This document discusses different methods for detecting pregnancy in animals. It covers clinical methods like rectal palpation, ultrasonography, radiography, and ballottement. It also discusses laboratory tests detecting hormones like progesterone and estrogens. Accuracy and time of detection for each method varies by species, from as early as day 18 for fetal fluid by ultrasonography in some species, to day 70-120 for visibility of the fetus by radiography in others. The goals of pregnancy detection methods are outlined as 100% accuracy, no false positives or negatives, and determining pregnancy as early as possible.

1. DIFFERENT METHODS OF PREGNANCY
DETECTION IN ANIMALS
SUBMITTER BY
MUHAMMAD RUMMAN ASLAM
2015-VA-215
SUBMITTED TO
DR ILYAS NAVEED

2. WHY?
 Improving reproductive performance (thompson et al., 1995)
 Earlier the pregnancy diagnosis performed, the more profitable is the return for dairy
cows and buffaloes (oltenacu, 1990; duggal et al., 2001a; youngquist,1997)
 Management
 Feeding
 Sale
 Monitor development of pregnancy
 Confirm absence of twin
 Monitor early embryonic death

3. GOALS
100% accuracy
No false positives
No false negatives
Determine the pregnancy as early as possible
The ability to age the conceptus
Be able to determine the viability of the conceptus
Possibly determine the sex of the fetus

4. • Non return to estrus
• Cocking of the tail [appears
14 to 15 days after fertile
mating and known to be
95% reliable (Banerjee,
1974; Banerjee et al., 1981)]
• Recto-genital palpation
• Ultrasonography
• Radiography
• fetal ballottement
• Essay of hormones
• Chemical tests
• Biological methods
Visual Laboratory Clinical

5. CLINICAL METHODS:
1.Recto-genital Palpation:

6. FETUS
 2 months mouse
 3 months rat
 4 months small cat
 5 months large cat
 6 months beagle dog
Crown rump length(measurement of the length of fetuses from the top
of the head (crown) to the bottom of the buttocks (rump)

7. BOVINES AND CATTLE
•Amniotic Vesicle 28 day
•Slip membrane test 33day
•Placentome 65-70 day
•Fetus itself 120 day

8. MARE
 Changes in the cervix, uterus and ovaries
 As early as 16 to 18 days after ovulation, the cervix of the pregnant marebecomes tightly closed, firm, slender,
and elongated
 Between 16 to 30 days the cervix can be with experience palpated on the floorof pelvis as a rigid firm structure
• Uterus
 The uterine tone appears at days 15-16 and continues up to day 48 to 55 (sertich, 1997)
 The bulge progressively increases in size and is palpable in most mares by day 30-35
• (approx 3-4 cm in diameter)
 By day 42-45, the conceptus occupies approximately half of the gravid horn and is5 to 7 cm in diameter. The
uterine wall over the bulge inthin.
 The pregnancy is like an elongated football and is nearly similar to a 60day

9.  Location of the uterus
 The uterus is located in the pelvic cavity or just at the pelvic brim until day90
 At 100-200 days, the gravid uterus is positioned cranial to the pelvic brim inthe
• abdominal cavity (sertich, 1997)
 Ovarian palpation
 Position of the ovaries up to 60 days of pregnancy is similar to that for thenon- 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 isa positive indication for pregnancy (3
to 5 months)
 Form 5 months of pregnancy onwards, the ovaries usually are not palpable asthey
• are under the broad ligaments

10. FEMALE CAMEL
 Persistence of the CL is one of the earliest sign of pregnancy as otherwiseluteal phase
is very short
 Left uterine horn is inherently longer than the right horn
 Earliest detection of uterine change (increase in diameter and appearanceof
fluctuation) is palpable at about 40 days
 Between 60-70 days left uterine horn is increased about twice to its non gravidsize
• has a thin wall and fluctuates
 Fetal membrane slip and placentome not seen

11. 2.ULTRASONOGRAPHY
 Vesicle/fetal fluid is visible in most species between days 18-22 except in themare in which it appears earlier (day 10-
16)
 Fetal heart beat can be seen between day 24-30.
 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 thebitch
 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 alittle 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)

12. Ultrasonographic features of early pregnancy in various species
Sonographic Structure
Appearance(days post
mating)
Cow Buffalo Mare Sheep/
Goat
Sow Bitch Camel Cat
Fetal fluid 18-20 18-22 10-16 20-25 18-20 18-20 17-18 10-16
Fetal Heartbeat 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 Curranet al.,
1986;
Pawsheet al.,
1994;
Karen et al.,
2007;
Ali and
Fahmy,
2008
Allenand
Goddard,
Garcia etal., 1993; Michael,
1988;
England and
Allen,
Vyaset
al., 2002
Zambelli
and Prati,
2006
Filteau and 1984; Buckrell et Holtz, 2008;
Des Ginther, al., 1986; 1982; Barr,
Coteaux, 1986; Karen etal., Almond et 2008
1998; Sertich, 2003;Santos al., 1985;
Curran, 1997; et al.,2007; Jackson,
1992; Pycock, Romanoand 1986
Pierson 2007; Christians,
and Holder, 2008;Harsh
Ginther, 2007 et al.,2008;
1984; Sugunaet

13. RADIOGRAPHY
 Sheep and goat, fetuses are visible by day 70 of gestation (grace et al.,1989; noakes, 1999)
 Bitches fetal skeletons are visible with high accuracy only by the sixth weekof pregnancy (toal et al., 2005)
 Fetal skulls are visible by day 45 and the entire fetal skeleton is visible by the endof seventh week of gestation.
 Signs of fetal death as seen by radiography include the spalding sign, (which isthe overlapping of the cranial
bones), gas shadows in the fetal heart and stomach and tightly flexed spine (seen in fetuses died for long time)
(jackson,2004)
 Fetal skeletons begin to calcify only after the sixth week in sows and hence radiography should be
performed only after this time for pregnancy diagnosisin sows (rapic, 1961)

14. Radiography of Fetuses.
Shille VM (1982)
Radiographic Pregnancy Diagnosis
Verstegen J (2009)

15. ABDOMINAL BALLOTTEMENT AND PALPATION
 Possible to some extent in cows during late gestation (7 monthsonward)
 Sheep and goats rectal abdominal palpation (by using a glass rod placed in the rectum to lift the uterus which is
palpated through abdomen) has been suggested(ott et al., 1981; hulet,1972; chauhan and waziri, 1991;)
 Similarly bimanual palpation for pregnancy diagnosis (palpation of uterus through fingers in the rectum and lifting
the abdomen) has been reported for smallruminants (chauhan and waziri, 1991; kutty, 1999)
 Palpation of fetuses through the abdomen is possible in sheep and goat onlybeyond 4 months of pregnancy
 In bitches beyond day 55 it is easy to diagnose pregnancy by the palpation ofthe fetuses
 At about 30-35 days the accuracy is high (87%) and depends on the palpationof tense conceptual swellings (6
to 30 mm in diameter)

16. RECTO-ABDOMINALPALPATION
Hulet CV (1972)

17. LABORATORY TESTS FOR PREGNANCY DIAGNOSIS
Progesterone hormoneassay
 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 embryonicdeath
Based on estrogen
 One of the earliest written records of a urine-based pregnancy test can be foundin 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 conceptus and therefore its presence in urine, milk, feces or blood is an
indicator of pregnancy

18. CUBONITEST
 Positive -dark,only greenfluorescentcolor in the lower sulfuric acid layer
 Negative- absenceof fluorescent color and presenceof abrownishcolor effective beyond 150days of
gestationand alsopredicts fetal viability
Phenolsulphonic acidtest
 Givesapink to cherry red color if the urine is from pregnant mares (mayer, 1944; benesch and wright,2001).
Mucin test
 Vaginalmucusfrom apregnantmare showed dark stainingcolumnarepithelial cells(pregnancycells)
(kurosawa,1931)
 Efficacyof 94%from day 70to end of gestation (miller and day,1938;day and miller, 1940)

19.  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)

20. Species Day of detection Reference
Mare Maternal estrogenhigh after 60days
Conjugatedurinary
estrogens high after150days
Sistet al. (1987);Cox
(1971);Bhavnaniand
Woolever,1978
Cow Day 100ofgestation Hamon etal. (1981);
Robertson etal., 1978
Goat Highafter day50of gestation Refsalet al (1991)Chaplin
and Holds worth(1982)
Sow Risestart at 20dayspeakat 25-30days
followed by adeclineat
45daysand againarise at 70-80daysto
term
Cunningham, 1982Seren
et al.(1983) Robertson et
al.(1978); Gutherie and
Deaver,1979
Bitch Slightly increasedat implantation and
remain constantly highfor
restof gestation and decline 2days
prepartum
Concannon etal. (1975)
Sheep Detectableby day70;rise thereafter till 2
daysprepartum
Illera etal., 2000;
Worsefold etal., 1986
Buffalo Appearat day150of gestation in the serum Prakash &Madan (1993);
Kamonpatana,1984
Camel Increasestart at day50andpeakfrom day
90-300
Skidmore etal., 1996
APPEARANCE OF ESTROGEN IN DOMESTIC ANIMALS DURING PREGNANCY

21. BIOLOGICALTESTS
 eCG continues to be secreted from day 40 to 120 days of pregnancy (Roserand Lofstedt, 1989)
 Aschiem zondek test (Miller and day, 1938)
 Friedman test (chicchini and chiacchiarini, 1963)
 On farm tests
some commercial kits are currently available which can detect the presence of ecgin blood of mares between 40-
100 days of pregnancy with 96-98%accuracy
 Pregnamare is one such kit which requires 5 drops of blood collected fromthe muzzle by using the lancet
provided with the kit

22. PREGNANCY ASSOCIATED GLYCOPROTEINS(PAG)
 Producedin various ruminant speciesincludingcattle, buffalo, sheepand goats (humblot et al., 1990;karenet
al., 2003;karenet al., 2007)
 Twopregnancyspecific proteins (PSP)Aand Bhavebeen isolated from bovine fetal membrane extracts
(butler et al., 1982)
 PSP-Awasidentified asaafetoprotein and PSP-Bwasfound to be specific to the placenta
 Thesemoleculesappear in the maternal circulation and canbe determined with accuracyfrom 29 to 30days
post breeding
 PAGcontinuesto be existent in maternal blood for the entire pregnancyand up to 100days postpartum
 Assayinvolvesradioimmunoassayon serum

23.  Sensitivity and specificity of PSPBbasedon RIAis known to be 92.0%and 82.6 to
91.9%from 29 to 30dayspost insemination(szenciet al.,1998)
 ELISAtechniques have been developed that detect the PAGmolecule in the
serumof cows(breed et al., 2009;green et al., 2005;green et al., 2009;silva et al.,
2007)
 Limitations to the wide spread useof this test is non availability of the protein in
milk or urine presence of PAGup to 100days postpartum (which interfere with
subsequentdetections)
 Non availability of cow sidecommercially availablekits for its detection
 Existence of PAGhasbeen documented in bovine milk (gajewskiet al., 2008)

24. EARLY PREGNANCYFACTOR
 Protein molecule was first identified in pregnant mice (morton et al., 1987)
 Sheep and cattle (nancarrow et al., 1981)
 Using the rosette inhibition bioassay EPF was detected in the serun 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)
 Developing embryo bears antigens foreign to the mother; hence immune rejection of the early embryo may occur
 Immunosuppressive early pregnancy factor (EPF) appears as early as 6 to 48 h of mating which functions to
suppress the maternal immune response thereby allowing for pregnancy to proceed (shaw and morton, 1980)
 Measurement of EPF activity may be useful as an indirect method of pregnancy diagnosis
 Commercially marketed kit is available in the US (ECF test, concepto diagnostics
knoxwille, TN)

25. RELAXIN ASSAY
 Pregnant bitches at 20-30 days of gestation, (steinetz et al,1989)
 Produced by the placenta in the bitch and cat and is thought to contribute toits maintenance by inhibiting
uterine activity.
 Established as a pregnancy-specific hormone (steinetz et al., 1987,1989)
 Cat it appears during the third week of pregnancy, with concentrations decliningjust before parturition (stewart
and stabenfeldt,1985)
 Molecule has also been detected and used successfully for pregnancydiagnosis
(carlson and gese, 2007; bauman et al., 2008)
 Commercially available canine relaxin enzyme-linked immunoassay (ELISA) (reprochek, ®a synbiotics
corporation, san diego, CA, USA)

26. CONCLUSION
 Pregnancy diagnosis by rectal palpation is the most frequently performedprocedure
(rosenbaum and warnick, 2004)
 Use of ultrasonography is growing (fricke,2002)
 Rapidly and accurately diagnosed with ultrasound at 26 days afterinsemination
under most on-farm conditions (filteau and descôteau,1998)
 The cost per pregnancy diagnosis was between $1.50 to $3.00 for palpationand
between $2.40 and $4.50 for ultrasound (rosenbaum and warnick,2004)
 Need pregnancy diagnosis kit available to farmer