The work focuses on development of digestive enzymes (amylase, total protease, trypsin, and chymotrypsin) and activity ratio of trypsin to chymotrypsin (T/C ratio) for digestive efficiency and growth, in blue swimming crab (
Blue swimming crab,
In decapod crustaceans, the herbivorous decapod larvae adapt to low food energy values with high enzyme activity levels, rapid food turnover, and low assimilation efficiency, while the carnivorous larvae exhibit low levels of enzyme activity but compensate by extending retention time of high-energy food to maximize assimilation efficiency [
The present study is a part of a project with the ultimate goal to establish feed formulae suitable for culture of
The healthy female broodstock
Approximately 1,000 first crab larvae from the hatchery were raised at 50 individuals per m2 in 5 m3 rectangular concrete tanks for 1 to 4 months (1M–4M). They were fed with a commercial shrimp feed with 35% protein for months 1 and 2 and with 30% protein for months 3 and 4, with 14% lipid and 30% carbohydrate.
An open system was used for culturing. Cleaned seawater using 25 ppm calcium hypochlorite was daily exchanged at 20% by volume for Z1 to Z2 stages and 30–60% for Z3 to C and adult stages. Seawater quality throughout the experimental period was maintained at 26-27°C and pH 7.8–8.2 with salinity 28 and total alkalinity 120–140 mg L−1.
Wild adult
All samples were collected in triplicate, and the crabs were anaesthetized by low temperature (on ice) before sampling. The whole body of crab larvae or hepatopancreas of adults were each pooled to 5 g per sample. Enzymes were extracted by homogenizing the sample with 1 : 3 (w/v) of 50 mM Tris buffer pH 7.0 on ice. The homogenate was centrifuged at 15,000 ×g at 4°C for 30 min. The upper lipid layer was discarded and the supernatant was then kept at –80°C for further determinations of specific activities of α-amylase, total protease, trypsin, and chymotrypsin. The protein content of the crude enzyme extracts was determined according to Lowry et al. [
The activity of α-amylase was determined by measuring the increase in reducing sugar by the hydrolysis of α-
Total protease activity was determined by measuring the increase in cleaved short chain polypeptides using azocasein as substrate, according to Areekijseree et al. [
Trypsin and chymotrypsin activities were determined by measuring the initial increasing of
Crude enzyme extracts of
Biochemical composition (%) of feed raw materials for
Raw materials | Moisture | Protein | Lipid | Fiber | Ash | Carbohydrate |
---|---|---|---|---|---|---|
For larval stages (Z1–C) | ||||||
|
4.5 | 42.0 | 6.2 | 7.9 | 25.4 | 14.0 |
|
2.3 | 38.0 | 4.6 | 19.2 | 19.4 | 16.5 |
|
5.6 | 36.8 | 5.5 | 1.7 | 22.7 | 27.7 |
|
1.3 | 52.2 | 18.9 | 14.8 | 9.7 | 3.1 |
|
1.4 | 56.4 | 11.8 | 12.1 | 13.4 | 4.9 |
|
4.2 | 40.4 | 4.4 | 1.1 | 1.8 | 48.1 |
|
3.6 | 50.1 | 4.8 | 1.8 | 3.1 | 36.6 |
|
4.2 | 46.4 | 7.0 | 9.0 | 10.0 | 23.4 |
Artemia flake | 1.3 | 55.7 | 20.0 | 11.0 | 11.4 | 0.6 |
Shrimp feed | 10.3 | 46.7 | 2.8 | 11.1 | 1.3 | 27.8 |
For adult stages (1M–4M) | ||||||
Fish meal | 6.8 | 61.0 | 8.6 | 1.0 | 21.0 | 8.4 |
Poultry meal | 5.5 | 64.0 | 13.0 | 2.2 | 14.4 | 6.4 |
Wheat gluten meal | 9.7 | 46.2 | 1.3 | 2.5 | 0.7 | 49.3 |
Corn gluten meal | 8.6 | 62.0 | 1.6 | 3.4 | 1.6 | 31.4 |
Soybean meal | 12.0 | 46.0 | 6.2 | 6.2 | 6.3 | 35.3 |
Cassava meal | 12.0 | 2.0 | 0.3 | 1.7 | 1.5 | 94.5 |
Rice bran meal | 10.7 | 12.0 | 15.0 | 7.1 | 7.1 | 58.8 |
Broken rice meal | 12.0 | 6.6 | 1.6 | 0.7 | 0.3 | 90.8 |
Yeast protein meal | 9.1 | 42.0 | 0.6 | 0.6 | 4.6 | 51.1 |
Bacterial protein meal (Bio-pro 480) | 9.0 | 42.2 | 1.5 | 0.4 | 6.1 | 49.8 |
The ages in days after hatching of
Experimental periods of
Metamorphosis stage | Z1 | Z2 | Z3 | Z4 | Mp | C | 1M | 2M | 3M | 4M |
|
||||||||||
Day after hatching | 1 | 4 | 7 | 10 | 13 | 18 | 48 | 78 | 108 | 138 |
Growth in weight and carapace width of
Developmental levels of specific activity of different digestive enzymes and T/C ratio of
Specific activities of digestive enzymes, amylase (
The experimental period and consumption rate at each stage during a molting period of
Experimental periods and fresh feed consumption rate (% of total weight) of adult
Molting stage | A | B | C1 | C2 | D1 | D2 | D3 | D4 |
---|---|---|---|---|---|---|---|---|
Experimental day | 1 | 3 | 8 | 15 | 22 | 27 | 32 | 35 |
Consumption rate | — | 5 | 10–15 | 15–20 | 15–20 | 10 | 5 | 1-2 |
Total weight and specific activities of digestive enzymes, amylase (
The correlation coefficients among different parameters studied during development of
Correlation coefficient (
Parameters |
Weight | % weight gain* | Carapace width | % carapace width gain* | Trypsin | Chymotrypsin | T/C ratio | Total protease |
---|---|---|---|---|---|---|---|---|
Weight | 1 | |||||||
% weight gain* | 0.180 | 1 | ||||||
Carapace width |
|
0.288 | 1 | |||||
% carapace width gain* | 0.016 |
|
−0.009 | 1 | ||||
Trypsin |
|
0.119 |
|
−0.165 | 1 | |||
Chymotrypsin |
|
0.011 |
|
−0.242 |
|
1 | ||
T/C ratio | 0.014 |
|
0.045 |
|
−0.134 | −0.295 | 1 | |
Total protease |
|
|
|
0.075 |
|
|
0.225 | 1 |
Amylase |
|
0.288 |
|
0.017 |
|
|
0.059 |
|
The correlation coefficients among different parameters studied during a molting period of wild adult
Correlation coefficient (
Parameters | Weight | % weight gain | Carapace width | % carapace width gain | Trypsin | Chymotrypsin | T/C ratio | Total protease |
---|---|---|---|---|---|---|---|---|
Weight ( |
1 | |||||||
% weight gain ( |
0.189 | 1 | ||||||
Carapace width ( |
|
0.078 | 1 | |||||
% carapace width gain ( |
0.077 | 0.351 |
|
1 | ||||
Trypsin ( |
|
0.023 | 0.130 | −0.063 | 1 | |||
Chymotrypsin ( |
0.106 | 0.358 | 0.043 | −0.117 |
|
1 | ||
T/C ratio ( |
|
− |
0.138 | −0.159 | 0.030 | − |
1 | |
Total protease ( |
0.296 | −0.011 | 0.150 | 0.111 |
|
|
−0.124 | 1 |
Amylase ( |
0.266 | 0.036 | 0.238 | 0.152 | 0.347 | 0.070 | −0.073 |
|
Among the 10 raw materials studied, live
Among the 10 raw materials studied, fish meal is the best raw material followed by poultry meal (Figure
Among the digestive enzymes studied, only the specific activities of chymotrypsin (
Decreases in trypsin and chymotrypsin specific activities during larval development were observed in
Several authors [
The
Specific activities of α-amylase, total protease, trypsin, and chymotrypsin were associated with growth during metamorphosis. The T/C ratio was associated with growth rate (% gain) during metamorphosis. Trypsin specific activity and T/C ratio were related with growth during molting cycle. Trypsin and chymotrypsin specific activities associated with consumption rate with especially high levels during late intermolt and early premolt stages. Carapace width gain and T/C ratio were highest at first crab stage.
Cassava meal could be used in the feed formula for juvenile stage, and fish and poultry meals are beneficial for adult stage of
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was financially supported by Thailand Research Fund Master Research Grants in Science and Technology 2006 and National Research Council of Thailand 2009. The authors wish to thank Department of Aquatic Science, Faculty of Science, Burapha University, and Department of Biochemistry, Faculty of Science, Kasetsart University, for the use of their facilities.