<i>Sargassum</i> sp. extract improve hematological profile of tilapia fish (<i>Oreochromis niloticus</i>)

Mohamad Gazali; Irwan Effendi; Amir Husni; Nurjanah Nurjanah; Sri Wahyuni; Ronal Kurniawan

doi:10.12688/f1000research.128819.4

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Research Article

Revised

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

[version 4; peer review: 2 approved]

Mohamad Gazali ¹, Irwan Effendi², Amir Husni³, Nurjanah Nurjanah⁴, Sri Wahyuni¹, Ronal Kurniawan ²

Mohamad Gazali ¹, Irwan Effendi², [...] Amir Husni³, Nurjanah Nurjanah⁴, Sri Wahyuni¹, Ronal Kurniawan ²

PUBLISHED 22 Apr 2024

Author details Author details

¹ Faculty of Fisheries and Marine Science, Teuku Umar University, Meulaboh, Indonesia
² Faculty of Fisheries and Marine, Universitas Riau, Pekanbaru, Indonesia
³ Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia
⁴ Faculty of Fisheries and Marine Science, Bogor Agricultural University, Bogor, Indonesia

Mohamad Gazali
Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Irwan Effendi
Roles: Conceptualization, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Amir Husni
Roles: Conceptualization, Project Administration, Visualization

Nurjanah Nurjanah
Roles: Conceptualization, Methodology, Writing – Review & Editing

Sri Wahyuni
Roles: Investigation, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Ronal Kurniawan
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Agriculture, Food and Nutrition gateway.

Abstract

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, LD50 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 (LD50). 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 1.34-2.11×106 cells/mm3, hematocrit 26.17-33.19%, hemoglobin 6.26-11.2 g/dL and total leukocytes 1.01-1.50×104 cells/mm3 and total erythrocytes 5.88-9.13×104 cells/ mm3.

Conclusions

A dose of 3000 ppm provided the highest health improvement against A. hydrophila bacterial infection.

Keywords

Sargassum sp. extract, hematology, A. hydrophila, fish health status

Associated Research Article Dr. Asma Khan, Dr. Zunera Hakim, Dr. Akbar Waheed, Dr. Naila Abrar » Impact of CYP3A4 rs2242480 polymorphisms on Imatinib HPLC determined trough concentration and response in Chronic Myeloid Leukaemia in Pakistani patients, F1000Research 2024, 13:310 (https://doi.org/10.12688/f1000research.146356.1)

Corresponding authors: Mohamad Gazali, Ronal Kurniawan

Competing interests: No competing interests were disclosed.

Grant information: This research was funded by Ministry of Education and Republic of Indonesia with Collaboration University Research scheme contract number: 037/UN59.7/PG.02.00.PT/2021 in July 6, 2022.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Gazali M et al. This is an open access article 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.

How to cite: Gazali M, Effendi I, Husni A et al. Sargassum sp. extract improve hematological profile of tilapia fish (Oreochromis niloticus) [version 4; peer review: 2 approved]. F1000Research 2024, 12:293 (https://doi.org/10.12688/f1000research.128819.4) First published: 16 Mar 2023, 12:293 (https://doi.org/10.12688/f1000research.128819.1) Latest published: 22 Apr 2024, 12:293 (https://doi.org/10.12688/f1000research.128819.4)

Revised Amendments from Version 3

Improvement of the language used in the manuscript.

To read any peer review reports and author responses for this article, follow the "read" links in the Open Peer Review table.

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^,³ To meet the demand, intensive aquaculture with high stocking density and artificial feed has become the standard in fish farming.⁴ Challenges 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^,⁷

Motile Aeromonas septicaemia (MAS) caused by Aeromonas hydrophila is a bacterial disease that can cause mass mortality in a culture.⁷^,⁸ The pathogenicity of A. hydrophila can cause mortality in cultured fish up to 80-100% within one to two weeks.⁹ Strategies 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^,¹¹

Understanding 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.¹² In 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.¹³ One of its most important functions is to provide a nursery habitat for juvenile fish and other marine animals.¹⁴ The floating mats of Sargassum provide shelter for young fish and other animals to hide from predators, feed, and grow.¹⁵ Sargassum sp. is a brown macroalga growing in the mid-littoral to sublittoral zone.¹⁶ It is mostly unexploited and has been found to contain phenols, alkaloids, triterpenoids,¹⁷ saponins, and flavonoids.¹⁸ According 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.²⁵ Therefore, 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).

Methods

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:

Oxytetracycline antibiotics as a positive control

D1: 100% macroalgae extract equivalent to 10,000 ppm

D2: 90% macroalgae extract equivalent to 9,000 ppm

D3: 80% macroalgae extract equivalent to 8,000 ppm

D4: 70% macroalgae extract equivalent to 7,000 ppm

D5: 60% macroalgae extract equivalent to 6,000 ppm

D6: 50% macroalgae extract equivalent to 5,000 ppm

D7: 40% macroalgae extract equivalent to 4,000 ppm

D8: 30% macroalgae extract equivalent to 3,000 ppm

D9: 20% macroalgae extract equivalent to 2,000 ppm

D10: 10% macroalgae extract equivalent to 1000 ppm

The parameters measured during the study were:

1. Clear zone
2. Minimum inhibitory concentration
3. LD₅₀ test of leaf extract of Rhizophora sp. in tilapia (O. niloticus).

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 5.20±0.03 g 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 × 30 × 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 10⁸ 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 hemocytometer²⁷ and analyzed.²⁸ Hematocrit 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.²⁹

The 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.

Results

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 6.5-15.0 mm, while doses of 10-20% no longer form inhibition zones. More details can be seen in Table 1.

Table 1. Observation of the inhibition zone of macroalga extracts against Aeromonas hydrophila bacteria.

Macroalga extract dosage (%)	Inhibition zone (mm)
Macroalga extract dosage (%)	Sargassum sp.
Oxytetracycline	27.3
100	15.0
90	11.5
80	10.0
70	9.7
60	9.0
50	8.3
40	7.5
30	6.5
20	0
10	0

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 155.33-215×10⁸ 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.

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

Extract dosage (%)	Repetition			The average number of bacterial colonies (CFU/mL)
Extract dosage (%)	1	2	3	The average number of bacterial colonies (CFU/mL)
0	∞	∞	∞	∞
20	218	213	125	215.33×10⁸
25	174	177	177	179.33×10⁸
30	153	155	158	155.33×10⁸

The LD₅₀ 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.

Figure 1. Calculation results of LD₅₀ determination for 96 hours.

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 1.57-1.93×10⁶ cells/mm³, when compared to fish given the feed without the addition of Sargassum sp. 1.40-1.67×10⁶ cells/mm³ 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%.

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

Observation time	Parameter	Treatment
Observation time	Parameter	NC	PC	F1	F2	F3
30 days of rearing	RBC (×10⁶ cell/mm³)	1.40±0.04^a	1.41±0.04^a	1.57±0.04^b	1,65±0.03^c	1.76±0.03^d
	Hemoglobin (g/dL)	7.20±0.20^a	7.27±0.12^a	7.67±0.12^b	7.73±0.12^b	7.93±0.12^b
	Hematocrit (%)	28.33±0.58^a	28.67±0.58^a	29.67±0.58^a	31.00±1.00^b	31.67±0.58^b
14 days post-challenge test	RBC (×10⁶ cell/mm³)	1.67±0.02^a	-	1.70±0.02^b	1.83±0.02^c	1.93±0.02^d
	Hemoglobin (g/dL)	7.47±0.12^a		8.00±0.20^b	8,27±0.12^c	8.73±0.12^d
	Hematocrit (%)	29.67±0.58^a	-	31.00±1.00^b	31.67±0.58^b	31.67±0.58^b

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 1.91-1.99×10⁴ cells/mm³ when compared to the control treatment 1.88-1.89×10⁴ cells/mm³ 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).

Table 4. Leukocytes of tilapia after feed containing extracts of Sargassum sp.

Observation Time	Parameter	treatment
Observation Time	Parameter	NC	PC	F1	F2	F3
30 days of rearing	WBC (×10⁴ cell/mm³)	1.89±0.01^ab	1.88±0.01^a	1.91±0.01^b	1.94±0.02^c	1.99±0.02^d
	Lymphocytes (%)	75.33±0.58^a	75.67±0.58^ab	76.67±0.58^bc	77.67±0.58^c	79.33±0.58^d
	Monocytes (%)	8.67±0.58	9.00±1.00	8.33±0.58	8.00±1.00	7.67±0.58
	Neutrophil (%)	8.33±0.58	8.00±1.00	7.67±0.58	7.00±1.00	6.33±0.58
	platelets (%)	7.67±0.58	7.33±0.58	7.33±0.58	7.33±0.58	6.67±0.58
14 days post-challenge test	WBC (×10⁴ cell/mm³)	1.92±0.01^a		2.01±0.01^b	2.04±0.01^c	2.10±0.02^d
	Lymphocytes (%)	76.67±0.58^a		79.00±1.00^b	81.33±0.58^c	83.33±0.58^d
	Monocyte (%)	8.00±1.00^b	-	7.67±0.58^b	6.67±0.58^ab	6.00±1.00^a
	Neutrophil (%)	7.67±1.15^b	-	7.33±0.58^b	5.67±0.58^a	5.33±0.58^a
	platelets (%)	7.67±0.58^b	-	6.00±1.00^a	6.33±0.58^a	5.33±0.58^a

After the challenge test, the total leukocytes count of the fish increased to between 2.01-2.10×10⁴ cells/mm³ (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).

Survival of tilapia (O. niloticus)

Tilapia 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 Figure 2.

Figure 2. Tilapia survival.

Discussion

Wayne ³⁰ categorizes 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 155.33×10⁸ CFU/mL – 215.33×10⁸ CFU/mL. At a dose of 20%, the average number of bacterial colonies was 215.33×10⁸ 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.³¹

The 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

In 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 1.34-2.11×10⁶ cells/mm³, hematocrit 26.17-33.19%,³⁵ hemoglobin 6.26-11.2 g/dL,³⁶ and total leukocytes 1.01-1.50×10⁴ cells/mm³,³⁷ 5.88-9.13×10⁴ cells/mm³.³⁸ Increased 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.³⁹ In 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.⁴⁰ Flavonoids 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^,⁴²

Morever, 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.⁴³ Hazzuli 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.⁴⁵ Sargassum 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^,⁵⁰ Overall, 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). 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/mm³, 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 2.10×10⁴ cells/mm³. Furthermore, a dosage of 3000 ppm resulted in the most significant health improvement against A. hydrophila bacterial infection.

Author roles

Gazali M: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation; Effendi I: Data Curation, Formal Analysis, Supervision, Methodology, Writing – Original Draft Preparation; Husni A: Data curation, Validation, Writing-Review & Editing; Nurjanah N: Data Curation, Validation, Writing-Review & Editing; Wahyuni S: Conceptualization, Investigation, Methodology, Writing – Original Draft Preparation; Kurniawan R: Conceptualization, Investigation, Methodology, Software, Writing – Original Draft Preparation

Data availability

Underlying data

Zenodo: Sargassum sp. extract improve Hematological profile of Tilapia fish (Oreochromis niloticus), https://doi.org/10.5281/zenodo.7595715.⁵¹

- Erytrogram, leucogram, survival.xlsx
- Figure 1. docx
- Table 1. docx
- TABLE CLEAR ZONE, COUNT BACTERIA, LD50.xlsx

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

We would like to thank the Laboratory of Fisheries Universitas Riau. This research was funded by the Ministry of Education and Republic of Indonesia with Collaboration University Research.

References

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51. Gazali M, Effendi I, Husni A, et al.: Sargassum sp extract improve Hematological profile of Tilapia fish (Oreochromis niloticus). [Data set]. Zenodo. 2022. Publisher Full Text

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Author details Author details

¹ Faculty of Fisheries and Marine Science, Teuku Umar University, Meulaboh, Indonesia
² Faculty of Fisheries and Marine, Universitas Riau, Pekanbaru, Indonesia
³ Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta, Indonesia
⁴ Faculty of Fisheries and Marine Science, Bogor Agricultural University, Bogor, Indonesia

Mohamad Gazali
Roles: Conceptualization, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Irwan Effendi
Roles: Conceptualization, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Amir Husni
Roles: Conceptualization, Project Administration, Visualization

Nurjanah Nurjanah
Roles: Conceptualization, Methodology, Writing – Review & Editing

Sri Wahyuni
Roles: Investigation, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Ronal Kurniawan
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation

Competing interests

No competing interests were disclosed.

Grant information

This research was funded by Ministry of Education and Republic of Indonesia with Collaboration University Research scheme contract number: 037/UN59.7/PG.02.00.PT/2021 in July 6, 2022.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (4)

version 4

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Published: 22 Apr 2024, 12:293

https://doi.org/10.12688/f1000research.128819.4

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Gazali M, Effendi I, Husni A et al. Sargassum sp. extract improve hematological profile of tilapia fish (Oreochromis niloticus) [version 4; peer review: 2 approved]. F1000Research 2024, 12:293 (https://doi.org/10.12688/f1000research.128819.4)

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Reviewer Report 29 May 2024

Malgorzata Witeska, Department of Ichthyology and Biotechnology in Aquaculture, Institute of Animal Science, Warsaw University of Life Sciences, Warsaw, Poland

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Wahidatul Husna Zuldin, Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia

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Reviewer Report 28 Mar 2024

Malgorzata Witeska, Department of Ichthyology and Biotechnology in Aquaculture, Institute of Animal Science, Warsaw University of Life Sciences, Warsaw, Poland

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Reviewer Report 17 Jan 2024

Malgorzata Witeska, Department of Ichthyology and Biotechnology in Aquaculture, Institute of Animal Science, Warsaw University of Life Sciences, Warsaw, Poland

Not Approved

https://doi.org/10.5256/f1000research.152629.r237564

The Authors performed a study of the antibiotic properties of Sargassum algae extract against Aeromonas hydrophila and toxicity of this alga as feed supplement to Nile tilapia. The results showed that concentrated extract of Sargassum had an antibiotic effect (however, ... Continue reading

The Authors performed a study of the antibiotic properties of Sargassum algae extract against Aeromonas hydrophila and toxicity of this alga as feed supplement to Nile tilapia. The results showed that concentrated extract of Sargassum had an antibiotic effect (however, weaker that oxytetracycline), addition of Sargassum as a feed supplement produced no toxic effect, and considerably increased (in a concentration-related way) fish survival after Aeromonas hydrophila challenge. This make Sargassum a potentially valuable antibacterial alternative or additive. However, I have some comments the intention of which is improving the quality of the work:
1. Explain, please, convincingly why no negative control (with 0% extract and only ethanol at 100% level) was not included in the MIC evaluation? This makes the results incomplete - we do not know what was the contribution of ethanol in bacterial growth inhibition.
2. Explain, please, how water quality was measured and maintained during the experiment? At 5% feeding rate and high stocking density water quality is supposed to deteriorate fast due to accumulation of uneaten food and feces.
3. In the paragraph Statistical analyses, "leukocyte differentiation" (which probably means: leukocyte differential count - the results of which are shown in Table 4) and Phagocytic Index (the results of which are not shown) are mentioned. No methods, however, of measurement of these parameters are given. Please, add.
4. Were dte data tested for ANOVA preconditions: normality of distribution and homogeneity of variance before the use of ANOVA?
5. Tables 3 and 4: titles should contain "Red blood parameters" and "White blood parameters" instead of "erythrogram" and "leukogram", and the 0 values for the parameters should be removed from the PC post-challenge since the values of the parameters were not "0" but no values were obtained due to the death of all fish.
6. The manuscript requires a careful language revision.

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?

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.

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Reviewer Report 25 Sep 2023

Wahidatul Husna Zuldin, Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia

Approved

https://doi.org/10.5256/f1000research.152629.r193687

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Reviewer Report 08 Jun 2023

Wahidatul Husna Zuldin, Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.141444.r166807

Abstract

The abstract was clearly written. Easy to comprehend with sufficient information on the research background, applied methods, and data produced from the study.

Introduction

The introduction section was nicely written. ... Continue reading

Abstract

The abstract was clearly written. Easy to comprehend with sufficient information on the research background, applied methods, and data produced from the study.

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.

Methods

Please clarify what features of Sargassum sp. were used in the study. Is it include the rhizoids/roots?

When does the author state Sargassum sp., and what potential species were used in the study?

Also, further, explain the extraction method. What solvent was used for the seaweed extraction?

Results and Discussion

The results section was clearly presented. Great work on that. The data are highly useful for the tilapia industry. The discussion section was nicely written with a recent literature review included to support the findings.

Conclusions

The conclusions are relevant and clearly stated.

Overall, the article is well written with minor revisions required.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Seaweed Culture, Macroalgal Extraction, Microalgae Culture, Seaweed Ecology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Version 1 16 Mar 23	read

Wahidatul Husna Zuldin, Universiti Malaysia Sabah, Kota Kinabalu, Malaysia
Malgorzata Witeska, Warsaw University of Life Sciences, Warsaw, Poland

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29 May 2024 | for Version 4

Malgorzata Witeska, Department of Ichthyology and Biotechnology in Aquaculture, Institute of Animal Science, Warsaw University of Life Sciences, Warsaw, Poland

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Approved

The Authors revised the manuscript according to the reviewer's comments.

Competing Interests

No competing interests were disclosed.

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Fish biology and hematology, toxicolofy and physiology

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Wahidatul Husna Zuldin, Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia

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Approved

Everything seems good and approved to be indexed.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Seaweed Culture, Macroalgal Extraction, Microalgae Culture, Seaweed Ecology.

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28 Mar 2024 | for Version 3

Malgorzata Witeska, Department of Ichthyology and Biotechnology in Aquaculture, Institute of Animal Science, Warsaw University of Life Sciences, Warsaw, Poland

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Approved With Reservations

The manuscript still requires a careful language revision.

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 confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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17 Jan 2024 | for Version 2

Malgorzata Witeska, Department of Ichthyology and Biotechnology in Aquaculture, Institute of Animal Science, Warsaw University of Life Sciences, Warsaw, Poland

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Not Approved

The Authors performed a study of the antibiotic properties of Sargassum algae extract against Aeromonas hydrophila and toxicity of this alga as feed supplement to Nile tilapia. The results showed that concentrated extract of Sargassum had an antibiotic effect (however, weaker that oxytetracycline), addition of Sargassum as a feed supplement produced no toxic effect, and considerably increased (in a concentration-related way) fish survival after Aeromonas hydrophila challenge. This make Sargassum a potentially valuable antibacterial alternative or additive. However, I have some comments the intention of which is improving the quality of the work:
1. Explain, please, convincingly why no negative control (with 0% extract and only ethanol at 100% level) was not included in the MIC evaluation? This makes the results incomplete - we do not know what was the contribution of ethanol in bacterial growth inhibition.
2. Explain, please, how water quality was measured and maintained during the experiment? At 5% feeding rate and high stocking density water quality is supposed to deteriorate fast due to accumulation of uneaten food and feces.
3. In the paragraph Statistical analyses, "leukocyte differentiation" (which probably means: leukocyte differential count - the results of which are shown in Table 4) and Phagocytic Index (the results of which are not shown) are mentioned. No methods, however, of measurement of these parameters are given. Please, add.
4. Were dte data tested for ANOVA preconditions: normality of distribution and homogeneity of variance before the use of ANOVA?
5. Tables 3 and 4: titles should contain "Red blood parameters" and "White blood parameters" instead of "erythrogram" and "leukogram", and the 0 values for the parameters should be removed from the PC post-challenge since the values of the parameters were not "0" but no values were obtained due to the death of all fish.
6. The manuscript requires a careful language revision.

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?

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.

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25 Sep 2023 | for Version 2

Wahidatul Husna Zuldin, Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia

14 Views Cite this report Responses(0)

Approved

The revised write-up are acceptable and recommended for being indexed.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Seaweed Culture, Macroalgal Extraction, Microalgae Culture, Seaweed Ecology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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21 Views

08 Jun 2023 | for Version 1

Wahidatul Husna Zuldin, Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia

21 Views Cite this report Responses(0)

Approved With Reservations

Abstract

The abstract was clearly written. Easy to comprehend with sufficient information on the research background, applied methods, and data produced from the study.

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.

Methods

Please clarify what features of Sargassum sp. were used in the study. Is it include the rhizoids/roots?

When does the author state Sargassum sp., and what potential species were used in the study?

Also, further, explain the extraction method. What solvent was used for the seaweed extraction?

Results and Discussion

The results section was clearly presented. Great work on that. The data are highly useful for the tilapia industry. The discussion section was nicely written with a recent literature review included to support the findings.

Conclusions

The conclusions are relevant and clearly stated.

Overall, the article is well written with minor revisions required.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Seaweed Culture, Macroalgal Extraction, Microalgae Culture, Seaweed Ecology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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

Abstract

Background

Methods

Results

Conclusions

Keywords

Revised Amendments from Version 3

Introduction

Methods

Time and location

Sensitivity test

Oral administration of Sargassum sp. extract to tilapia (O. niloticus)

Measured parameters

Statistical analyses

Ethical statement

Results

A. hydrophila bacteria sensitivity to Sargassum extract

Table 1. Observation of the inhibition zone of macroalga extracts against Aeromonas hydrophila bacteria.

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

Figure 1. Calculation results of LD50 determination for 96 hours.

The effect of adding Sargassum sp. extract on tilapia red blood parameter

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

The effect of adding Sargassum sp. extract on tilapia white blood parameters

Table 4. Leukocytes of tilapia after feed containing extracts of Sargassum sp.

Survival of tilapia (O. niloticus)

Figure 2. Tilapia survival.

Discussion

Conclusions

Author roles

Data availability

Underlying data

Acknowledgments

References

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Figure 1. Calculation results of LD₅₀ determination for 96 hours.