3.4.3. Hindgut

In the hindgut tissue, the VL and MS of coral trout were significantly higher in the 1 bl/s water flow velocity group than in the control and other water flow velocity groups (*p* < 0.05). In terms of both MS and VT, the MS of coral trout in the 2.5 bl/s flow velocity group was the lowest, and there was no significant difference in VT among the 1 bl/s water flow velocity group and the control group (*p* > 0.05, Table 4). At 2 bl/s water flow velocity, the goblet cells showed normal levels, whereas at 2.5 bl/s water flow velocity, the goblet cells were fragmented and unevenly distributed, with significantly reduced numbers compared to all other groups (Figure 6).


**Table 4.** Effect of water flow velocity on the index of hindgut tissue of coral trout.

The values are average ± standard deviation of four replications (*n* = 6). Values with different letters show significant difference among the groups (*p* < 0.05), and those with no letters showed no significant difference (*p* > 0.05).

**Figure 6.** The effect of water flow velocity on the hindgut structure of coral trout. (**a**) Hydrostatic control group: muscular thickness (MS), villus length (VL), and villus thickness (VT) were uniform, and there were more goblet cells (thick arrows); (**b**) 1 bl/s water flow velocity group: the muscle layer was thicker than in the control, the villus was the longest, and the goblet cells (thick arrows) were relatively large; (**c**) 2 bl/s water flow velocity group: the muscle layer was the thinnest, and the goblet cells (thick arrows) were evenly distributed and numerous; (**d**) 2.5 bl/s water flow velocity group: some of the epithelial cells at the end of the villus fell off, and the goblet cells (thick arrows) were broken and unevenly distributed in size. The muscle layer was thicker, and the number of goblet cells (thick arrows) was significantly reduced compared with other groups.

#### **4. Discussion**

The different water flow velocities manifested significant differences in the growth performances of the fish. In a study by Wei [11], the highest SGR and WGR values were found in the 1 bl/s water flow velocity group of *Epinephelus coioides* (42.54 ± 0.62 g), and 2 bl/s water flow velocity negatively affected fish growth. Turbot (*Scophthalmus maximus*) (average body length 20.10 cm) achieved higher SGR values by swimming and exercising at 0.9 bl/s water flow velocity [12]. This is consistent with the results obtained in the present study, where the 1 bl/s water flow velocity group had the highest WGR and SGR values and the fastest growth. The desire to feed was enhanced when the current exercised the fish [13]. The SGR value of coral trout tended to decrease when the water flow velocity exceeded 1 bl/s, reaching a minimum at 2.5 bl/s water flow velocity. Goldfish (*Carassius auratus*) (10.1 ± 0.2 cm) that received high-water flow velocity exercise showed reduced feeding compared to the control [14]. Fish are capable of spontaneous swimming exercises at suitable water flow velocities, which helps to improve the efficiency of energy metabolism and accelerate their growth. At high-water flow velocities, the fish are forced to swim, and they fatigue faster, thus affecting their growth. At a high-water flow velocity (4 bl/s), *Schizothorax prenanti* (average body length 9.70 cm) allocated more energy to swimming and showed reduced growth performance [15].

It has been suggested that there are significant differences in the morphological characteristics of fish at different water flow velocities [16]. In the present experiment, however, there was no significant difference in the fatness of the fish among the groups, with the highest CF at 1 bl/s water flow velocity. The HSI value was also not significantly different, but the VSI ratio was, with the VSI in each water flow velocity group being smaller than that in the hydrostatic control group, and the VSI in the 2 bl/s water flow velocity group being the smallest. The reason for this is the difference in the thickness and mass of the fat envelope between the organs. Liu [17] pointed out that, as the water flow velocity increased, *Micropterus Salmoides* (8.13 ± 0.82 cm) broke down body fat to provide energy for maintained swimming movement, and the appropriate water flow velocity (5.0 bl/s) could reduce the excessive accumulation of fat in the fish, enhance the absorption of nutrients, and improve the quality of fish meat. However, when the water flow velocity exceeds the tolerance limit, the fish become fatigued, and no longer swims.

GLU metabolism is an important physiological metabolic process in animals. GLU can provide energy and carbon sources for the growth and metabolism of the body and meet the basic needs of the body's life activities [18]. In this experiment, the GLU concentration of the water flow velocity group was lower than that of the control group, and those in the 2 bl/s and 2.5 bl/s water flow velocity groups were significantly different from those in the control group, far below the average level. Under transport stress, the GLU concentration of silver pomfret (*Pampus argenteus*) increased significantly, and the breakdown of liver glycogen and muscle glycogen in these fish provided energy to ensure that the fish withstood the stress [19]. Different from the results of this experiment, the GLU concentration of black porgy (*Acanthopagrus schlegelii*) (6.75 ± 0.03 cm) increased at a high-water flow velocity (4.0 bl/s) [20]. The reason for this difference may be related to the swimming ability and living habits of fish. In another study, the GLU concentration of largemouth bass decreased with increasing water flow velocity [17]. Under acute hyperosmotic stress, the Chinese mitten crab (*Eriocheir sinensis*) undergoes corresponding adaptive physiological and biochemical changes, preferentially using GLU for energy supply to regulate osmotic pressure, and subsequently using nutrients, such as proteins [21]. At a high-water flow velocity (2.0 bl/s), tinfoil barbs (15.1 ± 1.35 cm) require more GLU to provide energy, which takes priority over protein and fat [22]. When GLU supply is inadequate, the organism derives additional energy from the breakdown of fat and protein to meet the needs of forced swimming to the detriment of nutrient accumulation in fish, and experimental fish undergo greater energy expenditure at high-water flow velocities (≥2 bl/s), the specific regulatory and metabolic mechanisms of which require further study.

LD is commonly considered to be the main metabolic product produced by muscles after exercise, and that which cannot be processed in the muscles diffuses and is released into the hemolymph and blood for absorption and use by other tissues [23]. LD concentrations in the blood of black porgy (19.37 ± 1.88 cm) increased with exercise time, and fatigue was positively correlated with LD concentration [24]. In the present study, LD concentration in the blood showed a trend of decreasing and then increasing as the water flow velocity increased. A 1 bl/s water flow velocity correlated with the lowest LD concentration. At water flow velocities above 2 bl/s, the coral trout swimming activity was shown to involve anaerobic exercise, with higher LD concentrations than in the hydrostatic control group, thus requiring additional energy to maintain it. Swimming at high-water flow velocities leads to more energy expenditure, which is consistent with the findings in grass carp (*Ctenopharyngodon Idella*) (16.83 ± 0.96 cm) [25].

Cortisol is an important hormone secreted by the fish's body in response to external stimuli and is often used as an indicator of the physiological response to stress in fish. When the external environment causes stress in fish, it triggers the release of corticotrophinreleasing hormone (CRH) by the hypothalamus, which stimulates the secretion of adrenocortico-tropic-hormone (ACTH) from the anterior pituitary gland of the hypothalamus, which is transmitted to the interrenal tissues to aid in the production of corticosteroids. The degree of stress in fish is related to the duration and intensity of the stress [26]. Atlantic salmon (19.26 ± 0.08 cm) subjected to 24 weeks of exercise at different water flow velocities (0.5 to 2.5 bl/s) showed similar levels of COR across treatment groups [7]. The research reported that GLU and COR are usually elevated for an initial period following stress, and then gradually decrease until normal levels are restored due to the negative feedback mechanisms arising from homeostasis and adaptation of the hypothalamic–pituitary– interrenal axis [27]. In another study, chronic high-flow stress (4.0 bl/s) also had no significant effect on COR levels in the blood of largemouth bass (4.50 ± 0.36 cm) [28]. Under crowding stress, a trend was observed of increasing and then decreasing COR concentrations in the whole bodies of *Ancherythroculter nigrocauda* (2.71 ± 0.31 cm) [29]. In the present study, there were no significant differences in COR concentrations under different water flow velocity conditions, and the experimental fish showed a good adaptive capacity. In conclusion, although the fish species, stressors, and modes of action were different, no significant differences were seen in COR concentrations among the treatment groups as the time was extended, indicating that the fish had an excellent adaptive capacity to water flow velocity.

The intestine is an essential digestive organ for fish, and the level of digestive enzyme activity directly affects the digestion and absorption of bait in this fish. The protease, LPS, and AMS activities of Jiffy Tilapia (*Oreochromis niloticus*) (15.1 ± 0.21 cm) were significantly increased under prolonged water flow [30]. The AMS activity of juvenile *Rhynchocypris lagowskii* (2.21 ± 0.07 g) showed a trend of increasing and then decreasing with increasing water flow velocity, which is similar to the results of this experiment [31]. A 1 bl/s water flow velocity correlated with the highest AMS activity in the intestine of *Oreochromis niloticus*, and the α-AMS activities of the 2 bl/s and 2.5 bl/s water flow velocity groups were significantly lower than those of the control and 1 bl/s groups. The high-water flow velocity group showed a poor ability to absorb starch-containing feeds, reducing feeding motivation. The lower GLU concentration observed in the high-water flow velocity group in the present study may be partly due to the lower activity of α-AMS at this water flow velocity, which affects the digestion and absorption of feed. LPS was not affected by water flow velocity, and there were no significant differences among the groups.

In the intestine, the secretion ability of mucous cells increases from front to back, and the mucous digestion ability of the rectum is the strongest in the intestine, which is closely related to the physiological function of the rectum [32]. An increase in VL in the intestine can increase the contact area with food, thus expanding the absorption area and improving the digestive capacity of the intestine [33]. The intestinal VL of experimental fish in the 1 bl/s water flow velocity group was significantly lower than that of other groups, possibly due to the fact that stimulation via water flow velocity weakens the digestive ability of the foregut, enhancing the digestive ability of the midgut and hindgut, and enabling them to secrete more goblet cells to promote food digestion. The specific causes need further experimental investigation. In this experiment, the VL, MS, and VT values in the midgut of each group were lower than those in the foregut and hindgut, and the relative absorption capacity of the midgut was poor. Therefore, the absorption of nutrients by the coral trout is mainly concentrated in the foregut and hindgut. Under stimulation by water flow velocity, the MS is increased to a certain extent, which can increase the elasticity of the intestine and promote food peristalsis. At 1 bl/s water flow velocity, the structural parameters of the foregut were slightly lower than those of the control group and other water flow velocity groups; further, both the midgut and hindgut were superior to those of other groups. In general, the intestinal structure of the 1 bl/s water flow velocity group was better than that of other water flow velocity groups, and its digestive enzyme activity was also higher than that of other water flow velocity groups. Medium and high-water flow velocities (2 bl/s and 2.5 bl/s) have a specific adverse effect on the shape and quantity of goblet cells, even causing cell breakage and other conditions, and thus have an inhibitory effect on digestive and immune functions. This is also in line with the lower digestive enzyme activity in these groups compared with the control.

In this experiment, the SGR and WGR values of coral trout showed high consistency with intestinal digestive enzyme activity. At a water flow velocity of 2.5 bl/s, the digestive enzyme activity of coral trout is weakened, resulting in a decrease in growth performance, while the GLU content also reflects nutritional deficiencies from a lateral perspective. At this water flow velocity, there was no significant impact on VT, VL, or MS. However, the reductions in the number of goblet cells and the structural changes are the main reasons for the reduced digestive capacity that was observed.
