in-vivo study on effect of stocking density on growth and...

Indian Journal of Geo Marine Sciences Vol. 47 (06), June 2018, pp. 1228-1236

In-vivo study on effect of stocking density on growth and production of marine prawn Litopenaeus vannamei

Kaliannan Durairaj1,2, Palanivel Velmurugan2, Palaninaicker Senthilkumar1, Pachamuthu Vetriselvan3 Arunachalam Manimekalan4* & Byung-Taek Oh2*

1Department of Environmental Science, Periyar University, Salem, Tamil Nadu, India. 2Division of Biotechnology, Advanced Institute of Environmental and Bioscience, College of Environmental and

Bioresource Sciences, Chonbuk National University, Iksan, Jeonbuk 570-752, South Korea. 3IVC (India Vetriselvan Chitra) aqua farm, Kattumadi village, Pudukottai district, Tamil Nadu, India.

4Department of Environmental Sciences, Bharathiar University, Coimbatore, Tamil Nadu, India. [E-mail: [email protected] : [email protected] ]

Received 28 December 2015 ; revised 30 August 2016

In this study, various physico chemical parameters, feeding rate, water exchange rate, effect of stocking density and growth rate of Litopenaeus vannamei were evaluated using four ponds in in-vivo (direct field) for a period of 142 days. The first stage juvenile of L.vannamei were stocked at different density in to each pond and periodically harvested from 78th day to 142 days of culture for regular analysis. The average stocking density of shrimps reared at A1 pond was higher up to 11,093 kg/ 0.65 ha and lower at A4 pond up to 8, 379 kg/0.65 ha after142days of culture. In addition, survival rate of shrimps was higher in A1 pond up to 75%, where as a lower rate A2 pond up to 60% could be achieved. In the case of average body weight of the shrimps was found to be low at A2 pond (27.5 g) and higher at A3 pond (30 g). Consequently, stocking density of pond A1 was commend for culture of PL12 L.vannamei species in Pudukkottai district in the south India, Tamil Nadu under these direct field experimental conditions.

[Keywords: Litopenaeus vannamei, in-vivo, shrimp farming, stocking density, growth rate]

Introduction Aquaculture is the cultivation of aquatic animals

and plants, especially fish, shellfish, and seaweed, in natural or controlled marine or freshwater environments for the production of food1. The global aquaculture production has shown tremendous growth in recent years2, 3. Recently, aquaculture is the fast growing food production sector in many countries. However, China produces the bulk of the world’s aquaculture production: 67 %, followed by India 6% and Vietnam 3% in Asia by far is the dominant producer4. Based on most recent Food and Agriculture Organization (FAO) estimation of the global fish production from aquaculture has increased to about 10 million metric tons (MT) in 1984 and further more than 39 million tons in 1998, with an annual growth rate of about 10%5. On the other hand, the production from capture fisheries increased at less than 1% to 87 million tons in 19985. In 2004, total world aquaculture production was 59.4 million tons and had a value of US$70.3 billion6. Although aquaculture has been practiced for millennia, the main expansion of

aquaculture occurred only in the last three decades. Approximately, a 9-fold increase in total world aquaculture production was documented between 1980 and 20047. Aquaculture Production in 2012 is estimated at around 66.5 million tones. Full global statistics of aquaculture production has been collected by FAO and the outcome of the data will be released in early March 20148.

The regular use of antibiotics in aquaculture has resulted in the development of resistant strains, which have contributed to the inefficiency of antibiotics9, 10. A recent study conducted by NOAA (National Oceanic and Atmospheric Administration) indicates that aquaculture poses a low risk to the environment11. Aquaculture impacts are typically local and temporary, where as in some cases, aquaculture can benefit the environment12. In cases where filter feeding shellfish, such as oysters, are cultured in-situ, water quality in ponds and lakes can improve. Although there are documented problems associated with aquaculture, governmental agencies believe it is a long-range and sustainable solution to the world's

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wild marine fish populations1. Like a giant aquarium, land-based fish farms also the dirty water must be changed frequently to maintain the quality of the culture. Depending on the system's set-up, this can result in the discharge of significant amounts of wastewater containing feces, nutrients and chemicals into the environment. Nutrients can result in algae blooms which eventually remove dissolved oxygen in the receiving water way or eutrophication13 leads to depletion of oxygen in the water, which may cause death to aquatic animals. In addition, chemicals are commonly used in the aquaculture industry, such as antibiotics and water treatment agents14. To avoid this crisis, aquaculture wastewater must be treated prior to discharge.

Aquaculture is a part of shrimp production. The majority of global shrimp aquaculture production comes from Asia15. Throughout the 1980s, millions of dollars in funding were invested in Asian and Latin American aquaculture projects by these groups, providing the necessary capital to seed the aquaculture industry16. The scope and scale of these investments, together with the availability of suitable land and a ready labor force, created a comparative advantage for Asian and Latin American countries. In 1998, Asia accounted for nearly 36 million tons or more than 90% of global quantity and more than 80% value of aquaculture production. China is the dominant producer, accounting for more than 75% of total aquaculture production of Asia, followed by India (5.7%), the Philippines (2.7%), and Indonesia (2.3%). Shrimp are commonly cultivated in brackish water conditions. In the beginning of 1996 L.vannamei was introduced into Asia for commercial purpose and it’s been practiced till date. In India L. vannamei shrimp culture which has taken roots in Gujarat, Andhra Pradesh and Tamil Nadu, India has now expanded throughout many parts of India because of its rapid growth within short period in brackish water and freshwater. At the same time freshwater demand is highly increased.

Hence, this study was conducted in in-vivo at IVC (India Vetriselvan Chitra) aqua farm, Kattumavadi village, Pudukkottai district, Tamil Nadu, India (Latitude: 10.1700323 & longitude: 79. 0012499). Experiments were conducted during December 2013 to April 2014 in 142 days. Over all study deals with the effect of stocking density of shrimp species (L.vannamei) in four ponds on shrimp weight, biomass, growth rate, diet consumption rate, pond water quality and survival rate.

Materials and Methods Study area and duration

Pudukkottai is one of the small districts of Tamil Nadu, India. The district has an area of 4663 Sq. Km. with a coast line of 39 Kms. The district lies between 78.25' and 79.15' of the Eastern Longitude and between 9.50' and 10.40' of the Northern Latitude. It is bounded by Tiruchirappalli district in the North and West, Sivagangai district in the South, Bay of Bengal in the East and Thanjavur district in the North East. Study area is Kattumavadi coastal village (Lat. 10º4’N; Long. 79º12’E). Total 10 villages under 2 Taluks have been identified for the proposed evacuation shelters in this district. Village Kattumavadi (Lat. 10º4’N; Long. 79º12’E) Location was used as the study area and the satellite image was shown in Figure1a & b. Pond preparation

Scraping the surface of the pond up to 2 - 3 inch and tilling the pond with tractor followed by drying of pond for about 1 week. After a week, the sea water was pumped directly from sea in to the dried pond up to 150 cm height (Figure 2a - b) and sodium hypochlorite powder 10ppm was added each pond (Figure 2c & 3d - e). The water was allowed to with stand for 3 days for removal of odor in de-chlorinating pond water. Next, approximate amount of lime powder was added in to the pond and wait for one day. Later, probiotics were applied in to the pond for bloom development (Figure 3a - c). After bloom development, the L. vannamei seeds are introduced into the pond (Figure 2f insert) with regular monitoring of pH, DO, ammonia, salinity and feed consumption. Almost care was taken to maintain the salinity of the pond water up to 25 to 35ppt. Shrimp and experimental conditions

Juveniles of L.vannamei were obtained from the hatchery at Marakkanam, Villupuram District, Tamilnadu, India. Prior to start the experiment, the shrimps were acclimated to the culture environment for 3 weeks and fed with a commercial diet (44% crude protein, 8% crude lipid, Charoen Pokphand Group (CP) Aqua cultural [India] Private Limited, Chennai).

The experiments were conducted after 15 days of the pond preparation and each pond covers (0.65 hectare) and name as (A1, A2, A3 andA4) with initial stocking density of 5,10,000, 5,82,000, 4,56,000, and 4,20,000 Sp (Shrimp pieces) of similar size with rough body weight 0.58g were randomly distributed, respectively. Each pond was regularly fed

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with appropriate diet in same interval. All groups of shrimps were fed to apparent satiation four times daily at 07:00 AM, 12:00 PM, 17: 00 PM and 22:00 PM with 5–8% body weight per day for 8 weeks. Feed were put in a 25 x 25 cm feeding tray that was placed at the bottom of each pond after feeding time 2 hour to check the feeding tray to find out the consumption any uneaten feeds were collected by siphoning, then dried, weighed and used to calculate feed intake.

Physico-chemical parameters were analyzed using water analysis kit and APHA 196516 every week after introduction of shrimp seed. The water exchange treatment process was also started after 50 days of culture period and maintained for four pond (A1, A2,

A3 and A4) and water pH, DO, Ammonia and Salinity, feed consumption was also recorded. The DO (Method: Titration 200 determinations), and ammonia (Method: Colorimetric 200 determinations) were estimated using test kits (Aqua AM and Aqua D.O Purchased from Advanced Pharma Co., Ltd, Thailand). The pH and salinity were determined using pH meter/pH paper and refractometer, respectively. Water quality parameters were monitored bi-weekly in the laboratory. Experimental diet analysis

Feed with various nutrient composition were fed to shrimps depend upon the shrimp size, and the feed composition ratio. The characteristics of feed

Fig — 1a&b. Location of study area (a) and the satellite image (b)

Fig — 2a-c. Pumping water from sea source (a), aerator off & on condition (b) and sheletal desighn of pond (c)


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were classified into feed code, types, feed size mm (01-fine crumble (0.42 mm), 02-crumble (0.89 mm), 03-crumble (1.41 mm), 04-pellet (2035 x3.5 mm) and 05-pellet (2.3x4-5 mm)) to the level of growth of the shrimp, respectively. Feed requirement of each pond per day was calculated based on the feeding quantity17. Periodic survey of shrimp survival analysis

After 60 days of culture period, shrimp survival, shrimp weight, growth rate, and biomass were analyzed every week and the data were recorded in average. In case, the growth was found to be declining, water treatment process will be stared to increase the shrimp growth and production18. Finally, the harvesting of complete grown shrimps was carried out at the end of 142nd day. One square meter of net was laid in the pond water and 25 numbers of shrimp individuals were subjected for analysis for 14 days, after 14 days counted the shrimp quantity survival were calculated using following formula (1). Survival(%) = × 100

Data Collection After 60 days all shrimp species from each pond

were sampled randomly collected at two weeks’ interval to assess shrimp body weight, growth rate and

total biomass. The total biomass was estimated using following formula (2).

Bodyweight(gm) = 2kgNo. ofshrimppieces Totalbiomass(kg) = NumberofShrimppieces xBodyweight1000 GrowthRate/day = Biweeklygrowth(currentweight − Previousweight)No. ofdays

Statistical analysis Microsoft office excel-2010, two-way ANOVA

and cluster analysis were used to analyze the data. The results were presented as the means ±. The data were first tested for homogeneity, if the data had similar variances, then one-way ANOVA was used to test the main effect of dietary manipulation. When there were significant differences (P < 0.05), the group means were further compared using Duncan's multiple range test17. Results and Discussion Diet consumption

The rate of diet consumption by shrimps with described diet composition for each pond on each day was shown in Figure. 4. In regard to all the four ponds, shrimps at pond A2 consume more feed

Fig 3 — a-f. Photograph showing workers are preparing the chemicals for preparing the pond (a-e), grown shrimp (f) and juvanile of PL12

stage L.vannamei species from hatchery (f insert).


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(13,835 Kg) and shrimps at pond A4 (10,921 Kg) consumed low feed for the entire study period. However, a moderate feed consumption was recorded at other two ponds A1 (12,723 Kg) and A3 (11,149 Kg) 19, 20. This factor might be due to alteration in various pond environmental parameters. According to Table 1, the disturbed pond environment like pH, ammonia, oxygen and salinity in the pond might influence the low and high feed consumption. In addition, the unexamined parameters like CO2, temperature, NO2

-, hardness, H2S, and BOD (Biological Oxygen Demand) also may be the major cause for the unbalanced feed intake21, 22. Water quality analysis

The Physico-chemical parameters: pH, ammonia, DO, and salinity were monitored in the culture ponds

at regular intervals with continuous aeration and the results are shown in Table. 1 and expressed graphically in Figure 5a, b, c & d. The fluctuations of pH in all ponds were between pH 7.14 to 7.71 in average. In further, the pond A1 exhibits high pH range then all other ponds and found to be non-fluctuating till the end of the shrimp growth (Figure. 5a). The values of ammonia were fluctuated between 0.21 ppm to 1.79 ppm in all the ponds. Further, it was noted that the ammonia percentage increases only in middle of the shrimp growth period and high mortality problems were prominent in all the four ponds19. In context with ammonia, pond A4 has maximum value and minimum was recorded in pond A1 (Figure 5b). As said earlier, this variation is due to the disturbed environmental conditions of the physico-chemical parameters of the pond. Consequently, to avoid disturbance by increase in ammonia, the water exchange treatment was initiated for all the four ponds. The fluctuation of DO values was noted throughout the study among 3.75 to 4.25 in all ponds. The maximum DO value was noted in pond A4 and minimum in pond A1 (Figure 5c). The aerators were switched on when the DO level decreased in each pond.

The reason behind the DO fluctuation might be depends on the rate of shrimp growth21, 23, 24. The salinity values were fluctuating among 31.57 to 36.50

Fig 4 — Diet consumption of shrimp in each pond.

DOC

Table. 1 — Water quality parameters of four ponds in different days parameters

pH Ammonia(ppm) DO(ppm) Salinity (ppt)

Pond A1

Pond A2

Pond A3

Pond A4

Pond A1

Pond A2

Pond A3

Pond A4

Pond A1

Pond A2

Pond A3

Pond A4

Pond A1 Pond A2 Pond A3 Pond A4

0 7.00 7.00 7.00 7.00 0.00 0.00 0.00 0.00 4.00 4.00 3.50 4.00 27 28 26 28 10 7.20 7.00 7.25 6.75 0.00 0.25 0.00 0.00 4.00 4.50 4.00 3.50 30 31 31 30 18 7.50 7.50 7.00 7.25 0.00 0.50 0.00 0.25 4.25 4.00 4.50 3.50 33 32 32 33 26 7.25 6.50 8.25 8.00 0.25 0.00 0.25 1.00 3.50 3.50 4.00 4.00 34 32 34 34 34 8.00 7.25 8.00 8.25 0.50 0.50 0.25 0.00 3.00 3.50 4.00 4.50 32 30 32 32 42 8.75 7.75 6.50 7.75 0.25 0.25 0.25 0.00 4.00 3.50 4.50 4.50 34 33 35 34 50 7.50 6.50 8.00 7.50 0.50 1.00 1.75 0.50 3.50 4.50 4.00 4.00 34 35 34 35 58 7.75 7.25 7.25 7.50 1.75 2.00 2.00 2.00 3.50 4.00 4.50 4.00 33 34 35 34 66 7.50 7.50 7.50 7.25 2.00 2.00 1.00 2.00 4.00 4.00 4.00 4.50 34 35 36 35 74 8.25 7.25 7.00 7.50 1.00 1.75 1.75 1.00 3.50 3.50 4.00 4.00 33 34 33 36 82 8.00 8.00 7.75 7.50 1.75 1.75 1.75 2.00 4.00 4.00 3.50 4.00 34 35 34 37 90 7.25 7.25 8.00 7.25 2.00 2.00 1.75 1.75 4.50 4.50 4.00 4.50 35 33 36 34 98 7.50 8.00 7.25 7.50 1.00 1.00 2.00 2.00 4.00 4.00 4.50 4.00 35 33 34 35 106 7.00 7.50 7.50 7.25 1.75 2.00 1.00 2.00 4.00 4.00 4.00 4.50 36 35 34 37 114 7.75 7.25 7.00 7.50 1.75 1.75 1.75 1.00 3.50 4.50 4.00 4.00 37 36 35 36 121 7.50 7.25 7.00 7.50 1.50 1.00 1.00 1.00 4.00 4.00 4.00 4.00 37 36 37 37 128 7.50 8.00 7.50 7.25 2.00 1.75 2.00 1.75 4.00 4.00 4.50 4.00 34 35 34 37 135 7.00 7.50 7.25 7.50 1.00 2.00 1.00 2.00 3.00 3.50 3.50 4.50 35 37 36 35 142 7.50 8.00 7.50 7.00 2.00 1.00 2.00 1.00 4.00 3.50 4.00 4.00 36 37 36 37


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in all ponds. The maximum salinity value was found in pond A4 and minimum in pond A2 (Figure 5d). Whenever a notable decrease in the salinity value, it is mandatory that the water should be drained and the new water from the sea is pumped to the culture ponds to maintain the salinity25.

Average survival analysis of shrimp in each pond The four ponds were stocked with different

stocking density of shrimps26, 27, 28. The survival of the shrimp was analyzed based on the shrimp body weight; growth rate and biomass were indicated in Figure 6a & b and Table 2. Running mortality

10 to 30 % were occurred during the whole period of culture due to de-molding, high ammonia content and lack of DO. Shrimps survival was analyzed and summarize in supplementary Table. 1 - 4. The shrimp body weight; growth rate and biomass were analyzed from 78th day of growth until harvesting period. The most average weight of shrimp was found in pond A3 (30 g) and least in pond A2 (27.5 g). Considering the mortality rate, the pond A2 (40 %) was recorded maximum and 25% as in pond A1. Since, the high stocking density of shrimp, growth rate was decreased (high stocking density means contain high number of

Fig 5 — a-d. Effect of physico chemical parameters in each in culture pond pH (a), ammonia (b), DO (c) and salinity (d).

Fig 6 — a&b. Different stocking density overall production in sea water (a) and growth rate of shrimp in each ponds (b)

Table. 2 — Total Shrimp survival and different stocking density survival production in seawater analysis in each pond

Pond Area (hectors) Initial shrimp numbers &

Stocking density of shrimp pieces

Final shrimp numbers Survival % Shrimp production (kg)

A1 0.65 5,10,000 3,82,500 75% 11,093 A2 0.65 5,82,000 3,49,200 60% 9,603 A3 0.65 4,56,000 3,19,200 70% 9,576 A4 0.65 4,20,000 2,94,000 70% 8,379


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pieces there, so growth rate was decreased), whereas, growth rate was exponentially increased when the water exchange process was done. Different stocking density and overall production of shrimp for the culture period is summarized in the Table.2 and Figure.6.

Statistical analysis

Two-way ANOVA is performed to pick out from the significant ponds with effective stocking density of the shrimp affected by various growth parameters like pH, ammonia, DO, salinity, growth weight and growth rate using Excel data sheet analysis tool pack for significant variation between ponds and within ponds. Finally, the result is shown Table.3. Since, it differs among four ponds in different stocking density

of shrimp growth and within ponds no significant was found in water quality parameters. Hence, all the ponds were filled with same sea water. Cluster analysis – dendrogram

Pond similarity based on Physico – chemical parameters and shrimp growth parameters were analyzed and shown in Figure 7a - f. The obtained dendrogram is to conclude that, pond A1 / A4 was group together and pond A3 / A2 group together based on the hydrogen ion concentration levels in the pond for the complete growth stage (Figure 7a). The pond A3 / A1 were group together and pond A4 /A2 group together based on the ammonia concentration levels in the pond for the complete growth stage (Figure 7b). The pond A2 /A1 were group together and pond A3 / A4 were group together based on the DO concentration levels in the pond for the complete growth stage (Figure 7c). The pond A3 / A1 are group together and pond A2 / A4 group together based on the salinity concentration in the pond for the complete growth stage (Figure 7d). Pond A4 / A1 are group together and pond A2 / A3 group together based on the growth weight of shrimps in the pond for the complete growth stage (Figure 7e). The pond A2 / A1 are group together and pond A4 / A3 group to gather based on the growth rate levels in the pond for the complete growth stage (Figure 7f).

Table.3 — Anova results for all parameters in each pond

Parameters F – values P – values

pH Between ponds 1.13 0.35 Within ponds 0.88 0.55

Ammonia Between ponds 0.03 0.99 Within ponds 1.22 0.32

DO Between ponds 1.63 0.21 Within ponds 1.94 0.09

Salinity Between ponds 2.65 0.07 Within ponds 3.29 0.01

Growth weight Between ponds 59.07 5.47 Within ponds 512.13 8.79

Growth rate Between ponds 17.28 1.84 Within ponds 3.41 0.01

Fig 7 — a-g. Dendrogram of pond similarity based on Physico–chemical and shrimp growth parameters pH (a), ammonia (b), DO (c),salinity (d), growth weight (e), and growth rate (f).


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Conclusion Physico – chemical parameters were maintained in

four ponds consistently, however, ammonia is fluctuating due the consumption of the food by shrimp for its growth in the middle of culture period and persuades high mortality rate. Hence, ammonia quantity in the pond must be taken into consideration to reduce the mortality rate. To increase the shrimp body weight and growth rate, diet was maintained and it’s resulted in the final stage. Out of all the four ponds, pond A3 (survival 70%, production is 9,576 Kg) turns out with maximum weight and growth rate. The shrimp survival percent was appearing to be high in pond A1 but with moderate production. Finally, from the present investigation, it was being concluded that L.vannamei culture has the best outcome in pond A3 with successful usage of sea water and growth is directly related to less stocking density. This study will give a moral idea for the shrimp cultivators for better production and will predict the production.

Conflict of Interest There is no conflict of interest in this manuscript.

Acknowledgement

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