No striking changes in the feeding response, swimming pattern, and body colouration in O. niloticus upon dietary administration of FFC at the 1X and 3X doses were noted. The 1X and 3X groups’ feed intake abated significantly than the control in a dose-dependent fashion during the dosing tenure hinting at the reduced acceptability of FFC feed. The 1X group showed an increment in biomass by 1.08-folds of its original biomass. In contrast, the 3X group recorded a 1.04-folds biomass increment. With the termination of dosing, the juveniles showed no abnormalities and increased interest in feeding, though insignificantly, similar to a previous study (Bowker et al., 2013). The 1X group that received the therapeutic dose and dosage recorded 100% survival. Contrarily, the survival in the 3X group decreased during the dosing and post-dosing periods but the differences were insignificant compared to other groups. In similar experiments also, no mortalities were observed during the dosing period at the therapeutic or lower FFC dose levels (Elia et al., 2016; Bardhan et al., 2022). Gaikowski et al. (2013) observed three inconsequential mortalities in their works on Oreochromis spp., which were unrelated to FD.
The serum glucose level of the O. niloticus was concomitant with the values recorded by Bardhan et al. (2022). The 10 days of dosing increased the glucose levels significantly in both groups, insinuating the FFC prompted stress even at the therapeutic dose and altered carbohydrate metabolism similar to earlier studies (Julinta et al., 2019; Bardhan et al., 2022). These higher levels in the 1X group remained constant till day 14 PFD signifying the persistence of stress. The degree of glucose increment was, however, significantly higher in the 3X group. Though the serum glucose levels of the 1X group curtailed significantly on day 21 PFD, the levels were still significantly higher than those on day 0. The serum calcium levels reduced significantly on day 10 FD, indicating a discrepancy in osmolarity and ionic balance, possibly due to dosing. Nevertheless, the calcium levels normalized within 3 weeks of cessation of dosing. Even, the 3X group followed a similar trend. Likewise, the serum chloride levels showed an insignificant decline on day 10 in the 1X group. In contrast, the chloride levels of the 3X group showed a rapid decline on day 10 FD. These levels persevered for 2 weeks of cessation of dosing and reverted to their initial conditions on week 3. These data revealed that the FFC had a greater impact on calcium ions than on chloride ions. The results of our study proclaimed that freely available chlorine readily combines with FFC and transforms it for easy elimination. The recorded serum creatinine levels in the control were affiliated with the results of a previous study (Bardhan et al., 2022). The creatinine levels increased significantly in a dose-dependent manner, which implied nephropathy similar to oxytetracycline (OTC)-dosed O. niloticus (Julinta et al., 2019). The creatinine levels of the 1X group recouped within 2 weeks of suspension of dosing indicating the reversible nature of FFC induced stress on kidney functioning at the therapeutic dose. The post-dosing tenure creatinine levels in the 3X group also suggested only a slight improvement in the renal functions of O. niloticus and corroborate our earlier report (Bardhan et al., 2022).
The serum ALT and AST levels in the control are analogous to previous studies (Julinta et al., 2019; Bardhan et al., 2022), which got influenced by FD, thus indicating liver tissue deterioration or injury. The significant increase in ALT levels in both groups and the inability to recoup on day 21 PFD indicated the FFC induced hepatotoxicity. A dose-dependent increase in AST levels was observed confirming the hepatotoxic potential of FFC. The significant hike in AST levels in the 1X group during the dosing period also signified that the FFC at the therapeutic dose may impair the liver tissues as was observed in earlier studies (Julinta et al., 2019; Bardhan et al., 2022). Our results endorsed the findings of Er and Dik (2014), who documented an increase in AST levels upon FFC application in Oncorhynchus mykiss. The induced liver damage was indistinguishable with the increased ALP levels in the 1X and 3X groups on day 10 FD and day 1 PFD probably due to the accumulation of FFC-residues in the liver tissues upon dosing. The ALP levels did not reduce with the cessation of dosing, suggesting persisting liver inflammation in the dosed fish.
Alterations in O. niloticus haematology and haematological indices were observed considerably in the dosed groups in a dose-dependent fashion. The TECs of the 1X and 3X groups reduced significantly upon dosing although the reduction was similar. A similar reduction in TECs was observed on OTC fed Cyprinus carpio (Kondera et al., 2020) and O. niloticus (Omoregie and Oyebanji, 2002), gentamicin injected O. niloticus (Chen et al., 2004) and amoxicillin exposed Labeo rohita (Umamaheswari et al., 2019). Decreased TECs are often conveyed by a reduction in Hb and Ht (Kondera et al., 2020), who proposed antibiotic exposure harms the haematopoietic organs of fish and induces anaemic conditions. Most of the previous works on antibiotic impairment in fish haematology also documented similar observations (Omregie and Oyobanji, 2002; Chen et al., 2004; Ambili et al., 2013; Umamaheswari et al., 2019; Kondera et al., 2020). Our observation on the decrease in TECs indicated anaemia and impaired osmoregulation caused by FFC, which was supported by the histological observations of dosed fish. In the present study, a significant decline in Hb content was observed in the FFC-dosed O. niloticus, which may be due to insufficient oxygen supply and anaemic conditions. Haem content possesses a significant role in energy metabolism, the alterations of which may lead to behavioural changes and lethargy. Though such responses were not documented in the current study, the ablation of Hb levels could also imply the inhibitory effect of FFC on the enzyme system responsible for haemoglobin synthesis. The reduction in Hb content could also lead to the formation of hypochromic cells (Chaudhry and Kasarla, 2017) as observed in our study. Irregularly shaped erythrocytes and a reduced TEC could decline the Ht (%) of an organism (Umamaheshwari et al., 2019) and our findings corroborate their findings. The levels of erythrocyte indices revealed the normal or anaemic condition of the juveniles. The MCV values of the FFC-dosed O. niloticus were elevated, which could be due to the hypoxic and anaemic conditions created by the drug. Increased MCHC values also indicated a protective response of the O. niloticus against FFC toxicity. The highly lipophilic property of FFC may enable it to cross the membranes of erythrocytes and make them fragile and prone to disruption (Bardhan et al., 2022). Furthermore, the ionic imbalance leads to excessive energy demands, which may also affect the membrane of erythrocytes as was observed in the current study. The observed alterations in the number of erythrocytes may be due to a redeeming response of fish towards FFC toxicity (Iftikhar and Hashmi, 2021).
Antibiotic feeding is often accompanied by leucocytosis due to inductive stress and activation of the piscine immune system (Kondera et al., 2020). The present study observed a considerable increase in TLCs of FFC-dosed O. niloticus to protect against FFC-toxicity, which persevered even 3 weeks upon cessation of feeding. This stimulatory effect may have happened due to the probable increase in antioxidant levels by FFC (Shiroma et al., 2020). Lunden and Bylund (2002) opined that TLC is related to the immune response of the body, thus, in the presence of any foreign stress, the spleen produces new leucocytes. The observed leucocytosis depicted the reaction of O. niloticus to FFC-toxicity. The increase in TLCs was accompanied by a significant hike in lymphocytes and ablation in monocytes, which corroborate the findings of Lis et al. (2011) documented in mice upon FFC administration. Induced lymphocytosis as observed in our study is likely due to FFC-toxicity, which is often preceded by inflammatory conditions or suppressed immunity in fish (Limbu et al., 2020). Thrombocytosis (excessive platelet counts) is uncommon in fish upon antibiotic exposure. It is more likely a secondary condition observed in connection with stress. However, earlier studies reported a significant hike in TCs of OTC-dosed O. niloticus (Reda et al., 2013; El-Adawy et al., 2018). The platelets are important for blood clotting and elevated TCs can be an indicator of inflammation. As a part of their inflammation mediation, platelets can exert chemotaxis on leucocytes and activate them (Klinger, 1997). The hike in TCs as observed in our study consolidated the role of thrombocytes in O. niloticus by transferring essential information to leucocytes compensating for subdued immune response upon dosing.
In the present study, the alterations in the RBC indices such as MCV, MCH, and MCHC values indicated the stress responses of the fish to the dietary FFC. During hypoxic conditions, the carbon dioxide level may increase and the blood becomes acidic, leading to the rupture of erythrocytes (Ramesh et al., 2018). The observations on the rupture of erythrocytes together with several lobopodial and cytosolic projections might have altered the MCV levels in O. niloticus. Further, an increase in MCH values is an indication of an increase of irregularly shaped erythrocytes in circulation and swelling of RBCs (Bojarski and Witeska, 2020). The reduction in MCHC values can be attributed to the loss of Hb and damaged erythrocytes (Bojarski and Witeska, 2020). The results of our study indicated mild alterations in the haematological parameters at the therapeutic dose as a non-specific immune response to FFC toxicity. Nevertheless, the levels of haematological parameters and indices recouped more or less within 3 weeks.
The blood cell morphological changes were mostly restricted to the erythrocytes in our study. The alterations and anomalies within the erythrocytes were roughly segregated into erythrocytic nuclear abnormalities (ENA) and erythrocytic cellular abnormalities (ECA) (Shahjahan et al., 2018). The ENAs included notched nucleus, nuclear blebs, bi-nucleated nucleus, prominent micronucleus, dark stained nuclear chromatin, peripheral nucleus, karyolysis, and dark stained regions on the nuclear membrane periphery. The ECAs included vacuolations in the cytosol, cytosol projections, hypochromic cytosol, tear-drop-shaped, spindle-shaped, and longitudinally flattened cells. The predominant increase in lymphocytes in the dosing groups suggested FFC-toxicity, stress, and lymphoid cell proliferation (Gaikowski et al., 2013). Although there was no tangible substantiation of hematopoietic or lymphopoietic tissue degradation, the significant increase in lymphocytes hinted at the stress the fish endured. The RBCs when exposed to certain stimuli produce apparent morphological and molecular alterations called “shape-shifted RBCs (shRBCs)” (Chico et al., 2018; Bardhan et al., 2022). In our study, several alterations in erythrocytic shape, viz., tear-drop, spindle, and longitudinally flattened were documented, which further established the cytotoxic and genotoxic effect of FFC as observed in earlier studies (Botelho et al., 2015; Bardhan et al., 2022). Documentation of the increased number of damaged erythrocytes can be attributed to the FFC induced genotoxicity, which was prominent in ENAs. Dark stained nuclear chromatin was observed in many erythrocytes of the 3X group on day 10 FD. Karyolysis, peripheral nucleus, reduced hypochromic cytosol, and vacuolations were also observed at the therapeutic dose. The increased incidence of ruptured cell membranes at the 3X dose supported the results of Blaskó et al. (1986), possibly due to the attachment of FFC on the erythrocytic membrane and hindrance in the membrane transport system as confirmed by the reduction in serum calcium and chloride levels. It has also been reported that the rupturing of cell membranes is due to disruption of the lipid solubility of membranes of erythrocytes (Walia et al., 2013). The smudge cells were documented plenty in our study, which indicated lymphocytosis. Erythrocytic morphological alterations often change the intracellular Hb content (Shou et al., 2020) and our results on Hb supported their findings.
In poikilothermal organisms, the lipid bilayer micro-viscosity, the phase distribution of lipids, the micro-surrounding of proteins, the protein-lipid interactions, and other characteristics of the membrane structure promotes the efficient organization of erythrocytes (Shahjahan et al., 2020). The increased frequencies of ENAs can occur due to the increased production of lipid peroxidation in the blood cells of fish (Shahjahan et al., 2020). Florfenicol-dosing induced excessive lipid peroxidation in the kidney and liver tissues of O. niloticus (unpublished results) justifying our observations on erythrocytes. The formation of lobopodial and cytoplasmic projections in erythrocytes from one side as observed in the present study has also been noted in C. carpio exposed to pesticides (Massar et al., 2012) demarcating the FFC-toxicity in fish erythrocytes. Significantly increased percentile rate of erythrocytes with micronucleus notched nucleus and blebbed nucleus possibly indicated higher production of caspase-activated DNase and oxidative stress to mitochondrion causing breakage of cytoskeletal and nuclear proteins (Witeska et al., 2014; Qureshi et al., 2016). The appearance of micronucleus in the cell is an index of accumulated genetic damage and is one of the most suitable techniques to identify cytotoxicity in fish (Massar et al., 2012). However, the micronuclei frequency is dependent upon the metabolic capacity, oxidative stress, DNA repair efficiency, and defence mechanisms of fish (Seriani et al. 2011). We observed various nuclear and cellular abnormalities in erythrocytes of the 1X and 3X groups. It is assumed that such types of ECA may endure morphological alterations in the plasma membrane affecting surface deformability and making the erythrocytes more susceptible to burst when crossing small capillaries. Nevertheless, the therapeutic dose did not show any signs of cell rupture hinting at the safety of FFC. Upon cessation of dosing, the blood smear revealed increased prominence of mature erythrocytes and healthier cellular elements in the therapeutic group. The elimination of stress also terminated the formation of shRBCs and reversed the increased prominence of lymphocytes However, ENA was still observed in all the treatment groups, perhaps the fish may require more time to recoup. Though the FFC induced cytotoxicity and genotoxicity are reversible, its impact on genotoxicity needs further studies.
Histopathological investigations have long been accepted to be reliable biomarkers of stress in fish for several reasons (van der Oost et al., 2003). The overall toxic impact on organs like kidneys and liver may seriously affect the metabolic as well as physiologic activities and could impair the growth and behaviour of fish (Bojarski et al., 2020). A mild to moderate histological alterations like degeneration of renal tubular epithelium, widened lumen, renal tubules with vacuolation, inflammation, and glomerulopathy were observed in the kidneys probably indicating the nephrotoxic effect of FFC upon oral-dosing at 15–45 mg/kg biomass/day with increased intensity of damages at the higher dose. Likewise, Gaikowski et al. (2013) reported mild tubular degeneration in O. niloticus upon FFC administration at 15 mg/kg biomass/day. The presence of tubule degeneration, coupled with the absence of necrosis in the kidneys of O. niloticus of the present study indicated that the kidney suffered damage upon dosing, but the lower dose may have barred the establishment of necrosis in this organ. Glomerulopathy with dilated Bowman’s space is the indication of a defective glomerular filtration of blood and removal of excess wastes and fluids. The observations on inflammation and loss of structural integrity of the renal tubules in FFC-dosed fish possibly suggested the prolonged time for excretion. Nephrocalcinosis (mineralization) was observed in this study on day 10 FD in the 3X group corroborating the previous FFC safety study (Gaikowski et al., 2013), possibly due to the elevated ambient CO2 levels and dietary mineral imbalances. The observed histopathological changes in the kidney can also be correlated to elevated serum creatinine levels and impaired kidney functions, particularly the glomerular filtration rate. Nevertheless, medication-related serum creatinine hikes are usually delimited and tend to stabilize and return to baseline values following drug discontinuation (Choudhury and Ahmed, 2006), which conforms to the results of the present study.
There was a dose-related increased incidence of mild to marked hepatocyte vacuolation in FFC-dosed O. niloticus. Healthy, captive-raised fish are expected to have varying amounts of glycogen-type hepatocellular vacuolation, often dependent on the amount of lipid or carbohydrate present within the diet (Gaikowski et al., 2013). Glycogen depletion in the hepatocytes is usually found in antibiotic-stressed fish (Julinta et al., 2020) because the glycogen acts as a reserve of glucose to supply the higher energetic demand occurring in such situations (Wolf and Wheeler, 2018). The dose-dependent significant decrease in food consumption, especially during the latter part of the dosing period in the 1X and 3X groups, may have impacted the energy balance (Wolf and Wolfe, 2005), and thus possibly hepatocyte vacuolation in FFC-dosed fish. The 1X group documented eosinophilic bodies, which are indicative of apoptosis and are often accompanied by swelling of the hepatocytes (Abdel-Moneim et al., 2012). However, the observation of such bodies accompanied by clear parenchyma suggested the absence of necrosis, as the probability of a dying hepatocyte due to excessive vacuolation is not rare (Wolf and Wheeler, 2018). A dose-dependent varying degree of cytoplasmic degeneration was documented in FFC-dosed groups, suggesting depletion of the glycogen reserves in the hepatocytes and stress to fish. This FFC-induced hepatotoxicity was also confirmed by the significant alterations in the liver function biomarkers like serum AST, ALT, and ALP. The excessive vacuolation may have altered the permeability of the hepatocellular membrane and the release of these soluble enzymes. The magnitude of increase crudely reflects the number of affected hepatocytes. Conceptually, the ALP, ALT, and AST are believed to be hepatocellular “leakage” enzymes (Ramesh et al. 2021). Although the intensity of vacuolation recouped slightly, it was still observed on day 21 PFD.