Effect Of Solid Feed Provision Biology Essay

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This experiment was carried out to quantify the effect of solid feed provision on protein metabolism and urea recycling in milk-fed calves. The experiment was performed at the experimental accommodation 'De Haar' of Wageningen University and Research Center in Wageningen. Experimental procedures like care, handling and sampling of the calves, were in accordance with the Dutch law on experimental animals.

Forty-eight Holstein Friesian (HF) bull calves of approximately 6 weeks of age (54.7 ± 2.1 kg) were selected (on a veal calf farm) based on age and weight. Based on age, calves were divided into groups of three. Groups were randomly assigned to a treatment. In between treatments, the calves were group housed in slatted floored pens without bedding material. Furthermore, in the stable, light conditions were stable and climate was controlled.

The four dietary treatments (Table 4.1) were offered on top of a milk replacer diet. In treatments, solid feed is not fed (treatment A) or fed in three incremental quantities (treatments B, C and D) in addition to a fixed intake of a commercial milk replacer.

The solid feed consists of maize silage, chopped wheat straw, and concentrates in a proportion of 1:1:2 on a DM (Dry Matter) basis. The analyzed nutrient composition of the milk replacer, roughages and concentrates are given in table 4.2.

To quantify the effect of solid feed provision on the apparent fecal protein digestibility and the marginal protein deposition, calves pass through two 4-day balance periods in the climatic respiration chambers at 12 and 20 weeks of age. During the balance periods, solid feed is provided according to table 4.1 on top of a fixed and for all treatments equal intake (…dose of milk replacer) of milk replacer. In between the two balance periods, milk replacer schemes were adapted (isocaloric on ME basis) to avoid weight differences between treatments during measurements. Solid feed dosages were kept equal during and in between measurements (Figure 4.1).

Figure 4.1. Solid feed dosages for the 4 treatments during the measurement periods and in between measurement periods.

Housing

Each measurement period consisted of an adaptation period of 4-5 days (stable) and a balance period of 4 days in the respiration chambers. During the adjustment and balance periods, calves are housed individually in metabolic cages equipped with funnels and buckets below the slatted floor to enable a quantitative urine collection. The funnels and slatted floor of the cages were regularly sprayed with …acid to prevent bacterial growth and disturbance of the N content in urine. The buckets were filled with 95-97% sulfuric acid (H2SO4) and 500 ml water solution to reduce the pH below 2.5 to prevent evaporating and escaping of urinary N sources. Furthermore, the calves wear harnesses with manure bags to enable quantitative collection of manure.

Measurements & sample collection

The respiration chambers allow measurements of O2 consumption and CO2 production (every 9 minutes) and from that, via indirect calorimetric measurements, heat production is calculated. From that, protein (and energy) balances can be calculated. Furthermore, methane production can be measured (every 9 minutes), which is an indication for the amount of ruminal milk and fermentation of roughage and concentrates.

Manure bags were collected for treatments A and B twice a day (at feeding time) and for treatments C and D thrice a day to prevent overfilled manure bags and manure loss, and weighed and registered. Feed orts were collected, weighed and registered once a day. Orts and manure were immediately after collection stored in the freezer at -20 °C until the end of the balance period, when it was weighed, pooled, and sampled for further analysis. Urine was pooled and sampled directly after finishing the measurement periods per group and stored in the freezer at -20 °C.

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

During the whole experiment, calves were fed twice a day, at 7:00h and 16:00h. Feed intake was registered and calves were weighted weekly at Tuesday. Daily water consumption was registered per pen. Health of calves was checked twice a day and sick calves (based on health or behavior) were treated or taken out of the experiment. Hemoglobin levels of all calves are regularly controlled (blood samples taken every 2 weeks), whereby hemoglobin levels of 5.5 mmol/L at slaughter age is aimed.

At the end of the experiment, calves were slaughtered. 13 calves were euthanatized in the abattoir of the experimental facility, by means of an injection of …ml pentobarbital. The remaining 35 animals were slaughtered at the slaughterhouse 'Worst' (Nijkerk, NL) by means of stunning by captive bolt and killed by exsanguination. Those slaughter methods were chosen due to tissue collection of all parts of the digestive system for the purpose of another subproject of the experiment.

Experimental procedures urea recycling

The effect of solid feed provision on the urea recycling was quantified during the second balance period at 20 weeks of age of the calves. This is done by a stable isotope methodology based on previous findings of Sarrascesa et al. (1998). 47 calves are used for this measurement. One calf was sick.

Applicable urea recycling explained

With the stable isotope methodology, [13C] and [15N] urea isotopes are intravenously infused and could be followed independently from each other in the metabolism of a calf. Those two stable isotopes can be followed independently from each other in the metabolism. Urea from the blood can, via the kidneys, directly being excreted in the urine or it can flow to the rumen.

 [13C] urea

When [13C]urea flows into the rumen, it is transferred into ammonia and will then lose its [13C] label by means of forming 13CO2 (gas production), where it disappears with the belching. The amount (recovery) of [13C] urea in the urine (% of the infused dose) is the amount which is not transported to the rumen. The amount not measured in the urine, is the proportion which is transported to the rumen. These fluxes give an estimation of the proportional urea flux to the rumen.

[15N15N]urea

After infusion of the [15N15N]urea isotope, three urea isotopomer species, namely [15N15N], [14N15N] and [14N14N]urea are formed within the body and eliminated in the urine (Figure 1; Lapierre and Lobley, 2001).

Figure 4.2. Use of [15N15N] urea and isotopomer analysis of urinary [15N15N], [14N15N] and [14N14N] urea to quantify flows and fates of urea that enters the digestive tract. Part of the infused [15N15N]urea enters the digestive tract were it can be excreted in the faeces or is hydrolyzed to [15N]ammonia. This latter is either used by the microbial population to synthesize bacterial proteins ([15N]) or it is absorbed directly as [15N]ammonia. [15N]ammonia is removed by the liver were [15N14N]urea is formed. The ratio of [14N15N]:[14N14N]urea in the urine reflects the proportion of urea flux that is converted to ammonia in the digestive tract and returned directly to the hepatic ornithine cycle (Lapierre and Lobley, 2001).

The [15N15N]urea isotope can be excreted directly from the blood (from the infusion moment onwards) in the urine (so no recycling) or flow to the rumen (recycling possible). The direct flow into the urine can be measured as the amount of [15N15N]urea in the urine.

However, when the [15N15N]urea flows to the rumen, the label can return via absorption to the bloodstream. The [15N15N] urea is hydrolyzed into 2 * [15NH3].

The 15NH3 from the rumen can be absorbed directly from the rumen or from the intestine into the blood and in the liver it can be used for urea synthesis. Thus the liver transforms the toxic ammonia into urea. Thus, [15N14N]urea will appear in the bloodstream, resulting in:

Excretion in the urine [15N14N]urea

Recycling to the rumen  hydrolyzation into [14NH3] and [15NH3]  absorption ( possibly infinite recycling)

(2) The 15NH3 from the rumen can also be used for microbial protein synthesis, whereby the isotope is incorporated in a [15N] labeled protein:

[15N]protein remains undigested and appears in the feces, mainly in the form of [14N15N]urea/protein and [15N]proteins, because there is only a very small chance that two [15N] isotopes are combining into one urea/protein molecule again, this [15N15N]urea/protein in feces is not specified in the measurements (REFERENTIE WAAROM 15N15N NIET GESPECIFICEERD WORD).

[15N]proteins can be absorbed and deposited in body protein, which will not be measured in this experiment (anabolic fate)

[15N]proteins can be absorbed, deposited, oxidized and…

…be converted (liver) and excreted as [15N14N]urea or [15N]protein in urine (catabolic fate)

…be converted (liver) into [15N14N]urea and be recycled to the rumen

Measurements and sample collection urea recycling

The method used, exists out of [13C] and [15N15N]urea infusion coupled with excreta collection for 72h. Assumed is that the calves are in steady state or that the calves have equal starting and ending body urea pools (Lapierre and Lobley, 2001). Figure 4.2 shows a schematic overview of the time span of measurements taken regarding urea recycling during the second balance period.

Figure 4.2.Schematic overview of the time span of urea recycling measurements.

Two days before the infusion (day -2), calves are catheterized (9:00 - 16:00h). Two semi-permanent catheters are placed in the vena jugularis. One catheter is used for infusion 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 addition to the normal procedures (feeding, weighting, registering feed intake, manure weight, water consumption), temperature measurements of the catheterized calves were done twice a day during collection and replacement of the manure bags.

One day before the intravenous infusion starts (day -1), urine samples from all individual calves are taken to determine the 'regular background' enrichments/concentrations of [15N] and [13C].

At day 0, the infusion of the isotope mixture of 99.1 atom% [15N15N] urea (99.1 atom%; Isotec, Miamisburg, Ohio, USA) and 99 atom% [13C] urea (…; Isotec, Miamisburg, Ohio, USA) isotopes, prepared in sterile 0.15M saline (NaCl solution), started. After the catheters were checked on functionality, the calves received a priming dose of on average 18.05 ml to achieve steady state levels of the isotope. After the priming dose, an isotope solution in saline of on average 42.76 ml is continuously infused via the jugular vein for 24 hours. The infusion rates were on average 1.96 ml/h and the infusion was done by means of syringe infusion pumps. The predicted enrichment at the plateau level was an 'ape' of 0.15 atom%. Directly from the start of infusion, cumulative manure and urine are collected for 72 hours (day 0 to day 3) until no (extra) [15N] urea is secreted (Sarraseca et al., 1998).

After this collection period (day 4-6), one background sample of approximately 52 hours is obtained (double check background concentrations [15N]), which also serves for the balance measurement.

After completion of the second balance period, catheters are removed from the calves. Calves returned back to the group housing in the stable.

Analytical procedures

By means of chemical analysis of samples taken for purpose of the N balance and the urea recycling, nutrient contents can be determined.

Chemical analysis N balance

Samples of feces, feed and feed orts of the 2 balance periods are freeze dried and grinded on a 1 mm sieve prior to analysis. In the laboratory, the Dry Matter (DM), Crude Ash and Crude Protein (N) content in samples of feed, feed orts, urine, (fresh) feces, and aerial NH3 and NH4+ in condensed water are determined. In other subprojects of the experiment, the Crude Fat, starch and sugar and NDF content were determined. From the results, the apparent fecal protein digestibilities and protein deposition can be obtained.

DM content

DM content was determined by drying the freeze dried (air-dry) weighed samples at 103 °C (4 hrs) and fresh feces samples at 70 °C (16 hrs) and 103 °C (4 hrs) according to the standards 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 freeze dried (air-dry) weighed samples at 550 °C till constant weight was reached and according to the standards of ISO 5948 (ISO, 1978). Thereafter the remaining 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 case of repeated incinerations) of the dish after ashing (g)

W3 = the sample weight (g)

N content

The N content was determined according to the Kjeldahl method and the standards of ISO 5983 (ISO, 1998). First, the organic matter in the samples is digested by means of boiling with sulphuric acid in presence of a catalyst, in a digestion block (Gerhardt Kjeldahltherm with Variostat and Turbosog). Hereby, nitrogen in the samples is converted into ammonium salts. Then, in the distillation unit (Gerhardt Vapodest 6), the reaction mixture is made alkaline, distilled, and the collected ammonia is titrated. The crude protein content is obtained by multiplying the calculated nitrogen content by an international protein factor, 6.25 (6.38 for milk products).

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

In which:

V1 = ml sulphuric acid for titration

V2 = ml sulphuric acid for blank

c = concentration of the used acid (mol/L)

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

m = weight of the sample in grams

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 case of CP in milk replacer)

Chemical analysis urea recycling

By means of determination of the enrichment of [13C] and [15N] in urine and feces, different routes of urea can be quantified. The enrichment of the isotopes in feces and urine is determined by means of (GC-C-)Isotope Ratio Mass Spectrometry (IRMS). The principle of continuous flow IRMS is depicted in figure 4.3.

Figure 4.3 The principle of the continuous flow IRMS.

Analysis of [13C] and [15N]urea enrichment in feces by means of GC-C-IRMS

The [13C] and [15N]urea enrichment in air-dry (freeze-dried) feces is measured by according to 'Sample preparation guidelines for IRMS analyses'. Hereby is the principle that the Gas Chromatograph (GC) separates the compounds from an elemental analyzer (EA) which are online evaluated by the Isotope Ratio Mass Spectrometer (IRMS).

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

First, the freeze dried samples have to be ground to 200 µg particle size (talcum powder consistency) with a Retsch ball mill (MM2000) at the IRMS lab (amplitude: 90; 3 min). To obtain precise and accurate results, 1-2 mg is weighed in small aluminum cups, cups are closed, solidly folded in so that no air remains by the sample, and the weight has to be registered.

Then the aluminum clod can be put into the EA (DP 200 Series 2 Fisons). In the EA, organic matter is oxidized by means of introducing it into a copper oxide packed capillary furnace (combustion at 600 °C) and N- and C-containing organic compounds are converted into N2 and CO2, then undergoing a redox reaction in the reduction reactor (metallic copper) at 150 °C. This results in a separation of N2 and CO2 from other (ash, O2) components.

In the GC, N2 and CO2 are separated from each other based on the selective characteristics for the stationary and mobile phase. Thus N and C leave the column at a different moment and also enter the IRMS at a different moment. The IRMS measures the actual enrichment by measuring the total atomic mass of enriched atoms proportional to the total atomic mass of natural occurring atoms. Results 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 intensities at 44 ([12CO2]: 12+16+16) and 45 ([13CO2]: 13+16+16), respectively.

Atom% [15N] = [15N]

---------------- * 100

[14N] + [15N]

In which [14N] and [15N] are calculated from the mass signal intensities at 28 ([14N14N]: 14+14), 29 ([14N15N]: 14+15) and 30 ([15N15N]: 15+15).  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 means of GC-C-IRMS

The new described protocol to determine the [13C] enrichment of urea in urine is mainly based on the procedure described by Beylot et al. (1994). Beylot et al. (1994) concluded that [13C]urea enrichment during the infusion of (3-13C)lactate in humans could not be detected by gas chromatography/mass spectrometry (GC-MS), but could be easily measured by Gas Chromatography - Isotope Ratio Mass Spectrometry (GC-IRMS). Therefore, for purposes of this experiment, [13C]urea enrichment is determined by means of GC-C-IRMS.

The first part of the protocol is identical to and can be combined with the protocol to determine the [15N]urea enrichment in urine. Further on, the steps are dividing and have to be done apart.

First, 0.15 ml from the urine samples is sampled, put in Eppendorf tubes, acidified with 1.2 ml sulfosalicylic acid and centrifuged. After centrifuging, almost all of the supernatant is put gently on top of the Dowex column. The Dowex column (Dowex 50W-X8 cation exchange column) functions as a cation-anion exchanger, and thus separates the loaded particles from the unloaded compounds. By flushing the columns 4 times with 1 ml Millipore water, other unloaded compounds are washed out of the column and removed. Then the columns are flushed with 6 times 5 ml Millipore water and the eluate is collected and put in a tube for storage in the cooling or freeze dried. After total evaporation in the freeze dryer, the remained urea is dissolved in 4 times 1 ml. Of the in total 4 ml, 2 ml is transferred in glass test tubes for further [13C] analysis, while the other 2 ml is put in an Eppendorf tube for [15N] analysis. The glass test tubes are put in the Speedvac (60 °C, ±5 hours) for evaporation. After evaporation, urea is derivatized to its dimethylaminomethylene derivative ester by means of adding 200 µL of a solution of dimethylformamide dimethylacetal, acetonitrile and methanol (proportion 3:2:1). Thereafter, the tubes are placed for 1h in a water bath (70 °C) to form the dimethylaminomethylene (DAM) derivative. Then the samples are analyzed in the Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) (injector 280 °C, furnace 240 °C, interface 260 °C). For analysis by GC-C-IRMS, the vials are injected in a GC interfaced with an IRMS, whereby helium is used as a carrier gas.

Analysis of [15N]urea enrichment in urine by means of …IRMS

The new described protocol to determine the [15N] enrichment of urea in urine is mainly based on the procedure described by Marini et al. (2006). Marini et al. (2006)describe a technique which involves infusion (or injection) of [15N15N]urea, followed by isotopomer analysis of the three species [15N15N], [14N15N] and [14N14N] urea formed within the body and eliminated in the urine.

The first part of the protocol is identical to and can be combined with the protocol to determine the [13C]urea enrichment in urine. Further on, the steps are dividing and have to be done apart. First, 0.15 ml from the urine samples, which were already acidified (pH<2.5) is sampled, put in Eppendorf tubes and centrifuged. After centrifuging, almost all of the supernatant is put gently on top of the Dowex column. The Dowex column (Dowex 50W-X8 cation exchange column) functions as a cation-anion exchanger, and thus separates the loaded particles from the unloaded compounds. By flushing the columns 4 times with 1 ml Millipore water, other unloaded compounds are washed out of the column and removed. Then the columns are flushed with 6 times 5 ml Millipore water and the eluate is collected and put in a tube for storage in the cooling or freeze dried. After total evaporation in the freeze dryer, the remained urea is dissolved with 4 times 1 ml. Of the in total 4 ml, 2 ml is transferred in glass test tubes for further [13C] analysis, while the other 2 ml is put in an Eppendorf tube for further [15N] analysis. The urea is dissolved with Millipore water until the right concentration (1.5 mmol/L) is reached. After putting the eluates into Exetainers and bubbling with helium (±20 min), the tubes are slowly frozen. Once frozen, 100 µL of LiOBr (Lithium Hypobromite Oxidation Reagent) is added to oxidize urea. The oxidation of urea with LiOBr results in the monomolecular degradation of urea, which preserves the identity of the parent urea molecule. The following reaction takes place during the monomolecular degradation of urea:

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

The reaction takes place when the tubes are removed from the dry ice bath and heated by means of a heating block (Stove) at 65 °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 °C, furnace 240 °C, interface 260 °C).

Calculations urea recycling kinetics

By means of determination of the enrichment of [15N] and [13C] in feces and [15N15N], [14N15N] and [14N14N]urea in urine, different routes of urea can be quantified. Urea recycling kinetics are calculated according to Sarraseca et al. (1998) and Lobley et al. (2000, 2001), Dinh (2007).

 [13C] urea

The difference of the total infused [13C]urea and the amount of [13C]urea excreted in the urine, is the proportion [13C]urea which is transported to the rumen. The urea flux to the rumen is calculated by means of the following equation:

[13C]urea flux to rumen = total amount [13C]urea infused - [13C]urea in urine

GER = UER - UUE

[15N15N]urea

In the figures 4.4 and 4.5 are models of urea kinetics depicted.

Figure 4.4. Model of urea kinetics (Sarraseca et al., 1998). The model consists of the body urea pool and the urea in the digestive tract. The dose (D) can be partitioned between urinary excretion (u) and that which enters the digestive tract (1-u). The (1-u) flow can be further partitioned in a portion (r) which returns to the body urea pool, a portion which is lost in the feces (x) and a portion which is transferred to into microbial synthetic processes (s). GEHELE FIGUUR MISSCHIEN ERUIT LATEN?

Figure 4.5. Model of urea kinetics based on the infusion of [15N15N]urea (Lobley et al., 2000). The model consists of the body [15N]urea pool and the [15N]urea pool in the gastrointestinal tract (GIT). Solid lines represent the fates of [15N]urea direct from the [15N15N]urea infusion dose, D30. Stroked lines represent the fates of [15N]urea which are converted to NH3 in the GIT. Flows with subscript 30 represent flows of [15N15N]urea, 29 [14N15N]urea and 28 [14N14N]urea. D30 can be partitioned between urinary excretion (u) of UUE30, and the GER30 that enters the digestive tract (1-u). The (1-u) flow can be further partitioned in a portion (r) which returns to the body urea pool as ROC (return to ornithine cycle), a portion which is lost in the feces (f) as UFE ([15N]urea in feces), and a portion which is utilized for anabolism (a) as UUA, mainly via microbial synthetic processes. Thus the r, a and f are the transfers 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 bloodstream is assumed to be equal to total synthesis, and is determined from the isotopic dilution of [15N15N]urea in the urine 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]urea in the dose

EU30 = enrichment of [15N15N]urea in the urine respectively

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

Or by means of the calculation of Sarraseca et al. (1998):

UER = (…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 percentage of infusate N as [15N15N]urea

The infused [15N15N]urea dose (D30) can be excreted directly in the urine or flow into the rumen. The difference between the UER and the direct urinary urea-N elimination (UUE30) yields the GER of [15N15N]urea (GER30):

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

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

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

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

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

The fate of the Dose (D30) can be partitioned between excretion in the urine (u) and that which enters the GI tract (1-u). The proportion of D30 excreted directly in the urine 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]urea

UUE30 = urinary [15N15N]urea-N excretion, 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 cycle (ROC) and ROC proportional to GER (r) can be calculated as follows:

ROC = ρ*UER (Lobley et al., 2000)

r = ρ/(1-u) (Lobley et al., 2000)

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

ROC = urea returned to ornithine cycle, g N/d

r = fraction of GER which returns to the ornithine cycle

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

ρ = UUE29/(UUE29 + UUE30)

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

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

The fraction of the GER what is lost in the feces (f) 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 feces

UFE15 = [15N]urea lost in feces, g/d

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

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

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

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

a = 1 - f - r (Lobley et al., 2000)

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

a = fraction of GER used for anabolism

f = fraction of GER lost in feces

r = fraction of GER which returns to the ornithine cycle

UUA = urea used for anabolic purposes, 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 + f + r = 1 (Lobley et al., 2000)

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

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

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

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

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

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

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

ROC29&30 = r * GER29&30 (Lobley et al., 2000)

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

UFE29&30 = f * GER29&30 (Lobley et al., 2000)

However, when the [15N15N]urea flows to the rumen, the label can return via absorption to the bloodstream. The [15N15N] urea is hydrolyzed into 2 * [15NH3].

The 15NH3 from the rumen can be absorbed directly from the rumen or from the intestine into the blood and in the liver it can be used for urea synthesis. Thus the liver transforms the toxic ammonia into urea. Thus, [15N14N]urea will appear in the bloodstream, resulting in:

Excretion in the urine [15N14N]urea

Recycling to the rumen  hydrolyzation into [14NH3] and [15NH3]  absorption ( possibly infinite recycling)

(2) The 15NH3 from the rumen can also be used for microbial protein synthesis, whereby the isotope is incorporated in a with [15N] labeled protein:

[15N]protein remains undigested and appears in the feces, mainly in the form of [14N15N]urea/protein and [15N]proteins, because there is only a very small chance that two [15N] isotopes are combining into one urea/protein molecule again, this [15N15N]urea/protein in feces is not specified in the measurements (REFERENTIE WAAROM 15N15N NIET GESPECIFICEERD WORD).

[15N]proteins can be absorbed and deposited in body protein, which will not be measured in this experiment (anabolic fate)

[15N]proteins can be absorbed, deposited, oxidized and…

…be converted (liver) and excreted as [15N14N]urea or [15N]protein in urine (catabolic fate)

…be converted (liver) into [15N14N]urea and be recycled to the rumen

Statistical analysis

Data were analyzed using the GLM/Mixed models procedure of SAS (SAS-Institute, 2002).

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

Y = µ + dieti + … + ɛijk

Y = dependent variable

µ = the average experimental value

Diet = the effect of dietary treatment i; i = diet 1, 2, 3, 4

… = other important effects

ɛijk = the error term, related to treatment i, … j, and calve k (k = 1 - 48).

NOG NADER TE BEPALEN EN UIT TE WERKEN