Effect of Dietary Protein Levels on Growth, Body Composition, and Haematology of Tilapia in Biofloc Without Solid Management System

Abstract

ABSTRACT

This study investigated the effects of varying dietary protein levels on the performance of tilapia reared in biofloc culture system without solid management. Five experimental diets containing crude protein (CP) levels of 32, 28, 24, 20, and 16% were tested in a completely randomized design in triplicate. Tilapia fingerlings (mean initial weight of 40.82 ± 0.38 g) were randomly stocked in biofloc tanks (effective water volume of 300 L) at a stocking density of 65 fish m⁻³. After 13 weeks of feeding trial, significant differences were observed in final weight (g), daily growth (g day⁻¹), feed conversion ratio, and yield (kg m⁻³) (p < 0.05). Water quality parameters remained unaffected by dietary treatments (p > 0.5). Notably, fish fed with diets containing 32%, 28%, and 24% CP demonstrated similar growth performance. Based on weight gain, a linear response plateau model estimated the minimal dietary CP level of 24.5%. Dietary CP levels altered visceral index (p < 0.05), but not visceral fat index, hepatosomatic index, or spleen somatic index (p > 0.05). Similarly, dietary CP levels did not significantly affect the fish composition (p > 0.05) and the haematological parameters of the experimental fish (p > 0.05). These results suggest that dietary protein in a small‐scale biofloc system can be reduced from 32% to 24.5% without compromising fish health. However, appropriate solid management in biofloc systems is recommended to ensure optimal growth of fish.

Introduction

Aquaculture intensification often relies on protein-rich feeds, high stocking densities, and frequent water exchanges to maintain water quality. However, water exchanges are laborious, expensive, and raise environmental concerns. The biofloc technology (BFT) has emerged as a promising approach for sustainable aquaculture, offering solutions for managing water quality and biosecurity while minimizing water exchange needs (Ekasari et al. 2014; Abbaszadeh et al. 2019). The core principle of BFT involves manipulating the carbon:nitrogen (C:N) ratio to promote the growth of heterotrophic bacteria, which convert nitrogenous waste into biofloc, a new form of biomass (Azim and Little 2008; Avnimelech 2009; Ferreira et al. 2015; Abbaszadeh et al. 2019). Therefore, this process significantly reduces the need for water exchange by recycling water through waste conversion. BFT not only offers a sustainable solution but also provides a valuable nutrient source for cultured species. Biofloc produced by heterotrophic bacteria contains significant protein content and a favourable amino acid profile, making it a potential substitute for dietary protein in fish feed (Ekasari et al. 2014). Supporting this notion, previous studies have demonstrated that biofloc biomass can contribute to fish growth and production (Avnimelech 2007; Azim and Little 2008). Similarly, research suggests that biofloc can stimulate proteinase activities in shrimp and fish, leading to improved feed utilization, protein retention, and overall growth performance (Jatobá et al. 2014; Xu and Pan 2014; Yun et al. 2016). These findings suggest that filter-feeding fish reared in BFT systems may require lower dietary protein levels compared to those raised in traditional aquaculture systems.

Among the filter-feeding fish species, tilapia is a commercially important one with high consumer acceptance and ranks second globally in farmed fish production (FAO 2022). This species can grow from 10–30 g to marketable size of 150–250 g within 3–4 months in grow out ponds, while in biofloc systems, tilapia can grow from 50 to 250 g within 3 months (da Silva et al. 2018). Its rapid growth, high productivity, and disease resistance make it suitable for various aquaculture systems. Tilapia's ability to consume biofloc, a protein-rich microbial floc, makes it an ideal candidate for BFT (Avnimelech 2007). Although tilapia can consume and utilize biofloc efficiently, to attain maximum growth potential, it is suggested to provide a well-balanced feed, which shares a major cost in BFT system. The most expensive component of fish feeds is the protein content, which accounts for about 60% of the total feed cost (Hamidoghli et al. 2018; Zablon et al. 2022). Thus, reducing the amount of protein in the diet without compromising fish growth and health is one of the potential ways to lower production costs of BFT. Studies suggest that consumed biofloc can potentially substitute protein in supplemented feeds for tilapia (Avnimelech 2007; Azim and Little 2008). Therefore, tilapia raised in BFT systems likely have a lower protein requirement compared to those reared in other culture systems and can contribute to lowering the feed cost. Determining the minimal protein requirement for tilapia in BFT is thus crucial for formulating cost-effective as well as well-balanced diets.

To determine the minimal dietary protein requirement under BFT systems, several studies have investigated the effect of different protein levels on various tilapia species (Azim and Little 2008; Mansour and Esteban 2017; da Silva et al. 2018; Green et al. 2019). However, these studies recommend widely varying crude protein (CP) levels of feeds, ranging from 22% (da Silva et al., 2018) to 31% (Green et al. 2019). Furthermore, the impact of lowering protein content on tilapia health in BFT remains unclear. It is important to note that most of these studies employed an intensive BFT system with a side-stream settling chamber for periodic sludge removal (Green et al. 2019). While sludge management can improve production efficiency (Ray et al. 2010; Manduca et al. 2020), it is more energy-intensive and cost-prohibitive for small-scale peri-urban farmers, who are the primary BFT users in low- or middle-income countries like Bangladesh.

To make BFT more sustainable and cost-effective, empirical data are needed on the performance of tilapia fed with low but optimum protein content in user friendly and simpler BFT systems without complex sludge removal processes. Therefore, this study aimed to determine minimal dietary protein required for tilapia reared in indoor BFT systems without sludge removal through investigating growth performances, body composition, and haematological parameters.

Materials and Methods

Diets and Experimental Design

The experiment was conducted using a completely randomized design in triplicate for 13 weeks. Five diets with CP levels of 32%, 28%, 24%, 20%, and 16% were tested. Unlike conventional diets (using low or without fishmeal) for omnivorous fish species (Hardy 2010), fishmeal (FM) was used as the main protein source to formulate the control diets with 32% CP (Abdel-Tawwab et al. 2010; Green et al. 2019). This was done to ensure balanced amino acid compositions in control diet because no supplementary amino acids were added to diets separately considering small-scale peri-urban farmers. It was also done to compare the contribution of protein of produced floc in the growth of fish fed low protein diets. To create contrast in protein levels, other experimental diets were prepared by replacing FM with locally available corn flour. All diets were stored in a refrigerator at 4°C throughout the experiment. Compositions and nutrient levels of experimental diets are presented in Table 1.

TABLE 1 Formulations and proximate compositions of experimental feeds fed to tilapia in an indoor biofloc production system.

Ingredients (%)	Crude protein level (%)
32	28	24	20	16
Fish meal	32.8	26.8	20.8	14.7	8.7
Soybean meal	10.9	10.9	10.9	10.9	10.9
Corn flour	23.4	29.2	35.0	40.9	46.7
Rice polish	11.7	11.7	11.7	11.7	11.7
Wheat floor	11.7	11.7	11.7	11.7	11.7
Soybean oil	2.5	2.6	2.7	2.8	2.9
Binder	2.5	2.5	2.5	2.5	2.5
Vitamin + mineral premix	2.0	2.0	2.0	2.0	2.0
Dicalcium phosphate	2.0	2.1	2.2	2.3	2.4
Sodium chloride	0.5	0.5	0.5	0.5	0.5
Proximate composition (%)
Crude protein	32.30	28.00	25.40	20.91	17.01
Crude lipid	6.05	6.93	6.67	6.18	6.74
Ash	12.44	13.24	10.92	9.92	9.35
Moisture	10.35	10.41	12.39	10.74	11.88

Fish and Tank Management

Floc Development and Management

The experiment was conducted at the field laboratory complex (biofloc unit) of the Fisheries and Marine Resource Technology Discipline, Khulna University, Bangladesh. Floc volume was maintained at a desired level (30 mL L⁻¹) in each tank following the procedures described in Debnath et al. (2021). Briefly, fermented organic carbon (FCO) was prepared in 20 L disinfected groundwater in a plastic bucket. The FCO mixture typically included a commercial probiotic blend (containing Bacillus subtilis, Bacillus licheniformis, Trichoderma viride, Nitrosomonas europaea, Nitrobacter winogradskyi, Aspergillus oryzae, Pseudomonas denitrificans, and Pseudomonas oxalaticus) to promote beneficial bacterial growth, salt (NaCl) for salinity balance, molasses as a readily available carbon source for bacteria, and a small amount of fish feed (containing 30% protein) to inoculate the culture. The mixture was vigorously aerated with air stones for 5 days to facilitate the fermentation process. To initiate floc development in each culture tank, 100 mL of the prepared FCO was added. Subsequently, FCO was added to respective tanks whenever the floc volume decreased below the desired level (30 mL L⁻¹).

Fish and Tank Conditions

Mixed-sex genetically improved farmed tilapia (GIFT) from one batch (40 days old) were obtained from a local breeder and acclimated in laboratory conditions for 1 week with a commercial feed containing 32% protein. Following acclimatization, tilapia with a mean initial weight of 40.82 ± 0.38 g were randomly stocked into 15 biofloc tanks (effective water volume of 300 L) at a stocking density of 65 fish m⁻³. Before stocking, fish were starved for 24 h to empty their gastrointestinal tract.

C:N ratio of 15:1 was maintained in each tank by adding brown sugar as a carbon source to control total ammonia nitrogen (TAN) concentration according to Avnimelech (1999) and Pérez-Fuentes et al. (2016). All tanks were continuously aerated and agitated using air stones connected to an air blower.

Feeding Management

Fish were hand-fed a diet corresponding to 3% of their body weight twice daily. The total daily feed ration was divided into two equal portions and provided at 9.00 AM and 3.00 PM. The feeding rate was adjusted fortnightly based on a random sampling of more than 50% of fish from each tank. Before each sampling event, fish were not provided supplementary diets for 24 h to ensure accurate weight measurements.

Water Quality Monitoring

Water quality parameters in each tank were monitored throughout the experiment, with measurements taken each morning (8:30 AM). Water temperature (°C) was recorded using a Celsius thermometer. TAN, nitrite (NO₂⁻), nitrate (NO₃⁻), pH, and dissolved oxygen (DO) were measured using API test kits (Mars Fishcare North America, Inc., USA). Biofloc volume was measured by using an Imhoff Cone (Xu and Pan 2012; Khanjani et al. 2017).

Analytical Procedures

Growth Performances

Growth performances and feed utilization were evaluated by final weight, absolute growth (g day⁻¹), and feed conversion ratio (FCR). Survival rate was calculated as (N_f/N_i) × 100, where N_i is the initial number of fish stocked and N_f is the final number of fish at the end of the experiment. Absolute growth (GR_abs, g day⁻¹) was calculated as (W_f − W_i)/d, where W_i is the initial individual weight (g), W_f is the final individual weight (g), and d is the duration of the growth trial (days). FCR was calculated as F_tot/W_f − W_i, where W_f is the final weight of fish and W_i is the initial weight of fish and F_tot is the total feed given during the experiment.

Somatic Indices

At harvest, three fish from each tank were randomly selected to collect viscera, visceral fat, liver, and spleen. Before dissecting, fish were euthanized using 2-phenoxyethnanol at an overdose of 1 mL L⁻¹ according to approved ethical guidelines for animal research (Sloman et al. 2019). Somatic indices were calculated using the following equations: visceral index (VI) = (viscera weight/fish weight) × 100; visceral fat index (VFI) = (visceral fat weight/viscera weight) × 100; hepatosomatic index (HI) = (liver weight/fish weight) × 100; spleen somatic index (SSI) = (spleen weight/fish weight) × 100.

Haematological Parameters

Blood samples were collected from the caudal fins of four randomly selected fish per tank using a sterile syringe containing heparin and pooled into a vacuum tube. Fish selected for sample collection were handled carefully to reduce stress, which may have impact on blood parameters. Before blood collection, fish were mildly sedated using 2-phenoxyethanol (0.25 mL L⁻¹) according to approved ethical guidelines for animal research (Sloman et al. 2019). Collected blood samples were immediately transferred to a commercial laboratory for analysis of specific haematological parameters (e.g., red blood cell, white blood cell, haemoglobin levels, neutrophils, lymphocytes, monocytes, haematocrit, total platelet, and red cell distribution width). An automated haematology analyzer (Mythic-22, Germany) was used for these analyses.

Proximate Analyses

At harvest, four fish from each tank were randomly sampled for body composition analysis and stored at −20°C in a refrigerator. Floc samples from each tank were also collected at the end of the final sampling. Fish, feed, and floc samples were analyzed using the same methods. Frozen fish samples were ground and homogenized. Crude protein, crude fat, ash, and moisture content of fish, feed, and floc samples were determined according to AOAC (2005) methods. Briefly, protein content (N × 6.25) was determined by the Kjeldahl method. Lipid content was determined using the ether extraction method. Ash content was determined by complete incineration of samples in a muffle furnace at 800°C for 4 h until a constant weight was achieved. Moisture content was determined by oven drying at 105°C for 24 h until a constant weight was achieved.

Statistical Analyses

Statistical analyses were performed using Statistical Package for Social Science (SPSS, Version, 23.0, NY). Prior to analysis, normality of experimental data was checked by using Shapiro-Wilk test. Data were subjected to one-way analysis of variance to compare treatment means. If the main effects were significant, differences among treatments were tested using Tukey's multiple comparisons of means. Differences were considered significant at p < 0.05.

Results

Water Quality Parameters

Throughout the culture period in BFT tanks, water quality parameters such as temperature, pH, DO, total ammonia nitrogen, nitrite, and nitrate concentrations remained unaffected by the dietary treatments (p > 0.05). The measured values were all within the ranges considered optimal for aquaculture (Table 2).

TABLE 2 Water quality parameters (mean) in indoor biofloc tanks during a 13-week tilapia culture experiment fed with different protein levels.

	Crude protein levels (%)	Pooled SEM	p value	Reference value
Parameters	32	28	24	20	16
Temperature (°C)	27.52	27.94	27.65	27.48	27.76	0.158	0.297	27–32a
pH	8.32	8.28	8.34	8.27	8.31	0.039	0.771	6–9b
DO (mg L−1)	5.27	5.15	5.19	5.27	5.26	0.073	0.690	>4b
TAN (mg L−1)	0.72	0.66	0.76	0.65	0.73	0.077	0.826	<1a
NO2− (mg L−1)	0.12	0.11	0.14	0.11	0.10	0.049	0.979	<8a
NO3− (mg L−1)	19.25	17.83	15.81	17.72	17.78	2.611	0.921	<500c

Growth Performances and Somatic Indices

The growth performance and somatic indices of tilapia under the experimental conditions are summarized in Table 3. Survival was relatively high (90%) across all dietary treatments and was not significantly affected by the diets (p > 0.1). There was no significant difference in initial weight (g) among dietary treatments (p > 0.1). However, after 13 weeks of feeding, significant differences were observed in final weight (g), GR^abs (g day⁻¹), FCR, and yield (kg m⁻³). Fish fed with 32%, 28%, and 24% CP showed almost similar growth performances and had significantly higher growth compared to those fed feed with 20% and 16% CP (p < 0.05). Fish fed with 16% CP diet had the highest FCR (4.43), which was significantly higher than those fed with 24%–32% CP diets (p < 0.05). When a linear plateau model was applied to determine relationship between weight gain (%) and dietary protein level, the estimated minimal protein level required for the highest gain was 24.5% (Figure 1).

TABLE 3 Growth performance and somatic indices of tilapia-fed diets containing different levels of crude protein for 13 weeks in indoor biofloc tanks.

	Crude protein levels (%)	Pooled SEM	p value
Parameters	32	28	24	20	16
Survival (%)	93.33	96.67	93.33	90.00	93.33	2.981	0.294
Initial weight (g)	40.90	40.85	40.45	40.98	40.92	0.220	0.486
Final weight (g)	88.01a	86.11a	85.66a	77.27bc	71.00c	1.856	<0.001
GRabs (g day−1)	0.52a	0.50a	0.50a	0.41b	0.33b	0.021	<0.001
FCR	2.90a	2.94a	2.83a	3.69ab	4.43b	0.213	0.001
Yield (kg m−3)	5.48a	5.55a	5.33a	4.64b	4.41b	0.199	<0.001
Visceral index	10.55a	12.57ab	11.78ab	10.63a	13.25b	0.544	0.021
Visceral fat index	13.47	13.60	15.08	15.83	12.26	1.604	0.565
Hepatosomatic index	2.76	2.73	2.58	2.70	3.15	0.269	0.648
Spleen somatic index	0.18	0.19	0.21	0.21	0.23	0.046	0.940

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Dietary CP levels significantly altered visceral index (p < 0.05), while visceral fat, hepatosomatic, and spleen somatic index remained unaffected (p > 0.05). Tilapia juveniles fed with diets containing 16% CP had the highest visceral index, which was significantly higher than those fed with 32% and 20% CP (p < 0.05), while fish fed feeds with 32% CP had the lowest visceral index.

Proximate Composition and Volume of Bioflocs

The proximate composition (dry weight basis) and floc volume of bioflocs accumulated in different protein fed tanks are presented in Table 4. As shown in the table, dietary CP level did not affect the biochemical composition of floc (p > 0.1). The average CP, lipid, and ash contents of floc were 28.82%, 5.31%, and 19.43%, respectively.

TABLE 4 Proximate composition (%, dry weight basis) and volume of bioflocs accumulated in tanks with tilapia fed diets containing different protein levels.

	Crude protein levels (%)	Pooled SEM	p value
Compositions	32	28	24	20	16
Floc volume (mg L−1)	39.78	38.65	38.31	36.65	36.68	4.654	0.986
Crude protein	30.67	30.21	27.59	27.28	28.32	3.262	0.919
Crude lipid	5.27	5.79	5.28	4.95	5.25	0.580	0.888
Ash	20.85	19.64	20.38	18.07	18.22	2.952	0.943

Proximate Composition of Fish

Whole-body proximate compositions of fish fed feeds with different protein levels are presented in Table 5. Moisture, ash, and CP contents of whole body of experimental tilapia at the final harvest (fresh weight basis, %) remained unaffected by the CP levels of the experimental feeds (p > 0.1).

TABLE 5 Body composition (whole fish, % wet weight basis) of tilapia at harvest fed with diets containing different levels of crude protein (CP) for 13 weeks in indoor biofloc production tanks.

	Crude protein levels (%)	Pooled SEM	p value
Compositions	32	28	24	20	16
Crude protein	14.08	13.45	13.24	13.59	13.94	1.227	0.987
Crude lipid	3.35	3.26	3.98	3.48	3.43	0.470	0.836
Ash	3.66	4.01	3.85	4.15	4.26	0.417	0.853
Moisture	76.61	76.70	77.41	74.73	77.03	0.695	0.139

Haematological Parameters

Haematological parameters of fish fed feeds with different protein levels are presented in Table 6. Dietary CP levels did not affect haematological parameters of experimental tilapia reared in biofloc tanks (p > 0.1).

TABLE 6 Haematological parameters of tilapia fed with diets containing different levels of crude protein (CP) for 13 weeks in indoor biofloc production tanks.

	Crude protein levels (%)	Pooled SEM	p value
Parameters	32	28	24	20	16
Haemoglobin (g dL−1)	7.27	7.97	8.03	7.67	7.53	0.253	0.258
Total WBC (cmm)	127,100	130,733	131,367	127,800	130,100	1648	0.338
Neutrophils (%)	14.67	15.67	18.00	15.33	16.33	1.300	0.473
Lymphocytes (%)	78.33	77.00	74.67	78.00	76.33	1.438	0.435
Monocytes (%)	7.00	7.33	7.33	6.67	7.33	0.471	0.804
Total RBC (m µL−1)	1.92	1.96	2.12	1.98	1.91	0.090	0.519
Haematocrit (%)	31.33	31.23	32.50	32.30	31.93	1.269	0.934
Mean corpuscular volume (fL)	166.63	160.27	162.10	162.27	159.83	4.493	0.832
Mean cell haemoglobin (pg)	37.23	39.07	38.07	37.83	38.00	0.652	0.438
Mean corpuscular haemoglobin concentration (g dL−1)	22.63	24.93	23.57	23.90	23.60	0.552	0.138
Red cell distribution width (%)	18.37	16.57	15.33	17.53	16.90	1.501	0.692
Total platelet (cmm)	125,000	132,000	120,000	132,000	128,000	8326	0.823

Discussion

The results of the present study showed that the water quality parameters did not vary significantly throughout the experimental period suggesting that the biofloc system effectively maintained a suitable aquatic environment for tilapia growth. Exogenous organic carbon was added based on TAN concentration of the respective tank, which, together with uneaten feed and faeces, stimulated heterotrophic bacterial growth and subsequent reduction in NO₂⁻ and NO₃⁻ levels (Pérez-Fuentes et al. 2016). In agreement with the present study, Ekasari et al. (2015) also found reduction in TAN, NO₂⁻, and NO₃⁻ levels in BFT systems.

Tilapia reared in the BFT system had high survival (90%) in all treatments and remained unaffected by dietary treatments. Similar to our finding, da Silva et al. (2018) and Green et al. (2019) did not find any effect of dietary protein levels on tilapia survival. Biofloc system is considered one of the potential aquaculture techniques to control mortality (Ekasari et al. 2015; da Silva et al. 2018), which is attributable to immune boosting response of aquaculture animals through consuming bioflocs (Ekasari et al. 2015; Mansour and Esteban 2017).

Our findings revealed that tilapia fed diets containing 32%, 28%, and 24% CP exhibited similar and superior growth performance compared to those fed with 20% and 16% CP diets (Results, Table 3). This confirms the established importance of dietary protein for optimal fish growth (NRC 2011). The estimated minimum protein level required to achieve the highest weight gain was determined to be 24.5% using a linear plateau model (Results, Figure 1). Fish fed the 16% CP diet had the poorest growth performance, indicated by the lowest final weight and weight gain. This suggests that protein deficiency at this level likely impaired feed utilization efficiency. Findings of the present study revealed that tilapia fed with diets containing 32%, 28%, and 24% CP exhibited similar growth performances. This suggests that biofloc may have contributed to fish growth where fish were fed at a suboptimal rate (3% of their body weight) across all treatments. Previous research supports this notion. Azim and Little (2008) reported that Nile tilapia reared in BFT systems achieved 44%–46% higher weight gain and production compared to those in clean water systems, highlighting biofloc utilization by tilapia as a natural food source. The authors also found similar growth performance in tilapia fed diets containing 35% and 24% CP within a BFT system, with fish in the control system exhibiting a higher FCR. Likewise, Green et al. (2019) found no significant difference in tilapia production when fed with diets containing 36% and 31.3% protein within a BFT system.

While several studies have shown that biofloc significantly enhances fish growth and production, the present study observed comparatively lower production resulted in high FCR. The growth rate of tilapia in the present study (0.51 g day⁻¹) was lower compared to some previous studies such as 2.2 g day⁻¹ (Green et al. 2019) and 1.89 g day⁻¹ (da Silva et al. 2018), but comparable 0.48 g day⁻¹ (Azim and Little 2008). Several factors may have contributed to this discrepancy including the floc properties and floc sludge management as well as the initial weight of experimental fish. In the present study, solid management was not followed which could affect overall growth performances of fish.

Floc composition in BFT systems can vary widely depending on factors like microbial community (Ray et al. 2010), floc size (Ekasari et al. 2014), and C:N ratio (Xu and Pan 2013; Panigrahi et al. 2018). As has been mentioned, this study employed a simple smallholder-friendly BFT system that did not involve partial water exchange or periodic biofloc sludge removal. This might have limited the absolute feed intake by the fish due to reduced floc availability or poor water visibility. Moreover, fish reproduction was observed in some tanks, which can overall growth performance as reported by Azim and Little (2008).

Finally, the initial body weight of fish used in the present study (41 g) was considerably lower compared to some other studies. The proposition regarding the potential effect of fish size on floc consumption warrants further investigation, with larger fish possibly consuming floc more efficiently. The protein content of biofloc can vary significantly due to management practices, such as maintaining a specific C:N ratio through the addition of external carbon sources (Azim and Little 2008; da Silva et al. 2018; Green et al. 2019). Floc composition can also vary depending on the fish presence in the system. Azim and Little (2008) found that biofloc from tanks had better quality compared to tanks with fish. Interestingly, this study observed no significant difference (p > 0.05) in floc compositions among dietary treatments, suggesting that biofloc quality in individual tanks was independent of the dietary CP levels. This aligns with previous findings (Azim and Little 2008; da Silva et al. 2018).

Studies have shown that tilapia can utilize biofloc, absorbing 24%–32% of its nitrogen (Avnimelech and Kochba 2009; Ekasari et al. 2014; Green et al. 2019). It suggests that biofloc can potentially substitute a portion of dietary protein needs, allowing for a reduction in protein content in formulated feed. However, the extent of this substitution varies depending on fish life stage (Green et al. 2019). For instance, da Silva et al. (2018) reported successful reductions in dietary protein from 33% to 28% for tilapia weighing 10 to 60 g and 33% to 22% for those weighing 60 to 230 g. Similarly, Green et al. (2019) achieved a protein reduction from 36% to 31.3% for tilapia weighing 32 to 554 g. In the present study, we found a protein reduction from 32% to 24.5% for tilapia weighing 41 to 87 g.

The present study found no significant differences (p > 0.1) in VFI, HSI, and SSI among dietary treatments, while VI did vary significantly (p < 0.05). These observations are consistent with previous studies that reported no significant changes in HSI in tilapia fed with varying protein levels (Abdel-Tawwab et al. 2010; Kpundeh et al. 2015; da Silva et al. 2018). These findings may be attributed to the tilapia's ability to utilize carbohydrates efficiently (Kpundeh et al. 2015). Although not statistically significant, fish fed with the lowest protein diet had the highest numerical HSI value. Interestingly, a trend of increasing HSI with decreasing dietary protein level was observed, particularly from 24% to 16% CP, while the opposite trend was observed in fish fed with 32% to 24% CP diets. This discrepancy could be attributed to the role of biofloc as observed in growth performances; fish fed diets with 32% to 24% protein showed similar growth performance, suggesting a potential contribution of biofloc to growth and production of tilapia.

Fish body composition analysis revealed no significant differences across dietary treatments. This aligns with the findings of Azim and Little (2008) and Green et al. (2019) who observed no significant differences in body composition of tilapia reared in biofloc systems with different protein feeds. However, studies in clean water systems have reported trends of increasing whole-body protein and decreasing body lipid with increasing dietary protein levels (Abdel-Tawwab et al. 2010). Similarly, other studies suggest that reduction in protein levels can lead to increased lipid deposition (Abdel-Tawwab et al. 2010; Kpundeh et al. 2015). The present study did not observe a specific trend in lipid deposition as in the study of da Silva et al. (2018), who also did not find any significant difference in body lipid for tilapia with an initial weight of 50 g. However, they did observe a reduction in lipid deposition with increasing dietary protein levels in another experiment with tilapia having initial mean weight of 10 g. These findings highlight the complex interactions between dietary protein, fish size, and biofloc utilization in BFT systems.

Visual observations throughout the experiment revealed no physical abnormalities in the fish. Haematological parameters also showed no significant differences (p > 0.05) among dietary treatments, suggesting that biofloc may contribute to improving the immune system of fish fed lower protein diets. This aligns with the findings of Hisano et al. (2019) who observed no significant differences in haematological parameters reared in biofloc systems with different protein feeds (36% vs. 28%). To the best of our knowledge, no published studies have investigated the effects of BFT and varying dietary protein levels (up to 16%) on tilapia haematology, making direct comparisons challenging. In a clear water system, however, Abdel-Tawwab et al. (2010) found significant differences in haematological parameters of tilapia fed with different protein levels. Further research is needed to elucidate the specific effects of biofloc on tilapia immune function in BFT systems with varying dietary protein levels.

Apart from suggesting dietary protein reduction for tilapia reared in BFT system, this study entails some limitations. The present study did not compare fish performances in BFT with and without solid management units; therefore, it was not possible to examine the impact of solid management on fish performances. In the present study, protein deposition in fish was not determined, which could explain the protein utilization as well as contribution of bioflocs protein in fish reared in BFT system. Furthermore, the study did not measure fish length as well as compare condition factors, which could explain the overall fish welfare interacting with rearing environment. Although the aim of the present study was to reduce dietary protein for tilapia reared in BFT system, economic analysis and cost-benefit comparison were not conducted. Comparing cost–benefit analysis could strengthen the impact of reducing the dietary protein content for tilapia raised in the BFT system for small-scale farmer.

Conclusion

This study demonstrates the potential of biofloc technology to reduce dietary protein requirements in tilapia aquaculture. A protein reduction from 32% to 24.5% in a small-scale BFT system maintained fish growth and health, suggesting a cost-effective approach for tilapia production. Further protein reduction (20%–16%) resulted in lower growth performance, highlighting the importance of optimizing dietary protein levels within BFT systems. Interestingly, dietary protein levels did not significantly affect floc composition, fish body composition, or various haematological parameters of fish.

Since protein is the most expensive item in commercial aqua feeds, lowering protein content would reduce overall production cost in BFT. However, this study also highlights the need for further research on BFT management practices. While biofloc offers a natural protein source and reduces reliance on expensive commercial feeds, the accumulation of solids from exogenous carbon addition and nitrogenous waste can pose challenges. This study did not address solid management, which could potentially impact overall production efficiency through increased sludge removal and partial water exchange requirements. Further research should focus on developing cost-effective strategies for managing solids in low-cost BFT systems to optimize production and minimize environmental impact.

Author Contributions

Sudip Debnath: Conceptualization; methodology; writing—original draft; funding acquisition. Md. Shahin Parvez: Conceptualization; writing—review and editing; resources; project administration. Sayma Sadia: Investigation; data curation; writing—review and editing. K M Rakibur Hossain: Data curation; investigation; writing—review and editing. Md. Nazmul Ahsan: Conceptualization; methodology; writing—review and editing; supervision.

Acknowledgements

The authors would like to acknowledge the financial support provided by Khulna University Research Cell, Khulna University, Bangladesh, to conduct the experiment. The authors would also like to acknowledge the contribution of Anup Kumar Karmokar and Sk Riazul Islam during the experiment.

Ethics Statement

The study was carried out in accordance with the guidelines approved by Animal Ethics Committee of Khulna University, Khulna-9208. (Research Ref. no.: KUAEC-2021/03/06).

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data will be available from corresponding author upon reasonable request

Peer Review

The peer review history for this article is available at .

References

Abbaszadeh, A., V. Yavari, S. J. Hoseini, et al. 2019. “Effects of Different Carbon Sources and Dietary Protein Levels in a Biofloc System on Growth Performance, Immune Response Against White Spot Syndrome Virus Infection and Cathepsin L Gene Expression of Litopenaeus vannamei.” Aquaculture Research 50: 1162–1176. [DOI: https://dx.doi.org/10.1111/are.13991].