| Animal production systems(specifically ruminants)produce a large proportion of ammonia(NH3)and nitrous oxide(N2O)into the atmosphere,where more than 70%of feed nitrogen is excreted.The sources of gaseous nitrogen emissions are urine and feces discharged by animals during grazing or captivity.It has been reported that nutrition control and precise feeding can improve nitrogen utilization and efficiency of ruminants,especially reduce the dietary nitrogen level.The current research about the feeding strategy of dietary soluble protein levels is mainly focused on dairy cows.However,to date,few published studies have reported a suitable ratio of dietary soluble protein levels in sheep production.Therefore,this experiment combined with in vivo and in vitro experiments to explore the effects of different dietary SP ratios on nitrogen metabolism and rumen microorganisms of fattening Hu sheep.Experiment 1.Effect of substrate soluble protein levels on rumen microbial fermentation and nitrogen utilization microorganisms in vitroThis part of the experiment aims to study the effects of different SP substrates levels on rumen microbial fermentation and nitrogen utilization microorganisms in vitro.The rumen fluid was collected from three Hu sheep with permanent rumen fistula.In the experiment,four kinds of substrates with different SP proportions,namely S20,S30,S40 and S50,the corresponding SP(%CP)are 20,30,40,50 respectively,the CP content of the substrates in each group is relatively consistent.To detect the rumen fermentation parameters,the culture medium was collected at 2,4,8,12,and 24 h.The results showed that:1)The pH value gradually decreased with time,but the difference between the treatments was not significant(P>0.05);NH3-N concentration in S50 remained higher during the 4-24h period,the NH3-N concentration was higher than the other three treatment groups except for 8h(P<0.05),and at 24h,with substrate SP(%CP)increase,NH3-N concentration showed a quadratic effect(R2=0.6616,P<0.01);the concentrations of TVFA,acetic acid,propionic acid and valeric acid are all higher in S30 and S40(P<0.05).In addition,with the SP(%CP)increased,they showed a quadratic effect(P<0.05).2)The DMD at 24h ranked:S30>S40>S20>S50.On CPD,S30 and S40 were higher than S20 and S50(P<0.05),and with SP(%CP)increased,CPD showed a quadratic effect(P<0.05).3)The microorganisms in the culture fluid quantitatively show that Bacteroides,Protozoa,Prevotella and Ruminobacter amylophilus have significant differences among SP treatments(P<0.05),and with SP(%CP)increased,they showed a quadratic effect(P<0.05),which showed that S30 and S40 were relatively higher.In summary,when the substrate SP(%CP)is 30 or 40,the TVFA,acetic acid and propionic acid content are increased,DMD,CPD and the expression of nitrogen utilization-related microorganisms are increased.Experiment 2.Effects of soluble protein levels in low-nitrogen diets on growth performance and nitrogen metabolism of fattening Hu SheepThe experiment aimed to study the effects of reducing dietary protein level and changing SP level on growth performance,apparent digestibility and nitrogen metabolism of fattening Hu sheep.Thirty-two six-month-old healthy fattening Hu sheep with an initial body weight of 40.37 ± 1.18 kg(BW ± SD)were selected and randomly divided into four treatments:The control diet(CON)with a 15%CP contents constructed to meet all requirements,low Protein A(LPA),LPB and LPC contain 13.9%,13.8%and 13.8%of CP,where SP(%CP)are 22.2,28.5 and 33.3 respectively.The results showed that:1)There was no significant difference between the feed intake and growth performance of each treatment(P>0.05),and the platelet and creatinine showed a linear effect in different SP treatment groups(P<0.05);2)At 14d,DM digestibility was highest in LPC group,which was higher than CON and LPA groups(P<0.05).The CP digestibility of LPB was higher than CON and LPC(P<0.05).At 28d,DM and CP digestibility of LPB were higher than CON and LPA(P<0.05),3)The serum urea nitrogen in low protein treatments was lower than CON,while the urinary urea nitrogen of LPA and LPB were lower than LPC and CON(P<0.05).In terms of nitrogen retention and nitrogen utilization,the LPB was higher than CON and LPC(P<0.05).To sum up,low-protein diets could reduce serum urea nitrogen and without affecting the growth performance of fattening Hu sheep,however,the CP digestibility is higher in LPB,and the nitrogen retention and nitrogen utilization are higher,which indicated that SP(%CP)is 28.5%,there is an increasing trend in nitrogen utilization performance.Experiment 3.Effects of soluble protein levels in low-nitrogen diets on rumen fermentation and bacterial microbiota of Fattening Hu sheepBased on experiment 2,this part of study further explores the effects of different SP levels in low-protein diets on the rumen fermentation and bacterial microbiota of the fattening Hu sheep.The results showed that:1)The NH3-N concentration of the three low-protein treatment groups(LPA,LPB and LPC)was lower than CON group(P<0.05),and the NH3-N concentration increased linearly with dietary SP level(P<0.05);TVFA,acetic acid and valeric acid all decreased in the LPA group(P<0.05),but butyric acid,isovaleric acid and A/P in LPA were lower than CON and LPB(P<0.05),and with dietary SP increased,TVFA,acetic acid,butyric acid,isobutyric acid,isovaleric acid and A/P showed quadratic effects(P<0.05).2)The microbiome results showed that compared with other treatments,the LPB increased the ACE index and Chao 1 index(P<0.05),and with dietary SP increased,the ACE index and Chao 1 index showed quadratic effects(P<0.05).PCoA showed that rumen microbial population in LPB group was different from LPC(P<0.05),while no difference was found among other treatments(P>0.05);LEfSe analysis showed that p_Proteobacteria was enriched in LPA(LDA>3,P<0.05),p_Elusimicrobia was enriched in LPB(P<0.05),while p_Firmicutes was enriched in LPC(P<0.05);there was a strong correlation between rumen microbiota and rumen fermentation parameters,and the main factors were related to VFA.3)The prediction of nitrogen metabolism-related functions showed that the LPB group improved the degradation of valine,leucine and isoleucine,histidine metabolism and tyrosine metabolism(P<0.05),and there is also a strong correlation between the microbiota and nitrogen metabolism-related pathways(P<0.05).In summary,the low-protein diet treatment reduced NH3-N concentration,which linearly increased with the increase of SP(%CP);while SP(%CP)of 28.5 increased the concentration of TVFA,acetic acid and butyric acid;meanwhile,this proportion increased the Alpha diversity of rumen bacteria microbiota,and with the increase of SP level,the microbial structure gradually changes;the metabolism of amino acids mediated by rumen microorganisms increased with the increase of SP level,mainly including the degradation of valine,leucine and isoleucine;histidine metabolism and tyrosine metabolism.In conclusion,in vitro study has shown that when SP(%CP)of substrate is 30 or 40,there is a tendency to increase the nitrogen utilization;and animal feeding experiments have shown that when the SP(%CP)of the low protein diets is 28.5,it can increase the nitrogen retention.Meanwhile this proportion also increased the Alpha diversity of rumen bacteria,and the microbial structure changes with the level of SP.The amino acid metabolism mediated by rumen microorganisms is mainly reflected in valine,leucine and isoleucine acid degradation;histidine metabolism and tyrosine metabolism,which suggests that the changes in amino acid metabolism pathways may be a potential reason for improving nitrogen utilization. |
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