9. Extraneous Variables

Extraneous factors that influence research data increase the number of animals that are needed to achieve statistically significant findings (Home Office, 1989; Institute for Laboratory Animal Research, 1992; Brockway et al., 1993). This makes it an ethical imperative to examine and then control these factors as best as possible (National Research Council,1996; Öbrink and Rehbinder, 1999).

9,1. Exposure to Distressed Conspecifics

When you subject an animal to a distressing procedure, are the other animals in the room disturbed?

We have just finished a study on the effects on cage mates, when mice are subjected to one-hour restraint stress in the animal room and then returned to the cage. We did this once daily for 14 days. The cage mates were not touched and had implanted telemetry transmitters to monitor heart rate and temperature. While mice were being restrained, the heart rates of the untouched cage mates peaked at about 650/min, 15 minutes into the restraint period. Only by the end of the one hour restraint period, had heart rates of the untouched cage mates returned to baseline. The untouched mice's stress response did not show signs of adaptation within the 14-day study period. A similar pattern of stress response to witnessing restraint stress of the cage mate was found for the rise in body temperature.

Iimori et al. (1982), Fuchs et al. (1987), Pitman et al. (1988), De Laat et al. (1989) and Guhad et al. (2003) documented that rats show physiological stress responses when they are exposed to a conspecific who exhibits signs of distress during a handling procedure. Flow and Jaques (1997) took blood samples of long-tailed macaques while restraining them in their home cages with the squeeze-back. Serum cortisol and thyroid hormone concentrations differed between control animals and animals who, while waiting for their turn, witnessed how others were physically restrained and sedated for blood collection. The authors concluded that the difference might have been due to anxiety resulting from seeing restraint and sedation of other animals.

At our facility, only non-invasive procedures, such as weighing, can be performed in the animal rooms. Everything else has to be done in procedure rooms. This creates fewer disturbances for both animals and humans. I realize, there is some stress involved in temporarily moving individual animals away from their familiar quarters to procedure rooms, but I have the impression that this policy reduces the overall disturbance and stress that all animals of that particular room experience.

I agree, yet it has been shown in rats (Friedman and Ader, 1967; Brown and Martin, 1974; Euker et al., 1975; Dobrakovavá and Jurcovicova, 1984; Damon et al., 1986; Duke et al., 2001; Sharp et al., 2003), mice (Drozdowicz et al., 1990; Tuli et al., 1995; Tabata et al., 1998), guinea pigs (Fenske, 1990) and primates (Mitchell and Gomber, 1976; Line et al., 1987; Phoenix and Chambers, 1984; Coe and Scheffler, 1989; Crockett et al., 1993) that being removed from the familiar quarters is already a significant stressor, which changes the subjects' physiological equilibrium, thereby invalidating research data. So, whether we

stress seems to be an unavoidable variable, unless perhaps, we can train the research subject to voluntarily cooperate during the handling procedure in the familiar homecage.

I found in rhesus macaques that animals who cooperate during blood collection in the homecage show neither a cortisol response nor behavioral signs of stress that could possibly disturb the other animals in the room (Reinhardt et al., 1991; Reinhardt and Cowley, 1992).

Being exposed to a distressed conspecific changes the physiological equilibrium of an animal, without the animal necessarily showing this in his or her behavior.

9,2. Construction Noise

Our city is going to build a tunnel running underneath our primate facility. I am concerned that our animals will be affected during the digging, drilling and dynamiting. It is planned to move the rodents out of the building but keep the primates during the construction. How will our monkeys cope with the noise?

We had a similar experience and noticed that our macaques were very disturbed in the beginning, but seemed to get used to the occasional bursts of extreme noise very quickly. It helps the animals to remain calm during periods of extreme noise, when the attending caretaker stays in the room, talks to them and offers favored food or other items that they find attractive.

I work in a monkey facility where the floors in the hallway right outside the primate rooms were recently jack-hammered and a tunnel built underneath the building. The flooring project took four months and the tunnel is still not completed. In my opinion, the noise is not the biggest issue with our monkeys, though I did notice an increase in locomotor stereotypies during the jack-hammering. What really distressed the animals was the fact that we had to keep moving them around the building, so that the workers could access the hallways to take the flooring up. That did upset the animals quite a bit, and it took them several days to settle down in their new home cages.

Since the issue of noise has been brought up in reference to primates, it made me curious to know, if there is any published information on the effect of drilling and jack-hammering on rats and mice. We will be having drilling and jack-hammering in our facility for about three days in a few weeks. It was decided to stop data collection during that time and move the animals housed closest to that area to another room down the hall. I am wondering if that is enough to protect them from excessive stress?

When construction was being done on campus within an acre of our facility, the barely audible noise and vibration threw all our rodent breeding programs out the window for quite some time.

We had been in the middle of construction off and on for the past 10 years. Most of the construction has had few consequences. However, when they built the classroom building across the street from us, they had to drive pilings and tamp the ground because the building has no basement. For six weeks, the ground vibrated constantly for eight hours a day. We lost at least six months of breeding of the transgenic mice, and even the zebrafish stopped laying eggs.

Mice, rats and guinea pigs show a distinct withdrawal response to experimentally generated intense noise (Anthony et al., 1959), suggesting that the animals are stressed.

I checked the literature and found not a single article, assessing the impact of construction noise on the physiology of rodents, rabbits and primates in research labs.

Given the fact that noisy construction and remodeling work is a common event in biomedical research facilities, it is surprising that not a single article could be found in the scientific literature, assessing the impact of this uncontrolled variable on animals assigned to research.

9,3. Researcher

It is my experience that many principal investigators show little or no interest in how their animals are housed and handled, and if they do handle their animals themselves, they often lack proper skills and patience. How do you "train" such individuals to realize that their attitude defeats sound scientific methodology?

It is quite difficult to "train" researchers to do their work with the animals in a more considerate and compassionate manner. Unfortunately, investigators often see "the results" of the research as more important than the animals themselves. They are in a hurry to get results. Usually they do not take the time to get to know their animals, let alone work with them in a more relaxed, less stressful ambiance.

One thing that really bugs me about this business is that a lot of the time investigators do not know how to treat the animals as sentient beings. A big problem is that first-hand experience with animals is often not a requirement for the researchers and their technicians to be hired, and/or to receive funding for their research proposals. I wish everyone involved in animal research was an "animal person," but sadly, that is not the case.

I am working with investigators who do not know that the rats they are doing research with are nocturnal animals. I always love the statement "they seem happy to me." I actually hear that quite frequently from researchers. Usually they simply mean that the animal is moving about in the cage, but there is no comprehension about whether the movement is normal or indicative of stress or boredom or discomfort. Reese (1991) aptly observed in the book Animals in Biomedical Research:

It is so obvious for those who genuinely care for the animals and are concerned about valid scientific methodology, but it seems to be of little or no relevance for those who see the animal merely as a means to get publishable data.

Our investigators usually have grad students doing the research-related procedures. It is not very often that they will show up in the animal areas, while others I have yet to ever see. We had some grad students come in to work with mice, others to work with monkeys, but they had never actually worked with a mouse or a monkey before! Apparently, the principal investigators had failed to make sure that their students had received basic training and were actually qualified to work with the animals in an appropriate manner.

Time is a major factor when dealing with researchers and their attitude towards animals. Often, I have tried to help the researchers with a task involving animals, such as acclimatizing an animal to a restraint procedure, only to be told, "No, that would take too long." There are many things that we could do to help alleviate stress, and improve the well-being of the animals, but these refined techniques may take a bit longer than the traditional, often quite brutal methods. The researchers usually give the impression of being in a hurry to get their data as quickly as possible and, therefore, that there is no "extra" time for the animals themselves. It is my experience that it is exceedingly rare to find researchers who "get into the muck" and have some appreciation of what it requires to provide decent housing and handling conditions for their animals. We still have quite a few who do not even want to walk through our dirty cage area side to drop off empty caging.

It always strikes me that many investigators hardly ever show up in our animal area. Some of them probably have never seen the animals assigned to their projects. Yes, they are familiar with the IDs and the subjects' history, but that is often the end of the "touch." A prestigious biomedical scientist puts it in a nutshell when he concedes that:

Researchers, who pretend to be too busy to show an active interest in the welfare of the animals assigned to their studies, cannot assure that the data they are collecting will not be influenced by uncontrolled variables related to species-inadequate housing and species-inadequate handling prior, during and after procedures.

9,4. Workdays versus Weekends/Holidays

Are the animals in your charge less stressed on weekends and holidays than on workdays?

Our rhesus and stump-tailed macaques, but also our guinea pigs, rats and chickens are less restless, less alert and apprehensive on weekends and holidays than during workdays, when personnel can enter their room any time, catch them and subject them to a painful procedure.

I agree that the animals—I refer to rhesus—seem to be calmer, more relaxed on weekends and holidays. This is perhaps not surprising, because the personnel who do the more invasive parts of the research are here during the week, not on weekends and holidays. Hassler et al. (1989) and Schnell and Wood (1993) assessed cardiovascular stress parameters of rhesus macaques and marmosets and found that values are significantly lower on the weekend than during workdays. Entry of technical staff into the colony could be clearly identified in the heart rate and activity recordings of the animals. Schreuder et al. (2007) made similar findings in rats: The animals' heart rate, locomotor activity and blood pressure differed significantly on workdays versus weekends.

The stress level of animals is higher on workdays as compared to days when no personnel are around. This phenomenon has implications for the interpretation of stress-sensitive data, as these may not reflect normal resting values on ordinary workdays.

9,5. Multi-Tier Caging

The cages of small and medium-size animals—such as rodents, rabbits, cats, monkeys, birds—are traditionally stacked on top of each other to allow maximal usage of room space. Animals caged in lower rows live closer to the ground and in a less illuminated environment than animals caged in upper rows. These differences introduce extraneous variables that are usually not accounted for in scientific articles (Davis et al., 1973; Gamble et al., 1979; Reinhardt and Reinhardt, 2000). Is this an issue we have to be concerned about?

Hens on the bottom tier are often more reactive—frightened?—than hens from upper tiers. Presumably, the birds on the upper tiers have been exposed to the sight of human eyes more frequently and so are more habituated to human presence.

This probably also applies to pigeons. Those caged in the bottom row are definitely more fearful and emotional, especially when I bend down face to face with them. They are harder to extricate from the cage and, certainly, flap their wings more when I interact with them in any manner.

Ader et al. (1991) noticed the opposite effect in mice: Animals caged on the top of a rack are more fearful and more "emotional" than those caged on the middle or bottom shelf of the rack. Mice probably feel more secure and secluded in the relatively dark environment of lower shelves than on the top shelf that may expose them directly to bright light. Garner et al. (2004) found that barbering was significantly more severe in upper-row than in lower-row caged mice. Lagakos and Mosteller (1981) also studied mice and found that the incidence of certain tumors increases conspicuously from the bottom to the top shelf. Similar observations were made by Mantel (1980), Greenman et al. (1984) and Young (1987). These studies make it quite clear that shelf level is an important variable that needs to be taken into consideration in scientific research with mice.

It is my experience that macaques living in bottom-row cages show more behavioral stress responses—such as crouching in a back corner, alarm vocalization, hyperaggression—when an investigator, dressed up in protective garb and a surgical mask, enters the room than those living in upper-row cages. At the same time, lower-row caged animals tend to "escape" into transfer boxes readily, while upper-row caged animals often stubbornly resist leaving their cages and exiting into transfer boxes. In a quantitative study I did on 20 pair-housed cynos, the animals spent 94 percent of their waking time in the upper part of the vertically arranged double cage. All food was given in the bottom section, yet the animals would bring the food to the upper part and consume it there. The monkeys' preference along the gradient of height was unequivocal!

Your observation is similar to mine. Two pair-housed female rhesus macaques visited the top half of their double-cage significantly more often and spent significantly more time there than in the bottom half of their double-cage (Clarence et al., 2006). MacLean and Roberts-Prior (2006) concluded from detailed studies with rhesus, that the monkeys' consistent preference for the upper-row reflects the paramount importance of access to elevated space.

I observed squirrel monkeys in vertically arranged double cages and also found that the animals clearly preferred the upper half of their cages. The only time they went to the bottom half was when they retrieved a toy or picked through the bedding for treats.

When visiting facilities that have their pair-housed macaques in vertically arranged double-cages, I repeatedly got the impression that subordinate partners are disadvantaged in this caging system, with dominant animals preventing subordinates from spending as much time in the upper section as they would like to.

Your impression is right in many situations. I see this happening often with our pair-housed cynos. Most of our pairs get along great, and both partners usually sit together in the top section of the double cage. Some pairs, however, do not get along so well, and one monk is at the bottom of the cage most of the time looking very worried, while the other monk spends almost all the time in the top part of the cage. If this situation goes on for a few days, we separate the animals and match them up with other more compatible companions.

Salzen (1989) observed small groups of squirrel monkeys in vertically interconnected cage units. The animals showed a preference for the upper cages, and subordinate females were liable to stay in the lower cages. Obviously, the animals competed over access to the preferred upper cages, with subordinate animals obviously being disadvantaged.

Does anyone work in a facility that has successfully dealt with the illumination differences in cages arranged in multi-tier racks?

I do not think the researchers over here have given it any thought. I do not even think that it crosses their minds that the quantity and/or quality of light their mice are receiving could affect the findings of their studies with these animals.

The differences in illumination in upper- versus lower-tier cages are indisputable (Figure 64). Clough (1982) is probably not exaggerating when he states that light intensity in the cages is likely to be the most variable environmental factor in the average animal room.

Figure 64  In the standard double-tier caging system for macaques animals in the top row live in a quasi-arboreal bright environment, while animals in the bottom row live in a cave-like environment to which they are not biologically adapted. Photo by Evan MacLean.

The Animal Welfare Regulations of the US Animal Welfare Act admonish that:

This legal mandate cannot be met when animals are kept in the traditional multi-tier caging systems: While animals in the upper row live in well-illuminated quarters those in lower rows often live in a semi-gloomy environment often making it necessary for care personnel to use flashlights in order to identify individual animals and assure the adequate cleaning of the cage (Figure 64; Reasinger and Rogers, 2001).

The National Research Council (1996) advocates rotating cage position relative to the light source to account for the different housing environments of animals kept in upper-row versus lower-row cages. I very much question if this is an acceptable "trick" or if it simply "rotates" the problem without fixing it. If anything, rotating cage position is likely to make the methodological situation even worse, by introducing another source of variance.

The differences in light could be addressed for rodents, by providing all animals with a species-appropriate shelter or nest. The animals will hide and rest in these dark places most of the time, thus being exposed to much more equal illumination.

In order to bring more light into the lower-row cages of macaques, I had all solid side panels replaced with mesh walls, allowing more light reflecting into the cages. This modification more than doubled the light intensity in the lower-row cages (Reinhardt et al., 1992), but it did not eliminate the significant illumination difference between upper- and lower-row cages.

The "tier effect" is a variable that does not necessarily invalidate research findings, but it must be accounted for in the statistical analysis. Of course this does not address the welfare issue, which should also be carefully considered for each species.

9,6. Individually Ventilated Caging (IVC)

Those of you who have first-hand experience with both the individually ventilated caging system and the traditional caging system, which system is more animal welfare conducive? Economical factors should not influence your decision, please, only the standpoint of the caged animal.

I believe that in terms of animal welfare, the development and uptake of individual ventilated cage systems is one of the worst "advances" in laboratory animal housing. I am regularly told that environmental enrichment is a threat to biomedical research and that more studies have to be conducted before enrichment can be adopted by the research industry, but so many labs have gone over to one of the many, extremely various ventilated systems without raising this same objection. The lack of data in this subject boggles the mind, given the numerous variables that come along with this caging system, for example, sound attenuation, smell attenuation, sensory deprivation, vibration, ultrasound, reduced handling, and movement to less preferred bedding types.

In our lab, IVCs are used only when researchers need frequent access to animals who are immune compromised and, consequently, would not survive in open top cages, e.g., SCIDs (severe combined immunodeficieny disorder), nudes and several strains of knockout mice.

We have not observed any detrimental effects of this caging system on the mice. However, IVCs and the associated equipment are relatively expensive and very labor intensive, so they are not something we would choose on purely economic grounds. There are two other drawbacks: The contact between animals and care personnel is reduced, and the complicated technology creates a comparatively high risk that something goes wrong. My biggest fear is a power outage, where the emergency back up does not kick in and whoever is on duty does not realize the implications and forgets to phone me.

We have better success with trio-housing male mice in IVCs than in open bins. During a follow-up period of four months no fights were reported among males kept in the IVCs, while in a parallel study of trio-housed mice in open bins, six groups had to be split up due to fighting.

We, too, have more luck with group-housing males in IVCs than in the static cages. In addition, we have better reproduction rates in breeding colonies kept in IVCs, which I believe is due to the relatively infrequent handling of the female and her litter, plus the longer period of time that they can stay in one and the same nest.

Some strains will build their nest very close to the air valve, while others build it as far away from it as possible. The mice build elaborate nests over the two weeks between cage changing. We try not to discard the whole nest but move parts of it that are dry into the new cage, which will then already have the familiar scent of the old nest. As for environmental enrichment, we have found a way of providing shelters without interfering with the ventilation too much, by using pipette boxes that have been cut in half. We place these in the front of the cage, with the opening facing into the cage. The mice use these shelters for sleeping and nesting. The great thing about these is that the labs provide them to us for free, the carpenter shop cuts them for a nominal fee, and they are autoclavable and disposable.

If properly adjusted to the animals' behavioral needs, such as building nests and sleeping in a nest or shelter, and if properly and reliably serviced, an individually ventilated caging system can enhance animal welfare.

9,7. Restraint Tubes for Rodents

Rodents are often restrained for blood collection and injection, by coaxing the animals into little tubes. I wonder if this kind of enforced immobilization is not introducing stress as an uncontrolled variable into the data collected from such animals?

We had a group of visiting scientists who used tube-restraint as classical stressor for experimental purposes in their research facility. When they saw us working with our rats, they could not believe their eyes: Our rats were quite happy to crawl into the tubes, go to sleep and show no apparent signs that they had become stressed by the procedure. It may well be that our rats were particularly good-natured and laid back and/or were so well habituated to being 30-minute tube-restrained, that they calmly accepted the situation.

I also find that rats, mice and guinea pigs will enter restraint tubes quite happily, provided I am patient and gentle-and-firm the first couple of times when I prompt them to crawl into a tube. The initial experience associated with the tube is probably the determining factor in the restrained subject's response to subsequent restraint sessions. I encourage our researchers to handle their animals daily during the week prior to the actual studies. On these occasions they will also tube-restrain their animals without doing any other procedure that could possibly cause pain to the animals. This preliminary routine assures that the subjects not only will be familiar with the researcher, but that they also will be well acclimatized to the tube at the beginning of the study. I think this provides a good condition for the animals to experience little or no restraint-related stress during the experiment.

Either we use ordinary transparent restraining tubes that we cover with a paper towel, or we use opaque tubes made of red Perspex so that they become a dark, "safe" hiding place. Since the scent of a stranger adhering to the tube is likely to induce a negative reaction in rodents, we thoroughly rinse the tubes between cages. We did notice that the animals are more reluctant to crawl into the tube and tend to be restless in the tube when we skipped the rinsing. With a bit of "training" rodents do enter such tubes without appearing to be stressed, and as already pointed out, they will often fall asleep after a few minutes. They do give the impression of being relaxed, even though we take tail-cuff blood pressure readings, or withdraw blood from previously implanted cannulae at various time points. When I take the animals out of the tube after a procedure, they are not agitated and usually resume their routine business, such as exploring the environment and grooming themselves. I really believe that gently habituated rodents do not experience undue stress, or any stress at all, while they are restrained in dark tubes during noninvasive procedures. I should perhaps emphasize, it is very important to make sure that the animals do not get overheated while they are restrained in the tubes. They can get hyperthermic very easily, and this will certainly distress them; they will come out of the tubes in a state that I can only describe as "prostrated"—reluctant to move, panting, semi-conscious, damp or moist. This must be avoided, and it can be avoided by keeping the animals in the restrainer for only short periods at a time.

Would you recommend to always keep the restraint tube-environment dark, or has your experience shown that it does not make a noticeable difference whether a rodent is restrained in a transparent tube or a dark tube?

We use the typical transparent plastic tube, which I always cover with a surgical drape to darken it, so that the animals feel relatively secure. After all, their natural instinct is to seek a dark shelter in the event of danger, and being coaxed into a tube by a human hand must, indeed, be rather scary for them.

It is my experience that, if the tubes are red Perspex or covered with paper or surgical drape, the rodents seem to be relaxed and remain relaxed throughout the procedure, even if I draw a blood sample—which, I guess, must cause some discomfort despite the use of topical analgesics. When the tubes are transparent and uncovered, the animals will often wriggle about in what I presume is an attempt to get out—our tubes open at both ends, so the animals do not have to come out backwards unless they want to.

Enforced restraint is not an intrinsic stressor for rodents. If an animal has been well familiarized with the handling personnel and with the restraint tube, the tube kept dark and the duration of the restraint session short enough to forestall overheating, behavioral signs of stress can be avoided. It needs to be demonstrated whether physiological stress parameters reflect baseline values in animals who seem to accept tube-restraint.

10. Miscellaneous

10, 1. Marking Mice for Video Recording

We plan to video-record groups of black mice and would like to somehow identify individuals in the recordings. Can anybody share experiences on how to mark rodents for individual identification?

I use a human hair bleach to individually mark my dark C57Bl/6J mice. Contrary to what might be expected, this shows up very well under infra-red, under some circumstances even better than under white light. Because mouse hair grows so quickly, you will have to re-apply the marks every few weeks. Some mice do develop bald patches at the site of bleaching. We believe this is due to the hair follicle becoming slightly damaged and the hair falling out as a consequence of normal grooming. I do not consider this as a welfare problem, as the skin is not reddened, hence probably not inflamed.

I do not know if they will show up under infra-red, but we always use histological dyes—neutral red, malachite green and crystal violet made up as a concentrated solution in 70 percent alcohol—to identify mice. Applied with a cotton bud, these marks last at least a week.

What an excellent idea!

There are several possibilities for marking dark mice. The application of histological dyes seems to be a perfect option.

10,2. Mice Who do not Reproduce

We have been trying to breed wild forest mice for almost a year now, and no litters have been born. The animals are pair-housed in standard cages; they do have nesting material and shelters. Any suggestions as to what we can do to get these animals to reproduce?

Wild mice usually breed in lab conditions reasonably well. I would suggest to breed your mice in larger than standard-size cages. As a general rule, wild mice breed better the larger their cage. Trios—two females and one male—tend to reproduce better than pairs, probably because a wild female mouse likes to share a nest with another female. They need more nesting and bedding material than usual, as they like to bury their young. Try not to let them get too fat, and breed as young as possible. The breeding success can be enhanced when you try adjusting daylight length and light intensity to mimic a biologically natural light cycle; this will fool your mice—just as ours—into believing that it's time to mate.

At our facility, we keep colonies of wild mice in 2 m x 3 m large enclosures with 100 cm high metal walls. The floor is wood and covered with a layer of shavings and/or hay. The enclosure is provisioned with cardboard boxes, bricks and other objects behind which and in which the mice can hide. Overcrowding can quickly become a problem!

I am impressed and very pleased. It took only 32 minutes to receive your really good advice. Thank you!

Wild mice do reproduce very well when the housing and living conditions are mice-appropriate.

11. References

Table of Contents