“Detection of the pregnant/ non-pregnant cow/ buffalo, heifer, as soon as possible after a successful/ unsuccessful insemination, is important to ensure good reproductive and / productive performance in dairy and beef animals” -Omics revolution and integration of all -omics sciences with a systems approach could help find solutions towards finding early pregnancy biomarkers.

2. “Detection of the pregnant/ non-pregnant cow/
buffalo, heifer, as soon as possible after a
successful/ unsuccessful insemination, is important
to ensure good reproductive and / productive
performance in dairy and beef animals”

3. ∗ Ovulation : 20-36 h of 1st
detection of estrus
∗ Fertilization : within hours of ovulation
∗ Transport to Uterine horn : 3½ to 4 days
∗ Hatching: Day 7
∗ Maternal recognition of pregnancy: Day 14-19
∗ Implantation : Starts on Day 22 - completed by Day 40
∗ Pregnancy losses
∗ Fertilization losses : 5-10%
∗ Early embryonic mortality : 10-20%
∗ Late embryonic losses : very few (<5%)
∗ Post implantation losses : rare
∗ Most reliable indication of successful pregnancy: implantation i.e. Day 40+
Major Landmarks:
Bovine/ Buffalo Early Pregnancy

4. time……the commencement of prenatal care
Changes in management, feeding and health
care……to promote a healthy pregnancy/ early
pregnancy termination if so desired
improve reproductive efficiency and pregnancy
rate by
decreasing the interval between AI services
increasing AI service rate
Economic considerations…. to farmers, dairy
entrepreneurs, policy makers
Early pregnancy detection: why?

5. ∗ Cattle and buffalo husbandry…….. Taking shape of industry
∗ Intensification of reproductive management in dairy/ beef
herds and the implementation of AI are creating the need
for more accurate and timely diagnoses of pregnancy
∗ At the same time, there is a shortage of large animal
veterinarians/ para-veterinarians in many
regions…..reasons!!!
∗ Shortage………growing realization of pressure on limited
human resource to complete a large number of pregnancy
diagnoses
∗ In some cases, there is the desire to perform the pregnancy
exams sooner after insemination so that non-pregnant
cattle can be identified earlier and resynchronized for a
second AI
Early pregnancy detection: why?

6. ∗ The human pregnancy produces copious
amount of a hormone called hCG that passes
into the urine and can be detected by a simple
lateral flow ELISA test (Fletcher, 1986)
∗ Unfortunately cows/buffaloes do not make
bovine chorionic gonadotropin (or any such
molecule that is readily detectable in the
urine…..till now)
∗ Outcome………so a simple test that is similar
to the human test is not available!!
If cattle & buffalo were humans then the solution
would be simple ☻

7. “It creates an opportunity for the
identification of pregnancy markers for
application in chemical pregnancy testing
(for example, blood/ urine/ saliva/ other non-
invasive biological tests for pregnancy)”

8. • Direct methods: Rectal/ Trans-rectal / Per-rectal
palpation, Ultrasonography
• skill and experience / instrumentation
• Indirect methods
• Immunoassays based on Progesterone and PAGs
• Sensitivity, Specificity and Accuracy
• Pregnancy marker molecules???
• prerequisites
• specifically up- or down-regulated
• least affected due to non-animal factors
• present in easily accessible body fluids
• expressed over a considerable period
• No residue after pregnancy
Early Pregnancy detection

9. Accurate method of pregnancy
diagnosis only from day 30-45
post mating
Risk of iatrogenic embryonic
mortality
No information about the
viability of the embryo/fetus
during stages of pregnancy
Compared to cattle, rectal
palpation in buffaloes must be
gentle as the rectal mucosa is
more fragile and bleed easily
Transrectal Palpation

10. In cow/ buffalo can be adopted successfully from Day
28-30 after service
At Day 30 it is possible to observed the fetal heart
beats
The sensitivity reaching 100% from Day 31 after mating
onward
Main advantage-can give an accurate diagnosis
It is necessary to have a proficient operator
Transrectal ultrasonography

11. This test is based on the milk progesterone
concentration 21-24 days post breeding.
The milk progesterone parallels blood progesterone.
It is important to know that this can only be used with
known AI or breeding dates and the test cannot be
done randomly in a herd.
It is based on the fact that if progesterone is low 21
days post-breeding then there is no chance that the
cow can be pregnant.
Remember that a cow must have progesterone if
pregnant Therefore the test is 100% accurate at non-
pregnancy diagnosis when the progesterone is low.
Milk progesterone assay

12. If Progesterone is high 21 days after breeding the cow 'should' be
pregnant

13. ∗ If Progesterone is low 21 days after breeding (or anytime) the
cow cannot be pregnant
∗ However, if the progesterone is high, the test is only 80 % accurate
at diagnosing cows pregnant…..because there may be mistakes
and cows may be bred that are not actually in heat…….
∗ These cows would therefore have high progesterone 21 days later, yet
not be pregnant
∗ Also cows with short and long cycles can cause misinterpretation of
the test

14. ∗ A false positive if breeding was not done at estrus
∗ The progesterone 21 days later may be high and indicate a
pregnant cow
∗ The blue line indicates the 2 ng/ml threshold value for positive
∗ These cows would therefore have high progesterone 21 days later, yet
not be pregnant
∗ Also cows with short and long cycles can cause misinterpretation of the
test

15. ∗ Trend in serum progesterone (P4) related to pregnancy
outcome in buffalo cows artificially inseminated (day 0=AI).
(Barbato and Barile, 2012)

16. Oestrous Cycle in Buffalo
(Source: FAO)

17. Perioestrus endocrine
changes in the buffalo cow
(Seren et al., 1994)

18. ∗ The Early Pregnancy Factor (EPF) or Early Conception Factor (ECF) test was
reported accurate on day 2 but the test was later shown to be inaccurate at
any time. Other tests measure the biological response to interferon-t (day
16 to 18), progesterone in blood or milk (day 18 to 23), or pregnancy
associated glycoproteins (PAG; after day 25)
Some candidate molecules associated
with early pregnancy in cattle
(Lucy et al., 2011)

19. This protein molecule was first identified in
pregnant mice (Morton et al., 1987) and later in
sheep and cattle (Nancarrow et al., 1981) by
using the rosette inhibition bioassay
With this assay, EPF was detected in the serum
of all mammals tested within 24 to 48 h of
fertilization and disappeared within 24 to 48 h
after death or removal of embryo (Morton et
al.,1987)
Early Conception Factor (ECF)

20. A chemical test for pregnancy termed Early
Pregnancy Factor (EPF) or Early Conception Factor
(ECF) was proposed and marketed in the 1990’s
(Concepto Diagnostics, Knoxville,TN)
This molecule was supposedly present in the blood of
pregnant cattle within two days after conception
The exact nature of this molecule (an
immunosuppressive glycoprotein protein) and how it
got into circulation were not well defined
A kit for pregnancy diagnosis reached the market but
three different studies found that the kit was
unreliable for pregnancy diagnosis (Cordoba et al.,
2001; Gandy et al., 2001; Ambrose et al., 2007)

21. Pregnancy-associated glycoprotein (also called
pregnancy-specific protein B, pregnancy specific protein
60 and SBU-3 antigen) constitute a large family of
glycoproteins expressed in the outer epithelial cell layer
of the placenta of Eutherian species
They are synthesized by mono and binucleate
trophoblastic cells, some of them being secreted in
maternal blood from the moment when the conceptus
becomes more closely attached to the uterine wall and
formation of placentomes begins
Pregnancy-associated Glycoproteins

22. ∗ A 14 weeks buffalo fetus with visible placentomes (button-
shaped structures) characteristic of ruminant placentation

23. The PAG (Pregnancy-associated glycoprotein) family
were isolated from cotyledons of cow, ewe, goat,
buffalo, bison, moose and elk
PAG belong to the aspartic proteinase gene family
However, most PAG molecules are assumed to be
enzymatically inactive due to key mutations within their
binding cleft
Cattle, sheep and probably all ruminants possess many,
possibly 100 or more, PAG genes

24. In bovine, 22 boPAG genes (boPAG-1 to boPAG-22)
were cloned and fully sequenced
The number of PAG gene is lower in ovine (15 genes)
and caprine species (about 11 genes)
Several bovine, ovine, caprine and buffalo closely
related PAG molecules (63–87% N terminal amino acid
identities) have been made available and have been
used to produce antisera for radioimmunoassay (RIA)
development

25. 1. BioPRYN
BioTracking, LLC, Moscow, ID
http://www.biotracking.com/
2. DG29
Conception Animal Reproduction Technologies, Beaumont, QC
http://www.conception-animal.com/test_an.html
3. IDEXX Bovine Pregnancy Test
IDEXX Laboratories, Inc., Westbrook, ME,
http://www.idexx.com/view/xhtml/en_us/livestock-poultry/ruminant/lpd-bovine-
pregnancy-test.jsf
∗None of these tests are cow-side or on-farm, so blood samples must be collected
by farm personnel and sent by courier to a local or regional laboratory that runs the
assay. Results are then returned to the farm via email, usually within 24 to 72 h.
Currently, three non-pregnancy tests based on detection
of PAGs in maternal serum are commercially
available for use by dairy farmers

26. Bovine interferon tau (boIFN-t) is one of the principal
proteins secreted by the bovine conceptus from 16 to 25
days
It has been showed that the “embryonic signals”
emitted by the conceptus of the ruminants, initially
called trophoblastin or trophoblastic protein, constitute
actually the same subclass of interferon, pertaining to
the family of interferon alpha
In the ewe, the interferon tau (ovIFN-t) is produced
between 13th to 21st days of gestation
Interferon tau (IFN-t) regulates uterine Mx gene
expression in ewes; however, the only known role for
Mx gene is in the immune response to viral infection.
Interferon Tau

27. It is hypothesized that Mx gene functions as a conceptus-
induced component of the antiluteolytic mechanism
and/or regulator of endometrial secretion or uterine
remodeling during early pregnancy
Molecules belonging to the subclass of IFN-t were also
identified on the level of the trophoblastic cells of the
caprine conceptus from 14 to 17 days.
However from the 18th day, when the establishment
starts, the caprine IFN-t is not detected any more,
suggesting that in this species, other factors are
necessary from the 18th day of gestation to provide the
luteotropic function
These effects range from pregnancy recognition signaling
in ruminants through IFN tau to effects on cellular
functions of the uterus and uterine vasculature

28. CNAs (Circulating Nucleic Acids) can also
be used as markers for detection of
early pregnancy in cattle
(Mayer et al., 2012)

29. Relaxin can be determined in the peripheral circulation
of pregnant bitches at 20-30 days of gestation, whereas
it is absent in non-pregnant bitches at all stages of the
reproductive cycle
This relaxin is known to be produced by the placenta in
the bitch and cat and is thought to contribute to its
maintenance by inhibiting uterine activity
In the domestic dog it has been established as a
pregnancy-specific hormone
Relaxin assay…..not for
bovines/buffaloes ?

30. Ideal pregnancy biomarker characteristics
Specifically up-regulated or down-regulated during
pregnancy
Expressed over a considerable period of time to give
ample time for diagnosis
Present in easily accessible body fluids like serum, milk,
urine, vaginal discharges etc
Preferably to have the ability to reflect age as well as
viability of the conceptus
Least affected by non-animal factors like feed,
environment and drug interactions
Testing method to have the ability to reveal the result
immediately

31. None of the present day methods qualifies as an
ideal diagnostic due to limitations of accuracy, later
stages of applicability as well as the requirement for
elaborate instrumentation and laboratory set-up
Further investigations on developing novel early
pregnancy diagnostics for livestock species,
especially buffaloes, are in progress and hopefully,
very soon we should have a suitable marker /
diagnostic test for early pregnancy in livestock
species including buffalo

32. Roadmap through Integrated
Genomics…….

33. A. MCQ Type
1. Most reliable indication of successful pregnancy in buffaloes
occurs at __________ post insemination
(a) Day 15 + (b) Day 23 + (c) Day 30 + (d) Day 40+
2. if progesterone is low 21 days post-breeding then
(a) There is good chance of cow being pregnant
(b) There is no chance that the cow can be pregnant
(c) The corpus luteum is fully formed
(d) None
3. Which of the followings is not a biomarker associated with
early pregnancy in cattle
(a) EPF (b) insulin (c) PAG (d) IFN-tau
Questions

34. 4. Which of the followings regarding ECF is not true?
(a)It is a nucleic acid molecule
(b)It was first identified in pregnant mice
(c)It was identified by using the rosette inhibition bioassay
(d)It was detected in the serum of all mammals tested within 24 to
48 h of fertilization
5. Which of the followings are true for PAGs
(a)These constitute a large family of glycoproteins
(b)These belong to the aspartic proteinase gene family
(c)Most PAG molecules are assumed to be enzymatically inactive
due to key mutations within their binding cleft
(d)B and C only
(e)All of the above

35. 6. Which of the following statements is false:
(a)Bovine interferon tau (boIFN-t) is secreted by the bovine conceptus
from 16 to 25 days
(b)Interferon tau (IFN-t) regulates uterine Mx gene
(c)CNAs (Circulating Nucleic Acids) can also be used as markers for
detection of early pregnancy in cattle
(d)Relaxin is present in non-pregnant bitches and absent in pregnant
bitches at 20-30 days of gestation
B. Q. Answer the followings in maximum 2-3 sentences only.
(1)Early Pregnancy Biomarker
(2)Progesterone for pregnancy diagnosis
(3)Relaxin assay
(4)Mx Gene
(5)Name 3 Candidate molecules for early detection of pregnancy
(6) Early pregnancy diagnosis
(7)Name three non-pregnancy tests based on detection of PAGs in
maternal serum

36. C. Q. Short Notes (8 lines)
∗ (1) Ideal Pregnancy biomarker characteristics
∗ (2) Why early pregnancy biomarkers are required?
∗ (3) ECF
∗ (4) Interferon-tau
∗ (5) Pregnancy associated glycoproteins
D. Q. Long notes (3-4 pages)
1. Discuss in detail about the early pregnancy biomarkers for early
pregnancy diagnosis in livestock with reference to cattle and
buffaloes.

37. E. Write True/ False for the given statements:
1.Per-rectal palpation and Ultrasonography are indirect method of
pregnancy diagnosis (Answer: False)
2.If Progesterone is high 21 days after breeding the cow 'should' be
pregnant (Answer: True)
3.If Progesterone is low 21 days after breeding (or anytime) the cow
cannot be pregnant (Answer: True)
4.Transrectal palpation is an accurate method of pregnancy diagnosis
only from day 30-45 post mating (Answer: True)
5.In the domestic dog relaxin has been established as a pregnancy-
specific hormone (Answer: True)