The objective of this study was to evaluate the effect of milking frequency on milk production and composition, mammary cell proliferation, apoptosis, and gene expression. For this investigation, 10 Holstein cows that were being milked twice a day in mid lactation were selected. To study the effect of differential milking, 2 quarters were milked once daily and the other 2 were milked thrice daily for 8wk. After that period, twice-daily milking was resumed for all quarters, and data were collected for an additional 6wk. Mammary gland biopsies were taken 1wk before differential milking (wk -1) and after 4 and 8wk of differential milking. Milk samples were collected weekly throughout the experiment. Once-daily milking resulted in an immediate reduction in milk yield, whereas thrice-daily milking resulted in an increase in milk yield. During differential milking, the daily milk yield of the quarters milked once daily declined by 0.54kg/wk, on average, but remained constant in the quarters milked thrice daily. Part of the difference in milk yield between the glands pairs persisted after twice-daily milking was reinitiated. In the quarters milked once daily, milk BSA concentration increased, indicating an increase in tight junction leakiness, and zymographic analysis of milk enzymes showed increased activity of several proteases. Reducing the milking frequency also increased mammary cell apoptosis and, surprisingly, mammary cell proliferation. Interestingly, milk concentrations of stanniocalcin-1 and insulin-like growth factor-I and mammary gland expression of several genes were also modulated by milking frequency. For example, expression of insulin-like growth factor I receptor was downregulated during once-daily milking. Last, expression of the short and long isoforms of the prolactin receptor and of CSN2 (beta-casein) were upregulated during thrice-daily milking. Taken together, these data suggest that milking frequency not only affects mammary gland remodeling and the expression of paracrine factors but also modulates hormone sensitivity.

The objective of this study was to evaluate the effect of shortening the dry period on the mammary gland and the hormonal regulation of its functions. Holstein cows (n = 18) were assigned to a short dry period (SDP; 35 d; n = 9) or a conventional dry period (CDP; 65 d; n = 9). All cows were fed the same diets, with the exception that, during the dry period, the SDP cows received only the pre-calving diet for 35 d, whereas the CDP cows were fed a high-fiber diet from 65 to 28 d before calving and then received the same pre-calving diet as the SDP cows. Mammary gland functional capacity was evaluated at 70 days in milk, and mammary biopsies were taken in early and midlactation. Dry period length averaged 64.3 ± 1.1 and 31.9 ± 1.0 d for the CDP and SDP cows, respectively. The SDP cows had a lower milk yield and a lower energy-corrected milk yield compared with the CDP cows. The SDP cows also had a lower dry matter intake from wk 5 to 20 of lactation and tended to have lower plasma concentrations of β-hydroxybutyrate from wk 1 to 4. Prepartum serum progesterone and estradiol concentrations were unaffected by the dry period management. Serum growth hormone concentrations and milking-induced prolactin release were similar in both groups. However, during the period when the CDP cows were dry but the SDP cows were still being milked (wk -9 to -6), serum prolactin concentrations were higher in the SDP cows than in the CDP cows. The SDP cows had a lower milk BSA content than the CDP cows after the dry period and similar milk lactose concentrations, suggesting that their mammary tight junctions were closed following parturition and, therefore, that the later stage of their lactogenesis was not impaired by SDP management. In early and midlactation, mammary cell apoptosis and proliferation rates as well as mammary expression of genes involved in the function of this tissue were unaffected by the dry period management strategy. For cows in their second lactation, mammary gland functional capacity at 70 d in milk tended to be lower in the SDP cows. In conclusion, even though SDP management decreased milk production during the subsequent lactation, it did not affect mammary cell activity. Although direct evidence is still lacking, decreased mammary cell growth during the dry period is likely responsible for this negative effect. The higher prolactin concentrations in lactating cows during late gestation could be involved in this effect. More research is needed to test these hypotheses.

There is considerable evidence to indicate the existence of local control of mammary gland involution, but the exact nature of this control has yet to be defined. Stanniocalcin-1 (STC-1) is a newly discovered mammalian hormone that seems involved in the lactation process and may be implicated in the control of involution. As a first step in investigating this hypothesis, the change in STC-1 levels in milk and serum was measured during drying off. Nine Holstein cows in late lactation were milked twice daily on half the gland, while the other half was left unmilked for a 14-d period. Milk and blood samples and mammary biopsies were taken on d -7, 1, 2, 7, and 14 relative to the onset of the nonmilking period. The concentrations of STC-1 in serum and milk were determined by RIA. The albumin concentration and proteinase activity of the milk were determined. Apoptosis of the mammary epithelium was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Finally, the effects of milk on cellular activity and apoptosis were tested in vitro on mammary epithelial cells by measuring the turnover of tetrazolium salts and by counting the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells. The drying off of 2 quarters increased the milk production of the quarters that were milked by 30%. Milk proteinase activity and BSA and STC-1 concentrations increased in the nonmilked quarters, but remained unchanged in the milked quarters. Moreover, at d 2, the apoptotic rate of the mammary cells was higher in the nonmilked quarters than in the milked quarters (0.22 +/- 0.04 vs. 0.07 +/- 0.04%, respectively). Finally, in vitro experimentation demonstrated that mammary epithelial cells cultured in the presence of milk from involuting quarters had 3-fold more apoptotic cells as compared with those cultured in milk from the milked quarters at d 14. The metabolic rate was reduced by 14.6% for milk from d 7 and 23.6% for milk from d 14. Interestingly, the metabolic rate was negatively correlated with the STC-1 concentration in milk (r = -0.65). This study shows for the first time that STC-1 in milk is increased following milk stasis, although its exact role in the involution process remains to be clarified.

Although it is known that disruption of the cell junctions in the mammary gland induces a decrease in milk yield, the cellular mechanisms involved in milk secretion reduction during mammary cell junction disruption are not well understood. The aim of this study was to investigate the cellular regulations taking place after cell junction disruption in the mammary gland of goats. We performed intramammary infusions of Ca chelators to induce cell junction disruption. In a first group of 5 goats, intramammary infusions of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) in the right gland halves and saline as a control in the left gland halves were performed after 4 consecutive milkings. A second group of 4 goats received 4 intramammary infusions of citrate solution in the right gland halves and lactose solution as a control in the left halves. Intramammary infusion of EGTA and lactose induced a disruption of cell junctions, whereas citrate infusions failed to modify mammary epithelium integrity. The effect of the infused solutions was also tested in vitro via the measurement of transepithelial resistance, confirming mammary epithelium disruption by the EGTA, lactose, and citrate solutions at high concentrations. The disruption of mammary epithelium integrity by EGTA induced a decrease in the expression of the cell junction protein E-cadherin. Both the EGTA and lactose infusions induced a decrease in milk secretion that was accompanied by cellular modifications. We observed a decrease in milk casein, which was associated with a decrease in the mRNA level of kappa-casein in the lactose-infused glands, and a decrease in milk lactose, which was associated with a downregulation of alpha-lactalbumin transcripts in both the EGTA- and lactose-treated glands. Both the EGTA and lactose infusions increased terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) in the mammary tissue, indicating an induction of apoptosis. Lactose infusion increased the mRNA level of Bax, suggesting that apoptosis was regulated at the transcriptional level. The results obtained in these experiments suggest that disruption of mammary epithelium integrity was associated with both reduced synthetic activity and apoptosis induction in the mammary gland.