1. Animal welfare and standardization in Tecniplast IVCs: better research outcomes Stefano Gandolfi Junior Product Specialist Housing Division

2. Housing system must be designed in order to: Maximize animal welfare Maximize microbiological protection Minimize cross-contamination Maximize ergonomics Ensure consistency and standardization Refinement of the research Reduction of the number of animals Increased attention to the economic aspect Introduction Research and technologies evolution

3. Advantages of IVCs Reduced cage handling Microbiological protection Microbiological unit at cage level Possibility to set positive or negative intra-cage pressure The IVC system must be designed in order to standardize as much as possible the intra-cage conditions, without adding undesired variables LESS ANIMAL STRESS, LOW RISK OF CROSS-CONTAMINATION, HIGH PROTECTION MORE CONSISTENCY AND RELIABILITY OF THE RESEARCH

4. What does consinstency mean? - Avoid undesired variables affecting the experiments - Standardization of the intra-cage conditions - Standardization of the intra-cage conditions across different facilites Why is consinstency needed? - To have more reliable research outcomes - To reduce the number of animals employed for the experiment

5. GoalMeasurable Index Increase animal WELLNESS Body Weight Production Efficiency Index PEI Reduce animal STRESS Air Velocity at animal level Cage change Frequency Intra-cage environmental parameters (NH3, RH, CO2…) Positive behavioural patterns Motor Activity Maternal Behaviour Differences between strains Animal Welfare Poor animal welfare could act as undesired variable: it is important to maximize it as much as possible. How to evaluate it?

6. Number of Air Changes per Hour affects: - High NH3 concentration - Compromised health of mice Undesired variables Bad Environmental Conditions Milite G. & Martino P.: Microbiological load in substrate over a one and two week period for mice housed in IVC and open cages. Animal Technology and Welfare, The journal of the Institute of Animal Technology. April 2003, pag. 29-31. Wallace J., Milite G., Baldwin L., Bunce M. ”Comparative breeding performances and growth rate of mouse strains in IVCs and open cages” Animal Technology and Welfare, The journal of the Institute of Animal Technology. April 2003, pag. 28-29.

7. Number of Air Changes per Hour affects: - High NH3 concentration - Compromised health of mice Undesired variables Bad Environmental Conditions “Ammonia is an irritant with a very pungent odour and is highly toxic. […] this suggest that to maintain low NH3 concentrations 60- 80 ACH are required. “ Joanna Cruden: A comparison of various IVC systems: the comfort of the mouse. Animal Technology and Welfare, december 2007, 93-116. “Air quality can affect an animal overall’s health and well-being, perhaps more than any other environmental factor“ Di Vincenti et al: Effects of housing density on nasal pathology of breeding mice housed in individually ventilated cages. Lab Animal, 2012

8. Undesired variables Hypoxic Conditions Some IVCs system could create a slightly hypoxic environment: - Different condition than in open cages -Changes in mice’s physiology - This phenomena is not detected in Tecniplast cages “report a significant reduction in oxygen within IVC housing when compared to AEC housing” “Air oxygen was maintained at a concentration of 20.9% throughout the trial with minor fluctuation” “An expected physiologic response to reduced oxygen is increased red blood cell (RBC) mass and blood hemoglobin” York et al: Individually Ventilated cages cause chronic low- grade hypoxia impacting mice hematologically and behaviourally. Brain, behaviour and immunity, 2012.

9. Besides the number of ACH, the position of the air valves is very important: Undesired variables Stress and Anxiety “Direct exposure of animals to air moving at high velocity (drafts) should be avoided as the speed of air to which animals are exposed affects the rate at which heat and moisture are removed from an animal. For example, air at 20°C moving at 60 linear feet per minute (18.3 m/min) has a cooling effect of approximately 7°C (Weihe 1971).” Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Research – eighth edition, Division on Earth and Life Studies, National Research Council, National Academy Press, Washington D.C. 2011.

10. Besides the number of ACH, the position of the air valves is very important: Undesired variables Stress and Anxiety “Mice in ## system had a significant drop in weight at the beginning of the study compared to mice housed in Tecniplast system. […] Air blowing directly onto the animal should be avoided […]. The air entering at the bottom may cause a ‘wind tunnel’.” Joanna Cruden: A comparison of various IVC systems: the comfort of the mouse. Animal Technology and Welfare, december 2007, 93-116. “Different IVC housing systems can influence mouse behaviour, exhibiting more anxiety-related behaviour when housed in system Two (air entry at animal level)” O. Burman et al: The effect of two Individually Ventilated Cage systems on anxiety-related behaviour and welfare in two strains of laboratory mouse. Poster presented at FELASA 2013. “The location of the air supply influenced the choice of the mice, who preferred having the air supply in the cover than in the wall of the cage” Baumans Vera, Schlingmann Freek, Vonck Marlice and Van Lith Hein A.: Individually Ventilated Cages: Beneficial for Mice and Men?” Contemporary Topics, Jan 2002

11. "Draught seems to be an important factor when housing mice in IVC systems, as the mice clearly rejected the cages with a notable air speed. “This shows the importance of developing and selecting IVC systems for mice without any draught inside the cage at animal level" Besides the number of ACH, the position of the air valves is very important: Undesired variables Stress and Anxiety TC Krohn and AK Hansen: Mice prefer draught free housing. Laboratory Animals 2010; 44: 370-372.

12. Also the position of the blower is important: Undesired variables Vibrations “Attempts should be made to minimize the generation of vibration, including from humans, and excessive vibration should be avoided.” Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Research – eighth edition, Division on Earth and Life Studies, National Research Council, National Academy Press, Washington D.C. 2011. “Some mouse colonies that could not breed succesfully in racks with mounted blower ventilation, have bred effectively using this cage ventilation system” (HVAC integration). “Our suspicion is that the lack of vibration and/or sound was a significant factor” Dysco et al: Ten years of experience with building-integrated ventilated caging systems: What we’ve learned. Poster presented at FELASA 2013. THE MAGNITUDE OF VIBRATION IS NOT CONSTANT ACROSS THE RACK: NO STANDARDIZATION

13. ACH PRESSURE RACK DESIGNED IN ORDER STANDARDIZE THESE PARAMETERS Standardization of intra-cage conditions

14. TIZ-BIFO protocol Intra-cage pressures distribution: a) b)

15. Intra-cage ACH distribution: Maximum deviation: ± 15 % from the mean value Standardization of intra-cage conditions TIZ-BIFO protocol Maximum air speed at animal level: 0.2 m/s In order to avoid this:

16. Homogeneous pressure differental distribution Pressure differential value within the range – 40 / +40 Pa Homogeneous ACH distribution Low air speed at animal level INDIPENDENTLY CERTIFIED BY: Standardization of intra-cage conditions TUV certification

17. Standardization of intra-cage conditions across different facilities A long reference list ensures standardization of the experimental condition even across different facilities Refinement of the research Reliability of the results

18. Conclusions IVCs help to achieve high level of consistency Not all IVC systems are equivalent: there are many features and parameters that may affect performances and research outcome The standardization of the intra-cage conditions across the same IVC system is fundamental. There are specific certifications validating the performances of an IVC system - Avoid undesired variables affecting the experiments - Standardization of the intra-cage conditions - Standardization of the intra-cage conditions across different facilites

19. Thank you!