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 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.

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.

Determination of the concentrations of the steroids estradiol, progesterone and testosterone in bovine sera: comparison of commercial dissociation enhanced lanthanide fluorescence immunoassay kits with conventional radio and enzyme immunoassays.

The performance of three conventional enzyme and radioimmunoassays routinely used to detect residues of anabolic steroids in cattle sera were compared with dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) kits designed for the hospital market. Slight modifications to the kit reagents were required for the analysis of bovine sera. Owing to the large sample volumes used in conventional assays, detection limits were generally better than those obtained with DELFIA kits, however, assay reproducibility was enhanced using the DELFIA technology. Comparison of sera obtained from cattle implanted with anabolic steroids revealed a good correlation between alternate methods (r2 from 0.91 to 0.97). The DELFIA kits offer a faster method for measuring estradiol, progesterone and testosterone with adequate sensitivity and in a safer environment than that encountered using radioimmunoassays.

Effective laboratory monitoring for the abuse of the beta-agonist clenbuterol in cattle.

The use of the beta-agonist clenbuterol (CBL) as a growth promoter has been outlawed in European meat production. The detection of its illegal use is dependent on CBL residues persisting in animal tissues for longer than the withdrawal times given by abusers. A comparison of urine, bile and liver matrices indicated that analysis of the liver offered the best possibility for CBL detection. However, an experimental study showed that CBL detection following withdrawal could be further extended (up to 56 d) if the retina was used as the target tissue. Analysis of 703 retina and liver samples from cattle suspected of CBL medication revealed that 96 cattle had CBL residues present in their retinas, only 46 of these were liver positive. There were no instances of liver CBL residues being detected without the associated retina also being positive.

Tail painting technique as an aid to oestrus detection in cattle.

The value of heat detection by tail painting was assessed by simultaneous visual observation and regular plasma progesterone assay in grazing animals; 88.1 per cent of all heats were accompanied by a positive tail paint record and 30.1 per cent of positive tail paint records occurred in dioestrous animals. Conception rates to the first insemination were 57.6 per cent and 57.9 per cent for heifers and cows, respectively. Of seven animals which showed behavioural oestrus but had intact tail paint, three conceived following insemination. These results suggest that tail paint may be a useful aid in heat detection.

Vaginal electrical resistance in cows: 2. Relationship to milk progesterone concentrations during the reproductive cycle.

When the vaginal electrical resistance (VER) was measured in the anterior vagina in 4 cows during a total of 12 oestrous cycles, there was a close correlation between VER, milk progesterone levels and visual observations of oestrus. The cyclic changes in both VER and milk progesterone ceased in 2 cows which became pregnant during the study. In a pregnant cow, the VER was found to be constant both pre-partum and immediately post-partum. These results show that measurement of VER can be a useful aid in the confirmation of oestrus and suggest that the technique may also be applicable to the early diagnosis of pregnancy.

Pregnancy diagnosis in cattle by measuring vaginal electrical resistance.

The results of early pregnancy diagnosis using a milk progesterone assay and a measurement of electrical resistance of the vaginal mucosa made on 135 dairy cows in two herds are compared. The tests were carried out on the day of insemination and 21 days later. Ninety-eight cows were diagnosed as pregnant by both tests. Of these, 92 calved and there were 6 cases of suspected embryonic mortality. Thirty animals were diagnosed as non-pregnant by both tests and none calved. One cow, diagnosed as non-pregnant by the vaginal electrical resistance method, calved later. Of the remaining 6 cows, 4 had extended post-parturition anoestrus, one had cystic ovarian disease and another had a long oestrus cycle. The results of this study suggest that measurements of the electrical resistance of the vaginal mucosa in dairy cows may have a part to play in on-farm pregnancy diagnosis.

An assessment by progesterone assay of the accuracy of oestrus detection in dairy cows.

The use of a milk progesterone assay in determining the extent to which dairy cows are presented for insemination at the wrong time is reported. Milk samples were taken from cows at the time of insemination and were subjected to plasma progesterone assay. In a preliminary study samples were obtained from 96 cows in an experimental herd at insemination. The calving rate of these cows was compared with the milk progesterone level at insemination. Eight cows which had levels of progesterone much above the normal oestrus level 9 more than 0.3 ng per ml) failed to conceive. As the progesterone level at insemination rose, calving rate declined. A further 1177 milk samples taken from cows being inseminated in commercial dairy herds were also examined. Ninety-one animals (7.7%) had levels of milk progesterone in excess of those observed at normal oestrus. A negative correlation was confirmed between non-return rate and milk progesterone level at insemination. These investigations show that the proportion of cows from the national herd which have high progesterone levels when presented for insemination is similar to that found under experimental conditions and suggests that the problem of heat detection may be a relatively constant factor in reduced conception to artificial insemination.

Preparation and use of teaser bulls.

Bilateral partial epididymectomy was performed on 23 bulls. These bulls were used to detect oestrus under two different management systems. The results obtained were compared with simultaneous plasma progesterone assays, and indicated that teaser bulls can provide accurate detection of oestrus.

Pregnancy diagnosis in sows: a comparison of the vaginal biopsy and Doppler ultrasound techniques.

Two pregnancy diagnosis techniques, vaginal biopsy and ultrasonic detection, were applied to 190 sows following service. Of 172 sows diagnosed pregnant by vaginal biopsy, five did not farrow. Of 18 sows judged not pregnant, one produced a litter. With the ultrasound technique, 175 sows were diagnosed pregnant, but 11 did not farrow. Of the remaining 15 sows considered not pregnant by the 39th day after service, four farrowed. The biopsy result was usually available up to 48 hours earlier than the ultrasound result. It was concluded that where laboratory facilities are available, the biopsy technique is the method of choice.