Cutting Time and Regrowth Age Affect the Quality of Elephant Grass Silage

The objective of this study was to evaluate the effects of regrowth age (RA) and cutting time (CT) of elephant grass (Cenchrus purpureus cv. Cameroon) on the fermentation profile, microorganism population, and nutritive value of the silage in two simultaneous trials at different sites. A 2 × 2 factorial scheme with two CTs (08:00 and 14:00) and two RAs (8 and 16 weeks) at ensilage was used in a completely randomized design, with four replicates in each trial, totaling 16 experimental units per trial. Results showed that ammonia nitrogen to total nitrogen content (N-NH3/TN) ranged from 71.8 (14:00; 16 weeks) to 137.0 g kg -1 (14:00; 8 weeks). Elephant grass silage harvested at 14:00 at 8 weeks had the lowest pH (3.53) and highest lactic acid content (39.7 g kg dry matter). The in vitro dry matter digestibility (IVDMD) was higher (P < 0.05) in the silage of plants harvested at 8 weeks than in the silage harvested at 16 weeks (59.9 vs. 46.8%, respectively). In trial 2, elephant grass silage harvested at 14:00 had lower pH and acetic acid content. The IVDMD was higher in elephant grass silage harvested at 14:00, with values of 65.3% and 56.2% at 8 and 16 weeks, respectively. We recommend elephant grass harvested at 8 weeks of regrowth and cut at 14:00 to produce silage with better nutritive value and fermentative profile.


Introduction
Elephant grass [Cenchrus purpureus (Schumach.) Morrone; Basionym: Pennisetum purpureum Schumach.] is among the most popular forage grasses for silage production in tropical regions because of its high productivity and good nutritional value (Pereira et al., 2017). However, tropical grasses are characterized by high moisture content and low soluble carbohydrate content (Pholsen et al., 2016), which can negatively affect forage fermentation in the silo.
The concentration of soluble carbohydrates in plants is fundamental to the fermentation process. At low concentrations of soluble carbohydrates, the amount of lactic acid produced by bacteria is insufficient to reduce pH and inhibit the growth of deleterious microorganisms, resulting in low-quality silage (Pholsen et al., 2016;Wang et al., 2019).
concentrations of soluble carbohydrates in forage plants harvested after 11:00 am were observed by some authors in elephant grass cv. Napier (Cenchrus purpureus cv. Napier; De Oliveira et al., 2014;Guo et al., 2015), ryegrass (Lolium multiflorum Lam.; Guo et al., 2014), alfalfa (Medicago sativa L.; Tremblay et al., 2014), Marandu palisade grass (Urochloa brizantha cv. Marandu; De , and mixed birdsfoot trefoil (Lotus corniculatus), and timothy grass (Phleum pratense; Silva et al., 2020). Due to the higher soluble carbohydrate concentration in plants harvested at a later time of the day, there is greater substrate availability for lactic acid bacteria during the fermentation phase, resulting in higher lactic acid production and a rapid reduction in pH. Therefore, the harvest time of forage plants could be used as a management strategy to produce better quality silage.
The nutritional value of elephant grass is also significantly influenced by maturity (Haryani et al., 2018). Zailan et al. (2016) evaluated four elephant grass cultivars harvested at 4, 6, and 8 weeks and observed an increase in the dry matter (DM), neutral detergent fiber (NDF) content, and acid detergent fiber (ADF) content, and a decrease in the crude protein (CP) content and in vitro DM digestibility, with an increase in harvest age. In a study of elephant grass silage cv. BRS Capiaç u harvested at 50-110 days of growth, Lopes et al. (2021) also found a decrease in the CP content and an increase in the NDF content, as well as a decrease in silage pH.
Evidence in literature demonstrates the effects of cutting time (CT) and regrowth age (RA) of elephant grass on plant nutritive value and the fermentative profile of the silages produced. However, there are no studies on the effects of combining these two factors on the nutritive value and fermentative profile of elephant grass silage. Therefore, we hypothesized that CT and RA, or their interaction affects the nutritive value, especially soluble carbohydrate content, and fermentation of elephant grass during ensiling. We aimed to identify the best combination for silage production without the need for wilting and additive use.

Test Sites, Soil, and Climatic Conditions
Two trials (Trials 1 and 2) were conducted simultaneously at two different sites based on soil chemical characteristics at the Animal Science Department of the Federal University of Viç osa (DZO), UFV, at the Viç osa Campus, from September 2015 to April 2016. The municipality of Viç osa (20°45′20″ S, 42°52′40'″ W; altitude, 657 m) is in Zona da Mata, in the state of Minas Gerais. The climate is of the Cwa type according to the Köppen classification, with two defined seasons: dry, from May to October, and wet, from November to April. The soil is classified as eutrophic red-yellow Argisol clay at both sites .
Organic fertilizer in the form of dry bovine manure was applied to the elephant grass in site 1 and as bovine manure water (1:1) in site 2. The chemical characteristics of soil samples from the two sites are listed in Table 1. ISSN 2166-0379 2021

Ensiling and Experimental Design
The treatments of both trials were arranged in a 2 × 2 factorial scheme, with two CTs (08:00 and 14:00) and two RAs (8 and  The forage was chopped using a stationary forage machine and packed in piles of approximately 8 kg to fill each bucket. A sample from each pile was collected to assess the buffering capacity, pH, microorganism population, and chemical composition of elephant grass before ensiling.
To make the silage, 12 kg plastic buckets were used as experimental silos. After weighing the buckets with lids, they were filled, and the forage was compacted with the aid of wooden sockets. Silage densities varied between 141 and 182 kg/m³ DM, owing to the different DM content of the samples. The silos were then sealed, weighed, and stored in a covered area at an average temperature of 23 °C for 60 d.
The experimental silos were then weighed and opened, and the silage within each silo was homogenized. An aliquot of the material was removed and divided into two sub-samples; the first (25 g) was used to quantify the lactic acid bacteria (LAB) population, enterobacteria, fungi (molds and yeasts), and the fermentative profile (pH, organic acids, ammonia, and ethanol), and the second sample (300 g) was used for chemical composition analysis.

Quantification of Microbial Populations
To quantify the microorganism population, an aqueous extract was prepared containing 25 g of plant or silage sample, which was homogenized together with 225 mL of buffer solution (Ringer Solution ® ; Oxoid, Hampshire, United Kingdom) for 1 min in a blender to obtain a 10 -1 dilution (Kung Jr., 1996). Subsequently, 10 mL of the aqueous extract was subjected to serial dilutions ranging from 10 -2 to 10 -7 . The microorganisms were cultured in sterile Petri dishes in appropriate culture media for different microorganism groups. MRS agar ( Brazil). Plates that had 30-300 colony forming units (CFU) were counted. For data evaluation and interpretation, the results were converted to a logarithmic basis (log CFU/g) (Kung Jr. et al., 2003).

Fermentative Profile and Chemical Composition
A sample of approximately 150 g of chopped elephant grass was collected before ensiling to analyze soluble carbohydrate content (Nelson 1944) and buffer capacity (Playne and McDonald 1966).
The fermentative capacity (FC) of forages before ensiling was calculated according to the equation proposed by Kaiser et al. (2002): where DM is dry matter (%), SC is soluble carbohydrates (%), and BC is buffering capacity (e.mg of NaOH/100 g DM).
The pH of the initial aqueous extract (25 g silage/225 mL of Ringer Solution) of the samples was measured using a digital potentiometer (Tecnal), as described by Kung Jr. (1996).
A 10 mL aliquot was removed from the aqueous extract and placed in Falcon tubes containing a sulfuric acid solution (50%) and frozen for further analysis of organic acids (lactic, acetic, propionic, and butyric acids) and ethanol. Organic acid and ethanol analyses were conducted using a high-performance liquid chromatograph (HPLC; Accella PDA model; Thermo Fisher Scientific, Hemel Hempstead, UK) coupled with an ultraviolet (UV) detector at 210 nm wavelength, as described by Siegfried et al. (1984). The ammonia nitrogen to total nitrogen content (N-NH3/TN) was also quantified according to the methodology described by Okuda et al. (1965). ISSN 2166-0379 2021 To evaluate the chemical composition of the samples, partial moisture removal was carried out in an oven with forced-air ventilation at 55 º C until a constant weight was achieved. Subsequently, the samples were processed in a Model 4 Wiley Mill (Thomas Scientific, Swedesboro, NJ, USA) with a 1 mm sieve. The DM, CP, neutral detergent fiber corrected for ash and protein (NDFap), ADF, mineral matter, neutral detergent insoluble nitrogen (NDIN), and acid detergent insoluble nitrogen (ADIN) contents were determined according to the methodologies described by Detmann et al. (2012).

Journal of Agricultural Studies
For the in vitro dry matter digestibility (IVDMD) test, silage samples (0.5 g) were placed in ANKOM F57 ® filter bags (Ankom Technology, Macedon, NY, USA) and incubated in an ANKOM Daisy Incubator for 48 h in jars containing buffer solution and ruminal fluid. The ruminal fluid was collected from fistulated cattle as described by Holden (1999).
The quality of all elephant grass silages was estimated using "Flieg's point," calculated using the equation proposed by Kilic (1986), cited by Moselhy et al. (2015): According to this classification, the silage quality was considered "very bad" at an index <20; "bad" at an index between 21 and 40; "average" at an index between 41 and 60; "good" at an index between 61 and 80; and "very good" between 81 and 100.

Statistical Analysis
Data were analyzed according to a completely randomized design in a 2 × 2 factorial, using PROC MIXED of SAS version 9.4 (SAS Institute, Inc., Cary, NC, USA). The RAs, CTs, and interaction between these factors were considered fixed effects in the model: where Yijk is the dependent variable, μ is the general mean, RAi is the effect of RA, Hj is the effect of CT, (RA × H)ij is the interaction between factors, and eijk is the random error, assuming a normal independent distribution, (0; σ2ε). The treatment means were subjected to analysis of variance and comparison using the F-test, adopting 0.05 as the critical level of probability for type I error.

Chemical Composition, Buffer Capacity, pH, and Epiphytic Microflora of Elephant Grass Before Silage
Tables 2 (Trial 1) and 5 (Trial 2) show the results for chemical composition, buffer capacity, pH, and epiphytic microflora of elephant grass harvested at different CT and RA. The highest concentrations of soluble carbohydrates were observed in elephant grass harvested at 14:00 than at 08:00 at both RAs. The highest concentrations of soluble carbohydrates were observed for grass harvested at 8 weeks than at 16 weeks at both CTs. ISSN 2166-0379 2021

Trial 1
The results for Trial 1 are presented in Table 3. pH, lactic acid, and ethanol were significantly affected (P < 0.05) by CT and RA. The silages produced with elephant grass harvested at 14:00 or 8 weeks of regrowth showed the lowest pH values (P < 0.05) and the highest lactic acid and ethanol levels (P < 0.05).  ISSN 2166-0379 2021 The RA × CT interaction significantly affected (P < 0.05) the levels of acetic, propionic, and butyric acids, ammonia nitrogen, and populations of LAB and fungi. The silages produced with 16 week plants harvested at 14:00 had a lower concentration of acetic acid than those harvested at 8:00. Silages produced at 16 weeks showed lower propionic acid concentrations than those produced at 8 weeks at both CTs. The butyric acid concentration was higher in silages from plants harvested at 16 weeks and 08:00. Lower concentrations of ammoniacal nitrogen (NH3-N/TN) were obtained in silage produced with 16 week plants harvested at 14:00.

Journal of Agricultural Studies
Larger LAB populations were observed in elephant grass silages at 16 weeks compared with plant silages at 8 weeks at both CTs. Elephant grass silages harvested at 8 weeks at 08:00 contained no fungi (molds and yeasts). No enterobacteria populations were observed in the silages at the evaluated CTs and RAs.
Flieg's point values were significantly affected (P < 0.05) by the H × RA interaction (Table 2). Silage production using elephant grass harvested at 14:00 at both RAs resulted in higher Flieg's point values than elephant grass harvested at 08:00, and elephant grass silage harvested at a regrowth age of 16 weeks showed higher Flieg's Point values, compared with that harvested at 8 weeks at both CTs.

Trial 2
Trial 2 results are presented in Table 6. There was a significant RA × CT interaction (P < 0.05) effect on the variables pH, lactic, acetic, propionic, and butyric acids, and ammoniacal nitrogen. Higher pH value (P < 0.05) was observed when the ensiled plants were harvested at 08:00 than 14:00 with 8 weeks of regrowth.
Higher lactic acid levels (P < 0.05) were obtained in the silage of elephant grass harvested at 8 weeks compared with elephant grass harvested at 16 weeks of regrowth at the two CTs. Acetic acid production was lower (P < 0.05) in silage produced from elephant grass harvested at 14:00 than the silage from elephant grass harvested at 08:00, during the two RAs. Higher contents of acetic and butyric acids (P < 0.05) and ammoniacal nitrogen (P < 0.05) were observed in the silage of elephant grass harvested at 8 weeks at 08:00.
Ethanol content and LAB population were affected (P > 0.05) only by RA. Higher (P < 0.05) LAB populations and lower (P < 0.05) ethanol concentrations were obtained in silage produced from plants harvested at 16 weeks than from that harvested at 8 weeks. Enterobacteria and fungi (molds and yeasts) populations were not observed in these silages.
The Flieg's point values were affected (P < 0.05) by the CT × RA interaction (Table 5), being higher in silages produced at 14:00 than those produced at 08:00 at the two RAs. Whereas silage of elephant grass harvested at 16 weeks presented higher Flieg's point values than that harvested at 8 weeks at both CTs.

Trial 1
Trial 1 results are listed in Table 4. DM and CP levels were significantly affected (P < 0.05) by the CT × RA interaction. Silage produced from elephant grass harvested at 14:00 showed higher DM levels and lower CP levels than silages produced from elephant grass harvested at 08:00 at both RAs. Silage produced from elephant grass harvested at 16 weeks of regrowth had higher DM levels (P < 0.05) and lower CP levels than silage from elephant grass harvested at 8 weeks of regrowth at both CTs. The levels of NDFap, ADF, and the IVDMD coefficients varied significantly (P < 0.05) with RA, while NDIN and ADIN levels varied with time and age. The levels of NDF, ADF, NDIN, and ADIN were lower for silage from plants harvested at 8 weeks; consequently, the IVDMD of this silage was higher. Higher levels of NDIN and ADIN were observed in the silage of elephant grass harvested at 14:00.

Trial 2
The results of Trial 2 are presented in Table 7. The DM and NDFap variables were significantly affected by CT (P < 0.05) and by RA (P < 0.05). Higher DM levels were observed in the silage of elephant grass harvested at 16 weeks or 14:00, while lower NDF levels were obtained for elephant grass harvested at 8 weeks or 14:00. A significant CT × RA ISSN 2166-0379 2021 interaction (P < 0.05) effect was observed on CP, ADF, NDIN, and ADIN and IVDMD coefficients. The CP contents were higher, and ADF and ADIN contents lower in the silage of plants harvested at 14:00 than at 08:00 at both RAs. Thus, the IVDMD coefficients were higher in plant silages harvested at 14:00 than at 08:00 at both RAs.  ISSN 2166-0379 2021

Discussion
The initial concentration of soluble carbohydrates in the plant at the time of ensiling affects the organic acid production, since that are used as a substrate by LAB. Lactic acid causes the rapid decline in pH and maintains forage stability during storage in the silo by inhibiting or eliminating undesirable and low pH-intolerant microorganisms . As verified in the present study, in both trials, the highest concentration of soluble carbohydrates in elephant grass harvested at 14:00 was responsible for increasing lactic acid levels and reducing silage pH, consequently improving silage quality (Tables 2, 3, 5, and 6).
The silage from elephant grass harvested at 08:00, at 8 weeks, showed satisfactory lactic acid concentrations (34.52 g kg -1 DM; Trial 2) according to Kung Jr. et al. (2018), who reported lactic acid concentrations of 20 to 40 g kg -1 DM for well-fermented silages. However, these silages showed higher pH values (5.02) relative to the other treatments, which may be related to the high levels of butyric and acetic acids, and ammonia (NH3-TN; Table 3). These compounds produced through fermentation of soluble carbohydrates and protein degradation by clostridia and enterobacteria increase the buffering capacity of the forage in the silo, preventing pH from declining (Dong et al., 2018).
The lower pH values in silages produced from elephant grass harvested at 14:00 relative to those of silages produced from elephant grass harvested at 08:00 (Trial 1) corroborate the results presented by Guo et al. (2015), who observed a pH value of 4.07 in the silages produced from elephant grass harvested at 08:00, and 3.87 in silage produced from elephant grass harvested at 13:00. The same authors reported an increase in the concentration of soluble carbohydrates, from 80.6 to 107 g kg -1 DM in elephant grass harvested in the morning and afternoon, respectively.
The levels of acetic acid in both trials (except for silages produced with 8 week plants at 08:00 in Trial 2) were less than 10 g kg -1 DM, below the limits recommended by  (10-30 g kg -1 MS) and Gerlach et al. (2021) (17 g kg -1 MS) that are characteristic of well-fermented silage. The butyric acid levels observed in the trial 1 silage were considered adequate, consistent  at <1 g kg -1 DM (Table 3). However, in trial 2, silages from plants harvested at 8 weeks, at 08:00 am, had a high butyric acid concentration (12.35 g kg -1 DM), which can be explained by the occurrence of secondary fermentation mainly because of bacteria of the genus Clostridium (Driehuis et al., 2018). Clostridium-fermented silages, in addition to the presence of butyric acid, have a high pH and high concentrations of acetic acid and NH3-TN (Oladosu et al., 2016;Kung Jr. et al., 2018) (Table 6).
Generally, silages from plants harvested with a low DM content have higher soluble N and ammoniacal nitrogen concentrations than silages with a high DM content. In the present study, although the elephant grass DM values were below the recommended (25-35%), the silage (except at 8 weeks, at 08:00 in Trial 2) presented low NH3-TN concentrations (< 12 g kg -1 DM), which indicates good fermentation quality, according to the criteria proposed by Kung Jr. et al. (2018). The lower levels of NH3-TN could be attributed to the low proteolytic activity of microorganisms, such as bacteria of the genus Clostridium, which are inhibited because of the rapid decline in pH during the fermentation of soluble carbohydrates by the LAB epiphytic population. (Ni et al., 2017;Wang et al., 2019a).
The epiphytic LAB population present in forage is important for silage fermentation. For good forage fermentation, the required LAB population is 5 log CFU g -1 fresh weight (FW) (Pahlow et al., 2003). LAB populations >5 log CFU g -1 FW were observed in both trials, which benefited the fermentation process during ensiling (Tables 2 and 5). The variation in the epiphytic LAB population in elephant grass between CT and RA is probably associated with environmental conditions, such as humidity, temperature, and solar radiation (Guo et al., 2015). These authors did not observe differences in the LAB population in elephant grass silages produced from plants harvested at 08:00 and 13:00. In the present study, this behavior was observed only in trial 2 (Table 6), while in trial 1, there was an increase in the LAB population in silage produced from plants harvested at 8 weeks at 14:00. As seen in literature and the present work, further studies are needed to understand the behavior of LAB in silages produced with forages harvested at different times.
The low mold count in the silages of the two trials is probably related to the high mass compaction at the time of ensiling (141-182 kg m -³ DM), which favored rapid oxygen exhaustion inside the silos. In addition to compaction, acetic acid fermentation may have also contributed to reducing the fungal population since acetic acid has antifungal properties Muck et al., 2018). Molds and yeasts are the main ethanol-producing microorganisms during the fermentation period in the silo . Despite the low mold and yeast populations observed, ethanol concentrations in the silage produced in trial 2 (mainly after 8 weeks of regrowth, 16.21 g kg -1 DM) were higher than those in trial 1 (Tables 3 and 6). Some authors have reported that in addition to that produced from molds and yeasts, ethanol can be produced by enterobacteria, and heterolactic bacteria (Gomes et al., 2019;Wang et al., 2020;Ávila and Carvalho, 2020). This could explain the higher ethanol production observed in the silages in the present study, which was above the level suitable for tropical grass silages (5-10 g kg -1 DM) according to Kung Jr. et al. (2018).
Based on Flieg's point evaluation system, the silage fermentation in Trial 1 was classified as very good, indicating silage of adequate quality since all silages presented a Flieg's point > 80. In Trial 2, the quality of the silage produced with elephant grass harvested at 14:00 and 16 weeks was classified as having very good fermentation quality; silages produced with elephant grass harvested at 8 weeks at 14:00 and 16 weeks at 08:00 were considered good quality silages. Those produced with elephant grass harvested at 8 weeks at 08:00 were considered to be of poor quality.
Flieg's point, based on the DM content and the pH value of silages, has been used to evaluate silage quality, although there are some limitations in this scoring system. According to Yuan et al. (2017), Flieg's point does not consider organic acids, the extent of protein degradation, or the aerobic stability of the silage.
For good silage fermentation, it is recommended to cut grasses with a DM content of 25-35% . Although the DM content of plants harvested at 8 weeks was below the minimum requirement, the results obtained in the present study showed that it was possible to produce silages of adequate nutritional value (Tables 4 and 7). In a study with elephant grass cv Napier, De Oliveira et al. (2014) observed DM content of 20.5% and 16.8% when the cut was made in the afternoon and in the morning, respectively. In a similar study, De  also observed an increase in DM accumulation in Marandu palisade grass from 06:00 to 15:00. Guo et al. (2015) reported increased DM concentrations in plants and silages produced from elephant grass harvested in the afternoon (13:00) than at 8:00, as verified in the present study. The higher DM values observed in the silages (Tables 4 and 7) relative to the elephant grass at the harvest time (Tables 2 and 5) could be attributed to the effluent losses observed after opening the experimental silos; however, this was not quantified in the present study.
The reduction of concentrations of soluble carbohydrates in the plant increases the concentration of CP (De Oliveira et al., 2014;Wang et al., 2020a), which explains the greater attributions of this nutrient in the silages produced in the morning than from silages produced in the afternoon (Tables 2,4,5, and 7). De Oliveira et al. (2014) reported an 85% increase in soluble carbohydrates and a 12% reduction in N from 06:00 to 18:00 in Cenchrus purpureus cv. Napier. De , who evaluated the diurnal vertical and seasonal variations of non-structural carbohydrates in Marandu grass, observed a similar result. In trial 2, in the silage produced at 08:00 h at 8 weeks, significant reduction in CP and increase in N-NH3 contents, butyric and acetic acids (Table 6) indicate the occurrence of proteolysis in the silo, which is related to clostridial and/or enterobacterial activity (Wang et al., 2020), especially under conditions of high humidity.
Since nitrogen compounds are present in the cellular content of young plants, with increasing RA, there is a reduction or dilution of the CP content due to the accumulation of structural carbohydrates. Monç ã o et al. (2019) verified a linear reduction of 0.37 g kg -1 per day in the CP concentration in BRS Capiaç u, with an average of 123,2 g kg -1 in 30 days. A similar effect was observed in the present study since silages of elephant grass harvested at 16 weeks presented lower CP concentrations than silages of elephant grass harvested at 8 weeks. When evaluating chemical composition, before and after ensiling of elephant grass BRS Capiaç u at four regrowth ages (50, 70, 90, and 110 days), Lopes et al. (2021) verified linear decreases in CP content with an increase in RA.
In Trial 1, silage of elephant grass harvested at 16 weeks showed lower IVDMD coefficients, probably because of the increase in the NDF and ADF levels that occurred with advancing RA. A similar result was observed by Lopes et al. (2021), with the advancing RA of elephant grass cv. BRS Capiaç u. In Trial 2, the highest IVDMD coefficients in elephant grass silage harvested at 14:00 could be attributed to the lower levels of NDFap, ADF, NDIN, and ADIN.
A similar result was observed by De Oliveira et al. (2019) in a study with palisade grass harvested in the afternoon compared with that collected in the morning.
Earlier RA and later cutting of elephant grass improved the chemical composition of the grass and the fermentation process in the silo. Therefore, harvesting elephant grass at 8 weeks and 14:00 is recommended to produce better quality silage without wilting or additives. Additional studies are needed to evaluate the intake and performance of animals fed the tested silages.