Sex differences in the neural mechanisms underlying energy balance are a fundamental aspect of avian integrative physiology, yet remain poorly understood. The 26RFamide/pyroglutamylated RFamide peptide (26RFa/QRFP) is a hypothalamic orexigenic neuropeptide whose precise role and regulation in songbirds are unknown due to the lack of species-specific analytical tools. 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 a competitive enzyme-linked immunosorbent assay and cross-validated by the neuroanatomical colocalization of 26RFa/QRFP-like immunoreactive cell bodies and mRNA transcripts in the ventromedial and lateral hypothalamic areas. Using this validated platform, we identified striking female-biased dimorphism in the zebra finch diencephalon, with approximately 3.9-fold higher mature peptide content and 1.5-fold higher mRNA expression in females than in males (P < 0.05). These findings suggest that zebra finches employ sex-specific neuroendocrine strategies for metabolic regulation.
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