Early Pregnancy Diagnosis using Pregnancy-Associated Glycoproteins in the Serum of Pregnant Ruminants

Doulat Khan1, Hamayun Khan1, Nazir Ahmad2, Muhammad Tarique Tunio3, Muhammad Tahir2, Muhammad Saleem Khan2 and Rifat Ullah Khan1*

1Department of Animal Health, The University of Agriculture, Peshawar, Pakistan

2Department of Animal Nutrition, The University of Agriculture, Peshawar, Pakistan 3Department of Agricultural Sciences, Allama Iqbal Open University, Islamabad

ABSTRACT

Early pregnancy diagnosis is an essential tool in successful reproductive management of ruminants. We attempted to explore the effectiveness of a marketable ELISA test kit for assessment of Pregnancy Associated Glycoproteins (PAGs) in peripheral blood for early pregnancy identification in cattle, buffalo, goats and sheep. A total of 120 blood were taken from jugular vein of different breeds of cattle (Achai, Achai x Jersey, Holstein Friesian and Jersey), buffalo (Nilli Ravi, Aza Kheli and non-descript), goats (Beetal, Teddy and non-descript) and sheep (Bulkhi, Karri and non-descript). In cattle, the average sensitivity, specificity, false pregnancy prognostic value, false non-pregnancy prognostic value and accuracy of the PAG-ELISA test were 86.77%, 66.67%, 86.77%, 100% and 90%, respectively. Similarly, the current study indicated that in buffalo, average sensitivity, specificity, false pregnancy prognostic value, false non-pregnancy prognostic value and accuracy of the PAG-ELISA test were 62.22%, 74.6%, 62.22%, 88.57% and 76.67% respectively. The current study demonstrated that average sensitivity, specificity, false pregnancy prognostic value, false non-pregnancy prognostic value and accuracy of the PAG-ELISA test were 100%, 100%, 100%, 100% and 100% respectively in goat. Similarly, the results in sheep showed that the average sensitivity, specificity, false pregnancy prognostic value, false non-pregnancy prognostic value and accuracy were 95.23%, 91.67%, 95.23%, 100% and 100% respectively. The results showed that PAG is a valuable marker for early pregnancy diagnosis in ruminants.

Article Information

Received 27 May 2019

Revised 30 July 2019

Accepted 11 September 2019

Available online 13 February 2020

Authors’ Contribution

DK and HK designed the study. NA, MTT did statistical analysis. MT, MSK and RUK wrote the article.

Key words

Pregnancy-associated glycoproteins, Ruminants, Sensitivity

DOI: http://dx.doi.org/10.17582/journal.pjz/20190527080541

* Corresponding author: [email protected]

0030-9923/2020/0002-0785 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan

Pregnancy maintenance is a clear economic factor in dairy animals since most of the pregnancy losses occur during the early stages of pregnancy (Garcıa-Ispierto et al., 2013; Commun et al., 2016). Therefore, timely pregnancy diagnosis is very important for sound reproductive management in herd health (Green et al., 2005; Kaya et al., 2016). Several techniques are being used to diagnose pregnancy in dairy herd such as ultrasound, palpation per rectum, estrone sulfate, milk progesterone assay, and blood tests. However, in most of the instances, accurate pregnancy diagnosis is hardly achieved due to one or the other reasons (Commun et al., 2016). Therefore, a simple but reliable pregnancy test for domestic animals have long been sought.

Finally, pregnancy can also be diagnosed through specific pregnancy-associated glycoproteins (PAGs). PAGs are considered powerful markers of pregnancy diagnosis since they are expressed during the early period of gestation (Commun et al., 2016). Among them, Pregnancy Associated Glycoprotein (PAG-1) has been used for pregnancy diagnosis because this molecule can be found in maternal blood soon after implantation as a marker of fetal well-being (Zoli et al., 1992). Importantly, PAG-1 is an essential component of binucleate trophoblast cells (Green et al., 2005) and can be easily monitored in serum since it can be test after at the end of one month of pregnancy and secondly interpretation of the assay does not require much knowledge. The aim of the present experiment was to evaluate the PAG as non-invasive diagnostic tool for early pregnancy marker in cow, buffalo, sheep and goat.

Materials and methods

All procedures in this experiment were approved by Committee on Ethics and Animal Welfare, The University of Agriculture, Peshawar, Pakistan.

A total of 120 pregnant animals including buffaloes (Nilli-Ravi, Aza-Khelli and cross bred), cows (Achai, Holstein Friesian and cross bred), sheep (Bulkhi, Karri, and cross bred) and goats (Beetal, Teddy and cross bred) were randomly selected which were recently inseminated. Each group of species consisted of 30 animals. The animals were synchronized by intramuscular injection of PGF2α. and estrus was detected. Animals with standing heat were inseminated artificially.

About 10 ml blood sample was taken on day 30 of the pregnancy of each animal inseminated. Blood was centrifuged at 3000 rpm for 10 min and then serum was separated and stored -20°C.

Pregnancy was confirmed on day 60 of each inseminated animal through abdominal ultrasonogrphy using a real-time B-mode ultrasound scanner equipped with a 5/7.5-MHz linear endorectal transducer. Serum PAG-1 was determined through commercial ELISA kit (IDEXX Laboratories, Westbrook, USA). The manufacturer instructions given in the kit were strictly followed.

The findings of the current study were subjected to statistical analysis in SPSS. In the current study (a) accurate pregnancy detection was done when both PAGs-ELISA analysis and ultrasound check were found affirmative. Likewise, accurate non-pregnancy detection was done when PAGs-ELISA analysis were affirmative but ultrasound check were found false. Also our finding further explored the false pregnancy prognostic assessment when PAGs-ELISA analysis were found false whereas ultrasound check were found affirmative. Lastly our results indicated false non-pregnancy prognostic assessment when both PAGs-ELISA analysis and ultrasound check were found negative. Furthermore, the sensitivity (100 × a / a + c), specificity (100 × d / d + b), positive predictive value (100 × a / a + b), negative predictive value (100 × d / c + d), and accuracy (100 × (a + d) / (a + b + c + d)) of the PAGs-ELISA analysis were measured from the current findings (Kaya et al., 2016).

Results and discussion

The results of pregnancy diagnosis in different breeds of cattle buffalos, goats and sheep are given in Table I. The average sensitivity, specificity, false pregnancy prognostic value, false non-pregnancy prognostic value and accuracy of the PAG-ELISA test were 86.77%, 66.67%, 86.77%, 100% and 90% for cattle, 62.22%, 74.6%, 62.22%, 88.57% and 76.67%, respectively for buffalos, 100%, 100%, 100%, 100% and 100%, respectively for goats and 95.23%, 91.67%, 95.23%, 100% and 100%, respectively for sheep.

Early pregnancy diagnosis is an important tool in livestock species and has traditionally been performed through transrectal palpation or ultrasonography. However, due to the limitations of the skilled technician and high demand of accurate pregnancy diagnosis, chemical pregnancy diagnosis is gaining more importance. In the current study, the accuracy was found to be 90%, 76.67, 100 and 96.66% in cattle, buffalo, goat and sheep respectively. The finding of the current study associated with PAGs in peripheral blood as pregnancy markers are in line with the literature (Zoli et al., 1992; Silva et al., 2007; Kaya et al., 2016). The slight difference in the results may be due to the differences among commercial kits, user experience, and individual variability of PAG concentration causing the limitations of pregnancy diagnosis. After successful conception, PAG may be detected in pregnant animals as early as 3 weeks of insemination. The concentration of PAG increases gradually until before calving, therefore, the sensitivity and accuracy of the test also increases with the advancement of the pregnancy (Szenci et al., 1998; Kaya et al., 2016).

In the current study, inaccurate positive pregnancy results were found more in cattle compared to the other species. In addition, no false positive pregnancy results were found in goat. According to Kaya et al. (2016) incorrect positive pregnancy results are most likely found during the early period of pregnancy. Incorrect positive results are most likely to be the result of embryonic death leading to the disappearance of maternal PAG in the blood due to the short half life of about 2.7 to 7 days (Semambo et al., 1992; Szenci et al., 2003). Another reason of false positive results may be due to the residual PAGs from the previous pregnancy since it takes almost 45 days before the residues of PAGs are cleared from the maternal blood (Kaya et al., 2015). Some authors have also reported missing PAG in cows near 100 days after calving following confirmed pregnancy (Silva et al., 2007). We infer that the incorrect positive results may be related to the cross-reaction with protein other than PAG resulting in lower sensitivity.

On the average, the accuracy of positive pregnancy diagnosis was severely affected in case of buffalo in this study. The average accuracy was 76.67% in buffaloes. In the past, attempts for the isolation of PAG in buffalo serum have been conducted, however, only partial purification have been achieved with a success rate of 68% (Jerome, 2012). Karen et al. (2007) found that the sensitivity of PAG test in buffalo serum was 11.1% during 19-24 days of pregnancy and reached 100% on day 31. It indicates that the best results through PAG test could be achieved after 31 days in buffalo.

In case of ewes and goats, in the present study, the results were satisfactory in terms of accuracy. On the average, the accuracy was 96.66% and 100% in sheep and goats, respectively. In ewes and goats, the concentration of PAG increases rapidly from week 3 of gestation. Maximum concentration reaches during the 9th week and then declines (Haugejorden et al., 2006).

Table I. Assessment of pregnancy associated glycoproteins in plasma sample of different breeds of cattle, buffalos, goat, and sheep for early pregnancy diagnosis.

a, accurate pregnancy detection (pregnant); b, inaccurate pregnancy detection (non-pregnant); d, accurate non-pregnancy detection (non-pregnant); Se%, sensitivity percentage; Sp%, specificity percentage; FPPV, false pregnancy prognostic value; FNPV, false non-pregnancy prognostic value; ACC, accuracy.

Conclusion

The results indicated that Pregnant Associated Glycoproteins (PAG) is a potential biomarker in early pregnancy in domestic ruminants.

Acknowledgment

We gratefully acknowledge the the financial support of Dr. Bakht Daraz, Station Director, Livestock Research and Development Station (LRDS) Surezai, Peshawar for the provision of the Pregnancy-Associated Glycoprotein Kit for execution of the current research project.

Statement of conflict of interest

Authors declare no potential conflict of interest

References

Commun, L., Velek, K., Barbry, J.B., Pun, S., Rice, A., Mestek, A., Egli, C. and Leterme, S. 2016. J. Vet. Diag. Invest., 28: 207-213. https://doi.org/10.1177/1040638716632815

García-Ispierto, I., Almería, S., Serrano, B., de Sousa, N.M., Beckers, J.F. and López Gatius, F., 2013. Rep. Dom. Anim., 48: 613-618. https://doi.org/10.1111/rda.12134

Green, J.A., Parks, T.E., Avalle, M.P., Telugu, B.P., McLain, A.L., Peterson, A.J., McMillan, W., Mathialagan, N., Hook, R.R., Xie, S. and Roberts, R.M., 2005. Theriogenology, 63: 1481-503. https://doi.org/10.1016/j.theriogenology.2004.07.011

Haugejorden, G., Waage, S., Dahl, E., Karlberg, K., Beckers, J.F. and Ropstad, E., 2006. Theriogenology, 66: 1976-1984. https://doi.org/10.1016/j.theriogenology.2006.05.016

Jerome, A., 2012. J. Adv. Vet. Res., 2: 50-58.

Karen, A., Darwish, S., Ramoun, A., Tawfeek, K., Van Hanh, N., De Sousa, N.M., Sulon, J., Szenci, O. and Beckers, J.F., 2007. Theriogenology, 68: 1150-1155.

Kaya, M.S., KÖSE, M., Bozkaya, F., Mutlu, H., Uçar, E.H. and Atli, M.O., 2012. Turk. J. Vet. Anim. Sci., 40: 694-699. https://doi.org/10.3906/vet-1602-41

Kaya, M.S., Kose, M., Coban, S., Mutlu, H., Ucar, E.H., Guzeloglu, A. and Atli, M.O., 2015. Rep. Dom. Anim., 50: 41.

Kaya, M.S., KÖSE, M., Bozkaya, F., Mutlu, H., Uçar, E.H. and Atli, M.O., 2016. Turk. J. Vet. Anim. Sci., 40:694-699.

Semambo, D.K.N., Eckersall, P.D., Sasser, R.G. and Ayliffe, T.R., 1992. Teriogenology, 37: 741-748. https://doi.org/10.1016/0093-691X(92)90153-I

Silva, E., Sterry, R.A., Kolb, D., Mathialagan, N., McGrath, M.F., Ballam, J.M. and Fricke, P., 2007. J. Dairy Sci., 90: 4612-4622. https://doi.org/10.3168/jds.2007-0276

Szenci, O., Beckers, J.F., Humblot, P., Sulon, J., Sasser, G., Taverne, M.A.M., Varga, J., Baltusen, R. and Scheckk, G., 1998. Teriogenology, 50: 77-88. https://doi.org/10.1016/S0093-691X(98)00115-0

Szenci, O., Beckers, J.F., Sulon, J., Bevers, M.M., Börzsönyi, L., Fodor, L., Kovács, F. and Taverne, M.A.M., 2003. Vet. J., 165: 307-313. https://doi.org/10.1016/S1090-0233(02)00180-6

Zoli, A.P., Guilbault, L.A., Delahaut, P., Ortiz, W.B. and Beckers, J.F., 1992. Biol. Reprod., 46: 83-92. https://doi.org/10.1095/biolreprod46.1.83