Productivity , chemical composition and ruminal degradability of irrigated Napier grass leaves harvested at three stages of maturity

______________________________________________________________________________________ Abstract The effect of the stage of maturity on the productivity, chemical composition, and ruminal degradability of Napier grass (Pennisetum purpureum) leaves under irrigation was studied. Samples were collected at three stages of maturity, at intervals of four weeks starting from the fourth week until the twelfth week (April– July) after sprouting and then harvested to determine biomass. The stages of maturity had significant effects on longest leaf length per plant and plant height, but had no effect on number of leaves per plant and number of tillers. Dry matter (DM) increased significantly with maturity, while crude protein (CP) and fat content decreased significantly, with no significant effect on acid detergent lignin (ADL), ash, acid detergent fibre (ADF) and neutral detergent fibre (NDF). Maturity significantly decreased potassium, copper and sodium, while calcium and zinc increased significantly up to the intermediate stage, then declined toward the late stage of maturity. Maturity did not affect the magnesium, phosphorus, manganese and iron contents of the forage. Ruminal degradability of CP at 48, 72, 96 and 120 incubation hour decreased significantly with maturity, with no effect on all other incubation times for DM and CP. Maturity significantly decreased the soluble fraction (a) of DM, with no significant effect on other fractions of DM and CP. Effective degradability (%) of DM and CP decreased with maturity when the rate of passage increased from 2% to 8%. Napier forage should be harvested before maturity to retain its high nutrient content, and the degradability of DM and CP. ______________________________________________________________________________________


Introduction
Currently, there is increasing demand for ruminant animal products owing to the growing population and improved livelihoods (Dastagiri, 2004).However, feed shortages and the poor quality of forage in the dry season are major production constraints in the smallholder livestock sector.Climate change, and economic and anthropogenic factors put pressure on conventional feeds to remain unaffordable; hence smallholder farmers are seeking affordable alternatives (Chakeredza et al., 2007).Pasture and fodder remain the cheapest forms of animal feed.The ideal pasture and fodder species should be high yielding and palatable, and contain adequate levels of highly digestible nutrients to satisfy the requirements of livestock (Kisitu, 2010).
Napier grass (Pennisetum purpureum), also known as Elephant grass, has been the most promising high-yielding fodder, giving DM yields that surpass most other tropical grasses (Ansah et al., 2010).It originates from sub-Saharan tropical Africa (Clayton et al., 2013) and has been introduced in most tropical and subtropical regions worldwide as forage.According to Mdziniso (2012), Napier grass is a tall perennial grass that grows to 2-5 metres (m) tall, rarely up to 7.5 m, with leaves about 30-120 centimetres (cm) long and 1-5 cm broad.
The grass has been used by smallholder farmers in cut-and-carry feeding systems (Premaratne & Premalal, 2006;Nyambati et al., 2010).Research consistently indicates that as Napier grass matures, the DM yield increases, while CP declines (Kramberger & Klemencic, 2003;Bayble, 2007, Sultan et al., 2007, Ansah et al., 2010).Hence, the maturity stage of pasture grass at harvesting or grazing is considered a crucial management practice because this determines nutritional value of the grass (Jusoh et al., 2014).Information on the relationship between the quality and degradability of Napier grass at various stages of maturity is limited and is ultimately important for silage making.Therefore, the current study was aimed at determining the productivity, chemical composition and ruminal degradability of DM and CP of Napier grass harvested at three stages of maturity.

Materials and Methods
The study was conducted at University of Venda, School of Agriculture Experimental Farm, Thohoyandou (22°58'32"S, 30°26'45"E; altitude 596 m above sea level).Samples were analysed in the Animal Science Nutrition Laboratory, University of Venda.The average maximum and minimum temperatures are 31 o C and 18 o C, respectively (Tadross et al., 2005).The area receives annual rainfall of 500 mm, which falls predominantly in summer.The area is characterized by deep well-drained red clay soil (Soil Classification Working Group, 1991), and low organic carbon, and has a slightly acidic pH.Characteristics of this soil are summarized in Table 1.The trial was approved by University of Venda's Ethics Committee.The study was conducted from April to July 2014.Daily data for weather (Table 2) during the experimental period were extracted from Agricultural Research Council -Institute for Soil, Climate and Water (ARC-ISCW) station at the University of Venda.The site was ploughed with a tractor, harrowed, then demarcated into six plots, each 4 x 5 m in a completely randomised design.After land preparation, the plots were irrigated to field capacity.A basal fertilizer 2:3:2 (Nitrogen: Phosphorus: Potassium) was applied at the rate of 250 kg/ha, plus superphosphate at the rate of 300 kg/ha.Napier grass was planted manually at a spacing of 70 cm inter and intra rows using Napier cuttings with three nodes.At least two nodes were placed into the ground and above the ground at an angle of 30 -45°.All the plots were irrigated manually with water pipes during the 12 weeks of the experiment.The irrigation regime was designed to supplement rainfall to maintain field capacity.Weeding was done manually immediately after the weeds started to grow.For production parameter data, the number of tillers and leaves were counted, and the height of the grass and the longest leaf length (cm) were measured once every two weeks from the second week until the last cut of sample grass on the twelfth week.Four plants were randomly selected in the middle of each plot to avoid edge effect, and tagged for data collection.Dry biomass was measured on the twelfth week (late stage) after collecting the samples.
Approximately 1000 g of samples of whole Napier grass leaves were manually cut at random from the nodes using pruning scissors, and plucked by hand from each plot per stage of maturity.The grass was cut when the leaves were fully expanded at the early growing stage and at intervals of four weeks until the twelfth week after sprouting.During the sampling process, fodder leaves were cut into lengths of 1-3 cm using pruning scissors, and oven dried at 60 °C for 48 hours (h) to determine DM content (AOAC, 2005).The samples were milled to pass through a 2-mm screen.Ash content was determined by combusting at 550 °C overnight (AOAC, 2005).Nitrogen (N) content was analysed with the Kjeldahl procedure (AOAC, 2005) and converted to CP as N x 6.25.Fat content was determined with the soxhlet fat extraction method described by AOAC (2005).ADF, NDF and ADL composition were determined according to the method of Van Soest et al. (1991).Phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulphur (S), copper (Cu), zinc (Zn), manganese (Mn) and iron (Fe) were determined with inductively coupled plasma optical emission spectrometry (ICP-OES) (SOP, 2005).
Three mature Bonsmara steers were fitted with rumen cannulae of 8 cm internal diameter and were used to determine the DM and CP degradability profiles of Napier grass leaves harvested at the early, intermediate and late stages of plant maturity.During the experiment, the animals were housed in individual pens.They were fed commercial complete cattle finisher (Table 3) ad libitum, starting 21 days before the commencement of the incubation of bags.Clean drinking water was available in buckets throughout the whole experiment.Nylon bags (12 × 6 cm, pore size 46 µm) duplicated per incubation time were filled with approximately 5 g Napier grass leaves samples collected at the early, intermediate and late stages of plant maturity.The bags were attached with plastic bands to flexible vinyl plastic tubes, approx.40 cm long and of 6 mm outer diameter, and inserted into the rumen of the cannulated steers for incubations of 0, 6, 12, 24, 48, 72, 96 and 120 h.The bags were inserted at 0600hrs before the morning feeding time.After each incubation period, the bags were removed from the rumen and washed in running tap water without squeezing, until the water was clear.The zero hour incubation bags were washed without incubation in the rumen.The washed bags were dried in an oven at 60 °C for 48 h, weighed, and the residues analysed for N content.Nutrient degradation was calculated as the difference between the amounts in the control sample and in the degraded residues.
The degradability of DM and CP with time for each sample was described using the mathematical model of Ørskov & McDonald (1979): ) Where P = DM disappearance at time t, a = zero time intercept b = slowly degradable fraction c = rate of degradation Potential degradability (PD) was estimated as (a + b).Effective degradability (ED) was calculated at rumen fractional outflow rates (k) of 0.02, 0.05 and 0.08 per hour according to Ørskov & McDonald (1979): The degradation constants were estimated with Neway Excel version 6 (The Rowett Research Institute, Aberdeen, UK).Production parameters, chemical composition, DM and CP disappearance, and degradation constants were subjected to analysis of variance using the general linear model of Minitab software version 17 (2014).Treatment means were compared using Tukey's procedure at P <0.05.The model was Y ij = µ + M i + Ԑ ij ; where, Y ij = is the observation; µ = overall mean; M i = effect of the ith stage of maturity and Ԑ ij = random error.

Results
The growth parameters of Napier grass at three stages of maturity are presented in Table 4.The longest leaf length per plant and plant height differed (P <0.01) at all three stages.The number of tillers and leaves per plant did not differ (P >0.05).The longest leaf length per plant, plant height, number of leaves per plant and number of tillers increased with maturity of the grass.The cumulative biomass yield was 6.08 tons/ha for 12 weeks' growth.The chemical composition (g/kg DM) of Napier grass leaves at three stages of maturity is presented in Table 5.The DM content increased (P <0.01) with maturity, while CP and fat content decreased (P <0.01 and P <0.05 respectively), with no effect (P >0.05) on, ash, NDF, ADF and ADL.The macro-mineral (g/kg) and micro-mineral (mg/kg) compositions of Napier grass leaves harvested at three stages of plant maturity are presented in Table 6.Stage of maturity had an effect on Ca, Na and Zn (P <0.05), as well as Cu and K (P <0.01).No effects (P >0.05) were observed for Mg, P, Mn and Fe at three stages of maturity.Mg increased from 1.82 to 2.29 g/kg while K, Na, P and Cu decreased from 19.94 to 16.09 g/kg, 0.63 to 0.37 g/kg, 1.34 to 1.08 g/kg and 9.51 to 6.78 mg/kg, respectively, with progressed maturity (from early to late).In sacco DM and CP disappearance (%) of Napier grass leaves harvested at three stages of maturity are presented in Table 7.As expected, the disappearance of DM and CP increased with escalating time of incubation, but decreased with advanced stages of maturity.Ruminal degradability of CP decreased (P <0.05) with maturity at 48,72, 96 and 120 incubation h, while there were no differences for DM and CP at all other incubation times.The rumen degradability constants a, b and c at three rumen fractional outflow rates are given in Table 8.For DM, the a and c values decreased, while the b value fluctuated with increased maturity.For CP, the a value decreased, and the b and c values increased with increased maturity.However, effective degradability (%) decreased with maturity, when the rate of passage was increased from 2% to 8% for DM and CP.

Discussion
Growth parameters play a vital role in enhancing fodder yield (Imran et al., 2007).High correlations between production parameters such as plant height, leaf length and biomass yield have been reported (Tessema et al., 2003;Ndikumana & Kamidi, 2006;Imran et al., 2007;Nyambati et al., 2010).In the present study, leaf length, plant height, number of leaves and number of tillers per Napier grass plant increased with maturity.As expected, in the present, study linear development of longest leaf length per plant with maturity was observed.Plant height increased by approximately 53% from early to late stage.These results agree with those of Ansah et al. (2010), who reported increased plant height with increase in harvest days.However, in this study it was observed that with the increase in the stage of maturity, the greater the number of leaves produced from the newly emerging tillers.These results agree with those of Amin (2011), who reported that the number of leaves of maize fodder plant increased with maturity.The number of tillers per plant increased with grass maturity as the number of tillers developed.When the plants approached maturity, numerous fine branches appeared, growing out from the leaf axils of the main stems (Wilsie & Takahashi, 1934).This observation agrees with the results of Ansah et al. (2010).
The biomass yield obtained in the present study was higher than the results of Katuromunda (2010), who reported fodder DM yield of 5.93 t/ha after 11 weeks.The differences in DM yields could be attributed to varying cutting height, soil and climatic conditions (Zewdu et al., 2003).
Plant maturity is considered the primary factor that affects the nutritive value of forages.A number of factors affect the rate of change in nutrient composition with advancing plant development and maturity stages.These factors include plant type, climate, season, weather, soil type and fertility, soil moisture, leaf stem ratio, physiological and morphological characteristics, and may vary with annuals versus perennials, grasses versus legumes, etc. (Kilcher, 1981).
In the present study, the DM content increased as the grass matured, and higher DM was observed at the late stage of maturity.Kramberger & Klemencic (2003), Bayble (2007) and Ansah et al. (2010) reported that the DM content increased as Napier grass maturity increased.As expected, CP was highest in the early stage compared with the intermediate stage and late stage.This could be attributed mainly to dilution of the CP contents of the forage crops by the rapid accumulation of cell wall carbohydrates at the later stages of growth (Van Soest, 1994).It was observed that the CP level decreased by 16% from early to late stage.CP content serves as an important indicator of fodder quality.In other studies, the CP content of Napier grass also decreased with maturity (Kramberger & Klemencic, 2003;Bayble, 2007;Sultan et al., 2007;Ansah et al., 2010;Jusoh et al., 2014).In the present study, despite the decline in CP content with increasing stage of maturity, the final concentration exceeded the minimum CP level (7.5%) required for rumen function (Jusoh et al., 2014).This indicated the possibility of improving the feeding of animals in tropical regions by planting Napier grass, thus enhancing the quality of nutrients supplied to animals.
In the present study, fat content decreased with maturity.Similar results were observed by Karabulut et al. (2006).Conversely, studies by Kamalak et al. (2005) and Kanak et al. (2012) showed increased fat content with progressing stages of maturity.Generally, ash content gives an indication of the minerals present in the sample.In contrast with the findings of the present study, Aganga et al. (2005) and Kanak et al. (2012) reported increased ash content with stage of maturity.
In the present study, the NDF and ADF contents decreased from early to intermediate and then increased at the late stage.In contrast, Aganga et al. (2005), Ansah et al. (2010) and Salamone et al. (2012) reported that the NDF content rose consistently as forage maturity increased.Results for the present study were higher than the NRC recommendations for NDF and ADF contents of 300 and 190 g/kg DM for a ration, respectively.The findings for ADL agree with the studies of Bayble (2007) and Aganga et al. (2005).Bayble (2007) and Aganga et al. (2005) observed increased ADL with progressive stages of maturity.The late stage had the highest lignin content, implying that though Napier grass had high DM, the high lignin content would bind the cellulose and hemicellulose and prevent them from being digested and utilized efficiently by the rumen microbes (Ansah et al., 2010).Therefore, forages with lower ADL concentrations are more desirable.Aganga et al. (2004) observed fluctuations of all individual major and minor minerals with plant maturity.However, the observed decline in Ca content with maturity was reported by Kariuki (1989) and Kariuki et al. (1999).In the present study, the Ca concentration of Napier grass at all stages of maturity was adequate for maintenance, growth and lactation in sheep (1.2-2.6 g/kg) (ARC, 1980;McDowell et al., 1993;Khan et al., 2006).The Mg concentration was adequate for grazing animals, although early lactation ruminants require 2 g/kg (Gill et al., 2004).While the K concentration was above the 8 g/kg recommended for grazing animals at all stages of maturity, it may not meet the requirements (10 g/kg) for high-producing cows, under stress such as hear stress (Mirzaei, 2012).Similarly, Na concentration was lower than the recommended range (1-4 g/kg) for growing and finishing ruminants by Underwood (1981) and Gill et al. (2004).In addition, natural forages with a deficiency of Na have been reported in many regions of the world (Pastrana et al., 1991;Areghoere, 2002).P concentration was lower than the critical requirement of 1.9 g/kg for growing and finishing beef cattle (Gill et al., 2004).However, Kariuki (1989), Kariuki et al. (1999) and Suttle (2010) observed that P content of Napier grass decreased with maturity.
Zn concentration at all stages of maturity was within the recommended range of 12-20 mg/kg, which is adequate for growing ruminants (ARC, 1980;Gill et al., 2004).Cu concentration in the present study decreased with maturity.It is commonly suggested that the dietary requirements of ruminants for Cu range from 8 to 14 mg/kg (NRC, 1984;Gill et al., 2004;Khan et al., 2006).Mn concentration in the present study at all stages of maturity exceeded the recommended levels of 20 mg/kg for growing and finishing cattle and 40 mg/kg, the critical level of dietary Mn (Gill et al., 2004).In this investigation, Fe concentration was above the level of 50 mg/kg proposed as adequate for grazing animals (McDowell, 1985;Gill et al., 2004;Khan et al., 2005).Little information has been found regarding mineral composition of Napier grass at three stages of maturity.
The decline in DM and CP rumen degradability with plant maturity has been reported widely (Aufrère et al., 2003;Yu et al., 2004;Rodrigues et al., 2004;Silva et al., 2008;Belachew et al., 2013;Ribeiro Junior et al., 2014).This in disappearance could be explained by the decrease in protein content and increase in ADL content of grass with maturity.The disappearance of the DM contents in the leaves by the end of 48 h of incubation, generally considered equivalent to digestibility (Ehargava & Ørskov, 1987) and being the mean retention time of fibrous feeds in ruminants (Kimambo & Muya, 1991), revealed that Napier grass had less than 50% DM loss at all stages of maturity.
The high degradability of Napier grass leaves suggests that very low proportions of the a and b components were degraded beyond the rumen, as was supported by Aufrère et al. (2003).Therefore, the exact rate of passage is important for estimating effective degradability and the amount of undegraded protein passing to the small intestine (Mghen et al., 1996).The late stage of maturity recorded the highest outflow rate for DM and CP.This may explain the greater voluntary feed intake (Kusmartono et al., 1997).However, Ørskov & McDonald (1979) showed that with higher outflow rates, less is degraded.In the present study, the reduction in degradability was much greater when the rate of passage was increased from 2 % to 8 % and also when the grass matured.These results are in agreement with Baloyi et al. (2008), who observed a decrease in DM and CP with advanced stage of maturity of Cowpea and Silverleaf desmodium forage legumes.

Conclusion
The cultivation of Napier grass is recommended to ensure a continuous supply of quality feed for ruminants.The findings indicate that the stage of harvesting is critical to obtaining high quality forage.Napier forage should be harvested before maturity to retain its high nutrient content, and the degradability of DM and protein.However, in vivo studies are necessary to evaluate the effect of change in nutritive value with maturity on specific nutrient digestibility and animal performance.

Table 1
Physical and chemical characteristics of representative soil sample before planting (0-20 cm depth)

Table 2
Rainfall, temperature and relative humidity recorded during the experimental period April-July 2014

Table 3
Chemical composition of the commercial complete cattle finisher diet* *Supplied by Driehoek Feeds, Vaalwater, Waterberg, Limpopo, South Africa

Table 4
Production parameters of Napier grass at three stages of maturity **: P <0.01; (NS) non-significant: P >0.05 ab Column means with different superscripts differ significantly at P <0.05 nd: not determined, cm: centimetres; SEM: standard error mean

Table 5
Chemical composition (g/kg) of Napier grass leaves harvested at three stages of plant maturity Column means with different superscripts differ significantly at P <0.05.

Table 7
In sacco dry matter and crude protein disappearance (%) of Napier grass leaves harvested at three stages of plant maturity *: P <0.05; NS: non-significant: P >0.05 ab Column means with different superscripts differ significantly at P <0.05 DM: dry matter; CP: crude protein

Table 8
Degradability constants and calculated effective degradability at three passage rates for dry matter and crude protein of Napier grass harvested at three stages of plant maturity **: P <0.01 *: P <0.05; (NS) non-significant: P >0.05 ab Column means with different superscripts differ significantly at P <0.05 DM: dry matter, CP: crude protein, a: soluble fraction (%), b: insoluble but potentially degradable fraction (%), c: outflow rate of degradation (h -1 ), and k: rumen outflow rate (h -1 )