The most widely accepted ethical concept for the mitigation of harm to animals used in biomedical research is known as the 3Rs, which refer to replacement, reduction, and refinement. The aim of our study was to determine the ethical and regulatory criteria that researchers in Mexico consider when developing their animal research protocols and that members of the ethics committees use when they evaluate and approve these protocols. We circulated a survey to 300 individuals from different research institutions and received responses from 179 researchers and members of ethics committees on questions related to their knowledge, attitudes, and practices toward the use of animals in research based on the 3Rs. The responses obtained indicate that the respondents were aware of the 3R concept, and they claim to apply these principles. However, the responses revealed resistance to using alternatives for research, testing, and teaching (66%). Nineteen percent of the researchers reported that their institutions do not have an integrated Institutional Animal Care and Use Committee (IACUC). Around 80% of respondents were aware of Mexican regulations. The knowledge and application of the 3Rs by researchers and members of the IACUC is a fundamental concept in animal research. Such knowledge contributes the use of ethical standards, attitudes, and practices relevant to the use of animals in research.

Training personnel to work with animals presents a variety of challenges, both logistically and with regard to animal welfare. These issues make training an ideal opportunity to evaluate practices and to implement the 3R principles (refinement, replacement, and reduction). Cardiac blood collection from mice is a procedure that can compromise the 3Rs by requiring repeated practice and animal euthanasia. The development of a non-animal training model would promote the 3R principles. Our goals for the development of a new training model for cardiac blood collection from mice were to reduce the number of mice needed to achieve competency, improve our culture of care, and refine the training approach by improving competency. The training model was developed using commonly available materials. The total cost of the model was less than $15 USD per model. Two training curricula were conducted concurrently over a 5-mo period: 1) a curriculum in which trainees used the model before progressing to live mice and 2) the traditional curriculum, which used euthanized mice throughout. The measured variables included the total number of mice used, proportions of trainees who reached competency, the time needed to reach competency, method comprehension, quality of skill performance, trainer and trainee feedback, and training costs. The alternative group used at least 10 fewer mice per technician as compared with the traditionally trained group. The alternative group had a higher competency rate, with 82% (9 of 11 trainees) reaching competency compared with 60% (3 of 5 trainees) in the traditional group. Skill comprehension and quality were superior in the alternative group, as evidenced by fewer gross lesions at necropsy. Overall, personnel in the alternative group provided positive feedback with regard to the use of fewer mice, acquisition of both skill and confidence, and benefits for compassion fatigue. The use of this model is now our standard approach for training personnel in cardiac blood collection in mice. Our results demonstrate that the use of models in training curricula can enhance skill development and reduce the use of mice.

Hormonal contraception is an effective, reversible tool for managing birth rates in humans and nonhuman animals alike. However, manipulating reproductive hormones has behavioral consequences that can impact social and sexual behavior between conspecifics. First, we studied 18 pairs of nonreproductive titi monkeys (Plecturocebus cupreus) to test the efficacy of a novel method of hormonal contraception (deslorelin acetate implants) on reproductive hormone cycling in females and found significant reductions in urinary estrogens and progestagens among treated females compared to untreated controls. We then studied 35 nonreproductive pairs of coppery titi monkeys (Plecturocebus cupreus) to ascertain whether treating females with one of 2 different forms of hormonal contraception (deslorelin acetate implants (n = 17) or medroxyprogesterone acetate injections (n = 9)) would influence the relationship between pair mates compared to the relationship between untreated females and their vasectomized male mates (n = 9). Over a 5-month period, we found no differences in affiliative behaviors between pairs containing untreated females compared to pairs in which the female was treated with either deslorelin acetate or medroxyprogesterone acetate. Similarly, we found no differences in affiliation between pairs in the 2 treatment groups. This study is the first to examine behavioral consequences of hormonal contraception in a pair-bonding species. The results are encouraging for captive, managed breeding colonies of such social animals, especially those used in behavioral research.

Relatedness and kinship structure in matrilines are a potential source of social stability. The current study aimed to analyze the extant pedigrees of 6 living matrilines in different field cages to assess rates of cross-generational inbreeding and loss of genetic variation over time. All 6 matrilines showed increasing levels of inbreeding over generation time, although the rates of increase were different. The female-to-male-adult sex ratio was correlated with average matriline inbreeding levels, while the number of adult males was positively correlated with average matriline genetic diversity. Over five times more paternal half-sibs than maternal half-sibs were present because paternity had been restricted to a few males yearly. Therefore, the relatedness through the paternal lines was over five times greater than that of the maternal lines. Overall, each matriline lost low to moderate levels of genetic variation with time. The current rates of gene flow between field cages by cross-fostered infants have not stopped inbreeding within these matrilines or loss of diversity due to genetic drift. This situation probably developed because translocated animals, especially males, may not breed successfully. Only 4 of the 22 translocated individuals, all females, eventually reproduced, resulting in 13 offspring and generating an overall breeding success of 0.59 across all 6 study matrilines. However, even this low rate of reproduction by the translocated animals reduced inbreeding and kinship among matrilines and increased genetic heterogeneity in the matrilines. Based on this study, we propose several colony management strategies, including equalizing adult sex ratios to increase the effective population size in the field cages, increasing the number of cross-fostered infants, and relying more on multigenerational pedigree data to aid the alignment of genetic and behavioral management techniques.

The use of artificial plants as environmental enrichment for zebrafish (Danio rerio) in biomedical research facilities has been shown to provide benefits in animal welfare and care. Despite the benefits of artificial plants to zebrafish welfare, some research facilities are hesitant to incorporate them into their routine husbandry practices due to concerns about disease transmission and a lack of guidance on effective disinfection practices between tanks. Limited published information is available on how to adequately disinfect artificial plants, which creates concerns regarding their reuse between tanks in recirculating water systems. Proper sanitation and disinfection of these items is crucial to preventing the spread of disease in the system. We evaluated 2 disinfection methods– a commercial-grade laboratory glassware dishwasher and an ethylene oxide (ETO) sterilizer–by using ATP detection and bacterial culture of the artificial plants before and after the disinfection process. Plants were placed in the dirty sump of 2 separate recirculating systems (2,500 to 3,000 fish per system) for 2 wk before the start of the study. High ATP levels and various bacterial organisms were detected prior to disinfection. The commercial-grade labo- ratory glassware dishwasher and ETO sterilizer both significantly reduced ATP levels and resulted in complete eradication of live bacteria that were present before treatment. This study demonstrates 2 effective methods for disinfecting artificial plants in zebrafish facilities.

Environmental enrichment is a necessary component of all research vivarium settings. However, appropriate enrichment decisions vary greatly depending on the species involved and the research use of the animals. The increasing use of ferrets in research settings—notably for modeling the pathogenicity and transmissibility of viral pathogens that require containment in ABSL-2 to -4 environments—presents a particular challenge for veterinary and research staff to ensure that enrichment needs for these animals are met consistently. Here, we discuss the species-specific enrichment needs of ferrets, enrichment considerations for ferrets housed in research settings, and the challenges and importance of providing appropriate enrichment during experimentation, including when ferrets are housed in high-containment facilities. This article is organized to support the easy availability of information that will facilitate the design and implementation of optimal environmental enrichment for ferrets used in diverse research efforts in vivarium settings.

Sterility in male NHP has long been achieved through surgical castration or vasectomy. However, these techniques are irreversible, require a surgical procedure, and have potential consequences such as sperm granulomas and long recovery time. Deslorelin is a gonadotropin-releasing hormone agonist that temporarily and reversibly suppresses sex hormone secretion. Our goal in this study was to investigate the effects of deslorelin on testosterone secretion and testicular volume in male rhesus macaques (Macaca mulatta). Male macaques (n = 4) each received two, 4.7-mg deslorelin implants subcutaneously in the interscapular region. Serum testosterone and testicular volume were then monitored at specific time points until 10 mo after treatment. Testosterone suppression was defined as testosterone levels lower than 0.6 ng/mL for a sustained period of at least 30 d. After implantation, mean testicular volume was significantly reduced by day 121. Testosterone suppression was observed in all subjects. However, the time from implantation to testosterone suppression and duration of suppression varied. Two macaques were hormonally suppressed by day 26 after implantation and remained suppressed for at least 6 mo. The other 2 macaques were hormonally suppressed by 2 mo after implantation; of these two, one remained suppressed for 70 days while the other was suppressed for at least 245 days. We conclude that deslorelin can safely suppress testosterone secretion in male rhesus macaques, but individual variation in onset and duration of action should be considered when establishing reimplantation time points and potential return to reproductive activity.

This study compared the therapeutic effects in mice of 3 different formulations of buprenorphine. These formulations were standard buprenorphine hydrochloride (Bup-HCL) and 2 different extended-release buprenorphine formulations (Bup-ER and Ethiqa-XR [Bup-XR]). Drugs were evaluated based on their ability to attenuate thermal hypersensitivity in a mouse plantar incisional pain model. We hypothesized that Bup-HCL would attenuate postoperative thermal hypersensitivity at 20 min after administration, and that Bup-ER and Bup-XR would attenuate thermal hypersensitivity at 40 min after administration. Male C57BL6/J mice were randomly assigned to 1 of 4 treatment groups: 1) saline, 5 mL/kg SC, once; 2) Bup-HCL, 0.1 mg/kg SC, once; 3) Bup-ER, 1 mg/kg, SC, once; and 4) Bup-XR, 3.25 mg/kg, SC, once. Thermal hypersensitivity was assessed on the day before surgery and again on the day of surgery at 20, 40, 60, 90, and 120 min after drug administration. Thermal hypersensitivity after surgery was not different among the Bup-HCL, Bup-ER and Bup-XR groups at any timepoint. In addition, all buprenorphine treatment groups showed significantly less thermal hypersensitivity after surgery than did the saline group. Subjective observations suggested that mice that received Bup-ER or Bup-XR became hyperactive after drug administration (83 and 75% of mice tested, respectively). Our results indicate that Bup-HCL, Bup-ER, or Bup-XR attenuate thermal hyper- sensitivity related to foot incision by 20 min after administration.

Both the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and Regulations require animals in research to receive adequate analgesia unless an exception can be scientifically justified and IACUC approved. Extended- release buprenorphine (BUP-XR) is a pharmaceutical-grade formulation that is FDA-indexed for use in mice and rats. However, this new formulation has not been evaluated in adult Mongolian gerbils (Meriones unguiculatus). Our goal was to determine whether the extrapolated dose (1 mg/kg SC) would achieve plasma buprenorphine concentrations above the murine therapeutic threshold (> 1.0 ng/mL) in male and female gerbils. We hypothesized that BUP-XR administered at 1 mg/kg would achieve the murine therapeutic threshold in both male and female gerbils until at least 48 h after injection. Gerbils received one injection of BUP-XR (1 mg/kg SC) and underwent 4 serial blood collections (0.5, 1, 2, and 4, or 0.5, 24, 48, and 72 h after injection). The average plasma buprenorphine concentrations were above 1 ng/mL within 30 min of administration for both males and females. Plasma buprenorphine concentrations remained above 1.0 ng/mL for 48 h after administration. In males, plasma buprenorphine concentrations were significantly higher at 1 h after injection as compared with females; no other significant differences were observed between sexes. Mild to moderate injection-site granulomas were observed in five of nine gerbils, presumably due to the lipid matrix of the BUP-XR formulation. Our findings demonstrate that a single BUP-XR dose (1 mg/kg SC) achieves plasma buprenorphine levels that remain above the murine therapeutic threshold of 1.0 ng/mL for up to 48 h in both sexes.

The minimization of pain in research animals is a scientific and ethical necessity. Carprofen is commonly used for pain management in mice; however, some data suggest that the standard dosage of 5 mg/kg may not provide adequate analgesia after surgery. We hypothesized that a higher dose of carprofen in mice would reduce pain-associated behaviors and improve analgesia without toxic effects. A pharmacokinetic study was performed in mice given carprofen subcutaneously at 10 or 20 mg/kg. Plasma concentrations were measured at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after dosing (n = 3 per time point and treatment). At these doses, plasma levels were above the purported therapeutic level for at least 12 and 24 h, respectively, with respective half-lives of 14.9 and 10.2 h. For the efficacy study, 10 mice per group received anesthesia with or without an ovariectomy. Mice were then given 5 or 10 mg/kg of carprofen, or saline subcutaneously every 12 h. Orbital tightening, arched posture, wound licking, rearing, grooming, nesting behavior, and activity were assessed before surgery and at 4, 8, 12, 24, and 48 h after surgery. The von Frey responses were assessed before and at 4, 12, 24, and 48 h after surgery. The efficacy study showed that all surgery groups had significantly higher scores for orbital tightening, arched posture, and wound licking than did the anesthesia-only groups at 4, 8, 12, and 24-h time points. At the 8 h time point, the surgery groups treated with carprofen had significantly lower arched posture scores than did the surgery group treated with saline only. No significant differences were found between carprofen treatment groups for rearing, grooming, von Frey, activity, or nesting behavior at any time point. These results indicate that subcutaneous carprofen administered at these doses does not provide sufficient analgesia to alleviate postoperative pain in female CD1 mice.

Exposure to CO₂ gas is a common rodent euthanasia method. CO₂ activates nociceptors in rats and is painful to humans at concentrations equal to or greater than 32.5% The concentration of CO₂ at which rodents become unconsciousness is inadequately defined. We used loss of righting reflex (LORR) to identify the concentration at which CO₂ caused loss of consciousness in C57Bl/6, CD1 and 129P3J mice (16 females and 16 males per strain). We used a custom built, rotating, motorized cylinder to determine LORR as CO₂ concentrations were increased. Two LORR assessment methods were used: 1) a 1-Paw assessment in which the righting reflex was considered to be present if one or more paws contacted the cylinder after rotation into dorsal recumbency and 2) a 4-Paw assessment in which the righting reflex was considered to be present only if all 4 paws contacted the cylinder. LORR test data were analyzed with Probit regression and dose response curves were plotted. 1-Paw EC₉₅ values (CO₂ concentration at which LORR occurred for 95% of the population) were: C57Bl/6; 30.7%, CD1; 26.2%, 129P3J; 20.1%. The EC₉₅ for C57Bl/6 was significantly higher than that of the 129P3J mice, with no significant differences between other strains. Four-Paw EC₉₅ values were: C57Bl/6; 22.8%, CD1; 25.3%, 129P3J; 20.1%. Values for 129P3J mice were significantly lower than those of CD1 mice), with no significant difference between other strains. The EC₉₅ varied significantly between 1-Paw and 4-Paw methods only for C57Bl/6 mice. These results suggest a potential for nociception and pain to occur in some individuals of some mouse strains during CO₂ euthanasia.

Small animal physiology studies are often complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron radiation facilities. This is because these facilities are typically not designed specifically for biomedical research, and the animals and image detectors are located away from the researchers in a radiation enclosure. In respiratory X-ray imaging studies one challenge is the detection of respiration in free-breathing anaesthetised rodents, to enable images to be acquired at specific phases of the breath and for detecting changes in respiratory rate. We have previously used a Philtec RC60 sensor interfaced to a PowerLab data acquisition system and custom-designed timing hub to perform this task. Here we evaluated the Panasonic HL-G108 for respiratory sensing. The performance of the two sensors for accurate and reliable breath detection was directly compared using a single anesthetized rat. We also assessed how an infrared heat lamp used to maintain body temperature affected sensor performance. Based on positive results from these comparisons, the HL-G108 sensor was then used for respiratory motion detection in tracheal X-ray imaging studies of 21 rats at the SPring-8 Synchrotron, including its use for gated image acquisition. The results of that test were compared to a similar imaging study that used the RC60 for respiratory detection in 19 rats. Finally, the HL-G108 sensor was tested on 5 mice to determine its effectiveness on smaller species. The results showed that the HL-G108 is much more robust and easier to configure than the RC60 sensor and produces an analog signal that is amenable to stable peak detection. Furthermore, gated image acquisition produced sequences with substantially reduced motion artefacts, enabling the additional benefit of reduced radiation dose through the application of shuttering. Finally, the mouse experiments showed that the HL-G108 is equally capable of detecting respiration in this smaller species.