Dry matter, protein, and energy digestibility of diets for juvenile Pacific white leg shrimps (Litopenaeus vannamei) reared at different salinity levels / Digestibilidade da matéria seca, proteína e energia de dietas para camarão juvenil do Pacífico (Litopenaeus vannamei) cultivadas em diferentes níveis de salinidades

ABSTRACT: This study evaluated the effect of low, medium, and high-water salinity (5, 35, and 50 ppt) on the apparent dry matter, protein, and energy digestibility of two formulated and six commercial diets for juvenile whiteleg shrimp, Litopenaeus vannamei, in a 120-day trial. Digestibility was determined in vivo using chromic oxide as an inert diet marker. Hydrostability in pellets varied from 86.8% to 99.9%; dry matter digestibility varied from 49.1% to 64.1%; protein digestibility showed greater variations at all salinities (56.9%-85.8%); and energy digestibility ranged from 70.1 to 86.4%. Salinity had a significant effect on dry matter, protein, and energy digestibility. Using a principal component analysis (PCA) with a covariance matrix, our findings suggested that the E2 (fishmeal-based formulation) diet and 35 ppt salinity provided optimum hydrostability and digestibility to Pacific white leg shrimp juveniles.

RESUMO: Nós medimos o efeito de baixa, normal e alta salinidade (5, 35 e 50ppt) na digestibilidade aparente da matéria seca, proteína e energia em duas dietas formuladas e seis comerciais para camarão juvenil do Pacífico, Litopenaeus vannamei. Os coeficientes de digestibilidade aparente da matéria seca, proteína e energia foram determinados in vivo utilizando o óxido crômico como marcador inerte nas dietas (peso inicial médio de 4g), em um teste de 120 dias. Hidrostabilidade na dieta de 86,8% a 99,9%, os coeficientes de digestibilidade da matéria seca variaram de 49,1 a 64,1%, os coeficientes de digestibilidade da proteína apresentaram maiores variações em todas as salinidades (56,9-85,8%), enquanto os coeficientes de digestibilidade da energia variaram de 70,1 a 86,4%. Efeito significativo da salinidade na digestibilidade da matéria seca, proteína e energia foi encontrado. Esses resultados, usando a análise de componentes principais (PCA) com a matriz de covariância, sugerem que a dieta E2 (fórmula à base de farinha de peixe) e salinidade a 35 ppt é ideal para a hidrostabilidade e digestibilidade das dietas para juvenis de camarão-branco-do-Pacífico.

Reduction of spermatophore melanization in Litopenaeus vannamei shrimp fed Ulva clathrata during a commercial hatchery production.

Male broodstock (Litopenaeus vannamei, 36 ± 7 g, n = 600) reproductive performance, spermatophores and reproductive tract melanization, prostaglandin concentrations and biochemical composition were evaluated after including 3.8% Ulva clathrata meal in the diet (dry base) of a commercial hatchery during 45 days. Males fed Ulva had less melanized spermatophores (120 compared with 233, P < 0.01), less bacteria in the ductus deferens (P < 0.01), more sperm cells in testicles (P < 0.05), and increased courtship activity (839 compared with 689, P < 0.01), with no effect on mortality. Ulva-fed males had more arachidonic acid (20:4n-6) in the spermatophores (P < 0.05) but this did not affect the prostaglandin concentrations in response to diet. Males fed Ulva had more carotenoids in the hepatopancreas (0.08 ± 0.02 compared with 0.01 ± 0.01 mg/g, P < 0.05), and phenolic compounds in hepatopancreas (6.1 ± 0.7 compared with 1.8 ± 0.7 mg eq. phloroglucinol/g, P < 0.05) and muscle (0.4 ± 0.3 compared with 0.2 ± 0.1 mg eq. phloroglucinol/g, P < 0.05). Males fed the Ulva also had a lesser carbohydrate content in the hepatopancreas (P < 0.01) and muscle (P < 0.01). In conclusion, supplementing fresh maturation diets with a small dose of dried Ulva allowed for improvement of reproductive performance and to decrease melanization in spermatophores and the male reproductive tract.

Nutraceutical Potential of Five Mexican Brown Seaweeds.

In search of pharmaceutically active products to control type 2 diabetes, five brown seaweeds (Silvetia compressa, Cystoseira osmundacea, Ecklonia arborea, Pterygophora californica, and Egregia menziesii) from the Northwest Mexican Pacific coast were investigated. Proximate composition and total polyphenol content (TPC) as phloroglucinol equivalents (PGE) were determined for the five seaweed powders and their respective hydroethanolic (1 : 1) extracts. Extracts were screened for their radical scavenging activity (DPPH and ORAC) and glycosidase inhibitory activity. HPLC-DAD, HPLC-MS-TOF, and ATR-FT-IR methodologies were used to identify the most abundant phlorotannins and sulfated polysaccharides in the extracts. Hydroethanolic extracts contained minerals (17 to 59% of the dry matter), proteins (4 to 9%), ethanol-insoluble polysaccharides (5.4 to 53%), nitrogen-free extract (NFE) (24.4 to 70.1%), lipids (5 to 12%), and TPC (2.6 to 47.7 g PGE per 100 g dry extract). S. compressa and E. arborea dry extracts presented the lowest ash content (26 and 17%, respectively) and had some of the highest phenolic (47.7 and 15.2 g PGE per 100 g extract), NFE (57.3 and 70.1%), and soluble polysaccharide (19.7 and 53%) contents. S. compressa and E. arborea extracts had the highest antioxidant activity (IC50 DPPH 1.7 and 3.7 mg mL-1; ORAC 0.817 and 0.801 mmol Trolox equivalent/g extract) and the highest α-amylase and α-glucosidase inhibitory capacities (IC50 940 and 1152 µg mL-1 against α-amylase and 194 and 647 µg mL-1 against α-glucosidase). The most abundant phlorotannins identified in the extracts were phloretol, fucophloroethol, and two- and three-phloroglucinol unit (PGU) phlorotannins. Laminarin, fucoidan, and alginate were among the sulfated polysaccharides identified in the extracts. The bioactivities of S. compressa and E. arborea extracts were mainly related with their contents of three PGU phlorotannins and sulfated polysaccharides (e.g., fucoidan, laminarin, and alginate). These results suggest S. compressa and E. arborea are potential candidates for food products and nutraceutical and pharmaceutical preparations, and as additives for diabetes management.

Virucidal and Synergistic Activity of Polyphenol-Rich Extracts of Seaweeds against Measles Virus.

Although preventable by vaccination, Measles still causes thousands of deaths among young children worldwide. The discovery of new antivirals is a good approach to control new outbreaks that cause such death. In this study, we tested the antiviral activity against Measles virus (MeV) of Polyphenol-rich extracts (PPs) coming from five seaweeds collected and cultivated in Mexico. An MTT assay was performed to determine cytotoxicity effect, and antiviral activity was measured by syncytia reduction assay and confirmed by qPCR. PPs from Ecklonia arborea (formerly Eisenia arborea, Phaeophyceae) and Solieria filiformis (Rhodophyta) showed the highest Selectivity Index (SI), >3750 and >576.9 respectively. Both PPs extracts were selected to the subsequent experiments owing to their high efficacy and low cytotoxicity compared with ribavirin (SI of 11.57). The combinational effect of PPs with sulphated polysaccharides (SPs) and ribavirin were calculated by using Compusyn software. Synergistic activity was observed by combining both PPs with low concentrations of Solieria filiformis SPs (0.01 µg/mL). The antiviral activity of the best combinations was confirmed by qPCR. Virucidal assay, time of addition, and viral penetration evaluations suggested that PPs act mainly by inactivating the viral particle. To our knowledge, this is the first report of the virucidal effect of Polyphenol-rich extracts of seaweeds.

Synergistic Effects of Sulfated Polysaccharides from Mexican Seaweeds against Measles Virus.

Sulfated polysaccharides (SPs) extracted from five seaweed samples collected or cultivated in Mexico (Macrocystis pyrifera, Eisenia arborea, Pelvetia compressa, Ulva intestinalis, and Solieria filiformis) were tested in this study in order to evaluate their effect on measles virus in vitro. All polysaccharides showed antiviral activity (as measured by the reduction of syncytia formation) and low cytotoxicity (MTT assay) at inhibitory concentrations. SPs from Eisenia arborea and Solieria filiformis showed the highest antiviral activities (confirmed by qPCR) and were selected to determine their combined effect. Their synergistic effect was observed at low concentrations (0.0274 µg/mL and 0.011 µg/mL of E. arborea and S. filiformis SPs, resp.), which exhibited by far a higher inhibitory effect (96% syncytia reduction) in comparison to the individual SP effects (50% inhibition with 0.275 µg/mL and 0.985 µg/mL of E. arborea and S. filiformis, resp.). Time of addition experiments and viral penetration assays suggest that best activities of these SPs occur at different stages of infection. The synergistic effect would allow reducing the treatment dose and toxicity and minimizing or delaying the induction of antiviral resistance; sulfated polysaccharides of the tested seaweed species thus appear as promising candidates for the development of natural antiviral agents.

Innocuity and anti-Newcastle-virus-activity of Cladosiphon okamuranus fucoidan in chicken embryos.

This study evaluated the potential toxicity and antiviral activity of fucoidan from Cladosiphon okamuranus against Newcastle disease virus (NDV), one of the most serious threats to the poultry industry in the world. Toxicity was assayed on chicken embryo fibroblast (CEF) secondary cultures at concentrations ranging from 0.1 to 1500 µg per mL culture medium, assessing the cell viability by the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, and on 9-day-old embryonated chicken eggs by inoculation of 2 to 500 µg doses in the allantoic cavity, assessing the embryos morphology and liver histology. At 48 h post-inoculation, viability of CEF exposed to concentrations up to 10 µg/mL was not significantly affected, and the 50% cytotoxic concentration was estimated as of 1062 µg/mL; after exposure in ovo, some chick embryos showed liver steatosis when treated with fucoidan doses over 20 µg per egg (15 to 28% at 200 µg, 27 to 56% at 500 µg), but no change was detected in their size or aspect. Antiviral activity was tested by treating 9-day-old embryos via the allantoic route with 0.25 to 16 µg fucoidan doses that were applied at different times (-1, 0 and +1 h) relative to the inoculation of 10,000 folds the 50% Tissue Culture Infective Dose (TCID50) of the NDV, La Sota strain. At 72 h post infection, virus titration in the allantoic fluid by hemagglutination assay (HA) showed a considerable and significant inhibition of infectivity for all doses, the best result (a 90% decrease) being obtained in embryos treated with 1 µg fucoidan one hour before infection. Viral RNA semi-quantification in pooled liver and small intestine of embryos that had been treated with 4 and 16 µg fucoidan 1 h before the infection showed reductions of the virus replication by 60 and 99.8%, respectively. Since this high anti-NDV activity in ovo was obtained with quite innocuous doses, fucoidan from C. okamuranus could be a potential low-toxicity antiviral compound to be used in areas exposed to NDV.

Sulphated polysaccharides from Ulva clathrata and Cladosiphon okamuranus seaweeds both inhibit viral attachment/entry and cell-cell fusion, in NDV infection.

Sulphated polysaccharides (SP) extracted from seaweeds have antiviral properties and are much less cytotoxic than conventional drugs, but little is known about their mode of action. Combination antiviral chemotherapy may offer advantages over single agent therapy, increasing efficiency, potency and delaying the emergence of resistant virus. The paramyxoviridae family includes pathogens causing morbidity and mortality worldwide in humans and animals, such as the Newcastle Disease Virus (NDV) in poultry. This study aims at determining the antiviral activity and mechanism of action in vitro of an ulvan (SP from the green seaweed Ulva clathrata), and of its mixture with a fucoidan (SP from Cladosiphon okamuranus), against La Sota NDV strain. The ulvan antiviral activity was tested using syncytia formation, exhibiting an IC50 of 0.1 µg/mL; ulvan had a better anti cell-cell spread effect than that previously shown for fucoidan, and inhibited cell-cell fusion via a direct effect on the F0 protein, but did not show any virucidal effect. The mixture of ulvan and fucoidan showed a greater anti-spread effect than SPs alone, but ulvan antagonizes the effect of fucoidan on the viral attachment/entry. Both SPs may be promising antivirals against paramyxovirus infection but their mixture has no clear synergistic advantage.

In vitro anti-canine distemper virus activity of fucoidan extracted from the brown alga Cladosiphon okamuranus.

Canine distemper virus (CDV) is a morbillivirus related to measles virus that infects dogs and other carnivores. CDV has a significant global impact on animal health; however, there is no current antiviral treatment for CDV infection. In recent years, it has been demonstrated that sulfated polysaccharides exhibit antiviral properties both in vivo and in vitro, despite their low cytotoxicity to host cells. Fucoidan is a sulfated polysaccharide found in the cell wall matrix of brown algae. In this study, we evaluated in vitro anti-CDV activity of fucoidan, which was derived from Cladosiphon okamuranus. Fucoidan actively inhibited CDV replication in Vero cells at a 50 % inhibitory concentration (IC50) of 0.1 µg/ml. The derived selectivity index (SI50) was >20,000. This polysaccharide likely inhibits viral infection by interference in the early steps and by inhibiting CDV-mediated cell fusion. Fucoidan may be useful in development of pharmacological strategies to treat and control CDV infection.

Apparent carbohydrate and lipid digestibility of feeds for whiteleg shrimp, Litopenaeus vannamei (Decapoda: Penaeidae), cultivated at different salinities.

Whiteleg shrimp, Litopenaeus vannamei is one of the most commercially farmed species worldwide because of its fast growth, good survival rate at high farming densities, and osmoregulatory capacity, which makes it an excellent candidate for cultures at different salinities. The knowledge of shrimp nutritional requirements is critical in the formulation of diets to allow optimal growth at different environmental conditions and development stages. The effect of salinity on apparent digestibility of shrimp feed is not well known, and this information is required in shrimp diet formulation. For this purpose, the apparent digestibility coefficients of carbohydrates (ACD) and lipids (ALD) were determined for juvenile whiteleg shrimps under controlled culture conditions. We evaluated the apparent digestibility of six commercial (D1:37CP, D2:38CP, D3:39CP, D4:34CP, D5:35CP, and D6:37CP) and two experimental (E1:33CP and E2:33CP) diets for juvenile whiteleg shrimp cultivated at three salinities (5, 35 and 50 psu) in 60 L aquariums. ACD and ALD were determined in vivo using chromic oxide as an inert marker. Our results showed that ALD in most cases was over 80%, independent of salinity, except the E1:33CP diet which had 74.0% at 50 psu. Diet D3:39CP showed the highest ALD coefficient (90.1 and 90.6% at 5 and 35 psu, respectively). For ACD, differences were detected between commercial and experimental diets at every salinity level, although salinity effect on ACD was not significant. Diet D4:34CP had the highest coefficient (92.4%) at 5 psu, and E2:33CP at 35 and 50 psu (97.3 and 94.7%). This study demonstrated that there is no significant effect of saline variations on carbohydrate and lipid digestibility by juvenile whiteleg shrimp, under the experimental conditions.

Apparent carbohydrate and lipid digestibility of feeds for whiteleg shrimp, Litopenaeus vannamei (Decapoda: Penaeidae), cultivated at different salinities / Digestibilidad aparente de carbohidratos y lípidos en alimentos para camarón blanco, Litopenaeus vannamei (Decapoda: Penaeidae), cultivados en diferentes salinidades

Whiteleg shrimp, Litopenaeus vannamei is one of the most commercially farmed species worldwide because of its fast growth, good survival rate at high farming densities, and osmoregulatory capacity, which makes it an excellent candidate for cultures at different salinities. The knowledge of shrimp nutritional requirements is critical in the formulation of diets to allow optimal growth at different environmental conditions and development stages. The effect of salinity on apparent digestibility of shrimp feed is not well known, and this information is required in shrimp diet formulation. For this purpose, the apparent digestibility coefficients of carbohydrates (ACD) and lipids (ALD) were determined for juvenile whiteleg shrimps under controlled culture conditions. We evaluated the apparent digestibility of six commercial (D1:37CP, D2:38CP, D3:39CP, D4:34CP, D5:35CP, and D6:37CP) and two experimental (E1:33CP and E2:33CP) diets for juvenile whiteleg shrimp cultivated at three salinities (5, 35 and 50psu) in 60L aquariums. ACD and ALD were determined in vivo using chromic oxide as an inert marker. Our results showed that ALD in most cases was over 80%, independent of salinity, except the E1:33CPdiet which had 74.0% at 50psu. Diet D3:39CP showed the highest ALD coefficient (90.1 and 90.6% at 5 and 35psu, respectively). For ACD, differences were detected between commercial and experimental diets at every salinity level, although salinity effect on ACD was not significant. Diet D4:34CP had the highest coefficient (92.4%) at 5psu, and E2:33CP at 35 and 50psu (97.3 and 94.7%). This study demonstrated that there is no significant effect of saline variations on carbohydrate and lipid digestibility by juvenile whiteleg shrimp, under the experimental conditions. Rev. Biol. Trop. 61 (3): 1201-1213. Epub 2013 September 01.

El camarón blanco, Litopenaeus vannamei, es una de las especies más cultivadas comercialmente en el mundo, debido a su velocidad de crecimiento y tasa de supervivencia en altas densidades de cultivo, y su capacidad de osmoregulación, que lo hacen un excelente candidato para cultivo en diferente salinidades. El conocimiento de los requerimientos nutricionales del camarón es fundamental en la formulación de dietas que permita el crecimiento óptimo en diferentes condiciones ambientales y fases de desarrollo del animal. El efecto de la salinidad sobre la digestibilidad aparente de alimentos comerciales para camarones no está documentado. Esta información es necesaria en el cultivo de camarón para la formulación de los alimentos comerciales. Se determinó la digestibilidad aparente de los carbohidratos (ACD) y lípidos (ALD), en juveniles del camarón blanco L. vannamei cultivado en condiciones controladas. Los tratamientos fueron seis dietas comerciales (D1:37CP, D2:38CP, D3:39CP, D4:34CP, D5:35CP y D6:37CP) y dos dietas experimentales (E1:33CP y E2:33CP) para juveniles de camarón blanco en cultivo, a tres salinidades (5, 35 y 50ups) en acuarios de 60L. Los coeficientes de digestibilidad aparente de los carbohidratos (ACD) y los lípidos (ALD) fueron determinados in vivo utilizando óxido crómico como marcador inerte en la dieta. ALD en la mayoría de los tratamientos fue superior al 80%, independientemente de la salinidad, con excepción de la dieta E1:33CP que presentó 74.0% a salinidad de 50ups. La dieta D3:39CP presentó el mayor coeficiente ALD (90.1 y 90.6 en 5 y 35ups, respectivamente). En ACD, se presentaron diferencias entre dietas comerciales y experimentales en cada nivel de salinidad, aunque la salinidad no presentó un efecto significativo. La dieta D4:34CP presentó el coeficiente de digestibilidad más alto (92.4%) en la salinidad de 5ups, mientras que E2:33CP en salinidades de 35 y 50ups fue de 97.3 y 94.7%, respectivamente. En general, el presente estudio demuestra que en juveniles de camarón blanco la variación de salinidad no afecta significativamente la digestibilidad de lípidos y carbohidratos en las condiciones experimentales de este estudio.

In vitro characterization of the antiviral activity of fucoidan from Cladosiphon okamuranus against Newcastle Disease Virus.

BACKGROUND: Newcastle Disease Virus (NDV) causes a serious infectious disease in birds that results in severe losses in the worldwide poultry industry. Despite vaccination, NDV outbreaks have increased the necessity of alternative prevention and control measures. Several recent studies focused on antiviral compounds obtained from natural resources. Many extracts from marine organisms have been isolated and tested for pharmacological purposes, and their antiviral activity has been demonstrated in vitro and in vivo. Fucoidan is a sulfated polysaccharide present in the cell wall matrix of brown algae that has been demonstrated to inhibit certain enveloped viruses with low toxicity. This study evaluated the potential antiviral activity and the mechanism of action of fucoidan from Cladosiphon okamuranus against NDV in the Vero cell line. METHODS: The cytotoxicity of fucoidan was determined by the MTT assay. To study its antiviral activity, fusion and plaque-forming unit (PFU) inhibition assays were conducted. The mechanism of action was determined by time of addition, fusion inhibition, and penetration assays. The NDV vaccine strain (La Sota) was used in the fusion inhibition assays. PFU and Western blot experiments were performed using a wild-type lentogenic NDV strain. RESULTS: Fucoidan exhibited antiviral activity against NDV La Sota, with an obtained IS50 >2000. In time of addition studies, we observed viral inhibition in the early stages of infection (0-60 min post-infection). The inhibition of viral penetration experiments with a wild-type NDV strain supported this result, as these experiments demonstrated a 48% decrease in viral infection as well as reduced HN protein expression. Ribavirin, which was used as an antiviral control, exhibited lower antiviral activity than fucoidan and high toxicity at active doses. In the fusion assays, the number of syncytia was significantly reduced (70% inhibition) when fucoidan was added before cleavage of the fusion protein, perhaps indicating a specific interaction between fucoidan and the F0 protein. CONCLUSION: The results of this study suggest that fucoidan from C. okamuranus represents a potential low-toxicity antiviral compound for the poultry industry, and our findings provide a better understanding of the mode of action of sulfated polysaccharides.

Chemical composition of cultivated seaweed Ulva clathrata (Roth) C. Agardh.

Samples of cultivated Ulva clathrata were collected from a medium scale system (MSS, 1.5×1.5m tank), or from a large scale system (LSS, 0.8ha earthen pond). MSS samples were dried directly while the LSS sample was washed in freshwater and pressed before drying. Crude protein content ranged 20-26%, essential amino acids accounting for 32-36% of crude protein. The main analysed monosaccharides were rhamnose (36-40%), uronic acids (27-29%), xylose (10-13%) and glucose (10-16%). Some notable variations between MSS and LSS samples were observed for total dietary fibre (26% vs 41%), saturated fatty acids (31% vs 51%), PUFAS (33% vs 13%), carotenoids (358 vs 169mgkg-1dw) and for Ca (9 vs 19gkg-1), Fe (0.6 vs 4.2gkg-1), Cu (44 vs 14mgkg-1), Zn (93 vs 17mgkg-1) and As (2 vs 9mgkg-1). The chemical composition of U. clathrata indicates that it has a good potential for its use in human and animal food.