UNIVERSITI PUTRA MALAYSIA

MAHDI EBRAHIMI

FPV 2012 17

PRODUCTION OF OMEGA-3 POLYUNSATURATED FATTY ACID ENRICHED CHEVON USING TREATED OIL PALM

(ELAEIS GUINEENSIS JACQ.) FROND SILAGE

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PRODUCTION OF OMEGA-3 POLYUNSATURATED FATTY ACID- ENRICHED CHEVON USING TREATED OIL PALM

(ELAEIS GUINEENSIS JACQ.) FROND SILAGE

By

MAHDI EBRAHIMI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

November 2012

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DEDICATION

MY FATHER AND MOTHER, I Love You Forever

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

PRODUCTION OF OMEGA-3 POLYUNSATURATED FATTY ACID-

ENRICHED CHEVON USING TREATED OIL PALM (ELAEIS GUINEENSIS JACQ.) FROND SILAGE

By

MAHDI EBRAHIMI

November 2012

Chairman: Professor Mohamed Ali Rajion, PhD

Faculty: Veterinary Medicine There is a need to treat oil palm fronds (OPF) to improve its nutritive value and

digestibility to be used as small ruminant feed. A study was carried out in which fresh

OPF have been ensiled with lactic acid bacteria (LAB), cellulase enzyme and a

combination of LAB and cellulase enzyme to improve the fermentation quality and

nutrient values of the OPF. The dietary treatment methods comprised non-ensiled

fresh OPF (F), ensiled OPF with no additives (C, control), ensiled OPF with a

commercial lactic acid bacteria (LAB) inoculant (I), ensiled OPF with an exogenous

fibrolytic, cellulase enzyme (E) and ensiled OPF with a LAB inoculant-cellulase

combination (I+E). Ensiling was carried out with about 2 Kg samples stored in air-

tight glass jars at 25-30 oC for 12 wk after which the silage samples were subjected to

standard proximate analyses. The ensiling decreased significantly (P < 0.05) the DM

concentration of ensiled OPF with additives compared to the non-ensiled fresh OPF

and control silage. The LAB inoculant-cellulase combination reduced significantly (P

< 0.01) the NDF and ADF content compared to the control silage. Crude fat also

decreased significantly (P < 0.05) after ensiling compared to the non-ensiled fresh

OPF. All the treated silages had a lower pH (4.09-4.28) than the untreated silage

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(4.88). Acetic acid and propionic acid in the ensiled OPF were significantly (P < 0.01)

higher than the non-ensiled fresh OPF. The NH3-N concentration in the silage with

additives significantly decreased (P < 0.01) compared to the non-ensiled fresh OPF

and control silage. The LAB inoculant-cellulase combination increased the water

soluble carbohydrate (WSC) and ethanol production significantly (P < 0.01) compared

to the control silage. The population of LAB increased significantly (P < 0.05) in all

treatments with additives compared to the non-ensiled fresh OPF and control silage.

The in vitro dry matter digestibility, in sacco dry matter digestibility, in vivo dry

matter digestibility, in vitro gas production, in vitro fermentation kinetics and in vivo

fermentation kinetics of the silages were determined using rumen fistulated goats. The

OPF silage treated with cellulase or the LAB inoculant-cellulase combination showed

a significantly (P < 0.01) higher in vitro gas production and dry matter digestibility

(DMD) compared to the non-ensiled fresh and control silage. In fact, the in vitro gas

production, including maximum gas production and the rate of gas production of the

ensiled OPF were all increased by the treatment with either cellulase or the LAB

inoculant-cellulase combination.

The OPF silage treated with the LAB inoculant-cellulase combination showed

significantly higher (P < 0.01) rapidly soluble fractions (18.23%), insoluble but

fermentable fractions (43.86%) (P < 0.05) and fractional degradation rates (0.018 h−1)

(P < 0.05) than the other treated silages.

The in vivo dry matter digestibility of the OPF silage with the LAB inoculant-cellulase

combination was 4.12% higher (P < 0.01) than the control silage. Thus, the ensiled

OPF treated with the LAB-cellulase combination was selected to be the best treated

OPF to be used in the animal feeding trial.

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Ruminant products are known to contain high saturated fatty acids (SFA) and low

polyunsaturated fatty acids (PUFA ), compared to pork, chicken meat or fish. Long

chain n-3 PUFA are important in the prevention of modern diseases such as

cardiovascular diseases, obesity and cancer. A part of the study attempted to decrease

the SFA and increase n-3 PUFA in the chevon using the best treatment of OPF using

diets with different n-6: n-3 PUFA ratios. Isonitrogenous and isocaloric experimental

diets containing the best treated OPF silage were formulated and either sunflower,

palm kernel or linseed oil were used to adjust the dietary n-6: n-3 fatty acid ratios

(FAR) to be 2.27:1 (LR), 5.01:1 (MR), and 10.38:1 (HR). Twenty-one five-month old

male Boer goats weighing 13.66 ± 1.07 Kg (mean initial body weight ± standard error)

were allocated randomly to the three dietary treatment groups and fed for 100 days.

The longissimus dorsi (LD) muscle, liver, rumen fluid and rumen digesta were

sampled at slaughter.

The average daily gain (ADG) for all the experimental groups was similar (P > 0.05).

The dressing percentage, back fat thickness and rib eye area of the LR group were

significantly (P < 0.05) higher than the HR group.

The thiobarbituric acid reactive substance (TBARS) values for the LD muscle for all

treatment groups after a 6-day post-mortem aging period were significantly (P < 0.05)

higher than at day 1 and the highest TBARS value belonged to the LR group

compared to the HR treatment group after a 6-day post-mortem aging period.

Goats fed the LR diet had a higher (P < 0.01) concentration (14.55mM/L) of total

VFA in the rumen fluid compared to the HR diet (11.67mM/L).

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Increasing the dietary n-6: n-3 FAR lowered (P < 0.01) the concentration of C18:0 but

linearly increased (P < 0.01) C16:0, C18:1 trans-11, C18:2n-6, and cis-9 trans-11 CLA

in the liver. The LR diet increased significantly (P < 0.01) C18:3n-3, C20:5n-3;

C22:5n-3 and C22:6n-3, consequently decreasing the liver n-6: n-3 FAR compared to

the HR group. Increasing the dietary n-6: n-3 FAR increased (P < 0.01) the C20:4n-6

while the low n-6: n-3 FAR increased (P < 0.01) �-linolenic acid (C18:3n-3)

concentration in the LD muscle. The C20:5n-3, C22:5n-3 and C22:6n-3 showed

positive linear effects (P < 0.01) with decreasing the dietary n-6: n-3 FAR.

There was a linearly increased population of cellulolytic bacteria including the

Ruminococcus albus (P < 0.05) and Ruminococcus flavefaciens (P < 0.05) in the

rumen liquid from the LR group compared to the HR group.� � The� population of

Butyrivibrio fibrisolvens was lower (P < 0.05) in the LR group, which had decreased

by 31.67%, compared to the HR group.

There was a higher (P < 0.05) PPAR� and PPAR� gene expression in the LD muscle

for the LR group compared with the HR group, indicating that the low dietary n-6: n-3

FAR upregulated the PPAR� and PPAR� genes. On the contrary, the stearoyl-CoA

desaturase (SCD) gene expression showed a significant (P < 0.05) reduction in the LR

group compared to the HR group suggesting that the SCD gene was downregulated by

the LR dietary treatment.

In conclusion, ensiling the OPF with the LAB inoculant-cellulase combination

improved the nutritional value and digestibility of the OPF. Diets formulated

incorporating the treated OPF and adjusted to have a low dietary n-6: n-3 FAR

(2.27:1) did not adversely affect the growth performance of the Boer goats. In fact the

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chevon was enriched with higher proportions of the n-3 PUFA. The increased n-3 fatty

acids in the rumen fluid, rumen digesta, liver and LD was suggestive of a reduced

biohydrogenation possibly due to changes in the ruminal environment which included

a lowered pH, increased VFA concentrations and a drastic decrease in the Butyrivibrio

fibrisolvens population, the microbe which is actively involved in biohydrogenation in

the rumen. The impact of the results derived from this study include the potential

reduction in feed costs using the treated OPF as small ruminant feed and the

‘healthier’ omega-3 enriched chevon would be very appealing to the health-conscious

consumer.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PENGHASILAN DAGING KAMBING TERPERKAYA ASID LEMAK POLITAKTEPU OMEGA-3 MENGGUNA SILAJ TERPELAKU PELEPAH

KELAPA SAWIT (ELAEIS GUINEENSIS JACQ.)

Oleh

MAHDI EBRAHIMI

November 2012

Pengerusi : Profesor Mohamed Ali Rajion, PhD

Fakulti : Perubatan Veterinar

Adalah perlu untuk memperlakukan pelepah kelapa sawit (OPF) supaya dapat

meningkatkan nilai nutrien dan kebolehcernaannya untuk diguna sebagai makanan

ruminant kecil. Kaedah perlakuan ini merangkumi OPF segar bukan ensilaj (F), OPF

ensilaj tanpa bahan tambahan (C, kawalan), OPF ensilaj dengan inokulan bakteria asid

laktik komersial (LAB), OPF ensilaj dengan enzim fibrolisis eksogenus (selulase) (E)

dan OPF ensilaj dengan gabungan inokulan LAB-selulase (I+E). Pengensilajan

dilakukan dengan menyimpan lebihkurang 2 Kg sampel dalam balang kaca kedap

udara pada 25 - 30°C selama 12 minggu selepas mana sampel silaj ini menjalani

analisa proksimat. Pengensilajan mengurangkan secara tererti (P < 0.05) kepekatan

DM OPF ensilaj dengan bahan tambahan berbanding OPF segar and silaj kawalan.

Gabungan inokulan LAB-selulase mengurangkan secara tererti (P < 0.01) kandung

NDF dan ADF berbanding silaj kawalan. Lemak kasar juga menurun secara tererti (P

< 0.05) selepas penensilajan berbanding OPF segar. Semua silaj terperlaku

menunjukkan pH (4.09-4.28) yang lebih rendah daripada silaj tidak diperlaku (4.88).

Asid asetik dan propionik dalam OPF silaj lebih tinggi tererti (P < 0.01) daripada OPF

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segar. Kepekatan NH3-N dalam silaj dengan bahan tambahan menurun secara tererti (P

< 0.01) berbanding OPF segar dan silaj kawalan. Gabungan inokulan LAB-selulase

meningkatkan karbohidrat larut air (WSC) dan penghasilan etanol secara tererti (P <

0.01) berbanding silaj kawalan. Populasi LAB meningkat secara tererti (P < 0.05)

dalam semua perlakuan dengan bahan tambahan berbanding OPF segar dan silaj

kawalan.

Kebolehcernaan bahan kering in vitro, kebolehcernaan bahan kering in sacco,

kebolehcernaan bahan kering in vivo, penghasilan gas in vitro, kinetik penapaian in

vitro, dan kinetik penapaian in vivo silaj ditentukan mengguna kambing berfistula

rumen. Silaj OPF terperlaku dengan selulase atau gabungan inokulan LAB-selulase

menunjukkan penghasilan gas in vitro dan kebolehcernaan bahan kering (DMD) lebih

tererti (P < 0.05) daripada silaj segar dan kawalan. Malah, penghasilan gas in vitro,

termasuk penghasilan gas maksimum dan kadar penghasilan gas OPF silaj meningkat

dengan perlakuan dengan selulase atau gabungan inokulan LAB-selulase.

Silaj OPF terperlaku dengan gabungan inokulan LAB-selulase menunjukkan lebih

tinggi tererti pecahan larut pantas (18.23%) (P < 0.01) pecahan tak larut tetapi boleh

tapai (43.86%) (P < 0.05) dan kadar penguraian pecahan (0.018 h-1) (P < 0.05)

daripada silaj terpelaku lain.

Silaj OPF yang diperlaku dengan gabungan inokulan LAB-selulase menunjukkan

penghasilan asid lemak meruap (VFA) keseluruhan lebih tinggi tererti (P < 0.01)

selepas 3 jam pascapemberian makan berbanding silaj kawalan. Propionat meningkat

secara tererti (P < 0.01) selepas 3 jam pascapemberian makan dengan silaj berbanding

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silaj kawalan. Pengambilan makanan juga meningkat secara tererti (P < 0.05) apabila

diperlakuan silaj OPF dengan selulase atau gabungan inokulan LAB-selulase

berbanding silaj kawalan. Kebolehcernaan bahan kering in vivo silaj OPF dengan

gabungan inokulan LAB-selulase adalah 4.12% lebih tinggi (P < 0.01) daripada silaj

kawalan. Justeru, silaj OPF yang diperlaku dengan gabungan LAB-selulase telah

dipilih sebagai OPF terperlaku terbaik untuk diguna pada haiwan dalam percubaan

pemberian makan haiwan.

Diet isonitrogenus dan isokalori ujikaji mengandungi silaj OPF terperlaku terbaik

dirumuskan dan sama ada bunga matahari, isirong sawit atau minyak linseed diguna

untuk menyelaraskan nisbah n-6; n-3 asid lemak (FAR) kepada 2.27;1 (LR), 5.01:1

(LR), dan 10.38:1 (HR) dalam diet. Dua puluh satu ekor kambing Boer jantan berumur

lima bulan berat badan 13.66 ± 1.07 Kg (min permulaan berat badan ± ralat piawai)

diagihkan secara rawak kepada tiga kumpulan perlakuan diet dan diberi makan selama

100 hari. Pada masa penyembelihan karkas dipecahkan kepada dua bahagian (kanan

dan kiri). Otot longissumus dorsi (LD), hati, bendalir rumen dan digesta rumen

disampel. Otot dipek vakum dan disesuaikan selama 1, 3 dan 6 hari dalam pendingin

pada 4°C.

Purata kenaikan harian (ADG) untuk semua kumpulan ujikaji adalah serupa (P >

0.05). Berat badan haiwan pada penyembelihan dalam kumpulan LR (26.83 Kg) dan

MR (26.19 Kg) adalah tinggi sedikit daripada kumpulan haiwan HR (25.68 Kg). Tiada

perbezaan tererti (P > 0.05) didapati di antara semua kumpulan perlakuan dari segi ciri

karkas, sifat dan mutu daging. Bagaimanapun peratus lapah, tebal lemak belakang dan

kawasan mata rusuk untuk kumpulan LR adalah lebih tinggi tererti (P < 0.05) daripada

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kumpulan HR. Parameter mutu daging lain termasuk warna daging, kehilangan

memasak, kehilangan drip dan kelembutan tidak berbeza tererti (P > 0.05) antarqa

semua kumpulan perlakuan.

Nilai bahan reaktif asid tiobarbiturik (TBARS) otot LD bagi semua kumpulan

perlakuan selepas 6 hari tempoh penuaan post-mortem adalah lebih tinggi tererti (P <

0.05) daripada hari 1, dan nilai TBARS tertinggi berlaku pada kumpulan LR

berbanding kumpulan perlakuan HR selepas tempoh 6 hari penuaan post-mortem.

Purata pH bendalir rumen untuk kumpulan LR (6.08) tidak rendah tererti (P > 0.05)

daripada kumpulan HR (6.39).

Populasi protozoa rumen didominasi oleh Entodinium (95.12 %) dan kurang sedikit

oleh spesies Holotrich (4.87 %) yang diberi makan diet LR menunjukkan kepekatan

VFA keseluruhan (14.55mM/L) lebih tinggi bererti (P < 0.01) dalam bendalir rumen

berbanding diet HR (11.67mM/L).

Digesta rumen daripada kambing yang diberi makan diet LR mengandungi perkadaran

C18:0 lebih tinggi (7.86 %) berbanding dua lagi kumpulan perlakuan. Pengurangan n-

6:n-3 FAR dalam diet menurunkan secara tererti (P < 0.01) C18:2n-6 dan cis-9 trans-

11 asid linoleik terkonjugat (CLA). Jumlah PUFA n-3 keseluruhan dalam kumpulan

LR meningkat secara tererti (P < 0.01) berbanding kumpulan HR.

Peningkatan n-6:n-3 FAR dalam diet menyebabkan peningkatan tererti (P<0.001)

C20:4n-6 sambil n-6:n-3 FAR yang rendah itu meningkatkan (P < 0.01) kepekatan �-

asid linolenik (C18:3n-3) dalam otot LD kumpulan LR berbanding kumpulan HR.

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Akhirnya C20:5n-3, C22:5n-3 dan C22:6n-3 menunjukkan kesan linear positif (P <

0.01) dengan penurunan n-6:n-3 FAR.

Penyataan gen PPAR� dan PPAR� dalam otot LD dalam kumpulan LR lebih tinggi (P

< 0.05) berbanding kumpulan HR, menunjukkan n-6:n-3 FAR rendah dalam diet telah

menaik regulasi gen PPAR� dan PPAR�. Disebaliknya, penyataan gen stearoil-KoA

desturase (SCD) menunjukkan penurunan tererti (P < 0.05) untuk kumpulan LR

berbanding kumpulan HR, menyarankan gen SCD telah diturun regulasi oleh

perlakuan diet LR.

Populasi bakteria selulolisis termasuk Ruminococcus albus (P < 0.05) dan

Ruminococcus flavefaciens (P < 0.05) meningkat secara linear dalam bendalir rumen

kumpulan LR berbanding kumpulan HR. Populasi Butyrivibrio fibrosolvens adalah

lebih rendah (P < 0.05) dalam kumpulan LR yang menurum sebanyak 31.67 %

berbanding kumpulan HR.

Kesimpulannya, mengensilaj OPF dengan gabungan LAB-selulase meningkatkan nilai

nutrien dan kebolehcernaan OPF. Diet yang dirumus dengan memasukkan OPF

terperlaku dan tersesuai untuk mengandungi n-6:n-3 FAR (2.27:1) rendah tidak

memberi kesan buruk terhadap prestasi pertumbuhan, ciri karkas dan mutu daging

kambing Boer sedang membesar. Malah daging kambing menjadi terperkaya dengan

perkadaran n-3 PUFA lebih tinggi. Peningkatan asid lemak n-3 dalam bendalir dan

digesta rumen, hati dan otot LD menyarankan biopenghidrogenan yang kurang,

sebahagiannya disebabkan oleh perubahan dalam persekitaran rumen termasuk pH

lebih rendah, peningkatan kepekatan VFA dan penurunan mendadak populasi

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Butyrivibrio fibrosolvens yang terlibat dalam biopenghidrogenan dalam rumen. Impak

keputusan daripada kajian ini termasuk potensi pengurangan kos makanan

menggunakan OPF terlaku sebagai makanan ruminan kecil dan daging kambing

terperkaya omega-3 akan menjadi lebih menarik untuk pelanggan yang mementingkan

kesihatan.

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ACKNOWLEDGEMENTS

First and foremost, my honest appreciation to my supervisory committee, who

were involved in my training towards obtaining this degree. I am most grateful to

Professor Dr. Mohamed Ali Rajion, the chairman of my supervisory committee

for his patience, tireless support, willingness to help, encouragement, kindness and

guidance throughout the research and during the preparation of the thesis. I am very

much indebted to the members of my supervisory committee namely Associate

Professor Dr. Goh Yong Meng, Dr. Awis Qurni Sazili from the Faculty of Agriculture,

UPM and Dr. Thomas Schonewille from the Faculty of Veterinary Medicine, Utrecht

University, The Netherlands for their encouragement, constructive discussion,

excellent advice, comments, and suggestions throughout the project. Special thanks to

Professor Dr. Rasedee Abdullah for help with the Bahasa Malaysia abstract

translation.

I would like to extend my deepest and sincere appreciation to Universiti Putra

Malaysia who supported my candidature and the Ministry of Science and Technology

and Ministry of Agriculture and Agro-Based Industry, Malaysia for the E-Science

Research Grant which supported the study.

I wish to express my sincere gratitude to the Deans of the Faculty of

Veterinary Medicine and Faculty of Agriculture and Directors of the Institute of

Bioscience, Institute of Tropical Agriculture, Universiti Putra Malaysia for the use

of their facilities and the unlimited assistance from their staff during the course of

this study. I would also like to thank staff members of the Veterinary Physiology,

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Clinical Pathology and Nutrition Laboratories in particular Mr. Kufli Che Nor, Mrs.

Zainab Nasri and Mrs. Rosmawati Hanipah, for their technical assistance. I would like

to thank all MARDI staff including Mr. Yunus Ismail and Mr. Fadil Jantan who

helped me in the provision of OPF, preparation of silage and pelleting of the feed.

I will always cherish the friendship and help from fellow graduate students Ms Ai Li

Ta, and Dr. Suria Kumari.

I would also like to thank all of my past Professors from the University of

Tehran in Iran who have encouraged me well and I will be forever thankful. I want to

express my huge thanks to my closest friend Dr. Abdoreza Soliemani Farjam who

found for me this opportunity to continue my postgraduate education in UPM. I also

must thank my closest friends in Kuala Lumpur; Dr. Mohamadreza Sanaii, Dr. Arash

Javanmard, Dr. Mohammad Feseleh Jahromi, Dr. Ehsan Oskouian, Dr. Elham

Maroufian, Dr Hadi Hajarian, Dr Toktam Hajjr, Parvaneh Mehrbod, Ramin Jorfi, Dr.

Hasliza Abu Hassim, Saied Nikbin, Dr. Tang Siew Ching, Su Ting Yong, Hanini

Sahari, Dr. Azhary AE Sherydane, Dr. Ainul Yuzairi, Zuriiyaatii Ramly, Aisha Yusuf

and back home in Tehran; Seyed Mehdi Khatami, Seyed Javad Mirabdollahi, Bahman

Jalali, Seyed Ahmadreza Fakharzadeh, Dr. Hamid Dorodian, Dr. Mostafa Sadeghi and

Dr. Reza Taherkhani. I would also like to thank my Heavenly Father (Bahram) and

Mother (Sakineh) for providing so many opportunities and support for both my

physical and spiritual growth. They have strengthened me during the times when I

thought I could go no further. Without them, none of this would have been possible. I

also thank all my brothers (Amirhossein and Gholamreza) and sisters (Marzieh,

Mahdieh and Hadiseh) for their love, understanding and support.

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I certify that a Thesis Examination Committee has met on 30…. December 2012 to conduct the final examination of Mahdi Ebrahimi on his Doctor of Philosophy thesis entitled “Production Of Omega-3 Polyunsaturated Fatty Acid-Enriched Chevon Using Treated Oil Palm Fronds” in accordance with Universities and Unievrsity Colleges Act 1971 and the Constitutions of the Universiti Putra Malaysia [P.U. (A) 106] 15 March 1998. The Committee recommends that the student be awarded the degree of Doctor of Philosophy (PhD). Members of the Examination Committee are as follows:

Ismail Idris, PhD Associate Professor aaaaMAKE SURE YOU HAVE THE NAMES AND APELLING CORRECTFaculty of YOUAgriculture Universiti Putra Malaysia (Chairman)

Zainal Aznam Mohd Jelan, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner)

Mohamed Ali Rajion, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner)

Egil Robert Orskov, PhD Professor The Macaulay Land Use Research Institute Aberdeen University United Kingdom (External Examiner)

_____________________________ SEOW HENG FONG, PHD

Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows: Mohamed Ali Rajion, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Goh Yong Meng, PhD Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Awis Qurni Sazili, PhD Lecturer Faculty of Agriculture Universiti Putra Malaysia (Member) Thomas Schonwille Associate Professor Faculty of Veterinary Medicine Utrecht University The Netherlands (Member)

___________________________ BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

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DECLARATION

I hereby declare that the thesis is my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or other institutions. ___________________ MAHDI EBRAHIMI Date: 9 Novenber 2012

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TABLE OF CONTENTS

Page

ABSTRACT…………………………………………………………………… iii ABSTRAK…………………………………………………………………….. viii ACKNOWLEDGMENTS……………………………………………………. xiv APPROVAL…………………………………………………………………... xvi DECLARATION……………………………………………………………… xviii LIST OF TABLES…………………………………………………………… xxiii LIST OF FIGURES………………………………………………………….. xxv LIST OF PLATES……………………………………………………………. xxvi LIST OF ABBREVIATIONS………………………………………………...

xviii

CHAPTER

1. GENERAL INTRODUCTION 1

2. LITERATURE REVIEW 5�

The Small Ruminant Industry in Malaysia ....................................................... 5�Oil Palm Fronds as Feed Sources for Animals ................................................. 7�Post-harvest Treatment and Animal Feeding .................................................... 9�Conservation of OPF as Silage ....................................................................... 11�Silage……………………………………………………………………… 11�Silage Additives .............................................................................................. 12�Lactic Acid Bacteria (LAB) ............................................................................ 13�Cell Wall Degrading Enzymes........................................................................ 14�Combinations of Enzymes and Other Additives ............................................. 14�Local research on OPF as Ruminant Livestock Feed .................................... 15�Growth Performance and Carcass Characteristics of Goats ........................... 16�Carcass Characteristics ................................................................................... 16�Muscle lipids ................................................................................................... 17�Lipid Oxidation in Muscles ............................................................................ 17�Factors Affecting Meat Quality ...................................................................... 18�Meat Quality ................................................................................................... 19�Meat pH .......................................................................................................... 19�Meat Color ...................................................................................................... 20�Water Holding Capacity (WHC) .................................................................... 20�Drip Loss ......................................................................................................... 21�Cooking Loss .................................................................................................. 21�Factors Determining Fatty Acid Profiles in Ruminant Meat .......................... 22�Fat Sources and Types .................................................................................... 23�The Rumen Ecosystem ................................................................................... 25�

Rumen Biohydrogenation ................................................................... 26�Rumen Bacteria ............................................................................................... 28�

Rumen Protozoa ................................................................................. 28�Effects of Dietary Fatty Acids on Ruminant Digestion .................................. 29�Effects of Dietary Fatty Acids on Ruminal Flora ........................................... 29�The Digestion and Absorption of Fatty Acids in Ruminants .......................... 30�Meat Fatty Acids and Human Health.............................................................. 31�

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Alteration of the Fatty Acid Profiles in Ruminants ........................................ 32�Fatty Acids and Gene Expression ................................................................... 33�PUFA and the Regulation of Gene Expression............................................... 33�Meat Consumption Patterns in Malaysia ........................................................ 34�Summary and Conclusion ............................................................................... 35

3. GENERAL MATERIALS AND METHODS 37�

OPF Ensiling Procedure ..................................................................... 37�Animal Welfare ................................................................................... 38�Animal Management .......................................................................... 38�Chemical Analysis .............................................................................. 39�Fatty Acid Analysis ............................................................................. 41

4. EFFECTS OF ADDING LACTIC ACID BACTERIA AND CELLULASE ENZYME SINGLY AND IN COMBINATION ON THE FERMENTATION CHARACTERISTICS, CHEMICAL COMPOSITION AND FATTY ACID PROFILES OF OIL PALM (ELAEIS GUINEENSIS JACQ.) FROND SILAGE �

Introduction ..................................................................................................... 43�Materials and Methods .................................................................................... 46�

Silage Preparation and Chemical Analyses ........................................ 46�Statistical Analysis .............................................................................. 46�

Results ………………………………………………………………………. 47�Effect of Additives on the Chemical Composition of OPF Silage ..... 47�Effect of Additives on the Fermentation Parameters of OPF Silage .. 48�Changes in Microbial Population of OPF Silage ................................ 49�Effect of Additives on the Fatty Acid Profile of OPF Silage .............. 49�

Discussion ....................................................................................................... 51�Effect of Additives on the Chemical Composition of OPF Silage ..... 51�Effect of Additives on the Fermentation Parameters of OPF Silage .. 52�Effect of Additives on the Fatty Acid Composition of OPF Silage .... 58�

Conclusion ...................................................................................................... 60

5. IN VITRO, IN SACCO AND IN VIVO DIGESTIBILITY OF ENSILED TREATED OIL PALM FRONDS �

Introduction ..................................................................................................... 61�Materials and Methods .................................................................................... 63�

Animals ............................................................................................... 63�Preparation of Silage .......................................................................... 63�In vitro Rumen Degradation and Fermentation Substrates ................. 63�Rumen Fluid Collection and Incubation ............................................. 63�Bicarbonate and Phosphate Buffer Preparation .................................. 64�In vitro Incubation .............................................................................. 64�Gas Determinations after Incubation .................................................. 65�Calculations ........................................................................................ 66�In sacco Ruminal Degradation ........................................................... 66�

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Apparent In Vivo Dry Matter Digestibility and Fermentation Kinetics ........................................................................................ 67�

Statistical Analysis .............................................................................. 69�Results ………………………………………………………………………. 70�

In vitro Gas Production, Fermentation Parameters and Apparent Digestibility of the OPF Silages................................................... 70�

In sacco Ruminal Degradation ........................................................... 72�Apparent In Vivo Dry Matter Digestibility and Fermentation

Kinetics ........................................................................................ 74�Discussion ....................................................................................................... 75�

In vitro Gas Production, Fermentation Parameters and Apparent Digestibility of the Silages ........................................................... 75�

In sacco Ruminal Degradation ........................................................... 80�Apparent in vivo Dry Matter Digestibility and Fermentation

Kinetics ........................................................................................ 84�Conclusion ...................................................................................................... 86

6. GROWTH PERFORMANCE, CARCASS CHARACTERISTICS, MEAT QUALITY, TISSUE FATTY ACID PROFILES, RUMEN FERMENTATION PARAMETERS AND MICROBIAL POPULATIONS IN BOER GOATS FED DIETS INCORPORATED WITH TREATED OIL PALM FRONDS AND ADJUSTED DIETARY N-6: N-3 FATTY ACID RATIOS �

Introduction ..................................................................................................... 88�Materials and Methods .................................................................................... 92�

Animals, Diets, and Management ....................................................... 92�Slaughter Procedures and Measurement of Carcass Quality .............. 93�Meat Color Determination .................................................................. 96�Water Holding Capacity ..................................................................... 96�Drip Loss ............................................................................................ 96�Cooking Loss ...................................................................................... 97�Texture Analysis ................................................................................. 97�Lipid Peroxidation .............................................................................. 98�Chemical Analyses ............................................................................. 98�Rumen Fluid Sample Collection ......................................................... 99�Rumen Fermentation Parameters ........................................................ 99�DNA Extraction ................................................................................ 100�Quantitative Real-Time Polymerase Chain Reaction (PCR) for

Rumen Microbes ........................................................................ 101�Tissue Collection and RNA Extraction and Purification .................. 102�Complementary DNA Synthesis ....................................................... 103�Real-Time PCR for PPAR and SCD Genes ...................................... 103�Statistical Analysis ............................................................................ 104�

Results ……………………………………………………………………... 106�Composition of Experimental Diets ................................................. 106�Effects of Dietary Treatments on Growth Performance ................... 108�Carcass Characteristics and Traits .................................................... 108�Meat Quality of Chevon ................................................................... 110�Color ... 110�

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Water Holding Capacity ................................................................... 113�Drip Loss .......................................................................................... 113�Tenderness (Warner–Bratzler Shear Force) ...................................... 114�Lipid Oxidation in Chevon ............................................................... 115�Fatty Acid Composition of Experimental Diets ............................... 116�Fatty Acid Profiles of Rumen Digesta .............................................. 116�Fatty Acid Profiles of the Liver ........................................................ 118�Fatty Acids Profile of LD Muscle ..................................................... 119�Ruminal Fluid pH, Ammonia N, and VFA ....................................... 122�The Effects of Different Dietary n-6: n-3 FAR on Rumen

Microbial Population.................................................................. 123�Effect of Dietary n-6: n-3 FAR on mRNA Expression of PPAR�,

PPAR� and SCD in LD Muscle ................................................. 124�Discussion ..................................................................................................... 126�

Diets, Feed Intake, and Growth Performance ................................... 126�Carcass Traits and Characteristics .................................................... 127�Meat Quality ..................................................................................... 128�Fatty Acid Profiles of the Ruminal Digesta 131�Fatty Acid Profiles of the Liver ........................................................ 133�Fatty Acid Profiles of the LD Muscle ............................................... 135�Nutritional Quality of the Meat Fatty Acids ..................................... 139�Ruminal Fluid pH, Ammonia N, Protozoa and VFA ........................ 140�The Effects of Different Dietary n-6: n-3 FAR on Rumen

Microbial Population.................................................................. 143�Effect of Different Dietary n-6: n-3 FAR on mRNA Expression

of PPAR�, PPAR� and SCD in LD Muscle ............................... 146�Conclusion .................................................................................................... 151�

7. GENERAL DISCUSSION 153�

8. SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR FUTURE RESEARCH 163�

Recommendations and Future Research ..... 164�

REFERENCES 165�

APPENDIX A 202�

APPENDIX B 204�

BIODATA OF STUDENT 207�

LIST OF PUBLICATIONS 208�