This article was downloaded by: [Ondokuz Mayis Universitesine]

On: 10 November 2014, At: 12:48

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Archives of Animal Nutrition

Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gaan20

Influence of urea–calcium mixtures as

rumen slow-release feed on in vitro
fermentation using a gas production
technique
a a
Anusorn Cherdthong , Metha Wanapat & Chalong
a
Wachirapakorn
a
Tropical Feed Resources Research and Development Center
(TROFREC), Department of Animal Science, Faculty of
Agriculture , Khon Kaen University , Thailand
Published online: 21 Apr 2011.

To cite this article: Anusorn Cherdthong , Metha Wanapat & Chalong Wachirapakorn (2011)
Influence of urea–calcium mixtures as rumen slow-release feed on in vitro fermentation
using a gas production technique, Archives of Animal Nutrition, 65:3, 242-254, DOI:
10.1080/1745039X.2011.568277

To link to this article: http://dx.doi.org/10.1080/1745039X.2011.568277

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
 Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014
 Archives of Animal Nutrition
Vol. 65, No. 3, June 2011, 242–254

Influence of urea–calcium mixtures as rumen slow-release feed on in vitro

fermentation using a gas production technique
Anusorn Cherdthong, Metha Wanapat* and Chalong Wachirapakorn

Tropical Feed Resources Research and Development Center (TROFREC), Department of

Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

(Received 30 August 2010; accepted 6 January 2011)

In this experiment the effects of different urea products (urea [U] and urea–
calcium mixtures [UCM]) on rumen fermentation were investigated in dependence
of different energy sources by using in vitro techniques. The 7 6 2 factorial
arrangement followed a completely randomised design using seven urea products
(U100, U40CaCl2, U50CaCl2, U60CaCl2, U40CaSO4, U50CaSO4 and
U60CaSO4) in combination with cassava chips (CC) or corn meal (CM).
Compared with other treatments, the cumulative gas production (96 h) was
significantly increased for U60CaCl2 þ CC and U60CaSO4 þ CC (p 5 0.01),
which was combined with a higher in vitro true digestibility (p 5 0.01). In
addition, the concentration of volatile fatty acids in the fluid of U60CaCl2 þ CC
and U60CaSO4 þ CC was significantly higher than in other treatments. Urea
treatments (U100 þ CC and U100 þ CM) caused the highest concentration of
ruminal ammonia nitrogen (p 5 0.01), which was significantly decreased by all
UCM products in combination with CC, but not with CM. The highest levels of
total bacteria, Fibrobacter succinogenes and anaerobic fungi were found for
treatment U60CaCl2 þ CC and U60CaSO4 þ CC (p 5 0.05). The findings
revealed that the utilisation of U60CaCl2 and U60CaSO4 in combination with
cassava chips improved the ruminal fluid fermentation in terms of NH3-N and
volatile fatty acid concentration, digestibility of energy and increased the
fibrobacter concentrations.
Keywords: calcium chloride; calcium sulphate; energy sources; in vitro
digestibility; rumen microorganisms; urea

1. Introduction
Urea is a non-protein nitrogen (NPN) substance and a nitrogen (N) source for
microbial protein synthesis in the rumen, which is cost-saving as compared with true
protein feeds (Pfeffer et al. 2009; Taylor-Edwards et al. 2009; Calsamiglia et al. 2010;
Cherdthong and Wanapat 2010). However, the amount of urea that can be used is
limited because of the rapid hydrolysis to ammonia (NH3) in the rumen (Galo et al.
2003; Golombeski et al. 2006). This rapid break-down to NH3 can occur at a much
faster rate than NH3 utilisation by the rumen bacteria, resulting in accumulation and
escape of NH3 from the rumen (Satter and Roffler 1975). Attempts to achieve a slow
NH3 release from urea have been variable not only in method, but also in degree of

*Corresponding author. Email: metha@kku.ac.th

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

Ó 2011 Taylor & Francis
DOI: 10.1080/1745039X.2011.568277
http://www.informaworld.com
 Archives of Animal Nutrition 243

success (Highstreet et al. 2010). In the past, several urea-calcium mixtures (UCM)
have been developed and evaluated for ruminant nutrition, but no UCM or modified
NPN compound have been accepted under practical conditions.
Simultaneous carbohydrate fermentation is also essential for the incorporation of
urea into microbial protein (Wanapat et al. 2008; Poungchompu et al. 2009).
Therefore, the objectives of this study were to develop urea-calcium mixtures and to
evaluate their fermentation characteristics in the rumen by varying two energy
sources in vitro.

2. Materials and methods

2.1. Animals, treatments and experimental design
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

This study was conducted by using an in vitro gas technique at various incubation
times. The experiment was a 7 6 2 factorial arrangement in a completely
randomised design. The treatments included two kinds of energy sources and seven
kinds of urea products. The two energy sources were cassava chips (CC) and ground
corn (corn meal [CM]), while rice straw was used as a roughage source. The seven
urea products were: urea alone (U100), and different amounts of urea in
combination with CaCl2 or CaSO4. The compositions of the UCMs are shown in
Table 1. In brief, the production processes for UCM was as following: (1) making an
aqueous solution of CaCl2 or CaSO4 (salt:water, 1:1 w/w) and heating and agitating
the mixture at 508C for 10 min; (2) dissolving solid feed grade urea in the aqueous
CaCl2 or CaSO4 (despite the respective relation of urea and salt 17% water was
added, see Table 1); and (3) heating and agitating the mixture at 508C for 10 min,
then the temperature of the solution was reduced to about 258C. The final products
were kept at room temperature. The two energy sources were CC and CM, while rice
straw was used as a roughage source. As substrate, roughage and concentrates were
used in a ratio of 30:70. Finally, the fourteen treatments were as follows:
U100 þ CC; U40CaCl2 þ CC; U50CaCl2 þ CC; U60CaCl2 þ CC; U40CaSO4 þ
CC; U50CaSO4 þ CC; U60CaSO4 þ CC; U100 þ CM; U40CaCl2 þ CM;
U50CaCl2 þ CM; U60CaCl2 þ CM; U40CaSO4 þ CC; U50CaSO4 þ CM and
U60CaSO4 þ CM. Samples of roughage and concentrates were dried at 608C,
then ground to pass a 1-mm sieve and used for chemical analysis and in the in vitro
gas test. The samples were analysed for DM (dry matter), ash and CP (crude protein)

Table 1. Composition of substrates with different urea levels and calcium sources.

Composition [%]
{
Urea–calcium mixtures Urea CaCl2 CaSO4 Water Nitrogen content [%]
U100 100 0 0 0 45.8
U40CaCl2 40 43 0 17 17.8
U50CaCl2 50 33 0 17 22.6
U60CaCl2 60 23 0 17 27.3
U40CaSO4 40 0 43 17 18.1
U50CaSO4 50 0 33 17 22.4
U60CaSO4 60 0 23 17 27.2

Notes: {U, Urea; CaCl2, Calcium chloride dehydrate (CaCl2  2H2O); CaSO4, Calcium sulphate dehydrate
(CaSO4  2H2O).
 244 A. Cherdthong et al.

using the procedures of AOAC (1998), neutral detergent fibre (NDF) and acid
detergent fibre (ADF) according to Van Soest et al. (1991). The ingredients and
chemical compositions of concentrate used in the in vitro experiment are shown in
Table 2. Rice straw (Oryza sativa) contained 91% DM, 2.5% CP, 84.6% NDF and
61.3% ADF on a DM basis.
For incubation, substrates (0.5 g DM) were weighed to into 50 ml bottles. For
each substrate and sampling time triplicate sets were prepared. Thereafter, the
bottles were sealed (CO2 atmosphere) with rubber stoppers and aluminium caps and
were placed into the incubator at 398C for in vitro gas test.

2.2. Animals as donors of rumen inoculum

Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

Two male, rumen-fistulated native beef cattle (crossbred Brahman-Thai, BW [body

weight] 400 + 50 kg) were used as rumen fluid donors. The animals were kept in
individual pens and clean fresh water and mineral blocks were offered as free
choice. Rice straw was fed on ad libitum basis and concentrate (16% CP, 11.0 MJ
metabolisable energy (ME)/kg, consisting of: 70% CC, 5% rice bran meal, 7%
coconut meal, 10% palm kernel meal, 1% sulphur, 4% urea, 1% mineral premix
and 1% salt) was fed at 0.5% of BW in two equal portions, at 07:00 h and 16:00 h.
The animals received the diets for 20 d before the rumen fluid was collected. From
each animal 1000 ml rumen liquor was obtained before the morning feeding. The
rumen fluid was filtered through four layers of cheesecloth into pre-warmed thermo
flasks. Strict anaerobic techniques were used during rumen fluid collection
according to the method of Menke et al. (1979). It was then transported to the
laboratory.

2.3. Preparation of inoculums and incubation

Preparation of artificial saliva was done according to Menke and Steingass (1988),
but the medium did not include a nitrogen source in the buffer. The artificial saliva
and rumen fluid was mixed in a 2:1 ratio to a mixed rumen inoculum. One hour
before filling with 40 ml of the mixed rumen inoculums, the serum bottles with the
respective substrates were pre-warmed in a water bath at 398C. Thirty minutes after
starting the incubation, the bottles were gently mixed and then mixed three times
every 3 h. For each sampling time, three bottles containing only the rumen inoculum
were included within each run and the mean gas production values of these bottles
were used as blank. The blank values were subtracted from each measured value to
give the net gas production.

2.4. Sampling and analysis

The gas production was recorded at 0, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 48, 72 and 96 h of
incubation. Cumulative gas production data were fitted to the model of Ørskov and
McDonald (1979) as follows:

y ¼ ða þ bÞð1  eðctÞ Þ; ð1Þ

where a is the gas production from the immediately soluble fraction, b is the gas
production from the insoluble fraction, c is the gas production rate constant for the
 Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

Table 2. Ingredients and chemical compositions of concentrates used as substrates in the in vitro experiment.

Treatments
U100 U40 U50 U60 U40 U50 U60 U100 U40 U50 U60 U40 U50 U60
CaCl 2 CaCl2 CaCl2 CaSO4 CaSO4 CaSO4 CaCl2 CaCl2 CaCl2 CaSO4 CaSO4 CaSO4
þ CC þ CC þ CC þ CC þ CC þ CC þ CC þ CM þ CM þ CM þ CM þ CM þ CM þ CM
Ingredients [% DM]
Cassava chips (CC) 70.0 70.0 70.0 70.0 70.0 70.0 70.0 – – – – – – –
Corn meal (CM) – – – – – – – 70.0 70.0 70.0 70.0 70.0 70.0 70.0
Rice bran 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
Coconut meal 7.0 5.0 5.0 5.0 5.0 5.0 5.0 7.0 5.0 5.0 5.0 5.0 5.0 5.0
Palm kernel meal 10.0 6.0 8.0 9.3 6.0 8.0 9.3 10.0 6.0 8.0 9.3 6.0 8.0 9.3
Sulphur 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Premix{ 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Molasses 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Urea 4.0 – – – – – – 4.0 – – – – – –
Urea–CaCl2 – 10.0 8.0 6.7 – – – – 10.0 8.0 6.7 – – –
Urea–CaSO4 – – – – 10.0 8.0 6.7 – – – – 10.0 8.0 6.7
Salt 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Chemical composition{
DM [%] 95.2 94.2 96.3 94.1 93.6 95.3 94.4 95.1 95.1 94.5 95.4 96.1 95.2 95.5
OM [% of DM] 91.1 90.3 91.2 92.1 91.2 90.7 90.9 90.2 91.0 90.8 92.3 91.3 92.1 90.9
CP [% of DM] 16.1 16.3 16.2 16.2 16.0 16.4 16.2 16.2 16.3 16.2 16.0 16.4 16.4 16.2
NDF [% of DM] 12.8 11.4 11.8 12.0 11.4 11.8 12.0 12.1 10.7 11.1 11.3 10.7 11.1 11.3
ADF [% of DM] 7.6 6.5 6.8 7.0 6.5 6.8 7.0 6.1 5.1 5.4 5.6 5.1 5.4 5.6
ME [MJ/kg]{ 11.2 11.4 11.1 11.2 11.2 11.2 10.9 11.3 11.4 11.5 11.4 10.8 10.7 11.2

Notes: {Contained per kg concentrate: vitamin A, 100,000 IU; vitamin E, 700 IU; vitamin D, 16,000 IU; Fe, 0.5 g; Zn, 0.4 g; Mn, 0.4 g; Co, 1 mg; Cu, 0.1 g; Se, 1 mg; I, 5 mg.
{
Archives of Animal Nutrition

DM, Dry matter; OM, Organic matter; CP, Crude protein; NDF, Neutral detergent fibre; ADF, Acidic detergent fibre; ME, Metabolisable energy; {Calculated values.
245
 246 A. Cherdthong et al.

insoluble fraction (b), t is the incubation time, (a þ b) is the potential extent of gas
production and y is the gas produced at time t. The ruminal fluid of inoculums was
collected at 0, 0.5, 1, 2, 4, 6, and 8 h post inoculation. Rumen fluid samples were then
filtered through four layers of cheesecloth. Samples were divided into two portions;
the first portion was used for NH3-N analysis (at 0, 0.5, 1, 2, 4, 6, and 8 h post
inoculations) using the micro-Kjeldahl methods (AOAC 1998) and volatile fatty acid
(VFA) analysis (at 0, 2, 4, 6, and 8 h post inoculations) using high-performance
liquid chromatography (HPLC). The HPLC system consisted of a Shimadzu VP Series
with SpD10A detector and WINCHROM software. A 3.9 mm 6 300 mm stainless-
steel column, packed with ReproGel H and a pre-column, packed with the same
material were used. The mobile phase consisted of 10 mM H2SO4 (pH 2.5) and the flow
rate was 0.8 ml/min. The UV detector (at 259 nm) was employed for quantification. The
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

UV-visible spectra were recorded at the peak maxima and were corrected for the solvent
background. The results were determined, using the standard volatile acids (Merck,
India) as control. The final portion (at 0 and 4 h post inoculations) was stored at 7208C
for DNA extraction (Yu and Morrison 2004). At 48 h post inoculation one bottle of
each sample was determined in vitro true digestibility according to van Soest et al.
(1991). The true digestibility was used to calculate microbial mass according to the
method of Blümmel et al. (1997) and calculated as:

Microbial mass ½mg ¼ Substrate truly degraded ½mg  ðGas volume ½ml  2:2Þ:
ð2Þ

2.5. Quantitative analysis of microbial populations

Community DNA was extracted from 0.25 ml aliquots of each sample by the
repeated bead beating plus column (RBB þ C) method (Yu and Morrison 2004),
which was shown to substantially increase DNA yields. The quality and quantity of
these DNA samples were also determined by agarose gel electrophoresis and
spectrophotometry (Figure 1). In total, 56 samples belonging to 14 treatments, two
incubation times (0 and 4 h) and two replicates were extracted for genomic DNA.
The primers used for the real time PCR were as follows: primers for Fibrobacter
succinogenes, Fs219f (50 -GGT ATG GGA TGA GCT TGC-30 ) and Fs654r (50 -GCC
TGC CCC TGA ACT ATC- 30 ), were selected to allow amplification (446-bp
product) of all 10 F. succinogenes strains deposited in GenBank1. For Ruminococcus
albus primers were Ra1281f (50 -CCC TAA AAG CAG TCT TAG TTC G-30 ) and
Ra1439r (50 -CCT CCT TGC GGT TAG AAC A-30 ) (175-bp product). Rumino-
coccus flavefaciens primers, Rf154f (50 -TCT GGA AAC GGA TGG TA-30 ) and
Rf425r (50 -CCT TTA AGA CAG GAG TTT ACA A-30 ), were also selected to allow
species–species amplification (295 bp) of all seven R. flavefaciens strains deposited in
GenBank1. All these primer sets were previously published by Koike and
Kobayashi (2001). Quantification of total bacteria population, primer and condition,
was previously published by Kongmun et al. (2010). Regular PCR conditions for
F. succinogenes were as follows: 30 s at 948C for denaturing, 30 s at 608C for
annealing and 30 s at 728C for extension (48 cycles), except for 9 min denaturation
in the first cycle and 10 min extension in the last cycle. Amplification of 16S rRNA
for the other two species was carried out similarly, except an annealing
temperature of 558C was used. Quantification of anaerobic fungal population,
primer and condition, was previously published by Denman and McSweeney
 Archives of Animal Nutrition 247
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

Figure 1. Genomic DNA agarose gel electrophoresis to check the DNA quality
(1 kb ¼ ladder).

(2006). Four sample-derived standards were prepared from treatment pool set of
community DNA. The regular PCR was used to generate sample-derived DNA
standards for each real time PCR assay. Then the PCR product was purified using
a QIA quick PCR purification kit (Qiagen, Inc., Valencia, CA, USA) and
quantified using a spectrophotometer. For each sample-derived standard, copy
number concentration was calculated based on the length of the PCR product and
the mass concentration. Ten-fold serial dilution was made in Tris-EDTA prior to
real time PCR (Yu et al. 2005). In total, four real time PCR standards were
prepared. The conditions of the real time PCR assays of target genes were the
same as those of the regular PCR described above. Biotools QuantiMix Easy Syg
Kit (B&M Labs, Spain) was used for real time PCR amplification. All PCR were
performed in duplicate.

2.6. Statistical analysis

The collected data were further used to analysis of variance (ANOVA) according the
general linear model of SAS (1996). The following model was used:

yijk ¼ m þ ai þ bj þ abij þ eijk ; ð3Þ

where y is the observation, m is the overall mean, ai is the factor a effect (a is the type
of urea–calcium mixture; i, 1–7), bj is the factor b effect (b is the source of energy; j,
1–2); abij is the interaction effect and eijk is the error. Multiple comparisons among
treatment means, urea products, energy sources, urea levels and calcium sources
were performed by Duncan’s new multiple range test (Steel and Torrie 1980).
Differences among means with p 5 0.05 were accepted as representing statistically
significant differences.
 248 A. Cherdthong et al.

3. Results and discussion

3.1. Parameters of gas production, true digestibility and microbial biomass
The estimated parameters of gas production for the studied substrates are given in
Table 3. Cumulative gas production (96 h) was influenced by different products of
UCM and energy sources while the highest cumulative gas volumes were found in
U60CaCl2 þ CC and U60CaSO4 þ CC (60% urea in UCM þ CC) (115.7 and 119.8
ml/0.5 g DM substrate respectively). These findings suggest that the inclusion of
cassava chips as energy substrate results in an increased rate and extent of
fermentation of the inoculums. The significant highest rates of gas production
(c) were measured for treatments U60CaCl2 þ CC, U50CaSO4 þ CC and
U60CaSO4 þ CC. However, Ca sources had no effect on gas kinetics, true
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

digestibility and microbial mass from in vitro incubation. Since the in vitro gas
production technique has been used as a measurement of ruminal degradation of
feed (Menke and Steingass 1988; Poungchompu et al. 2009) or hay (Karabulut et al.
2007), high gas production indicated high digestibility of substrates. In the current
study, it was found that the treatments with higher gas production showed also a
higher in vitro true digestibility (Table 3). Moreover, supplementation of urea at 60%
of UCM in combination with CC significantly increased the in vitro true digestibility
and cumulative gas production. The differences between UCM-products in
combination with CC and CM could be attributed to the properties of corn starch,
which is slowly degradable. Rooney and Pflugfelder (1986) explained that low
digestibility of corn starch was a result of the protein matrix associated with starch
granules, the type and proportion of protein bodies found in the endosperm, and the
proportion of corneous endosperm that must be disrupted for release of starch. In
addition, the diet of the donor beef cattle for rumen fluid may affect differences
between CC and corn meal. In this study, the donor beef cattle were fed with CC-
based diet and this could also improved the degradation of CC, which were used as
energy source in the gas production test.
Higher in vitro true digestibility reflects higher microbial biomass (Blümmel et al.
1997). This was also confirmed in our study for treatments U60CaSO4 þ CC and
U60CaCl2 þ CC (Table 3). Possibly, UCM released ammonia more slowly than
urea, and can potentially be used more efficiently by rumen micro-organisms,
especially combined with CC as energy source (Zinn and DePerters 1991; Sommart
et al. 2000; Galo et al. 2003; Pfeffer et al. 2009).

3.2. Ammonia nitrogen and volatile fatty acids

NH3-N concentrations ranged for all treatments from 10.7–15.7 mg/dl (Table 4).
These values were above the optimum for rumen micro-organism growth (3–8 mg/
100 ml), as stated by Satter and Slyter (1974). Throughout this in vitro experiment,
urea treatments (U100 þ CC and U100 þ CM) produced the highest concentration
of NH3-N (14.5 and 15.7 mg/100 ml, respectively). Only UCM treatments in
combination with cassava chips reduced the NH3-N significantly. The reason could
be that UCM products lead to a slower releasing rate of NH3-N when compared
with urea treatment. These results agree with those of Huntington et al. (2006), who
found that urea calcium reduces the rate of NH3-N release and absorption when
steers rapidly consume urea that provided 0.322, 0.386 or 0.460 of the total daily N
intake. Moreover, Taylor-Edwards et al. (2009) and Cherdthong et al. (2011a,
 Archives of Animal Nutrition 249

Table 3. The effect of different urea–calcium mixtures and energy sources on gas kinetics,
true digestibility and microbial mass from in vitro incubation with rumen fluid.

Gas
Gas kinetics{ production True Microbial
(96 h) [ml/0.5 g digestibility mass
Treatment{ a b c aþb DM substrate] [%] [mg]
d d d
U100 þ CC –3.3 84.1 0.049 80.8d 83.3d 53.3d 23.1d
U40CaCl2 þ CC –4.4de 105.4e 0.041 101.1e
e
103.4e 55.5d 25.6e
U50CaCl2 þ CC –2.8d 93.3f 0.038e 90.5e 103.4e 54.0d 26.1e
U60CaCl2 þ CC –4.8e 116.7g 0.054f 111.9f 115.7f 59.7e 30.3f
U40CaSO4 þ CC –4.0de 102.8e 0.044d 98.7e 101.2e 55.1d 27.2ef
U50CaSO4 þ CC –5.2e 101.3e 0.055f 96.1e 100.8e 56.6de 26.4e
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

U60CaSO4 þ CC –5.3e 120.6g 0.053f 115.3f 119.8f 61.5e 32.1f

U100 þ CM –2.2f 75.0h 0.033g 72.8g 71.8g 47.5f 20.2df
U40CaCl2 þ CM –3.6d 91.5f 0.037e 87.9d 88.9d 50.9f 22.4df
U50CaCl2 þ CM –2.5f 88.6d 0.036g 86.2d 85.8d 49.7f 23.1d
U60CaCl2 þ CM –2.8d 97.6e 0.041e 94.7e 95.7de 53.3d 26.1e
U40CaSO4 þ CM –2.5f 82.2d 0.036g 79.7d 79.6d 47.6f 23.3d
U50CaSO4 þ CM –1.8g 85.4d 0.034g 83.5d 82.0d 48.9f 22.5f
U60CaSO4 þ CM –3.1d 99.6e 0.042e 96.5e 97.9de 53.4d 26.4e
SEM# 0.04 0.4 0.0003 0.3 0.4 0.03 0.34
Comparison
Energy sources * ** ** ** ** * *
Urea–Ca mixed ** ** ** ** ** ** *
Ca sources ns ns ns ns ns ns ns
Urea levels ns ns ns ns * * *
Interaction * ns ** * ** ns ns

Notes: {U, Urea; CC, Cassava chips; CM, Corn meal; {a ¼ Gas production from the immediately soluble
fraction; b ¼ Gas production from the insoluble fraction; c ¼ Gas production rate constant for the
insoluble fraction (b); a þ b ¼ Potential extent (omit gas); {Microbial mass [mg] ¼ Substrate truly
degraded [mg] 7 (Gas volume [ml]  2.2) (Blümmel et al. 1997); #SEM, Standard error of the mean. Means
in the same column with different superscripts are significantly different (p 5 0.05); *p 5 0.05;
**p 5 0.01; ns, Non-significant.

2011b) reported that urea products, which are slowly released, reduced the rapidity
of ammonia release in the rumen without affecting other metabolites of rumen
fermentation. According to Taylor-Edwards et al. (2009), it could be inferred that
diets with slowly released urea prolong microbial utilisation of additional N sources
during ruminal fermentation. Concentrations of NH3-N in rumen fluid decrease
when the content of nonstructural carbohydrates in the diet (e.g. starch) is increased
(Chanjula et al. 2004). Therefore, the synchronisation between ruminal-NH3 release
and carbohydrate availability from CC might be improved, which can result a higher
microbial mass.
The different treatments affected the production of total and individual VFA
(Table 4). Total VFA concentrations after treatment U60CaSO4 þ CC and
U60CaCl2 þ CC were significantly higher than for all other treatments. A significant
increase was only found in treatments containing CC, but not in those including corn
meal. Comparable results were reported by Sommart et al. (2000), who observed that
in rumen fluid the concentration of total VFA tended to increase with increasing CC
inclusion. In addition, the molar proportion of propionate after supplementation of
U60CaCl2 þ CC and U60CaSO4 þ CC was significantly increased (p 5 0.01). This
higher propionate production can be explained by the presence of glucogenic
 250 A. Cherdthong et al.

Table 4. The effect of different urea-calcium mixtures and energy sources on in vitro volatile
fatty acids (VFA), methane production and ruminal ammonia-nitrogen (NH3-N).

VFA [%]
Total VFA Acetate Propionate Butyrate Ratio NH3-N
Treatment{ [mM/l] (C2) (C3) (C4) C2:C3 [mg/100 ml]
U100 þ CC 48.7ab 68.7 20.1a 11.2 3.4 14.5a
U40CaCl2 þ CC 51.0b 66.8 22.1b 11.1 3.0 11.6b
U50CaCl2 þ CC 51.2b 66.8 22.0b 11.2 3.0 11.5b
U60CaCl2 þ CC 53.2c 66.5 23.2c 10.3 2.9 11.0b
U40CaSO4 þ CC 51.4b 66.8 22.7bc 10.5 2.9 11.6b
U50CaSO4 þ CC 51.0b 66.9 22.9bc 10.2 2.9 11.5b
U60CaSO4 þ CC 54.7c 66.2 23.4c 10.4 2.8 10.7b
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

U100 þ CM 44.2a 68.2 20.5a 11.3 3.3 15.7a

U40CaCl2 þ CM 45.6a 68.3 20.4a 11.3 3.3 13.1ab
U50CaCl2 þ CM 45.5a 68.1 20.5a 11.4 3.3 13.0ab
U60CaCl2 þ CM 47.2ab 67.2 22.5bc 10.3 3.0 13.2ab
U40CaSO4 þ CM 45.2a 67.8 20.7a 11.5 3.3 13.4ab
U50CaSO4 þ CM 45.4a 68.1 21.6b 10.3 3.2 13.1ab
U60CaSO4 þ CM 47.1ab 67.2 22.3b 10.5 3.0 13.4ab
SEM{ 1.4 3.4 0.3 1.3 0.1 0.05
Comparison#
Energy sources * ns ** ns ns *
Urea–Ca mixed ** ns ** ns ns **
Ca sources ns ns ns ns ns ns
Urea levels ns ns ns ns ns ns
Interaction ** ns * ns ns **

Notes: {U, Urea; CC, Cassava chips; CM, Corn meal; {SEM, Standard error of the mean; #Comparisons:
*p 5 0.05; **p 5 0.01. Means in the same column not sharing the same superscript are significantly
different (p 5 0.05); ns, Non-significant.

compounds of CC in these substrates. However, the proportions of acetic and

butyric acid and the C2:C3 ratio was not different among treatments. Additionally,
the ability of UCM products to modulate ruminal-NH3 release is possibly beneficial
to supply N to energy from starch fermentation. However, ruminal NH3-N and VFA
concentration were not affected by levels of urea and Ca sources in UCM
(p 4 0.05).

3.3. Population of total and specific bacteria

Bacterial numbers in the rumen are very high (4 1010 cells per g of contents),
and bacteria play a dominant role in all facets of ruminal fermentation (Hungate
1950). Early work indicated that the complexity of gastrointestinal tract (GIT)
bacteria was great, and molecular techniques have revealed even more diversity
(Russell and Rychlik 2001; Simon et al. 2005). Molecular-based, culture-
independent enumeration methods have also permitted in vitro studies to
elucidate interactions among cellulolytic species (Russell et al. 2009). The
accuracy of each real time PCR was validated by quantifying known numbers
of target species templates (total bacteria, F. succinogenes, R. flavefaciens, R. albus
and anaerobic fungi). Then, those templates were used for a generated standard
curve. In this study, the standard curves showed a high linear relationship
 Archives of Animal Nutrition 251

between cycle threshold (Ct) values and known number template dilution by a
high r2 for each species (Figure 2). The UCM treatments and levels of urea in
UCM have affected the total bacteria concentrations, levels of F. succinogenes and
number of anaerobic fungi population (p 5 0.05), whereas Ca source did not
influence these data (Table 5). Quantifying the predominant cellulolytic bacteria
in in vitro incubation provided additional interesting data. The population of F.
succinogenes was 107 copies/ml and were highest in U60CaCl2 þ CC and
U60CaSO4 þ CC respectively. Similarly, Wanapat and Cherdthong (2009), who
studied cellulolytic bacteria population in rumen using real time PCR, found that
F. succinogenes was also higher than R. flavefaciens and R. albus. It appears that,
once NH3 starts to accumulate, the growth of bacteria utilising NH3 is not
enhanced by increasing NH3 concentration (Satter and Slyter 1974; Lebzien et al.
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

Figure 2. Standard curves obtained by plotting the logarithm of DNA concentration for
total bacteria (A), F. succinogenes (B), R. flavefaciens (C), R. albus (D) and anaerobic fungal
(E) against the threshold cycle (Ct) for population quantification using real time PCR.
 252 A. Cherdthong et al.

Table 5. Effects of different urea-calcium mixtures and energy sources on total bacterial and
fungal populations after in vitro incubation with rumen fluid [copies/ml].

Total Anaerobic
bacteria F. succinogenes R. flavefaciens R. albus fungi
Treatment{ [109] [107] [106] [105] [106]
U100 þ CC 3.2a 2.6b 1.4 3.3 1.2a
U40CaCl2 þ CC 5.4bc 4.6d 2.1 4.7 2.4b
U50CaCl2 þ CC 5.8bc 4.8d 2.4 4.5 2.7b
U60CaCl2 þ CC 8.9d 7.1f 2.4 4.9 4.8c
U40CaSO4 þ CC 6.4c 4.7d 2.2 4.7 2.8b
U50CaSO4 þ CC 6.7c 4.0c 2.5 4.8 2.4b
U60CaSO4 þ CC 9.2d 7.2f 2.5 4.9 4.8c
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

U100 þ CM 2.3a 1.2a 1.1 2.8 0.8a

U40CaCl2 þ CM 4.2b 3.5bc 1.9 3.5 1.8ab
U50CaCl2 þ CM 4.4b 3.3bc 2.0 3.4 1.5ab
U60CaCl2 þ CM 6.1c 5.5e 2.2 3.7 2.7b
U40CaSO4 þ CM 4.5b 3.7c 2.1 3.6 1.6ab
U50CaSO4 þ CM 4.7b 3.2bc 2.0 3.8 1.7ab
U60CaSO4 þ CM 6.5c 5.7e 2.1 3.8 2.6b
SEM{ 0.5 0.2 2.7 3.6 0.3
Comparison#
Energy sources * * ns ns *
Urea–Ca mixed ** * ns ns *
Ca sources ns ns ns ns ns
Urea levels * * ns ns *
Interaction * ns ns ns ns

Notes: {U, Urea; CC, Cassava chips; CM, Corn meal; {SEM, Standard error of the mean; #Comparisons:
*p 5 0.05; **p 5 0.01. Means in the same column not sharing the same superscript are significantly
different (p 5 0.05); ns, Non-significant.

2006; Calsamiglia et al. 2010). These data indicate that products containing a
mixture of urea and Ca sources could be utilised in ruminant diets as slow-
released NH3 sources, which provide continuous NH3 for microbial growth.
Sudana and Leng (1986) showed that supplements must provide continuously
adequate levels of NH3 for persistent growth of both, fibrolytic and saccharolytic
micro-organisms.
Moreover, the enhancement of ruminal population of anaerobic fungi was
especially pronounced after treatment U60CaCl2 and U60CaSO4 with CC. These
results agreed with Chanjula et al. (2004) who also found that the number of
fungal zoospores were greater for cassava-based diets than for corn diets.
Although the concentrations of fungi are relatively low in comparison with those
of bacteria and ciliate protozoa, they possess a wide range of enzymes, which
are capable of hydrolysing most of the structural polysaccharides occurring in
plant cell walls (Orpin and Letcher 1979). In the current study, lower a
digestibility reflects reduced populations of ruminal anaerobic fungi after urea
treatment.
It can be concluded that studies on in vitro properties of UCM intended for use as
slow-release urea feeds provide valuable information for the prediction of in vivo
effectiveness. Especially the use of urea level at 60% of UCM combined with CaCl2
or CaSO4 seems to be effective. This study is an important tool in further product
development.
 Archives of Animal Nutrition 253

Acknowledgements
The authors would like to express sincere thanks to the Tropical Feed Resources Research and
Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture,
Khon Kaen University, Thailand and the Thailand Research Fund (TRF) through the Royal
Golden Jubilee Ph.D. Program for providing financial support for the research and the use of
the research facilities.

References
[AOAC] Association of Official Analytical Chemists. 1998. Official methods of analysis,
Vol. 2. 16th ed. Arlington (VA): AOAC.
Blümmel M, Makkar HPS, Becker K. 1997. In vitro gas production: a technique revisited.
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

J Anim Physiol Anim Nutr. 77:24–34.

Calsamiglia S, Ferret A, Reynolds CK, Kristensen NB, Van Vuuren AM. 2010. Strategies for
optimizing nitrogen use by ruminants. Animal. 4:1184–1196.
Chanjula P, Wanapat M, Wachirapakorn C, Rowlinson P. 2004. Effect of synchronizing
starch sources and protein (NPN) in the rumen on feed intake, rumen microbial
fermentation, nutrient utilization and performance of lactating dairy cows. Asian-Aust J
Anim Sci. 17:1400–1410.
Cherdthong A, Wanapat M. 2010. Development of urea products as rumen slow-release feed
on ruminant production: a review. Aust J Basic Appl Sci. 4:2232–2241.
Cherdthong A, Wanapat M, Wachirapakorn C. 2011a. Effects of urea-calcium mixture in
concentrate containing high cassava chip on feed intake, rumen fermentation and
performance of lactating dairy cows fed on rice straw. Livest Sci. 136:76–84.
Cherdthong A, Wanapat M, Wachirapakorn C. 2011b. Influence of urea calcium mixture
supplementation on ruminal fermentation characteristics of beef cattle fed on concentrates
containing high levels of cassava chips and rice straw. Anim Feed Sci Technol. 163:43–51.
Denman SE, McSweeney CS. 2006. Development of a real time PCR assay for monitoring
anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS
Microbiol Ecol. 58:572–582.
Galo E, Emanuele SM, Sniffen CJ, White JH, Knapp JR. 2003. Effects of a polymer-coated
urea product on nitrogen metabolism in lactating Holstein dairy cattle. J Dairy Sci.
86:2154–2162.
Golombeski GL, Kalscheur KF, Hippen AR, Schingoethe DJ. 2006. Slow-release urea
and highly fermentable sugars in diets fed to lactating dairy cows. J Dairy Sci. 89:4395–4403.
Highstreet A, Robinson PH, Robison J, Garrett JG. 2010. Response of Holstein cows to
replacing urea with a slowly rumen released urea in a diet high in soluble crude protein.
Livest Sci. 129:179–185.
Hungate RE. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriol Rev. 14:1–49.
Huntington GB, Harmon DL, Kristensen NB, Hanson KC, Spears JW. 2006. Effects of a
slow-release urea source on absorption of ammonia and endogenous production of urea
by cattle. Anim Feed Sci Technol. 130:225–241.
Karabulut A, Canbolat O, Kalkan H, Gurbuzol F, Sucu E, Filya I. 2007. Comparison of in
vitro gas production, metabolizable energy, organic matter digestibility and microbial
protein production of some legume hays. Asian-Aust J Anim Sci. 20:517–522.
Koike S, Kobayashi Y. 2001. Development and use of competitive PCR assays for the rumen
cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus
flavefaciens. FEMS Microbiol Lett. 204:361–366.
Kongmun P, Wanapat M, Pakdee P, Navanukraw C. 2010. Effect of coconut oil and garlic
powder on in vitro fermentation using gas production technique. Livest Sci. 127:38–44.
Lebzien P, Riemeier A, Flachowsky G. 2006. Investigations on the effect of the ruminal N-
balance on rumen metabolism, urea content in blood serum and milk as well as some liver
parameters of lactating cows. Arch Anim Nutr. 60:99–109.
Menke KH, Raab L, Salewski A, Steingass H, Fritz D, Schneider W. 1979. The estimation of
the digestibility and metabolizable energy content of ruminant feedstuffs from the
gas production when they are incubated with rumen liquor in vitro. J Agric Sci. 92:
217–222.
 254 A. Cherdthong et al.

Menke KH, Steingass H. 1988. Estimation of the energetic feed value obtained from chemical
analysis and gas production using rumen fluid. Anim Res Dev. 28:7–55.
Orpin CG, Letcher AJ. 1979. Utilization of cellulose, starch, xylan and other hemicelluloses
for growth by the rumen phycomycete Neocallimastix frontalis. Curr Microbiol. 3:
121–124.
Ørskov ER, McDonald I. 1979. The estimation of protein degradability in the rumen from
incubation measurements weighted according to rate of passage. J Agric Sci. 92:499–503.
Pfeffer E, Speckter H, Bornemann S, Holthausen A, Rodehutscord M. 2009. Kinetics of
endogenous urea in lactating goats and cows fed diets varying in their crude protein
concentrations. Arch Anim Nutr. 63:230–242.
Poungchompu O, Wanapat M, Wachirapakorn C, Wanapat S, Cherdthong A. 2009.
Manipulation of ruminal fermentation and methane production by dietary saponins and
tannins from mangosteen peel and soapberry fruit. Arch Anim Nutr. 63:389–400.
Rooney LW, Pflugfelder RL. 1986. Factors affecting starch digestibility with special emphasis
Downloaded by [Ondokuz Mayis Universitesine] at 12:48 10 November 2014

on sorghum and corn. J Anim Sci. 63:1607–1623.

Russell JB, Rychlik JL. 2001. Factors that alter rumen microbial ecology. Science. 292:
1119–1122.
Russell JB, Muck RE, Weimer PJ. 2009. Quantitative analysis of cellulose degradation and
growth of cellulolytic bacteria in the rumen. FEMS Microbiol Ecol. 67:183–197.
SAS. 1998. User’s guide: statistics. Version 6. 12th ed. Cary (NC): SAS.
Satter LD, Roffler RE. 1975. Nitrogen requirement and utilization in dairy cattle. J Dairy Sci.
58:1219–1237.
Satter LD, Slyter LL. 1974. Effect of ammonia concentration on rumen microbial protein
production in vitro. Brit J Nutr. 32:199–208.
Simon O, Taras D, Vahjen W. 2005. Nutritional impact on intestinal bacterial communities
of pigs studied by molecular biology techniques. J Anim Feed Sci. 14(Suppl. 1):575–578.
Sommart K, Wanapat M, Rowlinson P, Parker DS, Climee P, Panishying S. 2000. The use of
cassava chips as an energy source for lactating dairy cows fed with rice straw. Asian-Aust J
Anim Sci. 13:1094–1101.
Steel RGD, Torrie JH. 1980. Principles and procedure of statistics. A biometrical approach.
2nd ed. New York (NY): McGraw-Hill Book Company.
Sudana IB, Leng RA. 1986. Effects of supplementing a wheat straw diet with urea or urea-
molasses blocks and/or cottonseed meal on intake and liveweight change of lambs. Anim
Feed Sci Technol. 16:25–35.
Taylor-Edwards CC, Elam NA, Kitts SE, McLeod KR, Axe DE, Vanzant ES, Kristensen NB,
Harmon DL. 2009. Influence of slow-release urea on nitrogen balance and portal-drained
visceral nutrient flux in beef steers. J Anim Sci. 87:209–221.
van Soest PJ, Robertson JB, Lewis BA. 1991. Methods for dietary fiber neutral detergent fiber,
and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci. 74:3583–3597.
Wanapat M. 2009. Potential uses of local feed resources for ruminants. Trop Anim Health
Prod. 41:1035–1049.
Wanapat M, Cherdthong A. 2009. Use of real-time PCR technique in studying rumen
cellulolytic bacteria population as affected by level of roughage in Swamp buffalo. Curr
Microbiol. 58:294–299.
Wanapat M, Cherdthong A, Pakdee P, Wanapat S. 2008. Manipulation of rumen ecology by
dietary lemongrass (Cymbopogon citratus Stapf.) powder supplementation. J Anim Sci.
86:3497–3503.
Yu Z, Michel FC, Hansen Jr G, Wittum T, Morrison M. 2005. Development and application
of real-time PCR assays for quantification of genes encoding tetracycline resistance. Appl
Environ Microbiol. 71:6926–6933.
Yu Z, Morrison M. 2004. Improved extraction of PCR-quality community DNA from digesta
and fecal samples. BioTechniques. 36:808–812.
Zinn RA, DePeters EJ. 1991. Comparative feeding value of tapioca pellets for feedlot cattle.
J Anim Sci. 69:4726–4733.