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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
Forage availability depends on seasonality, and this may cause prob-
lems for farmers maintaining regular livestock productivity through-
out the year. Therefore, forage conservation as silage has become an
important and popular method of feed storing because it can mini-
mize the loss of nutrients after harvest during storage (Fulgueira,
Amigot, Gaggiotti, Romero, & Basílico, 2007). Sorghum [Sorghum bi-
color (L.) Moench] is a tropical plant that efficiently uses water and
it is a good alternative to maize crops for feeding ruminants, mainly
Peters, 2016). Compared with maize plants, sorghum plants require
less fertilization and less pest control and may grow in soils with a
low pH (AERC, 2007).
Several authors have reported good ensilability of sorghum
(Meeske, Ashbell, Weinberg, & Kipnis, 1993; Miron et al., 2006;
Ward, Readfern, McCormick, & Cuomo, 2001). The optimal amounts
of water-soluble carbohydrate (WSC) in maize and grass plants are
80–200 and 100–200 g/kg DM respectively (Pitt, 1990); in sorghum
plant, it is 110–280 g/kg DM. Frequently, sorghum and maize crops
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 |
decreases pH values to the extremely important range required for
quality: 3.53–4.2 (Miron et al., 2006). Despite a lower crude pro-
tein content and a higher fibre content compared with maize silage,
sorghum silage has the potential to support ruminant performance;
therefore, it is an acceptable candidate for extensive use in livestock
(Harper et al., 2017).
It is, however, important to point out that the large variation
in nutritive composition among the different commercial sorghum
cultivars can lead to differences in their individual silage. Further,
the harvest date can alter the silage quality (Miron et al., 2006).
Gaining greater knowledge about the biochemical processes that
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
of fermentation that have a large influence on it as well (Kung et al.,
2018). Still, a negligible amount of studies is available to provide data
about individual end products of silage fermentation related to their
initial composition (Mogodiniyai Kasmaei, Rustas, Spörndly, & Udén,
2013).
The aim of this study was to obtain prediction models for end
products of fresh sorghum fermentation. We hypothesized that
the composition of fresh sorghum could explain the fermentation
pattern of sorghum silage when performed under strictly anaerobic
conditions.
2 | M ATE R I A L S A N D M E TH O DS
Regarding ethical aspects, this paper did not use living animals.
Cannulated animals used as inoculum donors at analysis of in vitro
dry-matter digestibility were approved by the Bioethics Committee
of the College of Veterinary Medicine and Animal Science, University
of São Paulo (approval number: 8453300914).
2.1 | Experimental site, forage
production and ensiling
The trial was carried out at Department of Animal Nutrition and
Production of the College of Veterinary Medicine and Animal
Science of University of São Paulo, Brazil.
For ensiling, 11 different sorghum [Sorghum bicolor (L.) Moench]
varieties were used, including the silage (AGRI–001E—Agricom;
AGRI–002E—Agricom; Volumax—Agroceres), the graniferous (Ag
1080—Agroceres; Ag 1085—Agroceres; Ag 1090—Agroceres; BRS
373—Embrapa; BRS 380—Embrapa) and the sweet (BRS 511—
Embrapa; CB 7290—Ceres; CB 7521—Ceres) cultivars. These cul-
tivars were selected to provide variation in chemical composition,
which was important for this modelling study.
Sorghum cultivars were grown in 5 experimental fields. Two
farms were used, and the distance from one farm to another was
RODRIGUES et al.
Highlights
• Fresh sorghum chemical and nutritional components did
alter its silage quality.
• The more hemicellulose and WSC in fresh sorghum, the
less difference between fresh and ensiled dry-matter
digestibility.
• Silage ethanol concentration increased linearly accord-
ing to water-soluble carbohydrates (WSC) level.
• Lactic acid production and pH were optimized at 125 g
WSC/kg DM in fresh sorghum.
25 km. The experimental fields were divided into 25 plots (experi-
mental units—EU), with two repetitions per plot (25 EU × 2 repeti-
tions = 50 microsilos). Eight cultivars were grown in field 1; another
8 cultivars in field 2; 3 cultivars in field 3; 3 cultivars in field 4; and
another 3 cultivars were planted in field 5. All plots were cultivated
in the same month in the agricultural season of 2015–2016. All were
grown under the same environmental conditions. The soil type in
this region is red latosol, and the local climate is subtropical (type
Cwa according to Köppen). Fertilizer rate was maintained according
to soil test recommendations (120, 30 and 80 kg of N, P and K re-
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
range was important to increase dataset variability, according to the
purpose of this experiment.
After manual harvesting (stubble height = 10 cm) under clean
conditions, sorghum plants were transported to the laboratory,
where they were chopped (Chopper Nogueira, model EM-9F3B) to
obtain a mean theoretical length of cut = 14 mm. The evaluation of
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
silage additives. All these procedures were completed within one
hour.
Fifty sterile polyvinyl chloride (PVC) plastic buckets (2 rep-
etitions for each plot) with 22 cm of diameter × 30 cm of height
equipped with Bunsen valves for gas escape were used as ex-
perimental microsilos. Two kilograms of sand was placed in the
bottom of the buckets and covered with a nylon mesh screen
(500 µm) to drain effluents. The forage was placed inside each
microsilo and compacted manually using disposable sleeve gloves
and plastic boot covers until the density of 500 kg of sorghum
plant/m 3 . Microsilos were sealed with tight PVC lids, weighed and
vertically stored in a covered area at room temperature of 25°C
and opened between 60 and 90 days of storage (mean fermenta-
tion time = 68.8 ± 8.9 days). Plantations were harvested in varied
days of growth and microsilos were opened in different matura-
tion stages in order to try to achieve the diverse conditions that
RODRIGUES et al.
these two important steps represent as those in farm conditions
and also to increase the data variation required for this type of
study.
2.2 | Laboratory analysis
2.2.1 | Before ensiling
After chopping, one sample of fresh material was obtained and
stored at −20°C for later determinations of the following: dry
matter (DM), ash, organic matter (OM), ether extract (EE), crude
protein (CP), acid detergent fibre (ADF), acid detergent insoluble
nitrogen (ADIN), neutral detergent fibre with residual ashes as-
sayed with a heat stable amylase (aNDF), neutral detergent insolu-
ble nitrogen (NDIN), lignin, hemicellulose (Hemi), cellulose (Cell),
water-soluble carbohydrates (WSC), starch, in vitro dry-matter
digestibility (IVDMD), in vitro NDF digestibility (IVNDFD), buffer
capacity (BC), calcium (Ca), phosphorus (P), total digestible nutri-
ents (TDN) and net energy for lactation (NEl), according to meth-
odologies described below. After thawing, this fresh material was
analysed simultaneously with silages in order to avoid analytical
variation.
2.2.2 | After ensiling
Before opening, silos were weighed for determination of DM losses
during fermentation. After opening the silos, each mass was ho-
mogenized and a fraction was separated for determinations of DM,
ash, EE, CP, Ca and P according to AOAC (1995) and aNDF, ADF and
lignin according to Van Soest, Robertson, and Lewis (1991). Organic
matter (OM) was determined by the difference between DM and
ash content. Hemicellulose and cellulose content were obtained by
the difference between aNDF and ADF and also between ADF and
lignin respectively. Water-soluble carbohydrate was determined
according to Johnson, Balwan, Johnson, McClure, and Dehority
(1966), and the ADIN and the NDIN were determined as reported
by Van Soest and Robertson (1985). Buffer capacity has been
measured by the quantity in meq of H+ that is spent to decrease pH
values of forage from 6.0 to 4.0, which indicates the resistance of
organic and inorganic components to acidification during preser-
vation (McDonald, Henderson, & Heron, 1991). Additionally, TDN
and NEl were estimated by forage or silage chemical composition
as stated in NRC—Dairy Cattle (2001), considering a cow eating at
a 3× maintenance level.
One silage fraction was placed in hydraulic press for silage juice
extraction. Immediately after material pressing, 50 ml of silage juice
were used for pH determination with a portable digital pH meter
(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
formic acid and frozen at −20°C for further organic acids and etha-
nol concentration analysis. Both of these analyses were determined
| 3
by gas chromatography and according to methodology proposed by
Erwin, Marco, and Emery (1961), which uses a gas chromatographer
(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.
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-
monia nitrogen (NH 3-N) concentration by colorimetric assay, as
described by Kulasek (1972) and adapted by Foldager (1977).
Absorbance measurements were performed in spectropho-
tometer (Beijing Rayleigh AIC model VIS-7220) set at 630 nm.
Absorbance values were used to calculate NH 3-N concentrations
in mg of NH 3-N/100 ml by a linear regression equation obtained
through equipment calibration with standard solutions at differ-
ent concentrations.
Dry-matter (DM) losses during fermentation were calculated
as the difference between the weights of masses obtained at the
moments of filling the silo and the silo's opening, multiplied by the
respective DM contents. Dry-matter losses were transformed in
percentage of initial mass. Because the aim of the study was to un-
derstand the total DM losses, no separate analysis was performed
for gas losses or effluent losses.
2.3 | In vitro digestibility
In vitro dry-matter digestibility and IVNDFD were determined
in conformance with Tilley and Terry (1963). Filter bags (F-57;
50 × 55 mm; ANKOM Technology Corporation) were identified and
weighed. In the formula below, the bag was considered the inoculum
without sample. Approximately 0.5 g of forage samples previously
oven-dried and ground were weighed and inserted into filter bags in
duplicates. These bags were put into test tubes.
Into test tubes, 40 ml of McDougall solution (artificial saliva)
were added to 10 ml of rumen inoculum from 3 different steers
that had been kept at pasture grazing Brachiaria decumbens and
then supplemented with 3.0 kg of maize silage (DM basis) and ad
libitum mineral salt. Tubes were sealed with rubber corks contain-
ing a Bunsen gas release valve, immediately after flushing out with
CO2, and incubated in oven for 48 hr under controlled temperature
(39°C). They were agitated a minimum of 3 times during fermenta-
tion. The second IVDMD phase occurred after discarding the liquid
solution. A pepsin solution (1:10.000) at 0.2% (50 ml) was added to
each tube, followed by agitation at 39°C for another 48 hr. After
washing, drying and weighing the bags, calculations were performed
as the formula below:
[
g of DM in sample − (g of residual DM − g of DM of inoculum without sample)
]
IVDMD = 100 × .
g of DM in sample
Same procedure was performed in IVNDFD analysis, except
that the bags were submitted to aNDF washing procedures after
incubation.
4 |
2.4 | Statistical analysis
Data were analysed using the SAS 9.3 (SAS Institute Inc.). Before
the actual analysis, data were explored to seek disparate infor-
mation (“outliers”) and for normality of residuals by Shapiro–Wilk
test (PROC UNIVARIATE). An individual observation was consid-
ered outlier when the standard deviations in relation to the mean
or to the model were larger than + 3 or less than −3. Data which
did not attend to normality premise were subjected to logarith-
mic [Log (X + 1)] or by square root [RQ (X + 1/2)] transformation.
The values 1 and ½ were added in the Log and RQ equations in
order to avoid negative values in the analyses when values be-
tween 0 and 1 were involved. Initially, statistic treatments of data
were organized in 5 distinctive parts: (a) descriptive characteri-
zation of forage and silage regarding chemical composition and
fermentative pattern; (b) magnitude of changes in chemical and
nutritional plant parameters, referring to the ensiling process; (c)
forage composition influence on silage fermentative pattern; (d)
losses causes attributed to ensiling; and (e) changes in BC regard-
ing ensiling.
Ordinary descriptive statistics, as mean and standard deviations,
were performed. The mean of each duplicate was used. Data in fresh
forage and in silage were compared with a t test. If the difference
equal to zero hypothesis was rejected, the chemical composition of
silage for a specific component or nutrient was different from that in
forage. Principal component analysis was performed to illustrate the
observed differences.
Multiple regression analyses were performed for some selected
variables to determine the causes of change in nutritional value
during ensiling. For multiple regressions, the difference between si-
lage and forage nutritional value was regressed on fresh sorghum
composition. The best model was chosen using a stepwise selection
procedure of PROC REG (SAS).
The influence of forage composition on silage fermentative
pattern was determined by simple and multiple regressions. For
simple regressions analyses, polynomial (linear and quadratic), ex-
ponential (first order kinetic) or broken-line models were created
with PROC REG or PROC NLIN of SAS, depending on situation.
Causes of losses and BC changes were studied with multiple re-
gressions analyses. For all analyses, the significance level adopted
was 5%.
3 | R E S U LT S
3.1 | Sorghum chemical and nutritional changes
after ensiling
High variability among experimental units of both fresh and en-
siled sorghum was detected for chemical and nutritional charac-
teristics. For some components (e.g. CP, ADIN, NDIN, EE, starch
and lignin), minimum and maximum values differed more than 2.7
times.
RODRIGUES et al.
Harvest date and ensiling duration were excluded from the
modelling process because they were not correlated with other
variables.
Ensiling resulted in significantly higher contents of ash, CP, EE,
aNDF, ADF, lignin, Cell, Ca and P, compared with fresh sorghum
(p < .05). On the other hand, lower DM, OM, NDIN, WSC and Hemi
contents were detected (p < .05). Only ADIN and starch values did
not change after ensiling (p > .05). Nutritional characteristics were
also different between fresh and ensiled sorghum. Lower values for
IVDMD, IVNDFD, TDN and NEl were found in sorghum silage than in
fresh sorghum (p < .05). Despite the differences observed between
fresh sorghum and its silage, chemical and nutritional composition
was highly correlated for most components, except EE and hemicel-
lulose (Table 1).
Principal component analysis (PCA) for chemical and nutritional
compositions showed a strong association between DM concentra-
tions before and after ensiling (Figure 1). It was the same for ash, cell
wall components (except hemicellulose), CP, digestibility and energy.
Concentrations of hemicellulose, EE, BC and WSC from fresh sor-
ghum were weakly associated with those in ensiled sorghum in the
PCA analysis.
It was possible to observe two main groups of variables in PCA
analysis. One group on the right side of the graph is formed by DM,
ash, cell wall components (excluding hemicellulose) and ADIN. The
group on the left side is formed by digestibility, energetic items and
CP. Starch seemed to be an isolated group neither related to cell wall
components nor to energetic items. Principal component analysis
was useful for these data: 2 factors in PCA explained 78.4% of the
overall variation observed (64.7% was factor 1 and 13.7% was factor
2).
An important aim of this study was to indicate which nutrients
were most associated with quality loss during the ensiling process.
Total digestible nutrients and IVDMD differences before and after
ensiling were analysed by a stepwise model selection procedure
in order to include in their prediction equations the variables with
higher F value. For TDN difference, the best model included for-
age CP and lignin content (dTDN = −200.8 + 2.20 fCP + 1.38 fLIG;
p < .0001; adjR 2 = .8050, RMSE = 1.16).
The first complex selected model for IVDMD included fresh for-
age Hemi, WSC, DM, Ca and EE content (dIVDMD = −966.3 + 0.90
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
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
fWSC; p = .0025; adjR 2 = .5058, RMSE = 3.67). These predictive
variables presented the biggest partial adjusted R 2 (.2605 and .2453
respectively) compared with DM, Ca and EE adjusted R 2 (.1678,
.0779 and 0.1301 respectively). In order to simplify the model and
because the slopes for fresh forage Hemi and WSC were very close,
we proposed another equation, in which Hemi and WSC contents
are summed (dIVDMD = −369.1 + 1.00 (fHemi + fWSC); p < .0001;
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
fermentative pattern
After considering biological and mathematical parameters, even-
tually fWSC was chosen to be the predictor for lactic, acetic,
propionic and butyric acids, as well as for ethanol and pH concen-
trations. These were estimated in a simple regression equation. The
model created to estimate lactic acid production in silage, using
forage components as predictors by stepwise procedure, included
3 variables (fWSC, fBC and fCELL), and R 2 was .5218. A simple
regression using only fWSC as predictor for lactic acid production
originated a broken-line model: Lactic acid = 36.8 + 0.37 fWSC;
Joint in fWSC = 125.4 g/kg DM; Plateau in lactic acid concentra-
tion = 84.2 g/kg DM; R 2 = .3637; p = .0037. This model showed
that each increased unit of WSC resulted in an increase of 0.37
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.
of 125.0 g/kg DM of fWSC, when lactic acid reached the plateau
(Figure 3).
To estimate ethanol production in silage, the stepwise proce-
dure resulted in a very complex equation, which included 7 differ-
ent variables (fWSC, fCELL, fEE, fDM, fP, fHEMI and fCP) with a
2
high coefficient of determination (R = .9911). Still, WSC was the
component with the highest partial R 2 (.3602). Thus, the simple
regression using only fWSC as a predictor for ethanol production
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).
The concentrations of acetic, propionic and butyric acids in
silage were influenced by forage composition, but the model cre-
ated was substantially complex. Using simple regression, as WSC
increases in forage, both propionic (Propionic acid = 30.7. e(−0.037.
fWSC)
; R 2 = .6652; p < .0001) and butyric acid concentrations (Butyric
acid = 1.5. e(−0.021. fWSC); R 2 = .5139; p = .0004) decreased in an ex-
ponential way. This experiment demonstrated that water-soluble
carbohydrate content in forage was not a good predictor (p > .05)
for acetic acid production in sorghum silages.
Regarding pH estimation, broken-line model was chosen due to
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
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
DM, when pH reaches its lowest level at 3.62.
A graphic of principal component analysis was also created to
illustrate multiple relationships of fresh sorghum composition and
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)
| 7
its silage fermentation pattern, shown in Figure 4. Ethanol and lactic
acid were closer to the group located at the left side of the graphic,
formed especially by digestibility and energetic items, as well as WSC
and CP. Acetic, propionic and butyric acids were dispersed variables
among themselves, but they were more related to the forage cell
wall components located at the right side of the graphic. These two
factors explained 68.3% of overall variation observed (55.1% factor
1 and 13.2% factor 2).
3.3 | Causes of dry-matter losses during
fermentation
Using multiple regression analyses, it was observed among the vari-
ables that only ethanol and ammonia nitrogen concentrations in the
silage reflected DM losses (DM Losses = −34.3 + 1.33 Ethanol + 1.34
NH3; p = .0010; adjR 2 = .5359). It is notable that these two inde-
pendent variables had similar slopes (1.33 and 1.34) and were also
close in partial R 2 (.2890 for ethanol and .2469 for ammonia nitro-
gen), particularly taking into account that the ammonia nitrogen is
expressed in percentage of CP.
3.4 | Buffering capacity
The buffering capacity difference between silage and forage (sBC
minus fBC), caused by metabolite production during fermentation,
was influenced only by lactic acid and NH3-N concentrations (BC
difference = −7.13 + 0.332 Lactic acid + 0.116 sNH3; p = .0009;
8 |
adjR 2 = .5408). Considering that the units used in this experiment
were not molar, lactic acid had a slightly higher influence on buffer-
ing capacity than NH3-N, with greater partial R 2 (.3193 vs. .2215).
4 | D I S CU S S I O N
4.1 | Fresh and ensiled sorghum composition
Chemical and nutritional compositions as well as fermentative
characteristics found in this trial are compatible with those ex-
pected for fresh and ensiled sorghum found in the literature
(NRC, 2001), especially for WSC, aNDF, ADF, lignin, TDN and NH3-
N. Nevertheless, specific deviations (CP, ADIN, Ca, P, acetic acid
and ethanol) were found when compared to values found in other
studies (Badigannavar, Girish, Ramachandran, & Ganapathi, 2016;
Fernandes, Barrón, Torrent, & Fontes, 2004; Jambunathan & Singh,
1981; Kung et al., 2018; Roth & Undersander, 1995).
Although within the normal range, the low average of CP con-
tents found in this study could be because of factors such as the
substantial variability among sorghum genotypes (Badigannavar
et al., 2016; Jambunathan & Singh, 1981), agroclimatic conditions
where the crops were cultivated (Van Soest, 1994), harvest maturity
stage (Ayub, Nadeem, Tanveer, & Husnain, 2002), and rearrange-
ments caused by the amount of nitrogen fertilization applied (Ayub
et al., 2002; Waggle, Lambert, Miller, Farrel, & Deyoe, 1967).
The ADIN value was higher compared with the one proposed
by Roth and Undersander (1995). These authors found that in maize
silage, ADIN above 120 g/kg of total nitrogen indicates overheating
in the fermentative process of ensiling. The ADIN content is asso-
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
an issue in this trial because fresh and ensiled sorghum had similar
ADIN values. As suggested by Rodrigues, Pedroso, Melotti, Andrade,
and Lima (2002), it is possible that a nitrogen and fibre complexation
could occur during the drying process for DM analysis. In addition,
higher ADIN values can be inherent to forage cultivated in a tropi-
cal climate with high temperatures, as reported by Johnson, Reiling,
Mislevy, and Hall (2001) and Adesogan (2010). Also, it has recently
been shown that the ADIN content is overestimated due to contami-
nation with nitrogen from cetyl trimethylammonium bromide used in
the ADF analysis (Marcos, Carro, García, & González, 2018).
Mineral contents in the sorghum plant may be altered due to
crop genotype, weather conditions and even to mineral concen-
tration and its translocation rates in soil. Lower Ca and P contents
detected in this work must be analysed carefully, when they are com-
pared with values reported in the literature. The standard deviation
demonstrated in 131 observations is markedly high for NRC (2001)
data and shows a high variability among samples. Jambunathan and
Singh (1981) found high phenotypic and genotypic coefficients of
variation for Ca and other mineral contents in sorghum seed. This
confirms the differences of Ca content in sorghum plants. Tropical
RODRIGUES et al.
soils have a high capacity of retaining P due to its oxidic character-
istics, which leads to stable bonds between P and soil colloids and
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
forage. Acetic acid concentration (organic acid with antifungal activity)
was within the desired range of ideal fermentation patterns and lac-
tic acid values (Kung et al., 2018). It is commonly accepted that lactic
acid is the most desirable metabolite produced during ensiling because
it causes pH depression that mitigates undesirable fermentation
(McDonald et al., 1991). As a result of those two metabolites concentra-
tions, lactic acid:acetic acid ratio was higher than 3.0, which is the value
stated as indicative of proper fermentation. Silages with high levels of
lactic:acetic acid ratio can be characterized by having a heterofermenta-
tive or homofermentative pattern. In this last case, it is more aerobically
unstable, because low concentrations of acetic acid may not be suffi-
cient to inhibit lactate-assimilating yeasts (Kung et al., 2018).
Mean ethanol concentration in sorghum silage was 58.3 g/kg DM,
but its maximum value reached 121 g/kg DM. This is explained by the
maximum WSC content observed in sorghum fresh forage (157 g/kg
DM), which was also high, compared with other sorghum silage WSC
(Thomas, Foster, McCuistion, Redmon, & Jessup, 2013). This finding must
be due to the fact that sweet sorghum cultivars were used in this experi-
ment. Likewise, considerable levels of ethanol concentrations (≈175 g/kg
DM) are commonly observed in sugarcane silage, which has a high WSC
concentration (Siqueira et al., 2007). The yeast population present in this
kind of fermented forage converts sugars (soluble in water) into ethanol,
CO2 and water. This diminishes DM quantity and increases fibre con-
centration in silage, which impairs silage quality (Borgatti, Neto, Marino,
Meyer, & Rodrigues, 2015; Pedroso et al., 2005).
4.2 | Chemical and nutritional changes in sorghum
after ensiling
Dry-matter reduction during ensiling may be initially explained by
“metabolic water” production, as related by Zago (1991). Moreover,
volatile compounds, which are commonly present in large quantities
in silages, were lost throughout DM analysis, which were below the
values initially estimated (Rodrigues, Senatore, et al., 2002).
Gases produced by fermentable carbohydrates can increase the
CP concentration in silage. This is a slight increase and may be ex-
plained by NH3-N production, which is not detected by CP analy-
sis (Kung et al., 2018). Considerable reduction of the hemicellulose
content during the ensiling process can be explained by acid hydro-
lysis at low pH to which this kind of carbohydrate is vulnerable, as
demonstrated by Zhao et al. (2018).
Increases of unfermentable and not volatile fractions in sorghum
silage as ash, lignin, Ca and P were 18.9%, 11.2%, 20.3% and 29.7%
respectively. This can be explained by gas and effluent losses during
the fermentative process, which result in increased contents of those
components after ensiling. The increase in fibrous components (aNDF,
ADF and Cellulose) and also EE can be explained by the same reason,
RODRIGUES et al.
because these components are poorly degraded during silo fermenta-
tion (Udén, 2017).
Notably, WSC content decreased and BC increased as a re-
sult of organic acids produced during fermentation (Udén, 2017).
Besides DM losses, which always occur during the ensiling process,
there was also a reduction in nutritional value compared with the
fresh plant, as shown in IVDMD, IVNDFD, TDN and NEl (Borreani,
Tabacco, Schmidt, Holmes, & Muck, 2018).
Changes among different components owing to the ensiling pro-
cess depend on the base value (fresh forage content) used to calcu-
late the difference in percentage. Thus, the same difference value in
units might be lower or higher, depending on the component concen-
tration of the initial forage. For example, the higher change (38.2%)
observed in EE is related to its small concentration in the fresh plant.
Using principal component analysis, Figure 1 summarizes WSC, EE,
HEMI and BC items as the most altered by silage fermentation.
Total digestible nutrient differences, before and after the en-
siling process, decreased 2.2% units (U) for each increase in fresh
forage CP content. A similar response was observed for lignin, with
a 1.8% U of increase. No biological explanation was found for the
impact of CP and lignin concentrations on TDN differences.
Conversely, WSC is well known as the principal substrate for lactic
acid production, which is commonly associated with high-quality silage.
However, for each increase in Hemi or WSC content in fresh forage, it is
possible to observe a 1.0% U decrease in IVDMD difference. Thus, di-
gestibility differences with the ensiling process reach close to zero when
forage has higher amounts of hemicellulose and/or WSC. McDonald
et al. (1991) observed that the amount of organic acids produced during
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
by hemicellulases from bacterial origin. According to Henderson (1993),
hemicellulose is the main additional substrate source for fermentation,
which may contribute up to 40% of this fraction utilization, whereas cel-
lulose and lignin remain practically unchanged during ensiling (Morrison,
Akin, Himmelsbach, & Gamble, 1993). This information could explain
why both components (WSC and Hemi) are intrinsically related to avoid-
ing a decrease in sorghum quality during ensiling.
Total digestible nutrients and IVDMD have been selected as in-
dicators of sorghum quality change after ensiling because decreases
in energetic value and digestibility of fresh forage are undesirable
within the ensiling process. It is important to point out that during
the selection process of forage improvement, choosing a genetic
material that is less prone to quality reduction during silage fermen-
tation is the key to succeeding (Saballos, Ejeta, Sanchez, Kang, &
Vermerris, 2009; Santacruz, Gómez, Torres, & López, 2012).
4.3 | Forage composition influence on silage
fermentative pattern
Lactic acid is the most desirable fermentation product of silage, as
it is the strongest organic acid. Thus, this organic acid is associated
| 9
with high nutrient preservation over the ensiling process. In con-
trast, ethanol production is undesirable because this metabolite is
associated with epiphytic yeasts multiplication in silage and results
in DM losses through its sucrose conversion (Kung et al., 2018).
Lactic acid and ethanol are products closely related to highly fer-
mentable carbohydrates and for lactic acid and ethanol equations,
each incremental unit of fWSC, resulted in 0.37 unit of lactic acid
and 0.68 unit of ethanol on silage. Interestingly, the sum of these
slopes is close to 1.0, allowing the conclusion that each additional
WSC, until 125 g/kg DM in forage, converges to an increase of ap-
proximately 35% of lactic acid and 65% of ethanol during the fer-
mentation process.
The minimum possible content of WSC in sorghum forage is im-
portant to avoid poor fermentation (Kung et al., 2018). In contrast,
our data indicated it is possible to infer that lactic acid concentration
increased linearly only until approximately 125 g/kg DM of WSC.
After that, it likewise reached a plateau in its pH; however, ethanol
continued to increase in line with WSC increases, which is undesir-
able. This finding supports the premise that above 125 g/kg DM
of WSC it is not advisable to produce silage from fresh sorghum.
Identifying genes that influence sorghum plant composition helps to
manipulate its silage quality and enables the possibility to increase
overall feed efficiency. Genetic selection for WSC would be an in-
teresting approach to be allied with other genetic selections such as
yield and starch content; this has not yet been studied.
Organic acid concentrations in silage were suitable for this kind
of forage and demonstrated satisfactory fermentation (Kung et al.,
2018; McDonald et al., 1991).
4.4 | Causes of dry-matter losses during
fermentation
Although DM loss was not directly influenced by sorghum forage
chemical composition, it was significantly influenced by silage fer-
mentative characteristics. Soluble carbohydrates reactions result in
ethanol and CO2 production in silage, which is inversely related to
silage aerobic stability. Its control is closely related to management
factors such as speed of packing, pack density, type of additive used,
chopping length, covering management and silo management during
feed-out (Kung et al., 2018).
Clostridial organisms lead to large DM losses and poor recovery
of energy in silage. They act by converting lactic acid into butyric
acid to cause proteolytic activity (Pahlow, Muck, Driehuis, Elferink,
& Spoelstra, 2003) and justify NH3 inclusion in the DM losses equa-
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
are sensitive to both osmotic pressure and lower pH levels (Mills &
Kung, 2002). However, a low concentration of butyric acid in the
silage was detected in this study.
Despite the fact that our results showed ideal pH and lactic acid
levels at 125.4 g/kg DM of fresh forage WSC concentration, high
10 | RODRIGUES et al.
levels of ethanol were detected at this point. This indicates high DM
losses through yeast activity. Our result was 154 g/kg of DM losses,
which is above the recommended level (<100 g/kg, Lima-Orozco,
Castro-Alegría, & Fievez, 2013). Because ethanol production comes
from non-structural carbohydrates in forage, the results found in
this study were expected since we used sweet sorghum cultivars
with high content of WSC.
4.5 | Buffering capacity
Forage buffering capacity can be defined as the degree to which a
forage material resists to changes in pH. Legume silages with high
protein and ash contents and failures in fermentation process can
induce high buffering capacity, with pH values higher than expected.
In this study, silage pH was in accordance with the range found in
the literature (3.7–4.0), which indicates that fermentation had pro-
ceeded acceptably (Kung et al., 2018; McDonald et al., 1991).
The obvious explanation for reduced differences in BC values
according to lactic acid increase is due to its pKa value of 3.86, which
is 10 to 12 times stronger than other acids. However, no biological
explanation was found to NH3-N increase impact on BC difference
between fresh and ensiled sorghum.
5 | CO N C LU S I O N
Water-soluble carbohydrates and hemicellulose not only increased
the nutritional value of sorghum forage and sorghum, but also
helped to avoid quality decreases during the ensiling fermentation
process. Notwithstanding, we found that 125.4 g/kg DM of water-
soluble carbohydrates in fresh plant was required for a satisfactory
silage pH level and lactic acid content. This information should be
considered in genetic selection programmes of sorghum destined for
ensiling. In contrast, at this water-soluble carbohydrates level, etha-
nol content still increased.
AC K N OW L E D G E M E N T S
The authors express appreciation to the Brazilian funding agency
CAPES (“Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior”) and CNPq (“Conselho Nacional de Desenvolvimento
Científico e Tecnológico”) for providing the fellowships.
C O N FL I C T O F I N T E R E S T
None.
ORCID
Paulo Henrique Mazza Rodrigues http://orcid.org/0000-0002-4646-6805
Lerner Arévalo Pinedo https://orcid.org/0000-0001-8119-8626
Paula Marques Meyer http://orcid.org/0000-0001-9319-928X
Thiago Henrique da Silva https://orcid.org/0000-0002-0028-1127
Iuli Caetano da Silva Brandão Guimarães https://orcid.
org/0000-0003-1375-1148
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Ensiling as pretreatment of rice straw: The effect of hemicellulase

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