Effects of Extended Feeding of Florfenicol Coated Medicated Diets on the Safety, Serum Biomarkers and Blood Cells Morphology of Nile Tilapia Oreochromis Niloticus (L.) Juveniles

Tilapia is one of the most consumed farmed �sh, which requires the use of antibiotics in certain phases of its production. This study assessed the safety of 30 days of oral �orfenicol (FFC)-dosing at 0-10 times the therapeutic dose (1X: 10 mg/kg biomass/day) in Oreochromis niloticus juveniles. Behavioural changes, feed consumption, mortality and biomass were evaluated. Besides, the levels of serum glucose, calcium, chloride, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and blood cell morphology were determined at scheduled intervals. The 30 days of oral FFC-dosing caused 3.33% (1X) to 18.33% (10X) mortalities, reduced feed intake and biomass in a dose-dependent manner. The �sh fed the therapeutic dose recorded 1.25 folds increase in biomass; while the control group recorded 1.45 folds increase in 30 days. No signi�cant erythrocyte morphological alterations were observed in the 1X group compared to the control. However, marked morphological alterations like tear-shaped, spindle-shaped and degenerative erythrocytes in higher dosing groups indicated FFC cytotoxicity. All the serum biomarkers of O. niloticus increased signi�cantly on day 10 and day 30 FFC-dosing in a dose-dependent manner, except for calcium and chloride, which reduced signi�cantly during the dosing period. Within 2 weeks of suspension of FFC-dosing, the serum biomarker levels became normal except for alkaline phosphatase and creatinine. The recovery of biomass, feed intake, serum biomarker levels and erythrocyte morphological changes suggested that the FFC induced changes are reversible. This study has, thus, proclaimed the safety of FFC at the therapeutic dose in O. niloticus.


Introduction
Florfenicol (FFC) is a synthetic, broad-spectrum, uorinated analogue of thiamphenicol with the same mechanism of action as chloramphenicol (Fukui et al. 1987).Studies have shown that its activity is quite high when compared to either chloramphenicol or thiamphenicol and more bactericidal (Gaikowski et al. 2003(Gaikowski et al. , 2013;;Gaunt et al. 2010;Dowling 2013).In-vitro investigations with FFC have validated its potent activity against numerous sh pathogenic bacteria (Michel et al. 2003;Dowling 2013).Florfenicol is a relatively recent addition to aquaculture's approved antibiotic arsenal (USFWS 2015).Being highly lipophilic, it provides high enough concentrations to treat intracellular pathogens and cross some anatomic barriers.Florfenicol binds to the bacterial 50S ribosomal subunit and inhibits protein synthesis at the peptidyltransferase stage (Dowling 2013).The therapeutic dose of FFC is 10-15 mg/kg sh biomass/day administered as medicated feed for 10 consecutive days (USFWS 2015).In-vivo e cacy against furunculosis in Salmo salar and vibriosis in Gadus morhua have been demonstrated (Samuelsen and Bergh 2004;Higuera-Llantén et al. 2018).Florfenicol is well absorbed in all sh species tested and possesses bio-availabilities of 91 and 100% in G. morhua and S. salar, respectively (Horsberg et al. 1996; Samuelsen et al. 2003).Elimination of orfenicol from the plasma of G. morhua is slow compared to S. salar with a half-life of 43 h and 12-14 h, respectively (Samuelsen et al. 2003).
Tilapia is one of the most consumed farmed sh in the world and the most important farmed noncyprinid sh globally, with a total production of 6.6 million tonnes in 2019.It has the potential to play a leading role in the ght against food insecurity and malnutrition (FAO 2020).Nile tilapia Oreochromis niloticus contributes to 80% of the global tilapia production and has been the only cichlid species maintaining stable market prices across the globe (FAO 2020).The intensi cation of aquaculture has led to frequent disease outbreaks, which rely on antimicrobial therapeutics (Okocha et al. 2018).Target animal safety data are an indispensable part of the drug registration process.The recent years have seen a radical reduction in antibiotic use in some countries due to vaccination and improved husbandry practices, predominantly in Norway (Evensen 2016).Yet, antibacterial therapy remains the last stand to combat bacterial infections in aquaculture in many countries (Lulijwa et al. 2020).Florfenicol is one of the United States Food and Drug Administration (USFDA) approved and the most commonly used antibiotics in aquaculture (USFWS 2015; Evensen 2016).To curb illegal use, aquaculturally in uential countries have formed safety authorities, viz., Norwegian Food Safety Authority (NFSA), EU Council Regulations (EC), USFDA and the European Medicines Agency (EMA) for proper antimicrobial monitoring, whose regulations and policies are backed up with stringent monitoring (Lulijwa et al. 2020).In intensive tilapia cultivation, the FFC has often been recommended as a therapeutic agent to control several diseases (Shiroma et al. 2020).The safety of Aqua or®, a feed premix containing FFC at a maximum recommended dose of 15 mg/kg biomass/day in monosex O. niloticus (45.8 ± 10.5 g) for 20 consecutive days has been demonstrated (Gaikowski et al. 2013).Although reports are available on the use of FFC to control diseases in aquaculture (Gaikowski et al. 2003(Gaikowski et al. , 2013;;Gaunt et al. 2010;Bowker et al. 2013), the effects of oral administration of pure FFC powder in the tropical condition is imprecisely studied.Also, safety studies are not available on the use of FFC for top-coating onto the basal feed for aquaculture.
The surface coating of a drug with a palatable binder like vegetable oil increases feed consumption and its bioavailability (Ranjan et al. 2017).Since orfenicol and its residues are heat-labile (Filazi et al. 2015), top-coating is the most suitable way to medicate aquacultured sh (Ranjan et al. 2017).The present study, therefore, assessed the biosafety of top-coated FFC feeds in monosex Nile tilapia O. niloticus when fed at 0-10 times the lowest therapeutic dose of 10 mg/kg sh biomass/day for 30 consecutive days, i.e., 3 times the proposed 10-day dose duration.

Experimental sh and design
A single lot of healthy juvenile Nile tilapia Oreochromis niloticus (13.64 ± 0.52 g; 9.40 ± 0.37 cm) were procured from a grow-out farm located in Sonarpur (Lat 22°27′50.2158″N; Long 88°23′7.4004″E), West Bengal, India and transported to the laboratory in oxygen-lled bags.The sh were stocked in circular 500-L breglass reinforced plastic tanks at 50 numbers/tank and acclimated for 15 days.They were fed thrice daily at 2% of the bodyweight (BW) with a commercial pellet feed of 2.0 mm dia (CP Private Limited, India).The sh without any gross abnormalities from the acclimatized population were then collected and randomly allocated among 15 rectangular study tanks (L58 cm × H45 cm × W45 cm; Volume: 80 L) with 20 sh each before dosing (pre-dosing period: 7 days).The allotment was done in groups of 10, weighed and then transferred to the study tanks.This procedure was repeated twice until 20 sh were placed in each tank.The experimental sh were allotted into 5 groups, viz., group 1: 0X control, group 2: 1X FFC-diet (10 mg/kg sh biomass/day), group 3: 3X FFC-diet (30 mg), group 4: 5X FFC-diet (50 mg) and group 5: 10X FFC-diet (100 mg) in triplicate.About 50% of the water was replaced thrice weekly to avoid the accumulation of waste and excretory produces.The water quality parameters, viz., water temperature: 25.17-29.17°C;pH: 7.30-7.97;dissolved oxygen: 5.24-6.00mg/L; nitrite: 0.23-0.65 mg/L and nitrate: 0.25-0.61mg/L maintained optimally during the experimentation period.Water chemistry (temperature and pH) was measured daily concurrent with feeding.Dissolved oxygen, nitrite and nitrate were measured twice weekly as per APHA/AWWA/WEF (2017).

Florfenicol-diets preparation
The medicated FFC-diets were freshly prepared one week before the dosing period.The inclusion rate for the orfenicol (Tokyo Chemical Industry, CAS RN: 7321-34-2; Product Number: F0811-5g) was calculated to deliver an approximate dosage of 0-100 mg active ingredient/kg sh biomass/day for 30 consecutive days.The required amount of FFC was mixed with vegetable oil (5 mL/kg basal feed) and this emulsion was then used for top-coating as per de Oliveira et al. (2018).The top coated FFC-diets were prepared by vigorously mixing the required amounts of antimicrobial emulsion in feed to avoid antimicrobial loss by hydrosolubilization during feeding.Medicated diets were prepared in order of increasing FFC concentration.The feeds were air-dried overnight, stored in plastic sealable containers and kept in a cool place away from light.In the control feed (non-medicated feed) only 5 mL vegetable oil was added and vigorously mixed with the feed.Feeding rates and methods were identical for the control and dosing groups during the trial.The experimental protocols were approved by the Indian Council of Agricultural Research, Government of India, New Delhi under the All India Network Project on Fish Health and ful lled the ethical guidelines including adherence to the legal requirements of India (CPCSEA, 2021).

Dose administration
The 50-day study included 7-days acclimation (pre-dosing), 30-days FFC-dosing, and 13-days post-FFCdosing periods.During the pre-dosing and post-dosing periods, the sh groups were fed with a control diet.During the dosing period, FFC-diets were administered to the respective groups.The control group was administered the control diet.The feed ration, 2.0% of the BW, was allocated into three equivalent portions and the feed consumption was determined daily.Feeds remaining in the tank 1 h after each feeding was siphoned from the tank into a pre-weighed container, dried overnight, pooled tank-wise on daily basis and weighed.The sh biomass from each tank was determined periodically, i.e., day 10, 20, 30 FFC-dosing and day 13 post-FFC-dosing) in groups of 10, indiscriminately netted out, and calculated the difference in weight gain.The observations on sh behavioural changes, external changes, feeding behaviour and mortality were recorded daily.The feed ration was adjusted with the biomass accrual and mortalities.Behavioural responses and external observations were noted daily throughout the experimental regimen.Behaviours like swimming to the surface during feeding, aggressive feeding behaviour and distribution throughout the water column were considered normal.External bodily changes like pigmentation and gross lesions were also observed during feeding and before tank cleaning.Fish with abnormal external changes were taken out, anaesthetized, documented and returned to the respective tanks.Feeding activity was visually assessed thrice daily.

Blood sampling
The blood sampling was done on day 0, 10, 20 and 30 FFC-dosing, and day 13 post-FFC-dosing from each group.Before the blood collection, two sh from each tank of the respective groups were arbitrarily netted out and anaesthetized using clove oil (20 µL/L water).The blood was collected by caudal vein puncture (Roberts 2012) using a 2 mL sterile plastic syringe.Instantly, 2 drops of non-heparinized blood were taken on microscopic slides followed by blood smear preparation.The sh upon blood collection were released into the respective tanks.The non-heparinized blood in the syringe was allowed to clot by keeping the syringe in a slanting position and then incubated at 4°C overnight.The serum was collected by centrifugation at 1000×g for 15 min, transferred to Eppendorf tubes and stored at -20°C for further analysis.

Statistical analyses
The data were expressed as a mean ± standard deviation.One way ANOVA followed by Tukey HSD posthoc for the comparison of means was carried out to know the signi cance of differences in each of the parameters among the treatments and each of the speci c treatment among the days using Statistical Package for Social Sciences (IBM-SPSS) Version: 22.0, considering a probability level of P < 0.05.

Feeding behaviour
Belligerent feeding was observed throughout the experimental tenure in the control and 1X groups.The sh of 3X and 5X groups reduced their feeding aggressiveness with time.Increased rate of subdued behavioural responses including lounging at the tank bottom, no curiosity in feeding were noted in the 10X group.The data on feed intake during the oral FFC-dosing trial are presented in Table 2.A reduction of 1.29%, 4.25%, 13.07% and 22.88% feed intake during the 30 days of FFC-dosing was observed in the 1X, 3X, 5X and 10X groups, respectively compared to control.The decreased feed intake was also seen during the early post-FFC-dosing period.The feed intake in all the treatment groups during the FFC-dosing and post-FFC-dosing periods differed signi cantly (P < 0.05).Another noteworthy observation among the treatment groups was the increased faecal output in the 5X and 10X groups, which tend to excrete more during the dosing period.However, no colour changes in the excreta or intestine during necropsy were observed.

Mortalities
There were no mortalities during the pre-dosing period and post-dosing period.At the end of the 30-day FFC-dosing regimen, 3.33%, 6.67%, 10.00% and 18.33% mortalities were noted in the 1X, 3X, 5X and 10X groups, respectively (Fig. 1a).The differences in mortalities between 1X and 3X groups were insigni cant (P > 0.05).The mortalities recorded in the 10X group were high and differed signi cantly with 1X and 3X groups (P < 0.05).Signi cant differences existed in the mortalities among the day 10 and 30 FFC-dosing, and 13 post-FFC-dosing in the 10X group (P < 0.05).

Fold change in biomass
Fish of the control group showed a higher fold change in biomass than the treatment groups.The increase in biomass of the control group uctuated between 1.22 and 1.49 folds with days of culture.The 1X group showed an increment in biomass of 1.11-1.31folds of its original biomass.The 3X and 5X groups recorded a biomass hike of 1.129-1.289folds and 1.126-1.286folds, respectively.The 10X group recorded the least fold increase (1.109-1.190folds) in biomass (Fig. 1b).The differences in biomass from day 0 to the end were 65.13 ± 23.24 g, 43.53 ± 13.37 g, 39.27 ± 15.84 g, 38.93 ± 12.73 g and 26.15 ± 1.62 g in the control, 1X, 3X, 5X and 10X groups, respectively.A signi cant difference existed in the biomass of the control and 10X groups (P < 0.05).The fold change in biomass of the 10X group on day 10 and 20 FFC-dosing, and day 13 post-FFC-dosing differed signi cantly (P < 0.05).

Abnormalities
During the feeding trial, O. niloticus juveniles were distributed throughout the water column of the tanks.Abnormal behaviour was not observed during the pre-dosing period.No gasping, loss of equilibrium and abnormal behaviour were observed during the dosing and post-FFC-dosing periods.Most of the sh of 5X and 10X groups exhibited dark opercular pigmentation (Figs.2c and 2d) with hard intestine, enlarged spleen and lique ed kidney during the dosing regimen, which subsided during the post-FFC-dosing.Enlargement of the liver and gall bladder was also seen in some sh of 5X and 10X groups (Fig. 2e).Black peritoneum was observed in almost all the 5X and 10X dosed sh during sampling (Fig. 2f).

Serum glucose
The serum glucose levels of the control group ranged between 74.00 ± 5.01 and 79.33 ± 6.11 mg/dL.All the treatment groups showed an increment in glucose levels till day 30 of FFC-dosing and subsequently, the levels reduced (Fig. 3a).The glucose levels of the 1X group differed signi cantly between day 10 and day 30 FFC-dosing (P < 0.05).The glucose levels of the 1X group increased signi cantly and peaked on day 30 FFC-dosing and started reducing with the termination of dosing.A similar trend in the glucose levels was noted in other treatment groups.The glucose levels of all the treatment groups differed signi cantly (P < 0.05) on day 20 and day 30 FFC-dosing.Insigni cant differences (P > 0.05) in glucose levels were observed between 1X and 3X as well as 5X and 10X groups on day 10 FFC-dosing.On day 13 post-FFC-dosing, the glucose levels of the 5X and 10X differed insigni cantly (P > 0.05).

Serum calcium
The control group had a uctuation in serum calcium levels ranging from 15.73 ± 0.12 to 15.93 ± 0.15 mg/dL.No signi cant differences (P > 0.05) in calcium levels were observed among the FFC-dosed groups during the dosing regimen.In the 1X group, the calcium levels reduced signi cantly in 30 days of FFC-dosing (P < 0.05).The sh of the 3X-10X groups also followed a similar trend.The cessation of dosing led to an increase in calcium levels in all the groups.On day 13 post-FFC-dosing, the calcium levels reached almost normal in all treatment groups (Fig. 3b).

Serum chloride
The serum chloride levels of the control were in the range of 123.00 ± 3.46-124.33± 2.31 mmol/L.The chloride levels of the 1X-5X groups reduced but insigni cantly on day 30 FFC-dosing (P > 0.05).The chloride levels of the 10X group reduced signi cantly (P < 0.05) on day 10 FFC-dosing.The chloride levels of the 10X group were signi cantly (P < 0.05) lower than the control, 1X and 3X groups on day 10 FFCdosing (Fig. 3c).Also, its levels on day 13 post-FFC-dosing differed signi cantly (P < 0.05) with day 20 and 30 FFC-dosing in the 10X group.

Serum creatinine
The serum creatinine levels of the control group uctuated between 0.08 ± 0.02 and 0.09 ± 0.01 mg/dL.The creatinine levels of the 1X and 3X groups, though increased on day 10, differed insigni cantly (P > 0.05).Its levels reached a peak on day 30 FFC-dosing in all the treatment groups and started reducing with the suspension of dosing.On day 30 FFC-dosing, creatinine levels of the 10X group were signi cantly (P < 0.05) higher than the control and 1X groups.On day 13 post-FFC-dosing, no signi cant differences in creatinine levels were observed among 1X, 3X and 5X groups.However, its levels in the 10X group were signi cantly (P < 0.05) higher than in other treatment groups (Fig. 4a).

Serum alanine aminotransferase (ALT)
The serum ALT levels of the control group uctuated between 37.00 ± 1.73 and 38.67 ± 3.51 IU/L.On day 30 FFC-dosing, the ALT levels of the 1X group were signi cantly higher than the control or day 0 (p < 0.05).There was a signi cant hike in the ALT levels of the 3X group with days of FFC-dosing.The ALT levels of the 5X and 10X groups also increased signi cantly with a peak on day 30 FFC-dosing (P < 0.05).Insigni cant differences were observed in ALT levels (P > 0.05) of control, 1X, 3X and 5X groups between day 0 and day 13 post-FFC-dosing (Fig. 4b).

Serum aspartate aminotransferase (AST)
The serum AST levels of the control group ranged from 77.33 ± 0.58 to 80.33 ± 0.58 IU/L.In the 1X group, signi cantly higher AST levels (P < 0.05) were noted on day 30 FFC-dosing, which reduced thereafter.Likewise, a signi cant increase in the AST levels (P < 0.05) was observed in the 3X-10X groups till day 30 FFC-dosing, after which their levels reduced.The AST levels of the 3X group were signi cantly higher (P < 0.05) than the 1X group on 10, 20 and 30 days of FFC-dosing.Insigni cant differences (P > 0.05) were observed among the AST levels of 3X-10X groups both on day 20 and day 30 FFC-dosing.The AST levels of the 1X-5X groups returned to near normal on day 13 post-FFC-dosing, except for the 10X group, which had signi cantly higher (P < 0.05) AST levels (Fig. 4c).

Serum alkaline phosphatase (ALP)
The serum ALP levels of the control group uctuated between 12.33 ± 0.58 and 13 ± 2.65 IU/L.The ALP levels of the 1X group were signi cantly higher than the control on all days of observation (P < 0.05).A signi cant rise in ALP levels (P < 0.05) was seen in the 3X group on day 30 FFC-dosing followed by a signi cant decrease (P < 0.05) on day 13 post-FFC-dosing.A similar trend was seen in the 5X and 10X groups, reaching the peak levels on day 30 FFC-dosing and on day 13 post-FFC-dosing the levels subsided.No signi cant differences (P > 0.05) were observed among the ALP levels of the 1X-10X groups on day 10, 20 FFC-dosing and 13 post-FFC-dosing (Fig. 4d).

Morphological changes in sh blood cells
Erythrocytes of the FFC-dosed O. niloticus showed variations in size and shape and the descriptions of the morphological changes are depicted in Figs.5a,b.An increased incidence of immature erythrocytes at the therapeutic dose (1X) was observed.No signi cant morphological alterations were observed in the 1X group compared to the control.The blood smear examination of the 3X, 5X and 10X groups, however, showed erythrocytes with aberrant morphological changes at all sampling days.Erythrocytes with an increased nucleus to cytosol ratio were seen in all the treatment groups.At the higher dosing groups, the erythrocytes with rupturing cell membranes were prominent.The main deviant changes in erythrocytes were tear-shaped, spindle-shaped and degenerative erythrocytes.The tear-shaped and damaged erythrocytes increased on day 20 and day 30 FFC-dosing in the 3X, 5X and 10X groups and returned to almost normal on day 13 post-FFC-dosing.Immature erythrocytes and smudge cells were also observed in all the treatment groups.Blood smears of the 3X group showed changes in blood cells, which included peripheral nuclear erythrocytes and visibility of the micronucleus.The 5X and 10X groups showed high erythrocytic damages and an increased number of mature lymphocytes.The shapes of the erythrocytes were irregular with the nucleus at the extreme periphery.Some erythrocytes showed lysis with the nucleus bursting out.Blood smears of the 10X group also showed nuclear changes like irregular and bilobed nucleus.Although the blood cell morphological changes were higher among the treatment groups during the feeding regimen, the day 13 post-FFC-dosing blood samples showed almost normal erythrocytic and leucocytic morphology.Equally, the increased number of lymphocytes was still seen among all the treatment groups.

Discussion
Florfenicol is a palatable broad-spectrum antibiotic and has been conditionally approved by the USFDA for use in aquaculture (USFWS 2015).Florfenicol therapeutics differs from country to country but includes control of mortality due to diseases associated with the warm water bacterial pathogens like Edwardsiella ictaluri, Streptococcus iniae, S. agalactiae, Flavobacterium columnare, Francisella asiatica and Aeromonas hydrophila (Gaunt et al. 2004 2018) demonstrated the use of vegetable oil as a successful coating agent in the top-coating of FFC medicated feed.Since drug metabolism is considered temperature dependent, this study was conducted at the ideal growth temperature of 28.6 ± 1.68ºC.Our experimental results demonstrated the margin of safety of oral FFC administration to O. niloticus juveniles at 0-10 times the lowest therapeutic dose (10 mg/kg biomass/day) for 30 days.Our results are consistent with those found in similar studies that evaluated the safety of FFC administered in feed to other freshwater n sh (Straus et al. 2012;Gaikowski et al. 2013;Bowker et al. 2013).The feed consumption and growth of FFC-dosed O. niloticus were signi cantly reduced in a dose-related fashion, particularly in the 3X-10X groups.The sh consumed approximately most of the feed offered during the dosing period (0X: 100%, 1X: 98.71% and 3X: 95.75%), often breaking the surface of the water while feeding.The sh tend to feed by gulping the feed on the surface followed by releasing the feed inside the water column and subsequently re-gulping it.Previous works (Bowker et al. 2013;Gaikowski et al. 2013) also elucidated similar feeding behaviour among the experimental sh.Further, the extended feeding of FFC-diets beyond 10 days signi cantly decreased the feed consumption in the higher dosed groups.On the contrary, FFC therapy in S. salar (Inglis et al. 1991), Perca avescens (Bowker et al. 2013) and hybrid striped bass, female Morone chrysops x male M. saxatilis (Straus et al. 2012) did not alter feed consumption.No gross or microscopic lesions were observed during the experimental tenure.The observed signi cant differences in feed intake among the treatment groups during the dosing and postdosing regimen possibly related to the palatability and dose-dependent toxicity of FFC.The clinical implication of the declined feed consumption is likely of minimal importance as decreased feed consumption was only comprehended at higher levels and only after administration for longer than the proposed 10-day period.Nevertheless, the sh were able to mount biological responses during the postdosing period and the feed intake recovered in a dose-dependent manner.The FFC-dosing at the 1X dose did not cause loss of equilibrium, gasping, ashing and hyperactivity.Contrarily, the 5X and 10X dosed O. niloticus had opercular pigmentation and a black peritoneal layer.Likewise, Gaikowski et al. (2013) reported body discolouration in FFC fed O. niloticus.
The mortalities during the rst 10 days of dosing, i.e., therapeutic dosing period, were observed only in the 10X group possibly due to FFC-intoxication.The cumulative mortalities of 3.33% in 1X to 18.33% in 10X groups on day 30 of dosing corroborate the works of Gaikowski et al. (2013), who asserted the chances of FFC-intoxication due to prolonged FFC feeding.Hentschel et al. (2005) opined that any drug at a higher concentration than its permissible limit is toxic to the host organism thereby rendering several intoxication symptoms in sh.Our results on the elevated mortality with the increase in FFC-dose and dosing period supported the earlier observations (Hentschel et  period.The necropsy observation on the alterations in the internal organs such as swelled kidney and spleen in the 5X and 10X groups probably indicated the concern of FFC toxicity upon oral dosing.The therapeutic dose group (1X) revealed a slight decrease in biomass, i.e., 1.24 folds hike, on day 30 FFCdosing compared to the control (1.45 folds).An insigni cant dose-dependent decrease in fold change in biomass of the 1X, 3X and 5X groups compared to control was observed, which coincided with the decreased feed intake of the respective treatment groups.The biomass increased for all the treatment groups with the termination of FFC-dosing, thereby indicating the recovery of sh.
The mean serum glucose level of the O. niloticus (74.00 ± 5.00 mg/dL) was concomitant with the values recorded by Bittencourt et al. (2003).The 30 days of FFC-dosing increased the glucose levels signi cantly in all the treatment groups, indicating the FFC induced stress and altered carbohydrate metabolism (Sopinka et al. 2016;Julinta et al. 2019).Even at the lowest therapeutic dose, the 30 days of FFC-dosing raised the glucose levels signi cantly.On day 10 FFC-dosing, the glucose levels of the 1X groups were signi cantly high, which signi ed that the therapeutic FFC dose and dosing period (10 days) may be stressful to the normal O. niloticus.The degree of glucose increment was comparatively higher in 5X and 10X groups.Further, the elevated glucose levels in FFC doses higher than the therapeutic dose and the extended dosing period gave conclusive indications on FFC as a stress inducer.Though the glucose levels abridged signi cantly on day 13 post-FFC-dosing, the levels were still signi cantly higher than the initial levels recorded on day 0.These results suggested that the FFC-induced stress and the physiological changes persevered.Also, the sh could not recover fully even after 13 days of termination of FFC-dosing, which would in uence the growth and farm production of O. niloticus.Possibly, more time would be required for the sh to revert to their initial conditions, which is a cause for concern for the aquaculturists.
At the therapeutic dose, the serum calcium levels reduced signi cantly on day 30 FFC-dosing, indicating an imbalance in osmolarity and ionic concentration.Nevertheless, within 2 weeks of cessation of FFCdosing, the calcium levels recovered to normal.Even, the sh offered the higher doses followed a similar trend in a dose-dependent manner.Likewise, the chloride levels reduced but insigni cantly on day 30 FFCdosing in the 1X group and recovered completely on day 13 post-FFC-dosing.The higher dosed groups also followed a similar trend except for the 10X group, which failed to recover within the 2 weeks of termination of dosing.These results suggested that the effects of FFC were more on calcium than on chloride ions.Our results corroborate the observations of decreased serum calcium levels in pigs (Liu et al. 2003) and chicks (Klaudia and Alina 2015) when injected with FFC.Although aquaculture reports are not available on the relationship between serum calcium and FFC, antibiotics like tetracyclines tend to bind to calcium reducing its availability in serum (Guidi et al. 2018).The works of Zhang et al. (2016) on FFC established a relationship between FFC and free available chlorine (FAC), which according to them readily combines with FFC and transforms it for easy removal.The transformation kinetics of FAC, thus, hinted at a similar mechanism in sh blood.Noticeably, the increased output of faecal matter may have a close linkage with decreased serum calcium and chloride levels in O. niloticus.It has been documented that the increase in faecal output elevated the output of faecal carbonates and bicarbonates, leading to a disruption in osmotic balance and acid-base homeostasis (Wilson and Grosell 2003).
The recorded serum creatinine levels (0.08 ± 0.02-0.09± 0.01 mg/dL) in the control group were concomitant with the results of earlier studies (Julinta et al. 2019).The creatinine levels increased signi cantly in all the treatment groups with the maximum in the 5X and 10X groups.Subtle uctuations in creatinine levels (0.20 ± 0.01-0.22± 0.03 mg/dL) were observed in the 1X group between day 10 and day 30 FFC-dosing.The increase in serum creatinine levels implied kidney damage and a reduction or loss of renal function (Julinta et al. 2019).Contrarily, in an earlier study by Reda et al. (2013), the FFC at the growth-promoting dose (5 mg/kg sh) signi cantly reduced the serum creatinine levels in O. niloticus.The creatinine levels in the 10X group increased by almost 4.41 folds on day 10 and 5.75 folds on day 30 FFC-dosing signifying the nephrotoxicity of FFC.Although the creatinine levels reduced on day 13 post-FFC-dosing in all the treatment groups, the levels were still signi cantly higher than on day 0.These observations suggested only a slight improvement in the renal functions of O. niloticus in 2 weeks of suspension of FFC-dosing.Perhaps, the sh would require more time to recoup.
The alterations in serum ALT and AST levels are indicative of liver tissue impairment or damage caused by drugs or stress (Julinta et al. 2019;Bojarski et al. 2020).The current study documented serum ALT levels in the range of 37.00 ± 1.73 to 38.67 ± 3.51 IU/L in the control group, which are analogous to previous studies (Julinta et al. 2019;Dawood et al. 2020).The signi cant increase in ALT levels in the 1X group on day 20 and day 30 FFC-dosing indicated the FFC induced liver tissue impairment or damage upon extended FFC-dosing.Nevertheless, the hike observed on day 10 FFC-dosing at the therapeutic dose (1X) was insigni cant compared to control, suggesting minimal liver damage, similar to the observations of Reda et al. (2013).Contrarily, the dose-dependent elevated ALT levels as observed in the 3X-10X groups on day 10, 20 and 30 FFC-dosing hinted at the hepatotoxicity of FFC with increased dose and dosing period.The 10X group demonstrated about 5 folds increase in ALT levels on day 30 FFC-dosing.Yet, the ALT levels, more or less, recouped on day 13 post-FFC-dosing, except for the 10X group, which had signi cantly higher levels than on day 0. Notably, the pronounced impact of FFC on liver enlargement was noted during necropsy.The serum AST levels of control (77 ± 4.58-80.33± 0.58 IU/L) were concomitant with the studies of Hastuti and Subandiyono (2020).A signi cant increase in AST levels was observed in all the treatment groups with the highest in the 10X group on day 30 FFC-dosing, thus con rming the hepatotoxicity of FFC.The observed signi cant hike in AST levels in the 1X group during the FFC-dosing period also signi ed that the FFC even at the therapeutic dose may impair the liver tissues or cause metabolic damage.In contrast, Reda et al. (2013) observed a signi cant decline in serum AST levels when fed FFC at a lower dose (5 mg/kg sh) in O. niloticus.Although the higher doses (3X-10X) showed a signi cant rise in serum AST levels, there existed insigni cant differences among them, thus suggesting persevering hepatotoxicity of FFC at elevated doses and portentous hepatic dysfunction in O. niloticus.It has been reported that amphenicols (Memik 1975) and oxytetracycline (Julinta et al. 2019) are hepatotoxic so also our study with FFC.Also, the FFC can cause an increase in the weight of the liver (Elia et al. 2016).Our results con rm the ndings of Er and Dik (2014), who observed a hike in AST levels upon FFC application in Oncorhynchus mykiss.Except for the 10X group, all the treatment groups recuperated within 2 weeks of suspension of FFC-dosing.The serum ALP levels of control (12.00 ± 1.73-13.00± 2.65 IU/L) were similar to the observations of Hrubec and Smith (2000).The signi cant increase in ALP levels at the therapeutic dose (1X) on day 10 FFC-dosing hinted at the possibility of FFC induced liver in ammation and hepatotoxicity (Labarrère et al. 2013;Soltanian et al. 2018).The ALP levels increased in a dose-dependent fashion with the highest in the 10X group on day 30 FFC-dosing.These results are in agreement with the observations recorded in sh during the misuse of FFC (Shiry et al. 2020) and goat (Shah et al. 2016) upon FFC injection.Although the ALP levels reduced with the cessation of FFC-dosing in all the treatment groups, their levels were still signi cantly higher than on day 0.These observations suggested persisting liver in ammation in FFC-dosed sh.
The oral FFC-dosing not only affected the serum biomarkers of healthy O. niloticus but also induced relative cytotoxicity.Our study used giemsa and safranin stains for assessing the blood cellular morphological changes upon FFC-dosing.The results showed that the safranin staining method is a good alternative to the giemsa method and has the advantage that abnormalities in other blood elements, in particular WBCs and thrombocytes, are better identi ed.Giemsa staining did not provide for comprehensible granulations in leucocytes.The safranin staining granted e cient visualization of smudge cells and granulations in leucocytes.However, considering the diagnosis of erythrocytes in a blood smear, the giemsa stain proved to be much superior to safranin.In a comparative study of Leishman and giemsa staining, Sathpathi et al. (2014) suggested the superiority of giemsa staining for a thick blood smear.In the present study, the giemsa staining produced distinct and evident erythrocytic morphology with subsequent alterations and abnormalities.Deformities like rupturing of nuclear membrane and vacuolation were prominent with giemsa stain.However, this did not translate into reduced sensitivity or reduced accuracy of safranin in light microscopy.This comparison, alongside cellular morphological alterations, also hinted at the effectiveness of time.Staining with safranin is shorter, which in many scenarios can be helpful.
The blood cell morphological changes were mostly restricted to the erythrocytes in our study.The predominant increase in mature and immature lymphocytes in all the treatment groups suggested a toxic or stress-related effect of FFC on the lymphoid cells and cell proliferation (Gaokowski et al. 2013).
Although there was no direct evidence of hematopoietic or lymphopoietic tissue degradation, the sudden increase in lymphocytes hinted at the stress the sh endured.Likewise, Umamaheswari et al. (2019) in their studies with amoxicillin on Labeo rohita demonstrated a signi cant increase in lymphocytes.The haematopoietic tissues are normally sensitive to antibiotics.This could be the reason for the increased incidence of WBCs (phagocytic response).The works of Passantino et al. (2004) indicated the morphology of mature sh erythrocytes (more elongated) and immature erythrocytes (less elongated).Our study noted an increased incidence of immature erythrocytes in the therapeutic group throughout the dosing period.Chico et al. (2018) in their works on O. mykiss RBCs coined the term "shape-shifted RBCs (shRBCs)" for normal RBCs, which when exposed to certain stimuli produce apparent morphological and molecular alterations.These shape-shifting RBCs are also often observed in sh under the stressed conditions (Lewis et al. 2010;Chico et al. 2018).Such alterations in RBCs, viz., teardrop-shaped and spindle-shaped, were frequently observed in our study.The morphological changes in sh erythrocytes further con rmed the cytotoxic effect of FFC, which may result in chromosomal disparities (Ghaffar et al. 2015).The increased incidence of erythrocytes with eccentric nucleus was observed possibly due to the higher production of caspase-activated DNAase or oxidative stress to the mitochondrion causing disruption and breakage in the cytoskeleton (Ghaffar et al. 2018).Changes like extruding nucleus, reduced cytosol density (lighter staining) and vacuolations were also observed under light microscopy.Such changes are comparable to those of Chico et al. (2018).Similar to the ndings of this study with FFC, antibiotics like amikacin have been known to reduce cell size, increase deformability and osmotic fragility in erythrocytes (Lijana and Williams 1986).The increased incidence of ruptured cells at the higher doses in our study supported the results of Blaskó et al. (1986), possibly due to the attachment of antibiotics on the erythrocytic membrane and hindrance in cation transport.Smudge cells are associated with high lymphocyte counts and hence, the observations of increased incidence of smudge cells and lymphocytes are related.Cytotoxicity was prominent in the 5X and 10X groups with distinguishably irregular RBCs, ruptured cells and eccentric nuclei.Cytotoxicity of FFC at the higher concentrations has been well demonstrated in goats and reptiles (Saganuwan 2019).Nevertheless, the therapeutic dose did not show any signs of cell rupture hinting at the safety of FFC at the test dose.Upon cessation of FFCdosing, the blood smear produced healthier cellular elements.Increased prominence of mature erythrocytes was also seen.The erythrocytic abnormalities like eccentric nuclei were not seen on day 13 post-FFC-dosing in O. niloticus of the higher dosed groups.The elimination of stress also terminated the formation of shRBCs.Although such erythrocytic aberrations decreased, the nucleus to cytoplasm ratio was still high among erythrocytes of all the treatment groups.Also, the increased prominence of lymphocytes persevered.The number of mature lymphocytes was still high on day 13 post-FFC-dosing in all the groups.With the observance of such blood cell morphological changes particularly in sh erythrocytes, the impact of FFC on blood cells need further studies to elucidate the mechanisms.It is believed that due to such signi cant deformities in blood cell morphology, the sh erythrocytes could be targeted as an e cient blood biomarker for future studies on the safety of approved aquacultural antibiotics.

Conclusion
The dietary FFC in uenced the physiological state of O. niloticus in a dose-and time-dependent manner.This study presented a signi cant decrease in feed consumption and biomass at elevated doses for an extended duration (beyond 10 days).Mortalities on day 10 FFC-dosing were seen only in the 10X group.
The extended 20 days of medication produced 3.33% mortalities in the 1X group.The anomalies in serum biomarker levels and blood cell morphology are more likely due to the FFC usage even at the therapeutic dose.Nevertheless, the results of the present study indicated that the FFC administered feed when consumed to deliver the lowest dose of 10 mg/kg biomass/day for the therapeutic duration would be well tolerated by O. niloticus.Florfenicol ration: X=10 mg/kg biomass/day; a-d: Values sharing common alphabetical superscripts within a row for a particular dosing period differed insigni cantly (P>0.05);1-3: Values sharing common numeral superscripts within a column for a particular treatment (dose) differed insigni cantly (P>0.05).

Figures Figure 1 Figure 2
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Table 2
Ethics Approval The current study was performed in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.The experimental protocols were approved by the ICAR, Government of India, New Delhi under the All-India Network Project on Fish Health (F.No.CIBA/AINP-FH/2015-16 dated 16.7.2015)Feedintake in orfenicol (FFC)-dosed Oreochromis niloticus juveniles at 0-10 times the therapeutic dose of 10 mg/kg biomass/day for 30 consecutive days