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RESEARCH PAPER OPEN ACCESS

Study of in vitro rumen fermentation kinetics of sour grape

pomace ensiled with some additives in ruminants

Ali Asghar Yaghoubi1*, Abdoul Mansour Tahmasbi2, Reza Valizadeh2, Abbas Ali

Naserian2 and Mohsen Kazemi3

1Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad,

International Campus, Mashhad, Iran

2Department of Animal Science, Excellence Center for Animal Science, Faculty of Agriculture,

Ferdowsi University of Mashhad, P. O. Box 9177948974, Mashhad, Iran

3Torbat-e-jam College of Agriculture and Animal Science, Torbat-e-jam, Iran

Key words: Sour grape pomace, additive, in vitro, rumen.

http://dx.doi.org/10.12692/ijb/4.5.198-209

Article published on March 10, 2014

Abstract

This study was conducted to assess the impact of 3 types of additives and ensiling process on nutritive value of

sour grape pomace (SGP) by the in vitro techniques. In vitro gas production parameters and in vitro dry matter

disappearance (IVDMD) were determined for total ensiled SGP. The dry matter (DM), crude protein (CP), and

organic matter (OM) increased with supplementation of 3 additives to the silages than control group (p<0.05),

while neutral detergent fiber (NDF), acid detergent fiber (ADF), and ash decreased (p<0.05). The highest

microbial protein (MP), metabolizable energy (ME), short chain fatty acids (SCFA), IVDMD and in vitro

cumulative gas production (IVCGP; after 24, 48 and 96 h incubation) observed for SGP ensiled with 1% urea

(P<0.05). Supplementation of SGP with different additives affected chemical composition and in vitro

fermentation, but there was no significant affect on pH after opening the silages. All estimated parameters

(SCFA, MP, ME and DDM) were lowest for control group. This paper, suggests that farmers in Iran can

efficiently use different additives (especially molasses and urea) before ensiling of SGP to improve nutritive value

of it and finally ruminant’s productivity, if ensiled at good conditions.

* Corresponding Author: Ali Asghar Yaghoubi ayaghoubi8@gmail.com

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print) 2222-5234 (Online)

http://www.innspub.net

Vol. 4, No. 5, p. 198-209, 2014

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Int. J. Biosci. 2014

Introduction

Animal husbandries continually search for

economically feasible by-product feeds for livestock.

Sour grape is called before ripening of grape (with a

sour taste). Sour Grape processing by-products,

including seed, skin, pulp and stalk, commonly called

sour grape pomace (SGP). In Iran, approximately 3.2

million tonnes of grapes produced in 2012 was used

for inner consumption and export (MJAS 2012), but

there was scarce data about sour grape production in

Iran. Also city of Kashmar, with 3 thousand and 200

hectares of vineyard to produce fine grapes and

raisins, is named khorasan-e-Razavi pantry. About

25% of Kashmar grapes are eaten freshly and the

other 75% is converted into raisins. Annually,

decoction factories and home consumers use fraction

of sour grape for Verjuice production in Iran. In this

process, wet SGP is produced in high level. These by-

products are usually burned causing environmental

pollution. The potential use of these wastes in

ruminant rations will participate in reducing the

shortage of feedstuffs and subsequently increase milk

and meat production. Due to difficulties in

environmental pollution, they are usually ensiled at a

lower dry matter (DM) content than other by-

products. Also data regarding the ensilability of SGP

are limited. Silage additives are applied to enhance

ensiling velocity and to prevent silage losses during

ensiling, storage and after the opening of the silo.

Having a rich soluble fiber, SGP should have value as

a compartment of ruminant diets, especially in years

when climatic conditions limit the availability of other

feeds. Nutritional values of grape by-products vary

with method of strainer, type of grape (i.e. red versus

white; Ruberto et al., 2008), and the relative rations

of seeds, pulp, skin and stalk in the pomace

(Baumgartel et al., 2007). Because these parameters

can change chemical composition, they may also alter

digestibility of SGP. Although feeding grape pomace

at the time of production has more advantages, but

nutrient losses during preservation as a feed has led

to disposal of most pomace as organic fertilizer

(Bustamante et al., 2008). Hence, it is necessary we

identify a good method for preservation of SGP after

production. To increase the rate of ensiling

fermentation, having a good silage and to achieve a

quick drop in pH of the silage, additives, such as

molasses and organic acids (Jaakkola et al., 2006;

Kazemi et al., 2014; Ebrahim Pour et al., 2014) are

used. These parameters effect on the ensiling process,

which in turn can influence the feeding quality of the

silage through manipulation of fermentation process

in the rumen. Recently the in vitro gas test technique

has been used for determining fermentation kinetics

of ruminant feed (Bohra et al., 2008; Garg et al.,

2012; Eslamian et al., 2013). Many studies have been

done by nutritionist as sources of dietary fiber and

polyphenols in animal feeds on grape pomace

(Baumgartel et al., 2007; Alipour and Rouzbehan

2007; Spanghero et al., 2009). However, there was

limited data about nutritive value of SGP after

ensiling. Also limited and localized availability of

product, different nutrient content and the presence

of contaminants have limited use of grape by-

products in the livestock and food industries. Due to

the little information available on the nutritive value

of the SGP by-product, the present study was carried

out for determination the influence of some additives

and ensiling of SGP on chemical composition,

IVDMD and in vitro rumen fermentation kinetics

using the gas production technique.

Material and methods

Samples and Treated silages

During late spring of 2013, fresh sour grape pomace

(SGP) samples were collected immediately after

extracting from juice producing companies located in

the Kashmar, Khorasan-e-Razavi distinct of Iran. A

total of 15 pomace samples were obtained from local

grape cultivars (mainly a composition with local

names of Askari , Rezghi and Sepidari grapes; latin

name: Vitis vinifera L.). Samples of SGP were

collected from each cultivar during the harvest season

(i.e., early summer). Immediately, samples

transferred to the laboratory of Azad university of

Kashmar and then Wet SGP were treated by sulfuric

acid (H2SO4; pure of 98%; Merck Company,

Germany), Molasses and Urea (45.5% Nitrogen;

Trade mark). All silages treated at 0.5 and 1% of dry

matter (DM). Also a control group (without any

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additive) prepared. The chemically treated samples

were filled into nylon bags with 0.2 mm thickness,

then tied up and kept at room temperature on

cemented floor. After having stored for 60, silages

were opened, DM was determined by oven drying at

60oC (Memmert 854) for 48 h. After drying, the

samples were ground through a 1 mm screen for gas

test (Cyclotec 1883; Sample Mill, Memmert GmbH

Co, Germany).

Chemical analysis

Ash, organic matter (OM) and crude protein (CP) of

samples were determined by procedures of AOAC

(1999). The NDF and ADF concentrations were

determined using the methods of Van Soest et al.

(1991). The ammonia nitrogen (NH3-N) content of

silages was determined according to Anonymous

(1986). The pH value of the ensiled BDG was

determined by a glass electrode (Metrohm, Model

691, Metrohm AG Ltd. Switzerland) immediately after

opening according to the British standard method

(Anonymous, 1986).

In vitro gas production and in vitro dry matter

disappearance

Rumen fluid for the in vitro gas production of the

ensiled Sour grape pomace (SGP) was withdrawn

before the morning feed from 4 Baluchi male sheep

which have the following characterizations and

feeding strategies: animals were fed with 0.8 Kg DM

alfalfa hay and 0.4 Kg DM concentrate (165 g CP/Kg

DM/head/day (at maintenance). Animals were fitted

with permanent ruminal cannula about 150 days

before use as donor of ruminal inoculums. All animal

groups were fed twice daily at 07:00 and 16:00 h. All

animals had free access to clean water. Ruminal

fermentation activity was assessed in vitro using the

reading pressure technique ((PTB330, Env Company)

of Theodorou et al. (1994) with the modification of

Mauricio et al. (1999). Rumen fluid was collected

from multiple sites within the rumen of each animal

separately in pre-warmed thermos flasks and

transported immediately to the laboratory. Rumen

contents of each animal species was strained through

four layers of cheesecloth, and kept at 39 ◦C under a

CO2 atmosphere. Approximately 500 mg of ensiled

SGP sample was weighed into 120 ml serum bottles.

Using an automatic dispenser (Jencons, Hemel

Hemstead, England), 50 ml of buffer containing

micro- and macro-elements, a reducing agent and a

reduction indicator of resazurin, was transferred to

each serum bottle. Serum bottles without samples

(i.e., blanks) were also included to allow correction of

96 h degradability values for residual feed from

rumen fluid and three bottles of blank (containing

only rumen fluid inoculum were incubated as blanks

and used to compensate for gas production in the

absence of substrate) and three samples of alfalfa as

slandered were incubated each run. Once filled up, all

the bottles were closed with rubber stoppers, crimped

with aluminum seals, shaken and placed in the

incubator at 39 ◦C. Volume of gas produced was

recorded at several incubation times (2, 4, 6, 8, 10, 12,

24, 48, 72 and 96 h after inoculation time), using the

barometer. Ensiled SGP were incubated in

quadruplicate. Volume of gas (ml/500 mg DM)

produced after 24 h of incubation (GP24) was used as

an index of energy feed value of ensiled SGP. The

method for measurement of the in vitro dry matter

disappearance (IVDMD) was conducted similar to gas

test procedure, but only after 24 h of the incubation,

the bottles were respectively transferred to an ice bath

to stop fermentation and then opened to measure

medium pH using a pH meter (Metrhom pH meter,

Model 691). Then, each bottle content was filtered (42

μm pore size) and a 5 ml sample of each filtrate bottle

was taken and acidified with 5 ml of 0.2 N HCl and

frozen at -20°C for NH3-N analysis. The filtrated

residual was oven dried (60°C for 48 h) and used to

calculate IVDMD.

Calculation and statistical analysis

Cumulative gas production data were fitted to the

exponential equation y = b (1 – e-ct) (Osuji et al.,

1993), where b is the asymptotic gas production

(ml/500mg DM); c is the gas production rate

constant (ml/h); t is the incubation time (h) and y is

the gas production at time of t (ml). Metabolizable

energy (ME, MJ/kg DM) was estimated according to

Menke and Steingass (1988) as:

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ME (MJ/kg DM) = 2.20 + 0.136 GP24 (ml/200 mg

DM) + 0.057 CP where GP24 was 24 h gas volume

and CP (% DM) was that of the ensiled SGP. Short

chain fatty acids (SCFA) were estimated depending on

the relationship (R2 = 0.94) between gas production

at 24 h and SCFA concentration of tannin-containing

browses following the equation of Getachew et al.

(2002): SCFA (mmol) = −0.00425 + 0.0222 GP24

(ml/200 mg DM). Microbial protein was calculated as

19.3 g microbial nitrogen per kg IVDOM, according to

the method Czerkawski (1986). The in vitro digestible

organic matter (IVDOM) were calculated using

equations of Menke et al. (1979) as IVDOM (g kg-1

DM) = (14.88 + 0.889 × Gp + 0.45 × CP + 0.0651 ×

XA) × 10, where, CP is the crude protein (% of DM);

XA is the ash (% of DM) and Gp is the net gas

production (ml) from 200 mg (DM of sample), after

24 h of incubation. Data obtained from chemical

composition and in vitro study was subjected to

ANOVA as a completely randomized design with 4

replicates by the GLM procedure (SAS, 2002), and

treatment means were compared by the Duncan test.

Results and discussion

Chemical composition of ensiled sour grape pomace

Chemical composition of sour grape pomace (SGP) is

presented in Table 1. The DM, CP and OM contents

were less (P<0.05) for control group than other

treatments, but ash, NDF and ADF contents were

higher for control group (p<0.05). The DM, CP, ADF,

NDF, ash and OM contents in control group (ensiled

SGP without additives) were 19.22, 9.52, 46.67, 63.33,

5.32 and 94.67%, respectively. As a fraction of initial

dry weight (before ensiling), the pulp (stalk, skin and

pulp together) and seed comprised an average of 32

and 68 (with SE: 1.58) %, respectively, of the pomace

from sour white grapes. The DM content of grape

pomace has not been reported extensively. The DM

was 46.50 % in a study from Turkey (Can et al.,

2004), higher than the values of 19.22 to 21.00% in

our study that may have varied with sampling time,

climatic conditions and possibly, season of harvest. In

ensiling studies of grape pomace, its DM content was

higher than we measured (25%; Alipour and

Rouzbehan 2007) and, in contrast with our results.

Baumgartel et al. (2007) reported that, compared

with white grape pomace, red grape pomace had a

higher DM content (30.50 % versus 27.30 %).

Cultivars from California had a higher DM content to

ours (i.e., 33.00% DM), but pomace from red and

white grape cultivars from Italy had very more DM

(i.e., 47.00 % DM; Spanghero et al., 2009). Sour

Grape maturity, proportions of the sour grape found

in various pomace fractions (such as stalk, skin, seed

and pulp) and separation techniques alter the DM

content of pomace. In comparison to ripening grape,

data about SGP is scarce. So we have to compare the

SGP with previous research about grape pomace. Ash

content of SGP in our study, 4.75 to 5.32 %, was

approximately similar to values reported by Can et al.

(2004) and Ozduven et al. (2005) of 5.30 and 5.70 %

for grape pomace, but lower than values of 18.60%

from Saricicek and Kilic (2002a) and 10.70 % from

Alipour and Rouzbehan (2007). These differences

may be result of harvest methods, extracting methods

and grape varieties. Motta Ferreira et al. (1996)

reported that NDF and ADF contents of grape

pomace were 63.00 and 57.00 % respectively,

considerably higher than the mean values of 63.33

and 46.67 % in our study (for control group).

Baumgartel et al. (2007) reported that, red grape

pomace had higher NDF and ADF concentrations

than white grape pomace (50.70 versus 30.60; and

36.50 versus 25.70 % NDF and ADF respectively), un-

similar with our results. The difference between data

from Motta Ferreira et al. (1996) and our results, may

be due to differences in grape species (white or red),

extracting methods and the environment or harvest

maturity that alters the nutrient contents of grapes.

The NDF and ADF contents of treatments decreased

with increasing additives in silages. In a research, it

was found contents superior of fiber in neutral

detergent (55.9%) in silages with addition of urea

(0.5%) or urea (0.5%) plus CaCO3 (0.5%) (55.7%) in

relation to control silages (52.1%), in which the

authors relate the negative effect of urea in the

development and action of bacteria degraders of the

fiber portion of the forage (fibrolytic bacteria; Vieira

et al., 2004), that is un-continent with our results.

Basalan et al. (2011) reported that CP content of white

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grape pomace and red grape pomace were 8.31 and

10.84% respectively, relatively similar to our study

(9.52 to 11.47%). The composition of grape pomace

major constituents in peels and seeds has been

reported by several authors, with high polyphenolic as

well as dietary fiber contents (Valiente et al., 1995;

Bravo and Saura-Calixto, 1998). The CP content

differed among treatments (P<0.05; Table 1). Control

had the lowest CP (9.52%) followed by 0.5% acid

sulfuric (10.21%) then 1% sulfuric acid (10.30%).

Preserving the protein in alfalfa or corn silages as true

protein or peptides is the result of reducing protease

activity by rapidly decreasing pH (Guo et al., 2008).

Table 1. Chemical constituents of ensiled sour grape pomace (SGP).

Treatments Chemical constituents of SGP (%)

DM CP Ash NDF ADF

Control 19.22c 9.52c 5.32a 63.33a 46.67a

0.5%Urea 20.10abc 11.24a 4.79b 47.33c 37.33c

1%Urea 20.29abc 11.47a 4.76b 46.67c 32.67d

0.5%Molasses 19.60bc 11.30a 4.80b 54.00b 41.33b

1%Molasses 21.00a 10.64b 4.75b 55.33b 44.00ab

0.5%Sulfuric acid 20.50abc 10.21b 4.84b 60.67a 45.33a

1%sulfuric acid 20.56ab 10.30b 4.89b 56.67b 44.00ab

SEM 0.40 0.16 0.04 1.23 1.04

a, b, c means in the same column with different superscript differ significantly (P<0.05); DM = Dry matter; CP =

crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber; Control=ensiled sour grape pomace

without any additive; SEM= Standard error of mean.

NH3-N and pH of silages

The pH and NH3-N of ensiled sour grape pomace is

presented in Table 2. The concentration of NH3-N in

the control, 0.5 and 1% urea treatments (4.05, 4.17

and 4.25 % respectively) were greater than in the

other treatments. The NH3-N in silage reflects the

degree of protein degradation (Wilkinson, 2005), and

well-preserved silages contain less than 100 g NH3-

N/kg total nitrogen (McDonald et al., 2002). The pH

was difference between treatments and increased

slightly after ensiling (from 4.07 to 4.11; p>0.05). The

application of urea (0.5%) according to Pereira et al.

(2007), contributes with the elevation of the average

contents of pH, increase in the content of NH3-

N/total nitrogen and elevation on the contents of

crude protein in the silages. Vieira et al. (2004)

reported that the addition of urea (0.5%) in the silage

increases the content of crude protein in the average

of 40% in relation to the control silage. According to

Van Soest (1982), for silages with low DM, the pH

should be less than 4.4, but when the moisture

content is below 65.00%, pH is less important. The

low pH of fresh grapes is due their high content of

organic acids (mainly malic and tartaric acids,

Ribereau-Gayon et al., 1998), which are metabolized

during ensiling and this probably explains the slight

pH increase and low level pH of ensiled SGP. The

degradability of protein in tannin-containing feeds is

depressed, resulting in a low NH3-N concentration

(Al-Masri 2010). Butyric acid and NH3 suggest a

Clostridium-type fermentation, which converts lactic

to butyric acid (Van Soest 1982), probably due to

inadequate compression of the forage during ensiling.

The SGP ensiled with 1% molasses had the lowest

NH3-N (3.63 mg/dl) followed by 0.5% molasses (3.73

mg/dl) then 0.5% sulfuric acid (3.87 mg/dl). The

advantages of the application of urea (low unit cost

per protein, containing between 42 and 45% of

Nitrogen), as silage additive, according to Matos

(2008) and Freitas et al. (2002), is in the facility of

obtaining, management in the use of this additive and

the production of ammonia (NH3) in the presence of

urease (enzyme which catalyses the hydrolysis of the

urea into carbon dioxide and ammonia), due to the

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partial transformation of urea into ammonia in the

fermentation of the silage. Ammonia has

antimicrobial action, inhibiting the development of

yeasts and molds, which consequently reduces the

production of ethanol (or ethyl alcohol), generating

lower losses on DM and soluble carbohydrates

(Schmidt 2006), besides enhancing stabilization of

the ensiled matter and stimulating the lactic

fermentation. Schmidt et al. (2007) reported higher

ruminal concentration of the propionic (21.70%) and

butyric (12.10%) acid in silages treated with urea

(0.5%), being related to the higher content of crude

protein and NH3-N of these silages to the ruminal

microbial growth. Silage additives have been

documented to reduce DM losses and improve

fermentation (Henderson, 1993). The literature

review published by Muck and Kung (1997)

established that silage microbial inoculants can alter

different aspects of silage fermentation such as pH,

lactic and acetic acid concentrations, NH3-N, DM

losses, and DM and fiber digestibility, but the level of

effect is variable across studies.

Table 2. The pH and NH3-N of ensiled sour grape pomace.

Treatments Parameters

NH3-N(mg/dl) pH

Control 4.05abc 4.07

0.5%Urea 4.17ab 4.07

1%Urea 4.25a 4.10

0.5%Molasses 3.74de 4.05

1%Molasses 3.63e 4.09

0.5%Sulfuric acid 3.87cde 4.11

1%sulfuric acid 3.97bcd 4.10

SEM 0.08 0.02

a, b, c, d, e means in the same column with different superscript differ significantly (P<0.05); Control=ensiled

sour grape pomace without any additive; SEM= Standard error mean.

In vitro dry matter disappearance, media pH and

estimated parameters

Effect of different chemical additive on dry matter

disappearance (IVDMD), media pH and some

estimated parameters of ensiled sour grape pomace

are presented in Table 3. The IVDMD, metabolizable

energy (ME), in vitro digestible organic matter

(IVDOM), short chain fatty acids and microbial

protein (MP) were lowest (P<0.05) in control group

than those of other treatments (p<0.05), but these

parameters was highest for 0.5 and 1 % urea

treatments (p<0.05). There is little data available on

the nutritive value of grape by-product. The control

had the lowest media pH followed by 1% molasses

then 0.5% sulfuric acid. Although grape pomace is

low in ME, it has been applied in diets of ruminants

fed close to maintenance ME levels, especially in

sheep (Abel and Icking 1984). Textbook ME values for

grape pomace range from 4.2 to 5.4 MJ/kg DM

(INRA 2007 and DLG 1997, respectively) and are

relatively in contaminant with our study (5.03 to 6.30

MJ/kg DM). Ensiled SGP with 1% urea had the

highest pH value than other treatments. The lower

IVDMD of control group versus other treatments was

probably due to higher extent of NDF and ADF

contents (63.33 and 46.67% respectively). Higher

ADF or NDF proportion, and higher tannin contents,

which can reduce attachment of ruminal microbes to

feed particles (McAllister et al., 1994), as well as

inhibit microbial growth and enzyme activity or

intestinal bacterial activity (Salem et al., 2004) by

free-condensed tannins, so it can have potentially

negatively effects on IVDMD. Al-Masri (2003)

observed negative correlations (P<0.001) between

crude fiber and IVDOM (R =−0.88) of some shrubs.

Also Lu and Foo (1999) reported that grape pomace

tannins have adverse effects on nutrient utilization,

and are toxic at high intake levels (Reed 1995) due to

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Int. J. Biosci. 2014

their ability to bind proteins, minerals and

carbohydrates. Tannins are the most widely occurring

anti-nutritional factor in non-conventional feeds. So

it might to there was a relationship between additives

and decreasing of tannins in our treatments than

those of control group. Although Saricicek and Kilic

(2002b) reported value of 20.00% for DM

disappearance of grape pomace in a 72 h in situ

fermentation study, but IVDMD for ensiled SGP was

52.66 to 60.33% at 24 h of incubation in our study,

which was higher than those of reported for grape

pomace silage by Ozduven et al. (2005) and Basalan

et al. (2011). Based on a conventional digestion study,

Baumgartel et al. (2007) reported that DM and NDF

digestibilities were higher for white grape pomace

(58.00 and 21.00 % respectively) than for red grape

pomace (33.00 and 15.00 % respectively). Bocque et

al. (1984) and Larwance et al. (1985) reported a lower

digestion for grape pomace and it was confirmed also

by a more recent digestibility study with sheep

(Baumgartel et al., 2007) where, on the basis of

digestibility, the ME content of a red grape was about

5.80 MJ/kg DM. Ensiling of grape pomace

considerably reduced concentrations of the secondary

compound that have been considered effective in

positively influencing digestion in ruminants

(Mangan, 1988; Salem et al., 2006). Hagerman et al.

(1992) reported that tannins reduced CP digestibility.

Basalan et al. (2011) reported that as a fraction of

initial dry weight, the stalk, skin plus pulp, and seed

comprised an average of 33.2, 34.90, and 31.90 %,

respectively, of the pomace from white grapes and

20.70, 41.00 and 38.30 %, respectively, of the pomace

from red grapes. A major limitation in use of grape

pomace as a feedstuff is the presence of grape seeds

which are high in lignified fiber (D’Urso and Nicolosi

Asmundo, 1983) and are often largely undigested in

ruminants because few of the seeds are broken open

during eating or rumination thereby preventing the

grape seed oil from being digested. If the seeds could

be separated from the pomace, after it may have

potential as a feedstuff while allowing the former to

be used for other purposes (such as oil extracting).

Besharati and Taghizadeh reported that dried grape

by-products had a moderate IVDMD (52.60% for

readily fraction and 26.20% for slowly degraded

fraction).

Table 3. Effect of different chemical additive on dry matter disappearance (IVDMD), media pH and some

estimated parameters of ensiled sour grape pomace.

Treatments Parameters

pH IVDMD ME IVDOM SCFA MP

Control 6.67d 52.67c 5.03d 30.99c 0.28b 5.98c

0.5%Urea 6.78b 59.67a 6.13ab 36.64ab 0.40a 7.07ab

1%Urea 6.83a 60.33a 6.35a 37.64a 0.43a 7.26a

0.5%Molasses 6.76b 58.67a 5.98b 35.53b 0.38a 6.87b

1%Molasses 6.69cd 59.00a 5.94b 35.47b 0.38a 6.84b

0.5%Sulfuric acid 6.71cd 57.83ab 5.19cd 31.26c 0.28b 6.03c

1%sulfuric acid 6.72c 55.33cb 5.39c 32.22c 0.30b 6.22c

SEM 0.01 1.00 0.11 0.65 0.02 0.13

a, b, c, d means in the same column with different superscript differ significantly (P<0.05). IVDMD: In vitro dry

matter disappearance (%); ME: Metabolizable Energy (MJ/kg DM); IVDOM: In vitro Digestibility of Organic

Matter (%); SCFA: Short Chain Fatty Acids (mmol); MP: Microbial Protein (g/kg IVDOM); Control=ensiled sour

grape pomace without any additive; SEM= Standard error of mean.

In vitro gas production parameters

Effect of different chemical additive on in vitro gas

production parameters of ensiled sour grape pomace

is presented in Table 4. Gas production parameters

(cumulative gas production after 24 and 48h

incubation), potential gas production (asymptotic gas

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Int. J. Biosci. 2014

production; fraction b), and rate of gas production

(fraction c; exception of 1% molasses) were greater

(P<0.05) in 0.5 and 1% urea treatments than those of

control group (Table 4). The least values observed for

control group. Generally, Pre-treatment of SGP with

different additives prior to ensiling increased gas

production up to 24 and 48h of incubation times. The

widely variation among treatments in their

fermentation of gas production, mostly may be due to

their variable nutrient and effects of additives. In

vitro fermentation is largely affected by sour grape

species, proportion of fraction in the pomace,

environmental factors, and maturity stage of grape

(Spanghero et al., 2009). Having high gas production

parameters during ensiling with different additives,

suggest high extent and rate of feed fermentation with

a high energy utilization (SCFA, MP, IVDOM and

ME). It seems that, variable gas production within

treatments could due to nature and proportion of

nutrient as well as fiber content. Therefore, higher

nutritive value of 1% urea treatment during the

ensiling than in control group could mostly explained

by lower fiber fractions and/or ADF/NDF proportion

(46.67 and 32.67%, respectively). Ensiling caused a

reduction in gas production (12.00–14.00%) in seeds

and pulps of grape (Alipour and Rouzbehan, 2007).

grape seeds are high in secondary compounds, such

as phenolics and anthocyanins (Makris et al., 2007),

which can have potentially negative effects on rumen

fermentation. There were differences between our

study and reported gas production data by other

researcher (Spanghero et al., 2009; Alipour and

Rouzbehan, 2007). The method used for in vitro

rumen gas production among laboratories is similar

in principle, but there are unavoidable differences

introduced in the course of adopting the technique by

any laboratory. These differences often relate to the

species of the rumen fluid donor animal, and

composition of its diet, which can influence the extent

of microbial growth in the rumen fluid used for

inoculating the in vitro system. The soluble fraction is

responsible for the gas volume produced within the

first 15 h of fermentation, when the microbial mass

production is virtually independent of the cell wall

(Campos et al., 2004).

Table 4. Effect of different chemical additive on in vitro gas production parameters of ensiled sour grape

pomace.

Treatments Parameters

Bgas(ml) Cgas(h-1) 24h(ml) 48h(ml)

Control 24.28d 0.026dc 12.91b 16.83c

0.5%Urea 30.67ab 0.034ab 18.44a 24.23ab

1%Urea 32.57a 0.034ab 19.45a 25.70a

0.5%Molasses 30.67ab 0.029bcd 17.16a 23.05b

1%Molasses 28.94bc 0.036a 17.42a 23.03b

0.5%Sulfuric acid 26.74dc 0.023d 12.90b 17.75c

1%sulfuric acid 24.60d 0.032abc 13.94b 19.12c

SEM 0.97 0.002 0.73 0.80

a, b, c, d means in the same column with different superscript differ significantly (P<0.05); Bgas= the asymptotic gas

production (ml/500mg of DM); Cgas= rate of gas production (ml/h/500mg of DM); 24h=Cumulative gas

production after 24h incubation (ml); 48= Cumulative gas production after 48h incubation (ml); Control=ensiled

sour grape pomace without any additive; SEM: Standard error of mean.

Conclusion

The in vitro rumen gas technique can be used to

study the nutritional quality of ensiling by-products.

Chemical composition of sour grape pomace (SGP)

differed between treatments with various additives

during ensiling. The SGP treated with different

206 Yaghoubi et al.

Int. J. Biosci. 2014

chemical additives, increased gas production,

IVDMD, In vitro estimated parameters (ME, MP,

SCFA and IVDOM) than those of control group. In

general, SGP ensiled with 0.5 and 1% urea, appeared

nutritionally superior to other ensiled one. Ensiled

SGP has promise as a source of digestible nutrients

for ruminants, and non-ruminants with extensive

cecal fermentation. Because the supply of SGP varies

seasonally, handling and storage conditions for

preservation needs further research.

Acknowledgement

The author wishes to thank the Ferdowsi University

of Mashhad and Excellence Centre for Animal Science

for financial support of this research. Thanks to Dr.

M. M. Moheghi for his valuable assistance in

laboratory analysis.

References

Abel H, Icking H. 1984. Feeding value of dried

grape pomace for ruminants. Landwirtschaftliche

Forschung 37, 44–52.

Alipour D, Rouzbehan Y. 2007. Effects of ensiling

grape pomace and addition of polyethylene glycol on

in vitro gas production and microbial biomass yield.

Animal Feed Science and Technolology 137, 138–149.

http://dx.doi.org/10.1016/j.anifeedsci.2006.09.020

Al-Masri MR. 2003. An in vitro evaluation of some

unconventional ruminant feeds in terms of the

organic matter digestibility, energy and microbial

biomass. Tropical Animal Health and Production 35,

155–167.

http://dx.doi.org/10.1023/A:1022877603010

Al-Masri MR. 2010. In vitro rumen fermentation

kinetics and nutritional evaluation of Kochia indica as

affected by harvest time and cutting regimen. Animal

Feed Science and Technology 157, 55–63.

http://dx.doi.org/10.1016/j.anifeedsci.2010.01013

Basalan M, Gungor T, Owens FN, Yalcinkaya I.

2011. Nutrient content and in vitro digestibility of

Turkish grape pomaces. Animal Feed Science and

Technolology 169, 194–198.

http://dx.doi.org/10.1016/j.anifeedsci.2011.07005

Baumgartel T, Kluth H, Epperlein K,

Rodehutscord M. 2007. A note on digestibility and

energy value for sheep of different grape pomace.

Small Ruminant Research 67, 302–306.

http://dx.doi.org/10.1016/j.smallrumres.2005.11.002

Bocque CV, Fiems LO, Cottyn BG, Buysse FX.

1984. Feeding value of some wastes and by-products

of plant origin and their use by beef cattle. In:

Ketelaars, E.H., Boer Iwema, S. (Eds.), Animals as

waste converters. Proceedings of an international

SymposiumWageningen. Netherlands 30 Nov–2 Dec,

1983. PudocWageningen, 54–61 p.

Bohra B, Singh V, Sharma RJ, Jaiswal RS,

Kumar A. 2008. Nutritive value of different feed

and fodder samples available in the study areas of

Uttarakhand Mountains. Indian Journal of Animal

Sciences 78(7), 783–86.

Bravo L, Saura-Calixto F. 1998. Characterization

of dietary fiber and the in vitro indigestible fraction of

grape pomace. American Journal of Enology and

Viticulture 49, 135–141.

Bustamante MA, Moral R, Paredes C, Perez-

Espinosa A, Moreno-Caselles J, Perez-Murcia

MD. 2008. Agrochemical characterization of the

solid by-products and residues from the winery and

distillery industry. Waste Management 28, 372–380.

http://dx.doi.org/10.1016/j.wasman.2007.01.013

Can A, Denek N, Tufenk S. 2004. Effect of

different additives on silage quality and in vitro dry

matter digestibility of wet grape pomace. Harran

University. Ziraat Fakultesi Dergisi 8, 11–15 (in

Turkish).

Campos FP, Sampaio AAM, Bose MLV, Vieira

PF, Sarmento P. 2004. Evaluation of in vitro gas

production of roughages and their mixtures using the

207 Yaghoubi et al.

Int. J. Biosci. 2014

curves subtraction method. Animal Feed Science and

Technology, 116, 161-172.

http://dx.doi.org/10.1016/j.anifeedsci.2004.06.001

DLG, Deutsche Landwirtschafts-Gesellschaft.

1997. Feedstuffs Tables for Ruminants, seventh ed.

DLG-Verlags GmbH, Frankfurt, Germany.

D’Urso G, Nicolosi Asmundo C. 1983. La

composizione dei sottoprodotti dell’industria

enologica utilizzabili nell’alimentazione animale.

Vignevini, 3, 41–44.

Ebrahim Pour M, Haghparvar RU, Rowghani

E, Shojaian K, Ghaderi Zefrei M. 2014. The

effect of a bacterial inoculant, urea and molasses on

chemical composition, in vitro gas production and

energy content of ensiled pomegranate

(Punicagranatum L.) seeds and peel pulp. Research

opinions in animal & Veterinary Sciences 4(1), 85-

90.

Eslamian E, Naserian AA, Tahmasbi AM,

Valizadeh R, Kazemi M. 2013. Nutritional

evaluation of ensiled brewers’ grain by the in vitro

and in situ techniques. International Journal of

Biotechnology and Food Science 1(5), 84-89.

Freitas AWP, Pereira JC, Rocha FC, Costa MG,

Leonel FP, Ribeiro MD. 2006. Avaliacao da

qualidade nutricional da silagem de cana-de-açucar

com aditivos microbianos e enriquecida com resíduo

da colheita de soja. Revista Brasileira de Zootecnia

35(1), 38-47.

http://dx.doi.org/10.1590/S15163598200600010000

5

Garg MR, Kannan A, Shelke SK, Phondba BT,

Sherasia PL. 2012. Nutritional evaluation of some

ruminant feedstuffs by in vitro gas production

technique. Indian Journal of Animal Sciences 82(8),

898–902.

Getachew G, Makkar HPS, Becker K. 2002.

Tropical browses: contents of phenolic compounds, in

vitro gas production and stoichiometric relationship

between short chain fatty acid and in vitro gas

production. Journal of Agricultural Science 139, 341–

352.

http://dx.doi.org/10.1017/S0021859602002393

Hagerman AE, Robbins CT, Weerasuriya Y,

Wilson TC, McArthur C. 1992. Tannin chemistry

in relation to digestion. Journal of Range

Management 45, 57–62.

Henderson N. 1993. Silage additives. Animal Feed

Science and Technology 45, 35–56.

http://dx.doi.org/10.1016/0377-8401(93)90070-Z

INRA, Istitut National de la Recherque

Agronomique. 2007. Alimentation des bovines,

ovins et caprins. Besoins des animaux–Valeurs des

aliments, Editions Quæ, Paris.

Jaakkola S, Kaunisto V, Huhtanen P. 2006.

Volatile fatty acid proportions and microbial protein

synthesis in the rumen of cattle receiving grass silage

ensiled with different rates of formic acid. Grass and

Forage Science 61, 282-292.

http://dx.doi.org/10.1111/j.1365-2494.2006.00532.x

Kazemi M, Tahmasbi AM, Valizadeh R,

Naserian AA, Haghayegh GH, Esmaeil Jami Y.

2013. Studies on the effects of different chemical

additives on the nutritive value of ensiled barley

distillers' grain (BDG) using in vitro techniques.

Direct Research Journal of Agriculture and Food

Science 2(2), 19-24.

Larwance A, Hammouda F, Gaouas Y. 1985.

Valeur alimentaire des marcs de raisin. V.

Comportement alimentaire et vitesse de transit chez

le mouton. Annales De Zootechnie 34, 389–400.

http://dx.doi.org/10.1051/animres:19860201

Mangan JL. 1988. Nutritional effects of tannins in

animal feeds. Nutrition Research Reviews 1, 209–231.

http://dx.doi.org/10.1079/NRR19880015

208 Yaghoubi et al.

Int. J. Biosci. 2014

Matos BC. 2008. Aditivos químicos e microbianos

em silagens de cana de açúcar: acao sobre o padrão

fermentativo e degradabilidade ruminal da massa

ensilada e possíveis incrementos no desempenho

animal. Pubvet, Vol. 7, No. 24, Ed. 247, Art. 1631.

Mauricio RM, Mould FL, Dhanoa MS, Owen E,

Channa KS, Theodorou MK. 1999. A semi-

automated in vitro gas production technique for

ruminant feedstuff evaluation. Animal Feed Science

and Technology 79, 321–330.

http://dx.doi.org/10.1016/S0377-8401(99)00033-4

McAllister TA, Bae HD, Jones GA, Cheng K J.

1994. Microbial attachment and feed digestion in the

rumen. Journal of Animal Science 72, 3004–3018.

McDonald P, Edwards RA, Greenhalgh JFD,

Morgan CA. 2002. Animal Nutrition. 6th ed.

Longman scientific and Technical, Prentice Hall, New

Jersey, USA.

McSweeney CS, Palmer B, McNeill DM,

Krause DO. 2001. Microbial interactions with

tannins: nutritional consequences for ruminants.

Animal Feed Science and Technology 91, 83–93.

http://dx.doi.org/10.1016/S0377-8401(01)00232-2

Menke KH, Steingass H. 1988. Estimation of the

energetic feed value obtained from chemical analyses

and in vitro gas production using rumen fluid.

Animal Research and Development 28, 7–55.

MJAS, The Ministry of Jihad-e-Agriculture.

2012. Performance of garden products. Statistics,

Cultivation DataBase, Statistical Pocketbook, Iran.

Motta Ferreira W, Fraga MJ, Carabano R.

1996. Inclusion of grape pomace, in substitution for

alfalfa hay, in diets for growing rabbits. Animal

Science 63, 167–174.

http://dx.doi.org/10.1017/S135772980002840X

Muck RE, Kung JL. 1997. Effects of silage additives

on ensiling. In: Silage: Field to Feedbunk, NRAES-99,

Northeast Reg. Agric. Eng. Serv., Ithaca, NY, USA,

187–199 p.

Osuji PO, Nsahlai IB, Khalili H. 1993. Feed

evaluation. Ilca manual (International Livestock

Center for Africa), Addis Ababa, Ethiopia, 40-41 p.

Ozduven ML, Coskuntuna L, Koc F. 2005.

Determination of fermentation and feed value

characteristics of grape pomace silage. Trakya

University Journal of engineering sciences 6, 45–50

(in Turkish).

Pereira CA, Silva RR, Gonalves LC, Borges

ALCC, Borges I, Gomes SP, Rodrigues JAS,

Saliba EOS, Fereira JJC, Silva JJ. 2007.

Avaliacao da silagem do hibrido de sorgo [Sorghum

bicolor (L.)moench] br 601 com aditivos 1-pH,

nitrogenio amoniacal, material seca, proteina bruta e

carboidratos solvies. Revista Brasileira de Milho e

Sorgo 6(2), 211-222.

Reed JD. 1995. Nutritional toxicology of tannins and

related polyphenols in forage legumes. Journal of

Animal Science 73, 1516–1528.

Ribereau-Gayon P, Glories Y, Maujean A,

Dubourdieu D. 1998. Traite d’enologie. 2. Chimie

du vin. Stabilisation et traitements. Dunod

Publication, Paris, 3–19 p.

Ruberto G, Renda A, Amico V, Tringali C.

2008. Volatile components of grape pomaces from

different cultivars of Sicilian Vitis vinifera L.

Bioresource Technology 99, 260–268.

http://dx.doi.org/10.1016/j.biortech.2006.12.025

Salem AZM, Gohar YM, El-Adawy MM, Salem

MZM. 2004. Growth-inhibitory effect of some

antinutritional factors extracted from Acacia saligna

leaves on intestinal bacteria activity in sheep. In:

Proceedings of the 12th Scientific Conference of the

Egyptian Society of Animal Production (ESAP),

Mansoura, Egypt, 283–300 p.

209 Yaghoubi et al.

Int. J. Biosci. 2014

Salem AZM, Salem MZM, El-Adawy MM,

Robinson PH. 2006. Nutritive evaluations of some

browse tree foliages during the dry season: secondary

compounds, feed intake and in vivo digestibility in

sheep and goats. Animal Feed Science and

Technology 127, 251–267.

http://dx.doi.org/10.1016/j.anifeedsci.2005.09.005

Saricicek BZ, Kilic U. 2002a. A study on

determining in situ degradability of grape pomace.

Ataturk Universitesi Ziraat Fakultesi Dergisi, 33,

289–292 (in Turkish).

Saricicek BZ, Kilic U. 2002b. An investigation on

determining of feed values of grape pomace. OMU

Ziraat Fakultesi Dergisi 17, 9–12 (in Turkish).

SAS. 2002. SAS User’s Guide: Statistics. Ver 9.0. SAS

Institute, Cary, NC, USA, p. 956.

Schmidt P. 2006. Perdas fermentativas na

ensilagem, parametros digestivos e desempenho de

bovinos de corte alimentados com racoes contendo

silagens de cana-de-acucar. 228f. Tese (Doutorado

em Ciência Animal e Pastagens) - Escola Superior de

Agricultura Luiz de Queiroz, USP, Piracicaba.

Spanghero M, Salem AZM, Robinson PH.

2009. Chemical composition, including secondary

metabolites and rumen fermentability of seeds and

pulp of Californian (USA) and Italian grape pomaces.

Animal Feed Science and Technolology 152, 243–

255.

http://dx.doi.org/10.1016/j.anifeedsci.2009.04.015

Theodorou MK, Williams BA, Dhanoa MS,

McAllan AB, France J. 1994. A simple gas

production method using a pressure transducer to

determine the fermentation kinetics of ruminant

feeds. Animal Feed Science and Technology 48, 185–

197.

http://dx.doi.org/10.1016/0377-8401(94)90171-6

Valiente C, Arrigoni E, Esteban RM, Amado R.

1995. Grape pomace as a potential food fiber. Journal

of Food Science 60, 818–820.

http://dx.doi.org/10.1111/j.13652621.1995.tb06237.X

Van Soest PJ. 1982. Nutritional Ecology of the

Ruminant. Cornell University Press, Ithaca, NY. USA.

Wilkinson JM. 2005. Silage. Chapter 19: Analysis

and clinical assessment of silage. Chalcombe

Publications, UK.

198-208 p.

Lu Y, Foo LY. 1999. The polyphenol constituents of

grape pomace. Food Chemistry 65, 1–8.

http://dx.doi.org/10.1016/S0308-8146(98)00245-3