Maternal Age, Parity and Nursing Status at Fertilization Affects Postpartum Lactation Up to Weaning in Horses.

Nulliparity is associated with intra-uterine growth retardation and foal delayed catch-up growth. Older mares produce larger/taller foals than the precedents. Nursing at conception on foal growth had not been investigated yet. In any case, milk production conditions the foal's growth. This study aimed to determine effects of mare parity, age and nursing on subsequent lactation quantity and quality. Saddlebred mares and their foals (N = 43) run as a single herd over the same year were: young (6-7-year-old) primiparous, young multiparous, old (10-16-year-old) multiparous nursing at insemination time or old multiparous barren the previous year. No young nursing nor old multiparous mares were available. Colostrum was collected. Milk production and foal weight were monitored at 3-, 30-, 60-, 90- and 180-days postfoaling. The foal average daily weight gain (ADG) was calculated for each period between two measurements. Milk fatty acid (FA), sodium, potassium, total protein and lactose contents were determined. The primiparous versus multiparous colostrum was richer in immunoglobulin G, with lower production but greater FA contents in milk. The primiparous foals had a lower ADG for 3 to 30 days postpartum period. Old mares' colostrum contained more SFA and less polyunsaturated FA (PUFA) whereas their milk was richer in proteins and sodium and poorer in short-chain-SFA with a reduced PUFA/SFA ratio at 90 days. Nursing mares' colostrum was richer in MUFA and PUFA and late-lactation milk production was reduced. In conclusion, parity, age and nursing at conception affect mare's colostrum and milk production and foal growth and should be considered for broodmares' management.

Effect of feed restriction on dairy cow milk production: a review.

In the dairy cow, negative energy balance affects milk yield and composition as well as animal health. Studying the effects of negative energy balance on dairy cow milk production is thus essential. Feed restriction (FR) experiments attempting to reproduce negative energy balance by reducing the quantity or quality of the diet were conducted in order to better describe the animal physiology changes. The study of FR is also of interest since with climate change issues, cows may be increasingly faced with periods of drought leading to a shortage of forages. The aim of this article is to review the effects of FR during lactation in dairy cows to obtain a better understanding of metabolism changes and how it affects mammary gland activity and milk production and composition. A total of 41 papers studying FR in lactating cows were used to investigate physiological changes induced by these protocols. FR protocols affect the entire animal metabolism as indicated by changes in blood metabolites such as a decrease in glucose concentration and an increase in non-esterified fatty acid or ß-hydroxybutyrate concentrations; hormonal regulations such as a decrease in insulin and insulin-like growth factor I or an increase in growth hormone concentrations. These variations indicated a mobilization of body reserve in most studies. FR also affects mammary gland activity through changes in gene expression and could affect mammary cell turnover through cell apoptosis, cell proliferation, and exfoliation of mammary epithelial cells into milk. Because of modifications of the mammary gland and general metabolism, FR decreases milk production and can affect milk composition with decreased lactose and protein concentrations and increased fat concentration. These effects, however, can vary widely depending on the type of restriction, its duration and intensity, or the stage of lactation in which it takes place. Finally, to avoid yield loss and metabolic disorders, it is important to identify reliable biomarkers to monitor energy balance.

Oxytocin Induces Mammary Epithelium Disruption and Could Stimulate Epithelial Cell Exfoliation.

Mammary epithelial cells (MEC) are exfoliated from the epithelium into milk, influencing the number of MEC present in the udder. This process is associated with epithelium integrity. The release of oxytocin (OT) induced by milking causes myoepithelial cell contraction, which, in turn, may stimulate MEC exfoliation through mechanical forces. To investigate the role of OT in MEC exfoliation, we inhibited or induced myoepithelial cell contraction by injecting the OT receptor antagonist atosiban (Ato) or a supraphysiological dose of OT, respectively. Eight cows were assigned to 2 treatments during 2 milkings according to a crossover experimental design: Control+OT (cows were first milked to collect standard milk and then received 5 IU of OT to collect residual milk through a second milking) and Ato + OT (cows were injected with Ato (50 µg/kg of body weight) and milked to collect cisternal milk, then received 5 IU of OT to collect alveolar milk through a second milking). Milk MEC were purified to determine their concentration and number in milk. Mammary epithelium integrity was assessed by measuring the kinetics of plasma lactose concentration. Inhibiting myoepithelial cell contraction by Ato injection decreased the number of exfoliated MEC in milk. In contrast, OT injection increased the concentration of MEC in the residual milk and the number of MEC in the alveolar milk. Ato injection reduced plasma lactose concentration, whereas, in both treatments, OT injections increased it. Our results suggested that myoepithelial cell contraction caused by OT could stimulate MEC exfoliation into milk and was associated with epithelium disruption.

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments.

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.

Mammary epithelial cells isolated from milk are a valuable, non-invasive source of mammary transcripts.

Milk is produced in the udder by mammary epithelial cells (MEC). Milk contains MEC, which are gradually exfoliated from the epithelium during lactation. Isolation of MEC from milk using immunomagnetic separation may be a useful non-invasive method to investigate transcriptional regulations in ruminants' udder. This review aims to describe the process of isolating MEC from milk, to provide an overview on the studies that use this method to analyze gene expression by qRT PCR and to evaluate the validity of this method by analyzing and comparing the results between studies. In several goat and cow studies, consistent reductions in alpha-lactalbumin mRNA levels during once-daily milking (ODM) and in SLC2A1 mRNA level during feed restriction are observed. The effect of ODM on alpha-lactalbumin mRNA level was similarly observed in milk isolated MEC and mammary biopsy. Moreover, we and others showed decreasing alpha-lactalbumin and increasing BAX mRNA levels with advanced stages of lactation in dairy cows and buffalo. The relevance of using the milk-isolated MEC method to analyze mammary gene expression is proven, as the transcript variations were also consistent with milk yield and composition variations under the effect of different factors such as prolactin inhibition or photoperiod. However, the RNA from milk-isolated MEC is particularly sensitive to degradation. This could explain the differences obtained between milk-isolated MEC and mammary biopsy in two studies where gene expression was compared using qRT-PCR or RNA Sequencing analyses. As a conclusion, when the RNA quality is conserved, MEC isolated from milk are a valuable, non-invasive source of mammary mRNA to study various factors that impact milk yield and composition (ODM, feeding level, endocrine status, photoperiod modulation, and stage of lactation).

DNA methylation and transcription in a distal region upstream from the bovine AlphaS1 casein gene after once or twice daily milking.

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

Comparison of five different RNA sources to examine the lactating bovine mammary gland transcriptome using RNA-Sequencing.

The objective of this study was to examine five different sources of RNA, namely mammary gland tissue (MGT), milk somatic cells (SC), laser microdissected mammary epithelial cells (LCMEC), milk fat globules (MFG) and antibody-captured milk mammary epithelial cells (mMEC) to analyze the bovine mammary gland transcriptome using RNA-Sequencing. Our results provide a comparison between different sampling methods (invasive and non-invasive) to define the transcriptome of mammary gland tissue and milk cells. This information will be of value to investigators in choosing the most appropriate sampling method for different research applications to study specific physiological states during lactation. One of the simplest procedures to study the transcriptome associated with milk appears to be the isolation of total RNA directly from SC or MFG released into milk during lactation. Our results indicate that the SC and MFG transcriptome are representative of MGT and LCMEC and can be used as effective and alternative samples to study mammary gland expression without the need to perform a tissue biopsy.

Unilateral once daily milking locally induces differential gene expression in both mammary tissue and milk epithelial cells revealing mammary remodeling.

Once daily milking reduces milk yield, but the underlying mechanisms are not yet fully understood. Local regulation due to milk stasis in the tissue may contribute to this effect, but such mechanisms have not yet been fully described. To challenge this hypothesis, one udder half of six Holstein dairy cows was milked once a day (ODM), and the other twice a day (TDM). On the 8th day of unilateral ODM, mammary epithelial cells (MEC) were purified from the milk using immunomagnetic separation. Mammary biopsies were harvested from both udder halves. The differences in transcript profiles between biopsies from ODM and TDM udder halves were analyzed by a 22k bovine oligonucleotide array, revealing 490 transcripts that were differentially expressed. The principal category of upregulated transcripts concerned mechanisms involved in cell proliferation and death. We further confirmed remodeling of the mammary tissue by immunohistochemistry, which showed less cell proliferation and more apoptosis in ODM udder halves. Gene expression analyzed by RT-qPCR in MEC purified from milk and mammary biopsies showed a common downregulation of six transcripts (ABCG2, FABP3, NUCB2, RNASE1 and 5, and SLC34A2) but also some discrepancies. First, none of the upregulated transcripts in biopsies varied in milk-purified MEC. Second, only milk-purified MEC showed significant LALBA downregulation, which suggests therefore that they correspond to a mammary epithelial cell subpopulation. Our results, obtained after unilateral milking, suggest that cell remodeling during ODM is due to a local effect, which may be triggered by milk accumulation.

Changes in mammary uptake and metabolic fate of glucose with once-daily milking and feed restriction in dairy cows.

The aim of this review is to better understand the regulation of milk yield in response to once-daily milking and feed restriction. Glucose is the principal precursor for the synthesis of lactose (a major osmotic agent in milk), and participates in determining the milk volume produced. When applying these two breeding factors, reductions in milk yield are associated with a reduction in milk lactose yield and in the arterial flow of glucose, due to a decrease in the mammary blood flow. The ability of the udder to extract glucose is altered with once-daily milking but not necessarily with feed restriction. Lactose synthesis is down-regulated in response to once-daily milking and feed restriction but the percentage of the extracted glucose which is converted into lactose is differently affected in response to treatments. No marked change is observed with once daily milking whereas this would be increased with feed restriction and in contrast, depressed with fasting.

Regulation of genes encoding proteolytic enzymes during mammary gland development.

The mammary gland undergoes extensive tissue remodelling during each lactation cycle. During pregnancy, the epithelial compartment of the gland is vastly expanded (Benaud et al. 1998). At the end of lactation the epithelial cells undergo apoptosis and adipocyte differentiation is induced (Lilla et al. 2002). Ductal and alveolar growth during puberty and pregnancy, and the involution process require the action of proteolytic enzymes (including matrix metalloproteinases, plasminogen and membrane-peptidases) and the corresponding genes are activated during these periods (Benaud et al. 1998; Alexander et al. 2001). Matrix metalloproteinases (MMP) are expressed in several cell types of the mammary gland including stromal fibroblasts (e.g., MMP3, MMP2), epithelial cells (e.g., MMP7 or MMP9), adipocytes (e.g., MMP2) and lymphoid cells (e.g., MMP9) (Crawford et al. 1996; Lund et al. 1996; Wiseman et al. 2003). A number of knock-out mice, which are deficient for individual MMP genes (e.g., MMP2, MMP3) or plasminogen, display alterations to mammary gland structure and impairment of lactation (Lund et al. 1999; Wiseman et al. 2003).

Growth hormone increases Stat5 and Stat1 expression in lactating goat mammary gland: a specific effect compared to milking frequency.

In ruminants, both milking frequency and exogenous GH treatment affect milk production. In a previous report, we showed that the modulation of milk yield due to variations in milking frequency and GH treatment was associated with variations in mammary cell numbers. The aim of this study was to clarify the different mechanisms governing the effects of GH treatment and milking frequency on signal transducer and activator of transcription (Stat) expression and activation, and on the expression of genes involved in mammary cell differentiation. Six Saanen goats in late lactation were milked once daily from one half-udder and thrice daily from the other half-udder for 23 days. At the same time, the goats were divided into two groups: GH-treated versus control group. After slaughter of the goats, soluble mammary proteins and RNA were extracted from half-udder samples. Levels of Stat5, Stat3 and Stat1 proteins and the Stat activation by phosphorylation were analysed by Western blot. The amounts of Stat5 protein and mRNA were significantly elevated by GH treatment in all half-udders (milked once or thrice daily). Positive Stat5 immunoreactivity was principally localised in the nuclei of epithelial cells, with heterogeneous intensity between cells. No significant changes in Stat5 protein phosphorylation levels were observed. Furthermore, GH significantly increased Stat1 protein levels, without modifying the level of Stat1 tyrosine phosphorylation, and tended to reduce the abundance of Stat3 protein. In contrast, milking frequency failed to modify Stat gene expression, protein level and phosphorylation. Using Northern blot, we showed that levels of kappa casein and prolactin receptor mRNA were not affected by the treatments. These observations suggest that GH probably acts specifically on mammary cells by regulating the expression of Stat1, 3 and 5. In contrast, milking frequency does not act through this regulatory pathway.

The number and activity of mammary epithelial cells, determining factors for milk production.

The ability of ruminant mammary glands to produce milk is determined by the number of cells secreting milk and their level of activity. Changes in the number of cells in the udder occur during lactation. It has been shown that mammary cells proliferate during this process, while other cells die through apoptosis. The decline in milk production after peak lactation appears to be due to a gradual reduction in the number of milk-secreting cells, either through cell death or by the abrasion of epithelial cells during milk ejection. Other factors are also known to modify cell turnover in the udder, such as reproductive status, growth hormone treatment or milking frequency and nutrition. A description of the effects of husbandry practices makes it possible to envisage different processes for mammary tissue regeneration during lactation. Indeed, changes in milking frequency are capable of modifying the number of epithelial cells in an alveolus, while GH treatment acts on the total number of alveoli. Thus recent studies have demonstrated an heterogeneity of the processes of proliferation and cell death within the mammary gland. However, unanswered questions still remain concerning the presence of stem cells in ruminants, the lifespan of mammary epithelial cells or the relative rate of loss of mammary cells due to apoptosis and epithelial abrasion.

Potential uses of milk epithelial cells: a review.

Secretions collected from the mammary gland of different species contain heterogeneous populations of cells including lymphocytes, neutrophils, macrophages and epithelial cells in different species. Several factors influence the somatic cell count in milk and the distribution of cell types, such as species, infection status, physiological status and management practices. The epithelial cells are shed into milk during the lactation process. Most of them are viable and exhibit the characteristics of fully differentiated alveolar cells. Primary cultures of epithelial cells from colostrum and milk of humans, baboons, cows and goats together with established cell lines from human and goat milk, provide a good model for the study of lactogenesis, immunity transmission, cancer research and infection by viruses. The RNA extracted from milk cells have been shown to be representative of gene expression in the mammary gland and thus provide a source of material for molecular studies of gene expression and environmental interactions.