AIBIBULA Yim amu，HALIDAIRehemujiang，MASAAK IHanada and MEIJIOkamoto

1.College of Grassland and Environmental Science，Xinjiang Agricultural University，Urumqi830052，China；2.Department of Agro-environmental Science，Obihiro University of Agriculture and Veterinary Medicine，Hokkaido 080-8555，Japan

1 Introduction

The source of dietary N and energy fed to ruminants significantly influences the utilization of N and energy in the rumen and nutrient flow to the small intestine.Well-managed temperate pasture herbage is characterized by high N contentand degradability in the rumen（Aibibula et al.2003；Bryant et al.2013）.The dietary N degraded in the rumen（RDN）is synthesized tomicrobial protein or absorbed from the rumenwall asammoniawhen there is surplus microbial protein synthesis.Several researchers（Minson，1990；Holden et al.1994；Elizalde et al.1998；Ulyatt et al，1988）have reported of high protein intake in well-managed temperate pasture and high proportion of protein degradability（60-80%）in the rumen.Although intake and apparent absorption of dietary N from fresh herbagemay be high，the quantity of dietary protein actually absorbed from the small intestine is not always high.A limitation in ruminal available energy and／or an excess of the dietary N degraded in the rumen（RDN）restrict the conversion efficiency of RDN to microbial N.The organic matter truly digested in the rumen（OMTDR）has been used as one of the parameters to estimate the ruminalavailable energy（Beever and Siddons，1986；Kolver et al.，1998，McCarthy et al.，1989）.Beever and Siddons（1986）suggested that the ratio of RDN to OMTDR should be approximately 25g／kg for optimal N utilization in the rumen.Consequently，metabolizable protein and AA supply to the small intestinemay be a limiting factor in the performance of high yielding ruminants in a grazing system even with an increase in intake and degradability of dietary N （Leaver，1985）.Several reports（Wilkerson etal.，1993；Titgemeyer and Loest，2001）have indicated that lack of some essential amino acids limits animal production.Our objective was to study the effects of fresh herbage on N and amino acids nutrition of steers grazing temperate pasturewithout supplementary feeds.

2 M aterials and methods

2.1 Pasture and grazingmethodOrchardgrass and meadowfescue pastures located at the experimental farm ofObihiro University of Agriculture and Veterinary Medicine in Hokkaido，Japan，were used in an intensive rotational grazing.Fifty-two digestion data were carried out using Holstein steers fitted with rumen，proximal duodenum and distal ileum （average body weight：321kg）and grazed on each pasture without supplementary feed from May to October alternatively，in the pass three years.The pastures were divided into several paddocks by using electric fences and the steers grazed each paddock daily.The steerswere rotated to a new paddock at0900 of every day.Steerswere able to accesswater and mineral block at all times，but other supplementary feed were notgiven to the steers during the experimental period.Chromic dioxide（Cr2O3）was used to estimate duodenal，ileal digesta flow and fecal output of steers.Each steer received twice daily（0900 and 1700）a gelatin bag containing 4.0 g of Cr2O3inserted into the rumen during the experimental period.

2.2 Samp le collectionEach experimental period lasted 23 days；the first 14 days were used to allow the steers to adapt to each new pasture and the following9 dayswere used for collecting samples.Herbagemass and composition of the grass weremeasured and herbage sampleswere collected before grazing daily during the first5 days of the sample-collection period.Herbagemass wasmeasured by cutting herbage within four quadrates（0.5m×0.5m）to ground level using hand-held electric grass clippers in each paddock.Herbage samples，plucked by hand to adjusted grazing height from many different areas were composited.Fecal samplewere collected at0900，1500，2100 and 0300 during the5 days and were composited on an equalweight basis for each steer.The composited sample of herbage and feceswere dried at60℃for 48 hrs in an air forced oven and then ground to pass a 1-mm screen and stored for subsequent analysis.Duodenal and ileal digestawere collected every four hourson the last2 daysof the sample-collection period.Each of duodenal and ileal digesta sample wasmixed on the fresh weight basis.The composited sample was divided into two portions；one half was lyophilized for subsequent general analysis and the otherwas acidified with 50%H2SO4 and frozen at-20℃until measurement of ammonium N concentration.The samples of ruminal fluid were obtained every four hours on day 8 of the sample-collection period.The samples of ruminal fluid collected were immediately measured for ruminal pH and then each sample was strained through four layers of gauze and acidified with 50%H2SO4 and frozen at-20℃ until analyzed.Ruminal fluid was also obtained at 1200 on the last day in each experimental period to obtain rumen bacteria fraction.The bacterial fraction was isolated using the method of Smith and McAllan（1974）stored at-20℃ and freeze-dried until appropriate chemical analysis.

2.3 AnalyticalmethodsHerbage，duodenal and ileal digesta，and fecal sampleswere analyzed for drymatter（DM），ash，N（crude protein）（AOAC，1990），neutral detergent fiber（NDF）and acid detergent fiber（ADF）（Goering and van Soest，1970）.The neutral detergent insoluble N and acid detergent insoluble N fractions were determined using N analysisof the neutral detergent and acid detergent residue.Herbage was analyzed for non-protein N and soluble true protein using the procedures of Licitra et al.（1996）.The ammonium-N concentration in the rumen fluid and duodenal digesta was determined by colorimetric method（Okuda and Fujii，1968）and urea N in serum was determined using a kit（Urea N TestWAKO；278-04801，WAKO Chemicals，Osaka，Japan）.In vitro DM digestibility of herbage was determined according to themethod of Tilley and Terry（1963）.Chromium concentration of duodenal，ileal and fecal sampleswere determined by phosphoric acid and potassium reagent method （Morimoto，1970）.Purine contents in rumen bacterial fraction and duodenum digestawere determined using the technique of Zinn and Owens（1986）.Herbage，each digesta and rumen bacteria sampleswere hydrolyzed with HCl（6N）for 24 h at 110℃ and amino acids quantified using a JLC-500 amino acid analyzer.

2.4 Calculation and statistical analysisDM flow to duodenum，ileum and feces were calculated by dividing the amount of Cr dosed daily by Cr concentration of duodenal，ileal and fecal samples.Herbage DM intakewas estimated by dividing fecal DM output by DM indigestibility（1-in vitro DM digestibility）of herbage.Duodenal N flows ofmicrobial origin were calculated by dividing the amountof purine flowed to duodenum by the ratio of N to purine in rumen bacterial fraction.ApparentOM digestibility in rumen was corrected formicrobial contribution to calculate the OM truly digested in rumen.The dietary N degradability（RDN）in rumen was calculated by the equation：

RDN（%）＝N intake-（non-ammonia N flow to duodenum-microbial N flow to duodenum）.

Comparison of the differences in the averages obtained from OG and MF pastureswasmade by t-test.The relationship between nutritional variables of herbage such as chemical composition and nutrients degradability，and N utilization in the rumen were estimated by correlation analysis.

3 Results and discussions

3.1 The chem ical constituents of pastureHerbagemass and chemical compositions of herbage are summarized in Table 1.The pre-grazing average sward length of OG pasture was higher and herbagemass was lower than MF pasture.The herbage mass on OG and MF pastures ranged from 78 to 298 gDM／m2and from 92 to 294 gDM／m2，respectively.The herbage allowance on OG and MF pastures ranged from 159 gDM／metabolic body size（MBS）to 436gDM／MBSand from 213 gDM／MBS to525gDM／MBS，respectively.According to the Japanese Feeding Standard for beef cattle（MAFF，2000），the predicted DM intake of dairy steersweighing 350 kg and growing at the rate of0.8kg／day is86.3 g／MBS，so it was thought that herbage intake was not restricted by the herbage allowance in this study.The OM content in the OG and MF pastures averaged 88.8%and 88.1%，respectively and therewas no significant difference in OM content of herbage between pasture species.There were no significant differences in CP content in herbage between OG and MF pastures and the average content is 23.4±2.5%and 25.0±4.5%respectively.The CP content in herbage observed in this studywasalmost similar to the valuesobtained from OG pasture（Hoffman et al.，1993；Kolver et al.，1998），MF and timothy（Phleum pratense，L）pasture（Khalili and Sairanen，2000；Sudo etal.2001），perennial ryegrass（Lolium perene，L.）pasture（Beever et al.，1986；Ulyatt et al.，1988）and tall fescue（Festuca arundinancea，Schreb.）pasture（Berzaghi etal.，1996；Elizalde etal.，1998）.The NDF content of herbage on OG and MF pastures averaged 48.7±5.2%and 45.6±4.0%，respectively and these valueswerewithin the range of average NDF contentobtained from cool season grass pastures in the United States（Muller and Fales，1998）.

Table 1 Sward height，herbagemass and herbage allowance on OG and MF pasture and chem ical composition of herbage plucked by hand to a level of post-grazing sward height

3.2 OM intake and digestibilityThe results of DM intake and OM digestion in steers are shown in Table 2.The DM intake on OG and MF pastures averaged 91.8g／MBS／day and 87.5g／MBS／day，respectively and they exceeded the predicted DM intake of dairy steersweighing 350kg and growing at the rate of 0.8 kg／day（MAFF，2000）.There were no significant differences in OM intake between OG and MF pastures.Apparent and true OM digestibilities in the rumen were higher on MF pasture than on OG pasture（P＜0.01），but the amountof OM apparently or truly digested in the rumen did not differ between OG and MF pastures.The true digestibility of OM in the rumen（OMTDR）ranged from 51.4%to 66.4%in OG pasture and from 52.0%to 70.4%in MF pasture and therewasa negative relationship between OMTDR and the NDF content in herbage（r＝-0.87，P＜0.01）.The amount of OMTDR ranged from 26.4g／MBS／day to 68.8g／MBS／day on OG pasture and from 27.6g／MBS／day to 75.5g／MBS／day on MF pasture.The OMTDR obtained on MF pasture in this study tended to be higher than the results of lactating cows grazing tall fescue pasturewithout supplements（Berzaghi et al.，1996），and lower than the result of steers grazing tall fescue pasture without supplement（Elizalde et al.，1998）.

Table 2 Dry matter intake，organicmatter intake and digestion of steers grazing OG and MF pastures w ithout supplements

3.3 N and am ino acid intake and digestibilityIntake and passage of N，and ruminalmicrobial N synthesis in steers grazing on OG and MF pastures are shown in Table 2.Therewere no significant differences in N intake between OG and MF pastures，and the average N intake wasmore than twice asmuch as the N requirement for dairy steerweighing 350kg and growing at the rate of 0.8kg／day（1.56gN／MBS／day，MAFF，2000）.AA-N intake did notdiffer significantly between pasturesand itaccounted about 80%of total N intake.However，RDN was significantly higher（P＜0.01）in MF pasture than in OG pasture.The N degradability obtained in this study was lower than the result of steers grazing tall fescue pasture without supplement（Elizalde et al.，1998）.The ratio of RDN to ingested N was positively related with CP content in herbage（r＝0.52，P＜0.01）and negatively related to NDF content in herbage（r＝0.61，P＜0.01）.Although the amount of OMTDR did not differ between pastures，the ratio of RDN to OMTDR on MF pasturewas higher than that on OG pasture（P＜0.05）.RDN and the ratio of RDN to OMTDR could be estimated by amulti-regression equation as follows；

The conversion efficiency of RDN tomicrobial N on OG and MF pastures averaged 74.8%and 54.8%，respectively and there was a negative relationship between the conversion efficiency of RDN to microbial N and the ratio of RDN to OMTDR（r＝-0.74，P＜0.01）.The lower conversion efficiency of MF pasture compared with that of OG pasturewasmainly due to the high ratio of RDN to OMTDR and itwas thought that the high protein intake and degradability in the rumen and the low OMTDR intake were contributory factors to the low efficiency of N utilization in the rumen of cattle grazing temperate pasture.As has been the practice in the National Research Council（NRC，2001），the conversion efficiency of RDN to microbial N of 85%is used to determine RDN requirement assuming an apparent ruminal N balance of zero.The N apparently absorbed in the rumen was greater on MF pasture than on OG pasture（P＜0.01）and itwas positively related with the ratio of RDN to OMTDR（r＝0.834，P＜0.01，F(xiàn)ig.1-a）and ammonium N concentration in the rumen fluid（r＝0.831，P＜0.01，F(xiàn)ig.1-b）.These relationships showed that the amount of apparent N absorption in the rumen would be positive when the ratio of RDN to OMTDR was above 24.7g／kg or the ammonium N concentration in the rumen fluid was above 8.7mg／100ml.As the averages of the ratio of RDN to OMTDR and the ammonium N concentration in the rumen were 37.2g／kg and 15.6 mg／100ml，itwas suggested that some extentof N was usually absorbed in the rumen of cattle grazing OG or MF pasture without supplement.The ammonium N concentration in the rumen fluid on OG and MF pasture averaged 13.6mg／dL and 19.8mg／dl，respectively and there was higher on MF pasture than on OG pasture（P＜0.01），and it was positively related to the N concentration in the herbage（r＝0.84，P＜0.01）and the ratio of RDN to OMTDR（r＝0.87，P＜0.01）.Thus，it appeared that the ammonium N concentration in the rumen fluid may be a useful parameter for evaluating the N utilization in the rumen of cattle grazing OG and MF pastures.

Table 3 N intake，flow to duodenum and digestion in different segments of the digestive tract of steers grazing OG and MF pastures w ithout supp lements

The total N and NAN flows to the duodenum significantly（P＜0.01）increased and the proportion of duodenum NAN flow to ingested N was higher（P＜0.05）in OG than in MF pasture.N flow to the duodenum increased with the increase of N intake，but did not always increase when the N intake was above 4.0g／MBS／day（Fig.2），which was about 2.5 times the N requirement of dairy steersweighing 350kg and growing at the rate of 0.8kg／day（MAFF，2000）.Furthermore，these flows tended to decrease with the increase in the ratio of RDN to OMTDR（r＝0.52，P＜0.01）.The proportion of the duodenum N flows to the ingested N averaged 86.6%on OG pasture and 72.5%on MF pasture.These recoverieswas significantly higher（P＜0.0）on OG pasture than on MF pasture.Berzaghi et al.（1996）reported that the recovery of ingested N at the duodenum of lactating cows grazing tall-fescue pasture without supplementwas 75%and the N recovery was improved by corn supplements.On the other hand，Elizalde et al.（1998）reported that the recovery of the ingested N at the duodenum of steers grazing on tall fescue pasture was about 72%and the N recovery was not improved by the energy supplementation.The low recoveries of ingested N at the duodenum on MFwere probably due to the high ratio of RDN to OMTDR.There were negative relationships between the recovery of ingested N at the duodenum and the ratio of RDN to OMTDR（r＝-0.80，P＜0.01）or the ammonium N concentration in the rumen fluid（r＝-0.75，P＜0.01）.Beever et al.（1986）also observed the negative relationship between the recovery of ingested N at the duodenum and the ammonium N concentration in the rumen fluid of steers grazing perennial ryegrass or white clover（Trifolium repense）pasture.We observed the duodenal N flow greater than the N intake in this study when the herbage CP was below 19%in DM.Ulyatt et al.（1988）also reported that the duodenal NAN flow was greater than the N intake when cattle grazed re-growth perennial ryegrass containing 12.6%of crude protein.The higher recovery of ingested N at the duodenum may be attributed to the recycling of N derived from saliva or rumen wall as a result of low ammonium N concentration in the rumen fluid.The high duodenal N flow and low N intake associated with low ammonium N concentration in the rumen fluid was also observed in steers fed corn glutenmeal as amajor protein source（Hanada et al.，1993）and in lactating cows receiving high concentrate rations containing 15%of crude protein（McCarthy et al.，1989）.

AA-N flow to the duodenum was significantly related（P＜0.01，r2＝0.67，F(xiàn)ig.3）to duodenal NAN flows but it accounted for 60%of the NAN flows.Total AA flows to the duodenum were higher in OG than in MF but did not differ in AA composition（Table 4）.There were apparent differences in essential AA composition between intake and duodenal AA flows（higher methionine and lysine），apparently enhanced by the AA profile ofmicrobial protein outflow from the rumen.However，these essential AA seem to be insufficient formilk orgrowth because the AA profile of the duodenal digesta was less compared with those of the lean tissue and milk.The N digested post-ruminally was greater on OG pasture than on MF pasture（P＜0.01），however the apparent N digestibility through the lower gastrointestinal tract did not differ between pastures.The quantity of N digested post-ruminally did not increasewith the increase of N intake（Fig.4-a），but tended to decrease with increase in the ratio of RDN to OMTDR（Fig.4-b）or the ammonium N concentration in the rumen fluid.The apparent N digestibility through the lower gastrointestinal tract obtained in this trialwas higher than the result obtained from steers grazing tall fescue pasture without supplements（Elizalde et al.，1998）and lower than the resultobtained from lactating cows fed silage and concentratemixed ration（McCarthy etal.，1989）.

Table 4 The am ino acid intake and flow to duodenum and comparison of the am ino acid composition of duodenal digestion w ith that of lean body tissue and m ilk

The resultsof this study suggested that cattle grazed on these pasturesmay have lost some N in the rumen before reaching the duodenum.The recovery of ingested N at post-rumen depends more on the dietary N degraded in the rumen than the amountof N intake.Decrease the loss of N from the rumen and to supplymore N to the small intestines，it is necessary to reduce the intake of N degraded in the rumen to OM truly digested in the rumen ratio supplementwith grains feeds.Methionine and lysinewere themost limiting AA for animal production from cows grazing temperate pastures.

［1］［1］AOAC.Official Methods of Analysis（15th Edition）［M］.Association of Official Analytical Chemists，Arlington，VA，1990.

［2］Agriculture，F(xiàn)orestry and Fisheries Research Council Secretariat，MAFF.Japanese Feeding Standard for Beef Cattle（2000）［M］.Japan Livestock Industry Association.Tokyo，Japan，2000.

［3］Aibibula Y.，Hanada M.，Okamoto M.Effect of pre-grazing sward length and daily herbage allowance on N utilization in the rumen and supply to the duodenum of steers under rotational grazing［J］.Grassland Science，2002，49：217-221.

［4］Association of Official Analytical Chemists.Official Method of Analysis（15th edition）AOAC［M］.Arlington.USA，1990.

［5］Beever DE，Siddons RC.Digestion and metabolism in the grazing ruminants.In Control of Digestion and Metabolism in Ruminants（eds.L.P.Milligam，W.L.Grovum and A.Dobson）［M］.A Reston Book.Cliffs.USA，1986：479-497.

［6］Beever DE，Losada HR，Cammell SB，et al.Effect of forage species and season on nutrient digestion and supply in grazing cattle［J］.Journal of Nutrition，986，56：209-225.

［7］Berzaghi P，Herbein JH，Polan CE.Intake，site，and extentof nutrient digestion of lactating cows grazing pasture［J］.Journal of Dairy Science，1996，79：1581-1589.

［8］Elizalde JC，Cremin JD，F(xiàn)aulkner DB，et al.Performance and digestion by steers grazing tall fescue and supplemented with energy and protein［J］.Journal of Animal Science，1998，76（6）：1691-1701.

［9］Goering HK，Van Soest PJ.Forage Fiber Analyses（Apparatus regents，pro-cedures and some applications）［M］.Agriculture Handbook No.379.ARS USDA.Washington D.C.USA，1970：20.

［10］Hanada，M，Obitsu T，Taniguchi K.The effectof different starch sources and protein on site and the extentof N digestion und utilization［J］.Anim.Sci.Tech.（Jpn.），1993，64：275-287.

［11］Hoffman K，Muller LD，F(xiàn)ales SL，et al.Quality evaluation and concentrate supplementation of rotational pasture grazed by lactating cows［J］.J.Dairy Sci.，1993，76：2651-2663.

［12］Holden LA，Muller LD，Varga GA.Ruminal digestion and duodenalnutrient flows in dairy cows consuming grass as pasture，hay or silage［J］.J.Dairy Sci.，1994，77：3034-3042.

［13］Khalili H，Sairanen A.Effect of concentrate type on rumen fermentation and milk production of cows at pasture［J］.Anim.Feed.Sci.and Tech.，2000，84：199-212.

［14］Kolver E，Muller LD，Varga GA etal.Synchronization of ruminal degradation of supplemental carbohydrate with pasture N in lactating dairy cows［J］.Journal of Dairy Science，1998，81（7）：2017-2028.

［15］Leaver JD.Milk production from grazed temperate grassland［J］.J.Dairy Res.，1985，52（2）：313-344.

［16］Licitra G，Hernandez TM，Van Soest PJ.Standardization of procedures for N fraction on ruminant feeds［J］.Animal feed Science and Technology，1996，57（4），347-358.

［17］Morimoto，H.Animal nutrition examination method［Z］.Yokendo Ltd.，1971

［18］McCarthy RD Jr，Klusmeyer TH，Vicini JL，etal.Effectsof source of protein and carbohydrate on ruminal fermentation and passage of nutrients to the small intestineof lactating cows［J］.J.Dairy Sci.，1989，72（8）：2002-2016.

［19］Minson DJ.Forage in Ruminant Nutrition［M］.Academic Press.San Diego.USA.1990：162-207.

［20］Muller LD，F(xiàn)ales SL.Supplementation of cool season grass pastures for dairy cattle.In Grass for Dairy Cattle（eds.J.H.Cherney and D.J.R.Cherney）［Z］.CABI International.UK.1998：335-350.

［21］National Research Council.Nutrient Requirements of Dairy Cattle 7th ed，2001［M］.National Academy Press.Washington D.C.USA，2001.

［22］Okuda T，F(xiàn)ujii S.Estimation method of ammonia which removal the protein direct comparison.In：Clinical chemistry analysis-analysis library-3［M］.Japanese Analytical Chemistry Meeting.1968.

［23］Sudo K，Ochiai K，Ikeda T.Comparison ofmilk production from meadowfescue（Festuca pratensis Huds.）pasture and perennial ryegrass（Lolium perenne L.）pasture under intensive grazing［J］.Grassland Science，2001，47：386-392.

［24］Simith RH，McAllan AB.Some factors influencing the chemical composition ofmixed rumen bacteria［J］.Br.J.Nutr.，1974，31：27-34.

［25］Tilly JM，Terry RA.A two stages technique for in vitro digestion forage crops［J］.Br.J.Nutr.，1963，18：104-111.

［26］Titgemeyer EC，Loest CA.Amino acid nutrition：Demand and supply in forage-fed ruminants［J］.J.Anim.Sci.2001，79（E.Suppl.）：180-189.

［27］Ulyatt MJ，Thomson DJ，Beever DE，et al.The digestion of perennial ryegrass（Lolium perene cv.Melle）and white clover（Trifolium repense cv.Blanca）by grazing cattle［J］.Br.J.Nutr.，1988，60：137-149.

［28］Wilkerson VA，Klopfenstein TJ，Britton RA，et al.Metabolizable protein and amino acid requirements of growing cattle［J］.J.Anim.Sci.1993，71：2777-2784.

［29］Yoshida M，Kosaka K，Horii S.A new procedure for the determination of chromic oxidewith potassium phosphate reagent［J］.Jpn.J.Poultry Sci.，1967，4：24-29.

［30］Zinn RA，Owens FN.A rapid procedure for purinemeasurement and its use for estimating net ruminal protein synthesis［J］.Canadian Journal of Animal Science，1986，66（1）：157-166.

［31］Bryant RH，Dalley DE，Gibbs J，et al.Effect of grazingmanagement on herbage protein concentration，milk production and nitrogen excretion of dairy cows inmid-lactation［J］.Grass and Forage Science，2013.

［32］Belanche A，Weisbjerg MR，Allison GG，et al.Estimation of feed crude protein concentration and rumen degradability by Fourier-transform infrared spectroscopy［J］.Journal of Dairy Science，2013，96（12）：7867-7880.