Final Report for SW05-065
Ion availability in inland, saline waters where shrimp are being produced was thought to be limiting production. We conducted tests to evaluate the strategies of supplementing diets and pond water with potassium (K) and with magnesium (Mg) to improve the growth and osmoregulation ability of Pacific white shrimp Litopenaeus vannamei in low salinity water. The effects of supplemented diets were examined in replicated greenhouse trials were conducted with shrimp acclimated to low salinities (4-5 ppt) in raceways. Tests examining the effect of adding K to the pond water were evaluated in replicated tests with shrimp held in tanks. Supplementing the diets with either K or Mg did not result in better shrimp growth. Similarly, there were no significant differences in the growth of shrimp related to K content of the water. The osmolarilty of shrimp hemolymph was not affected by the supplemented diets or water. Our data do not support the use of this strategy for increasing shrimp production in inland, saline waters.
The overall goal of the project is to increase the viability of inland shrimp farms in the Western SARE region by improving farm productivity. The project objectives are: 1) to determine the concentrations of K and Mg, both in the pond water and feed, needed to reduce osmoregulation stress, and related mortalities, to shrimp in low-salinity ponds; and 2) to develop pond management strategies based on these results.
A developing industry in the Western SARE region and elsewhere in the United States is the farming of marine shrimp in inland areas where saline groundwater is available. There are numerous advantages of inland shrimp aquaculture, and these are well known. Chief among these is that the farms are located far from coastal areas. Inland farms can be developed in areas where land costs are much lower and where the risk of transferring diseases between farmed and wild stocks is virtually eliminated. The saline groundwater used for shrimp culture is abundant in the arid west and previously has not been considered a resource, because it is generally not suitable for agriculture. However, the marine shrimp currently being farmed can grow at the salinities characteristics of these groundwater sources. This is possible since the shrimp can regulate the hemolymph ionic concentrations, and can be acclimated to low salinities.
Osmoregulation by penaeid shrimp is influenced by several factors including size, temperature, and condition. Osmoregulatory capacity has been suggested for use as means of monitoring stress in farmed shrimp populations. Conditions adverse to the ability of the shrimp to osmoregulate can result in reduced growth and increased mortality.
Although these marine shrimp can be grown in low salinities, the current farms in Arizona have had problems with mortalities, especially at times of added stress, such as during molting, and at higher temperatures. These problems occur even at salinities the shrimp are known to tolerate. Similar problems are known from inland shrimp farms in other parts of the US. The problems have been attributed to the ionic composition of the groundwaters differing considerably from that of dilute (low salinity) seawater.
Analyses of the ionic composition of saline groundwaters have shown the relative concentrations of individual cations vary considerably among farms and are quite different from those in seawater. Of particular concern is potassium (K+), which is known to be important in shrimp osmoregulation and which is proportionately very low in saline groundwater in comparison to seawater. Magnesium is also of interest. However, from previous research, potassium levels have been suggested as being critical to shrimp survival in low-salinity groundwaters. In some cases it may be the ratio of potassium to other cations in the water that is of importance, rather than simply the potassium concentrations. In particular, the ratio of potassium to sodium (Na+) may be important because these are exchanged between the shrimp, via the gills, and the environment in order to balance critical levels in the shrimp hemolymph. These exchanges of these ions at specialized sites in the gills are made physiological pumps that require metabolic energy expenditure, so shrimp must have adequate caloric intake to meet these needs. Two general approaches have been taken to increase the availability of potassium to shrimp grown in inland farms. One approach is to add additional potassium to the pond water. However, adding sea salt to inland pond water to achieve an ionic balance is expensive and, therefore, not practical for commercial farms. Although some producers have had success with adding potassium, in the form of potash, to the pond water, critical levels have not been established. Also, because many variables involved in shrimp farming can affect production and mortality, it is difficult to draw meaningful conclusions based on short-term experiences of various farms. In addition, solutions derived through trial and error on one farm may not work on other farms. We propose to address this issue through carefully designed replicated experiments at two commercial farms.
Another approach is to provide critical minerals in the diet. There has been some work in this area, but the results in practical application have been inconclusive. We are using rigorous statistical designs in experimental trials to determine if increased magnesium and potassium in the shrimp feed will improve growth and survival at commercial farms in Arizona. This entails a series of replicated experiments both at farm locations and in carefully controlled laboratory/greenhouse experiments.
We sampled well waters from three shrimp farm sites in western Arizona and analyzed samples with regard to salinity and ionic composition. Salinity of the samples was determined with a Markson conductivity meter calibrated with standard concentrations of NaCl. Water salinities used for rearing marine shrimp in Arizona ranged from 1.5 to approximately 8 ppt.
We conducted independent, growth trials with shrimp held in a recirculating system inside a greenhouse at the Environmental Research Laboratory of the University of Arizona. The system was comprised of two, 800-L, fiberglass raceways (Red Ewald Inc., Karnes city, TX, USA). Each (244 cm x 61 cm x 30 cm) raceway was divided into 3 equal sections with plastic mesh partitions held in place by frames made of PVC pipe or with similarly constructed mesh cages. The two raceways shared a common water supply from a reservoir (61 cm x 46 cm x 46 cm) supplied with a submersible pump, a gravel filter, a submersible heater, and aeration. Water was pumped from the reservoir into the end of one raceway. The flow went through a standpipe at the other end and into the second raceway. Then the flow went through a standpipe in the second raceway back into the reservoir. For both growth trials, the environmental conditions were the same, with water temperature held at 26 degrees C and salinity at 4-5 ppt. The low-salinity water for the Mg enriched diet trials was prepared with commercially available artificial sea salts (Marine Enterprises International Inc., Baltimore, MD, USA). For the potassium supplemented diet trials we transported well water from one of the commercial farms to the raceways
Shrimp were obtained as post-larvae or juveniles and maintained in the fiberglass tanks and acclimated slowly to salinities of approximately 5 ppt prior to being used in the growth trails. Shrimp were obtained either from the stock of Aquaculture Pathology Laboratory at the University of Arizona (Tucson, AZ, USA). Shrimp; or were provided by Miami-Aquaculture Inc. (Boynton Beach, FL, USA).
We used slightly different designs for two growth trials with each of two dietary supplements, either Mg or K. The designs were to compare the growth of the shrimp among three diets, which differed in mineral content. In all trials, shrimp in each raceway section were fed one of the diets, with all three diets represented in each raceway (i.e. the diet treatments in one raceway were replicated in the other). In the first trial with each diet type, we compared the specific growth rates of individual shrimp that varied in initial size. Specific growth rate was calculated as: [(lnWf -lnWi)100]/days, where Wf is the final weight and Wi is the initial weight. In the second trials with each supplement, we compared the final weights of shrimp that were uniform in size (0.7 to 0.9 g) at the start. At the termination of the second trial, samples of 5 individual shrimp, selected through the use of a random number table, from each raceway section were oven dried (67 C)to a constant weight and analyzed for mineral content.
The test diets were prepared from mash used to produce shrimp grow-out feed (35% protein) that was supplied by a commercial feed manufacturer (Rangen Inc., Buhl, ID, USA). The control diet was made from the commercial mash without alteration. Each of the other four diets was composed of the commercial mix but supplemented either with MgCl2 or KCl. The supplements were mixed with the mash and water was added at 15% of the weight of the dry mash. The moist diets were mixed for 15 minutes in an industrial mixer then pelletized with a pellet mill (California Pellet Mill Company, Crawfordville, IN, USA). The pellets were dried in an oven overnight and stored at 4 degrees C. Analysis of the diets showed the final Mg contents to be 0.26. 0.37, and 0.47 %, and the final K contents were 0.5, 1.0, and 1.5 %/. Samples of each diet were sent to an analytical laboratory (IAS Laboratories, Tempe, AZ, USA) for analysis of mineral content.
At the end of the trials samples of 5-10 shrimp from each group were collected, dried at 67 degrees C to constant weight and analyzed for cation content. Also, upon termination of the first set of growth trials (with Mg supplements), hemolymph samples were taken from shrimp from each group and the osmolarity determined with a vapor pressure osmometer (Wescor Inc. Logan, UT, USA), to see if the enriched diets improved shrimp osmoregulation.
Effect of K enriched water
We conducted two sets of growth trials with shrimp to determine the effect of increasing the K content of the water. These trials were conducted in 6, covered 120-L aquaria supplied with gravel filters, and aeration. Water temperature was maintained at 28 degrees C by means of heaters with temperature controllers. Light was maintained on a cycle, of 12 hours of light alternating with 12 hours of darkness, by a timer wired into to the room lights. The shrimp were fed ad libitum once or twice per daily. For each feeding, commercial feed was placed in circular trays and uneaten feed was removed after two hours. Juvenile shrimp of assorted sizes were individually marked shrimp in each tank. Mortalities were counted daily and dead shrimp were removed immediately from the tanks. The weight of each shrimp was determined at the beginning of the experiment and at the end of the three-week growth trials.
Data from the growth trials with shrimp of assorted sizes were analyzed with an analysis of covariance (ANCOVA) comparing the log of the specific growth rates of individual shrimp among diet treatments. The log of the individual shrimp initial mass was the covariant. Log-transformed data were used in the analysis in order to make the relation between the dependent variable and the covariant linear. In the growth trial with shrimp of uniform initial size, groups were compared with a one-way ANOVA, with final weight as the dependent variable and diet as the independent factor. ANVOVA was used to compare groups with regard to the content of Ca, K, Mg, Na, and P in whole body samples of individual shrimp. Survival among groups was compared with Multi-sample Survival Analysis. Statistical analyses were conducted either with the Systat 11 statistical programs (Systat Software, Inc., Richmond, CA, USA) or with Statistix 9 statistical programs (Analytical Software, Talahassee, FL, USA).
At the end of the 2006, two farms quit producing shrimp and switched to raising fish. The remaining farm (Desert Sweet Shrimp) is not conducive to conducting pond trials, because it has few employees and the farm manager is concerned about possible contamination. Therefore, our emphasis since then has focused on greenhouse and laboratory trials. For this we: (1) set up two recirculating systems in an environmentally controlled greenhouse at the Environmental Research Laboratory to use in diet studies, (2) established a wet lab with environmentally controlled aquaria for replicated growth and physiology trials, (3) constructed a post-larval, system for rearing shrimp for the experiments; and (4) refurbished a pellet mill and feed laboratory at the Environmental Research Laboratory of the University of Arizona, allowing us to produce small lots of pelletized shrimp feeds with added K and Mg. We then focused our experiments on evaluating the effects of supplementing commercial shrimp feed with K and Mg of the growth and osmoregulation of shrimp reared in low-salinity water in the laboratory.
Mg enriched diet
Results of the first growth trial showed that the specific growth rate of the individual shrimp was strongly influenced by the initial size; and when log transformed data were used, the relation was linear. ANCOVA based on the log-transformed data showed a significant effect of diet (F2,25 = 4.162, p = 0.028) on the specific growth rates. The mean growth rates, adjusted for the covariant, were slightly lower for the group fed the diet with the highest Mg content (approximately 2.2%/day) than the shrimp fed the other two diets (both approximately 2.5%/day). Therefore, the Mg supplement did not improve growth rates of the shrimp over those fed with the commercial feed.
Similar results were obtained in the second growth trial. There was a significant effect of diet on the growth of individual shrimp (F2,51 = 3.496, p = 0.038) with the best growth with the non-supplemented (control) diet and with the worst growth on the diet with the highest Mg content. If an apparent outlier is removed from the analysis, the F value increases to 4.5 and the p value decreases to 0.016, indicating a highly significant effect of Mg content on shrimp growth.
Our studies were designed to see if supplementing a commercial shrimp diet would be result in improved growth of shrimp cultured in low-salinity water. Under our culture conditions it appears that this strategy is not effective. It is known from studies on fish that the protein content of the diet can affect the growth response to Mg in the diet so some differences in shrimp growth to dietary mineral content might also be expected with diets differing in protein or other constituents. It is also possible that Mg supplements might have an effect on osmoregulation of larger shrimp or may prevent other problems such as cramped muscle syndrome. However, although the underlying physiological mechanisms remain unclear, our results suggest that supplementing commercial shrimp diets with Mg leads to somewhat reduced growth, not the effect that we were hoping for.
Effect of supplementing diets with K.
We conducted a similar replicated test in the raceway to examine at the effect of increased K on shrimp growth. We started with shrimp that were the same size. We found no significant differences in growth among the groups (F2,57=2.98 and P=0.5 9). One caveat is that the K ions added to the diet were added as KCl, which is water soluble. We examined the leaching of K from the diets under our experimental conditions. We found that at the end of a two-hour feeding period the differences in K content of the diets were substantially reduced. For example the K content of the control diet decreased from 0.82 to 0.1 percent over the two-hour feeding period and the high level diet decreased from 1.5 to 0.3 percent over the same period. Therefore, the shrimp may not have received much of a nutritional advantage from the increased K in the diet. Based on our results, supplementing commercial diets with KCl will not improve shrimp growth in well waters used for production in Arizona. It is possible that K in another, less soluble, form may have different results. However, we had similar results from growth trials where KCl was added to the water directly, indicating that in these well waters, the K levels do not limit growth.
Effect of supplementing well water with K
The results of the trials comparing shrimp growth among treatments of supplementing well water with K were the similar. There were no significant differences relative growth (log-transformed ) among treatments (F2,20=0.63, P=0.54). There were no significant differences in survival among treatments (Chi-Square=0.7, P=0.72), and no significant differences in hemolymph osmolarity among groups (F2,21=1.41, P=0.27). So, addition of K to the water did not improve the shrimp’s ability to osmoregulate.
These results suggest that levels of K in saline groundwaters of Arizona are suitable for sustaining the growth of marine shrimp, and that supplementation with K is not necessary. The levels of K that become limiting to shrimp growth and survival in inland culture are not known.
Accomplishments and Milestones
1) We conducted replicated, growth trials with commercial feeds supplemented with K and Mg;
2) We conducted similar growth trials testing the effect of adding KCl to pond water on shrimp growth and osmoregulation;
3) Three graduate students and two undergraduate students have received training by participating in this project;
4) Some of the results were presented at an international symposium on shrimp culture and have also been accepted for presentation at the next meeting of World Aquaculture Society;
5) Manuscripts are being prepared for submission to appropriate journals.
The results of these studies contribute to clarification of a major issue in the development of inland shrimp aquaculture. The results may stimulate additional research in this area. In addition, the results will bring attention to potential of using saline groundwater as a resource for inland aquaculture.
Education and Outreach
The results of the research here have been presented at an international shrimp aquaculture conference held in Mexico and have been accepted for presentation at the annual meeting of the World Aquaculture Society. A poster summarizing some of the work has been produced for these purposes. In addition, two manuscripts are being developed for submission to appropriate journals.
Education and Outreach Outcomes
Areas needing additional study
For inland farms to be economically sustainable, producers need to find suitable markets for the locally produced shrimp products and means to become competitive with imported products. There has been some concern for some producers in Arizona of increased mortalities of larger shrimp in low-salinity ponds. Although, problems with osmoregulation was a suspected cause, other factors need to be examined. For example, high temperatures in the late summers and other potential stress factors need to be examined. Mortalities seem to occur primarily during molting, so factors contributing to physiological stress or stimulating premature moulting need to be elucidated.