Date Published: February 5, 2019
Publisher: Public Library of Science
Author(s): Shakil Ahmad Bhat, Syed Mudasir Ahmad, Eveline M. Ibeagha-Awemu, Basharat A. Bhat, Mashooq Ahmad Dar, Peerzada Tajamul Mumtaz, Riaz A. Shah, Nazir A. Ganai, Juan J Loor.
Jersey and Kashmiri cattle are important dairy breeds that contribute significantly to the total milk production of the Indian northern state of Jammu and Kashmir. The Kashmiri cattle germplasm has been extensively diluted through crossbreeding with Jersey cattle with the goal of enhancing its milk production ability. However, crossbred animals are prone to diseases resulting to unsustainable milk production. This study aimed to provide a comprehensive transcriptome profile of mammary gland epithelial cells at different stages of lactation and to find key differences in genes and pathways regulating milk traits between Jersey and Kashmiri cattle. Mammary epithelial cells (MEC) isolated from milk obtained from six lactating cows (three Jersey and three Kashmiri cattle) on day 15 (D15), D90 and D250 in milk, representing early, mid and late lactation, respectively were used. RNA isolated from MEC was subjected to next-generation RNA sequencing and bioinformatics processing. Casein and whey protein genes were found to be highly expressed throughout the lactation stages in both breeds. Largest differences in differentially expressed genes (DEG) were between D15 vs D90 (1,805 genes) in Kashmiri cattle and, D15 vs D250 (3,392 genes) in Jersey cattle. A total of 1,103, 1,356 and 1,397 genes were differentially expressed between Kashmiri and Jersey cattle on D15, D90 and D250, respectively. Antioxidant genes like RPLPO and RPS28 were highly expressed in Kashmiri cattle. Differentially expressed genes in both Kashmiri and Jersey were enriched for multicellular organismal process, receptor activity, catalytic activity, signal transducer activity, macromolecular complex and developmental process gene ontology terms. Whereas, biological regulation, endopeptidase activity and response to stimulus were enriched in Kashmiri cattle and, reproduction and immune system process were enriched in Jersey cattle. Most of the pathways responsible for regulation of milk production like JAK-STAT, p38 MAPK pathway, PI3 kinase pathway were enriched by DEG in Jersey cattle only. Although Kashmiri has poor milk production efficiency, the present study suggests possible physicochemical and antioxidant properties of Kashmiri cattle milk that needs to be further explored.
Mammary gland development and the physiological control of its dynamics are a vital part of the mammalian reproduction strategy [1–2]. Milk evolved as an essential source of nutrients and immune factors including immune-modulatory, anti-inflammatory and anti-microbial agents that offer protection against infections [3–4]. Milk yield and quality are important economic traits. An increase in the efficiency of milk synthesis both in terms of quality and quantity is a highly desirable goal for the dairy industry . The mammary gland displays a high level of developmental plasticity with the ability to undergo repeated cycles of growth and regression . Lactation is a dynamic physiological process characterized by an initial rapid increase in milk yield during early lactation, which peaks around 6 weeks into lactation, followed by a gradual decrease until the end of lactation . The knowledge of gene expression involved in lactation informs on the biological mechanisms underlying mammary morphogenesis and metabolic activities as well as enhances our understanding of milk composition [8–9]. The ability to manipulate lactation performance in less improved breeds is an area of increasing interest, and knowledge of the biological pathways and mechanisms that govern mammary gland development and lactation may help to increase the lactation performance of dairy animals. Recent developments in “omics” technologies like transcriptomics make it possible to comprehensively and systematically identify the potential factors or processes that may influence lactation [10–11]. Using high throughput RNA sequencing technique, a high number of genes were identified as differentially expressed between different stages of lactation, and the expression alterations may play crucial roles in the regulation of lactation [9–12]. Thus, a thorough and deeper understanding of the genes and biological networks that regulate bovine milk composition is required.
In this study, we investigated the transcriptome profile of bovine MEC at different stages of lactation in Kashmiri and Jersey cattle by the method of high throughput RNA sequencing. Purified RNA from MEC, which represents a non-invasive source of material for assessing gene expression in mammary gland  was used. The quality of RNA from milk isolated MEC can be sensitive to degradation (due to several long processing steps) resulting in a wide range of RIN values (4 to 9) [24,31,32]. Using RNA from MEC with RIN of 6, Canovas et al.  reported discrepancies in gene expression when compared with other sources of mammary RNA (mammary gland tissue, milk somatic cells, laser micro dissected mammary epithelial cells and milk fat globules) . Consequently, Boutinaud et al.  advised that the quality of isolated MEC RNA should be assessed before use in gene expression analysis. In this study, the RIN values of isolated MEC RNA ranged from 7.4 to 9.1 suggesting that the RNA was of high quality with minimal degradation. Therefore, the low RIN 6 reported by Canovas et al.  could explain the relatively low levels of CSN2, CSN3, CSN1S1 and CSN2S2 when compared with our data, suggesting that the antibody-captured milk MEC technique used by these authors was probably not optimal. The validity of gene expression results obtained by using purified MEC has been demonstrated in cows and buffalo [31, 33–36] and supported by our data. The advantages of purified MEC as a non-invasive source of RNA for mammary gland transcriptome analysis include but not limited to possibility for repeat sampling over a period of time on the same animals without causing damage to mammary tissue and ability to specifically study milk secreting cells.
This study represents a cohesive comparison of the milk epithelial cell transcriptome profiles at different stages of lactation between Kashmiri and Jersey cattle. The results revealed higher gene expression profiles of candidate genes for milk synthesis and yield traits in Jersey compared to Kashmiri cattle. More genes were differentially expressed between lactation days in Jersey cattle as compared to Kashmiri cattle. Sixteen pathways were significantly enriched by DEG in Jersey cattle while no pathway was found to be significantly enriched in Kashmiri cattle. On the other hand, varied numbers of GO terms were enriched between lactation stages in both Kashmiri and Jersey cattle. The presence of enriched GO terms like endopeptidase and antioxidant activity in Kashmiri cattle suggests special effects on the physico-chemical characteristics of milk from Kashmiri cattle. Such properties may lead to the development of certain niche products and thereby help in the conservation of this unique germplasm which has been diluted through extensive cross breeding programmes. The results provide a significant advance in our knowledge of Kashmiri cow lactating mammary gland gene expression and valuable information for future studies and breed improvement.