Feed restriction and once-daily milking (ODM) reduce milk yield in dairy cows and the amount of glucose taken up by the mammary gland. The modulation of mammary glucose uptake may be the consequence of modifications to glucose transport, capacity for lactose synthesis, and cell death in mammary epithelial cells (MEC). The aim was to demonstrate the usefulness of a new method to purify MEC from milk somatic cells and to examine the effects of feed restriction and ODM on mammary transcripts. Five Holstein cows were subjected to a 2 x 2 factorial arrangement of 2 milking frequencies and 2 feeding levels, during which the cows were milked once or twice daily while fed a diet providing either 98 or 70% of requirements. The cows were equipped to study net mammary balance of glucose. On d 7 of each experimental week, milk and lactose yields and mammary glucose uptake were measured. Cells were isolated from fresh milk by centrifugation to generate total milk cell samples. Mammary epithelial cells were separated from total milk cells by using magnetic beads associated with anticytokeratin 8 antibodies. Total RNA was extracted from both total milk cells and purified MEC samples. Real-time reverse transcription PCR was performed to determine mRNA levels in purified MEC under feed restriction and under ODM. Purified MEC samples revealed higher total RNA quality (RNA integrity number = 8) and were better suited to the measurement of mammary transcripts than total milk cell samples (RNA integrity number = 4). Significant correlations were obtained between mRNA levels and net glucose balance data (0.465 < r < 0.680), demonstrating the validity of results obtained by using purified MEC. Feed restriction induced a significant reduction (by half) in type 1 glucose transporter mRNA levels without any effect on alpha-lactalbumin (alpha-LA), galactosyltransferase, kappa-casein, bcl2, or bax mRNA levels. When compared with twice daily milking, ODM reduced kappa-casein (-86%) and alphaLA (-73%) mRNA levels and up-regulated bax and bcl2 mRNA levels (7- and 9-fold). The results suggest that the regulation of glucose uptake and milk yield is dependent on the transcription of glucose transporters under feed restriction and on the transcription of alphaLA under ODM. Further studies are required to con-firm the suggested onset of cell death after 7 d of ODM.

-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. ruminant / milk production / mammary gland / mammary cells / proliferation / apoptosis

Most dairy cows throughout the world are milked twice daily. In intensive dairying systems, however, it is not uncommon to increase milking frequency to between 3 and 6 times daily to increase milk production. Reducing milking frequency is much less common; however, once-daily milking of dairy cows, practiced either strategically during certain parts of the lactation or for the entire lactation, is not uncommon in key dairying countries where less emphasis is placed on milk production per cow. The practice fits well with more extensive dairy production systems, particularly those based on grazed pasture. A feature of once-daily milking is that it reduces milk yield by approximately 22%, depending on stage of lactation, breed, and parity, and it may adversely affect lactation length and persistency. However, it can offer several positive farm management options, especially related to labor requirements and farm working expenses. In addition, it may provide a tool to better manage the metabolism and energy balance of cows during early lactation or during periods of pasture deficit, and it may help to improve reproductive performance and animal health and welfare. Once-daily milking, representing one extreme of the mammary function spectrum, has attracted considerable research interest over the years. Consequently, substantial scientific information is available on its effects on mammary function, at both the physiological and molecular levels. This review focuses instead on the management of the cow milked once daily, covering the production response in relation to breed, stage of lactation, and parity, and its effect on energy status, reproduction, health and welfare, as well as on milk composition and processability.

This study analyzed the effect of propionate (C3) and casein (CN) on whole-body and mammary metabolism of energetic nutrients. Three multiparous Holstein cows fitted with both duodenal and ruminal cannulas were used in 2 replicated Youden squares with 14-d periods. Effects of CN (743 g/d in the duodenum) and C3 (1,042 g/d in the rumen) infusions, either separately or in combination as supplements to a grass silage diet, were tested in a factorial arrangement. The control diet provided 97% of energy and protein requirements. Within each period, blood samples were taken (d 11) from the carotid artery and the right mammary vein to determine net uptake of energetic nutrients. Plasma blood flow was calculated using the Fick principle (based on Phe and Tyr). On d 13, [6,6-(2)H(2)]glucose was infused in the jugular vein to determine whole-body glucose rate of appearance (Ra) based on enrichments in arterial plasma. Both C3 and CN treatments increased whole-body Ra (17% and 13%, respectively) but only CN increased milk (18%) and lactose (14%) yields, suggesting no direct link between whole-body Ra and milk yield. When CN was infused alone, the apparent ratio of conversion of CN carbon into glucose carbon was 0.31 but, when allowance was made for the CN required to support the extra milk protein output, the ratio increased to 0.40, closer to the theoretical ratio (0.48). This may relate to the observed increases in arterial glucagon concentrations for CN alone. Conversely, the apparent conversion of infused C3 carbon alone to glucose was low (0.31). With C3, mammary plasma flow increased as did uptakes of lactate, Ala, and Glu whereas the uptake for beta-hydroxybutyrate (BHBA) decreased. Mammary net carbon balance suggested an increase with C3 treatment in glucose, lactate, Ala, and Glu oxidation within the mammary gland. Mammary glucose uptake did not increase with CN treatment, despite an increase in glucose arteriovenous difference and extraction rate, because plasma flow decreased (-17%). Whereas CN, alone or in combination with C3, increased both lactose and protein yields, only mammary AA (and BHBA in CN alone) uptake increased because plasma flow decreased (-17%). These data suggest that the observed variations of milk lactose yield (and other milk components) are linked to metabolic interchanges between several energetic nutrients at both the whole-body and mammary levels and are not explained by increases in whole-body glucose availability.