| Distiller’s grains,a by-product of liquor-making process,are cheap,widely sourced,good palatability and rich in nutrients,and are traditional feedstuff for cattle and sheep.But the components of distiller’s grains(wet)are complex,besides rich nutrients,there are also toxic components such as ethanol,methanol,fusel oil,aldehydes and acids.In the production of cattle and sheep breeding,the use of distiller’s grains is large,feeding process is long,easy to cause chronic accumulation of toxic substances in distiller’s grains.It can lead to chronic poisoning of distiller’s grains,damage animal health,and seriously affect the benefit of breeding production.Because of the slow onset of chronic distiller’s grains poisoning in animals,the early symptoms of the disease are not obvious and it is difficult to make an early diagnosis.There are few records of the changes of blood biochemical indexes,blood metabolite types and contents in goats with chronic distiller’s grains poisoning,and the mechanism of the main toxic substances in distiller’s grains causing goat poisoning is not clear yet.In this study,goats were fed with 65%,75% fresh distiller’s grains and 65% stored distiller’s grains to duplicate the chronic distiller’s grains poisoning model,to investigate the mechanism of goat distiller’s grains poisoning and find out the key index of disease diagnosis,blood biochemical index,metabonomics analysis and detection of the expression level of dopamine key enzyme m RNA in the pathway of Alcoholism were used,this study provides some theoretical basis for the effective prevention and cure of distiller’s grains poisoning in ruminant breeding.The animals in this experiment were 24 local crossbred goats,4 months old,half male and half female,weight(27.02 ± 3.03 kg)and body condition were similar.The goats were randomly divided into I ~ IV Group and 6/Group by single factor design.Experiment I and II were fed with 65% and 75% fresh distiller’s grains,experiment III was fed with 65% stored distiller’s grains,and experiment IV was fed with control basal diet.Dietary energy and protein levels were designed according to the "feeding standard for mutton "(NY/T816-2004).Each goat is fed at 9:00 and 18:00,respectively,in a separate enclosure with a half-slotted floor and free water.Pre-trial period 14 days,formal trial period 90 days.During the regular vaccination,deworming,environmental disinfection.Blood samples were collected on the 15 th,30th,45 th,60th,75 th and 90 th day of the experiment,the contents of ethanol,acetic acid and lactic acid in serum were determined by microplate and Elisa.Blood samples were collected from goats in groups I,II and IV on the 90 th day,non-target metabonomics ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UHPLC-QTOFMS)platform and related analysis software were used for the determination and differential analysis of metabolites.The liver tissues of Group II and IV were collected at the 90 th day of the experiment.The expression levels of the key genes of TH,DDC,SLC18A2,MAOA and DAT were detected by q PCR.The results showed that during the trial period,goats developed diarrhea and constipation,bronchitis,abnormal nerve excitement,mouth foaming,ataxia and other digestive,respiratory and neurological symptoms.Pathological changes such as intestinal mucosal bleeding congestion,liver and kidney swelling,fragile texture and so on.After combining with the laboratory test method,the success of the mould making can be determined.(1)At the 15 th day,the contents of acetic acid and ethanol in groups I and II were significantly higher than those in Group IV,and at the 30 th day,the contents of UREA and acetic acid in groups I,II and III were significantly higher than those in Group IV,the content of Alb in Group I and II was significantly higher than that in Group IV,Crea in Group III was significantly higher than that in Group IV,ALB and UREA in Group I,II and III were significantly higher than that in Group IV at the45 th day,p content in Group II and III was significantly higher than that in Group IV,Crea content in group I and III was significantly higher than that in Group IV,and ethanol content in Group III was significantly higher than that in Group IV at the 60 th day,the content of UREA and acetic acid in group I,II and III were significantly higher than that in Group IV,the content of AST in Group II and III was significantly higher than that in Group IV,and the content of Crea in Group III was significantly higher than that in Group IV at the 75 th day,the contents of UREA,ethanol and acetic acid in group I,II and III were significantly higher than those in Group IV,the contents of Alt and AST in Group III were significantly higher than those in Group IV,and the contents of P in Group II were significantly higher than those in Group IV At the 90 th day,the contents of acetic acid in group I,II and III were significantly higher than those in Group IV,and the contents of ALT,AST and ethanol in Group III were significantly higher than those in Group IV.At the 45 th,60th,75 th and 90 th days,the content of Ca in Group I,II and III was significantly lower than that in Group IV.The results showed that adding fresh distiller’s grains could increase the content of ALB,adding fresh distiller’s grains and storing distiller’s grains could lead to disorder of calcium and phosphorus metabolism and damage of liver and kidney.(2)In the non-targeted metabonomics study,340 differential metabolites were found in group I and Group IV,of which 186 metabolites were up-regulated and 154 metabolites were down-regulated.Compared with Group IV,there were 315 differential metabolites in Group II,142 of which were up-regulated and 173 were down-regulated.The metabolites of dopamine and 2-furylglycine were significantly up-regulated and prostaglandin I2 was significantly down-regulated,potential biomarkers for diagnosis of chronic alcoholism in distiller’s grains of goats.KEGG metabolic pathway enrichment analysis,these differential metabolites involved 18 significant changes in the metabolic pathway,these include Synaptic vesicle cycle,Dopaminergic synapses,Alcoholism,Taurine and hypotaurine metabolism,Histidine metabolism,Phenylalanine,tyrosine and tryptophan biosynthesis,Tricarboxylic acid cycle,Sphingolipid signaling pathway,and c AMP signaling pathway.Among them,the two metabolic pathways of Alcoholism and Cocaine addiction were significantly changed in the three groups,and the impact were > 0.2.The results showed that the addition of fresh distiller’s grains had significant effects on the two metabolic pathways of alcoholism and cocaine addiction.(3)Compared with the control group,the expression of TH in group II decreased significantly(p < 0.05),the expression of DDC,MAOA and DAT also decreased,but there was no significant difference(P > 0.05);The expression level of SLC18A2 was similar between the two groups and there was no significant difference(P > 0.05).The results showed that fresh distiller’s grains could inhibit the expression of TH and DAT,accelerate the production of dopamine and increase the content of DA in the cells,and then lead to alcoholism and mental disorder in goats.Conclusion: high dosage of distiller’s grains can cause chronic poisoning,liver and kidney damage,disturbance of calcium and phosphorus metabolism and alcohol metabolism in goats.Non-targeted metabonomics analysis showed that chronic alcoholism of distiller’s grains resulted in significant up-regulation of dopamine and2-furanoylglycine,and significant down-regulation of prostaglandin I2,dopamine,2-furylglycine and prostaglandin I2 may be potential biomarkers for the diagnosis of the disease.Long-term addition of fresh distiller’s grains could inhibit the expression of TH and DAT m RNA in liver tissues of goats,increase the content of dopamine in cells,and cause the neurological disorder of goats poisoned by distiller’s grains. |
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