**Background:** Yellowtail kingfish (YTK) is a carnivorous fish farmed for its high-quality meat. As fishmeal is progressively replaced with more sustainable protein sources, it is critical to quantify the dietary requirements of essential nutrients like sulphur amino acids (SAAs) — methionine (Met), cysteine (Cys), and taurine (Tau). Previous research showed that sub- or supra-optimal levels of these SAAs impair growth, feed efficiency, and cause pathological conditions such as cataracts, enteritis, and liver abnormalities. However, the specific effects on liver and intestinal histology and biochemistry were not well characterized. This study aimed to evaluate how varying dietary Met, Cys, and Tau levels affect liver and posterior intestine health in juvenile YTK, using samples from two previously published requirement studies.
**Methods:** Samples were obtained from two feeding trials. The TauMet study used a factorial design with seven Tau levels (1.6–20.4 g/kg diet) crossed with two Met levels (10.9 or 17.2 g/kg) at constant Cys (~5.9 g/kg), resulting in 14 diets. The MetCys study used five Met levels (7.9–25.2 g/kg) crossed with two Cys levels (5.6 or 13.9 g/kg) at constant Tau (7.0 g/kg), plus a commercial diet. Fish were hand-fed to satiation for 45–54 days. After euthanasia, blood plasma was collected for biochemistry (cholesterol, TAG, AST, LD, ALP, bicarbonate, Ca, phosphate). Livers were photographed under standardized conditions for digital color analysis (CIE Lab, RGB, HSB) and then processed for histology (hematoxylin-eosin staining). Posterior intestines were processed for histomorphometry and histochemistry (AB-PAS staining). Semi-quantitative scoring of liver fattiness (0–3 scale) and quantitative measurements of bile duct wall (BDW) thickness, necrotic hepatocytes, large nuclei, cytoplasm eosinophilia, and marginated chromatin were performed. Intestinal measurements included total intestinal wall thickness, muscularis interna thickness, stratum compactum+granulosum thickness, villus area, lamina propria area, lamina epithelial area, total villus height, villus length, total villi count, and goblet cell mucus types (acidic AB+, neutral PAS+, mixed AB+PAS+), supranuclear vacuoles, and small PAS+ bullet-shaped bodies (S-PAS+). Statistical analyses used two-way ANOVA/ANCOVA with Tukey HSD post-hoc tests (P<0.05).
**Key Results:**
- **Liver histology:** Higher dietary Tau (20.4 g/kg) significantly reduced BDW thickness. YTK fed 17.2 g Met/kg had more necrotic hepatocytes, but increasing Tau at this Met level reduced necrosis. YTK fed 10.9 g Met/kg had more large hepatocyte nuclei (7.45 per 94 µm²) vs. 17.2 g Met/kg (5.05 per 94 µm²). Cytoplasm eosinophilia was highest in diet T+M 3 (14.5 per 94 µm²). Liver fattiness scores were not significantly different among treatments.
- **Liver surface color:** The a* (red-green) value was highest (reddest) in YTK fed diet T+M 3 (8.5 g Tau/kg, 10.9 g Met/kg; a*=34.2). Higher Met (17.2 g/kg) significantly increased R and B values. The average liver color was close to hex 'copper-red' (#CB6D51). No green liver syndrome was observed. BDW thickness correlated positively with a* (r=0.57) and negatively with b* (r=-0.41) and H (r=-0.56). Fattiness correlated positively with b* (r=0.89) and H (r=0.67). Plasma cholesterol correlated positively with R (r=0.57) and B (r=0.56); TAG correlated positively with L (r=0.54), R (r=0.60), and B (r=0.55).
- **Plasma biochemistry:** Cholesterol and TAG did not exceed 4.7 and 1.6 mmol/L, respectively. Higher Met (17.2 g/kg) increased AST and LD activity. Bicarbonate decreased from 10 to 5 mmol/L with increasing Tau; phosphate increased from 1.85 to 3.07 mmol/L; Ca increased from 2.7 to 3.3 mmol/L.
- **Posterior intestine (TauMet study):** YTK fed diets with higher Met and Tau (T+M 8, 11, 14) had increased posterior intestine circumference, total villus height, villus length, villus area, lamina epithelial area, total intestinal wall thickness, and muscularis interna thickness. YTK fed low Tau had more villi. Goblet cell mucus was predominantly acidic (AB+). Increasing Tau and Met decreased acidic mucus density and increased mixed and neutral mucus. Total goblet cell density was highest in T+M 7 (0.091 per µm²). Supranuclear vacuole density was highest in T+M 1 (0.055 per µm²) and lowest in T+M 7 (0.010 per µm²). S-PAS+ density was highest in T+M 8 (0.150 per µm²).
- **Posterior intestine (MetCys study):** YTK fed diet M+C 2 (24.7 g Met/kg, 5.9 g Cys/kg) had the longest villi, thickest muscularis interna, and more villi. High Cys (13.9 g/kg) reduced villus height and villus area. Goblet cell mucus shifted from acidic to mixed with higher Met and Cys. Supranuclear vacuole density was highest in M+C 2 (0.101 per µm²) and in commercial diet (0.264 per µm²). S-PAS+ density was lower than in TauMet study.
**Clinical Implications:** This study provides the first comprehensive quantitative histological and biochemical reference data for liver and intestinal health in juvenile YTK fed varying levels of Met, Cys, and Tau. Adequate dietary SAA levels (approximately 24.5 g Met+Cys/kg, with at least 14.3 g Met/kg, ≤13.9 g Cys/kg, and 7.7 g Tau/kg) improved liver color (redder, brighter), reduced bile duct wall thickness, increased intestinal absorptive surface area, shifted goblet cell mucus toward digestive/absorptive functions, and reduced lipid accumulation in enterocytes. These findings support the use of nutrient-based formulations that meet SAA requirements to optimize fish health and productivity in commercial YTK aquaculture. The quantitative methods described (digital liver color analysis, histomorphometry, histochemistry) can be used as diagnostic tools and may facilitate automation via deep learning algorithms. Further research is needed to understand the relationships between histology and organ function, particularly regarding goblet cell mucus, stratum granulosum, and immune responses.