Sargassum sp. extract improve hematological profile of tilapia fish ( Oreochromis niloticus)

Background Strategies to increase body resistance and prevent disease in aquaculture include using vaccines, antibiotics, and probiotics. Today, the use of antibiotics with natural ingredients is becoming a trend. Sargassum sp is a natural ingredient that contains high antioxidants and antibiotics. Methods This research was conducted from March to May 2022 at the Biotechnology Laboratory, Faculty of Fisheries and Marine, Universitas Riau, in two stages: 1) the sensitivity of extracts of Sargassum sp. and 2) the application of Sargassum sp. extract orally in tilapia ( O. niloticus). The parameters measured were clear zone, minimum inhibitory concentration, LD 50 test of leaf extract of Sargassum sp. in tilapia ( O. niloticus), hemoglobin levels, hematocrit, total leukocytes, total erythrocytes, leukocyte differentiation, and survival rate. Data on hematology parameters were tabulated and analyzed using a One-Way ANOVA followed by a Student Newman Keuls (SNK) test when deemed necessary. Results The results showed that the extract of Sargassum sp. inhibited the growth of Aeromonas hydrophila bacteria with a clear zone of 6.5-15.0 mm, which is classified as resistant. At doses of 2000, 2500, and 3000 ppm, it did not cause death in fish for 96 hours (LD 50). Hematological parameters can be a sign of the health status of fish. Tilapia given Sargassum sp. in different doses gave an effect between treatments after 30 days of rearing and post-test against A. hydrophila bacteria (p<0.05). The results showed that the hematology of fish fed with Sargassum sp. extract was in the normal or healthy range. Healthy tilapia had erythrocyte counts ranging from 1.34-2.11×10 6 cells/mm 3, hematocrit 26.17-33.19%, hemoglobin 6.26-11.2 g/dL and total leukocytes 1.01-1.50×10 4 cells/mm 3 and total erythrocytes 5.88-9.13×10 4 cells/ mm 3. Conclusions A dose of 3000 ppm provided the highest health improvement against A. hydrophila bacterial infection.


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Introduction
Aquaculture is the answer to meeting the global nutritional needs of a growing population and ensuring food safety from aquatic sources. 1 Tilapia (Oreochromis niloticus) is an omnivorous fish with advantages such as tolerance to different environments, high economic value, and high market demand. 2,3To meet the demand, intensive aquaculture with high stocking density and artificial feed has become the standard in fish farming. 4Challenges in this aquaculture include feed costs, environmental pollution, climate change, water quality, and pathogen infection, 5 which can reduce the productivity and profitability of aquaculture activities. 6,7tile Aeromonas septicaemia (MAS) caused by Aeromonas hydrophila is a bacterial disease that can cause mass mortality in a culture. 7,8The pathogenicity of A. hydrophila can cause mortality in cultured fish up to 80-100% within one to two weeks. 9Strategies aimed at enhancing body resistance and preventing diseases in aquaculture encompass various approaches such as vaccination, antibiotic administration, and probiotic supplementation.Antibiotics have historically served as a common tool in combating fish diseases, including MAS.Nevertheless, their widespread use poses significant environmental and human health concerns, contributing to bacterial multi-resistance and accumulating these compounds in food products. 10,11derstanding how specific and non-specific immune responses modulate fish health is key to increasing productivity and reducing losses in the intensive aquaculture sector.Nowadays, the use of antibiotics with natural ingredients is a trending.Some immunostimulants used as additives in feed can enhance the body's defense system and thus prevent losses from disease. 12In addition, enhancing the immune response with environmentally friendly materials is an effective strategy to promote sustainable cultivation.
Marine macroalgae are natural ingredients containing high antioxidants and antibiotics.Macroalgae contain primary metabolites such as vitamins, minerals, fiber, alginate, carrageenan, and agar, widely used as cosmetic ingredients for skin care.The brown macroalga Sargassum sp. has an essential role in marine ecosystems. 13One of its most important functions is to provide a nursery habitat for juvenile fish and other marine animals. 14The floating mats of Sargassum provide shelter for young fish and other animals to hide from predators, feed, and grow. 15Sargassum sp. is a brown macroalga growing in the mid-littoral to sublittoral zone. 16It is mostly unexploited and has been found to contain phenols, alkaloids, triterpenoids, 17 saponins, and flavonoids. 18According to Lee et al., 19 the extract of S. horneri, an additional immunostimulant in feed, showed significant results supporting its use in white shrimp culture.In addition, the macroalga Sargassum sp. has been used to enhance the immune response in several fish, including hybrid red tilapia, 20 tilapia, 21 rainbow trout, 22 Indian major carp, 23 spotted scat, 24 and great sturgeon. 25Therefore, it is necessary to research the effect of Sargassum sp. as a feed supplement for immune response and prevention of infection with A.hydrophila bacteria in tilapia (O.niloticus).

Time and location
This research was conducted from March to May 2022 at the Biotechnology Laboratory, Faculty of Fisheries and Marine, Universitas Riau.
This research was conducted in two stages, namely 1) the sensitivity test of extracts of Sargassum sp. and 2) the oral administration of extracts of Sargassum sp. in tilapia (O.niloticus).The experiments were carried out within the ethical guidelines provided by the research institution and national or international regulations.

Sensitivity test
The extraction of macroalgae Sargassum sp. was done using maceration method with ethanol solvent.A sensitivity test was performed using the Kirby Bauer disc method.To reduce the error rate, it was repeated three times.Determination of the dose used refers to the study of Ref. 26.The dose of macroalgae extract used is as follows: REVISED Amendments from Version 3 Improvement of the language used in the manuscript.
Any further responses from the reviewers can be found at the end of the article The observation of the inhibition zone of Sargassum sp.against bacteria A. hydrophila was conducted using the Kirby-Bauer disc method, using a blank disk with a 6 mm diameter.Initially, a solution of Sargassum sp.(50 μL) and oxytetracycline as a control were applied to separate blank discs using a micropipette.Subsequently, each blank disc was allowed to absorb the solution for approximately 3 minutes before being placed onto TSA media containing A. hydrophila bacterial inoculant.The discs were then incubated for 24 hours at 37°C.Following incubation, the inhibition zone was observed by measuring the diameter of the clear zone formed using a caliper.
The dose utilized in the MIC test was determined based on the extract dose that produced minimal inhibition to one that did not yield any to identify a minimum dose capable of inhibiting bacterial growth while ensuring safety for fish.Each extract dose was combined with 50 μL of bacterial suspension (with a bacterial density of 108 CFU/mL).The resulting solution was homogenized and incubated for 24 hours at 37 C.The number of colonies was assessed by isolating bacteria from a solution previously incubated for 24 hours and then adding them to 50 μL of TSA media before re-incubation.After 24 hours, bacterial colonies were counted in dishes containing 30-300 colonies.
The LD50 toxicity test commenced by preparing 120 tilapia fish in containers containing Sargassum sp.extract based on the treatment dose established in the MIC test.Each container was filled with 10 L of water, accommodating a stocking density of 1 fish per 1 L. LD50 observations were conducted over 24-96 hours, during which the behavior, clinical symptoms, and fish mortality reaching 50% were monitored.
Oral administration of Sargassum sp.extract to tilapia (O.niloticus) The immune response was observed through an experimental approach employing a one-factor, completely randomized design (CRD) with five treatment levels.The experiment was replicated three times to minimize error, requiring 15 experimental units.The treatment doses for this study were chosen based on preliminary testing, as outlined below: NC: Negative control (feeding without Sargassum sp.extract and without being infected with A. hydrophila bacteria) PC: Positive control (feeding without Sargassum sp.extract and infected with A. hydrophila bacteria) F1: Feed containing sargassum sp.extract at a dose of 2.0 g/kg feed F2: Feed containing sargassum sp.extract at a dose of 2.5 g/kg feed F3: Feed containing sargassum sp.extract at a dose of 3.0 g/kg feed

Measured parameters
The parameters measured were hemoglobin levels, hematocrit, total leukocytes, total erythrocytes, leukocyte differentiation, and survival rate.
This study used 300 fingerlings of tilapia, around 5.20AE0.03g BW.Fish specimens were chosen based on their observed characteristics, including active swimming behavior, absence of wounds, and absence of external parasites.Prior to treatment, the fish underwent a one-week acclimatization period.All experimental procedures were conducted in compliance with ethical guidelines established by the research institution and adhered to relevant national and international regulations.
The macroalgae Sargassum sp. was selected for its potential suitability in this study, given that all species within the Sargassum genus possess usable components in their leaves, stems, and roots/rhizoids.The macroalgae was processed by drying and crushing using a blender until a smooth consistency was achieved.Subsequently, extraction was performed, and the resulting products were incorporated into commercial feed at the prescribed dosage according to the experimental treatment.Following this, the mixture was pelletized and dried before undergoing proximate analysis to assess the nutritional content of the pellets.
The fish were reared in a 60 Â 30 Â 30 cm aquarium filled with 60 L fresh water, with the fish density of one fish/3 L water.A recirculation system and a filter (dacron) are added to the aquarium.The dacron filter is checked every two days; if dirty, it will be replaced with a new dacron.This aims to maintain water quality, supporting the growth and survival of fish.The fish was reared for 30 days and fed three times/day (08.00 AM, 01.00 PM, and 06.00 PM).The total feed provided was 5% of body weight per day.
The A. hydrophila strain (ATCC 35654) used in this study was obtained from the Fish Quarantine in Pekanbaru, Riau, Indonesia.Fish were infected with A. hydrophila (0.1 mL 10 8 of A. hydrophila culture).The negative control (Nc) was fish without treatment, while the positive control (Pc) were fish infected with A. hydrophila and not given Sargassum extract.A total of 15 fish from each treatment were studied.Blood sampling was conducted three times: at the beginning, middle, and end of the study period, with three fish from each aquarium.They were anesthetized using clove oil (5 drops/ L).While they were inactive and unresponsive to touch, blood was drawn from the tail vein by inserting a 10% moistened EDTA (Merck) syringe.Blood samples were stored in moistened EDTA vials in cold boxes filled with crushed ice.Total erythrocytes and leukocytes were counted using a Neubauer hemocytometer 27 and analyzed. 28Hematocrit levels were determined using heparin capillary micro-hematocrit and centrifuged at 12,000 rpm for three minutes.To calculate the hematocrit level, the length of the column of packed red cells or white cells was measured and divided by the length of the entire column of blood (cells and plasma) and multiplied the number by 100%.Hemoglobin levels were measured using Sahli's method. 29e calculate leukocyte differentiation was calculated based on the Blaxhall and Daisley method, 27 involving the preparation of fish blood smears on slides.Next, fixation was done using a 95% methanol solution, followed by staining with Giemsa 50% for 1 minute.Subsequently, the slides were washed with running water and allowed to air dry.Once dried, the preparations were examined under a microscope at 1000× magnification.Leukocytes identified included lymphocytes, monocytes, neutrophils, and platelets.Cell counts were conducted until 100 cells were enumerated, and the percentage was calculated using the formula: cell percentage = ∑ number of cells counted × 100%.

Statistical analyses
Data on hematology parameters (the total erythrocytes, hematocrit level, hemoglobin, total leucocytes, and leucocyte differentiation) and survival rate were tabulated and analyzed using SPSS 26.The normality of obtained data was confirmed, and the significant difference (p<0.05)among treatments was determined by one-way ANOVA followed by a Student Newman Keuls (SNK) test.

Ethical statement
All animals were treated according to animal welfare guidelines established and approved by the Dean of the Faculty of Fisheries and Marine Science, Teuku Umar University, Prof. M. Ali Sarong.Prof. Sarong serves as the ethical committee who approved the use of vertebrae animal in these experiments with approval number: 0674/UN59.3/TU.00.01/2022.

A. hydrophila bacteria sensitivity to Sargassum extract
The results of the sensitivity test showed that the use of Oxytetracycline and extracts of Sargassum sp. with doses of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, and 10% resulted in different inhibitory zones against the growth of A. hydrophila bacteria.Doses of 30-100% can inhibit the growth of A. hydrophila bacteria with an inhibition zone ranging from 6.5-15.0mm, while doses of 10-20% no longer form inhibition zones.More details can be seen in Table 1.
The minimum inhibitory concentration (MIC) test was conducted following the sensitivity test of Sargassum sp., identifying a dose yielding the minimum zone of inhibition, which was subsequently diluted to achieve concentrations of 30%, 25%, and 20%.The MIC test aimed to determine the minimum concentration to inhibit bacterial growth.The results showed that a dose of 20-30% produced an average number of colonies capable of inhibiting the growth of A. hydrophila bacteria ranging from 155.33-215Â10 8 CFU/mL.A dose of 20% was the minimum dose to inhibit the growth of A. hydrophila bacteria.On the other hand, in the control treatment (0%), the number of growing bacterial colonies was uncountable.Table 2. present the result of the MIC test.
The LD 50 toxicity test of macroalga extract was carried out to obtain a dose causing 50% death for 96 hours in 10 tilapia tested per aquarium.The doses used were based on the MIC test results obtained, namely 20% (2000 ppm), 25% (2500 ppm), and 30% (3000 ppm) and control.The results showed that tilapia immersed with Sargassum sp. did not die after 96 hours of experience, indicating that the Sargassum sp. was not toxic to fish. Figure 1 illustratres the results of the toxicity test can be seen in.The effect of adding Sargassum sp.extract on tilapia red blood parameter Measurements of erythrocyte cells of tilapia fed a diet containing extracts of Sargassum sp. with different doses can be seen in Table 3.The administration of Sargassum sp. in the feed with different doses improved in the immune response, as shown by the increase in total erythrocytes 1.57-1.93Â10 6cells/mm 3 , when compared to fish given the feed without the addition of Sargassum sp.1.40-1.67Â106 cells/mm 3 after 30 days of rearing and post-challenge test with A. hydrophila bacteria (p<0.05).Meanwhile, tilapia fed without Sargassum extract could not be examined for erythrocyte cell numbers due to mortality reaching 100%.
The increase in total erythrocytes was in line with the increase in hemoglobin and hematocrit levels in tilapia fed with Sargassum sp.extract after 30 days of rearing and post-test against A. hydrophila bacteria (p<0.05).
The effect of adding Sargassum sp.extract on tilapia white blood parameters Table 4 present the measurement of leukocyte cell of tilapia fed a diet containing extract of Sargassum sp. with varying doses.Feed containing extracts of Sargassum sp.improved the immune response of tilapia, indicated by an increase in total leukocytes 1.91-1.99Â10 4cells/mm 3 when compared to the control treatment 1.88-1.89Â10 4cells/mm 3 after 30 days of rearing (p<0.05).This range remained in normal conditions.In addition, it also affected the concentration of lymphocytes ranging from 76.67-79.33%(p<0.05).The concentration of monocytes, neutrophils, and platelets was not significantly different between treatments (p>0.05).
After the challenge test, the total leukocytes count of the fish increased to between 2.01-2.10Â10 4 cells/mm 3 (p<0.05),which falls within the normal range under these conditions.However, observation of white blood cells in the peripheral blood smear of the positive control group was not feasible due to 100% mortality.Feeding tilapia with extracts of Sargassum sp. at various doses significantly influenced the concentrations of lymphocytes, monocytes, neutrophils, and platelets (p<0.05).Remarks.WBC: White blood cell concentration; F1: extract dose 2000 ppm; F2: 2500 ppm, F3: 3000 ppm.Superscript letters on the same line show significantly different results between treatments (p<0.05).

Survival of tilapia (O. niloticus)
was 215.33Â10 8 CFU/mL, suggesting that this minimum dose effectively inhibited A.hydrophila growth.Optimal bacterial colony growth for inhibiting bacterial growth falls within the range of 30-300 colonies. 31e survival assessment of tilapia exposed to Sargassum sp.extracts at doses of 2000, 2500, and 3000 ppm revealed no instances of fish mortality.This finding suggests that Sargassum sp.extract is non-toxic.Moreover, this macroalga species contains active ingredients capable of inhibiting bacterial growth, thereby presenting a potential alternative for disease control in Indonesian aquaculture.
According to Tavares-Dias et al. and Fagbenro et al., 32,33 stress and nutritional imbalance can trigger changes in tilapia blood parameters.Hematological parameters are essential for assessing the health status of fish and evaluating fish physiology, feed impact, and other stressors.Environmental conditions, sex, age, feeding, and fish activity can affect hematological parameters. 34 this study, the hematological parameters (erythrogram and leukogram) of tilapia subjected to the addition of Sargassum sp. at different doses showed significant effects among treatments, both after 30 days of rearing and in post-tests against A. hydrophila bacteria (p<0.05).This indicates that the administration of Sargassum sp. can indeed impact fish health.Specifically, a dose of 3000 ppm resulted in the most significant improvement in health against A. hydrophila bacterial infection, likely due to the nutrients and secondary metabolites present in the feed, which are capable of fulfilling the requirements for the fish's immune system development.
Fish infected with the bacteria experience changes in the number of erythrocytes, hemoglobin, hematocrit, and total leukocytes.The results showed that the hematology of fish fed with Sargassum sp.extract was within the normal or healthy ranges.Healthy tilapia have erythrocyte counts ranging from 1.34-2.11Â10 6cells/mm 3 , hematocrit 26.17-33.19%, 35hemoglobin 6.26-11.2g/dL, 36 and total leukocytes 1.01-1.50Â10 4cells/mm 3 , 37 5.88-9.13Â10 4 cells/mm 3 . 38ncreased hemoglobin levels are associated with increased oxygen transport capacity and the body's defense mechanism against stress.
The secondary metabolite contents of Sargassum include vitamin C, fucoidan, and flavonoids.Vitamin C in Sargassum sp. can increase immunity and function and act as a fish immune system booster. 39In addition, fucoidan can stimulate the immune response by producing fish immune cells.Its ability to activate immune cells and induce cytokine production suggests its potential as an immune-boosting supplement. 40Flavonoids act as immunomodulators, affecting the quality and intensity of the immune response.They stimulate the immune system by sending intracellular signals to cell receptors, thereby enhancing cell performance.Particularly, flavonoids, among other active ingredients, expedite the activation of leukocytes and macrophages, facilitating rapid phagocytosis against foreign bodies. 41,42rever, vitamin C can increase the body's resistance to pathogens; its role in protein synthesis is necessary for immune responses and collagen biosynthesis to accelerate wound healing.Leukocytes, in addition to the thymus gland, spleen, and immune cells, store large concentrations of vitamin C. In stressed fish, the number of lymphocyte cells in the blood and lymphoid organs (bone marrow, lymph glands, and spleen) decreases. 43Hazzuli et al. 44 stated that fish would respond to vitamin C by increasing the activity and reactivity of cellular and humoral defense cells.Besides, it can increase fish's phagocytic activity (non-specific immune response).
Macroalgae serve as valuable sources of secondary metabolites that enhance immune responses and are utilized as immunostimulants in aquaculture fish feed. 45Sargassum sp.extract application on immune responses has been carried out in several types of aquatic biota such as tiger prawns, 46 mullet, 47 Asian sea bass, 48 and rainbow trout. 49,50Overall, studies suggest that Sargassum extract holds promise as an immunostimulant.

Conclusions
The present research result indicate that the extract of Sargassum sp.effectively inhibited the growth of A. hydrophila bacteria, as evidenced by an inhibition zone (clear zone) measuring 6.5-15.00. at 2000, 2500, and 3000 ppm dose, the extract exhibited resistance, and no mortality in fish was observed over 96 hours (LD50

Wahidatul Husna Zuldin
Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia The revised write-up are acceptable and recommended for publication.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?

Introduction
The introduction section was nicely written.However, the author is suggested to add more on past studies related to the relationship between macroalgal extract and fish hematological profile.
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Figure 1 .
Figure 1.Calculation results of LD 50 determination for 96 hours.

6 .
The manuscript requires a careful language revision.Is the work clearly and accurately presented and does it citethe current literature?Partly Is the study design appropriate and is the work technically sound?Partly Are sufficient details of methods and analysis provided to allow replication by others?Partly If applicable, is the statistical analysis and its interpretation appropriate?Partly Are all the source data underlying the results available to ensure full reproducibility?No Are the conclusions drawn adequately supported by the results?Partly Competing Interests: No competing interests were disclosed.Reviewer Expertise: Fish biology and hematology, toxicolofy and physiology I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.Reviewer Report 25 September 2023 https://doi.org/10.5256/f1000research.152629.r193687© 2023 Zuldin W. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table 2 .
The number of A. hydrophila colonies bacteria after being given Sargassum sp.

Table 1 .
Observation of the inhibition zone of macroalga extracts against Aeromonas hydrophila bacteria.
30lapia survivors were fed Sargassum sp.extracts with doses ranging from 95-98.33% for 30 rearing days (p>0.05).After the challenge test with A. hydrophila bacteria, tilapia survival ranged from 71.67-83.33%,while the PC (control feed and challenged A. hydrophila) experienced 100% mortality.More details are presented in Figure2.DiscussionWayne30categorizes the resulting inhibition zones into susceptible, intermediate, and resistant based on specific criteria.According to CLSI standards, susceptibility is determined by an inhibition zone diameter of 21 mm, intermediate status falls within the range of 16-20 mm, and resistance is indicated by a diameter of 15 mm.This study obtained an inhibitory zone of 6.5-15 mm against A. hydrophila bacteria, aligning the overall treatment group with the resistant criteria for Sargassum sp.against A. hydrophila bacteria.
Calculation of the growth of the number of bacterial colonies of A. hydrophila given a solution of the extract of Sargassum sp. with doses of 20%, 25%, and 30% resulted in the average number of colonies capable of inhibiting the bacterial growth ranging from 155.33Â10 8 CFU/mL -215.33Â10 8 CFU/mL.At a dose of 20%, the average number of bacterial colonies

Table 3 .
Red blood parameters of tilapia given feed containing extract of Sargassum sp.

Table 4 .
Leukocytes of tilapia after feed containing extracts of Sargassum sp.
). Hematological parameters serve as indicators of fish health status.Tilapia administered Sargassum sp.extract at various doses exhibited differential effects among treatments after 30 days of rearing and subsequent exposure to A. hydrophila bacteria (p<0.05).Specifically, the hematology of fish fed with Sargassum sp.remained within the normal or healthy range.Healthy tilapia displayed erythrocyte counts ranging from 1.40 to 1.93×106 cells/mm 3 , hematocrit levels between 28.33% to 31.67%, hemoglobin concentrations from 7.20 to 8.73 g/dL, and total leukocyte counts ranging from 1.88 to 2.10×10 4 cells/mm 3 .Furthermore, a dosage of 3000 ppm resulted in the most significant health improvement against A. hydrophila bacterial infection.