The effects of density and artificial substrate on intensive shrimp Litopenaeus vannamei nursery production

Recirculating aquaculture systems (RAS) can be installed indoors, allowing year-round production of tropical animals in nearly any climate. A nursery phase is commonly used in Litopenaeus vannamei production since it allows for enhanced biosecurity and better quantification of animals while reducing space requirements. However, it is unclear whether animal density and inclusion of artificial substrate may improve shrimp performance during the nursery phase. In this experiment, we compared shrimp production parameters in two stocking densities with or without the use of an artificial substrate by creating four treatments: low-density LD; 1500 PL/m⁻³, low-density with substrate LDS, high-density HD; 3000 PL/m⁻³), and high-density with substrate (HDS). The LDS and HDS treatments included 0.46-m² of high-density polyethylene 2.5-cm mesh as a substrate, which increased the tank surface area by 21 %. Each treatment was randomly assigned to four 160-l culture tanks, each with a biofilter. The shrimp had an initial weight of 4 mg and were grown for 50 days. The low-density treatments had significantly higher dissolved oxygen (DO) and pH than the high-density treatments (P ≤ 0.001). Specifically, LDS had the highest DO and pH followed by the LD, HD, and HDS treatments, respectively. High-density treatments had significantly higher NO₂-N levels than low-density treatments during week 2 of the experiment when an unusually high concentration of nitrite was observed. FCR was significantly lower in both low-density treatments than in high-density treatments. At harvest, the total biomass (kg m⁻³) was significantly higher in high-density treatments than in low-density treatments (P ≤ 0.001), and the HDS treatment had a significantly greater biomass output than HD. Producers should consider artificial substrate and higher densities during nursery production to maximize shrimp production; however, the effects on water quality should also be taken into account.     

Shrimp (Litopenaeus vannamei) production and stable isotope dynamics in clear‐water recirculating aquaculture systems versus biofloc systems

Closed recirculating aquaculture systems (RAS) offer advantages over traditional culture methods including enhanced biosecurity, the possibility of indoor, inland culture of marine species year‐round and potential marketing opportunities for fresh, never‐frozen seafood. Questions still remain regarding what type of aquaculture system may be best suited for the closed‐system culture of marine shrimp. In this study, shrimp (Litopenaeus vannamei) were grown in clear‐water RAS and in biofloc‐based systems. Comparisons were made between the system types with respect to water quality, shrimp production and stable isotope dynamics used to determine the biofloc contribution to shrimp nutrition. Ammonia and nitrite concentrations were higher, and shrimp survival was lower in the biofloc systems. Although stable isotope levels indicated that biofloc material may have contributed 28% of the carbon and 59% of the nitrogen in shrimp tissues, this did not correspond with improved shrimp production. Overall, the water column microbial communities in biofloc systems may be more difficult to manage than clear‐water RAS which have external filters to control water quality. Biofloc does seem to offer some nutritional contributions, but exactly how to take advantage of that and ensure improved production remains unclear.     

Replacement of fishmeal by vegetable meal mix in the diets of Litopenaeus vannamei reared in low‐salinity biofloc system: effect on digestive enzymatic activity

Biofloc (consortium of diverse microorganisms associated to suspending substrates) was developed from waste of shrimp Litopenaeus vannamei postlarvae culture under low salinity (5 g L^?1) to provide an additional nutritious biomass and reduce fishmeal inclusion in feeds in a 28‐day indoor shrimp nursery trial conducted in 15 experimental containers (250 L stocked at 600 org m^?3). Four experimental diets (isoproteic and isocaloric) containing different percentage of fishmeal: 0%, 10%, 20% and 30% substituted by vegetable meal mix (corn, sorghum and wheat) were formulated and elaborated. A control treatment consisted of a commercial feed. The main water quality parameters were monitored, and no significant differences were found among treatments. The growth and survival were similar among treatments. In general, digestive enzymatic activities showed differences being greater in the biofloc system compared with clear water. It was concluded that low‐salinity shrimp nursery could be successfully developed with minimum inclusion of fishmeal in feeds, without significant effect on production response. The adjustment of C : N ratio allowed the increase of microbial biomass in the bioflocs, which contributed to maintain good water quality, provide live food and enhance digestive enzymatic activity of cultured organisms.     

Dietary protein level and C/N ratio manipulation in zero‐exchange culture of Litopenaeus vannamei: Evaluation of inorganic nitrogen control,biofloc composition and shrimp performance

A 30‐day experiment was conducted to evaluate inorganic nitrogen control, biofloc composition and shrimp performance in zero‐exchange culture tanks for juvenile L. vannamei offered a 35% (P35) or 25% (P25) crude protein feed, each feed supplemented with additional carbohydrate to increase the C/N ratio to 20:1 (CN20) or 15:1 (CN15). Sucrose was used as a carbohydrate to manipulate the two C/N ratios based on the carbon and nitrogen content of both the feeds and sucrose. The four treatments were referred to as: P35 + CN20, P35 + CN15, P25 + CN20 and P25 + CN15. Each treatment consisted of four replicate tanks (125 L), each stocked with 28 shrimp (equivalent to 224 shrimp m^?3). Bioflocs formed and developed based on initial inoculation in all four treatments; and monitored water quality parameters were maintained within acceptable ranges for shrimp culture throughout the experiment. No significant effects (P > 0.05) of dietary protein level, C/N ratio or their interaction were observed on biofloc development (BFV, TSS and BFVI) and inorganic nitrogen (TAN, NO₂^?‐N and NO₃^?‐N) concentrations. At the end of the experiment, proximate analysis of the bioflocs collected from the four treatments showed crude protein levels of 21.3% ~ 32.1%, crude lipid levels of 1.6% ~ 2.8% and ash levels of 43.4% ~ 61.4%. Extracellular protease and amylase activities of the bioflocs were 9.9 ~ 14.4 U g^?1 TSS and 293.5 ~ 403.8 U g^?1 TSS respectively. Biofloc composition and enzyme activity were both affected by dietary protein level (P < 0.01) and C/N ratio (P < 0.05). Survival, per cent weight gain and protein efficiency ratio of shrimp were not affected (P > 0.05) by dietary protein level, C/N ratio or their interaction; however, the feed conversion ratios were significantly lower (P < 0.05) in treatments with high dietary protein (P35) compared with those in treatments with low dietary protein (P25). The results from this study demonstrate that dietary protein level and C/N ratio manipulation can have important implications for water quality, biofloc composition and shrimp performance in intensive, zero‐exchange biofloc‐based culture systems.     

Optimum dietary protein‐to‐energy ratio for juvenile whiteleg shrimp,Litopenaeus vannamei,reared in a biofloc system

An 8‐week feeding trial was conducted to evaluate the optimum dietary protein‐to‐energy (P/E) ratio in juvenile whiteleg shrimp, Litopenaeus vannamei. Six diets were formulated with two protein levels (30% and 35%) and three digestible energy levels (16, 17.5 and 19 kJ/g diet) at each protein level (₃₀P₁₆, ₃₀P_17.5, ₃₀P₁₉, ₃₅P₁₆, ₃₅P_17.5 and ₃₅P₁₉). Fifty shrimp averaging 0.97 ± 0.03g (Mean ± SD) were randomly distributed in biofloc tanks and fed one of the experimental diets. Weight gain (WG), specific growth rate (SGR) and feed efficiency (FE) of shrimp fed the ₃₅P_17.5 diet were significantly higher than those of shrimp fed ₃₀P₁₆, ₃₀P_17.5 and ₃₀P₁₉ diets (p < .05). Results for non‐specific immune responses showed that diet ₃₅P_17.5 caused higher lysozyme activity in shrimp comparing to lower protein diets. Thiobarbituric acid reactive substances (TBARS) of plasma were lower for shrimp fed on diets consisting of higher protein and energy levels than shrimp fed on the ₃₀P₁₉ diet. Analyses of digestive enzyme activities showed higher trypsin activities for shrimp fed on ₃₅P_17.5 and ₃₅P₁₉ diets comparing to ₃₀P₁₆ and ₃₀P_17.5 diets. Also, hepatopancreatic lipase activity of shrimp fed ₃₅P₁₆, ₃₅P_17.5 and ₃₅P₁₉ diets were significantly higher than those of shrimp fed the other diets. In conclusion, based on the results for growth performance, biochemical parameters, immune responses, oxidative stress and enzyme activities, 35% protein and 17.5 kJ/g digestible energy (₃₅P_17.5) could be considered as the optimum protein‐to‐energy ratio in the diet of juvenile whiteleg shrimp reared in a biofloc system.     

Effect of light limitation on the water quality,bacterial counts and performance of Litopenaeus vannamei postlarvae reared with biofloc at low salinity

The aim of this study was to evaluate the effect of light limitation on the water quality, bacterial counts and performance of Litopenaeus vannamei postlarvae reared with biofloc at low salinity (≈9 g L^?1). Two treatments were designed: T₁ = culture with natural sunlight and T₂ = culture in darkness. After 28 days, in both treatments, the final weight of shrimp was over 0.6 g with a specific growth rate over 7.4% d^?1, and a survival rate over 70%. In both treatments, Vibrio sp. concentration presented low values (culture with natural sunlight = 0.1 to 9.9 × 10² CFU mL^?1, culture in darkness = 0.4 to 11.7 × 10² CFU mL^?1) and Bacillus sp. had high values (culture with natural sunlight = 0.7 to 66.0 × 10⁴ CFU mL^?1, culture in darkness = 0.7 to 65.8 × 10⁴ CFU mL^?1). All water quality parameters remained within the ranges suitable for shrimp culture, except for alkalinity during the first stage of the study. Although in some sampling periods some significant differences were found in bacterial counts and water quality parameters, shrimp productive performance under culture with biofloc at low salinity was not affected significantly by light limitation.     

Effect of feeding frequency on growth and enzymatic activity of Litopenaeus vannamei during nursery phase in biofloc system

This study evaluated the zootechnical performance and enzymatic activity of Litopenaeus vannamei reared at different feeding frequencies during the nursery phase in biofloc system. The experiment consisted of four treatments, corresponding to the feeding frequencies of one, two, three and four times a day. Twelve‐day postlarvae (PL12) were stocked in 12 circular tanks at a density of 3,000/m² for 35 days. These tanks were connected to a recirculation system supplied by a matrix tank where biofloc management was carried out. Water quality remained within acceptable limits for the species over the experiment. Food frequencies had no influence on survival (88.5–92.7%) and feed conversion ratio (1.5–1.7), but the final mean weight (0.43–0.56 g) was significantly higher in shrimp fed three times a day. This fact is probably associated with amylase (14.58 U/mg) and trypsin (23.84 U/mg) activities, as well as the significant increase of chymotrypsin (11.74 U/mg) and lipase (1.27 U/mg) in shrimp of this treatment at the end of culture period. Feeding three times a day provided the highest enzymatic activity and the best zootechnical performance of L. vannamei during the nursery phase in biofloc system.     

An extender solution for the short‐term storage of Litopenaeus vannamei sperm to be used in artificial insemination

Some shrimp hatcheries use artificial insemination (AI) to improve the male to female ratio in their breeding populations. We describe a sperm extender solution, which allows the short‐term storage of diluted sperm in Litopenaeus vannamei, and its use in an artificial insemination process. We also evaluate its fertilization capacity. An AI experiment was designed using two, one, or half spermatophore segments. We tested four treatments involving three different male:female ratios: Natural mating (1:1), Regular and Regular diluted (1:2) and Half diluted (1:4). Data analysis revealed that the number of nauplii produced per mating was affected by treatment, with Regular (158 420) performing better than Half diluted (112 864) (P < 0.05), but with no differences between the latter and Regular diluted (130 340) (P > 0.05). A binomial variable named female success (FS) was defined as successful when the number of nauplii obtained per mate was ≥25 000. Analysis showed differences for FS across treatments (P < 0.001), but not between Regular (79.2%), the hatchery conventional AI technique and Half diluted (60.4%), maybe due to sample size. Since the number of nauplii per mate is crucial to consider AI successful, it is necessary to improve this AI technique before it can be used in the shrimp industry.     

Rearing of the Pacific white shrimp,Litopenaeus vannamei in a biofloc system: The effects of different food sources and salinity levels

The present study assessed the effects of different types of feeds and salinity levels on water quality, growth performance, survival rate and body composition of the Pacific white shrimp, Litopenaeus vannamei, juveniles in a biofloc system. Shrimp juveniles (2.56 ± 0.33 g) were cultured for 35 days in 300 L fibreglass tanks (water volume of 180 L) with a density of 1 g/L in six treatments. Three sources of feed (100% formulated feed, mixture of 66.6% formulated diet and 33.3% wet biofloc, and 100% wet biofloc) and two levels of salinity (10 and 32 ppt) were considered in two control groups and four biofloc treatments. Water quality parameters in the biofloc treatments were significantly better than control groups (p < .05). The highest increase in growth performance and survival rate were obtained in salinity of 32 ppt and mixed feed sources. Analysing the proximate composition of body shrimp indicates an increase in lipid and ash levels in biofloc treatments, which was more evident in the salinity of 32 ppt. In addition, the proximate analysis of shrimp body showed significant differences between biofloc treatments and control groups (p < .05). The highest FCR was found in the treatment with salinity level of 10 ppt and fed only with floc. Overall, it was found that the artificial diet supplemented with biofloc at the salinity of 32 showed better performance in the juvenile stage of Pacific white shrimp.     

Effect of no carbohydrate addition on water quality,growth performance and microbial community in water‐reusing biofloc systems for tilapia production under high‐density cultivation

A 56‐day experiment was conducted to investigate the effect of no carbohydrate addition applied to control water quality in water‐reusing biofloc systems for tilapia (GIFT Oreochromis niloticus) cultivation. Reusing water‐contained flocs was initially inoculated into six 300 L indoor tanks. Thirty fish (average individual weight 99.62 ± 7.34 g) were stocked in each tank. Glucose was extra added into three tanks (GLU‐tanks) according to biofloc technology, while other tanks were no carbohydrate added (NCA‐tanks). Concentrations of total ammonia nitrogen in GLU‐tanks and NCA‐tanks were fairly consistent and below 4.74 ± 0.35 mg/L. Nitrite concentrations in NCA‐tanks were significantly lower than GLU‐tanks, which were below 0.59 ± 0.10 mg/L during the later culture period. NCA‐tanks achieved a low relative abundance of denitrifiers and high concentrations of nitrate. Soluble reactive phosphorous in NCA‐tanks was consistently increased, which was decreased to a low level in GLU‐tanks. However, growth parameters in NCA‐tanks were similar to GLU‐tanks (p > .05) and reach a high finial density of 24.32 ± 1.04 kg/m³. Cetobacterium sp. was the first‐dominant bacterial genus in all tanks, which was a commonly indigenous bacterium in the intestinal tract of freshwater fish. The results demonstrate the feasibility of no carbohydrate addition in water‐reusing biofloc systems for tilapia.     

尼龙筛绢基质对对虾养殖水体细菌群落结构演替的影响

采用基于16S rDNA的PCR-DGGE（变性梯度凝胶电泳）技术,比较分析了添加尼龙筛绢网基质对凡纳滨对虾（Litopenaeus vannamei）精养系统中水体细菌群落结构演替的影响。结果表明：1）基质组水体菌群结构丰富度稍低于对照组;2）基质组水体菌群结构相似性始终高于对照组;随着时间的推移,基质组的相似性系数不断升高,而对照组出现了先升高后下降的波动;3）对照组水体养殖前期优势菌主要为变形菌门和未培养细菌,在养殖后期则以厚壁菌门芽孢杆菌属（Bacillus spp.）细菌为主,基质组水体菌群则在整个养殖周期中均以厚壁菌门芽孢杆菌属细菌和未培养细菌为主。该研究结果可为进一步阐明人工基质养殖系统中微生物的生态调控功能和作用机制提供参考。     

凡纳滨对虾不同养殖密度高位池水体细菌群落动态

通过对养殖水体环境基因组DNA中细菌16S rRNA基因V4–V5区的高通量测序和生物信息学分析,研究了两种养殖密度的凡纳滨对虾(Litopenaeus vannamei)高位池水体中细菌群落在养殖过程中的动态。结果显示,养殖过程中各菌群相对丰度变化明显,细菌多样性随时间逐渐提高,优势菌群为变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes)。随着养殖时间增长,蓝藻丰度所占比例逐渐减少,而变形菌、拟杆菌和浮霉菌丰度逐渐增大,同时养殖前期高密度池浮霉菌丰度显著高于低密度池(P0.01),而其他菌群无显著差异。结果表明,养殖期前50 d不同养殖密度水体细菌群落差异较大,而后30 d内细菌群落的时间异质性大于空间异质性,这意味着高位池水体菌相被划分为两类,到养殖后期菌相快速转变,养殖密度所带来的影响被减弱。     

Antioxidant effects of nanoencapsulated lipoic acid in tissues and on the immune condition in haemolymph of Pacific white shrimp Litopenaeus vannamei (Boone, 1931)

The antioxidant and oxidative damage responses and immune parameters in shrimp, Litopenaeus vannamei, reared in clear water (CW) and in a biofloc system (BFT) were evaluated. The animals were given feed supplemented with either nanocapsules containing the antioxidant lipoic acid (NCLA) or empty nanocapsules (NC). Antioxidant and oxidative stress analyses were evaluated by measuring glutathione S‐transferase activity (GST), reduced glutathione (GSH) concentration and lipid peroxidation (TBARS) in the gills, hepatopancreas and muscle. The immune parameters were then determined by the differential hemocyte count in the haemolymph. The final weight of the shrimp reared in both CW and BFT and treated with NCLA increased (p < .05). The shrimps reared in BFT showed increased GST activity in the gills and hepatopancreas after NCLA treatment (p < .05). Higher GSH levels were observed in the hepatopancreas with NCLA supplementation in CW and BFT (p < .05). Decreased levels of TBARS were observed in the gills and muscles of shrimp supplemented with NCLA and maintained in CW (p < .05). A decrease in the percentage of hyaline hemocytes and an increase in granular hemocytes were observed in shrimp treated with NCLA in CW and BFT (p < .05). The antioxidant treatment induced positive responses in terms of the zootechnical, biochemical and immunological parameters.     

Nutritional Contribution of Biofloc within the Diet of Growout and Broodstock of Litopenaeus vannamei,Determined by Stable Isotopes and Fatty Acids

The relative contributions of feed sources were determined through the isotopic signal (δ¹³C and δ¹⁵N) and fatty acid profile of feed items, shrimp muscle, and eggs of Litopenaeus vannamei reared in a biofloc system. In the growout phase, the isotope analysis showed the biofloc particle size class ≥250 μm contributed 55–100%; size ≥50 < 250 μm contributed 0–22%; and artificial feed contributed 0–45%. Principal component analysis applied to fatty acid profiles showed that biofloc ≥250 μm and artificial feed were the most important items in shrimp growout. For the egg production, isotope analysis suggested that the most important feed sources according to their relative contributions were polychaetes (0–100%), followed by artemia biomass (0–86%) and semi‐moist feed (0–66%), with lower contributions from squid, mussel, and the muscle of L. vannamei broodstock that had been cultured in biofloc. In terms of fatty acids, the most important items were artemia, polychaetes, and semi‐moist feeds. This work clarified the importance of feed sources for shrimp during culture in biofloc systems and during reproduction. Analysis of stable isotopes and fatty acids can be successfully used to trace the assimilation of nutrients during the nutrition of shrimp.     

Effect of salinity on natural community and production of Litopenaeus vannamei (Boone), within experimental zero-water exchange culture systems

Recent efforts have been made to culture marine shrimp in systems operating under low or zero‐water exchange and with decreased water salinity. The aim of this study was to investigate the impact of various salinity levels on qualitative and quantitative characteristics of the natural community and, more particularly, ciliated protozoa, and compare this information with shrimp growth and survival. Tanks with 9‰ salinity were characterized by a higher pH, but also by a significantly higher concentration of chlorophyll a (Chl a) per weight of suspended matter (1.93 ± 0.72 µg Chl a/mg TSS) than tanks with 18‰ (1.29 ± 0.68 µg Chl a/mg TSS) or 36‰ (1.37 ± 0.61 µg Chl a/mg TSS) salinity. Concentrations of ciliates (max 6000 cells mL^?1) showed considerable fluctuations over the sampling period, reflecting the impact of water salinity, dynamic interactions between ciliates and their diverse roles within the shrimp production system. There was no significant difference between survival rates of shrimp reared at 9‰, 18‰ or 36‰, but decreasing salinity from 36‰ to 9‰ led to a significant decrease in final shrimp body weight (from 13.40 ± 0.26 g to 10.23 ± 2.72 g). Future work should address the potential of ciliates as an indicator of aquaculture water quality, as is currently being done in the wastewater industry, and the contribution of ciliates as food sources.     

Water quality,production parameters and nutritional condition of Litopenaeus vannamei (Boone, 1931) grown intensively in zero water exchange mesocosms with artificial substrates

The effect of artificial substrates on the water quality, production parameters and nutritional condition was assessed in experimental intensive cultures of Litopenaeus vannamei grown in mesocosm units with zero water exchange. The initial stocking densities in triplicate 1000 L tanks were 600 and 800 g of juvenile shrimp (2.7 g) per unit, with (W) and without (WO) artificial substrates. There were no significant differences between the water quality parameters of the four treatments. In the tanks with the lower stocking biomass, the best survival (96.7% vs. 87.0%), growth rate (1.69 vs. 1.35 g week⁻¹), final biomass (1969.6 vs. 1516.0 g m³) and feed conversion ratio (1.30 vs. 1.90) were obtained with artificial substrates. Similar results were observed for the higher stocking biomass (90.9% vs. 74.5%, 1.50 vs. 1.13 g week⁻¹, 2221.6 vs. 1560.8 g m³, and 1.50 vs. 2.60 respectively). The nutritional condition of shrimp was improved using artificial substrates. The protein content in muscle was higher (21.6% and 20.4%) in ponds with substrates as compared with the control (21.2% and 15.9%).     

Butyrate and propionate improve the growth performance of Litopenaeus vannamei

Organic salts may improvement the animal performance, increasing the efficiency of nutrient utilization and modifying the intestinal microbiota. This study aimed to evaluate the effects of sodium butyrate and sodium propionate supplementation at different levels of dietary inclusions in the growth of Litopenaeus vannamei. In total, seven diets were evaluated: a control diet (without supplementation) and three diets from each sodium salt, propionate and butyrate, in concentrations of 0.5%, 1% and 2%. We used 21 tanks of 6000 L stocked with 150 shrimps (2.53 ± 0.03 g). The shrimps fed diets supplemented with propionate and butyrate, in all concentrations, increased their final weight. The feed efficiency, nitrogen retention, protein efficiency rate, survival and yield of shrimps fed the diet containing 2% butyrate were higher in comparison with control treatment. The shrimps supplemented with butyrate also showed lower counts of Vibrio sp. in the intestine. The shrimps fed the diet supplemented with butyrate and propionate also showed higher values of serum agglutination titre. Thus, it is possible to conclude that dietary supplementation with propionate and butyrate in different dietary concentrations modify the intestinal microbiota and improves the growth of L. vannamei.     

The effects of artificial substrate and stocking density on Pacific white shrimp (Litopenaeus vannamei) performance and water quality dynamics in high tunnel-based biofloc systems

The use of artificial substrates in shrimp aquaculture may allow for production of shrimp at increased densities while providing a growth medium for microbes that assist with water quality processes and provide supplemental nutrition for shrimp. Greenhouse-based shrimp production systems can extend the shrimp production season in temperate climates while conserving water and energy. For this study, we evaluated the effects of providing extra substrate and shrimp density on water quality and shrimp production in greenhouse-based biofloc systems. Four 11-m³, wood framed, and rubber-lined tanks were constructed in each of four high tunnel greenhouses (for a total of 16 tanks). Four treatments were evaluated: high-density stocking with substrate (HDS), high-density stocking with no substrate (HDNS), low-density stocking with substrate (LDS), and low-density stocking with no substrate (LDNS). Each treatment was randomly assigned to one tank in each tunnel to block for location. No artificial heat was used, and shrimp were grown for 120 days. High-density systems were stocked at 200 shrimp/m³ while low-density tanks had 100 shrimp/m³. Adding substrate increased total in-tank surface area by 13.4%. The addition of substrate had no significant effect on any shrimp production or standard water quality parameters. Shrimp had significantly greater final weight, faster growth rate, and lower feed conversion rate in low-density treatments (P ≤ 0.02 for all). Total shrimp biomass production was significantly higher in high-density treatments (HD: 4.0 kg/m³; LD: 2.3 kg/m³; P < 0.05). There were no significant differences in survival between densities (HD: 91.3%; LD: 94.5%; P = 0.43). Peak and overall mean nitrite levels were significantly higher in high-density treatments compared to low-density treatments. Dissolved oxygen levels and pH over the course of the study were significantly lower in high-density treatments, likely due to increased respiration rates in the water column. This project shows the feasibility of shrimp production in temperate climates with no artificial heat using high tunnel greenhouses, few impacts of added substrate on shrimp production, and increased shrimp density can result in much larger harvests with few negative impacts on production metrics.