Wickramasinghe et al. BMC Genomics 2012, 13:45 http://www.biomedcentral.com/1471-2164/13/45 detected in genes up regulated in late lactation milk. MetaCore pathway analysis of these two groups showed cytoskeleton remodeling pathway to have the most sig- nificant p value (p = 9.58 × 10-7) for enrichment for up regulated genes in transition milk, and leukocyte chemo- taxis pathway (p = 1.6 × 10-12) for up regulated genes in late lactation MSC. Late lactation showed significant enrichment of several pathways related to immune response and cell cycle. The complete list of significant pathways and their p values are provided in the Addi- tional File 4, Table S3. Peak and late lactation MSC showed more similarity in gene expression with only 238 genes with statistically significant expression differences (p ≤ 0.05, FDR q ≤ 0.3). Among these genes, 132 (55%) had higher expres- sion in peak lactation and 106 (45%) had higher expres- sion in late lactation. The BLAST2GO analysis of the up regulated genes showed similar enrichment of most of the GO terms. However, genes up regulated in late lac- tation had higher number of GO terms for cell prolifera- tion, death, immune system process and growth (Additional File 5, Figure S2). The cell killing biological process GO term was detected only in genes with high expression in late lactation. Molecular function categori- zation showed significant high expression of genes with antioxidant activity in peak lactation. This shows that compared to peak lactation, somatic cells in late lacta- tion milk were more involved in immune activity and involution process. MetaCore pathway analysis showed statistically signifi- cant enrichment of G protein coupled receptors (GPRCs) involved in the regulation of smooth muscle contraction pathway in peak lactation MSC (p = 0.0003). Effect of the oxytocin hormone is mediated by the G-protein coupled receptors present in mammary epithelial cells [32] and milk secretion from lactating mammary gland is stimulated by the binding of oxytocin to these specific receptors [33]. High production and secretion of milk from the mammary gland in peak lac- tation can be the reason for an enrichment of GPRCs in the regulation of smooth muscle contraction pathway in peak lactation MSC. Role of the anaphase promoting complex (APC) in cell cycle regulation pathway had the most significant p value (p = 2.9 × 10-5) for enrichment in late lactation. Anaphase promoting complex is responsible for inducing the progression and exit of cell cycle from mitosis by APC induced proteolysis of differ- ent cell cycle regulators [34]. Enrichment of APC path- way in cell cycle regulation is a reflection of the biological changes occurring in the involuting mammary gland during late lactation. The complete list of signifi- cant pathways and their p values are provided in the Additional File 6, Table S4. Page 6 of 14 In summary, the blast2GO and the pathway enrich- ment analysis showed significant differences in the func- tions performed by genes with different expression in each stage of lactation. Genes involved in immune func- tion, cell cycles and apoptosis showed significant higher expression towards the late lactation, where the mam- mary gland is in the initial stage of involution. This indi- cates that the MSC have the ability to adapt different molecular functions according to the biological need of the animal. However, we did not find enrichment of lac- tation specific pathways in any of the stages, most likely, these pathways are not well annotated in the gene ontol- ogy databases and pathways used for enrichment analysis. Expression of genes encoding for caseins, whey proteins and enzymes in lactose synthesis pathway Caseins (CN) and whey proteins are the two major milk proteins in milk. Caseins are encoded by CSN1S1, CSN1S2, CSN2 and CSN3, a cluster of genes located on bovine chromosome 6. These genes encode the milk proteins as1-CN, as2-CN, b-CN and -CN, respectively [35]. All the CN genes showed very high expression in transition lactation MSC and a significant decrease in expression along the course of lactation. The CSN2 gene had the highest expression among the CN gene family throughout lactation, which agrees with the composition of caseins in which b-CN constitutes up to 45% of the caseins [36]. The main whey proteins, b- lactoglobulin and a-lactalbumin, are encoded by the LGB and LALBA genes, respectively. These two genes also showed very high expression in transition lactation and, similar to CN genes, a significant decrease in expression of whey protein genes during the course of lactation (Figure 2). It is interesting that the percent of total milk proteins, whey proteins and casein along the course of lactation does not show a significant fluctuation along the lacta- tion period [37]. However, the mRNA gene expression profiles in our study indicate higher transcription rate for casein and whey proteins in transition lactation. One possibility for this discrepancy is that these abundant caseins and whey proteins are broken in to bio-active peptides and therefore their concentration is not reflected in the analysis of major milk component pro- teins. It has been identified that the bio-active peptides formed by cleavage of caseins and whey proteins are higher toward the beginning of lactation [38,39]. One other possible explanation is that even though there is high expression of the genes encoding caseins and whey, the protein synthesis may not be efficient in transition lactation animals that are in negative energy balance or essential amino acids may be limiting.