Morphological Analysis of the Intestine in Yangtze Sturgeon (Acipenser dabryanus) During Development

Abstract

This study aimed to investigate the histological features of the intestine of Acipenser dabryanus from 1 to 15 months of age via HE staining, AB-PAS staining, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The intestine of A. dabryanus comprises the duodenum, spiral valve intestine, and rectum. With age, the duodenal diameter and mucosal/muscular layer thickness increased, the spiral valve intestine’s mucosa thickened and protrusions formed networks, and the rectal diameter enlarged. Abundant mucus cells, predominantly type IV, were found in the duodenum, spiral valve intestine, and rectum of A. dabryanus at different ages by AB-PAS staining. Our study confirmed the presence of ciliated columnar cells (with ‘9 + 2’ cilia structure) with orderly arranged cilia at their apices in the mucosal epithelium of A. dabryanus’s duodenum, spiral valve intestine, and rectum for the first time, as shown by SEM and TEM. The presence of spiral valves and ciliated columnar cells in the intestinal structure of A. dabryanus highlights its unique features and evolutionary significance. These findings highlight A. dabryanus’s unique intestinal features and evolutionary significance, providing a basis for scientific feed formulation and enhancing our understanding of the histological characteristics of the sturgeon intestine.

1. Introduction

Sturgeons, ancient chondrostei with an evolutionary history of over 200 million years, hold a pivotal phylogenetic position in the transition from cartilaginous to bony fishes [1]. The Yangtze sturgeon (Acipenser dabryanus), an endemic species of the Yangtze River basin in China, has garnered significant attention due to its ecological importance and conservation status [2,3]. However, in recent decades, the natural population of A. dabryanus has experienced a sharp decline due to overfishing, habitat destruction, and dam construction, leading to its classification as an endangered species listed in the Key Protected Wild Animals in China [4,5]. Consequently, artificial breeding and propagation have emerged as crucial strategies for the conservation and restoration of this species [4].

The intestine, as a key organ for nutrient digestion, absorption, and immune defense in fish, plays a vital role in the growth, development, and survival of individuals [6,7,8]. The structural and functional maturation of the intestine in juvenile fish during early development significantly impacts their adaptation to the external environment, utilization of artificial feed, and resistance to pathogens [9]. The intestinal barrier is essential for preventing the infiltration of harmful substances and pathogens, as well as for preserving the internal environment’s homeostasis [10,11]. Consequently, comprehending the histological characteristics and developmental patterns of the A. dabryanus intestine is crucial for enhancing artificial breeding strategies, developing precise feed formulations, and enhancing the survival rate of juvenile fish.

To date, research on the intestinal histology of sturgeon species has made some progress. For example, Cataldi et al. [12] studied the fine structure of the alimentary canal of Acipenser naccarii and noted the presence of ciliated columnar cells in the gastric canal and postgastric alimentary canal, but the study lacked clear transmission electron microscopy (TEM) images and comprehensive documentation of all intestinal segments, resulting in insufficient understanding of the distribution and characteristics of ciliated cells in the entire intestinal tract. The study focused on Acipenser baerii and found that ciliated columnar cells are only distributed in specific regions of the digestive system, which is quite different from the distribution pattern of ciliated cells in other fish species [13]. In addition, Yang et al. [14] conducted a morphological and histochemical analysis of glycoproteins in the digestive tract of A. dabryanus, but the research mainly focused on the distribution of glycoproteins in the adult stage, and there was a lack of systematic research on the dynamic changes in intestinal histological structures (such as mucosal layer, muscular layer, and mucous cells) during the early developmental stages.

The spiral valve intestine is a unique structural feature of sturgeons and some other ancient fish species that is formed by the folding of the intestinal mucosa into the intestinal lumen [15]. This structure can significantly increase the surface area of the intestinal mucosa, thereby enhancing the efficiency of nutrient absorption [16]. In addition, ciliated columnar cells, as a primitive cell type in the intestinal epithelium, have been observed in the intestinal tract of some ancient fish lineages, and their rhythmic ciliary movement can promote the movement of intestinal contents and secretions [17]. For A. dabryanus, whether ciliated columnar cells exist in the entire intestinal tract, what their ultra-structural characteristics are, and what role they play in the development and function of the intestine are scientific questions that need to be answered urgently.

Thus, this study examined A. dabryanus juveniles aged 1 to 15 months, focusing on the histological features of the duodenum, spiral valve intestine, and rectum. The findings of this study may provide more theoretical support for scientific feed formulation, the refinement of optimal artificial breeding practices, and the conservation of A. dabryanus resources.

2. Materials and Methods

2.1. Fish and Sample Collection

The A. dabryanus used in this study were F2 (second filial generation) juveniles bred at the Fishery Institute of the Sichuan Academy of Agricultural Sciences in Chengdu, China. The A. dabryanus specimens were acclimated in indoor cylindrical tanks with the following water quality parameters strictly monitored and maintained: dissolved oxygen (DO) levels were kept at ≥8.0 mg/L, pH was maintained between 7.8 and 8.2, and ammonia nitrogen levels were controlled within the range of 0–0.1 mg/L. Water temperature was regulated at 16 ± 0.5 °C, and a standard natural photoperiod of 12 h light and 12 h dark was implemented. Daily water changes were carried out, replacing approximately one-third of the total water volume using a continuous inflow–outflow system. Stocking density was adjusted dynamically according to fish growth to avoid overcrowding. During acclimation and rearing, the fish were fed three times daily with a commercial compound feed, and feeding was stopped when the fish no longer actively competed for feed. We examined the histological features of the intestine in A. dabryanus across early developmental stages. The experimental fish were bred in March 2022, with the day the larvae hatched recorded as day 1. From 1 month of age to 15 months of age, 6 fish of similar growth status were randomly selected as experimental subjects each month.

2.2. HE Staining

To investigate the developmental characteristics of the intestine of A. dabryanus aged 1 to 15 months, we conducted histological analysis via HE staining. The intestines (duodenum, spiral valve intestine, and rectum) were removed after A. dabryanus were anesthetized using MS222 dissolved in rearing water at a concentration of 100 mg/L, with the anesthesia process closely monitored: adequate anesthesia was judged to be achieved when the fish exhibited signs of loss of body balance, slowed gill movement, and no response to gentle tactile stimulation. After being fixed with 4% PFA for 24 h, the samples were dehydrated with ethanol and embedded in paraffin. Standard procedures were followed to obtain 5 μm paraffin sections, which were then stained with hematoxylin and eosin (H&E) for histological investigation. The sections were observed and photographed via a light microscope from Leica Microsystems (Wetzlar, Germany). Whole-slide imaging was conducted via the slide scanning system SQS-40R (Shengqiang Technology, Shenzhen, China).

2.3. AB-PAS Staining

AB-PAS staining was also used to assess the presence of acid and neutral mucins in the duodenum, spiral-valved intestine, and rectum of A. dabryanus aged 1 to 15 months. The tissues were fixed in 4% PFA, dehydrated, and sectioned into 5 μm slices. The sections were then stained with Alcian blue/periodic acid–Schiff (AB/PAS; Solarbio, Beijing, China) and examined under a light microscope from Leica Microsystems (Wetzlar, Germany). Whole-slide imaging was performed via the SQS-40R slide scanning system (Shengqiang Technology, Shenzhen, China).

Starting from the base of the villi, three images were captured along the direction of the villi under a 400× field of view. The area of the mucosal epithelial layer was measured for each image. Previous studies categorized mucus cell types into four groups based on AB-PAS staining results: type I, red, and PAS-positive mucins, which primarily contained neutral mucins; type II, blue, and AB-positive mucins, which mainly consisted of acidic mucins; type III, purple-red and positive for both AB and PAS, which contained PAS-positive neutral mucins and a small amount of AB-positive acidic mucins; and type IV cells, bluish purple and positive for both AB and PAS, which primarily contained AB-positive acidic mucins with a minor presence of PAS-positive neutral mucins [18,19,20]. The number of mucous cells was counted, and the mucous cells per unit area of the mucosal epithelial layer (cells/mm²) were calculated using Image-Pro Plus 6.0. Furthermore, various types of mucous cells in the intestine of 15-month-old A. dabryanus were quantified. Statistical analysis was conducted using one-way analysis of variance (one-way ANOVA), and graphs were created using GraphPad Prism 9.

2.4. Ultrastructure Examination

Samples of the duodenum, valve intestine, and rectum from 15-month-old A. dabryanus were prepared for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis. For SEM analysis, the tissues were initially washed with phosphate-buffered saline (PBS) to eliminate surface contaminants and mucus and then fixed in 3% glutaraldehyde. Following fixation, the samples were treated with 1% osmium tetroxide for 1–2 h, dehydrated gradually with alcohol, and dried in Quorum K850 Critical Point Dryer (Quorum Technologies Ltd., Laughton, United Kingdom). Gold sputtering was subsequently performed via an ion sputtering instrument (Smart Coater; JEOL Ltd., Tokyo, Japan) before imaging with a JSM-IT700HR SEM (JEOL Ltd., Tokyo, Japan). For TEM analysis, the duodenum, valve intestine, and rectum samples were excised, fixed in 2.5% glutaraldehyde overnight at 4 °C, rinsed in PBS buffer, and postfixed with 1% osmium tetroxide for 2 h. After dehydration in acetone solutions, the samples underwent infiltration and embedding in Epox 812. Sections (60–90 nm) were sliced with a diamond knife, mounted on metal grids, and double-stained with uranyl acetate and lead citrate. Examination and imaging were carried out via a JEM-1400-FLASH TEM (JEOL Ltd., Tokyo, Japan).

3. Results

3.1. Histological Evaluation

The intestine of A. dabryanus can be divided into three distinct sections: the duodenum, the spiral valve intestine, and the rectum (Figure S1). The proximal end of the duodenum features the openings of the pyloric caecum, and it subsequently curves forwards in the middle section. The slightly thicker segment posterior to the duodenum is known as the spiral valve intestine. This region contains eight spiral valves formed by protruding intestinal walls, which could increase the area for digestion and absorption while also delaying the downwards movement of the food bolus. Following the spiral valve intestine, the thinner and shorter section was the rectum, which terminated at the anus and opened into the cloaca.

3.1.1. Histological Characteristics of the Duodenum

Examination of duodenal transverse sections from juvenile fish aged 3 to 15 months revealed morphological changes over time (Figure 1). At 3 months, densely packed columnar cells and elongated intestinal villi were observed in the mucosal layer, alongside a notable presence of mucous cells among the epithelial cells. By 5 months, mucosal protrusions began to interconnect, leading to a predominantly reticular structure of the intestinal villi. As the fish aged, there was a consistent increase in duodenal diameter, mucosal thickness, and muscular layer thickness. The connective tissue beneath the lamina propria appeared loose, while the inner circular muscle of the muscular layer was well developed, with the outer layer composed of longitudinal muscle. Particularly, the submucosa of the duodenum was thin and loosely structured.

3.1.2. Histological Characteristics of the Spiral Valve Intestine

In the spiral valve intestine, finger-like protrusions are arranged along the valve intestine and are created by folds that extend into the intestinal lumen (Figure 2). These protrusions display consistent widths and are occasionally connected by local cross-links. With advancing age, the mucosa of the valve intestine progressively thickens. At 3 months of age, the mucosal layer features numerous sparse finger-like protrusions of varying widths. By 5 months, these protrusions become interconnected, forming a network, whereas some independent finger-like protrusions remain locally visible. As aging continues, both the mucosa and the muscularis of the spiral valve intestinal wall gradually thicken. The muscularis is clearly differentiated into outer longitudinal muscles and inner circular muscles.

3.1.3. Histological Characteristics of the Rectum

As shown in Figure 3, the rectal wall presented morphological traits similar to those of the spiral valve intestine, with finger-like protrusions of varying widths in the mucosa at 3 and 5 months. By 7 months, distinctive folds reminiscent of tree branches had appeared in the mucosa alongside smaller finger-like protrusions. As age advanced, there was noticeable enlargement in the rectal diameter. However, by 9 months, both the thickness and diameter of the intestinal wall no longer consistently increased.

3.1.4. Distribution and Characteristics of Mucous Cells

Using AB-PAS staining, we investigated the distribution of intestinal mucus cells in A. dabryanus aged 3, 5, 7, 9, 12, and 15 months (Figure 4). Our findings revealed the presence of mucus cells among the epithelial cells of the duodenum (Figure 4a), spiral valve intestine (Figure 4b), and rectum (Figure 4c), with a relatively uniform distribution among these segments. There were no significant differences in mucus cell morphology across the three intestinal segments at any age, with cells predominantly displaying oval, nearly round, or pear-shaped forms. The mucous cells in the duodenum, spiral valve intestine, and rectum of A. dabryanus in all age groups are densely distributed within the intestinal epithelium. Predominantly, these cells are type IV mucus cells, characterized by a bluish purple stain, with occasional occurrences of type III (purple‒red) and type II (blue) positive cells.

Figure 5A illustrates the quantification of mucous cells in the duodenum, spiral valve intestine, and rectum of A. dabryanus at 3, 5, 7, 9, 12, and 15 months. The results indicated a significantly higher number of mucous cells in the spiral valve intestine compared to the duodenum and rectum at 3 and 5 months of age (p < 0.05). At 7 months, the spiral valve intestine still exhibited a significantly higher mucous cell count than the rectum (p < 0.05). Between 9 and 15 months, there were no significant differences in mucous cell numbers among the three sites, with the spiral valve intestine consistently having the highest count. Furthermore, in 15-month-old A. dabryanus, the quantification of different mucous cell types (type II, type III, type IV) revealed a significantly higher number of type IV cells compared to type II and type III (p < 0.05), while no significant difference was observed between type II and type III cells (Figure 5B).

3.2. Ultrastructure Observation

SEM and TEM analyses were performed on the duodenum, valve intestine, and rectal mucosal epithelium of 15-month-old A. dabryanus (Figure 6). The findings revealed the presence of ciliated columnar epithelial cells in all three examined regions. In the duodenum, the mucosal folds are relatively flat, with abundant microvilli and cilia on the epithelium. The cilia were densely packed in the intercellular grooves rather than on the cell surface, forming a tuft. TEM further confirmed the presence of ciliated columnar epithelial cells with the characteristic ‘9 + 2’ structure and observable ciliated basal bodies. Conversely, the valve intestinal mucosa displayed more convex folds with cilia and microvilli arranged in clusters, showing lower density than the duodenum. The rectal mucosa was predominantly covered with cilia that lacked microvilli, which hindered the clear identification of specific cell morphologies. Numerous ciliated columnar epithelial cells were also identified in the rectum through TEM.

4. Discussion

During the growth stage from 1 to 15 months of age, the intestinal structure of the A. dabryanus underwent orderly dynamic changes. Histological observations (HE and AB-PAS staining) were conducted on fish aged 1–15 months, but only representative stages (3, 5, 7, 9, 12, 15 months) were presented due to non-significant monthly changes in some periods; SEM/TEM analyses were limited to 15-month-old fish to ensure sufficient tissue sampling. At 3 months of age, the intestinal layers, including the mucosal layer, submucosal layer, muscular layer, and serosal layer, were present, albeit with a relatively thin mucosal layer. As the age increased, the mucosal layer thickened gradually, and the folds became more complex. The thickening of the mucosal layer and the increased complexity of folds greatly enhance the intestinal surface area [21,22]. The presence of spiral valves not only increases the digestive absorption area of the intestine, but may also regulate the digestion process, aiding the A. dabryanus in better utilizing food from various sources. In terms of changes in intestinal diameter, the diameters of the duodenum, spiral valve intestine, and rectum steadily increase from 1 to 15 months of age, following a trend consistent with the intestinal growth of most fish species.

The mucous cells of A. dabryanus are widely distributed throughout the intestine. These cells can secrete various substances, including mucins, enzymes, and immunoglobulins, which play crucial roles in immune defenses in fish, as well as in the digestion and absorption of nutrients [6,23]. Research has been conducted on the intestinal mucous cells of aquatic animals, with a focus on species such as Cynoscion guatucupa [24] and Oreochromis niloticus [25]. While previous studies have examined the distribution of mucous cells in the digestive tract of A. dabryanus [14], there is still a gap in understanding the changes in mucous cells across different developmental stages. This study examined the intestinal morphology and histology of juvenile A. dabryanus throughout ontogeny to characterize the mucins present in mucosal epithelial cells.

Our findings demonstrated the presence of numerous mucus cells interspersed among the epithelial cells in the duodenum, spiral valve intestine, and rectum of A. dabryanus. Remarkably, there was uniformity in the morphological characteristics of mucus cells across these three intestinal segments, irrespective of the age of the sample. The mucous cells mainly contained type IV mucous cells, and small amounts of type II and type III mucous cells are present at the same time. The predominant mucus cells found in the intestine of A. dabryanus were type IV, accompanied by a smaller proportion of type II and type III mucus cells. Many of the cells exhibited bluish-purple staining, indicating that the secretion of mixed mucins was more acidic than neutral. The results were essentially identical to those reported for the sturgeon. Mucins probably provide significant mechanical protection against coarse food particles and act as a physical and immunological barrier against invading microorganisms [26]. Neutral mucopolysaccharides primarily facilitate digestive processes, whereas acidic mucopolysaccharides provide protective functions for the epithelium [27]. During early development, the presence of acidic mucins is crucial for innate immune defense, as their acidic components effectively impede pathogen invasion.

The finding of SEM and TEM in this study contrasts with previous research on A. baerii, which identified ciliated columnar cells only in specific regions of the digestive system [16]. Although Acipenser naccarii has been noted to possess numerous ciliated columnar cells in its gastric canal and postgastric alimentary canal [12], earlier evidence was deemed inadequate owing to a lack of clear TEM images and comprehensive documentation of all intestinal segments. Ciliated columnar cells, a primitive trait observed in the intestinal epithelium of certain ancient fish lineages, such as Elasmobranchii fishes, exhibit rhythmic coordination that aids in the movement of secretions and granular substances along the epithelial surface [16,17]. In contrast, microvilli increase the cellular surface area, thereby optimizing absorptive capacity. The identification of ciliated columnar cells in the intestine of A. dabryanus highlights its distinctive digestive tract characteristics, underscoring its significant evolutionary position. However, the precise roles of these ciliated columnar cells in the intestine of A. dabryanus require further exploration.

5. Conclusions

The histological and ultrastructural features of the intestines of A. dabryanus (1–15 months old) were examined in this study using HE staining, AB-PAS staining, SEM, and TEM. The intestine comprises duodenum, spiral valve intestine, and rectum, whose structural parameters (diameter, mucosal/muscular layer thickness) increased orderly with age to enhance nutrient absorption. AB-PAS staining showed type IV mucous cells dominated all segments, supporting juvenile immune defense and digestion. SEM and TEM confirmed ciliated columnar cells (with ‘9 + 2’ cilia structure) in all three segments, reflecting the species’ unique evolutionary traits. These findings enrich our understanding of A. dabryanus intestinal development and provide theoretical support for targeted feed formulation and artificial breeding optimization of this endangered species.