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June 2013

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Croatian Journal of Fisheries

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GENEtska struktura PoPulaCiJa uGrožENoG CiPriNida Labeo calbasu (haMiltoN, 1822) istražENa PoMoću MikrosatElitskih MarkEra Md. Robiul Hasan, Md. Nahiduzzaman, Mostafa Ali Reza Hossain, Md. Samsul Alam

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PrEhraMbENa VriJEdNost kaliForNiJskE PastrVE (Oncorhynchus mykiss) uzGoJENE u JadraNskoM Moru Tibor Janči, Danijel Kanski, Marina Dulić, Nives Marušić, Helga Medić, Tomislav Petrak, Sanja Vidaček

65

broj 2

učiNCi hraNidbE soJiNiM brašNoM Na PErForMaNCu rasta i biokEMiJska sVoJstVa hEMoliMFE duNaVskoG raka (Astacus leptodacylus EsChsCholtz, 1823) Mahdi Banaee, Fatemeh Daryalaal, Mohammad Reza Emampoor, Maryam Yaghobi

58

Volumen 71

uGrožENE ribE sViJEta: Paracobitis rhadinaeus (rEGaN, 1906) (NEMaChEilidaE) Hamed Mousavi-Sabet, Ahmad Gharaei, Mostafa Ghaffari

Prilozi ribarstvenoj struci kako PoMoću ribarstVa sMaNJiti siroMaštVo? Santosh Kumar Mishra

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

CODEN RIBAEG ISSN 1330-061X

ORIGINAL SCIENTIFIC PAPER

EffEcts of soybEan MEal basEd diEt on Growth PErforMancE and hEMolyMPh biochEMical ParaMEtErs of narrow-clawEd crayfish (Astacus leptodacylus Eschscholtz, 1823) Mahdi banaee*, fatemeh daryalaal, Mohammad reza Emampoor, Maryam yaghobi Department of Aquaculture, Natural Resource and Environmental Faculty, Behbahan Khatam Alanbia, University of Technology, Behbahan, Iran; Postal Code: 6361647189; Tel.: +98 671 2221191; +98 9177011572; Fax: +986712231662. * Corresponding Author, E-mail: Mahdibanaee@yahoo.com

ARTICLE INFO

ABSTRACT

Received: 3 February 2013 Received in revised form: 7 May 2013 Accepted: 22 May 2013 Available online: 25 May 2013

Like other crustaceans, narrow-clawed crayfish (Astacus leptodacylus Eschscholtz, 1823) can change its diet to the available food during varied life cycles. Diet alteration can affect different biological indices of this species, therefore this study aims at studying changes in growth indices, hemolymph biochemical parameters and biochemical quality of its carcasses, which might occur during diet change of crayfish. The purpose of this experiment was to evaluate the effects of partial replacement of fish meal with soybean meal on growth performance, carcass quality and hemolymph biochemical parameters of narrow-clawed crayfish. 90 healthy adult narrow-clawed crayfish (W=35.50±4.05 g; TL=16.96±1.92 cm) were randomly distributed into 9 fiberglass tanks (200 L) and were fed for 45 days with three varied diets including: commercial shrimp diet, fishmeal-based diet (A) and soybean meal-based diet (B). The results show that changing the diet from animal protein to plant protein caused a significant decrease in the cholesterol and triglyceride levels in hemolymph, as well as carcass fat of the crayfish nourished with B diet when compared with the crayfish fed with A diet. No significant changes of hemolymph levels of glucose, AST and ALT were found between different treatments during this experimental period. In conclusion, it was found that though growth performance reduced, the increased rate of soybean meal in diet from 0.0% to 76% had no adverse effects on biochemical parameters.

Keywords: Crayfish Protein source Growth indices Carcass quality Biochemical parameters

introdUction Narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823) is the largest known invertebrate in freshwater ecosystems. It constitutes a significant biomass of benthic species (Holdich, 2002) and is an endemic crustacean in Iran inhabiting only the River Aras in West Azarbaijan, the Anzali Lagoon and also some rivers in Gilan province (Nekuie Fard et al., 2011). This species is also found in many

Eastern European countries, Turkey and Australia (Sepici-Dinçel et al., 2013). Most species of narrowclawed crayfish are nocturnal and find their fellows, food and prey via their chemical and mechanical sensors (Nystrom, 2002). Like other crustaceans, this species can change its diet alternatively at different times, which means that determining the status of this species in the food chain depends on the amount and type of available food. The natural food preference of crayfish depends on their age and 45

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

stage of life cycles. Their larvae are carnivorous animals feeding primarily on zooplankton, especially small crustaceans, while the post-larvae and adults are omnivorous animals feeding on algae, aquatic plants, carcasses of died aquatic animals, aquatic invertebrate animals such as molluscs, aquatic insects, worms and smaller crustaceans (Nystrom, 2002; Figueiredo and Anderson, 2003; Johnston and Robson, 2009). Studies show that the change of biological and non-biological factors may cause alterations in growth indices, hemolymph biochemical indices and biochemical analysis of their carcasses. The change of hemolymph biochemical indices of freshwater crayfish (Parastacus defossus) in varied seasons for both males and females (Buckup et al., 2008) confirms that biological conditions such as life cycle, sexual puberty and reproduction, molting and accessibility of food, and non-biological factors such as light period, temperature, pH and soluble oxygen in water can severely affect physiological and biological parameters of crustaceans (Rosa and Nunes, 2003a,b; Vinagre et al., 2007). However, the results of most studies show that feeding this species with plant protein sources, such as soybean, does not have an adverse effect on its growth indices (Thompson et al., 2005; McClain and Romaire, 2009). Among plant protein sources, soybean is regarded as a good candidate for substituting fish meal in aquaculture diet due to having favourable amino acid profile as well as abundance and appropriate price (Dersjan-Li, 2002; Watanabe, 2002). However, it is worth noting that, like any other plant protein source, soybean has many anti-nutritional compounds including trypsin inhibitors, lectins, oligosaccharides, soy antigens, phytoestrogens, phytic acid, antivitamins and saponins (Francis et al., 2001; Dersjant-Li, 2002) which can adversely affect the growth performance, food digestibility, physiological status and general health of aquatic species. In many cases, however, using soya in diet has no adverse effect on the growth and health of aquaculture (Khan et al., 2003a,b) and is favourable to aquacultures and its usage has increased in some species (Tomás et al., 2005; Ogunkoya et al., 2006). Recently, practical efforts have been done at the optimal utilization of soybean meal in the feeding of aquatic species. For example, soybean meal has been used in diets for sea turbout, Psetta maeotica, (Yigit et al., 2010), white shrimp, Litopenaeus schmitti, (Alvarez et al., 2007), sharpsnout sea bream, Diplodus puntazzo, (Hernandez et al., 2007), Cobia, Racycentron canadum, (Hsu, 2005), Japanese flounder, Paralichthys olivaceus, (Masumoto et al., 2001), Pacific white shrimp, Litopenaeus Vannamei, (Davis 46

and Arnold, 2000; Samocha et al., 2004; Suárez et al., 2009), European sea bass, Dicentrarchus labrax, (Tulli et al., 2000), gilthead sea bream, Sparus aurata, (Kissil et al., 2000), Dentex, Dentex dentex (Tomás et al., 2009), Catla catla (Priyadarshini et al., 2011) and red sea bream, Pagrus major (Takagi et al., 2001). Nevertheless, variation in diet formulation as well as individual features of the specific species are among the most important reasons for difference in the diet response of aquaculture to soybean in their diet. In other words, varied species sensitivity and their ability to use this protein source are so different (Refstie et al., 2000). Since our knowledge on nutrition requirements of cultured species of freshwater narrow-clawed crayfish is so limited, most farmers produce diet for this species based on their diet in nature. Therefore, with regard to freshwater narrow-clawed crayfish compatibility in using animal and plant protein sources and considering difficulties in supplying raw food materials, farmers prefer to use low-cost sources for cultivating this species. Due to lack of knowledge on the effects of using plant protein sources on growth index, biochemical and physiological factors and carcass quality of narrow-clawed crayfish, conducting studies on this issue is a necessity. Therefore, studying changes in growth indices, hemolymph biochemical indices, as well as carcass quality of this species can not only be effective in monitoring and evaluating the health of this species, but also it is effective in economic justification of cultivating this species with low-cost diets. Therefore, this study aims at investigating growth indices, hemolymph biochemical indices and carcass quality of narrow-clawed crayfish (A. leptodacylus Eschscholtz, 1823) treated with different proportions of animal and plant protein in diet.

MatErials and MEthods Animal Healthy adult crayfish (average weight and length 35.50±4.05 g; 16.96±1.92 cm, respectively) were purchased from Maku, West Azerbaijan Province, Aras Shrimp Company, Iran and were transferred to the aquaculture laboratory of the Natural Resource Faculty, Bahbahan University of Technology, Iran. The crayfish were randomly divided into nine groups (three treatment with triplicate) each consisting of 10 animals and were stocked in the 200 L fiberglass tanks with semi-closed water recirculating systems for at least two weeks to acclimate to laboratory conditions (17±2 °C; 7.4±0.2 pH; light regime of 8 hours ligh: 16 hours dark; 20% water exchange rate/day) prior to experiments. The floor

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

of the tanks was covered with gravel, broken bricks and lengths of drainpipe to act as shelters, and limestone chippings in order to maintain calcium concentrations in water. During acclimation, crayfish were fed with the fresh fish meal based on the recommendations of Aras Shrimp Co experts.

Diet preparation

ed from the pericardium using a 1 cc syringe. After bleeding, they were sacrificed and their exoskeleton was removed under sterile conditions and washed in normal saline solution. Then, crayfish carcasses (gutted body) were immediately pooled, minced, dried and ground to be analyzed for the final whole body composition after washing and weighting.

The feed ingredients chosen for preparing the experimental diets were fresh fish meal (Kilka fish, Clupeonella cultriventris) and soybean meal as protein source, wheat flour, corn flour and rice flour as carbohydrate source and sunflower oil as lipid source, albumen as blender and zeolite as filler. These materials were purchased from the local market. Supplementary minerals and vitamins were obtained from the Razak Damloran Drug Co. Tehran, Iran. The minced fresh fish meal and the dry pulverized feed ingredients such as soybean meal, wheat flour, corn flour and rice flour were accurately weighed and mixed in a blender. The mixture was moistened with water containing the required amount of gelatin and steam cooked for 30 minutes. After cooling, sunflower oil, vitamins and minerals were mixed uniformly in a kneader. The albumen was used as plate binder. The dough baked was passed through a meat grinder, producing extruded string shapes approximately 10 mm long which were dried in oven at 55 °C overnight. After drying, the pellets were broken into small crumbs and were packed in airtight containers and stored at -18 °C in a freezer until used. The prepared feeds were analyzed for proximate composition. Commercial formulated shrimp food used as formulated crayfish food in Iran was used as control (reference diet) with composition and proximate analysis reported in Table 1.

Table 1. Ingredients and proximate nutrient composition of experimental diets (values expressed as feed basis, g/100 g)

Feeding trial

Dietary reference componentes are written according to personal correspondence with experts from the Aras Shrimp Company, Iran. b Mineral mix (g/kg diet): 0.5 KCl; 0.5 MgSO4.7H2O; 0.09 ZnSO4.7H2O; 0.00234 MnCl2.4H2O; 0.005 CuSO4.5H2O; 0.005 KI; 0.00025 CoCl2.2H2O; 2.37 Na2HPO4. c Vitamin mix (unit in mg/kg except where given): 5000 IU retinol; 4000 IU cholecalciferol; 100 mg α-tocopherol acetate; 5 mg menadione; 60 mg thiamin; 25 mg riboflavin; 50 mg pyridoxine HCl; 75 mg pantothenic acid; 40 mg niacin; 1 mg biotin; 400 mg inositol; 0.2 mg cyanocobalamin; 10 mg folic acid.

At the end of this adaptation period, twelve crayfish were randomly sampled from each treatment for biometric analyses before initial feeding trial. Following adaptations, all animals were fed experimental diets (commercial shrimp pellets as reference diet, A diet and B diet) at 5% of their wet body weight, twice a day for 45 days (Table 1). Mortalities were recorded daily. Uneaten feed and feces were removed by siphoning as required. Animals were fed at least 24 hours before sampling. At the end of the experimental period, 12 crayfish per treatment were randomly captured, then the total length and total weight were determined for each individual crayfish; hemolymph was collect-

Ingredient Fish meal Soybean meal Corn grain Wheat flour Rice bran Safflower oil Vitamin premixb Mineral premixc Egg Spinach Zeolite DL-Methionine L-Lysine Proximate composition Dry material (%) Metabolic energy (Kcal/g) Crud protein (%) Ether extract (lipid) (%) Ash (%) Crude fiber (%) Carbohydrate

Reference dieta 60 0 10 12 0 2 1 1 0 0 2 0.159 5.7 ×10-3

A diet

B diet

51.5 12 6.5 17 6 2 1 1 1 0 2 0 0

18 57 6 5 6 2 1 1 1 1 2 0 0

92.54

93.44

93.31

350.24

323.16

354.58

40.22

40.74

40.26

10.49

8.04

13.3

7.86 5.79 27.56

9.83 7.10 27.85

7.13 8.70 24.26

a

Growth parameters All crayfish were deprived of food for 24 hours before weighing and sampling. Growth parameters, such as survival percentage, weight gain percent47

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

age, specific growth rate, feed conversion ratio, were calculated by following these formulas at the end of a 45 day trial.

Biochemical body composition analysis At the termination of the 45 day trial, survived crayfish from each experimental group were sampled and sacrificed for proximate analysis. The gutted bodies of crayfish were minced and weighed, then moisture was determined by drying pre-weighed samples in porcelain cups at 100 °C for 24 h. Ash was obtained by incinerating the dried samples at 500 °C for 12 h. Crude protein was determined according to the Kjeldahl method by measuring the total nitrogen content of the sample multiplied by the empirical factor 6.25. Soxhlet extraction with petroleum ethers was used for crude lipid at 60-80ºC for 12 h. All analyses were performed in triplicate.

Statistical analysis

Biochemical Analysis Plasma levels of glucose, total protein, cholesterol and triglycerides were determined in plasma by standard procedures used in clinical biochemistry laboratories based on manual biochemical kits (Pars Co, Iran). Lactate dehydrogenase (LDH) activity determination is based on measuring the conversion of pyruvate to L-lactate by monitoring the oxidation of NADH. Aspartate aminotransferase (AST) was assayed in a coupled reaction with malate dehydrogenase in the presence of NADH. In alanine aminotransferase (ALT) assay, the enzyme reacts with alanine and a-ketoglutarate to form glutamate and pyruvate. Pyruvate is converted by lactate dehydrogenase to make lactate and NAD+. All these activities were monitored by measuring the change in absorbance at 340 nm. Alkaline phosphatase (ALP) assay is based on the enzyme-mediated conversion of p-nitrophenol phosphate to nitrophenol in an alkaline buffer at 405 nm. All biochemical parameters were measured in duplicate by UV/Vis spectrophotometer in the biology laboratory.

Statistical analyses were performed using SPSS (Release 15) software (SPSS Inc. 2006). Data are presented as mean ± SD. All the data were tested for normality (Kolmogorov-Smirnov test). Data were analyzed by one-way of variance analysis (ANOVA). The significant means were compared by Tukey’s test and p< 0.05 was considered statistically significant.

rEsUlts The crayfish which were fed with diet (A) had bigger affinity for feed. Also, molting frequency and mortality percentage, as well as cannibalism was lower among the crayfish nourished with diet (A) compared with other groups. The final weight, specific growth rate (SGR) of the crayfish treated with diet (A) were significantly higher than of the crayfish fed with diet (B) (p<0.05). The results indicate a significant difference in food conversion ratio (FCR) among the crayfish fed with diet (A) and the crayfish nourished with diet (B) (p<0.05) (Table 2).

Table 2. Initial weight, final weight, weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR) and survival rate (SR) were calculated for the crayfish fed diets containing different levels of soybean Growth Parameters Initial weight (g) Final weight (g) WG (%) SGR FCR SR (%)

Reference diet 35.06±6.41a 47.09±7.32a 35.67±11.95ab 0.67±0.19ab 2.43±0.64ab 76.67

Diets A diet 36.39±3.54a 52.54±4.18b 44.04±11.27b 0.81±0.18b 1.92±0.53a 80.00

Values in the same row with different letter notation statistically significantly different at p<0.05;

48

B diet 35.05±2.21a 46.29±4.37a 32.06±8.88a 0.61±0.15a 2.68±0.74b 76.67

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

Table 3. Moisture, crude protein, lipid and ash content in gutted body were estimated for the crayfish fed diets containing different levels of soybean Biochemical parameters Moisture (%) Protein (%) Lipid (%) Ash (%)

Reference diet 83.53±1.78a 74.00±11.88a 23.95±2.71b 6.44±0.13a

Diets A diet 85.70±2.16a 78.67±6.77a 22.64±2.96b 7.63±0.21b

B diet 85.22±1.86a 75.15±10.32a 18.93±1.98a 7.52±0.32b

Values in the same row with different letter notation statistically significantly different at p<0.05;

Fig. 1. Enzyme activity levels in the hemolymph of the crayfish fed with diets containing different levels of soybean meal Carcass analysis of crayfish treated with varied diets shows no significant difference between moisture percentage and carcass protein percentage in different groups (p>0.05), while fat content in the crayfish treated with diet (B) and ash content in the crayfish treated with commercial shrimp diet compared with other groups were significantly lower (p<0.05); (Table 3). Alkaline phosphatase activity level in the hemolymph of the narrow-clawed crayfish treated with diet (B) compared with other experimental groups was reduced significantly (p<0.05). Lactate dehydrogenase activity level in the hemolymph of the crayfish treated with diet (A) was significantly higher than the activity level of this enzyme in the hemolymph of the crayfish treated with commercial shrimp diet (p>0.05). No significant change was observed in the activity level of aspartate aminotransferase and alanine aminotransferase

in the hemolymph of the crayfish treated with varied diets (p<0.05); (Figure 1). There is no significant difference in hemolymph glucose level of narrow-clawed crayfish in varied treatments (p>0.05). The results show that cholesterol level in the hemolymph of the narrow-clawed crayfish fed with diet (B) is significantly lower (p<0.05) compared with other groups. Triglyceride level in the hemolymph of the crayfish fed with diet (A) was significantly higher than the level of this biochemical index in the hemolymph of the crayfish fed with diet (B) (p<0.05); (Figure 2). Although total protein level in the hemolymph of the crayfish fed with diet (A) is significantly higher than that of the crayfish fed with commercial shrimp diet (p<0.05), there is no significant difference between total protein level in the hemolymph of the crayfish fed with diet (B) and that of other groups (p>0.05); (Figure 3). 49

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

Fig. 2. Hemolymph biochemical parameters of the crayfish fed with diets containing different levels of soybean meal

Fig. 3. Hemolymph biochemical parameters of the crayfish fed with diets containing different levels of soybean meal 50

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

discUssion Narrow-clawed crayfish is the only native freshwater crayfish species in Iran that has been a candidate for breeding species in aquaculture industry of Iran in recent years. Although, many attempts have been made to culture this species, the lack of knowledge about the nutritional needs and favourite foods of this species has led to failure of these efforts. The use of different food sources including animal protein, plant protein source or a combination of both in crayfish diet can help expand our knowledge in order to provide a proper diet for rearing this species. The optimal protein requirements for most cultivated crayfish have shown levels of 25 to 60% in the dry diet (Amaya et al., 2007). Fish meal is the most important protein ingredient used in diets of farmed fish and crustaceans, and also serves as a food ingredient in human diets. Fish meal contains high levels of essential amino acids and fatty acids. The soybean meal has high potential to be used as a supplement and substitute for fishmeal as a protein source in the diets of aquatic species (Dersjant-Li, 2002). Although the adverse effects of soybean meal in food diet of cultured aquatic species on growth and development, indices and biochemical parameters are verified (Wang et al., 2006; Førde-SkjÌrvik et al., 2006; Venou et al., 2006) in most studies, there is no report on crustaceans growth reduction or adverse effect of soybean as an alternative to fish meal (García-Ulloa, 2003; Muzinic et al., 2004; Thompson et al., 2005, 2006; Campana-Torres et al., 2005, 2006). Considering the amount of food remaining in the food trays, the crayfish which were fed with a higher percentage of fish meal were more interested in food, which might be due to balanced amino acid profiles in their diet. Lower molting, mortality and cannibalism were observed in groups fed with diets higher on fishmeal. Lack of some essential animal acids including methionine and cystine compared with fish meal (Peres and Lim, 2008), or the presence of some anti-nutritional materials in plant protein sources (Krogdahl et al., 2010), are accounted for certain deficiencies in species and can affect their appetite and increase tendency to cannibalism. It is worth noting that molting among crayfish is the major driving factor in increasing cannibalism in this species. However, by providing them shelters, cannibalism was resolved to a great extent. Final weight of the crayfish fed with diet A was significantly higher. Therefore at first it might seem that utilization of a diet having a higher percentage of animal protein is better, compared with a diet having a higher percentage of plant protein. Our results show that increasing the rate of soybean meal

in diet can decrease the weight gain percentage, special growth rate and increase the feed conversion ratio (FCR) (Table 2). The results of studies carried out by Changizi et al. (2010) and Ghiasvand et al. (2012) show that using varied levels of protein in diet of Astacus leptodactylys could have impressive effects on growth indices such as the increase of final weight growth, average body weight, food conversion ratio, protein efficiency and special growth rate. In contrast to our finding, using soybean as a plant protein source in diet of other crustaceans and cultured crayfish could have similar results to those when fish meal is used as an animal protein in diet (McClain and Romaire, 2009). For example, in the study done on growth indices of Australian red claw crayfish (Charas quadricarinatus) no significant difference was observed in a group which was fed with a diet containing 35% of raw protein of plant origin such as soybean and a group which was fed with protein of animal origin (Thompson et al., 2005). Since carcass quality determines marketability of a product, farmers pay attention to this factor. According to the obtained results, there is a significant difference between animal protein level in the diet and fat content in the carcass; i.e. with the increase of fish meal as an animal protein source in the diet, fat level of the carcass increases. This might be due to the fact that protein level might be more than a physiological need; therefore an extra amount of protein is stored in the form of fat or carbohydrate (Cowey and Sarjent, 1979). However, fat percentage in the carcass of the crayfish fed with a diet containing a higher ratio of soybean was significantly lower compared with that of other groups, which might be due to the influence of soybean compounds on the lipid metabolism process (Sugiyama et al., 1980). The analysis of crayfish carcass shows that by changing the diet, no significant change is observed in the moisture and protein percentage of carcasses (Table 3). Similar results were observed in studies done on Eriocheir sinesis; in these crustaceans the change of protein level and source had no impact on the protein level, and source had no impact on the protein level of young crayfish carcasses (Mu et al., 1998). However, in a similar study done on Eriocheir sinensis, the increase of protein level and fish meal proportional to soybean in the diet, protein level of carcass increased as well (Chen et al., 1994). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are found in varied tissues such as hepatopancreas, gill, muscles and the hemolymph of crustaceans (Berges and Ballantyne, 1991; Jiang et al., 2012). So, activity level of these enzymes was used as a biomarker to assess the hepato-pancreas health; because it is expected that 51

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

using plant protein source which contains anti-nutritional compounds might disturb the performance of hepatopancreas activity and affect the synthesis level and activity of AST and ALT. However, with regard to the absence of significant changes in the activity level of aspartate aminotransferase and alanine aminotransferase of the hemolymph in the crayfish fed with different diets, we can assert that the change of protein source had no effect on the activity level of this enzyme in the hemolymph of narrow-clawed crayfish (Figure 1). Lactate dehydrogenase is one of the most important active enzymes participating in anaerobic pathway of carbohydrate metabolism which is found in different tissues of crustaceans, such as hemolymph, ovary, gills, hepatopancreas, muscles and spermatheca (Chourpagar and Kulkarni, 2009; Sreenivasan et al., 2010; Banaee and Ahmadi, 2011). Therefore, any kind of change in the activity level of this enzyme illustrates a change in the process of anaerobic metabolism; in such a case the process of converting pyruvate to lactate and the regeneration of glucose is accelerated (Banaee and Ahmadi, 2011). However, there was a significant difference in the activity level of lactate dehydrogenase in the hemolymph of the crayfish fed with a diet containing a higher percentage of fishmeal compared with the activity level of the same enzyme in the hemolymph of the crayfish which were fed with commercial shrimp diet. But the absence of a significant difference between the activity level of LHD enzyme in the hemolymph of the crayfish which were respectively fed with animal and plant protein source indicates that the change of diet in this species had no effect on the activity of this enzyme (Figure 1). Alkaline phosphatase plays a significant role in phosphate hydrolysis and in membrane transport. It is also a good clinical biomarker of health status of aquatic species which is found in different tissue of crustaceans such as hemolymph, gills, muscle and hepatopancreas (Sreenivasan et al., 2010; Banaee and Ahmadi, 2011). ALP has a significant role in cell membrane and could be used as a proper biological index for assessing the health of cell membrane. Measuring the activity level of this enzyme in investigating the performance of varied tissues especially hepatopancreas is of a great importance (Banaee and Ahmadi, 2011). The present study suggests that we can increase the rate of soybean meal in diet without negative effects on the ALP level in the hemolymph of crayfish (Figure 1). Based on the obtained results, glucose level in the hemolymph of the crayfish treated with varied diets does not show a significant difference. Glucose is a basal monosaccharide which is found in the hemo52

lymph of crustaceans and has a great role in their physiology, including the synthesis of muco-polysaccharides, synthesis of chitin, ribosome and reduced nicotine amide dinucleotide phosphate (NADPH), the production of pyruvate, glycogen synthesis and is also an energy generation source (Chang and O’Conner, 1983). Stable level of glucose in hemolymph is essential for regulating the performance and activity of neural, muscular and reproductive system of crustaceans. Also, glucose can accumulate as glycogen in hepatopancreas and other tissues such as muscles and gill (Vinagre and Da Silva, 2002; Oliveira et al. 2003). Production and storage cycle of glycogen and glucose level of hemolymph are so varied and can change depending on varied factors: stages of growth, molting, season, diet, nutritional condition, circadian cycle, salinity and dissolved oxygen level in water (Oliveira et al., 2004a,b). Therefore, the absence of glucose level change of hemolymph indicates that the change of plant protein proportion to animal protein proportion of the diet did not have any effects on the level of this biochemical factor (Figure 2). The absence of fat tissue in crustaceans has made hepatopancreas the most important fat storage place, yet lipids can accumulate in muscle tissue and gonads of females (García et al., 2002). For instance, in burrowing crab Chasmagnathus granulatus the total lipid level was more than 20% of hepatopancreas weight (Kucharski and Da’Silva, 1991). Therefore any kind of change in the cholesterol level and triglyceride of hemolymph can act as an index in assessing the health of hepatopancreas. A lower level of cholesterol in the hemolymph of the narrow-clawed crayfish fed with diet B compared with commercial and diet A was found, as well as a significant decrease of triglyceride level in the hemolymph of the crayfish fed with diet B when compared with the triglyceride level in the hemolymph of the crayfish fed with diet A (Figure 2). Since arginine amino acids have higher ratio than lysine amino acids (the same is also true for glycine in comparison to methionine in soybean protein), a decrease of cholesterol in the crayfish which were fed with plant diets is expected. Likewise, a higher proportion of methionine and lysine amino acids in fish meal protein could lead to a higher cholesterol level in the blood (Sugiyama et al., 1980; Morita and Oh-Hashi, 1997). The presence of isoflavones such as genistine, daidzein and glyctin in soybean (Setchell and Raad, 2000) could decrease the fat level of blood in animals under diet, due to having anti-oxidant, anti-bacterial and anti-inflammatory features (Verdrengh et al., 2003). Although total protein level in the hemolymph of the crayfish fed with a higher proportion of ani-

Croatian Journal of Fisheries, 2013, 71, 45-57 M. Banaee et al.: Effects of Soybean Meal on Growth Performance of Narrow-clawed Crayfish

mal protein was significantly higher than that of the crayfish fed with commercial shrimp diet, there was no significant difference in the total protein level in the hemolymph of the crayfish treated with diets based on soybean plant protein, in comparison to other groups (Figure 3). In crustaceans, muscles are apparently the most important place for storing protein and the level of free amino acids in their tissues is several times higher than that of vertebrates (Chen et al., 1994). Studies show that amino acids have a role in osmoregulation and the control of cell volume (Chang and O’Conner, 1983; Schein et al., 2005). Since there is a balance between stored protein content in tissues and total protein level in hemolymph, with regard to the absence of carcass protein level in the crayfish which were fed with plant and animal protein-based diets, it can be concluded that the change of diet has no effect on the total protein level of hemolymph in crayfish.

conclUsion Although with the increased rate of soybean meal in diet weight gain and specific growth rate of crayfish reduced, no significant changes were observed in biochemical parameters. Crayfish can feed on both animal and plant protein sources, however, according to the results obtained in the present study, we recommend that the effect of soybean meal on growth performance and biochemical parameters of crayfish should be evaluated before empirical use of different ratios of soybean meal as a protein source in their diet.

acKnowlEdGEMEnts The authors gratefully acknowledge the Managing Director and staff of the Aras Shrimp Maku Company (West Azarbaijan Province, Iran) for their cooperation and assistance throughout the research.

Sažetak Učinci hranidbE sojiniM brašnoM na PErforMancU rasta i bioKEMijsKa svojstva hEMoliMfE dUnavsKoG raKa (Astacus leptodacylus Eschscholtz, 1823) Poput drugih vrsta rakova, dunavski rak (Astacus leptodacylus Eschscholtz, 1823) tijekom različitih životnih ciklusa može promijeniti svoju hranidbu ovisno o hrani koja je dostupna. Promjena hranidbe može utjecati na različite biološke indekse ove vrste,

stoga ova studija ima za cilj proučiti promjene koje se mogu pojaviti tijekom promjene hranidbe raka u indeksima rasta, biokemijskim svojstvima hemolimfe i biokemijskim svojstvima njihova mesa. Svrha ovog eksperimenta je procijeniti učinke djelomične zamjene riblje hrane sa sojinim brašnom na performancu rasta, kvalitetu mesa i biokemijska svojstva hemolimfe dunavskog raka. 90 zdravih adultnih dunavskih rakova (W=35,50±4,05 g; TL=16,96±1,92 cm) su raspoređeni nasumce u 9 bazena od fiberglasa (200 L) te su tijekom 45 dana hranjeni s tri različite vrste hrane, uključujući hranidbu komercijalnim škampima, ribljom hranom (A) i sojinim brašnom (B). Rezultati su pokazali da je promjena hranidbe životinjskim proteinom u hranidbu biljnim proteinom uzrokovala značajan pad razine kolestoerola i triglicerida u hemolimfi, kao i masti mesa rakova koji su jeli hranu B, u usporedbi s rakovima koji su jeli hranu A. Nisu zamijećene značajnije promjene razine glukoze hemolimfe, AST i ALT prilikom različitih tretmana tijekom ovog eksperimentalnog razdoblja. Kao zaključak, utvrđeno je da iako se smanjila performanca rasta, povećana hranidba sojinim brašnom od 0,0% do 76% nije imala nikakvih negativnih učinaka na biokemijska svojstva. Ključne riječi: dunavski rak, izvor proteina, indeksi rasta, kvaliteta mesa, biokemijska svojstva

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Croatian Journal of Fisheries, 2013, 71, 58-64 T. Janči i sur.: Prehrambena vrijednost pastrve uzgojene u moru T. Janči et al.: Nutritive value of trout farmed in sea

CODEN RIBAEG ISSN 1330-061X

IZVORNI ZNANSTVENI ČLANAK / ORIGINAL SCIENTIFIC PAPER

PrEhraMbEna vrijEdnost KalifornijsKE PastrvE (Oncorhynchus mykiss) UzGojEnE U jadransKoM MorU tibor janči1, danijel Kanski2, Marina dulić3, nives Marušić1, helga Medić1, tomislav Petrak1, sanja vidaček*1 Prehrambeno-biotehnološki fakultet, Pierottijeva 6, 10 000 Zagreb, Hrvatska / Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10 000 Zagreb, Croatia 2 Dalmar doo, Obala kralja Petra Krešimira IV, 64, 23 211 Pakoštane, Hrvatska / Dalmar doo, Obala kralja Petra Krešimira IV, 64, 23 211 Pakoštane, Croatia 3 Lavanda Mia d.o.o., Ilica 51, 10 000 Zagreb, Hrvatska / Lavanda Mia d.o.o., Ilica 51, 10 000 Zagreb, Croatia * Corresponding Author, E-mail: svidacek@pbf.hr 1

STATUS ČLANKA / ARTICLE INFO

SAŽETAK

Primljeno/Received: 15 February 2013 Korigirano/Received in revised form: 1 May 2013 Prihvaćeno/Accepted: 22 May 2013 Dostupno na internetskoj mreži/Available online: 25 May 2013

Cilj ovog rada bio je određivanje kemijskog sastava fileta svježe i hladno dimljene kalifornijske pastrve (Oncorhynchus mykiss) uzgojene u Jadranskom moru mjerenjem udjela vode, masti, proteina, soli, pepela, sastava masnih kiselina s naglaskom na eikozapentaensku (EPA) i dokozaheksaensku (DHA) masnu kiselinu te fizikalnih karakteristika mjerenjem pH i određivanjem boje. Analize su izvršene na homogeniziranom mišićju nakon odvajanja kože i kostiju. Određivanje udjela vode, pepela, ukupne količine masti i proteina izvršeno je prema referentnim metodama AOAC (1995). Za određivanje sastava masnih kiselina pripremljeni su metilni esteri prema metodi HRN EN ISO 5509 (2004) te su analizirani plinskom kromatografijom prema metodi HRN EN ISO 5508 (1999). Rezultati su pokazali da je istražena svježa pastrva uzgojena u moru odličan izvor proteina (21,21%) te da ima nešto niže vrijednosti udjela masti (5,21%) i omega-3 masnih kiselina (12,52%) od rezultata drugih istraživanja. Proces hladnog dimljenja ne uzrokuje gubitke na udjelu masti i omega-3 masnih kiselina. Fileti svježe i dimljene kalifornijske pastrve uzgojene u Jadranskom moru mogu se smatrati namirnicama visoke prehrambene vrijednosti.

Ključne riječi: Prehrambena vrijednost Omega-3 Pastrva uzgojena u moru Hladno dimljena riba

Uvod U posljednjih nekoliko godina porastao je interes za konzumaciju ribe, uvelike zbog činjenice da konzumacija ribe pogoduje zdravlju. Riba se tradicionalno preporučuje u prehrani kao značajan izvor visokovrijednih bjelančevina i masti, minerala i vitamina, osobito onih topivih u mastima. Blagodat ribe za zdravlje povezuje se i s visokim sadržajem dugolančanih polinezasićenih omega-3 masnih kiselina (polyunsaturated fatty acids - PUFA), posebice eikozapentaenske (eicosapentaenoic acid - EPA) i dokozaheksaenske (docosahexaenoic acid - DHA). 58

Dokazano je da su omega-3 masne kiseline bitne za neurorazvoj djece, in utero te tijekom prvih nekoliko godina života, a imaju i koristan učinak kod koronarne bolesti srca, hipertenzije, upala, aritmija, psorijaze, agresije, depresije i raka (AHA, 2011). Kako je svjetski trend povećanje potrošnje uzgojene ribe u odnosu na ulovljenu (FAO, 2012), brojna istraživanja se baziraju na ispitivanju utjecaja uzgoja na prinos i zdravlje riba, njihov kemijski sastav i okoliš. Uzgoj pastrve u moru pokazao se povoljnim s obzirom na dobar prirast ove vrste pri višem salinitetu okruženja (Austreng i sur., 1987). Osim povoljnog utjecaja na prirast, salinitet utječe i na

Croatian Journal of Fisheries, 2013, 71, 58-64 T. Janči i sur.: Prehrambena vrijednost pastrve uzgojene u moru T. Janči et al.: Nutritive value of trout farmed in sea

probavljivost hrane (Usher i sur., 1990; Storebakken i sur., 1998), metabolizam ugljikohidrata (Soengas i sur.,1993) te sastav masnih kiselina, posebno odnos omega-3 prema omega-6 masnim kiselinama za koji je utvrđeno da je puno niži kod riba uzgojenih u slatkoj vodi s obzirom na morsku (Steffens, 1997). Znanstveno dokazana na brojnim vrstama riba, pozitivna korelacija između omega-3 masnih kiselina u ribljoj hrani i njezina sadržaja u ribljem mesu, omogućuje uzgajivačima riba da ciljanom tehnologijom uzgoja određenih vrsta proizvode vrlo kvalitetno meso s visokim sadržajem omega-3 masnih kiselina (Brown i sur., 2010; Miller i sur., 2007; Turchini i sur., 2003). Sadržaj lipida ribljeg mesa u velikoj mjeri ovisi o sastavu masnih kiselina u hrani riba, a kod uzgojene ribe se može prilagoditi podešavanjem prehrambenog unosa. Kako riba iz uzgoja općenito ima veće ukupne razine lipida od divlje ribe, 100 g fileta uzgojene ribe može biti bolji izvor omega-3 PUFA (osobito EPA i DHA) od 100 g divlje ribe. Omega-3 masne kiseline su podložne oksidaciji tijekom pohrane ribe na ledu, zamrzavanja, toplinske obrade i prerade (Chaijan i sur., 2006; Guillén i Ruiz, 2004; Moradi i sur., 2011; Panpipat i Yongsawatdigul, 2008; Saldanha i Bragagnolo, 2008). Prooksidansi su prisutni u samoj ribi, kao npr. željezo, no značajniji utjecaj na brzinu oksidacije masti imaju oni vanjski, kao što su prisustvo zraka, sol, povišena temperatura. Kako su sol i povišena temperatura glavni antimikrobni čimbenici kod prerade većine riba (konzerviranje), tako se i udio omega-3 masnih kiselina smanjuje tijekom ovih postupaka prerade. U Republici Hrvatskoj riba se najčešće prodaje svježa i zamrznuta te prerađena u sterilizirane i soljene proizvode, konzervira se i soli, no na hrvatskom tržištu prisutni su i marinirani te dimljeni proizvodi. U svijetu je dimljenje vrlo rasprostranjena metoda konzerviranja ribe koja omogućuje dobivanje proizvoda zanimljivih tržištu. Potrošačima su dimljeni proizvodi zanimljivi prvenstveno zbog karakteristične arome (Fuentes i sur., 2010) i svojstvene boje (Torrissen i sur., 2000). Cilj ovog rada je određivanje fizikalno i kemijskih svojstava fileta svježe i hladno dimljene pastrve kao i sastava masnih kiselina, s naglaskom na eikozapentaensku (EPA) i dokozaheksaensku kiselinu (DHA) za procjenu prehrambene vrijednosti pastrve uzgojene u Jadranskom moru.

MatErijal i MEtodE rada U ovom istraživanju korišteni su fileti kalifornijske pastrve (Oncorhynchus mykiss) uzgojene u Velebitskom kanalu na uzgajalištu tvrtke „Jadran riba d.o.o.“ te izlovljene u svibnju i lipnju 2011. u

starosti od dvije godine. Tijekom uzgoja riba je hranjena ekstrudiranom ribljom hranom proizvođača „Skretting“ sa dvije linije hrane. U prvoj godini uzgoja u moru korištena je hrana „Royal Optima“ granulacije 3 mm i 4,5 mm sa omjerom proteina i masti od 45:27% i 43:30% i bez dodanih pigmenata. U posljednjih 6 mjeseci korištena je hrana „Focus Salmo“ granulacije 6mm, omjerom proteina i masti 40:24% s dodanim pigmentima. Pastrve su nakon izlova poleđene te su prenesene na Prehrambeno-biotehnološki fakultet u Zagrebu. Pojedinačna masa riba iznosila je 1,3±0,12 kg. Nakon 24 sata skladištenja u hladnjaku na temperaturi + 7 °C, uzorci riba su evisecerirani, filetirani te su fileti izvagani na laboratorijskoj vagi KB 2000-2N (Kern & Sohn GmbH). Masa fileta s kostima i kožom iznosila je 450 ± 25,0 g. Završna faza pripreme uzoraka obuhvaćala je odstranjivanje kože i kostiju, homogenizaciju očišćenog mesa te uzimanje uzoraka odgovarajuće mase za daljnje analize. Dimljenje fileta provedeno je prema internoj recepturi „BEK-pušnice d.o.o.“. Nakon prijema u pogon, ručnog filetiranja i pranja, fileti s kožom su se salamurili u otopini krupne i sitne soli te šećera. Salamurenje je trajalo 12-24 sata, ovisno o veličini ribe. Nakon salamurenja, fileti su dimljeni hladnim dimom (do 32 °C) dobivenim iz drveta bukovine i borovnice (80%/20%). Trajanje procesa dimljenja trajalo je minimalno 3 dana, ovisno o veličini fileta. Nakon završenog procesa dimljenja, pristupilo se završnoj obradi fileta, nakon čega su fileti pakirani u vakumu. Za analize su korišteni uzorci homogeniziranog mišićja nakon odvajanja kože i kostiju. Za istraživanje korišteni su uzorci svježih riba te uzorci vakumiranih dimljenih fileta. Istraživanje je provedeno na osam primjeraka iz svake kategorije, na 2 do 4 paralelna uzorka, ovisno o metodi. Određivanje udjela vode, pepela, ukupne količine masti i proteina izvršeno je prema referentnim metodama AOAC (1995). Mast dobivena ekstrakcijom korištena je za određivanje sastava masnih kiselina. Esterski vezane masne kiseline prevedene su u metilne estere masnih kiselina koji su pogodni za analizu plinskom kromatografijom (HRN EN ISO 5509, 2004). Sastav masnih kiselina određivan je metodom plinske kromatografije (HRN EN ISO 5508, 1999) uređajem CP-3800 (Varian, Palo Alto, CA, SAD). Za injektiranje je korišten TriPlus autosampler (Thermo Scientific, Augustin, TX, SAD). Temperatura injektora s mogućnošću djelomičnog unošenja uzorka je bila 250 oC a volumen injektiranja 1 μL uz omjer razdjeljenja 1:30. Uzorci su analizirani na kapilarnoj koloni DB-23 duljine 60 m, unutrašnjeg promjera kapilare 0,25 mm i debljine sloja selektivne tekućine 0,25 μm (Agilent, Walnut Creek, CA, SAD), a tem59

Croatian Journal of Fisheries, 2013, 71, 58-64 T. Janči i sur.: Prehrambena vrijednost pastrve uzgojene u moru T. Janči et al.: Nutritive value of trout farmed in sea

peraturni program kolone je bio: početna temperatura kolone 600C, brzina porasta temperature 70C / min do konačne temperature kolone 2200C koja je zadržana 15 min. Plin nosioc je bio helij uz protok od 1,5 mL/min. Temperatura plameno-ionizacijskog detektora je bila 2600C. Za obradu podataka korišten je računalni program Star GC Workstation Ver. 6.4 (Varian, Palo Alto, CA, SAD). Vrijednosti udjela pojedinih masnih kiselina prikazani su kao % od ukupnih masnih kiselina. Vrijednost pH mjerena je izravnim ubodom u mišić digitalnim pH-metrom (704 pH Meter, Metrohm, Švicarska) uz ubodnu staklenu elektrodu 6.0236.100, istog proizvođača. Za utvrđivanje boje korišten je spektrofotometar CM-700d/600d (Konica Minolta). Mjereni parametri boje bili su L* (svjetloća), a* (crveno-zeleni spektar, stupanj crvenila mesa) i b* (žuto-plavi spektar, stupanj žute boje mesa) prema CIELAB modelu (CIE, 1976). Udio natrijevog klorida utvrđen je titracijskom metodom po Mohru (AOAC, 1995). Statistička analiza (jednostruka ANOVA) provedena je u programu SPSS (SPSS Statistics 17.0).

rEzUltati i rasPrava Rezultati analize kemijskog sastava fileta pastrve prikazani su u tablici 1. Udio masti fileta svježe pastrve iznosio je 5,21 % , što je niže od udjela masti koji su dobili drugi autori na pastrvama uzgojenima u morima sjeverne Europe. Kennedy i sur. (2007) istraživali su pastrvu uzgojenu u Sjevernom moru prosječne mase oko 450 g hranjenu standardnom smjesom, a udio masti fileta iznosio je 7,2%. Udio masti fileta pastrve s kožom mase 650 g, uzgojene u Norveškom moru iznosio je 12,2 % (Krogdahl i sur., 2004). Udio proteina fileta u ovom istraživanju je visok (21,2%) s obzirom istraživanje Krogdahl i sur. (2004) gdje je udio proteina iznosio 17,1%, no kako je u navedenom radu analizirano mišićje sa kožom, udio proteina je niži nego što bi bio da su analize provedene samo na mišićju. Udio vode iznosio je 71,9 %, što je nešto niže od rezultata Vidaček i sur. (2011) dobivenih analizom fileta svježe kalifornijske pastrve uzgojene u slatkoj vodi gdje je srednja vrijednost iznosila 78,0%. Rezultati izmjera pH vrijednosti u filetima svježe pastrve slični su rezultatima koje su dobili Vidaček i sur. (2011). Dobivena pH vrijednost iznosila je 6,43 dok je u navedenom radu iznosila 6,25. Mjereni parametri boje pokazali su da je meso kalifornijske pastrve uzgojene u moru slične boje kao uzgojeni losos što je rezultat hranidbe, tj. odlaganja karotenoidnih pigmenata unesenih hranom u mi60

šićju (Rørå i sur., 1998). L* u ovom radu iznosio je 37,10 ± 3,04; a* 13,05 ± 2,37 i b* 14,62 ± 2,78. Rørå i sur. (2005) ispitivali su stupanj gubitka boje kod lososa pri različitim temperaturama dimljenja te su zaključili da nema promjene u boji kod hladnog dimljenja, a iznosi za L*, a* i b* iznosili su redom 43,0; 8,3 i 16,6. Usporedbom ovih podataka, može se zaključiti da fileti ribe ispitani u ovom radu imaju jaču crvenu boju od boje lososa u radu Rørå i sur. (2005). Tablica 1. Srednje vrijednosti ( ) i standardne devijacije (sd) kemijskih i fizikalnih parametara svježe i hladno dimljene pastrve uzgojene u moru n=16 Table 1. Average values ( ) and standard deviations (sd) of physical and chemical parameters of fresh and cold-smoked sea-farmed trout n=16 Fizikalno-kemijski parametar / Physico-chemical parameter Voda / Water (%) Mast / Fat (%) Proteini / Protein (%) Pepeo / Ash (%) NaCl (%) L* a* b* pH

Svježa riba / Fresh fish ±sd

Dimljena riba / Smoked fish ±sd

71,86 ± 0,88a 5,21 ± 1,25a

65,54 ± 1,93b 4,95 ± 2,62a

21,21 ± 0,63a

24,23 ± 0,26b

1,31 ± 0,15a 37,10 ± 3,04 13,05 ± 2,37 14,62 ± 2,78 6,43 ± 0,00

4,10 ± 0,59b 3,17 ± 0,88 -

Različita slova (a, b) pokazuju statističku razliku između svježih i dimljenih uzoraka (p<0,05) Different letters (a, b) show statistical difference betwen fresh and smoked samples (p<0.05)

Kemijski sastav hladno dimljenih proizvoda ovisi o sastavu sirovine i parametrima hladnog dimljenja. Hladno dimljeni riblji proizvodi obično sadrže udio natrijevog klorida 2,5-4,5% i vode 65-70% (Espe i sur., 2004; Karásková i sur., 2011; Rørå i sur.,

2005). Prema rezultatima određivanja kemijskog sastava dimljene pastrve u ovom radu prikazanih u tablici 1, udio vode (65,54%) i natrijevog klorida (3,17%) odgovaraju parametrima hladnog dimljenja. Identičan podatak za sadržaj natrijevog klorida u hladno dimljenom lososu dobili su i Rørå i sur. (2005). Sniženje vode uslijed procesa sušenja uzrokuje proporcionalno povišenje suhe tvari. Rezultati su pokazali povišene udjele proteina i pepela dimljenih uzoraka s obzirom na svježe, no udio masti nije se statistički značajno promijenio nakon dimljenja. Espe i sur. (2002) istražujući atlantskog lososa također su utvrdili da kemijski sastav dimljenih fileta općenito reflektira vrijednosti u svježem filetu, me-

Croatian Journal of Fisheries, 2013, 71, 58-64 T. Janči i sur.: Prehrambena vrijednost pastrve uzgojene u moru T. Janči et al.: Nutritive value of trout farmed in sea

đutim sadržaj masti se nije značajno promijenio nakon dimljenja niti u navedenom radu. Rezultati sastava masnih kiselina prikazani su u tablici 2. Najzastupljenije predstavnice SFA i MUFA su identične, no najzastupljenija PUFA kod drugih autora je DHA, čiji je udio u ukupnoj masti oko 2,5 puta viši nego u ovom radu i iznosi 16,5% (Haliloğlu i sur., 2004), odnosno 17,3% (Kennedy i sur., 2007) kod svježe pastrve. Razlog za tako niske vrijednosti za DHA u ovom istraživanju mogle bi se djelomično pripisati visokom salinitetu Jadranskog mora. Naime, Haliloğlu i sur. (2004) istraživali su sastav masti kod pastrve uzgojene u slatkoj vodi i Crnom moru, a hranjene identično. Njihovi rezultati su pokazali da utjecaj saliniteta nema utjecaja na omjere SFA, MUFA i PUFA, ali da postoji značajan utjecaj na omjer EPA i DHA te da je kod pastrve uzgojene u Crnom moru (salinitet 17‰) došlo do smanjenja DHA za 33% s obzirom na onu slatkovodnu. Jadransko more je izrazito slano, 38 39‰ (Janeković i sur., 2006) što je moguće utjecalo na tako niske vrijednosti za DHA. No, salinitet ne može u potpunosti objasniti rezultate ovoga rada, jer su i vrijednosti za EPA niže od onih iz literature, a prema Haliloğlu i sur. (2004), salinitet povećava sadržaj EPA. U ovome radu sadržaj EPA je 3,19%, što je oko dva puta manje nego kod drugih autora za pastrvu uzgojenu u moru gdje su dobivene vrijednosti iznosile 6.2% (Haliloğlu i sur., 2004) odnosno 8.7% (Kennedy i sur., 2007). Prema preporukama American Heart Association (AHA, 2011) unos EPA+DHA u prehrani ljudi trebao bi iznositi minimalno 500 mg/dan. Uzgojeni losos se smatra jednim od najboljih izvora EPA i DHA, te se vrijednosti ovih masnih kiselina obično kod lososa procjenjuju na oko 2000 mg u porciji od 180 g fileta, čime se dolazi do opće poznatih zdravstvenih preporuka za EPA i DHA u smislu prevencije kardivaskularnih bolesti, prema kojima je potrebno konzumirati masnu ribu dva puta tjedno (AHA, 2011). U ovome radu, uz faktor konverzije 0,9 (Greenfield i Southgate, 2003), dobivene vrijednosti za EPA+DHA iznose oko 900 mg za porciju od 180 g, što je dvostruko manje od lososa. Omjeri SFA, MUFA i PUFA kod svježih i dimljenih uzoraka prikazani su slikom 1 te ne odgovaraju omjerima u drugim istraživanima. Prema podacima iz Haliloğlu i sur. (2004), sadržaj SFA iznosio je 27,4%; MUFA 34,3% te PUFA 20,9%, dok su rezultati u ovom radu 23,14%; 40,11% te 35,53%. Kod Kennedy i sur. (2007), sadržaj SFA iznosio je 30,0%; MUFA 30,1% te PUFA 39,9%. Prema rezultatima sastava masnih kiselina kod dimljenih uzoraka, vidljivo je da nema velikih razlika u sadržaju masnih kiselina s obzirom na svje-

že uzorke (tablica 2). Statistička razlika (p ≤ 0,05) postoji samo kod oleinske masne kiseline čiji je sadržaj niži kod dimljenih uzraka, te kod linolne i DHA čiji je sadržaj viši kod dimljenih nego kod svježih uzoraka. Kod određivanja sastava masnih kiselina u takvom kompleksnom matriksu kao što je mišićno tkivo riba, prisutna je velika varijabilnost rezultata (Stołyhwo i sur., 2006) što je mogući razlog razlika u sadržaju ovih pojedinačnih masnih kiselina između svježih i dimljenih fileta. Omjeri SFA, MUFA i PUFA nisu značajno različiti kod dvije skupine uzoraka (slika 1). Povećani sadržaj DHA u dimljenom filetu pastrve u odnosu na svježe filete ribe djelomično odgovara rezultatima koje su dobili Espe i sur. (2002), a koji pokazuju da se sadržaj DHA (ali i EPA) povećao hladnim dimljenjem lososa. Tablica 2. Sastav masnih kiselina svježe i hladno dimljene pastrve uzgojene u moru (% od ukupnih masnih kiselina ± standardna devijacija) n=16 Table 2. Fatty acid content of fresh and cold-smoked sea-farmed trout (% of total fatty acids ± standard deviation) n=16 Masna kiselina/ Svježa riba/ Dimljena riba/ Fatty acid Fresh fish (%±sd) Smoked fish (%±sd) 2,97 ± 0,37a C14:0 2,73 ± 0,18a C15:0 0,23 ± 0,01a 0,24 ± 0,01a C16:0 14,05 ± 0,40a 13,42 ± 0,91a C16:1 3,75 ± 0,34a 3,86 ± 0,92a a C17:0 0,47 ± 0,08 0,56 ± 0,08a a C17:1 0,34 ± 0,06 0,41 ± 0,06a a C18:0 3,46 ± 0,21 3,38 ± 0,06a b C18:1c 30,40 ± 2,41 26,97 ± 2,16a b C18:2c 21,59 ± 0,53 22,75 ± 1,09a a C18:3n6 0,40 ± 0,04 0,42 ± 0,14a a C18:3n3 2,82 ± 0,15 2,85 ± 0,27a a C20:0 0,89 ± 0,12 0,95 ± 0,11a a C20:1 2,32 ± 0,21 2,10 ± 0,24a a C21:0 0,80 ± 0,10 0,81 ± 0,07a a C20:3n6 0,48 ± 0,07 0,48 ± 0,01a a C20:4n6 0,53 ± 0,04 0,57 ± 0,02a C22:0 0,51 ± 0,08a 0,54 ± 0,02a C20:5n3 3,19 ± 0,87a 4,05 ± 0,57a C22:1 1,77 ± 0,13a 1,65 ± 0,26a C24:1 1,54 ± 0,24a 1,87 ± 0,50a b C22:6n3 6,51 ± 0,27 7,53 ± 0,16a a n.i. 1,22 ± 0,25 1,61 ± 0,84a % omega-3 12,52 14,42 % omega-6 23,59 24,23 Različita slova (a, b) u istom redu pokazuju statističku razliku između svježih i dimljenih uzoraka (p<0,05) Different letters (a, b) in the same row show statistical difference betwen fresh and smoked samples (p<0.05)

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Croatian Journal of Fisheries, 2013, 71, 58-64 T. Janči i sur.: Prehrambena vrijednost pastrve uzgojene u moru T. Janči et al.: Nutritive value of trout farmed in sea

Dobro je poznato da je oksidacija masti jedan od najvećih problema u ribljim proizvodima, no faktori koji mogu utjecati na sastav masti tijekom dimljenja djeluju na različite načine na njih. Fenolne komponente dima su antioksidansi, a sol i povišena temperatura prooksidansi, a i same vrste ribe koje se uglavnom dime su uglavnom masnije i time podložne oksidaciji. Losos i pastrva su idealne riblje vrste za ovakav način prerade jer sadrže veliki postotak masti koja u procesu dimljenja upije dim, pa se smatra da takva riba ima bolji okus. Zbog navedenih faktora, u literaturi postoje kontradiktorni podaci o stabilnosti PUFA u dimljenoj ribi. Utjecaj različitih faktora na oksidaciju masti u ribljem tkivu detaljno je obrađen u Kołakowska i sur. (2002).

Slika 1. Udjeli zasićenih (SFA), mononezasićenih (MUFA) i polinezasićenih (PUFA) masnih kiselina kod svježih i hladno dimljenih uzoraka n=16 Fig. 1. Share of saturated (SFA), monounsaturated (MUFA) and poliunsaturated (PUFA) fatty acids in fresh and cold-smoked fish samples n=16 Najvjerojatniji razlog stabilnosti PUFA u ovom istraživanju antioksidativna su svojstva fenolne frakcije dima. Sastav fenolne frakcije, koja može sadržavati više od 200 različitih spojeva, primarno ovisi o vrsti drveta. Najaktivniji fenolni spojevi dima su pirogalol, resorcinol, 4-metil gvajakol, 4-vinil gvajakol i 4-trans-propenil siringol (Sikorski, 1990), te njihova prisutnost u dimu i na površini fileta rezultira stabilnim sastavom masti. Sastav fenolne frakcije dima ovisi i o temperaturi dimljenja (Stołyhwo i sur., 2006). Tako je sadržaj ukupnih fenola u filetima lososa hranjenog ribljim uljem ili sojinim uljem kod Rørå i sur. (2005) bio pozitivno koreliran s temperaturom hladnog dimljenja. Rezultati ovoga rada pokazuju da su parametri procesa dimljenja adekvatno optimizirani jer nije došlo do promjena u sastavu masti odnosno do oksidacije omega-3 masnih kiselina tijekom dimljenja. 62

zaKljUčaK Fileti svježe i dimljene kalifornijske pastrve uzgojene u Jadranskom moru sadrže visoke udjele proteina te nešto niže udjele masti i omega-3 masnih kiselina. Na sastav fileta svježe pastrve uzgojene u Jadranskom moru vjerojatno utječu hranidba i salinitet mora, a na sastav fileta dimljene pastrve sastav sirovine te parametri dimljenja.

Abstract nUtritivE valUE of troUt (Oncorhynchus mykiss) farMEd in thE adriatic sEa The aim of this study was to determine the chemical composition of fresh and cold smoked rainbow trout fillets (Oncorhynchus mykiss) farmed in the Adriatic sea by measuring water, fat, protein, salt and ash content, fatty acid profile with an emphasis on eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids. Physical characteristics were determined by pH and color measurements. Analysis was performed on homogenized fish muscles without skin and bones. Determination of moisture, ash, fat and protein was conducted according to AOAC (1995). Determination of fatty acid content of previously prepared methyl esters (HRN EN ISO 5509, 2004) was conducted by gas chromatography according to HRN EN ISO 5508 (1999). Results showed that fresh rainbow trout farmed in the Adriatic sea is an excellent protein source (21.21%) but has slightly lower fat (5.21%) and omega-3 fatty acid content (12.52%) compared to the results of other studies. Fat and omega-3 fatty acid content was not decreased by the process of cold smoking. Overall, fresh and smoked trout farmed in the Adriatic may be regarded as food high in nutritional value. Keywords: nutritional value, omega-3, sea farmed trout, cold smoked fish

litEratUra AHA (2011) Triglycerides and Cardiovascular Disease: A Scientific Statement From the American Heart Association. Circulation, 123, (20), 2292 – 2333. AOAC (1995): Official Methods of Analysis. 16th edn, Arlington, VA: Association of Official Analytical Chemists. Austreng, E., Storebakken, T., Åsgård, T. (1987): Growth rate estimates for cultured Atlantic

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salmon and rainbowtrout. Aquaculture, 60, 157– 160. Brown, T.D., Francis, D.S., Turchini, G.M. (2010): Can dietary lipid source circadian alternation improve omega-3 deposition in rainbow trout? Aquaculture, 300, 148–155. Chaijan, M., Benjakul, S., Visessanguan, W., Faustman, C. (2006): Changes of lipids in sardine (Sardinella gibbosa) muscle during iced storage.Food Chemistry, 99, 83–91. CIE, Commission Internationale de l’Eclairage (1976): Official recommendations on uniform colour spaces, colour differences equations and metric colour terms. Paris, France. Espe, M., Nortvedt, R., Lie, O., Hafsteinsson, H. (2002): Atlantic salmon (Salmo salar, L) as raw material for the smoking industry. II: Effect of different smoking methods on losses of nutrients and on the oxidation of lipids. Food Chemistry, 77, 41–46. Espe, M., Kiessling, A.,Lunestad, B-T.,Torrissen, O. J., Bencze Røra, A. M. (2004): Quality of cold smoked salmon collected in one French hypermarket during a period of 1 year. LWT - Food Science and Technology, 37, 6, 627–638. FAO, Food and Agriculture Organization (2012): The state of world fisheries and aquaculture. Rome, Italy. Fuentes, A., Fernández-Segovia, I., Barat, J. M., Serra, J. A. (2010): Physicochemical characterization of some smoked and marinated fish products. Journal of Food Processing and Preservation, 34, 83–103. Greenfield, H., Southgate, D.A.T. (2003): Food composition data, Production, management and use. FAO. Guillén, M. D., Ruiz, A. (2004): Study of the oxidative stability of salted and unsalted salmon fillets by 1H nuclear magnetic resonance. Food Chemistry, 86, 297–304. Haliloğlu, H. I., Bayir, A., Sirkecioglu, A. N., Aras, N.M. (2004): Comparison of fatty acid composition in some tissues of rainbow trout (Oncorhynchus mykiss) living in seawater and freshwater. Food Chemistry, 86, 55-59. HRN EN ISO 5508 (1999): Životinjske i biljne masti i ulja- Analiza metilnih estera masnih kiselina plinskom kromatografijom. HRN EN ISO 5509 (2004): Životinjske i biljne masti i ulja- Priprava metilnih estera masnih kiselina. Janeković, I., Antonić, O., Križan, J., Bukovec, D., Bakran-Petricioli, T. (2006): Modelling ba-

sic physical parameters in the Adriatic Sea as the basis for marine benthic habitats mapping. Ecological modelling, 194, 62–69. Karásková, P., Fuentes,A., Fernández-Segovia, I., Alcañiz, M., Masot,R., Barat, J. M. (2011): Development of a low-cost non-destructive system for measuring moisture and salt content in smoked fish products. Procedia Food Science, 1, 1195–1201. Kennedy, S.R., Bickerdike, R., Berge, R.K., Dick , J.R., Toche, D.R. (2007): Influence of conjugated linoleic acid (CLA) or tetradecylthioacetic acid (TTA) on growth, lipid composition, fatty acid metabolism and lipid gene expression of rainbow trout (Oncorhynchus mykiss L.). Aquaculture, 272, 489– 501. Kołakowska, A., Olley, J., Dunstan, G. A. (2002): Fish lipids. In: Sikorski,Z.E., Kołakowska, A. (eds.), Chemical and functional properties of food lipids. CRC Press, Boca Raton, Florida, USA, pp. 221-264. Krogdahl, Å., Sundby, A., Olli, J. J. (2004): Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) digest and metabolize nutrients differently. Effects of water salinity and dietary starch level. Aquaculture, 229, 335360. Miller, M. R., Nichols, P. D., Carter, C. G. (2007): Replacement of dietary fish oil for Atlantic salmon parr (Salmo salar L.) with a stearidonic acid containing oil has no effect on omega-3 longchain polyunsaturated fatty acid concentrations. Comparative Biochemistry and Physiology, Part B, 146, 197–206. Moradi, Y., Bakar, J., Motalebi, A. A., Syed Muhamad, S. H., Che Man, Y. (2011): A review on fish lipid: composition and changes during cooking methods. Journal of Aquatic Food Product Technology, 20, 379–390. Panpipat, W., Yongsawatdigul, J. (2008) Stability of potassium iodide and omega-3 fatty acids in fortified freshwater fish emulsion sausage. LWT – Food Science and Technology, 41, 483–492. Rørå, A. M. B., Birkeland, S., Hultmannc, L., Rustadc, T., Skåra, T., Bjerkeng, B. (2005): Quality characteristics of farmed Atlantic salmon (Salmo salar) fed diets high in soybean or fish oil as affected by cold-smoking temperature. LWT- Food Science and Technology, 38, 201-211. Rørå, A. M. B., Kvåle, A., Mørkøre, T., Rørvik, K. A., Steien, S. H., Thomassen, M. S. (1998): Process yield, colour and sensory quality of smoked Atlantic salmon (Salmo salar) in relation 63

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to raw material characteristics. Food Research International, 31, 601-609. Saldanha, T., Bragagnolo, N. (2008): Relation between types of packaging, frozen storage and grilling on cholesterol and fatty acids oxidation. In: Atlantic hake fillets (Merluccius hubbsi). Food Chemistry, 106, 2, 619–627. Sikorski, Z. E. (1990): Seafood: resources, nutritional composition, and preservation. CRC Press, Boca Raton, Florida, pp. 163–180. Soengas, J. L., Barciela, P., Fuentes, J., Otero, J., Andrés, M. D., Aldegunde, M. (1993): Changes in muscle carbohydrate metabolism in domesticated rainbow trout (Oncorhynchus mykiss) after transfer to seawater. Comparative Biochemistry and Physiology, Part B: Comparative Biochemistry, 104, 1, 173–179. Steffens, W. (1997): Effect of variation in essential fatty acid in fish feeds on nutritive value of freshwater fish for humans. Aquaculture, 151, 97–119. Stołyhwo, A., Kołodziejska, I., Sikorski, Z. E. (2006): Long chain polyunsaturated fatty acids in smoked Atlantic mackerel and Baltic sprats. Food Chemistry, 94, 589–595. Storebakken, T., Shearer, K .D., Refstie, S., Lagocki, S., McCool, J. (1998): Interactions between sa-

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linity, dietarycarbohydrate source and carbohydrate concentration on the digestibility of macronutrients and energy inrainbow trout Oncorhynchus mykiss. Aquaculture, 163, 347–359. Torrissen, O. J., Bencze-Rørå, A. M., Nortvedt, R., Espe, M., Jørgensen, L., Sørensen, N. K., Olsen, S. O. (2000): U: Program & Abstract. Atlantic salmon–quality and market responses. The Ninth International Symposium on Nutrition & Feeding in Fish. May 21–25, 2000, Miyazaki, Japan, pp. 75. Turchini, G. M., Mentasti, T., Froyland, L., Orban, E., Caprino, F., Moretti, V. M., Valfre, F. (2003): Effects of alternative dietary lipid sources on performance, tissue chemical composition, mitochondrial fatty acid oxidation capabilities and sensory characteristics in brown trout (Salmo trutta L.). Aquaculture, 225, 251–267. Usher, M. L., Talbot, C., Eddy, F. B. (1990): Effects of transfer to seawater on digestion and gut function in Atlanticsalmon smolts (Salmo salar L.). Aquaculture, 90, 85– 96. Vidaček, S., Janči, T., Marušić, N., Petrak, T., Vnučec, T., Medić, H. (2011): Utjecaj smrzavanja na impedanciju kalifornijske pastrve (Oncorhynchus mykiss). Ribarstvo, 69, 2, 38-50.

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

CODEN RIBAEG ISSN 1330-061X

ORIGINAL SCIENTIFIC PAPER

PoPUlation GEnEtic strUctUrE of an EndanGErEd KalibaUs, Labeo calbasu (haMilton, 1822) rEvEalEd by MicrosatEllitE dna MarKErs Md. robiul hasan*1, Md. nahiduzzaman2, Mostafa ali reza hossain3, Md. samsul alam3 Department of Fisheries and Marine Science, Noakhali Science and Technology University, Sonapur, Noakhali-3814, Bangladesh 2 Youth Development Department, Bangladesh 3 Department Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh * Corresponding Author, E-mail: rhasan_rony@yahoo.com 1

ARTICLE INFO

ABSTRACT

Received: 8 February 2013 Received in revised form: 10 June 2013 Accepted: 24 June 2013 Available online: 24 June 2013

The population genetic structure of kalibaus Labeo calbasu collected from four wild and a hatchery population was studied using microsatellite DNA marker analysis. Five heterologous microsatellite markers (Lr10, Lr21, Lr24, Lr26 and CcatG1) developed from rohu (Labeo rohita) and catla (Gibelion catla) were analyzed to test the genetic variability of kalibaus stocks. The number of alleles observed in the loci ranged from 2-10. The loci were found to be polymorphic (<P95) in all the populations. The average numbers of possessed alleles were higher by the four wild stocks than the hatchery stock. The average number of allele was the highest in the Jamuna population (5.8) and the least in the Hatchery population (4.8). The observed average heterozygosity (Ho) in the Jamuna population (0.776) was the highest followed by the Halda (0.667), the Haor (0.661) and the Padma (0.642) populations. Except loci Lr10 and Lr24 in the Halda and locus Lr10 in the Padma and Hatchery populations, significant deviations from Hardy-Weinberg Equilibrium (HWE) were detected in all cases. The FST values and the Nm values indicated high level of differentiation and a low level of gene flow between the populations. The largest genetic distance value (D = 0.543) was measured between the Jamuna and the Hatchery populations while the least value (D =0.124) was observed between the Padma and the Halda populations. The estimated genetic population structure and potential applications of microsatellite markers may assist the proper management of kalibaus populations in the wild.

Keywords: Cross species amplification Labeo calbasu endangered species microsatellite DNA markers

introdUction Kalibaus Labeo calbasu (Hamilton, 1822), is one of the four Indian major carps available in Bangladesh (family: Cyprinidae, order: Cypriniformes), possessing a karyotype with 25 pairs of diploid (2n) chromosomes (Reddy, 1990). L. calbasu is distributed in many countries namely Bangladesh - the Padmaâ&#x20AC;&#x201C;Brahmaputra, i.e. Padma, Jamuna, Arial

Khan, Kumar and Old Brahmaputra and the Halda River and Pakistan, India, Myanmar, Thailand and China (Reddy, 1990). L. calbasu has great commercial importance due to its adaptability to a wide range of environments, palatability and consumer preference. At the beginning of 1980s, when carp polyculture was started in Bangladesh owing to the success of artificial propagation, all four Indian major carps (Labeo ro65

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

hita, Gibelion catla, Cirrhinus mrigala and L. calbasu) were brought under aquaculture. However, due to slower growth (compared to other three) and unavailability of the fingerlings, farmers lost their interest in L. calbasu. Over the last few decades the abundance of this species has seriously been decreased in nature due to overfishing, habitat degradation, aquatic pollution and other anthropogenic and natural causes. L. calbasu is now enlisted as an endangered species from the biodiversity point of view (IUCN, 2000). Moreover, there has been a long-term decline in genetic quality of L. calbasu due to inbreeding, inter-species hybridization and improper brood management practice by the hatchery owners. A little amount of L. calbasu seed is presently being produced in a few hatcheries of Bangladesh without considering the genetic quality. Many hatcheries of Bangladesh practice hybridization of kalibaus with rohu (Labeo rohita) or catla (Gibelion catla). The sustainable utilization of genetic resources, including fish, is a vital part in improving the standard of living in a developing country like Bangladesh. Therefore, it is high time to save L. calbasu from extinction and bring it back in natural environments and culture systems. A number of studies reported that cultured fish stocks showed lower genetic diversity than wild populations (Frost et al., 2006). Both landmark-based morphometric analysis and microsatellite markers are recognized methods for stock selection and identification (Turan et al., 2006; Hossain et al., 2010). There have been few attempts to evaluate the population structure of L. calbasu using different methods based on phenotypic and genetic aspects. Genetic variation is used as a useful tool for characterization of different species or strains, comparison of farmed with wild populations. Maintenance of genetic variation is the most important concern for the management of hatchery stocks of any species. Molecular marker is a powerful tool which has been extensively used to evaluate genetic diversity and structure of farmed food fish species, such as Asian seabass (Zhu et al., 2006), oyster (Carlsson et al., 2006) and tilapia (Rutten et al., 2004), and used for identification of Quantitative Trait Loci (QTLs) and applied to assist in the breeding program and broodstock management (Jackson et al., 2003). In Bangladesh, microsatellite DNA marker has been used for some fish species including catla (Alam and Islam, 2005), silver carp and bighead carp (Mia et al., 2005), common carp (Mondol et al., 2006), walking catfish (Islam et al., 2007), mrigal (Hasnat, 2007) and rohu (Alam et al., 2009). Microsatellite marker is able to detect lower level of genetic variation than isozyme and mtDNA 66

RFLP (Desvignes et al., 2001). Microsatellite markers have added new dimension in the field of fisheries and aquaculture as many populations are subjected to bottleneck, inbreeding, genetic drift and as a consequence exhibit low variation that cannot be detected by other markers. As kalibaus is an endangered fish, it may be subjected to bottleneck, inbreeding, genetic drift, gene introgression etc. and exhibit low production performance in the aquaculture practices. Therefore, it is necessary to study genetic status of this endangered fish. The present study evaluated the genetic diversity and population structure of endangered carp, kalbaus L. calbasu from five different stocks – three major rivers, a haor (wetlands in the northeastern part of Bangladesh bowl or saucer shaped shallow depressions) and a hatchery using microsatellite markers.

MatErials and MEthods A total of 165 adult L. calbasu samples, 33 each from five different locations were collected during August 2007- October 2008 from five different locations: the Jamuna, the Padma, the Halda, the Tola Haor and a Hatchery (Fig. 1). The sampling sites were selected to cover genetic variation on a wide geographical distribution range of the species. Briefly, approximately 50 mg of caudal fin tissue was clipped from each individual in separate eppendorf tubes and stored at -18 °C.. The fin clips were cut into small pieces with scissors and ground with a tissue grinder in a 1.5 ml microcentrifuge tube. The tissue was digested overnight at 37oC in a 1.5 ml microcentrifuge tube containing 500 μl of extraction buffer (100 mM M Tris-HCl pH 8; 10 mM EDTA; 250 mM NaCl and 1% SDS) and 30 μl of Proteinase K (10 mg/ml). Total DNA was extracted with two washes of phenol: chloroforms: isoamyl alcohol (25:24:1) and one of chloroform: isoamyl (24:1) followed by ethanol precipitation. Finally, the DNA samples were stored in freezer at -18 °C. After completion of PCR, 2.5 μl loading dye was added to each PCR tube and mixed well and centrifuged briefly containing ethidium bromide in 1× TBE buffer. After confirmation on agarose gel, the gel containing 19:1 acrylamide:bis-acrylamide and 7 M urea. Electrophoresis was conducted using a SequiGen GT sequencing gel electrophoresis system (Bio-Rad Laboratories, Hercules, CA). The PCR products and 100 bp DNA ladder (GENEI, Bangalore, India) were preheated at 95 oC for 5 minutes and cooled immediately on ice before loading. The DNA ladder was loaded on either side of the gel and was run at 60 W for required length of time (1 hr 50).

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

rEsUlts Cross species amplification Developed from rohu, catla and common carp (Cyprinus carpio) were tested in this experiment for PCR amplification using kalibaus DNA as template of which nine primers were amplified successfully (Table 1). Out of nine loci, four microsatellite markers (Lr10, Lr21, Lr24 and Lr26) developed from rohu (Das et al., 2005) and one microsatellite marker (CcatG1) developed from catla (Naish and Skibinski, 1998), yielded consistently scorable bands. A total of thirty alleles were detected in the five loci examined.

Genetic variation

Fig. 1. Sampling sites of L. calbasu in Bangaldesh No microsatellite marker has been developed from L. calbasu. Twenty five (25) primers developed from other related carp species were tested of which nine were amplified. The experiment was conducted by five among nine amplified primers. Four microsatellite loci (Lr10, Lr21, Lr24 and Lr26) developed from rohu by Das et al. (2005) and one microsatellite locus (CcatG1) developed from catla by Naish and Skibinski (1998) were used in this experiment.

The microsatellite CcatG1 was most polymorphic with ten alleles while the locus Lr24 was least polymorphic with two alleles (Table 2). The average number of allele was highest in the Jamuna population (5.8), followed by the Padma (5.2), the Halda (5) and the Haor (5) populations. The Hatchery populations possessed the least number (4.8) of average alleles (Table 2). Therefore, the wild populations possessed higher average allele number than the cultured stock. The highest Ne (7.383) was found in locus CcatG1, while the locus Lr24 had the least Ne (1.832) (Table 2). The Jamuna stock possessed the highest average Ne (3.805) followed by the Haor (3.797), the Padma (3.785), the Halda (3.661) and the Hatchery (3.054) populations (Table 2). The observed average heterozygosity (Ho) in the Jamuna population (0.776) was the highest which was followed by the Halda (0.667), the Haor (0.661) and the Padma (0.642) populations. The hatchery stock had the lowest observed average heterozygosity (Ho) (0.370) (Table 2). The 1-Ho/He values

Table 1. Primers of microsatellite loci which developed from Labeo rohita, Gibelion catla and Cyprinus carpio tested for cross-species amplification in L. calbasu Resource Species Labeo rohita

Primer pairs tested (no.) 12

Gibelion catla

9

Cyprinus carpio

4

Total tested

25

Locus

Reference

Lr1, Lr3, Lr6, Lr10, Lr12, Das et al., 2005 Lr14a, Lr14b, Lr20, Lr21, Lr23, Lr24, Lr26 CcatG1 CcatG2, C catG3 Naish and Skibinski, 1998 Cc6, Cc 7, Cc 8, Cc 9, Cc10, McConnell et al., Cc 12 (2001) MFW 7, MFW 19, MFW 24, Crooijmans et al., 1997 MFW 26

Successful amplified result in L. calbasu (%) 5 (45) 3 (33)

1 (25) 9 (36)

67

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

Table 2. Allelic variations in five microsatellite loci in a sample of five populations of L. calbasu (N= Number of alleles, Ne= Effective number of alleles, Ho= Heterozygosity observed, He= Heterozygosity expected, inbreeding index (f = 1- Ho / He) Microsatellite loci Lr10

Parameters N Ne Ho He 1- Ho / He H-W test

Lr21

N Ne Ho He 1- Ho / He H-W test

Lr24

N Ne Ho He 1- Ho / He H-W test

Lr26

N Ne Ho He 1- Ho / He H-W test

CcatG1

N Ne Ho He 1- Ho / He H-W test

Average number of alleles Average Effective no. of alleles Average Ho over loci Average He over loci Polymorphism (P95)

Jamuna 4 3.170 0.788 0.685 -0.151 22.41** (6) 6 4.279 0.697 0.766 0.090 97.62*** (15) 5 2.789 0.788 0.641 -0.228 53.34*** (10) 4 3.463 0.818 0.711 -0.150 44.79*** (6) 10 5.325 0.788 0.812 0.030 175.93*** (45) 5.8 3.805 0.776 0.723 100

Padma 4 3.227 0.667 0.690 0.034 7.77 NS (6) 6 3.636 0.697 0.725 0.039 82.26*** (15) 2 1.832 0.697 0.454 -0.535 9.44** (1) 5 3.588 0.758 0.721 -0.050 93.78*** (10) 9 6.640 0.394 0.849 0.536 105.48*** (36) 5.2 3.785 0.642 0.688 100

Halda 4 3.419 0.818 0.708 -0.156 9.87 NS (6) 6 4.436 0.636 0.775 0.178 45.92*** (15) 2 1.936 0.515 0.483 -0.066 0.14 NS (1) 5 3.125 0.879 0.680 -0.292 56.36*** (10) 8 5.391 0.485 0.815 0.405 85.95*** (28) 5 3.661 0.667 0.692 100

Haor 4 3.212 0.485 0.689 0.296 37.56*** (6) 6 4.287 0.879 0.767 -0.146 36.83** (15) 2 1.832 0.636 0.454 -0.401 5.31* (1) 4 2.271 0.697 0.560 -0.245 78.75*** (6) 9 7.383 0.606 0.865 0.299 91.55*** (36) 5 3.797 0.661 0.667 100

Hatchery 3 2.450 0.606 0.592 -0.024 2.22NS (3) 4 2.401 0.273 0.584 0.533 34.95*** (6) 3 1.919 0.182 0.479 0.620 24.30*** (3) 4 1.561 0.182 0.360 0.494 22.01** (6) 10 6.936 0.606 0.856 0.292 105.05*** (45) 4.8 3.054 0.370 0.574 100

Statistically significant values are marked with asterisks. * P<0.05, ** P<0.01, *** P<0.001, NS=Not Significant

were negative in both the Jamuna and Halda populations at Lr10, Lr24 and Lr26; in the Padma at Lr24 and Lr26; in the Haor at Lr21, Lr24 and Lr26; and in the Hatchery population at Lr10 (Table 2). The 1-Ho/He values were negative which means that all the sampled populations were excess of heterozygosity. 68

Deviation from Hardy-Weinberg proportion The deviations from Hardy-Weinberg Equilibrium at Lr21, Lr24, Lr26 and CcatG1 in the Jamuna; Lr21, Lr26 and CcatG1 in the Padma and Halda; Lr10, Lr26 and CcatG1 in the Haor; Lr21 Lr24 and CcatG1 in the Hatchery and at Lr21 in the Jamuna were

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

very high (P<0.001). The deviations from HardyWeinberg Equilibrium at Lr10 in the Jamuna, Lr24 in the Padma, Lr21 in the Haor, Lr26 in the Hatchery populations were high (P<0.01). The deviations from Hardy-Weinberg Equilibrium at Lr24 in the Haor population was relatively low (P<0.05) (Table 2).

Inter-population genetic structure

Bottleneck test Since the H0 values were higher than He in L. calbasu populations, this shows that there might be bottleneck in some populations except the Hatchery population. The ratio (5:0) was significantly different from the expected ratio (1:1) for nonbottlenecked, equilibrium populations (Sign test: P = 0.064; Wilcoxon test: one tail for Hex, P = 0.015). On the other hand, the data sets from the Jamuna, the Padma and the Haor population that did not have a significant heterozygosity excess (Sign test: P = 0.678, 0.299 and 0.295 and Wilcoxon Table 3. Heterozygosity excess under two-phase mutation model at five microsatellite loci from each of five populations of L. calbasu Population

Jamuna Padma Halda Haor Hatchery

test: one tail for Hex, P = 0.406, 0.031 and 0.031 respectively), deviated toward an excess of heterozygosity as expected for bottleneck populations (nearly significant). Only the Hatchery population had significant excess of heterozygosity (Hex/Hd = 2/3) (Table 3).

Sign Test Hex/Hd

P

3/2 4/1 5/0 4/1 2/3

0.678 0.299 0.064 0.295 0.328

Wilcoxon Test P (one tail for Hex) 0.406 0.031 0.015 0.031 0.593

P = probability, Hex = heterozygosity excess, Hd = heterozygosity deficiency

The FST between the population pairs were compared and it was found that the population differentiation values between all the population pairs are significant. This result indicates that the populations are not homogeneous. The FST (population differentiation) value between the Jamuna population and the Hatchery population was the highest (0.109), while the FST value between the Padma population and the Halda population was the lowest (0.024). The Nm (gene flow) value between the Padma and the Halda across all loci was the highest (10.306) and the Nm value between the Jamuna and the Hatchery was the lowest (2.045) (Table 4). Analysis of molecular variance (AMOVA) revealed that 18% of the molecular variance existed among the populations and 82% existed within populations (Table 5).

Genetic distance A matrix of genetic distance (Nei, 1972) was built based on allelic frequencies of all loci (Table 6). The largest genetic distance value (D = 0.543) was measured between the Jamuna and the Hatchery population while the smallest value (D =0.124) was observed between the Padma and the Halda populations.

Table 4. Multilocus FST (below diagonal) and Nm (above diagonal) values between pairs of five populations of L. calbasu across all loci Populations Jamuna Padma Halda Haor Hatchery

Jamuna * 0.048 0.053 0.078 0.109

Padma 4.949 * 0.024 0.044 0.084

Halda 4.479 10.306 * 0.047 0.085

Haor 2.959 5.381 5.102 * 0.067

Hatchery 2.045 2.716 2.686 3.460 *

Nm = [(1/ FST)-1]/4

Table 5. Summary of analysis of molecular variance (AMOVA) among and within the populations Source Among Pops Within Pops Total

df 4 160 164

SS 119.079 589.455 708.533

MS 29.770 3.684

Est. Var. 0.790 3.684 4.475

% 18% 82% 100%

69

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

Table 6. Neiâ&#x20AC;&#x2122;s (1972) genetic distance in five populations of L. calbasu Populations Jamuna Padma Halda Haor Hatchery

Jamuna **** 0.251 0.295 0.455 0.543

Padma

Halda

Haor

Hatchery

**** 0.124 0.235 0.381

**** 0.247 0.379

**** 0.242

****

Dendrogram

Genetic variability

The UPGMA dendrogram based on Neiâ&#x20AC;&#x2122;s (1972) genetic distance resulted in two major clusters. The first cluster was separated into two sub-clusters: Jamuna stock alone in one cluster and remaining three stocks (the Padma, the Halda and the Haor) in the other cluster. Hatchery stock was in the second cluster (Fig. 2).

In the present study, all the five loci were found to be polymorphic in five populations of L. calbasu. Average effective number of alleles and gene diversity were different in the wild and hatchery stocks, suggesting significant difference of genetic variability between wild and hatchery stocks. Similar results were also reported from microsatellite and RAPD markers analyses (Bartfai et al., 2003 and Lal et al., 2004). Das et al. (2005) developed these microsatellite markers and Alam et al. (2009) obtained similar genetic polymorphism in an Indian farmed population of rohu. The Hatchery populations possessed the least number (4.8) of average alleles and thus they have the highest number of null alleles (6 each). Loss of allelic variation has also been reported for hatchery populations of catla by Alam and Islam (2005) and Hansen et al. (2006), and for Clarias batrachus hatchery populations by Islam et al. (2007). The higher proportion of polymorphic loci and gene diversity in the individuals of the Jamuna River are usually expected, because it is well known that the Jamuna is a large river. Therefore, in Bangladesh, genetically more diversified L. calbasu individuals can only be found in the Jamuna River. The hatchery population might have been founded with a small effective number of parents (Ne). However, it was not possible to confirm from the hatchery owners which Ne was maintained in the hatchery under study, as no record was maintained in the hatchery. It is, however, a common practice to maintain only a small number of broodfish in Bangladeshi hatcheries. The losses of alleles and heterozygosity may increase with bottlenecking and inbreeding through time in the hatchery stocks (Alam and Islam, 2005). Therefore, measures should be taken to prevent inbreeding in the commercial hatcheries through proper broodstock management practices.

Fig. 2. UPGMA dendrogram based on Neiâ&#x20AC;&#x2122;s (1972) genetic distance between five populations of L. calbasu, according to the microsatellite DNA analysis

discUssion This is the first extensive study of genetic diversity and population structure of endangered L. calbasu using microsatellite DNA markers in Bangladesh.

Cross species amplification Cross species amplification is dependent on the phylogenetic relationship of species for which microsatellites were originally developed (Hulak et al., 2010). Catla and rohu are closely related species to kalibaus under the same family and it was expected that the markers developed from rohu and catla should also work in kalibaus. These results suggest that time and money invested in evaluating the cross-species amplification of microsatellite loci in target species may be worth it. The cross-species amplification data indicated that allelic diversity was low in L. calbasu compared with the original species. Therefore, greater attention should be been given to primer optimization or redesign. 70

Population bottleneck Since the H0 values were higher than He in L. calbasu populations, this shows the possibility of bot-

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

tleneck in some populations. The tests of mutation drift equilibrium for detecting genetic bottlenecks revealed signs of population decline of the species. In the present study, the Hatchery population with a significant heterozygosity excess reveals risk of sever population bottlenecks. According to the statements of the hatchery owner, the hatchery was established seven years ago with brood fish collected from a hatchery. The samples of L. calbasu used in the present study were progeny of the fourth generation. At present, population is declining in the Halda River for many reasons such as overexploitation, habitat destruction, water pollution, siltation etc.

Population structure Except loci Lr10 and Lr24 in the Halda and locus Lr10 in the Padma population, deviation from Hardy-Weinberg were found at all loci. Deviation of all populations at Lr21 and CcatG1 loci from Hardy-Weinberg expectations were found to be relatively larger due to deficiency in heterozygosity and loss of alleles in all samples. Alam and Islam (2005) found that the hatchery population of catla deviated from Hardy-Weinberg equilibrium at a number of loci. Deviations from Hardy-Weinberg expectation might be due to inbreeding. Pair-wise FST estimates between the Jamuna and hatchery populations were higher than all other population pairs and significant population differentiation was observed between the Jamuna and the hatchery population of L. calbasu. Geological structures separate the Jamuna River from the hatchery populations and may limit the gene flow between the Jamuna and hatchery population. The effect of geographical distance on FST and gene flow (Nm) values has been reported in stream-living brown trout collected from different sections of the main stream in Jamtland, central Sweden (Carlsson et al., 1999). The genetic differentiation in the populations of L. calbasu may be caused by artificial selection or random genetic drift or founder effect in the hatchery stocks.

Genetic distance The UPGMA dendrogram based on Nei’s (1972) genetic distance resulted in two major clusters. Hatchery stock was in the second cluster. The clustering suggests that the status of hatchery population were unique where the origin of broods might be geographically distant from wild stocks. However, it is imperative that effective coordina-

tion of ecological and genetic approaches is helpful to conserve this endangered species.

Conclusion The result of the present study has proven to understand the intra and inter-population genetic variation and population status. For proper conservation of a species and effective management strategy, genetic monitoring is essential because a population may suffer the risk of genetic drift, bottleneck, inbreeding, founder effect etc. Hatchery owner should maintain good brood management system through collecting brood of different sources. It may be an important way to maintain diverse gene pool. However, further study with large number of populations, including all segments of the country with microsatellite DNA markers, is recommended in order to have more information about population genetic structure of this endangered fish species. Therefore, it is very essential to develop specific primer for L. calbasu.

Acknowledgements Financial support received from the USDA through the project “Ex situ conservation of some indigenous fishes of Bangladesh by selecting the best stock through DNA markers” (BGARS-120) is thankfully acknowledged.

Sažetak GEnEtsKa strUKtUra PoPUlacija UGrožEnoG ciPrinida Labeo calbasu (haMilton, 1822) istražEna PoMoćU MiKrosatElitsKih MarKEra Genetska struktura populacija ciprinida Labeo calbasu, prikupljena od četiri divlje i jedne uzgajane populacije, proučavana je koristeći mikrosatelitske markere. Pet heterogenih mikrosatelitskih markera (Lr10, Lr21, Lr24, Lr26 i CcatG1) sastavljenih za indijske ciprinide su proučavani da bi se istražila genetska varijabilnost L. calbasu populacija. Broj uočenih alela u lokusu se kretao od 2 do 10. U proučavanim populacijama lokusi su bili polimorfni (<P95). Prosječan broj alela je bio veći kod divljih nego kod uzgajanih populacija. Prosječan broj alela je bio najveći u Jamuna populaciji (5.8), te najmanji u uzgajanoj populaciji (4.8). Uočena prosječna heterozigotnost (Ho) u Jamuna populaciji (0.776) bila je najveća, slijedile su je populacije Halda (0.667), Haor (0.661) i Padma (0.642). Izuzev lokusa Lr10 i Lr24 u 71

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

populaciji Halda i lokusa Lr10 u Padma populaciji i uzgajanoj populaciji, zabilježeno je značajno odstupanje od Hardy-Weinbergovog zakona ravnoteže (HWE) u svim slučajevima. Vrijednosti FST i Nm ukazuju na visok stupanj diferencijacije i nizak stupanj protoka gena između populacija. Najveća genetska udaljenost (D = 0.543) je izmjerena između populacije Jamuna i uzgajane populacije, dok je najmanja vrijednost (D = 0.124) uočena između populacija Padma i Halda. Procijenjena genetska struktura populacije i moguća upotreba mikrosatelitskih markera mogu pomoći pri upravljanju populacija L. calbasu u otvorenim vodama. Ključne riječi: međuvrsna amplifikacija, Labeo calbasu, ugrožene vrste, mikrosatelitski DNA markeri

rEfErEncEs Alam, M. S., Islam, M. S. (2005): Population genetic structure of Catla catla (Hamilton) revealed by microsatellite DNA markers. Aquaculture, 246, 151-160. Alam, M. S., Jahan, M., Hossain, M. M., Islam, M. S. (2009): Population genetic structure of three major river populations of rohu, Labeo rohita (Cyprinidae: Cypriniformes) using microsatellite DNA markers. Gen. Genom., 31, 1, 43-51. Bartfai, R., Egedi, S., Yue, G. H., Kovacs, B., Urbanyi, B., Tama, G., Horvath, L., Orban, L. (2003): Genetic analysis of two carp broodstocks by RAPD and microsatellite markers. Aquaculture, 219, 157-167. Carlsson, J., Morrison, C. L., Reece, K. S. (2006): Wild and aquaculture populations of the eastern oyster compared using microsatellites. Heredity, 97, 595-598. Carlsson, J., Olsen, K. H., Nilsson, J., Averli, A., Stabell, O. B. (1999): Microsatellites reveal finescale genetic structure in stream-living brown trout. Journal of Fish Biology, 55, 1290-1303. Crooijmans, R. P. M. A., Vander, P. J. J., Groenen, M. A. M., Bierbooms, V. A. F., Komen, J. (1997): Microsatellite markers in common carp (Cyprinus carpio L.). Anim. Genet., 28, 129-134. Das, P., Barat, A., Meher, P. K., Ray, P. P., Majumdar, D. (2005): Isolation and characterization of polymorphic microsatellites in Labeo rohita and their cross species amplification in related species. Molecular Ecology Notes, 5, 231. Desvignes, J. F., Laroche, J., Durand, J. D., Bouvet, Y. (2001): Genetic variability in reared stocks of common carp (Cyprinus carpio L.) based on al72

lozymes and microsatellites. Aquaculture, 194, 291-301. Excoffier L., Laval, G. and Schneider, S. 2005. ARELEQUIN ver.3.0: An integrated software package for population genetics data analysis. Evol. Bioinformatics Online, 1, 47-50. Frost, L. A., Evans, B. S., Jerry, D. R. (2006): Loss of genetic diversity due to hatchery culture practices in barramundi (Lates calcarifer). Aquaculture, 261, 1056-1064. Hansen, M. M., Simonsen, V., Mensberg, K. L. D., Sarder, M. R. I., Alam, M. S. (2006): Loss of genetic variation in hatchery-reared Indian major carp, Catla catla. Fish Biol., 69, 229-241. Hossain, M. A. R., Nahiduzzaman, M., Saha, D., Khanam, M. U. H., Alam, M. S. (2010): Landmark based morphometrics of an endangered carp kalibaus, Labeo calbasu stocks. Zoological Studies, 49, 556-563. Hulak, M., Kaspar, V., Kozak, P., Burice, M., Filipova, L., Petrusek, A. (2010) Cross-species amplification of microsatellite markers in the invasive spiny-cheek crayfish (Orconectes limosus): assessment and application. Appl. Genet., 51, 1, 73–78. Islam, M. N., Islam, M. S., Alam, M. S. (2007): Genetic structure of different populations of walking catfish (Clarias batrachus L.) in Bangladesh. Biochem. Genet., 45, 647-662. Islam, M.S., Alam, M. S. (2004): Randomly amplified polymorphic DNA analysis of four different populations of the Indian major carp, Labeo rohita (Hamilton). J. Appl. Ichthyol., 20, 407-412. IUCN (2000): Red Book of Threatened Fishes of Bangladesh. Islam, M.A., M. Ameen and Nishat, A. (Eds.). The World Conservation Union, Dhaka, Bangladesh, pp. 116. Jackson, T. R., Martin-Robichu, D.J., Reith, M. E. (2003): Application of DNA markers to the management of Atlantic halibut (Hippoglossus hippoglossus) broodstock. Aquaculture, 220, 245259. Lal, K. K., Chauhan, T., Mandal, A., Singh, R. K., Khulbe, L., Ponniah, A. G. and Mohindra, V. (2004): Identification of microsatellite DNA markers for population structure analysis of Indian major carp (Cirrhinus mrigala) (HamiltonBuchanan, 1882). J. Appl. Ichthyol., 20, 87-91. Mia, M. Y., Taggart, J. B., Gimour, A. E., Gheyas, A. A., Das, T. K., Kohinoor, A. H. M., Rahman, M. A., Sattar, M. A., Hussain, M. G., Mazid, M. A., Penman, D. J., McAndrew, B. J. (2005): Detection of hybridization between Chinese carp species

Croatian Journal of Fisheries, 2013, 71, 65-73 R. Hasan et al.: Population genetic structure of Labeo calbasu

(Hypophthalmichthys molitrix and Aristichthys nobilis) in hatchery broodstock in Bangladesh, using DNA microsatellite loci. Aquaculture, 247, 167-273. McConnell, S. K. J., Leamon, J., Skibinski, D. O. F., Mair, G. C. (2001): Microsatellite markers from the Indian major carp species, Catla catla. Molecular Ecology Notes., 1, 115. Mondol, M. R. K., Islam, M. S., Alam, M. S. (2006): Characterization of different strains of common carp (Cyprinus carpio L.) (Cyprinidae, Cypriniformes) in Bangladesh using microsatellite DNA markers. Gen. Mol. Biol., 29, 4, 626-633. Naish, K. A., Skibinski, D. O. F. (1998): Tetranucleotide microsatellite loci for Indian major carp. Journal of Fish Biology, 53, 886-889. Nei, M. (1972): Genetic distance between populations. Am. Nat., 106, 283-292. Peakall, R., Smouse, P. E. (2005): GenAlEx V6.1: Genetic Analysis in Excel. Population genetic

software for teaching and research. Australian National University, Canberra, Australia. http:// www.anu.edu.au/ BoZo/GenAlEx. Reddy, P. V. G. K. (1990): Genetic resources of Indian major carps. FAO Fisheries Technical Papers - T387, pp. 76. Rutten, M. J. M., Komen, H., Deerenberg, R. M., Siwek, M., Bovenhuis H. (2004): Genetic characterization of four strains of Nile tilapia (Oreochromis niloticus L.) using microsatellite markers. Anim. Genet., 35, 93-97. Turan, C., Oral, M., Ozturk, B., Duzgunes, E. (2006): Morphometric and meristic variation between stocks of bluefish (Pomatomus saltatrix) in the Black, Marmara, Aegean and northeastern Mediterranean Seas. Fish. Res., 79, 139-147. Zhu, Z. Y., Lin, G., Lo, L. C., Xu, Y. X., Renee, C., Yue, G. H. (2006): Genetic analyses of Asian sea bass stocks using novel polymorphic microsatellites. Aquaculture, 256, 167-173.

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Croatian Journal of Fisheries, 2013, 71, 74-76 S. Dutta and S. Hazra: Mega Landing of Blacktip Sea Catfish

CODEN RIBAEG ISSN 1330-061X

SHORT COMMUNICATION

a rEPort on MEGa landinG of blacKtiP sEa catfish, Plicofollis dussumieri (valEnciEnnEs, 1840) froM frasErGUnjE fishinG harboUr, wEst bEnGal, india sachinandan dutta*, sugata hazra School of Oceanographic Studies, Jadavpur University, Kolkata 700 032, India *Corresponding Author, E-mail: duttasachi@gmail.com

ARTICLE INFO

ABSTRACT

Received: 18 April 2012 Received in revised form: 31 January 2013 Accepted: 22 May 2013 Available online: 27 May 2013

A significantly large landing of Blacktip Sea Catfish (Plicofollis dussumieri) was reported from Frasergunje Fishing Harbour of Sundarbans, West Bengal between 31 December 2011 and 6 January 2012. Around 320 tonnes of Blacktip Sea Catfishes were caught during this period. The mean length and weight of the catch were 665.19±75.83 SD mm and 3833±1336 SD g respectively. The length-weight relationship was showing the negative allometric growth. Unusual heavy landings are frequent on Indian coast, but the present landing is bigger than any other published. The important factors of aggregation of this fish species are the fog and temperature fluctuation during the winter season, though it is yet to be ascertained.

Keywords: Landing Blacktip Sea Catfish Plicofollis dussumieri Frasergunje Fishing Harbour Sundarban

introdUction

MatErials and MEthods

Blacktip Sea Catfish (Plicofollis dussumieri) belongs to the family Ariidae. It is a common species in the coastal waters of West Bengal. Blacktip Sea Catfish is a highly mobile species which undertakes long courses of migration and lives in different types of unstable environments, depending on the changes of monsoon, etc. (Menon et al., 1990). The species is purely marine and forms large shoals that move vertically in the column and horizontally along surface waters (Menon and Bande, 1987). The biology and ecology of this species is not well studied. The species appears to be under least concern (LC) in the ‘Red List’ of IUCN (http:// www.iucnredlist.org/details/169632/0, access on 10.02.12 at 1.55 PM). Heavy landings of this species were reported by Waghmare (1987) from Mumbai, Srinivasarengan (1988) and Madras, Sarang et al. (2004) at Arnala. The present report tries to explain heavy landing of this species during the four days in the winter season 2011 and 2012 with help of fishermen.

Frasergunje Fishing Harbour (21° 34’ 46.2” N, 88° 15’ 03.0” E) is one of the important fish landing centers of Western Sundarban, West Bengal (Fig. 1).

74

Fig. 1. Frasergunje Fishing Harbour and the zone of Blacktip Sea Catfish catch.

Croatian Journal of Fisheries, 2013, 71, 74-76 S. Dutta and S. Hazra: Mega Landing of Blacktip Sea Catfish

The fishing in this estuarine region is carried out in the shallow waters of the Bay of Bengal up to 70 km from shoreline. This area receives the perennial flow of nutrient rich freshwater from the GangaMeghna-Brahmaputra (GMB) basin which makes the salinity of the coastal water significantly low in Frasergunje. According to the information collected from fishermen, boat owners, field staff and fishermen’s association of Frasergunje, trawl nets were not used in Frasergunje. The positions of the catch were noted from the fishing boats. The total catch was provided by the boat owners and the fishermen’s association. During 36 hours, 20 fishing boats (six cylinder) could catch fish shoal from 10-12 m water depth. The fish was caught using gill nets with 120 mm mesh size. The length and weight of 130 specimens were measured after landing. Length was measured by a metric scale and weight was measured by a digital scale Wensar (TTB-10) with capacity of 10 kg (accuracy ± 0.5 g). Length was measured in millimeter (mm) and weight was measured in gram (g). The length-weight relationship was established by the equation W=a*Lb (Le Cren, 1951), where W is the weight of the fish, L is the total length of the fish, ‘a’ is the exponent describing the rate of change of weight by length and ‘b’ is the weight at unit length. The photographs were taken by a 12 megapixel digital camera (Cannon SX-13). The geographic position of the large catch was recorded by using a Garmin GPS 152 receiver present in the fishing boat. Coordinates were recorded as latitude and longitude in decimal degrees. Identification of the species was done using the keys provided by Talwar and Kacker (1984).

rEsUlts and discUssion A huge catch of Blacktip Sea Catfish (Plicofollis dussumieri) (Fig. 2) was reported on 31 December 2011 and 1, 5, 6 January 2012 from Frasergunje Fishing Harbour of West Bengal. About 320 tonnes of Plicofollis dussumieri were caught in these four days. During the first two days, 150 tonnes of the species were caught in the geographical position within 21° 02’ N - 21° 04’ N latitude and 88° 20’ E - 88° 21’ E longitude. The second catch of 170 tonnes was caught within the geographical position of 21° 07’ N - 21° 09’ N latitude and 88° 19’ E - 88° 20’ E longitudes. The length of the 130 measured individuals varied between 518 mm and 817 mm (mean length 665.19±75.83 SD mm) and the weight ranged between 1730 g and 6896 g (mean weight 3833±1336 SD g). The length weight relationship (Fig. 3) of this species was W=0.00001 L2.984, ‘a’ is 0.00001 and

Fig. 2. Blacktip Sea Catfishes unloaded from a fishing boat

Fig. 3. The length weight relationship of Blacktip Sea Cat fish (Plicofollis dussumieri) the ‘b’ value is 2.984 whereas the R2 value is 0.9687. The growth of P. dussumieri was negatively allometric but close to isometric in nature. Menon and Muthiah (1987) estimated the length-weight relationship of P. dussumieri as W=0.00001047 L2.6479. In this report, large fish landing could be explained by fishermen’s observations. During the winter season, they noticed that fish schools come close to shallow regions of the Bay of Bengal. The fishermen also observed that this fish school had appeared during foggy conditions in the winter season. Fog and temperature fluctuation are an important factor of aggregation of the fish, though it is yet to be ascertained.

ACKNOWLEDGEMENTS The authors are grateful to the fishermen and Frasergunje Fishermen’s Association for providing the data. We are also very grateful to the 75

Croatian Journal of Fisheries, 2013, 71, 74-76 S. Dutta and S. Hazra: Mega Landing of Blacktip Sea Catfish

Indian National Center for Ocean Information Services (INCOIS) of the Ministry of Earth Science, Government of India for funding the Potential Fishing Zone Validation Project. We are also thankful to Bibhas Koley and Baburam Bhowmik for their valuable information.

Sažetak

izvjEšćE o vEliKoM UlovU MorsKoG soMa, Plicofollis dussumieri (valEnciEnnEs, 1840) U ribarsKoj lUci frasErGUnjE, zaPadni bEnGal U indiji Prijavljen je veliki ulov morskog soma (Plicofollis dussumieri) u ribarskoj luci Frasergunje, Sundarbans, Zapadni Bengal od 31. prosinca 2011. do 6. siječnja 2012. Oko 320 tona morskog pauka je ulovljeno tijekom ovog razdoblja. Srednja dužina i masa ulova iznosila je 665,19±75,83 SD mm, odnosno 3833±1336 SD g. Dužinsko-maseni odnos je pokazivao negativni alometrijski rast. Iako su neobično veliki ulovi česti na obali Indije, navedeni ulov je veći od svih dosada objavljenih. Magla i fluktuacija temperature tijekom zimskog razdoblja su važni čimbenici sakupljanja ove vrste što je potrebno potvrditi. Ključne riječi: ulov, morski som, Plicofollis dussumieri, ribarska luka Frasergunje, Sundarban

76

rEfErEncEs Le Cren, E. D. (1951): The length-weight relationship and seasonal cycle in gonad weight and condition in the perch (Perca fluviatilis). Journal of Animal Ecology, 20, 201-219. Menon, N. G., Muthiah, C. (1987). Marine Cat fish Resource of India. Biology of the important species of catfish. Bulletin of Central Marine Fisheries Research Institute, Kochi, 40, 60-69. Menon, N. G., Bande, V. N. (1987): Taxonomic Considerations and General Distribution of Commercially Important Catfishes. Marine Catfish Resources of India Exploitation and Prospects. CMFRI bulletin no 40. Chapter two, pp. 5-11. Menon, N. G., Bande V. N., Balachandran, K. (1990): Population Dynamics of Tachysurus dussumieri In North Kerala. Journal of Marine Biological Association of India, 32, 1-2, 129-141. Sarang, J. D., Dias, J. R., Sundram, S., Baikar, K. K. (2004): Unusual heavy landing of Tachysurus dussumieri by dol net at Arnala. Marine Fisheries Information Service, Technical and Extension Series, 181. Srinivasarengan, S. (1988): Unusual Landing of Cat fish Tachysurus dussumieri at Madras. Marine Fisheries Information Service, Technical and Extension Series, 89, pp. 17. Talwar, P.K. and Kacker, R. K. (1984): Commercial sea fishes of India. Zoological Survey of India, pp. 997. Waghmare, K. B. (1987): Heavy landings of Cat fish by ‘Dol’ nets at Sassoon docks. Marine Fisheries Information Service, Technical & Extension Series, 78, pp. 21.

Croatian Journal of Fisheries, 2013, 71, 77-79 S. Khataminejad et. al.: First record of Alburnus atropatenae

CODEN RIBAEG ISSN 1330-061X

SHORT COMMUNICATION

first rEcord of Alburnus atropatenae (bErG, 1925) (cyPrinidaE) in naMaK basin, cEntral iran somaye Khataminejad1, hamed Mousavi-sabet*1, Masud sattari1, saber vatandoust2 Dept. of Fisheries Sciences, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Guilan, Iran. Tel./Fax: +98 182 3223600 2 Dept. of Fisheries Sciences, Babol Branch, Islamic Azad University, Mazandaran, Iran * Corresponding Author, E-mail: mousavi-sabet@guilan.ac.ir 1

ARTICLE INFO

ABSTRACT

Received: 29 January 2013 Received in revised form: 31 March 2013 Accepted: 22 May 2013 Available online: 27 May 2013

Alburnus atropatenae Berg, 1925 is reported for the first time from the River Ghareh-Chay in the Namak basin, central Iran. Previously, this species has only been reported from the Lake Orumiyeh basin, in northwest Iran. In this report twenty-nine specimens were caught on 8 August 2012. Morphometric and meristic characteristics of the specimens were similar to those reported elsewhere for the species.

Keywords: First record Alburnus atropatenae Namak basin River Ghareh-Chay

introdUction

MatErials and MEthods

Thirty-eight species are presently recognized in the European and west Asian genus Alburnus (Bogutskaya et al., 2000; Freyhof and Kottelat, 2007; Kottelat and Freyhof, 2007; Bzulug and Freyhof, 2007). Iran is an important geographic and zoogeographic area in the Middle East that has been divided into nineteen major drainage basins (Coad, 2013). Cyprinids which belong to genus Alburnus Rafinesque, 1820 have seven confirmed species recorded from Iran. Three species are in the southern Caspian Sea basin (Iranian part) including A. alburnus Linnaeus, 1758, A. chalcoides Güldenstaedt, 1772 and A. filippii Kessler, 1877. One species, A. atropatenae Berg, 1925 has been reported from the Lake Orumiyeh basin in northwest Iran. Alburnus zagrosensis is described from a stream in the River Karun basin of Chahar Mahall va Bakhtiari Province in the Zagros Mountains of Iran. The Zagros Mountains in Iran have a series of rivers draining westward and southward to the River Tigris or to the head of the Persian Gulf. Previously, only A. mossulensis Heckel, 1843 has been recorded from these rivers, although A. caeruleus Heckel, 1843 known from Iraq, may also occur there (Coad, 2013). Thus this study was conducted to confirm the presence of A. atropatena in the River Ghareh-Chay (Namak Basin).

The study was conducted in the River Ghareh-Chay (34°53´.250” N, 050°02´.251” E), one of the two permanent and big rivers in Markazi Province, which is almost 540 kilometers long and originates from the heights of Shazand. It flows in the direction of west to east of Markazi Province and finally flows into Qom’s Namak Lake (=salt lake) (Fig 1). Its surface area under sampling was 100 m with an average depth of 35 cm, width 230 cm, water temperature of the catching site was 22 °C and water velocity was 0.7 ms-1. The sampling site is 1198 m above sea level. The specimens were collected by electrofishing on 8 August 2012. Specimens were preserved in 4% formalin fluid and transported to the laboratory (University of Guilan) for further analysis. Two types of morphological characters (meristic traits and morphometric measurements) were examined. Meristic traits included the number of un-branched and branched fin rays in dorsal (D ub-b), anal (A ub-b), ventral/pelvic (V ub-b), pectoral (P ub-b) and caudal (C ub-b) fins. The number of scales in lateral line was counted in the complete lateral line, from the anterior scale next to the operculum to the posterior one on the caudal fin. A total of 28 morphometric characters were measured using a digital caliper to the nearest 0.1 mm including: to77

Croatian Journal of Fisheries, 2013, 71, 77-79 S. Khataminejad et. al.: First record of Alburnus atropatenae

tal length (TL), standard length (SL), head length (c), distance between head tip and anal aperture (pan), pre-anal distance (aA), pre-ventral distance (aV), pre-pectoral distance (aP), pre-dorsal distance (aD), caudal peduncle length (lpc), length of dorsal (lD), anal (lA), caudal (lC), pectoral (lP) and ventral (lV) fins, distance between ventral fin and anal aperture (Van), head depth (hc), maximum body depth (H) and caudal peduncle depth or minimum body height (h), head width (laco), maximum body width (lac), distance between eyes (io), eye diameter (o), pre-orbital distance (prO), postorbital distance (poO), dorsal fin base (db), anal fin base (ab), ventral fin base (vb), pectoral fin base (pb). The percentage ratios of morphometric characters in relations to SL and c were analyzed.

ously been recorded from the Namak basin and this constitutes a new record. See Table 1 and Table 2 for meristic and morphometric data of the new recorded species. Table 1. Meristic characteristics (Mean ± SD) of Alburnus atropatenae from the River Ghareh-Chay, Namak basin (SD=Standard Deviation; Min=Minimum; Max=Maximum; Number of specimens=29) Characters D ub-b C ub-b A ub-b V ub-b P ub-b Lateral line scales

Mean ± SD Min - Max 3.00-8.00 ± 0.00-0.00 3.0-3.0 - 8.0-8.0 17.00-20.34 ± 0.00-1.59 17.0-17.0 - 18.0-24.0 3.00-10.97 ± 0.00-0.19 3.0-3.0 - 10.0-11.0 2.00-8.10 ± 0.00-0.31 2.0-2.0 - 8.0-9.0 1.00-14.62 ± 0.00-0.56 1.0-1.0 - 14.0-16.0 51.48 ± 3.27 47.0 - 58.0

Table 2. Morphometric characteristics (Mean ± SD) of Alburnus atropatenae from the River Ghareh-Chay, Namak basin (SD=Standard Deviation; Min=Minimum; Max=Maximum; Number of specimens=29)

Fig. 1. a: Distribution of Alburnus atropatenae in Iran, the type locality “the lake Orumiyeh basin” (light grey) and the new locality “the Namak basin” (dark grey). b: Namak basin, River Chareh-Chay, new habitat for A. atropatenae in central Iran.

rEsUlts and discUssion In the present study one new fish species was found from one stream which flows into the Namak basin namely; Alburnus atropatenae. This species was captured from the River Ghareh-Chay on 8 August 2012 (Fig 1). The reported species bas not previ78

Characters TL (mm) SL (mm) Standard Length % TL C Pan aA aV aP aD lpc lD lA lC lP lV Van hc H h laco lac io o prO poO db ab vb pb

Mean ± SD 92.19±11.96 77.20±10.47

Min - Max 69.92-112.11 58.17-95.57

119.51±0.02 24.33±0.01 62.49±0.01 64.57±0.01 46.45±0.03 24.94±0.01 52.33±0.01 23.34±0.01 18.86±0.01 13.60±0.01 22.15±0.02 18.35±0.01 14.38±0.01 16.81±0.01 16.10±0.01 19.95±0.02 9.60±0.00 11.55±0.01 11.68±0.01 7.42±0.01 7.06±0.00 6.68±0.00 11.46±0.01 11.62±0.01 13.53±0.01 4.12±0.01 4.20±0.00

1.17-1.23 0.23-0.26 0.60-0.65 0.62-0.68 0.35-0.50 0.24-0.26 0.50-0.54 0.20-0.26 0.17-0.21 0.12-0.16 0.19-0.25 0.16-0.21 0.13-0.16 0.14-0.19 0.15-0.17 0.10-0.22 0.09-0.10 0.11-0.13 0.10-0.13 0.06-0.09 0.06-0.08 0.06-0.08 0.10-0.13 0.10-0.14 0.12-0.16 0.03-0.05 0.03-0.05

Croatian Journal of Fisheries, 2013, 71, 77-79 S. Khataminejad et. al.: First record of Alburnus atropatenae

rEfErEncEs

Table 2. Continued Characters Head Length % hc laco io o prO poO

Mean ± SD

Min - Max

66.19±0.02 47.47±0.02 30.52±0.02 29.00±0.01 27.45±0.01 47.10±0.02

0.62-0.72 0.44-0.50 0.27-0.34 0.27-0.31 0.25-0.30 0.40-0.51

This species is endemic to the Lake Orumiyeh basin and has been recorded previously from Kazim-Chai, the River Ozband, Talkheh, Ghalechai, Zarrineh and Tatavi Rivers in the basin (Günther, 1899; Berg, 1925; Abdoli, 2000). According to this study, this species is also represented in the Namak basin.

Sažetak Prvi nalaz Alburnus atropatenae (bErG, 1925) (cyPrinidaE) U slivU jEzEra naMaK, cEntralni iran Vrsta Alburnus atropatenae (Berg, 1925) je prvi put zabilježena u rijeci Ghareh-Chay u slivu jezera Namak u centralnom Iranu. Ranije je ova vrsta zabilježena jedino u slivu jezera Orumiyeh na sjeverozapadnom dijelu Irana. Dana 8. kolovoza 2012. ulovljeno je dvadeset devet jedinki. Morfometrijska i meristička svojstva bila su slična onima već zabilježenima za tu vrstu. Ključne riječi: prvi nalaz, Alburnus atropatenae, jezero Namak, rijeka Ghareh-Chay

Abdoli, A. (2000): The Inland Water Fishes of Iran. Iranian Museum of Nature and Wildlife, Tehran. pp. 378. (in Persian) Berg, L. S. (1925): Opisanie novogo vida roda Alburnus (Pisces) iz basseina Oz. Urmii ezhegodnik [Description of a new species of the genus Alburnus (Pisces) from the basin of Lake Urmia]. Zoologischekogo Instituta Akademii Nauk SSSR, 26, 213-214. Bogutskaya, N. G., Kucuk, F., Unlu, E. (2000): Alburnus baliki, a new species of cyprinid Fish from the Manavgat River system, Turkey. Ichthyological Exploration of Freshwaters, 11, 55-64. Bzulug, M., Freyhof, J. (2007): Re-diagnosis of four species of Alburnus from Turkey and description of two new species (Teleostei: Cyprinidae). Ichthyological Exploration of Freshwaters, 18, 233-246. Coad, B. W., (2013): Freshwater Fishes of Iran. Available at http://www.briancoad.com (accessed on 12 March 2013). Freyhof, J., Kottelat, M. (2007): Review of the Alburnus mento species group with description of two new species (Teleostei: Cyprinidae). Ichthyological Exploration of Freshwaters, 18, 213–225. Günther, A. (1899): Fishes, pp. 381-391, pl. 23-24. In: Günther, R. T. Contributions to the natural history of Lake Urmi, N. W. Persia, and its neighbourhood. Journal of Zoology of the Linnaean Society, 27, 345-453, pl. 21-30. Kottelat, M., Freyhof, J. (2007): Handbook of European Freshwater Fishes. Kottelat, Cornol and Freyhof, Berlin, XIV, pp. 646.

79

Croatian Journal of Fisheries, 2013, 71, 80-83 N. Antolović et. al.: Oocyte size on LHRHa induced ovulation in saddled bream

CODEN RIBAEG ISSN 1330-061X

SHORT COMMUNICATION

iMPact of oocytE sizE on lhrha indUcEd ovUlation and fErtilizEd EGG qUality in saddlEd brEaM Oblada melanura (linnaEUs, 1758) nenad antolović*, valter Kožul, nikša Glavić, jakša bolotin University of Dubrovnik, Institute for Marine and Coastal Research, Kneza D. Jude 12, 20000 Dubrovnik, Croatia * Corresponding Author, E-mail: nenad.antolovic@unidu.hr

ARTICLE INFO

ABSTRACT

Received: 20 September 2012 Received in revised form: 11 April 2013 Accepted: 22 May 2013 Available online: 27 May 2013

The objective of this study was to evaluate the effects of oocyte size and luteinizing hormone releasing hormone analogue (LHRHa) on ovulatory success in artificial fertilization. Vitellogenic females with maximum oocyte diameters 400-550 μm were repeatedly injected with LHRHa (20 μg kg-1 per injection). Fish with maximum oocyte diameters <500 μm failed to ovulate. In contrast, all fish with maximum oocyte diameters >500 μm spawned within 48-54 h. These results demonstrate that injected LHRHa is effective for ovulation of saddled bream with maximum oocyte diameters >500μm.

Keywords: Saddled bream Oblada melanura LHRHa artificial fertilization

introdUction Saddled bream Oblada melanura, (Linnaeus, 1758), is recently considered as a potential candidate for mariculture in the Adriatic Sea. The reports on characteristics of the species in captivity are scarce. There is also no data on spontaneous spawning in captivity. Control of reproductive function in captivity is essential and can be achieved by manipulating photoperiod, water temperature or spawning substrate (Mylonas et al., 2010). In captivity females of most fish species fail to undergo final oocyte maturation (FOM), ovulating and spawning (Zohar, 1989). Similar to most wild animals held in captivity, many fish of commercial interest to aquaculture industry exhibit reproductive dysfunctions (Zohar and Mylonas, 2001). Among other hormones, luteinizing hormone releasing hormone analog (LHRHa) has been successfully used to induce ovulation in fish (Mylonas et al., 2010; King and Pankhurst, 2007; Berlinsky et al., 2005; Firat et al., 2005).

MatErials and MEthods Broodstock of saddled bream were maintained under natural ambient conditions (water temperature 12-26 °C, salinity 37 gL-1). During the spawning 80

studies (June), photoperiod was held at 15L: 9D and water temperature at 19-23 °C. Females were selected for induced ovulation based on their ovarian development. Ovarian biopsy tissue was obtained using tygon cannula (1.88 mm o.d. x 1.11 mm i.d.) as described by Shehedeh et al. (1973) and measured to the nearest 25 μm using stereo microscope (Wild Heerbrugg) fitted with an ocular micrometer (Wild Heerbrugg type 325400). The experiment was conducted at the Institute for Marine and Coastal Research (June, 2008). Temperature of water was 21 °C, females were biopsied and 25-30 of the largest oocyte present were measured. Fish (n= 24; 210-260 g body weight (BW)) that exhibited pre-spawning morphology (distended abdomens; Mylonas et al., 1995) were divided into two groups: females with the oocyte size <500 μm (range 400-500 μm) and females with oocyte size >500 μm (range 500-550 μm). Fish were anesthetized in solution (1 mg l-1) of benzocaine, LHRHa was administered intramuscularly with injection (priming injection), 20 μg kg-1 BW. After hormonal treatment the fish were placed in separated 1500 l tank and beginning 24 h post injection. After 24 h fish received second injection (resolving injection) of 20 μg kg-1 BW. The eggs were fertilized by adding pooled milt (0.5 ml) from three anesthetized males and 35 ml of filtered ambient sea

Croatian Journal of Fisheries, 2013, 71, 80-83 N. Antolović et. al.: Oocyte size on LHRHa induced ovulation in saddled bream

water and gently mixed. Hormone treatments were evaluated using the following criteria: egg production, buoyancy, fertilization rate, hatching rate. The total number of ovulated eggs produced by each female was calculated by counting eggs in 1 ml subsample of egg mass. Total egg count per female was derived by multiplying the number of eggs in a subsample with the total egg volume. Buoyancy, fertilization and hatching rates were determined for each female (tree replicates of 300 eggs from each fish were randomly taken and used to estimate fertilization rate and hatch rate). These percentages were calculated using the following formula: Buoyancy (%) = 100 x no. of buoyant eggs/ no. of ovulated eggs Fertilization (%) = 100 x no. of fertilized eggs/ no. of buoyant eggs Hatching (%) = 100 x no. of hatched larvae/ no of fertilized eggs The percentage of fertilized eggs was determined 1h and 20 min after spawning (4-8 cell stage) by stereo microscopic examination.

Statistical methods All data sets were tested for normality using Kolmogorov-Smirnov normality test, while ANOVA model was used to examine the effects of LHRHa on oocyte size of females within each 6 hours. Multiple regression was used to model the relation of egg diameter to LHRHa treatment.

rEsUlts Prespawning females contained vitellogenic oocytes varying in oocyte diameters containing 400-550 μm. The ovulatory response to LHRHa was affected by oocyte size. No fish with the maximum oocyte diameters <500 μm ovulated. In contrast, 12 of 12 fish with uniformly, fully vitellogenic oocyte >500 μm ovulated substantial numbers of eggs. After 24 h of prime injection of LHRHa at the sea temperature of 21.8 °C, maximum oocyte diameters were 700 μm, oocyte range was from 600-750 μm with the mean diameter 718± 21μm and the formation of oil drop began. After 48 h of resolving injection, maximum oocyte diameters were >800 μm and range from 755-928 μm, oil drop was formed with range of 166-185 μm and fish were ready to spawn. All injected females with oocyte maximum diameters >500 μm were spawned at 48 – 54 h after the priming injection. The number of ovulated eggs, rate of buoyancy, fertilization and hatching for different females are shown in Table 1. The mature unfertilized eggs were pelagic and transparent with one oil globule, with the mean diameter 833±55 μm. Significant increase in egg diameter was shown (ANOVA, Wilks-lambda, 0.00027; p<0.001) after administering LHRHa. This study shows that oocyte with diameter of 500 μm or more grows to 833±55 μm 48h after the first injection of LHRHa at the mean temperature of 21.8 °C.

Table 1. Body weight (g), eggs production (x g -1 fish), buoyancy (%), fertilization rate (%) during the induction spawning of saddled bream Oblada melanura Spawned female number 1 2 3 4 5 6 7 8 9 10 11 12 Mean + SD

Body weight (g) 254 209 236 214 238 246 205 223 246 209 218 248 228.8 ±17.7

Number of ovulated Buoyancya (%) Fertilization rateb (%) eggs (x g -1 fish) 605 589 608 702 625 730 506 608 654 723 568 502 618.3 ± 74.8

35 54 49 57 62 38 39 46 52 57 52 29 47.5 ±10.1

46 54 49 52 62 71 72 58 64 75 48 52 58.5 ± 10.2

Hatching ratec (%) 52 58 61 72 59 64 58 63 68 58 54 70 61.4 ± 6.2

Buoyancy (%) = 100 x no. of buoyant eggs/ no. of ovulated eggs Fertilization (%) = 100 x no. of fertilized eggs/ no. of buoyant eggs c Hatching (%) = 100 x no. of hatched larvae/ no. of fertilized eggs a

b

81

Croatian Journal of Fisheries, 2013, 71, 80-83 N. Antolović et. al.: Oocyte size on LHRHa induced ovulation in saddled bream

discUssion Synthetic analogues of LHRHa are successful in spawning induction of saddled sea bream. LHRHa has been effectively used to induce ovulation and spawning in a number of commercially important finfish (Rasines et al., 2013; Tucker, 1994; Mylonas and Zohar., 2001; Glamuzina et al., 1998). In some species, such as spotted seatrout (Cynoscion nebulosus) and red drum (Sciaeops ocellatus) with fully grown oocytes, a single injection of LHRHa is sufficient for ovulation induction (Thomas and Boyd, 1989). In the present study, ovulation was induced in saddled bream just prior to the natural spawning season. Under this condition LHRHa administered with two injections of 20 μg kg-1 BW was sufficient to induce ovulation in saddled bream. Our study is in accordance whit Berlinsky et al., (2005) who suggested that oocyte diameters >550 μm have high potential for response to LHRHa. The latency period after hormonal injection was related to the initial oocyte diameter and water temperature. Fertilization percentage was 58.5% when eggs were fertilized 0-6 h after ovulation. Fertilization percentage of saddled bream, if compared with other sparids, was smaller. For example, in red porgy Pagrus major fertilization percentage was 70% (Mylonas et al., 2004), 88% for gilthead sea bream and 82% for sharpsnout sea bream, Diplodus puntazzo (Papadaki et al., 2008). Overall, saddled bream exhibits good fertilization percentages in captivity, but further studies are needed in order to examine the effect of various environmental and physiological parameters on egg quality variation.

ACKNOWLEDGEMENTS This study was supported by the Croatian Ministry of Science, Education and Sports (grant number 275-0000000-3628).

Sažetak djElovanjE lhrha za izazivanjE ovUlacijE Pri različitiM vEličinaMa oocita i KvalitEtE oPlođEnih jajašaca U UšatE Oblada melanura (linnaEUs, 1758) Cilj ovog rada bio je utvrditi veličinu oocita za uspješno djelovanje hormona LHRHa i uspješnost umjetne oplodnje nakon hormonskog tretmana. Vitelogene ženke s promjerom oocita 400-550 μm injektirane su hormonom LHRHa 20 μg/kg težine. Ženke s najvećim brojem oocita promjera <500 μm 82

nisu ovulirale, dok su ženke s najvećim brojem oocita promjera >500 μm ovulirale u roku 48 -54 sati nakon tretmana s LHRHa. Ovaj rad pokazuje da je hormon LHRHa djelotvoran za izazivanje ovulacije kod ušate kada se injektira pri veličini oocita iznad 500 μm. Ključne riječi: ušata, Oblada melanura, LHRHa, umjetna oplodnja

rEfErEncEs Berlinsky, D. L., King, V. W., Smith, T. I. J. (2005): The use of luteinizing hormone releasing hormone analogue for ovulation induction in black sea bass (Centropristis striata). Aquaculture, 250, 813-822. Firat, K., Saka, S., Suzer, C. (2005): Gonadal oocyte development in LHRHa hormone treated European sea bass (Dicentrarchus labrax L., 1758) broodstock. Turkish Journal of Veterinary and Animal Sciences, 29, 83-87. Glamuzina, B., Glavić N., Kožul V., Skaramuca, B. (1998): Induced sex reversal of the dusky grouper, Epinephelus marginatus (Love, 1834). Aquaculture Research, 29, 563-568. King, H. R., Pankhurst, N. W. (2007): Additive effects of advanced temperature and photoperiod regimes and LHRHa injection on ovulation in Atlantic salmon (Salmo salar). Aquaculture, 273, 729-738. Mylonas, C. C., Fostier, A., Zanuy, S. (2010): Broodstok management and hormonal manipulation of fish reproduction. General and Comparative Endocrinology 1, 165, 3, 516-34. Mylonas, C. C., Papadaki, M., Pavlidis, M., Divanach, P. (2004): Evaluation of egg production and quality in the Mediterranean red porgy (Pagrus pagrus) during two consecutive spawning seasons. Aquaculture, 232, 637-649. Mylonas, C. C., Zohar, Y. (2001): Use of GnRHa- delivery system for control of reproduction in fish. Reviews in Fish Biology and Fisheries, 10, 463-491. Papadaki, M., Papadopoulou, M., Siggelaki, I., Mylonas, C.C. (2008): Egg and sperm production and quality of sharpsnout sea bream (Diplodus puntazzo) in captivity. Aquaculture, 276, 187–197. Rasines, I., Gómez, M., Martín, I., Rodríguez, C., Mañanós, E., Chereguini, O. (2013): Artificial fertilisation of cultured Senegalese sole (Solea senegalensis): Effects of the time of day of hormonal treatment on inducing ovulation. Aquaculture, 392, 94-97.

Croatian Journal of Fisheries, 2013, 71, 80-83 N. AntoloviÄ&#x2021; et. al.: Oocyte size on LHRHa induced ovulation in saddled bream

Shehedeh, Z. H., Kuo, C. M., Milisen, K. K. (1973): Validation of an in vivo method for monitoring ovarian development in the grey mullet (Mugil cephalus L.). Journal of Fish Biology, 5, 479-487. Thomas, P., Boyd, N. (1989): Dietary administration of an LHRH analogue induces spawning of spotted seatrout (Cynoscion nebulosus). Aquaculture, 80, 363-370. Tucker, Jr. J.W. (1994): Spawning by captive serranid fishes: a reviw. Journal of the World Aquaculture Society, 25, 345-359.

Zohar, Y., Mylonas, C. C. (2001): Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture, 197, 99-136. Zohar, Y. (1989): Fish reproduction: its physiology and artificial manipulation. In: Shilo, M., Sarig, S. (Eds.), Fish Culture in Warm Water Systems: Problems and Trends. CRC Press, Boca Raton, 65-119.

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Croatian Journal of Fisheries, 2013, 71, 84-86 G. Özcan.: Threatened fishes: C. kinzelbachi

CODEN RIBAEG ISSN 1330-061X

SHORT COMMUNICATION

thrEatEnEd fishEs of thE world: Chondrostoma kinzelbachi KrUPP, 1985 (cyPrinidaE) Gulnaz Özcan Mustafa Kemal University, Faculty of Marine Scinces and Technology, 31200, Iskenderun, Hatay, Turkey * Corresponding Author, E-mail: gulnazozcan@yahoo.com

ARTICLE INFO

ABSTRACT

Received: 15 February 2013 Received in revised form: 12 April 2013 Accepted: 22 May 2013 Available online: 27 May 2013

Orontes nase, Chondrostoma kinzelbachi is an endangered freshwater fish species in Turkey and Syria. Populations are declining due to dam construction and water pollution. Orontes nase populations should be completely protected.

Keywords: Orontes nase Chondrostoma kinzelbachi Conservation

coMMon naMEs Asi kababurun baligi (Tr), Orontes nase (Eng) (Fig 1).

consErvation statUs IUCN International Red List: endangered ENB2ab(iii) (Crivelli, 2006); Turkey: Critically endangered (Fricke et al. 2007).

idEntification D III/ (8)9(10), A III/10-11, Ll. 63-69, gill rakers 24-28. The body is elongate and moderately compressed.

The dorsal profile of the head is slightly convex. The mouth is inferior, the anterior margin of the lower jaw is gently arched and covered with a sharp corneous layer, the upper lip is very thin, barbels are absent. The lateral line is complete. The upper margin of the dorsal fin is straight or slightly concave (Krupp, 1985).

distribUtion C. kinzelbachi is endemic to the River Orontes basin in Turkey and Syria (Krupp, 1985). Also, it is found in Gölbaşı Lake (36° 32’ N 36° 30’ E) in Hatay (Turkey) (Bostancı, 2006) (Fig 2).

Fig. 1. Orontes nase, Chondrostoma kinzelbachi (photo G. Özcan) 84

Croatian Journal of Fisheries, 2013, 71, 84-86 G. Özcan.: Threatened fishes: C. kinzelbachi

thrEats Populations of C. kinzelbachi are threatened by habitat loss, water extraction, eutrophication/pollution and destruction of shallow water habitats by the construction of dams (Crivelli, 2006; Fricke et al. 2007). The species is sensitive to chemical (ammonia) pollution which also reduces its populations (Crivelli 2006).

consErvation action The species is officially protected by the prohibition of fishing during spawning season in Turkey. Conservation action on the Syrian part of the river is still unknown (Crivelli, 2006).

consErvation rEcoMMEndations

Fig. 2. Map of distribution of existed nase populations in Turkey and Syria (1: Karasu, 2: Gölbaşı Lake, 3: Buhairat ar-Rastan, 4: Buhairat Hims)

Studies on biology and ecology of this species are required. Remaining populations of the species should be completely protected. Biological research for population size and structure, habitat requirement and reproduction ecology are required in Turkey and Syria. Pollution from industrial and domestic effluent should be reduced. Water extraction for agriculture and dam construction must be controlled.

rEMarKs abUndancE Restricted to the River Orontes basin. No data on population size, but according to a survey of the river and catchments, the species was found in only two locations in Turkey; the River Karasu and Gölbaşı Lake, and three locations in Syria (Krupp, 1985; Bostancı, 2006). This species is represented with low abundances in certain localities in Turkey. The population status of the Syrian part is unknown.

habitat and EcoloGy Mostly a riverine species, but lacustrine forms are also described. This species needs well-oxygenated water for spawning. Feeds on algae from rock surfaces (Crivelli, 2006). C. kinzelbachi occurs in gravel, stone and vegetated areas in Gölbaşı Lake (Bostancı, 2006).

C. kinzelbachi differs from all other members of this genus by a modal pharyngeal tooth formula of 7-7 (Krupp, 1985).

Sažetak UGrožEnE ribE svijEta: Chondrostoma kinzelbachi KrUPP, 1985 (cyPrinidaE) Sirijski podust, Chondrostoma kinzelbachi je ugrožena slatkovodna riblja vrsta u Turskoj i Siriji. Broj populacije je u opadanju zbog izgradnje brana i onečišćenja voda. Potrebno je u potpunosti zaštititi njihove populacije. Ključne riječi: sirijski podust, Chondrostoma kinzelbachi, očuvanje

rEProdUction The reproductive biology of Orontes Nase is unknown. 85

Croatian Journal of Fisheries, 2013, 71, 84-86 G. Özcan.: Threatened fishes: C. kinzelbachi

rEfErEncEs Bostancı, Z. (2006): Taxonomic investigation of freshwater fish fauna living Seyhan, Ceyhan and Asi River (in Turkey) M.Sc. Thesis, Karadeniz Technical University, Trabzon, pp. 113. (in Turkish with English abstract). Crivelli, A. J. (2006): Chondrostoma kinzelbachi. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2. (www.iucnredlist.org).

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Fricke, R., Bilecenoğlu, M., Sarı H. M. (2007): Annotated checklist of fish and lamprey species (Gnathostomata and Petrmyzontomorphii) of Turkey, including a Red Lisat of threatened and declining species. Stuttgarter Beiträge zur Naturkunde, Serie A (Biologie), 706, 1-169. Krupp, F. (1985): A new species of Chondrostoma from the Orontes River drainage basin of Turkey and Syria. Senckenbergiana biologica, 66, 1/3, 27-33.

Croatian Journal of Fisheries, 2013, 71, 87-89 H. Mousavi-Sabet et al.: Threatened fishes: P. rhadinaeus

CODEN RIBAEG ISSN 1330-061X

SHORT COMMUNICATION

thrEatEnEd fishEs of thE world: Paracobitis rhadinaeus (rEGan, 1906) (nEMachEilidaE) hamed Mousavi-sabet*1, ahmad Gharaei2, Mostafa Ghaffari2 Department of Fisheries Sciences, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, P.O. Box 1144, Guilan, Iran Department of Fisheries, Hamoun International Wetland Research Institute, University of Zabol, Zabol, Iran * Corresponding Author, E-mail: mousavi-sabet@guilan.ac.ir

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ARTICLE INFO

ABSTRACT

Received: 15 February 2013 Received in revised form: 12 April 2013 Accepted: 22 May 2013 Available online: 27 May 2013

Paracobitis rhadinaeus is an endemic Nemacheiline loach in the Sistan basin, southeast Iran. The population is declining probably due to habitat loss or degradation, damming, drought and poaching. Urgent habitat protection with bans on further regulation of the Hamoun wetland and related reservoirs is suggested. Captive breeding of the fish should be initiated. Fishing activities should be forbidden or limited. A detailed study of current population status, biology and ecology of P. rhadinaeus is required.

Keywords: Paracobitis rhadinaeus Nemacheilidae Sistan Basin Conservation

coMMon naMEs Mar-Mahi (Persian), meaning Snake-fish; this may refer to this species which has an elongate body. Sistanâ&#x20AC;&#x2122;s Loach (English) (Fig 1).

consErvation statUs IUCN Red List: not evaluated; Iran: unknown (Coad, 2013). Not protected in Iran nor Afghanistan.

idEntification DIII 7-81/2, AII 51/2, VI 6-71/2, PI 8-91/2, C 19-22; the snout is longer than the postorbital distance; body depth is 7-10 times its body length; head length 5.05.5 times its body length; the mouth cleft extends to

below the nostrils; lower lip interrupted medially; outer rostral barbel is as long as maxillary barbel reaching back to or beyond nostrils; dorsal fin origin nearer tip of snout than caudal fin base; caudal fin slightly emarginated; caudal peduncle 2.0-2.75 as long as deep, 5.0-5.3 in length of fish; scales are highly deciduous and not always present on old preserved material; the dorsal fin rounded; there is a well-developed post-dorsal fin crest and a slight ventral crest on the caudal peduncle; the pelvic fin origin lies just in front of the mid-point of the dorsal fin base; there is an adipose tissue flap at the pelvic fin base; the anterior nostril is a tube followed immediately by a horizontal slit; the bony upper jaw has a slight protuberance and the lower jaw is curved and not indented; size attains 288 mm TL (Coad, 2013).

Fig. 1. Paracobitis rhadinaeus, 166 mm TL (photo by Sahel Pakzad-Touchaei, Jun 2012). 87

Croatian Journal of Fisheries, 2013, 71, 87-89 H. Mousavi-Sabet et al.: Threatened fishes: P. rhadinaeus

distribUtion

consErvation rEcoMMEndations

This endemic species is restricted to the Sistan basin of Iran and presumably Afghanistan. The species was distributed in the Hamoun wetland (Sistan basin in southeast Iran), but ever since dam construction on the River Hirmand (the water supply of the wetland) in Afghanistan one decade ago, the wetland dried. After the wetland dried, the fish has remained in related reservoirs to the wetland. Bănărescu and Nalbant (1966) place this species in the Rivers Atrak and Sefid of the Caspian Sea basin, the River Abkhar of central Iran, probably most of Iran, the Helmand drainage and the River Tedzhen, evidently confusing it with P. malapterura and P. iranica. Abdoli (2000) lists as questionably from the Bejestan, Kerman-Na’in and Dasht-e Lut basins, from the middle and lower Rivers Halil and Bampur of the Hamun-e Jaz Murian basin, from the Simish and the river to its north in the River Mashkid basin.

Urgent habitat protection with bans on further regulation of the wetland and related reservoirs is suggested. A detailed study of current population status, biology (especially reproduction) and ecology of P. rhadinaeus is required in order to design a captive breeding protocol and stock management of the fish. Water rights of the wetland and related reservoirs should be respected (but are ignored by damming) to prevent drought. Education of local fishermen should be initiated. Fishing programs should be forbidden or at least should be limited in the habitat of P. rhadinaeus. Also fishing equipment and methods should be edited according to minimum bycatch.

abUndancE Sporadic; locally abundant.

habitat and EcoloGy Annandale and Hora (1920) note that the fish was healthy buried some inches in mud when cyprinids died in foul water above (the Hamoun wetland was turbid when the River Hirmand overflowed). Stomach contents include cyprinid fish remains and mayfly larvae (Annandale, 1921). Reproduction is unknown (Coad, 2013).

thrEats It is endangered by primary anthropogenic which lowers the quality of its limited habitats, dams (about 10 concrete dams on the River Hirmand in Afghanistan), natural drought in recent decades, also anthropogenic drought which was caused by damming and unintended poaching (Iranian people don’t eat the fish - so it is not economically or nutritionally important - but the population is damaged as incidental catch or bycatch in Cyprinid fishing in reservoirs).

consErvation action Not protected in Iran nor Afghanistan.

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rEMarKs Bănărescu and Nalbant (1995) as well as Nalbant and Bianco (1998) place this species in Paracobitis. The Catalog of Fishes (Eschmeyer, 2013) spells the trivial name as Nemacheilus macmahoni or Nemacheilus rhadinæus. Nemacheilus macmahoni Chaudhuri, 1909 described from the “affluents (= delta, an error for effluents) of the Helmand” is a synonym according to Bănărescu and Nalbant (1966) who refute the opinions of Nikol’skii (1947) and Berg (1949) who consider macmahoni to be identical to P. malapterura. Earlier Bănărescu and Nalbant (1964) placed fish from the Sistan and Caspian Sea basin of Iran as Nemacheilus malapterurus macmahoni. P. malapterura has both lips strongly furrowed, pelvic fin origin under the dorsal fin origin rather than behind, better developed scales which are also present on midflank, and a colour pattern of numerous oblique bands. P. rhadinaeus is distinguished from macmahoni by Annandale and Hora (1920) in having an extremely short posterior diverticulum and minute vesicle in the swimbladder, by the absence of scales, a more elongate body, smaller, narrower and less flattened head, and by differences in body profile. A syntype of Nemacheilus rhadinæus (ZSI F1240/1) is in the Zoological Survey of India, Calcutta, under the name Adiposia rhadinaea and the holotype of Nemacheilus macmahoni (ZSI F1222/1) is also there under the name Adiposia macmahoni (Menon and Yazdani, 1968). Two syntypes listed as Nemacheilus rhadinaeus from “Sistan” are in the Natural History Museum, London (BM (NH) 1905.11.29:28-29, 2, 137.8-209.1 mm standard length) (Coad, 2013).

Croatian Journal of Fisheries, 2013, 71, 87-89 H. Mousavi-Sabet et al.: Threatened fishes: P. rhadinaeus

Sažetak UGrožEnE ribE svijEta: Paracobitis rhadinaeus (rEGan, 1906) (nEMachEilidaE) Paracobitis rhadinaeus je endemski vijun iz Sistanskog slijeva na jugoistoku Irana. Populacija je u opadanju najvjerojatnije zbog gubitka i narušavanja staništa, podizanja brana, isušivanja i potapanja zemljišta. Predlaže se hitna zaštita staništa sa zabranom daljnje regulacije slijeva Hamun i njegovih akumulacijskih jezera. Potrebno je potaknuti kontrolirani mrijest ove vrste te zabraniti ili ograničiti ribolov. Nužna je detaljna studija sadašnjeg stanja populacije, biologije i ekologije vrste P. rhadinaeus. Ključne riječi: Paracobitis rhadinaeus, Nemacheilidae, Sistanski slijev, očuvanje

rEfErEncEs Abdoli, A. (2000): The Inland Water Fishes of Iran. Iranian Museum of Nature and Wildlife, Tehran, pp. 378. [In Persian] Annandale, N. (1921): The aquatic fauna of Seistan. A summary. Records of the Indian Museum, 18, 235-253. Annandale, N., Hora, S. L. (1920): The fish of Seistan. Records of the Indian Museum, 18, 151-203, pls. XV-XVII (includes:- Appendix. Note on the fisheries of the delta of the Helmand and on the use of shaped rafts of bulrushes in India and Seistan, by N. Annandale). Bănărescu, P. M., Nalbant, T. T. (1966): The 3rd Danish Expedition to Central Asia. Zoological Results 34. Cobitidae (Pisces) from Afghanistan and Iran. Videnskabelige Meddelelser fra Dansk naturhistorisk Forening, 129, 149-186, pl. XIXXXI.

Bănărescu, P. M., Nalbant, T. T. (1995): A generical classification of Nemacheilinae with description of two new genera (Teleostei: Cypriniformes: Cobitidae). Travaux du Muséum d’Histoire Naturelle Grigore Antipa, Bucurešti, 35, 429-496. Bănărescu, P. M., Nalbant, T. T. (1964): Süßwasserfische der Türkei. 2. Teil Cobitidae. Mitteilungen aus dem hamburgischen Zoologischen Museum und Institut, 61, 159-201. Berg, L. S. (1949): Presnovodnye ryby Irana i sopredel’nykh stran [Freshwater fishes of Iran and adjacent countries]. Trudy Zoologicheskogo Instituta Akademii Nauk SSSR, 8, 783-858. Coad, B. W. (2013): Freshwater Fishes of Iran (Available at http://www.briancoad.com) (accessed on 6 January 2013). Eschmeyer, W. N. (ed). (2013): Catalog of Fishes. California Academy of Sciences (Available at http://research.calacademy.org/research/ichthyology/catalog/fishcatmain.asp) (Electronic version accessed on 1 April 2013). Menon, A. G. K., Yazdani, G. M. (1968): Catalogue of type-specimens in the Zoological Survey of India. Part 2.-Fishes. Records of the Zoological Survey of India, 61, 1 & 2, 1-190. Nalbant, T. T., Bianco, P. G. (1998): The loaches of Iran and adjacent regions with description of six new species (Cobitoidea). Italian Journal of Zoology, 65 (Supplement), 109-123. (Proceedings of the Ninth Congress of European Ichthyologists (CEI-9) “Fish Biodiversity” organized in Naples at the University Federico II and held in Trieste - Italy, 24-30 August 1997). Nikol’skii, G. V. (1947): Gol’tsy besstochnykh vodoemov Turkmenii [The loaches of the inland waters of Turkmenia]. Byulleten’ Moskovskogo Obshchestva Ispytatelei Prirody, Otdel Biologii, 52, 3, 29-34.

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an invEstiGation into PovErty rEdUction throUGh fishEriEs activitiEs Note: Abridged version of the paper presented at the IIFET 2012 Tanzania: Conference on “Visible Possibilities: The Economics of Sustainable Fisheries, Aquaculture and Seafood Trade”, to be held at Dar es Salaam, Tanzania, during July 16-20, 2012. Authored by: Dr. Santosh Kumar Mishra (Ph. D.), Technical Assistant, Population Education Resource Centre (PERC), Department of Continuing and Adult Education and Extension Work, S. N. D. T. Women’s University, Patkar Hall Building, First Floor, Room. No.: 03, New Marine Lines, Mumbai - 400020, Maharashtra, India, Tel.: 91 022 22066892 (O), 09224380445 (M), 91 022 28090363 (R), Email: drskmishrain@yahoo.com, Web Link: http://sndt. ac.in/

1. introdUctory rEMarKs: From the earliest times and in practically all countries, fisheries have been of industrial and commercial importance. Fisheries and aquaculture contribute significantly to food security. Livelihoods of millions of people across the regions of the globe depend on healthy aquatic ecosystems. Achieving the Millennium Development Goals to eliminate extreme poverty by 2015 requires a concerted and unified effort by governments and the international community (United Nations, 2010). Many fishers are poor and so targeting development interventions at fishing communities can help reduce poverty. In order to increase the contribution that fisheries make to poverty reduction, management that ensures sustainability of fish stocks and equitable distribution of the benefits is needed. To achieve this, policy-makers must recognize the important contributions that fisheries make at both national and local levels (FAO, 1999). This paper aims to examine how fisheries can contribute to poverty reduction, with relevant examples.

2. thE issUE of PovErty: Poverty (defined as “the state of one who lacks a certain relative amount of material possessions or money”) is a complex phenomenon involving failure to meet a range of basic human needs and the denial of options that have consequences for opportunities to live long, healthy and 90

creative lives. Poverty in fishery dependent communities, therefore, is not solely related to the abundance of the catch, market opportunities or the state of the resource. It is also critically dependent on how the benefits from the use of fishery and other resources are used and whether a range of basic services (e.g. in health and education) are provided (FAO of the UN, 2005).

3. fishEriEs dEvEloPMEnt: Fisheries development is the process towards achieving the full potential of the sector through growth and improvement. A fishery is being developed if the biomass of the stock is being reduced by fishing, rebuilt (after depletion) or enhanced to increase its productivity. It is also developed if the quality of the catch or its value improves, not necessarily increasing the harvest. The main goal of an ecologically sustainable development is to improve the well-being of all the people engaged directly or indirectly in the fisheries sector, as well as the natural productive system. Under an ecosystem approach to fisheries, ‘development’ may be achieved by reducing the negative environmental impact and/or increasing resilience of the system to unexpected change, meeting broader societal objectives. As the full potential of wild fisheries resources has been achieved – and often ‘lost’ through ‘overfishing’ – the main objective and emphasis in capture fisheries development strategies has changed from increasing harvest (the main objective for three quarters of the last century) to establishing a more sustainable and optimal use of the available fisheries resources (particularly since UNCED in 1992). Concern for environmental management and sustainability appeared essentially during the 1990s (Abbot and Guijt, 1998).

4. EconoMic dEvEloPMEnt and fishEriEs: The fisheries sector is rarely a strategic sector for national economic development. Although it plays a prominent role in only a few countries such as Iceland, Namibia, the Maldives and other small island developing states rich in fishery resources relative to their populations, it is nonetheless an important economic activity, and very often a strategic

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one, in many coastal regions of the world. Indeed, in many countries, fish export is a major contributor to foreign exchange earnings, often ranking far higher than other agricultural commodities. The major trade flow underlines the significance of this sector for the trade balance of many developing countries. Licensing fees of foreign fishing fleets are another source of foreign exchange revenue from marine fishery resources, especially in West African and South Pacific countries. The more considerable and substantial contribution of fisheries worldwide is the supply of highly nutritious animal protein for human consumption and the employment and income generation in often-remote coastal areas (Centre for Science and Environment, 1985).

5. thE contribUtion of fishEriEs to PovErty rEdUction: 5.1 Fisheries are economically important: Fisheries provide an important source of revenue for many developing countries. Net fisheries exports amounted to US$17.4 billion in 2002 in foreign exchange earnings for developing countries, more than the net exports of coffee, cocoa, sugar and tea combined.

5.2 Fisheries are important to food security: Fisheries provide a key source of protein, micronutrients, essential fatty acids and minerals. They contribute to food security by providing an accessible and cheap protein source for the poor that complements other locally available food sources. Over one billion people worldwide rely on fish as their main source of animal protein. Fish are a particularly important source of protein in developing countries where protein intake may be low. For 2.6 billion people in developing countries, fish provides over 20% of total animal protein intake, compared with 8% in developed countries.

5.3 Fisheries form a key part of livelihoods: Fisheries are an important part of the livelihoods of millions of people around the world. They provide a source of food, employment and income. For many poor families, fishing is a way of reducing their vulnerability to risks by supplementing and diversifying their incomes. They also provide a ‘safety net’ for the poor when other economic opportunities or food sources are limited (Binswanger and von Braun, 1993).

6. contribUtion of sMall-scalE fishEriEs to EconoMic Growth and PovErty allEviation: The small-scale fisheries sector is generally described as being dynamic and evolving and is typically labour intensive, using relatively small amounts of capital and energy (OECD, 1972). Smallscale fishers use small vessels and often fish inshore for local and domestic markets although export-oriented production has increased significantly during the last couple of decades. The small-scale fisheries contribute to economic growth and poverty reduction by the following ways:

6.1 Foreign exchange: Small-scale fisheries can make significant contributions to national economies through the generation of foreign exchange derived from international trade.

6.2 Multiplier/GDP effects: Income multiplier effects can potentially “trickle up” to the national economy ensuring that small-scale fisheries can support national economic growth through contributions to gross domestic product (GDP).

6.3 Tax generation: Taxes provide national governments with an opportunity to assist both poverty reduction and poverty prevention initiatives. Taxes made available to national treasuries can be spent on re-distributive mechanisms aimed at targeted poverty prevention or on generic social support.

7. contribUtion of sMall-scalE fishEriEs to local EconoMiEs: Wealth generated by individuals, households or small enterprises can make significant contributions to local economies through income and employment multiplier effects. This is especially the case in fisheries because of the “cash crop” nature of the harvest. Fish may be one of the few products in some rural economies that can generate cash to spur and stimulate demand, because other food products may be more generally bartered or consumed within the household (Twopeny, 1983).

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8. EnhancinG thE contribUtion of fishEriEs to PovErty rEdUction: Fisheries contribute to poverty reduction in the following ways: o Effective management for sustainability, o Coherent policy objectives, o Governance and capacity building and o Distribution of benefits.

9. conclUdinG rEMarKs: Achieving the Millennium Development Goals to eliminate extreme poverty by 2015 requires a concerted and unified effort by governments and the international community. When fisheries are well managed, they can contribute to reducing poverty. The paper concludes that although much progress has been made towards understanding the contributions that fisheries can make to poverty reduction, and to achieving the Millennium Development Goals, further support is needed in the areas of: (a) strategies to maximize the benefits derived from fisheries and reduce the poverty and vulnerability of fishers; (b) establishing appropriate governance structures and strengthening fisheries institutions dealing with management, especially in their ability to manage fisheries sustainably; and (c) raising awareness of the importance and contribution of fisheries to poverty reduction. Keywords: fisheries, poverty reduction, development interventions, future work priorities, Millennium Development Goals

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rEfErEncEs: United Nations (2010): Making Millennium Development Goals Happen. New York, United Nations. FAO (1999): Indicators for Sustainable Development of Marine Capture Fisheries. Rome, FAO. FAO of the UN (2005): Increasing the Contribution of Small-Scale Fisheries to Poverty Alleviation and Food Security. FAO Technical Guidelines for Responsible Fisheries 10. Rome, FAO of the UN. Abbot, J., Guijt, I. (1998): Changing Views on Change: Participatory Approaches to Monitoring the Environment, SARL Discussion, Paper No. 2. London: IIED. Centre for Science and Environment (1985): The State of Indiaâ&#x20AC;&#x2122;s Environment 1984-85. New Delhi, Centre for Science and Environment. Binswanger, H. P., von Braun, J. (1993): Technological Change and Commercialization in Agriculture. Washington DC, World Bank/IFPRI. OECD (1972): Guiding Principles Concerning International EconomicAspects of Environmental Policies. Council Recommendation C, 72, 128. Paris: OECD. Richard Twopeny (1983): Town Life in Australia. London: Elliot Stock.

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Croatian Journal of

Fisheries Contents

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Original scientific paper

PoPulatioN GENEtiC struCturE oF aN ENdaNGErEd kalibaus, Labeo calbasu (haMiltoN, 1822) rEVEalEd by MiCrosatEllitE dNa MarkErs Md. Robiul Hasan, Md. Nahiduzzaman, Mostafa Ali Reza Hossain, Md. Samsul Alam

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Short communication a rEPort oN MEGa laNdiNG oF blaCktiP sEa CatFish, Plicofollis dussumieri (ValENCiENNEs, 1840) FroM FrasErGuNJE FishiNG harbour, wEst bENGal, iNdia Sachinandan Dutta, Sugata Hazra

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First rECord oF Alburnus atropatenae (bErG, 1925) (CyPriNidaE) iN NaMak basiN, CENtral iraN Somaye Khataminejad, Hamed Mousavi-Sabet, Masud Sattari, Saber Vatandoust

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iMPaCt oF ooCytE sizE oN lhrha iNduCEd oVulatioN aNd FErtilizEd EGG quality iN saddlEd brEaM Oblada melanura (liNNaEus, 1758) Nenad Antolović, Valter Kožul, Nikša Glavić, Jakša Bolotin

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thrEatENEd FishEs oF thE world: Chondrostoma kinzelbachi kruPP, 1985 (CyPriNidaE) Gulnaz Özcan

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June 2013

NutritiVE ValuE oF trout (Oncorhynchus mykiss) FarMEd iN thE adriatiC sEa Tibor Janči, Danijel Kanski, Marina Dulić, Nives Marušić, Helga Medić, Tomislav Petrak, Sanja Vidaček

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Number 2

EFFECts oF soybEaN MEal basEd diEt oN Growth PErForMaNCE aNd hEMolyMPh bioChEMiCal ParaMEtErs oF Narrow-ClawEd CrayFish (Astacus leptodacylus EsChsCholtz, 1823) Mahdi Banaee, Fatemeh Daryalaal, Mohammad Reza Emampoor, Maryam Yaghobi

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Volume 71

thrEatENEd FishEs oF thE world: Paracobitis rhadinaeus (rEGaN, 1906) (NEMaChEilidaE) Hamed Mousavi-Sabet, Ahmad Gharaei, Mostafa Ghaffari

Supplement to fishing profession aN iNVEstiGatioN iNto PoVErty rEduCtioN throuGh FishEriEs aCtiVitiEs Santosh Kumar Mishra