Pituitary Gland Extraction and Genetic Analysis

Pituitary glands were obtained from two fish farms. The first is located in São João da Boa Vista (São Paulo), and the processed material was used for studies regarding ag-LH and ag-FSH [16 (link)] and detection of a first partial region (~200 bp, “seq1”) of ag-GH, utilizing primer #1 and #2 (Table 1). Initially, the design of the two primers was based on conserved regions of the GH gene from three different fish species: Anguilla anguilla, Cyprinus carpio, and Ictalurus punctatus, available in GenBank under the accession numbers AY148493.1, M27000.1, and NM_001200245.1, respectively. For the selection of the species in this study, data previously obtained by our research group were used [16 (link),17 (link)]. These GH sequences were aligned and the region that presented the closest profile for the three species was chosen as a safe region for the design of the primers used in this first step. The second pituitary collection was from Canabrava do Norte, Mato Grosso State, within the facilities of the Raça fish farm, collaborating with our project. The animals weighed 15–40 kg and the collected pituitary glands were 70–100 mg. These were stored in 1 mL of RNALater™ (ThermoFisher Scientific, Waltham, MA, USA), and transported to our laboratory in São Paulo in dry ice.