Radiographic imaging and blood profile analysis of canine pyometra in a Swedish small animal clinic "A" Kalių sergančių piometra rentgenogramų ir kraujo tyrimų analizė Švedijos klinikoje "A"

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY MEDICINE PROGRAM

INTEGRAL STUDIES

Dr. Leonas Kriauceliunas small animal Clinic

Faculty of veterinary medicine Anna Mia Regina Lidén 6th year 10 group student

Radiographic imaging and blood profile analysis of canine

pyometra in a Swedish small animal clinic "A"

Kalių sergančių piometra rentgenogramų ir kraujo tyrimų analizė

Švedijos klinikoje "A"

MASTER’S THESIS

of Integrated Studies of Veterinary Medicine

Supervisor: VMD Brigita Grigaleviciene

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2 THE WORK WAS DONE IN THE DEPARTMENT OF

Dr. Leonas Kriauceliunas small animal Clinic

CONFIRMATION OF THE INDEPENDENCE OF DONE WORK

I confirm that the presented Master Theses “ Radiographic imaging and blood profile analysis of canine pyometra in a Swedish small animal clinic "A" ”.

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.

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

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

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CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESES

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THE MASTER THESES HAVE BEEN APPROVED IN THE DEPARTMENT/CLINIC

(date of approbation) (name, surname of the manager of department/clinic) (signature) Reviewers of the Master Theses:

1)_________________________________________________________________________

2)_________________________________________________________________________

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Evaluation of defence commission of the Master Theses:

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ABBREVIATIONS

ALT-Alanineamino transferase CL- Corpus luteum CL%-confidence limits at 95% Cm- centimeter Crp- C-reactive protein Dr.-Doctor DIC-Disseminated

FSH- Follicular stimulating hormone Hb- Hemoglobin

Hct- hematocrit

LH- Luteinizing hormone mAs- Milliamperes

MODS-Multiple organ disorder syndrome OHE- Ovario-hysterectomy

SIRS-Systemic inflammatory response syndrome V-Voltage

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SUMMARY

Radiographic imaging and blood profile analysis of canine pyometra in a Swedish small animal clinic "A"

Anna Mia Regina Lidén Master’s Thesis

The aim of the master thesis was to analyze and to compare radiographic measurements and its appearance, for canines suffering from pyometra. To compare and analyze the results from the analysis of blood profiles, and to compare parametric changes that will occur during the condition of pyometra during the period of 2016-2017. The objectives were to evaluate incidence and prevalence of pyometra (breed, weight, age, days after estrous and state of diagnosis) and to establish average size of uterine enlargement with maximum and minimum limits of affected females.

The research was executed in Sweden at a small animal clinic ”A” from the period of 1st of december 2016 until 10th of october 2017. During this period, a total number of 274 canines were collected, with the included number of 149 females. A collection from the total number of females could be selected from 41 females: 25 female canines with the diagnosis of pyometra and 16 female canines was selected as control group. For all of these participating females, a blood profile (Hb, Hct, WBC, CREA, GLU, ALT,Na+, K+ and CRP) and radiography has been performed and evaluated. The analytical statistics has been calculated and evaluated by the use of ”Microsoft Excel 2017”.

The results of the master thesis pronounced a prevalence of breed distribution with the labrador retriver, Golden retriever and German shepherd as the most frequently recruited breeds in this research. There were no significant correlation of breed-, age-, weight predisposition nor days after estrous in the affected females. Significance was observed concerning the size of uterus in correlation to the state of pyometra. Estimated average size of uterus was 3.39 cm. Blood profile showed that canines with pyometra tend to have elevations in the average of ALT (1.18 U/L) and CRP (65,35 g/dL), and lowered value of Hct (37.98 %) in comparison to those canines included in the control group.

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SANTRAUKA

Kalių sergančių piometra rentgenogramų ir kraujo tyrimų analizė Švedijos klinikoje "A" Anna Mia Regina Lidén

Magistro baigiamasis darbas

Darbo tikslas: palyginti ir išanalizuoti radiografijos naudą diagnozuojant kalių piometrą. Darbui buvo iškelti šie uždaviniai: išanalizuoti ir palyginti kraujo morfologinius ir biocheminius parametrus; palyginti piometra sergančių kalių kraujo parametrų pokyčius; įvertinti veislės, svorio, amžiaus, porujo laikotrapio ir piometros stadijos įtaką ligos pasireiškimui; radiografijs pagalba įšmatuoti gimdos skersmenį kalėms, kurioms diagnozuota piometra.

Tyrimas buvo atliktas 2016-12-10 – 2017-10-10 mėn., „A“ smulkiųjų gyvūnų klinikoje Švedijoje. Minėtu laikotarpiu klinikoje apsilankė 274 šunys, iš kurių buvo 149 kalės. Tyrimui pasirinkta 41 kalė, iš kurių 25 buvo diagnozuota piometra, o likusios 16 kalių buvo kliniškai sveikos – kontrilinė grupė. Visoms kalėms buvo atliekami kraujo morfologiniai ir biocheminiai tyrimai (Hb, HCT, WBC, CREA, GLU, ALT, Na, K, CPR) bei gimdos radiografija.

Atlikus tyrimus nustatyta, kad dažniausiai piometra sirgo Labradoro retriverių, Auksaspalvių retriverių ir Vokiečių aviganių veislės kalės, nors koreliacijos tarp susirgimų ir veislės, amžiaus, svorio, porujo laikotrapio ar piometros stadijos - nebuvo. Stiprus teigiamas koreliacijos ryšys pastebėtas tarp piometros stadijos ir gimdos skersmens. Sirgusiųjų piometra vidutinis gimdos skersmuo buvo 3,39 cm. Atlikus tyrimus nustatyta, kad sirgusių piometra kalių ALT (1,18 U/L) ir CRP (65,35 g/dL) padidėjo, o HCT (37,98 proc.) sumažęjo, lyginant su kalėmis kontrolinėje grupėje.

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INTRODUCTION

Objectives of the research

The aim of the master thesis is to analyze and to compare the importance of the radiographic measurements and its appearance, when dealing with canines suffering from pyometra. To compare and analyze the received results from the analysis of blood profiles, and to compare parametric changes that will occur during the condition of canine pyometra.

Research tasks

The canine pyometra is a common problematic that will affect our un-neutered female canines in today’s society. Investigation protocols and radiographic instruments have been optimized for efficiency while the significance of establishing measurable ranges of the canine uterus in determination of its prominence, in correlation to the importance of blood parameters, are left with minimal documented data. It is therefore interesting to analyze and evaluate these parameters with the help of radiography and blood analysis, to compare and to acknowledge the initial changes that is occurring during this condition. The significant ranges of an abnormal uterine size that can be measured by radiographic instruments in order to determine a correct diagnosis of pyometra, and to support this identification by some helpful results of an ongoing immunological response.

The main objective was to determine average size of an enlarged uterus and to investigate according to what parameters and factors that will become affected and/or serve as an influence during the state of the diagnosis. Based on the purpose, the following three tasks were investigated: 1 Incidence and prevalence of pyometra (breed, weight, age and state of diagnosis) within the

collected number of participants, by the examination of radiography and blood analysis.

2 Determination of an average size by the use of radiographic measurement of the uterus, in which the veterinarian was able to state a certain diagnosis of pyometra. The formulation of a maximum and minimum limit in which the uterus become prominent upon radiographic imaging.

3 Comparison of blood parameters in an affected vs. non-affected canine, and what their significance towards the diagnosis of pyometra will conclude.

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1. REVIEW OF LITERATURE

1.1 The basic principles of radiography

Radiographic examinations is a commonly used method in the determination of disease within the clinical practice of the veterinarian field. In order to obtain a radiograph that consist of a high-quality image, the veterinarian must understand and considerate the important principles and methods that is required when dealing with radiographic examinations. The first radiograph was discovered by Wilhelm Rontgen 1895. The radiograph is used by the measurement of V and mAs. In order to create a correct installation of these two units, the measurement of the abdominal diameter is needed (for giving the veterinarian the skills in calculating the preferable values for the examination). But nowadays these measurements are instructed and concluded by specific guidelines, containing all the necessary records of data and units that has been established for each specie and their abdominal size in cm (39).

The principle of radiography involves the recording of an X-ray beams that will become reflected on a film. Different object will possess different abilities in reflecting this beam. It can therefore be concluded that all objects will absorb the beam and reflect them with a different appearance onto the film. According to the density of the object, these beams are either scattered or absorbed. Terms describing these abilities can be noted as “radiolucent” or “radiopaque” properties. An object that will possess an increased radiopacity is simply explained as having the ability to inhibit any passage of the incidental radiographic beam. These subjects addresses the beam as a “white shadow” upon the reviewing of the film. Radiolucent subject on the other hand, is said to allow passage and will cause minimal incidence of this beam, resulting in a reflection of a “dark shadow”. Structures can therefore be listed according these abilities when measuring the most increased opacity of bone, mineral, fluid, soft tissue, fat and gases (with the structures listed from most radiopaque to the one with smallest and/or radiolucent abilities such as gas). Because of this, internal organs and structures can be evaluated for their size, location, number, shape and opacity (39,40).

When performing a radiograph it should be considered that following factors may have an impact of the quality of the image, including movement during the examination, the object distance (the proper distance should be 100cm between the beam and the examined object), properties of the film, faults of processing, distortion by improper positioning or presence of artifacts (39).

The non-gravid healthy uterus are not commonly visible during the radiograph of a healthy female, since they possess a homogenous minimized fluid opacity. In cases of pyometra the opacity of the uterus will become increase and contribute to an outlining of the enlarged organ. When dealing with canines that are believed to be affected by a pyometra, a recumbent projection is made. This can be done in two ways: left lateral recumbence or right lateral recumbence. In some clinics, the

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dorso-10 ventral or ventro-dorsal projections have also been included in the determination of the disease (39, 41).

1.2 Reproductive tract of the female canine

A common trait that we humans share with all of the animals on this planet, is the ability and the urge to reproduce. The canine anatomical structure together with its features and developmental characteristics are very similar in the comparison to us humans. Included in this unique tract of reproduction there are the vagina, uterus, oviducts and ovaries represented. In which all of them will starts its hormonal, structural and functional development immediately after the time of birth (1,5). In the abdominal cavity, situated dorsally to the small intestines, is the anatomical position of the uterus. The expected size for a normal and healthy dog has been estimated approximately to reach a diameter of 0.5-1 cm (2, 3). Although in another study when measuring the size of the uterus, the average size ranged between 0.3- 0.4 cm in diameter (4).

From a histologically point of view the uterine wall can be divided into three different layers. The outermost layer that has been referred as the perimetrium, which will proceed into the second layer called the myometrial layer. The last, and the innermost layer, is described accordingly as “endometrium”, which is the mucosa of this organ (5). The myometrium is the thickest layer of all of these structures, and consists of smooth muscles, which in turn is divided into three distinctive layers. The muscular layer is important for the uterine contractions, but also plays an important role in the ability to stretch out (5, 6). The endometrium, on the other hand, has a major role in the function of reproductive and cyclical abilities. Both the myometrium and the endometrium correspond in the cyclic changes. But presumably, the endometrium is the layer with the largest influence of a close contact system with the hormonal regulation, correspondingly provoked by the ovarian activities and stimulations (6, 7). During the end of each cycle the endometrium will become partly destroyed and sloughed off, which will cause the layer to become prone to infections and inflammations. As a response, inflammatory cells will be send to the place of the damaged endometrium (6).

The female’s reproductive tract involves a complex regulation and interplay, by sending and receiving hormonal information, between the hypothalamus, pituitary gland and reproduction organs (34). This allows the organs to work together and to quickly respond and to receiving necessary information, contributing to an availability for reproduction. As a contributing result of the companionship with the organs and systems working together, hormones are able to be released, produced or repelled (32-34).

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1.3 Estrous cycle and its hormonal changes

Dr. L Forsberg (18) states that dogs between nine and twelve years of age, that has retained 85 percent of their adult weight, will possess the abilities to start the reproduction at this stage of reaching a maturity. As the female is being introduced into its first estrous cycle, the reproductive organs have reached their full potential in their developmental stage. The dog enters the reproduction cycle that will extend generally throughout life (9,18).

The dogs’ reproduction cycle differs from the reproduction cycles in other mammals (9). In general the reproduction cycle is being expressed to consist of four phases. More specifically, these four phases will correlate with one another and thereby creating a complete reproductive process, which has been divided into the four listed phases (9,12,17):

 Proestrous

 Estrous

 Diestrous

 Anestrous

A significant characteristic that the female canines emphasizes, is when displaying the property of having a reproduction cycle with the longest duration of time (measured in days), when comparing to other domesticated animals (12-16).

Synchronously as the reproductive cycle is carrying on in different stages at different times, the production of hormones will fluctuate its production and inhibition. Hormones including in the estrous cycle involves primary the GnRH, FSH, LH, estrogen, inhibin and progesterone (22).

In order to understand the pathogenesis of the pyometra that will affect our un-neutered canines, it is crucial to understand the concept of the reproduction cycle, its hormonal influence and their stimulatory responses in general. The domesticated female canine have a monoestrous cycle, which can be simply explained by having one estrous during each cycle. The estrous cycle contain a spontaneous ovulation that will have a non-seasonal stimulatory response. Although there is a presence of a cyclic periodicity that will maintain a regulatory gap of approximately 6 to 8 month between each cycle (22).

1.3.1 Proestrous

Proestrous is the period prior to estrous phase, and is characterized by the event of ovarian follicles to persist and grow. The duration of this phase has been estimated from 6 to 11 days. This phase is included in the follicular phase, together with estrous phase, and will become evident by its clinical appearance of a vaginal bleeding (22). Hypothalamus has the main role in the production and reduction effects of hormones. During this time the follicles will tend to have an influence on the

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12 endocrine system by stimulating secretion of estrogens. Hypothalamus is inhibiting its generation of GnRH, as the level of estrogens and inhibin becomes successively increased. When the estrogen level increases it will have an impact on the circulatory system that will immediately become stimulated. As a response the endometrial glands will start to grow, as the endometrium increases in size and commences a secretion of mucous. The initial role of the secretion of mucous is to produce a satisfactory environment for the male sperms. The estrogen levels will reach its peak at the end of proestrous, and the female devolves into estrous. (11,23,24)

1.3.2 Estrous

During this phase the female will accept the act of mating. The hormonal changes with an increase in the estrogen levels and the low levels of progesterone during the phase of proestrous, hypothalamus acts in form of a response by suppressing the LH levels. The LH levels will still continue in sporadic pulses to stimulate some pre-ovulatory follicles, in which a selective number will become luteinized and prohibits to develop into the ovulation stage. The more follicles that will luteinize in the ovary, the more progesterone will be released in turn. The increase of the progesterone will permit an excessive release of FSH. The FSH in turn stimulate LH to gain its peak. The hormonal changes gives access to a development of the CL. Progesterone will continue to increase throughout the whole phase, contrary the estrogen that will diminish to its basal levels (23,24).

1.3.3 Diestrous

Characterized by its high level of progesterone in both non-pregnant and pregnant females. The uterus protrudes to increase in size until there will be a regression of CL. When CL regresses the progesterone levels will start to reduce to its basal level. As a response to the reduction of progesterone, it will trigger an elevation of estrogen (24).

1.3.4 Anestrous

Anestrous is expressed as the final stage of the estrous cycle, and is indicated by an inactivation of the ovaries and the endometrium. At this time the endometrium is small with few glands and is being repaired in prior to the next estrous cycle. An altered increase of the estrogen levels also corresponds with an alteration of inhibin, LH and FSH. The hormonal concentrations will vary by the time at a basal level, until the female starts a new estrous cycle within the period of a few months (22,24).

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1.4 Pyometra

Pyometra is one of the most frequent occurring diseases among the female canines. Its process and outcome can range from a mild state, but will always preserve the potential of drastically change into a life-threatening disease. The condition can become fatal for the ones suffering from that disease. It is therefore important to pay a complete attention to the signs and symptoms, that the female expresses during the state of this illness. The treatment of the pyometra can be consulted in two different ways (10,19):

 One way is to surgically correct the problem by the implementation of an OHE (by removing the affected uterus by the mean of a total castration),

 Another way may be to medically reduce the signs and symptoms of occurring pyometra (12,21,25).

1.4.1 Prevalence

According to a Swedish study that was performed under the provision of Svenska Lantbruks Universitetet, Dr S.Waernersson states (19) that the prevalence of the canine pyometra has been determined to be one out of four females, having no significant predisposition of either age nor breed. In another study that was performed at year 2001, in which a total number of 200.000 swedish dogs participated, it was indicated a prevalence of 2 % (20). In the same study it was concluded that 24 %, of the total number “affected females”, became affected before ten years of age. But the identification and estimation of the prevalence of the canine pyometra can come to differ in a wide range (21).

1.4.2 Epidemiology

It has been discussed if the diagnosis of pyometra is caused by an age-related influence by the canines developmental stages. For a long time it was believed that pyometra was only affecting bitches in an older age. Nowadays it has been documented that age is not the contributing factor to why the condition of pyometra develops. It has, instead, been considered to be a response from the hormonal stimuli occurring within the body (25). Pyometra arises when the female responds abnormally to a normal level of hormones (19). It can affect females in any ages, but most commonly at the age after 5 to 6 years. Bitches is considered to have a higher incidence of pyometra than queens. The predisposition of breed for developing pyometra has been shown in increased number of some particular breeds, including the Bernese Mountain dog, bull terrier, Irish wolfhound, Leonberger, Bouvier des Flandres, Great Dane, Rottweiler, New Foundland, Staffordshire and keeshound (26).

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14 1.4.3 Associated disorders

There has been a prevalence of other disorders in the association of a persisting pyometra, represented as genitourinary disorders and systemic disorders (26). In the first named disorder, vaginitis and UTI are the main complications of the genitourinary tract. The most commonly occurring strain of bacteria has been identified to be those bacteria’s belonging to the E.coli spp. In other less pronounced cases of further bacterial infestations, isolates of Proteus sp, Staphylococcus sp. and Streptococcus sp has also been represented (27-31). During the estrous cycle, the uterus becomes prone to bacterial infections. A possible derivative in enabling the bacterial growth is considered to be from the normal uterine micro flora or from the urinary tract (26).

When pyometra is correlated with systemic disturbances, the condition will result in a more serious and fatal outcome. Systemic disorders includes septic shock, SIRS, MODS, DIC, Acute kidney injury, peritonitis and hepatic injury (26).

1.4.4 Classification

Commonly the pyometra can be classified from the view of clinical sign. In this way the extent of vaginal discharges corresponds to a hypothesis in the determination wether the cervix is closed or not. When there is a present vaginal discharge , the pyometra is concluded as open. And in most cases, pyometra will be marked by evident clinical findings with the additional detection of an enlarged uterus upon administration of radiographic imaging, and not to forget to mention that ultrasonography may serve in these procedures also. The closed pyometra will in contrast become more severe, as the cervix will not permit the uterus to expel its uterine content (26). The appearance of these two classifications are described as followed:

 Open pyometra- have vaginal/vulvar discharge (seen as purulent, mucopurulent, hemorrhagic or haemopurulent discharge) ± signs of systemic illness.

 Closed pyometra: will not specifically show any sign of vaginal/vulvar discharge, but may become present in small amounts. There will be signs of systemic illness, and this classification are considered as more severe in comparison to open pyometra. The exudate will become trapped within the uterus, leading to a rapid filling of discharge within the uterus. This will eventually lead to a painful abdomen and the animal progresses into a state where it becomes prone to systemic disturbances such as sepsis (describes as previous).

Another classification were done by the reclamation of Dow’s principle, in which Dr Dow states a correlation with the endometrial hyperplasia. In the mentioned classification, CEH-pyometra complex with the presentation of four conditions, in which the fifth state explains the occurrence of a chronic endometrial inflammation, with the extent of hyperplastic endometrium (commonly

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15 referred as open pyometra). Dow´s principle focusing more or less on the character of affected endometrium. CEH can protrude into pyometra, but does not necessarily result into pyometra of canines (35,36).

1.5 Blood profiles

1.5.1 Red blood cell count

Red blood cells have the ability of carrying oxygen throughout the entire body. It is a crucial role of the red blood cells to transport the oxygen molecule successfully, by binding those to a component called hemoglobin that is an important constituent of all the red blood cells. Hemoglobin in turn will make it possible to transmit the oxygen to all of the cells within the organism. It is a constant cooperation between delivering the oxygen and exchanging the released oxygen molecule with the attraction of carbon dioxide. In this way, carbon dioxide can be brought back from the cells and be expelled at the site of the lungs. The bone marrow is the producer of the red blood cells, and when it will become compromised or suppressed this will become evident in the test analysis. Also the occurrence of a disease may require the support of instant infiltration of red blood cells. Red blood cells are included in defending the body against various bacteria and viruses (37).

There are two possible ways to determine the type and number of the red blood cells. This can be done by a so called complete blood count. A complete blood count is a test measuring the red blood cells, white blood cells and platelets. Hematocrit (Hct with normal range of 39-59%) serves as a guideline in measuring the number of the red blood cells. When hematocrit is at a lower range, the veterinarians would suspect an ongoing anemic state or overhydration. When the level of Hct is elevated, it is referred as erythrocytosis, that can have the common cause of dehydration, hypoxemia, renal lesions or splenic contractions (26,37,38).

Hemoglobin is also an important indicator when calculating the red blood cells. Hemoglobinuria and hematouria are causes of an abnormal high levels of hemoglobin within the circulatory system. The causes of hemoglobinuria are determined to be by the course of a severe intravascular hemolysis. It can also be detected as free in urine, in case of hematuria. Hematuria can implicate indication of urinary tract disorder, including hemorrhage, lesion, trauma, necrosis or inflammation etc. the normal ranges of hemoglobin are considered to range between 120-190 g/dl (26).

1.5.2 White blood cell count

White blood cells, also called leucocytes, works as a defender of the body. Leucocytes has been simply divided into two typed: granulocytes (eg. Neutrophils) and agranulocytes (eg. Monocytes and lymphocytes). When the amount of leucocytes becomes abnormally changed, for example

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16 elevated, there is a sign of a severe infection. The causes may be expected as a ongoing buildup of metabolic toxins by an acute failure in liver or renal function. In situation of elevated number of neutrophils, this can be an indicator for a bacterial infection that is termed as severe and acute. Situations that require an increased effort of the immune system may also cause elevations of neutrophils, by the mean of an acute inflammation process (referred as left shift). In contrast to this, a decrease will usually determine a viral influence. The normal ranges of neutrophils are expected as 3-11.5x103_{/uL (26,37,38).}

The elevation of monocytes is associated to the presence of cancer, and will works as a“cleaning-cell” when engulfing foreign bodies that are coexisting within the body. It also removes inflammatory tissue from its localization, in order to break it down. Normal ranges of monocytes are within 0.2-1.4x 103 /uL (37,38).

Lymphocytes are elevated in cases where there might be a concurring inflammatory disease, excitement, neoplasia or hypoadrenocorticism. When the lymphocytes will decrease in number there might be a result from a immunodeficiency, stress leukogram, acute inflammation or lymphatic flow interruption. Normal ranges of lymphocyte has been estimated to obtain a range from 1.4-4.8 x 103/uL (26,38).

1.5.3 Blood serum profile

Creatinine (CREA) is a degradation product that will become elevated in certain cases of renal problems and failure. The glomerular filtration rate may thereby be decreased, contributing to a renal failure that may progress into a systemic impaction. A decreased value of CREA in the biochemical blood analysis will be shown in animals with a decreased musculature, and a low body condition score. Normal ranges of CREA has been estimated within the range of 0-130 g/dL (26,38).

Glucose (GLU) possess the role as energy preservation for the body. Elevations of this parameter corresponds to diabetes mellitus, acute pancreatitis or stressful circumstances. When the levels are determine to become lower than usual, it may signify a chronic liver disease, malabsorption, sepsis or starvation. Normal ranges of the glucose levels have been established as 3.5-7.2 mmol/L (26,38).

ALT (alanine aminotransferase) is a liver enzyme which will become elevated in situations of liver damage (primarly or secondarily). Hepatotoxins may cause the elevation of the biochemical test results. The range of this liver enzyme is considered to be within 0-130 units (38).

High levels of potassium (K+_{) in the blood will give the information of a possible reduction in}

the renal excretion, gastrointestinal disorders, metabolic acidosis, kidney failure and urinary obstructions. Low levels of potassium can be in association with vomiting, diarrhea or postobstructive diuresis. The normal range of potassium within the circulatory system are 3.1-5.3 mmol/L (26,37,38).

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17 Sodium (Na+) ions may provide the informative signalement of kidney disease, peritonitis and pancreatitis in elevated numbers, as in contrast to nephrotic syndrome, hepatopathy, advanced kidney disease and nephropathy in low levels. The average range of normal value are listed as 137-152mmol/L (26,38,39)

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2. RESEARCH METHODS AND MATERIAL

2.1 Ethical perspective of collection

According to ethical principles and laws, all the participating animals has been accepted and proven to be suitable for radiological examination procedures by the small animal clinic, in combination with the permission of the animal’s representative, to participate in the study. Before the examination procedure were performed, owners or caretakers were asked and informed about this study. In this way, only animals with the allowance of collection and documentation of significant data, has been implied in this study. The participating animals were handled according to animal welfare and hygienic regulations at the Swedish small animal clinic.

2.2 Collection of participating Canines

In order to carry out the research, there have been a collection of a recruited group of canines (with the total number of 274 and included 149 females) that were recruited at the small animal clinic. All the participating dogs showed individual documentation of gender, age, breed, and diagnosis. From this total number of females, a sampling was done in order to collect two different, and more specified, groups for the study. In which both of them included females with various age and breed. For the control group (considerable healthy females without any impact on the uterus, but received a minor sign or symptom, local affection and thereby with no systemic effect involved) it included 16 females that was recruited by the small animal clinic “A” in the purpose of a general checkup or with a small complaint and recieved the result of no specific evidence of an internal ongoing disease. The group was given the name accordingly as Ynr for all included participants. A general clinical study,

included the assessment of the general condition, was performed before the collection of radiographic image and blood profiles.

The pyometra group (named Xnr), 25 females were recruited at the small animal clinic “A”

for the purpose of expressing the diagnosis of pyometra. These females were diagnosed by the means of a clinical examination, radiographic imaging and blood profiles. A major focus was put on the radiographic investigation and its possibilities in the determination of observing and measuring a pronounced enlargement of the uterus. The criteria for participation was made by diagnosis and by the final results from radiography and blood tests.

In this study there were an attendance of a total number of 25 in the pyometra group (diagnosed with pyometra by the diagnostic investigation and clinical appearance) and 16 in the control group (considerable healthy uterus by clinical and diagnostic investigation procedure)

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2.3 Radiographic measurements

During the radiographic imaging all participating canines was placed, with the help from veterinarians and nurses, under the beam of the radiographic instrument. The abdominal diameter were estimated by the measurement of abdominal width. The size (in centimeters) of the abdominal width were used primary for evaluating the proper indication of the radiographical settings. According to schematic instructions and its ranges, a correct installation of mAs and V could be established for the research.

The radiographical methods used in receiving proper and accurate images of the abdominal region of the animal, two projections were promoted. Ventro dorsal and latero-lateral projections were taken and evaluated on the computerized system of “Visbion-Pacs”, used at the small animal clinic. As the image has been evaluated, and uterus measured by the same system, the animal could be classified into a certain group. The animal were given antisedatives after the radiographic evaluation and the establishment of an accurate diagnosis.

The collected images received for this study shows latero-lateral projections, due to the lack of qualifying the criteria of an improved visualization property in the ventro-dorsal projection. Images has been selected and stored for all patients in the two groups, including all patients contributed in the control group and pyometra group.

2.4 Blood profiles

Blood sampling procedures were done prior to the radiographic imaging. The collection of blood was routinely obtained according to the method of intravenous sampling, by the injection at region of the vena cephalica. Only sterile equipment and materials were allowed during blood sampling procedures. The animal was properly prepared at the place of sampling (by the act of shaving, cleaning and disinfecting). The application of a venous catheter were individually collected and inserted into the vein of the animal, as the blood were collected separately into two different tubes. The tubes included for this study involved EDTA-containing and hematology tubes, specialized for each specific analyzing methods. The haematology tubes were measured and evaluated by the use of “IDEXX vet lab station” (aid in the additional results of Hb, Hct, WBC, lymphocytes, monocytes and neutrophils).

In the management of analyzing the tube with no additional EDTA, it is crucial to perform centrifugation prior to the analysis. This process enables the collection of blood plasma, which will become necessary in the analysis. Within the walls of the laboratory, different machines are used for certain calculations of parameters. The measurement of the acute phase protein, obtained by the method of using a specific instrument in the measurement referred simply as “SOLO”. Meanwhile

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20 the chemical blood results, dealing with the values of liver and kidney parameters, are evaluated and estimated by the use of a chemistry analyzer.

When values was evaluated and controlled, documentation was performed by the transmission of the result to the computerized system that has been particularly used for the documentation of all the patients data, expressed as “Vetvision”. This system stores all the information that has been recieved for each specific patient, recruited at the clinic. According to laws and regulations, the collected data will be stored throughout the animal’s life until 7 years after the time of death.

2.5 Statistics

The statistical measurements were estimated and evaluated in the statistical software of “Excel” and “SPSS”. P-value was selected as p<0.05 for the purpose of evaluating significance between groups in different age, weight, size and state of diagnosis. Data has been received and used by this statistical system, for the objective of calculating average, maximal and minimal limits, confidence limits and sample proportion with established with number and percentage. Formation of diagrams and figures was created by the interpretation of calculated numbers and percentage. P<0.05 is and indicator for significant values, and when receiving a value of P<0.001 it should be termed as very significant

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21

3. RESULT

3.1 Incidence and prevalence of pyometra

3.1.1 Prevalence of breed and gender

Among the total number of recruited animals (n=274) at clinic ”A” in Sweden, prevalence was calculated, showing within the total estimate a dominance of females (included castrated and non-castrated). The majority of the females that was included were uncastrated (Figure 1).

Figure 1 Illustrates a prevalence of gender expressed in percentage.

In the determination of frequency in accordance to the breed distribution, it has been concluded that a wide range of different breeds were represented in the study (n=79). The breed which secured the highest percentage, was the labrador retriever with 7% (n=8). The breed with the second highest percentage was the Golden retriever with 6% (n=7), that in turn was followed by the German shepherd (n=5) with 4%. The Chihuahua (n=4), Border collie (n=4), Poodle (n=4) and Bichon Frisé (n=4) all recieved a number of 3% . The other breeds had a low percentage, and was thereby generally described in the Figure 2 under the category ”Others”, that has been shown below. Some of these breeds, included in this column, (Basenji, Swedish elkhound, Irish wolfhound, Affenpinsher, Nova scotia, French bulldog and Beagle etc) had a percentage that ranged from 1-2%.

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22 Figure 2. The figure illustrates the breed distribution, noted by actual number and percentage within the selected group of females included in the population

3.1.2 Prevalence and incident of pyometra as a diagnosis

In the evaluation of diagnostic variations from the provided data, 10 frequently occuring diagnoses among both non-castrated and castrated females (n=149) has ben stated. The most common diagnosis for this study was marked by an increased frequence of pyometra (n=25), which contributed with as much as 17% among the represented cases . The following diagnoses (as listed from the highest to the lowest prevalence in percentage) was considered to be 12% of gastrointestinal (GI) disorder (n=17), 9% of health control/profylaxis (n=13), 8% of trauma/fracture (n=12), 7% of eye problems (n=10) and 7% of urinary tract disorders with the included complaint of polydipsia (n=10). The manifestation of remaining diagnoses, within the concluded total number of diagnoses (n=21), were exhibiting a lower number of cases and was thereby grouped together in the column of ”others” (Figure 3). The diagnoses involved in this column ranged from 1-6% (n= ranged from 2-9 cases for each) and involved for instance some immunological, toxic, parasitic and gynaecological complications. 8 7 ₅ ₄ ₄ ₄ ₄ ₃ ₃ ₃ 104 0 20 40 60 80 100 120 N UM BE R O F C A N IN ES NAME OF BREED

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23 Figure 3 Representing the prevalence of breed of the recruited group

3.1.3 Prevalence of weight

According to the collection of data for the selected females within the pyometra group (n=25), an average weight of 24.31 kg was obtained. The collected data provided the establishment of a wide range of weight, starting with the minimal weight at 2.4 kg, with an extending maximal weight of 47.9 kg (Figure 4). 25 17 13 ₁₂ 10 10 ₉ 7 7 ₆ 33 0 5 10 15 20 25 30 35 NUMB ER OF CA NINE S NAME OF DIAGNOSIS

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24 Figure 4 Illustrating prevalence of weight with trend, among affected canines in pyometra group

3.1.4 Prevalence of age

With the determination of the maximal and minimal ranges of age within the pyometra group (n=25), it was distinguished a minimal age of 5 years together with the maximal age of 13 years. The average age for all the female canines whoose suffered from pyometra, gave the estimated result of 8.84 years. The collected and measured data, with the involvement of only age and affected females, has been expressed. Showing the identified ages for each participating female below (Figure 5).

Figure 5 showing the prevalence of age among affected females.

0,00 10,00 20,00 30,00 40,00 50,00 60,00 X1 X2 X3 X4 X5 X6 X7 X8 X9 _X10 _X11 _X12 _X13 _X14 _X15 _X16 _X17 _X18 _X19 _X20 _X21 _X22 _X23 _X24 _X25 WE IG H T ( KG ) AFFECTED FEMALES 0 5 10 15 X1 X2 X3 X4 X5 X6 _X7 _X8 _X9 X10 _X11 _X12 X13 X14 _X15 _X16 X17 X18 _X19 _X20 X21 _X22 _X23 X24 X25 AG E (Y EAR) AFFECTED FEMALES

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25

3.2 Collection of data and radiography among the control group

3.2.1 Collection and analysis of data

Using the calculated data of the participating females of the ”control group” (n=16) the ranges of weight was indicated to scope from 2.9 kg until 31.1 kg. Which concluded in an average weight of 12.92 kg. The diversity of age itself, compassed from a minimum of 3 months to a maximum of 11 years. That proceeded correspondendly to the determination of 5.35 years for the measurement of average. The prevalence of the breeds in this group was ruled out in the specific calculations concerned, the enumerated data of the ”control group” (Group Ynr) was lacking and thereby excluded.

Thereby the meaurements has not been specified (Annex 1).

In the Table that has been expressed in Annex 1 below, illustrates the complete amount of collected data covering all the individually recieved information for each participating female (Y1-16).

Expressing the representation of breeds, age (years) and weight (kg). The evaluation of the radiography was documented together with the concurring complaint (including Polydipsia,health control, vomiting and with a small local affection etc) has been mentioned.

3.2.2 Radiographic analysis

Upon the radiographic evaluations, common trends in the control group has been distinguished. Marked for each member in the control group whoose exhibited the results as free from pyometra in its statement, when analyzing the status of the uterus. The evaluation in form of a description has been notified in the Annex 1, indicating no prominence of a uterine enlargement. The recieved information has been described in the table, with the provision that no members was excluded from the control group.

Within the control group (Y1-16), images has been stored and evaluated accordingly. Example

of radiography concerned within the control group has been presentated below as followed:

The Picture 1 illustrates a female canine that was recruited at the Clinic ”A”. Showing the radiography of an 11 year old Jack Russel(Y5), with a complaint of problematic in its movement. The picture shows the beginning of spondylosis with no influence on blood parameters. The vital organs are considered normal, with the main focus on: size of organs, localization and radiolpacity/radiolucency appearance within the abdominal region. There is no evident enlargement of the uterus, and no displacement of visible organs. The stomach is filled with engulfed content, and gas is contained within the intestines.

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26 Picture 1. Illustrating radiography of Y5 at clinic ”A”.

In the image below, showing radiographic image of Y11, a 6 year old Karelian bear dog, which

expresses a normal extended bladder, content-filled organs and normal distribution of the stomach. There is no evidence of uterine enlargement. The female canine suffered from a skin wound, at the position of the inner thigh of the right leg. Radiography was performed in order to control the risk of abdominal penetration (Picture 2).

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27

3.3 Collection of data and radiographic images among pyometra group

3.3.1 Collection and analysis of data

In the pyometra group, Xnr, (n=25) data has been described and explained in the Table.2.

Represending the documentation of the diagnostic statement as ”pyometra” and ”suspected pyometra” together with the age, weight and size of uterine enlargement.

The predisposition of breed has been considered as non-suitable for this study, as the pyometra group concluded a minimal amount of representing breeds, and has been estimated as not significant for the study. The average number of significant breed can thereby not be calculated (Annex 2).

3.3.1.1 Average size of uterus for pyometra group

The average size of the uterus within the pyometra group were calculated to 3.39 cm. The calculation of average size has included both statements in the X group (n=25) of ”pyometra” such as ”suspected pyometra”, with no values excluded from the measurement based on the values demonstrated in Annex 2.

3.3.1.2 Maximum and minimum size of diagnostic statement

The maximum limits in the determination of the highest value of uterine size were measured at 8 cm. With a minimum size of 1.17 cm, representing the minimal limits in which pyometra could be identified and detected, by the use of radiographic imaging, performed by software of ”Visbion”. The maximum limits for the determination of pyometra for the suspected diagnostic statement was determine 3.17 cm (Annex 2).

3.3.1.3 Prevalence of diagnostic statement of pyometra

The results that has been concluded in the collection of pyometra group (n=25), was maintained by the categorization for the evaluation state of diagnosis, including ”Pyometra” (n=15) and ”Suspected pyometra” (n=10). The percentage of the incidence between these two groups was estimated as 40% for ”Suspected pyometra” and 60 % for ”pyometra” (Figure 6).

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28 Figure 6. Demonstrating the prevalence of pyometra in percentage, as being classified according to the state of diagnosis

3.3.2 Radiographic analysis

Radiographic measurements in the size of uterine enlargement was estimated and calculated based on the values described in Annex 2. Upon the recieved data that was stored, pyometra was considered to affect all females that has been participating in the group (n=25). Thereby radiographic evaluation stated that none of the females, that has been included in this group, had to be excluded.

Examples of the images that was recieved at the clinic ”A”, with the involvement of ”suspected pyometra”, ”pyometra”and size has been demonstrated in the images as listed below.

A radiographic image of X4 showing a 6 year old Leonberger suffering from closed-cervix pyometra with an enlargement of 7.2 cm of a lobulated and filled uterus with the position of caudo-centrally to ventrally location within the abdomen. The enlargement resultet in that the colon was positioned in an upward direction,with other organs in a cranio-dorsal position against the diaphraghm (Picture 3).

60% 40%

Pyometra

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29 Picture 3. Showing radiographic image of X4

Imaging of the female X9 were showing a suspected pyometra of a 7 year old Lagotto Romangolo at the time of radiography. The clinical examination provided a detection of hemopurulent vaginal discharge. Vaginal swab was done and determined a growth of mixed micro-flora. Upon biochemical parameters the liver profile and the white blood cells were abnormally elevated. Was in heat for 3 weeks ago. Uterus 1.67 cm, and OHE provided a support in diagnosis(Picture 4).

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30 Radiographic image of X10 expressing a widely enlarged and distributed uterus within the abdominal cavity. Estimated size of 7.4 cm large lobes of the uterus. The lobes of the enlarged uterus are evident centrally within the abdominal cavity, with the extention dorso-caudally and ventrocaudally in the region towards the urinary bladder (Picture 5).

Picture 5. Showing radiographic image of X10

3.3.3 Analytical statistics

3.3.3.1 Sample proportion of age

The confidence limits in the age group were considered at the lower confidence limits of 95% as 6.4% in <7 years, 33.5% in 7-10 years and 17.21% in >10 years. The upper confidence limit (95%) were estimated as 34.65% (<7 years), 69.97% (7-10years) and 51.59% (>10 years). Sample proportion of these groups were 16%, 52% and 32% as followed (Table 1).

Table 1 Illustrating the confidence limits for sample proportion Age (year) Nr of females with pyometra Sample proportion % Lower 95% CL Upper 95% CL <7 4 16,0000 6.4 34.65 (7-10) 13 52,0000 33.5 69.97 >10 8 32,0000 17.21 51.59

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31 3.3.3.2 Sample proportion of size

Sample proportion in the calculation of uterine size and number of affected females, indicated 32% in cases where the size was evaluated as <2 cm, 36% from 2-4 cm and 32% for measurements >4 cm. Which will correspond to an evenly distribution within these 3 samples of ages (Table 2).

Table 2 Showing the sample proportion in percentage within pyometra group

Size (cm) Nr of females Nr of females %

<2 8 32

(2-4) 9 36

>4 8 32

Total: 100

The sample proportion for the size in comparison to the state of diagnosis has been calculated to obtain a value of 100%. In which the sample for <2 cm represented 2 females of “pyometra”(8%) and 6 females were for the “suspected pyometra” (24%). As the largest size of the uterus (>4 cm) contained only 8 females (32%), in which none of them received the suspected state. This sample will thereby have a sample proportion who’s differed in its ratio between the two groups. The females that received a size between 2 to 4 cm had a sample proportion with a more even ratio, in comparison with the other two proportions (Table 3)

Table 3. Representing the sample proportion of size within the pyometra group. Size (cm) Pyometra Suspected pyometra Pyometra % Suspected pyometra % <2 2 6 8 24 (2-4) 5 4 20 16 >4 8 0 32 0

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32 3.3.3.3 Sample proportion of weight

Estimation of the sample proportion, concerning the weight, showed an even ratio within the group of females exhibiting a weight more than 25 kg (28%). The proportion of females that weighted less than 10 kg (12 %) did not receive a number of females that were affected by the suspected form (Table 4).

Table 4. Showing sample proportion of weight Weight (kg) Pyometra Suspected pyometra Pyometra % Suspected pyometra % <10 3 0 12,00 0,00 (10-25) 5 3 20,00 12,00 >25 7 7 28,00 28,00 Total: 100

3.3.4 Calculation of significance value

The P-value of age in the relation to the state of pyometra was considered as non-significant (P-value=0.201). The weight in the consideration to the state of pyometra, showed an interpretation of P-value=0.272, also indicating a value without significance.

In the relation to weigh, age and state of pyometra it was statistically proven that measurement of size in relation to both weight (P>1) and days after estrous (P>1) were invalid and not significant. Due to the results of that data, the measurement and parameters were excluded from the study.

Although, a detection of significance was spot between the relation of size with the state of diagnosis (P=0.009). All of the P-values has been summarized in Table 5.

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33 Table 5 Illustrating the calculated P-values of parameters for the pyometra group (Xnr )

Parameters: Size (cm): Size (cm): Size (cm): Size(cm): State of diagnosis: State of diagnosis: <2 <2 <2 <2 Pyometra Pyometra (2-4) (2-4) (2-4) (2-4) Suspected pyometra Suspected pyometra >4 >4 >4 >4 State of diagnosis: Age (years): Weight (kg):

Days after estrous (months): Age (years): Weight (kg): Pyometra <7 <10 <1 <7 <10 Suspected pyometra (7-10) (10-25) (1-2) (7-10) (10-25) >10 >25 >2 >10 >25

P-value: 0,009 0,202 Invalid Invalid 0,201 0,272

3.4 Comparison of collected blood profiles between the test groups

The results from the collected data, when comparing the group Y together with group X, it has been determined that an average value of C-reactive protein (CRP) retained a higher number in group X. In consideration to group Y, this value shows a moderate increase.

In the evaluation and calculation of liver enzymes (ALT) it has been interpretated an elevation in the comparative measurement of females with pyometra, providing an average number of 1.18 U/I during the state of the diagnosis in the affected females (Xnr).

Hct was expressing a lower estimates in the group X contrary the group Y. The average values for this parameter has been calculated to 37.98, ranging from 29.8 to 44.7. In response, the control group received 43.26 when stating the average. Indicating that the comparative measurements will express a change supporting only a mild decrease in the group X of the parametric value for Hct (Table 6).

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34 Table 6. Showing the biochemical blood profiles and the results for the group X (Pyometra group) and Y (control group).

Parameter Average/Range for Xgroup Average/Range for Ygroup

(n=25) (n= 16) Hb 145.32 (108-164) 152.31 (142-177) HCT 37.98 (29.8-44.7) 43.26 (23.6-53.1) WBC 16.61 (6.9-42.2) 8.45 (6.3-15.5) CREA 61.76 (44-87) 75.06 (45-105) ALT 1.18 (0.3-7.2) 0.42 (0.25-0.89) GLU 9.32 (3.6-99) 4.89 (3.6-6.2) Na+ 144.12 (126-149) 147.63 (145-153) K+ 4.2 (3.6-4.8) 4.18 (3.6-4.8) CRP 65.35 (0-207.6) <10 (0-14)

A statistical measurement was performed showing a significance in CRP (P =0.001), ALT(P 0,034) and Hct (P =0,009). Other parameters were not possible to evaluate and has therefore been excluded from the study (Table 7).

Table 7. Showing the calculated values of P, by the evaluation of microsoft software Excel, between the parameters in the two test group of X and Y.

Parameters: P-value:

Hct normal range

Hct< 0,009

Hct>

ALT normal range

ALT< 0,034

ALT>

CRP normal range

CRP< 0,001

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35

4. DISCUSSION OF RESULTS

It has been concluded that a performed radiologic examination serves as an initial method for the determinion and identification of the prevalence in canines affected by pyometra. According to the radiographs, an incidence of pyometra was easily estimated in the calculation from the total number of females that has been recruited at the small animal clinic in Sweden. The results shows a prevalence of pymetra with 17%, which differed from the previous mentioned studies included in the literature review. Dr. Waernsson(19) stated that pyometra will recieve a number of 25%. Although it should be mentioned that this particular study included a higher amount of participants than provided in this study. The prevalence in other studies, such as the study written by Dr Egenwall, Hagman and Bonett (20), will also come to differ in the comparison with Dr.Waernssons study, where only 2% of the population size were showing a condition of pyometra. One reason that the ranges may come in a broad and wide range, can be due to that prevalence may depend on population size of the study, its location, frequence and possebilities of recruiting these kind of patients at a clinic. For instance, this study recieved a total number of 25 pyometra cases in a relative large animal clinic for only a short period of 6 months, and will thereby become very restricted when calculating. The numbers may be as well misleading to the actual number of cases recieved per year. Another factor contributing to the prevalence, is the skill of the veterinarian to analyze radiographic images. The veterinarian must possess a high competence when analyzing the radiographic images. To produce a high quality image will also have a key role in the investigation process.This study was performed together with a veterinarian with a working experience of more than 6 years, who was guiding and assisting in the formation of diagnosis and its process in the estimation.

Average size of the uterus represented in the pyometra group has been estimated to 3.39 cm. This study supports the theory that hyperplasia and prominent enlargement of the uterus will always become evident when canines are affected with a pyometra. Leading to the understanding of the importance for the radiographic images in the determination when stating a correct diagnosis. The size of the uterus, in all cases included, was concluded to retain a size above the normal range of 0.5-1 cm. Supporting the study of Dr E Luther (2), which considered that a normal size of the uterus will remain within that specific range. Also, There were no canines in the pyometra group, whose uterus was unable to measure upon the radiographic imaging. The human factor certainly plays a role when estimating and evaluating the sizes in all of the specific cases of pyometra. The software of Visbion for radiographic evaluation will always be dependent and/or prone to the person that are using this system. The exact size for any individual case of pyometra can, because of that, come to differ. But more or less, the radiographic estimations will provide a general view of the enlargement, and

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36 revealing the uterine size with quite a sensitive measurement. What was shown in this study is that the classification of pyometra will be ruled out as invalid, and can not be determine when evaluating the utrerine size, but is instead almost entirely dependent on the anamnesis of an incoming patient. The hypothesis given, expressing that a closed pyometra will retain a bigger size, is dependent on the duration of illness and the rate of pus that will be filled up within the uterus. The radiographic investigation thereby rules out and excludes the possibilities to classify the pyometra in these ways. Maximum and minimum limits were concluded in this study by the use of a computerized software of SPSS and Excel. Generally the values will not determine the exact range of when it becomes visible to detect an ongoing pyometra. As these values were satisfactory indications for this study, a concrete comparison with other studies will be impossible because of the fact that no studies has been performed for this specific reason. To conclude a statistical range by its relevance and significance, further studies must therefore be made. Not to forget to mention, that the sizes in which the state of diagnosis has been considered, was ranging. For instance a golden retriever recieved the state of a suspected diagnosis with a size of 1.02 cm, in contrast to another flatcoated retriever with a size of 2.8 cm whose also recieved the same state. Both of these females were within the same range of age and weight. A third patient, involving a flatcoated retriever with similar weight and age, as the two females previously mentioned, retained a uterine size of 1.5 cm was still identified with a certain diagnosis of pyometra (and not as suspected). Giving the conclusion that the maximum and minimum limits will not be significantly implemented as a true genrealized range for all female canines, but will instead remain an individual estimate for each cases under the influence of a proper radiography, position and skilled veterinarian.

Statistically, p-values were established for the identification of importance for each parameter. The breed in this study was excluded, and considered of no importamce due to the lack of collected data. The test group, such as the pyometra group, recieved a wide range of breeds represented with a minimal number of occurence. Because of that, the breeds were presented only one time, making the calculation of incidence with unsignificant values. The average age of the pyometra group showed a range between 5-13 years, with the average age of 9 years. In the literature review it was referred by the study made by Dr. Egenvall, Hagman, Bonnett, Hedhammar, Olson & Lagerstedt, who stated that there were 24% of the females pronouncing pyometra before the age of 10 years (20). Another data considering the incidence of age, expressed by Dr Cote (26) determine the diagnosis to be frequently occuring after the age of 5-6 years. This study will thereby be supported (but also supporting these mentioned studies) with a significance of the calculated ages ranging from 5-13 years. Why the females will obtain this diagnosis at a higher age is not yet completely understood. A hypothesis may include relevance of the hormonal stimulations, as well as the policy of neutering at young ages in different countries. Neutering is performed at a young age, and is

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37 frequently done among swedish dogs, inhibiting the estrous cycle to normally occur. Pyometra is not only a disease of older females, but will also be recognized in younger individuals too. As the estrous cycle will start to take its course, it will influencing the body to respond and to recieve hormones, thereby the risk of developing pyometra will become possible. This theory is supported by the studies made by Dr L. Forsberg (18).

This study prooves that pyometra will not be influenced by either age nor weight. But one interesting relationship was indicated in this study as ”very significant” (P<0.01), when comparing the size and state of the pyometra (P=0.009). This will explain that the significance of identifying the pyometra and its challenges upon radiographic imaging, is related to the uterine size. In practice, it was prooven that the ranges of size in determination of ”Suspected pyometra” and ”Pyometra” will come to differ for each individual. But statistically it will support the hypothesis of that the size of an enlarged uterus will correlate with the degree of visualization upon radiography, wheter it will be possible to state a certain diagnosis or not. Although, more studies have to be made in order to state this correlation. The test was performed only within a small group of females, with variable estimations and results, which will not be enough to draw the conclusion of considering it as rational and scientifically proven.

During the analytical statistics of the comparasion of blood parameters between the two test groups (pyometra group and control group), some of the parameters were evaluated as not relevant for this study. This was due to that these parameters (including Hb,GLU, Na, K and Crea) were not evidentally expressed in elevated or reduced values. Giving the result that there will be no difference between the groups, that contributed to an calculation that could not be done statistically. When focusing on the CRP and ALT parameters, it was shown an elevation for the pyometra group. The test group remained within normal ranges for all of the parameters. The P-value considered in CRP (P=0.001) and ALT (P=0.034) was representing to be influenced by the affect of pyometra. There were also evidence of decrased values of Hct (P=0.009) during the state of pyometra. As expressed by the literature, the study written by Dr (26) was compared with another study performed by Dr Falkenö,Hillström and Jones (37), in which both of these studies will explain a usual occurence of change in these three parameters. Both studies explains that the liver and immune system will become affected, which will result into an elevation. Dr. Verstegen and Dr. Dahliwahl (27) expressed in their studies that a secondary bacterial infection may be prominent in pyometra cases. This will also lead to a higher level of CRP, and may add a suggestion to why this parameter was commonly affected. A lowered value of Hct could be an indicator of overhydration or anemic state. In this situation, overhydration can be a common response to pyometra, as the females commonly drinks more water than usual as a clinical symptom. This theory was also shared with Dr Fredriksson (22).

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38 The theory of that a blood profile can become helpful in stating a diagnosis, should be considered at the veterinary practice. It should be understood that these parameters may not only be specific for pyometra, but also other diseases that may cause a response of the immune system and its vital organs.. This in turn will recieve the information that blood profiles should never be done alone when diagnosing pyometra, but always be used together with other investigation and examination methods (including radiography and/or ultrasonography). As already states, this study provided only a small group of females, and further studies are thereby necessary in determine these results as valid.