Effects of Refined Handling on Reproductive Indices of BALB/cJ and CD-1 IGS Mice.

Current mouse handling methods during cage change procedures can cause stress and potentially compromise animal welfare. Our previous study of breeding C57BL/6J mice found modest increases in pup production and a significant reduction in preweaning litter losses when mice were handled using a tunnel as compared with a tail-lift with padded forceps. The current study evaluated how these 2 handling methods affected reproduction by 2 additional mouse strains, BALB/cJ (a low- to intermediate-fecundity strain) and CD-1 IGS (a high-fecundity stock). We predicted that refined handling would have minimal effects on the high-fecundity line with a satisfactory production rate and greater effects on the low-fecundity line. Handling method (tunnel compared with tail-lift) was randomly assigned to monogamous breeding pairs of mice. Reproductive metrics (litter size at birth and weaning, numbers of litters, litter attrition, between-litter intervals, pup wean- ing weight, and sex ratio) were prospectively monitored for 80 BALB/cJ and 77 CD-1 pairs that were bred continuously for 6 mo. Both strains of mice were highly productive, exceeding previously published breeding data. However, neither strain demonstrated operational or statistically significant differences between handling methods for any reproduction metric. As we detected no negative effects in these 2 strains and the benefits are clear in other strains, refined handling should be considered for all breeding mice.

Unmasking the Adverse Impacts of Sex Bias on Science and Research Animal Welfare.

Sex bias in biomedical and natural science research has been prevalent for decades. In many cases, the female estrous cycle was thought to be too complex an issue to model for, and it was thought to be simpler to only use males in studies. At times, particularly when studying efficacy and safety of new therapeutics, this sex bias has resulted in over- and under-medication with associated deleterious side effects in women. Many sex differences have been recognized that are unrelated to hormonal variation occurring during the estrous cycle. Sex bias also creates animal welfare challenges related to animal over-production and wastage, insufficient consideration of welfare (and scientific) impact related to differential housing of male vs female animals within research facilities, and a lack of understanding regarding differential requirements for pain recognition and alleviation in male versus female animals. Although many funding and government agencies require both sexes to be studied in biomedical research, many disparities remain in practice. This requires further enforcement of expectations by the Institutional Animal Care and Use Committee when reviewing protocols, research groups when writing grants, planning studies, and conducting research, and scientific journals and reviewers to ensure that sex bias policies are enforced.

Using refined methods to pick up mice: A survey benchmarking prevalence & beliefs about tunnel and cup handling.

Refined handling improves laboratory mouse welfare and research outcomes when compared to traditional tail handling, yet implementation does not seem to be widespread. Refined handling includes picking up a mouse using a tunnel or cupped hands. The aim of this study was to determine the current prevalence of and beliefs towards refined handling using the theory of planned behavior. It was predicted that refined handling prevalence is low compared to traditional handling methods, and its implementation is determined by individual and institutional beliefs. Research personnel were recruited via online convenience sampling through email listservs and social media. A total of 261 participants in diverse roles (e.g. veterinarians, managers, caretakers, researchers, etc.) responded primarily from the USA (79%) and academic institutions (61%) Participants were surveyed about their current use, knowledge, and beliefs about refined handling. Quantitative data were analyzed via descriptive statistics and generalised regression. Qualitative data were analyzed by theme. Research personnel reported low levels of refined handling implementation, with only 10% of participants using it exclusively and a median estimate of only 10% of institutional mice being handled with refined methods. Individually, participants had positive attitudes, neutral norms, and positive control beliefs about refined handling. Participants' intention to provide refined handling in the future was strongly associated with their attitudes, norms, and control beliefs (p<0.01). Participants believed barriers included jumpy mice, perceived incompatibility with restraint, lack of time, and other personnel. However, participants also believed refined handling was advantageous to mouse welfare, handling ease, personnel, and research. Although results from this survey indicate that current refined handling prevalence is low in this sample, personnel believe it has important benefits, and future use is associated with their beliefs about the practice. People who believed refined handling was good, felt pressure to use it, and were confident in their use reported higher implementation. Increased refined handling could be encouraged through education on misconceptions, highlighting advantages, and addressing important barriers.

Perspective: Opportunities for advancing aquatic invertebrate welfare.

Welfare considerations and regulations for invertebrates have lagged behind those for vertebrates, despite invertebrates comprising more than 95% of earth's species. Humans interact with and use aquatic invertebrates for exhibition in zoos and aquaria, as pets, research subjects, and important food sources. Recent research has indicated that aquatic invertebrates, in particular cephalopod mollusks and decapod crustaceans, experience stress and may be able to feel pain. With this article, we present results of a survey on attitudes of aquatic animal health professionals toward aquatic invertebrate welfare and provide practical recommendations for advancing aquatic invertebrate welfare across four areas of opportunity: use of anesthesia, analgesia, and euthanasia; development of less invasive diagnostic and research sampling methods based on 3R principles; use of humane slaughter methods for aquatic invertebrates; and reducing impacts of invasive procedures in aquaculture and fisheries. We encourage consideration of these opportunities to achieve far-reaching improvements in aquatic invertebrate welfare.

'Invisible actors'-How poor methodology reporting compromises mouse models of oncology: A cross-sectional survey.

The laboratory mouse is a key player in preclinical oncology research. However, emphasis of techniques reporting at the expense of critical animal-related detail compromises research integrity, animal welfare, and, ultimately, the translation potential of mouse-based oncology models. To evaluate current reporting practices, we performed a cross-sectional survey of 400 preclinical oncology studies using mouse solid-tumour models. Articles published in 2020 were selected from 20 journals that specifically endorsed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) preclinical reporting guidelines. We assessed reporting compliance for 22 items in five domains: ethical oversight assurance, animal signalment, husbandry, welfare, and euthanasia. Data were analysed using hierarchical generalised random-intercept models, clustered on journal. Overall, reporting of animal-related items was poor. Median compliance over all categories was 23%. There was little or no association between extent of reporting compliance and journal or journal impact factor. Age, sex, and source were reported most frequently, but verifiable strain information was reported for <10% of studies. Animal husbandry, housing environment, and welfare items were reported by <5% of studies. Fewer than one in four studies reported analgesia use, humane endpoints, or an identifiable method of euthanasia. Of concern was the poor documentation of ethical oversight information. Fewer than one in four provided verifiable approval information, and almost one in ten reported no information, or information that was demonstrably false. Mice are the "invisible actors" in preclinical oncology research. In spite of widespread endorsement of reporting guidelines, adherence to reporting guidelines on the part of authors is poor and journals fail to enforce guideline reporting standards. In particular, the inadequate reporting of key animal-related items severely restricts the utility and translation potential of mouse models, and results in research waste. Both investigators and journals have the ethical responsibility to ensure animals are not wasted in uninformative research.

Preventing the Transmission of Murine Norovirus to Mice (Mus musculus) by Using Dry-heat Sterilization.

A critical component of an animal care biosecurity plan includes the sterilization of materials that come into direct contact with the animals. Dry-heat sterilization is gaining popularity in animal research facilities due to lower cost, less space utilization, no water usage, and the ability to sterilize water-sensitive materials. Currently, dry-heat sterilization ovens are validated against Bacillus atropheus spore strips with the assumption that a lack of sporulation is equivalent to successful sterilization. However, no published studies describe sterilization of rodent cages that contain relevant rodent pathogens by using this method. To determine if a dry-heat sterilizer can sterilize rodent cages and bedding against relevant rodent pathogens, we created murine norovirus (MNV)-contaminated cages by using mice with known MNV infection and shedding. The contaminated cages were either sterilized with the dry-heat sterilizer or not sterilized. Naïve, 4-wk-old, CD-1 mice were placed in the dry-heat-sterilized cages, contaminated unsterilized cages, or standard autoclaved cages for 2 wk. The mice were subsequently placed into clean, autoclaved cages for the remainder of the study. Fresh fecal pellets were collected at weeks 0, 12, and 16 and submitted for MNV PCR. Whole blood was collected for MNV serology at weeks 0, 8, 12, and 16. At week 16, all mice that had been in the unsterilized contaminated cages were positive for MNV by both fecal PCR and serology, whereas the mice in the dry-heat-sterilized and autoclaved cages were negative for MNV by both methods at all time points. Our study supports the use of dry heat sterilization as a viable sterilization method for rodent cages and bedding.

Effects of non-aversive versus tail-lift handling on breeding productivity in a C57BL/6J mouse colony.

Non-aversive handling is a well-documented refinement measure for improving rodent welfare. Because maternal stress is related to reduced productivity, we hypothesized that welfare benefits associated with non-aversive handling would translate to higher production and fewer litters lost in a laboratory mouse breeding colony. We performed a randomized controlled trial to examine the effects of a standard method of handling (tail-lift with forceps) versus non-aversive handling with transfer tunnels ('tunnel-handled') on breeding performance in 59 C57BL/6J mouse pairs. Intervention assignments could not be concealed from technicians, but were concealed from assessors and data analyst. An operationally significant effect of tunnel-handling (large enough differences to warrant programmatic change) was defined before study initiation as a 5% increase in productivity, or one extra pup over the reproductive lifetime of each pair. Pairs were randomly allocated to handling intervention and cage rack location, and monitored over an entire 6-month breeding cycle. For each group, we measured number of pups born and weaned, and number of entire litters lost prior to weaning. Differences between transfer methods were estimated by two-level hierarchical mixed models adjusted for parental effects and parity. Compared to tail-lift mice, tunnel-handled mice averaged one extra pup per pair born (+1.0; 95% CI 0.9, 1.1; P = 0.41) and weaned (+1.1, 95% CI 0.9, 1.2; P = 0.33). More tunnel-handled pairs successfully weaned all litters produced (13/29 pairs, 45% vs 4/30 pairs, 13%; P = 0.015), averaged fewer litter losses prior to weaning (11/29 pairs [38%] vs 26/30 pairs [87%]; P <0.001), and had a 20% lower risk of recurrent litter loss. The increase in numbers of pups produced and weaned with tunnel handling met threshold requirement for operational significance. These data and projected cost savings persuaded management to incorporate tunnel handling as standard of care across the institution. These data also suggest that overlooked husbandry practices such as cage transfer may be major confounders in studies of mouse models.

Developing Recommendations for Cumulative Endpoints and Lifetime Use for Research Animals.

Research animals are important for scientific advancement, and therefore, their long-term welfare needs to be monitored to not only minimize suffering, but to provide positive affective states and experiences. Currently, there is limited guidance in countries around the world on cumulative and experimental endpoints. This paper aims to explore current opinions and institutional strategies regarding cumulative use and endpoints through a scoping survey and review of current regulations and welfare assessment tools, and ultimately to provide recommendations for assessment of cumulative and lifetime use of research animals. The survey found that only 36% of respondents indicated that their institution had cumulative use endpoint policies in place, but these policies may be informal and/or vary by species. Most respondents supported more specific guidelines but expressed concerns about formal policies that may limit their ability to make case-by-case decisions. The wide diversity in how research animals are used makes it difficult for specific policies to be implemented. Endpoint decisions should be made in an objective manner using standardized welfare assessment tools. Future research should focus on robust, efficient welfare assessment tools that can be used to support planning and recommendations for cumulative endpoints and lifetime use of research and teaching animals.

Practical Euthanasia Method for Common Sea Stars (Asterias rubens) That Allows for High-Quality RNA Sampling.

Sea stars in research are often lethally sampled without available methodology to render them insensible prior to sampling due to concerns over sufficient sample quality for applied molecular techniques. The objectives of this study were to describe an inexpensive and effective two-step euthanasia method for adult common sea stars (Asterias rubens) and to demonstrate that high-quality RNA samples for further use in downstream molecular analyses can be obtained from pyloric ceca of MgCl2-immersed sea stars. Adult common sea stars (n = 15) were immersed in a 75 g/L magnesium chloride solution until they were no longer reactive to having their oral surface tapped with forceps (mean: 4 min, range 2-7 min), left immersed for an additional minute, and then sampled with sharp scissors. RNA from pyloric ceca (n = 10) was isolated using a liquid-liquid method, then samples were treated with DNase and analyzed for evaluation of RNA integrity number (RIN) for assessment of the quantity and purity of intact RNA. Aversive reactions to magnesium chloride solution were not observed and no sea stars regained spontaneous movement or reacted to sampling. The calculated RIN ranged from 7.3-9.8, demonstrating that the combination of animal welfare via the use of anesthesia and sampling for advanced molecular techniques is possible using this low-cost technique.

Intraperitoneal Alfaxalone and Alfaxalone-Dexmedetomidine Anesthesia in Sprague-Dawley Rats (Rattus norvegicus).

Due to their unpredictability and variable effects, injectable anesthetic regimens in laboratory rodent species warrant refinement. In our study we sought to evaluate alfaxalone, which has gained recent popularity in veterinary medicine, alone and in combination with dexmedetomidine to evaluate their anesthetic ability in Sprague-Dawley rats when administered intraperitoneally. Three doses of alfaxalone only and 4 dose combinations of alfaxalone-dexmedetomidine were tested in males and female rats. The time to induction, anesthetic duration, pulse rate, respiratory rate, temperature, and time to recovery were recorded by a blind observer. The level of anesthesia induced by the various anesthetic protocols was assessed by using pedal withdrawal reflex to a noxious stimulus and scored according to the response. Dependent on the treatment group, atipamezole or saline was administered intraperitoneally once animals reached 60 min of anesthesia. Regardless of the dose, alfaxalone alone achieved only a sedative level of anesthesia, whereas all alfaxalone-dexmedetomidine combinations led to a surgical level of anesthesia in all animals. Anesthesia regimens using alfaxalone alone and in combination with dexmedetomidine demonstrated sex-associated differences, with female rats maintaining longer durations of sedation or anesthesia than their male counterparts. Both male and female rats displayed decreases in physiologic parameters consistent with the effects of dexmedetomidine. Given the results described herein, we recommend 20 mg/kg alfaxalone for sedation and 30 mg/kg alfaxalone combined with 0.05 mg/kg dexmedetomidine for surgical anesthesia in female rats. Appropriate doses of alfaxalone only and alfaxalone-dexmedetomidine for male rats were not determined in this study and need further evaluation.

Butorphanol-Azaperone-Medetomidine for the Immobilization of Rhesus Macaques (Macaca mulatta).

Maximizing animal wellbeing by minimizing drug-related side effects is a key consideration when choosing pharmaceutical agents for chemical restraint in nonhuman primates. One drug combination that may promote this ideology is butorphanol (27.3 mg/mL), azaperone (9.1 mg/mL), and medetomidine (10.9 mg/mL; BAM). Based on results from a pilot study, 2 doses of BAM (16 and 24 µL/kg IM) were compared in healthy, 3-y-old rhesus macaques. Physiologic parameters and anesthetic quality were assessed and recorded every 5 min. Experimental endpoints were established for hypoxemia (85% or less peripheral oxygen saturation with oxygen supplementation), pulse rate (80 bpm or less for 2 consecutive readings), mean arterial pressure (MAP; 50 mm Hg or less), and hypothermia (97 °F or less); if any endpoint was achieved, medetomidine was reversed by using atipamezole (0.22 mg/kg IM). Both BAM doses resulted in immobilization of all animals with no clinically significant differences between groups. All animals initially exhibited hypoxemia that resolved with oxygen supplementation. Regardless of dose, most macaques (71%) reached established experimental endpoints for bradycardia (62 to 80 bpm) or hypotension (44 to 50 mm Hg MAP). Given the results of this study, our recommendation regarding the use of 16- or 24-µL/kg BAM for immobilizing rhesus macaques is dependent on caution regarding cardiopulmonary parameters and the provision of supplemental oxygen.

A Review of the Effects of Pain and Analgesia on Immune System Function and Inflammation: Relevance for Preclinical Studies.

One of the most significant challenges facing investigators, laboratory animal veterinarians, and IACUCs, is how to balance appropriate analgesic use, animal welfare, and analgesic impact on experimental results. This is particularly true for in vivo studies on immune system function and inflammatory disease. Often times the effects of analgesic drugs on a particular immune function or model are incomplete or don't exist. Further complicating the picture is evidence of the very tight integration and bidirectional functionality between the immune system and branches of the nervous system involved in nociception and pain. These relationships have advanced the concept of understanding pain as a protective neuroimmune function and recognizing pathologic pain as a neuroimmune disease. This review strives to summarize extant literature on the effects of pain and analgesia on immune system function and inflammation in the context of preclinical in vivo studies. The authors hope this work will help to guide selection of analgesics for preclinical studies of inflammatory disease and immune system function.

Evaluation of Analgesic Efficacy of Meloxicam and 2 Formulations of Buprenorphine after Laparotomy in Female Sprague-Dawley Rats.

Managing postoperative pain in rodents is an important part of any animal care and use program, and identifying an optimal analgesic plan for a surgical procedure is critical to providing for animal welfare. Opioids and NSAID are commonly used in rodents, but few studies have evaluated their efficacy in surgical models. The current study aimed to evaluate the therapeutic efficacy of clinically relevant doses of buprenorphine (2 formulations) or meloxicam used in combination with ketamine and xylazine anesthesia in a Sprague-Dawley rat ovariohysterectomy surgical model. Rats received either subcutaneous saline once daily for 3 d, low-dose (0.05 mg/kg SC) or high-dose (0.1 mg/kg SC) buprenorphine twice daily for 3 d, a single injection of sustained-release buprenorphine (1.2 mg/kg SC), or low-dose (1 mg/kg SC) or high-dose (2 mg/kg SC) meloxicam once daily for 3 d. Clinical analgesic efficacy was assessed over 8 d according to cageside observation scoring, body weight, and behavioral testing. Ovariohysterectomy was associated with 2 d of postoperative pain, and all 3 buprenorphine dosing strategies and both doses of meloxicam demonstrated varying amounts of analgesia. Given the results of the current study, we recommend 0.05 mg/kg SC buprenorphine at least twice daily or a single dose of 1.2 mg/kg SC of sustained-release buprenorphine for rats undergoing midline laparotomy with ovariohysterectomy. Alternatively, meloxicam at 1 to 2 mg/kg SC once daily could be used for this indication.

Xiphoid Surface Temperature Predicts Mortality in a Murine Model of Septic Shock.

Sepsis continues to be a major challenge for modern medicine. Several preclinical models were developed to study sepsis and each has strengths and weaknesses. The cecal slurry (CS) method is a practical alternative because it does not require surgery, and the infection can be dosed. However, one disadvantage is that the dosage must be determined for each CS preparation using survival studies. Our aim was to refine a survival protocol for the CS model by determining a premonitory humane endpoint that would reduce animal suffering. Mice become hypothermic in sepsis; therefore, we tested whether reductions in surface temperature (Ts), measured by noninvasive infrared thermometry, could predict eventual death. We injected 154 C57BL/6J mice with CS (0.9-1.8 mg/g) and periodically monitored Ts at the xiphoid process over 5 days. We used, as predictors, combinations of temperature thresholds (29°C -31°C) and times, postinjection (18-36 h). A receiver-operator curve, sensitivity, and specificity were determined. A Distress Index value was calculated for the threshold conditions. The optimum detection threshold (highest Youden index) was found at Ts ≤ 30.5°C at 24 h (90% specific, 84% sensitive). This threshold condition reduced animal suffering by 41% while providing an accurate survival rate estimate. Using this threshold, only 13 of 154 mice would have died from sepsis; 67 would have been euthanized at 24 h, and only 7 of 154 would have been euthanized unnecessarily. In conclusion, using a humane endpoint of Ts ≤ 30.5°C at 24 h accurately predicts mortality and can effectively reduce animal suffering during CS survival protocols.

To Treat or Not to Treat: The Effects of Pain on Experimental Parameters.

A common dilemma faced by all animal bioethics committees arises when exceptions are proposed to the use of analgesics in painful procedures. The committee and researcher must weigh the possible confounding effects of including additional drugs (analgesics) in their treatment regimen against the moral obligation to perform humane research. Often neglected in these considerations are the potential confounding effects of unrelieved pain and consistency with pain-relieving practices in human medicine. In this review, we summarize what is currently known regarding the molecular and physiologic effects of pain and analgesics in common animal models used across several therapeutic areas. This work is intended to help provide guidance and assurance that a comprehensive approach has been taken when contemplating how pain relief will be applied in animal research protocols.

Clinical efficacy of sustained-release buprenorphine with meloxicam for postoperative analgesia in beagle dogs undergoing ovariohysterectomy.

The goal of the current study was to compare the efficacy, adverse effects, and plasma buprenorphine concentrations of sustained-release buprenorphine (SRB) and buprenorphine after subcutaneous administration in dogs undergoing ovariohysterectomy. In a prospective, randomized, blinded design, 20 healthy adult female Beagle dogs underwent routine ovariohysterectomy and received multimodal analgesia consisting of meloxicam and one of two buprenorphine formulations. Dogs were randomly assigned to receive either SRB (0.2 mg/kg SC, once) or buprenorphine (0.02 mg/kg SC every 12 h for 3 d). Blinded observers assessed all dogs by using sedation scores, pain scores, temperature, HR, RR, and general wellbeing. Dogs were provided rescue analgesia with 0.02 mg/kg buprenorphine SC if the postoperative pain score exceeded a prede- termined threshold. Blood samples were collected, and mass spectrometry was used to determine plasma buprenorphine concentrations. Data were analyzed with a linear mixed model and Tukey-Kramer multiple comparison. Age, body weight, anesthetic duration, surgical duration, sevoflurane concentration, and cardiorespiratory variables did not differ significantly between groups. Dogs in both formulation groups had comparable postoperative sedation and pain scores. One dog from each formulation group had breakthrough pain requiring rescue analgesia. Plasma buprenorphine concentrations remained above a hypothesized therapeutic concentration of 0.6 ng/mL for 136.0 ± 11.3 and 10.67 ± 0.84 h for SRB and buprenorphine, respectively. Based on the results of this study, multimodal analgesic regimens consisting of meloxicam and either buprenorphine or SRB are equally efficacious in managing pain associated with an ovariohysterectomy and show comparable side effects.

Effects of weekly blood collection in male and female cynomolgus macaques (Macaca fascicularis).

This study was designed to evaluate the maximal amount of blood that can be safely collected in healthy, adult male and female cynomolgus macaques for 4 consecutive weeks with minimal effect on animal wellbeing. General guidelines for blood collection volumes in laboratory animals are not species-specific, and currently there are few evaluations of blood collection in macaques. In this study, blood was removed at 7.5%, 10%, 12.5%, 15%, or 17.5% of total blood volume (TBV) for 4 consecutive weeks. Hematologic parameters and body weights were evaluated immediately prior to each blood collection time point and for an additional 4 consecutive weeks following the last collection. Male and female macaques tolerated removal of as much as 15% TBV with minor clinical effects, whereas macaques in the 17.5% TBV group exhibited an increased incidence of emesis and anorexia during the first 24 h after blood collection. According to these results, we recommend collecting no more than 15% TBV weekly for 4 consecutive weeks from healthy, adult male and female cynomolgus macaques.

Pharmacokinetics of sustained-release and transdermal buprenorphine in Göttingen minipigs (Sus scrofa domestica).

The opioid buprenorphine has been shown to provide adequate postoperative analgesia in both companion and laboratory animals. However, its use is still hindered by the need for multiple parenteral injections to achieve continuous analgesia. The purpose of the current study was to conduct a pharmacokinetic analysis of 2 new long-acting formulations of buprenorphine-an injectable sustained-release buprenorphine (SRB) and a transdermal buprenorphine (TDB) patch-in healthy Göttingen minipigs by using liquid chromatography-electrospray ionization-tandem mass spectrometry. Administration of 0.18 mg/kg SC SRB and 30 µ g/h TDB achieved AUC(0-Tlast) of 221.6 ± 26.8 and 25.2 ± 3.9 ng × h/mL, respectively, compared with 9.7 ± 1.4 ng*h/mL for 0.02 mg/kg IV buprenorphine. By using a hypothesized therapeutic plasma buprenorphine concentration threshold of 0.1 ng/mL, therapeutic concentrations were achieved at the first study time point (5 to 30 min) and lasted an average of 8.0 ± 1.3 h for intravenous buprenorphine and 264.0 ± 32.2 h for SRB. TDB achieved therapeutic concentrations in 12 to 24 h after patch application, which lasted until the patch was removed at 72 h. The results of this study suggest that SRB and TDB are long-acting alternatives for pain management, and their use could decrease animal handling and stress, thereby simplifying pain management and improving welfare in laboratory swine.

Predictive observation-based endpoint criteria for mice receiving total body irradiation.

Total body irradiation of mice is a commonly used research technique; however, humane endpoints have not been clearly identified. This situation has led to the inconsistent use of various endpoints, including death. To address this issue, we refined a cageside observation-based scoring system specifically for mice receiving total body irradiated. Male and female C57BL/6 mice (age, 8 wk) received 1 of 3 doses of radiation from 1 of 2 different radiation sources and were observed for progression of clinical signs. All mice were scored individually by using cageside observations of their body posture (score, 0 to 3), eye appearance (0 to 3), and activity level (0 to 3). Retrospective analysis of the observation score data indicated that death could be predicted accurately with total scores of 7 or greater, and observation scores were consistent between observers. This scoring system can be used to increase the consistent use of endpoint criteria in total body murine irradiation studies and ultimately to improve animal welfare.

Endpoint refinement for total body irradiation of C57BL/6 mice.

Acute radiation syndrome is a life-threatening condition that has the potential to affect large populations of humans. Although several animal models of this syndrome are available, the total-body-irradiated mouse has emerged as an important tool to evaluate the efficacy of prospective prophylaxis, mitigation, and treatment compounds. Despite the widespread use of this model, humane endpoints have not been clearly identified. To address this issue, we developed a cageside observation-based scoring system specifically for total-body-irradiated mice to assess the progression of clinical signs associated with acute radiation syndrome. Male C57BL/6 mice (n=175; age, 8 to 9 wk) received an anticipated LD50 dose of radiation and were observed for progression of clinical signs of acute radiation syndrome for 30 d. All mice were scored individually through cageside observation of their body posture (score, 0 to 3), eye appearance (0 to 3), and activity level (0 to 3). Retrospective analysis of the score data indicated that death could be predicted accurately by using increasing cumulative scores (0 to 9). Total scores of 6, 7, 8, and 9 were associated with mortality rates of 78.6%, 86.4%, 93.3%, and 100%, respectively. Furthermore, scores of 6, 7, and 8 predicted death within 3, 1.5, and 0.5 d, respectively. The use of this scoring system provides investigators and IACUCs with predictive humane, surrogate endpoints for total-body-irradiated mice. This system allows preemptive euthanasia of mice before they become moribund, thereby minimizing pain and distress associated with acute radiation syndrome and improving animal welfare.