## 4 Discussion  
The use of macroalgae, namely the red seaweed _G. gracilis_, in animal feed and food has been widely sought after. In nutritional terms, these algae can be used, for example, in protein replacement or for nutritional enrichment [44; 45; 46; 47; 48; 49; 50; 51]. Additionally, the bioactive properties of this species allow us to foresee a series of benefits associated with its use in animal feed products with clear benefits for animal health and welfare [52; 53; 54; 55]. The sustainability associated with the collection of these algae in their natural environment or their production in aquaculture systems can play an important role in their biotechnological use. There are already studies that indicate both the optimal conditions for the cultivation of this species and the conditions that allow for the maximization of the production of some nutritional or bioactive compounds [56; 57; 58; 59; 30]. Regarding the inclusion of bioactive components, the presence of antioxidant and/or antimicrobial compounds in animal feed can lead to additional levels of protection against cellular oxidation mechanisms and pathogenic diseases, contributing to the improvement of the animal health and well-being, especially in fish from aquaculture [60; 61] that are exposed to several stress factors. These bioactive compounds can positively impact a  
Figure 6: Growth inhibition (%) of fish pathogenic bacteria in the presence of ethanol extracts of _G. gracilis_ in liquid media after 18 h of growth. The extracts were obtained at room temperature (RT, bars in shades of blue), 40 \({}^{\circ}\)C (40, bars in shades of green), or 70 \({}^{\circ}\)C (70, bars in shades of yellow/red) for 30 min, and chloramphenicol (CHL, black bars) was used as a positive control. Results are expressed as average values \(\pm\) SD (\(n=4\)). Figure 7: Growth inhibition (%) of fish pathogenic bacteria in the presence of aqueous extract of _G. gracilis_ in liquid media after 18 h of growth. The extracts were obtained at room temperature (RT, bars in shades of blue), 40 \({}^{\circ}\)C (40, bars in shades of green), or 70 \({}^{\circ}\)C (70, bars in shades of yellow/red) for 30 min, and chloramphenicol (CHL, black bars) was used as a positive control. Results are expressed as average values \(\pm\) SD (\(n=4\)). farm's profitability and the welfare of animals in captivity that would translate into a better-quality product. The use of macroalgae as an additive in feed can be achieved in the form of biomass or an extract, the latter being obtained with non-toxic and appropriate grade solvents with subsequent incorporation during the feed manufacturing process. Several studies demonstrate that the activities exhibited by crude extracts are different depending on the temperature, extraction time, and solvents used [26, 62, 63, 64], the most common including water, ethanol, hexane, methanol, and mixtures of different solvents. The different extraction solvents according to their polarity allow the fractionation and separation of different compounds such as pigments (chlorophyll a and b and carotenoids), alkaloids, and different classes of phenolic compounds, among others. The use of water, a highly polar solvent (polarity index of 10.2), allows for the obtaining of extracts not only enriched in soluble proteins and sugars but also pigments, starch, and minerals, in addition to the use of other water-soluble compounds [65]. Ethanol allows for the extraction of slightly polar compounds (polarity index of 5.2) such as polar lipids and fatty acids, some phenolic compounds, and carotenoids, among others [66, 67, 68, 62]. Generally, the phenolic compounds are better extracted with increased solvent polarity [64] and thus it is expected to find them in higher concentrations in highly polar solvents. In this study the use of conventional solid-liquid extraction processes with ethanol or water as solvents sought to obtain extracts with different compositions and bioactivities.