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ESTIMATION OF
THE DRY MATTER DIGESTIBILITY OF CORN SILAGE
technique for the in vitro estimation of the dry matter digestibility of corn
silage. Can. J. Anim. Sci. 60: 367-378.
The two-stage Tilley and Terry technique (incubation with rumen fluid followed by an
acid-pepsin digest) , used to estimate dry matter (DM) digestibility of forages in vitro,
was evaluated with oven-dried corn silage as a substrate. The effect of weight of
substrate (100-3000 mg), continuous shaking of incubations for the period of
incubation with rumen fluid, number of bacteria present in the inoculum, and the
contribution of bacterjal dry matter to residual feed DM was measured. Percent dry
matter digested decreased linearly as weight of substrate per incubation tube
increased. Continuous shaking, as opposed to intermittent mixing (twice daily) during
incubation with rumen fluid, increased the rate of DM disappearance and resulted in
higher digestibility coefficients. Both the volume of inoculum and the number of
bacteria present in that volume of inoculum influenced the percent DM digested.
For personal use only.
Bacteria contribute weight to residual feed DM unless steps are taken to remove them
by centrifugation or solubilization.
La technique en deux 6tapes Tilley et Terry (incubation avec du liquide ruminal suivie
d'une digestion d la pepsine acide) servant ) estimer la digestibilit6 de la matibre sbche
des fourrages in vitro a 6t6 6,valu6e au moyen d'ensilage de mais s6ch6 au four comme
substrat. On a ainsi mesur6 I'effet du poids du substrat (100 h 3000 mg), de I'agitation
continue des incubats pendant la p6riode d'incubation avec le liquide ruminal, le
nombre de bact6ries de I'inoculum et la contribution de la matidre sdche bact6rienne d
la teneur en matibre sdche r6siduelle de I'aliment. Le pourcentage de matibre sbche
dig6r6e d6croit de fagon inversement proportionnelle b I'augmentation du poids du
substrat par tube d'incubation. L'agitation continuelle, contrairement d intermittente
(deux fois parjour) au cours de I'incubation avec la liquide ruminal, accroit le taux de
consommation de la matibre sbche et am6liore les coefficients de digestibilit6. Le
volume de I'inoculum et le nombre de bact6ries qu'il contient influent sur la digestion
de la matibre sbche. Les bact6ries contribuent I accroitre la matibre sbche 16siduelle de
I'aliment si l'on ne prend pas les mesures n6cessaires pour les enlever par
centrifugation ou solubilisation.
A two-stage in vitro technique was de- Several factors influence the final in vitro
veloped by Tilley and Terry (1963) for the estimate of DM digestibility. The largest
estimation of the dry matter (DM) digestibil- single source of variability is the inoculum
ity of forages. The method has been widely (Johnson 1969), which is influenced by the
used for this purpose and has been type of diet fed to the donor animal (Knipfel
recommended by Tilley et al. (1960) and and Troelsen 1966). Additional variation is
TilleyandTerry(1963)fortheclassification due to the particle size of the substrate,
of groups of forage samples relative to one weight of substrate, ratio of volume of
another in terms of their DM digestibilities. inoculum to buffer (Mcleod and Minson
Can. J. Anim. Sci. 50: 367-378 (June 1980)
36'/
CANADIAN JOURNAL OF ANIMAL SCIENCE
I 969), and length of time for the first stage of Corn silage (33.07o dry matter, 7.387c crtde
the technique (Troelsen and Hanel 1966). protein on a DM basis) was oven-dried at 100'C
The Tilley and Terry (1963) method in its for 24 h. The oven-dried corn silage was ground
original form is still widely used and is one in a laboratory mill fitted with a l-mm screen.
Weighed quantites of dried corn silage, in
of the best methods for the prediction of in triplicate, were mixed with a modified
vivo dry matter digestibility (apparent McDougall's buffer (McDougall 1948). In the
digestibility) for forages (Scales et al. 1974: modified McDougal['s buffer, MgSO1.7H2O
Aerls et al. 1911; Barnes and Marten 1979). (0.12 glL) was substituted for MgCl2 (Johnson
Numerous modifications to the original
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silage as a substrate with regard to the was added, the head-speace of each incubation
tube was flushed with COz, and each incubation
relationship between in vitro DM disappear-
tube was sealed with a Bunsen gas release valve,
ance and ( 1 ) recovery of individual and total
and incubated anaerobically for 48 h in a water
volatile fatty acids and weight of substrate bath at 39'C. Incubation tubes were mixed
per incubation, (2) the effect of continuous manually with a swirling action after 3, 6,24 and
mixing during incubation with rumen fluid, 30 h of incubation for the first stage of the
(3) the volume of inoculum and the bacterial procedure. Unless otherwise indicated, this
counts in that inoculum, and (4) the procedure was used throughout.
contribution of bacterial DM to residual feed After the rumen fluid digest, the contents of
dry matter used in the calculation of percent each incubation tube were acidified directly with
DM digested. 4 x I mL of 3 N HCI containing a total of 0. I g
pepsin ( I : 10 000; Nutritional Biochemicals
Corporation, Cleveland, Ohio, U.S.A.). All feed
MATERIALS AND METHODS particles were washed from the sides of the
General Incubation Technique incubation tubes and from the stoppers. The
Dry matter digestibility was determined in vitro acid-pepsin digest was incubated aerobically for
using a modification of the two-stage technique 48 h in a water bath at 39'C. Undigested DM was
of Tilley and Terry (1963). Rumen fluid was recovered by filtering the acid-pepsin digest
obtained from a fistulated cow approximately 3 h through tared glass microfibre filters (Whatman
after feeding. The cow was fed either a diet GF/A). Filters plus residual DM were dried at
containing 4OVa corn silage,407c alfalfa hay and 55'C under vacuum for a minimum of48 h, and
2OVo haylage (alfalfa-grass mixture) on a DM weighed to determine the quantity of residual DM
basis (diet A) or a diet of badly wearhered poor recovereo.
quality alfalfa hay (diet B). Rumen fluid from the A minimum of four incubations of rumen fluid
cow when fed diet A was used in all studies plus buffer to a total volume of 50 mL were
reported in this paper; only when the relationship prepared as blank incubations. Average DM
between percent DM digested and bacterial recovered from the blank incubations was
counts per incubation was examined was substracted from the residual dry matter before an
inoculum used from the same cow but fed diet B. in vitro estimate of dry matter digestibility
BOILA ET AL. DIGESTIBILITY OF CORN SILAGE 369
-
(DMD) was calculated according to the fbllowing incubation) were calculated as a glucose
equatlon: equivalent; this estimate of DM disappearance
was compared to the DM residue (residual DM
DMD:[Substrate DM - (Residual DM - Blank minus blank DM) obtained after 96 h of
DM)l (100)/(Substrate DM) digestion. Nitrogen, crude fat and ash present in
the original weight of substrate were substracted
Volatile Fatty Acid Analysis from the DM digested before this comparison was
Filtered incubation media, with added intemal made. The stoichiometry between VFA reco-
standard (isocaproic acid), were injected directly vered and DM metabolized by rumen bacteria
onto a glass column (6 m long, 3 mm i.d.)
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
procedure described by Hungate et al. (1971). in the blank preparations was used to calculate
Rumen fluid, incubation media or filtrates of residual feed DM at all times except 0 and 12 h.
incubation media were homogenized for 2 min This arrangement of incubations was duplicated;
(90 volt setting on a rheostat) with an one set of incubations was mixed intermittently
Omni-Mixer (Ivan Sorvall Inc., Norwalk, Conn. by hand aI 3 , 6, 24 , 30, 49 and 72 h for the first
U.S.A.) before being diluted l:1 (vol/vol) with stage of the procedure; the second set of
8% formaldehyde. Appropriate dilutions were incubations was shaken continuously (55 oscilla-
made with 87o formaldehyde. Bacteria were tions/min) in an upright position, in addition to
stained with crystal violet and counted in a being mixed by hand at the times specified for the
Thoma counting chamber (Hawksley, England) first set of incubations during the rumen fluid
under oil immersion at a masnification of 1000 digest. The same rumen fluid was used for both
under phase contrast. sets of incubations.
The Effect of Weight of Substrate on Dry The Effect of Bacterial Numbers and Volume
Matter Digested and Volatile Fatty Acid of Inoculum
Production Inoculum was obtained from a fistulated cow fed
Total dry matter (residual DM minus blank DM) either diet A or diet B; each source of inoculum
digested per incubation, percent DM digested, was prepared separately. Inoculum volumes of
and individual and total volatile fatty acid (VFA) 1.0. 2.5. 5.0. 10.0. 15.0 and 20.0 mL were
recovered from each incubation were determined incubated with 250, 500, 1000 and 1500 mg of
forpreparations containing substrate at 100-3000 substrate. The total volume of inoculum plus
mg per incubation. The pH of samples was buffer was 50 mL. Bacterial counts per
measured after 48 h of incubation with rumen incubation were obtained by multiplying the
fluid. The volume of incubation medium was counts present in the original rumen fluid by the
determined by weighing incubation tubes with volume of rumen fluid included in each
and without the incubation medium present; incubation.
allowance was made for the weight of residual
DM present in each incubation. The millimoles of The Contribution ofBacteria to Residual Feed
individual VFA recovered at each weight of Dry Matter
substrate (minus millimoles present in a blank Duplicate incubation tubes were prepared with 0,
310 CANADIAN JOURNAL OF ANIMAL SCIENCE
100, 250, 500, 1000 and 1500 mg substrate. A in buffer and acid, and the retention of
lO-mI- aliquot from each tube was taken after bacterial mass in the filtered residue.
48 h of incubation, homogenized and prepared Mcleod and Minson (1969) observed that
for bacterial counts as described above. The DM digestibility decreased with increasing
remaining 40 mL was filtered through Whatman
sample size. In agreement with this, the
GF/A glass fibre filters, and the filtrate
present results show that the percent DM,
homogenized and prepared for bacterial counts.
Incubations (in triplicate) with substrate
digested decreased linearly when the amount
weights of
100, 250, 500, 1000 and 1500 mg of substrate per incubation was increased
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
u
F
o
U
d
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
u
F
F
o a
F
zu
o Y= 73.5-0.0097X
(r2=O.97: Sy.x= 1.5)
U
0 1000 2000
For personal use only.
3000
WEIGHT OF SUBSTRATE (mg)
Fig. I . The effect of weight of substrate dry matter present per incubation upon percent dry matter
digested, after completion of incubation with rumen fluid and an acid-pepsin digest. Each point
reDresents a mean of three values.
Table l. Calculation of individual volatile fatty acids recovered from incubations as a glucose equivalent
z 8.0
F z
3 z.o TOTAL MILLIIVOLES VFA RECOVERED 400 k
PER INCUBATION (SE = 0.16) o
l
= 200 z
-- 6.0
c(
o u
u
ooo ff
F
o
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
U
H +.0 800 :
>
= 1.0 200 a
F
a
F
Ftg. 2 .
The effect of weight of sub strate present per incub ation upon the total of volatile fatty acid s
recovered from each incubation and dry matter disappearance per incubation. Standard errors (SE)
were calculated using error mean square from a one-way analysis of variance of the data. Each point
represents a mean of three values.
z 2.O
tr
c0
1.0
=
q
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
0
U
U
0.6
u
E.
; 0.4
o.2
I
u
J
0
o
L 0.06
o
For personal use only.
o
U
0.04
E
f
E 0.02
of this mat of substrate. Since some and Sayre and Van Soest (1972). These
accumulation of substrate at the surface of authors observed that good DM digestibility
the incubation medium was also observed values could be obtained with only occa-
when incubation tubes were shaken continu- sional agitation when either screwcapped
ously, it was necessary to mix these tubes by tubes or 125-mL Erlenmeyer flasks were
hand. Mellenberger et al. (1970) found that used. From this it may be concluded that
with slow rotation of sealed incubation tubes continuous mixing of incubation tubes with
in a horizontal position, there was better Bunsen values, incubation in screwcapped
contact between substrate and medium with tubes in a horizontal position, or incubations
less mat formation. in 125-mL glass Erlenmeyer flasks will
A different approach to particle dispersion increase DM digestibility above that ob-
was used by Goering and Van Soest (1970) rained with the original Tilley and Teny
J t+ CANADIAN JOTIRNAL OF ANIMAL SCIENCE
Table 2. Effect of intermittent mixing and continuous Sy (Table 3) were found with 500 mg of
shaking of incubation tubes upon percent dry matter
substrate (Fig. a). A higherr2 and a lowerSj
disested in vitro
indicate that bacterial counts would account
Time lntermittent Continuous for a greater proportion of the variation in
(h) mixing shaking SE percent DM digested than would the volume
0 25.5 A of inoculum. At 1500 mg of substrate,
t2 39.6 Ba 43.5 Bb 1.17* factors other than inoculum volume or
.A 49.6 Ca 56.8 Cb 1.05+* bacterial counts per incubation might have
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
0 48't216 20
procedure. The eventual choice of method VOLUME OF INOCULUM (mll PER
INCUBATION
may well depend on the facilities available or
the preference of the investigator.
U
U
The Effect of Bacterial Numbers and
Volume of lnoculum 6
When the fistulated cow fed diet A was U
F
F
a
o
changed to diet B, the number of bacterial
cells decreased from 2.38 x 1010 to 1 .44 x /
E
1010 per mL of rumen fluid. Percent DM
digested in in vitro experiments at each zUF
weight of substrate was combined for a 4
polynomial regression analysis. When per- c
40
cent DM digested (dependent variable) was ttttll
01020304050
regressed upon bacterial counts per incuba- BACTERIAL coUNTs (Xlo-10) PER
tion, as opposed to volume of inoculum, as INCUBATION
the independent variable, the proportion of Fig. 4. The relationship between percent dry
the variation in the dependent variable matter digested (I) as the dependent variable and
attributed to its regression on the indepen- volume of inoculum per incubation (A, Y : 47 .7
dent variable (r2) was higher at weights of + 5.30x -0.41-r2 + 0.01 xs;12 :0.6a; Sy :
substrate of 250,500 and 1000 mg, but not at 4.75) or bacterial counts per incubation (B,Y :
47.5 + 2.55x -0.08912 * 0.00096x3;r2 - 0.'79:
1500 mg (Table 3). Standard errors for the
Si - 3.61) as the independent variable for500 mg
estimate of the dependent variable (Sy) were
as a weight of substrate per incubation. Inoculum
lower when bacterial counts per incubation was obtained from a fistulated cow fed corn
was the independent variable at weights of silage: alfalfa hay: haylage (closed circles) or a
250, 500 and 1000 mg, but not at 1500 mg poor quality hay (open circles). Each point
(Table 3). The largest differences for 12 and represents a mean of three values.
BOILA ET AL. DIGESTIBILITY OF CORN SILAGE 3'75
-
Table 3 . The 12 values and the standard errors of the estimate (Sj) from a polynominal regression analysis of degree
equal to three for the relationship between percent dry matter digested and volume of inoculum or bacterial counts p€r
rncuDanon.
ir2, the proportion of the variance ofthe dependent variable that can be attributed to its regression on the independent
variable.
tSy, standard error of the estimate of the dependent variable.
limited percent DM digested. It was were isolated from rumen fluid from the cow
concluded that percent DM digested de- when fed diet A, using the differential
pended to some degree upon the number of centrifugation procedure of Meyer et al.
bacteria added to each incubation. Mcleod (196'7). The relationship of bacterial DM to
and Minson (1969) did not do bacterial bacterial counts was 1.75 mg DM per 1010
counts but noted that there was an interaction bacteria. With 50O mg of substrate (Table 4),
between percent DM digested and volume of 3.43 x 10e bacteria per mL of incubation
inoculum. medium were retained with the residual DM
For personal use only.
Total bacterial counts per incubation do after the rumen fluid digest; bacteria from 50
not completely account for the differences in mL of incubation medium could contribute
DM digestibility (Figs. 4A and 48) because 30 mg DM to residual feed DM (1.75 x 10-to
when bacterial counts were equalized (Fig. mg per bacterium x 3.43 x lOe bacteria per
48). some differences due to the source of mL x 50 mL) after 48 h of incubation.
inoculum still persisted. These differences
could be due to changes in the rumen Table 4. Bacterial counts present in the original
microflora (Slyter and Weaver 1972), a incubation medium and recovered in the filtrate after a
requirement for added glucose or nitrogen in 48-h incubation with rumen fluid and percent dry matter
rumen fluid from cows fed a poor quality diet digested after the complete procedure
(Nelson et al. 1972), or the need for
(x 10s)
Bacterial counts
unspecified components that may be lacking at 48 hl
in the rumen fluid of such cows (Hungate
Wt of Original 7o dry
1960; Bryant 1973; Bales et al. 1916). matter digested
subskate incubation
f mo) medium Filtrate at 96 hl
Based on the composition of rumen bacteria Tinnimit and Thomas 1976) and is particu-
(Hoogenraad and Hird 1970) and the larly advantageous when an attempt is made
digestibility of bacterial protein in pepsin to automate the technique (Alexander and
(Bergen et al. 1968), the digestibility of the McGowan 1966). When subsequent
DM of rumen bacteria in acid-pepsin was between-laboratory comparisons are made,
estimated at 50Vo. In the residual DM it is important to recognize the potential error
recovered from 500 mg of substrate, introduced by the replacement of the
undigested bacteria could contribute l5 mg centrifugation step with direct acidification.
of DM, or account for a difference of A modification of the Tilley and Terry
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approximately 3 percentage points in percent (1963) procedure has been reported by Van
DM digested. Soest et al. (1966) and Goering and Van
Centrifugation of incubation tubes after Soest (1970). Centrifugation and acidifica-
48 h of incubation was a part of the original tion of incubations are replaced by an
technique developed by Tilley and Terry analysis for neutral detergent fibre wherein
(1963). After a low speed centrifugation step bacterial cells are solubilized and not
(18009 for 15 min), the supernatant, which recovered with the filtered residue. In vitro
contained most of the bacteria from the estimates of percent DM digested obtained
incubation medium, was discarded. The with the Van Soest modification are higher
precipitate, which contained only those than the estimates obtained with the Tilley
bacteria associated with feed particles, was and Terry procedure. This difference,
resuspended in acid-pepsin and digested attributed by Van Soest et al. (1966) to the
enzymatically. Since this centrifugation step recovery of bacterial DM with residual feed
For personal use only.
was time-consuming, and some forages did DM in the Tilley and Terry procedure, is
not sediment readily, the centrifugation step several-fold larger (Aerts et al. 1977) than
was replaced with a step where incubation the 3 percentage points calculated for the
media were acidified with pepsin in an HCI data at 500 mg of substrate in Table 4.
solution (3 N HCI was used in this study). Further studies are needed to establish the
Residual DM was recovered when the relationship between the Van Soest modifi-
incubation medium was filtered 48 h after cation and the original Tilley and Terry
direct acidification (total of 96 h for the procedure with respect to the composition of
procedure). With direct acidification at DM disappearance and residual DM.
48 h, DM of bacterial origin is recovered
with residual feed DM when filtration is
undertaken after 96 h (Table 4). This SUMMARY
bacterial mass can represent a difference of The implications of several modifications to
three percentage points in the in vitro the two-stage technique presented by Tilley
estimate of DM digestibility. and Terry (1963) have been studied with
Tilley and Terry (1963) found no oven-dried corn silage as a substrate.
consistent decrease in percent DM digested With 500 + 5 mg of substrate, there was
when direct acidification was included as a less variation among triplicate analyses than
part of their procedure. However, in with 100 or 250 mg of substrate, and the
agreement with out study, Schmid et al. buffering capacity of the incubation medium
(1975) found the percent DM digested to be was not exceeded.
reduced 2-3 percentase points for corn and When centrifuge tubes or other narrow
sorghum silages when direct acidification incubation vessels are used, continuous
was used after 48 h. Direct acidification of mixing of incubations is preferable to
incubation media, after the fermentation intermittent mixing. With continuous mix-
step, has been used (Troelsen and Hanel ing, the variance for percent DM digested
1966; O'Shea etal. 1972: Scales et al.1974 was smaller, and a higher DM digestibility
BOILA ET AL. DIGESTIBILITY OF CORN SILAGE 3'77
-
coefficient was measured with 48 h for the procedures and some applications). Agric. Res.
period of incubation with rumen fluid. Serv., U.S. Dep. Agric. Agric. Handb.379.
Both the volume of inoculum and the GRANT, R. J., VAN SOEST, P. J. and
number of bacteria present in that volume of McDOWELL, R. E. 1974. Influence of rumen
fluid source and fermentation time on in vitro true
inoculum contributed to the variability
present in the two-stage technique. A small
dry matter digestibility. J. Dairy Sci.57:
1201-1205.
amount of variability may be due to HOOGENRAAD, N. J. and HIRD, F. J. R.
components present in the rumen fluid. 1970. The chemical composition of rumen
The contribution of bacterial mass to
Can. J. Anim. Sci. Downloaded from www.nrcresearchpress.com by 36.79.36.240 on 03/11/20
ing forage quality. J. Dairy Sci. 55: 358-366. 1967. Statistical methods. The Iowa State
O'SHEA, J., WISON, R. K. and SHEEHAN, University Press, Ames, Iowa. pp. 593.
W. 1972. Prediction of silage digestibility by in STADTMAN, E. R., STADTMAN, T. C., and
vitro and chemical methods. Ir. J. Agric. Res. 11: BARKER, H. A. 1949. Tracer experiments on
l7 5-179. the mechanism of synthesis of valeric and caproic
SAYRE, K. D. and VAN SOEST, P. I. 19'72. acids by Clostridium kluyveri. J. Biol. Chem.
Comparison of types of fermentation vessels for 178: 677-682.
an in vitro artificial rumen procedure. J. Dairy TILLEY, J. M. A., DERIAZ, R. E. and TERRY,
Sci. 55: 1496-1498. R. A. 1960. The in vitro measurement of herbage
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SCALES, G. H., STREETER, C. L., DEN- digestibility and assessment of nutritive value.
HAM, A. H. and WARD, c. M. 1974. A Proc. Sth Int. Grassl. Congr. pp. 533-537.
comparison of indirect methods of predicting in TILLEY, J. M. A. and TERRY, R. A. 1963. A
vivo digestibility of grazed forage. J. Anim. Sci. two-stage technique for the in vitro digestion of
38: 192-199. forage crops. J. Br. Grassl. Soc. 18: 104-1 1 1.
SCHMID, A. R., GOODRICH, R. D., MAR- TINNIMIT, P. and THOMAS, J. W. 1976.
TEN, G. C,, MEISKE, J. C., JORDAN, R. M. Forage evaluation using various laboratory
and HALGERSON, J. L. 1975. Evaluation of techniques. J. Anim. Sci.43: 1058-1065.
laboratory methods for determining quality of TROELSEN, J. E. and HANEL, D. J. 1966.
corn and sorghum silages. I. Biological methods Ruminant digestion in vitro as affected by
for predicting in vivo digestibility. Agron. J. 67: inoculum donor, collection day, and fermenta-
243-246. tion time. Can. J. Anim. Sci. 46: 149-156.
SLYTER, L. L. and WEAVER, I. M. 1912. VAN SOEST, P. J., WINE, R. H. and MOORE,
Dietary influences on ruminal microbes at L. A. 1966. Estimation of the true digestibility of
constant pH. J. Anim. Sci. 35: 288. forages by the in vitro digestion of cell walls. Xth
For personal use only.
1. Dervan D.S.L. Bryan, Dawn A. Abbott, Henry L. Classen. 2018. Development of an in vitro
protein digestibility assay mimicking the chicken digestive tract. Animal Nutrition 4:4, 401-409.
[Crossref]
2. J.L. Argyle, R.B. Hespell. 1987. Digestion of Alfalfa Hay Using Washed Ruminal Bacteria. Journal
of Dairy Science 70:12, 2525-2533. [Crossref]
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