Approaches for Diagnosis of Pregnancy in Female Buffaloes

Early identification of non-pregnant dairy buffaloes post-breeding can improve reproductive efficiency by short-cycling the animals and rebreeding during the same breeding season. Thus early and accurate diagnosis of pregnancy seem important to shorten the calving interval in buffaloes [1] that show a profound effect of season on reproduction, coupled with poor estrus expression [2]. The availability of pregnancy diagnosis services appears to be extremely low in some locations [3,4] and at other places the diagnostic approaches seem to be far from perfect. Failure of pregnancy diagnosis leads to slaughter of pregnant buffaloes; with every fourth buffalo slaughtered being pregnant [5] resulting in heavy economic losses.

The attributes of an ideal pregnancy diagnostic test include i) high sensitivity (i.e., correctly identify pregnant animals) ii) specificity (i.e., correctly identify non-pregnant animals), iii) inexpensive, iv) simple to conduct under field conditions and v) ability to determine pregnancy status at the time the test is performed.

It is desirable to diagnose pregnancy as soon as possible after insemination but without having the diagnosis being confounded by subsequent embryonic mortality. Most direct (clinical) and indirect (laboratory) methods of pregnancy diagnosis include one or more of these attributes, but no test currently available or under development include all the attributes mentioned. Two clinical methods for pregnancy diagnosis in buffaloes have become popular; transrectal palpation is the oldest method and continues to be the most widely used method for pregnancy diagnosis in cattle and buffaloes [6]. However, this approach suffers from the drawback that diagnosis is not accurate before day 45 [7] and early embryonic death may occur possibly due to rough genital manipulation. Although transrectal ultrasonography procedures have gained popularity as a clinical method for pregnancy diagnosis in the buffalo [1,8] providing additional information about the fetus and its surroundings, their specificity and sensitivity is 100% only around Day 30 [1]. A large number of laboratory methods have been developed for pregnancy diagnosis in buffaloes including the detection of circulating hormones, pregnancy-associated molecules or chemical determination of these or other molecules in natural secretions (cervical mucus, urine or milk) from pregnant buffaloes, however, either their detection requires a specialized laboratory setup or their sensitivity and specificity is low limiting their widespread use.

In this chapter the authors have classified and discussed methods of pregnancy diagnosis in buffaloes under two subheadings

1. Clinical methods (Non-return to estrus, transrectal palpation, transrectal ultrasonography) and
2. Laboratory methods (Plasma and milk progesterone, plasma estrogen, pregnancy associated proteins and chemical tests).

1. Clinical Methods

1.1. Non-return to Estrus

After conception, the embryo inhibits the regression of the corpus luteum (CL) by secreting some molecules that suppress PGF2α secretion thus maintaining high progesterone concentrations that prevent the buffalo from returning to estrus. The non-return to estrus of a bred buffalo 18-24 days after breeding is an assumption by breeders that the animal is pregnant. Alternatively, if the buffalo is not pregnant after breeding, the uterus will secrete PGF2α that will lyse the CL, plasma progesterone concentrations will decrease and the buffalo will return to estrus after 21 to 23 days post-service.

The reliability of the method depends on the accuracy of estrus detection in the herd. This approach over-estimates pregnancy by 10-20% [9]. Gestational estrus in a small proportion of pregnant buffaloes also complicates the diagnosis [9-10]. Poor estrus signs and sub-estrus in some buffaloes [2] lead to their erroneous diagnosis of pregnancy. Moreover, reasons other than pregnancy such as chronic uterine infection or ovarian cysts, may prevent estrus, resulting in buffaloes being erroneously diagnosed as pregnant. Buffaloes bred during the time of increasing day length may fail to return to estrus although they are not pregnant [6]. Thus the reliability of non-return to estrus as an indicator of pregnancy is poor in the buffalo [9]. An increased incidence of embryonic mortality during periods of increasing day length has been recorded in the buffalo [11-12]. This can increase the interval from insemination to return to estrus in buffaloes that maintain a pregnancy for some time and then loose that pregnancy later when the breeding season is already over.

1.2. Transrectal Palpation

Transrectal palpation has been used for pregnancy diagnosis in cattle since early 1900s [13] and has been the customary field diagnostic procedure. Due to many similarities with cattle, the same method has probably been used in buffaloes as well. During rectal examination, the ovary and uterus are palpated through the rectal wall. The presence of a corpus luteum (CL) does not necessarily indicate pregnancy and its detection over the bubaline ovary is difficult, since it is mostly embedded deeper in the ovarian stroma and projects less on the ovarian surface [14]. The positive findings of pregnancy include palpation of uterine enlargement, amniotic vesicle, slipping of the fetal membranes, placentomes, fetus and middle uterine enlargement during different stages of gestation [6] (Table 1 & Fig. 1A, Fig. 1B, Fig. 2, Fig. 3, Fig. 4 and Fig. 5). The various changes during the 3 trimesters of pregnancy in buffalo have been mentioned previously [9].

Transrectal palpation is widely used and considered a safe method for pregnancy diagnosis in buffaloes. Palpations should be gentler in the buffalo as the rectal mucosa is more fragile and bleeds easily [6]. The accuracy of the rectal palpation depends on the experience of the practitioner. The sensitivity of transrectal palpation at 31 to 40 days was 60%, between Day 41 to 50 it was 92%and from Day 51 to 55 the accuracy was 100% [7]. Perera [15] reported that the gestation length in buffaloes is longer than that of cattle (315 vs. 285 days), the amount of fetal fluid at Day 31 to 40 may be smaller in buffalo and this may be the reason for the diagnosis of early pregnancy being more difficult by transrectal palpation in buffalo. Transrectal palpation as a method of early pregnancy diagnosis was considered accurate in buffalo from Day 46 after breeding [7]. The sequential growth characteristics of a bubaline fetus (Fig. 5A, Fig. 5B, Fig. 5C, Fig. 5D, Fig. 5E) and the fetal fluids and annexes (Table 2) lead to an increase in the uterine size. Fetal fluid accumulation is low up to Day 40 of pregnancy but thereafter there is a significant increase in the volume of fetal fluids and the fetal size with a consequent increase in the size of the uterus. The sequential increase in the size of the uterus is forward and upward both with the enlarging uterus moving towards the abdominal cavity. By the sixth month of gestation, the uterus is fully located in the abdominal cavity and only cotyledons, uterine distension and fremitus can be felt between 5-7 months of pregnancy.

Figure 5A. Day 70: The approximate size of fetus is 7.70 cm.

Figure 5B. Day 80: The approximate size of fetus is 9.50 cm.

Figure 5C. Day 90: The approximate size of fetus is 10.72 to 14.30 cm and weight of the fetus is 57.81 g.

Figure 5D. Day 100: The approximate size of fetus more than 20 cm.

Figure 5E. Day 150: The approximate size of fetus more than 24.92 to 34.50 cm and weight of fetus is 275.58 g.

Some researchers [25] reported that fetal membrane slip and fluctuation inside the uterine horn were detectable in buffalo from about Day 42 to 56 of gestation; it is difficult to distinguish between early pregnancy and pathological conditions causing asymmetry of the uterine horn by transrectal palpation. Agarwal and Tomer [9] reported that fetal membrane slip can be felt between 60-90 days of pregnancy and the amniotic vesicle can be palpated between 30-45 days of gestation in the buffalo. However, rectal palpation does not provide any information about the viability of the embryo/ fetus. Therefore, some buffaloes with a non-viable embryo/ fetus or in the process of a degenerating fetus might be diagnosed as pregnant. Pathological conditions such as chronic endometritis or accumulation of intrauterine fluid causing asymmetry of uterine horns might be the reasons for other false positive diagnosis made by transrectal palpation.

Differential Diagnosis of Pregnancy

Similar to normal pregnancy fluid accumulations might occur in some pathological conditions of the uterus like mucometra and pyometra and might create confusion about the existence of a pregnancy. These conditions have been reported in the bubaline species mostly from abattoir material [26-30]. Clinicians can differentiate such conditions by the bilateral presence of fluid in the uterus, absence of fetal membrane slip, cotyledons and fetus in mucometra and a thick walled uterus with a doughy feel in pyometra, however; ultrasonography would confirm the diagnosis in confusing cases. An ultrasound examination would reveal the presence of fluid or pus in the uterine lumen and the absence of a fetus. A urine filled and distended urinary bladder can sometimes be confused with pregnancy. Inducing urination will confirm or deny pregnancy. A rare case of delivery of a live bubaline fetus from a pyometra-affected buffalo has been recorded [31].

1.3. Transrectal Ultrasonography

Ultrasonography has gained tremendous popularity in recent years as a diagnostic tool in veterinary science. Diverse applications for ultrasonography, continued improvement in image quality, availability of portable ultrasound scanners and more cost-effective equipment have led to its general acceptance in veterinary practice.

Several reports on the application of real time ultrasonography for pregnancy diagnosis in buffalo are available. Ultrasonic anatomy of developing conceptus from Day 18 to 60 has been reported. Application and detailed methods for performing transrectal ultrasonography for reproductive research have been reviewed and described in detail in the buffalo [1,8,32-37]. Buffaloes are restrained in a chute and the rectum is evacuated of the feces. The ultrasound probe (5.0–7.5 MHz) enclosed in a sleeve and lubricated with gel is introduced in the rectum and both the ovaries and uterine horns are scanned sequentially.

The appearance of the different fetal and fetal annexes is gradual in the buffalo (Fig. 6). The earliest indication of pregnancy is the visualization of the embryonic vesicle on Day 19. This appears as an anechoic area (Fig. 7A) sometimes with an echogenic embryo being visible. The vesicle is first spherical and then becomes elongated and irregular in shape at around Day 26 (Fig. 7B). There is variability among buffaloes in the rate of elongation of the vesicle. The height of the embryonic vesicle increased from 11.7 mm on Day 19 to 33 mm on Day 40 and to 63.8 mm on Day 60 [8]; the embryo proper was first detected on Day 19 in buffalo. The growth of the embryo proper increased steadily from 4.2 mm on the first day of detection to 53.6 mm at Day 62.

Figure 6. Days of first ultrasonographic detection of identifiable characteristics of buffalo conceptus on day 18 to 60.

Figure 7A. Day 19: Embryo proper within the embryonic vesicle (arrow).

Figure 7B. Day 26: Embryo proper is 9.1 mm long and the allantois is visible (arrow).

On Day 34, the amnion is visible around the embryo proper (Fig. 7C). At Day 42 the embryo proper is visible to be differentiated with head, trunk and limb buds (Fig. 7D) that are more clearly visible at Day 44 (Fig. 7E). At day 50, the placental attachments can be clearly visualized (Fig. 7F). At Day 55, the bony ribs of the embryo are visible as white echogenic streaks (Fig. 7G) whereas on Day 62 the embryo proper is distinctly visible with head, optic area, forelimbs and hind limbs (Fig. 7H).

Karen et al., [1] reported that under field condition, the sensitivity of transrectal ultrasonography at Day 19 to 24 was 44.4%. A small amount of allantoic fluid appeared difficult to be detected between Day 19-24 of gestation resulting in higher frequency of false negative diagnosis. The sensitivity of the test increased from Day 25 and reached 100% from Day 31-35 of gestation.

Figure 7C. Day 34: Amnion as a white echogenic circle around the embryo proper is visible (arrow).

Figure 7D. Day 42: Embryo proper with differentiation of head trunk and limbs.

Figure 7E. Day 44: Embryo proper with clear differentiation of head, trunk and limbs.

During ultrasonography, the early identification of the embryo is difficult due to its position, which was very close to or in contact with the uterine wall [35-36]. In the buffalo, the detection of embryonic heartbeat is also an important step in ultrasonographic pregnancy diagnosis, as it allows us to evaluate the viability of the embryo [36].

Figure 7F. Day 50: Embryo proper with placental attachment.

Figure 7G. Day 55: Embryo proper with ribs and hindlimbs.

Figure 7H. Day 62: Embryo proper distinct visibility of complete buffalo fetus with head, optic area, forelimbs and hindlimbs.

The ultrasonographic appearance of different structures in different studies (Table 3) reveals that the fetus and fetal heartbeat are visible at Day 25 to 29 whereas other fetal structures become sequentially visible.

Real time (B-mode) ultrasound is a reliable and relatively simple method of diagnosing pregnancy as early as Day 25 to 30 in buffaloes. Two factors affect the speed at which ultrasound examination can be conducted on a dairy farm: 1) Operator proficiency and availability, and 2) restraint of buffalo. When both factors are optimized, the speed of the ultrasound examination can approach that of rectal palpation, while exceeding palpation in the amount of information gathered from each buffalo. The main advantage of ultrasound scanning is that it can give an accurate diagnosis earlier than rectal palpation can.

Although the accuracy of ultrasonography for pregnancy diagnosis in buffaloes is high at Day 30 [7], clinicians should consider re-examination of buffaloes evidencing positive signs of pregnancy at Day 30 again at Day 60 because of the possibilities of early embryonic death (that frequently occurs between Day 28-60) [12] which may lead to errors in diagnosis.

1.4. Vaginal Electrical Resistance

The conductivity towards flow of current in the vagina increases at estrus due to decrease in the resistance towards passage of low voltage current possibly because of increased blood flow and the increased hydration in the vaginal mucosa. Vaginal electrical resistance (VER) has been measured in many species by commercially available probes in an effort to detect estrus. Daily regular monitoring of VER in cattle [42-44] and buffaloes [45-49] revealed that VER declines at estrus. The fall in plasma progesterone was synchronous with a fall in VER, the correlation (0.65) between them being positive and significant [47]. Conception rates in buffaloes were highest when they were inseminated at VER between 26-30 Ohms [48]. When animals are probed on a daily basis, those returning to estrus show a fall in VER whereas pregnant animals continue to show a consistently high VER. However, due to many confounding variables and lower hydration in bubaline vagina during estrus and due to the requirement of daily probe measurements, such approach appears to be less practical and therefore has not gained popularity for pregnancy diagnosis in the buffalo.

2. Laboratory Tests

Indirect methods for early pregnancy diagnosis use qualitative or quantitative measures of reproductive hormones at specific stages after AI or detect conceptus specific substances in maternal body fluids as indirect indicators of the presence of a viable pregnancy. Research to develop commercial indirect methods for pregnancy diagnosis continues because these methods are non-invasive and the tests can be marketed and performed by dairy farmers or farm employees. Currently available methods or methods under development for indirect diagnosis of pregnancy include measurement of endocrine hormones such as progesterone, estrogen, and pregnancy specific proteins such as pregnancy-associated glycoproteins or the early pregnancy factor and a few chemical tests.

2.1.        Progesterone Hormone Assay

In the buffalo, the corpus luteum formed on the ovary subsequent to ovulation produces progesterone for maintenance of pregnancy throughout gestation [50]. The concentration of blood progesterone is at its nadir (0.1-0.3 ng/mL) during estrus and remains close to 1 ng/mL for the next 3-4 days [51]. The first significant increase in the progesterone concentration occurs about 7 days after estrus [52]. Peak progesterone values of 4.0 to 5.1 ng/mL [51,53-54] have been recorded about 15 days after estrus.

In a normal cycling buffalo the corpus luteum regresses consequent to the release of prostaglandins (PGF2α) produced by the uterine endometrium. If the buffalo is not pregnant, progesterone levels decrease. Therefore, low progesterone concentrations in maternal blood at 18 to 24 days post-breeding can predict that the buffalo is non-pregnant and high progesterone concentration at this time indicates a possible pregnancy. The low progesterone concentrations at 18 to 24 days post-breeding can accurately predict non-pregnancy in normal cyclic buffaloes, but most of the time high progesterone concentrations during this period is not a specific indicator of pregnancy due to the variation in estrous cycle length among buffaloes, embryonic mortality, uterine pathology resulting in persistence of corpus luteum and luteal cysts.

Both plasma/serum [51,55-66] and milk [67-77] progesterone assays have been used for diagnosis of pregnancy in buffaloes. The approaches have utilized both radioimmunoassay (RIA) [78-81] and enzyme immunoassay (ELISA) [82-83]. A longer estrus period and highly variable estrous cycle length contribute to a proportion of false diagnosis [70]. A 100% accuracy for diagnosis of non-pregnant buffaloes in a few reports [70,80,84] have not been validated in other studies (Table 4). The mean progesterone in serum/plasma during early pregnancy varies from 1.0 to 3.95 ng/mL on the day after pregnancy [9]. Milk progesterone is 4-5 times higher compared to progesterone in serum/plasma [85-86] (Table 4).

The specificity of the progesterone test at 21 days was only 66.70% or 75% [56,67]. Progesterone concentrations in milk or serum can be quantified using a laboratory RIA or ELISA procedure. Kaul and Prakash [73] reported the accuracy of pregnancy diagnosis in buffaloes to be 57%, 69.50% and 75% on Day 20, 22 and 24, respectively.

The main advantage of using plasma progesterone concentrations to diagnose pregnancy is that, this method allows detection of non-pregnant buffaloes soon after insemination. However, there are several disadvantages of progesterone estimation to diagnose pregnancy. The buffalo with reproductive problems, i.e. uterine infection or an ovarian luteal cyst, may have consecutively high progesterone concentration and can confuse the diagnosis. Sometimes progesterone estimation post-breeding gives a positive result for pregnancy, however, early embryonic death occurring between days 30 to 40 post-breeding lead to errors in diagnosis.

Although, quantitative progesterone assays conducted at regional laboratories as well as qualitative cow-side test kits have been widely available to the dairy industry for many years, progesterone testing as an indirect method for early diagnosis of non-pregnancy is not a widespread practice among buffalo breeders. Specific reasons for the limited implementation of this technology in cows and buffaloes may include its less than perfect accuracy and cost of the tests.

2.2. Estrone Sulfate

Estrone sulfate is a conjugated estrogen that has been used for diagnosis of pregnancy by using milk samples in cattle [92]. Unlike progesterone concentration, this is not directly related to pregnancy.

Estrone sulfate is produced by the feto-maternal axis or the conceptus and therefore its presence in urine, milk, feces or blood is an indicator of pregnancy [92]. Peak concentrations of estradiol (30-35 pg/mL) were detected in buffaloes on the day of estrus or one day before [93] followed by a decline to 5-10 pg/mL within 2 days. Circulating estradiol concentrations remain low during luteal phase with minor fluctuations (10-20 pg/mL) around Days 4 and 10 of the estrous cycle [93] in buffaloes.

The appropriate day at which estrone sulfate detection is possible in buffalo is at 150 days of gestation in serum [63,94]. In a previous study [95], a progressive increase in estrone sulfate concentrations in buffalo plasma after the 4th or 5th month of pregnancy was recorded. However, a recent study [96] depicted a decrease in plasma estradiol during the second trimester (3-6 months) of pregnancy compared to the first trimester of pregnancy. Estradiol profiles in pregnant (10.71 + 2.25 pg/mL) Murrah buffaloes were not significantly lower compared to non-pregnant (20.71 + 3.82 pg/mL) on Day 22 after breeding [97]. Estrogen levels remain elevated throughout the gestation period and reach a peak of 129.33 + 33.38 pg/mL towards parturition [91]. Estradiol rose considerably on the day of parturition followed by a steep and significant fall postpartum [98]. The estradiol profile in Jaffarabadi buffaloes during the first, second and third trimesters of gestation was 10.0 + 4.54, 43.43 + 18.36 and 101.53 + 32.79 pg/mL respectively [91].

Therefore, in buffaloes pregnancy diagnosis using an estrone sulfate assay is not reliable. Hamon et al., [99] reported that in cattle concentrations of estrone sulfate in maternal circulation are not reliably detectable until around 80 days of gestation and are not present in all pregnant cows until 100 days of gestation; estrone sulfate cannot compete with progesterone to assess pregnancy status early post-breeding.

2.3. Pregnancy Associated Proteins

Proteins produced and secreted specifically by the placenta during early pregnancy are obvious candidates for development of an early pregnancy test. Promising candidates that have been researched to date include the pregnancy-associated glycoproteins (PAG) and early pregnancy factor (EPF).

2.3.1. Pregnancy Associated Glycoproteins (PAG)

Pregnancy-Associated Glycoproteins (PAGs) are trophoblastic proteins belonging to the aspartic proteinase family secreted by different placental cells of many mammalian species. PAGs play a pivotal role in placentogenesis, feto-maternal unit remodeling and implantation [100-102]. They are synthesized by binucleate trophectoderm cells (BNC) which originate from the mononucleate chorionic epithelium [103]. The BNC invade the endometrial epithelium and secrete PAG continuously throughout gestation [104-105]. Therefore, these glycoproteins are good indicators of a live conceptus [106]. Similar to other ruminants, these molecules have also been isolated from blood and placenta of pregnant river and swamp buffaloes [107-111].

Two pregnancy specific proteins (PSP) A and B have been isolated from bovine fetal membrane extract [112]. Of these PSP-A was identified as a fetoprotein and PSP-B was found to be specific to the placenta. These molecules appear in the maternal blood circulation and can be determined with accuracy from 29 to 30 days post-breeding. The PAG continue to be present in maternal blood during the entire pregnancy and up to 100 days postpartum.

Karen et al., [1] studied PAG concentrations using heterologous double antibody RIA for diagnosis of pregnancy in buffaloes between Day 19 to 55 post-breeding and suggested the PAG-RIA test as highly accurate for detecting pregnancy in buffaloes from Day 31 onwards after breeding. The sensitivity and specificity of the test was 11.1% and 92.5% respectively on Day 19-24 but increased to 100.0% by Day 31-35. Barbato et al., [109] studied the concentrations of PAGs in buffaloes and observed that during pregnancy, concentrations of PAG were detected at week 6 (9.9 ± 07 μg/mL), with concentrations increasing gradually until week 28 (68.2±20.8 μg/mL). At parturition, the mean concentration was 84.70 ±10.6 μg/mL, thereafter concentrations decreased rapidly, reaching very low (<1.0 μg/mL) levels at one week postpartum. In one study [113], PSP-B were detectable in 33% of pregnant buffaloes at Days 20 to 25 of gestation, while 91% of pregnant buffaloes had PSP-B levels higher than the threshold for diagnosing pregnancy at Day 35 of gestation. Pregnancy proteins are synthesized by binucleate cells originating from the trophoblast [114], which start to migrate into the (endometrium) around the time of final attachment. The level of PAG was low at Day 19 to 24 of gestation, resulting in higher frequency of false negative diagnoses. The limitations to the widespread use of this test is the non-availability of the protein in milk or urine, and the presence of PAG up to 100 days postpartum which could interfere with subsequent detection of early pregnancy. Abdulkareem et al., [117] evaluated the use of a commercially available ELISA test for PAGs in pregnancy detection of buffaloes and found that by careful assay for PSP-B, if a buffalo was detected as non- pregnant, the test was correct over 99% of the time. If it was detected as pregnant, the test was correct 91-94% of the time. The difference in results is due to embryo loss occurring before a follow up test could be carried out.

2.3.2. Early Pregnancy Factor

This protein molecule was first identified in pregnant mice [118] and later in sheep and cattle [119,120] and in buffalo [121] by using the rosette inhibition bioassay. With this assay, early pregnancy factor was detected in the serum of all mammals tested within 24 to 48 h of fertilization and disappeared within 24 to 48 h after death or removal of embryo [118]. Because the rosette inhibition assay for EPF is indirect, substances that have similar effects may produce confusing results. A number of studies in the year after the discovery of EPF were unable to reproduce the consistent detection of EPF in post-conception females and the validity of the test was poor and needed to be substantially improved.

2.4. Chemical Tests

2.4.1. Sodium Hydroxide Test

This test consists in boiling 0.5 mL cervical mucus with 5 mL 10% sodium hydroxide for six minutes. The development of a light brownish color is indicative of pregnancy whereas a pale yellow indicates the absence of pregnancy. Following reports on the use of this test in cows [122-123], the test was done on 110 cervical mucus samples collected from abattoir derived genitalia of Murrah and Nagpuri buffaloes [124]. The test yielded only 40% accuracy in early pregnant buffaloes whereas 77.77% of non-pregnant buffaloes were correctly diagnosed by this test. A low accuracy of the test and difficulty in obtaining the cervical mucus limits its widespread use. Similarly a lower accuracy of the cervical mucus specific gravity test in buffaloes (which is presumed to be increased in pregnant buffaloes) [124] also precludes its widespread use.

2.4.2. Barium Chloride Test

The principle of the test is that progesterone present in the urine during pregnancy prevents precipitation of barium chloride while estrogens favor precipitation [125-126]. Five to six drops of 1% barium chloride solution is added to 5 mL of urine in a test tube and mixed well. If there is no precipitation, the test indicates pregnancy. Clear white precipitation indicates no pregnancy. The accuracy of the test was described to be 70-95% in earlier studies on cows [125,127-128] between 15 to 210 days of pregnancy; however, later studies on cows [129-130] recorded only 64-80% accuracy. In studies on buffaloes [124] the accuracy of the test was only 68-96% during early and 100% during late pregnancy. In non-pregnant animals the accuracy was only 52%. The low accuracy of the test has prevented its large scale use. An important limitation of this method is the presence of a persistent corpus luteum due to uterine infection which may result in a false positive result.

2.4.3. Milk Copper Sulfate Test

This test consists in adding 10 mL of 3% CuSO4 to 0.5 to 1 mL milk in a test tube. The coagulation of milk indicates pregnancy whereas if the milk remains homogeneous for several hours then it is indicative of non-pregnancy. The overall accuracy of the test on 249 milk samples was only 64.24% [124].