1. Introduction
Sturgeon is one of the most threatened fish species in the world due to the obstruction of spawning migrations, the construction of river dams, the destruction of natural spawning grounds, and overexploitation [1,2]. Due to the declining stocks of many natural sturgeon populations, sturgeon aquaculture is growing rapidly, and today most sturgeon products come from aquaculture. The sustainable use of natural resources is a challenge for the future development of this sector [3]. The scientific research on sturgeon culture has developed rapidly to support and optimize new breeding technologies [4,5] and nutritional requirements [6]. Compared to other sturgeon species, Siberian sturgeon (Acipenser baerii) has a faster growth rate and a relatively shorter reproductive cycle (7–8 years). It is a species more resistant to pathogens and environmental conditions [7,8] and one of the most valuable species in aquaculture, due to the production of caviar and high-quality meat for human consumption. The sustainable development of aquaculture involves an effective management of diseases and animal welfare, correlated with stress causing factors [9]. The scientific community is looking for alternatives to reduce the abuse of antibiotics, because they lower the immunity of the fish, damage the aquatic environment and can be found in fish tissue, affecting the health of the consumer [10,11].
Probiotics included in diets can improve growth parameters and disease resistance. They have the role of improving digestion and reducing permeability induced by pathogenic microorganisms [12]. Plant components used in feed can have negative effects on fish nutrition due to the anti-nutritional effects of secondary metabolites from plant, with negative consequences on the health and immunity of the fish [13]. By using probiotic supplements, these problems can be remedied by improving feed digestibility and gut micro biota, which are key components in the development and modulation of the immune system [14].
The objective of this study was to analyse the effect of probiotics on survival, health, growth indices, and blood composition in Acipenser baerii reared in a recirculating system and fed diets supplemented with Lactobacillus acidophilus (FLa) and Saccharomyces boulardii (FSb), separately and combined (FLa + Sb).
2. Materials and Methods
2.1. Experimental Design
The rearing system was composed of 4 rearing tanks, fabricated from fiberglass, with the dimensions 1.2 × 1.25 × 0.5 m and a water volume of 0.35 m3.
The water treatment system was composed from: decanter tank, pumping group (2 pumps), sedimentary filter with discs, 2 sand filters, 2 active carbon filters, one denitrifier, sterilization installation with UV and a filtered water storage tank.
The effect of probiotics was investigated on 2000 specimens of Acipenser baerii with the age of 60 days and an initial mean weight of 8.82 ± 1.22 g reared in the pilot station of artificial reproduction belonging to Institute of Research and Development for Aquatic Ecology, Fishing and Aquaculture, Galați for 8 weeks.
Fish were randomly distributed into four fiberglass tanks (control tank and three experimental tanks) with a capacity of 350 L and a feed rate of 4–8 L/min/tank.
Before sampling, fish were anesthetized by bathing for 5 min in a solution of 0.025 mL of clove oil and 1 L of water with a temperature of 23 °C, according to law no. 43/2014, on the protection of animals used for scientific purposes and Directive 2010/63/EU of the European Parliament and of the Council from 22 September 2010 on the protection of animals used for scientific purposes.
2.2. Fish Feeding Experiments
The control tank (C) (N = 500 specimens, initial mean weight = 8.52 ± 1.32 g) was fed a commercial feed (Nutra no. 3-Skretting, Italia), purchased form Dream Fish - Bucharest, România, with the granulation of 0.4–0.8 mm, without the addition of probiotic coded FC.
Swanson ProLacto Acidophilus and Saccharomyces Boulardii from Yamamoto® Research were used as probiotics. Both are dietary supplements in capsule form for human use, purchased from a drug store in Galați, România.
For the experimental tanks, the commercial feed was enriched with lactic acid bacteria and yeast as follows:
0.019% addition of Lactobacillus acidophilus coded with FLa, (6 × 109 CFU (187.0 mg)/kg feed) for tank T1;
0.030% addition of Saccharomyces boulardii coded with FSb, (6 × 109 CFU (300.00 mg)/kg feed) for tank T2;
0.009% addition of Lactobacillus acidophilus (3 × 109 CFU-93.75 mg) and 0.015% Saccharomyces boulardii (3 × 109 CFU–150.00 mg) in equal proportions, coded with FLa + Sb, (6 × 109 CFU/kg feed), for tank T3.
The three variants of feed with probiotic were prepared by dissolving each probiotic, or in a mixture, in a 2% gelatine solution. The probiotic solution was sprayed over the commercial feed, which was then dried at a temperature of 25 °C for 4 h, in a ventilated oven.
The feed administered daily was 0.5–1% of the biomass, measured weekly depending on water temperature and feed availability.
2.3. Physical and Chemical Parameters of the Water
An evaluation of the physico-chemical parameters of the water was performed for the purpose of monitoring the aquatic environment, with the possibility of intervening in case of deviations in water quality conditions from the allowed limits.
A portable multiparameter, model HQ40D-Hach, was used to measure the pH, temperature, dissolved oxygen, and oxygen saturation.
Nitrogen compounds (ammonia, ammonium ions, nitrite and nitrate ions) were determined spectrophotometrically according to Standard Methods for the Examination of Water and Wastewater, 2005, with a Hach Lange DR 1900 spectrophotometer using LANGE kits.
The determination of chemical oxygen consumption (CCO-Mn) was performed using the potassium permanganate method, expressed in mg KMnO4/L, according to the standard SR ISO 6060:1996. A mineralizer, model LT 200 from Hach Lange, was used to determine the organic matter.
The concentration of oxygen in the water varied between 6.82 ± 0.74 mg/L and 7.29 ± 0.62 mg/L. The pH values varied between a minimum of 7.18 upH and a maximum of 8.21 upH throughout the experiment, values that fall within the range for this species at this stage of development. The values of nitrogen compounds and organic matter fell within the limits established by Order no. 161/2006 [15] regarding the classification of surface water quality in order to establish the ecological status of water bodies throughout the experimental period.
2.4. Assessment of Growth Performance and Feed Efficiency
To evaluate the growth parameters, 50 specimens were randomly collected from each tank, both at the start and at the end of the experiment, which were measured and weighed (wet weight).
The fish condition factor represented by Fulton coefficient (K, %), individual weight growth (WGi, g), total weight growth (WGt, kg), feed conversion ratio (FCR, kg/kg) and Specific growth rate (SGR, %/day), were determined as follows:
K (%) = Weight (g)/Standard body length (cm) 3 × 100;
WGi = Final weight-Initial weight (g/fish);
WGt = Final lot weight-Initial lot weight (kg/total fish);
FCR = feed fed (kg)/weight gain (kg);
SGR = 100 × [(ln Final fish weight)-(ln Initial fish weight)]/experimental days;
2.5. Feed Composition
The feed analysis was performed using the procedures indicated by the standard methods of analysis for feed used in animals.
The moisture was determined by Standard Official Methods of the AOAC (1990).
The total ash was determined by Furnace Incineration described by AOAC (1990).
The crude protein contents of the samples were determined using the Kjeldah method of AOAC 17th edition, 2000, Official Method 928.08 Nitrogen in Meat (Alternative II), which involved protein digestion and distillation, where F (conversion factor) is equivalent to 6.25.
Both insoluble and soluble fibres were determined using the Gerhardt Fiberbags System, according to ISO 6541:1981 (Agricultural food products—Determination of crude fibre content—Modified Scharrer method) reference.
The total fats were determined using the Soxhlet method, equipped with Gerhardt Brand Multistate Controller, with modified ether extraction methods AOAC 960.39.
The total carbohydrate percentage was determined by the difference method. This method involved adding the total values of crude protein, lipid, moisture, ash, and fibre from the sample and subtracting it from 100.
2.6. Haematological Analysis
The collection of blood samples for haematological, biochemical, and immunological determinations was carried out only at the end of the experiment, motivated by the fact that at the beginning of the experiment the fish size was too small and presented a risk of mortality. From each tank, 5 fish were randomly chosen from which blood was collected by caudal vein puncture.
To separate the serum, blood was collected on anticoagulant (EDTA-ethylene-diamino-tetraacetic-acid). Samples were allowed to clot for 15–20 min at 4 °C before centrifugation (3000 rpm, 20 min). Fresh serum was then subjected to biochemical analysis using the Celercare VS Analyser and Health Checking Kit, in collaboration with the Royal Vet veterinary medicine office.
To determine the number of erythrocytes (mil.cel/µL blood), a Newbauer haemocytometer was used by counting directly under a microscope, the haemoglobin concentration (Hb, g/dL) was determined by the colorimetric method with Drabkin reagent, and the haematocrit value (PCV, %) was determined by the microhaematocrit method with a Dlab DM 1224 centrifuge.
Mean corpuscular volume (MCV, fl), mean corpuscular haemoglobin (MCH, pg), and mean corpuscular haemoglobin concentration (MCHC, g/dL) were determined as follows:
MCV = Hct × 10/no. erythrocyte;
MCH = Hb × 10/no. erythrocyte;
MCHC = Hb × 100/Hct.
Fresh serum was then subjected to biochemical analysis using the Celercare VS Automated Biochemical Analyser and Health Checking Kit following the manufacturer’s instructions. The studied parameters were Immunoglobulin M (IgM), total proteins (TP), albumin (ALB), globulin (GLO), glucose (GLU), cholesterol (CHOL), and the following enzymes: Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), and Creatine kinase (CK)
2.7. Statistical Analysis
Data are presented as mean ± standard deviation. A comparison of several samples was performed using the ANOVA single factor test followed by the Student’s t-test (p < 0.05).
3. Results
3.1. Fish Growth Parameters
Initial and final body length and weight are presented in Table 1. Compared to the control tank, the largest increase in body length was recorded in tank FLa + Sb (68.48%). The largest weight gain was recorded in tank FLa + Sb (37%), followed by tank FSb (31.3%) and tank FLa (16.48%), compared to the control tank.
The statistics of the studied specimens, at the end of the experiment, regarding the standard weight and length, indicated significant differences between the control tank and the three experimental tanks (p < 0.01).
Regarding body mass and length, between tanks FLa and FSb, respectively, tanks FLa and FLa + Sb, there were significant differences (p < 0.01), while between tanks FSb and FLa + Sb the differences were insignificant regarding weight (p = 0.34) but significant regarding standard length (p = 0.01).
The CV values at the beginning and at the end of the experiment regarding weight and length were below 15%. Therefore, the sample is homogeneous, the mean is representative, and data scattering is small.
In order to characterize the condition factor of the Siberian sturgeon, fed with added probiotics, somatic measurements were performed at the beginning and at the end of the experiment, based on which the values of the Fulton coefficient were calculated.
The final average values of the Fulton K coefficient were between 0.39 and 0.48 in the experimental tanks and 0.61 in the control tank.
The effect of probiotics on the growth parameters of the Siberian sturgeon is presented in Table 2.
The stocking density was between 12.17 and 13.07 kg/m3 (2.88–3.05 kg/m2). Motivated by the accumulation of biomass, at the end of the experiment, the density increased, being between 32.99 and 56.08 kg/m3 (7.70–13.09 kg/m2).
The results indicated an improvement in the individual growth rate in the experimental tanks that received the probiotic, compared to the control tank, the values being 16.91% (FLa), 31.32% (FSb), and 37.03% (FLa + Sb) higher.
Feed conversion ratio (FCR), represented by the quantity of feed consumed to obtain 1 kg of fish weight, registered higher values in the experimental tanks (2.22 for tank FLa, 1.91 for tank FSb, 1.58 for tank FLa + Sb) compared to control tank C (3.02) (Table 2).
Specific growth rate (SGR) in the experimental tanks was 2.27% (FLa), 2.45% (FSb), and 2.70% (FLa + Sb), compared to tank FC, 1.78%, that did not receive probiotic.
Survival was estimated by recording dead larvae daily. There were differences regarding the survival rate between the control tank (77%) and the experimental tanks (83.20% FLa, 85.40% FSb, 88.80% FLa + Sb).
3.2. Chemical Analysis of Feed
Nutrient concentrations found in the administered feed during the experiment were in accordance with the recommendations from the specialized literature for the 60-day-old Siberian sturgeon [16] and can be found in Table 3.
3.3. Haematological and Biochemical Profile of the Blood
The blood haematological profile of Acipenser baerii fry, reared in a recirculating system for 56 days and fed with diets supplemented with Lactobacillus acidophilus and Saccharomyces boulardii can be found in Table 4.
At the end of the experiment, the haematocrit, haemoglobin, and erythrocytes values were lower in the control tank, compared to the experimental tanks fed with probiotic addition. In tank FLa + Sb, the highest increase was recorded for haematocrit (20.19%), haemoglobin (65.82%) and erythrocytes (35.18%), compared to the control tank.
The statistical analysis revealed that there were no significant differences between the experimental tanks and the control tank regarding the values of haematocrit (p = 0.11) and erythrocytes (p = 0.067).
Regarding the haemoglobin values, significant differences were found between tank FC and the experimental tanks (p ˂ 0.05) and insignificant differences between tank FSb and FLa + Sb (p = 0.25).
From a statistical point of view, the average values of Ht and Hb RBC were representative for the analysed sample (CV < 15%), with the data scattering being low.
The RBC values had a larger spread around the mean value, the mean being still sufficiently representative (CV was between 16% and 20%).
The haematological examination also included the analysis of erythrocyte indices, which describe the morphology and properties of erythrocytes, such as MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin), and MCHC (mean corpuscular haemoglobin concentration) (Table 5).
The total number of leukocytes did not vary essentially, the values being between 21.62 and 22.74 × 103/μL. There were no statistically significant differences (p = 0.67). Of the total white blood cells (WBC), lymphocytes were found in the largest quantity, followed by neutrophils and monocytes in all tanks.
The differences between the control and the experimental tanks were insignificant from a statistical point of view, regarding the main leukocyte components: lymphocytes (p = 0.97), monocytes (p = 0.98), respectively, neutrophils (p = 0.50).
The results of the biochemical profile of the blood are presented Table 6. Total proteins, albumin, AST, and ALT increased compared to the control tank, but the differences were not statistically significant (p > 0.05). The addition of probiotics to the feed of fish from tanks FLa, FSb, and FLa + Sb caused a decrease in values for glucose and cholesterol compared to the control tank, with the differences being insignificant (p > 0.05).
Serum Immunoglobulin M (IgM) values in Siberian sturgeon fry, fed with added probiotic, were higher compared to the control tank. There were significant differences between the IgM values identified in the tank that received FLa + Sb and the control tank without probiotic addition (p = 0.005).
4. Discussion
Currently, there is an increased interest in aquaculture production, capable of increasing the amount of healthy food available for the population. Research is being undertaken on the effects of different feed additives on the growth performance of fish and their well-being. The addition of probiotics to the diet has been studied by many authors over time [17,18,19,20].
In this study, the addition of probiotics to the fish diet led to the improvement of growth parameters, compared to the control tank without probiotics. The tank T2, receiving Saccharomyces boulardii (Sb), showed a greater individual and total growth increase, compared to the tank receiving Lactobacillus acidophilus (La), suggesting that this yeast better stimulates the growth of Acipenser baerii. The tank that received a complex of yeasts and bacteria, T3, showed the highest biomass accumulation. Similar results were obtained by Ghaziani, S.D.; et al. [21] regarding specific growth rate (SGR) in Acipenser baerii. Positive results on growth performance due to probiotic addition were also obtained in common carp [22,23]. Yeast as a probiotic used by Lara-Flores et al. in feeding diets of tilapia fingerlings resulted in better growth [24]. Moreover, Pourgholam et al. (2017) [25] recommends diets enriched with probiotics to improve the growth and immune response of Siberian sturgeon. The use of lactic acid bacteria in rearing turbot larvae resulted in an increase in biomass accumulation and disease resistance [26].
The Fulton condition factor, K, usually used to characterize the well-being of fish, had lower values in the experimental tanks. This indicates the beneficial effect of the feed with added probiotic on the well-being of fish.
The lowest value of the Fulton coefficient (0.39), which characterizes the best health condition of the fish, is found in the tank fed with a complex of bacteria and yeasts. Therefore, this variant is the most favourable for obtaining a fish with a superior health condition.
The values of the condition factor (K) in the present experiment are lower than those identified by Pyka J. et al. [27], rearing Acipenser baerii in earthen ponds.
Similar to the results obtained in this study, in which the survival rate of the fish fed with added probiotics was higher than the control, Carnevali O. et al. [28] successfully used two strains of Lactobacillus to decrease mortality in sea bream larvae. Moreover, in experiments carried out by Gram et. al. [29], Pseudomonas fluorescens was used as a probiotic, decreasing the mortality of Oncorhynchus mykiss fry.
These results regarding the growth performance highlighted the efficiency of probiotics used in diets for Acipenser baerii, reared in a recirculating system.
The aim of the haematological analyses was to identify whether the two probiotics, introduced separately or together in the diets for Acipenser baerii, determined a change of the haematological and plasma indices.
In sturgeons reared in intensive systems, knowing the values of haematological parameters provides useful information regarding physiological disorders, changes in health status, as well as the prognosis and diagnosis of diseases.
The values of haematological indices in Acipenser baerii in the present experiment were within a normal range, characteristic of sturgeons in a good state of health, similar to those identified by Ramezani, S. et al. [30]
Haematological indices (Ht, Hb, and RBC) changed in relation to the diets. The mean values of Ht, Hb, and RBC, in the present experiment are lower compared to those obtained by Hassani, M.H.S. et al., [31], who studied the effect of probiotics on growth performance and haematological and immune indices in Acipenser baerii fry.
The results of this study, regarding the haematological indices, in which the values were within normal limits, without significant differences, can be explained by the adaptive response of the fish to a nutrition supplemented with probiotics, without adverse reactions in the rearing technology of Acipenser baerii. The probiotics introduced in the diets did not represent a stress factor that would negatively modify the values of the blood parameters.
The blood analysis values in Acipenser baerii are significantly different from those identified by Rouhollah, V. et al. [32], in Cyprinus carpio, with the mention that the two species belong to different trophic levels.
In our study, erythrocytes, which represent an important indicator of the stress state of the fish, had values similar to those identified by Hassani, M.H.S. et al., [31]. Among the white cells involved in the body’s defence system, lymphocytes, responsible for the production of antibodies, were found in the largest amount, followed by neutrophils (useful in the defence against bacteria and fungi) and monocytes (involved in the defence against bacterial infections). The percentage of lymphocytes, monocytes and neutrophils is slightly increased in the experimental tanks (FLa, FSb, FLa + Sb) fed with added probiotic compared to the control FC. The lower percentage of lymphocytes and neutrophils in the control tank may result in a less efficient immune system. The percentage of neutrophils in Acipenser baerii, from our experiment (16.76–18.43%) was higher than that identified by Salkova, E. et al., [33] in the three species of sturgeon reared in a recirculating system for a year. The addition of probiotics in the diets of Acipenser baerii fry caused an increase in the number of lymphocytes, monocytes and neutrophils to the detriment of eosinophils and basophils. Similar results were obtained by Dima F.M. et al. (2022) in carp fry by adding a mixture of BioPlus® 2B probiotics [34]. Neutrophils are the first immune cells to react by producing antibacterial molecules when a pathogen becomes active inside the fish body [35].
Total serum protein is considered the most stable blood component (Jácome et al. 2019) [36] and acts as an indicator of nutritional status, physiological state, stress, and fish health (Ahmed et al. 2019) [37]. The results obtained were in accordance with Dipanjan et al. [38], who studied the impact produced by Bacillus tequilensis used as an additive in feed or in water on haemato-immunological parameters in Labeo rohita.
The albumin/globulin ratio is well above the limit of 0.3. The increase in the A/G ratio is not very important, but the decrease of this ratio below this value indicates either a disease that reduces the synthesis of blood albumin, or a disease that increases the synthesis of globulins.
The decrease in albumin, below the normal limit, suggests a disturbance of the protein synthesis function of the liver, which is not valid for the fish involved in our experiment, the liver being considered the main target organ that responds quickly and bio-accumulates toxic substances.
IgM is the most common immunoglobulin with a role in both the innate and adaptive immune systems, producing specific antibody responses against various antigens [31,39]. In cartilaginous fish, IgM is a major serum protein accounting for more than 50% of serum protein [40].
Regarding the concentration of glucose, values in the experimental tanks that received probiotics were lower than those identified by Alireza et al., [41], who studied the influence of fish density on glucose and cholesterol in Siberian sturgeon fry.
Serum biochemical parameters in our experiment are similar to those identified by Ramezani et al., [30], studying serum biochemical parameters in Acipenser baerii fed a diet supplemented with Immunogen. CK and CRE values are within normal limits, which indicates the absence of lesions or atrophy in the muscles.
5. Conclusions
The results of this study concluded that the bacteria (Lactobacillus acidophilus) and yeasts (Saccharomyces boulardii), used as probiotics, increased the digestion process and feed conversion, improved health, increased disease resistance, decreased sensitivity to stress, and improved the maintenance status of fish.
The combined supplementation of the Siberian sturgeon diet with probiotic bacteria (Lactobacillus acidophilus) and probiotic yeast (Saccharomyces boulardii), through a synergistic action, modulates the physio-metabolic responses and the innate immune system more effectively than their individual supplementation.
Modulation of the gastrointestinal microbiome in Acipenser baerii, by administering probiotics in feed, is a potential strategy to improve the production of microbial metabolites, stimulate immune signalling, and increase defence mechanisms against pathogens.
Modulation of the gastrointestinal (GI) microbiota in Acipenser baerii by using probiotics is promising due to beneficial outcomes on survival and health status, but microbiome research is still limited compared to other hosts and requires further research.
Based on the results of the present study, the bacteria (Lactobacillus acidophilus) and yeast (Saccharomyces boulardii), used separately and together, could be probiotics in the rearing of Siberian sturgeon (Acipenser baerii).
Conceptualization, E.E.M.; methodology, E.E.M.; software, E.E.M., V.S. and M.D.P.; validation, E.E.M., V.S. and M.D.P.; formal analysis, E.E.M.; investigation, V.S. and M.D.P.; resources, F.M.D.; data curation, E.E.M., V.S. and M.D.P.; writing—original draft preparation, E.E.M., V.S. and M.D.P.; writing—review and editing, E.E.M., V.S. and M.D.P.; visualization, E.E.M., V.S. and M.D.P.; supervision, E.E.M.; project administration, F.M.D.; funding acquisition, F.M.D. All authors have read and agreed to the published version of the manuscript.
All procedures involving animals were conducted in line with the Romanian legislation (Low 43 of 11 April 2014) on the protection of animals used for scientific purposes, approved by the Ethical Commission of Veterinary Medicine Section of the Academy of Agricultural and Forestry Sciences (Decision No. 17 of 9 February 2021).
All data used in this study are presented in this article.
The authors declare no conflict of interest.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Fulton coefficient in Siberian sturgeon (n = 50) reared in a recirculating system, fed with the addition of probiotics for 56 days.
| Variable | Tank C | Tank T1 | Tank T2 | Tank T3 |
|---|---|---|---|---|
| Statistical Indices | FC | (Fla *) | (FSb **) | (FLa + Sb ***) |
| Initial Parameters | ||||
| Individual weight (g) | ||||
| Min. | 6.1 | 7.1 | 6.8 | 6.4 |
| Max. | 11.95 | 10.9 | 10.9 | 10.6 |
| Mean ± Standard Deviation | 8.52 ± 1.32 | 8.95 ± 1.13 | 9.15 ± 1.26 | 8.65 ± 1.15 |
| Variability coefficient, CV | 0.16 | 0.13 | 0.14 | 0.13 |
| Individual standard length (cm) | ||||
| Min. | 7.8 | 9.4 | 7.2 | 7.9 |
| Max. | 12.5 | 13.5 | 10.9 | 12.6 |
| Mean ± Standard Deviation | 10.50 ± 0.96 | 11.20 ± 0.92 | 9.80 ± 0.83 | 10.11 ± 1.07 |
| Variability coefficient, CV | 0.14 | 0.10 | 0.20 | 0.17 |
| Fulton coefficient, K (%) | 0.73 | 0.64 | 0.97 | 0.84 |
| Final Parameters | ||||
| Individual weight (g) | ||||
| Min. | 26.3 | 30.4 | 28.5 | 23 |
| Max. | 43.71 | 48.5 | 59.8 | 53.6 |
| Mean ± Standard Deviation | 34.47 ± 4.78 c | 39.29 ± 3.87 b | 43.23 ± 8.84 a | 44.21 ± 7.35 a |
| Variability coefficient, CV | 0.09 | 0.08 | 0.08 | 0.11 |
| Individual standard length (cm) | ||||
| Min. | 16.7 | 17.6 | 18.2 | 18.2 |
| Max. | 21.1 | 23.6 | 25.6 | 25.3 |
| Mean ± Standard Deviation | 17.80 ± 1.08 d | 20.20 ± 1.38 c | 21.50 ± 2.13 b | 22.40 ± 2.07 a |
| Variability coefficient, CV | 0.06 | 0.07 | 0.10 | 0.09 |
| Fulton coefficient, K (%) | 0.61 | 0.48 | 0.44 | 0.39 |
* Lactobacillus acidophilus (6 × 109 CFU/kg feed). ** Saccharomyces boulardii (6 × 109 CFU/kg feed). *** Lactobacillus acidophilus (3 × 109 CFU/kg feed) and Saccharomyces boulardii (3 × 109 CFU/kg feed). Different superscript in the same rows represent significant differences, by one-way ANOVA and t-test (p < 0.05).
Bioproductive indicators obtained by rearing Siberian sturgeon in recirculating system and fed for 56 days with added probiotic.
| Growth Parameters | UM | Tank C | Tank 1 | Tank 2 | Tank 3 |
|---|---|---|---|---|---|
| FC | (Fla *) | (FSb **) | (FLa + Sb ***) | ||
| Initial Parameters | |||||
| Number of Specimens | - | 500 | 500 | 500 | 500 |
| Mean individual weight | (g/specimen) |
8.52 ± 1.32 | 8.95 ± 1.13 | 9.15 ± 1.26 | 8.65 ± 1.15 |
| Initial Biomass | Kg | 4.26 | 4.48 | 4.58 | 4.33 |
| Density of the initial population | kg/m3 | 12.17 | 12.79 | 13.07 | 12.36 |
| Density of the initial population | kg/m2 | 2.84 | 2.98 | 3.05 | 2.88 |
| Final Parameters | |||||
| Number of Specimens | - | 335 | 406 | 417 | 444 |
| Mean individual weight | (g/specimen) |
34.47 ± 4.78 c | 39.29 ± 3.87 b | 43.23 ± 8.84 a | 44.21 ± 7.35 a |
| Final Biomass | kg | 11.55 | 15.95 | 18.03 | 19.63 |
| Density of the final population | kg/m3 | 32.99 | 45.57 | 51.51 | 56.08 |
| Density of the initial population | kg/m2 | 7.70 | 10.63 | 12.02 | 13.09 |
| Growth Parameters | |||||
| Number of days | days | 56 | 56 | 56 | 56 |
| Weight growth individual (WGi) | g | 25.95 | 30.34 | 34.08 | 35.56 |
| Weight growth total (WGt) | kg | 7.29 | 11.48 | 13.45 | 15.30 |
| Total Shared Food | kg | 22.00 | 25.50 | 25.70 | 24.20 |
| Feed Conversion Rate (FCR) | kg/kg | 3.02 | 2.22 | 1.91 | 1.58 |
| Daily growth rate (DGR) | g/day | 0.46 | 0.54 | 0.61 | 0.63 |
| Specific growth rate (SGR) | %/day | 1.78 | 2.27 | 2.45 | 2.70 |
| Survival | % | 77.00 d | 83.20 c | 85.40 b | 88.80 a |
* Lactobacillus acidophilus (6 × 109 CFU/kg feed). ** Saccharomyces boulardii (6 × 109 CFU/kg feed). *** Lactobacillus acidophilus (3 × 109 CFU/kg feed) and Saccharomyces boulardii (3 × 109 CFU/kg feed). Different superscript in the same rows represent significant differences, by one-way ANOVA and t-test (p < 0.05).
Composition of diets (Mean ± St. Dev.) used for Acipenser baerii, for 56 days.
| Feed Sample | Moisture |
Proteins |
Fats |
Carbohydrates |
Fibre |
Ash, |
Energy Value **** kcal/100 g |
|---|---|---|---|---|---|---|---|
| FC | 6.78 ± 0.03 | 54.85 ± 0.04 | 17.84 ± 0.06 | 8.18 ± 0.03 | 1.25 ± 0.01 | 10.85 ± 0.01 | 424.98 ± 0.85 |
| Fla * | 6.78 ± 0.08 | 54.71 ± 0.08 | 17.80 ± 0.13 | 8.13 ± 0.08 | 1.29 ± 0.01 | 10.96 ± 0.01 | 423.17 ± 1.86 |
| FSb ** | 6.79 ± 0.06 | 54.82 ± 0.03 | 17.82 ± 0.09 | 8.21 ± 0.14 | 1.31 ± 0.05 | 10.81 ± 0.01 | 424.15 ± 1.53 |
| FLa + Sb *** | 6.97 ± 0.03 | 54.67 ± 0.06 | 17.78 ± 0.07 | 8.26 ± 0.02 | 1.35 ± 0.01 | 10.79 ± 0.06 | 423.39 ± 0.98 |
| p-Value | 0.06 | 0.16 | 0.76 | 0.04 | 0.04 | 0.0002 | 0.15 |
* Lactobacillus acidophilus (6 × 109 CFU/kg feed). ** Saccharomyces boulardii (6 × 109 CFU/kg feed). *** Lactobacillus acidophilus (3 × 109 CFU/kg feed) and Saccharomyces boulardii (3 × 109 CFU/kg feed). **** Calories conversion factors used: for proteins 4.1 kcal/g, for lipids 9.3 kcal/g; for carbohydrates 4.1 kcal/g.
Variations of haematological indices in Acipenser baerii, reared in a recirculating system, fed for 56 days with diets supplemented with probiotic (n = 5).
| Haematological Indices/UM |
After 56 Days of Experiment. | |||
|---|---|---|---|---|
| Tank C | Tank 1 | Tank 2 | Tank 3 | |
| FC | (Fla *) | (FSb **) | (FLa + Sb ***) | |
| Haematocrit (Ht), % | 17.56 ± 2.60 a | 19.85 ± 2.32 a | 20.95 ± 1.46 a | 21.11 ± 1.59 a |
| CV **** | 0.15 | 0.12 | 0.07 | 0.08 |
| Haemoglobin (Hb) (g/dL), | 3.05 ± 0.28 c | 3.55 ± 0.15 b | 4.85 ± 0.65 a | 5.06 ± 0.61 a |
| Variability coefficient, CV | 0.09 | 0.04 | 0.13 | 0.12 |
| Erythrocyte (RBC), (105/µL) | 6.12 ± 1.08 a | 7.88 ± 1.60 a | 8.22 ± 1.40 a | 8.28 ± 1.34 a |
| CV **** | 0.18 | 0.20 | 0.17 | 0.16 |
| Mean Corpuscular Volume (MCV), (fl) | 29.40 ± 6.97 | 25.74 ± 3.95 | 26.28 ± 5.95 | 25.96 ± 3.78 |
| CV **** | 0.24 | 0.15 | 0.23 | 0.15 |
| Mean Corpuscular Haemoglobin (MCH), (pg) | 5.15 ± 1.23 | 4.69 ± 1.17 | 6.01 ± 1.09 | 6.22 ± 1.04 |
| CV **** | 0.24 | 0.25 | 0.18 | 0.17 |
| Mean Corpuscular Haemoglobin Concentration (MCHC), (g/dL) | 17.75 ± 3.49 | 18.17 ± 2.85 | 23.29 ± 3.86 | 24.18 ± 4.01 |
| CV **** | 0.20 | 0.16 | 0.17 | 0.17 |
* Lactobacillus acidophilus (6 × 109 CFU/kg feed). ** Saccharomyces boulardii (6 × 109 CFU/kg feed). *** Lactobacillus acidophilus (3 × 109 CFU/kg feed) and Saccharomyces boulardii (3 × 109 CFU/kg feed). **** Coefficient of variability. Different superscript in the same rows represent significant differences, by one-way ANOVA and t-test (p < 0.05).
Variation of leukocytes in Acipenser baerii, reared in a recirculating system, fed for 56 days with diets supplemented with probiotic (n = 5).
| Parameters | UM | After 56 Days of Experiment. | |||
|---|---|---|---|---|---|
| Tank C | Tank 1 | Tank 2 | Tank 3 | ||
| FC | (Fla *) | (FSb **) | (FLa + Sb ***) | ||
| WBC | (103/μL) | 21.62 ± 1.61 a | 21.73 ± 1.94 a | 22.75 ± 2.17 a | 22.33 ± 2.56 a |
| Lymphocytes | (%) | 74.29 ± 9.85 a | 74.63 ± 3.16 a | 75.02 ± 3.34 a | 75.01 ± 5.68 a |
| Monocytes | (%) | 5.41 ± 0.40 a | 5.53 ± 1.06 a | 5.76 ± 1.32 a | 5.85 ± 0.98 a |
| Neutrophils | (%) | 16.76 ± 1.80 a | 18.43 ± 1.87 a | 17.66 ± 2.23 a | 17.78 ± 1.95 a |
| Eosinophils | (%) | 3.14 ± 0.78 a | 1.11 ± 0.26 bd | 1.22 ± 0.24 bc | 1.01 ± 0.19 cd |
| Basophils | (%) | 0.40 ± 0.12 a | 0.30 ± 0.07 a | 0.34 ± 0.14 a | 0.35 ± 0.09 a |
* Lactobacillus acidophilus (6 × 109 CFU/kg feed). ** Saccharomyces boulardii (6 × 109 CFU/kg feed). *** Lactobacillus acidophilus (3 × 109 CFU/kg feed) and Saccharomyces boulardii (3 × 109 CFU/kg feed). Different superscript in the same rows represent significant differences, by one-way ANOVA and t-test (p < 0.05).
Serum biochemical parameters (Mean ± Standard Deviation) of Acipenser baerii fed for 56 days with diets supplemented with probiotic.
| Parameters | UM | After 56 Days of Experiment | p-Value | |||
|---|---|---|---|---|---|---|
| Tank C | Tank T1 | Tank T2 | Tank T3 | |||
| FC | (Fla *) | (FSb **) | (FLa + Sb ***) | |||
| Total serum protein (TP) | g/dL | 3.55 ± 1.26 a | 4.80 ± 1.63 a | 5.16 ± 1.15 a | 5.25 ± 1.00 a | 0.458171 |
| Serum albumin (ALB) | g/dL | 0.95 ± 0.44 a | 1.05 ± 0.43 a | 1.25 ± 0.37 a | 1.2 ± 0.33 a | 0.591325 |
| Globulin (GLO) | g/dL | 2.05 ± 0.74 a | 1.65 ± 0.70 a | 1.45 ± 0.76 a | 1.4 ± 0.45 a | 0.417054 |
| Albumin/globulin ratio (A/G) | - | 0.46 | 0.64 | 0.86 | 0.86 | - |
| Glucose (GLU) | mg/dL | 45.00 ± 7.87 a | 35.20 ± 5.45 a | 33.55 ± 5.59 a | 33.30 ± 8.14 a | 0.202443 |
| Cholesterol (CHOL) | mg/dL | 179.50 ± 28.84 a | 155.50 ± 16.44 a | 135.15 ± 9.94 a | 136.22 ± 21.15 a | 0.095441 |
| Aspartate aminotransferase (AST) | U/L | 151.22 ± 18.14 a | 155.85 ± 10.68 a | 155.12 ± 11.15 a | 156.25 ± 21.23 a | 0.839124 |
| Alanine aminotransferase (ALT) | U/L | 3.72 ± 1.07 a | 4.15 ± 1.35 a | 4.25 ± 1.15 a | 4.2 ± 1.18 a | 0.510627 |
| Creatine kinase (CK) | U/L | 33.00 ± 3.77 a | 31.90 ± 2.07 a | 32.50 ± 3.22 a | 30.14 ± 6.88 a | 0.911305 |
| Immunoglobulin (IgM) | mg/dL | 38.50 ± 4.32 ab | 43.60 ± 4.94 acd | 44.80 ± 6.57 bc | 55.25 ± 8.28 d | 0.023794 |
* Lactobacillus acidophilus (6 × 109 CFU/kg feed). ** Saccharomyces boulardii (6 × 109 CFU/kg feed). *** Lactobacillus acidophilus (3 × 109 CFU/kg feed) and Saccharomyces boulardii (3 × 109 CFU/kg feed). Different superscript in the same rows represent significant differences, by one-way ANOVA and t-test (p < 0.05).
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Abstract
The use of probiotics in aquaculture has become a necessity to increase disease resistance. Probiotics are also capable of increasing feed digestion and conversion, decreasing sensitivity to stress, and improving the health of the fish. The aim of this study was to analyse the effect of probiotics on survival, welfare, growth indices and blood composition in Siberian sturgeon (Acipenser baerii), reared in a recirculating system. Diets were supplemented with Lactobacillus acidophilus (FLa) and Saccharomyces boulardii (FSb), separately and combined (FLa + Sb). The effect of probiotics was investigated on 2000 fish with a mean weight of 8.82 ± 0.29 g/specimen. Fish were fed for eight weeks with four different diets: a control without probiotics, one with the addition of lactic acid bacteria (La), one with the addition of yeast (Sb), and one with a mixture of bacteria and yeast in equal proportions (50% La + 50% Sb). FLa + Sb showed a better condition factor (Fulton coefficient, K = 0.39%) and significantly higher growth performance in terms of individual growth (WGi = 35.56 g), total growth gain (WGt = 15.30 g), specific growth rate (SGR, 2.70%/day), and feed conversion ratio (FCR = 1.58), compared to the control. The concentration of lymphocytes, monocytes, and neutrophils was higher in the tanks fed with probiotics compared to the control. The presence of probiotics caused a decrease in cholesterol and glucose. There were significant differences between the Immunoglobulin M values identified in the tank fed with FLa + Sb and the control tank. The results showed that the addition of lactic acid bacteria and yeasts, introduced as probiotics in the diets of Acipenser baerii, reared in a recirculating system, improved the growth indicators, survival, and welfare of the fish.
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