Leaf blade nutritional quality of rhodes grass (Chloris gayana) as affected by leaf age and length

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1 CSIRO PUBLISHING Crop & Pasture Science, 2011, 62, Leaf blade nutritional quality of rhodes grass (Chloris gayana) as affected by leaf age and length M. G. Agnusdei A,D, O. N. Di Marco B, F. R. Nenning C, and M. S. Aello B A Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria Balcarce, CC 276 (7620), Balcarce, Buenos Aires, Argentina. B Facultad de Ciencias Agriarias, Universidad Nacional de Mar del Plata, CC 276 (7620) Balcarce, Buenos Aires, Argentina. C Instituto Nacional de Tecnología Agropecuaria (INTA), Estación Experimental Agropecuaria El Colorado, Av. Carlos Pellegrini. D Corresponding author. magnusdei@balcarce.inta.gov.ar Abstract. A greenhouse experiment was performed to determine the effect of leaf age and length on neutral detergent fibre (NDF) and in vitro NDF digestibility (NDFD) during a vegetative regrowth of Chloris gayana. Dense micro swards were grown in two plots under non-limiting conditions of water, phosphorus and nitrogen. Plants were harvested at seven consecutive leaf appearance intervals. Leaf blades were dissected from individual tillers and separated into five age categories (from early growing to pre-senescence). Leaf blade and sheath length were measured and leaves of the same category were bulked for NDF and NDFD analysis. The leaf lifespan (LLS) was determined in 15 marked tillers per plot. Linear and curvilinear functions were used to describe the relationships between NDF and NDFD with leaf age and length. The NDF concentration increased until half of LLS, concomitantly with leaf expansion, and remained stable thereafter. However, NDFD declined curvilinearly through the complete LLS. The final length of consecutively formed leaves increased through regrowth, this change being associated with an increase in NDF and a decline in NDFD. Results were consistent with previous findings for temperate species and highlight the importance of leaf length, in addition to leaf age, to determine leaf blade digestibility. Additional keywords: forage quality, leaf turnover, rhodes grass, tropical grasses. Received 27 June 2011, accepted 28 November 2011, published online 10 February 2012 Introduction The process of herbage accumulation in vegetative grass swards involves a continuous turnover of leaves (Lemaire et al. 2009) and an increase in the proportion of structural tissues needed to support and position leaves in the uppermost canopy layers to favour the interception of light (Gastal and Lemaire 2002). Structural tissues are composed of complex arrays of cell walls that limit to a variable extent the attack of ruminal microbes, and consequently the digestibility of the cell walls (or neutral detergent fibre digestibility, NDFD) (Buxton and Fales 1994; Wilson 1994). The importance of NDFD on forage DM digestibility, intake and animal performance has been addressed by different authors (Akin and Chesson 1989; Wilson 1993; Buxton and Fales 1994; Van Soest 1994; Jung and Allen 1995). Small increases in NDFD have been associated quantitatively with significant increases in DM digestibility, feed intake and animal performance (Oba and Allen 1999; Mertens 2009). However, current knowledge is still incomplete to properly understand how the processes of herbage accumulation can affect fibre content and fibre digestibility at specific developmental stages during pasture growth. In this context, the principles of pasture management Journal compilation CSIRO 2011 based on the proper leaf stage to optimise forage production, quality and persistence advocated by Fulkerson and Donaghy (2001), which corresponds to a certain number of accumulated leaves during leaf lifespan (LLS, Lemaire and Chapman 1996), offer a sound analytical and practical framework to study the posed issue. The underlying mechanisms that govern changes in herbage quality during leaf development have been comprehensively studied in a few temperate forage grasses. In a study on Italian ryegrass during a vegetative regrowth Groot and Neuteboom (1997) found that the indigestibility of the NDF (the complement of the NDFD) of consecutive leaf blades increased during LLS and with the progressive enlargement of the sheath tube that occurs with biomass accumulation. Likewise, Duru and Ducrocq (2002) reported that the decline of in vitro DM digestibility (DMD) of orchardgrass (Dactylis glomerata) was associated with the increase of the sheath tube length. Preceding information clearly indicates that leaves of temperate species are neither static nor uniform in their nutritional properties, even in the vegetative stage, and that changes in quality can be expected in accordance with the rhythms of leaf turnover, the stage of leaf development and the changes in leaf length during regrowth.

2 Rhodes grass leaf quality Crop & Pasture Science 1099 Interestingly, results obtained in vegetative and reproductive crops of perennial ryegrass by Groot et al. (2003) revealed that rising temperature accelerated plant development rate (i.e. leaf appearance rate) and NDFD decline rate during aging to the same extent, while the different plant fractions (i.e. leaf blades, stems and internodes) showed a similar pattern of NDFD decline with developmental stage. These findings highlight the relevance of leaf appearance rate as a plant developmental indicator to monitor and control forage quality under a wide range of environmental conditions, genotypes and physiological stages. In comparison with temperate grasses, tropical grasses have thicker-walled vascular and sclerenchyma tissues, higher lignin content or more complex cross links of lignin precursors with carbohydrates of the cell wall (Wilson 1976a, 1976b; Wilson and Hattersley 1989). Thus, a larger proportion of NDF in DM and a faster decline in NDFD during regrowth can be expected. The present study was conducted to evaluate patterns of leaf blade NDF content and NDFD in relation to leaf age and length of individual leaves consecutively formed on vegetative tillers of Chloris gayana, a long-leaved tropical grass broadly used in pasture-based grazing systems in warm areas of the world. Leaf blades were used instead of whole leaves, since it is expected that they would have higher digestibility, DM intake and lower variations in quality than leaf sheaths over a wide range of growth stages (Nelson and Moser 1994).The methodology was designed to separate the effects of morphological changes of leaves during pasture regrowth from those inherent to aging. For this purpose, leaf blade quality of consecutively formed leaves was evaluated at comparable stages of leaf development, from early growing to pre-senescent leaves. Materials and methods Experimental conditions The experiment was conducted in late spring of 2006 (28 November to 22 December) at Balcarce, Buenos Aires Province, Argentina (37845S, 58818W, 1 m a. s. l.) in a greenhouse during a period of 24 days with natural light and a daily average air temperature of C and solar radiation of MJ/m 2. Plants of C. gayana, cv. Finecut (rhodes grass) were planted in winter (July of 2006) in two wooden rafts (plots) 1. m long, 1.00 m wide and 1.00 m deep filled with Argiudol top soil. They were fertilised with urea and triple superphosphate ( and 6 g/m 2, respectively), and irrigated daily to reach a soil moisture content of %. Plants were regularly defoliated by clipping to obtain dense swards. Once this condition was achieved, the swards were cut at 5 cm stubble height to synchronise leaf growth. Afterwards, the pasture was regularly sampled during the following regrowth period. Maximum and minimum temperatures were measured daily and the accumulated growing degree-days (GDD) were calculated as the sum of daily mean temperatures above a base temperature for growth of 128C (Jones 1985). The GDD was used to express time of regrowth, leaf age, LLS and leaf expansion duration (i.e. from leaf emergence to ligule appearance). Measurements and sampling during the experimental period Each plot 1.8 m long and 1.0 m wide was divided in eight 1.0-mlong and 22.5-cm-wide strips. One strip was left uncut until the end of the experiment for measurements of leaf morphogenesis. Leaf expansion dynamics and turnover were determined by monitoring leaf appearance (leaf tip visible), ligule appearance and onset of senescence (decrease in green length) on 15 marked tillers every 2 days throughout the experimental period. From these data leaf appearance rate (interval between two consecutive leaf appearances), time when final leaf length was attained (ligule appearance), number of living leaves per tiller and the LLS (interval between leaf appearance and the onset of senescence) were estimated. The remaining seven strips were sequentially harvested at each leaf appearance interval following the methodology used by Groot and Neuteboom (1997). The frequency of harvesting was 3 5 days ( 65 GDD), depending on the average daily temperature. At each harvest, the accumulated herbage biomass was cut at the plant crown base and ~0 tillers were randomly selected within the population present at the beginning of the sampling period to prevent the inclusion of tillers differing in developmental stage. These tillers were identified by the presence of cut leaves remnant from the initial clipping. The material was immediately placed in plastic bags in liquid N and carried to the laboratory to be stored in a freezer at 208C for further processing. Frozen tillers were defrosted and separated into leaf blades and leaf sheaths. Leaf blades of the first three leaves (L1, L2, L3) were further separated for forage quality analysis in five leaf age categories: early growing, advanced growing, just expanded (ligule appearance), adult, pre-senescent (i.e. close to the end of the LLS) (Table 1). Samples for forage quality analysis were also obtained at ligule appearance for L4 and L5, and before ligule appearance for L6. Leaf blade and sheath lengths were measured, and leaf blades grouped by age category within each leaf appearance sequence (L1, L2, L3, L4, L5, L6) for laboratory analysis. Laboratory analysis Leaf blades samples were freeze-dried to constant weight (Lyophiliser Model CHRISS 336, Lyophilisation Techn. Inc., Caledonia, MI, USA) and ground to pass a 1-mm screen (CYCLOTEC sample mill 1093, FOSS TECATOR, Höganäs, Sweden). The NDF was determined according to Van Soest et al. (1991) with a heat stable a-amylase and sodium sulfite (ANKOM 200 fibre analyser, ANKOM Techn., New York, USA) and expressed inclusive of residual ash. Then, samples of ~0 mg DM were placed in Dacron bags F1020 (ANKOM) and incubated in vitro for 24 h in the Daisy II apparatus (ANKOM) with ruminal liquor from a donor steer fed a diet of 3 g/kg DM of lucerne hay, 3 g/kg DM of grass hay, 1 g/kg DM of ground corn grain and g/kg DM of sunflower meal. After incubation, bags were boiled in ND solution (ANKOM 200 fibre analyser) to measure residual NDF. From this parameter the NDF digestibility [NDFD = (NDF incubated NDF r )/NDF incubated ] was estimated (Goering and Van Soest 19) and expressed in percentage of DM. Statistical analysis Data of final leaf length (i.e. leaf length at ligule appearance) were compared among leaf generations by ANOVA using the MIXED procedure of SAS (2000) according to a completely randomised design with two replicates. Polynomial functions using the NLIN

3 1100 Crop & Pasture Science M. G. Agnusdei et al. Table 1. Harvest number, days of regrowth, leaf age in growing degree-days (GDD) and leaf age categories sampled for neutral detergent fibre (NDF) and in vitro NDF digestibility (NDFD) in Chloris gayana L1 to L6 denote the first to the sixth leaf appeared during regrowth, respectively. L1 to L3 were sampled at five age categories: early growing, advanced growing, just expanded(ligule appearance), adult and pre-senescent. L4 and L5 were only sampled at ligule appearanceand L6 at advancedgrowing.*leaves of L3 in the 6th harvest were discarded due to insufficient sample for laboratory analysis Harvest Days of Leaf age in GDD Leaf age categories sampled for NDF and NDFD analysis number regrowth L1 L2 L3 L4 L5 L L1 early growing L1 advanced growing, L2 early growing L1 just expanded, L2 advanced growing, L3 early growing L1 adult, L2 just expanded, L3 advanced growing L1 pre-senescent, L2 adult, L3 just expanded * 1 L2 pre-senescent, L3 adult, L4 just expanded L3 pre-senescent, L5 just expanded, L6 advanced growing procedure of SAS were fitted to describe variations of NDF and NDFD with leaf age and time of regrowth (both expressed in GDD), and between NDF and leaf blade length. Linear functions using the LIN procedure of SAS were fitted between leaf blade and sheath lengths, between NDFD with leaf blade length. Parameters of the curvilinear functions describing variations of NDF and NDFD with leaf age among leaf generations were compared by ANOVA using the MIXED procedure of SAS. A Tukey test was used for mean comparisons and significant differences were accepted if P < In the text mean values are presented the standard error of the mean. Results Leaf morphogenesis The experimental period comprised 4 GDD. All leaves under study (Table 1) were completely green, with no signs of senescence since they were collected within the LLS. The average leaf appearance interval and LLS were and GDD, respectively. During the LLS of the first appeared leaf, an average of six leaves per tiller were accumulated and two simultaneously growing leaves were visible. The leaf expansion period (i.e. period from leaf emergence out of the sheath tube until the final leaf blade length was attained) lasted on average GDD, corresponding to the moment of visible ligule appearance. Leaf blade length increased during the first half of the LLS to plateau at ligule appearance (Fig. 1a). The final leaf blade length increased for consecutive leaves up to L3 and no significant variations among this leaf and later appeared ones were observed. Leaf blade length was highly associated (P < 0.05) with the length of the leaf sheath tube of the preceding leaf (Fig. 1b). Variations of NDF and NDFD The pattern of variation of NDF and NDFD along the LLS, expressed in GDD, differed as shown in Fig. 2. Whereas no significant change in NDF was detected in L1, in L2 and L3 the Leaf blade length (mm) (a) (b) Leaf age (GDD from leaf emergence) Leaf sheath tube length (mm) Fig. 1. Evolution of leaf blade length of consecutively formed leaves during a regrowth of Chloris gayana, (a) with leaf age and (b) its relationship with the leaf sheath length of the preceding leaf. Symbols denote consecutive leaves, L1: ^, L2: & &, L3: ~ ~, L4: *, L5: +, L6:. Open and + symbols denote ligule appearance. L1 to L3 were sampled at five age categories: early growing, advanced growing, just expanded (ligule appearance), adult and pre-senescent. L4 and L5 were only sampled at ligule appearance, and L6 at advanced growing. The accumulated growing degree-days (GDD) were calculated as the sum of the average daily temperature above 128C. (a) Standard error of the mean = 2.3 at ligule appearance; L1 < L3 (P < 0.05), L1 = L2 (P > 0.05), L3 = L4 = L5 (P > 0.05); (b) y = 1.94x (R 2 = 0.85, P < 0.05).

4 Rhodes grass leaf quality Crop & Pasture Science 1101 (a) (b) NDF (%) NDFD (%) Leaf age (GDD from leaf emergence) Fig. 2. Relationships between the neutral detergent fibre (NDF) content (a) and the in vitro NDF digestibility (NDFD) (b) with leaf age (accumulated thermal time since emergence) in three sequentially appeared leaves in Chloris gayana. Symbols denote consecutive leaves, L1: ^, L2: & &, L3: ~~, L4: *, L5: +, L6:. Open and + symbols denote ligule appearance. L1 to L3 were sampled at five age categories: early growing, advanced growing, just expanded (ligule appearance), adult and pre-senescent. L4 and L5 were only sampled at ligule appearance, and L6 at advanced growing. The accumulated growing degree-days (GDD) were calculated as the sum of the average daily temperature above 128C. NDF L1, y = x x (R 2 = 0.81, P > 0.05); NDF L2, y = x x (R 2 = 0., P < 0.05); NDF L3, y = x x (R 2 = 0.94, P < 0.05); NDFD L1, y = x x (R 2 = 0.87, P < 0.05); NDFD L2, y = x x (R 2 = 0.89, P < 0.05); NDFD L3, y = x x (R 2 = 0.88, P < 0.05). NDF increased up to ligule appearance and remained stable afterwards (Fig. 2a). The intercepts did not differ among leaves, suggesting that the three leaves emerged with similar NDF (average 36.9%). The linear and quadratic terms were greater in L3 than in L1, whereas L2 showed an intermediate behaviour between both leaves (similar linear term to L1 and L3, and lower quadratic term than L3). These results indicate that the increase in NDF was more pronounced in later emerged leaves. For those leaves that were sampled only at ligule appearance (L4 and L5) or before ligule appearance (L6), no significant differences in NDF among them and L3 at the same stage of development were observed. The NDFD declined curvilinearly (P < 0.05) during LLS, showing an average reduction from the first to the last sampling of ~26% (Fig. 2b). The intercepts and linear terms did not differ among appeared leaves, but the quadratic term was greatest in L3. The analysis suggests that at emergence NDFD would be similar among leaves, with a subsequent decrease along LLS that was most pronounced in the case of L3. Later appeared leaves (i.e. L4 L6) showed no difference in NDFD with L3 at similar developmental stage (prior to and at ligule appearance, respectively). The increase in leaf blade length during the period of leaf expansion (i.e. up to ligule appearance), as well as the larger final blade length for consecutively formed leaves (Fig. 1a), was accompanied by a concomitant decrease in forage quality (Fig. 2a, b). These associations were properly described by curvilinear and linear equations fitted on NDF and NDFD against leaf blade length (Fig. 3a, b, respectively). It can be seen that NDF increased slowly and remained below 45% up to ~% of the maximum leaf blade length (~320 mm), to rapidly increase around 10 percentage units afterwards. Conversely, the NDFD showed a continuous and sharper decline all along the range of leaf blade lengths. This trend was consistent for growing as well as for already expanded leaves [i.e. previous or after ligule appearance (open and closed symbols, respectively)]. Curvilinear functions properly described (P < 0.05) the evolution of NDF and NDFD against thermal time of regrowth (Fig. 4). The NDF reached a plateau of around % at ~0 GDD, whereas NDFD continuously decreased until the end of the regrowth period (4 GDD). Discussion Forage quality evaluation approach Our aim was to generate knowledge to link the variations in forage quality with the pattern of leaf growth of vegetative pastures, as well as to eventually extend the pattern of NDFD variation observed in Italian ryegrass by Groot and Neuteboom (1997), an annual short-leaved temperate grass, to a long-leaf tropical grass (C. gayana). The study was carried out under adequate growth conditions with the aim to obtain sound genotypic benchmarks. Thus, caution should be taken to extend the obtained results to other environmental situations, particularly thermal due to the significant changes that temperature can induce in both NDF and NDFD (Akin et al. 1987; Wilson et al. 1991; Groot et al. 2003). The distinctive feature of the methodology used in the present study is that the NDF content and the NDFD were investigated in relation to leaf age and length of individual leaf blades during a complete regrowth cycle of a C 4 species. Leaf age and other time-dependent parameters (i.e. LLS and the period to ligule appearance) were expressed in accumulated thermal units above a base temperature for growth of 128C (GDD), in order to better describe changes in forage quality with plant development (Lemaire et al. 2009; Schut et al. 2010).

5 1102 Crop & Pasture Science M. G. Agnusdei et al. (a) (b) NDF (%) NDFD (%) Leaf blade length (mm) Fig. 3. Relationships between the neutral detergent fibre (NDF) content (a) and the in vitro NDF digestibility (NDF) digestibility (b) with leaf blade length in Chloris gayana. Symbols denote leaf age categories: ^ early growing, ~ advanced growing, * just expanded, & adult. NDF, y = 9.0E-05x x (R 2 = 0.87, P < 0.05); NDFD, y = 0.06x (R 2 = 0.85, P < 0.05). (a) (b) NDF (%) NDFD (%) Regrowth period (GDD) Fig. 4. Relationship between the neutral detergent fibre (NDF) content (a) and the in vitro NDF digestibility (NDF) digestibility (b) with the accumulated thermal time along a regrowth in Chloris gayana. Symbols denote consecutive leaves, L1:, L2: &, L3: ~, L4: *, L5: +, L6:. L1 to L3 were sampled at five age categories: early growing, advanced growing, just expanded (ligule appearance), adult and pre-senescent. L4 and L5 were only sampled at ligule appearance and L6 at advanced growing. The accumulated growing degree-days (GDD) were calculated as the sum of the average daily temperature above 128C. NDF, y = 4.0E- 07x E-04x 2 7.2E-03x (R 2 = 0.66, P < 0.05); NDFD, y = 9.0E-05x x (R 2 = 0.96, P < 0.05). The NDFD was determined at 24 h of in vitro incubation in accordance with the ruminal retention time reported by Poppi et al.(1981) for this grass species. In agreement, better predictions of the in vivo apparent DM digestibility (admd) of different cultivars of sorghum stover silages were obtained at 24 h incubation time as compared with the traditional period of 48 h (Di Marco et al. 2009). An important characteristic of NDFD as a forage quality trait is that it is not affected by variations of cell content that affect the admd (Oba and Allen 1999). These variations occur in response to environmental conditions like solar radiation, temperature or water and nutrient availability. To avoid such variations in relative quantities, the specific leaf weight of NDF (mg/cm 2 leaf area) instead of the NDF content per unit of DM would be a more consistent way to express changes in cell wall mass, as stated by Groot and Neuteboom (1997). However, the inherent methodological difficulties in measuring leaf area, particularly in narrow-leaved species like forage grasses, as well as the widespread use of NDF content per unit of DM in the nutritional field would limit the referred way of expression of the NDF. Leaf growth characteristics Both the LLS and the number of leaves per tiller (i.e. leaf stage) are key plant indicators of the rhythm of leaf turnover that are useful to determine the frequency of defoliation of pasture required to optimise production and quality and to avoid or minimise losses of leaf biomass by senescence (Fulkerson and Slack 1994; Fulkerson et al. 1999; Lemaire and Agnusdei 2000; Fulkerson and Donaghy 2001; Lemaire et al. 2009). The short leaf

6 Rhodes grass leaf quality Crop & Pasture Science 1103 appearance interval (61 GDD) and short LLS (355 GDD) observed in C. gayana indicates that this grass has a rapid leaf turnover as compared with values reported for a wide range of temperate and tropical species (100 2 and 0 8 GDD, respectively), by other authors (i.e. Lemaire and Chapman 1996; Lemaire and Agnusdei 2000; Lemaire et al. 2009; Da Silveira et al. 2010). Moreover, the relatively large number of living leaves (i.e. ~6 per tiller) observed in this species is a common trait in tropical grasses (Fulkerson et al. 1999; Cruz and Boval 2000; Da Silveira et al. 2010). Another important feature to remark is the presence of three simultaneously growing leaves (i.e. two visible appeared at an average interval of 61 GDD and one growing inside the sheath tube) during the 1 GDD required to achieve full expansion. This indicates that at least three intercalary meristems were active during the referred period, a feature that differs from most common temperate grasses which typically have one growing leaf at a time (Skinner and Nelson 1995). The final blade length was positively associated to the sheath length at the time of tip emergence, that is, to the sheath length of the preceding leaf (Fig. 1b). The slope of the linear regression indicates that blade length tended to double that of sheaths for all consecutively appeared leaves and that this ratio was relatively stable for the range of sheath lengths observed in the study. Interestingly, the relative increases in blade and sheath lengths between L1 and L3 were both ~25%. These morphological changes were not accompanied by an increase in leaf elongation duration out of the sheath tube, (which remained relatively stable at ~1 GDD among consecutive leaves). This means that the enlargement of the sheath length resulted in a concomitant increase in the elongation rate of consecutive leaves. Correspondingly, it has been demonstrated that the length of the sheath tube has a positive influence upon the length of the growing zone of the emerging leaf (Skinner and Nelson 1995) and, hence, on its elongation rate and final leaf length (Kavanová et al. 2006). The positive association between leaf blade elongation and leaf sheath length reported here is in agreement with other studies (i.e. Mazzanti et al. 1994; Kavanová et al. 2006; Agnusdei et al. 2007; Berone et al. 2007). However, sheath length enlargement has also been associated with an increase in leaf elongation duration and minor variations in leaf elongation rate (Groot and Neuteboom 1997; Duru and Ducrocq 2000, 2002). So, while genotypes can differ in the underlying mechanisms that determine the morphological plasticity of plants in dense canopies the evidence consistently reveals a strong association between leaf blade and sheath length along regrowth. Factors affecting the NDF and NDFD of leaf blades During the first half of the LLS, which corresponded to the phase of leaf growth previous to ligule appearance (<1 GDD), the NDF increased in two of the studied leaves and the NDFD declined in all cases (Fig. 2a, b, respectively). However, from ligule appearance to the end of the LLS, which corresponds to the maturity phase of the leaf blades, the NDF concentration remained constant and the NDFD continued to decline curvilinearly. Both trends are indicating that leaf blade quality, in terms of NDF and NDFD, was more affected during leaf growth phase than during leaf maturity. Groot and Neuteboom (1997) observed in individual leaves of Italian ryegrass similar patterns of leaf quality decline during leaf ontogeny, expressed in their case as the complement of the NDFD, that is the indigestible NDF. This concordance between ours and the referred experiment suggests that NDFD decline during LLS could be a generalised pattern for temperate and tropical grasses. The changes in NDF and NDFD observed during the phase of leaf expansion can be explained by two simultaneous processes: the progressive appearance of parts of the leaf with different tissues and anatomical constitution (Wilson and Hacker 1987; Wilson 1993), and concomitant changes in the chemical composition of fibre that reduce the digestibility of cell walls during aging (Akin and Chesson 1989; Engels and Schuurmans 1992; Wilson 1994; Grabber 2005). The first process reflects differences in leaf anatomy that result from the increase in size of the vascular system from the tip to the base of the leaf (Wilson 1993; Maurice et al. 1997). This anatomical variation determines that the consecutive fractions of leaf blade that appear out of the sheath tube during the visible phase of leaf expansion are progressively richer in structural tissues. This phenomenon entirely explains the increase in NDF observed during leaf expansion (Fig. 2a). In turn, the steeper decline in NDFD as compared with the rate of increase in NDF observed during this phase (Fig. 2b)reflects that the NDFD was additionally affected by the process of fibre aging. The following changes in NDFD that occurred after leaves attained their final length indicate that the process of fibre aging continued along the LLS. The increase in NDF and the decrease in NDFD as consecutive leaves became larger during leaf expansion and at adult stages of leaf ontogeny (Fig. 3a, b) suggest the existence of unequivocal relationships between leaf blade length and leaf tissue quality. Open and closed symbols in Fig. 3 differentiate two sources of variation of leaf tissue quality: a dynamic one that results from the process of leaf expansion and a static one corresponding to the increase in leaf blade length among consecutive leaves as mediated by the increase in the sheath tube length of the preceding leaf. As far as we know, these two aspects have not been explicitly noticed in previous evaluations of the relationship between leaf length and leaf tissue quality. The curvilinear trend described for NDF (Fig. 3a) was similar to the increase in mid rib weight with leaf weight reported for sweet sorghum by Lemaire and Gastal (1997), reflecting the more than proportional increase in structural tissues (e.g. bundle sheath, lignified vascular tissues) in larger leaves (Wilson and Hattersley 1989; Lemaire and Gastal 1997). Changes in NDFD with leaf blade length (Fig. 3b) have not been described in grasses. Present data suggest that the necessary structure invested by plants to keep larger leaf blades in a proper position to capture light in dense canopies would have a greater effect on NDFD than on NDF content. From a nutritional point of view this suggests that feed consumption could be highly affected during a period of regrowth in spite of minor changes in leaf NDF content (Fig. 3a), reflecting the importance of the NDFD on pasture quality evaluation (Jung and Allen 1995; Oba and Allen 1999; Mertens 2009). The strong associations between leaf and sheath tube length (Fig. 1b), as well as between NDF and NDFD with leaf blade length (Fig. 3) are indications that the nutritional quality of leaf blades are negatively affected by the enlargement of the sheath tube of the preceding leaf, independently of the leaf stage of development. Groot and Neuteboom (1997) and Duru and

7 1104 Crop & Pasture Science M. G. Agnusdei et al. Ducrocq (2002) attributed such a decline in forage quality to the longer period of leaf blade growth inside the sheath tube and, hence, to the fact that succeeding leaves were more aged at emergence from the sheath tube. In the present experiment this aging effect might have been counterbalanced, at least in part, by the positive effect of sheath length enlargement on leaf elongation rate (see previous discussion on leaf growth characteristics). In this sense, the effect of leaf blade length per se on forage quality should not be disregarded. The control of forage quality through plant indicators The number of appeared leaves within the LLS (i.e. leaf stage) is considered a useful principle to establish the readiness of temperate and tropical pastures to be defoliated in order to optimise forage net production and quality (Fulkerson and Slack 1994; Fulkerson et al. 1999, 2007). Our data provide additional information about the relevance of this principle as well as the sheath tube length of the stubble as criteria to help control fibre digestive characteristics and pasture quality. Further, the different patterns of variation observed for the NDF and the NDFD of consecutive leaves when data were plotted against time of regrowth (Fig. 4) stresses the value of complementing leaf stage and thermal time to estimate the evolution of forage quality. For example, Pembleton et al. (2009) suggested that a 4-leaf stage of regrowth would be an adequate defoliation interval to maintain C. gayana in vegetative stage, limiting stems build-up to counteract the consequent decrease in leaf : stem ratio in more advanced stages of regrowth. Present results indicate that this leaf stage would be achieved at ~% of LLS, or ~2 GDD of regrowth. At this thermal time, the expected values of leaf blade NDF and NDFD would be 49.4 and 62.1%, respectively, according to the equations fitted to data presented in Fig. 4. Then, an admd of 69.4% can be estimated by subtracting the metabolic factor 11.9 proposed by Van Soest (1994) from the true DM digestibility [tdmd = DM incubated (NDFr/DM incubated ), Goering and Van Soest 19]. According to the SCA (19), the referred admd would correspond to a leaf blade metabolisable energy density of 9.8 MJ/kg DM [ME = (%admd*0.17) 2.0]. This value is within the range of ME densities estimated by Fulkerson et al.(1999) for several tropical and temperate forage grasses. Further studies to verify these expectations from greenhouse data under field conditions are required since the association between leaf developmental stage and forage quality is likely to vary with several factors such as season of the year, level of carbohydrates, stubble height, among others (Fulkerson et al. 1999). Conclusions Leaf aging and length were important factors affecting the NDF content and the NDFD of leaves blades of vegetative plants of C. gayana. The rate of change of both quality parameters was faster during the phase of leaf expansion than during aging. The fact that the NDFD declined throughout LLS whereas, as expected, the NDF was stable after ligule appearance, reveals the importance of the NDFD to explain leaf blade quality variations during leaf ontogeny. In addition to the effect of aging, higher NDF content and lower NDFD of consecutively appeared leaf blades were associated to a concomitant increase in leaf length occasioned by the enlargement of the sheath length of the preceding leaf. In this context, the length of the sheath tube could be considered as an objective and simple plant trait to control the appropriate residual sward height required to manage forage quality in the field. These results corroborate for a tropical grass previous findings obtained in temperate forage grasses, confirming that the nutritional quality of herbage is not a static feature directly linked to phenological stages of the plant, but a dynamic process closely coupled with leaf ontogeny and morphogenesis. Acknowledgements The authors would like to thank three anonymous reviewers for comments that significantly improved the manuscript; Dr Fernando Lattanzi for valuable discussions at early stages of the work; Dr Michael Wade for English language revision. The research programme is funded by the Instituto Nacional de Tecnología Agropecuaria (INTA, PE AEFP-2492 and ), the Universidad Nacional de Mar del Plata (AGR 288/09) and the Agencia para la Promoción Científica y Tecnológica (PICT ). Fernando Nenning was supported by a graduate fellowship awarded by INTA on study leave from INTA El Colorado (Argentina). 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