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Husbandry Effects on Experiments

“I don't like the idea that I'm not in control of my life.”
Keanu Reeves, The Matrix. Village Roadshow Pictures.
In the past, experiments have been conducted on the effects of having an “enriched environment” (EE) on mice. Essentially, the investigators kept cages with a bare-bones, Spartan environment and compared it to an EE setting, which was loaded with toys like wheels and ladders that would provide its occupants with mental and sensory stimulation. EE animals have shown improved recovery from trauma and cellular degeneration. In a recent publication by Rattazzi et al., T cells from the EE mice showed decreased production of IFN-γ, but increased levels of IL-10 and IL-17 in vitro1. This demonstrates that in an EE setting, immune activity may turn from a proinflammatory Th1 state to one that promotes recovery instead (although it should be noted IL-17 has been associated with various proinflammatory functions).
Some mice got to live in luxury...
The purposeful introduction of toys for the animals led to some intriguing results. Rattazzi et al., like most other scientists, focused on creating the right controls for their experiment. Biological, fluorophore, and blocking controls (all of which are discussed in our new webpage) are enough to occupy the mind. But, what about the variables we can introduce into our animal work without even realizing it? How often do we stop to consider how animal husbandry might affect our test subjects?
Husbandry practices can create variables in experiments, affecting lab animals in unexpected ways. These variables can then affect reproducibility. We've discussed the difficulties associated with reproducibility in a previous blog, and BioLegend itself is contributing to the Reproducibility Initiative. Timo Nevalainen wrote a helpful review that provides details on several areas of husbandry that can influence experimental outcomes2.
Sensing the Days, Weekends, and Seasons

BBC's Talking Animals.
Animals are accustomed to a day/night cycle that determines their activity. Disrupting this natural cycle (whether by stress, light or even the tinting in cage walls) can affect melatonin and various hormones in rats. In fact, as little as 0.2 lux of light repressed normal nighttime melatonin levels in rodents (by comparison, a well-lit office is typically at 400 lux). Surprisingly, it also stimulated the metabolism and proliferation of a human breast cancer xenograft in these rodents. If dealing with albino rats, extra care may have to be observed, as 25 lux is typically the maximum recommended amount of light for these animals3.
In a separate study4, Ly-6Chi inflammatory monocytes exhibited diurnal variation, which controlled their trafficking to sites of inflammation. When lights were on, there was higher trafficking of these monocytes to the spleen, liver, and peritoneum. Consequently, if Listeria (a pathogen Ly-6Chi monocytes serve as a first line of defense for) was injected into mice during the day (lights on), the monocytes cleared the infection more effectively. On the other hand, if Listeria was injected during the night, a significantly higher bacterial load in those peripheral locations was observed due to decreased monocyte trafficking. This means that the time of day when a mouse is treated with an inflammatory drug or infectious agent may have profound effects on the immune response and the final readout.
Further, the typical human work cycle dictates that animal facilities are likeliest to be busy during the weekdays. Aside from the occasional graduate student running experiments, weekends tend to be quieter. The animals take notice of this as well. With less people and noise around, their blood pressure and locomotive activity tends to be lower overall. Whether you choose to sample animals every day of the week, or only on weekdays or weekends is a discussion you should have with your animal facility and principal investigator.
Despite being kept indoors in windowless rooms (and away from the Weather Channel), the internal clocks of rodents are still able to sense the seasons. Mice living with a 12 hour “day” had an enhanced chance to die of periotonitis (inflammation of the peritoneum), specifically during summer and autumn seasons. If you're aware of these types of variables, you could restrict your testing to seasons where this doesn't occur, but that isn't a very practical solution (and even the same season can vary from year to year). Control groups may help, but this situation could be unavoidable.
Even the mice can sense it...
Image from Late Night with Seth Myers.
Housing
Cages themselves can also influence results. Cages made of polycarbonate can leach Bisphenol A (BPA) upon high temperature or alkaline condition exposure, which can be common in washing procedures. BPA has been associated with lower uterine weight in prepubertal females and can affect estrogen-based studies overall. Polycarbonate and other polymer materials can offer an alternative. In the past, certain bedding material was also shown to have measurable endotoxin levels that could lead to inflammation in the lungs. Finally, the cages used to house rodents must be changed (often weekly), and this has been shown to increase blood pressure and heart rate, locomotive activity, and in some instances, induce aggressive behavior. Time should be allotted for the animals to get readjusted and to calm down after the cage change.
What's That Smell?
Animal odors play a key role in the rodent reproductive cycle: male urine pheromones have been known to synchronize female estrous cycles, and the absence of male scents can prolong them. The introduction of an unfamiliar male scent can actually terminate a pregnancy. Needless to say, controlling odors in your animal facility is important...including your own. It turns out, human scents and odors can also influence rodents. In an experiment by Mogil et al., they demonstrated that mice handled by men (or even being in proximity to a shirt worn by a man) had 35% lower pain scores when injected with an inflammatory drug, zymosan A. They hypothesized that men stressed the mice out more, demonstrated by the increased levels of corticosterone in the blood, resulting in an anti-inflammatory state. Mice also tended to demonstrate more anxiety when handled by men5. These results don't advocate that men stay out of the animal facilities, but rather that the sex of the scientist may also need to be reported, particularly if the studies focus on behavior or fear.
Seems men can influence rodents via scent alone.
There are a number of additional topics covered by Nevalainen that we haven't covered here in this blog, but hopefully we've shed a bit of light on some subjects you may have never considered. Consistency is key for your experiments, and ideally, you should control as many of the variables in your experiment as you can, whether it's biological controls in your samples or the amount of daylight” exposure your mice receive. Do you have any additional thoughts on how husbandry can affect your animals? Let us know at tech@biolegend.com.

When you finally get the hang of science...and controls...
The Matrix, Village Roadshow Pictures.
References:
  1. Impact of Enriched Environment on Murine T Cell Differentiation and Gene Expression Profile
  2. Animal Husbandry and Experimental Design
  3. Eliminating Animal Facility Light-at-Night Contamination...
  4. Circadian gene Bmal1 regulates diurnal oscillations of Ly6Chi inflammatory monocytes
  5. You smell, and mice can tell
Contributed by Ken Lau, PhD.
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