Methods of Pregnancy Diagnosis in Domestic Animals

Article ID: WMC001305
Methods Of Pregnancy Diagnosis In Domestic

Animals: The Current Status
Author(s):Dr. Govind Purohit
Corresponding Author:
Dr. Govind Purohit,
Associate Professor, Rajasthan University of Veterinary and Animal Sciences (Animal Reproduction,
Gynaecology and Obstetr, Department of Animal Reproduction Gynecology and Obstetrics, College of Veterinary
and Animal Science, Bijey Bhawan Bikaner Rajasthan, 334001 - India
Submitting Author:
Mr. Govind Purohit,
Associate Professor, Vet Obst & Gynec, Raj University of Vet & Anim Sci Bikaner Rajasthan, Deptt of Vet Obst &
Gynec Veterinary College Bikaner, 334001 - India
Article ID: WMC001305

Article Type: Review articles
Submitted on:09-Dec-2010, 08:07:23 AM GMT Published on: 10-Dec-2010, 07:37:00 PM GMT
Article URL: http://www.webmedcentral.com/article_view/1305
Subject Categories:REPRODUCTION
Keywords:pregnancy diagnosis, rectal palpation, ultrasonography, laboratory tests, clinical methods,
radiography
How to cite the article:Purohit G. Methods Of Pregnancy Diagnosis In Domestic Animals: The Current Status .
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Methods Of Pregnancy Diagnosis In Domestic

Animals: The Current Status
Abstract There is a need to educate farmers to get their

animals checked for pregnancy at an early date as it
has been shown that earlier the pregnancy diagnosis
performed, the more profitable is the return for dairy
Methods of pregnancy diagnosis in the domestic
cows and buffaloes (Oltenacu, 1990; Duggal et al.,
animals can be classified into visual, clinical and
2001a; Youngquist, 1997).
laboratory methods. In most domestic animals clinical
methods are currently used. Visual methods are far
from perfect in domestic animals. In animals like cattle,
buffaloes and mare’s recto genital palpation and
Review
trans-rectal ultrasonography continue to be the
methods of choice for an accurate and early
Methods of Pregnancy Diagnosis
pregnancy diagnosis. In sheep, goat, sow, bitch and
A variety of approaches have been evaluated and
cat ultrasonography is the only reliable method of
developed during the past and recent years, some of
pregnancy diagnosis. In the camel cocking of the tail is
which have some limitations to their wide scale use.
an effective visual method of pregnancy diagnosis
The methods of pregnancy diagnosis have been
and recto genital and ultrasonography are also useful.
classified into two (direct and indirect) or three
Laboratory methods used in past and developed in
categories i) Visual methods, ii) Clinical methods, iii)
recent years are described. The efficiency of a few of
Laboratory tests.
these procedures is likely to be improved in near
Visual Methods
future.
Non return to estrus
When an animal is mated and it does not return to
Introduction estrus the owner usually thinks that the animal has
become pregnant and hence has not returned to
estrus. This happens because during pregnancy, the
conceptus inhibits the regression of the corpus luteum
The diagnosis of pregnancy (cyesiognosis) has been
and thus, prevents the animal from returning to estrus.
sought since long by farmers for curiosity however, it
However, many a times the animal does not return to
is essential for profitable animal husbandry especially
estrus because of non regression of CL due to
in the productive animal species. For an economical
reasons other than pregnancy. Moreover, in the
dairy farm, cows must calve every year, and to
seasonally breeding species the animal may not return
maintain this sequence, identifying pregnant animals
to estrus (when mating is done during the end of the
at an early date seems imperative. In the current
breeding season) because the season was over.
systems of planned breeding, diagnosis of pregnancy
Anestrus, and the rare occurrence of gestational
would help to evaluate the therapies at an early date
estrus in cattle and buffaloes can affect the reliability
and devise alternative manipulations. In some
of non return to estrus as a method of pregnancy
situations in the pet practice pregnancy may not be
diagnosis. Moreover, difficulty in estrus detection and
desirable by the owners and an early diagnosis would
silent estrus render this method of pregnancy
help in termination of these unwanted pregnancies. An
diagnosis unsuitable for the buffalo. Likewise, estrus
early pregnancy diagnosis is essential in mares to
detection methods used for sheep, goat and mares
tease them if non pregnant, and try to get them
need to be properly designed so as to make efficient
pregnant in the same season. It therefore, appears
use of non-return to estrus as a method of pregnancy
that early diagnosis of pregnancy is essential in animal
diagnosis in these species. In dairy cows non-return
management for economic reasons. In many
rates usually over estimate true pregnancy diagnosis
developing countries, farmers often present their
(Kidder et al., 1954) and are also affected by the
animals for pregnancy diagnosis very late when much
detection procedure used (Foote, 1974). Moreover,
of their time is lost in maintaining non pregnant cows.

estrus expression appears to be reduced in intensity animal and its fetus when performed carefully. To a
and duration in the present day dairy cows leading to limited extent this method is used for pregnancy
lower estrus detection efficiency (Dransfield et al., diagnosis in pigs. The method is the technique of
1998; Lucy, 2001). Therefore, the probability of choice being taught to veterinary graduates and para
misdiagnosis of pregnant females by estrus veterinary staff. Transrectal palpation is considered to
observation appears to be increased. This may be be an accurate method of pregnancy diagnosis in
confounded by a small proportion of pregnant cows dairy cattle for a trained veterinarian after day 35 post
and buffaloes expressing estrus (Gilmore, 1952; breeding (Euler, 1930; Gotze, 1940; Roberts, 1971;
Agarwal and Tomer, 1998) and some cows expressing Zemjanis, 1970; Momont, 1990). A few studies point
prolonged inter-estrus intervals of around 24 days out that the procedure may increase the risk of
(Sartori et al., 2004). Therefore, non return to estrus is iatrogenic embryonic mortality in dairy cattle (Paisley
an unreliable procedure for pregnancy diagnosis in et al., 1978; Vaillancourt et al., 1979; White et al.,
most domestic animal species. 1989) while others and the author do not concur with
Besides the non-return to estrus a few of other visual this view. The procedure however, does not provide
signs of pregnancy appearing in late pregnancy any information about the viability of the embryo/fetus
include increase in the size of the abdomen, during earlier stages of pregnancy (Romano and
development of the udder specially in dairy heifers (4 Magee, 2001). Some of the basic principles which a
months onwards), slight vaginal discharge (from 4-5 palpator must understand and the precautions that he
months onward in dairy cows) and movements of the must observe are mentioned below and then the
fetus visible externally (specially in fed cows on the findings are described for cattle, buffaloes, mare,
right side of abdomen 6 months onwards). However, camels and pigs. Transrectal palpation of the amniotic
the accuracy of these visual diagnostic symptoms is vesicle as an aid in determining pregnancy status in
always low and a clinician must use them as a dairy cattle was described by Wisnicky and Cassida
supplement to clinical diagnosis. (1948), whereas slipping of the chorioallantoic
Cocking of the tail membranes between the palpator’s thumb and
The pregnant female dromedary camels exhibit a forefinger beginning on about day 30 of gestation was
characteristic behavior when approached by a male or described by Zemjanis (1970).
a person. The female assumes a stiffened posture Basic principle
with the head held high and tail curled upwards. The genital organs lie usually on the pelvic floor during
(Banerjee, 1974; Banerjee et al., 1981). This is known early pregnancy beneath the rectum in most species
as cocking of the tail. This behavior appears 14 to 15 and in the abdominal cavity during late gestation. The
days after fertile mating and known to be 95% reliable genital organs can thus be palpated indirectly by
for pregnancy diagnosis in quiet and calm dromedary placing the hand in the rectum evacuated of the feces.
females camels. However, many false positives can The growth of the conceptus in either of the uterine
be obtained in agitated females if the observer is horn leads to sequential increase in the size,
untrained. Tail cocking is also observed in the tenseness and palpable characteristics of the uterine
pregnant Bactrian camel although not with the same cornua. Thus, with experience the palpator can feel
intensity as in the dromedary female camel (Tibary these changes in the uterus of a pregnant animal and
and Anouassi, 1997). with fair to good accuracy predict pregnancy
Clinical methods of pregnancy diagnosis depending upon the species, stage of gestation and
Four clinical methods of pregnancy diagnosis are his experience. Two bottle necks appear to be
available for pregnancy diagnosis in the various significant while performing rectal palpation, i) the
domestic farm and pet animal species i) rectal peristalsis that occurs in the rectal musculature, which
palpation, ii) abdominal ballottement, iii) produces obstacles in palpation and ii) ballooning of
ultrasonography and iv) radiography. Each of these the rectal wall due to entry of air inside.
methods is discussed separately. The palpator must stop making movements of arm
Recto-genital palpation during a peristaltic wave (while still keeping his hand
Transrectal palpation is the oldest and most widely inside the rectum) wait for 1-2 minutes and then start
used method for early pregnancy diagnosis in dairy palpation again when the peristalsis has subsided.
cattle (Cowie, 1948). In most large domestic animal The ballooning of rectum can be easily appreciated, by
species like cattle, buffaloes, mares and female the finding that the operator can move his hand up and
camels recto-genital palpation (with some limitations) down in the rectum without resistance when the
is the easiest, cheapest and fastest method of rectum is ballooned. The operator must catch hold of a
pregnancy diagnosis with little or nil harm to the pinch of rectal mucosa and move his hand back and

forth (known as back racking) without completely Buffaloes also kick less frequently by hind legs but,
taking it out. This will push the air inside, to the sometimes excitable, nervous buffaloes do not allow
exterior and the rectal mucosa will then be closely rectal palpation by moving frequently. Such animals
over the operator’s hand. may rarely need little sedation, examination in a sitting
After proper restraint and wearing of proper position or examination at a later date. Palpators must
clothing and also proper lubrication, the operator must take every care to avoid damage to themselves by the
make a cone of his hand and push it inside the rectum. kick of the animal.
The anal sphincter dilates and the hand enters inside Thoroughbred mares are usually docile and
the rectum. The feces must be removed without taking can be easily made to enter a chute however; some
out the hand completely. The cervix which is a hard nervous mares or mares of other breeds need different
round to oval or sometimes caudally enlarged restraint procedures. It is often safe for the palpator to
disfigured structure is the land mark for location of make the mare stand on one side of a small wall (3 ft)
genital structures in cattle and buffaloes. This can be and the examiner stand on the other side of the wall.
located by sliding the hand in an arc like fashion from Stable wooden rails can sometimes serve the purpose.
dorsal to ventral side. The cervix is followed further to A good approach would be to tie both hind legs with
locate the uterine body and the uterine horns. These one fore leg using rope. A twitch on the lower lip, ear
structures can be pulled caudally when located at the or nose and/or lifting one fore leg may be required.
pelvic brim or further, by retracting the broad ligament Nervous mares sometimes move sideways or try to
or hooking the inter-cornual ligament by the index jump resisting any examination. Such mares need
finger. When the pregnancy is beyond 60 days this tranquillization.
cannot usually be done and the operator has to move Female camels are examined in a sitting
his hand further in the rectum, so as to locate the position with both hind, and forelegs tied together
intra-abdominally placed uterus and palpate other separately with ropes. An assistant holds the head
features diagnostic of pregnancy. tightly. Vicious females often require pressure on the
In the mare, the cervix is not easily palpable back by legs of persons standing on both sides to
and hence the ovaries are the land mark for rectal prevent side wise movements.
palpation especially for novice palpators. They are The examiners must wear proper clothing
located about 10-20 cm cranial to the shaft of the ilium including coveralls, gum boots and disposable plastic
bone and about 5 to 10 cm below the lumbar or rubber full arm sleeves. This is essential to protect
vertebrae in non-pregnant mares, and in mare during the examiner from contracting zoonotic disease and
early pregnancy. After locating one ovary the hand is spoiling his clothes. Separate trousers and shirts
passed down the utero-ovarian ligament to locate the made of dark coloured (green or blue) slightly thick
uterus. cloth are easier for working compared to a single
A striking feature of the genitalia of the female cover all. Plastic long sized aprons are used by many
camel is the shortness of the right uterine horn. This is clinicians in the field.
probably, because of the existence of exclusive left Sufficient lubrication must be used while
horn pregnancies in this species (Tibary and Anouassi, introducing the hand in the rectum. Non-irritating soap
1997). Recto-genital palpation is similar to that and water or liquid paraffin is a suitable lubricant.
described for cattle and buffalo as the cervix is easily Since, the feces of the mare are harder and the
palable. peristalsis stronger, more frequent lubrication is
Rectal palpation is only possible to a limited essential in the mare. Tail bandaging is also
extent in the large sized sows. It is barely possible in advantageous in the mare to avoid tail hairs enter the
gilts and in small sized breeds of sows. rectum and cause damage to the rectal wall.
Restraint and clothing Precautions during rectal palpation
The animal to be examined should be properly When performed gently and carefully rectal
restrained. Cows and buffaloes can be securely palpation is a non-invasive procedure. The following
restrained in a Travis or chute. At many situations points would be helpful in minimizing damage to the
when this is not available the hind legs of cows are animal and the examiner as well.
tied with a rope to avoid kicking and the head is held 1) Ruthless movements of the hand in the rectum
securely. The tail is held to one side by an assistant. should be avoided. Avoid palpations during a
Pressing on the back relaxes the pelvic structures and peristaltic wave.
reduces peristalsis. Buffaloes often resist tying ropes 2) Examiners must trim their nails and avoid using
on hind legs and ropes are usually tied either on both dirty soiled sleeves.
fore legs or one leg while folding it from the knee. 3) Rectal examination without a sleeve must be

avoided specially in mares to avoid contracting transrectal palpation, and because most cows within a
diseases and obnoxious odors. Sleeves must be herd are submitted for pregnancy examination, it is
replaced after examination of 2-5 animals, or better impossible for dairy producers and veterinarians to
after each examination. distinguish between iatrogenic losses occurring due to
4) Rectal palpation of an animal suffering from fever transrectal palpation and spontaneous losses that
should be extremely gentle or better avoided as the would normally have occurred in these cows.
blood vessels are more fragile and bleed easily. Palpable findings of pregnancy in cattle and
Similarly examining an animal with rectal tear or rectal buffaloes
fistula is hazardous. Whenever, a clinician notices Palpable findings of pregnancy have been
such conditions he must bring them to the notice of the described in detail previously (Roberts, 1985). The
owner or else he would blame the clinician. Rectal water buffalo has an approximately one month longer
fistulas are an emergency in a mare and immediate gestation period compared to cattle however; the
treatment including broad spectrum oral and parental parameters used in cattle are used in the water buffalo
antibiotics and anti-inflammatory drugs must be given for pregnancy diagnosis by rectal palpation.
along with laxatives or else the mare may develop a Two things must be kept in mind by clinicians
fatal peritonitis. The fistula/tear may be palpable as a in making positive diagnosis of pregnancy by rectal
blind pouch or a slit in the rectal mucosa that bleeds palpations in cattle and buffalo. The first is, that when
when the hand is forwarded through these openings. the palpator in unable to detect any of the palpable
5) Compared to cattle rectal palpation in buffaloes characteristics mentioned herein, he must neither
must be gentle as the rectal mucosa is more fragile comment positively or negatively as both would be
and bleed easily. frustrating both to the clinician and the owner on a
6) Clinicians must assure that even it the animal kicks later date. He must better admit the fact that he is not
it does not harm them, and so also the palpators must able to detect out properly and the animal must be
also be cautious that sudden sideways movement of re-submitted for examination 15-30 days later
the animal with the operators hand inside can cause preferably after a fasting. The second thing that
fracture of the operators arm and hence due care must clinicians must keep in mind is the accuracy of the
be exercised. gestation period (this is especially applicable for the
7) Uncareful palpation of the uterine horns with undue 5-8 month period in cattle and 5-9 months in buffalo).
pressure can cause rupture of the amniotic vesicle and An approximation of the gestation period must be
loss of an early pregnancy and hence this must be conveyed to the owner rather than an accurate period.
avoided. Although experienced clinicians can more precisely
Consequences of improper palpation comment on the gestation period after an examination
Because pregnancy in cattle can be terminated by it is usually safe to be approximate.
manual rupture of the amniotic vesicle (Ball and When pregnancy examinations are made early
Carroll, 1963), many studies have investigated the (Day 30-45) the possibility of an early embryonic death
extent of iatrogenic embryonic mortality induced by must be kept in mind and a confirmation of pregnancy
transrectal palpation. Several studies have suggested must be done only after re-examination at a later
that examining pregnant cows early in gestation by period (60-90 days).
transrectal palpation increases the risk of iatrogenic Owners often consult veterinarians on the fetal
embryonic mortality (Abbitt et al., 1978; Franco et al., viability during mid to late gestation. It is often difficult
1987; Paisley et al., 1978; Valliancourt et al., 1979; to comment positively by a single rectal palpation.
White et al., 1989), whereas other studies have Until unless characteristic changes in the uterus and
suggested that cows submitted for transrectal fetus are palpable negative comments must better be
palpation earlier during gestation had a decreased risk avoided. The usual test for fetal viability during this
for abortion or that palpation had no effect on period is the movement of the fetus in response to a
subsequent embryonic losses (Studer, 1969; stimuli by the examiners hand (movement of a fetal leg
Thurmond and Picanso, 1993). Although controversy when pressed by hand or suckling movements by the
still exists regarding the extent of iatrogenic embryonic calf when a finger is touched in the mouth) however,
mortality induced by transrectal palpation, other this may be sometimes misleading specially when the
factors have a greater influence on calving rates than fetus is depressed.
pregnancy examination by transrectal palpation Location of pregnant uterus
(Thompson et al., 1994). Furthermore, because the The pregnant uterine horn under goes
risk of embryonic mortality is high during the period of sequential changes in size, location and morphology
gestation when cows are diagnosed pregnant by and they form the basis for pregnancy diagnosis. The

early pregnant uterus lies in the pelvic cavity in heifers, palpated.

and just ahead of the pelvic brim in pleuriparous large Palpation of placentomes
sized cows. As it grows in size, its growth is forward so The presence of placentomes is another
it starts descending into the abdominal cavity (approx positive sign of pregnancy and is detectable from
3½ - 4 months). At approximately 4½ - 5 months it about 75 days to term. The period of pregnancy when
reaches the abdominal floor and at this time only the uterus has descended into the abdominal cavity
cervix is palpable within the pelvic cavity which is also and the fetus is not palpable, palpation of a
drawn forward. The growth is then forward and then placentome is the surest indication that the
again upwards. The entire uterus or the fetus is cow/buffalo is pregnant. Since there is great variation
therefore barely palpable during the 4 – 6½ months in size among individual placentomes (those nearest
period and diagnosis has to be dependent on other the fetus are the largest), their usefulness in aging a
features of pregnancy (placentomes or fremitus). After pregnancy is limited. In general, they can be detected
this period the fetal parts are usually palpable and as soft, thickened lumps in the uterine wall and are
clinicians find no difficulty in commenting whether the more easily detected as pregnancy advances.
animal is pregnant or non-pregnant. During early Palpation of the fetus
pregnancy (day 30-60) clinicians have to depend on The palpation of the fetus itself is a positive
finding of the fetal membrane slip or the palpation of sign of pregnancy. Depending on the skill of the
the amniotic vesicle. examiner and the location of the fetus, the fetus can
The definite signs of pregnancy in the cow as be palpated from the time of amniotic softening (65 to
determined by rectal palpation are i) palpation of 70 days) to term. However, in large sized cows the
enlarged uterine horn containing the placental fluids ii) abdomen should be lifted up by a bamboo held by two
palpation of the amniotic vesicle iii) slipping of the attendants on either side of the abdomen to palpate a
fetal membranes iv) palpation or ballottement of the fetus during mid gestation (4½ - 6½ months). The
fetus v) Palpation of the placentomes vi) palpation of whole of the fetus is palpable many a times only
enlarged thin walled “whirring” uterine arteries. during early gestation (2 to 4 months). The size of the
Uterine changes fetus is approximately that of a mouse or rat at 2 and 3
The increase in the diameter of the uterine months and it increases to the size of a small cat at 4
horns is characterized by a thinning of the uterine wall months, a large cat at 5 months and a beagle dog at 6
and the feeling of a fluid filled structure. By 40-90 days months respectively (Bon Durant, 1986). The
of pregnancy, the uterus feels like a thick rubber maximum fetal growth occurs during the last one to
balloon nearly filled with water. The volume of fluid one and a half month of gestation and estimates of
increases rapidly the first 5 months of pregnancy and predicting pregnancy status depend upon the
then increase slowly. With advancing pregnancy the experience of the clinician and location of the fetal
non pregnant horn may also increase in size slightly. parts. Beyond 8 months of gestation, fetal parts (legs,
The amniotic vesicle head) are palpable within the pelvic cavity or just
The amniotic vesicle can be palpated with due cranial to the pelvic brim. Palpation of a fetal extremity
care between 30-50 days of gestation as a movable is sufficient evidence for pregnancy if other uterine
oval object within the uterine lumen, many a times at findings are normal.
the apex of the cornua. The vesicle is turgid, early in Palpation of uterine artery fremitus
pregnancy but becomes flaccid with advancing The major supply of blood for the gravid uterus
gestation until days 65-70 when it is difficult to detect arrives via the uterine arteries, which enlarge
at all. The width of the vesicle is around 1 finger (1.5 considerably as pregnancy progresses. These bilateral
cm) at 40-42 days of pregnancy and increases to 4 vessels travel in the broad ligaments, just below and
fingers (9.0 cm) at 60-62 days of gestation (Roberts, anterior to the iliac shafts reflecting in a cranio ventral
1985). direction. They can thus, be felt by the hand directed
Slipping of the fetal membranes laterally towards the iliac shaft. These vessels are
The fetal membrane slip can be felt between freely movable. Enlargement of the uterine artery
35-90 days of gestation (Zemjanis, 1970). The entire ipsilateral to the pregnant horn is detectable after 80 to
uterine horn must be grasped in the palm and allowed 90 days of gestation. By approximately 120 days, the
to slip while the fingers compress it gently. The blood flow within the artery increases to a point where
allantois chorion, slip between the thumb and fingers the blood flow is palpable as a buzzing sensation, also
before the uterine wall escapes. It is felt like a called “thrill” or “fremitus”. By 7 to 8 months the
connective tissue band. The pressure must be gentle. fremitus is often palpable on the side of the
The entire diameter of each uterine horn must be non-pregnant uterine horn also. The detection of

fremitus is a positive sign of pregnancy. fremitus is absent. However, it is always safer to make
Palpation of ovaries a re-examination 1 to 2 months later when the clinician
As the pregnancy advances ovaries may be is not sure, or make use of ultrasonography to
dragged forward along with the pregnant uterus and differentiate the condition. Sonographic evaluation
may not be palpable beyond the four to five months. would depict anechogenic fluid without cotyledons and
Vaginal changes echogenic material accumulated. A clinical therapy to
During pregnancy the vagina usually develops terminate the corpus luteum on an assumption that the
a pale, dry sticky mucus membrane. The cervix is condition is pyometra could be hazardous and loss to
closed and the cervical (mucus) seal covers the the owner, if unfortunately the condition was a normal
external os by day 40 to 120 of pregnancy. Slight pregnancy. In mucometra, the positive findings of
degree of vaginal discharge is evident in some cows pregnancy are absent, but, contrary to pyometra the
beyond 5 months of pregnancy but the cervical seal uterine wall is thin. Ullrasonography can easily
liquefies only prior to parturition or abortion and is differentiate the condition from pregnancy. When
discharged in strings. these two conditions are to be differentiated from early
Differential Diagnosis of pregnancy pregnancy a characteristic feature found most often is
Owners are many times perplexed on the the bilateral enlargement of both uterine horns, which
outcome of a pregnancy that did not progress normally is not found in pregnancy. The fetus is not palpable in
and often present their animals for a definitive these two pathologies, and often there is a history of
diagnosis to a clinician. Some pathological conditions vaginal discharge.
mentioned in detail in subsequent chapters often Palpable findings of pregnancy in mares
should be identified to be different from a normal The early diagnosis of pregnancy in the mare
pregnancy. The palpable differences of these has its own significance. A limited breeding season in
conditions are briefly described below. some breed registries (for e.g. Thoroughbred)
Only rarely does a urinary bladder full of urine warrants that the non pregnant mares are diagnosed
creates confusion for the presence of an early early and steps be taken to breed the mare again in
pregnancy (2-3 months) in dairy cows. This can be the same breeding season. Some peculiarities of the
easily differentiated by the absence of palpation of equine pregnancy are that an ovarian source of
both uterine horns and the ease with which the animal progesterone is essential for maintenance of
urinates when the bladder is gently massaged leading pregnancy until approximately days 50 to 70;
to disappearance of the enlargement. Rarely tumors of thereafter, the fetal-placental unit begins producing
the genital tract can create confusion, but their sufficient progestagens to support pregnancy.
consistency and location is different. The differential (Bergfelt and Adams, 2007).
diagnosis of three conditions commonly encountered The endometrial cups form at about 35 days of
by clinician’s namely mummified fetus, macerated gestation and remain up to 150 days. Secondary,
fetus and/or pyometra/mucometra is described below: accessory and supplemental corpora lutea (day 40)
Mummified fetus form on the mare ovary and all corpora lutea regress
A uterus with mummified fetus has thick by day 180-210 (Bergfelt and Adams, 2007). The early
uterine walls, absence of fluid and placentomes and a equine fetomaternal axis secretes sufficient estrogen
hard palpable structure. The fetus is closely apposing and the equine embryo stabilizes by day 16.
the uterine wall. The fremitus is absent. Diagnosis of an early pregnancy by rectal
Maceration of the fetus palpation is one of the easiest and accurate means for
There is copious vaginal pus discharge. The experienced personnel. However, a diagnosis at day
uterine wall is thick and doughy. There is no dorsal 18-20 should be confirmed at day 45-60 because of a
bulging of the uterus and placentomes are not late implantation of the equine embryo and chances of
palpable. Parts of bones are sometimes palpable early embryonic death and reabsorption. The basis of
separately floating tacked up. The fremitus is absent. pregnancy diagnosis in the equine species lies on the
Pyometra and Mucometra changes that follow in the cervix, uterus and ovaries.
These two conditions are many times difficult Placentomes are not found in the equine species and
to differentiate from normal pregnancy especially when the fremitus or fetal membrane slip is not marked,
the pus or mucus is present in enormous quantity therefore some of the parameters used in cattle
(sometimes 20-40 liters) so that the uterus is largely cannot be used in the equine species directly.
enlarged and placed on the abdominal floor. In Cervix
pyometra the uterine wall is thick, uterus is doughy As early as 16 to 18 days after ovulation, the
and placentomes or fetus is not palpable and the cervix of the pregnant mare becomes tightly closed,

firm, slender, and elongated. Between 16 to 30 days positioned cranial and ventral to their normal positions
the cervix can be with experience palpated on the floor and closer together.
of pelvis as a rigid firm structure. By 5 to 7 months the uterus is positioned low in
Uterus the abdomen and it is difficult to thoroughly evaluate
Most pregnancy diagnosis in the mare by the conceptus by palpation per rectum. During the
rectal palpation is done by palpation of the uterus. The sixth and seventh months, the horns are
parameters used for a positive diagnosis are as approximately perpendicular to the dorsal cranial
follows- aspect of the uterine body. Beyond the seven to eight
Uterine tone and thickness months the fetus is easily palpable by rectal palpation.
One of the positive sign that a mare has Ovarian palpation
conceived to a breeding is the finding of increase in Both ovaries should be palpated. Both ovaries
uterine wall thickness and a marked tone. The usually are enlarged from 18 to 40 days as a result of
endometrial folds are no longer palpable as folds of follicular development and the CL is not palpable.
tissue. The uterus becomes tubular, smooth and firm. From 40 to 120 days, extensive ovarian activity with
The uterine tone appears at days 15-16 and continues ovulations, luteinization, and development of
upto day 48 to 55 (Sertich, 1997). This is diagnostic secondary corpora lutea is evident. Follicular activity
except in cases of endometritis or in mares bred at the decreases from 120 days to term, and the ovaries
foal heat, which are still undergoing uterine involution. become small and inactive. The position of the ovaries
Uterine size and embryonic vesicle up to 60 days of pregnancy is similar to that for the
The conceptus becomes positioned at the non-pregnant mare. From then on, they are drawn
base of one of the uterine horns at the junction cranially and medially but remain dorsal to the uterus.
between uterine horn and body. At 25-28 days, a small The finding of both ovaries nearer to each other and
bulge may be palpable in maiden mares. The bulge close to the pelvic floor is a positive indication for
progressively increases in size and is palpable in most pregnancy when the uterus or other structures are
mares by day 30-35 (approx 3-4 cm in diameter). A difficult to palpate (3 to 5 months). Form 5 months of
small notch can be readily appreciated on both sides pregnancy onwards, the ovaries usually are not
of the bulge. By day 42-45, the conceptus occupies palpable as they are under the broad ligaments.
approximately half of the gravid horn and is 5 to 7 cm Rectal palpation in the sow
in diameter. The uterine wall over the bulge in thin. To a limited extent rectal palpation has been
At 60 days of gestation, nearly the entire gravid described as a method of pregnancy diagnosis in the
horn and half of the uterine body are filled with the sow. (Cameron, 1977). Sows are examined while
conceptus but the non-gravid horn remains small. The standing in gestation crates or pens. This technique is
pregnancy is like an elongated football and is nearly based on examination of the cervix and uterus,
similar to a 60 day pregnancy in cattle. The tonicity is together with palpation of the middle uterine artery to
markedly reduced at this time. The 60 day conceptus assess size, degree of tone, and type of pulse. At
is approximately 8-10 cm in diameter and 12 to 15 cm around 21-30 days of gestation the bifurcation of the
in length. cornua is less distinct, the cervix and uterine walls are
Palpation of fetus flaccid and thin. The middle uterine artery is 5-8 mm in
The fetus is active after 40 days and mobile diameter and more easily identified. The uterus
after 70 days. Palpation of the turgidity of the becomes progressively thin walled and ill defined by
conceptus is absent by day 90, and the fetus is 31-60 days and fremitus can be identified at 37 days.
palpable, which feels like a small, heavy, submerged Beyond 60 days the fremitus is very strong, however,
but floating object as the hand contacts it. In most piglets can only be palpated towards the end of
mares it is usually possible to palpate the fetus per gestation. The procedure is often difficult in gilts and
rectum from the third month onward throughout the small sized breeds because of a small pelvis and too
gestation. In a few deep bodied and large sized mares small rectum. False positive diagnosis is likely if the
palpation of the fetus is difficult from the fifth to external iliac artery is mistakenly identified as the
seventh months of gestation. In these mares the middle uterine artery. The technique is however, not
location of uterus and ovaries would aid the diagnosis. popular at most locations.
Location of the uterus Rectal palpation in female camels
The uterus is located in the pelvic cavity or just The corpus luteum formed on the camel ovary
at the pelvic brim until day 90. At 100-200 days, the (ovulation is induced by mating) persists and is
gravid uterus is positioned cranial to the pelvic brim in necessary for the entire gestation. The persistence of
the abdominal cavity (Sertich, 1997). The ovaries are the CL is one of the earliest sign of pregnancy as

otherwise; the luteal phase is very short. The CL is out others) which when stimulated by a high voltage
of reach by day 90 of pregnancy. The left uterine horn current emits the ultrasound. The ultrasound is
is inherently longer than the right horn and this must transmitted to the patient from the transducer and
be kept in mind when making pregnancy diagnosis in propagates through the tissues. The ultrasound beam
female camels. The earliest detection of uterine is either reflected back, partially absorbed or entirely
change (increase in diameter and appearance of absorbed. The returning beam (echoes) meets back
fluctuation) is palpable at about 40-15 days. Between and deforms the crystals in the transducer. This
60-70 days, the left uterine horn is increased about mechanical energy is converted back to an electrical
twice to its non gravid size, has a thin wall and signal proportional to the strength of the echo and
fluctuates (Banerjee, 1974). delayed by a time roughly proportional to the distance
The gestational changes in camel have been traveled. The scan converter interprets the variations
described (Tibary and Anouassi, 1997). The uterus in brightness displayed on the cathode ray tube of a
becomes cranial and ventral after the third month of B-mode system (or as a variation in amplitude in
pregnancy. The cervix is pulled forward and lies just at A-mode oscilloscope screen) and also stores images
the pelvic brim by 4 months. At 5 months of pregnancy, when required. The ultrasound is emitted in a pulse –
the uterus is completely in an abdominal position with echo manner. A pulse of ultrasound is emitted and its
a small degree of fluctuation but the fetus is not reflection perceived prior to emission of the next pulse.
always palpable. The fetus becomes palpable again Types of instruments and some definitions
beyond the 6th month of pregnancy, first with For most diagnostic veterinary purposes
ballottement, and then, the head and legs become B-mode, real time ultrasonography is used employing
easily palpable by the 7-8 months as the fetus starts different types of transducers. Transducers used
its ascent. By the 9th month, movement can be commonly in veterinary reproductive practice are the
observed by inspection of the right flank of the animal. linear transrectal transducer (frequencies of 5-10 MHz)
At around 11 months the fetal legs can be easily found and the sector transabdominal transducer (frequencies
in the pelvic cavity. The precise estimation of the stage of 1-4.0 MHz). For most reproductive diagnostic work,
of pregnancy beyond 3 months, because of the linear array transrectal transducers are employed in
absence of cotyledons and difficulty in reaching the cattle, buffaloes, mares and female camels. Small
fetus in this species is difficult. The fetal membrane sized transrectal transducers are also used for early
slip is not seen in camels because of a diffuse pregnancy diagnosis in small ruminants (sheep and
placenta. goat).
Ultrasonography For bitches mostly transabdominal sector
During the last couple of years, transducers are useful for pregnancy diagnosis with
ultrasonography has gained popularity in veterinary frequencies from 2 to 4.0 MHz. The same transducers
medicine and has become the method of choice for can be used for pregnancy diagnosis in sheep and
diagnostic imaging of the various organs of the body, goat beyond day 40 of gestation. However, in order to
including reproductive organs. Ultrasound is a high visualize an early pregnancy or the non pregnant bitch
frequency sound wave. Sounds audible to the human uterus transducers of high frequency (5-7.5 or 10.0
ear vary between 20 to 20,000 Hertz (Hz) (Cycles per MHz) are essential.
second) while ultrasound waves are of frequency A wide variety of transducers with single, dual,
higher than this, and for most diagnostic applications or multiple frequency and multiple functions are
frequencies of 1-10 MHz are used. Ultrasound cannot available and clinicians must decide what type of
be propagated in vacuum and in gas, transmission is instrument and the transducer he must purchase
poor. Reflection of ultrasound occurs between depending upon his work. Advancement to
substances of different acoustic impedance (defined ultrasonongraphy includes diagnostic imaging using
as the product of the velocity of sound in a substance color doppler, magnetic resonance imaging and
and the density of the substance). Even the short readers must refer pertinent excellent texts available
distance between the transducer (which emits and elsewhere (Singh et al., 2003).
receives ultrasound signals) and the patient must be When performing ultrasonography it is
bridged by a suitable coupling gel. important for the sonographer to have basic
Basic principle knowledge of the anatomic location of the different
The ultrasound equipment basically consists of organs to be visualized and problems that can be
a transducer and a scan converter. The transducer is encountered in obtaining and interpreting the images
the ultrasound producing part. It is fitted with a obtained. A few of the common terms related to
piezoelectric crystal (Lead – zirconate – titanate or ultrasonography are described below:-

Anechoic (sonolucent) A tissue failing to reflect is covered with a condom with coupling gel put inside
the ultrasound beam produces no echoes (e.g. A fluid or a full arm disposable sleeve is used to cover the
filled follicle) and appears black. transducer. The operator keeps the transducer in his
A- mode Amplitude modulation. A one-element (one arm and takes it inside the rectum. The uterine horn
dimensional) display with time (distance) on the on one side is scanned to the entire length and the
horizontal axis. ovary of that side is also scanned. The operator then
B-Mode Brightness modulation. A compound A-mode moves his hand to the other uterine horn and ovaries.
scan with amplitude translated into a brightness scale. If the pregnancy is advanced the operator may have to
Location on the display is related to position and depth. take his hand deeper. When required the images seen
Doppler ultrasound When an ultrasound beam meets may be frozen and the diameter of the structures
a moving object the reflected ultrasound is either of measured by inbuilt calipers with the machine. The
increased or decreased frequency, depending upon amount of fluid and thickness can also be measured.
whether the motion is towards or away from the The transducer is then taken out and the perineum
transducer. washed again.
Echogenic A structure causing a marked reflection of Trans-abdominal (Transcutaneous)
the ultrasound beam. A change in echogenecity in a ultrasonography
homogeneous structure may indicate a pathological For reproductive trans-abdominal
change. ultrasonography in sheep and goats the hair must be
Gain The amplification level of a returned signal. clipped from just above the udder and 15 to 20 cm
Hperechoic Showing increased echogenecity. ahead of the udder on both sides of the abdomen. The
Hypoechoic Showing decreased echogenecity. transducer is placed above the udder between the
Linear array Distribution of piezoelectric crystals thigh and abdomen preferably the left side and moved
along the length of a scan head. The image produced in a ‘W’ shape from one side of the abdomen to the
is generally rectangular. other side. The procedure can be performed with the
M-Mode Motion mode. A rapidly updated one animal standing (sector scan) or in lateral or ventral
dimensional B-mode display with time on the second recumbency (linear scan) depending upon the type of
axis to allow study of moving structures. Used in transducer being used.
cardiology. For pregnancy diagnosis, sows are examined
Probe The transducer and its attachments. in a standing position when using sector scanners
Real time Images generated from reflected ultrasound (3.5-5.0 MH2 frequency) or doppler instruments (which
following sequential activation of transducer array are are more frequently used in pigs to detect fetal heart
displayed on the screen at sufficient speed to give the beat, fetal movements and uterine artery pulsations).
appearance of a live image. The transducers are placed over the abdomen just
Scatter When the ultrasound beam encounters a medial to the stifle skin fold, just at the level of second
small object in its path the beam energy is spread in last teat.
all directions. Trans-abdominal imaging of the uterus of a
Sector scan A pieslice/sector shaped image is bitch can be done with the bitch in the standing
produced on the screen. The initial signal is produced position after clipping the hair of the ventral abdomen.
by a single or small number of rotating piezoelectric However, dorsal and lateral recumbent positions may
crystals. also be used. The uterus lies dorsal to the bladder, but
Shadowing Caused by severe attenuation (decrease its position may vary with the extent of bladder filling,
in the power of ultrasound beam by absorption, scatter and the size of the uterus. During early pregnancy the
and reflection) such that it fails to penetrate sufficiently uterus has a more dorsal position in the abdomen of a
deeply. standing bitch, but with advanced pregnancy the
Transducer The piezoelectric crystal or element uterus is closer to the ventral lower abdominal wall.
which converts electrical to mechanical energy. Sonographic findings during pregnancy
Procedure for ultrasonography Interpretation of sonograms of the reproductive
Transrectal ultrasonography tract requires an understanding of the composition of
The detailed methods for performing the images and an awareness of the possible artifacts
transrectal ultrasonography in cattle have been which can occur and lead to misdiagnosis. For
described in detail previously (Goddard, 1995 Manion, example, acoustic enhancement will appear as a
2006). The animal to be examined is properly hyper echoic region deep to the fluid (anechoic) area.
restrained, the feces are evacuated from the rectum Gas and bone will totally reflect the sound waves and
and the perineum washed with water. The transducer produce the strongest of echo signals, leading to an

image which appears on the screen as near-white. So bitch. It therefore, appears that the fetal fluid, fetal
complete is the return of echoes in some cases that heart beat and the fetus become sonographically
sound waves do not penetrate deep to these areas, visible at nearly the same time in most species. The
resulting in a lack of imaging which manifests itself as most appropriate time for pregnancy diagnosis using
a black zone and is referred to as acoustic shadowing. ultrasonography with high accuracy in cattle, camel
This must not be interpreted as the anechoic image of and buffaloes appears to be day 28-30 using a trans
fluid filled structures. Another artifact found with gas rectal linear array probe of 5.0 to 7.5 MHz frequencies
filled viscera or bone is the rebounding of echoes back (Filteau and Des Coteaux, 1998; Vyas et al., 2002; Ali
and forth between the object and the transducer. With and Fahmy, 2008; Kahn et al., 1990). Using the same
each cycle of rebound there is loss of signal strength, probe pregnancy can be diagnosed with sufficient
and this is imaged on the screen as a series of layered accuracy a little earlier (24-25 days) in the mare
hyper echoic images repeating themselves between (Pycock, 2007). Recently, the use of a 3.5 MHz
the object and the transducer face. This is termed transcutaneous sector probe applied over the right
reverberation and is often encountered in trans-rectal flank has been suggested for pregnancy diagnosis in
scanning where gas filled viscera are present. When cows between 73 to 190 days of gestation. The fetal
the ultrasound strikes to a smooth and wide structure thoracic, abdominal and umbilical diameter was
(for e.g. a CL) there will be almost total return of significantly associated with gestational age (Humman
echoes where the sound waves strike at right angles, et al., 2009a, b,c). A trans rectal probe of 5.0 MHz
giving an intensified signal that appears on the screen frequency (usually a prostatic probe used in human
as a whiter shade of grey. This is referred to as beings) is required to diagnose pregnancy between
specular reflection, and is often seen in early days 25-30 in sheep and goats (and this can be used
pregnancy when imaging the embryonic vesicle. to study non pregnant uterus and ovaries) but under
With these basic things in mind one has also to field conditions a transabdominal probe (linear or
keep in mind that fluid filled structures appear black sector 3.5 to 5.0 MHz frequency) is generally used and
(anechoic), hard structures (like the bone) appear this can diagnose pregnancy earliest at day 40-50 in
white (hyperechoic) and other structures with their sheep and goats with reasonable accuracy as the fluid
structure midway between the bone and fluid appear and cotyledons are easily visible sonographically at
grey (hypoechoic). The basic diagnosis of pregnancy this time (Duggal et al., 2001b; Suguna et al., 2008).
lies in the identification of structures from black, grey The diagnosis of pregnancy in the bitch requires a
or white scale. transabdominal probe (of frequency 3.5 to 5.0 MHz) to
The earliest sonographic finding for pregnancy visualize pregnancy with accuracy from 25-30 days
in most animals is the appearance of anechoic fluid (Bondestam et al., 2008). The visualization of earlier
within the uterine lumen. This fluid goes on increasing pregnancy or the visualization of a non-pregnant
to a stage when the embryo proper becomes visible as bitches uterus necessitate the use of probes of higher
a hypoechoic structure floating within this fluid and frequency (7.5 to 10.0 MHz) as the uterus lie more
progressively the fetal structures become more clearly closer to the skin. Color Doppler ultrasonography in
visible along with the fetal membranes. The viability of the bitch can detect placental fetal circulation (Blanco
a growing fetus is ascertained when the fetal heart et al., 2008) and in sheep pregnancy can be
beat becomes visible as a hypoechoic flickering diagnosed with high accuracy at 76-90 days of
structure. gestation (Ganaie et al., 2009).
The methods of evaluation of pregnancies Doppler ultranongraphy and A-mode probes are
using ultrasonagraphy have been described for generally used in pigs for pregnancy diagnosis but
various species in sufficient detail by Goddard (1995). B-mode probes with frequencies of 3.5 to 5.00 MHz
The days of sonographic appearance of various can diagnose pregnancy with high accuracy between
structures in different domestic animals are mentioned days 25-30 post mating (Almond and Woodard, 1997;
in Table 1. On analyzing this table it is clear that with Williams et al., 2008).
different probes the amniotic vesicle/fetal fluid is Radiography
visible in most species between days 18-22 except in To a limited extent radiography has been used
the mare in which it appears earlier (day 10-16). The for pregnancy diagnosis in the small ruminants (sheep
fetal heart beat can be seen between day 24-30 and and goat), the companion animals (dog and cat) and
the fetus itself between days 25-30 in most species. rarely in pigs. The technique is known to be good in
The cotyledons are visible between day 30-40 and evaluating fetal numbers in the bitch and cat, but is
fetal extremity/bone by day 57-60 in cattle, day 70 in poor in evaluating fetal viability. Moreover, the high
sheep; however it is visible earlier (42-50 days) in the cost and the hazards of exposure to growing fetuses

to x rays limit the use of radiography as a routine only beyond 4 months of pregnancy by lifting the
procedure, and warrants its use in specialized cases. abdomen held between both hands and location of
Mostly, a single radiograph taken with the animal in bony fetal structures. However, sometimes bezoars in
lateral recumbency is sufficient however; sometimes a the rumen may confuse with pregnancy.
dorsal or a dorso ventral view may be required. In In cattle abdominal ballottement is performed
sheep and goat, fetuses are visible by day 70 of by placing the fist over the lower right abdominal wall
gestation (Grace et al., 1989; Noakes, 1999) with a and pushing it in an intermittent manner in a dorsal
high accuracy. The overall accuracy of the method in medial direction deeply. The fetus can be felt as a
detecting pregnancy increases with advancing hard solid object floating in fluid. This is usually
gestation: 52% between 66 and 95 days to 100% after possible in lean cows after the 7th month of gestation.
96 days (Richardson, 1972). The accuracy of The fetal movements can be seen at the same place
determining fetal numbers approaches 87% only by careful visual observation however, the method is
between day 91 and 110 (Ardran and Brown, 1964). applicable too late in diagnosis. Abdominal palpation
Therefore, radiography is suggested to be done only of pregnancy in bitches may be possible in small or
after day 90 in sheep and goat. medium sized bitches which are not too obese.
In bitches fetal skeletons are visible with high Moreover, bitches often tense their abdominal wall and
accuracy only by the sixth week of pregnancy although respire too fast making abdominal palpation often too
they may be sometimes visible as early as 23-25 days difficult to perform. At about 30-35 days the accuracy
of gestation (Toal et al., 2005). The fetal skulls are is high (87%) and depends on the palpation of tense
visible by day 45 and the entire fetal skeleton is visible conceptual swellings (6 to 30 mm in diameter) within
by the end of seventh week of gestation. The accuracy the uterine cornua but they must be differentiated from
of radiographic diagnosis is dependent on the quality feces in the colon and the palpator must have
of radiograph obtained. Radiography is generally expertise to differentiate or identify them correctly. The
suggested for bitches and cats, whenever there is a bitch’s foreparts must be slightly elevated. Beyond day
doubt about the nature of the abdominal contents at or 55 it is easy to diagnose pregnancy by the palpation of
near whelping. The number and position of the fetuses the fetuses. They may be palpable in the flank and
can be detected easily by radiography at this time. also in the lower abdomen. With the availability of
Signs of fetal death as seen by radiography include more effective methods like ultrasonsography for early
the spalding sign, (which is the overlapping of the pregnancy diagnosis with accuracy the use of
cranial bones), gas shadows in the fetal heart and abdominal palpation has been reduced as the palpator
stomach and tightly flexed spine (seen in fetuses died is many a time confused.
for long time) (Jackson, 2004). Fetal echocardiography
The fetal skeletons begin to calcify only after To a limited extent fetal echocardiography had
the sixth week in sows and hence radiography should been used in the past to diagnose in cattle, sheep and
be performed only after this time for pregnancy mares, but with the advent of ultrasonography its use
diagnosis in sows (Rapic, 1961). Because pregnancy has been limited.
can be diagnosed with high accuracy with other Vaginal electrical resistance
methods, radiography is seldom performed for The conductivity of the vaginal mucous
pregnancy diagnosis in sows. membranes changes at estrus due to increased
Abdominal Ballottement hydration, increased blood supply and other changes.
Abdominal palpation and abdominal When measured by ohm meters the vaginal electrical
ballottement of the fetus is possible to some extent in resistance (VER) is low at estrus. Hence when VER is
cows during late gestation (7 months onward). In measured constantly animals returning to estrus can
sheep and goats rectal abdominal palpation (by using be identified and thus those probably becoming
a glass rod placed in the rectum to lift the uterus which pregnant can be differentiated (Foote et al 1979; Mc
is palpated through abdomen) has been suggested Caughey, 1981; Gupta and Purohit, 2001; Meena et
(Ott et al., 1981; Hulet,1972; Chauhan and Waziri, al., 2003) however the accuracy of such estimations in
1991;). Similarly bimanual palpation for pregnancy diagnosis of pregnancy is limited due to false positive
diagnosis (palpation of uterus through fingers in the results and the problem related with daily probing of
rectum and lifting the abdomen) has been reported for animals.
small ruminants (Chauhan and Waziri, 1991; Kutty, Laparoscopy
1999) however, both the methods are inaccurate and Laparoscopy can be used as a method of
the first method is often invasive. Palpation of fetuses pregnancy diagnosis by directly visualizing the
through the abdomen is possible in sheep and goat genitalia in animals however, the invasive nature of

the technique, the high cost of equipment and clinic 1987). Since progesterone concentration in the
required, and the availability of non invasive peripheral blood of pregnant bitches is similar to those
techniques limits the use of this technique as a means of non pregnant bitches, and since there is no
of pregnancy diagnosis in most animals. placental progesterone produced in the pregnant bitch
Laboratory tests for pregnancy diagnosis (Verstegen-Onclin and Verstegen, 2008) hence
The various laboratory tests developed for progesterone assay cannot be used to diagnose
pregnancy diagnosis in domestic animals are indirect pregnancy in the bitch.
methods of pregnancy evaluation, and utilize To a limited extent plasma progesterone has
qualitative or quantitative measures of reproductive been used as a means of pregnancy diagnosis in the
hormones at specific stages after AI or mating, or female camel in which species the CL is required for
detect conceptus specific substances in maternal body the entire gestation (Abdel Rahim and El-Nazier,
parts or body fluids as indirect indicators of the 1987).
presence of a viable pregnancy. Unfortunately, none Estrone sulfate
of the methods developed so far in animals are as One of the earliest written records of a urine-based
accurate as is the detection of hCG in pregnant human pregnancy test can be found in an ancient Egyptian
females. However, the research to develop document. A papyrus described a test in which a
commercial indirect methods continues because these woman who might be pregnant could urinate on wheat
methods are non invasive and the tests can be and barley seeds over the course of several days: “If
marketed to and performed by the dairy farmers. The the barley grows, it means a male child. If the wheat
currently available methods are briefly described. grows, it means a female child. If both do not grow,
Progesterone hormone assay she will not bear at all.” Testing of this theory in 1963
The corpus luteum formed on the ovary found that 70 percent of the time, the urine of pregnant
subsequent to ovulation produces progesterone for women did promote growth, while the urine of
maintenance of pregnancy for a reasonable time non-pregnant women and men did not. Scholars have
period in some species and for entire gestation in identified this as perhaps the first test to detect a
other species like the cow, buffalo, goat and sow. unique substance in the urine of pregnant women, and
In normally cycling cows the CL is lysed have speculated that elevated levels of estrogens in
because of the effects of prostaglandins from the pregnant women’s urine may have been the key
uterus if the animal is not pregnant, and thus the to its success.
progesterone level goes down. Therefore, low A similar test of germination of wheat seeds when they
progesterone concentrations in maternal blood at 18 to are soaked in urine from pregnant cows which inhibits
24 days post breeding can predict that the animal is germination compared to urine from non-pregnant
non-pregnant and high progesterone gives an insight cows which stimulate germination has been described
that probably the animal is pregnant. The specificity of to be known as Punyakoti test (Nirmala et al., 2008).
progesterone tests conducted between 18 and 24 The differential germination is considered to be
days post breeding have shown a specificity of around because of the presence or absence of estrogens in
98% (Zaied et al., 1979; Laing et al., 1980; Waldman, the urine. However, controlled studies using estrogen
1993; Gowan et al., 1982; Pennington et al 1985; and progesterone failed to affect the germination rate
Nebel et al., 1987) and is the easiest proven method of wheat seeds (Nirmala et al., 2008).
for identifying non-pregnant (regularly cycling) animals The estrone sulfate is produced by the feto
post breeding. However, for the pregnant animals the maternal axis or the conceptus and therefore its
accuracy of the test is low (75%) because of early presence in urine, milk, feces or blood is an indicator
embryonic death which alter the results. Commercially of pregnancy.
available ELISA, plasma or milk progesterone assay The appropriate day, at which detection of estrone
kits have not become popular due to their high cost sulfate detection is possible in the body fluids or
and a low specificity. Non pregnant cows not returning secretions are mentioned in Table. 2 The detection of
to estrus and pregnant cows in which embryonic death estrogens depends on the availability of suitable
occurs at a later time can both give false results. laboratory and availability of commercial assay kits.
Likewise, in mares, sheep, goats, buffaloes, camels Laboratories evaluating concentrations of estrogens in
and sows assay of plasma or milk progesterone is not urine or serum usually are equipped with
very accurate for diagnosis of pregnancy (Zarkawi, radioimmunoassay, enzyme immuno-assay or other
1997; Kaul and Prakash, 1994; Sato, 1977; Fleming et more precise and specific diagnostic modalities for
al., 1990; Dionysius, 1991; Almond and Dial, 1986; assay of steroids in urine, serum, feces (Bamberg et
Ellendorf et al., 1976; Abdel Rahim and El-Nazier, al., 1984) or other body fluids. Evaluation of steroids

like estrogen from feces is especially helpful for zoo edema, appearance of cornified cells and mucus
and feral species where feces are the most easily discharge due to presence of estrogens in the
collected specimens (Bamberg et al., 1991; Hermann pregnant mare’s serum or urine (Allen and Doisey,
et al., 2005). Commercial kits have been developed for 1924). The phenolsulphonic acid test utilizes ether
pregnancy detection in mares by using on farm kits extraction of urinary estrogens subsequent to removal
like Wee-Foal-Checker® or Equitest ES® which of urinary pigments by hydrolysis and their conjugation
require urine or serum as the test material. These with phenolsulphonic acid reagent after evaporation of
commercially available tests are recommended to be the ether. The final reaction gives a pink to cherry red
performed only after 120 days of gestation and color if the urine is from pregnant mares (Mayer, 1944;
specially suggested for miniature horses and donkeys Benesch and Wright, 2001). The test is lengthy and
in which pregnancy diagnosis by rectal palpation or cumbersome and requires special precaution in
ultrasonography is extremely difficult. The test processing which has turned out this test of historic
procedures utilize an immunochromatographic importance only. These tests are 70-80% accurate
procedure to measure concentrations of pregnancy when performed between the 120 to 250 days of
specific steroids (estrogens) and manufacturers pregnancy.
recommendations should be strictly followed for Another test described previously include the mucin
optimum results. test in which the vaginal mucus from a pregnant mare
Chemical tests for pregnancy diagnosis showed dark staining columnar epithelial cells
Most chemical tests reported in the past appear to be (pregnancy cells) (Kurosawa, 1931) and known to
of historic importance only in current day pregnancy have an efficacy of 94% from day 70 to end of
diagnostic procedures. Some of the chemical tests gestation (Miller and Day, 1938; Day and Miller, 1940)
that utilize urinary estrogens, or other molecules as a however, these old tests have now been replaced
basis of pregnancy diagnosis in domestic animals are because of availability of better diagnostic, highly
described below: specific radio immunoassays and enzyme
Cuboni test: immunoassays.
This test was first developed by Cuboni (1934) and Barium chloride test: A test has been described for
modified later (Galina and Cox, 1969). The test is pregnancy diagnosis in the bovine species commonly
performed in the mare for detection of pregnancy known as the Barium chloride test. To 5 ml of urine
through assay of urinary conjugated estrogens. The from cows a few drops of 1% barium chloride is added
test is preformed as follows:- and warmed slightly. In non pregnant cows a white
“To 15 ml of urine 3 ml of concentrated hydrochloric precipitate is formed, whereas, in pregnant animals
acid is added and heated in a water bath for 10 the urine remains clear (Temblador and Landa, 1971).
minutes and then cooled under a tap. To this 18 ml of The accuracy of the test was described to be 70-95%
benzene is added and shaken vigorously for half a (Maslov and Smirnov, 1965; Elpakov and Cyganok,
minute. The supernatant (mainly benzene) is collected 1966; Akmadeev and Vasilev, 1967) from 15 to 210
in another tube and 3 to 10 ml of concentrated sulfuric days of pregnancy; however, a later study (Kavani,
acid is added and the mixture heated in a water bath 1976) noticed only a low accuracy (64%) with a high
at 80°C for 5 minutes, and then allowed to cool. A occurrence of false positive and false negative results.
positive (pregnant) test is indicated by the appearance In camels the test was considered to be 85% accurate
of a dark, only green fluorescent color in the lower between days 50-90 of pregnancy (Banerjee, 1974).
sulfuric acid layer and a negative (non-pregnant) is By and large the validity of such a test continues to be
characterized by absence of fluorescent color and controversial.
presence of a brownish color.” Some other previously described tests for pregnancy
The cuboni test is only effective beyond 150 days of diagnosis in cows include two tests on milk; i) milk
gestation, and also predicts fetal viability. alchohol coagulation test: in this test there is
Other chemical tests previously described for the coagulation of milk from pregnant cows when mixed
detection of urinary estrogens in mares include the with equal quantities of alchohol and allowed to stand
vaginal cornification, mouse or rat tests using for 1-3 hours (Linkes, 1930; Rutz, 1932; Stancev and
ovariectomised female rats or mice, and the Angelov, 1966; Kavani, 1976). ii) copper sulfate test: 1
phenolsulphonic acid test. (Based on kober mL of milk when mixed with a few drops of 3% copper
calorimetric test for estrogens) (Roberts, 1985; sulfate coagulates if the animal is pregnant (Tembldor
Benesch and Wright, 2001). In the mouse test, the and Acosta, 1971; Kavani, 1976). The accuracy of
serum or urine from pregnant mares when injected to these tests is considered to be low (52.0 to 64.2%)
ovariectomised mouse or rats would induce vaginal and many a times the tests are inconclusive and

confounded by breeds and disease in the udder Several biologic tests were developed for the
(Kavani, 1976). Moreover, no specific molecules were detection of eCG including the Aschiem Zondek Test,
determined in these studies and therefore their use in the Friedman test (rabbit test) or the Frog or toad test.
current diagnostic methodologies is limited and usually Aschiem Zondek test
of historic importance. Similar to these tests a Aschheim and Zondek described a test (known
previously described test documented the appearance as the A-Z test) which identified the presence of hCG
of deep yellow to orange coloration of cervical mucus in human urine. To test for pregnancy, a woman’s
from non-pregnant cows when boiled with 10-15% urine was injected into an immature rat or mouse. If
NaOH whereas cervical mucus (which is difficult to the subject was not pregnant, there would be no
obtain from pregnant cows) from pregnant cows reaction. In the case of pregnancy, the rat would show
evidenced a pale yellow or colorless state an estrous reaction (be in heat) despite its immaturity.
(Sokolovakaya et al., 1959; Williams, 1964; Kavani, This test implied that during pregnancy there was an
1976). increased production of the hormone.
Costa’s test: This test was developed by Costa (1927) A similar test in mares has been described wherein
for testing of pregnancy in human females and is serum from mares is injected to mice and the results
based upon sedimentation of haematin in the are read later (Miller and Day, 1938). The serum (0.5
presence of a solution of novocaine. To 1.5 mL of 2% ml SC daily for 2 to 4 days or 5 ml intraperitoneally)
novocaine 3 drops of blood are added and then 5% from test mare is injected to 2 to 3 rats (22 days of age)
solution of sodium citrate is added. The mixture is and rats are killed (72 hr when injected
centrifuged and 1 drop of formalin is added. In intraperitoneally and 96 to 120 h later when injected
pregnancy grey or grayish yellow color or precipitate subcutaneously) and a positive test is indicated by the
appears within 15 minutes. The test was experimented presence of multiple corpora haemorrhagica on the
in cows and the test was known to be positive after 38 ovaries and uterine edema. The test was considered
days of insemination with 65.3% accuracy in cows to be 90 percent accurate when performed between
(Kriisa, 1934, Bhattarcharya, 1967; Kavani, 1976) and the 60 to 100 days of pregnancy in the mare. The
inaccurate in camels (Banerjee, 1974). same test performed in camels revealed no changes
Kosjakovs test:This test apprehends that the sulfur in the mice (Banerjee, 1974; El-Azab and Musa, 2007)
content of hair in pregnant animals is increased due to lack of any gonadotropic molecule.
(Kosjakov, 1929). Hair from animals under test are Friedman Rabbit test
digested with 10% potassium hydroxide and boiled Serum from test mare is injected (2 ml given IV)
with 2 ml of distilled water and 1% methylene blue and to rabbits (14 to 20 weeks age) kept in isolation and
a few drops of 4% hydrochloric acid are added. The laparotomy performed 24 h later. A positive test is
blue color disappears in pregnant animals due to indicated by the presence of corpus haemorrhagicum
increased sulfur. The basis of the test was not verified and uterine edema (Chicchini and Chiacchiarini,
in subsequent research and hence the test is now 1963).
obsolete. Toad test
Assay of gonadotrophins Toads like Bufo valliceps or frog like Rana
The human female secretes the gonadotropin Pipiens are used in this test. The basis of this test is
hCG which is present in sufficient quantities in the the concept that the sperm cells are emitted by
urine of pregnant women and many simplified tests toads/frogs only when stimulated with female frogs or
have been developed to detect this molecule in urine gonadotrophins. Two male toads are taken and their
for an easy pregnancy diagnosis in women. The only cloaca is wiped with normal saline and examined for
animal species that secrete sufficient quantities of presence of spermatozoa. If no sperms are present
gonadotrophins that can be used as a marker the cloaca is cleaned and 1 ml of test serum from a
molecule for pregnancy diagnosis is the equine (Cole mare is injected into the dorsal lymph sac of 2 male
and Hart, 1930). Endometrial cups form as early as toads thrice at an interval of 1 hour. The cloaca is
day 35 and secrete the equine chorionic gonadtrophin examined for the presence of the sperms. A positive
(eCG) which can then be detected from serum and test is indicated by the presence of sperms in the
urine (Roser and Lofstedt, 1989) of pregnant equine cloaca within 1 to 6 hours after the last injection
females. The eCG continues to be secreted from day (Cowie, 1948; Neto, 1949) Similar test depicting the
40 to 120 days of pregnancy and is the basis of presence of a gametokinetic substance in the feces of
previously described biologic tests and the currently dairy cows, buffalo, goat and sheep has been depicted
available on farm tests. which also initiate a similar response in the cloaca of
Biologic tests the frog Rana Pipiens (Bhaduri and Bardhan, 1949;

Bhaduri, 1951; Banik and Reineke, 1955) although the popularity.

validity of the toad test in species other than the mare Pregnancy associated glycoproteins (PAG)
remains doubtful. Pregnancy specific proteins are known to be
The above mentioned older methods have now produced in various ruminant species including cattle,
been replaced by newer methods including buffalo, sheep and goats (Humblot et al., 1990; Karen
radioimmunoassay (Nett et al. 1975), radioreceptor et al., 2003; Karen et al., 2007). Two pregnancy
assay (Fay and Douglas, 1982), haemagglutination specific proteins (PSP) A and B have been isolated
inhibition test (Berthelot et al. 1987), ELISA (Squires et from bovine fetal membrane extracts (Butler et al.,
al 1983) and direct latex agglutination tests (Decoster 1982). Of these PSP-A was identified as a
et al 1980). Commercially available kits are in use at a-fetoprotein and PSP-B was found to be specific to
many places for these assays (Envet Servies, Omaha the placenta. These molecules appear in the maternal
NE). Pregnamare® is one such test, which can be circulation and can be determined with accuracy from
performed on blood between day 40 and 100 of 29 to 30 days post breeding. The PAG continues to be
pregnancy, however, false results may be obtained if existent in maternal blood for the entire pregnancy and
fetal death occurs after formation of the endometrial up to 100 days post partum. The assay involves
cups. radioimmunoassay on serum. The sensitivity and
On farm tests specificity of PSPB based on RIA is known to be
Some commercial kits are currently available which 92.0% and 82.6 to 91.9% from 29 to 30 days post
can detect the presence of eCG in blood of mares insemination (Szenci et al., 1998). More recently
between 40-100 days of pregnancy with 96-98% simple ELISA techniques have been developed that
accuracy. Pregnamare is one such kit which requires 5 detect the PAG molecule in the serum of cows (Breed
drops of blood collected from the muzzle by using the et al., 2009; Green et al., 2005; Green et al., 2009;
lancet provided with the kit. This color change test Silva et al., 2007). The limitations to the wide spread
requires about one hour completing. use of this test is non availability of the protein in milk
Other methods or urine, presence of PAG up to 100 days postpartum
Some of other methods described previously include (which interfere with subsequent detections) and the
onset of estrus by injections of estradiol valerate (2mg) non availability of cow side commercially available kits
at day 9-12 of service in cows (Jochle and Schilling, for its detection. Recently however, the existence of
1965; Kavani, 1976) and day 17-18 in sows (Laerum PAG has been documented in bovine milk (Gajewski
et al., 1974). The potential dangers of injecting et al., 2008).
estrogens to pregnant cows have refrained from the Early pregnancy factor
large scale use of these tests. Some years ago, a This protein molecule was first identified in
pregnancy diagnosis based in determining differences pregnant mice (Morton et al., 1987) and later in sheep
in cervical mucus using nuclear magnetic resonance and cattle (Nancarrow et al., 1981) by using the
was used (Merilan, 1983). This test looked promising rosette inhibition bioassay. With this assay, EPF was
at that time but no subsequent information is available. detected in the serun of all mammals tested within 24
Milk ejection by low dose prostaglandin to 48 h of fertilization and disappeared within 24 to 48
A method tested some years ago was the injection of h after death or removal of embryo (Morton et al.,
low dose prostaglandin F2 alpha (non-luteolytic dose) 1987). The developing embryo bears antigens foreign
in animals two weeks after breeding resulting into milk to the mother; hence immune rejection of the early
ejection. The animals detected further as pregnant embryo may occur. An immunosuppressive early
showed an increase in the pressure in the milk pregnancy factor (EPF) appears as early as 6 to 48 h
ejection and alveolar milk volume collected by a teat of mating which functions to suppress the maternal
probe in comparison with the non-pregnant cows immune response thereby allowing for pregnancy to
(Labussiere et al., 1982; Prakash et al., 1996) and proceed (Shaw and Morton, 1980). In cattle significant
ewes (Labussiere et al., 1983; Labussiere et al., differences in rosette inhibition titer were observed
1988). The intrajugular administration of a between pregnant and non pregnant cows on day
subluteolytic dose of PGF2 alpha induces a large 13-16 and 25 post breeding (Sakonju et al., 1993),
increase in intramammary pressure when given during suggesting that measurement of EPF activity may be
the luteal phase and this is directly correlated to the useful as an indirect method of pregnancy diagnosis.
plasma progesterone profiles (Labussiere et al., 1988). A commercially marketed kit is available in the US
However, due to potential dangers of inducing (ECF test, concepto Diagnostics Knoxwille, TN)
luteolysis by accidental over dosage, the use of this however its reliability is known to be poor (Adams and
technique of pregnancy diagnosis, could not gain wide Jardon, 1999; Des Coteaux et al., 2002) and need to

be substantially improved. reduced.

Relaxin assay
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Illustrations
Illustration 1
Tables
Table : 1 Ultrasonographic features of early pregnancy in various species

Sonographic Cow Buffalo Mare Sheep/ Sow Bitch Camel Cat
Structure Goat
appearance
(days post
mating)
Fetal fluid 18-20 18-22 days, 10-16 20-25 18-20 18-20 17-18 10-16
5th week
Fetal Heart beat 24 30 24-25 21-23 – 24 28-30 16-18
Fetus 28-30 20-26day, 20-22 25-30 25-30 – 23-25 16-20

4th week
Cotyledons 35-40 30-35 20-22 40-50 – – – 25

/Allantois
Fetal bones fetal 57-60 – – 70 – 42-50 40 days 30-33

buds
Fetal sex 57-60 10-18 60-70 60–90 – – – 38-43

determination weeks
Fetal movement 42-50 47-51 40-45 – 60 – – 30-34
Reference Curran et Pawshe et Allen and Garcia et al., Michael, England Vyas et Zambelli
al., 1986; al., 1994; Goddard, 1993; 1988; and Allen, al., 2002 and Prati,
Filteau and Karen et 1984; Buckrell et Holtz, 2008; 2006
Des al., 2007; Ginther, al., 1986; 1982; Barr,
Coteaux, Ali and 1986; Karen et al., Almond et 2008
1998; Fahmy, Sertich, 2003; Santos al., 1985;
Curran, 2008 1997; et al., 2007; Jackson,
1992; Pycock, Romano and 1986
Pierson 2007; Christians,
and Holder, 2008; Harsh
Ginther, 2007 et al., 2008;
1984; Suguna et
Nation et al., 2008
al., 2003

Table 2 Appearance of estrogen in domestic animals during pregnancy

Day of detection Reference
Species
Mare Maternal estrogen high after 60 days Conjugated urinary Sist et al. (1987); Cox
estrogens high after 150 days (1971); Bhavnani and
Woolever, 1978
Cow, Day 100 of gestation Hamon et al. (1981);

Robertson et al., 1978
Goats High after day 50 of gestation Refsal et al (1991) Chaplin

and Holds worth (1982)
Sows Rise start at 20 days peak at 25-30 days followed by a decline at Cunningham, 1982 Seren
45 days and again a rise at 70-80 days to term et al.(1983) Robertson et
al.(1978); Gutherie and
Deaver, 1979
Bitch Slightly increased at implantation and remain constantly high for Concannon et al. (1975)
rest of gestation and decline 2 days prepartum
Sheep Detectable by day 70; rise thereafter till 2 days prepartum Illera et al., 2000;
Worsefold et al., 1986
Buffalo Appear at day 150 of gestation in the serum Prakash & Madan (1993);
Kamonpatana, 1984
Camel Increase start at day 50 and peak from day 90-300 Skidmore et al., 1996

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Methods Of Pregnancy Diagnosis In Domestic

Abstract There is a need to educate farmers to get their

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early pregnant uterus lies in the pelvic cavity in heifers, palpated.

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Bhaduri, 1951; Banik and Reineke, 1955) although the popularity.

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be substantially improved. reduced.

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Table : 1 Ultrasonographic features of early pregnancy in various species

Fetal Heart beat 24 30 24-25 21-23 – 24 28-30 16-18

Fetus 28-30 20-26day, 20-22 25-30 25-30 – 23-25 16-20

Cotyledons 35-40 30-35 20-22 40-50 – – – 25

Fetal bones fetal 57-60 – – 70 – 42-50 40 days 30-33

Fetal sex 57-60 10-18 60-70 60–90 – – – 38-43

Fetal movement 42-50 47-51 40-45 – 60 – – 30-34

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Table 2 Appearance of estrogen in domestic animals during pregnancy

Cow, Day 100 of gestation Hamon et al. (1981);

Goats High after day 50 of gestation Refsal et al (1991) Chaplin

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