| Subacute rumen acidosis（SARA）is a common nutritional and metabolic disease in ruminants under intensive conditions.It usually induced by the rapid decrease of rumen pH caused by the rapid ruminal fermentation of starch under a high concentrate diet condition.Concurrently,the digestive tract microflora balance was destroyed,and the abnormal bacteria metabolites in the rumen was increased,resulting in inflammation.SARA is associated with nutritional and metabolic diseases,which harmful to animal health,it also leads to the decline of animal production performance.Several studies conducted to add exogenous substances to prevent the occurrence of SARA.In this study,in vivo and in vitro experiments were conducted to clarify whether pretreatment grain corn effectively prevent SARA or not,and to analyze their alternations in ruminal microbiome and metabolism levels.Three parts are included in this paper.Trail 1 Study on the parameter selection and effect of corn pretreated with tannic acid to alleviate rumen acidosis in vitro.The purpose of this experiment was to screen the suitable steeped time,temperature and concentration parameters of corn pretreated with the tannic acid solution.The treatment methods in this experiment are as follows:the corn samples were crushed and screened by a 2 mm sieve.The treatment factors included tannic acid concentration（0,1.5%,2.5%and 3.5%）,steeped time（12 h,24 h and 48 h）and treatment temperature（25℃ and 55℃）.Corn grain was steeped for 12 h,24 h and 48 h in an equal quantity（in a 1:1 ratio,wt/vol）of water,1.5%,2.5%and 3.5%tannic acid solution before being mixed with other ingredients of the substrate.After the treatment,the samples dried at room temperature and used for batch culture in vitro.Three parts are included in this experiment:Firstly,the effects of three factors of tannic acid pretreatment on rumen fermentation.Three goats with rumen fistula were used as rumen fluid donors.The ratio of concentrate to forage was 8:2 and the fermentation time was 24 h.The results showed that when tannic acid concentration was the main effect,tannic acid treatment significantly increased the pH value and the proportion of acetate,while significantly decreased the proportion of butyrate（P<0.05）.Compared with the control group,the content of total volatile fatty acids in the high corn substrate steeped in 2.5%and 3.5%tannic acid solution decreased significantly,and the proportion of acetate to propionate significantly increased（P<0.05）.When the steeped time was the main effect,the pH value at 12 h was significantly higher than that at 24 h and 48 h.There was no significant change in other fermentation parameters（P>0.05）.When the steeped temperature was the main effect,there was no significant difference in in vitro fermentation parameters among all treatments（P>0.05）.The results of interaction analysis of the two factors showed that tannic acid concentration and steeped time had interactive effects on pH value,the proportion of butyrate and lactate concentration.The pH value was the highest in the group that corn steeped in 2.5%and 3.5%tannic acid solution for 12 h,and there was no significant difference between the two groups（P>0.05）.Compared with water pretreated-corn group,the propotion of butyrate was significantly lower in tannic acid pretreated-corn group（P<0.05）.The results show that the concentration of lactate was the lowest in the group that corn steeped in water for 12 h and the group that corn steeped in 1.5%tannic acid solution for 48 h,but there was no significant difference between these groups and the group that corn steeped in 2.5%tannic acid solution for 12 h（P>0.05）.No interaction effect was found among treatments（P>0.05）.Secondly,the effects of tannic acid pretreatment on the rumen degradation parameters of starch and the changes of starch structure after rumen in-situ degradation of corn were determined.The results showed that tannic acid pretreatment significantly decreased the rumen slow degradation part and effective degradation rate of starch（P<0.05）.The results of electron microscope scanning showed no differences in the appearance of starch granules between the control group and the tannic acid-treated corn group after 2 h fermentation.However,the scanning electron microscope results of 12 h and 24 h after fermentation,it was found that the pores on the surface of starch granules treated with tannic acid were less compared with the control group.Finally,the effects of high corn substrates,corn steeped in 2.5%tannic acid solution for 12 h,on bacteria structure and their alternations in the metabolic pathway.The results showed that the relative abundance of Firmicutes in the control group was significantly higher than that in the TA group,on the contrary,the relative abundance of Bacteroidetes in the control group was significantly lower than that in the TA group（P<0.05）.There was no significant difference in the relative abundance of other bacteria among groups（P>0.05）.At the genus level,the relative abundance of ChristensinaceaeR7group,RuminococcaceaeNK4A214group,Ruminococcus2 and unclassifiedoClostridiales in the HG group was significantly higher than that in the TA group（P<0 05）.In contrast,the relative abundance of RikenellaceaeRC9gutgroup,Succiniclasticum and Quinella in the control group was significantly lower than that in the TA group（P<0.05）.The results of PICRUSt（KEGG Level3）showed that relative abundance of pyruvate metabolism,starch and sucrose metabolism,pentose phosphate pathway and pentose and glucuronic acid transformation in TA group were significantly lower than that in the control group（P<0.05）.In terms of amino acid metabolism,compared with the control group,the biosynthesis of lysine was decreased and the metabolism of phenylalanine was enhanced in the TA group（P<0.05）.Overall,this study showed that tannic acid pretreatment could improve the rumen resistance of starch,weaken the metabolism of pyruvate in microflora,reduce the content of VFA produced by rumen bacteria,and increase the pH value of rumen fluid in vitro.Trail 2 Effects of corn pretreated with tannic acid on rumen fermentation,the microbial community and fecal characteristic of goats fed a high-concentrate dietThe aim of this experiment was to determine the effect of tannic acid pretreated-corn on SARA induced by a high grain diet from the microbial level.Twelve Boer goats with an average body weight of 30.25±1.36 kg was randomly divided into one of two diets.The corn used in the（HG）diet of the control group was steeped in water for 12 h,while the corn used in the experimental group（TA）was steeped in 2.5%tannic acid for 12 h.The experiment lasted for 24 days,in which rumen fluid samples were collected on 24 d,and feces samples were collected on the last week of experimental peirod.The results showed that the average value of ruminal pH in the TA group was significantly lower than that in the HG group.In the TA group,the TVFA and butyrate were significantly decreased,while the acetate and the acetate to propionate ratio were significantly increased（P<0.05）.The concentrations of LPS,NH3-N and lactate in the TA group were significantly lower than those in HG group.At the phylum level,the Firmicutes decreased by 7.76%and the Bacteroides increased by 7.75%in the TA group.Compared with the HG group,the relative abundance of Prevotella and Lachn-ospiraceaeNK3A20group were significantly increased,but the relative abundance of Weissella,Streptococcus,Acetitomaculum were significantly decreased in the TA group,the relative abundance of Ruminococcus was tended to decreased（P<0.10）.The quantitative analysis of lactate producing and metabolizing bacteria in rumen fluid was determined using q-PCR.The results showed that the relative number of Streptococcus bovis in the TA group was significantly lower than in the HG group（P<0.05）,but there was no significant change in the relative abundance of Megasphaera elsdenii and Selenomonas ruminantium between the two groups（P>0.05）.Subsequently,the effect of tannic acid pretreated starch on acid production of ruminal Streptococcus bovis was determined.The results showed that the content of the total acid produced by Streptococcus bovis with tannic acid pretreated starch as substrate was significantly decreased than that of the control group（P<0.05）.The contents of lactate and formic acid produced by Streptococcus bovis were lower than that in the control group（P<0.05）.However,there was no significant difference in fermentation pattern between the two groups（P>0.05）.There was no significant difference in pH value,LPS concentration,starch content,apparent digestibility of starch,VFA concentration in feces between the two groups（P>0.05）.A total of 23 phylum of bacteria were detected in fecal samples.Among them,the dominant bacteria were Firmicutes（61.55%,65.87%）and Bacteroides（25.31%,24.19%）.At the genus level,there was no significant difference in the diversity of rumen bacteria between the two groups（P>0.05）.Compared with the HG group,the relative abundance of NK4A214group in the TA group was significantly increased（P<0.05）.Trail 3 Effect of corn pretreated with tannic acid on rumen and serum metabolic profile of goats fed a high-concentrate dietThe purpose of this experiment was to explore the regulation of tannic acid-pretreated corn on rumen microbial metabolism,serum metabolic profile and SARA-induced inflammation in goats fed a high-concentrate diet.The experimental design was the same as that of the second experiment.The rumen fluid and blood samples were collected on 24 d of the experimental period.A total of 30 ruminal differential metabolites were obtained.Compared with the HG group,the contents of D-（+）trehalose and D-allose was significantly decreased in the TA group（P<0.05）.The contents of amino acids and their related metabolites including L-alanine,L-glutamic acid,citrulline,trans-4-hydroxy-L-proline were significantly decreased（P<0.05）in the TA group when compared with the HG group.The niacin and urea contents were significantly decreased in the TA group when compared to the HG group（P<0.05）.A total 38 differential metabolites were obtained in positive mode and 64 differential metabolites were obtained in negative mode of serum sample.Compared with the HG group,the contents of N-acetyl-L-histidine and L-3-Methylhistidine were significantly decreased in the TA group（P<0.05）.The methionine and choline contents were significantly increased in the TA group compared with the HG group（P<0.05）.In addition,compared with the HG group,the contents of SAA,IL-1β and LPS were significantly lower in the TA group than those in the HG group.To sum up,the effective method with the potential of alleviating SARA were screened by batch culture in vitro.After that,the effect of tannic acid pretreated-corn on rumen metabolic disorder and inflammation caused by high concentrate diet in microbial and metabolic levels in vivo.Based on the present study results,we consider that corn pretreated with 2.5%tannic acid solution for 12 h can enhance corn resistance from the rumen microbes,and then alleviate the occurrence of SARA under a high-concentrate diet. |
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