LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Faculty of Veterinary Medicine

Meliza Timocin

KALIŲ ŠUNINGUMAS – VADOS DYDŽIO NUSTATYTMO TIKSLUMAS ULTRAGARSINIU TYRIMU

CANINE PREGNANCY – THE ACCURACY OF LITTER SIZE DETERMINATION USING ULTRASOUND

MASTER THESIS

of Integrated Studies of Veterinary Medicine

Supervisor: Prof. Dr. Gintaras Zamokas

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THE WORK WAS DONE IN THE DEPARTMENT OF SMALL ANIMAL CLINIC CONFIRMATION OF THE INDEPENDENCE OF DONE WORK

I confirm that the presented Master Theses “Canine pregnancy – The accuracy of litter size determination using ultrasound”

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.

Meliza Timocin

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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. Meliza Timocin

(date) (author’s name, surname) (signature) CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE

MASTER THESES

Gintaras Zamokas

(date) (supervisor’s name, surname) (signature) THE MASTER THESES HAVE BEEN APPROVED IN THE

DEPARTMENT/CLINIC

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Reviewers of the Master Theses

1) 2)

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Evaluation of defense commission of the Master Theses:

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SUMMARY

Canine pregnancy – Litter size determination using ultrasound

Meliza Timocin Master Thesis

This master’s thesis is performed as part of the department of Dr. L. Kriaučeliūno small animal clinic of Lithuanian University of Health Sciences in Kaunas. The thesis evaluates the accuracy of using ultrasound as a method for determining the litter size in canine.

Empirical data was collected through a survey study. In addition to collecting data on expected and actual outcome of the litter size, the survey also focused on a wide variety of factors such as size of the breed, gestation day and weight of the bitch. In total 221 cases were collected, but to ensure quality of data only 213 were further used in the analysis. The collected data comes from breeders that are registered in the Swedish Kennel Club (SKK) who in most cases breed pure-breeds.

The analysis studied the average deviation between expected litter size and actual outcome of litter size. The deviation was related to the additional data collected in the survey (e.g. size of breed, gestation day, weight etc.). Further, a sensitivity analysis was performed calculating deviation by 1 and 2 pups above and below the actual litter size to compare with the actual litter size outcome.

The analysis shows that the general average deviation of the litter size determination using ultrasound was 50% (comparing expected and actual outcome of litter size). Further sensitivity analysis by ±1 or ±2 pups within the actual litter size outcome shows an average litter size deviation of 31% and 24% respectively. The analysis also shows that factors such as size of the breed, gestation day and weight do not affect the accuracy of litter size determination using ultrasound (p>0,05).

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6 Each category in each factor (for example normal weight and overweight) were also analyzed and compared to each other, the result showed that there was no statistical significant difference between them either (p>0,05).

Each category in the factors were also analyzed with the sensitivity analysis of ±1 or ±2 pups within the actual litter size, and the results showed that most of these results had a statistical significant difference (p<0,001).

This study concludes a low accuracy of litter size determination using ultrasound and that the factors analyzed e.g. size of breed, gestation day etc. did not affect this result. The sensitivity analysis of ±1 or ±2 pups within the actual litter size outcome showed that is has a statistical significance, but this result should be seen with a care as a ±1 or ±2 pups adjustment to the actual outcome of the litter size, has a large relative effect on deviation outcome.

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SANTRAUKA

Kalių šuningumas – vados dydžio nustatytmo tikslumas ultragarsiniu tyrimu

Meliza Timocin Magistro darbas

Baigiamasis darbas rašytas Lietuvos Sveikatos Mokslų Universitete, Dr. L. Kriaučeliūno smulkiųjų gyvūnų klinikoje, Kaune. Magistriniame darbe buvo vertinamas ultragarsinio tyrimo patikimumas nustatant kalių vados dydį.

Tyrimo metu buvo atliktas empirinis tyrimas. Buvo renkama informacija apie prognozuojamos vados dydį ir kiek šuniukų atvesta, taip pat buvo atsižvelgiama ir į daugiau aspektų, kaip: veislės dydis, šuningumo diena, kalės svoris. Viso surinktas 221 atvejis, bet analizuoti buvo pasirinkta 213 atvejų. Surinkti duomenys kuriuos pateikė Švedijos šunų klube (SKK) registruoti veisėjai ir kurie dažniausiai veisia grynaveislius šunis.

Tyrimo metu buvo norima įvertinti paklaidą tarp tikimosi vados dydžio ir atvestos vados dydžio. Apklausos metu surinkti papildomi duomenys apie veislės dydį, vaikingumo dieną, kalės svorį taip pat turėjo įtakos galimai paklaidai. Toliau buvo atliktas patikimumo tyrimas skaičiuojant paklaidą ±1 ar 2 šuniukai nuo nustatytos vados dydžio ir jau atvestų šuniukų skaičiaus.

Tyrimo rezultatai parodė, kad nustatant vados dydį ultragarsiniu tyrimu, paklaida buvo 50 proc. (lyginant nustatytą vados dydį su atvestų šuniukų kiekiu). Tolimesnio tyrimo metu ±1 ir ±2 šuniukai lyginant su faktiniu vados dydžiu, patikimumas buvo atitinkamai tik 31 proc. ir 24 proc.. Rezultatai taip pat parodė, kad tokie veiksniai, kaip veislės dydis, svoris, vaikingumo diena, neturi įtakos ultragarsinio tyrimo rezultatų patikimumui (p>0,05).

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8 Kiekvieno veiksnio kategorijas išanalizavus ir palyginus tarpusavyje, buvo nustatyta, kad skirtumai tarp jų nėra statistiškai patikimi (p>0,05).

Analizuojant kiekvienos veiksnių kategorijos reikšmę patikimumui ±1 ir ±2 šuniukai pagal faktinį vados dydį, gauti rezultatai parodė, kad dauguma jų buvo statistiškai reikšmingi (p<0,001).

Tyrimai parodė, kad nustatant vados dydį ultragarsinis tyrimas nėra tikslus ir analizuoti veiksniai (veislės dydis, nėštumo diena ir kt.) rezultatų neįtakojo. Patikimumo analizė ±1 ar ±2 šuniukai lyginant su faktiniu vados dydžiu parodė, kad ji turi statistinę reikšmę, tačiau rezultatai turėtų būti vertinami atsargiai, nes ±1 ir ±2 šuniukai turi didelį santykinį poveikį nustatant vidutinę paklaidą.

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ABBREVIATIONS

LH – Luteinizing hormone

FSH – Follicle stimulating hormone

SKK – Svenska Kennelklubben (Swedish Kennel Club)

GnRH – Gonadotrophin releasing hormone

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INTRODUCTION

Ultrasound examination is non-invasive method that can be used as a non-invasive method to determine pregnancy, litter size and other important measures during pregnancy in dogs [1]. There have only been few studies performed on litter size determination using ultrasound even if new equipment and methods have developed [2]. By knowing the litter size, planning for the parturition and care of the puppies can be performed, which in turn give the veterinarian and the owner the most accurate results of litter size determination [2,5,6].

Studies have shown that the accuracy to detect the actual outcome of litter size is low and usually underestimated and factors such as embryonic or fetal reabsorption can affect the results [2,3]. The frequency of a loss of one or more fetuses in healthy bitches are 5-13% [2]. Other factors that might affect the results can be maternal colonic or intestinal gas, the cooperation of the patient, experience of the veterinarian and the patient´s weight, size, and age [2,3,4].

The recommendations and best results for litter size determination are obtained when ultrasound examination between day 28-35 of the gestation and when the litter is smaller than five offsprings [2,3]. The gestation day can be hard to decide exactly due to that the bitch releases immature oocytes that require some days to mature and the sperm can survive in the bitch’s reproductive tract for several days, which makes the exact ferilization day hard to know [5,6,7,8].

In other similar studies performed, the result show that the determination of litter size by using ultrasound was correct in 65%, 36 % and 32% of the cases and that the prediction within 1 pup above or below of the actual litter size was 90% [2,9,10]. One study did in particular analyze the bitch size in relation to the litter size. This, however showed no significant difference [2].

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AIM AND OBJECTIVES

The aim of the work:

To evaluate the accuracy of using ultrasonography as a method to determine the litter size in canines.

The objectives of the work:

1. To evaluate the general average deviation between expected and actual outcome of litter size determination using ultrasound and to perform a sensitivity analysis of the average deviation with 1 or 2 pups above or below the actual litter size.

2. To evaluate if the breed size of the bitch effects the general average deviation in litter size determination using ultrasound and to perform a sensitivity analysis of the average deviation with 1 or 2 pups above or below the actual litter size.

3. To evaluate if the gestation day effects the general average deviation in litter size determination using ultrasound and to perform a sensitivity analysis of the average deviation with 1 or 2 pups above or below the actual litter size.

4. To evaluate if the weight of the bitch effects the general average deviation in litter size determination using ultrasound and to perform a sensitivity analysis of the average deviation with 1 or 2 pups above or below the actual litter size.

5. To evaluate if the breed size of the bitch and gestation day together effects the general average deviation in litter size determination using ultrasound and to perform a sensitivity analysis of the average deviation with 1 or 2 pups above or below the actual litter size.

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1. REVIEW OF LITERATURE

1.1 Oestrus cycle in dogs

1.1.1 General about the oestrus cycle

The bitches average age for sexual maturity is around the age between 6-9 months of age, but this can vary depending on the size and breed [11]. For bitches the sexual maturity begins with the first oestrous cycle [11]. Dogs are mono-estrus which is explained as, during each period estrus cycle there is one period when the bitch will accept the male [11,12].

The estrus cycle affecting the bitch can be divided into a number of sub-phases, proestrus, oestrus, met-oestrous and anoestrus [11, 14].

The pro-oestrus lasts for about 9 days [11]. During this period oestrogen is secreted and the vagina thickens and becomes moist [11,13]. There will be an increase of red blood cells which gives a bloody vaginal discharge [11]. In the ovary the primary follicles will mature and form Graafian follicles [11]. The external signs seen on the bitch is an enlarged vulva, blood stained vaginal discharge and an increased frequency to urinate [11]. The bitch will flirt with male dogs but will not allow mating during this period, this can also be called “estrous behavior” [5,11].

The oestrus also lasts for about 9 days [11]. The estrogen levels will fall during this period and there will be a peak of luteinizing hormone (LH) which triggers the ovulation, this period can also be called the “endocrine estrus” [11,14]. The period of the LH surge can also be called the “endocrine estrus” [5]. Progesterone will be released by the luteinized corpus luteum [11]. The ovulation will occur on day 10 of the cycle and the remaining follicle tissue will form a corpus luteum [11]. The vagina becomes moist and thicker and, the blood in the vaginal discharge reduces [11]. The endometrium of the uterus will become thicker during the end of this period [11]. The external signs

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13 of the body on the bitch, are an enlarged vulva and a more straw colored vaginal discharge [11]. The bitch will flirt and stand still and allow mating [11,14].

The met-oestrous period can be divided into two phases [11]. The first phase of the met-oestrus last for about 20 days [11]. The progesterone levels in the blood will still be high and the formed corpus luteum will remain in the ovary through the whole period [11]. During this period the endometrium becomes thicker and glandular to be able keep the fertilized oocyte [11]. The vulva will start to shrink, and the discharge will end [11]. The behavior of bitch will slowly return to normal [11].

The second stage of the met-oestrus last for about 70 days [11]. During this period the progesterone levels will stay high until the corpus luteum is regressed at the end of the period [11]. The endometrium is thicker, and the mammary glands will get swollen [11]. If the bitch is unfertilized this will slowly return to normal [11]. The appearance and the behavior of the bitch will be normal [11].

The anoestrus is the longest phase and last for 3-9 months, with an average around 7 months [11,14]. One of the factors that can affect this interval is, if the dog had a litter in between the estrus cycle [12]. During this period the hormones levels are very low [11]. The follicle stimulating hormone (FSH) and LH levels are pulsating through the whole period [13]. At the end of this period the FSH will start to rise and the follicles will start to form. [11,13]. The estrogen levels will also rise, and a new cycle will soon start [11]. The reproductive tract will look normal and the behavior will be normal [11].

1.1.2 Hormones of the reproductive system

The oestrous cycle is driven by several different hormones, forming a complex system [11]. External factors, such as day length, temperature and pheromones, trigger receptors that stimulates the hypothalamus [11]. The hypothalamus is located in the forebrain and secretes gonadotrophin releasing hormone (GnRH) [11]. When GnRH is released the oestrus cycle starts [11]. It travels with the blood and reaches the pituitary gland, which then secretes FSH and LH [3,5]. FSH stimulates the follicular growth in

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14 the ovary allowing germ cells to develop into mature Graafian follicles [11]. The Graafian follicles in turn release oestrogen, which prepares the reproductive tract [11]. Oestrogen also works as an inhibitor for FSH and stimulates the release of LH from the anterior pituitary gland [11]. The LH affects the follicles and the more mature follicle cells will luteinize and begin to release progesterone [11]. In the end the follicles ruptures, and the oocytes are released and in turn forming corpus luteum [11]. The corpus luteum will release progesterone [11]. During this time the oestrogen levels will decrease and the LH levels will increase [11]. The progesterone has several functions, as it will yield the early signs of oestrus, prepare the reproductive tract for pregnancy, and inhibits GnRH secretion [11]. The inhibition of the GnRH will stop the formation of follicles and help to preserve the pregnancy [11]. If the bitch is not pregnant the corpus luteum will degenerate and progesterone will decrease, and the inhibition of GnRH will stop and the oestrous cycle will start again [11].

1.2 Ovulation

Ovulation can be defined as the process of releasing oocyte from the ovary [11]. The bitch is a spontaneous ovulator which means she will ovulate during each oestrous cycle regardless if she is mated or not [11]. Bitches release multiple oocytes each cycle, as canines are litter-bearing animals [11]. The follicles will reach full size during the end of the proestrus and after 38-44 hours after the LH peak the ovulation will occur, this is around day 10 of the estrus cycle [5,6,11,13,14,15,16]. During the end of the proestrus the follicles reach full size and rupture so that the oocyte can be released to the oviduct [11]. The released oocytes are primary oocytes, which means they are immature and cannot be fertilized immediately [6,14,15]. The oocyte will mature first after 48 hours and can then be fertilization [6,14]. They will then stand viable for 4-5 days before they degenerate [14].

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1.2.1 Determination of ovulation

To determine the time of ovulation can be very important to get a successful fertilization, especially when using insemination methods [7]. 65 days after the LH peak or 63 days after the ovulation parturition occurs [15]. The process of ovulation for bitches are complete within 24 hours [7]. To determine ovulation by just clinical signs, such as vulvar edema and discharge, and vaginal cytology and/or endoscopy is not accurate enough [7]. The LH and the ovulation can be more accurately determined by performing a hormonal assay of the progesterone levels in the serum and combined with the other mentioned methods [15,17]. The progesterone levels range from 2-3 ng/ml (or around 6.5 nmol/l) in the serum during the LH peak and increases to 4-11 ng/ml (or around 32.0 nmol/L) during ovulation and the optimal breeding time can be interpreted [6,7,8,13,14,16,17,18]. This method can fail when bitches have an early increase of the progesterone several days before ovulation [7]. A hormonal assay of the LH is also a method to determine the ovulation, but it is a difficult procedure which requires at least two blood samples per day, takes time to evaluate and it is very expansive [7,16,17]. Ultrasound examination can also be used as a method to determine ovulation, but due to the need of daily examination and determinations it is very unpractical [4].

1.2.2 Ultrasound examination to determine ovulation

Ultrasound examination can also be used as a method to determine ovulation, but due to the need of daily examination and determinations it is very unpractical [4,14]. To determine the ovulation time and examine ovaries in bitches is harder compared to other species [7]. During ovarian ultrasound a real-time diagnostic B-mode ultrasound can be used [14]. To be able to detect the time of ovulation and follicular growth professional experience is needed [14,19]. It is difficult to detect the ovulation due to several factors such as that the follicles not always collapsing, there can be echogenicity changes which not always being constant and that newly formed corpus lutea can be fluid filled cavities that resembles follicles [14]. In bitches the amount of follicular collapse occurs in a limited proportion, while it is normal for other species [12]. The antral follicles can be

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16 observed as a round black anechoic structures in the ovary [14,17]. At the beginning of the proestrus the ovaries are easy to see, and the diameter of these structures are about 2-3 mm [7,14,17]. In the end of the period they are about 5mm in diameter. [7,14,17]. They are located caudo-ventrally from the kidneys [7]. In the end of the proestrus the ovarian size will increase, and a large amount of anechoic fluid can be seen in the follicle [7]. During the LH peak and the day of ovulation the size of the follicles reaches 7-10 mm in diameter, but this can depend on the body size of the dog [7,14,17]. Follicles in the ovaries that don’t ovulate will stay visible up to three days after the ovulation [7].

1.3 Fertilization, gestation, and parturition

1.3.1 Gestation length and parturition

The gestation day is relatively constant, when measured from the ovulation rather than the breeding day [5]. The parturition for bitches occurs around day 65-65 after the LH peak that triggers the ovulation, where the LH peak is considered day 0, or 62-64 days after the ovulation [5,8,16,17,20,21]. (Ovulation occurs 2 days after the LH peak [5].)

When using mating as the reference point to calculate the gestation length and parturition day it can vary [5]. The gestation length can range from day 56 and even up to day 68 [5]. Longer gestation period can occur when the bitch is forced breed before the LH peak has occurred and shorter gestation period can occur in some cases when the bitch is still fertile 7-10 days after the ovulation and breed [5].

Other factors that have to be included when calculating in the gestations length are oocyte release in an immature state during ovulation and needs at least 2-3 days to mature to be able to be fertilized and dog sperm survival [5,6,7,8]. A dog sperm may survive in the bitch’s reproductive tract for up to 7-9 days, but for each day the number of sperms will die and lose their function to fertilize [5,8,16,17].

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17 The benefit of knowing the date of parturition is that it can help to minimize and prevent loss of newborns due to incorrectly delivery management [4,16,17]. In some cases, a planned Cesarean section can be performed, which can reduce losses of the newborns and injuries to the bitch [16].

The serum progesterone levels will decrease 12-40 hours before the first-born puppy and this will cause a lowering in the body temperature of the bitch [16,17]. Due to this the serum progesterone levels can be used a tool for determine the parturition [17].

1.3.2 Fertilization period and fertility

The fertilization period is described as the time when viable oocytes are located in the uterine tubes and mature to secondary oocytes that can be fertilized by spermatozoa, while the fertile period is the time during breeding that can result in a pregnancy [14]. The fertility period is considered to be from 3 days before the LH surge to 7 days after it [14].

As described before the fertilization can only occur when the oocytes are mature which is around 1-2 days after the ovulation, otherwise the sperm cannot penetrate the oocyte barrier [5,22].

As fertility is declining for each day, a mating before the LH peak is not recommended, the sperm need to survive 2 days to be able to penetrate the oocyte (in a mature state), and the chromatin needs to survive another 2-3 days so that the fuse between the male and female nucleus can occur and form a 1-cell-zygote [5]. If mating is performed 2 days after the oocyte mature (4-5 days after the ovulation or 6-7 days after the LH peak,) both litter size and pregnancy rate will decline [5]. This gives a 2-day opportunity for an optimal fertility to occur, and that’s why it is important to know the exact day of ovulation or LH peak [5]. Nevertheless, there can be individuals which have oocyte that are more fertile and can survive for longer periods and have successful mating after this 2-day opportunity [5,14]. When the oocyte matures late the bitch can

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18 give birth to litters with multiple sires when they are mated by different males before ovulation [5].

To determine the best mating time a clinical examination should include several vulvar palpations, vaginal cytology and endoscopy and measurement of the hormonal changes. [8,14,15]. Vaginal cytology and examination of vaginal smear can be used to determine the period of the oestrous cycle and the optimal breeding time [8,14,23]. When performing vaginal smear Wrights-Giemsa stain or Trichrome stain is used and a microscopical examination for exfoliated epithelial cells is performed to determine the oestrus period [14,23]. During late oestrus the smear is thick in consistency and contains a few large intermediate cells and plenty of erythrocytes and bacteria [14]. During pro-oestrus/mid-oestrus, as oestrogen is increasing, a thickening of the vaginal mucosa due to an increase in the number of cell layers occurs, and a change from low cuboidal cell into a keratinized squamous epithelium will occur [14,24]. The microscopical examination of the vaginal smear will show well cornified cells but in some cases the consistency can be watery and sometimes containing a few large intermediate cells and plenty of bacteria and erythrocytes [14]. The fertile period can be predicted by calculating the percentage of superficial epithelial cells [23]. During met-oestrus, well cornified epithelial cells will be seen, with some large and small intermediate cells and some parabasal cells [14]. At the end of the fertilization period, the oestrogen levels will decline while the progesterone levels will increase [23]. The vaginal epithelium is repelled and lost, and the number of cell layers will decrease [23].

Vaginal endoscopy is used to examine the surface of the vaginal mucosa by using a ridged endoscope [14]. When performing the examination, the mucosal fold contours, color and fluid presence is observed [14]. During the anoestrus the vaginal mucosa looks flat, dry and have a red color in appearance [14]. During proestrus and the rising of estrogen level, the mucosal folds gets enlarged, edematous and the color changes to a more pink and white color [14]. The folds are formed are filled with serosanguinous

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19 fluid [14]. During the late proestrus and early estrus, the LH surge occurs and during this period the folds start to shrink slowly, and the color gets pallor [14].

1.4 Pregnancy diagnosis

Ultrasound is a non-invasive and accurate method for pregnancy determination for both the veterinarian and the patient [1]. The first conformation of pregnancy can be performed as early as on day 14-18 after ovulation, but the recommendations for owners are to perform it after day 25-30 of gestation [1,25,26]. The gestational sac can be detected, 12-18 mm but the fetus cannot be visualized [25,26]. The heart beat can be seen around day 22-28 [1,26]. The fetus will have grown to be about 10 mm at day 32 [26]. An experienced veterinarian with a good ultrasound machine will be able to see the gender of the fetuses around day 62-67 [25,26] A study made by Yao Wan, 2014, showed a 100-positive accuracy rate when using ultrasound as a method to determine pregnancy [27].

Radiography can also be used to determine the pregnancy and it is also a method to determine accurate fetal count [8]. The skeleton of the fetus can be visible from day 44 of gestation and it has been 100% accurate to determine pregnancy in the late trimester [28]. This method also has a 93% accuracy to determine the litter size [28]. During the first trimester of the pregnancy the risk of ionizing radiation affecting the fetus is the highest, but later the radiation doesn’t affect the fetuses much more than the bitch [1]. Radiographic images will not be as accurate indicator of fetal vitality as with ultrasound examination [1].

Serum relaxin assay can be used to confirm pregnancy in canines [1]. It can be performed from day 19 and 38 after the LH surge, as the relaxin hormone will increase [4]. Relaxin is released by the canine placenta and is the nearest thing to a so called “pregnancy-specific” hormone in dogs [1].

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20 Digital palpation of the abdomen is a traditional method to diagnose pregnancy [1]. It can be performed between day 24-35 and has an accuracy of 87-88% [1,28]. Before day 28 the amniotic vesicles are to small and difficult to palpate and after day 35 the amniotic vesicles are more fluid field and harder to distinct [1]. Palpation to determine litter size by palpation is only assessed to 12% [1,28].

1.5 Ultrasound during pregnancy

1.5.1 Ultrasound during gestation

It is recommended to perform an ultrasound examination around day 30 after ovulation, but already on day 17-20 the anechogenic gestation sac can be seen through ultrasound, but the embryo and the heartbeat can first be seen at day 23 [2,3,4,20,25]. The structure of the embryo is described as an oblong structure and it is found opposed to the uterine wall in the chronic cavity [4]. The embryo vesicle diameter grows about 1mm per day from day 17-30 [20]. Between day 27-31 after ovulation, the shape of the embryo changes into a more bipolar and limb buds can be seen [4]. Between day 29-33 the diencephalo-telecephalic vesicle, thalamus (as a anechogenic area in the head) and the basal nuclei primordia can be seen, and in the abdomen the stomach and the urinary bladder can be detected [4]. The skeleton becomes more hyperechoic in structure between day 29-33 [4]. Fetal movement can first be observed between day 32-34 [4]. During day 34-36 the separation of the thorax and abdomen becomes visual, and the lungs will look more hyperechoic, compared to the liver which will appear as hypo-echoic compared to the other internal organs [4]. The kidneys can be detected on day 41-43 [4]. The last organ to be visualized if the fetus is the bowel and this can occur during day 57-63 [4].

1.5.2 Gestation age

Gestation age of a fetus can be determined by using different methods such as the measuring the embryonic vesicle diameter, crown-rump length, body diameter, and biparietal diameter of the fetus [2,8,17,20,29]. To get a more accurate measurement and

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21 result the examination should be performed around day 30 or it will hard to take any correct measurements [2,20]. Another method is to look for certain organ development in the fetus [3,8]. The first abdominal organ to develop is the stomach, around day 33, and the urine bladder, around day 34 [3]. The kidneys are only visible during the last 20 days of gestation [3].

1.5.3 Fetal sex determination

Ultrasound can also be used for fetal sex determination, but many factors must be aligned for this procedure to succeed [3]. The fetus has to have a specific position and it is possible to perfrom sex determination during day 55-58 of gestation [30]. The positions of the fetus have to be in ventral decubitus with the pelvic limbs flexed and with the ilieal wings parallel with each other [30]. A female gender during ultrasound examination is visualized as two hyperechoic lines which is joined anteriorly with a pyriform shape and a central hyperechoic line [30]. A male fetus is visualized as central hyperechoic line caudal to the inguinal region [30]. The accuracy for determining the sex has been 95% in a study performed by Gil et al. (2015) [30].

1.5.4 Litter size determination

Litter size determination can be hard to decide [3]. The number of embryonic vesicles is counted, and the accuracy of the number is more accurate in normal litter sizes (2-6 puppies) [4]. When performing the ultrasound examination, the amount of the maternal colonic or intestinal gas, patient’s cooperation, the experience of the veterinarian, and the bitch’s weight, size, and age, is aspects to considerate [2,3,4].

Another factor is that the during the pregnancy the number of fetuses can change due to embryonic and fetal resorption [2]. A loss of one or more fetuses during pregnancy is a frequent event that occurs in healthy bitches with a frequency of 5-13% [2]. Embryonic death is counted for when it occurs before the day 37 of gestation and a total resorption will occur without any clinical signs such as vaginal discharge [2,3]. Most commonly the number of fetuses is undercounted [15]. The recommendations and

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22 best results is are obtained when the ultrasound examination between day 28-35 of the gestation and when the litter is smaller than five offsprings [2,3].

In other similar studies performed, the result show that the determination of litter size by using ultrasound was correct in 65%, 36 % and 32% of the cases and that the prediction within 1 pup above or below of the actual litter size was 90% [2,9,10]. One study did in particular analyze the bitch size in relation to the litter size. This, however showed no significant difference [2].

In other similar studies performed, the result show that the determination of litter size by using ultrasound was correct in 65%, 36 % and 32% of the cases and that the prediction within 1 pup above or below of the actual litter size was 90% [2,9,10]. One study did in particular analyze the bitch size in relation to the litter size. This, however showed no significant difference [2]. These studies concluded that the accuracy for litter size determination is low [9,10].

1.5.5 Abnormalities

Ultrasound can also be used to identify abnormalities during pregnancy [3]. Fetal abortion can occur after day 35 of gestation [3]. Vaginal discharge will be seen after the death of the fetuses [3]. During early abortion an increase in the ecogenicity of the amniotic and allantoic fluid with echogenic structrues can be seen [3]. A thickning of the uterine wall and a abscess of a fetal heart is also a sign for fetal death [3].

Fetal death can be concluded when there is a no fetal heartbeat, no movement of the fetus, reduced volume and increased echogenicity of the fetal fluid and accumulation of gas in the stomach of the fetus [3].

Fetal abnormalities can be hard to see due to the amount of the fetuses [3]. In some cases, hydrocephalus, fetal anasarca, herniating of the ventral abdominal wall and fetal monsters has been detected [3]. Some abnormalities will need cesarean section to be able to deliver the offspring’s [3].

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23 The detection of the fetal distress can also be seen during an examination [3]. Signs of fetal distress and hypoxia can be suspected when the fetal heart rate is reduced to half the maternal rate [3,4].

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24

2. RESEARCH METHOD AND MATERIAL

2.1 Study population

Empirical data was collected through a web-based survey study between 2016 and 2017. The collected data comes from breeders that are registered in the SKK who in most cases breed pure-breeds. The survey included questions about the breeder, the dog, the ultrasound examination, and the actual outcome of the litter size after parturition. The survey can be seen in Annex 1 on page 52.

2.1.1 Cases and categorization

221 answers where collected but to ensure quality of data only 213 were further used in the analysis. In addition to collecting data on expected and actual outcome of the litter size, the survey also focused on a wide variety of factors such as size of the breed, gestation day and weight of the bitch. In total 221 cases were collected.

These cases resulted in a large variety of breed. To give a better overview of the cases, the dogs were divided into three groups depending on the breed size according to the SKK breed standard, small (S) medium (M) and Large (L) (Table 1).

An analysis was performed to calculate the average deviation between expected litter size and actual outcome of litter size. The average deviation was related to the additional data collected in the survey (e.g. size of breed, gestation day, weight etc.). Further, a sensitivity analysis was performed calculating the average deviation by 1 and 2 pups above and below the actual litter size to compare with the actual litter size outcome.

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25 Below, please find the overview of the full set of collected cases from the survey

2.2 Analysis of statistical data

The online survey results were downloaded to a Microsoft excel spreadsheet. Understanding of accuracy was established by calculating the average deviation between expected litter size and actual outcome of litter size. The deviation was further related to

Table 1. Dog breed analysis

Breeds according to size Number Number Number

of dogs of dogs of dogs

Small breeds n = 76 Medium breeds n = 94 Large breeds n = 43

American cocker spaniel 2 Airedale Terrier 2 Alaskan malamute 2

Australian terrier 4 American staffordshire Terrier 2 Beauceron 4

Basenji 9 Australian Shepherd 6 Berner Sennen 7

Beagle 2 Australien Kelpie 8 Bloodhound 3

Bedlingtonterrier 1 Barbet 2 Borzoi 5

Berger des Pyrénées à face rase 1 Basset Artesien Normand 2 Bouvier des flandres 2

Bichon Bolognese 2 Bearded Collie 2 Briard 2

Bichon Havanais 8 Belgian Sheperd 16 Broholmer 1

Border Terrier 9 Border Collie 12 Bullmastiff 3

Bostonterrier 2 Boxer 8 Cane Corso 3

Cavalier King Charles Spaniel 1 Breton 1 Cimarron Uruguayo 1

Chihuahua 6 Bullterrier 1 Coonhound 2

Coton De Tule'ar 1 Chodsky pes 2 Dobermann 1

Dachshund 1 Chow Chow 1 Dogue de bordeaux 1

French Bulldog 8 Clumber spaniel 2 German shepherd 2

Mix breed, small 3 Collie 2 Japanese akita 1

Perro sin pelo del Peru 1 Dalmatian 1 Riesenschnauzer 1

Pomeranian 1 Danish Swedish farmdog 6 Rottweiler 2

Poodle middle 1 Entlebucher sennenhund 2

Pug 1 Field spaniel 1

Pumi 1 Golden Retriever 1

Russkaya Tsvetnaya Bolonka 1 Gos d Atura Catala 1

Shetland sheepdog 5 Irish softcoated wheaten terrier 1

Shiba Inu 1 Labrador Retriever 3

Tibetan spaniel 3 Laekenois 1

Tibetan Terrier 1 Lagotto 1

Mix breed, medium 1

Nova scotia duck tolling retriever 1

Schnauzer 1

Shorthaired Vorsteh 2

Welsh Springer Spaniel 1

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26 size of breed, gestation day, weight etc. The p-value was calculated using two different methods. Student´s paired test (T-test) was used when calculating the p-value and the significance in each cart group, while student’s test for independent samples was used to compare the different chart group with each other to calculate the p-value and if there was a significance. The independent sample method was also used to compare the different factors analyzed in this research with the general average deviation of the litter size to see if the factor had a significant effect on the end result.

2.2.1 General average deviation of litter size

Understanding of accuracy was established by calculating the deviation between expected litter size and actual outcome of litter size. The average value was then calculated.

2.2.2 Breed size

The size of the breed where analyzed to see if this factor had an influence on the accuracy of the litter size determination. The breeds were divided into small (S), medium (M), and large (L) category. The small breeds are dogs with a maximum weight up to 15 kg, medium breeds are maximum up to 29 kg and large breeds are every breed with a minimal weight over 30 kg.

2.2.3 Weight of the bitch

Another factor that is investigated is if the weight of the dog has an effect on the result. The survey asked for the bitch’s weight before pregnancy and were categorized into normal weight and overweight. The weight given in the survey were compared to the normal weight of the breed given on the SKK breed standards.

2.2.4 Gestation day

To be able to determine if the gestations day can affect the general average deviation of litter size determination, the days were divided into four periods. The first

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27 period stated on less than 20 days (<20), after that 20-39 days, 40-60 days and the last was more than 60 days (>60).

2.2.5 Sensitivity analysis of difference in pups

A sensitivity analysis was performed calculating average deviation by 1 and 2 pups above and below the actual litter size to compare with the actual litter size outcome of the general average deviation and to each factor. This was performed to see how much the accuracy differs and if they are statistical significant.

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28

3. REASEARCH RESULTS

3.1 General average deviation of litter size determination

In table 2 below, the general average deviation of litter size determination using ultrasound is analyzed. Each chart pillar represents the 213 dogs used during this study. The general average deviation from the actual litter size outcome (labelled with 0 pups) was calculated to 50%, which means that the veterinarians had a 50% accuracy when determining the actual litter size outcome. The average deviation for 1 pup above or below the actual litter size was 31%, which is 19 percentage points (ppt) lower than the general average deviation from the actual litter size outcome (0 pups). This means in this case the accuracy of litter size determination is 69%. When 2 pups above or above were analyzed the average deviation is calculate to 24%, which is a difference of 26 ppt compared to the general average deviation from the actual litter size. This means that the accuracy of litter size determination in this case is 76%. The results showed that the factors had a statistically significantly difference from each (p<0,001).

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29

3.2 Average deviation of litter size determination depending on size of

breed

In Table 3 on the next page, the average deviation of litter size determination is analyzed with the breed size as a factor to compare if this influence the general average deviation. The categories are divided into large sized breed (n=43), medium sized breed (n=95) and a small sized breed (n=75). When analyzing the actual litter size (0 pups) for all different sizes of breeds, the average deviation for large breeds are 48%, for medium sized breeds 53% and for small sized breeds 47%. When comparing these numbers to the general average deviation for the actual litter size outcome (0 pups chart in table 2), the large sized breeds has a lower average deviation of 2 ppt, the medium sized breeds has a higher average deviation of 3 ppt, and small breeds have 3 ppt lower average deviation. This means that the large and small breeds have a higher accuracy of litter size determination, while in medium sized breed the accuracy is lower of litter size determination, compared to the general average deviation of the litter size determination (0 pups chart in table 2).

In the cases where the average deviation for 1 pup above or below the actual litter size outcome, the large sized breeds have an average deviation of 33%, medium sized breeds have 35% and the small sized breeds 24%. When comparing these numbers to the general average deviation of the complete data set (±1 pup chart in table 2), the large breeds has a higher average deviation of 2 ppt, the medium has a higher average deviation of 4 ppt and the small sized breeds has a lower average deviation of 7 ppt. This means that in small sized breeds has a higher accuracy, while the medium and large sized breeds have a lower accuracy, compared to the general average deviation of the litter size determination (±1 pup chart in table 2).

In the cases where the average deviation for 2 pups above or below the actual litter size the large sized breeds have an average deviation of 24%, medium sized breeds have 24% and the small sized breeds 23%. When comparing these numbers to the general actual litter size outcome for all sizes (±2 pups chart in table 2), the large breeds and the

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30 medium sized breeds has the same average deviation as the general average deviation, while the small sized breeds has a lower average deviation of 1 ppt. This means that in small sized breeds has a higher accuracy, while in medium and large sized breeds the accuracy is the same, compared to the general average deviation of the litter size determination (±2 pups chart in table 2).

There was no statistically significant difference between the average deviation on the litter size determination between the different sizes of the breeds, or between the general average deviation in litter size determination (table 2) and the size of breeds (p>0,05), but in each breed size category there was a statistical significantly between the difference in puppies (0, 1 and 2 pups) (p<0,001).

3.3 Average deviation of litter size determination depending on gestation

day

In table 4 on page 32, the average deviation of the litter size determination is analyzed with the gestation day as a factor to compare if this effects the general average

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31 deviation from the actual litter size. The categories are divided into <20 days (n=1), 20-39 days (n=109), 40-60 days (n=41) and >60 days (n=62). Less than 20 days will not be analyzed due to small number of cases. When analyzing the actual litter size (0 pups) in relation to gestation days, the average deviation for 20-39 days is 49%, for 40-60 days 46% and for >60 days 54%. When comparing these numbers to the general average deviation (0 pups in table 2), 20-39 days has a lower average deviation of 1 ppt, the 40-60 days has a lower average deviation of 4 ppt, and during >40-60 days there is a higher average deviation of 4 ppt. This means that in during day 20-39 and 40-60 the accuracy is higher, while during more than 60 days (>60) the accuracy is lower, compared to the general average deviation of the litter size determination (0 pups chart in table 2).

In the cases where the average deviation for 1 pup above or below the actual litter size the average deviation for 20-39 days is 30%, for 40-60 days 28% and for >60 days 34%. When comparing these numbers to the general actual litter size (±1 pups in table 2), 20-39 days has a lower average deviation of 1 ppt, the 40-60 days has a lower average deviation of 3 ppt, and during >60 days there was a higher average deviation of 3 ppt. This means that during day 20-39 and 40-60 days the accuracy is higher, and during more than 60 days (>60) the accuracy is lower, compared to the general average deviation of the litter size determination (±1 pup chart in table 2).

In the cases where the average deviation for 2 pups above or below the actual litter size the average deviation for 20-39 days is 24%, for 40-60 days 19% and for >60 days 26%. When comparing these numbers to the general actual litter size (±2 pups in table 2), 20-39 days has the same deviation as the average deviation of all gestation days, the 40-60 days has a lower average deviation of 5 ppt, and during >60 days there is a higher average deviation of 2 ppt. This means that during day 20-39 there is no difference, during day 40-60 the accuracy is higher and during more than 60 days (>60) the accuracy is lower, compared to the general average deviation of gestation days of the litter size determination (±2 pups chart in table 2).

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32 There was no statistically significant difference between the average deviation on the litter size determination between the gestation days, or between the general average deviation in litter size (table 2) and the gestation days (p>0,05), but in each gestation day interval there was a statistical significantly between the difference in puppies (0, ±1

and ±2 pups) (p<0,001).

3.4 Average deviation of litter size determination depending on the weight

of the bitch

In table 5 on page 35, the average deviation of the litter size determination is analyzed with the weight of the bitch as a factor to compare if this influences the general average deviation from the actual litter size. The categories are divided into normal weight (n=179) and overweight (n=34). When analyzing the actual litter size (0 pups) for the weight of the bitch, the average deviation for normal weight is 49% and for overweight is 55%. When comparing these numbers to general average deviation (0

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33 pups in table 2), normal weight has as a lower average deviation of 1 ppt, and overweight has a higher average deviation of 5 ppt. This means that for normal weight the accuracy is higher, while for overweight the accuracy is lower, compared to the general average deviation of the litter size determination (0 pups chart in table 2).

In the cases where the average deviation for 1 pup above or below the actual litter size the average deviation for normal weight is 29% and for overweight it is 37%. When comparing these numbers to the general actual litter size outcome for all bitch’s weight (±1 pup in table 2), normal weight has a lower average deviation of 2% and the overweight has a higher deviation of 6%. This means that for normal weight the accuracy is higher, while for overweight the accuracy is lower, compared to the general average deviation of the litter size determination (±1 pup chart in table 2).

In the cases where the average deviation for 2 pups above or below the actual litter size the average deviation for normal weight is 23% and for overweight it is 26%. When comparing these numbers to the general average deviation (±2 pups in table 2), normal weight has a lower average deviation of 1% and the overweight has a higher deviation of 2%. This means that for normal weight the accuracy is higher, while for overweight the accuracy is lower, compared to the general average deviation of the litter size determination (±2 pup chart in table 2).

There was no statistically significant difference between the average deviation on the litter size determination between the weight of the bitch, or between the general average deviation (table 2) and the weight of the bitch (p>0,05), but in each weight category there was a statistical significantly between the difference in puppies (0, 1 and 2 pups) (p<0,001).

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34

3.5 Average deviation of litter size depending on the size breeds and the

gestation day

3.5.1 Large breeds

In table 6 on page 37, the average deviation of the litter size determination is analyzed for large sized breeds depending the gestation day as factors to compare if this influence the general average deviation from the actual litter size (table 2) and to compare the results with the average deviation depending on gestation day (table 4). The charts are divided into 20-39 days (n=26), 40-60 days (n=4) and >60 days (n=13). When analyzing the actual litter size (0 pups) for all different gestation days, the average deviation for 20-39 days is 53%, for 40-60 days 51% and for >60 days 38%. When comparing these numbers to the general average deviation (0 pups in table 2), 20-39 days has a higher average deviation of 3 ppt, the 40-60 days has a higher average deviation of 1 ppt, and during >60 days there is a lower average deviation of 12 ppt. This means that during 20-39 and 40-60 of the gestation days categories the accuracy is lower and during more than 60 days the accuracy is higher, compared to the general average deviation of the litter size determination (0 pups chart in table 2).

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35 In the cases where the average deviation for 1 pup above or below the actual litter size the average deviation for 20-39 days is 38%, for 40-60 days 37% and for >60 days 22%. When comparing these numbers to the general average deviation (±1 pups in table 2), 20-39 days has a higher average deviation of 7 ppt, the 40-60 days has a higher average deviation of 6 ppt, and during >60 days there is a lower average deviation of 9 ppt. This means that during day 20-39 and 40-50 the accuracy is lower, and during more than 60 days (>60) the accuracy is higher, compared to the general average deviation of the litter size determination (±1 pup chart in table 2).

In the cases where the average deviation for 2 pups above or below the actual litter size the average deviation for 20-39 days is 27%, for 40-60 days 23% and for >60 days 19%. When comparing these numbers to the general average deviation (±2 pups in table 2), 20-39 days has a higher average deviation of 3 ppt, the 40-60 days has a lower average deviation of 1 ppt, and during >60 days there is a lower average deviation of 5 ppt. This means that during day 20-39 the accuracy is lower, during day 40-60 and during more than 60 days (>60) the accuracy is higher, compared to the general average deviation of the litter size determination (±2 pups chart in table 2).

A segmentation of average litter size deviation into gestation day (table 4) and large sized breed, show lower accuracy (higher average deviation) in the gestation day intervals 20-39 and 40-60 and a higher accuracy (lower average deviation) when days exceed 60 (table 6).

There was no statistically significant difference between the average deviation on the litter size determination, between the breed size depending on the gestation days, or between the general average deviation of the litter size determination (table 2) and, the breed size and gestation day (p>0,05), but in the gestation day category 20-39 and >60 days there was a statistical significantly between the difference in puppies (0, 1 and 2 pups) (p<0,001). There was no statistically significant difference between the average deviation depending on the gestation day (table 4) and the average deviation of litter size determination on large sized breeds depending on the gestation days (table 6) (p>0,05).

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36

3.5.2 Medium breeds

In table 7 on page 39, the average deviation of the litter size determination is analyzed for medium sized breeds depending on the gestation day as factors to compare if this influence the general average deviation from the actual litter size (table 2) and to compare the results with the average deviation depending on gestation day (table 4). The charts are divided into <20 days (n=1), 20-39 days (n=43), 40-60 days (n=19) and >60 days (n=32). Due to that the <20 category has only one case it is not included in the analyze. When analyzing the actual litter size (0 pups) in relation to gestation days, the average deviation for 20-39 days is 47%, for 40-60 days 43% and for >60 days 67%. When comparing these numbers to the general average deviation (0 pups in table 2), 20-39 days has a lower average deviation of 3 ppt, the 40-60 days has a lower average deviation of 7 ppt, and during >60 days there is a higher average deviation of 17 ppt. This means that during day 20-39 and 40-60 the accuracy is higher, and during more than 60 days (>60) the accuracy is lower, compared to the general average deviation of the litter size determination (0 pups chart in table 2).

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37 In the cases where the average deviation for 1 pup above or below the actual litter size the average deviation for 20-39 days is 30%, for 40-60 days 25% and for >60 days 48%. When comparing these numbers to the general average deviation (±1 pups in table 2), 20-39 days has a lower average deviation of 1 ppt, the 40-60 days has a lower average deviation of 6 ppt, and during >60 days there is a higher average deviation of 17 ppt. This means that during day 20-39 and 40-60 the accuracy is higher, and during more than 60 days (>60) the accuracy is lower, compared to the general average deviation of the litter size determination (±1 pup chart in table 2).

In the cases where the average deviation for 2 pups above or below the actual litter size the average deviation for 20-39 days is 17%, for 40-60 days 22% and for >60 days 36%. When comparing these numbers to the general average deviation (±2 pups in table 2), 20-39 days has a lower average deviation of 7 ppt, the 40-60 days has a lower average deviation of 2 ppt, and during >60 days there is a higher average deviation of 12 pp. This means that during day 20-39 and 40-60 the accuracy is higher and during more than 60 days (>60) the accuracy is lower, compared to the general average deviation of the litter size determination (±2 pups chart in table 2).

A segmentation of average litter size deviation into gestation day (table 4) and medium sized breed, show better accuracy (lower average deviation) in the gestation day intervals 20-39 and 40-60 and a lower accuracy (higher average deviation) when days exceed 60 (table 7).

There was no statistically significant difference between the average deviation on the litter size determination for these categories, between the breed size and gestation days or, between the general average deviation of litter size determination (table 2) and, the breed size and the gestation day (p>0,05), but in each gestation day category there was a statistical significantly between the difference in puppies (0, 1 and 2 pups) (p<0,001). There was no statistically significant difference between the average deviation depending on the gestation day (table 4) and the average deviation of litter size determination on medium sized breeds depending on the gestation days (table 7)

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38 (p>0,05).

3.5.3 Small breeds

In table 8 on page 41, the average deviation of the litter size determination is analyzed for small sized breeds depending on the gestation day as factors to compare if this influence the general average deviation (table 2) and to compare the results with the average deviation depending on gestation day (table 4). The charts are divided into 20-39 days (n=40), 40-60 days (n=18) and >60 days (n=17). When analyzing the actual litter size (0 pups) for all different gestation days, the average deviation for 20-39 days is 49%, for 40-60 days 48% and for >60 days 42%. When comparing these numbers to the general average deviation (0 pups in table 2), 20-39 days has a lower average deviation of 1 ppt, the 40-60 days has a lower average deviation of 2 ppt, and during >60 days there is a lower average deviation of 8 ppt. This means that during all gestation day categories the accuracy is higher, compared to the general average deviation of the litter size determination (0 pups chart in table 2).

In the cases where the average deviation for 1 pup above or below the actual litter size the average deviation for 20-39 days is 25%, for 40-60 days 28% and for >60 days 18%. When comparing these numbers to the general average deviation (±1 pups in table

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39 2), 20-39 days has a lower average deviation of 6 ppt, the 40-60 days has a lower deviation of 3 ppt, and during >60 days there is a lower average deviation of 13 ppt. This means that during all gestation day categories the accuracy is higher, compared to the general average deviation of the litter size determination (±1 pup chart in table 2).

In the cases where the average deviation for 2 pups above or below the actual litter size the average deviation for 20-39 days is 29%, for 40-60 days 18% and for >60 days 15%. When comparing these numbers to the general average deviation (±2 pups in table 2), 20-39 days has a higher average deviation of 5 ppt, the 40-60 days has a lower average deviation of 6 ppt, and during >60 days there is a lower average deviation of 9 ppt. This means that during day 20-39 the accuracy is lower and during day 40-60 and more than 60 days (>60) the accuracy is higher, compared to the general average deviation of the litter size determination (±2 pups chart in table 2).

When comparing the results from the average deviation of litter size determination on small sized breeds depending on the gestation days (table 8) with the average deviation depending on the gestation day (table 4), during day 20-39 the result is the same when comparing the actual litter size outcome (0 pups) from both results, 1 pup below or above the actual litter size there is a lower average deviation and 2 pups below or above has an higher average deviation. During day 40-60, the actual litter size outcome (0 pups) has a higher average deviation, 1 pup below or above the actual litter size has the same average deviation in both results and 2 pups below or above has a lower average deviation. During more days than 60 the average deviation is lower in all cases.

A segmentation of average litter size deviation into gestation day (table 4) and small sized breed, show that during day 20-39 the result is the same when comparing the actual litter size outcome (0 pups) from both results, 1 pup below or above the actual litter size shows better accuracy (lower average deviation) and 2 pups below or above has a lower accuracy (higher average deviation) (table 8). During day 40-60, the actual litter size outcome (0 pups) has a lower accuracy, 1 pup below or above the actual litter

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40 size has the same average deviation in both results and 2 pups below or above has a higher accuracy (lower average deviation). During more days than 60 the accuracy is higher (lower average deviation) (table 8).

There was no statistically significant difference between the average deviation on the litter size determination for these categories, between the breed size and gestation days, or between the general average deviation in litter size (table 2) and, the breed size and the gestation day (p>0,05), but in each gestation day category there was a statistical significantly between the difference in puppies (0, 1 and 2 pups) (p<0,001). There was no statistically significant difference between the average deviation depending on the gestation day (table 4) and the average deviation of litter size determination on small sized breeds depending on the gestation days (table 8) (p>0,05).

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41

4. DISCUSSION OF RESULTS

The results of this research show that the average deviation is 50% of determining the litter size by using ultrasound. When comparing the sensitivity analysis of 1 and 2 pups above or below the actual litter size the average deviation is 31% and corresponding 24%. Compared to the study performed by Lenard et al. (2007), where the result of the prediction of litter size was correct in 65% of the cases, and 90% in cases where the litter size was predicted to within 1 pup above or below the actual litter size and comparing to the similar studies with an accuracy of 36% and 32% [9,10]. This research showed an accuracy of 50% for the correct litter size and a 69% (31% deviation) correct determination on 1 pup below or above the actual litter size outcome.

The sensitivity analysis of 1 pup and 2 pups below and above the actual litter size outcome showed that is has a statistical significance, but this result should be seen with a care due to that a ±1 or 2 pups gives a big value difference when calculating the average deviation.

Lenard et al. (2007), also compared if the maternal size had any effect in the prediction, which showed a no statistical significant difference, but that in the large breed you could see a trend of lower accuracy in the prediction. The result of this research showed that the there was no big difference in the average deviation depending on the size of the bitches, but the larger sized breeds had a lower average deviation when a prediction of 1 or 2 pups above or below the actual litter size outcome. This means that the larger breeds had a more correct determination which is the opposite in the study performed by Lenard et al. (2007). One of the factors that can have affect the litter size determination is that the accuracy of the determination decreases when the litters are larger than 5 offspring’s, which are more common in larger breeds [2,3].

The recommendations for ultrasound to determine the litter size is on day 28-35 [2,3]. The result of this study showed that the prediction of correct litter size depending

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42 on the gestation day compered to the actual out come has a diffrence on 4 ppt (above or below). The results from <20 days are not analyzed due to that only one dog was examined so early. The lowest average deviation was seen between days 40-60. The highest average deviation was seen on <60 days on the actual litter size, and the lowest was also <60 days when a determination of 1 or 2 pups above or below the actual outcome. The factors that can lower the prediction accuracy is that before 37 days of gestation fetal and embryonic resorption can occur and in 5-13% of the cases when a bitch is pregnant a loss of one or more fetuses occurs [2]. Embryonic death occurs without any signs which makes it hard to determine if it has occurred or not without an ultrasound [2]. After day 35 fetal abortion can occur with clinical signs such as vaginal discharge which makes it easier to detect [3].

The weight of the bitches where also anlyzed to understand if they had any affect on the accuracy of the litter size determination. The result showed to have no statistical significance, but the results observed showed that the normal weight dogs had an lower average deviation than the overweight bitches compared to the general avarage deviation.

In table 6,7 and 8, the average deviation of litter size determination depending on the size of the breed and the gestation day were also analyzed to see if these factors together effects the general average deviation of litter size determination (table 2). The results were also compared to the average deviation depending on the gestation day (table 4). There was no material difference in the average deviation between the general average deviation (table 2) and the average deviation depending on the gestation day (table 4) and the results showed to have no statistical significance.

All the results and analysis can be affected by different aspects and factors. Some factors can depend on the veterinarian performing the ultrsound and his/her experience, the type of ultrasound machine, the amount of colonic or intestinal gas in the patient during the ultrasound examination, patient’s cooperation, and the bitch weight, size and age [2,3,4]. If the bitch has too much gas the view of the fetuses can be blocked and

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43 easily missed [2,3,4]. The day of fertilization and gestation are hard to get fully accurate, due to the bitch oocyte is released immature and the sperm can survive for many days in the bitch’s reproduction tract, this can make the recommended day for ultrasound examination for an accurate litter size determination easy to miss [5,6,7,8].