Studies On Distribution Of Digestive Enzymes, Nutrients Requirement And Physiological Responses To High Dietary Lipid Of Brachymystax Lenok Larvae

The purposes of this study were to investigate the structure of digestive tract and distribution of digestive enzymes,requirements of protein and lipid,regulating mechanism of pancreatic enzymes and responses of antioxidant enzymes to high dietary lipid levels in Brachymystax lenok larvae.The study composed of five trials,the description of results and discussion as follows:In Exp.1,the histological structure of stomach,intestine were observed with larvae post-hatch for 40d feeding living organism.Histological results showed that intact structure of intestinal villus has formed for digestive nutrients and extended into lumen to enhance efficiency for digestion and absorption through sufficient contact with thymus.Posterior segments of intestinal become thinker related with increasing movement of ehymus and formation of feces.Histochemical distribution of digestive enzymes revealed that there have been enzymes activities,including acid phosphatase, alkaline phosphatase,non-specific esterase,esterase and phopholipidase A2,along with intestine. The activities of digestive enzymes were mainly distributed in anterior segments and play a crucial role in digestion of nutrients,but that of posterior segments were weaker compared with anterior parts.Presence of acid phosphatase implied that endogenous digestion also play an important role of digestion in larvae.In trial 2,brush border membrane vesicle was prepared from 60d,mean weight 350±50 mg/fish and chloride magnesium as a precipitator in homogenate.Different centrifuging method was used to separate BBM,the centrifuged velocity maintained at 28,000 and 43,000g,respectively. Marker enzymes was used to represent different parts of epithelial enterocytes,AKP andγ-GT for BBM,Na⁺-K⁺-ATP for basal-lateral membrane,SDH for mitochondria.Evaluation for preparation was conducted with enzymes activities of accumulation between initial homogenate and centrifuged BBMV.Results showed that the preparation with 43,000g was better compared with 28,000.The former could not only removed basal-lateral membrane completely but prevent the contamination of organelle,and preparation with 28,000g maintained some extent contamination of organelle and lower concentrating efficiency of enzymes.Results elucidated that preparation of BBMV with 43000g is applicable for studying development of digestive enzymes and activities in larvae.In trial 3,diets were formulated in gradient with protein content of 40,48,55%and lipid content of 15,20,25,30%.Larvae post-hatched for 21d were fed with different diets mentioned before and calculation of growth,body length and survival rate was made on the basis of stage and final.Results indicated that groups fed with protein content 48%,lipid 25%and protein 55%,lipid 24%maintained maximum growth,body length and survival.Survival rate of control was 80%,but that was lower than 70%of protein 40%group,and lower than all groups of protein 48%and 55% (excluding protein 48%,lipid 30%).The result implied that dietary protein content affected larvae growth.Results of body length showed that body length of P48 and P55 was similar and enhancement of high dietary was obvious,these parameters revealed that high dietary lipid level improve nutrients absorption and promote formation of bone.In Exp.4,activities of TRY,LIP,AMS,AKP andγ-GT were determined with larvae fed diets containing 48%protein and 15,20,25,30%lipid after 35d culture.The aim of this trial was to investigate the response of pancreas and BBM to dietary lipid content and to evaluate the effects of lipid in facilitating development of digestive tract.Results showed that(1)activities of BBM increased significantly with lipid content higher than 15%.The optimal lipid level(25%) is significantly different with others and reveals that dietary lipid level enhance the expression of BBM hydrolyzed enzymes.(2)Activities of LIP reached a plateau demonstrated that secretion and hydrolyzing ability had nearly come to saturation level.(3)there were not regularity between groups although TRY activities was affected by dietary lipid level.Activities of initial stage in control and L1 decreased gradually and related with feeds (r_con²=0.9146,r₁²=0.9138),and activities of L1 increased in final stage,which may explain as secretion enhancement.Activity of L3 maintained at higher level during experiment may be revealed that dietary lipid level stimulated TRY secretion and induced synergistic effect to lipid digestion.Activity of L4 which was probably in inhibitory situation and increased in final stage may elucidate that disturbance broke the balance between LIP and TRY,LIP secreted fervently and TRY depressed.Recovery of TRY activity might be related with increasing secretion in final stage.(4)Activities of AMS were affected by dietary lipid levels.The situation may be related with digestibility of carbohydrate in fish.Activities of AMS were not changed significantly as LIP,but activities of AMS in L2 and L3 increased definitely which might be response to lipid level.Optimal lipid levels cold enhance digestibility of carbohydrate and higher level might cause depression.(5)Artificial diets could achieve better results in promoting growth.Minimum body weigh was L1 group.Difference among groups was nearly 15%in final body weight.Similarly,survival rate was affected by dietary lipid level.Low survival rate was observed in high and low dietary lipid level.Highest survival rate was L3,as the same as control.That there was no significantly difference in growth and significantly difference in survival rate revealed that a number of larvae might be in a uncomfortable situation.In Exp.5,Activities of SOD,CAT,GPX and content of MDA of brain,intestine,gill and muscle in diets contained protein 48%,lipid from 15,20,25to 30%were determined to investigate responses of antioxidant enzymes to peroxidation and establish the relation between antioxidant defense oxidation. Results showed that:(1) Activities of GPX were highest compared with other tissues,and significant difference was found between initial and final stage.Activities of GPX of increased gradually with time in different groups;no regularity was found in muscle,but the tendency was in declining situation.Activities of GPX of L1 group declined during initial stage and maintained constant in final stage,but values was lower than that of control.Activities of L3 were higher than that of control and significantly higher than control in final stage(p＜0.05).Activities of control declined after increased in initial stage.Brain GPX activities of control declined apparently. Activities of all groups declined in final stage except high value of L1 at 10d.Activities of L3 and L4 increased with time and highest value was obtained at 40d of control.Highest value observed in intestine reveals that activities of GPX may prevent fish from damaging of free radicals.(2) CAT activities of intestine of L4 were significantly higher than any other groups and declined after 10 days.No difference between L2 and L3 but higher than control of same sampling time and no difference compared with control's values in initial stage.CAT activities of muscle were significantly higher than that of control,the highest values were observed in L4 and significantly higher than that of any other groups in final stage.Activities of L1 and L3 decreased after increased for a short period and still higher than that of control,indicated that oxidation was at high level.CAT activities of gill were higher than that of control and values of L3 maintained at high level,but no difference was found among groups.CAT activities of control maintained constant during experiment and higher value obtained from other groups which highest value was from L4 and significantly higher than others.High activities were corresponded to high dietary lipid in gill.Optimal dietary lipid does not affect CAT activities but excessive lipid may facilitate production of CAT,which indirectly reveal that more hydrogen peroxides were produced.(3)SOD activities of intestine in final stage were higher than that of initial except L1 declination.Highest value was observed in L4 and significantly higher than any others.There were no difference among L1,L2,L3 and control.Activities of muscle of L3 and L4 maintained at high level and significantly higher than other groups.The highest value was observed from L4 and remained increasing.Activities of muscle maintain constant and were similar as that of L1. Activities of gill of L1 and L3 increased linearly and that of L2 and L4 decreased linearly.No difference among values of L2,L3,LA and significantly higher activities of L1,which indicated activities were positive with lipid content.Higher Activities were found in brain,300-500U/mg prot,comparing with that of control.The tendency of SOD activities of groups were maintain at upwards comparing with con and no difference among L2,L3 and L4 but lower of L1.All these results indicated that dietary lipid level was positive related with SOD activities in up-regulation mechanism.High dietary lipid content affected SOD activities of fish may be caused by increment of ROS and responded through SOD activities.(4)MDA content of control of intestine maintained constant,0.50～0.61 nmol/mg prot.Highest value was from L4 and all groups were higher than that of control with no difference each other. MDA content of muscle in groups were higher than that of control and in declining tendency except initial value of L1.The rest of L1 were significantly higher than others and were in upward situation.MDA content of control of gill kept steadily,0.34～0.37 nmol/mg prot.Sharp declination appeared in L1 and L2 at 10d and slightly rose at 20d but returned to low value at 30d.Values of L4 maintained at high level from beginning and significantly higher than other groups.MDA content of control of brain was slightly lower than groups and in declined tendency.Values of L4 were significantly higher than other groups at 20d and 30d.Values of L3 maintained at higher level but no difference with L2.Values of L4 kept steady and higher which indicated that high lipid level may affect peroxidation in brain.All these results revealed that dietary lipid level may affect production of MDA in intestine and peroxidation in brain.Oxidative damage may be induced in tissues except surpass defense of antioxidantive ability.High dietary lipid level may damage tissue and affect survival rate.In conclusion,digestive enzymes has been detected in digestive tract to utilize nutrients, optimal protein and lipid content were 48%and 20-25%,respectively,could facilitate secretion of digestive enzymes and development of GI tract with high growth and survival rate in Brachymystax lenok larvae.Determination of antioxidant enzymes demonstrates that fish can prevent peroxides damage with regulation of enzyme activities and excessive lipid may affect normal growth and survival.The possible mechanisms of dietary lipid are as follows:appropriate lipid level could stimulates secretion of digestive enzymes and improve development of BBM,enhance exogenous digestion and meanwhile promote antioxidantive ability of absorptive and storing location, facilitating absorption and storage of nutrients,and further to improve growth and survival.