Genetic polymorphism of the osteopontin gene and milk composition traits in Red Kandhari cattle

Introduction

India has emerged as the world's leading milk producer, recording 254.19 million tonnes of milk in 2022-23, a ~35.4% increase over 2018-19 (Anonymous, 2024-25). Dairy farming supports the livelihoods of millions of rural households; however, this impressive national output masks a critical challenge. Despite having the largest cattle population, milk yield per animal in India remains low, especially in indigenous cattle, which produce on average 4.03 kg of milk per day, compared with 11.36 kg in exotic and 8.32 kg in crossbred cows. Improving productivity in indigenous cattle is therefore essential to meet the growing demand for milk while preserving adaptability and sustainability. Conventional breeding approaches have contributed to genetic progress, but their effectiveness is limited by the late expression and moderate heritability of milk production traits. In recent years, molecular breeding strategies, such as marker-assisted and genomic selection, have provided powerful tools for accelerating genetic improvement. Among the available molecular markers, single nucleotide polymorphisms (SNPs) are particularly valuable for identifying genetic variations associated with economically important traits. Several candidate genes influencing milk yield and composition have been reported, with the osteopontin (OPN) gene receiving considerable attention. The bovine OPN gene, also referred to as ETA-1 or secreted phosphoprotein-1 (SPP-1), is located on chromosome 6 and consists of seven exons (Sodek et al., 2000) [27] . Osteopontin is a multifunctional phosphoglycoprotein expressed in various tissues and body fluids, including milk. Its expression is notably elevated during lactation, and it plays a key role in mammary gland development, cellular differentiation, and immune regulation, all of which are closely linked to milk production (Nagatomo et al., 2004) [17] . Previous studies have demonstrated significant associations between OPN gene polymorphisms and milk yield, milk composition, somatic cell count, and lactation persistency in exotic and crossbred cattle populations (Leonard et al., 2005 [15,10,1,5,7,11,12,24,16,4] . In contrast, similar information for Indian indigenous breeds remains limited, restricting the effective use of molecular tools in their improvement. Red Kandhari cattle, an important indigenous breed of the Marathwada region, are primarily distributed in the Latur, Nanded, and Parbhani districts of Maharashtra. Locally known as Lal Kandhari, this breed is valued for its hardiness, adaptability to harsh climatic conditions, disease resistance, and high breeding efficiency. Although traditionally valued as draught animals, Red Kandhari cows produce 400-600 kg of milk per lactation, with an average fat content of 4.6% (NBAGR, 2020) [18] , indicating clear potential for genetic enhancement of milk traits. The National Cattle Breeding Policy emphasizes genetic enhancement of indigenous cattle through selective breeding and modern biotechnological interventions. Early and accurate identification of elite germplasm carrying favourable alleles is essential for accelerating genetic progress. Given the economic and adaptive importance of Red Kandhari cattle and the proven role of the OPN gene in milk production traits, the present study was undertaken to explore the genetic variability of the OPN (intron-4) gene and its association with milk traits in this indigenous breed.

Materials and Methods

Animal selection and sample collection

The study was conducted at the Genetic Investigation Laboratory (GIL), Department of Animal Genetics and Breeding, Mumbai Veterinary College, Mumbai. A total of 30 lactating Red Kandhari cows maintained at a private herd in Sayala Khating village, Parbhani district, Maharashtra were selected for the study. Blood samples (4-6 ml) were collected aseptically in EDTA vacutainers and stored at 4°C until analysis. Milk samples (10 ml) were collected after complete milking and analyzed on the same day for fat, protein, SNF, and lactose using a Lactoscan milk analyser.

Genomic DNA extraction and quality assessment

Genomic DNA was isolated from blood samples using the phenol-chloroform-isoamyl alcohol method described by Sambrook and Russell (2006) [23] . The concentration and purity of DNA were assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA), while DNA integrity was verified by electrophoresis on 0.8% agarose gel. DNA samples with an OD260/280 ratio of 1.8-2.0 were used for further analysis and diluted to a working concentration of 50-100 ng/µl. [15] . PCR was performed in a 25 µl reaction volume containing PCR master mix (Takara Bio), primers, genomic DNA, and nuclease-free water, with a negative control included. Amplification was carried out using an initial denaturation at 95°C for 5 min, followed by 32 cycles of denaturation at 94°C for 45 s, touchdown annealing from 63°C to 53°C (-2°C per cycle) for 45 s, extension at 72°C

PCR amplification of the Osteopontin gene

for 45 s, and a final extension at 72°C for 7 min. PCR products were resolved on 1.8% agarose gel.

PCR-RFLP analysis

Genotyping of the OPN intron-4 region was performed using the BsrI restriction enzyme to detect polymorphism. PCR products were digested with BsrI at 65°C for 4.5 hours, followed by electrophoresis on 2.5% agarose gel. Restriction patterns were visualized under UV illumination and documented using a gel documentation system. Genotypes were determined based on fragment size comparison with a 100 bp DNA ladder.

Estimation of genotype and allele frequencies

Genotype and allele frequencies of the Osteopontin gene were calculated using standard population genetics methods based on observed genotype counts.

Results

The present study was conducted to evaluate polymorphism in the intron-4 region of the Osteopontin (OPN) gene in Red Kandhari cattle and to assess its association with milk composition traits. PCR amplification of the target region was successfully optimized using a touchdown annealing protocol ranging from 63 to 53 °C. The optimized PCR conditions resulted in clear and specific amplification with minimal non-specific products. All genomic DNA samples yielded a uniform PCR amplicon of 290 bp (Fig. 1) corresponding to the intron-4 region of the OPN gene. The consistency of amplification across all samples indicated the reliability and reproducibility of the assay. PCR-RFLP analysis of the amplified products using the BsrI restriction enzyme revealed two genotypes, TT and CT. The TT genotype was characterized by a single undigested fragment of 290 bp, whereas the CT genotype produced three fragments of 290 bp, 200 bp, and 90 bp (Fig. 2). Among the 30 Red Kandhari cows analyzed, 29 animals (0.97) exhibited the TT genotype, while only one animal (0.03) showed the CT genotype. The CC genotype was not detected in the studied population. Allelic frequency analysis showed a predominance of the T allele (0.983) over the C allele (0.017), indicating near fixation of the T allele at the OPN intron-4 locus in Red Kandhari cattle. Milk composition traits were recorded for all animals included in the study. Cows with the TT genotype showed mean fat, SNF, protein, and lactose percentages of 4.27 ± 0.05, 8.43 ± 0.04, 3.38 ± 0.03, and 4.40 ± 0.03, respectively. The single animal with the CT genotype exhibited slightly higher values for these traits; however, due to the presence of only one CT genotype animal, statistical association analysis between OPN intron-4 genotypes and milk composition traits could not be performed.

Shingane, 2024) [15,25,8,26] . The predominance of the TT genotype and the high frequency of the T allele observed in Red Kandhari cattle are in agreement with findings reported in several Indian indigenous breeds, including Gir, Gaolao, Dangi, Sahiwal, and Haryana cattle ( [25,26,8,9] , as well as East Anatolian Red cattle (Oztabak et al., 2008) [19] . In contrast, studies in exotic and crossbred cattle populations have [15,10,6,5] . The absence of the CC genotype and near fixation of the T allele in Red Kandhari cattle may be attributed to breed-specific evolutionary forces such as natural selection, adaptation to local agro-climatic conditions, restricted gene flow, or genetic drift.

The association between OPN intron-4 polymorphism and milk composition traits could not be statistically evaluated in the present study due to extremely low genotype variability, particularly the occurrence of only one CT genotype animal. Similar limitations have been reported earlier in Dangi cattle, where the presence of only a single CT genotype animal precluded meaningful association analysis (Karwa, 2023) [9] . Although the CT genotype cow exhibited slightly higher milk composition values, these observations remain indicative rather than conclusive. [15,10,5,11] . Conversely, other studies identified the T allele as favourable for milk fat percentage or milk yield (Pasandideh et al., 2015; De Mello et al., 2012) [20,7] . In contrast, numerous investigations reported no significant association between OPN intron-4 polymorphism and milk production traits in both indigenous and exotic cattle breeds (Salehi et [28,13,14,26] . These inconsistencies across studies may be attributed to differences in breed composition, sample size, production systems, statistical models, and linkage disequilibrium patterns between the investigated polymorphism and causative variants. [16,4,21,22,3] .

Conclusion

The present study confirmed the presence of polymorphism at the OPN intron-4 BsrI locus in Red Kandhari cattle, characterized by a predominance of the TT genotype and T allele and absence of the CC genotype. However, due to limited genotype variability, the association between OPN intron-4 polymorphism and milk composition traits could not be established. These findings emphasize the need for large-scale studies across the entire breeding tract of Red Kandhari cattle to identify additional polymorphisms and to better elucidate the role of the OPN gene in milk production and adaptation of indigenous cattle.

Acknowledgement

AcknowledgementAuthors are grateful to the Red Kandhari cattle owners for providing the samples for the research.

References

1. Alain K, Karrow NA, Thibault C, St Pierre J, Lessard M, Bissonnette N. Osteopontin: an early innate immune marker of Escherichia coli mastitis harbours genetic polymorphisms with possible links with resistance to mastitis. BMC Genomics. 2009;10:444.
2. Anonymous. Annual report 2024-25. Department of Animal Husbandry and Dairying, Ministry of Animal Husbandry, Dairying and Fisheries, Government of India [Internet]. 2025 [cited 2025 Jan 10]. Available from: https://dahd.nic.in/document/annual_report
3. Bauer EA, Kułaj D, Sawicki S, Pokorska J. Gene association analysis of an osteopontin polymorphism and ketosis resistance in dairy cattle. Scientific Reports. 2023;13:48771.
4. Bissonnette N. Genetic association of variations in the osteopontin gene (SPP1) with lactation persistency in dairy cattle. Journal of Dairy Science. 2018;101:456461.
5. Boleckova J, Matejickova J, Stipkova M, Kyselova J, Barton L. The association of five polymorphisms with milk production traits in Czech Fleckvieh cattle. Czech Journal of Animal Science. 2012;57(2):45-53.
6. De Mello F, Guimarães MFM, Cobuci JA, Gualberto MV. Analysis of genetic diversity of osteopontin gene in Girolando cattle. Revista Brasileira de Zootecnia. 2011;40(11):2374-2377.
7. De Mello F, Cobuci JA, Martins MF, Silva MVGB, Neto JB. Association of the polymorphism g.8514C>T in the osteopontin gene (SPP1) with milk yield in the dairy cattle breed Girolando. Animal Genetics. 2012;43:647-648.
8. Kate RD, Kale J, Singh J, Patil D, Bahiram K. Polymorphisms within intron-4 and exon-4 regions of the SPP1 gene and their association with milk traits in Gaolao cattle. Indian Journal of Animal Science. 2023;93(1):51-55.
9. Karwa S. Osteopontin gene polymorphism and its association with milk traits in Dangi cattle [M.V.Sc. thesis]. Nagpur (India): Maharashtra Animal and Fishery Sciences University; 2023.
10. Khatib H, Zaitoun I, Wiebelhaus-Finger J, Chang YM, Rosa GJM. The association of bovine PPARGC1A and OPN genes with milk composition in two independent Holstein cattle populations. Journal of Dairy Science. 2007;90:2966-2970.
11. Kulaj D, Pokorska J, Ochrem A, Dusza M, Makulska J. Effects of the c.8514C>T polymorphism in the International Journal of Advanced Biochemistry Research https://www.biochemjournal.com osteopontin gene on milk production, milk composition and disease susceptibility in Holstein-Friesian cattle. Italian Journal of Animal Science. 2019;18(1):546-553.
12. Lali A, Kulangara A, Aravindakshan TV. Effect of osteopontin gene variants on milk production traits in Holstein Friesian crossbred cattle of Kerala. Turkish Journal of Veterinary and Animal Sciences. 2020;44:695-701.
13. Leonard S, Khatib H, Schutzkus V, Chang YM, Maltecca C. Effects of the osteopontin gene variants on milk production traits in dairy cattle. Journal of Dairy Science. 2005;88:4083-4086.
14. Manzoor S, Nadeem A, Maryam J, Hashmi AS, Imran M, Babar ME. Osteopontin gene polymorphism association with milk traits and expression analysis in milk of riverine buffalo. Tropical Animal Health and Production. 2016;50(2):275-281.
15. Nagatomo T, Ohga S, Takada H, Nomura A, Hikino S, Imura M, et al. Microarray analysis of human milk cells: persistent high expression of osteopontin during the lactation period. Clinical and Experimental Immunology. 2004;138:47-53.
16. National Bureau of Animal Genetic Resources. Red Kandhari cattle breed profile [Internet]. Karnal (India): ICAR-NBAGR; 2020 [cited 2025 Jan 10]. Available from: https://nbagr.icar.gov.in/wpcontent/uploads/2020/02/Red-KandhariNBGR.pdf
17. Oztabak K, Cemal I, Dawit T, Iraz A, Ahmet M. Genetic polymorphisms of osteopontin, prolactin and PIT-1 genes in South and East Anatolian Red cattle. Animal Science. 2008;58:109-112.
18. Pasandideh M, Mohammadabadi MR, Esmailizadeh AK, Tarang A. Association of bovine PPARGC1A and OPN genes with milk production and composition in Holstein cattle. Czech Journal of Animal Science. 2015;60:97-104.
19. Raschia MA, Nani JP, Maizon DO. Single nucleotide polymorphisms in candidate genes associated with milk yield in Argentinean Holstein and Holstein × Jersey cows. Journal of Animal Science and Technology. 2018;60:31.
20. Raza SHA, Khan R, Pant SD, Shah MA, Quan G, Feng L, et al. Genetic variation in the OPN gene affects milk composition in Chinese Holstein cows. Animal Biotechnology. 2021;15:1-7.
21. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2006.
22. Salehi A, Nasiri K, Aminafshar M, Sayaadnejad MB, Sobhani R. Association of bovine osteopontin (OPN) gene polymorphism with milk production traits in Iranian Holstein bulls. Iranian Journal of Biotechnology. 2015;13:43-48.
23. Sharma M, Singh SP, Tiwari M, Sharma D, Kumar A, Goel R, et al. Fixation of T allele in C>T polymorphism in intron IV region of the SPP1 gene in Indian cattle breeds. International Journal of Livestock Research. 2019;9(9):116-121.
24. Shingne DF, Sawane MP, Chopade MM, Sorte SD, Pawar VD. Association of osteopontin gene with milk production traits in Gir native and HF crossbred cows. Indian Journal of Animal Science. 2024;94(11):948952.
25. Sodek J, Ganss B, McKee MD. Osteopontin. Critical Reviews in Oral Biology and Medicine. 2000;11:279303.
26. Zakizadeh S, Gholami H, Vakili R, Rohani MG. Effect of osteopontin gene polymorphism on milk production traits in Brown Swiss cattle. Journal of Animal Science. 2015;105:283-290.