Elsevier

Aquaculture

Volume 315, Issues 1–2, 1 May 2011, Pages 144-153
Aquaculture

A new method to increase and maintain the concentration of selenium in rotifers (Brachionus spp.)

https://doi.org/10.1016/j.aquaculture.2010.09.007Get rights and content

Abstract

Rotifers are used as the first feeding diet for the larvae of many commercially produced marine fish species. However, the nutritional requirements of marine fish larvae appear to be better fulfilled by their wild feed, copepods. Consequently, rotifers are fed diets that alter their body composition to better imitate copepod composition and this results in greater success when rearing rotifer fed marine fish larvae. Despite this, copepod fed fish larvae still have higher growth rates and survival than rotifer fed larvae. This may occur because of the less investigated mineral differences that exist between rotifers and copepods. The concentration of selenium (Se) in rotifers Brachionus sp. (0.08–0.09 mg Se kg 1 dry weight) can be over 30 fold lower than the concentration found in copepods (3–5 mg Se kg 1 dry weight). In this study, the enrichment and retention of Se in rotifers fed Se enriched yeast (Se-yeast) were investigated. Rotifer Se concentration increased linearly with increasing levels of Se-yeast, with rotifers reaching a maximum of 138 mg Se kg 1 dry weight. The use of Se-yeast was highly effective, with only 1% of the batch culture or short term enrichment diet needed to be replaced with Se-yeast to achieve copepod Se levels. At this feeding level there were no negative effects on rotifer egg ratio or population growth. Uptake of Se from Se-yeast was over 40 fold higher than obtained from using sodium selenite in short term enrichments (3 h). Se-yeast enriched rotifers had a high retention (100–85%) of Se for up to 10 h storage in clear water at cold (10 °C) or warm (20 °C) temperatures, while storage in green water (with algae) resulted in a slightly lower Se retention (65%) in a similar time period (8 h). Overall, rotifers enrichment with Se-yeast resulted in reproducible concentrations of Se that were then retained for extended periods of time. This will allow Se-yeast enriched rotifers to be used as a Se delivery method for fish larvae nutritional requirement or toxicological studies.

Introduction

Rotifers are widely used as the start feeding diet of marine fish larvae, but in the wild marine fish larvae feed mainly on copepods. Past experience has shown that feeding rotifers diets that change the rotifer nutritional composition to more closely imitate that of copepods can yield substantial increases in fish larvae quality. For example, rotifer diets that alter their lipid composition to better imitate copepod lipid composition (van der Meeren et al., 2008) have increased larval rearing success for numerous marine fish species (Rainuzzo et al., 1997, Rodriguez et al., 1997, Park et al., 2006). However, these lipid altered rotifer fed larvae still fall short of obtaining the growth and survival rates obtained by copepod fed larvae (Payne et al., 2001, Rajkumar & Kumaraguru Vasagam, 2006, Schipp, 2006, Wilcox et al., 2006, Olivotto et al., 2008, Koedijk, 2009, Busch et al., submitted for publication). This may be because a large amount of research has gone into addressing some nutrient differences between rotifers and copepods, such as lipids, but other potential nutrient deficiencies in rotifers have been largely overlooked. The nutritional difference between rotifers and copepods occurs across all areas including lipids, free and total amino acids (Drillet et al., 2006, van der Meeren et al., 2008) as well as in vitamins and minerals (Hamre et al., 2008a). Recent findings by Hamre et al. (2008a) demonstrate that one of the largest unaddressed nutritional differences between copepods and rotifers is in their mineral composition. While copepods have ample concentrations of essential minerals, often much higher than the national research councils' recommendations for juvenile fish (NRC, 1993), rotifers appear to contain many minerals at low and possibly deficient concentrations. The mineral found with the largest difference and thus potentially the most deficient in rotifers was selenium (Se). Selenium levels of rotifers fed a range of commonly used culture diets (0.08–0.09 mg kg 1 dry weight (DW)) were over 30 fold lower than copepod levels (3–5 mg kg 1 DW; Hamre et al., 2008a) and 3–8 fold lower than the Se requirements for juvenile fish studied so far; channel catfish Ictalurus punctatus 0.25 mg kg 1, rainbow trout Oncorhynchus mykiss 0.3 mg kg 1 and grouper Epinephelus malabaricus 0.7 mg kg 1, of dry feed (Hilton et al., 1980, Gatlin & Wilson, 1984, Lin & Shiau, 2005). Furthermore, several recent studies have demonstrated that rotifers provide insufficient Se to meet the Se requirement, as measured by Se-dependent enzyme mRNA expression and/or activity, of Atlantic cod Gadus morhua (Penglase et al., 2010) and Senegalese sole Solea senegalensis (Ribeiro et al., submitted for publication) larvae.

Selenium is an essential nutrient for vertebrates (Johansson et al., 2005) but can also be toxic. The speciation of Se affects both its bioavailability and its potential toxicity. Selenium occurs in foods in numerous forms but generally the supplemental inorganic selenate (Se6+) and selenite (Se4+), and the naturally present Se containing amino acids, selenomethionine (Se-Met) and selenocysteine (Se-Cys) are the most prevalent (Combs and Combs, 1986). Both Se-Met and Se-Cys differ from methionine and cysteine respectively, by containing a Se atom in place of the sulphur atom. While Se-Cys differs greatly in chemistry to cysteine (Johansson et al., 2005), Se-Met shares similar chemistry to methionine (Kohrle et al., 2005). Transfer-RNA does not distinguish between methionine and Se-Met, and so both are readily incorporated into proteins at methionine positions (Waschulewski & Sunde, 1988, Bell & Cowey, 1989, Kohrle et al., 2005). This may explain why Se-Met has a higher retention (Lorentzen et al., 1994, Jaramillo et al., 2009, Rider et al., 2009a, Rider et al., 2009b) and is more bioavailable (Wang and Lovell, 1997) than selenite in fish.

The window between Se requirement and toxicity is the smallest of any element (Chassaigne et al., 2002, Polatajko et al., 2006). For example, rainbow trout has a Se requirement of 0.3 mg kg 1 and a suggested chronic toxicity level of 3 mg kg 1 in dry feed (Hilton et al., 1980), a level only 10 fold higher. Selenium toxicity occurs through two main mechanisms. The first, is that Se can disrupt proteins via substituting as sulphur in sulphur bonds, resulting in incorrect protein shape and dysfunctional enzymes. The second mechanism is through oxidative stress caused by excess unbound Se (Lemly, 2002). Both mechanisms rely on Se in free forms, which explains why Se-Met which contains inert Se until catabolised (Ip, 1998), is regarded as less toxic than selenite and selenate. For example, while Hilton et al. (1980) suggested that 3 Se mg kg 1 supplied as Na-Se was chronically toxic for rainbow trout, Rider et al. (2009b) found no toxic effects of feeding 8 mg Se kg 1 supplied mainly as Se-Met via selenoyeast.

Interestingly, nutrient interactions between Se and other minerals can affect their retention. High dietary or waterborne Se concentrations have been shown to decrease the concentration of copper (Lorentzen et al., 1998, Lin & Shiau, 2007) and mercury (Belzile et al., 2006; Deng et al., 2008) in aquatic organisms. Therefore analysing other minerals alongside Se is necessary to identify any secondary effects of increasing rotifer Se concentration.

Selenoyeast (Se-yeast) is the common name for commercial bakers yeast Saccharomyces cerevisiae products which have been enriched with Se as a nutritional supplement. When cultured in the presence of high concentrations of inorganic Se, the yeast converts and then stores Se in organic forms (Polatajko et al., 2006). The exact speciation of Se within Se-yeast is unknown (Suhajda et al., 2000), and there is large variations in speciation between commercial products (Encinar et al., 2003). In general, around 70% of the Se in Se-yeast is in the form of Se-Met (Polatajko et al., 2006) and the remaining Se are inorganic forms such as selenite and selenate (Suhajda et al., 2000), or low molecular weight organic Se compounds other than Se-Met (Ip, 1998, Chassaigne et al., 2002). As rotifers can ingest and digest yeast (Rodriguez et al., 1996), feeding rotifers Se-yeast appears a logical method of increasing rotifer Se concentration.

Nutrients that are desirable for fish larvae can be increased in rotifers through their feed in both the culture phase, called long term (LT) enriching or for a short period before feeding to fish larvae, called short term (ST) enriching (Olsen et al., 1993). The culture diet has two purposes; to maintain healthy and high rotifer population growth rates, and to build up concentrations of nutrients in rotifers that are beneficial for fish larvae. Short term (ST) enrichment occurs for less than 24 h (Olsen et al., 1993), and aims to rapidly increase the concentration of desirable nutrients in the rotifer just prior to feeding to fish larvae. When combined, LT and ST enrichment results in higher levels of nutrients in rotifers and higher delivery of these nutrients to fish larvae than either enrichment technique alone.

Once enriched, rotifers are either fed directly to fish larvae, or stored in clean water and fed to fish larvae up to 24 h post enrichment. Rotifers are often stored after enrichment due to logistical constraints, where hatcheries may be limited in the number of batches of rotifers that can be enriched per day. In addition to storage, rotifers may remain uneaten in fish larvae tanks for extended periods of time, and fish larvae tanks may contain algae (Reitan et al., 1997). Rotifers metabolise and excrete ingested nutrients over time, which can result in large changes in rotifer nutrient composition after enrichment. For example rotifers have been shown to lose essential fatty acids (Rodriguez et al., 1996, Naz, 2008) and zinc (Matsumoto et al., 2009) after enrichment. Algae added to fish larvae tanks (green water technique) have also been shown to affect rotifer composition (Reitan et al., 1997, Yamamoto et al., 2009). Long term enrichment results in a greater uptake, assimilation and stabilisation of nutrients in the rotifer body than ST enrichment (reviewed by Dhert et al., 2001). Thus ST enriched nutrients are more prone to be lost quickly from rotifers, and hence are less likely to be passed on to fish larvae if the rotifers are not consumed within a short space of time (Olsen et al., 1993). The decrease of desirable nutrients in rotifers as they return to homeostasis is problematic for both marine fish larvae production and research. This loss of desirable nutrients lowers the nutritional value of rotifers to fish larvae, and also makes conclusions difficult on fish larvae nutrient intake and requirement studies.

There were two aims of this study. The first aim was to investigate the effect of LT and ST enrichment of rotifers with increasing concentrations of Se-yeast on rotifer Se concentration. As a sub factor of the first aim, rotifer egg ratio, population growth and mineral composition other than Se were also measured to determine the suitability for application of feeding Se-yeast to rotifers in commercial hatcheries. The second aim was to determine the rate of Se retention in rotifers enriched to copepod levels with Se-yeast, after storage in clear or green water.

Section snippets

Yeast specification

A commercially available Se-yeast (Sel-Plex® 2000, 2000 mg Se kg 1, Alltech, Lexington, KY) was used in the experiments. Speciation of Se in Sel-Plex consists of 63–66% Se-Met, 34–36% low molecular weight Se compounds and < 0.5% inorganic Se (S. Elliott, personal communication). The total Se level of the Se-yeast was analysed as per Section 2.3.

To determine yeast diameter, individual Se-yeast (n = 359) were measured using an Olympus BX51 binocular microscope fitted with an Olympus DP50 3.0 Camera

Yeast specification

The mean diameter of the Se-yeast was 5.0 ± 1.2 μm (mean ± SD). The total Se concentration determined in the Se-yeast was 1722 mg Se kg 1 (Table 3). There was no statistical difference between total water solubility of Se in crushed Se-yeast in either seawater or at pH 2 (p = 0.14) (Table 3). Approximately 26% of the Se in the crushed Se-yeast was water soluble. Approximately 25% of the Se in whole Se-yeast was leached within 1 min of dispersion in seawater while there was no significant leakage of Se

Se-yeast specification

The percentage and speciation of insoluble Se are important considerations when Se-yeast is fed to aquatic organisms, where soluble Se may be quickly lost to the environment. The present study found that approximately 74% of the Se in the Se-yeast is in a non water soluble form and remains associated with the crushed Se-yeast fragments after dispersal in either seawater or water at pH 2. This result is within range of the typical 75–85% Se remaining after aqueous extraction of various Se-yeast

Conclusion

This study concludes that Se-yeast is a suitable medium to increase Se levels in rotifers. Generally, less than 1% of the rotifer culture diet or ST enrichment must be replaced with Se-yeast to obtain copepod Se levels in rotifers. At this feeding rate, Se-yeast had no negative effect on rotifer egg ratio or population growth. Se-yeast enriched rotifers retained a high percentage of Se after extended periods of storage in both the presence and absence of food. The results show rotifers had 40

Acknowledgements

This work was financed by the Norwegian Research Council (project no. 185006/S40), Alltech and NIFES. We would also like to acknowledge Sagafjord Seafarms AS for providing rotifers, algae and technical expertise, technical staff at IMR (Austevoll) for rotifers, and technical staff at NIFES for skilled analytical assistance.

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