Factors Affecting Utilisation Of Carotenoids In Salmonid Fishes

TABLE OF CONTENTS

• ACKNOWLEDGEMENTS
• ABSTRACT
• SAMMENDRAG
• LIST OF PUBLICATIONS
• INTRODUCTION
• AIM OF THE STUDY
• GENERAL BACKGROUND
  • Carotenoids
  • Factors affecting the bioavailability of carotenoids
• METHODOLOGICAL CONSIDERATIONS
  • Measuring feed intake
  • Feeding regime
  • Digestibility estimates
  • Carotenoid analyses
  • Quantitative determination of carotenoids
  • Qualitative determination of carotenoids
  • Visual colour assessment
• MAIN RESULTS AND DISCUSSION
  • Digestion and absorption (Paper I and II)
    • Temperature
    • Feed intake
    • Carotenoid species
  • Carotenoid transport in plasma (paper I, II, IV and V)
  • Metabolism of carotenoids (paper I, III, IV and V)
  • Deposition of carotenoids in the muscle (paper III, IV and IV)
• CONCLUSIONS
• FUTURE PERSPECTIVES
• REFERENCES
• PAPERS I-V

ABSTRACT

Absorption from the intestine is a major limitation for the utilisation of carotenoids, and factors that influence gut absorption can potentially have a large effect on the amount of carotenoid retained in the body. The apparent digestibility (ADC) of astaxanthin (3,3´-dihydroxy-b,b-carotene-4,4´-dione) in Atlantic salmon (Salmo salar) was affected both by water temperature and by ration level (Papers I and II). By decreasing the water temperature from 12 to 8 °C the apparent digestibility of astaxanthin was reduced from 45 to 40 % (Paper I). The apparent digestibility of astaxanthin decreased with increasing feed intake in a linear fashion when the daily ration level ranged from 0.15-0.63 % of biomass for groups of Atlantic salmon (Paper II). The corresponding ADC ranged from 60 to 26 %. A positive linear correlation was observed between digested astaxanthin and ration level.

However, the ratio of mg digested astaxanthin /growth rate (TGC) decreased with increasing feed intake. Thus, a reduced muscle concentration of astaxanthin may be caused by a high feed intake. There was, however, no significant correlation between the individual meal sizes measured by radiographic methods and astaxanthin ADC although a weak trend trend was observed in one of the treatments (p =0.07, R²=0.22) (Paper I).

The plasma content of idoxanthin (3,3´,4´-trihydroxy-b,b-carotene-4-one), a reductive metabolite of astaxanthin, was used as an indicator of metabolic turnover. The concentration of idoxanthin in plasma of individual fish ranged from less than 0.5 to 70 % of total carotenoids and was not affected by temperature or feed intake (Paper I). However, the plasma concentration of carotenoids was positively correlated with feed intake both in individual fish and at the group level. Fish without faeces in the hindgut had a lower plasma carotenoid concentration than fish with faeces in the hindgut (Papers I and II). Thus, if plasma carotenoid concentrations are used an indictor of bioavailability it is important that the feed intake is monitored carefully.

4´-Hydroxyechinenone (4´-hydroxy-b,b-carotene-4-one), a reductive metabolite of canthaxanthin, was isolated from muscle from Atlantic salmon fed a diet supplemented with canthaxanthin and characterised by mass spectrometry, absorption maximum and cochromatography with authentic standard (paper III). The stereoselective reduction of canthaxanthin in favour of the (4´S)-isomer (comprising ca. 81 % of the total 4´- hydroxyechinenone) strongly indicates that it was a metabolic product of canthaxanthin.

The accumulation of carotenoids and lipid in muscle was studied in Atlantic salmon transferred to seawater as 0+ or 1+ smolts (paper III). The fish were fed a diet containing a mixture of canthaxanthin (30 mg kg^-1) and astaxanthin (30 mg kg^-1). The 0+ smolts had a higher muscle carotenoid content compared to 1+ smolts when the difference in body weight was corrected for. The higher carotenoid content in 0+ smolts was also reflected by a higher colourimetrically recorded redness (a*-value) and yellowness (b*-values) and lower lightness (L*) in 0+ than in 1+ smolts of a similar size.

The content of 4´-hydroxyechinenone in the muscle was low (< 3.1 % of total carotenoids) and declined with increasing fish size in both smolt types. The colour characteristics of the muscle (measured as a* and b*-values) mainly depended on the carotenoid concentration of the muscle and were only marginally affected by smolt production regime. Salmon transferred to seawater as 0+ smolts deposited carotenoids more efficiently than 1+ smolts during the summer and early fall. The higher muscle concentration in 0+ smolts during this period indicates that the deposition of carotenoids did not depend on the carotenoid level in the muscle. The condition factor was slightly higher in 0+ than in 1+ smolts but there were no differences in muscle lipid content when mass was used as a covariate to correct for the larger size of the 0+ smolts. Thus the results from this study show that the timing of smoltification may influence accumulation of carotenoids and body shape in Atlantic salmon.

When astaxanthin was injected into the abdominal cavity of Atlantic salmon, Atlantic cod and rainbow trout (Oncorhynchus mykiss) the astaxanthin concentrations in plasma and muscle increased in a dose dependent manner in all three species (Papers IV and V). The shape of the dose- response curves was species specific. In salmon and cod (Paper V) the plasma astaxanthin concentrations were much higher than concentrations reported after oral administration of astaxanthin (up to 90 and 43 mg l^-1 in individual fish respectively).

Furthermore, there was no indication of a saturation of the blood transport capacity because the dose-response curves in plasma were close to linear in both salmon and cod. In contrast, curvilinear dose-response curves with a relatively lower plasma and muscle response at higher doses injected were observed in both plasma and muscle of rainbow trout (Paper IV). As a result, the mean plasma and muscle concentrations of astaxanthin were higher in salmon (53 mg l^-1 and 18 mg kg^-1 respectively) than in rainbow trout (13 mg l^-1 and 11 mg kg^-1 respectively). The lowest muscle concentrations were found in cod (1 mg kg^-1) even though the mean plasma astaxanthin concentration was as high as 36 mg l^-1 in cod injected with 50 mg astaxanthin. However, the cod deposited more astaxanthin in the skin compared to Atlantic salmon and rainbow trout. The observed species specific in vivo distribution of astaxanthin after intraperitoneal administration indicates that the uptake of astaxanthin is regulated at the cellular level in the various tissues.

In conclusion, the results from this thesis may explain the reduced flesh pigmentation that has been reported after periods with rapid growth. It is also shown that the timing of seawater transfer can influence flesh pigmentation and other quality traits in Atlantic salmon. Finally, it is shown that neither the plasma transport capacity nor the muscle binding capacity seems to be limiting factors for flesh pigmentation of Atlantic salmon. Future work should focus on identifying the uptake mechanisms in muscle and other organs involved in carotenoid utilisation.

INTRODUCTION

The aquaculture industry is a major contributor to the Norwegian economy, and the total production of Atlantic salmon and rainbow trout was 590 000 metric tons in 2003 of which 93% was exported (Fiskeridirektoratet 2003). The delicate pink flesh colour unique to salmonid fishes is caused by deposition of carotenoids such as astaxanthin and canthaxanthin in the muscle. All animals, including fishes, are unable to synthesise carotenoids de novo and thus depend on a dietary supply. In Norway the main carotenoid sources are synthetically manufactured astaxanthin and canthaxanthin. The expenses related to pigmentation were approximately 0.6 billion NOK in 2004, assuming an average concentration of 50 mg kg-1 in the diet, a feed conversion ratio (FCR) of 1.2 and a cost of 18 000 NOK per kg astaxanthin. The Norwegian fish farming industry could save more than 5 million NOK each year by increasing the utilisation of astaxanthin by only 1 %.

Flesh colour is an important quality criterion for Atlantic salmon, and only fillet freshness was regarded as more important for consumer acceptance (Moe 1990; Sigurgisladottir et al. 1997). A mean astaxanthin level of 7 mg kg-1 in the muscle is considered necessary to obtain an acceptable flesh colour. To achieve a sufficient colouration of the flesh it has been common practice to fortify diets with 30-60 mg carotenoids per kg during the entire seawater phase. Approximately 12 % of the total feed costs in 2003 were related to pigmentation due to a combination of high price and poor utilisation of the carotenoid pigments.

The muscle retention of astaxanthin is typically between 5-12 % in Atlantic salmon (Wathne et al. 1998; Bjerkeng et al. 1999 a,b). A major reason for the poor utilisation of carotenoids is low absorption from the gut. The difference between the amount ingested and the amount absorbed is called the apparent digestibility (ADC) and for astaxanthin the ADC is normally between 40-50 % (Bjerkeng and Berge 2000). However, it is possible that the bioavailability of astaxanthin may be improved if the gut is bypassed by injecting the astaxanthin in the abdominal cavity (Maltby et al. 2003).

Only about 10 % of the ingested carotenoid is retained in the muscle. This is less than half of the absorbed astaxanthin. The rest is probably converted into colourless metabolites and excreated or deposited in other tissues. Other limiting factors could be transport processes in the blood and uptake and deposition in the muscle cells. Considering the large costs associated with pigmentation, it would be of great importance to improve the understanding of factors that may limit the absorption, and influence metabolism and deposition of carotenoids in salmonid fishes.

Production of underyearling smolts (0+) has become increasingly popular during the last decade. In the fall of 2005 about 70 million underyearling smolts were transferred to seawater in Norway. Advanced smolting is achieved by manipulating temperature and photoperiod (Duston and Saunders 1995; Duncan et al. 1998, 2002). Underyearling smolts can be transferred to seawater already in September, thereby shortening the freshwater production cycle by 7 months. This represents a major saving for the farmer, provided that the quality of fish transferred to seawater as 0+ is not inferior compared to that of fish transferred in spring (1+). Another advantage is the predictable all year supply of 3-4 kg fresh salmon.

Although the production of 0+ smolts is becoming an important production practice, the effcts of this practice on flesh quality parameters and carotenoid utilisation is not well documented. As a result of the different temperature- and photoperiod regimes experienced before and after seawater transfer, 0+ and 1+ smolts exhibit different growth profiles during their production cycle (Mørkøre and Rørvik 2001; Duncan et al. 1998, 2002; Lysfjord et al. 2004; Roth et al. 2005). Growth rate may have implications for flesh quality with respect to proximate composition, muscle fibre density, breaking strength, deposition of carotenoids and visual perception of colour (Einen et al. 1999; Mørkøre and Rørvik 2001; Johnston et al. 2000; Johnston et al. 2004).

The muscle pigmentation in Atlantic salmon may exhibit seasonal fluctuations during the production cycle. A drop in flesh carotenoid level may take place during spring and early summer in the southern parts of Norway (Torrissen et al. 1995; Mørkøre and Rørvik 2001; Nordgarden et al. 2003) and during fall in the north. A drop in muscle astaxanthin levels has been observed during periods of rapid growth in Atlantic salmon (Nordgarden et al. 2003). These fluctuations can be a result of a reduced uptake of carotenoids and/or an increased bioconversion or mobilisation of carotenoids. Temperature and photoperiod vary with season, which in poikilothermic animals such as fishes affect feed intake and growth rate.

Temperature may affect the digestibility of nutrients through its positive effects on passage time and diffusion rates. An increase in feed intake is also positively correlated with gastric evacuation rate leaving less time available for absorption (reviewed by Fänge and Grove, 1979), and there may be a negative correlation between feeding rate and nutrient absorption efficiency (reviewed by Jobling 1994). This may particularly affect compounds such as carotenoids that are absorbed slowly from the gut (Aas et el. 1999).

AIM OF THE STUDY

The major causes for the low utilisation of carotenoids in salmonids seem to be absorption from the intestine and metabolic transformation of carotenoids into colourless metabolites. Other limiting factors could be blood transport and uptake and deposition in the muscle cells. The work of the present thesis therefore focused on factors that may influence digestion, metabolism and deposition of carotenoids such as astaxanthin and canthaxanthin. The specific aims were:

1. To evaluate the effects of feed intake and temperature on apparent digestibility and metabolism of astaxanthin in Atlantic salmon.

2. To isolate and identify 4´-hydroxyechineon, a reductive metabolite of canthaxanthin, in muscle of Atlantic salmon.

3. To study carotenoid deposition, metabolism and flesh colour characteristics in Atlantic salmon transferred to seawater as 0+ or 1+ smolts.

4. To investigate whether the bioavailability of carotenoids may be improved when the gut is bypassed by administering astaxanthin intraperitoneally in Atlantic salmon, rainbow trout and Atlantic cod.

Further Information

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September 2006