Beyond a biased binary: A perspective on the misconceptions, challenges, and implications of studying females in avian behavioral endocrinology.

For decades, avian endocrinology has been informed by male perspectives and male-focused research, leaving significant gaps in our understanding of female birds. Male birds have been favored as research subjects because their reproductive behaviors are considered more conspicuous and their reproductive physiology is presumably less complex than female birds. However, female birds should not be ignored, as female reproductive behavior and physiology are essential for the propagation of all avian species. Endocrine research in female birds has made much progress in the last 20 years, but a substantial disparity in knowledge between male and female endocrinology persists. In this perspective piece, we provide examples of why ornithology has neglected female endocrinology, and we propose considerations for field and laboratory techniques to facilitate future studies. We highlight recent advances that showcase the importance of female avian endocrinology, and we challenge historic applications of an oversimplified, male-biased lens. We further provide examples of species for which avian behavior differs from the stereotypically described behaviors of male and female birds, warning investigators of the pitfalls in approaching endocrinology with a binary bias. We hope this piece will inspire investigators to engage in more comprehensive studies with female birds, to close the knowledge gap between the sexes, and to look beyond the binary when drawing conclusions about what is 'male' versus 'female' biology.

Neural endocannabinoid CB1 receptor expression, social status, and behavior in male European starlings.

Many species modify behavior in response to changes in resource availability or social status; however, the neural mechanisms underlying these modifications are not well understood. Prior work in male starlings demonstrates that status-appropriate changes in behavior involve brain regions that regulate social behavior and vocal production. Endocannabinoids are ubiquitously distributed neuromodulators that are proposed to play a role in adjusting behavior to match social status. As an initial step to provide insight into this hypothesis we observed flocks of male starlings in outdoor aviaries during the breeding season. We used quantitative real-time PCR to measure expression of endocannabinoid CB1 receptors in brain regions involved in social behavior and motivation (lateral septum [LS], ventral tegmental area [VTA], medial preoptic nucleus [POM]) and vocal behavior (Area X and robust nucleus of the arcopallium; RA). Males with nesting sites sang to females and displaced other males more than males without nesting sites. They also had higher levels of CB1 receptor expression in LS and RA. CB1 expression in LS correlated positively with agonistic behaviors. CB1 expression in RA correlated positively with singing behavior. CB1 in VTA also correlated positively with singing when only singing birds were considered. These correlations nicely map onto the well-established role of LS in agonistic behavior and the known role of RA in song production and VTA in motivation and song production. Studies are now needed to precisely characterize the role of CB1 receptors in these regions in the production of status-appropriate social behaviors.

Reward associated with singing behavior correlates with opioid-related gene expression in the medial preoptic nucleus in male European starlings.

Birdsong consists of species-specific learned vocal sequences that are used primarily to attract mates and to repel competitors during the breeding season. However, many birds continue to sing at times when vocal production has no immediate or obvious impact on conspecific behavior. The mechanisms that ensure that animals produce important behaviors in contexts in which the function of these behaviors is not immediate or obvious are not known. One possibility is that animals engage in such behaviors because they are associated with pleasure. Here we examined the hypothesis that male European starlings sing outside of the breeding season in part because the act of singing in this context is facilitated and/or maintained by opioid-mediated reward. We measured song-associated reward using a conditioned place preference (CPP) test in male starlings producing fall, non-breeding season-typical song. We used quantitative real time PCR to measure expression of the enkephalin opioid precursor preproenkephalin (PENK) and mu opioid receptors (MOR) in the medial preoptic nucleus (POM; a region in which opioids are implicated in both reward and starling fall song) and additionally the song control region HVC as a control. Starlings developed a strong preference for a place that had been paired previously with the act of producing fall-typical song, indicating that fall song production was associated with a positive affective state. Both PENK and MOR mRNA expression in the POM, but not HVC, correlated positively with both individual reward state (as reflected in CPP) and undirected singing behavior. These results suggest that singing induces opioid receptor and enkephalin expression in the POM and consequent reward, and/or that opioid release in the POM induced by individual or environmental factors (e.g., the presence of food, safety of a flock or the absence of predators) induces a positive affective state which then facilitates singing behavior.

Testosterone elevation and response to gonadotropin-releasing hormone challenge by male northern cardinals (Cardinalis cardinalis) following aggressive behavior.

There is much discrepancy about the relationship between testosterone (T) and male aggressive behavior. For example, in birds, males of many species significantly elevate T levels during inter-male conflict. However, this is not universal, and in species where males typically do not elevate T during aggressive interactions, concentrations of the hormone are often assumed to be circulating at maximum levels. We examined if male northern cardinals (Cardinalis cardinalis) significantly elevated T during simulated territorial intrusions (STIs). We also examined if individuals had the capacity to further elevate T levels in response to gonadotropin-releasing hormone (GnRH) injections immediately after an aggressive encounter. Our results indicate that male cardinals do not significantly elevate T levels in response to STIs, but have the physiological capacity to significantly elevate T in response to GnRH injections following aggressive interactions. This implies that T levels of individuals captured during STIs were not at maximum concentrations. However, additional findings in this study also suggest the possibility that prolonged social instability could elicit significant elevations in T in males of this species, warranting further investigation.

Non-breeding gonadal testosterone production of male and female northern cardinals (Cardinalis cardinalis) following GnRH challenge.

Yearly, testosterone (T) levels fluctuate as many vertebrates cycle through reproductive and non-reproductive periods. Among many temperate birds, it is well established that levels of T peak as gonads recrudesce for breeding and then fall as gonads regress prior to the non-breeding season. While the tissues producing breeding season T are well studied, the tissues responsible for non-breeding T have received less investigative attention. We examined the ability of male and female Northern Cardinals (Cardinalis cardinalis) to elevate gonadal T following standardized injections of gonadotropin-releasing hormone (GnRH) across three non-breeding seasons. Males and females were capable of significantly elevating gonadal T production following GnRH injections during periods of reproductive quiescence. The magnitude of T elevation varied across the non-breeding season, but not between sexes. To our knowledge, this is the first report of a significant increase in gonadal T production following GnRH injections administered in the non-breeding season.