Vitamin D and cholesterol share the same intestinal transporters. Thus, it was hypothesized that dietary cholesterol adversely affects vitamin D uptake. The current studies investigated the influence of cholesterol on the availability of oral vitamin D. First, 42 wild-type mice received a diet with 25 µg/kg labelled vitamin D3 (vitamin D3-d3), supplemented with either 0% (control), 0.2%, 0.4%, 0.6%, 0.8%, 1.0% or 2.0% cholesterol for four weeks to investigate vitamin D uptake. In a second study, 10 wild-type mice received diets containing 0% (control) or 1% cholesterol over four weeks to determine cholesterol-induced changes in bile acids. Finally, we investigated the impact of cholesterol versus bile acids on vitamin D uptake in Caco-2 cells. Surprisingly, dietary cholesterol intake was associated with 40% higher serum levels of vitamin D3-d3 and 2.3-fold higher vitamin D3-d3 concentrations in the liver compared to controls. The second study showed that cholesterol intake resulted in higher concentrations of faecal bile acids (control: 3.55 ± 1.71 mg/g dry matter; 1% dietary cholesterol: 8.95 ± 3.69 mg/g dry matter; P < 0.05) and changes in the bile acid profile with lower contents of muricholic acids (P < 0.1) and higher contents of taurodeoxycholic acid (P < 0.01) compared to controls. In-vitro analyses revealed that taurocholic acid (P < 0.001) but not cholesterol increased the cellular uptake of vitamin D by Caco-2 cells. To conclude, dietary cholesterol seems to improve the bioavailability of oral vitamin D by stimulating the release of bile acids and increasing the hydrophobicity of bile.

Toxoplasmosis is a significant public health concern with limited therapeutic options. The medicines for malaria venture (MMV) developed the pandemic response box (PRB) containing 400 drug-like molecules with broad pathogen activity. The aim of this work is to evaluate PRB compounds for their anti-Toxoplasma gondii activity and identify promising candidates for further evaluation. Screening identified 42 selective compounds with half effective concentration (EC50) ranging from 2.4 to 913.1 nm and half cytotoxic concentration (CC50) ranging from 6 μm to >50 μm. Selectivity index (SI) values (CC50/EC50) ranged from 11 to 17 708. Based on its in silico and in vitro profile and its commercial availability, RWJ-67657 was selected for further studies. Molecular docking analysis showed RWJ-67657 is predicted to bind to T. gondii p38 mitogen-activated protein kinase (TgMAPK). Oral administration of RWJ-67657 (20 mg kg day−1/10 days) significantly reduced parasite burden in chronically infected mice compared to mock-treated group (P < 0.01). These findings highlight the PRB as a promising source for anti-T. gondii compounds, with several showing favourable drug properties, including MMV1634492, MMV002731, MMV1634491, MMV1581551, MMV011565, MMV1581558, MMV1578577, MMV233495 and MMV1580482, firstly described here as anti-T. gondii agents. RWJ-67657 emerges as a valuable drug candidate for experimental chronic cerebral toxoplasmosis therapy.

Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.

Rodent control tends to involve methods that cause animal suffering, but little attention has been paid to the animal welfare implications of rodent control. The aim of the current study was to gain insight into the opinions and attitudes of clients of Dutch pest controllers, regarding liminal rodents, rodent control, and rodent welfare. A better understanding of their attitudes may contribute to more ethical rodent management programmes. An online survey among 248 clients of Dutch pest controllers was carried out. Respondents, especially those within the agricultural sector, have a relatively negative attitude towards rats and mice. Respondents in the agricultural subgroup do not consider the welfare of liminal rodents important. They also think that the welfare impact of commonly used control methods is limited, and they have low tolerance levels for the presence of rodents. Respondents from other sectors have a far more positive attitude towards rats and mice, consider their welfare to be of greater importance, have a greater estimation of the welfare impact of control methods and show greater tolerance levels towards rodents. The respondents from the latter subgroup have a similar attitude compared to Dutch pest controllers participating in a previous survey. The findings of the current study firstly provide useful information for the further development and practical implementation of preventive control methods. Secondly, they provide input for a more animal-friendly rodent control and for the development of an assessment framework to support ethical decision-making. Finally, they can be helpful for further research and the communication and co-operation between professional pest controllers and their clients.

Currently, no effective treatment is available for trichinellosis, a zoonotic parasitic disease caused by infection with the genus Trichinella. Kaempferol (KPF), a dietary flavonoid, has been documented to have anti-parasitic effects and various medicinal uses. Thus, this study aimed to investigate the efficacy of KPF in preventing and treating the intestinal and muscular phases of trichinellosis in mice compared with albendazole (ABZ). To achieve this, mice were divided into six groups: negative control; positive control; KPF prophylaxis; KPF treatment; ABZ treatment; and a combination of ABZ and KPF. Parasitological, histopathological and immunohistochemical evaluations were conducted to assess the effectiveness of the treatments. The parasitological assessment involved counting small intestinal adult worms and encysted muscle larvae. Additionally, the histopathological evaluation used the haematoxylin and eosin staining method for intestinal and muscular sections and picrosirius red stain for muscular sections. Moreover, the immunohistochemical expression of the intestinal NOD-like receptor-pyrin domain containing 3 (NLRP3) was evaluated. The group treated with combined drugs demonstrated a statistically significant reduction in the count of adults and encysted larvae (P < 0.05), a remarkable improvement in the inflammation of the intestines and muscles and a decrease in the thickness of the larvae's capsular layer. Additionally, the highest reduction in NLRP3 expression was observed in this group. Based on this study, KPF shows promise as an anti-trichinellosis medication that, when taken with ABZ, has a synergistic impact by modulating inflammation and larval capsule formation.

In nature, mice live in burrows with nest chambers where they breed and may hide from predators. In the laboratory, a shelter or refuge is an easily applicable form of enrichment which may enhance the welfare of laboratory mice by giving them more control over their environment. Six nest boxes made of different materials were evaluated in a preference test with male and female mice of two strains (C57BL/6J and BALB/c). In general, mice showed a preference for cages with a nest box made of grid metal as compared to clear or white perspex nest boxes, or no nest box. They also showed a preference for a cage with a nest box of perforated metal as compared to nest boxes made of grey PVC or sheet metal, or no nest box. When offered a nest box with one open side or a nest box with two open sides, most mice preferred the nest box with one open side and were observed to lie in it with their heads directed towards the opening.

The results of this study show that nest boxes may be used for enrichment purposes in caged mice, although it is not yet entirely clear what are the main features influencing the animals’ preferences. When providing nest boxes as shelters, the structure and design of this type of enrichment should be taken into account, because these may have an effect on the social structure of groups of mice.

Group housing of male laboratory mice often leads to welfare problems due to aggressive behaviour. From a welfare perspective, individual housing is not a preferred solution to these problems - and so we sought other ways of reducing aggression between male mice. Aggression peaks after disturbances such as cage cleaning. Transfer of olfactory cues during cage cleaning procedures has been repeatedly proposed as a means of reducing these peaks in aggression. In this study, the aggression-modulating properties of olfactory cues were studied by investigating the effects of their source and distribution on aggression after cage cleaning in groups of male BALB/c mice. The physiological effects of aggression on individuals within a group were also monitored.

Our results indicated that neither kinship nor distribution of urine marks affected aggression. Olfactory cues from nesting and bedding material, however, affected aggression to a marked degree: transfer of nesting material reduced aggression significantly, while transfer of sawdust containing urine and faeces seemed to intensify aggression. None of the physiological data revealed any differences between dominant and subordinate animals, nor any correlations with aggressiveness, except that dominant animals gained weight more rapidly than subordinate ones. We conclude that the transfer of nesting material will reduce aggression, or at least slow down its development, and thus aid the reduction of social tension due to cage cleaning.

Chromosome substitution strains (also called consomic lines or strains) are strains in which a single, full-length chromosome from one inbred strain — the donor strain — has been transferred onto the genetic background of a second inbred strain — the host strain. Based on the results obtained from behavioural tests with the two parental strains, the minimum number of animals from each of the host and consomic strains that are required for a successful behavioural genetic analysis can be estimated. Correct application of statistical knowledge can lead to a further reduction in the number of animals used in behavioural genetic experiments using chromosome substitution strains.

The ability to measure objectively how an animal perceives its home environment is essential for improving the housing and husbandry conditions of laboratory animals. Chewing at cage bars by a rodent may reflect the animal's desire to escape from its home cage and thus provide a measure of the relative aversiveness or inadequacy of different housing conditions from the animal's viewpoint. To assess whether bar chewing by laboratory mice is an escape behaviour, adult male and female ICR-(CD-1) mice were housed individually or in same-sex groups of three in modified shoebox-type cages. Cages had two sets of external bars in the side walls, an equivalent set of bars fixed internally and a Perspex lid. One set of external bars opened daily, allowing the mice to escape into a larger arena. All mice showed a strong preference for chewing at external bars over those that were internal to each cage. After one week of experience, mice also preferred the external bars that opened daily to those that did not open. Behaviour directed towards the cage lid declined over time as the mice experienced the new escape route in the cage side. Interest in the external bars correlated positively with time since last escape. Results confirm that bar chewing reflects an attempt to escape the cage and explore the surrounding area and may provide a suitable behavioural measure of perception of the cage environment for use in welfare assessment.

A device for analysing mouse behaviour which is based on recording the movements of the animal in a balance test cage has been developed. The amplitude patterns provoked by the movements of the animal correlate with behavioural patterns. The system not only allows differentiation between five behavioural categories: resting, grooming, eating, locomotion and climbing - but also indicates the location of the animal in its cage. Upon validation, the system has proven to be a reliable and time-saving device for the non-invasive recording of behavioural patterns in the mouse.

The widespread use of transgenic animals in research, and especially for generating models of human disease, has given rise to calls for a careful documentation of transgenic strains with particular emphasis on characterizing phenotypes and their animal welfare aspects. Based upon several published suggestions for general health monitoring of laboratory animals and upon a questionnaire we developed for the characterization of transgenic strains, score sheets for litter-wise and individual data collection were tested in a pilot study. The documentation concept and some of the most important parameters are presented and discussed with respect to their feasibility and usefulness. The use of score sheets can help in determining the application of humane end points at the individual level, phenotype characterization and assessment of animal welfare for the whole strain, and the development of better breeding strategies - apart from being of direct scientific interest. We suggest that these score sheets, available via the Internet at: www.bzl.unizh.ch/de/database/formtransg/index2.html, should be applied for routine and comprehensive phenotype characterizations of transgenic mouse strains used and kept for animal experimentation.

Enhancing the complexity of the environments of captive animals is often referred to as environmental enrichment, and aims to have positive effects on the animals’ well-being. Such enrichments may have consequences both for so-called ‘normal’ behaviour and for the pathophysiology of the animals in question. The effects of a lack of environmental complexity, including social isolation, on home cage behaviour and on pathophysiology in rats is considered in this review. Several preference tests on rats — choice tests and operant tests — indicate a preference for bedding, nesting material and social contact. Contradictory research results concerning the need for gnawing objects per se are more difficult to interpret, and it is argued that excessive gnawing may be indicative of primary frustration and hence reduced welfare. One disadvantage of providing environmental enrichment to laboratory animals is a possible increase in subject variability, resulting in the need to use a greater number of test animals. However, this increased variability seems to be inconsistent and is not very well documented. It is argued that in cases where the behavioural benefits of environmental enrichment justify the use of more animals, better welfare should be more highly valued than a reduction in the number of animals used.

Biotelemetry is a useful tool for the simultaneous measurement of several physiological and behavioural parameters in non-restrained, freely moving animals. However, the weight and volume of the implanted intra-abdominal transmitter may cause discomfort. The aim of this study was to assess body weight and behaviour of BALB/c and 129/Sv mice after implantation of an intra-abdominal transmitter. In order to measure more detailed behaviour, the automated behaviour observation analysis system (LABORAS™) was used. During the first days after surgery, body weight and the behaviours of climbing, locomotion and eating were found to decrease in both strains, whereas grooming and immobility increased. These changes were more pronounced in the transmitter animals than in the sham operated animals, however, indicating a temporary impairment in well-being. Within two weeks after surgery, the animals seemed to have fully recovered.

‘Environmental enrichment’ is often considered to improve captive animal welfare. However, some studies using male mice, Mus musculus, indicate that increasing cage complexity increases aggression. Limited evidence suggests that enrichment differs in its effects on behaviour and physiology between strains; but behaviour also differs between strains in non-enriched environments. Differences in enrichment type, evaluation methods, and strains used, have caused difficulty in interpreting the efficacy of environmental enrichment in improving welfare. Using enrichment suitable for commercial laboratories (nesting material and a Perspex tunnel), we compared within-cage behavioural and physiological responses among males of six strains housed in non-enriched standard polypropylene cages with those housed in ‘enriched’ cages. Outbred ICR(CD-l) and TO mice, and inbred BALB/c mice were more aggressive than C57BL/6, CBA/Ca and DBA/2 mice, which exhibited low levels of aggression typical of most inbred strains. Enrichment did not significantly affect aggression levels. Animals in enriched cages spent more time investigating the internal cage environment, eating and drinking, and in stereotypic behaviour patterns, although levels differed between strains. The greatest increase in stereotypy levels (bar-related stereotypies) with enrichment was found in DBA/2 mice. Higher testosterone levels were maintained over the study period in mice housed in enriched cages, and in more aggressive strains. IgG levels were also higher in mice housed in enriched cages, and in the outbred strains ICR(CD-l) and TO compared with inbred strains. The relationship between aggression, testosterone and ‘enrichment ‘ suggests that increasing complexity in laboratory cages may increase a naturally selected territorial response in some strains. The implications for strain-specific welfare are discussed.

Tests of emotionality conducted outside the home-cage show that rodents from standard laboratory housing are more anxious than animals from enriched housing; however, it is not known if this also indicates increased anxiety within the home-cage. We used a novel method, recording the self-administration of a psychoactive anxiolytic, to examine home-cage anxiety levels of laboratory mice (three per cage) in Standard (n = 10 cages), Unpredictable (n = 10 cages) and Enriched (n = 6 cages) housing. The mice were given a choice of drinking either non-drugged water or a solution of the benzodiazepine Midazolam. There was a significant effect of housing on the proportion of total fluid consumed from the bottle containing Midazolam solution (P = 0.02). Mice from Standard and Unpredictable cages drank a greater proportion than mice from Enriched cages. This indicates that mice from the Standard and Unpredictable laboratory caging may have been experiencing greater anxiety than mice from the Enriched cages. There was also a significant effect of bottle position (P = 0.002). Mice from the Standard and Unpredictable cages drank a greater proportion of total fluid from the bottle containing Midazolam solution when this was toward the rear of the cage rack, ie in a location that was less susceptible to extraneous disturbance. Monitoring self-administration of psychoactive drugs as a method of welfare assessment could be applied to a wide variety of housing conditions, husbandry practices, or experimental procedures that putatively induce negative mental states. The major finding is that standard cages for laboratory rodents may induce greater anxiety than enriched cages. This is discussed in terms of animal welfare and the validity of data from animals housed in minimalistic environments.

Isolating an animal refers to the situation where the animal is physically fully demarcated from conspecifics without physical, visual, olfactory and auditory contact. Animals housed in separate cages in the same room are, although deprived of physical and visual contact, still in olfactory and auditory contact, and thus not totally isolated. During the fifties and sixties several studies claimed to show physiological and behavioural differences between individually and group housed rats and mice. The so-called ‘Isolation Syndrome’ characterised by changes in corticosterone levels, metabolism, growth, and behaviour was introduced, rather as a model for psychoneurosis than through any concern for animal welfare. Today, it is often stated as common knowledge in laboratory animal science textbooks that individual housing as well as isolation of rats and mice has an effect on physiology and behaviour. It is, however, unclear whether this effect actually impairs animal welfare.

The aim of this paper is to analyse studies on individual housing of mice and rats to evaluate whether there is documented proof that individual housing affects welfare, and, alternatively whether it is possible to house these animals individually without negative impact on welfare, eg by providing special housing improvements.

A range of studies have shown that individual housing or isolation has effects on corticosterone, the open field behaviour, barbiturate sleeping time and the metabolism of different pharmaceuticals in the animals. However, this review of 37 studies in rats and 17 studies in mice showed divergence in test results difficult to explain, as many studies lacked basal information about the study, eg information on genetic strains and housing conditions, such as bedding, enrichment and cage sizes. Furthermore, test and control groups most frequently differed in cage sizes and stocking densities, and behavioural tests differed in ways which may very well explain the differences in results. Overall, there seemed to be an effect of individual housing, although it may be small, and it seems reasonable to assume that, through making small changes in the procedures and housing environments, the effects can be minimised or even eliminated. More well-controlled and standardised studies are needed to give more specific answers to the questions this issue poses.

Laboratory animals are frequently subjected to routine procedures, such as injections or the withdrawal of blood samples. Acute stress caused by such procedures is associated with physiological changes that can have a strong impact on experimental results. This study investigated the integrated effects of cage enrichment, social housing and handling on the acute stress response of animals subjected to routine experimental procedures. Female mice of two inbred strains (BALB/c and C57BL/6) were housed under either minimal husbandry conditions (MH: no cage enrichment, infrequent handling and a period of individual housing) or enriched husbandry conditions (EH: with cage enrichment, frequent handling and social housing at all times). One mouse in each cage was implanted with a radio-telemetry transmitter for measuring heart rate (HR) and body temperature (BT). The animals were subjected to intraperitoneal injections or short periods of restraint. In addition to telemetry measurements, thymus weight and tyrosine hydroxylase (TH) activity were assessed. It was found that individual housing under MH conditions, as compared with social housing under EH conditions, elevated both basal HR and BT, and significantly elevated the relative recovery time following routine experimental procedures. Thymus weight and TH activity suggested a long-term stress response under MH conditions following individual housing, although the influence of transmitter implantation and (repeated) acute stress remains to be investigated. The results emphasise that husbandry conditions should be taken into account when evaluating physiological measures after routine procedures.

Buprenorphine is a widely used analgesic for laboratory rodents. Administration of the drug in a desirable food item for voluntary ingestion is an attractive way to administer the drug non-invasively. However, it is vital that the animals ingest the buprenorphine-food-item mix as desired. The present study investigated how readily female and male mice (Mus musculus) of two different strains consumed buprenorphine mixed in a commercially available nut paste (Nutella®), and whether variation between genders and strains would affect the subsequent serum concentrations of buprenorphine. Buprenorphine at different concentrations mixed in Nutella® was given to male and female C57BL/6 and BALB/c mice in a complete cross-over study. Pure Nutella® or buprenorphine (1.0-3.0 mg kg−1 bodyweight [bw]) mixed in 10 g kg−1 bw Nutella® were given to the mice at 1500h. The mice were video recorded until the next morning, when blood was collected by submandibular venipuncture. The concentration of buprenorphine in the Nutella® mix did not affect the duration of ingestion in any of the groups. However, female mice consumed the Nutella® significantly faster than males. Repeated exposure significantly reduced the start time of voluntary ingestion, but not the duration of eating the mixture. These differences did not however affect the serum concentration of buprenorphine measured 17 h post administration.

When introducing environmental enrichment in laboratory animals, positive and/or negative effects on behavioural and physiological parameters should be evaluated. This three-step randomised controlled trial in male C57BL/6 mice investigated the effect of supplementing the environment with one or more hemp ropes. In part 1, the effect of a hemp rope on aggressive and social behaviour, stress and anxiety levels was assessed by social interaction test, elevated plus maze behaviour, and faecal corticosterone metabolites, respectively (n = 224 mice). In part 2, the effect of 1, 2 or 7 hemp ropes on aggressive behaviour in mice subjected to routine handling was evaluated by assessing the number of wounded companion animals and wounds per animal (n = 224). In part 3, climbing activity in the rope and amount of material shredded from the rope was assessed (n = 56). Mice housed with one hemp rope engaged in social behaviour for longer time than mice housed without a hemp rope, while no difference was detected in stress and anxiety levels. No difference was seen in the number of wounded animals or wounds per animal when adding 1, 2 or 7 hemp ropes to the existing environment in mice undergoing minimal human handling. The mice continuously shredded and climbed the rope, even when provided with a new rope, although time spent climbing decreased slightly over time. Thus, a hemp rope can be used as additional environmental enrichment amongst male C57BL/6 mice.

Managing and assessing well-being in laboratory mice (Mus musculus) is both challenging and necessary. Assessments intended to detect negative welfare states in mice are usually performed via observation of animals in the home cage, but a substantial amount of time and skill may be required to detect subtle behavioural changes. The Time to Integrate to Nest Test (TINT) is a simple, cageside assessment tool that identifies the presence or absence of a highly motivated normal behaviour in mice. The test is conducted by adding a small amount of new nesting material to a mouse cage. A positive outcome is achieved when this new material is integrated into the home nest within 10 min. This study examined whether housing condition or nesting experience affects TINT outcome. Single or group housing did not influence the TINT outcome, but a significant difference in latency to integration was found; singly housed mice took longer than group-housed mice to integrate TINT substrate. Mice which were raised naïve to nesting material had no significant delays when tested. However, experience with the TINT procedure showed increased speed to incorporate the testing substrate, indicating that previous experience to the paradigm prior to experimental testing may be necessary. These findings help to define the expected outcomes of the TINT, better positioning it for use as an assessment tool in varied research settings.