Introduction
Pearl millet [Pennisetum glaucum (L.) R. Br.] is an annual tropical grass that can be utilized for grain or forage production. It is highly drought tolerant and resistant to many diseases affecting other crops traditionally used for silage production (Mason et al., Reference Mason, Maman and Pale2015). The interest in annual forages (such as millet) has increased considerably over the years with the occurrence of constant droughts in various regions of the world, and these severe climate events have led to an urgent necessity of identifying efficient water-use plants that are adapted to the recent climate changes. It is believed that the increase in the use of such rain-fed crops may help to achieve sustainable agricultural development in areas that are more prone to extreme weather (FAO, 2008). The use of forage millet in Brazil has been mainly concentrated on the central-west and southern regions. However, forage millet cultivars adapted to Brazilian northeastern conditions (semi-arid) have also been developed, including the IPA BULK1-BF cultivar (Agronomic Institute of Pernambuco (IPA), 1999). Several studies were conducted to evaluate the field performance of IPA BULK1-BF cultivar (Bezerra et al., Reference Bezerra, Santos, Almeida, Pereira, Lima and Ferreira2011; Costa and Priesnitz, Reference Costa and Priesnitz2014; Santos et al., Reference Santos, Neves, Pereira, Sollenberger, Rodrigues, Tabosa, Verneque, Oliveira, Jayme and Gonçalves2015), and it was found that they could serve as a feed source for livestock during the dry season. However, knowledge of the phenological stages at harvest and performance of IPA BULK1-BF is still lacking. The stage of maturity at harvest is a major factor to determine the nutritive value of a silage. The effects of plant maturity on silage fermentation and animal performance have been investigated in corn and sorghum crops (Bernard and Tao, Reference Bernard and Tao2015; Peyrat et al., Reference Peyrat, Baumont, Le Morvan and Noziere2015), but the relative scarcity of data on pearl millet for silage production suggests a need for additional studies in this area of research.
As a part of an overall strategy to deal with this issue, the current study evaluated agronomic characteristics of the IPA BULK1-BF pearl millet cultivar harvested at four different growth stages and assessed its potential application in silage production. Finally, the impact of these forages on intake and digestibility in lambs was quantified.
Materials and methods
Experiments location and general information
The experiment was conducted from June to October 2012 at the Semi-Arid Experimental Station of the Brazilian Agricultural Research Corporation (Embrapa) in the municipality of Nossa Senhora da Glória, Sergipe State, Brazil (10°13′S, 37°25′W, 291 m asl). The soil type in this region is a eutrophic red-yellow podzol (Santos et al., Reference Santos, Jacomine, Anjos, Oliveira, Lumbreras, Coelho, Almeida, Cunha and Oliveira2013), with an average depth of 1.5 m. The climate is typically semi-arid with an annual rainfall of 710 mm and average maximum and minimum temperatures of 32 and 20°C, respectively. Precipitation in the region is low, erratic, and the balance between rainfall and evaporation rate can be negative in some months based on meteorological data from a weather station located about 400 m from the experimental site (Table 1 and Fig. 1). Seed of the pearl millet cultivar (IPA BULK1-BF) was supplied by the pearl millet breeding programmes of IPA.
Fig. 1. Rainfall distribution (mm) and average temperature (°C) at the experimental station of Embrapa Semi-Arid from June to September 2012.
Table 1. Meteorological data during the experimental period
RD, rainfall occurrence in days; RH, relative humidity; SR, solar radiation; wind average speed at 2 m height.
Agronomic characteristics
Treatments were the four harvesting intervals (35, 50, 65 and 80 days after sowing) replicated five times in a randomized complete block design (20 plots). In this experiment, plants were harvested when at least 60% of them reached the Zadoks's scale of 55, 65, 75 and 85, respectively, which correspond to the four harvesting intervals investigated in this study (35, 50, 65 and 80 days) (Zadoks et al., Reference Zadoks, Chang and Konzak1974). Plots measured 10.5 m2 (5 × 2.1 m), with seeds sown manually at a depth of 3 cm in four rows (on 0.70 m centres). The sowing date was 13 June 2012. The soil at the site had the following properties: pH (water): 5.8; phosphorus (P): 0.0071 cmolc/dm3; potassium (K): 0.32 cmolc/dm3; aluminium (Al): 0.05 cmolc/dm3; hydrogen (H) + Al: 1.89 cmolc/dm3; calcium (Ca): 1.4 cmolc/dm3; magnesium (Mg): 0.74 cmolc/dm3 and organic matter (OM): 10.54 g/kg. All plots were randomly allocated and fertilized at seeding with 150 kg N/ha as urea and with 300 kg P/ha and 41 kg K/ha in a mixed fertilizer according to soil test recommendations. Each treatment comprised c. 32 plants/m2, achieved by thinning plots 15 days after emergence. Additionally, it was applied 1 kg/ha of the pre-emergent herbicide atrazine.
Plants were harvested according to each treatment, which were represented by the days 35, 50, 65 and 80 after planting. Harvests were made manually and taken at 5 cm above ground level. Only the two central rows in each plot were harvested, with the remainder being discarded. The harvested crop from each plot was collected and weighed to estimate fresh biomass (yield/ha). After chopping (average of 1.5 cm long) a representative sample from each plot, a 400-g sub-sample was oven-dried at 55°C for 48 h to determine dry matter (DM) concentrations and biomass yield of the four treatments.
The agronomic characteristics studied included: plant height; population density; extent of lodging; DM partitioning of plant organs (panicle, stem and leaf); DM yield (DMY) (t/ha) and digestible DM yield (DDMY) (t/ha). The height of ten randomly selected plants within each plot was determined by measuring from the ground level to the top of the panicle using a tape measure. These plants were then separated into panicles, stems and leaves, with the mass of each of these fractions being determined after oven-drying at 65°C for 72 h. The whole plants and their respective fractions (panicles, stems and leaves) were not chopped, and thus the time of drying and the temperature required to process them were higher than those employed to analyse the material used to determine DM concentrations and biomass yield.
In the current study, scores of lodging were estimated from the percentage area of the plot that was lodged. These scores were determined from an angle of 10° taken from the perpendicular, such that stems scored as 10 were considered as lodged and stems scored as 90 were not lodged in the plots. Lodging for each plot was then calculated as: (% plot area lodged × angle of lodging from vertical)/90 as described by Bell and Fischer (Reference Bell and Fischer1994). The DDMY was estimated by multiplying the in vitro DM digestibility from each repetition by its respective DMY.
Ensiling procedures
At harvest, a silage harvester was used to chop plants within each treatment to an average of 1.5 cm long and transferred into plastic barrels with a capacity of 250 kg (large scale silos used in the animal feeding trial described below). Representative herbage samples from each plot were also packed manually into polyvinyl chloride mini-silos (four mini-silos × five replications for a total of 20 mini-silos; 10.5 cm diameter × 35.5 cm high, capacity of 2.5 kg and average density of 813.7 kg/m3) using a wooden pestle (Sebastian et al., Reference Sebastian, Phillip, Fellner and Idziak1996). The mini-silos and the large scale silos were thoroughly washed and sanitized before ensiling to avoid contamination of the silage fermentation. As gas release would be possible from both types of silos (mini-silos and the large scale silos), care was taken to seal them appropriately with plastic lids followed by storage at room temperature.
Mini-silos were opened following 90 days of ensiling, with forage samples (15 g) from both mini-silos and plastic barrels being homogenized for 1 min in 500 ml of distilled water to measure the pH using a pH meter. Aqueous extracts (10 ml) were acidified with 50 μl of 9.77 mol/l of sulphuric acid (H2SO4) (Kung and Ranjit, Reference Kung and Ranjit2001) and frozen before analysis. Thawed extract samples were centrifuged for 15 min at 10 000 × g at 4°C and analysed for acetic, propionic, lactic and butyric acids using a Varian high performance liquid chromatography system as described by Adams et al. (Reference Adams, Jones and Conway1984). Organic acids were separated using an Aminex HPX-87H column (300 × 7.8 mm) with a mobile phase of 0.013 M H2SO4 at a flow rate of 0.5 ml/min. Organic acids were quantified using an ultraviolet detector set at 210 nm.
Ammonia was determined using a phenol-hypochlorite reaction, as described by Weatherburn (Reference Weatherburn1967). Finally, silage sub-samples (500 g) were oven-dried at 60°C for 72 h, ground through a 1 mm screen using a Wiley mill and stored at room temperature until further analysis.
Intake and digestibility measurements
All lambs were cared for in accordance with the guidelines of the National Council for the Control of Animal Experimentation (CONCEA) (2008) and approved by the Ethical Committee on Animal Use (CEUA) of the Embrapa Semi-Arid (Permit Number: CEUA/CPATSA/002-2012). Apparent nutrient digestibility of silages was measured using 24 Santa Inês male lambs (initial body weight (BW): 24 kg ± 1.6 kg) over a 21-day period. Lambs were blocked by weight and assigned randomly to one of four treatments. The first 17 days were used to adapt lambs to the diets in individual metabolic cages equipped with a poly-ethylene sieve tray to separate faeces from urine. Lambs were fed pearl millet silage only (without concentrate) twice daily at 07.30 and 16.30 h in a manner that assured 0.15 orts at the morning feeding. Water and a trace mineralized salt mixture were available to lambs ad libitum.
Apparent digestibility was determined over 5 days, with lambs being fed pearl millet silage ad libitum as described by Silva and Leão (Reference Silva and Leão1979). During these 5 days, total faeces, feed and orts of each lamb were measured and sampled daily. Samples of the 5 days were mixed, sub-sampled (400 g fresh faeces, 400 g fresh feeds and 400 g fresh orts per lamb) and stored at −20°C until analysed. The total urine output of each animal was collected daily into plastic containers containing 100 ml of hydrochloric acid (HCl) with 2N concentration to prevent fermentation, degradation and N losses. During the 5-day collection phase, subsamples (10% from the total urine volume) were collected in the morning and stored at −20°C until further analysis.
Chemical analysis
Ground samples were analysed for DM and OM as described by the Association of Official Analytical Chemists (AOAC) (2005) (methods 942.05 and 934.01). A Leco combustion N analyser was used to measure N concentration. Crude protein (CP) was calculated as N × 6.25. Both neutral detergent fibre (NDF), which was determined by using heat stable α-amylase, and sodium sulphite (ash free), and acid detergent fibre (ADF) were quantified as described by AOAC (2005) (methods 2002.04 and 973.18) using an Ankom Fibre Analyser. Concentration of hemicellulose was determined by subtracting ADF from NDF. Ether extract (EE) was determined as described by AOAC (2005) (method 920.39) using an Ankom Fat Extractor.
Total carbohydrate (TC) and non-fibrous carbohydrates (NFC) were calculated as described by Sniffen et al. (Reference Sniffen, O'Connor, Van Soest, Fox and Russell1992): TCg/kg DM = 100 − (CP + EE + ash) and NFCg/kg DM = 100 − (CP + EE + ash + NDF).
In vitro DMD analysis of fresh forage and silage was conducted in 100 ml serum bottles and examined in a single run for each forage/silage with triplicate bottles being used per sample. Plant material (0.5 g) was incubated with 10 ml of rumen fluid mixed with 40 ml of McDougall's buffer (McDougall, Reference McDougall1948) for 48 h at 39°C. Samples were subsequently incubated with 0.1N HCl and 2 g/l pepsin for further 48 h (Tilley and Terry, Reference Tilley and Terry1963). Equal volumes of rumen fluids were collected immediately after feeding from three rumen-fistulated bulls fed a mixture of the four treatments silages. After stirring the three samples, the combined ruminal fluid was used in the IVDMD assay as described above.
Statistical methods
Experiments were analysed by a mixed model approach with maturity stage as a fixed effect, random effects of blocks (agronomic and silage quality trials) and lambs (digestibility study) and random residual error using the MIXED procedure of SAS Version 9.1 statistical program (SAS 2002). The model used was: Y ij = μ + T i + B j + E ij, where Y ij is the observation, μ the overall mean, T i the treatment, B j the block/animal and E ij the residual error. When the fixed effect of plant maturity was significant, the means were compared using Fisher's protected LSD tests (i.e., the DIFF option of the LSMEANS statement). Polynomial contrasts were used to determine linear and quadratic effects of maturity stage. Significance was declared at P < 0.05.
Results
Agronomic traits
Plant height, stem, leaves and panicles proportion increased linearly (P < 0.001) with the increasing plant maturity. At the same plant density for all treatments, DM yield and digestible DM yield increased linearly (P < 0.001) with the increase in plant maturity (Table 2).
Table 2. Performance, phenological traits and fermentation products concentrations (g/kg DM) of silages produced from cultivar IPA BULK1-BF harvested in different days after planting
DM, dry matter; SEM, standard error of mean; TN, total nitrogen.
Silage quality
Increasing plant maturity resulted in a linear increase (P < 0.001) in DM, OM, TC, NFC and lignin concentrations, while the CP and ADF concentrations decreased linearly (Table 3). There was a significant positive quadratic relationship between hemicellulose (P = 0.004) and NDF concentration (P = 0.005) with the age of harvest. However, the relationship was negative for IVDMD (P = 0.011) (Table 3). A larger variation in fermentation products was detected among treatments, with a linear effect for silage pH and concentrations of acetic and butyric acids (P < 0.001) being observed in the current study. A quadratic effect was observed for lactic acid concentration (P = 0.003), with the silage produced from plants harvested at 65 days after planting exhibiting the highest values. A quadratic effect (P < 0.001) was also observed for propionic acid and ammonia-N concentrations, with the lowest values noted in silages made of plants harvested at 65 and 80 days after sowing (Table 2).
Table 3. Chemical composition (g/kg DM) of silages produced from cultivar IPA BULK1-BF harvested in different days after planting
DM, dry matter; IVDMD, in vitro dry matter digestibility; SEM, standard error of mean.
Digestion study
Intake of DM, OM, CP, NDF and ADF decreased linearly (P < 0.005), while NFC and lignin intake increased quadratically (P < 0.05) in animals fed silages produced from plants harvested at later maturity stages (Table 4). Digestibility of all variables decreased linearly (P < 0.05), except for NFC, which exhibited an increase in its digestibility as the maturity stages of the plants advanced (Table 4).
Table 4. Intake (g/kg BW0.75), total apparent and true digestibility (fraction) of dietary components and nitrogen balance in lambs fed silage produced from cultivar IPA BULK1-BF harvested in different days after planting
SEM, standard error of mean; BW, body weight.
Discussion
Agronomic traits
Significant differences were observed among treatments for plant height, DM and DDM yield, panicles and stem proportions with respect to the different ages of harvest. All these parameters increased significantly with the delay in harvesting, as they showed the highest values at 80 days after planting, likely due to the completion of the required time for the plants to finalize their phenological development. The delay in the harvest date from 35 to 80 days after sowing resulted in an increase of nearly 2.4 cm per day in plant height, and this result is similar to the average plant height (2.8 cm per day) reported for three Pakistani cultivars of pearl millet grown in the Pakistani hot desert climate (Bukhari et al., Reference Bukhari, Ayub, Ahmad, Mubeen and Waqas2011).
The increase in DM and digestible DM yields associated with the increasing plant maturity is consistent with previous research won silage crops (Marsalis et al., Reference Marsalis, Angadi and Contreras-Govea2010; Atis et al., Reference Atis, Konuskan, Duru, Gozubenli and Yilmaz2012; Aoki et al., Reference Aoki, Oshita, Namekawa, Nemoto and Aoki2013). For example, Monks et al. (Reference Monks, Ferreira and Peske2005) reported that DM yield ranged from 2.3 to 11.5 t/ha for pearl millet harvested at 44 or 144 days after sowing and grown in a Brazilian sub-tropical region. The increase in DM yield associated with the increasing plant maturity is likely caused by the increase in stem and panicle proportions, which can reduce the leaf ratios as the plants age (Fig. 2).
Fig. 2. Participation of morphological components (%) in the total biomass of cultivar IPA Bulk1BF harvested in different days after planting.
Silage quality
The stage of maturity at harvest is a major factor to determine the nutritive value of silages (Johnson et al., Reference Johnson, Harrison, Hunt, Shinners, Doggett and Sapienza1999). Previous research (Johnson et al., Reference Johnson, Harrison, Davidson, Robutti, Swift, Mahanna and Shinners2002) has demonstrated that DM concentration of silages increases with the advance in maturity. In the current study, increasing plant maturity resulted in a 2.4% increase in DM concentration per week. In general, the nutritive value of forages declines dramatically with the increase in maturity, mainly due to the increase in concentrations of NDF and ADF and a decrease in CP (Blaser et al., Reference Blaser, Hammes, Fontenot, Bryant, Polan, Wolf, McClaugherty, Kline and Moore1986).
Hassanat et al. (Reference Hassanat, Mustafa and Seguin2007) reported that IVDMD of pearl millet stover decreased with the increasing plant maturity and was highly correlated with ADF and lignin contents. Although the reduction in IVDMD was associated with the increase in lignin concentrations in the current study, it was found that plant maturity did not affect NDF concentrations despite the linear decrease observed for ADF. Similar results were found by Morales et al. (Reference Morales, Alatorre, Becerra and Vazquez2014) who reported a reduction in ADF and IVDMD associated with increasing concentrations of lignin in pearl millet evaluated in four phenological stages (leaf 7th, flag leaf, boot stage and flowering). A possible explanation for those results is that increasing proportions of grains are typically observed in later maturity stages of pearl millet crops, and this increased proportion of grains may weaken the relationship between plant maturity and digestibility of the whole plant silage. Khan et al. (Reference Khan, Khan, Sarwar and Azim2007) studied maturity effects in maize, sorghum and pearl millet silage quality. Concentrations of NDF and ADF in the whole plant decreased as maturity proceeded from pre-heading to milk stage maturity. Despite the decline in NDF and ADF contents, in situ DM digestibility of the whole plant decreased progressively from early (63.0%) to late (54.1%) maturity. These data highlight the importance of assessing grain development and stover quality in conjunction with maturity management of pearl millet silages. Identification of pearl millet hybrids that maintain stover quality while increasing grain proportion at advanced stages of maturity is a logical strategy for improving pearl millet silage quality.
The highest levels of pH (5.9) and ammonia-N (4.8 g/kg TN) observed in silages produced from pearl millet harvested at 35 days after sowing are in agreement with the levels frequently observed in high pH silages, in which secondary fermentations usually lead to an increase in ammonia-N (McDonald et al., Reference McDonald, Henderson and Heron1991). The larger amount of ammonia-N in silages produced from plants harvested at 35 days of sowing is a reflection of its higher moisture and CP contents at the time of harvest. According to Ferraretto et al. (Reference Ferraretto, Shaver, Massie, Singo, Taysom and Brouillette2015), ammonia-N and soluble CP are good indicators of an efficient in vitro digestibility of starch present in the whole plant of corn silages. A reduction in butyric acid concentration with the increase in plant maturity was expected owing to an increase in the DM content of the mature forage, as was observed for silages made of plants harvested at 80 days after sowing. The silages produced from plants harvested after 65 days after sowing had the highest lactic acid concentrations and the lowest pHs as compared to plants harvested in other maturity stages. Despite the contribution of all acids formed during fermentation, lactate plays a critical role in the reduction of silage pH due to its low dissociation constant (Moisio and Heikonen, Reference Moisio and Heikonen1994). This is mainly relevant as indicative of silages made with plants less than 35 days after planting, which have a low lactic acid content and consequently low silage stability. As shown by Ferreira et al. (Reference Ferreira, Lana, Zanine, Santos, Veloso and Ribeiro2013), a higher lactic acid production leads to a lower rate of DM loss in silages that undergo active lactic acid fermentations. On the other hand, acetic and butyric fermentations are undesirable and need to be avoided as they favour DM losses in the form of gases released from the fermentative process.
Digestion study
Increased plant maturity resulted in decreased DM intake due to a decrease in digestibility, and this finding is corroborated by previous studies that investigated cereal crop silages (Helander et al., Reference Helander, Nørgaard, Zaralis, Martinsson, Murphy and Nadeau2015; Khan et al., Reference Khan, Yu, Ali, Cone and Hendriks2015). Lower intake of silages produced from plants harvested at later stages of maturity could be caused by higher stem proportions and more lignified stems, which in turn may result in a higher NDF and lignin concentrations in the silages. Overall, decreased digestibility of pearl millet silage decreased DM intake, and this agrees with the assumption that decreased digestibility decreases intake potential (Illius and Jessop, Reference Illius and Jessop1996). A reduction in DM digestibility with an increasing plant maturity was expected due to a reduction in plant cell wall digestibility. This observation was confirmed in the current study as shown by the linear decrease in NDF and ADF digestibility followed by the subsequent increase in lignin intake as the plant maturity increased. In fact, Davis et al. (Reference Davis, Freetly, Kuehn and Wells2014) demonstrated that DM digestibility was correlated with consumption, digestible DM intake and BW gain in multiple regression models.
The decrease in CP concentration with the increasing plant maturity has been observed in previous studies (Khan et al., Reference Khan, Khan, Sarwar and Azim2007; Guimaraes et al., Reference Guimaraes, Costa, Paludo, Santos, Rossi and Cruvinel2014) and can be associated with a higher proportion of stems in the plants, which could ultimately reduce the protein solubility. It is also important to emphasize that the CP concentrations were higher than 60 g/kg DM for all treatments, a level that is considered suitable to sustain optimal microbial activity for an efficient ruminal fermentation (Van Soest, Reference Van Soest1994). A decrease in CP concentration had no effect on CP intake, but the lambs examined in this study underwent a decrease in N intake. Regardless, N intake and N absorbed were on average larger than those recorded for Sipli lambs (8.7 and 4.5 g/day) fed pearl millet silage in Pakistan, as demonstrated by Khan et al. (Reference Khan, Shahzad, Nisa and Sarwar2011). It should be mentioned that the positive N balance and the lack of body reserve mobilization observed in all treatments suggest an adequate digestibility of dietary protein.
Therefore, the current study showed that the pearl millet cultivar IPA BULK1-BF has the potential to yield forage within a wide harvest window, which ranges between 50 and 80 days after sowing. According to the digestion and in vitro digestibility studies, the nutritive value of pearl millet silage decreases as the plant maturity advances. In contrast, the fermentation parameters were improved with the increasing plant maturity. The possibility of harvesting pearl millet forage in any phenological stage between 50 and 80 days after sowing allows for the implementation of harvest management strategies that reduce the risk of losses caused by droughts while ensuring forage availability for ruminant feeding programs in arid regions. However, the current study has limitations that need to be addressed in future research, which include investigations on key ensilage metrics of the cultivar IPA BULK1-BF (such as DM loss, the chemical composition of pre-ensiling) as well as the microbial dynamics and its impact on silage quality and ruminal fermentation. Additionally, investigations involving yield, DM intake and digestibility of IPA BULK1-BF compared with competing forages (e.g., sorghum, corn) cultivated in a similar fertilization scheme are warranted in future studies in order to harness the full potential of the inclusion of pearl millet in ruminant feeding programs.
Financial support
This work was implemented by Fundo Setorial do Agronegócio (CTAgro) as part of the project ‘Forrageiras e alimentos alternativos do Nordeste – Tabelas de composição de alimentos e estratégias de formulação de dietas de melhor benefício bioeconômico para bovinos leiteiros (CNPq/CTAgro No. 555708/2009-8)’, made possible by the support of the Banco do Nordeste.
Conflict of interest
The authors declare no conflict of interest.
Ethical standards
Not applicable.
