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“SIAF APRAS”

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SIAF APRAS

Updates from
Anthony C. Ostrowski, Ph.D.:

New Research at AF4

AF4 Research and Traceability Agenda

Tri-way Tests New Species



AF4: Successful Preliminary Trial of Pacific White Shrimp in APRAS

by:  A.C. Ostrowski, York Wong, and Vincent Feng

follow-up to:  AF4: New Research on Pacific White Shrimp

Abstract

A preliminary trial to raise L. vannamei (Pacific white shrimp) at AF4 in 2 x 50 m3 SIAF proprietary A-Power recirculating aquaculture system (APRAS) tanks was conducted for 77 days after nursery to 1.1 g. Shrimp reached an average 16 g (1.3 g/week = 0.19 g/d), 1.4 feed conversion ratio (FCR = dry feed fed/wet weight gain), 75% survival, and final harvest density of 3.0 kg/m3. Regimented use of a commercial probiotic added to water was effective at controlling harmful levels of Vibrio, Aeromonas, and Pseudomonas gram-negative bacteria. Installation of a drum filter effectively removed total suspended solids from the water column. Results also presumptively demonstrated 17% better growth in the tank with refuge versus the tank without refuge. Subsequent studies will determine the statistical significance of the use of refuges in tanks, and effect of density on growth and overall production in both 50 m3 and 150 m3 APRAS tanks. Biological targets are 2 g/week (0.28 g/d) growth to 5-10 kg/m3, 80% survival, and a 1.2 FCR for > 50% total profitability with this species in APRAS.

Introduction

A distinct advantage of the SIAF APRAS technology is its flexibility for use with a variety of species. Recent interest at SIAF to grow the Pacific white shrimp (L. vannamei) stems from rapidly rising consumer demand in China, coupled with the shortage of domestic supply. In 2017, China imported 400,000 MT of farmed shrimp (mostly from Vietnam) valued at $3.6 billion (Harkell, 2017). This is in addition to the estimated domestic supply of 700,000 MT farmed and 100,000 MT of wild caught marine shrimp.  Demand is so high that inland provinces are substituting crayfish for shrimp (both marine and freshwater species). 

Off-the- farm prices for live Pacific white shrimp in China can reach as high as $16 - 20/kg for the largest sizes (> 21 -25+ grams), with highest prices received during February – May. Prices for medium sized shrimp (16 – 20 g) can reach $14 - $16/kg. High prices are due to a particular lack of supply of live shrimp during the winter and early spring months. Most shrimp farming in China is done outdoors in open ponds, and operations shut down during winter (January – March) because air and water temperatures are too cold to raise this tropical species. Shrimp growth and feeding rate is directly related to temperature within physiological limits (Wyban et al., 1995); animals stop feeding at around 19oC.  The next season’s first crops are not ready until May, when prices begin to decline because of increased supply. 

Furthermore, China’s outdoor pond production is not expected to increase much in the future, primarily hampered by rampant disease issues. This results in frequent early, emergency harvests and a persistent paucity of the largest sizes in the marketplace even during the normal production season.  Indoor, year-round, bio-secure production would provide market advantage to SIAF for producing the largest shrimp and at times when domestic supply is limited.

A preliminary trial at AF4 was recently concluded to determine the feasibility of raising white shrimp in APRAS at low salinity. White shrimp are a saltwater species, isotonic with its environment at 18 ppt. In China, most shrimp are raised at 5 – 15 ppt in ponds. Research has shown that this species can be raised in salinities lower than 5 ppt if certain minerals in the water are amended (Boyd et al., 2002; Roy et al., 2007) if ambient water is lacking minimal levels and ratios. In RAS systems, as in ponds, management of the microbial environment is key to maximizing growth and minimizing the risk of non-excludable diseases, those primarily caused by gram-negative bacteria of the genus, Vibrio. Vibrios arise within tanks due to deteriorating water quality or high-density rearing conditions. This trial also examined the use of probiotic additions to water to control harmful Vibrio populations.

Methods

A single 100 m3 APM (A-Power Module), consisting of 2 x 50 m3 APRAS tanks serviced by a single baffled chamber for biofiltration and water remediation was used.  One tank had stacked refuges made of ½ inch (3.81 cm) PVC pipe and 1.5 cm diameter plastic meshed screen; the other tank had no refuge. The refuges nearly doubled that tank surface area from 65 m2 to 121 m2. The APM was housed in a 9,000 m2 covered building, with transparent roofing at regular intervals to allow entry of ambient light.

Water was recycled within the APM using 2 x 0.15 kw pumps. In addition, a 0.37 kw drum filter (10 m3 of water processed/hr) with 200 micron-mesh screening was added to the system 10 days after stocking to assist the passive APM mechanical filter with removal of total suspended solids (TSS).  The total 0.67 kw of pumping power yielded 3.12 turnovers of tank water volume/d. A commercial probiotic (INVE Sanolife™) was added to tank water one day prior to stocking and on a weekly regimen per manufacturer recommendations. Aeration was supplied using perforated flex-tubing.

Each 50 m3 tank was stocked with 13,000 shrimp that were previously raised from PL 10 (10-day old post-larvae) in a separate nursery system (100 m3 APM) for 42 days. These D42 shrimp were an average 1.1 grams. D42 animals were raised in the tanks for 77 days (total 119 days from PL10). Animals were fed a commercially pelleted shrimp feed (35% protein) four times daily based on a calculated 4% body weight per day, adjusted with each feeding to satiation using feeding trays for determination. Subsamples of (approx. 30 – 35) animals from each tank were weighed weekly. Tank bottoms were siphoned daily of uneaten feed, feces, and other settled solids (SS). Siphoning required water removal and addition, with remediation of salinity level.  Salinity was targeted at 3 ppt.

Water quality characteristics (temperature, pH, alkalinity, TAN (total ammonia nitrogen), nitrite, nitrite, salinity, potassium, magnesium) were measured either daily or as needed. Alkalinity was adjusted with sodium bicarbonate, and salinity adjusted with additions of refined aquarium seasalt. 

In addition, the presence of bacterial gram-negative, green (presumptive V. parahaemolyticus, V. vulnificus) and yellow (presumptive V. cholerae, V. alginolyticus) Vibrio colonies were monitored in addition to separate monitoring of other gram-negative bacteria of the genus, Aeromonas and Pseudomonas. Vibrio levels were kept in check with addition of the probiotic, which contained primarily gram-positive Bacillus, at a rate of 1 mg/m3 of water. An initial dose was provided prior to stocking, and every week three days in a row thereafter. Probiotics were also added when Vibrio spikes were observed. UV-light was added to the water exit chamber the second day after stocking in response to an early spike in Aeromonas and Pseudomonas populations.

At the end of the trial, shrimp were harvested and weighed en mass from each tank, and random aliquots were taken for size class determination and overall average weight and number determinations. A total 199 shrimp were sampled from the tank with refuge, and 125 from the tank without refuge. 

Results

Shrimp grew from 1.1 grams (g) to an average 15.9 g in 77 days, at an average 1.3 g/week, or 0.19 g/day (Figure 1). Subsample weight of shrimp in the refuge tank was consistently higher after 14 days and at the end of the trial. Final weight of shrimp in the tank with refuge was 17% higher (17.1 vs. 14.7 g).  Final overall density in the 100 m3 APM reached 3.0 kg/m3 (3.2 vs. 2.8 kg /m3, refuge vs. no refuge tanks). Overall survival averaged 75%, with no obvious difference between the two tanks (74% vs. 75%, refuge vs. no refuge tanks).  Feed conversion ratio (FCR) averaged 1.4 (1.3 vs.1.5, refuge vs. no refuge tanks).

A normal bell-shaped curve in weight classes was obtained for the entire population (Figure 2). This was slightly skewed to lower size classes in the tank without refuge. Approximately 47% of all shrimp sampled fell within 2 g of the mean. Over 15% of the population reached 19 g or higher, with the greater percentage of those (88%) coming from the tank with refuge.

Water quality parameters remained within acceptable levels for this species (Table 1). Water temperature was 27.2oC and rose to 30.1oC during the trial, in direct relation to increasing ambient air temperature within the building. Potassium and magnesium maintained minimal levels and ratios desired, without amendments. 

There was an early spike within 2 days of stocking in total ammonia nitrogen (TAN = 0.5 ppm), which lead to a spike in nitrite (0.15 - 0.2 ppm) two days later. High nitrate levels persisted for a total 9 days, then fell to 0.05- 0.01 ppm for the remainder of the trial. High TSS was also observed for the first two weeks, although TSS was not numerically quantitated. TSS diminished greatly within three days after installation of the drum filter 10 days after stocking. 

There was a similar spike in Aeromonas and Pseudomonas colonies (5,000 – 6,000 colonies/ml) on the second day after stocking (Table 2). Aeromonas quickly receded by the fourth day after UV-lights were installed within the baffle chamber on the second day (with addition of probiotics) and remained low (200 – 400 colonies/ml) throughout the rest of the trial. Pseudomonas counts also fell rapidly but remained between 1,000 – 2,000 colonies/ml for the rest of the trial.  

In contrast Vibrio levels were not affected by the early spike in TAN and TSS and remained low (< 450 colonies/ml) throughout the entire trial (Figure 3). Any increases or spikes noted in Vibrio levels were quickly remediated by addition of probiotics. 

Discussion

Results demonstrated that Pacific white shrimp can be raised successfully to at least 16 grams and 3 kg/m3 in 50 m3 APRAS tanks. Growth was linear after 1 g, typical of this species (Wyban et al., 1995). The 1.3 g/week rate obtained in this trial is within good limits. The normal bell-shaped curve of size classes was indicative of a healthy population. The 1.4 FCR obtained was as good or better than that achieved in other intensive shrimp culture systems (1.4 – 1.8) that rely on pelleted feeds for the primary source of nutrition (FAO, 2018).  Shrimp health and water quality appeared adequate to take the trial further to higher density but was curtailed because shrimp were pre-scheduled to be sold.

Most mortality in this trial likely occurred during the first two weeks when TAN and nitrite levels were excessively high.  Recommended (based on LD50 trials) safe levels of nitrite for post-larval L. vannamei at 1 – 3 ppt are 0.17 – 0.25 mg/l, respectively (Valencia-Castaneda et al., 2018). Observed nitrite levels in this trial (0.15 – 0.2 mg/l) during for the first 10 days of culture could have caused subclinical stress in the young and newly stocked shrimp, and latent or acute mortality.  Support for this is the rapid increase in TSS (not quantified) at this time, which was likely indicative of the dissolution of uneaten feed. Sub-clinical signs of stress (e.g. ammonia toxicity) include reduced or stopped feed intake. Persistent elevated TAN levels were also likely due to dead and dying shrimp. It is possible that the transfer of D42 shrimp from the nursery to growout tanks was overly stressful, resulting in reduced or stopped feeding with subsequent mortality that caused the cascade of poor water quality events.

The initial poor water conditions (in terms of TAN, nitrite, and TSS) also likely caused the rapid spike in Aeromonas and Pseudomonas populations at the same time. Aeromonas are ubiquitous in waters and considered pathogenic (Tomas, 2012), and may have caused additional latent (stress-related) mortality. Aeromonas are particularly prevalent in ambient waters in China (Nielsen et al., 2018). Pseudomonas counts were more than twice as high. However, the influence of Pseudomonas on shrimp health in this trial is unclear since the species was not characterized. Different stains of Pseudomonas can have both positive and negative effects on shrimp health (Zorriehzahra et al., 2016). Vibrio levels were unaffected by the increase in TAN suggesting the varying population dynamics of bacterial species in response to water quality conditions.

The combination of probiotics and UV-lights in APRAS were essential to maintain a healthy rearing environment for the shrimp, preventing dominance of the pathogenic bacterial species. In combination, they corrected and maintained low populations of Vibrio and other presumptive pathogenic (i.e. Aeromonas) bacteria. Control of type and quantity of bacterial populations is essential in high-density and closed (RAS) environments to prevent disease outbreaks. Vibriosis is a major bacterial disease in shrimp worldwide (Lightner et al., 1992). The early spikes in Aeromonas and Pseudomonas within the first few days after stocking shrimp were quickly remedied with addition of UV-lights. The lights also likely helped control Vibrio populations, although did not prevent the dramatic spikes observed. These spikes were best remediated (within a day or two) by spot additions of the probiotic. The weekly regimen of added probiotics also likely maintained low Vibrio colony counts throughout the trial and was certainly necessary since all bacteria (including the beneficial Bacillus which comprised most of the probiotic mix) that passed through the UV-lights were assumed affected.

In addition, the drum filter incorporated into the system during the second week of the trial proved effective at removing TSS. Shrimp excrete an encased fecal strand, which breaks up in the water column in APRAS tanks due to heavy aeration and water movement, resulting in increased TSS. Shrimp also masticate their feed, which causes small feed particles to be released into the water column. The passive mechanical filter of the APRAS design clogged within just a few hours and had to be repeatedly washed. Concerns were that the clogging would cause chamber water to backflow in tanks during the evening when staff were not present. The drum filter had a self-cleaning mechanism which greatly improved operation and staff time to maintain tanks, although the drum filter cleaning mechanism had to be run almost constantly near the end of the trial. A larger drum filter with greater screen surface area should reduce the number of times the self-cleaning pump is activated to improve cleaning efficiency and reduce electrical costs of operation.

The maintenance of minimum concentrations of potassium, magnesium, and sodium:potassium ratio is considered crucial for good growth of shrimp raised at low salinity of < 5 ppt (Roy et al., 2007). Concentrations of these minerals determined in the 3 ppt water in this trial were at minimum recommended levels. Adjusting the levels slightly higher could improve growth.

It was also encouraging to observe better (17%) growth in the tank with refuge, although this result needs to be scientifically validated with proper tank replication. Refuges provide respite places for shrimp during molt, making them less prone to cannibalism by congeners, and increased surface area for these primarily bottom-dwelling animals, especially in the deep APRAS tanks. Increased surface area also increases the proportion of bacteria that grow on any surface in the tank, including the refuges. Shrimp are continuous feeders and consume bacterial biofilm to gain additional nutrition and unknown growth factors (Emerenciano et al., 2013).  Shrimp in this trial were observed scraping tank sidewalls and the refuges ostensibly feeding on established biofilm. The increased surface area and bacteria provided by the refuges could also be partly responsible for improved growth in this tank.

Conclusion

In conclusion, this preliminary trial encouragingly demonstrated that Pacific white shrimp (L. vannamei) can effectively be raised in APRAS at low (3 ppt) salinity. The intent going forward is to ensure the economic viability of the approach. Targets include a growth rate of 2 grams/week (20 grams within 60 days of growout after nursery), at densities between 5-10 kg/m3.  Samocha et al. (2012) obtained up to 1.95 grams/week and 9.87 kg/m3 to 25 grams individual weight of fast-growth L. vannamei in zero-water exchange, 18 ppt salinity, high-density biofloc raceways in the U.S. Some well-managed intensive ponds in China harvesting between 3-5 kg/m3 have reportedly obtained growth rates of up to 2 grams per week after nursery stocking, and FCR’s closer to 1.2 – 1.4. Preliminary economic projections indicate that with this level and other minor assumptions, a total profit of well over 50% can be achieved in APRAS for this species. Better biological performance than observed in this study can likely be achieved with faster growth in the nursery, increased feeding rate to account for maximum growth, higher quality feed, higher salinity and/or mineral levels, and use of refuges, in addition to continued microbial management and improved management of the APRAS APM. Subsequent trials will address such issues and establish growth rates at various densities. 

 

References

 Boyd, C.E., T. Thunjai, and M. Boonyaratpalin. 2002. Dissolved salts in water for inland low salinity shrimp culture. Global Aquaculture Advocate. June 2002:40-45.

Emerenciano, M., G. Gaxiola, and G.Cuzon. 2013. Biofloc technology (BFT): A review for aquaculture application and animal food industry. Pp. 301 - 328. In: Miodrag Darko Matcovic (eds.), Biomass Now - Cultivation and Utilization, 2013. 

FAO. 2018. Cultured Aquatic Species Information Programme. Penaeus vannamei. Cultured Aquatic Species Information Programme. Text by Briggs, M. In: FAO Fisheries and Aquaculture Department [online]. Rome. Updated 7 April 2006. [Cited 31 August 2018].

Harkell, L. 2018. CAPPMA: China shrimp imports worth over $3.6 bn, growth to slow in 2018. Undercurrent News. June 4, 2018.

Lightner, D.V., T.A. Bell, R.M. Redman, L.L Mohney, J.M. Natividad, A. Rukyani, and A Poernomo. 1992. A review of some major diseases of economic significance in penaeid shrimps/shrimps of the Americas and Indo-Pacific. In: M. Shariff, R. Subasinghe and J.R. Arthur (eds.) Proceedings 1st Symposium on Diseases in Asian Aquaculture. Fish Health Section, Asian Fisheries Society, Manila, Philippines. pp. 57-80.

Nielsen, M.E., L. Hoi, A.S. Schmidt, D. Qian, T. Shamada, J.Y. Shen, J.L. Larsen. 2018. Is Aeromonas hydrophila the dominant Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China? Diseases of Aquatic Organisms 46 (1):23-29.

Roy, L.A., D.A. Davis, I.P. Saoud, and R.P. Henry. 2007. Effects of various levels of aqueous potassium and magnesium on survival, growth and respiration of Pacific white shrimp, Litopenaeus vannamei, raised in low salinity waters. Aquaculture 262:461-469.

Samocha, T., R. Schveitzer, D. Krummenauer, T.C. Morris, S. Woodring, and T. Hansen. 2012. Performance of fast-growth Litopenaeus vannamei in super-intensive zero exchange raceways. USMSFP ISG Meeting, Aquaculture America, Las Vegas Nevada, USA. February 28, 2012.

Tomas, J.M. 2012. The main Aeromonas pathogenic factors. Internationally Scholarly Research Notices. Volume 2012. Article ID 256261. 22p.

Valencia-Castaneda, G., M.G. Frias-Espericueta, R.C. Vanegas-Perez, J.A. Perez-Ramirez, M.C. Chavez-Sanchez, and F. Paez. 2018. Acute toxicity of ammonia, nitrite and nitrate to Litopenaeus vannamei postlarvae in low-salinity water. Bulletin of Environmental Contamination and Toxicology 101(2):229-234.

Wyban, J., W.A. Walsh, and D.A. Godin. 1995. Temperature effects on growth, feeding rate, and feed conversion of the Pacific white shrimp (Penaeus vannamei). Aquaculture 138 (1-4):267-279.

Zorriehzahra, M.J., S.T. Delshad, M. Adel, R. Tiwari, K. Karthik, K. Dhama and C. C. Lazado. 2016. Probiotics as beneficial microbes in aquaculture: an update on their multiple modes of action: a review, Veterinary Quarterly, 36:4, 228-241.

 

Figure 1. Growth of D42 post-nursery L. vannamei in 2 x 50 m3 APRAS tanks with and without refuge at 3 ppt and 27 - 30oC at AF4. Shrimp in the tank with refuge grew consistently better after 14 days, and final weight was 17% higher at the end of trial. This result, however. needs to be confirmed scientifically through replication. Final density achieved was 3.0 kg/m3 with water quality conditions remaining suitable for further increases. The trial was taken down early because of the scheduled sale of the crop.

Figure 2. Size class distribution of 324 harvested D119 (from PL10) L. vannamei raised in 2 x 50 m3 APRAS tanks with and without refuge at 3 ppt and 28oC at AF4. A normal bell-shaped curve for the entire population was achieved with 47% of shrimp falling within 2 g of the mean, and 15% higher than that.

 

Table 1. Water quality characteristics of the 100 m3 APRAS stocked with an initial 26,000 D42 (from PL10) L. vannamei for 77 days. (D.O. = dissolved oxygen). All water quality characteristics fell within acceptable ranges for this species.  Water mineral concentrations were considered minimum.

Temperature

27.2 – 30.1oC

pH

7.6 – 8.0

Salinity

2.5 – 3.5 ppt

D.O.

> 6.0 mg/l

TAN

< 0.5 mg/l

Nitrite

< 0.2 mg/l

Alkalinity

112.5 – 157.5 mg/l

Potassium

47 mg/l

Magnesium

30 mg/l

Sodium

850 mg/l

Sodium:

Potassium

30.5

 

 

Table 2. Aeromonas and Pseudomonas bacterial counts. Spikes of both species occurred on the second day of stocking shrimp and were correlated with spikes in total ammonia (TAN), nitrite, and total suspended solids (TSS) in tanks at the same time. Probiotics and addition of UV lights on the second day quickly reduced and kept both species at apparent ambient levels.

Day of Culture

Aeromonas (# of colonies/ml)

Pseudomonas (# of colonies/ml)

1

200

440

2

> 6,000

> 6,000

4

180

1200

7

140

1800

10

260

3400

16

200

1400

25

240

2000

32

180

1200

38

400

1400

46

420

960

52

300

800

59

380

1600

67

320

1200

72

420

1000

 

 

Figure 3Vibio (green and yellow colonies) population dynamics and use of probiotics. Probiotics were added at a rate of 1 mg/m3 of water. Probiotics were added weekly, three days in a row, after an initial start-up period, and when Vibro spikes were observed.

 

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