Study On Mechanism Of Nitrogen Utilization In Rumen Of Dairy Cows With Alfalfa As Hay Or Silage

Five experiments were conducted to investigate the effects of alfalfa hay or haylage on ruminal fermentation, microbial synthesis, nitrogen utilization, difference of rumen bacteria flora in dairy cows, to explore possible mechanism on utilization of dietary protein in dairy cows with alfalfa hay and alfalfa silage.Experiment1, Rumen fluids were collected from three Holstein heifers which fitted with permanent rumen fistulas, and an in vitro batch culture was conducted to investigate differences in the fermentation characteristics. Alfalfa hay (AH) mixed concentrate with different ratio (0:100,25:75,45:55,55:45and75:25, respectively) in terms of24h gas production, volatile fatty acid production, ammonia nitrogen, total purines, efficiency of microbial protein synthesis, concentration of endopeptidase and exopeptidase, rumen gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus. Results show that acetate/total volatile acid ratio, ammonia nitrogen content and total bacterial count were significantly increased (P<0.05) with adding alfalfa hay; total purines content, Prevotellabryantii and Ruminobacter amylophilus gene expression quantities significantly decreased (P<0.05) with alfalfa hay treatment, tripeptidyl-peptidase concentration also was affected by alfalfa hay (P<0.05). Cysteine peptidase concentration and24h gas production significantly increased with the increase of alfalfa hay level (P<0.05), the total bacteria, Butyrivibrio fibrisolvens, Prevotellabryantii and Ruminobacter amylophilus gene expression quantities also affected by forage-to-concentrate ratio. Total bacterial count, Butyrivibrio fibrisolvens count and Ruminobacter amylophilus count were highest on55:45(AH: concentrate).Experiment2, In vitro batch culture was conducted to investigate differences in the fermentation characteristics alfalfa silage (AS) mixed concentrate with different ratio (0:100,25:75,45:55,55:45and75:25, respectively) in terms of24h gas production, volatile fatty acid production, ammonia nitrogen, total purines, efficiency of microbial protein synthesis, concentration of endopeptidase and exopeptidase, Rumen gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus. Results show that24h gas production, efficiency of microbial protein synthesis, aspartic peptidase, cysteine peptidase, dipeptidyl-peptidase concentration and Prevotellabryantii gene expression quantity were significantly reduced (P<0.05) with alfalfa silage treatment; ammonia nitrogen concentration, total bacteria and Butyrivibrio fibrisolvens gene expression quantities significantly increased by alfalfa silage (P<0.05). Microbial protein synthesis, aspartic peptidase, cysteine peptidase concentration were significantly decrease, ammonia nitrogen concentration got higher (P<0.05) with the increase of alfalfa silage level (P<0.05). In this trial, ammonia nitrogen concentration, total bacteria, Butyrivibrio fibrisolvens and Prevotellabryantii gene expression quantities also affected by alfalfa silage and forage-to-concentrate ratio. Butyrivibrio fibrisolvens count, Streptococcus bovis count, Prevotellabryantii count and Ruminobacter amylophilus count were highest on45%alfalfa silage mixed55%concentrate.Experiment3, Fresh alfalfa was harvested from AnHui province and then prepared as alfalfa hay (AH) and alfalfa silage (AS). In vitro batch culture was conducted to investigate differences in the ammonia nitrogen, total purines and volatile fatty acid production and gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus between AH and AS. Rumen fluids were collected from three healthy Holstein heifers, fitted with permanent rumen fistulas. Results indicate that the total purines of AH was significantly higher (P><0.05) than those of AS, efficiency of microbial protein synthesis of AH also higher than that of AS even though there was not significantly affect (P>0.05).Experiment4, Fresh alfalfa was harvested from AnHui province and then prepared as alfalfa hay (AH) and alfalfa silage (AS). In situ ruminal dry matter, crude protein and amino acid degradation characteristics were determined for AH and AS with three Holstein heifers, fitted with permanent rumen fistulas. Effective crude protein degradability was significantly greater in AH than in AS (P<0.05). AH has higher aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isleucine, leucine, tyrosine, phenylalanine, histidine, arginine and tryptophan and cystine effective degradation rate (P<0.05) than AS. Lysine and methionine effective degradation rate in AH were higher than those in AS (P>0.05).Experiment5, Collected rumen fluid with alfalfa hay or alfalfa silage were incubated24h by using in vitro batch culture, tested the microbial flora distribution by using high-throughput sequencing technology after DNA were extracted. Results show that microorganisms OTUs class of alfalfa hay group had large cross with that of alfalfa silage group. Microorganism OTUs number of AH was higher than that of AS, AHgroup contains5386OTUs, AS group contains4633OTUs, including both cross3908OTUs with the cutoff value of0.10. AH contains19544OTUs, AS contains16099OTUs, including both cross8806OTUs with the cutoff value of0.03. Thus, microbial flora class level in AH and that in AS was similar, bacteroides, clostridia, β-proteobacteria and fibrobacteria were major microorganisms.