RSM‐Optimised Lactococcus lactis and Schizochytrium limacinum Combination Improves Immunity and Streptococcus agalactiae Resistance in GIFT Tilapia

This study optimised the dietary supplementation of Lactococcus lactis and Schizochytrium limacinum in genetically improved farmed tilapia (GIFT, Oreochromis niloticus ) to improve immunity and resistance to Streptococcus agalactiae infection. Single‑factor experiments and central composite design combined with response surface methodology (CCD‑RSM) were employed for sequential optimisation. A desirability function was developed as the optimisation criterion, integrating percentage survival of S. agalactiae ‑infected tilapia, non‑specific immune and antioxidant parameters (total antioxidant capacity, superoxide dismutase, catalase, alkaline phosphatase, and acid phosphatase), and liver health indices (reduced glutathione, alanine aminotransferase, and aspartate aminotransferase). Single‑factor experiments determined optimal individual supplementation levels of 1.5% for S. limacinum and 1.0 × 10 7 CFU/g for L. lactis . CCD‑RSM revealed that L. lactis significantly affected total antioxidant capacity, catalase, and alanine aminotransferase, while S. limacinum significantly influenced percentage survival of S. agalactiae ‑infected tilapia, superoxide dismutase, catalase, alkaline phosphatase, acid phosphatase, alanine aminotransferase, and reduced glutathione, with significant interactive effects between the two supplements on multiple parameters. The RSM‑optimised combination was 5.45 × 10 7 CFU/g L. lactis plus 0.91% S. limacinum . Tilapia were fed for 28 days with four dietary treatments: the RSM‑optimised combination, L. lactis alone, S. limacinum alone, and a control. The RSM group showed higher superoxide dismutase, catalase, and acid phosphatase activities, lower alanine aminotransferase and aspartate aminotransferase levels, and fewer hepatic vacuoles than other groups. Following S. agalactiae challenge (3 × 10 8 CFU/kg), the RSM group achieved the highest relative per cent survival (44.12%), followed by L. lactis alone (35.29%) and S. limacinum alone (26.47%). Collectively, the RSM‑optimised combination demonstrates promising potential for enhancing resistance to S. agalactiae infection in GIFT tilapia.