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The objective of this study was to systematically evaluate and compare the effect of selected spices on methanogenesis using in vitro cultures. Some of the spices selected are Allium cepa, piper nigrum. Aframonum melegueta, Dennttia tripetala,, syzygium aromaticum, Gongronema latifolium among others were dried and milled for chemical analysis and in vitro gas fermentation study. The results showed that the organic matter was highest in Allium crispum (97.98%) and lowest in Allium cepa (96.77%) ash content ranges from 4.2% to 2.34% for Allium cepa and Allium crispum respectively. The crude protein (CP) also was found to vary from 24.50% in Allium cepa to 11.19% for Gongroneme latifolium. NDF values ranges from 52.00% for Capsicum annuum to 14.43% for Allium ascalonium and ADF values was 40.50% for Capsicun annuum to 4.25% for Allium crispum, also hemicelluloses was noticed to be highest in Allium crispum (39.00%) and lowest in Allium cepa(white) (2.75%). The effect of spices was also observed on the dry matter digestibility (DMD), methane gas production and reduction and short chain fatty acid production. For digestibility shea butter gave the highest value of 96.15% while the lowest was noticed in Vernonia amygdalina (61.54%). Dennttia tripetala which had the least % methane production (25.02%) also had the highest percentage of methane reduction (47.69%) and short chain fatty acid (0.66mmol). This reduction in methane gas could be attributed to the bioactive substances such as tannin(0.09%) and saponin (0.20%) present in the spices. These results reveal that spices could be used to enhance digestibility, increases the energy value of feed and reduces methanogenesis which is of great concern worldwide. Spices such as Aframonum melegueta, Allium cepa, Vernonia amygdalina, Allium crispum, Cymbopogon citratus, Dennttia tripetala, Allium cepa(white), Syzygium aromaticum, Gongronema latifolium, Capiscum annuum that have the same degree of methane reduction potential like Dennttia tripetala are recommended for this purpose


Title Page – ——— i

Abstract – – – – – – – – – – – – – ii

Acknowledgement – – – – – – – – iii

Certification – – – – – – — v

Dedication ———-vi

Table of contents ———- vii

List of tables ———-ix

List of Plates———–xi



1.2Thesis Objectives——- –2


2.0Literature Review———3

2.1What is Tannin ———3

2.2Properties of Tannin ——–3

2.3 Tannin And Rumen Fermentation——-4


2.5Saponin And Rumen Fermentation——-6

2.6Spices ———-7

2.7In Vitro Gas Production Techniques ——10

2.7.1History of In Vitro Gas Production Techniques—–10

2.7.2Effect Of Venting Gas During The Incubation —–11

2.7.3Effect of Agitation of the Medium ——-12

2.7.4Effect of Inoculum ———12

2.7.5Use Of Blanks———13

2.7.6Effect of Medium Composition——-13

2.7.7Effect of Apparatus———14


3.0Materials And Methods ——–15

3.1Experimental Materials and Its Preparation Collection of Spices —15

3.2 Preparation for In Vitro study——-21

3.2.1 Preparation of Buffer——–21

3.2.2 Preparation of Inoculums ——–22

3.3Determination of Methane Gas ——-24

3.4 Cell Wall Fraction Determination (NDF & ADF)—–25

3.4.1 Crude Protein Determination——-26

3.4.2 Ash determination ———27

3.4.3 Tannin Content Determination ——-27

3.4.4 Saponin Content Determination ——-28

3.4.5 Moisture Content Determination ——-29

3.5Short chain fatty acids———30

3.6Experimental Design———30



4.1In Vitro Gas Production at Different Hours Of Incubation Treated With

Different Spices———31

4.2 Effect of Spices on Dry Matter Disappearance, Absolute Methane And Short

Chain Fatty Acid Production ——-34

4.3Chemical Composition of some of the spices used in the study Data of the

Chemical composition of the ten spices selected by grading are several in 4.3.36

4.4 Tannin and Saponin Content of Some of the Spices—–39


5.0 Discussion———-40

5.1In-Vitro Gas Production At Different Hours Of Incubation Treated

With Different Spices——–40

5.2Effect of spices on dry matter disappearance methane and short chain

fatty acid production——–40

5. 3Chemical Composition of Some of the Spices Used in the Study —41

5.4Tannin and Saponin Content of Some Of The Spices Used —-42


6.0Conclusion And Recommendation——-43



Table 2.1: List of spices and there active ingredients as well as medicinal uses -9

Table 3.1: While their pictures are shown in plate 3.1. —–15

Table 4.1: Effect of Spices on the Volume of Gas (ml/130mg) Produced at Different

Incubation Hours ——-33

Table 4.2: Effect Of Species On Dry Matter Disappearance, Methane And

Shortchain Fatty Acid Product ——-35

Table 4.3 Chemical Composition of Some of the Spices Used In the Study (%) –37

Table 4.4 Tannin and Saponin Content of Some of the Spices (%)—-38


Plate 2.1: Tannin powder———4

Plate 3. 2: Collection of rumen fluid from a goat with a suction tube into a


Plate 3.3: Filtration of rumen liquor with cheese cloth —-23

Plate 3.4: Measuring of 20ml of buffered solution into syringes —-24

Plate 3.5: Syringes containing buffered rumen fluid being kept in an incubator at 39 o C-24



Livestock is one of the longest sources of methane emission with 80 – 115 million tons produced per year, equivalent to 15 – 20% of total anthropogenic methane (IPCC, 2001).

Ruminants are major contributors to biogenic methane formation. It has been estimated that preventing methane formation from ruminants would stabilized atmospheric methane concentrations and improve animal performance (Johnson and Johnson, 1995). The global cattle population is responsible for 73% of methane emissions of all livestock and methane produced during ruminal fermentation represents a loss of 2-15% of gross energy intake and may also known as a great contributor to global warming which is a primary environmental concern world wide (moss et al., 2000).

Recent studies have shown that plant secondary metabolites such as tannin, essential oils and Saponin at lower concentration could be used to manipulate rumen fermentation favorably.

Plant bioactive (PB) or plant secondary compounds are chemicals synthesized in plants but are not involves in the primary biochemical processes of plant growth but acts as a protective agents against predators.

Plant extracts from spices and medicinal plants with high concentrations of secondary compounds such as tannin and saponin are good candidates for reducing ruminal methanogenesis (Teferedegne, 2000).

Therefore altering dietary formulation can have great impacts on environmental performance from dairy operations.

Many studies have reported that feeding forages containing tannin decreases ruminal protein degradation (Min et al., 2003) and also have the potential to reduce enteric CH4 emissions (Carulla et al., 2005; Animut et al., 2008).

It was also reported that saponins or saponin-like substances had the potential to suppress the methane emission, reduces protozoa counts and change fermentation patterns (Hristov et al., 1999). At appropriate dose tannin and saponin decreases methane production and increases the efficiency of microbial protein synthesis (Min et al., 2003).

Therefore this experiment is planned to ascertain the effect of plants extracts such as tannin and saponin from different species and their effect on methane are other in vitro fermentation parameter.

1.2Thesis Objectives

The study of tannin and saponin content of some species and its effect on in vitro rumen fermentation have been designed with the following objectives.

To determine the tannin, crude protein, cell wall and saponin, contents of some spices in Edo State, Nigeria.

To determine the effects of adding spices as additives on in vitro ruminal methane production and other fermentation parameters.

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