1. High density liver improvement For Aquaculture LiverCall GCG TM - product description

2. ▣ Culture environment of aquaculture fish In order to culture fish within limited period, the cultured fish usually are fed feeds containing more nutrients than they required and fish are cultured in high density in limited space, therefore arise the unbalanced metabolism and give fish many artificial stress. Because of these problems in aquaculture, liver is the most affected intestines and most of the parts in liver are wound or the metabolism can't be proceeded smoothly, then arise nutritional diseases or are easy to be infected by bacteria or virus because their immune resistance are depressed.

3. ▣ Function of liver (1) The center of metabolism  Liver is the place for metabolism of carbohydrate, protein and lipid  Liver has the important metabolism function of protein synthesis.  If the liver is wound or its function is depressed - the balance of body materials metabolism will be destroyed - Arise nutritional diseases, the normal metabolism could not be maintained. (2) Maintaining the balance of body hormones  Hormones are secreted by the endocrine organs.  If the disease of liver is serious, the secretion of hormones will be unbalanced and arise many problems of body functions.  If the liver function is depressed, such secretion of hormones will become unbalanced and decrease the growth of fish.

4. (3) Antidotal function  Liver is the organ that all medicines absorbed in fish body (antibiotic, formalin etc.) are transferred to other forms and the organ that could secrete ammonia produced from protein metabolism.  Specially in summer when water temperature is high, or fish are cultured in high density or fish were over fed, the concentration of ammonia in blood is easy to become high to be toxic to fish.  If the concentration of ammonia continuously accumulate, fish could become comatose or even die. (4) Immune function  Liver has the function of storing antibody to against abnormal materials.  However, if the function of liver is depressed, the immune resistance ability is also depressed, and the fish is infected by bacteria and virus.  In order to increase immune resistance ability, use the additives to improve its own immune resistance ability through improvement of liver function.

5. (1)LiverCall GCG could completely replace the existed liver improvement - glutathione. LiverCall GCG contains several kinds of amino acids, minerals and immune stimulants. (2) LiverCall GCG could improve the liver function, release stress and increase disease resistance through increase of immune resistance. (3) LiverCall GCG could greatly improve feed intake as feed additive to improve liver function, almost has no adverse effect and has high absorption. ▣ Specific character of LiverCall GCG

6. ▣ Merits of LiverCall GCG (1) Improve feed intake, growth and feed efficiency (2) Improve metabolism of protein and amino acids (3) Improve the synthesis of glutathione and glutathione peroxidase. (4) Prevent fat liver and improve antioxidation ability (protect body against oxygen free radical and peroxide) (5) Release stress (6) Strengthen disease resistance through improve immune resistance (7) Improve secretion of liver ammonia, heavy metal and antibiotic (8) Colorless through reducing of melanin color

7. ▣ The composition of LiverCall GCG (1) GCG could completely replace glutathione, could improve synthesis of glutathione, strengthen liver function and counteracting poison function, could improve body metabolism as a strong reducing agent (As the improvement of liver function, LiverCall GCG is in the application processing of patent) (2) Several kinds of essential nutrients : containing 18 kinds of essential and non-essential free amino acids, could improve growth and feed efficiency through increasing of feed intake, could increase disease resistance through increasing of non-specific immune resistance.

8. (1) Improve growth 1) Experimental method  Test animal : juvenile flounder flatfish (average weight 22.5 ± 0.1 g)  Test condition : test period 6 weeks  Experimental design The basal diet was used as control (Cont), GCG (liver improvement material of Liver Call) and GSH (glutathione) were supplemented in diets as 10 and 20 mg/kg fish/day (Cont, GCG10, GCG20, GSH10 and GSH20). ▣ Function of LiverCall GCG proved through experiment Figure 1. Weight gain of fish fed diets containing GCG and GSH for 6 weeks

9. (1) Improve liver function 1) Experimental method  Test animal : juvenile flounder flatfish (average weight 22.5 ± 0.1 g)  Test condition : test period 10 days  Experimental design The basal diet was used as control (Cont), GCG (liver function improvement material of Liver Call) and GSH (glutathione) were supplemented in diets as 10 and 20 mg/kg fish/day (Cont, GCG10, GCG20, GSH10 and GSH20). ▣ Function of LiverCall GCG proved through experiment 2) Experimental result Diets ContGCG10GCG20GSH10GSH20 GOT (IU/L) 56a45b42b43b47b GPT (IU/L) 13a8b9b13a12a Liver glutathione (micromole/g) 2.8b4.2a3.9a3.8a4.1a Liver glutathione peroxidase (micromole/(minute×mg protein)) 20b26a29a27a30a Liver antioxidation rate (%) 25b43a41a42a44a

10. Figure 2. Values of liver glutathione and glutathione peroxidase fed diets containing GCG and GSH for 10 days.

11. (2) Non-specific immune response 1) Experimental method  Test animal : juvenile flounder flatfish (average weight 22.5 ± 0.1 g)  Test condition : test period 10 days  Experimental design The basal diet was used as control (Cont), GCG (liver function improvement material of Liver Call) and GSH (glutathione) were supplemented in diets as 10 and 20 mg/kg fish/day (Cont, GCG10, GCG20, GSH10 and GSH20). 2) Experimental result Diets ContGCG10GCG20GSH10GSH20 Peak value of CL (mV) 446b631a617a472b469b Lysozyme activity (U/ml) 323b425a417a341b352b

12. (3) Stress response 1) Experimental method  Test animal : juvenile flounder flatfish (average weight 22.5 ± 0.1 g)  Experimental design After formalin bath, blood cortisol returned to normal level, and blood cortisol level of fish fed GCG and GSH returned to normal level after 6 hours of formalin bath. ▣ Function of LiverCall GCG proved through experiment 2) Experimental result Figure 3. Stress responses during 48 hours after formalin bath

13. Figure 4. Non-specific immune response of fish fed diets containing GCG and GSH for 10 days.

14. ▣ Discussion (1) Improve liver function  When liver cells are mysterious death or are infected - GOT and GPT could flow out to blood in high concentration - GOT and GPT will become high in blood quickly  GCG (improve liver function material in Liver Call) could help the metabolism of protein and amino acids in liver more smoothly than glutathione (GSH), and improve the function of liver.

15.  Glutathione (GSH) - Existed in all animals in nature and yeast - The important function in liver to prevent the lipid accumulation and the formation of fat liver and peroxides. - GSH are absorbed through the effect of digestible enzymes from each amino acids, it can't be absorbed as the GSH form. - After releasing from each amino acids, GSH becomes part of active, and SH radical of GSH has the effect to improve liver function.  From the above mentioned experimental results, it is obvious that the supplementation of GCG and GSH could improve the synthesis of glutathione.

16.  The supplementation of GCG and GSH - Improve the synthesis the glutathione peroxidase - Could improve the total antioxidation activity through glutathione and glutathione peroxidase  With the oxidation of accumulated lipid in liver - The toxic of produced peroxides become serious and destroy the liver of cultured fish - The glutathione peroxidase could prevent the formation of such peroxides and could be considered as an improtant enzyme in liver function

17. (2) Non-specific immune response  The function of liver is depressed - Immune resistance ability is also depress - The fish is easy to be infected by bacteria, parasite and virus  Non-specific immune resistance with the supplementation of GCG - The activities of Chemiluminescence (CL) and plasma lysozyme increased - Thus the disease resistance is also increased from the above mentioned experiments

18. (3) Stress response  The blood cortisol concentration - To be stressed, the cortisol level will be increased - Stress are released, the cortisol will recover to normal level again  After the formalin bath, the plasma cortisol levels returned to normal levels within 6 ∼ 12 hours in fish supplemented with GCG and GSH, and within 24 ∼ 48 hours in fish from control group.  GCG and GSH have strong effects on releasing stress.

19.  There is no difference in GCG (improve liver function material in Liver Call) and glutathione in liver function improvement.  Compared with the existed glutathione product (containing 2% of glutathione), the concentration of GCG in LiverCall GCG is high (containing 6% of GCG), similar results could be obtained even using 1/3 concentration of Liver Call.  It is a big burden for fish farmers to use glutathione due to its high price, however, only 1/3 of expense is possible if using Liver Call that could get the similar results in improving liver function and it is possible to use for long term. ▣ Economical merit of LiverCall GCG

20. ▣ How to use LiverCall GCG effectively Efficacy Medication amount (g/ton fish weight) Medication periodRemarks Improve liver function150-300g1-2 weeks Possible to extend or shorten medication period according to the status of fish Counteracting medicine poison and medicine bath 150-300g1-2 weeks When culture fish in high water temperature and high density 150-300g Medication biweekly Prevent liver disease 50-100g Continuously medication