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 weaning 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.Abstract
Low-stress handling methods have been studied in detail in mice, but relatively little research exists concerning preferred handling methods in rats. Most recommendations for low-stress handling of rats have been extrapolated from the mouse literature, despite known differences in handler interaction between the 2 species. The goal of the current study was to evaluate common methods of handling in rats, including application of recognized, low-stress handling methods from other species to rats, in order to determine relative stress levels associated with the handling methods. Seventy male and 70 female, 8-wk-old, Crl:CDSD rats, were housed either individually or in pairs, and were handled weekly or daily using one of the following methods: encircling of the torso (standard thoracic hold), handled using a tunnel, handled using a protective bite glove, handled using a soft paper towel, or tickled prior to being handled by the torso (n = 10 per sex per treatment group). Body weight and clinical observations were scored at each handling session, abbreviated functional observation batteries were performed every other week, and an interaction test and hematology were conducted prior to study and on the day of study termination. Rats that were socially housed and handled weekly using the standard thoracic hold showed the least evidence of stress, while those that were singly housed and handled weekly using a protective bite glove or tunnel showed the highest level of stress. These effects were predominantly seen in males. This study suggests that standard low-stress handling methods used for other species may not be optimal for rats, and that additional research is needed to identify alternative methods to the standard thoracic hold that would further reduce stress during handling in rats.Abstract
The Leibniz Institute on Aging has maintained killifish colonies for over 15 y. Our veterinarians, scientists, and animal technicians developed a fish health scoring system and routine colony health surveillance program for our colonies. Over a 4-y period, health data from the African turquoise killifish Nothobranchius furzeri colony were systematically collected and analyzed. The fish health assessment system facilitated categorization of clinical signs and differentiation of fish with mild clinical signs from fish that required euthanasia. This report provides new information on clinical signs and conditions that may occur in young and aged N. furzeri. To be comprehensive, a colony health surveillance program incorporates animal health at both the individual and the population levels. The quarterly routine health monitoring program identified Mycobacterium spp. as the most common agent in our facility and identified the killifish pathogen (Loma acerinae) for the first time. Taken together, these findings demonstrate the importance of a comprehensive colony health management system in a fish research facility. By improving the health and welfare of fish used for research, the scientific community will benefit from less variable and more reliably reproducible research results.Abstract
Although the Guide suggests changing rodent cage components every 2 wk, it states that “decreased sanitation frequency may be justified if the microenvironment in the cages, under the condition of use …, is not compromised.” The purpose of this study was to evaluate extended sanitation intervals of cage components (automated watering valve, wire bar lid, and filter top) of mouse individually ventilated caging (IVCs) at our institution. We hypothesized that there would be no significant difference in relative light units measured by ATP luminometry of these cage components at the control time point of 14 d as compared with each extended time interval: 28, 56, and 84 d. In addition, for automated watering valves, the study was extended to 168 d. We also hypothesized that time-and-motion studies performed by moving to a sanitation interval of 84 d for all components would result in substantial time and cost savings. The components of a total of 24 cages containing 4 or 5 mice each were swabbed, and an ATP luminometer was used to detect organic matter. We found no significant differences in organic matter load between 14 d and all other time points for all cage components. Our time- and cost-savings analysis found that extending the sanitation interval of cage components from every 2 wk (14 d) to every 3 mo (84 d) for every 10,000 cages would save about 3,000 technician hours annually, for a total annual labor cost savings of about $100,000. This study is the first to validate the extended sanitation interval of automated watering valves and confirms the findings of previous studies that validated the extended sanitation frequency of wire bar lids and filter tops of rodent IVCs. Overall, extending the sanitation frequency of cage components reduces workload of animal care staff without compromising the cage microenvironment.Abstract
Mechanical ventilation can be used in mice to support high-risk anesthesia or to create clinically relevant, intensive care models. However, the choice of anesthetic and inspired oxygen concentration for prolonged procedures may affect basic physiology and lung inflammation. To characterize the effects of anesthetics and oxygen concentration in mice experiencing mechanical ventilation, mice were anesthetized with either isoflurane or pentobarbital for tracheostomy followed by mechanical ventilation with either 100% or 21% oxygen. Body temperature, oxygen saturation, and pulse rate were monitored continuously. After 6 h, mice were euthanized for collection of blood and bronchoalveolar lavage fluid for evaluation of biomarkers of inflammation and lung injury, including cell counts and cytokine levels. Overall, both isoflurane and pentobarbital provided suitable anesthesia for 6 h of mechanical ventilation with either 21% or 100% oxygen. We found no differences in lung inflammation biomarkers attributable to either oxygen concentration or the anesthetic. However, the combination of pentobarbital and 100% oxygen resulted in a significantly higher concentration of a biomarker for lung epithelial cell injury. This study demonstrates that the combination of anesthetic agent, mechanical ventilation, and inspired oxygen concentrations can alter vital signs and lung injury biomarkers during prolonged procedures. Their combined impact may influence model development and the interpretation of research results, warranting the need for preliminary evaluation to establish the baseline effects.Abstract
Alfaxalone is a commonly used injectable anesthetic in dogs and cats due to its minimal cardiovascular side effects. Data for its use in mice are limited and demonstrate strain- and sex-associated differences in dose-response relationships. We performed a dose-comparison study of alfaxalone-xylazine-buprenorphine (AXB) in Crl:CFW(SW) mice. Subcutaneous injection of 50 mg/kg alfaxalone-10 mg/kg xylazine-0.1 mg/kg buprenorphine HCl consistently achieved a surgical plane of anesthesia (loss of toe pinch) for 48.6 ± 4.7 and 60.8 ± 9.6 min in females and males, respectively. The same dose and route of AXB induced a surgical plane of anesthesia in C57Bl/6NCrl (females: 42.3 ± 11.2 min; males: 51.6 ± 12.3 min), NCr-Foxn1nu (females: 76.8 ± 32.5 min; males: 80.0 ± 1.2 min), and NOD.Cg-PrkdcSCIDIl2rgtm1Wjl/SzJCr (females: 56.0 ± 37.2 min and males: 61.2 ± 10.2 min) mice. We found no significant difference in the duration of the surgical plane of anesthesia between males and females within the mouse strains Crl:CFW(SW), C57Bl/6NCrl, NCr-Foxn1nu, and NOD.Cg-PrkdcSCIDIl2rgtm1Wjl/SzJCr. We next performed an echocardiography study (n = 5 per group) of Crl:CFW(SW) mice (n = 5 per group) to compare subcutaneous AXB anesthesia with that produced by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine (KX). AXB induced significantly less bradycardia (295.4 ± 29 bpm) than KX (185.8 ± 38.9 bpm) did, with no significant differences in cardiac output, ejection fraction, end-diastolic volume, end-systolic volume, or fractional shortening. These results suggest that subcutaneous administration of AXB is a viable alternative to KX for inducing a surgical plane of anesthesia in Crl:CFW(SW), C57Bl/6NCrl, NCr-Foxn1nu, and NOD.Cg-PrkdcSCIDIl2rgtm1Wjl/SzJCr mice, regardless of sex. AXB may also be a better injectable anesthetic option as compared with KX for avoiding adverse cardiac effects in mice.Abstract
Neonatal rodents undergo anesthesia for numerous procedures and for euthanasia by anesthetic overdose. However, data regarding whether neonatal anesthesia is humane are limited. Hypothermia (cryoanesthesia) is the most commonly used anesthetic protocol for neonatal rats 10 d of age or younger. However, hypothermia has recently been restricted in several countries due to perceived painful effects, including pain on rewarming. Minimizing the potential pain and distress of neonates in research is imperative, although very challenging. Traditional validated and nonvalidated behavioral and physiologic outcome measures used for adult rats undergoing anesthesia are unsuitable for evaluating neonates. Therefore, we investigated the effects of several anesthetic methods on neonatal rats by using the innovative objective approaches of noninvasive ultrasonic vocalizations and more invasive neuroendocrine responses (i.e., serum corticosterone, norepinephrine, glucose). Our results show that hypothermia leads to heightened acute distress in neonatal rats as indicated by prolonged recovery times, increased duration of vocalizations, and elevated corticosterone levels, as compared with neonates undergoing inhalational anesthesia. We demonstrate that inhalational anesthesia is preferable to cryoanesthesia for neonatal rats, and researchers using hypothermia anesthesia should consider using inhalational anesthesia as an alternative method.Abstract
The primary objective of this study was to evaluate the pharmacokinetic profile of a new extended-release formulation of buprenorphine (BupBaseER) at a dose that would produce pain management of the desired duration. A secondary objective was to compare the incidence of injection site reactions between the original extended-release formulation (BupHClER) and BupBaseER, which uses a different proprietary polymer-based vehicle than does the BupHClER formulation. Eighteen cynomolgus macaques (M. fascicularis) were divided into 2 groups. Each macaque in the first group (n = 6) received a single subcutaneous injection of 0.06 mg/kg BupBaseER (10 mg/mL) followed at least 2 wk later by a single subcutaneous injection of 0.12 mg/kg. Animals in group 2 (n = 12) received 2 injections of each of 3 compounds—the original polymer matrix vehicle used in BupHClER, the modified polymer matrix vehicle used in BupBaseER, and 0.9% saline—in designated areas of the dorsoscapular region. The 0.06- and 0.12-mg/kg doses both maintained therapeutic levels that were 3 times higher than the hypothesized analgesic threshold of 0.1 ng/mL. These doses maintained therapeutic level for approximately 44 and 103 h, respectively. Based on these data, buprenorphine concentration likely remains well above the therapeutic threshold beyond the 120 h span of this study. During the 30 d after administration, one macaque had a mild skin reaction to BupHClER. None of the animals in either group had skin reactions to BupBaseER at either dosage. These findings support the use of BupBaseER to provide pain management, promote animal welfare, decrease animal stress, and simplify the postoperative management of NHP in research and zoological settings.Abstract
The research use of zebrafish has risen exponentially over the past decade while anesthetic options have remained largely unchanged.6 Tricaine methanesulfonate (MS-222) is widely accepted as an anesthetic for routine husbandry procedures, however it has limitations and safety concerns.5,11 A greater variety of effective anesthetic options for surgical procedures would be advantageous for the research community. Adult zebrafish were randomly assigned to one of the following groups (n = 10, 5 males and 5 females): 200 mg/L MS-222; 6-, 10-, 13-, and 16-mg/L alfaxalone, and control. All zebrafish in the MS-222 group reached a surgical plane of anesthesia within 95 ± 32 s. By contrast, only 2 of 10, 1 of 10, 0 of 10, and 0 of 4 of the 6, 10, 13, and 16 mg/L alfaxalone groups, respectively, reached a surgical plane of anesthesia within the allotted 10-min period. Recovery time was also significantly slower in the alfaxalone groups as compared with MS-222, with some fish taking greater than 10 min to recover. In addition, 33 of 34 zebrafish (the 16 mg/L group was not completed due to safety concerns) in the alfaxalone groups lost opercular movements for greater than one minute during their anesthetic event and had to be removed to the recovery tank. The results demonstrated that alfaxalone was unable to provide a reliable and safe surgical plane of anesthesia at any of the drug doses tested. Therefore, we recommend alfaxalone not be used as an anesthetic for painful procedures on zebrafish and conclude that MS-222 remains a more viable anesthetic for immersion anesthesia in zebrafish.Abstract
Selecting a method of euthanasia is an important step in designing research studies that use animals; euthanasia methods must be humane, cause minimal pain and suffering to the animal, and preserve the tissue architecture of the organs of interest. In this study, we evaluated the histomorphology of the internal organs (lung, spleen, heart, kidney, liver, brain, and adrenal gland) of rats submitted to five different methods of euthanasia, with the goal of determining which protocol caused the least alteration of histomorphology. Twenty adult Wistar Han rats (Rattus norvegicus) were divided into 5 groups of 4 rats each (2 females and 2 males) and were euthanized by CO2 or isoflurane inhalation, sodium thiopental or xylazine plus ketamine overdose, or decapitation. All euthanasia was performed in accordance with published guidelines and local legal requirements. Necropsy was performed immediately after euthanasia. Specific internal organs were removed and placed in formalin and submitted for routine histologic processing. Histomorphological examination of hematoxylin and eosin-stained tissues revealed circulatory alterations in multiple organs, predominantly congestion in multiple tissues, pulmonary hemorrhage, and hepatic degeneration. The euthanasia methods that induced the most severe alterations were exposure to CO2 and anesthetic overdose with xylazine plus ketamine or sodium thiopental. Euthanasia by overexposure to isoflurane caused less damage, and the alterations were of minimal severity. Decapitation resulted in the lowest incidence of lesions in multiple organs but due its traumatic nature, it caused the highest incidence of pulmonary hemorrhage. In selecting a method of euthanasia, factors to consider are the species of animal, the purpose of the research, and the practical ability to perform the procedure to achieve maximal animal welfare without iatrogenic changes that could compromise the outcome and reproducibility of the study.Abstract
In vitro fertilization (IVF), embryo cryopreservation, and embryo transfer (ET) are assisted reproductive technologies (ARTs) that are used extensively for the maintenance of mouse models in animal research. Inbred mouse strains with different genetic backgrounds vary in their reproductive performance. Cryopreservation can affect embryo quality and viability, and the genetic background of ET recipients can influence the ET result. In this retrospective study, we analyzed the outcomes of ETs performed in our facility during the last 6 y. We found that B6C3F1 mice with swollen ampullae show almost 3-fold higher pregnancy rates than mice with nonswollen ampullae when either freshly isolated or frozen-thawed embryos are implanted. Implantation of freshly collected embryos in recipients with swollen ampullae led to significantly higher pregnancy rates in comparison to implantation of frozen-thawed embryos, regardless of whether the latter were fertilized in vivo or in vitro. Moreover, we found a significant effect of genetic background on the birth rate; C57BL/6J mice and mice with a mixed genetic background had 34% higher birth rates than did C57BL/6N mice. Within the C57BL/6J group, the birth rates were significantly higher when using fresh in vivo-fertilized embryos, and cryopreservation negatively affected both in vivo- and in vitro-fertilized embryos. The success rate of obtaining one living pup was not significantly different between frozen-thawed and fresh embryos. Overall, a swollen ampulla is a strong indicator for a successful pregnancy, together with the embryo manipulation and genetic background. A better understanding of the factors that affect the reproductive outcome might lead to optimization of the ART protocols and contribute to a reduction in the number of mice used for these procedures.Abstract
The American Association for Laboratory Animal Science (AALAS) currently publishes two journals containing data-driven, peer-reviewed articles. The mission of Comparative Medicine (CM) is to disseminate high-quality, peer-reviewed information that expands biomedical knowledge and promotes human and animal health through the study of laboratory animal disease, animal models of disease, and basic biologic mechanisms related to disease in people and animals.General