Biofloc system avoid the negative effects of diets with suboptimal protein levels on zootechnical performance, intestinal histomorphometry and protein metabolism of Nile tilapia juvenile fed Spirulina biomass (Arthrospira platensis) as alternative protein source

doi:10.21203/rs.3.rs-3746739/v1

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Biofloc system avoid the negative effects of diets with suboptimal protein levels on zootechnical performance, intestinal histomorphometry and protein metabolism of Nile tilapia juvenile fed Spirulina biomass (Arthrospira platensis) as alternative protein source

https://doi.org/10.21203/rs.3.rs-3746739/v1

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This study aimed to evaluate the effect of the production system (clear water x bioflocs) and the replacement of fish meal with Spirulina biomass (Arthrospira platensis) on productive performance, intestinal histomorphometry, serum biochemistry, and oxidative stress of Nile tilapia juveniles (Oreochromis niloticus) fed suboptimal levels of protein. The design was completely randomized in a 4 x 2 factorial scheme with four replications, four treatments, and two cultivation systems. Four isoproteic (28% crude protein) and isoenergetic (4,000 crude energy kcal kg ^-1) diets were formulated with 0, 33, 66 and 100% replacement levels of fish meal by Spirulina biomass, supplied for 48 days. Juveniles (0.23 ± 0.01 g) were distributed in 16 circular tanks (70 L) with seven fish per tank. The tanks were divided into two independent recirculation systems (clear water x bioflocs) connected to larger tanks (850 L) equipped with a heating and aeration system. No interaction was observed between the evaluated systems and treatments. Rearing in the BFT system avoided the negative effects of diets with suboptimal protein levels on performance, intestinal histomorphometry and protein metabolism. Lower values lower lipid peroxidation and higher antioxidant capacity were observed in fish reared in the BFT system, showing evidence of improvements in antioxidant responses and lower levels of physiological oxidative stress. Spirulina biomass completely replaced fish meal in the diets of Nile tilapia juveniles without negative effects in intestinal morphometry, protein metabolism and antioxidant response. Replacing 66% of fish meal with Spirulina biomass improved the performance, regardless of the rearing system.

Animal nutrition

Arthrospira platensis

Biofloc system

Intestinal histomorphometry

Spirulina

World aquaculture produced 88 million tons in 2020, most of which were fish (FAO 2022). This significant growth in activity is explained by the increase in the world population and per capita consumption (FAO 2022). The growth in fish consumption is associated with increasing demand for healthier animal proteins (Henchion et al. 2017; Boer et al. 2020). To meet this demand, fish farming tends towards the adoption of intensive systems with greater demand for processed feed (Subashinge 2013; Oddsson 2020). In this context, the demand for quality feed and high food costs create bottlenecks for the continued expansion of fish production. The ingredients that constitute the protein fraction of fish diets are expensive and contribute significantly to the total expenses (Barbosa et al. 2020). In addition, excess protein can increase levels of nitrogen compounds in effluents (Herath and Satoh, 2015). Therefore, using feeds with lower protein levels and searching for alternative protein ingredients are strategies to reduce feed costs and increase the sustainability of the activity.

Cultivation in a biofloc system (BFT) is an alternative to reduce protein content in diets and feed costs in intensive fish production (Avnimelech, 2007; Azim and Little 2008). BFT is a closed cultivation system with low water renewal. Water quality is maintained with intensive aeration and management of heterotrophic and autotrophic bacterial populations (Monroy-Dosta et al. 2013). An organic carbon source is added to balance the relationship between carbon and nitrogen (Pérez-Fuentes et al. 2016; Silva et al. 2017). Therefore, the nitrogenous compounds are recycled, and bioflocs are formed, which serve as a food supplement for the fish (Avnimelech 2007; Avnimelech and Kochba 2009; Ekasari 2014). The microbial biomass produced in this system can supply up to 50% of the protein requirement of fish produced in this system (Avnimelech 2007; Azim and Little 2008; Da Silva et al. 2018; Durigon et al. 2020). Lower feed conversion rates can also be achieved (Luo et al. 2014) In general, the focus of protein reduction experiments in the BFT system is performance (Azim and Little 2008; Luo et al. 2014; Durigon et al. 2020). More studies are still needed to better understand the metabolism and physiology of fish receiving suboptimal levels of protein in a BFT system.

Fish meal is one of the most commonly used protein ingredients in fish feed. It is characterized by its high protein value, polyunsaturated fatty acids, considerable mineral and vitamin fractions and antioxidant properties (Masumoto et al. 1996; Pike 1999; Cho and Kim 2011). Fish meal exclusion from diets may decrease zootechnical indices in fish (Gomes et al. 1995; Glencross et al. 2011; Turchini et al. 2019). In addition, its high cost and reduced availability (World Bank 2020) have driven the search for cheaper protein alternatives. The microalgae Spirulina (Arthrospira platensis) is an alternative source of protein with the potential to replace fish meal (Hussein et al. 2013; Raji et al. 2018; Erdogan 2019; Raji et al. 2020; Mansour et al. 2021). It is considered a rich source of highly digestible protein and essential amino acids (Andrade et al. 2005) and contains various substances (fibrous carbohydrates, vitamins, minerals, carotenoid pigments, and polyunsaturated fatty acids) with antioxidant properties (Promya and Chitmanat 2011; Gutiérrez-Salmeán et al. 2015). Some studies demonstrate that Spirulina can also enhances zootechnical performance in comparison to fish meal (Hussein et al. 2013; El-Sheekh et al. 2014; Velasquez et al. 2015; Roohani et al. 2019; Raji et al. 2020). Improvements in biochemical (Belal et al. 2012; Mansour et al. 2021), histological (Abu-Elala et al. 2016), and oxidative stress parameters (Cao et al. 2018; Salah El-Din et al. 2021) have also been reported.

Nile tilapia (Oreochromis niloticus) is a prominent species in global fish farming (Prabu et al. 2019; FAO 2022). It is a rustic species that quickly adapt to intensive cultivation. The rearing of Nile tilapia in a BFT system has been previously reported with positive results (Lima et al. 2018; Martins et al. 2020; Picoli et al. 2022). Nile tilapia ingests bioflocs, enabling a reduction in the demand for protein (Avnimelech 2007; Avnimelech and Kochba 2009; Ekasari 2014.). Spirulina has also been evaluated in Nile tilapia feeding with good results (Belal et al. 2012; Hussein et al. 2013; Velasquez et al. 2015; Abdelkhalek et al. 2017). However, its use in a BFT system with suboptimal levels of protein in the diet has not been evaluated. Therefore, the objective of this study was to determine the effect of the culture system and the replacement of fish meal by Spirulina biomass (A. platensis) on performance, intestinal histomorphometry, serum biochemistry, and oxidative stress in Nile tilapia juveniles fed suboptimal levels of protein.

2.1 Experimental design

The experiment was carried out at the Aquaculture Laboratory of the State University of Santa Catarina, Campus Chapecó, Santa Catarina, Brazil, for 48 days. The Committee for Ethics in the Use of Animals approved the study under protocol number 6983180521. Four diets were formulated: 0% or control (without Spirulina biomass) and 33, 66 and 100% replacement level of fishmeal by Spirulina biomass. The design was completely randomized with four replications, in a 4 x 2 factorial scheme, with four treatments (0, 33, 66, and 100%) and two cultivation systems (clear water x BFT).

2.2 Experimental diets

The experimental diets were isoenergetic (4,300 GE kcal kg^− 1 – Hafedh 1999) and isoproteic (28% crude protein [CP]). They were formulated using SuperCrac® software to meet protein levels approximately 20% below the required for the species and life stage (35% CP [FAO 2017]). The proximal composition of the diets (Table 1) was analyzed according to methods described in the Association of Official Analytical Chemists (AOAC 2000).

The cultivation of Arthrospira platensis was carried out in raceway tanks arranged inside a greenhouse in a semi-continuous system at the Laboratory of Algae Cultivation and Biotechnology – LCBA UDESC, Laguna, Brazil (Table 2). The culture medium contained fresh water and was composed of 10 g L^− 1 of NaHCO₃, 1 g L^− 1 of commercial soluble fertilizer with a Nitrogen:Phosphate:Potassium ratio of 18:6:18 and a high amount of NaCl (30 g L^− 1). The cultivation was carried out under natural temperature and lighting conditions (not controlled) and the Spirulina biomass was collected three times a week by filtration with a 20 µm mesh. Subsequently, it was dried in a laboratory oven at 50 ° C for 24 hours and ground in a cryogenic spray mill (MA775, Marconi) before use in the experimental diets.

In addition to this ingredient, the formulated diets contained fish and soybean meal as protein sources and soybean and corn oil as energy sources (Table 1). All ingredients were ground in an industrial processor and sieved through a 0.71-mm mesh. Subsequently, they were mixed with the addition of water (50%), pelleted in sizes 0.1 (mash), 0.5 and 1-mm in “spaghetti” mixer (Malta®, Model 1036, nozzle no. 8), and placed in an oven (Cienlab®, Model CE-220/150) at 55°C for 48 h. Diets were stored in plastic packages and placed in refrigerators (4°C) until use.

Table 1

Composition of experimental diets (g kg^-1) based on dry matter.
Ingredients	Replacement percentage of fish meal
Ingredients	0%	33%	66%		100%
Fish meal	36.45	23.26	10.08		-
Spirulina biomass	-	26.25	52.50		72.57
Soybean meal	10.00	10.00	10.00		10.00
Corn	41.59	27.86	14.02		3.43
Soy oil	10.73	9.00	7.29		5.98
Premix	0.40	0.40	0.40		0.40
Limestone	0.64	1.65	2.53		3.20
Dicalcium phosphate	-	1.15	2.53		3.58
Table salt	0.06	0.30	0.53		0.72
Antioxidant	0.10	0.10	0.10		0.10
Total (%)	100	100	100		100
Analyzed composition (%)
DM	84.33	83.78		84.56		83.10
MM	8.07	8.55		8.80		8.89
EE	10.32	7.09		6.67		6.74
CP	27.64	28.76		27.65		28.26
GE (kcal kg^− 1)	4,596	4,380		4,395		4,425

Composition analyzed (AOAC 2000). DM – Dry Matter; MM – Mineral Matter; EE – Ethereal Extract; CP – Crude Protein. GE – Gross Energy; Premix - Folic Acid – 2,400 mg, Nicotinic Acid – 48 g, Pantothenic Acid – 24 g, Biotin – 96 mg, Vit. A – 2,400,000 IU, Vit. D3–400,000 IU, Vit. E – 24,000 IU, Vit. B1–9,600 mg, Vit. B2–9,600 mg, Vit. B6–9,600 mg, Vit. B12–9600 mg, Vit K3–4800 mg, Vit. C – 96 g, Iron – 100 g, Manganese – 40 g, Zinc – 6,000 mg, Cobalt – 20 mg, Iodine – 200 mg, Selenium – 200 mg, Antioxidant – 19.6 g.

Table 2

Proximal composition of fish meal and *Spirulina* biomass used in experimental diets.
Proximal composition	Fish meal	Spirulina biomass
DM (%)	93.33	84.74
MM (%)	25.79	16.56
EE (%)	7.52	2.60
CP (%)	55.02	32.01

Composition analyzed (AOAC 2000). DM – Dry Matter; MM – Mineral Matter; EE – Ethereal Extract; CP - Crude Protein.

2.3 Animals and facilities

Nile tilapia juveniles with an average initial weight of 0.23 ± 0.01 g were purchased from a commercial fish farm. The animals were distributed at seven fish per tank in circular polyethylene tanks (useful volume of 70 L), divided into two independent water recirculation systems (clear water x BFT). Each system consisted of 16 circular boxes interconnected with circular tanks (usable volume: 850 L) called “macrocosms.” The tanks were individually aerated through porous stone (1 x 2 cm) connected to a radial air compressor. Each macrocosm contained a peripheral water pump (Intech® model BP500), responsible for the distribution and recirculation of water in its respective system. Each macrocosm was equipped with a heater resistance (Nobre Brasil®, 3000 W) and connected to a digital temperature controller with a sensor (XH-W3001®, 12V, China), aiming to maintain the appropriate temperature for the species (El-Sayed 2006). A biomass of 2.25 kg m^− 3 of tilapia was also maintained in the BFT macrocosm, fed with commercial feed (Supra® Tilapia 32% CP) once a day (1:00 pm; 32% CP). The macrocosm of the clear water system was composed of a filtering system combined with a mechanical filter (60-µm perlon mesh) and biological particulate material (0.2 m³ of analog of low-density polyethylene bio-balls and 0.05 m³ of expanded clay stone). These materials served as a substrate for the nitrifying bacteria to carry out nitrification of the culture water.

The animals were fed throughout the experimental period at a rate of 10% of the biomass per day (Workagegn et al. 2014), divided into three feedings (08:00 am, 1:00 pm, and 5:00 pm). The biomass monitoring was performed every 16 days using biometrics of all experimental units.

At the beginning of the experiment, all tanks in the BFT system received a previously matured biofloc inoculum (10 L) to prevent unwanted variations in water quality (Krummenauer et al. 2014; Martins et al. 2020). Subsequently, dechlorinated water was added to complete the useful volume of 70 L. The same proportion (1:6) was used in the macrocosm. For the formation and maintenance of the microbial community and the adequate conditions of the culture medium, a C:N ratio of 10–15:1 was maintained (Pérez-Fuentes et al. 2016). Sugarcane molasses was used as an external source of carbon.

The water quality parameters of both cultivation systems were measured daily, including temperature, dissolved oxygen (ALFAKIT® oximeter, model AT170), and pH (pHmeter ALFAKIT®, model AT315). Three times a week, the measurement of total ammoniacal nitrogen (TAN) was performed for both systems, and the measurement of total sedimentable solids (TSS) using an Imhoff cone for the BFT system. According to the results obtained in these measurements (TAN > 1 mg L^− 1), molasses was or was not added to the BFT system. Weekly analyses of nitrite, nitrate, orthophosphate, and alkalinity of the culture medium were performed using a photocolorimeter (ALFAKIT®, model AT100P). The parameters remained within the recommended range for tilapia (El-Sayed 2006; Azim and Little 2008; Luo et al. 2014; Lima et al. 2015; 2018) (Table 3).

Table 3

Water quality parameters (mean ± standard deviation) of juvenile Nile tilapia grown in two culture systems (clear water x BFT) after 48 days of feeding with *Spirulina* biomass.
Parameters	Macrocosm
Parameters	AC	BFT
Tª (°C)	25.40 ± 1.28	25.90 ± 1.26
OD (mg L^− 1)	7.97 ± 1.10	7.72 ± 1.05
pH	7.53 ± 0.59	7.36 ± 0.30
N-NH₃ (mg L^− 1)	0.24 ± 0.64	0.60 ± 0.67
NO₂⁻ (mg L^− 1)	0.14 ± 0.15	0.31 ± 0.08
NO₃⁻ (mg L^− 1)	1.82 ± 0.09	1.32 ± 0.14
ALK (mg L^− 1 CaCO₃)	25.8 ± 20.62	41.83 ± 20.18
ORT (PO₄)	0.64 ± 0.03	3.41 ± 1.67
TSS (mg L^− 1)	-	13.35 ± 13.82

Tª = Temperature; °C = Degrees Celsius; pH = Hydrogenionic Potential; N-NH3 = Ammonia Nitrogen; mg L^− 1 = milligram per liter; NO2- = Nitrite; NO3- = Nitrate; ALK = Alkalinity; mg L^− 1 CaCO3 = milligram per liter of calcium carbonate; ORT = Orthophosphate; PO4 = Phosphate; TSS = total sedimentable solids; AC = clear water culture system; BFT = Biofloc culture system.

2.4 Zootechnical performance and hepatosomatic index

After the experimental period, the animals remained fasting for 24 hours and were individually weighed (0.01 g precision scale, Marte model ML 600, São Paulo, Brazil) and measured for the evaluation of zootechnical parameters: final weight; average weight gain = final weight (g) – initial weight (g); specific growth rate = ln final weight (g) – ln initial weight (g) / experimental period x 100 (% day^-1); apparent feed conversion = feed intake/weight gain; feed intake = feed consumed during the experimental period (g) and survival (S) = number of dead animals / total fish x 100 (%). Subsequently, 12 fish per treatment were sedated with eugenol and sacrificed by medullary section to collect the liver to measure the hepatosomatic index (HSI = liver weight / total weight x 100) and the gastrointestinal tract for further analysis.

2.5 Intestinal and liver histomorphometry

We collected fractions of the proximal intestine and liver for the histomorphometric analysis. The histological processing of the samples was performed at the UDESC Histology Laboratory, Campus Lages (Santa Catarina, Brazil), according to routine methods for preparing histological slides (Nunes and Cinsa 2016). The intestinal and liver fragments were placed in sterile plastic containers with a 10% formalin solution, identified, and fixed for 12–16 hours in 70% alcohol until the evaluation of the morpho-histological characteristics. The fractions were sectioned at 0.3 cm using the histology technique, diaphanization, and embedding in histological paraffin. Fractionation into 3-µm sections was performed using a semiautomatic microtome (Mello et al. 2013). Longitudinal sections were semi-serialized and stained using periodic acid-Schiff (Tolosa et al. 2003).

The samples were observed using light microscopy (Bioval L-2000ª microscope, São Paulo, Brazil). Using a camera attached to the microscope, the intestinal (10x) and liver (40x) images were digitized using “TCapture” software (Brand Laborana^®, São Paulo, Brazil). After the scans were completed, ten villi per animal were selected by integrity criteria (Picoli et al. 2019), as follows: i) VH – villus height (i.e., distance from the apex of the villi to the beginning of the muscle layer); ii) TVH – total height of the villi (i.e., height from the apex of the villi to the end of the serosa); iii) VW – villi width; iv) VT – villus epithelium thickness, according to Mello et al. (2013). All measurements were performed using Image J software (National Institute for Health, Bethesda, Maryland, United States of America). An area including 100 hepatocytes per animal was measured. The mean area of hepatocytes (MAH) per treatment was estimated using the same software, according to Picoli et al. (2019).

2.6 Serum biochemical and antioxidant analyses

After anesthesia of 12 animals per treatment, blood was collected by puncture of the caudal vein into syringes containing 10% EDTA. Due to the size of the fish in clear water, a "pool" of the animals' blood was analyzed. The collected blood was centrifuged for 10 min at 3,500 rpm, and supernatants (serum) were stored in microtubes at − 20 ºC until analysis. Total proteins (PTNA) and albumin (ALBU) were evaluated in a biochemical analyzer (Bio Plus® BIO-2000) using commercial kits (Gold Analisa®). Globulin levels (GLOBU) were calculated using the formula:

\(GLOBU{\text{ }}={\text{ }}PTNA{\text{ }}--{\text{ }}ALBU\)

Liver samples of 0.5 g were ground/homogenized (Turratec®) with saline solution (0.9%) and centrifuged at 7,000 rpm for 10 min. The supernatants were stored in microtubes at − 20 ºC until analysis of the following parameters: thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), and protein thiol (PSH). Lipid peroxidation was estimated in samples by measuring malondialdehyde (MDA). Tissue samples were incubated at 95°C for 60 minutes in an acid medium containing 8.1% sodium dodecyl sulfate, 0.5 ml acetic acid buffer (500 mM, pH 3.4), and 0.6% TBA (Ohkawa et al. 1978). Absorbances were measured at 532 nm and compared to the malondialdehyde standard curve. The results were expressed in nmol MDA/ml (Jentzsch et al. 1996).

Glutathione-S-transferase activity was measured kinetically using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The reaction mixture (2 ml) contained 0.1 M phosphate buffer, GSH (1 mM), CDNB (1 mM), and TPM (10%). The absorbance was determined at 340 nm at 37°C, and enzymatic activity was expressed as µmol CDNB/min/mg of protein (Habig et al. 1974).

Protein thiols (PSH) were measured using a method based on the use of DTNB (5,5-dithiobis-2-nitrobenzoic acid) (Ellman, 1959; Sedlak and Lindsay 1968), measured after deproteinization of the homogenates with trichloroacetic acid. The sediment formed by the precipitated protein was resuspended with a homogenization buffer to determine the PSH content. Results were expressed as µmol SH g^-1 of tissue.

2.7 Statistical analysis

Normality (Shapiro-Wilk Test) and homogeneity of variances (Bartlett Test) were verified, and the data were subjected to analysis of variance for a factorial scheme. When not significant, means were compared using Tukey's test at a 5% probability level. In the case of interaction, factorial splitting and means were compared using Tukey's test (5%). The analyses were performed using the R® 4.1.1 statistical analysis program.

3.1 Zootechnical parameters and organosomatic indexes

No interaction (P > 0.05) was observed between culture systems and levels of replacement of fish meal by Spirulina biomass for tilapia performance variables (Table 4). In the BFT system, the performance of the fish was better than those reared in the AC system (P < 0.05; Table 5). Spirulina biomass completely replaced fish meal without compromising performance (P > 0.05). Replacing 66% of fish meal with Spirulina biomass resulted in greater growth and improvement in feed conversion than the control treatment (0%; P < 0.05; Figs. 1 and 2). There was no effect of treatments on survival or hepatosomatic index (P > 0.05).

Table 4

Analysis of variance of performance variables of Nile tilapia juveniles reared in two culture systems (AC x BFT) after 48 days of feeding on Spirulina biomass.
		P-value
Variables	Systems (S)	Fish meal replacement level (T)	Interation (S x T)
FW (g)	< 0.0010*	0.0029*	0.0853 NS
AWG (g)	< 0.0001*	0.0029*	0,0851 NS
SGR (% day^− 1)	< 0.0001*	0.0028*	0.0849 NS
FC	0.0002*	0.0023*	0.0970 NS
FI (g)	< 0.0001*	0.8360 NS	0.5850 NS
S (%)	0.3640 NS	0.2590 NS	0.2590 NS
HSI (%)	0.1603 NS	0.5107 NS	0.4379 NS

S = Systems (BFT and AC); T = Treatments (0, 33, 66 and 100% replacement of fish meal by Spirulina; S x T = Interaction between systems and treatments; FW = Final Weight; AWG = Average Weight Gain; SGR = Specific Growth Rate; FC = Feed Conversion; FI = Feed Ingestion; S = Survival; HSI = Hepatosomatic Index; g = grams; % day^-1 = % weight gain per day; NS = Not significant; * P < 0.05.

Table 5

Mean values (± standard deviation; n = 12) of zootechnical parameters of Nile tilapia juveniles reared in two culture systems (AC x BFT) after 48 days of feeding on *Spirulina* biomass.
Variables	AC	BFT	0%	33%	66%	100%
FW	4.50 ± 1.58 B	16.50 ± 0.56 A	9.65 ± 5.99 b	10.03 ± 6.20 b	11.43 ± 6.50 a	10.89 ± 7.17 ab
SGR	0.45 ± 0.05 B	1.65 ± 0.15 A	0.96 ± 0.60 b	1.00 ± 0.62 b	1.14 ± 0.65 a	1.08 ± 0.71 ab
FI	4.91 ± 0.47 B	15.75 ± 1.60 A	10.56 ± 6.03	10.30 ± 5.77	10.44 ± 6.33	10.01 ± 5.49
S	93.84 ± 0.17	96.93 ± 0.13	90.75 ± 0.20	100.0 ± 0.00	96.93 ± 0.13	93.84 ± 0.18
HSI	1.94 ± 0.66	1.62 ± 0.23	1.75 ± 0.24	1.80 ± 0.78	1.77 ± 0.69	1.80 ± 0.11
AC = Clear water culture system; BFT = Biofloc culture system; T = Treatments (0, 33, 66 and 100% replacement of fish meal by Spirulina biomass (Arthrospira platensis); FW = Final Weight (g); SGR = Specific Growth Rate (% dia ^− 1); FI = Feed ingestion (g); S = Survival (%); HSI = Hepatosomatic index (%); g = grams; % day^− 1 = % weight gain per day; Means followed by different capital letters in columns, differ from each other by Tukey's test

(P < 0.05).

3.2 Hepato-intestinal histomorphometry

No interaction was observed between the factors in the tilapia hepato-intestinal histomorphometry (P > 0.05; Table 6). In the BFT system, the total villus height (TVH) was higher (P < 0.05) than in the AC system (Fig. 3). The treatments did not affect the other evaluated histomorphometric parameters (P > 0.05; Table 7).

Table 6

Analysis of variance of hepato-intestinal histomorphometry of Nile tilapia juveniles grown in two culture systems (AC x BFT) after 48 days of feeding with *Spirulina* biomass.
P-value
Variables	Systems (S)	Fish meal replacement level (T)	Interaction (S x T)
TVH (µm)	0.048*	0.871 NS	0.623 NS
VH (µm)	0.105 NS	0.728 NS	0.549 NS
VW (µm)	0.219 NS	0.950 NS	0.630 NS
VT (µm)	0.217 NS	0.868 NS	0.614 NS
MAH (µm²)	0.700 NS	0.211 NS	0.667 NS

S = Systems (BFT and AC); T = Treatments (0, 33, 66, and 100% replacement of fish meal by Spirulina biomass; S x T = Interaction between systems and treatments; TVH = Total Villi Height; VH = Villi Height; VW = Villi Width; VT = Villi Epithelium Thickness; MHA = Mean Area of Hepatocytes; NS = Not Significant; * = P < 0.05.

Table 7 - Mean values (± standard deviation; n= 12) of hepato-intestinal histomorphometry of Nile tilapia juveniles cultivated in two culture systems (AC x BFT) after 48 days of feeding with Spirulina biomass.

Variables	AC	BFT	0%	33%	66%	100%
VH (µm)	454.79 ± 208.32	592.35 ± 220.98	459.25 ± 171.29	515.92 ± 217.42	542.45 ± 239.84	584.67 ± 272.83
VW (µm)	158.07 ± 55.39	184.17 ± 53.72	172.67 ± 59.32	161.73 ± 46.91	171.38 ± 65.20	178.70 ± 58.30
VET (µm)	77.77 ± 30.62	95.43 ± 42.57	95.41 ± 58.69	79.83 ± 24.17	82.92 ± 32.79	88.23 ± 31.40
MAH (µm²)	502.51 ± 36.50	515.08 ± 91.79	470.34 ± 33.85	492.81 ± 35.69	529.81 ± 88.27	536.22 ± 86.40

AC = Clear water culture system; BFT = Biofloc culture system; S = Systems (AC x BFT); T = Treatments (0, 33, 66, and 100% replacement of fish meal by Spirulina biomass; VH = Villi Height; VW = Villi Width; VET = Villi Epithelium Thickness; MHA = Mean Area of Hepatocytes; NS = Not Significant; * = P <0.05. Means followed by different capital letters in the columns differ from each other by Tukey's test (P <0.05).

3.3 Serum biochemistry and oxidative stress

For the biochemical and oxidative stress parameters, no interactions (P > 0.05) were observed between the cultivation systems and the replacement levels of fish meal by Spirulina biomass (Table 8). In the BFT system, ALBU and GLOBU were higher in relation to AC (P < 0.05). (Table 9). Lower values for thiobarbituric acid reactive substances (TBARS) were observed in the BFT system compared to the AC system (Fig. 4). The opposite was observed for the enzyme GST (Fig. 5). Serum total protein (PTNA) and protein thiol (PSH) levels were unaffected by treatments and systems (P > 0.05) (Table 9).

Table 8

Analysis of variance of biochemical parameters and oxidative stress of Nile tilapia juveniles cultivated in two culture systems (AC x BFT) after 48 days of feeding with *Spirulina* biomass.
P-value
Variables	Systems (S)	Fish meal replacement level (T)	Interaction (S x T)
PTN (g dL^− 1)	0.189 NS	0.870 NS	0.150 NS
ALBU (g dL^− 1)	< 0.000***	0.826 NS	0.987 NS
GLOBU (g dL^− 1)	0.0475**	0.067 NS	0.129 NS
TBARS	< 0.0001	0.987 NS	0.830 NS
GST	< 0.0001	0.983 NS	0.600 NS
PSH	0.326 NS	0.676 NS	0.247 NS

S = Systems (BFT and AC); T = Treatments (0, 33, 66, and 100% replacement of fish meal by Spirulina biomass; S x T = Interaction between systems and treatments; PTN = Total proteins; ALBU = Albumin; GLOBU = Globulins; g/dL = gram per deciliter; TBARS = Thiobarbituric acid reactive substances (nmol MDA/mL); GST = Glutathione S-transferase (µmolCDNB/min/mg protein); PSH = Protein thiols (µmol SH/mg protein); NS = Not significant; * = P < 0.05.

Table 9

Mean values (mean ± standard deviation; n = 12) of biochemical parameters and oxidative stress of Nile tilapia juveniles grown in two culture systems (AC x BFT) after 48 days of feeding with *Spirulina* biomass.
Variables	AC	BFT	0%	33%	66%	100%
PTN	3.53 ± 0.57	4.37 ± 1.33	4.31 ± 0.78	3.86 ± 0.97	3.60 ± 0.42	4.07 ± 1.35
ALBU	0.88 ± 0.25 B	1.42 ± 0.47 A	1.27 ± 0.50	1.13 ± 0.59	1.09 ± 0.39	1.12 ± 0.40
GLOBU	2.65 ± 0.44 B	2.95 ± 0.47 A	3.04 ± 0.28	2.73 ± 0.48	2.51 ± 0.46	2.95 ± 0.52
PSH	1.33 ± 0.18	1.40 ± 0.21	1.36 ± 0.18	1.42 ± 0.22	1.30 ± 0.18	1.38 ± 0.24

S = Systems (BFT and AC); T = Treatments (0, 33, 66, and 100% replacement of fish meal by Spirulina biomass; S x T = Interaction between systems and treatments; PTN = Total proteins (g dL^− 1); ALBU = Albumin (g dL^− 1); GLOBU = Globulins (g dL^− 1); g/dL – gram per deciliter; PSH = Protein thiols (µmol SH/mg protein); Systems: Means followed by different capital letters in the columns, differ from each other by Tukey's Test (P < 0.05).

4.1 Zootechnical parameters and organosomatic indexes

In the clear water system, the supply of suboptimal levels of protein reduced fish growth. The specific growth rate was much lower (0.45% day^− 1) than observed in other studies (5.01% day^− 1, 1.9% day^− 1, 1.31% day^− 1, and 4.42% day^− 1) (Gullian-Klanian and Arámburu-Adame 2013, Luo et al. 2014, Tanjung et al. 2019 and Akter et al. 2021). This finding confirms that the protein supply was inadequate. By contrast, for fish reared in BFT the growth was not impaired, confirming that reduced protein levels in this system did not affects the perfomance (Azim and Little 2008; Luo et al. 2014; Durigon et al. 2020). Nile tilapia is able to supplement the diet by ingesting the bioflocs rich in nutrients, which can avoid possible deficiencies problems (Tacon 2002; Hargreaves 2006; Azim and Little 2008; Crab et al. 2012). It should be noted that the fish used in the experiment were small (0.23 ± 0.01 g) compared to those used in previous studies, demonstrating that the protein reduction in the BFT system can be applied for Nile tilapia in the first stages of development.

The performance results suggest that it is possible to replace fish meal completely with Spirulina biomass in Nile tilapia juvenile diets. Previous studies have demonstrated the viability of replacing fish meal with Spirulina biomass for tilapia and other species (Nandeesha et al. 1998; 2001; El-Sheekh et al. 2014; Velasquez et al. 2015; Cao et al. 2018; Raji et al. 2020), but this is the first work where replacement was evaluated in different production systems. The protein content, amino acid profile, and polyunsaturated fatty acid contents of this microalga are excellent (Albuquerque 2014; Zhang et al. 2020; Rutar et al. 2022), what can explain the positive results. In the present study, replacing fish meal with Spirulina biomass did not affect consumption. Generally, diets without animal-based proteins reduces feed intake by tilapia (Fontaínhas-Fernandes et al. 1999; Zhou and Yue 2010; Hernández et al. 2013). Spirulina has attraction-palatability properties (Jaime-Ceballos et al. 2005; 2007; Silva-Neto et al. 2011; Ali 2014) and it has already been demonstrated that its inclusion in replacement of fish meal may positively affect the feeds ingestion (Abdel-Tawwab and Ahmad 2009).

The 66% level of fish meal replacement by Spirulina biomass (52.50% inclusion) improved growth and feed efficiency compared to fish meal diet. Spirulina biomass can be used as a growth promoter in fish diets (Abdel-Tawwab and Ahmad 2009; Elsayed et al. 2012; Hernández et al. 2012; Jana et al. 2014). Previous studies demonstrated that the supplementation with Spirulina biomass in the diets improved the growth and feed conversion of Nile tilapia and its hybrids (Oreochromis niloticus x Oreochromis mossambicus) (Elsayed et al. 2012; El-Sheekh et al. 2014; El-Ward et al. 2016). It was not possible to relate the level of inclusion with the results obtained. The effects are possibly linked to Spirulina's composition. Spirulina is rich in various substances with bioactive properties, including fibrous carbohydrates, vitamins, minerals, carotenoid pigments, and polyunsaturated fatty acids (Promya and Chitmanat 2011; Gutiérrez-Salmeán et al. 2015). In the present study, the characterization of these compounds in Spirulina was not carried out. Further studies are still needed to explain how one or more of these compounds may positively impact performance.

4.2 Hepato-intestinal histomorphometry

The animals cultivated in the BFT system presented larger villi than those in the clear water system. The BFT system may improve gut health (Ahmad et al. 2017; Pilotto et al. 2018; Deng et al. 2022). The rich microbial community in bioflocs competes directly with pathogenic microorganisms in the environment and can mitigate possible health challenges (Monroy-Dosta et al. 2013; Ferreira et al. 2015). Furthermore, beneficial microorganisms can colonize the digestive tract and provide a possible probiotic effect, which may help explain the improvement in villus height (Aguilera-Rivera et al. 2014; Pilotto et al. 2018). Studies on the microbiology of the digestive tract of fish cultured in a BFT system demonstrated the multiplication of beneficial bacteria and colonization of the digestive tract (Pilotto et al. 2018; Deng et al. 2022). In addition to the benefits of the BFT system, it should be mentioned that in the clear water system there was a deficit of nutrients, which may have affected the integrity of the intestinal epithelium of the animals (Dawood 2020; Zarkasi et al. 2016; Zhu et al. 2020).

The replacement of fish meal by Spirulina biomass did not compromise intestinal histomorphometry of Nile tilapia. This result corroborates the performance results and reinforces the viability of Spirulina as a substitute for fish meal in tilapia diets. Other authors have demonstrated the feasibility of the replacement in relation to intestinal morphometry and also observed larger intestinal villi in Nile tilapia supplemented with Spirulina in the diet (Abu-Elala et al., 2016). In the present study, Spirulina's lack of positive effects on the villi may be related to the suboptimal levels of protein used. Nutrient deficiency can affect intestinal histomorphometry (Dawood 2020; Zarkasi et al. 2016; Zhu et al. 2020), masking the benefits of supplementation. On the other hand, this result was also observed in the BFT system where the bioflocs provided an extra supply of nutrients. More studies are needed to confirm these results.

4.3 Serum biochemistry

Albumin and globulin in the serum were lower in fish cultivated in the clear water system compared to BFT system. The levels of albumin and globulins were also above (0.88 ± 0.25 g dL^− 1 and 2.65 ± 0.44 g dL^− 1, respectively) than those normally observed for tilapia (1.20 g dL^− 1 and 4.0 g dL^− 1, respectively; Pereira et al. 2016). Albumin and globulins are plasma proteins directly linked to protein metabolism. In addition, they are essential markers of the nutritional status of animals (Sampaio et al. 2012). The supply of suboptimal levels of protein explains the decreased these markers and confirms the results obtained in the productive performance. Likewise, the highest values of albumin and globulin obtained in the BFT system confirm that the bioflocs served as a complementary supply of nutrientes and maintained the nutritional status.

The fish meal substitution for Spirulina did not affected the serum levels of albumin and globulin for Nile tilapia. In all treatments the values were similar (1.15 ± 0.47 g dL^− 1 and 2.80 ± 0.43 g dL^− 1, respectively) to those already observed for tilapia (1.20 ± 0.60 g dL^− 1 and 2.67 ± 0.33 g dL^− 1 - Pereira et al., 2016 and Tanekhy et al., 2016, respectively). Previous studies showed the beneficial effects of Spirulina on biochemical parameters in Nile tilapia (Belal et al. 2012; Abdekhalek et al. 2017; Abdel-Daim et al. 2020; Mansour et al. 2021; Soliman et al. 2021). However, as previously reported, the supply of suboptimal levels of protein may have altered some of these biomarkers, such as total protein and albumin (Ayat et al. 2011) and avoided the detection of effects of the protein source. Further studies using Spirulina in conventional diets are needed to confirm the possible effects of the protein source on markers of protein metabolism in tilapia grown in a BFT system.

4.4 Oxidative stress

Lower values of lower lipid peroxidation and higher values of glutathione S-transferase were observed in the BFT system compared to clear water system. These results indicate that there was less cell damage and antioxidant capacity was improved (Bacou et al. 2021). The BFT system avoided the negative effects of physiological oxidative stress and improved the antioxidant responses of the animals. These results might be explained by bioactive compounds (essential fatty acids, carotenoids, free amino acids and chlorophylls, among others), minerals, and vitamins) in microbial aggregates (Tacon et al. 2002; Ju et al. 2008; Crab et al. 2012. Collaborating with this hypothesis, in the clear water system the lower efficiency in the antioxidant defense mechanism was insufficient to prevent the increase in lipid peroxidation. Further evaluations of the composition of bioflocs are necessary to correlate the presence of bioactive compounds with the antioxidant response of animals.

The replacement of fish meal with Spirulina did not change lipid peroxidation levels or antioxidant capacity of Nile tilapia juveniles. Several authors observed an increase in the antioxidant response of Nile tilapia supplemented with Spirulina biomass (Abdelkhalek et al. 2017; Awed et al. 2020; Elabd et al. 2020; Abdel-Daim et al. 2020; Salah El-Din et al. 2021; Soliman et al. 2021). In the present study the lack of positive results can be explained by the nutritional composition and functional properties of fish meal and Spirulina biomass (Henrikson 1994; Masumoto et al. 1996; Wang et al. 2007; Kumar and Sibi 2020. Marine fish meal is rich in polyunsaturated fatty acids with antioxidant properties (Pike 1999; Cho and Kim 2011) which may explain the antioxidant response results similar to Spirulina. More studies are needed to confirm these results.

Cultivation in the BFT system avoided the negative effects of diets with suboptimal protein levels on zootechnical performance, intestinal histomorphometry, and protein metabolism of Nile tilapia juveniles. Lower values lower lipid peroxidation and higher antioxidant capacity were observed in fish reared in the BFT system, showing evidence of improvements in antioxidant responses and lower levels of physiological oxidative stress. Spirulina biomass can completely replace fish meal in the diets of Nile tilapia juveniles without negative effects is intestinal morphometry, protein metabolism and antioxidant response. Replacing 66% of fish meal with Spirulina biomass (52.50% inclusion) improved fish performance regardless of the culture system (clear water or BFT).

Acknowledgments: We thank the members of the UDESC laboratories in Chapecó (GT-AQUA) and Lages (LAPIS) and other partnerships.

Conflict of interest: The authors have no relevant financial or non-financial interests to disclose.

Ethical Approval: The authors observed the ethical policies in the journal's author guidelines and confirmed the approval of the Committee for Ethics in the Use of Animals (CEUA - UDESC) (CEUA protocol number 6983180521).

Author Contributions: Fernanda Picoli: Conceived and designed the analysis, collected the data, contributed data of analysis tools, performed the analysis and wrote the paper; Alana D. de Oliveira, Suelyn O. Marques and Deise C. Terhorst: Collected the data; Suélen Serafini and Luísa Nora: Contributed to the writing of the paper; Aleksandro S. da Silva, Fabio F. Neves and Maurício G. C. Emerenciano: Conceived and designed the analysis and contributed data of analysis tools; Diogo Luiz de Alcantara Lopes and Thiago El Hadi Perez Fabregat: Conceived and designed the analysis, collected the data, contributed data of analysis tools, performed the analysis and wrote the paper.

Funding: The University Scholarship Program of Santa Catarina (UNIEDU/FUMDES) financed this work.

Data Availability: Upon request to the corresponding author, data supporting this work are available.

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Biofloc system avoid the negative effects of diets with suboptimal protein levels on zootechnical performance, intestinal histomorphometry and protein metabolism of Nile tilapia juvenile fed Spirulina biomass (Arthrospira platensis) as alternative protein source

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Experimental design

2.2 Experimental diets

2.3 Animals and facilities

2.4 Zootechnical performance and hepatosomatic index

2.5 Intestinal and liver histomorphometry

2.6 Serum biochemical and antioxidant analyses

2.7 Statistical analysis

3. Results

3.1 Zootechnical parameters and organosomatic indexes

3.2 Hepato-intestinal histomorphometry

3.3 Serum biochemistry and oxidative stress

4. Discussion

4.1 Zootechnical parameters and organosomatic indexes

4.2 Hepato-intestinal histomorphometry

4.3 Serum biochemistry

4.4 Oxidative stress

5. Conclusion

Declarations

References

Additional Declarations

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