Influence of Fumaric Acid Fortified Diet on Growth Performance and Haematological Responses of Genetically Improved Farmed Tilapia
H. Manimaran1*, P. Chidambaram2, P. Yuvarajan1,3 and M. Joshna1
1Department of Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University - Dr. M.G.R. Fisheries College and Research Institute (Dr. M.G.R FC and RI), Ponneri, Thiruvallur-601 204, Tamil Nadu, India
2Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam 611002, Tamil Nadu, India
3Department of Aquaculture, Tamil Nadu Dr. J. Jayalalithaa Fisheries University - Dr. M.G.R FC and RI, Thalainayeru, Nagapattinam – 614 712, Tamil Nadu, India
ABSTRACT
An eight-week indoor feeding trial was carried out to determine the consequences of fumaric acid (FA) addition in the diet on growth performance, feed utilization and haematological responses of GIFT. Four isonitrogenous (28% of crude protein) diets were formulated with graded levels of dietary fumaric acid (0 (control), 0.5, 1.0 and 1.5 g/kg of diet). Each diet was allocated at random to three groups of thirty fish per experimental tanks, which were fed twice a day (09:00 and 17:00 h). Growth performance and feed utilization were improved in fish fed fumaric acid 1.5 g/kg of diet compared to control and other dietary treatments. Haemoglobin, RBC and haematocrit values were significantly (p<0.05) improved in fish fed fumaric acid 1.5 g/kg of diet compared to control. Overall, these findings suggest that fumaric acid (1.5 g/kg of diet) can be effectively utilized for improving growth performance and immunological status of GIFT, highlights its potential as a nutritional strategy for tilapia aquaculture.
Article Information
Received 05 April 2024
Revised 25 June 2024
Accepted 07 July 2024
Available online 25 November 2024
(early access)
Published 13 November 2025
Authors’ Contribution
HM: Conducted the experiment, data analysis, interpretation and prepared original manuscript draft. PC: Conceptualized, designed and supervision of the study. PY: Reviewed and edited the manuscript. MJ: Assistance during experimental trial.
Key words
Fumaric acid, Feed utilization, Genetically Improved Farmed Tilapia, Growth performance, Haematology
DOI: https://dx.doi.org/10.17582/journal.pjz/20240405062027
* Corresponding author: [email protected]
0030-9923/2025/0006-2921 $ 9.00/00
Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
INTRODUCTION
Tilapia is one of the most important candidate fish species cultured in more than 120 countries and contributes more than 10% of worldwide fish production (FAO, 2020). It can tolerate a wide range of climatic conditions. At the same time, it has poor genetic management and inbreeding problems. To mitigate these problems genetically improved farmed tilapia (GIFT) has been produced through selective breeding of tilapia by World Fish Centre. GIFT is an improved strain of Nile tilapia with an enormous benefit for boosting yields and increasing nutritional benefits (Eknath and Hulata, 2009). However, Intensive tilapia farming is susceptible to disease (Machimbirike et al., 2019). At present, various chemical disinfectants, antibiotics, and sulfa drugs are repeatedly used in this sector in large quantities to control aquatic diseases by traditional disinfection and sterilization methods. The usage of these chemicals has many undesirable effects like environmental contamination and accumulation of toxic substances in fish muscles which could have an impact on human health and increase the spread of antibiotic-resistant strains by transferring resistant bacteria from the aquatic environment (Dawood and Koshio, 2020; Romero et al., 2012; Hoseinifar et al., 2017).
As a result, fish immune responses to invasive infections must be strengthened immediately, and ecologically benign immunostimulants should be used instead of antibiotics (Wang et al., 2017; Dawood et al., 2016). In this context, various organic acids have been utilized as immunostimulants in fish farming by several authors (Aalamifar et al., 2020; Abdel-Tawwab, 2016; Ahmed and Sadek, 2015; Da Silva et al., 2013, 2016; Nhan et al., 2010; Owen et al., 2006; Omosowone et al., 2015). Among various organic acids, fumaric acid (FA) is non-toxic organic acid. Since 1946, FA has been utilized as a food additive (Malone, 2000; Xie et al., 2003), because of its ability to reduce bacteria in feed, it has been used in food preservation (Luckstadt, 2008). Dietary organic acids have been used as alternatives to other feed additives in aquaculture diets to improve growth performance of many fish species (Ramli et al., 2005; Owen et al., 2006; Freitag and Lückstädt, 2007; Ng et al., 2009; Luckstadt and Kuhlmann, 2011). But the use of FA as an alternative to antibiotics in GIFT farming is not yet studied. Since the GIFT strain has better feed utilization, the present study hypothesized that GIFT would reveal a better immune response than Nile tilapia. Hence, the present study is aimed to evaluate the influence of FA incorporated diet on the growth performance and immune response of GIFT.
MATERIALS AND METHODS
Experimental fish and feeding trial
Healthy and uniform sized 500 numbers of GIFT fry (average weight of 0.05±0.02 g and average length of 2.03±0.21 cm) were procured from Government Fish Seed Farm, Krishnagiri, Tamil Nadu, India and transferred to the Advanced Research Farm Facility, Madhavaram, Tamilnadu Dr. J. Jayalalithaa Fisheries University, Tamil Nadu, India. The fishes were acclimatized and conditioned for 30 days and fed with a commercial feed containing crude protein at 280 g/kg of diet. After acclimatization, 360 fishes with an initial mean body weight of 2.0±0.3 g were randomly distributed in 12 FRP tanks, with 30 fish per FRP tanks (500 lit capacity), with three replicates per diet. During the 60-day feeding trial, the fish were fed until apparent satiation two times per day (09:00 and 17:00). Water was exchanged at the rate of 10% every day in each FRP tank. Aeration was continuously provided throughout the experimental period using an air blower (Hailea hi-flow diaphragm air pump). During the feeding trial, water quality parameters were monitored daily and the mean values were as follows: water temperature 27±1oC, pH 8.4±0.6, dissolved oxygen 5±0.6 mg/L and ammonia –N 0.03±0.01 ppm.
Experimental diets
Using a vertical ingredient mixer (Jinan Sunpring Machinery Co Ltd, China), dietary ingredients were finely ground and fully blended and then extruded at 60 to 70oC to prepare 1.5 mm floating pellets using a twin-screw extruder. A total of four experimental diets were formulated in this present study including one control diet without inclusion of FA and three treatment diets with graded levels of FA at 0.5, 1.0 and 1.5 g/kg of diet which were named FA 0.5, FA 1.0 and FA 1.5, respectively (Table I). A commercially available FA was procured from Hi media. All the experimental diets were stored in air tight plastic containers and kept at room temperature.
Table I. Formulation and biocomposition of the experimental diets (Kg of diet).
|
Ingredients |
Control |
FA 0.5 g/kg |
FA 1.0 g/kg |
FA 1.5 g/kg |
|
Fish meal |
80 |
80 |
80 |
80 |
|
Soybean meal |
100 |
100 |
100 |
100 |
|
Corn gluten meal |
150 |
150 |
150 |
150 |
|
Corn flour |
180 |
180 |
180 |
180 |
|
Wheat flour |
100 |
100 |
100 |
100 |
|
Rice bran (Defat) |
388 |
387.5 |
387 |
386.5 |
|
Vitamin premixa |
1 |
1 |
1 |
1 |
|
Mineral premixb |
1 |
1 |
1 |
1 |
|
Fumaric acid |
0 |
0.5 |
1 |
1.5 |
|
Chemical composition (% dry matter) |
||||
|
Moisture |
8.6 |
8.8 |
8.7 |
8.8 |
|
Crude protein |
27.79 |
27.48 |
27.81 |
27.61 |
|
Crude lipid |
5.75 |
5.6 |
5.9 |
5.8 |
|
Crude fibre |
2.78 |
2.6 |
2.41 |
2.52 |
|
Ash |
8.78 |
8.56 |
8.5 |
8.8 |
aComposition of vitamin premix (quantity per kg): Vit. A – 1, 00, 00,000 IU, Vit. B1-5,000 mg, Vit. B2 – 5,000 mg, Vit. B3- 6,000 mg, Vit. B5 – 6,000 mg, Vit. B6 – 6,000 mg, Vit. C – 60,000 mg, Vit. D3 – 20,00,000 IU, Vit. E – 10,000 EU, Vit. H – 200 mg. bComposition of mineral premix (quantity per kg): Magnesium – 2,800 mg, Iodine – 7.4 mg, Iron – 7,400 mg, Copper – 1,200 mg, Manganese – 11,600 mg, Zinc – 9,800 mg, Chlorides Cobalt – 4 mg, Potassium – 100 mg, Selenium – 4 mg, Calcium Carbonate – 27.25%, Phosphorous – 7.45 mg, Sulphur – 0.7 mg, Sodium – 6 mg, Calpan – 200 mg, Aluminium – 1,500 mg and Choline Chloride – 10,000 mg. FA, Fumaric acid.
Growth parameter and nutrient utilization
At the end of trial, all the fishes were individually counted and weighed to estimate their weight gain (WG), feed conversion ratio (FCR), feed efficiency ratio (FER), average daily gain (ADG), specific growth rate (SGR), protein efficiency ratio (PER) and survival rate (SR) were calculated according to Raj et al. (2008).
Haematological assay
At the end of the feeding trial (60th day), blood sample was collected from the caudal vein of four fish from each treatment after they were starved for 24 h. The red blood cell (RBC) counts were determined by a Neubauer hematocytometer. Haemoglobin (Hb) concentrations were determined by Cyanmethaemoglobin method (Drabkin, 1946) and haematocrit (Ht) was determined by the microhematocrit method (Nelson and Morris, 1989). According to Wintrobe (1934) the erythrocyte indices such as mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC) were calculated.
Statistical analysis
All the data were presented as the mean values ± standard deviation (SD) of three replicates. All the percentage values were subjected to arcsine transformation prior to statistical analysis. One-way ANOVA, followed by Duncan’s test at the significant level of 0.05 was used to compare the differences among the four dietary groups. The data were statistically analysed by SPSS 20.0 for windows (SPSS Inc., Chicago, IL, USA).
RESULTS AND DISCUSSION
Growth performance and nutrient utilization
The dietary supplementation of FA in the diets of GIFT significantly improved the growth performance and feed utilization. FA plays an important role in improving the growth performance, nutrient utilization and immune enhancement (Owen et al., 2006; Omosowone et al., 2015; Luckstadt and Kuhlmann, 2011). Similarly in the present study, the highest mean WG of GIFT was recorded in the fish fed with FA 1.5 g/kg of diet followed by FA 1 g/kg and FA 0.5 g/kg. There was a significant WG from lower to higher concentration of FA 0.5 g/kg to FA 1.5 g/kg. The highest SGR of GIFT was recorded in the fish fed with FA 1.5 g/kg of diet followed by FA 1 g/kg and FA 0.5 g/kg. There was a significant difference between FA 0.5 and FA 1.5 g/kg and lowest was observed in control. Previously, Das Neves et al. (2021) reported an improvement in growth, feed efficiency of Nile tilapia juveniles fed up to 15 g/kg FA diets. Similarly, Abd-Elala and Ragaa (2015) reported that O. niloticus fed on 0.2% organic acid showed significant improvements in their feed intake, live WG, SGR, PER and FCR compared to control. Similarly enhanced nutrient and mineral digestibility leading to maximum growth performance in GIFT was reported by Luckstadt and Kuhlmann (2011).
Increase in the dietary FA level also resulted in a significant increase in growth rate and lower FCR. The FA 1.5 g/kg of diet produced better WG and lower FCR than FA 1.0 g/kg of diet in the present study given in Table II. In contradictory, Omosowone et al. (2015) observed better growth performance in African catfish Clarias gariepinus when the FA level is supplemented at FA 1.0 g/kg and growth rate decreased with increasing addition of FA. The study showed lowest growth performance was observed with the diet containing 0.5 mg/kg of FA in GIFT. Overall, the present study revealed best growth performance of GIFT at 1.5 g/kg of FA incorporated diet.
Haematology parameters
Haematological characteristics are used as an effective and sensitive index to screen physiological and pathological changes in fishes. The normal ranges of various blood parameters in GIFT have been reported by different investigators (Prabu et al., 2020; Sathishkumar et al., 2021). However, FA supplemented diet fed GIFT haematological response not yet documented. Hence, the present study also intended to analyse the haematological changes of GIFT when fed with FA incorporated diet.
The results of the FA diet showed increase in haematological value of GIFT fed with FA 1.5 g/kg of diet when compared to other experimental diets (Table III). FA 1.5 g/kg diet fed fish showed significantly (p<0.05) higher RBC, haematocrit, Hb, MCV and MCHC value compared to control, significant difference was noticed within the treatments.
Table II. Growth performances of GIFT fed graded levels fumaric acid (FA) supplemented diets.
|
Parameters |
Control |
FA 0.5 g/kg |
FA 1 g/kg |
FA 1.5 g/kg |
|
Initial weight (g) |
2.08±0.04 a |
2.09±0.06 a |
2.09±0.01 a |
2.08±0.04 a |
|
Final weight (g) |
6.33±0.31 b |
6.54±0.13 b |
6.56±0.17 b |
7.80±0.16 a |
|
Weight gain (WG, g) |
4.25±0.30 b |
4.45±0.14 b |
4.48±0.17 b |
5.72±0.17 a |
|
Feed conversion ratio (FCR) |
3.14±0.21 a |
2.98±0.09 a |
2.97±0.11 a |
2.32±0.07 b |
|
Feed efficiency ratio (FER) |
0.32±0.02 b |
0.34±0.01 b |
0.34±0.01 b |
0.43±0.01 a |
|
Average daily growth (ADG, g/animal/day) |
0.071±0.004 b |
0.074±0.002 b |
0.075±0.002 b |
0.095±0.002 a |
|
Specific growth rate (SGR) (%) |
1.85±0.08 b |
1.90±0.06 b |
1.90±0.05 b |
2.20±0.04 a |
|
Protein efficiency ratio (PER) |
1.16±0.08 b |
1.21±0.04 b |
1.23±0.05 b |
1.59±0.05 a |
|
Survival rate (%) |
95.00±5.00 a |
98.33±1.52 a |
96.33±3.21 a |
96.00±1.73 a |
Values were expressed as means ± SD of three replicate trough per treatment (n=3) and values with different superscripts indicate significant differences as determined by Duncan test (p < 0.05).
Table III. Hematological responses of GIFT tilapia fed graded levels of fumaric acid supplemented diets.
|
Parameters |
Control |
FA 0.5 g/kg |
FA 1 g/kg |
FA 1.5 g/kg |
|
RBC (million/ cu mm) |
1.39±0.21 b |
1.54±0.32ab |
2.04±0.35 a |
|
|
Haemoglobin (g/dl) |
5.02±0.68 c |
4.47±0.65bc |
5.80±0.66 ab |
|
|
Haematocrit (%) |
19.30±2.86b |
21.60±0.91 b |
26.40±1.93 a |
|
|
MCV (fl) |
134.83±6.75b |
138.43±4.68 ab |
137.37±5.76ab |
|
|
MCH (pg) |
33.77±1.60a |
34.47±1.76 a |
34.73±2.10 a |
|
|
MCHC (g/dl) |
24.56±1.73b |
23.17±1.30ab |
27.90±1.51 ab |
Values were expressed as means ± SD of three replicate troughs per treatment (n=3), and values with different superscripts indicate significant differences as determined by Duncan test (p <0.05).
The haematological findings of the present study is in agreement with Reda et al. (2016) reported in which inclusion of FA diet significantly increased total erythrocyte count, Hb, haematocrit and MCH in O. niloticus. In the current study, FA might have increased the immunomodulatory effect in GIFT body through the highest liberation of calcium, phosphorous, iron, and copper from the feed constituents, particularly FA complexes. Similarly, Omosowone et al. (2015) also reported that FA incorporated diet fed African catfish showed significantly (p<0.05) higher immune response.
CONCLUSION
The present study conclude that genetically improved farmed tilapia fed with 1.5 g/Kg of FA incorporated diet significantly improved the growth, nutrient utilization and haematological response. Further study is recommended to test the maximum inclusion level of FA in the diet of GIFT.
DECLARATIONS
Acknowledgement
The authors sincerely thank Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, Tamil Nadu, India for the grants and facilities offered. We also thank the Dean, Dr. M.G.R. Fisheries College and Research Institute, TNJFU, Ponneri, Tamil Nadu, India for providing the Indoor aquaculture facility to conduct the experiment.
Funding
The study was conducted with the financial support from Tamil Nadu Dr. J. Jayalalitha Fisheries University, Nagapattinam, Tamil Nadu, India.
IRB approval
This research work has been approved by Advisory committee and Institutional Review Board of Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Tamil Nadu, India.
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), Government of India on care and use of animals in scientific research. The study was approved by Ethical Committee of Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, Tamil Nadu, India.
Statement of conflict of interest
The authors have declared no conflict of interest.
REFERENCES
Aalamifar, H., Soltanian, S., Vazirzadeh, A., Akhlaghi, M., Morshedi, V., Gholamhosseini, A. and TorfiMozanzadeh, M., 2020. Dietary butyric acid improved growth, digestive enzyme activities and humoral immune parameters in Barramundi (Lates calcarifer). Aquac. Nutr., 26: 156-164. https://doi.org/10.1111/anu.12977
Abdel-Tawwab, M., 2016. Feed supplementation to freshwater fish: Experimental approaches. LAP Lambert Academic Publishing.
Adu-Elala, N.M. and Ragaa, N.M., 2015. Eubiotic effect of a dietary acidifier (potassium diformate) on the health status of cultured Oreochromis niloticus. J. Adv. Res., 6: 621-629. https://doi.org/10.1016/j.jare.2014.02.008
Ahmed, H.A. and Sadek, K.M., 2015. Impact of dietary supplementation of sodium butyrate and/or protexs in on the growth performance, some blood parameters, and immune response of Oreochromis niloticus. Int. J. agric. Res., 3: 985-991.
Da Silva, B.C., do Nascimento Vieira, F., Mouriño, J.L.P., Ferreira, G.S. and Seiffert, W.Q., 2013. Salts of organic acids selection by multiple characteristics for marine shrimp nutrition. Aquaculture, 384: 104-110. https://doi.org/10.1016/j.aquaculture.2012.12.017
Da Silva, B.C., Vieira, F.D.N., Mouriño, J.L.P., Bolivar, N. and Seiffert, W.Q., 2016. Butyrate and propionate improve the growth performance of Litopenaeus vannamei. Aquacult. Res., 47: 612-623. https://doi.org/10.1111/are.12520
Das Neves, S.C., da Silva, S.M., Costa, G.K., Correia, E.S., Santos, A.L., da Silva, L.C. and Bicudo, Á.J., 2021. Dietary supplementation with fumaric acid improves growth performance in Nile tilapia juveniles. Animals, 12: 8. https://doi.org/10.3390/ani12010008
Dawood, M.A. and Koshio, S., 2020. Application of fermentation strategy in aquafeed for sustainable aquaculture. Rev. Aquacult., 12: 987-1002. https://doi.org/10.1111/raq.12368
Dawood, M.A., Koshio, S., Ishikawa, M., Yokoyama, S., El-Basuini, M.F., Hossain, M.S. and Moss, A.S., 2016. Effects of dietary supplementation of Lactobacillus rhamnosus or/and Lactococcus lactis on the growth, gut microbiota and immune responses of red sea bream, Pagrus major. Fish Shellfish Immunol., 49: 275-285. https://doi.org/10.1016/j.fsi.2015.12.047
Drabkin, D.L., 1946. Spectrophotometric studies XIV. The crystallographic and optical properties of the hemoglobin of man in comparison with those of other species. J. biol. Chem., 164: 703-723. https://doi.org/10.1016/S0021-9258(17)41272-5
Eknath, A.E. and Hulata, G., 2009. Use and exchange of genetic resources of Nile tilapia (Oreochromis niloticus). Rev. Aquacult., 1: 197-213. https://doi.org/10.1111/j.1753-5131.2009.01017.x
Food and Agriculture Organization, 2020. Sustainability in action. State of World Fisheries and Aquaculture. Rome, pp. 200.
Freitag, M., 2007. Organic acids and salts promote performance and health in animal husbandry. In: Acidifiers in animal nutrition (ed. C. Luckstadt). Nottingham University Press, Nottingam, U.K. pp. 131-139.
Hoseinifar, S.H., Sun, Y.Z. and Caipang, C.M., 2017. Short-chain fatty acids as feed supplements for sustainable aquaculture: An updated view. Aquacult. Res., 48: 1380-1391. https://doi.org/10.1111/are.13239
Luckstadt, C., 2008. The use of acidifiers in fish nutrition. Persp. Agric. Vet. Sci. Nutr. Nat. Resour., 3: 1-8.
Lückstädt, C. and Kühlmann, K.J., 2011. The use of diformates in tilapia-ways to improve performance sustainably. Inf. Fish Int., 2: 22-25.
Machimbirike, V.I., Jansen, M.D., Senapin, S., Khunrae, P., Rattanarojpong, T. and Dong, H.T., 2019. Viral infections in tilapines: More than just tilapia lake virus. Aquaculture, 503: 508-518. https://doi.org/10.1016/j.aquaculture.2019.01.036
Malone, L.J., 2000. Basic concepts of chemistry, 6th edition. Wiley Inter Science, pp. 639.
Nelson, D.A. and Morris, M.W., 1989. Basic methodology: Hematology and coagulation, part IV. In: Clinical diagnosis, management by laboratory methods, 17th edn (eds. D.A. Nelson and J.B. Henry). Saunder Company, Philadelphia, pp. 578–62.
Ng, W.K., Koh, C.B., Sudesh, K. and Siti-Zahrah, A., 2009. Effects of dietary organic acids on growth, nutrient digestibility and gut microflora of red hybrid tilapia, Oreochromis sp., and subsequent survival during a challenge test with Streptococcus agalactiae. Aquacult. Res., 40: 1490-1500. https://doi.org/10.1111/j.1365-2109.2009.02249.x
Nhan, D.T., Wille, M., De Schryver, P., Defoirdt, T., Bossier, P. and Sorgeloos, P., 2010. The effect of poly β-hydroxybutyrate on larviculture of the giant freshwater prawn Macrobrachium rosenbergii. Aquaculture, 302: 76-81. https://doi.org/10.1016/j.aquaculture.2010.02.011
Omosowone, O., Dada, A. and Adeparusi, E., 2015. Effects of dietary supplementation of fumaric acid on growth performance of African catfish Clarias gariepinus and Aeromonas sobria challenge. Croat. J. Fish., 73: 13-19. https://doi.org/10.14798/73.1.782
Owen, M.A.G., Waines, P., Bradley, G. and Davies, S., 2006. The effect of dietary supplementation of sodium butyrate on the growth and microflora of Clarias gariepinus (Burchell 1822). In: Proceedings of the XII international symposium fish nutrition and feeding. 147.
Prabu, E., Felix, N., Uma, A., Ahilan, B. and Antony, C., 2020. Metabolic responses of juvenile GIFT strain of Nile tilapia (Oreochromis niloticus) to dietary L-tryptophan supplementation. Aquacult. Nutr., 26: 1713-1723. https://doi.org/10.1111/anu.13122
Raj, A.J.A., Haniffa, M.A., Seetharaman, S. and Appelbaum, S., 2008. Utilization of various dietary carbohydrate levels by the freshwater catfish Mystus montanus (Jerdon). Turk. J. Fish. aquat. Sci., 8: 31-35.
Ramli, N., Heindl, U. and Sunanto, S., 2005. Effect of potassium-diformate on growth performance of tilapia challenged with Vibrio anguillarum. In: World aquaculture society conference. Abstract, CD-Rom, Bali, Indonesia.
Reda, R.M., Mahmoud, R., Selim, K.M. and El-Araby, I.E., 2016. Effects of dietary acidifiers on growth, hematology, immune response and disease resistance of Nile tilapia, Oreochromis niloticus. Fish Shellfish Immunol., 50: 255-262. https://doi.org/10.1016/j.fsi.2016.01.040
Romero, J., Feijoó, C.G. and Navarrete, P., 2012. Antibiotics in aquaculture–use, abuse and alternatives. Hlth. Environ. Aquacult., 159. https://doi.org/10.5772/28157
Sathishkumar, G., Felix, N. and Prabu, E., 2021. Effects of dietary protein substitution of fish meal with bioprocessed poultry byproduct meal on growth performances, nutrient utilization, whole body composition and haemato biochemical responses of GIFT tilapia reared in floating cages. Aquacult. Res., 52: 5407-5418. https://doi.org/10.1111/are.15410
Wang, W., Sun, J., Liu, C. and Xue, Z., 2017. Application of immunostimulants in aquaculture: Current knowledge and future perspectives. Aquacult. Res., 48: 1-23. https://doi.org/10.1111/are.13161
Wintrobe, M.M., 1934. Variations in the size and hemoglobin content of erythrocytes in the blood of various vertebrates. Folia Haematol., 51: 32-49.
Xie, S., Zhang, L. and Wang, D., 2003. Effects of several organic acids on the feeding behavior of Tilapia nilotica. J. appl. Ichthyol., 19: 255-257. https://doi.org/10.1046/j.1439-0426.2003.00451.x