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Milk replacers (MR) formulated to contain more lactose than whole milk could potentially reduce insulin sensitivity in dairy calves. This study evaluated how partially replacing lactose in MR with fat on a weight/weight basis affects glucose-insulin kinetics in neonatal dairy calves fed high quantities of MR. Thirty-four Holstein bull calves were blocked by dam parity (1.6 ± 0.3) and randomly assigned to 1 of 2 treatments (n = 17): a high-lactose MR (HL; 46.1% lactose, 18.0% crude fat, and 23.9% crude protein on a dry matter basis) or a high fat MR (HF; 39.9% lactose, 24.6% crude fat, and 24.0% crude protein on a dry matter basis). Calves were individually housed and fed pooled colostrum at 1.5 h and 12 h postnatal at 18 and 9% of metabolic body weight (BW0.75), respectively. From 24 h postnatal until the end of the study (d 7), calves were transitioned to MR (prepared at 150 g/L) at 18% of BW0.75 twice daily at 0700 and 1900 h. On d 4, an insulin-modified intravenous glucose tolerance test was conducted 8 h after the morning meal by intravenous infusion of glucose (0.54 g/kg of BW0.75, min 0) and insulin (3.9 µg/kg of BW0.75, min 20). During postprandial sampling on d 6, abomasal emptying was assessed by dosing acetaminophen (0.13 g/kg of BW0.75) with MR and measuring its appearance within plasma. Sequential blood samples were collected and analyzed for plasma glucose, insulin, and acetaminophen concentrations. Calves were killed on d 7 and liver and pancreatic tissues were collected for histomorphological analysis. Partially replacing lactose in MR with fat increased body weight gain (679.6 vs. 462.0 ± 69.68 g/d) and gain:metabolizable energy intake (186.6 vs. 120.5 ± 20.21 g of body weight/Mcal) compared with feeding HL MR. Furthermore, abomasal emptying rate was reduced in calves fed HF MR. Calculated variables based on postprandial glucose and insulin concentrations, such as maximum concentration or area under the curve, were or tended to be lower in HF than in HL calves. Postprandial insulin sensitivity tended to be reduced in HL compared with HF, although insulin sensitivity estimated from the insulin-modified intravenous glucose tolerance test was not different. Calves fed HF MR showed enhanced glucose-stimulated insulin secretion. In conclusion, partially replacing lactose in MR with fat resulted in smaller fluctuations in postprandial glucose and insulin concentrations and tended to increase postprandial but not fasting insulin sensitivity in neonatal dairy calves.

A variety of measures are currently used to evaluate feed efficiency in livestock, with some of the most popular being the linear regression of nutrient intake on the various energy sinks of the cohort being evaluated. The adoption of novel on-farm technologies has greatly increased the breadth and depth of data being collected on a daily basis, which creates the opportunity for the implementation of more advanced feed efficiency models. The objectives of the current study were to leverage data collected using high-throughput biometric technologies to evaluate changes of early-lactation feed efficiency on a daily basis and to develop a novel measure of feed efficiency based on the NRC net energy model. Daily records of nutrient intake, fat- and protein-corrected milk yield, body weight, and body condition score were used to model 7 different indices of feed efficiency (gross feed efficiency, return over feed costs, residual feed intake, residual net energy intake, and net energy efficiencies of lactation, maintenance, and body reserve flux) in primiparous Holstein heifers (n = 40) from 0 to 150 d in milk. We observed that feed efficiency was dynamic and fluctuated over the course of the experimental period regardless of the efficiency measure used, but most measures followed similar trends in changes of efficiency on a day-to-day basis. Of the measures evaluated, return over feed costs would be most suited for management purposes due to practicality and simplicity, whereas other measures, such as the efficiency of net energy flux to and from body reserves, present potential targets for inclusion in breeding goals. Net energy efficiency of lactation was estimated to have higher repeatability compared with residual feed or energy intake, which may reduce the time required to estimate whole-lactation efficiency. Because decreased net energy balance was strongly correlated with increased efficiency for all indices evaluated, future work investigating the associations of feed efficiency with the magnitude and duration of negative energy balance is warranted.

Estimating single nucleotide polymorphism (SNP) effects over time is essential to identify and validate candidate genes (or quantitative trait loci) associated with time-dependent variation of economically important traits and to better understand the underlying mechanisms of lactation biology. Therefore, in this study, we aimed to estimate time-dependent effects of SNP and identifying candidate genes associated with milk (MY), fat (FY), and protein (PY) yields, and somatic cell score (SCS) in the first 3 lactations of Canadian Ayrshire, Holstein, and Jersey breeds, as well as suggest their potential pattern of phenotypic effect over time. Random regression coefficients for the additive direct genetic effect were estimated for each animal using single-step genomic BLUP, based on 2 random regression models: one considering MY, FY, and PY in the first 3 lactations and the other considering SCS in the first 3 lactations. Thereafter, SNP solutions were obtained for random regression coefficients, which were used to estimate the SNP effects over time (from 5 to 305 d in lactation). The top 1% of SNP that showed a high magnitude of SNP effect in at least 1 d in lactation were selected as relevant SNP for further analyses of candidate genes, and clustered according to the trajectory of their SNP effects over time. The majority of SNP selected for MY, FY, and PY increased the magnitude of their effects over time, for all breeds. In contrast, for SCS, most selected SNP decreased the magnitude of their effects over time, especially for the Holstein and Jersey breeds. In general, we identified a different set of candidate genes for each breed, and similar genes were found across different lactations for the same trait in the same breed. For some of the candidate genes, the suggested pattern of phenotypic effect changed among lactations. Among the lactations, candidate genes (and their suggested phenotypic effect over time) identified for the second and third lactations were more similar to each other than for the first lactation. Well-known candidate genes with major effects on milk production traits presented different suggested patterns of phenotypic effect across breeds, traits, and lactations in which they were identified. The candidate genes identified in this study can be used as target genes in studies of gene expression.

We performed genome-wide association analyses for milk, fat, and protein yields and somatic cell score based on lactation stages in the first 3 parities of Canadian Ayrshire, Holstein, and Jersey cattle. The genome-wide association analyses were performed considering 3 different lactation stages for each trait and parity: from 5 to 95, from 96 to 215, and from 216 to 305 d in milk. Effects of single nucleotide polymorphisms (SNP) for each lactation stage, trait, parity, and breed were estimated by back-solving the direct breeding values estimated using the genomic best linear unbiased predictor and single-trait random regression test-day models containing only the fixed population average curve and the random genomic curves. To identify important genomic regions related to the analyzed lactation stages, traits, parities and breeds, moving windows (SNP-by-SNP) of 20 adjacent SNP explaining more than 0.30% of total genetic variance were selected for further analyses of candidate genes. A lower number of genomic windows with a relatively higher proportion of the explained genetic variance was found in the Holstein breed compared with the Ayrshire and Jersey breeds. Genomic regions associated with the analyzed traits were located on 12, 8, and 15 chromosomes for the Ayrshire, Holstein, and Jersey breeds, respectively. Especially for the Holstein breed, many of the identified candidate genes supported previous reports in the literature. However, well-known genes with major effects on milk production traits (e.g., diacylglycerol O-acyltransferase 1) showed contrasting results among lactation stages, traits, and parities of different breeds. Therefore, our results suggest evidence of differential sets of candidate genes underlying the phenotypic expression of the analyzed traits across breeds, parities, and lactation stages. Further functional studies are needed to validate our findings in independent populations.

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, β-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.

Elevated planes of nutrition in the preweaning period of dairy calf management can increase average daily gain, shorten age at puberty, and increase milk yield. In a previous study, 12 Holstein calves were fed 2 meals/d of 4 or 2 L milk replacer up to 7 wk of age. The objective of the current study was to estimate parameters of abomasal emptying and glucose-insulin dynamics in these calves by fitting a mechanistic model to postprandial appearances of plasma glucose, insulin, and the abomasal emptying marker acetaminophen measured at 4 and 7 wk of age. Higher intake of milk replacer resulted in longer bouts of abomasal emptying at a slower rate. Parameters of glucose and insulin dynamics were not affected by milk replacer intake. However, older calves had decreased insulin-stimulated glucose utilization indicating impaired insulin sensitivity, as well as increased pancreatic responsiveness. Neither of these effects were apparent from i.v. glucose tolerance tests on the calves and may have been related to postprandial gut hormone release. Effects of age on parameters of glucose-insulin dynamics were larger than effects of milk replacer intake. Conversely, effects of milk replacer intake on abomasal emptying were larger than effects of age.

Made available in DSpace on 2020-12-12T01:12:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-01 Essential AA (EAA), particularly leucine, isoleucine, methionine, and histidine, possess signaling properties for promoting cellular anabolic metabolism, whereas methionine, lysine, and histidine are considered also to be substrate limiting AA. The objective of this study was to evaluate production responses to supplementation of 2 AA groups in a 2 × 2 factorial design. Eight cows (99 ± 18 days in milk) were assigned to 4 jugular infusion treatments consisting of saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL, in a replicated 4 × 4 Latin square design. Periods were 18 d in length, comprising 8 d of rest followed by 10 d of jugular infusion. Daily infusion amounts were 21 g of methionine, 38 g of lysine, 20 g of histidine, 50 g of leucine, and 22 g of isoleucine. Cows were ad libitum fed a common diet consisting of 15.2% crude protein and 1.61 Mcal/kg NEL on a dry matter basis that was predicted to meet rumen degradable protein requirements but was 15% deficient in metabolizable protein. Milk and energy-corrected milk yields increased by 2.3 kg/d and 1.9 kg/d, respectively, with infused IL, and no change was observed for MKH. Milk protein concentration increased by 0.13 percentage units for MKH, whereas milk protein yield increased for both MKH and IL by 84 g/d and 64 g/d, respectively. The milk protein yield increase for MKH+IL was 145 g/d versus CON. Gross feed efficiency tended to increase with IL infusion, and N efficiency tended to increase with MKH infusion. Aggregate arterial EAA concentrations less Met, Lys, and His declined by 7.2% in response to MKH infusion. Arterial EAA less Ile and Leu also declined by 6.2% in response to IL infusion. Net total AA (TAA) and EAA uptake by the udder tended to increase in response to MKH infusion, whereas mammary blood flow increased in response to IL infusion, but TAA and EAA net uptakes were unaffected. Apparent udder affinity increased for TAA and EAA less Met, Lys, and His in response to MKH infusion, whereas affinity for EAA less Ile and Leu increased for IL infusion. Venous Met and Leu concentrations increased by 192% and 35% from the MKH and IL infusions, respectively, compared with CON, which indicates that intracellular concentration of these EAA changed substantially. Increases in milk protein yield were observed from 2 groups of amino acids independently and additively, which contradicts the single limiting amino acid theory that a single EAA will limit milk protein yield. Department of Dairy Science Virginia Tech Perdue AgriBusiness LLC UNESP Department of Animal Biosciences University of Guelph UNESP

The objective of this study was to investigate how preweaning plane of milk replacer intake and age can affect insulin and glucose kinetics as well as abomasal emptying rate in dairy calves fed twice a day. A total of 12 female Holstein Friesian calves were blocked by cow parity, paired by colostrum origin, and were randomly assigned to a high plane of milk replacer intake (8 L/d, 1.2kg of milk replacer/d; n=6) or a low plane of nutrition (4 L/d, 0.6kg of milk replacer/d; n=6). All calves received 4 L of colostrum over 2 meals (1 and 6h after birth) and were then directly transferred to their assigned feeding plans until they were stepped-down from milk by 50% during wk 7 and weaned on wk 8. Milk replacer (24% crude protein, 18% crude fat) was fed at 150g/L twice daily (0700 and 1700h) and all calves had ad libitum access to pelleted calf starter, chopped wheat straw, and water. Jugular catheters were placed in all calves at 4, 7, and 10wk of age. Then, postprandial response to plasma glucose, insulin, and acetaminophen (supplied with the meal) were determined to measure abomasal emptying. The next day, a glucose tolerance test was conducted by infusing glucose via the jugular catheter. At 4 and 7wk of age, the rate constant (%/h) for abomasal emptying of the meal was lower in high calves (0.21±0.02 in wk 4; 0.27±0.02 in wk 7) compared with low (0.34±0.02 in wk 4; 0.47±0.02 in wk 7). The postprandial plasma insulin area under the curve over 420min was greater in high calves (18,443±7,329; low=5,834±739 µU/mL) compared with low. We found no differences in glucose tolerance test kinetics between the high and low dairy calves at 4, 7, or 10wk of age. The findings from this study suggest that feeding dairy calves an elevated plane of nutrition in 2 meals of milk replacer per day does not decrease insulin sensitivity.

To test which, if any, of the major milk precursors can elicit a rapid change in the rate of mammary blood flow (MBF) and to define the time course and magnitude of such changes, 4 lactating cows were infused with glucose, amino acids, or triacylglycerol into the external iliac artery feeding one udder half while iliac plasma flow (IPF) was monitored continuously by dye dilution. Adenosine and saline were infused as positive and negative controls, respectively, and insulin was infused to characterize the response to a centrally produced anabolic hormone. To test the roles of cyclooxygenase, NO synthase and ATP-sensitive K (KATP) channels in nutrient-mediated changes in blood flow, their respective inhibitors-indomethacin, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and glibenclamide-were infused simultaneously with glucose. Each day, 1 infusate was given twice to each cow, over a 20-min period each time, separated by a 20-min washout period. In addition, each treatment protocol was administered on 2 separate days. A 73% increase in IPF during adenosine infusion showed that the mammary vasodilatory response was quadratic in time, with most changes occurring in the first 5min. Glucose infusion decreased IPF by 9% in a quadratic manner, most rapidly in the first 5min, indicating that a feedback mechanism of local blood flow control, likely through adenosine release, was operative in the mammary vasculature. Amino acid infusion increased IPF 9% in a linear manner, suggesting that mammary ATP utilization was stimulated more than ATP production. This could reflect a stimulation of protein synthesis. Triacylglycerol only tended to decrease IPF and insulin did not affect IPF. A lack of IPF response to glibenclamide indicates that KATP channels are not involved in MBF regulation. Indomethacin and L-NAME both depressed IPF. In the presence of indomethacin, glucose infusion caused a quadratic 9% increase in IPF. Indomethacin is an inhibitor of mitochondrial function, so the glucose-induced increase in IPF was interpreted as feedback on mammary adenosine release from an anabolic response to glucose. Because NO synthase was not inhibited during indomethacin infusion, the feedback system is postulated to act through endothelial NO synthase. In the presence of L-NAME, glucose infusion had no effect on IPF, indicating that endothelial cyclooxygenase is not involved in glucose-induced changes in MBF.

Removing His from a postruminal AA infusion decreases milk protein and increases milk fat content. Feather meal is an inexpensive protein source, high in rumen undegradable protein but low in His. The objective of our study was to investigate dietary feather meal as a method for creating a His deficiency or imbalance to alter milk composition. Four dietary treatments were fed for 4 wk each to 8 multiparous mid-lactation Holstein cows in a replicated 4 × 4 Latin square design. A standard-protein control diet (SP-C) was formulated to provide 3,100g/d of metabolizable protein (MP). Feather meal was added to the control diet either to replace the MP isonitrogenously (SP-FM) or to increase the MP supply to 3,484 g/d (HP-FM). As an isonitrogenous control for HP-FM, a high-protein diet (HP-C) was formulated with His-adequate protein sources to provide the same MP content as HP-FM. Dry matter intake tended to decrease when feather meal was fed. Predicted flows of digestible His, Met, and Lys, and plasma concentrations of these AA were reduced on both feather meal diets. Predicted flows of total digestible essential AA were not different between HP-FM and SP-C. We concluded that the DMI depression on HP-FM prevented an imbalance of excess AA over His, and created a deficiency of His, Met, and Lys compared with SP-C. Milk production decreased on the 2 feather meal treatments, partly explained by a tendency for DMI to decrease. Milk yield was lowest on SP-FM at 30.3 kg/d and highest on HP-C at 37.9 kg/d. Milk fat yield was not affected by diet but protein and lactose yields were both lower with feather meal. Protein yields were 860 and 998 g/d, whereas lactose yields were 1,384 and 1,561 g/d for SP-FM and HP-FM, respectively. This resulted in a higher fat content and lower protein percentage on FM diets. The ratio of solids-not-fat:fat in milk was lowest on SP-FM at 2.11 compared with 2.56 on SP-C. Adding feather meal to the diet by replacing MP isonitrogenously was more effective at lowering the solids-not-fat:fat ratio than increasing the MP content with an imbalanced protein source.

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.

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H2O2 with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H2O2 to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H2O2 to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H2O2. In conclusion, SeMet supplementation protects BMEC from H2O2-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.

The nutritional and endocrine factors affecting protein translation in the bovine mammary gland, and the molecular mechanisms mediating their effects, are not well understood. The objective of this study was to assess the role of the mammalian target of rapamycin (mTOR) signaling pathway in the regulation of mammary protein synthesis by nutrients and hormones. Mammary epithelial acini isolated from lactating dairy cows were treated in medium containing, alone or in combination, a mixture of AA or glucose and acetate (GA) as energy substrates, or a combination of the lactogenic hormones hydrocortisone, insulin, and prolactin (HIP). Changes in the rate of mammary protein synthesis and the phosphorylation state of components of the mTOR signaling pathway were measured. Mammary protein synthesis was 50% higher with increased availability of AA in medium. The presence of GA or treatment of mammary acini with HIP alone did not affect mammary protein synthesis. The stimulation of mammary protein synthesis by AA was enhanced by HIP treatment, but not by the presence of GA in medium. Treatment of mammary acini with HIP induced the phosphorylation of protein kinase B. This effect was augmented in the presence of either AA or GA in medium. The stimulation of mammary protein synthesis by AA and its enhancement by HIP were associated with increased phosphorylation of the mTOR substrates, p70 ribosomal protein S6 kinase-1, and eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1), and dissociation of 4E-BP1 from eIF4E. The results suggest that nutrients and hormones may modulate mammary protein synthesis through the mTOR signaling pathway.

This study examined the localization of cellular glutathione peroxidase (GPx1) and extracellular glutathione peroxidase (GPx3) in lactating mammary tissue and in primary cultures of bovine mammary epithelial cells (BMEC). The effect of selenium as selenomethionine (SeMet) on the growth and viability of BMEC and GPx protein expression and activity were also studied. Single mammary epithelial cells were recovered by serial collagenase/hyaluronidase digestion from lactating bovine mammary tissue and cultured in a low-serum collagen gel system enriched with lactogenic hormones and 0, 10, 20, or 50 nM SeMet. Positive immunostaining with anti-cytokeratin and bovine anti-casein confirmed the epithelial nature and differentiated state of BMEC. Addition of SeMet to media facilitated rapid confluence of BMEC and formation of dome structures. Immunohistochemical and immunocytochemical staining revealed that both GPx1 and GPx3 are synthesized by BMEC and localized in the cytoplasm and nucleus. Up to 50 nM SeMet linearly increased BMEC number and viability over 5 d of culture. Bovine mammary epithelial cells cultured in SeMet-supplemented medium also exhibited markedly elevated GPx activity and linear increases in abundance of GPx1 and GPx3 proteins. It is apparent that SeMet degradation to release Se for synthesis of selenoproteins is carried out by BMEC. Results indicate that bovine mammary epithelial cells express GPx1 and GPx3 in vivo and in vitro; SeMet enhances expression of these selenoproteins in vitro and the growth and viability of BMEC.

The objective of this study was to determine the effects of supplementing glycerol and soybean oil in drinking water on feed and water intake, calculated energy balance, and production performance of periparturient dairy cows. Ninety multiparous Holstein dairy cows were randomly assigned to 1 of 3 treatments: 1) no nutrients supplemented in the drinking water (control); 2) 20 g/L of glycerin supplemented in the drinking water (glycerol); and 3) 10 g/L of soybean oil supplemented in the drinking water (SBO). The trial lasted from 7 d prepartum to 7 d postpartum. Cows were offered a close-up and milking cow TMR for ad libitum intake, pre- and postpartum, respectively. The dry matter intake of cows supplemented with glycerol and SBO was lower than for the control cows throughout the experimental period but not different from each other. Water intake for the control cows was greater than the average for the glycerol and SBO cows prepartum, and greater than for SBO cows but similar to that of glycerol cows postpartum. Glycerol cows consumed more water than SBO cows. There were no differences in energy intake and energy balance of the cows pre- and postpartum. Serum triacylglycerol concentration for glycerol cows was lower than for the control and SBO cows prepartum and was lower than for the SBO cows postpartum. There were no differences in the serum nonesterified fatty acids and glucose concentrations throughout the experiment. There were no differences in the serum beta-hydroxybutyrate (BHBA) concentrations at parturition, but serum BHBA concentration of the glycerol cows was greater than for control and SBO cows during the prepartum period. However, during the postpartum period, serum BHBA concentrations of the control cows were greater than for glycerol and SBO cows. There were no differences in calf birth weights or milk yield and composition. Although the glucogenic property of glycerol supplemented in the drinking water at 20 g/L may not have been sufficient to elicit a milk yield response, it did reduce the concentration of BHBA postpartum.

Experiments were conducted to investigate the use of added flavor in drinking water of Holstein calves and lactating dairy cattle to determine effects on dry feed intake. Nine calves were used in a replicated 3 x 3 Latin square design, and water offered was unflavored or flavored with orange or vanilla. All calves were offered commercial starter. Feed intake of the dry starter was increased in calves offered the orange flavor treatment compared with the control or the vanilla treatment. The increased dry feed intake agreed with the significant increase in weight gain measured in calves on the orange treatment. Further experiments were performed with 4 second-lactation cows using the addition of another orange flavor to the water compared with unflavored water under conditions of free access or time-restricted water access. No significant changes were found for dry matter intake, water consumption, or milk yield. These findings demonstrate an important finding that flavoring agents need not be added only to the starter feed for calves, but flavor can stimulate dry feed intake and BW gain when used in drinking water.

To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5 d with saline, 844 or 1,126 g/d of a complete essential AA mix, with and without the inclusion of 1,000 g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1 kg/d, milk protein by 256 g/d, milk fat by 95 g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

Deficiencies and imbalances of specific group II essential amino acids (EAA) were created in lactating cows by an infusion subtraction protocol to explore effects on milk production and abundance and phosphorylation state of regulators of mRNA translation in the mammary glands. Five lactating cows on a diet of 11.2% crude protein were infused abomasally for 5d with saline, 563 g/d of a complete EAA mix, or EAA mixes without the branched-chain amino acids (BCAA), Leu, or Lys in a 5 × 5 Latin square design. Milk protein yield was stimulated by EAA infusion and returned to saline levels upon subtraction of BCAA, Leu, or Lys. Mammary abundance of phosphorylated S6K1 was measured as an indicator of mammalian target of rapamycin complex 1 (mTORC1) activity and was found not to be affected by the complete EAA mix but was increased by the mixture lacking Lys. Total S6K1 abundances in mammary tissue were elevated by complete and BCAA-lacking infusions. All of the EAA treatments except the one lacking BCAA upregulated mammary eIF2Be and eIF2α abundances, which is stimulatory to global mRNA translation. Phosphorylation state of eIF2Be tended to decrease when complete or Lys-lacking EAA mixtures were infused. Phosphorylation state of eIF2α was not affected by treatment. We detected a correlation of 0.62 between phosphorylation state of S6K1 and total eIF2Be abundance, and a correlation of 0.58 between phosphorylation state of S6K1 and total eIF2α abundance, suggesting that mTORC1 activation may have upregulated eIF2Be and eIF2α expression. Despite maintenance of mammary eIF2Be and eIF2α abundances during Leu and Lys deficiencies, milk protein yield declined, suggesting that other factors are responsible for mediating effects of Lys and Leu. A deficiency of all 3 BCAA may impair milk protein yield through deactivation of mTORC1-mediated upregulation of eIF2Be and eIF2α abundances.

Previous work shows that mammary uptake of milk precursors from blood can be affected by the rate of blood flow (F) to the glands. The purpose of the current work was to test the ability of compartmental and cylindrical capillary models to account for the variation in mammary extraction and net uptake of plasma metabolites produced by perturbation of mammary F. The data for model fitting were obtained from a previous experiment in which mammary arteriovenous differences of acetate + β-hydroxybutyrate (2C), glucose, triacylglycerol (TAG), and long-chain fatty acids (LCFA) were measured in 4 cows before, during, and after intraarterial infusion of inhibitors of endothelial nitric oxide synthase and cyclooxygenase, which are 2 major systems of F control in the mammary glands. The 4 models tested were (1) constant extraction within each cow, (2) clearance from an extracellular compartment is a linear function of F with an intercept, (3) total capillary volume in a cylindrical representation is a linear function of F with an intercept, and (4) uptake from an extracellular compartment obeys Henri-Michaelis-Menten kinetics, where maximum velocity (Vmax) is a linear function of F with an intercept. According to prediction errors, model 4 fitted 2C extraction data best, accounting for 82% of the observed variation. The estimated Km (Henri-Michaelis-Menten constant) for venous 2C was 0.4 mM. For glucose clearance, a variant of model 2 with a positive effect of 2C uptake on clearance was identified as best, producing a coefficient of determination (R(2)) of 0.31. For TAG, model 2 with a positive effect of arterial TAG concentration on TAG clearance was best, with an R(2) of 0.22. For LCFA, model 2 with a positive effect of arterial LCFA on LCFA clearance was best, with an R(2) of 0.29. Models 2 and 3 fitted the extraction data with the same R(2)-values and prediction errors, so both compartmental and cylindrical approaches to describing the vascular bed were equally capable of describing the effect of F on mammary uptakes. A combined fit of all best-fit models to extraction data for all 4 metabolites at once explained 52, 42, 73, and 77% of variation in net uptakes of 2C, glucose, TAG, and LCFA, respectively. According to the fitted model, each 1 L/min increase in F increased the mammary volumes of distribution of 2C, glucose, TAG, and LCFA by 13, 14, 18, and 7%, respectively.

The objectives of this research were to determine the flux control coefficients for acetyl CoA carboxylase and fatty acid synthase using an in vitro preparation of bovine mammary homogenate. For an enzyme to be considered rate limiting with the use of metabolic control analysis, its control coefficient would be equal to unity. The hypothesis for this experiment was that the control coefficient for acetyl CoA carboxylase was not equal to unity, and that this enzyme was not, therefore, the rate-limiting step. Mammary tissue was isolated from lactating Holstein cows at slaughter and frozen in liquid nitrogen. Tissue was ground, homogenized, and centrifuged to obtain a postmitochondrial supernatant for use in in vitro incubations containing labeled acetate. Specific inhibitors for acetyl CoA carboxylase and fatty acid synthase were used to fractionally inhibit de novo synthesis for the calculation of flux control coefficients. The composition of fatty acids synthesized in the absence of enzyme inhibitors was similar to the composition of fatty acids in the presence of inhibitors. Calculations following avidin inhibition of acetyl CoA carboxylase determined the flux control coefficient was 0.63 +/- 0.15, which means that 63% of the control of fatty acid synthesis is exerted by acetyl CoA carboxylase. The remaining control (37%) was from fatty acid synthase, which indicates a significant degree of control over the flux of acetate in de novo synthesis resides with this enzyme. The rate-limiting status ascribed to acetyl CoA carboxylase was not supported, because the flux control coefficient was less than unity. Metabolic control analysis, through its use of pathway product measurements, allows for potential interactions in the pathway such as feedback inhibition contribution to the flux control coefficients, which would not otherwise be considered in studies measuring enzyme kinetics with purified enzymes.

Lactating cows were exposed to large amino acid imbalances and deficiencies by i.v. infusion to characterize responses in milk production and plasma concentrations of metabolites and hormones. Six cows in early lactation were fed a basal diet of 9% CP and infused continuously for 6 d with saline (negative control), 1.1 kg/d of a complete amino acid mix (positive control), or the equivalent mix lacking Met, Lys, His, or all 3 branched-chain amino acids. All cows received all treatments in 6 successive periods in a Latin square design. Infusion of the complete amino acid mix resulted in an increase in the plasma concentrations of several essential amino acids, insulin, and glucagon. Milk protein production was stimulated by 19%, which accounted for 10% of the infused amino acid. Plasma urea, acetate, and beta-hydroxybutyrate concentrations were increased. Compared with saline, the amino acid mixtures lacking Met, Lys, or His increased essential amino acids, glucose, insulin, and glucagon concentrations in plasma, and decreased growth hormone. Plasma concentration of the essential amino acid absent from the infusate fell 2-fold but milk protein yield remained within 12% of its basal value. Dry matter intakes were depressed 35% over the first 2 d of infusion of imbalanced mixtures but recovered thereafter. Milk fat yields were increased 258 and 320 g/d by mixtures devoid of Lys and His, respectively. Correction of a Met, Lys, or His deficiency did not affect hormone concentrations in plasma and milk protein yield increased 27% due entirely to increased concentration of the single amino acid in plasma. Although imbalance and deficiency generated similar amino acid profiles in plasma, it was concluded that endocrine responses to total amino acid supply during imbalance can override imperfections in the circulating amino acid profile to maintain milk protein yield at higher levels than expected from deficiency states. Both imbalance and deficiency were characterized by a low protein:fat ratio in milk. Infusion of a mix of amino acids lacking Val, Ile, and Leu, despite a decrease in plasma Leu to 58% of its basal value, increased milk protein and fat yields to the same extent as the complete amino acid mix.

In the dynamic modeling of dairy cow performance over a full lactation, the difference between net energy intake and net energy used for maintenance, growth, and output in milk accumulates in body reserves. A simple dynamic model of net energy balance was constructed to select, out of some common dry matter intake (DMI) prediction equations, the one that resulted in a minimum cumulative bias in body energy deposition. Dry matter intake was predicted using the Cornell Net Carbohydrate and Protein System, Agricultural Research Council, or National Research Council (NRC) DMI equations from body weight (BW) and predicted fat-corrected milk yield. The instantaneous BW of cows at progressive weeks of lactation was simulated as the numerical integral of the BW change obtained from the predicted net energy balance. Predicted DMI and BW from each DMI equation, using either of 2 equations to describe maintenance energy expenditures, were compared statistically against observed data from 21 herd average published full lactation data sets. All DMI equations underpredicted BW and DMI, but the NRC DMI equation resulted in the minimum cumulative error in predicted BW and DMI. As a general solution to prevent predicted BW from deviating substantially over time from the observed BW, a lipostatic feedback mechanism was integrated into the NRC DMI equation as a 2-parameter linear function of the relative size of simulated body reserves and week of lactation. Residual sum of squares was reduced on average by 52% for BW predictions and by 41% for DMI predictions by inclusion of the negative feedback with parameters taken from the average of all 21 least squares fits. Similarly, root mean square prediction error (%) was reduced by 30% on average for BW predictions and by 23% for DMI predictions. Inclusion of a feedback of energy reserves onto predicted DMI, simulating lipostatic regulation of BW, solved the problem of final BW deviation within a dynamic model and improved its DMI prediction to a satisfactory level.

Equations that predict daily dry matter intake (DMI) of a lactating cow could be evaluated by comparing the predicted accumulation of energy in body weight (BW) over the course of lactation with the observed BW evolution. However, to do so requires that first the energy balance calculations from observed DMI are evaluated. The purpose of the work reported here was to determine the degree of deviation of predicted from observed BW, according to net energy for lactation (NE(L)) balance calculated from weekly observations of DMI, BW, and fat-corrected milk production in 21 sets of full-lactation data, and to determine an appropriate correction of the NE(L) bias for subsequent DMI prediction evaluations. When the National Research Council maintenance equation 0.08 x BW(kg)(0.75) was used in energy balance calculation, BW was overpredicted with an increasing difference between the cumulative predicted BW and observed BW as lactation progressed. Placing all the error of BW prediction into maintenance energy expenditures resulted in a best-fit equation of 0.096 +/- 0.003 Mcal/kg of BW(0.75). A time-dependent equation was also developed, in which weekly maintenance expenditures were determined as the NE(L) expenditure to yield a zero NE(L) balance and could be described by a second-order polynomial equation related to week of lactation (WOL) where maintenance NE(L) = [-0.0227(+/- 0.0098) x WOL2 + 1.352(+/- 0.456) x WOL + 78.09(+/- 4.92) Mcal/kg of BW(0.75)] x 10(-3). Average maintenance energy expenditure at the onset of lactation was approximately 0.08 Mcal/kg of BW(0.75), and this value increased to a plateau at wk 15 of lactation of approximately 0.098 Mcal/kg of BW(0.75). Standard deviations between data sets of weekly maintenance parameter estimates throughout lactation were large but consistent at approximately 25% of the mean. Revision of the maintenance energy expenditure estimate substantially improved BW prediction by the energy balance model. On average, the 0.096 Mcal of NE(L)/kg of BW(0.75) equation resulted in the best BW predictions, although substantial variation existed around this value.

Essential amino acid (EAA) deficiencies and imbalances were created in lactating cows by using an infusion subtraction protocol to explore effects on milk protein yield and activity state of regulators of mRNA translation in the mammary glands. Six lactating cows on a diet of 11.2% protein were infused abomasally for 5 d with saline, 563 g/d of a complete EAA mix, or EAA without His, Met, Phe, or Trp in a 6 × 6 Latin square design. Infusion of complete and imbalanced EAA solutions increased mammalian target of rapamycin (mTOR) signaling in the mammary glands, as evidenced by higher ribosomal S6 kinase 1 (S6K1) phosphorylation compared with saline infusion. Total S6K1 abundance was decreased by imbalanced AA infusions. Except for the mixture lacking Phe, infusion of EAA, whether imbalanced or not, increased abundance of total eukaryotic initiation factor 2Be (eIF2Be). A correlation of 0.33 between phosphorylation state of S6K1 and total eIF2Be abundance suggests that an mTOR-mediated upregulation of eIF2Be translation occurred. Despite increased mTOR/eIF2Be signaling, milk protein yields increased only with the complete EAA mixture compared with saline. Low plasma concentrations of His, Met, and Phe during their respective imbalances likely interfered with protein synthesis. Total abundance and phosphorylation state of eukaryotic initiation factor 2α were not responsible for the interference. Further study of eIF2Be as a regulator of milk protein yield is warranted.

To test the effect of mammary blood flow on net uptakes of milk precursors by the mammary glands, inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) were infused into the mammary circulation of 4 lactating cows. Inhibitors were infused in a 4×4 Latin square design, where treatments were infusion for 1 h of saline, NOS inhibitor (N ω -nitro-l-arginine methyl ester hydrochloride), COX inhibitor (indomethacin), or both NOS + COX inhibitors into one external iliac artery. Para -aminohippuric acid was also infused to allow for estimation of iliac plasma flow (IPF), of which approximately 80% flows to the mammary glands. Blood samples were collected before, during, and after inhibitor infusion from the contralateral external iliac artery and ipsilateral mammary vein. Inhibition of COX and NOS each produced a decrease in IPF, although the NOS effect was smaller and IPF continued to be depressed throughout the recovery period. The combination of COX and NOS inhibition produced a 50% depression in IPF and there was no carryover into the recovery period. Treatments that depressed IPF also increased arterial concentrations of acetate, β-hydroxybutyrate (BHBA), and glucose. Similarly, arteriovenous differences of acetate, BHBA, and glucose were all increased during IPF depression. To correct for a potential effect of arterial concentration, arteriovenous differences were normalized to arterial concentration, producing an extraction percentage. Inhibition of COX increased glucose extraction and tended to increase acetate and BHBA extraction. Dual inhibition only increased BHBA extraction and had no effect on mammary extraction of other metabolites. These extractions did not increase because clearances of glucose and TAG decreased as IPF decreased, and clearances of acetate and BHBA tended to decrease. Net uptake of TAG was depressed by dual NOS/COX inhibition, whereas uptakes of acetate, BHBA, and glucose were not affected by any of the treatments. To separate effects of flow from effects of arterial concentration, uptakes were regressed against IPF and arterial concentration simultaneously. According to the slopes of the regressions, a 10% decrease in IPF from the mean observed during saline infusion resulted in 3.8, 7.3, and 10.4% decreases in uptakes of acetate, glucose, and triacylglycerol, respectively. These findings indicate that mammary blood flow affects milk precursor uptake, and that clearance should not be assumed constant to predict mammary uptakes of milk precursors in situations where blood flow is changing.