| The experiment 1 investigated the effects of dietary nitrate dosage on ruminal nitrate disappearance rate and fermentation parameters.The randomized block design was used and six Hu-sheep with rumen fistula were fed with different levels of potassium nitrate:0%(control), 1%,2%,3%,4%,5% of DM intake. The concentration of blood methemoglobin was determined 2.0 h after feeding from the jugular vein; rumen fluid was obtained continuous 2 days after 14-d adaptation. The concentration of nitrate, nitrite and fermentation parameters in rumen fluid were test before feeding and 0.5,1.0,1.5,2.0,4.0,6.0,8.0 h after feeding. Net gas production of experimental diets was compared in vitro with rumen fluid from control. The results showed that the content of methemoglobin significantly increased with the nitrate addition (P< 0.01), and increased to maximum value (0.95-2.14 g/L) at 1.0 h after feeding. Nitrate addition also significantly increased pH value, ammonia nitrogen concentration and acetic acid/propionic acid ratio 2.0 h after feeding (P< 0.01). Potassium nitrate addition level over 3% of DM intake reduced the concentration of microbial protein and total volatile fatty acids.2% potassium nitrate addition had the highest microbial protein synthesis and total TVFA which was beneficial to rumen fermentation. In vitro study showed that there was no significant difference in the net gas production, theoretical maximum gas volume and gas speed among groups, however, the net gas production and theoretical maximum gas volume of 5% group tended to declined(P<0.1).The experiment 2 was to evaluate the effect of dietary potassium nitrate on bacterial diversity in vivo in sheep. Total DNA of rumen fluid was extracted for 16S rDNA Illumina sequencing and Real-time PCR analysis. Paired-End sequencing of 16S rDNA gene targeting showed that Chaol index of 5% group was significantly higher than control. No difference in Shannon index was observed. At the phylum level, the total percentage of Bacteroidetes, Firmicutes, Proteobacteria, Fibrobacteres, Verrucomicrobia was more than 90%. At the genus level, Prevotella, Selenomonas, Succinivibrio were dominant genus in each group, the average proportion were 47.87%,5.76%,2.37%, respectively. Real-time PCR showed increase in the relative multiples of Campylobacter fetus with nitrate concentration increased (P<0.01). These results indicate that nitrate addition ranged from 0% to 5% variefy the abundance of bacteria, especially nitrate reduction bacteria, but do not change rumen bacterial diversity and advantages community structure.The objective of experiment 3 was to evaluate the effect of diet supplementation with potassium nitrate on rumen fungi and protozoa diversity in sheep. Total DNA of rumen fluid was extractedfor 18S rDNA Illumina sequencing. Paired-End sequencing of 18S rDNA gene targeting showed that no difference in Shannon index and Chao 1 index was observed. In fungi genus, the total percentage of Saccharomyces, Malassezia, Phaeosphaeri, Davidiella, Issatchenkia was the most. Saccharomyces relative abundance of control group had significant higher than group 2%,3%,5%. Other fungi had no significant difference in genus. In protozoa genus, Unculture relative abundance of group 2% was significant higher than other groups (P<0.01). The result show that mycoflora reduction of nitrate ability may be weak, but protozoa had strong ability to reduction nitrate.In conclusion,2% dietarynitratelevel was most benefit to ruminal fermentation. Nitrate do not change rumen bacterial diversity and advantages community structure, but increased abandence of C.fetus. Saccharomyces was inhibited by adding nitrate.2% potassium nitrate significantly increased relative abundance of Unculture. |
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