Animal Pairing and Partner Preference Test

At 8–9 wk of age, eight sexually naive males were individually partnered with an adult female for 18 h. Females were injected with 0.1 ml of 20 μg/ml estradiol benzoate (Fisher BioReagents) once a day for the 2 days before pairing to induce ovulation and receptivity toward males. At the end of the 18-h partnering phase, the strength of the male’s mate preference was assessed via a partner preference test (PPT) (see Fig. 7A) (Ahern et al. 2009; Slob et al. 1987; Williams et al. 1992). During the PPT, the partner female and an age-matched stranger female never before encountered by the male were tethered at opposite ends of a 0.6 × 0.15 × 0.3-m box. The male was placed in the middle of the cage, and his movements were recorded for 3 h. The time spent by the male in physical contact with his partner female, in physical contact with the stranger female, and alone was analyzed with TopScan (version 3.00), as previously described (Ahern et al. 2009). Partner preference scores were calculated by subtracting the time spent with the stranger from the time spent with the partner, following a published protocol (Slob et al. 1987). Testing was initiated between 8:00 and 8:30 AM. Between testing sessions, the corncob bedding was removed and the arena cleaned with 70% isopropyl alcohol. At the end of the test, each male was returned to his cage with his partner female. Rats did not undergo any behavioral experiments before death because they do not form pair bonds.

Vole partner preference test apparatus and behavior. A: top-down view of the partner preference test apparatus. A male vole was placed in the middle of a 0.6 × 0.15 × 0.3-m box, and his movements were recorded for 3 h (10,800 s). His partnered female and a stranger female never before encountered by the male were tethered at opposite ends of the box. The time spent by the male in physical contact with his partner female, in physical contact with the stranger female, and alone was analyzed. B: 4 of the 8 male voles spent the vast majority of time in physical contact with the partnered female, indicated by a gray circle around the respective data points. C: individual vole preference scores. Positive preference score indicates that male vole spent more time with partnered female than stranger female (these voles again indicated with gray circles). Negative score indicates that male spent more time with stranger female. Horizontal line in C indicates mean.