Relationships between urea dilution measurements and body weight and composition of lactating dairy cows.

The objective of the present study was to investigate the potential of the urea dilution technique, coupled with live animal measures to predict the body components of dairy cattle. The study involved 104 lactating Holstein-Friesian cows offered grass silage-based diets. Urea space volume (USV) was calculated from 2 collection periods of blood samples following infusion of urea at 12 (USV12, kg) and 30 (USV30, kg) min after infusion, and then as a proportion of live weight (LW) or empty body weight (EBW). All cows were slaughtered within 2 d of the USV trials. Large ranges existed in EBW and empty body concentrations of water, crude protein (CP), lipid, ash, and gross energy (GE). The USV12 and USV30 were both positively related to LW, EBW, and empty body component weights. The r2 values for USV12 were greater than USV30. The r2 values in the relationships of EBW and empty body composition with USV, however, were smaller than those with LW. Nevertheless, the relationships were improved when both USV and LW were used as predictors, rather than using either alone. Adding milk yield and body condition score as supporting predictors to prediction equations using USV and LW data for EBW, lipid, and GE contents further improved the relationships (r2 = 0.93, 0.66, and 0.77, respectively). Internal evaluation of one-third of the present data using equations developed from two-thirds of the present data indicated that using USV, live weight, and other live animal variables as predictors, rather than using USV alone, considerably improved the prediction accuracy. It was concluded that USV can be used to predict body composition, but the relationships with USV were poorer than those with LW. The USV can only be used as a supporting variable to live weight for prediction of body components in lactating dairy cows.

Fertility of dairy cows in Northern Ireland.

A comprehensive database was established on the milk production and reproductive performance of dairy cows in 19 selected herds in Northern Ireland, varying in size, management system and genetic merit. Data were obtained for 2471 cows, 1775 of which calved in a second year, and 693 were culled from the herd for specific reasons. The estimated mean rate of heat detection (assessed by the interheat interval during the main breeding season) in all the herds was 71 per cent, with a range from 53 to 92 per cent The average conception rate to first insemination was 37.1 per cent (range 21 to 66 per cent). The average calving interval for the retained cows was 407.2 days (range 359 to 448 days). Twenty-eight per cent of the cows that calved were culled, with infertility being the largest single reason (26.8 per cent of the cows culled). There were major differences in reproductive performance between the herds, but heat detection rate, conception rate and calving interval did not appear to be affeded by a herd's genetic merit. The herds with shorter calving intervals were characterised by better heat detection efficiency (83 v 61 per cent, P<0.01), a shorter interval from calving to first insemination (74 v 97 days; P<0.05), a higher conception rate to first insemination (45 v 34 per cent, P>0.10) and a lower removal rate (23 v 37 per cent, P<0.01). Furthermore, the cows in these herds had lower body condition scores (BCS) in the dry period (3.0 v 3.3; P<0.05) but lost less body condition in early lactation (0.3 v 0.6 BCS units, P<0.05). These results show that dairy herd fertility in Northern Ireland is generally low and similar to that previously reported for England and the USA, but that in some herds changes in herd management practices improved the cows' fertility.

Nortestosterone is not a naturally occurring compound in male cattle.

Nortestosterone (beta-NT) is a hormonal growth promoter banned from livestock production in the EU. Following injection, the major metabolite in cattle is the 17 alpha-epimer (alpha-NT). However, this also occurs naturally in pregnant cattle. It is not known whether alpha-NT is also endogenous to intact or castrated male cattle. Three surveys were undertaken to assess whether alpha-NT is naturally produced in this subset of the population. Bile samples from a total of 1,281 cattle (73 bulls and 1,208 steers) from 366 herds were collected at slaughter and initially screened by using a semi-automated EIA with multi-analyte immunoaffinity chromatography (IAC) clean-up. Bile samples from a further 38 male cattle (10 bulls and 28 steers) were analysed by high-resolution gas chromatography-mass spectrometry (GC-MS) with IAC pretreatment. Only samples containing more than 2 ng/ml alpha-NT were subjected to GC-MS. With 2 ng/ml alpha-NT as a threshold for confirmatory testing, the false positive rate of the screening EIA was 1.8%. Bulls (n = 16) and steers (n = 179) from government farms (n = 2) and which were not treated with exogenous beta-NT, did not have measurable concentrations of alpha-NT in their bile. Bulls (n = 35) and steers (n = 606) taken from herds (n = 204) which had no previous history of illegal growth promoter abuse also did not have alpha-NT in their bile. Of 32 bulls and 451 steers of unknown treatment history sampled from herds (n = 160), 56 steers from 19 herds contained GC-MS confirmed concentrations of alpha-NT higher than the limit of quantification of the assay LOQ (0.7 ng/ml). Of these animals, two had beta-NT-containing injection sites and five had residues of the beta-agonists clenbuterol and mabuterol. Examination of the animal movement and ownership histories of the 56 confirmed positive animals strongly suggested that exogenous beta-NT had been administered at the presenting farm. It is concluded that alpha-NT is not endogenous to this subset of the cattle population and that detection of this hormone in bile from bulls and steers constitutes evidence of abuse.

Comparison of the effects of injections of nortestosterone phenylpropionate at single and multiple sites in cattle on the detection of its residues in plasma, urine and bile.

The synthetic androgen 17 beta-19-nortestosterone (beta-NT) has been used illegally as a growth promoter in cattle production in the European Union. The elimination of beta-NT and its metabolites in plasma and urine was studied in cattle which had received intramuscular injections of its phenylpropionate ester (NTPP) at either single or multiple sites at a dose rate of 1 mg/kg bodyweight. In both groups, the plasma concentrations of beta-NT, measured by enzyme immunoassay, were consistently greater than the assay's limit of quantification (0.24 ng/ml) during days 1 to 7 of the study. The mean (sd) maximum plasma concentration (Cmax) was significantly greater in the multiply injected animals (4.4 [0.48] v 2.7 [0.15] ng/ml), but other plasma pharmacokinetic parameters, AUC, CL, T1/2 beta, Tmax and MRT, were not significantly different in the two groups. The equivalent urinary concentrations exceeded the limit of quantification of the assay (4.5 ng/ml) for up to 24 days after injection. In a second study, the biliary concentrations of beta-NT and its 17 alpha-epimer (alpha-NT) were measured by gas chromatography-high resolution mass spectrometry after cattle were injected intramuscularly at either single or multiple sites with NTPP. Only alpha-NT was detected in bile for up to 62 days after injection at concentrations above the limit of quantification of the assay (0.7 ng/ml). It is concluded that in some animals, intramuscular injections of NTPP at several sites may decrease the period after injection during which free beta-NT and its metabolites are detectable in plasma and urine. After the injection of NTPP, alpha-NT was detected in bile for longer than it was detected in plasma or urine.

Plasma, urinary and biliary residues in cattle following intramuscular injection of nortestosterone laurate.

The synthetic androgen 19-nortestosterone (beta-NT) has been used illegally as a growth promoter in cattle production in the European Union. Elimination of beta-NT and its metabolites in plasma, urine and bile was studied in three cattle with cannulated gallbladders following intramuscular injection at a single site of 500 mg of the laurate ester (NTL) containing 300.5 mg beta-NT. Using enzyme immunoassay quantification, plasma Cmax of free beta-NT was 0.5 +/- 0.15 microgram/L (mean +/- SEM). Concentrations of free beta-NT in plasma were consistently greater than the assay limit of quantification (0.12 microgram/L) for 32.7 +/- 13.42 days. Mean residence time for the beta-NT in plasma was 68.5 +/- 20.75 days. Following sample preparation by immunoaffinity chromatography, high-resolution GC-MS was used to quantify beta-NT and alpha-NT in urine and bile. beta-NT was detected irregularly in urine from two of the three animals post injection. The principal metabolite present in the urine, alpha-NT, was detected for 160.3 +/- 22.67 days post injection. Cmax for alpha-NT in urine was 13.7 +/- 5.14 micrograms/L. Mean urinary AUC0-183 days for alpha-NT was 845.7 +/- 400.90 (microgram h)/L. In bile, alpha-NT was the only metabolite detected for 174.3 +/- 8.67 days post treatment. Cmax for alpha-NT in bile was 40.8 +/- 12.70 micrograms/L and mean biliary AUC0-183 days for alpha-NT was 1982.6 +/- 373.81 (microgram h)/L. Concentrations of alpha-NT in bile samples were greater than those in urine samples taken at the same time. The mean ratio of biliary:urinary AUC0-183 days was 3.0 +/- 0.72. It is concluded that bile is a superior fluid for detection of alpha-NT following injection of NTL, owing to the longer period during which residues may be detected after administration.

Biliary elimination of endogenous nortestosterone by pregnant cows.

A temporal study of the biliary elimination of endogenous 19-nortestosterone during two successive pregnancies was made in three cows with cannulated gall bladders. Bile samples were analysed for 17 beta-19-nortestosterone (beta-NT) and the 17 alpha-epimer (alpha-NT) by using high resolution gas chromatography and mass spectroscopy. No beta-NT was detected in any of the samples analysed. However, alpha-NT was detected from around 120 days of gestation in each of the cows. Peak concentrations were observed in the last week before calving and ranged from 9.5 to 36.7 ng/ml. After parturtion, the concentrations of alpha-NT declined rapidly and were undetectable by seven days after calving, and it was not detected again until after 120 days of gestation. The biliary concentrations of alpha-NT detected subsequently were similar to those observed in cattle several weeks after an exogenous injection of the synthetic ester beta-NT phenylpropionate.

Contamination of animal feedingstuffs with undeclared antimicrobial additives.

A study was carried out on 397 feedingstuffs and 11 premixes on which 161 (39.5%) were declared free of medication and 247 (60.5%) were medicated. These were subjected to analysis for the presence of antibiotics. Of the 247 medicated feeds 87 (35.2%) contained undeclared antimicrobials of which 59 (23.9%) were at a concentration sufficient to allow quantification by HPLC. Of the 161 unmedicated feeds 71 (44.1%) were shown to contain detectable antimicrobials of which 42 (26.1%) contained concentrations which could be quantified by HPLC. The most frequently identified contaminating antimicrobials were chlortetracycline (CTC) (15.2%), sulphonamides (6.9%), penicillin (3.4%) and ionophores (3.4%). Four samples (ionophores--3, sulphadimidine--1) contained therapeutic concentrations and one sample a supra-therapeutic concentration (monensin). The remainder were sub-therapeutic. All the contaminating concentrations of sulphadimidine detected were sufficient to cause violative tissue residues if fed to animals immediately prior to slaughter.

Residues of nortestosterone esters at injection sites. Part 1. Oral bioavailability.

Injectable esters of 17 beta-19-nortestosterone (beta-NT) have been used illegally for growth promotion in European Union (EU) cattle production. There are no data on oral bioavailability of free beta-NT from beta-NT esters. Adult non-pregnant female Landrace pigs (n = 4) were fitted with jugular vein cannulae and were treated as follows with an appropriate 'flushing out' period between each treatment: an intravenous bolus of beta-NT at 0.1 mg kg-1 body weight (NTi.v.), 300 mg nortestosterone phenylpropionate (NTPP) in oil of arachis administered per os (NTPPoil) and 300 mg NTPP in aqueous suspension administered per os (NTPPaq). Blood samples were taken for up to 24 h and analysed for free beta-NT by enzyme immunoassay. Calculation of the area under the plasma time concentration curve (AUC), allowed absolute bioavailability estimations for both oral treatments. Mean bioavailability of beta-NT following NTPPaq was 0.35% (range 0.25-0.41%) compared to 2.25% (range 0.86-2.85%) for NTPPoil. Mean +/- standard error of mean time of maximum plasma concentration (Tmas) of free beta-NT occurred earlier (2.3 +/- 0.6 h) with NTPPoil compared to NTPPaq (10.3 +/- 1.03 h) and the maximum plasma concentration (Cmax) was also greater for NTPPoil compared to NTPPaq (36.1 +/- 6.49 vs. 3.2 +/- 0.31 micrograms l-1). It is concluded that the presence of arachis oil enhances the absorption of NTPP. Notwithstanding the possible effects that cooking and other food processing may have on such injection site residues, extrapolation of these results to man suggests that consumption of NTPP-containing injection sites may result in peak plasma concentrations of free beta-NT which are significantly greater than those observed following injection of NT esters.

Residues of nortestosterone esters at injection sites. Part 2. Behavioural effects.

Anecdotal evidence in man suggests that abuse of anabolic androgenic steroids (AAS) is linked with increased aggression. In order to test the hypothesis that administration of nortestosterone (beta-NT) esters may increase aggression, behavioural effects following beta-NT phenylpropionate (NTPP) administration were assessed in male mice using the isolation-induced aggression paradigm. Mice (n = 80) were individually caged and randomly assigned to 'resident' or 'intruder' status. Residents received one of four treatments: subcutaneous (s/c) injection of oil of arachis (OILINJ), s/c injection of NTPP in oil of arachis (NTINJ), oil of arachis per os (OILOS) and NTPP in oil of arachis per os (NTOS). After treatment, weight-matched intruders were introduced into each resident's cage and the encounter was videotaped to allow the frequency (f) and duration (d) of social, non-social, offensive and defensive behaviours displayed by residents and intruders to be measured. The latency of bite attack for residents was significantly shorter in NTOS compared to OILOS. There was no difference in either f or d of aggressive behaviours between NTINJ and OILINJ. Because OILINJ residents demonstrated similar levels of aggression to NTOS, it is possible that the aggression in NTOS mice may be mediated by a different biochemical pathway. Extrapolation of these results indicate that ingestion of beta-NT esters may have a role in the induction of aggression in consumers.

Evaluation of a modified EC Four Plate Method to detect antimicrobial drugs.

This paper describes a modification of the EC Four Plate Method based on microbial growth inhibition of Bacillus subtilis on agar medium at pH 6.0, 7.2 and 8.0 and Micrococcus luteus at pH 8.0 developed to cope with large numbers of samples. The method's performance was evaluated by determining the Minimum Inhibitory Concentrations of 66 commonly used drugs and determining the between-assay variation of antimicrobial control standards. The modified method proved particularly sensitive for beta-lactams, tetracyclines, quinolones, marcrolides and lincosamides and least sensitive for anticoccidials and nitrofurans. The pH 6.0 and 7.2 plates were more sensitive for 39 of the 66 antimicrobials (59%) whereas the two pH 8.0 plates (B. subtilis, M. luteus) were the most sensitive for 27 (41%). Muscle samples were taken from 1830 routine meat inspection investigations between 1994 and 1996. Of the 38 (2%) positive meat inspection carcasses, the following antimicrobials were confirmed above the MRL: penicillin G (10), oxytetracycline (16), sulphadimidine and sulphadiazine in combination (4) and chlortetracycline (1). The method as described is technically simple, cost effective, robust, suitable for large sample throughput and for frozen, thawed or fresh tissues. When all four plates are used the pattern of inhibition can reduce unnecessary confirmatory assays by indicating the antimicrobial group most likely to be present.

Determination of thyreostat residues from bovine matrices using high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) methodologies were evaluated for the detection and quantification of thyreostatic drug residues in cattle serum and thyroid tissue. The paper details a protocol, using a simple ethyl acetate extraction for the determination of thiouracil, tapazole, methyl thiouracil, propyl thiouracil and phenyl thiouracil in thyroid tissue. Using two sequential HPLC injections, and quantitative analysis, in two steps, all five thyreostats were detectable at concentrations greater than 2.45-4.52 ng/g. Modifications to a published method for detection of thyreostatic residues in serum involving the addition of mercaptoethanol and a freezing step are described. The modifications improved sensitivity and allowed detection of the five thyreostats at levels greater than 16.98-35.25 ng/ml. Young bulls were treated with thyreostats to demonstrate the validity of the methodologies described. Administered thyreostats were not absorbed equally by the test animals and the compounds were not all detected in the serum samples removed at 7 days following drug withdrawal. These experiments indicate the necessity to be able to detect thyreostat residues in a variety of matrices.

Production and biological activity of murine monoclonal antibodies against GnRH.

Immunization against GnRH represents a nonsurgical means of castrating domestic species. However, clear target antibody titres for bioactivity have not been established. The aims of this study were to produce characterized anti-GnRH monoclonal antibodies and to determine a threshold titre. Three murine monoclonals were developed which produced IgG2a class immunoglobulins and bound 50% I(125)-GnRH at a 10(6) to 10(7) dilution. The antibodies were specific to GnRH, showed a strong affinity (Ka values from 1.99 to 2.60 x 10(10) litres/mole), and were directed towards the amino terminus. In female mice all 3 antibody clones interrupted ovarian cyclicity, causing an extension in diestrus followed by prolonged estrus/metestrus (12 to 30 d). Throughout this period circulating titres were greater than 15% I(125)-GnRH binding at a 5 x 10(4) dilution. In male mice, immunization with 0.2 ml of ascites significantly reduced testes (P < 0.05), epididymides (P < 0.001) and seminal vesicle (P < 0.01) weights. A 0.1 ml dose (61.4 +/- 18.6% binding at a 10(6) dilution) was ineffective. A serial dilution study indicated that a titre of 50% binding at 2 x 10(6) dilution (antigen binding capacity of 268 +/- 35 ng/ml) was required to completely block GnRH activity. This is a higher tire than threshold levels determined previously. Identification of factors determining the titre required for bioactivity is needed.

Comparison and evaluation of the specificity and binding capacity of commercial and in house affinity columns used in sample preparation for analysis of growth-promoting drugs.

Immunoaffinity chromatography (IAC) and affinity chromatography (AC) are widely used for extraction of drugs from biological samples. Fifteen column types were purchased from five different manufacturers and their ability to bind specific drugs including beta-agonists and anabolic steroids over a range of analyte concentrations in fortified bovine urine samples was assessed. The performance data obtained from these columns were compared with columns produced in this laboratory (in house columns). The in house columns gave the highest recoveries, ranging from 92 to 100% at the 1 ng spiking concentration, for five of the seven analytes assessed. Forty percent (11 of 27) of all the commercial column assessments recorded recoveries of less than 50% even when the lowest spiking concentration was applied (1 ng). For one manufacturer, only one of seven different columns purchased delivered extraction efficiencies greater than 50%. The extraction efficiencies of the clenbuterol columns were the highest with all commercially prepared columns showing at least 50% binding of radiolabelled tracer. Recoveries of alpha-nortestosterone were the lowest. The variability of these products with respect to quality control requires constant monitoring.

Comparison of porcine urine and bile as matrices to screen for the residues of two sulfonamides using a semi-automated enzyme immunoassay.

Porcine urine enzyme immunoassays for sulfamethazine and sulfadiazine have previously been employed as screening tests to predict the concentrations of the drugs in the corresponding tissues (kidneys). If a urine was found positive (> 800 ng ml-1) the corresponding kidney was then analysed by an enzyme immunoassay and, if found positive, a confirmatory analysis by HPLC was performed. Urine was chosen as the screening matrix since sulfonamides are mainly eliminated through this body fluid. However, after obtaining a number of false positive predictions, an investigation was carried out to assess the possibility of using an alternative body fluid which would act as a superior indicator of the presence of sulfonamides in porcine kidney. An initial study indicated that serum, plasma- and bile could all be used as screening matrices. From these, bile was chosen as the preferred sample matrix and an extensive study followed to compare the efficiencies of sulfonamide positive bile and urine at predicting sulphonamide positive kidneys. Bile was found to be 17 times more efficient than urine at predicting a sulfamethazine positive kidney and 11 times more efficient at predicting a sulfadiazine positive kidney. With this enhanced performance of the initial screening test, the need for the costly and time consuming kidney enzyme immunoassay, prior to HPLC analysis, was eliminated.

The incidence and cause of lasalocid residues in eggs in Northern Ireland.

Lasalocid is a coccidiostat licensed for use in poultry, but not for use in egg-laying birds. Lasalocid residues were determined in an egg sample from each of 161 egg producers in Northern Ireland using liquid chromatography-electrospray ionization mass spectrometry, following reports from the Veterinary Medicines Directorate concerning the incidence of lasalocid residues in the United Kingdom. Approximately 66% of the eggs contained lasalocid residues at concentrations in excess of 0.3 ng/g. There was no apparent difference in the incidence of lasalocid residues between free range and battery eggs. Carry-over of lasalocid from medicated to unmedicated batches of both premix and feed, during milling processes, was identified as a possible cause of contamination. Subsequently, egg-laying birds were fed meal containing a range of lasalocid concentrations, similar to those found as a result of unintentional contamination at a feed mill (0.1-5.0 mg/kg). The concentrations of lasalocid, measured in their eggs, were similar to that found in the survey. Lasalocid persisted in eggs for 10 days after withdrawal of medicated feed and replacement with lasalocid-free feed.

Monitoring for clenbuterol abuse in N. Ireland 1989-1994.

Laboratory testing in N. Ireland for the illegal growth promoting agent, clenbuterol (CBL), is centralized at the Veterinary Sciences Division, Belfast. During the past 6 years a variety of testing schemes have evolved to determine the level of abuse of this drug in the local meat industry. The types of samples from cattle tested during this period altered as pharmacokinetic data for the compound increased. Initially, fluids such as urine and bile were used, however testing switched to more appropriate tissues such as liver, eyes, and hair. The first positive samples were detected in 1990, with 43 out of 121 samples tested showing detectable residues. In the following year, this number increased to 139 out of 286 tests. Despite substantial increases in the number of samples analysed over the succeeding years, the numbers of positive results steadily declined, thus giving strong evidence that abuse was also on the decline. From the data collected over the 6-year period, it became clear that the EU National Surveillance Scheme designed to detect abuse of illegal substances was ineffective and locally designed programmes were required to effectively tackle the problem.

The production of pig tissue sulphadimidine reference material.

The production of stable homogeneous reference materials containing the antimicrobial agent sulphadimidine in pig tissue is described. These were commissioned by the Community Bureau of Reference (BCR), established by the Commission of the European Communities, to promote improvements in analytical accuracy and to ensure uniformity of results determined by member states. Sulphadimidine-containing tissue powders (400 vials each of muscle, liver and kidney) were prepared by orally dosing pigs with drug, producing lyophilized tissue powders and blending these with negative tissues from unmedicated animals to achieve target concentrations. Details of the production process, the stabilizing procedure developed and the analytical assessments of homogeneity and stability are given.

Possible naturally occurring zeranol in bovine bile in Northern Ireland.

Zeranol and two Fusarium toxins, alpha-zearalenol and beta-zearalenol, were confirmed by gaschromatography/mass spectrometry (GC/MS) in bovine bile samples referred to this laboratory for analysis. No evidence of zeranol abuse was found on-farm. Given the recent suggestion that zeranol might arise from the metabolism of these Fusarium toxins, and the finding of zeranol in bovine and ovine urine across the EU, it was concluded that the residues had arisen as a result of natural metabolism.