Indian Journal of Animal Research

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Efficacy of in situ and ex situ Biofloc on Growth, Immune-physiological Responses, Nutrient Composition and Muscle Growth-related Gene Expression of Etroplus suratensis

A. Jackqulinwino1,*, B. Ahilan1, Cheryl Antony1, P. Chidambaram1, A. Uma1, P. Ruby1
1Dr. M.G.R. Fisheries College and Research Institute, Tamil Nadu Dr.J. Jayalalithaa Fisheries University, Nagapattinam, Ponneri-601 204, Tamil Nadu, India.

Background: A 90-day trial was carried out to investigate the effects of insitu and exsitu biofloc on the immune-physiological responses, nutrient composition and muscle growth-related gene expression.

Methods: The experimental groups consisted of IB-1, IB-2, IB-3 (insitu biofloc), EB-1, EB-2, EB-3 (exsitu biofloc). Capacity of 2.4-ton outdoor tanks were stocked with 4.36±0.1 g pearlspot (Etroplus suratensis) juveniles @ 150/m3. Organic jaggery was used as carbon source to maintain 12:1, 15:1, and 20:1 C/N (IB-1, IB-2, IB-3) ratio for insitu biofloc development and exsitu biofloc mass production and feed supplementation at 10, 20 and 30 % graded level in the diet (EB-1, EB-2, EB-3).

Result: At the end of the experiment, insitu biofloc system showed significantly better physiological performances in terms of SOD, RBT, MPO, GLU, CAT, and Serum protein compared to exsitu treatment. In exsitu system, biofloc supplemented at 20% significantly improved nutrient composition. There was a significant effect of insitu and exsitu biofloc treatments on whole-body amino acids like Phenylalanine, Leucine, Isoleucine, methionine, tryptophan, and lysine. However, the targeted muscle-growth-related myo D gene was significantly upregulated in IB-3 insitu and EB-2 exsitu treatments, while the myostatin was downregulated in IB-3 and EB-3. Therefore, this present study suggests the adoption of the insitu biofloc method to obtain better performance of Pearlspot, insitu biofloc at 20:1 C: N level could be used to improve overall health and muscle growth in Etroplus suratensis.

Over the past few decades, the unprecedented world population has explained various plausible scenarios for aquaculture expansion and fish intensification, which may dissipate the limited water resources and increase environmental deterioration (Wetengere, 2011). The major challenge in the sector is the periodical water exchange requirement to maintain optimum water quality, resulting in huge water consumption and thereby the release of nutrient and organic matter-loaded effluent. Biofloc Technology” (BFT) could be a potential and sustainable alternative that can reduce environmental impacts with zero water exchange and less feed input while increasing stocking density and production (Emerenciano et al., 2022).

The crucial factor for the ammonia pathway in BFT systems was carbon amendment at a high C: N ratio, heterotrophic bacteria are the most predominant species (Elaiyaraja et al., 2020). The jaggery is considered a simple carbon source with a higher dissolving capacity than other complex carbon sources. On the contrary, simple carbon sources can disperse quickly in water and help reduce ammonia compared to complex carbon sources (Dauda et al., 2017). Hence, the simple carbon sources like jaggery, glycerol and sugars can be efficiently utilized by heterotrophic bacteria for multiplication resulting in dispersion and quicker floc and paving the way for the heterotrophic bacteria to assimilate ammonia with decreased concentrations of TSS thereby maintaining ideal water quality (Felix et al., 2015).

To overcome the demand for aquatic food for the growing population, aquaculture needs to be projected through species diversification and technological advancements. Lates calcarifer, Chanos chanos, Etroplus suratensis and Mugil cephalus are already under domestication for the development of commercial aquaculture in India. As a medium to high-value species, the pearl spot, E. suratensis is considered one of the most suitable species for diversified freshwater and coastal aquaculture in India. This species is mostly produced in Kerala and Goa, although it has recently gained recognition in other parts of India due to high consumer preferences and market prices.

Feed costs in aquaculture can be reduced by the adoption of biofloc technology since the expenses with commercial diets account for more than 50% of the production cost. Biofloc contains up to 30% crude protein and about 2% lipids (Azim and Little, 2008; Ballester et al., 2010; Xu and Pan, 2012; Luo et al., 2014). The dried biofloc can be used possibly to replace fishmeal or soybean meal as cheaper sources of protein. The major objective of the study aimed to determine the intake of biofloc by different incorporation methods and its impact on animals’ immune-physiological and muscle growth performance.
Ethical statement
The experiment was conducted following the procedures of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), Ministry of Environment and Forests (Animal Welfare Division), Govt. of India on care and use of animals in scientific research. This study was approved by the ethical committee of Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, Tamil Nadu, India.
Experimental design
A completely randomized design (CRD) was followed for the experiments. The experiment was designed with in situ biofloc developed within the culture system (IB-1, IB-2, IB-3) and ex situ biofloc incorporation in diet (EB-1, EB-2, EB-3). The experimental trial was performed in an FRP tank, which consisted of eighteen circular tanks of 2,400 L capacity. The oxygen supply was maintained at an optimum level with a 1.5 HP air blower. Before stocking all the water quality parameters were analyzed.
Experimental fish
The Pearlspot fingerlings used for the experimental study were collected from Pulicat Lake, Pazhaverkadu and the fishes were disinfected with KMnO4 and stocked in 25 ppt brackish water. The fish were slowly acclimatized to 22 ppt by adding fresh water. Before the experiment, an average size of 4.36±0.1 g juveniles was used for experiments. During the trial, fish were fed @ 5% of fish body weight and feeding was done three times a day. The water quality parameters were monitored daily and the mean values were recorded as per standard procedure (APHA, 2005). The bio-growth sampling was done every fortnight.
Production of biofloc for ex situ feed preparation
The development of biofloc was carried out as per the protocol described by Avnimelach (2009). The development of floc was triggered with continuous aeration with the help of an air compressor. The C: N ratio of 20:1 was maintained by adding Jaggery as a carbon source. The amount of carbon required to reduce the ammonia content in the water was calculated according to Avnimelach (1999). The floc samples were collected and checked for floc development using of Imhoff cone. When floc volume reached 30ml/l, once in two days wet biofloc was collected from the tank with the help of 20μ mesh size hand net, centrifuged at 2000 rpm and washed twice with filtered freshwater to get rid of the traces of ammonia nitrogen if any, dried under shadow and then powered into fine particles<200 µm and stored in airtight containers for experimental feed preparation.
Experimental diets
The experimental diets were prepared with biofloc powder incorporation at 10,20,30% of the feed viz., EB-10, EB-20 and EB-30. The experimental feed comprised fishmeal, biofloc meal, soybean meal, wheat gluten, soy lecithin, vitamins, minerals and fish oil. The formulation of dietary ingredients and chemical composition of experimental diets is shown in Table 1. The wet-formulated feed was dried in sunlight to reduce moisture content (<10%) and stored in an airtight container for ex situ feeding trials. 0% without-biofloc incorporation feed was used for the in situ experimental trials.

Table 1: Formulation and chemical composition of experimental diets (g/kg of diet).

Growth analysis
The bio-growth parameters of fish like percentage weight gain, specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER) and survival rate were calculated (Ruby et al., 2022).
Immune-physiological assay
At the end of the 90-day trial, the fish (n=5) were anesthetized using clove oil to collect blood samples from a caudal vein. The blood was collected in EDTA-coated (anti-coagulant) vials and blood was allowed to clot and centrifuged to separate the serum.

The Respiratory Burst Activity was measured by Nitro blue tetrazolium assay (NBT as modified by Anderson and Siwiki (1995). Myeloperoxidase activity-Total MPO content present in serum was measured according to Quade and Roth (1997). The lysozyme assay was carried out with a few minor adjustments based on (Guardiola et al., 2019). The Superoxide dismutase (SOD) activity was estimated following the modified method of (Mishra and Fridovich, 1972). Catalase activity (CAT) was estimated according to the method of Takahara et al., (1960). The serum glucose Blood glucose (GLU), Serum cholesterol (CHO) and Triglyceride (TG) were estimated using a semi-automatic blood biochemical analyzer (Alpha chem. 100i). The total triglycerides were measured using an auto-analyzer (MERCK Selectra Junior, Germany).
Nutrient and amino acid analysis
To determine the carcass composition of pearlspot fingerlings (n=8) the fishes were cut into pieces, minced, homogenized and immediately frozen until further analysis and the Proximate and Amino acid were determined using AOAC (1995) method.  
Expression of muscle-related genes by real-time analysis (qRT-PCR)
Consecutively, after the end of the trial all fishes were fasted for 24 hours before the collection of samples (n=5 fish per treatment) of skeletal muscle (for MyoD and myostatin), Total RNA was extracted from muscle tissue using RNA iso-plus (Takara Bio Inc., Otsu, Shiga, Japan) according to the manufacturer’s protocol. The gene-specific primers of MyoD, myostatin, 18sRNA and â actin are shown in Table 2. The first strand complementary DNA (cDNA) was synthesized from 2 μg of total RNA using the first strand cDNA synthesis kit (Thermo Scientific, Vilnius, Lithuania) as following the manufacturer’s protocol. The Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) consisted of 20 ng of cDNA template, 10 μM of each primer (forward and reverse) and 1´ SYBR Green PCR Master Mix Kit (Takara Bio Inc., Otsu, Shiga, Japan), in a 20 μl of total volume (Table 3). The qRT-PCR was carried out in a C00 Touch thermal cycler-CFX96 Real-time PCR (Bio-Rad, Foster City, California, USA) programmed with an initial denaturation at 95°C for 10 min, followed by 40 cycles of 15 s denaturation at 95°C, annealing at 60 to 62°C (depends on the target genes) for 30 s, extension at 72°C for 30 s and ended with dissolution curve. The qRT-PCR was performed in triplicate and the threshold cycle (Ct) values obtained by amplification were measured and a relative expression level of the specific gene was presented as 2"ÄÄCt (Livak and Schmittgen, 2001). The expression of housekeeping genes 18S rDNA and β-Actin was used to compare the relative expression levels of the genes. PCR amplification was carried out using a 96-well Takara PCR System (Takara, Japan).

Table 2: Primers used for qRT-PCR analysis of selected genes of Etroplus suratensis reared in in situ and ex situ bio floc system.

Table 3: Growth performance of Etroplus suratensis reared in in situ and ex situ biofloc system for 90 days.

Statistical analysis
The data were analyzed by analysis of variance (ANOVA) using SPSS, VERSION 20.0 (SPSS Inc., Chicago, IL, USA). All data were tested for normality (Shapiro–Wilk test) and homogeneity of variance (Levene’s test) and transformed when the data did not show normal distribution. The significance of differences among the treatment groups was evaluated using a predetermined threshold p-value of <0.05.
Growth performance of pearlspot reared in insitu and ex situ biofloc system
The bio-growth parameters of pearlspot reared in in situ and ex situ biofloc systems are shown in Table 3. The results indicated that in situ groups significantly (P<0.05) performed well in terms of higher weight gain compared to ex situ groups (P<0.05). Improved weight gain was observed in the 20:1 in situ biofloc system due to the continuous availability of in situ biofloc in the system. Similar results were documented by Ezhilarasi et al., (2019) and Elaiyaraja et al., (2020). Various studies resulted in in situ biofloc performing higher growth in biofloc-raised tilapia (Crab et al., 2009; Azim and Little 2008; Elaiyaraja et al., 2020), L. rohita (Mahanand et al., 2013; Ahmad et al., 2016), Carassius auratus (Wang et al., 2015), Cyprinus carpio Najdegerami et al., (2016) and Etroplus suratensis Ruby et al., (2022) which indicates that biofloc can substitute aquatic animal feed and can promote the growth of diverse fish species.

The inclusion of biofloc as a dietary ingredient in the shrimp diet was found to improve the growth performance of L. vannamei (Ju et al., 2008; Kuhn et al., 2010). Among ex situ treatments, biofloc at 20% incorporation in the feed (EB-20) was recorded with higher weight gain. In contrast, shrimp fed higher biofloc incorporation in their diet had significantly lower growth performances which agrees with Anand et al., (2014) who reported that an increase in biofloc meal dietary supplementation in the diet does not increase proportionately the digestive enzyme activities and thereby the growth of shrimp. Our study revealed that enhanced growth performances were obtained within the biofloc in situ system compared to the ex situ system.
Immune-physiological responses
Immune-Physiological responses in E. suratensis reared with in situ and ex situ biofloc treatments are summarised in Table 4. The significant interaction (P<0.05) indicated that in situ treatment enhances the overall immune-physiological function of pearlspot.

Table 4: Immune-physiological responses of Etroplus suratensis reared in in situ and ex situ biofloc system for 90 days.

Increased Respiratory burst activity (RBA) was observed in IB-3 treatment (0.73±0.01) when compared to other in situ and ex situ groups. Significantly (p<0.05) higher levels of Myeloperoxidase, lysozyme, Catalase and SOD were recorded in fishes reared in in situ compared to ex situ groups. In addition, decreased glucose (80.30±0.8) and cortisol (65.2±0.9) values were observed in the in situ IB-3 treatment of Etroplus suratensis when compared with ex situ and other treatments. Respiratory burst activity is an indicator of oxygen-dependent bactericidal activities. The observed higher respiratory burst activity in the present study IB-3 was reported by Ekasari et al., (2014) who found a higher respiratory burst activity in the post-challenge of shrimp reared under molasses-supplemented biofloc compared to rice bran and tapioca-by-product-supplemented biofloc systems.

Myeloperoxidase (MPO) is an important enzyme having antimicrobial activity. The present study recorded the highest MPO values in the IB-3 (7.5±0.08) groups, followed by IB-2, EB-2, IB-1 and EB-3 least value was documented in the EB-1 (4.17±0.30) group which similar to the inference of (Long et al., 2015). The increased level of Lysozyme and catalase enzymes in the IB-3 and IB-2 agreed with the findings of Anantharaja et al., (2023) in the Hypselobarbus pulchellus. Superoxide dismutase (SOD) is a class of enzymes that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. The SOD values obtained in the present study were in the range (50.86±0.4 to 33.59±1.3) reported in Carassius auratus (Wang et al., 2015). In the present study, there was a significant increase in SOD levels among in situ biofloc-based treatments compared to ex situ groups, which indicates the need for SOD activity to scavenge the oxidative radicals produced by higher respiratory activity among the in situ biofloc treatments.

Serum cortisol is used as a general index for stress, which can be used to determine primary stress response in animals, including fish (Metwally and Wafeek, 2014). In the current investigation, ex situ treatments showed higher cortisol levels indicating more stress in this group. The results agreed with earlier findings in Nile tilapia (Azim and Little, 2008). In the present study, a reduced trend of glucose content was observed in biofloc treated with probiotics, followed by biofloc and the serum glucose content was found highest in ex situ which was highest among other treatments.
Whole body nutrient and amino acid composition
The whole-body crude lipid content were not significantly (p>0.05) affected by the in situ and ex situ groups (Table 5) However, higher crude protein content of the whole body was observed in fish reared in IB-3 followed by IB-2 and IB-1. Similar results were reported by (Yuvarajan et al., 2023). There were significant (p<0.05) differences observed in the whole-body essential amino acids’ profiles viz., Phenylalanine, lysine, isoleucine, methionine and tryptophane when compared with ex situ groups (Table 6). In the present study, E. suratensis in in situ biofloc showed a higher percentage of protein and lipid compared to ex situ groups. The findings of the present study are in agreement with the results reported by, Paul et al., (2024) in Ompok pabda and Hasbullah et al., (2018) in Nile tilapia.

Table 5: Whole body nutrient of Etroplus suratensis reared in in situ and ex situ biofloc system for 90 days.

Table 6: Amino acid composition of Etroplus suratensis reared in in situ and ex situ biofloc system for 90 days.

Muscle-related gene expression
The MyoD gene regulates muscle cell proliferation and differentiation during white skeletal muscle growth to create new hyperplastic fibres and hypertrophy (McCarthy et al., 2011). The relative expression of muscle-growth-related genes in the white skeletal muscle of Etroplus suratensis reared in in situ and ex situ biofloc with jaggery as a carbon source are illustrated in Fig 1A and B.

Fig 1: (A) Myo D muscle-related gene, (B) Myostatin in Etroplus suratensis reared in in situ and ex situ biofloc system for 90 days. Different letters indicate significant (p<0.05) differences among treatments determined by Tukey’s test.

El-Hawarry et al., (2021) reported that adding glycerol as a carbon source at Low stoking density downregulated Growth gene expression without affecting MSTN expression. Further, he concluded that the upregulation of both IGF-1 and GHR1 genes 5 and 2.1-fold, respectively and growth-related genes upregulated in fish under LSD with glycerol and molasses as carbon sources. MSTN is a skeletal muscle mass-negative regulator gene that suppresses muscle growth (Rodgers and Garikipati, 2008). To the best of our knowledge, there have been no previous investigations on the expression of muscle growth-related genes in Etroplus suratensis grown in an in situ and ex situ biofloc farming system.

The pattern of the targeted muscle genes expression in pearlspot reared in (IB-3) 5.40-fold change revealed a clear pattern of statistically significant (p<0.05) upregulation of Myo D. Similarly, in the ex-situ groups, EB-2 expressed higher Myo D 4.33-fold than other treatments. The MyoD was upregulated in IB-3 and EB-2, respectively. On the contrary, the relative expression of myostatin was downregulated in IB-3 0.99 and EB-2 1.28-fold changes. With an increase in the C: N ratio of carbon sources the relative muscle gene expression was upregulated.

However, these findings add to our understanding of the impact of in situ and ex situ biofloc technology in upregulating muscle growth gene expression which underlies the enhancement of fish growth. Future research should be done to investigate the role of in situ and ex situ biofloc in the expression and regulation of muscle growth-related genes.
This study suggests in terms of an in situ system with the use of a 20:1 C: N ratio could improve the biofloc formation and enhance the water quality resulting in higher biogrowth and immune status of Etroplus suratensis and it should be an appropriate C: N ratio for biofloc farming of pearlspot. Simultaneously, 20% bio floc meal ex situ incorporation in pearlspot feed may potentially improve the health status of fish with sustainability. This study and its findings are the first to know the effect of jaggery-based biofloc intake relating to the enhanced immunological performance of Etroplus suratensis with gene expression. These findings concluded that the jaggery carbon source at the C: N (20:1) ratio could be an effective potential source that provides higher production with improved water quality parameters.
The authors are thankful to the Dean, Dr. M.G.R. Fisheries College and Research Institute, Tamil Nadu Dr.J. Jayalalithaa Fisheries University, Ponneri 601 204, Tamil Nadu, India for rendering the facilities to conduct the present study.
Every step taken was compliant with the responsible parties’ ethical standards and the authors declare that they have no conflict of interest.

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