Impact of astaxanthin-enriched bacterium (Paracoccus carotinifaciens) on growth, immune response, and reproduction performance of broodstock Nile tilapia during winter season

Impact of astaxanthin-enriched bacterium (Paracoccus carotinifaciens) on growth, immune response, and reproduction performance of broodstock Nile tilapia during winter season | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of astaxanthin-enriched bacterium (Paracoccus carotinifaciens) on growth, immune response, and reproduction performance of broodstock Nile tilapia during winter season Paiboon Panase, Thitiwut Vongkampang, Eakapol Wangkahart, Nantaporn Sutthi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3218062/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2024 Read the published version in Fish Physiology and Biochemistry → Version 1 posted 7 You are reading this latest preprint version Abstract The growth, immune response, and reproductive performance of broodstock of Nile tilapia ( Oreochromis niloticus ) under winter stress conditions were investigated the effects of supplementary diets with astaxanthin-enriched bacterium, Paracoccus carotinifaciens . Throughout an eight-week period in the winter season, male and female tilapia were fed with diets containing different levels of P. carotinifaciens dietary supplementation: 0 g/kg (T1; control), 5 g/kg (T2), 10 g/kg (T3), and 20 g/kg (T4). Subsequently, a four-week mating system was implemented during the winter stress period. The results revealed that there were no significant differences observed in growth, hematological indices, and blood chemical profiles among all treatment groups for both male and female tilapia. However, a significant increase in cholesterol content was noted in both male and female tilapia fed with the T4 diet (p<0.05). The total carotenoid content in the muscle was evaluated, and significantly higher values were found in both male and female tilapia that fed T4 supplementation (p<0.05). Moreover, immunological parameters such as myeloperoxidase and antioxidant parameters in the liver including superoxide dismutase activity and catalase enzyme activity showed significant increases in tilapia fed with the T4 diet. The impact of P. carotinifaciens supplementation on broodstock tilapia indicated a significant increase in spermatozoa concentration in males and increased egg production in females after consumption of the T4 diet (p<0.05). Thus, this study highlighted that the presence of astaxanthin-enriched bacterium P. carotinifaciens in the diet of broodstock Nile tilapia can lead to the accumulation of carotenoids in their muscle tissue, improvement in antioxidant status, enhancement of immune function, and potential enhancement of reproductive capabilities, even under overwintering conditions. Astaxanthin-enriched diet Paracoccus carotinifaciens Winter season Reproductive performance Heath status Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Water temperature is crucial factor for fish's growth, reproductive capabilities, and metabolism, and can also impact their immune response (Azaza et al. 2008 ). Winter conditions directly affect Nile tilapia ( Oreochromis niloticus ) by reducing their feed intake, weakening the immune system, and inhibiting reproductive function. These challenges influence productivity and profitability in aquaculture, of this fish which is one of the most important warm-water aquaculture fish groups worldwide (Abd El-Hack et al. 2022 ). Nile tilapia is the third most cultivated species in global aquaculture, with a production of 4.5 million tonnes (FAO 2022 ). Moreover, it is predicted that total production will increase by 53% by 2030 (Maulu et al., 2021 ). In terms of food security, this fish species is an affordable source of protein for low-income people (El-Ouny et al. 2023 ). Due to metabolic dysfunctions and immunological disorders, tilapia are more susceptible to opportunistic infections at low temperatures (Panase et al. 2018 ). Mass mortality often occurs during winter cold fronts, and feed consumption declines at temperatures below 20°C, compromising growth performance (El-Sherif and Elfeky 2009 ). Nehemia et al. ( 2012 ) found that the low temperature level between 20 to 22°C could limit the growth development of tilapia by 30%, and the lethal minimum temperature for tilapia is 10 or 11°C. Moreover, high mortalities are related to severe winters with extended cold periods, which can induce pathogens, resulting in economic losses (Hurst 2007 ). Recently, many studies have concentrated on the energy requirements for maintaining growth or physiological responses during cold winter period of stress (Nobrega et al. 2017 ; Abdel-Ghany et al. 2019 ; Hassaan et al. 2019 ). However, these studies have given insight into the significance of energy intake. There has been much study of how to keep fish alive and growing, but much less on how winter stress affects their ability to reproduce and, more research is needed to fully emphasize the effect of winter conditions on the reproductive capacities of fish species. Reproductive processes may be interrupted or reduced due to oxidative stress during the winter months when organisms are already facing several physiological obstacles. The negative impacts of oxidative stress, including reduced gamete formation, changed hormone levels, and diminished fertility, could disrupt the delicate balance necessary for healthy reproduction (Aitken et al. 2022 ). Astaxanthin is a subgroup of carotenoid pigment which is found in various organisms, ranging from plants to animals, and are known for their diverse biological activities (Maoka 2020 ). It exhibits powerful antioxidant properties, neutralizing harmful reactive oxygen species (ROS) that can lead to oxidative stress and damage cellular structures (Brotosudarmo et al. 2020 ). Beyond its antioxidant capacity, research has highlighted a potential role of astaxanthin in supporting reproductive health (Liu et al. 2016 ; Li et al. 2022 ). Numerous studies have investigated the impact of dietary astaxanthin in farmed fish species on reproductive parameters e.g. gamete quality, hormone regulation, and fertility enhancement (Choubert et al. 1998 ; Ahmadi et al. 2006 ; Sheikhzadeh et al. 2012 ). Astaxanthin, in its role as a natural antioxidant, could potentially lessen the harmful effects of oxidative stress on reproductive cells and tissues (Li et al. 2022 ). Additionally, this carotenoid may influence hormone production and signaling pathways related to reproduction, ultimately influencing reproductive success and outcomes (Pasquariello et al. 2022 ). The precise mechanisms underlying astaxanthin's effects on reproductive processes are still being explored, but the existing evidence suggests its potential as a valuable component in the modulation of reproductive functions. Moreover, astaxanthin, a potent antioxidant with health benefits, can be obtained naturally or synthetically (Ambati et al. 2014 ). Although they share similar chemical characteristics, their structural compositions differ, raising concerns about long-term health effects and the scarcity of natural sources and their high-cost limit widespread utilization more than synthetic ones (Stachowiak and Szulc 2021 ). Natural astaxanthin, a carotenoid pigment discovered in specific microorganisms, plants, and marine creatures, is recognized for its potent antioxidant, anti-inflammatory, and immunomodulatory capabilities (Ambati et al. 2014 ; Martínez-Cámara et al. 2021 ). Moreover, natural astaxanthin demonstrates greater biological effectiveness compared to its synthetic counterpart (Aneesh et al. 2022 ). Main carotenoids are produced by microorganisms, such as Paracoccus carotinifaciens , which is gram-negative, aerobic microorganism characterized by its orange pigmented, rod-shaped structure and motility through peritrichous flagella (Tsubokura et al. 1999 ). It can produce astaxanthin which has been proven to be bioavailable and effective in enhancing the color of flesh in Coho salmon, Atlantic salmon, and rainbow trout (European Food Safety Authority 2007 ). Fillets of salmon fed with P. carotinifaciens have exhibited a more diverse composition of muscle carotenoids and a darker red color compared to conventional salmon (Lerfall et al. 2016 ) and feeding with P. carotinifaciens enhances the color of shrimp (Maoka et al. 2018 ). Panaferd®-AX, a natural source of carotenoids derived from P. carotinifaciens , was given conditional approval for use in salmonid aquaculture by the European Food Safety Authority in 2007 (European Food Safety Authority 2007 ). Moreover, it is used to provide antioxidants, nutrients, and color to salmonids and crustaceans (Oehlenschläger and Ostermeyer 2016 ). The efficacy of astaxanthin-rich carotenoid extracts from P. carotinifaciens has been explored through animal and clinical studies, indicating potential benefits for human nutrition and anxiety prevention (Hayashi et al. 2021 ). While previous research has examined the effects of P. carotinifaciens on numerous aspects of fish physiology, such as coloration, growth, and immunity (Lerfall et al. 2016 ; Maoka et al. 2018 ; Nakano and Wiegertjes 2020 ), relatively little is known about the reproductive response of fish, specifically tilapia, under winter stress conditions. Understanding the reproductive effects of P. carotinifaciens in this context is crucial for assessing its potential benefits or limitations in aquaculture practices. Winter stress poses unique challenges to fish health and reproduction. Therefore, the objective of this study was to investigate the reproductive response of tilapia fish to winter stress in the presence of P. carotinifaciens , and the study will provide valuable insight into the relationship between this microorganism and fish in challenging environmental conditions. Materials and Methods Fish and experimental design Each one hundred and twenty of broodstock, including male and female Nile tilapia ( Oreochromis niloticus ) of 6 months of age, weighing 161.70 ± 2.25 g of male and 126.54 ± 7.05 g of female, were purchased from Maha Sarakham Inland Fisheries Research and Development Center (Maha Sarakham, Thailand). The fish were separated by gender and placed in concrete ponds (size: 10×5×1.2 m.). They were allowed to acclimate for two weeks and were hand-fed twice a day with a commercial diet containing 32% protein (Betagro®, Thailand) during the acclimatization period. Subsequently, a total of 24 cages (size: 1.5×1×1 m.) were randomly stocked, with 12 cages assigned to male fish (n = 10 fish/cage) and 12 cages assigned to female fish (n = 10 fish/cage). The fish were then randomly divided into four groups for triplicate experiments. The control group (T1) was fed the basal diet without supplementation, while the other three groups were fed diets containing different levels of dried cells of an astaxanthin enriched bacterium Paracoccus carotinifaciens : T2 (5 g/kg feed), T3 (10 g/kg feed), and T4 (20 g/kg feed). The fish were fed twice daily at 09.00 and 16.30 h, with a feeding rate of 3% biomass per day, for a duration of 8 weeks from October to November. After the 8-week feeding trial, a total of seventy-two healthy fish were selected for the mating experiment with a male-to-female ratio of 1:2, conducting for a period of 4 weeks during the winter season in December. The water quality parameters dissolved oxygen, total ammonia nitrogen (TAN), and pH were monitored throughout the experimental period, with average values of 6.77 ± 0.37 mg/L, 0.22 ± 0.03 mg/L, and 8.09 ± 0.82, respectively. The water temperature during the experimental period is shown in Fig. 1 , with an average temperature of 22.18 ± 2.46°C, ranging from 17°C to 26°C, over a span of 12 weeks. Additionally, the water temperature during the mating period averaged 19.87 ± 1.45°C, ranging from 17.00°C to 23.00°C. Supplemental diet preparation Throughout the study, commercial fish feed pellets from Betagro® in Thailand were used. Analysis of the commercial feed was shown in Table 1 . These pellets contain a composition of 32% crude protein, 4% lipid, 12% moisture, and 6% fiber. The dried cells of an astaxanthin-enriched bacterium called Paracoccus carotinifaciens were obtained from the commercial product Panaferd® AX in Japan. In addition to this product, it contains astaxanthin (20 g/kg), adonirubin (8 g/kg), and canthaxanthin (2 g/kg) (Table 1 ). The procedures for preparation of feed supplementing with astaxanthin were conducted according to the methos by Sutthi and Thaimuangphol ( 2020 ). The astaxanthin-enriched bacterium was mixed with the basal diet in four different proportions: 0, 5, 10, and 20 g/kg of the diet. The mixture was thoroughly combined using a mixer machine and air-dried. Afterward, the pellets from all four groups were bound with 20 g of guar gum and top coated with a 4% agar solution at a rate of 20 mL/kg of the diet. The coated pellets were then subjected to another round of drying at 30°C for 24 hours using a hot air oven. The prepared diets were then stored at 4°C for future use. The proximate analysis of the experimental diets, including crude protein, crude lipid, moisture, and ash, was performed using standard analysis methods (AOAC 1995 ), and the results are presented in Table 2 . Table 1 Analysis of the commercial feed for Nile tilapia and Paracoccus carotinifaciens components. Feed Analyses 1 Composition Crude protein (%) 32 Crude lipid (%) 4 Crude fiber (%) 12 Moisture (%) 6 Paracoccus carotinifaciens components 2 Composition Astaxanthin (g/kg) 21 Adonirubin (g/kg) 8 Canthaxanthin (g/kg) 2 1 Commercial feed information (Betagro®, Thailand). Ingredients of the commercial fish feed: fish meal, soybean meal, corn, broken rice, rice bran, including vitamins and minerals. 2 Technical Information (Panaferd® AX, Japan) Table 2 Proximate analysis of the experimental diets (% dry matter). Analyzed chemical composition T1 (control diet) T2 (5 g kg − 1 diet) T3 (10 g kg − 1 diet) T4 (20 g kg − 1 diet) Crude protein (%) 32.86 31.71 33.38 33.97 Crude lipid (%) 4.08 4.17 4.29 4.74 Crude fiber (%) 4.04 4.37 4.32 5.06 Moisture (%) 13.18 13.86 14.12 13.52 Ash (%) 9.88 10.11 9.97 10.16 NFE (%) 1 49.14 49.64 48.04 46.07 GE (MJ/kg) 2 17.82 17.67 17.84 17.81 1 Nitrogen-free extract (NFE%) = 100 - (crude protein + crude lipid + ash + crude fiber). 2 Gross energy (GE) calculated based on 23.6, 39.5 and 17.2 kJ/g protein, lipid, and carbohydrates, respectively. Growth measurements The weight and length of each fish in all treatments were recorded at the beginning and the end of the experimental period. The growth rate of all fish was determined using the mathematical growth model with the following equations (Bagenal 1978 ): Weight gain (WG; g) = final weight (g) – initial weight (g); Average daily gain (ADG; g/day) = [final weight (g) – initial weight (g)]/days; Specific growth rate (SGR; %/day) = 100×[{Ln final weight (g) – Ln initial weight (g)}/days]; Feed conversion ratio (FCR) = total feed (g)/weight gain (g); Survival Rate (SR, %) = [number of survived fish/initial number of fish]×100. Samples collection After the 8-week feeding trial, blood samples were collected immediately from the caudal vein of the fish. Prior to collection, the fish were anesthetized using clove oil at a concentration of 100 mg/L. A volume of 0.5 mL of whole blood was taken and transferred into anticoagulant tubes for hematological analysis. Additionally, another part of blood samples of 1 mL was placed into sterile Eppendorf tubes without anticoagulant for serum collection. The serum was obtained by allowing the blood sample to clot at room temperature for 1 hour, followed by 4°C for 4 hours. Subsequently, the samples were centrifuged at 5000×g for 10 minutes at 4°C. All the samples were then stored in Eppendorf tubes at -20°C until further use. The serum samples were utilized for blood chemical analysis and immunological analysis. Total carotenoid extraction After feeding the experimental diet, the total carotenoid content in fish muscle (n = 3 per treatment) was measured using the method as per Rodriguez-Amaya and Kimura ( 2004 ). Briefly, the measurement of total carotenoid content was conducted following the procedure outlined in our previous study (Van Doan et al. 2022 ). It involved using a spectrophotometer at a wavelength of 450 nm, and the total carotenoid content was calculated using the formula provided by Rodriguez-Amaya and Kimura ( 2004 ): Total carotenoid content (µg g − 1 ) = \(\frac{\text{A}\times \text{v}\text{o}\text{l}\text{u}\text{m}\text{e}\left(\text{m}\text{l}\right)\times {10}^{4}}{{\text{A}}_{1\text{c}\text{m}}^{1\text{\%}}\times \text{s}\text{a}\text{m}\text{p}\text{l}\text{e} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \left(\text{g}\right)}\) Where A = absorbance; volume = total volume of extract; \({\text{A}}_{1\text{c}\text{m}}^{1\text{\%}}\) = absorption coefficient of β-carotene in petroleum ether (2592). Serum biochemical analysis The collected blood samples were sent to the Veterinary Central Lab located Mueang Khon Kaen District, Khon Kaen 40000, Thailand for blood chemical analysis. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), cholesterol, total protein, and albumin were determined using commercial reagent kits (ABX Pentra reagent, Horiba, France) following the manufacturer's instructions. The concentration levels of these parameters were measured using the colorimetry method with an ABX Pentra 400 Clinical Chemistry Analyzer (Horiba, France). The globulin level was calculated by subtracting the value of albumin from the total protein. Hematological parameters The blood samples collected were sent to the Veterinary Central Lab located Mueang Khon Kaen District, Khon Kaen 40000, Thailand for hematological analysis. The erythrocyte (RBC) and leukocyte (WBC) counts were determined using a Neubaeur hemocytometer (Rehulka, 2003 ). To obtain the differential leukocyte count, blood smears were prepared and stained with a combination of Giemsa/May-Grunwald (Davis et al. 2008 ). The hematocrit (Hct) was measured by centrifuging heparinized micro-hematocrit capillary tubes at 12,000×g for 5 minutes and reported as percentages (Zhao et al. 2018 ). The concentration of hemoglobin (Hb) was measured using a lysing reagent kit (ABX reagent, France) that enables the lysis of erythrocytes and cyanide-free determination of hemoglobin. The released hemoglobin was then oxidized and stabilized, and the resulting complexes were quantified by spectrophotometry at a wavelength of 550 nm using a hematology analyzer (ABX Micros EVS 60, France). Blood indices were calculated according to the formulas proposed by (Mansour et al. 2017 ): the following formulas: Mean cell volume (MCV) = Hct(%)×10/RBCs (10 6 mm − 3 ); Mean cell hemoglobin (MCH) = Hb (g/dl)×10/RBCs count (10 6 mm − 3 ); Mean corpuscular hemoglobin concentration (MCHC) = Hb (g/dl)/Hct (%). Lipid peroxidation and antioxidant enzymes activity Serum malondialdehyde (MDA) concentrations were determined by measuring the amount of thiobarbituric acid reactive substances (TBARS) using the method suggested by Aengwanich et al. 2011 with slightly modifications, as described in detail by Sutthi et al. ( 2018 ). The concentration of MDA was measured using a spectrophotometer at 532 nm. Catalase activity (CAT) in the liver was determined according to the method described by (Wangkaghart et al. 2021 ). The difference in absorbance was recorded after 20 s (A1) and after 80 s (A2) of incubation at 240 nm and room temperature. The CAT value was calculated as (A1 - A2) / 0.0008. Liver superoxide dismutase (SOD) activity was measured according to the methods described by (Mansour et al. 2017 ). The activity was calculated using the following formulas: The percent of inhibition (%) = 100- ((ΔA control-ΔA sample/ ΔA control) × 100). SOD activity (U g − 1 liver) = % inhibition × 3.75. Lysozyme activity (LZM) in serum was determined using a turbidimetric assay based on the lysis of the lysozyme-sensitive Gram-positive bacterium Micrococcus lysodeikticus (Sigma, USA). The method followed the approach of (Parry et al. 1965 ) with some modifications, as described in detail by (Wangkaghart et al. 2021 ). The myeloperoxidase (MPO) activity present in serum was measured following the method suggested by (Sahoo et al. 2020 ) with slight modifications, as described in detail by (Mansour et al. 2017 ). The MPO content was measured by absorbance at 450 nm using a microplate reader. Reproductive activity In male tilapia, milt parameters were collected before and after the experimental diet trial (8 weeks). The measurement followed the method described by (Marijani et al. 2019 ). Milt volume was randomly selected and collected from males by using hand stripping. The volume was measured with a pipette and expressed in milliliters (ml). The spermatozoa concentration was determined using a hemocytometer and expressed as the number of cells x 10 8 cells/ml. In females, seventy-two healthy male and female fish were selected for the mating experiment in a ratio of 1:2 (male (n = 2): female (n = 4)) per replication. The egg parameters were collected every week during the 4-week mating period in the winter season (December). The diameters of the oocytes were measured using a microscope equipped with an ocular micrometer. The hatching rate was calculated by counting the number of larvae 4 days after fertilization, following the methods described by De Lapeyre et al. ( 2010 ). The spawning rate was defined as the percentage of females that spawned over a 7-days period out of the total number of females in each group. The survival rate of larvae was calculated by counting the percentage of number of survival larvae 7 days after hatching egg. Statistical analysis The obtained data were performed using a one-way analysis of variance (ANOVA) followed by Duncan’s post hoc for multiple comparisons among the treatments. The significance level was set as P < 0.05. Results are expressed throughout as mean ± standard deviation (SD). Results Growth performances The effects of astaxanthin-enriched P. carotinifaciens supplementation in the feed for 8 weeks on the growth performance of broodstock tilapia are presented in Table 3 . The growth performance parameters, including weight gain (WG), average daily gain (ADG), specific growth rate (SGR), feed conversion ratio (FCR), and survival rate (SR), showed no significant differences (p > 0.05) among all treatment groups for both male and female tilapia. Table 3 Growth performances of Nile tilapia fed with P. carotinifaciens supplement in diet for 8 weeks. Parameters T1 (control) T2 (5 g kg − 1 diet) T3 (10 g kg − 1 diet) T4 (20 g kg − 1 diet) Male Weight gain (WG; g) 88.63 ± 12.68 82.30 ± 3.86 66.20 ± 14.11 80.07 ± 9.05 Average daily growth gain (ADG; g/day) 1.97 ± 0.28 1.83 ± 0.09 1.47 ± 0.31 1.78 ± 0.20 Specific growth rate; SGR (% /day) 0.96 ± 0.13 0.91 ± 0.12 0.77 ± 0.14 0.91 ± 0.12 Feed conversion ratio; (FCR) 3.03 ± 1.70 3.10 ± 0.68 3.24 ± 1.20 3.90 ± 1.55 Survival rate (%) 93.33 ± 5.77 96.67 ± 5.77 96.67 ± 5.77 90.00 ± 10.00 Female Weight gain (WG; g) 75.28 ± 14.83 82.44 ± 35.93 59.55 ± 22.27 67.22 ± 30.07 Average daily growth gain (ADG; g/day) 2.51 ± 0.49 2.75 ± 1.20 1.99 ± 0.74 2.24 ± 1.00 Specific growth rate; SGR (% /day) 1.55 ± 0.29 1.68 ± 0.68 1.21 ± 0.39 1.48 ± 0.59 Feed conversion ratio; (FCR) 2.78 ± 0.09 3.83 ± 1.04 3.15 ± 1.50 3.00 ± 0.19 Survival rate; SR (%) 70.00 ± 10.00 80.00 ± 10.00 86.67 ± 5.77 83.33 ± 5.77 Data are given as mean ± SD. Blood chemical profiles and hematological analysis The blood chemical profiles and hematological indices were examined at the end of the feeding period with experimental diets, and the results are presented in Table 4 and Table 5 . The cholesterol content in both male and female tilapia fed with astaxanthin-enriched P. carotinifaciens showed a significant increase compared to the control group (p 0.05) for total protein, albumin, globulin, aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN) levels. In the hematological analysis of male and female tilapia, there were no significant differences observed among all treatments (P > 0.05) in terms of red blood cell count, white blood cell count, hemoglobin level, hematocrit level, neutrophil count, and lymphocyte count. Table 4 Blood chemical profiles of Nile tilapia fed with P. carotinifaciens supplement in diet for 60 days. Parameters T1 (control) T2 (5 g kg − 1 diet) T3 (10 g kg − 1 diet) T4 (20 g kg − 1 diet) Male Total protein (g/dL) 2.9 ± 0.10 2.8 ± 0.09 2.8 ± 0.09 3.16 ± 0.32 Albumin (g/dL) 2.03 ± 0.05 1.96 ± 0.05 2.03 ± 0.05 2.26 ± 0.23 Globulin (g/dL) 2.03 ± 0.05 1.96 ± 0.05 2.03 ± 0.05 2.26 ± 0.23 Aspartate aminotransferase; AST (U/L) 71.00 ± 17.52 30.33 ± 8.50 64.33 ± 29.87 36.00 ± 7.21 Alanine aminotransferase; ALT (U/L) 42.00 ± 23.51 32.00 ± 7.00 65.33 ± 17.78 26.66 ± 11.01 Blood Urea Nitrogen; BUN (g/dL) 1.33 ± 0.57 1.16 ± 0.28 1.5 ± 0.5 1.33 ± 0.28 Cholesterol (g/dL) 126.66 ± 10.01 b 142.66 ± 14.0 ab 143.33 ± 22.36 ab 168.33 ± 9.07 a Female Total protein (g/dL) 2.63 ± 0.20 2.63 ± 0.23 2.83 ± 0.15 2.96 ± 0.25 Albumin (g/dL) 0.86 ± 0.15 0.8 ± 0.00 0.86 ± 0.11 0.86 ± 0.05 Globulin (g/dL) 1.76 ± 0.35 1.83 ± 0.23 1.96 ± 0.05 2.1 ± 0.2 Aspartate aminotransferase; AST (U/L) 55.33 ± 30.35 65.00 ± 35.51 62.66 ± 20.25 58.33 ± 26.57 Alanine aminotransferase; ALT (U/L) 39.33 ± 14.46 39.33 ± 8.02 24.00 ± 1.00 27.33 ± 17.38 Blood Urea Nitrogen; BUN (g/dL) 1.00 ± 0.00 1.16 ± 0.28 1.33 ± 0.16 1.33 ± 0.28 Cholesterol (g/dL) 112.33 ± 7.50 c 135.33 ± 6.50 b 149.00 ± 11.13 ab 163.66 ± 9.29 a Data are given as mean ± SD and different superscripts are significant differences (p < 0.05). Table 5 Hematological analysis of Nile tilapia fed with P. carotinifaciens supplement in diet for 60 days. Parameters T1 (control) T2 (5 g kg − 1 diet) T3 (10 g kg − 1 diet) T4 (20 g kg − 1 diet) Male RBC (x10 6 cells/mm 3 ) 2.04 ± 0.17 2.09 ± 0.15 1.96 ± 0.18 1.94 ± 0.02 Hemoglobin (g/dL) 8.66 ± 3.13 10.93 ± 1.27 9.93 ± 0.41 10.03 ± 0.90 Haematocrit (%) 25.66 ± 10.01 32.66 ± 4.61 29.66 ± 1.52 29.00 ± 2.64 WBC (x10 3 cells/mm 3 ) 9.09 ± 1.99 8.80 ± 3.71 10.49 ± 5.86 9.90 ± 3.91 Neutrophils (%) 31.33 ± 7.09 31.33 ± 5.50 29.66 ± 3.05 30.33 ± 2.08 Lymphocytes (%) 64.33 ± 6.65 64.66 ± 5.50 67.66 ± 2.30 65.00 ± 2.64 Female RBC (x10 6 cells/mm 3 ) 2.11 ± 0.10 1.97 ± 0.09 1.95 ± 0.17 2.11 ± 0.15 Hemoglobin (g/dL) 12.93 ± 0.23 11.30 ± 1.60 10.20 ± 0.45 10.96 ± 1.25 Haematocrit (%) 38.33 ± 1.52 32.66 ± 4.50 30.00 ± 1.00 33.00 ± 4.35 WBC (x10 3 cells/mm 3 ) 7.45 ± 1.36 8.51 ± 0.96 8.80 ± 2.06 8.47 ± 2.17 Neutrophils (%) 32.00 ± 8.54 37.66 ± 4.04 27.00 ± 2.64 32.00 ± 4.35 Lymphocytes (%) 63.33 ± 7.50 59.00 ± 5.19 69.00 ± 2.00 63.66 ± 4.72 Data are given as mean ± SD. RBC = red blood cell, WBC = white blood cell. The total carotenoid analysis The total carotenoid content in the muscle was assessed at the end of the experiment, and the results are presented in Fig. 2 . There was a significant difference (p < 0.05) in the values of total carotenoid content between male and female tilapia fed T4 diet compared to the control diet. Both male and female fish fed T4 diet exhibited the highest total carotenoid content in fish muscle. Antioxidant and immunological observations The liver antioxidant parameters of male and female Nile tilapia, which were fed astaxanthin-enriched P. carotinifaciens diets for 8 weeks, were presented in Fig. 3 . The activity of superoxide dismutase (SOD) obtained in fish fed with the experimental diets were illustrated in Fig. 3 . The highest SOD values were observed in female tilapia fed T4 diet, followed by the T3 and T2 diets. All these values (T3, T4, T5) were significantly higher than those of the control group. However, no significant differences (p > 0.05) in SOD levels were observed among the treatments for male tilapia (Fig. 3 a). There were no significant differences observed in MDA levels among all treatments for both male and female tilapia (p > 0.05) (Fig. 3 b). The CAT enzyme activity of both male and female tilapia fed the T4 diet was significantly higher compared to the control group (p < 0.05) (Fig. 3 c). The immunological parameters in the serum of male and female Nile tilapia, which were fed experimental diets, was depicted in Fig. 4 . The MPO in response to the dietary sources of astaxanthin-enriched P. carotinifaciens was shown in Fig. 4 a. It is observed that male tilapia fed the T4 diet exhibited a significant increase in MPO activity compared to the control group (p < 0.05). However, the supplementation of astaxanthin-enriched P. carotinifaciens in all diets did not have an impact on MPO activity in female tilapia (Fig. 4 a). There were no significant differences LZM activity observed among all groups for both male and female tilapia (p > 0.05) (Fig. 4 b). Reproductive activity The effect of dietary supplementation of P. carotinifaciens on sperm parameters were assessed in male tilapia as presented in Fig. 5 . Prior to the experimental diet, there were no significant differences in spermatozoa concentration among all groups (p > 0.05) (Fig. 5 a). However, after feeding the diet containing astaxanthin-enriched P. carotinifaciens , the spermatozoa concentration was significantly increased in fish fed T4 diet compared to the control and T2 diets (p < 0.05). Furthermore, there was a significant increase in spermatozoa count in fish fed T4 diet compared to before the diet started (p < 0.05) (Fig. 5 a). The milt volume before and after feeding male tilapia with P. carotinifaciens was presented in Fig. 5 b, and no significant differences were observed in milt volume among all treatment groups (p > 0.05). In the case of female tilapia, after feeding with astaxanthin-enriched P. carotinifaciens for 8 weeks, a mating experiment was implemented with a 1:2 ratio of males to females for four weeks in December. The reproductive performance in terms of egg quality is presented in Table 6 . Fish fed with the T4 diet showed significant improvements in the average number of eggs per week and the number of eggs per female, which were higher than on the control diet (p 0.05). Table 6 Female reproductive performances during 4 weeks in winter season after fed with P. carotinifaciens supplement in diet. Parameters T1 (control) T2 (5 g kg − 1 diet) T3 (10 g kg − 1 diet) T4 (20 g kg − 1 diet) Average egg numbers per week 411.41 ± 101.52 b 555.75 ± 53.11 b 768.00 ± 314.46 ab 995.75 ± 291.17 a Number of eggs per female 102.85 ± 25.38 b 138.93 ± 13.27 b 192.00 ± 78.61 ab 248.93 ± 72.79 a Spawning rate (%) 16.66 ± 11.78 14.58 ± 7.97 20.83 ± 4.81 25.00 ± 6.80 Diameter oocyte (mm) 2.64 ± 0.05 2.65 ± 0.20 2.62 ± 0.25 2.78 ± 0.10 Hatching rate (%) 86.21 ± 2.44 91.49 ± 2.68 91.69 ± 4.79 85.46 ± 2.81 Larvae survival rate (%) 86.29 ± 3.09 92.37 ± 5.52 92.34 ± 6.46 90.63 ± 11.91 Data are given as mean ± SD and different superscripts are significant differences (p < 0.05). Discussion The understanding of carotenoids in fish has expanded in recent times, revealing their diverse biological functions beyond muscle pigmentation. Carotenoids, such as astaxanthin, have been found to play a crucial role in promoting growth (Storebakken and Goswami 1996 ), enhancing broodstock performance (Watanabe and Vassallo-Agius 2003 ; Ahmadi et al. 2006 ; Sawanboonchun et al. 2008 ; Palma et al. 2017 ), improving antioxidant status (Wang et al. 2006 ; Fiedor and Burda 2014 ; Crupi et al. 2023 ), boosting immune function (Amar et al. 2001 ; Amar et al. 2004 ; Chew and Park 2004 ), and increasing disease resistance in fish (Amar et al. 2012 ; Bhatt and Patel 2020 ). Astaxanthin, the primary carotenoid pigment in aquatic animals, imparts vibrant colors to fish meat, crustacean shells, and bird feathers (Miki 1991 ; Breithaupt 2007 ). It is naturally produced by algae and converted by plankton crustaceans from precursor carotenoids (Stachowiak and Szulc 2021 ). Incorporating astaxanthin from natural sources, such as Haematococcus pluvialis , into commercial fish (red tilapia) feed has shown positive effects on color and growth parameters in some studies (Tuan Harith et al. 2022 ). Paracoccus carotinifaciens is a species of microorganism that serves as a natural source of carotenoids, providing antioxidants, nutrients, and color to salmonids like salmon and trout, as well as shrimp (European Food Safety Authority 2007 ; Oehlenschläger and Ostermeyer 2016 ; Maoka et al. 2018 ; Hayashi et al. 2021 ). This aerobic bacterium, characterized by its orange pigmentation and ability to produce astaxanthin, is widely distributed in both marine and freshwater fish (Tsubokura et al. 1999 ). Animals generally cannot synthesize carotenoids themselves, relying on their diet to acquire these pigments primarily in their skin and flesh (Galasso et al. 2017 ). While previous studies on tilapia have focused mainly on red tilapia, which were fed carotenoid or astaxanthin-rich diets derived from microalgae and plants, leading to enhanced redness in their skin and improved tissue carotenoid levels (Judan Cruz et al. 2021 ; Tuan Harith et al. 2022 ). However, carotenoids are less prevalent in muscle compared to the integument (Shahidi and Brown 1998 ). To determine which microorganisms produce the carotenoid in Nile tilapia, previous study discovered that the red yeast Sporidiobolus pararoseus can produce carotenoids (Chaiyaso and Manowattana 2018 ), and feeding Nile tilapia with this red yeast resulted in increased total carotenoid levels in muscle tissue compared to the control group (Van Doan et al. 2022 ). In a recent study, it was observed that Nile tilapia can accumulate carotenoids in their muscle tissue, as evidenced by increased total carotenoid levels when fish were fed with P. carotinifaciens at a concentration of 20 g/kg, irrespective of gender. This study is the first to report the utilization of P. carotinifaciens in Nile tilapia, highlighting the beneficial effects of supplementing their diet with astaxanthin-enriched P. carotinifaciens , leading to elevated total carotenoid levels in the fish's muscle tissue. In terms of promoting growth, our research utilizing carotenoids derived from the bacterium P. carotinifaciens , which produces astaxanthin, did not indicate any significant effects on the growth performance of male and female Nile tilapia. These outcomes might be attributed to the adult and maturation stage of the tilapia used in the study, as this fish typically observed a growth rate slowdown after reaching a certain size (Bwanika et al. 2007 ). Additionally, suboptimal temperature conditions during the research, with an average temperature of 22.18 ± 2.46°C and a range of 17°C to 26°C, could have contributed to these results. While Nile tilapia are generally tolerant of a wide temperature range (Dan and Little 2000 ), the optimal temperature for growth in most tilapia species is between 27 and 30°C (Nivelle et al. 2019 ). Thus, we suggest that the utilization of carotenoids from P. carotinifaciens may not be effective in enhancing the growth of fish when subjected to such environmental stressors. Low-temperature water stresses may affect the physiology and metabolism of fish, altering their receptivity to dietary supplements (Volkoff and Rønnestad 2020 ). It is essential that fish feed meets high nutritional standards in order to provide the essential nutrients for growth and health. In challenging environmental conditions, optimizing the nutritional value of fish feed becomes crucial. Dietary astaxanthin offers more than providing color and increasing carotenoid content in fish muscles (Brambilla et al. 2009 ). It exhibits antioxidant properties by neutralizing singlet oxygen, scavenging free radicals, and reducing lipid peroxidation (Naguib 2000 ; Hussein et al. 2006 ; Liu and Osawa 2007 ). Previous research has shown that incorporating astaxanthin-rich food sources in the diet of fish species such as rainbow trout improves fish fillet quality and reduces peroxide levels and transaminase activities in their serum (Nakano et al. 1995 ; Nakano et al. 1999 ; Rahman et al. 2016 ). Astaxanthin has exceptional antioxidant activity and the ability to enhance the protective capacity against oxidative stress (Sztretye et al. 2019 ). To assess antioxidant and immune function, various parameters were examined, including catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and lysozyme (LZM). CAT plays a crucial role in maintaining the physiological environment and innate immunity (Elvitigala et al. 2015 ), while SOD helps eliminate excessive reactive oxygen species and maintains the immune system's redox balance (Lin et al. 2009 ). MDA serves as an indicator of oxidative stress (Chen et al. 2017 ), and MPO is involved in activating immune cells during inflammatory responses (Noia et al. 2021 ). LZM is an essential component of the immune system (Costa et al. 2011 ). These enzymes, LZM and MPO activity, have been widely utilized as biomarkers for assessing innate immunity (Nhu et al. 2019 ; Bae et al. 2020 ). In this study, Nile tilapia fed with astaxanthin-enriched bacterium ( P. carotinifaciens ) exhibited enhanced SOD and CAT activity in the liver for both male and female fish. However, no significant difference was observed in MDA levels. Regarding immune function, dietary supplementation of carotenoid-rich bacteria significantly increased MPO activity, indicating immunostimulatory effects. Interestingly, it was not statistically significant in LZM activity, however, the outcome showed a potential enhancing health condition in tilapia. These findings align with previous reports demonstrating the ability of other microorganisms, such as S. pararoseus , Rhodotorula mucilaginosa and Rhodosporidium paludigenum , to improve antioxidant activity and immune response in fish species. For instance, Nile tilapia fed with S. pararoseus (Van Doan et al. 2022 ) and golden pompano fed with Rhodotorula mucilaginosa (Yu Wei et al. 2016 ) showed the increase of SOD activity; also shrimp fed with Rhodosporidium paludigenum exhibited increase of CAT and SOD activities (Yang et al. 2010 ). Based on these findings, carotenoids have the potential to enhance antioxidant activity and influence the immune response of tilapia. The mode of action was reviewed by Khalil et al. ( 2021 ), who showed that astaxanthin significantly affects the immune system. Numerous investigations have shown that astaxanthin has strong antioxidant effects in both in vitro and in vivo conditions. It was shown that astaxanthin significantly lowers the levels of pro-inflammatory cytokines such IL-6, TNF-α, IL-1β, and PGE2 and suppresses the activity of NF-κB, a key regulator of inflammation. Additionally, astaxanthin supports the recovery of Src homology 2 domain-containing protein tyrosine phosphatase 1, a protein that inhibits the signaling of inflammatory cytokines, restoring it to normal levels. As shown by Kishimoto et al. ( 2016 ), astaxanthin is also involved in lowering the release of reactive oxygen species (ROS) and inflammatory cytokines through the MAPK pathway. Hematological parameters and blood chemical profiles are the general reliable indicator to evaluate overall fish health (Kim et al. 2021 ; Casanovas et al. 2021 ). The hematological parameters and blood chemical profiles of male and female tilapia did not differ significantly across each treatment, according to our findings. However, both male and female tilapia fed astaxanthin-enriched P. carotinifaciens showed a significant increase in cholesterol levels compared to the control group. The correlation between plasma carotenoids and serum cholesterol levels is supported by the results of our research. This correlation can be explained by the absorption of lipophilic carotenoids along with dietary lipids and the transport of these substances by lipoproteins (Castellano et al. 2020 ). Moreover, the NPC1-like transporter 1 (NPC1L1) has been proposed as a possible candidate for carotenoid uptake, as indicated by the studies conducted by Davis and Altmann, ( 2009 ) and Reboul et al. ( 2011 ). Cholesterol undergoes esterification with free fatty acids upon absorption in the intestines, resulting in the formation of hydrophobic cholesterol esters (CEs). These CEs are then transported via bloodstream lipoproteins, which transport cholesterol to various metabolic and storage sites (Gonen and Miller 2020 ). Through receptor-mediated lipoprotein endocytosis, tissues acquire cholesterol from the bloodstream and either use it immediately or re-esterify it for storage (Maxfield and Wüstner 2002 ). While certain tissues, such as the testis, preferentially use de novo synthesized cholesterol as a substrate for steroid production (Hu et al. 2010 ), the majority of steroidogenic tissues, including the adrenal gland and ovary, acquire exogenous cholesterol via this process. In addition, the observed increase in cholesterol levels in the tilapia in our study can be attributed to their maturation, which resulted in elevated levels of sex hormones that are closely associated with cholesterol. Cholesterol is a precursor for a variety of steroid hormones, such as estrogens, androgens, and corticosteroids (Moon et al. 2016 ). As in other vertebrates, fish can acquire cholesterol via dietary consumption, release from intracellular depots, or de novo synthesis (Sharpe et al. 2006 ). Thus, understanding the relationship between carotenoids and cholesterol metabolism in fish is crucial, as cholesterol is a precursor for key steroid hormones involved in numerous physiological processes, including reproduction. The results of this study suggest that carotenoids may have an influent on the cholesterol metabolism of fish, thereby influencing their hormonal equilibrium and overall physiological state. The reproductive study was examined by exposing male and female subjects to astaxanthin-enriched P. carotinifaciens for a period of two months, followed by observing their mating behavior during the winter season. The water temperature during the study, specifically for a duration of four weeks in December, had an average of 19.87 ± 1.45°C (ranging from 17.00–23.00°C). Previous research has indicated that lower temperatures lead to developmental delays, while higher temperatures promote earlier spawning (Pankhurst and Munday 2011 ). Temperature plays a crucial role in various reproductive processes such as gametogenesis, development rate, recruitment, and gamete quality, affecting the metabolic pathways within the brain-pituitary-gonadal (BPG) axis (Migaud et al. 2013 ). For Nile tilapia, when temperatures are between 25 and 29°C, in this fish these processes are developed at their optimum level (Faruk et al. 2012 ). Below this temperature, reproduction comes to an end and feeding is limited below 20°C. Tilapia are easily damaged by temperatures below 10–12°C (Ernst et al. 1991 ; Dan and Little 2000 ; Costa-Pierce 2003 ; El-Sayed 2020 ). Apart from temperature conditions applied to captive broodstock, the incubation of eggs and larval rearing are known to impact gamete and larval quality (Migaud et al. 2013 ). Our study investigated the effects of feeding Nile tilapia with astaxanthin-enriched P. carotinifaciens on their reproductive parameters. Despite the low water temperature, which was unfavorable for hatchery purposes and optimal egg quality, our results suggest that the supplementation of P. carotinifaciens , particularly in the T4 group, had a significant improvement in the egg production, even when there was winter stress. For the quantity of spermatozoa, it is worth noting that there was a significant increase in spermatozoa concentration in the male group that received the enriched diet. These findings imply that the nutritional potential provided by P. carotinifaciens supplementation could positively impact broodstock, affecting the distribution of critical macro and micronutrients inside the eggs. This highlights the importance of effective broodstock feeding for ensuring optimal larval survival and early development, as previously suggested by other studies (Izquierdo et al. 2001 ). The consistency of our results with previous research adds further support to the potential benefits of carotenoid supplementation in reproductive processes. Studies on goldfish, Atlantic cod broodstock, ornamented convict cichlid, and discus fish have demonstrated significant improvements in osmolality, spermatocrit value, sperm concentration, egg quality, gonadal maturation, and the upregulation of vitellogenin gene expression upon the addition of carotenoids like astaxanthin and β-carotene to the diets (Sawanboonchun et al. 2008 ; Brown et al. 2014 ; Tizkar et al. 2015 ; Haque et al. 2023 ). Carotenoids, including both natural and synthetic forms of astaxanthin, are widely used as feed additives in aquatic animals. They have been shown to play a significant role in reproductive processes such as egg production, egg quality, and semen improvement (Tizkar et al. 2013 ; Palma et al. 2017 ). Carotenoids, in a role similar to that in mammals, exhibit a steroidogenic function and serve as regulators of folliculogenesis and oogenesis in fish. And the presence of retinol and retinoic acid, which are crucial for the development of mature sperm, further highlights the significance of carotenoids in reproductive functions (Pasquariello et al. 2022 ). Furthermore, astaxanthin serves as an essential source of retinol and 3,4-didehydroretinol, which are precursors of vitamin A and have crucial roles during the development of vertebrate embryos (Moren et al. 2002 ; Blomhoff and Blomhoff 2006 ; Duester 2008 ; Kin Ting Kam et al. 2012 ). In addition, retinoic acid, a carotenoid derivatve, plays an essential role in the development of larval structures such as the neural crest (Bohnsack and Kahana 2013 ) and the heart (Huang et al. 2011 ). Brown et al. ( 2013 ) discovered that fish diets supplemented with carotenoids increased the quantitative of carotenoids deposited in the gonads. Carotenoids substance is accumulated in the ovaries, where the eggs mature. In addition, it can convert into retinoids as a precursor for reproductive system (Levi et al. 2011 ). During embryonic development, retinoids, which belong to the vitamin A family, serve a function in a variety of cellular processes (Johnson and Scadding 1991 ). Based on our exploring data, we conclude that the astaxanthin-enriched bacterium ( P. carotinifaciens ) potentially improves reproductive capabilities in tilapia, even when they are under overwintering conditions. However, to improve the quality and viability of progeny, more research is necessary to clarify the mechanisms and dynamics of carotenoid metabolism and its role in reproduction. Conclusion The results demonstrated that the dietary inclusion of the astaxanthin-enriched bacterium Paracoccus carotinifaciens at a dose of 20.0 g/kg can lead to accumulation of carotenoids in muscle tissue, improve antioxidant status, boost immune function, and affect cholesterol metabolism in fish. This, in turn, has the potential to enhance tilapia's reproductive capabilities, particularly sperm quality and egg production, even under overwintering conditions. However, further research is needed to fully understand the potential benefits and limitations of P. carotinifaciens in aquaculture practices under challenging environmental conditions. Declarations Acknowledgements We gratefully thank Marine leader or.th for kindly supplying the bacterium ( Paracoccus carotinifaciens ) used for this research. We extend special thanks to Ms. Wachirintra Sanseemon and Ms. Piyapohn Kanhaseeya for their kind assistance during the preparation of this research. Author contributions Nantaporn Sutthi was involved in conceptualization, methodology, formal analysis, investigation, resources, data curation, writing-original draft preparation, visualization, writing - review & editing, project administration, and funding acquisition. Paiboon Panase was involved in methodology, writing - review & editing, and funding acquisition. Thitiwut Vongkampang was involved in methodology, writing-original draft preparation, writing - review & editing. Eakapol Wangkahart was involved in methodology, formal analysis, writing - review & editing. Funding This research project was financially supported by Mahasarakham University and this research work was partially supported by Unit of Excellence Physiology and Sustainable Production of Terrestrial and Aquatic Animals (FF66-UoE014), University of Phayao. Ethic Animal Institutional Animal Care and Use Committee, Mahasarakham University (IACUC-MSU), Thailand (IACUC-MSU-13/2021). Competing interest The authors declare no competing interests. Data availability All data generated or analyzed during this study are included in this published article. References Abd El-Hack ME, El-Saadony MT, Nader MM, Salem HM, El-Tahan AM, Soliman SM, Khafaga AF (2022). 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Cite Share Download PDF Status: Published Journal Publication published 21 Mar, 2024 Read the published version in Fish Physiology and Biochemistry → Version 1 posted Editorial decision: Revision requested 15 Feb, 2024 Reviews received at journal 24 Sep, 2023 Reviewers agreed at journal 23 Sep, 2023 Reviewers invited by journal 23 Sep, 2023 Editor assigned by journal 23 Sep, 2023 Submission checks completed at journal 01 Aug, 2023 First submitted to journal 30 Jul, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3218062","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":223346906,"identity":"d20fdb6a-d124-487d-98d3-0a4a1dd046ba","order_by":0,"name":"Paiboon Panase","email":"","orcid":"","institution":"University of Phayao","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Paiboon","middleName":"","lastName":"Panase","suffix":""},{"id":223346907,"identity":"72a0cfac-d91e-4099-a88d-f6fed1aa00d7","order_by":1,"name":"Thitiwut Vongkampang","email":"","orcid":"","institution":"Mahasarakham University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Thitiwut","middleName":"","lastName":"Vongkampang","suffix":""},{"id":223346908,"identity":"a8f2acb0-d2e9-4fb8-ac4c-7bf1f3361022","order_by":2,"name":"Eakapol Wangkahart","email":"","orcid":"","institution":"Mahasarakham University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Eakapol","middleName":"","lastName":"Wangkahart","suffix":""},{"id":223346909,"identity":"027e28d4-f530-4072-9b5a-912ede188eda","order_by":3,"name":"Nantaporn Sutthi","email":"data:image/png;base64,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","orcid":"","institution":"Mahasarakham University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Nantaporn","middleName":"","lastName":"Sutthi","suffix":""}],"badges":[],"createdAt":"2023-07-30 14:59:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3218062/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3218062/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10695-024-01331-8","type":"published","date":"2024-03-21T15:00:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":41183454,"identity":"0d5c8376-ab0e-4a3a-82a8-663c6213e606","added_by":"auto","created_at":"2023-08-07 14:07:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60156,"visible":true,"origin":"","legend":"\u003cp\u003eWater temperature (°C) during the 12 weeks experimental (fish culture 8 weeks and mating period 4 weeks) during the winter season (October to December).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3218062/v1/c784b6c89f2c5105a33a368b.jpg"},{"id":41182429,"identity":"daf9058b-90e4-4883-a907-a19cf8efd645","added_by":"auto","created_at":"2023-08-07 13:59:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48828,"visible":true,"origin":"","legend":"\u003cp\u003eTotal carotenoid content of broodstock Nile tilapia, which were fed different levels of \u003cem\u003eP. carotinifaciens\u003c/em\u003e for 8 weeks: T1 (control), T2 (5.0 g/kg diet), T3 (10.0 g/kg diet), and T4 (20.0 g/kg diet). Data are given as mean ± SD, and different superscripts indicate significant differences (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3218062/v1/4342ac4429d50d8cc22daa3e.jpg"},{"id":41182433,"identity":"d9dd2650-8de7-43a9-8b11-f6754f576fb1","added_by":"auto","created_at":"2023-08-07 13:59:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74621,"visible":true,"origin":"","legend":"\u003cp\u003eAntioxidant activity enzyme and lipid peroxidation parameter. (a) superoxide dismutase (SOD), (b) malondialdehyde (MDA), and (c) catalase (CAT) of broodstock Nile tilapia fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e for 8 weeks: T1 (control), T2 (5.0 g/kg diet), T3 (10.0 g/kg diet), and T4 (20.0 g/kg diet). Data are given as mean ± SD, and different superscripts indicate significant differences (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3218062/v1/1473e5e3714a9902bd96b0e5.jpg"},{"id":41182436,"identity":"05358b9c-d2fb-441c-97d1-4afa64e29643","added_by":"auto","created_at":"2023-08-07 13:59:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45968,"visible":true,"origin":"","legend":"\u003cp\u003eMyeloperoxidase (MPO) (a) and lysozyme activity (LZM) (b) of broodstock Nile tilapia fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e for 8 weeks: T1 (control), T2 (5.0 g/kg diet), T3 (10.0 g/kg diet), and T4 (20.0 g/kg diet). Data are given as mean ± SD, and different superscripts indicate significant differences (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3218062/v1/a4f0cb131c3b3cf836f09ec3.jpg"},{"id":41182431,"identity":"0b013371-ecd4-4589-847c-c0695b2bb827","added_by":"auto","created_at":"2023-08-07 13:59:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34166,"visible":true,"origin":"","legend":"\u003cp\u003eSperm parameters of male tilapia fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e for 8 weeks. Spermatozoa concentration (a) and Milt volume (b): T1 (control), T2 (5.0 g/kg diet), T3 (10.0 g/kg diet), and T4 (20.0 g/kg diet). Data are given as mean ± SD, different superscripts are significant differences (p\u0026lt;0.05), and * represents the student’s t-test for significant differences (p\u0026lt;0.05) between before and after feeding diet in the same group.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3218062/v1/a52b6474228ce5c50989dde1.jpg"},{"id":53403486,"identity":"a4d0c07e-2f02-4493-baaf-694041b355c2","added_by":"auto","created_at":"2024-03-25 15:10:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":781811,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3218062/v1/6c08a1ee-c2a5-40ce-9e74-ff2e6390331d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of astaxanthin-enriched bacterium (Paracoccus carotinifaciens) on growth, immune response, and reproduction performance of broodstock Nile tilapia during winter season","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWater temperature is crucial factor for fish's growth, reproductive capabilities, and metabolism, and can also impact their immune response (Azaza et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Winter conditions directly affect Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) by reducing their feed intake, weakening the immune system, and inhibiting reproductive function. These challenges influence productivity and profitability in aquaculture, of this fish which is one of the most important warm-water aquaculture fish groups worldwide (Abd El-Hack et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Nile tilapia is the third most cultivated species in global aquaculture, with a production of 4.5\u0026nbsp;million tonnes (FAO \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, it is predicted that total production will increase by 53% by 2030 (Maulu et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In terms of food security, this fish species is an affordable source of protein for low-income people (El-Ouny et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Due to metabolic dysfunctions and immunological disorders, tilapia are more susceptible to opportunistic infections at low temperatures (Panase et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Mass mortality often occurs during winter cold fronts, and feed consumption declines at temperatures below 20\u0026deg;C, compromising growth performance (El-Sherif and Elfeky \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Nehemia et al. (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) found that the low temperature level between 20 to 22\u0026deg;C could limit the growth development of tilapia by 30%, and the lethal minimum temperature for tilapia is 10 or 11\u0026deg;C. Moreover, high mortalities are related to severe winters with extended cold periods, which can induce pathogens, resulting in economic losses (Hurst \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecently, many studies have concentrated on the energy requirements for maintaining growth or physiological responses during cold winter period of stress (Nobrega et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Abdel-Ghany et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hassaan et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, these studies have given insight into the significance of energy intake. There has been much study of how to keep fish alive and growing, but much less on how winter stress affects their ability to reproduce and, more research is needed to fully emphasize the effect of winter conditions on the reproductive capacities of fish species. Reproductive processes may be interrupted or reduced due to oxidative stress during the winter months when organisms are already facing several physiological obstacles. The negative impacts of oxidative stress, including reduced gamete formation, changed hormone levels, and diminished fertility, could disrupt the delicate balance necessary for healthy reproduction (Aitken et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAstaxanthin is a subgroup of carotenoid pigment which is found in various organisms, ranging from plants to animals, and are known for their diverse biological activities (Maoka \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It exhibits powerful antioxidant properties, neutralizing harmful reactive oxygen species (ROS) that can lead to oxidative stress and damage cellular structures (Brotosudarmo et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Beyond its antioxidant capacity, research has highlighted a potential role of astaxanthin in supporting reproductive health (Liu et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Numerous studies have investigated the impact of dietary astaxanthin in farmed fish species on reproductive parameters e.g. gamete quality, hormone regulation, and fertility enhancement (Choubert et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Ahmadi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sheikhzadeh et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Astaxanthin, in its role as a natural antioxidant, could potentially lessen the harmful effects of oxidative stress on reproductive cells and tissues (Li et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, this carotenoid may influence hormone production and signaling pathways related to reproduction, ultimately influencing reproductive success and outcomes (Pasquariello et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The precise mechanisms underlying astaxanthin's effects on reproductive processes are still being explored, but the existing evidence suggests its potential as a valuable component in the modulation of reproductive functions. Moreover, astaxanthin, a potent antioxidant with health benefits, can be obtained naturally or synthetically (Ambati et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although they share similar chemical characteristics, their structural compositions differ, raising concerns about long-term health effects and the scarcity of natural sources and their high-cost limit widespread utilization more than synthetic ones (Stachowiak and Szulc \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Natural astaxanthin, a carotenoid pigment discovered in specific microorganisms, plants, and marine creatures, is recognized for its potent antioxidant, anti-inflammatory, and immunomodulatory capabilities (Ambati et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mart\u0026iacute;nez-C\u0026aacute;mara et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, natural astaxanthin demonstrates greater biological effectiveness compared to its synthetic counterpart (Aneesh et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Main carotenoids are produced by microorganisms, such as \u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e, which is gram-negative, aerobic microorganism characterized by its orange pigmented, rod-shaped structure and motility through peritrichous flagella (Tsubokura et al. \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). It can produce astaxanthin which has been proven to be bioavailable and effective in enhancing the color of flesh in Coho salmon, Atlantic salmon, and rainbow trout (European Food Safety Authority \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Fillets of salmon fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e have exhibited a more diverse composition of muscle carotenoids and a darker red color compared to conventional salmon (Lerfall et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and feeding with \u003cem\u003eP. carotinifaciens\u003c/em\u003e enhances the color of shrimp (Maoka et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Panaferd\u0026reg;-AX, a natural source of carotenoids derived from \u003cem\u003eP. carotinifaciens\u003c/em\u003e, was given conditional approval for use in salmonid aquaculture by the European Food Safety Authority in 2007 (European Food Safety Authority \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Moreover, it is used to provide antioxidants, nutrients, and color to salmonids and crustaceans (Oehlenschl\u0026auml;ger and Ostermeyer \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The efficacy of astaxanthin-rich carotenoid extracts from \u003cem\u003eP. carotinifaciens\u003c/em\u003e has been explored through animal and clinical studies, indicating potential benefits for human nutrition and anxiety prevention (Hayashi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). While previous research has examined the effects of \u003cem\u003eP. carotinifaciens\u003c/em\u003e on numerous aspects of fish physiology, such as coloration, growth, and immunity (Lerfall et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Maoka et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nakano and Wiegertjes \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), relatively little is known about the reproductive response of fish, specifically tilapia, under winter stress conditions. Understanding the reproductive effects of \u003cem\u003eP. carotinifaciens\u003c/em\u003e in this context is crucial for assessing its potential benefits or limitations in aquaculture practices. Winter stress poses unique challenges to fish health and reproduction. Therefore, the objective of this study was to investigate the reproductive response of tilapia fish to winter stress in the presence of \u003cem\u003eP. carotinifaciens\u003c/em\u003e, and the study will provide valuable insight into the relationship between this microorganism and fish in challenging environmental conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFish and experimental design\u003c/h2\u003e \u003cp\u003eEach one hundred and twenty of broodstock, including male and female Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) of 6 months of age, weighing 161.70\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 g of male and 126.54\u0026thinsp;\u0026plusmn;\u0026thinsp;7.05 g of female, were purchased from Maha Sarakham Inland Fisheries Research and Development Center (Maha Sarakham, Thailand). The fish were separated by gender and placed in concrete ponds (size: 10\u0026times;5\u0026times;1.2 m.). They were allowed to acclimate for two weeks and were hand-fed twice a day with a commercial diet containing 32% protein (Betagro\u0026reg;, Thailand) during the acclimatization period. Subsequently, a total of 24 cages (size: 1.5\u0026times;1\u0026times;1 m.) were randomly stocked, with 12 cages assigned to male fish (n\u0026thinsp;=\u0026thinsp;10 fish/cage) and 12 cages assigned to female fish (n\u0026thinsp;=\u0026thinsp;10 fish/cage). The fish were then randomly divided into four groups for triplicate experiments. The control group (T1) was fed the basal diet without supplementation, while the other three groups were fed diets containing different levels of dried cells of an astaxanthin enriched bacterium \u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e: T2 (5 g/kg feed), T3 (10 g/kg feed), and T4 (20 g/kg feed). The fish were fed twice daily at 09.00 and 16.30 h, with a feeding rate of 3% biomass per day, for a duration of 8 weeks from October to November. After the 8-week feeding trial, a total of seventy-two healthy fish were selected for the mating experiment with a male-to-female ratio of 1:2, conducting for a period of 4 weeks during the winter season in December. The water quality parameters dissolved oxygen, total ammonia nitrogen (TAN), and pH were monitored throughout the experimental period, with average values of 6.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 mg/L, 0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mg/L, and 8.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82, respectively. The water temperature during the experimental period is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, with an average temperature of 22.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46\u0026deg;C, ranging from 17\u0026deg;C to 26\u0026deg;C, over a span of 12 weeks. Additionally, the water temperature during the mating period averaged 19.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u0026deg;C, ranging from 17.00\u0026deg;C to 23.00\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSupplemental diet preparation\u003c/h2\u003e \u003cp\u003eThroughout the study, commercial fish feed pellets from Betagro\u0026reg; in Thailand were used. Analysis of the commercial feed was shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These pellets contain a composition of 32% crude protein, 4% lipid, 12% moisture, and 6% fiber. The dried cells of an astaxanthin-enriched bacterium called \u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e were obtained from the commercial product Panaferd\u0026reg; AX in Japan. In addition to this product, it contains astaxanthin (20 g/kg), adonirubin (8 g/kg), and canthaxanthin (2 g/kg) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The procedures for preparation of feed supplementing with astaxanthin were conducted according to the methos by Sutthi and Thaimuangphol (\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The astaxanthin-enriched bacterium was mixed with the basal diet in four different proportions: 0, 5, 10, and 20 g/kg of the diet. The mixture was thoroughly combined using a mixer machine and air-dried. Afterward, the pellets from all four groups were bound with 20 g of guar gum and top coated with a 4% agar solution at a rate of 20 mL/kg of the diet. The coated pellets were then subjected to another round of drying at 30\u0026deg;C for 24 hours using a hot air oven. The prepared diets were then stored at 4\u0026deg;C for future use. The proximate analysis of the experimental diets, including crude protein, crude lipid, moisture, and ash, was performed using standard analysis methods (AOAC \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), and the results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of the commercial feed for Nile tilapia and \u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e components.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeed Analyses\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eComposition\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude protein (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude lipid (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude fiber (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoisture (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eParacoccus carotinifaciens\u003c/b\u003e \u003cb\u003ecomponents\u003c/b\u003e\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eComposition\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAstaxanthin (g/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdonirubin (g/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCanthaxanthin (g/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003csup\u003e1\u003c/sup\u003e Commercial feed information (Betagro\u0026reg;, Thailand). Ingredients of the commercial fish feed: fish meal, soybean meal, corn, broken rice, rice bran, including vitamins and minerals.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003csup\u003e2\u003c/sup\u003eTechnical Information (Panaferd\u0026reg; AX, Japan)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProximate analysis of the experimental diets (% dry matter).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnalyzed chemical composition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003cp\u003e(control diet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003cp\u003e(5 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003cp\u003e(10 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ediet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003cp\u003e(20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude protein (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude lipid (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude fiber (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoisture (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNFE (%)\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e49.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e49.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGE (MJ/kg) \u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e1\u003c/sup\u003eNitrogen-free extract (NFE%)\u0026thinsp;=\u0026thinsp;100 - (crude protein\u0026thinsp;+\u0026thinsp;crude lipid\u0026thinsp;+\u0026thinsp;ash\u0026thinsp;+\u0026thinsp;crude fiber).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003e2\u003c/sup\u003eGross energy (GE) calculated based on 23.6, 39.5 and 17.2 kJ/g protein, lipid, and carbohydrates, respectively.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGrowth measurements\u003c/h2\u003e \u003cp\u003eThe weight and length of each fish in all treatments were recorded at the beginning and the end of the experimental period. The growth rate of all fish was determined using the mathematical growth model with the following equations (Bagenal \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1978\u003c/span\u003e):\u003c/p\u003e \u003cp\u003eWeight gain (WG; g)\u0026thinsp;=\u0026thinsp;final weight (g) \u0026ndash; initial weight (g);\u003c/p\u003e \u003cp\u003eAverage daily gain (ADG; g/day) = [final weight (g) \u0026ndash; initial weight (g)]/days;\u003c/p\u003e \u003cp\u003eSpecific growth rate (SGR; %/day)\u0026thinsp;=\u0026thinsp;100\u0026times;[{Ln final weight (g) \u0026ndash; Ln initial weight (g)}/days]; Feed conversion ratio (FCR)\u0026thinsp;=\u0026thinsp;total feed (g)/weight gain (g);\u003c/p\u003e \u003cp\u003eSurvival Rate (SR, %) = [number of survived fish/initial number of fish]\u0026times;100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSamples collection\u003c/h2\u003e \u003cp\u003eAfter the 8-week feeding trial, blood samples were collected immediately from the caudal vein of the fish. Prior to collection, the fish were anesthetized using clove oil at a concentration of 100 mg/L. A volume of 0.5 mL of whole blood was taken and transferred into anticoagulant tubes for hematological analysis. Additionally, another part of blood samples of 1 mL was placed into sterile Eppendorf tubes without anticoagulant for serum collection. The serum was obtained by allowing the blood sample to clot at room temperature for 1 hour, followed by 4\u0026deg;C for 4 hours. Subsequently, the samples were centrifuged at 5000\u0026times;g for 10 minutes at 4\u0026deg;C. All the samples were then stored in Eppendorf tubes at -20\u0026deg;C until further use. The serum samples were utilized for blood chemical analysis and immunological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTotal carotenoid extraction\u003c/h2\u003e \u003cp\u003eAfter feeding the experimental diet, the total carotenoid content in fish muscle (n\u0026thinsp;=\u0026thinsp;3 per treatment) was measured using the method as per Rodriguez-Amaya and Kimura (\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Briefly, the measurement of total carotenoid content was conducted following the procedure outlined in our previous study (Van Doan et al. \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It involved using a spectrophotometer at a wavelength of 450 nm, and the total carotenoid content was calculated using the formula provided by Rodriguez-Amaya and Kimura (\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2004\u003c/span\u003e):\u003c/p\u003e \u003cp\u003eTotal carotenoid content (\u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{\\text{A}\\times \\text{v}\\text{o}\\text{l}\\text{u}\\text{m}\\text{e}\\left(\\text{m}\\text{l}\\right)\\times {10}^{4}}{{\\text{A}}_{1\\text{c}\\text{m}}^{1\\text{\\%}}\\times \\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e} \\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t} \\left(\\text{g}\\right)}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWhere A\u0026thinsp;=\u0026thinsp;absorbance; volume\u0026thinsp;=\u0026thinsp;total volume of extract;\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{A}}_{1\\text{c}\\text{m}}^{1\\text{\\%}}\\)\u003c/span\u003e\u003c/span\u003e= absorption coefficient of β-carotene in petroleum ether (2592).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSerum biochemical analysis\u003c/h2\u003e \u003cp\u003eThe collected blood samples were sent to the Veterinary Central Lab located Mueang Khon Kaen District, Khon Kaen 40000, Thailand for blood chemical analysis. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), cholesterol, total protein, and albumin were determined using commercial reagent kits (ABX Pentra reagent, Horiba, France) following the manufacturer's instructions. The concentration levels of these parameters were measured using the colorimetry method with an ABX Pentra 400 Clinical Chemistry Analyzer (Horiba, France). The globulin level was calculated by subtracting the value of albumin from the total protein.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eHematological parameters\u003c/h2\u003e \u003cp\u003eThe blood samples collected were sent to the Veterinary Central Lab located Mueang Khon Kaen District, Khon Kaen 40000, Thailand for hematological analysis. The erythrocyte (RBC) and leukocyte (WBC) counts were determined using a Neubaeur hemocytometer (Rehulka, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). To obtain the differential leukocyte count, blood smears were prepared and stained with a combination of Giemsa/May-Grunwald (Davis et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The hematocrit (Hct) was measured by centrifuging heparinized micro-hematocrit capillary tubes at 12,000\u0026times;g for 5 minutes and reported as percentages (Zhao et al. \u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The concentration of hemoglobin (Hb) was measured using a lysing reagent kit (ABX reagent, France) that enables the lysis of erythrocytes and cyanide-free determination of hemoglobin. The released hemoglobin was then oxidized and stabilized, and the resulting complexes were quantified by spectrophotometry at a wavelength of 550 nm using a hematology analyzer (ABX Micros EVS 60, France). Blood indices were calculated according to the formulas proposed by (Mansour et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e): the following formulas: Mean cell volume (MCV)\u0026thinsp;=\u0026thinsp;Hct(%)\u0026times;10/RBCs (10\u003csup\u003e6\u003c/sup\u003e mm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e); Mean cell hemoglobin (MCH)\u0026thinsp;=\u0026thinsp;Hb (g/dl)\u0026times;10/RBCs count (10\u003csup\u003e6\u003c/sup\u003e mm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e); Mean corpuscular hemoglobin concentration (MCHC)\u0026thinsp;=\u0026thinsp;Hb (g/dl)/Hct (%).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLipid peroxidation and antioxidant enzymes activity\u003c/h2\u003e \u003cp\u003eSerum malondialdehyde (MDA) concentrations were determined by measuring the amount of thiobarbituric acid reactive substances (TBARS) using the method suggested by Aengwanich et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e with slightly modifications, as described in detail by Sutthi et al. (\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The concentration of MDA was measured using a spectrophotometer at 532 nm. Catalase activity (CAT) in the liver was determined according to the method described by (Wangkaghart et al. \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The difference in absorbance was recorded after 20 s (A1) and after 80 s (A2) of incubation at 240 nm and room temperature. The CAT value was calculated as (A1 - A2) / 0.0008. Liver superoxide dismutase (SOD) activity was measured according to the methods described by (Mansour et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The activity was calculated using the following formulas:\u003c/p\u003e \u003cp\u003eThe percent of inhibition (%)\u0026thinsp;=\u0026thinsp;100- ((ΔA control-ΔA sample/ ΔA control) \u0026times; 100).\u003c/p\u003e \u003cp\u003eSOD activity (U g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e liver) = % inhibition \u0026times; 3.75.\u003c/p\u003e \u003cp\u003eLysozyme activity (LZM) in serum was determined using a turbidimetric assay based on the lysis of the lysozyme-sensitive Gram-positive bacterium \u003cem\u003eMicrococcus lysodeikticus\u003c/em\u003e (Sigma, USA). The method followed the approach of (Parry et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e1965\u003c/span\u003e) with some modifications, as described in detail by (Wangkaghart et al. \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The myeloperoxidase (MPO) activity present in serum was measured following the method suggested by (Sahoo et al. \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with slight modifications, as described in detail by (Mansour et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The MPO content was measured by absorbance at 450 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eReproductive activity\u003c/h2\u003e \u003cp\u003eIn male tilapia, milt parameters were collected before and after the experimental diet trial (8 weeks). The measurement followed the method described by (Marijani et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Milt volume was randomly selected and collected from males by using hand stripping. The volume was measured with a pipette and expressed in milliliters (ml). The spermatozoa concentration was determined using a hemocytometer and expressed as the number of cells x 10\u003csup\u003e8\u003c/sup\u003e cells/ml.\u003c/p\u003e \u003cp\u003eIn females, seventy-two healthy male and female fish were selected for the mating experiment in a ratio of 1:2 (male (n\u0026thinsp;=\u0026thinsp;2): female (n\u0026thinsp;=\u0026thinsp;4)) per replication. The egg parameters were collected every week during the 4-week mating period in the winter season (December). The diameters of the oocytes were measured using a microscope equipped with an ocular micrometer. The hatching rate was calculated by counting the number of larvae 4 days after fertilization, following the methods described by De Lapeyre et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The spawning rate was defined as the percentage of females that spawned over a 7-days period out of the total number of females in each group. The survival rate of larvae was calculated by counting the percentage of number of survival larvae 7 days after hatching egg.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe obtained data were performed using a one-way analysis of variance (ANOVA) followed by Duncan\u0026rsquo;s post hoc for multiple comparisons among the treatments. The significance level was set as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Results are expressed throughout as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGrowth performances\u003c/h2\u003e \u003cp\u003eThe effects of astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e supplementation in the feed for 8 weeks on the growth performance of broodstock tilapia are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The growth performance parameters, including weight gain (WG), average daily gain (ADG), specific growth rate (SGR), feed conversion ratio (FCR), and survival rate (SR), showed no significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) among all treatment groups for both male and female tilapia.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth performances of Nile tilapia fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e supplement in diet for 8 weeks.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003cp\u003e(control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003cp\u003e(5 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003cp\u003e(10 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003cp\u003e(20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight gain (WG; g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e88.63\u0026thinsp;\u0026plusmn;\u0026thinsp;12.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e82.30\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e66.20\u0026thinsp;\u0026plusmn;\u0026thinsp;14.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e80.07\u0026thinsp;\u0026plusmn;\u0026thinsp;9.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage daily growth gain (ADG; g/day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecific growth rate; SGR (% /day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeed conversion ratio; (FCR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvival rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e93.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e96.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e96.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e90.00\u0026thinsp;\u0026plusmn;\u0026thinsp;10.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight gain (WG; g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e75.28\u0026thinsp;\u0026plusmn;\u0026thinsp;14.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e82.44\u0026thinsp;\u0026plusmn;\u0026thinsp;35.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e59.55\u0026thinsp;\u0026plusmn;\u0026thinsp;22.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e67.22\u0026thinsp;\u0026plusmn;\u0026thinsp;30.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage daily growth gain (ADG; g/day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecific growth rate; SGR (% /day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeed conversion ratio; (FCR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvival rate; SR (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e70.00\u0026thinsp;\u0026plusmn;\u0026thinsp;10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e80.00\u0026thinsp;\u0026plusmn;\u0026thinsp;10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e86.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e83.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBlood chemical profiles and hematological analysis\u003c/h2\u003e \u003cp\u003eThe blood chemical profiles and hematological indices were examined at the end of the feeding period with experimental diets, and the results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The cholesterol content in both male and female tilapia fed with astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e showed a significant increase compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The highest cholesterol content was observed in fed T4 diet for both males and females. However, no significant differences were observed among all treatment groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) for total protein, albumin, globulin, aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN) levels. In the hematological analysis of male and female tilapia, there were no significant differences observed among all treatments (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in terms of red blood cell count, white blood cell count, hemoglobin level, hematocrit level, neutrophil count, and lymphocyte count.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBlood chemical profiles of Nile tilapia fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e supplement in diet for 60 days.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003cp\u003e(control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003cp\u003e(5 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003cp\u003e(10 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ediet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003cp\u003e(20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal protein (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlbumin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlobulin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAspartate aminotransferase; AST (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.00\u0026thinsp;\u0026plusmn;\u0026thinsp;17.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.33\u0026thinsp;\u0026plusmn;\u0026thinsp;29.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlanine aminotransferase; ALT (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.00\u0026thinsp;\u0026plusmn;\u0026thinsp;23.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.33\u0026thinsp;\u0026plusmn;\u0026thinsp;17.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.66\u0026thinsp;\u0026plusmn;\u0026thinsp;11.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood Urea Nitrogen; BUN (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholesterol (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e126.66\u0026thinsp;\u0026plusmn;\u0026thinsp;10.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e142.66\u0026thinsp;\u0026plusmn;\u0026thinsp;14.0\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e143.33\u0026thinsp;\u0026plusmn;\u0026thinsp;22.36 \u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e168.33\u0026thinsp;\u0026plusmn;\u0026thinsp;9.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal protein (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlbumin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlobulin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAspartate aminotransferase; AST (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.33\u0026thinsp;\u0026plusmn;\u0026thinsp;30.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e65.00\u0026thinsp;\u0026plusmn;\u0026thinsp;35.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.66\u0026thinsp;\u0026plusmn;\u0026thinsp;20.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e58.33\u0026thinsp;\u0026plusmn;\u0026thinsp;26.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlanine aminotransferase; ALT (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.33\u0026thinsp;\u0026plusmn;\u0026thinsp;14.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27.33\u0026thinsp;\u0026plusmn;\u0026thinsp;17.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood Urea Nitrogen; BUN (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholesterol (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e112.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.50\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e135.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e149.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e163.66\u0026thinsp;\u0026plusmn;\u0026thinsp;9.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and different superscripts are significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHematological analysis of Nile tilapia fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e supplement in diet for 60 days.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003cp\u003e(control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003cp\u003e(5 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003cp\u003e(10 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ediet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003cp\u003e(20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBC (x10\u003csup\u003e6\u003c/sup\u003ecells/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e10.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e10.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHaematocrit (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.66\u0026thinsp;\u0026plusmn;\u0026thinsp;10.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e32.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e29.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e29.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC (x10\u003csup\u003e3\u003c/sup\u003ecells/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.49\u0026thinsp;\u0026plusmn;\u0026thinsp;5.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e9.90\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophils (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e31.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e31.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e29.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e30.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocytes (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e64.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e64.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e67.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e65.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFemale\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRBC (x10\u003csup\u003e6\u003c/sup\u003ecells/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHemoglobin (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e10.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHaematocrit (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e38.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e32.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e33.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC (x10\u003csup\u003e3\u003c/sup\u003ecells/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e8.47\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophils (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e32.00\u0026thinsp;\u0026plusmn;\u0026thinsp;8.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e37.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e32.00\u0026thinsp;\u0026plusmn;\u0026thinsp;4.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocytes (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e63.33\u0026thinsp;\u0026plusmn;\u0026thinsp;7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e59.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e69.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. RBC\u0026thinsp;=\u0026thinsp;red blood cell, WBC\u0026thinsp;=\u0026thinsp;white blood cell.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThe total carotenoid analysis\u003c/h2\u003e \u003cp\u003eThe total carotenoid content in the muscle was assessed at the end of the experiment, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. There was a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the values of total carotenoid content between male and female tilapia fed T4 diet compared to the control diet. Both male and female fish fed T4 diet exhibited the highest total carotenoid content in fish muscle.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant and immunological observations\u003c/h2\u003e \u003cp\u003eThe liver antioxidant parameters of male and female Nile tilapia, which were fed astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e diets for 8 weeks, were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The activity of superoxide dismutase (SOD) obtained in fish fed with the experimental diets were illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The highest SOD values were observed in female tilapia fed T4 diet, followed by the T3 and T2 diets. All these values (T3, T4, T5) were significantly higher than those of the control group. However, no significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in SOD levels were observed among the treatments for male tilapia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). There were no significant differences observed in MDA levels among all treatments for both male and female tilapia (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The CAT enzyme activity of both male and female tilapia fed the T4 diet was significantly higher compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe immunological parameters in the serum of male and female Nile tilapia, which were fed experimental diets, was depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The MPO in response to the dietary sources of astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. It is observed that male tilapia fed the T4 diet exhibited a significant increase in MPO activity compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the supplementation of astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e in all diets did not have an impact on MPO activity in female tilapia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). There were no significant differences LZM activity observed among all groups for both male and female tilapia (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eReproductive activity\u003c/h2\u003e \u003cp\u003eThe effect of dietary supplementation of \u003cem\u003eP. carotinifaciens\u003c/em\u003e on sperm parameters were assessed in male tilapia as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Prior to the experimental diet, there were no significant differences in spermatozoa concentration among all groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). However, after feeding the diet containing astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e, the spermatozoa concentration was significantly increased in fish fed T4 diet compared to the control and T2 diets (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, there was a significant increase in spermatozoa count in fish fed T4 diet compared to before the diet started (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The milt volume before and after feeding male tilapia with \u003cem\u003eP. carotinifaciens\u003c/em\u003e was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, and no significant differences were observed in milt volume among all treatment groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the case of female tilapia, after feeding with astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e for 8 weeks, a mating experiment was implemented with a 1:2 ratio of males to females for four weeks in December. The reproductive performance in terms of egg quality is presented in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Fish fed with the T4 diet showed significant improvements in the average number of eggs per week and the number of eggs per female, which were higher than on the control diet (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant differences were observed in spawning rate (%), oocyte diameter, hatching rate, or larvae survival rate compared to the control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFemale reproductive performances during 4 weeks in winter season after fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e supplement in diet.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003cp\u003e(control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003cp\u003e(5 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003cp\u003e(10 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003cp\u003e(20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e diet)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage egg numbers per week\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e411.41\u0026thinsp;\u0026plusmn;\u0026thinsp;101.52\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e555.75\u0026thinsp;\u0026plusmn;\u0026thinsp;53.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e768.00\u0026thinsp;\u0026plusmn;\u0026thinsp;314.46\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e995.75\u0026thinsp;\u0026plusmn;\u0026thinsp;291.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of eggs per female\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102.85\u0026thinsp;\u0026plusmn;\u0026thinsp;25.38\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e138.93\u0026thinsp;\u0026plusmn;\u0026thinsp;13.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e192.00\u0026thinsp;\u0026plusmn;\u0026thinsp;78.61\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e248.93\u0026thinsp;\u0026plusmn;\u0026thinsp;72.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpawning rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.66\u0026thinsp;\u0026plusmn;\u0026thinsp;11.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.58\u0026thinsp;\u0026plusmn;\u0026thinsp;7.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiameter oocyte (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHatching rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.69\u0026thinsp;\u0026plusmn;\u0026thinsp;4.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarvae survival rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86.29\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92.37\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.34\u0026thinsp;\u0026plusmn;\u0026thinsp;6.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e90.63\u0026thinsp;\u0026plusmn;\u0026thinsp;11.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and different superscripts are significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe understanding of carotenoids in fish has expanded in recent times, revealing their diverse biological functions beyond muscle pigmentation. Carotenoids, such as astaxanthin, have been found to play a crucial role in promoting growth (Storebakken and Goswami \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), enhancing broodstock performance (Watanabe and Vassallo-Agius \u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Ahmadi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sawanboonchun et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Palma et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), improving antioxidant status (Wang et al. \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Fiedor and Burda \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Crupi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), boosting immune function (Amar et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Amar et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Chew and Park \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and increasing disease resistance in fish (Amar et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Bhatt and Patel \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Astaxanthin, the primary carotenoid pigment in aquatic animals, imparts vibrant colors to fish meat, crustacean shells, and bird feathers (Miki \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Breithaupt \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It is naturally produced by algae and converted by plankton crustaceans from precursor carotenoids (Stachowiak and Szulc \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Incorporating astaxanthin from natural sources, such as \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e, into commercial fish (red tilapia) feed has shown positive effects on color and growth parameters in some studies (Tuan Harith et al. \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e is a species of microorganism that serves as a natural source of carotenoids, providing antioxidants, nutrients, and color to salmonids like salmon and trout, as well as shrimp (European Food Safety Authority \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Oehlenschl\u0026auml;ger and Ostermeyer \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Maoka et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hayashi et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This aerobic bacterium, characterized by its orange pigmentation and ability to produce astaxanthin, is widely distributed in both marine and freshwater fish (Tsubokura et al. \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Animals generally cannot synthesize carotenoids themselves, relying on their diet to acquire these pigments primarily in their skin and flesh (Galasso et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). While previous studies on tilapia have focused mainly on red tilapia, which were fed carotenoid or astaxanthin-rich diets derived from microalgae and plants, leading to enhanced redness in their skin and improved tissue carotenoid levels (Judan Cruz et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tuan Harith et al. \u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, carotenoids are less prevalent in muscle compared to the integument (Shahidi and Brown \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). To determine which microorganisms produce the carotenoid in Nile tilapia, previous study discovered that the red yeast \u003cem\u003eSporidiobolus pararoseus\u003c/em\u003e can produce carotenoids (Chaiyaso and Manowattana \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and feeding Nile tilapia with this red yeast resulted in increased total carotenoid levels in muscle tissue compared to the control group (Van Doan et al. \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In a recent study, it was observed that Nile tilapia can accumulate carotenoids in their muscle tissue, as evidenced by increased total carotenoid levels when fish were fed with \u003cem\u003eP. carotinifaciens\u003c/em\u003e at a concentration of 20 g/kg, irrespective of gender. This study is the first to report the utilization of \u003cem\u003eP. carotinifaciens\u003c/em\u003e in Nile tilapia, highlighting the beneficial effects of supplementing their diet with astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e, leading to elevated total carotenoid levels in the fish's muscle tissue.\u003c/p\u003e \u003cp\u003eIn terms of promoting growth, our research utilizing carotenoids derived from the bacterium \u003cem\u003eP. carotinifaciens\u003c/em\u003e, which produces astaxanthin, did not indicate any significant effects on the growth performance of male and female Nile tilapia. These outcomes might be attributed to the adult and maturation stage of the tilapia used in the study, as this fish typically observed a growth rate slowdown after reaching a certain size (Bwanika et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Additionally, suboptimal temperature conditions during the research, with an average temperature of 22.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.46\u0026deg;C and a range of 17\u0026deg;C to 26\u0026deg;C, could have contributed to these results. While Nile tilapia are generally tolerant of a wide temperature range (Dan and Little \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), the optimal temperature for growth in most tilapia species is between 27 and 30\u0026deg;C (Nivelle et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thus, we suggest that the utilization of carotenoids from \u003cem\u003eP. carotinifaciens\u003c/em\u003e may not be effective in enhancing the growth of fish when subjected to such environmental stressors. Low-temperature water stresses may affect the physiology and metabolism of fish, altering their receptivity to dietary supplements (Volkoff and R\u0026oslash;nnestad \u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is essential that fish feed meets high nutritional standards in order to provide the essential nutrients for growth and health. In challenging environmental conditions, optimizing the nutritional value of fish feed becomes crucial.\u003c/p\u003e \u003cp\u003eDietary astaxanthin offers more than providing color and increasing carotenoid content in fish muscles (Brambilla et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It exhibits antioxidant properties by neutralizing singlet oxygen, scavenging free radicals, and reducing lipid peroxidation (Naguib \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Hussein et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Liu and Osawa \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Previous research has shown that incorporating astaxanthin-rich food sources in the diet of fish species such as rainbow trout improves fish fillet quality and reduces peroxide levels and transaminase activities in their serum (Nakano et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Nakano et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Rahman et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Astaxanthin has exceptional antioxidant activity and the ability to enhance the protective capacity against oxidative stress (Sztretye et al. \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To assess antioxidant and immune function, various parameters were examined, including catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and lysozyme (LZM). CAT plays a crucial role in maintaining the physiological environment and innate immunity (Elvitigala et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), while SOD helps eliminate excessive reactive oxygen species and maintains the immune system's redox balance (Lin et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). MDA serves as an indicator of oxidative stress (Chen et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and MPO is involved in activating immune cells during inflammatory responses (Noia et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). LZM is an essential component of the immune system (Costa et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These enzymes, LZM and MPO activity, have been widely utilized as biomarkers for assessing innate immunity (Nhu et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bae et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, Nile tilapia fed with astaxanthin-enriched bacterium (\u003cem\u003eP. carotinifaciens\u003c/em\u003e) exhibited enhanced SOD and CAT activity in the liver for both male and female fish. However, no significant difference was observed in MDA levels. Regarding immune function, dietary supplementation of carotenoid-rich bacteria significantly increased MPO activity, indicating immunostimulatory effects. Interestingly, it was not statistically significant in LZM activity, however, the outcome showed a potential enhancing health condition in tilapia. These findings align with previous reports demonstrating the ability of other microorganisms, such as \u003cem\u003eS. pararoseus\u003c/em\u003e, \u003cem\u003eRhodotorula mucilaginosa\u003c/em\u003e and \u003cem\u003eRhodosporidium paludigenum\u003c/em\u003e, to improve antioxidant activity and immune response in fish species. For instance, Nile tilapia fed with \u003cem\u003eS. pararoseus\u003c/em\u003e (Van Doan et al. \u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and golden pompano fed with \u003cem\u003eRhodotorula mucilaginosa\u003c/em\u003e (Yu Wei et al. \u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) showed the increase of SOD activity; also shrimp fed with \u003cem\u003eRhodosporidium paludigenum\u003c/em\u003e exhibited increase of CAT and SOD activities (Yang et al. \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Based on these findings, carotenoids have the potential to enhance antioxidant activity and influence the immune response of tilapia. The mode of action was reviewed by Khalil et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who showed that astaxanthin significantly affects the immune system. Numerous investigations have shown that astaxanthin has strong antioxidant effects in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e conditions. It was shown that astaxanthin significantly lowers the levels of pro-inflammatory cytokines such IL-6, TNF-α, IL-1β, and PGE2 and suppresses the activity of NF-κB, a key regulator of inflammation. Additionally, astaxanthin supports the recovery of Src homology 2 domain-containing protein tyrosine phosphatase 1, a protein that inhibits the signaling of inflammatory cytokines, restoring it to normal levels. As shown by Kishimoto et al. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), astaxanthin is also involved in lowering the release of reactive oxygen species (ROS) and inflammatory cytokines through the MAPK pathway.\u003c/p\u003e \u003cp\u003eHematological parameters and blood chemical profiles are the general reliable indicator to evaluate overall fish health (Kim et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Casanovas et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The hematological parameters and blood chemical profiles of male and female tilapia did not differ significantly across each treatment, according to our findings. However, both male and female tilapia fed astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e showed a significant increase in cholesterol levels compared to the control group. The correlation between plasma carotenoids and serum cholesterol levels is supported by the results of our research. This correlation can be explained by the absorption of lipophilic carotenoids along with dietary lipids and the transport of these substances by lipoproteins (Castellano et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, the NPC1-like transporter 1 (NPC1L1) has been proposed as a possible candidate for carotenoid uptake, as indicated by the studies conducted by Davis and Altmann, (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and Reboul et al. (\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Cholesterol undergoes esterification with free fatty acids upon absorption in the intestines, resulting in the formation of hydrophobic cholesterol esters (CEs). These CEs are then transported via bloodstream lipoproteins, which transport cholesterol to various metabolic and storage sites (Gonen and Miller \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Through receptor-mediated lipoprotein endocytosis, tissues acquire cholesterol from the bloodstream and either use it immediately or re-esterify it for storage (Maxfield and W\u0026uuml;stner \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). While certain tissues, such as the testis, preferentially use \u003cem\u003ede novo\u003c/em\u003e synthesized cholesterol as a substrate for steroid production (Hu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), the majority of steroidogenic tissues, including the adrenal gland and ovary, acquire exogenous cholesterol via this process. In addition, the observed increase in cholesterol levels in the tilapia in our study can be attributed to their maturation, which resulted in elevated levels of sex hormones that are closely associated with cholesterol. Cholesterol is a precursor for a variety of steroid hormones, such as estrogens, androgens, and corticosteroids (Moon et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As in other vertebrates, fish can acquire cholesterol \u003cem\u003evia\u003c/em\u003e dietary consumption, release from intracellular depots, or \u003cem\u003ede novo\u003c/em\u003e synthesis (Sharpe et al. \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Thus, understanding the relationship between carotenoids and cholesterol metabolism in fish is crucial, as cholesterol is a precursor for key steroid hormones involved in numerous physiological processes, including reproduction. The results of this study suggest that carotenoids may have an influent on the cholesterol metabolism of fish, thereby influencing their hormonal equilibrium and overall physiological state.\u003c/p\u003e \u003cp\u003eThe reproductive study was examined by exposing male and female subjects to astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e for a period of two months, followed by observing their mating behavior during the winter season. The water temperature during the study, specifically for a duration of four weeks in December, had an average of 19.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u0026deg;C (ranging from 17.00\u0026ndash;23.00\u0026deg;C). Previous research has indicated that lower temperatures lead to developmental delays, while higher temperatures promote earlier spawning (Pankhurst and Munday \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Temperature plays a crucial role in various reproductive processes such as gametogenesis, development rate, recruitment, and gamete quality, affecting the metabolic pathways within the brain-pituitary-gonadal (BPG) axis (Migaud et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For Nile tilapia, when temperatures are between 25 and 29\u0026deg;C, in this fish these processes are developed at their optimum level (Faruk et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Below this temperature, reproduction comes to an end and feeding is limited below 20\u0026deg;C. Tilapia are easily damaged by temperatures below 10\u0026ndash;12\u0026deg;C (Ernst et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Dan and Little \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Costa-Pierce \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; El-Sayed \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Apart from temperature conditions applied to captive broodstock, the incubation of eggs and larval rearing are known to impact gamete and larval quality (Migaud et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Our study investigated the effects of feeding Nile tilapia with astaxanthin-enriched \u003cem\u003eP. carotinifaciens\u003c/em\u003e on their reproductive parameters. Despite the low water temperature, which was unfavorable for hatchery purposes and optimal egg quality, our results suggest that the supplementation of \u003cem\u003eP. carotinifaciens\u003c/em\u003e, particularly in the T4 group, had a significant improvement in the egg production, even when there was winter stress. For the quantity of spermatozoa, it is worth noting that there was a significant increase in spermatozoa concentration in the male group that received the enriched diet. These findings imply that the nutritional potential provided by \u003cem\u003eP. carotinifaciens\u003c/em\u003e supplementation could positively impact broodstock, affecting the distribution of critical macro and micronutrients inside the eggs. This highlights the importance of effective broodstock feeding for ensuring optimal larval survival and early development, as previously suggested by other studies (Izquierdo et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The consistency of our results with previous research adds further support to the potential benefits of carotenoid supplementation in reproductive processes. Studies on goldfish, Atlantic cod broodstock, ornamented convict cichlid, and discus fish have demonstrated significant improvements in osmolality, spermatocrit value, sperm concentration, egg quality, gonadal maturation, and the upregulation of vitellogenin gene expression upon the addition of carotenoids like astaxanthin and β-carotene to the diets (Sawanboonchun et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Brown et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tizkar et al. \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Haque et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Carotenoids, including both natural and synthetic forms of astaxanthin, are widely used as feed additives in aquatic animals. They have been shown to play a significant role in reproductive processes such as egg production, egg quality, and semen improvement (Tizkar et al. \u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Palma et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Carotenoids, in a role similar to that in mammals, exhibit a steroidogenic function and serve as regulators of folliculogenesis and oogenesis in fish. And the presence of retinol and retinoic acid, which are crucial for the development of mature sperm, further highlights the significance of carotenoids in reproductive functions (Pasquariello et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, astaxanthin serves as an essential source of retinol and 3,4-didehydroretinol, which are precursors of vitamin A and have crucial roles during the development of vertebrate embryos (Moren et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Blomhoff and Blomhoff \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Duester \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kin Ting Kam et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition, retinoic acid, a carotenoid derivatve, plays an essential role in the development of larval structures such as the neural crest (Bohnsack and Kahana \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and the heart (Huang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Brown et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) discovered that fish diets supplemented with carotenoids increased the quantitative of carotenoids deposited in the gonads. Carotenoids substance is accumulated in the ovaries, where the eggs mature. In addition, it can convert into retinoids as a precursor for reproductive system (Levi et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). During embryonic development, retinoids, which belong to the vitamin A family, serve a function in a variety of cellular processes (Johnson and Scadding \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Based on our exploring data, we conclude that the astaxanthin-enriched bacterium (\u003cem\u003eP. carotinifaciens\u003c/em\u003e) potentially improves reproductive capabilities in tilapia, even when they are under overwintering conditions. However, to improve the quality and viability of progeny, more research is necessary to clarify the mechanisms and dynamics of carotenoid metabolism and its role in reproduction.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results demonstrated that the dietary inclusion of the astaxanthin-enriched bacterium \u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e at a dose of 20.0 g/kg can lead to accumulation of carotenoids in muscle tissue, improve antioxidant status, boost immune function, and affect cholesterol metabolism in fish. This, in turn, has the potential to enhance tilapia's reproductive capabilities, particularly sperm quality and egg production, even under overwintering conditions. However, further research is needed to fully understand the potential benefits and limitations of \u003cem\u003eP. carotinifaciens\u003c/em\u003e in aquaculture practices under challenging environmental conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully thank Marine leader or.th for kindly supplying the bacterium (\u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e)\u003cem\u003e\u0026nbsp;\u003c/em\u003eused for this research. We extend special thanks to Ms. Wachirintra Sanseemon and Ms. Piyapohn Kanhaseeya for their kind assistance during the preparation of this research.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNantaporn Sutthi was involved in conceptualization, methodology, formal analysis, investigation, resources, data curation, writing-original draft preparation, visualization, writing - review \u0026amp; editing, project administration, and funding acquisition. Paiboon Panase was involved in methodology, writing - review \u0026amp; editing, and funding acquisition. Thitiwut Vongkampang was involved in methodology, writing-original draft preparation, writing - review \u0026amp; editing. Eakapol Wangkahart was involved in methodology, formal analysis, writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research project was financially supported by Mahasarakham University and this research work was partially supported by Unit of Excellence Physiology and Sustainable Production of Terrestrial and Aquatic Animals (FF66-UoE014), University of Phayao.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthic Animal\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInstitutional Animal Care and Use Committee, Mahasarakham University (IACUC-MSU), Thailand (IACUC-MSU-13/2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbd El-Hack ME, El-Saadony MT, Nader MM, Salem HM, El-Tahan AM, Soliman SM, Khafaga AF (2022). 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Path. 27\u003cstrong\u003e:\u003c/strong\u003e 741-45. https://doi.org/10.1007/s00580-018-2660-2.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Astaxanthin-enriched diet, Paracoccus carotinifaciens, Winter season, Reproductive performance, Heath status","lastPublishedDoi":"10.21203/rs.3.rs-3218062/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3218062/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe growth, immune response, and reproductive performance of broodstock of Nile tilapia (\u003cem\u003eOreochromis niloticus\u003c/em\u003e) under winter stress conditions were investigated the effects of supplementary diets with astaxanthin-enriched bacterium, \u003cem\u003eParacoccus carotinifaciens\u003c/em\u003e. Throughout an eight-week period in the winter season, male and female tilapia were fed with diets containing different levels of \u003cem\u003eP. carotinifaciens\u003c/em\u003e dietary supplementation: 0 g/kg (T1; control), 5 g/kg (T2), 10 g/kg (T3), and 20 g/kg (T4). Subsequently, a four-week mating system was implemented during the winter stress period. The results revealed that there were no significant differences observed in growth, hematological indices, and blood chemical profiles among all treatment groups for both male and female tilapia. However, a significant increase in cholesterol content was noted in both male and female tilapia fed with the T4 diet (p\u0026lt;0.05). The total carotenoid content in the muscle was evaluated, and significantly higher values were found in both male and female tilapia that fed T4 supplementation (p\u0026lt;0.05). Moreover, immunological parameters such as myeloperoxidase and antioxidant parameters in the liver including superoxide dismutase activity and catalase enzyme activity showed significant increases in tilapia fed with the T4 diet. The impact of \u003cem\u003eP. carotinifaciens\u003c/em\u003esupplementation on broodstock tilapia indicated a significant increase in spermatozoa concentration in males and increased egg production in females after consumption of the T4 diet (p\u0026lt;0.05). Thus, this study highlighted that the presence of astaxanthin-enriched bacterium \u003cem\u003eP. carotinifaciens\u003c/em\u003ein the diet of broodstock Nile tilapia can lead to the accumulation of carotenoids in their muscle tissue, improvement in antioxidant status, enhancement of immune function, and potential enhancement of reproductive capabilities, even under overwintering conditions.\u003c/p\u003e","manuscriptTitle":"Impact of astaxanthin-enriched bacterium (Paracoccus carotinifaciens) on growth, immune response, and reproduction performance of broodstock Nile tilapia during winter season","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-07 13:59:15","doi":"10.21203/rs.3.rs-3218062/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-15T12:35:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-09-24T13:29:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6dca31fd-6de3-44f6-8aec-a5e4110f2128","date":"2023-09-23T18:52:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-09-23T18:26:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-23T18:25:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-08-02T00:24:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fish Physiology and Biochemistry","date":"2023-07-30T14:43:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fish-physiology-and-biochemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fish","sideBox":"Learn more about [Fish Physiology and Biochemistry](https://www.springer.com/journal/10695)","snPcode":"10695","submissionUrl":"https://submission.nature.com/new-submission/10695/3","title":"Fish Physiology and Biochemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a263680b-f316-4389-b7a9-9527f02be76d","owner":[],"postedDate":"August 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-25T15:03:42+00:00","versionOfRecord":{"articleIdentity":"rs-3218062","link":"https://doi.org/10.1007/s10695-024-01331-8","journal":{"identity":"fish-physiology-and-biochemistry","isVorOnly":false,"title":"Fish Physiology and Biochemistry"},"publishedOn":"2024-03-21 15:00:46","publishedOnDateReadable":"March 21st, 2024"},"versionCreatedAt":"2023-08-07 13:59:15","video":"","vorDoi":"10.1007/s10695-024-01331-8","vorDoiUrl":"https://doi.org/10.1007/s10695-024-01331-8","workflowStages":[]},"version":"v1","identity":"rs-3218062","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3218062","identity":"rs-3218062","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}