Carbon stable isotope evidence for the contribution of supplemental diets to the growth of brown-marbled grouper (Epinephelus fuscoguttatus Forsskål 1775)

Abstract

The objective of this study was to evaluate the relative contribution of two supplemental diets (soybean meal [SM] and beer yeast [BY]) to the growth of juvenile brown-marbled grouper (Epinephelus fuscoguttatus Forsskål 1775). The juvenile groupers were fed fish meal (FM) as the main protein source, and 10% SM, 20% SM or 10% BY as supplemental diet with contrast carbon stable isotope (δ¹³C) ratios. The final δ¹³C ratios of the fish muscle tissue fell between those of FM and supplemental diet, suggesting that the supplemental diet was assimilated by fish. Estimates using a two-source isotope mixing model showed that the juvenile groupers used approximately equal amount of SM relative to the supplied amount at 10% level, but used less than the supplied amount when fed 20% SM. The fish also showed unbiased assimilation of BY relative to the supplied amount (10%).

Keywords:

• carbon stable isotopes
• brown-marbled grouper
• relative contribution
• supplemental diets

1. Introduction

Demands for aquatic protein have increased steadily worldwide due to rapid economic development (Naylor et al. 2009). Traditional intensive aquaculture systems require fish meal (FM) to support the growth of commercially cultivated aquatic animals. However, there has been increasing shortage of the supply of FM due to the limitation of marine resources and environmental pollution. Alternative protein sources such as plants- or microbes-based protein sources have the potential to replace the depleted FM resource. A number of studies have been conducted to evaluate the suitability of supplemental protein as fish diets (e.g., Regost et al. 1999; Zhou et al. 2004; Lunger et al. 2006).

2. Materials and methods

2.1. Experimental diets

Three compound diets were formulated using two protein sources: Peruvian anchovy (Engraulis ringens) as FM, a commercially defatted soybean meal (SM) and a commercial beer yeast (BY) provided by Zhanjiang Huanong Corp., Zhanjiang, China, and Guangdong Haida Group Co., Ltd. Each supplemental diet was added to FM and other ingredients to form a compound diet with the supplemental diet providing between 10% and 20% of dietary protein content. The control diet is 100% FM. Fish oil and soybean oil (V/V = 1:1) were added to keep the contents of lipid and energy constant in all treatments. All of the dry ingredients were thoroughly mixed in a Hobart-type mixer; water and lipid were added and thoroughly mixed. Pellets with 5.0 mm in diameter were wet-extruded, air-dried and sealed in vacuum-packed bags and frozen stored at −20°C until use. Protein and energy contents of the dietary ingredients were determined prior and after formulation according to Horwitz (2005).

2.2. Experimental design and stable isotope analysis

The experiment was conducted in the aquaculture facility of Long Hai Tian Science Garden, Guangdong, China, between July and October 2009. The experiment unit included 25 aquaria (70 cm × 50 cm × 70 cm). Each aquarium was filled with continuously aerated natural seawater prefiltered with a sand medium. Water quality was monitored using the standard methods (American Public Health Association [APHA] 1998). The experiment was conducted indoor using the natural dark:light cycle.

Epinephelus fuscoguttatus larvae with an average weight of 22.5 ± 3.2 g were stocked in 20 aquaria with 12 fish per aquarium. Five aquaria were used for each treatment. Fish were fed twice daily at 09:00 and 13:00 local time. Samples for stable isotope analysis were taken at day 0, 7, 14, 28, 42 and 56. Prior to sampling, fish were starved for 24 hours to vacant the food content. All 12 fish from a randomly selected aquarium were taken during each sampling event. About 10 grams of lateral muscle tissue were taken from each fish, dried and homogenised. About 1 milligram of sample was loaded into tin capsules and shipped to Stable Isotope Facility of University of California Davis for ¹³C analysis using a PDZ Europa ANCA-GSL elemental analyser interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). Samples were combusted at 1000°C in a reactor packed with chromium oxide and silvered cobaltous/cobaltic oxide. During analysis, samples were interspersed with several replicates of at least two different laboratory standards. The heavy-to-light isotope ratios (¹³C/¹²C) are reported as the conventional delta notation (δ) defined as carbon stable isotope (δ¹³C) = ([R_sample/R_standard] −1) × 1000, where R is the ratio of ¹³C/¹²C. Isotopic ratios of δ¹³C are expressed relative to isotope standard, Pee Dee Belemnite formation. The long-term standard deviation is 0.1‰ for ¹³C.

2.3. Data analysis

The relative contribution (%) of supplemental diet (f_SD) to juvenile groupers was estimated using a two-source isotope mixing model:

(1)

where δ¹³C_M, δ¹³C_FM and δ¹³C_SD are the δ¹³C ratios of fish muscle tissue at the end of experiment, FM and a supplemental diet, respectively. The final isotope ratios in consumers were corrected for isotope enrichment during assimilation of each diet. We did not conduct experiments to assess isotope enrichment between juvenile groupers and each supplemental diet due to concern on the poor growth of this carnivorous fish fed solely a plant-based diet. Previous studies demonstrated small isotope fractionation with an average of 0.3‰ during carbon assimilation by consumers (Post 2002). We corrected the isotope enrichment between juvenile groupers and a supplemental diet with a range of isotope ratios from 0 to 0.6‰ to examine the sensitivity of different isotope enrichment levels to the relative contribution of supplemental diet.

3. Results and discussion

Major water quality parameters measured during this study indicated good growth conditions. Water temperature ranged from 24 to 28°C, salinity from 29 to 31‰, pH from 7.9 to 8.2, dissolved oxygen >6 mg L⁻¹, ammonium <0.02 mg L⁻¹ and nitrate <2.0 mg L⁻¹. Fish survival rate was 100% in FM and 10% SM treatments and 96.7% for all other treatments (i.e., two fish mortalities in each treatment). Specific growth rate was highest in the FM treatment (56.9%) and lowest (48.8%) in the 10% SM treatment. The average wet weight of juvenile groupers was more than double by the end of the 56-day feeding experiment.

FM and the two supplemental diets had contrast δ¹³C ratios (Figure 1). The average δ¹³C ratio of the fish muscle tissue taken on day 0 was −18.6‰ and the δ¹³C ratios of the major and supplemental diets were −17.2‰ for FM, −25.2‰ for SM and −22.2‰ for BY, respectively (Figure 1). As FM and supplemental diets were incorporated into fish biomass, isotope composition of the fish muscle tissue changed accordingly. The δ¹³C ratios in all treatments increased, indicating that the growth of juvenile groupers was influenced more by FM which possessed higher δ¹³C ratios than those of the fish before experiment and the supplemental diets. The average δ¹³C ratios of fish muscle tissue measured on day 56 were −18.0‰ in the treatment with 10% SM and 90% FM, and −18.2‰ in treatment with 20% SM and 80% FM. This suggests that fish feeding on a diet with higher percentage of SM was also depleted in ¹³C than fish fed a diet with less SM. The average δ¹³C ratio of fish in treatment with 10% BY and 90% FM was −17.5‰ in day 56. The higher final δ¹³C ratio in this treatment than the two soybean treatments reflects the influence by the isotope composition of the BY which possessed higher δ¹³C ratio than the SM.

Figure 1. Changes in carbon stable isotope ratios of muscle tissue of juvenile Epinephelus fuscoguttatus fed various experimental diets during this study.SM: soybean meal; FM: fish meal; BY: beer yeast. The upper dash line represents the δ¹³C ratio of FM and the lower dash line (if presented) represents the δ¹³C ratio of SM or BY.

The estimated relative contribution from different supplemental diets varied slightly at 0 and 0.3‰ enrichment level (Table 1). When the enrichment level was double from the literature average, the estimated relative contribution from the supplemental diets varied from those estimated at 0.3‰ enrichment level by less than 5% (Table 1). This indicates that the use of estimated δ¹³C enrichment for supplemental diets did not result in significant variation in our estimate of the relative contribution from the supplemental diet. We will use the estimated relative contribution from supplemental diets based on the 0.3‰ enrichment in the following discussion.

Table 1. Estimates of proportion (%) of diet from soybean meal (SM) and beer yeast (BY) to the growth of Epinephelus fuscoguttatus using a two-source isotope mixing model.

		Estimated
Diet	Expected	0	0.3	0.6
10:90 SM:FM	10	10.0	9.0	8.6
20:80 SM:FM	20	12.5	15.9	15.3
10:90 BY:FM	10	6.0	11.1	10.5

FM: fish meal. A range of Δδ¹³C from 0 to 0.6‰ was used to assess the sensitivity of different isotope enrichments to the estimates.

Fish fed 10% SM and 90% FM showed basically unbiased contributions from the two diets, with SM contribution at 9.0% (Table 1). Similarly, the relative contribution of supplemental diet at 10% BY and 90% FM treatment was 11.1% which is virtually the same as the treatment level. However, in the 20% SM and 80% FM treatments, our estimate showed that fish assimilated 15.9% from SM, which is considerably below the 20% supplied to the compound diet. These results suggest at low level (10% SM in this case) of supplemental diet substitution, juvenile groupers were capable of using all supplied SM. However, when the supplemental level increased to 20%, fish assimilated dietary carbon in a way that is favoured for FM. This bias is likely because plant proteins such as soybean protein have amino acid imbalance (Alam et al. 2005). Our finding is consistent with that reported by Gamboa-Delgado and Le Vay (2009) who found preferential incorporation of FM to the growth of Pacific white shrimp (Litopenaeus vannamei) fed a diet with soybean and FM protein.

4. Conclusions

This study demonstrated using different dietary components with contrast δ¹³C ratios that dietary carbon from SM and BY in a composite diet with FM as the dominant protein source was assimilated by juvenile groupers. Unbiased assimilation of these supplemental diets at 10% level and reduced assimilation when fish fed 20% SM were revealed during this study.

Acknowledgements

We appreciate Xiaohui Dong for assistance in the design and formulation of the feed pellets. This study was supported by grants A200608C02, A200908D03, 2006B20201059 and 2009B020308005 from several funding agencies of the Guangdong province government, China.