Serum cardiac troponin I concentrations in dogs with generalised seizures.

OBJECTIVES: To determine if serum cardiac troponin I concentrations - measured with both a first-generation assay and a high-sensitivity assay - were greater in dogs with generalised seizures than in controls and to identify clinical variables associated with cardiac troponin I concentration. MATERIALS AND METHODS: Prospective study of 30 dogs with recent generalised seizures and 30 healthy controls. Serum cardiac troponin I concentration was measured using two commercially available assays, and the correlation of clinical factors with concentration was examined. RESULTS: Serum concentrations of cardiac troponin I were higher in dogs that had recent seizures compared to controls when measured by both assays. The predictors most clearly associated with cardiac troponin I concentration were number of seizures and age. Both predictors were positively associated with increasing concentrations of troponin I. CLINICAL SIGNIFICANCE: Serum cardiac troponin I concentration was significantly elevated in dogs that had recent generalised seizures when compared to controls, and concentrations were higher in dogs that experienced more seizures. This association may indicate that generalised seizures are associated with damage to the myocardium.

Ultrasound reduces the minimum effective local anaesthetic volume compared with peripheral nerve stimulation for interscalene block.

BACKGROUND: Previous studies have demonstrated that lower local anaesthetic (LA) volumes can be used for ultrasound (US)-guided interscalene brachial plexus block (ISB). However, no study has examined whether US can reduce the volume required when compared with nerve stimulation (NS) for ISB. Our aim was to do this by comparing the minimum effective analgesic volumes (MEAVs). METHODS: After ethics approval and informed consent, patients undergoing shoulder surgery were recruited to this randomized, double-blind, up-down sequential allocation study. The volume used for both US and NS was dependent upon the success or failure of the previous block. Success was defined as a verbal rating score of 0/10, 30 min after surgery. Ten needle passes were allowed before defaulting to the opposite group. Patients received general anaesthesia. Pain scores and analgesic consumption were assessed by a blinded observer. Statistical comparisons of continuous variables were performed using Student's t-test and Mann-Whitney U-test as appropriate. Categorical variables were analysed using χ² test. MEAV values were estimated using log-transformed up-down independent pairs analysis and probit regression. Significance was assumed at P<0.05 (two-sided). RESULTS: The MEAV required to provide effective analgesia was significantly (P=0.034) reduced to 0.9 ml [95% confidence interval (CI) 0.3-2.8] in the US group from 5.4 ml (95% CI 3.4-8.6) in the NS group. Fewer needle passes were needed to identify the brachial plexus with US (1 vs 3; P<0.0001) and patients had less pain at 30 min after surgery (P=0.03). CONCLUSIONS: US reduces the number of attempts, LA volume, and postoperative pain when compared with NS for ISB.

Effect of local anaesthetic volume (20 vs 5 ml) on the efficacy and respiratory consequences of ultrasound-guided interscalene brachial plexus block.

BACKGROUND: Interscalene brachial plexus block (ISBPB) is an effective nerve block for shoulder surgery. However, a 100% incidence of phrenic nerve palsy limits the application of ISBPB for patients with limited pulmonary reserve. We examined the incidence of phrenic nerve palsy with a low-volume ISBPB compared with a standard-volume technique both guided by ultrasound. METHODS: Forty patients undergoing shoulder surgery were randomized to receive an ultrasound-guided ISBPB of either 5 or 20 ml ropivacaine 0.5%. General anaesthesia was standardized. Both groups were assessed for respiratory function by sonographic diaphragmatic assessment and spirometry before and after receiving ISBPB, and after surgery. Motor and sensory block, pain, sleep quality, and analgesic consumption were additional outcomes. Statistical comparison of continuous variables was analysed using one-way analysis of variance and Student's t-test. Non-continuous variables were analysed using chi(2) tests. Statistical significance was assumed at P<0.05. RESULTS: The incidence of diaphragmatic paralysis was significantly lower in the low-volume group compared with the standard-volume group (45% vs 100%). Reduction in forced expiratory volume in 1 s, forced vital capacity, and peak expiratory flow at 30 min after the block was also significantly less in the low-volume group. In addition, there was a significantly greater decrease in postoperative oxygen saturation in the standard-volume group (-5.85 vs -1.50, P=0.004) after surgery. There were no significant differences in pain scores, sleep quality, and total morphine consumption up to 24 h after surgery. CONCLUSIONS: The use of low-volume ultrasound-guided ISBPB is associated with fewer respiratory and other complications with no change in postoperative analgesia compared with the standard-volume technique.

Enhanced vascular permeability in rat skin induced by sensory nerve stimulation: evaluation of the time course and appropriate stimulation parameters.

Activation of nociceptors causes them to secrete neuropeptides. The binding of these peptides to receptors on blood vessels causes vasodilation and increased vascular permeability that allows loss of proteins and fluid (plasma extravasation, PE); this contributes to inflammation. This study defines the relationship between electrical activation of nociceptors and PE and evaluates the time course of this response in the skin of rats. We measured the time course and extent of PE by digital imaging of changes in skin reflectance caused by leakage of Evans Blue (EB) dye infused in the circulatory system before stimulation. Stimulation of the exclusively sensory saphenous nerve caused the skin to become dark blue within 2 min due to accumulation of EB. While PE is usually measured after 5-15 min of electrical stimulation, we found that stimulation for only 1 min at 4 Hz produced maximum PE. This response was dependent on the number of electrical stimuli at least for 4 Hz and 8 Hz stimulation rates. Since accumulation of EB in the skin is only slowly reversible, to determine the duration of enhanced vascular permeability we administered EB at various times after electrical stimulation of the saphenous nerve. PE was only observed when EB was infused within 5 min of electrical stimulation but could still be observed 50 min after capsaicin (1%, 25 microl) injection into the hind paw. These findings indicate that enhanced vascular permeability evoked by electrical stimulation persists only briefly after release of neuropeptides from nociceptors in the skin. Therefore, treatment of inflammation by blockade of neuropeptide release and receptors may be more effective than treatments aimed at epithelial gaps. We propose, in models of stimulation-induced inflammation, the use of a short stimulus train.

Effect of storage of reconstituted recombinant human thyroid-stimulating hormone (rhTSH) on thyroid-stimulating hormone (TSH) response testing in euthyroid dogs.

Bovine thyrotropin (bTSH) stimulation testing has long been considered the gold standard for diagnosis of canine hypothyroidism. Unfortunately, bTSH is no longer commercially available. Recently, the use of recombinant human thyrotropin (rhTSH) to perform thyroid-stimulating hormone (TSH) stimulation testing in dogs was described. The cost of an rhTSH vial (1.1 mg) limits the practical use of this product. The study reported here was performed to determine the effects of storing rhTSH on the post-TSH increase of serum total (TT4) and free (FT4) thyroxine concentrations during TSH stimulation testing in 12 euthyroid Beagles in a crossover trial. Three TSH tests with recombinant human thyrotropin (rhTSH; 91.5 microg IV) were performed on each dog during 3 different periods: 1 with freshly reconstituted rhTSH (fresh); 1 with rhTSH, reconstituted and stored at 4 degrees C for 4 weeks (refrigerated); and 1 with rhTSH, reconstituted and frozen at -20 degrees C for 8 weeks (frozen). Blood samples for determination of TT4 and FT4 concentrations were collected before and 4 and 6 hours after rhTSH administration. There was no significant difference in TT4 or FT4 concentration after stimulation with fresh, refrigerated, and frozen rhTSH. Furthermore, there was no significant difference between TT4 or FT4 serum concentration observed 4 and 6 hours after rhTSH administration. In conclusion, reconstituted rhTSH can be stored at 4 degrees C for 4 weeks and at -20 degrees C for 8 weeks without loss of biological activity, allowing clinicians to perform more TSH response tests per vial.

Association of canine hypothyroidism with a common major histocompatibility complex DLA class II allele.

Dogs exhibit a range of immune-mediated conditions including a lymphocytic thyroiditis which has many similarities to Hashimoto's thyroiditis in man. We have recently reported an association in Doberman Pinschers between canine hypothyroidism and a rare DLA class II haplotype that contains the DLA-DQA1*00101 allele. We now report a further series of 173 hypothyroid dogs in a range of breeds where a significant association with DLA-DQA1*00101 is shown.

Cerebral and conjunctival haemorrhages associated with von Willebrand factor deficiency and canine angiostrongylosis.

A case of angiostrongylosis is described in a 14-month-old golden retriever bitch. Conjunctival haemorrhage and neurological signs, referable to a space-occupying cerebral lesion, were associated with defective primary haemostasis caused by low levels of von Willebrand factor. Full clinical recovery followed treatment with desmopressin, fresh whole blood transfusion, fenbendazole and supportive care. The magnetic resonance image of the suspected organising haematoma is described. Similarities to the human condition, acquired von Willebrand syndrome, and a possible role for aberrant larval migration in haematoma formation are suggested.

A comparative study of the sensitivity of the 3-induction and 9-induction Buehler test procedures for assessing skin sensitisation potential.

Assessment of skin sensitization potential is a mandatory requirement for the registration or notification of most types of chemicals and products. Until recently, two methods using the guinea pig as test model were the most widely accepted; the guinea pig maximisation test and the Buehler test. In the case of agrochemical formulations, which constitute the final end use product in contact with operators, industry and also some regulatory authorities consider the Buehler method more appropriate as the methodology is more relevant to likely exposure in the field. However, certain European regulatory authorities have become concerned about the sensitivity of the Buehler test for this purpose and have requested that a modified method is used in which additional applications of test materials are used during the induction phase of the protocol (a total of 9 rather than the normal 3). This study was designed to assess whether this modification was justified. Six reference substances (formaldehyde, alpha-hexylcinnamaldehyde, fragrance mix, thimerosal, mercaptobenzothiazole and phthalic anhydride); all mild to moderate skin sensitizing chemicals, were assessed in a study, which compared the use of 3 and 9 induction applications. The results of this study demonstrated that, although most of these sensitisers were detected by both protocols, the modified method (9 induction applications) was no more sensitive than the standard method (3 induction applications). As the modified protocol is also potentially more stressful to the animals, it is concluded that the use of additional induction applications in the Buehler test cannot be justified from either a scientific or an animal welfare perspective.

Toxicity and chemistry of aspen wood leachate to aquatic life: field study.

A dark, toxic leachate has been observed around woodpiles of trembling aspen (Populus tremuloides Michx.) cut in winter for pulp or structural lumber. We measured production of leachate from 18 m3 of harvestable aspen logs stacked in an open field near Dawson Creek, British Columbia, Canada. The logpile began producing leachate during the first winter thaw and continued to do so for the duration of the two-year study (mean, 250 L/collection). Aspen leachate was characterized by dark color, acidic pH (5.0-6.5), elevated conductivity (200-500 microS/cm), high to very high biochemical oxygen demand (500-5,000 mg/L) and total organic carbon concentrations (500-2,000 mg/L), variable levels of phenolic compounds (2-27 mg/L), and low dissolved oxygen tensions (<2 mg/L). In tests with rainbow trout (Oncorhynchus mykiss), Daphnia magna, and luminescent bacteria, the leachate varied from weakly toxic (median lethal concentration, >10%) to very toxic (median lethal concentration, <1%). The volume of leachate generated by the logpile was correlated with total precipitation (rain or snow) since the last collection. Loads of chemical constituents or toxicity (lethal concentration x volume) in the leachate did not decline over the duration of the study. Less than 10% of the total mass of leachable material in the aspen logs was removed during two years of exposure.

Optimisation of cell-based assays for medium throughput screening of oxidative stress.

Identification and development of cell-based assays adapted to medium throughput screening requirements is important when screening chemicals for their potential toxic properties. We describe here rapid fluorometer-based and spectrophotometer-based microplate assays which allow for the evaluation of oxidative stress in hepatocyte cell cultures by measurement of three markers: production of hydroperoxide assessed by the DCFH-DA probe, cellular antioxidant status by measurement of reduced glutathione and glutathione peroxidase activity and cytotoxicity and mitochondrial damage by evaluation of the mitochondrial transmembrane potential with rhodamine 123 fluorescent dye. The assays described here are rapid, simple and inexpensive, which are desirable when setting up screening assays. This system should be useful in selecting candidate compounds during the pre-development phase of agrochemicals or pharmaceuticals. It should also be of interest for retrospective and explanatory studies of mechanisms underlying toxicity observed in vivo.

Tumorigenic potential of carbaryl in the heterozygous p53 knockout mouse model.

The heterozygous p53 knockout mouse model was used to assess whether vascular tumors noted in a 2-year carcinogenicity study in CD-1 mice with carbaryl were induced through a genotoxic mechanism. This knockout mouse model was selected for carbaryl because of the high sensitivity of this model to genotoxic events and its low spontaneous incidence of tumors until 9-12 months of age. Carbaryl was administered continuously via the diet to groups of 20 male heterozygous p53 knockout mice at concentrations of 0, 10, 30, 100, 300, 1000 and 4000 ppm for 180 days. Histopathological examinations revealed no evidence of carbaryl-induced neoplasms of any type. In particular, no neoplastic or preneoplastic changes were noted in the vascular tissue of any of the organs examined. Only neoplasms, recognized as those that occur spontaneously in untreated mice of this strain, were sporadically observed in a few animals from the intermediate dose groups with no evidence of a dose- or treatment-related effect. Therefore, under the conditions of this study, the no-observed-effect level (NOEL) of carbaryl for neoplastic changes in male mice was 4000 ppm (around 716 mg/kg body weight/day). We conclude: (1) carbaryl does not appear to be a genotoxic carcinogen at least in male mice; (2) if the vascular tumors observed in the CD-1 mice are treatment-related, they could have been induced by a non-genotoxic mechanism; (3) the response in transgenic animals may provide useful complementary results to better assess carbaryl's potential genotoxic hazard to humans.

Extensive interbreed, but minimal intrabreed, variation of DLA class II alleles and haplotypes in dogs.

The DLA class II genes in the dog major histocompatibility complex are highly polymorphic. To date, 52 DLA-DRB1, 16 DLA-DQA1 and 41 DLA-DQB1 allelic sequences have been assigned. The aim of this study was to examine the intrabreed and interbreed variation of DLA allele and haplotype frequencies in dogs, and to ascertain whether conserved DLA class II haplotypes occur within and between different breeds. One thousand and 25 DNA samples from over 80 different breeds were DLA class II genotyped, the number of dogs per breed ranging from 1 to 61. DNA sequence based typing and sequence specific oligonucleotide probing were used to characterize dogs for their DLA-DRB1, DQA1 and DQB1 alleles. The high frequency of DLA class II homozygous animals (35%), allowed the assignment of many haplotypes despite the absence of family data. Four new DLA alleles were identified during the course of this study. Analysis of the data revealed considerable interbreed variation, not only in allele frequency, but also in the numbers of alleles found per breed. There was also considerable variation in the number of breeds in which particular alleles were found. These interbreed variations were found in all three DLA class II loci tested, and also applied to the three-locus haplotypes identified. Within this data set, 58 different DLA-DRB1/DQA1/DQB1 three-locus haplotypes were identified, which were all found in at least two different animals. Some of the haplotypes appeared to be characteristic of certain breeds. The high interbreed, and relatively low intrabreed, variation of MHC alleles and haplotypes found in this study could provide an explanation for reports of interbreed variation of immune responses to vaccines, viruses and other infections.

Critique of the paper: could pesticide toxicology studies be more relevant to occupational risk assessment? (by Ross et al., 2001).

The toxicology studies required for the registration of a pesticide are not necessarily well adapted to user safety assessment. This problem can be overcome in some cases by improving the guideline studies. In other cases the guidelines are based on animal models which are poor models for man; the most obvious example is rat percutaneous penetration. In other cases it may simply be impractical to have a model which can be extrapolated to all exposure situations. This is the case for intermittent exposure which is infinitely variable. The general conclusion is that the entire data package should be re-examined to make it more relevant to risk assessment.

Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

Thyroxine (T(4))-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague-Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T(4)-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T(4)-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), beta-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T(4)-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T(4)-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T(4)-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T(4)-UGT activities.

Setting of acute reference doses for pesticides based on existing regulatory requirements and regulatory test guidelines.

Reference doses (RfD) for the definition of tolerable food residues have traditionally been based on the lowest no observed adverse effect level (NOAEL), which usually comes from chronic toxicity studies. While this is generally agreed to be a safe approach to evaluate the overall significance of expected consumption it is clear that it is not appropriate for evaluation of the toxicological significance of residues in a single meal. Standard acute toxicity tests are not designed to generate an NOAEL, from which an RfD can be derived. They are more appropriate to evaluating risk following accidental high exposure to the product itself rather than to food residues. A typical toxicological database for a pesticide active substance contains studies which may be appropriate, on a case-by-case basis, to evaluate shorter term endpoints of interest for specific molecules, such as developmental or acute neurotoxicity studies. However, their specificity limits their scope of application. General toxicological endpoints are well covered by short-term, 28- or 90-day, guideline studies. However, neither of these studies is ideal for setting of an acute RfD (ARfD) as the treatment period is significantly longer than the duration of consumer exposure. This could be balanced by applying a reduced safety factor to the NOAEL to set the ARfD. Alternatively, a test guideline could be designed to generate a relevant acute NOAEL but the time necessary for development, validation and acceptance of such a guideline means that an interim approach is, in any case, necessary.

The development of methods for assessing the in vivo oestrogen-like effects of xenobiotics in CD-1 mice.

The increasing awareness and concern about the potential health risks posed to the ecosystem and to man by endocrine disrupting chemicals with oestrogen-like activity in the environment has focused attention on the need for developing sensitive and specific methods for identifying these xenobiotics and to evaluate their degrees of toxic effects. We have conducted dose response studies in immature (21 days old) CD-1 female mice treated with four compounds, diethylstilboestrol (DES) (0.1 microg to 25 mg/kg body weight), alpha-zearalanol (0.5 mg to 25 mg/kg body weight), methoxychlor (0.5 mg to 500 mg/kg body weight) and bisphenol A (10 microg to 100 mg/kg body weight) administered subcutaneously daily for 3 days, and measured a number of uterine markers in treated and control (vehicle treated) mice. These were, in addition to the commonly measured changes in relative uterus weight and histopathological examination of uterine tissue, three other markers indicative of uterotrophic effects, namely, uterine luminal epithelium BrdU labelling index over the last 24 hr, peroxidase activity and lactoferrin expression. All of these markers showed clear dose-related increases in DES- and methoxychlor-treated animals. In the case of alpha-zearalanol treatment, relative uterine weight, peroxidase activity and lactoferrin expression showed dose-related increases at all the doses investigated. BrdU incorporation (an index of cell proliferation) also progressively increased at dose levels ranging from 0.1 mg to 5.0 mg/kg body weight, but apparently decreased at 25 mg/kg body weight. In contrast to these findings, bisphenol-A treatment showed no consistent changes in any of the four markers at the dose levels investigated. Additionally, studies were also conducted on a number of chemicals in CD-1 mice at one dose level. The chemicals investigated were: bisphenol A (1 g/kg body weight/day), naringenin (1 g/kg body weight/day) o,p'-DDT (500 mg/kg body weight/day), genistein (1 g/kg/day), coumestrol (0.5 mg/kg/day) and chlordecone (20 mg/kg/day) administered subcutaneously daily for 3 days. There was some variability in response of the markers perhaps indicating that the chemicals did not all act in the same way. The findings of our exploratory in vivo studies in CD-1 mice suggest that the measurement of a range of uterine markers, in addition to organ weight and histopathology, would provide useful information on the potential oestrogenicity of chemicals.

The p53 heterozygous knockout mouse as a model for chemical carcinogenesis in vascular tissue.

Heterozygous p53 knockout mice were investigated as a potential model for vascular tumor carcinogenesis. Groups of 20 male mice were exposed by gavage for 6 months to the vascular carcinogen urethane at 1, 10, or 100 mg/kg body weight/day. Wild-type and heterozygous p53 knockout control groups were exposed by gavage to the vehicle alone. Another group of 20 male mice received d-limonene by gavage (d-limonene is noncarcinogenic in mice). The high dose of urethane caused early mortality in the majority of mice associated with histopathologic evidence of toxicity and tumors, including a high incidence of benign and malignant vascular tumors, in all animals. At the intermediate dose, toxicity was less marked and 3 of 20 mice had tumors; mice that received the low dose did not have signs of toxicity or neoplasia. The two control groups had no tumors and the d-limonene group had one tumor of the prostate, which was considered spontaneous. We conclude that the p53 knockout mouse is a useful tool for investigating vascular tumorogenesis.

Laying hens in large flocks in a perchery system: influence of stocking density on location, use of resources and behaviour.

1. ISA Brown laying hens (3000) were housed in a perchery in 10 pens, each with 300 birds. The pens varied in size to produce 4 different stocking densities: 9.9 birds/m2 (n = 3), 13.5/m2 (n = 2), 16.0/m2 (n = 2) and 19.0/m2 (n = 3). Observations began at 20 weeks of age and continued until 69 weeks to establish the spatial distribution of the birds, usage of the different resources and the expression of behaviour. 2. Overall, birds spent most time on the perch frame (47%), litter area (23%), slatted floor (17%) and nestbox area (9%). 3. There was no effect of density on the proportion of birds observed on the slatted floor or on the elevated perches but as density increased the proportion on the littered area decreased. 4. Space usage was determined vertically, horizontally and longitudinally. Individual birds were seen to use about 80% of the pen volume available to them. This value was similar for all densities and showed that individuals did not have separate home ranges. 5. Fewer vertical movements were made within the main perch frame at the upper than at the lower levels but movements between the perches of the main frame and the nestbox rails were relatively frequent. This may help birds move up and down through the main frame. 6. Behaviours which decreased in incidence with crowding included moving, foraging and dust-bathing. Behaviours which increased with crowding included standing. Behaviours which were unaffected included resting, preening, prelaying behaviour, comfort behaviour and the minor behaviours. 7. The proportion of birds engaged in feeding and drinking was unaffected by density, except each time the chain feeders (which operated intermittently) ran more hens were seen feeding at the lower densities. This suggests that food delivery stimulated feeding behaviour; there may have been some restriction at the higher densities on birds feeding when and where they wanted. 8. Stocking density had no effect on the frequency of agonistic interactions: threats, lunges, comb/head pecks, chases and fights. 9. The incidence of damaging pecking was low and not density dependent. 10. Increasing density within the range investigated inhibited the expression of a number of behaviours and limited the use of specific resources: bird welfare at 19 birds/m2 may have been very slightly impaired.

Treating Japanese quail with vitamin C does not facilitate their capture by the experimenter.

1. Vitamin C supplementation reduces fear of novel situations and of people. The present study examined its effects on the ease of capture of male Japanese quail by the experimenter. 2. At 20 d of age, quail received either vitamin C (ascorbyl-2-polyphosphate, APP, 1 g L-ascorbic acid/l) solution or untreated drinking water (UDW) for 24 h before they were mixed in 2 groups of 40 (20 APP + 20 UDW. All the birds in 1 group were caught individually by an unsighted experimenter whereas a sighted catcher captured the others. The bird's identity was noted each time. This capture/recapture procedure was repeated 6 times for each group (12 capture trials per bird) and an overall capture rank across all 12 trials was assigned to each bird. 3. Regardless of whether the catcher was sighted or unsighted, the mean ranks of neither APP nor UDW quail differed significantly from the value expected by chance. Neither were there any linear trends in the effects of repeated testing. Thus, prior treatment with vitamin C neither facilitated nor hindered capture. 4. Body weights were similar in both treatment groups and there were no significant intra-individual correlations between body weight and capture rank. 5. Highly significant tendencies were found for individual birds to be caught at similar stages of each capture trial regardless of treatment or test situation. This finding sounds a cautionary note for all studies involving putatively random sampling of a population.