Effects of Nesting Material on the Toxicologic Assessment of Cyclophosphamide in Crl:CD1(ICR) Mice

Abstract

The provision of nesting material benefits mice by reducing cold stress, improving feed conversion, increasing litter size, and improving adaptive immunity. The effects of toxins are sensitive to environmental changes, and the introduction of novel items can alter results in some toxicologic studies. We hypothesized that nesting material would reduce stress and positively alter immunologic parameters in Crl:CD1(ICR) mice, thus changing typical results from a well-studied immunomodulating drug, cyclophosphamide. A 13-wk study assessed the following treatments in a factorial design (n = 4; 32 cages total): nesting (0 or 10 g) and drug (50 mg/kg cyclophosphamide or 10 mL/kg saline; IP weekly). Detailed examinations and body weights were recorded weekly, and nests were scored twice weekly. Fecal pellets were collected at 0, 4, 6, and 12 wk for analysis of corticosterone metabolites. At study termination, clinical pathology and immune parameters were collected, a necropsy performed, and lymphoid organs and adrenal glands were submitted for histopathology. All expected results due to cyclophosphamide were observed. Nesting reduced the proportion of mice with piloerection, and body weights were highest in saline-nested male mice. No differences in hematology, clinical chemistry, or absolute lymphocyte counts were observed. Corticosterone metabolites in all nested groups were not different from baseline levels but all nonnested groups had higher levels than baseline. Nested cyclophosphamide-treated groups had significantly lower corticosterone levels than nonnested cyclophosphamide-treated groups. This study illustrates that nesting material does not alter the results of a standard toxicology study of cyclophosphamide but alleviates study-related stress and improves mouse welfare.

Figure 1.

Blood parameters evaluated at termination of the study.

Figure 2.

Percentage (least-square mean ± SE) of weekly detailed examinations during which clinical signs were present. Different letters indicate significant (P < 0.05) differences within clinical sign.

Figure 3.

Body weight averaged by cage throughout the experiment. Differences due to sex, drug treatment (S, saline; CY, cyclophosphamide), and nesting treatment are shown as least-square means ± SE. Different letters indicate significant (P < 0.05) differences within sex; bars indicate significant (P < 0.05) differences in body weight between sexes. No effect on body weight in female mice was observed.

Figure 4.

Average nest score throughout the experiment. Differences due to sex, drug treatment (S, saline; CY, cyclophosphamide), and nesting treatment are plotted as least-square means ± SE; different letters indicate significant (P < 0.05) differences within sex.

Figure 5.

Differences in corticosterone fecal metabolites from baseline values before the first injection of saline or cyclophosphamide averaged across the experiment. Mice were provided with bedding plus 10 g of crinkle paper nesting material (Nest) or bedding only (Control). Data are plotted as least-square means ± SE; different letters indicate significant (P < 0.05) differences within drug treatment, and bars indicate significant (P < 0.05) differences between drug treatments. Asterisks indicate significant differences according to t tests (values different from 0 or baseline; α corrected for 4 comparisons).

Figure 6.

Differences in corticosterone fecal metabolites from baseline values over the 13-wk experiment. Data are plotted as least-square means ± SE; different letters indicate significant (P < 0.05) differences within drug treatment according to Tukey pairwise comparison.