Synthesis of milk components involves different mammary metabolism adaptations in response to net energy and protein supplies in dairy cows.

Net energy for lactation (NEL) and metabolizable protein (MP) are the 2 main nutritional forces that drive synthesis of milk components. This study investigated mammary-gland metabolism in dairy cows in response to variations in the supply of NEL and MP. Four Holstein dairy cows were randomly assigned to a 4 × 4 Latin square design, in which each experimental period consisted of 14 d of dietary treatment. The diets provided 2 levels of NEL (low energy, 25.0 Mcal/d vs. high energy, 32.5 Mcal/d) and 2 levels of MP (low protein, 1,266 g/d vs. high protein, 2,254 g/d of protein digestible in the intestine) in a 2 × 2 factorial arrangement. Performance and dry matter intake (DMI) were measured during the last 5 d of each period, and the mammary net balance was measured on d 13 by collecting 6 sets of blood samples from the left carotid artery and left mammary vein. Mammary plasma flow was measured according to the Fick principle for Phe and Tyr. The mammary net balance of carbon equaled the uptake of nutrients expressed as carbon minus the output of lactose, fatty acids (FA) synthesized in the mammary gland, AA of milk protein, and glycerol-3P from triglyceride on d 13. Milk, lactose, fat, and protein yields increased when NEL and MP supplies increased. However, increasing the NEL supply increased FA synthesis more than increasing the protein supply did. In addition, FA secretion increased more than lactose secretion when the NEL supply increased. Increasing the NEL supply increased the left half-udder uptake of all major energy-yielding nutrients by increasing mammary plasma flow. However, nutrient uptake increased more than milk output did, which in turn increased carbon dioxide output. This increase in nutrient oxidation by the mammary gland decreased the mammary efficiency of nutrients utilization when the NEL supply increased. Increasing MP supply tended to increase glucose uptake through mammary clearance and increased mammary AA uptake with no change in mammary plasma flow. In addition, the protein supply did not change the mammary uptake of acetate or ß-hydroxybutyrate. The increase in milk-component secretions in response to either NEL or MP supplies occurred through different metabolic adaptations (increase in mammary plasma flow vs. clearances, respectively). These results suggest that the nutrient use by the mammary gland is highly flexible, which helps in maintaining milk and milk-component yields even with limiting nutrient supplies.

Effects of parity and week after calving on the metabolic, redox and immune status of dairy cows.

At the onset of lactation in dairy cows, inflammation and oxidative stress may occur and result in a risk of pathologies and lower milk yield. To propose an innovative management strategy for cows during this period, it is essential to better understand these physiological variations. Our objective was to evaluate the metabolic, redox and immune status of 7 primiparous and 8 multiparous Holstein cows during late gestation and the first months of lactation. Blood samples were collected between 3 weeks before calving until 12 weeks postpartum. Milk samples were also collected, but only at the time points after calving. The metabolic (nonesterified fatty acids (NEFA), BHB, glucose, urea, calcium) and redox (reactive oxygen metabolites (ROM), oxidative stress index (OSI), glutathione peroxidase activity, vitamin E) statuses were analyzed in plasma or erythrocytes. The expression of genes related to antioxidant functions was determined in leukocytes collected from milk. For immune status, plasma cytokine levels and the production of reactive oxygen species (ROS) in classical and regulatory neutrophils were measured in 2 whole blood ex vivo challenges. The data were analyzed using a mixed model that included the fixed effects of parity and week and their interaction. Milk yield, plasma NEFA and BHB in wk 2 and 4 after calving were higher in multiparous cows than in primiparous cows, whereas glucose and calcium tended to be lower. Plasma ROM and OSI levels in wk 8 were higher in multiparous than in primiparous cows. Multiparous cows also displayed higher glutathione peroxidase activity in erythrocytes, and antioxidant transcription factor and superoxide dismutase-1 expression levels in milk leukocytes. Moreover, multiparous cows had higher plasma concentrations of vitamin E but lower plasma levels of cytokines CXCL10, CCL2, IL1Rα and IFNγ. Following ex vivo whole blood stimulation with Escherichia coli, lower IL1α and TNFα levels were measured in multiparous than in primiparous cows. Intracellular ROS production by neutrophils was lower in multiparous than in primiparous cows. These results thus indicated marked physiological changes in wk 8 compared with wk 2 and 4 of lactation. These differences in the physiological status of primiparous and multiparous cows offer interesting perspectives for potential dietary strategies to prevent pathologies which take account of parity and week relative to calving.

MiRNome variations in milk fractions during feed restrictions of different intensities in dairy cows.

BACKGROUND: In dairy cows, diet is one factor that can affect their milk production and composition. However, the effect of feed restriction on milk miRNome has not yet been described. Indeed, milk is the body fluid with the highest RNA concentration, which includes numerous microRNA. Its presence in the four different milk fractions, whole milk, fat globules, mammary epithelial cells and extracellular vesicles, is still poorly documented. This study aimed to describe the effects of different feed restrictions on the miRNome composition of different milk fractions. RESULTS: Two feed restrictions were applied to lactating dairy cows, one of high intensity and one of moderate intensity. 2,896 mature microRNA were identified in the different milk fractions studied, including 1,493 that were already known in the bovine species. Among the 1,096 microRNA that were sufficiently abundant to be informative, the abundance of 1,027 of them varied between fractions: 36 of those were exclusive to one milk fraction. Feed restriction affected the abundance of 155 microRNA, with whole milk and milk extracellular vesicles being the most affected, whereas milk fat globules and exfoliated mammary epithelial cells were little or not affected at all. The high intensity feed restriction led to more microRNA variations in milk than moderate restriction. The target prediction of known microRNA that varied under feed restriction suggested the modification of some key pathways for lactation related to milk fat and protein metabolisms, cell cycle, and stress responses. CONCLUSIONS: This study highlighted that the miRNome of each milk fraction is specific, with mostly the same microRNA composition but with variations in abundance between fractions. These specific miRNomes were affected differently by feed restrictions, the intensity of which appeared to be a major factor modulating milk miRNomes. These findings offer opportunities for future research on the use of milk miRNA as biomarkers of energy status in dairy cows, which is affected by feed restrictions.

Milk yield loss in response to feed restriction is associated with mammary epithelial cell exfoliation in dairy cows.

In dairy cows, feed restriction is known to decrease milk yield by reducing the number of mammary epithelial cells (MEC) in the udder through a shift in the MEC proliferation-apoptosis balance, by reducing the metabolic activity of MEC, or both. The exfoliation of MEC from the mammary epithelium into milk is another process that may participate in regulating the number of MEC during feed restriction. The aim of the present study was to clarify the mechanisms that underlie the milk yield loss induced by feed restriction. Nineteen Holstein dairy cows producing 40.0 ± 0.7 kg/d at 77 ± 5 d in milk were divided into a control group (n = 9) and a feed-restricted group (n = 10). Ad libitum dry matter intake (DMI) was recorded during a pre-experimental period of 2 wk. For 29 d (period 1), cows were fed either 100 (control) or 80% (feed-restricted) of their ad libitum DMI measured during the pre-experimental period. Then, all cows were fed ad libitum for 35 d (period 2). Milk production and DMI were recorded daily. Blood and milk samples were collected once during the pre-experimental period; on d 5, 9, and 27 of period 1; and on d 5, 9, and 30 of period 2. Mammary epithelial cells were purified from milk using an immunomagnetic method to determine the rate of MEC exfoliation. Mammary tissue samples were collected by biopsy at the end of each period to analyze the rates of cell proliferation and apoptosis and the expression of genes involved in synthesizing constituents of milk. Feed restriction decreased milk yield by 3 kg/d but had no effect on rates of proliferation and apoptosis in the mammary tissue or on the expression of genes involved in milk synthesis. The daily MEC exfoliation rate was 65% greater in feed-restricted cows than in control cows. These effects in feed-restricted cows were associated with reduced insulin-like growth factor-1 and cortisol plasma concentrations. When all cows returned to ad libitum feeding, no significant difference on milk yield or MEC exfoliation rate was observed between feed-restricted and control cows, but refeeding increased prolactin release during milking. These results show that the exfoliation process may play a role in regulating the number of MEC in the udders of dairy cows during feed restriction without any carryover effect on their milk production.

Mammary epithelium disruption and mammary epithelial cell exfoliation during milking in dairy cows.

The presence of mammary epithelial cells (MEC) in the milk of ruminants indicates that some MEC are shed from the mammary epithelium; however, the mechanisms that regulate the MEC exfoliation process are not known. Through the release of oxytocin, prolactin, and cortisol and through oxytocin-induced mechanical forces on the mammary epithelium, milking could participate in regulating the MEC exfoliation process. The aims of the present study were to determine the rate of MEC exfoliation throughout milking and to investigate its relationship to mammary epithelium integrity and milking-induced hormone release. Milk samples from 9 Holstein dairy cows producing 40.6 ± 1.36 kg of milk/d were collected at the beginning (after 1 and 2 min), in the middle, and at the end of milking. Milk MEC were purified using an immunomagnetic method. Blood samples were collected before, during, and after milking, and the oxytocin, prolactin, and cortisol concentrations in the samples were measured. Tight junction opening was assessed by plasma lactose concentration and the Na+:K+ ratio in milk. The somatic cell count in milk varied during the course of milking; it decreased at the beginning of milking and then increased, reaching the highest values at the end of milking. Exfoliated MEC were present in all milk samples collected. The presence of MEC in the milk sample collected during min 1 of milking, likely corresponding to the cisternal milk fraction, suggests that MEC were exfoliated between milkings. The observed increase in the Na+:K+ ratio in milk and in the plasma concentration of lactose indicated that disruption of mammary epithelium integrity occurred during milking. The MEC exfoliation rate at milking was not correlated with the variables describing milking-induced prolactin release but was negatively correlated with cortisol release, suggesting that cortisol may play a role in limiting exfoliation. In conclusion, milking induced a disruption of the mammary epithelial barrier. Mammary epithelial cells may be continuously exfoliated between milkings or exfoliated during milking as a consequence of the oxytocin-induced mechanical forces and the disruption of mammary epithelium integrity.

Inhibiting prolactin by cabergoline accelerates mammary gland remodeling during the early dry period in dairy cows.

The inhibition of prolactin release using cabergoline, a dopamine agonist, is an effective strategy to accelerate the changes in mammary secretion composition after drying-off. The objective of this study was to determine how cabergoline may affect mammary tissue remodeling during early involution. Holstein dairy cows were treated with either a single i.m. administration of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n = 7) or placebo (n = 7) at the time of drying-off. Mammary biopsy samples were collected 1 wk before drying-off (d -6), after 30 h of milk accumulation (d 1), and again 8 d following drying-off (d 8) to determine changes in gene expression, lactoferrin content, and cell turnover. Blood and mammary secretion samples were collected at d -6 and again at d 1, 2, 3, 4, 8, and 14 following the abrupt cessation of lactation to evaluate indicators of blood-milk barrier integrity and other markers of mammary tissue remodeling. Cabergoline induced less SLC2A1, BAX, CAPN2, and IGFBP5 mRNA expression. In contrast, cabergoline did not modify changes in cell proliferation and apoptosis. Following the cessation of lactation, changes in mammary secretion composition (Na+ and K+) and blood lactose concentrations were indicative of a loss in the blood-milk barrier function in both treatment groups. Cabergoline treatment affected only Na+ and K+ concentrations at d 1, suggesting a moderate increase in tight junction permeability. The increase in the activity of MMP9 and in mammary epithelial cell concentration in mammary secretions was greater in cabergoline-treated cows than in control cows, suggesting more mammary tissue remodeling. The increase in lactoferrin immunostaining in the mammary tissue occurred earlier for cabergoline-treated cows than for control cows, and was essentially localized in the stroma. Changes in some key markers of mammary involution suggest that cabergoline accelerates mammary gland remodeling. Thus, a single injection of cabergoline after the last milking would facilitate drying-off by enhancing mammary gland involution.

New insights into the importance of prolactin in dairy ruminants.

In most mammals, prolactin (PRL) is essential for maintaining lactation, and the suppression of PRL inhibits lactation. However, the involvement of PRL in the control of ruminant lactation is less clear, because inconsistent effects on milk yield have been observed with the short-term suppression of PRL by bromocriptine. Therefore, several experiments have been conducted to assess the galactopoietic role of PRL. In an initial experiment, cows in early lactation received daily injections of the dopamine agonist quinagolide for 9 wk. Quinagolide reduced milking-induced PRL release and caused a faster decline in milk production. Quinagolide also reduced mammary epithelial cell activity, survival, and proliferation. In goats, cabergoline, another dopamine agonist, caused a 28% decrease in milk yield the day after injection. In another experiment, cows were injected for 5d with quinagolide, with quinagolide plus bovine PRL injected at milking time, or with vehicles only. Again, quinagolide reduced milk, protein, and lactose yields. Although PRL injections were not sufficient to restore milk yield, they tended to increase milk protein and lactose yields and increased the viability of mammary epithelial cells purified from milk. Recently, our team stimulated PRL secretion with daily injections of the dopamine antagonist domperidone for 5 wk. Milk production increased gradually and was greater in domperidone-treated cows during the last 4 wk of the treatment period. In most experiments where PRL secretion was manipulated, feed intake paralleled the changes of PRL concentration, supporting the idea that PRL increases feed intake to provide the nutrients necessary to support lactation in dairy ruminants. In late-lactation cows, quinagolide and cabergoline decreased milk production within the first day of treatment and induced more rapid changes in several markers of mammary gland involution after drying-off. In addition, quinagolide improved the resistance to intramammary infection, suggesting that PRL inhibition could be an alternative strategy for facilitating drying-off. Prolactin appears to directly affect mammary gland functions, but mammary gland responsiveness to PRL appears to be modulated by local and systemic factors. Therefore, the modulation of the number and isoforms of the PRL receptors as well as the expression of intracellular modulators of cell signaling in the mammary gland require further investigation. In conclusion, these data, combined with those from other studies, provide a good body of evidence that PRL is galactopoietic in dairy ruminants.

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.

Regulation of cell number in the mammary gland by controlling the exfoliation process in milk in ruminants.

Milk yield is partly influenced by the number of mammary epithelial cells (MEC) in the mammary gland. It is well known that variations in MEC number are due to cell proliferation and apoptosis. The exfoliation of MEC from the mammary epithelium into milk is another process that might influence MEC number in the mammary tissue. The rate of MEC exfoliation can be assessed by measuring the milk MEC content through light microscopy, flow cytometry analysis, or an immuno-magnetic method for MEC purification. Various experimental models have been used to affect milk yield and study the rate of MEC exfoliation. Reducing milking frequency from twice to once daily did not seem to have any effect on MEC loss in goat and cow milk after 7 d, but increased MEC loss per day in goats when applied for a longer period. An increase in MEC exfoliation was also observed during short days as compared with long days, or in response to an endotoxin-induced mastitis in cows. Other animal models were designed to investigate the endocrine control of the exfoliation process and its link with milk production. Suppression of ovarian steroids by ovariectomy resulted in a greater persistency of lactation and a decrease in MEC exfoliation. Administering prolactin inhibitors during lactation or at dry-off enhanced MEC exfoliation, whereas exogenous prolactin during lactation tended to prevent the negative effect of prolactin inhibitors. These findings suggest that prolactin could regulate MEC exfoliation. In most of these studies, variations of MEC exfoliation were associated with variations in milk yield and changes in mammary epithelium integrity. Exfoliation of MEC could thus influence milk yield by regulating MEC number in mammary tissue.

Dietary cation-anion difference and day length have an effect on milk calcium content and bone accretion of dairy cows.

Milk and dairy products are an important source of Ca for humans. Recent studies have shown fluctuations in cow milk Ca content during the year in France, with high values in winter and with corn silage diets, and a decrease during May and June and with grass diets. The aim of this study was to identify the reasons for this seasonal decrease in milk Ca content by testing the effect of 2 levels of dietary cation-anion differences (DCAD; 0 mEq/kg of dry matter for DCAD 0 and 400 mEq/kg for DCAD 400) and 2 day lengths (8 h of light/d for short days: SD; and 16 h/d for long days: LD) on the Ca balances of dairy cows. The DCAD treatments were designed to mimic diets based either on corn silage or on herbage. The cows were only illuminated by solarium lights providing UVA and UVB. The trial was conducted according to 2 simultaneous replicates of a 4×4 Latin square design using 8 dairy cows averaging 103±44 d in milk with 4 periods of 14 d. Data were analyzed by ANOVA with a model including treatment, cow, and period effects. No significant interaction was found between day length and DCAD treatments. With DCAD 400 compared with DCAD 0, blood pH increased and plasma ionized Ca content decreased, whereas the plasma total Ca content did not differ between treatments. Milk Ca content, however, increased with DCAD 400 compared with DCAD 0, in relation to a decrease in the amount of Ca excreted in urine. The DCAD had no significant effect on protein and casein contents and DCAD 400 tended to decrease milk yield. This illustrates that the udder did not decrease Ca uptake from the blood at high DCAD even though DCAD 400 decreased the mammary availability of Ca by decreasing the proportion of blood ionized Ca. Milk Ca and casein contents were significantly lower with LD compared with SD, whereas day length had no effect on milk yield after 14 d of treatment. Bone accretion of cows increased when the Ca content of milk increased (i.e., with DCAD 400 compared with DCAD 0 and with SD compared with LD). This work suggests that long and sunny days could explain part of the seasonal decrease in milk Ca content in summer and refutes the hypothesis that low milk Ca contents at grazing could be due to the high DCAD of herbage.

Cabergoline inhibits prolactin secretion and accelerates involution in dairy cows after dry-off.

Dairy cattle require a dry period between successive lactations to ensure optimal milk production. Because prolactin (PRL) is necessary for the initiation and maintenance of milk production, strategies that can inhibit PRL secretion might hasten the involution process. The objective of this study was to determine the effect of the PRL release inhibitor cabergoline on markers of mammary gland involution during the early dry period. To assess the effect of cabergoline treatment on mammary gland involution, 14 Holstein dairy cows in late lactation were treated with either a single i.m. administration of 5.6mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n=7) or placebo (n=7) at the time of dry-off. Blood samples and mammary secretion samples were collected 6d before dry-off and again 1, 2, 3, 4, 8, and 14d following the abrupt cessation of lactation. Blood samples were used to determine plasma PRL concentrations. Mammary secretion samples were used to determine somatic cell count, milk fat, lactose, true protein content, and concentrations of α-lactalbumin, lactoferrin, and citrate. Following the cessation of lactation, changes in mammary secretion composition indicated diminished milk synthesis, including reduced concentrations of α-lactalbumin, citrate, and lactose. In contrast, milk somatic cell count, percent total protein, percent fat content, and lactoferrin concentrations significantly increased as involution progressed. Cabergoline treatment decreased the plasma PRL concentrations during the first week of dry-off, compared with the control treatment. No significant differences in citrate, α-lactalbumin, or protein content were observed between treatment groups. The most dramatic changes in secretion composition as a consequence of cabergoline treatment occurred during the first week of the dry period, when lactose concentrations and the citrate:lactoferrin molar ratio were lower and lactoferrin concentrations higher than in the control cows. Cabergoline treatment also tended to increase fat content and somatic cell count more rapidly following dry-off compared with the control group. These changes in mammary secretion composition following the abrupt cessation of lactation indicate that cabergoline treatment facilitated dry-off and effectively accelerated mammary gland involution.

Differences during the first lactation between cows cloned by somatic cell nuclear transfer and noncloned cows.

Lactation performance is dependent on both the genetic characteristics and the environmental conditions surrounding lactating cows. However, individual variations can still be observed within a given breed under similar environmental conditions. The role of the environment between birth and lactation could be better appreciated in cloned cows, which are presumed to be genetically identical, but differences in lactation performance between cloned and noncloned cows first need to be clearly evaluated. Conflicting results have been described in the literature, so our aim was to clarify this situation. Nine cloned Prim' Holstein cows were produced by the transfer of nuclei from a single fibroblast cell line after cell fusion with enucleated oocytes. The cloned cows and 9 noncloned counterparts were raised under similar conditions. Milk production and composition were recorded monthly from calving until 200d in milk. At 67d in milk, biopsies were sampled from the rear quarter of the udder, their mammary epithelial cell content was evaluated, and mammary cell renewal, RNA, and DNA were then analyzed in relevant samples. The results showed that milk production did not differ significantly between cloned and noncloned cows, but milk protein and fat contents were less variable in cloned cows. Furthermore, milk fat yield and contents were lower in cloned cows during early lactation. At around 67 DIM, milk fat and protein yields, as well as milk fat, protein, and lactose contents, were also lower in cloned cows. These lower yields could be linked to the higher apoptotic rate observed in cloned cows. Apoptosis is triggered by insulin-like factor growth binding protein 5 (IGFBP5) and plasminogen activator inhibitor (PAI), which both interact with CSN1S2. During our experiments, CSN1S2 transcript levels were lower in the mammary gland of cloned cows. The mammary cell apoptotic rate observed in cloned cows may have been related to the higher levels of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) transcripts, coding for products that maintain the epigenetic status of cells. We conclude, therefore, that milk production in cloned cows differs slightly from that of noncloned cows. These differences may be due, in part, to a higher incidence of subclinical mastitis. They were associated with differences in cell apoptosis and linked to variations in DNMT1 mRNA. However, milk protein and fat contents were more similar among cloned cows than among noncloned cows.

In vivo inhibition followed by exogenous supplementation demonstrates galactopoietic effects of prolactin on mammary tissue and milk production in dairy cows.

It has been previously shown that the long-term inhibition of milking-induced prolactin (PRL) release by quinagolide (QN), a dopamine agonist, reduces milk yield in dairy cows. To further demonstrate that PRL is galactopoietic in cows, we performed a short-term experiment that used PRL injections to restore the release of PRL at milking in QN-treated cows. Nine Holstein cows were assigned to treatments during three 5-d periods in a 3×3 Latin square design: 1) QN: twice-daily i.m. injections of 1mg of QN; 2) QN-PRL: twice-daily i.m. injections of 1mg of QN and twice-daily (at milking time) i.v. injections of PRL (2µg/kg body weight); and 3) control: twice-daily injections of the vehicles. Mammary epithelial cells (MEC) were purified from milk so that their viability could be assessed, and mammary biopsies were harvested for immunohistological analyses of cell proliferation using PCNA and STAT5 staining. In both milk-purified MEC and mammary tissue, the mRNA levels of milk proteins and BAX were determined using real-time reverse-transcription PCR. Daily QN injections reduced milking-induced PRL release. The area under the PRL curve was similar in the control and PRL injection treatments, but the shape was different. The QN treatment decreased milk, lactose, protein, and casein production. Injections of PRL did not restore milk yield but tended to increase milk protein yield. In mammary tissue, the percentage of STAT5-positive cells was reduced during QN but not during QN-PRL in comparison with the control treatment. The percentage of PCNA-positive cells was greater during QN-PRL injections than during the control or QN treatment and tended to be lower during QN than during the control treatment. In milk-purified MEC, κ-casein and α-lactalbumin mRNA levels were lower during QN than during the control treatment, but during QN-PRL, they were not different from the control treatment. In mammary tissue, the BAX mRNA level was lower during QN-PRL than during QN. The number of MEC exfoliated into milk was increased by QN injections but tended to be decreased by PRL injections. Injections of PRL also increased the viability of MEC harvested from milk. Although PRL injections at milking could not reverse the effect of QN treatment on milk production, their effects on cell survival and exfoliation and on gene expression suggest that the effect of QN treatment on the mammary gland is due to QN's inhibition of PRL secretion.

Post-milking application of a Lacticaseibacillus paracasei strain impacts bovine teat microbiota while preserving the mammary gland physiology and immunity.

Bovine mastitis (BM) is a major disease in dairy industry. The current approaches - mainly antibiotic treatments - are not entirely effective and may contribute to antimicrobial resistance dissemination, rising the need for alternative treatment. The present study aims to evaluate the impact of post-milking application of Lacticaseibacillus paracasei CIRM BIA 1542 (Lp1542) on the teat skin (TS) of 20 Holstein cows in mid lactation, in order to reinforce the barrier effect of the microbiota naturally present on the teat. Treatment (Lp1542, iodine or no treatment) was applied post-milking twice a day on the 4 teats of healthy animals for 15 days. Blood and milk samples, and TS swabs were collected at day (D)1, D8, D15 and D26 before morning milking and at D15 before evening milking (D15E) to evaluate Lp1542 impact at the microbial, immune and physiological levels. Lp1542 treatment resulted in a higher lactic acid bacteria and total microbial populations on TS and in foremilk (FM) at D15(E) compared with iodine treatment. Metabarcoding analysis revealed changes in the composition of TS and FM microbiota, beyond a higher Lacticaseibacillus abundance. This included a higher abundance of Actinobacteriota, including Bifidobacterium, and a lower abundance of Pseudomonadota on TS of Lp1542 compared with iodine-treated quarters. In addition, Lp1542 treatment did not trigger any major inflammatory response in the mammary gland, except interleukin 8 production and expression which tended to be slightly higher in Lp1542-treated cows compared with the others. Finally, Lp1542 treatment had no impact on the mammary epithelium functionality (milk yield and composition) and integrity (epithelial cell exfoliation into milk and milk Na+/K+ ratio). Altogether, these results indicate that a topical treatment with Lp1542 is safe with regard to mammary gland physiology and immune system, while impacting its microbiota, inviting us to further explore its effectiveness for mastitis prevention.

Mammary cell activity and turnover in dairy cows treated with the prolactin-release inhibitor quinagolide and milked once daily.

To assess the regulation of mammary cell activity, survival, and proliferation by prolactin (PRL), 5 Holstein cows in early lactation received daily i.m. injections of 1mg of quinagolide, a suppressor of PRL release, for 9 wk, whereas 4 control cows received the vehicle (water) only. During the last week of treatment, one udder half was milked once a day (1×) and the other twice a day (2×). Mammary biopsies were harvested 1 wk before and 4 and 8 wk after the start of quinagolide treatment. The quinagolide injections reduced milk yield and resulted in lower levels of κ-casein and α-lactalbumin mRNA in the mammary biopsies at wk 4 compared with the control cows. In the mammary tissue of the quinagolide-treated cows at wk 8 of treatment, cell proliferation (as determined by proliferating cell nuclear antigen labeling) was lower and apoptosis (as determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay) was higher than in the mammary tissue of the control cows. During differential milking, mammary epithelial cells (MEC) were extracted from the milk by centrifugation and purified by immunocytochemical binding to allow variations in the levels of mammary transcripts to be observed. After 9 wk of treatment, levels of α-lactalbumin and κ-casein mRNA were lower in the MEC isolated from milk of the quinagolide-treated cows. This effect was associated with lower PRL receptor mRNA levels and a tendency toward lower viability in the milk-isolated MEC from the 2×-milked glands. The decrease from 2× milking to 1× milking also downregulated α-lactalbumin and κ-casein transcripts in the milk-isolated MEC. Viability was higher for the MEC collected from the 1×-milked udder halves compared with the 2×-milked halves. In conclusion, the reduction in milk yield after chronic administration of the PRL-release inhibitor quinagolide is associated with a reduction in mammary cell activity, survival, and proliferation in lactating dairy cows. Reduced milking frequency was also associated with a decrease in MEC activity.

Milk proteins as a feed restriction signature indicating the metabolic adaptation of dairy cows.

Milk production in dairy cows is affected by numerous factors, including diet. Feed restriction is known to have little impact on milk total protein content but its effect on the fine protein composition is still poorly documented. The objective of this study was to describe the effects of two feed restriction trials of different intensities on the milk protein composition of Holstein cows. One restriction trial was of high intensity (H: 8 mid-lactation Holstein cows) and the second of moderate intensity (M: 19 peak lactation Holstein cows). Feed restriction decreased the milk protein yield for caseins under the M trial and of all six major milk proteins under the H trial. These decreased yields lead to lower concentrations of αs1-, αs2- and ß-caseins during the H trial. The milk proteome, analyzed on 32 milk samples, was affected as a function of restriction intensity. Among the 345 proteins identified eight varied under the M trial and 160 under the H trial. Ontology analyses revealed their implication in carbohydrate, lipid and protein metabolisms as well as in the immune system. These proteins reflected adaptations of the animal and mammary gland physiology to feed restriction and constituted a signature of this change.

Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows.

In most mammals, prolactin (PRL) is essential for maintaining lactation, and yet the short-term suppression of PRL during established lactation by bromocriptine has produced inconsistent effects on milk yield in cows and goats. To assess the effect of the long-term inhibition of PRL release in lactating dairy cows, 5 Holstein cows in early lactation received daily intramuscular injections of 1mg of the PRL-release inhibitor quinagolide for 9 wk. Four control cows received the vehicle (water) only. During the last week of the treatments, one udder half was milked once a day (1×) and the other twice a day (2×). Blood samples were harvested at milking in wk -1, 1, 4, and 8. The daily injections of quinagolide reduced milking-induced PRL release but not the basal PRL concentration. Quinagolide induced a faster decline in milk production, which was about 5.3 kg/d lower in the quinagolide-treated cows during the last 4 wk of treatment. During wk 9, the inhibition of milk production by quinagolide was maintained in the udder half that was milked 2× but not in the half milked 1×. Milk production was significantly correlated with the quantity of PRL released at milking. Quinagolide did not affect the release of oxytocin at milking. Serum concentration of insulin-like growth factor-1 was not affected by treatment or correlated with milk production. Serum concentrations of leptin and the calciotropic hormone stanniocalcin were not affected by the treatment. In conclusion, the chronic administration of the PRL-release inhibitor quinagolide decreases milk production in dairy cows. The effect is likely the result of the reduced release of milking-induced PRL and is modulated at the level of the gland by milking frequency.

Effects of nutrient restriction on mammary cell turnover and mammary gland remodeling in lactating dairy cows.

The aim of this study was to investigate the effects of a severe nutrient restriction on mammary tissue morphology and remodeling, mammary epithelial cell (MEC) turnover and activity, and hormonal status in lactating dairy cows. We used 16 Holstein × Normande crossbred dairy cows, divided into 2 groups submitted to different feeding levels (basal and restricted) from 2 wk before calving to wk 11 postpartum. Restricted-diet cows had lower 11-wk average daily milk yield from calving to slaughter than did basal-diet cows (20.5 vs. 33.5 kg/d). Feed restriction decreased milk fat, protein, and lactose yields. Restriction also led to lower plasma insulin-like growth factor 1 and higher growth hormone concentrations. Restricted-diet cows had lighter mammary glands than did basal-diet cows. The total amount of DNA in the mammary gland and the size of the mammary acini were smaller in the restricted-diet group. Feed restriction had no significant effect on MEC proliferation at the time of slaughter but led to a higher level of apoptosis in the mammary gland. Gelatin zymography highlighted remodeling of the mammary extracellular matrix in restricted-diet cows. Udders from restricted-diet cows showed lower transcript expression of α-lactalbumin and kappa-casein. In conclusion, nutrient restriction resulted in lower milk yield in lactating dairy cows, partly due to modulation of MEC activity and a lower number of mammary cells. An association was found between feed restriction-induced changes in the growth hormone-insulin-like growth factor-1 axis and mammary epithelial cell dynamics.

Disruption of cell junctions induces apoptosis and reduces synthetic activity in lactating goat mammary gland.

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.

Evolution of milk composition, milk fat globule size, and free fatty acids during milking of dairy cows.

The objective of this study was to measure milk composition (fat, protein, and calcium contents; fatty acid profile), milk fat globule size, and free fatty acid content throughout milking. Composition was measured from milk samples collected every 1 min during morning milking in 2 previously published experiments. Experiments 1 and 2 used 9 and 6 dairy cows, respectively. From the beginning to end of milking in experiments 1 and 2, lactose content decreased (-0.45 percentage units), as did protein content (-0.28 and -0.17 percentage units, respectively). In contrast, fat content increased (+5.66 and +5.57 percentage units, respectively). Milk fat globule size increased (+1.51 and +0.43 µm, respectively), whereas free fatty acid content (measured after 24 h of storage at 4°C) decreased quickly during the first minutes (-0.45 mEq/100 g of fat from time point 1 to time point 4 in experiment 1, and -0.85 mEq/100 g of fat from time point 1 to time point 5 in experiment 2), and then largely stabilized, with a slight tendency to increase toward the end of milking period in experiment 2 (+0.32 mEq/100 g of fat). The evolution of milk fatty acid composition depended on the experiment. From the beginning to the end of milking, the concentration of C16:0 consistently increased (+3.4 wt/wt % in experiment 1 and +3.3 wt/wt % from time point 2 to time point 7 in experiment 2), whereas the C18:1/C16:0 ratio increased during the first minutes of milking and then slightly decreased (-0.050 in experiment 1 and -0.031 from time point 2 to time point 7 in experiment 2). Calcium content decreased in experiment 2 (-58 mg/kg). In conclusion, milk composition changed greatly during milking, suggesting that different mechanisms are involved in synthesis and excretion, depending on the type of milk component.