Article WMC001305

Article ID: WMC001305

Methods Of Pregnancy Diagnosis In DomesticAnimals: The Current StatusAuthor(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 Veterinaryand 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 .WebmedCentral REPRODUCTION 2010;1(12):WMC001305

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Methods Of Pregnancy Diagnosis In DomesticAnimals: The Current Status

Abstract

Methods of pregnancy diagnosis in the domesticanimals can be classified into visual, clinical andlaboratory methods. In most domestic animals clinicalmethods are currently used. Visual methods are farfrom perfect in domestic animals. In animals like cattle,buffaloes and mare’s recto genital palpation andtrans-rectal ultrasonography continue to be themethods of choice for an accurate and earlypregnancy diagnosis. In sheep, goat, sow, bitch andcat ultrasonography is the only reliable method ofpregnancy diagnosis. In the camel cocking of the tail isan effective visual method of pregnancy diagnosisand recto genital and ultrasonography are also useful.Laboratory methods used in past and developed inrecent years are described. The efficiency of a few ofthese procedures is likely to be improved in nearfuture.

Introduction

The diagnosis of pregnancy (cyesiognosis) has beensought since long by farmers for curiosity however, itis essential for profitable animal husbandry especiallyin the productive animal species. For an economicaldairy farm, cows must calve every year, and tomaintain this sequence, identifying pregnant animalsat an early date seems imperative. In the currentsystems of planned breeding, diagnosis of pregnancywould help to evaluate the therapies at an early dateand devise alternative manipulations. In somesituations in the pet practice pregnancy may not bedesirable by the owners and an early diagnosis wouldhelp in termination of these unwanted pregnancies. Anearly pregnancy diagnosis is essential in mares totease them if non pregnant, and try to get thempregnant in the same season. It therefore, appearsthat early diagnosis of pregnancy is essential in animalmanagement for economic reasons. In manydeveloping countries, farmers often present theiranimals for pregnancy diagnosis very late when muchof their time is lost in maintaining non pregnant cows.

There is a need to educate farmers to get theiranimals checked for pregnancy at an early date as ithas been shown that earlier the pregnancy diagnosisperformed, the more profitable is the return for dairycows and buffaloes (Oltenacu, 1990; Duggal et al.,2001a; Youngquist, 1997).

Review

Methods of Pregnancy DiagnosisA variety of approaches have been evaluated anddeveloped during the past and recent years, some ofwhich have some limitations to their wide scale use.The methods of pregnancy diagnosis have beenclassified into two (direct and indirect) or threecategories i) Visual methods, ii) Clinical methods, iii)Laboratory tests.Visual MethodsNon return to estrusWhen an animal is mated and it does not return toestrus the owner usually thinks that the animal hasbecome pregnant and hence has not returned toestrus. This happens because during pregnancy, theconceptus inhibits the regression of the corpus luteumand thus, prevents the animal from returning to estrus.However, many a times the animal does not return toestrus because of non regression of CL due toreasons other than pregnancy. Moreover, in theseasonally breeding species the animal may not returnto estrus (when mating is done during the end of thebreeding season) because the season was over.Anestrus, and the rare occurrence of gestationalestrus in cattle and buffaloes can affect the reliabilityof non return to estrus as a method of pregnancydiagnosis. Moreover, difficulty in estrus detection andsilent estrus render this method of pregnancydiagnosis unsuitable for the buffalo. Likewise, estrusdetection methods used for sheep, goat and maresneed to be properly designed so as to make efficientuse of non-return to estrus as a method of pregnancydiagnosis in these species. In dairy cows non-returnrates usually over estimate true pregnancy diagnosis(Kidder et al., 1954) and are also affected by thedetection procedure used (Foote, 1974). Moreover,



estrus expression appears to be reduced in intensityand duration in the present day dairy cows leading tolower estrus detection efficiency (Dransfield et al.,1998; Lucy, 2001). Therefore, the probability ofmisdiagnosis of pregnant females by estrusobservation appears to be increased. This may beconfounded by a small proportion of pregnant cowsand buffaloes expressing estrus (Gilmore, 1952;Agarwal and Tomer, 1998) and some cows expressingprolonged inter-estrus intervals of around 24 days(Sartori et al., 2004). Therefore, non return to estrus isan unreliable procedure for pregnancy diagnosis inmost domestic animal species.Besides the non-return to estrus a few of other visualsigns of pregnancy appearing in late pregnancyinclude increase in the size of the abdomen,development of the udder specially in dairy heifers (4months onwards), slight vaginal discharge (from 4-5months onward in dairy cows) and movements of thefetus visible externally (specially in fed cows on theright side of abdomen 6 months onwards). However,the accuracy of these visual diagnostic symptoms isalways low and a clinician must use them as asupplement to clinical diagnosis.Cocking of the tailThe pregnant female dromedary camels exhibit acharacteristic behavior when approached by a male ora person. The female assumes a stiffened posturewith the head held high and tail curled upwards.(Banerjee, 1974; Banerjee et al., 1981). This is knownas cocking of the tail. This behavior appears 14 to 15days after fertile mating and known to be 95% reliablefor pregnancy diagnosis in quiet and calm dromedaryfemales camels. However, many false positives canbe obtained in agitated females if the observer isuntrained. Tail cocking is also observed in thepregnant Bactrian camel although not with the sameintensity as in the dromedary female camel (Tibaryand Anouassi, 1997).Clinical methods of pregnancy diagnosisFour clinical methods of pregnancy diagnosis areavailable for pregnancy diagnosis in the variousdomestic farm and pet animal species i) rectalpalpat ion, i i ) abdominal bal lot tement, i i i )ultrasonography and iv) radiography. Each of thesemethods is discussed separately.Recto-genital palpationTransrectal palpation is the oldest and most widelyused method for early pregnancy diagnosis in dairycattle (Cowie, 1948). In most large domestic animalspecies like cattle, buffaloes, mares and femalecamels recto-genital palpation (with some limitations)is the easiest, cheapest and fastest method ofpregnancy diagnosis with little or nil harm to the

animal and its fetus when performed carefully. To alimited extent this method is used for pregnancydiagnosis in pigs. The method is the technique ofchoice being taught to veterinary graduates and paraveterinary staff. Transrectal palpation is considered tobe an accurate method of pregnancy diagnosis indairy cattle for a trained veterinarian after day 35 postbreeding (Euler, 1930; Gotze, 1940; Roberts, 1971;Zemjanis, 1970; Momont, 1990). A few studies pointout that the procedure may increase the risk ofiatrogenic embryonic mortality in dairy cattle (Paisleyet al., 1978; Vaillancourt et al., 1979; White et al.,1989) while others and the author do not concur withthis view. The procedure however, does not provideany information about the viability of the embryo/fetusduring earlier stages of pregnancy (Romano andMagee, 2001). Some of the basic principles which apalpator must understand and the precautions that hemust observe are mentioned below and then thefindings are described for cattle, buffaloes, mare,camels and pigs. Transrectal palpation of the amnioticvesicle as an aid in determining pregnancy status indairy cattle was described by Wisnicky and Cassida(1948), whereas slipping of the chorioallantoicmembranes between the palpator’s thumb andforefinger beginning on about day 30 of gestation wasdescribed by Zemjanis (1970).Basic principleThe genital organs lie usually on the pelvic floor duringearly pregnancy beneath the rectum in most speciesand in the abdominal cavity during late gestation. Thegenital organs can thus be palpated indirectly byplacing the hand in the rectum evacuated of the feces.The growth of the conceptus in either of the uterinehorn leads to sequential increase in the size,tenseness and palpable characteristics of the uterinecornua. Thus, with experience the palpator can feelthese changes in the uterus of a pregnant animal andwith fair to good accuracy predict pregnancydepending upon the species, stage of gestation andhis experience. Two bottle necks appear to besignificant while performing rectal palpation, i) theperistalsis that occurs in the rectal musculature, whichproduces obstacles in palpation and ii) ballooning ofthe rectal wall due to entry of air inside.The palpator must stop making movements of armduring a peristaltic wave (while still keeping his handinside the rectum) wait for 1-2 minutes and then startpalpation again when the peristalsis has subsided.The ballooning of rectum can be easily appreciated, bythe finding that the operator can move his hand up anddown in the rectum without resistance when therectum is ballooned. The operator must catch hold of apinch of rectal mucosa and move his hand back and



forth (known as back racking) without completelytaking it out. This will push the air inside, to theexterior and the rectal mucosa will then be closelyover the operator’s hand. After proper restraint and wearing of properclothing and also proper lubrication, the operator mustmake a cone of his hand and push it inside the rectum.The anal sphincter dilates and the hand enters insidethe rectum. The feces must be removed without takingout the hand completely. The cervix which is a hardround to oval or sometimes caudally enlargeddisfigured structure is the land mark for location ofgenital structures in cattle and buffaloes. This can belocated by sliding the hand in an arc like fashion fromdorsal to ventral side. The cervix is followed further tolocate the uterine body and the uterine horns. Thesestructures can be pulled caudally when located at thepelvic brim or further, by retracting the broad ligamentor hooking the inter-cornual ligament by the indexfinger. When the pregnancy is beyond 60 days thiscannot usually be done and the operator has to movehis hand further in the rectum, so as to locate theintra-abdominally placed uterus and palpate otherfeatures diagnostic of pregnancy. In the mare, the cervix is not easily palpableand hence the ovaries are the land mark for rectalpalpation especially for novice palpators. They arelocated about 10-20 cm cranial to the shaft of the iliumbone and about 5 to 10 cm below the lumbarvertebrae in non-pregnant mares, and in mare duringearly pregnancy. After locating one ovary the hand ispassed down the utero-ovarian ligament to locate theuterus. A striking feature of the genitalia of the femalecamel is the shortness of the right uterine horn. This isprobably, because of the existence of exclusive lefthorn pregnancies in this species (Tibary and Anouassi,1997). Recto-genital palpation is similar to thatdescribed for cattle and buffalo as the cervix is easilypalable. Rectal palpation is only possible to a limitedextent in the large sized sows. It is barely possible ingilts and in small sized breeds of sows.Restraint and clothing The animal to be examined should be properlyrestrained. Cows and buffaloes can be securelyrestrained in a Travis or chute. At many situationswhen this is not available the hind legs of cows aretied with a rope to avoid kicking and the head is heldsecurely. The tail is held to one side by an assistant.Pressing on the back relaxes the pelvic structures andreduces peristalsis. Buffaloes often resist tying ropeson hind legs and ropes are usually tied either on bothfore legs or one leg while folding it from the knee.

Buffaloes also kick less frequently by hind legs but,sometimes excitable, nervous buffaloes do not allowrectal palpation by moving frequently. Such animalsmay rarely need little sedation, examination in a sittingposition or examination at a later date. Palpators musttake every care to avoid damage to themselves by thekick of the animal. Thoroughbred mares are usually docile andcan be easily made to enter a chute however; somenervous mares or mares of other breeds need differentrestraint procedures. It is often safe for the palpator tomake the mare stand on one side of a small wall (3 ft)and the examiner stand on the other side of the wall.Stable wooden rails can sometimes serve the purpose.A good approach would be to tie both hind legs withone fore leg using rope. A twitch on the lower lip, earor nose and/or lifting one fore leg may be required.Nervous mares sometimes move sideways or try tojump resisting any examination. Such mares needtranquillization. Female camels are examined in a sittingposition with both hind, and forelegs tied togetherseparately with ropes. An assistant holds the headtightly. Vicious females often require pressure on theback by legs of persons standing on both sides toprevent side wise movements. The examiners must wear proper clothingincluding coveralls, gum boots and disposable plasticor rubber full arm sleeves. This is essential to protectthe examiner from contracting zoonotic disease andspoiling his clothes. Separate trousers and shirtsmade of dark coloured (green or blue) slightly thickcloth are easier for working compared to a singlecover all. Plastic long sized aprons are used by manyclinicians in the field. Sufficient lubrication must be used whileintroducing the hand in the rectum. Non-irritating soapand water or liquid paraffin is a suitable lubricant.Since, the feces of the mare are harder and theperistalsis stronger, more frequent lubrication isessential in the mare. Tail bandaging is alsoadvantageous in the mare to avoid tail hairs enter therectum and cause damage to the rectal wall.Precautions during rectal palpation When performed gently and carefully rectalpalpation is a non-invasive procedure. The followingpoints would be helpful in minimizing damage to theanimal and the examiner as well.1) Ruthless movements of the hand in the rectumshould be avoided. Avoid palpations during aperistaltic wave.2) Examiners must trim their nails and avoid usingdirty soiled sleeves.3) Rectal examination without a sleeve must be



avoided specially in mares to avoid contractingdiseases and obnoxious odors. Sleeves must bereplaced after examination of 2-5 animals, or betterafter each examination.4) Rectal palpation of an animal suffering from fevershould be extremely gentle or better avoided as theblood vessels are more fragile and bleed easily.Similarly examining an animal with rectal tear or rectalfistula is hazardous. Whenever, a clinician noticessuch conditions he must bring them to the notice of theowner or else he would blame the clinician. Rectalfistulas are an emergency in a mare and immediatetreatment including broad spectrum oral and parentalantibiotics and anti-inflammatory drugs must be givenalong with laxatives or else the mare may develop afatal peritonitis. The fistula/tear may be palpable as ablind pouch or a slit in the rectal mucosa that bleedswhen the hand is forwarded through these openings.5) Compared to cattle rectal palpation in buffaloesmust be gentle as the rectal mucosa is more fragileand bleed easily.6) Clinicians must assure that even it the animal kicksit does not harm them, and so also the palpators mustalso be cautious that sudden sideways movement ofthe animal with the operators hand inside can causefracture of the operators arm and hence due care mustbe exercised.7) Uncareful palpation of the uterine horns with unduepressure can cause rupture of the amniotic vesicle andloss of an early pregnancy and hence this must beavoided.Consequences of improper palpationBecause pregnancy in cattle can be terminated bymanual rupture of the amniotic vesicle (Ball andCarroll, 1963), many studies have investigated theextent of iatrogenic embryonic mortality induced bytransrectal palpation. Several studies have suggestedthat examining pregnant cows early in gestation bytransrectal palpation increases the risk of iatrogenicembryonic mortality (Abbitt et al., 1978; Franco et al.,1987; Paisley et al., 1978; Valliancourt et al., 1979;White et al., 1989), whereas other studies havesuggested that cows submitted for transrectalpalpation earlier during gestation had a decreased riskfor abortion or that palpation had no effect onsubsequent embryonic losses (Studer, 1969;Thurmond and Picanso, 1993). Although controversystill exists regarding the extent of iatrogenic embryonicmortality induced by transrectal palpation, otherfactors have a greater influence on calving rates thanpregnancy examination by transrectal palpation(Thompson et al., 1994). Furthermore, because therisk of embryonic mortality is high during the period ofgestation when cows are diagnosed pregnant by

transrectal palpation, and because most cows within aherd are submitted for pregnancy examination, it isimpossible for dairy producers and veterinarians todistinguish between iatrogenic losses occurring due totransrectal palpation and spontaneous losses thatwould normally have occurred in these cows.Palpable findings of pregnancy in cattle andbuffaloes Palpable findings of pregnancy have beendescribed in detail previously (Roberts, 1985). Thewater buffalo has an approximately one month longergestation period compared to cattle however; theparameters used in cattle are used in the water buffalofor pregnancy diagnosis by rectal palpation. Two things must be kept in mind by cliniciansin making positive diagnosis of pregnancy by rectalpalpations in cattle and buffalo. The first is, that whenthe palpator in unable to detect any of the palpablecharacteristics mentioned herein, he must neithercomment positively or negatively as both would befrustrating both to the clinician and the owner on alater date. He must better admit the fact that he is notable to detect out properly and the animal must bere-submitted for examination 15-30 days laterpreferably after a fasting. The second thing thatclinicians must keep in mind is the accuracy of thegestation period (this is especially applicable for the5-8 month period in cattle and 5-9 months in buffalo).An approximation of the gestation period must beconveyed to the owner rather than an accurate period.Although experienced clinicians can more preciselycomment on the gestation period after an examinationit is usually safe to be approximate. When pregnancy examinations are made early(Day 30-45) the possibility of an early embryonic deathmust be kept in mind and a confirmation of pregnancymust be done only after re-examination at a laterperiod (60-90 days). Owners often consult veterinarians on the fetalviability during mid to late gestation. It is often difficultto comment positively by a single rectal palpation.Until unless characteristic changes in the uterus andfetus are palpable negative comments must better beavoided. The usual test for fetal viability during thisperiod is the movement of the fetus in response to astimuli by the examiners hand (movement of a fetal legwhen pressed by hand or suckling movements by thecalf when a finger is touched in the mouth) however,this may be sometimes misleading specially when thefetus is depressed.Location of pregnant uterus The pregnant uterine horn under goessequential changes in size, location and morphologyand they form the basis for pregnancy diagnosis. The



early pregnant uterus lies in the pelvic cavity in heifers,and just ahead of the pelvic brim in pleuriparous largesized cows. As it grows in size, its growth is forward soit starts descending into the abdominal cavity (approx3½ - 4 months). At approximately 4½ - 5 months itreaches the abdominal floor and at this time onlycervix is palpable within the pelvic cavity which is alsodrawn forward. The growth is then forward and thenagain upwards. The entire uterus or the fetus istherefore barely palpable during the 4 – 6½ monthsperiod and diagnosis has to be dependent on otherfeatures of pregnancy (placentomes or fremitus). Afterthis period the fetal parts are usually palpable andclinicians find no difficulty in commenting whether theanimal is pregnant or non-pregnant. During earlypregnancy (day 30-60) clinicians have to depend onfinding of the fetal membrane slip or the palpation ofthe amniotic vesicle. The definite signs of pregnancy in the cow asdetermined by rectal palpation are i) palpation ofenlarged uterine horn containing the placental fluids ii)palpation of the amniotic vesicle iii) slipping of thefetal membranes iv) palpation or ballottement of thefetus v) Palpation of the placentomes vi) palpation ofenlarged thin walled “whirring” uterine arteries.Uterine changes The increase in the diameter of the uterinehorns is characterized by a thinning of the uterine walland the feeling of a fluid filled structure. By 40-90 daysof pregnancy, the uterus feels like a thick rubberballoon nearly filled with water. The volume of fluidincreases rapidly the first 5 months of pregnancy andthen increase slowly. With advancing pregnancy thenon pregnant horn may also increase in size slightly.The amniotic vesicle The amniotic vesicle can be palpated with duecare between 30-50 days of gestation as a movableoval object within the uterine lumen, many a times atthe apex of the cornua. The vesicle is turgid, early inpregnancy but becomes flaccid with advancinggestation until days 65-70 when it is difficult to detectat all. The width of the vesicle is around 1 finger (1.5cm) at 40-42 days of pregnancy and increases to 4fingers (9.0 cm) at 60-62 days of gestation (Roberts,1985).Slipping of the fetal membranes The fetal membrane slip can be felt between35-90 days of gestation (Zemjanis, 1970). The entireuterine horn must be grasped in the palm and allowedto slip while the fingers compress it gently. Theallantois chorion, slip between the thumb and fingersbefore the uterine wall escapes. It is felt like aconnective tissue band. The pressure must be gentle.The entire diameter of each uterine horn must be

palpated.Palpation of placentomes The presence of placentomes is anotherpositive sign of pregnancy and is detectable fromabout 75 days to term. The period of pregnancy whenthe uterus has descended into the abdominal cavityand the fetus is not palpable, palpation of aplacentome is the surest indication that thecow/buffalo is pregnant. Since there is great variationin size among individual placentomes (those nearestthe fetus are the largest), their usefulness in aging apregnancy is limited. In general, they can be detectedas soft, thickened lumps in the uterine wall and aremore easily detected as pregnancy advances.Palpation of the fetus The palpation of the fetus itself is a positivesign of pregnancy. Depending on the skill of theexaminer and the location of the fetus, the fetus canbe palpated from the time of amniotic softening (65 to70 days) to term. However, in large sized cows theabdomen should be lifted up by a bamboo held by twoattendants on either side of the abdomen to palpate afetus during mid gestation (4½ - 6½ months). Thewhole of the fetus is palpable many a times onlyduring early gestation (2 to 4 months). The size of thefetus is approximately that of a mouse or rat at 2 and 3months and it increases to the size of a small cat at 4months, a large cat at 5 months and a beagle dog at 6months respectively (Bon Durant, 1986). Themaximum fetal growth occurs during the last one toone and a half month of gestation and estimates ofpredicting pregnancy status depend upon theexperience of the clinician and location of the fetalparts. Beyond 8 months of gestation, fetal parts (legs,head) are palpable within the pelvic cavity or justcranial to the pelvic brim. Palpation of a fetal extremityis sufficient evidence for pregnancy if other uterinefindings are normal.Palpation of uterine artery fremitus The major supply of blood for the gravid uterusarrives via the uterine arteries, which enlargeconsiderably as pregnancy progresses. These bilateralvessels travel in the broad ligaments, just below andanterior to the iliac shafts reflecting in a cranio ventraldirection. They can thus, be felt by the hand directedlaterally towards the iliac shaft. These vessels arefreely movable. Enlargement of the uterine arteryipsilateral to the pregnant horn is detectable after 80 to90 days of gestation. By approximately 120 days, theblood flow within the artery increases to a point wherethe blood flow is palpable as a buzzing sensation, alsocalled “thrill” or “fremitus”. By 7 to 8 months thefremitus is often palpable on the side of thenon-pregnant uterine horn also. The detection of



fremitus is a positive sign of pregnancy.Palpation of ovaries As the pregnancy advances ovaries may bedragged forward along with the pregnant uterus andmay not be palpable beyond the four to five months.Vaginal changes During pregnancy the vagina usually developsa pale, dry sticky mucus membrane. The cervix isclosed and the cervical (mucus) seal covers theexternal os by day 40 to 120 of pregnancy. Slightdegree of vaginal discharge is evident in some cowsbeyond 5 months of pregnancy but the cervical sealliquefies only prior to parturition or abortion and isdischarged in strings.Differential Diagnosis of pregnancy Owners are many times perplexed on theoutcome of a pregnancy that did not progress normallyand often present their animals for a definitivediagnosis to a clinician. Some pathological conditionsmentioned in detail in subsequent chapters oftenshould be identified to be different from a normalpregnancy. The palpable differences of theseconditions are briefly described below. Only rarely does a urinary bladder full of urinecreates confusion for the presence of an earlypregnancy (2-3 months) in dairy cows. This can beeasily differentiated by the absence of palpation ofboth uterine horns and the ease with which the animalurinates when the bladder is gently massaged leadingto disappearance of the enlargement. Rarely tumors ofthe genital tract can create confusion, but theirconsistency and location is different. The differentialdiagnosis of three conditions commonly encounteredby clinician’s namely mummified fetus, maceratedfetus and/or pyometra/mucometra is described below:Mummified fetus A uterus with mummified fetus has thickuterine walls, absence of fluid and placentomes and ahard palpable structure. The fetus is closely apposingthe uterine wall. The fremitus is absent. Maceration of the fetus There is copious vaginal pus discharge. Theuterine wall is thick and doughy. There is no dorsalbulging of the uterus and placentomes are notpalpable. Parts of bones are sometimes palpableseparately floating tacked up. The fremitus is absent.Pyometra and Mucometra These two conditions are many times difficultto differentiate from normal pregnancy especially whenthe pus or mucus is present in enormous quantity(sometimes 20-40 liters) so that the uterus is largelyenlarged and placed on the abdominal floor. Inpyometra the uterine wall is thick, uterus is doughyand placentomes or fetus is not palpable and the

fremitus is absent. However, it is always safer to makea re-examination 1 to 2 months later when the clinicianis not sure, or make use of ultrasonography todifferentiate the condition. Sonographic evaluationwould depict anechogenic fluid without cotyledons andechogenic material accumulated. A clinical therapy toterminate the corpus luteum on an assumption that thecondition is pyometra could be hazardous and loss tothe owner, if unfortunately the condition was a normalpregnancy. In mucometra, the positive findings ofpregnancy are absent, but, contrary to pyometra theuterine wall is thin. Ullrasonography can easilydifferentiate the condition from pregnancy. Whenthese two conditions are to be differentiated from earlypregnancy a characteristic feature found most often isthe bilateral enlargement of both uterine horns, whichis not found in pregnancy. The fetus is not palpable inthese two pathologies, and often there is a history ofvaginal discharge.Palpable findings of pregnancy in mares The early diagnosis of pregnancy in the marehas its own significance. A limited breeding season insome breed registries (for e.g. Thoroughbred)warrants that the non pregnant mares are diagnosedearly and steps be taken to breed the mare again inthe same breeding season. Some peculiarities of theequine pregnancy are that an ovarian source ofprogesterone is essential for maintenance ofpregnancy until approximately days 50 to 70;thereafter, the fetal-placental unit begins producingsufficient progestagens to support pregnancy.(Bergfelt and Adams, 2007). The endometrial cups form at about 35 days ofgestation and remain up to 150 days. Secondary,accessory and supplemental corpora lutea (day 40)form on the mare ovary and all corpora lutea regressby day 180-210 (Bergfelt and Adams, 2007). The earlyequine fetomaternal axis secretes sufficient estrogenand the equine embryo stabilizes by day 16. Diagnosis of an early pregnancy by rectalpalpation is one of the easiest and accurate means forexperienced personnel. However, a diagnosis at day18-20 should be confirmed at day 45-60 because of alate implantation of the equine embryo and chances ofearly embryonic death and reabsorption. The basis ofpregnancy diagnosis in the equine species lies on thechanges that follow in the cervix, uterus and ovaries.Placentomes are not found in the equine species andthe fremitus or fetal membrane slip is not marked,therefore some of the parameters used in cattlecannot be used in the equine species directly.Cervix As early as 16 to 18 days after ovulation, thecervix of the pregnant mare becomes tightly closed,



firm, slender, and elongated. Between 16 to 30 daysthe cervix can be with experience palpated on the floorof pelvis as a rigid firm structure.Uterus Most pregnancy diagnosis in the mare byrectal palpation is done by palpation of the uterus. Theparameters used for a positive diagnosis are asfollows-Uterine tone and thickness One of the positive sign that a mare hasconceived to a breeding is the finding of increase inuterine wall thickness and a marked tone. Theendometrial folds are no longer palpable as folds oftissue. The uterus becomes tubular, smooth and firm.The uterine tone appears at days 15-16 and continuesupto day 48 to 55 (Sertich, 1997). This is diagnosticexcept in cases of endometritis or in mares bred at thefoal heat, which are still undergoing uterine involution.Uterine size and embryonic vesicle The conceptus becomes positioned at thebase of one of the uterine horns at the junctionbetween uterine horn and body. At 25-28 days, a smallbulge may be palpable in maiden mares. The bulgeprogressively increases in size and is palpable in mostmares by day 30-35 (approx 3-4 cm in diameter). Asmall notch can be readily appreciated on both sidesof the bulge. By day 42-45, the conceptus occupiesapproximately half of the gravid horn and is 5 to 7 cmin diameter. The uterine wall over the bulge in thin. At 60 days of gestation, nearly the entire gravidhorn and half of the uterine body are filled with theconceptus but the non-gravid horn remains small. Thepregnancy is like an elongated football and is nearlysimilar to a 60 day pregnancy in cattle. The tonicity ismarkedly reduced at this time. The 60 day conceptusis approximately 8-10 cm in diameter and 12 to 15 cmin length.Palpation of fetus The fetus is active after 40 days and mobileafter 70 days. Palpation of the turgidity of theconceptus is absent by day 90, and the fetus ispalpable, which feels like a small, heavy, submergedbut floating object as the hand contacts it. In mostmares it is usually possible to palpate the fetus perrectum from the third month onward throughout thegestation. In a few deep bodied and large sized marespalpation of the fetus is difficult from the fifth toseventh months of gestation. In these mares thelocation of uterus and ovaries would aid the diagnosis.Location of the uterus The uterus is located in the pelvic cavity or justat the pelvic brim until day 90. At 100-200 days, thegravid uterus is positioned cranial to the pelvic brim inthe abdominal cavity (Sertich, 1997). The ovaries are

positioned cranial and ventral to their normal positionsand closer together. By 5 to 7 months the uterus is positioned low inthe abdomen and it is difficult to thoroughly evaluatethe conceptus by palpation per rectum. During thesixth and seventh months, the horns areapproximately perpendicular to the dorsal cranialaspect of the uterine body. Beyond the seven to eightmonths the fetus is easily palpable by rectal palpation.Ovarian palpation Both ovaries should be palpated. Both ovariesusually are enlarged from 18 to 40 days as a result offollicular development and the CL is not palpable.From 40 to 120 days, extensive ovarian activity withovulations, luteinization, and development ofsecondary corpora lutea is evident. Follicular activitydecreases from 120 days to term, and the ovariesbecome small and inactive. The position of the ovariesup to 60 days of pregnancy is similar to that for thenon-pregnant mare. From then on, they are drawncranially and medially but remain dorsal to the uterus.The finding of both ovaries nearer to each other andclose to the pelvic floor is a positive indication forpregnancy when the uterus or other structures aredifficult to palpate (3 to 5 months). Form 5 months ofpregnancy onwards, the ovaries usually are notpalpable as they are under the broad ligaments.Rectal palpation in the sow To a limited extent rectal palpation has beendescribed as a method of pregnancy diagnosis in thesow. (Cameron, 1977). Sows are examined whilestanding in gestation crates or pens. This technique isbased on examination of the cervix and uterus,together with palpation of the middle uterine artery toassess size, degree of tone, and type of pulse. Ataround 21-30 days of gestation the bifurcation of thecornua is less distinct, the cervix and uterine walls areflaccid and thin. The middle uterine artery is 5-8 mm indiameter and more easily identified. The uterusbecomes progressively thin walled and ill defined by31-60 days and fremitus can be identified at 37 days.Beyond 60 days the fremitus is very strong, however,piglets can only be palpated towards the end ofgestation. The procedure is often difficult in gilts andsmall sized breeds because of a small pelvis and toosmall rectum. False positive diagnosis is likely if theexternal iliac artery is mistakenly identified as themiddle uterine artery. The technique is however, notpopular at most locations.Rectal palpation in female camels The corpus luteum formed on the camel ovary(ovulation is induced by mating) persists and isnecessary for the entire gestation. The persistence ofthe CL is one of the earliest sign of pregnancy as



otherwise; the luteal phase is very short. The CL is outof reach by day 90 of pregnancy. The left uterine hornis inherently longer than the right horn and this mustbe kept in mind when making pregnancy diagnosis infemale camels. The earliest detection of uterinechange (increase in diameter and appearance offluctuation) is palpable at about 40-15 days. Between60-70 days, the left uterine horn is increased abouttwice to its non gravid size, has a thin wall andfluctuates (Banerjee, 1974). The gestational changes in camel have beendescribed (Tibary and Anouassi, 1997). The uterusbecomes cranial and ventral after the third month ofpregnancy. The cervix is pulled forward and lies just atthe pelvic brim by 4 months. At 5 months of pregnancy,the uterus is completely in an abdominal position witha small degree of fluctuation but the fetus is notalways palpable. The fetus becomes palpable againbeyond the 6th month of pregnancy, first withballottement, and then, the head and legs becomeeasily palpable by the 7-8 months as the fetus startsits ascent. By the 9th month, movement can beobserved by inspection of the right flank of the animal.At around 11 months the fetal legs can be easily foundin the pelvic cavity. The precise estimation of the stageof pregnancy beyond 3 months, because of theabsence of cotyledons and difficulty in reaching thefetus in this species is difficult. The fetal membraneslip is not seen in camels because of a diffuseplacenta.Ultrasonography Dur ing the last couple of years,ultrasonography has gained popularity in veterinarymedicine and has become the method of choice fordiagnostic imaging of the various organs of the body,including reproductive organs. Ultrasound is a highfrequency sound wave. Sounds audible to the humanear vary between 20 to 20,000 Hertz (Hz) (Cycles persecond) while ultrasound waves are of frequencyhigher than this, and for most diagnostic applicationsfrequencies of 1-10 MHz are used. Ultrasound cannotbe propagated in vacuum and in gas, transmission ispoor. Reflection of ultrasound occurs betweensubstances of different acoustic impedance (definedas the product of the velocity of sound in a substanceand the density of the substance). Even the shortdistance between the transducer (which emits andreceives ultrasound signals) and the patient must bebridged by a suitable coupling gel.Basic principle The ultrasound equipment basically consists ofa transducer and a scan converter. The transducer isthe ultrasound producing part. It is fitted with apiezoelectric crystal (Lead – zirconate – titanate or

others) which when stimulated by a high voltagecurrent emits the ultrasound. The ultrasound istransmitted to the patient from the transducer andpropagates through the tissues. The ultrasound beamis either reflected back, partially absorbed or entirelyabsorbed. The returning beam (echoes) meets backand deforms the crystals in the transducer. Thismechanical energy is converted back to an electricalsignal proportional to the strength of the echo anddelayed by a time roughly proportional to the distancetraveled. The scan converter interprets the variationsin brightness displayed on the cathode ray tube of aB-mode system (or as a variation in amplitude inA-mode oscilloscope screen) and also stores imageswhen required. The ultrasound is emitted in a pulse –echo manner. A pulse of ultrasound is emitted and itsreflection perceived prior to emission of the next pulse.Types of instruments and some definitions For most diagnostic veterinary purposesB-mode, real time ultrasonography is used employingdifferent types of transducers. Transducers usedcommonly in veterinary reproductive practice are thelinear transrectal transducer (frequencies of 5-10 MHz)and the sector transabdominal transducer (frequenciesof 1-4.0 MHz). For most reproductive diagnostic work,linear array transrectal transducers are employed incattle, buffaloes, mares and female camels. Smallsized transrectal transducers are also used for earlypregnancy diagnosis in small ruminants (sheep andgoat). For bitches mostly transabdominal sectortransducers are useful for pregnancy diagnosis withfrequencies from 2 to 4.0 MHz. The same transducerscan be used for pregnancy diagnosis in sheep andgoat beyond day 40 of gestation. However, in order tovisualize an early pregnancy or the non pregnant bitchuterus transducers of high frequency (5-7.5 or 10.0MHz) are essential. A wide variety of transducers with single, dual,or multiple frequency and multiple functions areavailable and clinicians must decide what type ofinstrument and the transducer he must purchasedepending upon his work. Advancement toultrasonongraphy includes diagnostic imaging usingcolor doppler, magnetic resonance imaging andreaders must refer pertinent excellent texts availableelsewhere (Singh et al., 2003). When performing ultrasonography it isimportant for the sonographer to have basicknowledge of the anatomic location of the differentorgans to be visualized and problems that can beencountered in obtaining and interpreting the imagesobtained. A few of the common terms related toultrasonography are described below:-



Anechoic (sonolucent) A tissue failing to reflectthe ultrasound beam produces no echoes (e.g. A fluidfilled follicle) and appears black.A- mode Amplitude modulation. A one-element (onedimensional) display with time (distance) on thehorizontal axis. B-Mode Brightness modulation. A compound A-modescan with amplitude translated into a brightness scale.Location on the display is related to position and depth.Doppler ultrasound When an ultrasound beam meetsa moving object the reflected ultrasound is either ofincreased or decreased frequency, depending uponwhether the motion is towards or away from thetransducer.Echogenic A structure causing a marked reflection ofthe ultrasound beam. A change in echogenecity in ahomogeneous structure may indicate a pathologicalchange.Gain The amplification level of a returned signal.Hperechoic Showing increased echogenecity.Hypoechoic Showing decreased echogenecity.Linear array Distribution of piezoelectric crystalsalong the length of a scan head. The image producedis generally rectangular.M-Mode Motion mode. A rapidly updated onedimensional B-mode display with time on the secondaxis to allow study of moving structures. Used incardiology.Probe The transducer and its attachments.Real time Images generated from reflected ultrasoundfollowing sequential activation of transducer array aredisplayed on the screen at sufficient speed to give theappearance of a live image.Scatter When the ultrasound beam encounters asmall object in its path the beam energy is spread inall directions.Sector scan A pieslice/sector shaped image isproduced on the screen. The initial signal is producedby a single or small number of rotating piezoelectriccrystals.Shadowing Caused by severe attenuation (decreasein the power of ultrasound beam by absorption, scatterand reflection) such that it fails to penetrate sufficientlydeeply.Transducer The piezoelectric crystal or elementwhich converts electrical to mechanical energy.Procedure for ultrasonographyTransrectal ultrasonography The detailed methods for performingtransrectal ultrasonography in cattle have beendescribed in detail previously (Goddard, 1995 Manion,2006). The animal to be examined is properlyrestrained, the feces are evacuated from the rectumand the perineum washed with water. The transducer

is covered with a condom with coupling gel put insideor a full arm disposable sleeve is used to cover thetransducer. The operator keeps the transducer in hisarm and takes it inside the rectum. The uterine hornon one side is scanned to the entire length and theovary of that side is also scanned. The operator thenmoves his hand to the other uterine horn and ovaries.If the pregnancy is advanced the operator may have totake his hand deeper. When required the images seenmay be frozen and the diameter of the structuresmeasured by inbuilt calipers with the machine. Theamount of fluid and thickness can also be measured.The transducer is then taken out and the perineumwashed again.T r a n s - a b d o m i n a l ( T r a n s c u t a n e o u s )ultrasonography For reproductive trans-abdominalultrasonography in sheep and goats the hair must beclipped from just above the udder and 15 to 20 cmahead of the udder on both sides of the abdomen. Thetransducer is placed above the udder between thethigh and abdomen preferably the left side and movedin a ‘W’ shape from one side of the abdomen to theother side. The procedure can be performed with theanimal standing (sector scan) or in lateral or ventralrecumbency (linear scan) depending upon the type oftransducer being used. For pregnancy diagnosis, sows are examinedin a standing position when using sector scanners(3.5-5.0 MH2 frequency) or doppler instruments (whichare more frequently used in pigs to detect fetal heartbeat, fetal movements and uterine artery pulsations).The transducers are placed over the abdomen justmedial to the stifle skin fold, just at the level of secondlast teat. Trans-abdominal imaging of the uterus of abitch can be done with the bitch in the standingposition after clipping the hair of the ventral abdomen.However, dorsal and lateral recumbent positions mayalso be used. The uterus lies dorsal to the bladder, butits position may vary with the extent of bladder filling,and the size of the uterus. During early pregnancy theuterus has a more dorsal position in the abdomen of astanding bitch, but with advanced pregnancy theuterus is closer to the ventral lower abdominal wall.Sonographic findings during pregnancy Interpretation of sonograms of the reproductivetract requires an understanding of the composition ofthe images and an awareness of the possible artifactswhich can occur and lead to misdiagnosis. Forexample, acoustic enhancement will appear as ahyper echoic region deep to the fluid (anechoic) area.Gas and bone will totally reflect the sound waves andproduce the strongest of echo signals, leading to an



image which appears on the screen as near-white. Socomplete is the return of echoes in some cases thatsound waves do not penetrate deep to these areas,resulting in a lack of imaging which manifests itself asa black zone and is referred to as acoustic shadowing.This must not be interpreted as the anechoic image offluid filled structures. Another artifact found with gasfilled viscera or bone is the rebounding of echoes backand forth between the object and the transducer. Witheach cycle of rebound there is loss of signal strength,and this is imaged on the screen as a series of layeredhyper echoic images repeating themselves betweenthe object and the transducer face. This is termedreverberation and is often encountered in trans-rectalscanning where gas filled viscera are present. Whenthe ultrasound strikes to a smooth and wide structure(for e.g. a CL) there will be almost total return ofechoes where the sound waves strike at right angles,giving an intensified signal that appears on the screenas a whiter shade of grey. This is referred to asspecular reflection, and is often seen in earlypregnancy when imaging the embryonic vesicle. With these basic things in mind one has also tokeep in mind that fluid filled structures appear black(anechoic), hard structures (like the bone) appearwhite (hyperechoic) and other structures with theirstructure midway between the bone and fluid appeargrey (hypoechoic). The basic diagnosis of pregnancylies in the identification of structures from black, greyor white scale. The earliest sonographic finding for pregnancyin most animals is the appearance of anechoic fluidwithin the uterine lumen. This fluid goes on increasingto a stage when the embryo proper becomes visible asa hypoechoic structure floating within this fluid andprogressively the fetal structures become more clearlyvisible along with the fetal membranes. The viability ofa growing fetus is ascertained when the fetal heartbeat becomes visible as a hypoechoic flickeringstructure. The methods of evaluation of pregnanciesusing ultrasonagraphy have been described forvarious species in sufficient detail by Goddard (1995).The days of sonographic appearance of variousstructures in different domestic animals are mentionedin Table 1. On analyzing this table it is clear that withdifferent probes the amniotic vesicle/fetal fluid isvisible in most species between days 18-22 except inthe mare in which it appears earlier (day 10-16). Thefetal heart beat can be seen between day 24-30 andthe fetus itself between days 25-30 in most species.The cotyledons are visible between day 30-40 andfetal extremity/bone by day 57-60 in cattle, day 70 insheep; however it is visible earlier (42-50 days) in the

bitch. It therefore, appears that the fetal fluid, fetalheart beat and the fetus become sonographicallyvisible at nearly the same time in most species. Themost appropriate time for pregnancy diagnosis usingultrasonography with high accuracy in cattle, cameland buffaloes appears to be day 28-30 using a transrectal linear array probe of 5.0 to 7.5 MHz frequencies(Filteau and Des Coteaux, 1998; Vyas et al., 2002; Aliand Fahmy, 2008; Kahn et al., 1990). Using the sameprobe pregnancy can be diagnosed with sufficientaccuracy a little earlier (24-25 days) in the mare(Pycock, 2007). Recently, the use of a 3.5 MHztranscutaneous sector probe applied over the rightflank has been suggested for pregnancy diagnosis incows between 73 to 190 days of gestation. The fetalthoracic, abdominal and umbilical diameter wassignificantly associated with gestational age (Hummanet al., 2009a, b,c). A trans rectal probe of 5.0 MHzfrequency (usually a prostatic probe used in humanbeings) is required to diagnose pregnancy betweendays 25-30 in sheep and goats (and this can be usedto study non pregnant uterus and ovaries) but underfield conditions a transabdominal probe (linear orsector 3.5 to 5.0 MHz frequency) is generally used andthis can diagnose pregnancy earliest at day 40-50 insheep and goats with reasonable accuracy as the fluidand cotyledons are easily visible sonographically atthis time (Duggal et al., 2001b; Suguna et al., 2008).The diagnosis of pregnancy in the bitch requires atransabdominal probe (of frequency 3.5 to 5.0 MHz) tovisualize pregnancy with accuracy from 25-30 days(Bondestam et al., 2008). The visualization of earlierpregnancy or the visualization of a non-pregnantbitches uterus necessitate the use of probes of higherfrequency (7.5 to 10.0 MHz) as the uterus lie morecloser to the skin. Color Doppler ultrasonography inthe bitch can detect placental fetal circulation (Blancoet al., 2008) and in sheep pregnancy can bediagnosed with high accuracy at 76-90 days ofgestation (Ganaie et al., 2009).Doppler ultranongraphy and A-mode probes aregenerally used in pigs for pregnancy diagnosis butB-mode probes with frequencies of 3.5 to 5.00 MHzcan diagnose pregnancy with high accuracy betweendays 25-30 post mating (Almond and Woodard, 1997;Williams et al., 2008).Radiography To a limited extent radiography has been usedfor pregnancy diagnosis in the small ruminants (sheepand goat), the companion animals (dog and cat) andrarely in pigs. The technique is known to be good inevaluating fetal numbers in the bitch and cat, but ispoor in evaluating fetal viability. Moreover, the highcost and the hazards of exposure to growing fetuses



to x rays limit the use of radiography as a routineprocedure, and warrants its use in specialized cases.Mostly, a single radiograph taken with the animal inlateral recumbency is sufficient however; sometimes adorsal or a dorso ventral view may be required. Insheep and goat, fetuses are visible by day 70 ofgestation (Grace et al., 1989; Noakes, 1999) with ahigh accuracy. The overall accuracy of the method indetecting pregnancy increases with advancinggestation: 52% between 66 and 95 days to 100% after96 days (Richardson, 1972). The accuracy ofdetermining fetal numbers approaches 87% onlybetween day 91 and 110 (Ardran and Brown, 1964).Therefore, radiography is suggested to be done onlyafter day 90 in sheep and goat. In bitches fetal skeletons are visible with highaccuracy only by the sixth week of pregnancy althoughthey may be sometimes visible as early as 23-25 daysof gestation (Toal et al., 2005). The fetal skulls arevisible by day 45 and the entire fetal skeleton is visibleby the end of seventh week of gestation. The accuracyof radiographic diagnosis is dependent on the qualityof radiograph obtained. Radiography is generallysuggested for bitches and cats, whenever there is adoubt about the nature of the abdominal contents at ornear whelping. The number and position of the fetusescan be detected easily by radiography at this time.Signs of fetal death as seen by radiography includethe spalding sign, (which is the overlapping of thecranial bones), gas shadows in the fetal heart andstomach and tightly flexed spine (seen in fetuses diedfor long time) (Jackson, 2004). The fetal skeletons begin to calcify only afterthe sixth week in sows and hence radiography shouldbe performed only after this time for pregnancydiagnosis in sows (Rapic, 1961). Because pregnancycan be diagnosed with high accuracy with othermethods, radiography is seldom performed forpregnancy diagnosis in sows.Abdominal Ballottement Abdominal palpation and abdominalballottement of the fetus is possible to some extent incows during late gestation (7 months onward). Insheep and goats rectal abdominal palpation (by usinga glass rod placed in the rectum to lift the uterus whichis palpated through abdomen) has been suggested(Ott et al., 1981; Hulet,1972; Chauhan and Waziri,1991;). Similarly bimanual palpation for pregnancydiagnosis (palpation of uterus through fingers in therectum and lifting the abdomen) has been reported forsmall ruminants (Chauhan and Waziri, 1991; Kutty,1999) however, both the methods are inaccurate andthe first method is often invasive. Palpation of fetusesthrough the abdomen is possible in sheep and goat

only beyond 4 months of pregnancy by lifting theabdomen held between both hands and location ofbony fetal structures. However, sometimes bezoars inthe rumen may confuse with pregnancy. In cattle abdominal ballottement is performedby placing the fist over the lower right abdominal walland pushing it in an intermittent manner in a dorsalmedial direction deeply. The fetus can be felt as ahard solid object floating in fluid. This is usuallypossible in lean cows after the 7th month of gestation.The fetal movements can be seen at the same placeby careful visual observation however, the method isapplicable too late in diagnosis. Abdominal palpationof pregnancy in bitches may be possible in small ormedium sized bitches which are not too obese.Moreover, bitches often tense their abdominal wall andrespire too fast making abdominal palpation often toodifficult to perform. At about 30-35 days the accuracyis high (87%) and depends on the palpation of tenseconceptual swellings (6 to 30 mm in diameter) withinthe uterine cornua but they must be differentiated fromfeces in the colon and the palpator must haveexpertise to differentiate or identify them correctly. Thebitch’s foreparts must be slightly elevated. Beyond day55 it is easy to diagnose pregnancy by the palpation ofthe fetuses. They may be palpable in the flank andalso in the lower abdomen. With the availability ofmore effective methods like ultrasonsography for earlypregnancy diagnosis with accuracy the use ofabdominal palpation has been reduced as the palpatoris many a time confused.Fetal echocardiography To a limited extent fetal echocardiography hadbeen used in the past to diagnose in cattle, sheep andmares, but with the advent of ultrasonography its usehas been limited.Vaginal electrical resistance The conductivity of the vaginal mucousmembranes changes at estrus due to increasedhydration, increased blood supply and other changes.When measured by ohm meters the vaginal electricalresistance (VER) is low at estrus. Hence when VER ismeasured constantly animals returning to estrus canbe identified and thus those probably becomingpregnant can be differentiated (Foote et al 1979; McCaughey, 1981; Gupta and Purohit, 2001; Meena etal., 2003) however the accuracy of such estimations indiagnosis of pregnancy is limited due to false positiveresults and the problem related with daily probing ofanimals.Laparoscopy Laparoscopy can be used as a method ofpregnancy diagnosis by directly visualizing thegenitalia in animals however, the invasive nature of



the technique, the high cost of equipment and clinicrequired, and the availability of non invasivetechniques limits the use of this technique as a meansof pregnancy diagnosis in most animals.Laboratory tests for pregnancy diagnosis The various laboratory tests developed forpregnancy diagnosis in domestic animals are indirectmethods of pregnancy evaluation, and utilizequalitative or quantitative measures of reproductivehormones at specific stages after AI or mating, ordetect conceptus specific substances in maternal bodyparts or body fluids as indirect indicators of thepresence of a viable pregnancy. Unfortunately, noneof the methods developed so far in animals are asaccurate as is the detection of hCG in pregnant humanfemales. However, the research to developcommercial indirect methods continues because thesemethods are non invasive and the tests can bemarketed to and performed by the dairy farmers. Thecurrently available methods are briefly described. Progesterone hormone assay The corpus luteum formed on the ovarysubsequent to ovulation produces progesterone formaintenance of pregnancy for a reasonable timeperiod in some species and for entire gestation inother species like the cow, buffalo, goat and sow. In normally cycling cows the CL is lysedbecause of the effects of prostaglandins from theuterus if the animal is not pregnant, and thus theprogesterone level goes down. Therefore, lowprogesterone concentrations in maternal blood at 18 to24 days post breeding can predict that the animal isnon-pregnant and high progesterone gives an insightthat probably the animal is pregnant. The specificity ofprogesterone tests conducted between 18 and 24days post breeding have shown a specificity of around98% (Zaied et al., 1979; Laing et al., 1980; Waldman,1993; Gowan et al., 1982; Pennington et al 1985;Nebel et al., 1987) and is the easiest proven methodfor identifying non-pregnant (regularly cycling) animalspost breeding. However, for the pregnant animals theaccuracy of the test is low (75%) because of earlyembryonic death which alter the results. Commerciallyavailable ELISA, plasma or milk progesterone assaykits have not become popular due to their high costand a low specificity. Non pregnant cows not returningto estrus and pregnant cows in which embryonic deathoccurs at a later time can both give false results.Likewise, in mares, sheep, goats, buffaloes, camelsand sows assay of plasma or milk progesterone is notvery accurate for diagnosis of pregnancy (Zarkawi,1997; Kaul and Prakash, 1994; Sato, 1977; Fleming etal., 1990; Dionysius, 1991; Almond and Dial, 1986;Ellendorf et al., 1976; Abdel Rahim and El-Nazier,

1987). Since progesterone concentration in theperipheral blood of pregnant bitches is similar to thoseof non pregnant bitches, and since there is noplacental progesterone produced in the pregnant bitch(Verstegen-Onclin and Verstegen, 2008) henceprogesterone assay cannot be used to diagnosepregnancy in the bitch. To a limited extent plasma progesterone hasbeen used as a means of pregnancy diagnosis in thefemale camel in which species the CL is required forthe entire gestation (Abdel Rahim and El-Nazier,1987).Estrone sulfateOne of the earliest written records of a urine-basedpregnancy test can be found in an ancient Egyptiandocument. A papyrus described a test in which awoman who might be pregnant could urinate on wheatand barley seeds over the course of several days: “Ifthe barley grows, it means a male child. If the wheatgrows, it means a female child. If both do not grow,she will not bear at all.” Testing of this theory in 1963found that 70 percent of the time, the urine of pregnantwomen did promote growth, while the urine ofnon-pregnant women and men did not. Scholars haveidentified this as perhaps the first test to detect aunique substance in the urine of pregnant women, andhave speculated that elevated levels of estrogens inpregnant women’s urine may have been the keyto its success.A similar test of germination of wheat seeds when theyare soaked in urine from pregnant cows which inhibitsgermination compared to urine from non-pregnantcows which stimulate germination has been describedto be known as Punyakoti test (Nirmala et al., 2008).The differential germination is considered to bebecause of the presence or absence of estrogens inthe urine. However, controlled studies using estrogenand progesterone failed to affect the germination rateof wheat seeds (Nirmala et al., 2008). The estrone sulfate is produced by the fetomaternal axis or the conceptus and therefore itspresence in urine, milk, feces or blood is an indicatorof pregnancy.The appropriate day, at which detection of estronesulfate detection is possible in the body fluids orsecretions are mentioned in Table. 2 The detection ofestrogens depends on the availability of suitablelaboratory and availability of commercial assay kits.Laboratories evaluating concentrations of estrogens inur ine or serum usual ly are equipped withradioimmunoassay, enzyme immuno-assay or othermore precise and specific diagnostic modalities forassay of steroids in urine, serum, feces (Bamberg etal., 1984) or other body fluids. Evaluation of steroids



like estrogen from feces is especially helpful for zooand feral species where feces are the most easilycollected specimens (Bamberg et al., 1991; Hermannet al., 2005). Commercial kits have been developed forpregnancy detection in mares by using on farm kitslike Wee-Foal-Checker® or Equitest ES® whichrequire urine or serum as the test material. Thesecommercially available tests are recommended to beperformed only after 120 days of gestation andspecially suggested for miniature horses and donkeysin which pregnancy diagnosis by rectal palpation orultrasonography is extremely difficult. The testprocedures utilize an immunochromatographicprocedure to measure concentrations of pregnancyspecific steroids (estrogens) and manufacturersrecommendations should be strictly followed foroptimum results.Chemical tests for pregnancy diagnosisMost chemical tests reported in the past appear to beof historic importance only in current day pregnancydiagnostic procedures. Some of the chemical teststhat utilize urinary estrogens, or other molecules as abasis of pregnancy diagnosis in domestic animals aredescribed below:Cuboni test:This test was first developed by Cuboni (1934) andmodified later (Galina and Cox, 1969). The test isperformed in the mare for detection of pregnancythrough assay of urinary conjugated estrogens. Thetest is preformed as follows:-“To 15 ml of urine 3 ml of concentrated hydrochloricacid is added and heated in a water bath for 10minutes and then cooled under a tap. To this 18 ml ofbenzene is added and shaken vigorously for half aminute. The supernatant (mainly benzene) is collectedin another tube and 3 to 10 ml of concentrated sulfuricacid is added and the mixture heated in a water bathat 80°C for 5 minutes, and then allowed to cool. Apositive (pregnant) test is indicated by the appearanceof a dark, only green fluorescent color in the lowersulfuric acid layer and a negative (non-pregnant) ischaracterized by absence of fluorescent color andpresence of a brownish color.”The cuboni test is only effective beyond 150 days ofgestation, and also predicts fetal viability.Other chemical tests previously described for thedetection of urinary estrogens in mares include thevaginal cornification, mouse or rat tests usingovariectomised female rats or mice, and thephenolsulphonic acid test. (Based on kobercalorimetric test for estrogens) (Roberts, 1985;Benesch and Wright, 2001). In the mouse test, theserum or urine from pregnant mares when injected toovariectomised mouse or rats would induce vaginal

edema, appearance of cornified cells and mucusdischarge due to presence of estrogens in thepregnant mare’s serum or urine (Allen and Doisey,1924). The phenolsulphonic acid test utilizes etherextraction of urinary estrogens subsequent to removalof urinary pigments by hydrolysis and their conjugationwith phenolsulphonic acid reagent after evaporation ofthe ether. The final reaction gives a pink to cherry redcolor if the urine is from pregnant mares (Mayer, 1944;Benesch and Wright, 2001). The test is lengthy andcumbersome and requires special precaution inprocessing which has turned out this test of historicimportance only. These tests are 70-80% accuratewhen performed between the 120 to 250 days ofpregnancy.Another test described previously include the mucintest in which the vaginal mucus from a pregnant mareshowed dark staining columnar epithelial cells(pregnancy cells) (Kurosawa, 1931) and known tohave an efficacy of 94% from day 70 to end ofgestation (Miller and Day, 1938; Day and Miller, 1940)however, these old tests have now been replacedbecause of availability of better diagnostic, highlyspecif ic radio immunoassays and enzymeimmunoassays.Barium chloride test: A test has been described forpregnancy diagnosis in the bovine species commonlyknown as the Barium chloride test. To 5 ml of urinefrom cows a few drops of 1% barium chloride is addedand warmed slightly. In non pregnant cows a whiteprecipitate is formed, whereas, in pregnant animalsthe urine remains clear (Temblador and Landa, 1971).The 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 210days of pregnancy; however, a later study (Kavani,1976) noticed only a low accuracy (64%) with a highoccurrence of false positive and false negative results.In camels the test was considered to be 85% accuratebetween days 50-90 of pregnancy (Banerjee, 1974).By and large the validity of such a test continues to becontroversial.Some other previously described tests for pregnancydiagnosis in cows include two tests on milk; i) milkalchohol coagulation test: in this test there iscoagulation of milk from pregnant cows when mixedwith equal quantities of alchohol and allowed to standfor 1-3 hours (Linkes, 1930; Rutz, 1932; Stancev andAngelov, 1966; Kavani, 1976). ii) copper sulfate test: 1mL of milk when mixed with a few drops of 3% coppersulfate coagulates if the animal is pregnant (Tembldorand Acosta, 1971; Kavani, 1976). The accuracy ofthese tests is considered to be low (52.0 to 64.2%)and many a times the tests are inconclusive and



confounded by breeds and disease in the udder(Kavani, 1976). Moreover, no specific molecules weredetermined in these studies and therefore their use incurrent diagnostic methodologies is limited and usuallyof historic importance. Similar to these tests apreviously described test documented the appearanceof deep yellow to orange coloration of cervical mucusfrom non-pregnant cows when boiled with 10-15%NaOH whereas cervical mucus (which is difficult toobtain from pregnant cows) from pregnant cowsevidenced a pale yellow or colorless state(Sokolovakaya et al., 1959; Williams, 1964; Kavani,1976).Costa’s test: This test was developed by Costa (1927)for testing of pregnancy in human females and isbased upon sedimentation of haematin in thepresence of a solution of novocaine. To 1.5 mL of 2%novocaine 3 drops of blood are added and then 5%solution of sodium citrate is added. The mixture iscentrifuged and 1 drop of formalin is added. Inpregnancy grey or grayish yellow color or precipitateappears within 15 minutes. The test was experimentedin cows and the test was known to be positive after 38days of insemination with 65.3% accuracy in cows(Kriisa, 1934, Bhattarcharya, 1967; Kavani, 1976) andinaccurate in camels (Banerjee, 1974). Kosjakovs test:This test apprehends that the sulfurcontent of hair in pregnant animals is increased(Kosjakov, 1929). Hair from animals under test aredigested with 10% potassium hydroxide and boiledwith 2 ml of distilled water and 1% methylene blue anda few drops of 4% hydrochloric acid are added. Theblue color disappears in pregnant animals due toincreased sulfur. The basis of the test was not verifiedin subsequent research and hence the test is nowobsolete. Assay of gonadotrophins The human female secretes the gonadotropinhCG which is present in sufficient quantities in theurine of pregnant women and many simplified testshave been developed to detect this molecule in urinefor an easy pregnancy diagnosis in women. The onlyanimal species that secrete sufficient quantities ofgonadotrophins that can be used as a markermolecule for pregnancy diagnosis is the equine (Coleand Hart, 1930). Endometrial cups form as early asday 35 and secrete the equine chorionic gonadtrophin(eCG) which can then be detected from serum andurine (Roser and Lofstedt, 1989) of pregnant equinefemales. The eCG continues to be secreted from day40 to 120 days of pregnancy and is the basis ofpreviously described biologic tests and the currentlyavailable on farm tests.Biologic tests

Several biologic tests were developed for thedetection of eCG including the Aschiem Zondek Test,the Friedman test (rabbit test) or the Frog or toad test. Aschiem Zondek test Aschheim and Zondek described a test (knownas the A-Z test) which identified the presence of hCGin human urine. To test for pregnancy, a woman’surine was injected into an immature rat or mouse. Ifthe subject was not pregnant, there would be noreaction. In the case of pregnancy, the rat would showan estrous reaction (be in heat) despite its immaturity.This test implied that during pregnancy there was anincreased production of the hormone. A similar test in mares has been described whereinserum from mares is injected to mice and the resultsare read later (Miller and Day, 1938). The serum (0.5ml SC daily for 2 to 4 days or 5 ml intraperitoneally)from test mare is injected to 2 to 3 rats (22 days of age)and rats are k i l led (72 hr when in jectedintraperitoneally and 96 to 120 h later when injectedsubcutaneously) and a positive test is indicated by thepresence of multiple corpora haemorrhagica on theovaries and uterine edema. The test was consideredto be 90 percent accurate when performed betweenthe 60 to 100 days of pregnancy in the mare. Thesame test performed in camels revealed no changesin the mice (Banerjee, 1974; El-Azab and Musa, 2007)due to lack of any gonadotropic molecule.Friedman Rabbit test Serum from test mare is injected (2 ml given IV)to rabbits (14 to 20 weeks age) kept in isolation andlaparotomy performed 24 h later. A positive test isindicated by the presence of corpus haemorrhagicumand uterine edema (Chicchini and Chiacchiarini,1963).Toad test Toads like Bufo valliceps or frog like RanaPipiens are used in this test. The basis of this test isthe concept that the sperm cells are emitted bytoads/frogs only when stimulated with female frogs orgonadotrophins. Two male toads are taken and theircloaca is wiped with normal saline and examined forpresence of spermatozoa. If no sperms are presentthe cloaca is cleaned and 1 ml of test serum from amare is injected into the dorsal lymph sac of 2 maletoads thrice at an interval of 1 hour. The cloaca isexamined for the presence of the sperms. A positivetest is indicated by the presence of sperms in thecloaca within 1 to 6 hours after the last injection(Cowie, 1948; Neto, 1949) Similar test depicting thepresence of a gametokinetic substance in the feces ofdairy cows, buffalo, goat and sheep has been depictedwhich also initiate a similar response in the cloaca ofthe frog Rana Pipiens (Bhaduri and Bardhan, 1949;



Bhaduri, 1951; Banik and Reineke, 1955) although thevalidity of the toad test in species other than the mareremains doubtful. The above mentioned older methods have nowbeen replaced by newer methods includingradioimmunoassay (Nett et al. 1975), radioreceptorassay (Fay and Douglas, 1982), haemagglutinationinhibition test (Berthelot et al. 1987), ELISA (Squires etal 1983) and direct latex agglutination tests (Decosteret al 1980). Commercially available kits are in use atmany places for these assays (Envet Servies, OmahaNE). Pregnamare® is one such test, which can beperformed on blood between day 40 and 100 ofpregnancy, however, false results may be obtained iffetal death occurs after formation of the endometrialcups.On farm testsSome commercial kits are currently available whichcan detect the presence of eCG in blood of maresbetween 40-100 days of pregnancy with 96-98%accuracy. Pregnamare is one such kit which requires 5drops of blood collected from the muzzle by using thelancet provided with the kit. This color change testrequires about one hour completing.Other methodsSome of other methods described previously includeonset of estrus by injections of estradiol valerate (2mg)at day 9-12 of service in cows (Jochle and Schilling,1965; Kavani, 1976) and day 17-18 in sows (Laerumet al., 1974). The potential dangers of injectingestrogens to pregnant cows have refrained from thelarge scale use of these tests. Some years ago, apregnancy diagnosis based in determining differencesin cervical mucus using nuclear magnetic resonancewas used (Merilan, 1983). This test looked promisingat that time but no subsequent information is available.Milk ejection by low dose prostaglandinA method tested some years ago was the injection oflow dose prostaglandin F2 alpha (non-luteolytic dose)in animals two weeks after breeding resulting into milkejection. The animals detected further as pregnantshowed an increase in the pressure in the milkejection and alveolar milk volume collected by a teatprobe in comparison with the non-pregnant cows(Labussiere et al., 1982; Prakash et al., 1996) andewes (Labussiere et al., 1983; Labussiere et al.,1988). The intrajugular administration of asubluteolytic dose of PGF2 alpha induces a largeincrease in intramammary pressure when given duringthe luteal phase and this is directly correlated to theplasma progesterone profiles (Labussiere et al., 1988).However, due to potential dangers of inducingluteolysis by accidental over dosage, the use of thistechnique of pregnancy diagnosis, could not gain wide

popularity.Pregnancy associated glycoproteins (PAG) Pregnancy specific proteins are known to beproduced in various ruminant species including cattle,buffalo, sheep and goats (Humblot et al., 1990; Karenet al., 2003; Karen et al., 2007). Two pregnancyspecific proteins (PSP) A and B have been isolatedfrom bovine fetal membrane extracts (Butler et al.,1982). Of these PSP-A was identif ied as aa-fetoprotein and PSP-B was found to be specific tothe placenta. These molecules appear in the maternalcirculation and can be determined with accuracy from29 to 30 days post breeding. The PAG continues to beexistent in maternal blood for the entire pregnancy andup to 100 days post partum. The assay involvesradioimmunoassay on serum. The sensitivity andspecificity of PSPB based on RIA is known to be92.0% and 82.6 to 91.9% from 29 to 30 days postinsemination (Szenci et al., 1998). More recentlysimple ELISA techniques have been developed thatdetect the PAG molecule in the serum of cows (Breedet al., 2009; Green et al., 2005; Green et al., 2009;Silva et al., 2007). The limitations to the wide spreaduse of this test is non availability of the protein in milkor urine, presence of PAG up to 100 days postpartum(which interfere with subsequent detections) and thenon availability of cow side commercially available kitsfor its detection. Recently however, the existence ofPAG has been documented in bovine milk (Gajewskiet al., 2008).Early pregnancy factor This protein molecule was first identified inpregnant mice (Morton et al., 1987) and later in sheepand cattle (Nancarrow et al., 1981) by using therosette inhibition bioassay. With this assay, EPF wasdetected in the serun of all mammals tested within 24to 48 h of fertilization and disappeared within 24 to 48h after death or removal of embryo (Morton et al.,1987). The developing embryo bears antigens foreignto the mother; hence immune rejection of the earlyembryo may occur. An immunosuppressive earlypregnancy factor (EPF) appears as early as 6 to 48 hof mating which functions to suppress the maternalimmune response thereby allowing for pregnancy toproceed (Shaw and Morton, 1980). In cattle significantdifferences in rosette inhibition titer were observedbetween pregnant and non pregnant cows on day13-16 and 25 post breeding (Sakonju et al., 1993),suggesting that measurement of EPF activity may beuseful as an indirect method of pregnancy diagnosis.A commercially marketed kit is available in the US(ECF test, concepto Diagnostics Knoxwille, TN)however its reliability is known to be poor (Adams andJardon, 1999; Des Coteaux et al., 2002) and need to



be substantially improved.Relaxin assay Relaxin can be determined in the peripheralcirculation of pregnant bitches at 20-30 days ofgestation, whereas it is absent in non-pregnant bitchesat all stages of the reproductive cycle. (Steinetz et al,1989). This relaxin is known to be produced by theplacenta in the bitch and cat and is thought tocontribute to its maintenance by inhibiting uterineactivity. In the domestic dog it has been established asa pregnancy-specific hormone (Steinetz et al., 1987,1989). The site of synthesis in the bitch has beenelucidated (Tsutsui and Stewart, 1991; Klonisch et al.,1999) and primarily ascribed to the placenta, althoughthe hormone can also be traced in the ovary anduterus. These latter tissues may be areas influencedby the paracrine deposition of relaxin. A clinical studyof domestic dogs using the commercially availablecanine relaxin enzyme-linked immunoassay (ELISA)(ReproCHEK, ®a Synbiotics Corporation, San Diego,CA, USA) reported detection of the hormone inmaternal peripheral blood as early as 25 days afterovulation (Buff et al., 2001). In the cat it appearsdur ing the th i rd week of pregnancy, wi thconcentrations declining just before parturition(Stewart and Stabenfeldt, 1985). The molecule hasalso been detected and used successfully forpregnancy diagnosis in the non domestic species(Carlson and Gese, 2007; Bauman et al., 2008)Vaginal BiopsyHistological assessment of the number of layers of thestratified squamous epithelium of the vaginal mucosaobtained by biopsy can be used as a method ofdiagnosing pregnancy in the sow (Done and Heard,1968; Morton and Rankin, 1969) and to a limitedextent in sheep. (Richardson, 1972). The accuracy ofthis method between 18 and 22 days is 97% and 94%for the diagnosis of pregnancy and non-pregnancyrespectively in the sow (Mc Caughey, 1979). Thebasis for the test is the decrease in the layers of thestratum germinativum (vaginal epithelium cells: to 3 to4 layers at 18-25 days of pregnancy) under theinfluence of progesterone. The number of layers ishigh at estrus (around 20 layers) due to influence ofestrogen hormone. In sheep the technique was 91%accurate in diagnosing pregnancy after 40 days andthe accuracy increased to 100% after 80 days ofgestation (Richardson, 1972). The limitations of such a diagnosis are theinvasive nature of the test, poor results due toimproper sampling and improper tissue processing.With the availability of more precise techniques ofpregnancy diagnosis in sheep and sow the use ofvaginal biopsy for pregnancy diagnosis has been

reduced.

References

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Table : 1 Ultrasonographic features of early pregnancy in various species

Sonographic

Structureappearance(days postmating)

Cow Buffalo Mare Sheep/

Goat

Sow Bitch Camel Cat

Fetal fluid 18-20 18-22 days,5th week

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 20-26day,4th week

20-22 25-30 25-30 – 23-25 16-20

Cotyledons/Allantois

35-40 30-35 20-22 40-50 – – – 25

Fetal bones fetalbuds

57-60 – – 70 – 42-50 40 days 30-33

Fetal sexdetermination

57-60 10-18weeks

60-70 60–90 – – – 38-43

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

Reference Curran etal., 1986;Filteau andDesCoteaux,1998;Curran,1992;PiersonandGinther,1984;Nation etal., 2003

Pawshe etal., 1994;Karen etal., 2007;Ali andFahmy,2008

Allen andGoddard,1984;Ginther,1986;Sertich,1997;Pycock,2007;Holder,2007

Garcia et al.,1993;Buckrell etal., 1986;Karen et al.,2003; Santoset al., 2007;Romano andChristians,2008; Harshet al., 2008;Suguna etal., 2008

Michael,1988;Holtz,1982;Almond etal., 1985;Jackson,1986

Englandand Allen,2008;Barr,2008

Vyas etal., 2002

Zambelliand Prati,2006

Illustrations

Illustration 1

Tables



Table 2 Appearance of estrogen in domestic animals during pregnancy

SpeciesDay of detection Reference

Mare Maternal estrogen high after 60 days Conjugated urinaryestrogens high after 150 days

Sist et al. (1987); Cox(1971); Bhavnani andWoolever, 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) Chaplinand Holds worth (1982)

Sows Rise start at 20 days peak at 25-30 days followed by a decline at45 days and again a rise at 70-80 days to term

Cunningham, 1982 Serenet al.(1983) Robertson etal.(1978); Gutherie andDeaver, 1979

Bitch Slightly increased at implantation and remain constantly high forrest 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



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