Article | Colbert et al. 2 male offspring and 2 female offspring were assigned to this procedure. The litter was always considered the unit of analysis, and only 1 male and 1 female pair per litter was assigned to the play procedure. For the penile reflex procedure, 11, 7, 11, 10, and 6 male offspring from the 0-, 1.5-, 3-, 6-, and 12-mg/kg groups were assigned, respectively. Play behavior. We randomly selected two male and two female pups from each litter. Within each same-sex pair, one animal was designated as the “target” and the other ani- mal served as partner. Before data collection, target and partner animals were marked with a nontoxic marker for identiﬁcation; they were then separated from their littermates. Twenty- four hours later, the target animal and their same-sex partner were placed together for 10 min in a glass aquarium (12 in. wide × 24 in. long × 12 in. high) with clean cage bed- ding. We ﬁlmed their interactions under dim red light with a Canon XL1s digital video camera (Canon, Inc., Lake Success, NY) inter- faced to an iMac computer running iMovie software (Apple Computer, Inc., Cupertino, CA). All testing was conducted during the middle of the dark phase of the light/dark cycle. No other animals were present in the room during ﬁlming. We examined play behavior on PND22 and again on PND34 in the same animals. The assessment ages were chosen in order to examine play immediately after weaning on PND21 and immediately before the decline in same-sex play in male rats, which begins dur- ing PND36–40 (Meaney and Stewart 1981). A pair of trained observers later viewed the films using QuickTime software. (Apple Table 2. Mean ± SEM body weight (g) and AGD (mm) for male pups. Computer Inc.) Observers tabulated the fre- quency and distribution of the following ﬁve behaviors: nape attack (the snout of the target animal makes contact with the nape area of the partner animal; this behavior occurs fre- quently and often initiates a bout of play behavior); pounce (the target animal lunges forward with its forepaws extended and makes contact with the partner animal); pin (the tar- get animal is positioned on top of the partner animal with its forepaws placed on the part- ner; the partner animal lies on its back, fully exposing its ventral surface to the target ani- mal); wrestle (the target and partner animal roll and tumble with each other); and mount (a component of the adult male copulatory pattern where the target animal approaches the partner animal from the rear, clasps its ﬂanks, and mounts). Penile reﬂex. In rats, reﬂexive penile erec- tions and movements can be observed if the penile sheath is retracted with light pressure directed at the base of the penis (Hart and Melese-D’Hospital 1983; Sachs and Garinello 1978). Penile reflexes in the rat consist of erections (tumescence followed by detumes- cence), cupping (the end of the erect glans penis flares out), and flipping (rapid dorso- ﬂexion of the erect penis). Erections serve to extend the penis beyond the penile sheath, a function that is necessary to achieve vaginal penetration during copulation. Penile ﬂipping serves to stretch the vaginal wall and cupping serves to collect coagulating semen and seal the seminal plug against the cervix. We conducted all erection tests during the middle of the dark phase. Tests lasted for 20 min after the ﬁrst response or for 15 min in the absence of responses. During each test, ani- mals were restrained in a supine position with their head and upper torso positioned in a darkened, ventilated tube (8.5 × 5.5 × 20 cm) fastened to a plastic base. The darkened tube is anxiolytic, and rats rapidly habituate to brief periods of restraint. The penile sheath was retracted and held in place (Hart and Melese- D’Hospital 1983; Sachs and Garinello 1978). Typically, clusters of penile erections and dorsoflexions (movements or “flips”) begin spontaneously within 5–10 min after sheath retraction. Trained observers recorded the frequency and time distribution of three gradations of erections: E1, reddening and distension of glans; E2, tumescence of the base and tip of glans; and E3, intense erection accompanied by cupping of the tip of glans (Eaton et al. 1991; Hull et al. 1991; Warner et al. 1991). Penile movements, seminal emissions, latency to the ﬁrst reﬂex, and the number of response clusters were also determined. We defined a response cluster as any display of responses separated by ≥ 15 sec. Seminal emissions were deﬁned as the expulsion of seminal ﬂuid fol- lowed by a coagulating plug. Statistical methods. For the play proce- dure, the five behaviors were summed and analyzed as total play behaviors per session with repeated-measures analysis of variance (ANOVA). The individual behaviors were also analyzed separately. The litter always served as the statistical unit of analysis, with the expo- sure level as a between-litter factor and sex and PND as within-litter factors. In cases where there was a significant main effect or inter- action involving the exposure factor, Duncan’s No. of litters 12 7 13 13 7 No. if litters 12 7 13 13 7 702 Exposure group Control 1.5 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg Exposure group Control 1.5 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg Table 3. Mean ± SEM body weight (g) and AGD (mm) for female pups. End point Body weight AGD Body weight AGD Body weight AGD Body weight AGD Body weight AGD End point Body weight AGD Body weight AGD Body weight AGD Body weight AGD Body weight AGD PND1 6.9 ± 0.25 3.6 ± 0.39 6.6 ± 0.47 3.4 ± 0.15 6.7 ± 0.17 3.7 ± 1.0 7.4 ± 0.27 3.5 ± 0.11 6.9 ± 0.35 3.7 ± 0.10 PND1 6.6 ± 0.24 2.2 ± 0.04 6.7 ± 0.31 2.1 ± 0.09 6.4 ± 0.17 2.2 ± 0.04 6.8 ± 0.21 2.2 ± 0.12 6.3 ± 0.41 2.2 ± 0.09 PND4 10.4 ± 0.52 4.7 ± 0.19 10.4 ± 0.75 4.4 ± 0.17 10.0 ± 0.25 4.5 ± 0.14 11.0 ± 0.54 4.8 ± 0.16 9.6 ± 0.57 4.7 ± 0.11 PND4 9.8 ± 0.51 2.2 ± 0.04 10.6 ± 0.67 2.1 ± 0.09 9.6 ± 0.26 2.2 ± 0.04 10.3 ± 0.45 2.2 ± 0.12 9.0 ± 0.69 2.2 ± 0.09 PND8 18.4 ± 0.96 6.7 ± 0.18 16.6 ± 1.6 6.1 ± 0.52 17.7 ± 0.61 6.5 ± 0.14 19.2 ± 0.79 6.8 ± 0.20 19.2 ± 1.01 6.7 ± 0.11 PND8 17.7 ± 0.96 2.7 ± 0.10 17.9 ± 0.69 2.6 ± 0.03 17.4 ± 0.66 2.7 ± 0.09 18.0 ± 0.77 2.8 ± 0.07 17.1 ± 1.22 2.9 ± 0.13 PND12 28.3 ± 1.08 9.1 ± 0.29 25.5 ± 1.83 8.5 ± 0.57 26.9 ± 0.53 9.0 ± 0.25 28.4 ± 0.74 9.0 ± 0.24 27.8 ± 1.20 8.7 ± 0.30 PND12 27.3 ± 1.18 4.3 ± 0.16 27.2 ± 0.52 4.2 ± 0.12 26.3 ± 0.66 4.1 ± 0.14 26.9 ± 0.57 4.4 ± 0.13 25.2 ± 1.56 4.2 ± 0.13 PND16 36.5 ± 1.34 12.0 ± 0.23 33.3 ± 2.33 12.4 ± 0.87 35.2 ± 0.67 12.4 ± 0.23 37.7 ± 1.18 12.0 ± 0.30 36.7 ± 1.72 12.7 ± 0.54 PND16 35.1 ± 1.37 6.5 ± 0.23 35.2 ± 0.43 6.3 ± 0.22 34.2 ± 0.79 6.3 ± 0.21 34.4 ± 0.76 6.5 ± 0.15 33.8 ± 2.21 6.2 ± 0.18 PND20 48.4 ± 1.98 16.5 ± 0.46 43.6 ± 3.28 15.8 ± 1.25 45.9 ± 0.88 16.3 ± 0.48 47.5 ± 1.27 16.0 ± 0.47 47.5 ± 2.79 15.4 ± 0.41 PND20 46.2 ± 1.89 8.5 ± 0.21 45.8 ± 1.25 8.9 ± 0.16 44.3 ± 1.06 9.0 ± 0.25 44.9 ± 1.02 8.8 ± 0.19 43.8 ± 3.31 9.1 ± 0.34 VOLUME 113 | NUMBER 6 | June 2005 • Environmental Health Perspectives