Canine fetal bpd and kl measurement in predicting delivery time by ultrasound

Luận văn tốt nghiệp thú nhỏ về siêu âm so sánh giữa 2 phương pháp đo chiều dài lưỡng đỉnh và chiều dài thận của chó con trong việc dự đoán ngày sinh. Đề tại được thực hiện tại thành phố Hồ Chí Minh trên 16 cá thể chó mang thai. Kết quả cho thấy phương pháp tính ngày sinh bằng cách đo đường lưỡng đỉnh cho kết quả chính xác hơn so với phương pháp tính ngày dự sinh bằng phương pháp đo chiều dài thận của chó con.

MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY – HO CHI MINH CITY

FACULTY OF ANIMAL SCIENCE AND VETERINARY MEDICINE

*******************************

GRADUATE PROJECT REPORT

CANINE FETAL BIPARIETAL DIAMETER AND KIDNEY LENGTH MEASUREMENT FOR DELIVERY TIME

BY ULTRASOUND: A COMPARISON

Student: LE DANG KHOA Class: DH16TT (Advanced Program) Discipline: Veterinary medicine Duration: 2016 – 2022

Ho Chi Minh City, July 2022

MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY – HO CHI MINH CITY FACULTY OF ANIMAL SCIENCE AND VETERINARY MEDICINE

(Research Veterinary Elective)

The report is submitted as a requirement for Doctor of Veterinary Medicine degree

Supervisor:

ASSOC PROF LE QUANG THONG

Ho Chi Minh City, July 2022

In bitches, the ultrasonographic measurement of fetal structures allows theevaluation of gestational age and the prediction of the parturition term for thesecond half of the pregnancy The aim of this study was to compare equations based

on biparietal diameter (BPD) and kidney length (KL) for prediction of theparturition date in second half of pregnancy in the small and medium-size bitcheswith comparable accuracy The effects of bitches weight, age, litter size, and breed(Poodle) on the accuracy of the prediction were also investigated The researchconsists of 16 clinically healthy pregnant bitches of 5 breeds and cross-breeds.Bitches were divided into groups according to compared classifications In each ofthe bitches, ultrasonographic examination of pregnancy was performed on at least 2fetuses from opposite uterine horns with 4 measurements (2 fetal kidney lengths and

2 biparietal diameters) The predicting parturition dates were compared with theactual dates of delivery provided by the bitch owners The data of each structurewas calculated and the predicted day of parturition was obtained by the application

of equations derived from growth curves previously developed for different sizebitches

On the effect of gestational age, BPD measurement gained better reliabilitythan KL measurements within 2 days, while by KL measurement, parturitionprediction was highly accurate on week 7 of pregnancy Bitch size and litter size didnot affect the accuracy of parturition prediction by BPD and KL measurement

In the Poodle breed only, based on gestational age, BPD and KL measurements hadhigh precision within ±2 days at week 8 (75%), week 7 (100%), respectively.Comparison based on bitch size, BPD remained more reliable than KLmeasurement while litter size did not affect the accuracy of BPD and KLmeasurement

Keywords: canine, parturition time prediction, biparietal diameter, kidney length.

First of all, I would like to thank all the lecturers of the Faculty of AnimalScience and Veterinary Medicine, Nong Lam University, Ho Chi Minh City, whoplayed an indispensable role in my education The immense knowledge andexperience that have been imparted for almost 6 years make me mature and stronger

in future path

Next, I would like to express my sincere thanks to my dedicated mentor –Assoc Prof Le Quang Thong for guiding me to carry out the thesis He was alwayspatient with my shortcomings in knowledge and gave me the best guidance Mythesis would not have been possible without his constant support, invaluable adviceand encouragement

I would then like to acknowledge the support at every stage of the rest of mytime as an intern to all the veterinarians and staff of PIMA Veterinary clinic,especially Dr Gia Khang, Dr Lam Van, Dr Bao Uyen, Dr Hong Uyen and Ms.Nuong They gave me great pleasure working there with a friendly, professionalworking environment and modern medical equipment

Last but not least, I must express my deepest gratitude to my family, mybeloved girlfriend and classmates With the constant support of my family and theconstant encouragement throughout the study period and through the researchprocess, it was the motivation for me to complete the thesis

TABLE OF CONTENTS

LIST OF ABBREVIATIONS vi

LIST OF TABLES vii

LIST OF FIGURES viii

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW 3

2.1 Maternal reproductive endocrinology and events 3

2.1.1 Pre-implantation: period of pre-fertilization, fertilization and the early embryo ………4

2.1.2 Post-implantation: periods of the late embryo 5

2.1.3 Period of fetus to parturition 6

2.2 Prenatal development 7

2.2.1 The period of the ovum 7

2.2.2 The period of the embryo 8

2.2.3 The period of the fetus 8

2.3 Canine fetal skull and kidney development 10

2.3.1 Canine fetal skull development 10

2.3.2 Canine fetal kidney development 13

2.4 Parameters used for parturition day prediction 16

2.4.1 Maternal parameters 17

2.4.2 Embryonic and fetal parameters 19

2.5 Ultrasonography and use in canine pregnancy diagnosis 24

2.5.1 Principle 24

2.5.2 Technique 25

2.5.3 Use of ultrasonography in veterinary practice 25

2.5.4 Application in pregnancy diagnosis 26

2.6 Recent related articles review 27

CHAPTER 3 MATERIALS AND METHODS 30

3.1 Time, location, and patient 30

3.2 Materials and equipment 30

3.3 Methods 31

3.3.1 Content and objective 31

3.3.3 Method procedure 32

3.4 Statistical analysis 35

CHAPTER 4 RESULTS AND DISCUSSION 36

4.1 Studied bitches information (breed, age, and weight) 36

4.2 Accuracy (%) of the prediction of parturition between BPD and KL measurement based on gestational age 38

4.3 Accuracy (%) of the prediction of parturition between BPD and KL measurement based on bitch size 39

4.4 Accuracy (%) of the prediction of parturition between BPD and KL measurement based on litter size 40

4.5 Accuracy (%) of parturition day prediction between BPD and KL measurement in Poodle breed 41

CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 44

Conclusions 44

Recommendations 44

REFERENCES 46

MANUSCRIPT 51

CURRICULUM VITAE 67

MOTIVATION LETTER 71

FSH: Follicular stimulating hormone

ICC: Inner chorionic cavity

LIST OF TABLES

Table 2.1 Days of first appearance at ultrasound of fetal structures observed during

the second half of pregnancy reported and references

……….9

Table 2.2 Equations for prediction of gestational age (days after LH peak) or days

before parturition (dbp) in the bitch by ultrasonographic measurement of extra-fetalstructures ……… ……20

Table 2.3 Equations for prediction of gestational age (days after LH peak) or days

before parturition (dbp) in the bitch by ultrasonographic measurement of fetalstructures ……… ……22

Table 4.1 Descriptive statistics (minimum and maximum, average, median and

standard deviation) of fetal BPD and fetal KL measurements during

parturition……….37

Table 4.2 Accuracy of parturition day prediction between BPD and KL

age……… …38

Table 4.3 Accuracy of parturition day prediction between BPD and KL

size……….… 39

Table 4.4 Accuracy of parturition day prediction between BPD and KL

size……… ….40

Table 4.5 Accuracy of parturition day prediction between BPD and KL

measurement in Poodle breed on the basis of gestational age………

…….…….…41

Table 4.6 Accuracy of parturition day prediction between BPD and KL

measurement in Poodle breed on the basis of bitch size……….…

……… ….…41

Table 4.7 Accuracy of parturition day prediction between BPD and KL

measurement in Poodle breed on the basis of litter size

……… 42

LIST OF FIGURES

Figure 2.1 Schematic of reproductive hormones in the estrus cycle of the domestic

dog ……… …….…… 5

Figure 2.2 Section of a 500 µm dog blastocyst showing the inner cell mass Note

mitotic division (arrow) and several spermatozoan heads embedded in the zona pellucida, 11 days after breeding ……….……… ……7

Figure 2.3 Dorsal view of the skull in a 45-day Beagle fetus The fontanel between

the frontal and parietal bones will close before birth …… ……….… 11

Figure 2.4 Dorsal view of the skull of a 50-day, 105-mm Beagle fetus, calvaria

removed Note that the incus and fourth superior premolar are ossified

Figure 3.1 Vetus 8 Veterinary UltrasoundMachine……… 30

Figure 3.2 4-12 MHz linear transducer (A) and 3-11 MHz microconvex transducer

(B)……….31

Figure 3.3 Pregnant bitch was under image acquisition……… 32 Figure 3.4 A longitudinal view of fetal BPD……….………… 33 Figure 3.5 A longitudinal view of both two kidney in a canine fetus……… ……33 Figure 3.6 A longitudinal view of left fetal kidney with caliper……….34 Figure 4.1 Studied bitch number……… ………36 Figure 4.2 Studied bitch age (A) and weight (B) 36

CHAPTER 1 INTRODUCTION

Loss of neonates at the time of parturition would happen because of incorrectmanagement representing potential emotional and economic loss for pet owners.Parturition is a critical event, accurate determination of gestational age allows betterplanning for breeders and veterinarians to assist the whelping to reduce theperipartum losses of offspring Many studies have been published on determinations

of gestational age and the prediction of delivery date in dogs such as ovulationdetermination and hormonal assays However, these methods are only accuratewithin 1 or 2 days In case these methods are not possible, estimation of gestationalage can be based on ultrasonographic imaging of embryonic and fetal structures.Therefore, more accurate determination of gestational age requires new methods inlate pregnancy to help to decrease fetal loss and also the risk of premature C-section

In the last decade, several ultrasonographic examinations such as fetal headdiameter, liver, stomach, bladder, and heart have been reported in helping toaccurately predict delivery time In late pregnancy, biparietal diameter (BPD) is themost accurate fetal parameter for predicting delivery time (Luvoni and Beccaglia,2006) with good visualization of parietal bone after day 35 of gestational age(Kutzler, 2003) BPD is measured by drawing a line in longitudinal view when thefalx cerebri is visualized and two parietal bones are parallel This method has agood accuracy during the 5th week (95.2% within ±2 days) and 6th week (88.4%within ± 2 days) of pregnancy and decreases to 85.3% in the 8th week and lowaccuracy during the last week (50.9% within ±1 day and 69.8% within ±2 days)(Luvoni and Beccaglia, 2012)

In the dog, the kidney continues to develop during the first weeks after birth(Sinowatz, 2010), is different from other species, and nephrogenesis decreases

shortly after delivery It has been a hypothesis that ultrasonographically differentappearances occur during stages of gestation due to the different embryologicalstages of renal development In 2018, Gil et al published a study about fetal kidneylength, it has been reported to be strongly and positively correlated with pregnancyage The formula may be applied from 24 to 1 day before parturition (dbp), in whichthe best period for monitoring kidney length is from 15 to 11 dbp (between week 7and week 8) with accuracy, sensitivity and specificity are 99.98%, 99.95% and99.92% within ±1 day, respectively.

Therefore, this study aimed to investigate the comparable accuracy ofobtained equations based on two fetal BDP and KL measurements for delivery timeprediction in second half of pregnancy in small and medium-size bitches

CHAPTER 2 LITERATURE REVIEW

Dogs are monoestrous, typically non-seasonal, polytocous, spontaneousovulators Based on breed size, pubertal estrus occurs variably at 6–14 months Theinterval between estrus ranges from 5–12 months, usually 6–7 months, from veryvariable to frequent in bitches, and between pregnancy and nonpregnancy Ingeneral, the canine cycle is divided into four phases (Evans and Cole, 1931): a 5 to

20 day proestrus, 5 to 15 day estrus, 50 to 80 day metestrus (post-estrus portion ofluteal phase), and anestrus typically lasting 80–240 days These phases reflect,respectively, follicular phase rise in estrogen, the initial luteal phase rise inprogesterone and decline in estrogen, the remainder of the luteal phase, and theinterval between loss of luteal function and onset of the next cycle In pregnantdogs, after fertilization, it takes about 61 days for the fertilized oocytes to fullydevelop into newborn puppies (Pretzer, 2008) Phemister (1974) divided theprenatal development of dogs into three periods, including ovum (days 2–17),embryonic (days 19–35) and fetal (days 35 to birth) Ovulation period followsfertilization and is characterized by a blastocyst, which is free in the uterine tubeand migrates to the uterus Next, the embryonic stage begins with the implantation

of the blastocyst and ends with the completion of organogenesis The fetal period is

a time when characteristic features appear and develop rapidly

2.1 Maternal reproductive endocrinology and events

Endocrinologically, pregnancy in dogs can be divided into three periods: (1)the period of fertilization and the early embryo encompass the time fromfertilization to approximately days 20 to 22 post luteinizing hormone (LH) surgewhen implantation occurs, (2) late period of embryogenesis from implantation tofetal calcification/ ossification at 40 to 42 days after LH surge, and the onset of fetaldevelopment and organogenesis, and (3) the fetal period is from thecalcification/fetal calcification stage to delivery

2.1.1 Pre-implantation: period of pre-fertilization, fertilization and the early embryo 

Initially, in the pro-estrus period, increases in androstenedione andtestosterone reach peak concentrations (mean 300 and 800 pg/ml, respectively)accompanied by an increase in estradiol during estrus LH and FSH levels weregradually suppressed to the lowest concentrations of the cycle (1 ng/ml and <50ng/ml, respectively) (Figure 2.1) Oocyte maturation is estimated to occur 60 hoursafter the preovulatory LH surge, so it can occur any time from late proestrus to mid-estrus Ovulation time can be estimated by monitoring vulvar swelling, vaginalcytology, and through observation of changes in vaginal mucosa morphology,shape, and color by vaginoscopy

Next, preovulatory luteinization occurs and followed by LH surge There israpid and general proliferation of luteal cells throughout the periphery of thepreovulatory follicles, this was accompanied by an increase in serum progesteronelevels to 1.0–3.0 ng/mL at the time of LH surge and from 4 to 8 ng/mL at the time

of ovulation Primary oocyte ovulation is about 2 days after the LH surge (day 2),then it takes another 2 to 3 days to reach final maturation and secondary oocyteformation

After fertilization, the embryo will remain free in the uterine cavity, floatingand moving in the uterine horn, and at the same time absorbing the nutrientsproduced by the early developing endometrial glands, under the influence ofincreased progesterone levels from the developing corpus luteum (CL) Theblastocyst then adheres evenly to the endometrium around days 18 to 20 after LHsurge, with final implantation occurring around days 20 to 22 after LH surge.Uterine swelling at the observed implant sites may vary from day 14 to day 20.Histological observations for local inflammatory reactions (edema, neutrophils, andlymphocyte infiltrates) with congestion and hypervascularization, recorded as early

as day 10 to day 14 at different sites of the horn and body of the uterus, which arepossible sites for future implantation

2.1.2 Post-implantation: periods of the late embryo 

This period begins with endotheliochorial cell growth, zonary, modifieddeciduate placenta at approximately days 20 to 22 Marginal hematomas areobserved at the edges of the zonary band, and are formed from maternal bloodwhere hemoglobin is metabolized into uteroverdin The embryo acquires a number

of metabolites from these sites, particularly iron The progesterone test should never

be used to detect pregnancy during this time because the length of the luteal phaseand the rise in serum progesterone is similar in non-pregnant and pregnant femaledogs Progesterone is absolutely necessary to maintain pregnancy, with aconcentration of 2 ng/mL considered the minimum

During the gradual decrease in plasma progesterone concentrations,progesterone morphology does not appear to be related to changes in concentrations

of estradiol or the prostaglandin F metabolite (PGFM) The PGFM phenotype was

Figure 2.1 Schematic of reproductive hormones in the estrus cycle of the

domestic dog (Concannon, 2011)

significantly different between diestrous and pregnant bitches There is an apparentbut gradual increase in PGFM in pregnant bitches between days 30 and 60,followed by a large increase before parturition, and then a decrease inconcentrations shortly postpartum

2.1.3 Period of fetus to parturition

During the period of the fetus, the fetuses undergo dramatic development,ossification, and their body weights increase considerably The CL are extremelydependent on the trophic support of the pituitary gland Any changes in prolactin,

LH or hormonal balance can lead to loss of CL function and pregnancy failure

Parturition occurs due to a rapid drop in progesterone from 4 - 10 ng/mL to <

2 ng/mL over a period of 12–24 hours, beginning 1–2 days before whelping.Progesterone depletion occurs in response to an increase in prostaglandin toluteolytic concentrations, which in turn occurs in response to fetal pituitary-adrenalaxis maturation and an increase fetal glucocorticoid concentrations ProstaglandinF2a, together with the endocrine and endocrine activities of oxytocin, is responsiblefor stimulating uterine contractions, cervical softening, and birth canal relaxation Amarked increase in plasma vasopressin concentrations was also observed during theexpulsion of the first few fetuses, followed by a decrease before the end of labor,which is related to stress and pain

Progesterone depletion can be monitored and examined in the management

of bitches with an apparent or presumed protracted pregnancy, difficult delivery andduration of elective Cesarean section Prenatal progesterone depletion may beresponsible for the acute rise in prolactin just before parturition A decline inprogesterone causes the onset of milk production Clinically, body temperaturedrops of 1°C or more between 12 and 24 hours before birth, occurring 12 to 18hours after a sudden drop in progesterone

2.2.      Prenatal development

2.2.1 The period of the ovum 

Fertilization occurs in the cranial part of the uterine tube (oviduct), and thefertilized oocyte begins to divide within a few hours Canine zygotes take longer toreach the uterotubal junction than other species, 7–10 days versus 3–4 days,respectively The developing embryo can enter the uterus as early as the 16-cellstage, but is more common as the morula or even early blastocyst (Holst &Phemister, 1971) In utero, the morula develops into a spherical blastocyst, with aninner cell mass and a thin surface troblast surrounded by a zona pellucida (Figure2.2) Unattached blastocysts travel within the uterine horn and absorb nutrientsfrom the ''uterine milk'' produced by the developing endometrial glands The free-floating blastocyst stage lasts about 7 days (Holst & Phemister, 1971) Finally,

Figure 2.2 Section of a 500 µm dog blastocyst showing the inner cell mass.

Note mitotic division (arrow) and several spermatozoan heads embedded in thezona pellucida, 11 days after breeding (Holst & Phemister, 1971)

blastocysts attach to the endometrium around day 18 to 20 after LH surge, with finalimplantation occurring around day 20 to 22 after LH surge.

2.2.2 The period of the embryo

This stage begins with the gastrulation (formation of the germ layer), which

is the embryonic development stage in which the single-layer blastula istransformed into a trilaminar structure including an outer ectodermal, a middlemesodermal, and an inner endodermal layer The ectoderm differentiates into theepidermis of the skin and into neural tissue, organs and tissues of reproductiveimportance, mammary glands, hypothalamus, also pituitary, caudal vagina,vestibule, penis or the clitoris all originate from this layer The endoderm forms thelining of the gastrointestinal and respiratory tracts, and from the middle mesodermallayer the genitourinary, circulatory, and supportive muscular skeletal systems areformed (McGeady, 2017)

Evans (2012) found that at 23 days of gestation, the embryo is 10 mm in length, has

a prominent thoracic limb bud, otic placode and lens placode, and the mandibularand maxillary processes are distinct By 25 days, the embryo with its length of 14

mm has a mammary ridge present, vertebral elements are condrifying, and thedental lamina forms The 28 days embryo (17 mm) is the age at which the firstossification is seen in four bones: mandible, maxilla, frontal bone, and clavicle By

30 days (19 mm) the eyelids and external ear are forming, and sensory hairs on themuzzle, chin, and eyebrows develop The 33 days embryo (27 mm) has developingossification of nasal, incisive, palatine, zygomatic, and parietal bones The canineteeth are in the early cap stage at this point. 

2.2.3 The period of the fetus

Studies of embryonic and fetal development have greatly benefited fromultrasound (US), staged pregnancy such as early detection of anatomical structures.Many embryonic and fetal structures can be viewed in the US during dogdevelopment, from the blastocyst in early pregnancy to the fetal intestine during thelast week of pregnancy

Fetal structures First detection (Days

Fetal development progresses rapidly from day 30, giving the recognition oforganogenesis sonographically Table 2.1 represents days of first appearance duringsecond half of pregnancy of fetal structure observed Limb buds and fetal movementare recognized on about day 31 to 34 The fetal skeleton can be identified on day 33

to day 39 and is seen as hyperechoic structures with acoustic  shadows (England,1990) The head is detected first, followed by rapid mineralization of the thoracicspine and ribs, then the cervical  spine and appendicular skeleton The urinary

Table 2.1 Days of first appearance at ultrasound of fetal structures observed

during the second half of pregnancy reported and references

bladder and  stomach are the first abdominal organs identified sonographically, byday 33 to day 37, and appear as anechoic focal areas The lung and liver arerelatively isoechoic when  they are initially seen, without clear definition betweenthem The lungs become hyperechoic relative to the  liver as the fetus develops byday 36 to day 40 The kidneys and the eyes are seen by day 37 to day 45 Thekidneys are hypoechoic with prominent anechoic pelvis Over time, the renal cortexand medulla can be differentiated and the pelves become less dilated The heart is hypoechoic to anechoic, with linear septate echoes representing the chamber wallsand heart valves Several days later, the great cardiac vessels can be  imaged Theintestine is seen late, by day 57 to day 63.

2.3 Canine fetal skull and kidney development

2.3.1 Canine fetal skull development

The skull is considered as parachordal, trabecular, and branchial cartilages,which are derived from neural crest in early time The parachordal and trabecularcartilages enlarge and fuse together beneath the brain, they combine the sensecapsules (olfactory, optic, and otic) and form a cartilaginous skull called achondrocranium This cartilaginous structure is of definitive skull ossifyendochondrally ventral to the brain and combine with bone to form the membrane

of the dorsal surface of the desmocranium to the brain

The fetal skull ossification to be first facial at 34 mm on day 35 (maxilla,mandible), palatal (palatine, pterygoid), and calvarial (frontal, parietal) centers,followed by basicranial centers at 54 mm (basisphenoid, basioccipital) and finallythe otic capsule (89 mm) and hyoid apparatus (93 mm)

Then, the covering bone of mandibular cartilage (dentary) andpalatoquadrate cartilage (maxilla) begins to ossify on day 28 in the 19 mm embryo,

as is the frontal bone of the skull roof and the membranous part of the clavicle.Other membrane-formed bones of the skull are the nasal, incisive, palatine,zygomatic, and parietal form by day 32 in the 27-mm embryo

The branchial arches give rise to the cartilages of the jaw, the auditoryossicles, the hyoid apparatus, and the larynx The lower jaw, which is part of the

first branchial arch, is present as a rod-shaped mandibular cartilage (Meckel'scartilage) on each side by day 25 of gestation, when the embryo is between 13 and

16 mm in length The mandibular cartilages are joined rostrally, whereas caudallyeach lies within a middle ear cavity, where its articular cartilage is intended tobecome the malleus of the middle ear

Later developmental stages of the skull show that the sequence of primaryossification centers shifts from facial and calvarial centers to basicranial and oticcenters, followed by hyoid centers

Calvarial centers (skull roof)

The skull roof, or calvaria, is composed of bones that develop in membrane

as paired frontal and parietal centers Each bone shows a central trabecular networkthat spreads to cover the brain There is an unpaired interparietal bone lying moresuperficially (overlapping the edges of the parietal and supraoccipital bones),which fuses with the supraoccipital bone on day 45 of gestation and only rarelymaintains its separate identity

Figure 2.3 Dorsal view of the skull in a 45-day Beagle fetus The fontanel

between the frontal and parietal bones will close before birth (Evans, 2012)

Basicranial centers (skull floor, walls and sense capsules)

The chondrocranium ossifies to form the bones of the floor, walls, and sensecapsules of the braincase The process is gradual, and involves endochondral locithat spread to predetermined borders of homogeneous cartilaginous anlagen Thusthe basicranial axis is formed by a central ossification for the basioccipital bone, asimilar one for the basisphenoid bone, and paired ossifications for the presphenoidand ethmoid bones These median elements fuse with their lateral component wings

to form a sphenoidal complex The pre-sphenoid fuses with the orbitosphenoids oneach side to form a presphenoid with orbital wings, whereas the basisphenoid fuseswith the alisphenoids on each side to form a basisphenoid with temporal wings.The body of the basisphenoid first appears as diffuse ossifications on the midline.The temporal wings are the earliest portion of the sphenoid complex to ossify Theyform as a single plaque in each ala temporalis of the chondrocranium, and as growthproceeds they fuse with the median basisphenoid

The sequence of sphenoidal ossification centers is temporal wing at 35 days(41 mm C-R), body of basisphenoid at 40 days (59 mm C-R), preoptic root oforbital wing at 42 days (71 mm C-R), and metoptic root and body of presphenoid

at 43 days (76 mm C-R) The presphenoid and their orbital wings fuse when thefetus is approximately 108 mm C-R or at 50 days of gestation The pituitary orhypophysis rests in the sella turcica, formed by the basisphenoid with the help oflate ossifications called clinoid processes On the rostral end of this complex thelateral ethmoids fuse to form the sphenethmoids below the cribriform plate, and themesethmoid forms a median perpendicular plate

2.3.2 Canine fetal kidney development

The kidney components consist of tubular units and nephrons, whichfunction by selective filtration, reabsorption and eventually excretion of wasteproducts There is a formation of the pronephros, mesonephros and metanephros,that develop from cranial to caudal along the urogenital ridge of intermediatemesoderm in the urinary system development approach In the developing process,the pronephric and mesonephric tubules atrophy and the metanephros persists as thedefinitive functioning kidney

Pronephros

In the early developmental period, somites are present, intermediatemesoderm cells in the cervical region separate into outer parietal layer and inner

Figure 2.4 Dorsal view of the skull of a 50-day, 105-mm Beagle fetus, calvaria

removed Note that the incus and fourth superior premolar are ossified (Evans,2012)

visceral layer, forming nephrocoele between the two layers Cords of cells at eachsomite referred to as nephrotomes, which grow out from the dorsal wall of theintermediate mesoderm, later form pronephric tubules A duct which is a pronephricduct simultaneously forms from the mesothelium of the somatopleura and growscaudally on the edge of the intermediate mesoderm to enter metenteron (hindgut-cloaca)

Mesonephros

Subsequently, 70 to 80 pairs of mesonephric tubules form a renal corpuscle

on one end with a glomerulus that develops from branches of the aorta Venousdrainage is provided by branches of the various cardinal veins The other end ofthese embryonic nephrons attaches to the pronephric duct, which changes its name

to the mesonephric duct As these mesonephric nephrons develop, the pronephrictubule and the adjacent pronephric duct degenerate The functional period of themesonephros is brief and degeneration from cranial to caudal commences asformation of the metanephric kidney is initiated

Metanephros

Figure 2.5 Dorsal views of the developing pronephros, mesonephros and

metanephros (McGeady, 2017)

The metanephros is formed from two primordial structures: the ureteric bud,which is an outgrowth of the mesonephric duct, and the metanephric blastemalocated in the sacral region, which forms from the caudal end of the nephric ridge.Development of the metanephros begins with an outgrowth from the mesonephricduct close to its entrance into the urogenital sinus The ureteric bud grows dorsallyinto the intermediate mesoderm, the dilated portion of the ureteric bud gives rise tothe pelvis and further branching forms papillary ducts and collecting tubules Each

of the collecting tubules induces the adjacent intermediate mesodermal cells to form

a cluster of cells that will develop into a nephron Each nephron is associated with aglomerulus developed from branches of the aorta that supply the metanephricintermediate mesoderm The metanephric duct that originated as the uteric buds

become ureter, which enters the portion of the urogenital sinus that gives rise to thebladder.

The canine fetal kidney is first visualized ultrasonographically in the fetalabdomen between days 40-46 of gestation (England, 1990) Histologically, it isdistinguishable between renal cortex and medulla (Figure 2.10) Initially, theyappear hypoechoic with a dilated, anechoic renal pelvis (Yeager, 1992) As thegestation progresses, the renal cortex and medulla differentiate and the less dilatedpelvis

Recently, with high-resolution ultrasound imaging provided better assessmentallowing the identification of detailed changes in the appearances of fetal kidney

over period of gestational age According to Gil (2018), the study has performed

fetal nephrogenesis monitoring and all the fetuses had the same ultrasonographiccharacteristics of kidney development There are 4 developmental periods that weredefined according to the gestational age.

Period 1 (20-24 dbp): Fetal kidney with thickened and hyperechoic cortex, nocorticomedullary definition, renal pelvis is dilated with a "mushroom" shape andfilled with anechoic contents (Figure 2.6A)

Period 2 (16-20 dbp): Fetal kidney with thin cortex tending to hypoechoic, part ofmedulla - hypoechoic, early corticomedullary definition and renal pelvis is dilatedwith a tubular formation filled with anechoic contents (Figure 2.6B)

Period 3: (11-15 dbp): Fetal kidney with thin hypoechoic cortex, presence ofcorticomedullary definition and renal pelvis is slightly dilated in the shape of acanaliculus filled with anechoic content (Figure 2.6C)

Perid 4 (1-5 dbp): Fetal kidney with similar appearance to the adult organ: thinhypo/isoechoic cortex with corticomedullary definition and without dilation of therenal pelvis (Figure 2.6D)

2.4 Parameters used for parturition day prediction

In dogs, identification of the onset of diestrus through daily vaginal cytologyafter mating has been used to predict the parturition Parturition occurs 65 ±1 daysafter LH peak and 63 ±1 days after ovulation (Concannon, 1983) However, bothdetection of LH peak and the ultrasonographic identification of ovulation may bedifficult due to daily determination requirement Therefore, LH peak and ovulationcan be indirectly determined by progesterone rise Progesterone concentrationdecreases below 2 ng/ml approximately 24 hours before parturition and it can beindirectly recognized by the drop in the body temperature (Geiser et al., 2013).When mating or ovulation history are not available, the ultrasonographicmeasurement of embryonic and fetal parameters is fundamental in order to stagegestational age and estimate the delivery day The most studied embryonicparameters include inner chorionic cavity (ICC), outer uterine diameter (OUD) andplacental thickness Fetal parameters include crown-rump length (CRL), body

diameter (BD), biparietal diameter (BPD), deep portion of telencephalic vesicle (DPTV) and kidney length (KL) Data obtained by measuringthese structures have been used to calculate formulas for the determination ofgestational age or the number of days before parturition (dbp), which can be used inpractice to stage pregnancy when ovulation is unknown

diencephalo-2.4.1 Maternal parameters

Vaginal cytology

In the bitch, vaginal cytology is an important indirect method of monitoringhormonal events, in which exfoliative vaginal cells collection and study maydetermine the stage of the estrous cycle It can only be useful for indicating the firstday of diestrus – D1, after which gestation will last 57 ±3 days (De Cramer andNothling, 2018) Vaginal cytology could be an interesting method for delivery timeprediction in helping to identify D1 because it is cheap, rapid and reliable

Progesterone assay

Serum progesterone (P4) is an essential parameter for the detection of the LHpeak, ovulation and parturition time Rise of serum P4 begins at the onset of theluteal phase, and the first day P4 is >= 1.5 ng/ml is considered to indicate the LHpeak (Kutzler et al., 2003) On the ovulation day, 48-60 hours after LH peak, P4concentration reach values between 4-10 ng/mL (Beccaglia et al., 2016) Pregnancylength is reported to be 63 ±1 days after ovulation Kutzler et al (2003) found thatthe accuracy of delivery timing using pre-breeding P4 concentration is described to

be 67, 90 and 100% within 65 ±1, ±2 and ±3 days, respectively, and this accuracy isnot affected by litter size or bitches weight

In the prepartum, a decrease in serum P4 concentration occurs, reaching a valuelower than 2 ng/ml in the last 24 hours before parturition (Concannon, 2011)resulting from the PGF2a release and the consequent luteolysis that begins 36 hoursbefore parturition

Rectal temperature

Body temperature is related to circadian rhythm and serum P4 concentrationduring the prepartum period in the bitch (De Cramer and Nothling, 2018)

Progesterone is likely to be a thermogenic hormone, and its sudden decrease due toprepartum luteolysis could determine a rectal temperature drop greater than thecircadian rhythm variations To detect a significant prepartum variation in rectaltemperature, a temperature baseline should be defined, and for this reason it should

be measured at least twice a day during the last 14 days of pregnancy or from day

55 after mating Temperature declines by nearly 1◦C in the last 12–24 hours ofpregnancy, following the abrupt decrease of P4 In the last 6-18 hours beforeparturition, temperature could decrease from 1.1 to 1.7◦C below the baseline(Johnson, 2008) In small-size breeds, body temperature could reach lower values(as low as 35◦C) than in large-size breeds (as low as 37◦C)

18 cm deep into the vagina of the bitch during the last 3–8 days of pregnancy, orfrom day 56–61 after the first mating (Geiser et al., 2013) Vaginal temperatureshould be assessed twice a day with a thermometer (Geiser et al., 2013) Atemperature drop greater than or equal to 0.4◦C within 24 hours indicates thatparturition will start within 48 hours with 69% sensitivity and 88% specificity(Geiser et al., 2013) It should not be used alone for the prediction of parturitiondate or planning a C-section

2.4.2 Embryonic and fetal parameters

Appearance of embryonic/fetal structure using ultrasonographic examination

Canine gestational age can be evaluated by the specific embryonic and fetalstructures using ultrasonography or radiography (Lopate, 2008) Initially, theanechogenic gestational sac can be detected on day 18 after ovulation On day 23,the embryo with heartbeat is detected as an oblong structure in the chorionic cavity

on day 23 Between days 27 and 31 after ovulation, the embryo is bipolar in shapeand limb buds can be visualized While anechogenic diencephalon-telencephalicvesicle (DPTV) in the fetal head which represents the thalamus and basal nucleiprimordia can be detected between day 29 and 33.

On day 29 - 33 and 31 - 35, respectively, the stomach and urinary bladder arefirst abdominal organs detected The skeleton is a hyperechoic structure on day 29 -

33 and fetal movements are later detected on day 32 - 34 The abdomen and thoraxare distinct between day 34 - 36, the lung is hyperechoic in comparison withhypoechoic liver, compared with the rest of the abdomen on day 35 - 38 Thekidneys are first visualized on day 41- 43 (Beccaglia and Luvoni, 2004) The bowel

is referred to as the last organ detected by ultrasonography on day 57 - 63 days ofpregnancy (Gil et al., 2015) The identification of peristaltic contractions indicatesthe end of organogenesis However, researchers do not agree to use these features asthe sole parameters in indicating the fetus is full term

Inner chorionic cavity (ICC)

During early pregnancy (>25 days before parturition), the most suitableparameter is the measurement of the inner diameters of the chorionic cavity (Luvoniand Grioni, 2000) From its first detection at 45 days up to approximately 25 daysbefore parturition, this anechoic structure with clearly defined margins remainsspherical in outline and easy to measure (Luvoni and Grioni, 2000) Themeasurement of the ICC is determined by taking the mean of two ICC diametersmade at 90o angles from one side of the trophoblastic decidual reaction to the other

By the application of ICC equations, Luvoni and Grioni (2000) found that theaccuracy rate when predicting the delivery day (±1 day) was approximately 77% insmall and medium size bitches, while within ±2 days was 88% and 85%respectively (Beccaglia and Luvoni, 2006)

Outer uterine diameter (OUD)

Outer uterine diameter (OUD) is measured from the outer points of theimplantation site considering two orthogonal diameters of the gestational vesicle,which should be scanned when it appears as a round shape and reaching its larger

diameter (Michel et al., 2011) OUD leads to inaccurate parturition timing, because

of its not well defined measurement markers (Luvoni and Grioni, 2000) Themeasurement of ICC is done using the same scanning planes but measuring thedistance between the inner sides of the chorionic wall from trophoblastic decidualreaction sites (Vieira et al., 2020)

Placental thickness (PT)

The reliability of the placental ultrasonographic measurements for prediction

of parturition day is controversial (Son et al., 2001; Maldonado et al., 2012) Zonaryplacental length, measured as the distance between the two extremities of the zonaryplacenta in a longitudinal scan is not significantly correlated with gestational age insmall-sized dogs (Son et al., 2001), while placental thickness is positively correlatedwith gestational age, regardless of breed and body weight (Maldonado et al., 2012)

Table 2.2 Equations for prediction of gestational age (days after LH peak) or

days before parturition (dbp) in the bitch by ultrasonographic measurement ofextra-fetal structures

The CRL cannot be measured until delivery because after 45 days of gestation,flexion of the foetus both laterally and dorso-ventrally hinder its measurement(England et al., 1990)

Body diameter (BD)

In the second half of pregnancy, BD is measured by taking the two diametersmade at 90o angles in the transverse plane at the level of fetal liver and stomach(Yeager et al., 1992) This measurement is related to gestational age and thecombined regression formula with biparietal diameter (BPD) has been used toconstruct a prediction table (England et al., 1990) BPD may be measured from day

26 after LH peak, with its best accuracy being on day 30 of pregnancy (Kutzler etal., 2003) When two or more parameters are used to predict the same end point, theaccuracy of the prediction increases

Biparietal diameter (BPD)

The BPD is the most suitable measurement to predict the day of parturitionduring late pregnancy and it is taken on the same longitudinal section of CRL Atthis stage of pregnancy, fetal head is usually easily identified, but it can be difficult

to image it on a longitudinal scan, which is necessary to standardize measurements

In fact, the parietal bones of the skull have to be parallel in order to measure thecorrect distance between them The measurement of this structure is highlycorrelated with gestational age and the technical approach is relatively simpler thanwith other parameters Applying the BP equations previously obtained (Luvoni andGrioni 2000, Table 2.3), the accuracy of prediction of delivery day (±1 day) was75% and 63% in small and medium size bitches, while within ±2 days, it was 88%and 81% respectively (Beccaglia and Luvoni, 2006)

Crown-rump length

Days after LH peak =

24.64 + 4.54 x cm - 0.24

Beagles Yeager et al 1992

Table 2.3 Equations for prediction of gestational age (days after LH peak) or

days before parturition (dbp) in the bitch by ultrasonographic measurement offetal structures

2000dbp = (mm − 29.18)/0.7 Medium size

dbp = (mm − 30)/0.8 Large size Alonge et al., 2016

sizes Furthermore, its accuracy is higher in normal (two to six puppies in and five to nine puppies in medium- and large-size bitches) and large litter sizesthan in small ones (62.3%, 73.7% and 47.6% within ± 2 days, respectively), and itseems to be unaffected by fetal sex ratio (Beccaglia et al., 2008) In Table 2, anoverview of formulas of BP, BD, CRL, DPTV and the time at which they werecalculated is reported.

small-Kidney length

Recently, Gil et al (2018) studied canine fetal renal length in predictingdelivery time, and it was reported to be strongly and positively correlated withgestational age The proposed formula can be applied from 24 to 1 dbp, and the bestinterval for monitoring kidney length is between 15 and 11 dbp, at which time theaccuracy, ensitivity and specificity are 99.98%, 99.95% and 99.92% within ±1 day,respectively

2.5 Ultrasonography and use in canine pregnancy diagnosis

The use of ultrasound (US) has become a popular diagnostic method due tosmaller size, high level of autonomy, high image quality and accessible price.Ultrasound is a non-invasive diagnostic tool based on the principle of pulse andecho to image the inside of the body It is portable, has no radiation risks, and isrelatively inexpensive when compared to other imaging modalities, such asmagnetic resonance and computed tomography

2.5.1 Principle

Diagnostic ultrasound transducers (probes) produce sound waves of muchhigher frequency (above the threshold of human hearing frequency - 20KHz) in themegahertz (MHz) range The probe contains piezoelectric crystals, which can vary

in frequency and crystal configuration In general, the higher the resonantfrequency, the greater the resolution, but the lower the penetration of the soundbeam Various types of probes such as sector, linear and curved scanners have beenused for diagnostic purposes An sector scanner produces a fan-shaped beam andrequires a small contact surface, which is used for cardiac imaging while a linear

scanner produces a rectangular beam and a large contact area is needed Curvedlinear scanners have smaller scan heads and wider field of view.

Ultrasonic transducers contain many piezoelectric crystals and vibrate inresponse to an electric current Piezoelectric crystals can convert mechanicalpressure into voltage across their surface A high-frequency sound pulse(ultrasound) is transmitted into the body This pulse travels through the body until itreaches the reflecting surface, after which part of the ultrasonic pulse (echo) isreflected back to the source of the pulse Piezoelectric crystals, which generatesound in response to electronic stimulation and generate electronic signals inresponse to acoustic stimulation, are two-way paths between the computer and thepatient A computer tracks the elapsed time from the start of the pulse to themoment the echo is received, allowing the position of the reflecting surface to bedetermined in two-dimensional space viewable on a video monitor (Qiu et al.,2015) The amount of ultrasound pulse reflected determines the brightness of thespot produced in the two-dimensional image When the ultrasound beam encounterstissues or objects that have very different acoustic properties from general softtissues (eg: bone, air, metal), almost complete reflection will occur

Echoes reflected from a body part under test can also be displayed along atime-directed motion graph This display is called M-mode and is used most often

in cardiology to quantitatively display the size of the heart valves, heart chambers,heart wall and great vessels, as well as the movement of the ventricular walls, heartvalve and main ships Mode B is a similar technique, but the echo is displayed aspoints of different grayscale brightness corresponding to the magnitude (amplitude)

of each signal The other mode is mode A which is outdated due to limitedinformation transmission

2.5.2 Technique

Ultrasound image quality is determined by 3 factors: patient preparation, thetranducer selected and the gain settings on the machine The patient hair should beclipped over the area of concern Hair will trap air and this interferes with soundtransmission After clipping or moistening the hair, acoustic gel is used to ensure

good contact and sound transmission from the transducer to the animals (Sippel etal., 2011) The probe should be selected based on the density of the region to beexamined and, if possible, the probe focal area should match the depth of the region

of interest Decreasing transducer frequency correlates with increased depth ofultrasonographic penetration with an accompanying loss of resolution If the signal

is not strong enough (e.g the image is too black), the gain setting should beincreased or a lower frequency transducer selected If the signal is too bright, thegain should be decreased or a higher-frequency transducer selected The use of ahigh-frequency transducer at high gain settings to compensate for lack ofultrasonographic penetration produces artifacts that may result in incorrectinterpretation

2.5.3 Use of ultrasonography in veterinary practice

Ultrasound is commonly used to examine uterine anatomy, involution andpathology, detect pregnancy, monitor fetal growth, and determine fetal sex.Ultrasound provides information about the size, shape, and location of the structure

as well as information about the soft tissue structure of the structure or organ beingexamined (Abd El-Aty and Medan, 2010) It is best for distinguishing solidstructures from cavity (liquid-filled) structures and providing internal details notshown by radiographic imaging (Thrawat et al., 2012) Ultrasound was also used toassess postpartum uterine growth and ovarian function Ultrasound plays a hugerole in the clear diagnosis of digestive disorders in dogs The entire thickness of thestomach or intestinal wall can be visualized and measured, as well as adjacentstructures such as lymph nodes May also be evaluated stomach and intestinalmotility, observing the peristaltic movements in real time

In patients with pleural effusion, mediastinal mass, or cardiac disease,ultrasound provides information that may not be apparent on radiographs Althoughultrasound is not as useful for examining the skeleton or skull as surveyradiographs, some information can be obtained from ultrasound evaluation ofmuscles, tendons, and joints, as well as orbital and brain examination (in animals

with open fontanelles) (Qiu et al., 2015) In small animal reproduction, Dopplerultrasound has been used to diagnose and monitor pregnancy since 1970, usingDoppler ultrasound, can perform an accurate assessment of maternal and fetal bloodflow patterns from the vessels and chambers of the fetal and maternal heart.

2.5.4 Application in pregnancy diagnosis

Ultrasonographic imaging in canine has been used for monitoring fetaldevelopment, predicting gestational age and parturition time, diagnosis andmanagement of reproductive tract diseases, and breeding soundnessexaminations It has been also employed for the study of blood flow velocity andresistance indices in fetal arteries like uteroplacental and umbilical arterieswhich can be helpful in determining abnormal pregnancies and determining thewhelping time

In practice, ultrasonography is a most preferred modality for pregnancy diagnosis,assessment of fetal age, litter size and livability of foetus in canines and alsoaliments of pregnancy The litter size can be estimated using ultrasound, however,the number of fetuses can vary due to embryo/fetal abortion during pregnancy inhealthy, unaffected female dogs, loss of external embryos The first ultrasound ofthe fetal organs may be even more useful for estimating gestational age andpredicting delivery For example, the kidneys are only visible within the last 20days of gestation when imaged using a 5.0 MHz transducer (Yeager et al., 1992).Based on the ultrasound shape of the fetal external genitalia in utero in relation to afetal position, it has been reported that can determine sex between 55 and 58 days ofgestation (Gil et al., 2015) The ultrasonographic shape of an embryo or fetus canoften indicate their gestational age accurately, to allow the prediction of deliverytime with an accuracy of +/- 2 days (Son et al., 2001; Kutzler et al., 2003; Luvoniand Beccaglia, 2006)

In addition, aliments were also detected during pregnancy in female dogs.Embryonic ultrasound aspects of resorption are generally associated with decreasedvolume of the concept, increased echogenicity of embryonic fluid, absence ofembryonic heartbeat, disintegration of the embryo mass and finally, the collapse of

the concept with internal bulging of the uterine wall Fetal heart rate can bemeasured with mode B or M ultrasound, and it is widely suggested that a drop infetal heart rate to less than twice the maternal heart rate is a sign of fetal distress orhypoxia Color doppler ultrasound can be used to visualize and estimate patency inthe ovary

2.6 Recent related articles review

In last decade, there were studies have aimed to compare accuracy not onlyamong maternal, embronic and fetal parameters at different stages but also differentequations in each parameter for gestational age determination and parturition dayprediction Besides, there were some studies have investigated new parameters fordelivery time prediction in bitches This represents that an accurate parameterswhich is most correlated with gestational age should be figured out to help breeders

or veterinarians to assist the whelping bitches to reduce the peripartum losses ofoffspring and reduce risk of premature C-section

Beccaglia et al (2012) aimed to investigate whether the accuracy ofparturition date prediction by ultrasound is affected by week of gestational age Byretrospective analysis, the result were obtained on the pregnancy period (from week

4 to week 9) in 495 ultrasonographic examination of small and medium sizepregnant biches and 60 of pregnant queens Based on biparietal (BP) measurement,accuracy (±1 day) was similar at week 5 and 6 of pregnancy (78.6% vs 78.9%, p >0.05), whereas a significant decrease (p < 0.05) was observed at week 7 and 8compared to week 6 There was no difference in the accuracy of the prediction at ±2days based on BP was observed from week 5 to 8 of gestation (range from 82.3% to95.2%) Close to whelping day (week 9), the accuracies of the prediction based on

BP were significantly lower than those obtained at week 5 (±1 day: 50.9% vs78.6%, p < 0.05; ±2 days: 69.8% vs 95.2%, p < 0.05) In conclusion, the accuracy

of the prediction in bitches and queens is influenced by the gestational period whenultrasonographic measurements are performed, particularly for BP measurement