paper no. : 09 animal cell biotechnology module :18

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Animal Cell biotechnology

Pregnancy Diagnosis in Animals

Paper No. : 09 Animal Cell Biotechnology

Module :18 Pregnancy Diagnosis in Animals

Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director

The Energy and Resouurces Institute (TERI), New Delhi

Paper Coordinator: Dr. Minakshi, Professor & Head, Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

Content Writer: Dr. Hari Mohan, Assistant Professor, Maharshi Dayanand University, Rohtak

Paper Reviewer: Dr. Minakshi, Professor & Head, LalaLajpatRai University of Veterinary & Animal Sciences, Hisar

Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry

JamiaHamdard University, New Delhi

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Description of Module

Subject Name Biotechnology

Paper Name Animal Cell Biotechnology

Module Name/Title Pregnancy Diagnosis in Animals

Module Id 18

Pre-requisites Basic knowledge of female reproductive tract anatomy and physiology

Objectives 1. To understand the importance of pregnancy diagnosis in Animals

2. To know the events happening during pregnancy

3. Methods of pregnancy diagnosis

4. Future prospects of pregnancy diagnosis

Keywords Pregnancy diagnosis (PD), Corpus luteum, implantation, biomarkers, progesterone,

estrogen, ultrasonography

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Table of Content:

1.) Introduction

2 Events during pregnancy

3.) Methods of Pregnancy detection

3.1) By visualisation or external appearance

3.1.1) Visual confirmation

3.1.2) Condition of Vulva

3.1.3) Other Signs

3.2) Examination by per-rectal palpation of the reproductive tract

3.3) Examination by ultrasonography and radiology

3.4) Biological fluid like Milk and Blood based testing

3.4.1) Progestrone

3.4.2) Estrone sulphate

3.4.3) Pregnancy associated glycoproteins (PAG)

3.4.4) Equine chorionic gonadotropin

3.4.5) Early Conception Factor (ECF)

3.4.6) Interferon Tau (IFN-τ) and genes stimulated by IFN-τ

3.4.7) Relaxin

3.4.8) PCR based assay

4) Pregnancy diagnosis in small animals

5) Summary: The Challenges and future for Early Pregnancy Diagnosis

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1. Introduction:

Diagnosis of pregnancy, more desirably as earliest as possible, is essential need to increase

profitability in animal husbandry and veterinary related economy. It is always desirable by a

farmer or the herd manger to identify animal which is pregnant as soon as possible so that he

can manage both open (non-pregnant) and pregnant animal judicially to maximise profit from

its animals. For dairy farm, it is essential to get animal pregnant just after involution period,

i.e., 30-50 day of calf birth, to get maximum potential milk output from farm. There is

potential loss of Rs 4000-8000 per animal if we loses one estrous cycle (18-21 days) time in

conceiving the cow/ buffaloes in from potential loss of milk yield (If consider cattle give 8-10

L milk/day and sold @25-40rs/L) for same period, and also same amount of delay in getting

new calf. Thus, detection of the open animals at earliest possible, preferably before the onset

of next estrus (before day 21 post-insemination) and re-breeding of those animals can

significantly increase profitability from dairy farming. Similarly, in animal related with meat

production, getting new crop/ child from concerned species as earliest interval as possible

will lead more meat production by increasing the number of animal in farms, thus more

profitability to farm owner. Early pregnancy diagnosis is used to know which animal is non-

pregnant as soon as possible so that animal can re-bred to achieve pregnancy as earliest as

possible. In the same way, for pet owner like dog and cat, early pregnancy diagnosis will help

to avoid unwanted pregnancy from stray animals and also in better pre-parturition care. In

advance confirmation of pregnancy is desirable to understand progression of pregnancy so

that animal owner can take necessary action for welfare of animals. Apart from this,

diagnosis of pregnancy and its progression have following other benefits:

Timely culling of open cows (non-productive) to reduces the cost of feeding and

managing such cattle.

Aid in the culling decision for cows with other issues

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Breeding management- Grouping cows for feeding, calving, calving watches, other

management based on their stage of pregnancy at pregnancy diagnosis.

Preparing in advance for calving when term is near.

Avoiding complication during pregnancy by regular examination, by examining the

progression of pregnancy

Therefore, pregnancy diagnosis would help to evaluate the therapies at an early date and

devise alternative manipulations as per requirement to increase the potential output from

animals. Thus, pregnancy diagnosis is a widely practiced procedure to increase the

profitability of farm and has come to be accepted as a highly recommended management

technique in animal husbandry.

2. Events during pregnancy:

To understand the methods of pregnancy diagnosis at different phases of gestation,

understanding of events of pregnancy is prerequisite, more importantly those events which

are used or exploited to diagnose the pregnancy. So, here we will discuss first those events

which are used or explored at different time interval to know the status of pregnancy in all

animals in general and in cattle particularly.

The pregnancy starts with fertilization of ovum by sperm to form zygote in uterine tract. The

event of ovum release happens under influence of estrogen hormone and then after pregnancy

is maintained under influence of progesterone hormone. Progesterone is produced by corpus

luteum (CL) of ovary in most of species and in some species later on by placenta. Any

decrease of progesterone level during gestation period will lead to termination of pregnancy,

a higher level of progesterone is pre-requisite to carry pregnancy.The developing embryo

enters the uterus between day 2 and 5 after ovulation (depending on the species) and secrete

various chemicals which helps the mother to recognise success of pregnancy. This process is

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called as maternal recognition of pregnancy and occurs between 12-17days after ovulation in

species like sow, cattle, ewe and goat. Signals in form of pituitary luteotrophin (LH),

Pregnancy-Associated Glycoprotein (PAG), interferon Tau (IFN-τ) and genes stimulated by

IFN-τ viz. ubiquitin- 68 like modifier (ISG15) or ubiquitin cross-reactive protein (UCRP), 2',

5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance genes (MX1 and MX2) are

secreted by developing embryo to prevent lysis of CL by inhibiting release of PGF2 alpha so

that progesterone concentration remains high and to provide environment from mother for

nourishment and safety. This leads to cessation of estrum. The fertilised zygote further

continues to divide and attach to mother uterine tract, by forming placentome. This

attachment starts in the sow at about 11-20 days, in the ewe about 16-20 days, in the cow

about 30-35 days, and in the mare about 50-60 days. During this period development of

placenta start and depending upon type of species the type of placenta differs. The embryo

continues to develop and organogenesis starts. The development of organ and size of embryo

progresses with a fix pattern which can be recognised with the help of ultrasound. By using

Ultrasound the heart beat can be detected at 16th day in sow and 22nd day in cow at the

earliest. The position of embryo also varies within uterus and thus in cattle, will also help to

know the status of pregnancy.

Apart from these changes during pregnancy mother also changes its physical appearance, like

enlargement of abdomen size, fat deposition, mammary gland development and search of

isolation at end of term are key changes which is typical in all species during advance

pregnancy. Success of a useful pregnancy diagnosis method depends on how early and

accurately it can diagnose the pregnancy in animal and differentially diagnose with

conditions like pyometra, tumors, mummification of foetus, metritis and fetal maceration etc.

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3. Methods of Pregnancy detection:

Foetus is the only positive sign of pregnancy, so detecting signals exclusively produced by

foetus is the best way to confirm the pregnancy.The pregnancy diagnosis methods can be

divided in four major heads as per methodology involved:

1. By visualisation or external appearance

2. Examination by rectal palpation of the reproductive tract

3. Examination by trans-rectal real-time, linear array ultrasonography

4. Biological fluid like Milk and Blood based testing

3.1) By visualisation or external appearance

3.1.1) Visual confirmation: Non-return of oestrus is indirect method to confirm the

pregnancy. Observation of estrous in next cycle is one indication of non-pregnancy.

Generally, animal shows sign of estrous during normal estrous cycle when they did-not

conceived. Non-return of estrus is one sign of pregnancy but is generally misleading as

sometime herd-man can miss sign of estrous in animal or sign itself is weak (Silent heat

condition). Installation of CCTV and Pedometer in organised herd can minimise such misses,

but there are also disease condition like anoestrus either pathogenic or normal in which also

animal does not show sign of estrous. Similarly many animals particularly cows with history

of cystic ovaries shows estrous sign even though they are conceived. The condition further

aggravates due to silent heat symptom in buffaloes leading to increase in open period in these

animals.

3.1.2) Condition of Vulva: In pregnant animals, vulva becomes anaemic and wrinkled;

where as in non-pregnant animal it is hyperaemic, smooth and edematous.

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3.1.3) Other Signs: The size of abdomen will increase in animal under advance pregnancy,

but is not a reliable indicator in cattle. Multiparous animals can show it more reliably.

Movement of foetus in 10% of pregnant animals can be seen in abdominal area after 6 month

while become apparent in 80% cases after 8 months.

In heifers of first conception, the mammary gland size increases after 4-5months of

pregnancy in cattle. Apart from these, following signs can be seen in animals in advanced

pregnancy:

1. Tendency to fatten.

2. Gradual drop in milk yield

3. Gradual increase in weight.

4. Drooping quarters.

5. Increase in size of udder.

6. Waxy-appearance of teats in last month of pregnancy.

All these signs lack accuracy and often misleading as these signs are also present in other

cases also. Further these signs appear only in advance stage of pregnancy. So, there is need to

confirm early stage of pregnancy by using more sensitive methods.

3.2) Examination by per-rectal palpation of the reproductive tract

Rectal palpation of uterus, horn, ovaries and uterine vessel is most commonly used method in

pregnancy diagnosis of cattle, buffaloes and horse currently. By this method pregnancy can

be diagnosed as early as 35 days and more accurately at 45-55 days of gestation in cattle.

However, this method can lead abortion in upto 10% of pregnant animals when applied

before or at 35-41 days of gestation. Further, if proper precaution is not taken, it may lead to

a damage of mucosa layer and rectum wall of the animal causing infection and other

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complications. Also, these techniques required specialised veterinarian to do pregnancy

diagnosis as earliest.

When hand is inserted in rectum with all precautions, we can hold the whole uterine tract of

female in large animal by hand to study status of tonicity of uterus, gravidity or enlargement

of horns, which happen due to fluid accumulation after implantation, confirm presence or

absent of corpus-luteum, and to feel the foetus in later stage of gestation. Generally, during

the early diagnosis uterine tract is slightly tonic in nature. Signs of pregnancy observed

during rectal palpation are enlargement of uterine horn with placental fluid (can be seen from

32-90 days), slipping of foetal membrane where the horn is handed properly followed by

gentle pinching leads to feeling of two membranes slipping first alllanto-chorion followed by

uterine wall of horn. Palpation of placentome can be done from 60–70 days onwards and

these placentomes are generally located in the middle of the horn or near the attachment of

middle uterine. From 80 days onward palpation of uterine artery will give a feeling of

pulsation flow of blood with whirr-sound or we can feel some high amount of liquid is

flowing through vessel. However, this technique can be confused with other disease condition

like persistence CL which can occur in anestrum condition. Similarly, large horn can be seen

in some infectious conditions like pyometra or mucometra. However, the technique of

slipping membrane is very useful to differentiate these fluid filled infections from pregnancy.

3.3) Examination by ultrasonography and radiology:

Examination of pregnancy by ultrasonography is safer than per-rectal examination but, it is

more costly and need specialised veterinarian for performing the test. Further, one of the clear

pitfalls when using real-time ultrasound, involves improper timing for diagnosis as one

potential cycle of oestrus is already lost if, animal is open this is because PD by USG

generally done after 25day in animals.

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Real-time, B-mode ultrasonography can detect pregnancy earlier than transrectal palpation in

cattle and buffaloes and useful in those animals (dog, cat, pig, rabbit etc.) where we cannot go

for per- rectal examination.Technique is more accurate in assessment of ovarian structures,

diagnosis of twins, and most importantly it also allows for monitoring of foetal viability by

detection of foetal heartbeat. False positives are rare in this method. Apart from assessing the

foetal viability, its numbers and age of foetus, real time ultrasonography can also be helpful

in diagnosing disease of reproductive tract.

Real-time B-mode (brightness mode) ultrasonography displays a 2-dimensional image in gray

scale. The image is composed of dots that vary from white to light gray for very dense tissues

such as the uterus and skin, and from dark gray to black, for fluids and less dense tissues. For

pregnancy diagnosis, decisions are based on the appearance of fluid vesicles (black) within

the surrounding uterine tissue. Ultrasonography is performed trans-rectal in larger animals

like cow and buffaloes while in smaller animals like dog, cat, sow etc. is performed by trans-

abdominal positioning. Ultrasound result quality depended upon transducers type which

generate ultrasonic wave due to piezoelectric crystal in them. Ultrasonic wave type is

characterized by the distance it travels (wavelength), and the number of times the wave

repeats within a second (frequency). Generally 3.5 to 5.0 MHz frequency range is frequently

used for routine pregnancy diagnosis. The size of the crystalin USG determines the wave,

larger crystals, such as the 3.5 MHz, produces the longer the wavelength and the lower the

frequency. They can penetrate deep into the soft tissues of the animal but provide lower

image resolution (ability to distinguish between different structures). In contrast, the smaller

crystals of the 5.0-7.5 MHz transducers produce signals that travel shorter distances, but

produce higher image resolution, since fewer waves are lost.

Trans-rectal ultrasonography is an accurate method for the diagnosis of pregnancy in cattle

and buffaloes. It is also used in sheep and doe (female goat). Here the probes of ultrasound is

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inserted trans-rectaly as done in trans-rectal palpation method. The probe is guided over

ovary and other reproductive structure with help of hand. This method can detect pregnancy

as early as day 26 post-breeding. During examination, if a discrete, intrauterine, sharply

demarcated anechoic, round or oval structure which is basically a fluid- filled allantoic cavity

will be detected in uterine lumen or if an apparent conceptus (anechoic, elongated structure)

or embryo proper were detected in the uterine lumen these indicate pregnancy. Post 25 days

we can see heart-beat of foetus also. Similarly in sheep and doe at day 21 to 42 it is possible

to diagnose the pregnancy by trans-rectal ultrasonography. In equines, ultrasound analysis of

pregnancy can be performed as early as 12 to 18 days after ovulation. This is beneficial for

the identification and management of twins, scheduling of rebreeding in open mares, and

early detection of problems associated with pregnancy. However final confirmation of

pregnancy can be done only after a follow-up examination between 25 and 35 days of

pregnancy.

3.4) Biological fluid like Milk and Blood based testing

Recent advancements in the field of cellular and molecular basis of early pregnancy have

paved the way for development of more specific and sensitive methods of early pregnancy

diagnosis. These techniques does not need highly qualified person to perform and can

indicate the pregnancy before onset of new estrous cycle time. More-over it doesn’t require

per-rectal examination, operation of which has its own drawback. This help farmer to

inseminate the animal which is open in just next cycle without missing any cycle, or take

corrective measure regarding any reproductive disorder if persist, so that no further loss of

estrous cycle to conceive the animals. These techniques are based on either ELISA (enzyme

linked immune sorbent assay) or PCR based. In ELISA, an antibody is raised against targeted

molecule which will be linked to enzyme. If that particular molecule is present in biological

fluid, it will bind with antibody and colour will develop due to conversion of substrate. The

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development of colour and its intensity indicates the presence of molecule and its quantity in

biological fluid for which test have developed. The same principal with slight modification is

applied for lateral flow development. In humans, such lateral flow based kits are available

commercially for diagnosis of pregnancy. These kits are based on detection of human

chorionic gonadotropin (hCG) (hormone produced by the placenta after implantation) in

urine. This hormone is not secreted by animals. In PCR based assays, generally blood

leucocytes are used to study of expression of such molecules which are expressed early

during maternal recognition of pregnancy. We will discuss more about such compounds

which are used or have potential to be used as early pregnancy diagnosis molecules.

3.4.1) Progestrone: Progesterone is a hormone produced by the corpus luteum (CL) and is

required for maintenance of pregnancy. This CL is maintained after pregnancy under

the influence of interferon-τ produced by the embryo. This lead to maintain level of

progesterone in blood after 16 days post conception, otherwise level of progesterone

start to decrease and become minimum level at day 21 post coitus. The concentration

of progesterone can be measured in milk or blood (serum/ plasma) by ELISA kit

commercially available. Milk is a better choice as it is easy to collect it. But

progesterone based early pregnancy diagnosis lacks accuracy due to false positive

result occur mainly in the conditions where CL persist for longer due to disease

condition like anestrum (Breed et al., 2009). Further, it is good for large farm as it

needs lab facility equipped with instruments such as ELISA reader & washer.

3.4.2) Estrone sulphate: It is a conjugated estrogen that is produced specifically by the

conceptus and it has been used to diagnose pregnancy using milk samples in cattle

(Heap and Hamon, 1979). Thus, it directly confirms the pregnancy. In cows, equine

and buffaloes its concentrations increase from day 60 and plateau around day 150

after insemination, and during this time it is good indicator of pregnancy. This can be

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detected by ELISA and some reports have suggested that it can also be performed by

using urine sample.

3.4.3) Pregnancy-associated glycoproteins (PAG): Bovine pregnancy-associated

glycoproteins (PAG) were discovered through attempts to develop indirect early

pregnancy tests in dairy cattle (Xie et al., 1997). In 1982, two proteins, pregnancy-

specific protein (PSP) A and B, were isolated from bovine fetal membrane extracts

(Butler et al., 1982; Gajewski et al., 2008). Whereas PSPA was identified as α-

fetoprotein, PSPB was found to be specific to placenta (Butler et al., 1982).

Development of a specific RIA for PSPB (Sasser et al., 1986) allowed for

quantification of PSPB in maternal serum as an indirect method for pregnancy

diagnosis and embryonic mortality in dairy cattle (Humblot et al., 1988a, b). PAG,

can be used to detect pregnancy on days 25 to 30 (Silva et al., 2009; Szenci et al.,

1998; Sousa et al., 2006), however, due to its long half life, PAG is present in the

body fluids even after calving (till day 90 post- calving), thus, leading to false-

positive results in cattle bred around day 60 post-calving.

3.4.4) Equine chorionic gonadotropin (eCG): eCG is produced from groups of

specialized placental cells called endometrial cups and is only present in pregnant

mares from approximately 35 to 120 days of gestation. So, it is used as confirmatory

test either by ELISA or lateral flow methods. However it also gives false positive

result if blood sample is collected from a mare that lost her pregnancy after

development of endometrial cup. Similarly, false negative results will appear if assay

is performed prior to day 35 or after day 120 of pregnancy. But, this is better

technique as it does not need expert veterinarian which is needed to perform

ultrasound or per-rectal examination. But on other-side, it can be done only after day

35 post-coitus.

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3.4.5) Early Conception Factor (ECF): Early pregnancy factor (EPF, also known as early

conception factor—ECF) is a protein, present in the sera of most mammalian species

including humans, mice, sheep, cows, buffaloes, pigs, mares and also in some wild

pregnant animals. They are, detectable within 6 to 24 hours of fertilization and can

continue upto up to two-thirds of the gestation. EPF remains the earliest serum

benchmark for positive fertilization and hence successful conception. This novel

pregnancy-specific protein has high immunosuppressive ability which is demonstrated

by rosette inhibition test, a bioassay first demonstrated in pregnant mice. However,

EPF bioassay is based on the inhibition of rosette formation which is a time

consuming process with limited use in routine pregnancy testing (Smart et al., 1982).

Further, EPF is not just confined to pregnancy but is also detected in many types of

tumours.

3.4.6) Interferon Tau (IFN-τ) and genes stimulated by IFN-τ: Interferon Tau (IFN-τ) and

genes stimulated by IFN-τ viz. ubiquitin- like modifier (ISG15) or ubiquitin cross-

reactive protein (UCRP), 2', 5'-oligoadenylate synthetase 1 (OAS1) and myxovirus

resistance genes (MX1 and MX2) are found to be associated with maternal

recognition of pregnancy in sheep, buffaloes and cattle. These factors are present in

serum of cattle and buffalo at days 15-17 post-breeding. Assay of these proteins, more

importantly of MX2 had shown better potential for early diagnosis of pregnancy in

heifer at least. But no commercial kit related to them is available in the market till

now.

3.4.7) Relaxin: Dog need special mention as bitches do not produce a pregnancy-specific

hormone similar to human chorionic gonadotropin. In addition, progesterone is not a

useful indicator of pregnancy because all bitches secrete progesterone for two months

after heat, regardless of whether they were pregnant or not. Relaxin assay by lateral

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flow or ELISA is specific for dog pregnancy diagnosis. It is performed by using blood

sample and is useful for pregnancy detection 25 days post gestation. However, this

test is slightly late as by abdominal ultrasonography we can detect it earlier. Further

false negatives can occur also as sperm survive in bitch uterus for 6-7days while false

positive occur in early abortion cases. Further the test does not evaluate foetus

viability or give litter size.

3.4.8) PCR based assay: Apart from ELISA based assay PCR based assay can also used

more in organised farm. Semi quantitative PCR or real-time PCR can be performed

for gene which are upregulated and expressed in blood during early pregnancy (Paré

et al., 2008). Those genes like interferon Tau (IFN-τ) and genes stimulated by IFN-τ

viz. ubiquitin- like modifier (ISG15) or ubiquitin cross-reactive protein (UCRP), 2',

5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance genes (MX1 and

MX2), PAG are also expressed in blood and its m-RNA expression can be used to run

semi quantitative PCR with reference to standard non-pregnant animal will confirm

early diagnosis of pregnancy from day 14 onward.

4) Pregnancy diagnosis in small animals:

For pregnancy diagnosing in sow, and other small animals including goat and sheep trans-

abdominal palpation is done. In these animals the surface of the transducer is lubricated and

applied to contact the abdomen just lateral to nipple line and ahead of the rear leg. PD in sow

is more accurate by this method when performed between 24 and 35 days following breeding,

because of the timing and amount of fluid accumulation, combined with the timing of foetal

bone formation and calcification, resolution and prognosis is very high. The accuracy of the

equipment is >90% for identifying pregnant females in this period with an average time to

make a diagnosis between at less than 10 seconds per sow. Optimal diagnosis is based on

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fluid accumulation, which begins at day 18 but remains low until about day 24. Thereafter,

between days 24 to 35, fluid volume peaks in early pregnancy.

In bitch, best time to palpatate the feto-placental units is between 25 and 30 days gestation

however palpation technique lacks sensitivity and specificity. Before this, they are not clearly

palpable and afterwards the placental units lose their individual spherical shape. Bitch after

proper restraining is positioned in dorsal recumbent orientation before subjected to

ultrasound. Optimally, a full bladder prior to the examination serves as a useful landmark and

may facilitate examination of structures distal to the bladder. The normal uterus is best

located by scanning transversely between the urinary bladder and the colon. The cervix and

uterine body are seen as a continuous hypoechoic round structure dorsal to the anechoic

urinary bladder and ventral to hyperechoic, crescent shaped colon. The fetous heartbeat (@

>180-200/minute) is regularly seen even without doppler after 25-28 days gestation. At 30-32

days of pregnancy, the vesicle is larger than transverse small bowel, making differentiation

easy. Further at same time counting the number of vesicles (of foetuses) is very easy as they

are small enough to allow easy counting within each uterine horn. Later on (>45-50 days),

due to overlap of large intestine with the uterine horns correct count of growing fetus

becomes difficult.

Radiology is a good technique for determining/confirming the number of foetuses near term.

In large animals, it is not practised because of restraining issue in advance pregnancy, and

equipment cost. However in dog and cat it is practiced but its scope become limited after

ultrasonography uses. Once bone development or hard tissue development started it can be

used. However they cannot confirm for early diagnosis of foetal death but permits evaluation

of bitch's pelvic anatomy.

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5. Summary: The Challenges and future for Early Pregnancy Diagnosis

Although several direct and indirect methods for early pregnancy diagnosis are available for

animals, none of these methods fulfill all of the attributes of the ideal early pregnancy test.

Moreover there is need to develop test which can indicate pregnancy before onset of next

estrous cycle and can be performed at farm level itself. Combination of two or more assays

hold potential for early pregnancy diagnosis. For example a test combining progesterone and

MRP protein can be a potential solution for such cases in dairy animals. Further identification

of molecule exclusively expressed in early pregnancy by application of omics technique is

the need of hour in dairy and meat based animal. In the time of globalization, the farmers

have the tough and daunting task of feeding the growing world population and for this we

need more production of animal products. Due to this reason we cannot afford to keep animal

open which will lead to economic loss of farmers on one hand and cause potential production

loss other hand. Exploring of new proteins like proteins as anti-testosterone antibody light

chain, apolipoprotein A-II precursor, serum amyloid A, cytokeratin type II, component IV

isoform 1, which are have established roles in embryogenesis and further confirming of

already established protein in biological fluids more preferably in saliva and urine to develop

lateral flow based ELISA kit to detect pregnancy before onset of new cycle is need of hour

and future work can be toward these area.

paper no. : 09 animal cell biotechnology module :18

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