Bx40f4

At the end of 6 weeks trial, 6 fish per treatment were selected randomly and euthanized with an overdose of AQUI-S^® to excise liver, intraperitoneal fat and intestine for histological analysis. Fragments of all tissue samples were fixed in 10% neutral buffered formalin, dehydrated with a series of ethanol concentrations before infiltrating in xylene and embedding in paraffin wax, and finally sectioned at approximately 5 µm using a rotary microtome machine for staining with hematoxylin and eosin (H&E) following the standard histological procedures. Histological slides were digitally photographed under a light imaging microscope (BX40F4, Olympus, Tokyo, Japan).
Intestinal section were further subjected to two different stains including Periodic Acid-Schiff (PAS) and Alcian Blue (AB) pH 2.5 staining to identify neutral mucins and acidic mucins, respectively. Both type of mucins were counted from ten intact randomly selected villi, as described earlier by Elia, et al.^{1 (link)}. Ten intact villi were randomly selected to measure the intestine histometric in terms of villi height and width, enterocyte width, muscular wall, submucosa thickness and microvilli height and diameters of adipocyte were measured using ImageJ software.

After fixation with 10% buffered formalin, fragments of intestine, liver, kidney, spleen, and skin tissue samples were dehydrated with a series of ethanol concentrations. Dehydrated samples were subsequently cleared in xylene, embedded in paraffin blocks, cut into around 5 µm slices, and stained with haematoxylin and eosin (H&E) for histopathological assessment. Intestine and liver samples were stained with Periodic Acid-Schiff (PAS) to visualize neutral mucins and glycogen, respectively. Skin tissue was stained with AB-PAS stain to visualize goblet cells. Microphotographs of all histological slides were taken with a light imaging microscope (BX40F4, Olympus, Tokyo, Japan). The number of neutral mucins in intestine and skin was counted as described in our earlier study^{13 (link)}.
Scanning electron microscopy of distal intestine from four biological replicates were analysed according to the earlier study in our laboratory^{24 (link)}. Intestinal samples (5 mm) were washed for 30 s with 1% S-carboxymethyl-L-cysteine to remove mucus and then fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (0.1 M pH 7.2). Samples were processed as described elsewhere, screened with JSM 6610 LV (Jeol, Tokyo, Japan) SEM and analysed with Image J 1.46r (National Institute of Health, USA).

Six fish per treatment (two fish from each replicate) were randomly selected for the histological analysis. The dissected liver and distal intestine were then fixed in 10% buffered formalin for 72 h, after being cleaned with normal saline solution. Following a series of alcohol washes to dry the samples, they were cleaned in xylene and imbedded in paraffin wax for sectioning. The serial sections were cut to the thickness of 5 μm. The sections were stained with hematoxylin and eosin (H&E), covered with a coverslip, and magnified 400 times under a light microscope (Olympus, Germany). Using the onboard camera, the histopathology pictures were obtained (BX40F4, Olympus, Tokyo, Japan). To determine the intestinal micromorphology, including intestinal fold height (IFh), fold width (IFw), goblet cell number (IGCn), and number of adipocyte cells, ten intact intestinal folds from each dietary treatment were studied, as described previously [25 (link),26 (link)]. The sections were assessed for anomalies in structure (the relative area of vacuolation in the liver) using the ImageJ software.

The pH meter (Testo-205, Testo AG, Lenzkirch, Germany) was used to analyze the muscle pH value according to the method of the previous study [20 (link)]. Three tests were performed in each sample.
A texture analyzer (Food Technology Corporation, Sterling, VA, USA), which was equipped with an 8 mm cylinder probe and a 250 N weighing cell, was utilized to measure muscle texture according to the method of the previous study [21 (link)]. A double compression experiment with a compression ratio of 60% was carried out. During the experiment, the moving speed of the probe was 1 mm/s, and 2 s after the end of the first compression, the second compression was implemented. Hardness, adhesiveness, cohesiveness, springiness and chewiness of muscle were determined using a texture analyzer.
For histological analysis [22 (link)], muscle samples soaked in paraformaldehyde were dehydrated step by step in ethanol and xylene. Subsequently, paraffin wax was utilized to embed the dewatered samples. After the samples were sliced, hematoxylin and eosin (HE) were utilized to stain. The optical microscope, which was equipped with a camera system (BX40F4, Olympus, Tokyo, Japan) was utilized to observe the morphology of muscle and photograph. The diameter and density of myofiber were measured and calculated by ImageJ software according to the previous methods [22 (link)].

Liver, intraperitoneal fat, heart, and muscle from six euthanized (AQUI-S®, 175 mg l⁻¹) fish/treatment (two/replicate) at the end of the trial and also a similar set of skin samples 24-h post-challenge were collected and immediately preserved in 10% neutral buffered formalin. Following preservation, the fragments of all tissue sections were subsequently dehydrated with a series of ethanol washes, cleared by xylene, and embedded in paraffin wax. The tissue wax was then cut into 5-μm slices by microtome, stained with periodic acid–Schiff as per standard histological procedures, and photographed under a light imaging microscope (BX40F4, Olympus, Tokyo, Japan). The average of epidermis (Ep) thickness was measured from three regions of each section, and the number of epidermis goblet cells were counted in 1-mm length of the epidermis, following the methods of Heidarieh et al. (38 (link)) and Sheikhzadeh et al. (39 (link)).

In order to analyze the histopathological condition of liver, spleen, muscle, distal intestine, and histomorphological condition of the intestine, two fish from each replicate were examined (i.e. six juvenile barramundi per dietary treatment) from which blood had previously been extracted. Samples of all tissues were fixed in 10% buffered formalin, dehydrated in ethanol before equilibration in xylene and embedding in paraffin wax. Sections of approximately 5 µm were cut and stained with haematoxylin and eosin (H&E) for histological examination under a light microscope (BX40F4, Olympus, Tokyo, Japan). Digitalized histology images were analyzed using Image J software at different magnification for assessing the height of folds, enterocytes and microvilli according to the procedures described by Escaffre et al.^{60 (link)} with minor modifications. The number of goblet cells were counted in the highest 10 mucosal folds with the numbers expressed as average number of goblet cells per fold as described by Ramos et al.^{61 (link)}. For gut sample, three cross-sections were quantified for GC, hF, hMV and ECS of the distal intestinal samples.

For histological analysis, samples of muscle were fixed immediately in 10% buffered formalin, subsequently dehydrated with a series of ethanol, infiltrated in xylene and embedded in paraffin wax, as per standard histological protocols. A section of approximately 5 μm thickness was stained with hematoxylin and eosin (HE) and digitally photographed under a light microscope (BX40F4, Olympus, Tokyo, Japan).
For the analysis of fiber diameter, the muscle fiber was assumed to be cylindrical, and the diameter was calculated as s = πr² (where s is the muscle fire area and r is the muscle fire radius), according to the method described by [31 (link)]. A size limit for identifying fibers was set at fiber diameter ≥ 10 µm, since the optical resolution below this limit did not allow for sufficient identification and accuracy in the analyses. Muscle fiber density = number of muscle fibers/area selected.