Monthly Archives: October 2011

Honess, P.
Dept of Biomedical Services, University of Oxford, Oxford, UK and School of Veterinary Medicine and Science, University of Nottingham, UK

Fernandez, L.
Bioculture (Mauritius) Ltd, Riviere des Anguilles, Mauritius

Abstract

Although the use of wild-born primates in research is banned in some countries, in others it is commonplace. It has been demonstrated that not only do wild-born primates react more strongly to some stressors than those that are captive-born, but they also use inanimate enrichment less. Given our understanding of the consequences of elevated stress for animal welfare, as well as the quality of science, more consideration should be given to the enrichment, and even the use, of wild-born macaques in biomedical research.

Introduction

It is well-established that environmental enrichment programmes should be well structured, goal defined and targeted at the specific characteristics of the animals for whom improvement in captive conditions, and hence welfare, are desired (Bloomsmith et al. 1991; Young 2003; Honess & Marin 2006b). Important characteristics include the species identity, the age-sex class, and aspects of individual temperament. A characteristic that is not often considered as part of this is the origin or birth context of the animals; specifically whether they were wild- or captive-born.

In Europe, under existing or incoming regulation (e.g. Home Office 1986; EU 2010), the use of wild-born, and even first generation captive-bred, primates is prohibited (except where there is specific justification). Ostensibly, these measures are to protect wild populations through the creation of self-sustaining breeding colonies, and counter the disproportionate stress that captive conditions may impose on naïve animals. However, there are regulatory environments (including the USA) where the housing and scientific use of wild-born animals is permitted. It is therefore important for responsible managers to consider this characteristic as one which may require specific enrichment provision; either in the quantity or quality of the enrichment, or both.

Birth origin and stress

The first question to ask is:

Do wild-born primates react differently to captive environments and routines than captive-born animals? There is evidence from the literature that indeed they do, but not always in the predicted way (Honess & Marin 2006a). For example, a study by Carolyn Crockett and colleagues (2000) found that wild-born female pigtailed macaques (Macaca nemestrina) exhibited more appetite suppression after being moved between rooms than captive-born equivalents. The authors interpret this type of appetite suppression, particularly where it is associated with raised cortisol, to indicate stress. Perhaps more dramatically, other studies have shown that wild-born pigtails also suffer higher mortality associated with translocation (Ha et al. 2000). On the other hand, in rhesus macaques (M. mulatta), wild-born animals have been shown to exhibit less stress-indicative behaviour (self-directed aggression, stereotypies) than captive-born individuals when housed in historically small cages (0.288m3) (Paulk et al. 1977). Of course, lower levels of abnormal behaviour do not in themselves indicate the absence or magnitude of a stress response.

Birth origin and enrichment

So, given that there is evidence that wild-born macaques can react more strongly to some captive management routines, the next question is: Do they react differently to environmental enrichment provided to reduce their stress response?

It has been reported that among older rhesus macaques, those that were wild-born made less use than captive-born of enrichment (wooden sticks, Kong toys, plastic balls) made available to both (Line et al. 1991). Also, in a study comparing enrichment use (Kong toys) between single housed pigtail and long-tailed, or cynomolgus (M. fascicularis) macaques, the fact that the pigtails used the toys more was, at least in part, accounted for by more of them being captive-born, and therefore more familiar with such toys (Crockett et al. 1989).

Birth origin and housing context

Therefore, while there is some evidence both of a heightened stress response and lower use of enrichment in wild-born macaques, it is relatively limited. Nevertheless, what evidence there is might be in line with hypotheses that suggest that animals of such origins might experience significant challenges in adapting to captivity. Having said this, it is likely that the context in which the animal is housed may well be critical in determining the extent of these challenges and their manifestation in the magnitude of the stress response.

Most of the studies cited above involve study subjects that were housed in a socially- and spatially-restricted laboratory environment. Responses may be very different in a breeding facility where animals are housed in species-appropriate, socially-complex groups in expansive caging under ambient tropical conditions. These are the conditions at Bioculture in Mauritius where long-tailed macaques are bred. There is a mixture of wild- and captive-bred animals in this now closed (since 2009) colony and anecdotally there is no meaningful difference in the use of environmental enrichment between them. The extensive range of enrichment (perches, swinging devices, manipulanda and visual barriers, positive reinforcement training and familiarisation to humans) may well mean that there is something provided that appeals to all animals, irrespective of their origin. In the breeding groups with up to forty adults, there is significant social complexity. The housing of primates with compatible conspecifics is perhaps the single most important contribution to their welfare and its beneficial effect is likely to swamp that from inanimate enrichment (Schapiro et al. 1996).

Conclusion

The need for high welfare standards and reduced stress in laboratory animals is well-rehearsed and includes meeting public expectations, addressing the harm:benefit balance and securing the quality of the research model. Where animals have a sustained or significant stress response to captive conditions or research procedures, it not only constitutes a risk to their health but is a source of unwanted variation and confounding variables in research programmes (Poole 1997; Garner 2005), except where these are examining stress itself. Evidence suggests that not only are wild-born macaques likely to react more strongly to stressors but also that they may be more resistant to attempts to ameliorate that response with environmental enrichment, particularly inanimate options. Therefore, for model quality and study design reasons, as well as animal welfare, it makes sense for researchers and procurement staff to obtain captive-born animals for their studies. Such preference will encourage breeding facilities to become self-sustaining with benefits derived from reducing the pressure on threatened native (non-introduced) populations. One of the macaque species most commonly used in research is the long-tailed macaque. This species, that was previously abundant across its natural range in SE Asia, is now reported to be threatened due, in no small part, to uncontrolled removal of animals from the wild for biomedical research (Eudey 2008). Therefore confining primate use to those individuals that are captive-born may have appreciable benefits for animal welfare, the quality of science, and conservation.

References

Bloomsmith, M., Brent, L. and Schapiro, S. J. (1991). Guidelines for developing and managing an environmental enrichment program for nonhuman primates. Laboratory Animal Science 41(4): 372-377.

Crockett, C. M., Bielitzki, J., Carey, A. and Velez, A. (1989). Kong toys as enrichment devices for singly-caged macaques. Laboratory Primate Newsletter 28(2): 21-22.

Crockett, C. M., Shimoji, M. and Bowden, D. M. (2000). Behavior, appetite, and urinary cortisol responses by adult female pigtailed macaques to cage size, cage level, room change, and ketamine sedation. American Journal of Primatology 52(2): 63-80.

EU (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276: 33-79.

Eudey, A. A. (2008). The crab-eating macaque (Macaca fascicularis): Widespread and rapidly declining. Primate Conservation 23: 129-132.

Garner, J. P. (2005). Stereotypies and other abnormal repetitive behaviors: Potential impact on validity, reliability, and replicability of scientific outcomes. ILAR Journal 46(2): 106-117.

Ha, J. C., Robinette, R. L. and Davis, A. (2000). Survival and reproduction in the first two years following a large-scale primate colony move and social reorganization. American Journal of Primatology 50(2): 131-138.

Home Office (1986). The Animal (Scientific Procedures) Act. UK.

Honess, P. E. and Marin, C. M. (2006a). Behavioural and physiological aspects of stress and aggression in nonhuman primates. Neuroscience and Biobehavioral Reviews 30(3): 390-412.

Honess, P. E. and Marin, C. M. (2006b). Enrichment and aggression in primates. Neuroscience and Biobehavioral Reviews 30(3): 413-436.

Line, S. W., Morgan, K. N. and Markowitz, H. (1991). Simple toys do not alter the behaviour of aged rhesus monkeys. Zoo Biology 10: 473–484.

Paulk, H. H., Dienske, H. and Ribbens, L. G. (1977). Abnormal behaviour in relation to cage size in rhesus monkeys. Journal of Abnormal Psychology 86: 87-92.

Poole, T. B. (1997). Happy animals make good science. Laboratory Animals 31: 116-124.

Schapiro, S. J., Bloomsmith, M. A., Suarez, S. A. and Porter, L. M. (1996). Effects of social and inanimate enrichment on the behavior of yearling rhesus monkeys. American Journal of Primatology 40: 247-260.

Young, R. J. (2003). Environmental Enrichment For Captive Animals Oxford, UK, Blackwell Science Ltd.

Volume 9, October 2011

Greetings from Shanghai.

I am writing this message from China where I am working with 22 eager college students planning to pursue careers in laboratory animal science. Happily, we don’t need translators to help us understand their love of animals and their desire to learn as much as they can about the role of animal welfare in advancing the science. Our professional trainers introduced the concept of environmental enrichment on the first day of class in the context of encouraging species-typical behaviors, tying efforts to decrease stress with increasing the integrity of the research data.

It may be the jetlag or the view from the other side of the world, but we don’t quite “get” the message behind the words of “Environmental Enrichment of Laboratory Rodents: The Answer Depends on the Question” (Vol 61, No 4, 2011, Pages 314-321). The abstract for this article, which appeared in the August issue of Comparative Medicine, can be found on page 30. Perhaps the choice of the term “so-called” to qualify enrichment of the cage environment for rodents threw up an immediate reading roadblock.

We will leave it to our readers to comment on the article.

We encourage members of the research community to weigh in on the questions raised as well as the conclusions reached. Perhaps our website can serve as a home for a meaningful discussion among professionals that will advance our understanding of the considerations raised by the authors.

Jayne Mackta, Publisher
President & CEO, Global Research Education & Training, LLC (GR8)

Read...Report from Shanghai

Volume 9, October 2011

Environmental Enrichment of Laboratory Rodents:
The Answer Depends on the Question

Toth, Linda A.
Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois

Kregel, Kevin
Department of Health and Human Physiology, The University of Iowa, Iowa City, Iowa

Leon, Lisa
Thermal Mountain Medicine Division, US Army Research Institute of Environmental Medicine, Natick, Massachusetts

Musch, Timothy I.
Departments of Kinesiology, Anatomy & Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

Comparative Medicine, Volume 61

Number 4 • August • Pages 314-321

http://aalas.publisher.ingentaconnect.com/content/aalas/cm/2011/00000061/00000004/art00003

Efforts to refine the care and use of animals in research have been ongoing for many years and have led to general standardization of rodent models, particularly with regard to animal housing, genetics, and health status. Concurrently, numerous informal practices and recommendations have

been promulgated with the laudable intent of promoting general animal well-being through so-called enrichment of the cage environment. However, the variety of housing conditions fostered by efforts at environmental enrichment (EE) complicates the goal of establishing standardized or even defined environments for laboratory rodents. Many studies over the years have sought to determine whether or how various enrichment strategies affect the behavior and physiology of laboratory rodents. The findings, conclusions, and interpretations of these studies are mixed, particularly with regard to their application across rodent

species, strains, genders, and ages; whether or how they affect the animals and the science; and, in some cases, whether the effects are positive, negative, or neutral in terms of animal well-being. Crucial issues related to the application of EE in research settings include its poorly defined effect on the animals, the potential for increased variability in the data, poor definition across labs and in publications, and potential for animal or scientific harm. The complexities, uncertainties, interpretational conundrums, varying conclusions, and lack of consensus in the EE literature warrant careful assessment of the benefits and liabilities associated with implementing such interventions. Reliance on evidence, professional judgment, and performance standards are crucial in the development of EE strategies.

Volume 9, October 2011

Better Methods for the Reduction Part of the Three Rs

G. Scott Lett, Ph.D.
CEO, The BioAnalyics Group LLC

In past articles, I’ve highlighted research efforts that study the impact of environmental enrichment (EE) on research outcomes. There is mounting evidence that improved environmental conditions result in more relevant research results. But what challenges face researchers who want to make a change for the better? Published literature, when available, isn’t always clear and consistent about what constitutes best practice for a particular animal species, nor is it clear what the impact is in terms of research endpoints. Not all researchers can afford to take time out from their regular research to undertake a study using rigorous experimental design to answer these questions. How do we make the transition to EE and make sense from the growing mountain of literature? In this article, I write about meta-analysis, an approach to analyzing published experimental results from multiple prior studies.

The Opportunity and the Challenge
Environmental Enrichment Fights Cancer and Improves Research Results—What Now for the Biomedical Researcher?

In the October 2010 issue of The Enrichment Record, Emily Patterson-Kane and I reported on the research of Cao et al, published in the July 9 issue of Cell. The researchers spent 5 years, used 1500 mice and painstakingly demonstrated significant effects of environmental enrichment (EE) on cancer outcomes. Based upon Cao et al and other studies, the evidence now suggests that EE is not just a more humane option for research animals, but is necessary to develop better animal models of human diseases. However, we also pointed out some major challenges in making the transition to EE. For the researcher, it is important to understand how the change to EE will affect her results. It is also important to know “best practice” for the care of research animals, and that the standards have not been set for every species.

Not many researchers can afford to undertake a 5-year 1500-mouse study to determine best practices and measure the effects. In another study, Hanno Würbel (2007) used 432 mice and experiments run in replicate in multiple laboratories to support the conclusion that EE does not disrupt standardization of experiments. Undertaking such studies in every laboratory will produce valuable data, but seems to sacrifice one of the three Rs of animal testing (Reduction) in favor of another (Refinement). Small studies can help make the transition more affordable, but may miss significant effects, due to small sample size. Published studies may give inconclusive or conflicting results, causing us to wonder which results to believe.

Sample Size and Statistical Power

Before we discuss meta-analysis, let’s look at the relationship between sample size and the reliability of research results. It is well understood that there is a great deal of variability in biomedical research. There are both biological sources of variability and technical sources. It is no surprise that similar mice don’t all respond identically to the same treatment. A researcher cannot measure the response of all mice, so we use data from a small group of mice (the sample) to predict the behavior of all similar mice (the population). For example, a researcher may want to measure the startle response time of a group of mice. The distribution of startle response times of normal mice might look something like the traditional “bell curve” as seen in figure 1.

Figure 1: Hypothetical Distribution of Response Times in Mice. Most are near 7.5 milliseconds.

In this hypothetical sample, the average response time is 7.5 milliseconds. We can see that some mice have response times as high as 8.5 ms and more, but most tend to cluster around 7.5 ms. A researcher would like to take a small sample of mice and measure their response times in order to predict the response times of all similar mice. How many mice are required to get a good estimate of the responses? Suppose three researchers each measure the response times of 3 mice each

Here’s what their results might look like:

__________________________________________________

                     Steve      Amy       Ted

Mouse 1       7.8           7.5          7.7

Mouse 2       7.6           7.7          7.3

Mouse 3       7.5           7.2          7.4

Average       7.6           7.5           7.5

_______________________________________________________

Figure 2: Hypothetical Response Time Measurements: Researchers get different results from similar mice.

We see that Amy and Ted measured average response times of about 7.5 ms, both at the “true” mean of 7.5 ms. Not bad! Steve, on the other hand, measured an average response of about 7.6 ms. Does this mean he made a mistake in measurements? No, it is just the natural biological variability of this type of mouse. The expected variation for a sample size of 3 of these mice is 0.14 ms, so all three researchers were well within the expected error range.

The expected error goes down as the sample size goes up. If Steve had used 9 mice instead of 3, his expected error would go from 0.14 down to 0.08, and if he used 100 mice, his expected error goes down to 0.02 ms.

How many mice does Steve need? This is an important question of study design. Suppose we have two groups of mice; one group with standard cages and environment and the other group housed in EE conditions. The two populations might have slight but important differences in startle response times, but the difference is difficult to see in small studies because their “bell curves” overlap, as seen in figure 3, which illustrates a hypothetical example.

The average response for the EE group is 7.7 milliseconds, compared to 7.5 milliseconds for the standard group, but if Steve uses only 3 mice in each group, there’s a 75 percent chance he won’t detect a significant difference. In fact, there is a 17 percent chance the EE group will appear to have a SHORTER response

time than the standard mice! If Steve wants an 80 percent chance of detecting a significant difference, he must use at least 20 mice in each group. The probability of correctly detecting a true effect is called the statistical power of the study. Good study design attempts to balance the power of the study with the desire to conserve precious resources, like  animals, money and time.

Figure 3: Hypothetical Responses for Standard and EE Mice. Overlapping distributions make it more difficult to detect a difference.

Meta-analysis

In statistics, a meta-analysis combines the results of several published studies into a larger “meta-study.” In the simplest form, a meta-analysis identifies a common measure of effect size across all the studies, in order to get better estimates of the true effect size than those derived in a single study under a given single set of assumptions and conditions. Another aim is to identify small but important differences in effect sizes that might be missed in a single study.

Finally, meta-analysis can help to identify hidden biases in published studies. The idea is really quite simple: by combining the results of published studies, we might get a better picture of best practices and effects of EE than could be seen in any particular published paper.

Karl Pearson is credited with the first published meta-analysis in 1904, studying the effects of inoculation against enteric fever. Combining studies with small sample sizes, he attempted to overcome the problem of reduced statistical power caused by the small samples. Gene V. Glass is credited with first using the term “meta-analysis” and is widely recognized as the modern founder of the method.

Meta-analysis has been successfully used to study environmental enrichment. For example, Averos et al (Applied Animal Behaviour Science, 2010) studied the effects of enrichment on the performance of pigs, and Janssen et al (An enriched environment improves sensorimotor function post-ischemic stroke, Neurorehabil Neural Repair, 2010) were able to sort through conflicting reports and show efficacy of EE using meta-analysis.

The File Drawer Problem—Biased Published Results

One potential weakness of meta-analysis is the dependence on published studies, which may create exaggerated outcomes. It is very hard to publish studies that show no significant results. For any given research area, one cannot know how many studies have been conducted but never reported and the results filed away. Remember Steve’s study design with 20 sample mice in each group? 20 percent of the time he won’t detect a difference between standard and EE mice, and he may not be able to publish the results. If all the results were published, we expect to see a bell curve distribution of differences between EE and standard mice. However, if the insignificant results are never published, we see a distribution that looks more like figure 4.

Figure 4: Hypothetical Distribution of Results, showing only the “significant results.” 20 percent of the results are too insignificant to be published and remain in the researchers’ file drawers.

This file drawer problem results in the distributions that are biased, skewed or completely cut off, and the significance of the published studies can be overestimated. Savvy meta-analysts use techniques to detect these biases and correct for them, but it would be much better to retain these results.

Conclusions

Good study design can optimize precious resources, including animals, money and time. Since researchers cannot afford to size their samples to produce results 100% of the time, meta-analysis can help sort through existing data and develop best practices for EE. Meta-analysis can be an effective tool for moving to better animal care while practicing the “Reduce” of the 3 Rs. The “file drawer problem” can limit our ability to re-use data in meta-analysis. We call for public repositories, where the unpublished and published data can be made available to the research community, providing better information for future meta-analysis.

Volume 9, October 2011

Shelly DeBoer
Graduate Student of Animal Sciences, Purdue University, West Lafayette, IN, USA

Pigs May Use Mirror Enrichment as a Way of Coping With Social Isolation

Pigs are gaining popularity for use as models in many areas of biomedical research, such as toxicity, wound healing, dental, peptic ulcer, metabolic syndromes, instrumentation/ implantation surgical procedures, and organ harvesting studies. This increase in use is largely driven by societal pressure to reduce the number of primates and companion animals, such as dogs, used in such research. In addition, swine have many similar physiological and anatomical features to humans such as their skin, as well as digestive and cardiovascular system1.

It is not uncommon for research pigs to be housed individually due to experimental constraints, although it should be avoided if possible. In laboratory settings, a pig isolated from other pigs is often housed in a sterile yet barren enclosure. Naturally gregarious, isolated pigs may show behavioral and physiological signs of stress such as increased cortisol production2, decreased body temperature3, decreased Tumor Necrosis Factor-alpha (TNF-)4 and increased frequencies of behaviors associated with anxiety and stress5,6.

A common buffer for many stressors caused by confinement is the implementation of environmental enrichment. Preference tests have been used historically to analyze an animal’s partiality for enrichment objects. Our experiment included a preference test that allowed young isolated pigs access to three practical enrichments: a mat, a mirror, and a companion pig in a pen across an alleyway. A mat was chosen since it was known to reduce discomfort in gestating and lactating sows. A visible live companion was chosen because of the pig’s highly sociable nature. The mirror was selected as a possible replacement for a companion in situations when complete isolation was necessary. However, with only one previous published study on swine use of mirrors, the pigs’ behavioral outcome was difficult to hypothesize7.

Our experiment used fourteen farm-type, weaner pigs (Yorkshire x Landrace) housed individually with free access between 4 adjacent pens, 3 of them containing one enrichment and one control pen with no enrichment. Each tested animal was only able to access each enrichment item while in that enrichment’s pen. Pigs were video recorded 14 h/day for 7 days and these were analyzed by scan sampling every 10 minutes to determine location, posture and behavior. Differences in the enrichment preference of the pigs were tested using a GLM model in JMP.

Our results showed that pigs spent more time in the pen across from the companion than in the control pen, with time spent in the mat pen and mirror pen intermediate. Feeling that this first analysis did not fully grasp all that was occurring with the pigs’ preferences, a second analysis was performed on the data to investigate preferences in the presence or absence of a human in the room. The pens were then combined into 2 categories: social pens (companion and mirror) and nonsocial pens (mat and control). The probability of a pig choosing a social pen when a human was present was significantly higher than when absent. Within the social enrichments, the probability of the animal choosing either mirror or companion was equal.

Our results showed that the pigs’ preference was largely dependent upon their environments. Pigs showed an overall propensity to spend their time with the companion enrichment, but when a human was present, the mirror and the companion enrichment were equally preferred. Preference tests are often criticized for their results being highly specific to the particular conditions in which the test is carried out8,9. Due to the strikingly different enrichment uses when a human is present, our results confirm that preference studies are indeed sensitive to experimental conditions and using time as a cost associated with preference choice may not be a reliable indicator of importance.

One can only wonder what image the pig is seeing and how the visual image is interpreted. However, it is generally accepted that pigs have relatively poor eyesight with severe near-sightedness, utilizing olfactory cues as their primary sense. The way the pig processes its surroundings through these senses is most certainly the key to understanding our observations. One explanation of our results is that although both the companion and mirror enrichments offer something important to the pig, they also have drawbacks. The companion provides both vocal and olfactory feedback to the tested pig, but due to the separating alleyway, the clear visual and tactile components of social support are inaccessible. The mirror enables the pig to receive tactile stimulation by lying parallel to it, as well as clear visual feedback due to its close proximity within the pen, but no olfactory or auditory feedback.

Overall, the pig appears to value the companionship received in the companion pen due to an innate need for communal living, plausibly because neither the mat nor the mirror provides olfactory or vocal feedback to the tested animal. However, unexpectedly, the mirror is also significantly important to the pig during times of perceived stress. Mirror usage has been tested in a number of different species including rodents, chimpanzees, elephants, rabbits, horses, sheep, poultry and cattle. Many species have shown a preference to be with a mirror when given a choice. Also, many of these species seem to benefit from the supplementation of a mirror to their surroundings. For instance, the addition of a mirror in poultry chicks resulted in an increase in exploratory pecking and decrease in vocalization10. Isolated heifers exposed to a front-viewed mirror had decreased locomotion as well as reduced heart rate11. Mirrors placed in stables have shown to decrease stereotypic weaving in horses12.

Shelly DeBoer and a swine friend

Unfortunately, the benefit of mirror usage in laboratory animals has been limited to a handful of experiments, and is only commonly implemented as an enrichment addition with non-human primates. In addition, it is not known how many other experiments testing the mirror (or other preference choices for that matter) have mistakenly concluded a possibly benefiting enrichment to be “unpreferred” or “unimportant.” Therefore, mirror supplementation for naturally gregarious animals housed in isolation should be further investigated.

ENDNOTES:

1. Bollen, P, Hansen A., Rasmussen H., The Laboratory Swine, CRC Press LLC, 2002.

2. Stolba, A., Wood-Gush, D.G.M., 1989. The behavior of pigs in a semi-natural environment. Anim. Prod. 48, 419-425.

3. Ruis, M. A.W., te Brake, J.H.A., Engel B., Buist, W. G, Blokhuis, H.J., Koolhaa, J. M., 2001. Adaptation to social isolation: Acute and long-term stress responses of growing gilts with different coping characteristics. Physiology & Behavior 73, 541– 551.

4. Tuchscherer, Margret, Ellen Kanitza, Birger Puppea, Tuchscherer A., Stabenow B., 2004. Effects of postnatal social isolation on hormonal and immune responses of pigs to an acute endotoxin challenge. Physiology & Behavior 82, 503-511.

5. Herskin, M.S., Jensen K. H., 1991. Effects of different degrees of social isolation on the behavior of weaned piglets kept for experimental purposes. Animal Welfare 2000 9, 237-249

6. Tuchscherer, M., Kanitz, E., Puppe B., Tuchscherer A., 2006. Early social isolation alters behavioral and physiological responses to an endotoxin challenge in piglets. Hormones and Behavior 50, 753–761.

7. Broom D. M., Sena, H., Moynihan K. L., 2009. Pigs learn what a mirror image represents and use it to obtain information. Animal Behaviour 78, 1037–1041.

8. Kirkden R. D., Pajor E. A., 2006. Using preference, motivation and aversion tests to ask scientific questions about animals’ feelings. Applied Animal Behaviour Science 100, 29–47.

9. Duncan, I.J.H., 1978. The interpretation of preference tests in animal behavior. Applied Animo 1 Ethology 4, 197-209.

10. Montevechhi, W.A., Noel, P.E., 1978. Temporal Effects of Mirror-Image Stimulation on Pecking and Peeping in Isolate, Pair- and Group-Reared Domestic Chicks. Behavioral Biology 23, 531-535.

11. Piller, C.A.K., Stookey, J.M., Watts, J.M., 1999. Effects of mirror-image exposure on heart rate and movement of isolated heifers. Applied Animal Behaviour Science 63, 93–102.

12. Mills, D. S. Davenport, K., 2002. The effect of a neighbouring conspecific versus the use of a mirror for the control of stereotypic weaving behaviour in the stabled horse. Animal Science 2002 74, 95-101.

Volume 9, October 2011