Postnatal Development of the Craniofacial Skeleton in Male C57BL/6J Mice

C57BL/6J is one of the most commonly used inbred mouse strains in biomedical research, including studies of craniofacial development and teratogenic studies of craniofacial malformation. The current study quantitatively assessed the development of the skull in male C57BL/6J mice by using high-resolution 3D imaging of 55 landmarks from 48 male mice over 10 developmental time points from postnatal day 0 to 90. The growth of the skull plateaued at approximately postnatal day 60, and the shape of the skull did not change markedly thereafter. The amount of asymmetry in the craniofacial skeleton seemed to peak at birth, but considerable variation persisted in all age groups. For C57BL/6J male mice, postnatal day 60 is the earliest time point at which the skull achieves its adult shape and proportions. In addition, C57BL/6J male mice appear to have an inherent susceptibility to craniofacial malformation.Abbreviations: CVA, canonical variates analysis; FA, fluctuation asymmetry score; GPA, generalized Procrustes analysis; PCA, principal component analysisC57BL/6J is one of the most commonly used inbred mouse strains for biomedical research and is the first mouse strain that has its genome fully sequenced. In addition to its broad use in developmental biology and disease modeling, C57BL/6J mice are used in modeling craniofacial development and understanding the effects of teratogens,^3,6,16,17 particularly of ethanol, given the strain''s high tolerance to this chemical.^{7-9,11,15,20-22} Most of these studies used a morphometric technique to assess the differences in the shapes of the craniofacial structures. The selection of the age at which to evaluate the outcome is typically guided by factors of experimental design and cost. However, the age at which evaluation is conducted might influence the results or their interpretation. The current study used a cross-sectional study design to determine the postnatal age at which a C57BL/6J male mouse becomes ‘fully adult’ in terms of the craniofacial shape and proportions.     

Improvement in the Development of Oocytes from C57BL/6 Mice after Sperm Injection

The C57BL/6 mouse strain is used widely for producing transgenic and knockout strains. Sperm motility is extremely low after a freeze–thaw process. Although intracytoplasmic sperm injection (ICSI) can be used to produce embryos from sperm with low or even no motility, its success rate is poor in the C57BL/6 strain. In particular, the survival of C57BL/6 oocytes after ICSI is extremely low compared with that of hybrid strains. We found that the survival percentages of C57BL/6J oocytes (63% and 64%) were lower than those of B6D2F1 oocytes (80% and 80%) when B6D2F1 and C57BL/6J sperm were injected, respectively. For C57BL/6J mice, 87%, 72%, 64%, 56%, and 59% of oocytes survived after ICSI in media containing 61.62, 71.62, 81.62, 91.62, and 101.62 mM NaCl, respectively. In addition, 64%, 81%, and 79% of oocytes survived after ICSI in media with 4.83, 14.83, and 24.83 mM KCl, respectively. Our results suggest that the survival of C57BL/6J oocytes after ICSI is improved by using Na⁺-deficient and K⁺ -rich media.Abbreviation: ICSI, intracytoplasmic sperm injectionThe C57BL/6 mouse is used widely for producing transgenic and knockout strains. Although the sperm of these strains have been maintained by cryopreservation as genetic resources,⁹ sperm motility is extremely low after a freeze–thaw process.^15,19 Partial dissection of the zona pellucida by using a steel needle is one technique to facilitate fertilizing oocytes with sperm showing poor motility.¹⁴ However, this technique requires a considerable degree of technical skill. Microdissection of the zona pellucida by using a laser beam is an easy and simple technique to produce a large number of embryos from poorly motile sperm.^4,10 These are useful tools unless the sperm are immotile.Intracytoplasmic sperm injection (ICSI) is a technique to produce embryos regardless of sperm motility.^3,20 Moreover, using the ICSI technique yielded offspring from oocytes injected with sperm that had been freeze-dried without cryoprotectants.^5-8,21 Reports of successful ICSI have been published for many species of mammals.^16,22 Since the technique was first reported,¹² various improvements have been applied to mouse ICSI to increase its success rates.^13,18Although the C57BL/6 strain is important for biomedical research, the success rate of ICSI in this strain remains low.¹⁷ In particular, the survival of C57BL/6 oocytes after ICSI is extremely low, because the oocytes have very poor tolerance to the damage caused by injection, compared with that of oocytes from hybrid strains. Hybrid strains have been used in most previous studies.^12,13,18,20 However, improving the success rate of ICSI in the C57BL/6 strain is important for biomedical research using transgenic or knockout mice derived from C57BL/6 so that they can be maintained as genetic resources. Here we investigated the culture media used for ICSI with the aim of improving the survival of C57BL/6 oocytes after sperm injection.     

Environmental Enrichment Reduces the Likelihood of Alopecia in Adult C57BL/6J Mice

Barbering (incessant grooming) is an abnormal behavior causing alopecia and commonly affects various strains of laboratory mice, including C57BL/6J. Barbering-induced alopecia is a potential symptom of brain impairment and can indicate a stressful environment. We compared alopecia prevalence and severity in mice housed in enriched or standard cages. Providing an enriched environment delayed the onset and reduced the prevalence and overall severity of alopecia in C57BL/6J mice. Husbandry methods that reduce adult alopecia are likely to promote the wellbeing of the animals. We suggest that environmental enrichment is a simple and economic way to reduce alopecia in mouse colonies.Abbreviations: EE, environmental enrichment; PND, postnatal dayEnriching environments for laboratory rodents can influence CNS development and forebrain function^13,21 and improve welfare.^22,25,26 Environmental enrichment (EE) comes in many forms (for example, toys, tunnels, nesting material, larger cages, social), and generally includes anything that is preferred (not avoided) by captive animals and increases species-specific behavior or decreases abnormal behavior.^17,23 Research shows numerous benefits of enriched environments, including enhanced cognitive abilities,^7,24 reduced abnormal behavior,¹⁵ increased resistance to stressors, and reduced pathogenesis and progression of disease.¹² A recent study reports that EE can lead to greater external validity of results as compared with standard housing.²⁰ In addition, standard captive-housing conditions (for example, housing laboratory mice in small, single-sex cages of low complexity) can induce behavioral frustration, leading to chronic stress, ^22,26 whereas enriched environments can reduce stress.^3,23Excessive hair-pulling is an abnormal behavior that occurs in a range of species (for example, humans, primates, mice, and dogs), particularly in those subjects confined to captivity.¹⁹ In some laboratory strains, such as C57BL/6J, excessive hair-pulling is thought to cause alopecia (hair loss), appearing as asymmetrical patches primarily on the dorsum; whisker trimming is common also, and together are termed ‘barbering.’^4,5,14 For mice, whiskers are an important source of sensory information,¹¹ making their loss a welfare concern and a potential source of behavioral variation in research data.Barbering may have additional welfare implications: originally thought to be a form of dominance behavior,^14,16 with the remaining sole untrimmed mouse presumed ‘guilty’ and dominant, recent research suggests that barbering is an abnormal behavior that models the human hair-plucking disorder trichotillomania.^4,5,10 Humans with this disorder show signs of clinical distress,¹⁹ and they increase their compulsive hair-pulling behavior in stressful environments. ⁵ Similarly, stressful conditions can promote barbering in laboratory mice.⁸ Various husbandry factors associated with reduced stress (such as particular cage designs⁵ and delaying weaning ages⁶) are reported anecdotally to reduce barbering-induced alopecia. Providing various toys such as cat or bird toys, balls, climbing structures and replacing them every 2 wk has previously been reported to reduce alopecia in laboratory mice.² However, that study did not indicate the strain(s) of the mice used, mice were housed in large groups (n = 10), and statistical analyses were not performed.We evaluated alopecia in mice housed in enriched and nonenriched environments to investigate the effects of EE on barbering in C57BL/6J mice, one of the most commonly used and frequently affected laboratory strains. Because barbering increases with age,⁵ we assessed mice at 4 and 6 mo, to address effects of EE on both onset and progression of alopecia.     

链脲佐菌素诱导C57BL/6小鼠糖尿病瘙痒模型的建立

[目的]探讨链脲佐菌素(streptozotocin , STZ)诱导C57BL/6小鼠糖尿病瘙痒模型的最佳剂量.[方法]24只雄性8周C57BL/6野生型小鼠,体重20～28 g,随机分为四组: A组(对照组)单次腹腔注射等剂量柠檬酸钠溶液 10 mL/kg,pH=4.5;B组STZ单次大剂量腹腔注射STZ 160 mg/kg;C组STZ腹腔注射40 mg/kg,连续注射5 d,每次给药时间间隔24 h;D组STZ两次中剂量注射,腹腔注射STZ 85 mg/kg+65 mg/kg,两次间隔时间24 h.从给药前4周开始,A组正常饮食,B、C、D组高脂饮食喂养至实验结束.注射STZ前1 d、注射STZ后1、2、3、4周测定动物的空腹血糖(FBG)、体重,采用录像记录30 min搔抓次数.[结果]与A组相比,B、C、D组造模后血糖升高并在不同时间点达到糖尿病标准(>200 mg/dL)(P<0.05),B组体重较A、C、D组下降(P<0.05),D组在STZ注射后第2、3周搔抓次数较A、B、C增多(P<0.05).[结论]腹腔分两次、间隔24 h注射STZ 85 mg/kg+65 mg/kg并配合高脂饮食是建立糖尿病并发瘙痒小鼠模型的合适方法.     

The Response of C57BL/6J and BALB/cJ Mice to Increased Housing Density

Increased numbers of mice housed per cage (that is, increased housing density) is seen as 1 way to reduce the costs of conducting biomedical research. Current empirically derived guidelines are based on the area provided per mouse depending on body weight as documented in the Guide for the Care and Use of Laboratory Animals. The current study aimed to provide a more scientific basis for housing density by examining the response of C57BL/6J and BALB/cJ mice to increased housing density from weaning to 5 mo of age, to determine those parameters most useful for future larger-scale studies. A wide range of phenotypic characteristics—including growth rate, body composition, hematology, serum biochemistry, hormone and metabolite measurements, in-cage telemetry, behavior, and cage microenvironment—was examined at various time points. The parameters showing greatest changes were: growth rate, which was significantly reduced in animals at the highest density; adrenal gland size, the proportion of adrenal cortex, and concentration of fecal corticosterone metabolites, all of which were increased at higher densities; and anxiety and barbering, which were more pronounced at higher densities. Cage microenvironment deteriorated with increasing density, but the increases in measured parameters were small, and their biologic impact, if any, was not apparent. The current findings indicate that mouse housing density can be increased 50% to 100% above the current recommendations (as floor area per mouse) with no or few apparent affects on mouse overall wellbeing. However, weight gain, fecal corticosterone metabolite levels, and barbering differed significantly with housing density and therefore are suggested as good measures of the response to alterations in housing.Abbreviation: CM, corticosterone metabolites; DEXA, dual-energy X-ray absorptiometry; FSH, follicle-stimulating hormone; LH, luteinizing hormone; TSH, thyroid-stimulating hormoneWithin the USA, space requirements for laboratory animals are outlined in the Guide for the Care and Use of Laboratory Animals (the Guide)²⁰ that states animals “must have enough space to turn around and to express normal postural adjustments … and must have enough clean bedding or unobstructed area to move and rest in.” The Guide provides a sliding scale for the floor space requirements per mouse based on average body weight. The designated minimum floor space requirements (per mouse) are 6 in.² (38.7 cm²) for mice less than 10 g, 8 in.² (51.6 cm²) for mice 15 g or less, 12 in.² (77.4 cm²) for mice 25 g or less, and at least 15 in.² (96.8 cm²) for mice weighing more than 25 g. All cages must be at least 5 in. (12.7 cm) high, with no designated minimum cage size.²⁰ Furthermore, housing of mice in social groups rather than alone is encouraged whenever this practice is not in conflict with the study design or aims.Previous studies at our institution demonstrated that housing mice of 4 commonly used inbred strains (C57BL/6J, BALB/cJ, NOD/LtJ, and FVB/NJ) for 8 wk in increasing densities up to a density twice that recommended by the Guide had no significant detrimental effects on the animals’ grossly observable health or wellbeing as assessed by weight gain, food and water consumption, and urinary corticosterone.^45,46 Throughout the 8 wk of these studies, the cage microenvironment (air quality and temperature) was, with few exceptions, well within the limits suggested by the Guide and OSHA for environmental gas contaminants.Studies using C57BL/6Crl and BALB/cJ mice housed from 3 to 6 wk of age for 5, 6, or 7 wk demonstrated limited negative effects of increased housing density.^13,30 Floor area per mouse tested in the cited 2 studies was 5, 10, 15, and 20 in.² per mouse for mice housed in groups of 3. Mortality was lowest for those mice housed at the highest density, whereas plasma corticosterone levels, thought to reflect the level of stress experienced by animals, increased significantly with increased density. However, inhibitory effects on immune function, as might have been expected from increased corticosterone, were not detected.^11,40 Apart from increased plasma corticosterone, the studies did not detect indicators of reduced wellbeing even when mice were housed in one-third of the area recommended by the Guide, equivalent to a 3-fold increase in density. BALB/c was the strain most commonly used in earlier density studies, and because male BALB/c mice are known for their native aggression^23,53 their characteristic increased aggression was hypothesized to be a sensitive indicator of social stress associated with increased housing density. None of these previous studies concluded that increased housing density resulted in more aggressive behavior by male BALB/c mice.These 2 groups of studies^13,30,45,46 highlight 1 of the many difficulties in performing experiments to assess housing density: the conundrum of distinguishing between floor area per mouse (cage size) versus group size. In 1 group of studies,^45,46 cage size was kept constant while group size was increased, whereas in the other studies,^13,30 group size was kept constant at 3 mice per cage while the area per mouse (cage size) was decreased. In practical terms these, studies had 2 different aims; the investigators for the first set of studies^45,46 were interested in using commercially available caging systems to assess the response of mice to increased group size as a way to increase housing density to try to determine the most suitable numbers of mice for a given cage type. In contrast, the researchers for the second group of studies^13,30 essentially had already decided on the ‘ideal’ group size and attempted to assess the response of mice to decreased cage area alone; this technique is useful to help determine which parameters might be best for assessing changes in cage density but is not entirely practical, nor economically feasible, in most vivaria given the standard cage sizes available and limited mouse room space. Interestingly, the ‘ideal’ group size determined by the second group of researchers^13,30 was similar to that resulting in the least aggression by male BALB/c mice.^52,53In the current study, we opted for the more practical, economic, and perhaps more realistic approach and varied group size by using cages that are standard at our institution. The previous studies^45,46 indicated that housing densities might be increased above the Guide’s recommendations with no, or few, strain-specific negative effects on the wellbeing of mice. However, we considered this conclusion to be limited by other contradictory data in many published studies (Figure 1). Further, direct comparison is confounded by variability in study design, including duration of housing, cage and group size, and the test parameters used to assess changes in animal wellbeing and welfare. In addition, few studies examined the effect of increased housing density over the prolonged periods of time often required for housing mice for colony maintenance and use in research projects.Open in a separate windowOpen in a separate windowFigure 1.Summary of published effects of increased housing density on mice. Parameters that increased with increased density (reduced floor space) are shown in bold, and those that decreased with increased density are in italics.In establishing the design of the current study, we had several aims. First, the current study is the first phase of a multiphase project designed to test a broad range of parameters and to focus on those tests for which there were large and statistically significant changes. Although the current sample size was small, we were particularly interested in identifying any robust parameters that may be apparent or strongly suggestive of changes in response to housing density alterations. These tests will then be incorporated into subsequent larger studies involving larger sample sizes and a wider range of different strains. The present study was devised as a preliminary experiment to examine the nature of animal responses to altered housing density. This information would enable specific hypotheses to be subsequently tested in confirmatory studies in the approach outlined in a previous work.¹² Second, by using the measures referred to earlier, we hoped to shed light on how laboratory mice respond to alterations in housing density. Third, we sought to raise awareness of the effects of increased housing density and ways to measure stress and to contribute to the ongoing discussion of these important issues. As a consequence of these aims, the current study was by necessity small in terms of sample size but broad in the range of physiologic parameters measured.¹²Because the experiments were designed to detect possible alterations in physiologic characteristics and behavior in response to alterations in housing density, the only variable introduced into the study design was the number of mice in each cage. Changing this parameter not only changed the area of cage space available for each mouse but also altered the group size; variation in group size may have been a contributing factor to some of the outcomes observed.We selected 2 common inbred strains, C57BL/6J (B6) and BALB/cJ (BALB), for the current study. We chose B6 mice because they are the most commonly used inbred strain in biomedical research.^24,27 We selected BALB mice because of their frequent use in previous density studies and because BALB males are more aggressive toward cagemates compared with many other inbred strains.⁵³ We reviewed previous studies to identify parameters that demonstrated differences likely attributable to changes in housing density (Figure 1). We selected a subset of these parameters for measurement in the current study. In addition, we included other measurements to detect whether increased housing density is associated with chronic stress. Our ultimate goal was to establish a set of quantitative parameters that could reliably assess changes in the wellbeing of laboratory mice. Mice were housed from weaning for 4 mo at 3 different densities, which was achieved by varying the number of mice per housing unit. The effects of different housing densities on various physiologic and behavioral parameters are presented and discussed.     

Morphometric analysis of the C57BL/6J mouse brain

Magnetic resonance microscopy (MRM), when used in conjunction with active staining, can produce high-resolution, high-contrast images of the mouse brain. Using MRM, we imaged in situ the fixed, actively stained brains of C57BL/6J mice in order to characterize the neuroanatomical phenotype and produce a digital atlas. The brains were scanned within the cranium vault to preserve the brain morphology, avoid distortions, and to allow an unbiased shape analysis. The high-resolution imaging used a T1-weighted scan at 21.5 microm isotropic resolution, and an eight-echo multi-echo scan, post-processed to obtain an enhanced T2 image at 43 microm resolution. The two image sets were used to segment the brain into 33 anatomical structures. Volume, area, and shape characteristics were extracted for all segmented brain structures. We also analyzed the variability of volumes, areas, and shape characteristics. The coefficient of variation of volume had an average value of 7.0%. Average anatomical images of the brain for both the T1-weighted and T2 images were generated, together with an average shape atlas, and a probabilistic atlas for 33 major structures. These atlases, with their associated meta-data, will serve as baseline for identifying neuroanatomical phenotypes of additional strains, and mouse models now under study. Our efforts were directed toward creating a baseline for comparison with other mouse strains and models of neurodegenerative diseases.     

Variation in Airway Responsiveness of Male C57BL/6 Mice from 5 Vendors

Mice are now the most commonly used animal model for the study of asthma. The mouse asthma model has many characteristics of the human pathology, including allergic sensitization and airway hyperresponsiveness. Inbred strains are commonly used to avoid variations due to genetic background, but variations due to rearing environment are not as well recognized. After a change in mouse vendors and a switch from C57BL/6J mice to C57BL/6N mice, we noted significant differences in airway responsiveness between the substrains. To further investigate the effect of vendor, we tested C57BL/6N mice from 3 other vendors and found significant differences between several of the substrains. To test whether this difference was due to genetic drift or rearing environment, we purchased new groups of mice from all 5 vendors, bred them in separate vendor-specific groups under uniform environmental conditions, and tested male first generation (F1) offspring at 8 to 10 wk of age. These F1 mice showed no significant differences in airway responsiveness, indicating that the rearing environment rather than genetic differences was responsible for the initial variation in pulmonary phenotype. The environmental factors that caused the phenotypic variation are unknown. However, differences between vendor in feed components, bedding type, or microbiome could have contributed. Whatever the basis, investigators using mouse models of asthma should be cautious in comparing data from mice obtained from different vendors.Abbreviation: AHR, airway hyperresponsivenessIn studies of the mouse lung involving measurement of pulmonary function, investigators commonly use inbred strains of mice to ensure a common genetic background. When mice are genetically identical the effects of specific environmental or genetic perturbations can be studied independent of background genotype. However, the need to similarly control for the source of the inbred mice is not always so apparent. In an effort to reduce costs in ongoing studies involving lung function measurements, we switched vendors from The Jackson Laboratory to the National Cancer Institute. Initial studies with the C57BL/6NCr mice unexpectedly showed substantially less responsiveness of the airways compared with the C57BL/6J mice. This preliminary observation called into question the validity of comparisons between studies of C57BL/6 mice of different substrains purchased from different vendors. If the differences were due to genetic drift rather than environmental factors, the effect of this variation could extend to genetically engineered mouse models generated by using different C57BL/6 substrains.C57BL/6 substrains have a long history in the United States: they were so named because they originated as black offspring from female mouse number 57 and male mouse number 52 in a mating by Clarence Cook (CC) Little of Abbie Lathrop''s stock in 1921. CC Little subsequently founded the Jackson Laboratory, and the substrain C57BL/6 was established at The Jackson Laboratory prior to 1937.¹⁴ The sublines C57BL/6N and C57BL/6J were separated at NIH in 1951. Harlan and Charles River acquired their breeding colonies from NIH in 1974, Taconic in 1991, and the National Cancer Institute in 1996. These long passages of time would suggest that genetic mutations arising in different colonies could have resulted in genetically distinct substrains. However, several studies suggest only minimal differences exist.^29,32,44 Most recently, one study⁴⁴ evaluated 1449 single-nucleotide polymorphisms distributed over all 20 chromosomes in 10 C57BL/6 sources from Europe, Australia, and the United States. Of the 1449 single-nucleotide polymorphisms, only 12 were polymorphic between strains, and most could not be directly associated with a known gene. Although these single-nucleotide polymorphisms distinguished the B6/N substrains from the B6/J substrains, there were no differences within the 4 B6/N or the 3 primary B6/J sources, whereas a second group of 3 B6/J sources differed by 3 single-nucleotide polymorphisms from the primary B6/J sources.These minimal differences in genotype between B6 substrains suggested that environmental factors may have played the major role in the phenotypic differences we observed. Differences in phenotype attributable to environmental variation have previously been reported in several fields. For example, behavioral testing differences in inbred mice were attributed to different testing locations;¹¹ behavioral tests, tumor growth, and immunologic parameters were affected by veterinary treatments with fenbendazole,^15,17,25 and numerous research areas are affected by intercurrent infections.^9,14 In addition, differences in behavioral testing attributed to differences between B6/J and B6/N mice⁵ may have resulted from differences in rearing environment rather than genetic differences. However, to our knowledge, there have been no reports of differences in airway responsiveness in B6 mice from different vendors. To further describe this finding and to evaluate the differing roles of genetics and environment, we tested airway responsiveness in 5 substrains of male B6 mice from 5 different vendors in the United States and then repeated the tests in the male offspring of mice of the same substrains purchased from the same vendors but bred and maintained under uniform environmental conditions at our institution.     

Comparison of Gelatin Flavors for Oral Dosing of C57BL/6J and FVB/N Mice

Precise oral dosing in rodents is usually achieved by intragastric gavage. If performed incorrectly due to technical difficulties, inexperience, or animal resistance, oral gavage may have animal welfare implications such as esophageal and gastric rupture and aspiration. The stress that is induced by this procedure can also lead to confounding results. In several animal models, drug vehicles must be sugar-free, deliver drugs in a specific formulation, and sometimes supply water. Gelatin has all of these properties. The current study aimed to evaluate the use of gelatin vehicles with different sensory features as an alternative to oral gavage. We investigated the time taken by 2 different inbred mouse strains, FVB/N and C57BL/6J, to ingest sugar-free gelatin pellets of varying flavors. Results showed that FVB/N mice took more time to eat the unflavored, strawberry and diet-flavored gelatin pellets than did C57BL/6J mice. Both strains showed low preference for lemon flavor, with the same ingestion times after the second day. This study showed that the C57BL/6J mice are more likely to eat gelatin than are FVB/N mice, and that the 2 strains of mice show a lower preference for lemon flavoring as compared with other flavors. This method of voluntarily oral administration offers an alternative to gavage for studies that use oral dosing studies.Abbreviation: BF, broccoli flour

Drugs or other substances can be administered to laboratory rodents by several routes. When designing a study that includes drug administration, the optimal delivery route and animal welfare should be considered, especially when the substance has to be administered repeatedly for a long period. The refinement of procedures is an important ethical issue in an experimental protocol and helps to promote reliable results. When the animal is experiencing pain, discomfort, or even stress resulting from routine handling, the body temperature, blood pressure, heart rate, corticosterone, prolactin, and glucose levels increase,^3,7,17,36 and the behavior of the animal is also altered.³ These changes may have animal welfare implications and can compromise the experimental results.When administering substances to laboratory rodents, compounds can be incorporated into the diet or drinking water. However, animals may not ingest the required individual dose, or the test compound may not be suitable for incorporation in the food or water due to its chemical stability or solubility. For these reasons, oral administration is mostly done by oral gavage,¹⁴ which is fast and allows the delivery of the correct dose directly into the stomach. Nevertheless, this technique requires a trained and proficient technician due to the risks it presents to the animal`s welfare. One study⁷ showed that oral gavage increased the plasma levels of corticosterone in rats and that lipid vehicles delivered by gavage induced a similar response in a volume-dependent manner. Another study demonstrated that oral gavage increased fecal corticosterone metabolites and altered the blood pressure and heart rate for 3 to 5 h in mice.³⁶ The stress induced by the restraint and introduction of the gavage needle^6,21,29 can be reduced by precoating the gavage needles with sucrose.¹⁶ However, other serious physical injuries such as gastric distention, aspiration pneumonia, esophageal and gastric rupture, and even death may occur.^1,3,28,34 Therefore, refined methods of oral dosing and alternatives to oral gavage would be useful. Oral administration through voluntary ingestion of a gelatin vehicle was reported for glucose administration to mice during oral glucose tolerance test⁸ and for the administration of a cannabinoid-1 receptor antagonist in mice.³⁷ In rats, the voluntary ingestion of a buprenorphine jelly was tested for postsurgical analgesia¹¹ and palatable gelatin tablets were tested for delivery of the wake-promoting drug modafinil.⁹ However, none of these studies evaluated how long the animals took to ingest gelatin and how the sensory characteristics of the vehicles influences intake duration. The time that the animal takes to eat the entire gelatin pellet must be standardized because variation may alter the onset of effect and influence pharmacokinetic measures. The current study evaluated 2 different mouse strains, C57BL/6J and FVB/N, with regard to the acceptance of and time taken to consume a whole gelatin pellet of 4 different flavors (unflavored, strawberry, lemon, and diet-flavored); we subsequently used this methodology to test voluntary ingestion of broccoli flour.     

The Water Delivery System Affects the Rate of Weight Gain in C57BL/6J Mice during the First Week after Weaning

Facility planners, IACUCs, veterinary staff, and researchers make choices on water delivery systems for rodents on the basis of cost effectiveness, water quality, risk of malfunction, and potential effect on animal health and welfare. Here we compare biometrics, including weight trends, of newly arrived mice unfamiliar with automated watering; weight trends of weanlings; fecundity of mice; and risk of malfunction among 3 water delivery techniques: water bottle only, combination of automated delivery and water bottle, and automated system only. There was no statistically significant difference among the 3 experimental groups with respect to fecundity, mortality, and delivery malfunction. On the basis of body weight trends, the health and wellbeing of the mice used in these studies were not affected by the water delivery system or housing density after the first week; however, there was a significant difference in the growth rate at 21 to 28 d of age among the 3 groups of pups. The mice receiving both automated delivery and water bottles experienced higher growth rates from 21 to 28 d of age than did the other experimental groups. However, after 35 d of age, weight trends did not differ among the groups. Our results suggest that mice weaned into the same method of water delivery as their respective dams thrive equally well among the 3 tested water delivery systems.The Guide for the Care and Use of Laboratory Animals⁴ indicates that “animals should have access to potable, uncontaminated drinking water according to their particular requirements,” and, furthermore, that “animals sometimes have to be trained to use automatic watering devices.” Facility planners, IACUCs, veterinary staff, and researchers make choices on water delivery systems for rodents on the basis of cost effectiveness, water quality, risk of malfunction, and potential impact on animal health and welfare. Here we compare biometrics, including weight trends, of newly arrived mice unfamiliar with automated watering; weight trends of weanlings; fecundity of mice; and risk of malfunction between 3 water delivery techniques: water bottle only, combination of automated delivery and water bottle, and automated delivery only (

Home Improvement: C57BL/6J Mice Given More Naturalistic Nesting Materials Build Better Nests

Environmental enrichment of laboratory mice can improve the quality of research, but debate arises over the means of enrichment and its ability to be used in a sterile environment. One important form of enrichment is nesting material. Mice in the wild build dome-shaped, complex, multilayered nests, but this behavior is not seen in the laboratory, perhaps due to inappropriate nesting material rather than the nest-building ability of the mice. Here we focus on the use of naturalistic nesting materials to test whether they improve nest quality through the use of a ‘naturalistic nest score’ system; we also focus on materials that can be sterilized and easily used in existing housing systems. We first determined whether C57BL/6J mice build naturalistic nests when given shredded paper strips. We then compared these shredded paper strips with other commonly used nesting enrichments (facial tissues and compressed cotton squares). Nests were scored for 6 d. We found that the shredded paper strips allowed the mice to build higher quality nests than those built with any of the other materials. Nests built with tissues were of intermediate quality, and nests built with compressed cotton squares were of poor quality, similar to those built by the control group. These results suggest that C57BL/6J mice given appropriate nesting materials can build nests similar to those built by their wild counterparts.Abbreviation: GLM, generalized linear modelEnvironmental enrichment of laboratory mice has the potential to improve mouse wellbeing, the public perception of mouse research, and the quality of scientific data.^2,16,32 However considerable debate exists over how to provide enrichment for mice, as well as the benefits and costs of practical enrichments.^{4,16,24,27,31} This controversy stems from 2 related issues: most of the enrichments examined in the mouse literature are impractical in a real housing facility; and most commercial ‘enrichments’ have simply been assumed to be beneficial, without testing their effects on the animal. The concept of ‘biological relevance’ provides a way to address these issues as well as a framework for developing practical, effective enrichments.^16,27,32 Here we illustrate this approach, using the development of a nest-building enrichment as an example.The term environmental enrichment, at its broadest, is used for any change in husbandry or caging intended to benefit the animal''s wellbeing.⁵ Changes in husbandry intended to benefit the animal may not always do so for 2 main reasons.³² First, the animal may not respond to the enrichment in the way intended. An animal may simply fail to perceive an enrichment as meaningful, it may find it aversive (as is the case for defensive burying of objects placed in the cage¹⁴), or it may try to defend the enrichment from its cage mates. For example, providing mice with shelves and nest boxes (resources that are thought to be beneficial but can be monopolized) can lead to increased aggression, immunosupression, and prolonged infections.^1,23,27 Second, an enrichment may improve one measure of wellbeing but compromise another. For example, individually ventilated cages (IVC systems) reduce ammonia levels, airborne disease transmission, and the amount of human handling, but mice find the increased ventilations rates aversive,³ show elevated levels of fear or anxiety,²¹ and demonstrate immune suppression.²⁵Several authors have argued that only biologically relevant enrichments—those that allow animals to control stressors in their environment—will actually benefit wellbeing.^16,27,32 For example, when provided with nesting material in ventilated cages, mice no longer find ventilation aversive.³ Furthermore, by giving animals homeostatic control over these stressors, such enrichments should reduce variability and benefit scientific outcomes^16,32 because the environment in which the animal lives considerably affects all aspects of physiology. For example, unenriched or socially isolated animals typically show altered brain development and physiology,²⁸ and the behaviors associated with unenriched environments may indicate altered brain function.¹⁶ Various lines of evidence suggest enrichment renders brains more ‘normal,’ not merely ‘different,’ in comparison to standard-housed animals. For instance, marsh tits (Parus palustris), which are naturally food-storing birds, have a greater hippocampus size than do nonfood storing Parus species. In captivity, however, only marsh tits with food-storing experience show greater hippocampal volume and neuron number, as well as fewer apoptotic cells, compared with those maintained with standard housing.¹¹A subset of biologically relevant enrichments incurs unintended costs. For example, shelters are biologically relevant, but they can induce aggression in group-housed animals.^27,32 These and other conditionally beneficial enrichments may benefit certain types of animals under certain conditions—for example, shelters may provide excellent enrichment for singly housed mice.³² Although some forms of enrichment may be detrimental to some animals, providing nesting material to mice does not appear to incur these kinds of disadvantages ^27,32 (but also see reference ²⁰). Mice show strong preferences for cages that provide nesting material (reviewed in references ¹⁹ and ²⁷), and nest building plays a central role in the natural history of wild mice, suggesting that nesting material may be a highly beneficial enrichment.In the wild, pregnant females tend to build the most complex nests, but nest-building ability extends to males and nonpregnant females.^7,10,22 Mice build nests to provide shelter from the elements, predators, and competitors, but also as a way to compensate for changes in external temperatures.²² Therefore, nests provide external insulation and create a less thermally stressful habitat.¹² The nests typically are built in secluded areas, and their quality at or right after birth is critical for the survival of offspring.¹⁰ If the nests remain undisturbed from outsiders, they generally take on a bowl shape or a dome with an exit hole on 1 side; this shape is most conducive to the survival of offspring.¹⁰ However, nests also can resemble something as simple as a shelf, but this configuration is less advantageous to the success of litters.^6,7,10 Dense colonies of wild house mice can contain a communal nest consisting of several mothers with litters,⁷ and some females with communal nests share them not only with their own litters but also eventually with the litters of their daughters.¹³Therefore, mice in the wild are expert and flexible nest builders, and this behavior is central to their survival, particularly in terms of dealing with challenging environments. However, in the laboratory, the use of nesting enrichments and nest-building behavior can vary, particularly between strains.^8,9,30 In particular, C57BL/6J mice often build rather poor, flat nests when provided with commercial nest-building enrichments.¹⁵ We hypothesized that this variability does not reflect a lack of importance of nest building to C57BL/6J mice but rather that the enrichments provided are not biologically relevant (that is, the mice did not perceive such enrichments as suitable nest-building material). Accordingly, we predicted that if provided with more naturalistic nest-building material, C57BL/6J mice would build nests equivalent to those of their wild counterparts.We first performed a pilot study to determine whether C57BL/6J mice would build naturalistic nests when given shredded paper strips. Having identified a material that allowed mice to build naturalistic nests, we then compared these shredded paper strips with other nesting enrichments to test our hypothesis that poor nest quality reflects the material provided, not the nest-building ability of the mice. Throughout the study, we focused on materials that could be sterilized and easily used in existing housing systems. If these materials are found to be suitable for nest building, then their use is more likely to be implemented across laboratories.     

Berberine Blocks the Relapse of Clostridium difficile Infection in C57BL/6 Mice after Standard Vancomycin Treatment

Vancomycin is a preferred antibiotic for treating Clostridium difficile infection (CDI) and has been associated with a rate of recurrence of CDI of as high as 20% in treated patients. Recent studies have suggested that berberine, an alternative medical therapy for gastroenteritis and diarrhea, exhibits several beneficial effects, including induction of anti-inflammatory responses and restoration of the intestinal barrier function. This study investigated the therapeutic effects of berberine on preventing CDI relapse and restoring the gut microbiota in a mouse model. Berberine was administered through gavage to C57BL/6 mice with established CDI-induced intestinal injury and colitis. The disease activity index (DAI), mean relative weight, histopathology scores, and levels of toxins A and B in fecal samples were measured. An Illumina sequencing-based analysis of 16S rRNA genes was used to determine the overall structural change in the microbiota in the mouse ileocecum. Berberine administration significantly promoted the restoration of the intestinal microbiota by inhibiting the expansion of members of the family Enterobacteriaceae and counteracting the side effects of vancomycin treatment. Therapy consisting of vancomycin and berberine combined prevented weight loss, improved the DAI and the histopathology scores, and effectively decreased the mortality rate. Berberine prevented CDIs from relapsing and significantly improved survival in the mouse model of CDI. Our data indicate that a combination of berberine and vancomycin is more effective than vancomycin alone for treating CDI. One of the possible mechanisms by which berberine prevents a CDI relapse is through modulation of the gut microbiota. Although this conclusion was generated in the case of the mouse model, use of the combination of vancomycin and berberine and represent a novel therapeutic approach targeting CDI.     

Segmentation of the C57BL/6J mouse cerebellum in magnetic resonance images

The C57BL mouse is the centerpiece of efforts to use gene-targeting technology to understand cerebellar pathology, thus creating a need for a detailed magnetic resonance imaging (MRI) atlas of the cerebellum of this strain. In this study we present a methodology for systematic delineation of the vermal and hemispheric lobules of the C57BL/6J mouse cerebellum in magnetic resonance images. We have successfully delineated 38 cerebellar and cerebellar-related structures. The higher signal-to-noise ratio achieved by group averaging facilitated the identification of anatomical structures. In addition, we have calculated average region volumes and created probabilistic maps for each structure. The segmentation method and the probabilistic maps we have created will provide a foundation for future studies of cerebellar disorders using transgenic mouse models.     

Effects of Repeated Anesthesia Containing Urethane on Tumor Formation and Health Scores in Male C57BL/6J Mice

Repeated injection of urethane (ethyl carbamate) is carcinogenic in susceptible strains of mice. Most recent cancer studies involving urethane-induced tumor formation use p53^+/– mice, which lack one copy of the p53 tumor suppressor gene. In contrast, the same protocol elicits at most a single tumor in wildtype C57BL/6 mice. The effect of repeatedly injecting urethane as a component of a ketamine–xylazine anesthetic mixture in the highly prevalent mouse strain C57BL/6 is unknown. Male C57BL/6J mice (n = 30; age, 3 mo) were anesthetized once monthly for 4 mo by using 560 mg/kg urethane, 28 mg/kg ketamine, and 5.6 mg/kg xylazine. The physical health of the mice was evaluated according to 2 published scoring systems. The average body condition score (scale, 1 to 5; normal, 3) was 3.3, 3.3, and 3.4 after the 2nd, 3rd, and 4th injections, respectively. The visual assessment score was 0 (that is, normal) at all time points examined. Within 1 wk after the 4th injection, the mice were euthanized, necropsied, and evaluated histopathologically. No histopathologic findings were noteworthy. We conclude that repeated monthly injection with urethane as a component of an anesthetic cocktail does not cause clinically detectable abnormalities or induce neoplasia in C57BL/6J mice. These findings are important because urethane combined with low-dose ketamine, unlike other anesthetic regimens, allows for accurate recording of neuronal activity in both the brain and retina. Longitudinal neuronal recordings minimize the number of mice needed and improve the analysis of disease progression and potential therapeutic interventions.Abbreviations: ERG, electroretinogram; VEP, visual evoked potentialUrethane is classified as a chemical carcinogen¹⁵ and is used by many laboratories to induce tumor formation in mice. Critical parameters affecting urethane-induced tumor formation in mice include the mouse strain, urethane dose, and frequency of administration. For example, urethane induces tumors in albino strain A mice, which are naturally susceptible to tumor formation.^11,17,22 The delivery of urethane to Swiss or C57BL1 mice induces the formation of skin and lung tumors but only when a tumor-promoting agent, such as croton oil or X-ray irradiation, is given concurrently.^2-4,10,12,24 Within these strains, young mice appear to be more susceptible than are adults.^9,17One of the mouse lines most resistant to urethane-induced tumor formation is also the strain used most often in research studies: C57BL/6.²¹ Weekly injections of 1000 mg/kg urethane for 10 wk are required to induce tumor formation in this strain.²¹ The tumors typically induced in this model are pulmonary adenomas and hepatic hemangiomas or hemangiosarcomas.^8,9,11,13 Furthermore, 5 mo after the first injection, only half of the mice injected had developed tumors, at an average of 0.63 tumors per mouse.²¹ Another study showed that the strain variability may be due to genetic alterations at 3 separate loci.¹⁹ Despite the existence of more susceptible mouse strains, many studies use p53^+/– mice on a C57BL/6 background to obtain reliable and consistent results.⁸ Although these mice are inherently susceptible to tumor formation due to the lack of one copy of the p53 tumor suppressor gene, they still have to be injected repeatedly with high doses of urethane to induce tumor formation. For example, daily injection of 1mg/kg urethane for 180 d failed to induce tumor growth in p53^+/– mice;⁸ to achieve tumor formation in these mice, the urethane dosage must be increased to 10 to 100 mg/kg daily for at least 180 d.⁸Another use of urethane is as an anesthetic. Electrophysiologists use urethane-containing anesthesia during the recording of electrical activity from the brain or retina of rodents, including that during vision testing through electroretinography (ERG) and a light-evoked encephalographic evaluation known as the visual evoked potential (VEP). Inducing anesthesia by combining urethane (560 to 1000 mg/kg) with ketamine (25 to 40 mg/kg) and xylazine (5.6 to 10 mg/kg) is ideal in this context because it avoids the confounding influences of higher doses of anesthetics on electrical responses, yet maintains a sufficient depth of anesthesia to obtain readable electrical signals.^5-7,18,25,27 However, in light of concerns regarding urethane-induced tumor formation, these recordings typically are only performed once in each rodent subject, just prior to euthanasia. This practice greatly limits the amount of information that can be obtained from a single animal. Longitudinal ERG and VEP from the same animal are needed to understand the progression of disease and therapeutic efficacy of various treatments.Longitudinal assessments of vision in models of glaucoma, trauma to the eye or brain, or inherited retinal degenerations provide information on disease course and therapeutic windows (for review, see reference 29). In addition, these types of assessments would strengthen therapy studies by determining the duration of therapeutic efficacy.²⁹ Importantly, most studies of visual system degeneration use C57BL/6-based models, in which vision loss is induced either through mechanical or genetic manipulation. Although different mice can be assessed at each time point, this strategy decreases the power of statistical analysis and increases the total number of mice needed for each study. The hypothesis of the current study is that repeated injection of an anesthetic mixture containing 560 mg/kg urethane, 28 mg/kg ketamine, and 5.6 mg/kg xylazine does not decrease overall physical health according to 2 scoring systems or induce tumors in C57BL/6 mice.     

Effects of Oxygen Supplementation on Injectable and Inhalant Anesthesia in C57BL/6 Mice

Oxygen supplementation is rarely considered when anesthetizing laboratory mice, despite reports that mice become profoundly hypoxic under anesthesia. Little is known about the effects of hypoxia on anesthetic performance. This article focuses on the effects of oxygen supplementation on physiologic parameters and depth of anesthesia in male and female C57BL/6 mice. Anesthesia was performed via common injectable anesthetic protocols and with isoflurane. Mice anesthetized with injectable anesthesia received one of 3 drug protocols. Low-dose ketamine/xylazine (100/8 mg/kg) was chosen to provide immobilization of mice, suitable for imaging procedures. Medium-dose ketamine/xylazine/acepromazine (100/10/1 mg/kg) was chosen as a dose that has been recommended for surgical procedures. High-dose ketamine/xylazine/acepromazine (150/12/3 mg/kg) was chosen after pilot studies to provide a long duration of a deep plane of anesthesia. We also tested the effects of oxygen supplementation on the minimum alveolar concentration (MAC) of isoflurane in mice. Mice breathed supplemental 100% oxygen, room air, or medical air with 21% oxygen. Anesthetized mice that did not receive supplemental oxygen all became hypoxic, while hypoxia was prevented in mice that received oxygen. Oxygen supplementation did not affect the MAC of isoflurane. At the high injectable dose, all mice not receiving oxygen supplementation died while all mice receiving oxygen supplementation survived. At low and medium doses, supplemental oxygen reduced the duration of the surgical plane of anesthesia (low dose with oxygen: 22 ± 14 min; low dose without supplementation: 29 ± 18 min; medium dose with oxygen: 43 ± 18 min; medium dose without supplementation: 61 ± 27 min). These results suggest that mice anesthetized with injectable and inhalant anesthesia without supplemental oxygen are routinely hypoxic. This hypoxia prolongs the duration of anesthesia with injectable drug protocols and affects survival at high doses of injectable anesthetics. Because of variable responses to injectable anesthetics in mice, oxygen supplementation is recommended for all anesthetized mice.

Anesthesia is frequently required for mice used in biomedical research, but anecdotal communications suggest that mice receive significantly less anesthetic monitoring and supportive care than do other research species. Monitoring of anesthetized mice is often minimal due to lack of specialized monitoring equipment, and the fact that many rodent surgeries are performed by a single person who acts as both surgeon and anesthetist. Supportive care during anesthesia is limited by a lack of supporting experimental evidence. The lack of monitoring and supportive care may increase the mortality rate in anesthetized mice.Previous studies have shown that mice anesthetized with both inhalant and injectable anesthetics without supplemental oxygen become profoundly hypoxic.^{1,6,8,9,19,26,39,41} While mice in these studies appear to recover normally from anesthesia, little is known about the effects of hypoxia on physiologic parameters, anesthetic depth, and perioperative mortality. Respiratory complications, including hypoxia and hypoventilation, are second only to cardiovascular complications as a cause of perioperative mortality in veterinary species, and in humans, hypoxemia accounts for over 50% of deaths under anesthesia.⁴ To mitigate the risk of hypoxia under anesthesia, oxygen supplementation is commonly provided to anesthetized humans and animals, but is rarely provided to mice in research settings.^6,19All anesthetics affect respiratory function; ketamine and isoflurane are particularly known to cause respiratory depression in mice and rats by impairing the normal physiologic responses to hypoxemia and hypercapnia.^{9,12,20,23,28} The peripheral chemoreceptors, primarily in the carotid body, normally sense dropping arterial partial pressure of oxygen (PaO₂) while central chemoreceptors located in the medulla sense changes in pH and rising partial pressure of carbon dioxide (PaCO₂).^22,23,29,40 Both sets of chemoreceptors compensate by initiating increases in respiratory rate and tidal volume.^{23,28,31,34,40} Injectable and inhalant anesthetic agents depress the function of these chemoreceptors, preventing the increases in respiration that compensate for hypoxia and hypoventilation.^22,29Pulse oximetry is commonly used to monitor peripheral oxygen saturation and detect the presence of hypoxia. Pulse oximeters use the difference in light absorption of oxygenated hemoglobin and deoxygenated hemoglobin in arterial blood to provide an estimate of arterial oxygen content, abbreviated as SpO₂.¹⁷ An SpO₂ of less than 90% to 95% generally corresponds to a PaO₂ of less than 60 to 80 mm Hg, which is considered hypoxic in most species of mammals.^7,17 Because of the small size of mice, species-specific pulse oximetry equipment is necessary to obtain this measurement. Therefore, measurement of SpO₂ in anesthetized mice is not routinely performed, meaning that hypoxia under anesthesia generally goes unrecognized, and is likely more common than is appreciated by our field.The purpose of this study was to confirm that mice become hypoxic after receiving a ketamine/xylazine based anesthetic admixture or isoflurane, which are commonly used anesthetics in mice and to investigate the effects of oxygen supplementation on anesthetic depth, physiologic values, and anesthetic requirements in these mice.^9,35 We hypothesized that mice not receiving supplemental oxygen would be hypoxic, as indicated by lower SpO₂ while anesthetized, and that supplemental oxygen would correct this hypoxia. We also hypothesized that oxygen supplementation would increase the doses of injectable and inhalant anesthesia necessary to maintain mice at a surgical plane of anesthesia.     

Sperm Freezing and In Vitro Fertilization in Three Substrains of C57BL/6 Mice

Traditional protocols for sperm recovery, cryopreservation, and in vitro fertilization (IVF) have been considerably less efficient for inbred mouse strains, including C57BL/6, than for hybrid and outbred strains. We report here that 3 changes to published and widely used protocols markedly improved fertilization rates for both fresh and frozen–thawed sperm in 3 substrains of C57BL/6 mice (C57BL/6J, C57BL/6NCrl, and C57BL/6NTac). First, the traditional cyroprotective agent was modified by adding amino acids. Second, preincubation of sperm in a preincubation medium containing methyl-β-cyclodextrin and polyvinyl alcohol enabled collection of progressively motile sperm for IVF. Third, we evaluated 3 media for IVF: human tubal fluid (HTF), modified Krebs–Ringer bicarbonate medium (TYH), and minimal essential medium (MEM). HTF and TYH were modified by adding minimal essential amino acids. The methodology reported here increased the IVF rate of both fresh and frozen–thawed sperm and enabled efficient isolation of capacitated viable sperm. Fertilization rates greater than 65% and 40% were obtained with the 3 tested substrains when fresh and frozen–thawed sperm, respectively, were used for IVF. Higher fertilization rates were seen with frozen–thawed sperm from C57BL/6NCrl and C57BL/6NTac mice than from C57BL/6J mice. Among all strains, fresh sperm from C57BL/6NTac mice gave the highest fertilization rate. Of 190 two-cell embryos, 63 (33.2%) developed to term after transfer to pseudopregnant recipient mice. The protocol we detail here provides reliable cryopreservation and recovery of live mice in 3 substrains of C57BL/6, making sperm cryopreservation and IVF a viable choice for preservation and distribution of mouse lines.Abbreviation: CPA, cyroprotective agent, mCPA, modified cryoprotective agent, HTF, human tubal fluid, IVF, in vitro fertilization, MEM, minimal essential medium, PM, preincubation medium, TYH, modified Krebs–Ringer bicarbonate mediumAn exponential increase in the number of mouse lines with induced mutations (including transgenes, targeted mutations, and chemically-induced mutations) from laboratories and commercial suppliers around the world has resulted in a concomitant increase in lines that need to be preserved or distributed for biomedical research.^4,17,31,33 Yet, at the same time, the technical challenges and limitations associated with shipping live mice have increased dramatically due to increased airport security,² ethical concerns, and other impediments, thereby limiting efficient distribution of important mouse models of human disease. Successful and efficient collection, cryopreservation, and reanimation of mouse sperm would be an ideal solution for preservation and distribution of these important in vivo models, whether for biomedical research or for drug discovery and development. However, the current methods for these processes have proven inefficient, difficult to perform, and rate-limiting. Mouse sperm is very sensitive to diverse stresses including mechanical, osmotic, and oxidative conditions.^19-21,33 The raffinose–skim milk method^25,28,35,36 currently predominates in many laboratories worldwide that routinely cryopreserve and store mouse sperm.^22,23,38 However, high-efficiency reanimation appears to be restricted to mice with hybrid or outbred backgrounds and is considerably less effective for inbred mouse strains, including C57BL/6.³⁵ This situation has a considerable effect on mouse models used for biomedical research, given that many transgenic and knockout lines are backcrossed onto inbred backgrounds, most frequently C57BL/6.³⁵In the present study, we focused on 3 aspects for improvement. First, the traditional cryoprotective agent containing a cryoprotectant (raffinose) and membrane protectant (skim milk) was modified to provide better cryoprotection during the freezing and thawing process. Second, frozen–thawed sperm suspensions underwent a preincubation procedure to enrich for progressively motile sperm. Finally, we compared 3 fertilization media and 3 substrains by using established criteria to select an optimal in vitro fertilization (IVF) medium.     

Vascular reactivity of isolated thoracic aorta of the C57BL/6J mouse

We characterized the thoracic aorta from the C57BL/6J mouse, a strain used commonly in the generation of genetically altered mice, in response to vasoactive substances. Strips of aorta were mounted in tissue baths for measurement of isometric contractile force. Cumulative concentration-response curves to agonists were generated to observe contraction, or relaxation in tissues contracted with phenylephrine or prostaglandin F(2alpha) (PGF(2alpha)). In endothelium-denuded strips, the order of agonist contractile potency (-log EC(50) [M]) was norepinephrine > phenylephrine = 5-hydroxytryptamine > dopamine > PGF(2alpha) > isoproterenol > KCl. Angiotensin II and endothelin-1 were weakly efficacious (15% of maximum phenylephrine contraction), as were UK14,304, clonidine, histamine, and adenosine. In endothelium-intact strips, agonists still caused contraction and both angiotensin II and endothelin-1 remained ineffective. In experiments focusing on angiotensin II, angiotensin II-induced contraction was abolished by the AT(1) receptor antagonist losartan (1 microM) but was not enhanced in the presence of the AT(2) receptor antagonist PD123319 (0.1 microM), tyrosine phosphatase inhibitor orthovanadate (1 microM) or when angiotensin II was given noncumulatively. Prazosin abolished isoproterenol-induced contraction and did not unmask isoproterenol-induced relaxation. Angiotensin II and endothelin-1 did not cause endothelium-dependent or -independent relaxation in phenylephrine- or PGF(2alpha)-contracted tissues. Acetylcholine but not histamine, dopamine, or adenosine caused an endothelium-dependent vascular relaxation. These experiments provide information as to the vascular reactivity of the normal mouse thoracic aorta and demonstrate that the mouse aorta differs substantially from rat aorta in response to isoproterenol, angiotensin II, endothelin-1, histamine, and adenosine.