Effects of starvation and re-feeding on some hematological and plasma biochemical parameters of juvenile Persian sturgeon ,Acipenser persicus Borodin, 1897

Effects of starvation and re-feeding on some hematological and plasma biochemical parameters of juvenile Persian sturgeon ,Acipenser persicus Borodin, 1897

M.Yarmohammadi¹*, M.Pourkazemi², R.Kazemi¹, M.Pourdehghani¹, M.Hassanzadeh Saber¹, L.Azizzadeh¹

1. International Sturgeon Research Institute, Agricultural Research Education and Extension Organization (AREEO), Rasht, Iran

2. Iranian Fisheries Research Organization, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran

* Corresponding author: Mahtabyarmohammadi@gmail.com

(Received: Oct. 06.2014 Accepted: April. 06.2015)

INTRODUCTION

The feed restriction for fish occur both in nature and captive condition aquaculture. Wild populations, undergo feed restriction due to food limitation, migration, weather events, and during the reproductive cycles (Liu et al., 2011). In captive condition, fish species undergo periods of starvation because of solving water quality problems, reduce handling stress and

mortality due to disease outbreaks, temperature changes, and also save feed to increase farm profit (Gaylord and Gatlin III, 2000, Caruso et al., 2011, Wang et al., 2000). During starvation, fish may employ various behavioral, physiological, and structural responses to cover their metabolic needs (Navarro and Gutiérrez, 1995). However, the metabolic and endocrine responses depend on the species and the duration of the deprivation period (Pottinger et al., 2003, Mommsen et al., 1999, Navarro and Gutiérrez, 1995). There is growing information on physiological responses following feed deprivation and re-feeding in fish species due to interest in the compensatory growth phenomena. Compensatory growth followed by different feeding regimes, has been reported in several fish species (Abolfathi et al., 2012, N Montserrat et al., 2007, Núria Montserrat et al,. 2007, Zhu et al,. 2005, Tian and Qin, 2003, Hayward et al., 1997, Nicieza and Metcalfe, 1997). However, there are few studies on the sturgeons, that have shown the morpho-physiological responses following starvation and  re-feeding (Falahatkar, 2012, Furné et al., 2012, Yarmohammadi et al., 2012, Liu et al., 2011, Gillis and Ballantyne, 1996, Hung et al,. 1997).

The physiological responses depending on mobilization of energy reserves are different in fish species. The assessment of hematological and biochemical parameters of blood provides valuable information for evaluating the physiological responses followed by different feeding regimes in fish (Bani and Vayghan, 2011, Coles, 1986). However, the effects of feed deprivation and re-feeding on blood parameters of Persian sturgeon are not understood and therefore further investigation is needed. Regarding the physiological responses under different feeding regimes, changes in hematological factors such as hemoglobin (Hb), hematocrit (Hct), white and red blood cells (WBC and RBC), and corpuscle indices (Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin (MCH) have been reported in fish species such as Siberian sturgeon, A. Baerii(Morshedi et al., 2011) and Nile tilapia, Orechromis niloticus(Abdel-Tawwab, et al. 2006). Changes in plasma cortisol levels are different in fish species, and it has been shown from decrease to no consistent changes, or increase in blood cortisol concentration in feed deprived specimens (Mommsen et al., 1999, Pottinger et al., 2003, Polakof et al., 2007). The liver enzymes including alkaline phosphatase (ALP), Aspartat amino transfers (AST) and alanine aminotransferase are localized within the tissue cells of liver and other organs. However, ALP is mainly produced in the liver compare to other organs. Measurement of the these enzymes in the blood, is one of the most valuable diagnostic tools because they reflect the physiological conditions of fish (Shahsavani et al. 2010, Anver 2004). Elevation of liver enzyme activity appears to reflect liver disorders. The stock of the Persian sturgeon, Acipenser persicus (Borodin, 1897) has decreased over the past 20 years (Pourkazemi et al. 1999). Due to unique biological characteristics of sturgeons such as very long life span and late sexual maturation, make it a suitable species for studies of repetitive feeding regimes or compensatory growth. The Persian sturgeon as a native fish with a fast growth rate and delicious meat, it has become a candidate species for Iranian aquaculture in recent years (Mohseni et al., 2007). Persian sturgeon, like many other fish species, experience periods of starvation in nature and captive condition (Falahatkar, 2012). Despite several studies in teleost fishes regarding starvation effects on physiological functions, there is still much to be understood about the physiology of sturgeons species. The aim of the present study is to assess the metabolic strategies of Persian sturgeon exposed to different periods of starvation and followed by re-feeding or compensatory growth. In this study, the effect of starvation and re-feeding was monitored on some hematological and plasma biochemical parameters. Knowledge of how fish respond to starvation periods in this study could provide a basis for improved nutrition and rearing and thereby help to optimize Acipenser persicus culture.

MATERIALS AND METHODS

Test specimens and experimental conditions

Juveniles (7-month-old) Persian sturgeon (A. persicus) were provided by Shahid Beheshti Sturgeon Propagation and Rearing Complex (Rasht, Iran) between October and December 2009. Prior to the experiment, fish were acclimated to experiment conditions for 2 weeks while they fed three times a day (08:00, 13:00 and 18:00(on a commercial food (BIOMAR, INICIO Plus 868Nersac, France) containing 48% crude protein, 22% crude fat. Fish were maintained under a free-stress condition during the experimental period. Water quality parameters, such as dissolved oxygen (6–8 mg.L^-1), pH (7.5) and temperature (16.5–18.5oC) were monitored daily and did not change significantly between treatments during the experiment. After acclimation, 375 fish with a mean initial weight 108 ± 0.63 g were randomly assigned to five feeding groups. Three replicates were assigned for each group, consisting of 25 fish per tank in a flow-through system with water supplied from the Sefidrood River and aerated well. The experiment lasted for 8 weeks and was divided into two periods, starvation periods (weeks 1 to 4) and a re-feeding period (weeks 4 to 8). The control fish (C) were fed on the commercial diet to satiation three times a day throughout the experiment. Fish in other four groups were subjected to the following  four periods of starvation: 1 week (W1), 2 weeks (W2), 3 weeks (W3), and 4 weeks (W4) and then fed to satiation three times a day during the 4-week re-feeding period. Starvation was timed so that the end of the starvation period occurred in all groups at week 4. The W1 fish were starved only in week 4; the W2 fish were starved from week 3 to week 4; the W3 fish were starved from week 2 to week 4, and W4 fish were starved from week 1 to week 4. Fish from each group were fed by hand until apparent satiety throughout the experiment. The tanks were siphoned weeklyto exonerate excessive food and feces. Fish in all 15 tanks were weighed (g) and measured for total length (mm) individually at the beginning of the experiment and at the end of starvation and re-feeding periods.

Growth indices including final weight, body weight increase (BWI), and specific growth rate (SGR) were calculated according to the formulae below:

BWI= 100 × (FW –IW) / IW

SGR= 100 × [ln (FW- IW)] /t

Where FW stands for final body weigth, IW refers to initial body weigth, and t is days of experiment (Eroldoğan et al., 2008).

Blood sampling and biochemical analysis

Five fish were randomly sampled from each tank at the end of each period of experiment for blood sampling. The fish were killed quickly with blow to the head and blood was taken from the caudal vessels using heparinized syringes. Blood sampling took less than 1 min for each fish and then the blood was transferred to heparinized 1.5 ml Eppendorf tubes, and kept on ice. Theblood was divided into two parts. In one of them, the whole blood was suspended in the diluent (Natt &Herrick, 1952) for red and white blood cell levels using a hematocytometer. Hemoglobin concentration (Hb) was measured using a commercial kit by photometric assay of cyanomethemoglobin method. Hematocit (Hct%) was determined by centrifuging whole blood in heparinized microhematocrit capillary tubes at 3500×g for 10 min. Osterode, Germany). Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular hemoglobin (MCH) were calculated using the following formulaes (Houston, 1990):

MCV (fl) = (Hct / RBC) × 10

MCHC (%) = (Hb / Hct) ×100

MCH (fl) = (Hb / RBC) × 10

Differential WBC counts (lymphocytes, neutrophils, eosinophils and monocytes ) were conducted on Giemsa stained blood smears. Cells were identified on the basis of morphology and cell ultrastructure, as documented in previous fish leukocyte studies (Afonos et al., 1997, Rowley, 1990, Ellis, 1977).

For biochemical assays, blood samples were immediately centrifuged (5 min at 5,000 rpm, Hettich D7200, Germany) then plasma was separated and stored in 1.5 ml Ependorf tubes at -20 ºC for subsequent analysis of biochemical parameters.Plasma Cortisol was determined by radioimmunoassay (RIA) using the Coat-A-Count Cortisol kit (#TKCO2, Diagnostics Products Corporation, Los Angeles, CA, USA). The concentration of ALT (Alanine aminotransferase), AST (Apartat aminotransferase) and ALP (Alkaline phosphatase) of blood plasma samples were analyzed by auto-analyzer (Technicon, RA1000, USA) and diagnostic kits (Pars Azmoon Co., Tehran, Iran) based on the protocols described by Shahsavaniet al.,(2010).

Data analysis

Data was presented as mean ± SEM (standard error of mean). All statistical analyses were conducted using SPSS statistical software, version 17 (IBM, Chicago, IL, USA). Normality of data and homogeneity of variance were checked by using Kolmogorov-Smirnov and Leven's test, respectively. The starvation⁄re-feeding effects were analyzed with One-Way ANOVA followed by Duncan’s test where variances were homogeneous. Differences between fed and food deprived groups at each sampling time were estimated using a t-test. P

RESULTS

Growth performance

Means of fish initial weight did not significantly differ among treatments at the start of experiment (P>0.05). Throughout the experiment, fish survival was 100%. The mean final body weight of juveniles was significantly impacted by deprivation (Table 1). Mean body weight of W2, W3, and W4 treatments were significantly lower than those of control fish (P<0.05). After re-feeding for four weeks, the W1, W2, and W4 treatments were not significantly different compared to the final weight of control fish (Table 1).After re-feeding, body weight increased (BWI) and specific growth rate (SGR) in all four starvation treatments were significantly higher than in the control fish (P<0.05).

Hematological and biochemical indices

The effect of different starvation treatments and subsequent re-feeding on Persian sturgeon blood indices are displayed in Table 2. Starvation and re-feeding had no significant effect on some hematological parameters including Hematocrit, lymphocytes, neutrophils, eosinophils, and monocytes; while Hb, RBC, MCHC, MCV, and MCH significantly changed during starvation and showed the least value for W2 group compared to control. After a 4- week re-feeding, the hematological and biochemical values of all food deprived groups recovered to the normal level of full-fed control fish.Red blood cell (RBCs) levels significantly increased during starvation periods in W1, W3, and W4 treatments. After fish re-feeding, Hb and RBCs values returned to the normal level of full-fed control fish showing that the fish had recovered and became healthy.

Starvation did not considerably affect plasma cortisol levels. But after one month re-feeding period, cortisol concentration in W groups increased compared to control fish, so that in W2 group, its level reached maximum (P<0.05) (Table 2). Plasma alkaline phosphatase (ALP), alanine aminotransferase and aspartate aminotransferase (AST) increased in different W groups during starvation period (P<0.05). But after re-feeding period, their levels decreased in W groups and reached to the control level.

DISCUSSION

In this study, we investigated the effects of nutritional restriction and subsequent re-feeding on compensatory growth and physiological responses of Persian sturgeon. Starvation for 2, 3, and 4 weeks resulted in significant reduction in body weight. After re-feeding for one month , the final weight of fish starved for 1, 2 and 4 weeks did not differ from the control, while those starved for 3 weeks (W3) were significantly lower than the control groups (C). During re-feeding period, BWI and SGR were significantly higher in the starved fish than that in the control group, indicating that the experiment was ended during a phase when the fish were still undergoing GC. Findings showed that the juvenile Persian sturgeon starved for 1,2, and 4 weeks showed complete compensatory growth while fish starved for 3 weeks showed partial compensatory growth. Thus compensatory growth exists in the juveniles of Persian sturgeon, and the compensatory response depends on the length of feed deprivation.

Table. 1:Changes in final body weight, body weight increase (BWI), specific growth rate (SGR), and survival in Persian sturgeon reared under different feeding regimes. Values are mean±SEM. Different superscripts in the same rows indicate significant differences (P<0.05) between feeding strategies by One - Way ANOVA.

C: Control (fed three times daily to apparent satiation); W1: Treatment 1 (one week starvation and 4 weeks re-feeding), W2 (two weeks starvation and 4 weeks re-feeding), W3 (three weeks starvation and 4 weeks re-feeding), W4 (four weeks starvation and 4 weeks re-feeding)

Table 2:Hematological indices of juvenile Persian sturgeon under different starvation periods and subsequent re-feeding. MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin, MCHC, mean corpuscular hemoglobin concentration. Values are mean±S.E. Different superscripts in the same rows indicate significant differences (P<0.05) between feeding strategies by One - Way ANOVA.

Control (fed three times daily to apparent satiation); W1: Treatment 1 (one week starvation and 4 weeks re-feeding), W2 (two weeks starvation and 4 weeks re-feeding), W3 (three weeks starvation and 4 weeks re-feeding), W4 (four weeks starvation and 4 weeks re-feeding).