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Early Pregnancy Diagnosis in Graded Murrah Buffaloes Using Elisa Based Serum Progesterone Assay

Bethapudi Srilatha G. Venkata Naidu Manda Srinivas Vaddadi Devi Prasad
Vol 7(7), 200-205

The present investigation was conducted to study the accuracy of serum progesterone assay in diagnosing early pregnancy. Twenty healthy graded Murrah buffalo cows aged 4-8years were selected and inseminated using frozen semen upon observation of heat signs. They were subjected to early pregnancy diagnosis on 21d post insemination using ELISA based serum progesterone assay. Out of 20 buffaloes tested for serum progesterone concentration, 9 (45%) were diagnosed as pregnant and 11 (55%) as non pregnant. The animals were diagnosed pregnant and non pregnant if the progesterone values were 2.5 or >2.5 ng/ml and <2.5ng/ml, respectively and the progesterone concentrations in the buffaloes found pregnant ranged between 2.54 to 7.63 ng/ml and the same in non pregnant buffaloes ranged between 0.63 to 1.9 ng/ml. Serum progesterone concentration of pregnant and non pregnant animals differs significantly (P<0.05). From this study it was concluded that serum progesterone assay was 80 per cent accurate in diagnosing early pregnancy and 90 per cent for non pregnancy with an overall diagnostic accuracy of 85.0 per cent.

Keywords : Buffalo Early Pregnancy Serum Progesterone ELISA Rectal Palpation Accuracy


Measurement of progesterone is an indirect method for pregnancy diagnosis in many livestock species. Establishing the progesterone levels in milk or serum between days 18 and 24 post insemination is an immununological method, an indirect one for determining the pregnancy (Sasser and Ruder, 1987). The concentration of progesterone in blood and milk at 20-24 days post insemination has been used as a tool for an early diagnosis of pregnancy in cattle (Heap et al.,1973 and Ginther et al., 1976). Hence, the present study was undertaken with an objective to evaluate the accuracy of serum progesterone assay in diagnosing early pregnancy and to estimate the serum progesterone concentrations in pregnant and non pregnant animals on day 21 post insemination in buffalo cows.

Materials and Methods

Graded Murrah she buffaloes aged 4 to 8 years, between second to sixth lactation and weighing 400-500 kg presented to the Department of Veterinary Gynaecology and Obstetrics were utilized in the present study. Estrus buffaloes which were bred by artificial insemination at 8-12 hours after the onset of estrus using frozen semen were selected. Twenty inseminated buffaloes which did not return to estrus by day 19-21 were subjected to pregnancy diagnosis using ELISA based serum progesterone assay on day 21 post insemination.

Blood Sampling

Blood samples were collected from the inseminated buffaloes (n=20) and allowed to clot for 30 to 60 minutes at 15 to 200C and serum was separated by centrifuging the clotted blood at 3000 rpm for 15 minutes (Prakash et al., 1990). Serum was separated and stored at -200C in plastic bullets.

Technique of ELISA

The serum progesterone concentration was estimated using ELISA technique with the help of progesterone kits (Progesterone EIA, XEMA Co., ltd, Russia). The Xema Progesterone EIA is a 96 well plate, solid phase immunoassay, utilizing the competitive enzyme immunoassay principle. By using the optical density (y-axis) and concentration of the calibrators (x-axis) a standard curve was drawn and from that curve concentration of test samples were calculated. The concentration of the test samples which obtained in nanomoles were converted into nanograms by conversion factor (1 nmol = 0.318 ng/ml). Progesterone values 2.5 or >2.5ng/ml was taken as criteria for determination of pregnancy. Pregnant and non pregnant animals were diagnosed based on the serum progesterone values. Mean ± SE and test of significance between pregnant and non pregnant animals were calculated statisticallyAll the experimental animals that were subjected to early pregnancy diagnosis were later confirmed by per rectal palpation on days 45-60 by considering the definite signs of pregnancy viz., fetal ballotment, double membrane slip.

Accuracy in the form of sensitivity, specificity, positive and negative predictive values of the present technique was calculated as per the formulas given by Pieterse et al. (1990) and were depicted as follows-

Pregnant Nonpregnant
Diagnosis pregnant correct (a) Diagnosis nonpregnant correct (c)
Diagnosis pregnant incorrect (b) Diagnosis nonpregnant incorrect (d)

Number of pregnant animals = a+d; Number of nonpregnant animals = b+c

Sensitivity = a/ (a+d) x 100; Specificity = c/(c+b) x 100

Positive predictive value = a/ (a+b) x 100; Negative predictive value = c/(c+d) x 100

Overall diagnostic accuracy = a+c/ (a+b+c+d) x 100.

Results and Discussion

Out of 20 buffaloes tested for serum progesterone concentration, 9 (45%) were diagnosed as pregnant and 11 (55%) as non pregnant. Out of 11 non pregnant buffaloes diagnosed by this test, 9 (81.81%) were confirmed non pregnant and 2 (18.19%) as pregnant upon rectal palpation at day 45-60. Out of 9 pregnant buffaloes, 8 (88.8%) were confirmed as pregnant and 1 (11.2%) as non pregnant upon rectal palpation. In the present study, the mean values of serum progesterone concentrations in pregnant and non pregnant Graded Murrah buffaloes were observed as 4.63 ± 0.93 and 1.272 ± 0.14 ng/ml (Table 1), respectively on day 21 post insemination.

Table 1: Mean ± SE of serum progesterone levels (ng/ml) 21 days post insemination

S. No. Diagnostic Particulars Day 21 Post Insemination
No. of Buffaloes Progesterone Concentration
1. Pregnant correct 8 4.63 ± 0.93a
2. Pregnant incorrect 1 7.3
3. Non-pregnant correct 9 1.272 ± 0.141b
4. Nonpregnant incorrect 2 0.954 ± 0.318

P<0.05 Means with different superscripts differ significantly

The serum progesterone concentrations in buffaloes found pregnant ranged between 2.54 to 8.26 ng/ml and the same in buffaloes found non pregnant was in between 0.63 to 1.908 ng/ml at day 21 post insemination. Although two buffaloes having serum progesterone values of 0.636 and 1.272 ng/ml that were diagnosed non pregnant and 1 buffalo having 7.3 ng/ml of serum progesterone that was diagnosed as pregnant were subsequently confirmed pregnant and non pregnant, respectively by rectal palpation.

The serum progesterone concentrations (2.54 to 8.26 ng/ml) associated with pregnancy at day 21 was corroborating the earlier findings of Batra et al. (1979), Jain and Pandey (1991), Sarvaiya et al. (1991), Jain and Pandey (1992), Glatzel et al. (2000), Dugwekar et al. (2008) and Naikoo et al. (2013), who recorded 3.6, 2.50, >3.0, 6.0, 2-3, 3.23 ± 0.16 and 4.42 ± 0.52 ng/ml, respectively on day 20-23 post insemination in buffaloes. Similarly, the serum progesterone concentrations (0.63 to 1.90 ng/ml) for the buffaloes found negative for pregnancy were also in confirmation with the earlier findings of Jain and Pandey (1991 & 1992), and Nakhashi et al. (2010) who reported them as 1.50, 0.8 and <1.0 ng/ml. In the recent times, Naikoo et al. (2013) further confirmed the non pregnancy status on day 20 in buffaloes with progesterone concentration of 1.22 ± 0.6 ng/ml.

As per the above observations in this study, the sensitivity, specificity of pregnant and non pregnant buffaloes at day 21 post insemination was 80.00 and 90.00 per cent respectively (Table 2).

Table 2: Accuracy of pregnancy diagnosis using serum progesterone assay (n=20)

S. No Particulars 21d Post Insemination
No. of Animals Tested 20
1. Diagnosis pregnant correct (a) 8
2. Diagnosis pregnant incorrect (b) 1
3. Diagnosis nonpregnant correct (c) 9
4. Diagnosis nonpregnant incorrect (d) 2
5. Sensitivity (Se ; %) 100xa/(a+d) 80.0
6. Specificity (Sp ; %) 100xc/(c+b) 90.0
7. Positive predictive value (PPV ; %) 100xa/(a+b) 88.88
8. Negative predictive value (NPV ; %) 100xc/(c+d) 81.81
9. Overall diagnostic accuracy 85.0

The overall diagnostic accuracy in this study was 85.00 per cent with positive and negative predictive values as 88.88 and 81.81 per cent, respectively at 21 day post insemination. The per cent (80) accuracy in detecting early pregnancy using this test in the present study was comparable with the earlier findings of Nanda et al. (1984) and Nakhashi et al. (2010) who reported 83.3 and 86.2 per cent, respectively. The present finding was much higher than the observations of Perera et al. (1980), Rao et al. (1983) who reported 66.7 and 73.00 per cent, respectively. The accuracy of this test in detecting non pregnancy in the present study was 90.00 per cent which was lower than the findings of Nanda et al. (1984), who reported 100 per cent. In cows, the accuracy of this test was 100 per cent in detecting non pregnancy status as reported by Spano and Rose-e-Silva (1992), Muhammd et al. (2000) and Shelar et al. (2011).

From the results of the present study, it was concluded that the use of serum progesterone concentration to diagnose early pregnancy is advantageous but this method allows detection of non pregnant buffaloes soon after insemination. However, incorrect diagnosis of pregnancy in this test may be due to reproductive problems like uterine infections, ovarian luteal cyst which may have consequently high progesterone concentrations and can confuse the diagnosis. Elevated progesterone concentration post breeding sometimes gives positive result for pregnancy but there may be a chance for early embryonic death occurring between days 30 to 40 post breeding leading to errors in diagnosis (Pawshe and Purohit, 2013). A major constraint to adopt progesterone assay for pregnancy diagnosis could be done in cases where artificial insemination or breeding dates are known/ recorded and not randomly in the herd (Balhara et al., 2013).



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