Short term starvation and re-feeding in Nile tilapia (Oreochromis niloticus, Linnaeus 1758): Growth measurements, and immune responses

Aquaculture Reports 16 (2020) 100261

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Short term starvation and re-feeding in Nile tilapia (Oreochromis niloticus, Linnaeus 1758): Growth measurements, and immune responses

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Michael Essien Sakyia,b,c,d,e, Jia Caia,b,c,d,e,*, Jufen Tanga,b,c,d,e,**, Liqun Xiaa,b,c,d,e, Pengfei Lif, Emmanuel Delwin Abarikea,b,c,d,e,g, Felix Kofi Agbeko Kuebutornyea,b,c,d,e, Jichang Jiana,b,c,d,e a

College of Fishery, Guangdong Ocean University, Zhanjiang 524088, PR China Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Animals, Zhanjiang, 524088, PR China c Key Laboratory of Control for Diseases of Aquatic Animals of Guangdong Higher Education Institutes, Zhanjiang 524088, PR China d Shenzhen Research Institute of Guangdong Ocean University, PR China e Laboratory for Marine Biology and Biotechnology Qingdao National Laboratory for Marine Science Technology, Qingdao, PR China f Guangxi Key Lab for Marine Biotechnology, Guangxi Institute of Oceanography, Guangxi Academy of Sciences, Beihai, Guangxi, PR China g Department of Fisheries and Aquatic Resources Management, University for Development Studies, Tamale-Ghana, Ghana b

ARTICLE INFO

ABSTRACT

Keywords: Starvation Haematological indices Immunoglobulin Antioxidants Lysozyme

The effects of short term starvation and re-feeding to satiation on compensatory growth performance, haematological parameters, biochemical parameters, and immune responses were investigated in Oreochromis niloticus of average weights 96.10 ± 0.47 g and 96.44 ± 0.49 g for feeding group and starvation group respectively under the same rearing condition within 21 days period. The fish were divided into two groups namely control group (continuous feeding/ feeding group) and starvation group (days; 0, 3, 7, 10, 14, 21) and re-feeding was done immediately after starvation (days 7, 14, 21). The changes in growth performance, haematological indices, biochemical parameters, and immune responses of the fish were examined (days; 0, 3, 7, 10, 14, 21) and during re-feeding of the fish (days; 7, 14, 21). The results of the growth measurements decreased significantly during starvation and recover significantly after re-feeding (P < 0.05). The re-feeding group indicated a compensatory growth. Haematological indices such as WBCs, Hgb, Hct, and RBCs increased significantly (P < 0.05) after 14 days of starvation while re-feeding group decreases significantly within the successive times (P < 0.05). In the starvation group, blood glucfundose decreases significantly while the re-feeding group increases significantly within successive times (P < 0.05). The levels of Lactate dehydrogenase (LDH) and Pyruvate kinase (PK) increased significantly in the starvation group while the re-feeding group was maintained significantly within successive times. In the serum and liver, the levels of antioxidant enzymes activities in the starvation group were increased significantly while the re-feeding varied significantly within successive times. The immunological responses including lysozyme, immunoglobulin, and antiproteases increased significantly after 21 days of starvation but decrease significantly in the re-feeding group. The protease and lipase in the starvation group decrease significantly during starvation but increase after 21 days of re-feeding. These findings showed that short term starvation had significant effects on growth performance, haematological and biochemical parameters, and immunological parameters in Nile tilapia and recover positively after re-feeding. Taken together, the present findings provide new sight into the beneficial role of short term starvation and re-feeding in fish.

1. Introduction Starvation is a common phenomenon in fish culture because of

weather conditions, specific periods of their reproductive cycles and food restriction (Davis and Gaylord, 2011). However, short term starvation or food deprivation may be part of a strategy of feeding to solve

⁎ Corresponding authors at: Fisheries College, Guangdong Ocean University, No. 1 of Haida Road, Mazhang District, Zhanjiang 524088, Guangdong Province, PR China. ⁎⁎ Corresponding author at: Fisheries College, Guangdong Ocean University, No. 1 of Haida Road, Mazhang District, Zhanjiang 524088, Guangdong Province, PR China. E-mail addresses: [email protected] (J. Cai), [email protected] (J. Tang).

https://doi.org/10.1016/j.aqrep.2019.100261 Received 28 September 2019; Received in revised form 25 November 2019; Accepted 3 December 2019 2352-5134/ © 2019 The Author. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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problems of water quality, decrease harmful effects of stress due to handling (Davis and Gaylord, 2011), mortality reduction related to diseases, or preserve feed in order to the growth of farm profits (Caruso et al., 2011; Gaylord and Gatlin, 2000; Wang et al., 2000). In fish, starvation causes cessation of growth and stress. Starvation causes metabolic stress attributed by metabolic changes for higher energy production and activates the production of acute-phase proteins which, defends the fish from oxidative and cellular damages (Arjona et al., 2009). Starvation induces decrease in antioxidant stores, oxidative stress and an increase in the generation of oxygen free radicals, mainly in the liver (Robinson et al., 1997a). The liver which aids in detoxification and degradation of metabolic products is unceasingly challenged by endogenous and exogenous free radicals (Lee et al., 2015). Studies have shown that the oxidative metabolism of cells is a constant source of reactive oxygen species (ROS), that can damage most cellular components leading to cell death (Morales et al., 2004). The rate of generation of ROS is mainly regulated by antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), etc (Morales et al., 2004) which also play roles in the defense mechanism against the free radicals, hence enhancing the immune condition of the fish (Mohapatra et al., 2017). Recently, emerging evidence has been the focus on investigating the relationship between the fish haematological index and environmental stress. Haematological indices aid can be applied in detecting the structural and functional condition of fish subjected to toxicants (Sharma and Langer, 2014). Also, haematological parameters have been used to assess fish health status and circumstantial stress (He et al., 2015). Variation in white blood cells (WBCs) is considered as an analytical tool and a primary warning sign of the disturbance in homeostatic defence capabilities of fish (Brucka-jastrzebska and Protasowiecki, 2005). The alteration of fish blood constituents could be a sensitive indicator of stress and any physiological dysfunctioning of fish`s body (Sharma et al., 2017). Several research studies have reported on the effects of starvation and subsequent re-feeding which focus on muscle growth, metabolic responses, haematological indices and biochemical parameters such as Grey mullet (Akbary and Jahanbakhshi, 2016) Siberian sturgeon (Jafari et al., 2018) Persian sturgeon (Yarmohammadi et al., 2015) Nile tilapia fries (Moustafa and Abd El-Kader, 2017), Red porgy (Caruso et al., 2012b). Despite the changes in plasma T3 and hepatic type II iodothyronine deidinase (Geyten et al., 1998) and effects of starvation interval and re-feeding on growth and some haematological parameters on Nile tilapia (Moustafa and Abd El-Kader, 2017), the effects of starvation/fasting and re-feeding on Nile tilapia remain unclear. Starvation can improve the health of fish and may not be detrimental to growth (profit). Nile tilapia production is greatly hampered by the occurrence of stressors and diseases. Starvation has been applied as management measure in other fish but none in Nile tilapia as far as our search for literature is a concern. The present study is to assess the metabolic and antioxidant activities, blood indices and related immunological responses in starvation and re-feeding of Nile tilapia. Knowledge of how fish respond to starvation and re-feeding in this current study could provide a basis for improved guidelines in farming, management, metabolism, and physiological response; thereby help to optimize production of Oreochromis niloticus.

1.5 m) 1000 L containing 800 L of freshwater with 30% daily water exchanged. The fish were allowed to acclimatize for two weeks during which time they were fed to satiation twice daily (9 a.m. and 5 pm) with a commercial feed. The tanks were assigned in triplicates for two groups thus control group (continuous feeding/ feeding group) and experimental group (starvation for 21 days and re-feeding for 21 days). After acclimatization, the experiment was carried out in two phases. In phase 1, fish in the control group were continuously fed with commercial feed while the experimental group was starved for 21 days. In phase 2, fish in the experimental group were now fed together with the control group twice daily to satiation at 9 a.m. and 5 pm using the same commercial feed for another 21 days. The photoperiod was set as a 12:12 dark/light cycle. Water quality was maintained daily by renewing 30 % of the water, with a maintenance temperature of 29 ± 1.0 °C, pH of 6.6 ± 0.50, and dissolved oxygen concentration of 6.25 ± 0.75 mg/L (mean ± SEM). 2.2. Determination of growth parameters Growth of control fish and starved/ re-fed fish were measured by weight of the fish using an electronic balance to the nearest 0.1 g at 0, 3, 7, 10, 14, and 21 days in phase 1 and at 7, 14, and 21 days during the re-feeding of experimentation group in phase 2. The fish were weighed in group of seven (7) to reach the number of fish stocked. Also, the total length, (TL) was measured using a ruler. Biometric measurements including weight gain percentage (WG %), weight loss percentage (WL %), condition factor (CF), hepatic somatic index (HSI), and body weight (Q). The abovementioned measurements were calculated as follows: WG% = (final body weight − initial body weight) / initial body weight × 100, HSI = 100 × (liver weight / total body weight), and condition factor CF = Weight of fish (g) / (fish total length)3(cm)3 × 100 and WL % = (final body weight − initial body weight) / initial body weight × 100 (Akbary and Jahanbakhshi, 2016). 2.3. Sample collection At each sampling time, blood samples from ten (10) fish from each group were collected from the caudal vein. The small amount of blood (0.2 ml) was placed in anticoagulated tubes and sent to Guangdong Ocean University hospital (Zhanjiang, Guangdong Province, China) for analysis haematological parameters using haematology analyser machine (mind ray, BC-30 s). The rest of blood samples was placed in 1.5 ml of Eppendorf tubes and allowed to clot at room temperature for two (2) hours, stored overnight at 4 °C and the next day centrifuged at 2000 g for 10 min at room temperature (Sirimanapong et al., 2015). Also, liver tissues from the same fish were aseptically removed and stored 1.5 ml Eppendorf tubes at -80 °C for later use. 2.4. Analysis of haematological parameters The Mind Ray BC-30 s was used to analysed haematological parameters such as red blood cells (RBCs), mean cell volume (MCV), mean cell haemoglobin (MCH), mean corpuscular haemoglobin cell (MCHC), haemoglobin (Hgb), haematocrit (Hct) and white blood cells (WBCs). 2.5. Preparation of tissue samples

2. Materials and methods

The liver samples were weighed and homogenised in ice-cold sucrose solution (0.25 M) using tissue homogeniser to prepare 5 % homogenate. The homogenate was centrifuged at 10, 000 × g for 10 min at 4 °C and the supernatant was collected and stored – 20 °C for antioxidant enzymes (Dar et al., 2019).

2.1. Fish and experimental conditions Four hundred and twenty (420) healthy (without any symptoms such as haemorrhage, ragged fins, abdominal distension) monosex (i.e. all males) Nile tilapia of average initial weight of range 96.30 ± 0.58 g and 96.93 ± 0.38 g were obtained from Langye Animal Husbandry Company Limited, Gaozhou City, Guangdong Province, China. 70 fish were stocked in concrete tanks (each of dimensions 5 m by 2 m by

2.6. Analysis of antioxidant activities (SOD, CAT and T-AOC) The SOD, CAT and T-AOC, activity of serum and liver samples were 2

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determined using commercial test kits provided by Nanjing Jiancheng Bioengineering Institute, Jiangsu, China. The T-AOC activity of serum and liver samples were expressed as units per milliliter (U/mL) and units per milligram (U/mg) respectively. The CAT activity of serum and liver samples were expressed as units per milliliter (U/mL) and units per milligram protein (U/mgprot) respectively. The SOD activity of serum and liver samples were expressed as units per milliliter (U/mL) and units per milligram protein (U/mgprot) respectively.

ammonium bicarbonate buffer) were incubated with 125 μl of 100 mM ammonium bicarbonate buffer containing 2% azocasein (Sigma) for 24 h at 30 °C. 10% trichloroacetic acid (TCA) was added to stop the reaction and centrifuged (6,000 g, 10 min). The supernatants were pipetted to a 96-well plate in triplicate containing 100 μl well-1 of 1 N NaOH, and the OD read at 450 nm using a plate reader ((Enspire plate reader). With the positive control, serum was replaced by trypsin (5 mg/ml, Sigma), as 100% of protease activity while negative controls indicate 0% activity.

2.7. Analysis of metabolic activities The LDH and PK activities of serum were assessed using commercial test kits provided by Nanjing Jiancheng Bioengineering Institute, Jiangsu, China. The LDH activity is expressed as units per litre (U/L). The PK activity is expressed as units per litre (U/L). With glucose analysis, fresh blood samples collected from the fish by dipping a drop of fresh blood on the blood glucose test strip and then inserted in the glucose meter. Blood samples were collected from the caudal vein using the sterilized syringe. The fresh blood samples were analysed with a glucose meter (Sinocare Inc, Cofoe).

2.9.3. Analysis of serum lipase activity Following the colorimetric method, the serum lipase activity was quantified to assess the degradation of triglycerides to free fatty acids using a commercial test kit provided by Nanjing Institute of Bioengineering, Jiangsu, China. One unit of the serum lipase is defined by each litre of serum reacted with the substrate in the reaction system for one minute, each consumption of 1 μmol under 37 °C and the substrate is an enzyme activity unit. The lipase activity was expressed in unit per litre (U/L) and measured at 420 nm using a microplate reader (Enspire plate reader).

2.8. Determination of immune parameters

2.10. Statistical analysis

2.8.1. Analysis of serum lysozyme activity Following the turbid metric method, serum lysozyme (LYZM) activity was determined as described earlier (Ellis, 1990). Briefly, 50 μl of serum was added to 500 μl of Micrococcus lysodeikticus suspension (Sigma-Aldrich, St. Louis, MO, USA) (0.243 mg/ ml) in a 0.05 M PBS (pH 6.4). The optical density (OD) was recorded at 530 nm at 1 min and 20 min at 22 °C (using plate reader, Enspire plate reader) with one unit of lysozyme activity being defined as the amount of serum that caused a decrease in the optical density of 0.001 units/ min. The activity was expressed as units per litre (U/L).

One-way analysis of variance (ANOVA) was used to determine variations within each group at 95% confidence level and paired t-test was used to determine variation at 95% confidence level between the feeding and starvation groups and that of feeding group and re-feeding group within each successive times. Duncan's multiple-range post-hoc test was used to assess statistically significant (P < 0.05) differences within the successive times in each group. Data were expressed as mean ± SEM (standard error of the mean) of the mean.

2.8.2. Immunoglobulin m (IgM) determination The serum IgM was determined using commercial test kits provided by Nanjing Jiancheng Bioengineering Institute, Jiangsu, China. The IgM was expressed in gram per litre (g/L) and measured at 340 nm using a microplate reader (Enspire plate reader).

3.1. Growth measurements

3. Results

No mortality occurred either in the feeding group or starvation/refeeding group. At phase 1, the starvation group, decreased in body weight within successive times while the feeding group increased within successive times. There were significant differences between the feeding group and starvation group within successive times (P < 0.05) as shown in Table 1a. At phase 2, after starvation, the re-feeding group recorded an increasing trend and a significant between the feeding group and re-feeding group within successive times (P < 0.05) as illustrated in Table 1b. After 21 days of re-feeding, the re-feeding group did not achieve the same value of 42 days of feeding. The feeding group and starvation group recorded a weight gain of +22.85% and -33.35% from day 0 – day 21 respectively. The re-feeding group % and feeding group recorded weight of 43.39% and 41.38% from day 21 – day 42 respectively. At phase 2, the starvation group recorded a declining trend in HSI within successive times while the feeding group recorded an increasing trend within successive times. Day 21 and 0 recorded the highest value in a feeding group and starvation group respectively. There were significant differences between the feeding group and starvation group within successive times (P < 0.05) as shown in Table 1a. At phase 2, the re-feeding group recorded an increasing trend within successive times while a significant difference was recorded between the starvation group and re-feeding group after 21 days (P < 0.05) (Table 1b). Within the successive times, CF recorded an increasing trend and deceasing trend in the feeding group and re-feeding group respectively. Day 14 and 0 recorded the highest value in the feeding group and starvation group respectively. There were significant differences between the feeding group and starvation group within successive times (days: 10, 14 and 21) (P > 0.05) as shown in Table 1a. The re-feeding group recorded an increasing trend within successive times while

2.9. Digestive enzymes determination 2.9.1. Analysis of serum antiprotease activity Serum antiprotease activity was determined to have a slightly modified method described by Alexander et al. (2010) as cited in Abarike et al. (2019). 10 μl of serum was incubated with 10 μl of trypsin solution (0.1% trypsin porcine pancreas of a final concentration of 50 μg/ml, in 0.01 M Tris HCl, pH 8.2) for 10 min. After incubation, 500 μl of BAPNA (sodium-benzoyl-DL-arginine-p-nitroanilide HCl, Sigma) substrate was added into the reaction, and the volume made up to 1 ml with 0.1 M Tris HCl pH 8.2 and incubated at 22 °C for 25 min. Subsequently, 250 μl of 30% acetic acid (AA) was added to halt the reaction, and optical density (OD) was read in a plate reader (Enspire plate reader) alongside a blank (trypsin solution without serum) set at 450 nm. The inhibitory activity of antiprotease was expressed as the percentage of trypsin inhibition using the following formula OD; Percent trypsin inhibition =

( trypsin blank OD sample OD) × 100 trypsin blank OD 2.9.2. Analysis of serum protease activity Serum protease activity was measured using the azocasein hydrolysis assay according to the method described by (Guardiola et al., 2016). Aliquots of 100 μl of serum (previously diluted 1/10 in 100 mM 3

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decreasing trend (91.52 ± 0.61, 10^9/L - 73.54 ± 0.41, 10^9/L) and increasing trend (91.20 ± 0.72, 10^9/L - 86.52 ± 0.54, 10^9/L) respectively within successive times (0 day – 21 days). There was a significant difference between feeding group and starvation group on day 3 – day 21 (P < 0.05) (Table 2a). At phase 2, the re-feeding group and feeding group recorded a decreasing trend (86.52 ± 0.54, 10^9/L 76.30 ± 0.59, 10^9/L) and (73.54 ± 0.41, 10^9/L - 67.10 ± 2.03, 10^9/L) within successive times (21 days– 42 days). Significant differences were recorded between the feeding group and re-feeding group within successive times (P < 0.05) (Table 2b). After 21 days of refeeding, the re-feeding group did not achieve the same value of 42 days of feeding. At phase 1, the feeding group and starvation group of RBCs level recorded (1.57 ± 0.04, 10^12/L - 1.19 ± 0.02, 10^12/L) and (1.58 ± 0.02, 10^12/L - 1.71 ± 0.03, 10^12/L) within successive times respectively; significant differences between the feeding group and the starvation groups on day 7 - day 21 (P < 0.05). Decreasing and increasing trends were recorded in the feeding and starvation groups respectively. However, the starvation group depicted increasing trend after 14 days and decreased on day 21 (Table 2a). In phase 2, the refeeding group and feeding group recorded (1.71 ± 0.03, 10^12/L 1.25 ± 0.04, 10^12/L) and (1.19 ± 0.04, 10^12/L - 0.97 ± 0.03, 10^12/ L) within the successive times respectively. There were significant changes between the feeding group and re-feeding group on day 21 – day 42 (P < 0.05). The re-feeding group recorded a decreasing trend (Table 2b). After 21 days of feeding, the re-feeding group achieves lower value as compared to the 42 days of feeding. At phase 1, Hgb level ranged from (92.80 ± 0.58, g/L 65.80 ± 0.49, g/L) in the feeding group while the starvation group ranged from (93.80 ± 0.97, g/L - 86.60 ± 0.93 g/L). The starvation group recorded an increased after 14 days and decreased on day 21 while the feeding group recorded decreased trend within successive times. There was a significant difference between the feeding group and the starvation group on day 3 – day 21 (P < 0.05) (Table 2a). At phase 2, re-feeding group recorded a decreasing trend (86.60 ± 0.93 g/L – 70.40 ± 1.99 g/L) within successive times. The feeding group recorded a decreasing trend (65.80 ± 0.4, g/L – 60.00 ± 2.59, g/L). Significant differences were recorded between the feeding group and re-feeding group within successive times (day 21 – day 35) (P < 0.05) (Table 2b).

Table 1a Growth measurements of feeding group and starvation group. Phase 1 (0 day - 21 days) Bodyweight, g Time(Days) 0 3 7 10 14 21

Feeding group 96.10 (0.47)a 99.16 (0.41)ab 102.78 (1.47)bc 105.68 (1.40)cd 108 .78 (0.83)d 118.06 (0.64)e

Starvation group 96.14 (0.49)a 92.90 (0.74)e 88.56 (1.36)d 81.62 (0.80)b 77.52 (0.60)c 72.32 (0.77)e

Feeding vs. Starvation ns s s s s s

HSI, % Time(Days) 0 3 7 10 14 21

Feeding group 2.38 (0.05)a 2.39 (0.09)a 2.41 (0.02)ab 2.37 (0.05)a 2.34 (0.02)a 2.55 (0.09)b

Starvation group 2.35 (0.06)a 2.12 (0.02)c 2.05 (0.06)c 1.95 (0.02)c 1.46 (0.10)b 1.10 (0.06)d

Feeding vs. Starvation ns s s s s s

CF, K Time(Days) 0 3 7 10 14 21

Feeding group 1.59 (0.09)a 1.60 (0.19)a 1.67 (0.10)a 1.62 (0.10)a 1.61 (0.13)a 1.74 (0.07)a

Starvation group 1.57 (0.06)a 1.54 (0.08)bc 1.49 (0.12)bc 1.33 (0.05)ab 1.21 (0.06)a 1.14 (0.04)d

Feeding vs. Starvation ns ns ns s s s

Mean ± SEM values of the growth measurement calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d) in the same column indicate significant variations between successive times (n = 5).

significant differences were recorded between the starvation group and re-feeding group within the successive times (P > 0.05) as shown in Table 1b. 3.2. Haematological parameters At phase 1, the feeding group and starvation group of WBCs indicate Table 1b Growth measurements of feeding and re-feeding groups.

Phase 2 (21 days - 42 days) Time(Days)

Bodyweight, g Feeding group

0/21 7/28 14/35 21/42

118.06 123.98 130.84 135.86

HSI, % Time(Days) 0/21 7/28 14/35 21/42 CF, K Time(Days) 0/21 7/28 14/35 21/42

a

(0.64) (1.00)b (0.89)c (1.15)d

Re-feeding group a

Feeding vs. Re-feeding

72.32 (0.77) 85.74 (0.88)b 90.34 (0.44)c 103.70(2.09)c

s s s s

Feeding group 2.55 (0.09)a 2.91 (0.04)b 3.37 (0.04)c 3.61 (0.11)d

Re-feeding group 1.10 (0.06)a 2.05 (0.05)b 2.47 (0.03)c 3.05 (0.03)d

Feeding vs. Re-feeding s s s s

Feeding group 1.74 (0.07)a 1.80 (0.09)ab 1.91 (0.07)ab 2.02 (0.08)b

Re-feeding group 1.14 (0.04)a 1.20 (0.04)a 1.37 (0.06)b 1.44 (0.06)b

Feeding vs. Re-feeding s s s s

Mean ± SEM values of the growth measurement calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c) in the same column indicate significant variations between successive times (n = 5). 4

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Table 2a Haematological parameters of feeding group and starvation group. Phase 1 (0 day - 21 days) ^ 9/

Time(Days)

WBCs, 10 L Feeding group

0 3 7 10 14 21

91.52 85.44 84.76 81.44 79.74 73.54

RBCs, 10 ^ 12/L Time(Days) 0 3 7 10 14 21

Starvation group

Feeding vs. Starvation

91.20(0.72)a 95.30(1.18)c 96.78(0.53)cd 98.98(0.95)d 104.4(1.10)e 86.52(0.54)b

ns s s s s s

Feeding group 1.57 (0.04)a 1.49 (0.06)cd 1.37 (0.01)bc 1.32 (0.05)b 1.27 (0.02)ab 1.19 (0.04)d

Starvation group 1.58 (0.02)a 1.62 (0.03)a 1.71 (0.03)b 1.82 (0.03)c 2.00 (0.03)d 1.71 (0.03)b

Feeding vs. Starvation ns ns s s s s

HGB, g/L Time(Days) 0 3 7 10 14 21

Feeding group 92.80 (0.58)a 85.40 (0.25)e 81.80 (0.58)d 76.40 (0.93)c 72.60 (0.51)b 65.80 (0.49)f

Starvation group 93.80 (0.97)a 94.20 (0.37)b 96.60 (0.81)c 97.80 (0.49)c 100.20(0.37)d 86.60 (0.93)b

Feeding vs. Starvation ns s s s s s

HCT, % Time(Days) 0 3 7 10 14 21

Feeding group 27.94 (0.58)a 25.80 (0.68)cd 23.92 (0.54)bc 22.82 (0.72)b 22.14 (1.02)b 19.50 (0.76)d

Starvation group 26.20 (0.46)a 27.16 (0.29)a 28.78 (0.52)b 28.80 (0.59)b 30.12 (0.69)b 27.02 (0.50)a

Feeding vs. Starvation ns ns s ns s s

(0.61)a (0.39)c (0.91)c (0.30)b (1.74)b (0.41)d

Mean ± SEM values of the WBCs, RBCs, Hct, and Hgb calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d) in the same column indicate significant variations between successive times (n = 5).

feeding group achieve did not the same value as compared to the 42 days of feeding.

At the phase 1, the feeding group of the Hct level ranged from 27.94 ± 0.58, % - 19.50 ± 0.76,% while in the starvation group ranged from 26.20 ± 0.46,% - 27.02 ± 0.50, %). Also, the starvation group recorded an increasing trend (day 3 – day 14) and decrease on day 21. There were significant differences between the feeding group and starvation group on day 7, 14 and 21 (P < 0.05) (Table 2a). At phase 2, the re-feeding group and the feeding group recorded a decreasing trend (27.02 ± 0.50, % – 22.36 ± 0.30, %) and (19.50 ± 0.76, % – 15.18 ± 0.84, %) within successive times respectively. Significant differences were recorded between the feeding group and re-feeding group (P < 0.05) (Table 2b).

3.3.2. Pyruvate kinase (PK) and lactate dehydrogenase (LDH) At phase 1, in the feeding group, PK activity indicates maintenance level and recorded values (9.60 ± 1.09 U/L - 10.60 ± 0.15 U/L) (P < 0.05) with in successive times. A significant increment in the starvation group shown (P < 0.05) after 21 days and recorded values (9.46 ± 0.07 U/L - 23.61 ± 0.54 U/L) (Table 3a). The significant differences were recorded between feeding group and starvation group from day 7 – day 21 (P < 0.05). At phase 2, there was significant decrement in the re-feeding group (P < 0.05) and recorded values (23.61 ± 0.54 U/L - 12.75 ± 0.44) (Table 3b). The feeding group maintained its level and recorded value (10.61 ± 0.15 U/L – 10.82 ± 0.58) within successive times. Significant differences were recorded between feeding group and re-feeding group within successive times (day 21 – day 42) (P < 0.05). After 21 days of re-feeding, the refeeding group achieve did not the same value as compared to the 42 days of feeding. At the phase 1, the starvation group of the LDH activity increased significantly after 14 days and decreased on 21 days (P < 0.05) and recorded values (427.85 ± 0.74 U/L - 615.91 ± 0.94 U/L). There was a significant decreased in the feeding group (P < 0.05) and recorded values (426.82 ± 0.66 U/L - 107 ± 1.37 U/L) (Table 3a). Significant differences were recorded between the feeding group and starvation group from day 3 – day 21. At phase 2, the significant difference were recorded between the re-feeding group and feeding group within

3.3. Biochemical parameters 3.3.1. Blood glucose At phase 1, the blood glucose of feeding group and starvation group recorded increasing and decreasing trend (8.52 ± 0.29 mmol/L – 10.00 ± 0.25 mmol/L) and (8.34 ± 0.24 mmol/L – 6.02 ± 0.38 mmol/L) within successive times respectively. Significant differences were recorded between feeding group and starvation group within successive times (day 14–day 21) (P < 0.05) (Fig. 1a). At phase 2, the re-feeding group depicted an increasing trend from day 21 - day 35 and decrease on day 42 within successive times (6.02 ± 0.38 mmol/L – 6.60 ± 0.42 mmol/L) while the feeding group indicated a decreasing trend (10.00 ± 0.38 mmol/L – 4.08 ± 0.21 mmol/L) within successive times. Significant difference recorded (P > 0.05) (Fig. 1b). After 21 days of re-feeding, the re5

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Table 2b Haematological parameters of the feeding group and re-feeding group. Phase 2 (21 Days - 42 Days) ^ 9

Time(Days)

WBCs, 10 /L Feeding group

0/21 7/28 14/35 21/42

73.54 72.84 68.00 67.10

RBCs, 10 ^ 12/L Time(Days) 0/21 7/28 14/35 21/42

Re-feeding group

Feeding vs. Re-feeding

86.52(0.54)b 84.06(0.38)b 80.58(0.64)b 76.30(0.59)a

s s s s

Feeding group 1.19 (0.04)b 1.10 (0.03)b 1.09 (0.02)b 0.97 (0.03)a

Re-feeding group 1.71 (0.03)c 1.42 (0.07)b 1.40 (0.06)ab 1.25 (0.04)a

Feeding vs. Re-feeding s s s s

HGB, g/L Time(Days) 0/21 7/28 14/35 21/42

Feeding group 65.80 (0.49)a 64.20 (2.20)a 61.60 (2.58)a 60.00 (2.59)a

Re-feeding group 86.60(0.93)b 75.20(1.46)a 73.60(1.83)a 70.40(1.99)b

Feeding vs. Re-feeding s s s ns

HCT, % Time(Days) 0/21 7/28 14/35 21/42

Feeding group 19.50 (0.76)b 17.80 (1.00)ab 16.48 (1.27)ab 15.18 (0.84)a

Re-feeding group 27.02(0.50)c 25.24(0.60)b 23.86(0.42)b 22.36(0.30)a

Feeding vs. Re-feeding s s s s

(0.41)d (1.63)c (1.90)b (2.03)a

Mean ± SEM values of the WBCs, RBCs, Hct and Hgb, calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” shows no significant differences between the two groups within each successive time. The different letters (a, b, c, d) in the same column indicate significant variations between successive times (n = 5).

Fig. 1. (a) Mean ± SEM values of the blood glucose calculated in the two groups. The “*” indicates significant differences (P < 0.05) whiles “**” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d.e) in the same column indicate significant variations between successive times (n = 5). (b) Mean ± SEM values of the blood glucose calculated in the two groups. The “*” indicates significant differences (P < 0.05) whiles “**” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d) in the same column indicate significant variations between successive times (n = 5).

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Table 3a LDH and PK activities on feeding group and starvation group. Phase 1 (0day – 21 days) Time(Days)

Serum PK, U/L Feeding group

Starvation group

Feeding vs. Starvation

0 3 7 10 14 21

9.60 (0.05)a 10.51 (0.21)b 10.54 (0.11)b 10.78 (0.10)b 10.38 (0.17c 10.61 (0.15)b

9.46 (0.07)a 11.17(0.09)b 15.56(0.10)c 21.83(0.16))d 23.81(0.20)e 23.61(0.54)e

ns ns s s s s

Serum LDH, U/L Time(Days) 0 3 7 10 14 21

Feeding group 426.82 (0.66)a 427.90 (0.86)d 426.54 (0.66)d 396.01 (0.56)c 188.78 (0.70)b 107.98 (1.37)d

Starvation group 427.85(0.74)a 483.05(0.54)b 596.24(0.56)c 607.79(0.51)d 626.92(1.34)e 615.71(0.94)f

Feeding vs. Starvation ns s s s s s

Mean ± SEM values of the PK and LDH calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d, e, f) in the same column indicate significant variations between successive times (n = 5).

successive times (day 21 – day 42) (P < 0.05). The feeding group and re-feeding group recoded increasing and decreasing trend with values (107.98 ± 1.37 U/L – 100.80 ± 0.51 U/L) and (615.71 ± 0.94 U/L – 280.26 ± 7.88 U/L) (Table 3b). After 21 days of re-feeding, the refeeding group achieve did not the same value as compared to the 42 days of feeding

(12.75 ± 0.27 U/mL – 10.54 ± 0.28 U/mL) within successive times. The feeding group were maintained its level and recorded value (9.73 ± 0.38 U/mL – 9.33 ± 0.31U/mL) within successive times. Significant differences were recorded between the feeding group and re-feeding group within the successive times (day 21and day 35) (P < 0.05) (Table 4b). After 21 days of re-feeding, the re-feeding group did not achieve the same value as compared to the 42 days of feeding. At phase 1, the CAT level recorded decreasing trend and recorded values within the successive times (17.36 ± 0.27 U/mL – 8.69 ± 0.31 U/mL) in the feeding group while the starvation group recorded an increasing trend (16.16 ± 0.87 U/mL - 28.15 ± 0.34 U/mL) within the successive times (Table 4a). Significant differences were recorded between feeding group and starvation group within successive times (day 3 – day 21) (P < 0.05). At phase 2, the re-feeding group and feeding group recorded decreasing trend and maintained its level with recorded values (28.15 ± 0.34 U/mL – 14.18 ± 0.45 U/mL) and (8.69 ± 0.31, U/mL – 8.57 ± 0.15, U/mL) within the successive times respectively. Significant differences were recorded between the feeding group and re-feeding group within successive times (day 21 –day 42) (Table 4b). After 21 days of re-feeding, the re-feeding group did not achieve the same value as compared to the 42 days of feeding. At phase 1, the T-AOC levels recorded a variation trend and

3.3.3. Antioxidant activity both in serum and liver samples In order to examine the effect of feeding, starvation, and re-feeding on the antioxidant defenses of O. niloticus, we examine the activity of antioxidant enzymes such as CAT, T-AOC, and SOD in the serum and liver samples. 3.3.4. Antioxidant activity in the serum samples At phase 1, the SOD levels recorded decreasing trend and recorded values (10.46 ± 0.40, U/mL - 9.73 ± 0.28, U/mL) within successive times in the feeding group while the starvation group recorded an increasing trend and recorded values (9.96 ± 0.17, U/mL – 12.74 ± 0.27, U/mL) within successive times. Significant differences were recorded between the feeding group and starvation group within successive times (day 3, day 10 – day 21) (Table 4a). At phase 2, the refeeding group recorded a decreasing trend and recorded values Table 3b LDH and PK activities on the feeding group and re-feeding group.

Phase 2 (21 days – 42 days) Time(Days)

Serum PK, U/L Feeding group

0/21 7/28 14/35 21/42

10.61 10.68 11.35 10.82

(0.15)a (0.37)a (0.43)a (0.58)a

Serum LDH, U/L Time(Days) 0/21 7/28 14/35 21/42

Feeding group 107.98 (1.37)b 108.41 (0.66)b 102.23 (0.45)a 100.80 (0.51)a

Re-feeding group 23.61 20.47 17.17 12.75

d

(0.54) (0.10)c (0.85)b (0.44)a

Re-feeding group 615.71(0.94)d 345.06(7.08)c 317.06(3.54)b 280.26(7.88)a

Feeding vs. Re-feeding s s s s Feeding vs. Re-feeding s s s s

Mean ± SEM values of the PK and LDH calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c,) in the same column indicate significant variations between successive times (n = 5). 7

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Table 4a SOD, CAT and T-AOC activities on feeding group and starvation group in serum samples. Phase 1 (0 day - 21 days) Time(Days)

Serum SOD, U/mL Feeding group

Starvation group

Feeding vs. Starvation

0 3 7 10 14 21

10.46 (0.40)ab 10.32 (0.22)ab 11.39 (0.39)b 10.47 (0.88)ab 10.73 (0.46)ab 9.73 (0.28)a

9.96 (0.17)a 11.71(0.27)b 13.86(0.34)c 17.86(0.43)d 19.58(0.47)e 12.74(0.27)c

ns s ns s s s

Serum CAT, U/mL Time(Days) 0 3 7 10 14 21

Feeding group 17.36 (0.27)a 16.08 (0.79)d 12.38 (0.69)c 10.43 (0.70)b 9.08 (0.26)ab 8.28 (0.31)d

Starvation group 17.36(0.27)a 24.02(0.44)b 25.49(0.83)b 29.05(0.42)c 31.68(0.46)d 28.15(0.34)c

Feeding vs. Starvation ns s s s s s

Serum T-AOC, U/mL Time(Days) 0 3 7 10 14 21

Feeding group 1.12 (0.08)a 3.74 (0.18)c 3.33 (0.17)b 3.82 (0.06)c 3.38 (0.04)b 3.17 (0.09)b

Starvation group 1.03 (0.06)a 3.35 (0.12)b 5.13 (0.04)c 5.82(0.06)d 6.37 (0.04)e 5.73 (0.06)d

Feeding vs. Starvation ns s s s s s

Mean ± SEM values of the SOD, CAT and T-AOC calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d, e) in the same column indicate significant variations between successive times (n = 5). Table 4b SOD, CAT and T-AOC activities on the feeding group and re-feeding group in serum samples. Phase 2 (21 days - 42 days) Time(Days)

Serum SOD, U/mL Feeding group a

Re-feeding group c

Feeding vs. Re-feeding

0/21 7/28 14/35 21/42

9.73 (0.28) 10.33 (0.31)a 10.11 (0.50)a 9.33 (0.31)a

12.75 (0.27) 11.81(0.44)bc 11.12(0.38)ab 10.54(0.28)a

s ns s ns

Serum CAT, U/mL Time(Days) 0/21 7/28 14/35 21/42

Feeding group 8.69 (0.31)a 8.49 (0.34)a 8.45 (0.38)a 8.57 (0.15)a

Re-feeding group 28.15 (0.34)d 23.70 (0.31)c 19.80 (0.33)b 14.18 (0.45)a

Feeding vs. Re-feeding s s s s

Serum T-AOC, U/mL Time(Days) 0/21 7/28 14/35 21/42

Feeding group 3.17 (0.09)b 2.96 (0.08)ab 2.80 (0.06)a 2.76 (0.07)a

Re-feeding group 5.73 (0.06)c 2.86 (0.03)b 2.75 (0.03)b 2.39 (0.15)a

Feeding vs. Re-feeding s ns ns ns

Mean ± SEM values of the SOD, CAT, and T-AOC in serum samples calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c) in the same column indicate significant variations between successive times (n = 5).

recorded values (1.12 ± 0.08 U/mL–3.17 ± 0.09 U/mL) within successive times in the feeding group while the starvation group recorded an increasing trend and recorded values (1.03 ± 0.06 U/ mL–5.73 ± 0.06 U/mL) within the successive times. Significant differences were recorded between the feeding group and starvation group within successive times (day 3–day 21) (P < 0.05) as illustrated in Table 4a. At phase 2, the re-feeding group and feeding group recorded decreasing trend and recorded value (5.73 ± 0.06 U/mL -

2.39 ± 0.15 U/mL) and (3.17 ± 0.09 U/mL - 2.76 ± 0.07 U/mL) within the successive times respectively. No significant differences were indicated between the feeding group and re-feeding group within successive times (day 28 – day 42) (P < 0.05) (Table 4b). After 21 days of re-feeding, the re-feeding group achieve the same value as compared to the 42 days of feeding

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Fig. 2. (a) Mean ± SEM values of the serum lysozyme activity calculated in the two groups. The “s” indicates significant differences (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d, e) in the same column indicate significant variations between successive times (n = 5). (b) Mean ± SEM values of the serum lysozyme activity calculated in the two groups. The “*” indicates significant differences (P < 0.05) whiles “**” show no significant differences (P > 0.05) between two groups within each successive times. The different letters (a, b, c) in the same column indicate significant variations between successive times (n = 5).

3.4. Determination of immune parameters

Table 5a IgM activities in the feeding group and starvation group.

3.4.1. Serum lysozyme activity At phase 1, the serum lysozyme activity measured in the feeding group and starvation group was shown in Fig. 2a. The lysozyme activity in the serum of the feeding group was lower than in the starvation group. The concentration ranged from 11.82 ± 0.09 U/L – 15.69 ± 0.14 U/L in the feeding group while the starvation group ranged from 11.60 ± 0.16 U/L - 23.80 ± 0.71 U/L. The starvation group increased significantly while the feeding group varied significantly (P < 0.05). At phase 2, after re-feeding, decreased significantly in the re-feeding group and recorded values were 23.80 ± 0.71 U/L - 18.12 ± 0.14 U/L. The feeding group maintained it level significantly (P < 0.05). The re-feeding group did not achieve the same value as compared to the 42 days of feeding (Fig. 2b).

Time 0 3 7 10 14 21

IgM, g/L Starvation

Feeding a

0.0089(0.00045) 0.0316(0.00308)c 0.0121(0.00015)ab 0.0165(0.00169)ab 0.0187(0.00144)b 0.0093(0.00234)a

0.0084 0.0092 0.0107 0.0114 0.0143 0.0065

Feeding vs. Starvation ab

(0.00039) (0.00015)ab (0.00020)b (0.00032)b (0.0016)c (0.00036)a

ns s s s ns ns

Mean ± SEM values of the IgM calculated in the two groups. The “s” indicates significant (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups. The different letters (a, b, c) in the same column indicate significant variations between successive times (n = 5). Table 5b IgM activities in the feeding group and re-feeding group.

3.4.2. Serum immunoglobulin (IgM) At phase 1, the starvation group recorded an increasing trend after 14 days (0.0084 ± 0.00039 g/L – 0.0065 ± 0.00036, g/L) and increased significantly (P < 0.05) while the feeding group recorded a variation and a significant differences within successive times (P < 0.05) (Table 5a). Day 14 and day 3 recorded the highest value in starvation and feeding groups respectively. At phase 2, after re-feeding, the re-feeding group was maintained within successive times (0.0065 ± 0.00036, g/L – 0.0188 ± 0.00036, g/L) and no significant differences recorded (P > 0.05) as illustrated in Table 5b. The refeeding group did not achieve the same value as compared to the 42 days of feeding.

Time 0/21 7/28 14/35 21/42

IgM, g/L Re-feeding

Feeding a

0.0093(0.00234) 0.0545(0.03686)a 0.0176(0.00039)a 0.0316(0.00308)a

Feeding vs. Re-feeding a

0.0065 (0.00036) 0.01824 (0.00037)a 0.01878 (0.00045)a 0.0188 (0.00036)a

s ns ns s

Mean ± SEM values of the IgM calculated in the two groups. The “s” indicates significant (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups. The different letters (a, b, c) in the same column indicate significant variations between successive times (n = 5).

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Table 6a Antiprotease and protease activities in the feeding group and starvation group. Time 0 3 7 10 14 21

Protease, % Starvation

Feeding a

12.83(0.28) 19.71 0.31)b 21.93 0.38)c 22.63(0.39)d 28.90(0.37)e 34.42(0.22)f

Antiprotease, % Time Feeding 0 13.03(0.41)a 3 30.44(0.39)d 7 37.80(0.37)e 10 26.34(0.51)c 14 23.34(0.26)c 21 16.78(0.31)b

Table 6b Antiprotease and protease activities in the feeding group and re-feeding group.

Feeding vs. Starvation a

12.30 (0.20) 11.89 (0.20)d 10.25 (0.21)bc 9.88 (0.29)c 9.00 (0.26)b 4.68 (0.31)a

ns s s s s s

Starvation 12.43 (0.28)a 19.84 (0.36)b 36.35 (0.37)c 37.82 (0.36)c 50.9 (0.40)d 55.34 (0.31)e

Feeding vs. Starvation ns s s s s s

Time

Protease, % Re-feeding

Feeding a

a

Feeding vs. Re-feeding

0/21 7/28 14/35 21/42

34.42(0.22) 35.86(0.48)ab 37.64(0.81)b 40.02(0.59)c

4.68 (0.31) 10.76 (0.31)b 15.57 (0.39)c 18.53 (0.37)d

s s s s

Time 0/21 7/28 14/35 21/42

Antiprotease, % Feeding 16.78 0.31)c 14.75 0.51)b 13.92(0.37)b 11.90(0.37)a

Re-feeding 55.34 (0.31)d 36.71 (0.36)c 33.93 (0.40)b 26.24 (0.50)a

Feeding vs. Re-feeding s s s s

Mean ± SEM values of the antiprotease and protease activities calculated in the two groups. The “s” indicates significant (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups. The different letters (a, b, c, d) in the same column indicate significant variations between successive times (n = 5).

Mean ± SEM values of the antiprotease and protease activities calculated in the two groups. The “s” indicates significant (P < 0.05) whiles “ns” show no significant differences (P > 0.05) between two groups. The different letters (a, b, c, d, e) in the same column indicate significant variations between successive times (n = 5).

4. Discussion Starvation and re-feeding are identified to have robust effects on the physiology and muscle growth of fish (Navarro and Gutiérrez, 1995). In this study, we examined the effects of starvation and compensatory growth by re-feeding on growth indices, haematology, biochemical parameters, antioxidant enzymes, immune responses and digestive enzymes of Nile tilapia. However, starvation decrease tissue metabolic capacities but on the other hand, the food deprivation causes degradation of endogenous sources of energy (lipids, glycogen, and proteins) in order to maintain the fish physiological homeostasis, leading to weight loss (Zheng et al., 2015). Compensatory growth is the phase of rapid growth, greater than normal or control growth, which occurs upon adequate re-feeding following a period of malnutrition (Laizcarrión et al., 2012). In this study, the re-feeding group indicated growth compensation. After re-feeding for 21 days, the increase in body weight showed regain of their initial weight and growth indices. Similarly, the findings were observed in Persian sturgeon (Acipenser persicus) (Yarmohammadi et al., 2015, 2012) barramundi (Lates calcalifer) (Tian and Qin, 2003) and in hybrid tilapia, (O. mossambicus and O. niloticus) (Wang et al., 2000) and Atlantic cod (Gadus morhua) (Jobling et al., 1994) indicating compensatory growth after re-feeding. These suggest that most fish species have compensatory growth when normal feeding is applied after starvation. The simple measure of the level of energy reserves is known as condition factor (CF) (Goede and Barton, 1990). CF is also a quantitative parameter of the state of well-being of the fish because of its influence on growth, reproduction, and survival (Richter, 2007). Variations of fish health and its values may be indicated by the changes in the nutritional status of the fish (Caruso et al., 2012a). CF increased in values in the feeding group and the re-feeding group increased after starvation but decreased in the starvation group. However, the findings of the current study were similar to the finding on red porgy (Caruso et al., 2010) and grey mullet (Akbary and Jahanbakhshi, 2016). Low values of HSI may be attributed to the nutritional problem because of the relative size of the fish liver which correlated with nutritional status (Caruso et al., 2012a). In the present, a significant decrease of HSI was observed in the starvation group suggesting the importance of the liver reserves of lipids during short fasting periods in Nile tilapia. Similar results (Akbary and Jahanbakhshi, 2016) on grey mullet, (Olivereau and Olivereau, 1997) on Anguilla anguilla, (Vosylienė and Kazlauskienė, 1999) on Oncorhynchus mykiss and (Falahatkar, 2012) on Huso huso have been reported. Furthermore, a significant decrease in HSI in the starvation group may relate to restriction in

3.5. Digestive enzymes determination 3.5.1. Protease and antiprotease results on feeding group, starvation group, and re-feeding group At phase 1, the protease activity was measured in the feeding group and starvation group shown in Table 6a. In the starvation group, the protease activity level was lower than the feeding group. This indicates a decreasing trend (12.30 ± 0.20, % - 4.68 ± 0.31 %) in the starvation group while the feeding group showed an increasing trend (12.83 ± 0.28, % - 34.42 ± 0.22, %) within the successive times. At phase 2, the re-feeding group showed an increasing trend (4.68 ± 0.31 % - 18.53 ± 0.37 %) with a significant difference (P < 0.05) within the successive times as shown in Table 6b. Also, between the feeding group and re-feeding group shown significant differences within the successive times (P < 0.05). After 21 days of feeding, the re-feeding group achieves lower value as compared to the 42 days of feeding. At phase 1, the antiprotease activity was measured in the feeding group and starvation group shown in Table 6a. In the starvation group, the antiprotease activity level was higher than the feeding group. This indicates an increasing trend (12.43 ± 0.28, % - 55.34 ± 0.48, %) in the starvation group while the feeding group showed a variation (13.03 ± 0.41, % - 16.78 ± 0.31, %) within the successive times. The starvation group increased significantly within successive times (P < 0.05). At phase 2, the re-feeding group decreased significantly (P < 0.05) within the successive times (55.34 ± 0.48, % 26.24 ± 0.50 %) as shown in Table 6b. Also, between the starvation group and re-feeding group shown significant changes within successive times (P < 0.05). After 21 days of feeding, the re-feeding group achieves higher value as compared to the 42 days of feeding. 3.5.2. Serum lipase activity The feeding group indicated an increasing trend (13.37 ± 0.34 U/L - 32.86 ± 0.39 U/L) while the starvation group depicted a decreasing trend after 14 days (13.06 ± 0.43 U/L - 5.38 ± 0.58 U/L) (Fig. 3a). Both groups increased significantly within successive times (P < 0.05). Day 0 and day 21 recorded the highest values in the starvation group and feeding group respectively. At phase 2, after 21 days of re-feeding, an increasing trend was recorded (6.39 ± 0.34 U/L - 31.30 ± 0.58 U/ L) within the successive times and increased significantly (P < 0.05) (Fig. 3b). The feeding group maintained its level within the successive times. After 21 days of feeding, the re-feeding group achieves lower value as compared to the 42 days of feeding. 10

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Fig. 3. (a) Mean ± SEM values of the lipase calculated in the two groups. The “*” indicates significant differences (P < 0.05) whiles “**” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d, e, f) in the same column indicate significant variations between successive times (n = 5). (b) Mean ± SEM values of the lipase calculated in the two groups. The “*” indicates significant differences (P < 0.05) whiles “**” show no significant differences (P > 0.05) between two groups within each successive time. The different letters (a, b, c, d) in the same column indicate significant variations between successive times (n = 5).

described for Hct and Hgb value in fish because of starvation(Caruso et al., 2012b, 2011, 2010; Hernández et al., 2019) where the decline in both blood parameters have been addressed with a depression in erythropoiesis (McCue, 2010). The decline of RBCs is as a result of decrease in degenerating erythrocytes (Rios et al., 2005) which conforms to the current study. Re-fed O. niloticus, after 21 days of starvation, did not recover levels of RBCs, Hct and Hgb to feeding (control) levels. It is possible that erythropoiesis was reactivated during re-feeding period suggesting that there was restoration of degenerating of erythrocytes, increasing iron levels in the blood, repairing the impair oxygen transport in the tissues (Rios et al., 2005) and decrease in immune response (Jafari et al., 2018). Navarro and Gutiérrez (1995) reported that various fish species undergo short and long periods of starvation and then recover after refeeding. In the current study, the glucose levels have significant (P < 0.05) differences between the feeding group and starvation group. Research had shown that starvation of fish causes a decrease in blood glucose level in fish (Boujard et al., 2000; Caruso et al., 2010). The present study indicated that there was a decrease in the glucose level after 14 days starvation and followed by a sharp increase in refeeding phase. The findings were similar to (Waagbo et al., 2017) on Atlantic salmon, and Rohu (Labeo rohita) (Dar et al., 2019, 2018) found a decrease in glucose levels during starvation. This suggests that the decrease in glycemia caused by starvation might be due to the utilization of glucose as fuel during starvation (Li et al., 2018). In addition, the requirements of glucose may be induced by glycogenolysis (glycogen degradation) or de novo glucose synthesis through gluconeogenesis (Polakof et al., 2012). The present study did not consider liver glucose production. After starvation, glucose levels increased and maintained

protein synthesis which was in line with the result in an earlier study on Clarias gariepinus (Bassam AL-Salahy and Ibrahim, 2018). After starvation, HSI levels of the re-feeding group increased significantly indicating that there was no restriction in protein synthesis and normal feeding boosts their nutritional requirement resulting in an increase in the fish liver size. Haematological indices are one of the pathophysiological parameters that are used in describing the fish health and physiological conditions of fish (Lim et al., 2000). In this study, differences were found in haematological parameters of O. niloticus subjected to 21 days starvation. Blood is a sensitive indicator of stress (Sharma et al., 2017) and starvation can be form a stress. In mammals, high levels of WBCs are caused by some factors such as smoking, stress, exercise, medication on corticosteroids (doctors health press). Generally, fish WBCs functions as cell defence and immunity (Jafari et al., 2018; Sakyi et al., 2019). In the present study, there was elevated WBCs after 14 days of starvation conforming to similar research studies Grey mullet (Akbary and Jahanbakhshi, 2016) and Siberian sturgeon (Jafari et al., 2018). The present study result suggests that short-term starvation does not impair the health and immunity of Nile tilapia. Also, the increase in WBCs can be as result of starvation (stress), thus Nile tilapia can tolerate short term starvation. Generally, RBCs, Hct and Hgb function in blood gas transport (Sakyi et al., 2019). RBCs, Hct and Hgb were found to increase after 14 days of starvation showing a robust increase in response to stress and immune response. In the present study, starvation causes a significant decrease RBCs, Hct and Hgb levels which are similar to findings on Hoplias malabaricus (Rios et al., 2005) and spotted rose snapper, Lutjanus guttatus (Hernández et al., 2019). Numerous responses have been 11

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after 21 days of re-feeding. Glucose storage and glucose production are performed by nutritional and hormonal factors which are very significant for maintaining glucose homeostasis; primarily dependent on the regulation of activity and expression of key enzymes involved in the glycolysis and gluconeogenesis pathways (Pilkis and Granner, 1992). Therefore, the stable levels of glucose indicate that glycogenolytic activity thereby enhancing protein/ amino acids synthesis. The Pyruvate kinase (PK) is another glycolytic enzyme involved in the conversion of glucose into pyruvate for the production of adenine triphosphate (ATP) (glycolysis) (Johansen and Overturf, 2006; Salway, 2004), which key for glycolytic pathway and carbon metabolism assure the conversion of glycolysis and gluconeogenesis (Yuan et al., 2013). The nature of dietary carbohydrate can affect the activities of PK (Enes et al., 2009). In the current study, the PK levels increased significantly after 14 days in the starvation group, indicating an increase in fructose1-biphosphate generated during gluconeogenesis (Johansen and Overturf, 2006) which similar to the research study on Red sea bream, Pagrus major (Mohapatra et al., 2017). Also, the PK activity process may be up-regulated during starvation (Salway, 2004). This suggests that PK enzyme was produced by the liver into the bloodstream which was similar to the finding (Knox et al. 1980) as cited in (Enes et al., 2009). After re-feeding, PK activity was stable after 21 days which is similar to the finding of Gilthead sea bream (Metón et al., 2003) suggesting that after 20 days of the re-feeding stimulus of glycolysis metabolise the excess glucose towards pyruvate production. This means that high PK activity produces more energy metabolism (adenosine diphosphate, ADP) and vice versa. Lactate dehydrogenase (LDH) is an enzyme that catalyses the anaerobic glycolysis through cooperating with the other glycolytic enzymes (Zakhartsev et al., 2004). Conversely, the enzyme helps to remove lactate during aerobic recovery, particularly in tissues such as liver or heart (Zakhartsev et al., 2004). LDH is also present in important major organs systems and used for detecting cell damage or cell death caused by excess heat or cold, starvation, exposure to bacterial toxins or injury (Drent et al., 1996). In the present study, higher LDH levels were observed in the starvation group after 14 days suggesting that the shortterm food deprivation enhances autophagy which later aid in the clearance of cellular damage and promote cellular reforming (Madeo et al., 2010), which similar to the research study on red sea bream, Pagrus major (Mohapatra et al., 2017). In addition, the high levels of LDH after 14 days starvation is similar to (Furné et al., 2012) on sturgeon and rainbow trout and (Dar et al., 2018) on Labeo rohita fingerlings suggesting the mobilization of lactate as a substrate for gluconeogenesis during starvation. After starvation, the levels of LDH in the re-feeding group were lower than the starvation group. This may suggest that glycogenic pathways were resumed during re-feeding of the fishes. It has been well-known that SOD and CAT are vital antioxidant players in the fish antioxidant defence system (Furné et al., 2009). The participation of ROS generated by starvation can lead to oxidative stress (Robinson et al., 1997b). Data from the current study observed the increases of activity of antioxidant enzymes which was similar with brown trout (Salmo trutta) (Bayir et al., 2012), European sea bass (Antonopoulou et al., 2013), Mesopotamichthys shareyi (Najafi et al., 2014), Dentex dentex (Morales et al., 2004) and in Pseudosciaena crocea (Bayir et al., 2012; Zhang et al., 2008). Increment of antioxidant activities in our study may not only be due to increased H2O2 but also caused by diminishing in lipid peroxidation which may lead to oxidative stress (Najafi et al., 2014; Velisek et al., 2011). Also, oxidative agents may lead to enhancement of antioxidant enzymes as a defence mechanism (Parihar et al., 1997). After re-feeding of 21 days, the levels of SOD and CAT in both serum and liver samples decrease implying that there were a decrease in H2O2 and restoration of lipid peroxidation. Also, T-AOC measures the total metabolic capacity of both the enzymatic and non-enzymatic antioxidant system to stress (Mahfouz et al., 2009; Zhang et al., 2004). The increased T-AOC value in the starvation

group reflects boosted antioxidants level of the fish, which increase the chance of survival (Dubovskiy et al., 2008). Interestingly, the T-AOC levels in both serum and liver sample decrease in the re-feeding group implying that there was low production of free radicals of both the enzymatic and non-enzymatic antioxidant activities. LYZM is a kind of antibacterial enzyme that involve in the innate immune response (Harikrishnan et al., 2010). Similar to the researches of Chinese sturgeon Acipenser sinensis (Feng et al., 2011) and grey mullet Mugil cephalus (Akbary and Jahanbakhshi, 2016) increase significantly of LYZM was found in the starvation group of this research. The reason might be the innate response of fish could be activated by starvation (Akbary and Jahanbakhshi, 2016). IgM opsonizes microorganisms (bacteria) causing them to reduced and/ or damaged; become vulnerable to destruction by phagocytes in the host (Beck et al., 2015). The present study indicated an increased level of IgM after 14 days of starvation suggesting rise in IgM levels in serum aid as natural antibodies, and displaying the status of the immune system without uncovering the fish to a specific antigen (Estensoro et al., 2013; Magnadóttir, 2006). It appears this is the first to report on the changes of serum IgM after 21 days of starvation on Nile tilapia. Proteins charged with the hydrolysis of peptide bonds are known as proteases, while antiproteases have the ability to sustain the proteases present in tissues (Gonzalez-silvera et al., 2018). In addition, antiproteases function in the inhibition of the action of proteases either by binding to their active sites or by ‘trapping’ the protease to prevent protein hydrolysis (Laskowki and Kato, 1980). The correct balance between protease and antiprotease activities is to protect accurate functionality of any tissue or organ. Besides this, an imbalance of protease and antiprotease activities predispose fish to disease (Greene and McElvaney, 2009). Thus, the significance of antiproteases in serum in order to neutralize these proteases is evident. The protease activities of the starvation group were significantly lower than the feeding group. This result supports the suggestion of Paralichthys Olivaceus (Bolasina et al., 2005), Megalobrama pellegrinii (Zheng et al., 2015), Penaeus vannamei (Muhlia-Almazan and Garcıa-Carreno, 2002), and Metapenaeus ensis (Leung et al., 1989) These suggest high protease activity (trypsin) depicting protein catabolism (Johnston, 2003). The protease activities of the re-feeding groups were significantly higher after refeeding suggesting protein is the main source of energy during refeeding and main nutritional store (Rosas et al., 2002). The antiprotease activities of the starvation group were significantly higher than the feeding group. These suggest that there was an inference with the hydrolysis of proteins by a protease enzyme and prevent the digestion of proteins. After re-feeding, the antiprotease activities were lower in the re-feeding group suggesting proteins were catabolized and showing high protease activity. Functionally, lipases catalyse the hydrolysis and transesterification of other esters as well as the production of esters and display enantioselective properties (Thakur, 2012). Research had shown that during starvation the lipase activity had a decreased trend at the start of starvation in other fish species (Zheng et al., 2015). Lipase activity suggests lipid use (Johnston, 2003). The current study indicated that lipase activity decreases significantly after 14 days of starvation. The finding was supported by (Furné et al., 2008, 2005) on sturgeon and (Zheng et al., 2015) on Megalobrama pellegrinis with a declining trend after starvation During the commencement of starvation, energy-rich lipid reserves are utilized as starvation period increases the lipid-protein ratio are depleted (Zhang et al., 2010). This suggests that the presence of the lipase activity signified the use of lipids during starvation. After re-feeding, the lipase activity increased significantly in the present study. The result is similar to that of (Furné et al., 2008, 2005) and (Zheng et al., 2015) indicating a boost in the lipase activity after refeeding. This suggests that the re-feeding boosted the lipase activity but did not regain the value after 21 days compared to 21 days of feeding. 12

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5. Conclusion

hematological and non-specific immune parameters in two different size groups of grey mullet, Mugil cephalus (Linnaeus, 1758). Acta Ecol. Sin. 36, 205–211. https:// doi.org/10.1016/j.chnaes.2016.04.008. Alexander, C.P., Kirubakaran, C.J.W., Michael, R.D., 2010. Water soluble fraction of Tinospora cordifolia leaves enhanced the non-specific immune mechanisms and disease resistance in Oreochromis mossambicus. Fish Shellfish Immunol. 29, 765–772. Antonopoulou, E., Kentepozidou, E., Feidantsis, K., Roufidou, C., Despoti, S., Chatzifotis, S., 2013. Starvation and re-feeding affect Hsp expression, MAPK activation and antioxidant enzymes activity of European Sea Bass (Dicentrarchus labrax). Comp. Biochem. Physiol.-A Mol. Integr. Physiol. 165, 79–88. https://doi.org/10.1016/j. cbpa.2013.02.019. Arjona, F.J., Vargas-Chacoff, L., Ruiz-Jarabo, I., Goncalves, O., Pascoa, I., Rıo, D., Martın, M.P., Mancera, J.M., 2009. 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In the present study, starvation group loss their body weight and recover after re-feeding but the feeding group increase in weight after 42 days. The study has shown that, starving Nile tilapia for about 14 days can improve fish health through significant increases in blood parameters (WBCs, RBCs, Hct, and Hgb), anti-stress responses parameters (PK, LDH, SOD, CAT, and T-AOC) and immune parameters (lysozyme, protease, antiprotease and lipase) and their values relapsed to basal levels on re-feeding. Meanwhile, the glucose level decrease after 14 days of starvation and elevated after re-feeding. Also, the starvation group increased IgM after 14 days of starvation and after starvation; the re-feeding group had maintained value after 21 days. This study provides basic data regarding the growth, metabolism and immune parameters on starvation and re-feeding of O. niloticus. Funding source This work was supported by National Natural Science Foundation of China (Grant no. 31302226, 31,572,651), Technology Planning Project of Guangdong Province of China (grant no. 2015A020209181), National Key R&D Program of China (2018YFD0900501), Guangdong South China Sea Key Laboratory of Aquaculture for Aquatic Economic Animals, Guangdong Ocean University (No. KFKT2019YB11), Guangxi Key Lab for Marine Biotechnology, Guangxi Institute of Oceanography, Guangxi Academy of Sciences, Beihai, Guangxi, 536000, China and special foundation for “achieving the first class” of Guangdong Province (231,419,013). Submission declaration and verification This manuscript to be considered for publication has not been published and is not under consideration for publication elsewhere. Authors' contributions Cai Jia and Tang Jufen conceived and designed the experiment. Michael Essien Sakyi, Liqun Xia, and Felix K.A. Kuebutornye carried out field experiment. Michael Essien Sakyi and Pengfi Li carried out laboratory analysis of data. Emmanuel Delwin Abarike and Jian Jichang drafted and proofread the manuscript. Declaration of Competing Interest None. Acknowledgments The authors thank Langye Animal Husbandry and Fishery Breeding of the Science and Technology Company Limited, Gaozhou City, Guangdong Province, for providing fish, feed, and experimental tanks for this research. The authors thank the staffs of the biochemical and haematological units of Guangdong Ocean University Hospital, Zhanjiang, Guangdong Province, China. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aqrep.2019.100261. References Abarike, E.D., Tang, J., Cai, J., Yu, H., Chen, L., 2019. Traditional chinese medicine enhances growth, immune response, and resistance to Streptococcus agalactiae in Nile Tilapia. J. Aquat. Anim. Health. https://doi.org/10.1002/aah.10049. Akbary, P., Jahanbakhshi, A., 2016. Effect of starvation on growth, biochemical,

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