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Sorghum silage quality as determined by chemical–nutritional factors

Article in Grass and Forage Science · August 2020

DOI: 10.1111/gfs.12495

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Received: 28 May 2019 | Revised: 26 June 2020 | Accepted: 29 June 2020

DOI: 10.1111/gfs.12495

ORIGINAL ARTICLE

Sorghum silage quality as determined by chemical–nutritional

factors

Paulo Henrique Mazza Rodrigues1 | Lerner Arévalo Pinedo1 | Paula Marques Meyer2 |
Thiago Henrique da Silva3 | Iuli Caetano da Silva Brandão Guimarães1

1
Department of Animal Nutrition and
Production, College of Veterinary Medicine Abstract
and Animal Science, University of Sao Paulo, Sorghum [Sorghum bicolor (L.) Moench] is an ensilable tropical plant known as a good
Pirassununga, Brazil
2 alternative to maize crops in regions with scarce rainfall. The objective of this trial
Brazilian Institute of Geography and
Statistic (IBGE), Pirassununga, Brazil was to obtain prediction models based on nutritional contents and end products of
3
Department of Animal Science, School sorghum silage fermentation as related to the dry-matter composition of fresh plants
of Animal Science and Food Engineering,
University of Sao Paulo, Pirassununga, Brazil
before ensiling. Eleven different sorghum cultivars (including silage, graniferous and
sweet types) were used. Twenty-five sorghum plots were harvested between 80 and
Correspondence
Paulo Henrique Mazza Rodrigues,
120 days of growth. Fifty plastic buckets were used as experimental microsilos and
Department of Animal Nutrition and opened between 60 and 90 days of storage. Statistical modelling was used to create
Production, College of Veterinary Medicine
and Animal Science, University of Sao Paulo,
a prediction equation that could explain the impact of fresh sorghum composition
Duque de Caxias Norte, 225, Pirassununga, on the chemical and nutritional composition of its silage. A complex model was de-
SP 13635-900, Brazil.
Email: pmazza@usp.br
tected by stepwise multiple regression to predict the difference of in vitro dry-matter
digestibility (IVDMD) before and after ensiling, but a simpler model, which involved
only the sum of water-soluble carbohydrate (fWSC) and hemicellulose (fHemi) con-
centrations in the dry matter of fresh forage, was considered to more usable. It had
an acceptable coefficient of determination (0.51). The higher amount of WSC and
Hemi in fresh sorghum linearly decreased the difference between IVDMD before
and after the ensiling process. A WSC concentration of 125.4 g/kg DM in fresh sor-
ghum is recommended for an ideal silage fermentation when considering pH and
lactic acid levels, although ethanol levels continued to increase.

KEYWORDS

conservation, ensiling, fermentation, fresh sorghum, prediction models

in regions with low or irregular rainfall (Getachew, Putnam, Ben, &

1 | I NTRO D U C TI O N Peters, 2016). Compared with maize plants, sorghum plants require
less fertilization and less pest control and may grow in soils with a
Forage availability depends on seasonality, and this may cause prob- low pH (AERC, 2007).
lems for farmers maintaining regular livestock productivity through- Several authors have reported good ensilability of sorghum
out the year. Therefore, forage conservation as silage has become an (Meeske, Ashbell, Weinberg, & Kipnis, 1993; Miron et al., 2006;
important and popular method of feed storing because it can mini- Ward, Readfern, McCormick, & Cuomo, 2001). The optimal amounts
mize the loss of nutrients after harvest during storage (Fulgueira, of water-soluble carbohydrate (WSC) in maize and grass plants are
Amigot, Gaggiotti, Romero, & Basílico, 2007). Sorghum [Sorghum bi- 80–200 and 100–200 g/kg DM respectively (Pitt, 1990); in sorghum
color (L.) Moench] is a tropical plant that efficiently uses water and plant, it is 110–280 g/kg DM. Frequently, sorghum and maize crops
it is a good alternative to maize crops for feeding ruminants, mainly are associated with a high lactic acid concentration in silage, which

Grass Forage Sci. 2020;00:1–12. wileyonlinelibrary.com/journal/gfs© 2020 John Wiley & Sons Ltd | 1
2 | RODRIGUES et al.

decreases pH values to the extremely important range required for

quality: 3.53–4.2 (Miron et al., 2006). Despite a lower crude pro-
Highlights
tein content and a higher fibre content compared with maize silage,
• Fresh sorghum chemical and nutritional components did
sorghum silage has the potential to support ruminant performance;
alter its silage quality.
therefore, it is an acceptable candidate for extensive use in livestock
• The more hemicellulose and WSC in fresh sorghum, the
(Harper et al., 2017).
less difference between fresh and ensiled dry-matter
It is, however, important to point out that the large variation
digestibility.
in nutritive composition among the different commercial sorghum
• Silage ethanol concentration increased linearly accord-
cultivars can lead to differences in their individual silage. Further,
ing to water-soluble carbohydrates (WSC) level.
the harvest date can alter the silage quality (Miron et al., 2006).
• Lactic acid production and pH were optimized at 125 g
Gaining greater knowledge about the biochemical processes that
WSC/kg DM in fresh sorghum.
occur during sorghum fermentation into the silo environment is nec-
essary. The ensiling fermentative process and productive efficiency
of ruminants are not only affected by the initial roughage compo-
sition (Huhtanen, Nousiainen, Khalili, Jaakkola, & Heikkilä, 2003;
Huhtanen, Rinne, & Nousiainen, 2007), but also by the end products 25 km. The experimental fields were divided into 25 plots (experi-
of fermentation that have a large influence on it as well (Kung et al., mental units—EU), with two repetitions per plot (25 EU × 2 repeti-
2018). Still, a negligible amount of studies is available to provide data tions = 50 microsilos). Eight cultivars were grown in field 1; another
about individual end products of silage fermentation related to their 8 cultivars in field 2; 3 cultivars in field 3; 3 cultivars in field 4; and
initial composition (Mogodiniyai Kasmaei, Rustas, Spörndly, & Udén, another 3 cultivars were planted in field 5. All plots were cultivated
2013). in the same month in the agricultural season of 2015–2016. All were
The aim of this study was to obtain prediction models for end grown under the same environmental conditions. The soil type in
products of fresh sorghum fermentation. We hypothesized that this region is red latosol, and the local climate is subtropical (type
the composition of fresh sorghum could explain the fermentation Cwa according to Köppen). Fertilizer rate was maintained according
pattern of sorghum silage when performed under strictly anaerobic to soil test recommendations (120, 30 and 80 kg of N, P and K re-
conditions. spectively, per hectare). Eleven different harvest times were used in
this trial (87, 95, 97, 100, 101, 104, 107, 110, 111, 114 and 120 days;
from the late line-milk to farinaceous stage of the seed) and this
2 | M ATE R I A L S A N D M E TH O DS range was important to increase dataset variability, according to the
purpose of this experiment.
Regarding ethical aspects, this paper did not use living animals. After manual harvesting (stubble height = 10 cm) under clean
Cannulated animals used as inoculum donors at analysis of in vitro conditions, sorghum plants were transported to the laboratory,
dry-matter digestibility were approved by the Bioethics Committee where they were chopped (Chopper Nogueira, model EM-9F3B) to
of the College of Veterinary Medicine and Animal Science, University obtain a mean theoretical length of cut = 14 mm. The evaluation of
of São Paulo (approval number: 8453300914). mean theoretical particle size was performed by “Penn State Particle
Size Separator” proposed by Lammers, Buckmaster, and Heinrichs
(1996). Chopped material was manually mixed and ensiled without
2.1 | Experimental site, forage silage additives. All these procedures were completed within one
production and ensiling hour.
Fifty sterile polyvinyl chloride (PVC) plastic buckets (2 rep-
The trial was carried out at Department of Animal Nutrition and etitions for each plot) with 22 cm of diameter × 30 cm of height
Production of the College of Veterinary Medicine and Animal equipped with Bunsen valves for gas escape were used as ex-
Science of University of São Paulo, Brazil. perimental microsilos. Two kilograms of sand was placed in the
For ensiling, 11 different sorghum [Sorghum bicolor (L.) Moench] bottom of the buckets and covered with a nylon mesh screen
varieties were used, including the silage (AGRI–001E—Agricom; (500 µm) to drain effluents. The forage was placed inside each
AGRI–002E—Agricom; Volumax—Agroceres), the graniferous (Ag microsilo and compacted manually using disposable sleeve gloves
1080—Agroceres; Ag 1085—Agroceres; Ag 1090—Agroceres; BRS and plastic boot covers until the density of 500 kg of sorghum
373—Embrapa; BRS 380—Embrapa) and the sweet (BRS 511— plant/m 3 . Microsilos were sealed with tight PVC lids, weighed and
Embrapa; CB 7290—Ceres; CB 7521—Ceres) cultivars. These cul- vertically stored in a covered area at room temperature of 25°C
tivars were selected to provide variation in chemical composition, and opened between 60 and 90 days of storage (mean fermenta-
which was important for this modelling study. tion time = 68.8 ± 8.9 days). Plantations were harvested in varied
Sorghum cultivars were grown in 5 experimental fields. Two days of growth and microsilos were opened in different matura-
farms were used, and the distance from one farm to another was tion stages in order to try to achieve the diverse conditions that
RODRIGUES et al. | 3

these two important steps represent as those in farm conditions by gas chromatography and according to methodology proposed by
and also to increase the data variation required for this type of Erwin, Marco, and Emery (1961), which uses a gas chromatographer
study. (Finnigan, model 9001) equipped with silica glass column MEGABOR
(Ohio Valley, model OV-351) of 30 m × 0.53 mm and stationary
phase of 1.0 micron.
2.2 | Laboratory analysis Another 2 ml of silage juice subsample was added to 1 ml
of sulphuric acid 1 N and frozen at −20°C for analysis of am-
2.2.1 | Before ensiling monia nitrogen (NH 3-N) concentration by colorimetric assay, as
described by Kulasek (1972) and adapted by Foldager (1977).
After chopping, one sample of fresh material was obtained and Absorbance measurements were performed in spectropho-
stored at −20°C for later determinations of the following: dry tometer (Beijing Rayleigh AIC model VIS-7220) set at 630 nm.
matter (DM), ash, organic matter (OM), ether extract (EE), crude Absorbance values were used to calculate NH 3-N concentrations
protein (CP), acid detergent fibre (ADF), acid detergent insoluble in mg of NH 3-N/100 ml by a linear regression equation obtained
nitrogen (ADIN), neutral detergent fibre with residual ashes as- through equipment calibration with standard solutions at differ-
sayed with a heat stable amylase (aNDF), neutral detergent insolu- ent concentrations.
ble nitrogen (NDIN), lignin, hemicellulose (Hemi), cellulose (Cell), Dry-matter (DM) losses during fermentation were calculated
water-soluble carbohydrates (WSC), starch, in vitro dry-matter as the difference between the weights of masses obtained at the
digestibility (IVDMD), in vitro NDF digestibility (IVNDFD), buffer moments of filling the silo and the silo's opening, multiplied by the
capacity (BC), calcium (Ca), phosphorus (P), total digestible nutri- respective DM contents. Dry-matter losses were transformed in
ents (TDN) and net energy for lactation (NEl), according to meth- percentage of initial mass. Because the aim of the study was to un-
odologies described below. After thawing, this fresh material was derstand the total DM losses, no separate analysis was performed
analysed simultaneously with silages in order to avoid analytical for gas losses or effluent losses.
variation.

2.3 | In vitro digestibility

2.2.2 | After ensiling
In vitro dry-matter digestibility and IVNDFD were determined
Before opening, silos were weighed for determination of DM losses in conformance with Tilley and Terry (1963). Filter bags (F-57;
during fermentation. After opening the silos, each mass was ho- 50 × 55 mm; ANKOM Technology Corporation) were identified and
mogenized and a fraction was separated for determinations of DM, weighed. In the formula below, the bag was considered the inoculum
ash, EE, CP, Ca and P according to AOAC (1995) and aNDF, ADF and without sample. Approximately 0.5 g of forage samples previously
lignin according to Van Soest, Robertson, and Lewis (1991). Organic oven-dried and ground were weighed and inserted into filter bags in
matter (OM) was determined by the difference between DM and duplicates. These bags were put into test tubes.
ash content. Hemicellulose and cellulose content were obtained by Into test tubes, 40 ml of McDougall solution (artificial saliva)
the difference between aNDF and ADF and also between ADF and were added to 10 ml of rumen inoculum from 3 different steers
lignin respectively. Water-soluble carbohydrate was determined that had been kept at pasture grazing Brachiaria decumbens and
according to Johnson, Balwan, Johnson, McClure, and Dehority then supplemented with 3.0 kg of maize silage (DM basis) and ad
(1966), and the ADIN and the NDIN were determined as reported libitum mineral salt. Tubes were sealed with rubber corks contain-
by Van Soest and Robertson (1985). Buffer capacity has been ing a Bunsen gas release valve, immediately after flushing out with
measured by the quantity in meq of H+ that is spent to decrease pH CO2, and incubated in oven for 48 hr under controlled temperature
values of forage from 6.0 to 4.0, which indicates the resistance of (39°C). They were agitated a minimum of 3 times during fermenta-
organic and inorganic components to acidification during preser- tion. The second IVDMD phase occurred after discarding the liquid
vation (McDonald, Henderson, & Heron, 1991). Additionally, TDN solution. A pepsin solution (1:10.000) at 0.2% (50 ml) was added to
and NEl were estimated by forage or silage chemical composition each tube, followed by agitation at 39°C for another 48 hr. After
as stated in NRC—Dairy Cattle (2001), considering a cow eating at washing, drying and weighing the bags, calculations were performed
a 3× maintenance level. as the formula below:
One silage fraction was placed in hydraulic press for silage juice
extraction. Immediately after material pressing, 50 ml of silage juice [
g of DM in sample − (g of residual DM − g of DM of inoculum without sample)
]
IVDMD = 100 × .
were used for pH determination with a portable digital pH meter
g of DM in sample

(Procyon, model 310) that was calibrated with pH buffer solutions

of 4.0 and 7.0. Also, 2 ml of silage juice were added to 0.4 ml of Same procedure was performed in IVNDFD analysis, except
formic acid and frozen at −20°C for further organic acids and etha- that the bags were submitted to aNDF washing procedures after
nol concentration analysis. Both of these analyses were determined incubation.
4 | RODRIGUES et al.

2.4 | Statistical analysis Harvest date and ensiling duration were excluded from the
modelling process because they were not correlated with other
Data were analysed using the SAS 9.3 (SAS Institute Inc.). Before variables.
the actual analysis, data were explored to seek disparate infor- Ensiling resulted in significantly higher contents of ash, CP, EE,
mation (“outliers”) and for normality of residuals by Shapiro–Wilk aNDF, ADF, lignin, Cell, Ca and P, compared with fresh sorghum
test (PROC UNIVARIATE). An individual observation was consid- (p < .05). On the other hand, lower DM, OM, NDIN, WSC and Hemi
ered outlier when the standard deviations in relation to the mean contents were detected (p < .05). Only ADIN and starch values did
or to the model were larger than + 3 or less than −3. Data which not change after ensiling (p > .05). Nutritional characteristics were
did not attend to normality premise were subjected to logarith- also different between fresh and ensiled sorghum. Lower values for
mic [Log (X + 1)] or by square root [RQ (X + 1/2)] transformation. IVDMD, IVNDFD, TDN and NEl were found in sorghum silage than in
The values 1 and ½ were added in the Log and RQ equations in fresh sorghum (p < .05). Despite the differences observed between
order to avoid negative values in the analyses when values be- fresh sorghum and its silage, chemical and nutritional composition
tween 0 and 1 were involved. Initially, statistic treatments of data was highly correlated for most components, except EE and hemicel-
were organized in 5 distinctive parts: (a) descriptive characteri- lulose (Table 1).
zation of forage and silage regarding chemical composition and Principal component analysis (PCA) for chemical and nutritional
fermentative pattern; (b) magnitude of changes in chemical and compositions showed a strong association between DM concentra-
nutritional plant parameters, referring to the ensiling process; (c) tions before and after ensiling (Figure 1). It was the same for ash, cell
forage composition influence on silage fermentative pattern; (d) wall components (except hemicellulose), CP, digestibility and energy.
losses causes attributed to ensiling; and (e) changes in BC regard- Concentrations of hemicellulose, EE, BC and WSC from fresh sor-
ing ensiling. ghum were weakly associated with those in ensiled sorghum in the
Ordinary descriptive statistics, as mean and standard deviations, PCA analysis.
were performed. The mean of each duplicate was used. Data in fresh It was possible to observe two main groups of variables in PCA
forage and in silage were compared with a t test. If the difference analysis. One group on the right side of the graph is formed by DM,
equal to zero hypothesis was rejected, the chemical composition of ash, cell wall components (excluding hemicellulose) and ADIN. The
silage for a specific component or nutrient was different from that in group on the left side is formed by digestibility, energetic items and
forage. Principal component analysis was performed to illustrate the CP. Starch seemed to be an isolated group neither related to cell wall
observed differences. components nor to energetic items. Principal component analysis
Multiple regression analyses were performed for some selected was useful for these data: 2 factors in PCA explained 78.4% of the
variables to determine the causes of change in nutritional value overall variation observed (64.7% was factor 1 and 13.7% was factor
during ensiling. For multiple regressions, the difference between si- 2).
lage and forage nutritional value was regressed on fresh sorghum An important aim of this study was to indicate which nutrients
composition. The best model was chosen using a stepwise selection were most associated with quality loss during the ensiling process.
procedure of PROC REG (SAS). Total digestible nutrients and IVDMD differences before and after
The influence of forage composition on silage fermentative ensiling were analysed by a stepwise model selection procedure
pattern was determined by simple and multiple regressions. For in order to include in their prediction equations the variables with
simple regressions analyses, polynomial (linear and quadratic), ex- higher F value. For TDN difference, the best model included for-
ponential (first order kinetic) or broken-line models were created age CP and lignin content (dTDN = −200.8 + 2.20 fCP + 1.38 fLIG;
with PROC REG or PROC NLIN of SAS, depending on situation. p < .0001; adjR 2 = .8050, RMSE = 1.16).
Causes of losses and BC changes were studied with multiple re- The first complex selected model for IVDMD included fresh for-
gressions analyses. For all analyses, the significance level adopted age Hemi, WSC, DM, Ca and EE content (dIVDMD = −966.3 + 0.90
was 5%. fHEMI + 1.20 fWSC + 1.30 fDM + 39.80 fCa + 9.86 fEE; p < .0001;
adjR 2 = .8816, RMSE = 2.06). Nevertheless, DM depends on har-
vesting characteristics and fresh forage concentrations of Ca and
3 | R E S U LT S EE were low, so a simpler model was performed that included only
fresh forage Hemi and WSC (dIVDMD = −371.6 + 0.93 fHemi + 1.07
3.1 | Sorghum chemical and nutritional changes fWSC; p = .0025; adjR 2 = .5058, RMSE = 3.67). These predictive
after ensiling variables presented the biggest partial adjusted R 2 (.2605 and .2453
respectively) compared with DM, Ca and EE adjusted R 2 (.1678,
High variability among experimental units of both fresh and en- .0779 and 0.1301 respectively). In order to simplify the model and
siled sorghum was detected for chemical and nutritional charac- because the slopes for fresh forage Hemi and WSC were very close,
teristics. For some components (e.g. CP, ADIN, NDIN, EE, starch we proposed another equation, in which Hemi and WSC contents
and lignin), minimum and maximum values differed more than 2.7 are summed (dIVDMD = −369.1 + 1.00 (fHemi + fWSC); p < .0001;
times. adjR 2 = .5115, RMSE = 3.73; Figure 2).
RODRIGUES et al. | 5

TA B L E 1 Chemical, nutritional and

Fresh Silage
fermentative composition of fresh and
ensiled sorghum (g/kg DM, otherwise Item Mean SD CV Mean SD CV p-value r
stated)
Dry matter (g/kg) 260 28.9 11.1 237 29.6 12.5 <.0001 .85*
Ash 51 7 13.7 60.7 10.6 17.5 <.0001 .82*
Crude protein 49.1 17.8 36.3 51.8 16.9 32.6 .038 .93*
ADIN (g/kg N) 166 85.5 51.5 151 51.7 34.2 .168 .77*
NDIN (g/kg N) 255 80.3 31.5 212 59.5 28.1 .003 .60*
Ether extract 12.3 3.5 28.5 17 6.5 38.2 .005 −.14
Starch 46.5 21 45.2 44.1 29.8 67.6 .548 .80*
WSC 107 25.2 23.6 14.6 16.6 113.7 <.0001 .57*
aNDF 552 62.3 11.3 588 53.8 9.1 <.0001 .79*
ADF 359 35.9 10.0 423 60.4 14.3 <.0001 .86*
Lignin 53.7 17.3 32.2 59.7 20 33.5 .004 .87*
Hemi 192 41.3 21.5 165 22.2 13.5 .005 .087
Cell 306 23.5 7.7 363 43.3 11.9 <.0001 .71*
BC (meq/100 g 24.9 3.59 14.4 51.9 10 19.3 <.0001 .80*
DM)
IVDMD 603 90.2 15.0 534 91.1 17.1 <.0001 .83*
IVNDFD 407 110 27.0 341 106 31.1 <.0001 .85*
TDN 546 49.2 9.0 523 58.9 11.3 .0002 .90*
a
NEl (Mcal/kg 1.19 0.15 12.6 1.13 0.18 15.9 .0003 .91*
DM)
Ca 3.2 0.8 25.0 3.9 0.8 20.5 <.0001 .70*
P 1.2 0.2 16.7 1.5 0.2 13.3 <.0001 .48*
pH — — — 3.81 1.4 36.7 — —
NH3-N (g/kg CP) — — — 86.6 32.1 37.1 — —
Ethanol — — — 58.3 32.2 55.2 — —
Acetic — — — 24.4 5.2 21.3 — —
Propionic — — — 0.8 0 0.0 — —
Butyric — — — 0.2 0.2 100.0 — —
Lactic — — — 76.6 16.4 21.4 — —
Lactic/Acetic — — — 3.29 1.11 33.7 — —
DM losses — — — 154 77.8 50.5 — —

Abbreviations: ADF, acid detergent fibre; ADIN, acid detergent insoluble nitrogen; aNDF, neutral
detergent fibre assayed with a heat stable amylase; BC, buffer capacity; Cell, cellulose; CV,
coefficient of variation; Hemi, hemicellulose; IVDMD, in vitro dry-matter digestibility; IVNDFD,
in vitro NDF digestibility; NDIN, neutral detergent insoluble nitrogen; p-value, difference data
(bias) between silage and fresh sorghum using t test for mean equal zero; r, Pearson's correlation
coefficient between fresh and ensiled sorghum; SD, standard deviation; TDN, total digestible
nutrients; WSC, water-soluble carbohydrates.
a
Calculated net energy of lactation using nutrient digestibility values as presented in NRC (2001).
*p ≤ .01; **p ≤ .05.

3.2 | Fresh forage composition influence on silage forage components as predictors by stepwise procedure, included
fermentative pattern 3 variables (fWSC, fBC and fCELL), and R 2 was .5218. A simple
regression using only fWSC as predictor for lactic acid production
After considering biological and mathematical parameters, even- originated a broken-line model: Lactic acid = 36.8 + 0.37 fWSC;
tually fWSC was chosen to be the predictor for lactic, acetic, Joint in fWSC = 125.4 g/kg DM; Plateau in lactic acid concentra-
propionic and butyric acids, as well as for ethanol and pH concen- tion = 84.2 g/kg DM; R 2 = .3637; p = .0037. This model showed
trations. These were estimated in a simple regression equation. The that each increased unit of WSC resulted in an increase of 0.37
model created to estimate lactic acid production in silage, using units of lactic acid concentration in silage until a concentration
6 | RODRIGUES et al.

F I G U R E 1 Principal component
analysis for chemical and nutritional
composition of sorghum forage (f) and
silage (s). Lines between dots were placed
only to facilitate visual identification of
pairs

F I G U R E 2 Effects of forage
hemicellulose (fHemi) and water-soluble
carbohydrates (fWSC) concentration on
IVDMD difference (dIVDMD) between
original forage and silage sorghum

F I G U R E 3 The pH and
fermentation end products of
sorghum silage as affected by water-
soluble carbohydrate content before
ensiling (fWSC). 1Y = 36.8 + 0.37
fWSC; fWSC joint = 125.4 g/kg DM;
Plateau = 84.2 g/kg DM; R 2 = .3637;
p = .0037; 2Y = −14.7 + 0.68 fWSC;
R 2 = .3602; p = .0051; 3Y = 30.7.
e(−0.037. fWSC); R 2 = .6652; p < .0001;
4
Y = 1.5. e(−0.021. fWSC); R 2 = .5139;
p = .0004; 5Y = 4.44 – 0.0064 fWSC;
fWSC joint = 125.4 g/kg DM; pH
baseline = 3.62; R 2 = .9011; p < .0001;
6
Not significant
RODRIGUES et al. | 7

of 125.0 g/kg DM of fWSC, when lactic acid reached the plateau its silage fermentation pattern, shown in Figure 4. Ethanol and lactic
(Figure 3). acid were closer to the group located at the left side of the graphic,
To estimate ethanol production in silage, the stepwise proce- formed especially by digestibility and energetic items, as well as WSC
dure resulted in a very complex equation, which included 7 differ- and CP. Acetic, propionic and butyric acids were dispersed variables
ent variables (fWSC, fCELL, fEE, fDM, fP, fHEMI and fCP) with a among themselves, but they were more related to the forage cell
2
high coefficient of determination (R = .9911). Still, WSC was the wall components located at the right side of the graphic. These two
component with the highest partial R 2 (.3602). Thus, the simple factors explained 68.3% of overall variation observed (55.1% factor
regression using only fWSC as a predictor for ethanol production 1 and 13.2% factor 2).
was linear and showed that each increased unit of forage WSC re-
sulted in an increase of 0.68 units in ethanol concentration in silage
(Ethanol = −14.7 + 0.68 fWSC; R 2 = .3602; p = .0051). 3.3 | Causes of dry-matter losses during
The concentrations of acetic, propionic and butyric acids in fermentation
silage were influenced by forage composition, but the model cre-
ated was substantially complex. Using simple regression, as WSC Using multiple regression analyses, it was observed among the vari-
increases in forage, both propionic (Propionic acid = 30.7. e(−0.037. ables that only ethanol and ammonia nitrogen concentrations in the
fWSC)
; R 2 = .6652; p < .0001) and butyric acid concentrations (Butyric silage reflected DM losses (DM Losses = −34.3 + 1.33 Ethanol + 1.34
acid = 1.5. e(−0.021. fWSC); R 2 = .5139; p = .0004) decreased in an ex- NH3; p = .0010; adjR 2 = .5359). It is notable that these two inde-
ponential way. This experiment demonstrated that water-soluble pendent variables had similar slopes (1.33 and 1.34) and were also
carbohydrate content in forage was not a good predictor (p > .05) close in partial R 2 (.2890 for ethanol and .2469 for ammonia nitro-
for acetic acid production in sorghum silages. gen), particularly taking into account that the ammonia nitrogen is
Regarding pH estimation, broken-line model was chosen due to expressed in percentage of CP.
2
bigger coefficient of determination (R = .9011) compared with the
linear model (R 2 = .8396). The equation produced by this regression
(pH = 4.44–0.0064 fWSC; joint in fWSC = 125.4 g/kg DM; baseline 3.4 | Buffering capacity
in pH = 3.62; R2 = .9011; p < .0001) shows that pH decreases with
increasing content of fresh forage WSC until the level of 125.0 g/kg The buffering capacity difference between silage and forage (sBC
DM, when pH reaches its lowest level at 3.62. minus fBC), caused by metabolite production during fermentation,
A graphic of principal component analysis was also created to was influenced only by lactic acid and NH3-N concentrations (BC
illustrate multiple relationships of fresh sorghum composition and difference = −7.13 + 0.332 Lactic acid + 0.116 sNH3; p = .0009;

F I G U R E 4 Principal component analysis for chemical and nutritional composition of sorghum forage (circle) and end products of
fermentation of silage (triangle)
8 | RODRIGUES et al.

adjR 2 = .5408). Considering that the units used in this experiment soils have a high capacity of retaining P due to its oxidic character-
were not molar, lactic acid had a slightly higher influence on buffer- istics, which leads to stable bonds between P and soil colloids and
ing capacity than NH3-N, with greater partial R 2 (.3193 vs. .2215). renders unavailable to the plant (Fernandes et al., 2004).
Despite the variation, the fermentation pattern observed in sor-
ghum silage was similar to what is expected for this type of conserved
4 | D I S CU S S I O N forage. Acetic acid concentration (organic acid with antifungal activity)
was within the desired range of ideal fermentation patterns and lac-
4.1 | Fresh and ensiled sorghum composition tic acid values (Kung et al., 2018). It is commonly accepted that lactic
acid is the most desirable metabolite produced during ensiling because
Chemical and nutritional compositions as well as fermentative it causes pH depression that mitigates undesirable fermentation
characteristics found in this trial are compatible with those ex- (McDonald et al., 1991). As a result of those two metabolites concentra-
pected for fresh and ensiled sorghum found in the literature tions, lactic acid:acetic acid ratio was higher than 3.0, which is the value
(NRC, 2001), especially for WSC, aNDF, ADF, lignin, TDN and NH3- stated as indicative of proper fermentation. Silages with high levels of
N. Nevertheless, specific deviations (CP, ADIN, Ca, P, acetic acid lactic:acetic acid ratio can be characterized by having a heterofermenta-
and ethanol) were found when compared to values found in other tive or homofermentative pattern. In this last case, it is more aerobically
studies (Badigannavar, Girish, Ramachandran, & Ganapathi, 2016; unstable, because low concentrations of acetic acid may not be suffi-
Fernandes, Barrón, Torrent, & Fontes, 2004; Jambunathan & Singh, cient to inhibit lactate-assimilating yeasts (Kung et al., 2018).
1981; Kung et al., 2018; Roth & Undersander, 1995). Mean ethanol concentration in sorghum silage was 58.3 g/kg DM,
Although within the normal range, the low average of CP con- but its maximum value reached 121 g/kg DM. This is explained by the
tents found in this study could be because of factors such as the maximum WSC content observed in sorghum fresh forage (157 g/kg
substantial variability among sorghum genotypes (Badigannavar DM), which was also high, compared with other sorghum silage WSC
et al., 2016; Jambunathan & Singh, 1981), agroclimatic conditions (Thomas, Foster, McCuistion, Redmon, & Jessup, 2013). This finding must
where the crops were cultivated (Van Soest, 1994), harvest maturity be due to the fact that sweet sorghum cultivars were used in this experi-
stage (Ayub, Nadeem, Tanveer, & Husnain, 2002), and rearrange- ment. Likewise, considerable levels of ethanol concentrations (≈175 g/kg
ments caused by the amount of nitrogen fertilization applied (Ayub DM) are commonly observed in sugarcane silage, which has a high WSC
et al., 2002; Waggle, Lambert, Miller, Farrel, & Deyoe, 1967). concentration (Siqueira et al., 2007). The yeast population present in this
The ADIN value was higher compared with the one proposed kind of fermented forage converts sugars (soluble in water) into ethanol,
by Roth and Undersander (1995). These authors found that in maize CO2 and water. This diminishes DM quantity and increases fibre con-
silage, ADIN above 120 g/kg of total nitrogen indicates overheating centration in silage, which impairs silage quality (Borgatti, Neto, Marino,
in the fermentative process of ensiling. The ADIN content is asso- Meyer, & Rodrigues, 2015; Pedroso et al., 2005).
ciated with protein bonding with lignin, tannin–protein complexes
and Maillard reaction products, which confers its high indigestibil-
ity (Lima et al., 2017). However, overheating does not appear to be 4.2 | Chemical and nutritional changes in sorghum
an issue in this trial because fresh and ensiled sorghum had similar after ensiling
ADIN values. As suggested by Rodrigues, Pedroso, Melotti, Andrade,
and Lima (2002), it is possible that a nitrogen and fibre complexation Dry-matter reduction during ensiling may be initially explained by
could occur during the drying process for DM analysis. In addition, “metabolic water” production, as related by Zago (1991). Moreover,
higher ADIN values can be inherent to forage cultivated in a tropi- volatile compounds, which are commonly present in large quantities
cal climate with high temperatures, as reported by Johnson, Reiling, in silages, were lost throughout DM analysis, which were below the
Mislevy, and Hall (2001) and Adesogan (2010). Also, it has recently values initially estimated (Rodrigues, Senatore, et al., 2002).
been shown that the ADIN content is overestimated due to contami- Gases produced by fermentable carbohydrates can increase the
nation with nitrogen from cetyl trimethylammonium bromide used in CP concentration in silage. This is a slight increase and may be ex-
the ADF analysis (Marcos, Carro, García, & González, 2018). plained by NH3-N production, which is not detected by CP analy-
Mineral contents in the sorghum plant may be altered due to sis (Kung et al., 2018). Considerable reduction of the hemicellulose
crop genotype, weather conditions and even to mineral concen- content during the ensiling process can be explained by acid hydro-
tration and its translocation rates in soil. Lower Ca and P contents lysis at low pH to which this kind of carbohydrate is vulnerable, as
detected in this work must be analysed carefully, when they are com- demonstrated by Zhao et al. (2018).
pared with values reported in the literature. The standard deviation Increases of unfermentable and not volatile fractions in sorghum
demonstrated in 131 observations is markedly high for NRC (2001) silage as ash, lignin, Ca and P were 18.9%, 11.2%, 20.3% and 29.7%
data and shows a high variability among samples. Jambunathan and respectively. This can be explained by gas and effluent losses during
Singh (1981) found high phenotypic and genotypic coefficients of the fermentative process, which result in increased contents of those
variation for Ca and other mineral contents in sorghum seed. This components after ensiling. The increase in fibrous components (aNDF,
confirms the differences of Ca content in sorghum plants. Tropical ADF and Cellulose) and also EE can be explained by the same reason,
RODRIGUES et al. | 9

because these components are poorly degraded during silo fermenta- with high nutrient preservation over the ensiling process. In con-
tion (Udén, 2017). trast, ethanol production is undesirable because this metabolite is
Notably, WSC content decreased and BC increased as a re- associated with epiphytic yeasts multiplication in silage and results
sult of organic acids produced during fermentation (Udén, 2017). in DM losses through its sucrose conversion (Kung et al., 2018).
Besides DM losses, which always occur during the ensiling process, Lactic acid and ethanol are products closely related to highly fer-
there was also a reduction in nutritional value compared with the mentable carbohydrates and for lactic acid and ethanol equations,
fresh plant, as shown in IVDMD, IVNDFD, TDN and NEl (Borreani, each incremental unit of fWSC, resulted in 0.37 unit of lactic acid
Tabacco, Schmidt, Holmes, & Muck, 2018). and 0.68 unit of ethanol on silage. Interestingly, the sum of these
Changes among different components owing to the ensiling pro- slopes is close to 1.0, allowing the conclusion that each additional
cess depend on the base value (fresh forage content) used to calcu- WSC, until 125 g/kg DM in forage, converges to an increase of ap-
late the difference in percentage. Thus, the same difference value in proximately 35% of lactic acid and 65% of ethanol during the fer-
units might be lower or higher, depending on the component concen- mentation process.
tration of the initial forage. For example, the higher change (38.2%) The minimum possible content of WSC in sorghum forage is im-
observed in EE is related to its small concentration in the fresh plant. portant to avoid poor fermentation (Kung et al., 2018). In contrast,
Using principal component analysis, Figure 1 summarizes WSC, EE, our data indicated it is possible to infer that lactic acid concentration
HEMI and BC items as the most altered by silage fermentation. increased linearly only until approximately 125 g/kg DM of WSC.
Total digestible nutrient differences, before and after the en- After that, it likewise reached a plateau in its pH; however, ethanol
siling process, decreased 2.2% units (U) for each increase in fresh continued to increase in line with WSC increases, which is undesir-
forage CP content. A similar response was observed for lignin, with able. This finding supports the premise that above 125 g/kg DM
a 1.8% U of increase. No biological explanation was found for the of WSC it is not advisable to produce silage from fresh sorghum.
impact of CP and lignin concentrations on TDN differences. Identifying genes that influence sorghum plant composition helps to
Conversely, WSC is well known as the principal substrate for lactic manipulate its silage quality and enables the possibility to increase
acid production, which is commonly associated with high-quality silage. overall feed efficiency. Genetic selection for WSC would be an in-
However, for each increase in Hemi or WSC content in fresh forage, it is teresting approach to be allied with other genetic selections such as
possible to observe a 1.0% U decrease in IVDMD difference. Thus, di- yield and starch content; this has not yet been studied.
gestibility differences with the ensiling process reach close to zero when Organic acid concentrations in silage were suitable for this kind
forage has higher amounts of hemicellulose and/or WSC. McDonald of forage and demonstrated satisfactory fermentation (Kung et al.,
et al. (1991) observed that the amount of organic acids produced during 2018; McDonald et al., 1991).
silage fermentation was higher than the possible amount to be produced
from WSC available in ensiled material. A possible explanation was that
hemicellulose would be an extra source of carbohydrates, hydrolysed 4.4 | Causes of dry-matter losses during
by hemicellulases from bacterial origin. According to Henderson (1993), fermentation
hemicellulose is the main additional substrate source for fermentation,
which may contribute up to 40% of this fraction utilization, whereas cel- Although DM loss was not directly influenced by sorghum forage
lulose and lignin remain practically unchanged during ensiling (Morrison, chemical composition, it was significantly influenced by silage fer-
Akin, Himmelsbach, & Gamble, 1993). This information could explain mentative characteristics. Soluble carbohydrates reactions result in
why both components (WSC and Hemi) are intrinsically related to avoid- ethanol and CO2 production in silage, which is inversely related to
ing a decrease in sorghum quality during ensiling. silage aerobic stability. Its control is closely related to management
Total digestible nutrients and IVDMD have been selected as in- factors such as speed of packing, pack density, type of additive used,
dicators of sorghum quality change after ensiling because decreases chopping length, covering management and silo management during
in energetic value and digestibility of fresh forage are undesirable feed-out (Kung et al., 2018).
within the ensiling process. It is important to point out that during Clostridial organisms lead to large DM losses and poor recovery
the selection process of forage improvement, choosing a genetic of energy in silage. They act by converting lactic acid into butyric
material that is less prone to quality reduction during silage fermen- acid to cause proteolytic activity (Pahlow, Muck, Driehuis, Elferink,
tation is the key to succeeding (Saballos, Ejeta, Sanchez, Kang, & & Spoelstra, 2003) and justify NH3 inclusion in the DM losses equa-
Vermerris, 2009; Santacruz, Gómez, Torres, & López, 2012). tion. Butyric fermentation can be prevented by ensiling forage from
300 to 350 g/kg DM immediately at the time of harvest in order to
stimulate quick lactic acid production in the silo, because clostridia
4.3 | Forage composition influence on silage are sensitive to both osmotic pressure and lower pH levels (Mills &
fermentative pattern Kung, 2002). However, a low concentration of butyric acid in the
silage was detected in this study.
Lactic acid is the most desirable fermentation product of silage, as Despite the fact that our results showed ideal pH and lactic acid
it is the strongest organic acid. Thus, this organic acid is associated levels at 125.4 g/kg DM of fresh forage WSC concentration, high
10 | RODRIGUES et al.

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Ensiling as pretreatment of rice straw: The effect of hemicellulase

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