The Effect Of Glycyrrhizic Acid Monoammonium Salt On The Key Bacteria,Related Metabolites And Blood Physiological Indexes Of Methane Production In Sheep

In order to provide data support for saponin additives that can improve rumen environment,reduce methane emission and improve feed utilization rate of ruminants,this experiment was conducted to investigate the effects of glycyrrhizic acid monoammonium salt on key microbial groups,related metabolites and blood physiological indexes of rumen methanogens in Karakul Sheep,and to provide data support for the application of glycyrrhizic acid monoammonium salt in ruminants diets.In this experiment,the optimum concentration of glycyrrhizic acid monoammonium salt was selected for animal feeding experiment in vitro.The rumen internal environment index,blood physiological and biochemical index,methane emission,nutrient digestibility,rumen microorganism and methane metabolism function gene were determined by animal experiment.The pre feeding period was 15 days and the normal feeding period was 60days.The normal feeding period was divided into two sampling stages,the first stage（0-30 days）and the second stage（31-60 days）.The specific test results are divided into the following five parts:Test 1:Screening the optimal concentration of glycyrrhizic acid monoammonium salt by in vitro fermentationIn this experiment,the effects of different concentrations of ammonium glycyrrhizinate on rumen fermentation parameters and methane production in vitro were determined for the appropriate concentration in feeding experiment.Three Karakul sheep with permanent fistulas were selected as rumen fluid donors.The fermentation parameters and methane production of rumen fluid were determined by gas production method in vitro after adding different concentrations of monoammonium glycyrrhizinate.The basal diet was used as fermentation substrate,and the control group,group I,group II,group III,group IV and group V were set up.The concentration of glycyrrhizic acid monoammonium salt was added by 0,50,150,250,350and 450 mg/L respectively,with 3 replicates in each group.After incubation for 3,9,12,21 and24 hours,the p H,NH₃-N concentration,gas production,dry matter degradation rate,volatile fatty acid（VFA）concentration and methane production on rumen fermentation parameters were determined.The results showed that:in the fermentation 24 hours,the gas production of group III,group IV and group V was significantly lower than that of the control group（P<0.05）;the p H of each group showed a downward trend with the extending of time;the dry matter degradation rate gradually increased with the prolonging of time,but there was no significant difference between each experimental group and the control group（P>0.05）;the NH₃-N concentration of the experimental group decreased with the increase of monoammonium glycyrrhizinate concentration,among which group III,group IV and group V had a downward trend.The concentration of NH₃-N in group V was significantly lower than that in control group（P<0.05）;the concentrations of acetic acid,propionic acid and butyric acid in experimental group had a downward trend compared with that in control group,but the differences were not significant（P>0.05）;after 24hours of fermentation,methane production of treatment group IV and V was significantly lower than that of control group and other treatment groups（P<0.05）.According to the comprehensive evaluation of total indexes,the suitable concentration of ammonium glycyrrhizinate for feeding experiment was 350 mg/L.Test 2:The effect of glycyrrhizic acid monoammonium salt on the rumen environment of karakul sheep in vivoThe 12 Karakul sheep wether sheep with good body condition and similar weight with permanent rumen fistula were randomly divided into two groups（control group and treatment group）.The treatment group was injected monoammonium glycyrrhizate solution into the rumen fistula,and the control group injected distilled water into the rumen fistula.The gastric juice was collected at 0,2,4,6 and 8 hours after feeding on the first day of sampling period and 31-33 days,and was used to measure the p H,NH₃-N and VFA indexes of gastric juice.The results showed that p H of Karakul sheep decreased significantly after feeding（P<0.05）,then increased gradually.The rumen p H value was decreased after adding glycyrrhizic acid monoammonium salt.There was no significant difference in p H at each time point in stage I（P>0.05）,but the p H in stage II 0h treatment group was significantly lower than that in the control group（P<0.05）.Although glycyrrhizic acid monoammonium salt reduced the rumen p H,it was all in the normal range,and had no negative effect on the rumen environment.The NH₃-N concentration of the treatment group was lower than that of the control group（P<0.05）,but all of them were within the normal range,which did not have adverse effect on the rumen environment.The concentrations of acetic acid,propionic acid and butyric acid in the rumen of karakul sheep increased significantly in 0-4hours（P<0.05）.The concentration of acetic acid and acetic acid/propionic acid in rumen was decreased by adding glycyrrhizic acid monoammonium salt,and the concentration of propionic acid in rumen was increased.There was no significant difference between butyric acid and TVFA（P>0.05）.Test 3:The effect of glycyrrhizic acid monoammonium salt on digestion and metabolism and methane production of Karakul sheep was determinedThe experimental animals and groups were the same as the second part.Fecal samples were collected at 5-12 days and 35-42 days of feeding period to determine the apparent digestibility of nutrients,and SF₆tracer method was used to collect and determine methane emission from Karakul sheep at 15-25 days and 45-55 days of feeding period.The results showed that the apparent digestibility of DM,OM and CP of Karakul sheep was not significantly affected by the addition of glycyrrhizic acid monoammonium salt（P>0.05）.Compared with the control group,ADF had a decreasing trend,but the difference was not significant（P>0.05）;adding glycyrrhizic acid monoammonium salt significantly reduced the daily methane emission of sheep,the methane emission of stage I treatment group was significantly lower than that of the control group（P<0.05）,and the methane emission of stage II treatment group was lower than that of the control group,but the difference was not significant（P>0.05）.The addition of ammonium glycyrrhizinate also reduced the daily methane emission per unit dry matter intake.Test 4:The effect of glycyrrhizic acid monoammonium salt on rumen microorganisms and related methane pathway metabolism genes of Karakul sheepIn this experiment,protozoa count and metagenome sequencing were used to study the effect of glycyrrhizic acid monoammonium salt on the number and structure of rumen microorganisms in Karakul sheep,and to explore the microbial mechanism of reducing methane production by glycyrrhizic acid monoammonium salt.The experimental animals and groups were the same as those in the second part.Rumen fluid was collected twice for protozoa counting and metagenome sequencing.The results showed that the number of protozoa at 0,4,6 and 8 h after feeding was significantly reduced by adding glycyrrhizic acid monoammonium salt in stage I（P<0.05）,but only 6 h after feeding was added in stage II.The number of protozoa in treatment group was significantly lower than that of the control group（P<0.05）;the species abundance and functional gene abundance PCo A analysis showed that there was a significant difference between the treatment group and the control group in stage I（P<0.05）,but the difference between the treatment group and the control group in stage II gradually narrowed,and there was no significant difference between the two groups（P>0.05）.In phase I,the addition of ammonium glycyrrhizinate significantly inhibited the growth of Methanobrevibacter,Methanosphaera,Prevotella,Bacteroides and methane metabolism genes（P<0.05）,but in phase II,the inhibition of ammonium glycyrrhizinate gradually decreased Low.Test 5:The effect of glycyrrhizic acid monoammonium salt on physiological and biochemical indexes of Karakul sheep was determinedThe animals and groups were the same as the second part.Blood samples were collected from sheep at 0 d,29-30 d and 59-60 d of pre feeding period to determine the indexes of blood antioxidant capacity and blood immunity.The results showed that the activities of GSH-Px,SOD and T-AOC in serum increased with the addition of glycyrrhizic acid monoammonium salt.The activity of T-AOC in treatment group was significantly higher than that in control group and before pre feeding（P<0.05）,but there was no significant difference between GSH-Px,SOD treatment group and control group and before pre feeding（P>0.05）;The level of serum MDA was decreased by adding glycyrrhizic acid monoammonium salt.There was significant difference between stage I treatment group and control group and before pre feeding（P<0.05）,but no significant difference between stage II treatment group and control group and before pre feeding（P>0.05）.The content of Ig G,Ig M and IL-2 in serum was increased by adding glycyrrhizic acid monoammonium salt,but there was no significant difference between two groups（P>0.05）.ConclusionAbove all,,monoammonium glycyrrhizinate can improve rumen environment,reduce the concentration of NH₃-N,acetic acid and the ratio of ethylene to propylene,increase the concentration of propionic acid,reduce methane emission,reduce the abundance of rumen microorganisms and related metabolic function genes related to methane emission,and improve the antioxidant and immune capacity of Karakul sheep,but its effect on ruminants is time-dependent.