Milk Citrate Research Articles

Effects of intramammary infusions of casein hydrolysate, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and lactose at drying-off on mammary gland involution

The transition from the lactation to the dry period in dairy cows is a period of high risk for acquiring new intramammary infections. This risk is reduced when involution of mammary glands is completed. Consequently, strategies that accelerate the involution process after drying-off could reduce the incidence of mastitis. The objective of this study was to assess the effect of 3 different treatments on mammary gland involution. Each quarter of 8 Holstein cows in late lactation was randomly assigned at drying-off to an intramammary infusion of casein hydrolysate (CNH; 70mg), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA; 5.7g), lactose (5.1g), or saline 0.9% (control) solutions. Milk samples were collected on the last 2 d before and 1, 3, 5, 7, 10, and 14 d after the last milking for determining concentrations of mammary gland involution markers. Lactoferrin, somatic cell counts (SCC), BSA, and Na+ concentrations, as well as matrix metalloproteinase-2 and matrix metalloproteinase-9 activities gradually increased in mammary secretions during the first 2 wk following the last milking, whereas milk citrate and K+ concentrations decreased. As involution advanced, the Na+:K+ ratio increased, whereas the citrate:lactoferrin ratio decreased. Compared with mammary secretions from control quarters, mammary secretions of quarters infused with CNH had higher SCC on d 1, 3, 5, and 7, and greater BSA concentrations on d 1, 3, and 5. Similarly, the CNH treatment induced a faster increase in lactoferrin concentrations, which were greater than in milk from control quarters on d 3, 5, and 7 after drying-off. Milk citrate concentrations were unaffected by CNH but the citrate:lactoferrin ratio was lower in CNH-treated quarters on d 3 and 5 than in control quarters. Moreover, CNH treatment hastened the increase in Na+ concentration and in the Na+:K+ ratio on d 1. Infusion of CNH also led to an increase in proteolytic activities, with greater matrix metalloproteinase 9 activities on d 1 and 3. The EGTA infusion increased SCC above that of control quarters on d 1 and 3 but it had no effect on the other parameters. Lactose infusion had no effect on any of the involution markers. In this study, intramammary infusions of CNH were the most efficient treatment to accelerate mammary gland involution, suggesting a potential role of CNH as a local milk secretion inhibitor during milk stasis.

RNA sequencing to study gene expression and single nucleotide polymorphism variation associated with citrate content in cow milk

The technological properties of milk have significant importance for the dairy industry. Citrate, a normal constituent of milk, forms one of the main buffer systems that regulate the equilibrium between Ca2+ and H+ ions. Higher-than-normal citrate content is associated with poor coagulation properties of milk. To identify the genes responsible for the variation of citrate content in milk in dairy cattle, the metabolic steps involved in citrate and fatty acid synthesis pathways in ruminant mammary tissue using RNA sequencing were studied. Genetic markers that could influence milk citrate content in Holstein cows were used in a marker-trait association study to establish the relationship between 74 single nucleotide polymorphisms (SNP) in 20 candidate genes and citrate content in 250 Holstein cows. This analysis revealed 6 SNP in key metabolic pathway genes [isocitrate dehydrogenase 1 (NADP+), soluble (IDH1); pyruvate dehydrogenase (lipoamide) β (PDHB); pyruvate kinase (PKM2); and solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1 (SLC25A1)] significantly associated with increased milk citrate content. The amount of the phenotypic variation explained by the 6 SNP ranged from 10.1 to 13.7%. Also, genotype-combination analysis revealed the highest phenotypic variation was explained combining IDH1_23211, PDHB_5562, and SLC25A1_4446 genotypes. This specific genotype combination explained 21.3% of the phenotypic variation. The largest citrate associated effect was in the 3′ untranslated region of the SLC25A1 gene, which is responsible for the transport of citrate across the mitochondrial inner membrane. This study provides an approach using RNA sequencing, metabolic pathway analysis, and association studies to identify genetic variation in functional target genes determining complex trait phenotypes.

Effect of prolactin-release inhibition on milk production and mammary gland involution at drying-off in cows

The end of each lactation is a challenging period for high-yielding cows as they are often dried off while still producing significant quantities of milk and, consequently, are highly susceptible to new intramammary infections. Once involution is complete, the mammary gland becomes much more resistant to infection. Therefore, it is critically important to develop strategies aimed at reducing milk production before drying-off and to accelerate mammary gland involution. This study assessed the effect of inhibition of the lactogenic signal driven by prolactin (PRL) on milk production and concentrations of involution markers in mammary secretions. Sixteen Holstein cows in late lactation were assigned to treatments based on milk yield, somatic cell count, and parity. Of those cows, 8 received twice-daily intramuscular injections (2mg per injection) of quinagolide, a specific inhibitor of PRL release, from 4d before drying-off to 3d after (Quin). The other 8 cows received injections of the solvent (water, control). Blood and milk (mammary secretion) samples were collected on the last 5d before and on d 1, 3, 5, 7, 10, and 14 after the last milking. Additionally, on the day preceding the first injection and on the following day, several blood samples were collected around milking time. Quinagolide reduced basal serum PRL concentrations on all injection days as well as PRL released in blood during milking. The PRL inhibitor decreased milk production before drying-off, which averaged, over the last 3d of lactation, 19.3 and 15.5kg/d for the control and Quin cows, respectively. Quinagolide had no significant effect on milk citrate:lactoferrin and Na:K ratios, which decreased and increased, respectively, during the first 2wk of the dry period. Nevertheless, the increases in the number of somatic cells and bovine serum albumin concentration during early involution were greater and matrix metalloproteinase-2 activity tended to be greater in mammary secretions of the Quin cows compared with the control cows. This experiment shows that inhibition of PRL release decreases milk production of cows in late lactation. Changes in the composition of mammary secretions suggest that this approach also hastens mammary gland involution.

Bacterial Stripe of Hog Millet Caused by Acidovorax avenae subsp. avenae, a New Disease in Korea

In July 2011, bacterial stripe was observed on a commercial field of hog millet (Panicum miliaceum L.) in Chuncheon, Korea, with a disease incidence of 37% in the field. Symptoms on leaves included reddish-brown, long, narrow stripes that varied in length and were sharply delineated by uninfected adjacent vascular bundles. Eleven bacterial isolates (BC3107, BC3214 to BC3223) were recovered on trypticase soy agar from lesions surface sterilized in 70% ethanol for 1 min. The isolates, all obtained from different plants, were gram negative, oxidase positive, aerobic rods with two to four flagella. The isolates produced circular, cream-colored, nonfluorescent, butyrous colonies with entire margins on King's B medium. Using the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), the isolates were identified as Acidovorax avenae subsp. avenae with Biolog similarity indices ranging from 0.52 to 0.72 after 24 hr. Characters for differentiating between Acidovorax spp. were tested according to Schaad et al. (2). The isolates were positive for gelatin liquefaction, nitrate reduction, lipase production, utilization of D-mannitol, sodium citrate, and alkaline in litmus milk. The isolates were negative for utilization of D-arabitol and did not amplify with PCR primer sets Aaaf5, Aaaf3/Aaar2, and Aacf2/Aacr2. Colonies were V-, V+, and V+ for utilization of D-fucose, maltose, and ethanol, respectively. Regions of the 16S rRNA (rrs) and the IGS were sequenced to aid in the identification of the isolates using reported PCR primer sets (1,4). A 1,426 bp fragment of the rrs region shared 100% similarity with all strains of A. avenae available in GenBank. Pathogenicity tests were separately performed for the 11 isolates in different greenhouses located in Suwon (National Academy of Agricultural Science), and Chuncheon (Gangwondo Agricultural Research and Extension Services) in Korea. Pathogenicity was confirmed by clip inoculation with sterilized scissors dipped into cell suspensions containing 105 CFU/ml on three 8-day-old leaves of hog millet (two plants per isolate), rice (Oryza sativa L. cv. Hopyeong), and sweet corn (Zea mays L. cv. Daehak) in a greenhouse maintained at 28 to 32°C and 90% relative humidity. The isolates induced similar symptoms as those originally observed on hog millet 5 days after inoculation. No symptoms were observed on the control plants (hog millet, rice, and sweet corn), which were clipped with scissors dipped in sterilized distilled water. The identity of bacteria reisolated from the stripes on inoculated leaves was confirmed by analyzing sequences of the 16S-23S rRNA intergenic spacer region (IGS) (1). On the basis of physiological, pathological, and sequence data, the isolates were identified as A. avenae subsp. avenae. To our knowledge, this is the first report of bacterial stripe of hog millet caused by A. avenae subsp. avenae in Korea. The spread of the bacterial disease is expected to have a significant economic impact on hog millet culture in the fields of Gangwon Province in Korea. Nucleotide sequence data reported are available under accession numbers JQ743877 to JQ743887 for rrs of BC 3207 and BC3214 to BC3223, and JQ743877 to JQ743887 for IGS of BC3207 and BC3214 to BC3223.

Metabolic and production profiles of dairy cows in response to decreased nutrient density to increase physiological imbalance at different stages of lactation

Physiological imbalance (PI) is a situation in which physiological parameters deviate from the normal, and cows consequently have an increased risk of developing production diseases and reduced production or reproduction. Our objectives were to (1) determine the effect of stage of lactation and milk yield on metabolic and production responses of cows during a nutrient restriction period to experimentally increase PI; (2) identify major metabolites that relate to degree of PI; and (3) identify potential biomarkers in milk for on-farm detection of PI throughout lactation. Forty-seven Holstein cows in early [n=14; 49±22 d in milk (DIM); parity=1.6±0.5], mid (n=15; 159±39 DIM; parity=1.5±0.5), and late (n=18; 273±3 DIM; parity=1.3±0.5) lactation were used. Prior to restriction, all cows were fed the same total mixed ration ad libitum. All cows were then nutrient restricted for 4 d by supplementing the ration with 60% wheat straw to induce PI. After restriction, cows returned to full feed. Daily milk yield was recorded and composite milk samples were analyzed for fat, protein, lactose, citrate, somatic cells, uric acid, alkaline phosphatase, β-hydroxybutyrate (BHBA), and milk urea nitrogen. Blood was collected daily and analyzed for metabolites: nonesterified fatty acids (NEFA), BHBA, glucose, plasma urea nitrogen, and insulin. The revised quantitative insulin sensitivity check index (RQUICKI) was calculated for each cow. Liver biopsies collected before and during restriction were analyzed for triglycerides, glycogen, phospholipids, glucose, and total lipid content. A generalized linear mixed model was used to determine the effect of stage of lactation on responses during restriction. Regression analyses were used to examine the effect of pre-restriction levels on changes during restriction. Similar decreases in milk yield among groups indicate that the capacity of individual responses is dependent on milk yield but the coping strategies used are dependent on stage of lactation. Milk yield was a better predictor of feed intake than DIM. Plasma glucose decreased for all cows, and cows in early lactation had increased plasma BHBA, whereas cows in later lactation had increased NEFA during restriction. Milk citrate had the greatest increase (58%) during restriction for all cows. Results reported here identified metabolites (i.e., glucose, NEFA, BHBA, cholesterol) as predictors of PI and identified milk citrate as a promising biomarker for PI on farm.

Comparison of effects of dietary coconut oil and animal fat blend on lactational performance of Holstein cows fed a high-starch diet

Dietary medium-chain fatty acids (C8:0 through C12:0) are researched for their potential to reduce enteric methane emissions and to increase N utilization efficiency in ruminants. We aimed to 1) compare coconut oil (CNO; ∼60% medium-chain fatty acids) with a source of long-chain fatty acids (animal fat blend; AFB) on lactational responses in a high-starch diet and 2) determine the effect of different dietary concentrations of CNO on dry matter intake (DMI). In experiment 1, the control diet (CTRL) contained (dry basis) 40% forage (71% corn silage, and alfalfa hay and haylage), 26% NDF, and 35% starch. Isonitrogenous treatment diets contained 5.0% of AFB (5%-AFB), CNO (5%-CNO), or a 1-to-1 mixture of AFB and CNO (5%-AFB-CNO) and 0.8% corn gluten meal in place of corn grain. Thirty-two multiparous dairy cows (201±46 d postpartum; 42.0±5.5 kg/d 3.5% fat-corrected milk yield) were adapted to CTRL, blocked by milk yield, and randomly assigned to 1 of 4 treatment diets for 21 d with samples and data collected from d 15 through 21. Treatment 5%-CNO decreased DMI markedly and precipitously and was discontinued after d 5. In wk 3, 5%-AFB and especially 5%-AFB-CNO lowered total-tract NDF digested vs. CTRL (2.6 vs. 1.8 vs. 3.1 kg/d, respectively), likely because fat treatments reduced DMI and 5%-AFB-CNO impaired total-tract NDF digestibility. Milk fat concentrations were 3.10% (CTRL), 2.51% (5%-AFB), and 1.97% (5%-AFB-CNO) and correlated negatively to concentrations of C18:2 trans-10,cis-12 in milk fat. Additionally, 5%-AFB and 5%-AFB-CNO tended to lower milk yield and decreased yields of solids-corrected milk and milk protein compared with CTRL. Fat treatments decreased milk lactose concentration, but increased milk citrate concentration. Moreover, cows fed 5%-AFB-CNO produced less solids-corrected milk than did cows fed 5%-AFB. In experiment 2, diets similar to CTRL contained 2.0, 3.0, or 4.0% CNO. Fifteen multiparous cows (219±42 d postpartum; 42.1±7.0kg milk yield; mean ± SD) were blocked by DMI and randomly assigned to 1 of 3 treatment diets for an 8-d evaluation. Dietary concentration of CNO affected DMI, with the greatest depression at 4.0% CNO. Overall, dietary CNO depressed DMI and NDF digestibility of a high-starch diet compared with AFB. Feeding CNO to lactating cows equal to or greater than 2.5% decreased lactational performance or DMI.

The use of the Fourier Transform Infrared spectroscopy to determine adulterants in raw milk

The objective of this study was to develop calibrations to determine the concentration of some milk adulterants by using the automated methodology of Fourier Transform Infrared (FTIR). For construction of calibrations, samples were collected from 100 farms in the states of Sao Paulo and Minas Gerais. Samples were tainted with three different adulterants commonly used in the adulteration of raw milk: sodium bicarbonate (SB), sodium citrate (SC) and cheese whey (W). Each adulterant was used at three different concentrations (SB: 0.05, 0.10 and 0.25%; SC: 0.025, 0.050 and 0.075% and W: 5, 10 and 20%). For validation, 60 samples were collected in other farms, which were not considered at the development stage of calibration. Adulterants were added at the following concentrations: 0.03, 0.06, 0.10 and 0.12% for SB; 0.02, 0.04, 0.06 and 0.08% for SC and 5, 10 and 20% for W. Performance of each calibration was evaluated in terms of accuracy (Se), detection limit (DL) and determination coefficient (R2). All calibrations presented R2 higher than 0.91 with DL of 0.015%; 0.017% and 3.9% for SB, SC and W, respectively. Accuracy was 0.005%, 0.009% and 2.26% for SB, SC and W, respectively. Results show that the FTIR methodology can be used for determining the concentration of sodium bicarbonate, sodium citrate and whey in raw milk. Associated with automated equipment, it is a viable option for monitoring these adulterants, having low operational costs and high analytical performance as additional features.

Concentrations of bovine lactoferrin and citrate in milk during experimental endotoxin mastitis in early-versuslate-lactating dairy cows

Lactoferrin (Lf) is a molecule naturally present in bovine milk that affects the availability and transport systems of iron. Lf also binds endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria and modulates the immunological response. In the present study, concentrations of bovine Lf (bLf) and citrate in milk were determined in early (EL) and late (LL) lactating dairy cows, using an experimentally induced endotoxin mastitis model and a crossover design. Nine clinically healthy Finnish Ayrshire cows were challenged twice with 100 μg endotoxin infused into one udder quarter. Milk samples were collected from the challenged and control quarters of each cow before and after endotoxin infusion during 3 d, and bLf and citrate concentrations were measured. In all cows, clinical signs of mastitis were seen at both times of challenge, but the response was more severe in EL than in LL. Concentration of bLf in the milk started to rise approximately 8 h after endotoxin infusion and was still higher than normal on the third day, especially in the late-lactating cows. In milk of the LL group, concentrations of bLf were significantly higher than in the EL group. In contrast, concentrations of citrate were higher in milk of the EL cows compared with the LL cows. Concentration of bLf and citrate varied substantially among cows. The molar ratio of citrate to bLf before and after challenge was significantly higher during the EL period. The results of this study partly explain why cows in early lactation are more susceptible to intramammary infections and why mastitis is more severe in them.

Effects of Cooling and Supplemental Bovine Somatotropin on Milk Production relating to Body Glucose Metabolism and Utilization of Glucose by the Mammary Gland in Crossbred Holstein Cattle

Problem statement: The low milk yield and shorter persistency of lactation of dairy cattle is the major problem for the dairy practices in the tropics. High environmental temperatures and rapid decline of plasma growth hormone level can influence milk production. Regulation of the milk yield of animals is mainly based on the mechanisms governing the quantity of glucose extracted by the mammary gland for lactose biosynthetic pathways. The mechanism(s) underlying the effects of cooling and supplemental bovine somatotropin on milk production relating to body glucose metabolism and intracellular metabolism of glucose in the mammary gland of crossbred Holstein cattle in the tropics have not been investigated to date. Approach: Ten crossbred 87.5% Holstein cows were divided into two groups of five animals each. Animals were housed in Normal Shade barn (NS) as non-cooled cows and cows in the second group were housed in barn which was equipped with a two Misty-Fan cooling system (MF) as cooled cows. Supplementation of recombinant bovine Somatotropin (rbST) (POSILAC, 500 mg per cow) were performed in both groups to study body glucose metabolism and the utilization of glucose in the mammary gland using a continuous infusion of [3-3H] glucose and [U- 14C] glucose as markers in early, mid and late stages of lactation. Results: Milk yield significantly increased in both groups during supplemental rbST with a high level of mammary blood flow. Body glucose turnover rates were not significant different between cooled and non-cooled cows whether supplemental rbST or not. The glucose taken up by the mammary gland of both non-cooled and cooled cows increased flux through the lactose synthesis and the pentose cycle pathway with significant increases in NADPH formation for fatty acid synthesis during rbST supplementation. The utilization of glucose carbon incorporation into milk appeared to increase in milk lactose and milk triacylglycerol but not for milk citrate during supplemental rbST in both non-cooled and cooled cows in early and mid lactation. Conclusion: The present study demonstrated that local changes for biosynthetic capacity within the mammary gland would be a factor in identification of the utilization of substrates in the rate of decline in milk yield. The proportion of glucose was metabolized less for lactose synthesis, but metabolized more via the Embden-Meyerhof pathway and the tricarboxylic acid cycle as lactation advanced to late lactation in both cooled and non-cooled cows whether supplemental rbST or not.

Effects of long‐term exogenous bovine somatotropin on glucose metabolism and the utilization of glucose by the mammary gland in different stages of lactation of crossbred Holstein cattle

ABSTRACTThe effects of long‐term administration of recombinant bovine somatotropin (rbST) on glucose turnover and the utilization of glucose in the mammary gland using a continuous infusion of [3‐3H]glucose and [U‐14C]glucose in lactating crossbred Holstein Friesian(HF) cattle were investigated. Glucose turnover of rbST‐treated animals was significantly higher than those of control animals (P < 0.05) in mid lactation, while plasma glucose concentrations were not affected. The utilization of glucose of non‐mammary tissues of rbST‐treated animals significantly increased (P < 0.05) as lactation advanced. The glucose taken up by the mammary gland in early lactation increased flux through the lactose synthesis and the pentose cycle pathway with significant increases in NADPH formation for fatty acid synthesis during rbST administration. The utilization of glucose carbon incorporation into milk appeared to increase in milk citrate and milk triacylglycerol but not for milk lactose as lactation advances in rbST‐treated animals. The stimulant effect for milk yield by rbST treatment was transiently and significantly increased in early lactation and was decreased in late lactation even though there was a high level of udder blood flow. These findings demonstrate that the regulation of biosynthetic capacity within the mammary gland would be influenced more by local than by systemic factors. The proportion of glucose would be metabolized less for lactose synthesis, but metabolized more via the Embden‐Meyerhof pathway and the tricarboxylic acid cycle as lactation advances.

Effects of long‐term administration of recombinant bovine somatotropin on the concentration of metabolites in milk in different stages of lactation in crossbred Holstein cattle

ABSTRACTEffects of long‐term treatment with recombinant bovine somatotropin (rbST) on concentrations of cellular metabolites in the milk of 87.5% crossbred Holstein cattle were performed. The peak milk yield of rbST‐treated animals was 22% higher (P < 0.05) than that of the control animals in early lactation. The mammary glucose uptake of rbST‐treated animals increased in early lactation, but decreased in mid and late lactation, while plasma glucose concentrations were not affected. Lactose and milk triacylglycerol secretion of rbST‐treated animals significantly increased (P < 0.05) when compared with those of control animals in early lactation. The concentrations of milk glucose of rbST‐treated animals significantly increased in early and mid‐lactation (P < 0.05). The concentrations of milk galactose markedly increased (P < 0.05) whereas the concentrations of milk uridine 5′‐diphosphoglucose (UDP‐glucose) and UDP‐galactose showed no significant changes as lactation advances in both groups. The concentrations of isocitrate, 2‐oxoglutarate and citrate in milk from both groups showed no significant changes throughout experiment. The concentration of glucose‐6‐phosphate (G6P), glucose‐1‐phosphate and cyclic adenosine 3,5′monophosphate in milk from both groups markedly decreased as lactation advances exception in early lactation of rbST‐treated animals, which G6P was not affected. These findings suggest that prolonged rbST treatment exerts its galactopoietic action at least in early lactation through both intramammary and extra‐mammary changes. Increases in the concentrations of glucose and G6P in milk maintained the level of pretreatment in early lactation associated with increases in milk yields during rbST administration, reflect their concentrations in the cytosol or Golgi vesicles of mammary cells, which would be one of the factors regulating intermediary metabolites in the lactose biosynthetic pathway.

Baroprotection of vegetative bacteria by milk constituents: A study of Listeria innocua

The baroprotective effect of milk constituents on Listeria innocua 4202 treated at 350 or 500 MPa for 5 min was examined. High-pressure (HP) treatment of L. innocua 4202 (1×10 9 cfu mL −1) resulted in complete inactivation in simulated milk ultra-filtrate (SMUF), a ∼5.0 log reduction in phosphate-buffered saline and a ∼2.9 log reduction in milk. The addition of micellar casein to SMUF increased survival of the bacterium by 3 logs, compared with SMUF alone, but the protective effect was negated if the minerals associated with the casein micelles were removed. The colloidal minerals calcium (30 m m), magnesium (5 m m), citrate (10 m m) and phosphate (20 m m), suspended in SMUF increased survival by ∼3.3, ∼1.7, ∼3.3 and ∼3.5 logs, respectively. The buffering capacity of the suspending medium was found to be a key factor in microbial baroresistance. Buffering by phosphate and citrate in milk may protect microorganisms against changes in pH during HP treatment, whereas the divalent cations calcium and magnesium may protect cell membranes against HP.

Variation of Milk Citrate with Stage of Lactation and De Novo Fatty Acid Synthesis in Dairy Cows

Citrate is a normal constituent of milk that affects milk-processing characteristics. It is an intermediate in the tricarboxylic acid cycle and plays an indirect role in fat synthesis by providing reducing equivalents in the form of NADPH. The objective of this study was to investigate variation in citrate with stage of lactation and de novo fatty acid synthesis, without confounding dietary effects. Twenty-four cows were fed the same diet, and milk citrate and fatty acids were determined over a 10-d period. Eight cows were in early lactation [13±1.8 d in milk (DIM; mean ±standard error], 8 in midlactation (130 ±4.6 DIM), and 8 in late lactation (283 ±3.4 DIM). For cows in early, mid, and late lactation, milk yield was 34.4, 34.4, and 21.4 L/d [standard error of difference (SED) 1.78]; milk fat was 50.4, 40.3, and 41.4g/L (3.68); milk citrate was 11.3, 9.7, and 10.1 mmol/L (0.64); the ratio of 4–14C:18–20C fatty acids was 0.9, 1.3, and 1.2 (0.07). Activity of the fatty acid synthase enzyme system (EC 2.3.1.85) was calculated as acetate used for chain elongation (ACE); ACE (mol/d) for cows in early, mid, and late lactation, was 7.3, 11.1, and 8.1 (SED 1.05). For individual cows, citrate (mmol/L) = 14.3 − 0.44 ×ACE (r2 = 0.58). We propose that ACE provides a more accurate indication of synthase activity than do fatty acid ratios or yields. This study confirms the hypothesis that variation in milk citrate with stage of lactation is related to de novo synthesis of fatty acids and that the relationship is independent of diet and milk yield.

Calcium Secretion into Milk

Ionized calcium ([Ca(2+)]) is present in milk at concentrations around 3 mM, a concentration that drives the formation of complexes with citrate, phosphate, and casein, thereby generating compounds that carry the major portion of calcium in milk. In humans and cows, where it has been studied, changes in milk calcium appear to be regulated by the amount of citrate and casein in milk rather than changes in [Ca(2+)]. Most or all of the calcium in milk is likely derived through exocytosis of secretory vesicles derived from the Golgi compartment where a calcium ATPase mediates transport from the cytoplasm. The identity of the transporters is not yet certain but gene expression for the plasma membrane calcium ATPase, PMCA2bw, and the secretory pathway calcium ATPase, SPCA, is highly upregulated during lactation. Currently nothing appears to be known about the mechanisms that mediate transport of calcium across the basolateral membrane of the alveolar cell.

Optimization and Validation of a Rapid Method to Determine Citrate and Inorganic Phosphate in Milk by Capillary Electrophoresis

Quantification of phosphate and citrate compounds is very important because their distribution between soluble and colloidal phases of milk and their interactions with milk proteins influence the stability and some functional properties of dairy products. The aim of this work was to optimize and validate a capillary electrophoresis method for the rapid determination of these compounds in milk. Various parameters affecting analysis have been optimized, including type, composition, and pH of the electrolyte, and sample extraction. Ethanol, acetonitrile, sulfuric acid, water at 50°C or at room temperature were tested as sample buffers (SB). Water at room temperature yielded the best overall results and was chosen for further validation. The extraction time was checked and could be shortened to less than 1min. Also, sample preparation was simplified to pipet 12μl of milk into 1ml of water containing 20ppm of tartaric acid as an internal standard. The linearity of the method was excellent (R2>0.999) with CV values of response factors <3%. The detection limits for phosphate and citrate were 5.1 and 2.4 nM, respectively. The accuracy of the method was calculated for each compound (103.2 and 100.3%). In addition, citrate and phosphate content of several commercial milk samples were analyzed by this method, and the results deviated less than 5% from values obtained when analyzing the samples by official methods. To study the versatility of the technique, other dairy products such as cream cheese, yogurt, or Cheddar cheese were analyzed and accuracy was similar to milk in all products tested. The procedure is rapid and offers a very fast and simple sample preparation. Once the sample has arrived at the laboratory, less than 5min (including handling, preparation, running, integration, and quantification) are necessary to determine the concentration of citric acid and inorganic phosphate. Because of the speed and accuracy of this method, it is promising as an analytical quantitative testing technique.

Relationship between concentration of citrate and ketone bodies in cow's milk.

The authors' hypothesis is that the members of the tricarboxylic acid cycle (TCA cycle) such as citrate decrease in association with increased ketone body formation. To prove this hypothesis the connection between ketone bodies and citrate formation of milk was studied. A fluorimetric method was used to determine citrate and a headspace sampling gas chromatographic (GC) method was developed for determination of ketone bodies. Under real conditions of milk sampling, transport and storage, preserved milk samples of 119 clinically healthy dairy cows obtained in the 48 hours after milking were investigated. A low level of acetoacetate (ACAC) was found in all samples. This fact can be explained by the spontaneous decarboxylation of acetoacetate during sample storage (previously decarboxylised acetoacetate = pdACAC) and, consequently, the majority of the amount of acetoacetate in the samples (AC + pdACAC) appeared in the measured acetone concentrations. Based on the measured acetone concentration of milk samples two groups were formed retrospectively: HA (high-acetone) group (n = 41) with an AC + pdACAC concentration of > 0.4 mmol/l and a LA (low-acetone) group (n = 78) with an AC + pdACAC level of < or = 0.4 mmol/l. In the milk of cows of Group HA a positive correlation (r = +0.623) and linear connection between acetone (AC + pdACAC) and beta-hydroxybutyrate (BOHB) levels was found [BOHB = 2.491 + 0.586 x (pdAC + ACAC)]. Furthermore, in this group a negative correlation between citrate and BOHB and AC + pdACAC was also established (r = -0.579). Focusing on the results of this group the authors found a significant drop of AC + pdACAC and citrate during the metabolically critical first 1-4 weeks of lactation. For this reason they suggest that simple, easy, automated methods (i.e. flow injection analysis, Fourier transformation infrared analysis) should be introduced for the simultaneous determination of acetone and citrate concentration in milk to make the evaluation of the energy status of high-producing dairy cows easier and more certain.

Milk Composition and Apparent Digestibilities of Dietary Fatty Acids in Lactating Dairy Cows Abomasally Infused with Cis or Trans Fatty Acids1,2,3

Fat supplementation of diets for dairy cows produces changes in nutrient supply and milk composition. The effect of abomasal infusion of either cis-C18:1 or trans-C18:1 fatty acid isomers on the digestibility of fatty acids and milk composition was determined in lactating dairy cows. Six multiparous midlactation Holstein cows were used and fed a control diet containing 50% forage and 50% concentrate. Treatments were (per day): no infusion, infusion of a 630-g fat mixture high in cis-C18:1 isomers, and infusion of a 623-g fat mixture high in trans-C18:1 isomers using two 3 × 3 Latin squares with 4-wk experimental periods. Fat infusion did not affect total dry matter intake and increased apparent digestibilities of total fatty acids. Apparent digestibilities of C18 fatty acids were directly related to the number of double bonds within isomers, and cis-C18:1 isomers were slightly more digestible than trans-C18:1 isomers. The lower yield of C12:0, C14:0, and C16:0 fatty acids in milk fat and higher milk citrate observed when cows were infused with trans-C18:1 suggests a depressed de novo milk fatty acid synthesis. Effects of trans infusion on milk fat were independent of ruminal fermentation, fatty acid apparent absorption, and fatty acid plasma concentrations. Lower milk protein yield in cows infused with fat may have been caused by a decrease in milk protein synthesis.

Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains.

Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.

Quantification of Gallium-67 Citrate in Breast Milk

A woman, 6 months postpartum and nursing her child, had a Ga-67 scintigram. A sample of breast milk obtained at day 2 revealed 2.8% of the injected dose (ID) per liter. The percent ID/L was compared with literature values; the highest concentration was in a report of galactorrhea. Similarity of the gallate ion to phosphate, as a possible explanation for accumulation in the breast, was pointed out. An earlier recommendation that a patient given Ga-67 citrate should discontinue breast feeding for at least 2 weeks was re-examined.

Milk Fat Yield and Composition During Abomasal infusion of Cis or Trans Octadecenoates In Holstein Cows

The role of trans-C18:1 fatty acids in milk fat depression was examined. Six rumen-cannulated Holstein cows were assigned to two Latin squares with 21-d periods. The common basal diet contained 40% forage and 60% concentrate. Treatments were the uninfused control, 750g/d of a mixture of cis fat (65% high oleic sunflower oil and 35% cocoa butter), and 750 g/d of a mixture of trans fat (93% shortening and 7% corn oil) infused into the abomasum via a tube that passed through the rumen cannula. Milk yield was similar among treatments. Milk fat percentage and yield were lower, and milk citrate concentration was higher, for the trans than the cis treatment. Changes in the fatty acid composition of milk were similar for the cis and trans treatments compared with the control except for trans-C18:1. The concentration of trans-C18:1 was greater for the cis and trans treatments than for the control and was greater for the trans than for the cis treatment. These data clearly demonstrated that infusion of trans-C18:1 fatty acids into the abomasum depressed milk fat percentage and yield. We speculate that reduced synthesis of fatty acids and reduced activity of acyl transferase in mammary tissue contributed to depressed milk fat percentage for the trans treatment.