Effects Of Carbohydrate Source On Nitrogen Utilization,Urine Metabonomics And The Expression Of UT-B And Aquaporin In Rumen Epithelial Of Lactating Dairy Cows

Dietary carbohydrate as the main energy source of ruminants has different effects on nitrogen metabolism of lactating dairy cows because it'sdifferentcomponents.The study was conducted to discuss the effect of different sources of carbohydrates on nitrogen metabolism in lactating dairy cows from four aspects of production and utilization,rumen fermentation,urinemetabolomics and the expression of UT-B and aquaporin.And expected to inquiry the mechanism of carbohydrate influence on nitrogen metabolism in lactating dairy cows and explore thenew nutritional regulation approach of reducing nitrogen emission.Trial 1.Seventy-two healthy Holstein lactating cows in similar weight,parity and milk yieldwere randomly assigned to 9 groups to receive L₉?3⁴?orthogonal experimental design treatment to study the effect of different carbohydrates and rumen protected amino acid on lactation performance,energy metabolism and nitrogen utilization of Holstein lactating cows.The L₉?3⁴?orthogonal experimental design treatments consisted of dietary carbohydrate sources?flaked corn-A1,wheat bran-A2 and sugarcane molasses-A3?,additive amount of carbohydrates?0.5kg/d·head-B1,1.5kg/d·head-B2 and 3.0kg/d·head-B3?and rumen protected amino acids((RPMet 5g/d·head +RPLys 15 g/d·head-C1,(RPMet 10g/d·head +RPLys 30 g /d·head-C2,?RPMet 20g/d·head +RPLys60 g/d·head-C3?.The results showed thatthe diets added flaked corn compared to adding molassessignificantly improve the milk yield,4% fat corrected milk yield,milk fat yield,lactose yield and lactose rate?P<0.05?of lactating dairy cows.While the milk yield and milk protein yield inwheat bran group was significantly higher than that of molasses group?P<0.05?.The additive amount of carbohydrates had significant effect on milk protein percentage and lactose yield?P<0.05?.Different rumen protected amino acids added level had significantly effect on milk yield,4% fat corrected milk yield,milk protein percentage,milk protein yield,lactose percentage,lactose yield,milk total solid content?P< 0.01?,but no effect was observed on milk fat percentage?P>0.05?.Different carbohydrate sources had no significant effect on concentrations of plasma urea nitrogen,glucose,TG and NEFA,BHBA,insulin and IGF-1?P>0.05?,but plasma NEFA and BHBA concentrations were significantly influenced by the additive level of carbohydrate and rumen protected amino acid?P<0.05?.No effects were observed of carbohydrate source and add level as well as rumen protected amino acid on nitrogen intake,fecal nitrogen,nitrogen excretion and nitrogen apparent digestibility?P>0.05?.while in flaked corn and sugarcane molasses treatment,the concentration of MUN and UUN were decreased compared with the wheat bran treatment?P<0.05?and the flaked corn treatment group had greatest nitrogen apparent utilization and MNE?P<0.05?.Compared the other supplementation levels,when the carbohydrate supplementation level was 1.5kg/head·d,the nitrogen conversion efficiency was the highest?P<0.05?.Rumen protected amino acids added level had significant effect on milk nitrogen and MUN concentrations?P<0.05?,the UN and MUN was decresaed and the MNE was improved when added RPMet RPMet 10g/d·head +RPLys 30 g /d·head.Trial 2.On basis of the Trial 1,the treatment factor which had significant effect on MNE were selected and then designeddietary treatment FC group: 1.5kg/d/head flaked corn + based diets,WB groups: 1.5kg/d/head wheat bran + based diet,SM group: 1.5kg/d/head sugarcane molasses + based diets.The experimental design was an 3×3 Latin square with six permanent ruminally cannulated Chinese Holstein dairy cows to investigate the effects of carbohydrate source on rumen fermentation parameters and microbial population in lactating dairy cow.The test was conducted in 3 periods and the each period lasted 16 dwhich was the experiment period 12 d,and sampling period 4d.The results showed that different carbohydrate sources could significantly affect rumen fermentation parameters of lactating dairy cows,and the rumen p H value were decreased significantly?P<0.05?while the VFA molar concentration and ruminal NH₃-N concentration were increased significantly?P<0.05?in FC and WB treatment compared with the SM treatment.The molar concentration of acetate,propionate and butyrate in FC and WB treatment were significantly higher than those of molasses treatment group?P<0.01?,and the molar concentration of acetate in FC group were significantly higher than that of wheat bran treatment group?P<0.01?,propionate and butyrate molar concentration higher than that of wheat bran treatment group,but the difference was not significant?P>0.05?.SM treatment group compared with the other two groups are higher inacetate: propionateratio?P<0.05?.The populations of B.fibrisolvens? R.flavefaciens?R.albu?R.amylophilus?Prevotella ruminicola?S.bovis in rumen fluid were not affected by different carbohydrate sources.The population of protozoa in SM treatment was significantly higher than that of FC treatment group?P < 0.05?.Trial 3.The effect of different carbohydrate sources on the urinary metabolomes of lactating dairy cows was investigated using a GC-MS platform.Six Chinese Holstein dairy cows with permanent rumen fistula were chosen as the experimental animals and allocated using a 3 × 3 Latin square design.The animals received three dietary treatments: 1.5 kg/kg/d/head flaked corn + basal diet?group A?,1.5 kg/kg/d/head wheat bran + basal diet?group B?,and 1.5 kg/kg/d/head molasses + basal diet?group C?.The experiment was conducted in three stages,and each stage lasted 16 d,including a pre-trial period of 14 d and a sampling period of 2 d.The urinary metabolomes of the dairy cows displayed a good separation trend with the three dietary treatments,as revealed by principal component analysis,partial least-squares discriminant analysis,and orthogonal partial least-squares discriminant analysis.Differential metabolites were selected?VIP> 1,P<0.05 by t-test?,and 41 differential metabolites were obtained after merging of identical metabolites.The disturbed metabolic pathways were identified by KEGG pathway enrichment analysis of differential metabolites,including arginine and proline metabolism;glycine,serine,and threonine metabolism;tyrosine metabolism;tryptophan metabolism;purine metabolism;pyrimidine metabolism;glyoxylate and dicarboxylate metabolism;glycerolipid metabolism;and the pentose phosphate pathway.Trial 4.The effect of carbohydrate source on urea transporter UT-B andaquaporin AQP3 and AQP7 and AQP9 expression in rumen epithelial of lactating dairy cow were explored by Western blot.Six Chinese Holstein dairy cows with permanent rumen fistula were chosen as the experimental animals and allocated using a 3 × 3 Latin square design.The animals received three dietary treatments: 1.5 kg/kg/d/head flaked corn + basal diet?group A?,1.5 kg/kg/d/head wheat bran + basal diet?group B?,and 1.5 kg/kg/d/head molasses + basal diet?group C?.The experiment was conducted in three stages,and each stage lasted 16 d,including a pre-trial period of 15 d and a sampling period of 1 d.The results showed that the relative expression of UT-B in WB group was higher than that of FC and SMtreatment,but there were no significant differences among the treatment?P>0.05?.Different carbohydrate sourceshad significant effect on AQP3 expression in rumen epithelial?P<0.05?,the relative expression of AQP3 in WB and SM treatmentwas significantly higher than that of FC?P<0.05?,and relative expression between the two treatment had no significant difference?P>0.05?.No significant effect was observed in the relative expression of AQP7 and AQP9 in rumen epithelial of different sources of carbohydrates?P>0.05?.In summary,different sources of dietary carbohydrates have effect on the nitrogen utilization efficiency of lactating dairy cows.Among the flaked corn,wheat bran and sugarcane molasses,the flaked corn have the optimal effect on improving the nitrogen use efficiency,and the optimum addition was 1.5kg/head?d.Different carbohydrate sources could significantly affect rumen fermentation parameters of lactating dairy cows,but did not have a significant impact on the number of main functional bacteria in the rumen.The urinary metabolomes analysis showed that the disturbed metabolic pathways were identified by KEGG pathway enrichment analysis of differential metabolites,including arginine and proline metabolism;glycine,serine,and threonine metabolism;tyrosine metabolism;tryptophan metabolism;purine metabolism;pyrimidine metabolism;glyoxylate and dicarboxylate metabolism;glycerolipid metabolism;and the pentose phosphate pathway.Different carbohydrate sourceshad effect on the expression of UT-B and AQP3,AQP7 and AQP9 in rumen epithelial cells,while only the AQP3 expression in rumen epithelial has the significant difference.