| In nature,fish often face the impact of food shortage due to temperature changes,seasonal changes,reproductive behavior,diseases or uneven distribution of food factors.Overwinter is a necessary stage in aquaculture.With the arrival of winter,the water temperature decreases,which leads to the occurrence of starvation.During the period of overwintering,fish can only mobilize the energy stored in the body for consumption,and then survive the winter safely.Grass carp Ctenopharyngodon idellus is the single species with the largest output of freshwater aquaculture in China.During the period of overwintering,due to the lack of relevant scientific research data,it cannot effectively ensure the safety of grass carp after the end of overwintering,there is a lack of an adequate accurate nutrition strategy to quickly restore the health of grass carp,and even lack of nutrition enhancement strategy before overwintering to ensure the safety of grass carp Overwinter effectively.Therefore,in view of the above phenomena in practice,this study explored the physiological response mechanism of grass carp during overwintering.At the same time,the traditional nutrition methods were used to study the strategies of rapid recovery of grass carp after overwintering and strengthening the health before overwinter,so as to provide corresponding help and reference for production practice.The results of this study are as follows:1.Effects of overwintering on biological characters,serum biochemical indexes,routine components,antioxidant capacity and fatty acid composition of grass carpThe changes of biological characters,serum biochemical indexes,routine components,antioxidant capacity and lipidty acid composition of grass carp during overwintering were studied,the result showed that the body weight,hepatopancreas weight,lipidness,liver body ratio,visceral body ratio,intestinal body ratio and abdominal lipid index decreased significantly（P<0.05）.After one week of overwintering,the contents of routine components in muscle of grass carp changed significantly（P<0.05）;with the prolongation of overwintering time,serum triglyceride（TG）,glycerol and total protein were significantly decreased（TP）,total cholesterol（TCHO）and blood glucose（GLU）decreased significantly at first（P<0.05）,and then remained stable The results showed that the contents of acid in hepatopancreas,muscle and adipose tissue decreased significantly（P<0.05）;the contents of glycogen and muscle glycogen in hepatopancreas,muscle and adipose tissue were significantly decreased（P<0.05）;the three tissues with the greatest oxidative stress were adipose tissue,hepatopancreas and muscle;with the prolongation of overwintering time,the lipidty acid ratio of each tissue had a significant change,and the correlation analysis showed that the lipidty acid content of grass carp lipidty group was significantly higher than that of control group The results showed that SFA in weave,PUFA and MUFA in muscle and MUFA in hepatopancreas were positively correlated with oxidative stress and even body damage during the period of overwintering;muscle lipid in grass carp increased significantly within 2 weeks of overwintering,which may be related to LPL dependent lipid transport mechanism.The results showed that the physiological state of grass carp changed significantly during the period of overwintering,involving the morphological changes,energy mobilization,the role of oxidative defense system and other related changes.2.Transcriptomics of hepatopancreas of grass carp under overwintering stressThrough the high-throughput platform program,the hepatopancreas of grass carp was sequenced before and after starvation.A total of 241,305,604 clean and high-quality reads were obtained.After normalization,a total of 795 differential genes were found,including336 genes significantly up-regulated and 459 genes significantly down regulated.After the enrichment analysis of GO,KEGG and KOG of all the differentially expressed genes,68 go functional annotations were obtained from 759 differentially expressed genes.Among them,there were more differentially expressed genes in small molecule metabolism and lipid metabolism,followed by intracellular part and coenzyme binding pathway.The enrichment analysis of KEGG pathway showed that AMPK signaling pathway was the most abundant,which was annotated to 17 of the 24 differentially expressed genes were down-regulated and7 were up-regulated.Further classification of gene functions using KOG database showed that lipid transport and metabolic pathways were the most enriched,and the number of differentially expressed genes was up to 55.Combined with go,KEGG and KOG analysis,the results showed that AMPK signaling pathway was the main pathway in the process of overwintering,and its downstream pathway regulation genes were the main action genes.Lipid metabolism played a decisive role in the energy consumption of grass carp in response to overwintering,indicating that the hepatopancreas of grass carp could supply energy through lipid metabolism and adapt to overwintering.3.Characterization of AMPK gene in grass carp and its regulation on metabolic homeostasis under overwintering stressThe results of transcriptome analysis showed that AMPK signaling pathway played an important role in grass carp during overwintering.Nine subtypes of AMPK gene were identified by bioinformatics analysis,namely AMPKα1,AMPKα1,AMPKα2,AMPKβ1a,AMPKβ1b,AMPKβ2,AMPKγ1,AMPKγ2a,AMPKγ2B and AMPKγ3,and obtained their complete coding sequences.The AMPK genes of grass carp are highly conserved and highly homologous with other species.AMPK may play different roles in the energy mobilization of hepatopancreas and adipose tissue;AMPKγmay play a more important role than AMPKα/βin adipocytes in vitro.During the winter starvation period,the contents of ATP,ADP and AMP in serum were significantly decreased,and the ratio of ADP+AMP/ATP was significantly increased（P<0.05）;the expressions of AMPKα1 and AMPKα2 genes in hepatopancreas,muscle and abdominal lipid were significantly increased（P<0.05）,and the transcription level of downstream glucose-lipid and protein metabolism related genes increased significantly（including ATGL,HSL,CPT1α,CD36 and other lipolysis related genes;GK,PFK,PK and other glycolysis related genes;GLDH,IGF-1 and other proteolysis-related genes）or significantly down-regulated（ACC,FAS and other lipid synthesis-related genes;CREB,Fox O1,PGC-1α,PEPCK,G6Pase,GLUT2 and other gluconeogenesis related genes;TOR,S6K and other related to protein synthesis genes）（P<0.05）.The results showed that during the period of overwintering,AMPK pathway and its downstream genes were activated,glycolysis,lipid decomposition,fatty acidβ-oxidation,fatty acid transport and protein decomposition were accelerated,and glycogen synthesis,lipid synthesis and protein synthesis were inhibited,and the homeostasis of the body was maintained.4.Effects of different protein and lipid levels on growth performance,feed utilization,body composition,digestibility and health status of grass carp after overwinteringAfter overwintering stress,the actual demand of grass carp for feed nutrients may be different from the appropriate level under the normal breeding environment.Therefore,eight kinds of diets with different protein and lipid levels,including 25%,28%,31%,34%crude protein levels and 4%,8%crude lipid levels,were designed to feed grass carp after overwintering for 56 days.The results showed that the feed with protein content of 31%and lipid content of 8%significantly increased the final body weight,weight gain rate,visceral body ratio,intestinal body ratio and liver body index,and significantly increased the deposition rate of protein and lipid,and promoted the utilization of feed（P<0.05）.31%protein and 8%lipid significantly increased the content of digestive enzymes in hepatopancreas,promoted the repair of intestinal structure,and significantly improved the antioxidant capacity of various tissues（P<0.05）.Through regression analysis,it is suggested that the best repair effect is when the grass carp is fed with protein 30.32%-30.41%and lipid8%after overwintering.5.Effects of Schizochytrium oil and lipoic acid on growth performance,feed utilization,body composition,fatty acid composition and antioxidant capacity of grass carp after overwinteringThe above results showed that feeding 31%protein（30.32%-30.41%）and 8%lipid diet after starvation and overwinter had a good repair effect on the body.On the basis of this and previous research results in the laboratory,high and low content of n-3 HUFA and high and low content of lipoic acid were added respectively to strengthen the feed.The results showed that adding appropriate level（0.52%）n-3 HUFA and 0.1%lipoic acid significantly enhanced the growth performance and survival rate of grass carp after overwinter,improved the intestinal quality,reduced the feed coefficient,and inhibited the excessive accumulation of lipid in abdominal cavity（P<0.05）.The activities of cat,SOD and GST in hepatopancreas,muscle,foregut,adipose tissue and serum were significantly increased after adding appropriate level of n-3 HUFA（P<0.05）,and the contents of MDA and O2·-in each tissue were significantly decreased（P<0.05）.When 0.1%lipoic acid was added,the content of O2·-in hepatopancreas and muscle was significantly decreased,but the content of cat was significantly increased（P<0.05）.The appropriate level of n-3 HUFA and 0.1%lipoic acid treatment groups significantly changed the proportion of lipidty acids in various tissues,and PUFA ratio played a major role in the changes of lipidty acid composition.Finally,it is suggested that the feed containing 30.32%-30.41%protein and 8%lipid after overwinter should be supplemented with appropriate level（0.52%）n-3 HUFA and 0.1%lipoic acid,which has better repairing effect on grass carp.6.Effects of different protein and lipid levels on biological characteristics and fatty acid composition of grass carp before overwinteringSix kinds of diets with different protein and lipid levels,including 28%,31%,34%crude protein levels and 4%,8%crude lipid levels,were designed to carry out the 28 day winter feeding experiment before starvation.The results showed that the body weight loss of grass carp fed with 31%fortified protein and 4%lipid before overwinter was significantly inhibited（P<0.05）.After overwintering,the levels of TP,GLU and TG in serum were significantly increased in the 31%protein and 4%lipid diet group,and the oxidative stress was reduced during the overwinter period,which laid a good foundation for the health of the body after the winter starvation.The lipidty acid patterns of hepatopancreas and muscle were analyzed before and after starvation.It was found that the 31%protein and 4%lipid diet had the least effect on the change of lipidty acid ratio,which showed that the treatment group could effectively reduce the drastic changes of lipidty acid ratio during overwinter,and then reduce oxidative stress.Through regression analysis,31.53%protein and 4%lipid in the fortified feed before overwintering had the most obvious effect on the safe overwinter of grass carp.7.Effects of n-3 HUFA enriched diet before overwintering on body weight and antioxidant capacity of grass carpWe designed four kinds of feed treatment groups:31%protein 4%lipid group,31%protein 8%lipid group,31%protein 8%lipid group（0.52%n-3 HUFA）and 31%protein 8%lipid group（1.04%n-3 HUFA）,and then explored whether adding n-3 HUFA to the fortified diet before overwintering was helpful to grass carp overwinter.The results showed that dietary n-3 HUFA at 8%lipid level could not significantly inhibit the weight loss rate of grass carp before and after overwinter.It was also found that 31%protein and 4%lipid could significantly improve the quality and histological integrity of liver,pancreas and intestine of grass carp after overwinter,significantly increase the content of serum metabolites,and reduce the oxidative stress during overwintering,which laid the foundation for the rapid recovery of grass carp fed with feed after overwintering.The analysis of lipidty acid ratio of hepatopancreas and muscle before and after starvation in winter showed that adding high and low content of n-3 HUFA in feed increased the change of lipidty acid ratio pattern,increased lipid mobilization and metabolism,and then caused oxidative stress,which was not conducive to overwinter.Therefore,n-3 HUFA supplementation before starvation could not effectively improve the tolerance of grass carp to the adverse effects of overwintering.In conclusion:（1）During the overwintering period,grass carp spent energy by mobilizing energy in the body,and then survived the winter safely.During this period,the energy supply of lipid was the strongest,and the adipose tissue was subjected to the maximum oxidative stress;AMPK pathway and its downstream related genes jointly maintained the stability of grass carp’s body state;（2）after overwintering,the feed contained30.32%-30.41%protein,8%lipid and on this basis,adding 0.52%level of n-3 HUFA and0.1%content of lipoic acid had better effect on grass carp restoration;（3）strengthening the appropriate protein 31%and 4%lipid feed before overwintering could ensure grass carp to survive the winter safely,but adding n-3 HUFA had no such effect. |
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