Effects of hydroxyl methionine chelated copper, zinc, iron and manganese on growth and immunity of Red Swamp Crayfish
Procambarus Clarkii, Red Swamp Crayfish also known as freshwater crayfish is the most popular cultured freshwater crayfish species in the world. It is native to North America and introduced via Japan to China in 1929. Through advancement of breeding technology, it has evolved and became one of the major freshwater culture species resources in China having footprints in more than 20 provinces, and including Taiwan. Coupled with the advancement of "mix farming technology in rice paddy field" which is gaining very good traction in farm communities, freshwater crayfish culture practices have been growing very rapidly. Its value chain has been expanding enormously, driving the cultured unit productivity and total acreage growth in China.
Hydroxyl Methionine chelated copper, zinc, iron and manganese are new types of organic trace elements with the essential amino acid methionine as ligand. Their characteristics as additives include being safe, having high bioavailability, good stability, good palatability, minimal detrimental effects on other nutrients in the feed like vitamins and less lost within the culturing water mass. They are very effective in replenishing the methionine storage pool within the animal; promoting growth rate in aquaculture practices, improving feed efficiency, enhancing shell pigmentation, and boosting immunity and livability.
This experiment is to study the effects on growth performances and immunity of supplementation of Hydroxyl methionine chelated copper, zinc, iron and manganese to Red Swamp Crayfish diets.
1. Materials and Methods
1.1 Experimental materials
The Red Swamp Crayfish (Procambarus Clarkii) used in the experiment was from Hunan Huxin Aquaculture Technology Co., Ltd., and the hydroxyl chelated organic trace elements and other trace elements were provided by Changsha Xingjia Bio-Engineering Co., Ltd.
1.2 Experimental design
The purchased Red Swamp Crayfish juveniles (Procambarus Clarkii) were kept in the 30m3 cement pool for 2 weeks for reconditioning. 360 healthy juvenile crayfish with average body weight of circa 7.5g were selected. They were randomly allocated to two treatments of 6 replicates each. 30 juvenile crayfish were raised in each of the replicate aquariums having dimensions of 80cm x 60cm x 75cm.
1.3 Experimental diets
Experimental diets were custom-made using imported fishmeal and soybean meal as the main protein source and soybean oil as the source of lipid, to form a basal diet (crude protein: 31.8%, crude fat: 5.32%, lysine: 1.55%, crude fibre: 9.57%, total phosphorus: 0.98%). Please refer to following tables for detailed trace mineral supplementation compositions:
Table 1a: Experimental diets treatment description
Table 1b: Supplementation levels of trace minerals in mineral premix
1.4 Experimental execution
The trial started after seven days of acclimatisation in the experimental aquarium. Crayfish were fed with experimental diets twice a day (08:30, 16:00) at 3-5% of body weight. 30% and 70% of daily ration (adjusted as needed per growth and feeding situation) were fed in the morning and afternoon respectively.
The feeding amount guideline was that all feed had to be consumed within 1.5 hours. Water was filled to a tank height of 15-20 cm (maintained at temperature at 26±4℃) with some branches and cans being used as shelters or strongholds. Reservoir water was used to replace about 1/3 ～1/2 of the tank daily while providing 24-hour aeration. Excreta and feed residual were siphoned out regularly. The trial period was 40 days.
1.5 Sampling and procedure
1.5.1 Growth performances
When the trial period ended, all crayfish were fasted for 24 hours, then weighed to analyse feed intake, weight gain ratio, feed conversion coefficient, and livability.
Calculation of the parameters was as follows:
Weight gain rate (%) = [(final total weight (g) - initial total weight (g) + dead crayfish weight (g)) / initial total weight (g)] × 100
Feed conversion coefficient = total feeding amount (g) / (final total weight (g) - initial total weight (g) + dead crayfish weight (g))
Survival rate (%) = (number of crayfish at the end of the experiment / number of crayfish at the start of the experiment) × 100
1.5.2 Determination of blood biochemical indicators
10 crayfish were selected randomly from each group and filter paper was used to handle the dry crayfish body. Blood samples were taken using 1 ml disposable syringes using carapace heart puncture technique. Samples were then stored in 4°C refrigeration overnight. Supernatant from cold centrifuge of 5000 r / min for 10 min was collected and stored under -21°C for lysozyme, ceruloplasmin, alkaline phosphatase and total protein level determination.
1.6 Data Analysis
The experiment data were analysed by SPSS statistics16.0 statistical software. One-way ANOVA was employed and experimental data were expressed as mean ± standard deviation.
2. Results and Analysis
2.1 Effects of Hydroxyl Methionine chelated Copper, Zinc, Iron and Manganese on growth and immunity of Red Swamp Crayfish
Results in Table 2 shows that weight gain, survival rate and feed conversion coefficient of Group II (hydroxyl methionine chelate group) are significantly (P < 0.05) better than Group I (the inorganic group). The respective improvements are 19.79 %, 9.65% and 9.93%.
Table 2: Effects of Hydroxyl Methionine chelated Copper, Zinc, Iron and Manganese on growth and immunity of Red Swamp Crayfish
2.2 Effects of Hydroxyl Methionine chelated Copper, Zinc, Iron and Manganese on plasma biochemistry of Red Swamp Crayfish
Results in Table 3 show that ceruloplasmin and lysozyme contents of crayfish were significantly (P<0.05) increased in Group II when compared to Group I. Total protein concentration and phosphatase levels of Group II showed improvements but were not statistically significant (P>0.05).
Table 3: Effects of Hydroxyl Methionine Chelated Copper, Zinc, Iron and Manganese on plasma biochemistry of Red Swamp Crayfish
3. Results and Discussion
Although there have been many studies of chelated trace minerals in poultry and livestock applications, such studies in aquaculture were quite limited. To date such application study on Red Swamp Crayfish cannot be found in the literature. The results from this study show that supplementation of Hydroxyl methionine chelated copper, zinc, iron and manganese in Red Swamp Crayfish diets can significantly (P<0.05) improve weight gain, feed conversion efficiency and livability. It was also observed that treatments fed with Methionine chelated minerals have better colour pigmentation and better vitality behaviour compared with treatments fed with inorganic trace mineral only. This finding echoed with Kumar Katya's (2016) study on shrimp (Litopenaeus vannamei).
Better growth performance results yielded from the treatment group fed Methionine trace minerals could have been contributed from better uptake of amino acid chelates and its better metabolism; when compared to inorganic trace minerals, more prominent antagonistic effects might be seen.
Changes in plasma parameters can be good indicators to reflect metabolism, nutritional and health status of crayfish. Total protein concentration reflects crayfish nutritional and metabolism status, and also indirectly reflects the organism's immune status.
Comparing to Group I fed inorganic trace minerals, the total plasma protein concentration level from Group II (hydroxyl methionine chelates) tend to have higher value. Further studies could be conducted to find out how Methionine chelated trace minerals can contribute to beneficial protein metabolism and growth improvement.
Ceruloplasmin is a copper-containing protein in serum having oxidase functions and acts as an antioxidant clearing free radicals. Lysozyme can dissolve muco-polysaccharides within the bacteria cell membrane, hydrolyse acetyl amino-polysaccharides in Gram-positive bacteria, thereby destroying and eliminating foreign organisms especially pathogens entering the body system. Lysozyme is one of the most important non-specific immunity factors of an organism. Alkaline phosphatase is an important component of the crustacean's defence system, and a critical component of the phagocytosis process against foreign or pathogenic organisms, and thus it plays an important role in the immune system of crustaceans.
Results in this trial showed that hydroxyl methionine chelated trace minerals can significantly (P<0.05) improve Red Swamp Crayfish's plasma ceruloplasmin content and lysozyme activity. We believe that these improvements would boost the immune system of crayfish and eventually be reflected in significantly better livability of 9.65%.
The supplementation of hydroxyl methionine copper, zinc, iron and manganese to Red Swamp crayfish (Procambarus Clarkii) diets can significantly improve production performance. Comparing to diets supplemented with inorganic trace minerals, the methionine chelated trace minerals fed group showed significantly improved growth, FCR and livability by more than 19%, 9% and 9% respectively. The group also showed significantly improved parameters relating to nutritional and immune status like plasma ceruloplasmin and lysozyme content by 20% and 7 % respectively.
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Article made possible through the contribution of Xingjia Bio-Engineering