Preparation Of Probiotics And Enzyme Compound Additive And Its Effects On Growth Performance And Rumen Microbiota Of Hu Sheep

Probiotics and enzymes are widely used as feed additives to improve animal growth performance due to their good effects on livestock and poultry.In this study,Aspergillus oryzae,Candida utilis and cellulase were optimized to prepare probiotics and enzyme compound additive,and its effects on growth performance,rumen fermentation parameters and rumen microbiota of fattening Hu sheep were studied,so as to provide a basis for the development and application of probiotics and enzyme compound additive in Hu sheep.This study mainly includes the following four parts:（1）Optimization of fermentation conditions of Aspergillus oryzae,Candida utilis and cellulase;（2）Effects of probiotics and enzyme compound additive on growth performance,serum biochemical indexes and economic benefits of fattening Hu sheep;（3）Effects of probiotics and enzyme compound additive on rumen fermentation parameters and digestive enzyme activity of fattening Hu sheep;（4）Effect of probiotics and enzyme compound additive on rumen microbiota of fattening Hu sheep.The results are as follows:（1）The compound ratio of Aspergillus oryzae,Candida utilis and cellulase was designed by orthogonal test.The effects of Aspergillus oryzae,Candida utilis and cellulase on neutral detergent fiber（NDF）,acid detergent fiber（ADF）,cellulose and hemicellulose in straw were measured,and the optimal compound ratio was determined with the degradation rate of cellulose as the main index.The results showed that the optimum ratio of bacterial enzyme complex was Aspergillus oryzae 1×10⁶ CFU/g,Candida utilis 1×10⁶ CFU/g,cellulase 30 FPU/g air dry matter,the degradation rate of cellulose increased significantly（P < 0.05）,reaching 29.41%.The control group and the optimal compound proportion group were fermented for 7 days.The optimal fermentation time was determined by measuring the degradation rate of cellulose,cellulase activity,reducing sugar content,the number of Aspergillus oryzae spores and the number of live Candida utilis producing bacteria in the fermentation system at 1,3,5 and 7 days.The results showed that the degradation rate of cellulose was the highest on the third day of fermentation,reaching 31.91%,which was significantly higher than that on the first day of fermentation（P < 0.05）;The highest cellulase activity and reducing sugar content（P < 0.05）were the best fermentation time.（2）Effects of probiotics and enzyme compound additive on growth performance,serum biochemical indexes and economic benefits of fattening Hu sheep: 156 male fattening Hu sheep aged 60 days,weighing about 17 kg and in good health were randomly divided into 4 groups,with 3 repetitions in each group and 13 sheep in each repetition.Group A: control group,fed basic diet;groups B.C and D were added with 0.1%,0.2% and 0.3% probiotics and enzyme compound additive respectively.The results showed that compared with group A,the average daily gain（ADG）of group D at 30 ～ 60 days and 1 ～ 60 days increased significantly（P < 0.05）,and the feed weight ratio（F/G）decreased significantly（P < 0.05）.In terms of apparent digestibility of nutrients,the digestibility of ADF in group B,C and D increased significantly（P < 0.05）,and the digestibility of NDF in group B and C increased significantly（P < 0.05）.In terms of serum biochemical indexes,compared with group A,the content of alanine aminotransferase（ALT）in group B decreased significantly（P < 0.05）,and the content of ALT in group D increased significantly（P < 0.05）;The contents of aspartate transferase（AST）,total protein（TP）and globulin（GLO）in group C and group D increased significantly（P < 0.05）;The content of low density lipoprotein cholesterol（LDL-C）in group B and group C decreased significantly（P < 0.05）.The profit of adding 0.3% probiotics and enzyme compound additive is the highest.（3）Effects of probiotics and enzyme compound additive on rumen fermentation parameters and digestive enzyme activity of fattening Hu sheep: through the determination of rumen fermentation parameters and digestive enzyme activity,the results showed that compared with group A,rumen pH in group D decreased significantly（P < 0.05）,the content of ammonia nitrogen（NH₃-N）in group B,group C and group D increased significantly（P < 0.05）,the contents of acetic acid,propanoic acid and isobutyric acid decreased significantly（P < 0.05）,and the contents of butyric acid in group B and group D decreased significantly（P < 0.05）;The activities of protease,amylase and cellulase in rumen of group B,group C and group D were significantly increased（P < 0.05）.（4）Effect of probiotics and enzyme compound additive on rumen microbiota of fattening Hu sheep: 16 S rRNA was used to sequence rumen bacteria in group A and group D.,the results showed that probiotics and enzyme compound additive could change the structure and composition of rumen bacteria;Compared with group A,group D significantly reduced the abundance of Bacteroidetes and TM7（P < 0.05）,and significantly increased the abundance of Actinobacteria and Cyanobacteria（P < 0.05）;At the genus level,group D significantly increased the abundance of Olsenella（P < 0.05）and significantly decreased the abundance of YRC22（P < 0.05）;Correlation analysis showed that among the top 10 bacterial groups in rumen fluid,Treponema was significantly positively correlated with NDF digestibility and rumen NH₃-N content（P < 0.01）,Olsenella was significantly positively correlated with ADF digestibility（P < 0.05）,and negatively correlated with rumen pH and propanoic acid content（P < 0.05）.The rumen protozoa of group A and group D were sequenced by 18 S rRNA.The results showed that the Chao1 index and Observed＿species index in the Alpha diversity index of group D was significantly higher than that in group A（P < 0.05）,Compared with group A,at the genus level,the abundance of Entodinium,Polyplastron and Isotricha in group D increased significantly（P < 0.05）,and the abundance of Epidinium and Blepharocories decreased significantly（P < 0.05）;At the species level,group D significantly increased the abundance of Polyplastron sp.LDK-2011,Endodinium furca and Isotricha sp.LDK-2011（P < 0.05）,and significantly decreased the abundance of Epidinium ecaudatum,Endodinium bursa,Endodinium nanellum and Blepharocories angusta（P < 0.05）;The correlation analysis showed that among the top five protozoa at the rumen level,Entodinium was positively correlated with the ADG of fattening Hu sheep,and Epidinium was negatively connected with ADG;Polyplastron was favorably correlated with NDF digestibility（P < 0.01）,negatively correlated with the pH of rumen（P < 0.05）,and Isotricha was negatively correlated with the content of propanoic acid in rumen（P < 0.05）.In conclusion,probiotics and enzyme complex can improve the cellulose decomposition rate of straw in the fermentation system.Adding 0.3% probiotics and enzyme compound additive to the diet of fattening Hu sheep can improve the daily gain of fattening Hu sheep,reduce the feed weight ratio and improve the digestibility of ADF in the diet;increase the contents of TP and GLO in serum;reduce rumen pH and increase rumen NH₃-N content;reducing the abundance of Bacteroidetes in rumen and increasing the abundance of Olsenella were significantly positively correlated with ADF digestibility,which was helpful to improve the digestibility of fiber in diet;increasing the abundance of Polyplastron and Isotricha is helpful to improve the activities of cellulase and amylase in rumen and improve the utilization rate of diet.