Effect of supplemental selenium in fish feed boosts growth and gut enzyme activity in juvenile tilapia (Oreochromis niloticus)

2.4.1 Sample preparation

Six live fish pieces per treatment tank were randomly captured and euthanized to degut after every 30 days. The removed gut was homogenized in a chilled Tris-HCl solution, and homogenate was centrifuged at 9300 rpm at 4 °C for 20 min. The acquired supernatant was collected and stored at −4 °C as a crude source of enzymes.

2.4.2 Amylase estimation

The estimation of amylase activity was performed through starch-hydrolysis (Bernfeld, 1955), experimental details given in supplementary material. The formula used is mentioned as under.

Absorption of standard = A; Absorption of sample = Y; A absorption is due to 01 ml (0.01 mg of standard glucose in 01 ml (glucose 10%) therefore 01 mg glucose/10 ml. So, 01 is = 0.01/A.

Y absorption is due to = (0.01/A) × Y = Fwhere F is mg of amylase in the sample. Therefore, the final equation is

The specific amylase activity was calculated as ml of maltose released per gram for one hour.

2.4.3 Lipase estimation

The lipase activity was estimated by the method of Furne et al. (2005), the details of experimental protocol followed are given in supplementary material. The calculation was performed using the formula as under.$$\frac{\mathit{Unit}}{\mathit{ml}}ofenzyme=y=\frac{\mathit{Volume}ofNaOH\times NormalityofNaOH\times 40}{\mathit{Volume}ofenzymesampleused}$$Y = µM of oleic acid released/minute.$$\mathit{Enzyme}activity=Z(I{U}^{-min})=\frac{Y\times 1000}{250(MWofoleicacid)\times 30minutes}$$

whereas lipase activity unit was specified as the amount of free fatty acid (oleic acid) released from triacylglycerol per unit of time and estimated by the total amount of NaOH required for constant pH maintenance and denoted as milli-equivalents of alkali used.

3.1.1 Weight and length dynamics

The growth performance of the juvenile tilapia was recorded significantly higher (P < 0.05) in T₁ as opposed to T₂, T₃ and T₄. On the other hand, the significantly weak growth outcomes were displayed in T₃. However, arrested growth shown by the selenium-deficient (T₄) group. Although fish growth was displayed continuously in the T₂ and T₃, the weight increments during last three fortnights remained insignificant (Fig. 1). The T₁ presented significantly higher average gains in body weight (21.88 ± 5.62 g) and length (11.14 ± 1.04 cm). The statistical analyses revealed significant variations (P ≤ 0.05) in the average weight and length gains in all the treatments (Table 3). The detailed comparisons between the average weight and length gains on the basis of selenium-supplemented fish feed are presented in Table 3. The combined linear projections clearly explained the juveniles could demonstrate promising and isometric growth outputs, if dispensed with feed containing 2 mg/Kg and vice versa. It is also mention-worthy that T₄ witnessed irregular and weak growth patterns.

Close

Fig. 1

Comparisons of fortnight growth and length gains and their linear projections in juvenile tilapia based on supplemental selenium and selenium-deficient fish diets.

Export to PPT

The intrinsic importance of supplemental selenium has been illustrated in standard growth increments in juvenile O. niloticus. Like other micro-minerals (Cu, Fe, and Zinc), selenium is essential for the gradually improving and well-noticed growth in nearly all cultured species of the animals. Our results are in line with Lin and Shiau (2005), who reported higher growth rates in Epinephelus malabaricus, under selenium supplementation with a lower growth rate at 4 mg/kg. However, selenium could not be the single factor responsible for lower growth performance. The other potential factors could be the increased RBCs reducing fish immunity. Similarly, Abdel-Tawwab et al. (2010) reported that feed supplemented with 0.5 g OS/kg (5.54 mg/kg) improved growth in tilapia.

Nonetheless, Gaber (2007) reported mixed findings on higher growth in C. carpio, fed on sodium selenite supplemented feed 0.24–0.32 mg/kg (0.12–0.15 mg/kg). Our findings corroborated with El-Hammady et al. (2007), who determined higher growth in fish fed on 2 mg/kg. The other inclusion levels of selenium for species tested include African catfish (3.67 mg/kg; Abdel-Tawwab et al., 2007). However, it was greater than specified for rainbow trout (0.38 mg/kg; Hilton et al. (1980), channel catfish (0.25 mg/kg, Gatlin and Wilson, (1984), and groupers (0.77 mg/kg; Lin and Shiau, (2005). Higher growth was observed in fish at lower levels of supplemental selenium (T₁) and vice versa. However, Kim et al. (2003) reported that juvenile tilapia with selenium (0.2–0.5 mg/kg) did not display a substantial growth. Selenium has registered higher bioavailability when administered as Seleno-methionine rather than sodium selenite in catfish (Wang and Lovell, 1997) and Atlantic salmon (Lorentzen et al., 1994). The moderate concentrations of selenium have a beneficial effect on the development of common carp (Kallaya et al., 2011).

3.1.2 FCR, SGR and juvenile mortality

Better FCR was observed in T₁ (1.46 ± 0.23) and T₂ (1.59 ± 0.35), while SGR was the highest in T₁ (2.61 ± 0.071). SGR displayed non-significant variations among the higher selenium (>2 mg/kg) and selenium deficient treatments (Table 4). The juvenile tilapia did not show any mortalities during the entire study, indicating thereby no or sub-lethal toxicity of selenium during the investigation. However, T₄ FCR (1.67 ± 0.59) performed worse than T₂ (1.59 ± 0.35). This further cemented the essentiality of selenium to fish, while signifying the fine difference between selenium as growth enhancer and toxicant. Abdel-Tawwab and Wafeek (2010) reported 0.5 g OS/kg of fish diet (5.54 mg Se/kg) showed better FCR. The higher the FCR, the lower is the growth efficiency and inverse is the link between SGR and FCR. Hilton and Hodson (1983) reported that the growth rate reduced when rainbow trout (Salmo gairdneri) was fed on a higher selenium (10 mg/kg) dose. They also revealed an increased rate of renal calcinosis having potential influence on growth. Selenium, as a micronutrient in fish feed, is required approximately as 0.1–0.5 mg/g (Gatlin and Wilson, 1984). In fish, the signs of selenium deficiency display constricted growth, exudative diathesis, muscular dystrophy, increased death rates and higher incidence of anemia (Bell et al., 1985). The projected linear relationship revealed no or near-zero growth and length increments in T₄. Conversely, juvenile tilapia exhibited promising growth at a lower dose of selenium (2 mg Se/Kg). Therefore, juvenile tilapia growth showed indirect links between fish growth and selenium doses by confirming growth as inversely dosed-dependent. Paradoxically, the T₄ exhibited reasonable growth outputs as compared to T₂. This signified the toxic impact of higher selenium doses.

3.2.1 Amylase activity

Table 5 displayed the comparative variance between the amylases and lipases activity under the influence of selenium graded and selenium-deficient diets in juvenile tilapia. The amylase activity was recorded the highest in T₁ (2.41 ± 1.344), followed by T₂ (2.14 ± 1.5). Fig. 2 presents the monthly activity of the amylase in the juvenile tilapia. The linear projections indicated the highest activity was recorded in T₁ during third month. Conversely, in second and third months, the activity of amylase displayed a negative trend in T₂. However, it exhibited a positive or no trend in T₄, which established selenium vital at lower level and without external supplementation. However, higher (>2 mg/kg) selenium is detrimental to the regular activity of amylase. In fish, the difference of gut enzyme activity among fish species could be influenced by growth stage, feed composition and level of micronutrients in fish feeds (Kallaya et al., 2011). The activity of amylase is higher in liver than other organs, while protease and lipase are significantly higher in intestine. However, medium-sized fish displayed higher activity of lipases, while large-sized fish demonstrated higher activity of amylase (Peres et al., 1998).

Close

Fig. 2

Monthly variations in amylase activity and linear projections in juvenile tilapia based on supplemental selenium and selenium-deficient fish diets.

Export to PPT

3.2.2 Lipase activity

The highest lipase activity was observed in an inverse relationship with the selenium levels (Table 5). The highest observed activity was in the T₁ (107.66 ± 2.54) while the lowest in the T₄ (64.09 ± 1.17). A gradual decline in the lipase activity with the progressive increase in selenium-dose alluded to the adverse influence of higher selenium doses. Monthly lipase activity in juvenile tilapia was influenced by selenium level (Fig. 3). The highest lipase activity was observed in T₁, and the lowest T₃ and T₄. The decreasing trends in the lipase activity validated critical role of selenium in lipase activity.

Close

Fig. 3

Monthly variations in lipase activity and linear projections in juvenile tilapia based on supplemental selenium and selenium-deficient fish diets.

Export to PPT

Selenium is known for its potential role in enzyme activity manifested in enhanced growth and physiological functions. The lipase activity was higher in T₁ followed by T₂ and T₃. Lipase monthly activity revealed that it was maximum in T₁ with linear intensification. Although mild, but declining tendency in other treatments confirmed that higher selenium significantly reduced the lipase activity. Stable release of DE is directly associated with the health of gastrointestinal tract, which is linked to the presence of required microflora. The enzyme production is triggered by the presence of food (Hidalgo et al., 1999). Ramesh et al. (2014) investigated that enzymatic activity in Labeo rohita altered when exposed to sub-lethal (2.38 mg/L) concentration of sodium selenite. Our findings in T₂ also establish the limited activity of lipase like control (T₄) group. The pancreas drains several DE (lipase, amylase, trypsin, and chymotrypsin) into lumen of intestine. The action of Na + and K + ATPase further expands and generates Na + gradient driving the amino acids and vitamin into the cells. This step is extremely critical for the proper accomplishment of absorption of fluids from the intestine, which is mainly accomplished by the gut enzymes activity. Lipases participate in metabolism and transmission of various particles in organisms (Ramesh et al., 2014). They catalyze the hydrolysis of ester bonds in substrates, phospholipids, cholesteryl esters, and vitamin esters (Kurtovic et al., 2009).

The enhanced release of DE can improve digestibility and availability of nutrients from supplemented feedstuffs (Kallaya et al., 2011; Khan et al., 2018; Haider et al., 2018). The DE activity also corroborated with the growth indices in our study. In conclusion, better growth and elevated releases of the DE in the gut of juvenile tilapia have occurred in the fish fed on 2 mg/kg selenium. In contrast, generally, the enzymatic action follows the dose-dependent trend. As selenium has been reported as a paradoxical micronutrient (Lemly, 1993; Iqbal et al., 2020), it could illustrate better growth performances boosted by the elevated activity of gut enzymes. Our study advocates that if fed as appropriate or near to appropriate selenium dose to tilapia, it acts as an essential micronutrient. Better growth and higher activity of intestinal enzymes are inter-linked as well as to dependent on the adequate level of micronutrients like selenium.