This document summarizes information about pregnancy diagnosis in cattle. It discusses evaluating pregnancy through rectal palpation and ultrasound. Rectal palpation allows feeling the fetus after 40 days and determining the location of the uterus. Ultrasound can detect a vesicle/fetal fluid starting around 18-22 days. It also discusses several laboratory tests for pregnancy diagnosis based on measuring progesterone, estrogen, or other compounds in urine, milk, or blood that indicate the presence of a fetus. These tests include progesterone assays, Cuboni tests, phenolsulphonic acid tests, and barium chloride tests.

1. Pregnancy diagnosis
Submitted to:- Submitted by:-
Dr Mitesh Gaur Ashish Tanwar
Endocrinology
Pseudo-pregnancy

2. Endocrinology

3. •The endocrine system uses chemical messengers or
hormones to regulate slow body processes e.g.
growth and reproduction
•The classic definition of a hormone is a physiologic,
organic, chemical substance synthesized by a
ductless endocrine gland which passes into the
circulatory system for transport.
•Hormone inhibit, stimulates or regulate the functional
activity of the target organ.

4. The reproductive process in cattle is regulated by complex
cascade of combined activity of central nervous system, the
number of secretary tissue, target tissue and several hormones.
Three Regulatory System governs the reproductive process in
cattle –
1. Nervous system
2. Endocrine system
3. Cell messengers

5. 1. Nervous System –
A. CNS receives information from the animal’s environment (visual,
olfactory, auditory and tactile stimuli) and conveys information relevant
for reproduction to gonads via hypothalamo–pituitary–gonadal axis.
B. These glands not only act as hormone producer but they also act as target
organs. Pineal and hypothalamus are part of CNS but they also act as
endocrine organs.
C. Hypothalamus links the CNS to endocrine system via pituitary.

6. 2. Endocrine System –
Major endocrine glands involved in the control of female reproduction are
A. Pineal Gland
B. Hypothalamus
C. Pituitary
D. Adrenal Cortex
E. Ovary
F. Uterus

7. A) Pineal Gland –
1. It is part of brain which originates as neuro-epithelial evagination
from the roof of third ventricle and is main organ in seasonality of
breeding .
2. It secrets Melatonin which in turn control activity of GnRH in some
species.
3. Melatonin is produced and secreted during night (dark period) and
controls sexual behavior?.

8. Cells of the
Anterior
Pituitary
• LH
• FSH
• Prolactin
• STH
• TSH
• ACTH
Hypothalamus
Nerve
Cells
Superior
hypophyseal
artery
Hypophyseal
portal vessels
Capillary
plexus
Posterior
pituitary
Capillary plexus
Preoptic
nuclei cell

9. B) Hypothalamus –
1. Hypothalamus is a small part of the brain located on its ventral side.
2. It synthesizes and secrets certain neuro hormones.
3. Hypothalamus is connected with posterior pituitary through neural
connection (hypothalamo–hypophyseal neural tract) and with anterior
pituitary through vascular connection (hypothalamo–hypophyseal portal
system).
4. Hypothalamus secrets gonadotropine releasing hormone (GnRH,
dacapeptide) which acts on anterior pituitary and cause releases of
FSH & LH.
5. The venous flow from pituitary partly goes to the hypothalamus thereby
exposing the hypothalamus to the high concentration of the pituitary
hormones (FSH & LH). This blood flow provides negative feed back
mechanism to the pituitary by which the pituitary controls the secretion
and function of the hypothalamus. This feed back mechanism is termed as
“short-loop feed back”

10. 6. The hypothalamus have two GnRH releasing centers –
Tonic release center (ventromedial nucleus and arcuate
nucleus) and Surge release center (preoptic nucleus,
anterior hypothalamic area and suprachaismatic nucleus).
7. Tonic release occurs in pulses and is control by blood
concentration of FSH and LH while surge release is
controlled by the level of oestrogen (via positive feed
back mechanism) in blood.

12. C. Pituitary (hypophysis)
1. It located just below the hypothalamus in a bony depression
called sella turcica.
2. It has three parts/lobes –Anterior, intermediate and posterior.
3. The anterior part of the pituitary secretes FSH (follicle
stimulating hormone) and LH (leutinizing hormone) of major
significance for control of female reproduction.
4. FSH plays a major role in the initial development of follicles
while LH is responsible for final maturation,ovulation of
follicle and leutinization of post ovulation remanenant of
follicle for formation of corpus luteum (CL).

13. D.) Gonads
1. Every female animal contains a pair of ovaries which are
the primary sex organ.
2. Overies contains a large no. of primodial follicles which are
potentially capable of developing into ovulating follicle but
in each cycle only one follicle develops in to mature follicle
while a large no. undergo atrsia.
3. Ovarian follicle produces many hormones but Estrogen and
inhibin are of major significance.
4. Follicle post ovulation forms CL under the influence of the
LH (CL produces progestrone and oxytocin and relaxin). If
the animal becomes pregnant the CL persists and continues
to function till term , and is called corpus luteum verum.
While in cycling , non pregnant, animal CL is called corpus
luteum spurium because it is destined to regress.

14. ANTERIOR PITUITARY HORMONES
Follicle Stimulating Hormone (FSH)
○ Is a glycoprotein hormone.
○ affects granulosa cells of follicle in the ovary
○ causes initial folliculogenesis
○ stimulates estradiol synthesis
Luteinizing Hormone (LH)
○ Is a glycoprotein hormone
○ affects theca interna cells and granulosa cells (and luteal
cells) follicle in the ovary
○ It stimulates final maturation of follicle, it’s ovulation and
formation of CL
PITUITARY HORMONES

15. Follicular Hormone :
Estradiol (E2)
○ steroid
○ produced by granulosa cells (follicle & placenta)
 Sertoli cells also produce
○ FSH stimulated synthesis
○ uterine mass and contractility (during estrus)
○ sexual behavior
○ secondary sex characteristics
○ GnRH release (positive feedback from E2)
○ Ca metabolism
○ influences mammary gland ducts
Inhibin
○ glycoprotein
○ produced by granulosa cells of dominant ova in female
○ produced by Sertoli cells in male
○ inhibits FSH secretion without altering LH secretion
GONADAL HORMONES

16. GONADAL HORMONES
CL Hormone:
Progestins
○ progesterone (P4) – “pro-gestation”
○ produced by corpus luteum and placenta
○ uterine preparation
○ influences alveoli of mammary gland
○ inhibits GnRH
○ “maintenance of pregnancy”

17. GONADAL HORMONES
CL Hormone:
 Relaxin
 Made of 2 polypeptides that are connected with
disulfide bonds. It is similar in size and structure to
insulin.
 Secreted by CL during pregnancy.
 In some species it may be secreted by the uterus
and/or placenta.
 Generally requires tissue first be exposed to
estrogens for its effects.
 Functions
○ cervical dilation, pelvic ligament relaxation
○ inhibits uterine contractions

18.  CL Hormone:
 Oxytocin
○ paraventricular nuclei of hypothalamus and CL
○ stored in & released from post pit
○ octapeptide
○ oviductal contractions
○ uterine contractions
○ milk letdown (mammary alveolar contraction)
○ Oxytocin secreted from the CL causes release of
PGF2α from uterine endometrium when the
endometrium is under the influence of estrogen.

19. UTERINE HORMONES
PROSTAGLANDINS
 F2α and E2
 20-carbon fatty acids
 uterine endometrium
 corpus luteum regression (luteolysis)
 “breaks” progesterone block and thus causes
release of GnRH from hypothalamus and FSH
and LH from pituitary & thus stimulates
ovulation.
 enhances uterine contractility

20. Placental Hormones
1.)Progestrone
2.) eCG (equine chorionic gonadotropin ) or PMSG
3.) PL (placental lactogen)
4.) HCG (human chorionic gonadotropin )

22. 10/27/2018 22

23. Pregnancy
diagnosis
clinically
External
exmination
Rectal
palpation

24. Pregnancy diagnosis clinically

25. Rectal palpation

26.  Palpation of fetus
 After 40 days and mobile after 70 days
 Location of the uterus
 The uterus is located in the pelvic cavity or just at the pelvic brim until day 90
 At 100-200 days, the gravid uterus is positioned cranial to the pelvic brim in the
abdominal cavity .
 Ovarian palpation
 Position of the ovaries up to 60 days of pregnancy is similar to that for the non-
pregnant mare
 From then on, they are drawn cranially and medially but remain dorsal to the
uterus
 The finding of both ovaries nearer to each other and close to the pelvic floor is a
positive indication for pregnancy (3 to 5 months)
 Form 5 months of pregnancy onwards, the ovaries usually are not palpable as
they are under the broad ligaments

27. PREGNANCY DIAGNOSIS
BY UltrasounOGRAPHY

28. Ultrasound machine

29. Mare and cattle

30. Methods of holding the US linear probe
A
Controlled Method
Sonographers who prefer
not to manipulate uterus
often choose to hold probe
in a very controlled manner
B
Palming Method
Frees the fingers for
retraction of uterus

31. Transducer position in bovines

32.  Vesicle/fetal fluid is visible in most species between days 18-22 except in the
mare in which it appears earlier (day 10-16)
 Fetal heart beat can be seen between day 24-30 and the fetus itself between
days 25-30 in most species.
 Cotyledons are visible between day 30-40 and fetal extremity/bone by day 57-
60 in cattle, day 70 in sheep; however it is visible earlier (42-50 days) in the
bitch
 The most appropriate time for pregnancy diagnosis using ultrasonography
with high accuracy in cattle, camel and buffaloes appears to be day 28-30 using
a trans rectal linear array probe of 5.0 to 7.5 mhz frequencies (filteau and des
coteaux, 1998; vyas et al., 2002; ali and fahmy, 2008; kahn et al., 1990)
 Using the same probe pregnancy can be diagnosed with sufficient accuracy a
little earlier (24-25 days) in the mare (pycock, 2007)
 Bitch requires a transabdominal probe of frequency 3.5 to 5.0 mhz) to
visualize pregnancy with accuracy from 25-30 days (bondestam et al., 2008)
Ultrasonography

33. Ultrasonographic features of early pregnancy in various species
Sonographic
Structure
Appearance (days
post mating)
Cow Buffalo Mare Sheep/
Goat
Sow Bitch Cam
el
Cat
Fetal fluid 18-20 18-22 10-16 20-25 18-20 18-20 17-18 10-16
Fetal Heart beat 24 30 24-25 21-23 - 24 28-30 16-18
Fetus 28-30 20-26 20-22 25-30 25-30 23-25 16-20
Cotyledons
/Allantois
35-40 30-35 20-22 40-50 - - - 25
Fetal bones/ fetal
buds
57-60 70-75 70 - 42-50 40 30-33
Fetal sex
determination
57-60 60-70 60-70 60-90 - - - 38-43
Fetal movement 42-50 47-51 40-45 - 60 - - 30-34
Reference Curran et
al., 1986;
Filteau
and
Des
Coteaux,
1998;
Curran,
1992;
Pierson
and
Ginther,
Pawshe et
al., 1994;
Karen et
al., 2007;
Ali and
Fahmy,
2008
Allen and
Goddard,
1984;
Ginther,
1986;
Sertich,
1997;
Pycock,
2007;
Holder,
2007
Garcia et al.,
1993;
Buckrell et
al., 1986;
Karen et al.,
2003; Santos
et al., 2007;
Romano and
Christians,
2008; Harsh
et al., 2008;
Suguna et
Michael,
1988;
Holtz,
1982;
Almond et
al., 1985;
Jackson,
1986
England
and Allen,
2008;
Barr,
2008
Vyas
et
al.,
2002
Zambell
i
and
Prati,
2006

34. 30 - day - old embryo 40 - day - old embryo
Fetus on day 59 with the fi rst view of the ribs embryonic mortality observed at day
30
Cow

35. Day-10 Day 30:
Day 48: Day 60
Day 35
Twin pregnancy
Mare

36. Day 41 stomach
Day 35 Yolk
sac
Day 25, head
Day 55
Bitch

37. Sheep/Goat

38. Sow

39. Laboratory tests
for
pregnancy diagnosis

40. Progesterone hormone assay
 Specificity of around 98% (zaied et al., 1979; laing et al., 1980; waldman, 1993;
gowan et al., 1982; pennington et al 1985; nebel et al., 1987)
 Accuracy of the test is low (75%) because of early embryonic death
Based on estrogen
 One of the earliest written records of a urine-based pregnancy test can be
found in an ancient egyptian document
 Wheat seeds when they are soaked in urine from pregnant cows which inhibits
germination compared to urine from non-pregnant cows which stimulate
germination has been described to be known as punyakoti test (nirmala et al.,
2008).
 The estrone sulfate is produced by the fetomaternal axis or the conceptus and
therefore its presence in urine, milk, feces or blood is an indicator of pregnancy

41. Cuboni test
 Positive -dark, only green fluorescent color in the lower sulfuric acid
layer
 Negative- absence of fluorescent color and presence of a brownish
color
effective beyond 150 days of gestationand also predicts fetal viability
Phenolsulphonic acid test
 Gives a pink to cherry red color if the urine is from pregnant mares
(mayer, 1944; benesch and wright, 2001).
Mucin test
 Vaginal mucus from a pregnant mare showed dark staining columnar
epithelial cells (pregnancy cells) (kurosawa, 1931)
 Efficacy of 94% from day 70 to end of gestation (miller and day, 1938;
day and miller, 1940)

42.  Barium chloride test
 Non pregnant cows a white precipitate is formed
 Pregnant animals the urine remains clear (temblador and landa, 1971)
 Accuracy of the test was described to be 70-95% (maslov and smirnov, 1965;
elpakov and cyganok, 1966; akmadeev and vasilev, 1967) from 15 to 210 days
of pregnancy
 Camels the test was considered to be 85% accurate between days 50-90 of
pregnancy (banerjee, 1974)
 Milk alchohol coagulation test
 In this test there is coagulation of milk from pregnant cows when mixed with
equal quantities of alchohol and allowed to stand for 1-3 hours (linkes, 1930;
rutz, 1932; stancev and angelov, 1966; kavani, 1976)
 Copper sulfate test
 1 ml of milk when mixed with a few drops of 3% copper sulfate coagulates if
the animal is pregnant (tembldor and acosta, 1971; kavani, 1976)

43. Species Day of detection Reference
Mare Maternal estrogen high after 60 days
Conjugated urinary
estrogens high after 150 days
Sist et al. (1987); Cox
(1971); Bhavnani and
Woolever, 1978
Cow Day 100 of gestation Hamon et al. (1981);
Robertson et al., 1978
Goat High after day 50 of gestation Refsal et al (1991) Chaplin
and Holds worth (1982)
Sow Rise start at 20 days peak at 25-30 days
followed by a decline at
45 days and again a rise at 70-80 days
to term
Cunningham, 1982 Seren
et al.(1983) Robertson et
al.(1978); Gutherie and
Deaver, 1979
Bitch Slightly increased at implantation and
remain constantly high for
rest of gestation and decline 2 days
prepartum
Concannon et al. (1975)
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
Appearance of estrogen in domestic animals during pregnancy

44. Pseudo-Pregnancy
&
Its Treatment

45. Pseudo-pregnancy
 6-20 weeks after oestrum
 Maternal behaviour, nesting, digging,
mothering inanimate objects, lactation and
milk release, weight gain, abdomen
enlargement.

46. Treatment
 Elizabethan collar
 Water removal overnight for 5-7 days
 Frusemide 0.25 to 0.5 mg per Kg
 Bromocriptine
 Cabergoline for 10 to 14 days

47.  Dopamine agonist
 Dopamine is prolactin inhibitory hormone.
 Prolactin is major luteotropic hormones
throughout the luteal phase. In pregnant and
nonpregnant bitch.
 Bromocriptine and cabergoline are ergot
alkaloids, hve strong D 2 receptor activity
and suppress PRL secretion and thereby
progesterone levels.

48.  Bromocriptine 0.1mg per kg PO BID after
day 30. for minimum 6 days or until all
fetuses are expelled.
 Cabergoline 5 microgram per kg for 5 days
after 32 days