Histological evaluation of neutral buffered formalin and Bouin’s fixative: Liver and gills of Nile tilapia (Oreochromis niloticus)

Background: The initial fixation step is paramount in histology; the eventual quality of a tissue section hinges largely on the specific fixative chosen and how long the sample remains submerged. Aim: This study aimed to compare the histological quality of the liver and gills of Nile tilapia (Oreochromis niloticus) fixed in 10% neutral-buffered formalin (NBF) and Bouin’s solution over various immersion times (1, 7, 14, and 21 days). Methods: Histological quality, including membrane integrity, staining affinity, and cytoplasmic density, was assessed using hematoxylin–eosin staining and observed under a light microscope at 400× magnification. A qualitative scoring grade (1 = poor, 2 = fair, 3 = good, and 4 = excellent) was used, and data were presented as median (interquartile range). Statistical analysis was performed using the nonparametric Friedman test, Wilcoxon signed-rank test, and Mann–Whitney U test (p < 0.05). Results: A 1-day immersion in both NBF and Bouin’s solution yielded the highest histological quality for both organs. In liver tissue, 10% NBF provided superior preservation during the first week of immersion (Days 1–7; p = 0.008). However, by Day 21, Bouin’s solution exhibited significantly higher structural stability for the liver than NBF (p = 0.032). No significant differences were observed between NBF and Bouin’s solution in gill tissues until Day 14 (p > 0.05). However, a significant divergence occurred on Day 21, when Bouin’s solution outperformed 10% NBF (p = 0.008). Conclusion: Under the tested conditions, a 24-hour immersion duration provides the most consistent results for Nile tilapia liver and gills. While 10% NBF is effective for short-term liver preservation, Bouin’s solution offers better resilience for both organs during prolonged immersion.