Aquaculture of the common octopus, Octopus vulgaris, has attracted considerable interest in recent years due to the increasing demand for this species in global seafood markets, as well as its rapid growth and high nutritional quality. Despite substantial progress, there are still numerous challenges that need to be addressed for the successful farming of this species. Rearing paralarvae to juvenile stages is one of the most challenging aspects due to the massive mortality during the first life stage, which is the major bottleneck for commercial production, among other reasons, due to their specific nutritional requirements. Several research institutions have worked to improve the breeding, rearing, and feeding techniques of O. vulgaris in captivity, which has allowed significant advances in the field of nutrition and culture technology, resulting in a culture protocol that allows for improved growth and survival of the paralarvae (Tur et al., 2020; Varó et al., 2022). However, there are still numerous aspects that need to be improved, the main one being the reliance on live preys to feed the paralarvae. The impossibility of obtaining these unsustainable preys on a commercial scale, together with the difficulty of rearing them on an experimental scale, has led to the search for other alternatives for feeding paralarvae. The use of Artemia is considered the only viable alternative due to its ease of production and availability. However, its use has often resulted in poor performance, inconsistent growth, and a lack of settlement. Several studies have shown that the Artemia enrichment with long-chain polyunsaturated fatty acids (LC-PUFA) and marine phospholipids can improve the growth, survival and overall wellbeing of paralarvae (Garrido et al., 2018). This enhancement is likely attributable to the increased levels of docosahexaenoic acid (DHA) and polar lipids, both of which play essential roles in the paralarval physiology (Garrido et al., 2018). Despite these improvements, the effectiveness of Artemia as the exclusive diet for paralarvae remains limited, highlighting the need to explore additional enrichment strategies. In this regard, the integration of health-enhancing additives offers a promising opportunity to enhance the nutritional profile of paralarval diets. Consequently, we are considering the enrichment of Artemia with natural bioactive compounds as a promising approach to improve the survival and welfare of paralarvae.

Numerous studies have reported that plant extracts promote diverse biological effects in aquaculture species, including anti-stress activity, growth promotion, appetite stimulation, enhancement of tonicity, immunostimulation, maturation, and anti-pathogen properties in marine organisms, such as fish, shrimp and mollusc (Tadese et al., 2021). The plant extracts that have been tested on finfish and shrimp are numerous. Among them, black cumin seed oil, derived from Nigella sativa, is one of the promising plant-based compounds that has garnered attention in aquaculture in recent years. This oil is recognized for its potential health benefits for cultured organisms, as it may promote the health and growth of aquatic species (Abd El-Hack et al., 2021). It is rich in thymoquinone, thymol, and carvacrol, which are substantial pharmacologically active compounds (Al-Saleh et al., 2006). Thymoquinone accounts for 18.4–24% of the essential oil and is responsible for its antioxidant, anti-inflammatory and antibacterial activities. The strong antioxidant properties of thymoquinone have been shown to reduce oxidative stress and enhance the immunity in fish and shellfish (Latif et al., 2020, 2021; Nur et al., 2020; Mahboub et al., 2022; Niroomand et al., 2024). Some studies also suggest that the inclusion of black cumin oil into the diet may improve feed conversion efficiency, leading to better growth performance (Öz et al., 2018). Overall, research suggests that black cumin oil can be an effective natural supplement in aquaculture to reduce oxidative stress, improve welfare, and enhance growth performance.

On the other hand, olive fruit extract, which contains a high concentration of polyphenols such as hydroxytyrosol and tyrosol, is known for its strong antioxidant properties in fish and crustaceans, helping to neutralize free radicals and reduce oxidative stress and enhance the immune system (Dong et al., 2022a; Hazreen-Nita et al., 2022). Several studies have shown that the inclusion of hydroxytyrosol-rich olive extract can lead to improved growth capacity of the musculoskeletal system and feed utilization (Balbuena-Pecino et al., 2022, 2024; Xia et al., 2024), reduced fat mass by decreasing adipocyte size (Lutfi et al., 2017), reduced hepatic fat accumulation (Dong et al., 2020), attenuated oxidative stress, apoptosis, inflammation and mitochondrial dysfunction (Dong et al., 2022a, b). In addition, olive extract has been shown to be an effective antioxidant during Artemia enrichment, particularly due to its high hydroxytyrosol content (Viciano et al., 2017). Such an antioxidant protective role is reflected in the lipid profile of Artemia, as the addition of hydroxytyrosol preserved the DHA content of enriched metanauplii, potentially improving the nutritional profile, and therefore the health and growth of aquatic species that rely on Artemia as a food source (Viciano et al., 2017).

To the best of our knowledge, these bioactive compounds have not previously been utilized in cephalopod culture. Nevertheless, existing literature suggests that their incorporation into Artemia enrichment protocols may contribute to maintaining the nutritional quality of this live prey and reduce oxidative stress. This, in turn, could potentially improve growth, enhance antioxidant defense mechanisms, and support overall health and survival of octopus paralarvae. Therefore, the aim of this study was to evaluate the effect of black cumin seed oil and Hytolive^® syrup (the natural extract obtained from the olive), containing natural bioactive compounds, using Artemia as a vector, on growth performance, fatty acid profiles, antioxidant activities and lipid peroxidation in O. vulgaris paralarvae.

Despite the extensive research efforts in common octopus aquaculture, the culture of the first developmental stages (paralarva) has remained a significant challenge for commercial production. Feeding and nutrition are key factors for understanding its needs under culture conditions. It has been demonstrated that the administration of nutritional supplements, particularly natural bioactive compounds, is an unequivocally effective strategy for enhancing larval survival in aquaculture (Reverter et al., 2014). Medicinal plants have potential as growth promoters, immunomodulators and antioxidants in aquaculture. The beneficial properties of hydroxytyrosol-rich extracts or black seed oil have been extensively studied in mammals and have recently been investigated in fish.

It is well established that hydroxytyrosol and tyrosol do not accumulate in the organism but are rapidly excreted (López de las Hazas et al., 2016). However, the detection of hydroxytyrosol and tyrosol in Artemia supplemented with these compounds suggests that they exhibit some degree of stability during their passage through Artemia’s metanauplii digestive tract. Our results indicate that, after 4 hours of enrichment with LC60 and Hytolive, these compounds are not fully metabolized or excreted. This is the first experimental demonstration that Artemia can serve as a vehicle to deliver these bioactive compounds to paralarvae.

In paralarvae fed Artemia supplemented with HYT, it was found that hydroxytyrosol and tyrosol were fully metabolized. Nevertheless, secondary metabolites such as homovanillyl alcohol, caffeic acid (both cis- and trans-), p-coumaric acid (both cis- and trans-) and cinnamic acid were detected. It is important to note that these metabolites are not exclusively derived from the metabolism of hydroxytyrosol and tyrosol, as they could also originate from the metabolism of polyphenols present in the microalga I. galbana, which was used for Artemia growth. This observation is supported by the detection of the same metabolites in the control group of paralarvae (CON). However, HYT supplementation appears to enhance polyphenol availability in the paralarvae, which likely contributes to the observed beneficial effects against oxidation (lower levels of lipid peroxidation) in the HYT group.

In contrast, Artemia supplemented with black cumin oil (BCO) did not retain detectable levels of thymoquinone when analysed by GC-MS, suggesting complete metabolism of the compound within Artemia. Nonetheless, potential thymoquinone-derived metabolites, including 3-pentanone, nonane (3,7-dimethyl), heptane (3,4,5-trimethyl), and 1-dodecanol (2-octyl), were identified. These findings suggest that Artemia can transfer thymoquinone metabolites to paralarvae, which may explain the observed antioxidant effects in the BCO group.

Previous studies have shown that incorporating bioactive olive extracts into the diet can enhance growth in some crustaceans and fish species, such as sea bream (Sparus aurata) (Gisbert et al., 2017), Persian sturgeon (Acipenser persicus) (Hosseinnia et al., 2021), Blunt snout bream (Megalobrama amblycephala) (Dong et al., 2022a) and crayfish (Astacus and Astacus leptodactylus) (Parrillo et al., 2017; Sateriale et al., 2023). However, no previous studies have specifically evaluated the effects of olive extracts on cephalopods, including O. vulgaris. The findings from the current study indicate no significant improvement in growth performance in paralarvae of O. vulgaris supplemented with hydroxytyrosol (HYT) after 30 days of dietary inclusion. No effects on specific growth rate (SGR) or survival were observed. Although it is not possible the comparison with other studies on cephalopods, similar findings have been reported in other fish studies. For instance, rainbow trout (Oncorhynchus mykiss) fed diets with varying levels of olive leaf extract for 60 days showed no significant differences in growth performance values, such as weight gain, SGR, or feed utilization (Baba et al., 2018). Likewise, juvenile sea bream supplemented with a hydroxytyrosol-rich extract showed no changes in body weight or other biometric parameters after 4 or 8 weeks of treatment (Balbuena-Pecino et al., 2022).

Regarding paralarvae supplemented with black cumin oil (BCO), no significant differences in SGR and survival rates were also observed compared to the control group. Particularly, the dry weight (DW) of the BCO group was lower. Similar to HYT, no studies have previously investigated the impact of BCO on growth and survival in O. vulgaris paralarvae or other cephalopods. Using other crustacean and fish studies as a reference, some researchers have shown improved growth rates in BCO-supplemented species, including white leg shrimp (Litopenaeus vannamei) (Asadi et al., 2018), common carp (Cyprinus carpio) (Al-Dubakel et al., 2012), rainbow trout (O. mykiss) (Öz et al., 2018; Bektas et al., 2019), and Asian sea bass (Lates calcarifer) (Abdelwahab and El-Bahr, 2012). However, in other species such as tilapia (Oreochromis niloticus), BCO supplementation had no significant effects on growth or feed intake (Ahmed et al., 2018). In all these studies, supplementation levels varied widely, from 0.1% to 30%.

Considering all the data, the growth performance results suggest that the response to bioactive compounds, in terms of growth and survival, is strongly influenced by the species studied and the concentrations of the bioactive extracts used. The paralarvae of O. vulgaris are notable by their unique physiological traits, which are characterized by a high metabolic rate and specialized lipid metabolism, both of which are essential for supporting their rapid growth during early life stages. These paralarvae exhibit a highly aerobic metabolism, that requires substantial energy for processes such as neurosensory development, active pelagic locomotion, and tissue biosynthesis (Morales et al., 2017). Furthermore, the digestive system of paralarvae at this stage exhibits functional limitations, including limited and specific enzymatic activity with a preference for animal proteins and marine phospholipids, and a low capacity to hydrolyse and absorb complex plant lipids. Since these bioactive products are herbal plants, higher inclusion levels of herbal extracts have been reported to negatively impact the growth of other species due to elevated concentrations of anti-nutritional factors (Hazreen-Nita et al., 2022). Therefore, determining the optimum inclusion level of these bioactive compounds in feed formulation is crucially important. In the present study, the tested concentrations of bioactive extracts had no significant effects on SGR or survival of paralarvae. This could indicate that while growth was not enhanced, the supplementation levels used were not harmful, particularly in terms of anti-nutritional factors.

The fatty acid composition of paralarvae from different treatments showed that, although variable, there was a difference in the profiles of the experimental groups by age, with the 10-dph paralarvae having more saturated fatty acid profiles distinct from those of 20 and 30-dph. The paralarvae supplemented with HYT and BCO showed patterns more linked to polyunsaturated fatty acids as compared to the CON group, which can be associated with a protective effect of both dietary treatments against lipid oxidation (Barata et al., 2005).

In order to gain insight into the potential mechanisms through which diverse natural antioxidants influence the antioxidant status of O. vulgaris paralarvae, this study examined a range of biomarkers of oxidative stress. Lipid peroxidation is a well-established biomarker for assessing oxidative stress-induced damage in animal tissues. Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defenses (Benedetti et al., 2022). When ROS production exceeds an organism’s ability to scavenge them, oxidative damage to macromolecules such as lipids, proteins, and DNA occurs (Valgimigli, 2023). Membrane lipids are particularly susceptible to ROS attack, resulting in increased lipid peroxidation products. In fact, lipid peroxidation has been extensively employed as a biomarker of oxidative stress (Lushchak, 2011). In our study, dietary supplementation with the bioactive compounds HYT and BCO significantly reduced the level of lipid peroxidation in O. vulgaris paralarvae at 30dph, despite an overall increase in lipid peroxidation observed across all groups during development. This reduction suggests an improvement in antioxidant status in paralarvae supplemented with both bioactive compounds. These results are consistent with previous research on the effects of these bioactives in fish (Latif et al., 2020; Dong et al., 2022a). The bioactive compound thymoquinone present in BCO has been shown to inhibit iron-dependent lipid oxidation via the Fenton reaction in a concentration-dependent manner (Nagi and Mansour, 2000). Similarly, hydroxytyrosol and tyrosol from HYT Hydroxytyrosol and tyrosol from Hytolive^® have been described as being able to reduce MDA levels in aquatic organisms (Viciano et al., 2017; Dong et al., 2022a, b).

On the other hand, enzymatic and non-enzymatic antioxidant defenses play a key role in preventing oxidative stress by counteracting the effects of reactive oxygen species (ROS). Aquatic organisms use antioxidant enzymes such as SOD and CAT, along with detoxification enzymes like GST, to mitigate the harmful impact of ROS and prevent oxidative damage (Mates, 2000; Martínez-Álvarez et al., 2005). These antioxidants are configured to maintain low levels of ROS within cells, making them essential components of an organism’s defense response (Mates, 2000).

The response to the inclusion of bioactive compounds may vary between individuals and species, depending on factors such as genetics, previous nutritional status, and environmental conditions. In other studies, carried out with different marine vertebrate and invertebrate species, it has been observed that diets enriched with vegetable oils or natural extracts can induce significant changes in the activity of detoxifying enzymes (Xavier et al., 2021).

The results of the present study did not reveal changes in CAT or SOD levels in paralarvae supplemented with HYT or BCO. These enzymes maintained steady activity levels, possibly reflecting transient rather than sustained responses to supplementation (Barata et al., 2005).

However, GST activities were significantly reduced in BCO-supplemented PL after 20 and 30 days of culture compared to the control group. GST is a crucial enzyme involved in the biotransformation process of metabolites. It catalyzes the conjugation of breakdown products of lipid peroxides to glutathione (GSH), facilitating their excretion and decreasing their toxicity (Hellou et al., 2012). Previous studies have shown that GST activity increases in response to external stressors, particularly those that generate ROS (Barata et al., 2005). This increase in activity suggests a detoxification process where GST conjugates with its natural substrates to eliminate toxic metabolites (Townsend and Tew, 2003). Specifically, GST activity is closely linked to the response to oxidative stress in cephalopods, where increased activity has been observed under stress conditions (Semedo et al., 2012). Bioactive compounds in BCO can help restore redox balance by neutralizing free radicals, which in turn preserves antioxidant enzymes like GST (Masella et al., 2004). This mechanism may explain the significant reduction in GST levels in paralarvae supplemented with BCO compared to the control, as well as the maintenance of GST levels throughout development, in contrast to the other two groups. GSTs are upregulated in response to oxidative stress to detoxify byproducts such as lipid peroxides and 4-hydroxynonenal. When oxidative stress is low, the demand for GST-mediated detoxification decreases, resulting in lower GST activity (Awasthi et al., 2017; Sharma et al., 2004). The reduced GST activity in BCO supplemented PL may therefore be attributed to directly scavenging of reactive oxygen species (ROS), which in turn lowers oxidative stress and reduces the demand for GST activity.

To the best of our knowledge, this study is the first to demonstrate that the bioactive compounds hydroxytyrosol (HYT) and black cumin oil (BCO) used as supplements in Artemia are transferred to paralarvae, either directly as a parent compound (in the case of HYT) or as metabolites (in the case of BCO). Notably, paralarvae fed these bioactives exhibited an improved profile of long-chain polyunsaturated fatty acids (PUFA), potentially linked to protective effects against oxidative stress.

Furthermore, paralarvae supplemented with HYT and BCO showed lower lipid peroxidation levels (TBARS) compared to the control group after 30 days of supplementation. Additionally, reduced GST activity was observed in the BCO-supplemented group after 20 days, suggesting a potential modulation of oxidative stress responses.

It is reasonable to assume that bioactives may offer protective benefits during the early stages of development. This fact is of high relevance for future studies on the diet of O. vulgaris due to the high economic interest in this species and the recent advances in its culture. Future studies should focus on establishing the optimal dose of the selected bioactive compounds, testing higher concentrations without compromising the viability of the live prey. Furthermore, the combination of both bioactives should be investigated to decipher if any synergistic effects could boost the immune system of O. vulgaris. Finally, extending the evaluation to subsequent developmental stages would facilitate a more comprehensive understanding of their long-term impact.