| Sažetak (engleski) | INTRODUCTION
The digestive system consists of the digestive tract and associated glands. The digestive tract
of fish can be divided into anterior and posterior part. The posterior part of the digestive tract
consists of the foregut (esophagus and stomach), midgut (intestine) and hindgut (rectum). The
midgut refers to the intestine proper, and in most fish, it can be further subdivided into the
anterior, middle, and posterior parts. The intestine represents the longest part of the digestive
tract, thereby serving as a key location for enzymatic digestion and nutrient absorption.
For understanding of the physiology of the digestive system in fish, it is essential to have
knowledge of metabolism and digestion processes. Digestion is the foundation of metabolism.
The breakdown and absorption of nutrients determine their availability for all biological
processes. The function of digestive system involves the mechanical, chemical, secretory and
microbiological activities. Mechanical activities include the intake and processing of food into
the oral cavity, swallowing, as well as stomach and intestinal movements. Chemical activities
include the secretion and action of digestive enzymes. Secretory activities involve the secretion
from associated glands and intraepithelial mucous glands. Microbiological activities refer to
bacterial colonies that assist the digestive process and have significant role in certain fish
species. In general, between all mentioned activities, the degree of digestion highly depends on
the availability of enzymes. Based on the site of enzyme action, the digestion can be either
extracellular or cellular.
The study of the digestive system of fish is specific due to the wide diversity of species, their
habitats and diets. Accordingly, variations in the structure and function of the digestive system
are numerous. Although, there is comprehensive literature focusing on the physiology of the
digestive system, the physiology of many fish species remains insufficiently researched. This
gives rise to the question about the variations in structure and function of the digestive system
found in fish with common characteristics. Therefore, the fundamental assumption of this
research was that, among cartilaginous and bony fish, specific morphological differences in the
digestive system are expected, as well as variations in enzyme localization and activity,
regardless of their common characteristics.
MATERIAL AND METHODS
To determine variations in digestive system, this study encompasses two taxonomic groups of
fish, cartilaginous and bony fish. Within these groups, the predatory fish with similar diets and
habitats were selected. Two representatives from each group were chosen: the small-spotted
catshark (Scyliorhinus canicula) and the smooth-hound (Mustelus mustelus) as representatives
of cartilaginous fish, and the European hake (Merluccius merluccius) and the piper gurnard
(Trigla lyra) as representatives of bony fish.
Fish were caught with longline fishing in the Adriatic Sea as a part of scientific project
“Improvement of cooperation between fishermen and scientists for the purpose of introducing
advanced technologies of marking of fishing tools, fish health and environmental protection”.
After measurements, fresh, ice-cold fish were dissected and the digestive tract was removed.
In this study, 30 small-spotted catsharks, 30 European hakes and 30 piper gurnards were used.
Within each species, 10 fish were used for the descriptive (topographical) investigation of
macroscopic structure of the digestive tract, 10 for histological and histochemical research and
10 for the investigation of the localization and enzymatic activity in the digestive tract.
Additionally, 29 smooth-hounds were used: 9 for the descriptive (topographical) investigation,
10 for histological and histochemical research and 10 for the investigation of the localization
and enzymatic activity in the digestive tract.
The macroscopic structure of the posterior part of digestive tract was investigated descriptively
(topographically) and morphometrically. Organ parts of the digestive system were identified
and described. For morphometric research, the digestive tract was removed from the body
cavity. The intestine length was measured, and the relative intestinal length was calculated.
Morphometric research included all 119 fish. Sampling was carried out within the anatomical
borders of the organs, in accordance with variations in the morphology of the digestive system
in cartilaginous and bony fish.
For histological and histochemical research, the tissue samples were fixed in 10% neutral
buffered formalin, embedded in paraffin, and sectioned with microtome into 6 µm sections.
The sections were stained using five different methods: hematoxylin and eosin, Mallory
trichrome, Verhoeff-Van Gieson, Alcian Blue-PAS kit and the seven-reagent kit for
determining reticulin fibers. For the investigation of cellular digestive processes, the
distribution and intensity of alkaline phosphatase, acid phosphatase, non-specific esterase and
aminopeptidase were investigated. The tissue samples were taken, embedded in Cryofix gel
and sectioned with cryostat into 8 µm sections. To detect the enzymatic reactions, azo-coupling
methods were used and the optical density (OD) of the reactions was measured using ImageJ
software.
RESULTS
Regarding macroscopic structure, cartilaginous fish, the small-spotted catshark and the
smooth-hound share several similarities: a short esophagus, a bifurcated stomach shaped like
an "U," a short spiral intestine and a rectum that terminates in a cloaca. The bony fish, the
European hake and the piper gurnard, also share similarities in the esophagus and stomach
(short esophagus and bifurcated stomach shaped like a "Y"). The main differences in
macroscopic structure of the bony fish were observed in the part of the digestive system from
the pyloric caeca to the rectum (presence of pyloric caeca in the piper gurnard and greater
relative intestinal length compared to the European hake). Variations in intestinal length were
observed among the studied fish species, despite similar dietary habits. In relation to the total
body length, the intestine in cartilaginous fish is relatively short, and the median value of
relative intestinal length between the small-spotted catshark and the smooth-hound is similar.
The relative intestinal length is greater in the piper gurnard than in the European hake.
Regarding microscopic structure, the digestive systems of the small-spotted catshark and the
smooth-hound share many similarities: the same type of epithelium in the esophagus, stomach
and spiral intestine, unicellular mucous glands in the epithelium of all parts except the stomach,
glands in the lamina propria of the stomach, well-developed lamina muscularis of mucosa in
the stomach and spiral intestine, a propria-submucosa in the rectum and similarly developed
muscularis of the digestive tract wall. Differences in microscopic structure between these two
species were observed in the type of epithelium lining the rectum, the content of mucous
glands, and the presence of lamina muscularis of mucosa in the part of the esophagus below
the Leydig's organ. The digestive systems of the European hake and the piper gurnard share
many similarities: the same type of covering epithelia in all segments of the posterior part of
the digestive tract, unicellular mucous glands in the epithelium of all parts except the stomach,
glands in the lamina propria of the stomach, well-developed lamina muscularis of mucosa in
the stomach but its absence in the anterior and middle part of the intestine, similarly developed
muscularis in the walls of organs, and serosa as the outer layer of the digestive tract. Differences
in microscopic structure between these two species were observed in the content of mucous
glands and in the occurrence of lamina muscularis of mucosa in the esophagus.
In this study, the OD of alkaline phosphatase was measured in the brush border of the intestines
of all investigated fish species. Enzymatic activity was detected in the epithelium of the
esophagus of the smooth-hound, European hake, and piper gurnard, as well as in the epithelium
of the rectum of the piper gurnard. Additionally, enzymatic activity was found in the layers of
connective tissue and around the blood vessels in all investigated fish species. When comparing
the mean optical densities (MOD) between the cartilaginous and the bony fish, values were
higher in the esophagus of bony fish, being highest in the piper gurnard. In the rectum, activity
was found only in bony fish and was higher in the European hake. Although, higher OD were
measured in the small-spotted catshark, the difference in the MOD of alkaline phosphatase in
the spiral intestine of cartilaginous fish was not statistically significant. In the intestine of the
bony fish, the MOD of alkaline phosphatase was higher in the European hake than in the piper
gurnard.
In the brush border, the reaction of acid phosphatase was found only in the spiral intestine of
the small-spotted catshark (0,227 ± 0,117). The activity of acid phosphatase was found in the
epithelium and connective tissue of all parts of the digestive tract. Among the cartilaginous
fish, the highest MOD was detected in the epithelium of the esophagus of the small-spotted
catshark (0,283 ± 0,047). In the smooth-hound, MOD increases up to the spiral intestine, where
it is the highest (0,194 ± 0,078) and then it declines afterwards. In the bony fish, the MOD of
acid phosphatase in the European hake shows higher values compared to the piper gurnard. In
the European hake the highest MOD were measured in the epithelium of the anterior (0,327 ±
0,079) and posterior (0,327 ± 0,063) parts of the intestine. In the piper gurnard, MOD increases
up to the pyloric caeca (0,109 ± 0,015), after which its value decreases and then gradually
increases along the length of the intestine. The MOD of acid phosphatase reactions in the
connective tissue generally follows the increase of the value in the epithelium.
In the cartilaginous fish, the activity of non-specific esterase was found along the epithelium
of the entire posterior part of the digestive tract. A similar enzymatic distribution was also
observed in the piper gurnard. Unlike the enzymatic localization in the cartilaginous fish and
the piper gurnard, enzyme activity in the European hake was only found in the epithelium of
the anterior, middle, and posterior parts of the intestine. The localization of non-specific
esterase differs in the connective tissue of the digestive tract between the cartilaginous and the
bony fish. In the cartilaginous fish, the activity of non-specific esterase was found in the
connective tissue of the stomach and spiral intestine. In the piper gurnard, enzymatic activity
was found along the connective tissue, from the stomach to the rectum. In contrast, enzymatic
activity was not found in the connective tissue of the digestive tract in the European hake. In
general, higher values of MOD of non-specific esterase were observed in the small-spotted
catshark than in the smooth-hound. The highest value in the small-spotted catshark was measured in the stomach (0,250 ± 0,085), while in the smooth-hound, it was measured in the
spiral intestine (0,132 ± 0,039). In bony fish, the enzyme is significantly more active along the
digestive system of the piper gurnard. Despite that, higher values were measured in the intestine
of the European hake. In both species of bony fish, the enzyme activity increases up to the
posterior part of the intestine where its activity is the highest.
The OD of aminopeptidase was measured in the brush border of the intestines of all the
investigated fish species. Additional activity was also found in the epithelium of the esophagus,
as well as in the brush border of the pyloric caeca of the piper gurnard and in the rectum of the
European hake. In the cartilaginous fish, the MOD of aminopeptidase were higher and more
variable in the smooth-hound. The enzymatic distribution suggests that the spiral intestine is
the primary site for protein digestion and absorption in these fish. In the smooth-hound,
aminopeptidase activity was also measured in the basal part of the esophageal epithelium. In
the bony fish, higher MOD of aminopeptidase were found in the brush border of all parts of
the European hake's intestine. The highest value was measured in the brush border of the
middle part of the intestine proper (0,314 ± 0,093). Additional activity was also found in the
basal part of the esophageal epithelium, as well as in the brush border of the pyloric caeca of
the piper gurnard and in the rectum of the European hake.
CONCLUSIONS
The macroscopic structure of the posterior part of the digestive system shows similarities
between the small-spotted catshark and the smooth-hound, while it differs between the
European hake and the piper gurnard. The structure in the cartilaginous fish is significantly
different from that found in the bony fish. Despite similar dietary habits, variations in the
intestinal length were observed among individual specimens within the same species, as well
as between the studied fish species. In all studied fish, the intestine is shorter than the total
body length. In the piper gurnard, additional mechanisms have developed to aid the digestion
and absorption of nutrients due to the harder-to-digest chitin shells of crustaceans that dominate
in their diet.
The microscopic structure of the posterior part of the digestive tract differs between the
investigated fish species. The type of the covering epithelia in the esophagus and rectum shows
significant differences, as does the development of the lamina muscularis of mucosa along
various segments of the digestive tract. Mucous glands were found in all parts of the digestive
tract except the stomach. The composition of the secretions in different parts varies between
the investigated fish species.
In all investigated fish species, the intestines are the site of the most intense metabolism. In the
small-spotted catshark, the main site of fat digestion is stomach, while in the other studied fish,
it is the intestine. Although, the breakdown and absorption of proteins are intensive in the
intestine, they also occur in other parts of the digestive tract. |
