Over the past 50 years, image-guided procedures have been established for a wide range of applications. The development and clinical translation of new treatment regimens necessitate the availability of suitable animal models. The juvenile Göttingen minipig presents a favourable profile as a model for human infants. However, no information can be found regarding the vascular system of juvenile minipigs in the literature. Such information is imperative for planning the accessibility of target structures by catheterization. We present here a complete mapping of the arterial system of the juvenile minipig based on contrast-enhanced computed tomography. Four female animals weighing 6.13 ± 0.72 kg were used for the analyses. Imaging was performed under anaesthesia, and the measurement of the vascular structures was performed independently by four investigators. Our dataset forms a basis for future interventional studies in juvenile minipigs, and enables planning and refinement of future experiments according to the 3R (replacement, reduction and refinement) principles of animal research.
Little attention has been paid to the effects of fish microbiotas on the reproducibility and comparability of fish studies so far. Extrinsic and intrinsic factors, such as water quality, environmental microbial populations, diet, host genetic profile, gender, age and stress status, affect fish microbiotas and create significant inter- and intra-species variations. Fish microbiotas play critical roles in many key aspects of host physiology, such as protection against pathogens, digestion and development of the digestive tract and the local immune system. Thus, greater effort should be invested in standardizing the microbiological profiles of research fish. In this context, issues requiring consideration include the establishment of isogenic and isobiotic fish lines, the standardization of rearing conditions and the development of appropriate tests to adequately describe microbial populations. There are many challenges involved in each of these issues, and the research community must decide which aspects should be standardized for each species and each type of research. For all studies in which microbiota is expected to exert an influence, thorough reporting is of paramount importance. Every step towards standardization increases study quality and simultaneously contributes to reducing the number of fish used in research, which is a legal and ethical obligation.
This study aims to determine the ability of laboratory animal bacteria to resist desiccation and inactivation by hydrogen peroxide vapour (HPV) on paper bedding pieces. Bedding pieces were saturated with bacterial suspensions in water or 2% (w/v) bovine serum albumin (BSA) in water, and held in a mouse facility. Viable counts showed variable survival rates over time for the bacterial species used ([Pasteurella] pneumotropica, Muribacter muris, Pseudomonas aeruginosa, Acinetobacter redioresistens, Escherichia coli, Klebsiella oxytoca, Bordetella bronchiseptica, Bordetella hinzii, Enterococcus faecalis, β-haemolytic Streptococcus spp., Staphylococcus aureus and Staphylococcus xylosus). Overall, BSA increased bacterial survival in the bedding pieces. The survival rates of Bacillus safensis were not influenced by BSA but depended on sporulation. When bedding pieces and Petri dishes inoculated with E. coli, P. aeruginosa and S. aureus were subjected to HPV disinfection, all bacterial species on the bedding pieces inoculated with bacterial suspensions in water were readily inactivated. By contrast, S. aureus and P. aeruginosa, but not E. coli cells survived HPV treatment in high numbers when inoculated on bedding pieces as a BSA suspension. Notably, all three bacterial species were readily inactivated by HPV even in the presence of BSA when smeared on smooth surfaces. In conclusion, the suspension medium and the carrier can influence the environmental survival and susceptibility of bacterial species to HPV. Our results may help to develop standard protocols that can be used to ensure the microbiological quality of experimental rodent housing.
This study was undertaken to assess the suitability of fentanyl/fluanisone (‘Hypnorm’, VetaPharma; 0.315 mg/mL of fentanyl citrate and 10 mg/mL of fluanisone) alone or combined with midazolam in rhesus macaques. Fifteen rhesus macaques requiring sedation for veterinary procedures received an intramuscular (IM) dose range of Hypnorm from 0.01 mL/kg to 0.3 mL/kg either alone or combined with 0.5 mg/kg of midazolam. To reverse the sedation, flumazenil in combination with either naloxone, buprenorphine or butorphanol was administered intravenously (IV) or IM. Rhesus macaques were successfully sedated with 0.1 mL/kg of Hypnorm and 0.5 mg/kg of midazolam, and sedation was partially reversed by the administration of flumazenil and either naloxone or buprenorphine. However the primates remained slightly sedated and were only released into their home cage several hours post recovery. Butorphanol failed to induce recovery and caused marked respiratory depression. The neuroleptanalgesic combination, Hypnorm and midazolam, effectively immobilized rhesus macaques and was reversible with a combination of flumazenil and either naloxone or buprenorphine.
Pulmonary arterial hypertension is a life-threatening disease with a poor prognosis. Oral treatment with vasodilators is often limited by systemic hypotension. Inhalation of vasodilators offers the opportunity for selective pulmonary vasodilation. Testing selective pulmonary vasodilation by inhaled nitric oxide or alternative substances in animal models requires an increased pulmonary vascular tone. The aim of this study was to identify animal models that are suitable for investigating selective pulmonary vasodilation. To do so, a haemodynamic stable pulmonary hypertension was initiated, with a 30 min duration deemed to be a sufficient time interval before and after a possible intervention. In anaesthetized and mechanically-ventilated Sprague–Dawley rats pulmonary hypertension was induced either by acute hypoxia due to reduction of the inspired oxygen fraction from 0.21 to 0.1 (n = 6), a fixed infusion rate of the thromboxane analogue U46619 (240 ng/min; n = 6) or a monocrotaline injection (MCT; 60 mg/kg applied 23 days before the investigation; n = 7). The animals were instrumented to measure right ventricular and systemic arterial pressures. Acute hypoxia caused a short, and only transient, increase of pulmonary artery pressure as well as profound systemic hypotension which suggested haemodynamic instability. U46619 infusion induced variable changes in the pulmonary and systemic vascular tone without sufficient stabilization within 30 min. MCT provoked sustained pulmonary hypertension with normal systemic pressure values and inhalation of nitric oxide caused selective pulmonary vasodilation. In conclusion, out of the three examined rat animal models only MCT-induced pulmonary hypertension is a solid and reliable model for investigating selective pulmonary vasodilation.
Unlike non-steroidal anti-inflammatory drugs (NSAIDs), metamizole has poor anti-inflammatory effects; and is suitable for models where analgesia, but not anti-inflammatory effects, is desirable. Like opioids, these drugs produce perioperative analgesia while reducing anaesthetic requirements, but it remains unclear whether they may develop tolerance or hyperalgesia, and thus decrease in analgesic efficacy. The aim was to determine whether tolerance or hyperalgesia to metamizole occurred in rats, and whether the sevoflurane minimum alveolar concentration (MAC) was affected. In a randomized, prospective, controlled study, male Wistar rats (n = 8 per group) were administered metamizole (300 mg/kg, day 4). Previously, the following treatments were provided: daily metamizole for four days (0–3), morphine (10 mg/kg; positive control, day 0 only) or saline (negative control). The main outcome measures were mechanical (MNT) and warm thermal (WNT) nociceptive quantitative sensory thresholds. The baseline sevoflurane MAC and the reduction produced by the treatments were also determined. The mean (SD) baseline MAC [2.4(0.2)%vol] was decreased by morphine and metamizole by 45(11)% and 33(7)% (P = 0.000, both), respectively. Baseline MNT [35.4(4.5) g] and WNT [13.2(2.4) s] were decreased by morphine and metamizole: MNT reduction of 22(6)% (P = 0.000) and 22(7)% (P = 0.001), respectively and WNT reduction of 34(14)% (P = 0.000) and 24(13)% (P = 0.001). The baseline MAC on day 4 was neither modified by treatments nor the MAC reduction produced by metamizole (days 0 and 4; P > 0.05). In conclusion, repeated metamizole administration may produce hyperalgesia, although it may not modify its anaesthetic sparing effect. The clinical relevance of this effect in painful research models requiring prolonged analgesic therapy warrants further investigation.
Researchers performing experiments on animals should always strive towards the refinement of experiments, minimization of stress and provision of better animal welfare. An adequate analgesic strategy is important to improve post-operative recovery and welfare in laboratory rats and mice. In addition, it is desirable to provide post-operative analgesia using methods that are minimally invasive and stressful. This study investigated the antinociceptive effects of orally administered buprenorphine ingested in Nutella® in comparison with subcutaneous buprenorphine administration. By exposing the animal to a thermal stimulus using a hot plate, significant antinociceptive effects of voluntarily ingested buprenorphine administered in Nutella® were demonstrated. This was evident at doses of 1.0 mg/kg 60 and 120 min post administration (P < 0.01), although antinociceptive effects were not as marked as with subcutaneous administration, and had a later onset. It is advised to administer the oral formulation of buprenorphine in Nutella® in a 10-fold higher dose, as well as approximately 60 min earlier, than with the more commonly employed subcutaneous route of administration.
The pathogenesis of human autoimmune disorders is incompletely understood. This has led to the development of numerous murine models in which the pathogenesis of autoimmunity can be probed and the efficacy of novel therapies can be tested. One of the most widely-used murine models of autoimmunity is experimental autoimmune encephalomyelitis (EAE). To induce autoimmune pathology, mice are often immunized with an autoantigen alongside an adjuvant, typically complete Freund's adjuvant (CFA). Unfortunately, CFA causes significant inflammation at the site of administration. Despite the well-recognized complication of injection site inflammation, CFA with autoantigen immunization is widely used to induce central nervous system autoimmunity. We performed a literature review which allowed us to estimate that over 10,000 mice were immunized with CFA in published EAE studies in 2013. In this study, we demonstrated that subcutaneously administered myelin basic protein (MBP)-pulsed CD11c+ bone marrow-derived dendritic cells (BMDC) were as effective at inducing EAE as subcutaneously administered MBP plus CFA. Importantly, we also discovered that the CD11c+ BMDC caused significantly less injection site inflammation than MBP plus CFA immunization. This study demonstrated that the use of CD11c+ BMDC can enable the development of autopathogenic T-cells to be studied in vivo without the unwanted side-effects of long-lasting injection site inflammation. This model represents a significant refinement to existing EAE models and may lead to the improvement of the welfare of experimental mice used to study the development of autoimmunity in vivo.
Group housing is considered to be important for rats, which are highly sociable animals. Single housing may impact behaviour and levels of circulating stress hormones. Rats are typically used in the toxicological evaluation of insulin analogues. Human insulin (HI) is frequently used as a reference compound in these studies, and a comparator model of persistent exposure by HI infusion from external pumps has recently been developed to support toxicological evaluation of long-acting insulin analogues. However, this model requires single housing of the animals. Developing an insulin-infusion model which allows group housing would therefore greatly improve animal welfare. The aim of the present study was to investigate the suitability of implantable infusion pumps for HI infusion in group-housed rats. Group housing of rats implanted with a battery-driven pump proved to be possible. Intravenous infusion of HI lowered blood glucose levels persistently for two weeks, providing a comparator model for use in two-week repeated-dose toxicity studies with new long-acting insulin analogues, which allows group housing, and thereby increasing animal welfare compared with an external infusion model.
One limitation to housing rodents in individually ventilated cages (IVCs) is the ineffectiveness of traditional health monitoring programs that test soiled bedding sentinels every quarter. Aerogen transmission does not occur with this method. Moreover, the transmission of numerous pathogens in bedding is uncertain, and sentinel susceptibility to various pathogens varies. A novel method using particle collection from samples of exhaust air was developed in this study which was also systematically compared with routine health monitoring using soiled bedding sentinels. We used our method to screen these samples for the presence of murine norovirus (MNV), a mouse pathogen highly prevalent in laboratory animal facilities. Exhaust air particles from prefilters of IVC racks with known MNV prevalence were tested by quantitative reverse transcription polymerase chain reaction (RT–qPCR). MNV was detected in exhaust air as early as one week with one MNV-positive cage per rack, while sentinels discharged MNV RNA without seroconverting. MNV was reliably and repeatedly detected in particles collected from samples of exhaust air in all seven of the three-month sampling rounds, with increasing MNV prevalence, while sentinels only seroconverted in one round. Under field conditions, routine soiled bedding sentinel health monitoring in our animal facility failed to identify 67% (n = 85) of positive samples by RT–qPCR of exhaust air particles. Thus, this method proved to be highly sensitive and superior to soiled bedding sentinels in the reliable detection of MNV. These results represent a major breakthrough in hygiene monitoring of rodent IVC systems and contribute to the 3R principles by reducing the number of animals used and by improving experimental conditions.
The aim of this study was to evaluate a total intravenous anaesthesia (TIVA) protocol using propofol and sufentanil without neuromuscular blocking agents (NBAs) for a non-recovery lung pathology study in rabbits including 10 h of pressure-controlled ventilation. TIVA was started with 20 mg/kg/h propofol and 0.5 µg/kg/h sufentanil. The depth of anaesthesia was assessed by reflex testing and monitoring of spontaneous movements or respiratory efforts. Vital parameters were monitored to assess the effects of the TIVA protocol. The infusion rates were increased whenever reflex testing indicated inadequate depth of anaesthesia, and were reduced when vital parameters indicated unnecessarily deep levels. Median infusion rates of 35 mg/kg/h propofol and 2.0 µg/kg/h sufentanil were needed to ensure an adequate depth of anaesthesia. This protocol suppressed spontaneous movements, breathing and palpebral reflexes, but was unable to suppress corneal and pedal withdrawal reflexes. Since significant drops in arterial blood pressure (ABP) were observed and the animals were not exposed to painful procedures, positive corneal and pedal withdrawal reflexes were tolerated. In conclusion, propofol and sufentanil is a suitable combination for long-term anaesthesia in non-recovery lung pathology models in rabbits without painful procedures. ABP must be monitored carefully because of the circulatory side-effects, but it is an inappropriate surrogate marker for depth of anaesthesia. Due to the lack of neuromuscular blockade this TIVA protocol allows the adjustment of infusion rates based on reflex testing. The resulting decreased risk of unnoticed awareness is a decisive refinement in anaesthesia for similar studies including long-term mechanical ventilation in rabbits.
Intraperitoneal (i.p.) injection is the most frequently used method for implementing parenteral therapies in rats and mice. Whether the caecum is located in the right caudal quadrant or left caudal quadrant in the abdominal cavity is not clear. For that reason, we have developed a method for identifying the location of the caecum in rats and mice and thus revealed the most reliable location for i.p. injection in these animals. Two hundred Wistar albino rats and 100 BALB/c mice were used. The location of the caecum was determined by revealing the intra-abdominal organs immediately following euthanasia, photographing the organs, and archiving the images. Both digital photographic images and computed tomographic (CT) sections were analysed in terms of caecum morphology and location. In both rats and mice, the caecum was most commonly located on the animal’s left side. It was less frequently located on the right side or in the centre. The caecum was typically comma-shaped, but it was round or S-shaped in some animals. The direction of rotation of the caecum from the basis to the apex was mostly counterclockwise. Additionally, the apex showed a tendency to be evenly centred. This study demonstrated that the caecum was mostly located on the animal’s left side; and for that reason, the most suitable location for i.p. injection in these animals was understood to be the right caudal quadrant. Furthermore, when we compared the CT images and autopsy findings, the caecum did not change location in the abdominal cavity postmortem.
Urethane is a widely used anesthetic in animal lower urinary tract research. Our objective was to investigate the quality of anesthesia and the correlation between bladder (voiding) contractions, micturition pressure, bladder capacity and urethane dosage and body weight. Urethane was given subcutaneously and/or intraperitoneally (1.0–1.2 g/kg). The bladder was filled with saline and the bladder pressure was recorded continuously. Animals in which the subcutaneous/intraperitoneal ratio was higher needed less urethane. Heavier animals needed less extra urethane. In animals, in which no bladder contractions could be evoked, the total amount of urethane given was similar to that in those that did show contractions. In the animals that did void, the bladder never emptied completely and residual volumes remained. There was no relationship between animal weight or total amount of urethane and mean capacity. Anesthesia lasted up till 14 h, during which bladder (voiding) contractions could be recorded. Considering all results, we conclude that urethane is a well suited anesthetic for acute lower urinary tract physiological research in the intact rat.
The aim is to create a new and safe experimental model of radiation-induced neurovascular histological changes with reduced morbidity and mortality for use with experimental microsurgical techniques. Seventy-two Sprague–Dawley rats (250–300 g) were divided as follows: Group I: control group, 24 rats clinically evaluated during six weeks; Group II: evaluation of acute side-effects (two-week follow-up period), 24 irradiated (20 Gy) rats; and Group III: evaluation of subacute side-effects (six-week follow-up period), 24 irradiated (20 Gy) rats. Variables included clinical assessments, weight, vascular permeability (arterial and venous), mortality and histological studies. No significant differences were observed between groups with respect to the variables studied. Significant differences were observed between groups I vs II–III regarding survival rates and histological changes to arteries, veins and nerves. Rat body weights showed progressive increases in all groups, and the mortality rate of the present model is 10.4% compared with 30–40% in the previous models. In conclusion, the designed model induces selective changes by radiotherapy in the neurovascular bundle without histological changes affecting the surrounding tissues. This model allows therapeutic experimental studies to be conducted, including the viability of microvascular and microneural sutures post radiotherapy in the cervical neurovascular bundle.
Rodent models produce data which underpin biomedical research and non-clinical drug trials, but translation from rodents into successful clinical outcomes is often lacking. There is a growing body of evidence showing that improving experimental design is key to improving the predictive nature of rodent studies and reducing the number of animals used in research. Age, one important factor in experimental design, is often poorly reported and can be overlooked. The authors conducted a survey to assess the age used for a range of models, and the reasoning for age choice. From 297 respondents providing 611 responses, researchers reported using rodents most often in the 6–20 week age range regardless of the biology being studied. The age referred to as ‘adult’ by respondents varied between six and 20 weeks. Practical reasons for the choice of rodent age were frequently given, with increased cost associated with using older animals and maintenance of historical data comparability being two important limiting factors. These results highlight that choice of age is inconsistent across the research community and often not based on the development or cellular ageing of the system being studied. This could potentially result in decreased scientific validity and increased experimental variability. In some cases the use of older animals may be beneficial. Increased scientific rigour in the choice of the age of rodent may increase the translation of rodent models to humans.
As part of routine husbandry, an increasing number of laboratory mice receive nesting material in addition to standard bedding material in their cages. Nesting material improves health outcomes and physiological performance in mice that receive it. Providing usable nesting material uniformly and efficiently to various strains of mice remains a challenge. The aim of this study was to determine how bedding particle size, method of nesting material delivery, and processing of the nesting material before delivery affected nest building in mice of strong (BALB/cAnNCrl) and weak (C3H/HeNCrl) gathering abilities. Our data suggest that processing nesting material through a grinder in conjunction with bedding material, although convenient for provision of bedding with nesting material ‘built-in’, negatively affects the integrity of the nesting material and subsequent nest-building outcomes. We also found that C3H mice, previously thought to be poor nest builders, built similarly scored nests to those of BALB/c mice when provided with unprocessed nesting material. This was true even when nesting material was mixed into the bedding substrate. We also observed that when nesting material was mixed into the bedding substrate, mice of both strains would sort their bedding by particle size more often than if it were not mixed in. Our findings support the utility of the practice of distributing nesting material mixed in with bedding substrate, but not that of processing the nesting material with the bedding in order to mix them.
Ketamine administration has been associated with controversial behavioural impairments and psychotic episodes. Even though ketamine alone and in combination with midazolam or dexmedetomidine are frequently used in laboratory animals, the side-effects of such protocols are not well known. Therefore, our aim was to evaluate the effects of ketamine alone and in combination with midazolam or dexmedetomidine on emotional reactivity, as well as the effects on learning and memory in adult rats at least 48 h after anaesthesia. The evaluation of the potential influence of 100 mg/kg ketamine administered alone and in combination with midazolam (5 mg/kg), or dexmedetomidine (0.25 mg/kg) on spatial learning and recognition memory was studied in adult Wistar rats using the radial maze as well as object recognition and location tests. The influence of these combinations on emotional reactivity was investigated using the new exploration test and the elevated plus maze. Results showed that ketamine alone or in combination with midazolam or dexmedetomidine affected neither spatial and recognition memory, nor emotional reactivity. These results reinforce the safe clinical use of ketamine and its combinations in rats in a research context since the administration of these anaesthetic combinations did not produce significant changes with regard to spatial and recognition memory or emotional reactivity. Furthermore, these results indicate that the quality of scientific data produced in adult rat neurobehavioural research is not jeopardized by the use of these anaesthetic protocols.
It has been shown that embryonated chicken eggs can be used as animal models for experimental infections. The aim of the present study was to investigate pigeon embryonated eggs as animal models for experimental neosporosis. An infection with Neospora caninum Nc1 isolate was conducted in chicken and pigeon embryonated eggs to evaluate LD50. After calculation of LD50, 2LD50 of tachyzoites were injected into the eggs. Macroscopic changes of each embryo were observed, and immunohistochemistry (IHC) and molecular methods were used to investigate the parasitic distribution in the tissues. In the present study, histopathological changes were considered, and sections of those used for histopathological examination including the heart, liver, brain and chorioallantoic (CA) membrane were also subjected to IHC. Pigeon embryos showed more macroscopic changes than chicken embryos. A hemorrhage of the CA membrane was the main gross lesion. Microscopic examination of tissues revealed acute neosporosis due to hemorrhage, necrosis and infiltration of mononuclear inflammatory cells. Based on IHC and molecular results, the parasite DNA was detected in the liver, heart and CA membrane. As with chicken embryonated eggs, these results reinforce the susceptibility of pigeon embryonated eggs to N. caninum, and provide new insights into using an inexpensive and available animal model for N. caninum research. The results of the present study suggest that pigeon embryos may be a good choice for studying the biology of N. caninum in living organisms.
Genotyping of genetically-engineered mice is necessary for the effective design of breeding strategies and identification of mutant mice. This process relies on the identification of DNA markers introduced into genomic sequences of mice, a task usually performed using the polymerase chain reaction (PCR). Clearly, the limiting step in genotyping is isolating pure genomic DNA. Isolation of mouse DNA for genotyping typically involves painful procedures such as tail snip, digit removal, or ear punch. Although the harvesting of hair has previously been proposed as a source of genomic DNA, there has been a perceived complication and reluctance to use this non-painful technique because of low DNA yields and fear of contamination. In this study we developed a simple, economic, and efficient strategy using Chelex® resins to purify genomic DNA from hair roots of mice which are suitable for genotyping. Upon comparison with standard DNA purification methods using a commercially available kit, we demonstrate that Chelex® efficiently and consistently purifies high-quality DNA from hair roots, minimizing pain, shortening time and reducing costs associated with the determination of accurate genotypes. Therefore, the use of hair roots combined with Chelex® is a reliable and more humane alternative for DNA genotyping.
Fetal growth restriction (FGR) is a common and potentially severe pregnancy complication. Currently there is no treatment available. The guinea pig is an attractive model of human pregnancy as placentation is morphologically very similar between the species. Nutrient restriction of the dam creates growth-restricted fetuses while leaving an intact uteroplacental circulation, vital for evaluating novel therapies for FGR. Growth-restricted fetuses were generated by feeding Dunkin Hartley guinea pig dams 70% of ad libitum intake from four weeks before and throughout pregnancy. The effect of maternal nutrient restriction (MNR) on dams and fetuses was carefully monitored, and ultrasound measurements of pups collected. There was no difference in maternal weight at conception, however by five weeks post conception MNR dams were significantly lighter (P < 0.05). MNR resulted in significantly smaller pup size from 0.6–0.66 gestation. Ultrasound is a powerful non-invasive tool for assessing the effect of therapeutic interventions on fetal growth, allowing longitudinal measurement of fetuses. This model and method yield data applicable to the human condition without the need for animal sacrifice and will be useful in the translation of therapies for FGR into the clinic.
The cannulation of the cisterna magna in rats for in vivo sampling of cerebrospinal fluid serves as a valuable model for studying the delivery of new drugs into the central nervous system or disease models. It offers the advantages of repeated sampling without anesthesia-induced bias and using animals as their own controls. An established model was retrospectively reviewed for the outcomes and it was hypothesized that by refining the method, i.e. by (1) implementing pathophysiological-based anesthesia and analgesia, (2) using state-of-the-art peri-operative monitoring and supportive care, (3) increasing stability of the cement–cannula assembly, and (4) selecting a more adaptable animal strain, the outcome in using the model – quantified by peri-operative mortality, survival time and stability of the implant – could be improved and could enhance animal welfare. After refinement of the technique, peri-operative mortality decreased significantly (7 animals out of 73 compared with 4 out of 322; P = 0.001), survival time increased significantly (36 ± 14 days compared with 28 ± 18 days; P < 0.001), as well as the stability of the cement–cannula assembly (47 ± 8 days of adhesion compared with 33 ± 15 days and 34 ± 13 days using two other cement types; P < 0.001). Overall, the 3R concept of Russell and Burch was successfully addressed and animal welfare was improved by (1) the reduction in the total number of animals needed as a result of lower mortality or fewer euthanizations due to technical failure, and frequent use of individual rats over a time frame; and (2) improving the scientific quality of the model.
The use of animals is essential in biomedical research. The laboratory environment where the animals are housed has a major impact on them throughout their lives and influences the outcome of animal experiments. Therefore, there has been an increased effort in the refinement of laboratory housing conditions which is explicitly reflected in international regulations and recommendations. Since housing conditions affect behaviour and brain function as well as well-being, the validation of an animal model or paradigm to study the brain and central nervous system disorders is not complete without an evaluation of its implication on animal welfare. Here we discuss several aspects of animal welfare, comparing groups of six rats living in the PhenoWorld (PhW), a recently developed and validated paradigm for studying rodent behaviour, with standard-housed animals (in cages of six rats or pair-housed). In this study we present new data on home-cage behaviour showing that PhW animals have a clearer circadian pattern of sleep and social interaction. We conclude that, by promoting good basic health and functioning, together with the performance of natural behaviours, and maintaining animals’ control over some of their environment but still keeping some physical and social challenges, the PhW stimulates positive affective states and higher motivation in rats, which might contribute to an increased welfare for animals living in the PhW.
Each different gas that is used to induce a pneumoperitoneum (PP) exhibits individual effects within the peritoneal cavity. This might include adverse effects such as pain and/or inflammatory reactions. The acute effects of ozonized oxygen (O3/O2), a highly oxidative gas mixture, after being insufflated into the peritoneal cavity are analysed in this study. Using the abdominal constriction response (‘writhing’) assay of chemical nociception in C57BL6/N mice, O3/O2-PP was found not to be associated with visible pain responses and did not alter the c-fos expression in the spinal cord. In addition, mRNA expression levels of the pro-inflammatory cytokines, interleukin (IL)-1β and IL-6, were found unaltered in the spleen 2 h after insufflation. In conclusion, O3/O2-PP is free of adverse pain and does not trigger inflammatory immune responses.
Previous studies have established that 7,12-dimethylbenz(a)anthracene (DMBA) can initiate skin tumourigenesis in conventional furred mouse models by acting on hair follicle stem cells. However, further cancer progression depends on repeated applications of tumour promoter agents. This study evaluated the timeline involved in skin tumourigenesis and progression in immunocompetent hairless SKH1-hr mice with dysfunctional hair follicles using only DMBA with no additional tumour promoter agents. The results showed that topical application of 30 µg (117 nmol) of DMBA over the back and flank regions of the mouse once a week and 15 µg (58.5 nmol) twice a week produced skin tumours after 7–8 weeks. However, by week 14 a heavy benign tumour load required the mice to be euthanized. Lowering the DMBA dose to 15 µg (58.5 nmol) once a week produced tumours more slowly and allowed the mice to be studied for a longer period to week 23. This low-dose DMBA regimen yielded a high percentage of malignant tumours (58.8%) after 23 weekly applications. Additionally DMBA-treated skin showed an increase in mean epidermal thickness in comparison to untreated and acetone-treated skin. Despite the aberrant hair follicles in SKH1-hr mice, this chemically driven skin cancer model in hairless mice can serve as a suitable alternative to the ultraviolet-induced skin cancer models and can be reliably replicated as demonstrated by both the pilot and main experiments.
Ultrasound is a powerful, low-cost, non-invasive medical tool used by laboratory animal veterinarians for diagnostic imaging. Sonohysterography and transvaginal ultrasound are frequently used to assess uterine anomalies in women presenting with abnormal uterine bleeding (AUB). In the present study, we have evaluated the abdominal ultrasound of bonnet monkeys (n = 8) showing spontaneous ovulatory (n = 5) and anovulatory (n = 3) AUB. The ovulatory (n = 5) macaques showed cyclic AUB for 7–8 days. The anovulatory (n = 3) macaques had irregular AUB with menstrual cycles of 40–45 days. The B-mode abdominal, colour Doppler and 3D ultrasound scans were performed during the proliferative phase of the menstrual cycle. Ultrasound examination revealed endometrial polyps in five macaques and endometrial hyperplasia in three animals. The width and length of endometrial polyps was around 0.5–1 cm (average 0.51 ± 0.23 cm x 0.96 ± 0.16 cm) with significant increase in endometrial thickness (P < 0.0002). 3D ultrasound also showed a homogeneous mass in the uterine cavity and colour Doppler ultrasound showed increased vascularity in the endometrial polyps. Endometrial hyperplasia characteristically appeared as a thickened echogenic endometrium (P < 0.0002). This study demonstrates the use of non-invasive ultrasound techniques in the diagnosis of AUB in macaques.
This study describes a lateral thoracotomy approach for thoracic duct cannulation and lymphatic fluid collection in a feline model. The thoracic duct was cannulated via a left lateral intercostal thoracotomy in 12 cats. Lymphatic fluid was collected for up to 16 days and analyzed on days 3, 9 and 16. The volume collected and duration of cannula patency were recorded. Contrast imaging of the thoracic duct was performed if fluid ceased to flow or at the end of the 16-day study period. In two cats, the cannula became dislodged within 24 h. For the remaining 10 cats, mean daily volume collected was 43.7 mL (median 41.0, range 2.3 to 152.4 mL), and mean duration of cannula patency was 8.2 days (median 6.5, range 3 to 16 days). Contrast imaging revealed that the cannula was patent in three cats, obstructed in two cats, and the thoracic duct had ruptured or had extravasation of contrast outside the duct in five cats. Cytological examination of lymphatic fluid from the three time points revealed normal appearing small lymphocytes (97%) and few (3%) non-degenerate neutrophils, macrophages, eosinophils, and plasma cells. Based on the results of this study, lateral thoracotomy approach for thoracic duct cannulation is a feasible technique for collecting lymphatic fluid in cats. This technique may have application as a model for short-term evaluation of thoracic fluid in cats; however, cannula patency was unpredictable and should be considered when utilizing this technique.
Injection anaesthesia is commonly used in laboratory mice; however, a disadvantage is that post-anaesthesia recovery phases are long. Here, we investigated the potential for shortening the recovery phase after injection anaesthesia with fentanyl–midazolam–medetomidine by antagonization with naloxone–flumazenil–atipamezole. In order to monitor side-effects, the depth of anaesthesia, heart rate (HR), core body temperature (BT) and concentration of blood gases, as well as reflex responses, were assessed during a 50 min anaesthesia. Mice were allowed to recover from the anaesthesia in their home cages either with or without antagonization, while HR, core BT and spontaneous home cage behaviours were recorded for 24 h. Mice lost righting reflex at 330 ± 47 s after intraperitoneal injection of fentanyl–midazolam–medetomidine. During anaesthesia, HR averaged 225 ± 23 beats/min, respiratory rate and core BT reached steady state at 131 ± 15 breaths/min and 34.3 ± 0.25℃, respectively. Positive pedal withdrawal reflex, movement triggered by tail pinch and by toe pinch, still occurred in 25%, 31.2% and 100% of animals, respectively. Arterial blood gas analysis revealed acidosis, hypoxia, hypercapnia and a marked increase in glucose concentration. After anaesthesia reversal by injection with naloxone–flumazenil–atipamezole, animals regained consciousness after 110 ± 18 s and swiftly returned to physiological baseline values, yet they displayed diminished levels of locomotion and disrupted circadian rhythm. Without antagonization, mice showed marked hypothermia (22 ± 1.9℃) and bradycardia (119 ± 69 beats/min) for several hours. Fentanyl–midazolam–medetomidine provided reliable anaesthesia in mice with reasonable intra-anaesthetic side-effects. Post-anaesthetic period and related adverse effects were both reduced substantially by antagonization with naloxone–flumazenil–atipamezole.
Certain special biological characteristics of Japanese quails (Coturnix coturnix japonica) could enable quicker and less expensive laboratory experiments than similar studies using mice or chickens. Moreover, due to some physiological similarities with humans, quails have been recognized as an ideal laboratory model for experiments in some fields such as immunology, endocrinology, and reproductive biology. The aim of this study was to acquire normal ultrasonographic images of reproductive organs of female Japanese quails in order to facilitate laboratory experiments on reproductive biology and to determine a non-invasive in-life alternative to carcass analysis. Thirty female Japanese quails of different ages were used for ultrasound scanning of ovaries, follicles, ova, oviducts and eggs. In addition, five quails were euthanized for experiments using a direct standoff imaging technique. It was possible to identify different sizes of follicles ranging from small white follicles (0.14 cm) to large yellow follicles (2.57 cm), post-ovulatory follicles (POFs), ova at various stages of development, oviducts, and also eggs within the oviducts. These findings demonstrate that ultrasound scanning of female Japanese quail reproductive organs might be a beneficial diagnostic tool for applications such as studying reproductive physiology, anticipating the beginning or the end of the breeding season, detecting any pathological disorders easily, and identifying non-productive females which could lead to more cost-efficient laboratory procedures.
This study was designed to determine whether perineural injections of local anaesthetics decreases intraoperative nociception and improves postoperative analgesia in New Zealand White rabbits undergoing experimental stifle arthrotomy. All animals were anaesthetized using isoflurane and received morphine intramuscularly. The sciatic and femoral nerves of the leg to be operated on were located using a nerve stimulator (1 Hz, 0.5 mA). Rabbits were assigned to a treatment group (LB; n = 12) or a placebo group (P; n = 12) in a randomized blinded fashion. Group LB received lidocaine 2% (1 mg/kg) combined with bupivacaine 0.5% (0.25 mg/kg) injections around the sciatic and femoral nerves (0.1 mL/kg total volume per site) and subcutaneous infiltration of the incision site with lidocaine 1% (1.25 mg/kg). Group P received the same volume of 0.9% NaCl. Rabbits in group P required higher doses of intraoperative fentanyl and propofol to reduce heart rate and suppress increase in systolic blood pressure, and maintain an adequate anaesthetic plane. Interventional analgesia (buprenorphine and carprofen) was required significantly earlier in rabbits in group P (2 and 6 h after the first nerve blockade, respectively) based on assessment of their pain-related behaviour and range of motion. Using a visual analogue scale (0 mm= no pain to 100 mm= maximal possible pain), rabbits in group LB were judged to show significantly less pain compared with rabbits in group P (14 ± 10 mm and 37 ± 25 mm, respectively) 2 h after nerve blockade. In conclusion, this technique of perineural analgesia combined with incision site infiltration reduced intraoperative fentanyl requirements and improved postoperative analgesia in rabbits.
Olfaction plays a crucial role in mouse communication, providing information about genetic identity, physiological status of conspecifics and alerting mice to potential predators. Scents of animal origin can trigger physiological and behavioural responses that could affect experimental responses and impact positively or negatively on mouse welfare. Additionally, differing olfactory profiles could help explain variation in results between laboratories. A survey was sent to animal research units in the UK to investigate potential transfer of scents of animal origin during routine husbandry procedures, and responses were obtained from animal care workers and researchers using mice in 51 institutions. The results reveal great diversity between animal units regarding the relevant husbandry routines covered. Most [71%] reported housing non-breeding male and female mice in the same room, with 76% reporting that hands were not washed and gloves not changed between handling male and female mice. The most commonly reported species housed in the same facility as mice was the rat (91%), and 41% of respondents were aware that scents from rats could affect mice. Changing of gloves between handling mice and other species was reported by 79% of respondents. Depending on the aspect considered, between 18 and 33% of respondents believed human and non-human animal odours would strongly affect mouse physiology, behaviour or standardization, while approximately 32–54% believed these effects would be weak. This indicates uncertainty regarding the significance of these factors. Understanding and controlling these practices could reduce unwanted variability in experimental results and maximize welfare.
Clinical chemistry is an essential analytical tool in many areas of research, drug assessment and development, and in the evaluation of general health. A certain amount of blood is required to evaluate all blood analytes. Experiments where mice are used, it is difficult to measure all analytes due to the small amount of blood that can be obtained from a single animal. To overcome this problem, separate cohorts of animals are used in toxicity studies for hematology and biochemistry analysis. This requires the use of extra animals and additional resources. Hence interpretation of results derived from using these different animals can be unreliable. This study was undertaken to explore the possibility of using diluted plasma for measuring various biochemistry analytes. Plasma from mice was diluted to 3, 5 and 10-fold with Water for Injection, and various biochemistry analytes were analyzed using an automated analyzer. Results of diluted and undiluted plasma from the same mouse were compared. Most of the analytes from the diluted plasma were found to be well within the ranges of the undiluted plasma except for sodium, potassium and chloride. Diluting plasma to analyze some analytes also freed up undiluted plasma for analyzing electrolytes. In conclusion, in order to obtain reliable and interpretable data from a single mouse it is worthwhile considering diluting the plasma, which should reduce the number of animals used in an experiment.
Subarachnoid hemorrhage (SAH) from rupture of an intracranial arterial aneurysm is a devastating disease affecting young people, with serious lifelong disability or death as a frequent outcome. Large animal models that exhibit all the cardinal clinical features of human SAH are highly warranted. In this pilot study we aimed to develop a non-craniotomy model of SAH in pigs suitable for acute intervention studies. Six Norwegian Landrace pigs received advanced invasive hemodynamic and intracranial pressure (ICP) monitoring. The subarachnoid space, confirmed by a clear cerebrospinal fluid (CSF) tap, was reached by advancing a needle below the ocular bulb through the superior orbital fissure and into the interpeduncular cistern. SAH was induced by injecting 15 mL of autologous arterial blood into the subarachnoid space. Macro- and microanatomical investigations of the pig brain showed a typical blood distribution consistent with human aneurysmal SAH (aSAH) autopsy data. Immediately after SAH induction ICP sharply increased with a concomitant reduction in cerebral perfusion pressure (CPP). ICP returned to near normal values after 30 min, but increased subsequently during the experimental period. Signs of brain edema were confirmed by light microscopy post-mortem. None of the animals died during the experimental period. This new transorbital injection model of SAH in the pig mimics human aSAH and may be suitable for acute intervention studies. However, the model is technically challenging and needs further validation.
Several questions have been raised in recent years about the euthanasia of laboratory rodents. Euthanasia using inhaled agents is considered to be a suitable aesthetic method for use with a large number of animals simultaneously. Nevertheless, its aversive potential has been criticized in terms of animal welfare. The data available regarding the use of carbon dioxide (CO2), inhaled anaesthetics (such as isoflurane, sevoflurane, halothane and enflurane), as well as carbon monoxide and inert gases are discussed throughout this review. Euthanasia of fetuses and neonates is also addressed. A table listing currently available information to ease access to data regarding euthanasia techniques using gaseous agents in laboratory rodents was compiled. Regarding better animal welfare, there is currently insufficient evidence to advocate banning or replacing CO2 in the euthanasia of rodents; however, there are hints that alternative gases are more humane. The exposure to a volatile anaesthetic gas before loss of consciousness has been proposed by some scientific studies to minimize distress; however, the impact of such a measure is not clear. Areas of inconsistency within the euthanasia literature have been highlighted recently and stem from insufficient knowledge, especially regarding the advantages of the administration of isoflurane or sevoflurane over CO2, or other methods, before loss of consciousness. Alternative methods to minimize distress may include the development of techniques aimed at inducing death in the home cage of animals. Scientific outcomes have to be considered before choosing the most suitable euthanasia method to obtain the best results and accomplish the 3Rs (replacement, reduction and refinement).
Lactating mice in laboratory conditions are thought to be under considerable stress. Dams may manifest this stress through a decrease in milk yield and/or increase in infanticide. The purpose of this study was to examine the effect of access to an environmental enrichment device called the RatLoft on milk yield, circulating serotonin, and pup mortality using both wild-type mice and mice genetically deficient in tryptophan hydroxylase 1 (TPH1–/–; the rate-limiting enzyme in the non-neuronal serotonin synthesis pathway). Presence or absence of the RatLoft did not affect milk yield or circulating serotonin concentrations overall, but serotonin concentrations decreased throughout the 21-day lactation period. Serotonin concentrations were increased in TPH1-deficient mice with access to the RatLoft compared with those without the RatLoft on day 21. Pup mortality tended to decrease for dams with access to the RatLoft as compared to no RatLoft. Within the TPH1–/– groups, dams with access to the RatLoft tended to kill less pups per dam than dams without the RatLoft. These results demonstrate that access to the RatLoft during lactation decreases pup infanticide by dams. This environmental enrichment may be particularly beneficial to TPH1–/– dams that, in addition to decreased infanticide, had increased serotonin concentrations, possibly indicating enhanced well-being. Use of the RatLoft could prove beneficial to researchers working with lactating mouse models to increase the number of pups weaned and positively impact the welfare of the dam.
The vaginal epithelium of the adult female laboratory rodent changes from mucous secretion to cornification over the course of the estrous cycle. The morphophysiological changes occur with such regularity, accuracy and precision that the specific stage of the estrous cycle in the rat can be determined by inspection of the vaginal opening and/or exfoliative vaginal cytology. However, in the mouse, post-mortem vaginal histology is often required to determine the estrous cycle stage for ensuring the required level of reliability. Consequently, an excess number of female adult mice are needed to allow for the delivery of sufficient numbers of mice in a desired estrous cycle stage. In this study, we demonstrate that the standard procedure for oocyte superovulation and collection in the laboratory mouse (e.g. injection of equine chorionic gonadotropin followed 48 h later by human chorionic gonadotropin) can also be reliably used to induce changes in the epithelium of 3.5-week-old mouse vaginas in an estrous cycle stage-specific manner (e.g. establishment and replacement of a mucous secreting epithelium with a cornified epithelium; induction of cornification-associated loricrin expression). The superovulation protocol thus allows for the efficient and economic induction of estrous cycle stage-specific characteristics in the Müllerian duct-derived vagina thereby avoiding the necessity of post-mortem identification of the estrous cycle stage. In addition, our study indicates that the laboratory mouse vagina is an excellent organ for studying the sequence of events leading to cornification.
An essential aspect of genetically-engineered mice (GEM) is the ability to produce live animals after the appropriate injection procedure. Animals are produced by implantation of manipulated embryos into pseudopregnant females for gestation, parturition, and growth to the weaning stage. This study was carried out to test whether the anesthesia used during surgery could affect the number of pups produced. Anesthetics commonly used for implant surgery include tribromoethanol (Avertin) delivered by intraperitoneal (IP) injection, IP-injected ketamine:xylazine or ketamine:medetomidine mix, and inhaled isoflurane. To determine if the anesthesia used might affect the number of animals produced, each anesthetic agent was tested in implant surgeries and the numbers of pups produced using both wild-type and GEM embryos were assessed. Parallel studies were conducted in institutions in the EU and in the USA. Based on a direct comparison of pregnancy status, number of pups born, and number of pups weaned for each agent, we found no statistical differences among the three anesthetics. We conclude that all three anesthetic agents tested are equally useful for implantation surgery.
With their highly sensitive olfactory system, the behaviour and physiology of mice are not only influenced by the scents of conspecifics and other species, but also by many other chemicals in the environment. The constraints of laboratory housing limit a mouse’s capacity to avoid aversive odours that could be present in the environment. Potentially odorous items routinely used for husbandry procedures, such as sanitizing products and gloves, could be perceived by mice as aversive or attractive, and affect their behaviour, physiology and experimental results. A survey was sent to research institutions in the UK to enquire about husbandry practices that could impact on the olfactory environment of the mouse. Responses were obtained from 80 individuals working in 51 institutions. Husbandry practices varied considerably. Seventy percent of respondents reported always wearing gloves for handling mice, with nitrile being the most common glove material (94%) followed by latex (23%) and vinyl (14%). Over six different products were listed for cleaning surfaces, floors, anaesthesia and euthanasia chambers and behavioural apparatus. In all cases Trigene™ (now called Anistel™) was the most common cleaning product used (43, 41, 40 and 49%, respectively). Depending on the attribute considered, between 7 and 19% of respondents thought that cleaning products definitely, or were likely to, have strong effects on standardization, mouse health, physiology or behaviour. Understanding whether and how these odours affect mouse welfare will help to refine mouse husbandry and experimental procedures through practical recommendations, to improve the quality of life of laboratory animals and the experimental data obtained.
The pathogenesis of Alzheimer’s disease (AD), a type of neurodegenerative disease characterized by learning and memory impairment, is often associated with pathological features, such as amyloid-beta (Aβ) accumulation and insulin resistance. The transgenic mouse, APPswePS1dE9 (APP/PS1), is one of the most commonly used animal models in pathogenesis studies of AD. The purpose of this study is to investigate the sex differences between APP/PS1 mice in the pathogenesis of AD. The impairment of glucose and insulin tolerance was found to develop earlier in male APP/PS1 mice than in females. Plasma insulin levels were significantly decreased in male APP/PS1 mice, while total cholesterol levels in male APP/PS1 mice were higher than those in females. Triglyceride levels in male mice in both the wild-type (WT) and APP/PS1 groups were higher than in their female littermates. Soluble and insoluble Aβ levels in female APP/PS1 mouse brains were higher than those in males. And the learning and memorizing abilities of female APP/PS1 mice were poorer than those of males. Our results concluded that there were sex differences in Aβ formation, pancreatic islet function and insulin sensitivity between male and female APP/PS1 mice during the pathogenesis of AD.
Production of genetically-modified mice is strongly dependent on environmental conditions. Mice are commonly housed at 22℃, which is significantly lower than their thermoneutral zone. But, when given a choice, mice often seem to prefer higher ambient temperatures. In the current study we investigated the effect of higher ambient temperature on the production of transgenic mice, with emphasis on embryo and sperm yield and quality. Mice (C57BL/6JOlaHsd) were housed under four different ambient temperatures (22, 25, 28 and 30℃). Female mice were superovulated, and mated with males. As indicators for reproductive fitness, the success of the mating was observed, including embryo yield and quality, as well as sperm count, motility and progressivity. Female mice were found to produce high amounts of high quality embryos from 22 to 28℃. Sperm count dropped continuously from 22 to 30℃, but sperm motility and progressivity remained high from 22 to 28℃. We conclude that mice can be housed at significantly higher temperatures than is commonly recommended without compromising embryo production and quality, or sperm quality. These results could lead to fundamental changes in how mouse facilities are built and operated – especially in warmer climates whereby energy consumption and therefore costs could be significantly reduced.
Various types of restraint collars have been used for research animals, and the Elizabethan collar (E-collar) is the most commonly used. However, animals can be choked by the E-collar or they tend to remove it; furthermore, repeated rubbing and scratching of the collar may chafe the neck. We developed a new restraint collar with a vest to overcome these limitations. The vest-collar (V-collar) can be worn similarly to a vest, in contrast to the E-collar, which is fixed around the neck. A cone-shaped collar is attached to the vest in the V-collar and is made of Eva foam to surround the chest softly, accompanied by a transparent polyvinyl chloride (PVC) film for visibility. To evaluate the performance of the V-collar, we conducted experiments with mice wearing the V-collar and the E-collar. Both groups showed normal weight gain and food intake. Glucose and stress hormone levels showed no significant differences, and no stress-associated leukocyte profiles were observed during the experiments. However, despite the short experimental duration, more than half of the mice in the E-collar group showed injury to the skin on the neck, with increased thickness of the epidermal and keratin layers. Moreover, inflammatory cell counts were higher in the E-collar group than in the V-collar group. In conclusion, the V-collar, in contrast to the E-collar, does not cause skin injuries in animals and is thus beneficial for animals and investigators. Investigators can effectively use the V-collar to enhance laboratory animal welfare.
Correct identification of bacteria is crucial for the management of rodent colonies. Some bacteria are difficult to identify phenotypically outside reference laboratories. In this study, we evaluated the utility of 16S ribosomal DNA (rDNA) sequencing as a means of identifying a collection of 30 isolates of rodent origin which are conventionally difficult to identify. Sequence analysis of the first approximate 720 to 880 bp of the 5'- end of 16S rDNA identified 25 isolates (83.33%) with ≥99% similarity to a sequence of a type strain, whereas three isolates (10%) displayed a sequence similarity ≥97% but <99% to the type strain sequences. These similarity scores were used to define identification to species and genus levels, respectively. Two of the 30 isolates (6.67%) displayed a sequence similarity of ≥95 but <97% to the reference strains and were thus allocated to a family. This technique allowed us to document the association of mice with bacteria relevant for the colonies management such as Pasteurellaceae, Bordetella hinzii or Streptococcus danieliae. In addition, human potential pathogens such as Acinetobacter spp., Ochrobactrum anthropi and Paracoccus yeei or others not yet reported in mouse bacterial species such as Leucobacter chironomi, Neisseria perflava and Pantoea dispersa were observed. In conclusion, the sequence analysis of 16S rDNA proved to be a useful diagnostic tool, with higher performance characteristics than the classical phenotypic methods, for identification of laboratory animal bacteria. For the first time this method allowed us to document the association of certain bacterial species with the laboratory mouse.
Postsurgical oral self-administration of analgesics in rodents is an interesting technique of providing analgesia, avoiding the negative effects of manipulation. Several strategies, using gelatin or nutella, have already been described. However, rodents require some habituation period to reach a good intake because of their neophobic behavior. The current study aimed to explore whether buprenorphine when mixed with an extruded diet offers a potential treatment option in the pain management of mice using a triple approach: by measuring the spontaneous intake in healthy animals; by using the hot-plate test; and finally by assessing the drug’s ability to provide postoperative analgesia in a surgical intervention of moderate severity (intra-utero electroporation). Mice consumed during 20 hours, similar amounts of extruded diet alone, mixed with glucosaline, and mixed with buprenorphine (0.03 mg per pellet) or meloxicam (0.25 mg per pellet) both of which were diluted in glucosaline, showing that no neophobia was associated with these administrations. Relative increase from baseline latency (% maximal possible effect) in the hot-plate test at 20 h of administration was significantly higher for oral buprenorphine in diet 0.03 mg/pellet, and diet 0.15 mg/pellet, compared with placebo and no differences were found between those oral administrations and subcutaneous buprenorphine 0.1 mg/kg measured 3 h later. The treatment was also effective in attenuating the reductions in food consumption and body weight that occur after surgery. These data suggest that providing buprenorphine with the diet is a feasible and effective way of self-administration of analgesia in mice and does not cause neophobia and may easily contribute to the refinement of surgical procedures.
A proper cage environment is essential for the welfare of laboratory mice, especially for females during the energy demanding lactation period and for pups during early development and growth. The most common housing system for laboratory mice is individually ventilated cages (IVCs) of which there are different layouts and ventilation strategies available on the market. The present study investigates the impact of cage environment in three different IVC types, on the maternal performance of females, and pup development and growth in C57BL/6NCrl and Crl:NMRI Foxn1nu mice. The results show differences in in-cage climate, female body weight, pup growth, feed and water consumption, and nest quality between cage types. There was a distinct effect of genotype in these differences, with the main effects found in NMRI NU mice. The results indicate that IVC systems might need to be managed differently for mice of different types and/or different physiological status. Many of the differences seen between cage systems could be drawn to the physical construction of the cage, such as location of feed hopper and location of air inlet and outlet. In conclusion, IVC in-cage climate affects the maternal performance of female mice and pup growth, but with differences between the two strains tested.
The mechanisms of production, and gross, microscopic and electrocardiograhic findings of surgically-induced complete heart block (CHB) in the adult rat are presented. This is an effective in vivo model for establishing alternative methods to electronic pacemakers and for providing detailed information aimed at replacement, reduction and refinement of the technique. Sternal thoracotomy was employed to identify the epicardial fat pad by the aortic root, used as a landmark for cauterization of the atrioventricular (AV) node. Stable CHB was produced in 60 rats with a 70% survival rate. The best survival rate was observed in 8-week-old animals weighing 221 ± 27.6 g. Heart rate before cauterization was 387 ± 55 bpm, reduced after cauterization to 126 ± 40 bpm in the survival and to 65 ± 19 bpm in the non-survival groups. At 30 days findings were: elevated left ventricular end-diastolic pressure (21 ± 5.4 mmHg, P < 0.05); maximal rate of rise of left ventricular pressure (LVP) during isovolumetric contraction (2192 ± 235 mmHg/s, P < 0.05); maximal rate of decrease of LVP (–1658 ± 191 mmHg/s, P < 0.05); isovolumetric relaxation constant (5.7 ± 0.8 ms, P < 0.05) with wet-to-dry lung–weight ratio (78.1 ± 0.4, P < 0.05); heart weight/body weight (0.6 ± 0.1, P < 0.05); heart volume (1.8 ± 0.3 mL, P < 0.05); longitudinal diameter (20.2 ± 1.91 mm, P < 0.05); and transversal diameter (17.0 ± 1.4 mm, P < 0.05) with supported dilated cardiomyopathy which culminated in chronic heart failure. CHB hearts had increased preload and replacement of myofibrils by collagen. CHB was achieved reproducibly by cauterization of the rat AV node and/or His bundle. This led to electrophysiological, hemodynamic, and structural remodeling, and could be useful in long-term cardiac remodeling assessments and potential therapy development.
Expansion of physiological knowledge increasingly requires examination of processes in the normal, conscious state. The current study describes a novel approach combining surgical implantation of radio-telemeters with vascular access ports (VAPs) to allow repeated hemodynamic and pharmacological measures in conscious rats. Dual implantation was conducted on 16-week-old male lean and obese Zucker rats. Continued viability one month after surgery was observed in 67% of lean and 44% of obese animals, giving an overall 54% completion rate. Over the five-week measurement period, reliable and reproducible basal mean arterial pressure and heart rate measures were observed. VAP patency and receptor-independent vascular reactivity were confirmed by consistent hemodynamic responses to sodium nitroprusside (6.25 µg/kg). Acutely, minimal hemodynamic responses to repeated bolus administration of 0.2 mL saline indicated no significant effect of increased blood volume or administration stress, making repeated acute measures viable. Similarly, repeated administration of the β-adrenoceptor agonist dobutamine (30 µg/kg) at 10 min intervals resulted in reproducible hemodynamic changes in both lean and obese animals. Therefore, our study demonstrates that this new approach is viable for the acute and chronic assessment of hemodynamic and pharmacological responses in both lean and obese conscious rats. This technique reduces the demand for animal numbers and allows hemodynamic measures with minimal disruption to animals’ welfare, while providing reliable and reproducible results over several weeks. In conclusion, dual implantation of a radio-telemeter and VAP introduces a valuable technique for undertaking comprehensive studies involving repeated pharmacological tests in conscious animals to address important physiological questions.
Haemarthrosis is the most common clinical manifestation of haemophilia and is responsible for significant morbidity in haemophilic patients. The murine experimentally-induced knee bleeding model is an important model in haemophilia research but it is currently unknown if the use of analgesia in this model might impact on the inflammatory response. The aim was to investigate the inflammatory response after a needle induced knee bleed in haemophilia A mice treated with buprenorphine or saline. One hundred and sixty mice were randomized into two groups to blindly receive buprenorphine or saline. All the mice were anaesthetized and knee injury was induced by inserting a 30 G needle into the right knee joint. At t = 6, 24, 48 and 72 h, 20 mice from each group were terminated and the following parameters were assessed: change in body weight and joint diameter, visual bleeding score (VBS), white blood counts, haematocrit, platelet concentrations, haemoglobin, plasma haptoglobin and plasma and synovial fluid levels of 23 cytokines. Twenty mice were terminated at t = 0 receiving no injury or treatment to provide baseline measures. Twenty-one cytokines in plasma and 22 cytokines in synovial fluid, joint diameter change, VBS and blood parameters were not significantly altered by the administration of buprenorphine. Slight alterations of plasma haptoglobin at t = 48 h, body weight, plasma and synovial eotaxin and plasma G-CSF were found in buprenorphine-treated mice. We demonstrated that buprenorphine does not overall impact on the inflammatory response, and the use of buprenorphine in the knee bleeding model in haemophilic mice should be continued.
Large mouse litters are often culled based on the premise of better survival and growth for the remaining pups. To test whether the culling of litters does provide the benefits of improved survival and growth, mortality and growth were measured in 468 litters of C57BL/6J x 129S1/SvImJ F1 hybrid mice that were unculled or culled to four or six pups per litter. In addition, a limited number of weanlings were evaluated until three months of age for growth, health and physiological measurements. Most measurements did not differ among the cull groups. These included mortality, organ weights (adrenals, kidneys and testes), bone mineral density, percent fat, 12 of the 17 blood parameters, and three of the seven electrocardiographic (EKG) parameters. Several parameters, including five of the 17 blood parameters and four of the seven EKG parameters, showed statistical differences, but all values were physiologically normal. Unculled weanlings showed a reduced weight of 4%, but this weight difference disappeared by three months. These results suggest that mice in culled litters do not demonstrate improved health compared with those in unculled litters.
The effects of streptozotocin (STZ) were studied in eight high-health herd-certified Yorkshire x Swedish Landrace pigs (32.5 ± 2.6 kg initial body weight [BW]), and an insulin treatment protocol was developed to re-establish their metabolisms. A single intravenous dose of 150 mg STZ/kg BW successfully induced hyperglycaemia and alterations in their fat and protein metabolisms. Within 13 h post-STZ treatment blood glucose concentration had fallen to a range of 1.3 to 4.7 mmol/L. Hypoglycaemia was promptly treated with 0.5 g glucose/kg BW intravenously. All the pigs became hyperglycaemic with blood glucose concentrations >23 mmol/L within 48 h post-STZ. Two days post-STZ serum C-peptide concentrations fell below 60 mol/L in all the pigs and remained below 96 mol/L for five weeks until the end of the study. The pigs were left untreated for one week after STZ injection. At the end of this week 13-fold and nine-fold increases in serum concentrations of triglycerides and non-esterified fatty acids, respectively, were observed. Also, at this time-point a three-fold increase in the concentration of branched-chain amino acids (BCAA) was observed, and alanine and taurine were decreased by approximately 70% and 40%, respectively. During the week when the pigs were untreated, a reduced weight gain was observed, but after the onset of insulin treatment the daily weight gain was at least as good as that of conventional high-health pigs. Then a subcutaneous treatment with short-acting insulin was initiated. The initial dose of 2/3 IU/kg BW daily, divided between two doses, was gradually increased to 1 IU/kg BW. Within three weeks, the insulin treatment restored the metabolic changes in carbohydrate, fat and protein metabolisms produced by the STZ. In conclusion, the results underscore the usefulness of this animal model in translational research as insulin treatment re-establishes the changes in carbohydrate, fat and amino acid metabolisms observed in STZ-diabetic pigs and resolves clinical signs of disease similar to those in humans.
The purpose of the study was to optimize postoperative comfort in lambs anaesthetized for the surgical creation of scoliosis while maintaining the ewe–lamb relationship to miminize rejection rates. The first management plan produced six scoliotic lambs but intraoperative hypoventilation and hypovolaemia followed by postoperative dyspnoea, pain, monitoring and nursing difficulties, hypothermia, hypoglycaemia and tympany were encountered. Three of eight lambs (38%) were rejected by their ewes. Perioperative management was amended which, combined with improved surgical technique, produced 16 scoliotic lambs. The lambs recovered more rapidly (mean time to standing after discontinuation of anaesthesia was reduced from 12 h to 70 min) and appeared to be more comfortable. No rejections occurred after the refinements were implemented.
The 3Rs principle of replacement, reduction, and refinement has increasingly been endorsed by legislators and regulatory bodies as the best approach to tackle the ethical dilemma presented by animal experimentation in which the potential benefits for humans stand against the costs borne by the animals. Even when animal use is tightly regulated and supervised, the individual researcher’s responsibility is still decisive in the implementation of the 3Rs. Training in laboratory animal science (LAS) aims to raise researchers’ awareness and increase their knowledge, but its effect on scientists’ attitudes and practice has not so far been systematically assessed. Participants (n = 206) in eight LAS courses (following the Federation of European Laboratory Animal Science Associations category C recommendations) in Portugal were surveyed in a self-administered questionnaire during the course. Questions were related mainly to the 3Rs and their application, attitudes to animal use and the ethical review of animal experiments. One year later, all the respondents were asked to answer a similar questionnaire (57% response rate) with added self-evaluation questions on the impact of training. Our results suggest that the course is effective in promoting awareness and increasing knowledge of the 3Rs, particularly with regard to refinement. However, participation in the course did not change perceptions on the current and future needs for animal use in research.
Mice with genetic alterations are used in heart research for the extrapolation of human diseases. Echocardiography is an essential tool for evaluating cardiac and hemodynamic functions in small animals. The purpose of this study was to compare the effect of different anesthetic regimens and the conscious state on the evaluation of cardiac function by echocardiography. Mice were examined in the conscious state after three days of training, and then for a 7 min period after a single intraperitoneal injection of ketamine at 100 mg/kg, etomidate at 10, 20 or 30 mg/kg, or after inhalation of isoflurane at 1.5% with or without a short period of induction with isoflurane 3%. Intra- and inter-observer variabilities were assessed. The operator’s comfort was also assessed. Heart rate, left ventricular end diastolic diameter, fraction shortening and cardiac output were measured using echocardiography. Ketamine at 5 and 7 min after induction and isoflurane at 3, 5 and 7 min after induction provided good anesthetic conditions and a quick awakening time, and did not influence cardiac performance, whereas the conscious state was associated with a non-physiological sympathetic activation and other anesthetic drugs induced a significant decrease in heart rate. Etomidate 10 mg/kg and 20 mg/kg were not enough to provide adequate anesthesia. Etomidate 30 mg/kg induced a good anesthetic condition but influenced cardiac performance and had a long awakening time. Our results indicate that ketamine and isoflurane with a short induction period are better anesthetic drugs than isoflurane without induction or etomidate for evaluating cardiac function in healthy mice.
In vivo assessment of ventricular function in rodents has largely been restricted to transthoracic echocardiography (TTE). However 1.5 T cardiac magnetic resonance (CMR) and transoesophageal echocardiography (TOE) have emerged as possible alternatives. Yet, to date, no study has systematically assessed these three imaging modalities in determining ejection fraction (EF) in rats. Twenty rats underwent imaging four weeks after surgically-induced myocardial infarction. CMR was performed on a 1.5 T scanner, TTE was conducted using a 9.2 MHz transducer and TOE was performed with a 10 MHz intracardiac echo catheter. Correlation between the three techniques for EF determination and analysis reproducibility was assessed. Moderate-strong correlation was observed between the three modalities; the greatest between CMR and TOE (intraclass correlation coefficient (ICC) = 0.89), followed by TOE and TTE (ICC = 0.70) and CMR and TTE (ICC = 0.63). Intra- and inter-observer variations were excellent with CMR (ICC = 0.99 and 0.98 respectively), very good with TTE (0.90 and 0.89) and TOE (0.87 and 0.84). Each modality is a viable option for evaluating ventricular function in rats, however the high image quality and excellent reproducibility of CMR offers distinct advantages even at 1.5 T with conventional coils and software.
An impaired intestinal epithelial barrier is thought to be a major factor in the pathogenesis of human inflammatory bowel disease (IBD). IBD is frequently investigated by inducing a damaged barrier in murine models of colitis. This can be done by feeding mice with dextran sulfate sodium (DSS) polymers in their drinking water. Refinement measures should focus on alleviating unnecessary suffering during this probably painful condition. Appropriate parameters are needed to decide when to terminate the experiments. Our aim was to investigate whether a change in burrowing behaviour is a sensitive measure of animal welfare in murine models of colitis. Acute colitis was induced in C57BL/6 mice with 2.0% DSS over nine days. The burrowing test is based on the species-typical behaviour of mice to spontaneously displace items from tubes within their home cage. As a burrowing apparatus, a water bottle (250 mL, 150 mm length, 55 mm diameter) filled with 138–142 g of pellets of the animal’s diet was used. The presence of intestinal inflammation as a result of acute DSS-induced colitis was confirmed by a decrease in body weight, colon length and an increase of murine endoscopic index of colitis severity, histological score and spleen weight in the group receiving DSS as compared with the control group. An onset of intestinal inflammation correlated with a significant decrease in burrowing behaviour (P < 0.05). Altered adrenal gland histology indicated stress as a result of acute colitis. Our findings provide evidence that changes of spontaneous burrowing behaviour correlate with the onset of inflammation in acute DSS-induced colitis.
The Desert hamster, Phodopus roborovskii, is one of the lesser-known laboratory animal models and therefore knowledge of its reproductive system and physiology is limited. This study investigated the time course of vaginal and behavioural oestrus cycles by means of vaginal smear cytology, serum luteinizing hormone (LH) levels, wheel-running activity patterns, and pairing tests. The oestrus cycle lasts between four and six days and follows a pattern rather similar to that described for other rodent species. An important finding of this study is the existence of a fifth cycle stage between pre-oestrus and oestrus, the so-called early oestrus. Early oestrus differs from pre-oestrus by a complete lack of leucocytes in the vaginal cytology and a dramatic increase of serum LH levels just before activity onset. The early oestrus stage lasts 4–6 h, but was not observed in every female. When present it affects the length of the whole oestrus cycle. With early oestrus the pre-oestrus stage lasts only 14–18 h and the total length of the oestrus cycle length is four days. Without early oestrus, the pre-oestrus is prolonged to 18–36 h and the oestrus cycle length varies between four and six days. Desert hamsters showed only subtle oestrus-correlated changes in wheel-running activity, i.e. they failed to show the characteristic scalloping of activity onset, but showed prolonged activity during early oestrus. Pairing tests revealed characteristic changes in the relative frequencies of socio-positive, neutral, aggressive and sexual behaviour during the course of the oestrus cycle, with an elevated level of sexual behaviour during oestrus.
Streptozotocin (STZ) is a selective pancreatic β cell toxin used to generate experimental hyperglycemia in rodent models. Several laboratory animal protocols suggest that STZ be administered to fasted rodents to minimize competition between STZ and glucose for low affinity GLUT2 transporters on β cells. However, whether the diabetogenic effects of multiple low dose (MLD)-STZ administration are enhanced by fasting has not been addressed. Given that repeated bouts of fasting can cause undue metabolic stress in mice, we compared the efficacy of MLD-STZ injections (50 mg/kg body weight daily for 5 days) to induce experimental hyperglycemia in both NOD/SCID/chainnull and C57BL/6J mice that were either ad libitum fed (STZ-Fed) or that had been fasted for 6 h (STZ-Fasted) prior to the time of STZ administration. Both STZ-Fed and STZ-Fasted mice had significantly worse glucose tolerance than vehicle-treated control mice 10 days after initiation of the MLD-STZ regimen. In C57BL/6J mice, fasting glucose levels, serum insulin levels, β cell mass, and glucose disposal during intraperitoneal glucose tolerance tests (IPGTTs) were indistinguishable between STZ-Fed and STZ-Fasted mice 20 days after MLD-STZ. The glucose intolerant phenotypes persisted for 20 weeks thereafter, irrespective of whether C57BL/6J mice were fed or fasted at the time of STZ injections. However, STZ-Fasted C57BL/6J mice experienced significant weight loss during the repeated bouts of fasting/re-feeding that were required to complete the MLD-STZ protocol. In summary, induction of experimental hyperglycemia can be achieved using the MLD-STZ protocol without repeated bouts of fasting, which have the potential to cause metabolic stress in laboratory mice.
Mice are widely used in studies investigating the effect of diet on metabolic risk factors, such as lipid profiles and plasma glucose levels. An important factor that is usually not taken into account is the biological age of the experimental models. The up-to-date identified experimental confounders do not cover all the parameters that may affect the results of animal studies. The aim of this study was to investigate the effects of a high-fat diet on the metabolic profile, hepatic and renal function in mice of differing ages. For this purpose two groups of male C57BL/6J mice were used, consisting of 10-week-old mice and 54-week-old mice in each group. Both groups followed identical high-fat diets for 12 weeks. The younger mice showed smaller increases in body weight, serum total cholesterol, glucose and urea levels while they had higher increases in high-density lipoprotein cholesterol levels than the older mice. Our results indicate the necessity to consider an experimental animal’s age as a confounding factor when researching or interpreting metabolic studies. Age adjustment is warranted in all animal research while a uniform approach regarding the age of the animal models should be applied in experimental studies.
Ad libitum (AL) feeding of rats leads to obesity and increased result variability, as well as premature morbidity and mortality. It may also alter metabolism and responses to foreign compounds. Moderate dietary restriction (DR) reduces these untoward effects without compromising the sensitivity of rodent bioassays. The diet board (DB) is a novel method for achieving moderate DR in group housing. Food pellets are firmly attached into grooves in an aspen board, and rats have to gnaw the wood in order to eat. Food is available continuously, but due to the effort involved rats eat less. This study simulated a chronic safety test to assess the long-term effects of DB feeding. A total of 146 male and female outbred Sprague-Dawley rats, nine weeks old at onset, were housed in groups of three and fed either AL or with DBs for two years. Food and water consumption were measured at six time points. The rats were weighed every one to two weeks. Body and tibial lengths and epididymal fat weight were measured at necropsy. Modified body mass index was calculated at five time points after one year of age. DB feeding reduced body weight and fat tissue moderately, more so in males. DB males ate less than AL males, but no differences were seen in the total food consumption in the females. There was no consistent difference in the within-group variations of the measured parameters. DB is a workable DR method, albeit some modification could enhance and standardize its DR effects, especially in female rats.