https://orcid.org/0000-0002-6012-1774
Figures
Abstract
The current study evaluated the effects of dietary supplementation of Triphala (TR) on yellow perch (Perca flavescens) growth performance, immune response, related gene expression, and intestinal histological structure. The experimental design included four groups: one control group (0% TR/ kg diet) and three TR-supplemented groups with 2, 4, and 6%/kg diet for four weeks and each group was allocated in triplicates with 30 fish each. Sampling included three fish from each replicate for evaluating immune response and gene expression. Findings showed that Triphala markedly improved growth performance, Immunoglobulin M (IgM) levels, lysozyme activity, and Nitric Oxide (NO) activity with the most significant (p < 0.05) results for 6% TR/kg diet group. The TR groups also showed significantly decreased glucose and cortisol concentrations with the lowest values for the 6% TR/kg diet group. Moreover, TR-incorporated groups revealed significantly upregulated expression (p < 0.05) of growth [Insulin-Like Growth Factor-1 (IGF-1)] and immune [Alpha 2 Macroglobulin (A2M), Serum Amyloid A (SAA) and Complement Component C3 (CCC3)] genes in incorporated groups, specially the 6% TR group. Moreover, the intestinal morphometric histological analysis revealed that villus length was increased in a dose-dependent manner, coping with other enhanced parameters. Current results endorse the positive effects of Triphala incorporation on yellow perch farming as a safe alternative option to enhance growth performance, immune response, related gene expression, and intestinal histology.
Citation: Elabd H, Wang H-P, Othman R, Yao H (2025) Effect of Triphala on growth, immunity, related gene expression and intestinal morphometry of yellow perch (Perca flavescens). PLoS ONE 20(3): e0315305. https://doi.org/10.1371/journal.pone.0315305
Editor: Eman Zahran, Mansoura University, EGYPT
Received: August 22, 2024; Accepted: November 24, 2024; Published: March 4, 2025
Copyright: © 2025 Elabd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the U.S. Department of Agriculture awarded to the Corresponding Author, Han-Ping Wang (Award No. 2021-7007-34785). Salaries and research support were provided by state and federal funds appropriated to The Ohio State University. There was no additional external funding received for this study.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Aquaculture faces several challenges that can compromise fish health status including infections and management of stressful conditions [1–5]. Yellow perch (Perca flavescens) is one of the most common fish species in the eastern part of North America [6]. Currently, attention is being paid toward the use of natural phytogenic compounds (i.e., natural plant herbal compounds) as efficient alternatives to antibiotics and chemical therapies that are proven to promote growth, antioxidative, and immune responses, resistance [7–9]; and manipulate gut histology [10–14]. The intestinal histology could be indicative to the effect of natural dietary supplementation on fish health status through assessing the immune response, growth performance, and biochemical profiles [9,12,13,15].
In the current study we focused on addressing the effect of dietary Triphala incorporation in fish diets, which is a natural polyherbal formula prepared by mixing three dried powder herbs at rate of 1:1:1 (namely, Emblica officinalis, Terminalia chebula and Terminalia bellerica), on growth, physiology and immune responses in yellow perch. Triphala possess growth and immune-promoting effect, detoxification, and diseases resistance in fish [16,17]. This growth-promoting effect could be due to the effect of its content of active herbal ingredients, specially rich amino acids, fatty acids, carbohydrates [18,19].
In terms of evaluating those responses, IGF-I is a key indicator for growth performance and regulation [7,20], while IgMtetramer is the predominant immunoglobulin in teleost and contains eight antigenic combining sites [21]. Alpha 2-macroglobulins (A2M) are proteinase inhibitors that are present in the plasma of vertebrates and invertebrates. They also function as a binding protein to numerous growth factors, cytokines and hormones [12,21]. Serum amyloid A (SAA) plays an important role in inflammatory cellular response as a member of acute phase proteins that recruits chemotactic cells in the inflammatory sites. Complement Component C3 (CCC3) is also a complement protein that helps in generation of chemotactic factors, immune responses and complexes [20,22,23].
As far as we know, no previous studies addressed the effect of Triphala on yellow perch or on its intestinal morphometry. Thus, the current study was designed to evaluate the effect of Triphala dietary supplementation in yellow perch diets on growth performance, immune response, gene expression, and intestinal histological analysis.
2. Materials and methods
The current experiment was performed according to the guidelines of The Ohio State University for laboratory animal care and use. All procedures assured that all applied management practices were done while avoiding stress exposure and reducing whenever possible its level to the minimal during the experiment. All personnel were adequately trained on fish handling and different husbandry procedures.
2.1. Diets
Triphala® was obtained from Banyan Botanicals (New Mexico, USA). AquaMax grower 400® (Purina Animal Nutrition, MO, USA; Crude Protein Minimum 45.0%, Crude Fat Minimum 16.0%, Crude Fiber Maximum 3.0%, Calcium Minimum 2.2%, Phosphorous (P) Minimum 1.2%, Sodium (NA) Minimum 0.6%) was divided into four portions. The first was kept as a control without any additives. The remaining portions were mixed with 2, 4, and 6% Triphala/kg, respectively. 100 ml water was added/kg basal diet, mixed thoroughly and extruded (2 × 3 mm) using a XLS turbo force extruder 3000 (XLS International, NE, USA). After that, pellets were spread on trays and left for 24 h air drying, then dried pellets were stored in plastic Ziploc bags at −20°C.
2.2. Experimental fish and design
Yellow perch of average initial weight 18.1 ± 2.9 g and initial length 11.1 ± 0.7 cm were obtained from the hatchery of the Ohio Center for Aquaculture Research and Development at The Ohio State University South Centers, Ohio, USA and randomly distributed into four groups in 50 L fiberglass round tanks in triplicates (90 fish/group, 30 fish/replicate). Acclimation to the experiment conditions took two weeks and fish were fed to satiation two times daily (09.00 am and 16:00 pm) for four weeks. Throughout the experiment, water temperature was 20.83 ± 0.3°C and dissolved oxygen content was 7.56 ± 0.4 mg/L. Both parameters were measured twice daily and photoperiod was set to 12 h light/12 h darkness cycle. We confirm that the entire experimental flow and fish samples were in compliance with the experimental and animal care procedures that were approved by the Institutional Animal Care and Use Committee of the Ohio State University.
2.3. Sampling
At the end of the four weeks, fish anesthetization was performed using 250 ppm MS222 (Syndel Laboratories Ltd., British Columbia) and blood samples were collected (n = 9/group, 3 samples/replicate) from caudal vessels using a 1 ml syringe (Becton Dickinson, USA) containing EDTA into EDTA-coated tubes and kept on ice until 5 minutes centrifugation at 3,600 rpm and 4 °C to obtain plasma following our laboratory protocols (Elabd et al., 2020, 2017, 2016a, 2016b) for the evaluation of glucose, cortisol, IgM levels, lysozyme activity, and nitric oxide activities.
Liver samples were carefully isolated from same fish, washed with phosphate-buffered saline (PBS, pH 7.4), homogenized in 50 mM PBS (containing 1 Mm EDTA, pH 7.4) as mentioned previously by Elabd et al. (2020, 2017, 2016a, 2016b), centrifuged (15,000 × g, 10 minutes, 4ºC), and the supernatant was stored at −80 ºC until evaluating IgM and nitric oxide levels. Another same set of liver samples (n = 9/group, 3 samples/replicate) were collected in RNAlater (Ambion, USA) and stored at −80 °C for subsequent gene expression study.
2.4. Growth indices
At the stocking and after four weeks, specific growth rate (SGR), body mass gain (BMG), length gain rate (LGR), feed conversion ratio (FCR), and hepatosomatic index were measured as follows: (SGR % day−1) = [(ln final body mass (g)) - ln initial body mass (g))/number of experimental days] X 100, BMG % = 100 × [final body mass (g) - initial body mass (g)]/ initial body mass (g)], LGR % = 100 × [average terminal body length (cm) – average initial body length (cm)]/average initial body length (cm), FCR = F/(Wf _ Wi); where F is the given feed weight, Wf is the fish final weight, and Wi is the fish weight at stocking and HSI = [liver weight (g)/total body weight (g) × 100].
2.5. Immune-related parameters
IgM, lysozyme activity, nitric oxide, and myeloperoxidase activity levels were measured using a Microplate spectrophotometer (Epoch™, USA). IgM levels were measured at 450 nm using Fish IgM ELISA kit (MyBioSource Inc., USA). Lysozyme activity was measured kinetically by recording the decrease in A450 for 5 min using a Lysozyme detection kit (Sigma-Aldrich, USA). Nitric oxide was assayed at 540 nm using commercial kit (BioVision Inc., USA) according to the manufacturer’s protocol.
2.6. Glucose and cortisol assays
Plasma glucose and cortisol concentrations assays were performed in triplicates at 450 nm using (Epoch™ Spectrophotometer, USA). Glucose level was measured following the glucose assay kit’s protocol (Biovision, USA) as previously mentioned in our studies (Elabd et al., 2017, 2016a, 2016b) and cortisol according to (MyBioSource Inc., USA) manufacturer’s protocol.
2.7. Expression of IGF-1 and immune related genes
Total RNA was extracted from liver samples (n = 9/group, 3 fish/replicate) after four weeks feeding Triphala-incorporated diets using TRIzol (Invitrogen, USA) following the company’s protocol. Extracted RNA was DNase treated and concentration was checked using a Nano-Drop spectrophotometer (Thermo Scientific, USA) and purity by OD 260/280 nm ratio that ranged 1.80: 2.00; and then reverse transcripted using a high-Capacity cDNA Reverse Transcription Kit (Invitrogen, USA), following Elabd et al. (2020, 2017a, 2016a, 2016b) according to the manufacturer’s protocol for 20 μL volume. cDNA yield was stored at −20°C. IGF-1, SAA, CCC3, A2M and β-actin genes primers (Table 1) were previously designed and tested (Elabd et al., 2017, 2016a) using NCBI’s Primer–BLAST. 20 μL volume Real-time PCR analysis was performed in triplicates in 7500 Real-Time PCR System (Applied Biosystems®, USA) using SYBR select Master Mix (Applied Biosystems, USA).
Relative expression level of the test sample’s target gene to that of the calibrator was calculated using the “ΔΔCt” method mentioned by [9,12,20,21,24].
2.8. Intestinal villi absorptive capacity morphometrical assessment
Intestinal samples were submitted for histological analysis (nine samples/group, three fish/ replicate). Samples were collected in Prefer Fixative (Fisher scientific, USA), and then dehydrated, embedded in paraffin at 60 °C, sectioned after cooling down (5 μm), stained with hematoxylin-eosin (H&E), and microscopically evaluated. The histomorphometric examination was accomplished using Image J analysis software (National Institutes of Health, USA), including the whole length of mucosa (villus’s tip - the muscular layer), villus’s width (from the villus’s middle), and villus’s height (villus’s tip - crypt junction) [25,26].
3. Results
3.1. Growth performance
Table 2 shows the growth performance results of yellow perch groups incorporated with 2, 4, and 6% Triphala (TR). Triphala-supplemented groups revealed better growth performance compared to the control. The group supplemented with 6% Triphala gave the maximum significant improved average body weight, specific growth rate, body mass gain and length gain rate followed by 4 and 2% incorporated groups compared to the control group (Table 2). Feed conversion ratio and hepatosomatic index showed the best and most significant values (P < 0.05) for TR group over the control (Table 2).
3.2. Immune response
Immune-related parameters are shown in Figs 1–4. Markedly higher IgM levels in both plasma and liver homogenate were recorded in all TR-supplemented groups compared with the control. The most significant increase (p < 0.05) was for the 6% group followed by 4% and then 2% (Fig 1), and in plasma both the 6 and 4% group showed the highest result (Fig 1).
N = 9 and Means ± SEM with different superscript letters are significantly different (p < 0.05).
Results are described as mean ± SEM (n = 9). Values with different superscript letters are significantly different at (p < 0.05).
Lysozyme activity significantly increased in all TR groups with the 6% group in first place (p < 0.05); followed by 4% then 2% over the control group (Fig 2).
NO levels in plasma and liver were significantly higher (p < 0.05) in TR groups compared to the control (Fig 3). The 6% TR incorporation gave the most significant (p < 0.05) increase followed by 4% and 2% groups, which gave similar significantly higher results (Fig 3).
3.2. Glucose and cortisol assays
The TR groups significantly (p < 0.05) decreased glucose and cortisol concentrations with the most significant (p < 0.05) decrease for the 6% TR group compared to the control (Table 3).
3.3. Expression of targeted genes
The group supplemented with 6% TR showed the highest (p < 0.05) upregulation of IGF-1, SAA, CCC3, and A2M genes expression than the control group (Fig 4 A–D). The group fed with 4% TR diet gave significant upregulation (p < 0.05) in both IGF-1 and CCC3 genes expression (Fig 4A and D) and the group supplemented with 2% TR showed significant upregulation (p < 0.05) of only IGF-1 gene expression (Fig 4A).
3.4. Histological findings
The dietary incorporation with TR showed significant (p < 0.05) improvement in intestinal villi length with the most marked increase for the 6% TR group (Figs 5 and 6).
Values are expressed as mean value (n = 9) ± SE. Mean values with different superscript letters are significantly different (p < 0.05).
A) Middle intestine of yellow perch control group showing villi lined with pseudostratified epithelium, B) Middle intestine of yellow perch supplemented with 2% Triphala showing an increase of intestinal villi length, C) Middle intestine of yellow perch supplemented with 4% of Triphala showing marked increase of intestinal villi length, and D) Middle intestine of yellow perch supplemented with 6% Triphala showing marked increase of intestinal villi length and branches. H&E, X100, bar = 50 µm.
3. Discussion
Current study revealed promising growth-promoting activity of Triphala on yellow perch, as it effectively improved the growth performance indices with the best results for the 6% TR group. Triphala is a well-known Indian poly-herbal formula consisting of a combination of three herbal plants Emblica officinalis, Terminalia bellerica, and Terminalia chebula belonging to the Euphorbiaceae, Combretaceae, and Combretaceae families, respectively. Triphala improves growth through the synergistic effect of the active ingredients of its herbal structure, specially its high content of amino acids, fatty acids, carbohydrates [18,19]. This, in turn, can promote good digestion and absorption of food (Baliga et al., 2012; [13,17,27]. Similarly, [16,28] reported maximum significant growth for Oreochromis mossambicus fed on 0.8% Triphala for 60 days over the control group. In the same instance, Emblica officinalis which is one of Triphala’s three herbs, resulted in significantly higher FCR, SGR, condition factor, and weight gain in Rohu (Labeo rohita) fingerlings [29]. Current results are also in accordance with the current IGF-1 growth-related gene expression.
In the current study, significantly increased Immunoglobulin M, lysozyme activity, Nitric Oxide, and Myeloperoxidase, specifically in the 6% TR group, conferred the immunostimulating effect of Triphala in yellow perch. Those properties could be linked to the ability of Triphala to mediate immune response, and this is hypothesized to be related to its immunomodulatory and antioxidant properties because of its constituents from potent antioxidants (tannins, gallic acid, and chebulinic acid) [16–18]. Lysozyme, NO, and IgM are essential components for fish’s immune response and their defense capability [30,31]. Triphala showed improvements in the immune response of Nile tilapia [16]. Triphala also showed significant higher lipid peroxidation levels and improved cell-mediated immune response at a concentration of 1% for 48 days in rats [27].
Glucose and cortisol levels are among the biochemical indices that can be used for evaluating fish status and condition [32–34]. In this instance, glucose and cortisol concentrations were markedly decreased in TR-supplemented groups, with the most significant result for 6% TR. This can be due to the ability of Triphala to decrease blood glucose concentration through inhibiting its absorption [17]. This attribution is supported by [35,36], who reported the ability of the most important Triphala components to significantly decrease blood glucose level.
The expression of growth-related gene IGF-I revealed upregulation for the TR groups over the control group with the most noticeable upregulation for the 6% TR group, and this supports our current growth performance findings. This is related to the growth-promoting properties of Triphala and its ability to improve the feed uptake, utilization, and absorption through its active ingredients of amino acids, fatty acids, and carbohydrates [18,19]. In addition, the expression profiles of immune-related genes SAA, CCC3, and A2M showed the maximum expression for the 6% TR-incorporated group, which are also in agreement with our current results on immune response. This upregulation is also due to the active components of the positive synergestic activity between the herbal content of Triphalaa, which are well known to stimulate immune and anti-oxidative responses [17,18]. Additionally, it has been reported that Triphala effectively mitigated alterations in lipid peroxidation at concentration of 1 g for 48 days in rats [37]. Moreover, Triphala at a dose of 3 mg increased the lysozyme activity in treated mice [38].
Intestinal histology reading is correlated with growth performance and immune response parameters data, revealing the most improved findings for the group enriched with 6% TR. This revealed a remarked development of the intestinal villi length that continued to progress over time throughout the entire experimental period compared to the control group. This finding may be due to the ability triphala to sustain the intestinal contour and restore exhausted protein concentrations on the brush border of villi; and this was obvious in data reported by [16] who found significantly improved intestinal villi length in Nile tilapia incorporated with triphala compared to the control group. Similarly, [12] reported improved intestinal histology in groups supplemented with curcumin and chitosan. In addition, other herbal natural supplementations were proved to significantly enhance the intestinal morphometry of Oreochromis mossambicus and Clarias gariepinus [39]. These results are consistent with the growth and antioxidants readings, which confirm the triphala positive properties.
With the arising issue of antimicrobial resistance that results from chemical and antibiotic usage, the need for safe, natural alternatives becomes a must. Accordingly, our current findings from the yellow perch study concluded that Triphala gave promising results for growth, immune response, related gene expression, and gut morphometry. With the lack of previous studies of the effect of Triphala on yellow perch and its gut morphometry and the effective lowering of cortisol level results, more investigations addressing its antioxidant activity are required.
Acknowledgments
Authors wish to thank Dean Rapp and Paul O’Bryant for their assistance and Bradford Sherman for his comments on the manuscript.
References
- 1. Abdel-Daim MM, Eissa IAM, Abdeen A, Abdel-Latif HMR, Ismail M, Dawood MAO, et al. Lycopene and resveratrol ameliorate zinc oxide nanoparticles-induced oxidative stress in Nile tilapia, Oreochromis niloticus. Environ Toxicol Pharmacol. 2019;69:44–50. pmid:30953933
- 2. Aly SM, Zohairy MAA, Rahmani AH, Fathi M, Atti NMA. Trials to improve the response of Orechromis niloticus to Aeromonas hydrophila vaccine using immunostimulants (garlic, Echinacea) and probiotics (Organic Green TM and Vet-Yeast TM). Afr J Biotechnol. 2016;15:989–94. Available from: https://www.ajol.info/index.php/ajb/article/view/137159
- 3. Cain K. The many challenges of disease management in aquaculture. J World Aquaculture Soc. 2022;53(6):1080–3.
- 4. Dayana Senthamarai M, Rajan MR, Bharathi PV. Current risks of microbial infections in fish and their prevention methods: a review. Microb Pathog. 2023;185:106400. pmid:37863271
- 5. Irshath AA, Rajan AP, Vimal S, Prabhakaran V-S, Ganesan R. Bacterial pathogenesis in various fish diseases: recent advances and specific challenges in vaccine development. Vaccines (Basel). 2023;11(2):470. pmid:36851346
- 6. Giraudo M, Mercier L, Gendron A, Sherry J, Houde M. Transcriptome analyses in juvenile yellow perch (Perca flavescens) exposed in vivo to clothianidin and chlorantraniliprole: possible sampling bias. PLoS One. 2024;19(4):e0302126. pmid:38625968
- 7. Elabd H, Youssuf H, Mahboub HH, Salem SMR, Husseiny WA, Khalid A, et al. Growth, hemato-biochemical, immune-antioxidant response, and gene expression in Nile tilapia (Oreochromis niloticus) received nano iron oxide-incorporated diets. Fish Shellfish Immunol. 2022;128:574–81. [cited 2022 Aug 24]. pmid:36007828
- 8. Elabd H, Wang H-P, Shaheen A, Matter A. Nano spirulina dietary supplementation augments growth, antioxidative and immunological reactions, digestion, and protection of Nile tilapia, Oreochromis niloticus, against Aeromonas veronii and some physical stressors. Fish Physiol Biochem. 2020;46(6):2143–55. pmid:32829476
- 9. Elabd H, Wang H-P, Shaheen A, Yao H, Abbass A. Anti-oxidative effects of some dietary supplements on Yellow perch (Perca flavescens) exposed to different physical stressors. Aquaculture Reports. 2017;8:21–30.
- 10. Balcázar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Múzquiz JL. The role of probiotics in aquaculture. Vet Microbiol. 2006;114(3–4):173–86. pmid:16490324
- 11. Ray C, Bujan N, Tarnecki A, Davis D A, C B, CR A. Analysis of the Gut Microbiome of Nile Tilapia Oreochromis niloticus L. Fed Diets Supplemented with Previda® and Saponin. Fisheries Sciences. 2017;11(2).
- 12. Elabd H, Mahboub H, Salem S, Abdelwahab A, Alwutayd K, Shaalan M, et al. Nano-curcumin/chitosan modulates growth, biochemical, immune, and antioxidative profiles, and the expression of related genes in Nile tilapia, Oreochromis niloticus. Fishes. 2023;8(7):333.
- 13. Gad FA, Emam MA, Shourbela RM, Younis EM, Abdelwarith AA, Davies SJ, et al. The influence of various doses of Moringa oleifera extract on the antioxidant trait, cytokines, reproductive hormones performance, and gonadal histological profiles of Nile tilapia. Aquacult Int. 2024;32(6):7103–18.
- 14. Suphoronski SA, Chideroli RT, Facimoto CT, Mainardi RM, Souza FP, Lopera-Barrero NM, et al. Effects of a phytogenic, alone and associated with potassium diformate, on tilapia growth, immunity, gut microbiome and resistance against Francisellosis. Sci Rep. 2019;9(1):6045. pmid:30988331
- 15. Nayak SK. Role of gastrointestinal microbiota in fish. Aquacult Res. 2010;41(11):1553–73.
- 16. Jastaniah SD, Hassoubah SA, Aljahdali N, Albaqami NM, Nasr S, Almohmadi NH, et al. Triphala involved in reducing the susceptibility of Nile tilapia (Oreochromis niloticus) fingerlings to Saprolegnia ferax infection by boosting immune and antioxidant responses, improving growth performance, histological improvement, and gene expression indicators. Aquacult Int. 2024;32(4):5125–47.
- 17. Peterson CT, Denniston K, Chopra D. Therapeutic Uses of Triphala in Ayurvedic Medicine. J Altern Complement Med. 2017;23(8):607–14. pmid:28696777
- 18. Belapurkar P, Goyal P, Tiwari-Barua P. Immunomodulatory effects of triphala and its individual constituents: a review. Indian J Pharm Sci. 2014;76(6):467–75. pmid:25593379
- 19. Parveen R, Shamsi TN, Singh G, Athar T, Fatima S. Phytochemical analysis and In-vitro Biochemical Characterization of aqueous and methanolic extract of Triphala, a conventional herbal remedy. Biotechnol Rep (Amst). 2018;17:126–36. pmid:29619331
- 20. Elabd H, Wang H-P, Shaheen A, Yao H, Abbass A. Feeding Glycyrrhiza glabra (liquorice) and Astragalus membranaceus (AM) alters innate immune and physiological responses in yellow perch (Perca flavescens). Fish Shellfish Immunol. 2016;54:374–84. pmid:27129627
- 21. Elabd H, Faggio C, Mahboub HH, Emam MA, Kamel S, El Kammar R, et al. Mucuna pruriens seeds extract boosts growth, immunity, testicular histology, and expression of immune-related genes of mono-sex Nile tilapia (Oreochromisniloticus). Fish Shellfish Immunol. 2022;127:672–80. pmid:35817363
- 22. Wang J, Yang Y, Zhang A, Zeng L, Xiao S, Ma H, et al. Serum amyloid protein (SAA) as a healthy marker for immune function in Tridacna crocea. Fish Shellfish Immunol. 2022;122:495–500. pmid:35202805
- 23. Mokhtar D, Zaccone G, Alesci A, Kuciel M, Hussein M, Sayed R. Main components of fish immunity: an overview of the fish immune system. Fishes. 2023;8(2):93.
- 24. Elabd H, Wang H-P, Shaheen A, Yao H, Abbass A. Astragalus membranaceus (AM) enhances growth performance and antioxidant stress profiles in bluegill sunfish (Lepomis macrochirus). Fish Physiol Biochem. 2016;42(3):955–66. pmid:26729192
- 25. Ismail T, Nassef E-D, Hegazi E-S, Bakr A-N, Moustaf EM, Abdo W, et al. The modulatory effect of dietary betaine on intestinal absorptive capacity, lipogenesis and expression of lipid metabolism- and growth-related genes in Nile tilapia fed on soybean meal-based diet. SVR. 2019 [cited 2024 Jun 4. ].
- 26. Othman R, Wang H-P, Elabd H, Xie D-K, Yao H, O’Bryant P, et al. The effect of density on sex differentiation, sexual dimorphism, stress, and related gene expression in yellow perch. PLoS One. 2022;17(5):e0267904. pmid:35507560
- 27. Baliga MS, Meera S, Mathai B, Rai MP, Pawar V, Palatty PL. Scientific validation of the ethnomedicinal properties of the Ayurvedic drug Triphala: a review. Chin J Integr Med. 2012;18(12):946–54. pmid:23239004
- 28. Sruthi MV, Nair AB, Arun D, Thushara VV, Sheeja CC, Vijayasree AS, et al. Dietary curcumin influences leptin, growth hormone and hepatic growth factors in Tilapia (Oreochromis mossambicus). Aquaculture. 2018;496:105–11.
- 29. Tamta M, Saxena A. Effect of Amla (Emblica officinalis) on the hematology and serum biochemical parameters of Rohu fingerlings in Tarai conditions of Uttarakhand. Int J Curr Microbiol App Sci. 2018;7(10):2188–94.
- 30. Chakrabarti R, Srivastava PK, Verma N, Sharma J. Effect of seeds of Achyranthes aspera on the immune responses and expression of some immune-related genes in carp Catla catla. Fish Shellfish Immunol. 2014;41(1):64–9. pmid:24657259
- 31. Magnadóttir B. Innate immunity of fish (overview). Fish Shellfish Immunol. 2006;20(2):137–51. pmid:15950491
- 32. Mahboub HH, Shahin K, Zaglool AW, Roushdy EM, Ahmed SAA. Efficacy of nano zinc oxide dietary supplements on growth performance, immunomodulation and disease resistance of African catfish Clarias gariepinus. Dis Aquat Organ. 2020;142:147–60. pmid:33331282
- 33. Osman AGM, Koutb M, Sayed AE-DH. Use of hematological parameters to assess the efficiency of quince (Cydonia oblonga Miller) leaf extract in alleviation of the effect of ultraviolet--A radiation on African catfish Clarias gariepinus (Burchell, 1822). J Photochem Photobiol B. 2010;99(1):1–8. pmid:20206545
- 34. Zhu W, Zhang Y, Zhang J, Yuan G, Liu X, Ai T, et al. Astragalus polysaccharides, chitosan and poly(I:C) obviously enhance inactivated Edwardsiella ictaluri vaccine potency in yellow catfish Pelteobagrus fulvidraco. Fish Shellfish Immunol. 2019;87:379–85. pmid:30690155
- 35. Rajan SS, Antony S. Hypoglycemic effect of triphala on selected non insulin dependent diabetes mellitus subjects. Anc Sci Life. 2008;27(3):45–9. pmid:22557278
- 36. Yang MH, Vasquez Y, Ali Z, Khan IA, Khan SI. Constituents from Terminalia species increase PPARα and PPARγ levels and stimulate glucose uptake without enhancing adipocyte differentiation. J Ethnopharmacol. 2013;149(2):490–8. pmid:23850833
- 37. Srikumar R, Parthasarathy NJ, Manikandan S, Narayanan GS, Sheeladevi R. Effect of Triphala on oxidative stress and on cell-mediated immune response against noise stress in rats. Mol Cell Biochem. 2006;283(1–2):67–74. pmid:16444587
- 38. Rasool M, Sabina EP. Antiinflammatory effect of the Indian Ayurvedic herbal formulation Triphala on adjuvant-induced arthritis in mice. Phytother Res. 2007;21(9):889–94. pmid:17533629
- 39. Mbokane EM, Moyo NAG. Use of medicinal plants as feed additives in the diets of Mozambique tilapia (Oreochromis mossambicus) and the African Sharptooth catfish (Clarias gariepinus) in Southern Africa. Front Vet Sci. 2022;9:1072369. pmid:36590800