Mastitis has a cumulative and lasting effect on milk yield and lactose content in dairy cows.

Milk lactose content (LC) physiologically decreases with parity order in dairy cows, but also after udder health inflammation(s) and/or in presence of elevated milk SCC in subclinical cases. Therefore, the progressive decrease in milk LC observed along cows' productive life can be attributed to a combination of factors that altogether impair the epithelial integrity, resulting in weaker tight junctions, e.g., physiological aging of epithelium, mechanical epithelial stress due to milking, and experienced clinical or subclinical mastitis. Mastitis is known to affect the udder synthesis ability too, so our intention through this study was to evaluate if there is a cumulative and lasting effect of mammary gland inflammation(s) on milk yield (MY) and LC. For this purpose, we used diagnoses of clinical mastitis and milk data of Austrian Fleckvieh cows to evaluate the effect of cumulative mastitis events on LC and MY. Only mastitis diagnoses recorded by trained veterinarians were used. Finally, we investigated if cumulative mastitis is a heritable trait and whether it is genetically correlated with either LC or MY. Estimates were obtained using univariate and bivariate linear animal models. A significant reduction in LC and MY was observed in cows that suffered from mastitis compared with those that did not experience udder inflammation. The h2 of cumulative mastitis is promising and much greater (0.09) than the h2 of the binary event itself (≤0.03). The genetic correlations between cumulative mastitis with LC and MY were negative, suggesting that cows with a great genetic merit for MY and LC are expected to be more resistant to repeated inflammations and less recidivist. When we used number of lifetime SCC peaks (≥200,000 or 400,000 cells/mL) to calculate cumulative inflammation events, h2 was even higher (up to 0.38), implying that also subclinical mastitis has a relevant negative impact on both LC and MY. Finally, the present study demonstrated how repeated mastitis events can permanently affect the mammary gland epithelial integrity and synthesis ability, and that the number of cumulative mastitis is a promising phenotype to be used in selection index in combination with other indicator traits toward more resistant and resilient mammary glands.

Molecular Characteristics of JAK2 and Its Effect on the Milk Fat and Casein Synthesis of Ovine Mammary Epithelial Cells.

In addition to its association with milk protein synthesis via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, JAK2 also affects milk fat synthesis. However, to date, there have been no reports on the effect of JAK2 on ovine mammary epithelial cells (OMECs), which directly determine milk yield and milk contents. In this study, the coding sequence (CDS) region of ovine JAK2 was cloned and identified and its tissue expression and localization in ovine mammary glands, as well as its effects on the viability, proliferation, and milk fat and casein levels of OMECs, were also investigated. The CDS region of ovine JAK2, 3399 bp in length, was cloned and its authenticity was validated by analyzing its sequence similarity with JAK2 sequences from other animal species using a phylogenetic tree. JAK2 was found to be expressed in six ovine tissues, with the highest expression being in the mammary gland. Over-expressed JAK2 and three groups of JAK2 interference sequences were successfully transfected into OMECs identified by immunofluorescence staining. When compared with the negative control (NC) group, the viability of OMECs was increased by 90.1% in the pcDNA3.1-JAK2 group. The over-expression of JAK2 also increased the number and ratio of EdU-labeled positive OMECs, as well as the expression levels of three cell proliferation marker genes. These findings show that JAK2 promotes the viability and proliferation of OMECs. Meanwhile, the triglyceride content in the over-expressed JAK2 group was 2.9-fold higher than the controls and the expression levels of four milk fat synthesis marker genes were also increased. These results indicate that JAK2 promotes milk fat synthesis. Over-expressed JAK2 significantly up-regulated the expression levels of casein alpha s2 (CSN1S2), casein beta (CSN2), and casein kappa (CSN3) but down-regulated casein alpha s1 (CSN1S1) expression. In contrast, small interfered JAK2 had the opposite effect to JAK2 over-expression on the viability, proliferation, and milk fat and milk protein synthesis of OMECs. In summary, these results demonstrate that JAK2 promotes the viability, proliferation, and milk fat synthesis of OMECs in addition to regulating casein expression in these cells. This study contributes to a better comprehension of the role of JAK2 in the lactation performance of sheep.

Leucine and arginine enhance milk fat and milk protein synthesis via the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways.

BACKGROUND AND AIMS: Amino acids (AAs) not only constitute milk protein but also stimulate milk synthesis through the activation of mTORC1 signaling, but which amino acids that have the greatest impact on milk fat and protein synthesis is still very limited. In this study, we aimed to identify the most critical AAs involved in the regulation of milk synthesis and clarify how these AAs regulate milk synthesis through the G-protein-coupled receptors (GPCRs) signaling pathway. METHODS: In this study, a mouse mammary epithelial cell line (HC11) and porcine mammary epithelial cells (PMECs) were selected as study subjects. After treatment with different AAs, the amount of milk protein and milk fat synthesis were detected. Activation of mTORC1 and GPCRs signaling induced by AAs was also investigated. RESULTS: In this study, we demonstrate that essential amino acids (EAAs) are crucial to promote lactation by increasing the expression of genes and proteins related to milk synthesis, such as ACACA, FABP4, DGAT1, SREBP1, α-casein, ß-casein, and WAP in HC11 cells and PMECs. In addition to activating mTORC1, EAAs uniquely regulate the expression of calcium-sensing receptor (CaSR) among all amino-acid-responsive GPCRs, which indicates a potential link between CaSR and the mTORC1 pathway in mammary gland epithelial cells. Compared with other EAAs, leucine and arginine had the greatest capacity to trigger GPCRs (p-ERK) and mTORC1 (p-S6K1) signaling in HC11 cells. In addition, CaSR and its downstream G proteins Gi, Gq, and Gßγ are involved in the regulation of leucine- and arginine-induced milk synthesis and mTORC1 activation. Taken together, our data suggest that leucine and arginine can efficiently trigger milk synthesis through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways. CONCLUSION: We found that the G-protein-coupled receptor CaSR is an important amino acid sensor in mammary epithelial cells. Leucine and arginine promote milk synthesis partially through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 signaling systems in mammary gland epithelial cells. Although this mechanism needs further verification, it is foreseeable that this mechanism may provide new insights into the regulation of milk synthesis.

Effects of the timing of protein infusion on the daily rhythms of milk synthesis and plasma hormones and metabolites in dairy cows.

Milk synthesis exhibits a daily rhythm that is modified by the timing of feed intake. However, it is unknown how specific nutrients entrain this daily rhythm. Amino acids have an important role in milk synthesis, and may have a role in entrainment of mammary circadian rhythms. The objective of this study was to determine the effects of intestinally absorbed protein on daily rhythms of milk and milk component synthesis and key plasma hormones and metabolites. Nine lactating Holstein cows were assigned to 1 of 3 treatment sequences in a 3 × 3 Latin square. Treatments included abomasal infusions of 500 g/d of sodium caseinate either continuously throughout the day (CON), for 8 h/d from 0900 to 1700 h (DAY), or for 8 h/d from 2100 to 0500 h (NGT). Cows were milked every 6 h during the final 8 d of each period. A 24-h rhythm was fit to data using cosine analysis and the amplitude and acrophase were determined. Night infusion of protein decreased the daily milk yield and milk protein yield by 8.2% and 9.2%, respectively. Milk fat yield was increased 5.5% by DAY and milk fat concentration was increased 8.8% by NGT. Milk yield exhibited a daily rhythm in all treatments, with NGT increasing the amplitude of the daily rhythm 33% compared with CON. Milk fat concentration fit a daily rhythm in CON and NGT, but not DAY, whereas milk protein concentration fit a daily rhythm in CON and DAY, but not NGT. Moreover, DAY abolished the daily rhythm of plasma glucose concentration, but induced rhythms of plasma insulin and nonesterified fatty acid concentrations. Results suggest that feeding increased protein levels during the early part of the day may increase milk fat yield and modify energy metabolism through increased daily variation in insulin-stimulated lipid release, but additional research focused on feeding multiple diets across the day is required.

Dietary supplementation with monosodium glutamate enhances milk production by lactating sows and the growth of suckling piglets.

This study was conducted to test the hypothesis that increasing dietary content of glutamate through addition of monosodium glutamate (MSG) enhances milk production by lactating sows and the growth of their offspring. Thirty multiparous sows (Landrace × Large White) were assigned randomly into one of three dietary groups: control (a corn- and soybean meal-based diet), the basal diet + 1% MSG, and the basal diet + 2% MSG. Diets were made isonitrogenous by the addition of appropriate amounts of L-alanine. Lactating sows had free access to drinking water and were fed twice daily their respective diets. The number of live-born piglets was standardized to 9 per sow at day 0 of lactation (the day of farrowing). On days 3, 15, and 29 of lactation, body weight and milk consumption of piglets were measured, and blood samples obtained from sows and piglets at 2 h and 1 h after feeding, respectively. Feed intake of sows did not differ (P > 0.05) among the three groups of sows. Concentrations of aspartate, glutamine, citrulline, arginine, tryptophan, proline, branched-chain amino acids, and glutamate were greater (P < 0.05) in the plasma of MSG-supplemented sows and their piglets than for controls. When compared with the control, dietary supplementation with 1-2% MSG increased (P < 0.05): concentrations of many free amino acids (including glutamate plus glutamine) and all protein-bound amino acids in milk; the milk intake of piglets by 14-25%; and daily weight gains of piglets by 23-44%. These results indicate that dietary supplementation with 1-2% MSG to lactating sows enhances milk production to support the growth of sow-reared piglets.

CRISPR-Cas9-mediated knockout of GCN2 reveals a critical role in sensing amino acid deprivation in bovine mammary epithelial cells.

The objective of this study was to determine the role of GCN2 in the response to AA deprivation of primary bovine mammary epithelial cells (BMEC). Cells were isolated from the mammary tissue of 2 lactating Holstein cows by enzymatic digestion, expanded, and induced to differentiate for 5 to 7 d. Relative mRNA expression was measured by real-time quantitative PCR. Protein abundance and site-specific phosphorylation were measured by immunoblotting. Knockout of GCN2 in BMEC was accomplished by lentiviral delivery of a targeted single guide RNA and endonuclease Cas9. To investigate the role of GCN2, we treated lactogenic differentiated BMEC with either culture medium lacking Arg, Leu, and Lys combined or lacking only one of the 3 AA of interest, in comparison to a control with a full complement of AA. Activation of GCN2 was inferred by the phosphorylation status of its downstream target eIF2α Ser51. We found that GCN2 was activated by both the deprivation of Arg, Leu, and Lys combined and of Arg alone, as shown by a 2.73- and 2.82-fold increase in phosphorylated eIF2α Ser51 after 1 h of deprivation, respectively. In addition, activation of GCN2 as measured by increased phosphorylation of eIF2α Ser51 during the deprivation of Arg, Leu, and Lys combined and of Arg alone was sustained for up to 8 h of deprivation. Phosphorylated eIF2α selectively upregulates translation of transcription factor ATF4, among others, during AA deprivation, which then targets genes necessary for restoring AA homeostasis. Therefore, we investigated the expression of ATF4 transcriptional targets, AA enzyme ASNS and AA transporters SLC7A1 and SLC38A2. We found that ASNS was upregulated in response to combined AA deprivation and by Arg deprivation alone by 3.6- and 4.51-fold, respectively, at 24 h of treatment. We found that SLC7A1 was upregulated in response to combined AA deprivation and deprivation of Arg alone by 2.0- and 2.36-fold, respectively, at 8 h of treatment. To establish the role of GCN2 (encoded by EIF2AK4) in the response to AA deprivation, we ablated GCN2 in BMEC using clustered regularly interspaced short palindromic repeats-Cas9. We showed that BMEC transduced with single guide RNA targeting EIF2AK4 were not as responsive to combined AA deprivation, compared with BMEC transduced with nontargeting single guide RNA. Taken together, our results demonstrate a critical role for GCN2 in sensing AA deprivation in BMEC.

Night-restricted feeding of dairy cows modifies daily rhythms of feed intake, milk synthesis and plasma metabolites compared with day-restricted feeding.

The timing of feed intake can alter circadian rhythms of peripheral tissues. Milk synthesis displays a daily rhythm across several species, but the effect of feeding time on these rhythms is poorly characterised. The objective of this experiment was to determine if the time of feed intake modifies the daily patterns of milk synthesis, plasma metabolites and body temperature in dairy cows. Sixteen lactating Holstein dairy cows were randomly assigned to one of the two treatment sequences in a cross-over design with 17 d periods. Treatments included day-restricted feeding (DRF; feed available from 07.00 to 23.00 hours) and night-restricted feeding (NRF; feed available from 19.00 to 11.00 hours). Cows were milked every 6 h on the last 7 d of each period, and blood samples were collected to represent every 4 h over the day. Peak milk yield was shifted from morning in DRF to evening in NRF, while milk fat, protein and lactose concentration peaked in the evening in DRF and the morning in NRF. Plasma glucose, insulin, NEFA and urea nitrogen concentration fit daily rhythms in all treatments. Night feeding increased the amplitude of glucose, insulin and NEFA rhythms and shifted the daily rhythms by 8 to 12 h (P < 0·05). Night feeding also phase-delayed the rhythm of core body temperature and DRF increased its amplitude. Altering the time of feed availability shifts the daily rhythms of milk synthesis and plasma hormone and metabolite concentrations and body temperature, suggesting that these rhythms may be entrained by food intake.

Annual rhythms of milk synthesis in dairy herds in 4 regions of the United States and their relationships to environmental indicators.

The annual rhythms of milk and milk component yields are not well described and are important to dairy management. Recent analysis of federal milk marketing orders in the United States observed that the amplitude and time at peak (acrophase) of the rhythms of milk fat and protein concentration differ among regions, but the rhythms of milk and milk component yields are not well described. Our objective was to determine the annual rhythms of milk and milk component production from 4 US regions at the herd level and examine potential environmental factors entraining these rhythms. Monthly Dairy Herd Improvement Association records of all available herds in Pennsylvania (PA), Minnesota (MN), Texas (TX), and Florida (FL) from the years 2003 to 2016 were obtained from Dairy Records Managements Systems. Milk yield, fat and protein yield, and fat and protein concentration were fit to the linear form of the cosine function with a 12-mo period using a linear mixed effects model. Additionally, the fit of models containing either the cosine function or environmental temperature were compared using an F-test. Milk yield and fat and protein yields and concentrations fit a cosine function in all 4 states, indicating an annual rhythm. The amplitude (peak to mean) of the rhythm of milk yield varied by state and was lower in PA (1.2 kg) and MN (1.2 kg) compared with TX (3.1 kg) and FL (3.3 kg). Fat and protein yields similarly showed greater amplitudes in the southern versus northern states. The amplitudes of the rhythms of fat and protein concentration were opposite by region, with greater amplitudes occurring in MN and PA than in TX and FL. The acrophases of milk yield and milk fat and protein yields and concentrations also varied by state, but all peaked between October and March. An annual rhythm fit the data better than changes in environmental temperature for all responses in all states, except for fat and protein concentrations in FL, which exhibited lower amplitude seasonal rhythms. The yearly pattern of milk yield closely followed the fixed yearly pattern of the day to day changes in day length, whereas the rhythms of milk fat and protein concentrations followed the yearly pattern of absolute day length. Results suggest that the region of the United States in which a herd is located affects their annual rhythms of production, with a greater yearly variation in milk, fat, and protein yields occurring in the southern United States. The consistency of annual rhythms across years and herds allowed development of regression equations to adjust expectations across the year to account for the annual rhythm.

Meta-analysis of mammary RNA seq datasets reveals the molecular understanding of bovine lactation biology.

A better understanding of the biology of lactation, both in terms of gene expression and the identification of candidate genes for the production of milk and its components, is made possible by recent advances in RNA seq technology. The purpose of this study was to understand the synthesis of milk components and the molecular pathways involved, as well as to identify candidate genes for milk production traits within whole mammary transcriptomic datasets. We performed a meta-analysis of publically available RNA seq transcriptome datasets of mammary tissue/milk somatic cells. In total, 11 562 genes were commonly identified from all RNA seq based mammary gland transcriptomes. Functional annotation of commonly expressed genes revealed the molecular processes that contribute to the synthesis of fats, proteins, and lactose in mammary secretory cells and the molecular pathways responsible for milk synthesis. In addition, we identified several candidate genes responsible for milk production traits and constructed a gene regulatory network for RNA seq data. In conclusion, this study provides a basic understanding of the lactation biology of cows at the gene expression level.

Cyclase-associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells.

Adenylyl cyclase-associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K-mTOR/SREBP-1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.

Invited review: Accelerating mammary gland involution after drying-off in dairy cattle.

Bovine mammary gland involution, as a part of the reproductive cycle in dairy cows, is a very important remodeling transformation of the mammary gland for the subsequent lactation. There is considerable incentive to accelerate mammary gland involution to improve udder health, shorten the dry period, and simplify the management process by reducing dietary changes. The complex process of mammary involution is characterized by morphological changes in the epithelial cells and mammary tissue, changes in the composition of mammary secretions, and changes in the integrity of tight junctions. Involution is facilitated by elements of the immune system and several types of proteases and is coordinated by various types of hormones. This review first describes the involution process and then argues for the need to accelerate it. Last, this review focuses on various intervention methods for accelerating involution. Our aim is to provide a comprehensive overview of bovine mammary gland involution as well as potential techniques and new opinions for dry cow management.

Annual rhythms of milk and milk fat and protein production in dairy cattle in the United States.

An annual pattern of milk composition has been well recognized in dairy cattle, with the highest milk fat and protein concentration observed during the winter and lowest occurring in the summer; however, rhythms of milk yield and composition have not been well quantified. Cosinor rhythmometry is commonly used to model repeating daily and annual rhythms and allows determination of the amplitude (peak to mean), acrophase (time at peak), and period (time between peaks) of the rhythm. The objective of this study was to use cosinor rhythmometry to characterize the annual rhythms of milk yield and milk fat and protein concentration and yield using both national milk market and cow-level data. First, 10 yr of monthly average milk butterfat and protein concentration for each Federal Milk Marketing Order were obtained from the US Department of Agriculture Agricultural Marketing Service database. Fat and protein concentration fit a cosine function with a 12-mo period in all milk markets. We noted an interaction between milk marketing order and milk fat and protein concentration. The acrophase (time at peak) of the fat concentration rhythm ranged from December 4 to January 19 in all regions, whereas the rhythm of protein concentration peaked between December 27 and January 6. The amplitude (peak to mean) of the annual rhythm ranged from 0.07 to 0.14 percentage points for milk fat and from 0.08 to 0.12 percentage points for milk protein. The amplitude of the milk fat rhythm generally was lower in southern markets and higher in northern markets. Second, the annual rhythm of milk yield and milk fat and protein yield and concentration were analyzed in monthly test day data from 1,684 cows from 11 tiestall herds in Pennsylvania. Fat and protein concentration fit an annual rhythm in all herds, whereas milk and milk fat and protein yield only fit rhythms in 8 of the 11 herds. On average, milk yield peaked in April, fat and protein yield peaked in February, fat concentration peaked in January, and protein concentration peaked in December. Amplitudes of milk, fat, and protein yield averaged 0.82 kg, 55.3 g, and 30.4 g, respectively. Milk fat and protein concentration had average amplitudes of 0.12 and 0.07, respectively, similar to the results of the milk market data. Generally, milk yield and milk components fit annual rhythm regardless of parity or diacylglycerol O-acyltransferase 1 (DGAT1) K232A polymorphism, with only cows of the low-frequency AA genotype (5.2% of total cows) failing to fit rhythm of milk yield. In conclusion, the yearly rhythms of milk yield and fat and protein concentration and yield consistently occur regardless of region, herd, parity, or DGAT1 genotype and supports generation by a conserved endogenous annual rhythm.

Effect of rumen-protected branched-chain amino acid supplementation on production- and energy-related metabolites during the first 35 days in milk in Holstein dairy cows.

Essential AA are critical for multiple physiological processes. Branched-chain AA (BCAA) supplementation has beneficial effects on body weight, lipogenesis, and insulin resistance in several species. The BCAA are used for milk and body protein synthesis as well as being oxidized by the tricarboxylic acid cycle to produce ATP during catabolic states. The objective was to evaluate the effect of rumen-protected BCAA (375 g of 27% l-Leu, 85 g of 48% l-Ile, and 91 g of 67% l-Val) with or without propylene glycol (PG) oral administration on milk production, dry matter intake, nonesterified fatty acids, ß-hydroxybutyrate, and plasma urea nitrogen during the first 35 d in milk (DIM) in dairy cattle. Multiparous Holstein cows were enrolled in blocks of three 28 d before expected calving and assigned randomly to either the control or 1 of 2 treatments. The control (n = 26) received 200 g/d of dry molasses, the BCAA treatment (n = 23) received BCAA mixed with 200 g/d of dry molasses from calving until 35 DIM, and the BCAA plus PG (BCAAPG) treatment (n = 25) received BCAA mixed with 200 g/d of dry molasses from calving until 35 DIM plus 300 mL of PG once daily from calving until 7 DIM. Postpartum, dry matter intake least squares means (LSM; 95% confidence interval) were 20.7 (19.9, 21.7), 21.3 (20.4, 22.3), and 21.9 (20.9, 22.8) kg for control, BCAA, and BCAAPG, respectively. Milk yield (1-35 DIM) LSM were 41.7 (39.4, 44.0), 42.7 (40.3, 45.0), and 43.7 (41.4, 46.0) kg for control, BCAA, and BCAAPG, respectively. Energy-corrected milk LSM were 50.3 (46.8, 53.7), 52.4 (48.9, 55.8), and 52.9 (49.5, 56.4) kg for control, BCAA, and BCAAPG, respectively. Milk urea nitrogen LSM in milk for control, BCAA, and BCAAPG were 8.60 (8.02, 9.22), 9.70 (9.01, 10.45), and 9.75 (9.08, 10.47) mg/dL. Plasma urea nitrogen concentrations LSM for control, BCAA, and BCAAPG were 8.3 (7.7, 8.9), 10.1 (9.4, 10.9), and 9.6 (9.4, 10.3) mg/dL, respectively. The numbers of plasma samples classified as hyperketonemia were 77, 44, and 57 in control, BCAA, and BCAAPG, respectively. The BCAA supplementation increased plasma urea nitrogen and milk urea nitrogen, free valine concentration in plasma, and decreased hyperketonemia events during the postpartum period.

Mammary gland metabolite utilization in response to exogenous glucose or long-chain fatty acids at low and high metabolizable protein levels.

We investigated mammary gland metabolism in lactating dairy cattle in response to energy from glucogenic (glucose; GG) or lipogenic (palm olein; LG) substrates at low (LMP) and high (HMP) metabolizable protein levels. According to a 6 × 6 Latin square design, 6 rumen-fistulated second-lactation Holstein-Friesian dairy cows (97 ± 13 d in milk) were abomasally infused with saline (LMP-C); isoenergetic infusions (digestible energy basis) of 1,319 g/d glucose (LMP-GG), 676 g/d palm olein (LMP-LG), or 844 g/d essential AA (EAA; HMP-C); or isoenergetic infusions of 1,319 g/d glucose + 844 g/d EAA (HMP-GG) or 676 g/d palm olein + 844 g/d EAA (HMP-LG). Each experimental period consisted of 5 d of continuous infusion followed by 2 d of rest. A total mixed ration (42% corn silage, 31% grass silage, and 27% concentrate on a dry matter basis) formulated to meet 100 and 83% of net energy and metabolizable protein requirements, respectively, was fed at 90% of ad libitum intake by individual cow. Arterial and venous blood samples were collected on d 5 of each period. Infusing GG or LG at the HMP level did not affect milk yield or composition differently than at the LMP level. Neither GG nor LG infusion stimulated milk protein or lactose yield, but fat yield tended to decrease with GG and tended to increase with LG. Infusion of GG increased arterial plasma concentrations of glucose and insulin and decreased concentrations of ß-hydroxybutyrate (BHB), nonesterified fatty acids, long-chain fatty acids (LCFA), total AA, EAA, and group 2 AA. Infusion of LG increased arterial triacylglycerides (TAG) and LCFA but did not affect EAA concentrations. Compared with the LMP level, the HMP level increased arterial concentrations of BHB, urea, and all EAA groups and decreased the concentration of total non-EAA. Mammary plasma flow increased with GG and was not affected by LG or protein level. Uptake and clearance of total EAA and group 2 AA were affected or tended to be affected by GG × AA interactions, with their uptakes being lower and their clearances higher with GG, but only at the LMP level. Infusion of LG did not affect uptake or clearance of any AA group. The HMP level increased uptake and decreased clearance of all EAA groups and decreased non-EAA uptake. Infusion of GG tended to increase mammary glucose uptake, and tended to decrease BHB uptake only at the LMP level. Infusion of LG increased mammary uptake of TAG and LCFA and increased or tended to increase clearance of TAG and LCFA. We suspect GG increased mammary plasma flow to maintain intramammary energy and AA balance and stimulated lipogenesis in adipose, accounting for depressed arterial BHB and group 2 AA concentrations. Mammary glucose uptake did not cover estimated requirements for lactose and fat synthesis at the HMP level, except during HMP-GG infusion. Results of this study illustrate flexibility in mammary metabolite utilization when absorptive supply of glucogenic, lipogenic, and aminogenic substrate is increased.

Symposium review: Amino acid uptake by the mammary glands: Where does the control lie?

Milk protein yield responses to changes in the profile of essential amino acids absorbed by the gastrointestinal tract or circulating in blood plasma do not follow the classic limiting amino acid response, in part because of an ability of the mammary glands to modify their blood flow rate and net clearance of amino acids out of plasma. The hypothesis that mammary blood flow is locally regulated to maintain ATP balance accounts for observed changes in flow due to postruminal glucose, insulin, and essential amino acid (EAA) infusions. An additional hypothesis that net mammary uptakes of metabolites from blood are affected by perturbations in their respective arterial concentrations and the rate of mammary blood flow also appears to hold for the energy metabolites glucose, acetate, ß-hydroxybutyrate, and fatty acids. However, net EAA uptakes by the mammary glands are poorly predicted by models considering arterial concentrations and blood flow rates only. Evidence points to intramammary protein synthesis and secretion as the determinant of net EAA uptake. The intracellular signaling network anchored by the mechanistic target of rapamycin complex 1 stands as an excellent candidate to explain nutritional effects on milk protein synthesis because it integrates information on physiological and nutritional state to affect protein synthesis and cell metabolism, growth, proliferation, and differentiation in many cell types. In mammary cells in vitro and in vivo, the mechanistic target of rapamycin complex 1, integrated stress response, and glycogen synthase kinase-3 networks that contribute to regulation of initiation of mRNA translation are responsive to acute changes in nutrient supply and EAA profile. However, after several days of postruminal infusion of balanced and imbalanced EAA profiles, these signaling networks do not appear to continue to account for changes in milk protein yields. Gene expression evidence suggests that regulation of components of the unfolded protein response that control biogenesis of the endoplasmic reticulum and differentiation of a secretory phenotype may contribute to effects of nutrition on milk protein yield. Connections between early signaling events and their long-term consequences should be sought.

Maintenance of plasma branched-chain amino acid concentrations during glucose infusion directs essential amino acids to extra-mammary tissues in lactating dairy cows.

The objectives of this study were to investigate the effects of branched-chain AA (BCAA) supplementation when glucose is infused postruminally into lactating dairy cows consuming a diet low in crude protein (CP) and to test the hypothesis that low BCAA concentrations are responsible for the poor stimulation of milk protein yield by glucose. Twelve early-lactation Holstein cows were randomly assigned to 15% and 12% CP diets in a switchback design of 6-wk periods. Cows consuming the 12% CP diet received 96-h continuous jugular infusions of saline and 1 kg/d of glucose with 0, 75, or 150 g/d of BCAA in a Latin square sequence of treatments. Compared with saline, glucose infusion did not affect dry matter intake but increased milk yield by 2.2 kg/d and milk protein and lactose yields by 63 and 151 g/d, respectively. Mammary plasma flow increased 36% during glucose infusion compared with saline infusion, possibly because of a 31% decrease in total acetate plus ß-hydroxybutyrate concentrations. Circulating concentrations of total essential AA and BCAA decreased 19 and 31%, respectively, during infusion of glucose, yet net mammary uptakes of AA remained unchanged compared with saline infusion. The addition of 75 and 150 g/d of BCAA to glucose infusions increased arterial concentrations of BCAA to 106 and 149%, respectively, of the concentrations in saline-infused cows, but caused a decrease in concentrations of non-branched-chain essential AA in plasma, as well as their mammary uptakes and milk protein yields. Plasma urea concentration was not affected by BCAA infusion, indicating no change in catabolism of AA. The lack of mammary and catabolic effects leads us to suggest that BCAA exerted their effects on plasma concentrations of the other essential AA by stimulating utilization in skeletal muscle for protein accretion. Results indicate that the glucose effect on milk protein yield was not limited by low BCAA concentrations, and that a stimulation of extra-mammary use of non-branched-chain essential amino acids by BCAA led to a decrease in milk protein yield.

Milking time and risk of over-milking can be decreased with early teat cup removal based on udder quarter milk flow without loss in milk yield.

Increasing the milk flow rate at which milking is terminated can shorten milking time and increase milking efficiency. The effects on milk yield and composition have not been fully investigated when the take-off is set at the udder quarter level and independent of feeding during milking. The objective of this study was to investigate the effect of 3 take-off levels at the udder quarter level (0.06, 0.3, and 0.48 kg/min) applied with or without feeding during milking on milking time, milk yield, the degree of udder emptying, milk composition, and free fatty acids. In this study, 30 cows were allocated into 6 groups, balanced by lactation number, lactation stage, and milk yield, and subjected to a 3 × 2 factorial arrangement of treatments using a Latin square design. Treatments were applied for 1 wk each. This study demonstrated milking time could be reduced by applying up to a take-off level of 0.48 kg/min on udder quarter level without losing milk yield or compromising milk composition or udder health.

Addition of glycerol to lactating cow diets stimulates dry matter intake and milk protein yield to a greater extent than addition of corn grain.

The objective of this study was to determine if the addition of glycerol to the diet of dairy cows would stimulate milk protein yield in the same manner as the addition of corn grain. Twelve multiparous lactating dairy cows at 81 ± 5 d in milk were subjected to 3 dietary treatments in a replicated 3 × 3 Latin square design for 28-d periods. The diets were a 70% forage diet considered the basal diet, the basal diet with 19% ground and high-moisture corn replacing forages, and the basal diet with 15% refined glycerol and 4% added protein supplements to be isocaloric and isonitrogenous with the corn diet. Cows were milked twice a day and samples were collected on the last 7 d of each period for compositional analysis. Within each period, blood samples were collected on d 26 and 27, and mammary tissue was collected by biopsy on d 28 for Western blot analysis. Dry matter intake increased from 23.7 kg/d on the basal diet to 25.8 kg/d on the corn diet and 27.2 kg/d on the glycerol diet. Dry matter intake tended to be higher with glycerol than corn. Milk production increased from 39.2 kg/d on the basal diet to 43.8 kg/d on the corn diet and 44.2 kg/d on the glycerol diet. However, milk yield did not differ between corn and glycerol diets. Milk lactose yields were higher on the corn and glycerol diets than the basal diet. Milk fat yield significantly decreased on the glycerol diet compared with the basal diet and tended to decrease in comparison with the corn diet. Mean milk fat globule size was reduced by glycerol feeding. Milk protein yield increased 197 g/d with addition of corn to the basal diet and 263 g/d with addition of glycerol, and the glycerol effect was larger than the corn effect. The dietary treatments had no effects on plasma glucose concentration, but plasma acetate levels decreased 27% on the glycerol diet. Amino acid concentrations were not affected by dietary treatments, except for branched-chain amino acids, which decreased 22% on the glycerol diet compared with the corn diet. The decreases in plasma acetate and branched-chain amino acid concentrations with glycerol and the larger effects of glycerol than corn on milk protein and fat yields suggest that glycerol is more glucogenic for cows than corn grain.

Exogenous essential amino acids stimulate an adaptive unfolded protein response in the mammary glands of lactating cows.

The phosphorylation of mammalian target of rapamycin complex 1 (mTORC1) components and integrated stress response networks in the mammary glands of lactating cows have not accounted for the stimulation of milk protein yield by chronic supplementation with AA or glucose. Faster milk protein synthesis could be a consequence of increased milk protein mRNA per cell, the number of ribosomes per cell, the secretory capacity of cells, or the mammary cell number. To investigate these 4 possibilities using a translational and transcriptional approach, we performed protein and gene expression analyses of mammary and longissimus dorsi tissue collected from lactating dairy cows after 5 d of abomasal infusion with saline or 844 or 1,126 g/d of an essential AA (EAA) mixture, with and without 1,000 g/d glucose. Infusion with EAA increased milk protein yield but did not affect the phosphorylation of mTORC1-related proteins in the mammary gland. In skeletal muscle, phosphorylation of 4EBP1 (eIF4E-binding protein 1) increased in response to both EAA and glucose, and phosphorylated S6K1 (70-kDa ribosomal protein S6 kinase) increased with glucose. In response to EAA, mammary mRNA expression of the marker genes for milk proteins, ribosome biogenesis, and cell proliferation were not upregulated. Instead, reciprocal regulation of 2 arms of the unfolded protein response occurred. Infusion of EAA for 5 d activated XBP1 (X-box binding protein 1) mRNA, encoding a transcription factor for endoplasmic reticulum biogenesis, and it decreased the mRNA expression of genes encoding pro-apoptotic protein CHOP (C/EBP homologous protein) and downstream GADD34 (growth arrest and DNA damage-inducible 34). These findings implicate non-stress-related, adaptive capabilities of the unfolded protein response in the long-term nutritional regulation of milk protein yield in lactating dairy cows.

Biosynthesis of milk fat, protein, and lactose: roles of transcriptional and posttranscriptional regulation.

The demand for high-quality milk is increasing worldwide. The efficiency of milk synthesis can be improved by taking advantage of the accumulated knowledge of the transcriptional and posttranscriptional regulation of genes coding for proteins involved in the synthesis of fat, protein, and lactose in the mammary gland. Research in this area is relatively new, but data accumulated in the last 10 years provide a relatively clear picture. Milk fat synthesis appears to be regulated, at least in bovines, by an interactive network between SREBP1, PPARγ, and LXRα, with a potential role for other transcription factors, such as Spot14, ChREBP, and Sp1. Milk protein synthesis is highly regulated by insulin, amino acids, and amino acid transporters via transcriptional and posttranscriptional routes, with the insulin-mTOR pathway playing a central role. The transcriptional regulation of lactose synthesis is still poorly understood, but it is clear that glucose transporters play an important role. They can also cooperatively interact with amino acid transporters and the mTOR pathway. Recent data indicate the possibility of nutrigenomic interventions to increase milk fat synthesis by feeding long-chain fatty acids and milk protein synthesis by feeding amino acids. We propose a transcriptional network model to account for all available findings. This model encompasses a complex network of proteins that control milk synthesis with a cross talk between milk fat, protein, and lactose regulation, with mTOR functioning as a central hub.