A non-invasive method for detecting the metabolic stress response in rodents: characterization and disruption of the circadian corticosterone rhythm

Plasma corticosterone (CORT) measures are a common procedure to detect stress responses in rodents. However, the procedure is invasive and can influence CORT levels, making it less than ideal for monitoring CORT circadian rhythms. In the current paper, we examined the applicability of a non-invasive fecal CORT metabolite measure to assess the circadian rhythm. We compared fecal CORT metabolite levels to circulating CORT levels, and analyzed change in the fecal circadian rhythm following an acute stressor (i.e. blood sampling by tail veil catheter). Fecal and blood samples were collected from male adolescent rats and analyzed for CORT metabolites and circulating CORT respectively. Fecal samples were collected hourly for 24 h before and after blood draw. On average, peak fecal CORT metabolite values occurred 7-9 h after the plasma CORT peak and time-matched fecal CORT values were well correlated with plasma CORT. As a result of the rapid blood draw, fecal production and CORT levels were altered the next day. These results indicate fecal CORT metabolite measures can be used to assess conditions that disrupt the circadian CORT rhythm, and provide a method to measure long-term changes in CORT production. This can benefit research that requires long-term glucocorticoid assessment (e.g. stress mechanisms underlying health).     

In vivo solid-phase microextraction for monitoring intravenous concentrations of drugs and metabolites

This protocol for in vivo solid-phase microextraction (SPME) can be used to monitor and quantify intravenous concentrations of drugs and metabolites without the need to withdraw a blood sample for analysis. The SPME probe is inserted directly into a peripheral vein of a living animal through a standard medical catheter, and extraction occurs typically over 2-5 min. After extraction, the analytes are removed from the sorbent and analyzed by, for example, liquid chromatography-tandem mass spectrometry. It has been validated in comparison with conventional blood analysis, and we describe here the in vitro experiments typically conducted during method development. The new-generation biocompatible SPME probes are designed specifically for extraction of semi-volatiles and nonvolatiles directly from aqueous samples and can be steam sterilized. Sorbents are coated on fine-gauge surgical steel wire (200-μm diameter), which is more rugged and biocompatible than conventional fibers (100-μm fused silica fiber). They incorporate a binding agent that resists fouling by the biological matrix and does not cause an immune response in the experimental animal. The sorbents used (coating thickness of ～50 μm) are selected for their affinity for the types of small molecules of interest. The procedure is illustrated by the analysis of benzodiazepines with polypyrrole-coated wires inserted into peripheral blood vessels of beagles, although it can be adapted for use in smaller animals. The in vivo sampling can require as little as 1 min, in which case the entire procedure from sampling to instrumental analysis can take as little as 30 min.     

Tail docking in pigs: acute physiological and behavioural responses

Tail docking of piglets is a routine procedure on farms to control tail-biting behaviour; however, docking can cause an acute stress response. The objectives of this research were to determine the stress responses to tail docking in piglets and to compare two methods of tail docking; cautery iron (CAUT) and the more commonly used blunt trauma cutters (BT). At approximately 6 days of age, piglets were tail docked using CAUT (n = 20), BT (n = 20) or sham tail docked with their tails remaining intact (CON; n = 40). Blood samples were taken prior to tail docking and at 30, 60 and 90 min after tail docking to evaluate the effect of tail docking on white blood cell (WBC) measures and cortisol concentrations. The above experiment was repeated to observe behaviour without the periodic blood sampling, so as not to confound the effects of blood sampling on piglet behaviour. Piglet behaviour was recorded in the farrowing crate using 1 min scan-samples via live observations for 60 min prior to and 90 min after tail docking. Total WBC counts were reduced (P > 0.05) among BT and CAUT compared with CON piglets 30 min after tail docking. Cortisol concentrations were higher (P < 0.01) among BT compared with CON and CAUT piglets 60 min after tail docking. Cautery and BT-docked piglets spent more (P < 0.05) time posterior scooting compared with CON piglets between 0 and 15 min, and 31 and 45 min after tail docking. Piglets tail docked using CAUT and BT tended to spend more (P < 0.07) time sitting than CON piglets between 0 and 15 min post tail docking. Elevated blood cortisol can be reduced by the use of the CAUT rather than the BT method of tail docking. Although the tail docking-induced rise in cortisol was prevented by using CAUT, the behavioural response to BT and CAUT docking methods was similar.     

Comparative analysis of ACTH and corticosterone sampling methods in rats

A frequently debated question for studies involving the measurement of stress hormones in rodents is the optimal method for collecting blood with minimal stress to the animal. Some investigators prefer the implantation of indwelling catheters to allow for frequent sampling. Others argue that the implantation of a catheter creates a chronic stress to the animal that confounds stress hormone measures and therefore rely on tail vein sampling. Moreover, some investigators measure hormones in trunk blood samples obtained after anesthesia, a practice that may itself raise hormone levels. To address these controversies, we 1) compared plasma ACTH and corticosterone (Cort) concentrations in pre- and poststress rat blood samples obtained via previously implanted vena cava catheters, tail vein nicks, or clipping the tip off the tail and 2) compared plasma ACTH and Cort in rat blood samples obtained by decapitation with and without anesthesia. Rats sampled via indwelling catheters displayed lower prestress ACTH levels than those sampled by tail vein nick if the time to acquire samples was not limited; however, elevated basal ACTH was not observed in samples obtained by tail clip or tail nick when the samples were obtained within 3 min. Baseline Cort levels were similar in all groups. After restraint stress, the profile of the plasma ACTH and Cort responses was not affected by sampling method. Decapitation with prior administration of CO2 or pentobarbital sodium increased plasma ACTH levels approximately 13- and 2-fold, respectively, when compared with decapitation without anesthesia. These data indicate that tail vein nicking, tail clipping, or indwelling venous catheters can be used for obtaining plasma for ACTH and Cort during acute stress studies without confounding the measurements. However, the elevation in basal ACTH seen in the tail vein nick group at baseline suggests that sampling needs to be completed rapidly (<3 min) to avoid the initiation of the pituitary stress response. Death by CO2 and pentobarbital sodium injection before trunk blood collection cause significant stress to animals, as reflected in the elevated plasma ACTH levels. These results support the use of either chronic vascular cannulas or sampling from a tail vein. However, collection of blood under pentobarbital sodium or CO2 anesthesia is likely to confound the results of stress studies when ACTH is an important endpoint.     

Comparative application of the coefficient of variation and range-based statistics for assessing the taxonomic composition of fossil samples

Dental variation has been used commonly to assess taxonomic composition in morphologically homogeneous fossil samples. While the coefficient of variation (CV) has been used traditionally, range-based measures of variation, such as the range as a percentage of the mean (R%) and the maximum/minimum index (I_max/min) have recently become popular alternatives. The current study compares the performance of these statistics when applied to single- and pooled-species dental samples of extant Cercopithecus species. A common methodology for such problems of species discrimination has been to simply compare the maximum value of a variation statistic observed in extant samples with that observed in the fossil sample. However, regardless of what statistic is used, this approach has an unknowable Type I error rate, and usually has low power to detect multiple species. A more appropriate method involves a formal hypothesis test. The null hypothesis is that the level of variation in the fossil sample does not exceed what might be expected in a sample drawn randomly from a reference population, taking into account sampling error and the size of the fossil sample. Previous research using this method with the CV has indicated that it offers considerable power at an acceptable Type I error rate. In the current study, the data of primary interest were posterior dental dimensions for single- and pooled species samples from extant Cercopithecus species. In addition, the study also investigated the relative performance of variation statistics when applied to highly dimorphic canine dimensions, since much recent work has employed sexually dimorphic dental dimensions for assessing single-species hypotheses. The results indicate that the CV consistently out-performed the range-based statistics when using posterior dental dimensions to test a single-species hypothesis. Regardless of which statistic was used, tests on sexually dimorphic dimensions offered minimal power. In consideration of these results and the problem of studywise Type I error rates, we recommend against the use of multiple measures of variation to test for multiple species composition, and advocate the CV as the statistic of choice when using the method of Cope & Lacy (1992). For similar reasons, we argue for careful selection of dental variables for inclusion in such analyses, and in particular recommend against including sexually dimorphic dimensions when testing for multiple species composition.     

A non‐invasive stress assay based upon measurement of free cortisol released into the water by rainbow trout

A procedure previously used for sex steroids was adapted to extract free cortisol and cortisone from water samples taken from rainbow trout Oncorhynchus mykiss tanks. Both corticosteroids could be readily detected by radioimmunoassay (RIA), with cortisol being predominant. All stages of the sampling, extraction and RIA procedure were validated for cortisol. An intermittent problem with poor replication was traced to the use of diethyl ether during the extraction procedure, and was overcome by the use of ethyl acetate. Other modifications were also introduced to speed up the procedure. The concentration and time course of release of both corticosteroids were shown to be related to the degree of stress that the fish had been subjected to. It was confirmed that cortisol concentrations in water and estimated cortisol release rates increased in response to handling stress, and that both were correlated with plasma cortisol concentrations. The potential for using water cortisol concentration and release rates to assess the primary stress response of fishes as a non‐invasive alternative to blood sampling is discussed.     

The physiological response of the Caribbean reef shark (Carcharhinus perezi) to longline capture

Longline fishing is the most common elasmobranch capture method around the world, yet the physiological consequences of this technique are poorly understood. To quantify the sub-lethal effects of longline capture in the commonly exploited Caribbean reef shark (Carcharhinus perezi), 37 individuals were captured using standard, mid-water longlines. Hook timers provided hooking duration to the nearest minute. Once sharks were landed, blood samples were taken and used to measure a suite of physiological parameters. Control data were obtained by sampling an additional three unrestrained Caribbean reef sharks underwater at an established shark feeding site. The greatest level of physiological disruption occurred after 120-180min of hooking, whereas sharks exposed to minimal and maximal hook durations exhibited the least disturbed blood chemistry. Significant relationships were established between hooking duration and blood pH, pCO(2), lactate, glucose, plasma calcium and plasma potassium. Longline capture appears more benign than other methods assessed to date, causing a shift in the stress response from acute at the onset of capture to a sub-acute regime as the capture event progresses, apparently facilitating a degree of physiological recovery. Continued investigation into the physiological response of elasmobranchs to longline capture is vital for the effective management of such fisheries.     

Adaptation of corticosterone-but not beta-endorphin-secretion to repeated blood sampling in rats

Effects of short-term repeated blood sampling on the secretion of corticosterone (CORT) and beta-endorphin (beta-END) were evaluated in male Wistar rats. Blood was drawn from the tail vein of conscious rats four times within 2 h both at the peak and trough period of the diurnal corticosterone secretion cycle. All rats were well accustomed to the procedure. The main findings were: (1) At both sampling intervals, CORT increased significantly in response to the first sampling and declined to baseline values in successive samples. (2) beta-END also increased significantly in response to the first sampling but remained elevated in successive samples. (3) Intensities of initial CORT and beta-END responses correlated positively with each other and with the baseline beta-END values. Feedback inhibition of CORT secretion with sustained elevation of beta-END titres suggests a moderate stress intensity of the repeated blood sampling procedures. In general, due to lack of short-term feedback inhibition, beta-END seems to reflect the effects of repeated administration of moderate intense stressors more closely than CORT.     

Measuring and controlling data quality in biological assemblage surveys with special reference to stream benthic macroinvertebrates

1. Biological assemblage surveys primarily aim to characterise species composition and relative abundance at one or more spatial or temporal scales. Data interpretation and conclusions are dependent on how well samples characterise the assemblage of interest. 2. Conventional measures of data quality, e.g. standard deviations or coefficients of variation, were designed for single variable estimation, and they are either insufficient or invalid for assessing the quality of data describing entire assemblages. Similarity indices take species composition and relative abundance into account and may be used to effectively measure and control the quality of data used to characterise assemblage structure. 3. The average Jaccard coefficient (JC) calculated across multiple pairs of replicate samples, i.e. autosimilarity JC (AJC), is conceptually and numerically related to the average coefficient of variation in the densities of all species recorded, a measure of sampling precision, and to the proportion of total species richness sampled, a measure of sampling accuracy. 4. We explored how AJC can be used to assess the effect of different potential sources of error on the quality of assemblage survey data, including the sampling effort used both within regions and at individual sites, the individuals collecting samples, sub‐sampling procedures, and consistency of taxon identification. 5. We found that the autosimilarity‐based approach overcomes most weaknesses associated with conventional measures of data quality and can be used to effectively measure and control the quality of assemblage survey data.     

A refined method for sequential blood sampling by tail incision in rats

Levels of endogenous or administered substances can be estimated by blood sampling. This allows an evaluation of the relationship between clinical signs, physiological parameters, pharmacological treatments and behaviour of the animal. We show that blood samples can be taken occasionally as well as sequentially by means of a small incision at the end of the rats' tails. Up to 300 microl of blood can be collected within 90 s. The advantages of this method are: (i) anaesthesia and surgery or restraint of the animal are not necessary; (ii) the procedure can be considered stress-free as indicated by the low, basal levels of the stress hormone corticosterone, even with frequent sequential blood sampling over 3 h; and (iii) it can be used for longitudinal studies allowing intra-individual comparisons over months and even years. Blood samples collected via an intravenous catheter and, at the same time, by our tail incision method resulted in comparable amounts of corticosterone. Moreover, we consider the tail incision method for rats to be 'animal-friendly' and a real alternative to other conventionally used blood sampling techniques.     

Adjoint multi-start-based estimation of cardiac hyperelastic material parameters using shear data

Cardiac muscle tissue during relaxation is commonly modeled as a hyperelastic material with strongly nonlinear and anisotropic stress response. Adapting the behavior of such a model to experimental or patient data gives rise to a parameter estimation problem which involves a significant number of parameters. Gradient-based optimization algorithms provide a way to solve such nonlinear parameter estimation problems with relatively few iterations, but require the gradient of the objective functional with respect to the model parameters. This gradient has traditionally been obtained using finite differences, the calculation of which scales linearly with the number of model parameters, and introduces a differencing error. By using an automatically derived adjoint equation, we are able to calculate this gradient more efficiently, and with minimal implementation effort. We test this adjoint framework on a least squares fitting problem involving data from simple shear tests on cardiac tissue samples. A second challenge which arises in gradient-based optimization is the dependency of the algorithm on a suitable initial guess. We show how a multi-start procedure can alleviate this dependency. Finally, we provide estimates for the material parameters of the Holzapfel and Ogden strain energy law using finite element models together with experimental shear data.     

Non-invasive measurement of faecal glucocorticoid metabolites in Upland Geese <Emphasis Type="Italic">Chloephaga</Emphasis><Emphasis Type="Italic">picta</Emphasis>

Glucocorticoid (GC) hormones rise in response to stressors, including natural events including weather or predator presence, and human activities, such as hunting, scientific research or recreational visits. However, because blood sampling itself causes stress and is dangerous or even impossible in some wildlife species, feedback-free methods for GC determination are needed to assess stress in these animals. Faecal GC analyses have thus gained interest. Here, we validate a non-invasive method to estimate the physiological stress in the Upland goose Chloephaga picta. An adrenocorticotropin hormone (ACTH) challenge was conducted in captive adults (female and male), and droppings were collected before, during and after the experiment. Corticosterone metabolite (CM) secretion in response to the ACTH challenge was measured with several enzyme immunoassays (EIA) to find the most appropriate test. We used CM levels during the periods before and after the experiment as control data. An EIA for 11-oxoetiocholanolone achieved the highest response to the ACTH challenge and also reflected a stress response to unfamiliar environment. Furthermore, CM concentrations of dry samples were highly correlated with the corresponding non-dried (frozen) samples. The data suggest that this method is appropriate to measure the stress in Upland geese, and that samples can be stored either frozen or dry form.     

Effectiveness of saliva collection and enzyme-immunoassay for the quantification of cortisol in socially housed baboons

Circulating cortisol levels are often used to assess the biological stress response in captive primates. Some methods commonly used to collect blood samples may alter the stress response. As such, noninvasive means to analyze cortisol levels are increasingly being developed. We adapted an existing collection method to simultaneously obtain saliva from multiple socially living hamadryas baboons (Papio hamadryas hamadryas) and validated an enzyme-immunoassay kit to quantify cortisol within the saliva samples. Over a period of 12 months, saliva samples were regularly collected from approximately half of the 18-member colony, representing younger monkeys who were more willing to participate. The assay met the four criteria typically used to assess the effectiveness of a new analytical technique: parallelism, precision, accuracy, and sensitivity. Cortisol levels were also proportional to those expected given published plasma levels of cortisol in baboons. Further, salivary cortisol levels increased in individuals following significant stress-related events, such as removal from the group, indicating biological validation. The technique provided a reliable and effective means to assess a physiological indicator of stress in a social group without initiating a stress response owing to handling or sedation, and provided a real-time assessment of cortisol levels and reactivity.     

A refined blood collection method for quantifying corticosterone

In many rodent laboratories, blood samples are collected from rats using the tail vein nick procedure and analyzed to quantify blood corticosterone levels as an indicator of stress. The standard method of corticosterone quantification often requires the collection of a relatively large volume of blood, followed by the extraction of the blood plasma. An alternative blood sampling method requires the collection of only a drop of blood on paper (the 'drop' method), minimizing handling of the animals, and does not require plasma extraction. The authors aimed to validate the drop method of blood sampling for use in corticosterone quantification. They induced stress in rats by cerebral ischemia, collected blood samples at various intervals using both the drop method and the plasma extraction method and then quantified corticosterone by radioimmunoassay. Corticosterone levels of the ischemic rats were compared with those of sham-operated rats and those of ischemic rats that had been given metyrapone, a glucocorticoid synthesis inhibitor, prior to vessel occlusion. Blood corticosterone levels in the samples obtained from the same animal using the two different methods were highly correlated for all rats. The authors further provide a regression model that can be used to predict plasma corticosterone values from those obtained from the drop blood samples. Quantification of corticosterone from only a small drop of blood has many practical and ethical advantages and should be considered as an alternative to standard methods.     

Individual Monitoring of Immune Response in Atlantic Salmon Salmo salar following Experimental Infection with Infectious Salmon Anaemia Virus (ISAV)

Monitoring the immune response in fish over the progression of a disease is traditionally carried out by experimental infection whereby animals are killed at regular intervals and samples taken. We describe here a novel approach to infectiology for salmonid fish where blood samples are collected repeatedly in a small group of PIT-tagged animals. This approach contributes to the reduction of animals used in research and to improved data quality. Two groups of 12 PIT-tagged Atlantic salmon (Salmo salar) were i.p infected with Infectious Salmon Anaemia Virus (ISAV) or culture medium and placed in 1 m³ tanks. Blood samples were collected at 0, 4, 8, 12, 16, 21 and 25 days post infection. The viral load, immune and stress response were determined in individual fish by real-time quantitative PCR (QPCR) on the blood cells, as well as the haematocrit used as an indicator of haemolysis, a clinical consequence of ISAV infection. “In-tank” anaesthesia was used in order to reduce the stress related to chase and netting prior to sampling. The data were analysed using a statistical approach which is novel with respect to its use in fish immunology. The repeated blood collection procedure did not induce stress response as measured by HSP70 and HSP90 gene expression in the un-infected animals. A strong increase in viraemia as well as a significant induction of Mx and γIP gene expression were observed in the infected group. Interleukin 10 was found induced at the later stage of the infection whereas no induction of CD8 or γ IFN could be detected. These results and the advantages of this approach are discussed.     

Identification of transgenic mice by direct PCR analysis of lysates of epithelial cells obtained from the inner surface of the rectum

Conventional screening protocols for transgene integration in mice employ tail tips or blood samples as sources to obtain genomic DNA preparations. We have developed a simple alternative non-surgical method. Epithelial cells are scraped off the inner surface of the rectum with a sterile plastic inoculation loop and are lysed with Kawasaki buffer. The lysate can be directly examined in a polymerase chain reaction (PCR) analysis without any need for further DNA purification. This procedure causes minimal harm and stress to the animals and repeated samples can be obtained as often as necessary. This technique has been used successfully to identify transgenic mice from a number of different lines. The method allows quick screening of numerous animals and contributes to a reduction of the number of surgical biopsies required     

Searching for the Optimal Sampling Solution: Variation in Invertebrate Communities,Sample Condition and DNA Quality

There is a great demand for standardising biodiversity assessments in order to allow optimal comparison across research groups. For invertebrates, pitfall or flight-interception traps are commonly used, but sampling solution differs widely between studies, which could influence the communities collected and affect sample processing (morphological or genetic). We assessed arthropod communities with flight-interception traps using three commonly used sampling solutions across two forest types and two vertical strata. We first considered the effect of sampling solution and its interaction with forest type, vertical stratum, and position of sampling jar at the trap on sample condition and community composition. We found that samples collected in copper sulphate were more mouldy and fragmented relative to other solutions which might impair morphological identification, but condition depended on forest type, trap type and the position of the jar. Community composition, based on order-level identification, did not differ across sampling solutions and only varied with forest type and vertical stratum. Species richness and species-level community composition, however, differed greatly among sampling solutions. Renner solution was highly attractant for beetles and repellent for true bugs. Secondly, we tested whether sampling solution affects subsequent molecular analyses and found that DNA barcoding success was species-specific. Samples from copper sulphate produced the fewest successful DNA sequences for genetic identification, and since DNA yield or quality was not particularly reduced in these samples additional interactions between the solution and DNA must also be occurring. Our results show that the choice of sampling solution should be an important consideration in biodiversity studies. Due to the potential bias towards or against certain species by Ethanol-containing sampling solution we suggest ethylene glycol as a suitable sampling solution when genetic analysis tools are to be used and copper sulphate when focusing on morphological species identification and facing financial restrictions in biodiversity studies.     

A high-throughput core sampling device for the evaluation of maize stalk composition

Background

A major challenge in the identification and development of superior feedstocks for the production of second generation biofuels is the rapid assessment of biomass composition in a large number of samples. Currently, highly accurate and precise robotic analysis systems are available for the evaluation of biomass composition, on a large number of samples, with a variety of pretreatments. However, the lack of an inexpensive and high-throughput process for large scale sampling of biomass resources is still an important limiting factor. Our goal was to develop a simple mechanical maize stalk core sampling device that can be utilized to collect uniform samples of a dimension compatible with robotic processing and analysis, while allowing the collection of hundreds to thousands of samples per day.

Results

We have developed a core sampling device (CSD) to collect maize stalk samples compatible with robotic processing and analysis. The CSD facilitates the collection of thousands of uniform tissue cores consistent with high-throughput analysis required for breeding, genetics, and production studies. With a single CSD operated by one person with minimal training, more than 1,000 biomass samples were obtained in an eight-hour period. One of the main advantages of using cores is the high level of homogeneity of the samples obtained and the minimal opportunity for sample contamination. In addition, the samples obtained with the CSD can be placed directly into a bath of ice, dry ice, or liquid nitrogen maintaining the composition of the biomass sample for relatively long periods of time.

Conclusions

The CSD has been demonstrated to successfully produce homogeneous stalk core samples in a repeatable manner with a throughput substantially superior to the currently available sampling methods. Given the variety of maize developmental stages and the diversity of stalk diameter evaluated, it is expected that the CSD will have utility for other bioenergy crops as well.