Jocelyne Guinard-Flament

Milking and Feed Restriction Regulate Transcripts of Mammary Epithelial Cells Purified from Milk

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.

Whole-body glucose metabolism and mammary energetic nutrient metabolism in lactating dairy cows receiving digestive infusions of casein and propionic acid

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.

Lactose in blood plasma and the ability of dairy cows to tolerate once-daily milking in terms of milk loss and milk recovery.

This experiment described the variability among cows with the aim of studying the ability of dairy cows to tolerate once-daily milking (ODM) in terms of milk losses and milk recoveries observed when cows are switched from twice- to once-daily milking and then back to twice-daily milking (TDM). It also aimed to investigate whether or not lactose in blood plasma, which indicates the mammary epithelium permeability, is correlated with milk losses and recoveries and, consequently, a potential candidate assessor of dairy cow tolerance to ODM. The study used 86 crossbred dairy cows (Holstein × Normande) split into 5 groups over 2 yr. The trial consisted of 3 successive periods: a 1-wk control period of TDM, then 3 wk of ODM, followed by 2 wk of TDM. Blood samples were collected 1 to 1.5h before the morning milking and 5 to 6h after milking on d -3, 0, 1, 3, 7, 21, 23, and 28, where d 0 is the last d of the control period. Milk losses measured as kilograms per day were higher in cows with highest control-period milk yields (r=-0.66). When expressed in relative terms (%), milk losses were weakly correlated with the control-period milk yield (r=-0.22). When switched back to TDM, cows recovered only 47% of the milk lost. Milk recovery (kg/d) was weakly correlated with control milk yield (r=0.34) and not correlated with ODM milk yield. Milk recovery was correlated with milk yield losses: the higher the milk yield losses (kg/d or %), the higher the milk recovery (kg/d; r=-0.59 and -0.52, respectively). Rate of milk recovery expressed as the quantity of milk recovered per kilogram of milk lost, was not correlated with milk loss expressed as a percentage. This means that to be considered well-adapted to ODM scheduling, a cow must be well adapted to milk loss, and be well adapted to milk recovery. Blood plasma lactose concentration (log(10)-transformed data) was not correlated with milk production levels in either control, ODM, or back-to-TDM periods. It was poorly correlated with milk loss but was positively correlated with milk recovery (kg/d) and rate of milk recovery. We conclude that blood plasma lactose needs to be coupled with other indicators for evaluating dairy cow tolerance to ODM.

Changes in mammary metabolism in response to the provision of an ideal amino acid profile at 2 levels of metabolizable protein supply in dairy cows: Consequences on efficiency

The aim of this study was to compare the modifications in mammary gland metabolism by supplying an ideal versus an imbalanced essential AA (EAA) profile at low and high metabolizable protein (or PDIE, its equivalent in the INRA feeding system). Four lactating, multiparous Holstein cows received 4 treatments composed of 2 basal diets containing 2 levels of PDIE (LP or HP) and 2 different infusions of AA mixtures (AA- or AA+) in the duodenum. The AA+ mixture contained Lys, Met, Leu, His, Ile, Val, Phe, Arg, Trp, and Glu, whereas the AA- mixture contained Glu, Pro, and Ser. The infusion mixtures were iso-PDIE. The diet plus infusions provided 13.9 versus 15.8% of crude protein that corresponded to 102 versus 118g/kg of dry matter of PDIE in LP and HP treatments, respectively. The treatments were designed as a 2×2 crossover design of 2 levels of PDIE supply (LP vs. HP) with 28-d periods. Infusions of AA in the duodenum (AA- vs. AA+) were superimposed to diet within each 28-d period according to 2×2 crossover designs with 14-d subperiods. Increasing the PDIE supply tended to increase milk protein yield; however, the efficiency of PDIE utilization decreased and the plasma urea concentration increased, indicating a higher catabolism of AA. The AA+ treatments increased milk protein yield and content similarly at both levels of protein supply. This was explained by an increase in the mammary uptake of all EAA except His and Trp. The mammary uptake of non-EAA (NEAA) was altered to the increase in EAA uptake so that the total AA uptake was almost equal to milk protein output on a nitrogen basis. The ratio between NEAA to total AA uptake decreased from 46% in LPAA- to 40% in LPAA+, HPAA-, and HPAA+ treatments. The PDIE efficiency tended to increase in the AA+ versus the AA- treatments because the NEAA supply and the amount of NEAA not used by the mammary both decreased. Nevertheless, our AA+ treatments seemed not to be the ideal profile: the mammary uptake-to-output ratio for Thr was higher than 1 in LPAA-, but it decreased to 1 in all the other treatments, suggesting that Thr was deficient in these treatments. Conversely, an excess of His was indicated because its uptake was similar in AA+ and AA- treatments. In conclusion, balancing the EAA profile increased milk protein yield and metabolizable protein efficiency at both levels of protein supply by increasing the mammary uptake of EAA and altering the NEAA uptake, leading to less AA available for catabolism.

Alteration of the nutrient uptake by the udder over an extended milking interval in dairy cows

Little is known about modifications of the mammary utilization of nutrients circulating in blood plasma when milk yield is strongly decreased by once-daily milking. A trial was carried out to describe the mammary nutritional adjustments linked to the downregulation of milk synthesis as milk accumulated over an extended milking interval in the bovine udder. Three Holstein dairy cows yielding 34.0 kg/d of milk were fitted with an ultrasound flow probe around the left external pudic artery and with catheters inserted into the left carotid and milk vein to estimate mammary blood flow (MBF) and mammary uptake of acetate, β-hydroxybutyrate, nonesterified fatty acids, glycerol, glucose, O(2), and CO(2) release. The trial was carried out over 2 consecutive weeks, with wk 2 repeating wk 1. Cows were milked twice daily at 12-h milking intervals. On d 3, cows were milked at 0630 h and were not milked for 36 h until d 4 at 1830 h. Over the following days, twice-daily milking was resumed using 12-h milking intervals. Each half-udder was milked separately. Secretion rates of milk and milk proteins decreased 67% during the 12-to-36-h interval of milk accumulation, whereas that of milk fat fell 30%. Timing of changes in MBF and lactose levels in blood plasma was concomitant and significant after 19.5 and 21.5h of milk accumulation in the udder, respectively. The MBF decreased, most likely because the usual increases in MBF no longer occurred when the udder was full of milk. After 24h of milk accumulation, MBF did not increase further when cows lay down, and did not increase as usual 3h after a meal, suggesting a possible physical effect of milk accumulated in the udder on MBF, complementing metabolic regulation. Mammary uptake or release of nutrients was lowered before 24h for glucose, acetate, and β-hydroxybutyrate and after 24h for total glycerol, O(2), and CO(2), mostly associated with the impaired MBF. However, these decreases ranged from 12 to 17%, and cannot entirely explain the -45 and -20% decreases in milk secretion rates observed during the entire 36 h of milk accumulation, thus confirming the primary role of intramammary metabolic regulation in the downregulation of milk secretion. The larger amount of nutrients taken up by the udder could explain the enhanced milk fat levels, involving a strongly modified metabolic fate of nutrients.

Effect of duration of milk accumulation in the udder on milk composition, especially on milk fat globule

Our objective was to study the effect of duration of milk accumulation on milk fat globules (MFG) secretion to better understand relationships between milk yield, milk fat, and MFG secretion. The modification of the milk accumulation duration in the udder is a tool to increase milk fat content. Four milking frequencies were studied on 6 dairy cows averaging 118±22 d in milk: 2 milkings/d separated by 11- and 13-h or by 4- and 20-h intervals and 1 milking/d. The experimental trial was a double Latin square 3×3 with 2-wk periods. Postexperiment, a milking frequency of 36-h was repeated twice. Compared with 2 milkings with 11- and 13-h frequencies, 1 milking/d reduced milk and milk fat yields and increased fat content, without any effect on the size of MFG. Two milkings with 4- and 20-h intervals had no significant effect on milk fat yield and content but tended to increase the size of the MFG. Lipolysis, measured on morning milk, was weaker with 1 milking/d. When data were analyzed according to milk accumulation duration (4, 11, 13, 20, 24, and 36h), the highest fat content and the largest diameters of MFG were obtained on milks from 4 and 36h milkings (62.8g/kg, 4.15 µm and 57.7g/kg, 4.09 µm, respectively). Such observations could have 2 origins: the richness in residual milk of the 4-h milk and the coalescence of MFG related to the long milk accumulation duration in the 36-h milk. For each duration of milk accumulation, a relationship exists between MFG size and fat yield. The positive relation between MFG size and fat content was confirmed at each duration of milk accumulation. Rate of secretion of milk fat (milk accumulation of 4h excluded) was also well correlated with MFG size. For the 36-h milk, this relationship was also observed but with a significantly different slope, assuming phenomena of MFG coalescence in response to the supposed increased intramammary pressure or to slower secretion rate and, hence, fusion events of microlipids droplets in the cytoplasm. Duration of milk accumulation joined with large increases in milk fat content induces changes in MFG size.