Growth Performance, Liver and Intestinal Histology, and Immune Response in Gilthead Seabream (Sparus aurata) Juveniles Fed with Diets Supplemented with Niaouli (Melaleuca viridiflora) Essential Oil

doi:10.21203/rs.3.rs-4806307/v1

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Growth Performance, Liver and Intestinal Histology, and Immune Response in Gilthead Seabream (Sparus aurata) Juveniles Fed with Diets Supplemented with Niaouli (Melaleuca viridiflora) Essential Oil

https://doi.org/10.21203/rs.3.rs-4806307/v1

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This study investigated the growth performance, liver and intestinal histopathology, and immune response in gilthead seabream (Sparus aurata) juveniles fed diets supplemented with Niaouli (Melaleuca viridiflora) essential oil. The study was conducted in two phases. In the first phase, a feeding trial was carried out to monitor the effects of the Niaouli essential oil-supplemented diet on fish growth, while in the second phase, the impact on blood parameters and liver-intestinal histopathology was observed in seabream groups fed with the oil-supplemented diet and control groups fed only with pellet feed after administration of a Vibrio anguillarum vaccine. The Niaouli oil was added to the feed at four different levels (0, 1, 1.5, and 3%). The study was replicated three times. Six hundred seabream juveniles, with an initial weight of 11.770 ± 0.006g, were randomly stocked in 12 tanks (50 fish per tank) and fed for 105 days. At the end of the study, feed conversion ratio (FCR), weight gain (WG), and specific growth rate (SGR) were found significantly higher in the group supplemented with 1% Niaouli oil compared to other groups. No pathological changes were observed in the intestinal histology. Similarly, no pathological disorders were detected in the livers of the control groups subjected to feeding and vaccination. However, varying degrees of hepatocyte degeneration from mild to severe were observed in all groups treated with the Niaouli essential oil additive, and the severity of degeneration was determined to be dose dependent. Most liver epithelial cells in the group supplemented with 1% Niaouli oil group appeared close to normal, but some hepatocytes had a granular appearance in their cytoplasm. It was also determined that there was less hydropic degeneration in the group supplemented with 1% Niaouli oil compared to other groups. In the second phase of the study, when vaccinated and unvaccinated groups were compared and assessed in terms of feeding parameters, the vaccine was observed to decrease the feed evaluation rate, increase live weight gain, and enhance the specific growth rate. Hematological values also indicated that the vaccine increased the red blood cell count, stimulated red blood cell production in the bone marrow, and induced erythrocytosis. In conclusion, further research is recommended, involving trials on different fish species with various oil ratios and vaccination timings, to update the literature.

Niaouli

Gilthead seabream

growth

histology

Vibrio anguillarum

immune response

Changes in water quality values in the culture environment of fish and the related stocking density directly impact fish stress, growth parameters, and the immune system. To control all these factors, various chemical interventions such as antibiotics and hormones are administered directly to the fish or through their feed (Tuna Keleştemur, 2023). These chemicals can lead to resistance against the active ingredient, residues in the aquatic environment, and issues that adversely affect sustainable fisheries and indirectly human health (Aslan and Gül, 2018). To counteract these issues, alternative feed additives are being researched and tested. Within the principles of sustainability, medical and aromatic plants and their essential oils, which are harmless to the aquatic ecosystem and possess antimicrobial properties, are being used in the industry as growth-supportive antimicrobials, antivirals, and antifungals (Tuna Keleştemur, 2023). These additives activate immune system components such as T and B lymphocytes, phagocytic cells, complement, and lysozyme (Küçükgül, 2023). It is known that their use in various concentrations enhances feed efficiency and quality while reducing the cost of the product obtained.

In a study conducted by Aktaş et al. (2022), the effectiveness of different levels of thyme leaf and oil as feed additives was evaluated in the cultivation of white shrimp (Litopenaeus vannamei). The experiment included groups fed with feeds supplemented with 1% and 2% thyme leaf (%1 ThyL and %2 ThyL), 0.5% and 1% thyme oil (%0.5 ThyO and %1 ThyO), along with a control group. The results showed that the highest increase in body weight was achieved by the group receiving 1% thyme oil (ThyO1%), which displayed a statistically significant difference compared to the control group (p < 0.05). This group also exhibited the best feed conversion ratio. On the other hand, no difference was found in survival rates among all groups, and histological examinations of hepatopancreatic tissues showed no significant difference in tissue morphology between groups. The previous study indicated that feed supplemented with 1% thyme oil has the potential to support the growth of white shrimp.

There are studies in the literature on the effects of different essential oils on aquatic animals. Niaouli essential oil has not been previously reported in the literature for use in aquaculture. This essential oil is known to be used in humans as an antioxidant and antifungal. Studies have shown that it contains a wide range of compounds. It has been reported to possess compounds that are used in antiseptic, antibacterial, antifungal, antiviral, and anti-inflammatory treatments and are also applied in skin care for treating acne, minor wounds, and burns (Gürer and Tunç, 2022). Evaluating it as a natural feed additive for gilthead seabream, determining its suitable dosages, introducing it to the aquaculture industry, and studying its effects on hematological parameters is considered to make a unique contribution to the literature as an original study.

Effective vaccination is crucial for healthy and sustainable fisheries. It is important to develop the best antigen and effective vaccination strategy specific to the species, considering the physiological factors in fish. If the application method and antigen selection are not properly established, a limited-time low antibody titer results (Nasr-Eldahan, 2021). The Vibrio genus alone, including the former Vibrio anguillarum, now known as Listonella anguillarum, encompasses more than 140 species (Steinum et al., 2021). Most fish species cultured in the Mediterranean are affected by vibriosis (Boynukara et al., 2022). Therefore, many studies are being conducted on fish immunology to achieve efficiency from vaccination (Dönmez, 2023).

Experimental Location and Plan

The research was conducted at the İskenderun Technical University Aquaculture Production Application and Research Center (İSTE-APARC). Six hundred gilthead seabream with an average weight of 11.77 ± 0.06 g, sourced from a farming facility in Karaağaç, were equally distributed across 12 tanks filled with seawater. In the study, each experimental tank housed 50 fish. Before starting the trial, the fish were held in incubation at the farming facility for 15 days for environmental adaptation and infection monitoring, during which they were fed with commercial pellet feed. A daily lighting pattern of 12 hours light and 12 hours dark was implemented in the experimental unit at the farming facility. The fish in the trial groups were randomly assigned to each tank, with 50 fish per tank. The first phase of the study, a 105-day feeding trial, involved three tanks per group including a control group (K), a group fed feed supplemented with 1 mL/kg Niaouli essential oil (N1), a group with 1.5 mL/kg (N1.5), and a group with 3 mL/kg (N3), totaling 12 tanks. Throughout the trial, the fish were fed twice daily. The feeds were specially prepared for each treatment group, calculated as 3% of the total fish weight in each tank. Fish weights were measured biweekly with a precision digital scale (Kern, PCB 2000-1, Germany) to an accuracy of 0.1 g. Water temperature and other water quality parameters in the tanks were measured and monitored daily. Water changes were performed daily using a siphon to remove organic matter. At the end of 105 days, blood, liver, and intestinal samples were taken from 25 fish, after which the fish were fasted for 48 hours. On day 107, the second phase of the study commenced, a 30-day vaccination trial, organized into two groups of 20 fish each, both vaccinated (K, N1, N1.5, and N3) and unvaccinated (K, N1, N1.5, and N3). Addition of Niaouli oil to feed the feeds used in the trial were obtained from the Normfeed feed factory (Konak/Izmir) and stored at + 4°C. The proximate analysis contents of the feed are provided below. The essential oil used in the research was sourced from Art De Huile Aromatherapy (Sarıyer/Istanbul). Due to the volatile nature of Niaouli oil, it was fixed in sunflower oil (10 mL/kg) as a carrier oil for the control group. Niaouli essential oil at concentrations of 1 mL/kg, 1.5 mL/kg, and 3 mL/kg was added to the feed by spraying into 10 mL/kg of sunflower oil. Additionally, given the volatile nature of Niaouli oil, the experimental feeds were prepared biweekly and stored in capped jars at + 4°C throughout the trials.

Growth performance

Live weight measurements were conducted at the start of the trial and at biweekly intervals using a precision scale sensitive to 0.1 grams. The amount of feed consumed periodically was calculated separately for each group. The total feed consumption per period was divided by the number of fish in the tank to calculate the average feed intake per fish using the following formulas:

Weight Gain (WG) = Final Average Weight - Initial Average Weight

Specific Growth Rate (SGR) = 100 x [(Ln Final Weight - Ln Initial Weight) / Number of Days]

Feed Conversion Ratio (FCR) = Total Consumed Feed (g) / Total Weight Gain

Liver and intestinal histology

For histopathological examination, five fish were randomly selected from all treatment groups. Prior to necropsy, the fish were treated with phenoxiethanol at a dose of 400 ppm, and their liver and intestinal tissues were collected in sterile stock bottles containing 10% neutral formaldehyde solution. Following embedding in paraffin blocks, sections of 4–5 µm thickness were taken from the blocks, stained with hematoxylin and eosin, and then fixed and examined under a light microscope and photographed.

Hematological parameters examination

At the end of the first and second phases of the trial, excess water from the fish samples was removed with blotting paper, and blood was collected from the caudal vein using the tail fin cut method into 0.5 mL and 2 mL vacuum tubes containing K3 EDTA. The blood samples were analyzed using an automated blood counter (Vega Vet MS4S, Melet Schloesing, FRANCE), which was calibrated for fish species. The hemogram values measured included white blood cell count (WBC), red blood cell count (RBC), mean corpuscular volume (MCV), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), and hemoglobin (Hb).

Vaccination application

For the second phase of the study, the Vaccine-Niaouli trial model, each group was organized into single tanks of vaccinated and unvaccinated control groups, one vaccinated and one unvaccinated group fed with 1 mL Niaouli oil supplemented feed, one vaccinated and one unvaccinated group with 1.5 mL Niaouli essential oil, and one vaccinated and one unvaccinated group with 3 mL Niaouli oil, totaling eight tanks. Vaccination was carried out according to the manufacturer's instructions using a commercial inactive vaccine (VR/PB Icthiovac Hipra, Spain). The ratio was 1:10 (vaccine to water): a solution was prepared by diluting one bottle (1 liter) of vaccine with 9 liters of seawater from the culture tank. The fish groups were immersed in the vaccine solution for 60 seconds each. Vaccination by immersion was applied to the groups treated with Niaouli essential oil and the control groups, both vaccinated and unvaccinated, while ensuring proper aeration and monitoring water quality parameters during immersion.

Statistical analyses

Before significance tests, all data were assessed for assumptions of parametric tests. Descriptive statistics were presented as "Mean ± standard error." Differences in fish weights by group, time, and their interactions were determined using two-way mixed ANOVA. Where significant differences were found, post-hoc tests with Bonferroni correction for simple effects analysis were applied. Additionally, the effects of group, time, vaccination status, and their interactions on fish weights were analyzed using a linear mixed model. Significant findings were further analyzed with a Bonferroni-corrected simple effects test. Differences in growth parameters such as FCR, WG, and SGR among groups were determined using one-way ANOVA, and significant results were further tested using the Duncan test. Additionally, the effects of group, vaccination status, and their interactions on hemogram, FCR, WG, and SGR parameters were analyzed using two-way ANOVA, with significant parameters subjected to the Duncan multiple range test. The significance level was set at p < 0.05. All statistical analyses were performed using IBM SPSS Statistics 23.0.

Growth performance

The average weights of the fish per group at the beginning of the trial and the average live weights obtained at the end of the trial are presented in Table 1. At the end of the first phase of the trial, when assessed for live weight gain, statistical differences were identified between the trial groups. It was observed that the highest live weight gain occurred in the N1 group. Significant differences were found between the treatment groups in terms of Specific Growth Rate (SGR), with the most significant and highest value found in the N1 group (p < 0.05). Regarding the Feed Conversion Ratio (FCR), the control group was found to be different from the other groups. No significant differences were observed between the groups with oil added to the feed (p > 0.05).

Table 1

Growth performance values of the fish (Mean ± Standard Deviation)
Parameter	Control	N1	N1.5	N3
IW (g)	11.770 ± 0.006^b	11.840 ± 0.006^b	11.740 ± 0.006^b	11.870 ± 0.006^b
FW (g)	46.930 ± 0.006^a.D	62.350 ± 0.006^a.A	60.220 ± 0.006^a.C	60.670 ± 0.006^a.B
FCR	2.210 ± 0.045^a	1.783 ± 0.043^b	1.717 ± 0.047^b	1.693 ± 0.020^b
WG (g)	35.160 ± 0.006^d	50.510 ± 0.006^a	48.480 ± 0.006^c	48.800 ± 0.006^b
SGR (%/day)	1.317 ± 0.001^d	1.581 ± 0.001^a	1.557 ± 0.001^b	1.553 ± 0.001^c

a, b, c, d: Lowercase letters on the same row indicate differences over time. A, B, C, D: Uppercase letters on the same row indicate differences between groups.

In the second phase of our study; among the vaccinated groups, the lowest feed conversion ratio, the highest live weight gain, and the highest specific growth rate were found in the vaccinated control group, with significant differences observed compared to all other groups (p < 0.05) (Table 2). Among the unvaccinated groups, the lowest feed conversion ratio, the highest live weight gain, and the highest specific growth rate were found in the unvaccinated control group, with significant differences observed compared to all other groups (p < 0.05).

Table 2

Growth performance values of fish according to vaccinated and unvaccinated groups (Mean ± Sd.)
	Parameter	Control	N1	N1.5	N3
V	IW (g)	46.930 ± 0.169^C.y.*	62.350 ± 0.169^A.y.*	60.220 ± 0.169^B.y.*	60.670 ± 0.169^B.y.*
	FW (g)	114.500 ± 0.169^C.a.#	121.000 ± 0.169^A.a.#	112.500 ± 0.169^D.a.#	116.500 ± 0.169^B.a.#
	FCR	0.62 ± 0.01^b.C	0.95 ± 0.01^b.B	1.03 ± 0.01^b.A	0.97 ± 0.01^b.B
	WG (g)	67.57 ± 0.01^a.A	58.65 ± 0.01^a.B	52.28 ± 0.01^a.D	55.83 ± 0.01^a.C
	SGR (%/day)	2.97 ± 0.01^a.A	2.21 ± 0.01^a.B	2.08 ± 0.01^a.D	2.17 ± 0.01^a.C
UnV	IW (g)	47.680 ± 0.169^C.x.*	63.100 ± 0.169 ^A.x.*	60.970 ± 0.169 ^B.x.*	61.420 ± 0.169 ^B.x.*
	FW (g)	109.500 ± 0.169^A.b.#	109.750 ± 0.169^A.b.#	102.250 ± 0.169^B.b.#	98.000 ± 0.169 ^C.b.#
	FCR	0.69 ± 0.01^a.D	1.21 ± 0.01^a.C	1.32 ± 0.01^a.B	1.51 ± 0.01^a.A
	WG (g)	61.82 ± 0.01^b.A	46.65 ± 0.01^b.B	41.28 ± 0.01^b.C	36.58 ± 0.01^b.D
	SGR (%/day)	2.77 ± 0.01^b.A	1.84 ± 0.01^b.B	1.72 ± 0.01^b.C	1.55 ± 0.01^b.D

V: vaccinated, UnV: Un-vaccinated, A, B, C, D: Uppercase letters in the same column indicate differences between groups. A, b: Differences between the final measurements within vaccination groups. x, y: Differences between the initial measurements within vaccination groups. *, #: Indicate differences between the initial and final measurements within the vaccinated and unvaccinated groups, respectively.

Liver and intestinal histology

No pathological changes were observed in the intestinal histopathology of any group. Similarly, no pathological disorders were detected in the livers of the control groups in the feeding and vaccination models. However, varying degrees of hepatocyte degeneration from mild to severe were observed in all groups treated with Niaouli essential oil, and the severity of degeneration was determined to be dose-dependent on the essential oil applied. In the groups treated with 1 mL/kg of Niaouli essential oil, the majority of the liver epithelial cells were observed to be nearly normal, but some hepatocytes had a granular appearance in their cytoplasm. In the groups supplemented with 1.5 mL/kg Niaouli essential oil, although the degeneration was milder compared to the groups treated with 3 mL/kg, severe irregular hydropic degeneration was observed in the hepatocytes of the group treated with 3 mL/kg Niaouli essential oil.

Hematological findings

While no statistically significant difference was observed between the groups for leukocyte values, the highest increase was detected in the vaccinated control group. Among the vaccinated groups treated with different concentrations of Niaouli essential oil, the highest increase in leukocytes was observed in the N1 group. In the unvaccinated groups, the highest value was observed in the N3 group. An increase in mean corpuscular volume was only observed in the vaccinated control groups, and no significant difference was found when compared with the unvaccinated group. The highest mean corpuscular volume in the unvaccinated oil treatment groups was observed in the N1.5 group. Upon examining the red cell distribution width values, lower values were detected in the vaccinated K and N1 groups and in the unvaccinated N1.5 and N3 groups, and these values were found to be statistically significant (p < 0.05). When comparing hemoglobin values for the vaccinated and unvaccinated groups, only the vaccinated K group showed an increase in hemoglobin compared to the unvaccinated K group, and while the hemoglobin values in all the unvaccinated groups treated with Niaouli essential oil were higher than in the vaccinated groups, this was not statistically significant. The highest hemoglobin value within the unvaccinated groups treated with Niaouli oil was found in the N3 group. No significant increase in hematocrit values was detected in the vaccinated K and N1.5 groups, while the highest hematocrit value was found in the N3 group among the unvaccinated groups.

The gilthead seabream, a carnivorous marine fish, is a significant cultured fish species predominantly raised in net cages in our country. One of the major challenges in the cultivation of carnivorous marine fish is the availability and sustainability of feed raw materials. In this context, the development of high-energy feeds to increase productivity and obtain healthy individuals also boosts the demand for vegetable and animal oils. Research has shown that vegetable oils are among the biggest sources of alternative resources. However, it has been observed that most of the vegetable oils studied so far are rich in n-6 and n-9 fatty acids. The alternative vegetable oils used in fish diets not only have a high n-3/n-6 ratio but also a high digestibility rate. Gilthead seabream requires 40–55% protein and 15–22% fat in their diets for optimum growth and development. Numerous studies have shown that stock density, water quality parameters, and biotic and abiotic factors affect the growth, reproduction, and immune systems in aquaculture (Öntaş et al., 2020). This study aims to investigate the effects of Niaouli (Melaleuca viridiflora) essential oil, a medicinal and aromatic plant, on the growth, liver-intestinal histopathology, and vaccine-related hematological parameters of gilthead seabream.

In the feeding study that constituted the first phase of the trial, the final live weights of gilthead seabream juveniles, initially weighing 11.770 ± 0.006 g, were determined to range from 46.930 to 62.350 ± 0.006 g. The highest final weight, live weight gain, and specific growth rate were observed in the N1 group, with the lowest values found in the control group. When examining the feed conversion ratio, the control group was found to differ from other groups. No significant difference was found between the oil treatment groups (p < 0.05). Diler et al., (2017) found that the addition of Origanum vulgare L. essential oil at 1.5; 2.5, and 3.0 mL/kg significantly increased growth values in rainbow trout (Oncorhynchus mykiss) groups compared to the control group (p < 0.05). Acar et al. (2019), when applying bergamot peel oil in European seabass, recorded the best growth performance at a low rate (BPO0.5) similar to this thesis study. Kesbiç (2018) demonstrated in a feeding trial with carp that groups fed with juniper oil significantly improved growth performance data. These results indicate that essential oil components can enhance digestion and absorption, thereby increasing growth performance. Essential oils have been reported to have a positive effect on the physiological state and live weight gain of fish, possessing stress-reducing and antimicrobial properties. The features of essential oils have attracted researchers' attention to the concentrations used in feed additions for both marine and freshwater organisms. In aquaculture, the importance of the ratio of essential oil addition in the diet has been emphasized. In this research, similar to previous studies with different fish species and botanical essential oils, Yazıcı et al. (2020) found that the best growth performance at the end of the trial was in the group supplemented with 1% Grobiotic-A in the feeds of juvenile seabass, and that this rate improved growth performance and feed conversion ratio.

In this thesis study, when comparing vaccinated and unvaccinated groups and evaluating all feeding parameters, it was found that vaccination reduced the feed conversion ratio, and increased live weight gain and specific growth rate. Particularly, when compared to other groups, the vaccinated control group had the lowest feed conversion ratio. The effect of vaccination on nutrition and development was evaluated by Özkesici et al., (2016), in a study conducted on European seabass (Dicentrarchus labrax L.,1758), where vaccination positively impacted growth and feed evaluation parameters and significantly reduced mortality rates. Therefore, while the relationship between vaccination and cultivation parameters aligns with some research (Soltani et al., 2019; Viale et al., 2006), it should also be considered that the trial fields such as cultivation conditions, fish species, and habitat have different environmental and managerial dynamics.

In this study, when evaluating the effects of the Vibrio anguillarum vaccine administered along with Niaouli essential oil on the liver-intestinal histopathology of fish, no pathological changes were observed in the intestines of any groups. Similarly, no pathological disorders were detected in the livers of the control groups within the vaccination models. However, varying degrees of hepatocyte degeneration from mild to severe were observed in all groups treated with Niaouli essential oil (1%, 1.5%, and 3%), and it is believed that the severity of degeneration is dependent on the dosage of the essential oil applied. According to Yazıcı et al., 2020, when GroBiotic®-A was added at four different ratios (0.1%, 2%, 3%) to the feed of juvenile seabass, an increase in hepatocyte degeneration, necrosis, and fat vacuole numbers was reported as the dosage increased.

Compared to previous studies (Acar et al., 2019; Altınterim et al., 2018), differences in hematological values have been noted in this research due to varying parameters such as the type of essential oil, the active ingredient component, the dosage applied, the species of fish, and the conditions of cultivation. Acar et al. (2019) researched the effects of various doses of essential oil extracted from bergamot (Citrus bergamia) peel (0%, 0.5%, 1%, 2%) on the growth, hematology, and immune response of European seabass (Dicentrarchus labrax) juveniles and reported no statistically significant differences in red blood cell count, hematocrit, and hemoglobin values at the end of their feeding trial. In another study, researchers reported increases in leukocytes, granulocytes, hemoglobin, hematocrit, MCH, MCHC, PLT, PDW, and PCT values compared to the control group when different concentrations of green tea oil (Camellia sinensis) (0.25%, 0.5%, 1.0%) were used in a feeding trial on rainbow trout (Oncorhynchus mykiss) (Altınterim et al., 2018). Acar et al. (2018) examined some blood parameters in rainbow trout at various levels of pomegranate seed oil, finding significant differences (p < 0.05) in red blood cell count, hemoglobin concentration, mean red cell volume, and mean red cell-hemoglobin concentration in the PSO5 and PSO10 supplemented groups compared to the control. Kaplan et al. (2022) investigated the effects of adding propolis to feed on the fatty acid profile and blood parameters in gilthead seabream (Sparus aurata L. 1758), finding higher hematological parameters such as red blood cell count, hemoglobin, and hematocrit in the P20 group compared to other groups, though no significant difference was found between the trial groups (p > 0.05). Kesbiç (2019) studied the effects of juniper oil (JBO) on growth performance and blood parameters in Cyprinus carpio diets, noting no significant effects on hemoglobin amount, hematocrit rate, and related erythrocyte indices across concentrations of 0 (JBO-0), 5 (JBO-5), and 10 (JBO-10) ml/kg (p > 0.05). Ngugi et al. (2017) worked with feeds supplemented with C. limon fruit peel EO extract at ratios of 1%, 2.5%, and 8%, and a control group fed a diet without C. limon fruit peel EO, reporting that hemato-immunological parameters including red and white blood cell counts, hematocrit, mean cell hemoglobin level, mean cell hemoglobin concentration, and neutrophils showed increasing ratios from 1–5%. They also noted that a 5% inclusion of C. limon fruit peel EO extract in the feed formulation significantly enhanced the biochemical, hematological, and immunological responses in juvenile fish and resulted in lower mortality rates compared to the untreated group. Ebrahimi et al. (2020), in their trials with control groups, 0.01%, 0.1%, 1% and 2% rosemary essential oil (RO) and 0.003% oxytetracycline (OT) treatment groups, reported positive effects of rosemary essential oil supplemented feed on some hematological parameters in mullet fish.

In this study, although no statistically significant differences were observed between groups regarding white blood cell (WBC) counts, the highest increase was detected in the vaccinated control group (K). The highest leukocyte increases among the groups treated with Niaouli essential oil, both vaccinated and unvaccinated, were observed in groups N1 and N3, respectively. Li et al. (2015), in their research assessing different vaccination methods against Vibrio alginolyticus in silver seabream (Sparus sarba), reported that despite no significant increase in serum hematocrit and hemoglobin levels, there was a significant increase in circulating lymphocyte numbers and a decrease in serum cortisol levels in vaccinated fish. Soltani et al. (2019) evaluated the effect of a probiotic, Lactobacillus plantarum, on the growth performance and hematological indices of rainbow trout vaccinated with a bivalent streptococcus/lactococcosis vaccine and found significantly higher (p < 0.05) leukocyte counts in vaccinated groups fed a normal diet with added probiotics compared to unvaccinated groups fed with and without added probiotics. In this thesis, the increase in leukocytes in the vaccinated N1 group is thought to be a result of immune system activation following vaccination, while the increase in the unvaccinated N3 group may have been caused by inflammation due to the essential oil.

In this research, although high erythrocyte values were observed in the vaccinated groups N1, N1.5, and control, no statistically significant values were found in the vaccinated and unvaccinated groups treated with essential oil. Among the unvaccinated groups treated with essential oil, the highest value was observed in group N3. When examining red cell distribution width (RDW) values, low erythrocyte distribution widths were determined in the vaccinated control and N1 groups, as well as in the unvaccinated oil treatment groups N1.5 and N3, and these values were found to be statistically significant. The hemoglobin values in the vaccinated and unvaccinated oil treatment groups were only higher in the vaccinated control group compared to the unvaccinated control group, but this was not statistically significant. The highest hemoglobin value among the unvaccinated groups treated with Niaouli essential oil was found in group N3. An increase in MCV values was observed only in the vaccinated control groups, but no statistically significant difference was found when compared with the unvaccinated group. The highest MCV value in the unvaccinated essential oil groups was observed in group N1.5. No significant increase in hematocrit values was detected in the vaccinated control and N1.5 groups, while the highest hematocrit value was found in the N3 group among the unvaccinated oil treatment groups.

This study particularly underscores the significant impact of Niaouli essential oil on erythrocyte, hemoglobin, and MCV values. In a vaccination study by Soltani et al. (2019), comparing vaccinated groups fed a normal diet and a diet supplemented with probiotics, as well as unvaccinated groups fed with supplemented and normal diets, and control groups, erythrocyte, hemoglobin, MCH, MCHC levels were significantly higher (p < 0.05), whereas there was no significant difference in MCV levels (p > 0.05). It is also known that hematological parameters, which are important indicators of overall health status in fish, can vary between different fish species, and even within the same species due to factors such as diet, water quality, season, size, sex, stocking density in their environment, stress factors, and blood sampling method (Kaplan et al., 2022).

In line with the sustainable ecosystem approach, productivity strategies in aquaculture are continuously evolving. This changing approach is observed to be shifting towards more sustainable aquaculture practices that cause less harm to ecosystems. Although the concept of sustainability is increasingly emphasized, the impacts of ecological damage and potential solutions are still being researched. Aquaculture is rapidly developing due to its ability to meet the increasing food demand driven by population growth and economic expansion, alongside the advantages it offers in terms of utilization areas and export opportunities. Additionally, factors such as industrial pollution in water sources and the decline of natural fish stocks due to unregulated and excessive fishing have facilitated the growth of aquaculture. This ongoing development and production in the aquaculture sector are accompanied by disease issues. Various chemicals and antibiotics are used to prevent and treat these diseases due to the economic and environmental adverse effects they may cause. The intensive use of these medications leads to antibiotic resistance and efficacy loss, also causing residue problems in aquatic ecosystems. The most crucial aspect of high-quality aquaculture is feeding, which utilizes organic materials, various amino acids, vitamins, probiotics, prebiotics, minerals, antioxidants, immunostimulants, and natural feed additives like plant extracts.

Additives incorporated into feed are used to maximize yield from the cultivated species, reduce cultivation costs, and maintain the health of the cultivated species without harming it, thereby preserving the principle of sustainability. The Niaouli plant is among the medicinal and aromatic plants. In this research examining the effects of essential oil derived from the Niaouli plant on growth performance, tissue histopathology, and immune response parameters in gilthead seabream, it has been shown that Niaouli essential oil added to their feed positively affects growth parameters in gilthead seabream. This observation can be expressed based on data obtained by adding 1 mg/kg of the oil to the feed. However, further detailed studies are needed to evaluate the growth parameters, histopathological, and immunomodulatory effects using different doses of Niaouli essential oil, across different fish species and trial models. Vaccination is the most effective method of protection and control against human and animal diseases caused by highly pathogenic microbial agents. In aquaculture, vaccines against vibriosis, furunculosis, pasteurellosis, and yersiniosis are frequently applied via injection, bath, and oral routes. This study has observed positive effects of vaccination on feeding and immune markers. It is recommended to evaluate the positive effects of vaccination on growth, feed conversion ratio, and blood parameters in other fish species, seasons, and cultivation conditions.

Ethical Approval

This material is the authors' original work, which has not been previously published elsewhere. The article is not under consideration for publication elsewhere. The article faithfully and completely reflects the authors' research and analysis. The article correctly mentions the significant contributions of co-authors and co-investigators. Results are appropriately placed in the context of previous and existing research. All sources used are properly cited. Copying of a text must be indicated as such using quotation marks and giving the appropriate reference. The authors have been personally and actively involved in the substantial work leading up to the writing of the article and will take public responsibility for it. The authors have been personally and actively involved in the substantial work that led to the writing of the article and will publicly assume responsibility for its content. Violating the rules of the ethics declaration can have serious consequences. We agree with the above statements and declare that this submission complies with the policies of Thalassas: An International Journal of Marine Sciences, as described in the “Authors' Guide” and “Ethics Statement”. The training program certificate on the use of experimental animals received from Hatay Mustafa Kemal University Animal Experiments Ethics Committee (Certificate No: 2009/031) is shared in the attachment.

Conflict of Interest

The authors of this work declare that they have no conflicts of interest.

Funding

This article is a publication of a doctoral study funded by a scholarship from The Council of Higher Education (YÖK), entitled 100/2000 YÖK PhD Scholarship. It was also supported by the scientific research project (BAP/2021LTP-06) of Iskenderun Technical University. The niaouli essential oil used in the research was provided by Art de Huile company. The fish vaccine applied in the research was developed and provided by HIPRA.

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No competing interests reported.

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Growth Performance, Liver and Intestinal Histology, and Immune Response in Gilthead Seabream (Sparus aurata) Juveniles Fed with Diets Supplemented with Niaouli (Melaleuca viridiflora) Essential Oil

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Abstract

Introduction

Materials and Methods

Experimental Location and Plan

Growth performance

Liver and intestinal histology

Hematological parameters examination

Vaccination application

Statistical analyses

Results

Growth performance

Liver and intestinal histology

Hematological findings

DISCUSSION

Declarations

References

Additional Declarations

Status:

Version 1