| Copper is an essential trace mineral nutrient element for all animals,including fish,which is involved in the formation of a variety of enzymes and functional proteins in the body,and thus plays a physiological role in immunity,antioxidant and energy metabolism regulation.Previous studies have shown that under artificial breeding conditions,fish mainly obtain the copper required by the body from feed.Deficiency of copper in feed can cause growth retardation and developmental malformation of cultured fish.Excessive copper in the feed will accumulate overly in the body and cause the peroxidation of biological macromolecules,resulting in a reduction in the survival rate,growth rate and fertility of the fish.Therefore,the research and development of feed with appropriate copper level is of great significance for fish cultivation.In addition,copper is a relatively common heavy metal element in the pollution of breeding waters,which is mostly in the form of copper ions,and can cause the growth of cultured fish to be blocked or even poisoned to death.Nutritional requirements for copper in various economic fish species and safe concentrations of copper in water have been reported,but no such studies have been seen on ricefield eel.This article conducted a preliminary study on the nutritional requirements of copper in ricefield eel and the safe concentration of copper in the water,aiming to provide a reference for the preparation of ricefield eel’s environmental friendly compound feed and healthy farming.The main findings are as follows:1.Study on demand and toxicity of ricefield eel for copper in feed.In the experiment,copper pentahydrate sulfate（CuSO4·5H2O）was used as the copper source,and the contents of 0,10,36.8,135.7 and 500 mg/kg of Cu2+were added to the basic feed by adopting the method of equal logarithmic spacing（LOG10）gradient.The measured copper levels of the 5 groups were 14.21,23.95,37.01,135.63,499.63 mg/kg,fed ricefield eel（62.09±1.07g）for 60 days,and the effects of copper level on the growth performance,tissue copper accumulation,serum and liver biochemical indexes,and intestinal and liver micro structure of ricefield were analyzed.Results:（1）The group with dietary copper level of 37.01 mg/kg achieved the best growth performance,with the highest weight gain rate and the lowest feed coefficient.（2）The copper level in feed had no significant effect on morphology parameters of ricefield eel and the moisture,crude protein and crude fat of whole fish（P>0.05）,but the high dietary copper can lead to a significant increase in ash content of whole fish（P<0.05）.（3）The accumulation rule of copper in ricefield eel was:liver>intestine>kidney>skin>spleen>muscle.Increased copper levels in feed can cause the copper accumulation in liver,intestine,kidney,skin and spleen to increase significantly（P<0.05）,but had no significant effect on copper accumulation in muscle（P>0.05）.Copper was mainly stored in liver.In the 499.63 mg/kg group,the copper accumulation in liver of ricefield eel could exceed the national safety standard of copper≤50 mg/kg in aquatic products.（4）The activity of alanine aminotransferase in serum of ricefield eel in the high copper group（135.63,499.63 mg/kg）was significantly higher than that in the low copper group（14.21,23.95,37.01 mg/kg）.The activity of CuZn superoxide dismutase and total antioxidant capacity in serum of ricefield eel increased significantly（P<0.05）when the dietary copper level was higher than 37.01 mg/kg.The serum ceruloplasmin activity of the ricefield eel in the 37.01 mg/kg group was the highest,which was significantly higher than that of the other 4 groups（P<0.05）.The 499.63 mg/kg group had the highest serum lactate dehydrogenase activity,which was significantly higher than the other 4 groups（P<0.05）.The content of malondialdehyde in serum of ricefield eel in 14.21,23.95 and 499.63 mg/kg groups was significantly higher than the other two groups（P<0.05）.The activities of liver superoxide dismutase and CuZn superoxide dismutase in the groups of 37.01,135.63 and 499.63 mg/kg were significantly higher than those in the other two groups（P<0.05）.The liver lactate dehydrogenase activity and malondialdehyde content of ricefield eel in the high-copper group were significantly higher than those in the low-copper group（P<0.05）.（5）There was no significant effect of dietary copper on the activities of three digestive enzymes（amylase,lipase and protease）in the stomach,intestines and liver of ricefield eel.（6）Low-copper diets had little effect on the liver and intestinal structure of ricefield eel.High dietary copper increased the number of mucous cells in the intestinal mucosa of eel,the hepatic endoplasmic reticulum was broken and coiled in a free vesicular distribution,mitochondria were swollen and ruptured,lysosomes increased in number and volume,and a large number of lipid droplets were observed in the cell.The results showed that the feed with appropriate copper level could promote the growth of ricefield eel,while the feed with insufficient or excessive copper level could inhibit the growth performance.Taking weight gain rate and feed coefficient as evaluation indexes,the copper demand of ricefield eel with average weight of 62.09±1.07g in feed was 44.29～45.84 mg/kg.Elevated copper levels in feed can enhance the antioxidant ability of ricefield eel,but the excess of copper will aggravate the degree of lipid peroxidation.Copper was mainly deposited in the liver,intestines and kidney of ricefield eel,and had no significant effect on the nutritional composition and food safety of muscle.High content of copper can cause excessive accumulation of copper in liver,resulting in changes and damage of liver and intestinal structure.The specific manifestations were:intestinal villi were damaged,the physiological structure and function of mitochondria and endoplasmic reticulum in liver cells were destroyed,and hepatocytes were lytic.The intestine of the ricefield eel enhanced the excretion of copper ions by increasing the number of mucus cells to promote the secretion of intestinal mucus,and showed a certain resistance to high dietary copper.2.Effect of dietary copper level on gut microbiota in ricefield eel.Based on the test of copper nutritional requirements,three groups of ricefield eel with copper levels of 14.21（group A）,37.01（group C）and 499.63 mg/kg（group E）were cultured and sampled at 120d.Four intestinal mucosa mixed samples of ricefield eel were selected from each experimental group for full-length and high-throughput sequencing of 16SrDNA,and the sequencing results were statistically analyzed.Results:（1）At the genus level,there was no significant difference in the composition of gut microbiota between groups A and C（P>0.05）The intestinal microbial diversity in group E increased,among which the relative abundance of immunologically related Romboutsia,Streptococcus and Ochrobactrum containing more opportunistic pathogens were significantly higher than that of group A and group C（LDAscore>3.5）.（2）Analysis of the KEGG metabolic pathway showed that there was no significant difference between group A and group C,and between group A and group E（P>0.05）.However,the enrichment degree of group E in the Excretory system and Metabolism of other amino acids pathways was significantly higher than that of group C（P<0.05）.（3）The analysis of BugBase functional prediction showed that the relative abundance of microbes with stress tolerance in group E was significantly higher than that in group A and C（P<0.05）,while there was no significant difference in other phenotypes.（4）The prediction of COG protein function showed that the Energy production and conversion,Cell cycle control,Cell division,Chromatin and dynamics functions of gut microbiota in group E were significantly lower than those in groups A and C（P<0.05）.The results showed that the intestinal microbial structure and function of ricefield eel did not change significantly within the appropriate range of copper content.However,excessive levels of copper in feed will lead to the increase of intestinal microbial diversity and functional change of ricefield eel.High dietary copper can increase the abundance of pathogenic microorganisms and harm the intestinal health.The excretory system pathway of intestinal microbiota was enhanced and the function of growth-related proteins was decreased.Although the intestinal pressure tolerance was enhanced,it was generally unfavorable to the growth of ricefield eel,so the copper level in feed should not be added in excess.3.A preliminary study on the toxicity of copper in water to ricefield eel.CuSO4·5H2O was used to prepare the test solution of Cu2+ with contents of 0,0.25,0.5,1,2,4 and 8mg/L,respectively（hardness:60 mg/kg（CaCO3）,pH7.2）.The hydrostatic biological test method was used to study the toxicity of Cu2+ to the juvenile ricefield eel,and the mortality of juveniles at different Cu2+concentrations was counted.The half-lethal concentration（LC50）of Cu2+ at 24,48,72,and 96 h in juvenile ricefield eel was linearly fitted,and the safe concentration（SC）of Cu2+ in eel was calculated.Results:（1）The symptoms of acute copper poisoning in juveniles are as follows:swimming away from the bottom of the water without regularity,secreting a lot of mucus on the body surface,red and swollen anus,straightening the body and sticking the snout out of the water,bleeding in the gill cavity.（2）The half-lethal concentrations of Cu2+ for ricefield eel at 24,48,72 and 96 h were 6.764,4.971,1.930 and 1.029 mg/L,respectively,and SC was 0.1092 mg/L.The results showed that the copper in water was highly toxic to ricefield eel,and the safe concentration was 0.1092 mg/L.It is suggested to avoid the use of copper sulfate in the process of breeding and to use copper-containing products with caution. |
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