Here we describe stress-like responses in mice caused by routine husbandry and experimental procedures, and focus on how such practices affect mouse physiology, behaviour and data quality. We aimed to identify sources and manifestations of stress, reduce stress through refinements to working practices and utilise the effects of stress to our advantage when the cause is unavoidable.
In this study we have demonstrated that BG and Tc measurements activate the cardiovascular system which declines between tests but is re-activated to the same level by a subsequent measurement. We show that an overnight fast was associated with significant and sex-specific changes in CV and locomotor activities. The procedure-induced changes observed in behavioural and physiological parameters were more prevalent in females than in males. We have also extended and confirmed previously published observations on the impact of BG and Tc measurement [18,21]
, ip sham injections [22,23]
, cage transport [18,24]
and individual housing [25–27]
on BW, BG and Tc parameters.
We have measured cardiovascular response to BG and Tc sampling methods by HR and SAP, which are sensitive indices of stress, activated via the sympathoadrenal system 
. Previous studies in mice subjected to Tc 
and BG measurement 
procedures have reported marked increases in catecholamines and corticosterone, confirming the stressful nature of these manipulations. These findings indicate that sampling methods can elicit physiological responses that may confound studies where repeat BG measurements are performed.
To minimise the influence of blood sampling on glucose tolerance test (GTT) results, a two hour recovery period following tail cut before obtaining the first blood sample has been recommended 
. However, in our experience the wound may seal after 2 h necessitating the removal of a scab or additional cutting to collect a blood sample. Furthermore, the dose of glucose injected during GTT (2 mg/g) exerts a dominant effect over the increase in BG caused by the sampling procedure alone. Taking into consideration these findings we adopted a protocol for the GTT where the first measurement is taken immediately following tail tip excision.
To our knowledge, this is the first study to document the effects of an overnight fast on CV function and locomotor activity in group-housed mice of both sexes. We show significant increases in HR, SAP and locomotor activity in group housed females and males immediately after initiation of the fast. The magnitude and duration of these responses were similar to those recorded after cage change procedure alone, suggesting that increases in CV parameters and locomotor activity during the first 2 h of fasting are likely due to stress caused by cage transfer. In females but not in males, significantly elevated HR and locomotor activity were recorded during the dark phase and a marked reduction in SAP and HR during the final 2 h of fasting was evident. The fasting-induced attenuation in CV locomotor activity observed in females is consistent with previous observations 
and may be due to decreased metabolic rate and activation of the parasympathetic system.
Our study adds to the literature reporting stress-like reactivity in laboratory animals caused by cage changes. Although this is well reported in rats [16,17]
, in mice this has been little studied 
. In our study the magnitude of HR and SAP responses to cage change was no different from the response to more invasive procedures such as BG and Tc sampling. However, the duration of the CV changes exceeded those triggered by the sampling procedures. Long-lasting CV responses were paralleled by increased exploratory activity, believed to be triggered by the novel environment 
. In accordance with previously published findings in rats 
, our results suggest that mice do not habituate to cage change despite the regularity of the procedure. Taking into consideration current and previous observations 
it is recommended that a 2 h acclimatisation period is required after cage change before recording baseline CV parameters. In our phenotyping pipeline, the husbandry protocols, sequence and intervals between tests have been selected to minimise the possibility of confounding influences between tests. This is achieved by acclimatisation to the procedure room, performing cage changes within testing procedures, testing in order of perceived invasiveness and providing enough recovery time between tests.
Another important finding was the differences between sexes in physiological responses to the experimental and husbandry procedures, females showing a greater and more prolonged reduction in BW following brief individual housing, and more sustained increases in CV parameters in response to blood sampling, cage change and during overnight fast. The increase in arousal during overnight fasting was also more prolonged and pronounced in females. Consistent with our findings, Hoppe et al. 2008 
reported a more protracted elevation in mean arterial pressure in females after placing C57BL/6J; 129sv mice in a new metabolism cage. Greater female CV and arousal responses to experimental and husbandry procedures have also been observed in the rat [35,36]
. The mechanisms underlying sex-specific differences in CV stress-responses can be explained by differences in autonomic and neuroendocrine control 
. These findings may have important implications for the interpretation of results of phenotyping tests since it indicates that potentially confounding effects of stress are more prevalent in female mice.
In view of our results on individual housing, the increasing body of literature reporting a preference for social housing in rodents [38,39]
and how this can improve welfare and aid post-operative recovery [40,41]
we attempted to re-house former cage-mate mice 3 days after telemetry implantation surgery. This was previously avoided, particularly in males, due to the aggressive nature of the mouse strain in use. Applying knowledge of rodents' reliance on olfactory cues 
and the effect of environmental enrichment 
we designed a protocol to minimise the likelihood of aggression. Following re-grouping, active exploratory behaviour with elements of non-aggressive play-like skirmishing was observed in females. Brief instances of aggressive behaviour were seen in some males. In general it took a shorter time for females to settle and become dormant than for males (1 h vs. 2 h). Body weight data suggests social housing had a positive effect on postoperative recovery as weight gain began shortly after re-grouping. Our findings and those previously reported 
suggest that social housing is the least stressful of housing conditions and reveals that mice can be re-housed after short term isolation and/or surgery 
Some of the most stressful events in husbandry result from changes in familiar cage environment. Whilst frequent cage changing is good for hygiene, it can be disruptive as rodents rely heavily on olfactory cues for recognising and communicating with cage-mates 
. In our study an increased ammonia level was recorded in cages changed once every 14 days in comparison to those changed once every 7 days. However, none of the parameters evaluated indicated a detrimental effect of the prolonged cage-change interval. These findings are in agreement with previous studies reporting the lack of effect of cage change frequency (weekly vs. fortnightly) on ICR females 
and C57BL/6J mice of both sexes 
housed in IVC. Our results suggest that the changing of bedding every 14 days in IVC housing may represent a balance between maintaining good inter-cage hygiene whilst reducing the disturbance to mice.
Stress induced hyperthermia is a robust and reproducible phenomenon observed in mice and rats 
. An increase in body temperature is elicited by removal from group housing, or by the introduction of any other stressor, such as the Tc measurement procedure itself. We have refined our working practice by using this phenomenon to assess the stress response in mice as part of our large-scale phenotyping screen by introducing a second sampling of Tc, 15 min after the initial measurement. The magnitude of Tc increase is then interpreted as a read-out for stress responsiveness, providing additional data without requiring additional animals.