Effect of supplemental phospholipase A₂ antibody on the growth performance and selected immune criteria of rainbow trout (Oncorhynchus mykiss)

Rainbow trout, Oncorhynchus mykiss (Walbaum), is a major aquaculture species in Europe and North America and knowledge on its nutritional requirements and feeding practices is extensive (Hardy 2002). Altered gut inflammatory processes can be caused by ingestion of specific feedstuffs, toxins and microorganisms carried with the feed exposing the organism to allergens and pathogens that can cause disease or depressed growth (Heikkinen, Vielma, Kemiläinen, Tiirola, Eskelinen, Kiuru, Navia-Paldanius & von Wright 2006; Knudsen, Jutfelt, Sundh, Sundell, Koppe & Frøkiær 2008). The gastro-intestinal tract acts as a physical and chemical barrier in combination with an effective mucosal immune system. When the immune system is activated, an array of specialized cells, proteins and signalling molecules are produced and mobilized to repel any threat, real or perceived. However, such a constant vigilance status has a metabolic cost (Colditz 2002), reallocating nutrients and energy from other processes such as somatic growth. Phospholipase A₂ (PLA₂) constitutes a large family of lipolytic enzymes that catalyze hydrolysis of the fatty acid ester at the sn-2 position of phospholipids, leading to the production of lyso-phospholipids and free fatty acids. When the latter is arachidonic acid, it originates potent pro-inflammatory mediators, prostaglandins, thromboxanes, leukotrienes and lipoxins, collectively known as eicosanoids (Kudo & Murakami 2002). Inhibiting PLA₂ could hinder inflammatory response (Meyer, Rastogi, Beckett & McHowat 2005; Yedgar, Cohen & Shoseyov 2006), and thus potentially alter energy allocation in farmed animals. Dietary supplementation with an anti-phospholipase A₂ antibody (aPLA₂) was found to significantly enhance growth performance of rainbow trout (Barry & Yang 2008). On the other hand, aPLA₂ supplementation had no effect on growth of trout fed a soybean meal-rich diet (Sealey, Barrows, Smith & Hardy 2010). The objective of the present work was to assess the effect of supplemental dietary levels of aPLA₂ on the overall growth performance of rainbow trout fed plant protein based diets and its consequences on selected non-specific immune response criteria.

Twelve groups of 80 rainbow trout (mean initial body weight: 25.3 ± 0.9 g) were stocked in 250 L fibreglass tanks and fed the various experimental diets over 42 days. A commercial feed for rainbow trout (Aquagold Trout Aquasoja, Sorgal S.A, Ovar, Portugal; 45.7% crude protein, 29.8% crude fat, 9.9% ash, 2.5% crude fibre, 1.5% phosphorus) containing low fishmeal level (15% fishmeal LT70 according to manufacturer disclosure; marine derived-proteins represent about 24% of dietary crude protein) was finely ground. This control mash was then supplemented with the commercial aPLA₂ product (Big Fish, supplied by Aova Technologies, Madison, WI, USA) at two doses (0.6% and 0.9%) These aPLA₂ doses were already used with success for other farm species by the Aova Company. Control mash and those supplemented with aPLA₂ were dry-pelleted without steam using a laboratory pellet press (CPM, C-300 model, San Francisco, CA, USA) with a 3 mm die. The proximate composition of the various experimental diets was similar, with 45.7% crude protein, 29.9% fat and 25.9 kJ g⁻¹ gross energy. Fish were fed to apparent satiety, by hand, three times a day and feed intake was recorded on a weekly basis. Each diet was tested in quadruplicate tanks, supplied with flow-through aerated ground water (temperature: 15 ± 1°C; oxygen maintained above 70% saturation) and subjected to natural photoperiod. Fish from the initial stock and from each tank at the end of the trial were sampled for analysis of whole-body composition. Proximate composition analysis of the diets and whole fish was made by the following procedures: dry matter (105°C for 24 h; ID 934.01), ash by combustion (550°C for 12 h; ID 942.05), crude protein (Kjeldahl method, Königswinter, Germany N × 6.25; ID 984.13) after acid digestion, lipid content by petroleum ether extraction (at Soxhlet 40–60°C; ID 920.39) and gross energy in an adiabatic bomb calorimeter (Werke C2000 IKA, Staufen, Germany). All analyses were run in duplicates and performed following AOAC (2006) procedures.

During the experiment, no mortality was observed in any of the groups. At the end of the 42 days of experimental feeding, the overall growth performance can be considered as good (SGR values ranging from 2.43% to 2.65% day⁻¹), and if converted to the thermal unit growth coefficient, within the normal range for rainbow trout of this size (Dumas, France & Bureau 2007). In the best performing treatment, fish had a 3-fold increase of their initial body weight. Given the relatively high and constant feed intake values (3.9–4.0% IBW day⁻¹) found for all treatments, it seems clear that within the tested doses, the aPLA₂ supplemental product had no detrimental effect on feed palatability. Growth performance was significantly affected by dietary treatments (Table 1). In comparison to trout fed the CTRL diet, those fed with the aPLA₂ supplemented diets showed a significant increase on FBW and SGR (P < 0.011 and P < 0.002, respectively). Additionally, trout fed the highest aPLA₂ 0.9% diet showed a significantly higher (P < 0.002) SGR than fish fed the aPLA₂ 0.6% diet. When expressed as a percentage relative to CTRL, it was found that after 42 days of experimental feeding, the FBW was increased by 5.9% and 6.7% for treatments supplemented with aPLA₂ at 0.6% and 0.9%, respectively. Feed conversion ratio (FCR) ranged from 0.84 to 0.94 and was also significantly affected by the various dietary treatments. In comparison to fish fed the CTRL diet, those fed the aPLA₂ 0.9% diet showed a significant reduction (P < 0.034) of FCR. The protein efficiency ratio (PER) was significantly higher (P < 0.031) in fish fed the aPLA₂ 0.9% diet than in those fed the CTRL diet. Within the dietary treatments supplemented with the aPLA₂ product (diets aPLA₂ 0.6% and aPLA₂ 0.9%), FCR and PER values were similar.

Recently, a 0.3% aPLA₂ supplementation in a plant-protein rich diet failed to show any beneficial effects on trout growth performance (Sealey et al. 2010). Similarly to our findings, Barry and Yang (2008) have reported that dietary aPLA₂ supplementation, at 0.15% and 0.3% doses in a commercial ‘Silver Cup steelhead’ diet, significantly increased rainbow trout weight gain and FCR. In the current study, no differences were found between dietary treatments in terms of whole-body composition of fish (Table 1). Protein and fat retention were not significantly affected by the various dietary treatments and values were within the range previously reported for the species (Valente, Bandarra, Figueiredo-Silva, Rema, Vaz-Pires, Martins, Prates & Nunes 2007). Thiobarbituric acid reactive substances (TBARS) levels, determined according to Richards and Hultin (2002), ranged between 0.12 and 0.77 μg MDA·mg protein⁻¹ in dorsal muscle homogenates (Table 1). TBARS levels measured in trout fed the aPLA₂ 0.6% diet were significantly higher (P < 0.003) than those found in fish fed the CTRL diet. Given the lack of an aPLA₂ dose response effect it is difficult to establish an eventual relation between the inhibition of the PLA₂ pathway and lipid oxidation. However, PLA₂ inhibitors have shown to reduce TBARS levels in mammalian systems (Al-Mehdi, Dodia, Jain & Fisher 1993).

In a complementary assessment the non-specific immunological response was studied; two groups of 25 trout (average body weight: 174.6 ± 26.4 g) were fed either the CTRL diet or an aPLA₂ mega-dose diet (3%) over 5 weeks, in rearing conditions identical to those previously described for the growth trial. At the end of the experimental feeding period, fish fed the aPLA₂ mega-dose were injected peritoneally with 100 μL of casein (12%), while trout fed the CTRL diet were injected with 4 mL of phosphate buffer saline. Eight fish from each group were sampled (blood and peritoneal exudate) at injection time (T0), day 2 (T2) and 7 days (T7) after injection. Methodologies used to process, identify and count intraperitoneal cells are described by Afonso, Silva, Lousada, Ellis and Silva (1998). Irrespective of the sampling time, the dietary aPLA₂ mega dose (3%) had no significant effect (P > 0.05) on the various inflammatory cell numbers in the peritoneal exudate. Similarly, aPLA₂ supplement did not affect significantly (P > 0.05) serum lysozyme or complement (alternative pathway), which are the criteria commonly chosen to evaluate the immune fish response in fish (data not shown), assayed by methodologies described by Ellis (1990) and Oriol Saunyer and Tort (1995), respectively. Despite preliminary, our data suggests the lack of an immune modulatory effect of a dietary aPLA₂ supplementation. In a previous study, Sealey et al. (2010) reported a downregulation of heat-shock proteins mRNA expression in trout fed aPLA₂-supplemented diet, but only at high soybean meal inclusion levels. According to research by Aova Company, the anti-PLA₂, when added to feed, works directly on the microbial (mPLA₂) and mucosal cellular (cPLA₂) PLA₂. In this way the anti-inflammatory cascade is not activated. Overall our results suggest that a short-term dietary aPLA₂ supplementation at 0.6% improves rainbow trout growth performance, without detrimental effects on selected innate immune criteria such as lysozyme or alternative complement system. A long term assessment of an aPLA₂ dietary supplementation strategy, with and without stressful events, and comprising a more robust set of immune status indicators is required to fully validate such results.