Research On Immune Cells And Immune Genes Of Grass Carp: Natural Killer Cell Activities And The MRNA Expression Of Lysozyme Genes

Medication residual,environment and food safety confront the traditionalmeasures in treatment of fish diseases. This research focused on the natural killeractivities and the expressions of lysozyme genes in order to get acquainted with thefunction and the importance of the application of nutrition and feed additives-dietarylysozyme to enhance the immune response to infections, preventing and treatingdiseases and to explore the immunologic potency and immune-regulating mechanism ofgrass carp. The main contents of this paper were as follows.Starvation is a situation undergone and tolerated by many fish species in the naturalenvironment. Starvation is also an important environment factor for fish and it can affectfish growth, metabolism, behavior, reproduction, development and survival, as well ascauses fish to infect diseases even to death. In order to estimate effects of long-termstarvation on growth, physiological, biochemical parameter and non-specific immuneparameters of grass carp, fish with body weight(31.86±1.47)g were randomly dividedinto two groups(the starvation group, the control group), the starvation group wasstarved for15,30,45,60d and the control group fed diet which met the nutritionalrequirements of grass carp Ctenopharyngodon idellus.1The results showed, with the prolonged time of starvation, hepatosomatic indexand viserosomatic index decreased significantly compared with the control group(P<0.05). During the starvation, protein and lipid contents in muscle markedly declined,and moisture content increased (P<0.05).2The contents of total protein, triacylglycerol, total cholesterol, ALT and ALB inthe serum fluctuated in wave-like curves and decreased as a whole during starvation. Asone of the important index reflecting the growth of fishes, RNA:DNA ratio in musclewas no significant difference, but there were significant differences in thehepatopancreas tissue compared with the control group (P<0.05).3In order to estimate the effects of starvation on the protein metabolism, theactivity of glutamate dehydrogenase was determined in serum, muscular and hepatopancreas tissue. The results showed that the activity of glutamate dehydrogenasewas increased with starvation time prolonging in the hepatopancreas, not as for the othertissues.Glutamic dehydrogenase in serum and hepatopancreas was regarded as anoperative index of protein catabolic metabolization compared with those in muscletissue.4Under starvation stress the natural killer activity was significantly lower instarvation group than that in control group in the kidney and spleen of the grass carpfingerlings(P<0.05),and the activity no longer prominently decreased as starvation timeextended. The activity of lysozyme in the serum and hepatopancreas showed the trend offirst-decrease-then-increase with the increase of the starvation time and the activity ofalkaline phosphatase in the serum was significantly lower compared to the control groupon15d,45d,60d, which maintained at a constant level after30d. Natural Killer cellactivity was one of the critical indexes to evaluate the cellular immunity, which wasmore specific and sensitive than the activity of lysozyme or alkaline phosphatase.The tested fish used the substances (lipid and protein) of body fluctuated inwave-like curves during60-day starvation period. Under long-term starvation stress thenon-specific immune level of grass carp fingerlings decreases.Aquaculture is one of the fastest growing food-producing sectors in the world. Theintensive culture practices render increase in transmission of infectious diseases.Lysozymes are important enzymes of the innate immunity. Little workers have shownthat signifcant correlation exist between disease resistance of fsh and dietarylysozyme.An experiment was conducted to evaluate the use of the dietary lysozyme as afeed additive for Ctenopharyngodon idellus.Fish with initial body weight（6.93±0.01）gwere randomly divided into five groups and fed the corresponding feeds with0,100,200,300,400mg/kg dietary lysozyme respectively for60days. Natural killer activitiesin head-kidneyand spleen and lysozyme activities were studied at30d,45d,60d infeed. G-type, c-type lysozyme gene expression were determined in the fourtissues(intestine, hepatopancreas,head-kidney,spleen) by Real-time Quantitative PCR, inorder to probe into the effect of dietary lysozyme levels on g-type, c-type lysozymemRNA spatio-temporal expression in grass carp.1Along with the increase of the dietary lysozyme, the special growth rate did notpresented the increase trend significantly. At the end of4-week test, the special growthin D2and D3groups were not significantly different from those of the control group.The feed conversion rate in D3(200mg/kg) group decrease markedly compared with the control group(P<O.05for all).2At the thirtieth day of the feeding trial, the natural killer cell activities andlysozyme activities were significantly higher than those in the control group;As thefeeding time went by，the high dose improved the natural killer cell activities. Lysozymeactivities in high level groups were dramatically greater than those in the control groupbetween two first periods, except that in the head-kidney at45d; Lysozyme activitieswere not changed or decreased compared with the control group in the final phase. Theactivities of the two parameters above were relative to the intaking dose, tissues and thefeeding time.3Effects of the dietary lysozyme on the expression of the lysozyme genes weredifferent with the tissues, gene type and the feeding time. On the genotype side, g-typelysozyme showed a regularity to a certain degree and the increase expression; At the endof the feeding trial, the highest expression was present in D5(400mg/kg) group and itindicate that the expression of the c-type lysozyme exist remarkably different ondifference tissues and periods.The expressions in the head-kidney and spleen werestronger than those in the intestinal and hepatopancreas from tissues aspect. In terms ofthe feeding time, its transcripts were found at lower level in the second phase.4Expressions were significantly up-regulated in response to the dietary lysozymeon the transciriptional level; The protein level acted in accord with the mRNA level inthe first phase and those in the next two stages were not so.All in all, in growth parameters, the natural killer cell activities and lysozymeactivities, it was proved that the optimal addition level of lysozyme product was200mg/kg. However, it was400mg/kg from the NK cell activities and the g-typelysozyme expressions in the last two phases.