| The first study was conducted to evaluate the effects of various weights on the digestive enzyme and intestinal brush border enzyme activities in Jian carp. Based on the first study, other studies of the effects of Trp on production performance, nutrient deposition, amino acid metabolism, digestion and absorption capacity, immunity and protein synthesis regulatory factor TOR expression in Jian carp were carried out. Using fish primary cell culture, we investigated the effects of Trp on the proliferation and differentiation, protein synthesis, and TOR expression of intestinal cell of Jian carp. The main researches and results are as follows:1. The effects of various weights on digestive enzyme activity and intestinal brush border enzyme activities in Jian carpA total of 750 Jian carp (initial mean weight,18.0±0.2g) were randomly distributed into five groups of each three replicates, feeding the same diet, for the 0,14th day,28th day, 42nd day and 56th day sampling, to explore the effects of various weights on digestive enzyme and intestinal brush border enzyme activities in Jian carp. The results indicated that:The hepatopancreas and intestinal protein content, digestive enzyme activity was great significantly (P<0.01) or significantly (P<0.05) increased with the increasing levels of body weights from 18.0g to 74.7g. When Jian carp weighed 74.7g, the hepatopancreas and intestinal protein content were significantly higher than the other treatments (P<0.05), hepatopancreas trypsin and chymotrypsin activities, hepatopancreas and intestinal amylase activities were great significantly higher than the other treatments (P<0.01). When the body weights levels up to 58.6,40.1,58.6 and 58.6g, the hepatopancreas lipase activity, intestinal trypsin, chymotrypsin and lipase activities were all significantly increased, respectively (P<0.05), and remained nearly constant thereafter (P>0.05). The effects of the body weights of juvenile Jian carp on the intestinal brush border enzyme activity also showed great significantly (P<0.01) or significantly (P<0.05) increased. When Jian carp weighed to 74.7g, the distal intestinal Na+, K+-ATPase activity, proximal, mid and distal intestinal alkaline phosphatase (AKP), and the proximal y-glutathione transaminase (y-GT) activities were great significantly higher than other groups (P<0.01), the y-GT activity in mid intestine was significantly higher than the other groups (P<0.05). The intestinal creatine kinase (CK) activity, proximal and mid intestinal Na+, K+-ATPase and distal intestinal y-GT activities were all increased with the increasing weights levels up to 58.6g (P<0.05), and no differences were found with further increase of zinc levels (P>0.05). The results indicated that following the body weight of Jian carp from 20 g to 80 g, the increased secretary capacity of hepatopancreas to secret trypsin, chymotrypsin, lipase and amylase, was due to the growth and development of the hepatopancreas; the improvement of intestinal absorption capacity was due to the growth and the development of the intestine.2. Effects of Trp on the growth performance, body composition and the GPT and GOT activities in serum, hepatopancreas and muscle of the juvenile Jian carpA total of 1050 Jian carp (7.73±0.03g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60% and 0.69%) for 60 days to investigate the effects of Trp on growth performance, body deposition and the GPT and GOT activities in serum, hepatopancreas and muscle. The results showed that the growth performance, body deposition, the GPT and GOT activities in serum, hepatopancreas and muscle and plasma ammonia levels were great significantly (P<0.01) or significantly (P<0.05) affected by Trp. When the Trp levels increased from 0.11% to 0.38%, the specific growth rate (SGR), feed intake, body protein content, protein production value (PPV), protein efficiency ratio, body lipid content, lipid production value (LPV), ash production value (APV), hepatopancreas and the activities of GOT and GPT in muscle were great significantly (P<0.01) or significantly (P<0.05) increased, the maximum value was on 0.38% Trp, and with further increase of Trp levels, were great significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.38%, the FCR, body water content, body ash content, ammonia content, serum GOT and GPT activities were great significantly (P<0.01) or significantly (P<0.05) decreased, and the results of 0.38% Trp were the minimum, and with further increase of Trp levels, were great significantly (P<0.01) or significantly (P<0.05) increased. Further correlation analysis showed that there were great significantly (P<0.01) or significantly (P<0.05) positive correlation between the percent weight gain and the protein, lipid and ash production value, and feed intake; between the FCR and the weight gain, protein and lipid production value, there showed great significant negative linear correlation (P<0.01). Results indicated that Trp could promote the growth of juvenile Jian carp, improve body protein and fat deposition ability, and enhance the amino acid metabolism of the hepatopancreas and muscle. Based on the percent weight gain, the requirement of Trp in juvenile Jian carp (7.73-32.67g) was 0.35% or 1.09/100g protein3 Effects of Trp on the growth and development of fish digestive organs, digestive enzyme and intestinal brush border enzyme activities of the juvenile Jian carpA total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%, 0.17%,0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days. The results showed that, when the Trp levels increased from 0.11% to 0.38%, the hepatopancreas weight, hepatosomatic index, hepatopancreas protein content, intestinal weight, intestinal length, intestinal protein content, intestosomatic index and intestinal length index and proximal, mid and distal intestinal folds height, hepatopancreas and intestinal trypsin, lipase and amylase activities, fintestinal creatine kinase activity, proximal, mid and distal intestinal alkaline phosphatase (AKP), Na+, K+-ATPase enzyme and y-GT enzyme activities were great significantly (P<0.01) or significantly (P<0.05) increased, the highest value was on 0.38% Trp, and thereafter, were great significantly (P<0.01) or significantly (P<0.05) decreased. Intestinal chymotrypsin activity was the highest at levels of 0.25% Trp (P<0.05). There was no effect on hepatopancreas chymotrypsin activity (P>0.05). Further correlation analysis showed that there were great significantly (P<0.01) or significantly (P<0.05) positive correlation between the PPV and the intestinal trypsin, hepatopancreas trypsin, and the activities of GOT and GPT in hepatopancreas and muscle. Meanwhile, there were significant positive linear correlation between the LPV and the hepatopancreas lipase (P<0.05). Finally, between the FCR and the hepatopancreas trypsin and lipase, intestinal trypsin and chymotrypsin, proximal and distal intestinal Na+, K+-ATPase, proximal, mid and distal intestinal AKP and y-GT, CK activities, it was showed great significantly (P<0.01) or significantly (P<0.05) linear negative correlation. These results indicated that Trp could improve digestion and absorption capacity of Jian carp. The improvement of digestion and absorption capacity of fish by Trp were due to the enhanced growth and development of hepatopancreas and intestinal, improving the protease, lipase and amylase secretion ability, increasing intestinal absorption areas and the intestinal absorption ability.4. Effects of Trp on the intestinal microflora, disease resistance, specific immunity and non-specific immunity in the juvenile Jian carpA total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days to measure intestinal microflora, red and white blood cell counts and immune organ weights. After the growth experiment, fish was injected with Aeromonas hydrophila for 17 days to examine the effects of Trp on the disease resistance and immune function of Jian carp. The results showed that, followed the Trp levels increased from 0.11% to 0.25%, intestinal lactic acid bacteria was significantly{P<0.01) or significantly (P<0.05) increased, there was no significant difference(P> 0.05) between 0.25% group and 0.38% group, and thereafter, it was great significantly (P<0.01) or significantly (P<0.05) decreased. Intestinal E. coli showed the opposite model. There was no significant effect on intestinal Aeromonas hydrophila (P>0.05). Trp was great significantly (P<0.01) or significantly (P<0.05) increased head kidney, behind kidney, spleen weights and the indexes, when the Trp level of 0.38%, these values reached the maximum, and thereafter, the head kidney, behind kidney, spleen weights and the indexes was great significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.38%, the red blood cells and white blood cell counts were great significantly (P<0.01) or significantly (P<0.05) increased,, when the Trp level of 0.38%, these values reached the maximum, and then were significantly (P<0.01) or significantly (P<0.05) decreased. When the Trp levels increased from 0.11% to 0.25%, the survival rate of post-infection was great significantly increased (P<0.01),0.25%-0.49% was on the plateaued (P>0.05), and then decreased significantly (P<0.05). When the Trp levels increased from 0.11% to 0.49%, the serum IgM was great significantly (P<0.01) or significantly (P<0.05) increased, and then great significantly decreased (P<0.01). Serum anti-Aeromonas hydrophila titre was increased with the increase Trp levels up to 0.38%, and then decreased gradually (P<0.05). When the Trp levels increased from 0.11% to 0.38%, the phagocytic activity (PA) of leucocytes, serum complement C3, C4, lysozyme activity and acid phosphatase enzyme activity, and total iron-binding capacity were great significantly (P<0.01) or significant (P<0.05) increased, and then were great significantly (P<0.01) or significantly (P<0.05) reduced, however, the serum haemagglutination titre had no significant effect (P>0.05). Correlation analysis found that between the survival rate of carp after injection and the immune organ weights, antibody titer, leukocyte phagocytosis rate, ACP, C3, C4, and total iron binding capacity, it showed great significantly (P<0.01) or significantly (P<0.05) linear positive correlation. The relationship between the phagocytic activity (PA) of leucocytes and the red and white cell counts, and complement C3, C4 levels was great significantly (P<0.01) or significantly (P<0.05) positive linear correlation; the red and white cell counts and the immune organ weights showed great significantly (P<0.01) or significantly (P<0.05) linear correlation. These results indicated that Trp could enhance disease resistance and improve intestinal micro-ecological balance in Jian carp. The enhanced resist disease was related to the improvement of the specific immune and non-specific immunity in Jian carp. The improvement of non-specific immunity was related to the enhanced the ability of chemotaxis, adherence, regulation, sterilization and bacteriostasis by Trp in fish.5. Effects of Trp on intestinal and muscle TOR mRNA expression in the juvenile Jian carpA total of 1050 Jian carp (7.73±0.03 g) were randomly distributed into seven groups of each three replicates, feeding diets containing graded levels of Trp (0.11%,0.17%, 0.25%,0.38%,0.49%,0.60%, and 0.69%) for 60 days to study the effects of Trp on interstinal and muscle TOR mRNA expression. The results showed that the expression of proximal and mid intestinal TOR mRNA was decreased with the increase of tryptophan levels, when the Trp levels reached 0.49% and 0.38%, the values were the minimum, respectively, and then increased gradually (P<0.05). When the Trp levels increased from 0.11% to 0.17%, the expression of the distal intestinal TOR mRNA was also significantly decreased (P<0.05), when Trp levels of 0.17% and 0.25%, the mRNA expression was the lowest, and followed by a gradual increase to the highest with the further increase Trp levels (P<0.05). The mRNA expression of muscle TOR was the lowest in the levels of Trp on 0.38% and 0.49%(P<0.05), low levels (0.11%) and high levels (0.60% and 0.69%) of Trp got the highest TOR mRNA expression (P<0.05). These results indicated that the dietary Trp deficiency or excessive improved fish proximal, mid and distal intestinal and muscle TOR mRNA expression.6. Effects of Trp on the proliferation and differentiation of Jian carp intestinal epithelial cell (IEC)The present study explored the effects of Trp on the proliferation and differentiation of carp intestinal epithelial cell (IEC). IEC coming from 80g carp were treated with Trp (0, 16,36,66,96, and 126mg/L Trp) with 4 replicates of each treatment for 96h. The results showed that with the increase Trp levels up to 36mg/L-66mg/L, the IEC counts were increased significantly, and formed a large colony and into the monolayer film, and with the further increase Trp levels, the cell counts were decreased. When the Trp levels increased from 0 to 36mg/L, the MTTOD of IEC was significantly increased (P<0.05), 36mg/L and 66mg/L Trp had no significant difference effect on MTT OD (P> 0.05), and declined thereafter (P<0.05). The protein content, GOT, GOT, AKP, Na+, K+-ATPase activities of IEC were elevated with increase Trp levels up to 36,36,66,36 and 16mg/L, respectively (P<0.05), and then decreased gradually thereafter (P<0.05). The ammonia content and lactate dehydrogenase activity of the culture medium were decreased with elevated Trp levels up to 96 and 66mg/L (P<0.05), and then decreased gradually thereafter (P<0.05). These results indicated that Trp could promote the proliferation and differentiation, and ensure the function of IEC.7. Effect of Trp on the protein synthesis and TOR mRNA expression of IEC in the juvenile Jian carpThis study was carried out three small trials. (1) IEC were treated with two levels of Trp (0mg/L and 36mg/L Trp) with 8 replicates of each treatment for 240min to research the effects of Trp on the protein synthesis of IEC. (2) IEC were treated with 5 levels of time (15min,30min, 100min,170min and 240min) with 6 replicates of each treatment to study the effects of time on the TOR mRNA expression of IEC using 36mg/L Trp DMEM. (3) IEC were treated with two levels of Trp (0mg/L and 36mg/L Trp) with 8 replicates of each treatment for 100min to research the effects of Trp on TOR expression of IEC. The results showed that the protein synthesis rate for 36mg/L Trp group was significantly higher than the control group which without added Trp (Omg/LTrp) (P<0.05). The second trail found that the TOR mRNA expression level on 30min was significantly lower than 15min (P<0.05), and after 30min, it followed by a gradual increase up to the highest on the 170min and 240min (P<0.05). The third trail found that the TOR mRNA expression for 36mg/L Trp group was significantly higher than the control group which without added Trp (Omg/L Trp) (P<0.05). These results indicated that Trp could promote protein synthesis and enhance the TOR mRNA expression of IEC.In summary, following the body weight of Jian carp from 20 g to 80 g, the increased secretary capacity of hepatopancreas to secret trypsin, chymotrypsin, lipase and amylase, was due to the growth and development of the hepatopancreas; the improvement of intestinal absorption capacity was due to the growth and the development of the intestine. Trp could promote the growth of juvenile Jian carp, improve body protein and fat deposition ability, and enhance the amino acid metabolism of the hepatopancreas and muscle. Trp could improve digestion and absorption capacity of Jian carp. The improvement of digestion and absorption capacity of fish by Trp were due to the enhanced growth and development of hepatopancreas and intestinal, improving the protease, lipase and amylase secretion ability, increasing intestinal absorption areas and the intestinal absorption ability. The dietary Trp deficiency or excessive improved the fish proximal, mid and distal intestinal and muscle TOR mRNA expression. Trp could promote the proliferation and differentiation, and ensure the function of IEC. Trp could promote protein synthesis and enhance the TOR mRNA expression of IEC. Trp could enhance disease resistance and improve intestinal micro-ecological balance in Jian carp. The enhanced resist disease was related to the improvement of the specific immune and non-specific immunity in Jian carp. The improvement of non-specific immunity was related to the enhanced the ability of chemotaxis, adherence, regulation, sterilization and bacteriostasis by Trp in fish. Based on the percent weight gain, the requirement of Trp in juvenile Jian carp (7.73-32.67g) was 0.35% or 1.09/100g protein. |
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