Effects of Dietary Thiamine Supplementation on Growth Performance, Digestive Enzymes’ Activity, and Biochemical Parameters of Beluga, Huso huso , Larvae

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Introduction
Te aquaculture sector is responsible for producing food for human consumption, and as such, this industry is rapidly expanding to meet the growing global demand for protein. However, one of the major challenges in aquaculture practices is the process of larviculture, as fsh larvae are highly susceptible to adverse conditions, which can result in signifcant mortality rates and economic losses [1]. Terefore, the study on this life stage is crucial to improve the performance of fsh larvae. Nutrition is very important in larval stage in fsh, because the larvae have less ability for swimming and predation and their digestive system is still developing [2,3]. Insufcient quantities or subpar quality of larvae feed can result in a rapid decline in the population. It is imperative to provide fsh larvae with high-quality feed that is suitable for their developing digestive systems. Failure to do so can impede proper digestion, leading to a decrease in energy intake and potential gastrointestinal issues [4]. Fish larvae have thin skin and large surface area, compared to juveniles and adults; hence, they are more vulnerable to waterborne pathogens and need to have a well-functioning immune system [5]. Moreover, fsh larvae have high metabolic and oxygen consumption rate, compared to the juveniles and adults [6], so they should have a strong antioxidant system to protect them from harmful pro-oxidants. A well-formulated diet can optimize the performance of the larvae. Among the nutrients, thiamine defciency has been found to cause serious problems and mortality in fsh larvae [7][8][9]. Tiamine is an essential nutrient that cannot be synthetized by fsh body, thus must be supplied via diet. Tiamine has various important physiological roles in animals [10]. In fsh, thiamine defciency has been reported to cause hampered antioxidant capacity, immune strength, digestive enzymes' activities, and growth rate [11][12][13][14]. Terefore, adequate thiamine supply can be crucial for fsh larval stages, as mentioned previously.
Sturgeons are important fsh species with huge global demand in aquaculture industry [15,16]. Tiamine defciency in these fsh can reduce their performance at larval stage, as shown in sterlet sturgeon, Acipenser ruthenus [9]. Tey represented that thiamine defciency caused reduction in feed intake which lead to insufcient supply of adequate nutrients. Also, diseases symptoms such as yolk sac deformation, irregular swimming, and loss of equilibrium were reported in that investigation. Beluga, Huso huso, is an important aquaculture candidate due to its higher growth rate and valuable meat and caviar [17]. Terefore, it is artifcially propagated in diferent countries. Te survival rate of sturgeon larvae during the initial feeding stage is signifcantly lower compared to many other freshwater farmed fsh. Low fry survival is generally ascribed to fsh husbandry practices, especially feeding practices. However, diet quality may also be a determinant of sturgeon fry survival [18,19]. While there are numerous studies on beluga diet formulations (for example, Razeghi Mansour et al. [20]), limited research has been conducted on the nutritional needs of beluga larvae [21,22]. As a result, it is crucial to improve the diets of sturgeon larvae with nutritional supplements, as doing so can have a positive impact on their health and survival. Considering the importance of dietary thiamine in larval stage of fsh, the present study aimed at assessing the efects of dietary thiamine supplementation on growth performance, digestive enzymes activity, and biochemical/immunological parameters of beluga larvae.

Larvae Maintenance and Feeding Live Food.
Tis study was conducted in International Sturgeon Research Institute, Rasht, Iran. Te larvae of beluga were provided by fertilization of cultured male and female fsh in that organization. Fertilized eggs were incubated in the McDonald incubator at constant water exchange of 0.13 L/s. After 7 days, the larvae hatched. Ten, larvae were distributed in 12 fberglass rectangular tanks (200 × 70 × 30 cm size) with a volume of 210 L of water and a water fow of 0.13 L/s to design 4 treatments as follows. Water was aerated constantly. Te storage density was 250 larvae per tank. Te larvae yolk sac absorption lasted 6 to 7 days. Semiactive feeding of larvae was started approximately one week posthatching when 80% of the larvae were actively swimming. Te larvae were frst fed with Artemia nauplius for 12 times a day with an interval of 2 hours. Ten, Chironomidae and Artemia biomass were added to the larvae diet in addition to the Artemia nauplius. Te larvae were fed until satiety.

Diets and Larviculture.
From the 18th day after hatching, the live foods were gradually replaced by artifcial diets. Two experimental diets were formulated as dough and pellet according to specifcations outlined in Tables 1  and 2. Te main sources of protein were casein and fsh meal (the Caspian Sea sprat low-temperature processed). Fish oil and soybean oil were the main sources of lipids in the diets. To prepare experimental diets containing 5 (TS5), 10 (TS10), and 20 (TS20) mg thiamine per kg, thiamine hydrochloride (Sigma-Aldrich, Stuttgart, Germany; T4625, reagent grade, 99%) was added to other ingredients in amounts of 6.36, 12.7, and 25.4 mg/kg, respectively. Tese levels were selected based on the only study available on sturgeons [9,23] and studies on fsh species fed energydense diets that feed on high energy diets [14,24]. A control diet without thiamine incorporation was also included (TS0). All dry ingredients were completely grounded by laboratory mill (Iran Damikar Co, Tehran, Iran) to about 100 μm. Tiamine-free vitamins and premixes of minerals were primarily mixed with wheat four for 15 min and then added to other ingredients. Finally, all the dry ingredients were thoroughly mixed for 10 min by an electric mixer (Raybony, Garma Electric, Amol, Iran). Te thiamine supplement was dissolved in water, and then, a blend of water and oils was added to the mixture, resulting in a paste. Te prepared dough was divided into small circular pieces and placed in several parts of the rearing tank to feed the larvae.
Since 27th day after hatching, the pellet diet was gradually added to the larvae diet. From 29th after hatching until the end of the experiment (44th day after hatching), the larvae were just fed with the pellet diet, until apparent satiation. Te prepared dough, which was formulated ingredients in Table 2, was transferred to a pelletizing machine (CPM, model CL series; USA) to generate 1-mm diameter feed. Eventually, the pellets were dried at 70°C (in an oven) for 18 h and stored at refrigerator to use.

Diet Tiamine Content.
Tiamine content of the diets was measured by fuorometric method. Tiamine is oxidized to thiochrome, which is a fuorescent compound, by alkaline potassium ferricyanide. Te thiochrome was extracted in isobutyl alcohol and measured in a fuorometer (Elico fuorometer) at 366 nm. Te standard substance used in the fuorometer was made by dissolving 50 mg of thiamine hydrochloride in 500 ml of 0.1 N sulfuric acid containing 25% alcohol (100 μg/mL) [25].

Sampling, Survival, and Growth Calculation of Larvae.
At the end of the experiment (44th day posthatching), the number of larvae in each tank was counted to calculate the survival rate. Ten, twelve larvae were randomly sampled from each tank and anesthetized at 400 mg/L clove powder extract and sacrifced by spinal cord transection. Following formula was used to calculate growth and survival rates: survival percentage: 100 × survived fish initial fish . (1)

Sampling Arrangement for Enzymatic and Biochemical
Assay. All of the parameters were measured at the end of the experiment (third sampling) in four thiamine concentrations (TS0, TS5, TS10, and TS20) except digestive enzymes. Digestive enzymatic analysis was performed in three times during the experiment to assay the larvae digestive development trend. Te three sampling times were performed as follows: frst sampling (FS): when the larvae weight was 34 mg (1 day after semiactive feeding or 8 days after hatching), second sampling (SS): when the larvae weight was 50 mg (10 days after semiactive feeding or 18 days after hatching), and third sampling (TS): when the larvae weight was 3 g (26 days after semiactive feeding or 44 days after hatching). As the larvae were fed with artifcial diets from 18th until 44th day after hatching, third sampling was divided into four treatments: feeding with diets contain 0, 5, 10, and 20 mg thiamine per kg which were, respectively, called TS0, TS5, TS10, and TS20.

Preparation of Larvae Extract.
To prepare the larvae extract, four out of twelve larvae which collected per tank were homogenized in an electric homogenizer to provide sufcient tissue for determination of required indices [26]. For enzymatic extraction, 2 gr of the homogenized tissue was separated while the rest was used to determine the proximate composition.

Diet and larvae Proximate Composition.
Kjeldahl method was used to measure the protein of diet and larvae, by determining the amount of nitrogen content (N × 6.25) through digestion in strong acid, distillation, and titration. Soxhlet extraction method was used to determine the lipid content of diet and larvae. Te moisture content of the diet and larvae was measured by drying the samples in an oven at 105°C for 24 hours to reach a constant weight. To measure the diet and larvae ash content, the samples were combusted at 550°C in a furnace for 3 h [27].

Enzymatic Analysis.
Te homogenized tissues of each sampling were mixed at a ratio of 1 : 5 with 50 mM phosphate bufer for 1.5 minutes at 4°C, pH 7.4. Te homogenates were centrifuged at 11200 g for 20 minutes at 4°C. Ten, the supernatant was removed and was used for the analysis. Total soluble protein was calculated by the Bradford method using bovine serum albumin as a standard [28]. Lactate dehydrogenase (LDH) enzyme activity was calculated using Pars Azmoun quantitative assay kit (product code: 122400-Karaj, Iran) by colorimetric method using pyruvate substrate at a wavelength of 340 nm for 3 minutes. Alanine aminotransferase (ALT) activity was measured using Pars Azmoun quantitative assay kit (product code: 119400-Karaj, Iran) by colorimetric method using L-alanine and 2-oxoglutarate substrates at a wavelength of 340 nm for 3 minutes. Alkaline phosphatase (ALP) activity was determined using Pars Azmoun quantitative assay kit (product code: 102400-Karaj, Iran) by colorimetric method using para-nitrophenyl phosphate substrate at a wavelength of 450 nm for 3 minutes [29]. Zinc sulfate precipitation method was used to measure IgM [30]. Lysozyme activity was measured by turbidimetric assay based on Hoseini et al. [31]. Alternative complement (ACH50) activity was measured based on the sheep red blood cell hemolysis method [31].
Te activity of amylase was measured using the method described by Bernfeld [32] using soluble starch as the substrate. Te increase in reducing power of bufered starch solution was measured with 3-5 dinitro salicylic acid (DNS) at 540 nm. Lipase activity was determined spectrophotometrically by hydrolysis of p-nitrophenyl myristate at 37°C and wavelength 580 nm [33]. Te activity of trypsin was calculated at 410 nm wavelength using DL-arginine-pnitroanilide as substrate [34]. Chymotrypsin activity was evaluated spectrophotometrically by monitoring the hydrolysis of N-benzoyl-L-tyrosine ethyl ester (BTEE) [35]. Absorbance was measured at 256 nm for 20 min at 1-min intervals. Determination of the pepsin activity was performed using 2% hemoglobin solution (in 0.3 N HCl at pH 2.0) at 280 nm. One unit of pepsin activity was equivalent to μg tyrosine per mL in one min [36].

Statistical Analysis.
All data of the experiment were subjected to one-way analysis of variance (ANOVA). Te activity of digestive enzymes was calculated among three times of sampling to estimate the efect of larvae stage and also among four thiamine concentrations. Duncan's test was used to compare the mean values among individual treatments. Te level of signifcant diference was set at P ≤ 0.05. Statistical analysis was carried out using the SPSS v.19 software, and the results are presented as mean ± SD. Table 3, diets containing diferent levels of thiamine led to the improvement of the growth performance and survival of beluga larvae, so that with the increase in thiamine concentration, the growth indicators including fnal weight, weight gain, and specifc growth rates increased signifcantly compared to the control group (P < 001). Although there was no signifcant diference in these indicators in TS10 and TS20 treatments, the percentage of survival also increased signifcantly by increasing the concentration of thiamin in the diet from 0 to 20 mg/kg.

Whole Body Composition.
Signifcant diferences were noticed in terms of body composition levels in the experimental treatments (P < 0.001). Percentage moisture of the control larvae was signifcantly higher than the thiamine fed larvae. Te body fat content was similar to the moisture status; however, it was signifcantly higher in TS5 treatment than TS10 and TS20 treatments. Te protein content of fsh fed thiamine increased considerably compared to the control group, while the highest amount was in the TS10 treatment. According to the results, the ash content of fsh fed thiamine signifcantly increased compared to the control group, and the highest level was in the TS20 treatment (Table 4).

Before Receiving
Tiamine. Pepsin, lipase, and amylase activities increased signifcantly with beluga growth and development, which was the highest at TS (P < 0.001). According to the results, trypsin activity increased signifcantly in SS compared to the FS and then decreased signifcantly in TS stage. Te activity of chymotrypsin in diferent stages of beluga nutritional development was completely opposite to that of trypsin, and the highest level of chymotrypsin activity was evaluated in TS.

After Receiving Tiamine.
Results show that the activity of digestive enzymes was signifcantly increased in the fsh fed with thiamine supplemented artifcial diets (TS5-20) in comparison with the fsh fed artifcial diets without thiamine supplementation (TS0). However, thiamine had no efect on the trypsin activity. Te activity of pepsin and chymotrypsin of fsh fed with TS5-20 was the same, and they were signifcantly higher than the TS0 treatment (P < 0.001). Te highest activity of lipase was estimated in TS10 (P < 0.031). Amylase activity of fsh fed TS10-20 was signifcantly higher than that of in TS0 and TS5 treatments, while the lowest one was in TS0 treatment (P < 0.001). Table 5, there were no diferences among the ALP activities of TS5-TS20 treatments, and they were signifcantly lower than the TS0 treatment (P < 0.013). Adding 10 and 20 mg/kg thiamine to the diets caused a signifcant reduction in ALT activity compared to the TS0 treatment (P < 0.027); however, there was no diference between TS5 and TS0. Dietary thiamine supplementation had no efect on LDH, IgM, ACH50, and lysozyme.

Discussion
Te activity and profle of digestive enzymes undergo changes throughout the ontogeny and development of fsh larvae. In sturgeons, it has been demonstrated that these enzymes are detectable after hatching, aiding the larvae in utilizing yolk nutrients [3,37]. Pepsin, trypsin, and chymotrypsin are main proteolytic enzymes that involve in fsh digestive tract [38]. Te sturgeon larvae have been observed to transition from alkaline protein digestion, facilitated by trypsin and chymotrypsin to acidic protein digestion facilitated by pepsin, during early ontogeny. Tis shift occurs as the larvae transition from endogenous feeding to exogenous feeding and from live food to formulated diets [3,19]. Accordingly, the increase in pepsin activity from FS to TS can be due to the development of gastric gland as suggested by Babaei et al. [3] and Asgari et al. [19]. Te increase in chymotrypsin activity at TS is in line with Asgari et al. [19], in this species, but opposite to fndings in other sturgeons such as Persian sturgeon, Acipenser persicus Babaei et al. [3] and stellate sturgeon, Acipenser stellatus [39]. Te observed variations may be specifc to the species and highlight the signifcance of chymotrypsin (or chymotrypsin-like enzymes) in the protein digestion process of beluga. Furthermore, studies using transcriptomic and inhibitor tools can address this topic in beluga. Te increase in lipase activity may be due to the importance of lipids in energy source to spare protein for growth performance, as previously observed in this species [19], as well as other sturgeons [3,39]. Moreover, it may be due to increase in total daily fat intake, when the fsh feed on the concentrated diets [40]. A previous study on beluga has shown that amylase activity increases immediately after exogenous feeding and during the early stage of cofeeding remains stable but shows an upsurge during full feeding on formulated diet [19]. Amylase activity showed similar pattern, and this might be increment in the diets carbohydrate of daily carbohydrate intake.
Te improved growth rate in thiamine-treated fsh is in line with previous studies on mrigal carp, Cirrhinus mrigala [41], Jian carp, Cyprinus carpio var. Jian [42], and grass carp, Ctenopharyngodon idella [43]. Such an improvement can be partly a consequence of increase in the digestive enzymes' Journal of Applied Ichthyology 5 activity in the present study. Te exact mechanisms of improvement in digestive enzymes' activity by thiamine are not known in fsh; however, studies on mammals have demonstrated that thiamine is necessary for proper functioning of a certain region of the central nervous system that controls the gastrointestinal tract function [44]. A decline in hydrochloric acid secretion by the gastric glands has been reported as a symptom of thiamine defciency in mammals [45,46]. Terefore, it is speculated that the increase in pepsin activity in the treatments fed with thiamine may be due to the increase in hydrochloric acid secretion by the gastric glands and, as a result, the increase in the conversion rate of pepsinogen to pepsin. Moreover, the highest growth performance was accompanied by the highest amylase and/or lipase activities, suggesting a potential better carbohydrate and lipid digestion and absorption. Such elevations in these pancreatic enzymes (as well as chymotrypsin) can be due to proper functioning of the pancreatic cells. Supporting this, mammalian studies have revealed that pancreatic cells are the richest cells in the body [47], and thiamine defciency leads to impaired dysfunction of these cells. Tiamine supplementation increased protein content in the whole body, which can be due to higher protein synthesis. Increase in the whole body protein content after dietary thiamine supplementation has been reported in golden pompano, Trachinotus ovatus [14] and Nile tilapia, Oreochromis niloticus [48]. Tis can be another explanation for improved growth rate in the thiamine-treated fsh. A previous study has shown that thiamine defciency leads to lower growth rate parallel to lower RNA/DNA ratio in mrigal carp [41], which is an indicator of hampered protein synthesis. Tis can be due to stress in the endoplasmic reticulum that decreases protein synthesis, as suggested in human [49]. Unlike previous studies on other fsh species, dietary thiamine supplementation signifcantly increased the whole body ash content in beluga, which its exact reason is not clear. Although thiamine is primarily known for its role as a coenzyme in energy metabolism and proper nervous system functioning in fsh, recent studies suggest that it may also have a non-coenzyme role in bone structuring and strength. Research conducted on mice has supported this theory [50]. However, further evaluations are needed to determine the extent of thiamine's impact on bone health in fsh.
ALT, ALP, and LDH are cytosolic enzymes and indicators of tissues' health [51]. Tere are limited studies on the efects of thiamine on tissues' health in fsh. A study on common carp, Cyprinus carpio, has shown that thiamine can mitigate elevations in ALT and ALP, when the fsh exposed to waterborne lead, suggesting potential hepatoprotective properties of thiamine [52]. Tiamine defciency can decrease NADPH concentration, a radical scavenger, due to decrease in transketolase activity [53]. Moreover, thiamine defciency can hamper antioxidant signaling pathways (Nrf2 and Keap1) [12]. So improvement in tissues' health can be due to boosted antioxidant capacity in the thiamine-treated beluga.
Te innate immune system of fsh has an important role in preventing the occurrence of diseases in fsh [54]. Tiamine treatment increased fsh survival rate, which is in line with previous studies showing thiamine defciency is detrimental for fsh early life stages [7][8][9]. Tis may be attributed to the higher metabolic rate during early life stages, which increases the demand for thiamine [14]. However, despite the higher survivability, no benefts of thiamine have been observed on immunological parameters. In contrast, thiamine has been found to improve various immune parameters in diferent fsh species. For example, thiamine defciency signifcantly decreased immune-related genes' expression, lysozyme, and complement activities in the gills and gut of grass carp [12,13]. Moreover, thiamine defciency signifcantly decreased serum immunological parameters in Jian carp [55]. Leukocytes functions decreased in lake trout, Salvelinus namaycush, when experienced a thiamine defciency state [56]. Terefore, it is speculated that no signifcant efects of thiamine supplementation on beluga immunological parameters may be due to measuring them in the whole body extract.   Amylase (U/mg pr) Figure 1: Comparison of digestive enzymes in beluga fngerling fed with the diets containing thiamine. FS: frst sampling, SS: second sampling, TS: third sampling. TS0, TS5, TS10, and TS20 were related to third sampling of larvae were fed with diets contain 0, 5, 10, and 20 mg thiamine per kg diet. Values are presented as (mean ± SD). Diferent lowercase and uppercase letters indicate signifcant diference among diferent feeding stages without thiamine and diferent thiamine levels in artifcial diet stage, respectively (P < 0.05). Values are presented as (mean ± SD). Diferent show signifcant diference between pair groups at P < 0.05.
In conclusion, thiamine supplementation is benefcial in rearing beluga larvae. Tiamine supplementation improves the growth rate of the larvae, possibly due to an increase in digestive enzymes' activity and/or protein retention. Additionally, thiamine supplementation boosts larvae survival rate, possibly due to improve tissues' health, as evidence by lower ALT and ALP activities. For optimal results, it is recommended that thiamine be included at a concentration of 10-20 mg/kg during the beluga weaning stage until the fngerling stage.

Data Availability
Te data of this study are available upon reasonable request from the corresponding author.

Conflicts of Interest
Te authors declare they have no conficts of interest.