| Zn?Zn?is an essential trace element required by fish.It functions as a cofactor in several enzyme systems.It is involved in many important physiological functions,including improving growth,development,immune function,and defense against free radicals.Signs of impaired growth,increased mortality,cataracts,short body dwarfism,and low tissue Zn may occur in fish fed a Zn-deficient diet.On the other hand,excessive Zn in the diet may negatively affect the nutritional status of other elements,such as iron?Fe?,cadmium?Ca?and magnesium?Mg?.Also,excessive Zn means unnecessary Zn release into aquatic water,this pollution may bring great impacts on fish.Therefore,it becomes necessary not only to ensure the minimal dietary levels to meet the requirement but also to make sure that the levels do not exceed the maximal limit which can affect the growth and other responses in fish.Blunt snout bream?Megalobrama amblycephala?belongs to the Cyprinidae family,which is one of the largest families of fishes in the world.In the year of 2013,its production reached about 0.73 million tons,which is the seventh freshwater-cultured fish species in China?Bureau of Fisheries of the Ministry of Agriculture 2014?.To date,the blunt snout bream has already became an important and popular freshwater species.However,reported data on Zn requirement for blunt snout bream range from 20 to 184 mg/kg of the diet.So 2 studies were employed to prove the dietary Zn requirements of blunt snout bream.And the effects of dietary Zn on testis development were evaluated by RNA-seq-based transcriptome analysis.At the last,a 1H NMR-based metabolomics approach was used to explore the impact of dietary Zn on the serum metabolic profile of blunt snout bream The results as follows: 1.Dietary Zn requirement of juvenile blunt snout bream,megalobrama amblycephalaA 12-week growth experiment was conducted to quantify the optimum dietary Zn requirement of juvenile blunt snout bream,and to evaluate its effects on antioxidant responses for this fish species.Seven experimental diets were formulated to contain graded levels of Zn?7.4,20.3,32.1,51.0,84.4,169.7 and 332.4mg/kg diet;supplied as Zn SO4·7H2O;casein and gelatin were used as dietary protein sources?.Each diet was assigned to three replicate groups of 40 blunt snout bream?initial body weight = 3.6±0.1 g?which were cultured in 500 L aquaria?r=0.9 m,h=0.75 m?.The results showed that,with the increase in dietary Zn level,weight gain rate of blunt snout bream first increased linearly then remained nearly unchanged at high Zn treatments?32.1 mg/kg or more?.The results of special growth rate and fish Zn content presented the similar tendency with the result of weight gain rate.Whole body moisture content of blunt snout bream in the supplemented groups were significantly decreased than that in the un-supplemented group?P<0.05?,while whole body crude protein content in the supplemented groups were significantly increased than that in the un-supplemented group?P<0.05?.No significant difference was observed on feed conversion rate among all the treatments?P>0.05?.The serum chemistry analysis showed that dietary Zn had significant effects on the content of total protein,urea nitrogen,high density lipoprotein cholesterol,total cholesterol,and triglyceride?P<0.05?;but no effect on the albumin content and alkaline phosphatase activity?P>0.05?.Hepatic malonaldehyde content was significantly decreased?P<0.05?,while superoxide dismutase and catalase were not changed significantly with increases in dietary Zn supplementation?P>0.05?.The broken-line regression analysis showed that the dietary Zn requirement of blunt snout bream was estimated to be 32.6 mg/kg for maximum growth,47.6 mg/kg for maximum fish Zn concentration,respectively.In order to observe the zinc deficiency,we had prolonged the breeding weeks to 48 weeks.At 24 weeks,part of the fish fed the diet with 7.4 mg/kg Zn had primary skeletal deformities.At the end of experiment,about 20% fish exhibited obvious skeletal deformities symptoms,damaged caudal fin and ventral fin.The results increased our knowledge on Zn requirement and its versatile functions of blunt snout bream,and provided the evidences for enhancing the quality of blunt snout bream feed.2.Dietary Zn requirement of adult blunt snout bream,megalobrama amblycephalaA growth experiment was conducted to investigate the effects of dietary Zn on growth,antioxidant responses and testis development of adult blunt snout bream,megalobrama amblycephala.Six experimental diets were formulated to contain graded levels of Zn?0,20,40,80,160,320 mg/kg,respectively?supplied as Zn SO4·7H2O,providing the actual dietary Zn values of 7.8?control?,32.7,50.3,87.2,165.4 and 328.5 mg/kg diet,respectively.Each diet was assigned to three replicate groups of 15 fish?mean initial body weight: 128.60±0.74g?for 12 weeks.The results showed that the weight gain?WG?and whole body Zn content increased linearly with the increase in dietary znic level,then remained nearly unchanged when znic levels is up to 50.3 and 87.2 mg/kg diet,respectively.Fish fed with the control diet had significantly higher feed conversation rate than those fed on diets supplemented with Zn?P<0.05?.However,no significant difference was observed among Zn–supplemented groups?P>0.05?.Dietary Zn supplementation had no significant effect on the protein contents of whole body.Whole body moisture of fish fed with Zn supplemented diets were lower than that of the control group,while ash content of the control group was significantly higher than those of Zn–supplemented groups?P<0.05?.The lipid content of whole body was reduced with increasing levels of Zn up to 165.4 mg/kg of diet?P<0.05?.The hepatic antioxidant indices and serum biochemical indices analysis showed that dietary Zn had significant effects on hepatic malonaldehyde?MDA?contents,superoxide dismutase?SOD?,catalase?CAT?,total antioxidant capacity?T-AOC?,glutathione peroxidase?GSH-Px?activities,but no effect on serum glucose?GLU?,high density lipoprotein cholesterol?HDLC?or total cholesterol?TCHO?content.Serum alkaline phosphatase?ALP?activity increases significantly?P<0.05?,whereas triglyceride?TG?content decreased significantly with the increase in dietary Zn level?P>0.05?.At 7.8mg/kg and 328.5 mg/kg group,liver histological sections showed a certain number of hepatocytes with empty vacuoles degeneration,nuclear migrations and cytoplasm disappearances.The broken-line regression analysis showed that dietary Zn requirement of blunt snout bream was 52.1 mg/kg for maximum WGR and 86.2 mg/kg for maximum whole body Zn content.3.Effects of dietary Zn on testis development of adult blunt snout bream,megalobrama amblycephalaWe observed the testis histotomy and sperm motility of blunt snout bream which were selected from the above test.The results showed that mature spermatozoa were visible in each group.The 7.8 and 32.7 mg/kg groups were lightly stained with hematoxylin,while the other four groups were darkly stained.Mesenchyme were relatively shriveled in the 7.8 mg/kg Zn-treated group;however,the opposite phenomenon was exhibited in the 50.3,87.2,165.4,and 328.5 mg/kg groups.The mesenchyme of these latter groups were plump,the nucleolus of the leydig cells were clear,and the nuclei were big and round.The sperm viability and normal morphological rate of the control group were lowest,as well as the percentage of fast-moving sperm,with increases in Zn concentration.The stated parameters gradually rose and then subsequently declined.In terms of sperm viability,the 50.3,87.2,165.4,and 328.5 mg/kg groups were distinctly higher than the controls?P<0.05?.Furthermore,the normal morphological rate and fast-moving sperm of groups 50.3,87.2,and 165.4 mg/kg were distinctly higher than that of other groups.All data were statistically significant?P<0.05?.Regarding sperm motility,it was shown that Zn did not influence the action of wobble?WOB?and straightness?STR?.However,average curvilinear velocity?VCL?,average straight linear velocity?VSL?,velocity average path?VAP?,velocity average path?ALH?,beat cross frequency?BCF?,linearity?LIN?,and mitral annular displacement?MAD?were seriously affected by dietary Zn?P<0.05?.With the increase of dietary Zn,sperm VCL,VSL,VAP,ALH,LIN,and MAD significantly increased and then subsequently decreased?P<0.05?.The increase of Zn concentration induced a reduction of BCF,with the 50.3,87.2,165.4,and 328.5 mg/kg groups obviously lower than that of other groups?P<0.05?.The results of this study imply that dietary Zn enhance spermary development by promoting the development of leydig cells and excess Zn has negative effects on sperm quality.4.Effect of dietary Zinc on testis transcriptomic responses of juvenile blunt snout bream,megalobrama amblycephalaIn this study,three levels of Zn content diets?8.5,30.9,328.1 mg/kg,respectively marked as ZNL,ZNM and ZNH?were feed Megalobrama amblycephala with initial average body weight of 1.7g.In order to understand the molecular mechanisms of zinc on the development of testis of Megalobrama amblycephala,testis transcriptomes profiles of these three groups of experimental fish were sequenced,respectively.The results showed that the totals of 67497 Unigenes were obtained by sequencing these three libraries.Annotation of 35,999 Unigenes in Nr,Swiss-Prot,KEGG and COG / KOG was successfully completed,respectively.The matching rate of Unigenes with zebrafish reference genome was the highest 39.71%.Compared these three groups Unigenes,there were 39963 differential expressed genes.Among them,34122 genes?20200 up-regulated genes and 13922down-regulated genes?were differential expression comparing ZNM with ZNL;22875 genes?19763 up-regulated genes and 3112 down-regulated genes?were differential expression comparing ZNM with ZNH;and 30315 genes?18294 up-regulated genes and 12021 down-regulated genes?were different expression comparing ZNH with ZNL.These genes involved multiple biological processes including single-organism process,developmental process,growth and immune system process,regulation of biological process,etc.By KEGG pathway annotations,30 signaling pathways were found to be significantly enriched.These pathways mainly include that Neuroactive ligand-receptor interaction,Oxidative phosphorylation,Ribosome biogenesis in eukaryotes RNA polymerase,Nucleotide excision repair,DNA replication,Cell adhesion molecules,Complement and coagulation cascades,Intestinal immune network for Ig A production,Hematopoietic cell lineage.These results indicate that zinc can cause many genes expression in different biological processes to vary in testis of M amblycephala.The results of this study can provide a theoretical basis for screening sensitive biomarkers to zinc,as well as the zinc nutritional requirements.5.1H NMR-based metabolomics studies on the effect of Zn in blunt snout breamA 1H NMR-based metabolomics approach was used to explore the impact of dietary Zn on the serum metabolic profile of blunt snout bream.Fish were fed diets containing graded levels of Zn?7.4,32.1,and 332.4 mg/kg diet;supplied as Zn SO4·7H2O?for 12 weeks.We had been identified 51 metabolites in serum including 22 amino acids or intermediary metabolites,16 organic acid,4 nucleic acid,3 sugars,5 vitamins and 2 others.Multivariate data analyses?PCA,PLS-DA and VIP?showed dietary Zn affected the metabolic profile impartially and these metabolites?such as proline,lactate,glucose,alanine,lysine,glutamine,creatine,trans-4-Hydrox,taurine,serine?were identified as charactisteristic metabolites.These metabolites are mainly increased in the group?7.4 and 332.4 mg/kg diet?compared with the group?32.1 mg/kg?,suggesting that Zn deficiency and excessive inhibited amino acid and carbohydrate metabolism.In conclusion,proline,lactate and glucose were found to have potentials for being new biomarkers for Zn nutritional status of blunt snout bream. |
PDF Full Text Request