Int. J. Aquat. Biol. (2013) 1(3): 132-137 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2013 Iranian Society of Ichthyology Original Article Age and sex specific variation in hematological and serum biochemical parameters of Beluga (Huso huso Linnaeus, 1758) Reza Akrami1, Ahmad Gharaei*2,1Roghayeh Karami2 1Department of Fisheries, Islamic Azad University, Azadshahr branch, P.O. Box: 30, Iran. 2Department of Fisheries, International Hamoun Wetland Research Institute, University of Zabol, P.O. Box: 98615-538, Iran. Article history: Received 26 April 2013 Accepted 28 May 2013 Available online 2 0 June 2013 Keywords: Sturgeon Biochemical parameters, Hematology Feeding rate Abstract: In the present study, the age- and sex-specific changes of various haematological and blood serum biochemical parameters of Beluga (Huso huso) were investigated. Blood samples were collected from 4-, 6-, 7-, and 8-year-old beluga (n = 7 for each sex and age). The specimens were fed at a rate of 0.5-3% body weight per day. AST and LDH levels in 7- and 8-year-old fish of both sexes were significantly higher (P<0.05) than those in 4- and 6-year-old individuals. The mean ALT were significantly different (P<0.05) in both sexes of 4-, 6-, and 7-year-old sturgeon. However, the 6-, 7-, and 8-year-old female sturgeon had higher ALP levels (P<0.05). Additionally, mean RBC, PCV, and Hb values were significantly higher (P<0.05) in 7- and 8-year-old females and males than the others. Two-tailed Pearson’s correlation between the biochemical and haematological parameters obtained for beluga sturgeon indicated significant positive correlations between AST and ALP, AST and LDH, ALP and LDH, RBC and Hb, RBC and PCV, Hb and PCV, MCH and MCHC, and MCV and MCH. However, significant negative correlations were found between RBC and MCV and MCH. These results suggest that the blood parameters of beluga are influenced by age- and sex-specific factors. Introduction Analysis of serological and hematological parameters is one of the most informative methods to monitor physiological status because these parameters are specific among different species and sexes (Celik, 2004; Asadi et al., 2006). The physiological parameter provides a diagnostic tool to assess possible dysfunction and detect the metabolic disturbances, infectious and non-infectious etiologies (Aldrin et al., 1982). There are many studies concerning the hematological and serum enzyme activities of different sturgeon species (Slynko, 1976; Shahsavani et al., 1999, 2001, 2010; Falahatkar et al., 2005; Asadi et al., 2006; Gharaei et al., 2010; Rajabipour et al., 2006, 2010; Ahmadifar et al., * Corresponding author: Ahmad Gharaei E-mail address: agharaei551@gmail.com Tel: +985422232600 2011). However, fluctuations in serum component levels and hematological indices during the artificial conditions have not been investigated yet. Sturgeons, the producers of valuable black caviar, live exclusively in the northern hemisphere. The Caspian Sea is the habitat of four commercial species of sturgeon (Keyvanshokooh et al., 2009). The giant sturgeon (Huso huso) is the largest species of Acipenseriformes and one of the most important species of sturgeon in the Caspian Sea (Jalali et al., 2008). This species is suitable for aquaculture because of its fast growth, ease of reproduction, high tolerance to adverse environmental conditions (Keyvanshokooh et al., 2009). The aim of this study was to (1) determine the normal fluctuations of the serum biochemical properties of 133 Akrami et al./ Int. J. Aquat. Biol. (2013) 1(3): 132-137 Beluga reared in ponds until maturity, (2) compare the parameters between sexes, and (3) provide a basis for future comparative investigations. Materials and methods This experiment was carried out on 4-, 6-, 7-, and 8- years old Beluga. The fish were reared in the earthen ponds supplied with ground water (19 ± 3°C) from the larval stage at the Shahid Marjani Breeding and Rearing Centre, Iran, from May 2005 to May 2009. They were fed a commercial feed containing 39% protein, 12% lipid, 8% ash, 3% fiber, 0.8% phosphorus and 11% moisture (Abizan™, Iran). Following sampling from each age group, total length and body weight were measured and recorded (Table 1). To measure serum biochemical factors during the culture period, blood samples (n = 7 for each sex and age) were taken on 4-, 6-, 7-, and 8-years old fish. All blood samples were collected from behind the anal fin using a 5 mL plastic syringes with 23 gauge needles. Prior to sampling, the fish were anesthetized with 200 mg L-1 MS 222. Blood sera were obtained by centrifuging the sample at 3,000 rpm for 10 min (using a Heraeus Labofuge 400) and the sera were removed with a disposable transfer pipette (Shakoori et al., 1996; Wood et al., 1996). The sera samples were analyzed using an auto-analyzer (Technicon RA1000, USA) and diagnostic kits (Pars Azmoon Co., Tehran, Iran). To study the hematological parameters, blood samples (n = 7 for each sex and age) were collected in heparinized tube prior to anaesthesia of the fish to prevent haemolysis. The following parameters were subsequently determined according to Akrami et al. (2009): red blood cell (RBC), white blood cell (WBC), hematocrit (PCV), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). Statistical analysis: Data were tested for normality and homogeneity of variance before the analysis. The data were examined using a one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test (Duncan, 1995) to rank the groups using SPSS (Version 10). The Pearson’s correlation coefficient was used to examine the correlation between each hematological index and serum biochemical parameters. Results The fluctuations in serum enzyme activity (i.e. AST, ALP, ALT, and LDH) in female and male Beluga are listed in Table 2. AST levels were significantly higher in 7- and 8-years old males and females than in 4- and 6-years old individuals (P<0.05). Mean ALT levels were significantly different (P<0.05) in both sexes of 4-, 6-, and 7-year-old fish. 6-8 years old females had higher ALP levels than 4 years old females (P<0.05). However, 7- and 8-year-old males and females exhibited significantly higher LDH levels than the others (P<0.05). There were significant positive correlations between AST, ALP, and LDH (Table 3). The hematological parameters of both sexes and all ages of the fish are listed in Table 4. The mean values of RBC, PCV, and Hb in 7- and 8-year-old females and males different significantly from those of the other age group. Age (year) Sex Weight (kg) (Mean±SD) Length (cm) (Mean±SD) 4+ Female 7.60 ± 0.84 101.20 ± 8.30 Male 7.80 ± 0.59 102.20 ± 8.90 6+ Female 12.43 ± 0.97 111.14 ± 12.86 Male 13.12 ± 1.23 114.37 ± 15.92 7+ Female 19 ± 1.11 139.75 ± 8.12 Male 23.25 ± 2.21 140.05 ± 16.17 8+ Female 25 ± 2.41 142.04 ± 12.65 Male 26.50 ± 2.36 145.25 ± 9.71 Table 1. The age, mean weight and total length of female and male (n = 7 for each sex and age) Beluga. 133 134 Akrami et al./ Int. J. Aquat. Biol. (2013) 1(3): 132-137 There were significant positive correlation between RBC and Hb, RBC and PCV, Hb and PCV, MCH and MCHC, and MCV and MCH. There were significant negative correlations between RBC, and MCV and MCH (Table 5). Discussion Many studies have documented changes in hematological and biochemical parameters in fish according to age or size (Satheeshkumar et al., 2011). However, these investigations are limited to the comparisons between juvenile and adult fish or examination of only wild-caught fish, in which other factors may influence blood variables (Terry et al., 2001). Few studies have investigated blood values from sturgeon of known ages (Gharaei et al., 2012; Terry et al., 2001), which makes comparison of our results with those on other fish species difficult. The mean levels of AST and LDH in the sera of 7- and 8-years old Beluga were significantly higher than those of 4- and 6-year old. The activity of serum enzymes can be increased in two ways; leakage from damage, or induction that increases the amount of the enzyme and hence the amount that is also likely to leak. It is also important to mention that such an induction may occur when the organism mobilizes its energy sources, including amino acids, to manage stress and repair damage caused by metals (Masola et al., 2008). However, Asadi et al. (2006) reported that mature female A. persicus exhibited higher AST activity than immature females. LDH is very nonspecific and is located in the cytoplasm of the most cells. LDH catalysis the conversion of lactate to pyruvate and is therefore an important enzyme for generating energy in cells (Shahsavani et al., 2010). In this study, the higher AST and LDH levels in the older Beluga may have been due to the beginning of the sexual maturation. It should also be noted that LDH levels are directly correlated with growth rate (Rajabipour et al., 2010). ALT and AST are non-plasma-specific enzymes located within the cells of the liver, heart, gills, kidneys, muscles, and other organs (Gharaei et al., 2010). In Beluga, simultaneous increases of ALT and AST enzyme activity with age may be related to changes in physiological metabolites due to sexual development, feeding management and differences in size (Trivedi et al., 2001; Cech et al., 2000). Shahsavand et al. (2010) measured the activity of AST in the sera of mature Beluga and stated that the Parameter (Ul-1) 4+ years 6+ years 7+ years 8+ years Male Female Male Female Male Female Male Female AST a 20 ±297.4 a 76.2 ±315.7 a 51.23 ±280.8 a 40.5 ±274.5 b 5 15 ±666.5 b 113 ±656.2 b 81.6 ±565.6 b 124 ±570.2 ALT b 1.14 ±3.4 b 0.92 ±3.8 a 0.69 ±1.1 a 0.55 ±1.6 c46 3. ±10.3 c 4.1 ±10.3 bc 1.51 ±6.1 bc 1.1 ±6.3 ALP a 28 ± 184.8 a 41 ±184 a 39.4 ±165.8 b 46.5 ±266 a6 51. ±184.7 b 32.6 ±208 a 37.4 ±157 b5 33. ±204.3 LDH a 103 ± 487.4 a 204 ±628.3 a 214 ±600 a 176.4 ±717.5 c 673 ±3392 c 712 ±4094 b 580.2 ±1852 b 634 ±2219 Table 2. The values of serum enzymes activity of female (n = 7) and male (n = 7) Beluga in difference ages. Values followed by different superscript letters in each row are significantly different (P<0.05). Parameter AST ALT ALP LDH AST p value 1 0 -0.06 0.939 *0.911 <0.001 *0.865 <0.001 ALT p value 1 0 -0.033 0.842 -0.053 0.753 ALP p value 1 0 *0.836 <0.001 LDH p value 1 0 Table 3. Pearson correlation and p value between serum enzymes activity of Beluga. * showed significantly correlation between parameters. 135 Akrami et al./ Int. J. Aquat. Biol. (2013) 1(3): 132-137 significant increase in AST levels may be related to sexual maturation. ALP exhibited increased serum activity in 6-, 7-, and 8-years old female Beluga. ALP induces activity transfer and is normally secreted in bile from the liver. This enzyme mainly leaks from the lining of the bile canaliculi and sinusoidal surfaces of hepatocytes (Gottelli et al., 1985). Based on our results, the increase of ALP activity may be due to an increase in biliary canal or osteoblast activity, or feeding conditions (Moraes et al., 2005; Cech et al., 2000). The activities of enzymes that play roles in amino acid catabolism differ based on sex and stage of maturity. Direct significant correlations were obtained between AST, ALP, and LDH, suggesting that individuals with higher AST levels also tend to have higher ALP and LDH levels. RBC, PCV, and Hb values were significantly higher in both sexes of 7- and 8-years old Beluga, suggesting that these parameters are age-specific (Table 4). The direct significant correlations found among some hematological parameters suggest that individuals with higher RBC and Hb levels tended to have the parameters that have greater correlations with each other. RBC and HB concentration tended to increase with length and age of the fishes (Satheeshkumar et al., 2011). However, the significant negative correlations between RBC, MCV and MCH could be argumentative. * showed significantly correlation between parameters. Parameters 4+ years 6+ years 7+ years 8+ years Male Female Male Female Male Female Male Female (fl) MCV 8.6± 314.3 10.4±317.9 9.65 ±288.6 11.32 ±295.7 12.9 ±283.2 27 ±300.8 19.14 ±294 16.11 ±293.8 MCH(pg) 4.5 ± 103.8 11.17±101.3 8.1 ±92.2 12 ±94.8 8.5 ±92.3 13.7 ±98.5 14.3 ±95.9 8.3 ±94.56 MCHC (%) 1.21 ±33 1.4±32 0.96± 32 1.3±32.1 0.95 ±32.6 1.44 ±32.5 0.91 ±32.6 1.63 ±32.1 RBC (106 mm) a 0.1± 0.82 a 0.14 ±0.79 a 0.2 ±0.85 a 0.13 ±0.84 b 0.19 ±1.13 b 0.13 ±0.97 b 0.17 ± 1.02 b 0.03 ±0.99 PCV (%) a 2.9 ±22.51 a 4.4 ±21.96 a 3.4 ±24.1 a 2.1 ±24.8 b 4.3 ±28.9 b 3.1 ±27.6 b 3.4 ±29.6 b 2.2 ±29.2 Hb (g dl-1) a 0.87 ±7.3 a 1.24 ±7.52 a 1.2 ± 7.74 a 0.67 ±7.62 b 1.19 ±10.1 b 1.2 ±9.93 b 1.12 ±9.7 b 1.01 ±10.4 Leukocyte (mm3) 1.8 ±24.01 1.5±24.3 2.3± 23.6 1.88 ±24.2 1.12 ±25.7 1.19 ±23 0.84 ±25.4 1.84 ±24.3 Lymphocyte (%) 2.94 ±68.2 3.4±72.5 3.5 ±71 3.14 ±69.6 3.8 ±70.3 3.33 ±71 3.69 ±70.25 3.43 ±70.2 Neutrophil (%) 1.8 ±18.2 1.16±19.1 2.14± 19.4 1.87 ±20.5 2.21 ±20.4 2.9 ±19.2 2.45 ±19 2.1 ±20.3 Eosinophil (%) 1.2 ±5.8 1.3±5.4 1.58 ±6 1.4± 6.3 1.16 ±6 1.87± 6.2 1.12 ±6.5 1.11 ±5.5 Monocyte (%) 0.86± 4.2 0.77±3.41 0.62± 3.6 0.46 ±3.3 0.57 ±3.75 1.01 ±3.66 0.94 ±4.01 0.34 ±4.00 Values followed by different superscript letters in each row are significantly different (P<0.05). Table 4. Hematological parameters of female (n = 7) and male (n = 7) Beluga in difference ages. parameter Leukocyte )3(mm RBC mm) 6(10 Hb )1-(g dl PCV (%) MCV (fl) MCH (pg) MCHC (%) )3Leukocyte (mm p value 1 0 0.224 0.144 0.214 0.164 0.259 0.090 0.011 0.944 0.059- 0.702 0.200- 0.194 mm) 6RBC (10 p value 1 0 *0.832 <0.001 *0.873 <0.001 *0.489- 0.002 *0.465- <0.001 0.77- 0.619 )1-Hb (g dl P value 1 0 *0.982 <0.001 0.026 0.869 0.096 0.535 0.183 0.236 PCV (%) P value 1 0 0.017- 0.915 0.000 0.998 0.003 0.978 MCV (fl) P value 1 0 *0.949 <0.001 0.064 0.681 MCH (pg) P value 1 0 *0.373 0.013 MCHC (%) P value 1 0 Table 5. Pearson correlation and p value between hematological parameters of Beluga. 135 136 Akrami et al./ Int. J. Aquat. Biol. (2013) 1(3): 132-137 In this study, we determined the age- and sex- specific changes in biochemical and hematological parameters of the serum in cultured Beluga. 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