Dietary astaxanthin levels affect growth, carotenoid digestibility and the deposition of specific carotenoid esters in the Giant Tiger Prawn, Penaeus monodon

The predominant carotenoid in prawn tissues is astaxanthin (Axn) and its role in pigmentation has been well studied. However, the effects of dietary Axn on other prawn physiological performance measures are uncertain and dietary carotenoid uptake and tissue deposition are poorly understood. This study fed juvenile prawns (Penaeus monodon) diets that contained 0, 25, 50 or 100 mg kg-1 Axn for 6 weeks. Animals fed carotenoid free diets had significantly reduced growth than those fed carotenoids, but survival was unaffected. Carotenoid uptake (digestibility) improved as dietary carotenoid levels increased, and was 98.5% in animals fed 100 mg kg-1 Axn. After 6 weeks, whole body carotenoid levels were significantly depleted in 0 or 25 mg kg-1 fed animals, compared with those fed 50 or 100 mg kg-1. Specific fatty acid esters of Axn accumulated in epithelial tissue, with mono-esters enriched saturated fatty acids, while di-esters were enriched with monounsaturated and polyunsaturated fatty acids. These data suggest a minimum dietary requirement of 25 mg kg-1 Axn in clear water systems to maintain growth performance, and 50 mg kg-1 or more to avoid depletion of body carotenoid levels and improve efficiency of utilisation. These results also implicate specific fatty acids in the function of carotenoid esters within prawn tissues. Introduction Carotenoids as an essential nutrient for prawns are poorly understood. Crustaceans cannot synthesise carotenoids and must obtain them from their diets (Goodwin, 1952), although they are able to inter-convert different carotenoids (Schiedt et al., 1993, Castillo et al., 1982). All wild and cultured crustacean species report the presence of free and esterified forms of various carotenoids, predominantly astaxanthin (Axn) (Castillo et al., 1982, Lenel et al., 1978, Tanaka et al., 1976, Sachindra et al., 2005). The distribution of these forms of carotenoids also varies with species, life history stages, developmental stage, moult stage and the organ or tissue of the animals (Ribeiro et al., 2001, Lenel et al., 1978, Sachindra et al., 2005, Okada et al., 1994, Pan and Chien, 2000, Dall, 1995, Petit et al., 1998, Pan et al., 1999, Valin et al., 1987, Katayama et al., 1971, Petit et al., 1997). Much of the work on carotenoid nutrition in prawns has focussed on optimising pigmentation levels, where dietary levels of Axn have been recommended at between 80-100 mg kg-1 for Penaeus monodon (P. monodon) (Menasveta et al., 1993, Niu et al., 2012, Okada et al., 1994, Yamada et al., 1990, Wade et al., 2015). Reports of the effect of dietary carotenoid supplementation on growth and survival in P. monodon have been mixed, where some studies recorded no effects (Pan et al., 2001, Boonyaratpalin et al., 2001), while others recorded improvements recorded in growth, survival or both (Darachai et al., 1998, Niu et al., 2012, Niu et al., 2014). Post-larvae of P. monodon greater survival when fed algal Axn (Haematococcus pluvialis) supplemented diets (Darachai et al., 1998). The two studies performed with P. monodon juveniles recorded higher growth and survival when fed dietary carotenoids (Niu et al., 2012, Niu et al., 2014), but used no less than 100 mg kg-1 Axn. Carotenoid digestibility was also shown to be very high, greater than 90% in Penaeus monodon for diets exceeding 100 mg kg-1 Axn (Niu et al., 2012, Niu et al., 2014), but has not been assessed below this level. The detection of performance differences in shrimp fed dietary carotenoids between early work and more recent studies is potentially reflective of improvements in trial maintenance, animal health, quality of feed ingredients, system design and animal husbandry. However, the requirement for carotenoids in prawn diets is not widely accepted, required levels are poorly characterised and carotenoid digestibility has not been investigated below 100 mg kg-1. Other than pigmentation, the function of carotenoids in prawn tissues have been mainly inferred from the innate oxygen free radical scavenging or photoprotective properties of Axn (Britton, 2008, Miki, 1991). Carotenoids and carotenoid esters appear in early juvenile stages (Berticat et al., 2000, Mantiri et al., 1995, Petit et al., 1991), and are predominantly thought to play a role in the storage of carotenoids in various tissues and organs. Other carotenoid functions include conversion to retinoids that play a prominent role in prawn development and differentiation (Linan-Cabello et al., 2002). Increased levels of dietary carotenoid resulted in the accumulation of carotenoid esters in crustaceans (Yamada et al., 1990, Supamattaya et al., 2005, Boonyaratpalin et al., 2001, Barclay et al., 2006, Kumar et al., 2009, Wade et al., 2008). The esterification of carotenoids has been implicated in the regulation of pigmentation in several species (Wade et al., 2005, Wade et al., 2012, Tume et al., 2009). Three studies in other crustacean species have analysed the specific fatty acid attachments of carotenoid esters. In the red crab langostilla Pleuroncodes planipes, C16:0 and C18:1n-9 saturated fatty acids accumulated in astaxanthin diesters and polyunsaturated C20:5n-3 and C22:6n-3 accumulated in astaxanthin mono-esters (Coral-Hinostroza and Bjerkeng, 2002). Of the major astaxanthin esters identified in the shrimp Pandalus borealis, both the astaxanthin mono-esters and diesters were found to contain both C12:0 and C18:1 fatty acids, with di-ester forms predominating (Breithaupt, 2004). The carapace of the spiny lobster Panulirus japonicus was found to contain 42% and 12% astaxanthin di-esters and monoesters, respectively, with both monoesters and diesters composed of predominantly C16:0 or C18:0 fatty acids (>40%), and a further 30% of monoesters containing C16:1 and C18:1 fatty acids (Maoka and Akimoto, 2008). Other than a role in storage, functional roles that fatty acid esterification may provide to the carotenoid molecule are poorly understood. This study sought to define the effects of a range of dietary Axn inclusion levels on growth and carotenoid digestibility in P. monodon. Animals were fed for 6 weeks on one of four diets that contained 0, 25, 50 or 100 mg kg-1 Axn. We also quantified whole body carotenoid levels, and identified specific fatty acids that form esters of Axn in the epithelial tissue of P. monodon. Materials and Methods Animal Handling Live shrimp (P. monodon) were obtained from commercial farms and maintained at CSIRO laboratories at Bribie Island. For all trials, filtered seawater was heated then pumped through the tanks at 1.2 L min-1 maintaining water temperatures at 28 ̊C and salinity at 35 g L-1. Animals were held in a total of 60 red tanks that held 80 L seawater in each. The experiment was conducted indoors under low artificial light conditions and a 12-12 light dark photoperiod. The experimental treatments were composed of a total of twelve replicate tanks for each of the four diets, each of which contained six individually eyetagged P. monodon. Animals across the experiment were within the size range 7.25 ± 1.34 g. Growth Trial Nine tank replicates were used to assess growth over time, with three further replicate tanks used to assess nutrient digestibility in week 7. Animals were fed once per day for 6 weeks on one of four formulated diets that differed only in the amount of Axn (Table 1) in the form of Carophyll Pink (DSM Nutritional Products). Amount of feed consumed was calculated as the amount of feed offered less the uneaten feed that was recorded from the number of pellets remaining after 24 hours. Animal weights and feed intake were collected at day 0, 14, 28 and 42. An initial sample of three groups of six shrimp was taken at random from the pool of individuals used to stock the experiment and stored at 20 ̊C until processing. A single animal was sampled at random from each of the 9 tank replicates at day 42 and pooled into three groups for whole animal carotenoid analysis and similarly stored at -20 ̊C until processed.