Optimum Protein Requirements And Effects Of Chinese Herbs On The Non-Specific Immune Functions Of Juvenile Myxocyprinus Asiaticus

This study was conducted on juvenile Chinese sucker, Myxocyprinus asiaticus. In the first part, the dietary protein requirement of juvenile Chinese sucker was determined. In the second part, the effects of three traditional Chinese medicines (TCM) formulated from propolis and Herba Epimedii extracts, Radix Astragali and Glycyrrhiza uralensis extracts, Tangerine Peel extract respectively, on non-specific immune response of Chinese sucker were investigated. The results were as follows.(1) Growth performance and proximate composition of juvenile Chinese sucker fed the experimental diets containing different protein levels: Six fishmeal-based experimental diets containing various crude protein levels including 30%, 34%, 38%, 42%, 46% and 50% were fed to triplicate groups of 20 fish each (initial weight 13.5±1.1 g) for 56 days. Weight gain (WG) and specific growth rate (SGR) increased with increasing dietary protein level up to 46% (P < 0.05). Based on broken-line regression analysis of SGR against dietary protein levels the optimal dietary protein requirement for juvenile Chinese sucker was estimated to be close to 46.5%. Survival was not affected by dietary protein level (overall survival 71 - 90%). Feed conversion rate (FCR) generally showed a decline with higher protein levels (from 1.62 in 30% protein group to 1.13 in 50% protein group). Protein efficiency ratio (PER) showed gradual improvements with increasing dietary protein up to 46%. A similar trend was found for protein productive value (PPV). Among the proximate compositions of the whole body and muscle, crude protein contents all increased significantly with increasing dietary protein levels (P < 0.05). Conditon factor (CF), hepatosomatic index (HSI) and viscerosomatic index (VSI) generally showed an increase with higher protein levels. It was estimated that the optimal dietary protein requirement for juvenile Chinese sucker was close to 46.5%.(2) Effects of combination of propolis and Herba Epimedii extracts on the non-specific immune response and disease resistance of Chinese sucker: Propolis extract (Total flavone content: 37.1%) and Herba Epimedii extract (Total flavone content: 42.6%) was extracted using alcohol and mixed at the ratio of 3:1 (w/w). Fish were fed diets containing 0 (control), 0.1%, 0.5% or 1.0% TCM extracts for 50 days. Head-kidney and blood samples were collected at 10 d, 20 d, 30 d, 40 d and 50 d, respectively. Macrophages in head-kidney were separated by discontinuous density gradient centrifugation. The phagocytic activity of macrophage was measured using neutral red. The respiratory burst activity of macrophage was measured by flow cytometry (FCM). The lysozyme, bactericidal and natural haemolytic complement (ACH₅₀) activities in blood serum were measured using Micrococcus lysoleikticus, Aeromonas hydrophila and RaRBC, respectively. Results of this study showed that feeding the fish with 0.5% TCM extracts significantly increased the phagocytic activity after 40 days (P < 0.05). The enhanced phagocytic activity remained as compared to the control until the end of the experiment. Fish fed with 1.0% TCM extracts showed enhanced phagocytic activity on the 30 days compared to the control. The respiratory burst activity of macrophage showed a statistically significant increase on the 30 and 40 days in 0.5% dose group compared to the control (P < 0.05). A significant increasing of lysozyme activity was found after 10 days in group fed with 1.0% TCM extracts compared to the control (P < 0.05). Significant differences were also measured in 0.5% dose group on the 20 and 40 days and 0.1% dose group on the 40 days and the end of the experiment. Feeding the fish with 0.5% TCM extracts significantly increased the bactericidal activity after 40 days (P < 0.05). There were no significant changes in ACH₅₀ during feeding. After 50 days of feeding fish were challenged with A. hydrophila and cumulative mortality was registered during one week. A cumulative mortality over the experimental period in the control group was 75%. All dosage of treated groups showed reduced mortality compared to the control. Cumulative mortalities in fish fed with 0.1% and 1.0% TCM extracts reached 45% and 55%, respectively. Feed containing 0.5% TCM extracts was the most effective with the mortality of the fish significantly reduced by 35% compared to the control. The results indicate that propolis and Herba Epimedii extracts in combination enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila. The optimal dosage added to feed was 0.5% (w/w). (3) Effects of combination of Radix Astragali and Glycyrrhiza uralensis extracts on the non-specific immune response and disease resistance of Chinese sucker: Radix Astragali extract (Astragalus polysaccharides content: 38.4%) and Glycyrrhiza uralensis extract (Glycyrrhizic acid content: 31.2%; total flavone content: 20.5%) was extracted using alcohol and mixed at the ratio of 10:6 (w/w). Fish were fed diets containing 0 (control), 0.1%, 0.5% or 1.0% TCM extracts for 50 days. Head-kidney and blood samples were collected at 10 d, 20 d, 30 d, 40 d and 50 d, respectively. Macrophages in head-kidney were separated by discontinuous density gradient centrifugation. The phagocytic activity of macrophage was measured using neutral red. The respiratory burst activity of macrophage was measured by FCM. The lysozyme, bactericidal and ACH₅₀ activities in blood serum were measured using M. lysoleikticus, A. hydrophila and RaRBC, respectively. Results of this study showed that the phagocytic activity of macrophage showed a statistically significant increase on the 30 and 40 days in 1.0% dose group, 40 days in 0.5% dose group and 50 days in 0.1% dose group compared to the control (P < 0.05). Feeding the fish with 1.0% TCM extracts significantly increased the respiratory burst activity of macrophage after 40 days. The enhanced activity remained as compared to the control until the end of the experiment (P < 0.05). A significant increasing was also found on the 20 and 40 days in 0.5% dose group compared to the control. Significant differences of lysozyme activity was measured in 1.0% dose group on the 20 and 50 days, 0.5% dose group on the 30 days and 0.1% dose group on the 40 days compared to the control (P < 0.05). Feeding the fish with 0.5% TCM extracts significantly increased the bactericidal activity after 40 days until the end of the experiment (P < 0.05). A significant increasing was also found on the 30 and 40 days in group fed with 1.0% TCM extracts compared to the control. The ACH₅₀ activity showed a statistically significant increase on the 40 and 50 days in 1.0% dose group, 30 days in 0.5% dose group and 50 days in 0.1% dose group compared to the control (P < 0.05). After 50 days of feeding fish were challenged with A. hydrophila and cumulative mortality was registered during one week. A cumulative mortality over the experimental period in the control group was 75%. All dosage of treated groups showed reduced mortality compared to the control. Cumulative mortalities in fish fed with 0.1% and 0.5% TCM extracts reached 60% and 45%, respectively. Feed containing 1.0% TCM extracts was the most effective with the mortality of the fish significantly reduced by 40% compared to the control (P < 0.05). The results indicate that Radix Astragali and Glycyrrhiza uralensis extracts in combination enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila. The optimal dosage added to feed was 1.0% (w/w).(4) Effects of Tangerine Peel extract on the non-specific immune response and disease resistance of Chinese sucker: Tangerine Peel extract (Total flavone content: 3.3%; Hesperidin content: 1.6%) was extracted using alcohol. Fish were fed diets containing 0 (control), 0.1%, 0.5% or 1.0% Tangerine Peel extract for 50 days. Head-kidney and blood samples were collected at 10 d, 20 d, 30 d, 40 d and 50 d, respectively. Macrophages in head-kidney were separated by discontinuous density gradient centrifugation. The phagocytic activity of macrophage was measured using neutral red. The respiratory burst activity of macrophage was measured by FCM. The lysozyme, bactericidal and ACH₅₀ activities in blood serum were measured using M. lysoleikticus, A. hydrophila and RaRBC, respectively. Results of this study showed that the phagocytic activity of macrophage had a statistically significant increase on the 50 days in 1.0% dose group compared to the control (P < 0.05). Significant differences of respiratory burst activity was measured in 1.0% dose group on the 40 days and 0.5% dose group on the 30 days compared to the control (P < 0.05). Feeding the fish with 1.0% Tangerine Peel extract significantly increased the lysozyme activity in blood serum after 20 days (P < 0.05). There were no significant changes in bactericidal and ACH₅₀ activities comparing to the control during the whole experiment. After 50 days of feeding fish were challenged with A. hydrophila and cumulative mortality was registered during one week. A cumulative mortality over the experimental period in the control group was 75%. Cumulative mortalities in fish fed with 0.1%, 0.5% and 1.0% Tangerine Peel extract reached 65%, 55% and 50%, respectively. The results indicate that Tangerine Peel extract enhances the non-specific immune response and disease resistance of Chinese sucker against A. hydrophila.(5) Effects of TCM extracts on respiratory burst activity of head-kidney macrophage of Chinese sucker in vitro: Macrophages in head-kidney of Chinese sucker were separated and purified by discontinuous density gradient centrifugation. Then head-kidney macrophages were incubated in a 96-well plate with different doses of TCM extracts (combination of propolis and Herba Epimedii extracts, combination of Radix Astragali and Glycyrrhiza uralensis extracts), 0, 25, 50, 100, 200 and 400μg/mL, respectively, for 24 h. The respiratory burst activity of macrophage was measured by nitroblue tetrazolium (NBT). Significant differences of respiratory burst activity was found in combination of propolis and Herba Epimedii extracts with doses of 25, 50 and 100μg/mL and combination of Radix Astragali and Glycyrrhiza uralensis extracts with doses of 100 and 200μg/mL compared to the control (P < 0.05). The results indicate that combination of propolis and Herba Epimedii extracts and combination of Radix Astragali and Glycyrrhiza uralensis extracts could modulate the immune response of Chinese sucker in vitro.