Infections resulting from A. hydrophila and Pseudomonas fluorescens in fish have been reported to lead to high mortality, which is a pivotal concern in fish culture, due to high economic losses (Plumb and Hanson 2011; Noga 2012; Hardi et al., 2019). The present study demonstrated the antibacterial activities of crude PAE against A. hydrophila, P. aeruginosa and P. fluorescens, with the methanol extract exhibiting similar activity to that of the synthetic antibiotics used in aquaculture. Our observations in the present study are similar to those of previous authors (Okigbo and Igwe, 2007; Oluwafemi and Debiri, 2008; Bhat et al., 2015; Biswas et al., 2020; Adeniyi et al., 2021b) who observed significant antimicrobial effects of Phyllanthus amarus against several pathogenic and food poisoning organisms.
The growth-promoting potential of PAE was demonstrated in the present study. The enhanced growth performance might have been due to the ability of P. amarus to stimulate nutrient digestibility through the presence of several phytochemical compounds, including the lignin glycosides called phyllanthostatin (Pettit et al., 1990); flavonoids like quercetin and kaempferol (El-Mekkawy et al., 1995); ellagitannins, such as geraniin and furosin (Miguel et al., 1996); niruroidine (Quian-Cutrone et al., 1996); and nirtetralin A (Wei et al., 2012). Although there are few studies on the influence of P. amarus on fish performance, growth-promoting effects of this species have been reported in aquatic crustaceans (Kalaiselvi et al.., 2019; Ngo et al.., 2020) while Sunitha et al. (2017) and Olusola et al. (2021) also observed growth-promoting effects of the leaf of another species, P. niruri, on common carp and African catfish. Adeniyi et al (2021a, 2023) also reported that the growth performance and/or nutrient digestibility of C. gariepinus increased with dietary tamarind and Euphorbia heterophylla foliage extracts. The greater survival and growth exhibited by the fish fed PAE-supplemented diets in the present study was similar to the observations of Sunitha et al. (2017). The enhanced survival might also be associated with the improved condition factor, which could be attributed to the enhanced uptake of nutrients and the antimicrobial activities of the supplement.
Red and white blood cell counts were enhanced in Clarias gariepinus fed fortified P. amarus in the present study, while the white blood cell differential counts did not differ significantly. Annalakshmi et al. (2013) and Olusola et al. (2021) also observed increased RBC and WBC counts in Labeo rohita fed with P. amarus and P. niruri infected with Aeromonas hydrophila and P. aeruginosa, respectively. Hematological parameters such as Hb, PCV, MCV, MCH and MCHC are particularly known to indicate erythrocytic status and oxygen carrying capability in fish. The increased in the levels of these parameters might indicate the stimulation of erythropoiesis, hence increasing the capacity for oxygen transport and strengthening the defense mechanisms against physiological stress (Gabriel et al., 2015). The increase in the WBC and lymphocyte counts in fish fed PAE-supplemented diets might be attributed to the immunostimulatory property of the plant. Medicinal herbs and their active metabolites have potent therapeutic and prophylactic effects on a wide array of immunological disorders by modulating the immune response (Mohamed et al., 2017).
Blood biochemical parameters are widely used tools for evaluating the safety of feed additives in animal and fish nutrition. The increase in protein synthesis; and decrease in AST, ALT, ALP and BUN in fish fed 1.0 and/or 1.5 g PAE diets in the present study could indicate the protective effect of these diets on hepatocytes and glomeruli (Brien and Walterson, 2009; Reda et al., 2013). Our the results obtained in this study is in line with several studies that have demonstrated hepatoprotective effects of genus Phyllanthus leaf in mammals (Bhattacharjee and Sil, 2007; Naaz et al., 2007; Pramyothin et al., 2007; Igwe et al., 2007; Faremi et al., 2008; Bakhtiary et al., 2012) and fish (Olusola et al.., 2021) in relation to reduced ALT and AST. The increase in antioxidant enzymes (SOD, GPx and GST) and reduction of MDA in the present study coincide with the earlier reports on the in vitro (Kumaran and Karunakaran, 2007; Devi et al., 2016) and in vivo antioxidative activity of this plant in terrestrial animal (Harikumar and Kuttan, 2007; Karuna et al., 2009; Akporowhe and Onyesom, 2016) and shrimp (Ngo et al., 2020). The antioxidant activity of PAE could be attributed to the presence of flavonoids (rutin, astragalin, kaemferol, quercetin) and ellagitannins (amariin, repandusinic, phyllanthusiin D (Londhe et al., 2008; Ghosh et al., 2022; Motou et al., 2023) in the plant.
Lysozyme, phagocytic and respiratory burst activities have been used widely to evaluate the immunostimulatory effects of dietary herbal additives in both farmed fishes and shrimps (Punitha et al., 2008; Sunitha et al., 2017; Ngo et al., 2020, Adesina et al., 2021, Adeniyi et al., 2023). Lysozyme is an important enzyme that contributes immensely to the innate immune system in animals. The enzyme plays an antimicrobial role by preventing the invasion of bacteria through disruption of the bacterial wall and promoting destruction of the wall through phagocytic cells, while respiratory burst causes release of reactive oxygen species that kill pathogenic bacteria (Saurabh and Sahoo, 2008; Abarike et al., 2019; Hampton et al., 2020). The enhanced activities of these immune parameters in the fish fed PAE-fortified diets further support the basis for utilization of the plant in traditional medicine. The enhanced immune responses are in line with the response of grouper (Punitha et al., 2008) and shrimp (Ibrahim et al., 2015; Ngo et al., 2020) to diets containing Phyllanthus extract and might have contributed to the enhanced fish survival. The enhanced survival is consistent with earlier reports on fishes fed herbal-supplemented diets due to their antimicrobial agents and immune-stimulating effects against fish pathogens (Hardi et al., 2012, 2019; Adesina et al., 2021; Adeniyi et al., 2023). The in vivo antibacterial activity observed in the present study could be attributed to the aforementioned lignans, flavonoids, alkaloids, and terpenoids, among others, in the P. amarus extract, which might justify the wide utilization of this plant in traditional medicine to treat different ailments.
In conclusion, the current study investigated the effects of dietary Phyllanthus amarus on the growth, blood profile, antioxidative status and immune response of Clarias gariepinus. It could be concluded that dietary Phyllanthus amarus leaf and flower mixture extract significantly promoted growth performance; hemoglobin and lymphocytes levels; and SOD, GPx, GST, lysozyme, phagocytic and respiratory burst activities at the 1.0-1.5 g inclusion level. Therefore, the inclusion of 1.0 g Phyllanthus amarus is recommended in the diet of Clarias gariepinus for enhanced growth and physiological status.