The Effects of Perioperative Analgesia on Litter Size in Crl:CD1(ICR) Mice Undergoing Embryo Transfer

The objective of this study was to evaluate the effect on litter size of 2 analgesics used perioperatively during mouse embryo transfer surgery. Day 2.5 pseudopregnant CD1 mice (n = 96) were divided equally into 2 analgesic treatment groups and a saline control group. Each mouse received a single, subcutaneous dose of buprenorphine hydrochloride (0.1 mg/kg), flunixin meglumine (2.5 mg/kg), or saline immediately after induction of anesthesia with 2.5% isoflurane. Each mouse then was prepared for aseptic surgery. Blastocysts had previously been collected from C57BL/6NCrl female mice that were synchronized and superovulated by using pregnant mare serum gonadotropin and human chorionic gonadotropin and mated with C57BL/6NTac male mice 3.5 d before collection. Viable blastocysts were pooled, and 8 were selected arbitrarily and transplanted into the right uterine horn of each pseudopregnant CD1 mouse. Mice were monitored throughout pregnancy, and the number of pups at birth was documented. No statistically significant difference was found between the 3 groups. These results indicate that perioperative analgesic treatment with buprenorphine or flunixin in the CD1 mouse undergoing embryo transfer is not associated with increased embryonic loss.Abbreviation: NSAID, nonsteroidal antiinflammatory drugsEmbryo transfer in the mouse is widely used in the production of genetically engineered mice, in vitro fertilization, intracytoplasmic sperm injection, rederivation, and reconstitution of mouse lines after embryo cryopreservation.^2,5,14,30 Embryo transfer has become a principal surgical procedure in many animal care programs involving mice and is routinely performed in our program.The Guide for the Care and Use of Laboratory Animals²¹ defines major survival surgery as a procedure that penetrates and exposes a body cavity or produces substantial impairment of physical or physiologic functions. According to this definition, the laparotomy performed for embryo transfer constitutes a major survival surgery.^1,21 The attending veterinarian must provide oversight to surgical programs, and part of that responsibility includes preventing or alleviating pain associated with surgical procedures with the use of appropriate analgesics.^1,21Several classes of analgesics are used perioperatively in rodent surgical procedures, including opioids and nonsteroidal antiinflammatory drugs (NSAID).⁷ Buprenorphine is a commonly used opioid analgesic in rodent surgery.²⁵ NSAID including flunixin, aspirin, and phenylbutazone have been used extensively in veterinary medicine.¹⁰Some investigators are concerned that the administration of analgesics perioperatively during embryo transfer adversely affects embryonic survival. However, only one study has examined the effects of perioperative analgesics on litter size in mice.¹⁵ The purpose of the present study was to evaluate the effect of perioperatively administered analgesics, buprenorphine (Buprenex, Reckitt Benckiser Pharmaceuticals, Richmond, VA) and flunixin (Banamine, Schering-Plough, Union, NJ), on embryonic survival after embryo transfer in CD1 mice.     

Refinements in the Cryopreservation of Mouse Ovaries

Here we describe a new technique for cryopreserving mouse ovaries by using 0.5-mL straws. One advantage of this method is that it uses the same controlled-rate freezer and programming routinely used for the cryopreservation of mouse embryos. Using a 0.5-mL French straw loaded in the same way as for embryo freezing (for example, the one-step dilution method) with 1 M sucrose as an osmotic buffer and 2 M propylene glycol as the cryoprotectant containing the ovary sample, we further standardized the 2 methodologies. Applying this technique, 11 ovarian halves were cryopreserved in straws and stored under liquid nitrogen. Straws containing the frozen ovarian halves were thawed in a water bath at room temperature and the recovered ovaries orthotopically implanted into 11 recipient female mice; 8 of the 11 frozen ovarian halves resulted in functional ovaries. The 73% pregnancy rate resulted in a total of 53 pups born, of which 38 (72%) were generated from cryopreserved ovaries. Ovarian cryopreservation has been demonstrated to be a valid option for banking mouse genetic resources. Unlike frozen embryos, cryopreservation of ovarian tissue preserves haploid gametes. Despite this limitation, ovarian cryopreservation is the only technique that can be used to preserve oocytes from aged or problematic breeders. This advantage is especially important in situations where the only males available in the line are infertile, aged, or problematic breeders.Since the first publications on ovarian cryopreservation,^3,11 many variations on the methodology have been developed.^{2,5, 6,16,17} Today we can freeze the ovary at different periods of development^1,3,5,6,9,10 and have our choice of freezing methods involving vitrification,^{2,5,8,10,16,17} rapid freezing,⁸ or slow cooling.^9,13,15 Through these techniques, coupled with appropriate thawing methodology and surgical implantation of frozen–thawed ovarian tissue, recipient female mice can produce viable oocytes for subsequent fertilization.Currently various methodologies are available for cryopreservation of mouse ovarian tissue, and most of them require the use of vials rather than straws, which typically are used for the cryopreservation of embryos and sperm. One goal of the present study was to design a practical ovarian freezing method that approximated the methodology routinely used for the cryopreservation of embryos. Standardization of the cryopreservation methodologies would eliminate the need to reprogram equipment and stock unique supplies. Because few laboratories still use the classic slow-freezing methods that involve a final temperature of –80 °C or lower, we have described an adaptation of the 2-step freezing method^4,18 using an alcohol-based rate-controlled freezer and straws.     

Cage Change Influences Serum Corticosterone and Anxiety-Like Behaviors in the Mouse

Environmental variables and husbandry practices can influence physiology and alter behavior in mice. Our study evaluated the effects of cage change on serum corticosterone levels and anxiety-like behaviors in C57BL/6 male mice. We examined the effects of 3 different methods of performing cage transfer and of transferring mice to a clean or a dirty familiar cage microenvironment. The 3 different handling methods were forceps transfer, gentle transfer with gloved hands, and a passive transfer technique that did not involve active handling. Active handling methods and transfer to both clean and dirty cage microenvironments significantly increased serum corticosterone 15 min after cage change; however, at 60 min after cage change, levels were comparable to those of unmanipulated mice. Although the effects were transient, cage change altered anxiety-like behaviors in the open field when behavioral testing was performed on the same day. These results demonstrate that the timing of cage change can influence behavioral results, an effect that is an important consideration for rodent behavioral studies.General husbandry practices can have myriad effects on animal physiology and behavior. As the number of studies that incorporate behavioral testing into their molecular and genetic profiling increases, the epigenetic effects of the housing environment are becoming increasingly evident. Seemingly negligible variables, such as enrichment, can have profound effects on brain structure, function, and neurophysiology.^14,17-19,23 Alterations of the light cycle, noise levels, rack position of the home cage, and home cage disruption have been shown to affect breeding, physiologic or behavioral measures of anxiety, and occasionally experimental outcome.^{1,4,10,13,20,21,24,25} Husbandry practices that induce physiologic changes in stress hormones can have both positive and negative implications for behavioral testing. Previous behavioral studies have demonstrated that elevated corticosterone levels can either impair or improve cognitive performance, depending on the individual circumstance.^8,22,30Historically cage change has been used to create a model of hypertensive stress in mice,^15,16,29 and multiple studies have demonstrated a correlation between cage change and physiologic stress responses in rodents.^{1,4,5,7,21,26} The components of cage change that contribute to rodent stress and anxiety are multifaceted. Cage change most often takes place during the light phase of the light:dark cycle, at a time when mice are less active or resting. Animal husbandry staff manipulate or handle the mice during the cage transfer process. After cage transfer, the new clean cage microenvironment no longer contains the urine scent markers or pheromones mice use for social identification and hierarchical perception. Male mice respond with increased activity levels and fighting,^27,28 and breeding mice have been reported to have increased pup mortality²¹ and a higher incidence of cannibalism⁴ after cage change.Whether handling or providing a clean cage microenvironment is of equal or greater significance to mice is debatable. Handling consistently generates a corticosterone response in rodents,¹ and mice, unlike rats, habituate poorly to handling.^1,9,12 In contrast, aggression between male mice after cage change is reduced if nesting material is transferred from the dirty cage,²⁷ suggesting that the loss of scent markers is an important factor.We designed a 2-part study to further examine the effects of cage change on the physiology and behavior of C57BL/6 male mice. Our study assessed 3 different cage-change handling techniques: handling with forceps, gentle handling with gloved hands, and a passive transfer technique. Forceps transfer was performed in the same efficient manner used by the animal care staff. Gloved transfer consisted of a gentler and deliberately slower transfer technique than was forceps transfer. A passive transfer technique was devised to eliminate direct handling and consisted of using a pair of forceps to gently herd mice between 2 tilted cages. Depending on their experimental group, mice were either transferred to a clean cage, with no residual pheromones or urine scent markers, or mice were returned to their original dirty home cage.Part one of our study evaluated serum corticosterone levels at 15 and 60 min after cage change. We hypothesized that serum corticosterone levels would be higher for experimental groups of mice subject to forceps transfer than they would be for groups of mice manipulated with slower or gentler handling techniques. We also hypothesized that mice would have higher serum corticosterone levels if they were transferred to a clean cage microenvironment. The second part of our study used 2 behavioral tests—the open-field test and elevated-plus maze—to assess anxiety-like effects of cage change on behavior. Our hypothesis was that mice would be more likely to exhibit behaviors consistent with anxiety after cage change. We predicted that anxiety-like behaviors would be most evident in groups of mice that underwent forceps transfer or were transferred to clean cages. The results of our study revealed transient elevations in serum corticosterone in all groups of mice, and anxiety-like behaviors were altered when mice were assessed behaviorally on the same day of cage change, regardless of handling method.     

Improving the Quality of EDTA-treated Blood Specimens from Mice

Nonterminal blood sampling in laboratory mice is a very common procedure. With the goal of improving animal welfare, different sampling sites and methods have been compared but have not achieved a consensus. Moreover, most of these studies overlooked the quality of blood specimens collected. The main preanalytical concern with EDTA-treated blood specimens for hematology analyses is platelet aggregation, which is known to cause analytical errors. Our objective was to find a nonterminal blood sampling method with minimal adverse effects on mice and few or no platelet aggregates. We tested and compared 2 collection sites, 4 sampling methods, and 3 antithrombotic drugs in 80 C57BL6/j male and female mice by evaluating platelet aggregates on blood smears and platelet, WBC, and RBC counts. In addition, the blood collection process was carefully evaluated, and adverse effects were recorded. Platelet aggregation was lower in specimens collected from the jugular vein than from the facial vein, with no effect of the sampling device or the presence of an antithrombotic additive. Highly aggregated specimens were significantly associated with lower platelet counts, whereas aggregation had no effect on WBC or RBC counts. Adverse events during sampling were significantly associated with more numerous platelet aggregates. The jugular vein is thus a satisfactory sampling site in mice in terms of both animal welfare and low platelet aggregation. Using antithrombotic agents appears to be unnecessary, whereas improving sampling conditions remains a key requirement to ensure the quality of EDTA-treated blood specimens from mice.

Industrial and academic research often require hematology analyses of mouse blood. Consequently, many terminal and nonterminal techniques have become available for blood sampling in mice.^{12,21,27,40,42,53} Preanalytical variation in clinical pathology is known to be a major issue.^5,45,49 Although the effects of the blood sampling method on animal welfare have been the subject of many preanalytical hematology and biochemical analyses,^{1,6,8,9,15,16,18,24-26,36,47,50-52,54} no agreement has been reached regarding the optimal method for nonterminal blood collection in mice and, to our knowledge, only a few investigations^1,8,15,16,18 have addressed the quality of the resulting blood specimens.Our own experience of hematology measurements from nonterminal mouse EDTA-blood specimens is that some specimens show both visible clots and platelet aggregation, the latter being detected only from microscopic examination of blood smears.³³ Whereas specimens with visible clots can be eliminated, microscopic platelet aggregates can also interfere with hematology analyses or cause analytical errors, as has been reported in other species including cats.^13,22,31,39 These abnormalities require repeat sampling when possible; otherwise, the number of validated results is decreased. EDTA-treated mouse blood is especially prone to platelet aggregation and clotting.^14,28,43 This characteristic leads to errors in platelet counts (pseudothrombocytopenia) and possible misidentification of platelet aggregates as eosinophils, resulting in false leukocytosis and eosinophilia.¹⁴ In vitro platelet aggregation in mice is due to high platelet counts^34,43 and is influenced by numerous preanalytical factors including the sampling method, collection site, specimen processing, anticoagulant used, the blood:anticoagulant ratio, the mouse strain and genetic alterations.^19,28,30,43 The literature on the influence of preanalytical factors on the quality of CBC analyses in mice is scant,⁴³ and no agreement has yet been reached regarding the optimal method for nonterminal blood collection in mice. In humans and various animal species, platelet aggregation can be reduced by adding platelet aggregation inhibitors that act at different steps of aggregation. To our knowledge, the addition of such inhibitors to mouse whole blood has not been tested as a means to improve the quality of mice EDTA-treated blood specimens.The aim of this study was therefore to identify the best preanalytical conditions for nonterminal blood collection in mice, based on animal welfare, scores of platelet aggregation, and platelet, RBC, and WBC counts. The hypotheses we tested were that 1) adding an antithrombotic drug (or multiple such drugs) to the EDTA-treated blood specimen would prevent or at least significantly lower platelet aggregation, 2) the site and the method of collection influence in vitro platelet aggregation, and 3) high-quality blood sampling is a key to reducing platelet aggregation in blood specimens.     

Continuous Observation of Rabbit Preimplantation Embryos In Vitro by Using a Culture Device Connected to a Microscope

We developed a compact culture device that maintains developing embryos in vitro under constant temperature and CO₂ concentration. Using this device, we cultured rabbit embryos from the pronuclear stage to the hatched blastocyst stage and recorded their development digitally for 7 d. Recorded images were converted to a movie, and the developmental movement of individual embryos was analyzed. With this culture system, we can observe embryonic development in a suitable environment continuously for several days; similar long-term observation is not possible in the conventional system. The proportion of embryos that developed from the pronuclear stage to the blastocyst stage was the same in the new system (73.1%; 38 of 52) as in the conventional (control) system (77.6%; 38 of 49). Compaction of embryos occurred from the 8-cell to the morula stage at 32.5 ± 0.71 h after insemination. The time of blastocyst formation (77.2 ± 3.2 h after insemination) varied somewhat between embryos. Average hatching time was 98.7 ± 4.4 h after mating. Therefore, the cleavage, blastomere movement, and hatching processes of blastocysts can be followed clearly and recorded by using this new culture system.Reproductive characteristics of the rabbit, such as easy manipulation of ovulation and the formation of blastocysts with far more inner cell mass cells than those of mice, make the rabbit an excellent model for the study of embryology, developmental biology, and genetic engineering.²⁶ The preimplantation rabbit embryo undergoes a rapid series of cell divisions resulting in a blastocyst with 128 cells by day 3 after fertilization.^1,5,11 Rabbit 1-cell embryos have successfully been cultured to blastocysts in a variety of complex media, including largely defined conditions.^4,12,13 Embryonic cells are sensitive to environmental changes: during in vitro culture of mammalian embryos, even a slight change in the culture conditions has lasting effects on the offspring.²¹ In addition to the medium composition, the gas phase and temperature of the medium are important factors influencing embryo development.Continuous observation of developing embryos in vitro is difficult. Heat stress during the critical stage of early embryo development increases the incidence of early embryonic death.²³ Therefore, a suitable atmospheric environment is necessary for normal embryonic growth in vitro.^12,14,23 An effective gaseous environment for culturing rabbit zygotes in synthetic medium is 10% CO₂ combined with 5% O₂.⁶ In conventional culture systems, cells and tissues are maintained in culture dishes, which are not airtight, in an incubator filled with an appropriate mixture of O₂ and CO₂. However, when the culture dish is removed from the incubator for examination of embryos, the CO₂ in the embryos’ environment is lost rapidly, thus increasing the pH of the medium, an effect that is likely to be detrimental to embryos. Continuous observation or recording of embryo growth requires maintaining a constant CO₂ concentration of the culture medium under the microscope.Here we describe a small airtight chamber that fits over the stage of the microscope and provides CO₂ gas perfusion. The incoming gas is humidified and warmed to the desired temperature before being passed over the cells. This apparatus, combined with a computerized digital image analysis system, enabled us to record the cell movement of rabbit preimplantation embryos continuously for 7 d. The device can be used for examination of virtually any type of cell over extended periods of time. Therefore, preimplantation embryos that are suitable for embryo transfer can be selected by close examination of the developmental pattern of the embryos.     

Dose Regimens,Variability, and Complications Associated with Using Repeat-Bolus Dosing to Extend a Surgical Plane of Anesthesia in Laboratory Mice

Extending a surgical plane of anesthesia in mice by using injectable anesthetics typically is accomplished by repeat-bolus dosing. We compared the safety and efficacy of redosing protocols administered either during an anesthetic surgical plane (maintaining a continuous surgical plane, CSP), or immediately after leaving this plane (interrupted surgical plane, ISP) in C57BL/6J mice. Anesthesia was induced with ketamine, xylazine, and acepromazine (80, 8, and 1 mg/kg IP, respectively), and redosing protocols included 25% (0.25K), 50% (0.5K), or 100% (1.0K) of the initial ketamine dose or 25% (0.25KX) or 50% (0.5KX) of the initial ketamine–xylazine dose. In the ISP group, the surgical plane was extended by 13.8 ± 2.1 min (mean ± SEM) after redosing for the 0.25K redose with 50% returning to a surgical plane, 42.7 ± 4.5 min for the 0.5K redose with 88% returning to a surgical plane, and 44.3 ± 15.4 min for the 1.0K redose, 52.8 ± 7.2 min for the 0.25KX redose, and 45.9 ± 2.9 min for the 0.5KX redose, with 100% of mice returning to a surgical plane of anesthesia in these 3 groups. Mortality rates for ISP groups were 0%, 12%, 33%, 12%, and 18%, respectively. Mice in CSP groups had 50% mortality, independent of the repeat-dosing protocol. We recommend redosing mice with either 50% of the initial ketamine dose or 25% of the initial ketamine–xylazine dose immediately upon return of the pedal withdrawal reflex to extend the surgical plane of anesthesia in mice, optimize the extension of the surgical plane, and minimize mortality.Abbreviations: CSP, continuous surgical plane; ISP, interrupted surgical plane; KXA, ketamine–xylazine–acepromazine; PWR, pedal withdrawal reflex; RR, righting reflexThe goals of achieving a surgical plane of anesthesia are to provide immobility, unconsciousness, analgesia, amnesia, and attenuation of autonomic responses to noxious stimuli⁵ without adversely compromising physiologic variables such as heart rate, respiratory rate, body temperature, and blood pressure.^1,5 For prolonged surgical procedures, inhaled anesthetics generally are recommended because of rapid onset of and recovery from anesthesia, efficacy in attaining and maintaining a surgical plane of anesthesia, and relative safety.¹⁵ In laboratory animal practice, there are situations when inhaled anesthetics cannot be used due to technical or experimental limitations.^6,13,15,43 In these cases, an intraperitoneal injectable anesthetic protocol, typically including the combination of ketamine–xylazine–acepromazine (KXA), is used in mice to achieve a surgical plane of anesthesia.^3,8,22,47 If the procedure requires a duration of anesthesia beyond what a single dose of these drugs can provide, repeat-bolus dosing is the default method to extend the duration of anesthesia in mice. Although previous studies have evaluated the safety and efficacy of a single bolus of injectable anesthetics in rodents, no study has evaluated these parameters in the repeated dosing of injectable anesthetics to extend the duration of anesthesia, resulting in minimal information and guidance for researchers and veterinarians beyond anecdotal experience.^{3,8-11,14,16,17,22,25,30,39}When considering repeat-bolus dosing, it is essential to factor in the dose as well as the timing of administration to the animal. Knowledge of pharmacokinetics of each drug, as well as of drug interactions with other combined agents, is imperative for deciding how much to administer and when to provide the repeat dose to avoid either overdosing or underdosing the animal. The elimination half-life of ketamine in various strains of mice is approximately 13 min when administered by the intraperitoneal route,²⁸ making it a preferred drug for redosing, because systemic concentrations are likely to be diminished at the time of dissipation of the surgical plane of anesthesia. Potential negative effects of ketamine include respiratory depression, arrhythmias, seizures, tremors, muscle hypertonicity, and erratic or prolonged anesthetic recovery.^32,42 Xylazine has a half-life of 1.2 to 1.6 h in rats, and although it has not been evaluated in mice,^44,45,48 the half-life of xylazine in mice is presumably longer than that of ketamine. Due to the lack of information on the half-life of xylazine in mice, caution should be taken with any redosing scenario to prevent overdosing the drug. Adverse effects of xylazine in multiple species include hypotension, bradycardia, decreased gastrointestinal motility, and respiratory and cardiovascular depression.^32,42 The metabolism of xylazine is likely unaffected by concurrent ketamine administration, according to in vitro data.²⁶ Acepromazine has a relatively long half-life in nonrodent species, for example 7.1 h in dogs and 50 to 183 min in horses,^21,37 and is not routinely redosed in mice.The timing of drug administration is another critical factor to consider when addressing anesthetic redosing. Ideally, an effective repeat-dosing regimen achieves an uninterrupted, continuous surgical plane of anesthesia with a predictable anesthetic recovery. The advantage of redosing prior to the animal leaving the surgical plane of anesthesia is that a continuous surgical plane of anesthesia is maintained, eliminating concerns over patient awareness and perception of pain. The main disadvantage of this redosing approach is that adding more drug to the system while there is potentially still a considerable amount affecting the animal increases the risk of adverse side effects. Conversely, dosing at the time of emergence from a surgical plane of anesthesia decreases the risk of anesthetic overdose but may increase the possibility of a patient''s transient conscious awareness of the procedure.The aim of the present study was to identify a safe and effective dosing regimen to extend the surgical plane of anesthesia in mice by using an injectable anesthetic combination of KXA. We hypothesized that the ideal anesthetic regimen would permit the administration of a second dose of anesthetic before the mouse emerged from a surgical plane of anesthesia, thus facilitating an uninterrupted extension of that plane of anesthesia while minimizing mortality.     

Assessment of Routine Procedure Effect on Breathing Parameters in Mice by Using Whole-Body Plethysmography

We used whole-body plethysmography to investigate the effect of restraint, ear marking, tail vein and retroorbital blood sampling, and tail clipping on respiration in Balb/c × TCR-HA^+/– F1 hybrid mice (F1h). Baseline values of breathing parameters were determined. During the experiment, mice experienced a procedure and then plethysmographic recordings were obtained immediately and at 4, 24, and 48 h afterward. Baseline breathing parameters showed significant differences between sexes. Restraint affected minute volume differently than did handling in male mice and to a lesser extent in female mice. Ear marking significantly changed minute volume compared with handling but not restraint in male mice and in the opposite manner in female mice. Tail vein blood sampling changed minute volume in a significant manner compared with restraint but not compared with handling in both sexes. Retroorbital blood sampling significantly changed minute volume compared with values for both handling and restraint in male mice but only compared with handling in female mice. Tail clipping modified minute volume significantly compared with handling in male mice and compared with restraint in both sexes. Analysis of data showed that routine procedures affect minute volume in mice depending on invasiveness of maneuver and in a sex-biased manner for as long as 24 h after the procedure. Our experiment shows that procedures performed on laboratory mice can change respiratory parameters and can be investigated by plethysmography.Abbreviation: F1h, Balb/c × TCR-HA^+/– F1 hybrid miceHandling, restraint, identification methods, and blood or tissue sampling are regarded as routine procedures in animal experiments. This aspect is particularly important when working with transgenic animals, where the need for genotyping demands the frequent use of these procedures, which can readily yield DNA samples.⁵ The effect of routine procedures on physiologic parameters varies depending on method, frequency, and duration, but these procedures generally are considered to be acute stressors.^2,5,26,27 A large body of research has focused on the effect of routine procedures on animals’ physiology. These effects were assessed by studying changes in behavior,^1,23,36,37 body weight,^29,37 food and water intake,^29,37 body temperature³⁵ and heart rate by radiotelemetry,^{5,10,19,26,27} corticosterone^{11,12,24,25,29} and glucose levels,³³ and blood variables.^29,36 Several differences in the response of laboratory animals to stress have been correlated to breed,^2,33 strain,^11,14,37 age,²⁴ and sex.^12,23,24 Some authors²⁴ have noted that female mice show a lower overall stress level due to the modest effect of social competition. Another study¹² showed a different, sex-specific response to hypoxic ventilation in rats, which was higher in male than female rats. In addition, these authors¹² suggested that female ovarian hormones are prime candidates for stress regulation.Whole-body plethysmography is a noninvasive, indirect method of studying respiratory function in conscious, unrestrained animals. This method has been used particularly in pharmacologic and toxicologic studies in diverse animal species including mice,^3,9,14,15,38 rats,^8,20 cats,²² dogs,^16,34 and pigs.¹³ The present study was designed to investigate the extent to which routine procedures affect breathing parameters in a transgenic mouse colony. Male and female mice were exposed to 4 routine procedures that are used for DNA sampling and identification of transgenic mice (ear marking, tail clipping, and tail vein and retroorbital blood sampling). Respiratory parameters were recorded by whole-body plethysmography immediately and at 4, 24, and 48 h after the procedure. Handling and restraint were used as control procedures.     

Progression of Ulcerative Dermatitis Lesions in C57BL/6Crl Mice and the Development of a Scoring System for Dermatitis Lesions

Ulcerative dermatitis (UD) is a common, spontaneous condition in mice with a C57BL/6 background. Although initial lesions may be mild, UD is a progressive disease that often results in ulcerations or debilitating fibrotic contractures. In addition, lesions typically are unresponsive to treatment. Euthanasia is often warranted in severe cases, thereby affecting study outcomes through the loss of research subjects. Because the clinical assessment of UD can be subjective, a quantitative scoring method and documentation of the likely time-frame of progression may be helpful in predicting when animals that develop dermatitis should be removed from a study. Such a system may also be helpful in quantitatively assessing success of various treatment strategies and be valuable to clinical laboratory animal veterinarians. In this 1.5-y, prospective cohort study, we followed 200 mice to monitor the development and course of UD. Mice were examined every 2 wk. A clinical sign (alopecia, pruritus, or peripheral lymphadenopathy) was not identified that predicted development of UD lesions in the subsequent 2-wk period. Once UD developed, pruritus, the character of the lesion (single or multiple crust, coalescing crust, erosion, or ulceration), and the size of the lesion were the only parameters that changed (increased) over the course of the disease. Pruritus was a factor in the rapid progression of UD lesions. We used these findings to develop a quantitative scoring system for the severity of UD. This enhanced understanding of the progression of UD and the quantitative scoring system will enhance the monitoring of UD.Abbreviation: UD, ulcerative dermatitis; S number, scratching number; COL, character of lesionsUlcerative dermatitis (UD) is an idiopathic, spontaneous, debilitating syndrome of laboratory mice that is typically a disease of aged^1,19,43,46 C57BL/6 mice or genetically engineered mice on a C57BL/6 background.^1,19,43,44 Some reports discuss a similar condition in young, weanling mice that presents initially as alopecia.^24,42,44,45 Prevalence rates of UD between 4.1% to 21% have been reported.^1,6,19 Although no etiology has been identified, environmental factors,^{6,19,41,42,44} diet,^{5,29,41,42,46} season,^19,41,43,44 age at weaning,⁴² alopecia,^24,42,44,45 sex,^19,39,41,43 immune complex vasculitis,¹ follicular dysplasia,⁴⁴ lesion location,²⁰ and deficiencies in vitamin A metabolism⁴⁴ have all been implicated as predisposing factors for disease development. In addition, oronasal pain and chronic inflammation may lead to self-mutilation as a result of, initiating an “itch” response.¹⁰ UD is diagnosed by ruling out other causes of dermatitis in laboratory mice, such as fur mites,⁹ infections, fight wounds,¹⁷ strain phenotype,^15,35,40,49 and experimentally induced dermatitis.^4,50 Other diagnostic criteria are based on professional judgment and may include strain (C57BL/6 background),^1,19,44 lesion location (head and dorsal thorax),^1,19,43,44 intense pruritus,^1,19,44 peripheral lymphadenopathy,^6,19,39 and failure to respond to treatment.¹⁹ The rapid progression of UD lesions results in significant morbidity in laboratory mice.^6,19,44 Typically, the lesions progress to large, irregularly shaped, confluent ulcerations on the dorsal cervical and thoracic region.^1,19,39,44 As the lesions heal, contracted scar tissue forms, which can impair species-typical behaviors and mobility.^39,41,43,44 The presence of large dermal ulcerations or debilitating contractures affect animal welfare and typically necessitate euthanasia of affected mice. Although reports on the later stages of UD have been consistent,^{1,19,39,41,43,44} information on the initiation and progression of UD lesions is conflicting. Pruritus,^1,19,44 pain,¹⁰ and genetic predisposition^1,19,43,44 have been implicated as initiators of the disease. Alopecia, pruritus, erythema, and single or multiple(s) crust have all been reported as early signs of the disease.^{1,19,39,42-44} However, the majority of this information has been collected retrospectively, at timed necropsies, or based on anecdotal reports.Scoring systems are useful tools to evaluate clinical diseases in laboratory animals. For example, scoring systems have been published for tumors,^14,28 body condition,^14,28,47 and neurologic phenotype¹³ in mice to aid in assessment of clinical disease severity. Even though the progressive and severe nature of UD typically warrants eventual euthanasia, determining the severity of disease has typically been based on professional judgment,^1,39 subjective scoring,^{12,15,19,40,43,48,49} or postmortem histology.^40,44 A quantitative scoring system for UD in live mice has not been described and could greatly aid laboratory animal veterinarians and researchers in determining the severity of the disease and response to treatment.The purpose of the current study is to investigate clinical parameters that reflect the progression of UD to facilitate management and veterinary care of mice with UD. We followed 200 mice from 3 wk of age until the development of UD to determine the initial signs and progression of UD lesions. We hypothesized that mice will first develop signs of pruritus prior to any clinical lesion. From there, we predicted that clinical lesions will progress stepwise though the following stages of severity, with or without alopecia: (1) excoriations; (2) a single, small punctuate crust; (3) multiple, small punctuate crusts; (4) coalescing crust; (5) erosion; and (6) ulceration. In addition, we hypothesized that a quantitative, validated, and reliable UD scoring system can be created that is based on physical examination parameters that do, in fact, predict development and progression of UD. Having a more thorough understanding of the initiation and progression of ulcerative lesions likely will enhance our ability to predict the outcome for a given mouse and develop earlier end-points for that mouse. Furthermore, use of this scoring system will enable accurate monitoring of UD lesions.     

Effects of Buprenorphine, Meloxicam, and Flunixin Meglumine as Postoperative Analgesia in Mice

C57BL/6NCrl male mice (n = 60; age, 6 to 7 wk) underwent partial hepatectomy or no surgery and were given 1 of 3 analgesics pre- and postoperatively. Food and water consumption, body weight, running wheel activity, locomotor activity, and serum corticosterone concentrations were measured before and after surgery. Mice that were surgically manipulated weighed significantly less on days 1 through 3 after surgery than did mice not manipulated surgically. On the day of surgery, the surgery groups consumed significantly less feed (–1.5 ± 0.35 g) than did nonsurgery groups. There were no differences in water consumption on any day between surgery and nonsurgery groups or among the 3 analgesic groups. For running wheel activity, significant decreases in the surgery groups were seen at day 1 after surgery compared with baseline. Surgery groups that received buprenorphine and meloxicam returned to baseline activity levels on day 2 after surgery. Open-field testing revealed no significant differences in locomotor activity in any groups; however, posttreatment locomotor activity in the buprenorphine nonsurgery group was increased compared with baseline, and posttreatment locomotor activity in the flunixin meglumine surgery group was decreased compared with baseline. Serum corticosterone concentrations were within normal limits regardless of treatment in all groups. Comparison of the overall results indicated that meloxicam and buprenorphine, at the dose given, appear to be suitable postoperative analgesics for partial hepatectomy in mice. Flunixin meglumine at the given dosage (2.5 mg/kg) may not provide adequate analgesia for partial hepatectomy.Abbreviation: NSAID, nonsteroidal antiinflammatory drugLaboratory animals commonly undergo procedures that may cause pain and distress. The Guide for the Care and Use of Laboratory Animals states, “An integral component of veterinary medical care is prevention or alleviation of pain associated with procedural and surgical protocols. Pain is a stressor and, if not relieved, can lead to unacceptable levels of stress and distress in animals.”²⁰ Such deleterious effects, while adversely affecting an animal''s wellbeing, may also confound research data and impede research study outcome. As a result, prevention or recognition and minimization or alleviation of pain and distress are integral components of an animal care and use program. In rats, unrelieved pain and distress after a surgical procedure has been shown to affect locomotor activity, body weight, and food and water intake.^12,31,32 Although mice comprise the majority of research animals, few studies have evaluated analgesia effectiveness in this species. Studies in mice have shown that the lack of postoperative analgesia can affect locomotor activity¹⁵ and food and water intake.¹⁹Locomotor activity testing involving an open field has been used as a method to assess exploratory and locomotor activity in mice.^{17,21,37,38,41,46,49,55} The assessment of unconditioned locomotor behavior in rodents has become one of the most widely used behavioral paradigms to determine the effects of various experimental manipulations ranging from genetic changes to pharmacologic challenges.^41,46 Levels of locomotor activity are measured by using a variety of methods based on the frequency of photocell beam breaks or distance traveled as assessed by video tracking.Running wheel activity has been used in research for a variety of experiments.^8,30,48 Running wheels have been used to study the hypothalamic–pituitary–adrenocortical axis, circadian rhythms, and social-stress-affected sleep in mice.^28,53 This parameter has also been used to monitor voluntary activity levels of mice by measuring distance traveled.^8,48Commonly used analgesics include buprenorphine hydrochloride, flunixin meglumine, and meloxicam. Buprenorphine hydrochloride is used in rodent surgical models^{13,15,22,26,32,45} primarily due to its longer duration of action over other opioid drugs. It is a partial µ opiate agonist and has a maximum biologic effect achieved regardless of the maximal dose given (ceiling effect).^7,34 The duration of effect in most species is 6 to 12 h.⁴⁴ The duration of buprenorphine in mice has been documented as 3 to 5 h.¹⁴ As a controlled substance, buprenorphine is regulated by the Drug Enforcement Administration, which requires strict storage and record keeping.Flunixin meglumine and meloxicam are nonsteroidal antiinflammatory drugs (NSAID) that reduce inflammatory pain by the inhibition of prostaglandin synthesis.^10,42 Because NSAID are not controlled substances, federal licensure and other restrictive measures are not necessary. Although the drugs are considered safe, NSAID-associated gastrointestinal bleeding is listed as a side effect reported with chronic use.⁴² A recent study by our laboratory found that flunixin meglumine did not interfere with embryo implantation when used in mice undergoing embryo transfer.¹⁸ We wanted to further investigate the properties of flunixin meglumine as an analgesic agent in a different mouse surgical model. Because meloxicam is a long-acting NSAID requiring once-daily dosing, we were interested in evaluating the effects of meloxicam in a mouse surgical model.Evaluations of physiologic parameters and results of behavioral tests are often used to assess pain in rodents; however, there are very few reports on assessing pain relief in mice. Various parameters have been used to monitor rodents during the postoperative period, including ethographic scoring,⁶ body weight changes, corticosterone concentrations, and food and water consumption.^{3,12,16,31,32} Our laboratory previously evaluated whether isoflurane anesthesia would alter the body weight, voluntary exercise or open-field locomotor activity of mice administered either flunixin, meloxicam, or buprenorphine. We found that isoflurane anesthesia had no effect on these parameters with any of the 3 analgesics.The purpose of the current study was to evaluate the effects of buprenorphine hydrochloride, flunixin meglumine, and meloxicam in mice after partial hepatectomy. We hypothesized that there would be no differences in physiologic parameters in mice with the use of the 3 analgesics. Partial hepatectomy is a surgical procedure performed frequently in our facility for research studies, and we were interested in investigating the effect of the 3 analgesics in this animal model. These mice were used specifically for this study to refine the analgesic protocol for this surgical procedure. Voluntary running-wheel activity, open-field locomotor activity, and serum corticosterone concentrations were used in conjunction with body weight and food and water consumption as objective parameters for assessment of analgesic effectiveness.     

Assessment of Immune Activation in Mice before and after Eradication of Mite Infestation

Mite infestation of mice remains a persistent problem for many institutions, leading to numerous health problems and creating unknown and unwanted variables for research. In this study, mice with mite infestation demonstrated significantly higher levels of inflammatory cytokines, both at draining lymph nodes (axillary) and systemically, as compared with mice without mites. In addition, histologic evaluation revealed significant inflammation in mite-infested mice. Inflammatory changes were still present in the skin of mice at 6 to 8 wk after treatment, despite absence of detectable infestation at that time. Because these significant and lasting local and systemic changes have the potential to alter research findings, eradication of mites infestations should be an important goal for all institutions.Abbreviation: KC, keratinocyte-derived chemokine; MIP, macrophage inflammatory proteinLaboratory mice can harbor several species of acarids (fur mites), including Myobia musculi, Radfordia affinis, Myocoptes musculinus, and Psorergates simplex.^{11,14,29,40,45} Fur mites are an excluded pathogen in most research facilities, particularly within barrier suites, and in order to control or avoid mite infestations, many facilities, including those with ongoing infestations, will not accept infested animals from outside sources. Such policies can prevent or halt collaborative research between investigators in different institutions because mite infestation is a sporadic or endemic problem in many facilities that house mice under conventional conditions, despite attempts at eradication.^{12,22,25,43,62,69}Mite infestations cause several health problems in mice, including ulcerative dermatitis, amyloidosis, and other immune system alterations.^{2,12,22,27,29-31,37,44,45,61} For example, mite infestations are associated with increased serum concentrations of IgE and IgG in mice.^30,44,48 Alterations in immune responses could alter research data and thereby perhaps alter the associated conclusions.^36,65,66,70 Mice with mite infections often develop dermatitis, which can lead to bacterial infection and additional changes in immune status.^{15,30,31,45,61} Because any pathogenic infection can cause variability and alter basal measures of immune function, clinical chemistry, and behavior in mice, maintaining laboratory rodents in a disease-free state is crucial to their use for the collection of valid research data.⁵¹The eradication of external parasites is a difficult process. Many reports have been published that attempt mite eradication using various drug treatments,^{5-7,17,18,23,24,35,39,41-43,46,47,49,50,57,59,67} with each method having distinct advantages and disadvantages. Some, but not all, of these treatment regimens have been compared directly.¹⁰ The mite life cycle complicates treatment, because eggs and larvae can be less susceptible to drugs than are adult parasites.^2,19,20,55 In addition, mite eggs can contaminate the environment, providing a source for re-infection of treated animals.^20,63,64 Some drugs (for example, ivermectin) have been associated with toxicity and death in mice, especially among specific transgenic lines.^{8,12,28,53,55,69} Other drugs may require frequent or repeated treatment of the mice. Furthermore, the drugs themselves may have properties that alter physiology or immune function in animals.^2,13,60 The development of new veterinary drugs for treatment of parasites has increased the available therapies for rodent acariasis. Compounds such as fipronil and selamectin provide good efficacy against external parasites with limited side effects in mammals.^9,22,68Our facility housed a large colony of mice that occupied several rooms and were infested with Myocoptes musculinus and Myobia musculi. Although the majority of mite-infested mice had mild or no dermatitis, some infected mice had severe dermatitis. The goal of this study was to evaluate the local and systemic immune response in mice infested with mites. To our knowledge, this study is the first to comprehensively compare cytokine levels and histologic findings in mite-infested, treated, and mite-negative mice. We hypothesized that the immune response would be altered in mite-infested mice as demonstrated by significantly elevated cytokine levels in the draining lymph nodes or spleen as compared with mice that had never been infested with mites. In addition, we hypothesized that significant pathologic changes in the epidermis, dermis, and subcutaneous tissues would be present in response to mite infestation.     

Adverse Effects of Vapocoolant and Topical Anesthesia for Tail Biopsy of Preweanling Mice

Tail biopsy of laboratory mice for genotyping purposes has been studied extensively to develop refinements for this common procedure. Our prior work assessed tail vertebral development in different mouse strains (age, 3 to 42 d) and analyzed behavior and activity in mice (age, 21 to 45 d) biopsied under isoflurane anesthesia. To assess the effects of biopsy on preweanling mice, we here evaluated BALB/cAnNCrl mice (n = 80; age, 18 to 21 d) that received topical vapocoolant (ethyl chloride), topical anesthetic (Cetacaine), or isoflurane anesthesia before undergoing a 5-mm or sham biopsy. Control mice did not receive any anesthetic intervention. Regardless of the anesthetic used, acute observation scores indicative of distress were increased at 10 min after biopsy, and locomotor activity was decreased, in biopsied compared with control mice. Acute observation scores at 10 min after biopsy were higher in mice that received ethyl chloride compared with isoflurane or no anesthesia. Microscopic analysis revealed that inflammatory changes in the distal tail remained elevated until 7 d after biopsy and were higher in tails exposed to ethyl chloride. Our findings indicate that vapocoolant, topical anesthesia, and inhaled isoflurane do not enhance the wellbeing of preweanling mice undergoing tail biopsy. Due to the lack of appreciable benefits and the presence of notable adverse effects, using vapocoolants or Cetacaine for this tail biopsy procedure in laboratory mice is unadvisable and we encourage the removal of these agents from institutional tail biopsy guidelines.Abbreviations: DT, distal tail; PT, proximal tailLaboratory mice can be genotyped by analyzing samples derived from multiple sources, including distal tail biopsies,^{4,11,21,25,31,46,60,61,65,74} blood,^{4,14,17,28,33,57} buccal^36,53,74 or rectal⁴⁵ swabs, fecal pellets,¹⁰ hair,^8,57,64 and toe^12,49 and ear pinnal biopsies.^17,24,25,62 Of all available options, distal tail biopsy remains the most widely used method for obtaining tissue from both preweanling and older mice, likely due to superior aspects of DNA quantification, accuracy, reproducibility and practicality of this source.^9,24 The tail biopsy procedure has not been linked to measurable outcomes indicative of prolonged distress or considerable pain, beyond the stress associated with capture and restraint.^3,18,29,55 Regardless of genetic background, the tail vertebrae of all mice have calcification and ossification, with mature vertebral endplates, within the distal 5 mm of tail prior to typical weaning age (21 d).³⁰ Therefore, contemporary recommendations are to harvest a minimal amount of distal tail tissue (less than 5 mm) from mice, to limit discomfort from the procedure and maximize DNA yield from highly cellular cartilaginous (nonossified) tissue.^24,30,60,61In our earlier work, we advocated for the application of anesthesia or topical analgesia for biopsies in weanling mice.³⁰ We elected to determine whether isoflurane anesthesia is an appropriate refinement for added comfort during the procedure of tail collection.²⁹ Due to its high safety profile for both the user and patient, isoflurane is one of the most commonly used inhalant anesthetics in the laboratory animal setting; therefore, isoflurane is frequently recommended in guidance regarding tail biopsy procedures. Unexpectedly, we demonstrated that brief (less than 1 min) exposure of isoflurane anesthesia to mice aged 21 to 45 d. resulted in significant locomotor and behavior deficits that persisted until 5 h postinhalation.²⁹ In addition, measurable benefits (for example, diminished anxiety-like responses to tail biopsy and normal activity levels after isoflurane exposure) were not evident when we compared sham and biopsied mice. A growing body of literature has identified additional undesirable outcomes of isoflurane on neonatal and young adult mice. This inhalant has been shown to have neurodegenerative effects and can impact cognitive function, spatial learning, and memory.^{15,19,37,43,47,67} Continued approaches to management of perceived discomfort after tail biopsy in laboratory mice have provoked controversy regarding the type, timing, and application of anesthesia and analgesia for the procedure. Therefore, for potential pain relief from biopsy, the topical application of different types of local anesthetics (vapocoolants, liquid analgesics, and alcohol-based agents) prior to tail and digit biopsy has been a recent topic of examination.^{23,38,50,51,58,59}To extend our assessment of refinements to the current practices for tail biopsy in mice, we compared inhaled isoflurane with topical treatments, including the vapocoolant spray ethyl chloride and the topical anesthetic Cetacaine, both of which are recommended specifically by the NIH and other institutions^{34,39,56,69,70} as anesthetic options for young mice. We determined whether local anesthesia was effective at mitigating the acute behavioral response to tail biopsy in preweanling (age, 18 to 21 d) mice, as well as whether there were any influences on locomotor activity. In addition, we assessed effect of these topical therapies on the tail by performing histologic analyses of biopsied tissues.     

Soiled Bedding Sentinels for the Detection of Fur Mites in Mice

Identification and eradication of murine fur mite infestations are ongoing challenges faced by many research institutions. Infestations with Myobia musculi and Myocoptes musculinus can lead to animal health problems and may impose unwanted research variables by affecting the immune and physiologic functions of mice. The purpose of this study was to evaluate the utility and efficacy of soiled bedding sentinels in the detection of fur mite infestations in colony mice. Female young-adult CRL:CD1(ICR) mice (n = 140) were exposed over a 12-wk period to various volume percentages of soiled bedding (11%, 20%, 50%, and 100%) from fur-mite–infested animals. Mice were tested every 2 wk with the cellophane tape test to identify the presence of fur mite adults and eggs. At the end of 12 wk, all mice exposed to 11%, 20%, and 50% soiled bedding tested negative for fur mites. One of the 35 mice (3%) receiving 100% soiled bedding tested positive for fur mites at the end of the 12-wk follow-up period. These findings suggest that the use of soiled bedding sentinels for the detection of fur mite infestations in colony mice is unreliable.Ectoparasite infestations present an ongoing threat to barrier facilities. Murine acariasis in laboratory mice frequently is caused by Myobia musculi, Myocoptes musculinus, and Radfordia affinis.^{1,13,17,40,41} These infestations can be challenging to identify and control and often lead to animal health problems and research complications. For this reason, many institutions strive to exclude these parasites from their barrier facilities.^1,17,18,41 Infestations can further compromise ongoing research by disrupting collaboration with institutions affected by sporadic or endemic mite infestations in their facilities.¹⁸Myocoptes musculinus is the most common fur mite identified among laboratory mice, although mixed infections with Myobia musculi are common.¹⁷ The life cycles of Myocoptes and Myobia are 14 and 23 d, respectively.^2,17 Myobia mites most frequently are found to inhabit the head and neck of mice, whereas Myocoptes are reported to have a predilection for the back, ventral abdomen, and inguinal regions.^2,17 Mite infestations in live animals are often diagnosed by using cellophane tape tests.^5,14,25 A clear piece of cellophane tape is pressed against the fur of the mouse, affixed to a slide, and examined microscopically for the presence of eggs or adult mites. Pelage collection and examination and skin scraping are 2 other common diagnostic methods. These tests have been shown to have increased sensitivity when compared with the tape test, but they have the disadvantage of requiring an anesthetized or recently euthanized animal.^2,5,17Fur mites feed on the superficial skin tissues and secretions of the animals they infest.^1,2,17 Mite infestations in mice have been associated with numerous health problems. Common clinical manifestations of acariasis include alopecia, pruritis, and scruffiness.^{1,2,10,15,17-20,22,26,31,42,44} Severe health problems including ulcerative dermatitis, hypersensitivity dermatitis, and pyoderma can develop also.^1,2,10,17,41 Infested mice may also be prone to secondary infections, reduced life span, and decreased body weight.^2,17,42 Several studies have analyzed the potential research complications associated with murine acariasis.^{10,15,18-20,22,26,31,42,44} Mite infestations have been shown to cause elevations in IgE, IgG, and IgA levels; mast cell degranulation; increased levels of inflammatory cytokines; and lymphocytopenia.^{18-20,22,26,31,44} The changes in the immunologic function of affected mice can persist even after mite eradication.¹⁸Multiple chemical treatment modalities have been proposed for the eradication of fur mites in infested animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} Conflicting information exists regarding the success of many of these treatment regimes. In addition, several of the proposed treatments have been associated with toxicity, adverse health effects in mice, and alterations in the physiologic or immune function of the animals.^{2,3,5,8,12,14,17,25,29,30,32,36,43} The complications associated with identifying an effective treatment for murine acariasis while minimizing toxicity and the introduction of unknown research variables highlight the importance of rapid and effective detection of mite infestations in barrier facilities.Many institutions rely on soiled bedding sentinels for their primary source of information on colony health status.^9,21,33,35 Several studies have demonstrated the efficacy of soiled bedding sentinels to detect common murine pathogens such as mouse hepatitis virus, mouse norovirus, Helicobacter spp., and pinworms.^{4,7,24,28,37,38} However, not all pathogens are easily transmitted through soiled bedding exposure. Agents that are not routinely identified through soiled bedding sentinels include those that are shed in low numbers, are susceptible to environmental factors, or are not easily transmitted through the fecal–oral route.^6,21,33 Examples of pathogens that are not easily transmitted or detected through soiled bedding exposure include mouse Sendai virus, Pasteurella pneumotropica, lymphocytic choriomeningitis virus, and cilia-associated respiratory bacillus.^7,9,11,16,35 In addition, the sensitivity of soiled bedding sentinel programs varies with the number of animals affected within the colony.^27,38In 2008, our institution faced a fur-mite outbreak that affected more than 25 rooms in a single barrier facility. Animals positive for Myobia musculi, Myocoptes musculinus, or both were identified through either health check requests for itching and scratching animals and by testing of animals scheduled for export to other institutions. Despite the extent of this outbreak, the soiled bedding sentinels in all mite-positive rooms consistently tested negative on cellophane tape tests for fur mites.To our knowledge, only one study has specifically examined the efficacy of soiled bedding sentinels in the detection of fur mites in mice.³⁴ A separate study, examining the transmission of mouse hepatitis virus to soiled bedding sentinels,³⁸ demonstrated that 75% of cages (3 of 4) exposed to soiled bedding from colony animals tested positive for fur mites after 19 wk of exposure. That previous study used 8 cages of 12 mice each; 4 cages received soiled bedding from colony animals, whereas the other 4 cages received clean nonsoiled bedding. In that study,³⁸ 56.3% of colony mice were known to be mite-positive. Other literature suggests that spread of mites to naïve animals requires direct contact and that soiled bedding does not serve as an effective mechanism for transmission.^1,17,23,39 However, we were unable to identify any research or experiments that substantiated these conclusions.The purpose of the present study was to evaluate whether CRL:CD1(ICR) mice housed in static microisolation caging on soiled bedding from mice with Myobia and Myocoptes infestations can be used as sentinels for the detection of fur mites and to determine how the efficacy of these soiled bedding sentinels for fur-mite detection varies with the prevalence of fur-mite infestation among colony animals.     

Prevalence of Murine Helicobacter spp. Infection Is Reduced by Restocking Research Colonies with Helicobacter-Free Mice

Most academic research colonies of mice are endemically infected with enterohepatic Helicobacter spp. (EHS). We evaluated EHS prevalence in surveillance mice before and after a 10-y period of requiring that imported mice be free of EHS by embryo transfer rederivation or purchase from approved vendors. In 2009, composite fecal samples from CD1 surveillance mice representing colony health in 57 rooms located in 6 facilities were evaluated for EHS infection by using PCR assays. Fecal samples were screened with primers designed to detect all known EHS, and positive samples were further assayed by using primers specific for H. hepaticus, H. bilis, H. rodentium, and H. typhlonicus. Most EHS were detected in surveillance mice within the first month of dirty bedding exposure, with prevalence ranging from 0% to 64% as monoinfections or, more commonly, infections with multiple EHS. Compared with 1999 prevalence data, EHS remained endemic in colonies importing the lowest number of EHS-free mice. EHS were absent or the prevalence was greatly reduced in colonies receiving the highest percentage of EHS-free mice. This study demonstrates that the management decision to require exclusive importation of EHS-free mice reduced EHS prevalence on an institutional scale without intensive labor and expense associated with other techniques or interference with research objectives.Abbreviation: EHS, enterohepatic Helicobacter spp.; ET, embryo transfer; Hb, H. bilis; Hh, H. hepaticus; Hm, H. mastomyrinus; Hr, H. rodentium; Ht, H. typhlonicusEnterohepatic Helicobacter spp. (EHS) infections are endemic in the majority of research mouse colonies. In 2007, 84% of mice shipped from academic institutions worldwide for embryo transfer (ET) rederivation at our institution were PCR-positive for EHS. H. hepaticus (Hh) was detected in 64% of the mouse shipments either as a monoinfection or in combination with other EHS including H. bilis (Hb), H. rodentium (Hr), H. typhlonicus (Ht), and H. mastomyrinus (Hm).³⁰ Although EHS generally cause subclinical infection in immunocompetent mice, opportunistic infections have the potential to confound experimental data in mouse models.^9,17,34 Importantly, chronic EHS infection in immunodeficient and select inbred strains of mice can induce liver¹⁰ and lower bowel carcinoma,¹³ typhlocolitis, and rectal prolapse,^16,21,28 and reduce reproductive performance.²⁵ In addition, EHS-induced inflammatory responses may alter host immune responses to unrelated experimental infections (for example, promoting elevated systemic IFNγ responses).^3,20Key challenges to eradication of EHS from rodent colonies are determining infection status, eliminating endemic infections, and instituting management practices that prevent reinfection. EHS are disseminated through fecal–oral transmission within a colony and are transmissible to surveillance mice through dirty-bedding exposure.^1,19,24,32 For routine surveillance, PCR assay of feces or cecal mucosal scrapings for genus-specific Helicobacter 16S rRNA genes is the most efficient means of detecting EHS infection, with speciation (if desired) of positive results by culture, restriction fragment length polymorphism analysis, species-specific PCR, or sequence analysis.³⁴ In 1999, as determined by species-specific PCR assays of cecal scrapings from 59 surveillance mice exposed to dirty bedding from colony mice in 26 rooms representing 4 mouse facilities, EHS were endemic on our campus, with prevalence in surveillance mice of 41% for Hh, 82% for Hr, and 6% for Hb.³² Husbandry practices used to minimize cage-to-cage transmission of EHS included microisolation caging, sanitized forceps to transfer mice, and a cage change order from known Helicobacter-free mice to mice of unknown or known EHS infection status (that is, clean to dirty traffic flow of personnel and equipment).³² Although EHS eradication potentially could be accomplished campus-wide by using labor-intensive antibiotics^7,15 and cross-fostering,^4,29,31 we hypothesized that a more cost-effective approach, without confounding experimental data, would be to restrict importation of mice to EHS-free sources. Vendors were screened to establish that production colonies were SPF for EHS, and a new requirement was instituted for embryo transfer (ET) rederivation of mice obtained from random sources, typically other academic institutions, replacing traditional quarantine practices. This study used PCR data from 1999 and 2009 to evaluate the success of this approach, which was defined as a marked decrease in the prevalence of EHS infection over time.     

Testing Alternative Surface Disinfection Agents for Zebrafish (Danio rerio) Embryos

Pathogen transmission into zebrafish colonies is controlled through vigilant biosecurity practices. One such practice is embryo surface disinfection, which often uses sodium hypochlorite. However, if sodium hypochlorite is used at an inappropriate pH, concentration, or exposure time, zebrafish embryos can experience significant mortality and morbidity. Reagent-grade sodium hypochlorite is often used for embryo surface disinfection because commercial-grade sodium hypochlorite has additional ingredients that may have deleterious effects on the embryo. In addition, chlorine dioxide and the combination of sodium chloride and potassium peroxymonosulfate (SCPP) are effective equipment disinfectants; however, the effects of these chemical agents on zebrafish embryos during surface disinfection are unknown. In this study, we exposed strain 5D zebrafish embryos (ages, 6 and 24 h after fertilization) to 4 chlorine-containing agents (reagent-grade sodium hypochlorite [bleach], commercial-grade sodium hypochlorite [bleach], SCPP, and chlorine dioxide) at either 50- or 100- ppm for 5 or 10 min. All groups were evaluated at 5 d after fertilization for survival, hatching rate, and morphologic defect rate. The experimental group with the highest survival and hatching rates and the lowest morphologic defect rate was the 24-h postfertilization embryos exposed to 50 ppm SCPP for 5 min. The survival, hatching rate, and defect rate did not differ significantly among age-matched controls; however, the hatching rate after exposure to 50 ppm SCPP was significantly higher than that of embryos exposed to 50 ppm reagent-grade sodium hypochlorite for 5 min (100% compared with 23% respectively). SCPP solution may provide an alternative surface disinfectant for zebrafish embryos because it maximizes survival and hatching rates and minimizes morphologic defect rates. However, in vivo efficacy against common zebrafish pathogens requires further testing. Use of chlorine dioxide at 50 ppm or greater is not recommended for zebrafish embryo surface disinfection due to significant mortality among 6 and 24 h postfertilization embryos.

The use of zebrafish (Danio rerio) as a research model is steadily on the rise. According to a PubMed search, the number of publications with ‘zebrafish’ as the keyword has increased from fewer than 500 hits in 1997 to more than 4000 hits in 2020. Over the last 20 y, zebrafish have become an ideal animal model to study embryonic development and mutagenesis due to their transparent embryonic chorion and external fertilization.⁸ Zebrafish have also been used as a model for human diseases in multiple fields of study, including oncology, musculoskeletal disorders, and biologic processes, such as tissue regeneration.⁸In the zebrafish scientific community, husbandry practices are implemented across institutions to minimize the introduction of opportunistic pathogens. As part of importation practices and inhouse breeding, researchers commonly perform embryo surface disinfection to reduce the viability of potentially pathogenic organisms, such as the bacterial agent Mycobacteria spp. and microsporidia Pseudoloma neurophilia, which can reside on the chorion, affecting the health of the current colony and decreasing embryo survival.^3,6,15,16 When considering disinfectant use to optimize the effectiveness of embryo surface disinfection protocols, many factors have to be weighed, including targeted zebrafish pathogens, germicidal effects of the agent, and the age and line of zebrafish embryos. Several Mycobacteria spp. have been identified as common pathogens that can cause disease outbreaks in zebrafish colonies.⁴ Mycobacteriosis has a variable disease manifestation that ranges from subclinical to severe epizootic outbreaks with significant colony mortality.⁵ Consequently, successful elimination of Mycobacteria spp. from the embryo’s surface has been the focus of most zebrafish embryo surface disinfection studies. Currently, many zebrafish embryo surface disinfection protocols use sodium hypochlorite.^4,12,24Sodium hypochlorite, the active ingredient in bleach, has germicidal effects that vary based on concentration, exposure time, and pH. Recent publications have shown that concentrations greater than 100 ppm are required to reduce survival of various chlorine-resistant organisms, such as some Mycobacteria spp. and P. neurophilia.^5,8,10 However, these higher concentrations of sodium hypochlorite can also negatively affect embryo survival and increase the incidence of chlorine-associated developmental malformations and embryo mortality.¹² Although one study used unbuffered chlorine solutions (pH 8.0 to 9.0) at 100 ppm for 10 min for embryos at 6 h postfertilization (HPF) of age and 5 min for 24-HPF embryos,¹² solutions with chlorine concentrations below 100 ppm are commonly used during surface disinfection to maximize embryo survival.Sodium hypochlorite is available as commercial-grade and reagent-grade products at various concentrations. Many zebrafish embryo surface disinfection protocols use reagent-grade bleach.^12,19 Reagent-grade solutions have ingredients of high purity and are typically solutions or dilutions of American Chemical Society-grade products. Commercial-grade products (that is, chlorine disinfectants) typically contain additional ingredients that may be of lower quality than reagent-grade components. In addition to sodium hypochlorite, some chlorine disinfectants also contain sodium chloride, sodium carbonate, sodium chlorate, sodium hydroxide, and sodium polyacrylate. These ingredients, which have yet to be studied specifically in an embryo disinfection protocol, may have deleterious or toxic effects on embryos.Aside from specific brands of disinfectants, other chlorine-containing disinfectants have different formulations. Chloride dioxide and the combination of sodium chloride and potassium peroxymonosulfate (SCPP) both reduced the number of relative bacterial light units when aquatic fishnets were soaked in solution for at least 5 min.^5,6 These varied formulations might allow embryos to tolerate higher concentrations of the chloride-containing solutions with lower incidence of morphologic defects and death. However, to our knowledge, studies using alternative agents for zebrafish embryo surface disinfection have yet to be investigated.Here we evaluated the toxicity of 4 formulations of chlorine-containing disinfectants in zebrafish embryos: reagent-grade sodium hypochlorite (bleach), commercial-grade bleach, chlorine dioxide, and SCPP solutions. We hypothesized that commercial-grade bleach would cause more embryo mortality and malformations than reagent-grade bleach. We also hypothesized that both chlorine dioxide and SCPP solutions would be at least as effective as reagent-grade bleach but without causing an increase in mortality or malformations.     

Efficacy and Safety of 5 Anesthetics in Adult Zebrafish (Danio rerio)

Although the safety and efficacy of tricaine methanesulfonate (MS222) for anesthesia of fish are well established, other anesthetics used less commonly in fish have been less extensively evaluated. Therefore, we compared gradual cooling, lidocaine hydrochloride (300, 325, and 350 mg/L), metomidate hydrochloride (2, 4, 6, 8, and 10 mg/L), and isoflurane (0.5 mL/L) with MS222 (150 mg/L) for anesthesia of adult zebrafish. The efficacy and safety of each agent was evaluated by observing loss of equilibrium, slowing of opercular movement, response to tail-fin pinch, recovery time, and anesthesia-associated mortality rates. At 15 min after anesthetic recovery, we used a novel-tank test to evaluate whether anesthetic exposure influenced short-term anxiety-like behavior. Behavioral parameters measured included latency to enter and number of transitions to the upper half of the tank, number of erratic movements, and number of freezing bouts. Behavior after anesthesia was unaltered regardless of the anesthetic used. Efficacy and safety differed among the anesthetics evaluated. Gradual cooling was useful for short procedures requiring immobilization only, but all instrumentation and surfaces that come in contact with fish must be maintained at approximately 10 °C. MS222 and lidocaine hydrochloride at 325 mg/L were effective as anesthetic agents for surgical procedures in adult zebrafish, but isoflurane and high-dose lidocaine hydrochloride were unsuitable as sole anesthetic agents due to high (30%) mortality rates. Although MS222 remains the best choice for generating a surgical plane of anesthesia, metomidate hydrochloride and gradual cooling were useful for sedation and immobilization for nonpainful procedures.Abbreviation: MS222, tricaine methanesulfonateSeveral publications review anesthesia in fish.^1,16,20 Buffered tricaine methanesulfonate (MS222) is the anesthetic typically used to provide surgical level anesthesia in zebrafish (Danio rerio),^8,15 and most researchers use MS222 at a concentration of 164 mg/L.^15,25,26 However, reported side effects of MS222 that are dependent on dose and exposure duration include respiratory acidosis, cardiac depression, cardiac failure, and death.^10,19,21,24 MS222 increases blood glucose, plasma cortisol, lactate, and blood chemistry values in zebrafish and other species.^1,6,23,24 Because of these and other undesirable side effects of MS222, other anesthetics have been used to anesthetize adult zebrafish.^{1,5,6,10,15,19,21,24}The use of several less common anesthetic agents has been described in ornamental fish, including zebrafish.¹⁶ Lidocaine hydrochloride is a local anesthetic that has previously been used for anesthesia in medaka (Oryzias latipes),^17,23 in which it produced a surgical plane of anesthesia. Metomidate hydrochloride is an imidazole-based nonbarbiturate hypnotic that is used to sedate fish for handling and to reduce the trauma and stress associated with transportation.^{1,2,4,5,12,14,16,22} Isoflurane is a hydrocarbon that can be used as an anesthetic immersion bath.^16,23 Alone, isoflurane provides variable anesthesia and analgesia in fish, but when combined with MS222, it has been reported to produce a surgical plane of anesthesia for more than 20 min in zebrafish.^10,16 Gradual cooling has been shown to be useful for short-term procedures, such as intraperitoneal injections in adult zebrafish.^6,13,15Little information is available regarding the suitability of these agents as anesthetics for invasive surgical procedures in the zebrafish. Therefore, we investigated the effects of lidocaine hydrochloride, metomidate hydrochloride, isoflurane, and gradual cooling on zebrafish because the anesthetic solutions require only a simple one-step preparation and because these agents potentially could provide a longer duration of surgical anesthesia than does MS222, with shorter induction and recovery times. This study is the first to compare multiple uncommon anesthetic agents in the adult zebrafish. We hypothesized that lidocaine hydrochloride, metomidate hydrochloride, and gradual cooling would be as efficacious as MS222 in providing surgical level anesthesia, but isoflurane alone would be ineffective, given that previous literature suggests this agent may be beneficial only in combination with MS222.⁹In addition, we performed anxiety-like behavior tests to determine the effects of these anesthetic agents on the behavior of adult zebrafish. We sought to determine whether anesthesia-related behavioral effects in zebrafish are similar between anesthetic agents, and we hypothesized that anxiety-like behavior would be altered in fish recovering from isoflurane anesthesia, similar to what is seen in mice.⁸     

Euthanasia Method for Mice in Rapid Time-Course Pulmonary Pharmacokinetic Studies

To develop a means of euthanasia to support rapid time-course pharmacokinetic studies in mice, we compared retroorbital and intravenous lateral tail vein injection of ketamine–xylazine with regard to preparation time, utility, tissue distribution, and time to onset of euthanasia. Tissue distribution and time to onset of euthanasia did not differ between administration methods. However, retroorbital injection could be performed more rapidly than intravenous injection and was considered to be a technically simple and superior alternative for mouse euthanasia. Retroorbital ketamine–xylazine, CO₂ gas, and intraperitoneal pentobarbital then were compared as euthanasia agents in a rapid time-point pharmacokinetic study. Retroorbital ketamine–xylazine was the most efficient and consistent of the 3 methods, with an average time to death of approximately 5 s after injection. In addition, euthanasia by retroorbital ketamine–xylazine enabled accurate sample collection at closely spaced time points and satisfied established criteria for acceptable euthanasia technique.Matching the attributes of the euthanasia method to different applications and study designs is an important consideration in selecting the euthanasia method for an in vivo study. Methods of euthanasia should adhere to the AVMA Guidelines on Euthanasia, ACLAM Task Force Guidelines on Euthanasia, and other references reiterating similar principles.^1,3,7,11,16, According to these guidelines, an acceptable euthanasia method is characterized by: (1) rapid loss of consciousness; (2) reliability; (3) safety of personnel; (4) irreversibility; (5) compatibility with study requirements; (6) minimal negative emotional effect on observers and personnel; and (7) compatibility with subsequent evaluation, examination, or use of tissue sample.^1,7 The purpose of the current set of studies was to compare commonly accepted means of euthanasia in mice with a novel method: retroorbital ketamine–xylazine euthanasia.Ketamine–xylazine is a commonly used combination for anesthesia and euthanasia in mice.^4,14,28 In our experience, ketamine–xylazine is most often given intraperitoneally as an anesthetic combination. When used for euthanasia purposes, typically an overdose of the anesthetic is administered intraperitoneally followed by a secondary means of euthanasia, such as exsanguination, thoracotomy, or cervical dislocation.For intravenous drug administration in mice, the retroorbital injection method is a technically simple, easily learned, reproducible, and rapid procedure, particularly as compared with intravenous tail vein dosing. Retroorbital injection has been shown to be interchangeable with the intravenous tail vein injection technique when parenteral access is desired in the mouse.^{9,10,12,18,20,22,29} The variability, technical demand, and other negative aspects of intravenous tail vein dosing in mice make the retroorbital method desirable.^{9,10,12,18,20,22,29} To minimize any potential associated pain or distress, retroorbital injections typically are given to anesthetized mice.¹⁵ This practice is feasible when mice are intended to recover after the injection; however, use of the retroorbital technique for euthanasia has not been documented. One goal of the current study was to demonstrate the adherence of retroorbital injection of ketamine–xylazine to the previously stated principles regarding euthanasia, with emphasis on the humaneness of the technique.We developed the retroorbital ketamine–xylazine euthanasia technique to support rapid time-course mouse pulmonary pharmakokinetics studies in drug development. For these types of studies, which involve direct delivery of compounds to the lungs, intratracheal dosing is often the preferred method because of its reproducibility, reliability, and translatability to the clinic setting.^{19,21,23,24,26,27} One key factor that affects the efficacy and potency of these drug candidates is their residence time in the lungs. Pharmacokinetics studies focus on the distribution, clearance, and metabolism of chemical or drug entities that are introduced into the body. Pulmonary pharmacokinetics parameters are often assessed in serum, lung tissue, and bronchoalveolar lavage fluid. Because of rapid local clearance of the compounds, the quality of these data relies on the precision of sample collection, particularly from early time points that often are within minutes of each other.When pulmonary pharmacokinetics studies are performed in mice, groups of animals are euthanized at specific time points after dosing to enable collection of tissue samples for concentration measurements over a time course. The method of euthanasia chosen for these studies must be nontraumatic and incorporate the attributes of rapid onset, ease of execution, reproducibility, and the ability to preserve tissues and samples. Currently, pulmonary pharmacokinetics studies use a variety of euthanasia techniques, including CO₂ exposure, intraperitioneal barbiturate overdose, cervical dislocation, and decapitation.^13,19,23,26 These methods, when used in rapid time-point pharmacokinetics studies, have attributes that can confound the results.^6,8,11,17 Cervical dislocation and decapitation result in rapid death but are traumatic in nature. These techniques damage the trachea and cervical region, making it difficult or impossible to lavage the lungs after the procedure. These methods also confound the results by causing hemorrhage into various tissues and contaminating the lung tissue. Other euthanasia methods, such as CO₂ exposure and intraperitoneal pentobarbital overdose are less traumatic, but the time to death is delayed and variable, thus preventing precise timing for tissue harvest.^2,5,8,11,17 Therefore, the goals of this study were to examine the utility of a novel method of euthanasia, retroorbital administration of ketamine–xylazine for euthanasia of mice. We here demonstrate its positive effect on the quality of in vivo data, and show that retroorbital administration of ketamine–xylazine meets the criteria for acceptable euthanasia. Retroorbital administration of ketamine–xylazine for euthanasia of mice is ideal for pulmonary pharmacokinetics studies and for any study needing a rapid, nontraumatic means of euthanasia.     

Effect of Enrichment Devices on Aggression in Manipulated Nude Mice

Agonistic behavior in group-housed male mice is a recurring problem in many animal research facilities. Common management procedures, such as the removal of aggressors, are moderately successful but often fail, owing to recurrence of aggressive behavior among cagemates. Studies have incorporated enrichment devices to attenuate aggression, but such devices have had mixed results. However, these studies did not include research manipulations when assessing the benefits of various enrichment devices. We obtained 100 male athymic nude mice and studied the efficacy of various enrichment devices, including cotton squares, paper rolls, shredded paper, nylon bones, and a mouse house and wheel combination in the reduction of fighting during an ongoing study that involved randomization followed by prostate and intratibial injections. Groups were evaluated according to a numerical grading system for wound assessment. Examination of the data revealed that the enrichment devices had no effect on the presence of wounds, thus none of the devices tested affected fighting in nude mice. However, when mice began experimental use, fight wounds increased significantly at cage change and after randomization, reflecting a disruption of existing social hierarchies. Therefore, in the context of an actual research study that involves common manipulations, the specific enrichment device had less effect on aggression in male nude mice than did the destruction and reconstruction of social structures within each group.Much is known about the normal behavior of mice in captivity. Mice prefer the company of conspecifics and are highly social.^6,29 Singly housed mice may exhibit signs of isolation syndrome that include stereotypies, anxiety, restlessness, and aggression; this syndrome can result in physiologic changes that alter the immune system and result in increased pathology.^15,53,63,64 Therefore, mice should be group-housed unless there is a strong scientific justification for doing otherwise.Mice are a territorial species.⁵⁶ In the wild, groups generally live within a territory and consist of social units composed of a dominant male, several females, their progeny, and, occasionally, subordinate males.^{17,26,35,36,58} A single dominant mouse often controls a self-identified territory, which often has a range of several hundred square meters.¹⁹ Other male mice respond to olfactory cues, primarily urine marking within and around the borders of the territory, that are dispersed by the dominant mouse and therefore avoid the territory and the risk of attack from its possessor.²⁶ Nevertheless, even when living in a small, limited space, such as a cage, male mice prefer the companionship of a dominant male over a solitary existence.⁵⁸ This behavior further supports the importance of social housing for the wellbeing and social structure development of mice.The importance of social housing for the wellbeing and social structure development of mice has been well documented.^{5,15,29,53,58,63,64} The social organization of mice in the wild varies between 2 types: dominance hierarchy and territorial organization.¹³ When balanced, dominance hierarchies mitigate additional aggression and allow for priority access for the most-dominant animals to limited resources such as food, territory, and mates.³ In addition, dominance hierarchies serve to prevent high levels of intermale aggression, which may be detrimental to the social group in the form of reduced foraging and reproduction and increased injury and death.³Therefore, in the laboratory setting, housing in groups meets the needs of the mice; however, the typical housing of multiple males or multiple females within the confines of a cage may frustrate their natural preference for small family groups and their attempts to develop the requisite social structure.^4,5,29,56 This situation results in agonistic behavior, one component in the repertoire of normal mouse behaviors that is demonstrated in the wild but that has become amplified and extreme.Agonistic behavior is any social behavior related to aggression, including fighting, defending oneself, reconciliation, and avoidance.^11,12,22,47 Aggression has several functions: the development and maintenance of dominance hierarchies, acquisition of limited resources, and competition for mates. The primary causes of direct physical conflict in mice are defense of territory by males and defense of pups by females.⁴⁰ Perhaps most importantly, physical aggression is an essential means of ascension to and preservation of male dominance status in a territorial group.^11,12,22,47 Clearly, the limited territory within the cage environment impedes access to the typical channels for the resolution of aggression. As a result of the limited living space, a dominant mouse has easy access to the subordinate, and the subordinate has no means of escape from the odor of marking scents deposited by aggressors or from direct attack by an aggressor. Furthermore, subordinates are prevented from demonstrating typical behaviors of retreat during conflicts with conspecific cagemates.^5,26,41 The inability to control the social and physical environment can lead to an escalation in aggressive behavior.⁵⁸A mouse''s proclivity for aggression is affected by past experiences. Past combative experiences result in learning, which influences future aggression; for example, aggressive behavior decreases after the experience of defeat.⁴⁴ Perhaps reduced experience with aggression limits the learning of aggressive behaviors. Consequently, if all studies could use siblings and mice that are familiar with each other and have established dominance hierarchies, aggression would likely be reduced. However, in many research studies, mice are assigned randomly to new groups, causing them to be removed from a cage and group of familiar mice and placed into a cage of unfamiliar mice.Ultimately, to meet the highest standards of animal welfare and produce reliable scientific data, a solution must be found to minimize aggression in mice within the confines of the research environment. Currently, the routine response to the observation of wounded mice in a cage is to remove the aggressor. This practice temporarily resolves the existing issue, but aggression often recurs as social hierarchies are reestablished. In addition, relocating mice involves an investment of resources, including time, effort, and increased housing costs for each additional cage.Intermale aggression is affected by modifications in the cage environment,²⁰ and therefore, one possible solution for agonistic behavior in mice—and more specifically, fighting—is environmental enrichment. Environmental enrichment is the modification of the microenvironment of the mouse through the addition of social groupings and physical structures to reduce undesirable behavior and stress while promoting and facilitating normal, positive species-typical behaviors with the goal of improving animal wellbeing.^8,42,52,64 Enrichment must address physiologic needs as well as behavioral needs of mice, which include social behavior, foraging, burrowing, nest construction, and exploration.⁸ Ideally, appropriate devices should satisfy other common behaviors also, including exercising, burrowing, foraging, digging, gnawing, climbing, and nesting.^5,29 Commonly used devices include manipulanda, nesting materials, shelters, tubes, platforms, running wheels, and enlarged cage space.^8,42 However, the efficacy of enrichment to attenuate agonistic behavior in mice remains unclear.Environmental enrichment studies have had varied outcomes regarding the effect of enrichment on aggression in mice. Many studies have demonstrated an increase in aggression resulting from enrichment.^{7,10,20,21,23,27,38,53,65} In contrast, multiple other studies have found that environmental enrichment reduces aggression in mice.^{2,4,14,45,55,57-59} Still other studies have demonstrated no effect of enrichment on aggression.^{21,38,55,57,58}Numerous explanations can be provided for the different outcomes in environmental enrichment studies. Variations are likely due to differences in mouse strain, age, or sex; population density in the experimental cages; variety of enrichment device or duration of enrichment; assessment criteria for aggression; and experimental design.^37,58 For instance, enrichment has a greater effect on males and variable effect on different strains, such that the choice of sex and strain in a study could have a significant bearing on results.^{15,38,53,61,64,65} One thing that studies of the effects of enrichment on aggression in male mice have in common is that they have not taken into account the use of strategies to ameliorate aggression during actual research studies and mouse manipulation, particularly after randomization of mice.We sought to fill this gap in knowledge by examining the effect of various enrichment devices on nude mice that proved to be aggressive during a research study involving randomization and surgery at our institution. To this end, we compared the efficacy of various enrichment devices with cotton squares, the standard enrichment device used in our facility. Previous research has indicated that, compared with other enrichment items, nesting material is most preferred by mice and effectual, but whether this preference and efficacy remains after the stressors experienced during a research study is unknown currently.^42,50,54 We hypothesized that alternative enrichment devices would prove more beneficial to reduce aggression in nude mice before and after manipulation.     

Effects of Maternal Fenbendazole on Litter Size,Survival Rate,and Weaning Weight in C57BL/6J Mice

Fenbendazole is a broad-spectrum benzimidazole commonly used in laboratory animal medicine as an anthelmintic for elimination of pinworms. This drug is generally regarded as safe, with minimal side effects. Some data in rodent species indicate multiple physiologic effects of fenbendazole, including changes in immune parameters and behavior, but no studies to date have evaluated possible effects on reproduction in mice. The purpose of the current study was to determine the effects of several treatment regimens of fenbendazole on reproductive parameters in C57BL/6J mice. Uninfected mice were given fenbendazole-treated feed continuously or every other week until pups were born or weaned. This treatment also was combined with environmental decontamination. No significant differences in litter size, survival rate, or weaning weight were detected between groups. Under the conditions of this study, fenbendazole treatment does not affect reproduction in C57BL/6J mice.

Pinworms of the order Oxyurina (Syphacia obvelata, Syphacia muris, and Aspiculuris tetraptera) are one of the most commonly reported parasites in the modern rodent facility.^{3,4,7,11,15,17,18} A 2009 study indicated a pinworm prevalence rate of 0.25% among North American and European samples submitted to a large commercial diagnostic lab over a 5-y period.⁴⁸ A 1996 survey indicated that approximately 67% of the responding institutions from the top 100 institutional recipients of NIH funds reported previous pinworm infestation in their laboratory rodents.²⁶ Like many large institutions, our university has had pinworm outbreaks that require increased prophylactic testing and financial resources to prevent spread of the infestation. Although infestations typically are subclinical, heavy pinworm burdens can lead to rectal prolapse, enteritis, and intussusception in immunodeficient rodents or those that have other comorbidities.^8,14,15,56 Infested mice may show decreased weight gain, diminished growth rates, increased caloric demands for basal metabolism, and compromised overall nutritional status.^15,25,45,47 Parasite load can be influenced by animal age, weight, and sex.^4,7,8,12,14 Furthermore, pinworm infestation of mice may confound experimental results—most notably in immunologic research—by altering data collected from the mice.^{1,15,24,28,29,36,37,45,47,53,59,62}Pinworms have a direct life cycle: embryonated nematode eggs are ingested by the rodent host and then hatch and develop in the gastrointestinal tract.^7,8,15 Immature eggs are passed in the feces or laid on the hair surrounding the perianal region; transmission occurs through fecal–oral contact or fomites.^8,38 Pinworm ova are persistent in the environment and can recontaminate treated colonies.^8,14,21,22 Pinworms can be diagnosed by PCR analysis of feces, perianal cellophane tape testing for Syphacia spp., or fecal floatation for Aspiculuris.^8,14,34 Postmortem diagnostic testing is considered more reliable than premortem testing and involves direct examination of the colonic or cecal contents for adult worms.^35,48Several strategies have been used to eradicate pinworms, although treatments themselves can effect research outcomes.^8,47 Rederivation is the ‘gold standard’ for eliminating parasites from rodent colonies, but this approach is expensive and time-consuming.^28,40,47 The most common pharmacologic treatments for rodent pinworms include benzimidazoles, such as fenbendazole and avermectins, including ivermectin.^8,48 Benzimidazoles are versatile anthelmintics due to their wide range of effectiveness against gastrointestinal nematodes without reliance on systemic drug concentrations. In addition, benzimidazoles act through the inhibition of microtubule polymerization by binding to β-tubulin.⁵⁷ Benzimidazoles have adulticidal, larvicidal, and ovicidal properties that make these drugs an attractive treatment choice.^30,32 Many treatment regimens have been used; one such regimen for mice includes ad libitum diet that contains fenbendazole, fed continuously or every other week and combined with environmental decontamination.^5,9,20,22,63 Fenbendazole is often used for treatment because of its wide margin of safety, ease of administration, and documented effectiveness, although little is known about its effects on the physiology and behavior of rodents.⁴²Many facilities prophylactically treat incoming rodents with fenbendazole during the quarantine period regardless of their health status.²⁹ These mice are generally shipped to and from other institutions as part of a collaborative effort, typically with the intent to breed. At our institution, researchers are often concerned about the effects of fenbendazole on fecundity. To the best of our knowledge, only a few studies involving rats have been published,^2,28 and none exist regarding the reproductive effects of fenbendazole on mice. In general, toxic effects of fenbendazole have not been reported at therapeutic levels but potentially may alter or interfere with ongoing research experiments, such as effects on immune parameters.^6,17,43,61 Fenbendazole did not affect pain perception or behavior studies.^{2,8,11,39,51,64} In an unpublished and non-peer-reviewed study referenced by WHO, rats showed reduced fertility and severe signs of toxicosis in pups (for example, decreased survival indices, decreased body weights at birth, and slower lactational growth) at a dose of 45 mg/kg.⁶⁴ A 1988 study found that mice given mebendazole (a drug in the benzimidazole family) had reduced litter size and female growth rate and increased incidence of kinked tails in the offspring.³ Another study concluded that treatment with fenbendazole decreased litter size in Sprague–Dawley rats but not GEPR-9 rats.²⁸ However, fenbendazole did not affect pregnancy indicators in rats, including maternal weight gain, water consumption, number of pups born, and pup birth weights.²The purpose of this study was to evaluate the effects of fenbendazole on fecundity in mice. To assess the effects of fenbendazole on reproductive parameters, we evaluated 2 common treatment schedules: an alternating schedule, during which mice received medicated diet for 1 wk followed by 1 wk of nonmedicated diet for a total of 5 treatments over a 9-wk period, and a 5-wk continuous application of treated feed. We chose to compare the effects of continuous feeding of the medicated diet as compared with an alternate-week approach regardless of treatment duration because of the breeding timeline. Continuous treatment provides a shorter treatment duration, greater cost effectiveness, and less labor.¹⁹ Although using the alternating week regimen requires a longer duration of treatment, this regimen may be necessary for infestations of A. tetraptera because of their prolonged prepatent period.¹⁹ Mice in the current study were not exposed to pinworms to reduce the likelihood of confounding effects and to isolate the pharmacologic effects of fenbendazole.     

Cryopreservation and Preparation of Thawed Spermatozoa from Rhesus Macaques (Macaca mulatta) for In Vitro Fertilization

Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.

Nonhuman primates (NHPs) are critical for biomedical research due to their close genetic and physiologic relationship to humans. In addition, according to the classification criteria proposed by the International Union for the Conservation of Nature, approximately 63% of the 702 species and subspecies of NHP in the world are at some level of risk of extinction.^21,31 Thus, efficient assisted reproductive technologies (ARTs) are required to 1) understand primate reproductive processes, 2) advance NHP biomedical model development and 3) advance species conservation efforts.The rhesus macaque (Macaca mulatta) is one of the most commonly used NHP species for research studies relevant to human health and disease.⁴² Advances in the use of ARTs in this important species include cryopreservation of sperm;⁴¹ artificial insemination;³⁴ in vitro fertilization (IVF);³ and intracytoplasmic sperm injection (ICSI).³⁰ Establishing effective and reproducible rhesus macaque ARTs is critical in allowing the birth of live offspring after blastomere nuclear transfer,²⁸ somatic cell nuclear transfer,¹⁰ mitochondrial replacement in oocytes,⁴³ and autologous grafting of prepubertal rhesus testis, followed by ICSI and embryo development.¹⁶ Moreover, recent advances in gene editing now allow NHP genome manipulation in one cell embryos (zygotes) for the subsequent generation of transgenic models of human disease.¹¹Genetic resource banking or cryobanking, (the low temperature storage of gametes, embryos, and tissues) is crucial to preserving genetic material for future use in ARTs. Cryobanking allows the preservation of material from genetically valuable individuals independent of the long-term goal for its use (biomedical models, domestic species breeding programs, species conservation, etc.).⁶ Moreover, cryobanking makes sperm readily available when needed, without the need to maintain live male sperm donors or personnel who are trained to collect fresh sperm.However, spermatozoa cryopreservation and recovery of viable sperm after thawing is still a major concern in NHP ARTs and would benefit from further improvement.⁵⁰ Although several studies have investigated the optimal conditions for primate sperm preservation,^{12-14,25,33,39-41,53,54} major gaps exist in our understanding of what is critical for promoting the survival of sperm after thawing. For example, IVF success using fresh sperm varied between 50% to 61%^50,51 with 55% of the embryos reaching the blastocyst stage,⁵¹ whereas IVF with cryopreserved sperm resulted in 82 ± 13% of the ova developing into embryos, of which only 39 ± 21% reached the blastocyst stage.⁴¹ One process that needs further optimization in rhesus macaques is sperm vitrification. Vitrification is the process of obtaining a glass-like solid to avoid ice crystal formation; it is achieved either by rapid cooling or using additives.⁵² Although successful vitrification of human sperm has been reported,^19,20 the only study that assessed sperm vitrification in rhesus macaques reported no motility after thawing.²⁹ Therefore, further studies are necessary on how to successfully cryopreserve sperm from rhesus macaques.An important factor to consider with regard to sperm cryopreservation is how to isolate good quality sperm after thawing (that is, progressively motile sperm with an intact plasma membrane and acrosome and low levels of DNA fragmentation). Several studies have compared different sperm preparation methods in various species, including bulls,^2,23 humans,¹⁵ and dogs,²² but no studies to date have systematically compared various methods in rhesus macaques.Therefore, the objectives of the current study were to 1) compare 2 methods for cryopreservation of sperm from rhesus macaques with regard to sperm viability characteristics after thawing; 2) compare 4 different methods of removal of cryopreservation reagents and sperm isolation after thawing, and (3) evaluate the overall efficacy of the 2 best sperm isolation methods for use in IVF.