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.