,are the most common ingredients of commercial pet foods , as they are a good source of starch (4,17). Heat processing and finely grounding the cereal grains , increases the digestibility and accessibility of starch as an energy source(4,19,20). Even though dietary fiber cannot be digested, due to the lack of specific enzymes in the gastrointestinal tract of the dog, it can be fermented by microbes found in the large intestine (4,17).

The fermentation has as a byproduct short chain fatty acids which are necessary for the healthy maintenance of the epithelial cells lining the intestines. But also promotes normal peristalsis of the intestines and minimizes the gastrointestinal transit time (4,17).

1.7.2 Protein

Protein is considered to be, a major and an essential building block of the body (21). Alike carbohydrates, protein consists of carbon, oxygen, hydrogen , and additionally , nitrogen and sulfur (17). The basic building units of a protein are amino acids, which are held together by peptide bonds to form protein chains. These protein chains, can be classified to simple or complex forms, according to their molecular yield after hydrolysis starts (17). Hydrolysis of dietary protein, occurs during digestion, and its absorbed by the body through the intestinal lining of the small intestine (22). There are approximately 22 amino acids constituting protein, twelve of these are considered non-essential or dispensable amino acids which can be synthesized by the body, and the remaining ten amino acids are considered essential. The essential amino acids cannot be synthesized by the body, and must be provided within the pet’s diet (4,17).

The body requires dietary protein, as it is used for protein synthesis by the use of the essential amino acids, but also to supply adequate nitrogen for the synthesis of non-essential amino acids and other nitrogen containing compounds (17). Protein as the major building block of the body (21), is a structural component of hair, skin, connective tissue, enzymes, hormones, the body’s musculoskeletal and immune system (4). Hence, an adult dog has maintenance protein requirement. As its body needs essential amino acids and a certain level of nitrogen, to synthesize new proteins from amino acids, because the body eventually catabolizes and replaces all of its protein molecules. As animals are impaired from storing excess amino acids, the additional amino acids are metabolized for energy, which yields to approximately 5.65 kcal ME/g ,or instead converted into glycogen and/or fat for energy storage (17). Another useful function of protein is to increase the palatability and acceptability of food by the dog, as different flavors are created when proteins are cooked in the presence of carbohydrates and fat (6,17). Majority of commercial food companies try to avoid protein deficiencies in the diet, as this would cause the loss of body weight and muscle mass (4). Usually when protein

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16 deficiency occurs is due to the owner who has chosen a low- quality and poorly formulated ration feeds, to economize on expenses. Both the quality and digestibility of the protein within the diet, affects the degree of which a dog can use this protein as a source of amino acids and nitrogen. Protein which is highly digestible and contains all of the essential amino acids, according to the needs of a dog, are considered to be high quality proteins (17,23). These protein sources contain meat or meat by- products which are listed as the first ingredient in the food label provided by the manufacturer (4,6). The greater the quality of protein within the diet, the smaller amount of feed is necessary to cover all of the essential amino acid needs of an adult dog (17,24). High quality commercial feeds contain protein which its digestibility levels range from 80 till 90 %, which is in contrast to the digestibility of low quality commercial pet foods, whose digestibility levels range from 70 till 75 % (4). It is greatly advisable to select feeds which contain a mixture of protein sources in order to provide a bigger variety of amino acids (4,25).

1.7.3 Fat

Dietary fat belongs and is a part of the heterogeneous group of compounds named lipids. In this group there are several forms of fat (4,26). One of them are Triglycerides, which are considered the most important lipid compounds in a dog’s nutrition. Triglycerides are composed of three fatty acids molecules bonded with glycerol (17,26). Fatty acids have a major role in metabolism, as they are considered as a metabolic fuel for storage and transportation of energy, as well as a necessary component for all cell membranes, a forerunner for several hormones. As well as a component for several parts of the skin, which prevent the loss of water and integrity. Another role is the insulation and protection of the body from losing heat, and by a covering the vital organs with a protective layer of fat to prevent physical injury (5,27,36). Therefore, all dogs have a bodily requirements for fatty acids. The three most vital and essential fatty acids (EFAs), include linoleic, gamma-linoleic and arachidonic acid, which all belong in the group of omega -6 fatty acids. As long as there is an adequate amount of linoleic acid provided by the dog’s diet, then there is no need for other two EFAs to be provided, as they can be synthesized from linoleic acid within the body (17). Another fatty acid requirement that dogs may have, is for alpha-linoleic acid, which belongs in the omega -3 fatty acid family, and has similar properties as EFAs, as well as some therapeutic value. The greatest source for linoleic acid (omega-6 fatty acids) in pet foods, are vegetable oils such as corn , soybean and safflower oils, as well as fats from animals raised by consuming plants rich in linoleic acid (4,16,17) . Terrestrial plants like flax seed and marine oils such as algal, krill and fish oil, are the best sources for alpha- linoleic acid (omega-3 fatty acids) (16). As fats are an important energy source for dogs is advisable to contain a wide range within their diets, as fat can provide 8.5 kcal ME/g which is twice the amount of energy which carbohydrates and protein can provide (4,17,27,28). EFA s deficiency is not common, but when it occurs is due to poorly formulation or the improper storage of feed. Some of

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17 the signs which a dog can indicate is hair loss, dry-dull coat and skin lesion which are easily infected (4). In case of high fat density feeds, it may lead some dogs to gain weight and become obese due to the high palatability and energy density of the feed (17,28,29).

1.7.4 Vitamins

Vitamins are required by the body for growth and maintenance, as they sustain life by being used for many body’s metabolic processes. These organic compounds are not used as an energy source and neither as a structural compounds. Vitamins are classified into fat-soluble vitamins and the water-soluble vitamins. Vitamins A, D, E, and K belong to the fat soluble group, and B-complex vitamin as well as vitamin C belong to the water soluble group (4,16).

1.7.5 Minerals

Minerals are inorganic elements which are vital to the body’s metabolic mechanisms. They are classified into two categories, macro minerals and micro minerals otherwise known as trace elements.

Macro minerals account for most of the body’s mineral content and they include elements such as calcium, phosphorus, magnesium, sulphur, iron and electrolytes sodium, potassium, chloride. These elements have several function like activating enzymatically catalyzed reaction, contribute in nerve impulse transmission and muscle contractions, provide skeletal support, and have major roles as components transport proteins and hormones. In addition, they maintain and control the water and electrolyte balance within the body. There are vital interrelationship existing among the mineral elements which if are disturbed the can affect the organisms absorption, metabolism and overall functioning (4,16,17).

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2 RESEARCH METHODS AND MATERIALS

2.1 Investigated shelters and dogs

The Lithuanian shelter “X” was an indoor facility equipped with several sizes of cages, which had a maximum capacity of two hundred dogs total. During the winter and summer season the facility is thermo regulated. The Cypriot shelter “Y” is an outdoor fenced facility, which contained a total of twenty identically sized cages, and had a maximum capacity of forty dogs. During the summer and winter the facility is not thermo regulated.

A total of 23 shelter dogs from Lithuania and Cyprus, participated in this research. Blood samples were taken from clinically healthy dogs from Lithuania (n=14) and from Cyprus (n=9), with an average age 3.93 years old (Lithuania), and 5 years old (Cyprus). In the Lithuanian dog shelter, there was a total number of 9 female dogs, and a total of 5 male dogs, all of which were neutered. In contrast, from the dogs in Cyprus, only 5 out of 7 females were spayed and only 1 out of the 2 males had been neutered. All the dogs from each shelter consumed the same type of feed.

2.2 Physical examination and determination of Body condition score

According to the American Animal Hospital Association Nutritional Assessment Guidelines for Dogs and Cats and the European Pet food Industry Federation guidelines(2,30), During the physical and visual examination of the dogs’, behaviour, locomotion, condition of fur and skin (moist, shiny, not easily plucked hair, nor shedding excessively, skin shouldn’t be scaly, dry or thin no fleas or ticks), as well as their oral and palpebral mucous membranes were examined. The determination of the body condition score, was done according to a scale of 1 to 5, with 2.5 to 3 being the ideal, which yields to the recommended 12 to 20 % of total body fat (24) (Annex 1).

2.3 Evaluation of shelter facilities and Environment

The examination of the shelter facilities and environment required the inspection of the location, of the cages, flooring, drainage system, bedding, cleaning and the overall conditions in which the animal lived and expressed their normal behavior. In order to be able to inspect, whether the shelters complied with the regulations of the Code of Practiceor the welfare of dogs for conditions of boarding a dog in a shelter(31). Information from both shelters was gathered according to the guidelines of assessment from the Code of Practice for the welfare of dogs and the American Animal Hospital Association Nutritional Assessment Guidelines for Dogs and Cats (2,32).

2.4 Evaluation of nutrition

Along the information provided by the self-conducted questionnaire (Annex 2) filled by the employees of the shelter, and the information gathered from the food label used in each shelter, about the type, composition, analytical constituents, energy density, as well as quantity consumed by the dogs, and additionally the snacks, supplements and water availability provided by the shelter. We

(19)

19 will be able to determine whether the quality and amount of feed provided is sustainable to provide the shelter dogs with the necessary nutrients and energy to maintain a proper health status and meet their daily energy requirements.

2.5 Collection of blood samples and medical history

The main biochemical parameters were evaluated through serum biochemical analysis, in order to assess wellness of dogs affected by the feed quality which was provided by the shelters, for a minimum period of 8 weeks. All of the samples of each country were taken on the same day, and none of the dogs had been fed in the 12 hours prior to sampling. According to the shelters’ health records, none of the participating dogs had received any kind of medications 10 days prior to the blood sampling. Moreover, all the animals which were younger than the age of 1 years old or older than 13 years old, as well as dogs which were recently diagnosed with a systemic or metabolic disease were excluded from the sampling.

Prior to blood sampling, all dogs underwent physical examination and the medical history of each individual was examined in order to gather information about their current health status. Whether they were spayed or neutered, and if they were suitable candidates to participate in the testing.

Furthermore, along with the physical examination, an assessment of their body condition score was performed.

All the samples were collected from the cephalic vein using a 23 or 25 gauge needle and a 5 ml syringe. Approximately 2 to 3 ml of whole blood was withdrawn from the vein and placed into serum (clot activator) tubes aseptically.

2.6 Transportation and Storage of samples

The serum separating tubes, with the blood samples which were collected from the Lithuanian dog shelter were left in room temperature to coagulate for approximately 30 minutes, while they were being transported to the Biochemistry department in the Veterinary Academy. In the laboratory, the samples spun in the centrifuge at 3500 rpm for 10 to 13 minutes. The serum was isolated in plain tubes and frozen to -18°C until further investigation. In Cyprus the samples were transported to a local veterinary clinic after 30 minutes from sampling, and they were treated in the same manner as the samples in Lithuania. The only exception is that the samples from Cyprus, after a 48 hour period of being placed in the freezer, were shipped to Lithuania within 24 hours, by use of cold shipping packaging, which maintained the temperature between a range of 2 to 8°C for an additional 48 hours.

As soon as the samples arrive at the laboratory of the Biochemistry department they were refrozen to -18 ° C until further investigation.

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20 2.7 Determination of selected blood parameters

All the serum biochemical parameters have been determined by biochemical analyzer DiaLab in the Biochemistry department, in Lithuania, in two consecutive days. A total of 15 parameters were analyzed (Annex 3). In order to provide information about the health condition of the dogs in the shelter of Lithuania and Cyprus. All the dogs of each shelter consumed the same feed for at least one month before the sampling.

2.8 Questionnaire and statistical investigation

A self-conducted questionnaire for the employees of both shelters (in Lithuania and in Cyprus) was conducted according to the American Animal Hospital Association Nutritional Assessment Guidelines for Dogs and Cats (2). The workers were asked a total of 8 questions, which inquired information regarding feeding management (type and amount of feed, snacks, supplements etc.), shelter facilities and environment. As well as, data about the physical activities of the dogs and about the cleaning and disinfection protocols which are applied to the shelters (Annex1).

The statistical analysis was made using Microsoft Excel 2011. ANOVA, P-value etc.

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21

3 RESEARCH RESULTS

3.1 Nutrition and feed assessment

The information about type, composition, analytical constituents, energy density, quantity, feeding schedule, snacks and supplements, as well as water availability (Table 1) was gathered according to the questionnaire, answered by the shelter” X” located in Lithuania, and shelter “Y”

located in Cyprus.

Table 1 Nutrition and feed assessment of Lithuania "X" shelter and Cyprus "Y" shelter

Nutrition and feed assessment

Lithuania (n=14) Cyprus (n=9)

Type Complete pet food for adult dogs

of all breeds – Dry food Extruded, and Complete pet food for adult dogs of all breeds – Dry food Composition PER KILO: Maize, salmon meal

(min. 8%), rice (min. 8%), wheat bran, triticale, poultry fat, poultry meal, sugar beet pulp, linseed, minerals.

Nutritional additives: vitamin A (retinol E672) – 9000 IU, vitamin D₃ (cholecalciferol E671) – 750 IU,

vitamin E (α-tocopherol 3a700) – 90 mg,

iron (E1) – 25 mg, iodine (E2) – 0,8 mg,

copper (E4) – 2,5 mg, manganese (E5) – 10 mg, zinc (E6) – 45 mg, Selenium (E8) – 0, 05 mg.

Technological additives:

antioxidants, preservatives.

PER KILO: High quality poultry meal, cereals, oil &Fat, Soya meal ,Wheat –bran, Sugar –beet Pulp, Fish meal, Minerals & vitamins,

permitted antioxidant (BHA) Nutritional additives:

Vat. A 15.000 IU D3 2.000 IU E 130 Mg K 35 Mg B1 10 Mg B2 8Mg Vit C 0.5 Mg B12 15μg

Energy Density No information provided on label ME 3250 Kcal / kg

Quantity

Large dogs two cups Medium/Small dogs one cup (approximately 200gr each cup)

All sized dogs get 2 cups (Approximately 200gr each cup)

Feeding schedule Once per day ,If dog cachectic then twice per day

Once per day

Snacks Commercial dog treats e.g.

Biscuits, sticks .During weekends the get 2- pieces of dog treats by visitors

Commercial dog treats e.g.

Biscuits, sticks. Each dog 2 dog treats everyday

Supplements Fish oil No supplements given

Water Available all the time Available all the time

(22)

22 Figures 4-5 represent the analytical constituents of the feed provided to the dogs by the aforementioned shelters, based on the information given by the shelters and according to feed labels.

Figure 4 Analytical feed constituents of dry feed provided by the Lithuanian Shelter “X”

Figure 5 Analytical feed constituents of dry feed provided by the Cypriot Shelter “Y”

The Lithuanian shelter provided feed which had a Crude protein of 22%, and the Cypriot shelter provided feed which had a Crude protein of 25 %. The Cypriot shelter feed also had an extra supplementation in the feed of, L-Lysine 1.10%, which is an essential amino acid. The Lithuanian

22%

8%

6%

4%

1%

0,95%

0,55% 1,60%

Analytical feed constituents-Lithuania

Crude protein Crude oils and fats Crude ash

Crude fibers Calcium Phosporus

Omega 3 fatty acids Omega 6 fatty acids

25%

12%

3%

7%

1,30% 0,20%1,10% 0,80%

Analytical feed constituents-Cyprus

Crude Protein Crude oil &Fat Crude Fibers Crude Ash

Calcium Sodium L-Lysine Phosphorus

(23)

23 shelter provided feed that had a Crude fats & oils value of 8%, and the Cypriot shelter provided feed that had a Crude fats & oils of 12%.

In Lithuania the composition of the feed provided, had the following list of ingredients in descending order, according to the prevalence of weight per kilo: Maize, Salmon Meal (min. 8%), Rice (min. 8%), Wheat Bran, Triticale, Poultry Fat, Poultry Meal, Sugar-Beet Pulp, Linseed, Minerals. In Cyprus the composition of the feed provided also had the following list of ingredients in descending order, according to the prevalence of weight per kilo: High Quality Poultry Meal, Cereals, Oil & Fat, Soya Meal, Wheat–Bran, Sugar–Beet Pulp, Fish Meal, Minerals & Vitamins, Permitted Antioxidant (BHA).

3.2 Shelter facilities and Environment

Based on the guideline(32) there are set standards and criteria, which differentiate adequate facilities from those that do not meet the proper requirements for the appropriate welfare of shelter dogs. In Table 2, the differences between the two shelters’ adequacy are evaluated in agreement to those set criteria presented in the guideline.

Table 2 Lithuania and Cyprus comparison standards for adequacy of care in shelter "X" and shelter "Y" ”+”meets the recommendations, “-“ does not meet recommendations, “+/-”inclusive parameters, “N/A” not applicable

Standards of Care in Dog Shelters Lithuania Cyprus

Cage

Location Indoors Outdoors

Safe Location (No hunting area, No cliffs, No

hazardous environment surrounding the shelter etc…)

+ +

Quite (No construction

sites, no busy road, etc.) + +/-

Size (large enough to allow dogs express normal behavior.)

- +

Dogs per cage

(No minimum has been recommended for dogs, as long as all dogs within the cage are able to express a variety of normal

behaviors.)

2-3(depending on the

dogs sizes) 2

Minimal risk of injury + +/-

Minimal risk of escape + +

Durable, good state and well maintained

(No cracks, on porous surfaces etc.)

+/- +/-

(24)

24 Flooring Type (Smooth, impervious

and able to disinfect) Epoxy coated flooring

+/- Concrete

+/- Drainage

system

No accumulation of fluids + +

Cleaning Daily. Use of suitable, safe, effective cleaning products.

Disinfection minimum 1 per week

Cleaned once per day, high pressure washer hose and chlorine used for disinfection

(everyday)

Cleaned twice per day only with high pressure washer hose.

Disinfection only once per week with chlorine Bedding Type (Soft blankets, dog

houses, dog beds, carpets, etc.)

No bedding provided Small wooden houses ,with a blanket for each dog individually

Easily disinfected N/A +/-

Clean, dry N/A -

Outdoors

access Each day for min 20 minutes (running, playing, walking, etc.)

10-30 min once per

day 10 min twice per day

Toys Must be provided Not provided within the cage , only during outdoors access

Not provided within the cage , only during outdoors access

3.3 Comparison of main biochemical parameters

Several biochemical parameters relating to nutrition were examined. The mean concentration of each parameter from both the shelters, in Cyprus and Lithuania were calculated (Table 4). The analysis showed that the average difference regarding concentration of several biochemical parameters, in Cyprus-sheltered dogs, in comparison to the Lithuania-shelter dogs, were more by 220.8% of Triglycerides (P<0.05), by 12.82% of Glucose (P<0.05), by 80.31% of Alkaline phosphatase (P<0.05), and by 2.675% of Aspartate aminotransferase (P>0.05). In addition Cyprus- sheltered dogs had higher average difference values in the case of, Protein total by 32.10%, of Phosphorus by 55.18% with positive significance (P<0.05). As well as, of Urea by 26.16% and of Magnesium by 32.38% with a great significance (P<0.01).

On the contrary, the average difference of some parameters of the Lithuanian-sheltered dogs related to Cyprus–sheltered dogs, were more by 24.17% of Alanine aminotransferase(P>0.05), by 20.22% of Cholesterol(P>0.05), by 5.68%Calcium(P>0.05), and by 7.76% of Potassium (P>0.05).

Conversely, the average difference of the Lithuania-Sheltered dogs is more by 98.13% of Creatinine (P<0.01), by 188.4% of Gamma-glutamyltransferase (P<0.01), by 303.03% of Sodium (P<0.05), when compared to Cyprus-sheltered dogs.

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25 Table 3 Mean concentration of main biochemical parameters from the shelter in Lithuania and Cyprus

No. Mean concentration of main

biochemical parameters Reference

value Lithuania Cyprus

1. Triglycerides mmol/L 0.32-3.3 6.125±1.083 19.647±14.269

2. Glucose mmol/L 3.9-7.7 4.186 ±0.162 4.723 ± 0.310

3. Creatinine μmol/L 44.2-141.4 105.572±21.861 209.17± 29.32

4. Urea mmol/L 2.9-10 15.026± 0.691 19.406±1.265

5. Protein total g/L 50-74 73.05± 2.31 96.5 ±10.06

6. Alkaline phosphatase U/L 5-131 82.285±32.049 45.633±11.602 7. Gamma-glutamyltransferase

U/L 1-12 7.947±0.807 2.755 ± 1.409

8. Aspartate aminotransferase

U/L 15-66 43.578±4.533 44.744 ±9.720

9. Alanine aminotransferase U/L 12-118 55.942±7.458 45.055±9.243

10. Cholesterol mmol/L 2.38-8.38 3.199±0.147 2.732±0.342

11. Phosphorus mmol/L 0.8-1.93 1.716±0.116 2.663±0.414

12. Magnesium mmol/L 0.5-0.8 0.386±0.0136 0.511±0 .0481

13. Calcium mmol/L 2.22-2.85 1.544±0.036 1.461± 0.185

14. Sodium mmol/l 139-154 184.15± 48.00 139.91±26.50

15. Potassium mmol/L 3.6-5.5 4.881±0.831 4.543±0.197

3.4 Comparison of main biochemical parameters according gender

3.4.1.1 Triglycerides, glucose and cholesterol

In Figure 6 the mean concentration of Triglycerides is shown, as it was measured, and it indicates that female dogs in Cyprus average difference is more by 298.58% of Triglycerides concentration (P>0.05), when compared to Lithuania female sheltered dogs. In Lithuania the male sheltered dogs average difference is more by 21.80% of Triglycerides mean concentration (P>0.05), in comparison to Cyprus male sheltered dogs.

(26)

26 Figure 6 Triglycerides mean concentration according to gender in Lithuania and Cyprus(Reference value 0.32-3.3 mmol/L)

In case of Glucose mean concentration, as it’s represented according to genders in Figure 7, the average difference of Female dogs coming from Cyprus, is more by 13.72% (P>0.05), when compared to Lithuanian female sheltered dogs. For the male sheltered dogs from Cyprus the average difference in comparison to the Lithuanian male sheltered dogs, is more by 9.55% (P>0.05).

Figure 7 Glucose mean concentration according to gender in Lithuania and Cyprus (Reference value 3.9-7.7 mmol/L)

Shown in Figure 8, Cholesterol’s concentration average difference is more by 26.51 %(

P>0.05), for female dogs originating from Lithuania when compared to the female sheltered dogs of Cyprus. But in the case of male sheltered dogs cholesterol’s concentration average difference, of Cyprus originating dogs is more by 7.19% (P>0.05), related to the male sheltered dogs of Lithuania

5,960

23,755

6,422 5,273

LITHUANIA CYPRUS

CONCENTRATION

TRIGLYCERIDES mmol/L

Female Male

4,188

4,763 4,185

4,585

LITHUANIA CYPRUS

CONCENTRATION

GLUCOSE mmol/L

Female Male

(27)

27 Figure 8 Cholesterol mean concentration according to gender in Lithuania and Cyprus (Reference value 2.38-8.38 mmol/L)

3.4.1.2 Kidneys function parameters

Creatinine’s mean concentration represented in Figure 9, indicates that the average difference of Female sheltered dogs from Cyprus, in comparison to the Lithuanian female sheltered dogs, is more by 78.99%(P>0.05). As well as, in the case of male sheltered dogs coming from Cyprus,whose average difference of Creatinine is more by 157.12%(P<0.05), in comparison to Lithuanian male sheltered dogs.

Shown in Figure 10, Urea’s mean concentration of female sheltered dogs originating from Cyprus, had an average difference of concentration higher by 29.02 %( P<0.05) when compared to the Lithuanian female sheltered dogs. Correspondingly, male sheltered dogs originating from Cyprus, had an average difference higher by 35.19 %( P<0.05) related to the Lithuanian male sheltered dogs.

Figure 9 Creatinine mean concentration according to gender in Lithuania and Cyprus (Reference value 44.2-141.4 μmol/L)

3,22

2,54

3,17 3,39

LITHUANIA CYPRUS

CONCENTRATION

CHOLESTEROL mmol/L

Female Male

110,731

198,200 96,288

247,576

LITHUANIA CYPRUS

CONCENTRATION

CRE AT ININE μmol /l

Female Male

(28)

28 Figure 10 Urea mean concentration according to gender in Lithuania and Cyprus (Reference value 2.9-10 mmol/L)

The average difference of Protein Total (Figure 11) concentration is more by 38.69% (P<0.05) for female sheltered dogs originating from Cyprus. In the case of male sheltered dogs originating from Cyprus, the average difference of Protein Total concentration is more by 4.76% (P>0.05), related to the corresponding concentration from Lithuanian male sheltered dogs.

Figure 11 Protein Total mean concentration according to gender in Lithuania and Cyprus (Reference value 50-74 g/L)

3.4.1.3 Liver function parameters

Contrastingly as shown in Figure 12, the average difference of alkaline phosphatase concentration is more by 119.76 %( P>0.05), in the case of Lithuanian female sheltered dogs. But in

14,621

18,865 15,755

21,301

LITHUANIA CYPRUS

CONCENTRATION

URE A mmol/L

Female Male

74,10

102,77

71,16 74,55

LITHUANIA CYPRUS

CONCETRATION

PROTEIN TOTAL g/L

Female Male

(29)

29 the case of male sheltered dogs, the average difference of alkaline phosphatase concentration of Cyprus originating dogs is more by 9.55% (P>0.05).

Figure 12 Alkaline phosphatase mean concentration according to gender in Lithuania and Cyprus (Reference value 5-131 U/L)

Shown in Figure 13 the average difference of Gamma-glutamyltransferase concentration is more by 125.03 %( P<0.05) for the Female sheltered dogs of Lithuania. Likewise, for the male sheltered dogs of Lithuania, their average difference is more by 788500% (P<0.05), in comparison to Cyprus male sheltered dogs.

Figure 13 Gamma -glutamyltransferase mean concentration according to gender in Lithuania and Cyprus (Reference value 1-12 U/L)

Aspartate aminotransferase’s concentration average difference is more by 2.72% (P>0.05), for the female sheltered dogs originating from Lithuania compared to the female sheltered dogs of Cyprus. But, for the male sheltered dogs, originating from Lithuania is less by 24.70 %( P>0.05), when compared to the Cyprus male sheltered dogs. (Figure 14)

4,30

2,40 2,50

4,40

LITHUANIA CYPRUS

CONCENTRATION

AL KALINE PHO SPHATASE U/ L

Female Male

7,98

3,54 7,89

0,00

L I T H U A N I A C Y P R U S

CONCENTRATION

GAMMA-GL UTAMYLT RANSFERASE U/L

Female Male

(30)

30 Figure 14 Aspartate aminotransferase mean concentration according to gender in Lithuania and Cyprus (Reference value 15-66 U/L)

In case of Alanine aminotransferase concentration (Figure 15), the average difference is more by 13.90% (P>0.05) for Lithuania Female sheltered dogs compared to Cyprus Female sheltered dogs.

Yet, for male sheltered dogs origination from Lithuania, the average difference of their Alanine aminotransferase concentration is more by 31.19% (P>0.05), related to the male sheltered dogs from Cyprus.

Figure 15 Alanine aminotransferase mean concentration according to gender in Lithuania and Cyprus (Reference value 12-118 U/L)

3.4.1.4 Minerals

In Figure 16 the mean concentration of Phosphorus is shown, and its indicated that female sheltered dogs from Cyprus had an average difference which is more by 63.85%(P<0.05), when

38,66 37,63

52,44

69,65

LIT HUA NIA CY PRUS

CONCENTRATION

ASPARTATE AMINOTRANSFERASE U/L

Female Male

49,01 43,03

68,42

52,15

LIT HUA NIA CYPRUS

CONCENTRATION

ALA NINE AMINOTRANSFE RASE U/L

Female Male

(31)

31 compared to Lithuanian female sheltered dogs. Furthermore, in Cyprus the male sheltered dogs average difference of Phosphorus is more by 28.11% (P>0.05), in comparison to Lithuanian male sheltered dogs.

Figure 16 Phosphorus mean concentration according to gender in Lithuania and Cyprus (Reference value 0.8-1.93 mmol/L)

The magnesium concentration as shown in Figure 17, had an average difference of concentration higher by 34.42 % (P<0.05) in female sheltered dogs originating from Cyprus, when compared to the female sheltered of Lithuania. Respectively, the male sheltered dogs of Cyprus average difference of magnesium concentration, is more by 21.92 % (P>0.05), in comparison to the Lithuanian male sheltered dogs.

Figure 17 Magnesium mean concentration according to gender in Lithuania and Cyprus (Reference value0.5-0.8 mmol/L)

In case of calcium concentration as shown in Figure 18, the average difference is more by 9.02% (P>0.05) for female sheltered dogs originating from Lithuania, in comparison to the female

1,700

2,785

1,746 2,237

LIT HUAN IA CY PRUS

CONCENTRATION

PHOSPHORUS mmol/L

Female Male

0,391

0,525 0,380

0,464

LIT HUA NIA CY PRUS

CONCENTRATION

MAGNESIUM mmol/L

Female Male

(32)

32 sheltered dogs of Cyprus. On the contrary, the average difference of calcium concentration is more by 4.32% (P>0.05), in the case of male sheltered dogs of Lithuania, when compared with male sheltered dogs of Cyprus

Figure 18 Calcium mean concentration according to gender in Lithuania and Cyprus (Reference value2.22-2.85)

The average difference for Sodium concentration is more by 44.98% (P>0.05), for female sheltered dogs originating from Lithuania, when compared to female sheltered dogs from Cyprus.

Moreover, in the case of male sheltered dogs the sodium concentration average difference is more by 21.77% (P>0.05) in Lithuania, in comparison to male sheltered dogs from Cyprus (Figure 19).

Figure 19 Sodium mean concentration according to gender in Lithuania and Cyprus (Reference value139-154 mmol/L)

In Figure 20 the mean concentration of Potassium is shown, and it indicates that female sheltered dogs from Lithuania had an average difference which is more by 18.29 % (P>0.05), when

1,550

1,422 1,536

1,602

LITHUANIA C YPRUS

CONCERNTATION

CALCIUM mmol/L

Female Male

172,00

118,63 196,30

161,20

LIT HUANIA CYPR US

CONCENTRATION

SODIUM mmol/l

Female Male

(33)

33 compared to Cyprus female sheltered dogs. Furthermore, in Cyprus the male sheltered dogs average difference of Potassium concentration is more by 4.27% (P>0.05), in comparison to Lithuanian male sheltered dogs.

Figure 20 Potassium mean concentration according to gender in Lithuania and Cyprus 3.6-5.5 mmol/L

5,547

4,689

4,215 4,398

LITHUANIA CY PRUS

CONCENTRATION

POTASSIUM mmol /L

Female Male

(34)

34

4 DISCUSSION OF RESULTS

According to Buckland 2014(33), a consistent and reliable diet should be provided to all the dogs which are accommodated to shelters, rather than a variety of mixed products. Providing a consistent diet decreases gastrointestinal disorders, and inappetance linked to frequent diet alterations. As it promotes the use of a constant appropriate quantity and prevents overfeeding or underfeeding. In Lithuania and Cyprus, both of the shelters provided dry feed, which was classified as complete pet food for all ages and breeds. This type of diet should be formulated in a way, which serves the optimal quantities of all vital nutrients, when any breed or dog of any age consumes it. If the appropriate amount of feed is consumed , then it should cover all the daily energy requirements of the dog (4). The feed provided to the Lithuanian dog shelter had cereals and corns as the most prevalent ingredients, followed by animal origin by-products. Contrastingly in Cyprus, the shelter provided a feed, which had as its main ingredient, an animal originating by-product, with less cereal and corn concentration. Which were not ,as digestible carbohydrates as, the ones provided in the Lithuania feed, such as rice and sugar beet-pulp etc.(34). In both feeds, the concentration of all the nutritional additives is above the minimum recommendation, making both of the feeds adequate as a source of minerals and vitamins (5, 24, 28).

The body requires dietary protein, as it uses it for protein synthesis, but also to supply adequate nitrogen, which will be used for the synthesis of non-essential amino acids and other nitrogen containing compounds (24). Hence, an adult dog, has as a minimum requirement of 18% Crude protein of total caloric distribution (5,24,28). Both of the shelters had Crude protein percentage values which were higher than the minimal recommendation. The higher the quality, and quantity of Crude protein percentage within the feed provided, the smaller amount of feed is necessary, in order to cover all of the essential amino acid needs of an adult dog (24,31).

Oils and Fats have a major role in metabolism, therefore, all dogs have a bodily requirement of minimum 5.5% of Crude fats (30). In order to have adequate essential fatty acids, to function as carrier of fat-soluble vitamins, to enhance palatability of the feed, and to supply an adequate caloric density(35). Both of the shelters had a percentage value of Crude fats & oils, higher than the minimum recommended value(30).

The group of omega -6 fatty acids (linoleic, gamma- linoleic and arachidonic acid), and omega -3 fatty acids (alpha – linoleic acid), are considered essential for a complete diet. The effects which omega-3 fatty acids have, depend on the level, but also on the ratio of omega-6 to omega-3 fatty acids.

According to Giorgia et al. 2019 (36), high levels of omega-3 fatty acids can deteriorate cellular immunity, especially in the of a low level of omega-6 fatty acids concentration. The minimum recommendation for Linoleic acid(omega 6 fatty acids) is 1.1% (30). A minimum requirement has

(35)

35 not yet been determined for omega-3 fatty acids. But several studies, have indicated that a ratio of 6:1 omega-6: omega-3 fatty acid, reduce the incidence of cancer and sudden cardiac death (2). In the case of Lithuanian shelter provided feed it contained omega 3 – 0.55% and omega 6 – 1.6%. (3:1 ratio), which is still considered adequate (2,30). There was no information provided for the concentration and ratio of omegra-6 and omega -3 fatty acids, in the feed provided in the Cypriot shelter.

A smaller source of energy, when compared with Fats in a dog’s nutrition, is Carbohydrates (26, 27). The major sources of carbohydrates in dog foods are cereals, legumes, and other plant originating products (5, 26,27). Absorbable carbohydrates, are directly absorbed, as they do not need to be digested by enzymes of the gastrointestinal tract of the dog. Digestible carbohydrates are easily broken down by intestinal tract enzymes. In the case of Dietary fibers or otherwise known as fermentable carbohydrates, they cannot be digested, by the gastrointestinal tract of the dog, but, some of the microbes found in the large intestine, can ferment them (5, 24). Dietary fibers are crucial for the healthy maintenance of the epithelial cells, which constitute the mucosa of the intestines (5.26).

These type of non-fermentable fibers are reported as crude fiber, and it reports the insoluble portion added within the feed. A recommended rage of Crude fiber is from 2.5-4.5%, both of the shelters had feeds which had over the minimum recommended concentration.

According to the guidelines for Standards of Care in Animal Shelters(31)the primary enclosure needs to be structurally durable and well maintained, in order to properly confine animals, and have the minimal risk of injury. Furthermore, the size of the enclosure needs to be large enough, to allow dogs to express normal behaviour (e.g. to turn freely and stand, sit, stretch, move their head with adequate space, in order not to touch the top of the enclosure, lie down in a comfortable position with limbs extended, extra space to move about and assume a comfortable posture during feeding, drinking, urinating and while defecating) (31,32,37).

A combination of cleaning and disinfecting of all the areas of the shelters, but especially of the enclosure and drainage system, is a requirement for the destruction of all microbes and their spores.

True sterilization of cage and shelter surfaces cannot be fully accomplished, but improper cleaning could promote the increase of pathogen transmission within the shelter (31,38).The Lithuanian and Cypriot shelter complied according to these standards.

Serum biochemical profiles of dogs, are essential for the evaluation of their health condition (39). The levels of most of the values, which were obtained from the shelter in Cyprus and Lithuania, were within the presented range in Atata, 2018 and Ihedioha et al., 2013 (39,40). This implies, that the shelter dogs which participated from the Lithuanian shelter, and the Cypriot shelter had normal acid base balance, with no indication of renal or hepatic dysfunction. Abnormally high values of Urea(P<0.05), creatinine(P<0.05), total protein (P<0.05) were obtained from both populations of the

COMPARISON OF THE IMPACT OF NUTRITION AND ENVIRONMENTAL FACTORS ON THE WELFARE OF SHELTER DOGS IN LITHUANIA AND

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Faculty of veterinary medicine

Anastasia Stylianidou

COMPARISON OF THE IMPACT OF NUTRITION AND ENVIRONMENTAL FACTORS ON THE WELFARE OF SHELTER DOGS IN LITHUANIA AND

CYPRUS

MITYBOS IR APLINKOS VEIKSNIŲ ĮTAKOS

PRIEGLAUDŲ ŠUNŲ GEROVEI LIETUVOJE IR KIPRE PALYGINIMAS

Of Integrated Studies of Veterinary Medicine

Supervisor: Dr. Vaida Andrulevičiūtė

KAUNAS 2019

TABLE OF CONTENTS

SUMMARY

SANTRAUKA

ABBREVIATIONS

INTRODUCTION

1 LITERATURE REVIEW

1.1 The types of commercial pet food

1.2 Regulation of pet food

1.3 Food labelling

1.4 Energy utilization

1.5 Energy requirements in a healthy adult dog

1.6 Assessment guideline for energy requirements

1.7 Energy intake

2 RESEARCH METHODS AND MATERIALS

3 RESEARCH RESULTS

Analytical feed constituents-Lithuania

Analytical feed constituents-Cyprus

3.2 Shelter facilities and Environment

3.4 Comparison of main biochemical parameters according gender

5,960

23,755

6,422 5,273

TRIGLYCERIDES mmol/L

4,188

4,763 4,185

4,585

GLUCOSE mmol/L

3,22

2,54

3,17 3,39

4 DISCUSSION OF RESULTS