A Comparison of LED with Fluorescent Lighting on the Stress, Behavior, and Reproductive Success of Laboratory Zebra Finches (Taeniopygia guttata).

There are limited evidence-based husbandry recommendations for laboratory zebra finches (Taeniopygia guttata), including appropriate light sources. Light-emitting diode (LED) technology has been shown to improve circadian regulation and reduce stress in some laboratory animal species, such as mice and rats, when compared with cool-white fluorescent (CWF) lighting, but the effects of LED lighting on zebra finches have not been published. We compared the effects of broad-spectrum, blue-enriched (6,500 Kelvin) CWF and flicker-free LED lighting on the behavior, stress, and reproductive outcomes of indoor-housed zebra finches. Using breeding pairs housed in cubicles illuminated with either CWF or LED lighting, we compared the reproductive output as determined by clutch size, hatching rate, and hatchling survival rate. We also compared the behavior of group-housed adult males, first housed under CWF followed by LED lighting, using video recordings and an ethogram. Fecal samples were collected from these males at the end of each recording period, and basal fecal corticosterone metabolite (FCM) levels were compared. A FCM assay for adult male zebra finches was validated for efficacy and accuracy using a capture-restraint acute stress response and parallelism analysis, respectively. The breeding pairs had no significant difference in the clutch size or percent hatching rate, but percent hatchling survival improved under LED with an increased proportion achieving 100% survival. There was no significant difference in FCM between the lighting treatments. However, the activity budgets of the birds were altered, with a reduction in flighted movement and an increase in enrichment manipulation under LED. Overall, these results support the use of blue-enriched, broad-spectrum flicker-free LED as a safe alternative to CWF lighting for breeding and nonbreeding indoor-housed zebra finches.

Bibliometric analysis on cannibalism/infanticide and maternal aggression towards pups in laboratory rodents.

Animal welfare has evolved during the past decades to improve not only the quality of life of laboratory rodents but also the quality and reproducibility of scientific investigations. Bibliometric analysis has become an important tool to complete the current knowledge with academic databases. Our objective was to investigate whether scientific research on cannibalism/infanticide is connected with maternal aggression towards the offspring in laboratory rodents. To carry out our research, we performed a specific search for published articles on each concept. Results were analyzed in the open-source environment RStudio with the package Bibliometrix. We obtained 253 and 134 articles for the first search (cannibalism/infanticide) and the second search (maternal aggression towards the pups) respectively. We observed that the interest in infanticide/cannibalism started in the 1950s, while researchers started showing interest in maternal aggression towards the pups 30 years later. Our analyses indicated that maternal aggression had better citations in scientific literature. In addition, although our results showed some common features (e.g. oxytocin or medial preoptic area in the brain), we observed a gap between cannibalism/infanticide and maternal aggression towards the pups with only 14 published articles in common for both the searches. Therefore, we recommend researchers to combine both concepts in further investigations in the context of cannibalism for better dissemination and higher impact in laboratory rodents' welfare research.

The grimace scale: a useful tool for assessing pain in laboratory animals.

Accurately and promptly assessing pain in experimental animals is extremely important to avoid unnecessary suffering of the animals and to enhance the reproducibility of experiments. This is a key concern for veterinarians, animal caretakers, and researchers from the perspectives of veterinary care and animal welfare. Various methods including ethology, immunohistochemistry, electrophysiology, and molecular biology are used for pain assessment. However, the grimace scale, which was developed by taking cues from interpreting pain through facial expressions of non-verbal infants, has become recognized as a very simple and practical method for objectively evaluating pain levels by scoring changes in an animal's expressions. This method, which was first implemented with mice approximately 10 years ago, is now being applied to various experimental animals and is widely used in research settings. This review focuses on the usability of the grimace scale from the "cage-side" perspective, aiming to make it a more user-friendly tool for those involved in animal experiments. Differences in facial expressions in response to pain in various animals, examples of applying the grimace scale, current automated analytical methods, and future prospects are discussed.

Evaluation of Vibration as an Extrinsic Variable in In Vivo Research.

Vibration is inherent in research animal facilities due to the mechanical systems and practices required for animal care and use. Ample evidence indicates that vibration can change behavior and physiology in multiple species, potentially altering the results of research studies. Although one cannot eliminate environmental vibration, its control is important in research animal environments to decrease the possibility of introducing a research variable due to vibration effects. To assess the potential for a vibration source to alter experimental results and variability, one must understand the principles of vibration, its likely sources, and control methods. The literature regarding the effects of vibration, as it applies in a practical sense, can be challenging to interpret because the vibration frequencies tested to date have often not been within or near the most sensitive ranges of the species being tested. Some previous studies have used unrealistic vibration magnitudes and provided insufficient detail to duplicate or build upon conclusions. Standardization is essential for research examining the effects of vibration on animals to validate knowledge of this extrinsic variable in animal research and identify ways to mitigate the variable in research facilities.

Light: An Extrinsic Factor Influencing Animal-based Research.

Light is an environmental factor that is extrinsic to animals themselves and that exerts a profound influence on the regulation of circadian, neurohormonal, metabolic, and neurobehavioral systems of all animals, including research animals. These widespread biologic effects of light are mediated by distinct photoreceptors-rods and cones that comprise the conventional visual system and melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs) of the nonvisual system that interact with the rods and cones. The rods and cones of the visual system, along with the ipRGCs of the nonvisual system, are species distinct in terms of opsins and opsin concentrations and interact with one another to provide vision and regulate circadian rhythms of neurohormonal and neurobehavioral responses to light. Here, we review a brief history of lighting technologies, the nature of light and circadian rhythms, our present understanding of mammalian photoreception, and current industry practices and standards. We also consider the implications of light for vivarium measurement, production, and technological application and provide simple recommendations on artificial lighting for use by regulatory authorities, lighting manufacturers, designers, engineers, researchers, and research animal care staff that ensure best practices for optimizing animal health and well-being and, ultimately, improving scientific outcomes.

Physiological differences between wild and captive animals: a century-old dilemma.

Laboratory-based research dominates the fields of comparative physiology and biomechanics. The power of lab work has long been recognized by experimental biologists. For example, in 1932, Georgy Gause published an influential paper in Journal of Experimental Biology describing a series of clever lab experiments that provided the first empirical test of competitive exclusion theory, laying the foundation for a field that remains active today. At the time, Gause wrestled with the dilemma of conducting experiments in the lab or the field, ultimately deciding that progress could be best achieved by taking advantage of the high level of control offered by lab experiments. However, physiological experiments often yield different, and even contradictory, results when conducted in lab versus field settings. This is especially concerning in the Anthropocene, as standard laboratory techniques are increasingly relied upon to predict how wild animals will respond to environmental disturbances to inform decisions in conservation and management. In this Commentary, we discuss several hypothesized mechanisms that could explain disparities between experimental biology in the lab and in the field. We propose strategies for understanding why these differences occur and how we can use these results to improve our understanding of the physiology of wild animals. Nearly a century beyond Gause's work, we still know remarkably little about what makes captive animals different from wild ones. Discovering these mechanisms should be an important goal for experimental biologists in the future.

Microbial experience through housing in a farmyard-type environment alters intestinal barrier properties in mouse colons.

To close the gap between ultra-hygienic research mouse models and the much more environmentally exposed conditions of humans, we have established a system where laboratory mice are raised under a full set of environmental factors present in a naturalistic, farmyard-type habitat-a process we have called feralization. In previous studies we have shown that feralized (Fer) mice were protected against colorectal cancer when compared to conventionally reared laboratory mice (Lab). However, the protective mechanisms remain to be elucidated. Disruption of the protective intestinal barrier is an acknowledged player in colorectal carcinogenesis, and in the current study we assessed colonic mucosal barrier properties in healthy, feralized C57BL/6JRj male mice. While we found no effect of feralization on mucus layer properties, higher expression of genes encoding the mucus components Fcgbp and Clca1 still suggested mucus enforcement due to feralization. Genes encoding other proteins known to be involved in bacterial defense (Itln1, Ang1, Retnlb) and inflammatory mechanisms (Zbp1, Gsdmc2) were also higher expressed in feralized mice, further suggesting that the Fer mice have an altered intestinal mucosal barrier. These findings demonstrate that microbial experience conferred by housing in a farmyard-type environment alters the intestinal barrier properties in mice possibly leading to a more robust protection against disease. Future studies to unravel regulatory roles of feralization on intestinal barrier should aim to conduct proteomic analyses and in vivo performance of the feralized mice intestinal barrier.

Behavioral analysis in laboratory rats: Challenges and usefulness of 50-kHz ultrasonic vocalizations.

Many rodent species emit and detect vocalizations in the ultrasonic range. Rats use three classes of ultrasonic vocalizations depending on developmental stage, experience and the behavioral situation. Calls from one class emitted by juvenile and adult rats, the so-called 50-kHz calls, are typical for appetitive and social situations. This review provides a brief historical account on the introduction of 50-kHz calls in behavioral research followed by a survey of their scientific applications focusing on the last five years, where 50-kHz publications reached a climax. Then, specific methodological challenges will be addressed, like how to measure and report 50-kHz USV, the problem of assignment of acoustic signals to a specific sender in a social situation, and individual variability in call propensity. Finally, the intricacy of interpreting 50-kHz results will be discussed focusing on the most prevalent ones, namely as communicative signals and/or readouts of the sender's emotional status.

An assessment of ambient noise and other environmental variables in a nonhuman primate housing facility.

Acoustic noise and other environmental variables represent potential confounds for animal research. Of relevance to auditory research, sustained high levels of ambient noise may modify hearing sensitivity and decrease well-being among laboratory animals. The present study was conducted to assess environmental conditions in an animal facility that houses nonhuman primates used for auditory research at the Vanderbilt University Medical Center. Sound levels, vibration, temperature, humidity and luminance were recorded using an environmental monitoring device placed inside of an empty cage in a macaque housing room. Recordings lasted 1 week each, at three different locations within the room. Vibration, temperature, humidity and luminance all varied within recommended levels for nonhuman primates, with one exception of low luminance levels in the bottom cage location. Sound levels at each cage location were characterized by a low baseline of 58-62 dB sound pressure level, with transient peaks up to 109 dB sound pressure level. Sound levels differed significantly across locations, but only by about 1.5 dB. The transient peaks beyond recommended sound levels reflected a very low noise dose, but exceeded startle-inducing levels, which could elicit stress responses. Based on these findings, ambient noise levels in the housing rooms in this primate facility are within acceptable levels and unlikely to contribute to hearing deficits in the nonhuman primates. Our results establish normative values for environmental conditions in a primate facility, can be used to inform best practices for nonhuman primate research and care, and form a baseline for future studies of aging and chronic noise exposure.

Food composition can influence how much alcohol your animal model drinks: A mini-review about the role of isoflavones.

Standard laboratory diets used have similar concentrations of proteins, carbohydrates and fat, but the concentration of some micronutrients can vary considerably. For example, the concentration of isoflavones can vary between 20 mg and 600 mg per gram of diet. Exposure to different concentrations of isoflavones interacts with alcohol (EtOH) intake, thereby influencing the results of alcohol research. In this mini-review, we describe correlations between isoflavone concentrations and alcohol intake based on data from previously published work. Although the administration of low doses of isoflavones can decrease alcohol intake in rats, there is a positive correlation between the isoflavone content in diets and alcohol intake in mice. This interaction seems to depend on the dose, route of administration, and time of exposure to isoflavones and may be related to specific neurobiological mechanisms. The literature also indicates that isoflavones can interact with some of alcohol's molecular targets and with neural pathways crucial to the alcohol reward process. Given these findings, more attention should be given to the different types of laboratory diets used in alcohol studies to allow better comparison and replication of animal research.

Natural versus Laboratory World: Incorporating Wild-Derived Microbiota into Preclinical Rodent Models.

Advances in data collection (high-throughput shotgun metagenomics, transcriptomics, and metabolomics) and analysis (bioinformatics and multiomics) led to the realization that all mammals are metaorganisms, shaped not only by their own genome but also by the genomes of the microbes that colonize them. To date, most studies have focused on the bacterial microbiome, whereas curated databases for viruses, fungi, and protozoa are still evolving. Studies on the interdependency of microbial kingdoms and their combined effects on host physiology are just starting. Although it is clear that past and present exposure to commensals and pathogens profoundly affect human physiology, such exposure is lacking in standard preclinical models such as laboratory mice. Laboratory mouse colonies are repeatedly rederived in germ-free status and subjected to restrictive, pathogen-free housing conditions. This review summarizes efforts to bring the wild microbiome into the laboratory setting to improve preclinical models and their translational research value.

LiftPose3D, a deep learning-based approach for transforming two-dimensional to three-dimensional poses in laboratory animals.

Markerless three-dimensional (3D) pose estimation has become an indispensable tool for kinematic studies of laboratory animals. Most current methods recover 3D poses by multi-view triangulation of deep network-based two-dimensional (2D) pose estimates. However, triangulation requires multiple synchronized cameras and elaborate calibration protocols that hinder its widespread adoption in laboratory studies. Here we describe LiftPose3D, a deep network-based method that overcomes these barriers by reconstructing 3D poses from a single 2D camera view. We illustrate LiftPose3D's versatility by applying it to multiple experimental systems using flies, mice, rats and macaques, and in circumstances where 3D triangulation is impractical or impossible. Our framework achieves accurate lifting for stereotypical and nonstereotypical behaviors from different camera angles. Thus, LiftPose3D permits high-quality 3D pose estimation in the absence of complex camera arrays and tedious calibration procedures and despite occluded body parts in freely behaving animals.

Evaluation of an enrichment programme for a colony of long-tailed macaques (Macaca fascicularis) in a rescue centre.

Long-tailed macaques are highly social primates that are commonly used in biomedical research as animal models. The aim of this study was to evaluate the effects of different kinds of enrichment on the behaviour and faecal cortisol metabolite (FCM) level in a colony of ex-laboratory long-tailed macaques during a programme of rehabilitation. The research was carried out in three periods, divided into two sessions each. Every period was composed of one control session (SC) and one session characterised by one type of enrichment: feeding enrichment (FE), manipulative enrichment (ME), and the last session during which manipulative and feeding enrichment were provided every day but in a mixed way (MIX). The results showed that manipulative and mixed enrichments caused positive changes to the activity budget of the colony, with a decrease in abnormal behaviour rates and an increase in play compared with control sessions. The rate of affiliative behaviours and low rate of aggression were probably because the group was composed mostly of females and it was stable, with a well-defined hierarchy. The research underlines the importance of a well-studied enrichment programme for the welfare of captive animals, which should exploit species-specific motivations.

Biochemistry, Doppler echocardiography, and electrocardiography evaluation in captive owl monkeys (Aotus sp).

BACKGROUND: The cardiovascular system of owl monkeys has been studied due to frequent postmortem findings of heart disease in asymptomatic animals. The silent aspect and the difficulty of early diagnosis intensify the importance of studying the cardiovascular system in this species. METHODS: Echocardiogram evaluation was carried out on 60 animals, grouped into suspect or non-suspect of having heart diseases, and evaluated through electrocardiogram, hematology, and biochemical tests. RESULTS: Doppler echocardiography indicated two animals with suspicion of left ventricular hypertrophy and eight with dilated cardiomyopathy. Suspect animals had higher cardiac measurements and reduced shortening fraction. Troponin I was detectable in two animals (0.128 ng/mL and 0.584 ng/mL), and serum albumin concentration was significantly higher in non-suspect animals (P < .05). CONCLUSIONS: The importance of echocardiographic measurements of IVSd, IVSs, LVIDd, LVIDs, LVPWd, LVPWs, LA, EF, and FS in the cardiac evaluation of captive owl monkeys was evidenced.

The acoustic startle reflex as a tool for assessment of odor environment effects on affective states in laboratory mice.

Apart from self and conspecific odors, odors from other species also influence the affective states in laboratory mice (Mus musculus musculus) in their home cages and during experimental procedures, possibly inducing confusion and inconsistency in experimental data. Thus, it is important to detect the types of animal odors associated with housing, husbandry, and laboratory practice that can arouse different types of affective changes in mice. Here, we aimed to test the effectiveness of the acoustic startle reflex (ASR) in detecting changes in the affective states of laboratory mice due to animal-derived-odor as it has a non-zero baseline, and can be enhanced or attenuated by positive or negative affective shifts, respectively. We used ASR to examine the affective changes in mice that were induced by bedding odors and an alarm pheromone. The odor of bedding obtained from the mice' home cages significantly attenuated the ASR, suggesting positive affective shifts in the test mice, whereas that from bedding obtained from rat cages significantly enhanced the ASR, suggesting negative affective shifts. No significant changes in ASR were observed in mice presented with the odor of bedding obtained from cages of unfamiliar conspecifics. In contrast, there was significant ASR enhancement in mice exposed to volatile components of alarm pheromones trapped in water, suggesting negative affective shifts. Thus, our findings show that ASR may be a valuable tool in assessing the effects of odors on the affective states in laboratory mice.

Short communication: Domesticated and wild fathead minnows differ in growth and thermal tolerance.

Many populations have evolved in response to laboratory environments (lack of predators, continual food availability, etc.). Another potential agent of selection in the lab is exposure to constant thermal environments. Here, we examined changes in growth, critical thermal maximum (CTmax), and food consumption under constant (25 °C) and fluctuating (22-28 °C and 19-31 °C) conditions in two populations of fathead minnows, Pimephales promelas: one that has been kept in a laboratory setting for over 120 generations (~40 years) and a corresponding wild one. We found that under thermal fluctuations, domesticated fathead minnows grew faster than their wild counterparts, but also exhibited lower thermal tolerance. Food consumption was significantly higher in the lab population under the constant and large fluctuation thermal treatments. Our results suggest that the lab population has adjusted to the stable conditions in the laboratory and that we should carefully apply lessons learned in the lab to wild populations.

Impact of three commonly used blood sampling techniques on the welfare of laboratory mice: Taking the animal's perspective.

Laboratory mice are the most frequently used animals in biomedical research. In accordance with guidelines for humane handling, several blood sampling techniques have been established. While the effects of these procedures on blood quality and histological alterations at the sampling site are well studied, their impact on the animals' welfare has not been extensively investigated. Therefore, our study aimed to compare three commonly used blood sampling techniques regarding their effects on different indicators of animal welfare, including physiological and behavioural response stress parameters, including pain measures, home-cage behaviour and nest-building as well as exploratory activity and neophobia. Male C57BL/6J mice were subjected to a single blood collection from either the vena facialis, the retrobulbar sinus or the tail vessel, or were allocated to the respective control treatment. While all blood sampling techniques led to an acute increase in plasma corticosterone levels, the response was strongest in animals that underwent sampling from the vena facialis and the retrobulbar sinus. Similar results were observed when the time-course of adrenocortical activity was monitored via corticosterone metabolites from faecal samples. Blood collection from the vena facialis and the retrobulbar sinus also decreased exploration of novel stimuli, resulted in decreased nest-building activity and induced higher scores in the Mouse Grimace Scale. Moreover, locomotor activity and anxiety-related behaviour were strongly affected after facial vein bleeding. Interestingly, tail vessel bleeding only induced little alterations in the assessed physiological and behavioural parameters. Importantly, the observed effects in all treatment groups were no longer detectable after 24 hours, indicating only short-term impacts. Thus, by also taking the animal's perspective and comprehensively assessing the severity of the particular sampling procedures, the results of our study contribute to Refinement within the 3R concept and allow researchers to objectively select the most appropriate and welfare-friendly blood sampling technique for a given experiment.

Seasonal weight changes in laboratory ferrets.

Ferrets (Mustela putorius furo) are a valuable animal model used in biomedical research. Like many animals, ferrets undergo significant variation in body weight seasonally, affected by photoperiod, and these variations complicate the use weight as an indicator of health status. To overcome this requires a better understanding of these seasonal weight changes. We provide a normative weight data set for the female ferret accounting for seasonal changes, and also investigate the effect of fluid regulation on weight change. Female ferrets (n = 39) underwent behavioural testing from May 2017 to August 2019 and were weighed daily, while housed in an animal care facility with controlled light exposure. In the winter (October to March), animals experienced 10 hours of light and 14 hours of dark, while in summer (March to October), this contingency was reversed. Individual animals varied in their body weight from approximately 700 to 1200 g. However, weights fluctuated with light cycle, with animals losing weight in summer, and gaining weight in winter such that they fluctuated between approximately 80% and 120% of their long-term average. Ferrets were weighed as part of their health assessment while experiencing water regulation for behavioural training. Water regulation superimposed additional weight changes on these seasonal fluctuations, with weight loss during the 5-day water regulation period being greater in summer than winter. Analysing the data with a Generalised Linear Model confirmed that the percentage decrease in weight per week was relatively constant throughout the summer months, while the percentage increase in body weight per week in winter decreased through the season. Finally, we noted that the timing of oestrus was reliably triggered by the increase in day length in spring. These data establish a normative benchmark for seasonal weight variation in female ferrets that can be incorporated into the health assessment of an animal's condition.

High laboratory mouse pre-weaning mortality associated with litter overlap, advanced dam age, small and large litters.

High and variable pre-weaning mortality is a persistent problem in laboratory mouse breeding. Assuming a modest 15% mortality rate across mouse strains, means that approximately 1 million more pups are produced yearly in the EU to compensate for those which die. This paper presents the first large study under practical husbandry conditions to determine the risk factors associated with mouse pre-weaning mortality. We analysed historical records from 219,975 pups from two breeding facilities, collected as part of their management routine and including information on number of pups born and weaned per litter, parents' age and identification, and dates of birth and death of all animals. Pups were counted once in their first week of life and at weaning, and once every one or two weeks, depending on the need for cage cleaning. Dead pups were recorded as soon as these were found during the daily cage screening (without opening the cage). It was hypothesized that litter overlap (i.e. the presence of older siblings in the cage when new pups are born), a recurrent social configuration in trio-housed mice, is associated with increased newborn mortality, along with advanced dam age, large litter size, and a high number and age of older siblings in the cage. The estimated probability of pup death was two to seven percentage points higher in cages with litter overlap compared to those without. Litter overlap was associated with an increase in death of the entire litter of five and six percentage points, which represent an increase of 19% and 103% compared to non-overlapped litters in the two breeding facilities, respectively. Increased number and age of older siblings, advanced dam age, small litter size (less than four pups born) and large litter size (over 11 pups born) were associated with increased probability of pup death.