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 41.4 g/L (3.68); milk citrate was 11.3, 9.7, and 10.1 mmol/L (0.64); the ratio of 4-14 C:18-20 C 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 xACE (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.

Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts.

Investigations into the development of a prototype electrochemical immunosensor for estradiol (E(2)) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E(2)) were immobilised by passive adsorption onto the surface of screen-printed carbon electrodes (SPCEs). A competitive immunoassay was then performed using an alkaline-phosphatase (ALP)-labelled E(2) conjugate. Calibration plots for E(2) buffer standards, performed colorimetrically on the SPCEs using a para-nitrophenyl phosphate substrate solution, were in good agreement with ELISA calibration plots. Electrochemical measurements were then performed using differential pulse voltammetry (DPV) following the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current versus E(2) concentration showed a measurable range of 25-500 pg/ml with a detection limit of 50 pg/ml. A coefficient of variation of between 13.0 and 15.6% was obtained for repeat measurements. The immunosensor was applied to the determination of E(2) in spiked serum, following an extraction step with diethyl ether. A mean recovery for the method of 102.5% was obtained with a CV of 19.1%. The options available for further development of the sensor regarding precision, limit of detection and direct sample analysis are discussed.

Quantitative analysis of the response of an electrochemical biosensor for progesterone in milk.

An electrochemical biosensor for progesterone in cow's milk was developed and used in a competitive immunoassay by Hart et al. (1977, Studies towards a disposable screenprinted amperometric biosensor for progesterone, Biosens. Bioelectron. 12, 1113-1121). The sensor was fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs) which were coated with rabbit anti-sheep IgG (rIgG). This sensor was operated following the steps of competitive binding between sample and conjugate (alkaline-phosphatase-labelled progesterone) for the immobilised mAb sites and measurements of an amperometric signal in the presence of p-nitrophenylphosphate using either colorimetric assays or cyclic voltammetry. The hook effect of the progesterone biosensor was found in the concentration range of milk progesterone between 0 and 5 ng/ml when the sensor was fabricated using a loading of 25 ng rIgG per electrode of a diameter of 3 mm and a 1/50 dilution of mAb. A computer model has been developed in this study to simulate the operation of this progesterone biosensor with consideration of the fabrication processes. This paper presents the results of validating the computer model and the model has predicted the hook effect as observed in tests. The model thus reveals that the hook effect is determined by the total number of binding sites available and the rates of labelled and unlabelled progesterone diffusing towards the sensor surface and the binding rates.

An electrochemical immunosensor for milk progesterone using a continuous flow system.

An electrochemical biosensor for cow's milk progesterone has been developed and used in a competitive immunoassay under thin-layer, continuous-flow conditions. Single-use biosensors were fabricated by depositing anti-progesterone monoclonal antibody (mAb) onto screen-printed carbon electrodes (SPCEs). Three operational steps could be identified: (1) Competitive binding of sample/conjugate (alkaline-phosphatase-labelled progesterone, AP-prog) mixture, (2) establishment of a steady-state amperometric baseline current and (3), measurement of an amperometric signal in the presence of enzyme substrate (1-naphthyl phosphate, 1-NP). In the thin-layer cell, the enzyme product, 1-naphthol, showed electrochemical behaviour consistent with bulk conditions and gave a linear amperometric response under continuous-flow conditions (E(app)=+0.3 V vs. Ag/AgCl) over the range 0.1-1.0 microg/ml. After pre-incubating biosensors with progesterone standards, signal generation within the cell (substrate concentration=5 mM) was recorded amperometrically as rate (nA/s) or maximum current (i(max), nA). Response values for milk standards were approximately 50% of those prepared in buffer. In both cases, calibration plots over the range 0-50 ng/ml progesterone were obtained. By conducting sample binding under flowing conditions, only 7% of the previous response was obtained, even at a substrate concentration of 50 mM, resulting in low signal:noise ratio. Using a stop-flow arrangement (i.e. quiescent sample binding, followed by continuous flow), low-noise amperograms were obtained at [1-NP]=5 mM. Calibration plots were obtained over the range 0-25 ng/ml, with a coefficient of variation of 12.5% for five replicate real milk samples.

Assessment of rumen processes by selected-ion-flow-tube mass spectrometric analysis of rumen gases.

This work investigated the potential to use measurement of the concentration of certain gases in the rumen headspace to gain information about rumen processes and as a potential diagnostic tool. We used new equipment (selected-ion-flow-tube mass spectrometer) that allows rapid and precise analysis of many of the gases present in a sample. Samples of rumen headspace gas and corresponding samples of rumen liquor were taken from three lactating cows, prepared with rumen fistulae, at intervals after receiving their morning feed allocation (grass silage and concentrates). Hydrogen sulfide, methyl sulfide, and dimethyl sulfide, were the predominant gases that were measured in the rumen headspace by this technique. The concentrations of these sulfur compounds declined over the interval after feeding, mirroring ammonia concentrations measured in rumen liquor, reflecting their common dependence on the fermentation of sulfur amino acids. Ammonia concentrations in rumen headspace gas varied in the opposite direction to the concentration of ammonia in rumen liquor and likely depend more on the pH of rumen liquor. Consideration of the pKa of ammonia suggests that ammonia concentrations in rumen gas will be very low below pH 6, representing a useful diagnostic for subacute ruminal acidosis. Low concentrations of volatile fatty acids were detected in rumen gas. The molar proportions of volatile fatty acids were similar in gas and liquor samples, with rumen gas containing slightly less acetic acid and disproportionately more valeric and caproic acids.

An immunosensor with potential for the detection of viral antigens in body fluids, based on surface second harmonic generation.

Field methods of assessing the immune status of animals are required to optimise vaccination programmes to control bovine viral diarrhoea (BVD) virus. An optoelectronic immunosensor was evaluated for the detection of viral antigens in a crude cell lysate in a pilot study. Binding of (BVD) virus antigen by two monoclonal antibodies immobilised on two different media (ELISA plate wells, and glass coverslips) was detected and quantified using the laser induced surface second harmonic generation (SSHG) technique. The results for both assays were correlated with an enzyme-linked immunoassay (ELISA) used for the diagnosis of BVD virus infection in cattle (ELISA plate; R(2)=0.86, coverslips; Exp. 1; R(2)=0.75, Exp. 2; R(2)=0.67). The method will allow rapid detection of antigens in the body fluids of farm animals.

An assay for the enzyme N-acetyl-beta-D-glucosaminidase (NAGase) based on electrochemical detection using screen-printed carbon electrodes (SPCEs).

An electrochemical assay for the enzyme N-acetyl-beta-D-glucosaminidase (NAGase) is described, using bare screen-printed carbon electrodes (SPCEs). The enzyme substrate, 1-naphthyl-N-acetyl-beta-D-glucosaminide, was added to the NAGase-containing sample under hydrodynamic conditions and was hydrolysed to 1-naphthol, which was monitored amperometrically at an Eapp of +650 mV versus SCE. A pH study revealed the apparent Vmax for the assay to occur at pH 4.5. corresponding to an apparent substrate Km of 0.28 mM. In order to be compatible with the analysis of biological fluids, a final operating pH of 5.4 was selected, and, using a data recording time of 100 s post-substrate addition, the assay gave a linear response (r2 = 0.988) over the range 3.1 to 108 mU ml(-1) NAGase (RSD = 15.4%). This assay has the potential to monitor NAGase levels in a number of application areas.