In social species like the feral horse (Equus caballus), changes in individual behavior are likely to affect associated animals. On Shackleford Banks, North Carolina, USA, mares treated with the contraceptive agent porcine zona pellucida (PZP) demonstrate decreased fidelity to their band stallions. Here, we assess the effects of such decreased mare fidelity on male behavior and address potential interactions with habitat visibility, a component of the environment shown to significantly affect feral horse behavior. We compared the frequency and escalation of male-male contests, rates of aggressive and reproductive behaviors directed toward females, and the percentage of time spent vigilant among males experiencing varying levels of mare group changing behavior. We found that regardless of habitat visibility, males experiencing more female group changes engaged in contests at a higher rate (P = 0.003) and escalation (P = 0.029) and spent more time vigilant (P = 0.014) than males experiencing fewer group changes. However, while visibility had a positive effect on aggression directed by stallions toward mares (P = 0.013), female group changing behavior did not influence male-female aggressive or reproductive behaviors (P > 0.1), showing that decreases in mare fidelity altered male-male but not male-female interactions. These results have important implications for feral horse management; PZP-contracepted mares demonstrating prolonged decreases in stallion fidelity may have a disproportionate effect on male behavior. Moreover, our results shed light on the relative influences of female behavior and environmental factors like habitat visibility on male behavior. Such findings can ultimately improve our understanding of how the social and physical environments interact to shape male-male and male-female interactions.

Stress responses can be triggered by several physical and social factors, prompting physiological reactions including increases in glucocorticoid concentrations. In a population of feral horses (Equus caballus) on Shackleford Banks, North Carolina, females previously immunized with the immunocontraceptive agent porcine zona pellucida (PZP) change social groups (bands) more often than unimmunized females, disrupting the social stability within the population. We assessed the effects of increased female group changing behavior (or female turnover) on individual male stress by comparing fecal cortisol metabolite (FCM) concentrations among stallions experiencing varying amounts of female group changing behavior. FCM concentrations did not significantly correlate with female turnover. Similarly, FCM concentrations were not dependent upon the timing of female group changing behavior. These findings suggest that female turnover rate has little influence on physiological measures of stress in associated stallions. That said, Shackleford stallions experiencing increased female turnover do engage in behaviors typically associated with stress (increased vigilance, highly escalated male-male conflicts). Future work should compare FCM concentrations across time within populations and among populations managed under different strategies to better isolate factors influencing stallion stress physiology. Such studies are especially important if we are to determine how changes in female behavior related to immunocontraception impact physiological and behavioral indicators of stress for non-target animals. Finally, our study highlights the importance of considering both physiological and behavioral measures when investigating animal responses to potentially challenging situations.

Management tools like immunocontraception can alter the behavior of target animals, but the extent to which they affect non-target individuals has received less attention. The feral horse (Equus caballus) population on Shackleford Banks, North Carolina is an ideal system with which these questions may be explored, as management of the population with the immunocontraceptive agent porcine zona pellucida (PZP) has resulted in an increased propensity for females to change social groups and thus, decreased social stability. During the study, on average, females made 1.4 group changes per day (range = 0-18.5 group changes per day): females previously treated with PZP made 1.8 group changes per day, while females that had never been treated made 1.2 group changes per day. Between May and August, 2017, we used playbacks of aggressive male vocalizations (squeals) and human voices (reciting "hello horse") to assess changes in stallion responses to male rivals versus socially irrelevant stimuli in the context of female turnover. Over the course of the study, males were observed for 9.4 hours on average (range = 2.4-20.5 hr).Males spent more time vigilant (estimate = 12.431, P = 0.016, 𝑥̅ squeal = 30 s, 𝑥̅ control = 19 s) and were more likely to approach the speaker following squeal playbacks than controls (estimate = 2.325, P = 0.039). Males' latency to return to normal behaviors varied depending on whether the playback was conducted in the weeks before, during, or after group changes occurred (P = 0.025, 𝑥̅ before = 26 s, 𝑥̅ during = 39 s, 𝑥̅ after = 53 s). Male responses were not affected by the total number of female group changes a male experienced during the observation period (P > 0.4), suggesting the effects are more context-dependent and not long-lasting. These findings suggest mare turnover can impact stallion responsiveness to potential rivals. As previously contracepted mares change groups more often than untreated mares and stallions exhibit prolonged responses to aggressive vocalizations after experiencing a female group change, contraception-induced changes to mare behavior may lead to increased male aggression in response to intruding rivals, which could be associated with greater energy expenditure. Finally, our work demonstrates that playback experiments are a useful tool for studying feral horse behavior in the wild. As the need for population control of different species continues to expand, rigorous investigations of immunocontraception's effects on non-target animals are critical if agencies are to manage populations most effectively.

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