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Discussion and conclusion

All the probiotics-supplementation diets resulted in growth performance higher than of the control diets. Suggesteing that the addition of probiotics optimized the feed consumption (Lara-flores et al., 2003). This resulted in better fish performance, with better growth results in the diets supplemented with probiotics microorganisms. Similar results

were observed by Vazquez-Juarez et al. (1993) when the yeast isolated from the intestines of wild raibow trout was introduced in tnto the digestive tracts of domestic raibow trout, producing a significant increase in the growth of the cultured trout. Bacillus subtilis and B. circulans supplemented in diets of rohu (Labeo rohita) fingerlings, the final body weight, relative gain ratio (RGR) and SGR significantly increased (Bairagi et al., 2004). Noh et al. (1994) obtained the satisfactory results in study of the effect of supplementing common carp feeds with different additives, including antibiotics, yeast S. cerevisiae and bacteria Streptococcus faecium. Lara-flores et al. (2003) indicated that the use of the bacteria S. faecium and the yeast S. cerevisiae as a promoters in Nile tilapia (Oreochromis niloticus) increased the growth parameters. Similar results were reported by Bogut et al. (1998), who fed common carp (Cyprinus carpio) diets supplemented with S. faecium, reporting that the bacterium has a better probiotic additive for carp than yeast. In conclusion, the experiments showed that the probiotic bacillus highly increase the growth performances in Common Carp larvae. Furthermore, different levels of probiotic bacillus indicated different performance.

References

Bairagi, A., Ghosh, KS., Sen, SK. and AK. Ray. 2004. Evaluation of the nutritive value of Leucaena leucocephala leaf meal , inoculated with fish intestinal bacteria Bacillus subtilis and Bacillus circulans in formulated diets for rohu, Labeo rohita (Hamilton) fingerlings . Aquaculture Research. 35: 436 - 446.

Bogut, I., Milakovic, Z., Bukvic, Z., Brkic, S. and R. Zimmer. 1998. Influence of probiotic Streptococcus faecium M74 on growth and content of intestinal microfolora in carp cyprinus carpio.Czech J.Anim.Sci, 43:231-235.

Ghosh, k., Sen, S.k., and A. k. Ray. 2003. Supplementation of an isolated fish gut bacterium, bacillus circulans , in Formulated diets for Rohu, Labeo rohita, Fingerlings. Aquaculture - Bamidgeh. 55(1): 13-21.

Lara-Flores, M., Olvera-Novoa, Miguel A., Guzman-Mendez, Beatriz E., and W.Lopez-Madrid. 2003. Use of the bacteria Streptococcus faecium and Lactobacillus acidophilus, and the yeast Saccharomyces cerevisiae as growth promoters in Nile tilapia (Oreochromis niloticus). Aquaculture. 216:193-201.

Noh, S.H., Han,K., Won, T.H. and Y.J. Choi .1994. Effect of antibiotics, enzyme, yeast culture and probiotics on the growth performance of Israeli carp.Korean

J.Anim.Sci. 36:480-486.

Olsen,Y. 1997. Larval rearing technology of marine species in Norway. Hydrobiologia

358: 27-36.

Ringo, E and T.H. Birkbeck.1999. Intestinal microflora of fish and fry. Aquaculture

Research. 30: 73 - 93.

Vazquez-juarez, R., Ascensio, F., Andlid, T., Gustafsson,L.,Wadstrom,T., 1993. The expression of potential colonization factors of yeast isolated from fish during different growth conditions. Can. J. Microbiol. 39:1135-1141.

Effects of feeding frequency on larval quality and survival rate of freshwater prawn (Macrobrachium rosenbergii)

Kamran Rezaei Tavabe1, Gholamreza Rafiee1, Michael Frinsko2, Harry Daniels3

1 Fisheries Department, Natural Resources Faculty, University of Tehran, Karaj, Iran.

2 North Carolina Cooperative Extension Service, North Carolina State University,

3Trenton, NC, USA.

3 Department of Biology, North Carolina State University, Raleigh, NC, USA.

Abstract

Optimum daily feeding frequency by artemia nauplii for Macrobrachium rosenbergii was evaluated. For the experimental design of the study, 3 treatments of feeding frequency with 1, 2 and 3 times daily feeding were set up triplicate. A static water system with partial exchange of water by siphoning was used for the treatments. Initial larval density was fixed at 100 larvae l-1 and larval quality parameters such as larval stage index (LSI), survival rate and time to reach postlarvae (PL) were determined. The results revealed that different feeding frequency of had a pronounced effect on larval quality. The results showed that feeding frequency at day has strongly effect on LSI, survival rate and time to reach PL. The results also showed that 2 times feeding a day is the best policy for M. rosenbergii feeding by artemia nauplii and at this feeding frequency survival rate was 42±6 % at the end of the larval period.

Keywords: Feeding frequency, Macrobrachium rosenbergii, Larval stage index, Larviculture.

The giant freshwater prawn (Macrobrachium rosenbergii) is one of the most important aquaculture species in tropical and subtropical regions of the world. The natural origin of this species is Asian southeastern countries and during past decades has been introduced to different countries in North and South America, Africa, Europe and Asia (New, 2000). The most recent statistics (FAO, 2011), show that global production in 2009 had already risen to 229 419 t, 80 times as much as in 1980. Despite this, one of the impediments to culture this species has been variable larval survival and larval quality during past decades (Bart & Yen 2003). The increasing demand on prawn production has caused in many efforts to decrease the cost and production of high quality larvae under controlled conditions.

Feeding policy plays an important role in larvae rearing of freshwater prawn. It affects survival, metamorphosis rate and larval quality as well. Nowadays, a variety of larval feeds are currently used during the hatchery phase of production of the giant freshwater prawn. Most hatcheries in the world depend on the use of live food such as artemia, in combination with various costly feed formulations. The food for culture of M. rosenbergii larvae consists basically of newly hatched artemia nauplii (Lavens et al., 2000) with different density in various larval stages (Valenti and Daniels, 2000).

The current study was carried out to investigate the effects of different feeding frequency on larval quality and survival of M. rosenbergii. The main objective of the study was to determine the optimum feeding regime of the larvae by artemia nauplii.

Material and Methods

The broodstock were obtained from a private farm in Kenly, North Carolina where they were then moved to the Lake Wheeler hatchery of the Department of Biology at North Carolina State University. In broodstock tanks, water quality factors, photoperiod and breeder feeding were maintained in accordance with recommendations for prawn

breeders (New, 2003; Nhan et al., 2010). Larvae collection was conducted based on Menasveta and Piyatiratitivokul (1980). Because of necessity to maintain a constant macroelements concentration, the rearing systems were selected with minimum water exchange. Water quality parameters were adjusted following Nhan et al. (2009). NH4-N and NO2-N were maintained below 0.2 and 0.1 mgl-1 respectively. Gentle aeration was applied in all rearing containers and glasses. Average water temperature and dissolved oxygen were 30±1 °C and 7±1 mgl-1. The larvae were fed Artemia franciscana (Great Salt Lake strain) nauplii. They were fed at the ideal density for each stage of larval period based on Barros and Valenti, (2003).

For the experimental design of the study, 3 treatments of feeding frequency with 1, 2 and 3 times feeding a day were set up triplicate based on New (2003) artificial brackishwater. A static water system with partial exchange of water by siphoning was used for the treatments. Various indices are expressed as larval quality parameters. At the present study LSI, survival rate and time to reach PL indices were assessed. Survival rate (%) was calculated at postlarvae stage LSI was determined on 5, 10, 15 and 20 days after hatching based on the description by Uno and Kwon

(1969).

LSI = 2 St /N Where: Si is the stage of the larvae (i= 1 to 11). N is number of larvae examined.

Evaluated parameters such as survival rate, LSI and time to reach postlarvae were analyzed by analysis of variance (one-way ANOVA) and significant differences between the means were found (P < 0.05) by Duncan's test in SPSS.

Results

During the study, the average value of the most important larviculture water physico-chemical parameters such as temperature, pH, dissolved oxygen, ammonia-N and nitrite-N were recorded 30±1 °C, 7-7.6, 7±1 mgl-1, D 0.2 mgl-1 and D 0.1 mgl-1 respectively.

Table 1. LSI (mean±SD) of different SAR treatments. Different letters denote

significant differences (P < 0.05). Small and capital letters denote different comparison in columns.

Feeding frequency at a day

LSI at different dph

Time to Reach PL

(Day)

Survival

(%)

 

5

10

15

20

 

 

1

3.5±0.5

6.2±0.5

8.3±0.4AB

9±0.3y

29±1.5b

35±7ab

2

3.9±0.4

6.5±0.3

8.7±0.5A

9.5±0.2x

26.2±1.2a

42±6a

3

3.7±0.3

6.4±0.4

8±0.3B

8.9±0.4y

31±1b

31±5b

In different treatments of feeding frequency, LSI at 5 and 10 dph did not show significant differences (P D 0.05) among the treatments while at 10 and 15 dph this parameter showed significant differences among the treatments (P D 0.05) and the treatment of 2 times frequency a day showed the highest level with 9.2±0.2 (Table 1). The results also showed that time to reach PL had significant difference (P D 0.05) among the treatments and the longest (31±1) and the shortest (26.2±1.2) larval period were in treatments 2 and 3 respectively (Table 1). The highest survival rate was record for 2 times feeding with 42±6 % during the larval period.

Discussion

Larval development and survival of freshwater prawn are affected by several factors. In the current study, larval growth and survival rate were affected by feeding regime. However, the results of the different experiments also depended on other factors such as brood stock, quality of the newly-hatched larvae, the scale of the experimental system used and environmental conditions during the experiment. The results showed that LSI at 5 and 10 dph did not show any significant differences but at 15 and 20 dph 2 times feeding a day showed significant differences and the highest values among the treatments. Aquacop (1983) suggests supplying 5 nauplii/larvae/day for M. rosenbergii from the second day of culture and increasing 5 nauplii/larvae every 1 or 2 days with 3 times feeding a day. Thus, more than 40 nauplii/larvae/day would be supplied only from the 12th day on, when the larvae have attained stages V and

VI. Feed consumption by larvae is dependent upon numerous factors related to culture conditions specific for site, and scientific knowledge is not enough to base an adequate feeding schedule. Hatcheries, in general, either maintain a constant artemia supply (about 5 nauplii/ml/day) or increase the density over culture period (Daniels et al., 1992; Valenti et al., 1998). Although these common practices yield positive results, larval ingestion rates may vary, thus presenting peaks and lows of ingestion at certain stages. Consequently, overfeeding of nauplii in some phases of culture may occur, generating waste and elevating the concentration of toxic nitrogen compounds. On the other hand, the larvae may be underfed during some phases, thus resulting in reduced growth rate and cannibalism. The results also showed that time to reach PL and survival rate values for 2 times feeding frequency were the highest among the treatments. overfeeding of artemia nauplii in larval rearing systems will generate waste and elevate the concentration of toxic nitrogen compounds; In contrast, underfeeding will reduce growth rate and induce cannibalism (Valenti and Daniels, 2000; Nhan et al., 2010). A comparison between maximum values obtained in the present work with the values reported by Aquacop (1983) indicate that in the initial stages, the amounts were underestimated by the latter, because values close to 40 nauplii/larvae/day are needed from stage II onward. Between stages V and VI, 55 nauplii/larvae/day would be necessary, which is lower than the value reported by Daniels et al. (1992). To establish an adequate feeding schedule for M. rosenbergii larviculture, the minimum concentration of artemia necessary to maximize capture efficiency and daily amount of nauplii ingested by the larvae needs to be considered. Considering the current great cost of artemia cysts, a multistage culture should be performed to improve larvae capture efficiency and more effectively use the live feed.

As a result of the present investigation, feeding frequency at day has strongly effect on LSI, survival rate and time to reach PL. The study results showed that 2 times feeding a day is the best policy for M. rosenbergii feeding by artemia nauplii and at this feeding frequency survival rate was 42±6 % at the end of the larval period.

Aquacop, 1983. Intensive larval rearing in clear water of Macrobrachium rosenbergii (De Man, Anuenue stock) at the Centre Oce'anologique du Pacifique, Tahiti. In: Mcvey, J.P., Moore, J.R. (Eds.), 1983. CRC Handbook of Mariculture, Crustacean Aquaculture, vol. 1. CRC, Florida, pp. 179- 187.

Barros, H.P., Valenti, W.C., 2003. Ingestion rates of Artemia nauplii for different larval stages of Macrobrachium rosenbergii. Aquaculture 217, 223-233.

Barros, H.P.D., Valenti, W.C., 2003. Food intake of Macrobrachium rosenbergii during larval development. Aquaculture 216, 165-176.

Bart A.N., Yen P.T. (2003) Comparison of larval performance between Thai and Vietnamese freshwater giant prawn, Macrobrachium rosenbergii (de Man): a preliminary study. Aquaculture Research 34, 1453-1458.

Daniels, W.H., D'abramo, L.R., De Parseval, L., 1992. Design and management of a closed, recirculating "clearwater" hatchery system for freshwater prawns, Macrobrachium rosenbergii, De Man, 1879. J. Shellfish Res. 11, 65-73.

FAO (2011) Fishstat Plus (v, 2.32) issued 07.04.2011. FAO, Rome, Italy.

Kitsawat P. & Chuchot S. (1991) Acute toxicity of formalin to giant freshwater prawn (Macrobranchium rosenbergii). King Mongkut's Agricultural Journal 9, 45-52.

Lavens, P., Thongrod, S., Sorgeloos, P., 2000. Larval prawn feeds and the dietary importance of Artemia. In: New, M.B., Valenti, W.C. (Eds.), Freshwater Prawn

Culture. Blackwell, Oxford, pp. 91- 111.

Menasveta, P., Piyatiratitivokul, S., 1980. A comparative study on larviculture techniques for the freshwater prawn, Macrobrachium rosenbergii (De Man).

Aquaculture 20, 239-249.

New, M.B., 2000. History and global status of freshwater prawn farming. In: New, M.B., Valenti, W.C., (Eds), Freshwater prawn culture: the farming of Macrobrachium rosenbergii. Oxford, Blackwell Science, pp. 1-11.

New, M.B., 2003. Farming freshwater prawns: a manual for the culture of the giant river prawn (Macrobrachium rosenbergii). FAO Fisheries Technical Paper No. 428. FAO, Rome, Italy, pp. 11-27.

Nhan, D.T., Wille M., Hung, L.T., Sorgeloos, P., 2009. Comparison of reproductive performance and offspring quality of giant freshwater prawn (Macrobrachium rosenbergii) broodstock from different region. Aquaculture 298, 36-42.

Nhan, D.T., Wille M., Hung, L.T., Sorgeloos, P., 2010. Effects of larval stocking density and feeding regime on larval rearing of giant freshwater prawn (Macrobrachium rosenbergii). Aquaculture 300, 80-86.

Uno, Y., Kwon, C.S., 1969. Larval development of Macrobrachium rosenbergii (de Man) reared in the laboratory. Journal of the Tokyo University Fisheries 55, 179­190.

Valenti, W.C., Daniels, W.H., 2000. Recirculation hatchery systems and management. In: New, M.B., Valenti, W.C., (Eds), Freshwater prawns culture. Oxford,

Blackwell, pp. 69-90.

Valenti, W.C., Mallasen, M., Silva, C.A., 1998. Larvicultura em sistema fechado dina'mico. In: Valenti, W.C. (Ed.), Carcinicultura de A ' gua Doce: Tecnologia para a Produc_a~o de Camarefes. FAPESP e IBAMA, Sa~o Paulo e Brasilia, pp.

112-139.

Evaluation of seaweed extracts as a supplement or alternative culture medium for microalga

Chaetoceros muelleri

Rohani-Ghadikolaei Kiuomars1*, Abdolalian Eisa1, Foroghifard Hojatallah1, Moezi Maryam, Dehghani Reza1

1Persian Gulf and Oman Sea Ecological Research Institute, Aquaculture Department,

79167-93165 Bandar Abbass, Iran.

*To whom all correspondence should be addressed:

Aquaculture Department, Persian Gulf and Oman Sea Ecological Research Institute, 79167-93165 Bandar Abbass, Iran, Telephone number: +98-761 3333390 E-mail: roohani2001ir@ yahoo. com

Abstract

Aqueous extracts of green [Ulva lactuca and Enteromorpha intestinalis], brown [Sargassum illicifolium and Colpomenia sinuosa] and red [Hypnea valentiea and Gracilaria corticata] seaweeds from the Persian Gulf of Iran were examined for their potential usefulness as components of microalga Chaetoceros muelleri culture medium against conventional f/2 medium. Each seaweed extract was added at a concentration of 0.2 mg mL-1 in 2.5 L of sterilized seawater with or without f/2 culture medium. All microalga cultures were grown under the same conditions. Chaetoceros muelleri was successfully cultured using the tested seaweed extracts (SWE) and exhibited higher or similar cell density and biomass when SWE were used as a supplement or an alternative medium, respectively. When the SWE were used as an alternative medium, microalgal protein, lipid and ash content were similar compared to control. Since no major changes were found in most of the measured growth parameters, proximate biochemical composition, and chlorophyll a content following culture of the microalga with

SWE as an alternative media, particularly extracts of U. lactuca, E. intestinalis and G. corticata, it was concluded that these SWE are able to provide the necessary nutrients for microalgae growth and could be used as a possible substitute to reduce microalgae production costs in establishing microalgal populations to use in aquaculture operations.

Keywords:       Seaweed   extract;   alternative medium; biochemical composition; Persian Gulf

Introduction

In mariculture, a supply of food with high nutritional quality offered to feed aquatic animals is an important parameter to be monitored as well as factors related to their culture growth rate and size appropriateness for ingestion (Brown et al., 1997). Zhang (1997) demonstrated that seaweed extract (SWE) contains growth stimulating components such as phytohormones (especially cytokinins) which may enhance nutrient uptake, regulate plant growth substances, and increase chlorophyll content, protein synthesis and cell division. To date, little is known about the effects of SWE as a culture medium on the growth rate and biochemical composition of marine microalgae. Therefore, the objective of the present study was to investigate the effect of different SWE on the growth rate, and the proximate compositions of the microalga Chaetoceros muelleri. This is the first report on the use of locally sourced SWE as organic fertilizer for the Iranian mariculture industries.

Material and methods

Sample collection and preparation of seaweed extract

In total, six seaweeds—two green [Ulva lactuca and Enthromorpha intestialis], two brown [Colpomenia sinuosa and Sargassum illicifolium], and two red [Gracilaria corticata and Hypnea valentiea] were collected from the northern coast of the Persian Gulf of Iran for the present study. The cleaned seaweed samples were freeze-dried. SWE was prepared

according to the method of Cho et al. (1999). All chemical analyses of dried seaweed samples were carried out in triplicates.

Microalgae Culture

Two separate experiments were conducted concurrently to test the effects of different SWE in water solution on the growth rate and biochemical composition of the microalga and were compared with a Control treatment with only f/2 culture medium during the same time period.

Experiment 1: SWE as a supplement added to f/2 culture medium on the first day.

Experiment 2: SWE as an alternative media to f/2 culture media added to sterilized seawater on the first and every 4th day thereafter.

The stock solutions of SWE were added at a concentration of 0.2 ug mL-1 to the respective microalga culture. All microalga cultures were grown under the same conditions (22-25°C, 30 mg L-1 salinity and at pH 8 under a 12:12 h light: dark cycle). The microalga cell counts were performed using a Neubauer haemacytometer (Germany). Specific growth rate was calculated as u= ln (N1/N0)/ (trfe), where N1=biomass at time of harvest t1 and N0=biomass at time zero, t0 (Guillard 1973).

Analysis of biochemical composition

Microalga samples were harvested during the exponential growth phase (day 8) and concentrated by centrifuge at 5000 rpm for 10 min and then freeze-dried before being stored at -25° C until biochemical analysis. Total protein was determined by the method of Lowry et al. (1951); total lipid using the Bligh and Dyer (1959) procedure and carbohydrate was estimated by following the method of Dubois et al. (1956). Microalgal cell weight and ash content (pg cell-1) were determined following the method of AOAC (1997). Dry biomass (mg L-1) was calculated by multiplying cell density (per L) by cell weight (per mg) at harvest time.

The specific growth rate (cell day-1), cell weight (pg cell biomass dry (mg L-1) and chlorophyll a content (pg cell-1) of C. muelleri grown with different SWE as a supplement and as an alternative to f/2 culture media, are shown in Table 1. The proximate biochemical composition— protein, carbohydrate, lipid and ash (% of dry weight) in C. muelleri cultured for eight days with the water extract of different seaweeds as a supplement or as an alternative to f/2 medium, are shown in Table 2. No significant differences (P < 0.05) were found in the protein, lipid, carbohydrate and ash content of C. muelleri following culture with SWE either as a supplement or as an alternative media compared to control.

Table 1: Growth rate, cell density and cell weight at harvest, and chlorophyll-a content of two microalgae I. galbana and C. muelleri grown with the water extract of different seaweeds 1

Growth rate Cell density Cell weight Biomass dry Chlorophyll a

(day -')_xlQ 6 (Cell ml-1)_(pg cell -1)_(mg l-1)_(pg cell-1)

Control

0.20 b

0.20 a

5.62 c

5.62 a

38.92

38.92

218.8 b

218.8ab

0.97

0.97

 

SWEsup

SWEalt

SWEsup

SWEalt

SWEsup

SWEalt

SWEsup

SWEalt

SWEsup

SWE

U. lactuca

0.23 a

0.19 a

6.58 a

5.50 a

38.69

38.66

261.7 a

219.3 ab

1.19 b

1.02

E. intestinalis

0.22 ab

0.19 a

6.69 a

5.66 a

38.71

38.68

259.2 a

215.3 ab

1.14 a

1.05

S. ilicifolium

0.20 b

0.19 a

6.06 b

5.47 a

38.51

38.47

233.2 b

204.8 b

1.03 a

0.97

C. sinuosa

0.21 b

0.17 b

6.11 b

5.45 b

38.68

38.64

236.3 b

195.3 c

0.98 b

0.96

G. corticata

0.22 a

0.19 a

6.64 a

5.65 a

38.93

38.89

257.9 a

235.0 a

1.23 a

1.04

H. valentiae

0.20 b

0.16 b

6.17 b

5.43 b

38.56

38.52

237.9 b

190.6 c

0.97 b

0.94

1 Data are mean contents of triplicate samples

Different superscript letters in row indicate significant differences in means (P < 0.05)

Table 2- Protein, carbohydrate, lipid and ash (% dry weight) of C. muelleri cultured for eight days with the water extract of different seaweeds as a supplement (SWEsup) and

alternative (SWEalt) media1._

Treatments Protein Lipid Carbohydrate Ash

Control 39.2 17.8 1 14.1 20.1

 

SWEsup

SWEalt

SWEsup

SWEalt

SWEsup

SWEalt

SWEsup

SWEalt

U. lactuca

39.6

39.1

17.9

17.8

14.5

14.8

20.2

19.9

E. intestinalis

39.5

39.0

17.6

17.5

14.8

15.1

20.4

19.8

S. ilicifolium

39.3

39.4

17.3

17.4

14.5

#2

19.8

19.9

C. sinuosa

39.1

38.2

17.7

17.5

14.7

#2

20.8

20.1

G. corticata

39.5

38.6

17.8

17.9

14.5

15.2

20.2

19.8

H. valentiae

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