Estimating volumes from common carp hepatocytes using design‐based stereology and examining correlations with profile areas: Revisiting a nutritional assay and unveiling guidelines to microscopists

Abstract

Assessing fish liver status is common in aquaculture nutrition assays. This often implies determining hepatocytes profile areas in routine thin (5–7 μm) histological sections. However, there are theoretical problems using planar morphometry in thin sections: inherent sampling cells biases, too small numbers of sampled cells, under/overestimation of size, measuring size as areas when cells are three‐dimensional (3D) entities. The gold standard for assessing/validate cell size is stereology using thick sections (20–40 μm). Here, we estimated the volume of hepatocytes and their nuclei by the nucleator and optical disector stereological probes (in thick sections), and, innovatively, in thin sections too (using single‐section disectors). The liver of common carp eating feed containing either low or high level of lipids was targeted. Results were compared with prior profile areas from planar morphometry using thin sections, and with profile areas estimated here with the two‐dimensional (2D) nucleator. Ratios between nucleus and cell/cytoplasm (N/C) areas and volumes were calculated and compared. There was high positive correlation between volumes in thin and thick sections (r = .85 to .89; p < .001), empirically validating the single‐section disector. Strong correlations existed between profile‐derived versus 2D‐nucleator areas (r = .74 to .83; p < .001). There was systematic underestimation of cells and nucleus size using planar morphometry. The N/C ratios derived from the 2D‐nucleator data were higher than those from planar morphometry. Despite theoretical premises for using simple planar morphometry in thin sections are flawed, our results support that such morphometry on carp/fish hepatocytes may offer some valid biological conclusions. Anyway, we advanced guidelines for implementing proper methods.

Dietary supplementation of ginger (Zingiber officinale) essential oil exhibits positive immunomodulatory effects on the Neotropical catfish Pseudoplatystoma reticulatum without negative effects on fish liver histomorphometry

“…Fish undergoing various types of nutritional stressors, such as feed restriction (Souza et al 2001), unbalanced diets (Rašković et al 2019) and lower fee-ding frequency (Rodrigues et al 2017), or exhibiting nutritional pathologies due to certain plant-derived feed additives (Zakęś et al 2008), were found to exhibit smaller hepatocyte nuclei. Accordingly, the essential oil supplementation at the levels evaluated seem to not have been a stressor in our study, as the measures of hepatocyte nuclear size (i.e.…”

Performance of Co-Housed Neon Tetras (Paracheirodon innesi) and Glowlight Rasboras (Trigonostigma hengeli) Fed Commercial Flakes and Lyophilized Natural Food

“…The nuclear area (NA) and cytoplasmic area (CA) of liver cells was measured for 100 cells per fish (20 hepatocytes × 5 fields of view × 5 fish per feeding regime; group n = 500), which is the minimal amount required to neglect the error generated by such planar morphometric approach, as suggested by Rašković et al [ 80 ]. The nucleo-cytoplasmic index (NCI) was calculated separately for each cell using the following formula [ 71 ]: NCI = 100 × NA × CA −1 .…”

Special Issue on the Histopathology of Aquatic Animals

“…Often, new ingredients in fish feeds will not cause significant histopathological alterations but will have an effect on the appearance of cells involved in the absorption or transformation of molecules that originate from the feed, such as enterocytes in the intestine or hepatocytes in the liver, respectively [3]. Their surface area or volume can be determined using various histological techniques, and a decreased size of cells (or their nuclei) is a marker for metabolic and physiological differences of organs in fish that are fed alternative feeds [4]. However, some antinutritional factors in plant-based fish feed have inflammatory effects on the distal intestine of fish, causing enteritis.…”