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Keywords: Kutum, Larvae, Zooplankton, Artificial feed, Artemia nauplii Growth
Rutilus frisii kutum (Kamenskii, 1901) is one of the most important and economic aquatics of Caspian Sea which devoted approximately 44% of bony fish catch in 2006 (9631 tons). Due to high captured fish, its transportation and maintaining has special importance. Iranian Fisheries Organization (Shilat) produce up to 200 million fry (1-2 g body weight (b.w.) to restock the Caspian Sea population annually(Caspian Environment. org., 2007). This species is distributed from the mouth of the Volga River up to Astrabadskiy bay but the main population of kutum is found in Iranian waters (Abdoli, 1999). This species has a great economics importance for the Iranian fishing industry in the southern Caspian Sea with more than 900 km of coastline. The average annual catch of kutum was about 9600 tonnes in 1991-2001 in Iran (FAO, 2003). Fish growth is dependent on numerous factors, such as food quantity and quality, temperature, salinity, body size and oxygen concentration (Wootton 1990; Bry et al. 1991 and references therein).
Success of larval rearing depends on mainly on the availability of suitable diets that are readily consumed, efficiently digested and that provide the required nutrients to support good growth and health (Giri et al. 2002). In spite of many efforts to develop artificial diets, the culture of fish larvae during the primary nursery phase still depends heavily on natural food (Van Speybroeck, 2005). Live food seems to provide a good source of exogenous enzymes, and also helps in chemoreception and visual stimuli (Verreth et al., 1993; Kolkovski et al., 1995).
Zooplankton, especially rotifers, cladocerans and copepods are the most important food items in freshwater aquaculture (Szlauer andSzlauer, 1980). Also rotifers and copepod nauplii are an important part of the diet oflarval fish reared in earthen ponds and often stocking of ponds with larvae is done when largeblooms of rotifers are present (Thurstan and Rowland, 1995; Li et al., 1996; Ludwig, 2000).
Brine shrimp (Artemia sp.) nauplii or de-capsulated cysts are also widely recognized as being excellent starter feeds for larval fish (Leger et al., 1986; Lavens and Sorgeloos, 1996). However, Artemia cyst price is
high, therefore limiting their use in most developing countries (ImorouToko et al., 2008). A number of studies were carried out to find a satisfactory, formulated diets that would substitute for natural food (rotifers, Artemia sp.) in larval rearing of various fish species (Jones et al., 1993; Pearson-Le Ruyet et al., 1993; Wang et al., 2005). Limited success has been achieved in larvae were fed with artificial diet from first feeding. Nevertheless, it has been reported that some freshwater fish species, can be exclusively reared on artificial diets from the start of exogenous feeding (Kujawa, 2003; Hamackova et al., 2009).
The aims of this study were to evaluate (1) the potential of live foods and an artificial dry feed in rearing Rutilus frisii kutum larvae on growth and survival of kutum larvae and also to find the optimal time to transfer kutum larvae from live food to the artificial feed.
Materials and methods
1. Experimental conditions
The experiment was carried out at laboratory of aquaculture located in Sari Agricultural and Natural Sciences University. One-day-old Kutum larvae were transported from ShahidRajaee restocking center to the laboratory in aerated plasticbags by car (30 min). Larvae were held in one polyethylene tank for one day prior to the start of the study. The experiment began on day 3 post- hatch (the onset of external feeding) and lasted for 21 days. The initial mean weight of the fry was 4.5 ±0.22 mg and their mean length was 8.9 ± 0.4 mm at the beginning of the experiment. Random groups of 75 larvae were stocked into 9 (3 treatments x 3 replicates) 2 l cylindrical plastic containers. Fry were reared in static water. Water quality parameters were checked daily; pH was 8.1 ± 0.2 and water temperature was 18.5 ± 1 ° C. oxygen ranged from 5.2 to 5.6 mg l-1 oxygen ranged from 5.2 to 5.6 mg l-1. The photoperiod for this experiment was set at 12L: 12D cycle.
2. Preparation of live food and artificial diet
Zooplankton (predominantly consisting of rotifers and copepod nauplii) was harvested using a plankton net with a 60 um mesh size between 8:00 and 9:00 h from a pond (2000 m2, mean depth 1.3 m), which is located 30 km north-west of Sari, Iran. Harvested zooplankton was transferred to a 10 1 plastic container through a sieve of 1 mm mesh size (to exclude large predatory insects like dragonflies). In laboratory, zooplankton was again passed through a sieve (250 um) to separate adult copepods and from rotifers and copepod nauplii (adult copepods were retained on sieve). Then zooplankton retained by a 80 um sieve and transferred to a 40 1 fiberglass tank and diluted with dechlorinated tap water. Mean density and numerical percentage composition of zooplankton in the stock (fiberglass tank) was estimated by thoroughly mixing the water, taking triplicate Sub-samples (2 ml), and counting the organisms under a light microscope. Then, zooplankton was used as live food for larvaeArtemiaurmiana cysts (from Urmia Lake, Iran) were incubated and hatched in a transparent cylindro-conical container under optimal conditions (water temperature=28±1 °C; salinity= 33 ppt; continuous strong aeration; illumination of 2000 lux; PH= 8-8.5; cyst density= 2 g l-1). After 24 h incubation, newly hatched Artemianauplii were separated from the unhatched cysts by stopping aeration in the hatching container. Then, the nauplii were siphoned and used for larvae feeding.
In this experiment, SFK starter feed produced by Mazandaran Feed Manufacturing Company (Kaboli, Sari, Iran), was used as artificial feed for kutum larvae. SFK commercial diet is used for rearing of kutum larvae in earthen ponds at restocking centers.
3. Proximate composition
Proximate composition of live foods and artificial diet were analyzed following the standard methods of AOAC (1990). Dry matter was determined by weight loss after drying the samples in an oven at 100 ° C for 24 h. and crude protein (CP) content by the Kjeldahl method and calculated as nitrogen content multiplied by 6.25. Ash content was determined by incinerating dried samples in a muffle furnace at 600 ° C
for 12 h. Crude lipid was analyzed by ether extraction in a Soxhelt apparatus using soxhlet apparatus and Total carbohydrate was calculated by difference, i.e. total carbohydrate%= 100- (CP% + CL% + ash%).
4. Feeding experiment
There were three feeding treatments (A, B and C) in this experiment. The group A (control group) were fed with Artemia nauplii during the entire experiment (21 days); the group B were fed with mixed zooplankton during the entire experimentand then the group C fed artificial feed to the end of the experiment. All groups of larvae were fed in excess four times daily (7:00, 11:00, 15:00 and 19:00 h). Feeding in excess was performed in such a manner that a small reminder of uneaten food could always be seen in the containers. Two-thirds of the water volume in each container was renewed twice daily with the stored water.
5. Sampling and growth study
Every week, 10 kutum larvae from each replication (30 larvae per treatment) were sampled. After larvae were anaesthetized, the total length was measured to the nearest 0.1 mm using a dial caliper and individual wet weight was determined by precision balance (0.1 mg sensitivity). The number of dead fry was recorded daily.
At the end of the experiment, indices such as weight gain, length increment, specific growth rate and survival rate were calculated for all treatments according to the following formulae:
Weight gain = Final weight - Initial weight
Length increment = Final length- Initial lengt
Specific growth rate = 100 x (Ln final weight- Ln initial weight)/ days Survival rate = 100 x (Final number of fry / Initial number of fry)
6. Statistical analysis
All statistical analysis was performed using statistical package SPSS, version 16. Descriptive statistic (means and standard deviations) of analysis results were calculated for each treatment. The homogeneity of variances of means was tested by Levene's test.
When data were normally distributed, the mean values for each parameter of different treatments were analyzed by one-way analysis of variance (ANOVA). When significant differences among treatments were found (P<0.05), the means were compared with Duncan's multiple range test.
In the investigation period, the density of zooplankton in the zooplankton stock was 80- 100 numbers/ml water. During the first 10 days of experiment rotifers was the dominant group of zooplankton, contributing 60- 70% of the total zooplankton density. Copepod nauplii were the dominant group of zooplankton in the second half of experiment. The proximate composition of live foods and artificial diet are given in Table 1.
Table 1 Proximate composition of mixed zooplankton and artificial diet
Dry matter (DM)
Crude protein (% of DM)
Crud lipid (% of DM)
Ash (% of DM)
a Zooplankton used for first 10 days of experiment
b Zooplankton used from day 11 to day 21 of experiment
The mean body weight and total length of kutum larvae under different treatments over a 3- week period (Fig. 1) showed significant differences from week 1 onwards. The mean body weight and total length of larvae fed during the whole period of rearing with live food (groups A and B) were significantly higher (P<0.05) than those of the other treatment (Fig. 1). Similarly, the weight gain and length increment of these larvae were also significantly higher (P<0.05) than those of the other treatment (Table 2). Indeed, The lowest mean body weight and total length were found in larvae fed over the whole period with artificial feed. When duration of initial feeding of kutum larvae with zooplankton increased the length and weight of larvae also gradually grew up (Fig. 1).
Table 2 Growth and survival of Rutilus frisii larvae in relation to feeding duration with live food and artificial feed
11.72 ± 1.71a
5.02 ± 0.62b
67.74 ± 9.4a
20.1 ± 5.07b
13.18 ± 0.62a
8.01 ± 1.1b
91.60 ± 5.2a
69.16 ± 10.1b
Values are mean ± SD of triplicates (n = 3 tanks per diet). Means followed by different letters with in a row are significantly different (P<0.05)
A: Artemianauplii for 21 days; B: zooplankton alone 21 days; C: artificial feed alone for 21 days. LI length increment; WG weight gain; SGR specific growth rate.
The lowest specific growth rate (8.01% day-1) was found in the group fed with artificial diet from first feeding to the end of the experiment (group C). The highest specific growth rate (13.58% day-1) was found in larvae fed during the whole period of rearing with Artemianauplii (group A), but it did not differ significantly (P>0.05) from that of larvae fed with mixed zooplankton (13.18% day-1) from first feeding to the end of experiment (Table 2).
The highest survival rates were obtained in the groups of larvae fed during the whole period of rearing with Artemia nauplii (group A, 92.5%) or zooplankton (group B, 91.6%), but it did not differ significantly (P>0.05). The larvae fed artificial feed only (group C) displayed the lowest survival rate (69.16%).
Larvae, especially first-feeding larvae, generally depend on live food. It is evident from the present study that live foods are more effectively utilized by the kutum larvae than the artificial feed. This phenomenon was attributed by various researchers to the presence of digestive enzymes in live foods that hasten the digestive processes in first-feeding larvae (Verreth et al., 1993; Kolkovski et al., 1995). On the other hand, the lower growth and survival of kutum larvae fed the artificial diet alone (treatment C) could be related to the incomplete development of the digestive system and poor efficiency in digestion and assimilation. Nevertheless, it has been reported that some freshwater fish species,
including Vimba (Vimbavimba) and Barbel (Barbusbarbus), can be exclusively reared on artificial diets from the start of exogenous feeding (Kujawa, 2003; Hamackova et al., 2009). Furthermore, the significantly lower growth response of kutum larvae in groups were fed with dry feed may be due to nutrient deficiency in artificial diet, for example the protein content of artificial diet was approximately 37%, which seems to be low for optimal growth of larvae.
1 7 14 21
Fig. 2 Mean body weight (a) and total length (b) of Rutilus frisii larvae fed with live food and artificial feed.
Values are mean ± SD. A: Artemianauplii for 21 days; B: zooplankton alone 21 days; C: artificial feed alone for 21 days
In the present study, larvae fed mixed zooplankton alone (treatment B) showed comparable results for growth and survival in comparison with larvae fed Artemianauplii alone (treatment A). The results indicate that mixed zooplankton represent a suitable food source for the kutum larvae. Various studies also have shown successful rearing of larval fish using live zooplankton (Dabrowski, 1984; Fermin et al., 1991).The present study also showed that the protein content of mixed zooplankton varied from 57.40- 63.28% dry matter. So mixed zooplankton could serve as a good source of protein for kutum larvae and other freshwater fish larvae because larval fish generally have high demand for dietary protein due to rapid growth rates and extensive catabolism of amino acids for production of metabolic energy (Srivastava et al., 2006). At the present study, the total protein content of Artemianauplii was around 61.3% dry matter. In spite of high nutritive value of Artemianauplii for larval fish the use of Artemianauplii as a starter feed in larviculture in most developing countries is limited by several factors such as their unavailability and high cost (ImorouToko et al., 2008). Therefore, the use of pond grown zooplankton is justifiably gaining more importance in the hatcheries in different regions (Mitra et al., 2007).
In this study, the better growth performance observed in larvae fed on live feed was mainly due to not only the nutritive and digestibility values of live mixed zooplankton but also because of the fact that from the start of active feeding kutum larvae showed an innate predatory behavior to capture zooplanktons (rotifers and copepod nauplii). Various studies also have shown successful rearing of larval fish using live zooplankton (Dabrowski 1984; Li et al. 1996; Ludwig 2000).
The present study also showed that growth of kutum larvae increased significantly when period of feeding with zooplankton increased. The determination of the optimal time to transfer kutum larvae from live food to the artificial feed differs according to aims and facilities of hatcharies. At the present study, based on growth performance, the most suitable way of rearing kutum larvae was exclusively with live food (mixed zooplankton) over the whole period of the experiment (21 days), however Considering data of growth and morphological development, kutum larvae could be progressively weaned around 10-12 day post hatching. the rearing of kutum larvae for 12 days on food zooplankton and then on artificial feed was relatively acceptable. In other words, if using live food (zooplankton) for whole period of rearing not possible, at least for the
first 10-12 days, live foods should be used. Kujawa (2003) recommended initial feeding with live food for 8-12 days for the larvae of some cyprinids such as Leuciscusidus, Leuciscusleuciscus and Leuciscuscephalus. Hamackova et al. (2007) also observed similar results for larvae of ide (Leuciscusidus).
In conclusion, this study showed that live foods were more effectively utilized by the kutum larvae and shown to have the best effect on growth parameters.. Poor growth of kutum larvae given artificial feed proved that starter feeds (dry diets) are not suitable for first feeding. This by no means excludes the use of artificial diets in rearing kutum larvae, which can support its growth, but with lower rate. The rearing of kutum larvae with artificial feed from the start of exogenous feeding might be considered suitable only in the case of absolute inaccessibility of live food for various reasons, or it might be anticipated in the future when better starter feeds might be commercially available.
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Effects of replacing fish meal with poultry by- product on Growth rate and body composition of fingerling Beluga (Huso huso)
Mir I lamed Sayed Hassani1, Davoud Talebi Haghighie2, Nemat Paykaran Mana1, Mohamad ALI Yazdani Sadati1, Hamid Reza Pour ali1
1 International Sturgeon Research Institute, Department of rearing and propagation, Rasht, Iran,
2 The central nutrition live food of Caspian sea
A 8 weeks Feeding trail was conducted to evaluate the potential of replacing fish meal (FM) with poultry by product meal (PBM) in practical diet for fingerlings Huso huso. Six Isocaloric (20 mg/j) and isonitrogenous (47%CP) diets were formulated. Fish mail was main source protein in control diets. In other treatment fish meal protein replaced with poultry by product at 20, 40, 60, 80 and 100% level (PBM20), (PBM40), (PBM60), (PBM80) and (PBM100), respectively. One Hundred eighteen fingerling Huso huso (initial body weight, 34.5 ±0.43 gr) were stocked to eighteen fiberglass tanks. Fish were fed to satiation. The result showed that there were not significant differences between the final weight, increase body weight and specific growth rate of fish fed to (control), (PBM20), (PBM40) and (PBM60) at the end of experimental period (P>0.05), but growth performance were depressed significantly for Huso huso fed higher levels of PBM diets (PBM80 and PBM100) compared with the fish fed to (control), (PBM20), (PBM40) and (PBM60) (P <0.05). Replacements up to 80 % were not negative effect on feed conversation ratio and protein efficiency ratio. FCR and PER varied between1.21-1.29 and 1.67-1.79 respectively. Lipid compositions were about 8 to 9 % in dry matter and were not influenced from diet containing
poultry by product supplemented. There were no significant differences found in body protein fishes fed (PBM0), (PBM20), (PBM40), (PBM60) and (PBM80)., The result showed that up to 60% fish meal can be replaced by poultry by product with no adverse effect on growth and feed conversion ratio and a suitable source for replacement with fish meal for nutrition Huso huso in fingerling period
Key words: Huso huso, Poultry by product, Replacement, Growth rate, Feed conversation ratio
Fish meal "a variable price product (Tacon et al ., 2008), dependent on capture of pelagic fish, a main source for feeding animal aquatic" is expensive food for aquaculture (Gill, 2000). reduction of capturing pelagic fish, increase of oil price, development of aquaculture industry and increasing demand of market in aquaculture and ranching sector led to increasing price of fish meal in the world annually (Tacon and Mtian
Poultry by product for reasons, high protein content (55 to 68%) (NRC, 1998) relative similar profile amino acid composition compare with fish meal (Yu et al .,2004), optimum coefficient digestibility in animal aquatic (Bureau et al ., 1999; Yang et al ., 2006; Liu et al ., 2008), lack of antinutritional factors present in plant protein (Francis et al., 2001), widespread industry processing poultry by product and having cheap price (Janmohamadi et al.,2009) is one of the suitable source for replacement fish meal in sturgeon diet compare with other present animal and plant protein source in Iran. PBM could be replaced at a level 50% dietary fish meal in Chinook salmon (Fowler, 1991), oncohynchus mykis (Steffen, 1994), African catfish (Abdel-Warith et al ., 2001), Black Sea turbot ( yigit et al ., 2006), 75 % in humpback grouper (Shapawai et al., 2007) and up to 100 % in Sparus aurata (Nengas et al .,1999), but there is no information about use of PBM in the diets of Huso huso, therefore
this study carried out for research about possibility replacement of fish meal with poultry by- product in commercial diet Huso hoso and compared effect on growth, feed efficiency and body composition.
Material and Method
The poultry by product had 68 % crude protein with a fat content of about 8.5% and ash content was about 12.5%. Estimated moisture, fiber and carbohydrate were approximately 3.4, 2.12 and 7.5% respectively. 6 experimental diets were formulated by Microsoft Office Excel 2003 to contain levels of protein (47%) and energy (21 MJ kg-1) which fish meal constituted half of diet and was provided 63 % protein in control diet. five other diets were formulated to be isonitrogenous and isocaloric as the basal d control diet , in which the fish meal protein was substituted in proportion 20, 40, 60, 80, and 100% by poultry by product protein. one hundred eighty fish, mean weight 27 to 28 gr were stocked in 18 fiberglass tanks (500 L water, volume2 L min -1 water flow). Three tanks were randomly assigned to each diet. The mean initial weight of fish in all tanks were 28.42 ± 0.17 and did not differ significantly between treatments (P >0.05). Fish reared for 80 days. Biometry carridout in 15 days Physicochemical parameter such as, dissolved oxygen, Water temperature and pH were recorded three times a day and ammonia was measured weekly. After 80 dayes feeding trail, 30% total population fish (three fish from each tank) were sampled randomly, killed by nail implant in back bone, then liver was removed and weighted and batch of fish body, mixed, frizzed and were transported for body composition analysis to laboratory . Proximate composition of diet ingredients, experimental diets and body composition analysis were performed according to the AOAC (1995) methods: dry matter (drying at 105 0C for 6 h to constant weight) ; ash ( AOAC , 1995) by burning on electrical furnace in in 55 to 60 0C for 9 hour ; crude fat (Soxhelet extraction method); protein (Kjeldahl using a selenium catalyst [N x 6.25]) and energy (bomb calorimeter). Using data on length and weight of fish in each tank, also liver and visceral weight, the following were determined
condition factor (CF), Body increase weight (Bwi) ,feed conversation ratio (FCR), specific growth rate (SGR), protein efficiency ratio (PER), Hepatosomatic index (HSI) and visceral index (VSI). One way and the correlations analysis were used to determine the relation- ship between the data at 95% confidence interval by SPSS software for windows (version 14.0).
At the end of 8- week trail, replacement of 20, 40 and 60% fish meal with poultry by product were not negative effect on final weight, weight gain and specific growth rate fishes where were not significantly different among fish fed dietary FM, PBM0, PBM20, PBM40 and
PBM60 (P >0.05), but final weight, weight gain and specific growth rate fish fed PBM 80 and PBM 100, replacement by 80% poultry by product or fish meal remove completely in diet were significantly lower than fish
fed dietary FM, PBM 20, PBM40 and PBM60 (P<0.05). Total
replacement of fish meal with poultry by product wasn't led to significant increase lipid content body compare with FM group. No significant difference was found in whole body lipid content for fish fed the different diet (P>0.05), also, whole body protein content for fish fed PBM 20, 40, 60 and 80% was similar to fish fed diet without fish meal (FM) (P>0.05), But elimination fish meal in diet (PBM 100) led to significant increase in whole body protein (P<0.05).
Poultry by product has high contain protein and dependent on processing method have a proper amino acid profile compared with fish meal that has introduced a good protein source for fish nutrition (Yigit et
if high quality PBM are used, many species tolerate up to 100% replacement (Steffens 1994; Nengas et al. 1999; Kureshy et al. 2000; Webster et al. 2000) Our study showed, final weight, weight gain and specific growth rate were not significantly differences among fish fed
control, PBM 20, PBM40 and PBM 60. Our result agreement with result of Alexis et al. (1985) who stated that FM can be partially replaced without any depression in growth performance in diets for rainbow trout by poultry by-products.
condition factor (0.44-0.47), Weight gain (1040- 1133 %), specific growth rate (3.6- 3.74 %/ day ), feed conversion ratio (1.21-1.26) and protein efficiency ratio (1.72-1.79) obtained on the present study were comparable to values reported by Mohseni et al (2005) about Determining nutritional requirements in Beluga ( Huso huso) from larval stage up to marketable size that were reared fingerling Huso huso by a formulated diets basis as fish meal (crude protein, 45% and gross energy 21.1 Mj/ kg-1) and were obtained a specific growth rate , protein efficiency ratio and feed conversion ratio about (2.83% / day ,1.29 and 1.55) respectively
. As a conclusion, according to the result obtained from growth rate, feed efficiency ratio and body composition, we suggested the fish meal in the diets of fingerling Huso huso can be replaced by 60 %PBM without no adverse on growth rate and body composition.
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1 Department of Natural Resources, Isfahan University of Technology, Isfahan, 8415683111, Iran.
This work was carried out in order to examine the effects of two anesthetic dosages (500,750ppm) clove powder on some hematological parameters (WBC,RBC,Hct,Hb,...) in common carp, Cyprinus carpio. Blood samples were taken just after complete sedation Results indicated that Hct and Hb Increased significantly in fish which anesthetized using 750ppm of clove powder. The rest of indices didn't show any differences between two groups. It was concluded, the recommended dose will be 500 ppm without to elicit hypothalamus-pituitary-internal axis (HPI).
Anesthetics are used with increasing frequency in aquaculture, especially to reduce the stress and mechanical injury to aquatic animals during handling (2). The use of anesthetic agents is particularly common during routine tasks such as biometry (3), transportation, and broodstock management (4).
Anesthesia is a biological state with the partial or complete loss of sensation or loss of voluntary neuromotor control included by chemical or nonchemical means (1). Increased time of exposure or concentration to anesthetic, first sedate an animal, than cause progressive loss of mobility, equilibrium, finally block reflex actions (4). Choosing an anesthetic must be attributed to several characteristics including its efficiency, availability, cost effectiveness, ease of use and safety for the
user including fish, humans and the environment (7).
It is generally accepted that anesthesia maybe a useful way to maintain fish stress-free during essential manipulations in aquaculture. However, these agents are also known to cause stress (3), thus stimulating blood indicators of stress (8). In fact, anesthesia generally involves a cessation of breathing which, in turn, reduces gas transfer leading to hypoxia (1). As a result, it has been reported that anesthesia may lead to stress response through changing blood parameters in fish
Clove powder is a dark brown powder from flowers, stalks and leaves of the clove tree Eugenina caryophyllata. Active ingredient is eugenol which has been widely used for several purposes such as antioxidant, antibacterial as well as anesthetic (2). Clove powder has some of characteristics considered for an ideal anesthetic agent. Eugenal is a phenolic compound which inhibits the prostaglandin H synthesis and result in analgestic effects of clove powder (7). Eugenal-based anesthesia is effective at low dosages, inexpensive, easily obtained and safe for both environment and user. Therefore it could be a promising anesthetic agent in aquaculture, thus it is essential to investigate its anesthetic optimum dosage and effects on physiological parameters such as haematological characteristics at least in commercial fish species such as common carp, Cyprinus carpio.
The aim of this study is to compare some hematological indices such as red blood cell, RBC, White blood cell, WBC, hematocrit, hemoglobin between two anesthetic dosages in common carp as one of the most important commercially produced fish in Iran.
Materials and method
The experiment conducted on juvenile common carp which were obtained from Isfahan culture and breeding centre, transported to the experimental aquarium in department of natural resources, Isfahan University of technology. Ten common carp weighting (mean weight 50.46 g mean length 15.45 cm) were housed in experimental aquarium
and acclimated to it for a one week. Throughout the acclimatization period and during experiment, environmental conditions were monitored and maintained within optimum range of variable. A total 10 fish divided into two groups. In group 1 and 2, fish were anesthetized respectively using 500 and 750 mg/L clove powder. There were 5 fish in each group. These two different concentrations of clove powder (500 and 750 ppm) were chosen according to our pervious pilot study (not published data).
In each experimental group, fish were individually anesthetized with mentioned dosages. The observation of stage 3 anesthesia was considered as complete sedation (7, 10). Blood samples were taken just after complete sedation. Then, the fish was placed in freshwater tank for recovery. Sample preserved in disodium salt of ethylene diamine tetra-acetic acid (EDTA) bottles for analysis. The count of erythrocytes and leukocytes were enumerated in an improved neubaeur hemocytometer, using hayem and truck diluting fluids (10). The amount of hemoglobin was determined according to cyanomethemoglobin procedure (7, 10). Hematocrit was determined by the standard microhematocrit method and expressed in percentage. The hematological indices (MCV, MCH, and MCHC) were calculated according to Svobodova et al. 1991 (11). The results are expressed as mean values + S.D. The data from experiments were analyzed by one-way ANOVA by using SPSS V.17 followed by student's T-test to compare the results of two experimental groups. In all cases, differences were considered at p<0.05.