Effects Of Nitrate On Rumen Fermentation, Microbe Diversity,Blood Biochemical And Antioxidative Ability

Nitrate could not only provide nitrogen for ruminants as non-protein nitrogen, but also inhibit methane production effectively. However, the possible nitrite toxicity hindered nitrate utilization in ruminant production system. Therefore, it is very important to determine the safety additional dose of nitrate and its effects on rumen fermentation, feed degradability and microbes. In this study, four experiments were conducted to study effects of nitrate addition level on rumen fermentation, microbe diversity, and animal health of nitrate-adapted steers to provide useful information for long-term application of nitrate in ruminant.Expt.2was carried out to evaluate influence of nitrate on rumen fermentation parameters, nutrient degradability and main digestive enzymes’activity. Nine rumen-fistulated steers (450±20kg) were fed diets supplemented with0%(Control, ONR),1%(1NR), and2%(2NR) nitrate (on dry matte basis) randomly. After two-week nitrate adaptation, rumen fluid was collected to determine fermentation parameters and enzyme activity. Besides, nutrient degradability of Chinese wildrye, steam-flaked corn and soybean meal were studied with the nylon bag technique. Results showed that nitrate supplementation linearly increased total volatile fatty acids (L;.P=0.01) and acetate (L; P=0.01) concentration obviously, and butyrate (Q; P=0.01) and isovalerate (Q;P=0.04) concentration quadraticly. Effective degradability (ED) of DM (EDDM)(L; P=0.01) and ED of NDF (L; P＜0.01) of Chinese wildrye were linearly increased by nitrate. Treatment1NR greatly changed rapidly degradable fraction of DM (aDM)(Q; P＜0.01) and potentially degradable fraction of DM (bDM)(Q; P=0.01) of steam-flaked corn, and its ED of CP (EDCP) were linearly raised by nitrate (L; P＜0.01). The EDDM (P<0.01) and EDCP (.P＜0.01) of soybean meal were linearly increased with nitrate addition level increasing. Xylanase activity was greatly increased by nitrate (P<0.05) and β-glucanase activity of2NR was much higher than that of the control (P＜0.01). Protease-6h activity of rumen fluid were obviously decreased by nitrate (P=0.04). Treatment1NR obviously increased amylase-6h activity (P=0.03). In conclusion, rumen fermentation of nitrate-adapted steers was promoted by nitrate addition. Nitrate obviously increased cellulose activity (xylanase activity, CMCase activity and β-glucanase activity), resulting in the increased DM and NDF degradability of Chinese wildrye. The DM degradation components of steam-flaked corn were changed by nitrate, which was related to the changes of amylase activity. Nitrate inhibited rumen fluid protease activity, while CP degradation of stem-flaked corn and soybean meal was greatly enhanced by nitrate addition.Expt.2was conducted to examine changes of rumen microbe abundance and bacteria biodiversity caused by nitrate addition. Six rumen-fistulated steers (450±20kg) were fed diets supplemented withO%(Control, ONR),1%(1NR), and2%(2NR) nitrate (on dry matter basis) in succession. Results showed that total bacteria number was not changed (.P=0.06), while protozoa abundance were obviously increased in nitrate treatments (L; P=0.01). Fungi number in2NR was much higher than that in the control (P<0.01. Nitrate increased Ruminobacter flavefaciens (P<0.01) and Fibrobacter succinogenes (P=0.02) abundance. Butyrivibrio fibrisolvens amount was declined by nitrate (P＜0.01). The alpha (P>0.05) and beta biodiversity of ruminal bacteria were not affected. Firmicrutes and Bacteroidetes were two main groups in the rumen, accounting for88.23%of total bacteria. Ruminobacter and Sphaerochaeta abundance were raised by1%nitrate addition level. Campylobacter fetus (.P<0.01), Selenomonas sruminantium (P=0.02) and Mannheimia succiniciproducens (P=0.04) were core nitrate reducing bacteria in steers and their abundance were increased linearly along with nitrate addition level. Potential nitrate reducer in the rumen, Campylobacter genus (P<0.01) and Cyanobacteria phyla (P=0.01) were significantly raised by nitrate. In conclusion, nitrate significantly increased rumen protozoa and fungi abundance. Bacteria abundance was not affected while its composition was greatly changed: cellulolytic bacteria (R. flavefaciens and F. succinogenes) abundance was improved by nitrate addition; low nitrate addition level increased non-fiber degrader genus (Ruminobacter and Sphaerochaeta). C. fetus, M. succiniciproducens and S. ruminantium were main nitrate reducing bacteria in nitrate-adapted steers.Expt.3was to examine effects of nitrate on rumen methane production, methanogen abundance and composition. Experimental design was the same as Expt.2. After two-week adaptation, rumen gas composition, methanogen abundance and composition were studied. Results showed that nitrate linearly decreased rumen in vitro6h,12h, and24h methane production (L; P<0.01). Compared with the control, methanogen number in1NR and2NR treatments were decreased by4.47%and25.82%respectively. Methanomicrobiales (L; P=0.05) and Methanoplanus (L;.P<0.05) abundance were linearly decreased by nitrate, and Methanasarcinales abundance was increased (L; P＜0.01). Methanosphaera and Methanimicrococcus abundance in2NR were much higher than that in the control. In conclusion, nitrate decreased rumen methane production significantly and methanogen abundance numerically, and methanogen composition was changed by nitrate. The inhibition of methane production was related with the reduction of methanogen number and changes of methanogen composition.Expt.4was conducted to study the effect of nitrate on blood biochemical, non-specific immunity and antioxidant indexes of nitrate-adapted steers. Experimental design was the same as Expt.1. After two-week adaptation, blood samples were collected at four hours after morning-feeding. Results showed that the non-specific immunity and antioxidant characteristics were not negatively affected by nitrate.2NR greatly increased red blood cells number (P=0.07), hematocrit value (P<0.01), hemoglobin (P＜0.01) and methemoglobin (P＜0.01). However, methemoglobin/hemoglobin ratio of nitrate treatments were not significantly different from the control (P=0.28). No difference were found in blood glucose (P=0.33), triglyceride (P=0.19) and cholesterol (P=0.96) concentration among three treatments. Total serum protein (p=003) and aspartic transaminase activity (P=0.02) of2NR were higher than that of the control. In conclusion, nitrate addition (less than2%) would not result in ruminant nitrite toxicity, and it did not show negative effect on non-specific immunity and antioxidant characteristics of nitrate-adapted steers.