Introduction
In recent years, several studies have been developed regarding the use of immunostimulants, probiotics and prebiotics to enhance the fish immune system response towards different pathogens (Cruz et al., 2012; Carbone & Faggio, 2016). There are several sources of immunostimulant chemical compounds that have been proved to be effective in fish disease prevention, namely those that are extracted from marine algae (Barman et al., 2013). Some extracts from marine algae can possess antiviral and antibacterial activity showing encouraging results as fish feed supplementation, specially the red algae (Sahu et al., 2008; Rizzo et al., 2017; Hoseinifar et al., 2018). Gracilaria gracilis, is a multi-product source, having several interesting components such as lipids, proteins, carbohydrates, phycobiliproteins, phenols and biologically active phytochemicals (Francavilla et al., 2013). The present study intends to analyse G. gracilis potential as fish feed supplementation for growth as well as its protective effect against a Photobacterium damselae subsp. piscicida (Phdp) infection on seabass.
Materials and methods
The study was performed at CETEMARES (Politécnico de Leiria) facilities, with seabass (mean wet weight 17.5 ± 6.1 g) juveniles obtained from the Aquaculture Research Station-EPPO (IPMA, Olhão, Portugal). Fish were randomly distributed as a completely randomized design into 12 tanks of 60 l closed recirculation systems for the feeding trial (i.e. triplicates per experimental conditions). Using standard seabass feed, SPAROS Lda (Olhão, Portugal) manufactured five different diets: a control diet, G. gracilis extract (0.35%) supplemented diet, and two G. gracilis powder (2.5% and 5%) supplemented diets. Fish were weighed before and after the feeding trial (51 days), and then subjected to Phdp challenge, the same amount of fish were sham injected with phosphate saline buffer. At each sampling point, fish were anaesthetized with 2-phenoxyethanol and blood samples were collected for haematological procedures such as total and differential counting of peripheral leukocytes and total circulating erythrocytes. The remaining blood was centrifuged and plasma was collected for the assessment of innate humoral immune parameters. Liver was dissected out from the fish to evaluate some parameters of oxidative stress.
Results
No differences were registered about the effect of the different diets on the growth performance. White blood cell (WBC) counts were consistently reduced in infected fish 24h post-infection (p<0.05). However, fish supplemented with algae powder recovered faster, as can be seen by the differences noted between the WBC counts at 24h and 48h post-infection in the 2.5% supplemented diet (p<0.05). Almost all red blood cell (RBC) counts decreased when compared with the 0h counts, even the not infected fish, as well as the haematocrit (p<0.05). However, the fish fed with no supplementation had a significant decrease in both parameters when infected with the bacteria from 24h to 48h post-infection (p<0.05). Lysozyme also showed differences, as seen by the lower concentration seen in both injected groups when the fish were fed with the base feed (p<0.05). Catalase activity, only measured before the infection, was higher on fish fed with the 5% algae powder supplementation, compared to the 0.35% algal extract supplementation (p<0.05).