Animal Research

Introduction

Animal research within CSU’s CVMBS includes research studies that use laboratory animal species and wildlife species as well as client-owned animals in clinical trials. In this section, we summarize the different ways in which animal research can impact the environment including energy use, water use, chemistry, and waste. Other sections in this guide, such as Fieldwork, provide additional information related to specific areas of animal research.

When considering making changes to the ways in which we conduct animal research, the care and welfare of research animals must always be a top priority. That being said, there are several areas where sustainability and animal welfare have shared goals. When considering animal welfare, two of the three Rs of humane use of laboratory animals (replacement, reduction, and refinement) are inherently in line with sustainability practices.1 The first of these is replacement, which is the use of other technologies in place of laboratory animals.1 Performing in vitro experiments, such as using cancer cell lines or “organs-on-a-chip” (i.e., 3D microfluidic cell cultures), are one useful example.2 One can also consider “partial replacement,” which involves substituting “an invertebrate or an immature form of a vertebrate for an animal, where suffering may be an issue.”1 The second R is reduction, which involves using the fewest animals necessary to reach impactful study outcomes. Supporting these practices, the Guide for the Care and Use of Laboratory Animals, USDA, as well as institutional animal care and use committees (IACUCs), which review animal research protocols, require justification of the species and number of animals proposed for a study using statistics whenever possible. These humane welfare concepts minimize the number of animals used for research.1 With fewer animals being used for research, there is lower usage of resources: less energy is spent on animals and less waste is generated. Animal research is an energy intensive practice. In fact, in a standard laboratory space, the air is exchanged twelve times per hour whereas “an animal research facility can be up to twenty air exchanges per hour.”2 It is the responsibility of each researcher to use the least number of animals that is practical for the research goals to be met. Meeting with a statistician and/or performing your own power calculation to determine the optimal animal number before designing your experiment is one way to reduce the number of animals used and to help ensure the animals and other resources that are used contribute to useful outcomes.

 

Energy Use

When we think of animal laboratories, we typically think of them as unchangeable in terms of green usage or energy maintenance due to specific protocols and animal welfare requirements. However, thorough assessment of these spaces can identify energy saving opportunities:

  • Lighting: Many of the building spaces in the animal facilities do not actually house animals, and the conditions in these rooms can be adjusted to be more energy efficient. Instead of having lights on the 12/12hr on/off cycle in the non-animal spaces, switches in these rooms could be turned on and off by researchers when needed; training and signage can be used to ensure the lights are turned off when no longer needed.
  • Autoclaves: Running autoclaves when they are full helps save energy (and water). A schedule/protocol can be developed to help ensure autoclaves are run when completely full as much as possible. See the Water section (hyperlink to Water) for more information.
  • HVAC: when feasible, adjusting building temperatures to be closer to environmental temperatures will save energy. Most animal species have an acceptable temperature range. For example, according to Jackson Labs, the temperature range for mice is 65-75°F (~18-23°C) with 40-60% humidity. If it does not negatively impact animal health and welfare or experimental results and in accordance with veterinary oversight, assessing whether it may be possible to slowly adjust temperatures to be closer to current environmental temperatures and still stay within a safe range for the animals can help decrease energy usage.
  • Cold storage: following best practices for cold storage of samples including optimizing sample storage and “chilling up” ULT freezer to –70°C can minimize the energy used by ULT freezers.
  • PPE: When handling animals, we utilize gloves and other PPE such as scrubs and lab coats to protect us and the animals. Having clean gloves is imperative and in many situations such as inside the ABSL-3 and biosafety cabinet cannot be changed. However, there are some situations that can. Critically evaluate current protocols to determine if there are ways to ensure the safety and quality of research and optimize the use of gloves and other PPE. For example, if you have multiple assays in a day and are working in an ABSL-2 or lower, you may be able to hang your lab coat/scrubs and use it/them again for the second part of your experiment instead of getting new and creating more laundry1.

Key Energy Saving Tips

  • Seasonal temperatures
  • Turn off lights
  • Fill autoclaves before running
  • Practice best practices for cold storage
  • Optimize use of PPE

Water

While it is difficult to estimate exactly how much water CSU Lab Animal Resources (LAR) and others use for animal research, the primary sources of water usage are drinking water and sterilizing cage materials. These are clearly critical functions for animal survival and preventing cross-contamination between experiments. Used for the sterilization of cages, autoclaves use massive amounts of water to generate the required steam. Ensuring autoclaves are full when running and optimizing the use of supplies that need to be autoclaved can help minimize water use when possible. Generally, this involves optimizing the number of animals, housing conditions, and sharing resources whenever possible. For example, co-housing animals when possible (in accordance with regulations) decreases the number of cages and other materials that need to be autoclaved.

 

Green Chemistry

The chemicals used for downstream processing of animal tissues in histology and pathology laboratories pose significant health risks to both the operators and the environment. Formaldehyde is a known human carcinogen and is highly toxic to aquatic life.7,8 Xylene is another commonly used chemical that has known risks to human health and aquatic life.9 The use of these chemicals requires deactivation for proper disposal and the use of energy-costly fume hoods to protect the user.10,11 Green alternatives for histological chemicals have come onto the market in recent years from places like Milestone Medical (https://www.milestonemedsrl.com/wp-content/uploads/2018/11/Histology-Goes-Green.pdf). My Green Lab® has a collated list of green alternatives for common processing agents including formaldehyde, xylene, decalcifying agents, and others. Although these products are available, they are not widely used. This is attributed to a lack of evidence-based research validating the use of these alternatives. Of the few publications, there has been some validation performed on glyoxal fixatives, such as “Prefer.”10 The researchers found that for most cases, glyoxal performed equally as well as formalin as a fixative. Despite these positive findings and the wide availability of these compounds, there are still more hurdles to employing them in laboratories. One of these hurdles is the lack of validated immunohistochemistry and downstream techniques for non-FFPE tissues. Without validated protocols for immunohistochemistry, this switch will be slow to happen among researchers. Companies providing antibodies and reagents for downstream analysis have thus far not made the effort to validate their protocols while using more sustainable and less hazardous fixatives. It is likely that there will be a need to be an incentive for these companies to complete the validation studies, which may have to start as a bottom-up approach via researchers and histopathology cores requesting these protocols from companies.

 

Waste

The disposal of animal tissues and carcasses is an area where environmental sustainability could be greatly improved. There is much opportunity to repurpose the non-hazardous, nutrient-rich animal waste produced through animal research and diagnostics as things like fertilizers, compost, rendered products, or even biofuels. Current protocols at the CSU Diagnostic Medicine Center send non-hazardous, domestic animal carcasses to the landfill, and hazardous or infectious carcasses are digested in a tissue digester via alkaline hydrolysis. Other institutions use incineration techniques to remove biohazardous waste.12 While it is difficult to avoid the more environmentally taxing disinfection of hazardous animal waste, alternatives to nonhazardous carcass and tissue disposal aside from the landfill should be considered.

One alternative use for non-hazardous carcasses is compost systems. Composting these types of waste holds great value for the ecosystem and is a low-cost method of carcass disposal.13 Although these techniques have been employed by some livestock operations, composting has yet to be widely employed for animals coming from research. When developing IACUC protocols, the method of euthanasia should be considered carefully for laboratory animals. Avoiding the use of chemicals that are toxic environmental contaminants, such as sodium pentobarbital, could permit these animals to enter a compost waste stream rather than becoming hazardous waste.14 Additionally, disinfected products from tissue digesters could be added to this compost stream as a highly nitrogenous additive.15 Composting also reaches high temperatures of 55°C or higher for long periods of time, which can serve to inactivate potential pathogens.16 Large scale composting systems are largely not used in the research institution setting but could serve as a more sustainable and safer option than landfills that also provides a useful end product.

 

Conclusion

As with any sustainability topic, there is no single pathway to improving sustainability in animal research.  Ultimately, individual researchers should take a close look at their protocols and use the suggestions in this guide to identify areas where they might conduct research more sustainably while ensuring animal welfare and upholding regulations.  Importantly, consultation and coordination with LAR and IACUC are recommended and likely required in any area that affects animal care and use.

 

Authors: McKenzie Fletcher, Laurel Haines, Heidi Kloser, and Deb Green

 

References:

  1. Russell, W.M.S., Burch, R.L. The Principles of Humane Experimental Technique.  Chapters 5,6,7. https://www.caat.jhsph.edu/principles/chap5e
  2. Groff, K., Bachli, E., Lansdowne, M., Capaldo, T., Review of Evidence of Environmental Impacts of Animal Research and Testing. Environments 2014, 1, 14-30; https://doi:10.3390/environments 1010014
  3. https://www.jax.org/jax-mice-and-services/customer-support/technical-support/breeding-and-husbandry-support/mouse-room-conditions
  4. https://www.fm.colostate.edu/energy/
  5. https://app.energycap.com/app/buildings
  6. Bachmanov, A. A., Reed, D. R., Beauchamp, G. K., & Tordoff, M. G. (2002). Food intake, water intake, and drinking spout side preference of 28 mouse strains. Behavior genetics, 32(6), 435–443. https://doi.org/10.1023/a:1020884312053
  7. Lan, Q., Smith, M. T., Tang, X., Guo, W., Vermeulen, R., Ji, Z., . . . Zhang, L. (2015). Chromosome-wide aneuploidy study of cultured circulating myeloid progenitor cells from workers occupationally exposed to formaldehyde. Carcinogenesis, 36(1), 160-167. doi:10.1093/carcin/bgu22
  8. Hohreiter, D. W., & Rigg, D. K. (2001). Derivation of ambient water quality criteria for formaldehyde. Chemosphere, 45(4-5), 471-486. doi:10.1016/s0045-6535(01)00061-3
  9. Kandyala, R., Raghavendra, S. P., & Rajasekharan, S. T. (2010). Xylene: An overview of its health hazards and preventive measures. J Oral Maxillofac Pathol, 14(1), 1-5. doi:10.4103/0973-029x.64299
  10. DeJarnatt, V., & Criswell, S. L. (2021). Glyoxal: a proposed substitute for formalin in H&E and special stains. J Histotechnol, 44(1), 37-45. doi:10.1080/01478885.2020.1830664
  11. Kongoletos, J., Munden, E., Ballew, J., & Preston, D. J. (2021). Motion and Sash Height (MASH) alarms for efficient fume hood use. Scientific reports, 11(1), 21412. doi:10.1038/s41598-021-00772-y
  12. Shearer, J. K., Griffin, D., & Cotton, S. E. (2018). Humane Euthanasia and Carcass Disposal. Vet Clin North Am Food Anim Pract, 34(2), 355-374. doi:10.1016/j.cvfa.2018.03.004
  13. Kalbasi, A., Mukhtar, S., Hawkins, S. E., & Auvermann, B. W. (2005). Carcass Composting for Management of Farm Mortalities: A Review. Compost Science & Utilization, 13(3), 180-193. doi:10.1080/1065657X.2005.10702239
  14. Cooney, K., & Titcombe, L. (2022). Lessons and Recommendations from a Pentobarbital Shortage: US and Canada 2021. Animals : an open access journal from MDPI, 12(3). doi:10.3390/ani12030365
  15. Das, K. C. (2008). Co-composting of alkaline tissue digester effluent with yard trimmings. Waste Manag, 28(10), 1785-1790. doi:10.1016/j.wasman.2007.08.027
  16. Reuter, T., Xu, W., Alexander, T. W., Gilroyed, B. H., Inglis, G. D., Larney, F. J., . . . McAllister, T. A. (2010). Biocontained carcass composting for control of infectious disease outbreak in livestock. Journal of visualized experiments : JoVE(39). doi:10.3791/1946

 

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CVMBS Green Labs Resource Guide Copyright © 2022 by MIP 700 FA 2022 Green Labs Class. All Rights Reserved.

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