Pebble to the Metal: A Boulder Approach to Enrichment for Danio rerio.

Zebrafish are an established and widely used animal model, yet there is limited understanding of their welfare needs. Despite an increasing number of studies on zebrafish enrichment, in-tank environmental enrichment remains unpopular among researchers. This is due to perceived concerns over health/hygiene when it comes to introducing enrichment into the tank, although actual evidence for this is sparse. To accommodate this belief, regardless of veracity, we tested the potential benefits of enrichments presented outside the tank. Thus, we investigated the preferences and physiological stress of zebrafish with pictures of pebbles placed underneath the tank. We hypothesized that zebrafish would show a preference for enriched environments and have lower stress levels than barren housed zebrafish. In our first experiment, we housed zebrafish in a standard rack system and recorded their preference for visual access to a pebble picture, with two positive controls: visual access to conspecifics, and group housing. Using a crossover repeated-measures factorial design, we tested if the preference for visual access to pebbles was as strong as the preference for social contact. Zebrafish showed a strong preference for visual access to pebbles, equivalent to that for conspecifics. Then, in a second experiment, tank water cortisol was measured to assess chronic stress levels of zebrafish housed with or without a pebble picture under their tank, with group housing as a positive control. Cortisol levels were significantly reduced in zebrafish housed with pebble pictures, as were cortisol levels in group housed zebrafish. In fact, single housed zebrafish with pebble pictures showed the same cortisol levels as group housed zebrafish without pebble pictures. Thus, the use of an under-tank pebble picture was as beneficial as being group housed, effectively compensating for the stress of single housing. Pebble picture enrichment had an additive effect with group housing, where group housed zebrafish with pebble pictures had the lowest cortisol levels of any treatment group.

Burlap and buddies: the effects of social enrichment (preweaning mixing) and object enrichment (burlap) on piglet performance, behavior, and welfare in the preweaning environment.

When weaned in commercial operations, piglets are not only separated from their sow but also mixed with unfamiliar pigs in an unfamiliar environment with a new diet. These abrupt changes can be stressful for piglets, often having negative welfare consequences. Our objective was to study the effects of early-life preweaning socialization and object enrichment in the preweaning environment. We compared piglet performance, behavior, and welfare across six treatments that combined multi-litter group size (1 vs. 2 vs. 4 litters) and burlap (yes vs. no). We recorded piglet behavior and lesion-scored sows and piglets. Normal conforming data, expressed per experimental unit (and behavior data were averaged over time), were analyzed by ANOVA. When given the opportunity in the sow barn, piglets in multi-litter groups socialized with other litters. Burlap use (P = 0.08) was observed in < 5% of the observations but tended to increase in mixed litter groups. Cross-sucking was observed in ~12% of the observations within mixed litter groups and tended to increase with mixed group size (P = 0.08). While there was no difference in the proportion of piglets nursing (P > 0.10), piglets were less active in the single crate groups and most active in the groups of two litters mixed (P = 0.03). Additionally, observed piglet/sow biting behaviors did not differ between treatments; however, piglet-piglet biting (P = 0.07), and pen object manipulation (P = 0.07) tended to be observed more frequently in non-enriched groups. Piglet displacements were observed more often in non-enriched groups around the pen (P = 0.03) but tended to be observed more often in enriched groups at the teat (P = 0.07). Preweaning socialization and object enrichment had no impact on the average number of piglets weaned per sow or total piglet mortality (P > 0.10). However, the proportion of laid-on piglets decreased as the number of mixed litters increased (P = 0.02). The average number of lesions per piglet did not differ between treatments. Although the final lesion scores of sow teat and udder condition did not differ between treatments (P > 0.10), sow udder scores tended to worsen more in the single litters than in the mixed litters (P = 0.08). Overall, social and object enrichment allows piglets to socialize at a younger age and to redirect their attention toward an object (burlap) which does not negatively impact piglet or sow performance, or behavior, and may improve piglet welfare around weaning.

Burlap and buddies: the effects of social enrichment (preweaning mixing) and object enrichment (burlap) on piglet behavior and welfare in the postweaning environment.

The process of weaning piglets in commercial swine operations subjects them to numerous abrupt and stressful changes often resulting in negative welfare consequences. The objective was to study the postweaning effects of early-life (1 to 3 d of age) preweaning socialization in multi-litter groups as well as object enrichment (burlap sheet) in the pre- and postweaning environment by comparing six treatments that combined mixing of one vs. two vs. four litters mixed preweaning with and without burlap provision. An ANOVA linear model was run on all normal data, expressed per experimental unit (and behavior data were averaged over time), while non-normal data were analyzed using the Kruskal-Wallis test. Non-enriched groups of piglets were observed manipulating pen objects more often than the enriched groups (P = 0.005). Biting behaviors, including the chewing of ears and tails of pen-mates but excluding fighting, were observed the least in groups of pigs of four litters mixed preweaning, while piglets that were not mixed preweaning were observed biting the most (P = 0.03). Piglets who were not mixed preweaning also manipulated the burlap more frequently than the piglets from groups of 4 litters mixed preweaning (P = 0.02). Biting (P < 0.001) and displacements (P = 0.03) and fighting (P = 0.002) throughout the pen were observed less in the enriched groups. There were fewer lesions per pig in the enriched groups vs. non-enriched groups initially (P = 0.07) and 1 wk after weaning (P = 0.10). Furthermore, pigs mixed in groups of four litters preweaning also tended to have lower lesion scores (P = 0.07) 1-wk postweaning compared to the other treatments. However, there were no differences between treatments in the proportion of piglets resting, eating/drinking, being active, or using the burlap, or for the observed frequency of displacements at the feeder, social behaviors, or belly nosing (P > 0.10). Overall, social enrichment encourages socialization with unfamiliar conspecifics at a younger age while object enrichment allows pigs to redirect their attention toward objects such as burlap. Both may improve pig behavior and welfare after weaning.

Practice does not make perfect: Juvenile object play does not improve the predation skills of adult mink in "simulated prey" tests.

In carnivores, juvenile object play is hypothesized to improve the development of adult predation ability. We tested this hypothesis in a model carnivore, the American mink (Neovison vison). Play was induced via the provision of diverse "enrichment" objects to 32 litters from 4 to 15 weeks of age on a rotating schedule; control mink (32 litters) were instead provided with one static object. Behavioral observations confirmed that enriched juveniles engaged in more object play than control juveniles (p < .01). Tenacity, strength, and chasing motivations were assessed in 52 mink at 27-29 weeks of age using five "simulated prey" tests. These tests showed internal consistency, with measures from the same test being weakly to very strongly correlated (r or ρ: |.33-.95|). Across tests assessing similar skills, some measures of tenacity and strength were also moderately correlated. Treatment had little effect, however, other than on maximum force during vertical pulling, in which control mink unexpectedly scored higher (1.01 ± 0.06 kg vs. 0.84 ± 0.06 kg, p = .0458). Although we did not find evidence to support the hypothesis that object play leads to improved predatory skills, the tests developed have the potential to be used in humane, prey-free future investigations of predation abilities.

Social status and previous experience in the group as predictors of welfare of sows housed in large semi-static groups.

Mixing gestating sows implies hierarchy formation and has detrimental consequences on welfare. The effects of social stress on the most vulnerable individuals may be underestimated and it is therefore important to evaluate welfare between individuals within groups. This study aimed at investigating the impact of social status and previous experience in the group on well-being of sows housed in large semi-static groups. We assessed aggression (d0 (mixing), d2, d27, d29), body lesions (d1, d26, d84) and feeding order on 20 groups of 46-91 animals. Social status was based on the proportion of fights won during a 6-hr observation period between d0 and d2. Dominants (29%) were those who won more fights than they lost, Subdominants (25%) won fewer fights than they lost, Losers (23%) never won any fight in which they were involved while Avoiders (23%) were never involved in fights. Resident sows (70%) were already present in the group in the previous gestation while New sows (30%) were newly introduced at mixing. Subdominants and Dominants were highly involved in fights around mixing but this was more detrimental for Subdominants than Dominants, Losers and Avoiders since they had the highest body lesion scores at mixing. Avoiders received less non-reciprocal agonistic acts than Losers on d2 (P = 0.0001) and had the lowest body lesion scores after mixing. However, Avoiders and Losers were more at risk in the long-term since they had the highest body lesions scores at d26 and d84. They were followed by Subdominants and then Dominants. New sows fought more (P<0.0001), tended to be involved in longer fights (P = 0.075) around mixing and had more body lesions throughout gestation than Resident sows. Feeding order from one-month post-mixing was influenced both by the previous experience in the group and social status (P<0.0001). New sows, especially with a low social status, are more vulnerable throughout gestation and could serve as indicators of non-optimal conditions.

The Role of Genetic Selection on Agonistic Behavior and Welfare of Gestating Sows Housed in Large Semi-Static Groups.

Confinement of gestating sows is becoming banished in favor of group-housing in countries worldwide, forcing breeding companies to develop genetic lines adapted for social living. This study aimed at assessing the influence of two genetic lines selected for high performance (HP1, HP2, derived from Landrace × Yorkshire) on welfare and reproductive performance of sows housed in large semi-static groups (20 groups of 46-91 animals) across several parities. To address this, agonistic behaviors were recorded on d0, d2, d27, and d29 post-mixing while body lesions were scored on d1, d26, and d84. Sows' individual and reproductive performances were also recorded. HP2 sows were more aggressive than HP1 sows since they fought (p = 0.028) and bullied (p = 0.0009) pen-mates more frequently on d0-d2. HP2 sows had more total body lesions throughout gestation than HP1 sows at higher parities (p < 0.0001). Regarding reproductive performance, HP2 sows lost less piglets (p < 0.0001) and tended to wean more piglets (p = 0.067) than HP1 sows. In conclusion, while HP2 sows were the most aggressive, HP1 sows had piglets with lower survivability, which raises ethical issues in both cases and points to the need of considering social aspects when developing genetic lines for group-housing.

The epidemiology of fighting in group-housed laboratory mice.

Injurious home-cage aggression (fighting) in mice affects both animal welfare and scientific validity. It is arguably the most common potentially preventable morbidity in mouse facilities. Existing literature on mouse aggression almost exclusively examines territorial aggression induced by introducing a stimulus mouse into the home-cage of a singly housed mouse (i.e. the resident/intruder test). However, fighting occurring in mice living together in long-term groups under standard laboratory housing conditions has barely been studied. We performed a point-prevalence epidemiological survey of fighting at a research institution with an approximate 60,000 cage census. A subset of cages was sampled over the course of a year and factors potentially influencing home-cage fighting were recorded. Fighting was almost exclusively seen in group-housed male mice. Approximately 14% of group-housed male cages were observed with fighting animals in brief behavioral observations, but only 14% of those cages with fighting had skin injuries observable from cage-side. Thus simple cage-side checks may be missing the majority of fighting mice. Housing system (the combination of cage ventilation and bedding type), genetic background, time of year, cage location on the rack, and rack orientation in the room were significant risk factors predicting fighting. Of these predictors, only bedding type is easily manipulated to mitigate fighting. Cage ventilation and rack orientation often cannot be changed in modern vivaria, as they are baked in by cookie-cutter architectural approaches to facility design. This study emphasizes the need to invest in assessing the welfare costs of new housing and husbandry systems before implementing them.

Somatosensory corticospinal tract axons sprout within the cervical cord following a dorsal root/dorsal column spinal injury in the rat.

The corticospinal tract (CST) is the major descending pathway controlling voluntary hand function in primates, and though less dominant, it mediates voluntary paw movements in rats. As with primates, the CST in rats originates from multiple (albeit fewer) cortical sites, and functionally different motor and somatosensory subcomponents terminate in different regions of the spinal gray matter. We recently reported in monkeys that following a combined cervical dorsal root/dorsal column lesion (DRL/DCL), both motor and S1 CSTs sprout well beyond their normal terminal range. The S1 CST sprouting response is particularly dramatic, indicating an important, if poorly understood, somatosensory role in the recovery process. As rats are used extensively to model spinal cord injury, we asked if the S1 CST response is conserved in rodents. Rats were divided into sham controls, and two groups surviving post-lesion for ~6 and 10 weeks. A DRL/DCL was made to partially deafferent one paw. Behavioral testing showed a post-lesion deficit and recovery over several weeks. Three weeks prior to ending the experiment, S1 cortex was mapped electrophysiologically, for tracer injection placement to determine S1 CST termination patterns within the cord. Synaptogenesis was also assessed for labeled S1 CST terminals within the dorsal horn. Our findings show that the affected S1 CST sprouts well beyond its normal range in response to a DRL/DCL, much as it does in macaque monkeys. This, along with evidence for increased synaptogenesis post-lesion, indicates that CST terminal sprouting following a central sensory lesion, is a robust and conserved response.

Spinal cord injury transiently alters Meissner's corpuscle density in the digit pads of macaque monkeys.

Meissner's corpuscles (MCs) are cutaneous mechanoreceptors found in glabrous skin and are exquisitely sensitive to light touch. Along with other receptors, they provide continuous sensory feedback that informs the execution of fine manual behaviors. Following cervical spinal deafferentation injuries, hand use can be initially severely impaired, but substantial recovery occurs over many weeks, even when ~95% of the original input is permanently lost. While most SCI research focuses on central neural pathway responses, little is known about the role of peripheral receptors in facilitating recovery. We begin to address this by asking the following: (1) What is the normal pattern of MCs in the distal pads of all five digits in the macaque monkey (with hands similar to humans)? (2) What happens to these receptors 4-5 months following either a dorsal column lesion (DCL) or a combined dorsal root/dorsal column lesion (DRL/DCL), when functional recovery is largely complete? (3) What happens chronically, 12-14 months later? Our findings show that in normal monkeys, MCs are densest in the distal pads of the opposing thumb and index finger, with the greatest concentration on the thumb. This reflects a close functional relationship between receptor density and precision grip. At 4-5 months post-injury, there was a (~30%) loss of MCs on the deafferented digits of the injured hand compared with the contralateral side. However, 12-14 months after a DRL/DCL, receptor densities had returned to normal levels. Our findings indicate a complex peripheral response and highlight the importance of the periphery in shaping central changes.

Automated monitoring of mouse feeding and body weight for continuous health assessment.

Routine health assessment of laboratory rodents can be improved using automated home cage monitoring. Continuous, non-stressful, objective assessment of rodents unaware that they are being watched, including during their active dark period, reveals behavioural and physiological changes otherwise invisible to human caretakers. We developed an automated feeder that tracks feed intake, body weight, and physical appearance of individual radio frequency identification-tagged mice in social home cages. Here, we experimentally induce illness via lipopolysaccharide challenge and show that this automated tracking apparatus reveals sickness behaviour (reduced food intake) as early as 2-4 hours after lipopolysaccharide injection, whereas human observers conducting routine health checks fail to detect a significant difference between sick mice and saline-injected controls. Continuous automated monitoring additionally reveals pronounced circadian rhythms in both feed intake and body weight. Automated home cage monitoring is a non-invasive, reliable mode of health surveillance allowing caretakers to more efficiently detect and respond to early signs of illness in laboratory rodent populations.

Adaptive developmental plasticity in rhesus macaques: the serotonin transporter gene interacts with maternal care to affect juvenile social behaviour.

Research has increasingly highlighted the role that developmental plasticity-the ability of a particular genotype to produce variable phenotypes in response to different early environments-plays as an adaptive mechanism. One of the most widely studied genetic contributors to developmental plasticity in humans and rhesus macaques is a serotonin transporter gene-linked polymorphic region (5-HTTLPR), which determines transcriptional efficiency of the serotonin transporter gene in vitro and modifies the availability of synaptic serotonin in these species. A majority of studies to date have shown that carriers of a loss-of-function variant of the 5-HTTLPR, the short (s) allele, develop a stress-reactive phenotype in response to adverse early environments compared with long (l) allele homozygotes, leading to the prevalent conceptualization of the s-allele as a vulnerability allele. However, this framework fails to address the independent evolution of these loss-of-function mutations in both humans and macaques as well as the high population prevalence of s-alleles in both species. Here we show in free-ranging rhesus macaques that s-allele carriers benefit more from supportive early social environments than l-allele homozygotes, such that s-allele carriers which receive higher levels of maternal protection during infancy demonstrate greater social competence later in life. These findings provide, to our knowledge, the first empirical support for the assertion that the s-allele grants high undirected biological sensitivity to context in primates and suggest a mechanism through which the 5-HTTLPR s-allele is maintained in primate populations.

Play and optimal welfare: Does play indicate the presence of positive affective states?

Play is commonly used to assess affective states in both humans and non-human animals. Play appears to be most common when animals are well-fed and not under any direct threats to fitness. Could play and playfulness therefore indicate pre-existing positive emotions, and thence optimal animal welfare? We examine this question by surveying the internal and external conditions that promote or suppress play in a variety of species, starting with humans. We find that negative affective states and poor welfare usually do suppress play (although there are notable exceptions where the opposite occurs). Furthermore, research in children suggests that beyond the frequency or total duration of play, poor welfare may additionally be reflected in qualitative aspects of this heterogeneous behaviour (e.g. display of solitary over social play; and the 'fragmentation' of play bouts) that are often overlooked in animals. There are surprisingly few studies of play in subjects with pre-existing optimal welfare or in unambiguously highly positive affective states, making it currently impossible to determine whether play can distinguish optimal or good welfare from merely neutral welfare. This therefore represents an important and exciting area for future research.

Introducing Therioepistemology: the study of how knowledge is gained from animal research.

This focus issue of Lab Animal coincides with a tipping point in biomedical research. For the first time, the scale of the reproducibility and translatability crisis is widely understood beyond the small cadre of researchers who have been studying it and the pharmaceutical and biotech companies who have been living it. Here we argue that an emerging literature, including the papers in this focus issue, has begun to congeal around a set of recurring themes, which themselves represent a paradigm shift. This paradigm shift can be characterized at the micro level as a shift from asking "what have we controlled for in this model?" to asking "what have we chosen to ignore in this model, and at what cost?" At the macro level, it is a shift from viewing animals as tools (the furry test tube), to viewing them as patients in an equivalent human medical study. We feel that we are witnessing the birth of a new discipline, which we term Therioepistemology, or the study of how knowledge is gained from animal research. In this paper, we outline six questions that serve as a heuristic for critically evaluating animal-based biomedical research from a therioepistemological perspective. These six questions sketch out the broad reaches of this new discipline, though they may change or be added to as this field evolves. Ultimately, by formalizing therioepistemology as a discipline, we can begin to discuss best practices that will improve the reproducibility and translatability of animal-based research, with concomitant benefits in terms of human health and animal well-being.

Play in juvenile mink: litter effects, stability over time, and motivational heterogeneity.

Mink are potentially ideal for investigating the functions of play: deleterious effects of early social isolation suggest a crucial developmental role for play; and huge numbers of highly playful juvenile subjects can be studied on farms. We collected descriptive data on 186 pairs from 93 litters, half provided with play-eliciting environmental enrichment objects in their home cages, to test three hypotheses: (1) play frequency is subject to litter effects; (2) relative playfulness is stable over time; (3) play sub-types share a single, common motivational basis. We found weak litter effects that were driven by stronger litter effects on general activity, and weakly stable individual differences in both total and rough-and-tumble play. Experimentally increasing object play did not inhibit rough-and-tumble play, showing these sub-types are not motivational substitutes. Frequencies of these sub-types were also uncorrelated, and changed differently with time of day and age, further supporting this conclusion.

Mixed-strain housing for female C57BL/6, DBA/2, and BALB/c mice: validating a split-plot design that promotes refinement and reduction.

BACKGROUND: Inefficient experimental designs are common in animal-based biomedical research, wasting resources and potentially leading to unreplicable results. Here we illustrate the intrinsic statistical power of split-plot designs, wherein three or more sub-units (e.g. individual subjects) differing in a variable of interest (e.g. genotype) share an experimental unit (e.g. a cage or litter) to which a treatment is applied (e.g. a drug, diet, or cage manipulation). We also empirically validate one example of such a design, mixing different mouse strains -- C57BL/6, DBA/2, and BALB/c -- within cages varying in degree of enrichment. As well as boosting statistical power, no other manipulations are needed for individual identification if co-housed strains are differentially pigmented, so also sparing mice from stressful marking procedures. METHODS: The validation involved housing 240 females from weaning to 5 months of age in single- or mixed- strain trios, in cages allocated to enriched or standard treatments. Mice were screened for a range of 26 commonly-measured behavioural, physiological and haematological variables. RESULTS: Living in mixed-strain trios did not compromise mouse welfare (assessed via corticosterone metabolite output, stereotypic behaviour, signs of aggression, and other variables). It also did not alter the direction or magnitude of any strain- or enrichment-typical difference across the 26 measured variables, or increase variance in the data: indeed variance was significantly decreased by mixed- strain housing. Furthermore, using Monte Carlo simulations to quantify the statistical power benefits of this approach over a conventional design demonstrated that for our effect sizes, the split- plot design would require significantly fewer mice (under half in most cases) to achieve a power of 80%. CONCLUSIONS: Mixed-strain housing allows several strains to be tested at once, and potentially refines traditional marking practices for research mice. Furthermore, it dramatically illustrates the enhanced statistical power of split-plot designs, allowing many fewer animals to be used. More powerful designs can also increase the chances of replicable findings, and increase the ability of small-scale studies to yield significant results. Using mixed-strain housing for female C57BL/6, DBA/2 and BALB/c mice is therefore an effective, efficient way to promote both refinement and the reduction of animal-use in research.

Benefits of a ball and chain: simple environmental enrichments improve welfare and reproductive success in farmed American mink (Neovison vison).

Can simple enrichments enhance caged mink welfare? Pilot data from 756 sub-adults spanning three colour-types (strains) identified potentially practical enrichments, and suggested beneficial effects on temperament and fur-chewing. Our main experiment started with 2032 Black mink on three farms: from each of 508 families, one juvenile male-female pair was enriched (E) with two balls and a hanging plastic chain or length of hose, while a second pair was left as a non-enriched (NE) control. At 8 months, more than half the subjects were killed for pelts, and 302 new females were recruited (half enriched: 'late E'). Several signs of improved welfare or productivity emerged. Access to enrichment increased play in juveniles. E mink were calmer (less aggressive in temperament tests; quieter when handled; less fearful, if male), and less likely to fur-chew, although other stereotypic behaviours were not reduced. On one farm, E females had lower cortisol (inferred from faecal metabolites). E males tended to copulate for longer. E females also weaned more offspring: about 10% more juveniles per E female, primarily caused by reduced rates of barrenness ('late E' females also giving birth to bigger litters on one farm), effects that our data cautiously suggest were partly mediated by reduced inactivity and changes in temperament. Pelt quality seemed unaffected, but E animals had cleaner cages. In a subsidiary side-study using 368 mink of a second colour-type ('Demis'), similar temperament effects emerged, and while E did not reduce fur-chewing or improve reproductive success in this colour-type, E animals were judged to have better pelts. Overall, simple enrichments were thus beneficial. These findings should encourage welfare improvements on fur farms (which house 60-70 million mink p.a.) and in breeding centres where endangered mustelids (e.g. black-footed ferrets) often reproduce poorly. They should also stimulate future research into more effective practical enrichments.