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Effects of Eucalyptus (E. camaldulensis) leaf powder (ELP) on rumen fermentation, feed digestibility and methane production in ruminants by using in vitro gas production technique

 

N S Manh, L V Hung, N T Long, N V Don and N T Huyen*

 

National Institute of Animal Sciences,
* Hanoi University of Agriculture
nguyensucmanh@gmail.com

 

Abstract

 

This study was conducted to determine effects of Eucalyptus leaf powder (ELP) supplementation on characteristics of ruminal fermentation of a 40:60 mixture of rice straw and concentrate, using the in vitro gas production technique.  Rumen fluid was collected from two rumen fistulated beef cattle with 215 ± 15 kg of body weight. The substrates were supplemented with ELP at levels of  0, 1, 2, 3, 4, 5 and 6%. The volume of gas was recorded after incubation periods of 3, 6, 9, 12, 24, 48, 72 and 96 h. In vitro true digestibility (IVTD) was determined after 12, 24 and 48h of incubation.  Methane production was measured after 48 h of incubation using the indirect method of carbon dioxide absorption as well as directly by gas chromotography.

 

The cumulative gas production and the rate of gas production, as well as the in vitro digestibiliity,  were reduced as the level of ELP was increased. The methane content of the gas decreased linearly with level of added ELP to a recorded maximum of 28% with addition of 6% ELP. The indirect measurement of methane by absorption of carbon dioxide gave values about 10% higher than the values recorded by gas chromotography; however, the trends with increasing levels of ELP were almost identical for the two methods.ion.

 

Keyword: Climate change, essential oil, fermentation, greenhouse gases

 

Introduction

 

Methane is one of the major products of anaerobic fermentation of feeds in the rumen. Reducing CH4 production is an important goal of ruminant nutritionists as it represents a significant loss of energy for the host animal and contributes to global warming (Moss et al 2000). Many attempts have been made to depress rumen methanogenesis through the use of feed additives such as ionophores, halogen compounds, unsaturated fatty acids and organic acids.

 

Currently numerous studies have attempted to exploit plant secondary metabolites as natural feed additives to improve the efficiency of rumen fermentation such as enhancing protein metabolism and decreasing methane production (McIntosh et al 2003). Recently, many reviews have been published on plant extracts such as saponins, tannins and essential oils (EOs) as rumen modifiers (Calsamiglia et al 2007). Plant derived EOs may be a useful means to improve efficiency of nutrient utilization in ruminants and reduce the impact of their production on the environment (Benchaar et al 2008). The Eucalyptus is a tall evergreen tree with many species available and these can be found in many parts of the world, which produce a wide variety of oil. Akin et al (2010) reported that the major components of Eucalyptus camaldulensis oil were ethanone (13.73%), eucalyptol (25.36%) and caryophyllene (11.55%). Eucalyptol (1.8- cineole) is the main active ingredient in oils from E. Camaldulensis (Sallam et al 2009a). Recently, in vitro studies have demonstrated that EOs or their components have the potential to favorably alter rumen metabolism (Busquet et al 2006). However, there are few experimental data on effects of the Eucalyptus leaf powder on rumen digestion and rumen fermentation.

 

Therefore, the objective of this study was to evaluate effects of different levels of E camaldulensis) leaf powder (ELP) supplementation on feed digestibility and methane production in ruminants by using in vitro gas technique.

 

Materials and methods

 

Location and climate of the study site

 

The study was carried out at the Animal Feed Station belonging to National Institute of Animal Sciences. The station is located in the Red River Delta at 105o47’E longitude and 21o05’N latitude, with a mean altitude of 2 m above sea level. The climate is tropical monsoon with wet season between April and November and dry season from December to March.

 

Experimental design and fermentation technique

 

This study was conducted using an in vitro gas technique with different incubation times. The treatments in a completely randomized design (CRD) were seven levels of Eucalyptus meal powder: ELP0=0 (control); ELP1= 1%, ELP2=2%, ELP3= 3%, ELP4= 4%, ELP5= 5% and ELP6= 6% added to a basal substrate containing ratio of 40:60 roughage (rice straw) and concentrate (14% crude protein). Eucalyptus powder was prepared by drying fresh Eucalyptus leaves at 60°C. Substrate and dried Eucalyptus leaves were milled to pass through a 1mm screen prior to mixing the ELP with the rest of the substrate at the pre-determined levels. Samples (200 mg of substrate mixture DM) were put into 100ml syringes for the incubations.  The ingredients of the substrate and their chemical composition are shown in Table 1.

 

Two, male 3-year-old, rumen fistulated, Laisind cattle with initial BW of 215 ± 15 kg were used as rumen fluid donors. They were fed the same roughage: concentrate mixture as was used in the incubations. dietary ingrediets as those used in the incubations.  The rumen liquor was obtained from each animal before the morning feeding.  It was filtered through four layers of cheese cloth into pre-warmed thermos flasks. Preparation of artificial saliva was done according to the procedure detailed in Menke and Steingass (1988). The artificial saliva and rumen fluid were mixed in a 2:1 ratio to prepare the inoculum. The syringes with the mixtures of substrates were prewarmed in a water bath at 39°C for 1 hour before adding 30 ml of rumen inoculum mixture to each one. During the incubation, the gas production was recorded at 3, 6, 9, 12, 24, 48, 72 and 96h. Cumulative gas production data were fitted to the model of Ćrskov and McDonald (1979) as follows:

 

y = a + b(1-e(-ct))

 

Where a = the gas production from the immediately soluble fraction, b = the gas production from the insoluble fraction, c = the gas production rate constant for the insoluble fraction (b), t = incubation time, (a+b) = the potential extent of gas production. y = gas produced at time “t”.

 
Fermentation parameters
 
In vitro true digestibility (IVTD)

 

Dry mater digestibility and organic matter digestibility were estimated at 12, 24, 48 hours after fermentation. The residual solutions were filtered by gravity, using Whatman No 4 filter paper, and the residues dried at 105°C for 24 h, for the determination of IVDMD. The dry residues were incinerated at 550°C for measuring in vitro OM disappearance (IVOMD).

 
Methane production

 

Two methods were compared.

 

Gas chromotography

Immediately after the incubation at 48h, 1ml of the gas phase was sampled from the syringe and analysed for methane by gas chromatography (GC17A, Detector FID) according to Van Nevel et al (1970). 

 

Carbon dioxide absorption

 

Immediately after removal of the syringes from the water bath, 4 ml of 10M NaOH were introduced using  a 5 ml capacity syringe. The content was inserted into the silicon tube, which was fastened to the 100 ml capacity syringe. The clip was then opened while the NaOH was gradually released. The content was agitated while the plunger began to shift position to occupy the vacuum created by the absorption of CO2. The volume of methane was read on the calibrated body of the syringe. To determine the actual gas produced, the average of the volume of gas produced from the blanks was deducted from the volume of gas produced per sample (Demeyer 1988; Fievez et al 2005).

 

Statistical analysis

 

Data were analyzed by using the General Linear Model (GLM) procedure of SAS (1998). The model was

 Yij = µ + Ti + εij,

where Yij is the observed treatment i, for replication j; µ the overall mean; Ti the mean of each treatment; and εij the residual effect. Multiple comparisons among treatment means were performed by Duncan’s New Multiple Range Test (DMRT) (Steel and Torrie 1980).

 

Results and discussion

 
Chemical composition of feeds

 

The composition of the concentrate and the analysis of the ingredients used in the substrates are in Table 1. The ELP was low in  crude protein and relatively high in NDF and ADF, the values being similar to those reported by Sobhy et al (2010) and Sallam et al (2010).

Table 1: Ingredient and chemical composition of experimental diets

 

Ratio

Concentrates

Rice Straw

ELP

Ingredients

--------------- % DM--------------

Cassava chip

58

 

 

Rice bran

11

 

 

Corn meal

13

 

 

Palm meal

15

 

 

Urea

3

 

 

DM, %

93.4

95.8

96.7

 

--------------% DM-----------------

OM

94.2

83.5

91.5

Ash

5.8

16.5

8.5

CP

14

3.8

7.5

NDF

26.7

75.6

55.8

ADF

18.5

47.2

42.5

ELP= Eucalyptus leaf powder; DM = dry matter, OM = organic matter, CP = crude protein, NDF = neutral-detergent fiber, ADF = acid-detergent fiber.

 

Kinetic analysis of gas production

 

The cumulative gas production (a+b) and the rate of production (c) were reduced as the level of ELP was increased (Table 1; Figure 1).  According to Sallam et al (2009a),  eucalyptus oils decreased gas production with increasing level of oil.

 

Table 2: The effects of ELP on gas kinetics

Gas kinetic

Fermentation kinetic values

SEM

ELP0

ELP1

ELP2

ELP3

ELP4

ELP5

ELP6

a

-7.47bc

-7.6bc

-9.20d

-5.37ab

-4.88a

-8.41cd

-6.37ab

0.497

b

64.92a

64.75a

63.06ab

59.56bc

57.51cd

58.81bcd

54.58d

1.35

c

0.057a

0.055ab

0.053b

0.052b

0.046cd

0.046c

0.044d

0.001

a+b

57.44a

57.13a

53.85ab

54.19ab

52.62b

50.39bc

48.21c

1.28

Gas 96 h (ml/200mg substrate)

58.15a

57.74a

54.45ab

54.80ab

51.80bc

49.26cd

47.16d

1.28

a-d Values in the same row with different superscripts differ (p<0.05).

  

Figure 1: Effect of ELP on cumulative gas production at different time of incubation

 

In vitro digestibility

 

The in vitro digestibility of DM and OM decreased as the level of ELP was increased (Tables 3 and 4; Figures 2 and 3).  Similar results were reported by Sallam et al (2009a) in in vitro experiments with Eucalyptus oil supplementation. Related results were presented by Soltan (2009) who observed that essential oil mixtures reduced DM and OM digestibility, and roughage intake, in growing heifers. Oh et al (1967) and Nagy and Tengerdy (1968) observed that Eucalyptus oil markedly inhibited activity of ruminal bacteria in vitro.  Russell and Strobel (1989) found that supplementation with Eucalyptus oil led to the inhibition of ruminal cellulolytic bacteria, such as Cellulotytic ruminococci and Butyrivibrio fibrisolvens. Eucalyptus leaves contain tannins, flavonoids and volatile oils. Inhibitory interactions between terpenes, as well as other plant secondary compounds, may inhibit the activity of rumen protozoa and methanogenic bacteria (Sallam et al 2009b).

 

Table 3: Effect of ELP on IVDMD at 12, 24, 48h of incubation

Incubation time (h)

Treatments

SEM

ELP0

ELP1

ELP2

ELP3

ELP4

ELP5

ELP6

12

58.2ab

59.6a

53.4ab

54.7ab

53.6ab

53.0ab

51.0b

1.99

24

64.4a

64.9a

57.9b

58.9b

55.0bc

55.5bc

52.2c

1.47

48

66.1a

65.7a

63.2ab

63.5ab

61.3ab

57.1bc

54.5c

1.81

a-d Values in the same row with different superscripts differ (p<0.05).

 

Figure 2: Effect of ELP on DM digestibility at different time of incubation

 

Table 4: Effect of ELP on IVOMD at 12, 24, 48h of incubation

Incubation time (h)

Treatments

SEM

ELP0

ELP1

ELP2

ELP3

ELP4

ELP5

ELP6

12

55.8a

53.4ab

55.8a

52.4ab

49.6bc

47.0c

46.7c

1.4

24

66.7a

61.8ab

61.8ab

57.7bc

53.6dc

52.5dc

51.0d

1.54

48

68.3a

67.9a

61.9b

62.9b

57.4c

54.2c

53.6c

1.26

a-dValues in the same row with different superscripts differ (p<0.05).

 

 

Figure 3: Effect of ELP on DM digestibility at different time of incubation

 

Methane production

 

The methane content of the gas decreased linearly with level of added Eucalyptus leaf meal (Table 5; Figure 4). One component of Eucalyptus oil - p-cymene - decreased methane by 29% at a concentration of 20 mg/liter according to Chaves et al (2008).  Sallam et al (2009a) found that eucalyptus oils inhibited methane production by 29.6 - 90.3 %  and that protozoal numbers were also decreased.  Similar findings were reported by  Kumar et al (2009).  

 

The indirect measurement of methane by absoirption of carbon dioxide gave values about 10% higher than the values recorded by gas chromotography; however, the trends with increasing levels of ELP were almost identical for the two methods which indicates that the absorption method could be used for comparative purposes, when direct measurement of methane is not possible through lackof appropriate equipment. 

 

Table 5. Effect of ELP on methane production at 48h of incubation

Method

Methane content in gas (%)

SEM

ELP0

ELP1

ELP2

ELP3

ELP4

ELP5

ELP6

Naxide OH

40.75a

39.45a

36.75ab

36.95ab

34.25abc

30.95bc

29.25c

1.86

GC

37.32a

35.62ab

33.83b

33.65b

30.41c

27.76d

26.65d

0.57

a-dValues in the same row with different superscripts differ (P<0.05).

 

Figure 4: Comparison of gas chromotography and carbon dioxide absorption as methods for measurement of methane

 

Conclusions  

Acknowledgements

 

The authors would like to express their sincere thanks to the MEKARN program financed by the Sida-SAREC project for providing the opportunity and the budget to do the research;  and to the National Institute of Animal Sciences for providing research infrastructure support.

 

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