Journal of Agricultural Science, Page 1 of 15.

© Cambridge University Press 2014

doi:10.1017/S0021859614001130

CROPS AND SOILS RESEARCH PAPER

Carbon and nitrogen reserves in marandu palisade grass subjected
to intensities of continuous stocking management

S. C. DA SILVA 1 *, L. E. T. PEREIRA 1 , A. F. SBRISSIA 2 AN D A. HERNANDEZ-GARAY 3

1
Escola Superior de Agricultura ‘Luiz de Queiroz’, Departamento de Zootecnia, Av. Pádua Dias, 11; C.P. 09, CEP: 13418-
900, Piracicaba, SP, Brazil
2
Universidade do Estado de Santa Catarina (UDESC-CAV), CEP: 88520-000, Lages, SC, Brazil
3
Colegio de Postgraduados em Ciencias Agrícolas, Montecillo, Texcoco, México

(Received 30 January 2014; revised 28 May 2014; accepted 2 October 2014)

SUMMARY
Plant organic reserves and sward leaf area index (LAI) influence plant growth, persistency and herbage accumu-
lation in grazed swards. The present study was conducted to describe patterns of variation in herbage accumu-
lation and carbohydrate and nitrogen (N) reserves in shoot and root of marandu palisade grass subjected to
intensities of continuous stocking management throughout the year. Treatments corresponded to four levels of
grazing intensity – severe (S), severe/moderate (S/M), moderate (M) and lenient (L) – and were implemented in
the field using bands of sward surface height (SSH – 10, 20, 30 and 40 cm ± 10%, respectively) maintained
through continuous stocking and variable stocking rate. Total N concentration was higher in the shoot relative
to the root compartment during autumn, early and late spring. On the other hand, the concentration of non-struc-
tural carbohydrates (NSC) and soluble N was higher in the root compartment, regardless of grazing intensity and
season of the year. When taking into account the pool of C and N reserves, the shoot compartment represented
the main storage organ, since it corresponded to the largest pool of NSC (averages of 0·102 ± 0·0038 and 0·201 ±
0·0088 kg/m2 for root and shoot, respectively) and soluble N (averages of 2·7 ± 0·26 and 5·3 ± 0·59 kg/m2 for root
and shoot, respectively). During late spring, the time of active plant growth, there was a clear contrast in herbage
accumulation and sward LAI among grazing intensities, particularly between the severe and lenient grazing treat-
ments. The results show that even with larger pools of soluble N and NSC in the shoot compartment, herbage
accumulation was limited by the reduced leaf area of swards subjected to the severe grazing treatment, indicating
that under continuous stocking growth seems to be sustained by current assimilates instead of organic reserves.
Therefore, targets of grazing management for maximizing herbage accumulation throughout the year should
provide adequate combinations between quantity and quality of sward leaf area. This condition was obtained
in the severe/moderate and moderate grazing intensities, and corresponded to sward heights between 20 and
30 cm for marandu palisade grass.

INTRODUCTION underground and ground-level organs, which

include roots, rhizomes, stolons, stem bases and
Plant organic reserves are nitrogen (N) and carbon (C)
crown (Volenec et al. 1996; Avice et al. 2001),
compounds elaborated and stored in permanent
although they may also be stored temporarily in all
organs of the plant, mainly those remaining after
parts of the plant (White 1973; Perry & Moser 1974).
herbage removal, and used as substrates for mainten-
In legumes such as alfalfa, C and N compounds are
ance during periods of stress and early re-growth of
stored and remobilized from crown and taproot to
tissues after defoliation (White 1973; Perry & Moser
support shoot growth (Avice et al. 2001). In perennial
1974). Those compounds are generally stored in
grass tillers, non-structural carbohydrates (NSC)
* To whom all correspondence should be addressed. Email: silada-
located in the lower region of the shoots are utilized
silva@usp.br as an energy source to initiate growth after defoliation
2 S. C. Da Silva et al.
until the minimum leaf area required to produce
enough photosynthates to sustain plant respiration
and growth is achieved (White 1973). In this sense,
the contribution of each storage organ to shoot re-
growth is reported to differ among species, strategies
of grazing management and seasonal climatic con-
ditions (White 1973; Perry & Moser 1974).
The ability of plants to use internal stores of C and N
to quickly re-establish photosynthetically active leaf
area and restore assimilate supply to meet the
demand of the remaining organs is one of the key
factors affecting plant survival during early re-growth
(Volenec et al. 1996). Although the importance of
NSC as an energy reserve during re-growth of peren-
nial temperate grasses has been established previously
(Hume 1991; Donaghy & Fulkerson 1998), there is
some debate regarding the relative contribution of
NSC and N reserves to plant growth (Mousel et al.
2005). The mobilization of reserve compounds may
be limited more by N than C stores, since N uptake
can be severely reduced or even interrupted for
several days after defoliation (Clement et al. 1978;
Jarvis & MacDuff 1989). According to Gloser et al.
(2007), N reserves can support re-growth of plants
after defoliation even under fluctuating external N
availabilities. In contrast, Turner et al. (2006b)
pointed out that N reserves were found to play a
minor role during re-growth of cocksfoot (Dactylis glo-
merata L.), indicating that the reliance on N as a plant
reserve has not been conclusively established.
Although they are used as substrates in respiration,
N reserves are still not as important as carbohydrate
reserves in supporting re-growth (White 1973).
However, Ourry et al. (1988, 1989) and Gloser et al.
(2007) concluded that N compounds are important
components interfering with restoration of sward leaf
area index (LAI) and growth.
Strategies of grazing management have been devel-
oped aiming at controlling timing, severity, frequency
and selectivity of defoliation to obtain optimal or
desired responses of the vegetation and the grazing
animal (e.g. rotational stocking), and considered feas-
ible only if they cause minimal impact on plant vigour
(Turner et al. 2007). Under those circumstances, plant
vigour has often been related to the level of available
carbohydrates and N reserves stored above and below
ground (Reece et al. 1997). Bell & Ritchie (1989) and
Fulkerson & Donaghy (2001) indicated that defolia-
tion interval is generally more important than defolia-
tion severity in determining re-growth. In cocksfoot,
Turner et al. (2006b) found that more frequent
defoliation resulted in reduced NSC assimilation
and, therefore, reduced leaf, root and tiller dry
weight during re-growth. On the other hand, defolia-
tion severity may interfere with plant re-growth and
persistence (Fulkerson & Slack 1995) by interfering
with NSC levels in plant stores. A decrease in NSC
levels with low cutting heights was observed on vege-
tative tillers of prairie and orchard grass by Turner
et al. (2007), highlighting the importance of determin-
ing minimum defoliation heights below which restor-
ation of photosynthetic tissues and growth is impaired
and persistence decreased. However, under rigid con-
tinuous stocking management, relatively stable LAI is
maintained and the physiological role of organic
reserves may be questioned, since plants would be
capable of sustaining a continuous flux of current
assimilates. In that context, organic reserves could
be important only when swards are maintained
below a minimum LAI and/or when plants are sub-
jected to limiting growth conditions. These are
questions fundamentally related to plant physiology
and would need adequately controlled experiments
to be answered. In the current literature there is
virtually no information on carbohydrate and N
reserves of forage plants subjected to continuous
stocking management, particularly for tropical
forage grasses.
Brazil has c. 196 million ha of permanent pasture
(FAO 2011), of which c. 0·56 are comprised of culti-
vated species (IBGE 1996), with marandu palisade
grass (Brachiaria brizantha (Hochst. ex A. Rich.)
cvar Marandu) representing more than 0·50 of the
total (Santos-Filho 1996) and normally used under
continuous stocking management. In spite of the
importance of marandu palisade grass to pastoral
systems of animal production, basic information
regarding its morphological and physiological
responses to grazing is scarce, particularly those
related to the relationship between plant organic
reserves (NSC and N compounds) and sward LAI
and its influence on plant growth and herbage
accumulation. Against that background, and
bearing in mind the limitations of a field experiment
to deal with this issue, the objective of the present
study was to determine a grazing management
target for continuously stocked marandu palisade
grass that ensures herbage production without
depleting plant organic reserves through evaluations
of the patterns of variation in LAI, herbage accumu-
lation and carbohydrate and N reserves in shoot
and root throughout the year.
 C and N reserves in marandu palisade grass 3

Table 1. Monthly solar radiation (SR), rainfall (RF) and averages of maximum, minimum and mean air temp-
eratures on the experimental site from November 2001 to December 2002

Temperatures (°C)
SR RF
Month/year (MJ/m2 per day) (mm) Maximum Minimum Mean

Nov 2001 20·1 152·4 30·7 18·7 24·7

Dec 2001 19·2 204·2 29·2 18·7 24·0
Jan 2002 17·9 320·2 29·8 19·3 24·6
Feb 2002 17·7 187·9 29·0 18·8 23·9
Mar 2002 20·3 274·6 32·0 19·5 25·8
Apr 2002 18·5 27·2 31·8 17·6 24·7
May 2002 13·0 112·4 26·8 14·5 20·7
Jun 2002 13·5 0·0 27·9 12·6 20·3
Jul 2002 12·0 23·4 25·2 10·2 17·7
Aug 2002 14·4 79·6 28·9 14·3 21·6
Sep 2002 16·0 44·8 27·5 13·6 20·6
Oct 2002 20·2 49·4 33·8 18·8 26·3
Nov 2002 19·5 176·5 30·6 18·8 24·7
Dec 2002 20·7 166·1 31·2 19·7 25·4

MATERIALS AND METHODS (30:00:20); 115 kg N/ha on 7 March 2002, and 45

Experimental site
The experiment was conducted at E.S.A. ‘Luiz de
Queiroz’ (ESALQ), University of São Paulo,
Piracicaba, SP, Brazil (22°42’S, 47°37’W and 550 m
a.s.l.), on a marandu palisade grass pasture
(Brachiaria brizantha (Hochst. ex A. Rich.) Stapf.
cvar Marandu) established in 2001 on a Eutroferric
Red Nitosol. The average soil chemical characteristics
for the 0–20 cm layer were: pH CaCl2: 5·6; organic
matter (OM) = 41 dg/dm3; phosphorus (P) (ion-
exchange resin extraction method) = 67 mg/dm3;
calcium (Ca) = 74 mmolc/dm3; magnesium (Mg) = 19
mmolc/dm3; potassium (K) = 6·5 mmolc/dm3; hydro-
gen (H)+aluminium (Al) = 36 mmolc/dm3; sum of
bases = 99 mmolc/dm3; cation exchange capacity =
134·8 mmolc/dm3; base saturation = 74%. Climate is
sub-tropical with dry winters, with an average
annual rainfall of 1328 mm (CEPAGRI 2012).
Rainfall, solar radiation and average mean,
minimum and maximum temperatures are shown in
Table 1 and soil water balance in Fig. 1. A total of
301 kg N/ha and 42 kg K/ha were applied throughout
the experimental period according to the following
dates and rates: 66 kg N/ha on 7 November 2001,
using ammonium nitrate; 30 kg N/ha and 17 kg K/ha
on 1 December 2001; 45 kg N/ha and 25 kg K/ha on
10 January 2002, using a commercial N:P:K formula
kg N/ha on 30 October 2002, using urea.
Treatments corresponded to four levels of grazing
intensity – severe (S), severe/moderate (S/M), moder-
ate (M) and lenient (L) – implemented in the field
using bands of sward surface height (SSH – 10, 20,
30 and 40 cm ± 10%, respectively) maintained
through continuous stocking and variable stocking
rate. These were allocated to experimental units
(1200 m2 paddocks) according to a randomized com-
plete block design with four replications. Grazing was
carried out by 2-year-old Nelore (Bos taurus indicus)
and Canchim (5/8 Charolais (Bos taurus taurus) × 3/8
Zebu (Bos taurus indicus)) heifers with an initial
body weight of 280–320 kg. The experimental
period started on 8 January and finished on 17
December 2002, totalling 343 days.
Prior to the beginning of measurements, on 28 and
29 August 2001, paddocks were grazed and sub-
sequently mowed to c. 8 cm in preparation for the
experiment. All paddocks had animals grazing at the
end of October 2001, ensuring a minimum period of
60 days under the grazing regimes imposed before
the commencement of measurements on 8 January
2002. Sward height was monitored twice a week (3
and 4 day-intervals) on 20 points per paddock using
a thin acetate sheet and ruler (Fagundes et al. 1999).
Average sward height on paddocks was allowed to
vary within a range of 0·1 around the target, with
4 S. C. Da Silva et al.
Fig. 1. Monthly soil water balance (calculated at 10-day intervals considering a soil water storage capacity of 50 mm) from
November 2001 to December 2002 (arrows correspond to fertilizer application dates and rates).
animals being added or removed when the sward
height was close to the upper or lower end of the
range, respectively.
Measurements
Herbage accumulation, herbage mass and sward leaf
area index
Herbage accumulation was determined within four
exclosure cages (1·40 × 0·70 m) per experimental
unit at 28-day intervals. After each sampling, cages
were rotated and anchored on new areas that were
considered to be representative of sward condition
at the time of sampling (visual assessment of height
and herbage mass). Herbage accumulation was calcu-
lated as the difference in herbage mass in the cage
between the first and last day of exclusion (Davies
et al. 1993). These were determined using calibration
curves between sward height and herbage mass, pro-
duced in a concomitant set of measurements being
made in the same experimental area (Da Silva et al.
2013) following the procedure described by Da Silva
& Cunha (2003). Estimates of sward herbage mass
were made on a monthly basis using data from the
measurements of sward height and the calibration
equations.
Leaf area index was determined every 4 weeks for
herbage samples collected within four metallic
frames (0·30 × 0·37 m) located in areas representative
of sward condition at the time of sampling (visual
assessment of height and herbage mass). Cuts
were made at ground level and a sub-sample was
hand-dissected into leaf (leaf laminae), stem (leaf
sheath + stem), dead material and weeds. The leaves
had their area estimated using the LAI-3100 leaf area
integrator (LICOR, Lincoln, Nebraska, USA). After sep-
aration, the components were dried in a forced
draught oven at 65 °C until constant weight and the
results used to calculate LAI.
Additional herbage samples were harvested from
the upper layer of the swards (mainly comprised of
leaves – Da Silva et al. 2013) every month, dried in
a forced draught oven at 65 °C until constant weight,
ground using 1 mm sieve and stored for N content
determination and inferences on the N nutrition
status of plants (Lemaire et al. 2008). Total N (TN)
was determined using the volumetric method of
micro Kjeldhal (AOAC 1995).
Root and shoot mass and organic reserves
Root and shoot samples were harvested every 4 weeks
using a cylinder with 15-cm internal diameter placed
onto the plant’s crown and introduced into the soil to a
depth of c. 20 cm. Six cylinders were harvested per
experimental unit from points representative of
sward condition at the time of sampling (visual assess-
ment of height and herbage mass). The resulting cylin-
ders (soil containing roots and herbage mass above
ground) were taken immediately to the laboratory,
where their depth was standardized to 10 cm from
ground level by removal and disposal of excess soil
and roots. The above-ground herbage was cut at
5 cm from ground level, leaving only the base of the

stems (from this point onwards denominated shoot)
and discarding the excess material. From the standar-
dized cylinders, the base of stems was cut at ground
level to form the shoot samples. Because of variations
in the contents of reserve carbohydrate and nitrogen-
ous compounds in storage organs throughout the
day, samples were consistently harvested between
06:00 and 10:00 h.
Shoot samples were washed using running water,
divided into two sub-samples and placed on alu-
minium trays, which were put aside to dry; one
under shade and the other in a forced draught oven
at 100 °C during the first hour (in order to stop respir-
ation and enzymatic processes) and at 65 °C after-
wards until constant weight. The remaining part of
the cylinder, containing soil and roots, was washed
on 300 mm sieves using pressurized water in order
to separate roots from dirt. After washing, roots were
also divided up into two sub-samples and placed on
aluminium trays, which followed the same drying pro-
cedure described above for shoot samples. Root and
shoot mass were calculated using the diameter of
the sampling cylinder as reference.
After drying, shoot and root samples were weighed,
ground using a 1 mm sieve and stored for chemical
analysis. Non-structural carbohydrates were deter-
mined using acid digestion of samples by sulphuric
acid and precipitation of carbohydrates by copper sul-
phate (Smith 1969 modified by Silva 1981). Total N
was determined using the volumetric method of
micro Kjeldhal (AOAC 1995). Soluble N was deter-
mined indirectly as the difference between total N
and insoluble N. Insoluble N content was determined
after washing of samples to eliminate soluble N, using
the same procedure described above for total N.
Data processing and statistical analysis
Data were collected monthly but results were pooled
into seasons of the year as follows: summer (January to
March), autumn (April to June), winter (July and
August), early spring (September and October) and
late spring (November and December). Grouping
using 2-month data from July onwards was done as
a means of separating September and October into a
single group, since the pattern of variation in
herbage accumulation rates in those months was
very distinct. Analysis of variance was carried out on
the grouped data using the Mixed Procedure of
SAS® (SAS Inst., Cary, NC, USA) and the restricted
maximum likelihood (REML) method. For the data
C and N reserves in marandu palisade grass 5
set comprised of sward structural characteristics –
LAI, herbage mass and herbage accumulation rates –
season of the year was analysed as repeated
measure and the correlation/covariance matrices
tested were: compound symmetric (CS), first-order
autoregressive (AR(1)), heterogeneous first-order auto-
regressive (ARH(1)), first-order autoregressive moving-
average (ARMA(1,1)) unstructured (UN), Huynh-Feldt
(HF), toeplitz (TOEP), heterogeneous toeplitz (TOEPH)
and standard variance components (VC). The model
used considered grazing intensity, blocks and season
of the year as variation causes. For the dataset com-
prised of organic reserves – NSC and total and
soluble N (concentration and content) – season of
the year and plant compartment (root and shoot)
were analysed as doubly repeated measures (Piepho
et al. 2004) and the correlation/covariance matrices
tested were: UN@AR(1), UN@CS and UN@UN. In
this case the model used considered grazing intensity,
blocks, plant compartment and season of the year as
variation causes. The subject was defined by nesting
the treatment factor into blocks. The choice of the cor-
relation/covariance matrix was made using the
Schwarz’s Bayesian Criterion (SBC or BIC in SAS)
(Littell et al. 2000). The correction for degrees of
freedom was made by the Kenward & Roger (1997)
method (SAS command: DDFM = KR) and the analysis
performed considering grazing intensity, plant com-
partment (root and shoot), season of the year and
their interactions and blocks as fixed effects (Piepho
et al. 2004). The SLICE command was used in cases
of significant interactions and, when appropriate,
means were calculated using the ‘LSMEANS’ state-
ment and comparisons made using the Student test
at P < 0·05.
RESULTS
A summary of the statistics and the results from the
analysis performed in the data set is presented in
Table 2. Additionally, information is given regarding
the choice and type of correlation/covariance
matrices used for each response variable studied.
Sward herbage mass and nitrogen content of the
upper layer
Throughout the experiment, sward herbage mass
progressively decreased as grazing intensity (GI)
increased. For the severe grazing treatment, herbage
mass was higher during summer and early spring,
6 S. C. Da Silva et al.

with lower similar values during autumn, winter and

P < 0·001

P < 0·001

P < 0·001
Table 2. Summary of the statistical analysis, matrices used for modelling correlation/covariance structures and values of the Schwarz’s Bayesian Criterion

UN@UN
P < 0·05

−345·7
late spring. For swards subjected to the severe/moder-

NSC

NS

NS

NS
ate grazing treatment, the highest and lowest values

(kg/m2)
were recorded during early and late spring, respect-
Soluble N ively, while intermediate similar values were recorded

UN@UN
during the remaining seasons of the year. For swards

P < 0·01
P < 0·01

P < 0·01
P < 0·05
P < 0·05
P < 0·05

492·5
subjected to the moderate and lenient grazing treat-

NS
ments, herbage mass increased from summer to
early spring, decreasing during late spring when the
lowest values were recorded (Table 3).
P < 0·001

P < 0·001

UN@UN
P < 0·01

P < 0·05

P < 0·01
The N concentration of the upper layer was higher

542·9
NSC

NS during late spring and summer, lower during winter

NS
and early spring and intermediate during autumn
(24·0, 18·7, 14·7, 14·9 and 20·4 ± 0·49 g/kg for
(g/kg of DM)

UN@AR(1) summer, autumn, winter, early and late spring,

Soluble N

P < 0·001

P < 0·001
P < 0·05

P < 0·05

P < 0·01

357·0 respectively) (Fig. 2). The N concentration of the

severe grazing treatment was similar to that of the
NS

NS

severe/moderate grazing treatment, but higher than

that of the moderate and lenient grazing treatments
P < 0·001

P < 0·001

P < 0·001

UN@UN

(20·4, 18·6, 18·1 and 17·3 ± 0·64 g/kg for S, S/M, M

P < 0·05
Total N

−448·9

and L treatments, respectively).

NS

NS

NS

Sward leaf area index and rates of herbage

(kg DM/ha/day)

accumulation
P < 0·001
P < 0·001

Sward LAI was larger for swards subjected to the

ARH(1)

lenient grazing treatment throughout the year, with

485·3
HAR

NA
NA
NA
NA
NS

similar values recorded for those subjected to the

moderate grazing treatment during summer and
early spring. The largest values were recorded in
(t DM/ha)

P < 0·001
P < 0·001
P < 0·001

summer for swards subjected to the severe/moderate,

TOEP
43·2

moderate and lenient grazing treatments, and

HM

NA
NA
NA
NA

decreased progressively until early spring, when the

lowest values were recorded. For swards subjected
to the severe grazing treatment, the largest values
N Up layer

P < 0·001
P < 0·05

were recorded in summer and winter, followed by

270·6

those recorded in autumn, early spring and late

NA
NA
NA
NA
NS

CS

spring. The LAI started to increase in late spring,

although recorded values were still lower than those
NS, not significant; NA, not applicable.

recorded in summer. It increased by 57 and 54%

P < 0·001
P < 0·001
P < 0·001

ARH(1)

from early to late spring for swards subjected to

146·1
LAI

NA
NA
NA
NA

lenient and moderate grazing treatments, contrasting

(SBC) used to choose them

with only 6·6 and 13·1% for those subjected to the

severe/moderate and severe grazing treatments
Plant compartment (PC)

(Table 3).
Grazing intensity (GI)

A seasonal effect was observed on rates of herbage

accumulation (HAR) for all intensities of grazing, with
larger values recorded during summer and late spring
PC × GI × S

SBC value
Season (S)

and lower values recorded during winter and early

PC × GI

Matrix
PC × S
GI × S

spring. There was no difference among GI treatments

during summer. For autumn, winter and early spring,
 C and N reserves in marandu palisade grass 7

Table 3. Leaf area index, sward herbage mass (t DM/ha) and herbage accumulation rate (kg DM/ha per day) of
marandu palisade grass subjected to intensities of continuous stocking management throughout the year. S.E.D.,
standard error of difference; degrees of freedom for the grazing intensity x season of the year interaction = 12 for
all variables; n = 4

Grazing intensity

Season of the year Severe Severe/moderate Moderate Lenient S.E.D.

LAI
Summer 2·62 5·11 7·19 8·33 0·733
Autumn 1·47 3·11 4·90 6·72 0·396
Winter 2·24 3·11 4·90 6·72 0·415
Early spring 1·22 1·97 2·19 2·76 0·309
Late spring 1·38 2·10 3·37 4·34 0·408
Sward herbage mass
Summer 6·51 8·44 10·17 11·87 0·179
Autumn 5·96 8·39 10·80 12·62 0·179
Winter 5·88 8·20 10·78 12·80 0·179
Early spring 6·32 9·00 11·22 13·07 0·179
Late spring 5·97 7·88 9·74 11·70 0·179
Herbage accumulation rate
Summer 127·6 141·7 133·2 136·6 17·05
Autumn 64·7 67·4 46·1 16·1 12·72
Winter 14·8 10·0 −2·0 −16·4 4·63
Early spring 38·0 24·4 8·0 −12·4 4·13
Late spring 88·4 92·3 157·6 155·6 17·08

lower values were recorded for swards subjected to
the lenient grazing treatment. However, swards sub-
jected to the moderate and lenient grazing treatments
showed larger values than those subjected to the
severe and severe/moderate grazing treatments
during late spring (Table 3).
Concentration of nitrogen compounds
Total N concentration for the whole plant was higher
for swards subjected to the severe grazing treatment
(6·4 ± 0·29 mg/g DM), with no differences observed
among severe/moderate, moderate and lenient
grazing treatments (5·2 ± 0·29, 5·5 ± 0·29 and 5·6 ±
0·29 mg/g DM to severe/moderate, moderate and
lenient, respectively). The highest values of TN con-
centration in roots (TNR) were recorded in winter
and early spring, decreasing 11·2% in late spring.
Total N concentration in shoot (TNS) increased from
summer to early spring, when the highest value was
recorded (7·8 ± 0·25 mg/g), and decreased by around
25% in late spring. Higher values of TN were observed
in shoot during autumn, early and late spring, with
similar values between plant compartments during
the remaining seasons of the year (Fig. 3(a)).
The concentration of soluble nitrogen (SN) was
slightly higher in the root than in the shoot compart-
ment for all seasons of the year, except for swards sub-
jected to the severe grazing treatment during winter.
Similar concentrations of SN in root (SNR) among GI
treatments were observed during summer, winter
and late spring. During autumn and early spring differ-
ences were observed only between swards subjected
to the severe and lenient grazing treatments, with
higher values recorded for the former. The values of
SNR in swards subjected to the severe grazing treat-
ment decreased from summer to winter, but increased
91·7% in early spring. In this season, the highest
values of SNR were observed for all GI treatments.
From early to late spring, values decreased by c. 20
and 30% in swards subjected to the severe and mod-
erate grazing treatments, respectively, but remained
stable for the severe/moderate and lenient grazing
treatments.
There was no difference in the concentration of SN
in shoot (SNS) among GI treatments during summer
8 S. C. Da Silva et al.
Fig. 2. Nitrogen concentration (g/kg of DM) of the upper layer of marandu palisade grass swards subjected to intensities of
continuous stocking management throughout the seasons of year. Dotted lines represent N critical level from 14·4 to 22·0
g/kg (Batista & Monteiro 2007). Bars represent S.E.M., n = 4.
(average of 2·0 ± 0·40 mg/g) and autumn (average of
2·5 ± 0·33 mg/g), but a clear contrast between the
severe and severe/moderate grazing with the lenient
grazing treatment was recorded in winter, early and
late spring, with lower values recorded for swards sub-
jected to the lenient grazing treatment (Table 4).
Values of SNS increased from summer to early spring
for swards subjected to the severe and severe/moder-
ate grazing treatments, and decreased by c. 24% in
late spring on those subjected to the severe grazing
treatment. For the moderate grazing treatment,
higher values were observed during early spring com-
pared to autumn and late spring. No differences were
registered during summer, autumn, winter and early
spring for the lenient grazing treatment, but values
decreased by 55·9% in late spring.
Concentration of non-structural carbohydrates
The concentration of NSC was higher in the root
than in the shoot compartment from summer to
early spring, with similar values between compartments
during late spring (Fig. 3(b)). Higher values were
observed during winter for the root, and winter and
early spring for the shoot compartment. For both com-
partments, lower values were recorded during summer
and autumn. During summer and autumn, lower
values were recorded for swards subjected to the
severe grazing treatment. There were no differences
among GI treatments (Table 5) during winter
(157 ± 4·1 mg/g), early (142 ± 4·0 mg/g) and late
spring (107 ± 3·3 mg/g).
Pools of non-structural carbohydrates and soluble
nitrogen
The pool of NSC (kg/m2) in the root compartment was
similar among the GI treatments. On the other hand, in
the shoot compartment larger values were recorded
for swards subjected to the severe compared to
those subjected to the moderate and lenient grazing
treatments. The pool of NSC was larger in the
shoot than in the root compartment regardless of GI
treatments and season of the year (Fig. 4(a) and (b)).
In relation to the seasons of the year, the pools of
NSC were 36·6, 78·6, 108·4, 123·7 and 103·9%
larger for the shoot relative to the root compartment
in summer, autumn, winter, early and late spring,
respectively. In relation to the grazing intensities, the
pools of NSC were 147·3, 99·0, 71·6 and 72·1%
larger for the shoot relative to the root compartment
for the severe, severe/moderate, moderate and
lenient grazing treatments, respectively. For both

Fig. 3. Concentration of total N (a) and NSC (b) of marandu
palisade grass subjected to intensities of continuous stocking
management throughout the seasons of year. Bars represent
S.E.M., n = 16.
compartments, the values increased from summer to
winter, remaining stable until late spring.
The pool of soluble N (kg/m2) in the root compart-
ment was similar among the GI treatments during
summer, winter and early spring. In autumn and late
spring, larger values were recorded for swards sub-
jected to the severe compared to those subjected to
the lenient grazing treatment. Larger values were
recorded during early spring for the severe and
severe/moderate grazing treatments. There was no
difference among seasons of the year for the moderate
grazing treatment. For the lenient grazing treatment,
differences were observed only in autumn and early
spring, with lower values for the former (Fig. 5(a)).
The pool of soluble N in the shoot compartment was
similar among the GI treatments during summer
only. For autumn, winter and late spring larger
values were recorded for the severe relative to the
lenient grazing treatment, and in early spring larger
values were recorded for the severe/moderate relative
C and N reserves in marandu palisade grass 9
to the lenient grazing treatment. For swards subjected to
the severe and severe/moderate grazing treatments,
values increased from summer to winter, remaining
stable from then onwards. For swards subjected to the
moderate grazing treatment, the lowest value was
recorded during summer, with no differences among
the remaining seasons of the year. There was no differ-
ence among seasons of the year for swards subjected to
the lenient grazing treatment (Fig. 5(b)). The pool of
soluble N was larger in the shoot than in the root com-
partment for the severe, severe/moderate and moderate
grazing treatments, with differences between plant
compartments increasing as intensity of grazing
increased. For the lenient grazing treatment, larger
values were recorded for the shoot compartment
during autumn and winter and for the root compart-
ment during summer and late spring. In early spring
there was no difference between plant compartments.
DISCUSSION
Under intermittent stocking, forage plants have a
well-known temporal pattern of mobilization and util-
ization of organic reserves, and the critical role of
carbohydrates and N reserves from root and shoot as
substrates for re-growth was advocated long ago
(Morvan-Bertrand et al. 1999). For such a grazing
method, the severity of defoliation affects root
growth although the effect may be compensated by
increased N uptake per unit of root weight in certain
species (Thornton & Millard 1997). For Lolium
perenne L., increasing defoliation severity increased
the relative contribution of roots in supplying mobilized
N to growing leaves and decreased the relative contri-
bution of adult leaves (Lestienne et al. 2006). Turner
et al. (2006a) demonstrated that frequent defoliation
of cocksfoot (Dactylis glomerata L.) resulted in
reduced water-soluble carbohydrate assimilation and,
therefore, leaf, root and tiller dry matter accumulation
during subsequent periods of re-growth. However,
under continuous stocking the relative importance of
storage organs (roots and shoot) and compounds (C or
N) to the supply of assimilates and maintenance of
growth for contrasting intensities of defoliation (targets
of grazing management) are not clearly described in
the literature, particularly for tropical forage species.
According to White (1973), the major storage areas
of carbohydrate reserves in perennial grasses are
usually the lower regions of the stems (stem bases),
stolons, crowns and rhizomes. However, Perry &
Moser (1974) demonstrated that the relative
10 S. C. Da Silva et al.

Table 4. Concentration of SN (mg/g DM) of root and shoot in marandu palisade grass subjected to intensities of
continuous stocking management throughout the year. S.E.D., standard error of difference; degrees of freedom for
the grazing intensity x season of the year x plant compartment interaction = 12; n = 4

Grazing intensity

Season of the year Severe Severe/moderate Moderate Lenient S.E.D.

Root
Summer 4·0 3·8 2·5 1·8 1·14
Autumn 4·5 3·6 3·0 1·9 0·93
Winter 2·9 3·3 2·8 2·6 0·81
Early spring 5·5 5·0 4·2 3·4 0·93
Late spring 4·4 4·4 2·9 2·7 1·14
Shoot
Summer 2·2 2·4 2·3 1·1 1·14
Autumn 3·1 2·7 2·6 1·4 0·93
Winter 3·7 3·2 2·8 1·4 0·81
Early spring 4·7 3·9 3·2 1·7 0·93
Late spring 3·6 3·3 1·8 0·8 1·14

Table 5. Concentration of NSC (mg/g DM) of marandu palisade grass subjected to intensities of continuous
stocking management throughout the seasons of year. S.E.D., standard error of difference; degrees of freedom for
the grazing intensity × season of the year interaction = 12; n = 8

Grazing intensity

Season of the year Severe Severe/moderate Moderate Lenient S.E.D.

Summer 53 71 86 98 6·7
Autumn 59 85 88 96 7·6
Winter 153 154 151 170 11·5
Early spring 151 136 136 147 11·3
Late spring 107 98 108 113 9·4

importance of plant parts as storage organs vary with
plant species in relation to both NSC concentration
and organ size. The results from the present study
show that although the NSC (Fig. 3) and soluble N
(Table 4) concentration were higher in the root com-
partment, the shoot compartment represented the
major pool of NSC and soluble N regardless of the
grazing intensities used (Figs 4 and 5).
Under intermittent stocking, the shoot includes fully
expanded leaf material (mainly leaf sheaths) as well as
the (enclosed) basal, immature parts of expanding
leaves and leaf primordia at the apex of the tiller
base. The former may serve as a source for mobilized
C and N, whereas the latter generate the new foliage,
and may act as sinks (Schnyder & De Visser 1999). In
that context, the role of shoot as storage organ
decreases as defoliation severity increases (Lestienne
et al. 2006), with initial growth becoming more
dependent on reserves mobilized from roots. Based
on the available information in the literature for
forage plants under intermittent stocking, it was
expected that increases in grazing intensity should
result in increasing importance of the root compart-
ment as reserve supplier. However, since under con-
tinuous stocking a relatively constant proportion of
the sward leaf area is removed (Mazzanti & Lemaire
1994), the remaining leaf area may be sufficient for
supplying assimilates. This is in line with the results
of Carvalho et al. (2001) for cultivars of Cynodon sp.
managed under continuous stocking at 5, 10, 15 and
20 cm using a similar protocol to the one used in the
current experiment: their results showed larger

Fig. 4. Pool of NSC in shoot and roots of marandu palisade
grass subjected to intensities of continuous stocking
management throughout (a) according grazing intensities
(n = 8). Bars represent S.E.M. and (b) the seasons of year
(n = 16)
depletion of NSC pool in shoot during periods of
active plant growth (November to March), while the
NSC pool in roots was not affected by the grazing
intensities used, only by season of the year.
The LAI was lower on swards subjected to the more
intense grazing regimes (severe and severe/moderate),
but there was no difference among GI treatments in
relation to the pool of NSC in the root compartment.
In addition, similar values to the pool of soluble N in
the root were verified during summer, winter and
early spring (Fig. 5(a)). On the other hand, the pool
of NSC in shoot was larger on swards subjected to
the severe and severe/moderate grazing treatments
relative to those subjected to the moderate and
lenient grazing treatments (Fig. 4(b)). Sbrissia et al.
(2010), in a concomitant experiment in the same
experimental area, reported higher rates of tiller
appearance and larger tiller population density for
swards subjected to the severe and severe/moderate
grazing treatments. This indicates that under severe
C and N reserves in marandu palisade grass 11
Fig. 5. Pool of SN of marandu palisade grass subjected to
intensities of continuous stocking management throughout
the seasons of year. (a) SN of root, and (b) SN of shoot.
Bars represent S.E.M., n = 4.
grazing, swards were capable of compensating the
reduced leaf area through a larger population of
younger tillers and, probably, higher photosynthetic
efficiency. The integration of results from the current
experiment and those from Sbrissia et al. (2010)
suggests that under continuous stocking and no soil
fertility limiting conditions, herbage accumulation is
sustained mainly by current assimilates produced by
the shoot rather than organic reserves mobilized
from the root, a condition in which the quality of the
sward leaf area is crucial for maintaining plant
growth. This hypothesis is corroborated by the larger
pool of SN in the root compartment of swards sub-
jected to the severe grazing treatment relative to
those subjected to the lenient grazing treatment in
autumn and late spring, and in the shoot compartment
of swards subjected to the severe and severe/moderate
grazing treatments from autumn until late spring.
The accumulation of organic reserves in plants is
seasonal, increasing during autumn and early winter
and rapidly declining with the beginning of the new
growing season in spring (Corre et al. 1996). During
12 S. C. Da Silva et al.
summer, the concentration of NSC was lower on
swards subjected to the severe and severe/moderate
grazing relative to those subjected to the moderate
and lenient grazing treatments. However, they were
capable of restoring NSC levels during autumn and
winter, with no differences among GI treatments
during winter, early and late spring. A similar seasonal
pattern of response was reported by Carvalho et al.
(2001) in an analogous experiment with Cynodon
sp. subjected to intensities of continuous stocking
management.
The N concentration of the upper layer of swards
decreased from summer to early spring. Although
the decrease was observed for all GI treatments,
values were smaller than 14·4 g/kg, the critical level
for N nutrition of marandu palisade grass (Batista &
Monteiro 2007), on swards subjected to the moderate
and lenient grazing treatments during winter and early
spring (Fig. 2). As the plant grows and herbage mass
increases, the N concentration in plant tissues
decreases, even when there is an unlimited supply
of N (Lemaire et al. 2008). Such a decrease is the
result of larger deposition of structural tissues than
necessary to support plant weight and architecture,
which have low N concentration. This is in agreement
with the observed variations in concentration of SN in
shoot among the GI treatments, according to which
swards subjected to the severe and severe/moderate
grazing treatments showed higher values during
winter, early and late spring relative to those subjected
to the lenient grazing treatment. Limiting climatic con-
ditions, particularly soil water deficit, interfere nega-
tively with N uptake and mineralization of soil
organic matter by reducing microbial activity. In that
context, since the supply of N is limited, internal recy-
cling may become the main source of nutrients to
plants. Under those conditions, proteins and free
amino acids may be used as reserve compounds.
Considering that the largest proportion of N is immobi-
lized as structural components due to the higher
herbage mass, and given the limited N supply at that
time of the year, the negative impact on plant N nutri-
tion was larger on swards subjected to the lenient
grazing treatment. This is in line with the results of
herbage accumulation, since recorded values were
lowest during winter and early spring and even nega-
tive for swards subjected to the moderate and lenient
grazing treatments. In late spring, LAI and HAR
increased relatively more on swards subjected to the
moderate and lenient grazing relative to those sub-
jected to the severe and severe/moderate grazing
treatments, demonstrating recovery of their growth
potential when growth conditions were restored and
the N was supplied via fertilization (45 kg N/ha in
30 October 2002).
On the other hand, since plants were not stimulated
to grow in winter and early spring, carbohydrates were
concentrated in plant tissues, resulting in the highest
recorded values of NSC concentration throughout
the experiment (Fig. 3(b)). Under those conditions,
plant growth was favoured on swards subjected to
the severe and severe/moderate grazing treatments,
since they showed the highest values of HAR.
Swards subjected to the moderate and lenient
grazing treatments had larger LAI than those subjected
to the severe grazing treatment, a condition that, in
theory, would favour photosynthesis and growth
(Booysen & Nelson 1975; Grant et al. 1981).
However, during winter and early spring the low
values of HAR of swards subjected to the moderate
and lenient grazing treatments indicate that leaves
were generally old and less photosynthetically
active, therefore unlikely to contribute substantially
to growth (Gifford & Marshall 1973; Woledge 1977;
Gay & Thomas 1995). Generally, when management
of continuously stocked swards is not adequate,
canopy photosynthesis is reduced because of
reduced photosynthetic efficiency of old leaves
(Parsons 1988; Hernandez-Garay et al. 2000) and
accumulation of stem and senescent material in
sward herbage mass.
Marandu palisade grass is known for presenting
very slow growth during the transition from winter to
early spring. Although SN concentration increased
from winter to early spring in both compartments,
the concentration of NSC in the root compartment
decreased by c. 12·5%. A larger reduction in the con-
centration of NSC was recorded for the lenient grazing
treatment (Table 5). The decrease in NSC concen-
tration was the result of the reduction in the NSC
pool mainly in root (which decreased by 11·7%) rela-
tive to shoot compartment (decrease of 5·3%). That
could be associated with mobilization of organic
reserves from the roots, including root death, a con-
dition in which the dependence of plants on the exter-
nal supply of nutrients (fertilization) would be greater
due to the reduction in root mass and exploited soil
volume.
The proportion of SN relative to TN in the shoot
compartment remained relatively stable throughout
the seasons of the year (values varying from 38 to
46%), but increased in the root compartment from

winter to early spring (59, 65, 50, 79 and 71% during
summer, autumn, winter, early and late spring). Free
amino acids have been identified as the major
storage form of N in roots. In the Euphorbiaceae
family, proteins also accumulate during the autumn
and winter months, although the pool of free amino
acids is larger (Bewley 2002). These may be the
more predominant source of N during the resumption
of spring growth, a time of intense turnover in the tiller
population (Sbrissia et al. 2010) as well as in roots, as
the result of the improving environmental conditions
and higher N mineralization. Under those conditions,
increases in soluble N in roots relative to TN may rep-
resent higher N uptake from the soil, highlighting the
importance of adequate fertilization programmes.
In late spring, clear contrasts for LAI and HAR were
recorded, particularly for the severe and lenient
grazing treatments. The results indicate that even
when maintaining higher soluble N concentration in
shoot, herbage accumulation was limited by the
reduced leaf area of the severe grazing treatments.
Therefore, under continuous stocking, targets of
grazing management that maximize herbage accumu-
lation throughout the year must ensure an adequate
balance between quantity and quality of sward leaf
area. This condition was obtained by the severe/mod-
erate and moderate grazing intensities, and corre-
sponded to management heights between 20 and
30 cm.
Thanks are due to Dr Gerson Barreto Mourão, from
University of São Paulo, for the valuable comments
and advices regarding the statistical analysis of the
data, to Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP) and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq)
for the financial support and to the research team at
USP/ESALQ, Brazil.
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