The study was conducted on the cattle (n=77) of different breeds (Sahiwal, Tharparkar and Crossbreds) which included pregnant (n=45) and non-pregnant (n=32) cattle. Pregnant animals were further divided into four groups i.e. 7-10D (n=11), 17-20D (n=8), 30-35D (n=16) and 45-50D (n=10). Non-pregnant animals included i.e. Heifer (n=6), Cyclic Non-AI (n=13) and Post-partum (PP; n=13).

Pregnancy was determined by tans-rectal ultrasound on two occasions i.e. about 30D and 45D post AI using 7.5 MHz linear probe through trans-rectal real time B mode USG (EXAGO ECM^TN, France), while it was retrospectively confirmed in 7-10D and 17-20D groups in pregnant cattle.

Blood samples were collected aseptically from the both pregnant and non-pregnant cattle of C&B Farm, ICARIVRI, Izatnagar and other dairy farms including field samples. Approximately, 5-7 ml of blood from each selected cattle was collected by jugular venipuncture in vacutainers containing clot activator. The vacutainers containing blood sample were kept at 37ºC for coagulation of blood. The sera were separated by centrifugation at 800 x G for 15 minutes. The serum samples were labeled properly and stored at−20ºC till use.

Serum samples were assayed for PAG-1 (Biospes; Lot no.: E06d/2020M) and PSP-B (Biospes; Lot no.: E06e/2020M) using two different ELISA kits (Chongquing Biospes Co. Ltd, China) according to manufacturer’s instructions. PAG-1 and PSP-B estimations were done only in two groups of pregnant animals i.e. 30-35D (n=16) and 4550D (n=10), based on the availability of the assay points. Reading in terms of optical density (OD) for all the test samples and provided standards were taken at 450 nm in multimode plate reader (Biorad, Imark, USA). The sensitivities of the kits were 1.0 and 0.3 ng/ml for PAG1 and PSP-B protein, respectively. Relative OD was calculated for each well through provided formula in instruction manual and subsequently standard curve was generated for both the kits (Fig.1). Concentration of PAG1 and PSP-B were determined using equation of standard curve and eventually pregnancy status was determined for each sample.

Progesterone concentrations in the sera were estimated by RIA using standard diagnostic kits (Immunotech, France). Progesterone estimation was done adopting the procedure described by the manufacturer. The radioactive count of all tubes measured in the gamma counter (Packard Cobra II Gamma Counter) calibrated for I¹²⁵ for one minute and programmed to evaluate the concentration of progesterone in the serum samples from the standard curve drawn using values of known standards. The values were obtained in ng/ml. The analytical sensitivity of the kit was 0.05 ng/ml and the intra-assay and inter-assay coefficients of variation were d” 6.5% and d” 7.2%, respectively.

Ultrasonographic (USG) examination was done to screen pregnant and non-pregnant animals. Embryonic vesicle, conceptus, embryonic membranes and embryonic fluid were observed to confirm the pregnancy, while empty uterine horns with rosette shape structure in cross-section or empty tube-like structure in longitudinal section were appreciated to confirm the non-pregnancy in experimental cattle (Fig. 2). Animals were considered doubtful, if failed to appreciate any of mentioned ultrasound features and the particular animal was not included in the study.

Further, early pregnant groups (7-10D and 17-20D) were confirmed retrospectively through USG and animals found non-pregnant were dropped from the study groups. The accuracy of pregnancy diagnosis through ultrasound examination may be considered 100% in controlled experimental conditions (Szenci et al., 1995) and therefore used as a reference method in the present study with 100% sensitivity and specificity.

Assessment of comparative diagnostic evaluation of three immunoassay kits was done for early pregnancy diagnosis using USG as reference method having ideal sensitivity and specificity of 100%. Cut-off value was decided for three assays to categorize the result into pregnant and non-pregnant animals. ROC curve (AUC: 0.929) was generated for data of serum progesterone and 4.82 ng/ ml concentration was decided as cut-off value through obtaining point farthest to x-axis and nearest to y-axis. ROC curve for other two biomarkers PAG-1 and PSP-B could not be generated as the values for non-pregnant groups were not available. Therefore, cut-off value to discriminate pregnant and non-pregnant was decided using available literature (Sasser et al., 1986; Piechotta et al., 2011). Cut-off value was 1.5 and 1.0 ng/ml for PAG-1 and PSP-B, respectively. Subsequently, true positive, false negative, true negative and false positive samples were screened based on cut off value for these immunoassay kits, eventually diagnostic parameters such as sensitivity (Sn), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), accuracy and Youden’s Index (J) were calculated to interpret comparative diagnostic performance using USG as reference method.

The mean±SEM concentrations of PAG-1 and PSP-B proteins in serum samples of cattle in the two pregnant groups (30-35D and 45-50D) are presented in Table 1.

Standard curve for the PAG-1 kit was obtained in straight line (R² = 0.979) with y = 0.006x + 0.163 equation, where x-axis represents the concentration of PAG-1 and y-axis represents respective OD₄₅₀. The mean concentration of serum PAG-1 in two pregnant groups viz., 30-35D and 45-50D were 17.28±2.70 and 16.58±3.19 ng/ml, respectively. Lowest PAG-1 concentration 1.0 ng/ml was reported in group 45-50D, while 30-35D group showed highest concentration (38.67 ng/ml). Standard curve for the PSP-B kit was obtained in straight line (R² = 0.997) with y = 0.011x + 0.231 equation, where x-axis represents the concentration of PSP-B and y-axis represents respective OD₄₅₀. The mean concentration of serum PSP-B in two pregnant groups viz., 30-35D and 45-50D were 15.23±1.94 and 19.92±2.21 ng/ml, respectively. Both lowest (0.09 ng/ml) and highest (30.09 ng/ml) PSP-B concentration was reported in group 30-35D. Linear trend for mean concentration was non-significant (P>0.05) and mean values showed non-significant difference (P>0.05) across the groups.

Blood concentrations of PAGs/PSP-B were initially measured by radioimmunoassay (Humblot et al., 1988; Zoli et al., 1992; Szenci et al., 1998), and the results have been compared with those of ultrasonography (Szenci et al., 1995). However, ELISA based assays for PAG/PSP-B became commercially available later (Friedrich et al., 2010). Twenty-two PAG genes have been reported till date and it was observed that all the PAGs are not expressed simultaneously throughout the pregnancy rather some are expressed early while others at the different point of time with the progression of pregnancy (Ushizawa et al., 2004). The wide variation in PAG levels limits PAG testing as a reliable indicator of pregnancy until about 26 to 30 day post-breeding (Zoli et al., 1992; Humblot, 2001). The concentration of PAGs measured through RIA was recorded 1.0-6.0 ng/ml around 28 to 30 days of gestation (Zoli et al., 1992; Karen et al., 2014; Gajewski et al., 2008), which increased steadily up to day 36 (8.0-16.0 ng/ml). Our findings are in accordance with these reports. Green et al. (2005) used homologous ELISA with monoclonal PAG antibodies and reported average concentration of PAG about 8.75 and 12.3 ng/ml at 4^th and 5^th week postbreeding, respectively. However, Gajewski et al. (2008) measured PAG concentration in cattle at different stages of pregnancy using RIA and reported 3.47 ng/ml on 41 day, which is comparatively lower than the value recorded in the present study on 45-50 day (16.58 ng/ml) using ELISA kit for PAG-1. The differences in the values as compared to reported ones might be attributed to the period of blood collection, different breeds, method of estimation and type of kits. In addition, individual variation in placental production of different PAG molecules could also be a reason for these variations (Piechotta et al., 2011).

We have recorded very high concentration of PSP-B (1517 ng/ml) around day 30-50 of gestation in contrast to 3 ng/ml by earlier report (Sasser et al., 1986). The first blood based PSP-B assay using RIA in ruminants was described by Sasser et al. (1986) and Humblot et al. (1988). Sasser et al. (1986) developed double antibody radioimmunoassay for the serological detection of PSP-B and observed 1 ng/ml around day 30 sera which increased up to 9 ng/ml around 90 days of gestation. Present study reported comparative higher value at around 30 to 50 days of gestation, however, our values are subjected to ELISA. Variable affinity of respective antibodies against targeted bio-molecules may be attributed to the recorded variations (Piechotta et al., 2011). In addition, effect of period of blood collection, breed differences, method of estimation and type of kits could not be ruled out.

The mean±SEM concentrations of serum progesterone in different pregnant as well as non-pregnant groups of cattle are presented in Table 2. The mean concentration of serum P₄ in different pregnant groups viz., 7-10D, 17-20D, 3035D and 45-50D were 4.18±0.41, 7.75±0.79, 9.27±0.98 and 10.78±1.28ng/ml, respectively, while values for non-pregnant groups viz., Heifer, Non-AI and PP were 0.13±0.03, 1.76±0.46, 1.31±0.55 ng/ml, respectively. Lowest P₄ concentration was observed in heifer group (0.02 ng/ml) while 30-35D group showed highest concentration (16.37 ng/ml). However, all the non-pregnant groups sera have low level of progesterone. Linear trend for mean concentration was significant (P<0.05) and mean values showed significant difference (P<0.05) across the groups.

High levels of progesterone in serum or milk between days 18 to 24 post-insemination forms the basis of establishment of pregnancy in cattle (Shemesh et al., 1973; Sasser and Ruder, 1987). Mean plasma progesterone concentration in pregnant and non-pregnant cows were recorded 3.05.8 and 0.22-2.0 ng/ml, respectively around 19-22 days post-breeding or post-estrus (Shemesh et al., 1973) and we also observed similar trend in both pregnant as well as non-pregnant groups. Progesterone concentration was observed 2.0-6.2 ng/ml in non-pregnant cows and pregnant cows showed about 10.0 ng/ml concentration around day 24 post-conception (Humblot et al., 1988). We observed comparatively lower progesterone level in present study for both the groups. Serum progesterone level in early gestation around 4^th week post-conception ranged between 4.7-5.5 ng/ml in dairy cow (Lobago et al., 2009). Further, mean progesterone concentration was recorded 7.2±2.2, 6.6±2.8, 6.6±2.4, 7.8±3.0, and 6.0±3.4 ng/ml at 26-29, 3033, 34-37, 38-41 and 42-58 days of gestation, respectively in dairy cows (Piechotta et al., 2011). However, we have recorded relatively higher level of serum progesterone during the same window. Variations among the findings might be due to the difference in the method of estimation, type of kits, days of blood collection and breeds.

In another study, serum progesterone level during the first three weeks post-partum period was reported about 0.280.75 ng/ml (Khatti et al., 2017) and our findings showed close agreement to this for PP group of animals. Most of the reports for estimation of progesterone to monitor the pregnancy are milk based (Zaied et al., 1979; Nebel et al., 1987; Kamboj and Prakash, 1993; Simersky et al., 2007; Samsonova et al., 2015). In addition, we did not find much reports showing level of progesterone around day 7 post-breeding in serum samples (Noakes et al., 2009). However, milk progesterone around day 9-10 postconception reported to be around 11.1 ng/ml in dairy cows (Zaied et al., 1979), which is comparatively higher to the values observed in present study as level of progesterone in milk is known to be higher because of fat content (Noakes et al., 2009).

In this study, all three immunoassay kits viz., PAG-1, PSP-B and P₄ showed high rate of sensitivity i.e. 92.31%, 96.15% and 84.44%, respectively. We could not calculate specificity of PAG-1 and PSP-B kits as the values for non-pregnant samples were not available. However, progesterone kit showed specificity of 90.63%. Positive predictive value, negative predictive values, accuracy and Youden’s index for P₄ kit were 0.93, 0.81, 87.01 and 75.07, respectively. These diagnostic parameters could not be calculated for other two immunoassay kits.

The sensitivity of tests reported in this study for PAG-1 (92.31%), PSP-B (96.15%) and progesterone (84.44%) is comparable to previous reports (Romano and Larson, 2010; Piechotta et al., 2011; Sah et al., 2017; Northop et al., 2019; Masello et al., 2020) which also showed about 90 to 97% sensitivity around fourth week of pregnancy. Specificity of progesterone estimation was observed highest (90.63%) as compared to the earlier studies i.e. 57% by Sah et al. (2017) and 83% by Masello et al. (2020). Sah et al. (2017) reported 80% accuracy for serum progesterone estimation through ELISA kit, while present study reported about 87% using RIA kit in dairy cow. Muhammad et al. (2000) reported 71.4% and 100% accuracy for positive and negative samples, respectively for estimation of blood plasma progesterone in Holstein cows. Further, Masello et al. (2020) reported 85% and 96% PPV and NPV, respectively. However, we have reported 93% and 81% PPV and NPV, respectively. All these differences in recorded values might be attributed to the different method of detection, variation in sample size, days of blood collection, and type of kits used and breed of animals.