Prediction of apparent protein digestibility of ingredients and diets by in vitro pH-stat degree of protein hydrolysis with species-specific enzymes for juvenile Pacific white shrimp Litopenaeus vannamei

Aquafeed production faces global issues related to availability of feed ingredients. Feed manufacturers require greater flexibility in order to develop nutritional and cost-effective formulations that take into account nutrient content and availability of ingredients. The search for appropriate ingredients requires detailed screening of their potential nutritional value and variability at the industrial level. In vitro digestion of feedstuffs by enzymes extracted from the target species has been correlated with apparent protein digestibility (APD) in fish and shrimp species. The present study verified the relationship between APD and in vitro degree of protein hydrolysis (DH) with Litopenaeus vannamei hepatopancreas enzymes in several different ingredients (n = 26): blood meals, casein, corn gluten meal, crab meal, distiller's dried grains with solubles, feather meal, fish meals, gelatin, krill meals, poultry by-product meal, soybean meals, squid meals and wheat gluten. The relationship between APD and DH was further verified in diets formulated with these ingredients at 30% inclusion into a reference diet. APD was determined in vivo (30.1 ± 0.5 °C, 32.2 ± 0.4‰) with juvenile L. vannamei (9 to 12 g) after placement of test ingredients into a reference diet (35 g kg^− 1 CP; 8.03 g kg^− 1 lipid; 2.01 kcal g^− 1) with chromic oxide as the inert marker. In vitro DH was assessed in ingredients and diets with standardized hepatopancreas enzymes extracted from pond-reared shrimp. The DH of ingredients was determined under different assay conditions to check for the most suitable in vitro protocol for APD prediction: different batches of enzyme extracts (HPf5 or HPf6), temperatures (25 or 30 °C) and enzyme activity (azocasein): crude protein ratios (4 U: 80 mg CP or 4 U: 40 mg CP). DH was not affected by ingredient proximate composition. APD was significantly correlated to DH in regressions considering either ingredients or diets. The relationships between APD and DH of the ingredients could be suitably adjusted to a Rational Function (y = (a + bx)/(1 + cx + dx²), n = 26. Best in vitro APD predictions were obtained at 25 °C, 4 U: 80 mg CP both for ingredients (R² = 0.86; P = 0.001) and test diets (R² = 0.96; P = 0.007). The regression model including all 26 ingredients generated higher prediction residuals (i.e., predicted APD − determined APD) for corn gluten meal, feather meal, poultry by-product meal and krill flour. The remaining test ingredients presented mean prediction residuals of 3.5 points. A model including only ingredients with APD > 80% showed higher prediction precision (R² = 0.98; P = 0.000004; n = 20) with average residual of 1.8 points. Predictive models including only ingredients from the same origin (e.g., marine-based, R² = 0.98; P = 0.033) also displayed low residuals. Since in vitro techniques have been usually validated through regressions against in vivo APD, the DH predictive capacity may depend on the consistency of the in vivo methodology. Regressions between APD and DH suggested a close relationship between peptide bond breakage by hepatopancreas digestive proteases and the apparent nitrogen assimilation in shrimp, and this may be a useful tool to provide rapid nutritional information.     

In vivo and in vitro protein digestibility in juvenile bagrid catfish Mystus nemurus (Cuvier and Valenciennes 1840) fed soybean meal‐based diets

In vivo and in vitro protein digestibility of 0% to 60% soybean meal protein (SBM) substitution from fishmeal protein was conducted for bagrid catfish Mystus nemurus juveniles. Seven experimental diets containing 35% protein and 15% lipid were fed to bagrid catfish to determine the in vivo protein digestibility. In vitro methods were determined using pH stat, pH shift, spectrophotometric assay and sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) with different enzyme mixtures (crude intestinal extract of bagrid catfish, Lazo 1‐enzyme, Hsu 3‐enzyme, Saterlee 4‐enzyme). In vivo and in vitro protein digestibility decreased with increased SBM protein substitution with highest tolerance level of 10% (in vivo). pH stat and pH drop methods showed the highest degree of hydrolysis (DH) and relative protein digestibility (RPD) using Saterlee 4‐enzyme system. However, pH stat method showed highest correlations (r² = 0.9263) with the in vivo results using crude intestinal enzyme extract compared to other enzyme systems. The highest correlation of the in vitro methods using crude intestinal enzyme extracts of bagrid catfish was determined using spectrophotometric assay (r² = 0.9284) followed by pH stat (r² = 0.9263), SDS‐PAGE (r² = 0.8348) and pH drop (r² = 0.6777). All the in vitro methods tested are suitable to rapidly determine protein digestibility for bagrid catfish except for pH drop.     

Prediction of culture performance of juvenile Litopenaeus vannamei by in vitro (pH-stat) degree of feed protein hydrolysis with species-specific enzymes

Rapid in vitro methods for measuring digestibility may be useful in analysing aqua feeds if the extent and limits of their application are clearly defined. The pH‐stat protein digestibility routine with shrimp hepatopancreas enzymes was previously related to apparent protein digestibility with juvenile Litopenaeus vannamei fed diets containing different protein ingredients. The potential of the method to predict culture performance of shrimp fed six commercial feeds (T3, T4, T5, T6, T7 and T8) with 350 g kg^?1 declared crude‐protein content was assessed. The consistency of results obtained using hepatopancreas enzyme extracts from either pond or clear water‐raised shrimp was further verified in terms of reproducibility and possible diet history effects upon in vitro outputs. Shrimps were previously acclimated and then maintained over 56 days (initial mean weight 3.28 g) on each diet in 500‐L tanks at 114 ind m^?2, clear water closed system with continuous renewal and mechanical filtering (50 μm), with four replicates per treatment. Feeds were offered four times daily (six days a week) delivered in trays at feeding rates ranging from 4.0% to 7.0% of stocked shrimp biomass. Feed was accessible to shrimp 4 h daily for 1‐h feeding period after which uneaten feed was recovered. Growth and survival were determined every 14 days from a sample of 16 individuals per tank. Water quality was monitored daily (pH, temperature and salinity) and managed by water back flushing filter cleaning every 7–10 days. Feeds were analysed for crude protein, gross energy, amino acids and pepsin digestibility. In vitro pH‐stat degree of protein hydrolysis (DH%) was determined for each feed using hepatopancreas enzyme extracts from experimental (clear water) or pond‐raised shrimp. Feeds resulted in significant differences in shrimp performance (P < 0.05) as seen by the differences in growth rates (0.56–0.98 g week^?1), final weight and feed conversion ratio (FCR). Shrimp performance and in vitro DH% with pond‐raised shrimp enzymes showed significant correlation (P < 0.05) for yield (R² = 0.72), growth rates (R² = 0.72–0.80) and FCR (R² = ?0.67). Other feed attributes (protein : energy ratio, amino acids, true protein, non‐protein nitrogen contents and in vitro pepsin digestibility) showed none or limited correlation with shrimp culture performance. Additional correlations were found between growth rates and methionine (R² = 0.73), FCR and histidine (R² = ?0.60), and DH% and methionine or methionine+cystine feed contents (R² = 0.67–0.92). pH‐stat assays with shrimp enzymes generated reproducible DH% results with either pond (CV ≤ 6.5%) or clear water (CV ≤ 8.5%) hepatopancreas enzyme sources. Moreover, correlations between shrimp growth rates and feed DH% were significant regardless of the enzyme origin (pond or clear water‐raised shrimp) and showed consistent R² values. Results suggest the feasibility of using standardized hepatopancreas enzyme extracts for in vitro protein digestibility.     

Evaluation of Three In Vitro Enzyme Assays for Estimating Protein Digestibility in the Pacific White Shrimp Penaeus vannamei1

Abstract— In vitro enzyme assays are rapid, inexpensive techniques for estimating protein digestibility of feed ingredients. Three assays—the Lazo single-enzyme assay with porcine trypsin; the Hsu multi-enzyme assay with porcine trypsin, α-chymotrypsin, and peptidase; and the Satterlee multi-enzyme assay with porcine trypsin, α-chymotrypsin, peptidase, and bacterial protease—were used to estimate relative protein digestibility (RPD) of selected feed ingredients used in diets for the Pacific white shrimp Penaeus vannamei. Ingredients tested were casein, gelatin, rice bran, shrimp meal, soybean meal, wheat gluten, and six varieties of fish meal. A highly significant, inverse, linear relationship existed between final pH in each of the enzyme assays and in vivo apparent protein digestibility of the ingredient in P. vannamei feeding trials. Ranking of RPD coefficients obtained in the Lazo single-enzyme assay followed closely the ranking of published in vivo apparent protein digestibility coefficients for the same ingredients in P. vannamei digestibility trials. Results indicated that a single-enzyme assay with porcine trypsin identified differences in protein digestibility of feedstuffs in vitro as well as the two multi-enzyme assays tested. The porcine-trypsin, single-enzyme assay appears to be a potentially useful tool for evaluating protein quality and relative protein digestibility of feedstuffs for P. vannamei.     

Apparent and true availability of amino acids from common feed ingredients for South African abalone (Haliotis midae L.)

Apparent (APD) and true (TPD) protein digestibility and apparent (AAAA) and true (TAAA) amino acid availability of fish meal and plant protein ingredients utilized in the South African animal feed industry (corn gluten meal, soya bean meal, cottonseed meal, sunflower meal, canola meal, peanut meal, lupins and faba beans), incorporated at 450 g kg^?1 into single protein diets, were determined with juvenile South African abalone (Haliotis midae). Soya bean meal (96.34%) and lupins (96.51%) presented the highest APD values, with corn gluten meal (76.08%) the lowest value, while values for sunflower meal (92.21%), canola meal (93.94%) and faba beans (93.17%) were above 90%. APD values for cottonseed meal (86.28%) and peanut meal (86.73%) were comparable with that of fish meal (82.94%). A correlation coefficient (r) of 0.99 was found between ADP and mean AAAA. Furthermore, amino acid availabilities were relatively consistent within various feed ingredients. The mean amino acid secretion from a protein‐free diet was 19.08 mg 100 g^?1 dry diet, resulting in a mean increase of 1.88% units over all feed ingredients when comparing TAAA with AAAA. Calculated APD according to APD coefficients determined with single protein diets underestimated determined APD in several compound diets by 1.10–6.50%, while mean AAAA was underestimated by 6.93%.     

Gluconeogenesis in trout (Oncorhynchus mykiss) white muscle: purification and characterization of fructose-1,6-bisphosphatase activity in vitro

Studies of the enzyme fructose-1,6-bisphosphatase (FBPase) of rainbow trout (Oncorhynchus mykiss) have been undertaken in order to illuminate aspects of skeletal muscle gluconeogenesis in these animals. Maximal activities in crude homogenates of several organs suggest that the liver possesses the greatest FBPase activity on a unit g^–1 tissue basis but that the white muscle, owing to its bulk, contributes substantially to whole body FBPase activity. Studies of fructose-6-phosphate-1-kinase (PFK) and FBPase in crude homogenates of several organs suggests an important role for intracellular pH in regulating the relative carbon flux through the FBPase/PFK locus in vivo. Furthermore, a three-step purification scheme is described for trout white muscle FBPase by which a stable and homogeneous (by SDS PAGE) enzyme preparation (isoelectric point = 7.2; molecular weight = 37.6 kd) was obtained. Kinetic studies of the purified enzyme were undertaken at 20°C under conditions reflective of "rest" and "exercise/recovery" intramuscular pH in vivo. Affinity for substrate (F-1,6-P₂) was increased (Km = 6.88 versus 2.44 mol 1-^–1 as was enzyme activity when pH was lowered from 7.0 to 6.5. Various inhibitor metabolites are identified including F-2,6-P₂ (mixed-type inhibitor, K_i = 0.201 mol 1^–1, pH 7.0) and AMP (non-competitive inhibitor, K_i = 0.438 mol 1^–1, pH 7.0). Inhibition by F-2,6-P₂ was strongly alleviated by a reduction in pH from 7.0 to 6.5 (I₅₀ increased from 0.14 to 0.32 mol 1^–1). AMP on the other hand was a more potent inhibitor at pH 6.5 but this inhibition was totally reversed under conditions of citrate, NH₄ ⁺ and AMP typical of muscle during recovery from exercise in vivo. In purified white muscle enzyme preparations, FBPase demonstrated maximal activity at pH 6.5 whereas the optimal pH of PFK was 7.0 or greater. Indeed, it appears from these in vitro data that regulation by metabolite levels as well as pH are required for net FBPase flux in vivo. It is concluded, therefore that trout white muscle FBPase demonstrates the potential to play an important enzymatic role in the control of intramuscular gluconeogenesis in these animals. The results are discussed in relation to present knowledge regarding the metabolic responses of trout white muscle to, and its subsequent recovery from, exhaustive exercise.     

Experimental Studies to Evaluate Larval Survival of the Fire Shrimp, Lysmata debelius, to the Juvenile Stage

Many coral reefs are threatened because of anthropogenic impacts such as destructive fishing methods for marine ornamentals. The marine ornamental industry is currently almost completely dependent upon wild collections. The development of commercially feasible methods to culture ornamental species could help to reduce the need for wild‐collected specimens and reduce pressure on coral reefs. Advances in larval rearing and nutrition would help overcome the bottlenecks that impede the commercial production of many marine ornamental species. This article focuses on research for improving the production of the fire shrimp, Lysmata debelius. Experiments were performed with larval fire shrimp to evaluate (1) the importance of essential fatty acid enrichment of live prey (rotifers and Artemia) and (2) feeding penaeid shrimp muscle tissue to improve survival to metamorphosis. We also provide the fatty acid composition of Day‐1 posthatch fire shrimp larvae. The enrichment of live prey with docosahexaenoic acid did not improve larval growth, survival, or time to metamorphosis, but larvae fed live prey together with pureed penaeid shrimp muscle had survival rates of 9.8 ± 1.2%. The data and protocols developed in this study provide a foundation and baseline for future fire shrimp larval studies.     

Effect of proteolytic enzyme addition to a practical feed on growth of the Pacific white shrimp, Litopenaeus vannamei (Boone)

Four commercially available proteolytic enzymes active over a wide pH range were assessed as shrimp feed additives. Feeds were formulated to contain 1% of the test enzymes by weight. A feed of the same composition with no enzyme addition served as a control. In vitro feed protein digestibility of the feeds was determined using a modified pepsin digestibility method. In vitro protein digestibility results indicated that commercially available proteases added to a shrimp feed formulation and then hot pelleted/dried did retain enzyme activity. Feeds exhibited low weight losses through leaching (≤ 10.9%) and low or no losses of protein content (≤ 2.7%), indicating that the test feeds were available in their entirety to the shrimp. An 8-week feeding trial using the enzyme-supplemented feeds was conducted to evaluate shrimp growth. Mean shrimp survival was high for all treatments (> 90%). One-way analysis of variance did not detect significant differences (P > 0.05) among feeds in terms of survival, weight gain, specific growth rates and feed–weight gain ratios. Although in vitro digestibility indicated the presence of active enzymes in the feed, the feeding trial revealed that shrimp growth was not enhanced by the inclusion of proteases in these feeds. This study provides sufficient proof to emphasize the importance of in vivo investigations before enzyme-supplemented feeds are used with shrimp.     

CONSUMPTION OF FROZEN AND LIVE Artemia BY PROTOZOEA OF Penaeus setiferus

The role of an animal component in the diet of protozoeal penaeid shrimp larvae is not known. Short-term experiments were performed on the larvae of Penaeus setiferus (L.) to determine the ability of each protozoeal substage to consume frozen and live Artemia nauplii. Significant consumption of frozen Artemia took place during the protozoea 2 and 3 substages at rates of 1.0 and 1.6 Artemia nauplii/penaeid larva/hour respectively. Consumption of live Artemia occurred in the protozoea 3 substage, at a rate of 0.7 Artemia nauplii/penaeid larva/hour. Data suggesting that there is a beneficial role of an animal component in the diet of protozoeal penaeid shrimp larvae are discussed.     

Biosecurity and Genetic Improvement of Penaeid Shrimp: Applications to the Marine Ornamental Industry

The marine ornamental industry relies almost exclusively on wild-caught fish, invertebrates, and live rock to meet the market demands of aquarium hobbyists. For the industry to expand, appropriate technologies are needed to mitigate disease problems and reduce the dependence on wild stocks. Biosecurity protocols and techniques for genetic improvement have recently been applied to the culture of penaeid shrimp for human consumption, and these approaches may be applicable to an emerging marine ornamental industry. Biosecure production systems for penaeid shrimp are being developed in response to disease problems and growing concerns about environmental pollution from pond effluent. Biosecure systems rely on minimal water exchange and provide shrimp farmers with an opportunity to move production away from the coastline. In addition to the development of biosecure production systems, researchers at the Oceanic Institute have established a selective breeding program for the Pacific white shrimp, Litopenaeus vannamei. To date, significant improvements in shrimp growth and disease resistance have been made through selective breeding. It is likely that advances in biosecure technologies and genetic improvement will be applicable to the culture of marine ornamental shellfish and finfish and will contribute to an economically viable and environmentally sustainable industry.     

The efficacy of ingredients included in shrimp feeds to stimulate intake

The inclusion in feed formulations of ingredients that act as attractants and feeding incitants or stimulants has been proposed as a means of increasing feed consumption, and hence growth, of farmed shrimp. Squid, crustacean and krill meals, fish and krill hydrolysates and a betaine product (Finnstim) were examined to assess their relative effectiveness in increasing the feed intake of black tiger shrimp Penaeus monodon. These presumed feeding effectors were added to a base feed at between 5 and 50 g kg^?1. Given a choice between the base feed and one containing one of the test ingredients, P. monodon showed a significantly greater preference for the feeds containing crustacean or krill meal. Four of the presumed feeding effectors were further evaluated in a growth response experiment. There was no significant difference in the amount the shrimp consumed of any of the feeds. However, the growth rate of the shrimp was about 20% faster on the feeds containing crustacean meal or krill meal. Although ingredients such as crustacean meal or krill meal can improve feed intake, in practical feed formulations that contain significant amounts of terrestrial protein but relatively low levels of marine ingredients, there does not appear to be need for additional feeding effectors. However, crustacean meal and krill meal do provide a significant nutritional benefit, as seen by the improved growth rates in this study.     

Stringed bed suspended bioreactors (SBSBR) for in situ nitrification in penaeid and non-penaeid hatchery systems

For establishing nitrification in prawn (non-penaeid, salinity 10–15 ppt) and shrimp (penaeid, salinity 30–35 ppt) larval production systems, a stringed bed suspended bioreactor (SBSBR) was designed, fabricated, and validated. It was fabricated with 5 mm polystyrene and low density polyethylene beads as the substrata for ammonia and nitrite oxidizing bacterial consortia, respectively, with an overall surface area of 684 cm². The reactors were activated in a prototype activator and were transported in polythene bags to the site of testing. Performance of the reactors activated with the nitrifying bacterial consortia AMONPCU-1 (ammonia oxidizers for non-penaeid culture) and NIONPCU-1 (nitrite oxidizers for non-penaeid culture) was evaluated in a Macrobrachium rosenbergii larval rearing system and those activated with AMOPCU-1 (ammonia oxidizers for penaeid culture) and NIOPCU-1 (nitrite oxidizers for penaeid culture) in a Penaeus monodon seed production system. Rapid setting up of nitrification could be observed in both the static systems which resulted in a higher relative per cent survival of larvae.     

Nutritional status of a nereidid polychaete cultured in sand filters of mariculture wastewater

This study examined the nutritional composition of the intertidal marine polychaete Perinereis helleri (Nereididae) when artificially cultured in sand filters treating mariculture wastewater. Moisture levels in harvested P. helleri ranged from 758 to 855 g kg^?1, and ash, from 23 to 61 g kg^?1 wet matter (WM). Stocking density and graded size after harvest significantly affected their composition. Higher total lipid contents were found in large (>0.6 g) P. helleri (16–19 g kg^?1 WM) and those grown at the lowest density (1000 m^?2: 18 g kg^?1 WM) than in small (≤0.6 g) ones (14 g kg^?1 WM) and those grown at the highest densities (4000–6000 m^?2: 13–16 g kg^?1 WM). Several fatty acids within a very broad profile (some 30 identified) reflected this pattern, yet their ARA/EPA/DHA ratios were relatively unaffected. Feeding the polychaete‐assisted sand filters (PASF) with fish meal to increase worm biomass productivity significantly increased their DHA content. Other components (e.g. protein, phospholipids, cholesterol, carbohydrate, amino acids, nitrogen, minerals and bromophenols) and nutritional factors (e.g. maturity, feeding seaweed and endemic shrimp viral content) were also investigated. Results suggest that PASF‐produced P. helleri have a well‐balanced nutritional profile for penaeid shrimp and fish broodstock.     

Prediction of digestible protein and lipid contents of crustacean feeds

Due to distinctive feeding habits and digestive physiology determination of nutrient digestibility is more demanding with crustacean than fish species. A study was conducted to validate the use of linear prediction equations established with fish species to predict apparent digestible protein (ADP) and lipid (DL) contents [g kg^?1 dry matter (DM)] of feed ingredients (ADP = ?10.0731 + 0.8942 CP, DL = ?1.5824 + 0.8654 CL) and compound diets (ADP = ?51.4001 + 0.9872 CP, DL = ?2.7303 + 0.9123 CL) from dietary crude protein (CP) and lipid (CL) contents for crustaceans (shrimp, lobster, crab). Observed values (n = 91) obtained in 17 studies, which evaluated CP digestibility of 26 feed ingredients for eight crustacean species, presented a linear relationship (R² = 0.9213, RMSE = 54.1245) with predicted values. Predicted values overestimated observed values with a mean prediction error (MPE) of 0.1315. However, observed DL values of 17 feed ingredients (n = 31; studies = 5; species = 5) were overestimated with a MPE of 0.4500. Similar trends than above were found with prediction of the ADP (n = 185; studies = 32; species = 11; R² = 0.8047; MPE = 0.1002) and DL (n = 64; studies = 11; species = 3; R² = 0.6907; MPE = 0.2372) contents of compound diets.     

Comparative study of the intracellular superoxide anion production in different penaeid species through the NBT‐reduction assay

The capacity of reactive oxygen intermediates production upon haemocyte stimulation is one of the most important immunoparameter utilized to assess the health status in cultivated shrimps. In the present study, we compared oxidative stress potential, by measuring the superoxide anion production in three penaeid shrimps: two wild Atlantic species, the pink shrimp Farfantepenaeus paulensis and the white shrimp Litopenaeus schmitti and one cultivated Pacific species, the white shrimp, Litopenaeus vannamei, through the nitro‐blue‐tetrazolium‐reduction assay. We also proposed an optimized experimental protocol for this assay, that produces rapid and consistent results with low levels of basal superoxide anion (O₂^?) production by unstimulated haemocytes and high levels of this oxygen radical after cell stimulation. Among the different cell elicitors used (zymosan, laminarin, lipopolysaccharide and phorbol myristate acetate), laminarin (β‐1,3‐glucans – 2 mg mL^?1) was the most potent cell activator for the haemocytes of all three penaeids and we recommend this immunostimulant to routinely evaluate shrimp respiratory burst. In general terms, the most elevated levels of O₂^? production, after cell stimulation with microbial components, were detected in L. schmitti. Interestingly, the stimulation profile of the haemocytes of L. vannamei was more similar to F. paulensis, than to L. schmitti, which is more phylogenetically related.     

Evaluation of Tilapia Effluent with Ion Supplementation for Marine Shrimp Production in a Recirculating Aquaculture System

Reuse of fish effluent for the culture of marine shrimp, such as Pacific white shrimp, Litopenaeus vannamei, could provide an opportunity for the US shrimp farming industry to ease constraints (e.g., environmental concerns and high production costs) that have limited them in the past. In this study under laboratory‐scale conditions, the feasibility of culturing L. vannamei in effluents derived from a commercial facility raising tilapia in recirculating aquaculture systems (RAS), supplemented with various salt combinations, was compared to the shrimp’s survival and growth in well water supplemented with 17.6 (control) and 0.6 (freshwater treatment) g/L synthetic sea salt. Three independent trials were conducted in RAS in which survival and growth in the control, the freshwater treatment, and two effluent treatments were compared. Water quality during this study was within safe levels and no differences (P < 0.05) between treatments were observed for dissolved oxygen, nitrite, pH, total ammonia nitrogen, and temperature. However, average nitrate and orthophosphate levels were consistently more than an order of magnitude greater in the effluent treatments compared to the control and the freshwater treatments. Survival and growth of shrimp over 6‐wk periods did not vary significantly between the control and the freshwater treatments; however, shrimp tested in the tilapia effluents often exhibited significant effects (P < 0.05) depending on the salts added. In the low‐salinity waters, correlations (P < 0.05) were observed between Ca²⁺, Mg²⁺, Ca²⁺ and Mg²⁺, K⁺, Na⁺ : K⁺ and Ca²⁺ : K⁺, and shrimp survival and growth. The results of this study revealed that L. vannamei can be raised in tilapia effluent when supplemented with synthetic sea salt (0.6 g/L), CaO (50 mg/L Ca²⁺), and MgSO₄ (30 mg/L Mg²⁺).     

Growth,absorption and assimilation efficiency by mature cuttlefish (<Emphasis Type="Italic">Sepia officinalis</Emphasis>) fed with alternative and artificial diets

The effects of feeding three natural frozen diets, grass shrimp (Palaemonetes sp.), crayfish (Procambarus clarkii) and fish (Sardina pilchardus) and two semi-humid artificial diets (based on fish powder) to mature cuttlefish, Sepia officinalis, were analysed. Growth and feeding rates (GR and FR, % BW day⁻¹), food conversions (FC, %), and total protein and lipid composition of the diets were determined. Digestive gland to body weight ratio and absorption efficiency were calculated for each diet. Cuttlefish fed shrimp and crayfish grew larger (1.5 and 1.1% BW day⁻¹, respectively) compared to the other diets. Shrimp promoted the highest FC, followed by crayfish, and sardine. The highest FR was obtained for cuttlefish fed crayfish (8.4% BW day⁻¹). Although both artificial diets were accepted, none produced growth. A positive correlation (r = 0.96) between cuttlefish ingestion rate and digestive gland weight was obtained. Some cannibalism occurred among cuttlefish fed the artificial diets during the last week of the experiment. According to the results obtained, P. clarkii could be used as an alternative prey to shrimp for rearing adult mature (>50 g) S. officinalis.     

Replacement of fish oil with alternative lipid sources in plant‐based practical feed formulations for marine shrimp (Litopenaeus vannamei) reared in outdoor ponds and tanks

The objective of this study was to demonstrate the feasibility of partial substitution of menhaden fish oil by alternative lipid sources (soybean oil, poultry grease and flaxseed oil) in non‐marine protein‐based shrimp production diet, its influence on fatty acid profile and its final product flavour. Results for the pond study (17 weeks) showed no differences (P ≥ 0.05) in shrimp production among different test diets. Production ranged from 5070 to 5363 kg ha^?1; mean final weight, 18.0 to 21.6 g; weekly growth, 1.04 to 1.25 g; survival, 65.6 to 75.4%; and FCR, 1.37 to 1.45. Results from the tank trial (12 weeks) confirmed these findings, final weight, 13.8–14.8 g; weekly growth, 1.1–1.2 g; survival, 92.5–98.3%; FCR, 1.05–1.11; and final standing crop, 4738–5024 kg ha^?1. The fatty acids profiles of edible tail muscle reared on the various diets displayed a similar fatty acid profile to that of the diets. The sensory test showed no statistical differences in texture, appearance, aroma and flavour between the shrimp fed diets containing menhaden fish oil and soybean oil. These studies demonstrated that practical shrimp feeds containing non‐marine protein ingredients and a percentage of fish oil replaced by alternative lipid sources had no negative impact on mean final weight, weekly growth, survival, FCR, final standing crop, fatty acids profile and organoleptic properties of Litopenaeus vannamei.     

Viscera of Labeo rohita: A Potential Source of Trypsin for Industrial Application

ABSTRACT

The serine protease trypsin was isolated and purified from the digestive system of carp Labeo rohita rohu by ammonium sulphate precipitation, ion exchange, and affinity chromatography. The purified enzyme showed high activity between pH 7.0 and 9.0. The activity was maximum at 40°C. Incubation of the purified enzyme with CaCl₂ (2 mM) stabilized the enzyme activity for 8 h. The enzyme showed stability at 30 and 40°C for 1 h, but above 40°C, enzyme activity was reduced. The kinetic constants were recorded as K_m (0.104 mM), k_cat (44.25 s^?1), and catalytic efficiency (427.54 s^?1 mM^?1). Monovalent, bivalent, and trivalent ions (Li⁺, K⁺, Hg²⁺, Al³⁺, Mg^2+, Cd^2+, Co^2+, Zn²⁺, and Al³⁺) influenced the enzyme activity. Phenylmethylsulfonylflouride, soybean trypsin inhibitor, and N-α-p-tosyl-_L-lysine chloromethyl ketone completely inhibited the enzyme activity, while ethylenediaminetetraacetate caused partial inhibition. Molecular mass of the purified enzyme was 22.46 kDa. The pH and temperature stability of enzyme may be useful for its industrial applications.     

Response of Pacific White Shrimp,Litopenaeus vannamei,to Three Sources of Solvent Extracted Soybean Meal

Three sources of soybean meal (SBM) were biochemically characterized and evaluated in diets for juvenile Litopenaeus vannamei. Three biological techniques were utilized to evaluate the nutritional quality of the meals including an outdoor growth trial as well as determination of in vivo apparent digestibility and in vitro digestibility including pepsin digestibility (0.0002%), pH Stat degree of hydrolysis, and immobilized digestive enzyme assay (IDEA). The growth trial was conducted over a 10‐wk period using soybean meal as the primary protein source with each meal being included at 54–58% of the diet based on an equal protein inclusion. At the conclusion of the growth trial there were no significant differences between the test diets indicating equivalent biological availability under practical conditions. The in vivo digestibility trial utilized chromic oxide as the inert marker and 70:30 replacement techniques resulted in typical results. Apparent protein digestibility (APD) and apparent energy digestibility (AED) of the diets ranged from 66.8 to 80.2% and 65.4 to 74.7%, respectively. Ingredient digestibility values for protein and energy ranged from 78.8 to 93.5% and 60.5 to 85.0%, respectively. One of the three SBM evaluated had numerically lower protein digestibility and significantly lower energy digestibility than the other two meals .