Laboratory Animals Volume 43, Number 3, July 2009

ORIGINAL ARTICLES Kasanen et al. The diet board: welfare impacts of a novel method of dietary restriction in laboratory rats, pp. 215—223

SUMMARY: the authors explore the possible short-term welfare impact of using dietary restriction in outbred Wistar rats when using the so-called "diet board". The board is made of aspen wood and contains holes where food pellets (chow) are inserted. The rats have to gnaw the wood to get to the pellets. The authors used the following physiological parameters as indicators of stress: serum corticosterone, faecal secretion of IgA, weight and adrenaline and noradrenaline content of adrenal glands, presence of stomach ulcers. Two groups of siblings (n=30 per group) were compared: those fed restrictively with the diet board and those fed ad-libitum. After 10 weeks, the rats fed on the diet board weighed 15% less than rats fed ad-libitum. They also had higher serum corticosterone levels and lower faecal IgA than rats fed ad-libitum, although the latter had significantly heavier left adrenal glands, with higher adrenaline and noradrenaline contents. No gastric ulcers were found in either group. The results suggest that the use of the diet board triggers some sort of a stress response in rats. The authors speculate as to whether this stress response is beneficial or detrimental to the animals but are of the firm opinion that the diet board, in the long-term, is a husbandry refinement.

QUESTIONS: 1. Which of the following is not considered an indicator of stress in rodents? a. High serum corticosterone levels b. High faecal secretion of IgA c. Heavier adrenal glands d. Presence of stomach ulcers 2. Which of the following is false with respect to controlled dietary restriction in rodents? a. It has been shown to promote longevity b. It reduces the incidence of neoplasia c. It prevents obesity d. It increases interindividual variation 3. What is the main advantage of using the diet board as a method of dietary restriction in rats? a. It allows group-housing and maintenance of normal diurnal eating rhythms b. It allows the animals to keep their incisor teeth short and sharp c. It is relatively non-expensive d. It is environmentally friendly 4. Which of the following is true with respect to the use of faecal IgA levels in rats? a. They are positively correlated with faecal corticosterone secretion b. They are a better indicator of chronic stress rather than acute stress c. The method is cumbersome for the animals d. Extraction of IgA cannot be done on frozen faecal pellets

ANSWERS: 1. b. High faecal secretion of IgA 2. d. It increases interindividual variation 3. a. It allows group-housing and maintenance of normal diurnal eating rhythms 4. b. They are a better indicator of chronic stress rather than acute stress

Diesch et al. Electroencephalographic responses to tail clamping in anaesthetized rat pups, pp. 224—231

Task 1: Recognize pain and/or distress. Primary species: rat (rodent).

Introduction: A functional state of consciousness in the brain and animal with capacity for sentient are prerequisites for experience any sensation. Timing of the onset of the capacity for pain perception may be expected to vary between species. The rat pup is the focus of the present paper after the publication of peripheral pain mechanisms. The aim of this study was to investigate cerebral responses to noxious stimulus in young rat pups before, during and after encephalographic (EEG) differentiation to determine the age at which they are likely to experience pain.

Material and methods: 43 outbreed Sprague-Dawley rat pups of either gender were weaned at 20 days and assigned to three groups. Experimental procedures were conducted during daylight and animals were anaesthetized with halothane. Needle electrodes were placed transdermally or at a shallow angle through the cranium. Then animals stabilized for 2 min prior recording a baseline EEG/ECoG. Tail was stimulated for 10 s by application of a hemostat. Pup behavior, movements and vocalizations were recorded following the placement of electrodes and the clamping of the tail. EEG recordings were using amplifiers set to a gain of 1000 and pass band of 0.1-0.5 kHZ EEG/ECoG analysis. Median frequency (F50) and spectral edge frequency (F95) of the power spectrum in the range of 1-3o Hz were determined before and after clamping.

Results: A postnatal increase in EEG power with isoelectric traces before clamping in pups aged 5-7 days. These pups did not exhibit EEG response after clamping. Pups aged 12-14 days showed intermittent EEG activity before clamping and after clamping there was a significant decrease.

Discussion and conclusions: The cerebral responses to noxious stimulation of pup’s behavior at 18-20 days suggest that animals older than about 18 days are normally capable of conscious perception and pain experience. The conclusion is that rat pups aged 5-7 days and younger are not likely to perceive pain and that the ability to perceive pain develops gradually between postnatal ages 12-14 days and 21 days.

QUESTIONS 1. Cerebral cortex and associated structures are known to be involved in the perception of pain. These structures are: a. Brainstem. b. Thalamus. c. Cortex. d. All of them. 2. Changes in cerebral activity associated with the cognitive perception of pain are thought to be reflected in electroencephalographic (EEG) spectra. True or False 3. Tail clamping by application of a hemostat closed to the first ratchet and held for 10 seconds is not considered a supramaximal stimulus in rat pups: True or False. 4. ARAS means: a. Ascending reticular activating system. b. Activated reticular ascending system. c. Ascending regular activated systems. d. None of the previous options. 5. Well-established developmental changes in the central nervous system during postnatal period are: a. Myelination. b. Proliferation of neurites. c. Increases in synaptic vesicles. d. Cortical synaptogenesis. e. All of them are correct.

ANSWERS: 1. d. 2. True. 3. False. 4. a. 5. e.

Coulter et al. Reported analgesic administration to rabbits, pigs, sheep, dogs and non-human primates undergoing experimental surgical procedures, pp. 232—238

Domain 2 - Management of Pain and Distress; T2. Minimize or eliminate pain and/or distress.

Multiple species- Rodents, Rabbits, Pigs, Sheep, Dogs and NHPs.

SUMMARY: The authors of this article attempted to investigate analgesic administration to dogs, pigs, nonhuman primates, rabbits and sheep (and also comment on rodents) following experimental surgery, and determine whether species differences in analgesic administration to animals used in scientific procedures exist. They carried out a literature review (using Scirus search engine) of studies published between 2000 and 2001 and from 2005 to 2006. Their aim was to review the frequency of use of analgesics, the agents used and to determine if the use of analgesics was related to the severity of the experimental procedure.

The investigation was originated by hypothesis of “speciecism” in the lab animal setting– where larger lab animal species were favored by animal care staff and because of general public concerns and opinions.

One hundred and fifty papers, 75 from each time period were selected from 61 journals for inclusion in the review.

The reported administration of systemic analgesics to dogs, pigs, non-human primates, rabbits and sheep increased from 50% in 2000–2001 to 63% in 2005–2006. There was an increase in the reported analgesic administration for all of the species examined between the two time periods with the exception of pigs, where the reported systemic analgesic administration was the same for both time periods, 67%. The largest increase in analgesic use was seen for non-human primates where there was an increase in reported analgesic administration from 40% to 67%. In NHPs species, the authors found reports of Multimodal analgesia (combining analgesics from different classes) and reported this as and improvement in analgesic techniques on the research field.

As per rodents, authors discussed that there are less reports of analgesics use on these lower species and attribute this situation to the lack of and/or the special challenges in observing signs of pain in these species (i.e. species-specific factors and group housing).

Authors also reported wide variability in analgesics dosages and treatment duration suggesting suboptimal analgesic treatments in all species.

QUESTIONS: 1. What is multimodal analgesia and give an example? 2. What is the most commonly use analgesic in large animal laboratory species?

ANSWERS: 1. Concurrent administration of different classes of analgesics aiming at better pain treatment. NSAIDs and opioids. 2. Buprenorphine. de Segura et al. Determination of the minimum alveolar concentration for halothane, isoflurane and sevoflurane in the gerbil, pp. 239—242

SUMMARY: This study aimed to determine MAC for the three commonly used gaseous anaesthetic agents in adult female gerbils. Anaesthesia was performed by chamber induction prior to endotracheal intubation and connection to an anon-rebreathing circuit. Parameters measured included body temperature, blood pressure, heart and respiratory rates and end tidal volatile anaesthetic levels. MAC was determined by applying a noxious stimulus (tail clamp). A positive response being noted when there was a purposeful movement of the head, limbs and /or body. If a negative response was seen the agent concentration as reduced until the negative response became positive. At 1x MAC the cardiovascular parameters did not differ significantly between anaesthetics but the respiratory rate was higher in the halothane group. Spo2 and pH and partial oxygen did not differ. The study suggested that MAC in gerbils for these gaseous agents is lower than the average MAC values for rats and mice.

QUESTIONS 1. T/F. MAC is defined as the concentration at one atmosphere that prevents movement in response to a standard noxious stimulus in 50% of animals. T/F. 2. T/F. In gerbils there are significant differences between halogenated anaesthetic agents in terms of their effect on the cardiovascular system. 3. T/F. In gerbils the MAC for these commonly used agents is higher than in other rodents such as rats and mice.

ANSWERS 1. T 2. F- There are no significant differences. 3. F- It is lower. Schöffmann et al. Haemodynamic changes and stress responses of piglets to surgery during total intravenous anaesthesia with propofol and fentanyl, pp. 243— 248

Domain: 1 & 2 Species: Pig - primary

SUMMARY: Porcine models are often used in cardiovascular research due to their anatomical and physiological similarities to humans. Total intravenous anesthesia (TIVA) with fentanyl and propofol has been used in porcine models, but its effect on the stress response of this species has not been evaluated. Surgery causes a nociceptive stimulus that initiates an endocrine-metabolic stress response leading to adrenocorticotropic hormone release from the adrenal cortex. Cortisol release is affected by anesthetics and severity of surgical trauma; cortisol concentrations have been used to evaluate stress response in pigs. Acute phase proteins are indicators of general health and physical welfare of animals; changes in serum concentrations correlate with tissue damage and internal changes (i.e. infections, inflammation). Serum Amyloid A is a fast reacting acute phase protein that is only present at low concentrations under normal conditions. This study assessed the stress response (serum amyloid A and serum cortisol concentrations) and hemodynamic changes (blood pressure and heart rate) associated with surgery in piglets undergoing total intravenous anesthesia with propofol and fentanyl. This study was conducted in conjunction with an ECMO (extracorporeal membrane oxygenation) project and the soft tissue surgery involved cannulation of various peripheral vessels (external jugular, common carotid, femoral). Animals were premedicated with midazolam, ketamine and butorphanol intramuscularly and induced with propofol IV prior to intubation. The animals were maintained on TIVA of 8 mg/kg/hr of propofol and 35 ug/kg/hr of fentanyl. Anesthesia was maintained for 300 minutes following the end of surgery and the animals were euthanized at the end of the anesthesia. Mean serum amyloid A concentrations increased from before the surgery to 300 minutes after the end of the procedure, while serum cortisol concentrations decreased significantly over that time period. Lactate concentration, blood pressure and heart rate did not change significantly over this time period. Based on the findings, TIVA with propofol and fentanyl suppresses the stress response in piglets undergoing surgery with minimal cardiovascular side effects and may be useful in cardiovascular research.

QUESTIONS: 1. Which of the following statements is false regarding serum amyloid A (SAA)? a. It is present in the bloodstream at a very low concentration under normal conditions. b. It is a fast reacting acute phase protein. c. Its concentration increases dramatically after stimulation. d. A rise in SAA concentration occurs within 4 hrs following surgical stress in pigs. e. All of the above are true. 2. T/F Butorphanol can be used to reverse the respiratory depression caused by fentanyl in rats, rabbits and humans, but it will compromise the post-operative analgesia. 3. T/F Propofol & fentanyl administered via constant rate infusion are suitable anesthetics for animals involved in cardiovascular research.

ANSWERS: 1. e. 2. False 3. True

Jensen-Waern et al. Effects of streptozotocin-induced diabetes in domestic pigs with focus on the amino acid metabolism, pp. 249—254

Primary Species: pig

SUMMARY: Streptozotocin was administered (150mg/kg) intravenously to 6 health- certified growing SPF Yorkshire/Landrace pigs fed ad libitum on a commercial finisher diet and monitored for 6 wks. One pig was euthanized due to poor health following STZ administration with centrolobular liver necrosis and pale kidneys observed on necropsy. During the third week following STZ injection, all 5 pigs had reduced skeletal muscle mass and were moderately emaciated at the end of the study with decreased muscle mass and subcutaneous fat reduction. Baseline glucose values were between 5.4 mM and 7.5 mM prior to injection and rose to 9.2-15.1mM at 24 hrs, and >25mM at 72 hours. Serum C-peptide (indicator of B-cell destruction) was highest at 24 hrs (0.24ug/L- 1.0ug/L) and remained between 0.25 + 0.16 ug/L for the rest of the study. No changes in WBC were observed throughout the study. Liver enzymes were normal for 3 pigs, with 2 other pigs showing elevations in ALT, GGT, and GLDH between 1-3 wks post injection. 2 days after injection, leucine, isoleucine, and valine concentrations (all BCAAs) were increased and remained elevated for 3 weeks. Although alanine was markedly decreased one wk post injection, no significant changes were noted in the total concentration of gluconeogenic amino acids (all amino acids except lysine, leucine, and taurine). Taurine remained decreased between 1-3 wks after injection. Insulin levels were low in all skeletal muscle and pancreatic tissues sampled at time of necropsy.

The decreased weight gain resembles stunted growth in adolescents with juvenile diabetes. In this study, analysis of amino acids revealed a marked decreased in serum alanine and a 4-5 fold increase in BCAAs, which is similar to the rat model of STZ- induced diabetes. The decrease in taurine was similar to that found in diabetic patients. Taurine when supplemented in diabetic patients has shown to be beneficial in treating diabetic complications, such as cardiomyopathy, retinopathy, and nephropathy.

QUESTIONS (T or F): 1. Lysine, leucine and taurine are non-gluconeogenic amino acids. 2. C-peptide is an indicator of beta-cell destruction 3. Alanine levels were markedly increased and BCAAs were decreased post injection.

ANSWERS: 1. T 2. T 3. F

Heimann et al. Blood collection from the sublingual vein in mice and hamsters: a suitable alternative to retrobulbar technique that provides large volumes and minimizes tissue damage, pp. 255—260

Domain 3: Research Primary species: Mouse Secondary species: Hamster

SUMMARY: In rodent, retrobulbar venous plexus puncture is a commonly used method for obtaining a blood sample. Although the technique yields large volumes of blood, the disadvantage is that it can lead to severe tissue damage. The aim of this study was to develop the puncture of the sublingual vein as a suitable alternative for retrobulbar collection. The authors were successful in developing a sublingual vein collection technique for hamsters and mice. The authors also compared retrobulbar and sublingual methods using male CD-1 mice. The sublingual method showed less tissue destruction, with a decreased mean severity in the histological examination.

QUESTIONS: 1. Which of the following rodents lack a peripherally located sublingual vein? a. Mus musculus b. Rattus norvegicus c. Mesocricetus auratus d. Cavia porcellus 2. Sublingual blood collection cannot be performed in which of the following species? a. Rats b. Mice c. Hamsters d. Guinea pigs

ANSWERS: 1. D 2. D

Xiang et al. An in vivo magnetic resonance imaging technique for measurement of rat lumbar vertebral body blood perfusion, pp. 261—265

Primary Species: Rat

SUMMARY: The authors carried out a study to investigate the technical feasibility of dynamic contrast-enhanced magnetic resonance imaging (MRI) measurement of rat vertebral blood perfusion using a 1.5 T clinical scanner and a small surface coil. Nine male 7-month-old Wistar-Kyoto rats were used. Computed tomographic assessment of lumbar vertebral bone mineral density (BMD) and MRI assessment of lumbar vertebral blood perfusion were performed twice with 8 weeks’ interval. A 4.7 cm surface coil was used for receiving radiofrequency signal. Gadolinium-DOTA (0.3 mmol/kg) was injected through the tail vein of the rat as a quick bolus and dynamic MRI scan was carried out on the central sagittal plane of the lumbar spine for 8 min with a temporal resolution of 0.6 s. Blood perfusion parameters of wash-in rate, maximum enhancement and wash- out rate were derived from the dynamic MR images. No vertebral body size and vertebral BMD difference was detected between the two time points (P ¼ 0.222 and 0.123, respectively). Wash-in rate was 0.105 (+0.016)% and 0.111 (+0.018)%, maximum enhancement 126.5 (+10.6)% and 129.2 (+13.4)%, and wash-out rate 0.788 (+0.182)% and 0.792 (+0.182)% for the two time points. With these parameters, no difference was found between the two time points with a P value of 0.575, 0.889 and 0.754, respectively. They conclude that dynamic contrast-enhanced MRI measurement of rat vertebral blood perfusion using a 1.5 T clinical scanner and a small surface coil is feasible and reliable and that this technique offers a non-invasive means to probe vertebral blood perfusion changes associated with rat disease models.

QUESTIONS: Answer True (T) or False (F) 1. Recent clinical studies have shown that, both in male and female subjects, magnetic resonance imaging (MRI)-derived parameters of vertebral marrow blood perfusion are significantly decreased in the osteoporotic subjects compared with those of the osteopenic subjects and normal bone density subjects. 2. Until the advent of MR perfusion imaging, there has been no straightforward means of non-invasively measuring intraosseous blood flow and perfusion to probe microcirculation changes of rat vertebrae, or other bone tissues. 3. Blood perfusion rates in sites consisting primarily of cancellous bone and marrow are smaller than in regions composed of cortical bone. 4. The wash-out rate (λ) is related to the backflow of contrast agent from interstitial space to blood flow and being eliminated from the animal body via the kidney. 5. The injection speed of Gd contrast (Gadolinium-DOTA 0.3 mmol/kg) is not expected to affect the wash-in rate.

ANSWERS: 1. T 2. T 3. F: Blood perfusion rates in sites consisting primarily of cancellous bone and marrow are greater than in regions composed of cortical bone, which is consistent with the denser vascular network and higher metabolic rates of cancellous bone and marrow. 4. T 5. F: The injection speed is expected to affect the wash-in rate, but not to a great extent of that of the maximum enhancement and wash-out rate. Ideally, the Gd contrast agent injection should be carried out via an automatic injector. However, no automatic injector capable of injecting such a small volume used accurately in this study was available and the injection of contrast agent was completed manually within less than 1 second; the authors expect the influence of injection rate on perfusion parameters to be small.

Leitner et al. Longitudinal as well as age-matched assessments of bone changes in the mature ovariectomized rat model, pp. 266—271

ACLAM Task: Research, T1-3, education T2 Primary Species: Rat

SUMMARY: Ovariectomized rats are the most common animal model of osteoporosis, exhibiting pathology including bone resorption >bone formation, loss of cancellous bone>cortical bone, and response to treatment in a similar manner to that of humans. There are two models: aged and mature (still growing, reproductively active). Due to the bone growth in the mature model, sham and age matched assessments are essential for research studies with this group. This study obtained pQCT scans of the femurs 21 female 12 week old ‘mature’ Wistar rats (8 shams, 13 OVX) pre ovariectomy and 4 weeks post ovariectomy. Rats were also imaged by micro CT after euthanasia at 4 weeks post ovariectomy, and the results of the scans compared. pQCT is the preferred method of assessment, as it may be used longitudinally, whereas both histology and conventional micro CT are in vitro techniques. The volume of interest (VOI) was determined at the most proximal aspect of the distal femoral growth plate for trabecular and cortical bone, and trabecular bone alone. Sham animals had significantly increased bone mineral density over the 4 week period, and as expected, OVX animals significantly lost bone mineral density. Micro CT and pQCT results were well correlated, validating the pQCT methodology as a valid alternative to in vitro techniques, and asserting that pQCT technology permits each animal to act as its own control, minimizing animal numbers needed on study. The authors note that the longitudinal bone growth of 10% in the sham animals was in contrast to the literature reports, and claim that this is due to their more consistent geographical measurements.

QUESTIONS: 1. Cortical bone is lost in greater amounts than cancellous bone in the OVX mature rat model of osteoporosis – T/F 2. pQCT of sham animals revealed ______(increased/decreased) bone mineral density over 4 weeks on study.

ANSWERS: 1. False 2. Increased

Gruber et al. A new small animal model for the study of spine fusion in the sand rat: pilot studies, pp. 272—277

Task 9 - Collaborate on the Selection and Development of Animal Models K1 - Animal models (spontaneous and induced) Tertiary species - other rodents

SUMMARY: Spinal fusion is an important surgical treatment for severe traumatic or degenerative disc disease, particularly in the lumbar and cervical spine. There is controversy over whether fusion causes (or increases the severity of) disease in adjacent spinal segments. Further studies are needed to investigate this question. The authors investigated the use of the sand rat as an economical small animal model in this pilot study. One of the reasons this species was chosen is that these animals develop age-related degenerative disc disease spontaneously.

An abdominal approach was made to access the ventral lumbar spine in animals of varying ages and a lumbar disc was removed by curettage to induce fusion. 1-26 months later (mean=10.9 months), the animals were euthanized and the spines analyzed with radiographs and histology (n=28). The authors found that at the time of harvest, the radiographic scores of adjacent disc spaces were significantly greater than before surgery. However, some caution must be taken in comparing this to human disease, as disease in the sand rat progresses at a faster rate than in humans. Histological features are shown and described. The authors conclude that the fat sand rat is a useful model for studying fusion.

QUESTIONS: 1. What is the scientific name of the sand rat? 2. What disease are sand rats prone to develop when fed ad lib standard rodent chow? 3. How was spinal fusion induced in this model? ANSWERS: 1. Psammomys obesus 2. Diabetes 3. Surgical removal of a lumbar intervertebral disc

Thon et al. An interview study of phenotypic characterization of genetically- modified mice, pp. 278—283

SUMMARY: An interview study was carried out with the aim of clarifying the reasons for the limited use of phenotypic characterization of genetically-modified mice (GMM) and identifying issues hindering its implementation. The investigation confirmed that few animals were systematically phenotyped and an observational approach was found to be widespread. The primary interest of the interviewees was phenotyping for impaired animal welfare.

The concept of phenotyping was widely understood and perceived as a scientific advantage. The comprehensiveness of the protocols and the resources required for phenotyping were seen as problematic. Phenotyping was predicted to become much more widespread in the future and its success was expected to depend on the development of reliable, fast and inexpensive methods. The contradiction between the limited use of characterization and its advantages was also discussed and proposals for the improvement of future phenotyping strategies are formulated.

QUESTIONS: 1. Which was one of the first systematic phenotyping protocols, and what kind of test does it include for adult mice? 2. But currently, which one is the most updated validated protocol collection? 3. After this interview study, they learned that a very commonly applied phenotyping approach was observational, but, what was this method based on? 4. It was stated that characterization of a strain might create a dilemma; why could it be, and what solutions would you propose? 5. The research projects of the EU have indisputably focused on the central unit phenotyping idea reflected by three projects, which are these projects? 6. Say TRUE or FALSE: a. In case of oral reporting it was possible and very easy to compare recent and historical findings b. None of the participants in the interview study were aware that phenotyping was commercially available c. Gaining knowledge about new and perhaps unexpected traits was not seen as an advantage d. For several participants, a comprehensive characterization was seen as too time- consuming and detrimental to publication of up-to-date results e. The need for expertise to perform several of phenotyping tests was seen as an advantage f. Possessing the know-how to choose the relevant parameters for characterization was seen as an obstacle g. The development of relatively inexpensive, high-throughput, dependable tests for future phenotyping are crucial for its improved implementation.

ANSWERS: 1. SHIRPA protocol 2. EMPReSS protocol 3. It was based on the expertise of the animal technicians: the animals were typically observed once per day when they were handled for other purposes and any deviation from normal was noticed and reported to the user. 4. You could find that your model was not the best for your particular experiment. You would then have to decide whether you wanted to use it anyway or spend time/resources on finding a better-suited model. 5. Eumorphia, EUMODIC and EuroPhenome projects. 6. a. F, b. T, c. F, d. T, e. F, f. T, g. T

Zhu et al. Limited sampling strategy in rats to predict the inhibited activities of hepatic CYP3A, pp. 284—290

Domain 1: Management of Spontaneous and Experimentally Induced Diseases and Conditions. K1: diagnostic procedures

Domain 3: Research. K1 (biomethodology techniques) and K12 (replacement, reduction and refinement techniques).

Primary Species: rat

SUMMARY: The use of probe substrates is a widely accepted method for evaluating the activity of cytochrome P450 (CYP) enzymes in individuals. Midazolam (MDZ) is commonly used as a probe to determine CYP3A activity in humans and animals. The measurement of the systemic clearance of MDZ, however, usually requires the determination of the area under the plasma concentration time curve (AUC) following its administration, for which multiple blood samples collection over several hours is often necessary. In the Limited Sampling Strategy (LSS) only two or three blood samples are collected. To achieve the aim of this study, 40 male Wistar rats were divided into 4 groups according the administration of ketoconazole (selective inhibitor of CYP3A): 1.2, 10, 30 mg/kg and vehicle (as control group) respectively, followed by subsequent constant infusion (0.7, 2.0 and 3.0). Two hours after starting the ketoconazole 10 mg/kg MDZ was injected. A volume of 0.5 ml blood was collected at 2, 5, 10, 20, 30, 45, 60, 90 and 120 min after the injection of MDZ. The results indicated that one (90 min), two (60- 90) or three-sampling (30-60-90 or 30-60-120) LSS is the model that had been developed using the training set, in which the values of errors were smaller than 8%, provided good estimates for the values of clearance in the validation set. It means that LSS can reliably predict Cls of MDZ and can be used to predict hepatic metabolism.

QUESTIONS 1. Which drugs are used to predict hepatic metabolism? a. Ketoconazole b. Diazepam c. Midazolam d. Fluconazole 2. T/F. LSS reduces the number of animals but still maintains the quality of the science. 3. T/F. LSS refines the procedure because it improves the welfare of individual animals. 4. T/F. The application of LSS has large-cost and time-saving benefits. ANSWERS 1. a 2. T 3. T 4. T

Coppens and Moens. Cardiac arrhythmias related to continuous thermodilution cardiac output measurement in an anaesthetized sheep, pp. 291—294

Domains 1 and 3 Secondary species, sheep

SUMMARY: This is a case report of cardiac arrhythmias related to continuous cardiac output (CCO) in a sheep. A nine month old Suffolk mixed breed male sheep of 37 kg was used for an experimental laparoscopic cystotomy in order to study the pharmacokinetic effects of lidocaine and ventilation changes. The animal received lidocaine 2mg/kg IV three different times during the procedure. Anesthesia was induced with thiopental and maintained with isoflurane. Cardiac output was measured with both the iced bolus thermodilution technique (BTD) and the CCO technique with products from Edwards Lifesciences. The users noticed that about an hour after CCO measurements, a ventricular extrasystole (VES) was observed. The incidence of VES increased and bursts were noticed. The VES ceased when the CCO was turned off. Peaks in the filament temperature were noticed at the same time as the VES, so filament temp, rather than Swan location, was implicated in the arrhythmias. No hypotension or other irregularities occurred during the VES episodes. The cause of the arrhythmias was supposed to be an area of thermally irritated myocardium.

QUESTIONS: T/F 1. Who makes the Swan Ganz catheter? a. Baxter b. Cook c. Edwards Lifesciences 2. What does the Swan Ganz catheter measure? a. Temperature b. Arterial oxygen c. Cardiac output 3. Where is the Swan Ganz placed? a. Right atrium b. Pulmonary artery c. Left ventricle

ANSWERS: 1. c 2. c 3. b

Pallares and Gonzalez-Bulnes. A new method for induction and synchronization of oestrus and fertile ovulations in mice by using exogenous hormones, pp. 295— 299 SUMMARY: Four consecutive experiments were conducted to design and establish the effectiveness of different protocols for induction and synchronization of oestrus and ovulation in mice. Results showed that the highest synchronization degree and the highest fertility rates were obtained using two intraperitoneal doses of 0.5 mg of cloprostenol, three days apart, plus a single subcutaneous dose of 3 mg of progesterone coincidentally with the first injection of cloprostenol; hence the proposed protocol arises as an adequate alternative for reproductive management in mice.

QUESTIONS: 1. A detailed analysis of the results found in the different experiments performed indicates, in the first trial, that the use of two luteolytic doses of prostaglandin analogues induces appearance of oestrus and ovulation. But if you compare it with male effect, what could you see? 2. Analyzing the results of the first experiment (Comparison of the effect obtained by the administration of either progestative or luteolytic hormones) we could say that these results would be very useful for the design of a simple and effective protocol for reproductive management of mice in practice; however, which is the main problem? 3. What effect could we expect with the use of a single dose of progesterone coincidentally with a prostaglandin treatment? 4. The comparison of the results found by applying single doses of progesterone and prostaglandin in the second and third experiments showed that there are two peaks of appearance of vaginal plugs, at 24 h and 96 h. So, what could we conclude? 5. Say TRUE or FALSE: a. In ruminants, the appearance and quality of ovulations are known to be independent on the stage of follicle development at the time of male introduction b. Administration of exogenous PGF2a, or its analogues, induces a rapid and controlled luteolysis c. The administration of progesterone in a single intravenous dose is used in large animals from early studies on oestrus synchronization d. Induction and/or synchronization of oestrous cycle is routinely achieved, in animal species, by supplying different combinations of hormones; luteolytic (prostaglandin F2a, PGF2a and its analogues) and progestative (progesterone and its analogues) factors are commonly used.

ANSWERS: 1. That the results are quite similar because most of the animals showed vaginal plugs around 72 h after the second injection. 2. The use of subcutaneous devices has the inconveniences derived from the necessity of a surgical approach for applying/removing the tool under the skin. 3. It would mimic the presence of a secretory corpus luteum and would unify the ovarian cycle and the response in the entire group of animals. 4. That it could be determined by the stage of oestrous and ovarian cycle at treatment. 5. a. FALSE b. TRUE c. FALSE d. TRUE

Sánchez-Morgado et al. Mycobacterium gordonae infection in a colony of African clawed frogs (Xenopus tropicalis), pp. 300—303

SUMMARY: This article describes an outbreak of nodular lesions on the skin of Xenopus tropicalis frogs bred and kept in a research facility. The lesions presented as raised cutaneous nodules of various sizes on different body locations. Affected frogs were culled and extensively sampled. Apart from the skin lesions, no other pathology was found. The lesions were described as localized granulomatous foci, predominantly composed of macrophages and lymphocytes. Numerous acid-fast bacilli were also present. Extensive molecular and microbiological characterization of these organisms was able to identify them as Mycobacterium gordonae, an opportunistic pathogen found in soil and water. Reports in the literature have linked this organism with cutaneous infections and granulomatous lesions in humans, especially immunocompromised ones. This is the first report of a Mycobacterium gordonae infection in amphibians. The source of this outbreak could not be determined.

QUESTIONS: 1. Which of the following statements is FALSE with respect to atypical mycobacteria in amphibians? a. They are widely present in aquarium environments b. They always present as skin infections c. They are a potential zoonotic risk d. Clinical manifestation is usually sporadic 2. Mycobacterium gordonae is: a. A non-pathogenic commensal b. Commonly found in soil and water c. An occasional human pathogen d. All of the above 3. Which of the following statements is TRUE with respect to the M. gordonae found in this outbreak? a. Their isolate did not completely fit with the phenotypic characteristics of the species b. It grew slowly on culture media c. It didn't grow on N-medium d. It was present in all of the skin lesions submitted for examination 4. Which of the following methods can be used to characterize atypical mycobacteria? a. PCR for 16S ribosomal RNA b. Sequencing of the hsp65 gene c. Sequencing of the ITS genomic region d. Biochemical properties e. All of the above 5. Best stain for detecting acid-fast organisms is: a. Gram stain b. Ziehl-Neelsen stain c. Giemsa stain d. Methenamine Silver stain

ANSWERS: 1. b. They always present as skin infections 2. d. All of the above 3. a. Their isolate did not completely fit with the phenotypic characteristics of the species 4. e. All of the above 5. b. Ziehl-Neelsen stain

CASE REPORT Alworth et al. Clostridial typhlitis associated with topical antibiotic therapy in a Syrian hamster, pp. 304—309

Domain: 1 Management of spontaneous and experimentally induced diseases and conditions; Task: 1 Secondary Species: hamster

SUMMARY: A case report describing C. difficile typhlitis in a Syrian hamster (Mesocricetus auratus) treated for fight wounds with topical triple antibiotic ointment (bacitracin, neomycin, polymyxin). The authors give a thorough summary of antibiotic- induced clostridial enterotoxemia in hamsters, and mention that rabbits and guinea pigs are also highly susceptible species. Pathogenesis of the disease is as follows: disruption of the normal intestinal flora occurs, allowing increased proliferation of Clostridial sp. and toxin formation. Clostridia are Gram positive, spore-forming anaerobic bacteria; exposure to spores is the most common means of infection. The authors suspect that the animal ingested the antibiotic during grooming activities, or transperitoneal absorption. The hamster was either exposed to C. difficile around the time of antibiotic administration, or harbored the organism prior to treatment.

QUESTIONS: 1. Which Clostridial species is most commonly isolated from rabbits affected with enterotoxemia? a. C. difficile b. C. spiroforme c. C. piliforme d. None of the above 2. In this report, the authors were able to make the diagnosis of C. difficile enterotoxemia based on all of the following except: a. Anaerobic culture and isolation of the organism b. ELISA for presence of toxins c. IFA d. Ruling out other infectious organisms as the cause for the illness (i.e. negative cultures for other pathogens) 3. T/F: Most hamsters normally harbor C. difficile in their GI tract. 4. Hamsters are a model for which human Clostridial disease? a. Inflammatory bowel disease b. Crohn’s disease c. Ulcerative colitis d. Pseuodomembranous colitis

ANSWERS: 1. B 2. C 3. False 4. D