Antioxidative and immunostimulatory effect of dietary cinnamon nanoparticles on the performance of Nile tilapia, Oreochromis niloticus (L.) and its susceptibility to hypoxia stress and Aeromonas hydrophila infection

Accepted Manuscript Antioxidative and immunostimulatory effect of dietary cinnamon nanoparticles on the performance of Nile tilapia, Oreochromis niloticus (L.) and its susceptibility to hypoxia stress and Aeromonas hydrophila infection Mohsen Abdel-Tawwab, Fatma Samir, Asmaa S. Abd El-Naby, Mohamed N. Monier PII:

S1050-4648(17)30774-X

DOI:

10.1016/j.fsi.2017.12.033

Reference:

YFSIM 5018

To appear in:

Fish and Shellfish Immunology

Received Date: 24 August 2017 Revised Date:

26 October 2017

Accepted Date: 20 December 2017

Please cite this article as: Abdel-Tawwab M, Samir F, Abd El-Naby AS, Monier MN, Antioxidative and immunostimulatory effect of dietary cinnamon nanoparticles on the performance of Nile tilapia, Oreochromis niloticus (L.) and its susceptibility to hypoxia stress and Aeromonas hydrophila infection, Fish and Shellfish Immunology (2018), doi: 10.1016/j.fsi.2017.12.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Antioxidative and immunostimulatory effect of dietary cinnamon nanoparticles on the performance of Nile tilapia, Oreochromis niloticus (L.) and its susceptibility to hypoxia

RI PT

stress and Aeromonas hydrophila infection

Mohsen Abdel-Tawwab1*, Fatma Samir2,

1

SC

Asmaa S. Abd El-Naby2, and Mohamed N. Monier1

Department of Fish Biology and Ecology and 2 Department of Fish Nutrition and Feed

M AN U

Processing, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia 44662, Egypt

Running title: Antioxidative and immunostimulatory effect of dietary cinnamon

EP

*Corresponding author:

TE D

nanoparticles on Nile tilapia

AC C

Mohsen Abdel-Tawwab

Department of Fish Biology and Ecology, Central Laboratory for Aquaculture Research, Abbassa, Abo-Hammad, Sharqia, Egypt. Emails: [email protected], [email protected]

ACCEPTED MANUSCRIPT

Abstract An experiment was conducted to evaluate the effect of dietary cinnamon nanoparticles (CNP) on growth performance, antioxidant and digestive enzymes activities, and innate

RI PT

immunity of Nile tilapia, Oreochromis niloticus (L.). Fish (9.7 ± 0.3 g) were fed on diets enriched with 0.0, 0.25, 0.5, 1.0, 3.0, 5.0, and 10.0 g CNP/kg diet for 8 weeks. After the feeding trial, fish were challenged against hypoxia stress or pathogenic bacteria (Aeromonas hydrophila) infection.

SC

Fish performance was significantly improved with increasing CNP levels over the control diet. Furthermore, only crude protein contents in whole-fish body were significantly higher in CNP-

M AN U

fed fish than those fed the control diet. Antioxidant-stimulated activity was observed with dietary CNP where malondialdehyde (MDA) level and activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly, whereas glutathione peroxidase (GPx) activity decreased significantly in CNP-fed fish. Likewise, CNP supplementation induced the secretion of protease,

TE D

lipase, and amylase, which were maximized at 3.0 – 10.0 g CNP/kg diet. All innate immunity variables i.e. nitrous oxide (NO), nitro blue tetrazolium (NBT), and lysozyme activity were significantly higher in CNP-fed fish than the control one. No fish mortality was observed during

EP

hypoxia stress among all treatments, but CNP administration protectd fish against A. hydrophila infection. No mortality was observed in fish fed 3.0 - 10.0 g CNP/kg diet after bacterial

AC C

challenge; meanwhile the mortality of fish fed the control diet was 66.7%. This study evoked that dietary CNP enhanced performance, antioxidant and digestive enzymes activity, and innate immunity of Nile tilapia and its optimum level is 3.0 g CNP/kg diet.

Keywords: Nile tilapia, Cinnamon nanoparticle, Growth performance, Biochemical variables, Antioxidant activity, Digestive enzymes, Innate immunity, Aeromonas hydrophila.

ACCEPTED MANUSCRIPT

1. Introduction Nile tilapia, Oreochromis niloticus (L.), is a popular fish species among fish farmers all over the world due to its good growth and high-marketing value [1]. Its intensive culture is

RI PT

regarded as the most appropriate approaches in modern aquaculture during which fish may be stressed by deteriorated water quality and hypoxia. This, in turn, would suppress immune system and increase the risk of aeromonoid disease caused by the Gram-negative bacteria Aeromonas

SC

hydrophila. This bacterium is an opportunistic pathogen, which may cause serious economic loss in marine and freshwater aquaculture [2][3]. The Aeromonas septicemia would be induced when

M AN U

fish are under stressful condition by high water temperature, bad water quality, hypoxia, parasitic infections, high stocking density, rough handling, and transportation [4][5]. Conventional disease avoidance and treatment strategies, such as vaccines and drugs, have major obstacles and restrictions [6][7][8][9]. Additionally, the usage of antibiotics to prevent and control bacterial

TE D

diseases in aquaculture may lead to antibiotic-resistant bacterial strains [10][11]. Therefore, there is a great interest in developing feed additives as cost-effective alternatives to traditional means of combating diseases in order to sustain environmentally friendly aquaculture.

EP

It is imperative to enhance the feed quality via using feed additives to support fish growth, health, immunity, and productivity [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]

AC C

[25] [26] [27] [28]. There are many attempts to use medicinal herbs as feed supplements, which have developing therapeutic effects with low side effects [29][30][31]. Among these herbs is cinnamon, Cinnamomum zeylanicum, which has been known as one of the most common spices for human nutrition since ancient times because it has potent antiemetic, anti-diarrheal, antiflatulent, and stimulant activities [32][33].

ACCEPTED MANUSCRIPT

Cinnamon possesses broad spectrum of biological activities because it contains large amounts of bioactive molecules, including essential oils (cinnamic aldehyde and cinnamyl aldehyde), polyphenol, tannins, saponins, flavonoids and carbohydrates [34][35]. Most of these

RI PT

compounds act as reactive oxygen and nitrogen species scavengers, redox-active transition metal chelators and enzyme modulators [36][37]. The bioactive properties of cinnamon have been studied in a wide range of biological functions including anti-inflammatory [38], antioxidant

SC

[39], and antimicrobial [40][39]. Consequently, the use of cinnamon or any of its derivatives as a natural feed supplement is becoming useful for fish where it enhanced fish growth, feed

M AN U

utilization, or resistance to pathogenic bacteria (A. hydrophila) infection [41][42][43]. Since materials at the nanometer dimension exhibit novel properties [44] and the nanomaterials remain in the blood stream for long period facilitating its good bioavailability [45], it is hypothesized that the use of cinnamon nanoparticles (CNP) may improve feed

TE D

characteristics, and in turn, may improve fish performance, health, and immunity. Therefore, this study was conducted to investigate the effect of dietary CNP on growth performance, antioxidant and digestive enzymes activity, as well as innate immunity of Nile tilapia. Fish challenge against

EP

hypoxia stress and pathogenic bacteria A. hydrophila infection was also evaluated. 2. Materials and Methods

AC C

2.1. Diet preparation and fish culture Cinnamon, C. zeylanicum, was obtained from a local market and its nanoparticles was

prepared by mechanical milling according to Bello et al. [46] and Mashkouri et al. [47] using a planetary ball mill (RETSCH PM 400, Haan, Germany) at 400 rpm for 8 hours. The central occurrence in mechanical milling is the ball–powder–ball collision, where the dried powder was trapped between the colliding balls during milling with high speed producing fine powder in

ACCEPTED MANUSCRIPT

nanoscales. Particles size was measured using a Zetasizer Nano-ZS-90 (Malvern Instruments, Malvern, UK) where 0.5 g from the prepared CNPs from the ball mill was dispersed in 25 ml distilled water for measuring the particles size distribution, and the maximum average particle

RI PT

size was about 300 nm.

Seven diets containing 0.0, 0.25, 0.5, 0.1, 3.0, 5.0, and 10.0 g CNP/kg diet were

formulated to contain 30% crude protein (Table 1). However, CNP of each diet was suspended in

SC

100 mL per kg diet and blended with the other ingredients for 30 min to make a paste of each diet. The pastes were separately passed through a grinder and pelleted through 1-mm diameter

M AN U

paste extruder. The diets were oven-dried at 55 oC for 24 h and stored in plastic bags at – 2 oC for further use.

Nile tilapia, O. niloticus (L.), fingerlings were obtained from the nursery ponds, Central Laboratory for Aquaculture Research (CLAR), Abbassa, Abo-Hammad, Sharqia, Egypt. Fish

TE D

were kept in an indoor aerated fiberglass tank for two weeks for adaptation to the laboratory condition and light-dark regime was maintained at 12 - 12 using fluorescent tubes as a light source. Fish (9.7 ± 0.3 g) were randomly distributed at a rate of 20 fish per 100-L aquarium in

EP

quadruplicates. Each aquarium was supplied with compressed air via air-stones using aquarium's air pump. Fish were fed diets up to satiation twice daily at 9:00 and 14:00 h for 8 weeks. Settled

AC C

fish waste along with three-quarters of an aquarium’s water was siphoned daily, which was replaced by clean and well-aerated water from a storage tank. Fish mortality was recorded daily and dead fish were removed. 2.2. Water quality parameters Water samples were collected weekly from each aquarium to monitor different water quality parameters. Water temperature and dissolved oxygen were measured in site using a portable oxygen meter (Jenway, London, UK). The pH value was measured using a pH-meter

ACCEPTED MANUSCRIPT

(Digital Mini-pH Meter, model 55, Fisher Scientific, Denver, CO, USA). The unionized ammonia (NH3) was measured using a Multi-parameters Ion Analyzer (HANNA Instruments, Rhode Island, USA).

RI PT

Water quality parameters did not show any significant differences due to CNP

supplementation and the ranges of water temperature was 27.8 – 28.7 oC, dissolved oxygen was 5.2 – 5.9 mg/L, pH was 7.7 – 7.8, and unionized ammonia concentration was 0.24 – 0.42 mg/L.

2.3. Growth and feed utilization parameters

SC

All these ranges are within the acceptable ranges for fish farming [48].

M AN U

After the feeding trial, fish were collected, counted, and bulk-weighed. Growth performance was determined and feed utilization was calculated as follows: Weight gain = W2 – W1;

Specific growth rate (SGR) = 100 [Ln W2 (g) – Ln W1 (g)] / T; where W2 is final weight, W1 is

TE D

initial weight, and T is the experimental period (day);

Feed intake = the summation of the offered feed to fish throughout the experiment; Feed conversion ratio (FCR) = feed intake / weight gain.

EP

2.4. Proximate chemical analyses

The proximate chemical analyses of diets and whole-fish body were carried out according

AC C

to the standard methods [49] for moisture, crude protein, total lipids, and total ash. The moisture content was estimated by drying the samples at 85 oC in a heat-oven (GCA, model 18EM, Precision Scientific group, Chicago, Illinois, USA) for 48 h. Nitrogen contents were measured using a microkjeldahl apparatus (Labconco, Labconco Corporation, Kansas, Missouri, USA) and crude protein contents were estimated by multiplying nitrogen content by 6.25. Lipid contents were determined by ether extraction in multi-unit extraction Soxhlet apparatus (Lab-Line Instruments, Inc., Melrose Park, Illinois, USA) for 16 h. Total ash contents were determined by

ACCEPTED MANUSCRIPT

combusting dry samples in a muffle furnace (Thermolyne Corporation, Dubuque, Iowa, USA) at 550 oC for 6 h. 2.5. Antioxidant and digestive enzymes assay

RI PT

At the end of the feeding trial, fish were not fed during the 24 h immediately prior to blood sampling and blood was collected from the caudal vein via heparinized syringe. The collected blood was centrifuged at 5000 x g for 15 min at room temperature. The collected

SC

plasma was stored at –20 0C for further assays. The activities of antioxidant enzymes in fish plasma were measured using the diagnostic reagent kits according to the manufacturer’s

M AN U

instructions (MyBioSource Inc., San Diego, California, USA). Malondialdehyde (MDA) level was analyzed by thiobarbituric acid method [50]. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured spectrophotometrically according to methods described by McCord and Fridovich [51], Aebi [52], and Paglia and

TE D

Valentine [53], respectively.

Activities of digestive enzymes in fish plasma were measured using the diagnostic reagent kits according to the manufacturer’s instructions (Cusabio Biotech Co. Ltd., Wuhan, Hubei,

EP

China). Protease, lipase, and amylase activities were measured according to methods of Ross et al. [54], Shihabi and Bishop[55], and Bernfeld [56], respectively.

AC C

2.6. Innate immunity assay.

The production of superoxide ion by leukocytes was assayed by the reduction of Nitro

Blue Tetrazolium (NBT, Sigma-Aldrich Chemical, St. Louis, MO, USA) according to Rook et al. [57]. Lysozyme activity of fish plasma was determined by turbidometric assays as described by Caruso et al. [58]. The concentration of nitrous oxide (NO) in culture supernatants was determined by the Griess reaction [59] using total nitrous oxide assay kit.

ACCEPTED MANUSCRIPT

2.7. Hypoxia stress challenge At the end of the feeding trial, fish at each treatment were randomly distributed at a density of 10 fish per 100-L aquarium in duplicates. Fish were exposed to hypoxia stress for three

RI PT

days by decreasing aeration level as described by Abdel-Tawwab et al. [60] where level of

dissolved oxygen was around 1.0-1.5 mg/L. Fish were kept under observation to record the daily fish mortality.

SC

2.7. Bacterial challenge

Fish at each treatment were randomly were stocked at a density of 10 fish per 100-L

M AN U

aquarium in duplicates. The challenge test was carried out using A. hydrophylla isolated previously in Department of Fish Disease, CLAR, Abbassa, Abo-Hammad, Sharqia, Egypt. The first subgroup was challenged with pathogenic A. hydrophila using a sublethal dose as described by Abdel-Tawwab et al. [19] where a 0.1 ml dose of 24-h broth from virulent A. hydrophila

TE D

(5x105 CFU/mL) was intraperitoneally injected. Fish were kept under observation for 10 days to record any abnormal clinical signs and the daily fish mortality. 2.8. Statistical analysis

EP

The obtained data were subjected to one-way ANOVA to evaluate the effect of CNP supplementation. Differences between means were tested at the 5% probability level using

AC C

Duncan test as a post-hoc test. The optimum CNP level was determined using a polynomial regression analysis. All the statistical analyses were done using SPSS program version 20 (SPSS, Richmond, VA, USA) as described by Dytham [61]. 3. Results

It is noticed that dietary CNP has a positive effect on fish growth and feed intake, which were significantly higher when fish fed CNP-enriched diets as compared to those fed the control

ACCEPTED MANUSCRIPT

diet (P < 0.05; Table 2). The relationship between final weight and CNP levels (Figure 1) was best expressed by the second-order polynomial regression equations as follows: Y = - 0.3869 X2 + 3.6917 X + 14.486

RI PT

This figure showed that the optimum CNP level for Nile tilapia is 3.0 g/kg diet. Moreover, fish fed on diets containing 3.0 g CNP/kg consumed more diet (20.6 g feed/fish) than the other

treatments; meanwhile no significant change in FCR values was observed and its range was 1.46

SC

- 1.54. Throughout the feeding period, fish in all experimental groups were in good health as observed from their general activity and fish survival ranged from 98.3 to 100% with no

M AN U

significant difference among the different fish groups (P > 0.05; Table 2). This result suggests that dietary CNP has no toxic effect on fish.

Data in Table 3 show that CNP supplementation significantly affected only contents of crude protein and total ash in the whole-fish body (P > 0.05). It is noticed that content of crude

TE D

protein was significantly higher, whereas total ash content was significantly lower in CNP-fed fish than those fed the control diet. On the other hand, no significant differences were observed in moisture and lipid contents in whole-fish body among the different treatments.

EP

Regarding the antioxidant activity, it is noticed that plasmatic MDA, SOD, and CAT levels were significantly induced; meanwhile GPx was significantly prohibited by CNP

AC C

supplement especially at levels of 3.0 – 10.0 g/kg diet (P < 0.05; Table 4). Likewise, dietary CNP promoted the secretion of digestive enzymes (protease, lipase, and amylase) and innate immunity (NO, NBT, and lysozyme activity) as compared with the control group and their highest values were obtained at 3.0 - 10.0 g/kg diet (P > 0.05; Tables 5 and 6). Regarding hypoxia stress, no significant mortality was observed in fish under hypoxia stress at all treatments. On the other hand, dietary CNP improved the fish challenge against A.

ACCEPTED MANUSCRIPT

hydrophila infection where no fish mortality was observed among fish fed 1.0 – 10.0 g CNP/kg diet; meanwhile fish fed CNP-free diet showed highest mortality (66.7%; Table 6 and Figure 2). 4. Discussion

RI PT

The present study indicates that dietary CNP could be a potential feed additive to enhance fish performance and innate immunity. These results may be because the inclusion of CNP in fish diet enhanced diets digestion and nutrient digestibility leading to improved nutrient utilization,

SC

which in turn improved fish growth. This hypothesis was supported by the high activities of digestive enzymes represented herein. On the other hand, CNP may inhibit potential pathogens in

M AN U

the digestive tract, may enhance the population of beneficial microorganism, and/or may enhance the microbial enzyme activities that consequently improve feed digestibility and nutrient absorption. In this respect, Ahmad et al. [41] and Sivagurunathan and Innocent [42] found that Nile tilapia fed diets containing 1% cinnamon powder and 1% cinnamon flour, respectively,

TE D

resulted in significantly greater fish performance than the other diets. Setiawati et al. [43] found that supplementation of 0.1% extract and 1% powder of cinnamon leaf in diet improved growth performance, feed efficiency, and protein retention of Asian catfish (Pangasianodon

EP

hypophthalmus) as compared to the control diet.

The optimum CNP level herein is 3.0 g/kg diet; meanwhile, a study of Ahmad et al. [41],

AC C

which was carried out in the same Lab using the same fish strain and Lab facility found that the optimum level of traditional cinnamon was 10.0 g/kg diet. This comparison indicates that cinnamon nanoform is more efficient than its ordinary form. These results may be because the CNP remained in the blood stream for long period facilitating its good bioavailability. In a similar study, Korni and Khalil [62] used traditional ginger and its nanoparticle in diets for common carp, Cyprinus carpio. They found that fish growth, immunity, and challenge against Aeromonas septicemia were more efficient with ginger nanoparticles than the traditional form.

ACCEPTED MANUSCRIPT

The present results indicate that the dietary CNP induced protein deposition alone in wholefish body. This result may because cinnamaldehyde (the main constituent of cinnamon) is capable to activate the insulin-like growth factor (IGF-1), which enhanced the biosynthesis of

RI PT

protein and collagen in the body tissues [63], thereby increasing the protein deposition. These results are in concomitant with Rolin et al. [64] who found that administration of 1% cinnamon extract in Asian catfish diet increased protein retention by 24% compared to the control diet. The

SC

present study showed that dietary CNP did not improve lipid deposition in fish body, whereas Setiawati et al. [43] reported that the addition of 1% cinnamon leaf improved flesh fat content,

M AN U

cholesterol, and triglycerides deposition in Asian catfish. These conflicting results may be linked with changes in their synthesis, deposition rate in muscle, and different growth rates [65][66]. Activity of antioxidant enzymes and lipid peroxidation (indicated by MDA) products are indicators of oxidative cell damage and examples of the toxic mechanisms of reactive oxygen

TE D

species (ROS), which are involved in pathological processes and in the aetiology of many fish diseases [68]. Thus, they are commonly used as biomarkers and quick responses to ROS generation [69][70]. The present study evoked that dietary CNP had antioxidant activity where

EP

MDA level and activities of SOD and CAT increased significantly, whereas GPx decreased significantly in fish fed CNP-enriched diets. These results may be because cinnamon contains

AC C

large amounts of bioactive molecules including essential oils (cinnamic aldehyde and cinnamyl aldehyde), polyphenol, tannins, saponins, flavonoids and carbohydrates [34][35], which show antioxidant properties [36][37]. Many studies reported that cinnamon has been documented to show in vitro and in vivo antioxidant activity [71][72][39][73]. The increased activities of digestive enzymes due to CNP supplementation indicated that dietary CNP stimulates digestive enzymes production. In this regard, Wang et al. [75] found higher intestinal lipase activity and lower intestinal amylase activity in yellow catfish

ACCEPTED MANUSCRIPT

(Pelteobagrus fulvidraco) fed diet supplemented with some Chinese herb medicines for 12 weeks compared with the control. Awad et al. [76] found modulatory increases in amylase, lipase, and pepsin activity in stomach and intestine of rainbow trout fed 2% lupin (Lupinus perennis), mango

RI PT

(Mangifera indica), and stinging nettle (Urtica dioica) for two months as compared with the control. Zahran et al. [77] investigated effects of Astragalus polysaccharides (APS) on digestive enzymes of Nile tilapia. They found significant increases in amylase and lipase activity between

SC

APS-fed fish and the control one. Rahimi Yadkoori et al. [78] reported that feeding binni fish, Mesopotamichthys sharpeyi on ginger extract enhanced the activity of digestive enzymes

M AN U

differently where amylase activity improved, but trypsin activity not affected. The innate immunity of Nile tilapia fed different CNP levels was examined by evaluating NO, NBT, and lysozyme activity. In the present study, those variables were significantly induced when fish fed CNP-enriched diets. It is known that activities of NBT and lysozyme have

TE D

important roles in the non-specific immune defense system [79][80]. Nitrous oxide is highly reactive molecule, which has antimicrobial activity and plays a central role in the physiology and pathology such as immune system damage and cell apoptosis [81][77]. Similar results were noted

EP

in common carp fed turmeric-supplemented diet [17], or Nile tilapia fed diets enriched with green tea [21], and ginger [62].

AC C

The results of the present study indicate that dietary CNP had a protective effect against

A. hydrophyla infection in fish. Similar results were obtained by Ahmad et al. [41] and Rattanachaikunsopon and Phumkhachorn [82] who found a protective role of cinnamon powder and cinnamon oil against A. hydrophyla and Streptococcus iniae infection on Nile tilapia, respectively. The protective role of cinnamon against pathogenic A. hydrophila may be due to cinnamaldehyde (the main component of cinnamon oil), which has antibacterial, antifungal, antiviral, antidiabetic, and antioxidative properties [83]. It has been also reported to be able to

ACCEPTED MANUSCRIPT

inhibit both Gram-positive and Gram-negative bacteria including Escherichia coli O157:H7, Helicobacter pylori, Listeria monocytogenes, and Salmonella choleraesuis [84][85]. Conclusion

RI PT

The present study evoked that dietary CNP may be had a novel properties that enhanced fish performance with optimum level of 3.0 g/kg diet. Moreover, dietary CNP enhanced

antioxidant and digestive enzymes activity as well as innate immunity. It could also protect fish

SC

against Aeromonas septicemia. Acknowledgments

M AN U

This study was funded and supported by the Central Laboratory for Aquaculture Research (CLAR), Abbassa, Abo-Hammad, Sharqia, Egypt. The authors would like to thank Dr. Samy M. Shaban, Egyptian Petroleum Research Institute, Nasr City, Cairo, Egypt, for doing cinnamon nanoparticles used in the present study and Dr. Somayah Awad, Department of Fish Disease,

5. References

TE D

CLAR, for doing the bacterial challenge test.

A.-F.M. El-Sayed, Tilapia culture, CABI, 2006.

[2]

E.J. Noga, Fish disease: diagnosis and treatment, John Wiley & Sons, 2011.

[3]

J.W. Pridgeon, P.H. Klesius, Virulence of Aeromonas hydrophila to channel catfish

AC C

EP

[1]

Ictaluras punctatus fingerlings in the presence and absence of bacterial extracellular products, Dis. Aquat. Organ. 95 (2011) 209–215. [4]

M.J. Saavedra, S. Guedes-Novais, A. Alves, P. Rema, M. Tacão, A. Correia, A. Mart’\inez-Murcia, Resistance to $β$-lactam antibiotics in Aeromonas hydrophila isolated from rainbow trout (Onchorhynchus mykiss), Int. Microbiol. 7 (2004) 207–211.

[5]

D. Stratev, O.A. Odeyemi, An overview of motile Aeromonas septicaemia management,

ACCEPTED MANUSCRIPT

Aquac. Int. 25 (2017) 1095–1105. [6]

A. Brian, A. Dawn, Bacterial Fish Pathogens: Diseases of Farmed and Wild Fish, Springer Publishers, 2007. M. Dadar, K. Dhama, V.N. Vakharia, S.H. Hoseinifar, K. Karthik, R. Tiwari, R. Khandia,

RI PT

[7]

A. Munjal, C. Salgado-Miranda, S.K. Joshi, Advances in Aquaculture Vaccines Against Fish Pathogens: Global Status and Current Trends, Rev. Fish. Sci. Aquac. 25 (2017) 184–

[8]

SC

217.

M.S. Llewellyn, S. Boutin, S.H. Hoseinifar, N. Derome, Teleost microbiomes: the state of

Front. Microbiol. 5 (2014). [9]

M AN U

the art in their characterization, manipulation and importance in aquaculture and fisheries,

A.G. Murray, E.J. Peeler, A framework for understanding the potential for emerging diseases in aquaculture, Prev. Vet. Med. 67 (2005) 223–235.

D.J. Alderman, T.S. Hastings, Antibiotic use in aquaculture: development of antibiotic

TE D

[10]

resistance--potential for consumer health risks, Int. J. Food Sci. Technol. 33 (1998) 139– 155.

M. Teuber, Veterinary use and antibiotic resistance, Curr. Opin. Microbiol. 4 (2001) 493– 499.

M. Abdel-Tawwab, The use of American ginseng (Panax quinquefolium) in practical diets

AC C

[12]

EP

[11]

for Nile tilapia (Oreochromis niloticus): growth performance and challenge with Aeromonas hydrophila, J. Appl. Aquac. 24 (2012) 366–376. [13]

M. Abdel-Tawwab, The use of American Ginseng (Panax quinquefolium) in practical diets for Nile tilapia (Oreochromis niloticus): Resistance to waterborne copper toxicity, Aquac. Res. 46 (2015) 1001–1006. doi:10.1111/are.12237.

[14]

M. Abdel-Tawwab, Incorporating Roasted Coffee Bean into Nile Tilapia Diets Does Not

ACCEPTED MANUSCRIPT

Improve Growth Performance, J. Appl. Aquac. 27 (2015) 87–93. doi:10.1080/10454438.2015.1007021. [15]

M. Abdel-Tawwab, Interactive effects of dietary protein and live bakery yeast,

RI PT

Saccharomyces cerevisiae on growth performance of Nile tilapia, Oreochromis niloticus (L.) fry and their challenge against Aeromonas hydrophila infection, Aquac. Int. 20 (2012) 317–331. doi:10.1007/s10499-011-9462-8.

M. Abdel-Tawwab, Feed Supplementation to Freshwater Fish : Experimental Approaches, Lambert Academic Publishing, 2016.

M. Abdel-Tawwab, F.E. Abbass, Turmeric Powder, Curcuma longa L., in Common Carp,

M AN U

[17]

SC

[16]

Cyprinus carpio L., Diets: Growth Performance, Innate Immunity, and Challenge against Pathogenic Aeromonas hydrophila Infection, J. World Aquac. Soc. 48 (2017) 303–312. doi:10.1111/jwas.12349.

H.S. Hamed, M. Abdel-Tawwab, Ameliorative effect of propolis supplementation on

TE D

[18]

alleviating bisphenol-A toxicity: Growth performance, biochemical variables, and oxidative stress biomarkers of Nile tilapia, Oreochromis niloticus (L.) fingerlings, Comp.

EP

Biochem. Physiol. Part C Toxicol. Pharmacol. 202 (2017) 63–69. doi:10.1016/j.cbpc.2017.08.001. M. Abdel-Tawwab, A.M. Abdel-Rahman, N.E.M. Ismael, Evaluation of commercial live

AC C

[19]

bakers’ yeast, Saccharomyces cerevisiae as a growth and immunity promoter for Fry Nile tilapia, Oreochromis niloticus (L.) challenged in situ with Aeromonas hydrophila, Aquaculture. 280 (2008) 185–189. doi:10.1016/j.aquaculture.2008.03.055. [20]

M. Abdel-Tawwab, M.H. Ahmad, Live Spirulina (Arthrospira platensis) as a growth and immunity promoter for Nile tilapia, Oreochromis niloticus (L.), challenged with pathogenic Aeromonas hydrophila, Aquac. Res. 40 (2009) 1037–1046.

ACCEPTED MANUSCRIPT

doi:10.1111/j.1365-2109.2009.02195.x. [21]

M. Abdel-Tawwab, M.H. Ahmad, M.E.A. Seden, S.F.M. Sakr, Use of Green Tea, Camellia sinensis L., in Practical Diet for Growth and Protection of Nile Tilapia,

41 (2010) 203–213. doi:10.1111/j.1749-7345.2010.00360.x. [22]

RI PT

Oreochromis niloticus (L.), against Aeromonas hydrophila Infection, J. World Aquac. Soc.

M. Abdel-Tawwab, K.M. Sharafeldin, M.N.M. Mosaad, N.E.M. Ismaiel, Coffee bean in

SC

common carp, Cyprinus carpio L. diets: Effect on growth performance, biochemical status, and resistance to waterborne zinc toxicity, Aquaculture. 448 (2015) 207–213.

[23]

M AN U

doi:10.1016/j.aquaculture.2015.06.010.

D. Carbone, C. Faggio, Fish & Shell fi sh Immunology Importance of prebiotics in aquaculture as immunostimulants . Effects on immune system of Sparus aurata and Dicentrarchus labrax, Fish Shellfish Immunol. 54 (2016) 172–178.

[24]

TE D

doi:10.1016/j.fsi.2016.04.011.

S.H. Hoseinifar, M.Á. Esteban, A. Cuesta, Y.-Z. Sun, Prebiotics and fish immune response: a review of current knowledge and future perspectives, Rev. Fish. Sci. Aquac. 23

[25]

EP

(2015) 315–328.

S.H. Hoseinifar, E. Ringø, A. Shenavar Masouleh, M.Á. Esteban, Probiotic, prebiotic and

[26]

AC C

synbiotic supplements in sturgeon aquaculture: a review, Rev. Aquac. 8 (2016) 89–102. S.H. Hoseinifar, Y.-Z. Sun, C.M. Caipang, Short-chain fatty acids as feed supplements for sustainable aquaculture: an updated view, Aquac. Res. (2016). [27]

S.K. Song, B.R. Beck, D. Kim, J. Park, J. Kim, H.D. Kim, E. Ringø, Prebiotics as immunostimulants in aquaculture: a review, Fish Shellfish Immunol. 40 (2014) 40–48.

[28]

H. Van Doan, S.H. Hoseinifar, M.A.O. Dawood, C. Chitmanat, K. Tayyamath, Effects of Cordyceps militaris spent mushroom substrate and Lactobacillus plantarum on mucosal,

ACCEPTED MANUSCRIPT

serum immunology and growth performance of Nile tilapia (Oreochromis niloticus), Fish Shellfish Immunol. (2017). T. Citarasu, Herbal biomedicines : a new opportunity for aquaculture industry, (2010) 403–414. doi:10.1007/s10499-009-9253-7. [30]

S.K. Dügenci, N. Arda, A. Candan, Some medicinal plants as immunostimulant for fish, J. Ethnopharmacol. 88 (2003) 99–106.

R. Harikrishnan, C. Balasundaram, M.-S. Heo, Impact of plant products on innate and

SC

[31]

RI PT

[29]

adaptive immune system of cultured finfish and shellfish, Aquaculture. 317 (2011) 1–15. L. Su, J.-J. Yin, D. Charles, K. Zhou, J. Moore, L.L. Yu, Total phenolic contents, chelating

M AN U

[32]

capacities, and radical-scavenging properties of black peppercorn, nutmeg, rosehip, cinnamon and oregano leaf, Food Chem. 100 (2007) 990–997. [33]

S.F. Nabavi, A. Di Lorenzo, M. Izadi, E. Sobarzo-Sánchez, M. Daglia, S.M. Nabavi,

TE D

Antibacterial effects of cinnamon: From farm to food, cosmetic and pharmaceutical industries, Nutrients. 7 (2015) 7729–7748. [34]

H.-K. Kwon, W.K. Jeon, J.-S. Hwang, C.-G. Lee, J.-S. So, J.-A. Park, B.S. Ko, S.-H. Im,

EP

Cinnamon extract suppresses tumor progression by modulating angiogenesis and the effector function of CD8+ T cells, Cancer Lett. 278 (2009) 174–182. J. Gruenwald, J. Freder, N. Armbruester, Cinnamon and health, Crit. Rev. Food Sci. Nutr.

AC C

[35]

50 (2010) 822–834. [36]

C. Rice-Evans, N. Miller, G. Paganga, Antioxidant properties of phenolic compounds, Trends Plant Sci. 2 (1997) 152–159.

[37]

W. Łuczaj, E. Zapora, M. Szczepański, K. Wnuczko, E. Skrzydlewska, Polyphenols action against oxidative stress formation in endothelial cells., Acta Pol. Pharm. 66 (2009) 617– 624.

ACCEPTED MANUSCRIPT

[38]

S.H. Lee, S.Y. Lee, D.J. Son, H. Lee, H.S. Yoo, S. Song, K.W. Oh, D.C. Han, B.M. Kwon, J.T. Hong, Inhibitory effect of 2?-hydroxycinnamaldehyde on nitric oxide production through inhibition of NF-$κ$B activation in RAW 264.7 cells, Biochem. Pharmacol. 69

[39]

RI PT

(2005) 791–799.

B. Shan, Y.-Z. Cai, J.D. Brooks, H. Corke, Antibacterial and antioxidant effects of five spice and herb extracts as natural preservatives of raw pork, J. Sci. Food Agric. 89 (2009)

[40]

SC

1879–1885.

N. Matan, H. Rimkeeree, A.J. Mawson, P. Chompreeda, V. Haruthaithanasan, M. Parker,

M AN U

Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions, Int. J. Food Microbiol. 107 (2006) 180–185. [41]

M.H. Ahmad, A.M.D. El Mesallamy, F. Samir, F. Zahran, Effect of cinnamon (Cinnamomum zeylanicum) on growth performance, feed utilization, whole-body

(2011) 289–298. [42]

TE D

composition, and resistance to Aeromonas hydrophila in nile tilapia, J. Appl. Aquac. 23

A. Sivagurunathan, B.X. Innocent, Immunomodulatory effect of dietary cinnamon in

EP

growth and haematology of tilapia challenged with Pseudomonas aeruginosa, Int. J. Pharm. Phytopharm. Res. 3 (2017) 277–280. M. Setiawati, D. Jusadi, S. Laheng, M.A. Suprayudi, A. Vinasyiam, The enhancement of

AC C

[43]

growth performance and feed efficiency of Asian catfish, Pangasianodon hypophthalmus fed on Cinnamomum burmannii leaf powder and extract as nutritional supplementation., Aquac. Aquarium, Conserv. Legis. J. Bioflux Soc. (AACL Bioflux). 9 (2016). [44]

A. Thulasi, D. Rajendran, S. Jash, S. Selvaraju, V.L. Jose, S. Velusamy, S. Mathivanan, Nanobiotechnology in Animal Nutrition, (2013).

[45]

M. Nair, R.D. Jayant, A. Kaushik, V. Sagar, Getting into the brain: potential of

ACCEPTED MANUSCRIPT

nanotechnology in the management of NeuroAIDS, Adv. Drug Deliv. Rev. 103 (2016) 202–217. [46]

S.A. Bello, J.O. Agunsoye, S.B. Hassan, Synthesis of coconut shell nanoparticles via a top

RI PT

down approach: Assessment of milling duration on the particle sizes and morphologies of coconut shell nanoparticles, Mater. Lett. 159 (2015) 514–519. [47]

S. Mashkouri, N. Arsalani, H. Mostafavi, Wet mechanochemical approach assistance to

SC

the green synthesis of graphene sheet at room temperature and in situ anchored with MnO 2 in a green method, J. Alloys Compd. 715 (2017) 486–493.

C.E. Boyd, C.S. Tucker, Pond aquaculture water quality management, Springer Science & Business Media, 2012.

[49]

M AN U

[48]

D. Firestone, Official methods of analysis of the Association of Official Analytical Chemists, Arlington, USA,. (1990).

H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by

TE D

[50]

thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [51]

J.M. McCord, I. Fridovich, Superoxide dismutase an enzymic function for erythrocuprein

EP

(hemocuprein), J. Biol. Chem. 244 (1969) 6049–6055. H. Aebi, [13] Catalase in vitro, Methods Enzymol. 105 (1984) 121–126.

[53]

D.E. Paglia, W.N. Valentine, Studies on the quantitative and qualitative characterization of

AC C

[52]

erythrocyte glutathione peroxidase, J. Lab. Clin. Med. 70 (1967) 158–169. [54]

N.W. Ross, K.J. Firth, A. Wang, J.F. Burka, S.C. Johnson, Changes in hydrolytic enzyme activities of naive Atlantic salmon Salmo salar skin mucus due to infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation, Dis. Aquat. Organ. 41 (2000) 43–51.

[55]

Z.K. Shihabi, C. Bishop, Simplified turbidimetric assay for lipase activity, Clin. Chem. 17

ACCEPTED MANUSCRIPT

(1971) 1150–1153. [56]

P. Bernfeld, Enzymes of carbohydrate metabolism, Methods Enzymol. 1 (1955) 149–158.

[57]

G.A.W. Rook, J. Steele, S. Umar, H.M. Dockrell, A simple method for the solubilisation

by $γ$-interferon, J. Immunol. Methods. 82 (1985) 161–167. [58]

RI PT

of reduced NBT, and its use as a colorimetric assay for activation of human macrophages

D. Caruso, O. Schlumberger, C. Dahm, J.-P. Proteau, Plasma lysozyme levels in sheatfish

SC

Silurus glanis (L.) subjected to stress and experimental infection with Edwardsiella tarda, Aquac. Res. 33 (2002) 999–1008.

H. Schmidt, M. Kelm, Determination of nitrite and nitrate by the Griess reaction, Methods Nitric Oxide Res. 497 (1996).

[60]

M AN U

[59]

M. Abdel-Tawwab, A.E. Hagras, H.A.M. Elbaghdady, M.N. Monier, Effects of dissolved oxygen and fish size on Nile tilapia, Oreochromis niloticus (L.): growth performance,

TE D

whole-body composition, and innate immunity, Aquac. Int. 23 (2015) 1261–1274. doi:10.1007/s10499-015-9882-y.

C. Dytham, Choosing and using statistics: a biologist’s guide, John Wiley & Sons, 2011.

[62]

F.M.M. Korni, F. Khalil, Effect of ginger and its nanoparticles on growth performance,

EP

[61]

cognition capability, immunity and prevention of motile Aeromonas septicaemia in

[63]

AC C

Cyprinus carpio fingerlings, Aquac. Nutr. (2017). N. Takasao, K. Tsuji-Naito, S. Ishikura, A. Tamura, M. Akagawa, Cinnamon extract promotes type I collagen biosynthesis via activation of IGF-I signaling in human dermal fibroblasts, J. Agric. Food Chem. 60 (2012) 1193–1200. [64]

F. Rolin, M. Setiawati, D. Jusadi, Evaluasi pemberian ekstrak daun kayu manis Cinnamomum burmannii pada pakan terhadap kinerja pertumbuhan ikan patin Pangasianodon hypophthalmus Sauvage, 1878 [Evaluation of the addition of cinnamon

ACCEPTED MANUSCRIPT

Cinnamomum burmannii leaves extract in diet for growth per, J. Iktiologi Indones. 15 (2017) 201–208. [65]

B. Fauconneau, Protein synthesis and protein deposition in fish, Nutr. Feed. Fish. (1985)

[66]

RI PT

17–45.

M. Abdel-Tawwab, Y.A.E. Khattab, M.H. Ahmad, A.M.E. Shalaby, Compensatory

Growth, Feed Utilization, Whole-Body Composition, and Hematological Changes in

SC

Starved Juvenile Nile Tilapia, Oreochromis niloticus (L.), J. Appl. Aquac. 18 (2006) 17– 36. doi:10.1300/J028v18n03.

O.A. Asimi, N.P. Sahu, A.K. Pal, Antioxidant activity and antimicrobial property of some

M AN U

[67]

Indian spices, Int J Sci. Res. Publ. 541 (2013). [68]

J.P. Kehrer, Free radicals as mediators of tissue injury and disease, Crit. Rev. Toxicol. 23 (1993) 21–48.

K.B. Storey, Oxidative stress: animal adaptations in nature, Brazilian J. Med. Biol. Res. 29 (1996) 1715–1733.

[70]

TE D

[69]

M. Hermes-Lima, Oxygen in biology and biochemistry: role of free radicals, Funct.

[71]

EP

Metab. Regul. Adapt. 1 (2004) 319–366.

C.-C. Lin, S.-J. Wu, C.-H. Chang, L.-T. Ng, Antioxidant activity of Cinnamomum cassia,

[72]

AC C

Phyther. Res. 17 (2003) 726–730. K.S. Azab, A.-H.A. Mostafa, E.M.M. Ali, M.A.S. Abdel-Aziz, Cinnamon extract ameliorates ionizing radiation-induced cellular injury in rats, Ecotoxicol. Environ. Saf. 74 (2011) 2324–2329. [73]

A. Eidi, P. Mortazavi, M. Bazargan, J. Zaringhalam, Hepatoprotective activity of cinnamon ethanolic extract against CCI4-induced liver injury in rats, Excli J. 11 (2012) 495.

ACCEPTED MANUSCRIPT

[74]

K. Platel, K. Srinivasan, others, Influence of dietary spices and their active principles on pancreatic digestive enzymes in albino rats, Nahrung/Food. 44 (2000) 42–46.

[75]

J. WANG, C. QI, A. CHENG, Y. YAN, W. LI, Y. YAN, Effects of Dietary Radix

RI PT

Astragali, Radix Polygoni Multiflori and Fructus Crataegi on Growth and Digestibility in juvenile yellow catfish (Pelteobagrus fulvidraco)[J], Chinese J. Fish. 1 (2008) 7. [76]

E. Awad, B. Austin, A. Lyndon, Effect of dietary supplements on digestive enzymes and

SC

growth performance of rainbow trout (Oncorhynchus mykiss, Walbaum), J. Am. Sci. 8 (2012) 858–864.

E. Zahran, E. Risha, F. Abdelhamid, H. Allah, Fish & Shell fi sh Immunology Effects of

M AN U

[77]

dietary Astragalus polysaccharides ( APS ) on growth performance , immunological parameters , digestive enzymes , and intestinal morphology of Nile tilapia ( Oreochromis niloticus ), Fish Shellfish Immunol. 38 (2014) 149–157. doi:10.1016/j.fsi.2014.03.002. N. Rahimi Yadkoori, N. Zanguee, S.M. Mousavi, M. Zakeri, Effects of Ginger (Zingiber

TE D

[78]

officinale) Extract on Digestive Enzymes and Liver Activity of Mesopotamichthys sharpeyi Fingerlings, (2015).

B. Grinde, Lysozyme from rainbow trout, Salmo gairdneri Richardson, as an antibacterial

EP

[79]

agent against fish pathogens, J. Fish Dis. 12 (1989) 95–104. A.E. Ellis, Lysozyme assays, Tech. Fish Immunol. 1 (1990) 101–103.

[81]

D.-H. Kim, B. Austin, Innate immune responses in rainbow trout (Oncorhynchus mykiss,

AC C

[80]

Walbaum) induced by probiotics, Fish Shellfish Immunol. 21 (2006) 513–524. [82]

P. Rattanachaikunsopon, P. Phumkhachorn, Potential of cinnamon (Cinnamomum verum) oil to control Streptococcus iniae infection in tilapia (Oreochromis niloticus), Fish. Sci. 76 (2010) 287–293.

[83]

E. Schmidt, L. Jirovetz, G. Buchbauer, G.A. Eller, I. Stoilova, A. Krastanov, A.

ACCEPTED MANUSCRIPT

Stoyanova, M. Geissler, Composition and antioxidant activities of the essential oil of cinnamon (Cinnamomum zeylanicum Blume) leaves from Sri Lanka, J. Essent. Oil Bear. Plants. 9 (2006) 170–182. P. Lopez, C. Sanchez, R. Batlle, C. Ner’\in, Vapor-phase activities of cinnamon, thyme,

RI PT

[84]

and oregano essential oils and key constituents against foodborne microorganisms, J. Agric. Food Chem. 55 (2007) 4348–4356.

SC

O. Senhaji, M. Faid, I. Kalalou, Inactivation of Escherichia coli O157: H7 by essential oil

EP

TE D

M AN U

from Cinnamomum zeylanicum, Brazilian J. Infect. Dis. 11 (2007) 234–236.

AC C

[85]

ACCEPTED MANUSCRIPT

Table 1. Nutrients contents and proximate chemical composition (on dry matter basis) of the tested diets differed in cinnamon nanoparticles powder levels. Cinnamon nanoparticles levels (g/kg diet) 0.25 0.5 1.0 3.0 5.0

10.0

11.0 42.5 14.9 19.3 3.0 2.3 1.0 2.0 4.0 0.0 100

11.0 42.5 14.9 19.05 3.0 2.3 1.0 2.0 4.0 0.25 100

11.0 42.5 14.9 9.3 3.0 2.3 1.0 2.0 4.0 10.0 100

11.0 42.5 14.9 18.3 3.0 2.3 1.0 2.0 4.0 1.0 100

11.0 42.5 14.9 16.3 3.0 2.3 1.0 2.0 4.0 3.0 100

11.0 42.5 14.9 14.3 3.0 2.3 1.0 2.0 4.0 5.0 100

RI PT

11.0 42.5 14.9 18.8 3.0 2.3 1.0 2.0 4.0 0.5 100

SC

Herring fish meal 1 Soybean meal 2 Wheat bran Ground corn Corn oil Cod liver oil Vitamin premix 3 Mineral premix 4 Starch Cinnamon nanoparticles Total

0.0

M AN U

Ingredients

AC C

EP

TE D

Chemical composition (%) Dry matter 93.0 92.9 92.8 92.7 92.5 92.3 92.3 Crude protein 30.8 30.9 31.2 31.2 31.5 31.7 31.8 Ether extract 7.1 7.1 7.1 7.2 7.2 7.2 7.2 Crude fiber 5.2 5.1 4.9 4.9 4.9 4.8 4.7 Total ash 7.2 7.2 7.2 7.1 7.1 7.1 7.1 49.7 49.7 49.6 49.6 49.3 49.2 49.2 Nitrogen free extract 5 Gross energy (kcal/100 g) 6 444.7 445.7 446.2 447.5 448.4 449.1 449.5 1 Danish fish meal 72.0% protein obtained from TripleNine Fish Protein, DK-6700 Esbjerg, Denmark. 2 Egyptian soybean flour 45.0% protein obtained from National Oil Co., Giza, Egypt. 3 Vitamin premix (per kg of premix): thiamine, 2.5 g; riboflavin, 2.5 g; pyridoxine, 2.0 g; inositol, 100.0 g; biotin, 0.3 g; pantothenic acid, 100.0 g; folic acid, 0.75 g; para-aminobenzoic acid, 2.5 g; choline, 200.0 g; nicotinic acid, 10.0 g; cyanocobalamine, 0.005 g; α-tocopherol acetate, 20.1 g; menadione, 2.0 g; retinol palmitate, 100,000 IU; cholecalciferol, 500,000 IU. 4 Mineral premix (per kg of premix): CaHPO4.2H2O, 727.2 g; MgCO3.7H2O, 127.5 g; KCl 50.0 g; NaCl, 60.0 g; FeC6H5O7.3H2O, 25.0 g; ZnCO3, 5.5 g; MnCl2.4H2O, 2.5 g; CuCl2, 0.785 g; CoCl3..6H2O, 0.477 g; CaIO3.6H2O, 0.295 g; CrCl3.6H2O, 0.128 g; AlCl3.6H2O, 0.54 g; Na2SeO3, 0.3 g (Juauncey and Ross 1982). 5 Nitrogen free extract (NFE) = 100 – (protein % + lipid % + total ash % + crude fiber %). 6 Gross energy was calculated according to NRC (1993) as 5.65, 9.45, and 4.11 kcal/g for protein, lipid, and carbohydrates, respectively.

ACCEPTED MANUSCRIPT

RI PT

25 24 23

SC

22

M AN U

Fish weight (g)

21 20

2

Y = - 0.3869 X + 3.6917 X + 14.486 2 R = 0.9786

19

TE D

18 17

15

EP

16

AC C

Control

0.25

0.5

1

3

5

10

Cinnamon nanoparticles (g/kg diet)

Figure 1. The relationship between final weight (g) of Nile tilapia and different levels of cinnamon nanoparticles.

ACCEPTED MANUSCRIPT

RI PT

Table 2. Growth performance and feed utilization of Nile tilapia fed diets supplemented with different levels of cinnamon nanoparticles (CNP) for 8 weeks. Cinnamon nanoparticles levels (g/kg diet) 0.5 1.0 3.0

0.25

5.0

10.0

Initial weight (g)

9.7±0.023

9.6±0.05

9.7±0.06

9.6±0.03

9.7±0.03

9.6±0.05

9.6±0.03

Final weight (g)

18.1±0.23 c

21.9±0.42 b

22.3±0.17 b

22.3±0.26 b

23.3±0.12 a

22.2±0.18 b

21.9±0.18 b

Weight gain (g)

8.5±0.23 c

12.3±0.42 b

12.6±0.18 b

12.7±0.29 b

13.7±0.14 a

12.6±0.21 b

12.3±0.16 b

Weight gain %

87.3±2.43 c

SGR (%g/day)

1.12±0.023 c

Feed intake (g feed/ fish) FCR

13.0±0.75 d

128.2±4.55 b 1.47±0.036 b 17.9±0.46 c

1.54±0.053

1.46±0.061

130.9±2.21 b 1.49±0.017 b 19.0±0.62 abc 1.50±0.023

132.0±3.28 b 1.50±0.025 b 19.6±0.53 ab

141.5±1.91 a 1.57±0.007 a 20.6±0.64 a

130.7±2.76 b 1.49±0.021 b 19.7±0.57 ab

127.5±1.31 b 1.47±0.012 b 18.8±0.44 bc

1.54±0.045

1.51±0.041

1.57±0.052

1.53±0.036

98.3±1.68

100.0±0.00

98.3±1.68

TE D

M AN U

SC

0.0

AC C

EP

Fish survival (%) 100.0±0.00 100.0±0.00 98.3±1.68 98.3±1.68 Means having the same letter in the same row are not significantly different at P < 0.05.

ACCEPTED MANUSCRIPT

1

Table 3. Proximate chemical composition (%; on fresh weight basis) of whole-body of Nile tilapia fed different levels cinnamon nanoparticles (CNP) for 8 weeks. Moisture

Crude protein

Total lipids

0.0

70.8±0.61

17.2±0.15 b

5.2±0.13

0.25

70.8±0.43

17.5±0.11 b

5.4±0.19

0.5

70.9±0.37

18.1±0.28 ab

5.2±0.19

1.0

71.0±0.22

18.3±0.15 a

5.3±0.07

5.4±0.15 b

3.0

70.9±0.23

18.8±0.31 a

5.1±0.04

5.2±0.23 b

5.0

70.8±0.61

18.6±0.55 a

5.2±0.09

5.4±0.28 b

10.0

70.6±0.86

18.6±0.63 a

5.3±0.09

5.5±0.46 b

6.8±0.33 a

6.3±0.35 ab 5.8±0.47 b

M AN U

SC

(g/kg diet)

Total ash

RI PT

CNP levels

TE D

Means having the same letter in the same column are not significantly different at P < 0.05.

6

CNP levels

MDA

SOD

CAT

GPx

(g/kg diet)

(nmol/L)

(IU/L)

(IU/L)

(IU/L)

0.0

1.45±0.038 d

11.5±0.66 d

10.5±1.59 d

24.5±0.58 a

0.25

2.10±0.021 c

AC C

22.5±0.58 c

15.5±1.96 c

22.8±0.38 a

0.5

2.56±0.133 b

24.6±0.69 bc

21.6±1.21 b

18.9±0.61 b

1.0

2.65±0.041 b

25.9±2.28 b

23.5±1.18 b

16.7±0.52 c

3.0

3.21±0.136 a

27.4±1.67 ab

26.4±0.72 ab

12.5±0.69 d

5.0

3.80±0.124 a

30.7±1.13 a

29.5±0.93 a

12.1±0.75 d

10.0

3.50±0.165 a

29.8±1.56 a

29.6±1.44 a

12.4±0.87 d

EP

4 5

Table 4. Changes in malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) of Nile tilapia fed diets supplemented by different levels of cinnamon nanoparticles (CNP) for 8 weeks.

Means followed by the same letter are not significantly different at P < 0.05. 27

2 3

7 8 9

10

ACCEPTED MANUSCRIPT

Table 5. Changes in digestive enzymes of Nile tilapia fed different levels cinnamon nanoparticles for 8 weeks. Protease

Lipase

Amylase

(g/kg diet)

(U/L)

(U/L)

(U/L)

0.0

11.2±0.64 d

34.0±1.15 c

136.0±10.97 e

0.25

21.8±0.26 c

38.0±3.46 c

0.5

22.6±0.43 c

41.5±5.48 c

1.0

26.2±0.78 b

61.5±4.33 b

3.0

31.5±0.98 a

68.5±3.17 ab

601.0±13.28 a

5.0

34.1±1.51 a

74.0±2.89 a

639.0±26.76 a

10.0

33.4±1.18 a

RI PT

CNP levels

266.0±14.62 d 404.5±8.94 c

SC

507.5±12.99 b

M AN U 71.5±4.33 a

614.0±25.41 a

Means followed by the same letter are not significantly different at P < 0.05.

13 14 15

TE D

Table 6. Changes in nitrous oxide, nitroblue tetrazolium (NBT), lysozyme, and post-challenge mortality of Nile tilapia fed different levels cinnamon nanoparticles for 8 weeks. Nitrous oxide

NBT

Lysozyme

(g/kg diet)

(Umol/L)

(mg/mL)

(unit/mg protein)

EP

CNP levels

(%)

0.112±0.0064 d

0.119±0.0144 c

3.74±0.15 c

66.7±5.0 a

0.25

0.233±0.0124 c

0.121±0.0041 c

3.79±0.42 c

20.0±5.0 b

AC C

16 17 18

Post-challenge fish mortality

0.0

0.5

0.242±0.0084 bc

0.135±0.0101 bc

4.03±0.32 bc

15.0±5.0 b

1.0

0.252±0.0101 bc

0.160±0.009 b

5.03±0.23 b

0.0±0.0 c

3.0

0.266±0.0072 b

0.189±0.0157 a

6.73±0.59 a

0.0±0.0 c

5.0

0.300±0.0064 a

0.183±0.0076 a

6.25±0.51 a

0.0±0.0 c

10.0

0.307±0.0104 a

0.183±0.0078 a

6.23±0.75 a

0.0±0.0 c

Means followed by the same letter are not significantly different at P < 0.05. 28

11 12

19

ACCEPTED MANUSCRIPT

20 21 22

0.25 g CNP/kg diet

0.5 g CNP/kg diet

1.0 g CNP/kg diet

3.0 g CNP/kg diet

5.0 g CNP/kg diet

RI PT

80

0.0 (Control)

SC

10.0 g CNP/kg diet

M AN U

60 50 40

TE D

Cumulative fish mortality (%)

70

30

EP

20 10

AC C

0

1

2

3

4

5

6

7

8

9

10

Days post-challenge 23

Figure 2. Cumulative mortality rate (%) of in Nile tilapia fed increasing levels of cinnamon nanoparticles for 8 weeks and post-challenged by A. hydrophila infection for 10 days.

29

24 25 26 27