Growth performance, real-time gizzard pH and calcium solubility in the gut of broiler chickens is dependent on the interaction between dietary calcium concentration and limestone particle size.

1.The purpose of this study was to determine the effect of limestone particle size and dietary Ca concentration on performance, real-time gizzard pH, and Ca and P solubility in the gastrointestinal tract of 21d broiler chickens.2. A total of 576, one-day-old Ross 308 male broilers were randomly allocated among 4 treatments, with 8 replicate pens, and 18 birds per pen. Treatments were arranged as a 2 × 2 factorial consisting of two particle sizes of limestone (coarse, CL, 1200 µm; fine, FL, 44 µm) and two Ca concentrations (9.6 or 6.0 g/kg). On d 19 and 20, four birds per treatment were administered Heidelberg pH capsules and readings monitored for 3 h.3. Reducing Ca concentration from 9.6 to 6.0 g/kg had no effect on d 21 weight gain or FCR of birds fed CL; however, feeding FL at 9.6 g/kg Ca increased weight gain by 10% and reduced FCR by 5% compared to FL at 6.0 g/kg Ca (P < 0.001).4. Average gizzard pH readings ranged from pH 0.67 to 3.01 across all treatments. Birds fed CL at 6.0 g/kg Ca had lower average gizzard pH compared to birds fed 9.6 g/kg Ca, while birds fed FL at 6.0 g/kg Ca had higher gizzard pH than birds fed CL at both Ca concentrations, but was comparable to birds fed FL at 9.6 g/kg Ca (P < 0.001).5. For birds fed CL at 6.0 g/kg Ca, soluble Ca in the gizzard was approximately 40% lower (P < 0.05) than all other treatments, and reduced by 44% (P < 0.05) in the small intestine compared with birds fed FL at 6.0 g/kg Ca.6. These findings demonstrated that the effect of Ca concentration on gizzard pH, Ca solubility and broiler performance was dependent on limestone particle size and suggested that solubility per se had little relevance to performance.

Exogenous phytase and xylanase exhibit opposing effects on real-time gizzard pH in broiler chickens.

1. The current study was conducted to evaluate the influence of high phytase doses and xylanase, individually and in combination, on performance, blood inositol and real-time gastric pH in broilers fed wheat-based diets. 2. In a 42-d experiment, a total of 576 male Ross 308 broiler chicks were allocated to 4 dietary treatments. Treatments consisted of a 2 × 2 factorial arrangement, with 500 or 2500 FTU/kg phytase and 0 or 16 000 BXU/kg xylanase, fed in two phases (starter 0-21; grower 21-42 d). Heidelberg pH capsules were administered to 8 birds from each treatment group, pre- and post-diet phase change, with readings captured over a 5.5-h period. 3. At 21 and 42 d, birds fed 500 FTU/kg phytase without xylanase had on average 127 and 223 g lower weight gain than all other treatments, respectively (P < 0.05). At 21 d, feed conversion ratio (FCR) was reduced (P < 0.01) by 2500 FTU/kg phytase or xylanase; however, 42-d FCR was unaffected by enzyme treatment. Inositol content of plasma was twice that of the erythrocyte (P < 0.001), with 2500 FTU/kg phytase tending to increase (P = 0.07) inositol content in both blood fractions. 4. Across all treatments, capsule readings ranged from pH 0.54 to 4.84 in the gizzard of broilers. Addition of 2500 FTU/kg phytase to the grower diet reduced (P < 0.05) average gizzard pH from 2.89 to 1.69, whilst feeding xylanase increased (P < 0.001) gizzard pH from 2.04 to 2.40. In contrast, digital probe measurements showed no effect of xylanase on gizzard pH, while addition of 2500 FTU/kg phytase increased (P = 0.05) pH compared to 500 FTU/kg phytase with or without xylanase. 5. These findings suggested that xylanase and high phytase doses have opposite effects on real-time gastric pH, while similarly improving performance of broilers.

Superdosing phytase reduces real-time gastric pH in broilers and weaned piglets.

1. The current study was conducted to investigate the effect of high phytase doses on growth performance and real-time gastric pH measurements in broiler chickens and pigs. 2. In the first experiment, 576 male Ross 308 broilers were fed in two phases (0-21 and 21-42 d) with 4 treatment groups, with diets meeting nutrient requirements containing 0, 500, 1500 or 2500 FTU/kg phytase. In the second, 64 Landrace weaners were fed on diets meeting nutrient requirements with or without phytase (0 or 2500 FTU/kg) in two phases (0-21 and 21-42 d). Heidelberg pH capsules were administered to 7 broilers and approximately 13 pigs per treatment group, pre- and post-phase change, with readings monitored over several hours. 3. Addition of phytase into an adequate Ca and P diet had no significant effect on broiler performance although phytase tended (P < 0.07) to improve feed conversion in pigs over the entire experimental period. Real-time pH capsule readings in broilers demonstrated an increase (P < 0.05) in gizzard pH when phytase was dosed at 500 or 1500 FTU/kg, while higher doses of 2500 FTU/kg phytase lowered pH to a level comparable to control birds. Gastric pH increased (P < 0.01) when animals were exposed to dietary phase change, signifying a potential challenge period for nutrient digestibility. However, pigs fed 2500 FTU/kg were able to maintain gastric pH levels through diet phase change. In contrast, spear-tip probe measurements showed no treatment effect on gastric pH. 4. These findings demonstrate dietary manipulation of gastric pH and the value of real-time pH capsule technology as a means of determining phytase dose response.

Effect of diet phase change, dietary Ca and P level and phytase on bird performance and real-time gizzard pH measurements.

In this study, a novel capsule technique was used to capture real-time pH readings from the gizzard over several hours, in response to different dietary treatments. 1. The first experiment was a preliminary study into capsule administration and pH recordings using 9 male Ross 308 broilers from 20 d. In the second experiment, broilers (576) were fed in two phases (0-21 and 21-42 d) with 4 treatment groups; low and adequate Ca and AvP diets with and without Quantum Blue phytase (1500 FTU/kg). Capsules were administered to 8 birds from each treatment group, pre and post diet phase change, with readings captured over a 2.5 h period. 2. Phytase addition improved body weight gain (BWG) and feed conversion ratio (FCR) of birds fed low dietary Ca, while having no significant effect on birds fed adequate Ca diets. Unexpectantly, diets with higher Ca levels gave a lower average gizzard pH compared to the low Ca diet. Phytase addition, irrespective of Ca level, increased average gizzard pH. Fluctuations in gizzard pH (0.6-3.8) were observed across all treatment groups. Higher frequencies of pH readings below pH 1.0 were seen in birds fed an adequate Ca diet and with phytase supplementation of a low Ca diet. 3. These results signify the potential use of capsule techniques to monitor real-time pH changes. The implication on gastric protein and fibre hydrolysis as a result of large fluctuations in pH should be considered.

Animal board invited review: precision livestock farming for dairy cows with a focus on oestrus detection.

Dairy cows are high value farm animals requiring careful management to achieve the best results. Since the advent of robotic and high throughput milking, the traditional few minutes available for individual human attention daily has disappeared and new automated technologies have been applied to improve monitoring of dairy cow production, nutrition, fertility, health and welfare. Cows milked by robots must meet legal requirements to detect healthy milk. This review focuses on emerging technical approaches in those areas of high cost to the farmer (fertility, metabolic disorders, mastitis, lameness and calving). The availability of low cost tri-axial accelerometers and wireless telemetry has allowed accurate models of behaviour to be developed and sometimes combined with rumination activity detected by acoustic sensors to detect oestrus; other measures (milk and skin temperature, electronic noses, milk yield) have been abandoned. In-line biosensors have been developed to detect markers for ovulation, pregnancy, lactose, mastitis and metabolic changes. Wireless telemetry has been applied to develop boluses for monitoring the rumen pH and temperature to detect metabolic disorders. Udder health requires a multisensing approach due to the varying inflammatory responses collectively described as mastitis. Lameness can be detected by walk over weigh cells, but also by various types of video image analysis and speed measurement. Prediction and detection of calving time is an area of active research mostly focused on behavioural change.

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.

Biosensors in the livestock industry: an automated ovulation prediction system for dairy cows.

Biosensor technology can offer the livestock industry new types of monitoring and measuring devices of which the specificity, sensitivity, reproducibility, speed and ease of use exceed the current technology. Biosensors can be applied to the detection and identification of infectious diseases in livestock, contaminants and toxins in feed, therapeutic drug residues in animal husbandry and oestrus detection. Our team is applying biosensor technology to the livestock industry by developing a fully automated ovulation prediction system for dairy cows. The results from field-tests show that the progesterone biosensor can characterize the ovulation cycles of cows and detect pregnancy.

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.

Detection of ketosis in dairy cows by analysis of exhaled breath.

In four healthy cows an elevation of ketone bodies was induced by reduction of feed intake. Two cows became clearly ketotic while the other two cows showed only slight increases in ketone body concentrations in serum and milk. Acetone concentrations in exhaled breath were measured by gas chromatography combined with mass spectrometry. These values were correlated with concentrations of serum beta-hydroxybutyric acid (r = 0.81) and milk acetoacetate+acetone (r = 0.70). It is concluded that the ketotic state of dairy cows can be detected by analysis of exhaled breath. This offers a potential non-invasive method of determining the metabolic state of dairy cows.