Re-evaluation of methionine and isoleucine requirements in juvenile hybrid grouper fed protein-bound and crystalline amino acids diets

This study determined the methionine (Met) and isoleucine (Ile) requirements of juvenile hybrid grouper ( Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂ ) using graded protein-bound amino acid (PBAA) diets. Two feeding trials were conducted, each including a crystalline amino acid (CAA) diet at the same dose as the corresponding PBAA diet for direct comparison. Seven PBAA-Met (8.2–17.1 g/kg) and six PBAA-Ile (15.3–20.7 g/kg) diets were tested, together with two CAA-based diets (Met-14.9 g/kg; Ile-20.0 g/kg). A total of 266 fish (initial weight: 12.0 ± 0.1 g/fish) were fed to apparent satiation for seven (Met trial) or eight (Ile trial) weeks. The optimal Met and Ile requirements for PBAA-fed fish were estimated at 11.26 g/kg and 19.87 g/kg, respectively. Fish fed CAA-Met showed lower protein efficiency ratio (PER) and protein productive value (PPV), but higher hepatic glutamate-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) activity than those fed PBAA-Met. Conversely, fish fed CAA-Ile showed significantly lower weight gain (WG) ( p < 0.001) and significantly higher ammonia nitrogen excretion ( p < 0.05), along with a decreasing trend in PER, and PPV, and a higher feed conversion ratio (FCR), compared to fish fed PBAAD-Ile. The postprandial essential amino acid (EAA) profile revealed rapid spikes and declines in serum AA levels within 6 h in fish fed CAA diets, indicating rapid, yet transient absorption and increased catabolism, corroborated by elevated hepatic GPT and GOT activities. In contrast, fish fed PBAA diets exhibited increased hepatic expression of mtor and igf1 , suggesting enhanced protein synthesis and nutrient utilization efficiency. Additionally, the expression of intestinal pept1 and slc1a1 was significantly upregulated in fish fed 10.4 g/kg PBAAD-Met, but not in those fed CAA-Met 14.9, correlating with improved amino acid absorption and feed efficiency. These findings demonstrate that CAA-based formulations overestimate Met requirements due to transient metabolic imbalances and increased oxidation, whereas Ile metabolism appears less affected by the AA form. The study also highlights that serum free AA dynamics may serve as potential biomarkers for AA bioavailability, providing a framework for optimizing precision nutrition strategies and improving nitrogen utilization efficiency in aquaculture feed formulation. • Hybrid grouper (HG) require 11.26 g/kg Met and 19.87 g/kg Ile in protein-bound diets. • Crystalline amino acids impaired growth, feed efficiency, and liver enzyme activities in HG. • HG fed crystalline amino acids show rapid fluctuations in serum amino acids post-feeding. • Ammonia nitrogen levels were higher in tanks with HG fed CAAs. • Hepatic expression of mtor and igf1 were mechanistically linked to protein-bound diets.