Introduction

Among the many nutrients provided in dairy milk and dairy products, milk protein is an important part of daily protein intake in the human diet. β-casein is the most extensively studied milk protein gene. Milk being produced in the udder by Mammary Epithelial Cells (MECs), they are a valuable source of mammary transcripts. Milk protein gene expression is regulated at multiple levels within MECs and depends on concentrated action of hormones, local growth factors, cell-cell interactions and cell-extracellular matrix (ECM) interactions that modulate the function of specific transcription factors (Qian and Zhao, 2014). β-casein gene regulation involves two principal cis-regulatory regions, a proximal promoter and a distal enhancer (Rijnkels et al., 2003). The proximal promoter of β-casein has lactogenic response elements that harbor multiple or a single binding site(s) for transcription factors, mainly including signal transducer and transcription activator 5 (STAT5), Runt-related transcription factor 2 (RUNX2), CAAT/enhancer binding protein β (C/EBPβ) and the repressive transcription factor Yin Yang 1 (YY-1) (Qian and Zhao, 2014). Knowledge about the regulation of expression of the different milk protein genes during lactation can be a great tool for the genetic improvement of milk composition and milk yield. Sigl et al. (2012; 2014) applied the immunomagnetic isolation method of pBMEC from fresh milk, to determine milk protein and its transcription regulatory genes expression profiles in the same animal over the course of lactation for the first time. Surti buffalo is considered an economical producer of milk and butter fat with an average lactation milk yield of 1667 kg with 7.02 percent fat during the lactation (NBAGR). Considering the importance of buffalo milk composition in the preparation of dairy products like cheese and the stage wise variation in the milk composition, it was decided to study the expression profile of the β-casein milk protein and its transcription regulating genes during 15^th and 60^th day pp in Surti buffaloes.

Materials and Methods

Fifteen hundred milliliter of the whole milk from 10 selected Surti buffaloes, maintained at Livestock Research Station, NAU, Navsari was collected during milking into a sterile bucket and was subjected for cell isolation. The method for isolation of somatic cells and pBMEC from buffalo milk samples were adapted from Sigl et al. (2012) using 2.5 μl primary mouse monoclonal antibody against cytokeratin 8 antibody (clone C-43). Total RNA was extracted from the purified pBMEC applying the miRNeasy MiniKit as per the manufacturer’s (Qiagen) protocol. After quantity and quality analysis of the RNA, 500 ng of the samples were subjected to reverse transcription for cDNA synthesis using QuantiTect® Reverse Transcription kit. Relative expression of β-casein (CSN2), its transcription regulatory (C/EBPβ, RUNX2, STAT5A and YY1) and keratin (KRT) genes was carried out using primers reported in a previous study in HF cows (Sigl et al., 2014) and quantified by Applied Biosystems 7500 Real Time PCR. The housekeeping gene ribosomal protein S9 (RPS9) was used as reference index and used for normalization. Quantitative cycle (Cq) values were calculated by Applied Biosystems 7500 software v2.0.5. The ΔCq values were calculated as ΔCq = Cq target gene transcript – Cq reference gene transcript (Pfaffl, 2001). Additionally the amount of target normalized to an endogenous reference gene and relative to a calibrator was obtained by following equation:

Fold increase/ decrease in target =  (Livak and Schmittgen, 2001)

The data on number of somatic, epithelial cells and expression analysis were subjected to the t test analysis using R (Version 3.3.0) software. The ΔCq values were normalized individually in relation to the housekeeping gene index of RPS9 prior to statistical analysis.

Results and Discussions

Somatic Cell Count, pBMECs Recovery and RNA Yield

The Mean ± SEm values (× 1000/ml milk) of Somatic Cell Count (SCC), pBMECs, pBMECs recovery percent and RNA yield are presented in Table 1.

Table 1: Mean somatic cells, primary bovine mammary epithelial cells and RNA yield in Surti buffalo at day 15 and 60 pp

* Significant at p≤0.05, ** highly significant at p≤0.01

Somatic cells isolated at early stage of lactation were significantly (p < 0.01) higher as compared to 60^th day pp. The overall mean SCCs, in the present study for both the groups were slightly higher than the average SCC of 1.99 ± 0.03 × 10⁵ cells/ml of milk reported from healthy quarters of buffaloes (Nandi, 2010). A decrease in the mean SCC in Surti buffalo milk from day 15 pp to day 60 pp has also been reported by Tyagi (2016) and Janmeda (2016), which is in accordance with the present finding. Patil et al. (2015) reported higher mean SCC of 3.21 ± 0.18 × 10⁵ cells/ml in the milk from normal buffalo as compared to the present study. Contrary, Kavitha et al. (2009) observed lower mean normal SCC of 1.6 × 10⁵ cells/ml in the milk of Murrah cross buffalo. The significant decrease in SCC with advancement of lactation in S60 group compared to S15 group was in accordance with rapid decrease in SCC reported for early lactating cows (Muggli, 1995; Sigl et al., 2012).

The mean pBMEC values (× 1000/ml) and pBMEC recovery percent obtained from total somatic cells were found to be similar in S15 and S60 groups. Tyagi (2016) and Janmeda (2016) could recover 4.99 ± 0.52 x 10³ to 6.30 ± 0.29 x 10³ pBMEC from buffalo milk, which is comparable with the range of present findings. However, lower pBMEC ranging from 1.10 ± 0.06 × 10³ to 1.40 ± 0.03 × 10³ cells/ml milk at different stage of lactation had also been reported in cows (Sigl et al., 2012). Significant differences at different stages of lactation in recovery percentage of pBMEC in relation to total milk cells were reported in HF cows (Sigl et al., 2012) and Surti, Mehsani and Jaffarabadi buffaloes (Tyagi, 2016; Janmeda, 2016). An optical density 260/280 ratio (OD 260/280) ~ (1.80 to 2.03) was observed in the samples of the present study indicating purity of RNA. The mean RNA yield (μg) was observed significantly high by 198.70 percent in S15 as compared to that of S60 group. Tyagi (2016) has also reported variation in the range from 2.13 ± 0.06 to 6.01 ± 0.59 μg in Surti and Mehsani buffaloes. While, Janmeda (2016) could obtain 3.89 ± 0.90 to 7.56 ± 0.81 μg of total RNA from Surti and Jaffarabadi buffaloes. Higher RNA yield in S15 group in this study may be attributed to the breed, various non genetic factors like stage of lactation, quality of the sample at the time of RNA isolation etc.

Gene Expression Analysis

The mean relative expressions, fold increase / decrease and correlations among CSN2, C/EBPβ, RUNX2, STAT5A, YY1 and KRT8 genes for day 15 and 60 pp in Surti buffaloes are given in Table 2, Table 3 and Table 4, respectively.

Table 2: Mean relative expression of β-casein milk protein, its transcription regulatory and keratin genes at day 15 and 60 pp in Surti buffaloes

Values in parenthesis are mean -∆Cq values

The results revealed that relative expression of genes under study are not affected by the stage of lactation in Surti buffaloes. Tyagi (2016) has also not found any difference in the mean relative expression of CSN2 in Surti and Mehsani buffaloes at various stages of lactation. However, significant changes in relative expression of CSN2 gene at various stages of lactation have been reported in various cattle breeds (Sigl et al., 2012; Sigl et al., 2014; Verma and Agastian, 2013) and Murrah buffaloes (Anonymous, 2014). Sigl et al. (2014) reported a significant increase in mRNA abundance of C/EBPβ from day 8 to day 57 pp, which was not observed in the present study.

Table 3: Folds increase/decrease in β-casein milk protein, its transcription regulatory and keratin gene transcripts at day 15 and 60 pp in Surti buffaloes

Table 4: Correlation coefficients between relative expression of β-casein milk protein and its transcription regulatory genes in Surti buffaloes at day 15 (above diagonal) and day 60 post partum (below diagonal)

* Significant at p≤0.05, ** highly significant at p≤0.01

Constant relative expression of RUNX2, STAT5A and YY1 genes during first 155 days of lactation has also been reported by Sigl et al. (2014), which is in agreement with the findings of the present study. KRT8 almost constantly expressed irrespective of the stage of lactation. This might be the result of common source of RNA obtained under the present study exclusively from pBMEC. The epithelial keratins had earlier been also found to be useful markers for epithelial cells (Taylor-Papadimitriou et al., 1989). Transcript abundance of KRT8 gene was also found to be constant at different lactation stages in Holstein Friesian cows (Sigl et al., 2012; Sigl et al., 2014) and buffaloes (Tyagi, 2016; Janmeda et al., 2017).

Highly significant and positive correlation was found between the relative expression of CSN2 gene and that of YY1 at early stage of lactation. The significant and moderately positive correlation of relative expression of C/EBPβ gene with RUNX2 and STAT5A at day 60 pp, suggests the combined role of these genes in β-casein synthesis in Surti buffalo milk. Similarly, highly significant and positive correlation between mean relative expression of RUNX2, STAT5A and YY1 as well as between STAT5A and YY1 at 60 days pp in the present study, suggests their interactive role in transcription regulation of β-casein milk protein gene.   

Conclusion

In the present study, the total RNA extracted from pBMEC obtained non-invasively from buffalo milk can be utilized for studying relative expression of β-casein and its transcription regulatory genes using primers adapted from the studies previously conducted on cows. No significant differences were found between mean relative expression of β-casein and its transcription regulatory genes at both the stages of lactation. The steady expression of KRT8 gene obtained among both the groups under present study makes it a gene of choice as epithelial cell marker.

Acknowledgement

The authors are very much thankful to the Dean, Veterinary College, NAU, Navsari for providing necessary financial support and infrastructural facilities to carry out this study.

References

1. 2014. Final Report: NAIP – ICAR. Analysis of Mammary Gland Transcriptome and Proteome during Lactation and Involution in Indigenous Cattle and Buffalo for Identification of Probable Mammary Biomarkers. National Dairy Research Institute, Karnal. pp-2.
2. Janmeda M, Kharadi V, Pandya G, Brahmkshtri B, Ramani U and Tyagi K. 2017. Relative gene expression of fatty acid synthesis genes at 60 days postpartum in bovine mammary epithelial cells of Surti and Jafarabadi buffaloes. Veterinary World. 10(5): 467-476.
3. Janmeda M. 2016. Relative gene expression study of genes associated with fatty acid synthesis in bovine mammary epithelial cells of Surti and Jafarabadi buffaloes, Ph.D. thesis. Navsari Agricultural University, Navsari.
4. Kavitha KL, Rajesh K, Suresh K, Satheesh K. and Syama Sunder N. 2009. Buffalo mastitis- Risk factors. Buffalo Bulletin. 28(3): 134-137.
5. Livak KJ and Schmittgen TD. 2001. Analysis of relative gene expression data using RealTime Quantitative PCR and the Methods. 25: 402–408.
6. Muggli J. 1995. Influence of stage of lactation on somatic cell counts. Animal Breeding Abstracts. p.1996.
7. Nandi S. 2010. Enzymatic alteration in buffalo milk related to udder health status, M.V.Sc thesis. Maharashtra Animal and Fishery Sciences, Nagpur, Maharashtra.
8. NBAGR 2017. http://www.nbagr.res.in/
9. Patil MP, Nagvekar AS, Ingole SD, Bharucha SV and Palve VT. 2015. Somatic cell count and alkaline phosphatase activity in milk for evaluation of mastitis in buffalo. Veterinary World. 8(3): 363-366.
10. Pfaffl MW. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research. 29(9): e45.
11. Qian X and Zhao F. 2014. Current Major Advances in the Regulation of Milk Protein Gene Expression. Critical Reviews™ in Eukaryotic Gene Expression. 24(4): 357–378.
12. Rijnkels M, Elnitski L, Miller W and Rosen JM. 2003. Multispecies comparative analysis of a mammalian-specific genomic domain encoding secretory proteins. Genomics. 82(4): 417–432.
13. Sigl T, Meyer HHD and Wiedemann S. 2012. Gene expression of six major milk proteins in primary bovine mammary epithelial cells isolated from milk during the first twenty weeks of lactation. Czech Journal of Animal Science. 57(10): 469–480.
14. Sigl T, Meyer HHD and Wiedemann S. 2014. Gene expression analysis of protein synthesis pathways in bovine mammary epithelial cells purified from milk during lactation and short-term restricted feeding. Journal of Animal Physiology and Animal Nutrition. 98: 84-95.
15. Taylor-Papadimitriou J, Stampfer M, Bartek J, Lewis A, Boshell M, Lane EB and Leigh IM. 1989. Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. Journal of Cell Science. 94(3): 403-413.
16. Tyagi KK. 2016. Relative gene expression analysis of milk protein genes in primary milk epithelial cells of Surti and Mehsani buffaloes, Ph.D. thesis. Navsari Agricultural University, Navsari.
17. Verma S and Agastian P. 2013. Expression profile of important milk producing genes in cattle breeds from India. International Journal of Advanced Biotechnology and Research. 4(1): 976-980.