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 degrees 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 degrees 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 degrees C, 4 U: 80 mg CP both for ingredients (R(2) = 0.86: P = 0.001) and test diets (R(2) = 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 (R(2) = 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, R(2) = 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. (C) 2009 Elsevier B.V. All rights reserved.

Ecological distribution of the shrimp Litopenaeus schmitti (Burkenroad, 1936) (Decapoda, Penaeoidea) in Ubatuba Bay, Sao Paulo, Brazil

The penaeidean Litopenaeus schmitti, popularly known as white shrimp, is a species of great economic importance, being a target of fishing fleets in the southeast region of Brazil. It is distributed through the western Atlantic, from Cuba to Brazil, until Rio Grande do Sul. Adults are found from shallow depths up to 30 m and have been found to depths of 47 m in the state of Rio de Janeiro, while juveniles are located in bays and estuaries. The studied species is seasonally distributed in the region of Ubatuba. The objectives of this study are to analyze the abundance and ecological distribution of L. schmitti and to assess if and when juveniles use the Indaia estuary during their life cycle. Furthermore, the hypothesis was tested that the main period of recruitment in the bay coincides with the period of closure of fisheries defined by the Instruction of IBAMA. To that end, samples were taken monthly from July 2005 through June 2007, both in Ubatuba Bay and in the estuary formed by the Rio Indaia. At each sampling station, salinity, temperature (bottom), depth, organic matter content (%), and grain size of sediments were measured. We found that the largest catches in the estuary were in late spring and early summer. In Ubatuba Bay, peak catches occurred during winter and early spring, whereas in the second year, already in May, there was a high peak capture. The variation in the number of individuals was correlated with some environmental factors both in the estuary and in the inlet (p < 0.05). In the estuary, abundance was positively correlated with temperature (p = 0.008) and organic matter (p = 0.025) and negatively with depth (p = 0.009). Regarding the Ubatuba Bay, only temperature (p = 0.034) and depth (p = 0.013) were significantly associated with the distribution of the shrimp, both being negative relations. The shrimp L. schmitti uses both the estuarine as well the shore environment, particularly the Ubatuba estuary and its adjacent bay, to complete its life cycle. The proposed period of fisheries closure (between March to May in the state of Sao Paulo) for this and other shrimps coincides with individuals capable of reproduction entering the inlet and thus are being protected.

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 mu m), 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 (R-2 = 0.72), growth rates (R-2 = 0.72-0.80) and FCR (R-2 = -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 (R-2 = 0.73), FCR and histidine (R-2 = -0.60), and DH% and methionine or methionine+cystine feed contents (R-2 = 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 R-2 values. Results suggest the feasibility of using standardized hepatopancreas enzyme extracts for in vitro protein digestibility.

Reproductive biology of the ghost shrimp Lepidophthalmus bocourti (A. Milne-Edwards, 1870) (Decapoda: Axiidea: Callianassidae): A tropical species with a seasonal reproduction

Based on the hypothesis that reproduction is a continuous process in tropical habitats, we analysed reproductive periodicity and egg production in the callianassid ghost shrimp Lepidophthalmus bocourti, one of the most common species in mangrove systems along the Pacific coast of Central America. During one year (May 2008 to April 2009), individuals of L. bocourti (N = 499) were collected nearshore Gulf of Nicoya, Pacific coast of Costa Rica. Observations were made on presence or absence of incubated embryos, and gonad activity of females was analysed as gonadosomatic index (GSI). Our results revealed that L. bocourti has a marked seasonal breeding period, which contradicts previous reports regarding coastal marine decapods from the tropics. Ovigerous females were found only from June to August, while high GSI values were obtained from March to July. The increase of GSI and appearance of ovigerous females were associated with a concomitant decrease of salinity, but not with temperature. We assume that reproduction of L. bocourti is adapted to local changes of environmental conditions, and that a decrease in salinity during rainy season may serve as a triggering factor for ovarian development. Compared to other ghost shrimps, L. bocourti produced on average more (2002 +/- 1365) and smaller (0.87 +/- 0.109 mm) eggs, which seems to suggest that this species does not have an abbreviated larval development as reported for other species of genus. The deviation from the generalization of constant reproduction in the tropics for shallow water marine invertebrates and its probable cause are adequately discussed.

Ontogenetic and evolutionary change of external morphology of the neotropical shrimp potimirim (Holthuis, 1954) explained by a molecular phylogeny of the genus

A phylogenetic analysis of a fragment of the mitochondrial gene 16S was used to test the monophyletic status of Potimirim. Existing doubts on the taxonomic status of brasiliana (once P glabra) and P potimirim (once P mexicana) were clarified. Potimirim mexicana and P potimirim are distinct species according to molecular data and appendix masculina morphology. A new species (Potimirim sp. 1) from Puerto Rico was revealed with molecular data, and it is evolutionarily related to P potimirim and P mexicana according to our analysis. We found out three distinct species under the name P glabra. Then, we recommend the application of the name P glabra for the populations of the Pacific slope of Central America and revalidation of P brasiliana for the Brazilian ones. The need for a new name to those "P glabra" of the Caribbean is highlighted, and it was provisionally referred as Potimirim sp. 2. The ontogenetic (juveniles to adults) development of the appendix masculina of P brasiliana was observed and compared to the other species of Potimirim (adults). In the light of our phylogenetic hypothesis, we postulate a pattern of character addition for the evolution of the appendix masculina of Potimirim. This hypothesis is plausible for two key reasons. First. Potimirim is a monophyletic group according to our hypothesis. Second, the shape of appendix masculina found in adults of P. americana is similar and comparable to those found in the earliest juvenile stages of P brasiliana, a derived species according to our phylogeny (P americana, ((P mexicana, Potimirim sp. 1. P potimirim), (P glabra, (brasiliana, Potimirim sp. 2)))). As so, the basal P americana retain the ancestral morphological state of the appendix masculina when compared to the other species of Potimirim. In our interpretation the ontogeny of the appendix masculina recapitulated the proposed phylogeny, giving further support to it.