Effects Of Hepcidin And Its Agonist On Growth,Immunity And Intestinal Health Of Grass Carp

Grass carp is the most important freshwater farmed fish in China.With the continuous development of high-density intensive culture mode in recent years,the incidence of disease outbreaks has increased dramatically,among which bacterial disease outbreaks have caused serious economic losses to the grass carp farming industry.In recent years,the abuse of antibiotics has led to increasing bacterial resistance,and there is an urgent need to find new treatment options.Hepcidin,an antibacterial peptide and iron regulator secreted by the fish itself,is considered an ideal alternative to traditional antibiotics because of its various effects including immune enhancement.This study investigated the effects of hepcidin and its agonists on growth performance,immunity and intestinal health of grass carp,with the aim of developing green and safe antibiotic alternatives to prevent bacterial diseases in fish.The main research contents and results are as follows:1.Effects of different feeding methods of hepcidin on growth,immunity and gut microbes of grass carpWe divided 450 grass carp into 5 groups.The grouping was as follows: the control group(Control)was fed with basal diet on daily basis;the Ci Hep group(Ci Hep-3d)was fed with Ci Hep for three days on end followed by three-day interval;the Ci Hep group(Ci Hep-7d)was fed with Ci Hep for seven days on end followed by seven-day interval;the Ci Hep+chitosan group(Ci Hep+CS-3d)was fed with Ci Hep and chitosan for three days on end followed by three-day interval;and the Ci Hep+chitosan group(Ci Hep+CS-7d)was fed with Ci Hep and chitosan for seven days on end followed by seven-day interval.The rest of the time is fed with basal feed.The test period was 8 weeks and samplings were performed on day 0 and 7 of the attack.We analyzed the clinical effects of hepcidin and chitosan for the prevention of bacterial infections by testing growth performance,protection rate,iron metabolism,tissue damage,immune gene changes and gut microbe changes.The results showed that oral administration of Ci Hep and CS could effectively promote the growth of grass carp,improve the serum immune indexes and survival rate.The survival rate of Ci Hep+CS-3d group was the highest at 55% and significantly higher than that of the control group.Oral administration of Ci Hep and CS also reduces serum iron levels in the body and promotes the accumulation of iron in the hepatopancreas,thus limiting the growth and reproduction of bacteria.In addition,oral administration of Ci Hep and CS can effectively modulate the inflammatory response and significantly reduce gill tissue damage.Also,Ci Hep and CS could change the composition and diversity of grass carp gut microbiota and significantly increase the proportion of beneficial microbiota.Most importantly,the growth and innate immune indexes in Ci Hep-3d group(feeding with Ci Hep for 3 days interval 3 days pattern)and Ci Hep+CS-3d group(feeding with Ci Hep and chitosan for 3 days interval 3 days pattern)are higher than those in other groups.The mortality rate in Ci Hep+CS-3d group is the lowest.These results indicated that the protective effect of consecutive 3-day feeding followed by 3-day interval is better than that of consecutive 7-day feeding followed by 7-day interval,and that the protective effect of Ci Hep in combination with chitosan is better than that of Ci Hep alone.Our findings optimized the feeding pattern to better oral administration of Ci Hep in aquaculture.2.Effects of hepcidin herbal agonists on growth,immunity and intestinal health of grass carpWe selected four herbal medicines: berberine hydrochloride(BBR),grape seed proanthocyanidins(GSP),rosmarinic acid(RA),and astragalus polysaccharide(APS).The four herbal medicines were applied to L8824 cells separately,and the expression of hepcidin gene and ferrtin gene was detected by q RT-PCR.The cells were co-incubated with A.hydrophila and herbal medicines,and the survival of the cells was determined by crystalline violet staining to detect the effect of the herbal medicine prevention.Then the selected Chinese herbal medicine was fed to grass carp,and the experimental grass carp were divided into 4 groups.The grouping was as follows: basal diet group(Control),proanthocyanidin group(GSP),berberine hydrochloride group(BBR),and proanthocyanidin plus berberine hydrochloride group(GSP+BBR).The experimental period was 4 weeks and samplings were performed on day 0 and 3 of the attack.At the cell level,GSP and BBR can significantly up-regulate the expression of hepcidin and ferrtin genes.The survival of L8824 cells infected with bacteria was significantly improved by GSP and BBR,and the best survival was achieved by GSP+BBR,as revealed by crystalline violet staining.The results suggest that GSP and BBR can be used as agonists of ferroregulators and that the combination of the two can more effectively improve the survival rate of cells after infection.At the individual level,we analyzed the clinical effect of GSP and BBR in preventing bacterial infections by testing growth performance,protection rate,tissue load,serum immunity,tissue damage and immune gene changes.The results showed that oral administration of GSP and BBR effectively promoted the growth of grass carp,significantly improved the serum innate immunity index and significantly increased the survival rate of grass carp.Oral administration of GSP and BBR also lowered cholesterol and other indicators of lipid metabolism.In addition,oral administration of GSP and BBR significantly reduced the bacterial load in the head kidney,spleen,hepatopancreas and hindgut tissues of grass carp,increased the expression levels of immune genes IL-1β,IL-6 and TNF-α in the spleen and head kidney tissues after the attack,and significantly reduced intestinal tissue damage.In summary,these results demonstrate that GSP and BBR as feed additives can effectively promote the growth of grass carp,improve immune status,enhance lipid metabolism,and reduce the probability of disease,thus proanthocyanidins and berberine hydrochloride are of great value as natural hepcidin agonist in grass carp culture.