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This experiment was carried out to quantify the consequence of solid provender proviso on protein metamorphosis and urea recycling in milk-fed calves. The experiment was performed at the experimental adjustment ‘De Haar ‘ of Wageningen University and Research Center in Wageningen. Experimental processs like attention, managing and sampling of the calves, were in conformity with the Dutch jurisprudence on experimental animate beings.

Forty-eight Holstein Friesian ( HF ) bull calves of about 6 hebdomads of age ( 54.7 A± 2.1 kilograms ) were selected ( on a veal calf farm ) based on age and weight. Based on age, calves were divided into groups of three. Groups were indiscriminately assigned to a intervention. In between interventions, the calves were group housed in slatted floored pens without bedding stuff. Furthermore, in the stable, light conditions were stable and clime was controlled.

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The four dietetic interventions ( Table 4.1 ) were offered on top of a milk replacer diet. In interventions, solid provender is non fed ( intervention A ) or fed in three incremental measures ( interventions B, C and D ) in add-on to a fixed consumption of a commercial milk replacer.

The solid provender consists of maize silage, chopped wheat straw, and dressed ores in a proportion of 1:1:2 on a DM ( Dry Matter ) footing. The analyzed alimentary composing of the milk replacer, fibers and dressed ores are given in table 4.2.

To quantify the consequence of solid provender proviso on the evident faecal protein digestibleness and the fringy protein deposition, calves pass through two 4-day balance periods in the climatic respiration Chamberss at 12 and 20 hebdomads of age. During the balance periods, solid provender is provided harmonizing to table 4.1 on top of a fixed and for all interventions equal consumption ( aˆ¦dose of milk replacer ) of milk replacer. In between the two balance periods, milk replacer strategies were adapted ( isocaloric on ME footing ) to avoid weight differences between interventions during measurings. Solid provender doses were unbroken equal during and in between measurings ( Figure 4.1 ) .

Figure 4.1. Solid provender doses for the 4 interventions during the measuring periods and in between measuring periods.

Housing

Each measuring period consisted of an version period of 4-5 yearss ( stable ) and a balance period of 4 yearss in the respiration Chamberss. During the accommodation and balance periods, calves are housed separately in metabolic coops equipped with funnels and pails below the slatted floor to enable a quantitative urine aggregation. The funnels and slatted floor of the coops were on a regular basis sprayed with aˆ¦acid to forestall bacterial growing and perturbation of the N content in piss. The pails were filled with 95-97 % sulphuric acid ( H2SO4 ) and 500 ml H2O solution to cut down the pH below 2.5 to forestall evaporating and escaping of urinary N beginnings. Furthermore, the calves wear harnesses with manure bags to enable quantitative aggregation of manure.

Measurements & A ; sample aggregation

The respiration Chamberss allow measurings of O2 ingestion and CO2 production ( every 9 proceedingss ) and from that, via indirect calorimetric measurings, heat production is calculated. From that, protein ( and energy ) balances can be calculated. Furthermore, methane production can be measured ( every 9 proceedingss ) , which is an indicant for the sum of ruminal milk and agitation of fiber and dressed ores.

Manure bags were collected for interventions A and B twice a twenty-four hours ( at feeding clip ) and for interventions C and D thrice a twenty-four hours to forestall overfilled manure bags and manure loss, and weighed and registered. Feed orts were collected, weighed and registered one time a twenty-four hours. Orts and manure were instantly after aggregation stored in the deep-freeze at -20 °C until the terminal of the balance period, when it was weighed, pooled, and sampled for farther analysis. Urine was pooled and sampled straight after completing the measuring periods per group and stored in the deep-freeze at -20 °C.

Twice a hebdomad, diets per group were weighted and prepared. Besides feed samples were taken and stored at -20 °C. At the terminal of the experiment, samples were pooled and prepared for analysis.

During the whole experiment, calves were fed twice a twenty-four hours, at 7:00h and 16:00h. Feed consumption was registered and calves were leaden hebdomadal at Tuesday. Daily H2O ingestion was registered per pen. Health of calves was checked twice a twenty-four hours and ill calves ( based on wellness or behaviour ) were treated or taken out of the experiment. Hemoglobin degrees of all calves are on a regular basis controlled ( blood samples taken every 2 hebdomads ) , whereby haemoglobin degrees of 5.5 mmol/L at slaughter age is aimed.

At the terminal of the experiment, calves were slaughtered. 13 calves were euthanatized in the butchery of the experimental installation, by agencies of an injection of aˆ¦ml Nembutal. The staying 35 animate beings were slaughtered at the abattoir ‘Worst ‘ ( Nijkerk, NL ) by agencies of stupefying by confined bolt and killed by exsanguination. Those slaughter methods were chosen due to weave aggregation of all parts of the digestive system for the intent of another subproject of the experiment.

Experimental processs urea recycling

The consequence of solid provender proviso on the urea recycling was quantified during the 2nd balance period at 20 hebdomads of age of the calves. This is done by a stable isotope methodological analysis based on old findings of Sarrascesa et Al. ( 1998 ) . 47 calves are used for this measuring. One calf was ill.

Applicable urea recycling explained

With the stable isotope methodological analysis, [ 13C ] and [ 15N ] urea isotopes are intravenously infused and could be followed independently from each other in the metamorphosis of a calf. Those two stable isotopes can be followed independently from each other in the metamorphosis. Urea from the blood can, via the kidneys, straight being excreted in the piss or it can flux to the first stomachs.

A [ 13C ] carbamide

When [ 13C ] urea flows into the first stomachs, it is transferred into ammonium hydroxide and will so lose its [ 13C ] label by agencies of organizing 13CO2 ( gas production ) , where it disappears with the belching. The sum ( recovery ) of [ 13C ] carbamide in the urine ( % of the infused dosage ) is the sum which is non transported to the first stomachs. The sum non measured in the piss, is the proportion which is transported to the first stomachs. These fluxes give an appraisal of the relative urea flux to the first stomachs.

[ 15N15N ] urea

After extract of the [ 15N15N ] urea isotope, three urea isotopomer species, viz. [ 15N15N ] , [ 14N15N ] and [ 14N14N ] carbamide are formed within the organic structure and eliminated in the piss ( Figure 1 ; Lapierre and Lobley, 2001 ) .

Figure 4.2. Use of [ 15N15N ] urea and isotopomer analysis of urinary [ 15N15N ] , [ 14N15N ] and [ 14N14N ] carbamide to quantify flows and destinies of urea that enters the digestive piece of land. Part of the infused [ 15N15N ] urea enters the digestive piece of land were it can be excreted in the fecal matters or is hydrolyzed to [ 15N ] ammonium hydroxide. This latter is either used by the microbic population to synthesise bacterial proteins ( [ 15N ] ) or it is absorbed straight as [ 15N ] ammonium hydroxide. [ 15N ] ammonium hydroxide is removed by the liver were [ 15N14N ] carbamide is formed. The ratio of [ 14N15N ] : [ 14N14N ] carbamide in the urine reflects the proportion of urea flux that is converted to ammonia in the digestive piece of land and returned straight to the hepatic ornithine rhythm ( Lapierre and Lobley, 2001 ) .

The [ 15N15N ] urea isotope can be excreted straight from the blood ( from the extract minute onwards ) in the piss ( so no recycling ) or flux to the first stomachs ( recycling possible ) . The direct flow into the piss can be measured as the sum of [ 15N15N ] carbamide in the piss.

However, when the [ 15N15N ] urea flows to the first stomachs, the label can return via soaking up to the blood stream. The [ 15N15N ] carbamide is hydrolyzed into 2 * [ 15NH3 ] .

The 15NH3 from the first stomachs can be absorbed straight from the first stomachs or from the bowel into the blood and in the liver it can be used for urea synthesis. Thus the liver transforms the toxic ammonium hydroxide into urea. Therefore, [ 15N14N ] carbamide will look in the blood stream, ensuing in:

Elimination in the urine [ 15N14N ] carbamide

Recycling to the first stomachs i? hydrolyzation into [ 14NH3 ] and [ 15NH3 ] i? soaking up ( i? perchance infinite recycling )

( 2 ) The 15NH3 from the first stomachs can besides be used for microbic protein synthesis, whereby the isotope is incorporated in a [ 15N ] labeled protein:

[ 15N ] protein remains undigested and appears in the fecal matters, chiefly in the signifier of [ 14N15N ] urea/protein and [ 15N ] proteins, because there is merely a really little opportunity that two [ 15N ] isotopes are uniting into one urea/protein molecule once more, this [ 15N15N ] urea/protein in fecal matters is non specified in the measurings ( REFERENTIE WAAROM 15N15N NIET GESPECIFICEERD WORD ) .

[ 15N ] proteins can be absorbed and deposited in organic structure protein, which will non be measured in this experiment ( anabolic destiny )

[ 15N ] proteins can be absorbed, deposited, oxidized andaˆ¦

aˆ¦be converted ( liver ) and excreted as [ 15N14N ] carbamide or [ 15N ] protein in urine ( katabolic destiny )

aˆ¦be converted ( liver ) into [ 15N14N ] carbamide and be recycled to the first stomachs

Measurements and sample aggregation urea recycling

The method used, exists out of [ 13C ] and [ 15N15N ] urea extract coupled with excreta aggregation for 72h. Assumed is that the calves are in steady province or that the calves have equal starting and stoping organic structure urea pools ( Lapierre and Lobley, 2001 ) . Figure 4.2 shows a conventional overview of the clip span of measurings taken sing urea recycling during the 2nd balance period.

Figure 4.2.Schematic overview of the clip span of urea recycling measurings.

Two yearss before the extract ( twenty-four hours -2 ) , calves are catheterized ( 9:00 – 16:00h ) . Two semi-permanent catheters are placed in the vein jugularis. One catheter is used for extract of the stable isotopes of urea, while the other catheter is used for blood sampling ( the latter is used for another subproject in the experiment ) . In add-on to the normal processs ( feeding, burdening, registering provender consumption, manure weight, H2O ingestion ) , temperature measurings of the catheterized calves were done twice a twenty-four hours during aggregation and replacing of the manure bags.

One twenty-four hours before the endovenous extract starts ( twenty-four hours -1 ) , urine samples from all single calves are taken to find the ‘regular background ‘ enrichments/concentrations of [ 15N ] and [ 13C ] .

At twenty-four hours 0, the extract of the isotope mixture of 99.1 atom % [ 15N15N ] carbamide ( 99.1 atom % ; Isotec, Miamisburg, Ohio, USA ) and 99 atom % [ 13C ] carbamide ( aˆ¦ ; Isotec, Miamisburg, Ohio, USA ) isotopes, prepared in unfertile 0.15M saline ( NaCl solution ) , started. After the catheters were checked on functionality, the calves received a priming dosage of on mean 18.05 milliliter to accomplish steady province degrees of the isotope. After the priming dosage, an isotope solution in saline of on mean 42.76 milliliter is continuously infused via the jugular vena for 24 hours. The extract rates were on mean 1.96 ml/h and the extract was done by agencies of syringe extract pumps. The predicted enrichment at the tableland degree was an ‘ape ‘ of 0.15 atom % . Directly from the start of extract, cumulative manure and piss are collected for 72 hours ( twenty-four hours 0 to twenty-four hours 3 ) until no ( excess ) [ 15N ] carbamide is secreted ( Sarraseca et al. , 1998 ) .

After this aggregation period ( twenty-four hours 4-6 ) , one background sample of about 52 hours is obtained ( dual cheque background concentrations [ 15N ] ) , which besides serves for the balance measuring.

After completion of the 2nd balance period, catheters are removed from the calves. Calfs returned back to the group lodging in the stable.

Analytic processs

By agencies of chemical analysis of samples taken for intent of the N balance and the urea recycling, alimentary contents can be determined.

Chemical analysis N balance

Samples of fecal matters, provender and provender orts of the 2 balance periods are freeze dried and grinded on a 1 millimeter screen prior to analysis. In the research lab, the Dry Matter ( DM ) , Crude Ash and Crude Protein ( N ) content in samples of provender, provender orts, piss, ( fresh ) fecal matters, and aerial NH3 and NH4+ in condensed H2O are determined. In other subprojects of the experiment, the Crude Fat, amylum and sugar and NDF content were determined. From the consequences, the evident faecal protein digestiblenesss and protein deposition can be obtained.

DM content

DM content was determined by drying the freezing dried ( air-dry ) weighed samples at 103 °C ( 4 hour ) and fresh fecal matters samples at 70 °C ( 16 hour ) and 103 °C ( 4 hour ) harmonizing to the criterions of ISO 6496 ( ISO, 1983 ) . Thereafter samples are air-equilibrated and weighed.

DM content is calculated as follows:

W3 – W2

— — — — — — – * 1000 = DM ( g/kg )

W1

In which:

W1 = the weight of the sample

W2 = the weight of the empty container

W3 = the weight of the container after drying

Ash content

Ash content was determined by incineration of freezing dried ( air-dry ) weighed samples at 550 °C till changeless weight was reached and harmonizing to the criterions of ISO 5948 ( ISO, 1978 ) . Thereafter the staying ash is air-equilibrated and weighed.

Ash content is calculated as follows:

W2- W1

— — — — — — * 1000 = Ash ( g/kg )

W3

In which:

W1 = the weight of the empty dish ( g )

W2 =the lowest weight ( in instance of repeated incinerations ) of the dish after ashing ( g )

W3 = the sample weight ( g )

N content

The N content was determined harmonizing to the Kjeldahl method and the criterions of ISO 5983 ( ISO, 1998 ) . First, the organic affair in the samples is digested by agencies of boiling with sulfuric acid in presence of a accelerator, in a digestion block ( Gerhardt Kjeldahltherm with Variostat and Turbosog ) . Hereby, N in the samples is converted into ammonium salts. Then, in the distillment unit ( Gerhardt Vapodest 6 ) , the reaction mixture is made alkaline, distilled, and the collected ammonium hydroxide is titrated. The petroleum protein content is obtained by multiplying the deliberate N content by an international protein factor, 6.25 ( 6.38 for milk merchandises ) .

N content is calculated as follows: N ( g/kg ) = ( V1 – V2 ) * hundred * F * M/m

In which:

V1 = milliliter sulfuric acid for titration

V2 = milliliter sulfuric acid for space

degree Celsiuss = concentration of the used acid ( mol/L )

F = valency of the used acid ( 1 for HCl, 2 for H2SO4 )

m = weight of the sample in gms

M = 14.008, molar mass ( g/mol )

Crude Protein = CP ( g/kg ) = N ( g/kg ) * 6.25

Crude Protein = CP ( g/kg ) = N ( g/kg ) * 6.38 ( in instance of CP in milk replacer )

Chemical analysis carbamide recycling

By agencies of finding of the enrichment of [ 13C ] and [ 15N ] in urine and fecal matters, different paths of urea can be quantified. The enrichment of the isotopes in fecal matters and piss is determined by agencies of ( GC-C- ) Isotope Ratio Mass Spectrometry ( IRMS ) . The rule of uninterrupted flow IRMS is depicted in figure 4.3.

Figure 4.3 The rule of the uninterrupted flow IRMS.

Analysis of [ 13C ] and [ 15N ] urea enrichment in fecal matters by agencies of GC-C-IRMS

The [ 13C ] and [ 15N ] urea enrichment in air-dry ( lyophilized ) fecal matters is measured by harmonizing to ‘Sample readying guidelines for IRMS analyses ‘ . Hereby is the rule that the Gas Chromatograph ( GC ) separates the compounds from an elemental analyser ( EA ) which are on-line evaluated by the Isotope Ratio Mass Spectrometer ( IRMS ) .

The maximal enrichment that can be measured is A± 2 atom % . Furthermore, both the [ 13C ] and [ 15N ] urea enrichment can be measured within one tally. Samples are analyzed in duplo.

First, the freezing dried samples have to be ground to 200 Aµg atom size ( talcum pulverization consistence ) with a Retsch ball factory ( MM2000 ) at the IRMS lab ( amplitude: 90 ; 3 min ) . To obtain precise and accurate consequences, 1-2 milligram is weighed in little aluminium cups, cups are closed, solidly folded in so that no air remains by the sample, and the weight has to be registered.

Then the aluminium ball can be put into the EA ( DP 200 Series 2 Fisons ) . In the EA, organic affair is oxidized by agencies of presenting it into a Cu oxide packed capillary furnace ( burning at 600 °C ) and N- and C-containing organic compounds are converted into N2 and CO2, so undergoing a redox reaction in the decrease reactor ( metallic Cu ) at 150 °C. This consequences in a separation of N2 and CO2 from other ( ash, O2 ) constituents.

In the GC, N2 and CO2 are separated from each other based on the selective features for the stationary and nomadic stage. Thus N and C leave the column at a different minute and besides enter the IRMS at a different minute. The IRMS measures the existent enrichment by mensurating the entire atomic mass of enriched atoms relative to the entire atomic mass of natural happening atoms. Consequences are expressed in atom % , which are calculated as follows:

Atom % [ 13C ] = [ 13C ]

— — — — — — — — * 100

[ 12C ] + [ 13C ]

In which [ 12C ] and [ 13C ] are calculated from the mass signal strengths at 44 ( [ 12CO2 ] : 12+16+16 ) and 45 ( [ 13CO2 ] : 13+16+16 ) , severally.

Atom % [ 15N ] = [ 15N ]

— — — — — — — — * 100

[ 14N ] + [ 15N ]

In which [ 14N ] and [ 15N ] are calculated from the mass signal strengths at 28 ( [ 14N14N ] : 14+14 ) , 29 ( [ 14N15N ] : 14+15 ) and 30 ( [ 15N15N ] : 15+15 ) . i? KLOPT NIET, TOCH ENKEL HET ATOOM % GEMETEN, HOE DAN TERUG REKENEN NAAR VOORGAANDE ISOTOPOMEREN? OF WORDT [ 15N15N ] GENEGEERD IN DE MEST?

Analysis of [ 13C ] urea enrichment in urine by agencies of GC-C-IRMS

The new described protocol to find the [ 13C ] enrichment of carbamide in piss is chiefly based on the process described by Beylot et Al. ( 1994 ) . Beylot et Al. ( 1994 ) concluded that [ 13C ] urea enrichment during the extract of ( 3-13C ) lactate in worlds could non be detected by gas chromatography/mass spectroscopy ( GC-MS ) , but could be easy measured by Gas Chromatography – Isotope Ratio Mass Spectrometry ( GC-IRMS ) . Therefore, for intents of this experiment, [ 13C ] urea enrichment is determined by agencies of GC-C-IRMS.

The first portion of the protocol is indistinguishable to and can be combined with the protocol to find the [ 15N ] urea enrichment in piss. Further on, the stairss are spliting and have to be done apart.

First, 0.15 milliliter from the urine samples is sampled, put in Eppendorf tubings, acidified with 1.2 milliliters sulfosalicylic acid and centrifuged. After centrifugating, about all of the supernatant is put gently on top of the Dowex column. The Dowex column ( Dowex 50W-X8 cation exchange column ) maps as a cation-anion money changer, and therefore separates the laden atoms from the unloaded compounds. By blushing the columns 4 times with 1 milliliters Millipore H2O, other unloaded compounds are washed out of the column and removed. Then the columns are flushed with 6 times 5 milliliters Millipore H2O and the eluate is collected and put in a tubing for storage in the chilling or freezing dried. After entire vaporization in the freezing drier, the remained carbamide is dissolved in 4 times 1 milliliter. Of the in entire 4 milliliter, 2 milliliter is transferred in glass trial tubing for farther [ 13C ] analysis, while the other 2 milliliter is put in an Eppendorf tubing for [ 15N ] analysis. The glass trial tubings are put in the Speedvac ( 60 A°C, A±5 hours ) for vaporization. After vaporization, urea is derivatized to its dimethylaminomethylene derivative ester by agencies of adding 200 AµL of a solution of dimethylformamide dimethylacetal, acetonitrile and methyl alcohol ( proportion 3:2:1 ) . Thereafter, the tubings are placed for 1h in a H2O bath ( 70 A°C ) to organize the dimethylaminomethylene ( DAM ) derived function. Then the samples are analyzed in the Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry ( GC-C-IRMS ) ( injector 280 A°C, furnace 240 A°C, interface 260 A°C ) . For analysis by GC-C-IRMS, the phials are injected in a GC interfaced with an IRMS, whereby He is used as a bearer gas.

Analysis of [ 15N ] urea enrichment in urine by agencies of aˆ¦IRMS

The new described protocol to find the [ 15N ] enrichment of carbamide in piss is chiefly based on the process described by Marini et Al. ( 2006 ) . Marini et Al. ( 2006 ) depict a technique which involves extract ( or injection ) of [ 15N15N ] carbamide, followed by isotopomer analysis of the three species [ 15N15N ] , [ 14N15N ] and [ 14N14N ] carbamide formed within the organic structure and eliminated in the piss.

The first portion of the protocol is indistinguishable to and can be combined with the protocol to find the [ 13C ] urea enrichment in piss. Further on, the stairss are spliting and have to be done apart. First, 0.15 milliliter from the urine samples, which were already acidified ( pH & lt ; 2.5 ) is sampled, put in Eppendorf tubings and centrifuged. After centrifugating, about all of the supernatant is put gently on top of the Dowex column. The Dowex column ( Dowex 50W-X8 cation exchange column ) maps as a cation-anion money changer, and therefore separates the laden atoms from the unloaded compounds. By blushing the columns 4 times with 1 milliliters Millipore H2O, other unloaded compounds are washed out of the column and removed. Then the columns are flushed with 6 times 5 milliliters Millipore H2O and the eluate is collected and put in a tubing for storage in the chilling or freezing dried. After entire vaporization in the freezing drier, the remained carbamide is dissolved with 4 times 1 milliliter. Of the in entire 4 milliliter, 2 milliliter is transferred in glass trial tubing for farther [ 13C ] analysis, while the other 2 milliliter is put in an Eppendorf tubing for farther [ 15N ] analysis. The carbamide is dissolved with Millipore H2O until the right concentration ( 1.5 mmol/L ) is reached. After seting the eluates into Exetainers and bubbling with He ( A±20 min ) , the tubings are easy frozen. Once frozen, 100 AµL of LiOBr ( Lithium Hypobromite Oxidation Reagent ) is added to oxidise urea. The oxidization of urea with LiOBr consequences in the monomolecular debasement of carbamide, which preserves the individuality of the parent urea molecule. The undermentioned reaction takes topographic point during the monomolecular debasement of carbamide:

CO ( NH2 ) 2 + 8 NaOH + Br2 i? 6 NaBr + Na2CO3 + 6 H2O + N2

The reaction takes topographic point when the tubings are removed from the dry ice bath and heated by agencies of a warming block ( Stove ) at 65 A°C ( 20-25 min ) to degrade urea into N2 gas. Within 4 hours, the samples have to be analyzed in the GC-C-IRMS ( injector 280 A°C, furnace 240 A°C, interface 260 A°C ) .

Calculations urea recycling dynamicss

By agencies of finding of the enrichment of [ 15N ] and [ 13C ] in fecal matters and [ 15N15N ] , [ 14N15N ] and [ 14N14N ] carbamide in urine, different paths of urea can be quantified. Urea recycling dynamicss are calculated harmonizing to Sarraseca et Al. ( 1998 ) and Lobley et Al. ( 2000, 2001 ) , Dinh ( 2007 ) .

A [ 13C ] carbamide

The difference of the sum infused [ 13C ] carbamide and the sum of [ 13C ] urea excreted in the piss, is the proportion [ 13C ] carbamide which is transported to the first stomachs. The urea flux to the first stomachs is calculated by agencies of the undermentioned equation:

[ 13C ] urea flux to rumen = entire sum [ 13C ] carbamide infused – [ 13C ] carbamide in piss

GER = UER – UUE

[ 15N15N ] urea

In the figures 4.4 and 4.5 are theoretical accounts of urea dynamicss depicted.

Figure 4.4. Model of urea dynamicss ( Sarraseca et al. , 1998 ) . The theoretical account consists of the organic structure urea pool and the carbamide in the digestive piece of land. The dosage ( D ) can be partitioned between urinary elimination ( u ) and that which enters the digestive piece of land ( 1-u ) . The ( 1-u ) flow can be farther partitioned in a part ( R ) which returns to the organic structure urea pool, a part which is lost in the fecal matters ( ten ) and a part which is transferred to into microbic man-made procedures ( s ) . i? GEHELE FIGUUR MISSCHIEN ERUIT LATEN?

Figure 4.5. Model of urea dynamicss based on the extract of [ 15N15N ] carbamide ( Lobley et al. , 2000 ) . The theoretical account consists of the organic structure [ 15N ] urea pool and the [ 15N ] urea pool in the GI piece of land ( GIT ) . Solid lines represent the destinies of [ 15N ] urea direct from the [ 15N15N ] urea extract dosage, D30. Stroked lines represent the destinies of [ 15N ] carbamide which are converted to NH3 in the GIT. Flows with inferior 30 represent flows of [ 15N15N ] carbamide, 29 [ 14N15N ] urea and 28 [ 14N14N ] carbamide. D30 can be partitioned between urinary elimination ( u ) of UUE30, and the GER30 that enters the digestive piece of land ( 1-u ) . The ( 1-u ) flow can be farther partitioned in a part ( R ) which returns to the organic structure urea pool as ROC ( return to ornithine rhythm ) , a part which is lost in the fecal matters ( degree Fahrenheit ) as UFE ( [ 15N ] carbamide in fecal matters ) , and a part which is utilised for constructive metabolism ( a ) as UUA, chiefly via microbic man-made procedures. Thus the R, a and degree Fahrenheit are the transportations of ROC, UUA and UFE proportional to GER.

The Urea Entry Rate ( UER, g N/d ; dominated by urea synthesis in the liver ) in the blood stream is assumed to be equal to entire synthesis, and is determined from the isotopic dilution of [ 15N15N ] carbamide in the piss compared to the blood.

UER = { ( ED30/EU30 ) – 1 } D30 ( Dinh, 2007 )

UER = ( D30/UUE30 ) * UUE FOR SINGLE DOSE STUDIES ( Lobley et al. , 2000 )

UER = urea entry rate, g N/d

ED30 ( 98 % ) = enrichment of [ 15N15N ] carbamide in the dosage

EU30 = enrichment of [ 15N15N ] carbamide in the piss severally

D30 = dosage of [ 15N15N ] carbamide ( ~47 g/d? ? ( 1.96 ml/h ) )

Or by agencies of the computation of Sarraseca et Al. ( 1998 ) :

UER = ( aˆ¦96.45 ape ) * urea-N infused ( mol N/d ) * 14 ( Sarraseca et al. , 1998 )

( Corrected m/z 30 ape )

UER is the Urea Entry Rate, g N/d

96.45 is the per centum of infusate N as [ 15N15N ] carbamide

The infused [ 15N15N ] urea dosage ( D30 ) can be excreted straight in the piss or flow into the first stomachs. The difference between the UER and the direct urinary urea-N riddance ( UUE30 ) yields the GER of [ 15N15N ] carbamide ( GER30 ) :

GER30 = UER – UUE30 ( Lobley et al. , 2000 )

GER30 = gut entry rate of carbamide, g N/d

UER = urea entry rate ( urea production ) , g N/d

UUE30 = urinary [ 15N15N ] urea-N elimination, g N/d

IS GER30 HETZELFDE ALS GER29 EN GER? ? ? i? WAARSCHIJNLIJK WEL, WANT EEN RATE IN G/D

The destiny of the Dose ( D30 ) can be partitioned between elimination in the piss ( u ) and that which enters the GI piece of land ( 1-u ) . The proportion of D30 excreted straight in the piss can be calculated as follows:

u = UUE30/UER ( Lobley et al. , 2000 )

GER30 = ( 1-u ) * UER ( Lobley et al. , 2000 )

u = fraction of urinary [ 15N15N ] carbamide

UUE30 = urinary [ 15N15N ] urea-N elimination, g N/d

UER = urea entry rate ( urea production ) , g N/d

( 1-u ) = fraction of [ 15N15N ] urea-N that enters the GIT

The absolute return to ornithine rhythm ( ROC ) and ROC proportional to GER ( R ) can be calculated as follows:

ROC = I?*UER ( Lobley et al. , 2000 )

R = I?/ ( 1-u ) ( Lobley et al. , 2000 )

ROC = R * GER ( Lobley et al. , 2000 )

ROC = carbamide returned to ornithine rhythm, g N/d

R = fraction of GER which returns to the ornithine rhythm

( 1-u ) = fraction of [ 15N15N ] urea-N that enters the GIT

I? = UUE29/ ( UUE29 + UUE30 )

UUE29 = urinary [ 14N15N ] urea-N elimination, g N/d

UUE30 = urinary [ 15N15N ] urea-N elimination, g N/d

The fraction of the GER what is lost in the fecal matters ( degree Fahrenheit ) is calculated as follows:

f = U ( UFE15 ) / [ ( 1-u ) ( UUE29 + UUE30 ) ] ( Lobley et al. , 2000 )

UFE = f * GER ( Lobley et al. , 2000 )

f = fraction of GER lost in fecal matters

UFE15 = [ 15N ] carbamide lost in fecal matters, g/d

( 1-u ) = fraction of [ 15N15N ] urea-N that enters the GIT

UUE29 = urinary [ 14N15N ] urea-N elimination, g N/d

UUE30 = urinary [ 15N15N ] urea-N elimination, g N/d

The fraction of the GER what is used for constructive metabolism ( a ) is calculated as follows:

a = 1 – degree Fahrenheit – R ( Lobley et al. , 2000 )

UUA = a * GER ( Lobley et al. , 2000 )

a = fraction of GER used for constructive metabolism

f = fraction of GER lost in fecal matters

R = fraction of GER which returns to the ornithine rhythm

UUA = carbamide used for anabolic intents, g N/d

Furthermore:

UER = UUE + GER ( Lobley et al. , 2000 )

UUE = u * UER ( Lobley et al. , 2000 )

GER = ( 1-u ) * UER ( Lobley et al. , 2000 )

GER = UUA + UFE + ROC ( Lobley et al. , 2000 )

a + degree Fahrenheit + R = 1 ( Lobley et al. , 2000 )

D30 = UUE30 + GER30 ( Lobley et al. , 2000 )

ROC29 & A ; 30 = UUE29 + GER29 ( Lobley et al. , 2000 )

UUE30 = u * D30 ( Lobley et al. , 2000 )

UUE29 = u * ROC29 & A ; 30 ( Lobley et al. , 2000 )

GER30 = ( 1-u ) * D30 ( Lobley et al. , 2000 )

GER29 = ( 1-u ) * ROC29 & A ; 30 ( Lobley et al. , 2000 )

GER29 & A ; 30 = GER29 + GER30 ( Lobley et al. , 2000 )

ROC29 & A ; 30 = R * GER29 & A ; 30 ( Lobley et al. , 2000 )

UUA29 & A ; 30 = a * GER29 & A ; 30 ( Lobley et al. , 2000 )

UFE29 & A ; 30 = degree Fahrenheit * GER29 & A ; 30 ( Lobley et al. , 2000 )

However, when the [ 15N15N ] urea flows to the first stomachs, the label can return via soaking up to the blood stream. The [ 15N15N ] carbamide is hydrolyzed into 2 * [ 15NH3 ] .

The 15NH3 from the first stomachs can be absorbed straight from the first stomachs or from the bowel into the blood and in the liver it can be used for urea synthesis. Thus the liver transforms the toxic ammonium hydroxide into urea. Therefore, [ 15N14N ] carbamide will look in the blood stream, ensuing in:

Elimination in the urine [ 15N14N ] carbamide

Recycling to the first stomachs i? hydrolyzation into [ 14NH3 ] and [ 15NH3 ] i? soaking up ( i? perchance infinite recycling )

( 2 ) The 15NH3 from the first stomachs can besides be used for microbic protein synthesis, whereby the isotope is incorporated in a with [ 15N ] labeled protein:

[ 15N ] protein remains undigested and appears in the fecal matters, chiefly in the signifier of [ 14N15N ] urea/protein and [ 15N ] proteins, because there is merely a really little opportunity that two [ 15N ] isotopes are uniting into one urea/protein molecule once more, this [ 15N15N ] urea/protein in fecal matters is non specified in the measurings ( REFERENTIE WAAROM 15N15N NIET GESPECIFICEERD WORD ) .

[ 15N ] proteins can be absorbed and deposited in organic structure protein, which will non be measured in this experiment ( anabolic destiny )

[ 15N ] proteins can be absorbed, deposited, oxidized andaˆ¦

aˆ¦be converted ( liver ) and excreted as [ 15N14N ] carbamide or [ 15N ] protein in urine ( katabolic destiny )

aˆ¦be converted ( liver ) into [ 15N14N ] carbamide and be recycled to the first stomachs

Statistical analysis

Datas were analyzed utilizing the GLM/Mixed theoretical accounts process of SAS ( SAS-Institute, 2002 ) .

Complete randomized block design. Experimental unit = group calves. ANOVA

Y = Aµ + dieti + aˆ¦ + E›ijk

Y = dependant variable

Aµ = the mean experimental value

Diet = the consequence of dietetic intervention I ; i = diet 1, 2, 3, 4

aˆ¦ = other of import effects

E›ijk = the error term, related to intervention I, aˆ¦ J, and break up K ( k = 1 – 48 ) .

NOG NADER TE BEPALEN EN UIT TE WERKEN

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