| Megalobrama amblycephala is one of the main cultured freshwater economic fishes in China,and has extremely high economic value.Increasing the carbohydrates level in diets can effectively reduce the cost of feed,but high levels of carbohydrates in diets can have a negative impact on fish growth and their liver health.Betaine supplement in high-carbohydrate diets can effectively improve the negative effects caused by high-carbohydrate.In experiment 1,we found that high-carbohydrate diets can reduce plasma taurine content in Megalobrama amblycephala,and the addition of betaine can significantly improve this phenomenon,but the mechanism is unclear now.Therefore,in experiment 2,based on a high-carbohydrate diet,we added different concentrations of betaine to it,then according to growth performance,liver condition,gut microbial diversity and metabolites changement in plasma,the optimum level of betaine was figured out in the high-carbohydrate diet.Combined with the changes of plasma metabolites and key enzyme gene expression between the control group and the optimal betaine addition group,the possible pathway were explored by which the betaine supplement in high-carbohydrate diets could increase plasma taurine.The main results are as follows:1.Effect of betaine in high-carbohydrate diets on the content of taurineIn experiment 1,300 juvenile Megalobrama amblycephala with an average body weight of 5.47±0.09 g were randomly divided into 4 groups,each group had 3 repetitions,each repetition had 25 fish,and there was no significant difference in the initial body weight among 4 groups.The CD(control)group was fed with standard diet,HCD(high-carbohydrate)group was fed with high-carbohydrate diet,LSB(long-term betaine)group was fed with high-carbohydrate+1% betaine diet and the SBD(short-term betaine)group was fed with 12 weeks high-carbohydrate diet and 4 weeks high-carbohydrate+1%betaine diet.The trial was lasted 16 weeks.After the trial,the plasma taurine content of the fish was detected.The results showed that the content of plasma taurine in HCD group was significantly decreased when compared to CD group(p<0.05),while the content ofplasma taurine in LSB group was significantly increased when compared to HCD group(p<0.05).The results suggested that high-carbohydrate diets resulted in the decrease the content of taurine in Megalobrama amblycephala,and betaine supplement in high-carbohydrate diets could increase the plasma taurine content.In experiment 2,450 healthy juvenile Megalobrama amblycephala with an average body weight of 6.75±0.10 g were randomly divided into 5 groups,each group had 3repetitions,each repetition had 30 fish,and there was no significant difference in the initial body weight among 5 groups(p>0.05).The five groups were fed a high-carbohydrate diets with betaine levels of 0%,0.2%,0.4%,0.8%,and 1.6%,respectively.The trial was lasted 79 days.After the trial,the growth performance,liver health status,intestinal microbial community diversity and plasma metabolites were measured.The result of the content of taurine shown that there was no significant difference between CD group(control,0% betaine addition)and 0.2B group(0.2%betaine addition)(p>0.05).They were significantly increased(p<0.05)in other three groups when compared with CD group.Among them,the content of taurine in 0.8B(0.8% betaine addition)was the highest,which was consistent with the results of experiment 1.2.Optimum level of betaine supplement in high-carbohydrate dietsThe results of experiment 2 showed that the weight gain rate(WGR)of the 0.8B group was the highest,significantly higher than that of the 0.2B group(p<0.05).Compared with the CD group,feed conversion ratio(FCR)and hepatosomatic index(HSI)in the 0.8B group were significantly reduced(p<0.05).The liver histology indicated that high-carbohydrate diet exhibited the elevated lipid accumulation in the CD group,and0.8% or 1.6% betaine supplementation can significantly improve the lipid accumulation in hepatocytes.The intestinal microbiological results showed that after the digestion of Hae III,compared with the CD group and 0.2B group,the Shannon-wiener index(H′)in the 1.6B group(1.6% betaine addition)was significantly increased(p<0.05),and the Eveness index(E′)of the 1.6B group were significantly increased comparing to 0.2B group(p<0.05);after Msp I digestion,compared with the 0.2B group,H′ and E′ of the1.6B group were significantly increased(p<0.05).Besides,microbial community indices suggested that the higher the amount of betaine added in the high-carbohydrate diet(0.2%<betaine addition<1.6%),the more abundant and stable of the gut microbial species in Megalobrama amblycephala,and the more balanced the number of individuals among each species.Among them,although the effect of betaine addition was the best in group1.6B,there was no significant difference between 0.8B and 1.6B groups(p>0.05).The principal component analysis showed that the samples of the CD group and the 0.2B group had higher variability,and the samples of the 0.4B(0.4% betaine addition),0.8B,and 1.6B groups were similar;cluster analysis showed that whether they were digested by Hae III or Msp I,the samples from the CD and 0.2B groups were divided into one category,while 0.4B,0.8B,and 1.6B were divided into another category,this shown that under the condition of high-carbohydrate diet,high levels of betaine addition(>0.4%)caused a similar and important transformation in the microbial communities,and high levels of betaine had a positive effect on the gut flora.The plasma metabolites results showed that,compared with the CD group,the contents of methionine,sarcosine,glycine,serine,hypotaurine and taurine in the 0.8B group were significantly increased(p<0.05),and were the highest among 5 groups.The contents of cysteic acid and cystine in 0.8B group were not significantly different from that of other groups(p>0.05).The content of homocysteine in the 1.6B group was significantly increased compared to CD group and0.8B group(p<0.05),however,the excessively high level of homocysteine in the blood results in homocysteinemia,which is not conducive to animal health,so it was the 0.8B group that had the best betaine addition effect.Combining the status of growth performance,liver histology,intestinal microbial community,and plasma metabolites,it can be concluded that the optimal level of betaine addition in high-carbohydrate diet is 0.8%.That is,when the total carbohydrate content in the diet is 43%,the optimum level of betaine supplement in the feed of Megalobrama amblycephala is 0.8%.3.Metabolic pathway of betaine in high-carbohydrate diet increasing plasma taurine contentTaking the CD group(control,0% betaine addition)and the 0.8B group(the best effect of betaine addition,0.8% betaine addition)as the research object,the gene expression of key enzymes related to taurine synthesis was analyzed,then combined with changes in plasma metabolites,the metabolic pathway were explored by which betaine addition in high-carbohydrate diets could increase plasma taurine content.Plasma metabolites results showed that betaine,methionine,sarcosine,glycine,serine,taurine,and taurine were significantly increased in the 0.8B group compared with the CD group(p<0.05),and there were no significant changes in the levels of homocysteine,cystine,cystathionine,and cysteic acid(p>0.05).Compared with the CD group,the m RNA expression of cysteamine dioxygenase a(ADOa),cysteamine dioxygenase b(ADOb),and cysteine sulfinate decarboxylase(CSAD)were significantly increased in the 0.8B group(p<0.05),the expression of cysteine dioxygenase 1(CDO1)remained unchanged(p>0.05),and the expression of glutamate decarboxylase(GAD)was significantly decreased(p<0.05).All these results showed that:(1)Betaine could regulate the taurine synthesis of Megalobrama amblycephala through the cysteamine(CS)pathway and cysteine sulfinate(CSA)pathway,but the cysteic acid(CA)pathway was not the main pathway for betaine to regulate taurine production;(2)Betaine increased the content of hypotaurine and taurine by enhancing the m RNA expression of ADO and CSAD,rather than changing the key metabolite contents of cystathionine,cystine and cysteic acid during the synthesis of taurine.(3)In the CSA pathway,betaine promoted taurine production by enhancing the m RNA expression of CSAD,while that of GAD did not participate in taurine biosynthesis,or its role was very little;in the CS pathway,betaine promoted taurine synthesis by enhancing the m RNA expression of ADO,but it was not determined which played a major role between ADOa and ADOb in this process. |
