in Revista MVZ Córdoba
Biological, nutritional, and hematoimmune response in juvenile Cherax quadricarinatus (Decapoda: Parastacidae) fed with probiotic mixture
Abstract
Objective. To evaluate the effect on biological, nutritional, and hematoimmune indicators of juvenile Cherax quadricarinatus were cultivated and fed with a probiotic mixture. Materials and methods. A completely randomized design (DCA) with six treatments: 0 (control), 1×10. µL, 2×10. µL, 3×10. µL, 4×10. µL and 5×10. µL of a probiotic mixture (Bacterol Shrimp Forte), with three repetitions each, 18 experimental tanks of diameter 1.7 m and area of 2.26 m. were used, with a density of 20 juveniles (0.95 ± 0.10g and 7.78 ± 0.77mm) per tank for 60 days. Biological (weight, length, weight gain, weight increase, specific growth rate, length gain, length increase and survival), nutritional (feed conversion, feed efficiency and protein efficiency rate) and hematoimmune (total of hemocytes, differential hemocytes, phagocytic rate, superoxide dismutase and hypoxic stress) parameters were measured.Results. For biological indicators, the best results (p<0.05) were obtained when using 4×10. µL of the probiotic (final weight: 9.11 g; final length: 68.95 mm; specific growth rate: 3.74). Regarding the nutritional parameters, the best results were found with for 3×10. µL (feed conversion: 1.09, feed efficiency: 0.91, and protein efficiency: 2.61); although there were no differences between 3×10. and 4×10. µL. For the hematoimmune response, there were differences (p<0.05) for all the indicators under study, with a better performance for 4×10. µL of the probiotic mixture. Conclusions. The probiotic mixture induces the hematoimmune, biological, and nutritional response with the best response for concentrations of 3×10. µL, 4×10. µL.
Main Text
INTRODUCCIÓN
Aquaculture has become one of the fastest growing food production industry in Latin America (20%) and the world (7%) per year, significantly contributing to global food security and this growth has been extending continuously for more than two decades (1,2). After fish, crustaceans are placed as the second world aquaculture product.
Redclaw crayfish Cherax quadricarinatus is one of the cultivated species. It is native of northern Australia and to the southeast in Papua New Guinea (3), and it was introduced in various Latin America countries in the 90s. It is a specie adaptable to a wide range of weather conditions, with notable advantages due to its omnivorous eating habits, high growth rate, easy handling, adequate organoleptic characteristics, good meat quality rich in amino acids and fatty acids (4). It is appreciated in the market both for ornamental purposes and for human consumption (5).
However, in aquaculture, it is required to control stress and diseases and regardless of the species, the purpose of this is to ensure their health and productive yield (6). The use of prophylactic and therapeutic treatments (antibiotics) has been used to maximize the health and performance of cultured organisms. However, antibiotics have proven to have some disadvantages, as they penetrate into body tissues reducing their quality for consumption and causing health problems (7,8).
Antibiotics are also widely restricted for their impact on the environment (9) and because eventually, many pathogenic microorganisms become resistant (7,10). In this sense, the use of environmentally friendly food additives, such as probiotics, have becoming a good option as safe dietary supplements in the aquafeed industry, and are among the many strategies and alternatives to reduce the excessive use of chemotherapeutic agents such as antibiotics, developing the capacity for tolerance under stress conditions and improving the resistance and immunity of the host (11,12). They can also take advantage from their invertebrate host by fighting pathogens through a competitive exclusion mechanism (7,13), as well as promoting growth, survival and healthier animals (11,14). Probiotics commonly used in aquaculture include Gram-positive, Gram-negative bacteria, bacteriophages, yeasts, and single-celled algae (10,13,15).
Several studies have attributed an improvement in animal growth to the nutritional benefits of probiotic bacteria since they produce digestive enzymes and vitamins and make available minerals and trace elements (9). However, all the modes of action discussed above require that the particular probiotic that want to be utilized, can successfully colonize the region in where its effect can occur successfully (10,12).
In recent years, Bacillus spp. (16) and yeasts (17) have been tested frequently in crustacean aquaculture. In the case of Bacillus spp., this microorganism can sporulate, grow rapidly and tolerate a wide range of conditions in host. They also have been proven very useful to improve water quality, reduce harmful bacteria amount in the culture and maximize the host response capacity without the addition of antibiotics (18). Furthermore, oral administration of yeast species, in particular Saccharomyces cerevisiae, has been shown to enhance the immune response in crustaceans (19).
Previously, many research involving the application of beneficial bacteria as probiotics have been carried out (13,14,15). From these researchs is evident that a mixture of several probiotics might produce better results (20). In this sense, the adding of mixed probiotics to the food has provided benefits in species such as Macrobrachium rosembergii (6). However, the dose-effect relationship of a probiotic must be carefully determined to avoid an overdose, which can cause a decrease in its groeing promoting effects and an increase in costs, or conversely, the use of very low doses could drastically reduce its efficiency on the cultured animals (14,20).
Despite the scientific advances, current knowledge on the use of probiotics as feed additives and the immune response in aquaculture is scarce for the initial stages of development in many species, like Cherax quadricarinatus. Considering the above, the purpose of this research was to evaluate several biological, nutritional, and hematoimmune indicators of juvenile redclaw Cherax quadricarinatus fed with a probiotic mixture.
MATERIALS AND METHODS
Study site. This research was executed in the Aquaculture Laboratory, of the Universidad Técnica Estatal de Quevedo (UTEQ), Quevedo, Los Ríos, Ecuador (01°03’18’’S, 79°25’24’’W), with an altitude of 120 meters above sea level with an average temperature of 24 °C.
Ethical considerations. The study was carried out strictly following the Standard Operating Procedures (SOP) for the Use of Experimental Animals of the UTEQ.
Treatments and feeding: Six treatments were used, which consisted of increasing doses of probiotic mixture: 0 (control), 1×102 µL, 2×102 µL, 3×102 µL, 4×102 µL and 5×102 µL / 100 L of water, respectively. The probiotic mixture (Bacterol Shrimp Forte, Dukay SA, CL) utilized contained strains of Saccharomyces cerevisiae (5×10⁸ CFU / mL), Lactobacillus acidophilus (5×10⁸ CFU / mL) and Bacillus spp. (5×10⁸ CFU / mL). The mixture was incubated with molasses and water in a ratio of 1.5:10:3×103, respectively, for 24 hours, before being supplied directly into the water in the culture tanks every three days.
Juveniles were fed daily 5% of its live weight with basal pelleted diet, distributed in two portions (50% at 9:00 am and 50% at 5:00 pm). Basal pelleted diet was formulated (Table 1) and prepared according with Méndez-Martínez et al. (4,21) and the proximal analysis (dry matter, proteins, ethereal extract, nitrogen-free extract, ash, and crude energy) was performed according to methods of AOAC (22).
Experimental design: Juveniles C. quadricarinatus (0.95 ± 0.10 g and 7.78 ± 0.77 mm) were obtained under laboratory (UTEQ) conditions, acclimatized for a week before starting the experiment with a duration of 60 days.
A completely randomized design was applied of six treatments with 3 replications (experimental tanks) with 20 juveniles of C. quadricarinatus per tank, for a total of 360 juveniles and eighteen circular plastic tanks (diametric: 70 cm and area: 9.39 cm2) filled with freshwater (200 L), respectively. The water in the experimental tanks were replaced (40%) every three days, to eliminate feces and food remains, and water quality parameters were monitored; to determine water temperature, a mercury thermometer was used, dissolved oxygen (DO) was determined with a digital oximeter (55-DO, YSI Incorporated, Yellow Springs, OH, USA). The pH, ammonia (NH3 / NH4), nitrites (NO2-) and nitrates (NO3-) were measured with colorimetric kits (Saltwater Master Test, OH, USA), respectively (21).
Temperature was maintained between 27.5 and 30 °C with the help of built-in thermostats (JAD Aquarrium Co., Guangdong, CN), respectively (21). The pH was kept between 7.0 and 8.0 and dissolved oxygen (DO) between 4.7 and 6.2 mg / L, ammonium from 0 to 0.25 ppm, nitrite at 0.01 ppm and nitrate with values from 0 to 10 ppm.
Biological and nutritional parameters. To determine the weight in juveniles, a digital scale was used (PE 3600 Mettler-Toledo, ± 0.01 g, Columbus, OH, USA), the length was evaluated with a vernier caliper (GT-MA15 Gester, ± 0.001 mm, Xiamen, CN). Animals were previously anesthetized for biometrics. The following variables were calculated (23):
Increased weight (%) = (final weight - initial weight) × 100
Weight gain (g) = (final weight - initial weight)
Specific growth rate = (logarithm of final weight - logarithm of initial weight / number of days) × 100
Longitude gain = (final length - initial length)
Length increase = (final length- initial length) × 100
Survival = (number of final organisms / number of initial organisms) × 100
Feed efficiency = (weight gain / feed consumption) × 100
Protein efficiency = (weight gain / protein consumption)
Feed conversion = (feed intake / weight gain)
Hematoimmune responses. At the end of the feeding trial, .. quadricarinatus juveniles were fasted during 14 hours prior to the extraction of hemolymph. For this, they were placed in dorsoventrally, exposing the ventral hemolymphatic sinus. The surface was disinfected with alcohol (90%). The hemolymph extraction was carried out with sterile syringes (27G × 13 mm) with a hypodermic needle between the first pair of pleopods. SIC-EDTA was used as anticoagulant (450 mM NaCl, 10 mM KCl, 10 mM Hepes, 10 mM EDTA, pH 7.3) at 4 °C, to preserve a 2:1 ratio (2 volumes of SIC-EDTA per each extracted volume of hemolymph). The needle was inserted upwards, the hemolymph was extracted and immediately homogenized to prevent coagulation (24).
Then, the samples were placed in sterile 1.5 mL Eppendorf polypropylene microtubes and labeled. Subsequently, they were diluted in a ratio of 3:1 (150 µL of 4% formaldehyde per 50 µL of the anticoagulant and hemolymph mixture), which were stored at 4 °C to perform the total count of hemocytes and differentials (hyaline, semi-grainy and grainy) (25). For the analysis of phagocytic activity, superoxide dismutase (SOD) and oxyhemocyanin, hemolymph without formaldehyde was used.
Phagocytic activity was measured in agreement with Hauton (26) and Chen et al. (27). Fresh hemolymph (40 µL) was spread on glass slides. Then, slides were incubated until the hemolymph was dried out. Meanwhile, a zymosan working solution was prepared by dissolving 0.0125 g of powdered zymosan in 25 mL of sterile seawater. Zymosan solution (40 µL) was added to the dried hemolymph samples on the slide and air dried. Then, they were treated with a 10% formaldehyde solution (seawater solvent) for 20 min. Subsequently, the glass slides were transferred to a GIEMSA solution and incubated during 20 min for cell staining of hemocytes.
To determine the oxyhemocyanin concentration, 20 µL of hemolymph diluted with 80 µL of SIC-EDTA was added and the absorbance at 335 nm was read with a spectrophotometer (28,29). The SOD enzyme activity was determined using 10 μL of hemolymph and a commercial kit (Ransel, Randox, Crumlin, Antrim, UK) based on the principle of the oxidation of glutathione (GSH) by cumene hydroperoxide catalyzed by GPx with glutathione reductase (GR) and NADPH. The absorbance was read at 340 nm.
At the end of the bioassay, hypoxia stress was determined. For this, 18 plastic containers of 200 mL each and six organisms were used for each test. Each container was considered a repetition. The elimination of dissolved oxygen (<0.1 mg / L) in the water was assured by adding sodium bisulfite (NaHSO.) (0.15 g / 500 ml). Juveniles were exposed to hypoxia for 1 h. Samplings were carried out every 5 minutes and the number of living and dead organisms were recorded.
Statistical analysis. Bartlett and Kolmogorov-Smirnov tests were applied. A simple Analysis of Variance (ANOVA) (. ≤ 0.05) was then applied to the different levels of probiotic mixture, which was the only source of variation. For the analysis of differences between means, a Tukey’s test was applied. All statistical processing (. ≤ 0.05) were performed with the statistical program 14.0v (InfoStat®, Cordova, AR).
RESULTS
Biological indexes in present research for tested juveniles are presented in Table 2. Final weight, weight increase, weight gain, specific growth rate, final length, length increase, and length gain in treatments using 3×102 µL and 4×102 µL of the probiotic mixture there was no statistical difference, but there was a significant difference (p < 0.05) compared to the control and the others treatments, respectively. Survival was significantly higher (p < 0.05) (88.33%) for the treatment with 3×102 µL of the probiotic mixture.
Nutritional parameters (feed conversion, feed efficiency and protein efficiency) are presented in Table 2. No significant differences were found (p<0.05) between the treatments with 3×10. µL and 4×10. µL of the probiotic mixture, but there were differences between the control and all others.
Significant differences on hematoimmune response are presented in Table 3. Total hemocytes (9.27 and 11.74 million cells / mL) and hyaline hemocyte cells (28.30 and 25.86%) showed a better response (p<0.05) with in the treatments with 3×10. and 4×10. µL of the probiotic mixture, respectively. A higher granulocyte hemocyte cell count (33.17%) and phagocytosis rate (38.28%) was observed (p<0.05) in the 4×10. µL treatment, respectively. While semi-granulocytes were higher in the control with 60.79%. For oxyhemocyanin, the highest (p<0.05) values were 0.84 and 0.82 mmol / L, when 2×10. µL and 3×10. µL of a probiotic mixture were applied, higher (p<0.05) in comparison with the control.
When analyzing hypoxic stress survival percentages of SOD activity (Figures 1 and 2), significant differences (p<0.05) were found depending on dosage. The highest hypoxia survival values were found in the 4×102 µL treatment with 76.19%. It should be noted that increases of 19.05, 57.15, 47.62, 66.67 and 57.15% (1, 2, 3 and 4×102 µL of probiotic mixture) were obtained in comparison with the control. While the enzyme SOD maintained a similar trend. Its highest value were with the concentration of 2×102 µL (41.18 Unit / mL), although with this concentration did not had significant differences in comparison with 4×102 µL of the probiotic mixture.
DISCUSSION
During aquaculture practices, the accumulation of organic matter affects water quality and cause pollution. It also allow pathogens that grow inside the culture. Water quality in our study were in agreement with the standards for freshwater decapod crustaceans including C. quadricarinatus (23,30,31,32).
Biological and nutritional parameters in present research were influenced by the dose of the probiotic mixtures as a result of the synergistic effect between the strains and its secondary metabolites. This effect can be beneficial since probiotic strains not only synthesize extracellular enzymes such as proteases, amylases, and lipases, but also other compounds required for growth (vitamins, amino acids, and fatty acids), which contribute to the absorption of nutrients more efficiently (15). This has been previously proven by Seenivasan et al (6), when supplementing with different doses of probiotic, reported survival (90%), growth (1.04 g), growth rate (0.88%), feed conversion (1.57 g), and protein efficiency (1.38%) significantly higher regarding the control treatment. Some other studies have shown positive effects of probiotics, such is the case of Bacillus sp. and Clostridium sp. on the growth of M. rosenbergii, where these food additives have been demostrate to improve digestion, assimilation, and metabolism of nutrients in crustaceans and fish by promoting the synthesis of digestive enzymes, improving growth and survival (33).
Some previous research had tested the use of probiotics with C.quadricarinatus, where Amrullah and Wahidah (14) found increases in body weight up to 7.30 g, with 2.63 g higher than the control treatment and with a survival of 73% while feeding the species with diets supplemented with three different concentrations of the probiotic mixture of Micrococcus spp. This effect can be caused by the symbiotic behavior of probiotics, besides stimulating the microbiota of the digestive tract with a more efficient intestinal biota and modifying the selection of bacterial enzymes (11).
Pérez-Chabela et al (33) found that using a mixture with strains of Bacillus subtilis, B. licheniformis and B. subtilis, supplied in the food as probiotics to Litopenaeus vannamei juveniles, improves growth rates by increasing the concentration of the probiotic mixture. Madani et al (34), when evaluating the effect of mixing probiotics (Bacillus subtilis and Bacillus licheniformis) in .. vannamei larvae, found that the addition of the probiotic mixture to the food had a positive effect on growth, which was confirmed by Zhao et al (35) in Macrobrachium rosenbergii, finding beneficial effects on biological and nutritional parameters, as well as an improvement on the action of digestive enzymes and optimization of costs.
Table 2 and 3 shows that probiotics improved growth and nutrient uptake because of their ability to stimulate beneficial intestinal microbiota, also with hematoimmuno-regulatory effects (10). In C. quadricarinatus, Amrullah and Wahidah (14), found that mixtures of probiotics with Micrococcus spp., increased total hemocytes, hyaline hemocytes, semi-granulocytes and granulocytes, as well as phagocytosis activity. This is a reason of an increasing in immunological activity caused by the activation of the non-specific immune response. Hemocytes are a reliable indicator to determine and prevent diseases, as well as a marker of the physiological status of the animal, since hemocytes in crustaceans is the basis of the immune system as they perform phagocytosis, encapsulation and lysis of unwanted cells (16).
Hemocytes play a vital role in defense, hyaline cells are responsible for phagocytosis, semi-granules also play a role in phagocytosis, encapsulation and in the release of the prophenoloxidase system. Furthermore, they synthesize and release peneidins and peptides, the granulated cells store the enzymes that constitute the prophenoloxidase system at a higher level than the semi-granular ones, just as they synthesize and store the peneidins, they intervene in encapsulation; which have been demonstrated to be an important part of the innate immune system (17). Zhao et al (35), when using different concentrations of Bacillus pumilus in M. rosenbergii, they did not find differences in the total hemocyte count, while for phagocytic activity (37%), differences were highly significant.
Valipour et al (36), used Lactobacillus plantarum in Astacus leptodactylus to evaluate its effect on the immune response, finding increases of 1.2×10. cells / mL and 1.4×10. cells / mL for total hemocyte and hyaline hemocyte cell count, respectively. Azad et al (37) evaluated the effect of probiotics on the immunological competence of .. rosenbergii against Vibrio spp. and Aeromonas spp., finding that prawns treated with probiotics improved the hematoimmune parameters and total hemocyte count (11×10. cells / mL), hyaline hemocyte cells (79%) and semigranulocyte hemocyte cells (19%). Such results differ from those obtained in this work, which can be attributed to species, experimental conditions, type of probiotic or experimental design. Results obtained indicate a greater capacity to prevent the invasion of foreign particles. It is known that hemocytes are also involved in different physiological functions, including carbohydrate metabolism, transport and storage of proteins and amino acids, stress regulation, leading to a disease resistance (13,15).
In present study, SOD activity was significantly influenced (p<0.05) by the probiotic concentration. Since nutritional status is the most important factor influencing immune defense mechanisms, low or high quantities of nutrients can alter the immune system, causing cell stress (32). In this sense, Soberanes-Yepiz et al (38), found that high antioxidant activity are the consequence of multiple oxidative reactions and therefore, an index of a high production of free radicals. In present research, the use of probiotics can be an important tool as an immunostimulant to prevent diseases, given the role played by beneficial bacteria in the prevention of the spreading of pathogens.
Ranjit-Kumar et al (39), when using different concentrations of Bacillus licheniformis as a probiotic, found an increased superoxide dismutase (SOD) and antibacterial activity (76%), which confirms such assumptions. In decapoda crustaceans, the level of SOD decreases during pathogenic infection, since its effect on the delay of the normal activity of cells of the hepatopancreas and hemocytes. Hence, the increase in SOD activity with probiotic supplementation is an index of positively regulated immune status.
In conclusion, probiotic mixtures induce an hematoimmune response, improving the biological and nutritional indicators. Such effect is best at at concentrations of 3×102 µL and 4×102 µL.
Conflict of interests
All authors declare that during the preparation and preparation of this work there was no conflict of interest.
Abstract
Main Text
INTRODUCCIÓN
MATERIALS AND METHODS
RESULTS
DISCUSSION