Female-biased 26RFa/QRFP peptide content and transcript levels in the zebra finch ( Taeniopygia guttata ) diencephalon

Sex differences in the neural mechanisms underlying energy balance remain a fundamental yet poorly understood aspect of avian integrative physiology. The 26RFamide/pyroglutamylated RFamide peptide (26RFa/QRFP) is a hypothalamic orexigenic neuropeptide; however, its precise role and regulation in songbirds have been hindered by a lack of species-specific analytical tools. In this study, we established a robust methodological framework to investigate the 26RFa/QRFP system in the zebra finch (Taeniopygia guttata). We developed and validated a novel, high-affinity rabbit antiserum directed against a specific fragment of zebra finch 26RFa/QRFP. The specificity of this reagent was rigorously confirmed through competitive ELISA and further cross-validated by the neuroanatomical colocalization of 26RFa/QRFP-like immunoreactive cell bodies and mRNA transcripts in the ventromedial and lateral hypothalamic areas. Leveraging this validated platform, we identified a striking female-biased dimorphism in the zebra finch diencephalon: mature peptide content was approximately 3.9-fold higher and mRNA expression was 1.5-fold higher in females than in males (P < 0.05). Our findings suggest that zebra finches employ sex-specific neuroendocrine strategies for metabolic regulation.