Experimental model of congestive heart failure induced by transverse aortic constriction in BALB/c mice.

INTRODUCTION: Murine transverse aortic constriction (TAC) is a frequently used model of pressure overload-induced left ventricular (LV) remodeling. However, there is considerable variability in disease progression to overt heart failure (HF) development in the most commonly used strain of mice (i.e., C57BL/6J). Studies have shown that C57BL/6J mice are more resistant than BALB/c mice to congestive HF development following myocardial infarction or angiotensin II-induced hypertension. Therefore, we tested the hypothesis that BALB/c mice may be a better research model to study TAC-induced progressive HF. METHODS: Following sham or TAC surgery in both C57BL/6J (n = 29) and BALB/c (n = 32) mice, we evaluated cardiac dimensions and function by echocardiography at 2, 4, 8, and 12 weeks and monitored survival throughout the study. In a separate cohort of BALB/c mice, we repeated the study in the presence of the angiotensin converting enzyme inhibitor enalapril or a vehicle initiated 2 weeks post-TAC and administered for 6 weeks. At the end of the studies, we assessed the heart weight, lung weight, and plasma brain natriuretic peptide (BNP) concentration. RESULTS: Following comparable TAC, both C57BL/6J and BALB/c mice showed significant LV remodeling compared with the sham control mice. BALB/c mice progressively developed systolic dysfunction, LV dilation, lung congestion, and significant mortality, whereas C57BL/6J mice did not. In the separate cohort of BALB/c TAC mice, enalapril significantly reduced the heart weight, lung weight, and plasma BNP concentration and improved survival compared with the vehicle control. DISCUSSION: BALB/c mice uniformly developed congestive HF post-TAC. Enalapril was effective in improving survival and reducing lung congestion in this model. The data suggest that BALB/c mice may be a better research tool than C57BL/6J mice to study TAC-induced disease progression to HF and to evaluate novel therapies for the treatment of chronic HF with reduced ejection fraction.

Evaluation of Blast Overpressure Exposure Effects on Concentration of Antibiotics in Mice.

OBJECTIVE: Infection as sequelae to explosion-related injury is an enduring threat to our troops. There are limited data on the effects of blast on antibiotic pharmacokinetics (PK), pharmacodynamics (PD), and efficacy. The observational study presented here is our Institute's first attempt to address this issue by combining our existing interdepartmental blast, infection modeling, and in vivo PK/PD capabilities and was designed to determine the PK effects of blast on the first-line antibiotic, cefazolin, in an in vivo mouse model. METHODS: A total of 160 male BALB/c mice were divided to sham and blast (exposed to blast overpressure of 19 psi) in two biological replicates. At 1 hour after blast/sham exposure, the animals received IV injection of cefazolin (328 mg/kg). Animals were euthanized at 3 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, or 10 hours after the injection. Plasma and liver were analyzed for concentration of cefazolin using mass-spectrometry. RESULTS: We observed increases in the concentration of cefazolin in the plasma and liver of blast exposed animals at later time points and increase in elimination half-life. CONCLUSION: Our results indicate that blast-induced physiologic changes significantly influence cefazolin PK and suggest that efficacy could be affected in the context of the blast; assessment of efficacy and PD effects require further investigation. Metabolic changes resulting from blast may influence other classes of antibiotics and other therapeutics used with these injuries. Therefore, this may have important treatment considerations in other areas of military medicine.

Color matters: They would choose if they could (see)!

Concerning standardization of laboratory animal husbandry, only exiguous changes of habitat can potentially influence animal physiology or results of behavioral tests. Routinely, mice chow is dyed when different types of diets are dispensed. Given the fact that the dye itself has no effects on food odor or flavor, we wanted to test the hypothesis that the color of chow has an impact on food uptake in mice. Twelve-week-old male mice of different strains (C57BL/6J, DBA/2J, C3H/HeJ, BALB/cJ; n = 12/strain) were single-housed in PhenoMaster® cages. After acclimatization standard mice chow in different colors was administered. Food intake was monitored as a two-alternative choice test of different color combinations. All animals had an average food intake of 3 g/d and no preferences were observed when a combination of identically colored food was offered. Preference tests yielded significant aversion to blue food and significant attraction to yellow and green food in C57BL/6 and DBA/2J mice. In C3H/HeJ and BALB/cJ mice no color-related pattern occurred. Selected mice strains have known differences concerning functionality of their visual sense. C57BL/6 and DBA/2 mice are considered to be normal sighted at testing age, BALB/c is representative for albino strains and C3H mice carry mutations resulting in retinal alterations. Results suggesting that normal-sighted mice would be selective concerning food color when given the choice. Nevertheless, this does not influence overall quantity of food intake when animals were provided solely with food colored with a single dye. Moreover, visually impaired mice showed no color-related food preferences.

Behavioural characterization of C57BL/6N and BALB/c female mice in social home cage - Effect of mixed housing in complex environment.

Developing reliable mouse models for social behaviour is challenging. Different tests have been proposed, but most of them consist of rather artificial confrontations of unfamiliar mice in novel arenas or are relying on social stress induced by aggressive conspecifics. Natural social interaction in home cage in laboratory has not been investigated well. IntelliCage is a fully automated home-cage system, where activity of the group-housed mice can be monitored along with various cognitive tasks. Here we report the behavioural profile of C57BL/6N (B6) and BALB/c (BALB) female mice in IntelliCage when separated by strain, followed by monitoring of activity and formation of 'home-base' after mixing two strains. For that purpose, 3 cages were connected. Significant differences between the strains were established in baseline behaviour in conventional tests and in IntelliCage. The B6 mice showed reduced anxiety-like behaviour in open field and light-dark box, slightly enhanced exploratory activity in IntelliCage during initial adaptation and clearly distinct circadian activity. Mixing of two strains resulted in reduction of body weight and anhedonia in B6 mice. In addition, the B6 mice showed clear preference to previous home-cage, and formed a new home-base faster than BALB mice. In contrast, BALB mice showed enhanced activity and moving between the cages without showing any preference to previous home-cage. It could be argued that social challenge caused changes in both strains and different coping styles are responsible for behavioural manifestations. Altogether, this approach could be useful in modelling and validating mouse models for disorders with disturbed social behaviour.

Impact of bedding volume on physiological and behavioural parameters in laboratory mice.

The standard housing temperature in animal facilities is substantially below the lower critical temperature of mice. This does not only endanger animal welfare, it can also jeopardize scientific research as cold stress has a major impact on mouse physiology. There is some evidence that deep bedding, comparable to nesting material, can help mice to reduce heat loss. Whenever changes are applied to the cage environment, the potential impact on experimental results, including variation, needs to be assessed. An increased variation can result in a conflict between reduction and refinement, when more animals are needed for significance due to the housing design. The aim of this study was to assess the impact of different bedding volumes (0.5 L, 1.5 L and 6 L per type III cage) on mean values and coefficient of variation (CV) of physiological (pentobarbital sleeping time, blood and anatomical parameters) and behavioural parameters (open-field and novel object recognition tests) of group-housed female and male BALB/c and C57BL/6 mice. A larger bedding volume did not interfere with the CVs, but influenced mean values of organ weights and tail lengths. Mice housed on deeper bedding showed a significant reduction in adrenal, liver, kidney and heart weights as well as an increase in tail lengths; these anatomical changes are akin to warm adaptation, and were previously observed for mice housed under warmer environments. A larger bedding volume appears to be a sensible way to reduce cold stress for laboratory mice without increasing variation in experimental results.

Duration and timing of daily light exposure influence the rapid shifting of BALB/cJ mouse circadian locomotor rhythms.

Photic entrainment of the murine circadian system can typically be explained with a discrete model in which light exposures near dusk and dawn can either advance or delay free-running rhythms to match the external light cycle period. In most mouse strains, the magnitude of those phase shifts is limited to several hours per day; however, the BALB/cJ mouse can re-entrain to large (6-8hour) phase advances of the light/dark cycle. In this study, we demonstrate that the circadian responses of BALB/cJ mice are dependent on duration as well as timing of light exposure, with significantly larger phase shifts resulting from >6-hour light exposures, yet loss of entrainment to photoperiods of <2-3hours per day or to skeleton photoperiods. Intermittent light exposures of the same total duration but distributed differentially over the same period of time as that of a 6-hour phase advance of the light cycle yielded phase shifts of different magnitudes depending on the pattern of exposure. Both negative and positive masking responses to light and darkness, respectively, were exaggerated in BALB/cJ mice under a T7 light cycle, but were not responsible for their rapid re-entrainment to chronic phase shifting of the light dark cycle. These results collectively suggest that the innately jetlag-resistant BALB/cJ mouse circadian system provides an alternative murine model in which to elucidate the limitations of photic entrainment observed in other commonly used strains of mice.

Wheat grain consumption and selection by inbred and outbred strains of mice.

Food selection and avoidance are driven primarily by orosensory cues. Previous studies with C57BL/6J mice indicated marked differences in selection and consumption of individual grains of different wheat varieties when presented in binary mixtures. The present study examined the patterns of mouse grain selection across four strains of laboratory mice: two inbred, BALB/c and C57BL/6J, and two outbred, Swiss-Webster and CD1. Four pairs of wheat varieties that were known to vary a priori for consumption preference or seed coat ('bran') color were tested. Two variety pairs were near-isogenic (>98% similar) with contrasting red and white seed coat coloration/pigmentation. All four mice strains exhibited similar preferences between wheat variety pairs, whereas consumption was not highly related to mouse body weight. This result indicates a more generalized phenomenon regarding how mice select and then consume individual wheat grains. The study supported the continued use of C57BL/6J as an effective strain model system to study food perception.

Effects of Enrichment and Litter Parity on Reproductive Performance and Behavior in BALB/c and 129/Sv Mice.

We examined the effect of adding species-appropriate environmental enrichment items to breeding cages of BALB/cAnNCrl and 129S2/SvPasCrl mice. The 3 enrichment conditions were: 1) cotton nesting material; 2) nesting material plus a paper shelter and rolled paper bedding; and 3) an igloo dome with an exercise wheel in addition to the shelter-group enrichments. We measured litter size, litter survival to weaning age, average pup weight at 21 d, and the interlitter interval to evaluate reproductive performance. A random subset of the first- or second-litter offspring from each enrichment condition and strain was assessed in multiple behavioral tests. Enrichment significantly affected anxiety-like behavior and sociability, with the direction of change dependent on strain and sex. Litter parity had greater effects on some reproductive parameters than did the enrichment condition, and this effect was not solely due to a difference between the first compared with subsequent litters. The significant effects of litter parity on the number of pups born and weaned, female pup weight, and interlitter interval were dependent on the enrichment condition in BALB/c but not 129/Sv mice. Offspring from the first or second litter were included in a generational component to investigate whether enrichment effects on reproduction persist in adult offspring after transfer to a different facility for breeding. Natal cage enrichment had no effect on any reproductive parameter in the transferred mice. Overall, additional enrichment beyond nesting material had a beneficial effect on the interlitter interval in BALB/c mice and on the number of pups weaned in 129/Sv mice.

Generating Chimeric Mice by Using Embryos from Nonsuperovulated BALB/c Mice Compared with Superovulated BALB/c and Albino C57BL/6 Mice.

The reliable generation of high-percentage chimeras from gene-targeted C57BL/6 embryonic stem cells has proven challenging, despite optimization of cell culture and microinjection techniques. To improve the efficiency of this procedure, we compared the generation of chimeras by using 3 different inbred, albino host, embryo-generating protocols: BALB/cAnNTac (BALB/c) donor mice superovulated at 4 wk of age, 12-wk-old BALB/c donor mice without superovulation, and C57BL/6NTac-Tyr(tm1Arte) (albino B6) mice superovulated at 4 wk of age. Key parameters measured included the average number of injectable embryos per donor, the percentage of live pups born from the total number of embryos transferred to recipients, and the number of chimeric pups with high embryonic-stem-cell contribution by coat color. Although albino B6 donors produced significantly more injectable embryos than did BALB/c donors, 12-wk-old BALB/c donor produced high-percentage (at least 70%) chimeras more than 2.5 times as often as did albino B6 mice and 20 times more efficiently than did 4-wk-old BALB/c donors. These findings clearly suggest that 12-wk-old BALB/c mice be used as blastocyst donors to reduce the number of mice used to generate each chimera, reduce the production of low-percentage chimeras, and maximize the generation of high-percentage chimeras from C57BL/6 embryonic stem cells.

Strain commonalities and differences in response-outcome decision making in mice.

The ability to select between actions that are more vs. less likely to be reinforced is necessary for survival and navigation of a changing environment. A task termed "response-outcome contingency degradation" can be used in the laboratory to determine whether rodents behave according to such goal-directed response strategies. In one iteration of this task, rodents are trained to perform two food-reinforced behaviors, then the predictive relationship between one instrumental response and the associated outcome is modified by providing the reinforcer associated with that response non-contingently. During a subsequent probe test, animals can select between the two trained responses. Preferential engagement of the behavior most likely to be reinforced is considered goal-directed, while non-selective responding is considered a failure in response-outcome conditioning, or "habitual." This test has largely been used with rats, and less so with mice. Here we compiled data collected from several cohorts of mice tested in our lab between 2012 and 2015. Mice were bred on either a C57BL/6 or predominantly BALB/c strain background. We report that both strains of mice can use information acquired as a result of instrumental contingency degradation training to select amongst multiple response options the response most likely to be reinforced. Mice differ, however, during the training sessions when the familiar response-outcome contingency is being violated. BALB/c mice readily generate perseverative or habit-like response strategies when the only available response is unlikely to be reinforced, while C57BL/6 mice more readily inhibit responding. These findings provide evidence of strain differences in response strategies when an anticipated reinforcer is unlikely to be delivered.

Long-Term Provision of Environmental Resources Alters Behavior but not Physiology or Neuroanatomy of Male and Female BALB/c and C57BL/6 Mice.

Few studies have evaluated the long-term effects of providing environmental resources to mice. This consideration is important given that mice are often maintained in vivaria for months. We evaluated the effects of providing simple cage resources (wood wool, cotton nesting material, a plastic tunnel, and oat cereal) compared with standard housing (solid-bottom cage with hardwood chips) to group-housed adult male and female C57BL/6 and BALB/c mice (n = 20/sex/strain/group) over 6 mo to determine whether these resources had a lasting effect on animal physiology, anatomy, and behavior. Body weights increased in all groups over time but were proportionately higher in male and female BALB/c mice housed in resource-supplemented environments. Throughout the study, adding environmental resources had no effect on hematology and lymphocyte subsets, fecal corticoid metabolite levels, response to LPS injection, or dendritic spine length or density. Strain- or sex×environmentspecific changes occurred in dark-light activity and thermal nociceptive responses. Dominant agonistic behaviors, abnormal conspecific sexual behaviors, and social nonagonistic behaviors demonstrated sex and strain×environment interactions such that fewer maladaptive social behaviors were noted in mice that were provided with environmental resources. This association was particularly evident in male mice of both strains in resource-supplemented environments. A small but significant increase in brain weight:body weight ratios occurred in mice in resource-supplemented environments. Under the conditions evaluated here, consistent use of simple environmental resources had a positive long-term effect on the behavioral wellbeing of male and female BALB/c and C57BL/6 mice yet minimally affected other aspects of murine physiology and neuroanatomy.

Behavioral characterization of a model of differential susceptibility to obesity induced by standard and personalized cafeteria diet feeding.

Despite the increase in obesity prevalence over the last decades, humans show large inter-individual variability for susceptibility to diet-induced obesity. Understanding the biological basis of this susceptibility could identify new therapeutic alternatives against obesity. We characterized behavioral changes associated with propensity to obesity induced by cafeteria (CAF) diet consumption in mice. We show that Balb/c mice fed a CAF diet display a large inter-individual variability in susceptibility to diet-induced obesity, such that based on changes in adiposity we can classify mice as obesity prone (OP) or obesity resistant (OR). Both OP and OR were hyperphagic relative to control-fed mice but caloric intake was similar between OP and OR mice. In contrast, OR had a larger increase in locomotor activity following CAF diet compared to OP mice. Obesity resistant and prone mice showed similar intake of sweet snacks, but OR ate more savory snacks than OP mice. Two bottle sucrose preference tests showed that OP decreased their sucrose preference compared to OR mice after CAF diet feeding. Finally, to test the robustness of the OR phenotype in response to further increases in caloric intake, we fed OR mice with a personalized CAF (CAF-P) diet based on individual snack preferences. When fed a CAF-P diet, OR increased their calorie intake compared to OP mice fed the standard CAF diet, but did not reach adiposity levels observed in OP mice. Together, our data show the contribution of hedonic intake, individual snack preference and physical activity to individual susceptibility to obesity in Balb/c mice fed a standard and personalized cafeteria-style diet.

Optical measurement of mouse strain differences in cerebral blood flow using indocyanine green.

C57BL/6 mice have more cerebral arterial branches and collaterals than BALB/c mice. We measured and compared blood flow dynamics of the middle cerebral artery (MCA) in these two strains, using noninvasive optical imaging with indocyanine green (ICG). Relative maximum fluorescence intensity (Imax) and the time needed for ICG to reach Imax in the MCA of C57BL/c were lower than that in BALB/c mice. Moreover, the mean transit time was significantly lower in C57BL/6 than in BALB/c mice. These data suggest that the higher number of arterial branches and collaterals in C57BL/6 mice yields a lower blood flow per cerebral artery.

Response, use and habituation to a mouse house in C57BL/6J and BALB/c mice.

Animal welfare depends on the possibility to express species-specific behaviours and can be strongly compromised in socially and environmentally deprived conditions. Nesting materials and refuges are very important resources to express these behaviours and should be considered as housing supplementation items. We evaluated the effects of one item of housing supplementation in standard settings in laboratory mice. C57BL/6JOlaHsd (B6) and BALB/cOlaHsd (BALB) young male and female mice, upon arrival, were housed in groups of four in standard laboratory cages and after 10 days of acclimatization, a red transparent plastic triangular-shaped Mouse House™ was introduced into half of the home cages. Animals with or without a mouse house were observed in various contexts for more than one month. Body weight gain and food intake, home cage behaviours, emotionality and response to standard cage changing procedures were evaluated. The presence of a mouse house in the home cage did not interfere with main developmental and behavioural parameters or emotionality of BALB and B6 male and female mice compared with controls. Both strains habituated to the mouse house in about a week, but made use of it differently, with BALB mice using the house more than the B6 strain. Our results suggest that mice habituated to the mouse house rather quickly without disrupting their home cage activities. Scientists can thus be encouraged to use mouse houses, also in view of the implementation of the EU Directive (2010/63/EU).

The BALB/c mouse: Effect of standard vivarium lighting on retinal pathology during aging.

BALB/cJ mice housed under normal vivarium lighting conditions can exhibit profound retinal abnormalities, including retinal infoldings, autofluorescent inflammatory cells, and photoreceptor degeneration. To explore the sensitivity of the outer retina to cyclic lighting during aging, a cohort of BALB/cJ mice was evaluated with Scanning Laser Ophthalmoscopy (SLO), Spectral-Domain Optical Coherence Tomography (OCT) and conventional histopathology. Mice were bred and reared in a low-illuminance (extracage/intracage: 13 lx/1 lx) vivarium under cyclic light (14 h light: 10 h dark). Retinal imaging (around postnatal day 70) was performed to screen for any pre-existing abnormalities and to establish a baseline. Mice with normal retinas were separated into groups (A, B, C) and placed on bottom (Groups A & B) or top (Group C) of the cage racks where cage illumination was <10 & 150 lx respectively. Experimental groups B & C were imaged multiple times over a 17 month period. Mice from group A (controls) were imaged only once post-baseline at various times for comparison to groups B & C. Mice were assessed by histology at 8, 15, 20, 36, and 56 weeks and immunohistochemistry at 15 weeks post-baseline. SLO and OCT retinal images were measured and the resulting trends displayed as a function of age and light exposure. Retinal lesions (RL) and autofluorescent foci (AFF) were identified with histology as photoreceptor layer infoldings (IF) and localized microglia/macrophages (MM), respectively. Few RL and AFF were evident at baseline. Retinal infoldings were the earliest changes followed by subjacent punctate autofluorescent MM. The colocalization of IF and MM suggests a causal relationship. The incidence of these pathological features increased in all groups relative to baseline. OCT imaging revealed thinning of the outer nuclear layer (ONL) in all groups at 1 year relative to baseline. ONL thinning followed an exponential rate of change but the decay constant varied depending on intensity of illumination of the groups. Advanced age and top row illuminance conditions resulted in significant photoreceptor cell loss as judged by decreased thickness of the ONL. Photoreceptor loss was preceded by both retinal infoldings and the presence of autofluorescent inflammatory cells in the outer retina, suggesting that these changes are early indicators of light toxicity in the BALB/cJ mouse.

Lifetime reproductive efficiency of BALB/c mouse pairs after an environmental modification at 3 mating ages.

The aim of this study was to evaluate the effects of an environment change and the age at which mating pairs were formed on the lifetime reproductive performance of BALB/c mice. We assigned 60 monogamous pairs to a randomized design in a 2 × 3 factorial arrangement (with or without an environmental modification and with 3 mating ages: 28, 45, or 60 d). Autoclaved cardboard tubes (length, 10 cm; diameter, 4 cm) were used as the environmental modification. Data were collected from a total of 456 litters over a period of 10 mo. The mice tore the cardboard tube and used its parts both as shelters and as nesting material. The presence of a cardboard tube decreased the preweaning litter mortality rate in the first 6 reproductive cycles. Mating at 28 or 45 d of age also decreased the preweaning mortality rate in the first 6 reproductive cycles, compared with monogamous pairs formed at 60 d of age. Treatments did not affect age at first parturition, number of litters, time between litters, or litter size and weight at birth and weaning. In addition to contributing to animal wellbeing, providing a cardboard tube improved productivity by decreasing the preweaning mortality rate. BALB/c siblings should be paired for mating when no older than 28 d, to reduce preweaning mortality of the offspring.

Temporal characteristics of stress-induced decrease in benzodiazepine reception in C57BL/6 and BALB/c mice.

We studied the duration of the drop of specific (3)H-flunitrazepam binding by synaptosomal membranes from the brain of C57Bl/6 and BALB/c mice after open-field and "contact with predator" tests. It was found that reduced benzodiazepine reception in BALB/c mice after open-field test persisted for 1.5 h, but no changes of this parameter were found in C57Bl/6 mice. After contact with predator, the binding capacity of the benzodiazepine site of GABAA receptor was reduced for 8 h in BALB/c mice and for 24 h in C57Bl/6 mice.

Effects of vaginal septa on the reproductive performance of BALB/cByJNarl mice.

From March through December 2010, the incidence of vaginal septa in our SPF breeding colony of BALB/cByJNarl mice was 14.2%. In general, septa obstructed half of the vaginal orifice. Here we sought to determine the effect of this defect by comparing the reproductive performance of affected (septate) mice with that of unaffected (nonseptate) mice. Our results showed that the rates of both copulatory plugs and pregnancy were significantly lower in septate mice than in nonseptate mice. Specifically, 23 of 45 bred septate female mice (51%) had vaginal plugs compared with 49 of 68 bred nonseptate females (72%). Only 12 septate female mice (27%) had successful pregnancies, compared with 37 nonseptate females (54%). Septate mice had a 1-logfold fewer intrauterine sperm after mating than did nonseptate mice. Three cases of dystocia were noted among septate mice whereas none occurred in nonseptate mice. Septate dams had a higher percentage of septate pups (15.5%) than did nonseptate dams (6.1%). Our findings indicate that vaginal septa affect the reproductive performance of laboratory mice and that such a defect should be considered as an exclusion criterion for the selection of future breeders in a mouse colony.

The effect of cage shelf on the behaviour of male C57BL/6 and BALB/c mice in the elevated plus maze test.

The effect of environmental enrichment in the form of a cage shelf on the behaviour of male C57BL/6 and BALB/c mice was compared. Male C57BL/6 and BALB/c mice were randomly allocated into 12 cages per strain. The mice were kept in control conditions or exposed to a cage shelf for two, four, six or eight weeks, and thereafter assessed with the elevated plus maze. C57BL/6 mice displayed a trend of being less anxious than the BALB/c mice. A cage shelf increased the number of entries made into the open arms and the total number of entries only in the case of the C57BL/6 mice, but had no effect on the behaviour of the BALB/c mice. In conclusion, the effect of a cage shelf on the elevated plus maze behaviour of mice depends on the strain of the animals.

Assessment of routine procedure effect on breathing parameters in mice by using whole-body plethysmography.

We used whole-body plethysmography to investigate the effect of restraint, ear marking, tail vein and retroorbital blood sampling, and tail clipping on respiration in Balb/c × TCR-HA +/- F1 hybrid mice (F1h). Baseline values of breathing parameters were determined. During the experiment, mice experienced a procedure and then plethysmographic recordings were obtained immediately and at 4, 24, and 48 h afterward. Baseline breathing parameters showed significant differences between sexes. Restraint affected minute volume differently than did handling in male mice and to a lesser extent in female mice. Ear marking significantly changed minute volume compared with handling but not restraint in male mice and in the opposite manner in female mice. Tail vein blood sampling changed minute volume in a significant manner compared with restraint but not compared with handling in both sexes. Retroorbital blood sampling significantly changed minute volume compared with values for both handling and restraint in male mice but only compared with handling in female mice. Tail clipping modified minute volume significantly compared with handling in male mice and compared with restraint in both sexes. Analysis of data showed that routine procedures affect minute volume in mice depending on invasiveness of maneuver and in a sex-biased manner for as long as 24 h after the procedure. Our experiment shows that procedures performed on laboratory mice can change respiratory parameters and can be investigated by plethysmography.