http://dx.doi.org/10.11594/jtls.13.03.12
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Research Article
Amylase Activity in Various Digestive Organs and Blood Urea Levels of Osphronemus gouramy with Chlorella vulgaris Feed Supplementation and Reared in Different System
Sorta Basar Ida Simanjuntak *, Gratiana Ekaningsih Wijayanti, Maditaningtyas Hawwa Zuwanda, Elly Tuti Winarni
Biology Faculty, University of Jenderal Soedirman, 53122, Indonesia
Article history:
Submission February 2023 Revised July 2023 Accepted July 2023
ABSTRACT
Osphronemus gourami, a promising Indonesian fish, requires optimal nutritional feed and water quality in a culture system to ensure successful commercialization. How- ever, there is a lack of research on the effect of combining Chlorella vulgaris supple- mentation with biofloc and non-biofloc culture systems on amylase activity and blood urea levels in gourami fish. This study aims to investigate the impact of Chlorella vulgaris supplementation with different culture systems on the amylase activity of various digestive organs and blood urea levels in Osphronemus gourami. The experi- ment was conducted using a Factorial Completely Randomized Design with two fac- tors. Gourami fish were fed with C. vulgaris at levels of 0, 2, 3, 4, and 5 g.kg-1 for a period of 28 days, and they were divided into biofloc and non-biofloc groups for the culture systems. Amylase activity was measured using three pH buffers, and blood urea levels were analyzed using a urea kit. The results of the study demonstrated that amylase activity significantly increased when supplemented with C. vulgaris at a level of 5 g.kg-1 feed using the biofloc system, leading to a decrease in blood urea levels.
These findings suggest that C. vulgaris supplementation, combined with the biofloc system, can be an effective improvement. In conclusion, this research provides valu- able insights into the use of Chlorella vulgaris supplementation and the biofloc system as alternative strategies for enhancing the amylase activity of various digestive organs and reducing blood urea levels in O. gourami, thereby improving their overall nutri- tion and water quality.
Keywords: Amylase activity, Biofloc system, Blood urea, Chlorella vulgaris, Gourami
*Corresponding author:
E-mail: [email protected]
Introduction
The protein content in commercially farmed fish feed is one of the most expensive feed constit- uents. Feed protein content of 40% shows the highest growth performance and indicators of feed utilization. If the protein content is more than 40%, it will reduce growth. Feed protein content can modulate the activity of digestive enzymes in catfish [1]. According to Aslam et al. [2], amylase is a digestive enzyme in herbivorous fish with the highest activity, including in Gourami. Therefore, amylase activity is important for response-ability to digest carbohydrates. The digestibility and nu- tritional content are the determinants of feed qual- ity. It is necessary to increase feed nutrition
through supplementation, and various alternative materials have been used to supplement the nutri- tional content of the feed, one of which is micro- algae.
Chlorella vulgaris is a green alga with high nutritional value. It is used in the food, health, and aquaculture industries as a feed additive. The dry biomass weight contains protein (43-58%), lipids (5-58%), carbohydrates (12-55%), and pigment [3]. Gouveia et al. [4] reported that C. vulgaris is a rich source of carotenoids (astaxanthin, lutein, β- carotene, lycopene and cantaxanthin) about 0.4%
dry weight. C. vulgaris also contains trace ele- ments (selenium, magnesium, phosphorus, zinc,
calcium, and aluminum), and vitamins (ascorbic acid, thiamine, B1, B2, B6, D, E, and K) [5]. The supplementation can be an alternative solution for increasing nutritional value because it has high di- gestibility [6].
The accumulation of metabolic wastes and feed in fish culture systems can lead to elevated levels of ammonia, which negatively impacts wa- ter quality. Ammonia is known to induce environ- mental stress and can adversely affect fish metab- olism, causing tissue damage, particularly in the kidney [7]. Blood urea levels have been suggested as an indicator of fish stress, with studies indicat- ing that elevated blood urea levels are associated with stressful conditions. Therefore, in this study, blood urea levels were utilized as a parameter to assess the condition of fish in relation to water quality in both biofloc and non-biofloc systems [8].
Biofloc system is an aquaculture technology in water quality management that uses a population of heterotrophic bacteria by maintaining the C/N ratio. Heterotrophic bacteria can convert nitrogen waste into biomass (floc), used as natural food for fish [9]. Another advantage of the biofloc system is no water exchange, which can minimize costs and reduce wastewater discharge (effluent). The growth of heterotrophic bacteria is activated by adding daily rice bran and molasses to develop bi- ofloc and maintain heterotrophic bacteria [10].
Previous research of the biofloc system mainly focused on shrimp [10, 11, 12] and many species of fish, namely tilapia (Oreochromis niloticus) [13, 7], catfish (Clarias sp.) [14], and goldfish (Carassius auratus) [15]. However, there is only limited information about the biofloc system on Osphronemus gourami [16, 17]. Research on C.
vulgaris supplementation has been carried out on Gourami outside the biofloc system [18].
The present study aims to investigate the im- pact of Chlorella vulgaris supplementation on the amylase activity of different digestive organs and blood urea levels. By exploring the effects of C.
vulgaris supplementation in different culture sys- tems, this research sheds light on the intricate re- lationship between supplementation, amylase ac- tivity, and blood urea levels. The study aims to identify the optimal feed combination of C. vul- garis supplementation that enhances amylase ac- tivity in various digestive organs while reducing blood urea levels. Through this research, we strive to uncover valuable insights into the potential ben-
efits of C. vulgaris supplementation and its role in promoting digestive health and improving meta- bolic functions.
Material and Methods
The experimental research followed a Facto- rial Completely Randomized Design with a 5 x 2 factorial pattern. The first factor involved the sup- plementation of Chlorella sp. in the fish feed at five different levels: P0 represented the control group, where Gourami was fed commercial feed without any supplementation; P1, P2, P3, and P4 represented the Chlorella sp. supplementation lev- els of 2 g.kg-1, 3 g.kg-1, 4 g.kg-1, and 5 g.kg-1 of feed, respectively. The second factor focused on the culture system and consisted of two levels: B0 represented fish rearing without the biofloc sys- tem, and B1 represented fish rearing in the biofloc system. Each treatment was replicated four times, resulting in a total of 40 fiber aquariums. The pa- rameters measured included amylase activity in various digestive organs (expressed as U.mg-1 pro- tein) and blood urea levels (expressed as mg.dL-1).
The fish were monitored and treated over a dura- tion of 28 days.
Preparation of maintenance tanks and supple- mented feed
The maintenance was carried out in fiber tanks measuring 60 × 40 × 60 cm3, with an aeration and a recirculation system. Furthermore, fiber tanks are prepared for biofloc and non-biofloc systems.
In the biofloc system preparation, 10 mL of probi- otics EM4 and 40 mL of molasses were added and stirred for seven days using strong aeration. Fish was included to utilize the remaining feed and fe- ces as the source of N, and the floc density was measured using Imhoff Cone to ensure its for- mation. Environmental factors, such as tempera- ture, pH, and dissolved oxygen content, were rou- tinely controlled weekly to maintain optimal maintenance media conditions.
The feed supplementation with C. vulgaris was prepared following the methods described by Simanjuntak et al. [19]. A total of 2 g of dried Chlorella vulgaris was added to a glass beaker, followed by the addition of 100 mL of distilled water. The mixture was then stirred until it became homogeneous. Additionally, 1 kg of commercial pellets, Pf1000 combined with C. vulgaris solu- tion, was placed on a tray and flipped slowly until mixed homogeneous. Feeds supplemented with C.
vulgaris were dried in the sun and stored in a closed container. The same procedures were re- peated for C. vulgaris supplementation levels of 3 g.kg-1, 4 g.kg-1, and 5 g.kg-1 feed.
Feeding treatment
The gourami fish used were 13.06 ± 0.70 cm in total length and 32.37 ± 2.47 g in weight. The fish originated from Sidabowa, Patikraja Subdis- trict, Banyumas Regency, and were put into a fiber tank with seven individuals/tank density. Feeding was according to treatment with a feeding rate (FR) of 3% of the weight of fish and given twice a day for 28 days. Fish body weight measurements were carried out on day 14 for adjustments to feed.
Data retrieval
A sampling of amylase enzymes was con- ducted at the end of the experimental period. The activity was measured at three different buffer so- lutions, namely pH 5, 7, and 10 in the liver, stom- ach, and intestines. In the blood urea parameters, blood samples were sucked using a sterilized sy- ringe of 1 mL and placed in microtubes. Further- more, the serum was separated from each blood sample by centrifuge at 4,000 g for 20 minutes, and blood serum was frozen at -20°C. Blood urea was measured using the procedure from the urea analysis kit (Urea FS-Diasys: 1 3101 99 10 021).
Preparation of the enzyme extracts
The digestive organs isolated from Gourami were crushed using an electric homogenizer in 50 mMTris-HCl buffer (pH 7.2-8.0) cold with a 1:10 (w/v) ratio. The homogenate was centrifuged (CT 15 E, himac, Hitachi, Japan) at 12,000 g for 10 minutes, and the supernatant obtained was used as an enzyme extract to test the amylase activity. Fur- thermore, the Lowry method measured the super- natant protein [20]. Protein supernatant was calcu- lated using a standard albumin curve with a con- centration of 0.50, 1.00, 2.00, 4.00, and 8.00 mg mL-1. The amylase activity test was according to the method of Summer (1921) as described by Rick and Stegbauer [21], using a reaction mixture of 1% (w/v) starch, phosphate buffer, and enzyme extract. The reaction was stopped with 1% di- nitrosalicylic acid (DNS), which detected the re- ducing sugars produced by the action of hydrolase enzymes on carbohydrates. Amylase activity was calculated using a standard maltose curve with a concentration of 1.00, 2.00, 4.00, 8.00, and 16.00
μmol mL-1. It was expressed as units of maltose produced (µmol) in the hydrolysis reaction of car- bohydrates per minute of incubation time per mg supernatant protein.
Data analysis
Data on blood urea levels and amylase activity in the liver, stomach, and intestine were analyzed using analysis of variance (ANOVA) with a con- fidence level of 95%, and the Duncan test fol- lowed the significant results.
Results and Discussion
Amylase activity in various digestive organs of Osphronemus gouramy
The results of the measurement of amylase ac- tivity in various digestive organs of Gourami with different levels of Chlorella vulgaris supplemen- tation are presented in Figures 1, 2, and 3. The pH value reflects the optimal environmental condi- tions for amylase activity in each organ. The find- ings indicated that the highest amylase activity was observed at pH 7, while it decreased at pH 5 and 10. Amylase activity in buffer solution with pH 5 was found in the liver, stomach, and intes- tines. It was 0.016 – 0.296 U.mg-1, 0.000 – 0.140 U.mg-1, 0.011 – 0.154 U.mg-1, 0.000 – 0.192 U.mg-1, and 0.007 – 0.169 U.mg-1 protein in the liver, stomach, anterior intestine, middle intestine, and posterior intestine, respectively (Figure 1).
These results show that amylase activity was still found at very low levels under an acidic buffer so- lution.
There was no interaction between C. vulgaris supplementation with the culture system on amyl- ase activity as measured at pH 5, but it was signif- icantly different in each treatment factor. These re- sults showed that amylase activity on C. vulgaris supplementation levels 3 g.kg-1, 4 g.kg-1, and 5 g.kg-1 feed differed between the levels 0 g.kg-1 and 2 g.kg-1 in the liver and middle intestine. The in- creasing activity was due to high carbohydrate content, which became the amylase substrate. It is presumed that the acid pH affects the enzyme, and the measurement is not optimum. These results are consistent with the research of Gioda et al. [22] in some freshwater fish species where amylase activ- ity decreased at acidic pH 2-5 compared to neutral pH 7.
The results of a buffer solution with pH 7 in- dicated amylase activity in the liver, stomach, and intestines, as seen in Figure 2. The activity was
higher at pH seven compared to 5 and 10, it works optimally in a neutral buffer solution. At buffer pH 7, the activity in the liver, stomach, anterior intes- tine, middle intestine, and posterior intestine was 0.303 – 0.739 U.mg-1, 0.011 – 0.416 U.mg-1, 0.057 – 1.030 U.mg-1, 0.095 – 0.637 U.mg-1, and 0.022 – 0.410 U.mg-1 protein. Additionally, measurement at pH 7 is optimum for amylase activity because it has the same conditions in the body. This is sup- ported by the findings of Zhang et al. [23], who reported that pH values in the gastrointestinal tract of fish ranged from 6.38 to 7.82. Furthermore, Solovyev et al. [24] stated that the optimal pH for amylase activity varies among fish species but generally falls within the neutral pH range of 6.5- 8.0. Additionally, Gioda et al. [22] observed that the highest activity of amylase in Ctenopharyngo- don idella (herbivorous) and Leporinus obtusidens (omnivorous) fish occurred at pH 7.
Based on ANOVA, amylase activity was sig- nificantly different between the supplementation treatments and the culture system in the anterior
intestine. The interaction of C. vulgaris supple- mentation with the culture system affected the am- ylase activity at the optimum pH. The results of further tests indicated amylase activity, and the highest Gourami was found in the anterior intes- tine of level 5 g.kg-1 feed in the biofloc system 1.030 U.mg-1 protein. This was significantly dif- ferent compared to others in the non-biofloc sys- tem. Supplementation of C.vulgaris 5 g.kg-1 feed in the biofloc system improved feed nutrition, es- pecially carbohydrates as a substrate in the hydrol- ysis reaction of starch by amylase. The biofloc system provides water quality to support various metabolic processes, including digestive activi- ties. According to Lundstedt et al. [25], a factor that can increase amylase activity is substrate con- centration. Azhari et al. [26] further emphasized that maintaining good water quality is crucial for supporting the overall health and optimal meta- bolic processes of fish. Previous research by Khani et al. [27] found that supplementation of C.
vulgaris 5 g/kg increases amylase activity in the Figure 1. Amylase activity of O. gouramy at buffer solution with pH 5. Note: P0 = C. vulgaris supplementation
level of 0 g.kg-1 feed, P1 = C. vulgaris supplementation level of 2 g.kg-1 feed, P2 = C. vulgaris supple- mentation level of 3 g.kg-1 feed, P3 = C. vulgaris supplementation level of 4 g.kg-1 feed, P4= C. vulgaris supplementation level of 5 g.kg-1 feed. Values in the same organ with a different letter show significant differences between treatments (Mean ± SD, n =P < 0.05).
hepatopancreas and intestines of koi fish (Cypri- nus carpio). Xu et al. [28] also showed that Chlo- rella supplementation increases in the hepatopan- creas and intestines of Carassius auratus gibelio.
Meanwhile, Long et al. [13] found that the amyl- ase activity in the intestines of tilapia (Oreo- chromis niloticus) was significantly higher in the biofloc system compared to the control.
The measurement results using an alkaline buffer solution revealed suboptimal amylase activ- ity, with lower levels observed at pH values below 5. Notably, the activity was observed in the liver and intestines but not in the stomach. At a buffer pH of 10 (Figure 3), the activities recorded in the liver, anterior intestine, middle intestine, and pos- terior intestine ranged from 0.000 to 0.092 U.mg-
1, 0.000 to 0.070 U.mg-1, 0.000 to 0.046 U.mg-1, and 0.000 to 0.019 U.mg-1 protein, respectively.
These findings align with the research conducted
by Gioda et al. [22], which indicated a decrease in amylase activity at alkaline pH levels, particularly at pH 10 compared to neutral pH conditions.
Based ANOVA, there was no interaction bet- ween C. vulgaris supplementation with the culture system as measured at pH 10 for each treatment factor. These results indicate that the amylase ac- tivity is not significantly different between C. vul- garis supplementation and culture systems in al- kaline pH. Almost all organs in the C. vulgaris supplementation level 0 g.kg-1 feed did not find any amylase activity. The supplementation of C.
vulgaris, particularly at levels of 4 g.kg-1 and 5 g.kg-1 of feed, exhibited notable amylase activity in the liver and intestines. These findings align with the research conducted by Yunida et al. [29], who also observed amylase activity in the liver and intestines at pH 10.
Furthermore, significant differences were ob- Figure 2. Amylase activity of O. gouramy at buffer solution with pH 7. Note: P0 represents C. vulgaris supple-
mentation level of 0 g.kg-1 feed, P1 represents C. vulgaris supplementation level of 2 g.kg-1 feed, P2 represents C.a vulgaris supplementation level of 3 g.kg-1 feed, P3 represents C. vulgaris supplementa- tion level of 4 g.kg-1 feed, and P4 represents C. vulgaris supplementation level of 5 g.kg-1 feed. Values within the same organ with different letters indicate significant differences between treatments (Mean ± SD, n = P < 0.05).
Figure 3. Amylase activity of O. gouramy at buffer solution with pH 10. Note: P0 represents C. vulgaris sup- plementation level of 0 g.kg-1 feed, P1 represents C. vulgaris supplementation level of 2 g.kg-1 feed, P2 represents C. vulgaris supplementation level of 3 g.kg-1 feed, P3 represents C.a vulgaris supple- mentation level of 4 g.kg-1 feed, and P4 represents C. vulgaris supplementation level of 5 g.kg-1 feed.
Values within the same organ with different letters indicate significant differences between treat- ments (Mean ± SD, n = P<0.05). Amylase activity in the stomach of fish reared in both the biofloc and non-biofloc systems was not detected.
served in the study conducted by Long et al. [13], where the biofloc treatment resulted in signifi- cantly higher amylase activity in the intestines.
The amylase activity in the intestines was higher because carbohydrate digestion occurred in the in- testines, while the stomach was more involved in digesting protein. Previous research by Wu et al.
[30] also indicated that the activity in Boleophthal- mus pectinorostris (herbivorous fish) is higher in the intestines compared to the stomach.
Blood urea levels of O. gouramy
Measurement results of blood urea levels of Gourami with C. vulgaris supplementation in dif- ferent culture systems are presented in Figure 4.
Gourami fish reared in the biofloc system had lower blood urea than in the non-biofloc at all lev- els of C. vulgaris supplementation.
Based on analysis of variance (ANOVA), there was no interaction between C. vulgaris supple- mentation with the culture system on blood urea levels of Gourami. The biofloc system resulted in better water conditions, as reflected in the blood urea levels of fish which were significantly lower than those in non-biofloc systems. Meanwhile, fish in non-biofloc systems had higher blood urea levels. Poor water quality is due to high ammonia, causing fish stress and higher blood urea levels.
Blood urea levels of Gourami in the biofloc system were lower than in the non-biofloc, presumably due to the conversion of ammonia waste which improved water quality and reduced the fish stress condition. This is consistent with the research of Ahmed et al. [7] that tilapia (O.
niloticus) treated with the biofloc system had lower blood urea levels at 26.5 – 38 mg.dL-1 than those without the biofloc at 38 – 85 mg.dL-1. The research also proved a decrease in ammonia levels from 9.6 mg.L-1 to 0.7 mg.L-1 in the biofloc system treatment. Therefore, the application of the biofloc system can improve water quality by controlling ammonia levels.
According to Choudhury et al. [8], high levels of ammonia cause stress that induces blood urea levels in Labeo rohita. Fish become stressed in poor aquatic environmental conditions and even cause tissue damage. Tayel et al. [31] reported that an increase in blood urea levels might come from kidney damage caused by poor water quality, such as the toxicity of ammonia waste. Kidney is one of the main detoxification organs in the fish body and the most affected by changes in water quality. Ac- cording to Ajeniyi et al. [32], urea is a byproduct generated by the liver during the conversion of protein into nitrogen. It is transported through the blood vessels to the kidneys, where the blood, con- taining a minimal concentration of urea, under- goes filtration.
Conclusion
The findings of this study demonstrate that the combination of C. vulgaris supplementation with the biofloc system enhances the amylase activity in the digestive organs of Gourami. Additionally, the biofloc system proves effective in reducing ammonia levels and improving the overall condi- tion of the fish. The supplementation of C. vul- Figure 4. Comparison of blood urea levels in Osphronemus gouramy between the biofloc system and the non-
biofloc system. Values with different letters indicate significant differences between treatments (Mean
± SD, n = P<0.05).
a
b
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00
Biofloc Non-biofloc
Blood urea levels (mg.L-1)
Culture systems
garis in gourami feed, along with the implementa- tion of the biofloc system, shows promise in en- hancing nutrition and water quality in fish culture.
These results indicate that the inclusion of C. vul- garis in the gourami diet, coupled with the adop- tion of the biofloc system, has a positive impact on the amylase activity in various digestive organs.
Moreover, the utilization of the biofloc system leads to a reduction in blood urea levels, further contributing to the overall health and well-being of the fish.
Acknowledgment
This research was funded by Jenderal Su- dirman University (BLU Unsoed) for the Higher Education Excellence Research (Development) scheme for the Fiscal year 2021. The authors are grateful to the Chancellor for the research funding assistance. The authors also thank Rizka Yunida and Ilham Semaruci for their help.
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