The effect of daily disturbance on the breeding performance of mice

The United Kingdom Home Office Code of Practice for the housing and care of breeding animals requires that, 'the general well-being of all animals must be checked at least once daily'. However, excessive daily disturbance of rodent breeding colonies could be counter-productive to animal welfare if it increases pre-weaning mortality. An experiment involving 100 breeding cages of BALB/c mice compared daily inspection of the mouse cages, but without disturbing the mice within the nest, with daily inspection in which every individual was studied even if this involved removing the cage lid and disturbing the nest. No statistically significant differences were found between the two groups in breeding performance or pre-weaning mortality, though the disturbed group produced marginally fewer offspring and had slightly higher mortality. Average weaning weight did not differ between the groups, but sexual dimorphism at weaning was significantly increased in the disturbed group. It is concluded that there are unlikely to be any welfare benefits in an inspection regimen that involves disturbance of breeding mice, provided the cage is inspected daily.     

Aspen wood-wool is preferred as a resting place, but does not affect intracage fighting of male BALB/c and C57BL/6J mice

Aspen wood-wool, provided as nesting material, was evaluated as a possible improvement of cage environment for 10-14-week-old inbred male mice maintained in groups of six (BALB/c n = 72 and C57BL/6J n = 36). The daily behaviour of mice was video recorded and their body weight, food consumption, weights of some organs and serum corticosterone concentrations were measured. Aggressive interactions between cage mates and against a strange intruder as well as the number of wounds on the back of the animals was monitored in order to evaluate the effect of nesting material on intermale aggression. Nesting material did not affect the daily active/passive behaviour patterns of mice, although animals clearly preferred it as a resting place. BALB/c mice given nesting material showed less weight gain and smaller brown adipose tissue weights than animals without nesting material. The other characteristics measured were not affected by the presence of nesting material in either strain. The presence of nesting material had no effect on fighting in cages. C57BL/6J mice were more aggressive than BALB/c mice according to the number of wounded animals in a cage. Wounded BALB/c mice had enlarged spleens and decreased epididymal adipose tissue weights. In conclusion, the nesting material used in this study did not adversely affect the animals. On the other hand, the material was clearly preferred to conventional bedding as a resting place. These findings suggest that nesting material may improve the cage environment of laboratory mice. Furthermore, there was an indication of strain differences in aggressive behaviour. It could be suggested that C57BL/6J mice are less tolerant towards intruders and housing six mice per cage is not suitable for this strain.     

Nesting material and number of females per cage: effects on mouse productivity in BALB/c, C57BL/6J, DBA/2 and NIH/S mice

Two different materials-aspen wood-wool and paper towel-were compared as nesting material for three inbred mouse strains (BALB/c, C57BL/6J and DBA/2) housed in barrier conditions. In addition, the effect of varying the number of females per cage (one to three per cage) of these three strains and with NIH/S outbred mouse stock was studied. The number of litters, litter size and neonatal mortality were determined, as well as age, sex and weight of weanlings. The type of nesting material did not affect the characteristics monitored. In all strains, the number of weanlings per female was greatest in singly-housed females. In terms of the number of weanlings per cage, two females per cage gave the best result. In DBA/2 mice, neonatal mortality increased when several females were caged together.     

Effects of housing density and cage floor space on three strains of young adult inbred mice

Some recommendations in the Guide for the Care and Use of Laboratory Animals (the Guide) are based on best professional judgment. Our current efforts are directed toward replacement with data-driven standards. We demonstrated earlier that young adult C57BL/6J mice could be housed with half the floor space recommended in the Guide without discernable negative effects. This report extends that work by examining optimal housing densities for young adult male and female BALB/cJ, NOD/LtJ, and FVB/NJ mice. These 8-week studies were initiated with 3-week-old BALB/cJ and NOD/LtJ mice and 3- to 5-week-old FVB/NJ mice housed in three cage types. We adjusted the number of mice per cage to house them with the floor space recommended in the Guide (approximately 12 in2 [ca. 77 cm2] per mouse) down to 5.6 in2 [ca. 36 cm2] per mouse. Early-onset aggression occurred among FVB/NJ male mice housed at all densities in cages having 51.7 in2 (ca. 333 cm2) or 112.9 in2 (ca. 728 cm2) of space. FVB/NJ male mice housed in shoebox (67.6 in2 [ca. 436 cm2]) cages did not exhibit aggression until the fifth week. Urinary testosterone output was density-dependent only for BALB/cJ male mice in shoebox cages (output decreased with increasing density) and FVB/NJ male mice. We conclude that all but FVB/NJ male mice can be housed with half the floor space specified in the Guide. The aggression noted for male FVB/NJ mice may have been due to their age span, although this did not impact negatively on the female FVB/NJ mice.     

Leishmania major: culture media, mouse strains, and promastigote virulence and infectivity

Promastigotes of Leishmania major were isolated from an infected mouse in two media, blood agar and Schneider's medium + 30% fetal calf serum, and maintained continuously for over 1 year. Infectivity studies in two strains of mice, outbred CD1 strain and inbred BALB/c strain, showed that promastigotes grown in Schneider's medium maintained infectivity to BALB/c mice throughout the period of cultivation. Infectivity to CD1 strain mice was progressively lost. Promastigotes grown in blood agar medium, however, lost infectivity to both strains of mice at a faster rate than promastigotes grown in Schneider's medium.     

Susceptibility of five strains of mice to Babesia microti of human origin.

One outbred (CF1) and four inbred (BALB/c, C57, CBA and C3H) strains of mice were tested for susceptibility to Babesia microti of human origin. Of these, intact C3H mice developed higher parasitemia than all other intact mice, while BALB/c mice developed the highest parasitemia among splenectomized mice. Susceptibility was not related to H-2 haplotype in any obvious way. Because C3H and BALB/c mice developed relatively high initial peak parasitemias, the parasite was serially passaged in both of these mouse strains in an attempt to increase parasite virulence. After 30 passages in BALB/c and 49 passages in C3H mice over a period of 12 months, maximum parasitemias were 50 times higher than those observed initially. After the peak parasitemias of these two mouse-adapted parasites had stabilized, the relationship between onset and level of maximum parasitemia and number of parasites inoculated was determined. With both C3H- and BALB/c-adapted parasites, as inoculum size increased, the time required to reach maximum parasitemia decreased and the level of maximum parasitemia increased. Studies involving infection of either mouse strain with parasites adapted to the heterologous mouse strain indicated that C3H mice were more susceptible than BALB/c mice to homologous or heterologous parasites. These data suggest that the virulence of B. microti to the mouse can be increased by prolonged passage in this host. Once adaptation to this host species has occurred, virulence appears to be more dependent on the innate susceptibility of the mouse strain than on adaptation of the parasites to a particular strain of mouse.     

Mouse strain differences in resident peritoneal cells: a flow cytometric analysis

A flow-cytometric study of resident peritoneal cells among 8 mouse strains showed a more than twofold variation in the ratio of macrophages to macrophages plus lymphocytes, ranging from 27% in A/J to 62% in C57B/L10, with significant strain differences in a number of other cellular parameters. There was a particular deficiency of lymphocytes in strain CBA/N, which carries the xid mutation. Studies of the phagocytosis of fluorescent beads also revealed large differences in the number of beads taken up, ranging from 0.99 per cell in MFI to 1.64 per cell in BALB/c mice in a 20-min period. The total number of peritoneal cells collected also varied between strains, ranging from 2.75 x 10(6) in CBA/Ca to 5.85 x 10(6) in MF1. The total yield of macrophages per mouse ranged from 0.93 x 10(6) in A/J to 3.16 x 10(6) in C57BL/10. These differences should be taken into account when designing experiments which use resident peritoneal cells.     

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).     

Mouse strain differences in Gurmarin-sensitivity of sweet taste responses are not associated with polymorphisms of the sweet receptor gene, Tas1r3

Gurmarin (Gur) is a peptide that selectively inhibits responses of the chorda tympani (CT) nerve to sweet compounds in rodents. In mice, the sweet-suppressing effect of Gur differs among strains. The inhibitory effect of Gur is clearly observed in C57BL/6 mice, but only slightly, if at all, in BALB/c mice. These two mouse strains possess different alleles of the sweet receptor gene, Sac (Tas1r3) (taster genotype for C57BL/6 and non-taster genotype for BALB/c mice), suggesting that polymorphisms in the gene may account for differential sensitivity to Gur. To investigate this possibility, we examined the effect of Gur in another Tas1r3 non-taster strain, 129 X 1/Sv mice. The results indicated that unlike non-taster BALB/c mice but similar to taster C57BL/6 mice, 129 X 1/Sv mice exhibited significant inhibition of CT responses to various sweet compounds by Gur. This suggests that the mouse strain difference in the Gur inhibition of sweet responses of the CT nerve may not be associated with polymorphisms of Tas1r3.     

Effect of enrichment on variation and results in the light/dark test

Several confounding factors may influence the outcome of an experiment and the extent of inter-individual variation. The aim of this study was to investigate if cage enrichment induces an effect on experimental mean values and on inter-individual variation in the light/dark paradigm using diazepam as the anxiolytic drug. The behaviour of 216 naive adult male mice of two different strains (BALB/c and C57BL/6) was studied. The animals were housed in groups of four in 'non-enriched', 'enriched' (nesting material) or 'super-enriched' (nest-box, nesting material, wooden gnawing stick and PVC tube) cages. After 5 weeks the animals were assigned to one of three treatments: control (no injection), sham (saline injection i.p.) or diazepam (1 mg/kg bw i.p.) and tested in the light/dark test for 5 min. Variation data were analysed using three different methods (mean absolute deviation, coefficient of variation and power analysis). The C57BL/6 mice scored higher than BALB/c mice in activity related measurements and showed a less 'emotional' behaviour profile in the pharmacological control situation of the light/dark test. In this study the anxiolytic effect of diazepam was clear in BALB/c mice but absent in C57BL/6 mice. Mice housed in enriched and super-enriched cages gained more weight than mice in non-enriched cages, although food intake was not affected. Generally, the strain of mouse had the greatest impact on both mean values and variation. However, there was no consistent increase for one particular strain. The choice of statistical method for analysing variation may influence the interpretation of within-group variability, but none of the methods showed any significant differences between standard and enriched conditions on variability in any of the parameters measured.     

Legionella pneumophila growth in macrophages from susceptible mice is genetically controlled

Growth of the intracellular opportunistic bacterium Legionella pneumophila in macrophages from A/J mice is a vigorous as growth in macrophages from susceptible guinea pigs and human monocytes, whereas growth is inhibited in macrophages from other mouse strains, such as nonpermissive BALB/c mice. Permissiveness versus nonpermissiveness of macrophages from A/J versus BALB/c mice appeared to be controlled by a genetic mechanism dependent upon a single gene or a closely clustered family of genes. Susceptibility versus resistance of macrophages from F1 offspring of these two strains of mice and macrophages from backcrossed mice prepared from F1 hybrids and the original parental strain showed a segregation of permissiveness for growth of Legionella in vitro, consistent with genetic control.     

Helicobacter sp. MIT 01-6451 infection during fetal and neonatal life in laboratory mice

Helicobacter sp. MIT 01-6451 has been detected in SPF mice kept in Japan. To characterize strain MIT 01-6451, its infection route during fetal and neonatal life and effects on pregnancy were investigated using immunocompetent and immunodeficient mouse strains (BALB/c, C57BL/6, and SCID). MIT 01-6451 was detected in the uterus, vagina, and mammary glands of 50% of infected SCID mice, whereas these tissues were all negative in immunocompetent mice. No fetal infections with MIT 01-6451 were detected at 16–18 days after pregnancy in any mouse strain. In newborn mice, MIT 01-6451 was detected in intestinal tissue of C57BL/6 and SCID mice at 9–11 days after birth, but not in BALB/c mice. The IgA and IgG titers to MIT 01-6451 in sera of C57BL/6 female mice were significantly lower than those of BALB/c mice. Although no significant differences in the number of newborns per litter were observed between MIT 01-6451-infected and MIT 01-6451-free dams, the birth rate was lower in infected SCID mice than in control SCID mice. The present results indicated that MIT 01-6451 infects newborn mice after birth rather than by vertical transmission to the fetus via the placenta and that MIT 01-6451 infection shows opportunistically negative effects on the birth rate. In addition, the maternal immune response may affect infection of newborn mice with MIT 01-6451 through breast milk.     

Effect of Salmonella enteritidis 11RX infection on two-stage skin carcinogenesis in mice.

S. enteritidis 11RX infection inhibits the growth of a number of transplantable tumours in mice. In addition, oral infection of mice with S. enteritidis 11RX inhibits colon carcinogenesis by 1,2 dimethylhydrazine. This study has examined the effect of S. enteritidis 11RX infection on two-stage skin carcinogenesis in mice using 7,12 dimethylbenz(a)anthracene (DMBA) as initiator and croton oil as promoter. No protection was observed in either LACA or (BALB/c x C57Bl/6J)F1 mice when live 11RX was repeatedly administered i.v. during promotion. When a protein antigen extract from S. enteritidis 11RX was administered i.v. to previously immunised mice during skin carcinogenesis, significant protection was observed both in terms of the number of mice with papillomas and the number of papillomas per mouse. However, the protection was weak and transient. LACA mice were much more susceptible to skin carcinogenesis by DMBA and croton oil than were (BALB/c x C57B1/6J)F1 mice. A preliminary study indicated that BALB/c, C57B1 and CBA mice were also relatively resistant to skin carcinogensis.     

Horizontal transmission of the mouse mammary tumor virus in cage mates of the same and opposite sex of low and high mammary cancer strain mice

Horizontal transmission of mouse mammary tumor virus (MTV) was investigated in cage mates of the same and opposite sex of low (BALB/c) and high mammary cancer strains (DD/Tbr, SHN and GR). By MTVp27 and MTVgp52 radioimmunoassay, MTV antigen expression was found in the salivary glands, mammary glands and secondary male genital organs of the MTV-free BALB/c strain. Infectivity and oncogenicity were also found in DDf or BALB/c mice by inoculating extracts of salivary gland and/or seminal vesicle in high mammary cancer strains. It is suggested that the primary source of the infectious agent in cases of caged animals of the same sex is saliva, while the primary source in cases of caged animals of the opposite sex is the seminal fluid, although additional infection through saliva cannot be ruled out in the latter case.     

Temporal transmission studies of mouse parvovirus 1 in BALB/c and C.B-17/Icr-Prkdc(scid) mice

Fecal shedding and transmission of mouse parvovirus 1 (MPV) to naive sentinels, breeding mates, and progeny were assessed. Neonatal SCID and BALB/c mice inoculated with MPV were evaluated over 24 wk; several mice from each strain were mated once during this period. Fecal MPV loads for each cage were determined weekly by quantitative polymerase chain reaction (PCR) analysis, and all mice were evaluated by quantitative PCR analysis of lymphoid tissues and seroconversion to MPV antigens in immunocompetent mice. Results indicated persistently high fecal shedding of MPV in SCID mice throughout the evaluation period sufficient to allow transmission to sentinels, naive breeding partners, and the progeny of infected male mice and naive partners. Lymphoid tissue viral loads in the progeny of infected female SCID mice were high at weaning but low at 6 wk of age. Infected BALB/c mice shed high levels of MPV in feces for 3 wk postinoculation, with seroconversion only in sentinels exposed during the first 2 wk postinoculation. Thereafter the feces of infected BALB/c mice and the lymphoid tissues of sentinels, naive breeding partners, and progeny intermittently contained extremely low levels of MPV DNA. Although pregnancy and lactation did not increase viral shedding in BALB/c mice, MPV exposure levels were sufficient to induce productive infection in some BALB/c progeny. These data indicate that the adaptive immune response suppresses, but does not eliminate, MPV shedding; this suppression is sufficient to inhibit infection of weanling and adult mice but allows productive infection of some progeny.     

Climbing on the cage lid, a regular component of locomotor activity in the mouse.

The aim of the study was to obtain base values of climbing behaviour in mice maintained under standardized conditions in Makrolon-cages. Therefore three adult male mice each of the inbred strains BALB/cJ and C57BL/6J were kept separately and two C57BL/6J females as a group in Makrolon-cages type III. In addition, the same BALB/c mice were later kept in a cage with an eightfold floor area. Behavioural observations were carried out by video technique using a light-sensitive camera and a time-lapse recorder. Locomotor activity on the cage floor and climbing on the top of the cage were measured over a period of 48 h for each animal. The duration of locomotion on the ground ranged from 24-65 min/day, climbing between 49-122 (males) and 159 min/day (females) respectively. Climbing showed a more pronounced daily periodicity than locomotion, especially in the case of the BALB/cJ strain, where the average duration of climbing was about 28 min/h during the first hour after light off. In the mouse, climbing is obviously a regular component of activity, which deserves not only attention in the discussion concerning the needs of laboratory animals, but also in measurements of locomotor activity.     

幽门螺旋杆菌ATCC 43504感染BALB/c小鼠动物胃炎模型的建立及分析

利用幽门螺旋杆菌标准菌株建立稳定可靠的幽门螺旋杆菌(Helicobacter pylori,Hp)感染小鼠胃炎模型对Hp疫苗研制、Hp致病机制的研究及抗Hp药物的筛选具有重要意义。通过灌胃国际标准菌株幽门螺旋杆菌Hp ATCC 43504,构建了BALB/c小鼠动物胃炎模型。利用小鼠胃部Hp尿素酶活性检测、Hp的定量培养、PCR检测、组织病理学等多种方法鉴定BALB/c小鼠动物胃炎模型。通过Hp诊断试剂盒检测,造模组小鼠的胃组织能使Hp尿素酶试剂变色,呈现玫瑰红色,而健康小鼠的胃组织不能使Hp尿素酶试剂变色,依旧呈现黄色;通过小鼠胃组织匀浆液培养Hp,造模组小鼠的胃组织匀浆液均可在BHI血平板长出Hp菌落,而对照组小鼠的胃组织匀浆液不能培养出Hp菌落;利用PCR对造模组和对照组BALB/c小鼠的胃组织进行Hp检测,造模组小鼠胃组织可扩增出150 bp的DNA产物,而对照组小鼠胃组织无扩增产物;通过HE染色法观察小鼠胃部病理变化和炎症情况,与健康BALB/c小鼠比较,造模组小鼠胃粘膜和粘膜下层有大量白细胞浸润,胃部炎症明显。利用国际标准菌株Hp ATCC 43504菌株成功构建了BALB/c小鼠胃炎模型,为评价防治Hp感染药物的效果奠定了实验基础。     

Maternal-fetal transmission of Toxoplasma gondii in interferon-gamma deficient pregnant mice

Toxoplasma gondii infection is generally asymptomatic in immunocompetent persons but can be life-threatening in immunocompromised persons and for fetuses in the case of maternal-fetal transmission. The effect of interferon (IFN)-gamma, which plays a crucial role in the protective immunity against T. gondii infection, on maternal-fetal transmission of T. gondii was analyzed by quantitative competitive polymerase chain reaction targeting T. gondii-specific SAG1 gene. T. gondii loads were obvious in uterus and placenta of wild type (WT) C57BL/6 (B6, susceptible strain) but not BALB/c (resistant strain) pregnant mice. Higher levels of T. gondii were detected in uterus and placenta of IFN-gamma knock-out (GKO) B6 and BALB/c than in those of WT mice. Furthermore, T. gondii was detected in fetus of GKO B6 but not GKO BALB/c, WT B6, or WT BALB/c mice. Thus, not only IFN-gamma but also genetic susceptibility to T. gondii infection was important for the protective immunity of maternal-fetal transmission of T. gondii to fetus via placenta. T. gondii-infected WT mice displayed a low delivery rate with high IFN-gamma production, whereas infected GKO mice did not. Additionally, mean body weight of neonates from T. gondii-infected GKO BALB/c pregnant mice was significantly lower than that of unaborted neonates from WT BALB/c pregnant mice, suggesting the effects of T. gondii infection on intrauterine growth retardation of fetus in pregnant GKO mice.     

Effects of housing density and cage floor space on C57BL/6J mice

The Guide for the Care and Use of Laboratory Animals (the Guide) is widely accepted as the housing standard by most Institutional Animal Care and Use Committees. The recommendations are based on best professional judgment rather than experimental data. Current efforts are directed toward replacing these guidelines with data-driven, species-appropriate standards. Our studies were undertaken to determine the optimum housing density for C57BL/6J mice, the most commonly used inbred mouse strain. Four-week-old mice were housed for 8 weeks at four densities (the recommended approximately 12 in2 [ca. 77.4 cm2]/mouse down to 5.6 in2 [ca. 36.1 cm2]/mouse) in three cage types with various amounts of floor space. Housing density did not affect a variety of physiologic parameters but did affect certain micro-environmental parameters, although these remained within accepted ranges. A second study was undertaken housing C57BL/6J mice with as little as 3.2 in2/mouse (ca. 20.6 cm2). The major effect was elevated ammonia concentrations that exceeded limits acceptable in the workplace at increased housing densities; however, the nasal passages and eyeballs of the mice remained microscopically normal. On the basis of these results, we conclude that C57BL/6J mice as large as 29 g may be housed with 5.6 in2 of floor space per mouse. This area is approximately half the floor space recommended in the Guide. The role of the Guide is to ensure that laboratory animals are well treated and housed in a species-appropriate manner. Our data suggest that current policies could be altered in order to provide the optimal habitation conditions matched to this species' social needs.     

Differences between BALB/c and C57BL/6 mice in mouse hepatitis virus replication in primary hepatocyte culture.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) resulted in acute hepatic failure accompanying extremely elevated viral growth in the liver in interferon-gamma-deficient BALB/c (BALB-GKO), but not C57BL/6 (B6-GKO) mice. To examine the basis of the strain difference against MHV infection in interferon-gamma-deficient mice, viral replication in primary hepatocyte cultures from BALB/c and B6 mice with or without the IFN-gamma gene was compared in vitro. The MHV replication in BALB/c hepatocytes with or without the IFN-gamma gene was significantly higher than that in B6 hepatocytes with or without the IFN-gamma gene, suggesting that there is a strain difference in MHV replication in hepatocytes. Since a significant difference in MHV replication in hepatocytes was not observed between wild type and IFN-gamma-deficient mice of the same genetic background, the phenomenon is thought to be independent of IFN-gamma. However, pretreatment of hepatocytes with recombinant mouse interferon-gamma inhibited MHV replication in a dose-dependent fashion. The results are discussed with respect to the pathology of MHV infection in mice with or without the IFN-gamma gene.