IntroductionTop

Shrimp aquaculture is one of the main economic activities in tropical and subtropical areas, which supply this product at a world scale reaching more than four million tons. The success of white shrimp (Litopenaeus vannamei Boone 1931) production depends on reducing the impact of diseases and epizootics (Zhang et al., 2016; Dewangan et al., 2017), mainly during its larval and juvenile stages. Among the most important pathogens that cause diseases in larval and juvenile stages of P. vannamei are White Spot Syndrome Virus (WSSV) and Taura Syndrome Virus (TSV), and Vibrio species (Cheng et al., 2011; Rajkumar et al., 2017). Intensification of culture applications has led to diseases causing up to 80% economic losses in hatcheries (mainly within 60–80 days post-stocking) (Kibenge, 2019)); e.g. total losses caused just by WSSV have amounted to 3 billion US dollars per year (Yang et al., 2016). For decades, the use of antibiotics has been a common practice for the prevention of shrimp diseases (Bermúdez-Almada & Espinosa-Plascencia, 2012); however, it is well documented that the application of these chemicals is a management practice that involves dangers and risks to the environment and human health (Thornber et al., 2020). Therefore, the search for alternative compounds that are environmentally-friendly, harmless to human health, and do not compromise shrimp production are a priority (Romero et al., 2012).

The use of alternative substances and microorganisms with antimicrobial and immunostimulant activities to reduce diseases impact (Peraza-Gómez et al., 2014; Trejo-Flores et al., 2016) and diminish the application of antibiotics (Cabello, 2006) in the shrimp industry has been increasing. Allelopathic powders and plant extracts are immunomodulator and immunostimulant additives commonly used in safeguarding animal health (Tiwari et al., 2018). Among these products, the phenolic compounds-based extracts have been described as potential immunostimulants, antipathogenic, and antistress agents in aquaculture (Chakraborty & Hancz, 2011).

The Maclura tinctoria tree is a deciduous, semideciduous, and even perennial arboreal native species of dry and wet tropical regions from Mexico to Argentina (Berg, 2001). Although antibacterial, antioxidant, and immunostimulant benefits of M. tinctoria extracts have been proved in laboratory assays (Lamounier et al., 2012; Matson-Robles et al., 2015), their chemical properties are commonly reduced by temperature, light, and environmental conditions (Santos-Buelga et al., 2019). The microencapsulation technique represents a viable alternative (Ozkan et al., 2019) to preserve the allopathic properties of M. tinctoria’ extracts. There are few studies on the use of some phenolic compounds in shrimp culture (Sudheer et al., 2011; Chandran et al., 2016; Tomazelli-Júnior et al., 2018), but so far, supplementation of microencapsulated phenolic extracts of M. tinctoria in diets for L. vannamei juvenile has not been described yet. In the present work, we studied the effect of microencapsulated phenolic compound extracts of M. tinctoria on growth performance (final weight gain, specific growth rate, and survival) and humoral immunity markers (protein concentration in plasma and hemocytes, phenoloxidase activity, and superoxide anion concentration) of white shrimp L. vannamei juveniles.

Material and methodsTop

Plant material

Three samples of M. tinctoria fruits were obtained from 20 reproductive trees (60 samples) from two tropical dry forest areas of Mexico (19°56´32” N, 90°22´32” W, and 24° 52´42.39” N, 107°17´47.53” W). Dried samples were pulverized with a domestic blender (Sunbeam®, USA), then, 5.5 g powder per sample were extracted with 30 mL of 50% ethanol (Baker®) and kept isolated from light in amber jars until laboratory analysis. For microcapsules, the core materials were M. tinctoria fruit extracts (MTBE) and fish oil (Pescadería Mora®, Mex), wall materials consisted of food grade gum arabic E 414 (Alfred L. Wolf®, Mex) and maltodextrin DE® 10 (Chemistry LEFE®, Mex), according to (González-Ocampo et al., 2016).

Extraction, identification, quantification, and microencapsulation of M. tinctoria phenolic compounds

To obtain the MTBE microcapsules, the bark powder was mixed with ethanol, stirred, and kept in the dark for 24 h. Thereafter, a solution was prepared with 183 g of bark powder and 1 L of absolute ethanol (50%) FAGALAB®. The solution was stirred for 24 h, filtered through an analytical sieve with a mesh (90 μm) (WS Tyler®, USA) to remove solids, and subsequently kept in amber jars (Almaraz-Abarca et al., 2013).

The microencapsulation technique was developed in the Aquaculture Laboratory of the CIIDIR-IPN Sinaloa Research Center, and is being processed for a patent (MX/a/2016/007504) at the Instituto Mexicano de la Propiedad Intelectual, IMPI (González-Ocampo et al., 2016).

Phenolic content

The phenolic composition of MTBE was determined through high performance liquid chromatography, HPLC (Perkin-Elmer®, Series 200 HPLC system, Shelton, CT, USA) using a Perkin Elmer® Brownlee Analytical C18 column (4.6 × 250 mm, 5 µm) and diode array-detection (DAD) system (Perkin Elmer Series 200, USA), with a modified gradient method (Campos & Markham, 2007). Maximal absorbance was achieved employing a gradient method with mobile phase, which consisted of a mixture of water, 0.05% orthophosphoric acid (2.5 pH, Baker®, USA) (A), and acetonitrile (J. T. Baker®, USA) (B) as follows: 100(A):0(B) 0-12 min; 87(A):13(B) 12-20 min; 67(A):33(B) 20-32 min, and 57(A):43(B) 32-42 min, at a flow rate of 1 mL min-1. The identified phenolic chroma-tograms were registered at 260 nm and structural infor-mation of compounds was compared with the reference of compounds and its retention time (RT) from Apin Che-micals Limited® (Abingdon, Oxon, UK) standard: caffeic acid (RT: 53.13 min; λmax), p-coumaric acid (RT: 37.2 min; λmax: 294sh, 308), quercetin (RT: 45.95 min; λmax: 260, 268sh, 299sh, 370), rutin (quercetin-3-O-[rhamnosyl(1-6)glucoside]; RT: 33.74 min; λmax: 255, 264sh, 294sh, 355), apigenin (RT: 59.60 min, 267, 290sh, 335), quercitrin (quercetin-3-O-rhamnoside, RT: 38.54, λmax: 255, 264sh, 295sh, 348), morin (RT: 45.4, λmax: 254, 264sh, 298sh, 354), hesperidin (RT: 39.34, λmax: 284, 335sh), and na-ringenin (RT: 52.25, λmax: 289, 335sh). Spectral informa-tion was obtained from the phenolic compounds’ spectral data described by Mabry et al. (1970) and Campos & Markham (2007).

Antioxidant capacity

The total antioxidant capacity (TAC) was calculated by the ferric reducing antioxidant power (FRAP) me-thod (Oyaizu, 1986) and the molybdate VI reduction to molybdate V assay, modified by Prieto et al. (1999). The FRAP assay is based on the reduction power of the plant extracts. The ferric ion (Fe3+) is reduced to ferrous ion (Fe2+) forming the blue complex (Fe2+ TPTZ-1), which increases the absorption at 593 nm; samples and standard (ascorbic acid) were read after 30 min. Results are ex-pressed as the extract concentration required to reach an absorbance value of 0.5 EC50 (effective concentration at 50%) in equivalent milligrams of ascorbic acid per milli-liter of extract (mg EAA mL-1) and equivalent milligrams of ascorbic acid per gram of microcapsules (mg EAA g-1). The calibration curve of ascorbic acid (Eq. 1) was prepa-red with four concentrations of ascorbic acid (10-40 µL combined with a volume of methanol to reach 1 mL as final volume). The absorbance values of the samples were substituted in the equation and the results were adjusted to the dilution factor used and expressed in equivalent milligrams of gallic acid per gram of microcapsules (mg GAE g-1).

TAC was determined by the method of reducing molybdate VI to molybdate V, following the procedure described Prieto et al. (1999). Each sample of 100 μL was mixed with 1000 μL of molybdate solution and incubated in a thermoblock (Thermolyne® Dry Bath DB28125) for 90 min at 95 °C. Once all samples had been cooled to room temperature (in the dark), the absorbance at 695 nm was recorded. A calibration curve (A695 = 0.0729-0.0747 [gallic acid], correlation coefficient r = 0.9947) (Eq. 2) was prepared with different concentrations of gallic acid (0.5, 1.0, 2.5, 5.0, 7.5. and 10 mg mL-1).

The absorbance values of the sample were substituted in the equation, and the results were adjusted to the used dilution factor and expressed in mg GAE mL-1.

Bioassay

Juveniles of L. vannamei (0.5 ± 0.2 g) were obtained from the “Cuate Mechado” shrimp farm (Guasave, Sina-loa, Mexico). Experimental units consisted of 120-L plas-tic tanks with 80 L of seawater (34-35 mg L-1) supplied with constant aeration. Stocking density was adjusted at 10 shrimp per tank. MTBE treatments were tested as a fe-eding additive with three replicates and coded as follows: CTRL, control group fed with commercial feed (Purina®, 35% protein); T1, 0.5 g MTBE/kg of feed; T2, 1 g MTBE/kg of feed; and T3, 2.5 g MTBE/kg of feed. Shrimps were fed twice a day (9:00 and 17:00 h) at a feeding ration of 7% of total biomass per tank, for 30 days. The tanks were cleaned (residues of food and wastes) by siphoning the bottom every three days. Clean seawater was added to re-place water loss by evaporation and siphoning up to 120 L from each thank every five days.

Growth and survival of L. vannamei

The final weight gain, specific growth rate, and survival of Litopenaeus vannamei juveniles were evaluated at the end of the study. Final weight gain (%) was obtai-ned following the procedure of Amaya et al. (2007). The specific growth rate (SGR) (% d-1) was calculated in percentage using the Ziaei-Nejad et al. (2006) equation (Eq. 3):

where t is the bioassay period (d), lnW0 is the natural logarithm of the initial shrimp´s weight (g), and lnWt is the natural logarithm of the final weight (g). Survival (%) was calculated according to Ziaei-Nejad et al. (2006).

Hemolymph collection

At the end of the bioassay, the hemolymph was extracted from the ventral area of the shrimp’s cephalothorax by using insulin syringes (27G × 13 mm) containing SIC-EDTA Na2 (450 mM NaCl, 10 mM KCl, 10 mM Hepes, and 10 mM EDTA, Na2) pH 7.3 (Hernández-López et al., 1996) as anticoagulant, at a proportion of 2:1 (anticoagulant:hemolymph).

Hemolymph analysis

Hemocytes from plasma and lysate supernatant (HLS) were obtained following the method of Vargas-Albores et al. (1993). Protein concentration in HLS was determined using the Bradford (1976) method using bovine serum albumin (BSA, Sigma®) as standard. The Bradford method involves the binding of Coomassie Brilliant Blue G-250 to protein and the absorbance is determined at 595 nm.

Phenoloxidase, prophenoloxidase, and superoxide anion

Humoral response of L. vannamei juvenile was determined with the activity of phenoloxidase (PO) and prophenoloxidase (proPO) enzymes and the superoxide anion concentration. PO for small samples was measured spectrophotometrically (490 nm min-1 g protein-1) according to Hernández-López et al. (1996), by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) and the amount of inactive proPO in samples containing also PO activity was calculated as total available proPO minus PO activity before trypsin treatment. The superoxide anion (O2-) was quantified (630 nm) using the methodology of Song & Hsieh (1994) based on SIC-EDTA buffer to wash hemocyte pellets; the cytoplasmic formazan was read at 630 nm in a Thermo Spectronic Genesys 2 spectrophotometer.

Physicochemical seawater parameters

Salinity, pH, dissolved oxygen, and temperature were determined in each tank before cleaning, siphoning, and water replacement. Nitrites, nitrates, and ammonium (Strickland & Parsons, 1972) were measured at the beginning and end of the bioassay and throughout the study remained within the optimum levels for shrimp culture (Brock & Main, 1994).

Statistical analysis

ANOVA was applied to determine the differences of protein in plasma and HLS, PO and proPO activities, SGR, and superoxide anion concentration (mean ± SD) among treatments. Results of SGR were log10 transformed to normalize its distribution. If significant differences were found (p < 0.05), a post-hoc Tukey honestly significant difference (HSD) with multiple comparisons of Scheffé, Bonferroni and Holm was used to identify the differences.