
# Research Plan

## Problem

Same-sex affiliative relationships are common in humans and social animals, forming one of the bases of group living. While the neuropeptide vasopressin (VP) and its receptors have been shown to mediate these relationships in mammals and birds with gregarious and colonial social structures, there is a significant gap in understanding how these relationships form and are maintained in species with strict dominance hierarchies.

Specifically, we lack understanding of whether triadic interactions promote affiliative relationships between dominant and subordinate individuals, and whether the VP system is involved in such affiliations. Previous theoretical research suggests that social interactions among three individuals differing in "power" and "activity" should be the minimal group size to produce coalition/alliance formation, but experimental evidence for this hypothesis is limited.

Large-billed crows present an ideal model to investigate these questions, as non-breeding males form strict linear dominance hierarchies while also being capable of forming affiliative relationships characterized by allopreening, food sharing, and conflict aiding. We hypothesize that cohabitation of three male non-breeder large-billed crows could facilitate the formation of affiliative relationships between specific dominant and subordinate individuals, and that vasopressin 1a receptors (V1aR) are involved in the maintenance of these relationships.

## Method

We will conduct two sequential experiments using 18 adult male large-billed crows aged 4-7 years. Prior to experimentation, we will establish dominance relationships between all potential dyads through three trials of dyadic social encounters, where winners and losers are determined based on aggressive and submissive behaviors.

**Experiment 1** will test whether triadic cohabitation facilitates affiliative relationship formation. We will introduce eight groups of three male crows (each with established dominance relationships but no affiliative relationships) into an aviary for two weeks. To ensure that any observed affiliative relationships result from triadic rather than dyadic interactions, we will conduct pre-test dyadic cohabitation for 3 days before triadic cohabitation, followed by post-test dyadic cohabitation for 3 days after the triadic period.

**Experiment 2** will investigate the role of V1aR in maintaining established affiliative relationships through pharmacological manipulation. We will systematically administer a V1aR antagonist (SR 49059) at both high (0.3 mL × 1 mg/mL) and low doses (0.3 mL × 0.1 mg/mL) via intramuscular injection, compared with vehicle control, to either dominant or subordinate individuals in both affiliated and unaffiliated dyads.

## Experiment Design

**Experiment 1 Design:**
- Pre-test: House all possible dyads from each triad separately for 3 days to establish baseline
- Triadic cohabitation: House three birds together for 2 weeks in experimental aviary
- Post-test: Re-house dyads separately for 3 days to confirm relationship persistence
- Video record behavior for 30 minutes daily during pre/post-tests and during early (days 1-3), middle (days 7-9), and last (days 12-14) periods of triadic cohabitation
- Define affiliative relationships as dyads showing reciprocal allopreening with both individuals initiating ≥10 bouts per 30 minutes in any 3-day block
- Use discriminant analysis with leave-one-out cross-validation to validate exclusive formation of affiliative relationships

**Experiment 2 Design:**
- Test birds that formed affiliative relationships in Experiment 1
- Each trial: 30-minute pre-administration observation, drug/vehicle injection to focal bird, 1-hour isolation, 30-minute post-administration observation
- Each bird receives 8 trials: 3 high-dose, 3 low-dose, 2 vehicle control
- Counterbalance trial order of dominance position and drug conditions within and between dyads
- Test both affiliated dyads and unaffiliated dyads (using third bird from original triad)
- Measure allopreening, aggressive behaviors, and submissive vocalizations
- Use Generalized Linear Mixed Models (GLMMs) with Poisson distribution to analyze effects of dominance status, drug condition, and phase on social behaviors
- Include trial number and dyad identity as random factors

We will code all behaviors from video recordings using established ethological criteria, with allopreening defined as passing the bill through another bird's feathers for >2 seconds. Statistical significance will be determined at the 5% level, with post-hoc pairwise comparisons performed for significant interactions.