Comparative Protein Profiling of Blood and Milk of Early Pregnant Buffaloes Using SDS-PAGE

Samridhi  Singh; Ratan K Choudhary; Navdeep  Singh

doi:10.48165/aru.2023.4.1.3

Authors

Samridhi Singh College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India
Ratan K Choudhary College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India
Navdeep Singh Directorate of Livestock farms, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana- 141004, Punjab, India

DOI:

https://doi.org/10.48165/aru.2023.4.1.3

Keywords:

Buffalo, Milk, Blood, SDS-PAGE, Pregnancy diagnosis

Abstract

Conventional methods for detecting pregnancy in buffalo are inefficient before 30-days after artificial insemination. A proteomic approach for high-resolution analysis of blood plasma and milk proteins by SDS polyacrylamide gel electrophoresis (PAGE) is of great importance. Blood and milk samples were collected on days 0 (before insemination) and 20 and 25 after insemination from retrospective pregnant (n = 6) and non-pregnant (n = 6) animals. An equal volume of samples was loaded in (12%-4%) SDS gel to resolve major plasma and milk proteins. Plasma proteins showed three distinct zones – high, medium, and low molecular weight (MW). In the high MW zone (135 - 250 kDa), ~208 kDa and ~190 kDa proteins were visible. In the moderate MW zone (60 -75 kDa), 5-6 proteins like albumin (~70 kDa), 75 kDa, and 63 kDa consisting of pregnancy-associated glycoproteins, ~73 kDa being dominant. In the low MW zone (25 – 35 kDa), a clear band of 27 kDa was visible. Proteins in milk supernatants showed four distinct zones of proteins – very high, high, medium, and low molecular weight (MW) zones. In the very high MW zone (135 - 250 kDa), ~208 kDa and ~190 kDa proteins were visible. In the high MW zone (60 -75 kDa), proteins like lactoferrin (~78.2 kDa), 73 kDa, albumen (~66 kDa), and a heavy chain of immunoglobulin (IgG) (~54 kDa) were detected. In the medium MW zone (25 – 35 kDa), clear bands of milk caseins, namely thick alpha S2 casein (29 kDa), and other caseins. In the low MW zone (11 – 20 kDa), two thick bands of 18 kDa and 12 kDa were visible. The mean gray values (sum of the gray values of all the pixels in the peak divided by the number of pixels) of the 73 kDa protein peak were analyzed in two-way ANOVA using GraphPad (ver 8.0). Repeated measures of ANOVA were selected where pregnancy status (pregnant and non-pregnant) defined column factor and time (0-, 20- and 25-day) defined row factor. The expression of 73 kDa protein in blood plasma was higher (P = 0.04) in pregnant women than in non-pregnant buffaloes at 20- and 25-days after insemination. However, in milk, there was no difference in the expression of the 73 kDa protein between pregnant and non-pregnant buffaloes across time. Variation in animals within the groups was significantly high. The differential expression of ~73 kDa protein in plasma and precise identification of the protein can be a diagnostic marker for early pregnancy detection in buffaloes.

References

https://www.dahd.nic.in/division/provisional-key-results-20th-livestock-census

Abd El-Salam, M. H., & El-Shibiny, S. (2011). A comprehensive review on the composition and properties of buffalo milk. Dairy science & technology, 91, 663-699.

Bahuguna, C., & Sharma, M. (2022). Proteomic analysis of serum protein during early pregnancy. Journal of Proteins and Proteomics, 13(1), 1-15.

Balhara, A. K., Gupta, M., Singh, S., Mohanty, A. K., & Singh, I. (2013). Early

pregnancy diagnosis in bovines: Current status and future directions. The Scientific World Journal,2013,1-10.

Basic Animal Husbandry Statistics. Government of India (GOI). Ministry of Agriculture. Department of Animal Husbandry, Dairying and Fisheries. New Delhi. (2018).

Bella A, Sousa N M, Dehimi M L, Watts J and Beckers J F. (2007). Western analysis of pregnancy associated glycoprotein family (PAG) in placental extract of various mammals. Theriogenology.68: 1055–66.

Becskei, Z., Savić, M., Ćirković, D., Rašeta, M., Puvača, N., Pajić, M., ... & Paskaš, S. (2020). Assessment of water buffalo milk and traditional milk products in a sustainable production system. Sustainability, 12(16), 6616.

Buragohain, L., Nanda, T., Ghosh, A., Ghosh, M., Kumar, R., Kumar, S., ... & Balhara, A. K. (2017). Identification of serum protein markers for early diagnosis of pregnancy in buffalo. Animal Science Journal, 88(8), 1189-1197.

de Carvalho, N. A. T., Soares, J. G., & Baruselli, P. S. (2016). Strategies to overcome seasonal anestrus in water buffalo. Theriogenology, 86(1), 200-206.

Fricke, P. M. (2002). Scanning the future—Ultrasonography as a reproductive management tool for dairy cattle. Journal of dairy science, 85(8), 1918-1926.

Han, R. X., Kim, H. R., Diao, Y. F., Lee, M. G., & il Jin, D. (2012). Detection of early pregnancy-specific proteins in Holstein milk. Journal of proteomics, 75(11), 3221-3229.

Inyawilert, W., Rungruangsak, J., Chanthi, S., Liao, Y. J., Phinyo, M., Tang, P. C., & Nfor, O. N. (2019). Age-related difference changes semen quality and seminal plasma protein patterns of Thai native rooster. Int. J. Agric. Technol, 15(2), 287-296.

Johnston, D., Estepa, I. M., Ebhardt, H. A., Crowe, M. A., & Diskin, M. G. (2018). Differences in the bovine milk whey proteome between early pregnancy and the estrous cycle. Theriogenology, 114, 301-307.

Kalleshwarappa, G. M., Malakar, D., Chauhan, M. S., & Majumdar, A. C. (2009). Isolation, identification and immunogenic characterization of secretory proteins of embryos and in blood circulation during estrus and in early pregnant buffalo. Indian Journal of Animal Sciences, 79(8), 764-772.

Kausar, R., Hameed, A., Qureshi, Z. I., & Muhammd, G. (2017). Comparative Protein Profiling of Milk of Nili-Ravi Buffaloes, Sahiwal and Cross Bred Cows by SDS-PAGE. Pakistan Veterinary Journal, 37(1).

Korhonen, H., Marnila, P., & Gill, H. S. (2000). Milk immunoglobulins and complement factors. British Journal of Nutrition, 84(S1), 75-80.

KUMAR, P., SAXENA, A., Singh, S. K., & Agarwal, S. K. (2014). Glycoproteins profiles of placental extract-A step towards development of biomarkers of pregnancy in water buffalo. Indian Journal of Animal Sciences, 84, 424-426.

Lacy-Hulbert, S. J., Woolford, M. W., Nicholas, G. D., Prosser, C. G., & Stelwagen, K. (1999). Effect of milking frequency and pasture intake on milk yield and composition of late lactation cows. Journal of dairy science, 82(6), 1232-1239.

Malakar, D., & Majumdar, A. C. (2005). Isolation, identification and characterization of secretory proteins of IVMFC embryos and blood circulation of estrus and early pregnant goat.43:693- 701.

Mir, M. R., Pampori, Z. A., Iqbal, S., Bhat, J. I., Pal, M. A., & Kirmani, M. A. (2008). Hemato-biochemical indices of crossbred cows during different stages of pregnancy. International Journal of Dairy Science, 3(3), 154-159.

Momont, H. (1990). Rectal palpation: safety issues. The Bovine Practitioner, 122-123.

Ningtyas, I. K., damayanti Lestari, T., & Hermadi, H. A. (2019). Characterization of pregnancy-associated glycoprotein as a biomarker of pregnancy in Etawa crossbred goat. World's Veterinary Journal, 9(3), 181-186.

Noce, A., Qanbari, S., González-Prendes, R., Brenmoehl, J., Luigi-Sierra, M. G., Theerkorn, M., ... & Hoeflich, A. (2021). Genetic diversity of Bubalus bubalis in Germany and global relations of its genetic background. Frontiers in genetics, 11, 610353.

Saadullah, J. H., Ghani, U., Au, M. I. & Akhtar, N. (1999). Economic losses due to slaughtering of pregnant bufaloes at Peshawar abattoir. Pak. Vet. J. 19, 218–219

Salam S. (2016). ‘Purification and characterization of bovine urinary proteins during early gestation.’ MVSc. thesis, G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand.

Sweety, Nirmal Sangwan, Komal (2020). Depletion of high abundance proteins in plasma of buffalo calves by SDS-PAGE of magnetic isolated anionic fraction. Journal of Entomology and Zoology Studies,8(1):918-919.

Ward, P. P., Uribe-Luna, S., & Conneely, O. M. (2002). Lactoferrin and host defense. Biochemistry and Cell Biology, 80(1), 95-102.

Zhang, M., Sun, X., Cheng, J., & Guo, M. (2022). Analysis and comparison of nutrition profiles of canine milk with bovine and caprine milk. Foods, 11(3), 472.

Zicarelli, L. (2011). Enhancing reproductive performance in domestic dairy water buffalo (Bubalus bubalis). Reproduction in Domestic Ruminants VII; Lucy, M., Pate, J., Smith, M., Spencer, T., Eds, 443-455.

Žvorc Z, Matijatko V, Beer Ljubic B, Foršek J, Bedrica L, Kučer N (2000). Blood serum proteinograms in pregnant and non-pregnant cows. Veterinarski arhiv , 70 (1), 21-30.