Introduction

Bangladesh is a sub-tropical country, most suitable for aquaculture [1]. Fish production from natural resources has gradually been declined due to the degradation of their ecological balance. Consequently, the aquaculture industry has expanded very rapidly in the last three decades and Bangladesh ranked 5th in global aquaculture production [2]. Nevertheless, recently, the low quality as well as increased price of fish feed is alarming for aquaculturists. In aquaculture, fish feed requires around 50 to 60% of the total operational cost [3, 4]. In fact, increased price of feed ingredients (fishmeal, fish oil, cereal, etc.) complicated the easy availability of aqua-feeds to many fish farmers. On the other hand, sustainability of aquaculture depends on proper nutrition content and cost-effective feed [4].

Nutrition is one of the most important factors in aquaculture. Aquaculturists must have a clear knowledge about the actual amount of feeds with proper nutrition for better growth, health status, and reproduction of fish. Among different trace elements, chromium (Cr) plays a significant role in the fish nutrition and physiology [5, 6]. Cr has various positive impacts on human health as well as several farmed animal species [7]. Cr in the feed participates in the metabolism of various nutrients, such as protein, fat, and carbohydrate [6, 8]. It has been reported that Cr in diet improve the metabolism of carbohydrates and thus enhance the feed utilization and growth of Nile tilapia, Oreochromis niloticus [9], and common carp, Cyprinus carpio [10]. Dietary carbohydrates in association with organic Cr act as energy currency that helps in smooth operation of various biological processes and exhibits superior growth of fish [11]. It has been reported that chromodulin role of Cr amplify the process of insulin signaling and successively influence the metabolic rate of various nutrients as well as growth performance [7, 12]. Supplementation of Cr improves growth performance of animals through increasing energy metabolism. Cr supplementation in certain dosage positively stimulates the growth performance of rainbow trout (Oncorhynchus mykiss) [13], Indian major carp (Labeo rohita) [14], and hybrid tilapia (Oreochromis niloticus) [15]. Cr changed the serum fatty acid profile through the metabolism of fatty acid in rats [16] and finishing pigs [17, 18]. Kucukbay et al. [5] reported that Cr decreases the blood cholesterol in the rainbow trout. Cr supplementation in the diets decreased serum cholesterol, low-density lipoprotein cholesterol, and triglycerides, while increased serum high-density lipoprotein cholesterol in the broiler chicken [19, 20]. Similar result was reported in the grass carp (Ctenopharyngodon idella) fed with Cr-supplemented diet [6].

The health status of fish is checked by measuring different blood parameters [21,22,23,24]. Usually, the oxygen is transported to different tissues by hemoglobin (Hb). Thus, the blood Hb content is commonly used to know the abnormalities and pathological signs of fish health [25, 26]. Similarly, variations in the white blood cell count and blood glucose content are also used as a biochemical immunosuppressive sign for fish [26,27,28]. On the other hand, morphological alterations of blood cells are examined to assess the health condition of fish [28, 29]. For example, several authors studied the frequency of micronucleus (MN) formation in the erythrocytes to know the stress due to environmental contaminants [29,30,31]. The MN is a small mass of cytoplasmic chromatin created outside of the central nucleus during the nuclear division of the chromosome [32].

Striped catfish (Pangasianodon hypophthalmus) is an important fish species for aquaculture in Bangladesh, which was introduced from Thailand in 1989. This is a fast growing fish species with high adaptability to diverse environmental conditions. Therefore, its culture practice has been expanded throughout the country. In Bangladesh, the annual production of this fish species is 0.45 million metric tons, representing 11% of the total fish production [33]. It is considered as a good source of protein and calories. It has a high consumer value due to its availability in the market as a live condition. Recently, the profit has been decreasing day by day due to increased feed cost and improper nutrition. However, there is no available information about the role of Cr as a nutritional component in the striped catfish. Therefore, the present investigation was designed to know the effects of Cr addition in the diet to growth and feed utilization in the striped catfish.

Materials and Methods

Experimental Diets

The ingredients (Table 1) used to formulate the diets were procured from the local market. In the experimental diets, rice and wheat brans were used as a source of carbohydrate. Four experimental diets were formulated containing 0, 2, 4, and 8 mg kg−1 of chromium (Cr). For preparation of the test diets, dry ingredients were mixed thoroughly with molasses and cold distilled water. Then, wet extrusion of the pellets was done with the help of a pelletizer. Finally, the diets were air-dried and stored at −20°C in airtight polythene bags until use in the experiment. The proximate composition (Table 1) of the formulated diets was analyzed following standard protocol [34]. A flame atomic absorption spectrophotometer (Model Shimadzu AA-7000) was used to determine the final concentrations of Cr in the basal diets and the concentrations of Cr in the basal diets were found as per the desired level.

Table 1 Formulation and proximate composition experimental diet (% dry matter basis)
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Experimental Fish

Apparently, healthy and active striped catfish (P. hypophthalmus) fingerlings of body weight (18.48 ± 1.67 g) and total length (13.81 ± 0.83 cm) were collected from a private hatchery of Sadar Upazila, Mymensingh. The fingerlings were transported to the Fish Ecophysiology Laboratory, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh. In the laboratory, fish were kept in aquaria (75 × 45 × 45 cm) containing 100 L of clean tap water. Before starting the experiment, fish were acclimatized for 21 days to the laboratory conditions. During the acclimatization process, the fish were fed twice daily with commercial grower feed (CP Bangladesh Co., Ltd.) and each aquarium was monitored daily to check fish mortality and dead fish were removed from each aquarium. We maintained all guidelines provided by the Animal Welfare and Ethical Committee, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh (approval number: BAU-FoF/2019/003).

Experimental Design

Four diets with chromium (0, 2, 4, and 8 mg kg−1) were fed to striped catfish in aquaria with triplicate groups for 60 days. After acclimatization, fish were distributed into 12 glass aquaria (75 ×45 × 45 cm) filled with 100 L of water. In each aquarium, 15 fingerlings of striped catfish were stocked. Aerators were used to provide sufficient aeration to the aquaria throughout the experiment that help to balance the dissolved oxygen level. The aquaria were divided into four dietary treatment groups, each with three replications. Throughout the experimental period, fish was fed with diets at 5% of their body weight two times in a day (9 am and 5 pm). After determining the new body weights of the fish, the rations were adjusted in every week. Uneaten feed and feces in each aquarium were siphoned in every morning before feeding.

Survival, Growth, and Feed Utilization

Total number of the survived fish was counted and weighed from each aquarium at the end of experiment. Survival, growth parameters (weight gain, WG; %WG; specific growth rate, SGR), and feed utilization (feed intake, FI; feed efficiency, FE; protein efficiency ratio, PER; feed conversion ratio, FCR) were calculated using the following formulae:

$$ \mathrm{Survival}\ \mathrm{rate}\ \left(\%\right)=\left[\mathrm{number}\ \mathrm{of}\ \mathrm{fish}\ \mathrm{harvested}/\mathrm{number}\ \mathrm{of}\ \mathrm{fish}\ \mathrm{stocked}\right]\times 100 $$
$$ \mathrm{Weight}\ \mathrm{gain}\ \left(\mathrm{WG}\right)=\mathrm{final}\ \mathrm{weight}-\mathrm{initial}\ \mathrm{weight} $$
$$ \mathrm{Percentage}\ \mathrm{weight}\ \mathrm{gain}\ \left(\%\mathrm{WG}\right)=\left[\left(\mathrm{final}\ \mathrm{weight}-\mathrm{initial}\ \mathrm{weight}\right)/\mathrm{initial}\ \mathrm{weight}\right]\times 100 $$
$$ \mathrm{Specific}\ \mathrm{growth}\ \mathrm{rate}\ \left(\mathrm{SGR}\right)=\frac{\ \ln\ \mathrm{final}\ \mathrm{weight}\left(\mathrm{g}\right)-\ln\ \mathrm{initial}\ \mathrm{weight}\ \left(\mathrm{g}\right)}{\mathrm{number}\ \mathrm{of}\ \mathrm{days}\ \mathrm{reared}}\times 100 $$
$$ \mathrm{Feed}\ \mathrm{in}\mathrm{take}\ \left(\mathrm{FI},\mathrm{g}/100\mathrm{g}/\mathrm{BW}/\mathrm{day}\right)=\mathrm{dry}\ \mathrm{feed}\ \mathrm{fed}\times 100/\left[\left(\mathrm{final}\ \mathrm{weight}+\mathrm{initial}\ \mathrm{weight}\right)/2\times \mathrm{experimental}\ \mathrm{duration}\ \mathrm{in}\ \mathrm{days}\right] $$
$$ \mathrm{Feed}\ \mathrm{efficiency}\ \left(\mathrm{FE}\right)=\left(\mathrm{final}\ \mathrm{weight}\ \left(\mathrm{g}\right)-\mathrm{initial}\ \mathrm{weight}\ \left(\mathrm{g}\right)\right)/\mathrm{dry}\ \mathrm{feed}\ \mathrm{intake}\ \left(\mathrm{g}\right) $$
$$ \mathrm{Feed}\ \mathrm{conversion}\ \mathrm{ratio}\ \left(\mathrm{FCR}\right)=\mathrm{dry}\ \mathrm{feed}\ \mathrm{fed}/\mathrm{live}\ \mathrm{weight}\ \mathrm{gain} $$

Protein efficiency ratio (PER) = live weight gain / crude protein fed

Hemato-biochemical Study

To assess the hemato-biochemical parameters, ten fish (n = 10) from each treatment were sacrificed at the end of the feeding trial. The hemoglobin (Hb; g/dL) and glucose content (mg/dL) of blood were directly measured by using a digital EasyMate® GHb double monitoring system (Model ET 232, Bioptic technology Inc., Taiwan 35057) with hemoglobin and glucose strips, respectively. For counting red blood cell (RBC) and white blood cell (WBC), blood was collected from the caudal vein and preserved in the tubes containing 20-mM EDTA anticoagulants. The preserved samples were further used for counting RBC and WBC using a Neubauer hemocytometer under a microscope.

Frequency Analysis of Micronucleus Formation

The procedures for analysis of frequency of micronucleus (MN) formation were described in detail by Islam et al. [35]. In brief, blood was smeared on glass slides immediately after collection. The slides were air-dried for 10 min, fixed in methanol for 10 min, and finally stained 5% Giemsa stain. The MN was scored blindly on randomized coded slides under a light microscope (MICROS MCX100LED, Austria) connected to a camera (AmScope 1000). Three slides were prepared from the blood of each fish and 2000 cells were scored from each slide.

Water Quality Parameters

Dissolved oxygen (DO), pH, free CO2, total alkalinity, and ammonia were measured fortnightly during the experimental period. Temperature, DO, and pH were monitored using a mercury thermometer, DO meter (DO5509, Lutron, Taiwan), and portable pH meter (RI 02895, HANNA Instruments Co.), respectively. Titrimetric method with phenolphthalein indicator and 0.0227N sodium hydroxide (NaOH) titrant was used to assess the free CO2 of water. Total alkalinity of water was also assessed by titrimetric method where methyl orange was used as indicator and 0.02N standard sulfuric acid (H2SO4) was used as titrant. Ammonia was determined by ammonia test kit solution. For these, the test vital was rinsed with a water sample and filled up to the 5mL of measuring cylinder. Then, 5 drops of ammonia test kits solution-1 was added, mixed, and waited for 1 min. After sometimes, 1 spoon of ammonia test kit powder-2 was added and mixed until dissolved. Then, 5 drops of solution ammonia test kit solution-3 were added, mixed, and waited for 3–5 min. Finally, it was compared with the standard color chart for ammonia (ppm).

Data Analysis

All values are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to test the statistical variation (p < 0.05) of growth parameters, feed utilization, hemato-biochemical parameters, and frequency of micronucleus (MN) of fish fed with different Cr supplemented feed. The polynomial regression analysis based on WG and FE was used to determine the optimum dietary Cr requirement. The SPSS version 14 for Windows (SPSS Inc., Chicago, IL, USA) was statistical analysis.

Results

Survival, Growth, and Feed Utilization

The survivability of fish was very high (98 to 99%), not affected by dietary treatments (Table 2). Weight gain (WG), %WG, and specific growth rate (SGR) were significantly (p < 0.05) higher in the fish fed with 2 and 4 mg Cr kg−1 containing diets compared to those in fish fed with 0 and 8 mg Cr kg−1 containing diets (Table 2). These growth parameters were decreased in the fish fed with 8 mg Cr kg−1 containing diets. In case of feed intake (FI), no distinct change was observed among different dietary treatments. On the other hand, the feed efficiency (FE) and protein efficiency ratio (PER) were significantly (p < 0.05) higher in the fish fed with 2 and 4 mg kg−1 Cr-based diets (Table 2). Consequently, the feed conversion ratio (FCR) was significantly (p < 0.05) lower in case of fish fed with 2 and 4 mg kg−1 Cr-based diets (Table 2). Based on WG and FE, the polynomial regression analysis showed the optimum dietary Cr requirements for striped catfish 2.82 and 2.75 mg Cr kg−1, respectively (Fig. 1). During the analysis of the polynomial regression, the equations were y = –344.89x2 + 310.4x + 94.06 (R2 = 0.99) and y = –1.7394x2 + 1.528x + 0.4892 (R2 = 0.99), respectively.

Table 2 Growth performances of striped catfish fed diets containing various concentrations of chromium for 60 days
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Fig. 1
figure 1

The effect of dietary Cr on (a) weight gain (WG) and (b) feed efficiency (FE) in striped catfish. Each point represents the mean (± SD) of three groups of fish (n = 3), with 15 fish per group. The analyzed dietary Cr concentrations were log-transformed for better visualization. Requirement derived with the polynomial regression method for WG and FE was 2.82 and 2.75 mg kg−1 Cr, respectively

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Hematological Parameters

At the end of the feeding trial, Hb, RBC, and WBC were measured in the blood of fish. The values of Hb (g/dL), RBC (×106/mm3), and WBC (×103 mm3) ranged from 10.5 ± 0.8 to 12.7 ± 0.9, 1.30 ± 0.09 to 2.51 ± 0.19, and 1.67 ± 0.18 to 1.87 ± 0.23, respectively, in the fish blood exposed to different concentrations of Cr. The values of Hb and RBC significantly (p < 0.05) decreased in the fish fed with 8 mg kg−1 Cr-based diet compared to others (Fig. 2A and B), while the value of WBC did not vary among the different Cr-based diets fed fish (Fig. 2C).

Fig. 2
figure 2

Changes in hematological parameters of striped catfish-fed diets containing various concentrations of chromium after 60 days. A. hemoglobin levels (g/dL); B. number of RBC (×106/mm3); and C. number of WBC (×103/mm3). Values accompanied by different alphabets are indicating statistically significantly different (p < 0.05) among treatments. All values expressed as mean ± SD (n = 10)

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Biochemical Parameters

At the end of the feeding trial, glucose levels (mg/dL) were measured in the blood of fish. There was a significant (p < 0.05) reduction in the levels of blood glucose in the fish fed with 8 mg kg−1 Cr-based diet compared to others (Fig. 3). The highest levels of blood glucose were observed in the fish fed with 4 mg kg−1 Cr-based diet followed by 2 and 0 mg kg−1 Cr-based diets (Fig. 3).

Fig. 3
figure 3

Changes in blood glucose levels (mg/dL) of striped catfish-fed diets containing various concentrations of chromium after 60 days. Values accompanied by different alphabets are indicating statistically significantly different (p < 0.05) among treatments. All values expressed as mean ± SD (n = 10)

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Formation of Micronucleus

There was a significant (p < 0.05) increase in the frequency of formation of MN in the blood of fish fed with 8 mg Cr kg−1 based diet. No change in the frequency of formation of MN was observed in the blood of fish fed with 0, 2 and 4 mg kg−1 Cr-based diets (Fig. 4).

Fig. 4
figure 4

Frequency of micronucleus (MN) in the erythrocytes of striped catfish-fed diets containing various concentrations of chromium after 60 days. Values with different alphabetical superscripts differ significantly (p < 0.01) among different diets. All values are expressed as mean ± SD. Three slides were prepared from blood of each fish and 2000 cells were scored from each slide. MN is shown by an arrow

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Water Quality Parameters

Water quality parameters (mean ± SD), dissolved oxygen (mg L−1), free CO2 (mg L−1), pH, total alkalinity (mg L−1), and ammonia (mg L−1) values are presented in Table 3. There were no distinct changes recorded for any water quality parameters irrespective of any treatments throughout the experimental period. All the parameters were within suitable range for the growth of fish.

Table 3 Water quality parameters (mean ± SD) during the study periods
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Discussion

Chromium (Cr) plays an important role in the metabolism of fat, protein, and carbohydrate. Cr-supplemented diets (2 and 4 mg kg−1) in the present study significantly enhanced WG, %WG, SGR, FCR, FE, and PER of striped catfish fingerlings. It has been reported that diets containing Cr improved the growth of the rohu, Labeo rohita [14], tilapia, Oreochromis niloticus × Oreochromis aureus [15], grass carp, Ctenopharyngodon idellus [6], yellow croaker, Larimichthys crocea [36], and golden pompano, Trachinotus ovatus [37]. In pigs, Cr-supplemented diets improved carcass composition, quality, and weight of muscle [38]. Considering the previous reports, the present study indicates that the growth might be enhanced due to the increased utilization of feed.

Remarkably, the growth parameters and feed utilization were declined in the fish fed with the diet containing more than 4 mg Cr kg−1, which indicated that high Cr supplementation probably led to toxicity and depressed the growth of striped catfish. In common carp (Cyprinus carpio), growth increased at 0.5 mg kg−1 dietary Cr supplementation, while growth decreased at 2 mg Cr kg−1 diet [39]. The palatability of feed reduces when the concentrations of Cr nanoparticles cross the optimum levels that may cause decrease of growth on higher levels of supplementation [40]. It is noted that several fish species, such as gilthead seabream [41], rainbow trout [42], and Nile tilapia [43], did not show any response to Cr-based diets. These variations are not surprising as many factors, such as form and dose of Cr, duration of experiment, and behavior of concerned species, determined the actions of supplemented Cr in the diets.

Health conditions, as well as the physiological status of fish, are frequently evaluated by the hematological studies. Hence, the obtained results revealed that Hb and RBC significantly decreased in the highest (8 mg kg−1) Cr in diets. There was a significant change in Hb content and RBC in broiler birds supplemented with vitamin E, zinc (Zn), and Cr [44] are good agreement with our study. Cr-picolinate-supplemented diet increased Hb values in Japanese quail [45]. The changes of RBC and Hb content may have resulted from degeneration of the erythropoietic tissues by Cr, which obstructs the viable condition of the cell. Cr impairs the metabolism and storage of iron that leads to a significant reduction in the iron-binding capacity of the serum, and ferritin and hemoglobin content of fish blood [46]. In this study, blood glucose levels were increased significantly in the fish fed with up to 4 mg Cr kg−1diets, whereas significantly declined in the fish fed with 8 mg Cr kg−1 diet. Increased Cr-based diet reduced the blood glucose levels in rainbow trout [5], hybrid tilapia [47], and Nile tilapia [43] which supported the findings of the presents study.

Significantly increased formation of micronucleus (MN) at 8 mg kg−1 Cr-based diet also indicates that increased Cr level causes toxicity and depressed the growth performance of striped catfish. The frequency of MN increased significantly in the erythrocytes of fish after exposure to cadmium (Cd), copper (Cu), and lead (Pb) [48]. MN as well as other erythrocytic abnormalities was observed in the erythrocytes of common carp exposed to Cu [49]. The frequency of nuclear abnormalities significantly increased in the erythrocytes of mosquitofish (Gambusia affinis) as a result of Cd and Cu toxicity [50]. MN frequency increased in the erythrocytes of tilapia, Oreochromis mossambicus [51], and spotted snakehead, Channa punctatus [52], exposed to arsenic. There was a linear connection among different heavy metals (Ni, Cd, Cu, and Pb), MN, and other nuclear abnormalities of the erythrocytes in mullet, Mugil cephalus, and catfish, Clarias gariepinus [53]. Therefore, high Cr supplementation probably led to toxicity and depressed the growth of striped catfish.

Cr supplementation in the feed enhances the metabolism of protein, fat, and carbohydrate [6, 8]. Basically, energy from carbohydrate depends on its digestibility, metabolic enzymes, and finally absorption [54]. It has been reported that dietary Cr increased the carcass crude protein content of Nile tilapia [43, 55]. Cr significantly influences the activities of several enzymes. For instant, Cr supplementation through diet enhances the liver enzymes such as glycolytic enzyme and lipogenic enzyme that are related with the preliminary step of glycolysis and lipogenesis pathways [10] which clarify the mechanisms regulating the utilization of carbohydrates [56, 57]. It has been reported that dietary Cr supplementation positively enhanced the growth and homeostasis of blood glucose levels through influencing the expression of glucose metabolism related genes (pyruvate kinase PK, phosphoenolpyruvate carboxykinase PEPCK, glucose-6-phosphatase G6Pase, and glycogen synthase GS) and lipogenesis-related genes (sterol regulatory element binding protein-1 SREBP1 and fatty synthase FAS) in Juvenile Blunt Snout Bream, Megalobrama amblycephala [58]. However, the role of dietary Cr on the enzymatic activities in striped catfish warrants further investigation.

In summary, the striped catfish fed with 2 and 4 mg kg−1 Cr-based diets had substantial higher growth and utilization of feed. In contrast, a diet with 8 mg Cr kg−1 suppressed the growth and feed utilization. The values of hemato-biochemical parameters and formation of MN also indicated that 8 mg Cr kg−1 supplementation causes toxicity, which decreased growth performance of striped catfish. Polynomial regression analysis based on WG showed that 2.82 mg Cr kg−1 is optimum for the diet of striped catfish. This level of Cr may be recommended as a feed supplementation for striped catfish farming as well as the factories involved in the preparation of fish feed. More studies are needed to know the role of Cr on reproductive physiology as well as stages of maturation to enlarge the scopes of Cr to be used as an efficient nutrient.