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 aim of this study was to gain a clearer understanding of the different levels of regulation involved in the reduction in milk yield in response to once-daily milking and feed restriction. The treatments were designed as a 2 x 2 factorial arrangement of 2 milking frequencies (once- or twice-daily milking) and 2 feeding levels (70 or 98% of requirements determined 1 wk before the trial). The cows were surgically prepared to study the net mammary balance of the nutrients that are precursors of milk components. Mammary efficiency in synthesizing milk components was estimated using a milk output:mammary uptake ratio. No interaction was observed between the effects of milking frequency and feeding level on milk and blood parameters except for milk protein yield, milk fatty acid profile, and nonesterified fatty acids metabolism. Once-daily milking and feed restriction reduced milk yield by 5.1 and 2.9 kg/d and fat-corrected milk yield by 4.2 and 4.1 kg/d, respectively. Both treatments induced a decrease in mammary blood flow. Once-daily milking led to a reduction in the extraction rate of glucose but no changes to the lactose output:glucose uptake ratio. Feed restriction did not change the glucose extraction rate but tended to improve the lactose output:glucose uptake ratio. Under once-daily milking, the slight increase in milk fat content (0.34 percentage units) was linked to a depressed uptake of glucose and acetate but without any variations in the uptake of beta-hydroxybutyrate and total glycerol and in the efficiency of acetate and beta-hydroxybutyrate conversion to short- and medium-chain fatty acids in milk. The decline in milk fat and protein contents (-0.43 and -0.23 percentage units, respectively) under feed restriction was associated with relatively similar reductions in the mammary uptake of all nutrients and with enhanced conversion of the glucose taken up by the mammary gland and used for lactose synthesis. As a result, once-daily milking and feed restriction seem to affect milk yield through mechanisms that may be different and relatively independent.

Whole-body glucose rate of appearance (Ra) responses and milk lactose secretion were compared in dairy cows receiving duodenal infusions of glucose (Glc), a mixture of 5 nonessential amino acids (NEAAm), or ruminal infusions of propionic acid (C3). Four mid-lactation Holstein cows, fitted with both duodenum and rumen cannulas, were used in a 4 x 4 Latin square design with 14-d periods. Cows were fed a grass silage-based diet (Ctrl) that provided 88% of net energy of lactation and 122% of protein requirements. Concentrate was formulated with wheat (21.5%) and barley (20%) containing some starch. Isoenergetic infusions (5.15 Mcal/d of digestible energy) of Glc into the duodenum (7.7 mol/d), C3 into the rumen (14.1 mol/d), or NEAAm into the duodenum (in mol/d; Ala: 1.60; Asp: 0.60; Glu: 5.94; Gly: 1.22; Ser: 2.45) were given as a supplement to the Ctrl diet. During each period on d 13, [6,6-(2)H(2)]glucose was infused into one jugular vein and blood samples were taken from the other jugular vein to measure glucose enrichment and determine Ra. Dry matter intake decreased slightly with the infusions (6%), but did not differ among them. Whole body glucose Ra averaged 502, 745, 600, and 576 mmol/h for Ctrl, Glc, C3, and NEAAm, respectively. It increased with the increase in energy supply (Ctrl vs. infusions) and differed according to the nutrients infused. The Ra response was higher with Glc and C3 than with NEAAm and higher with Glc than with C3. Plasma concentrations of insulin were not affected, but insulin-like growth factor 1 increased with infusions. Plasma glucagon increased with NEAAm, which could favor the increased Ra. Overall, milk lactose yield (137, 141, 142, and 130 mmol/h for Ctrl, Glc, C3, and NEAAm, respectively) was not modified by the infusions, but was lower with NEAAm compared with Glc and C3. Changes in lactose yield did not parallel the increase in Ra, and therefore the ratio of lactose yield to Ra decreased with the infusions and was lower in Glc compared with C3, suggesting a shift of glucose utilization away from lactose synthesis toward other pathways, including mammary metabolism. Intestinal Glc was the most efficient nutrient in terms of increasing glucose Ra; however, there was no direct link between the increases in whole body glucose Ra observed with the 3 types of nutrients and milk lactose yield.

Increasing the milking interval decreases milk yield and modifies milk composition. To gain a clearer understanding of the regulation of milk yield and composition, a study was conducted to establish the response curves of nutrient extraction by the mammary gland and mammary epithelial permeability in response to increasing milking intervals. Four multiparous lactating dairy cows were milked at 8-, 12-, 16-, or 24-h intervals over a period of 7 d using a Latin square design. Between the 8- and 24-h milking intervals, milk yield and milk protein levels fell curvilinearly from 38.2 to 29.2 kg/d and from 1,086 to 827 g/d, respectively. Milk fat yield decreased linearly from 1,475 to 1,235 g/d. Indicators of the opening of tight junctions increased linearly with increasing milking intervals: milk BSA increased from 148 to 207 mg/L and plasma lactose increased from 22.9 to 32.0 mg/L. The mammary gland's ability to extract nutrients decreased with increasing milking intervals. Extraction rates of glucose, beta-hydroxybutyrate, and total glycerol decreased significantly (from 27.2 to 23.3%, from 42.3 to 34.4%, from 36.6 to 30.8% between 8- and 24-h milking intervals, respectively), and not significantly for alpha-amino nitrogen (from 23.2 to 20.0%). The extraction rate of acetate remained constant. Moreover, the extraction of milk fat precursors appeared to be less regulated than those of the precursors of milk protein and lactose, which could partly explain why milk yield and milk protein yield decreased more than milk fat yield. The arteriovenous differences of beta-hydroxybutyrate and total glycerol remained constant, whereas those of glucose decreased significantly from 0.98 to 0.87 +/- 0.05 mmol/L and not significantly from 0.74 to 0.64 +/- 0.12 mmol/L for alpha-amino nitrogen. As a result, the mammary gland's ability to extract nutrients appears to be downregulated explaining partly the decrease in daily milk yield observed in response to increased milking intervals.