Dietary protein levels for juvenile matrinxã Brycon amazonicus produced in biofloc and clear water systems

doi:10.21203/rs.3.rs-4319897/v1

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Dietary protein levels for juvenile matrinxã Brycon amazonicus produced in biofloc and clear water systems

https://doi.org/10.21203/rs.3.rs-4319897/v1

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This study evaluated potential for rearing the matrinxã, Brycon amazonicus, under different crude protein (CP) levels, using production performance, water quality and animal welfare as indicators. To achieve this, 720 juvenile fish (3.65 ± 0.17 g) were distributed across 24 experimental units (200 L) and fee study lasted 60 days. The study followed a factorial design (2x4), with two production systems: Biofloc technology (BFT) and clear water (CW), and four levels of crude protein (CP = 24%, 30%, 36%, and 42%) in the diet, with three repetitions for each. The study found that the BFT system showed a lower total ammonia level. However, the production system did not have any effect on pH or nitrite concentration. Oxygen levels were found to be lower in the 42% CP group. Fish produced in the BFT exhibited better performance of apparent feed conversion, productivity, and survival. The production system affected total leukocytes and neutrophils in the blood. There was no significant difference in carcass crude protein content and the ether extract content was higher in the animals from the BFT system. Nutritional composition of the biofloc showed differences for ash, with lower levels in the 42% CP treatment. In conclusion, juvenile matrinxã can be fed a diet containing 30% CP in both production systems. Furthermore, the results demonstrated that biofloc did not confer a direct benefit to matrinxã juveniles in terms of their feeding, however BFT represented a promising alternative for increasing the survival and improved animal welfare during the rearing phase.

Amazonian fish

production systems

crude protein

feed management

BFT

Biofloc technology can increase the survival rate of juvenile matrinxã.

It is recommended to feed juvenile matrinxã with diet containing 30% crude protein.

The production performance of matrinxã was better with the BFT system.

Matrinxã did not consume sufficient microbial flocs to compensate for their growth.

Among current production technologies, the biofloc system (BFT) can be considered sustainable in terms of the use of water, physical space, and generation of effluents (Souza et al. 2019; Jatobá et al. 2019; Durigon et al. 2020; Khanjani et al. 2024a). This technology guarantees an additional source of food for aquatic organisms through the conversion of nitrogenous waste into microbial flocs (Silva et al. 2018; Khanjani et al. 2022; Dos Santos et al. 2021; 2023a, b; McCusker et al. 2023), in addition to contributing to the improvement of water quality, having a direct influence on the performance, reproduction and greater productivity of aquatic organisms (Dos Santos et al. 2023a; Khanjani et al. 2024b). The water in the system can be reused in subsequent production cycles, due to the presence of a microbiota that acts in the cycling of nitrogenous waste, and which, when kept in suspension in the water column by continuous aeration, can be used as a nutritional source for the farmed animals (Wasielesky-Jr et al. 2013;Vadhel et al. 2020; Dos Santos et al. 2023a; McCusker et al. 2023).

When consumed as food, microorganisms in the BFT system can reduce the crude protein content of the feed and/or the feed supply, making the system more economical and contributing to fish performance (Avnimelech 2007; Crab et al. 2010; Wang et al. 2015; Lara et al. 2017; Dos Santos et al. 2023a, b; Khanjani et al. 2023). The potential application of this technology has been evaluated on juvenile tambaqui Colossoma macropomum, a fish native to the Amazon, as demonstrated by Dos Santos et al. (2021, 2023a, b). The use of the BFT system can be a valuable production strategy for increasing the production of fish species with greater economic interest. This method has already been successfully implemented in other countries for shrimp species, as demonstrated by Emerenciano et al. (2013b, c), for Pacific white shrimp (Litopenaeus vannamei), red tilapia (Oreochromis niloticus), and mussels (Perna viridis) (Ekasari et al. 2014).

In this sense, the matrinxã has high commercial interest due to its good acceptance in the consumer market and easy adaptation to confined systems (Nascimento et al. 2020), According to Mattos et al. (2013), it has great potential in the Amazon region and other regions of Brazil due to its omnivorous feeding habits and to its excellent production performance rates in various farming systems. However, despite being a species with great productive/economic potential, its production technology still needs to be better structured (IBGE 2021), especially in the larviculture and rearing phase of the species, which presents intraspecific cannibalism and aggressive behavior (Carvalho et al. 2018; Ferreira et al. 2020; Izel-Silva et al. 2024). It can be demonstrated that the reduction in luminosity due to the turbidity of the water in the biofloc system contributes to a reduction in aggressive behaviour and an increase in the productivity of the species, as previously demonstrated by Izel-Silva et al. (2024). A reduction in light intensity in the water leads to an increase in melatonin production, which in turn reduces the aggressive behaviour of matrinxã larvae and juveniles (Lopes et al. 2018; Amaral et al. 2020; Souza-Cornélio et al. 2021). It may also contribute to the reduction of protein levels in the feed, as already studied with tilapia (Oreochromis sp.) (Day et al. 2016; Luo et al. 2017; Silva et al. 2018) and carp (Cyprinus sp.) (Castro et al. 2016; Najdegerami et al. 2016) in the juvenile phase.

Furthermore, matrinxã production is still in its early stages despite the species' market potential. It is important to study production management strategies, such as the BFT system, which can increase productivity and reduce production costs by providing additional nutrients to the fish in situ, such as microbial protein. Dos Santos et al. (2023a, b) discovered that it is feasible to decrease the crude protein levels in tambaqui feed without compromising performance or animal welfare, as well as improving the digestibility and absorption of food. Although the matrinxã is not a filter-feeding species like the tambaqui, it does consume zooplankton in its juvenile stage, and in the natural environment, fish between 25 and 50 mm standard length, for example, can have up to 80% of their diet made up of zooplankton microorganisms (mainly microcrustaceans) and insect larvae (Leite 2004). As a result, it is not really known how much the matrinxã could consume the microorganisms present in the BFT or how significant this consumption could be in reducing the food offered or the protein in the diet. Thus, the aim of this study was to determine the optimal percentage of crude protein in the diet of juvenile matrinxã that would promote the best zootechnical performance and welfare for the species, without compromising water quality in BFT system.

2.1 Ethical approval and experimental design

The experiment was carried out at the Laboratory of Physiology Applied to Fish Farming (LAFAP), of the Aquaculture Station of the Technology and Innovation Coordination (COTEI) from the National Amazon Research Institute – INPA, Manaus – AM; and was approved by the Ethics Committee on the Use of Animals – CEUA of INPA (Nº 040/2020, SEI 01280.001374/2020-68).

Juvenile matrinxã weighing approximately 0.5 g were obtained from a commercial fish farm and acclimatized for 40 days in 2000 L fiberglass tanks with Bioflocs and constant aeration. The study was conducted for 60 days in a factorial design (2×4), containing two production systems (bioflocs – BFT and clear water – CW) and four levels of crude protein in the diet (24%, 30%, 36% and 42%), with three repetitions each. Juvenile matrinxã (720 fish; 3.6 ± 0.1 g) were distributed in 24 circular polyethylene tanks of 500 L (filled with 200 L of water) with intense and continuous aeration, using microperforated hoses to suspend particulate matter in the water column. Each experimental unit (EU) received 30 matrinxã juveniles (equivalent to 150 fish/m³).

The experimental units of treatments in the BFT system received an inoculum of bioflocs matured in the laboratory, corresponding to 50% of the volume used in each experimental unit, following the Krummenauer et al. (2012) methodology. In CW treatments, 50% water exchange occurred daily throughout the experimental period.

2.2 Experimental diets

The feeds (Table 1) used in the experiment were isocaloric, the ingredients of each diet were ground to 1 mm, homogenized in a mixer and processed in a commercial extruder, model INBRA MX-80 (Inbramaq, Ribeirão Preto, Brazil), and the pellets were dried by forced ventilation (55°C). The size of the pellets was 2–4 mm. The animals were fed three times a day (8 am, 12 pm and 5 pm) until apparent satiety, with the amount adjusted daily by observing the food leftovers after 15 minutes of offering the food.

Table 1

Formula and proximal composition of the experimental diets with different levels of crude protein for feeding *Brycon amazonicus* juvenile in biofloc and clear water systems for period of 60 days.
Ingredients (%)	Crude protein levels
Ingredients (%)	24%	30%	36%	42%
Soybean meal	10.45	25.35	29.72	30.58
Corn grain	44.42	30.48	16.17	3.49
Corn gluten	12.00	8.00	13.00	19.50
Corn starch	10.00	10.00	10.00	10.00
Offal bran	6.96	11.50	15.00	19.50
Fish meal	5.00	5.00	5.00	5.00
Meat and bones bran	2.00	2.00	2.00	2.00
Wheat flour	2.00	2.00	2.00	2.00
Soy oil	2.80	1.77	1.34	0.50
Dicalcium phosphate	1.80	1.05	0.60	0.00
Limestone	1.05	0.83	0.65	0.46
Inert	0.00	0.50	3.00	5.45
L-Lysine	0.60	0.60	0.60	0.60
DL-Methionine	0.40	0.40	0.40	0.40
Mineral supplement	0.50	0.50	0.50	0.50
BHT	0.02	0.02	0.02	0.02
Total	100.00	100.00	100.00	100.00
Centesimal composition (%)
Dry matter	91.24	90.23	94.13	90.64
Crude protein	24.40	30.20	36.90	42.80
Ethereal extract	6.80	4.80	4.20	4.00
Crude fiber	1.30	1.50	1.90	2.00
Ash	4.80	4.58	6.84	6.82
Gross energy (Kcal)	4,086.85	4,086.85	4,086.85	4,086.84

Premix: folic acid (minimum) 100.00 mg/kg; nicotinic acid (minimum) 10.00 g/kg; pantothenic acid (minimum) 5,000.00 mg/kg; BHT (minimum) 5,000.00 mg/kg; biotin (minimum) 50.00 mg/kg; cobalt (minimum) 30.00 mg/kg; copper (minimum) 1,000.00 mg/kg; choline (minimum) 80.00 g/kg; iron (minimum) 7,500.00 g/kg; iodine (minimum) 250.00 mg/kg; inositol (minimum) 4,000.00 g/kg; manganese (minimum) 2,000.00 mg/kg; magnesium (minimum) 600.00mg/kg; potassium (minimum) 2,00.00 mg/kg; selenium (minimum) 70.00 mg/kg; vitamin A (minimum) 2,000,000.00 IU/kg; vitamin B1 (minimum) 2,000.00 mg/kg; vitamin B2 (minimum) 4,000.00 mg/kg; vitamin B12 (minimum) 10,000.00 mcg/kg; vitamin C (minimum) 80.00 g/kg; vitamin B6 (minimum) 5,000.00 mg/kg; vitamin D3 (minimum) 600,000.00 IU/kg; vitamin E (minimum) 15,000.00 IU/kg; vitamin K3 (minimum) 700.00 mg/kg; zinc (minimum) 7,500.00 mg/kg.

2.3 Water quality analyses

Temperature, pH, dissolved oxygen and salinity were checked daily using a multiparameter probe (model Akso AK88). The total ammonia concentration (Verdouw et al. 1978) and nitrite (Boyd and Tucker 1992) in water was assessed three times per week. Nitrate (adapted from Papaspyrou et al., 2014) and turbidity (NTU; portable turbidimeter - Hanna, HI93703C) were measured once a week.

Total suspended solids levels were checked every week by filtering 20 mL of water through GF 50-A glass fiber filters (Strickland and Parsons 1972). The volume of settleable solids was monitored twice a week, using ImHoff cones (Avnimelech 2007). The alkalinity (mg/L of CaCO₃) was monitored three times a week through acid-base titration, using the methyl-orange indicator (APHA 1998), with a target of maintaining a level above 50 mg/L; and when necessary, corrections were made with sodium bicarbonate (Avnimelech 2009). In the CW system, the alkalinity was always adjusted after changing the water in the system.

2.4 Zootechnical performance analyses and somatic indices

To evaluate productive performance, the weight of each fish was measured using biometric assessments at the beginning and end of the experiment. For this, the animals were anesthetized in a benzocaine solution (100 mg/L) and from the weight obtained the following was calculated: average final weight (FW) = final weight of all fish / number of fish; daily weight gain (DWG) = (average final weight (g) – average initial weight (g)) / days; specific growth rate (SGR) = [(ln final average weight (g) – ln initial average weight (g)) / days] × 100; apparent feed conversion (AFC) = feed intake (g) / weight gain (g); survival (S%) = (number of animals at the end of the experiment / number of animals at the beginning of the experiment) × 100 and productivity (g/m³) = (total biomass/tank volume) / 1,000; protein efficiency ratio (PER%) = average weight gain/(crude protein in the diet × average feed intake).

For the analysis of somatic indices, three fish from each experimental unit were euthanized in a benzocaine solution (300 mg/L) for dissection of the total viscera and liver, for subsequent weighing and following calculations: viscerosomatic index (%) = (viscera weight / live weight) × 100; and hepatosomatic index (%) = (liver weight / live weight) × 100.

2.5 Blood collection and determination of blood parameters

To evaluate blood parameters, three fish from each experimental unit were previously anesthetized in a benzocaine solution (100 mg/L) and had blood collected through puncture of the tail vein using heparin anticoagulant (10 µL). The following were analyzed: hematocrit (Ht), using the microhematocrit method; hemoglobin concentration ([Hb]) by the cyanmethemoglobin method and using commercial reagents; erythrocyte count (RBC), using a Newbauer© camera and formaldehyde-citrate solution. The total and differential leukocyte count occurred through blood samples previously stained with May-Grünwald-Giemsa-Wright (MGGW) according to Tavares-Dias and Moraes (2003). The number of leukocytes and thrombocytes were quantified from the 2000 erythrocyte count and for the differential blood cell count, 200 leukocytes were identified and classified as lymphocytes, monocytes, and neutrophils.

From the Ht, [Hb] and RBC results, the Wintrobe indices were calculated: mean corpuscular volume (MCV) = hematocrit × 10 / number of erythrocytes (fl); mean corpuscular hemoglobin (MCH) = [Hb] × 10 / RBC (pg); and mean corpuscular hemoglobin concentration (MCHC) = [Hb] × 100 / Ht (g/dL). The values of cholesterol, triglycerides and glucose in plasma were determined by the enzymatic colorimetric method using commercial kits; and total proteins were determined by the modified biuret method.

2.6 Nutritional fish and biofloc composition assessments

To determine the proximal composition of the fish, three animals were collected per experimental unit, euthanized in benzocaine (300 mg/L) and subsequently stored in a -18°C freezer. The samples were dehydrated in a ventilated oven at 55°C until constant weight and crushed in a multiprocessor to obtain uniform samples. Dry matter (%) was determined in an oven at 105°C, crude protein was obtained using the micro-Kjehldal method, ether extract analyzed through continuous extraction with petroleum ether in a Soxleth extractor, and ash collected through burning in a muffle furnace at 550°C (AOAC 2012).

For proximal analysis of the flocs, at the end of the experiment, 80 L of suspended bioflocs were collected from each experimental unit and transferred to conical tanks with a capacity of 100 L. After decanting for 3 hours, approximately one liter of sedimented material was collected and stored at -18°C. For analysis, this material was dried in a ventilated oven at 55°C and macerated to obtain uniform samples for determination of crude protein, ether extract and ash (AOAC 2012).

2.7 Statistical analysis

The results of water quality, productive performance and hematological parameters were checked for data normality using the Shapiro-Wilk test with the InfoStat statistical program, and homogeneity using the Bartlett test with “software R” for the appropriate analyses. For the variables that met the assumptions, two-way analysis of variance (two-way ANOVA) was performed, and the means were compared using the Tukey test (CV < 10%), with a significance level of 5%. For the proximal composition of the flocs, one-way analysis of variance (one-way ANOVA) was performed.

3.1 Water quality

No significant temperature difference was observed between the treatments. The concentration of total ammonia in the water was only affected by the production system, with lower levels observed in the biofloc system. The production system had no effect on pH or nitrite concentration. Salinity was higher and dissolved oxygen (DO) was lower in BFT. There were no significant differences in pH and nitrite levels at the three lowest crude protein (CP) levels. OD was higher in the water with fish fed 24% CP and the two lowest CP levels resulted in lower salinity (Table 2).

Table 2. Mean ± standard deviation of water quality variables from biofloc (BFT) and clear water (CW) systems verified over 60 days during the production of Brycon amazonicus matrinxã juveniles fed with different levels of crude protein in the diet (24, 30, 36 and 42%).

Variables	T (ºC)	DO (mg.L^− 1)	pH	Salinity (ppt)	Ammonia (mg.L^− 1)	Nitrite (mg.L^− 1)
Production system
BFT	27.04 ± 0.13^A	6.03 ± 0.10^B	7.19 ± 0.28^A	1.45 ± 0.13^A	0.45 ± 0.15^B	0.49 ± 0.22^A
CW	27.04 ± 0.12^A	6.16 ± 0.11^A	7.27 ± 0.18^A	0.06 ± 0.03^B	1.16 ± 0.50^A	0.38 ± 0.30^A
Protein levels
24%	26.98 ± 0.14^A	6.26 ± 0.10^A	7.43 ± 0.06^A	0.69 ± 0.68^B	0.53 ± 0.25^A	0.22 ± 0.09^B
30%	27.08 ± 0.08^A	6.08 ± 0.11^B	7.26 ± 0.08^AB	0.75 ± 0.76^AB	0.80 ± 0.35^A	0.39 ± 0.17^AB
36%	27.06 ± 0.12^A	6.07 ± 0.12^B	7.20 ± 0.14^AB	0.83 ± 0.82^A	0.83 ± 0.50^A	0.46 ± 0.27^AB
42%	27.03 ± 0.17^A	5.98 ± 0.08^B	7.03 ± 0.24^B	0.79 ± 0.79^AB	1.07 ± 0.77^A	0.68 ± 0.28^A
Two-way ANOVA
System	NS	< 0.0015	NS	< 0.0001	< 0.0001	NS
Protein	NS	< 0.0002	< 0.0016	< 0.0391	NS	< 0.0196
Interaction	NS	NS	NS	NS	NS	NS

Abbreviations: T: temperature; DO: dissolved oxygen; NS: not significant.

Means followed by different letters in the same column differ according to ANOVA and Tukey 's test (p < 0.05).

The study found a significant interaction between CP level and production system in relation to nitrate, alkalinity, total suspended solids (TSS), settleable solids (SS), and turbidity. However, only alkalinity was affected by the different levels of CP in clear water. On the other hand, reducing the CP level in the diet resulted in lower concentrations of nitrate, TSS, SS, and turbidity, and an increase in the concentration of alkalinity in BFT (Table 3).

Table 3. Mean ± standard deviation of water quality variables from biofloc (BFT) and clear water (CW) systems verified over 60 days during the production of Brycon amazonicus matrinxã juveniles fed with different levels of crude protein in the diet (24%, 30%, 36% and 42%).

	Nitrate (mg.L^-1)	Alkalinity CaCO₃ (mg.L^-1)	TSS (mg.L^-1)	SSV (mg.L^-1)	Turbidity (UNF)
WC Protein level (%)
24	7.76±0.58^Ba	48.11±1.64^Ba	19.67 ± 10.67^Ba	0.10 ± 0.10^Aa	3.53 ± 1.41^Ba
30	12.84±8.01^Ba	48.32±0.80^Ba	25.00 ± 7.05^Ba	0.20 ± 0.001^Ba	4.16 ± 1.08^Ba
36	9.11±0.53^Ba	44.04±2.24^Aab	27.67 ± 2.93^Ba	0.23 ± 0.06^Ba	5.14 ± 1.09^Ba
42	8.74±2.17^Ba	40.87±1.86^Ab	29.00 ± 2.63^Ba	0.23 ± 0.06^Ba	6.07 ± 0.64^Ba
BFT Protein level (%)
24	96.49±10.76^Ab	63.79±1.78^Aa	141.00 ± 27.37^Ad	3.30 ± 1,04^Ac	50.32 ± 8.88^Ad
30	104.35±19.68^Ab	56.07±1.28^Ab	177.50 ± 24.07^Ac	10.90 ± 4.35^Ab	72.74 ± 6.78^Ac
36	152.29±20.00^Aa	46.05±4.95^Ac	207.00 ± 19.83^Ab	12.90 ± 2.19^Ab	86.73 ± 8.79^Ab
42	166.82±32.80^Aa	36.48±4.64^Ad	244.67 ± 15.73^Aa	21.50 ± 9.87^Aa	96.08 ± 5.09^Aa
Two-way ANOVA
System	< 0.0001	< 0.0003	< 0.0001	< 0.0001	< 0.0001
Protein	< 0.0037	< 0.0001	< 0.0002	< 0.0082	< 0.0001
Interaction	< 0.0027	< 0.0001	< 0.0010	< 0.0092	< 0.0001

Abbreviations: TSS: total suspended solids; SSV: settleable solids volume; NS: not significant.

Means followed by different letters, uppercase for comparation in production systems and lowercase for protein levels, differ according to ANOVA and Tukey 's test (p < 0.05).

3.2 Zootechnical performance and somatic indices

No significant differences were found in final weight (FW), specific growth rate (SGR), daily weight gain (DWG), protein efficiency ratio (PER) and feed consumption between the production systems. However, fish produced in the biofloc system showed higher productivity and survival rates, and lower apparent feed conversion (AFC) (Table 4).

The 24% CP level was generally the worst performer, while there was no significant difference between the 30% and 42% CP levels. Additionally, the CP levels did not affect animal survival. In the biofloc system, the hepatosomatic (HSI) and somatic index (GVI) were higher. CP levels did not have a significant effect on the HSI. However, juveniles that were fed with 42% CP had a lower GVI compared to those fed with 24% CP (Table 4).

Table 4. Mean ± standard deviation of productive parameters and somatic indices of Brycon amazonicus matrinxã juveniles produced in biofloc (BFT) and clear water (CW) systems fed with different levels of crude protein in the diet (24%, 30%, 36% and 42%), for 60 days.

Variables

FW (g)

SGR (%/day)

DWG (g/day)

AFC

Productivity (g/m³)

S (%)

Feed consumption (g)

VSI (%)

HSI (%)

BFT

66.16±18.33^A

4.78±0.53^A

1.04±0.30^A

1.06±0.15^B

7.68±2.38^A

76.95±9.15^A

1458.72±374.34^A

11.27±2.94^A

2.45±0.48^A

64.34±24.83^A

4.62±0.75^A

1.02±0.41^A

1.16±0.13^A

4.57±1.39^B

50.00±12.31^B

929.48A±309.71^A

8.32±2.25^B

1.59±0.44^B

Protein Levels

24%

41.23±19.19^B

3.94±0.67^B

0.62±0.32^B

1.25±0.07^A

3.60±1.41^B

60.00±14.46^A

758.20±328.86^B

12.30±3.82^A

2.13 ±0.94^A

30%

71.81±15.436^A

4.95±0.36^A

1.14±0.26^A

1.09±0.13^AB

6.69±2.05^A

62.78±15.83^A

1303.68±285.43^A

9.56±2.28^AB

2.04±0.48^A

36%

66.88±17.10^AB

4.78±0.49^A

1.06±0.28^AB

1.06±0.10^B

6.74±2.27^A

68.33±18.23^A

1295.29±452.90^A

9.59±2.55^AB

2.03±0.64^A

42%

81.07±12.38^A

5.13±0.72^A

1.29±0.21^A

1.04±0.17^B

7.46±2.46^A

62.78±23.52^A

1419.23±381.00^A

7.73±1.22^B

1.89±0.51^A

Two-way ANOVA

System

< 0.0424

< 0.0001

< 0.0042

< 0.0006

Protein

< 0.0082

< 0.0052

< 0.0083

< 0.0092

< 0.0001

< 0.0007

< 0.0181

Interaction

PER* (%)

Production system

Protein Level

Two-way ANOVA

24%

30%

36%

42%

System

BFT

3.38±0.18^Aa

3.46±0.32^Aa

2.64±0.10^Ab

2.51±0.26^Ab

Protein

< 0.0001

3.38±0.02^Aa

2.91±0.28^Bb

2.74±0.10^Abc

2.11±0.20^Ac

Interaction

< 0.0197

Abbreviations: FW: final weight; SFR: specific growth rate; DWG: daily weight gain; AFC: apparent feed conversion; S: survival; VSI: viscerosomatic index; HIS: hepatosomatic index; NS: not significant.

Means followed by different letters in the same column differ according to ANOVA and Tukey’s test (p < 0.05).

* Means followed by different letters, uppercase for comparation in production systems and lowercase for protein levels, differ according to ANOVA and Tukey 's test (p < 0.05).

3.3 Blood parameters

The blood parameters of the fish reared in the two systems (BFT and CW) are shown in Table 5 and 6. No significant differences were found between the systems. The blood parameters of the juveniles fed different CP levels in the diet also showed no significant differences, except for the haemoglobin (Hb) concentration variable. The 42% CP level in the diet showed a higher haemoglobin concentration than those fed a 24% CP diet, while there was no difference between the three highest CP levels.

The study found an interaction between production systems and CP level for the haematocrit variable. Juveniles produced in the BFT system had a lower average haematocrit than those produced in the CW system. Haematocrit was not affected by CP levels. However, Table 5 showed that there was no significant difference in higher CP levels in BFT. Table 6 showed the profile of defense cells and were observed the statistical differences only between the BFT and CW systems in terms of total leucocytes and neutrophils, with higher averages in matrinxã juvenile reared in BFT compared to CW.

Table 5. Mean ± standard deviation of blood parameters of Brycon amazonicus matrinxã juveniles produced in biofloc (BFT) and clear water (CW) systems fed with different levels of crude protein (24%, 30%, 36% and 42%) for 60 days.

Variables

Hb (gd/L)

RBC (×106/μL)

MCV (fl)

MCH (pg)

MCHC (g/dL)

Cholesterol (mg/dL)

Triglycerides (mg/dL)

Glucose (mg/dL)

Total protein (g/dL)

Production system

BFT

8.80±2.42^A

1.71±0.22^A

202.23±35.57^A

54.75±16.37^A

26.74±8.18^A

201.71±33.62^A

613.75±166.58^A

107.11±9.06^A

3.11±0.42^A

8.91±1.01^A

1.72±0.27^A

215.23±44.87^A

53.10±10.41^A

24.90±3.03^A

204.53±47.07^A

585.40±188.19^A

114.62±11.7^A

3.31±0.09^A

Protein Levels

24%

8.26±3.88^B

1.71±0.20^A

197.00±34.56^A

53.50±20.16^A

27.96±11.24^A

222.74±43.59^A

689.39±145.04^A

103.86±4.14^A

3.08±0.44^A

30%

8.25±0.93^AB

1.78±0.21^A

205.75±26.82^A

50.08±6.39^A

24.51±2.82^A

197.78±46.39^A

599.84±195.99^A

103.98±2.35^A

3.17±0.30^A

36%

9.02±1.11^AB

1.65±0.29^A

216.93±56.70^A

53.77±14.54^A

26.30±3.16^A

193.82±35.05^A

566.03±160.22^A

113.03±16.29^A

3.09±0.16^A

42%

9.28±0.78^A

1.71±0.26^A

223.23±37.65^A

55.33±9.63^A

24.98±3.19^A

198.13±32.58^A

543.03±180.70^A

122.59±3.19^A

3.51±0.33^A

Two-way ANOVA

System

Protein

< 0.0262

Interaction

Hematocrit* (%)

Production system

Protein Level

Two-way ANOVA

24%

30%

36%

42%

System

BFT

30.06±2.05Bb

36.56±5.54Aa

35.17±4.64Aab

37.39±1.75Aa

Protein

< 0.0092

36.39±3.58Aa

36.33±4.04Aa

32.39±3.33Aa

37.56±4.88AA

Interaction

< 0.0223

Abbreviations: Ht: hematocrit; Hb: hemoglobin; RBC: red blood cell count; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration.

Means followed by different letters in the same column differ according to ANOVA and Tukey’s test (p < 0.05).

* Means followed by different letters, uppercase for comparation in production systems and lowercase for protein levels, differ according to ANOVA and Tukey 's test (p < 0.05).

Table 6. Mean ± standard deviation of the total and differential leukocytes counts of Brycon amazonicus matrinxã juveniles produced in biofloc (BFT) and clear water (CW) systems fed with different levels of crude protein in the diet (24%, 30%, 36% and 42%) for 60 days.

	Thrombocytes (×10³ μL^‑1)	Total Leukocytes (×10³ μL^‑1)	Lymphocytes (×10³ μL^‑1)	Monocytes (×10³ μL^‑1)	Neutrophils (×10³ μL^‑1)
Production system
CW	1.11±0.89^A	29.74±9.74^B	24.83±8.03^A	4.82±2.48^A	0.09±0.22^B
BFT	1.21±1.45^A	36.31±12.54^A	29.35±9.38^A	6.16±3.47^A	0.81±1.11^A
Protein Levels
24%	1.12±1.46^a	31.28±11.38^a	26.15±7.51^a	4.62±3.57^a	0.51±1.27^a
30%	1.15±0.97^a	29.17±13.04^a	23.70±9.62^a	4.99±3.49^a	0.48±0.82^a
36%	0.74±0.78^a	34.63±10.70^a	28.18±8.25^a	6.26±2.99^a	0.19±0.35^a
42%	1.60±1.42^a	37.42±10.86^a	30.60±9.78^a	6.20±2.01^a	0.63±0.83^a
Two-way ANOVA
System	NS	< 0,0001	NS	NS	< 0,0001
Protein	NS	NS	NS	NS	NS
Interaction	NS	NS	NS	NS	NS

Means followed by different letters in the same column differ according to ANOVA and Tukey’s test (p < 0.05). NS: not significant

3.4 Nutritional fish and biofloc composition

Table 7 displays the average composition values of the juvenile matrinxã at the end of the study. There was no significant difference in crude protein content in the carcass between the treatments. However, the content of ether extract was higher in the juveniles from the BFT system. Additionally, a significant interaction was observed for dry matter and ash. It was found that there was no difference in the dry matter content in the nutritional composition of matrinxãs reared in a BFT system and fed CP levels of 30 to 42%. The nutritional composition of the flake did not show any significant differences in CP and ether extract at different levels of crude protein. However, the mineral matter content was lower when the percentage of CP in the diet was higher (Table 8).

Table 7. Mean ± standard deviation of the proximal composition of Brycon amazonicus matrinxã juveniles produced in biofloc (BFT) and clear water (CW) systems and fed with different levels of crude protein in the diet (24%, 30%, 36% and 42%) for 60 days.

	Dry matter*	Crude protein	Ethereal extract	Ash*
BFT	-	54.31±1.52^A	22.04±2.36^A	-
CW	-	55.03±1.70^A	20.35±1.69^B	-
Protein Levels (%)
24	-	53.94±1.07^a	21.18±2.88^a	-
30	-	54.35±1.90^a	21.27±1.53^a	-
36	-	55.50±1.34^a	20.90±2.49^a	-
42	-	55.09±1.77^a	21.50±1.95^a	-
BFT
24	88.44±0.96^Ab	-	-	8.37±0.47^Aa
30	91.27±2.09^Aa	-	-	7.84±0.39^Bb
36	90.07±1.24^Aab	-	-	8.76±0.80^Aa
42	91.97±0.50^Aa	-	-	8.07±0.43^Aab
CW		-	-
24	90.24±1.92^Aa	-	-	8.45±0.85^Aa
30	91.23±1.97^Aa	-	-	8.94±0.74^Aa
36	89.09±2.50^Aa	-	-	8.80±0.70^Aa
42	89.40±2.20^Ba	-	-	8.84±0.65^Ba
Two-way ANOVA
System	NS	NS	< 0.0058	< 0.0473
Protein	< 0.0389	NS	NS	NS
Interaction	< 0.0365	NS	NS	< 0.0327

Values in % dry basis (db). NS: not significant.

Means followed by different letters in the same column differ according to ANOVA and Tukey's test (p<0.05), or Duncan's test (p<0.05).

* Means followed by different letters, uppercase for comparation in production systems and lowercase for protein levels, differ according to ANOVA and Tukey's test (p<0.05), (p<0.05).

Table 8. Mean ± standard deviation of the proximate composition of the biofloc collected in the biofloc system with the production of Brycon amazonicus matrinxã juveniles fed with different levels of crude protein in the diet (24%, 30%, 36% and 42%) for 60 days.

Protein levels	Crude protein (%)	Ethereal extract (%)	Ash (%)
24%	31.07±0.76^a	4.30±0.52^a	28.87±1.33^a
30%	32.07±1.78^a	5.30±0.46^a	29.37±1.05^a
36%	32.30±2.17^a	4.50±0.56^a	29.67±3.70^a
42%	33.57±0.38^a	4.70±0.62^a	24.53±2.16^b
One-way ANOVA	NS	NS	< 0.0003

Values in % dry basis (db). NS: not significant.

Means followed by different letters in the same column differ according to ANOVA and Tukey’s test (p < 0.05).

4.1 Water quality

The benefits of BFT technology include improved performance, welfare, water quality maintenance, and a source of protein (Khanjani and Sharifinia 2020; Mugwanya et al. 2021; Ogello et al. 2021; Dos Santos et al. 2023a, b). This system allows for a reduction in crude protein levels in feed and an increase in productivity (Dos Santos et al. 2023a).

The treatments with higher levels of crude protein (30, 36 and 42%) resulted in a lower concentration of dissolved oxygen in the water because of crude protein in diet and the accumulation of nitrogenous excreta. The bioflocs contain heterotrophic bacterial biomass, which, along with the oxidation processes of organic matter, consumes dissolved oxygen and reduces its concentration (Wasielesky et al. 2013; Manduca et al. 2021). However, the dissolved oxygen levels remain within the acceptable range for fish farming (Boyd, 1982).

The disparity in pH values between the protein levels can be attributed to the consumption of alkalinity by the chemoautotrophic bacteria and the alkalinity adjustments made with sodium bicarbonate to maintain optimal bacterial activity within the system. Furthermore, the metabolic processes of the animals and microorganisms within the BFT result in elevated CO2 levels, which subsequently lead to a reduction in pH. (Ebeling et al. 2006; Vinatea et al. 2010; Widanarni et al. 2012). However, these pH values remained within the ideal range for production systems, maintaining homeostasis, animal performance, and the nitrification process (Ebeling et al. 2006; Emerenciano et al. 2017; Figueroa-Espinoza et al. 2022).

The use of sodium bicarbonate to stabilize water alkalinity and pH in BFT systems (Poli et al. 2015; Emerenciano et al. 2017) resulted in an increase in salinity in this system. The alkalinity concentrations in both the BFT and clear water systems were influenced by the level of crude protein in the fish feed, in addition to the compound mentioned. The decrease in alkalinity values in the CW system may be attributed to the daily water renewal, which causes ion losses, unlike the BFT system (Tidwell 2012; Dos Santos et al. 2023a). The variation in alkalinity values among the studied systems is likely attributed to their distinct characteristics (Dos Santos et al. 2023a). The study, which tested different levels of CP in the feed in two production systems (BFT and clear water) using tambaqui, found higher alkalinity values in BFT.

Frequent monitoring of the concentration of total ammonia is necessary as it tends to increase with the amount of feed provided, biomass in the tanks, and the level of protein in the diet (Leira et al. 2017). Total ammonia results from the decomposition of organic matter, feed, and excreta. However, the study found no statistically significant difference in the average concentration of ammonia in the water across the different levels of crude protein in the diet. This may be attributed to the assimilation and oxidation efficiency of multitrophic bacteria (Ebeling et al. 2006; Hargreaves 2013). The BFT system showed statistically lower levels of ammonia, indicating the efficiency of ammonia assimilation by nitrifying and heterotrophic bacteria in the system (Ebeling et al. 2006). Similar results were reported by Dos Santos et al. (2021, 2023a).

There were no significant differences in nitrite concentrations between the BFT and CW production systems. This may be attributed to the change in water and the stabilization of nitrifying bacteria. However, higher average values were observed in the BFT system and in diets with higher protein levels, likely due to increased nitrogen intake from the feed. The interaction between the factors reflected these effects, with the BFT system providing a greater amount of nitrate compared to clear water. This was due to the presence of chemoautotrophic bacteria, which promoted a greater conversion of nitrogen compounds. The effect was intensified by the greater amount of nitrogen in the fish feed. Nitrite values, however, remained within the ideal range for production (Souza-Bastos et al. 2017; Luo et al. 2020; Manduca et al. 2021). The nitrate concentrations in the factors studied (Table 3) support the findings of Mansour and Esteban (2017) and Dos Santos et al. (2023a), who reported higher nitrate concentrations in the BFT system compared to AC. This was observed in tilapia raised under different carbon sources and protein levels, as well as tambaqui in two production systems (BFT and CW) and three crude protein levels, respectively.

In a BFT system, it is crucial to maintain levels of total suspended solids (TSS) below 500 mg/L and volume of settleable solids (SS) below 50 ml/L to ensure proper system function (Hargreaves 2013). The experiment's average values remained within the range used for other species, such as Nile tilapia raised in a biofloc system (Avnimelech 2011) and juvenile tambaqui (Dos Santos et al. 2023a). These species did not experience any mortality or clinical-behavioral changes. The BFT system used in this study showed that higher protein diets led to an increase in TSS and SS, resulting in greater nitrogen excretion and microbial floc formation (Azim and Little 2008; Silva et al. 2018). The reduction of light entering the BFT system had the effect of increasing the turbidity of the water. This may suggest better animal welfare, as lower light intensity has been shown to reduce aggressive behaviour in the species (Silva 2016; Lopes et al. 2018; Amaral et al. 2020; Souza-Cornélio et al. 2021).

4.2 Zootechnical performance, somatic indices, and blood parameters

Several studies have shown that the BFT system can enhance the productive performance of fish. This is because the microorganisms present in the biofloc system become bioavailable as a complementary food source (Silva et al. 2019; Dos Santos et al. 2021, 2023a, b; Pires et al. 2022). The study found that the performance of juvenile matrinxã in the two production systems evaluated was mainly related to the formation of microbial flocs in the BFT system. This suggests that the fish may be utilizing the flocs and experiencing better well-being, resulting in better performance compared to those reared in the clear water system (Dos Santos et al. 2023a).

The apparent feed conversion of the juveniles in the BFT system was found to be lower. This may have been due to the animals exhibiting a more optimal use of the food provided, given that the system's high turbidity reduces aggressive behaviour and stress among the animals, which also contributes to a higher survival rate of the specie. (Poli et al. 2015; Amaral et al. 2020; Souza- Cornélio et al. 2021; Izel-Silva et al. 2024). Furthermore, the presence of microorganisms and nutrients in the BFT can facilitate digestion and contribute to weight gain in the fish (Azim e Little 2008). This supports the findings of Luo et al. (2014), who observed a decrease in the feed conversion rate of Nile tilapia from 1–4 in a recirculating water system (RAS) to 1.2 in a biofloc system, resulting in a 22% increase in the final weight of the fish. Nevertheless, the outcomes of this study indicate that the matrinxã, despite not being filter feeders like the tilapia, may ingest particles from the BFT system. This is evidenced by the higher productivity and survival of the fish in the BFT system compared to the AC system. The quantities of BFT consumed by the matrinxã are insignificant for their growth or for the reduction of protein in the diet. However, there are potential benefits for the health and well-being of the animals, as indicated by the survival rate and some of the physiological parameters discussed below.

Based on the performance results, it is evident that a diet containing 24% crude protein does not meet the nutritional requirements of juvenile matrinxã in both systems. This leads to lower feed consumption and productivity, as well as higher feed conversion. Mattos et al. (2018) evaluated the effect of different levels of crude protein (30, 35, 40 and 45%) on juvenile matrinxã (approximately 3 g) in a continuous flow system. The study found no significant difference in the performance of the animals fed the three highest levels of crude protein in the diet. The difference observed in this study may be related to the production system and the composition of the nutrients in the diet. Sgnaulin et al. (2021) evaluated the impact of reducing protein in the diet on the production of Piaractus mesopotamicus. The researchers observed that fish reared in the BFT system had a lower requirement for crude protein in their feed due to the utilization of nutrients from the bioflocs. However, the present study found no interaction between the production system and the level of protein in the feed. This suggests that the juvenile matrinxã did not consume enough microbial flakes to increase or compensate for their growth in relation to the diets. Despite this, there was a noticeable improvement in feed conversion.

In addition to zootechnical performance parameters, body indices have been used to assess factors affecting nutrient assimilation and nutritional status in fish (Silva et al. 2018; Silva et al. 2019). This study found that the BFT and WC systems had a statistically significant effect on the viscerosomatic and hepatosomatic indices of juvenile matrinxã. The animals fed higher levels of protein had a lower viscerosomatic index, which resulted in better zootechnical performance. However, this effect did not affect the differences in the hepatosomatic index. This demonstrates that even when the animals were fed the lowest levels of protein, the levels of energy reserves in their livers were similar between treatments.

The blood parameters of juvenile matrinxã in the BFT and clear water systems did not indicate any damage to the fish's homeostasis. This suggests that the water quality and microbiological action did not induce stress in the animals. These results support the findings for Nile tilapia (O. niloticus) and tambaqui (C. macropomum) in BFT systems compared to clear water systems (Long et al. 2015; Dos Santos et al. 2021; 2023b; Figueroa-Espinoza et al. 2022). The reduction in protein levels in the diet did not harm the physiological homeostasis of the fish, which supports the findings of Dos Santos et al. (2023b). They also observed no significant effects on the indicators of metabolism and/or well-being of juvenile tambaqui when reducing protein levels from 32 to 24% in BFT and CW systems.

However, the fish showed a significant reduction in haemoglobin levels when they received the feed with the lowest protein content. This suggests a nutritional insufficiency that may have decreased oxygen demand and, as a result, reduced weight gain. Furthermore, while the number of erythrocytes and the mean corpuscular volume remained similar across production systems and protein levels, the haematocrit level exhibited an interaction between the two factors. Specifically, the CW system intensified the effects of protein restriction in the diet, resulting in an alteration of the haematocrit level in fish produced with the lowest protein level diet.

Total leucocytes and neutrophils showed statistical differences only between the juveniles from BFT and CW systems, with higher averages in matrinxã reared in BFT. As observed by Dos Santos et al. (2023b), this difference can be attributed to prolonged contact with the microorganisms present in the BFT, who found not only a higher number of monocytes in tambaqui (C. macropomum) raised in BFT, but also an increase in their respiratory activity.

4.3 Nutritional fish and biofloc composition

The nutritional composition of the food of aquatic organisms can be a determinant of nutrient assimilation (Silva et al. 2019). The biofloc system can achieve a balance between protein, lipids, and ash in food (Crab et al. 2012; Marinho-Pereira et al. 2020). The production of microorganisms in the system is affected by the type of feed and the crude protein level of the diets (Pires et al. 2022). The study shows that the nutritional composition of the animals was not affected by the production system. The levels of composition were similar between the BFT and AC systems in terms of crude protein. The results suggest that the CP in the feed can be reduced without compromising the efficiency of bioflocs as a feed supplement in BFT systems (Dos Santos et al. 2023a). The results for crude protein are comparable to those reported by Eid et al. (2021), but lower than the values reported by Figueroa-Espinoza et al. (2022) and Ferreira et al. (2011) who evaluated juvenile Nile tilapia in a biofloc system with experimental diets.

The bioavailability of protein, lipids, mineral matter, and microorganisms in microbial flocs within the BFT system can vary depending on the feed, stage of development of the cultivated organisms, and production management in the environment (Crab et al. 2012; Pires et al. 2022; Figueroa-Espinoza et al. 2022). The protein levels in the flakes, ranging from 31.0 to 33.5 percent, fall within the literature's recommended range of 12 to 49 percent (Emerenciano et al. 2013a; Dos Santos et al. 2023). Therefore, the protein content in bioflocs can vary depending on the farming system (age and C:N ratio) and the formulation of the added feed (Figueroa-Espinoza et al. 2022).

The study found that juvenile matrinxã can be fed with a diet containing 30% PB in both the BFT and AC systems. The results demonstrated that microbial flocs did not confer a direct benefit to matrinxã juveniles in terms of their feeding. However, the presence of flocs and increased turbidity in the water led to an increase in the survival rate of the juveniles and improved animal welfare. Consequently, it is crucial to monitor the system to ensure that it is functioning optimally and to prevent future losses, thereby promoting greater availability of juveniles in the BFT system for production.

Funding

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – [grant numbers 88887.510257/2020-00 - PDPG-Amazônia Legal/CAPES]; Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) [grant numbers 062.01300/2018 – Amazonas Estratégico, and grant numbers 01.02.016301.03071/2022-11 – POSGRAD 2022/2023];

Author Contribution

Rafaelle Borges Cunha: Formal analysis, investigation, methodology, project administration, writing-original draft preparationElizabeth Gusmão Affonso: Conceptualization, funding acquisition, supervision, writing-review and editingMarcos Antônio da Silva: Investigation, methodology, supervision, writing-review and editingSabrina Medeiros Suita: Supervision, writing-review and editingHigo Andrade Abe: Supervision, writing-review and editingThiago Mendes de Freitas: Conceptualization, supervision, writing-review and editing

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Dietary protein levels for juvenile matrinxã Brycon amazonicus produced in biofloc and clear water systems

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Abstract

Highlights

1. Introduction

2. Material and Methods

2.2 Experimental diets

2.3 Water quality analyses

2.4 Zootechnical performance analyses and somatic indices

2.5 Blood collection and determination of blood parameters

2.6 Nutritional fish and biofloc composition assessments

2.7 Statistical analysis

3. Results

4. Discussion

5. Conclusion

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References

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