Effects Of Mulberry Leaf Extract On Growth Performance, Intestinal And Liver Functions Of Rana Nigromaculat

Mulberry leaf extract(MLE)is a natural and green feed additive that is rich in various functional active substances and has multiple physiological activities.It is widely used in the fields of poultry,livestock,and aquaculture.Previous studies have found that MLE can be used as a functional feed additive for aquaculture,reducing the use of antibiotics and effectively improving fish health and meat quality.Previous research has found that mulberry leaf extract can serve as a functional feed additive for aquatic animals,as it reduces the use of antibiotics and effectively improves fish health and meat quality.Furthermore,this study explores the potential use of MLE as a replacement for antibiotics in Rana nigromaculata farming and investigates the appropriate amount of MLE to be added to artificial frog feed,as well as its impact on the health of Rana nigromaculata.This provides theoretical support for the application of MLE in healthy Rana nigromaculata farming.In this experiment,six different diets were prepared by adding 0,300,600,900,1200,and 1500 mg of mulberry leaf extract(MLE)per kilogram of Rana nigromaculata base feed.MLE and base feed were mixed with bentonite to create these six iso-nitrogenous and iso-caloric experimental feeds,denoted as control group C and experimental groups M03,M06,M09,M12,and M15.The initial weight of the Rana nigromaculata used in the study was recorded as 2.7±0.2g,and the experiment lasted for a period of56 days.During the experiment,the growth and disease status of the frogs were recorded,and at the end of the experiment,samples were collected and analyzed for changes in the growth performance,body measurements,and body composition of the frogs across the different groups.Furthermore,indicators related to the health of the frog’s intestines and liver were analyzed.The results demonstrated that:1.The effects of Morus leaf extract(MLE)on growth performance,body morphology indicators,and body composition of Rana nigromaculataIn terms of growth performance,the average weight,weight gain rate,and specific growth rate of the M09 group were significantly lower than those of the control group C(P<0.05),while the average weight,weight gain rate,and specific growth rate of the M03 and M12 groups were significantly higher than those of the control group(P<0.05).When the amount of MLE added was within the range of 0.3～0.9%,the survival rate,final average weight,weight gain rate,and specific growth rate of all groups showed a trend of first increasing and then decreasing with the increase in the proportion of MLE added.The intake rate and feed conversion ratio of the M09 group did not differ significantly from those of the control group(P>0.05),while the intake rate and feed conversion ratio of other groups were significantly higher than those of the control group(P<0.05).Moreover,a trend of decreasing and then increasing was observed for these indicators.By using second-order regression analysis,the optimal addition amount of MLE was determined to be within the range of 0.34～0.42%.In terms of physical indicators,the degree of fatness and liver-body ratio of the M09 group were significantly lower than those of the control group(P<0.05),while the visceral-body ratio of the M12 group was significantly higher than that of the control group(P<0.05),and the carcass ratio was significantly lower than that of the control group(P<0.05).When the amount of MLE added was within the range of 0.3～0.9%,the degree of fatness and carcass ratio showed a decreasing trend with the increase in the proportion of MLE added,while the visceral-body ratio showed an increasing trend.The hind leg index of the MLE added group did not differ significantly from that of the control group(P>0.05).In terms of body composition,the moisture content of the carcass in the MLE added group was significantly higher than that in the control group(P<0.05).The crude protein and crude fat content in the M09 group were significantly lower than those in the control group(P<0.05),and showed a linear decreasing trend with the increase in the proportion of MLE added within the range of 0.3～0.9%.There was no significant difference in the crude ash content of the carcass between the MLE added group and the control group(P>0.05).In terms of lipid-body ratio and liver lipid content indicators,there was no significant difference in the lipid-body ratio between the MLE added group and the control group(P>0.05),but the lipid-body ratio showed an increasing-decreasing trend with the increase in the level of MLE added.The crude fat content of the liver in the M03,M06,and M12 groups were significantly lower than those in the control group(P<0.05),while those in the M09 and M15 groups were significantly higher than those in the control group(P<0.05).Furthermore,as the proportion of MLE added increased within the range of 0.3～0.9%,the crude fat content of the liver showed a decreasing-increasing trend.2.The impact of mulberry leaf extract(MLE)on the intestinal and hepatic functions of Rana nigromaculata is assessed.In terms of intestinal digestive enzyme activity indicators,the addition of MLE resulted in significantly higher levels of protease and amylase activity than the control group(P<0.05).Additionally,the M06 and M09 groups saw significantly lower levels of lipase activity than the control group(P<0.05).In regards to antioxidant capability indicators in the intestine,the MLE added group showed significantly lower levels of MDA compared to the control group(P<0.05),and the T-AOC values were significantly higher in the MLE added group than in the control group(P<0.05).The SOD in the M15 group of the intestinal tract was significantly higher than that in the control group(P<0.05);furthermore,as the proportion of MLE added increased within the range of 0.3～0.9%,the intestinal SOD showed an increasing-decreasing trend.Histological changes in the intestinal morphology were observed by making paraffin sections and H&E staining in the midgut of the control C,M03 and M09 groups.The height and number of intestinal villi first increased and then decreased with an increase in the amount of MLE added,with the M03 group showing significantly higher villi height than the control C(P<0.05);the number of intestinal villi in the M06 group increased slightly from the control group,while the M09 group was significantly lower than the control group.Furthermore,the intestinal cavity rate in the M03 and M09 groups was lower than that in the control group,with a decreasing-then-increasing trend as the amount of MLE added increased.The villi in the midgut of the M03 and M09 groups were denser than those in the control C group,and the remaining cavity area was smaller than that in the control group.However,the villi height in the anterior gut of the M09 group was lower than that in the control group,and the cavity area was larger than that in the control group.This suggests that the addition of 0.3% mulberry leaf extract is more conducive to the development of intestinal cells and tissue structure integrity of the frogs,and the frog’s intestinal tract is healthier with the addition of 0.3% mulberry leaf extract compared to 0.9%.Analyzing the biochemical indicators of plasma,as the proportion of MLE added increased,the values of TC and LDL showed a trend of first decreasing and then increasing,with the M03 group showing significantly lower values of TC and LDL compared to the control(P<0.05).There was no significant difference(P>0.05)between the MLE added group and the control group in terms of TG and HDL.The ALT of the M06 and M12 groups were significantly higher than the control(P<0.05).The AST of the M12 group was significantly higher than the control(P<0.05).In terms of liver antioxidant capacity indicators,there was no significant difference(P>0.05)between the MLE added group and the control group in terms of liver MDA and T-AOC.However,the SOD activity in the liver of the MLE added group was higher than the control,with the SOD values of the M03,M06,and M15 groups showing significant differences compared to the control(P<0.05).When the amount of MLE added was within the range of 0.3～0.9%,MAD showed a trend of first decreasing and then increasing,while T-AOC and SOD showed a trend of first increasing and then decreasing.Furthermore,the values of these indicators were all higher than those of the control group.Paraffin sections of the liver were made and HE stained in the control C,M03,and M09 groups to observe the morphological structure of the liver cells of the Rana nigromaculata.The results showed that there was severe fatty degeneration in the liver cells of the control group,with irregular shapes,most cell nuclei deviating from the center of the cell and close to the cell membrane,severe vacuolization,and multiple large bright areas appearing in the cytoplasmic matrix.In addition,the liver sinus became narrower and the arrangement of liver cords was also disordered.The degree of fatty degeneration in the liver cells gradually decreased in the M03 and M09 groups with the addition of MLE,and vacuolization was reduced.This indicates that the addition of MLE can improve the phenomenon of liver cell hollowing.3.The impact of Mulberry Leaf Extract(MLE)on the intestinal microbiota of Rana nigromaculataThe intestinal contents of the frogs were collected,with 6 replicates per group and 3frogs per replicate.After immediately being frozen in liquid nitrogen,they were sent to Bio Miao Ke for intestinal microbial diversity sequencing.The results showed that adding a suitable amount of MLE to the feed could reduce the diversity and richness of the intestinal microbiota in Rana nigromaculata,but it had a positive effect on improving the structure of the intestinal microbiota.Compared to the control group and other MLE addition groups,adding 0.3% MLE showed the lowest abundance of pathogenic bacteria in the intestines of Rana nigromaculata,with a higher relative abundance of beneficial bacteria,and could improve lipid metabolism.Summary: This experiment indicates that the appropriate amount of MLE to be added to the artificial feed of Rana nigromaculata is 0.34-0.42%.Adding MLE can significantly improve the growth performance and feed utilization of Rana nigromaculata,enhance the digestive and absorptive functions of protein,fat,and carbohydrates in the frog’s intestines;while also increasing the antioxidant capacity of liver and intestinal tissues,improving liver lipid metabolism.Moreover,an addition of0.3% MLE can increase the proportion of beneficial bacteria in the intestines of Rana nigromaculata,improve intestinal lipid metabolism,and thus promote frog growth.Therefore,we believe that MLE from mulberry leaves can serve as a green additive in the feed of Rana nigromaculata,playing a positive role in improving intestinal health and promoting liver function of the frog.