Article ID: WMC001305 ISSN 2046-1690

Methods Of Diagnosis In Domestic Animals: The Current Status

Author(s):Dr. Govind Purohit Corresponding Author: Dr. Govind Purohit, Associate Professor, Rajasthan University of Veterinary and Animal Sciences (Animal Reproduction, Gynaecology and Obstetr, Department of Animal Reproduction Gynecology and Obstetrics, College of Veterinary and Animal Science, Bijey Bhawan Bikaner Rajasthan, 334001 - India Submitting Author: Mr. Govind Narayan P, 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: 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 Domestic Animals: The Current Status

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Bhaduri, 1951; Banik and Reineke, 1955) although the popularity. validity of the toad test in species other than the mare Pregnancy associated glycoproteins (PAG) remains doubtful. Pregnancy specific proteins are known to be The above mentioned older methods have now produced in various ruminant species including cattle, been replaced by newer methods including buffalo, sheep and goats (Humblot et al., 1990; Karen radioimmunoassay (Nett et al. 1975), radioreceptor et al., 2003; Karen et al., 2007). Two pregnancy assay (Fay and Douglas, 1982), haemagglutination specific proteins (PSP) A and B have been isolated inhibition test (Berthelot et al. 1987), ELISA (Squires et from bovine fetal membrane extracts (Butler et al., al 1983) and direct latex agglutination tests (Decoster 1982). Of these PSP-A was identified as a et al 1980). Commercially available kits are in use at a-fetoprotein and PSP-B was found to be specific to many places for these assays (Envet Servies, Omaha the placenta. These molecules appear in the maternal NE). Pregnamare® is one such test, which can be circulation and can be determined with accuracy from performed on blood between day 40 and 100 of 29 to 30 days post breeding. The PAG continues to be pregnancy, however, false results may be obtained if existent in maternal blood for the entire pregnancy and fetal death occurs after formation of the endometrial up to 100 days post partum. The assay involves cups. radioimmunoassay on serum. The sensitivity and On farm tests specificity of PSPB based on RIA is known to be Some commercial kits are currently available which 92.0% and 82.6 to 91.9% from 29 to 30 days post can detect the presence of eCG in blood of mares insemination (Szenci et al., 1998). More recently between 40-100 days of pregnancy with 96-98% simple ELISA techniques have been developed that accuracy. Pregnamare is one such kit which requires 5 detect the PAG molecule in the serum of cows (Breed drops of blood collected from the muzzle by using the et al., 2009; Green et al., 2005; Green et al., 2009; lancet provided with the kit. This color change test Silva et al., 2007). The limitations to the wide spread requires about one hour completing. use of this test is non availability of the protein in milk Other methods or urine, presence of PAG up to 100 days postpartum Some of other methods described previously include (which interfere with subsequent detections) and the onset of estrus by injections of estradiol valerate (2mg) non availability of cow side commercially available kits at day 9-12 of service in cows (Jochle and Schilling, for its detection. Recently however, the existence of 1965; Kavani, 1976) and day 17-18 in sows (Laerum PAG has been documented in bovine milk (Gajewski et al., 1974). The potential dangers of injecting et al., 2008). estrogens to pregnant cows have refrained from the Early pregnancy factor large scale use of these tests. Some years ago, a This protein molecule was first identified in pregnancy diagnosis based in determining differences pregnant mice (Morton et al., 1987) and later in sheep in cervical mucus using nuclear magnetic resonance and cattle (Nancarrow et al., 1981) by using the was used (Merilan, 1983). This test looked promising rosette inhibition bioassay. With this assay, EPF was at that time but no subsequent information is available. detected in the serun of all mammals tested within 24 Milk ejection by low dose prostaglandin to 48 h of fertilization and disappeared within 24 to 48 A method tested some years ago was the injection of h after death or removal of embryo (Morton et al., low dose prostaglandin F2 alpha (non-luteolytic dose) 1987). The developing embryo bears antigens foreign in animals two weeks after breeding resulting into milk to the mother; hence immune rejection of the early ejection. The animals detected further as pregnant embryo may occur. An immunosuppressive early showed an increase in the pressure in the milk pregnancy factor (EPF) appears as early as 6 to 48 h ejection and alveolar milk volume collected by a teat of mating which functions to suppress the maternal probe in comparison with the non-pregnant cows immune response thereby allowing for pregnancy to (Labussiere et al., 1982; Prakash et al., 1996) and proceed (Shaw and Morton, 1980). In cattle significant ewes (Labussiere et al., 1983; Labussiere et al., differences in rosette inhibition titer were observed 1988). The intrajugular administration of a between pregnant and non pregnant cows on day subluteolytic dose of PGF2 alpha induces a large 13-16 and 25 post breeding (Sakonju et al., 1993), increase in intramammary pressure when given during suggesting that measurement of EPF activity may be the luteal phase and this is directly correlated to the useful as an indirect method of pregnancy diagnosis. plasma progesterone profiles (Labussiere et al., 1988). A commercially marketed kit is available in the US However, due to potential dangers of inducing (ECF test, concepto Diagnostics Knoxwille, TN) luteolysis by accidental over dosage, the use of this however its reliability is known to be poor (Adams and technique of pregnancy diagnosis, could not gain wide Jardon, 1999; Des Coteaux et al., 2002) and need to

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be substantially improved. reduced. Relaxin assay Relaxin can be determined in the peripheral References circulation of pregnant bitches at 20-30 days of gestation, whereas it is absent in non-pregnant bitches at all stages of the reproductive cycle. (Steinetz et al, 1.bdel-Rahim SEA, Nazier AE (1987). Estimation of 1989). This relaxin is known to be produced by the progesterone level in camels (Camelus dromedarius) placenta in the bitch and cat and is thought to milk and its application in pregnancy diagnosis. Br Vet contribute to its maintenance by inhibiting uterine J 143:555-559. activity. In the domestic dog it has been established as 2.Abitt BL, Ball G, Kitto P et al. (1978). Effect of three a pregnancy-specific hormone (Steinetz et al., 1987, methods of palpation for pregnancy diagnosis per 1989). The site of synthesis in the bitch has been rectum on embryonic and fetal attrition in cow. J Am elucidated (Tsutsui and Stewart, 1991; Klonisch et al., Vet Med Assoc 173:973-977. 1999) and primarily ascribed to the placenta, although 3.Adams CS, Jardon PW. (1999). Evaluation of the the hormone can also be traced in the ovary and early conception factor test in cows 3-7 days post uterus. These latter tissues may be areas influenced breeding. Proc Am Assoc Bov Pract. 32: 340 – 241. by the paracrine deposition of relaxin. A clinical study 4.Agarwal SK, Tomer OS (1998). Ed Pregnancy of domestic dogs using the commercially available Diagnosis In: Reproductive Technologies in Buffalo. canine relaxin enzyme-linked immunoassay (ELISA) Indian Vet Res Instit Res Publ 37 : pp 41 – 49. (ReproCHEK, ®a Synbiotics Corporation, San Diego, 5.Akmadeev AN, Vasilev GT (1967). Results of a field CA, USA) reported detection of the hormone in trial of methods of diagnosing pregnancy in cows. maternal peripheral blood as early as 25 days after Veterinariya Moscow. 431:77-78. ovulation (Buff et al., 2001). In the cat it appears 6.Ali A, Fahmy S (2008). Ultrasonographic fetometry during the third week of pregnancy, with and determination of fetal sex in buffaloes (Bubalus concentrations declining just before parturition bubalis). Anim Reprod Sci 106:90-99. (Stewart and Stabenfeldt, 1985). The molecule has 7.Allen E, Doisey EA et al (1924). The hormone of the also been detected and used successfully for ovarian follicle. Am J Anat 34:133. pregnancy diagnosis in the non domestic species 8.Allen WE, Goddard PJ (1984). Serial investigations (Carlson and Gese, 2007; Bauman et al., 2008) of early pregnancy in pony mares using real time Vaginal Biopsy ultrasound scanning. Equine Vet J 16: 509-516. Histological assessment of the number of layers of the 9.Almond GW, Bosu WTK, King GJ (1985). Pregnancy stratified squamous epithelium of the vaginal mucosa diagnosis in swine: A comparison of two ultrasound obtained by biopsy can be used as a method of instruments Can Vet J 26:205. diagnosing pregnancy in the sow (Done and Heard, 10.Almond GW, Dial GD (1986). Pregnancy diagnosis 1968; Morton and Rankin, 1969) and to a limited in swine: A comparison of the accuracies of extent in sheep. (Richardson, 1972). The accuracy of mechanical and endocrine tests with return to estrus. J this method between 18 and 22 days is 97% and 94% Am Vet Med Assoc 189:1567. for the diagnosis of pregnancy and non-pregnancy 11.Almond GW, Woodard TO (1997). Diagnosis of respectively in the sow (Mc Caughey, 1979). The pregnancy. In: Youngquist RS ed. Current Therapy in basis for the test is the decrease in the layers of the Large Animal Theriogenology. WB Saunders Co stratum germinativum (vaginal epithelium cells: to 3 to Philadelphia. P 710-715. 4 layers at 18-25 days of pregnancy) under the 12.Ardran GM, Brown TH (1964). X ray diagnosis of influence of progesterone. The number of layers is pregnancy in sheep with special reference to the high at estrus (around 20 layers) due to influence of determination of the number of fetuses. J Agric Sci estrogen hormone. In sheep the technique was 91% Camb 63: 205-207. accurate in diagnosing pregnancy after 40 days and 13.Ball L, Carroll EJ. (1963). Induction of fetal death in the accuracy increased to 100% after 80 days of cattle by manual rupture of aminotic vesicles. J Am gestation (Richardson, 1972). Vet Med Assoc142: 373 – 374. The limitations of such a diagnosis are the 14.Bamberg E, Choi HE, Mostl E et al (1984). invasive nature of the test, poor results due to Enzymatic determination of unconjugated estrogens in improper sampling and improper tissue processing. feces for pregnancy diagnosis in mares. Equine Vet J With the availability of more precise techniques of 16: 537. pregnancy diagnosis in sheep and sow the use of 15.Bamberg E, Mostl E, Patzl M et al. (1991). vaginal biopsy for pregnancy diagnosis has been Pregnancy diagnosis by enzyme immunoassay of

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Illustrations Illustration 1

Tables

Table : 1 Ultrasonographic features of early pregnancy in various species

Sonographic Cow Buffalo Mare Sheep/ Sow Bitch Camel Cat

Structure Goat appearance (days post mating)

Fetal fluid 18-20 18-22 days, 10-16 20-25 18-20 18-20 17-18 10-16 5th week

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

Fetus 28-30 20-26day, 20-22 25-30 25-30 – 23-25 16-20 4th week

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

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

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

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

Reference Curran et Pawshe et Allen and Garcia et al., Michael, England Vyas et Zambelli al., 1986; al., 1994; Goddard, 1993; 1988; and Allen, al., 2002 and Prati, Filteau and Karen et 1984; Buckrell et Holtz, 2008; 2006 Des al., 2007; Ginther, al., 1986; 1982; Barr, Coteaux, Ali and 1986; Karen et al., Almond et 2008 1998; Fahmy, Sertich, 2003; Santos al., 1985; Curran, 2008 1997; et al., 2007; Jackson, 1992; Pycock, Romano and 1986 Pierson 2007; Christians, and Holder, 2008; Harsh Ginther, 2007 et al., 2008; 1984; Suguna et Nation et al., 2008 al., 2003

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Table 2 Appearance of estrogen in domestic animals during pregnancy

Day of detection Reference Species

Mare Maternal estrogen high after 60 days Conjugated urinary Sist et al. (1987); Cox estrogens high after 150 days (1971); Bhavnani and Woolever, 1978

Cow, Day 100 of gestation Hamon et al. (1981); Robertson et al., 1978

Goats High after day 50 of gestation Refsal et al (1991) Chaplin and Holds worth (1982)

Sows Rise start at 20 days peak at 25-30 days followed by a decline at Cunningham, 1982 Seren 45 days and again a rise at 70-80 days to term et al.(1983) Robertson et al.(1978); Gutherie and Deaver, 1979

Bitch Slightly increased at implantation and remain constantly high for Concannon et al. (1975) rest of gestation and decline 2 days prepartum

Sheep Detectable by day 70; rise thereafter till 2 days prepartum Illera et al., 2000; Worsefold et al., 1986

Buffalo Appear at day 150 of gestation in the serum Prakash & Madan (1993); Kamonpatana, 1984

Camel Increase start at day 50 and peak from day 90-300 Skidmore et al., 1996

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Reviews Review 1

Review Title: the review of the article "methods of pregnancy diagnosis in domestic animals: The current status".

Posted by Dr. Sumant Vyas on 11 Jan 2011 11:48:06 PM GMT

1 Is the subject of the article within the scope of the subject category? Yes 2 Are the interpretations / conclusions sound and justified by the data? Yes 3 Is this a new and original contribution? No 4 Does this paper exemplify an awareness of other research on the topic? Yes 5 Are structure and length satisfactory? No 6 Can you suggest brief additions or amendments or an introductory statement that will increase No the value of this paper for an international audience? 7 Can you suggest any reductions in the paper, or deletions of parts? No 8 Is the quality of the diction satisfactory? Yes 9 Are the illustrations and tables necessary and acceptable? Yes 10 Are the references adequate and are they all necessary? Yes 11 Are the keywords and abstract or summary informative? Yes

Rating: 7 Comment: Was it possible to include photos? The photographs of ultrasound images and visual behaviour like cocking of tail may improve the article.

Competing interests: No Invited by the author to make a review on this article? : Yes Experience and credentials in the specific area of science: I am working in area of animal reproduction since 1988.

Publications in the same or a related area of science: Yes References: It is already included. How to cite: Vyas S.the review of the article "methods of pregnancy diagnosis in domestic animals: The current status".[Review of the article 'Methods Of Pregnancy Diagnosis In Domestic Animals: The Current Status ' by ].WebmedCentral 2011;2(1):WMCRW00360

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Review 2

Review Title: Over all review of article entitled

Posted by Dr. Nitin Bhatia on 11 Dec 2010 07:18:38 PM GMT

1 Is the subject of the article within the scope of the subject category? Yes 2 Are the interpretations / conclusions sound and justified by the data? Yes 3 Is this a new and original contribution? No 4 Does this paper exemplify an awareness of other research on the topic? Yes 5 Are structure and length satisfactory? Yes 6 Can you suggest brief additions or amendments or an introductory statement that will increase Yes the value of this paper for an international audience? 7 Can you suggest any reductions in the paper, or deletions of parts? No 8 Is the quality of the diction satisfactory? Yes 9 Are the illustrations and tables necessary and acceptable? Yes 10 Are the references adequate and are they all necessary? Yes 11 Are the keywords and abstract or summary informative? Yes

Rating: 7 Comment: The article provides a review of available data on pregnancy diagnosis in Farm animals. The data compiled is based on available information and author expertise in the area of Animal Reproduction

Competing interests: Veterinary Medicine Invited by the author to make a review on this article? : Yes Experience and credentials in the specific area of science: I am myself a Post graduate Vet and a editor of Intas Polivet, a Veterinary Journal from the Indian domain abstracted and reviewed nationally and internationally

Publications in the same or a related area of science: No How to cite: Bhatia N.Over all review of article entitled [Review of the article 'Methods Of Pregnancy Diagnosis In Domestic Animals: The Current Status ' by ].WebmedCentral 2011;1(12):WMCRW00229

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