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Potential utility of RSAD2 transcript and protein in early detection of pregnancy in buffaloes

Published online by Cambridge University Press:  02 October 2024

Samridhi Singh
Affiliation:
Animal Stem Cells Laboratory, College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India
Ratan Kumar Choudhary*
Affiliation:
Animal Stem Cells Laboratory, College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India
*
Corresponding author: Ratan Kumar Choudhary; Email: [email protected]

Abstract

This study investigates a novel early pregnancy marker in water buffaloes, focusing on RSAD2 mRNA expression, known to be upregulated by interferon-tau (IFNT) during pregnancy. While RSAD2 is primarily recognized for its antiviral effect, we hypothesized its role as a conceptus-induced component in regulating pregnancy in buffaloes. Given its differential expression compared to other IFNT-induced genes in cows, RSAD2 may serve as a biomarker for early pregnancy detection in buffaloes. RNA, cDNA, and plasma samples were obtained from archived samples collected before insemination (d0) and at d20, d25 and d40 after insemination. Twelve RNA samples, having optimal optical density and concentration, from six pregnant and six non-pregnant buffaloes were selected. The cDNA was analyzed to measure the abundance of RSAD2 mRNA using real-time quantitative PCR (RT-qPCR) and plasma for protein expression analysis using Western blot. The RT-qPCR analysis showed a transcript of RSAD2 increased significantly by 7-fold and 6-fold on d20 and d25, compared to both d0 and d40 in the pregnant group only. At d20, the sensitivity of RSAD2 was 100% and the specificity was 83.3%, and at d25-d both the sensitivity and specificity was 100%, indicating low incidences of misdiagnosing early pregnancy in buffaloes. In the non-pregnant group, RSAD2 expression remained low and did not change after insemination. Western blot analysis revealed an immunoreactive RSAD2 protein band. Densitometry analysis of the RSAD2-specific protein band, based on gray mean value, showed significantly increased expression of RSAD2 at d25 compared to d0 in the pregnant group. In conclusion, these results indicated that RSAD2 expressions at both the mRNA and protein levels show promising potential for detecting pregnancy at d25 post-insemination.

Type
Research Article
Copyright
Copyright © The Author(s), 2024. Published by Cambridge University Press on behalf of Hannah Dairy Research Foundation

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References

Aba, MA, Sumar, J, Kindahl, H, Forsberg, M and Edqvist, LE (1998) Plasma concentrations of 15-ketodihydro-PGFf(2α), progesterone, oestrone sulphate, oestradiol-17β and cortisol during late gestation, parturition and the early post partum period in llamas and alpacas. Animal Reproduction Science 50, 111121.CrossRefGoogle Scholar
Abdulkareem, TA, Al-Sharifi, S, Ishak, MA, Eidan, SM, Alnimr, MA, Passavant, CW, Branen, JR and Sasser, RG (2011) Early pregnancy detection of Iraqi riverine buffalo (Bubalus bubalis) using the BioPRYN enzyme-linked immunosorbent assay for PSPB and the progesterone assay. Reproduction in Domestic Animals 46, 455462.CrossRefGoogle ScholarPubMed
Barbato, O, Menchetti, L, Sousa, NM, Malfatti, A, Brecchia, G, Canali, C, Beckers, JF and Barile, VL (2017) Pregnancy-associated glycoproteins (PAGs) concentrations in water buffaloes (Bubalus bubalis) during gestation and the postpartum period. Theriogenology 97, 7377.CrossRefGoogle ScholarPubMed
Barbato, O, Menchetti, L, Brecchia, G and Barile, VL (2022) Using pregnancy-associated glycoproteins (PAGs) to improve reproductive management: from dairy cows to other dairy livestock. Animals (Basel) 12, 2033.Google ScholarPubMed
Barile, VL, Menchetti, L, Casano, AB, Brecchia, G, Melo de Sousa, N, Zelli, R, Canali, C, Beckers, JF and Barbato, O (2021) Approaches to identify pregnancy failure in buffalo cows. Animals 11, 117.CrossRefGoogle ScholarPubMed
Campanile, G, Neglia, G and D'Occhio, MJ (2016) Embryonic and fetal mortality in river buffalo (Bubalus bubalis). Theriogenology 86, 207213.CrossRefGoogle 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, 3068.CrossRefGoogle 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, 383401.CrossRefGoogle Scholar
Chin, KC and Cresswell, P (2001) Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirus. Proceedings of the National Academy of Sciences of the United States of America 98, 1512515130.CrossRefGoogle ScholarPubMed
Forde, N, Carter, F, Spencer, TE, Bazer, FW, Sandra, O, Mansouri-Attia, N, Okumu, LA, McGettigan, PA, Mehta, JP, McBride, R, O'Gaora, P, Roche, JF and Lonergan, P (2011) Conceptus-induced changes in the endometrial transcriptome: how soon does the cow know she is pregnant? Biology of Reproduction 85, 144156.CrossRefGoogle Scholar
Gallo-Oller, G, Ordoñez, R and Dotor, J (2018) A new background subtraction method for western blot densitometry band quantification through image analysis software. Journal of Immunological Methods 457, 15.CrossRefGoogle ScholarPubMed
Green, JC, Okamura, CS, Poock, SE and Lucy, MC (2010) Measurement of interferon-tau (IFN-tau) stimulated gene expression in blood leukocytes for pregnancy diagnosis within 18–20d after insemination in dairy cattle. Animal Reproduction Science 121, 2433.CrossRefGoogle ScholarPubMed
Helbig, KJ, Lau, DTY, Semendric, L, Harley, HAJ and Beard, MR (2005) Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector. Hepatology 42, 702710.CrossRefGoogle 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, 11501155.CrossRefGoogle ScholarPubMed
Katze, MG, He, Y and Gale, M (2002) Viruses and interferon: a fight for supremacy. Nature Reviews. Immunology 2, 675687.CrossRefGoogle ScholarPubMed
Kizaki, K, Shichijo-Kizaki, A, Furusawa, T, Takahashi, T, Hosoe, M and Hashizume, K (2013) Differential neutrophil gene expression in early bovine pregnancy. Reproductive Biology and Endocrinology: RB&E 11, 6.CrossRefGoogle 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 (San Diego, Calif.) 25, 402408.CrossRefGoogle ScholarPubMed
Mamo, S, Mehta, JP, Forde, N, McGettigan, P and Lonergan, P (2012) Conceptus-endometrium crosstalk during maternal recognition of pregnancy in cattle. Biology of Reproduction 87, 6.CrossRefGoogle ScholarPubMed
Panda, B. S. K., Mohapatra, S. K., Chaudhary, D., Alhussien, M. N., Kapila, R. and Dang, A. K. (2020). Proteomics and transcriptomics study reveals the utility of ISGs as novel molecules for early pregnancy diagnosis in dairy cows. Journal of Reproductive Immunology 140, 103148.CrossRefGoogle ScholarPubMed
Pohler, KG, Pereira, MHC, Lopes, FR, Lawrence, JC, Keisler, DH, Smith, MF, Vasconcelos, JLM and Green, JA (2016) Circulating concentrations of bovine pregnancy-associated glycoproteins and late embryonic mortality in lactating dairy herds. Journal of Dairy Science 99, 15841594.CrossRefGoogle 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, 112.CrossRefGoogle ScholarPubMed
Rawat, P, Bathla, S, Baithalu, R, Yadav, ML, Kumar, S, Ali, SA, Tiwari, A, Lotfan, M, Naru, J, Jena, M, Behere, P, Balhara, AK, Vashisth, R, Singh, I, Dang, A, Kaushik, JK, Mohanty, TK and Mohanty, AK (2016) Identification of potential protein biomarkers for early detection of pregnancy in cow urine using 2D DIGE and label free quantitation. Clinical Proteomics 13, 15.CrossRefGoogle ScholarPubMed
Rocha, CC, Cristina, S, De Melo, GD, Motta, IG, Coutinho, LL, Maria, A, Diaza, G and Binelli, M (2020) Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers. Scientific Reports 10, 13733.CrossRefGoogle ScholarPubMed
Rocha, CC, Martins, T, Silva, FACC, Sponchiado, M, Pohler, KG and Binelli, M (2023) Viperin (RSAD2) gene expression in peripheral blood mononuclear cells of pregnant crossbred beef cows is altered by Bos indicus genetics. Theriogenology 209, 226233.CrossRefGoogle ScholarPubMed
Sarangi, A, Ghosh, M, Sangwan, S, Kumar, R, Balhara, S, Phulia, SK, Sharma, RK, Sahu, S, Kumar, S, Mohanty, AK and Balhara, AK (2022) Exploration of urinary metabolite dynamicity for early detection of pregnancy in water buffaloes. Scientific Reports 12(1), 117.CrossRefGoogle ScholarPubMed
Schneider, CA, Rasband, WS and Eliceiri, KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671675.CrossRefGoogle ScholarPubMed
Schoggins, J. W. (2019). Interferon-stimulated genes: what do they all do?. Annual Review of Virology 6, 567584.CrossRefGoogle Scholar
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, 142145.CrossRefGoogle ScholarPubMed
Shirasuna, K., Matsumoto, H., Kobayashi, E., Nitta, A., Haneda, S., Matsui, M., Kawashima, C., Kida, K., Shimizu, T. and Miyamoto, A. (2012). Upregulation of interferon-stimulated genes and interleukin-10 in peripheral blood immune cells during early pregnancy in dairy cows. The Journal of Reproduction and Development 58, 8490.CrossRefGoogle ScholarPubMed
Silva, E, Sterry, RA, Kolb, D, Mathialagan, N, McGrath, MF, Ballam, JM and Fricke, PM (2007) Accuracy of a pregnancy-associated glycoprotein ELISA to determine pregnancy status of lactating dairy cows twenty-seven days after timed artificial insemination. Journal of Dairy Science 90, 46124622.CrossRefGoogle ScholarPubMed
Singh, S, Choudhary, RK and Singh, N (2024) Comparative protein profiling of blood and milk of early pregnant buffaloes using SDS-PAGE. Animal Reproduction Update 4, 1116.CrossRefGoogle Scholar
Sofia, HJ, Chen, G, Hetzler, BG, Reyes-Spindola, JF and Miller, NE (2001) Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Research 29, 10971106.CrossRefGoogle Scholar
Song, G, Bazer, FW and Spencer, TE (2007) Pregnancy and interferon tau regulate RSAD2 and IFIH1 expression in the ovine uterus. Reproduction (Cambridge, England) 133, 285295.CrossRefGoogle ScholarPubMed
Tadeo, RD, Atabay, EP, Atabay, EC, Matias, DD, Fajardo, ZP, Apolinario, JPR, Cruz, CF and Tilwani, RC (2021) Early pregnancy diagnosis in water buffaloes through detection of pregnancy-associated glycoprotein (PAG) in milk using enzyme-link immunosorbent assay. Thai Journal of Veterinary Medicine 51, 3541.CrossRefGoogle Scholar
Tirumurugaan, KG, Pawar, RM, Raj, GD, Thangavelu, A, Hammond, JA and Parida, S (2020) RNAseq reveals the contribution of interferon stimulated genes to the increased host defense and decreased PPR viral replication in cattle. Viruses 12, 463.CrossRefGoogle Scholar
Toji, N., Koshi, K., Furusawa, T., Takahashi, T., Ishiguro-Oonuma, T., Kizaki, K. and Hashizume, K. (2018) A cell-based interferon-tau assay with an interferon-stimulated gene 15 promoter. Biomedical Research (Tokyo, Japan) 39, 1320.CrossRefGoogle ScholarPubMed
Van Hanh, N, Nghia, SH, De Sousa, NM, Barbato, O and Beckers, JF (2020) Validation of buffalo PAG antisera for early pregnancy diagnosis in bovine. Indian Journal of Animal Research 9, Article Id: B-1135.Google Scholar
Wiltbank, MC, Baez, GM, Garcia-Guerra, A, Toledo, MZ, Monteiro, PLJ, Melo, LF, Ochoa, JC, Santos, JEP and Sartori, R (2016) Pivotal periods for pregnancy loss during the first trimester of gestation in lactating dairy cows. Theriogenology 86, 239253.CrossRefGoogle 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, 188193.CrossRefGoogle ScholarPubMed
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