Laboratory Animals

The primary aim of this report is to assist scientists in selecting more reliable/suitable identification (ID) methods for their studies. This is especially true for genetically altered (GA) animals where individual identification is strictly necessary to link samples, research design and genotype. The aim of this Federation of European Laboratory Animal Science Associations working group was to provide an update of the methods used to identify rodents in different situations and to assess their implications for animal welfare. ID procedures are an indispensable prerequisite for conducting good science but the degree of invasiveness differs between the different methods; therefore, one needs to make a good ethical evaluation of the method chosen. Based on the scientific literature the advantages and disadvantages of various methods have been presented comprehensively and this report is intended as a practical guide for researchers. New upcoming methods have been included next to the traditional techniques. Ideally, an ID method should provide reliable identification, be technically easy to apply and not inflict adverse effects on animals while taking into account the type of research. There is no gold standard method because each situation is unique; however, more studies are needed to better evaluate ID systems and the desirable introduction of new and modern approaches will need to be assessed by detailed scientific evaluation.

Positron emission tomography (PET) provides a means of studying physiological and pharmacological processes as they occur in the living brain. Mice, rats, dogs, cats, pigs and non-human primates are often used in studies using PET. They are commonly anaesthetized with ketamine, propofol or isoflurane in order to prevent them from moving during the imaging procedure. The use of anaesthesia in PET studies suffers, however, from the drawback of possibly altering central neuromolecular mechanisms. As a result, PET findings obtained in anaesthetized animals may fail to correctly represent normal properties of the awake brain. Here, we review findings of PET studies carried out either in both awake and anaesthetized animals or in animals given at least two different anaesthetics. Such studies provide a means of estimating the extent to which anaesthesia affects the outcome of PET neuroimaging in animals. While no final conclusion can be drawn concerning the ‘best’ general anaesthetic for PET neuroimaging in laboratory animals, such studies provide findings that can enhance an understanding of neurobiological mechanisms in the living brain.

The ketamine/midazolam association of a dissociative with a sedative agent is used for the induction and maintenance of anaesthesia in laboratory animals. Anaesthesia may interfere with research results through side-effects on the nervous system, such as memory impairment. It is known that ketamine and midazolam affect cognition; however, their effects have not been clarified when used in a context of balanced anaesthesia. Thus, this study evaluated the effects of ketamine/midazolam on the acquisition of motor and of a spatial memory task in adult mice. Twenty-eight C57BL/6 adult male mice were divided into three groups: untreated control, treated with ketamine/midazolam (75 mg/kg / 10 mg/kg) and treated with midazolam (10 mg/kg) groups. Respiratory rate, heart rate and systolic pressure were measured every 5 min in the animals treated with ketamine/midazolam, as this was the only group that exhibited loss of the righting reflex. One day after treatment, animals were tested in the open field, rotarod and radial arm maze. There were no differences between treatments regarding open-field activity, rotarod performance or number of working and reference memory errors in the radial arm maze task. In conclusion, the learning process of spatial and motor tasks was not disrupted by the anaesthetic combination of ketamine/midazolam. These results suggest its safe use in adult mice in projects where acquisition of a spatial and motor task is necessary.

The possibility of modifying the genome in mice has led to an exponential increase in the number of strains that have been developed for biomedical research. This will continue during the next few decades because international programmes plan to develop genetically-modified strains for every known mouse gene. Due to our own experiences and that of colleagues we know that the reproductive performance of many of these modified stains is impeded, despite that the modification is independent from genes that control reproduction. In some cases the spermatogenesis might be disturbed. The reason presumably lies in a defective endocrine function of the testes. This can cause reduced and/or abnormal sperm production. In livestock as well as in humans these disorders can be treated with gonadotropins. One treatment period lasts for the duration of spermatogenesis of the respective species. Up to now, nothing is known about such treatments in laboratory mice to restore or increase reproduction of genetically-modified strains. Spermatogenesis in the mouse lasts approximately 35 days. Therefore, we treated sexually mature male mice of C57BL/6 and BALB/c strains with gonadotropins for this period. The aim of this study was to test the principle suitability of such treatment for the improvement of sperm count, sperm motility, fertilization ability and reproduction.

It has previously been shown that high-calorie diet alters the function of the mammalian circadian clock and that obesity has an influence on circadian organization of hormone secretion. That prompted us to test whether inbred Wistar Ottawa Karlsburg W (RT1^u) (WOKW) rats developing facets of the metabolic syndrome show changes in their metabolic profiles under different feeding conditions (high-fat, high-sugar versus control diet) and under two different 12 h:12 h light–dark (LD) cycles. At the age of four weeks, these rats were divided into four groups. Groups 1 and 2 were kept under initial LD cycle (lights on at 05:00 h). Group 1 was fed with a normal rat diet while group 2 received a high-fat, high-sugar diet from 10 up to the age of 21 weeks. Groups 3 and 4 were kept under a shifted LD cycle (lights on at 11:00 h). Group 3 was given a normal diet while group 4 received a high-fat, high-sugar diet from an age like groups 1 and 2. Several metabolic traits were studied during the observation period of 21 weeks. The blood samples were obtained 2 h before lights off. Body weight gain (P < 0.001), leptin (P < 0.001), triglycerides (P < 0.001) and cholesterol (P < 0.05) were significantly reduced in group 4 versus group 2, but comparable between control groups (1 versus 3). The insulin concentrations were reduced in groups 3 and 4 versus groups 1 and 2 without effect of diet. In conclusion, the results provide evidence that light conditions influence diet induced changes in phenotypic traits like body weight gain, lipids as well as hormone levels (insulin and leptin) in WOKW rats.

This study was undertaken to characterize the effects of monotonous training at lactate minimum (LM) intensity on aerobic and anaerobic performances; glycogen concentrations in the soleus muscle, the gastrocnemius muscle and the liver; and creatine kinase (CK), free fatty acids and glucose concentrations in rats. The rats were separated into trained (n = 10), baseline (n = 10) and sedentary (n = 10) groups. The trained group was submitted to the following: 60 min/day, 6 day/week and intensity equivalent to LM during the 12-week training period. The training volume was reduced after four weeks according to a sigmoid function. The total CK (U/L) increased in the trained group after 12 weeks (742.0 ± 158.5) in comparison with the baseline (319.6 ± 40.2) and the sedentary (261.6 ± 42.2) groups. Free fatty acids and glycogen stores (liver, soleus muscle and gastrocnemius muscle) increased after 12 weeks of monotonous training but aerobic and anaerobic performances were unchanged in relation to the sedentary group. The monotonous training at LM increased the level of energy substrates, unchanged aerobic performance, reduced anaerobic capacity and increased the serum CK concentration; however, the rats did not achieve the predicted training volume.

Non-alcoholic fatty liver disease (NAFLD) is a common problem with a wide variety of phenotypes. While its pathogenesis is still not fully understood, several risk factors for disease progression have been identified. Therefore, defining adequate animal models may serve to unreveal the pathogenesis in NAFLD. We studied Lewis and Sprague-Dawley rats of both genders (n = 6) fed standard (Std) or high-fat (HF) diet for three weeks. Disease stage was assessed by haematoxylin–eosin, Azan Heidenheim and Oil-Red staining, apoptosis by single-stranded DNA (ssDNA) detection and liver regeneration by Ki-67 staining. Serum markers of liver injury and lipid metabolism including adipocytokines were analysed. Livers of both strains and genders fed with HF diet demonstrated evidence of steatosis. Lewis rats developed microvesicular steatosis whereas Sprague-Dawley rats presented macrovesicular steatosis accompanied by pronounced fibrosis. Female gender of both strains was associated with lower steatosis grade and higher proliferation rate (P < 0.05). Gender-specific differences were most prominent in Lewis rats on a HF diet, where females showed lower alkaline phosphatase, cholesterol, triglyceride and leptin levels and a more favourable low-density lipoprotein/high-density lipoprotein ratio than males (P < 0.05). Reverse transcriptase-polymerase chain reaction analysis was performed to demonstrate changes in expression of various genes important for liver regeneration, fibrosis and steatosis. HF diet induced downregulation of proangiogenic genes such as vascular endothelial growth factor receptor 1 and 2 (P < 0.05) in males was not present in females. In conclusion, strain and gender served major roles in disease progression. These differences should be considered when designing studies and may offer new ways to advance therapeutic strategies.

Short-term storage of embryos at low temperature induces developmental arrest of the embryo and would appear to be a valuable aid in embryo-transfer techniques to avoid wasting embryos. Embryo storage at 4°C was examined to allow synchronization with embryo-transfer recipients using the microinjection technique. Superovulation was induced in female Japanese White donor rabbits four days before mating with males. At the same time, control recipients were injected with human chorionic gonadotropin (hCG) to allow synchronization (R1); the hCG injections were delayed by 24 h in the experimental group (R2). DNA constructs for expressing human C-reactive protein or apolipoprotein AII were microinjected into the male pronuclei of the ova. The microinjected embryos were immediately transferred to recipients (R1) or stored at 4°C in phosphate-buffered saline containing 10% fetal bovine serum. After 17–20 h, the stored embryos were incubated at 37°C for one hour, and the morphologically normal embryos were transferred to recipients (R2). In the R1 rabbits, 855 embryos were transferred to 29 recipients, and 72.4% of the recipients became pregnant. Seven of the 84 offspring were transgenic. In the R2 rabbits, 478 embryos were transferred to 16 recipients, and 62.5% of the recipients became pregnant. Two of the 39 offspring were transgenic. There were no differences in pregnancy rate, litter size and transgenic integration rate between R1 and R2. These results suggest that the short-term 4°C storage of microinjected embryos can be a valuable method for synchronization with recipients, and reducing wastage of embryos and the sacrifice of rabbits.

Articular cartilage has a limited capacity for self-repair in adult humans, and methods used to stimulate regeneration often result in re-growth of fibrous cartilage, which has lower durability. No current treatment option can provide complete repair. The possibility of growth factor delivery into the joint for cartilage regeneration after injury would be an attractive treatment option. A full thickness osteochondral defect of 4 mm in diameter and 2 mm deep was created by mechanical drilling in the medial femoral condyle in 20 female adult New Zealand White rabbits. In an attempt to improve regeneration a hyaluronic hydrogel system, with or without bone morphogenetic protein-2 (BMP-2) was delivered intraarticularly. The contralateral joint defect was treated with saline as control. Throughout the study, rabbits were clinically examined and after 12 (n = 6) or 24 (n = 9) weeks, the rabbits were euthanized and the joints evaluated by histology. The defects healed with fibrocartilage like tissue, and the filling of the defects ranged from less than 25% to complete. The healing of the defects varied both inter- and intra-group wise. Treatment with hyaluronan gel with or without BMP-2 had no effect on cartilage regeneration compared with controls. Instead, severe ectopic bone formation was found in seven joints treated with BMP-2. In conclusion, the present study shows that neither treatment with hyaluronic gel alone, nor in combination with BMP-2, improves the healing of an induced cartilage defect in rabbits. It further shows that BMP-2 can induce ectopic bone formation, which severely affects the functionality of the joint.

The absorption of medetomidine released by continuous infusion from an osmotic pump in the abdominal cavity was studied in pregnant sheep during the 24 h postoperative period. Additionally pain and sedation was assessed. Eleven sheep were studied: six were treated with a medetomidine loaded osmotic pump delivering 10 μL/h (3 μg/kg/h medetomidine); and five with a saline loaded osmotic pump (control). Serial blood samples were taken and analysed to determine plasma medetomidine levels. Medetomidine was absorbed from the peritoneal cavity and a steady plasma concentration was achieved within 10 h, mean (SD) peak concentration was 2.87 (0.22) ng/mL. Sheep receiving medetomidine analgesia had significantly lower pain scores at 10 h than controls. Four control sheep required rescue analgesia, compared with 0 in the treatment group. Delivery of 3 μg/kg/h medetomidine by an intraperitoneal osmotic pump to pregnant sheep in the 24 h postoperative period provides adequate plasma concentrations of medetomidine for analgesia without sedation.

Pinworms (Nematoda: Oxyurida) are common contaminants in most laboratory rodent colonies. The aim of the study was to monitor the transmission of Syphacia muris eggs in laboratory rat breeding facilities. Dust in a breeding room was investigated using special grids (free fallout, or through the help suction chamber). Furthermore, the ventilation system, breeding cages and the hands of the laboratory technical staff were examined. In the case of free fallout, the percentage of positive grids increased slightly over time: from 5.5% (after 24 h) to 8.2% (72 h). Similar values were also found when using the suction chamber (7.6%). Many more pinworm eggs were found in samples collected every second month from suction holes of the ventilation system (28.7%). One-half of the samples taken from the breeding cages (before washing) exhibited pinworm eggs (50.8%). Examination of the hands of technical staff showed positive detection in 37.9% of cases. In this study, certain transmission factors (dust, unclean cages and technicians) were proved to be significant in the distribution of pinworm infection in laboratory rodent facilities.

When establishing animal models of viral respiratory infection, the optimal dose and route of delivery are critical to ensure reproducible outcomes. The mouse model for influenza infection is widely used due to the small animal size and simplicity of viral inoculation. During establishment of a mouse model of influenza A infection we observed a marked shift in morbidity when identical influenza A inoculum doses were delivered in less than 35 μL. We show for the first time that mice challenged with a 25 μL inoculum volume readily recovered following infection with an infectious dose of influenza A virus that was fatal when inoculated in 35 or 50 μL volumes.