4.1. Histological Investigations
One of the limiting factors of including insect meal in fish feed is their chitin content, which is able to determine gut and liver histological changes [16
]. In particular, chitin could affect liver lipid accumulation [20
] and may induce intestinal inflammation [52
]. The histological effects on liver and gut by HIM utilization in diets for different fish species have already been characterized, with different results being obtained until now [16
]. Our results showed that up to 37.5% of highly defatted HIM can be included in Siberian sturgeon feed without any negative effects on spiral valve and liver histology.
No histopathological alterations were observed in the liver of Siberian sturgeons fed HIM based diets. This result is in agreement with those observed in Japanese seabass [29
], Atlantic salmon [30
], zebrafish (Danio rerio
], and rainbow trout [16
] fed with HIM meal. On the contrary, Li et al. [20
] observed significant alterations in liver (decrease hepatopancreas fat deposition and increase in mild necrosis and apoptosis of hepatocytes) in Jian carp (Ciprinus carpio
var. Jian) fed 7.9% inclusion level of HIM. Another recent study shows that inclusion level up to 40% of mopane worm meal (Imbrasia belina
) worsened liver degradation, probably caused by the high fiber content in the mopane worm [24
In most fish species, the dynamics of absorption decrease proceeding towards the posterior tract of the intestine. Since in sturgeon the maximum nutrient absorption takes place in the posterior intestine (spiral valve) [53
], this segment was herein considered. In the present study, no significant differences were observed between the Vh and the number of GC between HIM0, VEG, and HIM-fed sturgeons. This is in agreement with what was reported in rainbow trout [16
], post smolt Atlantic salmon [30
], and Japanese seabass [29
] fed with HIM meal. Another study conducted in Siberian sturgeon fed with HIM-based diet [18
] showed that in the proximal intestine, the inclusion of 15% of full-fat HIM did not affect the villus height, but the authors reported a reduction in the thickness of mucosa and increase in muscle layer thickness—changes that have been associated with an enhancement of the digestion and absorption process.
An increase in the number of GC in the distal intestine is usually associated with an immune reaction during the inflammation process and a decrease in nutrient absorption [35
]. These gut mucin dynamics are frequently related to high inclusion levels of VP sources in fish diets [7
]. In particular, the use of VP may cause morphological alterations capable of affecting the optimal nutrient absorption [6
]. However, the effects of VP usually vary on varying the species, the protein source, and the level of inclusion [35
]. Based on the authors’ knowledge, the effects of VP on the liver and gut histology of sturgeon have been only characterized by Kittel et al. [59
]. In particular, the authors formulated four experimental diets for shovelnose sturgeon (Scaphirhynchus platorhynchus
) containing increasing levels of soybean meal (from 0 to 51.23%) as partial replacement of FM and highlighted histopathological signs of distal enteritis in fish fed soybean meal-based diets confirming what was reported by previous studies [6
4.2. Oxidative Stress Biomarkers
The dietary inclusion of HIM meal in substitution of 25% and 50% of FM and VEG diet did not significantly alter the histological traits of liver and distal intestine sections suggesting no adverse effect on digestive capacity of Siberian sturgeons. However, all the experimental diets (HIM25, HIM50 and VEG) have led to a disturbance of some oxidative stress indicators in liver and kidney of sturgeons.
Both kidney and liver tissues chosen for this study have high potential for ROS production and response of the oxidative stress biomarkers in fish is tissue-specific [60
Each biomarker, under chemical or physical pressure, is prone to change and, within the different treatments, the response of all biochemical parameters can be dissimilar. In order to deepen the knowledge on the effects of HIM and VEG diets on sturgeons, a panel of physiological and biochemical markers was thus applied. Fish fed the HIM (25% and 50% substitution) and VEG diets showed increased levels of several biomarkers in liver and especially in kidney. Although HIM25 diet led to sporadic changes in renal enzymes activity, altered detoxifying response occurred more frequently in sturgeon fed HIM50. The GPx showed lowest value in both liver and kidney of specimens following HIM50 diets. However, no marked lipid peroxidation was observed through the experiment.
In the present study, the altered activity of several antioxidant enzymes, mainly in sturgeon fed HIM50, may be related to the diet’s composition. Hepatic increased levels of antioxidant enzymes, such as CAT, GPx, and GR, have been previously associated to protein- or lipid-rich FM diets in Adriatic sturgeon Acipenser naccarii
]. Although HIM25 and HIM50 were isoproteic, isolipidic, and isoenergetic, differences in fatty acid profile were found compared to HIM0 diet [19
]. Indeed, the levels of total fatty acids (TFA) were consistently higher in HIM50 compared to HIM0 (8025.84 vs. 6276.23 mg/100 g dry matter (DM), respectively), as well as saturated fatty acids (SFA—2794.26 vs. 1989.84 mg/100 g DM, respectively), and monoinsatured fatty acids (MFA—3580.15 vs. 2545.82 mg/100 g DM, respectively). Moreover, the ratio PUFA/SFA in HIM50 diet was found lower than control diet (0.59 vs. 0.87 mg/100 g DM, respectively). Sturgeon fed HIM50 also showed a reduced feed consumption compared to fish fed HIM0 (2823.15 and 3003.04 g DM, respectively), resulting in a diminished final body weight (141.94 g and 159.32 g, respectively), weight gain (117.73 g and 135.12 g, respectively), and specific growth rate (1.48% and 1.59%/day, respectively) [19
]. Starvation was previously associated with a triggered antioxidant capacity in Siberian sturgeon, mainly increasing the activity of SOD [65
]. Similarly, in our study a higher SOD activity was found in specimen fed HIM50. We may thus assume that the raised SOD and GR levels in liver and kidney of A. baerii
may be related to the different lipid composition and lower HIM consumption.
In addition, the polysaccharide chitin, one of the centerpieces of insect exoskeleton and present at higher levels (up to 1.92 g/kg) in HIM substituted diets, deserves attention. Chitin has been recognized as an antioxidant molecule, also preventing deleterious effects in various diseases [66
] and boosting the SOD activity in orange-spotted grouper (Epinephelus coioides
]. Chitin role is very complex and, as for other antioxidants, such as selenium [69
], may act as pro-oxidant when the optimal threshold is encompassed. In the present study, significantly higher chitin doses (1.92 g/kg) were found in HIM50 diet and may be related to the lower GPx activity in both liver and kidney of sturgeon. This outcome is not surprising and was previously discussed in rainbow trout (Oncorhynchus mykiss
) as associated with the ability of polysaccharide to bind selenium present in the diets, both in inorganic (selenite and selenite) and organic (selenomethionine and selenocysteine) form [16
]. In particular, selenocysteine is essential for GPx functioning since it contains a selenocysteine residue in its structure and its unavailability following chitin bond may have reduced the enzyme activity.
In the present study, GST was affected by both the included diets (HIM25, HIM50) and VEG, although different responses were observed. Similarly to our previous results in rainbow trout [16
], GST was enhanced only in kidney of sturgeon fed the higher substituted diet. This outcome and the concomitant increase of EROD suggest a strengthening of the detoxifying ability in groups fed HIM50 and an important role in the biotransformation of lipophilic compounds. Increase in GST activity has been previously reported in African catfish liver fed with cricket meal (Grillus bimaculatus
], while no changes were measured in carp fed with domestic fly larva meal [44
]. On the contrary, the sturgeon fed VEG showed a decreased GST level. A previous study showed that β-conglycinin, one of the major allergenic proteins present in soybean [72
], can decrease GST activity in intestine and enterocytes of carp, concomitant with an increased expression of the main antioxidant genes [73
]. The dropped hepatic GST levels measured in our study may suggest a pointing scenario, since the reactive intermediates produced by the phase I enzyme (CYP) may not be adequately removed.
Nevertheless, the unchanged MDA levels in both liver and kidney in sturgeon fed with both the categories of substituted diets indicate preserved antioxidant efficiency. The lack of lipid peroxidation following vegetable meal is not surprising. Peng et al. [74
] measured a reduction in lipid peroxidation in fish fed with soybean meal, probably correlated with an increased storage of hepatic α-tocopherol. Likewise, Wang et al. [29
] found a lower concentration of MDA in Japanese seabass fed H, revealing that this diet may improve the antioxidant status of fish. On the contrary, Ji et al. [75
] showed that the amino acid deficiencies in diets substituted with higher silkworm pupae doses caused lipid peroxidation and oxidative damage, also altering the intestinal microvilli and hepatocytes structure in juvenile Jian carp. This outcome suggests that lipid peroxidation may be related to fish species and/or protein source.
In conclusion, the inclusion of a highly defatted HIM and VP does not significantly affect the histology of liver and distal intestine of Siberian sturgeon. Nevertheless, as unfavorable effects on antioxidant response were reported at 37.5% of HIM inclusion, an inclusion level up to 18.5% is recommended for sturgeons.
Considering the high longevity of the sturgeons, further investigations are required to observe the long-time effects of insect and vegetable meals on gut and liver histology and the oxidative stress biomarkers of Siberian sturgeon.