Animal Feed Science and Technology 208 (2015) 3343

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Animal Feed Science and Technology

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Silages of Brachiaria brizantha cv. Marandu harvested at

two regrowth ages: Microbial inoculant responses in
silage fermentation, ruminant digestion and beef
cattle performance
Andrea Santos Cezrio a , Karina Guimares Ribeiro a ,
Stefanie Alvarenga Santos b , Sebastio de Campos Valadares Filho a ,
Odilon Gomes Pereira a,
a
b

Universidade Federal de Vicosa, Departamento de Zootecnia, 36571-000 Vicosa, MG, Brazil

Universidade Federal da Bahia, DMVPPA, 40170-110 Salvador, BA, Brazil

a r t i c l e

i n f o

Article history:
Received 16 December 2014
Received in revised form 24 June 2015
Accepted 25 June 2015
Keywords:
Acetic acid
Lactic acid
Microbial efciency
Nitrogen balance

a b s t r a c t
Brachiaria brizantha cv. Marandu silages (BBS) were ensiled at two (35 and 70 d) regrowth
ages (RA), with and without microbial inoculant (MI). No differences were observed
regarding the chemical composition and fermentation parameters, with the exception of
volatile fatty acids, which were affected by MI and RA interaction. No effects were observed
on total nutrient digestibility in beef steers. The ruminal digestibility of ether extract and
crude protein and the NH3 -N concentration were affected by treatments. No effects were
observed on total nutrient intake and beef steers performance. These ndings were investigated by conducting three trials. Silage preparation and quality attributes were evaluated
using a 2 2 factorial scheme in a completely randomized design. Rumen degradation and
nutrient outow to abomasum from silages were evaluated using four Nellore steers with
a 267 12 kg body weight (BW), which were stulated in the rumen and abomasum and
distributed in a 4 4 Latin square design. Beef steers performance was evaluated using a
total of 32 Holstein Zebu steers with a 364 20 kg BW, which were distributed into eight
randomized blocks. In a brief view, an earlier harvest of B. brizantha cv. Marandu grass, at
35 d of regrowth, did not increased the nutritional value of silage, nutrient digestibility,
microbial or nitrogen use efciency and beef cattle performance, when compared with a
later harvest, at 70 d. The addition of microbial inoculant did not improve DM recovery,
fermentation process, and did not inuenced beef cattle nutrient intake and performance.
Also, this inoculant was unable to reduce fermentation losses.
2015 Elsevier B.V. All rights reserved.

Abbreviations: BBG, Brachiaria brizantha cv. Marandu grass; BBS, Brachiaria brizantha cv. Marandu silages; RA, regrowth age; MI, microbial inoculant;
BW, body weight; NH3 -N, ruminal ammonia nitrogen; ADG, average daily gain; DRSi, initial dressing; DRSf, nal dressing; IBW, initial body weight; FBW,
nal body weight; CADG, carcass average daily gain; DM, dry matter; OM, organic matter; CP, crude protein; EE, ether extract; aNDFom(n), neutral detergent
ber assayed with a heat-stable amylase and corrected for ash and nitrogenous compounds; NFC, non-brous carbohydrates; TDN, total digestible nutrients.
Corresponding author at: Universidade Federal de Vicosa, Departamento de Zootecnia, 36570-900 Vicosa, MG, Brazil. Tel.: +55 31 3899 2265;
fax: +55 31 3899 2275.
E-mail address: odilon@ufv.br (O.G. Pereira).
http://dx.doi.org/10.1016/j.anifeedsci.2015.06.025
0377-8401/ 2015 Elsevier B.V. All rights reserved.

34

A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

1. Introduction
Grasses belonging to the Brachiaria genus are distributed throughout the entire tropical zone worldwide and they grow
in a highly varied range of habitats, although predominantly occurring in the African savannas. In South America, especially
in Brazil, Brachiaria grasses has been used as forage since the 1950s and occupies approximately 85% of the 180 million ha
of cultivated pastures (Macedo, 2004), demonstrating the high productive potential of this forage crop for ensiling.
Tropical forages are known to exhibit low water soluble carbohydrates content, which compromises the rapid lowering of
the silage pH. Additionally, the low DM content of silage favors the Clostridiums development in the ensiled material when
it is harvested young. However, the Brachiaria grasses have a higher proportion of leaves and higher DM content compared
with other tropical grass species. Thus, it is necessary to know whether the harvest of young silage is advantageous from
a nutritional standpoint and whether the use of a microbial inoculant (MI) can improve the fermentation efciency of the
ensiled material. According to Kung et al. (2003), the use of MI may increase the lactic/acetic acid ratio and reduce proteolysis
and protein deamination, allowing better utilization of the water-soluble carbohydrates and, consequently, increasing DM
recovery. Additionally, there is a need to further know the growing period that would be allowed to maximize silage quality,
which would be considered the ideal regrowth age.
Therefore, it was hypothesized that an earlier harvest of B. brizantha cv. Marandu grass, at 35 d of regrowth, can increase
the nutritional value of silage compared with a later harvest, at 70 d, which may be directly reected in ruminant performance.
Also, the use of a MI can improve DM recovery and the fermentation process, without inuencing in ruminant performance.
Thus, this study evaluated the fermentation prole and chemical composition of BBS harvested at two RA (35 and 70 d), each
with and without added MI, and assessed silage digestibility, nutrient intake, and performance in cattle fed these silages.
2. Materials and methods
2.1. Location and management of B. brizantha cv. Marandu grass
The experiment was performed at the Research and Extension Center of the Tringulo Mineiro (Central de
Experimentaco, Pesquisa e Extenso do Tringulo Mineiro CEPET) of the Federal University of Vicosa (Universidade Federal de Vicosa UFV) in the city of Capinpolis (18 41 S latitude, 49 34 W longitude average altitude 620.2 m), Minas Gerais
State, Brazil. According to Kppen (1948), the climate is classied as type Aw, hot and humid, with coldest monthly temperatures above 18 C; annual average rainfall between 1400 and 1600 mm, rainy season in the summer (OctoberMarch) and
dry season (AprilSeptember) in the winter. The growing period for the grass was comprised between January and march
2008; the mean rainfall for this period was 264.6 mm, maximum temperature 28.2 C and minimum 20.2 C.
A total area of 6 ha with BBG was seeded and fertilized with 60 kg/ha of P2 O5 for crop establishment using simple
superphosphate as phosphorus fertilizer source. The area was divided into two plots, one measuring 2.75 ha and the other
3.25 ha. The grass in the plots was harvested to a height of approximately 20 cm via beef steers grazing. After the animals
were removed, the plots were top-dressed with 60 kg/ha of N and 60 kg/ha of K2 O, using a 20-0-20 NPK fertilizer. Access to
both plots was restricted for 70 and 35 d corresponding to the RA.
2.2. B. brizantha cv. Marandu grass harvest and additive application
The BBG was harvested at 35 and 70 d of regrowth and chopped into approximately 2 cm pieces using a forage machine
(JF 92 Z10, JF Agricultural Machinery, Itapira, SP, Brazil) tted with a Sahara 100 forage harvester platform (Haramaq, Serto,
RS, Brazil). The grass was harvested 10 cm above ground level.
Immediately before ensiling, the MI (Sil All C4, Alltech, Araucria, PR, Brazil) was applied to half of the forage harvested
at 35 and 70 d of regrowth by manually spraying with a 0.250 L solution prepared according to the manufacturer (250 g of
inoculant diluted in 50 L of water). The MI used contained a basic composition of cellulolytic and hemicellulolytic enzymes
and 80% dextrose and exhibited the following guaranteed analysis: Lactobacillus plantarum (10 billion CFU/g), Pediococcus
acidilactici (1 billion CFU/g) and Enterococcus faecium (10 billion CFU/g). The other half of the forage harvested at 35 and 70
d was ensiled without MI.
The management and care of animals was performed in accordance with the guidelines and recommendations of the
Committee of Ethics on Animal Studies at the Federal University of Vicosa (UFV), MG, Brazil.
2.3. Silage quality
A 2 2 factorial scheme was used in a completely randomized design with ve replicates. The factors corresponded to
experimental bucket silos with the BBS at the two RA (35 and 70 d) and with or without MI. The BBG was ensiled in 18 L
plastic buckets after harvesting and applying the additive. Buckets were tted with Bunsen valves on the lids to allow the
gases escape. At the bottom of the silos, cloth bags containing 4 kg of dry sand were placed for the estimation of efuent
production. The silos were sealed with adhesive tape, weighed and stored in a covered area at room temperature. The
experimental silos were opened and weighed at 60 d after ensiling to quantify the losses of DM, gases and efuent.

A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

35

Table 1
Ingredient proportion in the concentrates used in diets based on silages prepared with tropical grass Brachiaria brizantha cv. Marandu harvested at 35 or
70 regrowth days.
Ingredient

Concentrate 35 d (g/kg DM)

Concentrate 70 d (g/kg DM)

Ground corn grain

Soybean meal
Urea/ASa
Mineral mixb

828.5
129.0
8.2
34.3

824.6
128.4
10.6
36.4

Urea and ammonium sulphate at the ratio 9:1.

Composition: lime (731 g/kg); Cooper sulphate (225 g/kg), cobalt sulphate (14 g/kg), zinc sulphate (754 g/kg), iodine sulphate (5 g/kg), sodium selenite
(2 g/kg).
b

Table 2
Chemical compositions (g/kg DM) of the tropical grass silages prepared with Brachiaria brizantha cv. Marandu harvested at 35 and 70 d, with or without
microbial inoculant, and concentrates.
Silages

Concentrates

35 d

DM
OM
CP
aNDFom(n)
NFC
EE

70 d

Control

Inoculant

Control

Inoculant

201
906
53
632
212
8.6

207
900
57
633
202
7.5

220
912
44
696
163
9.0

220
904
47
696
154
6.8

35

70

888
954
188
97
638
31

885
959
199
103
630
27

DM: dry matter; OM: organic matter; CP: crude protein; aNDFom(n): neutral detergent ber assayed with a heat-stable amylase and corrected for ash and
nitrogenous compounds; NFC: non-ber carcorhydrates; EE: ether extract.

After opening, approximately 500 g of silages were dried in a forced-air oven at 60 C for 72 h, ground in a Wiley-type
knife mill (TE-625, TECNAL, Piracicaba, So Paulo, Brazil) with a 1-mm mesh, and stored in properly sealed plastic containers
to proceed later chemical analysis. Another silage samples were collected for pH and ammonia nitrogen determinations
according to Bolsen et al. (1992). Silage pH was determined with a glass electrode after homogenization of 25 g of fresh
silage with 100 mL of distilled water. Another 25 g of silage was mixed with 200 mL of H2 SO4 solution (0.2 N) and left
undisturbed in a refrigerator for 48 h. This mixture was passed through a quantitative lter paper and the ltrate was frozen
for subsequent determination of NH3 -N content.
To determine volatile fatty acids and acid lactic, 10 g of silage was weighed and then mixed with 90 mL of distilled water
in a blender for 1 min. An aliquot of the homogenized sample was then strained through 2 layers of cheesecloth, and the
extract was treated with 10% metaphosphoric acid for subsequent determination of lactic, propionic and butyric acids.
2.4. Rumen degradation and nutrient outow to abomasum
Ruminal degradation and nutrient outow were measured using four Nellore Steers (267 12 kg BW) tted with rumen
and abomasal stulas. The steers were distributed in a 4 4 Latin square design in which the four treatments of the
experiment were dened by the factorial combination of the two RA (35 or 70 d) of BBS with the presence or absence
of MI.
A concentrate was formulated for each forage RA (Tables 1 and 2). The diets were formulated for ADG of 12 g/kg BW
according to the Brazilian System for Nutritional Requirements of Pure and Crossbred Zebu Cattle (BR-CORTE), as described
by Valadares Filho et al. (2010), adopting a 50:50 forage:concentrate ratio on DM basis.
Each experimental period lasted 17 d (four periods in all), with 10 d for adaptation and seven for collecting samples,
which included ruminal uid, abomasal digesta, feces, feed provided, and refusal feed. The samples of the feed provided and
orts were collected daily between 11th and 17th d of each experimental period, placed in previously labeled plastic bags
and stored at 15 C for later analysis. At the end of each experimental period, the feed samples, orts, feces, and abomasal
digesta were thawed and subjected to same drying and milling processing.
To determine fecal dry matter and abomasal ow of dry matter, chromic oxide was infused via rumen cannula in a pulse
dose of 15 g at 11:00 h on 4th to 14th d of each experimental period. Feces and abomasal digesta were collected every 26 h,
starting at 08h00 on 11th d and ending at 18h00 on 15th d of each period. The feces samples, directly collected from the
rectum of each animal, and the abomasal digesta samples were placed in labeled plastic bags and stored at 15 C for later
analysis.
Ruminal uid was collected to measure the pH and NH3 -N concentrations. These samples were collected at 0, 2, 4, and
6 h after the morning feeding on the 16th d of each experimental period. The pH was immediately measured after collection;
1 mL of 1:1 sulfuric acid (H2 SO4 ) was added to the sample, which was then stored in the freezer at 18 C for later NH3 -N
concentration analysis.

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A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

On 17th d 50-mL urine spot samples were obtained for each steer. The urine was ltered, and 10-mL aliquots were
removed and immediately diluted in 40 mL of 0.036 N H2 SO4 to prevent bacterial destruction of the purine derivatives
and uric acid precipitation, according to Valadares et al. (1999). These samples were stored at 15 C for later analysis of
creatinine, allantoin, and uric acid. An undiluted urine sample was stored for determining the total nitrogen compound
yields and urea concentration.
2.5. Performance of beef cattle
Thirty-two Holstein Zebu crossbred bulls with 364 20 kg BW were distributed into eight randomized blocks to evaluate the intake and productive performance. The initial BW of the animals was used as a criterion for distributing them
among the treatments. Treatments were dened by the factorial combination of the two RA (35 or 70 d) of BBS with the
presence or absence of MI.
Four 30-t surface silos were lled with BBG: two silos for each RA, treated or not with MI. The silos were opened 60 d
after ensiling. A concentrate was formulated for each forage RA (Tables 1 and 2). The diets used in the previous experiment
were adopted with a 50:50 forage:concentrate ratio on DM basis.
This trial lasted 99-d and was divided into a 15-d adaptation period and three experimental periods of 28-d each. Individual stalls measuring approximately 10 m2 and tted with feeders and water troughs were used to house the animals.
Five steers were slaughtered at the end of the adaptation period to estimate the initial carcass weights of all 32 steers at
the beginning of the experiment. The nal carcass weight of each animal minus the average initial carcass weight of the
reference group was used the estimate carcass gain.
Feed was provided in two daily meals at 7h00 and 15h00, and the orts were weighed daily to allow a maximum of 510%
of total feed provided as fresh feed. The orts were sampled, placed in labeled bags, and stored in the freezer for later analysis.
All steers were slaughtered at the end of the experiment to determine dressing percentage (DRS), which was calculated as
the percentage between the hot carcass weight and nal body weight (FBW) after fasting. Thus, CADG was calculated by
using the following equation:



CADG (kg/day) =

FBW % DRSf
100

 

IBW % DRSi
100



2.6. Chemical analyses

Water extracts of each bucket silos stored with H2 SO4 were analyzed for NH3 -N according to the colorimetric method
described by Weatherburn (1967). The other one stored with metaphosphoric acid were used to determine lactic, propionic
and butyric acids in a high performance liquid chromatography (HPLC) (model SPD-10A VP, Shimadzu, Kyoto, Japan) coupled
to an ultraviolet (UV) detector using 210-nm wavelengths according to the method described by Kung (1996).
Fresh forage, silage and concentrate samples, orts, feces, and abomasal digesta from all of the three experiments were
analyzed for DM (Method 934.01; AOAC, 1990); ash (Method 924.05; AOAC, 1990); CP obtained by determining total N, using
the micro-Kjeldahl technique (Method 920.87; AOAC, 1990) and xed conversion factor (6.25); EE content, determined gravimetrically after extraction with petroleum ether in a Goldsh device (Method 920.85; AOAC, 1990). The neutral detergent
ber was corrected for ash and protein according to Mertens (2002) and Licitra et al. (1996), assayed with a heat-stable amylase [aNDFom(n)]. Fresh forage, silage and concentrate samples were subjected to analysis of neutral-detergent-insoluble
nitrogen and the acid-detergent-insoluble nitrogen according to Licitra et al. (1996); acid detergent ber (ADF) (Method
973.18; AOAC, 1990); and lignin (sa) in 13.51 M sulfuric acid (Robertson and Van Soest, 1981).
The chromium contents of the feces and abomasal digesta were determined according to Willians et al. (1962), using
an atomic absorption spectrophotometer. The NH3 -N of ruminal uid samples was determined by using the colorimetric
technique described by Chaney and Marbach (1962).
Creatinine analysis was performed with acid picrate using a commercial kit (Labtest Diagnstica, Uria CE, Lagoa Santa,
Minas Gerais, Brazil), according to the modied diacetyl method. Daily creatinine excretion was estimated based on the
27.76 mg/kg BW recommendations (Renn et al., 2000). Daily urine volume was estimated by dividing daily creatinine
excretion by its concentration in the urine spot sample. The urinary urea nitrogen was performed with an enzymatic colorimetric method using a commercial kit (Labtest Diagnstica, Urea CE, Lagoa Santa, Minas Gerais, Brazil). The nitrogen balance
was calculated in g/d as follows: N intake (fecal N + urine N).
Analyses of purine derivatives as allantoin and uric acid were performed using a colorimetric method described by Chen
and Gomes (1992). Purine derivatives excretion was calculated by multiplying the urine volume, which was estimated within
24 h, by the purine derivatives concentration of the spot urine samples. Absorbed purines (Y, mmol/d) were calculated from
the purine derivatives excretion (X, mmol/d) by using the equation Y = 0.85X + 0.385 BW0.75 , in which 0.85 is the recovery of
absorbed purines as purine derivatives and 0.385 BW0.75 is the endogenous contribution to purine excretion (Verbic et al.,
1990). The NMIC was calculated as:
Microbial nitrogen (g N/d) =

70 absorbed purine derivatives

0.93 100 0.137

A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

37

Table 3
Experimental bucket silos obtained with tropical grass Brachiaria brizantha cv. Marandu harvested at 35 or 70 d (age) and ensiled during 60 d with or
without (inoc) microbial inoculant.
Control

Fermentation parameters
Efuent losses (kg/t)
Gases losses (g/kg FM)
DM recovery (g/kg FM)
pH
NH3 -N (g/kg N)
Lactic acid (g/kg DM)
Propionic acid (g/kg DM)
Butyric acid (g/kg DM)
Chemical composition (g/kg)
DM
OM
aNDFom(n)
ADF
CP
EE

Inoculant

SEM

35 d

70 d

35 d

70 d

27.5
162
838
4.9
91
45.4
7.7
2.1

36.5
144
856
4.9
95
18.5
13.4
1.2

29.3
147
853
4.9
83
24.1
10.9
1.4

45.8
146
854
4.8
95
18.8
11.4
1.4

203
869
618
371
39
19

209
838
605
371
38
19

205
875
612
368
38
16

215
794
621
387
35
23

P-value
Age

Inoc

Age*inoc

8.1
15
15
0.01
15
1.8
0.7
0.2

0.13
0.56
0.56
0.73
0.64
<0.01
<0.01
0.03

0.50
0.68
0.68
0.48
0.81
<0.01
0.39
0.40

0.65
0.58
0.58
0.58
0.80
<0.01
<0.01
0.04

6.4
5.6
12.2
11.7
3.4
3.5

0.21
0.33
0.88
0.42
0.59
0.28

0.60
0.74
0.70
0.57
0.55
0.97

0.73
0.67
0.39
0.46
0.64
0.33

FM: fresh material; NH3 -N: ruminal ammonia nitrogen; DM: dry matter; OM: organic matter; CP: crude protein; aNDFom(n): neutral detergent ber
assayed with a heat-stable amylase and corrected for ash and nitrogenous compounds; NFC: non-ber carcorhydrates; EE: ether extract.

where 70 is the N content of purines (mg N/mmol), 0.93 represents the true digestibility of purines, and 0.137 is the average
N-purines:N-total ratio in the bacteria that were isolated from the rumen (Barbosa et al., 2011).
2.7. Statistical analyses
The silage fermentation prole were subjected to ANOVA in a completely randomized design and a 2 2 factorial scheme
with four treatments, namely control 35 d, inoculant 35 d, control 70 d and inoculant 70 d, with ve replicates. The digestibility, pH and nitrogen usage were subjected to ANOVA in a Latin square design with four treatments and four replicates. To
evaluate pH and ruminal NH3 -N, the same treatments aforementioned were considered, in addition to collection time and the
interaction among all the effects described. The animal and period factors were also considered random effects. A repeated
measures model (Littell et al., 1998) was used when data collections were repeated in time (0, 2, 4 and 6 h after feeding)
within each experimental unit, which was composed by each animal within each period. To obtain the linear, quadratic and
cubic effects relative to the pH and ruminal NH3 -N collection times, quantitative orthogonal contrasts were used. The nutrient intake and animal performance analyses were subjected to ANOVA in a randomized block design with four treatments
and eight replicates.
All of the data were analyzed using the MIXED procedure of the Statistical Analysis System (SAS Institute Inc., Cary, NC,
USA) software (version 9.1). For all experiments the homogeneity of the treatment variances was assumed, and the degrees
of freedom were estimated using the KenwardRoger method. The SLICE command of the SAS software (version 9.1) was
used to further examine the interactions when necessary. The statistical procedures were conducted using 0.05 as the critical
level for the probability of a type I error.
3. Results
3.1. Silage quality
No effects (P > 0.05) of the RA, microbial inoculation or their interaction were observed on the chemical composition, the
fermentation parameters, the gasses and efuent losses or the DM recovery of the silages evaluated (Table 3).
The lactic acid content (P < 0.01) was affected by the interaction between the studied factors (Table 3). After examining
the interactions further (Fig. 1), the silages prepared with the forage harvested at 35 d were found to have higher lactic acid
values when was not inoculated (P < 0.01). However, among the silages prepared with forage harvested at 70 d, there was no
effect of the inoculant (P = 0.91). When comparing the uninoculated silages (control), those prepared with forage harvested
at 35 d had higher lactic acid contents than those harvested at 70 d (P < 0.01). For the inoculated silages, there was a nearly
signicant tendency (P = 0.06) for a higher lactic acid content in the silages prepared with forage harvested at 35 d compared
with 70 d.
The propionic acid content (P < 0.01) was affected by the interaction between the factors studied (Table 3). After examining
the interaction further (Fig. 1), the silages prepared with the forage harvested at 35 d were found to contain more propionic
acid when inoculated (P < 0.01) than when uninoculated. However, among the silages prepared with forage harvested at 70
d, there was a trend for an effect of the inoculant (P = 0.06), with a higher propionic acid content in the uninoculated silages.

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A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

Fig. 1. Interaction slicing for organic acids quantied in experimental bucket silos obtained with tropical grass Brachiaria brizantha cv. Marandu harvested
at 35 or 75 d and ensiled during 120 d with or without microbial inoculant (EXP 1).

When comparing the uninoculated silages (control) prepared with forage harvested at 70 and 35 d, the silages harvested at
70 d had the higher propionic acid content (P < 0.01); however, the inoculated silages were not affected by the RA (P = 0.61).
The butyric acid content (P = 0.04) was affected by the interaction between the factors studied (Table 3). After examining
the interactions further (Fig. 1), the silages prepared with forage harvested at 35 d were found to have higher butyric acid
values when uninoculated (P = 0.04) than when inoculated. However, among the silages prepared with forage harvested at
70 d, there was no effect of the inoculant (P = 0.33). When comparing the uninoculated silages (control), the butyric acid
content was higher in those prepared with forage harvested at 35 d compared with 70 d (P < 0.01). By contrast, the inoculated
silages were not affected by the RA (P = 0.94).
3.2. Rumen degradation and nutrient outow to abomasum
In the cannulated steers, there were no effects (P > 0.05) of the RA, the MI or their interaction on the intake of DM or total
digestible nutrients (TDN) in g/kg LW (Table 4).
There was no effect (P > 0.05) of the MI or its interaction with the RA on the total digestibility of either DM or the other
nutrients. However, the RA affected the EE digestibility (P < 0.05). There were also trends toward an increase in the digestibility of DM (P = 0.072), OM (P = 0.068) and non-brous carbohydrates (NFC) (P = 0.061) and in the TDN content (P = 0.075) of

A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

39

Table 4
Nutrient digestibility of tropical grass silage prepared with Brachiaria brizantha cv. Marandu harvested at 35 or 70 d (age) and ensiled during 60 d with or
without (inoc) microbial inoculant for feeding cannulated Nellore beef steers.
Control
35 d
Nutrient intake (g/kg LW)
1.7
DM
0.9
TDN
Total apparent digestibility (g/kg)
DM
547
OM
567
428
aNDFom(n)
665
EE
559
CP
703
NFC
557
TDN
Ruminal digestibility (g/kg)
623
DM
OM
688
851
aNDFom(n)
134
EE
316
CP
652
NFC
Intestinal digestibility (g/kg)
377
DM
PB
328
595
EE

Inoculant
70 d

35 d

1.6
1.0

SEM
70 d

1.5
0.8

1.7
1.0

P-value
Age

Inoc

Age*inoc

0.1
0.1

0.51
0.09

0.55
0.57

0.30
0.41

619
635
447
787
543
803
617

559
578
415
613
523
761
562

605
624
435
752
508
808
604

2.7
2.7
3.9
5.0
3.1
3.6
2.5

0.07
0.07
0.63
0.03
0.52
0.06
0.07

0.96
0.99
0.76
0.39
0.35
0.37
0.86

0.65
0.69
0.99
0.86
0.85
0.44
0.72

624
679
849
447
172
724

636
691
944
427
244
703

644
772
848
391
644
837

5.9
6.7
6.1
15.4
8.7
8.1

0.92
0.23
0.47
0.06
0.12
0.02

0.73
0.13
0.45
0.06
0.03
0.04

0.94
0.15
0.46
0.03
0.01
0.38

375
425
847

364
334
729

355
236
804

5.9
11.6
6.5

0.92
0.99
0.04

0.73
0.39
0.48

0.94
0.36
0.19

DM: dry matter; OM: organic matter; CP: crude protein; aNDFom(n): neutral detergent ber assayed with a heat-stable amylase and corrected for ash and
nitrogenous compounds; NFC: non-ber carcorhydrates; EE: ether extract; TDN: total digestible nutrients.
Table 5
Efciency of dietary nitrogen usage in the metabolism of Nellore beef steers fed with tropical grass silage prepared with Brachiaria brizantha cv. Marandu
harvested at 35 or 70 d (age) and ensiled during 60 d with or without (inoc) microbial inoculant.
Control

NB (g/day)
N intake
N fecal
N urine
NB
NB (%)a
Microbial efciency
NMIC b
Ec NMIC c
a
b
c

Inoculant

P-value

35d

70d

35d

70d

SEM

Age

Inoc

Age*inoc

504
232
39
233
47

429
195
25
209
45

452
210
33
209
48

456
229
55
172
37

42.0
33.1
7.8
26.1
4.7

0.39
0.80
0.63
0.17
0.41

0.75
0.85
0.17
0.18
0.12

0.34
0.42
0.06
0.78
0.22

53
131

41
121

40
117

56
162

6.2
22.9

0.73
0.28

0.94
0.41

0.06
0.12

NB: nitrogen balance in percentage of N intake.

g/day.
g CP per kg TDN ingested.

the experimental feed composed of the BBS harvested at 70 d compared with 35 d. The total crude protein digestibility was
not affected (P > 0.05) by the RA.
The apparent ruminal digestibility of DM, OM, and aNDFom(n) were not affected by the RA, MI or by their interaction
(P > 0.05). The ruminal digestibility of the EE and CP contents were affected (P < 0.05) by the interaction between the factors.
Further examination of the interaction revealed that the EE was signicantly affected (P < 0.05) by the presence of inoculant
in forage harvested at 35 d of regrowth, with digestibility being higher for the feed containing the MI. There was also an effect
(P < 0.05) of RA in the uninoculated treatment, with the ruminal EE digestibility higher for the feed prepared with forage
harvested at 35 d of regrowth than at 70 d. For ruminal CP digestibility, examination of an interaction between the treatment
factors indicated a signicant effect (P < 0.05) of the presence of inoculant in the forage harvested at 70 d of regrowth, with
a higher digestibility of the feed containing MI. Within the uninoculated treatment, there was also an effect (P < 0.05) of
RA, with a higher ruminal CP digestibility of the feed prepared using the forage harvested at 70 d compared with 35 d of
regrowth.
The ruminal digestibility of NFC (Table 4) was signicantly affected (P < 0.05) by both the RA and the MI but not by the
interaction between these treatment factors (P > 0.05). The ruminal digestibility of NFC was higher for the feed containing
forage harvested at 70 d compared with 35 d (P < 0.05). The silages containing MI also exhibited the same pattern (P < 0.05).
The variables regarding nitrogen balance and microbial efciency (Table 5) were not affected (P > 0.05) by the RA, MI or
the interaction between the factors.

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A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

Table 6
Ruminal ammonia nitrogen (NNH3 ) and pH over sampling time (T) of rumen uid in Nellore beef steers fed with tropical grass silage prepared with
Brachiaria brizantha cv. Marandu harvested at 35 or 70 d (age) and ensiled during 60 d with or without (inoc) microbial inoculant.
Control

NH3 -Na
pHb

Inoculant

SEM

35 d

70 d

35 d

70 d

6.6
6.56

8.1
6.38

29.8
6.53

9.9
6.44

11.4
4.5

P-value
Age

Inoc

A*I

A*T

I*T

A*I*T

<0.01
0.04

<0.01
0.85

<0.01
<0.01

<0.01
0.39

<0.01
0.98

0.01
0.67

<0.01
0.56

a
Slice effect of time inside age*inoc: P = 0.9560 for 35 d-control; P < 0.001 for 70 d-control; P = 0.9566 for 35 d-inoculant; P = 0.4812 for 70 d-inoculant.
Time effect: P = 0.0155 for linear; P = 0.3068 for quadratic.
b
Time effect: P = 0.0017 for linear; P = 0.0963 for quadratic.

Table 7
Nutrient intake and performance of crossbred Holstein Zebu beef steers fed with tropical grass silage prepared with Brachiaria brizantha cv. Marandu
harvested at 35 or 70 d (age) and ensiled during 60 d with or without (inoc) microbial inoculant.
Control
35 d
Nutrient intake (kg/d)
9.7
DM
9.1
OM
aNDFom(n)
5.1
0.21
EE
CP
1.2
2.5
NFC
Nutrient intake (g/kg BW)
23.6
DM
12.5
aNDFom(n)
Performance and carcass evaluation
ADG
1.1
Dressing
556
121
GE
0.84
CG

Inoculant
70 d
9.9
9.1
4.8
0.21
1.1
2.9

35 d
9.1
8.7
5.0
0.21
1.1
2.4

SEM
70 d

P-value
Age

Inoc

Age*inoc

10.1
9.5
5.5
0.22
1.2
2.4

0.4
0.4
0.5
0.01
0.6
0.5

0.15
0.36
0.81
0.55
0.34
0.73

0.69
0.98
0.63
0.93
0.84
0.58

0.37
0.38
0.43
0.79
0.18
0.72

0.8
1.2

0.64
0.94

0.80
0.69

0.31
0.41

0.1
14.0
9.1
0.1

0.54
0.28
0.64
0.45

0.80
0.75
0.61
0.66

0.20
0.80
0.13
0.74

23.1
11.5

22.5
11.9

23.8
13.1

1.3
537
131
0.80

1.3
558
140
0.89

1.2
545
122
0.81

DM: dry matter; OM: organic matter; CP: crude protein; aNDFom(n): neutral detergent ber assayed with a heat-stable amylase and corrected for ash and
nitrogenous compounds; NFC: non-ber carbohydrates; EE: ether extract; ADG: average daily gain (kg/d); Dressing: g/kg of shrunk body weight (SBW);
GE: gain efciency (g gain/kg DM consumed); CG: carcass gain (kg/d).

The concentration of NH3 -N in the rumen (Table 6) was affected (P < 0.05) by the 3-way interaction among the RA, the
MI and the sampling time. Further examination of the interaction indicated that the sampling time had a positive linear
relationship with the concentration of NH3 -N in the rumen (P = 0.0155) and was only signicant for the animals that were
fed the inoculated silage harvested at 70 d (P < 0.001). The other interactions involving sampling time were not signicant
(P > 0.05). An investigation of the interaction between the presence or absence of the inoculant and the forage harvested
at 35 or 70 d of regrowth showed that the concentration of NH3 -N in the rumen was higher for the animals fed the silage
harvested at 35 d and containing the MI (P < 0.05). The other interactions relative to these two factors were not signicant
(P > 0.05).
Ruminal pH was affected by the factors RA and sampling time. The pH was higher (P < 0.05) in the rumen of the animals
fed the forage harvested at 35 d of regrowth compared with 70 d, and the pH exhibited a negative linear relationship with
the sampling time (0 h = 6.64, 2 h = 6.54, 4 h = 6.31 and 6 h = 6.24).
3.3. Performance of beef cattle
There were no effects (P > 0.05) of the RA, the MI or their interaction on the intake of DM and other nutrients (in kg/d and
g/kg BW, respectively) or on the animal performance variables (Table 7).
4. Discussion
4.1. Silage quality
Adding MI did not improve the fermentation characteristics of BBS. The inefciency of many commercial inoculants in
wet tropical grass silages may result from the inclusion of inappropriate species of lactic acid bacteria or species unable
to effectively compete with epiphytic ora when applied at low doses (Muck, 2010). Variation in the population of wild
bacteria and fungi pre-existing in the forage could interact with MI, and then alter the response.
This lack of response may be related to the reduced variation in the means obtained for pH values of the different silages
(SEM = 0.01) because this result suggests that the inoculant used was unable to break the buffering effect characteristic
of the fermentation process of grass silages. It is known that grass silages, especially tropical grass, stabilize at higher pH

A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

41

values (Santos et al., 2011). Coan et al. (2005) tested the effect of inoculation with L. plantarum, E. faecium and Pediococcus
(1.5 105 bacteria/g of forage) in Tanzania and Mombasa grass silages at two RA (35 and 60 d) and found that the presence
of the MI did not change the pH values or the lactic acid and NH3 -N concentrations in the treatments. Bernardes et al. (2008)
also found no change in the pH and NH3 -N concentrations in Marandu grass silages treated or untreated with L. plantarum
and Propionibacterium.
In the absence of the MI, the silages had higher lactic and butyric acid contents when harvested at 35 d than at 70 d,
most likely because of the higher water soluble carbohydrates content found at the earlier stage (Table 2). Thus, the MI was
ineffective in controlling undesirable populations of bacteria because the inoculant was unable to lower the butyric acid
content produced during clostridia growth in the ensiled mass. The growth of such microorganisms is undesirable in that
they consume lactic acid, thus increasing the pH and reducing the nutritional value of the silage due to proteolysis (Mcdonald
et al., 1991). With the lack of response to the inoculant, there were no reduced fermentation losses in the BBS.
It was also noted that the two different ages of forage regrowth did not affect silage quality. This growth interval was
likely insufcient to cause signicant alterations in cell wall lignication. These facts reinforce the possibility of harvesting
at 70 d of regrowth, when there is increased fresh biomass production per hectare. It is important to highlight that tropical
grasses harvested at older ages generate better silage fermentation patterns (Coan et al., 2008; Santos et al., 2011).
This presupposition could be better observed by taking into consideration ADF content in silages, which did not differ
between treatments. Possibly, even at 70 d maturity, lignication was not too pronounced and would not reach a high rate
at this age.
4.2. Rumen degradation and nutrient outow to abomasum
The absence of an effect of the inoculant on total nutrient digestibility may be explained by the absence of an effect of
the inoculant on silage quality. In testing the digestibility of Marandu grass inoculated or uninoculated with MI in crossbred
steers, Bergamaschine et al. (2006) found no difference in the total digestibility of DM, CP, aNDFom(n) and carbohydrates.
However, a trend indicating that the forage RA caused differences in the total digestibility of DM, OM and NFCs and in the
TDN content in the diets was observed. The digestibility of these characteristics was higher in the silages prepared with forage
harvested at 70 d compared with that harvested at 35 d. Therefore, this trend may have resulted from the digestibility of the
water soluble carbohydrates in the harvested forage because the digestibility of aNDFom(n) and CP remained unaffected.
The positive coefcients for the ruminal digestibility of CP indicated that ammonia was absorbed in the rumen and that
the feeds likely contained excess rumen degradable protein relative to available energy because an adequate balancing
of CP should have resulted in values close to zero. The inclusion of more urea in this diet combined with the presence of
the inoculant may have increased the rate of ruminal nitrogen degradation. Several studies hypothesize that the bacteria
present in inoculants can cause a probiotic effect or interact with rumen bacteria (Weinberg and Ashbell, 2003). The negative
coefcient for the apparent ruminal digestibility of EE contents results from microbial lipid synthesis in the rumen, which
allows for more lipids to reach the abomasum than the amount ingested.
The similar pattern of response regarding nitrogen balance and microbial nitrogen among the diets may partially result
from the absence of differences in nutrient intake, especially CP intake. According to Clark et al. (1992), only an increase in
DM intake, with a consequent increase in the ruminal passage rate, would increase microbial passage to the small intestine,
reducing the recycling of energy and N in the rumen. Microbial protein synthesis largely depends on the availability of
carbohydrates and nitrogen in the rumen, such that microbial growth increases with the synchronization between the
availability of fermentable OM and of degradable nitrogen in the rumen (NRC, 2001).
The ruminal concentrations of NH3 -N, regardless of the diet and sampling time, were higher than the 6.24 mg of NH3 N/dL of rumen uid established by Sampaio et al. (2009) and the 5 mg of NH3 -N/dL reported by Satter and Slyter (1974) as
the minimum concentration necessary for maximum rumen fermentation. However, Van Soest (1994) suggested a ruminal
concentration of 10 mg of NH3 -N/dL as the optimal level, noting that this value should not be considered xed because the
rate of carbohydrate fermentation directly affects ammonia uptake by bacteria and, consequently, the microbial synthesis
capability. The animals fed inoculated silages had higher mean NH3 -N in their rumen uid, most likely because of the action of
the inoculant microorganisms on protein degradation, a fact that was evident in these trials. However, there are insufcient
data to determine the nature of the interactions of the inoculant microbes with the diets provided. The mean ruminal pH of
6.5 is higher than the values established by Hoover (1986) as inhibitory to the development of cellulolytic microorganisms.
Thus, the activity of the cellulolytic microorganisms and the consequent ber digestion were not affected by diets.
4.3. Performance of beef cattle
The absence of an effect of treatments on the DM intake is likely due to the similar chemical composition of the diets. The
similarity in the DM intake may also explain the similarity in the nutrient intake. These results corroborate those of silage
quality because no difference in DM intake would be expected unless the MI modied the fermentation prole of the silages.
Similar to the MI, the different RA did not inuence the fermentation prole and produced silages of equivalent quality. The
nutritional quality of the silage likely affected the DM intake, which did not differ among the treatments.
Paziani et al. (2006) and Santos et al. (2011) did not observe responses in studies with tropical grass silages. According
to Muck (2010), one of the causes of the high variability in responses to the inoculants in grass silages is the variation in the

42

A.S. Cezrio et al. / Animal Feed Science and Technology 208 (2015) 3343

composition and abundance of the population of epiphytic bacteria in the forage, which may dominate the fermentation,
inhibiting the bacteria in the inoculant.
The intake of DM and of the other nutrients in turn impacted the animal performance. According to Van Soest (1994),
animal performance is predetermined by nutrient intake, which is important for meeting the maintenance and production
requirements of ruminants. Thus, the absence of an effect of the treatments on animal productive performance reects the
pattern observed for the nutrient intake.
Muck (2010) discussed several reasons to explain why the inoculant does not alter fermentation and, subsequently,
animal performance. First, the inoculant may be ineffective or misused, as tests are not usually performed to verify the
number of lactic acid bacteria claimed by the manufacturer. Second, inoculation with an additional additive to supply WSC
for forage with low sugar levels, such as grasses, may be necessary. There is also the need to quantify the number of epiphytic
bacteria in the crop to be ensiled, which may be so high that the inoculant bacteria never dominate the fermentation. Thus,
similar fermentation among silages would, in turn, result in similar performance among the animals that consume them.
5. Conclusion
An earlier harvest of Brachiaria brizantha cv. Marandu grass, at 35 d of regrowth, did not increased the nutritional value
of silage, nutrient digestibility, microbial or nitrogen use efciency and beef cattle performance, when compared with a
later harvest, at 70 d. Therefore, the silage of this grass is recommended for use at 70 d of regrowth because this age allows
for higher forage accumulation and likely lower production cost than a shorter regrowth period. The addition of microbial
inoculant based on L. plantarum (10 billion CFU/g), P. acidilactici (1 billion CFU/g) and E. faecium (10 billion CFU/g) did not
improve DM recovery, fermentation process, and did not inuenced beef cattle nutrient intake and performance. Also, this
inoculant was unable to reduce fermentation losses.
Conicts of interest
We wish to conrm that there are no known conicts of interest associated with this publication and there has been no
signicant nancial support for this work that could have inuenced its outcome.
Acknowledgements
The authors wish to thank the Conselho Nacional de Pesquisa e Desenvolvimento Cientco e Tecnolgico (CNPq),
Fundaco de Amparo Pesquisa do Estado de Minas Gerais (FAPEMIG), and Instituto Nacional de Cincia e Tecnologia
Cincia Animal (INCT-CA) for nancial support.
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