Effects Of Dihydropyridine Addition On Rumen Fermentation And Ruminal Bacterial Community Of Dairy Cows In Different Stress Levels

When cows are feed in central China, the relatively high temperature and humidity in here as well as the extra heat produced by the rumen fermentation process can induce a significant decrease in DMI, which can subsequently cause sever stress to Holstein cattle originated in cold northern Europe. The present study was conducted to investigate the influence of dihydropyridine (DHP) on antioxidant status, rumen fermentation and ruminal bacterial community of dairy cows in different stress levels. The four experiments of this study are as follow.1 Effects of DHP on antioxidant status of dairy cows in different stress levelsThe THI72 indext, metabolin and enzyme content in serum and rumen liquid were tested to determine the anti-oxidative status in dairy cows. There were four periods in this research:unstressed without DHP (NS-ND), unstressed with DHP (NS-D), stressed without DHP (S-ND), stressed with DHP (S-D). Cows were fed with DHP for 3.0g/d per cow. With increasing levels of stress, serum catalase, total antioxidant capacity, the level of serum glutathione peroxidase and superoxide dismutase decreased, with a significant difference between NS-ND and S-ND period (P<0.01), while the serum malondialdehyde was increased in S-ND period (P<0.01). Compare to the NS-D period, serum superoxide dismutase were higher than in NS-ND, S-ND, S-D period (P<0.01), enhanced by 6.50U//mL、37.76tU/mL、18.48U/mL. The level of serum glutathione peroxidase was highest in NS-D period (P<0.01). There was a tendency for increasing of total antioxidant capacity due to DHP in NS-D and S-D period (P=0.08). DHP tended to decrease malondialdehyde value (P=0.14) and increase catalase activity (P=0.20). The results indicated that the antioxidant parameters had a similar trend in the rumen, as in the body, they were more sensitive. From the present study, it is inferred that inclusion of DHP in the diets could increase antioxidant ability in dairy cows.2 Influence of DHP and stress on the rumen fermentation of dairy cowsThe rumen fluids were assessed to determine the effects of DHP and stress on rumen fermentation. The results indicated that there was a decrease tendency of ruminal pH values with the increase of the environmental THI and the DHP addition, but all values were in the normal range. Compared to the NS-ND period, NH3-N concentration was higher than in the S-ND period (P<0.01). DHP addition decreased ruminal NH3-N concentration in spring and summer (P<O.01), reduced by 29.8%,29.4%. Concentration of total VFA in the rumen was reduced significantly in summer (P<0.01). Addition of DHP significantly increased VFA production (P<0.01). The proportion of acetic acid among other VFA was reduced throughout the stress shifts (P>0.05), while propionic and butyric acids, demonstrated the same tendency, with the former and the latter increasing with the environmental stress rising (P>0.05). Accordingly, the ratio of acetic acid to propionic acid was decreasing with the stress increased (P=0.13). The proportion of propionic acid was increased significantly in NS-D and S-D period (P<0.05), while proportion of propionate decreased (P<0.05) and butyrate increased (P>0.05) in S-D period. The ratio of acetic acid to propionic acid tended to increase due to DHP addition (P<0.01). It is indicated that DHP can accelerate the uptake rate of rumen on the NH3-N and improve rumen fermentation pattern.3 Effects of feeding DHP on rumen microbial populations of dairy cows in different stress levelsThe rumen microbial communities were analyzed by Real-Time PCR technique. Populations of rumen Fungi, Protozoa, Methanogens, B.fibrisolvens, C.proteoclasticum, F.succinogenes, R.albus and R.flavefaciens were expressed as a proportion of total rumen bacterial 16S rDNA. The results indicated that the population of B.fibrisolvens relative to total bacterial 16S rDNA was inhibited markedly in S-ND period compared to NS-ND period (P<0.01). The quantity of C.proteoclasticum was reduced (P<0.01) by 83.0%. An interaction between stress and DHP was observed on B.fibrisolvens and C.proteoclasticum (P<0.01). Protozoa population was decreased markedly in S-ND compared NS-ND (P<0.01). Supplementation of DHP significantly increased the populations of B.fibrisolvens, C.proteoclasticum and Protozoa relative to total bacterial 16S rDNA. Both stress (P=0.04) and DHP (P<0.01) had great effect on Methanogens reduced, indicated that both of them had significant inhibitory effect on it. There was a tendency for increasing of the quantity of R.albus (P=0.83) and R.flavefaciens (P=0.60) due to DHP in NS-D period. Both F.succinogenes and Fungi was decreased (P<0.01) in S-ND period compared to NS-ND period. Supplementation of DHP significantly increased the populations of F.succinogenes (P<0.05) and Fungi (P<0.01) relative to total bacterial 16S rDNA in NS-D period, an interaction between stress and DHP was observed on F.succinogenes and Fungi (P<0.05). It is concluded that the stress may result in inferior effect on rumen microorganism, while the DHP addition could stimulated the fermentation activity of cellulolytic bacteria and biohydrogenation associated bacteria, whereas reduced the activity of the rumen Methanogens.4 Dynamics of bacterial community associated with DHP and stress in the rumen analyzed by PCR-DGGE analysisMolecular methods based on the 16S rDNA gene were used to study the effects of dihydropyridine addition on ruminal bacterial community of dairy cows in different stress levels. The analysis of DGGE profile revealed that, as compared with the summer teams, the spring teams showed a higher degree of bacterial diversity. The DHP treatments increased new bands in NS-D and S-D periods. A clone library was created from 16S rDNA. From the library,9 clones had their V6-V8 regions matched predominant bands on the DGGE gel and their 16S rDNA were then sequenced and subjected to an online similarity search. Eight of the clones showed their similarities over 99% with database sequences, with the other one was similar to unidentified rumen bacteria, and this one had its similarities over 99% with database sequences. Substrates-associated bacterial community showed that the majority of clones fell into the Proteobacteria and Firmicutes. As compared with the control, Lysobacter and Xanthomonas became the new predominant bands after DHP addition. Results indicated that addition of DHP was effective in stimulating the growth of some ruminal bacteria.