Carbon Dioxide, Oxygen, and Ammonia Levels in Mouse and Rat Disposable IVC Removed from Mechanical Ventilation

Abstract

Maintenance of an appropriate microenvironment for rodents used in research is of paramount importance because changes in environmental parameters such as O₂ and humidity can influence animal health and welfare and potentially alter research results. Here we evaluated the microenvironment of mouse and rat disposable cages after removal from mechanical ventilation in order to guide recommendations for their use. Cages with sealed IVC lids, unsealed lids (partially ajar), and lids without the exhaust filter (for rats) or static lids (for mice) were removed from the ventilated rack and were thereafter monitored CO₂, O₂, and NH₃ levels. For mice, effects were investigated under both standard (set point of 72°F/22°C) and thermoneutral (set point of 82°F/28°C) temperatures. When IVC with sealed lids and group-housed C57BL/6J male mice were removed from ventilation under standard temperatures, CO₂ started at 6,600 ± 265 ppm at 0 h and rose to 42,500 ± 7,263 ppm at 1 h, with mice showing a visibly elevated respiratory rate in 1 of the 3 cages; CO₂ stabilized at 26,150 ± 3,323 ppm at 8 h. In contrast, CO₂ levels in cages with single mice were stable after 1 h (1,350 ± 409 ppm at 0 h, 9,367 ± 802 ppm at 1 h, and 8,333 ± 1,115 ppm at 8 h). Findings were similar at thermoneutral temperatures: sealed group-housed mice cages started at 3,617 ± 475 ppm at 0 h and rose to 39,333 ± at 5,058 ppm at 1 h, whereas sealed cages with 1 mouse started at 1,117 ± 247 ppm at 0 h and were 7,500 ± 1,997 ppm at 8 h. IVC with sealed lids and pair-housed Crl:CD(SD) female rats rose to 48,000 ± 2,828 ppm CO₂ and over 70% humidity within 1 h. By 3 h, IVC with sealed lids and singly housed rats had 40,167 ± 5,132 ppm CO₂, and rats were displaying a visually elevated respiratory rate. O₂ levels had an inverse relationship with CO₂ levels. Removing the rat lid exhaust filter was not helpful. However, leaving the IVC lid ajar ameliorated the rise in CO₂ and fall in O₂ for both species. Therefore, IVC with sealed lids and group-housed mice should not be removed from ventilation more than 1 to 2 h; IVC containing pair- or singly-housed rats IVC should not be removed for more than 1 or 3 h, respectively. Whenever possible, such cages should be fitted with static lids, left partially ajar and monitored, or replaced on ventilation.

Figure 1.

Representative images of mouse lid–cage conditions after removal from mechanical ventilation. (A) Mouse IVC lid. (B) Mouse IVC lid held ajar approximately 1/2 in. by using disposable cloth wipe. (C) Mouse static lid. Note: water bottles were removed temporarily and then restored after data acquisition.

Figure 2.

Representative images of rat lid–cage conditions after removal from mechanical ventilation: (A) Rat IVC lid. (B) Rat IVC lid held ajar approximately 1/2 in. by using disposable cloth wipe. (C) Rat IVC lid with filter removed. Note: water bottles were removed temporarily and then restored after data acquisition.

Figure 3.

Representative images of gas sampling procedures for (A) mouse cage with CO_2–NH₃ meter and (B) rat cage with O₂ meter. Note: water bottles removed were temporarily and then restored after data acquisition.

Figure 4.

(A and C) CO₂ and (B and D) O₂ conditions in mouse caging after removal from ventilation over time according to cage occupancy and lid type at standard temperatures (A and B) and thermoneutral temperatures (C and D). Data are shown as mean ± SEM. Circled numbers indicate the observation of clinical signs and removal of group IVC lid cages from study at that time point. Colors indicate significant differences between conditions (black (group) and gray (single), sealed IVC lid compared with static lid; green (group) and light green (single), sealed IVC lid compared with unsealed IVC lid; blue (group) and light blue (single), unsealed IVC lid compared with static lid): *, P < 0.05; †, P < 0.01; ‡, P < 0.001; §, P < 0.0001.

Figure 5.

(A) CO₂ and (B) O₂ conditions of rat caging after removal from ventilation according to cage occupancy and lid type at standard temperatures. Data are shown as mean ± SEM. Circled numbers indicate the observation of clinical signs and removal of cages that were paired housing with IVC lid, paired housing with exhaust filter removed, and single housing with IVC lid cages at that time point. Colors indicate significant differences between conditions (black (pair) and gray (single), sealed IVC lid compared with exhaust filter removed; green (pair) and light green (single), sealed IVC lid compared with unsealed IVC lid; blue (pair) and light blue (single), exhaust filter removed compared with unsealed IVC lid): *, P < 0.05; †, P < 0.01; ‡, P < 0.001; §, P < 0.0001.