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In the present study, the addition of different levels of Betaine supplementation had no significant effects on feeding efficiency. At our observation (under viewed loop) this study demonstrated that feeding larvae with artificial food (Bio Optimal and different levels of Betaine supplementation) was consumed less than 20% by pikeperch larvae after 19 dph. On other hands, live food treatment was ranged more than 50%. Sadeghi, (2004) indicated positive effect of Betaine on feed conservation ratio of rainbow trout. In addition, FCR was decreased in the presence of 3% Betaine supplementation (1.26 in 3% Betaine versus 1.86 in the other treatments). Kasper et al. (2002) reported that addition of 0.5% Betaine to tilapia diet led to a significant increase in feed consumption. Betaine acted as a dietary feeding attractant. As Betaine supplementation resulted

by increase in feed consumption and growth as shown by Can and Sener (1992) in trout fingerlings and Mackie and Mitchell (1982) in dover sole (Solea solea).

The results of the present study demonstrated that the highest survival rate observed in larvae fed live food while larvae fed artificial diet (i.e Bio Optimal and different levels of Betaine supplementation) had no effect on survival. The highest mortality rate was observed on 12-26 dph. Hamza et al, (2008) described this critical period of early stage of larval development of pikeperch as the end of post-larval II and the beginning of juvenile stage (16-21 dph). Indeed, at this stage, after yolk sac and oil globule disappearance, nutrient reserves of fish larvae are exhausted and exogenous feeding must be effective or the larvae quickly starve. Between 16 and 21 dph, the relatively high mortality probably concerned the larvae that did not ingest enough feed and reached the point of no return, while in the last 2 weeks of the experiment the mortality was essentially due to cannibalism. In the same way, Szkudlarek and Zakes (2007) reported three phases of mortality in pikeperch larval rearing which were related to the beginning of the exogenous feeding (4-6 dph), swim bladder inflation (8-14 dph) and cannibalism (22-34 dph). Rearing pikeperch larvae on live feed (Artemia sp. nauplii and/or freshwater zooplankton) does not pose any major difficulty but artificial feeds have proven unsatisfactory, resulting in low survival rates and high mortality (over 90%) (Szkudlarek and Zakes, 2007). In a recent study, Ostazewska et al, (2005) maintained that pikeperch larvae can be fed from 5 dph with high quality commercial dry diet as unique food source without significant reduction of survival (more than 50%) when compared with controls fed Artemia nauplii.

The highest cannibalism found in the groups of larvae fed live food and had significantly more than the other treatments. Cannibalism has been identified in a number of vertebrate species including fish (Elgar and Crespi, 1992; Baras, 1998; Kestemont et al., 2003). In our artificial feeding trial, cannibalistic behavior was not found until 26 dph. The large fish attacking and ingesting the smaller fish in live food treatment were observed on 19-26 dph and this cannibalistic behavior gradually

decreased after 26 dph. An early study of pikeperch rearing reported cannibalism on small size fish (less than 30 mm) fed in laboratory experiments using artificial diets (Hilge, 1990), and cannibalism ceased at about 5 cm body length with 1.2 g body weight (Hilge and Steffens, 1996). These findings are in agreement with our observations. The low rate of cannibalism observed in larvae fed artificial diet at 19 dph can be explained by the small size larvae and acceptance of formulated feed as their unique food while feeding live food can encourage cannibalistic behavior in pikeperch. It has been demonstrated that cannibalism is influenced by food type and availability, but also positively by the size heterogeneity among the population (Kestemont et al., 2007).

The present study showed that there is a need for further studies regards weaning of pikeperch larvae to artificial diet. These results need to be verified in future investigations on a larger sample of fish. It was understood that larvae fed live food had significantly higher growth, feed efficiency indices and survival rate compare to other treatments and Betaine supplementation did not improve growth and survival S. lucioperca, but it had maybe positive effects on growth in juvenile or developed larvae of pikeperch.

Acknowledgments

This study was supported by a grant of the Faculty of Natural Resource, University of Tehran, Iran. The authors would like to thank Mr. Mecknatkhah, Jalali and Ms. Hemmati for their technical assistance as well as Mr. Sohaii and Shoaii for his support and encouragement.

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Dietary vitamin C requirements in fish larvae

Forouzan Bagherzadeh Lakani

Department of Fisheries, Faculty of Natural Resources, Urmia University, Iran. Email: f.bagherzadeh@urmia.ac.ir

Abstract

Vitamin requirements are species-specific and depend on the environment, physiological needs and developmental stage. Due to the high growth and metabolic rate, it has been suggested that larval fish could deplete the storage of vitamins faster than juveniles. Therefore, vitamin requirements for larval fish might be higher than those for juveniles. Vitamin C (L-ascorbic acid, AA) supplied by diet is crucial for fish larvae development, since most of the species are unable to synthesise this vitamin .AA roles in several biological processes such as immunoactivity and stress response. AA deficiency caused reduced growth rate, spinal deformities, hemorrhages, anemia, hypertyrosinemia, slow wound healing and sensitivity to infections. AA levels lower than 30 mg/kg diet and as high as 400 mg AA/kg diet generated severe deformities of the jaws and caudal fin, specially affecting to the epurals, uroneural and specialized neural arch. Low AA levels also caused cartilage damage, characterized by unformed haemal arches and cartilaginous vertebrae, pugheadness and the lost of one vertebra, whereas 400 mg AA/kg diet induced the formation of one extra vertebra. Only a few percent of larvae affected by skeletal malformation can survive after larval development. This leads to significant loss of money for the hatchery. In addition, fish growing with malformations are sold at a depressed price. Deformities still develop, usually as a consequence of suboptimal/sublethal conditions.

Keywords: Vitamin C, Larvae, deformity, malformation.

Fish have specific requirements for quantitative and qualitative proteins, amino acids, fatty acids, vitamins and minerals (Cowey and Sargent 1979, Millikin 1982), which are derived from their diet. Several chemical compounds, characterizing the quality of food, are also known to influence the survival and development of fish larvae (e.g. Millikin 1982, Falk-Petersen et al. 1989). Knowledge of larval nutritional needs in the fish farming industry is limited due to the fast changing needs of the larval requirements during ontogeny.

Vitamin requirements vary according to their physiological function, are species-specific and depend on the environment (i.e., rearing conditions), physiological needs and developmental stage. Indeed, nutrient requirements change throughout the larval period in line with ontogenesis (molecular, cellular and tissue development), this being also reported for vitamins C (Merchie et al., 1997; Darias et al., 2010).

Due to the high growth and metabolic rate, it has been suggested that larval fish could deplete the storage of vitamins faster than juveniles. Therefore, vitamin requirements for larval fish might be higher than those for juveniles (Dabrowski, 1992). The content in AA rapidly declines during embryonic development in fish (Cowey et al., 1985). AA supplied by diet is crucial for fish larvae development, since most of the species are unable to synthesise this vitamin. AA roles in several biological processes such as immunoactivity and stress response (Dabrowski, 1992; Xie et al., 2006; Azad et al., 2007).

Vitamin C (L-ascorbic acid, AA) is necessary for the formation of collagen and cartilage, as well as bone formation and remodeling (Wilson and Poe, 1973; Kraus et al., 2004; Darias et al., 2011). AA detected in skin, caudal fin, head, jaws, cartilage of gills and collagen-areas of the bone in fish (Halver, 1972). AA also has many non-enzymatic actions. It is a powerful water-soluble antioxidant which protects low density lipoproteins from oxidation, reduces harmful oxidants in the stomach and promotes iron absorption (Darias et al., 2011).

The importance of AA in fish nutrition was first reported by McLaren et al. (1947) who observed arrest of growth and pathologies in the rainbow trout. Since then, numerous studies have shown that AA is an

indispensable micronutrient for the teleost fish.

The signs of AA deficiency, such as reduced growth rate, spinal deformities, hemorrhages, anemia, hypertyrosinemia, slow wound healing and sensitivity to infections, have been described in cyprinids (Dabrowski et al. 1988) and cichlids (Soliman et al. 1986, 1994; Shiau and Hsu 1995). Dabrowski et al. (1988) reported a deficiency in dietary ascorbic acid induced gill arch pathology and caudal fin erosion in carp larvae. Merchie et al. (1995) showed that high ascorbic acid levels increased growth rate and stress resistance in African catfish larvae.

First-feeding common carp larvae need vitamin C (Dabrowski et al., 1988; Gouillou-Coustans et al., 1998). The minimum requirement for optimal growth and survival is about 45 mg/kg dry diet. For maximal tissue concentration, a level of above 350 mg/kg diet was found to be necessary (Gouillou-Coustans et al., 1998). In newly hatched mrigal, Cirrhina mrigala an Indian major carp (Mahajan and Agrawal, 1980) found the optimum requirement to be 650-700 mg/kg diet, based on weight gain, mortality or behavioural and morphological criteria. In the Mexican native cichlid, Cichlasoma urophthalmus a minimum level of 110 mg/kg diet was found necessary to produce normal growth and lack of disease (Chavez de Martinez, 1990).

Most malformations develop during skeletogenesis, therefore larval nutrition has been suggested to have a key role in skeletogenesis (Cahu et al., 2003; Lall and Lewis-McCrea, 2007).The influence of vitamins on the appearance of larval deformities has been already demonstrated (i.e., Dedi et al., 1997; Takeuchi et al., 1998; Villeneuve et al., 2005; Fernandez et al., 2008; Mazurais et al., 2009). Unbalanced dietary vitamins induced skeletal deformities (Villeneuve et al., 2006; Fernandez et al., 2008; Mazurais et al., 2009; Darias et al., 2010). However, the mechanisms underlying the development of malformations are still unknown. AA levels lower than 30 mg/kg diet and as high as 400 mg AA/kg diet generated severe deformities of the jaws and caudal fin, specially affecting to the epurals, uroneural and specialized neural arch. Low AA levels also caused cartilage damage, characterized by unformed haemal arches and cartilaginous vertebrae, pugheadness and the lost of

one vertebra, whereas 400 mg AA/kg diet induced the formation of one extra vertebra (Darias et al., 2011)

Few studies exist on the role of dietary vitamins C on the development of skeletal deformities in fish. It has been shown distortion of gill filament cartilages and short opercula in juvenile tilapias (Soliman et al., 1986) and Mexican native cichlid (Chavez de Martinez, 1990). Andrades et al. (1996) showed that only a few percent of larvae affected by skeletal (lordotic) malformation can survive after larval development. This leads to significant loss of money for the hatchery. In addition, fish growing with malformations are sold at a depressed price. Deformities still develop, usually as a consequence of suboptimal/sublethal conditions (including nutrition).

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Darias, M.J., Mazurais, D., Koumoundouros, G., Cahu, C.L., Zambonino-Infante, J.L., 2011. Overview of vitamin D and C requirements in fish and their influence on the skeletal system, Aquaculture, 315. 49-60.

Darias, M.J., Mazurais, D., Koumoundouros, G., Glynatsi, N., Christodoulopoulou, S., Huelvan, C., Desbruyeres, E., Le Gall, M.M., Quazuguel, P., Cahu, C.L., Zambonino-Infante, J.L., 2010. Dietary vitamin D3 affects digestive system ontogenesis and ossification in European sea bass (Dicentrachus labrax, Linnaeus, 1758). Aquaculture 298, 300-307.

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336-341.

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152-157.

Allometric growth patterns of a triploid sturgeon (Acipenser baeri) x (Huso huso) during early development

Mariam Bahrami Ziarani1*, Soheil Eagderi1, Hadi Pourbagher1, Hamid Farahmand1

1 Department of Fisheries, Faculty of Natural Resources, the University of Tehran,

P.O. Box: 31585-4314, Karaj, Iran * Corresponding author: maryambahrami65@Gmail.com

Introduction

Early ontogeny of fishes is a rapid and complex process of growth and differentiation including morphogenesis, body shape changes, metabolism, swimming abilities and behavior, controlled by the genome (Osse and van den Boogart, 1995; Van Snik et al., 1997; Gisbert, 1999). Changes in body shape leads to the formation of characteristic morphologies and allometric growth patterns (Gisbert et al., 2002). Allometric process is an important factor during the growth of fishes and can display body structures develop according to their importance for primary functions (Osse and van den Boogaart, 1995). This research studied a triploid sturgeon i.e. (Acipenser baeriS) x (Huso huso^) as a suitable candidate in aquaculture. Information about the growth patterns of a fish may allow better understanding the patterns underlying the early life stages, their priorities during early growth and provides insight into fish biology, behavior, ecology and aquaculture (Gisbert, 1999; Koumoundouros et al., 1999).

Material and methods

Triploid specimens (Acipenser baeriS) x (Huso huso^) were obtained from Shahid Dadman International Institute of Sturgeon Fishes. Ten specimens were sampled every day from each tank, anaesthetized with MS 222 and preserved in 10% buffered formalin. The specimens were photographed using a dissecting microscope equipped with a Cannon

camera with a 5 MP resolution. The following parameters were measured using the software ImageJ (version 1.240): total Length, head length, trunk length, tail length, snout length, eye diameter and height of the yolk sac. Regression analysis was used to calculate the allometric growth. Allometric growth was calculated as a power function of total length using non-transformed data: Y=axb where Y was the independent variable, x the dependent variable, a the intercept and b the growth coefficient. Isometric growth, positive and negative allometric growth are indicated by b = 1, b > 1 and b <1, respectively. The Inflexion points of growth curves were determined according to van Snik et al (1997). All analyses were performed using MS-Excel 2007 (Microsoft Corporation).

Results

The growth of the head region was biphasic, with an inflexion point at 23 day post hatch (dph; 31.44 mm, TL). There was positive allometric growth before the inflexion point but turned to nearly isometric growth after the point. Growth of the eye diameter was biphasic with an inflexion point at 20 dph (25.65mm, TL), which was positive allometric in first phase of the growth and negative allometric in the second phase. The growth in the snout region was biphasic, with inflexion point at the 17 dph (23.47mm, TL) being positive allometric in first phase turning to side isometric growth in the second phase. The growth of the trunk region was biphasic with an inflexion point at 25 dph (28.59mm, TL). In first phase, there was negative allometric growth and in the second phase, there was nearly isometric growth. The growth of the tail region was biphasic, with an inflexion point at the 17 dph (23.47 mm, TL), which was positive allometric and nearly isometric in the first and second growth phases, respectively. The yolk sac size changed from the 1 to 14 dph, with an inflexion point at 5 dph (15.81mm, TL) with the growth being negative allometric growth and isometric in the first and second growth phases, respectively.

At hatching, the most functional systems of tripoloid sturgeon larva are incompletely differentiated. Differentiation and morphogenesis are complex processes during pre-larva and larval periods that influenced by environmental factors and genes (Osse and van den Boogart, 1995). The growth of head in the triploid sturgeon was positive allometric before the inflexion point but nearly isometric afterward. While, during the early development of the green sturgeon, the head length showed a positive allometric growth and in the Siberian sturgeon (Gisbert, 1999) and beluga (Asgari, 1391) were biphasic being positive allometric before the inflexion point but isometric after the point. Positive allometric growth of the head is a common feature in the early ontogeny of many fishes, including sturgeon (Van snik et al., 1997) that is concomitant with the development of the brain, sensory, respiratory organs and feeding systems (Gisbert and Doroshov, 2006).

Figure 1: Allometric growth equations and the relationship between different selected head regions and organs with total length in the triploid sturgeon (Acipenser baeri) x (Huso huso) during early stages of development (from hatching up to day 50). The dotted line represents the inflexion point of growth.

The growth of the eye in the tripoloid sturgeon was biphasic, with an inflexion point at 20 dph, which was positive allometric before the inflexion point, turned to negative after the inflection point. Growth of the in beluga and Siberian sturgeon were biphasic, with an inflexion point at 6 dph in beluga and 3 dph in Siberian sturgeon. Both displayed a positive allometric growth that changed to isometric pattern later. However, the green sturgeon displayed isometric growth during the entire period of larval development (Gisbert and Doroshov, 2006). The growth of trunk region tripoloid larvae was biphasic with negative allometric pattern and isometric growth at the first and second growth phases, respectively.

The trunk growth of green sturgeon and beluga are also biphasic (Gisbert, 1999). The Trunk inflexion point of green sturgeon is at 6 dph and in beluga is at 28 dph. There is no inflexion point in the Siberian sturgeon and the growth is almost isometric pattern (Gisbert, 1999). Morphogenesis such as differentiation and growth of myotome and sclerotome and digestive is occurred in the trunk region (Gisbert and Dorosho, 2003). The tail region of tripoloid larvae had a biphasic growth, with an inflexion point at 17 dph, being positive allometric and then almost isometric. The inflexion point of this parameter is nearly matched with absorption of the yolk sac that can also be seen in beluga. The inflexion points of green and Siberian sturgeon are coincided with beginning of the external feeding. Similarly, the growth of snout region in the tripoloid sturgeon is coincided with the absorption of the yolk sac and the start of active feeding. However, the growth of snout in beluga larva is 3 phasic with two inflexion point at 5 and 28 dph (Asgari, 1391). The growth of snout in Siberian sturgeon larva is always positive allometric and there is no inflexion point (Gisbert, 1999). Growth of yolk sac was biphasic with inflexion point at 5 dph being negative allometric and then isometric. From 1 to 5 dph, the yolk sac was consumed slowly but with a higher rate later. The inflexion point in beluga larva was about 6-7 dph showing negative allometric growth pattern that change to isometric later. The inflexion points of yolk sac, snout length and eye diameter were coincided together. According to studies of osteology that

were performed on beluga, it appears that the teeth inflexion point is matched that of yolksac indicating the preparation for external feeding (Asgari, 1391). The present study showed that the pattern of allometric growth in tripoloid sturgeon larva is distinctive in compare to their parent's pattern. This different allometric pattern may be related to different environmental, nutritional and genetic factors.

References

Asgari, R., 1391. Ontogeny of Bluga (Huso huso) from hatching time till 50th dph; survey of ontogeny of growth, skeletal system, histology and enzyme activity of digestive system to improve of biotectic of Bluga larviculture. Doctoral Dissertation, University of Tehran.

Gisbert, E. 1999. Early development and allometric growth patterns in Siberian sturgeon and their ecological significance. Journal of Fish Biology. 54, 852-862.

Gisbert, E., Merino, G., Muguet, B., Bush, D., Piedrahita, RH., Conklin, DE., 2002. Morphological development and allometric growth patterns in hatchery-reared California halibut larvae. Journal of Fish Biology. 61, 1217-1229.

Gisbert, E., Doroshov, S.I. 2006. Allometric growth in green sturgeon larve. Journal of Applied Ichthyology. 22, 202-207.

Koumoundouros, G., Divanach, P., Kentouri, M. 1999. Ontogeny and allometric plasticity of Dentex dentex (Osteichthyes: Sparidae) in rearing conditions. Marine Biology. 135, 561-572.

Osse, J.W.M., van den Boogart, J.G.M. 1995. Fish larvae, development allometric growth, and the aquatic environment. ICES Marine Science Symposium. 201, 21-34.

Rodriguez, A., Gisbert, E. 2002. Eye development and the role of vision during Siberian sturgeon early ontogeny. Journal of Applied Ichthyology. 18, 280-285.

Van Snik, G. M. J., van den Boogaart, J.G.M., Osse, J.W.M. 1997. Larval growth patterns in Cyprinus carpio and Clarias gariepinus with attention to finfold. Journal of Fish Biology 50, 1339-1352.

The effect of salinity on hatchability and larval survival rate in molly (Poecilia sphenops)

Bahremand, M., Naserizadeh, M., Pirbeygi, A., Nematollahi, M. A.

Department of Fishery,Faculty of Natural Resource, University of Tehran, Karaj, Iran Bahremand.m@ut.ac.ir

Abstract

Breeding and larval survival trials were carried out with Poecilia sphenopsin different salinities (0.5(freshwater), 6%c) and effects on gestation period, fry production, and fry survival rate were examined. Results showed that while P. sphenopssuccessfully spawned in 6%c salinities, there was a significant difference in fry production among two treatments. The minimum gestation period was 24 days in treatment B (6%c); the maximum fry production was obtained in 6%c. Fry survival rate was highest in 6%c and significantly differed (p<0.05). The results suggest that the optimum salinity for breeding and rearing P. sphenopsare around 6%c respectively.

Keywords: Poecilia sphenops, hatchability, salinity.

Introduction

Ornamental fish keeping is one of the most popular hobbies in the world today. An estimated trade with a turnover of US$ 5 billion and an annual growth rate of 8 percent offer a lot of scope for its development

(Sudha, 2012).

Among livebearers, Poecilia reticulata (Peters), Poecilia sphenops (Valenciennes), Xiphophorus helleri (Heckel) and Xiphophorus maculatus (Gunther) are the most important and familiar species (Ghosh et al., 2008).Poecilia sphenops, an omnivorous ornamental fish, was

chosen for the present study as the experimental animal.It gives birth to young ones directly(Sudha,2012).Many researchers demonstrated the remarkable tolerance of poeciliid fishes in wide ranges of temperature (Bennett and Beitinger,1997) and salinity (Haney and Walsh, 2003).

Salinity is a major influencing factor on reproductive physiology in fishes (Claireaux and Lagardere, 1999). Since the rate of hatchability in P. sphenopsis low, and since this fish tolerate a wide range of salinity, have been decided to assess the effect of saliniti's amount on the rate of hatchability in this fish.

Material and methods

One-hundred 4-months-old(broodstock)P.sphenops were prepared and maintained inalaboratory in oxygenated bags, and acclimatized to laboratory conditionsin two 160L tanks.In this stage accordingto avoid size differences60 broodstocks were selected and stocked in two 160Ltanks(30 per each tank),after that,was added 2ppt marine saltto oneof them as a treatment(treatment B) and every day was added 2ppt salt until salinity reached to 6ppt after 3 days, while there was no change in salinity in the other treatment(treatment A).After this stage,broodstockswere weighed to stocking and the mean weight of any of them was 4.21+0.58g.After that,was used three replicates for one of them. For treatment A was used of freshwater and for treatmentB was used of 6 ppt salinity.During the experiment Water was exchanged at 30% per two days and water quality parameters were:dissolved oxygen was saturation, temperature:25-27°C, photoperiod: 12 h light-12 h dark. salinity was measured regularly after water exchanged.

As diet, was used of BIOMAR pellet and live adult brine shrimp in three rations per day(at 08:00, 12:00, and 18:00) 5% of body weight.Two hours after feeding, fecal matter and unconsumed feed were siphoned from the tank bottom and discarded.

Statistical analysis. Data were analyzed by a one-way ANOVA using the Statistical Analysis Software Program of SPSS 17. Duncan's Multiple Comparison Test was used to determine differences between

treatment means (Duncan, 1955).Results were considered statistically significant if p<0.05.

Results

At the end of the experiment, the results showed this fish successfully acclimatized to 6ppt salinity. Swimming and feeding behavior were normal in two treatments. Fish released young in two treatments (Fig.1). The earliest fry were released in the replicate2 of treatment(B) after ten days. Fish kept in salt water ingested more food than those in fresh water and had better coloration.

Hatched larvae Unhatchcd eggs

Fig.1- Number of larvae & eggs in treatment A & B

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