Introduction

Feeding most finfish larvae for aquaculture still depends on live feeds during the earliest phases of

development (Conceição et al., 2010a), despite the fact that progress has been achieved in the

production of inert diets for some fish larvae (Cahu and Infante, 2001; Koven et al., 2001; Lazo et al.,

2000). The reason is that when newly hatched marine larvae start to feed they are characterized by

a small and simple digestive system (Ronnestad et al., 1999; Watanabe and Kiron, 1994), where

there is no stomach and much of the protein digestion takes place in hindgut epithelial cells (Govoni

et al., 1986). The larval digestive system is incapable of processing formulated diet as efficiently as

live feeds, and consequently limits larval growth and survival when compared to larvae fed on live

feeds (Conceição et al., 2010a). The two main live foods offered to marine fish larvae in culture are

rotifers, Brachionus plicatilis,and Artemia species.Average body size is 50-200 µm for rotifers and

200-500 µm for Artemia, depending on the strain and age (Conceição et al., 2010a; Watanabe and

Kiron, 1994). The advantages of using live feeds in aquaculture, in addition to their high digestibility

for larvae (water content > 80%), are their small size, which makes it easier for the larvae to prey

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Despite the simple characteristics of the larval gut, larval fish require sufficient balanced nutrients to

support normal development. The nutritional profile of cultured rotifers and Artemia do not meet

these requirements for many fish species. However, dietary manipulation to improve the nutritional

profile of the cultured live feed is accomplished by a post-culture enrichment process (Conceição et

al., 2010a; Lubzens and Zmora, 2007). Enrichment is through bioencapsulation where live feeds are

cultured in a medium rich in fatty acids, amino acids, vitamins or other substances such as hormones

or vaccines (Conceição et al., 2010a; Coutteau and Sorgeloos, 1997; Dhert et al., 2001). The non-

selective feeding behaviour of the live prey enables them to feed on the correctly sized diffused

particles and incorporate them into the digestive tract and assimilate them into their bodies

depending on enrichment periods and temperature (Conceição et al., 2010a; Sorgeloos et al., 2001;

Sweetman, 2004). The enrichment of rotifers and Artemia to deliver important nutrients including

lipids, amino acids, vitamins and minerals to the marine larvae to support normal development is

widely used in aquaculture (Aragao et al., 2004; Coutteau and Sorgeloos, 1997; Sargent et al., 1999).

Vitamins such as A, C, D, E and K play an important role to support the optimal growth and

development of fish larvae (Brown et al., 2005; Demartinez, 1990; Haga et al., 2004b; Hamre et al.,

2010; Lall and Lewis-McCrea, 2007; Lock et al., 2010; Mazurais et al., 2008; Roy and Lall, 2007;

Villanueva et al., 2009; Waagbø, 2010). Vitamin A (VA), also known as retinoid, in fish larvae plays a

key role in vision, immunity, differentiation of epithelial tissue, morphogenesis, tissue homeostasis

and skeletogenesis (Blomhoff and Blomhoff, 2006; Combs, 2008; Fernández et al., 2009; Ross et al.,

2000; Srinivas and Chethankumar, 2007; Thompson et al., 1995). Retinoids also have a role in

establishing body and organ axes in conjunction with other nutrients such as vitamin D and fatty

acids (Balmer and Blomhoff, 2002; Hamre et al., 2010; Villeneuve et al., 2006). Vitamin A deficiency

in fish leads to retarded growth, blindness, restlessness, abnormal movement, exophthalmia and

haemorrhages of the eye, fins or skin, decreased immunity and harm to the intestinal epithelia

(Goswami and Basumatari, 1988; Goswami and Dutta, 1991; Moren et al., 2004; Saleh et al., 1995;

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patterning defects, skin haemorrhage, abnormal pigmentation and abnormal bone formation in the

fish (Dedi et al., 1997; Fernández et al., 2008; Haga et al., 2002; Hernandez-H et al., 2006; Hilton,

1983; Martinez et al., 2007; Suzuki et al., 1999; Takeuchi et al., 1998; Tarui et al., 2006).

The aim of this research Chapter was to examine the enrichment process of live feeds, rotifers and

Artemia,with VA in the retinyl palmitate form, and to determine the concentration of retinoids over

time in both live feeds. The research reported here forms the basis for the enrichment protocols

used to enrich live feeds with VA. For later Chapters, VA-enriched live feeds were fed to Striped

Trumpeter, Latris lineata, larvae in dose-response experimental designs (Chapters 3, 4 and 5). My

thesis was designed to assess the effect of dietary VA delivered in live feeds on skeletal

malformations and determine the VA requirement for optimal development of L. lineata. Since

retinoids are unstable compounds and can be oxidized and/or isomerised to other compounds,

especially in the presence of oxidants including air, light or heat (Barua and Furr, 1998), the effect of

the absence or presence of light on the enrichment process was examined. The effect of light on the

enrichment process of live feeds with VA has been examined by Haga et al. (2006). He found that

light affected the enrichment process of rotifers, with the concentration of retinoids being higher in

dark conditions, but not the Artemia. I also examined the lipid profile of the VA-enriched rotifers to

determine if they met L. lineata requirements for fatty acids, based on previous research (Bransden

et al., 2004, 2005b).

Latris lineata culture is optimal with the addition of the microalga Nannochloropsis oculata into the

larval rearing tanksduring the rotifer feeding period (Battaglene and Cobcroft, 2007; Cobcroft et al.,

2001; Shaw, 2006), known as the “greenwater technique”. This microalga contains small amounts of

retinol (< 0.25ng mg-1) and 290 ± 40 ng mg -1 β-carotene (Brown et al., 1999), where β-carotene is a

major dietary precursor of VA (Ross and Ternus, 1993). I also assessed the effect of adding rotifers

enriched with VA into the larval rearing tanks with the presence or absence of microalgae, in

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rotifers was examined with respect to the water type, and the time after transfer from the

enrichment.