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Early pregnancy detection in buffaloes targeting interferon-stimulated genes in duplex RT-qPCR

Published online by Cambridge University Press:  15 October 2025

Paramjeet Sharma ,
Navdeep Singh Ratta ,
Shanti Choudhary ,
Yashpal Singh Malik  and
Ratan Kumar Choudhary Open the ORCID record for Ratan Kumar Choudhary [Opens in a new window]
Paramjeet Sharma
Affiliation:
Animal Stem Cells Lab, Department of Bioinformatics, College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India
Navdeep Singh Ratta
Affiliation:
Directorate of Livestock Farms, College of Veterinary Sciences, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India
Shanti Choudhary
Affiliation:
Animal Stem Cells Lab, Department of Bioinformatics, College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India
Yashpal Singh Malik
Affiliation:
Animal Stem Cells Lab, Department of Bioinformatics, College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India ICAR-Indian Veterinary Research Institute, Mukteshwar, India
Ratan Kumar Choudhary*
Affiliation:
Animal Stem Cells Lab, Department of Bioinformatics, College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India ICAR-National Research Centre on Camel, Bikaner, Rajasthan, India
*
Corresponding author: Ratan Kumar Choudhary; Email: vetdrrkc@gmail.com
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Abstract

Early pregnancy diagnosis in buffaloes enhances reproductive efficiency and dairy production. This study aimed to develop and validate a duplex TaqMan-based RT-qPCR assay using two interferon-stimulated genes (ISGs), ISG15 and LGALS3BP, for early pregnancy diagnosis in buffaloes. Whole blood samples were collected from artificially inseminated buffaloes before (day 0) and after (days 20, 25 and 40) artificial insemination (AI). On 40 days, post-insemination ultrasonography was performed to categorize animals as pregnant or non-pregnant. RNA from peripheral blood mononuclear cells (PBMCs) was isolated and converted into cDNA. A duplex TaqMan-based RT-qPCR assay was developed to predict pregnancy based on the expression of ISG15 and LGALS3BP in PBMCs. Gene expression was evaluated in a test dataset at various time points. The assay's performance was validated using two additional datasets, under which blood samples were collected randomly at 20 ± 2 days to evaluate its utility for predicting pregnancy. Colour Doppler ultrasonography was utilized to confirm pregnancy at 40 days post-insemination for all the animals. Significantly different abundance of transcripts of ISG15 and LGALS3BP, in pregnant buffaloes, was observed on day 20 post-insemination. A statistically significant fold change (p < 0.05) of ISG15 and LGALS3BP transcripts was observed between pregnant and non-pregnant buffaloes. The receiver operating characteristic curve of validation datasets demonstrated the AUC = 0.95 and AUC = 0.90. The negative predictive value and positive predictive value range from 90% to 95% and 75% to 85%. In conclusion, the developed duplex RT-qPCR-based assay demonstrates high sensitivity and specificity in detecting early pregnancy in buffaloes.

Keywords

biomarkersbuffaloesISG15 and LGALS3BPpregnancy diagnosisRT-qPCR

Information

Type
Research Article
Information
Journal of Dairy Research , First View , pp. 1 - 8
Copyright
© The Author(s), 2025. Published by Cambridge University Press on behalf of Hannah Dairy Research Foundation.

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Footnotes

Equal contributions by the first and second authors.

References

Ali, A and Fahmy, S (2008) Ultrasonographic fetometry and determination of fetal sex in buffaloes (Bubalus bubalis). Animal Reproduction Science 106(1–2), 90–99. doi:10.1016/J.ANIREPROSCI.2007.04.010CrossRefGoogle ScholarPubMed
Baba, NA, Panigrahi, M, Verma, AD, Sadam, A, Sulabh, S, Chhotaray, S, Parida, S, Krishnaswamy, N and Bhushan, B (2019) Endometrial transcript profile of progesterone-regulated genes during early pregnancy of Water Buffalo (Bubalus bubalis). Reproduction in Domestic Animals 54(1), 100–107. doi:10.1111/RDA.13315CrossRefGoogle ScholarPubMed
Barile, VL, Menchetti, L, Casano, AB, Brecchia, G, de Sousa, M, Zelli, C, Beckers, JF and Barbato, O (2021) Approaches to identify pregnancy failure in buffalo cows. Animals 11(2), 1–17. doi:10.3390/ANI11020487CrossRefGoogle ScholarPubMed
Barile, VL, Terzano, GM, Pacelli, C, Todini, L, Malfatti, A and Barbato, O (2015) LH peak and ovulation after two different estrus synchronization treatments in Buffalo cows in the daylight-lengthening period. Theriogenology 84(2), 286–293. doi:10.1016/J.THERIOGENOLOGY.2015.03.019CrossRefGoogle ScholarPubMed
Bauersachs, S, Ulbirch, SE, Gross, K, Schmidt, SEM, Meyer, HH, Wenigerkind, H, Vermehren, M, Sinowatz, F, Blum, H and Wolf, E (2006) Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity. Reproduction 132(2), 319–331. doi:10.1530/REP.1.00996CrossRefGoogle ScholarPubMed
Bottomley, C, Van Belle, V, Kirk, E, Van Huffel, S, Timmerman, D and Bourne, T (2013) Accurate prediction of pregnancy viability by means of a simple scoring system. Human Reproduction 28(1), 68–76. doi:10.1093/HUMREP/DES352CrossRefGoogle ScholarPubMed
Bustin, SA, Benes, V, Garson, JA, Hellemans, J, Huggett, J, Kubista, M, Mueller, R, Nolan, T, Pfaffl, MW, Shipley, GL, Vandesompele, J and Wittwer, CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55(4), 611–622. doi:10.1373/clinchem.2008.112797CrossRefGoogle ScholarPubMed
Campanile, G, Neglia, G and D'Occhio, MJ (2016) Embryonic and fetal mortality in river Buffalo (Bubalus bubalis). Theriogenology 86(1), 207–213. doi:10.1016/J.THERIOGENOLOGY.2016.04.033CrossRefGoogle ScholarPubMed
Casano, AB, Barile, VL, Menchetti, L, Guelfi, G, Brecchia, G, Agradi, S, De Matteis, G, Scatà, MC, Grandoni, F and Barbato, O (2022) Interferon tau (IFNt) and interferon-stimulated genes (ISGs) expression in peripheral blood leukocytes and correlation with circulating pregnancy-associated glycoproteins (PAGs) during peri-implantation and early pregnancy in Buffalo cows. Animals 12(22), 3068. doi:10.3390/ani12223068CrossRefGoogle ScholarPubMed
Casano, AB, Menchetti, M, Trabalza-Marinucci, M, Riva, F, De Matteis, G, Brecchia, G, Inglesi, A, Rossi, E, Signorelli, F, Barile, VL and Barbato, O (2023) Gene expression of pregnancy-associated glycoproteins-1 (PAG-1), interferon-tau (IFNt) and interferon stimulated genes (ISGs) as diagnostic and prognostic markers of maternal-fetal cellular interaction in Buffalo cows. Theriogenology 209, 89–97. doi:10.1016/J.THERIOGENOLOGY.2023.06.028CrossRefGoogle ScholarPubMed
Cheng, L, Xiang, M, Hu, X, Yu, J, Xia, Y, Tao, B and Zhao, S (2019) Duplex quantitative polymerase chain reaction of ISG15 and RSAD2 increases accuracy of early pregnancy diagnosis in dairy cows. Annals of Animal Science 19(2), 383–401. doi:10.2478/AOAS-2019-0001CrossRefGoogle Scholar
Ferraz, PA, Filho, CASG, Rocha, CC, Neto, AL, de Andrade Bruni, G, Oshiro, TSI, Baruselli, PS, Lima, FS and Pugliesi, G (2021) Feasibility and accuracy of using different methods to detect pregnancy by conceptus-stimulated genes in dairy cattle. JDS Communications 2(3), 153–158. doi:10.3168/JDSC.2020-0062CrossRefGoogle ScholarPubMed
Fontes, PLP and Oosthuizen, N (2022) Applied use of Doppler ultrasonography in bovine reproduction. Frontiers in Animal Science 3, 912854. doi:10.3389/FANIM.2022.912854/BIBTEXCrossRefGoogle Scholar
Gnemmi, GM, Maraboli, CVA, Gnemmi, B, Saleri, R and De Rensis, F (2022) Use and adequacy of non-pregnancy diagnosis in cow. Which Future? Reproduction in Domestic Animals 57(S5), 45–52. doi:10.1111/RDA.14206CrossRefGoogle ScholarPubMed
Han, H, Austin, KJ, Rempel, LA and Hansen, TR (2006) Low blood ISG15 mRNA and progesterone levels are predictive of non-pregnant dairy cows. The Journal of Endocrinology 191(2), 505–512. doi:10.1677/JOE.1.07015CrossRefGoogle ScholarPubMed
Kanazawa, T, Seki, M and Iga, K (2022) Early pregnancy diagnosis based on luteal morphology and blood flow on Days 17-21 post-artificial insemination in Japanese Black cattle. Theriogenology 181, 69–78. doi:10.1016/J.THERIOGENOLOGY.2022.01.002CrossRefGoogle ScholarPubMed
Karen, A, Darwish, S, Ramoun, A, Tawfeek, K, Van Hanh, N, de Sousa, NM, Sulon, J, Szenci, O and Beckers, JF (2007) Accuracy of ultrasonography and pregnancy-associated glycoprotein test for pregnancy diagnosis in buffaloes. Theriogenology 68(8), 1150–1155. doi:10.1016/J.THERIOGENOLOGY.2007.08.011CrossRefGoogle ScholarPubMed
Karen, AM, Darwish, S, Ramoun, A, Tawfeek, K, Van Hanh, N, de Sousa, NM, Sulon, J, Szenci, O and Beckers, JF (2011) Accuracy of transrectal palpation for early pregnancy diagnosis in Egyptian buffaloes. Tropical Animal Health and Production 43(1), 5–7. doi:10.1007/S11250-010-9675-2CrossRefGoogle ScholarPubMed
Lang, R, Li, H, Luo, X, Liu, C, Zhang, Y, Guo, S, Xu, J, Bao, C, Dong, W and Yu, Y (2022) Expression and mechanisms of interferon-stimulated genes in viral infection of the central nervous system (CNS) and neurological diseases. Frontiers in Immunology 13, 1008072. doi:10.3389/fimmu.2022.1008072CrossRefGoogle ScholarPubMed
Liu, C, Wei, X and Wang, F (2024) The predictive value of ultrasound markers for pregnancy outcomes in recurrent pregnancy loss: a retrospective study. Scientific Reports 14(1), 1–10. doi:10.1038/s41598-024-67744-wGoogle ScholarPubMed
Liu, FT and Stowell, SR (2023) The role of galectins in immunity and infection. Nature Reviews Immunology 23(8), 479–494. doi:10.1038/s41577-022-00829-7CrossRefGoogle ScholarPubMed
Livak, KJ and Schmittgen, TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4), 402–408. doi:10.1006/meth.2001.1262CrossRefGoogle ScholarPubMed
Margadant, C, Van Den Bout, I, Van Boxtel, AL, Thijssen, VL and Sonnenberg, A (2012) Epigenetic Regulation of galectin-3 expression by β1 integrins promotes cell adhesion and migration. Journal of Biological Chemistry 287(53), 44684–44693. doi:10.1074/JBC.M112.426445CrossRefGoogle ScholarPubMed
Nag, BSP, Arunmozhi, N, Sarath, T, Asokan, SA and Vijayarani, K (2018) Interferon stimulated gene 15 and myxovirus resistance 2 genes are upregulated during early pregnancy in buffaloes. International Journal of Current Microbiology and Applied Sciences 7(12), 1755–1762. doi:10.20546/ijcmas.2018.712.204CrossRefGoogle Scholar
Pawshe, CH, Appa Rao, KBC and Totey, SM (1994) Ultrasonographic imaging to monitor early pregnancy and embryonic development in the Buffalo (Bubalus bubalis). Theriogenology 41(3), 697–709. doi:10.1016/0093-691X(94)90179-MCrossRefGoogle ScholarPubMed
Pugliesi, G, Miagawa, BT, Paiva, YN, França, MR, Silva, LA and Binelli, M (2014) Conceptus-induced changes in the gene expression of blood immune cells and the ultrasound-accessed luteal function in beef cattle: how early can we detect pregnancy? Biology of Reproduction 91(4). doi:10.1095/BIOLREPROD.114.121525CrossRefGoogle ScholarPubMed
Rocha, CC, da Silva Andrade, SC, de Melo, GD, Motta, IG, Coutinho, LL, Gonella-Diaza, AM, Binelli, M and Pugliesi, G (2020) Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers. Scientific Reports 10(1), 1–15. doi:10.1038/s41598-020-70616-8CrossRefGoogle ScholarPubMed
Sangwan, S, Vikram, R, Hooda, E, Choudhary, R, Jawla, J, Somagond, YM, Balhara, S, Phulia, SK, Khan, MH, Girish, PS, Datta, TK, Mitra, A and Balhara, AK (2024) Urinary metabolomics reveals potential biomarkers for early detection of pregnancy in mithun (Bos frontalis) cows. Journal of Proteomics 306, 105259. doi:10.1016/J.JPROT.2024.105259CrossRefGoogle ScholarPubMed
Santos, JEP, Thatcher, WW, Chebel, RC, Cerri, RLA and Galvão, KN (2004) The effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs. Animal Reproduction Science 82–83, 513–535. doi:10.1016/j.anireprosci.2004.04.015CrossRefGoogle ScholarPubMed
Sharma, P, Choudhary, RK, Ratta, NS and Singh, ST (2023) Investigation of conceptus stimulated gene expression in Buffalo peripheral blood mononuclear cells as potential diagnostic markers of early pregnancy. Journal of Dairy Research 90(2), 142–145. doi:10.1017/S0022029923000304CrossRefGoogle ScholarPubMed
Singh, S and Choudhary, RK (2024) Potential utility of RSAD2 transcript and protein in early detection of pregnancy in buffaloes. J Dairy Research 1-6, doi:10.1017/S0022029924000360.Google Scholar
Walsh, SW, Williams, EJ and Evans, and AC O (2011) A review of the causes of poor fertility in high milk producing dairy cows. Animal Reproduction Science 123(3–4), 127–138. doi:10.1016/j.anireprosci.2010.12.001CrossRefGoogle ScholarPubMed
Yoshino, H, Toji, N, Sasaki, K, Koshi, K, Yamagishi, N, Takahashi, T, Ishiguro-Oonuma, T, Matsuda, H, Yamanouchi, T, Hashiyada, Y, Imai, K, Izaike, Y, Kizaki, K and Hashizume, K (2018) A predictive threshold value for the diagnosis of early pregnancy in cows using interferon-stimulated genes in granulocytes. Theriogenology 107, 188–193. doi:10.1016/j.theriogenology.2017.11.014CrossRefGoogle ScholarPubMed