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Small Ruminant Research 112 (2013) 49– 55

Contents lists available at SciVerse ScienceDirect

Small Ruminant Research

jou rn al h om epa ge: www. elsev ier .com/ locate /smal l rumres

he relationship between microbial N synthesis and urinary excretionf purine derivatives in Dorper × thin-tailed Han crossbred sheep

ao Maa, Kaidong Dengb, Chenggang Jianga, Yan Tua, Naifeng Zhanga, Jie Liua,iguang Zhaoa, Qiyu Diaoa,∗

Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaCollege of Animal Science, Jinling Institute of Technology, Nanjing, Jiangsu 210038, China

r t i c l e i n f o

rticle history:eceived 13 April 2012eceived in revised form 21 August 2012ccepted 3 September 2012vailable online 20 September 2012

eywords:5Nurine basesurine derivativesicrobial N yield

a b s t r a c t

This study aimed to investigate the relationship between microbial N (MN) yield and urinaryexcretion of purine derivatives (PD) from the rumen in Dorper × thin-tailed Han crossbredsheep. Twelve Dorper × thin-tailed Han crossbred, non-castrated male lambs (41.3 ± 2.8 kgbody weight) fitted with ruminal and duodenal cannulae were randomly assigned to oneof three levels of DM intake: ad libitum intake, 70%, or 50% of the ad libitum intake, withfour lambs at each level. The MN yield from the rumen was measured using 15N or purinebases (PB) as a microbial marker, and urinary PD were determined by colorimetric methods.Microbial components (OM, N, PB and ratio of PB to N) were not affected (P > 0.05) by dietarytreatment. The MN yield measured using PB was slightly lesser and more variable than using15N. No difference in MN yield was found between markers. The efficiency of MN synthe-sis (g N/kg organic matter apparently digested in the rumen) was not affected (P > 0.05)by feed intake regardless of the marker used. Urinary PD decreased significantly (P < 0.05)with decreasing level of feed intake and the molar recovery of duodenal PB measured withdigesta markers as urinary PD was 0.83 (r2 = 0.89, P < 0.001). Urinary PD excretion was lin-

early correlated with DOMI (r2 = 0.94, P < 0.001), and MN yield estimated by using either15N or PB (r2 = 0.91 and 0.80, respectively, P < 0.001). These results suggest that urinary PDexcretion is an accurate indicator of MN synthesized in the rumen, and an equation, MN(g/d) = 0.030 + 0.741 × PD (mmol/d), is proposed to predict the MN yield from the rumen ofChinese sheep.

. Introduction

Protein supply is of great value for livestock becausef the important role of protein in animal nutrition asell as the relatively high cost of protein feeds. The pro-

ein requirement of ruminants is expressed in terms ofetabolizable protein (MP) to depict the true protein

bsorbed from the small intestine including microbial pro-ein from the rumen and undegraded dietary protein (AFRC,998; CSIRO, 2007; NRC, 2007). Microbial protein usually

∗ Corresponding author. Tel.: +86 108210 6055; fax: +86 106216 9105.E-mail address: diaoqiyu@mail.caas.net.cn (Q. Diao).

921-4488/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.smallrumres.2012.09.003

© 2012 Elsevier B.V. All rights reserved.

accounts for a significant part of MP, as over 50% of theamino acid absorbed by the small intestine is of micro-bial origin (AFRC, 1992). However, quantifying microbialprotein from the rumen is of great complexity and uncer-tainty (NRC, 2001). Two microbial marker systems wererecommended to quantify microbial protein, includingpurine bases (PB) method and 15N method (Broderick andMerchen, 1992). However, use of these markers requirescannulated animals, and the number of experimental ani-mals is therefore limited due to the relatively high cost of

maintaining cannulated animals and increasing concernsabout animal welfare (Tas and Susenbeth, 2007). For in vivostudy, number of experimental animal is an importantfactor causing variation in results (Nocek, 1988). Besides,

ant Res

50 T. Ma et al. / Small Rumin

Titgemeyer (1997) reviewed that in order to identify treat-ment differences of 10%, number of animals needed in thestudy of efficiency of microbial yield was at least two timesmore than that needed in the study of digestibility. Urinaryexcretion of purine derivatives (PD) is an effective indic-tor of microbial protein synthesized in the rumen (Toppsand Elliott, 1965). Furthermore, the prediction of microbialprotein yield from urinary PD is non-invasive, convenient,and relatively accurate (Tas and Susenbeth, 2007).

The Dorper × thin-tailed Han crossbred sheep is awidely reared breed for mutton production in northernChina. The current study therefore aimed to investigate thevalidity of use of urinary PD as a predicator of microbialN (MN) yield from the rumen of Dorper × thin-tailed Hancrossbred sheep.

2. Materials and methods

2.1. Animals and dietary treatments

This study was conducted at the Experimental Station of the ChineseAcademy of Agricultural Sciences (CAAS), Beijing, China from August 2010to October 2010. The experimental procedures were approved by the Ani-mal Ethics Committee of CAAS, and humane animal care and handlingprocedures were followed throughout the experiment.

Twelve 6-month-old Dorper × thin-tailed Han crossbred, non-castrated male lambs (41.3 ± 2.8 kg body weight) were surgically fittedwith ruminal and duodenal cannulae and used in this study approximately4 weeks after the surgery. The lambs were used in a completely random-ized design and assigned to three levels of feed intake: ad libitum, 70% and50% of ad libitum, with four lambs at each level. The diet (Table 1) wasoffered as a single pelleted mixture (6.0 mm of diameter) and fed oncedaily at 08.00 h. The lambs had free access to clean water at all times.

The trial lasted totally for 25 d. For the first 7 d, the lambs were kept inindividual pens for adaption to the different level of feed intake, and the adlibitum intake was determined as the lowest feed intake in the ad libitumgroup. Lambs were then moved into individual metabolism crates for 12 dincluding 7 d of adaption and 5 d of a digestibility trial. Then duodenaland ruminal digesta were sampled consecutively for 6 d. All lambs were

weighed at the beginning and end of the digestibility trial.

Yb and Co were used as the markers for particulate and liquid phasesin the rumen, respectively, to determine the flow of digesta from therumen to the duodenum. Additionally, 15N was used as an external micro-bial marker. All markers were incorporated into the concentrate prior

Table 1Ingredients and chemical composition of the experimental diet.a

Item

Ingredientsb (g/kg, as fed)Chinese wildrye hay 550Corn 295Soybean meal 140Calcium carbonate 8.4Salt 5.0Mineral/vitamin premixc 1.6

Composition (g/kg DM, determined)DM (g/kg, as fed) 906OM 916CP 112NDF 620ADF 241

OM, organic matter; CP, crude protein; NDF, neutral detergent fibre; ADF,acid detergent fibre.

a Experimental diet was fed ad libitum, 70% or 50% of the ad libitumintake (DM basis).

b All ingredients were pelleted (6.0 mm in diameter).c The premix contained (per kg): 22.1 g Fe, 13.0 g Cu, 30.2 g Mn, 77.2 g

Zn, 19.2 g Se, 53.5 g I, 9.10 g Co, 56.0 g vitamin A, 18.0 g vitamin D3, and170 g vitamin E.

earch 112 (2013) 49– 55

to pelleting as described by Koenig et al. (2007). Approximately 300 gYbCl3·6H2O, 1.3 kg Li–Co–EDTA (880 g C4H6O4·Co·H2O, 1.03 kg EDTA and150 g LiOH were required according to Udén et al. (1980)) and 200 g(15NH4)2SO4 (10 atom percentage 15N) were dissolved in 6 l water andsprayed on 300 kg concentrate diet before pelleting.

From days 8 to 25, the lambs were fed the marked diet. Prior tooffering the labelled feed, a priming dose that contained half of the dailymarker intake was administered through the rumen cannula of each ani-mal (Koenig et al., 2007). The markers were dissolved in deionised water.The priming doses of YbCl3·6H2O, Li–Co–EDTA, and (15NH4)2SO4 were 1.5,6.5, and 1 g for the lambs fed ad libitum; 1, 4.33, and 0.67 g for the lambsfed at 70% of the ad libitum intake; and 0.75, 3.25, and 0.5 g for the lambsfed at 50% of the ad libitum intake.

2.2. Measurements and sample collection

2.2.1. Feed, orts, faeces, and urine samplesIn the digestibility trial, faeces were collected daily from days 15 to 20.

Faeces were weighed daily, and then a sample of 10% was collected andpooled across days for each animal, dried at 65 ◦C, and ground through a1-mm sieve for analysis. Urine from each lamb was also collected daily ina bucket containing 100 ml of 7.2 N H2SO4. The volume was measured andthen diluted to 5 l with tap water, and a sample of 20 ml was collected,pooled across days for each animal, and stored at −20 ◦C for analyzingtotal N and PD. Samples of feed were also collected daily, combined, driedat 65 ◦C for 72 h, and ground through a 1-mm sieve. Feed refusals wereweighed, sampled, dried, ground, and combined for each lamb beforeanalysis.

2.2.2. Duodenal digesta collectionFrom days 21 to 23, a 100-ml sample of duodenal digesta was collected

every 6 h, moving the collection time forward 2 h each day to obtain thesamples at 2-h intervals. The samples were combined for each lamb andfrozen at −20 ◦C before analysis.

The duodenal digesta were thawed before analysis, and a volumeof 800 ml was separated into particulate and liquid fractions by cen-trifugation at 1000 × g, 4 ◦C for 15 min (Sigma 3-18K, Sigma LaboratoryCentrifuges Co., Germany). Samples of whole and particulate digesta werefreeze-dried for analysis of DM and then ground through a 1-mm sieve.Nutrient concentrations and markers were also determined in each frac-tion for determining the apparent ruminal digestibility of nutrients andthe digesta flow from the rumen to the duodenum. An additional sam-ple of the duodenal digesta (10 ml) was centrifuged at 20,000 × g, 4 ◦C for20 min, and the supernatant was stored for analysis of ammonia N.

2.3. Ruminal fermentation parameters and microbial protein yield

Samples of ruminal digesta (300 ml) were collected at 6-h intervalsfrom days 24 to 25 for the determination of ruminal fermentation param-eters and bacterial N yield (08.00, 14.00, and 20.00 h on day 24; and 02.00,05.00, 11.00, 17.00, and 23.00 h on day 25). The ruminal samples wereobtained through the cannula from 5 different sites within the rumenusing a syringe attached to a plastic tube (20-mm internal diameter). ThepH of the digesta was measured immediately using a pH metre (Sarto-rius PB-10, Sartorius Co., Germany). Thereafter, approximately 50 ml ofthe digesta were filtered through one layer of cheesecloth. A 10-ml sam-ple of the strained fluid was collected, acidified with 2 ml of 25% (w/v)metaphosphoric acid, and stored frozen at −20 ◦C for analysis of volatilefatty acids (VFA) and ammonia N. The remaining 250 ml of digesta weremixed with an equal amount of 0.9% saline and homogenized in a Waringblender (Waring Products, CT, USA) for 1 min to dislodge particulate-associated bacteria. The homogenate was then strained through four lay-ers of cheesecloth. The filtrate was centrifuged at 1000 × g, 4 ◦C for 10 minto remove feed particles and protozoa. The supernatant was then cen-trifuged at 20,000 × g, 4 ◦C for 45 min to obtain the bacterial pellet (Koeniget al., 2007). The bacterial pellet obtained at each sampling time werepooled for each animal and then transferred into a centrifuge tube with aminimum amount of distilled water and stored at −20 ◦C for analysis.

2.4. Chemical analysis

Feed and orts were analyzed for DM, organic matter (OM), and N. Drymatter was determined by drying samples in an oven at 135 ◦C for 2 h(AOAC, 1995); Organic matter was measured as the difference between

ant Research 112 (2013) 49– 55 51

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Table 2Average daily gain, intake of dry matter, digestible organic matter, organicmatter apparently digested in the rumen, apparent digestibility of nutri-ents, and N balance in Dorper × thin-tailed Han crossbred lambs fed a totalmixture diet at 3 levels of feed intake (mean values with their pooledstandard errors, n = 4/dietary treatment).

Item Level of feed intake* SE

AL 70% 50%

ADG (g/d) 324a 148b 29c 36.42Intake (g/d)

DM 1622a 1163b 815c 99.70DOM 1038a 750b 573c 53.52OMADR 708a 463b 320c 47.68

Apparent digestibility (%)Total tract

DM 60.1b 62.9ab 66.0a 0.91OM 64.6b 67.5b 70.9a 0.95N 61.8b 63.3b 67.7a 0.76

RumenDM 52.9 51.5 52.8 1.31OM 43.3b 45.4ab 48.0a 0.69N 82.1 79.3 80.1 1.51

N balance (g/d)N intake 31.23a 21.97b 15.99c 3.42Urinary N 7.87a 5.50b 4.20c 0.81Faecal N 11.92a 8.07b 5.16c 1.19N retention 11.44a 8.40b 6.63c 1.64

ADG, average daily gain; DOM, digestible organic matter; OM, organicmatter; OMADR, organic matter apparently digested in rumen.

T. Ma et al. / Small Rumin

M and the ash content (g/kg DM) (AOAC, 1995). Nitrogen was deter-ined by the Kjeldahl method, using Se as a catalyst, and crude proteinas calculated as 6.25 × N. Purine bases from isolated bacteria pellets

nd duodenal content were determined according to the method of Zinnnd Owens (1986). Urinary PD including allantoin, uric acid, xanthine,nd hypoxanthine were quantified separately by the colorimetric methodescribed by Chen and Gomes (1995). Ruminal VFA were measured by aame ionization detector in a GC (GC522, Wufeng Instruments, China)sing a 15-m semicapillary glass column (0.53 mm in diameter) packedith Chromosorb 101, with N2 as carrier gas at a column temperature

f 120 ◦C. Before analysis, caproic acid was added to each sample as annternal standard (Cao and Yang, 2011). Ammonia N was assessed byhe colorimetric method of Chaney and Marbach (1962). The isotopicbundance of 15N in bacterial N and duodenal fractions was determinedy isotope ratio mass spectrometry (Finnigan Mat 251, Thermo Fishercientific Inc., USA). The concentration of Yb and Co in both feed andigesta were separately determined by inductively coupled plasma emis-ion spectrometry (X series 2 ICP-MS, Thermo Fisher Scientific Inc., USA).

.5. Calculations and statistical analysis

Digested DM, OM, and N were calculated as the difference betweenntake and faecal output. The duodenal flows of nutrients and PB wereetermined by reconstitution of the duodenal digesta based on Yb ando concentrations and the content of the nutrients in the particulate andhole fractions (Faichney, 1975). Microbial N in the duodenum was deter-ined by using 15N or PB as a microbial marker. For the calculation oficrobial N yield with 15N as the marker, the microbial contribution to

on-ammonia N (MN/NAN) flow to the duodenum was estimated as:

MNNANdigesta

= E − NANdigesta

E − Nbacteria,

here (E − NANdigesta) and (E − Nbacteria) are the 15N enrichments of digestaAN and rumen bacterial N, respectively. Based on the observation that

he natural abundance of 15N from ruminal microbes and duodenal digestaas similar (Ahvenjärvi et al., 2002), ruminal samples were analyzed

or 15N before using marked feed, and an average value of 0.3671% wasbtained as 15N background for both microbes and duodenal digesta. Forhe calculation of microbial N yield with PB as the marker, microbial

contribution to duodenal NAN was calculated from the PB/N ratio inigest NAN compared to that in the rumen bacterial pellets, assuminghat duodenal PB flows were only of bacterial origin.

The data were analyzed as a completely randomized design using SASSAS version 9.1; SAS Institute, Inc., Cary, NC, USA). All results were ana-yzed using PROC GLM and the comparison of the means was performedsing the least squares means option of SAS. Microbial N obtained for eacharker was subjected to paired t-test. Besides, the linear regressions anal-

ses were conducted with PROC REG. Statistical significance was acceptedt P < 0.05.

. Results

.1. Average daily gain (ADG), intake, nutrientigestibility, and N balance

Mean values of ADG, DM intake, digestible OM (DOM),M apparently digested in the rumen (OMADR), and nutri-nt digestibility are presented in Table 2. ADG was highlyorrelated with DM intake (r2 = 0.96, P < 0.001). The appar-nt digestibility of DM, OM, or N was negatively correlatedith DM intake (r2 = −0.79, −0.81, and −0.67, respectively,

< 0.001). A significant difference (P < 0.05) in the appar-nt digestibility of DM only existed between lambs fed adibitum and those fed at 50% of the ad libitum intake. Forither OM or N, the apparent digestibility was significantly

reater (P < 0.05) for lambs fed 50% ad libitum while no dif-erence (P > 0.05) was found between those fed ad libitumnd 70% ad libitum. No difference (P > 0.05) in the ruminaligestibility of DM or N was detected among all groups of

a,b,cMean values within a row with different superscript letters were sig-nificantly different (P < 0.05).

* Ad libitum (AL) or restricted to 70% or 50% of the ad libitum intake.

animals. Ruminal OM digestibility was significantly greaterfor lambs fed 50% ad libitum than those fed ad libitum. Alllambs maintained a positive N balance, and N retentionwas highly correlated with N intake (r2 = 0.92, P < 0.001).The excretion of either faecal N or urinary N was reduced(P < 0.05) by feed restriction.

3.2. Ruminal characteristics

Ruminal pH values, VFA, and ammonia N concentra-tions are presented in Table 3. Feed restriction had no effect(P > 0.05) on ruminal pH values. The ruminal concentrationof total VFA was greater (P < 0.05) in lambs fed ad libitumthan in those fed at 70% of the ad libitum intake. No dif-ferences in molar proportions of VFA were found amongtreatments but lambs fed at 70% of the ad libitum intakehad a greater ratio of acetate to propionate (P < 0.05) thanthose fed ad libitum or at 50% of the ad libitum intake.The ruminal ammonia N concentration decreased (P < 0.05)with decreasing feed intake.

3.3. Bacterial composition, duodenal flow, MN yield, andmicrobial efficiency

Bacterial composition; duodenal flow of total N, NAN,and PB; MN yield; and efficiency of MN synthesis arepresented in Table 4. No effect (P > 0.05) of feed restric-

tion on bacterial composition was found. Duodenal flowsof total N, NAN, and PB decreased significantly (P < 0.05)with decreasing level of feed intake. Microbial N mea-sured using PB was more variable than using 15N although

52 T. Ma et al. / Small Ruminant Research 112 (2013) 49– 55

Table 3Ruminal pH, volatile fatty acids, and ammonia N concentrations in rumi-nal fluids of Dorper × thin-tailed Han crossbred lambs fed at 3 levels offeed intake* (mean values with their pooled standard errors, n = 4/dietarytreatment).

Item Level of feed intake† SE

AL 70% 50%

Ruminal pH 6.07 6.28 6.13 0.08Total VFA (mmol/l) 62.1a 55.9b 60.5ab 4.57VFA (mol/100 mol)

Acetate 62.7 64.9 64.7 1.24Propionate 20.8 18.4 21.1 0.98Butyrate 10.3 10.7 8.2 0.45Branched chain andlonger acids

6.24 6.03 6.04 0.21

Acetate/propionate 3.13b 3.53a 3.07b 0.15Ammonia N (mg N/l) 102.7a 84.2b 66.0c 4.69

VFA, volatile fatty acids.a,b,cMean values within a row with different superscript letters were sig-nificantly different (P < 0.05).

* Ruminal fermentation characteristics were determined at 8 time-

Table 5Urinary volume, excretion of purine derivatives (PD), and ratio of each PDcomponent to total PD in Dorper × thin-tailed Han crossbred lambs fedat 3 levels of feed intake (mean values with their pooled standard errors,n = 4/dietary treatment).

Item Level of feed intake* SE

AL 70% 50%

Urine volume (l/d) 1.36 1.38 1.54 0.19PD excretion (mmol/d)

Allantoin 14.36a 10.24b 6.33c 1.05Uric acid 2.52a 2.11b 1.73c 0.14Xanthine + hypoxanthine 1.54a 1.12b 0.98b 0.10Total PD 18.42a 13.74b 9.04c 1.24

Ratio of each componentAllantoin/total PD 0.78a 0.76b 0.71c 0.01Uric acid/total PD 0.13b 0.16ab 0.19a 0.008(Xan-thine + hypoxanthine)/total PD

0.08 0.08 0.10 0.005

PD, purine derivatives.a,b,cMean values within a row with different superscript letters were sig-

points (02.00, 05.00, 08.00, 11.00, 14.00, 17.00, 20.00, 23.00 h) from days19 to 20 and averaged for each sheep.

† Ad libitum (AL) or restricted to 70% or 50% of the ad libitum intake.

the means for both markers were similar. Microbial effi-ciency expressed as g N/kg OMADR slightly increased withdecreasing level of feed intake while no significant differ-ence was detected (P > 0.05) for either 15N or PB.

3.4. Urinary purine derivatives

Urine volume, urinary excretion of PD, and the per-centage of each PD component are presented in Table 5.

No difference (P > 0.05) in urine output was found amonglambs. The lambs fed ad libitum had a greater (P < 0.05)total PD excretion than those fed at 70% or 50% of the ad

Table 4Bacterial composition, duodenal flows of total N, non-ammonia N, andpurine bases, microbial N yields, and microbial efficiency estimated using15N, purine bases, and purine derivatives as markers (mean values withtheir pooled standard errors, n = 4/dietary treatment).

Item Level of feed intake* Average

AL 70% 50% SE

Bacteria componentOM (g/100 g DM) 85.9 86.3 86.5 0.07N (g/100 g OM) 9.46 9.30 9.23 0.06PB (�mol/g OM) 12.32 12.21 12.30 0.04PB/N (mmol/g) 1.30 1.31 1.32 0.006

Duodenal flowTotal N (g/d) 28.38a 23.47b 17.25c 1.41NAN (g/d) 27.23a 22.76b 16.80c 1.30PB (mmol/d) 20.70a 15.45b 10.98c 1.12

Microbial N (g/d)15N 14.40a 9.60b 6.78c 0.97 10.26PB 13.71a 10.17b 7.14c 1.51 10.37

Microbial efficiency (g N/kg OMADR)15N 20.34 20.73 21.19 0.45 20.83PB 19.36 21.95 22.30 0.95 21.25

PB, purine bases; NAN, non-ammonia N; OM, organic matter; OMADR,organic matter apparently digested in rumen.a,b,cMean values within a row with different superscript letters were sig-nificantly different (P < 0.05).

* Ad libitum (AL) or restricted to 70% or 50% of the ad libitum intake.

nificantly different (P < 0.05).* Ad libitum (AL) or restricted to 70% or 50% of the ad libitum intake.

libitum intake. Allantoin excretion decreased (P < 0.05) withthe decreasing feed intake. Uric acid excretion was lesser(P < 0.05) in lambs fed at 55% of ad libitum feed intake thanin those fed ad libitum and 70% of ad libitum. No signif-icant difference was found (P > 0.05) in the excretion ofxanthine and hypoxanthine between those fed 70% and 50%ad libitum. As feed intake decreased, the ratio of allantoin tototal PD decreased from 0.78 to 0.71 (P < 0.05), whereas theratio of uric acid and xanthine plus hypoxanthine to totalPD increased from 0.13 to 0.19 and 0.08 to 0.10 (P < 0.05),respectively. Urinary PD excretion was found linearly cor-related with DOMI (r2 = 0.84, P < 0.001, Fig. 1). The molarrecovery of duodenal PB measured with digesta markersas urinary PD was 0.83 (r2 = 0.89, P < 0.001, Fig. 2). Besides,A linear correlation was found between urinary PD excre-

tion and MN yields measured by using either 15N (r2 = 0.91,P < 0.001) or PB (r2 = 0.80, P < 0.001).

Fig. 1. Relationship between urinary excretion of total PD (mmol/d)and DOMI (kg/d) of Dorper × thin-tailed Han crossbred lambs at 3 lev-els of feed intake. PD (mmol/d) = −1.81(±0.035) + [21.41(±0.042) × DOMI(kg/d)], r2 = 0.84, RMSE = 0.941, n = 12.

T. Ma et al. / Small Ruminant Res

Fig. 2. Relationship between urinary excretion of total PD (mmol/d)ab[

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nd duodenal PB flow (mmol/d) of Dorper × thin-tailed Han cross-red lambs at 3 levels of feed intake. PD (mmol/d) = 0.8241(±0.041) +0.8288(±0.100) × PB (mmol/d)]. r2 = 0.89, RMSE = 1.431, n = 12.

. Discussion

.1. Intake and digestibility of nutrients, N balance, anduminal fermentation

In our study, ADG was highly correlated with DM intake,nd the apparent digestibility of DM, OM, and N was neg-tively correlated with DM intake. As feed intake waseduced, the apparent digestibility of DM increased from.60 to 0.66 in our study. These results are similar to find-

ngs in goats (Fernandes et al., 2007) and Texel crossbredheep (Galvani et al., 2009), although the values of DMigestibility from the latter two studies were greater. Thepparent N digestibility increased from 61.8% to 67.7% withecreasing feed intake, which is consistent with the findingf Galvani et al. (2009). The ratio of N retained to N intakeanged from 0.37 to 0.41, with a mean value of 0.38 over the

feeding levels in our study. This value is lesser than theange (0.45 to 0.46) reported by Galvani et al. (2009), whichould be explained in part by the greater N digestibility0.70–0.73) measured in the latter study. Ruminal pH wasot affected by feed intake in our study, which is in accor-ance with findings in dairy cows (González Ronquillot al., 2004).

.2. Bacterial composition and duodenal PB flow

The composition of mixed bacteria in the rumen was notffected by levels of feed intake, which is consistent withhe results reported by Cecava et al. (1990) and Brodericknd Merchen (1992). The ratio of PB to N ranged from 1.30o 1.32, which is close to the results (1.28–1.31) reportedy Cecava et al. (1990) and those (1.28–1.32) reported byroderick and Merchen (1992) in steers and dairy cows,espectively.

The distribution of Yb in different digesta phase wasependent on the method (filtration or centrifugation)sed to separate digesta. Separation by filtration couldesult in a similar distribution of Co and Yb concentrations

earch 112 (2013) 49– 55 53

between phases (Ahvenjärvi et al., 2003). In this trial, theratio of concentration of Co to Yb in solid and fluid digestaobtained by centrifugation was 0.47 (s.e. 0.10) and 0.93 (s.e.0.03), respectively, which showed a preferential associa-tion of Yb with particulate matter according to Siddons et al.(1985). In our study, the recovery duodenal PB flow mea-sured with dual marker as urinary PD was 0.83 (r2 = 0.89),greater than that reported by Pérez et al. (0.78; 1996), andChen et al. (0.77; 1990) in sheep, and close to that reportedby Orellana Boero et al. (0.84; 2001) in cows. This discrep-ancy could be explained by the difference in errors inducedby the application of flow markers (Martín-Orúe et al.,2000). Nevertheless, urinary PD responded sensitively toexogenous purine input (Balcells et al., 1991, 1993; Chenet al., 1990; Pérez et al., 1996; Martín-Orúe et al., 2000) andthus could be an effective indicator of changes in duodenalPB flow.

4.3. Urinary excretion of PD

In our study, total PD excretion decreased significantlyby feed restriction. Chen et al. (1992) also found thatPD excretion or microbial protein yield from the rumenof sheep was proportional to DM intake. Furthermore,González Ronquillo et al. (2004) reported that the uri-nary excretion of allantoin and uric acid in dairy cowsalso decreased significantly with decreasing feed intake.The urinary excretion of PD (from 9.04 to 18.42 mmol/d)measured in our study is within the range reported by pub-lished studies in sheep (Pérez et al., 1996; Yu et al., 2002;Webster et al., 2003). In sheep and goats, purine bases areconverted into allantoin, uric acid, xanthine, and hypoxan-thine in metabolic processes. Allantoin is dominant PD inboth sheep (Yu et al., 2002) and goats (Belenguer et al.,2002). In our study, the ratios of allantoin, uric acid, andthe sum of xanthine plus hypoxanthine to total PD were onaverage 0.75, 0.16, and 0.08, respectively; these values areconsistent with those reported by Chen and Gomes (1995)(0.60–0.80, 0.10–0.30, and 0.05–0.10, respectively).

4.4. Prediction of MN from urinary excretion of PD

In our study, a linear relationship was found betweenurinary PD and MN measured from 15N or PB. Strong cor-relation between MN and urinary excretion of PD or othercomponents (allantoin or uric acid) has been found inmany studies using different animals (Table 6). Amongthose studies, PB was a commonly used marker. In thistrial, the regression coefficient was much greater when15N (r2 = 0.91) was used as the microbial marker comparedto PB (r2 = 0.80), and this difference could be explained bythe lesser variability in MN measurement achieved by 15N(SE = 0.97) compared to PB (SE = 1.51). Greater variabilityin the calculation of MN from PB than from 15N was alsofound in other studies (Calsamiglia et al., 1996; Carro andMiller, 2002; Reynal et al., 2005). Theoretically, 15N hasmany advantages in measuring MN. For example, 15N is

not present in the diet above natural enrichment and canbe evenly distributed within microbial cells; therefore, lossof cytoplasmic contents during isolation does not influ-ence microbial enrichment (Broderick and Merchen, 1992;

54 T. Ma et al. / Small Ruminant Research 112 (2013) 49– 55

Table 6Comparison of different methods used to predict microbial N flow (g/d) from urinary excretion of PD (mmol/d).

Experiment Animal species Number of animals Models R2 Marker used

Puchala and Kulasek (1992) Ewe 10 MN = exp(0.747 + 1.817 × PD) – PBMN = exp(0.830 + 2.089 × Allantoin)a –

Johnson et al. (1998) Cow 4 MN = 75.834 + 1.048 × Allantoin 0.58 PBMN = 5.774 + 0.212 × Uric acid 0.82

Moorby et al. (2006) Cow 4 MN = 19.9 + 0.689 × PD 0.79 PBThis trial Sheep 12 MN = 0.030 + 0.741 × PD 0.91 15N

a PD and Allantoin are expressed as purine derivatives nitrogen (g/d).

Reynal et al., 2005). These advantages enable 15N to yield amore precise and accurate estimate of MN than other mark-ers (Reynal et al., 2005). In contrast, when PB is used as amicrobial marker, factors such as undegraded dietary PB(Broderick and Merchen, 1992), possible loss of PB dur-ing analysis procedures (Calsamiglia et al., 1996; Reynalet al., 2005), and lack of representative isolation of ruminalmicrobes (Firkins et al., 1987; Pérez et al., 1997) may affectthe accuracy of MN estimates. Therefore, 15N is a more idealmarker for quantifying MN.

5. Conclusions

In this study, a linear relationship existed between theMN yield from the rumen measured by using 15N or PB andurinary PD excretion. The MN yield measured from 15N wasmore accurate and less variable than that measured fromPB. As a result, the equation MN (g/d) = 0.030 + 0.741 × PD(mmol/d) is proposed to predict MN yields from urinary PDexcretion in Chinese sheep.

Conflicts of interest statement

The authors state that there are no conflicts of interest.

Acknowledgements

This study was conducted as part of the National Tech-nology Program for Meat Sheep Industry of China (ProgramNo.: nycytx-39 and No.: 200903006) and funded by theMinistry of Agriculture of the People’s Republic of China.We thank Prof. Z.C. Feng for his professional help with sur-gical operations of sheep. We also thank Y.C. Wang andY.F. Zhang for their technical assistance. All authors partic-ipated in the writing of the final draft of the manuscript andagreed with the final format.

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