The effect of the source of nitrogen supplementation on nitrogen balance,

rates of plasma leucine turnover, protein synthesis and degradation in

sheep

Hiroaki Sano*, Sachi Shibasaki and Hirotaka Sawada

Department of Animal Sciences, Faculty of Agriculture, Iwate University, Morioka, Japan

(Received 28 November 2008; accepted 6 March 2009)

Combined experiments of the isotope dilution method of [1-13C]leucine, open-circuit calorimetry and nitrogen (N) balance test were used to determine theeffect of the source of N supplementation on N balance, whole body proteinsynthesis (WBPS) and degradation (WBPD) in sheep. The experiment wasperformed in a replicated 3 6 3 Latin square design. The control diet consistedof timothy hay, ground maize and soybean meal. The urea diet was the controldiet supplemented with 1.5% urea. The SBM diet contained the same N andmetabolisable energy as the urea diet, which was reached by changing groundmaize and soybean meal weights of the control diet. Nitrogen retention wasgreater (p 5 0.05) for the urea diet than the control and SBM diets. Plasmaurea concentrations were highest for the SBM diet, followed by the urea diet,and the control diet was lowest. The WBPS and WBPD did not differ betweendiets, but were numerically lower for the urea and SBM diets. These resultssuggest that in sheep, urea supplementation influenced N retention without clearchanges in WBPS and WBPD.

Keywords: urea; feed supplements; soybean oil meal; amino acid metabolism;protein synthesis; protein degradation; sheep

1. Introduction

In ruminants, dietary protein utilisation is more complex than in monogastricanimals because microorganisms in the rumen play an important role for thedigestion of diets. The rumen microorganisms synthesise microbial protein fromboth dietary protein and non-protein nitrogen (N) compounds. However, littleinformation is currently available focusing on the effect of the source of Nsupplementation on amino acid and protein metabolism in ruminants. In sheep,increased dietary crude protein (CP) intake did not influence whole body proteinsynthesis (WBPS) but decreased whole body protein degradation (WBPD), resultingin increased N retention (Sano et al. 2004).

Recently, soybean production has not been sufficient for both human food andanimal diets, which has become a serious problem. Moreover, soybean meal, one ofthe most important dietary protein sources, is costly, largely degraded in the rumen

*Corresponding author. Email: sano@iwate-u.ac.jp

Archives of Animal Nutrition

Vol. 63, No. 5, October 2009, 401–412

ISSN 1745-039X print/ISSN 1477-2817 online

� 2009 Taylor & Francis

DOI: 10.1080/17450390903052698

http://www.informaworld.com

and less absorbed than other protein sources in lactating dairy cattle (Santos et al.1984). Urea, a non-protein N, is available at a low cost (Mahouachi et al. 2003).Urea supplementation is widely applied and has benefits for the production of cows(Tedeschi et al. 2002; Zinn et al. 2003). It was hypothesised that dietary Nsupplementation as urea and soybean meal might influence amino acid metabolismand protein retention in ruminants by increasing microbial protein synthesis in therumen (Archimede et al. 1999). Therefore, this experiment was designed to measureplasma leucine (Leu) turnover rate (LeuTR), as measured by the isotope dilutionmethod using a stable isotope, and to calculate WBPS and WBPD combined withdetermination of Leu oxidation (LeuOX) and N balance in sheep fed a basal dietsupplemented with urea and soybean meal.

2. Materials and methods

2.1. Animals and diets

The surgery and experimental procedures were reviewed and approved by theAnimal Care Committee of Iwate University. All experiments were carried outwithout noticeable stress to the animals.

Six crossbred (Corriedale6 Suffolk) shorn sheep (3 ewes and 3 rams), aged 1 to 2years and 45+2 kg BW were used. The sheep were surgically prepared underanaesthesia with a skin loop enclosing the left carotid artery at least three monthsbefore the start of the experiment. The sheep were assigned to three dietarytreatments. The basal diet (control diet) consisted of timothy hay (metabolisableenergy [ME] 9.2 kJ/g air DM, 6.1% CP, 57.7% neutral detergent fibre [NDF]),ground maize (ME 13.8 kJ/g air DM, 5.4% CP) and soybean meal (ME 13.3 kJ/g airDM, 36.2% CP). For the second diet (urea diet) the control diet was supplementedwith urea (0.86 g � kg BW70.75 � d71), and N intake was designed to be 150% ofmaintenance. The third diet (SBM diet) was mixed with the same N content as theurea diet by changing ground maize and soybean meal weights of the control diet.Metabolisable energy and metabolisable protein (MP) intakes assumed from theAgricultural and Food Research Council (AFRC 1993) were similar for all dietarytreatments and were slightly above the maintenance levels, as listed in Table 1. The

Table 1. Diet formulation and intakes of crude protein (CP), metabolisable protein (MP)and metabolisable energy (ME) of the dietary treatments.

Diet#

CONT UREA SBM

Timothy hay [g � kg BW70.75 � d71] 35.0 35.0 35.0Ground corn [g � kg BW70.75 � d71] 15.6 15.6 8.6Soybean meal [g � kg BW70.75 � d71] 5.4 5.4 12.4Urea [g � kg BW70.75 � d71] 0 0.86 0CP intake [g � kg BW70.75 � d71] 4.9 7.3 7.1MP intake* [g � kg BW70.75 � d71] 4.2 4.2 4.5ME intake* [kJ � kg BW70.75 � d71] 609 609 606

Notes: #CONT, basal diet; UREA, basal diet plus urea; SBM, soybean meal-rich diet. *Assumed fromAFRC (1993).

402 H. Sano et al.

experiment utilised a replicated 3 6 3 Latin square design with three periods of29 d. Animals were housed in individual pens in an animal room during thepreliminary period and the first three weeks of the experiment. The sheep were thenmoved to metabolic cages in a controlled environment chamber at an airtemperature of 23+18C, with lighting present from 06:00 to 22:00 h. The animalsreceived feed once daily at 14:00 h, which was commonly eaten within 1 h, but themanger was not removed until the next morning. Water was available ad libitum. Thesheep were weighed on day 1, 15 and 29 of each dietary treatment.

2.2. Nitrogen balance test and rumen fluid collection

Nitrogen balance was determined over five successive days of the last week of each29 day treatment. Faeces were collected for each 24 h, dried in a forced air oven(608C, 48 h) and weighed at 5 d after placement at room temperature. An aliquotwas ground and stored until analysis. Urine was collected for each 24 h in a bottlecontaining 100 ml of 6 N H2SO4 and an aliquot was stored (7308C). On day 27 ofeach treatment, rumen fluid (50 ml) was collected through a stomach tube at 2 hafter feeding. Immediately after determination of pH of rumen fluid by a pH meter(HM-10P, Toa Electronics Ltd., Japan), the rumen fluid was centrifuged at 1000 gfor 10 min at 48C (RS-18IV, Tomy, Japan). An aliquot (2 ml) acidified for ammoniadetermination and residuals of the rumen fluid were stored at 7308C until furtheranalysis.

2.3. Isotope dilution method

A catheter for infusion was inserted into a jugular vein on day 27 and a catheter forblood sampling was inserted into the skin loop of the carotid artery in the morningon day 28. Catheters were filled with a sterile solution of 3.8% trisodium citrate. Thecombined experiment of an isotope dilution method of [1-13C]Leu and open-circuitcalorimetry was conducted to determine plasma LeuTR, LeuOX, WBPS and WBPDas reported by Sano et al. (2004). At 11:00 h, the sheep were fitted with a clear headchamber (approximately 0.2 m3) for collecting gaseous samples throughout thesamplings of blood and exhaled gas. After collecting blood and gaseous samples atthe pre-infusion period, 10 mmol/kg BW0.75 of [1-13C]Leu (L-leucine-1-13C, 99 atom% excess 13C; Isotec Inc., A Matheson, USA Co., USA) and 3.5 mmol/kg BW0.75 ofNaH13CO3 (sodium bicarbonate-13C, 99.2 atom % excess 13C; Isotec Inc., AMatheson, USA Co., USA) dissolved in saline solution (0.9% sodium chloride)were injected into the catheter for infusion as a priming dose. Then, [1-13C]Leu(4 mmol/l in saline) was continuously infused by a multichannel peristaltic pump(AC-2120, Atto, Japan) at a rate of 10 mmol � kg BW–0.75 � h71 through the samecatheter for 8 h. Blood samples were taken from the catheter for blood samplingimmediately before (10 ml) and at 30-min intervals (5 ml) during the last 90 min of[1-13C]Leu infusion. Samples were transferred into centrifuge tubes containingheparin sodium and were chilled until centrifugation. Blood samples werecentrifuged at 10,000 g for 10 min at 48C, and the plasma was stored at 7308Cuntil further analyses.

Open-circuit calorimetry (Metabolic Monitor, Coast Electronics, UK) was usedto determine carbon dioxide (CO2) production and exhaled 13CO2 enrichmentsthroughout the isotope dilution method. The CO2 production rate was continuously

Archives of Animal Nutrition 403

determined for 15 min at 5.5 h of [1-13C]Leu infusion and immediately after the endof [1-13C]Leu infusion. An aliquot of exhaled CO2 was collected in 4 ml of 1 NNaOH for 30 min immediately before and four times half hourly during the last 2 hof [1-13C]Leu infusion to determine the isotope enrichments of exhaled 13CO2 andthe NaOH solution was stored at 7308C. The catheters were removed aftercollections of all blood and gas samples.

2.4. Chemical analyses

Nitrogen contents in diets, faeces, and urine were determined by a colorimetricmethod for ammonia N (Weatherburn 1967) after Kjeldahl digestion. Theconcentrations of plasma Leu and a-ketoisocaproic acid (a-KIC) and enrichmentsof plasma [1-13C]Leu and a-[1-13C]KIC were determined by gas chromatographymass spectrometry (QP-2010, Shimadzu, Japan) by the procedures of Rocchiccioliet al. (1981) and Calder and Smith (1988) as described previously (Sano et al. 2004).The isotopic abundance of exhaled 13CO2 was determined with a gas chromato-graphy-combustion-isotope ratio mass spectrometric system (DELTAplus, ThermoElectron, USA). Concentrations of plasma free amino acids and urea at the pre-infusion period were determined with an automated amino acid analyser (JLC-500/V, JEOL, Japan). Plasma insulin concentration at the pre-infusion period wasdetermined by a radioimmunoassay kit (IRI ‘Eiken’, Eiken Chemical, Japan). Theintra- and interassay coefficients of variation were 6 and 9%, respectively.Concentrations of ammonia in the rumen fluid and plasma ammonia weredetermined using a diagnostic kit (Ammonia-test, Wako, Japan). Concentrationsof total volatile fatty acids (VFA) in the rumen fluid (5 ml) were determined bytitration using 0.1 mol/l NaOH during steam distillation. The molar ratio ofindividual VFA was then analysed by gas chromatography (HP-5890, HewlettPackard, USA) equipped with a 30 m 6 0.25 mm DB-FFAP capillary column (J &W Scientific, USA). The conditions included the injector and detector temperature of1708C. Oven temperature was raised from 1008C to 1658C by 88C/min gradient withcarrier gas (He) flow of 1.0 ml/min.

2.5. Calculations

Mean values with standard errors of the mean are given. Plasma LeuTR and LeuOXwere calculated using the equations by Krishnamurti and Janssens (1988):

LeuTR ½mmol � kgBW�0:75 � d�1� ¼ I � ð1=EKIC � 1Þ;

where I is the infusion rate of [1-13C]Leu [mmol � kg BW70.75 � h71] and EKIC is theisotope enrichment of plasma a-[1-13C]KIC during the steady states (ratio of[1-13C]KIC to [1-12C]KIC), and:

LeuOX ½mmol � kgBW�0:75 � d�1� ¼ ½ECO2=0:81Þ � VCO2�=EKIC;

where ECO2is the isotope enrichment of exhaled 13CO2 (ratio of 13CO2 to

12CO2) andVCO2 [mmol � kg BW–0.75� d–1] is the CO2 production rate. The recovery fraction ofthe exhaled CO2 production in the animal body was estimated to be 0.81 as describedby Allsop et al. (1978) and Wolfe et al. (1982). Whole body protein synthesis (WBPS)

404 H. Sano et al.

and WBPD were calculated from the following equations as described byKrishnamurti and Janssens (1988):

WBPS g � kg BW�0:75 � d�1� �

¼ LeuTR � LeuOXð Þ=Leu concentration in carcass protein,

WBPD g � kg BW�0:75 � d�1� �

¼WBPS�N balance g � kg BW�0:75 � d�1� �

� 6:25:

Leucine concentration in carcass protein (6.6%) was used as described by Harriset al. (1992).

2.6. Statistics

All data were analysed with the MIXED procedure of SAS (1996). The fixed effect inthe model was period, diet, and period 6 diet interaction and the random effect wassheep. The sex effect was also tested. Results were considered significant at thep 5 0.05 level. If the effect of diet was significant, the Tukey adjustment was used tocompare diets and was considered significant at p 5 0.05. Repeated statement wasused for the time course of parameters during the isotope dilution method and thedifference in least square means with the Tukey adjustment was used (p 5 0.05).

3. Results

The sheep consumed all the diets supplied; therefore, ME and MP intakes wereestimated to be similar among the dietary treatments. Nitrogen intake and Nexcretion in urine differed (p 5 0.001 and p 5 0.01, respectively) between diets(Table 2). Nitrogen intake was highest (p 5 0.05) for the urea diet, but urinary Nexcretion was highest (p 5 0.05) for the SBM diet. Nitrogen excretion in faeces did

Table 2. Effects of the source of nitrogen (N) supplementation on N balance, N digestibilityand body weight change in sheep (n ¼ 6).

Diet{

SE#

Significance

CONT UREA SBM Period Diet Period 6 diet Sex

N intake [g � kgBW70.75 � d71]

0.81c 1.18a 1.14b 0.08 NS *** NS NS

N in faeces [g � kgBW70.75 � d71]

0.21 0.19 0.20 0.02 NS NS NS NS

N in urine [g � kgBW70.75 � d71]

0.44c 0.62b 0.75a 0.08 NS ** NS NS

N retention [g � kgBW70.75 � d71]

0.16b 0.36a 0.18b 0.07 NS ** NS NS

N digestibility [%] 74.5b 83.6a 82.0a 2.6 NS ** NS NSBody weightchange [g/d] 33 51 56 10 NS NS NS NS

Notes: {CONT, basal diet; UREA, basal diet plus urea; SBM, soybean meal-rich diet. #SE, standard errorof the mean. ***p 5 0.001; **p 5 0.01. NS ¼ not significant. Means with different superscripts in thesame row differ (p 5 0.05).

Archives of Animal Nutrition 405

not differ between diets. Nitrogen retention differed (p 5 0.01) between diets, andwas higher (p 5 0.05) for the urea diet than the control and SBM diets. Nitrogendigestibility was higher (p 5 0.05) for the urea and SBM diets than the control diet.Body weight change did not differ between diets.

At 2 h after feeding pH values in the rumen fluid differed (p 5 0.001) betweendiets, and were higher (p 5 0.05) for the urea and SBM diets than the control diet(Table 3). Ammonia concentrations in the rumen fluid differed (p 5 0.01) betweendiets, and were higher (p 5 0.05) for the urea diet and lower (p 5 0.05) for thecontrol diet than other diets. Concentrations of total VFA, acetate, propionate,n-butyrate, isovalerate and n-valerate in the rumen fluid did not differ between diets.Concentrations of isobutyrate differed (p 5 0.01) between diets, and were higher(p 5 0.05) for the SBM diet than other diets. The period effect was significant inisobutyrate (p 5 0.01) and n-butyrate (p 5 0.05).

Plasma amino acids, ammonia, urea and insulin concentrations determined at thepre-infusion period of the isotope dilution method were listed in Table 4. Of plasmaamino acids, glutamine concentrations differed (p 5 0.001) between diets and werelower (p 5 0.05) for the urea and SBM diets than for the control diet. Other aminoacid concentrations did not differ between diets. Plasma urea concentrations differed(p 5 0.01) between diets, and were higher (p 5 0.05) for the SBM diet and lower(p 5 0.05) for the control diet than other diets. Plasma ammonia and insulinconcentrations did not differ between diets. Concentrations of plasma isoleucine,Leu, phenylalanine, lysine, serine, arginine, tyrosine and proline were significantlyhigher for the rams compared with the ewes.

Concentrations of plasma Leu and a-KIC and enrichments of plasma [1-13C]Leuand a-[1-13C]KIC and exhaled 13CO2 were almost constant during the latter periodsof the isotope dilution method for each treatment (data not shown). The meancoefficients of variances of isotope enrichments during the corresponding periodwere 6.4, 4.9 and 19.6% for plasma [1-13C]Leu and a-[1-13C]KIC and exhaled 13CO2,respectively. Plasma Leu and a-KIC concentrations for the urea diet did not differfrom those for the control diet, but plasma Leu concentrations for the SBM diet

Table 3. Effects of the source of nitrogen supplementation on ruminal characteristics insheep (n ¼ 6).

Diet{

SE#

Significance

CONT UREA SBM Period Diet Period 6 diet Sex

pH 6.53b 6.88a 6.70a 0.07 NS *** NS NSAmmonia [mmol/l] 22.5c 51.6a 33.2b 6.3 NS ** NS NS

Volatile fatty acid concentrations [mmol/l]Total 84.6 89.2 90.6 3.0 NS NS NS NSAcetate 53.8 58.0 58.5 2.0 NS NS NS NSPropionate 15.9 17.6 16.4 0.8 NS NS NS NSiso-butyrate 0.8b 0.9b 1.1a 0.1 ** ** ** NSn-butyrate 10.7 9.6 10.9 0.6 * NS NS NSiso-valerate 2.1 1.8 2.3 0.3 NS NS NS NSn-valerate 1.3 1.3 1.5 0.1 NS NS NS NS

Notes: {CONT, basal diet; UREA, basal diet plus urea; SBM, soybean meal-rich diet. #SE, standard errorof the mean. ***p 5 0.001; **p 5 0.01; *p 5 0.05. NS ¼ not significant. Means with differentsuperscripts in the same row differ (p 5 0.05).

406 H. Sano et al.

were lower (p 5 0.05) than the other two diets (Table 5). Plasma LeuTR, LeuOX,WBPS and WBPD did not differ between diets, but the values of LeuTR, WBPS andWBPD were numerically lower for the urea and SBM diets compared with thecontrol diet. Plasma LeuTR was greater (p 5 0.01) for the rams compared with theewes, but no other gender difference was detected in parameters of proteinmetabolism including the WBPS.

4. Discussion

The present experiment demonstrated that in mature sheep dietary N sourceinfluenced N retention without clear changes in WBPS and WBPD. Ureasupplementation benefits ruminant production and improves the function ofmicroorganisms in the rumen (Tedeschi et al. 2002; Zinn et al. 2003; Wallace et al.1987). Tedeschi et al. (2002) reported that in growing and finishing cattle,supplemental urea (0.4–1.2% of diet DM) improved average daily gain and feedconversion. Zinn et al. (2003) reported that in steers, average daily gain increasedlinearly with increasing urea level (0, 0.4, 0.8 and 1.2% DM basis), whereas feedefficiency remained unchanged. Wallace et al. (1987) reported that microbialproteolytic activities of rumen fluid in sheep fed diets supplemented with urea werehigher than with casein and was similar to that with egg albumin. The level of urea

Table 4. Effects of the source of nitrogen supplementation on plasma amino acid, urea,ammonia and insulin concentrations in sheep (n ¼ 6).

Diet{

SE#

Significance

CONT UREA SBM Period Diet Period 6 diet Sex

Plasma amino acids [mmol/l]Arginine 185 181 202 13 NS NS NS NSHistidine 79 79 73 4 NS NS NS NSIsoleucine 93 96 100 6 NS NS NS **Leucine 137 145 138 9 NS NS NS **Lysine 134 139 151 9 NS NS NS *Methionine 24 22 20 3 NS NS NS NSPhenylalanine 57 59 58 4 NS NS NS **Threonine 267 259 270 23 NS NS NS NSValine 195 211 223 14 NS NS NS NSAlanine 183 194 190 22 NS NS NS NSAspartic acid 3.6 3.1 4.2 0.8 NS NS NS NSGlutamic acid 83 79 80 4 NS NS NS NSGlycine 709 629 597 42 NS NS NS *Proline 140 141 126 9 NS NS NS NSSerine 168 144 145 14 NS NS NS **Asparagine 64 64 67 5 NS NS NS *Glutamine 345a 298b 299b 19 NS * NS NSTyrosine 82 83 75 9 NS NS NS *Tryptophan 47 41 42 2 NS NS NS NS

Ammonia [mmol/l] 85 92 85 5 NS NS NS NSUrea [mmol/l] 6.9c 8.6b 10.1a 0.7 NS ** NS NSInsulin [mU/ml] 35 40 29 7 NS NS NS NS

Notes: {CONT, basal diet, UREA, basal diet plus urea, SBM, soybean meal-rich diet; #SE, standard errorof the mean; **p 5 0.01; *p 5 0.05; NS ¼ not significant. Means with different superscripts in the samerow differ (p 5 0.05).

Archives of Animal Nutrition 407

supplementation (1.5% of diet) in the present experiment was comparable to thosedescribed above. Plasma urea concentrations were higher for N supplemented diets,but plasma ammonia concentrations were comparable between the dietarytreatments. When urea was supplemented to the basal diet, the postprandial plasmaammonia concentrations increased markedly because a large part of the ammoniaproduced from supplemental urea in the rumen was directly absorbed from therumen (Sinclair et al. 2000) and ammonia-N concentrations did not differ betweenwith and without urea supplementation by 4 h after feeding (Mahouachi et al. 2003).Protein degradability of soybean meal in the rumen was greater and availability ofamino acids for absorption was less than corn gluten meal, wet brewers’ grains anddistillers’ dried grains with solubles in lactating cattle (Santos et al. 1984). Therefore,degradability of urea and protein from the urea and SBM diets in the rumen wasfaster than the control diet, but urea utilisation would still be activated even at 22 hafter feeding without the effect on plasma ammonia concentrations.

In the present experiment, all dietary treatments showed positive N balance;therefore, CP intake may be above the maintenance requirements. Although bodyweight change did not differ between the urea and SBM diets, N retention wasgreater for the urea diet compared with the SBM diet due to the differences inurinary N excretion. The urea recycling rate into the gut tended to increase ureainfusion into the rumen of sheep (Obara and Dellow 1993). Therefore, the urearecycling rate would be increased for the urea diet and result in lower plasma ureaconcentrations than the SBM diet. The higher N digestibility for the urea and SBMdiets agreed with the observation by Ferrell et al. (1999). They suggested that theapparent N digestibilities for urea, soybean meal and ruminally undegraded proteinsupplementation to diets should be interpreted with caution, because most of thefaecal N loss was attributed to metabolic faecal N. Moreover, the higher N retention

Table 5. Effects of the source of nitrogen supplementation on plasma leucine (Leu) and a-ketoisocaproic acid (a-KIC) concentrations, rates of Leu turnover and Leu oxidation, wholebody protein synthesis and degradation in sheep (n ¼ 6).

Diet{

SE#

Significance

CONT UREA SBM Period Diet Period 6 diet Sex

Leu [mmol/l] 108a 108a 106b 4 NS ** * NSa-KIC [mmol/l] 15.8a 14.1ab 13.1b 1.3 NS * NS NSLeu turnover rate[mmol � kgBW70.75 � d71]

8.8 8.4 8.2 0.2 NS NS NS **

Leu oxidation rate[mmol � kgBW70.75 � d71]

1.1 1.2 0.8 0.3 NS NS NS NS

Protein synthesis[g � kgBW70.75 � d71]

15.2 14.4 14.7 0.6 NS NS NS NS

Protein degradation[g � kgBW70.75 � d71]

14.2 12.1 13.6 0.9 NS NS NS NS

Notes: {CONT, basal diet, UREA, basal diet plus urea, SBM, soybean meal-rich diet. #SE, standard errorof the mean. **p 5 0.01; *p 5 0.05. NS ¼ not significant. Means with different superscripts in the samerow differ (p 5 0.05).

408 H. Sano et al.

for the urea diet accorded with the results obtained in sheep (Kempton et al. 1979;Archimede et al. 1999). Kempton et al. (1979) reported that in growing lambs fed alow-protein cellulose diet, urea supplementation improved digestibility and retentionof N. Archimede et al. (1999) also reported that in rams fed a low quality ofroughage diet, supplemental urea and sugar (2.3 and 5.0% of hay, respectively)improved N balance and stimulated the microbial proliferation and activity. On theother hand, Bernard et al. (2003) reported that in Jersey cows fed whole cottonseedcoated with combinations of corn starch (2.5 and 5%) and urea (0.25 and 0.5%)duodenal flow rates of total and bacterial N were not influenced by urea treatment.The inconsistency of the effect could partly be related to the level of supplementalurea and species used. Net hepatic uptake and portal release of a-amino N increasedwith both urea and soybean meal supplementation in sheep (Ferrell et al. 1999).Therefore, increased N digestibility for the urea and SBM diets and N retention forthe urea diet in the present experiment may be partly related to increased microbialprotein synthesis from dietary N.

Zinn et al. (2003) reported that in steers fed twice daily, urea supplementation didnot influence total VFA concentrations and molar percentages of acetate andpropionate in the rumen fluid. Hsu et al. (1991) observed the similar results ofruminal VFA in defaunated sheep. In the present experiment concentrations ofacetate and propionate, the major VFA, in the rumen fluid were not influenced by Nsupplementation. Therefore, it seems that fermentation of carbohydrates is notmodified by N supplementation, when ME intake is constant. Ruminal pHdetermined at 2 h after feeding, the only spot sample from a stomach tube, waswithin the normal range for all the dietary treatments and was higher for the ureaand SBM diets than the control diet. Higher ruminal pH would be reflected by bothunchanged VFA production and the alkalising effect of supplemental urea in therumen (Zinn et al. 2003).

In the present experiment, enrichments of plasma a-[1-13C]KIC and exhaled13CO2 from plasma [1-13C]Leu were used for calculation of whole-body proteinmetabolism instead of those of plasma [1-13C]Leu and exhaled 13CO2, becauseenrichments of plasma a-[1-13C]KIC represent the true kinetics (Magni et al. 1994).The enrichment ratio of plasma a-[1-13C]KIC to plasma [1-13C]Leu was 70+1%,agreeing with those obtained previously (Sano et al. 2004; Al-Mamun et al. 2007).Whole body protein synthesis was close to those reported in sheep using the sameexperimental methods (Krishnamurti and Janssens 1988; Sano et al. 2004), and wasalso similar to those in goats using the [2H5]phenylalanine model (Fujita et al. 2007).However, WBPS in sheep was considerably lower than growing steers (Lapierre et al.1999), even when the data was compared based on the metabolic body size. Sanoet al. (2004) reported that in sheep WBPS and WBPD were changed towardreduction with increased CP intake using both enrichments of plasma [1-13C]Leu anda-[1-13C]KIC. The present experiment observed a similar trend, but plasma LeuTR,WBPS and WBPD did not differ significantly between diets. Whole body proteinsynthesis would be more influenced by dietary energy and MP intakes than N intake(Lapierre et al. 2002; Fujita et al. 2006).

Plasma amino acid concentrations were not significantly influenced by Nsupplementation, except that plasma glutamine concentrations were lower than thecontrol diet. Ferrell et al. (1999) reported that in sheep plasma a-amino Nconcentrations remained unchanged with both urea and soybean meal supplementa-tion. It is possible that decreased plasma glutamine concentrations were attributable

Archives of Animal Nutrition 409

to reduced proteolysis in the body protein. In the present experiment, the gendereffect was significant in eight plasma free amino acid concentrations and LeuTR.Similar results were obtained in healthy humans as Caballero et al. (1991) reportedpostabsorptive plasma concentrations of plasma valine, leucine and isoleucine weresignificantly lower in young women than in young men. The difference would partlybe reflected by secretion of gonadotropins (Zurek et al. 1995).

Unchanged plasma insulin concentrations suggested that insulin release was notmodified by N supplementation, although plasma insulin concentrations were onlydetermined at the pre-infusion period. Concerning insulin action on proteinmetabolism, intravenous insulin infusion reduced WBPS, WBPD and endogenousLeu appearance rate with the isotope dilution method of [1-14C]Leu in fed and fastedlambs, and lactating and dry goats (Oddy et al. 1987; Tesseraud et al. 1993).Bequette et al. (2002) also reported that in lactating goats insulin infusion reducedwhole-body and hind-leg protein metabolism with the isotope dilution method of[15N, 1-14C]Leu and a hyperinsulinemic-euglycemic clamp. Insulin sensitivity wasinfluenced by CP intake in sheep (Sano and Terashima 2001). The impact onincreased N balance for the urea diet may partly be related to enhanced action ofinsulin which reduces WBPS and WBPD.

In conclusion, N supplementation as urea and soybean meal did not influenceprotein synthesis and degradation in sheep clearly. The difference in N balancebetween N supplementation would be related to the urea recycling and insulinaction.

Acknowledgements

The authors are grateful to Kim Taylor, University of Guelph, Canada, for his kind commentson the manuscript.

References

Agricultural and Food Research Council (AFRC). 1993. Energy and protein requirements ofruminants. An advisory manual prepared by the AFRC technical committee on responsesto nutrients. Wallingford, UK: CAB International.

Allsop JR, Wolfe RR, Burke JK. 1978. Tracer priming the bicarbonate pool. J Appl Physiol.45:137–139.

Al-Mamun M, Ito C, Sato A, Fujita T, Sano H. 2007. Comparison of the [2H5]phenylalaninemodel with the [1-13C]leucine method to determine whole body protein synthesis anddegradation in sheep fed at two levels. Asian Austral J Anim Sci. 20:1517–1524.

Archimede H, Aumont G, Saminadin G, Depres E, Despois P, Xande A. 1999. Effects of ureaand saccharose on intake and digestion of a Digitaria decumbens hay by black belly sheep.Anim Sci. 69:403–410.

Bequette BJ, Kyle CE, Crompton LA, Anderson SE, Hanigan MD. 2002. Protein metabolismin lactating goats subjected to the insulin clamp. J Dairy Sci. 85:1546–1555.

Bernard JK, West JW, Trammell DS, Parks AH, Wedegaertner TC. 2003. Ruminalfermentation and bacterial protein synthesis of whole cottonseed coated with combina-tions of gelatinized corn starch and urea. J Dairy Sci. 86:3661–3666.

Caballero B, Gleason RE, Wurtman RJ. 1991. Plasma amino acid concentrations in healthyelderly men and women. Am J Clin Nutr. 53:1249–1252.

Calder AG, Smith A. 1988. Stable isotope ratio analysis of leucine and ketoisocaproic acid inblood plasma by gas chromatography/mass spectrometry. Use of tertiary butyldimethyl-silyl derivatives. Rapid Commun Mass Spectro. 2:14–16.

Ferrell CL, Kreikemeier KK, Freetly HC. 1999. The effect of supplemental energy, nitrogen,and protein on feed intake, digestibility, and nitrogen flux across the gut and liver in sheepfed low-quality forage. J Anim Sci. 77:3353–3364.

410 H. Sano et al.

Fujita T, Kajita M, Sano H. 2006. Responses of whole body protein synthesis, nitrogenretention and glucose kinetics to supplemental starch in goats. Comp Biochem Physiol.144B:180–187.

Fujita T, Kajita M, Sano H. 2007. Effects of non-protein energy intake on whole body proteinsynthesis, nitrogen retention and glucose turnover in goats. Asian Austral J Anim Sci.20:536–542.

Harris PM, Skene PA, Buchan V, Milne E, Calder AG, Anderson SE, Connell A, Lobley GE.1992. Effect of food intake on hind-limb and whole-body protein metabolism in younggrowing sheep: Chronic studies based on arterio-venous techniques. Brit J Nutr.68:389–407.

Hsu JT, Fahey GC, Jr, Clark JH, Berger LL, Merchen NR. 1991. Effects of urea and sodiumbicarbonate supplementation of a high-fiber diet on nutrient digestion and ruminalcharacteristics of defaunated sheep. J Anim Sci. 69:1300–1311.

Kempton TJ, Nolan JV, Leng RA. 1979. Protein nutrition of growing lambs. 2. Effect onnitrogen of supplementing a low-protein-cellulosic diet with either urea, casein orformaldehyde-treated casein. Brit J Nutr. 42:303–315.

Krishnamurti CR, Janssens SM. 1988. Determination of leucine metabolism and proteinturnover in sheep, using gas-liquid chromatography-mass spectrometry. Brit J Nutr.59:155–164.

Lapierre H, Bernier JF, Dubreuil P, Reynolds CK, Farmer C, Ouellet DR, Lobley GE. 1999.The effect of intake on protein metabolism across splanchnic tissues in growing beef steers.Brit J Nutr. 81:457–466.

Lapierre H, Blouin JP, Bernier JF, Reynolds CK, Dubreuil P, Lobley GE. 2002. Effect ofsupply of metabolizable protein on whole body and splanchnic leucine metabolism inlactating cows. J Dairy Sci. 85:2631–2641.

Magni F, Arnoldi L, Galati G, Galli Kienle M. 1994. Simultaneous determination of plasmalevels of a-ketoisocaproic acid and leucine and evaluation of a-[1-13C]ketoisocaproic acidand [1-13C]leucine enrichment by gas chromatography-mass spectrometry. Anal Biochem.220:308–314.

Mahouachi M, Haddad L, Kayouli C, Thewis A, Beckers Y. 2003. Effect of the nature ofnitrogen supplementation on voluntary intake, rumen parameters and ruminaldegradation of dry matter in sheep fed oat silage-based diet. Small Ruminant Res.48:181–187.

Obara Y, Dellow DW. 1993. Effects of intraruminal infusions of urea, sucrose or urea plussucrose on plasma urea and glucose kinetics in sheep fed chopped lucerne hay. J Agric Sci.(Camb). 121:125–130.

Oddy VH, Lindsay DB, Barker PJ, Northrop AJ. 1987. Effect of insulin on hind-limb andwhole-body leucine and protein metabolism in fed and fasted lambs. Brit J Nutr.58:437–452.

Rocchiccioli F, Leroux JP, Cartier P. 1981. Quantitation of 2-ketoacids in biological fluidsby gas chromatography chemical ionization mass spectrometry of o-trimethylsilyl-quinoxalinol derivatives. Biomed Mass Spectrom. 8:160–164.

Sano H, Kajita M, Fujita T. 2004. Effect of dietary protein intake on plasmaleucine flux, protein synthesis, and degradation in sheep. Comp Biochem Physiol.139B:163–168.

Sano H, Terashima Y. 2001. Effects of dietary protein level and cold exposure on tissueresponsiveness and sensitivity to insulin in sheep. J Anim Physiol Anim Nutr. 85:349–355.

Santos KA, Stern MD, Satter LD. 1984. Protein degradation in the rumen and amino acidabsorption in the small intestine of lactating dairy cattle fed various protein sources. JAnim Sci. 58:244–255.

SAS. 1996. SAS/STAT1 Software: Changes and enhancements through release 6.11. Cary:SAS Inst Inc.

Sinclair KD, Sinclair LA, Robinson JJ. 2000. Nitrogen metabolism and fertility in cattle: I.Adaptive changes in intake and metabolism to diets differing in their rate of energy andnitrogen release in the rumen. J Anim Sci. 78:2659–2669.

Tedeschi LO, Baker MJ, Ketchen DJ, Fox DG. 2002. Performance of growing and finishingcattle supplemented with a slow-release urea product and urea. Can J Anim Sci.82:567–573.

Archives of Animal Nutrition 411

Tesseraud S, Grizard J, Debras E, Papet I, Bonnet Y, Bayle G, Champredon C. 1993. Leucinemetabolism in lactating and dry goats: effect of insulin and substrate availability. Am JPhysiol. 265:E402–E413.

Wallace RJ, Broderick GA, Brammall ML. 1987. Protein degradation by ruminalmicroorganisms from sheep fed dietary supplements of urea, casein, or albumin. ApplEnviron Microbiol. 53:751–753.

Weatherburn MW. 1967. Phenol-hypochloride reaction for determination of ammonia. AnalChem. 39:971–974.

Wolfe RR, Goodenough RD, Wolfe MH, Royle GT, Nadel ER. 1982. Isotope analysis ofleucine and urea metabolism in exercising humans. J Appl Physiol. 52:458–466.

Zinn RA, Barrajas R, Montano M, Ware RA. 2003. Influence of dietary urea level ondigestive function and growth performance of cattle fed steam-flaked barley-basedfinishing diets. J Anim Sci. 81:2383–2389.

Zurek E, Foxcroft GR, Kennelly JJ. 1995. Metabolic status and interval to first ovulation inpostpartum dairy cows. J Dairy Sci. 78:1909–1920.

412 H. Sano et al.