Research Article

Growth Performance, Biochemical, Antioxidant Status and Histopathology Changes of Clarias Gariepinus, Raised in a Biofloc Medium Utilizing Natural Carbon Sources

Ikele*, Chika Bright, Ujunwa Aghaji, Mgbenka, Benard Obialo

Department of Zoology and Environmental Biology, University of Nigeria, Nsukka.

Received | January 22, 2022; Accepted | March 01, 2022; Published | June 01, 2022

*Correspondence | Ikele, Department of Zoology and Environmental Biology, University of Nigeria, Nsukka; Email: chika.ikele@unn.edu.ng

Citation | Ikele, Bright C, Aghaji U, Mgbenka, Obialo B (2022). Growth performance, biochemical, antioxidant status and histopathology changes of clarias gariepinus, raised in a biofloc medium utilizing natural carbon sources. J. Anim. Health Prod. 10(2): 168-182.

DOI | http://dx.doi.org/10.17582/journal.jahp/2022/10.2.168.182

ISSN | 2308-2801

 

 

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Aquaculture has persistently developed globally under best practices. A good health status from the function point of view is preponderant for both cultured and wild fish species in early development (Mota et al., 2019). Poor health precludes the reduced exponent of the fish to perform its physiological functions, conform to stressful conditions, and prevent disease (Caipang et al., 2015). Lofty intake of protein above 25% by both fin and shellfish is remarkably excreted as ammonia-nitrogen and influence the quality of water. When inorganic nitrogen, total suspended solids (TSS) are high in concentration in culture water, it leads to death of culture species, diseases, accumulation of harmful residues in culture systems (Karunaarachchi et al., 2018), and obstruction of the growth of global aquaculture market (Caipang et al., 2015). Enforcement of agro-derived resources and aggrandize natural food sources towards intensifying aquaculture systems (Azim and Little, 2006) is preeminent. To promote efficient fish production, recycling aquaculture system though expensive to maintain, is most preferable. Therefore, it is apropos to utilize non-expendable ecological clues to resolve the problems associated with cost, poor fish production, etc. However, inappropriate husbandry conditions such as poor water quality, crowding leading to accentuated social interactions between similar schemes present in the culture strategies are a potential threat to animal health/welfare.

Therefore, biofloc technology (BFT) provides an alternate solution as a consequence of practicing zero-water exchange, waste nutrient recycling (Bossier and Ekasari, 2017) regulating water qualities by microbial communities, promoting fish growth (Hamidaghli et al., 2019; Mota et al., 2019; Lima et al., 2018; Zapata-Lovera et al., 2017, Yun et al., 2016, Avnimelech, 2007) with reduced environmental impact (Bossier and Ekasari, 2017) for sustainable aquaculture. It is a more beneficial system when heterotrophic bacteria, a nitrogen conversion agent is stimulated by adjusting the C:N ratio in the culture system by the addition of carbonaceous plant-based material or equivalent inorganic carbons for sustainable fish production to achieve sustainable development goals (Bossier and Ekasari, 2017). It has also offered practical solutions to effective water quality control (Panigrahi et al., 2017; Avnimelech, 2007) and improvement of fish growth in the healthy culture system (Lananan et al., 2014). In recent times, the technology is considered best back-up towards increased protein rich food. (Karunaarachchi et al., 2018). Proportionate carbon source agents utilized to intensify profitable microbial production as in aquaculture systems includes; yucca flour (Castro et al., 2018), dextrin (Hamidoghli et al., 2019, molasses (Bakhshi et al., 2018, Correia et al., 2010; Crab et al., 2010), glucose (Caipang et al., 2015; Ekasari et al., 2010), and plant-based, corn starch (Bakhshi et al., 2018), wheat flour (Caipang et al., 2015), sweet potato flour (Caipang et al., 2015), broken wheat and rice grain (Zaki et al., 2020), etc. There is a growing interest due to attributes associated to the choice of natural carbonaceous substrate for bioflocculation which includes; availability, low cost, solubility, and biodegradable as a replacement for protein utilized by fish. Organic and inorganic carbon sources are the key to the composition, formation, and nutriment values of bioflocs such as vitamin C, carotenoid, fatty acids, and trace minerals (Bossier and Ekasari, 2017). Considering the high stocking density, increased feed input utilized in rearing C. gariepinus in intensive aquaculture system, with a negative impact on water quality, it is imperative to adopt a better water quality control strategy in production systems (Popoola and Miracle, 2022). The bioflocculation is determined by the adjusted C:N ratio and qualities of substrate to correspond to fish species (Nguyen and Ly, 2018). Manipulation of the C:N ratio above 20 can promote the formation of microbial aggregates (protozoa, zooplankton, phytoplankton, autotrophic/heterotrophic bacteria), and yeast (Mato et al., 2019). Popoola and Miracle (2022) reported positive welfare of C. gariepinus raised in a biofloc for 72 days. CN 14:1 and CN 30:1 produced beneficial microbial floc for the growth of Litopenaeus vannamei (Silva et al., 2017). C:N ratio of 10 is beneficial for Clarias gariepinus farming, while C:N ratio below 10 (Liu et al., 2018) is essential in profit-making feeds but requires additional carbon for bioflocculation. There is need to develop more literatures on the effect of BFTs in rearing African Catfish considering its high values and possible acceptability among both fish farmers and consumers. However, the efficacy of different carbon sources (local cassava variety and commercial wheat flour) with constant availability on the growth performance, biochemicals, antioxidant activities and histological changes of African catfish, Clarias gariepinus raised in biofloc tank by steering different C/N ratios were evaluated.

MATERIALS AND METHODS

Experimental Fish

Two hundred and seventy healthy C. gariepinus fingerlings of mean weight 1.24±0.02g and length, 3.72±0.01 cm were procured from the catfish multiplication center, Aquafish Nigerian Limited, Awka, Nigeria. The fish were transported to the Department of Zoology and Environmental Biology, University of Nigeria Nsukka. The fish was acclimatized for two (2) weeks before the commencement of the study in 30 L tap water ((NO3- 0.91, Cl- 7.4, SO42- 15.2, Ca 2+ 4.01, total phosphorus 0.03 and mg2+ 9.72, Na+ 4.7, pH, 6.9, DO, 4.2) and was changed daily during acclimatization.

Procurement and Processing of Local Carbon Sources

5kg local variety cassava Manihot esculenta and 5kg wheat flour were both obtained from Ogige Market, Enugu State, Nigeria due to their biodegradable potential and availability to farmers. The cassava was peeled, washed, chopped (4cm long, 2mm thick), and dried in a Heraeus vacutherm oven at 40°C and ground into powder.

Experimental Design

A total of Two hundred and seventy juveniles C. gariepinus fingerlings were randomly distributed into 6 plastics tanks (54x54x67cm) (A-F) triplicated to 18 tanks at an initial density of 15 fish per tank. The experimental design adopted a complete randomized design. The C:N ratios of the carbohydrates (cassava and wheat) sources in the culture system was adjusted (CN 10:1 and CN 20:1), respectively, and evaluated following the protocol of Avnimelech (2007) and Piedrahita (2003). Group A (CE) and B (NE) were maintained as controls with inoculum + no carbon addition, and no inoculum + no carbon addition, respectively with 50% water exchange every week. Two different natural carbon sources, were maintained at two different C:N ratio 10 and 20. Group C cassava (CA 10:1 + inoculum), group D cassava (CA 20:1, inoculum), Group E wheat flour (WH 10:1 + inoculum) and group F wheat flour (WH 20:1 + inoculums) while water was added when necessary to make up for loss caused by evaporation during aeration. The plastic tanks were sealed up using mosquito nets (0.024 inches) to prevent drosophila entrance and constantly aerated using 45L/min 200 W electromagnetic aquarium oxygen pump, model ACO-010, China) with air blowers distributed in all the tanks. Aeration was supported by using petroleum motor spirit (PMS) generator (Elemaq 3600) due to power failure. The plastic tank was maintained at 30 L volume of tap water (temperature 24.4° C to 25.1°C, pH 6.7-6.9). The experiment lasted for 28 days, but prior to the growth yield determination, the fish were starved overnight.

Inoculum Preparation, Biofloc, water quality and growth yield

Natural inoculum consisted of 200ml of Chlorella lewinni (cultivated in BG-11 medium in g/L of distilled water (pH 6.9) NaNO3 1.5, K3HPO4.3H20 0.04, CaCL2.H20 0.036, MgSO4.7H20, Na2CO3 0.02, citric acid 0.006, C6H807 Fe3, NH3,0.006, Na2 EDTA 0.001 and 1.0 ml trace metal solution (H3BO3 2.860, ZnSO4.7H2O 0.222, MnCl2.4H20 1.8, CuSO4.5H2O 0.079, Na MOO4.2H20 0.390 and C0(NO3)2.6H2O 0.0494) was prepared and supplemented with 30ml of filtered algae concentration obtained from pond water (pH, 5.5, TAN, 0.67, Temperature, 24.7°C, TDS 207 mgL-1, dissolved oxygen 5.26 mgL-1), put into each biofloc tank with 30 L of tap water.

Cassava and wheat flour were added together with coppens feed (45% crude protein) to support the build-up of the microbial community against the nitrogenous waste product. In consequence, CN 10:1 and CN 20:1 gave 1.08g and 2.16g, cassava, and wheat, respectively, and administered to induce heterotrophic medium for optimum biofloc production.

The floc volumes (ml) were obtained and measured weekly using improvised imhoff cone (seven observations) for 28 days. A 100 ml of heterogeneous aggregates were obtained at intervals, put in an improvised Imhoff cone, and flocculated for 10 minutes. The presence of floccules was observed and obtained by opening the central point of the improvised imhoff cone. Re-suspension of particles due to gas formation was avoided. The harvested flocs were measured in triplicates in a 100 ml measuring cylinder, to obtain the floc volume. The harvested flocs were examined and observed under an optika binocular microscope using X40 objectives.

Physicochemical parameters such as dissolved oxygen (mg/L), temperature (°C), pH, nitrite (mg/L), nitrate (mg/L), total suspended solids (mg/L), and total ammonia nitrogen (mg/L) were measured daily between 9:00 and 11:00 hours following the protocol of (APHA, 1995) throughout the experiment.

The growth parameters were determined throughout the experiment, by the measurement of both initial and final body weight (g) and length (cm) of fish. Meanwhile, the growth rate (%), length gain, percentage length gain (%), weight gain was determined. Other growth parameter includes;

WG=

Logarithmic transformation of length-weight relationship was determined

Log W=log a + b log L

Where W=weight of the fish (g)

L=length of the fish (g)

a=constant

b=exponent

Specific growth rate was determined in percentage;

Fulton Condition factor was expressed in percentage as K=

Survival rate was expressed in percentage (%) as;

X100

Heterotrophic Bacteria

Biofloc samples from each bioreactor were taken and approximately 1-2g flocs (net weight equivalent) were suspended in 10ml sterile saline solution (0.85% NaCl) and peptone water (Merck). After the preparation of ten-fold serial dilution (1ml of sample in 9 ml of diluent), 0.1 ml of the dilution was spread on the surface of sterile Petri dishes containing Tryptone Soy Agar (TSA) and De Mann-Rogosa and Sharpe Agar (MRS) in triplicate. Inoculated plates were incubated at 30ºC for 48h aerobically and anaerobically for TSA and MRS cultures, respectively. Bacterial colonies formed on the incubated plates were counted and calculated against the cultured volume and dilution factor to get the colony forming units per ml (cfu/ml) thus;

C = n

VD

Where:

C = concentration in cfu/ml

n = number of colonies formed on culture plate

V = volume cultured

D = Dilution factor.

Total heterotrophic bacteria (THB count) expressed as colony-forming units (CFU) were observed within 21 days

Biochemicals

Aspartate aminotransferase and alanine aminotransferase Alkaline phosphatase, creatinine, Urea and total protein activities in the serum were assayed following the protocol of Reitman and Frankel, (1957) using randox kit.

Antioxidants

Antioxidants activities in the homogenate of fish gills (n=3) were used to assay for Glutathione peroxidase (GPx) (Lawrence and Buck, 1976) expressed as u/mg protein. Lipid peroxidation (TBARS) expressed as nanomoles of TBARS formed/mg protein (Sharma and Krishna-Murti, 1968). Catalase activity (CAT) assay (Aebi, 1984), and superoxide dismutase activity (SOD) in the gill tissue was measured based on the auto-oxidation of adrenalin due to the presence of superoxide anion (Misra and Fridovich, 1972).

Proximate composition of harvested flocs

The proximate composition of the flocs in triplicates was measured following the protocol of AOAC (1989).

Histopathology

The histological procedure of liver sections was carried out following the protocol of Bancroft and Cook (1994).

Statistical Analysis

Data were analyzed for significant differences (p<0.05) using analysis of variance (ANOVA). Also, All analyses were done using Statistical Package for Social Sciences windows version 20.0 (IBM Corporation, Armonk, USA).

RESULTS

Physicochemical

The simultaneous increase in temperature in CE and NE followed a clear-cut trend within 21 days. In all the setups, the temperature changes differed significantly at p<0.05 following the trend day 21>day 28>day14> day 7> baseline, respectively. The fluctuated pH varied between groups, especially in CA 10:1 and WH 20:1. The pH values significantly (p<0.05) increased in CA 10:1, WH 10:1, and WH 20:1 on day 14, followed by CE and CA 20:1. The dissolved oxygen levels maintained a static concentration throughout the study period, with a bit of variation in values in CA 10:1. Total suspended solids fluctuated among the groups, and 324.67mg/L peaked at WH 10:1. Inorganic nitrogen TAN concentration in the biofloc tanks depended on the components that produced the flocs duration-dependent. Generally, no significant difference was observed in the TAN in all BFTs groups. Irrespective of minor fluctuations in TAN, 0.31mg/L TAN was reached. Changes in nitrogen, nitrate, nitrite, and phosphate depended on the study duration and medium. No clear cut trend was observed in the variation of inorganic parameters (Table 1). The observed physicochemical parameters such as temperature, pH, and dissolved oxygen were still within the normal range for the rearing of C. gariepinus.

Growth Performance and Survival of C. gariepinus reared in Biofloc

The growth parameters of C. gariepinus to conditions of BFTs at the end of the experiment are presented in Table 2. The difference between weight gain, growth rate, specific growth rate, and the length-weight was insignificant (p>0.05). The growth performance normalized in the BFTs group at the end of the experiment with a significant condition factor of 0.88 observed in group B. At the end of the experiment, the length-weight relationship showed ‘b’ < 1 in all the groups (Table 3). The ‘b’ value of C. gariepinus ranged from 0.210 to 0.2597 throughout the study. The best relationship in the length-weight was observed in group C (log W=0.2101L0.7873, a, 6.128, b, 0.210, R=0.873, R2= 0.762, p<0.001) (Table 3). The peak growth rate, the percentage weight gain (214±26.80), the specific growth rate (3.78±0.26) of C. gariepinus showed significantly higher values among the treatment (p<0.05) compared to CA and NE.

Survival was not 100% in any of the biofloc tanks. Mortality was recorded in groups that provided carbon sources compared to non-carbon sources (CE and NE). The survival percentage was most minuscule in NE and highest in WH 20:1. Similar magnitudes of survival were observed in WH 10:1 and CA 10:1 (Table 2). The survival differentials between groups ranged from 28.25-60.4% (CE) and 53.25-85.64 (NE). There was a significant difference in the survival of C. gariepinus in the BFTs groups (F=14.982, p<0.001).

Antioxidants

The antioxidant markers, catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) activities in the gills of C. gariepinus were dependent on the biofloc medium and duration of the study. A significant variation in CAT, SOD, and GPx activities were noticed on day 14 between the groups, while MDA activities were substantial (p<0.05) on day 21. Also, CA 20:1 and WH 10:1 increased CAT activities (P<0.05). The activities of SOD were not significant in all the groups, but the GPx activities in the gill changed significantly in CA 10:1, CA 20:1, WH 10:1, and WH 20:1 throughout the study compared to the baseline. Lipid peroxidation activities observed in the fish exposed to WH 20:1 declined in concentration; the disparate was insignificant to CA

 

Table 1: Physicochemical parameters of the culture system

Parameter

Groups

Baseline

Day 7

Day 14

Day 21

Day 28

Temperature (°C)

CE

25.30 ± 0.00a1

25.80 ± 0.00a2

26.00 ± 0.00b3

27.07 ± 0.03b5

26.70 ± 0.00a4

NE

25.30 ± 0.00a1

25.70 ± 0.00a2

26.07 ± 0.03ab3

27.13 ± 0.035ab5

26.70 ± 0.00a4

CA 10:1

25.30 ± 0.00a1

25.77 ± 0.03a2

26.07 ± 0.00ab3

27.17 ± 0.03a5

26.67 ± 0.17a4

CA 20:1

25.30 ± 0.00a1

25.83 ± 0.09a2

26.07 ± 0.03ab3

27.20 ± 0.00a5

26.50 ± 0.06a4

WH 10:1

25.30 ± 0.00a1

25.77 ± 0.03a2

26.10 ± 0.00a3

27.17 ± 0.03a5

26.63 ± 0.07a4

WH 20:1

25.30 ± 0.00a1

25.80 ± 0.00a2

26.07 ± 0.03ab3

27.20 ± 0.00a5

26.70 ± 0.00a4

pH

CE

8.00 ± 0.90a1

7.40 ± 0.10a1

7.10 ± 0.06c1

7.37 ± 0.03a1

7.37 ± 0.03a1

NE

8.00 ± 0.90a1

7.27 ± 0.09a1

7.57 ± 0.03a1

7.47 ± 0.12a1

7.37 ± 0.03a1

CA 10:1

7.73 ± 0.09a2

7.43 ± 0.09a1

7.37 ± 0.03b1

7.43 ± 0.07a1

7.53 ± 0.03a12

CA 20:1

7.73 ± 0.09a2

7.50 ± 0.10a12

7.10 ± 0.06c1

7.43 ± 0.09a12

7.27 ± 0.07a1

WH 10:1

7.73 ± 0.09a2

7.43 ± 0.07a1

7.36 ± 0.03b1

7.53 ± 0.19a1

7.30 ± 0.10a1

WH 20:1

7.73 ± 0.09a2

7.37 ± 0.03a1

7.27 ± 0.06b1

7.50 ± 0.21a1

7.37 ± 0.18a1

DO (mgL-1)

CE

5.45 ± 0.09a1

5.37 ± 0.18a1

5.57 ± 0.18a1

4.93 ± 0.18b1

5.43 ± 0.52a1

NE

5.45 ± 0.09a1

5.73 ± 0.38a1

5.33 ± 0.09a1

4.97 ± 0.26ab1

6.00 ± 0.37a2

CA 10:1

5.55 ± 0.09a12

5.77 ± 0.15a2

5.80 ± 0.31a2

5.07 ± 0.03ab1

5.87 ± 0.15a2

CA 20:1

5.55 ± 0.09a1

5.53 ± 0.23a1

5.40 ± 0.17a1

5.30 ± 0.26ab1

5.67 ± 0.15a1

WH 10:1

5.55 ± 0.09a1

5.73 ± 0.49a1

5.27 ± 0.20a1

5.53 ± 0.32ab1

5.13 ± 0.20a1

WH 20:1

5.55 ± 0.09a1

5.83 ± 0.24a1

5.63 ± 0.32a1

5.73 ± 0.24a1

5.43 ± 0.35a1

TSS (mgL-1)

CE

311.50 ± 12.99a1

222.00 ± 84.01ab1

156.00 ± 103.71a1

166.67 ± 38.44a1

196.67 ± 39.23a1

NE

311.50 ± 12.99a1

194.67 ± 96.45ab1

294.67 ± 14.71a1

220.67 ± 40.70a1

154.70 ± 45.19a1

CA 10:1

270.50 ± 6.63b2

144.67 ± 43.46ab1

280.67 ± 40.83a2

193.33 ± 23.22a12

197.33 ± 24.34a12

CA 20:1

270.50 ± 6.63b2

111.33 ± 21.46b1

270.67 ± 46.34a2

269.33 ± 38.68a2

108.73 ± 81.83a1

WH 10:1

270.50 ± 6.63b2

240.00 ± 23.86ab2

167.43 ± 68.49a2

346.67 ± 12.72b1

188.67 ± 44.13a2

WH 20:1

270.50 ± 6.63b2

324.67 ± 13.68a2

226.00 ± 58.41a12

177.33 ± 25.57a1

197.67 ± 17.32a1

TAN (mgL-1)

CE

0.22 ± 0.00a1

0.23 ± 0.01a1

0.23 ± 0.01b1

0.24 ± 0.00a1

0.24 ± 0.01ab1

NE

0.22 ± 0.00a1

0.24 ± 0.00a12

0.27 ± 0.02ab2

0.26 ± 0.02a12

0.24 ± 0.02ab12

CA 10:1

0.23 ± 0.00a1

0.24 ± 0.02a2

0.26 ± 0.01ab12

0.24 ± 0.00a12

0.24 ± 0.00ab12

CA 20:1

0.23 ± 0.00a1

0.26 ± 0.02a2

0.26 ± 0.03ab1

0.26 ± 0.03a1

0.23 ± 0.01b1

WH 10:1

0.23 ± 0.00a1

0.26 ± 0.02a1

0.31 ± 0.03a1

0.23 ± 0.01a1

0.26 ± 0.04ab1

WH 20:1

0.23 ± 0.00a1

0.27 ± 0.02a12

0.27 ± 0.01ab12

0.25 ± 0.02a12

0.30 ± 0.01a2

Nitrite (mgL-1)

CE

1.83 ± 0.34a2

1.40 ± 0.10b12

1.21 ± 0.09c1

1.39 ± 0.12a12

1.28 ± 0.03a12

NE

1.83 ± 0.34a2

1.23 ± 0.06b1

1.41 ± 0.09c12

1.36 ± 0.08a12

1.31 ± 0.07a12

CA 10:1

1.19 ± 0.03a1

1.78 ± 0.16a2

1.45 ± 0.09c12

1.38 ± 0.03a1

1.50 ± 0.15a12

CA 20:1

1.19 ± 0.03a1

1.52 ± 0.13ab1

1.91 ± 0.23ab2

1.33 ± 0.04a1

1.30 ± 0.08a1

WH 10:1

1.19 ± 0.03a1

1.27 ± 0.09b1

1.55 ± 0.14bc1

1.60 ± 0.21a1

1.34 ± 0.13a1

WH 20:1

1.19 ± 0.03a1

1.47 ± 0.08ab12

2.27 ± 0.14a2

1.45 ± 0.57a12

1.52 ± 0.03a12

Nitrate (mgL-1)

CE

0.15 ± 0.00a1

0.15 ± 0.01a1

0.14 ± 0.01c1

0.20 ± 0.04a1

0.13 ± 0.01a1

NE

0.15 ± 0.00a1

0.14 ± 0.00a1

0.14 ± 0.01c1

0.16 ± 0.01a1

0.13 ± 0.01a1

CA 10:1

0.14 ± 0.00b1

0.18 ± 0.06a1

0.16 ± 0.01c1

0.12 ± 0.01a1

0.17 ± 0.03a1

CA 20:1

0.14 ± 0.00b1

0.20 ± 0.02a2

0.20 ± 0.01ab2

0.32 ± 0.17a12

0.17 ± 0.02a12

WH 10:1

0.14 ± 0.00b1

0.17 ± 0.01a1

0.18 ± 0.03abc1

0.15 ± 0.03a1

0.15 ± 0.03a1

WH 20:1

0.14 ± 0.00b1

0.21 ± 0.03a12

0.23 ± 0.02a2

0.15 ± 0.02a12

0.20 ± 0.02a12

Values as mean ± S.E. Values with different alphabet within a column were significantly different (p < 0.05). Values with different numeric superscript across a row were significantly different (p < 0.05). Group A (CE); Group B (NE), Group C (CA 10:1), Group D (CA 20:1) Group E (WH10:1), Group F (WH 20:1). DO, dissolved oxygen; TAN, total ammonia nitrogen; TSS, total suspended solid.

 

Table 2: Effects of different organic carbons on weight gain, growth rate, specific growth rate, condition factor and survival differences of Clarias gariepinus after 28 days growth trial under varied C:N ratios in a biofloc tank.

Parameters	CE	NE	CA 10:1	CA 20:1	WH 10:1	WH 20:1
Initial weight (g)	1.38 ± 0.03a	1.38 ± 0.03a	1.38 ± 0.03a	1.38 ± 0.03a	1.38 ± 0.03a	1.38 ± 0.03a
Initial length (cm)	5.78 ± 0.06a	5.78 ± 0.06a	5.78 ± 0.06a	5.78 ± 0.06a	5.78 ± 0.06a	5.78 ± 0.06a
Final Weight (g)	3.11 ± 0.25a	3.24 ± 0.31a	4.05 ± 0.30a	3.46 ± 0.23a	4.09 ± 0.32a	4.19 ± 0.30a
Weight gain (g)	1.80 ± 0.25a	1.93 ± 0.24a	2.69 ± 0.30a	2.10 ± 0.24a	2.73 ± 0.33a	2.82 ± 0.31a
Weight gain (%)	139.42 ± 20.30a	146.77 ± 13.85a	204.25 ± 24.32a	162.18 ± 19.46a	211.61 ± 29.75a	214.91 ± 26.80a
GR (%)	6.42 ± 0.90a	6.88 ± 0.86a	9.59 ± 1.09a	7.50 ± 0.85a	9.75 ± 1.18a	10.06 ± 1.12a
SGR (%)	2.99 ± 0.26a	3.20 ± 0.21a	3.74 ± 0.24a	3.22 ± 0.24a	3.73 ± 0.28a	3.78 ± 0.26a
Final Length (cm)	7.21 ± 0.17b	7.16 ± 0.26b	8.15 ± 0.11a	7.70 ± 0.17ab	8.15 ± 0.19a	8.22 ± 0.18a
LG(cm)	1.54 ± 0.18b	1.56 ± 0.32b	2.36 ± 0.15a	1.92 ± 0.19ab	2.36 ± 0.21a	2.44 ± 0.19a
LG (%)	27.40 ± 3.42b	28.21 ± 5.82ab	41.53 ± 2.91ab	33.78 ± 3.49ab	41.33 ± 3.95ab	42.93 ± 3.54a
CF (%)	0.82 ± 0.03ab	0.88 ± 0.06a	0.72 ± 0.03b	0.75 ± 0.02b	0.73 ± 0.02b	0.73 ± 0.02b
Survival (%)	38.89±7.35b	13.89±7.35	77.78±7.35a	75.10±12.73a	77.78±2.78a	88.89±2.78a
Survival differences *CE *NE	0 25.00±10.64	-25.00±10.64 0	38.89±10.64 63.89±10.64	36.11±10.64 61.11±10.64	38.89±10.64 63.89±10.64	50.00±10.64 75.00±10.64
Mean survival	4.67±-0.88b	1.67±0.88c	9.33±0.88a	9.00±1.53a	9.33±0.33a	10.67±0.33a

Values as mean ± S.E. Values with different alphabet superscript across a row were significantly different (p < 0.05). GR, growth rate; SGR, specific growth rate; LG, length gain, CF, condition factor. Group A (CE); Group B (NE), Group C (CA 10:1), Group D (CA 20:1) Group E (WH10:1), Group F (WH 20:1)

 

Table 3: Length-weight relationship of C. gariepinus raised in a biofloc tank after 28 days growth trial

	Groups	Logarithmic equation	A	b	R	R2	P
Baseline		LogW = 1.319Log L – 0.8699	0.134927	1.319	0.540	0.292	0.000
Day 7	CE	LogW = 1.3244LogL – 0.7775	0.16799	1.324	0.579	0.335	0.001
	NE	LogW = 0.9622LogL-0.5371	0.290335	0.962	0.477	0.228	0.008
	CA 10:1	LogW = 2.5973LogL - 1.8175	0.015223	2.597	0.925	0.852	0.000
	CA 20:1	LogW = 1.7867LogL-1.1834	0.065554	1.787	0.747	0.558	0.000
	WH 10:1	LogW = 1.4296LogL-0.8647	0.136553	1.430	0.608	0.370	0.000
	WH 20:1	LogW = 2.1463LogL-1.4276	0.037359	2.146	0.813	0.661	0.000
Day 14	CE	LogW = 1.7162LogL-1.0684	0.085428	1.716	0.740	0.548	0.000
	NE	LogW = 1.859Log-1.2188	0.060423	1.859	0.734	0.539	0.001
	CA 10:1	LogW = 1.5605LogL-0.9289	0.117788	1.561	0.696	0.485	0.000
	CA 20:1	LogW = 1.985LogL-1.301	0.050003	1.986	0.731	0.534	0.000
	WH 10:1	LogW = 1.7075LogL-1.0433	0.090511	1.708	0.707	0.500	0.000
	WH 20:1	LogW = 2.2317LogL-1.4872	0.031827	2.232	0.796	0.634	0.000
Day 21	CE	LogW = 1.8218LogL-1.1097	0.077678	1.822	0.821	0.675	0.000
	NE	LogW =1.2646LogL-0.7051	0.197197	1.265	0.655	0.429	0.021
	CA 10:1	LogW = 2.5071LogL-1.7029	0.01982	2.507	0.953	0.908	0.000
	CA 20:1	LogW = 1.9171LogL-1.2097	0.061702	1.917	0.741	0.549	0.000
	WH 10:1	LogW = 2.5697LogL-1.7731	0.016862	2.570	0.929	0.863	0.000
	WH 20:1	LogW = 2.0387LogL-1.275	0.053088	2.039	0.888	0.788	0.000
Day 28	CE	LogW = 0.2727LogL+0.7258	5.318633	0.273	0.826	0.682	0.000
	NE	LogW = 0.3426Logl+0.6813	4.80065	0.343	0.830	0.689	0.082
	CA 10:1	LogW = 0.2101LogL+0.7873	6.127735	0.210	0.873	0.762	0.000
	CA 20:1	LogW = 0.356LogL+0.6988	4.998043	0.356	0.911	0.830	0.000
	WH 10:1	LogW = 0.3226LogL+0.7193	5.239623	0.323	0.917	0.840	0.000
	WH 20:1	LogW = 0.3024LogL+0.7325	5.401321	0.302	0.889	0.790	0.000

Group A (CE); Group B (NE), Group C (CA 10:1), Group D (CA 20:1) Group E (WH10:1), Group F (WH 20:1).W, weight; L, length

 

Table 4: Responses of oxidative stress biomarkers in Clarias gariepinus reared in a different BFT conditions

Parameters	Groups	Baseline	Day 7	Day 14	Day 21
CAT (U/mg protein)	CE	1.13 ± 0.02a12	1.17 ± 0.04a2	1.16 ± 0.04ab2	1.06 ± 0.01b1
	NE	1.23 ± 0.08a1	1.25 ± 0.03a1	1.18± 0.05ab1	1.08 ± 0.00b1
	CA 10:1	1.08 ± 0.11a1	1.25 ± 0.03a1	1.20 ± 0.07ab1	1.10 ± 0.01ab1
	CA 20:1	1.30 ± 0.21a1	1.19 ± 0.04a1	1.25 ± 0.03ab1	1.14 ± 0.00a1
	WH 10:1	1.14 ± 0.14a1	1.23 ± 0.03a1	1.13 ± 0.00b1	1.14 ± 0.03a1
	WH 20:1	1.17 ± 0.01a12	1.17 ± 0.06a12	1.38 ± 0.14a2	1.09 ± 0.01ab1
SOD (U/mg protein)	CE	10.50 ± 0.26a1	10.92 ± 0.03a12	11.32 ± 0.20a2	11.22 ± 0.03b2
	NE	10.66 ± 0.12a1	10.89 ±0.14a1	10.88 ± 0.20b1	11.52 ± 0.16a2
	CA 10:1	10.83 ± 0.12a1	10.98 ± 0.09a12	11.04 ± 0.03ab12	11.24 ± 0.03b2
	CA 20:1	10.77 ± 0.28a1	10.94 ± 0.08a12	11.05 ± 0.05ab12	11.22 ± 0.07b2
	WH 10:1	10.95 ± 0.06a1	10.93 ± 0.06a1	11.01 ± 0.06ab1	11.37 ± 0.03ab2
	WH 20:1	10.97 ± 0.10a12	10.87 ± 0.10a1	10.91 ± 0.02ab1	11.22 ± 0.07b2
GPx (ng/mg protein)	CE	11.09 ± 0.21a2	10.06 ± 0.29a1	9.91 ± 0.25b1	9.91 ± 0.25b1
	NE	11.58 ± 0.15a3	11.34 ± 0.39a23	10.35 ± 0.50ab12	9.91 ± 0.25b1
	CA 10:1	11.78± 0.21a2	10.92 ± 0.29a12	10.78 ± 0.25ab12	10.35 ± 0.50b1
	CA 20:1	11.30 ± 0.48a1	10.63 ± 0.29a1	10.78 ± 0.25ab1	11.64 ± 0.25a1
	WH 10:1	11.82 ± 0.39a2	10.34 ± 0.50a1	11.21 ± 0.50a12	10.34 ± 0.00b1
	WH 20:1	11.04 ± 0.22a1	10.06 ± 0.58a1	10.77 ± 0.25ab1	9.91 ± 0.25b1
MDA (U/mg protein)	CE	1.16 ± 0.02a1	1.22 ± 0.04a1	1.27 ± 0.12a1	1.29 ± 0.12ab1
	NE	1.17 ± 0.07a1	1.16 ± 0.07a1	1.50 ± 0.07a2	1.58 ± 0.07a2
	CA 10:1	1.20 ± 0.05a1	1.22 ± 0.16a1	1.44 ± 0.08a1	1.25 ± 0.06ab1
	CA 20:1	1.17 ± 0.04a1	1.21 ± 0.08a1	1.41 ± 0.01a2	1.14 ± 0.08ab1
	WH 10:1	1.16 ± 0.16a1	1.23 ± 0.07a1	1.29 ± 0.05a1	1.16 ± 0.03ab1
	WH 20:1	1.27 ± 0.08a1	1.13 ± 0.06a1	1.22 ± 0.12a1	1.05 ± 0.02c1

Values as mean ± S.E. Values with different alphabet superscript within a column were significantly different (p < 0.05). Values with different numeric superscript across a row were significantly different (p < 0.05). CE; NE, CA 10:1 carbon (cassava) 10:1; CA 20:1 carbon (cassava) 20:1; WH10:1, carbon (wheat) 10:1; WH20:1, carbon (wheat) 20:1. CAT, catalase, SOD, superoxide dismutase; GPx, glutathione peroxidase, MDA, malondialdehyde

 

Table 5: Changes in the liver biomarkers of C. gariepinus raised in biofloc tank.

Parameters	Groups	Baseline	Day 7	Day 14	Day 21
ALT (IU/L)	CE	8.03± 0.34a1	7.24 ± 0.51b1	8.53 ± 0.61b1	8.45 ± 0.52b1
	NE	8.39± 0.10a1	8.99 ± 0.31a12	10.37 ± 1.08a2	8.78 ± 0.14ab12
	CA 10:1	8.43± 0.41a1	8.12 ± 0.85b1	12.05 ± 0.28a2	9.44 ± 0.11a1
	CA 20:1	8.16± 0.21a1	9.11 ± 0.25a2	11.82 ± 0.38a3	8.94 ± 0.23ab12
	WH 10:1	8.26± 0.41a1	9.38 ± 0.42a1	11.90 ± 0.29a2	8.96 ± 0.13ab1
	WH 20:1	8.25± 0.22a1	9.03 ± 0.36a1	10.89 ± 0.41a2	8.86 ± 0.19ab1
AST (IU/L)	CE	11.24± 0.78a1	10.90 ± 0.37ab1	11.82 ± 0.14b1	11.07 ± 0.06b1
	NE	11.10± 0.35a1	12.33 ±0.22a2	12.27 ± 0.21a2	11.87 ± 0.37a12
	CA 10:1	11.53± 1.39a1	11.60 ± 0.23a1	11.79 ± 0.12b1	11.99 ± 0.28a1
	CA 20:1	10.83± 0.58a1	12.29 ± 0.15a2	11.00 ± 0.08c1	11.38 ± 0.03ab12
	WH 10:1	11.11± 0.80a1	11.19 ± 0.19ab1	11.75 ± 0.09b1	11.52 ± 0.05ab1
	WH 20:1	10.66± 0.40a1	10.59 ± 0.89b1	11.42 ± 0.05b1	11.20 ± 0.08b1
ALP (IU/L)	CE	52.49± 4.04a1	45.47 ± 2.32a1	46.30 ± 1.31bc1	42.42 ± 6.57b1
	NE	54.07± 0.66a2	46.60 ± 2.84a1	49.90 ± 1.67b12	52.90 ± 2.48a12
	CA 10:1	49.42± 2.00a1	51.73 ± 5.33a1	56.70 ± 1.10a1	55.10 ± 1.67a1
	CA 20:1	47.42± 3.04a1	47.00 ± 4.08a1	45.80 ± 2.19bc1	50.00 ± 0.46ab1
	WH 10:1	49.90± 1.82a12	51.93 ±1.88a2	45.20 ± 0.58c1	50.80 ± 1.27ab2
	WH 20:1	51.36± 2.82a2	46.00 ± 3.36a12	41.80 ± 0.69c1	46.30 ± 0.29ab12

Values as mean ± S.E. Values with different alphabet superscript within a column were significantly different (p < 0.05). Values with different numeric superscript across a row were significantly different (p < 0.05). CE (normal control); NE,; CA 10:1 carbon (cassava) 10:1; CA 20:1 carbon (cassava) 20:1; WH10:1, carbon (wheat) 10:1; WH20:1, carbon (wheat) 20:1. ALT, alanine aminotransferase, AST, aspartate aminotransferase; ALP, alkaline phosphatase

10:1, CA 20:1, and WH 10:1. The MDA level in the fish reared in NE was significantly different (p<0.05) (Table 4).

Biochemicals

Alanine aminotransferase (ALT) and alkaline phosphatase (ALP) indicated variations dependent on the media harbouring the fish and the duration of the experimental setup. AST only showed a duration-dependent variation in concentration. In the cassava carbon source (CA 10:1 and CA 20:1) and wheat carbon source (WH 10:1 and WH 20:1) groups, ALT on day 14 was significantly higher than day 14 and baseline concentrations (p < 0.05) (Table 5). The ALT level in these groups declined on day 21 to day 7 level. ALT in NE appeared to follow the same trend as the groups that provided carbon sources. No significant difference occurred in ALT in CE between baseline and day 21 (p > 0.05). AST concentration in groups provided carbon sources were within the same range except WH 20:1 with reduced AST level compared to CA 20:1 (p < 0.05). The AST level was significantly lower in CA 20:1 than all other groups, while NE was considerably higher (p < 0.05) on day 14.

Fluctuations in the concentration of ALP for the duration of the study only occurred in groups provided wheat carbon source (WH 10:1and WH 20:1). The significant variations did not follow a clear cut pattern in both groups. The marked difference in ALP activities between the groups occurred on days 14 and 21. On day 14, ALP was significantly higher in CA 10:1 group compared to the other groups (p < 0.05). But ALP in CA 10:1 was within the same limits as other groups except for CE on day 21 (Table 5).

The total protein level on days 7, 14 and 21 were within the baseline range. Complete protein within any group was not significantly different between days 7, 14 and 21 (p > 0.05). On day 21, marked differences occurred in total protein level between the groups (Figure 1); WH 10:1 and WH 20:1 total protein concentration was significantly higher than NE (p < 0.05). Creatinine concentration in all the groups dropped from day 7, through day 14 to day 21, except CA 10:1 group (Figure 2). On day 21, creatinine concentration in CA 10:1 was significantly higher.

Bars with different alphabet label were significantly different between the groups on days 7, 14 and 21(p < 0.05). Bars with different numeric superscript were not significantly different based on duration (p < 0.05).

Variations in urea concentration were dependent on biofloc media used and the setup duration. The level of urea in C. gariepinus in CE, NE, CA 10:1 and CA 20:1 were in the same range as the baseline level through the experiment (Figure 3). From day 7 to day 21, urea concentration in C.

 

Table 6: Floc volume (ml) of different carbon sources (n=7, observations)

Groups	Duration (Days)
	7	14	21	28
A (CA 10:1)	17.33 ± 1.33c1	37.33 ± 2.67b2	17.33 ± 1.33b1	29.33 ± 7.06c12
B (CA 20:1)	56.00 ± 4.62a1	69.33 ± 5.33a1	52.00 ± 6.23a1	50.67 ± 7.06ab1
C (WH 10:1)	32.00 ± 2.31b1	60.00 ± 6.93a2	37.33 ± 10.67ab1	36.00 ± 4.00bc1
D (WH 20:1)	50.67 ± 2.67a1	72.00 ± 8.00a1	65.33 ± 13.92a1	55.33 ± 2.91a1

Values as mean ± S.E. Values with different alphabet within a column were significantly different (p < 0.05). Values with different numeric superscript across a row were significantly different (p < 0.05). Group A (CE); Group B (NE), Group C (CA 10:1), Group D (CA 20:1) Group E (WH10:1), Group F (WH 20:1).

 

Table 7: Nutritional composition of flocs produced by local cassava variety and wheat flour

Composition	CA 10:1	CA 20:1	WH 10:1	WH 20:1
Moisture	65.55 ± 3.94b	69.40 ± 1.46a	69.64 ± 1.06a	63.31 ± 0.47a
Ash	0.47 ± 0.05a	0.38 ± 0.02ab	0.29 ± 0.03c	0.31 ± 0.01c
Fats	0.05 ± 0.02a	0.06 ± 0.01a	0.04 ± 0.01a	0.05 ± 0.01a
Protein	20.95 ± 1.79a	18.65 ± 1.31ab	20.46 ± 1.52ab	21.17 ± 1.62b
Fibre	0.58 ± 0.06a	0.55 ± 0.02a	0.55 ± 0.04a	0.64 ± 0.05a
Carbohydrate Vitamin A Vitamin E Potassium Calcium Sodium	12.40 ± 5.64a 0.51±0.01a 0.24±0.02a 0.32±0.02a 0.47±0.01a 0.28±0.02a	10.96 ± 0.13a 0.47±0.01a 0.25±0.02a 0.27±0.01a 0.39±0.02a 0.24±0.02a	9.02 ± 0.48a 0.51±0.01a 0.21±0.02a 0.34±0.01a 0.42±0.01a 0.22±0.01a	14.52 ± 1.39a 0.46±0.01a 0.30±0.04a 0.28±0.02a 0.43±0.02a 0.19±0.01b

Values as mean ± S.E. Values with different alphabet superscript across a row were significantly different (p < 0.05)

gariepinus from WH 10:1, and WH 20:1 did not change significantly (p >0.05). There was no significant difference in urea concentration between the groups on day 7.

Bacteria Counts in different BFT Media

The total heterotrophic bacteria count (THBC) in the baseline was similar. On day 14, there was no significant

 

Bars with different alphabet label were significantly different between the groups on days 7, 14 and 21(p < 0.05). Bars with different numeric superscript were not significantly different based on duration (p < 0.05).

difference between the groups for THBC (p < 0.05). The apparent discrepancies between the THBC among the groups occurred on day 21 (Figure 4). Meanwhile, THBC was significantly higher in CA 20:1, while WH 10:1 produced the highest (p<0.05) THBC.

 

Floc Volume under Different Carbon Sources

After 28 days of growth trial (Table 6), the volume of floccules harvested after flocculation was higher in WH 20: 1 than WH 10:1 on days 7 to 28. The most minor floc volumes were recorded with a cassava carbon source (CA 10:1) compared to other harvested floc volumes from other groups (p < 0.05) on day14. Meanwhile, the floc volume of CA 20:1 and WH 20:1 was within the same range but significantly higher than CA 10:1 and WH 10:1 (p < 0.05) on day 7 only.

 

Proximate composition of harvested flocs

Significant changes in the proximate and mineral composition of the floc produced by different carbon sources at the end of the experiment were observed. The fats, fibre, carbohydrate, and ash composition were similar between carbon sources (Table 7).

Microscopic view of biofloc developed by local cassava variety and wheat flour at different C:N ratios

Biofloc developed by other C:N ratios of carbon sources (cassava and wheat flour) within 28 days showed increased macroaggregates biofloc size mostly in the WH 20:1. Small developed bioflocs were observed in the CA 10:1 (Figure 5) followed by CA 20: 1.

Histopathology

The hepatocytes, polyhedral in shape, were arranged in two cell thick laminas/hepatic chords separated by engorged sinusoid containing nucleated red blood cells. Hepatic parenchyma is not organized into distinct lobules with indistinguishable interlobular connective tissues. At the end of the experiment, the liver sections in the groups showed similar indistinct cords of hepatocytes composed of branching anastomosing, two cell thick laminae of hepatocytes radially arranged around the central veins non-obvious portal triad. Cytoplasmic vacuolation with a consequent eccentric displacement of the nucleus was observed in the NE group (Figure 6-7).

Discussion

Physicochemical, growth performance and Biofloc Media

Biofloc technology system supported the regulation of physicochemical parameters of culture medium preventing the accumulation of toxicants and monitoring the pollution status of the medium. From the preceding, the temperature range obtained from the present study ranged between 25 ºC and 27 ºC, which is within the optimal degree of catfish fingerling production. The observed temperature in the experimental group supported the optimal temperature for juvenile Clarias gariepinus is 25 -35 ºC. The pH range 7-8 was optimal for the production and survival of Clarias gariepinus, which suggested pH stability within the biofloc treatment (Popoola and Miracle, 2022). Uzoka et al. (2015) reported pH 7.8 acceptable for fish production (FAO, 1996). The observed dissolved oxygen concentration from the present study suggested that increased microbial biomass may affect the DO in biofloc culture (Popoola and Miracle, 2022). The continuous stability of DO concentration was attributed to the C:N ratio variation. The TSS was within the range of 21.95-338.35 mg/L. TSS level of 400-600 mg/L was preferred for Litopeneaus vannamei production. More so, Ekasari et al. (2016) reported a much higher range of TSS for biofloc (188-1250 mg/L) and non-biofloc (160-1150 mg/L). TAN was generally within the acceptable limits. The initial increase was probably returned to baseline levels in groups provided carbon sources by activities of heterotrophic bacteria that assimilate nitrogen (Hargreaves, 2013). From the present study, TAN was between the ranges of 0.22-0.30mg/L. The TAN level was below 0.35-22.8 mg/L, usually considered toxic for freshwater organisms. The initial increase in TAN between 7 and 14 days was due to metabolic activities, decompositions of leftover feed, and initial lag in establishing heterotrophic and nitrification bacteria (Hagreaves, 2013). The nitrite concentration in the present study was high (1.12-2.41 mg/L) compared to recommended levels for biofloc media, 0.07-0.42 mg/L (Ekasari et al., 2016; Roques et al., 2015) in Clarias gariepinus fingerlings culture. The impact of nitrate concentration in the present study is uncertain, especially as the nitrate concentration in control and biofloc media were similar. The activities of the biofloc media did not facilitate the nitrification processes, which would have lowered the nitrite concentration by the production of nitrates. This is the cause of the observed drop of nitrate concentration >0.35 mg/L in all the groups.

Fish cultured in biofloc media did not clearly show a better growth performance than the standard control. Several studies have reported better growth performances in fish (Kuhn et al.,. 2009; Ekasari et al., 2016) and shrimps (Hussain et al., 2015; Sakkaravarthi and Sankar, 2015) cultured using BFT or fed with biofloc formulated feeds compared to those cultured in non-biofloc media or fed commercial feeds. The length-weight relationship of C. gariepinus for the duration of the study and in all the treatment groups showed a negative allometric growth pattern. Factors possibly responsible for this observation were physicochemical properties of the water (Nehemia et al., 2012), insufficient feed, lack of suitable feed types, poor feeding (Khatoon et al, 2016), pathogenic stress (da Silva et al, 2016), including deficiencies in experimental setups (Goana et al., 2011). The quantity of microbial biomass generated in proportion to the number of fish per group was insufficient to support growth. Provision of additional carbon sources may serve as feed sources even though inadequate to correct the deficiency. The suitability of microbial biomass generated as feed for the C. gariepinus fingerlings is yet another factor that may have affected growth performance and the Length-weight relationship of the fish. The microbial biomass is unsuitable for the fish, so they may have avoided it or fed less on it. Despite choosing cost-effective biomaterials and using bioflocs as food by C. gariepinus, the contribution to the growth was comparable to the control.

Floc volume and TSS are valid indicators of biofloc formation (Avnimelech, 2007). In the present study, the increased high floc volume observed in CN 20:1 was in agreement with the report of El-Husseiny et al. (2018), who recorded the highest floc volume with wheat bran. The floc volume was a little above the safe range of 5-50 ml/L, according to Avnimelech (2011). Meanwhile, the flocs produced may depend on the carbon sources utilized (Zhou et al., 2002). This indicated that the nature of carbohydrates affects the quantum of biofloc produced by the application of simple sugars.

Biochemicals

The biochemical liver markers such as AST, ALT and ALP did not change significantly in all the groups, indicating non-hepatoxic conditions. The levels of these enzymes increased in the NE, which may indicate possible exposure to pollution stress than other experimental groups. Provision of aeration and sometimes additional carbon sources by BFT caters to water pollution and the need for water exchange (Ekasari et al., 2016; Crab et al. 2007); in NE, the problem of water pollution was obvious. The observed increase in creatinine and urea in NE compared to other groups may indicate higher absorption of nitrogen-related toxicants (e.g. ammonia and nitrite).

Production of reactive oxygen species (ROS) and antioxidant capacity was due to oxidative stress in C. gariepinus caused by chemical or physical stressors. Under normal conditions, animal cells produced ROS such as H2O2, OH- and O-, damaging cellular components leading to cell death. From the present study, the catalase activity played a crucial role in the antioxidant defence of cells by reducing H2O2 and O2 was critical for scavenging free radicals (Bello et al., 2000). The BFT increased the CAT activities of fish which supported the report of Popoola and Miracle (2022), who stated that BFT increased the CAT and GPx activities of Clarias gariepinus. Meanwhile, SOD activities catalyzed the destruction of superoxide radicals, and their activity indicated the tissues coping with oxidative stress (Misra, 1972). The increased SOD level in the BFTs group was attributed to lower oxidative stress caused by the bioflocs. These findings supported the report of Long et al. (2015), Popoola and Miracle (2022). Glutathione peroxidase catalyzes the reduction of hydrogen peroxides by glutathione and protects the body from oxidation and damaging effect of endogenously formed hydrogen peroxides. The observed changes in the GPx activities were due to the sequestering potentials of GPx in eliminating and scavenging free radicals from the body of the fish raised in the Biofloc tank. Lipid peroxidation occurred when fatty acids came in contact with ROS produced a series of reactive aldehyde, including MDA. The changes in the MDA levels of the fish raised in biofloc may be due to a reduction in the level of radicals produced.

Nutritional composition

Floc biomass provided a complete source of nutrition and various bioactive compounds (Akiyama et al., 1992). Different factors such as carbohydrate source, microbial community, floc density etc., affect the nutritional value of bioflocs (Hargreaves, 2006). Meanwhile, the type of carbon source also influences the palatability and digestibility of the cultured organisms (Crab et al., 2009; Crab, 2010a). The protein content of biofloc in the present study is in the same range with findings of Soares (2004), who reported a crude protein level of 16-20%, while Hende et al. (2014) wrote 15.8-27% crude protein. A reduced level of biofloc crude protein was reported by Magondu (2012) and Megahed (2014). Bakhshi et al. (2018) said crude protein of 21-25% in biofloc is utilized for carp farming. Hence, the nutritional composition of the floc obtained in the present study (18.65%-21.17% protein, 9.02-14.52% carbohydrate, and 0.04-0.06% fat) was appropriate for fingerlings, despite the reduced lipid content observed. The reduced lipid content was because biofloc is a poor source of lipid (Azim et al., 2008). The variations in the nutrient composition of harvested flocs from biofloc tanks were due to the cultured species’ different carbon sources and feeding preferences. These findings agreed with the reports of Khanjani et al. (2016), who opined that wheat flour resulted in high protein levels as observed in the biofloc produced by WH 20:1.

Histopathology

The liver is an essential organ in nutrient metabolism, and tissue changes are considered a reliable indicator in the evaluation of toxic conditions (Bakhshi et al., 2015). No histological changes were observed in the control (CE) and biofloc media in the present study. This result supported the assertion of Bakhshi et al. (2016), who confirmed no changes in proteolytic enzymes, liver histology in common carp raised in biofloc with zero water exchange. He further reported no change in the liver tissues. Meanwhile, in the NE, slight changes in the liver tissues characterized by cytoplasmic vacuolation indicated hepatoxicity, supported by the reports of Stefan et al. (2008).

Conclusion

The adjustable C:N ratio of cassava and wheat flour in flocculation is essential, efficient, and indicated a positive reduction of inorganic nitrogen and noxious metabolites produced in the culture system of African catfish, Clarias gariepinus. It is pertinent to observe the peculiar attributes of natural carbohydrates sources such as locally available, not harmful to the cultured fin and shellfish, and cheap. Fish culture using BFT is more beneficial for better growth performances with a reduced pathophysiology changes. CA 10: 1 and WH 20:1 is better utilized in Clarias gariepinus culture compared to CA 20:1 and WH 10: 1. There is a need to investigate other cheaper sources of carbohydrates such as white yam tuber (Dioscorea rotundata), water yam (Dioscorea alata), cocoyam (Xanthosoma sagittifolia, Colocasia esculenta) and relevant agricultural by-product as natural carbon sources in culture of finfish in a biofloc media.

Authors Contributions

Chika Bright Ikele designed the experiment, proof read the manuscript and provided technical information of the research. Uju, Aghaji, conducted the experiment by routinely monitoring the experiment. Mgbenka, Obialo proof read the manuscript and analyzed the data.

Ethical Approval

The fish were handled following the approved regulatory guidelines of the University of Nigeria, Nsukka Senate Committee on Medical and Research Ethics (UNN-ACC, Protocol No. 0764/2015) and does not contain clinical or patient data.

Conflict of interest

The authors declare no conflict of interest.

Data Availability

Research Data are not shared.

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