Historically, pre-pubertal development of the bovine mammary gland (MG) has received little attention compared to later development. Recent evidence suggests not only that this period represents a very active time in the development of the MG but also that the first 90 days of life can partially dictate future productivity of the lactating cow. The MG, often considered quiescent during early life (first 3 months), is now known to increase in size by over 60-fold in the same period. The importance of sex steroids in MG development is well classified, but a complex signaling network exists among estrogen, progesterone and other growth factors and hormones. Complicating our understanding of this developmental period further is the discovery that pre-weaning nutrition of the calf not only influences the growth of the mammary parenchyma but may also alter the way in which it responds to mammogenic stimuli. Recent data suggest that feeding calves a higher plane of nutrition improves the ability of the mammary epithelium to respond to estradiol and also alters the way in which the mammary parenchyma and fat pad communicate. It is clear that early life nutrition, although able to influence the MG, is still poorly understood mechanistically. For example, additional evidence suggests that increased feeding rates in early life alter the morphology of myoepithelial cells in the mammary epithelium. Further data have also suggested a role for other cell types, such as immune cells, in the penetration of the mammary parenchyma into the fat pad during the early life development of the MG suggesting that mammary development is not only controlled by the local tissue population (parenchyma and fat pad) but perhaps systemically by other tissue types (i.e., immune system). Understanding the roles of these various stimuli and signaling pathways as they relate to the development of the MG in early life may hold the key to unlocking the potential for the optimal development of this crucial organ and, in turn, may lead to improvements in other phases of mammary development and milk yield potential.

Previous studies in prepubertal heifers suggest that the magnitude of reduction in mammary parenchymal growth in response to ovariectomy varies with the age at which surgery is performed. We hypothesized that ovarian secretions are essential for initiating mammary development but not required to maintain allometric mammary growth in prepubertal dairy heifers. The objectives of this study were to determine the effect of staged ovariectomy during the prepubertal period on mammary growth and tissue composition and the expression of selected genes. Prepubertal Holstein heifers at 2, 3 or 4 months of age were randomly assigned to one of two treatments, ovariectomized (OVX; n = 12) or sham operated (INT; n = 12). Mammary parenchyma (PAR) and fat pad (MFP) were harvested 30 days after surgery. Proximate composition of PAR and MFP (DNA, protein and lipid) as well as expression of the selected estrogen-responsive genes stanniocalcin1 (STC1), tissue factor pathway inhibitor precursor (TFPI) and proliferating cell nuclear antigen (PCNA) were determined in PAR and MFP by quantitative real-time PCR. The relative amount of epithelium and proportion of epithelia cell nuclei expressing the proliferation marker Ki67 were determined by histological and immunohistochemical analyses, respectively. MFP mass was not impacted by treatment but was decreased with age as was lipid content and concentration (P ⩽ 0.01). The mass of mammary PAR was reduced in OVX and increased with age (P ⩽ 0.01). Parenchymal tissue tended to have less total DNA, protein and lipid in OVX heifers. Parenchymal tissue concentrations of protein and DNA were increased with age and there was an age × treatment interaction. Treatment had no effect on either the Ki67 labeling index or percent epithelial area. The relative abundances of STC1, TFPI and PCNA mRNA in PAR were reduced in OVX. We did not find a significant impact of ovariectomy on mRNA expression when surgery was performed at 2 months compared with surgery at 3 or 4 months of age. However, having nearly undetectable PAR in two heifers ovariectomized at the earliest period (2 months of age) suggests that early ovariectomy is especially detrimental to subsequent parenchymal development.

Environmental variables such as photoperiod, heat, stress, nutrition and other external factors have profound effects on quality and quantity of a dairy cow's milk. The way in which the environment interacts with genotype to impact milk production is unknown; however, evidence from our laboratory suggests that circadian clocks play a role. Daily and seasonal endocrine rhythms are coordinated in mammals by the master circadian clock in the hypothalamus. Peripheral clocks are distributed in every organ and coordinated by signals from the master clock. We and others have shown that there is a circadian clock in the mammary gland. Approximately 7% of the genes expressed during lactation had circadian patterns including core clock and metabolic genes. Amplitude changes occurred in the core mammary clock genes during the transition from pregnancy to lactation and were coordinated with changes in molecular clocks among multiple tissues. In vitro studies using a bovine mammary cell line showed that external stimulation synchronized mammary clocks, and expression of the core clock gene, BMAL1, was induced by lactogens. Female clock/clock mutant mice, which have disrupted circadian rhythms, have impaired mammary development and their offspring failed to thrive suggesting that the dam's milk production was not adequate enough to nourish their young. We envision that, in mammals, during the transition from pregnancy to lactation the master clock is modified by environmental and physiological cues that it receives, including photoperiod length. In turn, the master clock coordinates changes in endocrine milieu that signals peripheral tissues. In dairy cows, it is clear that changes in photoperiod during the dry period and/or during lactation influences milk production. We believe that the photoperiod effect on milk production is mediated, in part by the ‘setting’ of the master clock with light, which modifies peripheral circadian clocks including the mammary core clock and subsequently impacts milk yield and may impact milk composition.

The peripartal period is characterized by dramatic alterations in metabolism and function of key tissues such as liver, adipose and mammary. Metabolic regulation relies partly on transcriptional control of gene networks, a collection of DNA segments, which interact with a transcription factor or nuclear receptor, as a mechanism controlling the concentration of key enzymes in cells. These ‘global’ interactions can govern the rates at which genes in the network are transcribed into mRNA. The study of the entire genome, sub-networks or candidate genes at the mRNA level encompasses the broad field of genomics. Genomics of peripartal metabolic adaptations has traditionally been focused on candidate genes and more recently, using microarrays, on the broader transcriptome landscape. The candidate gene approach has expanded our knowledge on the functional adaptations of ureagenesis, fatty acid oxidation, gluconeogenesis, inflammation and growth hormone signaling in liver. More recent work with peripartal mammary tissue has used a gene network approach to study milk fat synthesis regulation as well as a candidate gene approach to study lipid transport, glucose uptake and inflammatory response. Network and pathway analysis of microarray data from cows fed different levels of dietary energy pre partum has revealed unique clusters encompassing functional categories including signal transduction, endoplasmic reticulum stress, peroxisome proliferator-activated receptors (PPARγ) signaling, PPARα signaling, immune or inflammatory processes and cell death in subcutaneous adipose tissue as well as liver. Of interest from a nutritional perspective is the potential to alter PPARγ signaling in adipose and PPARα signaling in liver as a means to enhance insulin sensitivity as well as fatty acid oxidation post partum. Major advances in understanding the metabolic adaptations of peripartal cows will come from using a systems biology approach to integrate data generated at the mRNA, protein, metabolite and tissue level across different nutritional management approaches and with cows of different genetic merit. This will allow the assembly of the important components needed to improve existing metabolic models of the peripartal cow and provide the tools to manipulate complex processes that could have significant long-term economic impact including lactation persistency, fertility and efficiency. An important goal of the future will be to apply additional experimental tools (e.g. gene silencing) and bioinformatics (e.g. transcription factor binding site identification) to studies focused on peripartal cows.

The MAC-T cell line has been used extensively to investigate bovine mammary epithelial cell function. A lactogenic phenotype is generally induced in this cell line by a combination of dexamethasone, insulin and prolactin and has typically been assessed by milk protein production. Few studies have focused on identifying other factors that may affect milk protein synthesis in the MAC-T cell line, and none have considered the lipid class distribution of MAC-T cells as a component of the lactogenic phenotype. Growth hormone (GH) has been shown to increase milk protein synthesis both in vivo and in mammary cell models, and has been shown to alter the lipogenic profile of mammary explant models. We tested the hypothesis that MAC-T cells would respond directly to GH and that the response would include alterations to the lipid class distribution as well as to milk protein gene expression, leading to a more appropriate model for mammary cell function than treatment with dexamethasone, insulin and prolactin alone. Differentiated cells expressed GH receptor mRNA, and addition of GH to the differentiation medium significantly induced production of α-s1 casein and α-lactalbumin mRNA. GH also significantly affected the proportion of triacylglycerol and sphingomyelin. These results indicate that GH is an important factor in inducing a lactogenic phenotype in the MAC-T cell line, and support the possibility of a direct effect of GH on milk synthesis in vivo.

Immune cells in the milk are most important in combating pathogens that invade the mammary gland. This study investigated the immune competence and viability of somatic milk cells that are already resident in milk and udders free of infection. Cells were studied in freshly removed milk to simulate conditions in the udder. Effects of incubation, cell preparation, and immunological stimulation with 0·5 μg/ml lipopolysaccharide (LPS) from Escherichia coli were analysed. Viability and differential counts of milk cells between high and low somatic cell count (SCC) quarters, and cisternal and alveolar milk with and without LPS stimulation were compared. Incubation and preparation of cells caused a cell loss which further increased with time independently of SCC and milk fraction. The viability of these cells was stable until 3 h post incubation and decreased until 6 h. Cell populations differed between both investigations, but did not change during the course of the experiment. mRNA expression of immune and apoptosis factors of the cells, measured by qPCR, did not change substantially: mRNA expression of caspase 3, Toll like receptor 4, and GM-CSF did not change, whereas the expression of the death receptor Fas/APO-1 (CD95), lactoferrin and lysozyme was decreased at 6 h. Cyclooxygenase-2 and TNF-α mRNA expression were decreased after 6 h of LPS treatment. In comparison with other studies in vivo or in vitro (in cell culture), in this study where cells are studied ex vivo (removed from the udder but kept in their natural environment, the milk) resident milk cells seem to be more vulnerable, less viable, less able to respond to stimulation, and thus less immune competent compared with cells that have freshly migrated from blood into milk after pathogen stimulation. The cell viability and differential cell count differed between high- and low-SCC milk and between cisternal and alveolar milk depending on the individual cow. In conclusion, the results support the view that for a most effective defence against invading pathogens the mammary gland is reliant on the recruitment of fresh immune cells from the blood.

In addition to its economic value, milk fat is responsible for many of milk’s characteristics and can be markedly affected by diet. Diet-induced milk fat depression (MFD) was first described over a century ago and remains a common problem observed under both intensive and extensive management. The biohydrogenation theory established that MFD is caused by an inhibition of mammary synthesis of milk fat by specific fatty acids (FA) produced as intermediates in ruminal biohydrogenation. During MFD, lipogenic capacity and transcription of key lipid synthesis genes in the mammary gland are down-regulated in a coordinated manner. Our investigations have established that expressions of sterol response element-binding protein 1 (SREBP1) and SREBP-activation proteins are down-regulated during MFD. Importantly, key lipogenic enzymes are transcriptionally regulated via SREBP1. Collectively, these results provide strong evidence for SREBP1 as a central signaling pathway in the regulation of mammary FA synthesis. Spot 14 is also down-regulated during MFD, consistent with a lipogenic role for this novel nuclear protein. In addition, SREBP1c and Spot 14 knock-out mice exhibit reduced milk fat similar to the magnitude and pattern of MFD in the cow. Application of molecular biology approaches has provided the latest chapter in the regulation of milk fat synthesis and is reviewed along with a brief background in nutritional regulation of milk fat synthesis in ruminants.