Study On Lipid Nutritional Requirements And Dietary Lipid Sources Of Onychostoma Macrolepis

Onychostoma macrolepis is a rare freshwater economic fish in China,commonly known as money fish,belonging to Cyprinidae and Barbinae.It is commonly found in the Yangtze River,Yellow River and its tributaries,and is the northernmost species of Barbinae.Qinling Mountains are one of the most important geographical distribution areas of O.macrolepis.O.macrolepis was once a tribute to the ancient court.Because of its rich nutrition and delicious taste,it is loved by the majority of consumers and the market demand is increasing.In recent years,due to overfishing and changes in water environment,the wild resources of this fish have been gradually decreasing.It has been listed as Grade II(only for wild species)in the National List of Key Protected Wild Animals in the 2021 edition,which is of great value in the protection,development and utilization of resources.Approved by the Ministry of Agriculture of China,two "National Aquatic Germplasm Resources Protection Areas for O.macrolepis" have been set up in Zhouzhi Heihe and Ziyang Renhe in Shaanxi Province.It is of great significance to carry out artificial cultivation for the protection,development and utilization of this fish resources.At present,there is a lack of special compound feed for O.macrolepis in the market.With the continuous expansion of breeding scale,the research and development of special compound feed for O.macrolepis is imperative.Few studies have been conducted on the nutritional requirements of O.macrolepis,while no studies on the lipid nutritional requirements and dietary lipid sources have been reported.In this study,O.macrolepis was used as the experimental object.With the muscle fatty acid compositions of O.macrolepis and natural bait lipid content analysis as the foundation,the effects of dietary lipid levels,essential fatty acids and lipid sources on growth,nutritional value and lipid metabolism-related gene expression of O.macrolepis were studied.In addition,the body fat status and its regulatory mechanism of O.macrolepis were studied under the nutritional restriction during the overwintering period.In this paper,the lipid nutritional characteristics of O.macrolepis were discussed from different perspectives,which provided valuable reference for the development of special compound feed for O.macrolepis and the conservation of resources.The main results are as follows:1.Seasonal variation of fatty acid composition and analysis of natural bait in O.macrolepis.Fatty acid biomarkers were used to analyze the fatty acid composition and seasonal variation in the natural feed of O.macrolepis.(1)The content of polyunsaturated fatty acids(PUFA)in muscle of wild O.macrolepis was higher than that of saturated fatty acids(SFA)and monounsaturated fatty acids(MUFA),and seasonal change had no significant effect on the contents of SFA,MUFA and PUFA in muscle of O.macrolepis.(2)In muscle of O.macrolepis,20:5n-3(6.04mg/g-6.47mg/g)and 22:6n-3(7.06mg/g-7.55mg/g)were the two kinds of PUFA with the highest contents.(3)The natural bait of O.macrolepis include plant feeds such as phytoplankton and benthic algae,and animal feeds such as zooplankton and benthic animals.(4)The contributions of diatoms was significantly in spring and autumn(P< 0.05),green algae and zooplankton contributed significantly in summer(P < 0.05).(5)The range of fat content in natural bait was 2.28%-13.19%.2.Effects of dietary lipid levels on growth,fatty acid composition,antioxidant status and lipid metabolism in juvenile O.macrolepis.Five diets of different lipid levels,3%,6%,9%,12% and 15%(L3,L6,L9,L12 and L15),were designed and fed to juvenile O.macrolepis for 8 weeks.(1)The juvenile O.macrolepis grew better at L9 than at L3,L6 and L15(P < 0.05).The viscera index(VSI)and hepatopancreas index(HSI)increased as the dietary lipid contents improved.(2)In serum,the contents of triglyceride(TG)and high density lipoprotein(HDL)decreased when the dietary lipid content ranged from 3.01% to 9.01%(P < 0.05).(3)Hepatic histology showed that the fish fed on L15 diet had more hepatic lipid droplets than those fed on the other diets.(4)Fish fed on L9 diet showed increases in superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)activities but decreases in malondialdehyde(MDA)content in hepatopancreas(P < 0.05).(5)The principle component analysis of fatty acid showed that n-3 long chain-polyunsaturated fatty acid(LC-PUFA)was mostly enriched in the fillet.(6)There were lower transcript levels of fatty acid synthetase(FAS),acetyl-Co A carboxylase 1(ACC1)and sterol regulatory element binding protein1(SREBP1)found in the hepatopancreas of fish with increased dietary lipid levels,whereas carnitine palmitoyl transferase 1(CPT-1),peroxisome proliferators-activated receptor ?(PPAR?)and adipose triglyceride lipase(ATGL)expressions were elevated(P < 0.05).These results suggested that diets with a proper dietary lipid level of 9.01%–11.95%(optimum 9.68% was based on the specific growth rate)could improve the growth performance and health status of juvenile O.macrolepis.3.Effects of dietary essential fatty acids requirements on growth,fatty acid composition,biochemical parameters,antioxidant response and lipid related genes expression in juvenile O.macrolepis.Seven isonitrogenous and isoenergetic purified diets containing tristearin(control group),2% linoleic acid(LA),2% ?-linolenic acid(LNA),1% LA + 1% LNA,1% EPA,1%DHA and 0.5% EPA + 0.5% DHA were formulated to feed juvenile O.macrolepis for 56 days.(1)The specific growth rate(SGR)and feed efficiency(FE)of the fish were significantly higher in the LNA group and the EPA + DHA group than in the control group(P < 0.05).The growth and feed utilization of the fish in the LNA group were similar to those in the EPA + DHA group.(2)The highest levels of 18:2n-6,18:3n-3,20:5n-3and 22:6n-3 in muscle and liver were found in the LA group,the LNA group,the EPA group and the DHA group,respectively.(3)The highest concentrations of serum cholesterol(CHOL)and TG were observed in the fish fed on the LA diet,and there were no significant differences in serum CHOL and TG between the LA group and the LA + LNA group.(4)Serum MDA content was observed to be the highest in the EPA group,DHA group and EPA + DHA group(P <0.05).In liver,the SOD activities and the MDA contents were found to be significantly the lowest and the highest in the EPA,DHA and EPA + DHA groups(P < 0.05).(5)The body fat content of the fish fed on the LA diet was significantly higher than those of the control ones(P < 0.05).The FAS,ACC1 and SREBP-1genes involved in the anabolic pathway had the highest expression levels in the LA group.The ATGL,CPT1 and PPAR? genes involved in the catabolic pathway had the lowest expression levels in the control group.These results showed that the increase in fish body fat content induced by the LA diet was related to the up-regulation of some lipid biosynthesis metabolism genes.This study recommended that the dietary requirement of fatty acids for the optimal growth and health of juvenile O.macrolepis be 2% LNA or 0.5% EPA + 0.5% DHA(approximately 9% dietary fat).4.Effects of dietary fish oil replacements with three vegetable oils on growth,fatty acid composition,serum parameters,antioxidant capacity and expression of lipid metabolism related genes in juvenile O.macrolepis.Five isonitrogenous diets with soybean oil(SO),linseed oil(LO),algae oil from Schizochytrium sp.(AO),mixed oil(MO,SO:LO:AO = 1:1:1)and fish oil(FO,control group)were selected to feed juvenile O.macrolepis(initial weight 1.86 ± 0.07 g)for eight weeks.(1)The results showed that in the LO and FO groups fish grew best.There was no significant difference in SGR and FE between the MO and FO groups(P < 0.05).(2)The highest contents of 18:2n-6,18:3n-3 and 22:6n-3 in liver and muscle were found with the SO,LO and AO groups,respectively(P < 0.05).(3)There appeared the highest concentrations of serum glucose(GLU),CHOL and TG in the SO group.(4)Compared with the control group,MDA content in serum and liver of fish in SO and LO groups was significantly decreased(P< 0.05).(5)The expressions of lipid anabolism genes were significantly up-regulated by dietary SO and LO(P < 0.05).The expressions of lipid catabolism genes were significantly higher with the AO,MO and FO groups(P < 0.05).This study recommended that LO or MO as a better oil source for juvenile O.macrolepis.5.Body fat status and regulatory mechanism of O.macrolepis in the overwintering periods.Samples of O.macrolepis were collected in the early(0 week,1G),middle(12 weeks,2G)and late(24 weeks,3G)of overwintering period,and high-throughput sequencing was performed on the liver of the fish.The results showed that,(1)With the duration of overwintering,the body weigh,VSI,HSI and intraperitoneal fat index(IPFI)of the fish showed an overall downward trend.The crude fat content of whole fish,muscle and liver in O.macrolepis after overwintering was significantly lower than those of the fish before overwintering(P < 0.05).(2)Compared with before overwintering,there were significant differences in the contents of SFA,MUFA and PUFA in liver after overwintering(P < 0.05),but there were no significant differences in the contents of SFA,MUFA and PUFA in muscle before and after overwintering.(3)A total of 4630,3976 and 2311 differently expressed genes(DEGs)were found in 1G versus 2G group,2G versus 3G group and 1G versus 3G group,respectively.It suggested that different stages of the overwintering period have significant effects on the gene expression of O.macrolepis,and the effect degree decreases gradually with the extension of hibernation.(4)The results of Gene ontology(GO)enrichment showed that DEGs were mainly related to metabolism and immunity,and most of these DEGs were down-regulated.(5)In addition,the KOG enrichment results showed that many of the DEGs associated with lipid transport and metabolism were down-regulated in the overwintering period.These observations suggested the down-regulation of lipid transport and metabolism-related DEGs might lead to the decrease of crude lipid content in body and tissues of O.macrolepis.Slowing metabolism and delaying immunity might be the overwintering adaptation strategy of O.macrolepis.In conclusion,(1)The muscle of O.macrolepis was rich in EPA and DHA,and had high nutritional value.The fat content of its natural bait ranges from 2.28% to 13.19%.(2)The diet with a lipid level of 9.01% to 11.95% was beneficial to the growth,antioxidant capacity and lipid metabolism of juvenile O.macrolepis.(3)Supplementation of 2% LNA or 0.5%EPA + 0.5% DHA in the diet is beneficial to the growth and health of juvenile O.macrolepis.(4)LO or MO was a better oil source in the diet of juvenile O.macrolepis.(5)During the overwintering period,the down-regulation of lipid transport and metabolism-related DEGs might lead to the decrease of crude lipid content in body and tissues of O.macrolepis.Slowing metabolism and delaying immunity might be the overwintering adaptation strategy of O.macrolepis.