Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science NOV. 2016 VOL. 39 (4) NOV.

Pertanika JTAS Vol. 39 (4) Nov. 2016 i 459 503 541 551 515 421 483 491 393

ield of

Y

A., and

Review A A.

Three Oil gy of r

ater Use and

W

A. raits of Rainfed Upland Rice in raits of Rainfed Upland Rice Review A. Samsudin, R. Mohamad and aters of Kudat, Sabah, T

etunde O. A

A. Q. Sazili and H. L. Foo Contents W

A.

Akit, richoderma harzianum SNRS3 T Amal, M. N. Vol. 39 (4) Nov. 2016 39 (4) Nov. Vol. Alawode, Y echniques on the Growth Rate of Marine echniques on the Growth Rate of Marine Acids Supplementation on Layer Hen di T

A. and Apparent Metabolisable Ene ferent Levels of Low Crude Protein Diets with eerasamy Sejian, Madiajagan Bagath and eerasamy Sejian, Madiajagan Bagath f . C. Loh, H. the Amino

. C. Loh, H. L. Foo, A., Ismail, their Combination in Broiler Chickens Adesola L. and

daus-Nawi and M. Zamri-Saad daus-Nawi and M. , Abdulla, T Articles enesa, T Analysis of Panicle and Grain Pertanika Journal of Tropical Agricultural Science Tropical of Journal Pertanika

Articles fect of Cultivation ropical Ecologies of Nigeria [email protected] T Sophia Inbaraj, V Raghavendra Bhatta D. Khodadadi Dehkor M. T Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. Rahnama, N., Shah, U. K. M., Foo, H. A. B. Ariff A. R. Raha Sule, H. N. R. Nassir Nayan Deep S. Kanwal M. Fir eword ferent Levels of fect of Higher Density Planting on Irrigation fects of Feeding Di Note Comparing Review of the Ichthyofauna of Malaysian Peat Swamp Forest Review of the Ichthyofauna wo Pertanika Editorial O ce, Journal Division Editorial O ce, Journal Pertanika (R&I) Chancellor Vice the Deputy O ce of II Tower IDEA Floor, 1st Centre UPM-MTDC Technology Malaysia Putra Universiti 43400 UPM Serdang Selangor Darul Ehsan Malaysia http://www.pertanika.upm.edu.my/ E-mail: +603 8947 1622 / 1619 Tel: Short Communication A Impact of Heat Stress on Immune Responses of Livestock: Impact of Heat Stress on Immune Responses Microalgae from the Coastal Dif Production and Characterisation of Cellulase from Solid State Production and Characterisation of Cellulase Fermentation of Rice Straw by Ef Sugarcane Ef Performance Regular Stability A Sources and T The Ef Review of Immunity: Major Components For Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science itself. the drive to realise exciting new horizons that will benefit not only the academic community, but society Weare continuously improving archives, access journal our to content, and research services. haveWe Future vision Guide). EBSCOhost,Cabell’sDOAJ,Agricola,& EBSCO Scholar,Directories,Google (Journal Rubriq & ISC MyAIS, being JTAS Pertanika in resulted has abstracted and knowledge indexed accumulatedin SCOPUS (Elsevier), this Thomson (ISI) Web old; of Knowledge years [BIOSIS & 38 CAB Abstracts], almost is Pertanika Abstracting andindexingof to publication for thearticles averages 5-6months. submission from publishing. time elapsed The weeks. 14 journal within decision a receive to guaranteedto are Submissions approach professional and responsible a by sustained excellence, for aim We Quality Our goal isto bringthehighest qualityresearch to thewidest possibleaudience. Goal of journal inMalaysia now focuses ontropical agricultural research anditsrelated fields. After 37 years, as an interdisciplinary journal of Agriculture, the revamped Journal, a leading agricultural the with aligned study of areas Social interdisciplinary strengths in oftheuniversity. journals of specialised Journal for need and Technology, the meet & to Humanities Science & of Sciences Journal Science, Agricultural Tropical of Journal as Pertanika was founded in 1978. A decision was made in 1992 to streamline Pertanika into three journals History economic importance, andveterinary medicine. pathology and management, physiology, plant and sciences, production of plants and of biotechnology,biochemistry, biology, ecology, fisheries, forestry, microbiology, genetics, sciences, food research quality high for agriculturalinclude: journal forumrelatedthe tropical agriculturaltoresearch.of relevantscopeAreas the to a provide to aims Science Agricultural Tropical of Journal Pertanika Aims andscope The Journalisavailable world-wide. world regardless ofthenationality. JTASa is tothe pertaining to tropical agriculture anditsrelated fields. devoted journal interdisciplinary peer-reviewed publication of original papers, leading it serves as a forum for the practical approaches to improving quality in as issues internationally Recognized the scientific outputs.Itneitheraccepts nor commissionsthird party content. published by UPM Press. It is an open-access online scientific journal which is free of charge. It publishes Pertanika Journal of Tropical Agricultural Science (JTAS) is the official journal of Universiti Putra Malaysia Overview About theJournal Journal ofTropical Agricultural Science original articles as per its scope. The journal publishes in publishes journal The scope. its per as articles original Pertanika quarterly ( February, May, AugustNovember and Pertanika English proia ta cnies o publication for considers that periodical ) and it is open toopen is it and authors aroundthe Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science , and . section reports section for full details, or details, full for Results (Online) iscussion. IMRAD is simply on of receipt a manuscript, D Pertanika J. Trop. Agric. Sci. Trop. J. Pertanika Code of Ethics nd, A , but the editors will make the final the make will editors the but , ; ISSN 2231-8542 esults, Manuscript ID Manuscript suggest suggest names of at least three potential R (Print) Title, Authors, Keywords, Abstract, Conclusions Abstract, Keywords, Title, Authors, section explains meaning and significance of the results the of significance and meaning explains section process. Manuscripts deemed suitable for publication at the back of this journal. at . Discussion aterials aterials and Methods, describes how the study was conducted; the conducted; was study the how describes M Authors are encouraged to encouraged are Authors http://www.pertanika.upm.edu.my/code_of_ethics.php ntroduction, ntroduction, I double-blind peer-review double-blind Instructions to Authors to Instructions explains the scope and objective of the study in the light of current knowledge on the knowledge study ofthe and inscope the objective the of explains light current Instructions to Authors to Instructions Materials and Methods Materials . Additionally, some papers include Acknowledgments and Appendices. include Acknowledgments some papers . Additionally, Introduction reviewers at the time of submission of their manuscript to Pertanika to manuscript their of submission of time the at reviewers these suggestions. bound by choice. The editors are not, however, Editorial process are Authors notified with a an containing acknowledgement publication. decision regarding and upon the editorial Pertanika follows a are usually sent to reviewers. research research papers; the missing parts of a paper are: References The the subject; the and study; the in found was what according prepared be must manuscript The research. of directions future for suggestions provides and the Journal’s to Manuscript preparation Pertanika’s to Refer Most scientific papersare prepared according to format calleda IMRAD. The term represents the first letters of the words a more ‘defined’version of the “IBC” [Introduction, Body, Conclusion] format usedwriting.rather IMRAD than a format indicates or pattern complete list ofa for headings or components of allacademic A decision on acceptance or rejection of a manuscript isreached in 3to 4 14 weeks). months (average months. 5-6 averages for the articles to publication submission time from The elapsed Authorship Authors are not permittedto add orremove any names from the authorship providedat the time of Editor. Executive Chief of the Journal’s consent initial submission without the An ISSN is an 8-digit code used to identify periodicals such as journals of all kinds and on all media–print media–print all on and kinds all of journals as such periodicals identify to used code 8-digit an is ISSN An as an e-ISSN. ISSN as well journals have All Pertanika and electronic. Science: ISSN 1511-3701 Agricultural Journal of Tropical Lag time Code of Ethics responsibility of all of its seriously the takes Malaysia Putra Journals and Universiti The Pertanika are editors ethics. Thus all journals and journal in publication the highest reflect to journal publications Pertanika’s to Refer ethics. of codes the Journal’s abide by to expected link at web visit the Journal’s (ISSN) International Standard Serial Number Publication policy consideration concurrent for manuscript same the submitting from author an prohibits policy Pertanika by two manuscript or of more publications. publication It permit prohibits not aswell does publication also of It any manuscript elsewhere. that part has alreadysubstantial been or whole in either published has been published in full in Proceedings. that Citing journal articles Citing journal Science is Agricultural Tropical of Journal Pertanika for The abbreviation Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science What happens to a manuscript once it is submitted to Operating andreviewprocess most appropriate andhighest qualitymaterial for thejournal. weaknesses Peer reviewers are experts chosen by journal editors to provide written assessment of the manuscripts. In the peer-review process, three referees independently evaluate the scientific quality of the submitted The Journal’s peer-review given at thebackofthisjournal. Pertanika’sfirst-4 in format page the in described as 2 page on only placed be should paper the of authorship identify might that material reviewed, double-blind are articles As accepted conditionally, pendinganauthor’s revision ofthematerial. are manuscripts cases, most In guaranteed. not is manuscripts solicited of Publication manuscript. of receipt the to from weeks fourteen ten within provided is usually decision editorial of the Notification editorial review process: 2. 1. 6. 5. 4. 3. about revising themanuscript. provides guidelines for attending to the reviewers’ suggestions and perhaps additional advice editor the indicated, is revision a author.If the to forwarded are author) the reviewers’ (to exception, comments without Almost revision. instances, no rare almost In with publication. accepted is for manuscript material the any refuse to right the reserves Edito- who the in-Chief, with rests rejection or acceptance Final reviews. the additional resubmitseek and or revisemanuscript, to author(s) the invite manuscript, the reject to whether decides executivechief editor,The editor-in-chief, the with consultation in and reviews the examines literature. to the contribution potential its and work the of significance the of nature the in are editor the to Comments manuscript. the of strengthening for suggestions include often Reviewers conclusions. and discussion, or and theoretical results the method, review,literature of framework, adequacy conceptual and appropriateness the about are authors to Comments them to complete thereview inthree weeks. asks editor executive chief The article. the by represented matter subject the in specialists are Others board. editorial Journal’s the from is these of Typically,one reviewers. three been removed, to having information the article-identifying sends editor executive chief The the appropriate, not If reviewed. be manuscript isrejected outright andtheauthorisinformed. should and journal the for appropriate is it whether determine to paper the examine board editorial the and Journal’sexecutive editor chiefThe the paperisready to bepublished,needsanotherround ofrevisions, orshouldberejected. with the editorial board and the editor-in-chief examine consultation their comments and decide whether in editor executive chief the work, their completed have reviewers the When the originalreviewers willbeasked to examine thearticle. chiefexecutive The out the paper for revised editor sends re-review. Typically, of least one at certain comments with author disagrees the provided by reviewer(s). when need especially a submit is also may there if author(s) rebuttal The a form. tabular a in usually the concerns editor, the and have answered’ reviewers they the of how describing information specific with along editor the editor’s concerns. The authors return a revised version of the paper to the chief executive The authors decide whether and how to address the reviewers’ comments and criticisms and of written research, with the aim of improving the reporting of research and identifying the Pertanika ? Typically, there are seven steps to the Instructions to AuthorstoInstructions strengths and

Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science Journal of Tropical Agricultural Science only . Finally, the article in appears the. pagesFinally, of the Journal and is

The Publisher ensures that the paper list, and adheres tables and are typical reference The grammar). areas of authors are concern, clarity, to the correct style (in-text citations, asked the to respond to any minor queries by the Following Publisher. these corrections, page proofs are mailed to the corresponding authors for their final approval.At this point, essentialchanges are accepted on-line. posted If the decision is to accept, an acceptance letter is sent to all the author(s), the paper is sent to sent is paper the author(s), the all to sent is letter acceptance an accept, to is decision the If months. three in approximately print should appear in The article the Press. 7.

JTAS Journal of Tropical Agricultural Science AN INTERNATIONAL PEER-REVIEWED JOURNAL EDITOR-IN-CHIEF EDITORIAL BOARD Mohd. Zamri-Saad, Malaysia 2015-2017 Veterinary Pathology Baharuddin Salleh Kadambot H.M. Siddique, Son Radu Plant pathologist / Mycologist, FTSE Food safety, Risk assessment, Molecular Universiti Sains Malaysia, Malaysia. biology, CHIEF EXECUTIVE EDITOR Crop and environment physiology, Universiti Putra Malaysia, Malaysia. Germplasm enhancement, Nayan Deep S. Kanwal Chee-Kong Yap The University of Western Australia, Biology, Ecotoxicology, Australia. Srini Kaveri Environmental Issues – Landscape Universiti Putra Malaysia, Malaysia. Veterinary, Immunology, Plant Modelling Applications INSERM, Centre de Recherche Cordeliers, Leng-Guan Saw Paris, France. David Edward Bignell Botany and Conservation, Plant Ecology, UNIVERSITY PUBLICATIONS Soil biology and termite biology, Forest Research Institute Malaysia University of London, UK. (FRIM), Kepong, Malaysia. Suman Kapur COMMITTEE Biological Sciences, Agricultural and Animal Biotechnology, Mohd Azmi Mohd Lila, Chair Eric Standbridge Mohd. Azmi Ambak Microbiology, Molecular genetics, Birla Institute of Technology and Science Fisheries, BITS-Pilani, Hyderabad, India. Universiti of California, USA. Universiti Malaysia Terengganu, EDITORIAL STAFF Malaysia. Ghizan Saleh Tan Soon Guan Journal Officers: Plant breeding and genetics, Molecular Population Genetics, Nor Aini Ab-Shukor Universiti Putra Malaysia, Malaysia. Kwan Lee Yin, ScholarOne Universiti Putra Malaysia, Malaysia. Tree improvement, Forestry genetics & Kanagamalar Silvarajoo, ScholarOne biotechnology, Idris Abd. Ghani Universiti Putra Malaysia, Malaysia. Wen-Siang Tan Lim Ee Leen, ScholarOne Entomology Molecular biology, Virology, Protein and biodiversity, Integrated pest chemistry, Richard T. Corlett Universiti Putra Malaysia, Malaysia. Editorial Assistant: management, Biological control, Biological Sciences, Terrestrial Ecology, Biopesticides, Zulinaardawati Kamarudin Climate Change, Conservation Biology, Universiti Kebangsaan Malaysia, Biogeography, Yusof Ibrahim Malaysia. National University of Singapore, Agricultural entomology, COPY EDITORS Singapore. Universiti Pendidikan Sultan Idris, Jamilah Bakar Malaysia. Doreen Dillah Food Science and Technology, Food Shamshuddin Jusop Crescentia Morais Quality / Processing and Preservation, Soil science, Soil mineralogy, Zora Singh Pooja Terasha Stanslas Universiti Putra Malaysia, Malaysia. Universiti Putra Malaysia, Malaysia. Horticulture, Production technology and post-handling of fruit crops, Curtin University, Australia. PRODUCTION STAFF Pre-press Officers: Nik Khairul Azizi Nik Ibrahim Kanagamalar Silvarajoo Layout & Typeset: Sarwani Padzil Noor Sholihah Mohd Daud

WEBMASTER INTERNATIONAL ADVISORY BOARD Mohd Nazri Othman 2013-2016 PUBLICITY & PRESS RELEASE Alexander Salenikovich Jane M. Hughes Syed M. Ilyas Forestry, Wood and Forest Sciences, Genetics, Project Director, National Institute Magdalene Pokar (ResearchSEA) Université Laval, Canada. Griffith University, Australia. of Rural Development, Post Harvest Florence Jiyom Engineering and Technology, Indian Council of Agricultural Research, Banpot Napompeth Malcolm Walkinshaw Hyderabad, India. Entomology, Biochemistry, EDITORIAL OFFICE Kasetsart University, . University of Edinburgh, Scotland. JOURNAL DIVISION Tanveer N. Khan Plant Breeding and Genetics, Office of the Deputy Vice Chancellor (R&I) Denis J. Wright Manjit S. Kang The UWA Institute of Agriculture, st Pest Management, Plant Breeding and Genetics, 1 Floor, IDEA Tower II The University of Western Australia, Imperial College London, UK. Louisiana State University Agric. Center, Australia. UPM-MTDC Technology Centre Baton Rouge, USA. Universiti Putra Malaysia Graham Matthews 43400 Serdang, Selangor Malaysia. Pest Management, Imperial College London, UK. Peter B. Mather Gen Enq.: +603 8947 1622 | 1619 | 1616 Ecology and Genetics, E-mail: [email protected] Queensland University of Technology, Australia. URL: www.journals-jd.upm.edu.my

PUBLISHER Kamariah Mohd Saidin UPM Press Universiti Putra Malaysia 43400 UPM, Serdang, Selangor, Malaysia. Tel: +603 8946 8855, 8946 8854 Fax: +603 8941 6172 ABSTRACTING/INDEXING E-mail: [email protected] Pertanika URL: http://penerbit.upm.edu.my is now over 38 years old; this accumulated knowledge has resulted the journals being indexed in SCOPUS (Elsevier), Thomson (ISI) Web of Knowledge [BIOSIS & CAB Abstracts], EBSCO, DOAJ, Google Scholar, AGRICOLA, ISC, Citefactor, Rubriq and MyAIS.

The publisher of Pertanika will not be responsible for the statements made by the authors in any articles published in the journal. Under no circumstances will the publisher of this publication be liable for any loss or damage caused by your reliance on the advice, opinion or information obtained either explicitly or implied through the contents of this publication. All rights of reproduction are reserved in respect of all papers, articles, illustrations, etc., published in Pertanika. Pertanika provides free access to the full text of research articles for anyone, web-wide. It does not charge either its authors or author-institution for refereeing/publishing outgoing articles or user-institution for accessing incoming articles. No material published in Pertanika may be reproduced or stored on microfilm or in electronic, optical or magnetic form without the written authorization of the Publisher. Copyright © 2016-17 Universiti Putra Malaysia Press. All Rights Reserved.

Pertanika Journal of Tropical Agricultural Science Vol. 39 (4) Nov. 2016

Contents

Foreword Nayan Deep S. Kanwal i

Review Articles Major Components of Fish Immunity: A Review 393 M. Firdaus-Nawi and M. Zamri-Saad

A Review of the Ichthyofauna of Malaysian Peat Swamp Forest 421 Sule, H. A., Ismail, A. and Amal, M. N. A.

Impact of Heat Stress on Immune Responses of Livestock: A Review 459 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

Short Communication A Note Comparing the Apparent Metabolisable Energy of Three Oil 483 Sources and their Combination in Broiler Chickens N. R. Abdulla, T. C. Loh, H. Akit, A. Q. Sazili and H. L. Foo

Regular Articles Stability Analysis of Panicle and Grain Traits of Rainfed Upland Rice in 491 Two Tropical Ecologies of Nigeria Nassir, Adesola L. and Alawode, Yetunde O.

The Effect of Cultivation Techniques on the Growth Rate of Marine 503 Microalgae Species from the Coastal Waters of Kudat, Sabah, Malaysia Nurzafirah Mazlan and Ridzwan Hashim

Production and Characterisation of Cellulase from Solid State 515 Fermentation of Rice Straw by Trichoderma harzianum SNRS3 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A., and A. B. Ariff

Effect of Higher Density Planting on Irrigation Water Use and Yield of 541 Sugarcane D. Khodadadi Dehkordi

Effects of Feeding Different Levels of Low Crude Protein Diets with 551 Different Levels of Amino Acids Supplementation on Layer Hen Performance M. Tenesa, T. C. Loh, H. L. Foo, A. A. Samsudin, R. Mohamad and A. R. Raha Characterisation of Plant Growth-Promoting Bacteria from Kacip Fatimah 565 (Labisia pumila) under Natural Tropical Forest Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A.

Impact of Daily Supplement of Probiotic on the Production Performance 585 of Akar Putra Chickens Hasan, S. A. Jawad, Lokman, I. H., Saad, A. Naji, Zuki, A. B. Z. and Kassim, A. B.

The Effects of Fermented Feed on Broiler Production and Intestinal 597 Morphology Saad A. Naji, I. F. B. Al-Zamili, Hasan, S. A. Jawad and J. K. M. Al-Gharawi

Characteristics and Potential Usage of Dissolved Silica in Rice Cultivation 609 in Sumani Watershed, Sumatra, Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga Foreword

Welcome to the Fourth Issue 2016 of the Journal of Tropical Agricultural Science (JTAS)!

The JTAS is an open-access journal on Tropical Agricultural Science published by Universiti Putra Malaysia Press. It is independently owned and managed by the university and runs on a non-profit basis for the benefit of the scientific community.

This issue contains thirteen (13) articles of which three are review articles, one a short communication while nine are regular papers. The authors of these articles hail from different countries, namely Malaysia, Nigeria, India, Iraq, Iran, Pakistan, Japan and Indonesia.

The first review paper briefly discusses on the major components available infish immunity, such as the adaptive and innate immunity (M. Firdaus-Nawi and M. Zamri- Saad). The second review paper focuses on the ichthyofauna of the peat swamp forests (PSFs) of Malaysia. It provides a checklist of blackwater fish species in Malaysia from published literature, and addresses their importance to the economy, their conservation status and problems of PSFs (Sule, H. A., Ismail, A. and Amal, M. N. A.). The third review paper discusses impact of heat stress on Immune responses of livestock as well as the impact of climate change on the immune function of the animal (Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta). In the short communication, the authors provide a report, comparing the apparent metabolisable energy (AME) of three oil sources, namely palm oil (PO), soybean oil (SO), linseed oil (LO) and blend oil (BO), and their combination in broiler chickens diet (N. R. Abdulla, T. C. Loh, H. Akit, A. Q. Sazili and H. L. Foo).

The nine research articles cover a wide range of topics: A report on the stability analysis of panicle and grain traits of rainfed upland rice in two tropical ecologies of Nigeria (Nassir, Adesola L. and Alawode, Yetunde O.); the effect of cultivation techniques on the growth rate of marine microalgae species in the coastal waters of Kudat, Sabah, Malaysia (Nurzafirah Mazlan and Ridzwan Hashim); production and characterisation of cellulose from solid state fermentation of rice straw by Trichoderma harzianum SNRS3 (Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A., and Ariff, A.); effects of higher density planting on irrigation water use and yield of sugarcane (D. Khodadadi Dehkordi); effects of feeding different levels of low crude protein diets with different levels of amino acids supplementation on layer hen performance (M. Tenesa, T. C. Loh, H. L. Foo, A. A. Samsudin, R. Mohamad and A. R. Raha); characterisation of plant growth-promoting bacteria from Kacip Fatimah (Labisia pumila) under natural tropical forest (Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Qurban, A. P.); impact of daily supplement of probiotic on the production performance of Akar Putra chickens (Hasan, S. A. Jawad, Lokman, I. H., Saad, A. Naji, Zuki, A. B. Z. and Kassim, A. B.); the effects of fermented feed on broiler production and intestinal morphology (Saad A. Naji, I. F. B. Al-Zamili, Hasan, S. A. Jawad and J. K. M. Al-Gharawi); and characteristics and potential usage of dissolved silica in rice cultivation in Sumani watershed, Sumatra, Indonesia (Hiroaki Somura, Drmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga).

You will find the discussions intriguing, thought-provoking and useful in reaching new milestones in your own research. Please recommend this journal to your colleagues and students.

I would also like to express my gratitude to all the contributors, namely the authors, reviewers and editors, who have made this issue possible. Last but not least, we record our appreciation to the staff at the journal division for their editorial assistance.

The JTAS is currently accepting manuscripts for upcoming issues based on original qualitative or quantitative research that opens new areas of inquiry.

Chief Executive Editor Nayan Deep S. KANWAL, FRSA, ABIM, AMIS, Ph.D. [email protected] Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Review Article Major Components of Fish Immunity: A Review

M. Firdaus-Nawi and M. Zamri-Saad* Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

ABSTRACT Fish are fascinating creatures with a certain degree of immunity comparable to those of mammals. The fish’s immune system consists of two major components, innate and adaptive immunities. Innate immunity is non-specific and acts as the primary line of protection against pathogen invasion while adaptive immunity is more specific to a certain pathogen/ following adaptation. Innate immunity consists of the non-specific cellular and the non- specific humoral components. The non-specific cellular component consists of toll-like receptors (TLRs), macrophages, neutrophils, eosinophils and non-specific cytotoxic cell while the non-specific humoral component involves lysozyme, the complement, interferons, C-reactive proteins, transferrins and lectins. They work together at the initial stage to prevent pathogen invasion. On the other hand, the adaptive immune system consists of highly specilised systemic cells and processes that are separated into two main components: the humoral and cellular components. Three types of antibodies, the IgM, IgD and IgT, are the major constituents of the humoral immunity, which act on invaded extracellular pathogens. The cytotoxic T-lymphocyte cells are the major component of the cellular immunity that frequently kills virus-infected and intracellular bacterial or parasitic-infected cells. Both innate and adaptive immunities complement each other in the host’s attempt to prevent infection.

Keywords: Immunity, component, mechanism, fish

INTRODUCTION

ARTICLE INFO The fish is an organism that comes in Article history: various shapes, and the general picture of Received: 1 August 2016 Accepted: 2 September 2016 a fish is of a water creature with scales that E-mail addresses: coat the whole body and which has fins to [email protected] (M. Firdaus-Nawi), [email protected] (M. Zamri-Saad) swim. However, these are not always true * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press M. Firdaus-Nawi and M. Zamri-Saad since some species of fish lack scales such and react against any microbe that enters as clingfish, while others lack fins, such the host by mobilising cells and molecules as the . Furthermore, some fish like in the blood stream. Failure of the system the lungfish can survive for several hours to react leads to immunodeficiency while outside of water. Berra (2001) defined fish over-reacting against foreign microbes as a poikilothermic with gills as causes autoimmunity that can cause tissue the major respiratory organ. Scales and/or damage. Principally, the immune system is mucus protects the whole body. regulated by sophisticated and complicated Fish are classified in the paraphyletic mechanisms (Lydyard et al., 2000) and any group of creatures that comprises all gill- failure leads to infection, disease and death. bearing aquatic vertebrates lacking limbs This review describes the major components with digits. Thus, hagfish, lamprey and of the fish immune system as well as its cartilaginous and bony fish and the various working mechanism in protecting the fish extinct related groups are included in this from invasive pathogens. class. Generally, fish are cold-blooded or ectothermic i.e. its body temperature follows THE FISH IMMUNE SYSTEM the ambient temperature, and some giant Similar to mammals, the fish immune system fish such as tuna and white shark are able is built with two major parts i.e. the innate to hold a higher core temperature (Carey & and adaptive immunities. Innate immunity Lawson, 1973; Goldman, 1997). Fish live in reacts to invading pathogens by recognising various water bodies of the world and they the germ’s line-encoded molecules. TLRs have been discovered in almost all watery and phagocytosis are the key components of environments, from the streams of high innate immunity that protect the host against mountains, where gudgeon and char may be foreign invaders by recognising and finally found to the deepest oceans where anglerfish destroying the phagocytised cells (Silva et and gulpers live. Thus, fish demonstrate the al., 2002). Adaptive immunity, on the other greatest species diversity compared to other hand, recognises pathogens via molecules classes of vertebrates on earth. So far over that are generated by somatic mechanisms 32,000 species of fish have been identified (Medzhitov & Janeway, 1997) followed by (Fish Base, 2011). humoral and cellular responses via B- and The immune system is a vital T-lymphocytes (Dixon & Stet, 2001). physiological mechanism that prevents The immune organs vary with the type infection and preserves internal of fish (Zapata et al., 1996). Lamprey, homoeostasis. Therefore, the immune system hagfish and other jawless fish lack a true acts as a shield to the fish and provides lymphoid organ. They depend only on the protection from attacks by a broad spectrum lymphoid tissues that are found within of invading microorganisms. The system has other non-lymphoid organs. Thus, plasma various specialised organs designed to detect cells, macrophages and erythrocytes are

394 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review fabricated within the pronephros or anterior lymphatic system as mammals (Kutcler et kidney and certain parts of the gut, where al., 2006). maturation of the granulocytes occurs. A more advanced immune system is observed Innate Immunity in cartilaginous fish such as rays and sharks. The innate immune system is the first Plasma cells, lymphocytes and granulocytes layer of host defence against pathogenic are housed in Leydig’s organ, the spiral organisms or invaders. It responds in a non- valve of the intestine and the epigonal specific manner before the specific adaptive organs. These lymphoid organs are unique immune system is ready to take over the to the chondrichthyes and do not exist in defence work (Holland & Lambris, 2002). other types of fish. Thymus and spleen, the Unlike the adaptive immune system, the vital immune organs where lymphocytes, innate immune system identifies and reacts plasma cells and macrophages are stored to invaders in a general manner. Thus, the and mature are also found in cartilaginous protection provided is shorter and weaker fish. However, for paddlefish, sturgeon and than that provided by adaptive immunity bichir of the chondrostean sub-class, the (Alberts et al., 2002). major site for fabrication of granulocytes In fish, the innate immune response is is located within the meninges. Covering a crucial initial component in preventing the heart of the chondrostean is a tissue infection due to slow lymphocyte that comprises reticular cells, lymphocytes proliferation and a limited antibody and some macrophages. Nevertheless, the repertoire that leads to a delay in the macrophages, granulocytes, erythrocytes adaptive immune response (Magnadottir, and lymphocytes develop inside the 1998). Therefore, the innate immune kidney, an important hemopoietic organ of response acts as an alarm that allows the chondrostean. adaptive immune system time to mount a Similarly, the vital hemopoietic organ for response (Fearon & Locksley, 1996). bony fish or teleost is the kidney, specifically, The innate immunity is subdivided into the anterior kidney where various types of cellular and humoral immune responses. immune cells develop (Anderson, 1977). The cellular immune response provides a The teleost fish also have lymphoid organs physical barrier in the form of mucus and in the form of the spleen, thymus and the epithelial cells that line the skin, gills and scattered lymphoid cells found within the stomach, responsible for preventing invasion mucosal tissues such as the gonads, gut, of microorganisms into the body. If the skin and gills. Lymphocytes are the major pathogen passes these barriers, specialised immune cells in the thymus as well as cells like granulocytes, monocytes or neutrophils, erythrocytes and granulocytes macrophages and the non-specific cytotoxic (Chilmonczyk, 1992). Zebrafish are one of cells are ready to kill and digest the the teleost species reported to have a similar pathogens. Non-specific immune cells are

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 395 M. Firdaus-Nawi and M. Zamri-Saad recruited into the site of infection primarily innate immunity. Therefore, exposure to by inflammatory cytokines. The humoral a similar pathogen does not lead to better component of the innate immune system and quicker secondary immune response. employs a wide variety of proteins and However, the cells that are involved in the glycoproteins that are capable of destroying non-specific cellular immunity may interact or inhibiting the growth of microorganisms with the cells of the adaptive immunity (Aoki et al., 2008). system and can be recruited by them or their Non-specific cellular immunity.Many products (Secombes, 1996). types of leukocyte are involved in the Toll-like receptors (TLRs). Toll- innate, non-specific cellular immunity like receptors (TLRs) are one of the vital of fish. They include toll-like receptors components of innate immunity. They are (TLRs), granulocytes, macrophages and able to recognise the pathogen’s unique non-specific cytotoxic cells (NCCs). TLRs molecules. The word ‘toll’ originated from are the small protein molecules that have vernacular German and means fantastic ability to recognise the conserved molecules or super (Chtarbanova & Imbler, 2011). of microbes. Granulocytes and macrophages Nu¨sslein-Volhard first discovered TLRs in are mobile phagocytic cells found circulating the early 1980s following his mutagenesis in the blood and within the secondary studies of the fruit fly Drosophila lymphoid tissues. Both cells play vital roles melanogaster (Anderson et al., 1985). in inflammatory reaction, which actually is However, some years later in 1996 Lemaitre the cellular immune response to any invaders et al. (1996) discovered that this receptor or tissue injuries. Eosinophilic granular cells also played major roles in adult-fly immunity (EGCs) are less mobile granulocytes that as well as a key role in the mammal’s innate target parasites. EGCs are the host’s innate immune system (Chtarbanova & Imbler, cellular immune response against helminth 2011). infestation at the mucosal sites such as the The first report of TLRs in fish was by gut and gills. Similarly, protozoa and virus- Stafford et al. (2003) in goldfish,Carassius infected host cells are the targets for NCCs, auratus auratus, followed by Oshiumi et making them appear in mucosal sites, blood al. (2003) in pufferfish, Jault et al. (2004) circulation and lymphoid tissues. They are in zebrafish while Takano et al. (2011) able to spontaneously kill the affected cells identified 11 types of TLR homologues in through apoptotic and necrotic mechanisms Japanese flounder (Paralichthys olivaceus). (Secombes, 1996). As in mammals, TLRs of fish work by Innate immunity lacks specificity to recognising the unique conserved molecules the pathogen, thus making innate immunity of the microbes, known as pathogen- cells to mobilise quickly in large numbers. associated molecular patterns (PAMPs). Unlike the specific immune system, there This recognition stimulates an inflammatory is no memory component in non-specific response that initiates the innate immunity (Akira et al., 2006).

396 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

Macrophages. Macrophages are lymphoid organs, blood and peritoneal mononuclear, non-specific esterase positive cavity (Secombes, 1990). and peroxidase-negative leukocytes. They Granulocytes. Fish granulocytes have are avidly phagocytic and emit nitrogen- a distinctive structure and are sometimes free radicals and oxygen that kill various referred to as the polymorphonuclear (PMN) pathogens (Secombes, 1990). Macrophages leukocytes. Their cytoplasm contains have both complement and antibody (Fc) numerous fine granules that give rise to the receptors (Secombes & Fletcher, 1992) three types of granulocytes, the neutrophils, and express the class II MHC molecules basophils and eosinophils. The neutrophil (Secombes, 1994). Since they belong to is stainable by neutral dyes but has no cellular innate immunity, macrophage- affinity for acidic or basic dyes. It is the specific antibodies are not being made in most abundant granulocyte that migrates fish, although antibodies to a related cell from blood into the affected tissues to in the brain (the glial cells) exist (Dowding engulf bacteria. On the other hand, acid et al., 1991). Their actions usually rely on dyes like eosin are suitable for staining another immune component, the antibody the eosinophil that plays a primary role in (Thuvander et al., 1992). allergic inflammatory reactions as well as Production of macrophages in fish destruction of internal parasites. Finally, occurs during primary hematopoiesis. The basophil can be stained with basic dyes and resident populations are self-maintained is found only in low numbers (Secombes, with contribution from monocytes that are 1996). circulating in blood to mature as tissue The granulocytes are distributed macrophage (Hodgkinson et al., 2015). differently in the different parts of fish. Macrophages are commonly found in Fletcher (1986) concluded that many factors both layers of fish thymus, the cortex and influence the distribution of granulocytes in medulla. Three kinds of macrophages blood, tissues and other body fluids. These have been observed in the thymus of factors include season of the year, disease, the teleost, the melanomacrophages environmental pollutants and the various (Gorgollon, 1983; Pulsford et al., 1991), stressors. Lowe-Jinde (1986) and Steinhagen the monocytes (Castillo et al., 1990) and the et al. (1990) supported this and revealed an multinucleated giant cells (Pulsford et al., increased numbers of leukocytes, especially 1991). Macrophages are strongly positive the granulocytes following infection. Lamas for non-specific esterase, acid phosphatase and Ellis (1994) reported that the numbers and 5’-nucleotidase (Castillo et al., 1990). of granulocytes in the blood were greatly Other than in the thymus, macrophages increased within 24 hours of stressing the are also found scattered throughout the fish. As macrophages, the granulocytes can area in between the inner and outer zones also be isolated from the lymphoid tissues, of the pharyngeal epithelium and in the blood and the peritoneal cavity (Lamas & Ellis, 1994).

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 397 M. Firdaus-Nawi and M. Zamri-Saad

Non-specific cytotoxic cells. Non- Lysozymes are also broadly distributed in specific cytotoxic cells (NCC) of fish invertebrates, bacteriophages, microbes and are considered to be similar to that of plants (Jollès & Jollès, 1984). Lysozyme mammalian natural killer (NK) cells. They is a leukocyte-released enzyme and has share several similarities, particularly a broader activity in fish compared to in the competent lytic cycle, the target mammals (Demers & Bayne, 1997). It has cells for lysis, recognition of target cell an antibiotic effect and is normally used and the effecters to lyse the infectious as an indicator of non-specific immune microorganisms (Jaso-Friedmann et al., functions. 1993). However, there are also differences, Neutrophils are the major producers of which include the kinetics of killing and lysozyme in fish (Ellis, 2001) but monocytes the morphology and specificity of the target can also produce lysozyme (Fletcher & cells (Evans & Jaso-Friedmann, 1992). White, 1973). Therefore, fish lysozyme Studies on NCCs of teleosts found that is mainly distributed in the leukocyte- they tend to target various cells including rich organs, especially the head kidney tumor cells, virus-transformed cells and and at sites of antigenic invasion such as some protozoa (Whyte, 2007). NCCs are the gills, skin, gastro-intestinal tract and reported to be most active in the head of eggs (Murray & Fletcher 1976; Lie et al., kidney of teleosts but spleen and peripheral 1989). Lysozyme is also detected in the blood leukocytes (PBL) also demonstrate body mucus, peripheral blood and various the cytolytic abilities (Evans et al., 1984). tissues of both freshwater and marine In sharks, however, macrophages are the (Fletcher & Grant, 1968; Ebran et al., 2000; cells that are responsible for spontaneous Fagan et al., 2003). cytotoxicity (McKinkey et al., 1986). Lysozyme works by further disrupting Non-specific humoral immunity. the bacterial cell wall after an earlier Teleost fish have been shown to have disruption of the outer wall by the substances of non-specific humoral defence. complement and other enzymes (Yano, These substances include the lysozyme, 1996; Saurabh & Sahoo, 2008). Therefore, alkaline phosphatase, complement, fish lysozyme attacks the lipopolysaccharide interferon, C-reactive protein, transferrin, layer leading to damage of the outer cell lectin and several other substances. They are membrane, allowing additional lysozymes extremely important for fish (Ingram, 1980) to reach and injure deeper structures (Day and play significant roles in maintaining et al., 1978; Iacono et al., 1980), increasing homoeostasis (Saurabh & Sahoo, 2008). permeability that results in the loss of cell Lysozyme. Lysozyme involves in viability without lysis. Therefore, fish mediating defence against invasion by lysozyme has substantial antibacterial pathogens. It is one of the major substances activity over the mammalian lysozymes in the saliva, mucus and blood of vertebrate. against both Gram-positive and Gram-

398 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review negative bacteria (Itami et al., 1992). important in the defence mechanism of fish Furthermore, lysozyme plays an important (Ellis, 2001; Holland & Lambris, 2002). role in preventing vertical transmission of The classic pathway is more common in some bacterial pathogens of fish (Yousif et mammals, involving the formation of a al., 1994). complex blend of antigen and antibody Alkaline phosphatase. Alongside with (Gasque, 2004). This pathway is activated lysozyme, alkaline phosphatase (AP) is also by the binding of the Fc portion of the IgG an important enzyme in fish, especially in to the C1q component of the C1 complex their innate immune system. It is a lysosomal (Muller-Eberhard, 1986; Kishore & Reid, enzyme and can be found in various body 2000; Pangburn & Rawal, 2002). The lectin secretions such as body mucus, intestinal pathway requires interaction between lectins mucus and blood serum (Nigam et al., of the complement with sugar moieties 2012). Concentration of AP increases when found on the surface of microbes (Turner, the host in stress making it as a potential 2003; Fujita et al., 2004), activating lectin- stress indicator (Ross et al., 2000). Fast et associated enzymes, the MBL-associated al. (2002) in their study of Atlantic salmon serine proteases (MASPs) that enhance the found that the activity of mucus AP increases complement activation (Chen & Wallis, following parasitic infections and suggested 2004). Microbes that are fixed with the that AP is one of the important enzymes in complement are readily phagocytosed and the innate immune system. Another study on lysed by the macrophages or the cytotoxic indicates high activity of AP during cells. skin regeneration due to wound healing, Interferons. Interferons (IFNs) are demonstrating the role of AP as a protective potent cytokines that act as key effectors of enzyme (Rai & Mittal, 1983). antiviral activity in the vertebrates (Castro The complement. The complement et al., 2008). They are secreted proteins or is one of the major mechanisms of the glycoproteins that induce antiviral capability humoral component of the immune system. in cells and defends against virus infection It is involved in both initiation of the by inhibiting viral replication (Yano, 1996; innate immune response and mounting of Samuel, 2001). IFN-like activity was first an adaptive immune response (Alvarez- detected in fish in 1965 and has since been Pellitero, 2008; Nakao et al., 2011) using detected in cells and organs of many fish its more than 35 soluble proteins (Sunyer species infected with virus (Robertsen, & Lambris, 1999; Gasque, 2004). The 2006). The first IFN gene of fish was complement works via a combination of cloned in 2003 (Robertsen et al., 2003). three pathways: the alternative, the lectin Nevertheless, IFNs production has been and the classic pathways. The alternative confirmed in bony but not cartilaginous fish pathway is active in the serum of fish than (Yano, 1996). in that of mammals (Yano, 1996), and is

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 399 M. Firdaus-Nawi and M. Zamri-Saad

It is now established that fish cells rainbow trout (Winkelhake & Chang, 1982; secrete IFN-α and IFN-β molecules in Murai et al., 1990), lumpsucker, Cyclopterus response to virus infection (Kelly & Loh, lumpus (Fletcher & Baldo, 1976; White 1973; Rio et al., 1973; Okamoto et al., et al., 1978), tilapia, Tilapia mossambica 1983; Snegaroff, 1993). Type I IFNs are (Ramos & Smith, 1978) and murrel fish involved in the first line of defence against (Mitra & Bhattacharya, 1992). However, virus infection (Robertsen, 2006). They there are fish species that lack CRP such as have five exon and four intron genes that flounder, Platichthys flesus while bacterial are not found in the classic type I IFNs of endotoxin (LPS) was found to be able to birds and mammals (Lutfalla et al., 2003; stimulate the production of CRP following Robertsen et al., 2003). Now, fish type I exposure to fish (White et al., 1981; White IFNs has been shown to have the same exon/ & Fletcher, 1985). intron structure as the IL-10 and IFN-λ gene Transferrin. Transferrin (Tf) is a multi- families (Lutfalla et al., 2003). functional protein or bi-lobed monomeric C-reactive protein. C-reactive protein iron-binding glycoprotein actively involved (CRP) is the first protein to exist in the in iron metabolism that is associated blood plasma of humans and most animals with innate immune response (Garcia- as a response to tissue damage, infection Fernandez et al., 2011). The primary role and inflammation. It was first found of Tf is transporting iron in a safe state reacted with the C-polysaccharide (CPS) from absorption, utilisation or storage sites of Pneumococcus bacterium in the serum of around the body (Gomme & McCann, 2005). patients with acute inflammation, and was Although iron is a vital element for growth thus named C-reactive protein. and survival, excess free iron is toxic to the The liver, in response to factors released cells (Kohgo et al., 2008). Therefore, tight by fat cells, synthesises CRP. It is a member regulation of iron metabolism maintains a of the pentraxin family of proteins (Pepys & balance between beneficial and toxic effects Hirschfield, 2003) and was the first pattern and this is accomplished by the interactions recognition receptor (PRR) to be identified of several genes, such as the iron transporter (Mantovani et al., 2008). Since the first transferrin, that are also involved in the discovery in 1930, CRP has been found in response to infection (Neves et al., 2009). many animal species, horseshoe crab and Transferrin is synthesised in the liver mollusk, Achatina fulica (Yano, 1996). and secreted into the blood but also found in Baldo and Fletcher (1973) reported CRP the brain and central nervous system, testes, that binds to pneumococcal CPS in plaice ovary, spleen, mammary gland and the serum. CRP has also been isolated from kidney (Lambert et al., 2005). Transferrin the smooth dogfish,Mustelus canis (Robey contributes to the immune system through & Liu, 1983), Japanese eel (Nunomora, binding to iron, creating a low iron 1991), channel catfish (Szalai et al., 1994), environment where few microorganisms

400 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review can survive and the infectivity of pathogenic Ingram (1980), Ellis (1981) and Fletcher microorganisms becomes limited (Suzumoto (1982) found many antipathogenic materials et al., 1977; Chen et al., 2009; Jurecka et al., in the fish mucus, including lectins. Lectins 2009a, b). have also been isolated from the skin mucus Tf has been detected in almost all fish of scaleless hagfish, freshwater eel, moray species (Yano, 1996), including the Pacific eel, , sea catfish, ayu, cusk eel, dragonet hagfish (Aisen et al., 1972) and the lamprey and flounders (Yano, 1996), suggesting that (Boffa et al., 1967; Macey et al., 1982). For lectin is produced by club cells (Al-Hassan cartilaginous fishes, Tf has been detected in et al., 1986). Furthermore, lectin was also the cat shark, Scyllium stellare (Got et al., isolated from the eggs of many species of 1967) and the lemon shark (Clem & Small, fish such as lamprey, herring, carp, loach, 1967). In bony fish, Tf has been detected Japanese catfish, smelts, ayu, salmonid in more than 100 species of fish (Turner & fishes, sea bass, perch, porgy and flounder Jamieson, 1987; Jamieson, 1990). (Yano, 1996). Lectins. Lectins are primordial There are many studies on the function molecules that have multiple functions. of fish lectins. Kamiya and Shimizu They have existed in fish and other animals (1980) reported the ability of lectins from for decades and were initially identified as windowpane flounder skin mucus to hemagglutinins (Russell & Lumsden, 2005) agglutinate marine yeast, Metschnikowia as they bind carbohydrate and agglutinate reukafii. Kamiya et al. (1990) revealed the cells (Ewart et al., 2001). Lectins comprise same ability of conger eel skin mucus lectins at least two sugar-binding sites but the to agglutinate Vibrio anguillarum. Blue monosaccharide or glycosaccharide that lectins were reported to agglutinate inhibits lectin-induced agglutination or fish pathogen Aeromonas hydrophila and precipitation provides lectins’ specificity at low concentrations (<1 ng/ml) promoted (Goldstein et al., 1980). Lectins have been phagocytosis of the same bacterium (Fock divided into several types, which include et al., 2001). A mannan-binding lectin in the the C- and S-type lectins (Yano, 1996). The plasma of the Atlantic salmon was showed C-type is calcium-dependent. to bind to fish pathogensVibrio anguillarum A number of lectins have been reported and Aeromonas salmonicida in a calcium- in fish, but most have been characterised dependent manner (Ewart et al., 1999) and to only in terms of agglutination activity increase phagocytosis and killing following and carbohydrate specificity (Ewart et al., incubation with A. salmonicida (Ottinger 2001). In fish, C-type lectins, galectins et al., 1999). Voss et al. (1978) reported and pentraxins have been identified from Chinook salmon egg lectins inhibited the the earliest jawed vertebrate (sharks) to growth of pathogenic bacteria such as Vibrio the more advanced teleost species such as anguillarum, Yersina ruckeri, Aeromonas salmon and carp (Vasta et al., 2004). hydrophila and Edwardsiella tarda. Fish

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 401 M. Firdaus-Nawi and M. Zamri-Saad egg lectins were suggested to provide antigen receptors that are uniquely expressed some protection to the developing egg and on each individual lymphocyte. This gene re- to prevent the transmission of pathogenic arrangement leads to an irreversible change organisms from mothers to their offspring. in the DNA of each cell and all progenies of that cell inherit the genes that encode the Adaptive Immunity same receptor specificity, including Memory Adaptive immunity or specific immune B and Memory T cells, which are the key to system is the third line of the immune long-lived specific immunity. system that invaders face after surviving the physical barrier and the innate immunity. Humoral Immunity The adaptive immune system is composed Humoral immunity refers to antibody of highly specialised, systemic cells secretion and the accessory processes and processes that eliminate or prevent that accompany it. These include the pathogenic growth. The term adaptive Th2 activation and cytokine production, refers to the differentiation of specific from germinal centre formation and isotype non-specific and the tailoring of response switching, affinity maturation and memory to a particular foreign invader. Adaptive cell generation. The humoral immunity immunity is activated by the non-specific involves substances found in the humours or innate immunity (Rubio-Godog, 2010). or body fluids, which include pathogen and Adaptive immunity consists of two toxin neutralisation, complement activation, major components: the antibodies and opsonin promotion of phagocytosis and lymphocytes, or often called the humoral pathogen elimination (Janeway, 2001). and the cell-mediated immune response, Thus, humoral immune response is one of respectively (Uribe et al., 2011). Cells of the branches of adaptive immunity that are the adaptive immunity are the lymphocytes, mediated by secreted antibodies produced both B and T cells. The B cells, derived by B lymphocyte lineage or the B cells. The from the bone marrow, become the cells B cells transform into plasma cells, which that produce antibodies. The T cells, which produce and secrete antibodies. The CD4+ mature in the thymus, differentiate into T-helper cells provide co-stimulation that cells that either participate in lymphocyte aids this entire process, allowing the secreted maturation or kill virus-infected cells. A key antibodies to bind to the antigens located on feature of adaptive immunity is ‘memory’, the surface of the invading microorganisms which differentiates it from innate immunity. and send them for destruction (Pier et al., Adaptive immunity is highly adaptable 2004). due to the mechanisms of somatic Humoral immunity in fish is variable hypermutation and V(D)J recombination. and quite different from other animals. It These mechanisms allow a small number of depends on the external conditions and genes to generate a huge number of different the species of fish (Lukjanenko, 1971).

402 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

However, the fish humoral immune response of fish IgM becomes extremely important does share several basic characteristics for effective prevention and control of with that of mammals. These include the various fish diseases (Magnadottir, 1998). basic immunoglobulin (Ig) structure, the IgM is tetrametric in teleost (Acton et al., cellular requirement for stimulation of 1971) but pentametric in higher vertebrates antibodies and the functions of antibodies and cartilaginous fish (Kobayashi et al., in neutralisation, complement fixation and 1984). Since isotypic repertoire of Ig is opsonisation of antigen. limited in fish (Kaattari et al., 1998), the Antibodies. Antibodies, also known degree of similarity between mucus and as immunoglobulins (Ig), are the primary serum derived Ig is unknown. However, humoral component of the adaptive immune there are reports that monoclonal antibodies system (Magnadottir et al., 2005). The Ig developed against serum of carp, Cyprinus molecule has a dual functions i.e. as antigen carpio L do not react with the mucus Ig receptor on the surface of B-cells and as (Rombout et al., 1993a) due likely to the an antibody secreted into blood and other existence of varied redox forms of Ig body fluids. Thus, there are two forms of in some teleost species (Kaattari et al., H-chains in the immunoglobulins, one 1998) or perhaps the existence of different with a hydrophobic C-terminal peptide glycosylation patterns (Kenneth et al., that can bind to a cell membrane and the 2000). Kenneth et al. (2000) revealed the other with a hydrophilic N-terminal region different protein band patterns between the that is secreted. The same gene encodes mucus and the serum Ig where the mucus Ig the two forms and processing of the pre- possesses four primary bands, the 72, 68, 43 mRNA determines which form should be and 28 kDa, while the serum Ig possesses synthesised. The N-terminal on both H and two primary bands; the 72 and 28 kDa. L chains is called the variable (V) domain Recently, scientists have discovered and is the structure of the antibody that binds the existence of IgD and IgT isotypes in to the antigen (Pilstrom & Bengten, 1996). teleost but not as abundant as the IgM The most prevalent immunoglobulin in (Tian et al. 2009). Wilson et al. (1997) first the serum of teleosts is the IgM tetramer with discovered IgD that was homologous with eight antigen-combining sites. It has been the mammalian IgD. Then, Hanzen et al. detected in many species of fish including (2005) discovered IgT, sometimes referred chondrichthyean and osteichthyean. It to as IgZ, in rainbow trout while Danilova consists of 70 kDa heavy chain and 22- et al. (2005) reported the occurrence in 25 kDa light chains (Tort et al., 2003). In zebrafish. However, unlike IgM, the roles of general, fish Igs are of lower affinity and these new Igs are still obscure. Nevertheless, diversity than those of mammals and birds IgD might be involved in innate immunity (Du Pasquier, 1982). Therefore, better as Edholm et al. (2010) found that the understanding of the structure and function IgD secreted by channel catfish lacked the

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 403 M. Firdaus-Nawi and M. Zamri-Saad antigen-specific V domain and could bind to immune response. The primary functions basophils to stimulate the pro-inflammatory of B cells are to produce antibodies against cytokines. According to Zhang et al. (2010), antigens, to perform the role of antigen- IgT might involve in the interactions presenting cells (APCs) and finally, to between the host intestinal mucosa and the develop into memory B cells after activation microflora. by antigenic interaction. The head of kidney Immunoglobulins of fish are found in (HK) or pronephros is the source of B cells the skin mucus, gut, gill mucus, bile and in teleost fish, making HK the primary systemically in the blood plasma (Morrison lymphoid tissue (Zapata et al., 2006). The & Nowak, 2002). The presence of Ig on the spleen is considered secondary lymphoid skin and gill surface is important since these tissue in which plenty of B cells are found in organs are consistently exposed to a wider teleost fish. Bromage et al. (2004) revealed natural environment. The systemic and that the spleen is a site for B cell activation, mucosal immune responses are autonomous plasmablast formation and differentiation because specific antibodies against certain into plasma cells. Plasma cells then migrate antigens can be elicited from the skin, gills to the HK, which explains the presence and gut. However, intravenous injection of few Ig-secreting cells in the spleen of antigen stimulates little activity in the compared to HK. mucus (Lobb & Clem, 1981), indicating Other than the lymphoid tissues, B cells that the mucosal Ig is exclusive from the are also found in various organs and tissues systemic plasma cells. Grabowski et al. including the intestine, skin and gills. In (2004), on the other hand, showed the the intestine, the distribution of B cells is stimulation of the mucus antibody response low and variable among different species of following intraperitonium vaccination with fish. Studies in sea bass, carp and rainbow sonicated formalin killed Flavobacterium trout demonstrated between 2% and 12% columnare. Similarly, Firdaus-Nawi et al. of the leukocytes in the intestine were (2011) demonstrated increasing mucus IgM-positive, mainly in the lamina propria antibody following oral vaccination with of both anterior and posterior intestines. killed Streptococcus agalactiae. This pattern However, a small number of these cells were was also observed with Flavobacterium also detected in the epithelium (Salinas et psychrophilum (LaFrentz et al., 2002), al., 2011). B cells are also detectable in the indicating that the systemic antibodies may skin of cartilaginous and teleost fish (Wolfle disseminate to mucosal sites from blood et al., 2009) and in the epithelium of carp circulation (Di Conza & Halliday, 1971; skin (Rombout et al., 1993b). Another study St. Louis-Cormier et al., 1984; Cain et al., in rainbow trout revealed the large numbers 2000). of B cells in the basement membrane area B cells. B cells are a type of lymphocyte followed by the epithelial layer and the cells that plays an important role in the humoral in the dermis or sub-epidermal layer (St.

404 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

Louis-Cormier et al., 1984). Furthermore, immunisation with sheep red blood cells Zhao et al. (2008) reported that the skin (Pontius & Ambrosius, 1972). Fletcher and of channel catfish contains B cells and White (1973) reported increased antibody antibody secreting cells (ASC), which most titers within the intestinal mucus of plaice likely serve as the major source of mucosal upon oral immunisation with heat-killed antibody. On the other hand, Grove et al. Vibrio anguillarum. Similarly, Firdaus- (2006) reported a large number of IgM- Nawi et al. (2011) demonstrated increased positive cells in the stratified epithelium of antibody titers in the intestinal mucus of red the gill arch and filaments of Atlantic halibut tilapia following oral immunisation against fish, while Grøntvedt and Espelid (2003) Streptoccoccus agalactiae. reported an abundance of B cells in primary Mucosal immunity also gives protection gill lamellae and filaments along the blood against parasitic infestation. A study by Sitja- vessels of spotted wolfish. Bobadilla et al. (2006) using co-habitation challenge of turbot with Enteromyxmum Mucosal Immunity scophthalmi resulted in leukocyte infiltration Mucosal immunity is vital because it is in the intestine. The infiltration consisted the first line of adaptive humoral defense of lymphocytes but no specific IgM was that effectively blocks or neutralises the detected in the serum. On the other hand, pathogen. However, fish lack secretory Zhang et al. (2010) demonstrated the IgA, Peyer’s patches and tonsils that play unchanged numbers of IgM-positive cells important role in mammalian mucosal in the gut of surviving trout that were immunity (Kaattari & Piganelli, 1996). naturally infected with parasite Ceratomyxa Instead, massive intraepithelial lymphocytic shasta. However, parasite-specific IgM were aggregations are observed in the central detected in the serum. This suggests that region of the spiral intestine of elasmobranch different fish species respond differently to (Tomonaga et al., 1986). They are believed different parasites. to play a similar role as the Peyer’s patch of mammals. Furthermore, minor subepithelial Cellular Immunity lymphoid accumulations were reported in Cellular immunity, also referred to as cell- the intestine of roach and perch (Zapata mediated immunity, is a specific immune & Solas, 1979; Rombout & van den Berg, response that involves macrophages, natural 1989). A recent study by Firdaus-Nawi et killer cells (NK), mast cells, basophils, al. (2011) demonstrated aggregations of eosinophils and neutrophils (Broere et al., lymphoid cells in the lamina propria of red 2011). Various cytokines are released in tilapia following oral immunisation against response to the antigen. Cellular immunity Streptoccoccus agalactiae. Antibody- protects the body by activating the antigen- secreting cells (ASCs) were observed in specific cytotoxic T-lymphocytes that induce the lamina propria of perch following apoptosis of cells that display epitopes of

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 405 M. Firdaus-Nawi and M. Zamri-Saad foreign antigen on their surface. These as well as the adaptive immune system include virus-infected cells, intracellular (Nakanishi et al., 2015). The presence of an bacteria-infected cells and cancerous antigen-specific receptor or T-cell receptor cells. Cell-mediated immunity is directed (TCR) on the cell surface distinguishes primarily at the pathogen that survived them from other lymphocytes (Manning & phagocytosis and the pathogens that infect Nakanishi, 1996). They are called T cells non-phagocytic cells. It is most effective because they mature in the thymus. They are in removing virus-infected cells, but also also sometimes called thymocytes. participates in protection against fungi, There are several types of T cell, which protozoans, cancer cell and intracellular include T helper cells (TH cell), cytotoxic T bacteria (Kerry & Hansen, 2011). cells (CTL), memory T cells, regulatory T Cellular immunity also plays a major cells (Treg cell) and gamma delta T cells. role in transplant rejection. Graft-versus- Naive CD4+ T cells can differentiate into host reaction (GVHR) is a representative the five types of effector T cells (Th1, phenomenon of cell-mediated immunity Th2, Th17, Th9 and Th22), three subsets involving CD4 and CD8 T-lymphocytes. of regulatory T cells (Treg, Th3, Tr-1) and Nakanishi and Ototake (1999) employed memory T cells (Annunziato & Romagnani, a model system of clonal triploid ginbuna 2009; Wan & Flavell, 2009). Thus, these and tetraploid ginbuna-goldfish, Carassius CD4+ T cells play vital roles in regulation of auratus hybrids to demonstrate the presence the immune system, immune pathogenesis of GVHR in a teleost fish. The sensitised and host defense mechanism. According triploid cells were injected into tetraploid to Zhu and Paul (2010), CD4+ T cells are recipients and a typical GVHR was induced characterised by their plasticity in addition that led to the death of the recipients within to heterogeneity. Fischer et al. (2006) one month. Post-mortem conducted during detected T-cell-related genes such as TCR, the course of the clinically apparent graft- CD3, CD4 and CD8 as well as MHC class versus-host disease (GVHD) showed several I and class II genes in several fish species. pathological changes including enlargement Additionally, mRNA expression of T of the spleen, infiltration of mononuclear cell surface marker genes in alloantigen cells and focal necrosis particularly in the or virus-specific effector cells has been skin, liver and lymphoid tissues. Most reported in several fish such as TCRβ and features of acute GVHR are similar to those CD8α in ginbuna (Somamoto et al., 2006) found in mammals and birds, providing and rainbow trout (Fischer et al., 2013) and evidence for the presence of allo-reactive TCR in channel catfish (Stuge et al., 2000). cytotoxic T cells in teleosts (Manning & This suggests the presence of CD4+ helper Nakanishi, 1996). T cells and CD8+ CTL in fish similar to their T cells. T cells are lymphocytes that presence in higher vertebrates (Fischer et play a vital role in cell-mediated immunity al., 2013).

406 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

Overall Working Mechanisms of Fish are displayed by macrophages on the Class Immune System II MHC (MHC II). This turns macrophages In an attempt to prevent establishment into antigen presenting cells (APC) and of infection, both innate and adaptive activates the adaptive immunity where immunities work in complement. Upon helper T-cells (Th) attract and bind to exposure to pathogenic organism, the innate the MHC II of APC by T-cell Receptors immunity is activated to prevent the invasion (TCR). Formation of APC-Th complex through TLRs that recognise the pathogen- releases signalling molecules known as associated molecular patterns (PAMPs). The cytokines by the activated Th that triggers first hurdle faced by the microorganism is the proliferation and maturation of B cells the efficient physical barrier in the form of as well as other immune mechanisms. mucus and epithelial cells of the mucosal Maturation of B cell leads to formation organs such as the intestines, gills and skin of two types of cell, the plasma cells that (Figure 1). Should the pathogen successfully produce specific antibody and the memory pass these barriers, invasion starts. B cells that remember the specific antigen Successful invasion by pathogens for certain periods of time (Kum & Sekkin, stimulates two major mechanisms i.e. the 2011) (Figure 3). The released antibodies innate cellular and humoral immunities, act to disable the pathogen through the and the specific adaptive immunity. Innate mechanism of opsonisation or neutralisation immunity involves granulocytes, phagocytes before the disabled pathogen is destroyed and the non-specific cytotoxic cells that by the complement system and cleared by are employed by inflammatory cytokines macrophages via phagocytosis. to kill and digest the invading pathogens Intracellular antigens (Ag) such as through a process known as phagocytosis virus undergo another effective mechanism (Secombes & Fletcher, 1992). At the same of elimination by the body. The pathogen time, the humoral component of the innate is phagocytised and processed before immune system employs a wide variety being displayed on Class I MHC (MHC of proteins and glycoproteins described I) of APC, which attracts the CD8+ cells earlier that are capable, either alone or to bind to the MHC I of APC via CD8+ in collaboration with the cellular innate receptors. Then the activated CD8+ cells immunity, of destroying or inhibiting the start to clone themselves into two types of growth of the microorganisms (Aoki et al., cell, the memory T cell and the Cytotoxic 2008). The phagocytosis process especially T-Lymphocyte (CTL) cell that destroy the by residence macrophages results in full virus-infected cells using various enzymes elimination of an invading pathogen (Figure and cell apoptosis (Figure 4). 2). Subsequent exposure to the same After the degradation process, selected antigen predisposes the pathogen to the small protein fragments from the pathogen same physical barrier of the mucosal organs.

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 407 M. Firdaus-Nawi and M. Zamri-Saad

Figure 1. Intestine and gills of the fish are coated with mucus layer produced by goblet cells (GC) as primary innate protection, followed by the layer of epithelial cells (EC) (A & B). Similarly, the mucus also covers the skin of the fish and the epidermal layer is made up of epithelial cells (C) that provide both a physical and chemical barrier against invading pathogens.

Figure 2. Process of phagocytosis by macrophages starting with attachment of pathogen such as bacterium followed by ingestion before it is killed and lysed into small fragments by the enzyme lysosome. Then the degradation process takes over.

408 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

Figure 3. Mechanism of B cell activation and maturation by helper T-cell (Th) resulted in formation of specific antibody secreting plasma cells and memory B cells that have the ability to remember the infection.

Figure 4. Elimination of virus-infected cell by the Cytotoxic T-Lymphocyte (CTL). This mechanism does not involve antibody production and is also known as Cell-Mediated Immunity (CMI).

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 409 M. Firdaus-Nawi and M. Zamri-Saad

At the same time, the Memory B and T cells improve the health and disease protection located in the mucosal layer of the exposed of fish. fish stimulate production and release of specific antibodies and/or cytotoxic cells ACKNOWLEDGEMENTS against the pathogen onto the mucosal The authors wish to thank Dr. Annas Salleh surface to prevent adhesion and invasion for the technical assistance. of the pathogen. This forms the basis for mucosal immunity. REFERENCES Inability of the mucosal layer to Acton, R. T., Weinheimer, P. F., Hall, S. J., efficiently prevent invasion leads to a second Niedermaier, W., Shelton, E., & Bennett, J. C. invasion of the pathogen into the host. As (1971). Tetrameric immune macroglobulins described earlier, this invasion stimulates in three orders of bony fishes. Proceedings of the non-specific cellular and humoral the National Academy of Sciences USA, 68(1), innate immunities to kill and remove the 107–111. invading pathogen. Simultaneously, this Al-Hassan, J. M., Thomson, M., Summers, B., & second invasion activates processing of the Criddle, R. S. (1986). Purification and properties pathogen by phagocytes to be presented of a hemagglutination factor from Arabian Gulf catfish Arius( thalassinus) epidermal secretion. to the adaptive immune system for the Comparative Biochemistry and Physiology Part Memory B cells to enhance production and B, 85(1), 31–39. release of antibodies specific to the pathogen Aisen, P., Leibman, A., & Sia, C. L. (1972). Molecular (humoral immunity) or the Memory T cells weight and subunit structure of hagfish to enhance the cytotoxic T-lymphocytes transferrin. Biochemistry, 11(18), 3461–3464. (cell-mediated immunity), depending on Akira, S., Uematsu, S., & Takeuchi, O. (2006). the type of invading pathogen. These form Pathogen recognition and innate immunity. Cell, the basis for vaccination against diseases. 124(4), 783–801.

Alberts, B., Johnson, A., Walter, P., Raff, M., Roberts, CONCLUSION K., & Lewis, J. (2002). Molecular biology of the The immune system of fish is not as complex cell. Taylor & Francis. as that of mammals because of the absence of Alvarez-Pellitero, P. (2008). Fish immunity and several components. However, the immune parasite infections: From innate immunity to system of fish is adequate in providing immunoprophylactic prospects. Veterinary protection against antagonistic pathogens Immunology and Immunopathology, 126(3), from the surroundings. Recent findings in 171–198. fish immunology and its components help Anderson, D. P. (1977). Fish immunology. S. F. to provide a better understanding of fish Snieszko & H. R. Axelrod (Eds.). Hong Kong: immunology as well as to pave the way for TFH Publications, Inc. Ltd. further research. This will help in efforts to

410 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

Anderson, K. V., Bokla, L., & Nusslein-Volhard, C. Castillo, A., Razquin, B. E., Lopez-Fierro, P., Alvarez, (1985). Establishment of dorsalventral polarity in F., Zapata, A. G., & Villena, A. J. (1990). Enzyme the Drosophila embryo: The induction of polarity and immunohistochemical study of the thymic by the toll gene product. Cell, 42(3), 791–798. stroma in the rainbow trout, Salmo gairdneri, Richardson. Thymus, 15(3), 159–173. Annunziato, F., & Romagnani, S. (2009). Heterogeneity of human effector CD4+ T cells. Castro, R., Martin, S. A. M., Bird, S., Lamas, J., Arthritis Research and Therapy, 11(6), 257. & Secombes, C. J. (2008). Characterization of γ-interferon responsive promoters in fish. Aoki, T., Takano, T., Santos, M. D., Kondo, H., & Molecular Immunology, 45(12), 3454–3462. Hirono, I. (2008). Molecular innate immunity in teleost fish: review and future perspectives. In Chtarbanova, S., & Imler, J. L. (2011). Microbial Fisheries for Global Welfare and Environment, sensing by toll receptors: A historical perspective. Memorial Book of the 5th World Fisheries Arteriosclerosis, Thrombosis, and Vascular Congress (pp. 263-276). Terrapub: Tokyo, Japan. Biology, 31(8), 1734–1738.

Baldo, E. A., & Fletcher, T. C. (1973). C-reactive Chen, C. B., & Wallis, R. (2004). Two mechanisms protein-like precipitins in plaice. Nature, for mannose-binding protein modulation of the 246(5429), 145–146. activity of its associated serine proteases. Journal of Biological Chemistry, 279(25), 26058–26065. Berra, T. M. (2001). Freshwater fish distribution. San Diego, California: Academic Press. (pp. 604). Chen, J., Wang, J., Meyers, K. R., & Enns, C. A. (2009). Transferrin-directed internalization and Boffa, G. A., Fine, J. M., Drilhon, A., & Amouch, cycling of transferrin receptor 2. Traffic, 10(10), P. (1967). Immunoglobulins and transferrin in 1488–1501. marine lamprey sera. Nature, 214, 700–702. Chilmonczyk, S. (1992). The thymus in fish: Broere, F., Apasov, S. G., Sitkovsky, M. V., & Development and possible function in the van Eden, W. (2011). T-cell subsets and T immune response. Annual Review of Fish cell-mediated immunity. In Principles of Diseases, 2, 181–200. immunopharmacology. ISBN: 978-3-0346- 0135-1. Danilova, N., Bussmann, J., Jekosch, K., & Steiner, L. A. (2005). The immunoglobulin heavy- Bromage, E. S., Kaattari, I. M., Zwollo, P., & chain locus in zebrafish: Identification and Kaattari, S. L. (2004). Plasmablast and plasma expression of a previously unknown isotype, cell production and distribution in trout immune immunoglobulin Z. Nature Immunology, 6(3), tissues. Immunology, 173(12), 7317–7323. 295–302. Cain, K. D., Jones, D. R., & Raison, R. L. Day, D. F., Marceau-Day, M. L., & Ingram, J. M. (2000). Characterization of mucosal and (1978). Protein-lipopolysaccharide interactions systemic immune responses in rainbow trout I. The reaction of lysozyme with Pseudomonas (Oncorhynchus mykiss) using surface plasmon aeruginosa LPS. Canadian Journal of resonance. Fish and Shellfish Immunology, 10(8), Microbiology, 24(2), 196–199. 651–666. Demers, N. E., & Bayne, C. J. (1997). The immediate Carey, F. G., & Lawson, K. D. (1973). Temperature effects of stress on hormones and plasma regulation in free-swimming bluefin tuna. lysozyme in rainbow trout. Developmental and Journal of Comparative Biochemistry and Comparative Immunology, 21(4), 363–373. Physiology Part A: Physiology, 44(2), 375–392.

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 411 M. Firdaus-Nawi and M. Zamri-Saad

Di Conza, J. J., & Halliday W. J. (1971). Relationship Evans, D. L., & Jaso-Friedmann, L. (1992). of catfish serum antibodies to immunoglobulin Nonspesific cytotoxic cells as effectors of in mucus secretions. Australian Journal of immunity in fish. Annual Review of Fish Disease, Experimental Biology and Medical Science, 2, 109–121. 49(5), 517–519. Ewart, K. V., Johnson, S. C., & Ross, N. W. (1999). Dixon, B., & Stet, R. J. (2001). The relationship Identification of a pathogen-binding lectin in between major histocompatibility receptors and salmon serum. Comparative Biochemistry and innate immunity in teleost fish. Developmental Physiology, Part C, 123(1), 9–15. and Comparative Immunology, 25(8), 683–699. Ewart, K. V., Johnson, S. C., & Ross, N. W. (2001). Dowding, A. J., Maggs, A., & Scholes, J. (1991). Lectins of the innate immune system and their Diversity amongst the microglia in growing and relevance to fish health.ICES Journal of Marine regenerating fish CNS: Immunohistochemical Science, 58(2), 380–385. characterization using FL. 1, an anti-macrophage Fagan, M. S., O’Byrne-Ring, N., Ryan, R., Cotter, monoclonal antibody. Glia, 4(4), 345–364. D., Whelan, K., & Mac Evilly, U. (2003). A Du Pasquier, L. (1982). Antibody diversity in lower biochemical study of mucus lysozyme, proteins vertebrates – Why is it so restricted? Nature, and plasma thyroxine of Atlantic salmon (Salmo 296, 211–213. salar) during smoltification. Aquaculture, 222(1), 287–300. Ebran, N., Julien, S., Orange, N., Auperin, B., & Molle, G. (2000). Isolation and characterization of novel Fast, M. D., Sims, D. E., Burka, J. F., Mustafa, A., glycoproteins from fish epidermal mucus: & Ross, N. W. (2002). Skin morphology and Correlation between pore-forming properties humoral non-specific defence parameters of and their antibacterial activities. Biochimica et mucus and plasma in rainbow trout, coho and Biophysica Acta, 1467(2), 271–280. Atlantic salmon. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Edholm, E. S., Bengten, E., Stafford, J. L., Sahoo, M., Physiology, 132(3), 645–657. Taylor, E. B., Miller, W. N., & Wilson, M. (2010). Identification of two IgD+ B cell populations in Fearon, D. T., & Locksley, R. M. (1996). The channel catfish, Ictalurus punctatus. Journal of instructive role of innate immunity in the Immunology, 185(7), 4082–4094. acquired immune response. Science, 272(5258), 50−54. Ellis, A. E. (1981). Stress and the modulation of defence mechanisms in fish. In A. D. Pickering Firdaus-Nawi, M., Noraini, O., Sabri, M. Y., Siti- (Ed.), Stress and fish (pp.147–169). London: Zahrah, A., Zamri-Saad, M., & Latifah, H. Academic Press. (2011). The effects of oral vaccination of Streptococcus agalactiae on stimulating gut- Ellis, A. E. (2001). Innate host defence mechanism of associated lymphoid tissues (galts) in tilapia fish against viruses and bacteria.Developmental (Oreochromis spp.). Pertanika Journal of and Comparative Immunology, 25(8), 827–839. Tropical Agricultural Science, 34(1), 137–143. Evans, D. L., Carlson, R. L., Graves, S. S., & Hogan, Fischer, U., Koppang, E. O., & Nakanishi, T. (2013). K. T. (1984). Nonspecific cytotoxic cells in fish Teleost T and NK cell immunity. Fish Shellfish (Ictalurus punctatus). IV. Target cell binding Immunology, 35(2), 197–206. and recycling capacity. Developmental and Comparative Immunology, 8(4), 823–833.

412 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

Fischer, U., Ototake, M., & Nakanishi, T. (2006). Gasque, P. H. (2004). Complement: A unique innate Effect of environmental temperature on in vitro immune sensor for danger signals. Molecular cell-mediated cytotoxicity (CMC) and graft- Immunology, 41(11), 1089–1098. versus-host reaction (GVHR) in ginbuna crucian Goldman, K. J. (1997). Regulation of body carp, Carassius auratus langsdorfii. Fish and temperature in the white shark, Carcharodon Shellfish Immunology, 9(3), 233–236. carcharias. Journal of Comparative Physiology FishBase. (2011, February). Update. (http://www. B Biochemical Systemic and Environmental .org/search.php) Retrieved on 2011, Physiology, 167(6), 423–429. October 24. Goldstein, I. J., Hughes, R. C., Monsigny, M., Osawa, Fletcher, T. C. (1982). Non-specific defence T., & Sharon, N. (1980). What should be called mechanisms of fish. Development and a lectin? Nature, 285, 66. Comparative Immunology, 2, 123–132. Gomme, P. T., & McCann, K. B. (2005). Transferrin: Fletcher, T. C. (1986). Modulation of nonspecific Structure, function and potential therapeutic host defenses in fish.Veterinary Immunology & actions. Drug Discovery Today, 10(4), 267–273. Immunopathology, 12(1-4), 59–67. Gorgollon, P. (1983). Fine structure of the thymus in Fletcher, T. C., & Grant, P. T. (1968). Glycoproteins the adult cling fish Sicyases sanguineus (Pisces in the external mucous secretions of the plaice, Gobiesocidae). Journal of Morphology, 177(1), Pleuronectes platessa and some other fishes. 25–40. Biochemistry, 106, 12pp. Got, R., Font, J., & Goussault, Y. (1967). Etude sur Fletcher, T. C., & White A. (1973) Lysozyme une transferrine de selacien, la grande roussette activity in the plaice (Pleuronectes platessa L.). (Scyllium stellare). Comparative Biochemistry Experientia, 29(10), 1283–1285. and Physiology, 23(2), 317–322.

Fock, W. L., Chen, C. L., Lam, T. J., & Sin Y. M. Grabowski, L. D., La Patra, S. E., & Cain, K. D. (2001). Roles of an endogenous serum lectin (2004). Systemic and mucosal antibody response in the immune protection of blue gourami, in tilapia, Oreochromis niloticus (L.) following Trichogaster trichopterus (Pallus) against immunization with Flavobacterium columnare. Aeromonas hydrophila. Fish and Shellfish Journal of Fish Diseases, 27(10), 573–581. Immunology, 11(2), 101–113. Grøntvedt, R. N., & Espelid, S. (2003). Vaccination Fujita, T., Matsushita, M., & Endo, Y. (2004). The and immune responses against atypical lectin-complement pathway – Its role in innate Aeromonas salmonicida in spotted wolffish immunity and evolution. Immunology Review, (Anarhichas minor Olafsen) juveniles. Fish and 198(1), 185–202. Shellfish Immunology, 16(3), 271–285.

Garcia-Fernandez, C., Sanchez, J. A., & Blanco, Grove, S., Johansen, R., Reitan, L. J., & Press, C. G. (2011). Characterization of the gilthead M. (2006). Immune and enzyme histochemical seabream (Sparus aurata L.) transferrin gene: characterisation of leukocyte populations within Genomic structure, constitutive expression and lymphoid and mucosal tissues of Atlantic halibut SNP variation. Fish and Shellfish Immunology, (Hippoglossus hippoglossus). Fish and Shellfish 31(4), 548–556. Immunology, 20(5), 693–708.

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 413 M. Firdaus-Nawi and M. Zamri-Saad

Hansen, J. D., Landis, E. D., & Phillips, R. B. Jollès, P., & Jollès, J. (1984). What’s new in lysozyme (2005). Discovery of a unique Ig heavy-chain research? Always a model system, today as isotype (IgT) in rainbow trout: Implications yesterday. Molecular and Cellular Biochemistry, for a distinctive B cell developmental pathway 63(2), 165–189. in teleost fish. Proceedings of the National Jurecka, P., Irnazarow, I., Stafford, J. L, Ruszczyk, A., Academy of Sciences USA, 102(19), 6919–6924. Taverne, N., Belosevic, M. … Wiegertjes, G. F. Hodgkinson, J. W., Grayfer, L., & Belosevic, M. (2009b). The induction of nitric oxide response (2015). Biology of bony fish macrophages. of carp macrophages by transferrin is influenced Biology, 4(4), 881–906. by the allelic diversity of the molecule. Fish and Shellfish Immunology, 26(4), 632–638. Holland, M. C. H., & Lambris, J. D. (2002). The complement system in teleosts. Fish & Shellfish Jurecka, P., Wiegertjes, G. F., Rakus, K. Ł., Pilarczyk, Immunology, 12(5), 399–420. A., & Irnazarow, I. (2009a). Genetic resistance of carp (Cyprinus carpio L.) to Trypanoplasma Iacono, V. J., MacKay, B. J., DiRienzo, S., & Pollock, borreli: Influence of transferrinpolymorphisms. J. J. (1980). Selective antibacterial properties Veterinary Immunology and Immunopathology, of lysozyme for oral microorganisms. Infection 127(1-2), 19–25. Immunity, 29(2), 623–632. Kaattari, S., Evans, D., & Klemer, J. (1998). Varied Ingram, G. A. (1980). Substances involved in the redox forms of teleost IgM: An alternative to natural resistance of fish to infection-a review. isotypic diversity? Immunological Reviews, Fish Biology, 16(1), 23–60. 166(1), 133–142. Itami, T., Takehara, A., Nagano, Y., Suetsuna, K., Kaattari, S., & Piganelli, J. D. (1996). The specific Mitsutani, A., Takesue, K., & Takahashi Y. immune system: humoral defense. Fish (1992). Purification and characterization of Physiology, 15, 207-254. lysozyme from a ayu skin mucus. Nippon Suisan Gakkaishi, 58(10), 1937–1944. Kamiya, H., Muramoto, K., & Goto, R. (1990). Purification and properties of agglutinins from Jamieson, A. (1990). A survey of transferrin in 87 conger eel, Conger myriaster (Brevoort), teleostean species. Animal Genetics, 21(3), skin mucus. Developmental and Comparative 295–301. Immunology, 12(2), 309–318. Janeway, C. A. Jr. (2001). How the immune system Kamiya, H., & Shimizu, Y. (1980). Marine protects the host from infection. Microbes and biopolymers with cell specificity. II. Purification Infection, 3(13), 1167–1171. and characterization of agglutinins from mucus Jaso-Friedmann, L., Leary III, J. H., & Evans, of windowpane flounderLophopsetta maculate. D. L. (1993). Nonspesific cytotoxic cells in Biochimica et Biophysica Acta, 622(2), 171–178. fish: Antigenic cross-reactivity of a function- Kelly, R. K., & Loh, P. C. (1973). Some properties of associated molecule with the intermediate an established fish cell line from Xiphophorus filament vimentin.Cellular Immunology, 148(1), helleri (red swordtail). In Vitro, 9(2), 73–80. 208–217. Kenneth, D. C., Darren, R. J., & Robert, L. R. Jault, C., Pichon, L., & Chluba, J. (2004). Toll-like (2000). Characterization of mucosal and receptor gene family and TIR-domain adapters systemic immune responses in rainbow trout in Danio rerio. Molecular Immunology, 40(11), (Oncorhynchus mykiss) using surface plasmon 759–771.

414 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

resonance. Fish and Shellfish Immunology, 10(8), Lamas, J., & Ellis, A. (1994). Atlantic salmon (Salmo 651–666. salar) neutrophil responses to Aeromonas salmonicida. Fish and Shellfish Immunology, Kerry, J. L., & Hansen, J. D. (2011). Fish T cells: Recent 4(3), 201–219. advances through genomics. Developmental and Comparative Immunology, 35(12), 1282–1295. Lambert, L. A., Perri, H., Halbrooks, P. J., & Mason, A. B. (2005). Evolution of the transferrin family: Kishore, U., & Reid, K. B. M. (2000). C1q: Structure, conservation of residues associated with iron and function and receptors. Immunopharmacology, anion binding. Comparative Biochemistry and 49(1-2), 159–170. Physiology Part B: Biochemistry and Molecular Kobayashi, K., Tomonaga, S., & Kajii, T. (1984). Biology, 142(2), 129–141. A second class of immunoglobulin other than Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. IgM present in the serum of cartilaginous M., & Hoffmann, J. A. (1996). The dorsoventral fish, the skate, Raja kenojei: Isolation and regulatory gene cassette spätzle/Toll/cactus characterization. Molecular Immunology, 21(5), controls the potent antifungal response in 397–404. Drosophila adults. Cell, 86(6), 973–983. Kohgo, Y., Ikuta, K., Ohtake, T., Torimoto, Y., & Lie, O., Sovensen, A., & Froysadal, E. (1989). Study Kato, J. (2008). Body iron metabolism and on lysozyme activity in some fish species. pathophysiology of iron overload. International Diseases of Aquatic Organisms, 6(1), 1–5. Journal of Hematology, 88(1), 30–35. Lobb, C. J., & Clem, L .W. (1981). Phylogeny of Kum, C., & Sekkin, S. (2011). The immune system immunoglobulin structure and function X. drugs in fish: Immune function, immunoassay, Humoral immunoglobulins of the sheephead, drugs, recent advances in fish farms. Faruk Archosagus probatocephalus. Developmental Aral (Ed.). ISBN: 978-953-307-759-8, InTech, and Comparative Immunology, 5(2), 271–282. Retrieved from http://www.intechopen.com/ books/recent-advances-in-fish-farms/the- Lowe-Jinde, L. (1986). Hematological changes immune-system-drugs-in-fishimmune-function- in Crytobia-infected rainbow trout (Salmo immunoassay-drugs. gairdneri). Canadian Journal of Zoology, 64(6), 1352–1355. Kutchler, A. M., Gjini, E., Peterson-Maduro, J., Cancilla, B., Wolburg, H., & Schulte-Merker. Lukjanenko, V. I. (1971). Immunobiology of fish. (2006). Development of the zebrafish lymphatic Moscow: Pishchevaya Promyshlennost. (p. 364.) system requires VegFc signalling. Current Lutfalla, G., Roest Crollius, H., Stange-Thomann, Biology, 16(12), 1244–1248. N., Jaillon, O., Mogensen, K., & Monneron, D. La Frentz, B. L., La Patra, S. E., Jones, G. R., (2003). Comparative genomic analysis reveals Congleton, J. L., Sun, B., & Cain, K. D. (2002). independent expansion of a lineage-specific Characterization of serum and mucosal antibody gene family in vertebrates: The class II cytokine responses and relative percent survival in receptors and their ligands in mammals and fish. rainbow trout, Oncorhynchus mykiss (Walbaum), BMC Genomics, 4(1), 29. following immunization and challenge with Lydyard P. M., Whelan A., & Fanger M. W. (2000). Flavobacterium psychrophilum. Journal of Fish Overview of the immune system. Instant Notes in Diseases, 25(12), 703–713. Immunology (pp.1–11). New Delhi, India: Viva Books Private Limited.

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 415 M. Firdaus-Nawi and M. Zamri-Saad

Macey, D. J., Webb, J., & Potter, I. C. (1982). Iron Murai, T., Kodama, H., Nakai, M., Mikami, T., & levels and major iron binding proteins in the Izawa, H. (1990). Isolation and characterization of plasma of ammocoetes and adults of the southern rainbow trout C-reactive protein. Developmental hemisphere lamprey Geotria australis Gray. and Comparative Immunology, 14(1), 49–58. Comparative Biochemistry and Physiology – Murray, C. K., & Fletcher, T. C. (1976). The Part A: Molecular and Integrative Physiology, immunohistochemical localization of lysozyme 72(2), 307–312. in plaice (Pleuronectes platessa L.) tissues. Magnadottir, B. (1998). Comparison of Journal of Fish Biology, 9(4), 329–334. immunoglobulin (IgM) from four fish species. Nakanishi, T., Shibasaki, Y., & Matsuura, Y. (2015). Icelandic Agricultural Sciences, 12, 47–59. T-cells in fish.Biology, 4(4), 640–663. Magnadottir, B., Lange, S., Gudmundsdottir, S., Nakanishi, Y., & Ototake, M. (1999). The graft- Bogwald, J., & Dalmo, R. A. (2005). Ontogeny versus-host reaction (GVHR) in the ginbuna of humoral immune parameters in fish.Fish and crucian carp, Carassius auratus langsdorfii. Shellfish Immunology, 19(5), 429–439. Developmental and Comparative Immunology, Manning, M. J., & Nakanishi, Y. (1996). The specific 23(1), 15–26. immune system: Cellular defenses. In G. Iwama Nakao, M., Tsujikura, M., Ichiki, S., K. Vo, T., & & T. Nakanishi (Eds.), The fish immune system. Somamoto, T. (2011). The complement system organism, pathogen and environment. San in teleost fish: Progress of post-homolog-hunting Diego, CA: Academic Press. researches. Developmental & Comparative Mantovani, A., Garlanda, C., Doni, A., & Bottazzi, Immunology, 35(12), 1296–1308. B. (2008). Pentraxins in innate immunity: From Neves, J. V., Wilson, J. M., & Rodrigues, P. N. S. C-reactive protein to the long pentraxin PTX3. (2009). Transferrin and ferritin response to Journal of Clinical Immunology, 28(1), 1–13. bacterial infection: The role of the liver and McKinkey, E. C., Haynes, L., & Droese, A. L. brain in fish. Developmental & Comparative (1986). Macrophage-like effector of spontaneous Immunology, 33(7), 848–857. cytotoxicity from the shark. Developmental & Nigam, A. K., Kumari, U., Mittal, S., & Mittal, A. K. Comparative Immunology, 10(4), 497–508. (2012). Comparative analysis of innate immune Medzhitov, R., & Janeway, C. A. Jr. (1997). Innate parameters of the skin mucous secretions immunity: The virtues of a nonclonal system of from certain freshwater teleosts, inhabiting recognition. Cell Press, 91(3), 295–298. different ecological niches. Fish Physiology and Biochemistry, 38(5), 1245–1256. Mitra, S., & Bhattacharya, S. (1992). Purification of C-reactive protein from Channa punctatus Nunomora, W. (1991). C-reactive protein in (Bloch). Indian Journal of Biochemistry and eel: Purification and agglutinating activity. Biophysics, 29(6), 508–511. Biochemical and Biophysical Acta, 1076(2), 191–196. Morrison, R. N., & Nowak, B. F. (2002). The antibody response of teleost fish. Seminars in Avian and Okamoto, N., Shirakura, T., Nagakura, Y., & Sano, Exotic Pet Medicine, 11(1), 46–54. T. (1983). The mechanism of interference with fish viral infection in the RTG-2 cell line. Fish Muller-Eberhard, H. J. (1986). The membrane attack Pathology. 18(1), 7–12. complex of complement. Annual Review of Immunology, 4(1), 503–528.

416 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

Oshiumi, H., Tsujita, T., Shida, K., Matsumoto, Ramos, F., & Smith, A. C. (1978). The C-reactive M., Ikeo, K., & Seya, T. (2003). Prediction of protein (CRP) test for the detection of early the prototype of the human Toll-like receptor disease in fishes.Aquaculture, 14(3), 261–266. gene family from the pufferfish,Fugu rubripes, Rio, G. J., Magnavita, F. J., Rubin, J. A., & Beckert, genome. Immunogenetics, 54(11), 791–800. W. H. (1973). Characteristics of an established Ottinger, C. A., Johnson, S. C., Ewart, K. V., Brown, goldfish Carassius auratus cell line. Journal of L. L., & Ross, N. W. (1999). Enhancement Fish Biology, 5, 315–321. of anti-Aeromonas salmonicida activity in Robertsen, B. (2006). The interferon system of teleost Atlantic salmon (Salmo salar) macrophages fish. Fish and Shellfish Immunology, 20(2), by a mannose-binding lectin. Comparative 172–191. Biochemistry and Physiology Part C, 123(1), 53–59. Robertsen, B., Bergan, V., Røkenes, T., Larsen, R., & Albuquerque, A. (2003). Atlantic salmon Pangburn, M. K., & Rawal, N. (2002). Structure interferon genes: Cloning, sequence analysis, and function of complement C5 convertase expression, and biological activity. Journal of enzymes. Biochemical Society Transactions, Interferon Cytokine Research, 23(10), 601–612. 30(6), 1006–1010. Robey, F. A., & Liu, T. Y. (1983). Synthesis Pepys, M. B., & Hirschfield, G. M. (2003). C-reactive and use of new spin-labeled derivatives of protein: A critical update. Journal of Clinical phosphorylcholine in a comparative study Investigation, 111(12), 1805–1812. of human, dogfish, and Limulus C-reactive Pier, G., Lyczak, J., & Wetzler, L. (2004). Cell mediated proteins. Journal of Biological Chemistry, immunity in Blackwell publishing. Immunology, 258(6), 3895–3900. Infection and Immunity. Washington, D. C.: Rombout, J. H. W. M., Taverne, P., Thiele, A. J., ASM Press. & Valenna, M. J. (1993b). The gut associated Pilstrom, L., & Bengten, E. (1996). Immunoglobulin lymphoid tissue (GALT) of carp (Cyprinus in fish – Genes, expression and structure. Fish carpio L.): An immunocyto-chemical analysis. and Shellfish Immunology, 6(4), 243–262. Developmental and Comparative Immunology, 17(1), 55–66. Pontius, H., & Ambrosius, H. (1972). Contribution to the immune biology of poikilothermic Rombout, J. H. W. M., Taverne, N., van de Kamp, vertebrates. IX. Studies on the cellular M., & Taverne-Thiele, A. J. (1993a). Differences mechanism of humoral immune reactions in in mucus and serum immunoglobulin of carp perch (Perca fluviatilis L.). Acta Biologica et (Cyprinus carpio L.). Development and Medica Germanica, 29(2), 319–339. Comparative Immunology, 17(4), 309–317.

Pulsford, A., Tomlinson, M. G., Lemaire-Gony, Rombout, J. H. W. M., & Van Den Berg, A. A. S., & Glynn, P. J. (1994). Development and (1989). Immunological response of the second immunocompetence of juvenile flounder gut segment of carp. I. Uptake and processing Platichthys flesus L.. Fish and Shellfish of antigens by epithelial cells and macrophages. Immunology, 4(), 63–78. Journal of Fish Biology, 35(1), 13–22.

Rai, A. K., & Mittal, A. K. (1983). Histochemical Ross, N. W., Firth, K. J., Wang, A., Burka, J. F., & response of alkaline phosphatase activity during Johnson, S. C. (2000). Changes in hydrolytic the healing of cutaneous wounds in a catfish. enzyme activities of naive Atlantic salmon Experientia, 39(5), 520–522. Salmo salar skin mucus due to infection with

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 417 M. Firdaus-Nawi and M. Zamri-Saad

the salmon louse Lepeophtheirus salmonis Sitja-Bobadilla, A. Redondo, M. J., Bermudez, and cortisol implantation. Diseases of Aquatic R., Palenzuela, O., Ferreiro, I., Riaza, A. … Organisms, 41(1), 43–51. Alvarez-Pellitero, P. (2006). Innate and adaptive immune responses of turbot, Scophthalmus Rubio-Godog, M. (2010). Teleost fish immunology: maximus (L.), following experimental infection Review. Revista Mexicana de Ciencias with Enteromyxum scophthalmi (Myxosporea: Pecuarias, 1(1), 47 –57. Myxozoa). Fish and Shellfish Immunology, Russel, S., & Lumsden, J. S. (2005). Function 21(5), 485–500. and heterogeneity of fish lectins. Veterinary Snegaroff, J. (1993). Induction of interferon Immunology and Immunopathology, 108(1), synthesis in rainbow-trout leukocytes by 111–120. various homeotherm viruses. Fish and Shellfish Salinas, I., Zhang, Y. A., & Sunyer, J. O. (2011). Immunology, 3(3), 191–198. Mucosal immunoglobulins and B cells of Somamoto, T., Yoshiura, Y., Sato, A., Nakao, M., teleost fish. Developmental and Comparative Nakanishi, T., & Okamoto, N. (2006). Expression Immunology, 35(12), 1346–1365. profiles of TCRbeta and CD8alpha mRNA Samuel, C. E. (2001). Antiviral actions of interferons. correlate with virus-specific cell-mediated Clinical Microbiology Review, 14, 778–809. cytotoxic activity in ginbuna crucian carp. Virology, 348(2), 370–377. Saurabh, S., & Sahoo, T. K. (2008). Lysozyme: An important defence molecule of fish innate St. Louis-Cormier, E. A., Osterland, D. K., & immune system. Aquaculture Research, 39(3), Anderson, P. D. (1984). Evidence for a cutaneous 223–239. secretory immune system in the rainbow trout (Salmo gairdneri). Developmental and Secombes, C. J. (1990). Isolation of salmonid Comparative Immunology, 8(1), 71–80. macrophages and analysis of their killing activity. Techniques in Fish Immunology, 1, Stafford, J. L., Ellestad, K. K., Magor, K. E., 134–154. Belosevic, M., & Magor, B. G. (2003). A toll- like receptor (TLR) gene that is up-regulated in Secombes, C. J. (1994a). Macrophage activation in activated goldfish macrophages.Developmental fish. Modulation of Fish Immune Responses, and Comparative Immunology, 27(8), 685–698. 1, 49–57. Steinhagen, D., Kruse, P., & Körting, W. (1990). Secombes, C. J. (1996). The nonspecific immune Some haematological observations on carp, system: Cellular defenses. In the fish immune Cyprinus carpio L., experimentally infected system: Organism, pathogen and environment. with Trypanoplasma borreli Laveran & Mesnil, (G. Iwama & T. Nakanishi Eds.). San Diego: 1901 (Protozoa: Kinetoplastida). Journal of Fish Academic Press. Disease, 13(2), 157–162. Secombes, C. J., & Fletcher, T. C. (1992). The role Stuge, T. B., Wilson, M. R., Zhou, H., Barker, of phagocytes in the protective mechanisms of K. S., Bengten, E., & Chinchar, G. (2000). fish.Annual Revision of Fish Disease, 2, 53–71. Development and analysis of various clonal Silva, J. R. M. C., Staines, N. A., Hernandez-Blazquez, alloantigen-dependent cytotoxic cell lines F. J., Porto-Neto, L. R., & Borges, J. C. S. (2002). from channel catfish. Immunology, 164(6), Phagocytosis and giant cell formation at 0° C 2971–2977. by macrophage (MO) of Notothenia coriiceps. Journal of Fish Biology, 60(2), 466–478.

418 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Major Components of Fish Immunity: A Review

Sunyer, J. O., & Lambris, J. D. (1999). Complement. Turner, M. W. (2003). The role of mannose- Encyclopedia of Life Sciences. New York: binding lectin in health and disease. Molecular MacMillan. Immunology, 40(7), 423–429.

Suzumoto, B. K., Schreck, C. B., & McIntyre, J. D. Turner, R. J., & Jamieson, A. (1987). Transferrins in (1977). Relative resistance of three transferrin fishes.XXth International Conference on Animal genotypes of coho salmon (Oncorhynchus Blood Groups and Biochemical Polymorphism. kisutch) and their hematological responses to Animal Genetics, 18(suppl. I), 70–71. bacterial kidney disease. Journal of the Fisheries Uribe, C., Folch, H., Enriquez, R., & Moran, G. Research Board of Canada, 34(1), 1–8. (2011). Innate and adaptive immunity in teleost Szalai, A. J., Bly, J. E., & Clem, L. W. (1994). fish: A review. Veterinarni Medicina, 56(10), Changes in serum concentrations of channel 486–503. catfish (Ictalurus punctatus Rafinesque) Vasta, G. R., Ahmed, H., & Odom, E. W. (2004). phosphorylcholine-reactive protein (PRP) Structural and functional diversity of lectin in response to inflammatory agents, low repertoires in invertebrates, protochordates and temperature shock and infection by the fungus ectothermic vertebrates. Current Opinion in Saprolegnia sp. Fish and Shellfish Immunology, Structural Biology, 14(5), 617–630. 4(5), 323–336. Voss Jr., E. W., Fryer, J. L., & Banowetz, G. M. (1978). Takano, T., Kondo, H., Hirono, I., Endo, M., Saito- Isolation, purification and partial characterization Taki, T., & Ao, T. (2011). Toll-like receptors of a lectin from Chinook salmon ova. Archives in teleosts. Diseases in Asian Aquaculture VII. of Biochemistry and Biophysics, 186(1), 25–34. Fish Health Section, Asian Fisheries Society, Malaysia, 197-208. Wan, Y., & Flavell, R. A. (2009). How diverse – CD4 effector T-cells and their functions. Journal of Thuvander, A., Johannison, A., & Grawe, J. (1992). Molecular Cell Biology, 1(1), 20–36. Flow cytometry in fish immunology.Techniques in Fish Immunology, 2, 19–26. White, A., & Fletcher, T. C. (1985). The influence of hormones and inflammatory agents on C-reactive Tian, J., Sun, B., Luo, Y., Zhang, Y., & Nie, P. (2009). protein, cortisol, and alanine aminotransferase Distribution of IgM, IgD and IgZ in mandarin in the plaice (Pleuronectes platessa L.). fish,Siniperca chuatsi lymphoid tissues and their Comparative Biochemistry and Physiology, transcriptional changes after Flavobacterium 80(1), 99–104. columnare stimulation. Aquaculture, 288(1-2), 14–21. White, A., Fletcher, T. C., Pepys, M. B., & Baldo, B. A. (1981). The effect of inflammatory agents Tomonaga, S., Kobayashi, K., Hagiwara, K., on C-reactive protein and serum amyloid Yamaguchi, K., & Awaya, A. (1986). Gut- P-component levels in plaice (Pleuronectes associated lymphoid tissue in Elasmobranchs. platessa L.) serum. Comparative Biochemistry Zoological Science (Tokyo), 3(3), 353–358. and Physiology, 69(2), 325–329. Tort, L., Balasch, J. C., & Mackenzie, S. (2003). Fish White, A., Fletcher, T. C., Towler, C. M., & Baldo, immune system. A crossroads between innate B. A. (1978). Isolation of a C-reactive protein- and adaptive responses. Immunologia, 22(3), like precipitin from the eggs of the lumpsucker 277–286. (Cyclopterus lumpus L.). Comparative Biochemistry and Physiology, 61(2), 331–336.

Pertanika J. Trop. Agric. Sci. 39 (4): 393 - 420 (2016) 419 M. Firdaus-Nawi and M. Zamri-Saad

Whyte, S. K. (2007). The innate immune response of Zapata, A. G., Chiba, A., & Vara, A. (1996). Cells finfish – A review of current knowledge. Fish and tissues of the immune system of fish. In G. and Shellfish Immunology, 23(6), 1127–1151. Iwama & T. Nakanishi (Eds.), The fish immune system: organism, pathogen and environment. Wilson, M., Bengte´n, E., Miller, N. W., Clem, L. W., Fish immunology series (pp. 1–55). New York: Du Pasquier, L., & Warr, G. W. (1997). A novel Academic Press. chimeric Ig heavy chain from a teleost fish shares similarities to IgD. Proceedings of the National Zapata, A. G., & Solas, M. T. (1979). Gut-associated Academy of Sciences USA, 94(9), 4593–4597. lymphoid tissue (GALT) in reptilia: Structure of mucosal accumulations. Developmental and Winkelhake, J. L., & Chang, R. J. (1982). Acute phase Comparative Immunology, 3, 477–487. (C-reactive) protein-like macromolecules from rainbow trout (Salmo gairdneri). Developmental Zapata, A., Diez, B., Cejalvo, T., Gutierrez-De Frias, and Comparative Immunology, 6(3), 481–489. C., & Cortes, A. (2006). Ontogeny of the immune system of fish. Fish and Shellfish Immunology, Wolfle, U., Martin, S., Emde, M., & Schempp, C. 20(2), 126–136. (2009). Dermatology in the Darwin anniversary. Part 2: evolution of the skin-associated immune Zhang, Y., Salinas, I., Li, J., Parra, D., Bjork, S., system. Journal of the German Society of Xu, Z. … Sunyer, O. J. (2010). IgT, a primitive Dermatology, 7(10), 862–869. immunoglobulin class specialized in mucosal immunity. Nature Immunology, 11(9), 827–835. Yano, T. (1996). The nonspesific immune system: Humoral defense. In Hoar, W. S., Randall, Zhao, X., Findly, R. C., & Dickerson, H. W. (2008). D. J., Iwama, G., & Nakanishi, T. (Eds.), The Cutaneous antibody- secreting cells and B cells fish immune system: Organism, pathogen and in a teleost fish.Developmental and Comparative environment. Fish immunology series. New Immunology, 32(5), 500–508. York: Academic Press. Zhu, J., & Paul, W. E. (2010). Peripheral CD4+ Yousif, A. N., Albright, L. J., & Evelyn, T. P. T. (1994). T-cell differentiation regulated by networks of In vitro evidence for the antibacterial role of cytokines and transcription factors. Immunology lysozyme in salmonid eggs. Diseases of Aquatic Revision, 238(1), 247–262. Organisms, 19(1), 15–19.

420 Pertanika J. Trop. Agric. Sci. 39 (4) 393 - 420 (2016) Pertanika J. Trop. Agric. Sci. 39 (4): 421 - 458 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Review Article A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

Sule, H. A.1,2, Ismail, A.1* and Amal, M. N. A.1 1Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2Department of Integrated Science, School of Science, Kogi State College of Education, PMB 1033 Ankpa, Nigeria

ABSTRACT A review of literature showed that numerous intensive surveys have been carried out on the ichthyofauna of the peat swamp forests (PSFs) of Malaysia. This review aim to provide a checklist of blackwater fish species in Malaysia from available published literature, and address their economical importance, conservation status and problems of PSFs. A total of 198 peat swamp fish species from 32 families have been recorded in Malaysia. From this number, a total of 114 species from 23 families, representing about 40% of the known fish fauna in Peninsular Malaysia, were recorded from north Selangor PSF. Meanwhile, a total of 49 species belonging to 18 families, 13 species from seven families, 58 species belonging to 19 families, and nine species from five families were recorded from the peat swamps of Perak, Johor, Pahang and East Peninsular Malaysia (part of Pahang and Terengganu), respectively. Meanwhile, 31 species from 12 families and 40 species belonging to 13 families were recorded from Sabah and Sarawak, respectively. Family has the highest recorded species, followed by Osphronemidae, and . The IUCN Red List revealed 12 threatened species facing risk of extinction. The importance of conserving PSFs was outlined and suggestions made in line with the objectives of conservation. Findings from literature revealed that Malaysia’s PSFs are rich in fish diversity, contrary to previous belief, and should therefore be conserved and protected

ARTICLE INFO to ensure the richness of their fish diversity. Article history: Received: 18 June 2015 Keywords: Ichthyofauna, peat swamp forest, Accepted: 27 May 2016 conservation, Malaysia E-mail addresses: [email protected] (Sule, H. A.), [email protected] (Ismail, A.), [email protected] (Amal, M. N. A.) * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press Sule, H. A., Ismail, A. and Amal, M. N. A.

INTRODUCTION of considerable size in Peninsular Malaysia Peat swamp forests (PSFs) are one of the are now restricted to north Selangor, central most unusual and harsh ecosystems in the Terengganu and Pahang (Ng et al., 1994). In tropical rainforest biome. The PSFs of Peninsular Malaysia, the fish fauna of the Peninsular Malaysia are one of the most PSF of north Selangor is comparably well threatened (illegal logging, irrigation of oil studied and recorded. palm and paddy fields), yet one of the most The unique characteristics of black poorly understood biotopes (Ng et al., 1994). waters (dark colour, low dissolved These swamps derive their name from oxygen and high acidity) led to an initial their substrate of peat consisting of plant misinterpretation of ‘habitat inhospitality’, detritus, which gradually release tannins and the conclusion that such habitats will and organic acids into poorly buffered water sustain very poor faunal diversity. One of and contribute to its characteristically low the earliest surveys of the fish fauna of pH (Ng et al., 1994; Yule, 2010; Posa et al., Malaysia peat swamps was by Johnson 2011; Wantzen et al., 2011). Peat swamps (1967ab, 1968) who recorded only 26 are typically deficient in oxygen, which is species in black waters, of which only one one consequence of plant decay (Beamish is stenotopic to black water. However, the et al., 2003). Peat swamps are generally first well-recognised documentation of referred as ‘black waters’, while waters blackwater fish species of the north Selangor originating from PSFs are highly acidic peat swamp forest (NSPSF ) was done by with pH values ranging from 3.6 to 5.9, Davies and Abdullah (1989). Numerous tea-coloured when seen against transmitted other surveys have also been conducted on light, and black when seen en masse via the ichthyofauna of Peninsular Malaysia reflected light (Johnson, 1967ab, 1968). (Shiraishi et al., 1972; Lim et al., 1982; Generally, the acidity of black waters is due Mizuno & Furtado, 1982; Zakaria-Ismail, to the high concentrations of humic acids 1990; Ng & Lim, 1991; Ng et al., 1992, and other phenolic acids (Goltenboth, 2006; 1994; IPT-AWB, 1993; Lee & Ng, 1994; Irvine et al., 2013). Zakaria et al., 1999; Lee, 2001; Beamish et Most of the black waters of Singapore al., 2003; Rezawaty, 2004; Shah et al., 2006; and Peninsular Malaysia recorded by Ahmad et al., 2013; Ismail et al., 2013; Siow Wyatt-Smith (1959, 1964), Johnson (1967a, et al., 2013). 1968), Anderson (1983) and Whitmore The objectives of this review are to (1984, 1988) have been converted to provide a checklist of black water fish agriculture, industrial states and residential species in Malaysia, assess their economic areas (Ng et al., 1994). The peat swamp importance, update the conservation status forests of Johor, which were of staggering of the species and address the conservation vastness in Peninsular Malaysia, are almost problems of PSFs. An exhaustive search completely degraded. Peat swamp forests of published literature was conducted to

422 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest collate lists and determine the distribution 11 families (see Table 1). A lot of scientific of the peat swamp fishes from surveys in names, from family to species level, have Malaysian PSF sites (Figure 1). Fish species been changed over the past decade. The provided in this review were recorded most recent comprehensive list of such from the surveys between 1989 and 2015. changes is outlined in Kottelat’s catalogue of Reference was made to Kottelat’s catalogue the fishes of (Kottelat, 2013). of the fishes of southeast Asia (Kottelat, An intensive survey in 1992 by IPT- 2013) to verify and update taxonomic AWB (1993) recorded 76 fish species revisions of species. belonging to 24 families (23 with exception of Belontiidae, now included in the family FISH SPECIES RECORDED IN Osphronemidae). This is one of the most MALAYSIAN PEAT SWAMP successful surveys in NSPSF in terms of FORESTS diversity. Ng et al. (1994) recorded 47 Peat swamp forests have unique water species of fish living in the black waters of quality and as a result, fish species that NSPSF, and 14 species of the fishes were can survive and breed in the black water stenotopic to acidic black waters. Six unique environment are limited. Researchers have fish species relatively new to science were reported many species of fish in PSFs that first recorded at NSPSF in 1989 (Ng et al., are not found in other habitats (Davies 1994). & Abdullah, 1989; Ng et al., 1994). A Studies by Beamish et al. (2003) comprehensive list and a summary of the conducted from 1997 to 1998 recorded 35 fish species of Malaysia PSFs are given in fish species from NSPSF belonging to 14 Table 1 and Table 2. families (actually 13 because Belontiidae and Luciocephalidae which are now included in North Selangor Peat Swamp Forest Osphronemidae, were recorded separately), The NSPSF has been well studied relative to while Giam et al. (2012) recorded eight other PSFs in Malaysia. The most extensive blackwater fish species belonging to the six surveys of the fish fauna of NSPSF were families from NSPSF. done by Davies and Abdullah (1989), IPT- Recently, during the Selangor Scientific AWB (1993), Ng et al., (1994), Beamish et and Biodiversity Peatland Expedition of al., (2003), Ahmad et al., (2013), Ismail et 2013, Ahmad et al. (2013), Ismail et al. al., (2013) and Siow et al., (2013). (2013) and Siow et al. (2013) recorded 30 Davies and Abdullah (1989), in a species from 13 families, 41 species from survey of freshwater fish of NSPSF in 13 families, and 24 species 10 families, 1989, recorded 42 fish species (actually 41 respectively. The comprehensive list of fish species, with the exception of species recorded from NSPSF is given in hasselti, the of O. vittatus recorded Table 1. as another different species) belonging to

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 423 Sule, H. A., Ismail, A. and Amal, M. N. A. 6- Segama River, Sabah 6- Segama River, 7- Maliau basin, Sabah 8- Rajang basin, Sarawak 9- Black water sites, Batang Kerang and Sadong, Sarawak 10- Black water sites, Nanga Merit area, Sarawak Location of PSFs and black water sites surveyed 1- North Selangor peat swamp forest (NSPSF), 2- Paya Beriah peat swamp forest (PBPSF), north Perak 3- Black water sites, west Johor 4- Southeast Pahang peat swamp forest (SEPPSF) Terengganu 5- Black water sites, part of Pahang and Key: 1. Figure

424 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

References Abdullah, 1989 Davies & 1993 IPT-AWB, 1993 IPT-AWB, 1993; IPT-AWB, Ng et al., 1994 Abdullah, 1989; Davies & 1993; Rezawaty, IPT-AWB, Ahmad et al., 2005; 2004; Shah et al., 2006; Khairul- Ahmad Adha et al., 2009; et al., 2013; Ismail 2013; Siow et al., 2013 1998 Tan, Martin-Smith & Zakaria et al., 1999; 2004; Shah et Rezawaty, al., 2006 1998 Tan, Martin-Smith & 1998 Tan, Martin-Smith & 1998 Tan, Martin-Smith & 1998 Tan, Martin-Smith & Sarawak Sarawak PSFs x Sabah Sabah PSFs x x x x x EP PSFs SEPPSF x Johor Johor PSFs PBPSF x x NSPSF x x x x x IUCN IUCN Status NE NE LC NE DD VU LC NE NE NE NE

Alfred, Alfred, (Bleeker, (Bleeker, Herre, 1940 (Hamilton, (Hamilton, Popta, 1924 (Bloch, 1792) hendricksoni cf. Species ischnosoma Acrochordonichthys 1858 Bleeker, rogosus Acrochordonichthys 1846) (Bleeker, Akysis 1986 Parakysis verrucosus Anabas testudineus Anguilla borneensis Aplocheilus panchax 1822) Cryptarius truncatus 1840) (Valenciennes, Batrachocephalus mino (Hamilton, 1822) Cephalocassis borneensis 1851) (Bleeker, Setipinna melanochir 1849)

Family Akysidae Anabantidae Aplocheilidae Engraulididae Table 1 Table Checklist of black water fish in Malaysia

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 425 Sule, H. A., Ismail, A. and Amal, M. N. A.

IPT-AWB, 1993; 1993; IPT-AWB, Ahmad et al., 2005 1993 IPT-AWB, 1993 IPT-AWB, Tan, Ng et al., 1994; & 1999; Beamish et al., 2003; Giam et al., 2012; Ahmad et al., 2013 Ahmad et al., 2005; Giam et al., 2012 Abdullah, 1989 Davies & Siow et al., 2013 Hassan et al., 2010 Abdullah, 1989; Davies & 1998 Tan, Martin-Smith & Ahmad et al., 2005 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Khairul-Adha et al., 2009; Shah et al., 2006; Jimmy et al., 2010; Ismail 2013; Siow et al., 2013 Hassan et al., 2010 1998 Tan, Martin-Smith & x x x x x x x x x x x x x x x x x x x x NE DD NE DD NE NE NE NE NE LC NE NE LC

(Alfred, (Alfred, (Regan, (Regan, (Popta, (Popta, (Bleeker, (Bleeker, (Inger & (Inger &

(Alred, (Alred, Roberts, 1989 nebulosa cf. Homaloptera 1969) Homaloptera ogilviei 1967) Homaloptera zollingeri Neohomaloptera johorensis (Herre, 1944) Barbucca diabolica hypselopterus 1852) (Bleeker, Bagrichthys macrocanthus 1854) (Bleeker, 1854) (Bleeker, Hemibagrus baramensis 1906) Hemibagrus capitulum 1904) Hemibagrus nemurus 1840) (Valenciennes, Hemibagrus planiceps 1840) (Valenciennes, Hemibagrus sabanus Chin, 1959) 153) Table 1 Table Balitoridae Barbuccidae Bagridae cont’d

426 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

Ng & Kottelat, 1998; Ng & Kottelat, 1998; Ahmad et al., 2005; Giam et al., 2012 Abdullah, 1989 Davies & Ng et al., 1994; Beamish et al., 2003 Siow et al., 2013 1993 IPT-AWB, 1993; IPT-AWB, Ng et al., 1994; Beamish al., 2003; Giam et 2012; Ahmad et al., 2013; Ismail et al., 2013 Abdullah, 1989; Davies & 1993; Shah et IPT-AWB, al., 2006; Hassan et 2010; Ismail et al., 2013; Siow et al., 2013 1998 Tan, Martin-Smith & Giam et al., 2012 1993; IPT-AWB, Ng et al., 1994 Ahmad et al., 2005 Giam et al., 2012 x x x x x x x x x x x x x x x x x NE NE NE NE NE NE LC NE NE NE NE NE

(Inger & (Inger &

(Bleeker, (Bleeker, (Weber & & (Weber (Duncker, (Duncker,

Bleeker, 1852 Bleeker, (Volz, 1904) (Volz, Kottelat & Kottelat & (Valenciennes, (Valenciennes, Kottelat & Lim, Kottelat & Lim, Hyalobagrus ornatus 1904) leiacanthus Leiocassis micropogon 1852) Leiocassis poeciloptera 1840) (Valenciennes, Leiocassis stenomus 1839) (Valenciennes, bimaculatus Mystus nigriceps 1840) Pseudomystus robustus bankanensis Bihunichthys monopteroides (Kottelat & Lim, 1994) Chendol keelini 1994 Nagaichthys filipes Lim, 1991 de Beaufort, 1912) Chin, 1959) Table 1 Table Chacidae Chaudhuriidae cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 427 Sule, H. A., Ismail, A. and Amal, M. N. A. IPT-AWB, 1993; Ng et al., 1993; Ng et al., IPT-AWB, Tan, 1994; Martin-Smith & 1998; Beamish et al., 2003; Ahmad Ahmad et al., 2005; et al., 2013; Ismail 2013 Ng et al., 1994 Abdullah, 1989; Davies & Ng et al., 1994; Zakaria al., 1999; Beamish et Ahmad et al., 2005; 2003; Parenti & Lim, 2005; Shah et al., 2006; Khairul-Adha Ahmad et al., et al., 2009; 2013; Ismail et al., 2013 Ahmad et al., 2005 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003 Abdullah, 1989; Davies & Ahmad et al., 2005 Abdullah, 1989; Davies & 1993; Beamish IPT-AWB, et al., 2003; Rezawaty, Ahmad et al., 2005; 2004; Ahmad et Shah et al., 2006; al., 2013; Siow et 2013 Shah et al., 2006 1998 Tan, Martin-Smith & x x x x x x x x x x x x x x x x x NE LC LC LC LC LC LC - NT (Bleeker, (Bleeker, (Bleeker, (Bleeker, (Cuvier, (Cuvier, (Bleeker, 1851) (Bleeker, (Hamilton, 1822) (Bloch, 1797) (Cuvier, 1831) (Cuvier, sp.? Channa bankanensis 1853) Channa gachua Channa lucius Channa marulioides 1851) Channa melosoma Channa micropeltes 1831) Channa striata Channa mossambicus *Oreochromis (Peters, 1852) Table 1 Table Channidae Cichlidae cont’d

428 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

IPT-AWB, 1993; Khairul- IPT-AWB, Adha et al., 2009; Siow al., 2013 Abdullah, 1989; Davies & Ng et al., 1994; Beamish Ahmad et al., et al., 2003; Adha et al., 2005; Khairul- Ahmad et al., 2013 2009; Ahmad et 2004; Rezawaty, al., 2005; Khairul-Adha et al., 2009; Siow et 2013 Ng et al., 1994; Ismail al., 2013 1993; Ng et IPT-AWB, al., 1994; Beamish et Ahmad et al., 2005; 2003; Khairul-Adha et al., 2009; Ismail et al., 2013 2000; Giam et Tan, Ng & al., 2012 1993; Ng et al., IPT-AWB, 1994; Giam et al., 2012 Giam et al., 2012 Giam et al., 2012 1993 IPT-AWB, 1993; IPT-AWB, 2004 Rezawaty, Giam et al., 2012 x x x x x x x x x x x x x x x x x x x x x x x LC NE NT LC LC NE CR CR VU LC LC NE

Ng & Ng & Ng & Ng & Ng & Ng & (van (van Ng & Ng & Günther, Günther, Bleeker, 1851 Bleeker, (Kottelat & (Kottelat & (Bleeker, (Bleeker, (Linnaeus, (Linnaeus, (Valenciennes, (Valenciennes, dialuzona cf. batrachus 1758) Clarias leiacanthus Clarias macrocephalus 1864 Clarias meladerma 1846) Clarias nieuhofii 1840) Encheloclarias baculum Lim, 1993 Encheloclarias curtisoma Lim, 1993 Encheloclarias kelioides Lim, 1993 Encheloclarias prolatus Lim, 1993 Acanthopsoides molobrion (Seibert, 1991) Acantopsis Hasseltt, 1823) Kottelatlimia katik Lim, 1992) Table 1 Table Clariidae cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 429 Sule, H. A., Ismail, A. and Amal, M. N. A. IPT-AWB, 1993; Ng et al., 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003 1993 IPT-AWB, Ahmad et al., 2005 Ahmad et al., 2005 Ahmad et Ng et al., 1994; al., 2013 Ahmad et al., 2005 Ahmad et al., 2005 1998; Tan, Martin-Smith & Jimmy et al., 2010 1998 Tan, Martin-Smith & Abdullah, 1989; Davies & 2004; Siow et Rezawaty, al., 2013 Abdullah, 1989; Davies & 1993; Rezawaty, IPT-AWB, 2004; Ismail et al., 2013 Ismail et al., 2013 1998 Tan, Martin-Smith & 1993 IPT-AWB, x x x x x x x x x x x x x x x x NE NE NE NE NE LC NE NE NE LC LC NE LC NE

(Kuhl (Kuhl

(Bleeker, (Bleeker, (de (de (Bleeker, (Bleeker, (Roberts, (Roberts, (Duncker, (Duncker, (Liao & Tan, (Liao & Tan, (Popta, 1903) (Herre, 1940) oblongus (Herre, 1933) cf. (Valenciennes, (Valenciennes, Kottelatlimia pristes 1989) Pangio kuhlii 1846) Pangio muraeniformis Beaufort, 1933) Pangio shelfordii Lepidocephalichthys tomaculum (Kottelat & Lim, 1992) Boraras maculatus 1904) Barbodes banksi Barbodes sealei balleroides 1842) (Valenciennes, Barbonymus gonionotus 1949) Barbonymus schwanefeldii 1854) (Bleeker, cheeya Brevibora 2011) cobitis Crossocheilus Crossocheilus & van Hasselt, 1823) 1854) Table 1 Table Cyprinidae cont’d

430 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest Davies & Abdullah, 1989; Abdullah, 1989; Davies & 1993; Zakaria et IPT-AWB, 2004; al., 1999; Rezawaty, Ahmad et al., 2005; Shah al., 2006; Ismail et 2013 1993; Hassan et IPT-AWB, al., 2010 1993; Martin- IPT-AWB, 1998 Tan, Smith & 1993; Ng et IPT-AWB, al., 1994; Zakaria et 1999; Beamish et al., 2003; Ahmad et 2004; Rezawaty, al., 2005; Shah et 2006; Ahmad et al., 2013; Ismail et al., 2013 1993; Ng et IPT-AWB, al., 1994; Kottelat, 1996; Beamish et al., 2003; Ahmad et al., 2005; Parenti & Lim, 2005; Giam et al., Ahmad et al., 2013; 2012; Ismail et al., 2013; Siow al., 2013 Parenti & Lim, 2005 Abdullah, 1989; Davies & 1993; Ismail et IPT-AWB, al., 2013 1998 Tan, Martin-Smith & 2004 Rezawaty, x x x x x x x x x x x x x x x x x x x LC LC LC NE NE NE LC NE NE

(Kuhl & (Kuhl & (Weber & & (Weber Inger & Chin, Inger & Chin, Cyclocheilichthys apogon 1842) (Valenciennes, Cyclocheilichthys heteronema 1853) (Bleeker, Cyclocheilichthys repasson 1853) (Bleeker, Desmopuntius hexazona de Beaufort, 1912) Desmopuntius johorensis 1904) (Duncker, Desmopuntius pentazona 1894) (Boulenger, Hampala macrolepidota van Hasselt, 1823) Hampala sabana 1962 Hypophthalmichthys molitrix 1844) (Valenciennes, Table 1 Table cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 431 Sule, H. A., Ismail, A. and Amal, M. N. A. Zakaria et al., 1999; Zakaria et al., 1999; 2004 Rezawaty, 1998; Tan, Martin-Smith & Hassan et al., 2010 Abdullah, 1989; Davies & 2004; Shah et Rezawaty, al., 2006 1998 Tan, Martin-Smith & 1998 Tan, Martin-Smith & Abdullah, 1989 Davies & Abdullah, 1989; Davies & 1993 IPT-AWB, 1998; Tan, Martin-Smith & Sade & Biun, 2012 1998 Tan, Martin-Smith & 1993 IPT-AWB, 1998 Tan, Martin-Smith & Sade & Biun, 2012 Ng et al., 1994; Beamish Ahmad et al., et al., 2003; 2005; Giam et al., 2012 Abdullah, 1989; Davies & 1993; Zakaria et IPT-AWB, 2004; al., 1999; Rezawaty, Ahmad et al., 2005; Shah al., 2006 x x x x x x x x x x x x x x x x x x x x x x NE NE LC NE NT LC NE NE LC LC LC NE NE LC

(Bleeker, (Bleeker, Boulenger, Boulenger, Popta, 1905 (Inger & (Inger & (Bleeker, (Bleeker,

Karnasuta, Karnasuta, (Axelrod, (Axelrod, Karnasuta, 1993 (Popta, 1904) leptocheilus Kottelatia brittani 1976) Lobocheilos bo ** 1842) (Valenciennes, Labiobarbus sabanus Chin, 1962) Luciosoma pellegrinii macrochirus Macrochirichthys 1844) (Valenciennes, Mystacoleucus marginatus 1842) (Valenciennes, Nematabramis everetti Osteochilus chini Osteochilus enneaporos Osteochilus ingeri 1993 Osteochilus microcephalus 1842) (Valenciennes, Osteochilus spilurus 1851) Osteochilus vittatus 1842) (Valenciennes, 1894 1852) Table 1 Table cont’d

432 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest IPT-AWB, 1993 IPT-AWB, Abdullah, 1989; Davies & 1993; Shah et IPT-AWB, al., 2006; Khairul-Adha et al., 2009 Giam et al., 2012 Giam et al., 2012 Giam et al., 2012 Ahmad et al., 2005 Abdullah, 1989; Davies & 1993; Ismail et IPT-AWB, al., 2013 Abdullah, 1989; Davies & 1993; Martin- IPT-AWB., 1998 Tan, Smith & Abdullah, 1989; Davies & 2004; Shah et Rezawaty, al., 2006 Abdullah, 1989 Davies & Ahmad et al., 2005 1998 Tan, Martin-Smith & Ahmad et 1993; IPT-AWB, al., 2005 2004 Rezawaty, x x x x x x x x x x x x x x x x x x LC NE - - LC LC DD LC LC LC NE NE LC LC (Fowler, (Fowler, (Bleeker, (Bleeker, (Bleeker, (Bleeker, (Bleeker, (Bleeker, Smith, 1934 (van (van (Bleeker, (Bleeker, (Valenciennes, (Valenciennes, (Jerdon, 1849) (Bleeker, 1851) (Bleeker, sp. “North Selangor” sp. “Pahang” Osteochilus enneaporos anomalura Hasselt, 1829) micromegethes 2006 Witte, & Tan Kottelat, Britz, Paedocypris Paedocypris hypophthalmus 1860) (Bleeker, Parachela oxygastroides 1852) bulu binotatus 1842) Puntius fasciatus Puntigrus partipentazona 1934) argyrotaenia 1849) Rasbora bankanensis 1853) Rasbora borapetensis 1852) Table 1 Table cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 433 Sule, H. A., Ismail, A. and Amal, M. N. A. Davies & Abdullah, 1989; Abdullah, 1989; Davies & 1993; Zakaria et IPT-AWB, 2004; al., 1999; Rezawaty, Shah et al., 2006; Sade & Biun, 2012; Siow et al., 2013 Ahmad et Ng et al., 1994; al., 2005 1993; Ng et al., IPT-AWB, 1994 1993; Beamish IPT-AWB, Ahmad et al., et al., 2003; 2005 Abdullah, 1989; Davies & 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad Ahmad et al., 2005; et al., 2013; Ismail 2013; Siow et al., 2013 Abdullah, 1989; Davies & 1993; Ng et IPT-AWB, al., 1994; Zakaria et 1999; Beamish et al., 2003; Ahmad et 2004; Rezawaty, al., 2005; Shah et 2006; Ahmad et al., 2013; Ismail et al., 2013 1993; Ismail et IPT-AWB, al., 2013 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad Ahmad et al., 2005; et al., 2013; Ismail 2013 x x x x x x x x x x x x x x x x NE NE NE NE NE NE NE NE (Bleeker, (Bleeker, (Bleeker, (Bleeker, (Duncker, (Duncker, (Bleeker, (Bleeker, (Bleeker, (Bleeker, (Bleeker, (Bleeker, (Duncker, (Duncker, (Kottelat, 1991) sumatrana cf. Rasbora 1852) Rasbora dorsiocellata Rasbora maculata 1904) Rasbora gracilis Rasbora kalochroma 1851) Rasbora cephalotaenia 1852) Rasbora dusonensis 1850) Rasbora einthovenii 1851) 1904) Table 1 Table cont’d

434 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest Ahmad et al., 2005 Ahmad et al., 2005 Parenti & Lim, 2005 Zakaria et al., 1999; Ahmad et 2004; Rezawaty, al., 2005; Shah et 2006 Zakaria et al., 1999; 2004; Shah et Rezawaty, al., 2006; Ismail et 2013 Giam et al., 2012 Abdullah, 1989 Davies & 1993; Ng et al., IPT-AWB, 1994; Zakaria et al., 1999 ; Ahmad et al., 2005; Khairul Ahmad Adha et al., 2009; et al., 2013; Ismail 2013 Ismail et al., 2013; Siow al., 2013 1993; Zakaria IPT-AWB, Ahmad et al., et al., 1999; 2005 Abdullah, 1989; Davies & 1993; Rezawaty, IPT-AWB, 2004 1998 Tan, Martin-Smith & 1993 IPT-AWB, x x x x x x x x x x x x x x x x x x x x x LC LC LC LC NE NE LC NE NE LC NE - NE

Conway, Conway,

(Bleeker, (Bleeker, (Bleeker, (Bleeker, Ahl, 1935 (Kottelat, (Kottelat, Steindachner, Steindachner, (Duncker, (Duncker, Ahl, 1922 Volz, 1903 Volz, sp. Rasbora elegans Rasbora paucisqualis Rasbora tornieri Rasbora trilineata 1870 Striuntius lineatus 1904) Sundadanio margarition 2011 Tan, Kottelat & thynnoides 1842) (Valenciennes, pauciperforatum Trigonopoma & de Beaufort, 1916) (Weber gracile Trigonopoma 1991) heteromorpha Trigonostigma 1904) (Ducker, Oxyeleotris marmorata 1852) Gobiid Pseudobiopsis oligactis 1875) Table 1 Table Eleotrididae Gobiidae cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 435 Sule, H. A., Ismail, A. and Amal, M. N. A. IPT-AWB, 1993; Ng et 1993; Ng et IPT-AWB, 2004; al., 1994; Rezawaty, Ahmad et al., 2005; Shah et al., 2006; Khairul-Adha Ahmad et al., et al., 2009; 2013; Ismail et al., Siow et al., 2013 1993 IPT-AWB, 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad et al., 2013; Ismail et al., 2013 Shah et al., 2006 1993; Ng et IPT-AWB, al., 1994; Zakaria et 1999; Beamish et al., 2003; Ahmad Ahmad et al., 2005; et al., 2013 Ahmad et al., 2005; Zakaria et al., 1999; Beamish 2003; Ismail et al., 2013 Abdullah, 1989 Davies & Ahmad et 1993; IPT-AWB, al., 2005; Shah et 2006 Abdullah, 1989; Davies & 2004; Shah et Rezawaty, al., 2006; Ismail et 2013 x x x x x x x x x x x x x x x x x x x LC LC LC DD NT LC LC NE LC

(La (La (Bloch, (Bloch, (Pallas, (Pallas, (Cuvier, (Cuvier, (Gray, 1835) (Gray, (Hamilton, 1822) (Bleeker, 1851) (Bleeker, Helostoma temminkii 1829) aculeatus Macrognathus circumcinctus Macrognathus (Hora, 1924) Mastacembelus armatus Nandus nebulosus Nemacheilus selangoricus 1904) (Duncker, Chitala chitala Chitala lopis Notopterus notopterus 1976) 1787) Cepède, 1800) Table 1 Table Helostomatidae Mastacembelidae Nandidae Nemacheilidae Notopteridae cont’d

436 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest Davies & Abdullah, 1989; Abdullah, 1989; Davies & 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad et 2004; Rezawaty, al., 2005; Shah et 2006; Ahmad et al., 2013; Ismail et al., 2013 Parenti & Lim, 2005 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad Ahmad et al., 2005; et al., 2013; Ismail 2013 Giam et al., 2012 Ahmad et Ng et al., 1994; al., 2013; Ismail et 2013 Giam et al., 2012 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad Giam et al., 2012; et al., 2013; Ismail 2013 1993 IPT-AWB, Giam et al., 2012 Abdullah, 1989; Davies & Zakaria et al., 1999; Beamish et al., 2003; Ismail et al., 2013; Siow al., 2013 Giam et al., 2012 2004; Shah et Rezawaty, al., 2006 x x x x x x x x x x x x x x x x NE NE LC NE VU NE EN LC CR NE NE VU Ng & Kottelat, Ng & Kottelat, Witte & & Witte Schaller, 1986 Schaller, (Cuvier, 1831) (Cuvier, Regan, 1910 Tan & Ng, 2004 Tan Regan, 1910 Tan & Tan, 1996 Tan, & Tan (Ladiges, 1975) (Cantor, 1849) (Cantor, (Sauvage, 1884) (Ng & Kottelat, 1992) hasselti akarensis Betta bellica Betta brownorum Schmidt, 1992 Betta hipposideros 1994 Betta ibanorum Betta livida Betta imbellis Betta persephone Betta pugnax Betta pulchra Betta splendens Table 1 Table Osphronemidae cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 437 Sule, H. A., Ismail, A. and Amal, M. N. A. Rezawaty, 2004 Rezawaty, Giam et al., 2012 Ahmad et al., 2005; Giam et al., 2012 Ahmad et 1993; IPT-AWB, al., 2005; Giam et 2012 Abdullah, 1989; Davies & 1993; Ng et IPT-AWB, al., 1994; Zakaria et 1999; Beamish et al., 2003; Ahmad et al., 2005; Parenti Ahmad et al., & Lim, 2005; 2013; Ismail et al., Siow et al., 2013 2004 Rezawaty, Giam et al., 2012 Giam et al., 2012 Zakaria et al., 1999 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad Giam et al., 2012; et al., 2013; Ismail 2013 Ahmad et al., 2005; Giam et al., 2012 Giam et al., 2012 x x x x x x x x x x x x x x x x x x x NE VU NE NE NE LC NE NE NE NE NE EN Kottelat Kottelat (Brown, (Brown, Kottelat Kottelat Brown, Brown, Schaller, Schaller, (Gray, (Gray, La Cepède, La Cepède, Regan, 1910 Schaller, 1985 Schaller, (Krummenacher, (Krummenacher, Ng & Kottelat, 1994 waseri

Betta taeniata Betta tomi Betta tussyae Betta 1987) Luciocephalus pulcher 1830) goramy Osphronemus 1801 alfredi Parosphromenus & Ng, 2005 allani Parosphromenus filamentosus Parosphromenus 1981 Vierke, harveyi Parosphromenus nagyi Parosphromenus 1985 tweediei Parosphromenus & Ng, 2005 1987 1987) Table 1 Table cont’d

438 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest IPT-AWB, 1993; Ng et 1993; Ng et IPT-AWB, al., 1994; Zakaria et 1999; Beamish et al., 2003; 2004; Ahmad Rezawaty, Ahmad et al., et al., 2005; 2013; Ismail et al., Siow et al., 2013 Abdullah, 1989; Davies & 1993; Ng et al., IPT-AWB, Ahmad et al., 2005; 1994; Ahmad et al., 2013; Ismail et al., 2013 1993; Ng et IPT-AWB, 2004; al., 1994; Rezawaty, Shah et al., 2006; Khairul- Adha et al., 2009 1993; Ng et al., IPT-AWB, Tan, 1994; Martin-Smith & 1998; Beamish et al., 2003; Ahmad et 2004; Rezawaty, al., 2005; Shah et 2006; Khairul-Adha et al., 2009; Ahmad et al., 2013; Ismail et al., 2013; Siow 2013 1993; Ng et al., IPT-AWB, Ahmad et al., 2005; 1994; Ahmad et al., 2013; Ismail et al., 2013; Siow 2013 Hassan et al., 2010 1998; Tan, Martin-Smith & Ahmad et al., 2005 x x x x x x x x x x x x x x x x x x NE NT LC LC NE LC DD

(Pallas, (Pallas, (Bleeker, (Bleeker, (Regan, (Regan, Roberts, Roberts, (Bleeker, 1852) (Bleeker, (Cuvier, 1831) (Cuvier, osphromenoides (Canestrini, 1860) leerii Trichopodus pectoralis Trichopodus trichopterus Trichopodus 1770) vittata Trichopsis lithostoma 1989 Pseudolais micronemus 1910) 1846) Table 1 Table Pangasiidae cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 439 Sule, H. A., Ismail, A. and Amal, M. N. A. IPT-AWB, 1993; Ng et al., 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ismail et al., 2013 Ahmad et 2004; Rezawaty, al., 2005; Shah et 2006; Siow et al., 2013 Abdullah, 1989; Davies & 1993 IPT-AWB, Hassan et al., 2010 Abdullah, 1989; Davies & 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad Ahmad et al., 2005; et al., 2013; Ismail 2013 Siow et al., 2013 Abdullah, 1989; Davies & Shah et al., 2006 Beamish et al., 2003; Siow et al., 2013 1993; Ng et al., IPT-AWB, 1994 1998 Tan, Martin-Smith & Zakaria et al., 1999; Shah et al., 2006 Ahmad et al., 2005 1998 Tan, Martin-Smith & x x x x x x x x x x x x x x x x x NE LC LC NE LC - NT VU DD NE - LC NE

(Inger (Inger (Bleeker, (Bleeker,

(Bleeker, (Bleeker, (Bloch, 1794) (Bleeker, (Bleeker, (Bleeker, (Bleeker, (Bleeker 1852) Inger & Chin, Inger & Chin, (Tan & Ng, 1996) (Tan sp.? sp.? Pristolepis grootii Pristolepis fasciata 1851) bircirrhis 1839) (Valenciennes, Kryptopterus limpok 1852) Kryptopterus macrocephalus 1858) (Bleeker, Kryptopterus Ompok bimaculatus Ompok fumidus Ompok leiacanthus 1853) Ompok sabanus 1959 Ompok apogon 1851) parvanalis Phalacronotus & Chin, 1959) Table 1 Table Pristolepididae Siluridae cont’d

440 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest IPT-AWB, 1993; Ng et al., 1993; Ng et al., IPT-AWB, Ahmad et al., 2005 1994; Beamish et al., 2003; Giam Ahmad et al., et al., 2012; 2013; Ismail et al., 2013 Giam et al., 2012 Ahmad et al., 2005; Siow al., 2013 1998 Tan, Martin-Smith & 1993; Ng et al., IPT-AWB, 1994; Beamish et al., 2003; Ahmad et al., 2005; Shah al., 2006; Siow et 2013 1993 IPT-AWB, 1993 IPT-AWB, Abdullah, 1989 Davies & 1998 Tan, Martin-Smith & Shah et al., 2006 Hassan et al., 2010 Hassan et al., 2010 Hassan et al., 2010 Hassan et al., 2010 x x x x x x x x x x x x x x x x x x x NE NE NE NE NE LC NE NE NE NE NE NE NE NE NE (Volz, (Volz,

Tan & & Tan (Bleeker, (Bleeker, (Peters, (Peters, (Bleeker, (Bleeker, Kuhl & van Kuhl & van Inger & Chin, Inger & Chin, (Hamilton, (Hamilton, (Zuiew, 1793) (Zuiew, Bleeker, 1851 Bleeker, (Bleeker, 1850) (Bleeker, Silurichthys hasseltii 1858) Silurichthys indragiriensis 1904) Silurichthys phaiosoma 1851) leeri Wallago maculatus Wallago 1859 Monopterus albus deokhatoides 1854) (Bleeker, Doryichthys martensii 168) Pao leirus chatareus Toxotes 1822) Dermogenys pusilla Hasselt, 1823 byssus Hemirhamphodon kapuasensis Hemirhamphodon Collett, 1991 kuekenthali Hemirhamphodon 1901 Steindachner, phaiosoma Hemirhamphodon 1852) (Bleeker, Lim, 2013 Table 1 Table Symbranchidae Tetraodontidae Toxotidae cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 441 Sule, H. A., Ismail, A. and Amal, M. N. A. IPT-AWB, 1993; Ng et 1993; Ng et IPT-AWB, al., 1994; Zakaria et 1999; Beamish et al., 2003; 2004; Ahmad Rezawaty, Ahmad et al., et al., 2005; 2013; Ismail et al., 2013 = East Peninsular Malaysia (part of x x x LC

Hemirhamphodon pogonognathus Hemirhamphodon 1853) (Bleeker, IUCN, (2015) Table 1 Table cont’d * Introduced species **Confusing taxonomy IUCN Status source: = Extinct in the Vulnerable, EN = Endangered, CR Critically EW VU = Threatened, = Near NE = Not Evaluated, DD Data Deficient, LC Least Concern, NT EX = Extinct Wild, NSPSF = North Selangor peat swamp forest, PBPSF Paya Beriah SEPPSF Southeast Pahang EP PSFs = Peat Swamp Forests Terengganu), Pahang and

442 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

Table 2 Summary of black water fish species recorded in Malaysia

Peat swamp sites Location Region No. of No. of Reference families species NSPSF Selangor Peninsular 23 114 Davies & Abdullah, Malaysia 1989; IPT-AWB, 1993; Beamish et al., 2003; Ahmad et al., 2013; Ismail et al., 2013; Siow et al., 2013 PBPSF Perak Peninsular 18 49 Zakaria et al., 1999; Malaysia Rezawaty, 2004; Shah et al., 2006 Black water ditches and Johor Peninsular 7 13 Kottelat, 1996; Ng & ponds of west Johor and Malaysia Kottelat, 1998; Giam et Ambat al., 2012 SEPPSF Pahang Peninsular 19 58 Ahmad et al., 2005 Malaysia Endau drainage, black Pahang and Peninsular 5 9 Kottelat, 1996; Ng & water sites including Terengganu Malaysia Tan, 1999; Giam et al., ponds and ditches 2012 Segama River, Maliau Sabah Malaysia 12 31 Martin-Smith & Tan, Basin 1998; Sade & Biun, 2012 Rajang Basin, Sadong, Sarawak Malaysia 13 40 Parenti & Lim, 2005; Batang Kerang, Nanga Borneo Khairul-Adha et al., Merit 2009; Hassan et al., 2010; Giam et al., 2012; Tan & Lim, 2013

Paya Beriah Peat Swamp Forest, North The most recognised and well-documented Perak surveys in PBPSF were done by Zakaria et The Paya Beriah peat swamp forest al. (1999), Rezawaty (2004) and Shah et al. (PBPSF), which is located near Bukit (2006). Merah reservoir, Perak, has a total area Zakaria et al. (1999) conducted a study of 5,500 hectares that are bordered by of the swamp-riverine fish populations of dykes, roads and railway tracks. Like most two spatially isolated fresh water swamp PSFs in Malaysia, it has been significantly ecosystems: Beriah Kiri River, which was impacted through conversion to residential, mistakenly identified as Beriah Kanan River industrial and agricultural uses (Ismail & (Shah et al., 2006), located in northern Ali, 2002). Nonetheless, the fish population Peninsular Malaysia, and the Ulu Sedili and its biodiversity in PBPSF are not well River in southern Peninsular Malaysia. The documented, in comparison with NSPSF. Beriah Kiri and Ulu Sedili river systems are separated by an extensive mountain

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 443 Sule, H. A., Ismail, A. and Amal, M. N. A. range and a north-south distance of about Fishes Recorded from Southeast 900 km, creating an effective dispersal Pahang Peat Swamp Forest and/or migration barrier (Johnson, 1967a; Ahmad et al. (2005) recorded 58 fish species Prentice & Parish, 1992; Krebs, 2009). (with an additional unidentified species Zakaria et al. (1999) reported a total of 24 belonging to the family Mastacembelidae) fish species identified from their study, of from 17 families (19 following the recent which 20 species were from Beriah Kiri and taxonomic revisions detailed in Kottelat’s 10 species from Ulu Sedili swamp-riverine catalogue) during a survey in Bebar River, area. Eight families were represented a large river that flows out of the southeast from the Beriah Kiri swamp-riverine area. Pahang peat swamp forest (SEPPSF, which However, only the fish species recorded is also known as Pekan peat swamp forest). from the Beriah Kiri swamp-riverine are This survey, which was conducted along outlined in this review. Bebar River and Serai River (a tributary of A study from Rezawaty (2004) reported Bebar River), brought the total fish species a total of 30 fish species from 12 families known in SEPPSF to 65 species (see Ahmad in a study carried out on the entire Sungai et al., 2005) (see Table 1). Beriah drainage system, while during an intensive survey of the PBPSF, specifically Fishes Recorded from the Peat Swamps in Beriah Kanan, Beriah Kiri and Beriah of East Peninsular Malaysia rivers, Shah et al. (2006) recorded a total Kottelat (1996) and Ng and Tan (1999) of 32 fish species belonging to 13 families recorded S. lineatus and Neohomaloptera (Table 1). johorensis respectively from east Peninsular Malaysia. Similarly, Giam et al. (2012) Fishes Recorded from the Peat Swamps recorded nine fish species from six families of Johor from the black waters of east Peninsular Information on the ichthyofauna of PSFs Malaysia comprising of part of Malaysia’s of the state of Johor is relatively patchy Johor, Pahang and Terengganu (Table 1). compared to the NSPSF. Kottelat (1996) and Ng and Kottelat (1998) recorded Fish Species Recorded in the Peat Systomus lineatus and Hyalobagrus ornatus Swamp Forests of Sabah respectively from PSF of Johor. Giam et al. Sabah, together with Sarawak, Kalimantan (2012) recorded 13 fish species belonging Barat, Kalimantan Timur and Brunei is to seven families from black water ditches, situated in Borneo, the world’s third largest streams, pools, remnants of PSF and PSFs island with some 743,107 km2 of land area. of West Johor and Ambat (Table 1). As a result of “…difficulties of access to the interior part of tropical rainforest, a

444 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest lack of reliable dating of igneous rocks, Malaysia. The ichthyofauna of Sarawak is poorly fossiliferous sedimentary rocks generally neglected in comparison to the and an absence of a coherent stratigraphic other political divisions of Borneo for which scheme for many parts of the island” (Hall detailed checklists have been documented & Nichols, 2002), there is only fragmentary (Parenti & Lim, 2005). information on the geology and fish fauna of Parenti and Lim (2005) carried out a the island (Parenti & Lim, 2005). The fish study of the ichthyofauna of the Rajang species of Sabah are relatively less surveyed Basin in Sarawak, Malaysia, Borneo, and and documented than those of Peninsular presented a checklist of 164 fish species Malaysia. Even when intensive surveys recorded from the headwaters all the way are carried out, they have not been widely to the brackish waters, including Belaga and published by the researchers. the Balui River, Kapit and the Baleh River, Martin-Smith and Tan (1998) carried and Sibu area. A total of seven blackwater out an intensive collection of freshwater fish species were recorded from blackwater fishes over a period of two years from the ditches and pools along Teku River and catchment of the upper Segama River near remnant of PSF behind the old Sibu airport. Danum Valley Field Centre, headwater A previous study from Khairul-Adha streams in the catchment of the Kuamut et al. (2009) recorded 36 species of fish River and from the lower Segama River. belonging to 13 families from brownish They reported a total of 65 species from and blackish water habitats of Batang 20 families. A total of about 30 fish species Kerang in Balai Ringin, Sarawak, during were recorded in blackwater ditches, ponds wet and relatively dry seasons. Thirty-two and streams from the lower Segama River species from 12 families were recorded in (Martin-Smith & Tan, 1998). the brownish water, while only 12 species Also, Sade and Biun (2012) studied from seven families were recorded in the the ichthyofauna of Maliau Basin, a black water habitat. In a study by Hassan saucer-shaped depression enclosed by a et al. (2010), seven out of 15 fish species mountainous rim in the remote part of recorded from 11 families in Nanga Merit Sabah, with an undisturbed flora and fauna. area were from a water body with peat-like Although 15 fish species were recorded from characteristics. the study, only three species were recorded Recently, Tan and Lim (2013) from acidic tea-coloured blackwaters in the recorded four species of fish of the zone (Table 1). Hemirhamphodon from blackwater ditches and ponds in Sarawak, Malaysia, Borneo, Fish Species Recorded in the Peat while Giam et al. (2012) recorded 13 fish Swamp Forests of Sarawak species belonging to five families from Like Sabah, the ichthyofauna of the peat the peat swamps of Rajang and Sadong in swamps of Sarawak has received little Sarawak (Table 1). attention in comparison to that of Peninsular

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 445 Sule, H. A., Ismail, A. and Amal, M. N. A.

TAXONOMIC NOTES and Ahmad et al. (2005). The Parakysis Some of the taxonomic names of the verucosus of the family Akysidae was also fish species outlined in Table 1 and their classified under Parakysidae by IPT-AWB placement within families are slightly or (1993) and Ng et al. (1994), while Siow et completely different from the originally al. (2013) recorded Pristolepis fasciata of recorded names from the surveys, as a result the family Pristolepididae as belonging to of the recent changes in the taxonomy of the family Nandidae. Also, a recorded fish the fish species. A comprehensive list of species belonging to the genus Kryptopterus taxonomic changes is given in Kottelat’s was not designated a species name and catalogue (Kottelat, 2013). Davies and the reason was not stated appropriately. Abdullah (1989) recorded O. vittatus, Considering there are about 17 distinct along with its synonym O. hasselti, as two species in the genus Krptopterus (Kottelat, completely different species. The species 2013), it is possible that the sample could Belontia hasselti now classified under be a newly discovered species. the family Osphronemidae was placed The species recorded as Puntius lineatus in the Anabantidae family. Members of is now named Striuntius lineatus, which Osphronemidae family were placed in is a “preferred” name or “authority”, the the family Belontia by IPT-AWB (1993) latter being a synonym. Another species and Ng et al. (1994). In the same vein, recorded as Labiobarbus lineatus, which Luciocephalus pulcher was classified is excluded from Kottelat’s catalogue was under the family Luciocephalidae. regarded as a preferred name for the species Following the work of Britz (1994), and Dangila lineata in Fish Base, Species 200 Kottelat and Whitten (1996), the earlier and ITIS Catalogue of Life: April 2013, families of Osphronemidae, Belontiidae and the NCBI Taxonomy (Torrres, 2000; and Luciocephalidae were constituted EOL, 2015). Dangila lineata was also as a single family, Osphronemidae. recorded as a synonym of L. leptocheilus Recently, Beamish et al. (2003) classified in Kottelat’s catalogue (Kottelat, 2013), Nemacheilus selangoricus under the family suggesting that both names may actually Nemacheiliidae in the family Bagridae, along be referring to the same species. However, with N. johorensis. In the same vein, Ahmad Rainboth et al. (2012) considered that L. et al. (2005) classified N. selangoricus of lineatus is a distinct species. There has been the family Nemacheilidae and Barbucca some confusion about the taxonomy of the diabolica of the family Barbuccidae species L. leptocheilus (L. lineatus). In the under the family Balitoridae. Meanwhile, taxonomic outline provided in this review, Hemirhamphodon progonognathus of the the species is presented as L. leptocheilus in family Zenarchopteridae was placed in the line with Kottelat’s catalogue. Also, the fish family Hemiramphidae by IPT-AWB (1993), earlier recorded as Mystus micracanthus is Ng et al. (1994), Beamish et al. (2003) actually M. nigriceps (Roberts, 1993; Ng, 2002; Kottelat, 2013).

446 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

ECONOMIC IMPORTANCE OF PEAT and distribution fragmentation (Mace et al., SWAMP FISHES 2008; Biodic, 2013; IUCN, 2014). The Black water peat swamps are important categories are “Extinct (EX), Extinct in the catchment areas. The peat has great water Wild (EW), Critically Endangered (CR), retention ability and serves as reservoir of Endangered (EN), Vulnerable (VU), Near rain water, which is utilised in agriculture for Threatened (NT), Least Concern (LC), irrigating the rice fields adjacent the NSPSF Data Deficient (DD) and Not Evaluated (Low & Balamurugan, 1989). The fish of (NE)” (IUCN, 2001; InfoNatura, 2007; NSPSF are harvested for consumption and IUCN, 2012; IUCN, 2015; IUCN, 2014). A also for the aquarium trade due to their species is EX when there is no reasonable ornamental value (Ng et al., 1994; Ismail doubt that the last individual has died, as et al., 2013) (Table 4). Most of the species a result of failure to record an individual harvested for consumption by the local through exhaustive surveys in known and/ people are sold at very low prices relative to or expected habitat, at appropriate times their actual worth (Ng et al., 1994). (diurnal, seasonal, annual), throughout its A breeding study has been carried historic range. Extinct in the Wild is used out on some of the ornamental fish of to refer to species that are known only to peat black water such as pearl gourami survive in cultivation, in captivity or as a (Trichogaster leeri), chocolate gourami naturalised population(s) well outside the (Sphaerichthys osphromenoides), clown past range (InfoNatura, 2007; Biodic, 2013; rasbora (Rasbora kalochroma), six-banded IUCN, 2014). barb (P. johorensis), bellicose Betta (Betta Out of the 198 black water fish species bellica), giant fighting fish (B. waseri) and recorded from Malaysia, 106 (53.54%) pygmy rasbora (R. maculata), with the aim species belonging to the category NE of conserving the species and improving indicate that their conservation status has their production (FFRC, 1995). not been evaluated against the criteria. Seven (3.53%) species are DD as a result of CONSERVATION STATUS OF PEAT inadequate information on their distribution SWAMP FISH SPECIES IN MALAYSIA and/or population status. Sixty-two species The conservation status of a species indicates (31.31%) belonging to the LC category, if it still exists and how likely it is to become indicate lowest risk, widespread and extinct in the near future (InfoNatura, 2007). abundant species. Six species (3.03%) The IUCN Red List of Threatened Species are NT, signifying likelihood to become is the best known conservation status listing endangered in the future. Six (3.03%) system in the world, which classifies species and two (1.01%) species are VU and EN, into nine categories based on the rate of respectively, indicating a highly and very decline of the population, population size, highly risk of extinction in the wild. Besides geographical range, and degree of population that, three species (1.52%) are CR and

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 447 Sule, H. A., Ismail, A. and Amal, M. N. A. facing an extremely high risk of extinction Welcomme, 1979). Therefore, conservation in the wild, while six species (3.03%) were surveys should be exhaustive, in all known not fully classified and so exempted from all and/or expected habitats, at appropriate the categories (Table 1). times covering seasons of high and low A total of only 12 species (6.70%) water levels over an extended period of are threatened, i.e. under CR, EN and VU time before a species is pronounced as rare category. The IUCN classification may not or threatened. be a true expression of the conservation Considering the extensive destruction status of black water fishes, particularly of tropical PSFs worldwide and the decline in Malaysia as so many of the species in PSF biodiversity, more black water fish (57.54%) remain unevaluated against the species will fall into the threatened (CR, criteria for classification of threatened EN and VU) category if evaluated against species. In data-poor situations, it is not the criteria. However, it is important to uncommon for listing errors of species that note that the category of threat simply otherwise should be classified as threatened provides an assessment of the extinction (Gärdenfors, 2000; Keith et al., 2000; risk under current circumstances, and is not Gärdenfors et al., 2001; Keith et al., 2004). necessarily sufficient to determine priorities For instance, five of the ten black water for conservation action (Mace & Lande, fish species B.( brownorum, Sundadanio 1991). margarition, B. ibanorum, Parosphromenus allani and H. ornatus), listed as “most VU” CONSERVATION OF PEAT SWAMP by Giam et al., (2012) using the criteria of FORESTS decline in population, geographical range Peat swamp forests are an important and basin extinction, were classified under component of the world’s wetlands, the NE in Red List category (Tables 1 and 3). providing a wide variety of goods and It is important to point out that the services in the form of carbon sequestration, species recorded as ‘rare’ at a distinct time flood mitigation and globally important may be more abundant at other times. For biodiversity reservoirs (Parish, 2002; example, the abundance of individual fish UNDP, 2006; Posa et al., 2011; Ismail et al., and species composition vary during the 2013), with many endemic species (Kottelat wet and dry seasons, which may be related & Ng, 2005; Kottelat & Widjanarti, 2005; to variations in migratory movements of fish UNDP, 2006; Tan & Kottelat, 2009; Conway species (Ng et al., 1994; Renato et al., 2000; et al., 2011; Conway & Kottelat, 2011; Posa Khairul-Adha et al., 2009). Some species et al., 2011; Giam et al., 2012; Taskforce migrate from downstream to upper reaches REDD, 2012; PIU-SERT, 2013; Muchlisin of a river during high water levels for et al., 2015). breeding or food, then migrate back to the Malaysian PSFs make up about 75% lower reaches after spawning as the water of the country’s total wetlands, with 80% level reduces (Lowe-McConnell, 1975;

448 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

Table 3 Peat swamp fishes listed as Threatened in IUCN Red List

Family Species Status Reference Anguillidae Anguilla borneensis VU Martin-Smith & Tan, 1998 Clariidae Encheloclarias CR IPT-AWB, 1993; Ng et al., 1994; Giam et al., 2012 curtisoma Encheloclarias CR Giam et al., 2012 keliodes Encheloclarias VU Giam et al., 2012 prolatus Osphronemidae Ompok fumidus VU Beamish et al., 2003; Siow et al., 2013 Betta hipposideros VU Ng et al., 1994; Ahmad et al., 2013; Ismail et al., 2013 Betta livida EN IPT-AWB, 1993; Ng et al., 1994; Beamish et al., 2003; Giam et al., 2012; Ahmad et al., 2013; Ismail et al., 2013 Betta splendens VU Rezawaty, 2004; Shah et al., 2006 Betta tomi VU Giam et al., 2012 Betta persephone CR Giam et al., 2012 Parosphronemus EN IPT-AWB, 1993; Ng et al., 1994; Beamish et harveyi al., 2003; Giam et al., 2012; Ahmad et al., 2013; Ismail et al., 2013 found in east Malaysia (Sabah 8%; Sarawak The main threats of PSFs in Malaysia 72%) and 20% in Peninsular Malaysia. Peat are forestry (overexploitation), illegal swamp forests in Malaysia have undergone logging, pollution (including oil, industrial, severe degradation over the years. For nutrient and sedimentation), waste example, the PSF cover of NSPSF was disposal, airports, land reclamation, large estimated to be 0.67 million hectares in scale land conversion for agriculture, 1981 but reduced drastically to 0.34 million industrialisation and settlement-urbanisation hectares in the 1990s (UNDP, 2006) and (Ng & Shamsudin, 2001; Chong et al., there has been further destruction since 2010). In addition, draining and clearing then. As the nation becomes aware of the of the PSFs for agriculture and palm oil need to conserve PSFs, some percentage of plantations have resulted in peat land fires PSFs have been protected within Permanent (Parish, 2002; Page et al., 2011), which Forest Reserves and stateland forests contribute to the annually recurring episodes (UNDP, 2006). However, this protection is of transboundary haze pollution in the inadequate. Agricultural conversion and fire Southeast Asian region (Lo & Parish, 2013). have destroyed PSF within these protected Several studies on the diversity of areas and furthermore, unless the entire fish in PSFs have revealed the existence forests are protected, any drainage around of economically important fishes the edges impacts the entire forest. for consumption, aquarium trade and

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 449 Sule, H. A., Ismail, A. and Amal, M. N. A.

Table 4 Commonly consumed and ornamental black water fish species in Malaysia

Consume Ornamental Anabas testudineus Betta bellica Belontia hasselti Betta livida Channa bankanensis Betta hipposideros Channa lucius Belontia hasselti Channa melosoma Channa gachua Clarias meladerma Helostoma temminkii Clarias nieuhofii Hemirhamphodon progonognathus Clarias leiacanthus Krvptopterus macrocephalus Helostoma temminkii Leiocassis micropogon Kryptopterus macrocephalus Luciocephalus pulcher Monopterus albus Macrognathus circumcinctus Mystus bimaculatus Mystus bimaculatus Macrognathus circumcinctus Osteochilus spilurus Hemibagrus nemurus Parosphromenus harveyi Pristolepis grootii Desmopuntius hexazona Trichopodus leerii Desmopuntius johorensis Trichopodus pectoralis Rasbora cephalotaenia Trichopodus trichopterus Rasbora dorsiocellata Rasbora einthovenii Rasbora gracilis Rasbora kalochroma Rasbora maculata Trigonopoma pauciperforatum Silurichthys hasseltii Sphaerichthys osphromenoides Trichopodus leerii Trichopodus trichopterus endangered species (IPT-AWB, 1993; use types vary in their ability to support Ng et al., 1994; Lee, 2001; Beamish et forest biodiversity, generally having a al., 2003), where the majority of fish are reduced capacity for biodiversity support highly dependent on the rivers in the PSFs than in their original unconverted form. (Beamish et al., 2003; Yule, 2010). The Giam et al. (2012) predicted the number degradation of PSFs is expected to affect of fish species that would become extinct the fish community structure in such a way under different land-use conditions using that extinction of some already endangered the Matrix-Calibrated Species-Area Model species in the near future is probable. Gibson (MCSAM) (Koh & Ghazoul, 2010) and et al. (2011) reported that converted land- Mote Carlo simulations to project PSF

450 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest basins extinctions. Under the scenario, the soil carbon storage (Page et al., 2011). 10 most vulnerable species (Encheloclarias Large scale conversion of this carbon sink, prolatus, B. brownorum, S. goblinus, S. as is happening today, is expected to and margarition, B. ibanorum, B. burdigala, E. will severely impact the earth’s climate tapeinopterus, Paedocypris progenetica, (Couwenberg et al., 2009; van der Werf P. allani and H. ornatus) were recognised et al., 2009; Miettinen et al., 2012) unless and predicted to be extinct by 2050, if the drastic measures are put in place to conserve present rate of PSFs conversion continues. PSFs of the world. Meanwhile, Beamish et al. (2003) In the discussion of conservation of PSFs reported 22 species of fish from 43 sites in fish biodiversity, safe fishing techniques the NSPSF in 1998 as against 33 species have always been ignored. Researchers, from 27 sites during the preceding year. and fishermen alike, employ varying fishing The PSF was largely forested during the techniques in PSFs and have always been 1997 survey, but the area was cleared and more concerned with increase in catchability planted with oil palms in 1998. Thirteen than safety and conservation of the fishes. species caught in 1997 were not caught in One fishing technique, electro-fishing, that 1998. This supports the prediction by Giam poses a threat to the health of fish has been et al. (2012) the extinction of fish species employed in several surveys in Malaysia following PSF land conversion. Generally, PSFs (Martin-Smith & Tan, 1998; Beamish riparian vegetation acts as a source of energy et al., 2003; Shah et al., 2006; Siow et al., and matter (Kindler, 1998), and contributes 2013). Electro-fishing establishes an electric matter to the PSF ecosystem through field in the water (Lamarque, 1990) creating production of leaf litter (Tabacchi et al., an epileptic response in fish resulting 1998). Modifications in riparian vegetation from electric shock to the central nervous through logging affect the structure and system (Reynolds & Kolz, 1993; Sharber processes within the peat swamps. It leads to & Black, 1999). The reported health effects the alteration of the swamp characteristics, on fish include internal haemorrhaging and reduction of food resources (Tabacchi et skin discolorations (Dalbey et al., 1996; al., 1998; Wright & Flecker, 2004) and Kocovsky et al., 1997; Muth & Ruppert, subsequent loss of biodiversity (Bruenig & 1997; Thompson et al., 1997; Ainslie et al., Droste, 1995). 1998; Habera et al., 1999), spinal injuries Peat swamp forests also serve as an (Kocovsky et al., 1997), lactacidosis and important global carbon storehouse (Parish, disturbance of the inter-renal stress response 2002; Chimer & Ewel, 2005; Jauhiainen et (Mitton & McDonald, 1994), retarded al., 2005; Rydin & Jeglum, 2006; UNDP, growth (Dalbey et al., 1996; Thompson et 2006). The PSFs of Malaysia and Indonesia al., 1997; Ainslie et al., 1998; Hughes, 1998), alone store 67 gigatons of carbon in peat, and low gamete viability (Muth & Ruppert, which represents 75% of total tropical peat 1997; Koupal et al., 1997). Although the

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 451 Sule, H. A., Ismail, A. and Amal, M. N. A. effects of electro-fishing on PSF biodiversity swamp forests in Malaysia are totally is minor compared to the enormous loss due inadequate (and in any case, they are largely to PSF drainage, clearance and fire, it is ignored). appropriate that only safe fishing techniques such as the use of scoop nets, cast nets, etc. REFERENCES should be employed in a unique habitat such Ahmad, A. B., Ahmad, M. F., & Nek, S. A. R. as peat swamps. T. (2013). Freshwater fishes recorded from north Selangor peat swamp forest, Selangor. CONCLUSION Proceedings of the Seminar on Scientific Biodiversity Expedition, North Selangor Peat It is apparent from the ongoing that one of Swamp Forest, 28th November 2013, Quality the strongest justifications for conservation Hotel, Shah Alam, Selangor, Malaysia. pp.72–76. of peat swamps is the existing ichthyofauna. Ahmad, A., Mazlan, A. G., Syed Ahmad Rizal, T. Therefore, maintaining fish populations of N., Abd. Rahman, A. R., Jamil, M., Jacqueline, PSFs is very important as this will require L., & Abdullah, S. (2005). Freshwater fishes of maintaining the natural water tables of the Sungai Bebar and adjacent areas. In Summary swamps with seasonal flooding. Apart from Findings PSF Technical Series No. 4. Peat the necessity to maintain appropriate aquatic Swamp Forest Project. UNDP, GEF, Pahang Forestry Department and Universiti Kebangsaan. habitats for the fish, another important pp.145–148. reason is that when peat dries out due to drainage, it is extremely inflammable Ainslie, B. J., Post, J. R., & Paul, A. J. (1998). Effects of pulsed and continuous DC electrofishing on and thus, becomes highly vulnerable to juvenile rainbow trout. North American Journal destruction by fire (Langner et al., 2007; of Fisheries Management, 18(4), 905–18. Langner & Siegert, 2009; Page et al., 2009; Ali, A. (1999). A case study on the conservation Posa et al., 2011). and management of freshwater wetlands in A review of literature on PSF fish Malaysia. Proceedings of Wetlands, Awareness, and also the IUCN Red List revealed Local People and the Ramsar Convention in the the likelihood of many important fishes River Basin. In O. Callagham (Ed.), becoming extinct in the near future if Can Local People Play a Role in the Wise Use of the present PSFs degradation continues. Wetlands (pp. 22–32). Phnom Penh, . Moreover, there is also an inevitable risk Anderson, J. A. R. (1983). The tropical peat swamps of increased global warming when the tons of western Malesia. In A. J. P. Gore (Ed.), of carbon stored in peat lands is released Systems of the World 4B. Mires: Swamp, Bog, Fen and Moor (pp. 181–99). Amsterdam: through degradation. Consequently, a Elsevier Sci. Publ. concerted effort for conserve regional PSFs is essential, and requires action Azmi, M. I., Cullen, R., Bigsby, H., & Awang, N. A. G. (2009). The existence value of peat swamp forest plans involving all stakeholders (Azmi et in Peninsular Malaysia. Paper presented at New al., 2009). The present measures aimed Zealand Agriculture and Resource Economics at protecting only some parts of the peat Society (NZARES) Conference. 27–28 August

452 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

2009. Tahuna Beach Resort, Nelson, New Ichthyological Exploration of Freshwaters, Zealand. 22(3), 251–288.

Beamish, F. W. H., Beamish, R. B., & Lim, S. L. (2003). Couwenberg, J., Dommain, R., & Joosten, H. (2009). Fish assemblages and habitat in a Malaysian Greenhouse gas fluxes from tropical peatlands in blackwater peat swamp. Environmental Biology Southeast Asia. Global Change Biology, 16(6), of Fishes, 68(1), 1–13. 1715–1732.

Biodic. (2013). Threatened Species. Retrieved from Dalbey, S. R., McMahon, T. E., & Fredenberg, W. www.biodic.go.jp. (1996). Effect of electrofishing pulse shape and electrofishing-induced spinal injury on long-term Britz, R. (1994). Ontogenetic features of Luciocephalus growth and survival of wild rainbow trout. North (Perciformes, ) with a revised American Journal of Fisheries Management, hypothesis of anabantoid intrarelationships. 16(3), 560–569. Zoological Journal of the Linnaean Society, 112(4), 491–508. Davies, J., & Abdullah, A. R. (1989). Freshwater fish survey of the north Selangor peat swamp forest. Bruenig, E. F., & Droste, H. J. (1995). Structure, Asian Wetland Bureau Publication No. 46, IPT dynamics and management of rainforests on Asian Wetland Bureau/WWF Malaysia, Kuala nutrient-deficient soils in Sarawak. In R. B. Lumpur. Primack & T. E. Lovejoy (Eds.), Ecology, Conservation and Management of Southeast Encyclopedia of Life. (2015). Synonyms of Asian Rainforests (pp. 41–53). Yale University Labiobarbus lineatus. EoL Online. Retrieved Press. from http://eol.org/pages/210213/names/ synonyms. Chimer, R. A., & Ewel, K. C. (2005). A tropical freshwater wetland: II. Production, Freshwater Fisheries Research Centre (FFRC). decomposition and peat formation. Asean (1995). Annual report. FFRC Online. Retrieved Wetland Bureau/WWF Malaysia. Kuala Lumpur, from http://www.fri.gov.my/pppat/page11-1. Malaysia. html.

Chong, V. C., Lee, P. K. Y., & Lau, C. M. (2010). Gärdenfors, U. (2000). Population viability analysis Diversity, extinction risk and conservation of in the classification of threatened species: Malaysian fishes.Journal of Fish Biology, 76(9), Problems and potentials. Ecological Bulletins, 2009–2066. 48, 181–190.

Conway, K. W., & Kottelat, M. (2011). Boraras Gärdenfors, U., Hilton-Taylor, C., Mace, G. M., & naevus, a new species of miniature and sexually Rodríguez, J. P. (2001). The application of IUCN dichromatic freshwater fish from Peninsular Red List criteria at regional levels. Conservation Thailand (Ostariophysi: Cyprinidae). Zootaxa, Biology, 15(5), 1206–1212. 3002, 45–51. Giam, X., Koh, L. P., Tan, H. H., Miettinen, J., Tan, H. Conway, K. W., Kottelat, M., & Tan, H. H. (2011). T. W., & Ng, P. K. L. (2012). Global extinctions Review of the Southeast Asian miniature of peat swamp fishes follow peatland conversion cyprinid genus Sundadanio (Ostariophysi: in Sundaland. Frontiers in Ecology and the Cyprinidae) with descriptions of seven Environment, 10(9), 465–470. new species from Indonesia and Malaysia.

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 453 Sule, H. A., Ismail, A. and Amal, M. N. A.

Gibson, L., Lee, T. M., & Koh, L. P. (2011). Primary peat swamp forest. Proceedings of the Seminar forests are irreplaceable for sustaining tropical on Scientific Biodiversity Expedition, North biodiversity. Nature, 478(7369), 378–381. Selangor Peat Swamp Forest, 28th November 2013, Quality Hotel, Shah Alam, Selangor, Goltenboth, F. (2006). Ecology of Insular Southeast Malaysia. pp.57–71. Asia: The Indonesian Archipelago. In F. Goltenboth, K. H., Timotius, P. P., Milan, & J. Ismail, W. R., & Ali, A. (2002). Managing our Margraf (Eds.). Elsevier. wetlands: Some results of the function of a wetland in Perak, Malaysia. Malaysian Journal Habera, J. W., Strange, R. J., & Saxton, A. M. (1999). of Environmental Management, 3, 83–105. AC electrofishing injury of large brown trout in low-conductivity streams. North American IUCN. (2001). IUCN Red List Categories and Journal of Fisheries Management, 19(1), Criteria. Version 3.1. Gland, Switzerland 120–126. and Cambridge, UK: IUCN Species Survival Commission. Hall, R. & Nichols, G. (2002). Cenozoic sedimentation and tectonics in Borneo: Climatic influences on IUCN. (2012). IUCN Red List Categories and orogenesis. In S. J. Jones & L. Frostiock (Eds.), Criteria. Version 3.1. Gland, Switzerland and Sediment Flux to Basins: Causes, Controls and Cambridge, UK: IUCN. Consequences (pp. 5–22), Special Publications. IUCN. (2014). Guidelines for using the IUCN Red London: Geological Society. List Categories and Criteria (Version 11). IUCN Hassan, R., Esa, F. A., & Harith, M. N. (2010). Standards and Petitions Subcommittee. Fish assemblages in Nanga Merit area, Kapit, IUCN. (2015). IUCN Red List of Threatened Species. Sarawak. The Sarawak Museum Journal, 88, Retrieved from www.iucnredlist.org. 274–284. Jauhiainen, J., Takahashi, H., Heikkinen, J. E. P., Hughes, N. F. (1998). Reduction in growth due Martikainen, P. J., & Vasanderet, H. (2005). to electrofishing and tagging may change Carbon fluxes from a tropical peat swamp interannual movement behavior of stream forest floor. Global Change Biology, 11(10), salmonids: Evidence from Arctic grayling in 1788–1797. an interior Alaskan stream. Transactions of the American Fisheries Society, 127(6), 1072–1077. Johnson, D. S. (1967a). Distributional patterns of Malayan freshwater fish. Ecological Society of InfoNatura. (2007). About the data: Conservation America, 48(5), 722–730. status. Retrieved from www.natureserve.org. Johnson, D. S. (1967b). On the chemistry of IPT-Asian Wetland Bureau. (IPT-AWB) (1993). Fish freshwaters in southern Malaya and Singapore. sampling demonstration in north Selangor peat Archiv für Hydrobiologie, 63(4), 477–496. swamp forest. AWB Publication No. 89. Johnson, D. S. (1968). Malayan blackwaters. In R. Irvine, K., Vermette, S., & Mustafa, F. B. (2013). Misra & B. Gopal (Eds.), Proceedings of the The “black waters” of Malaysia: tracking water Symposium on Recent Advances in Tropical quality from the peat swamp forest to the sea. Ecology (pp. 303–310). International Society for Sains Malaysiana, 42(11), 1539–1548. Tropical Ecology, Varanasi. Ismail, A., Amal, M. N. A., Abdullah, T., Johari, S., Keith, D. A., Auld, T. D., Ooi, M. K. J., & Mackenzie, Azizul, A., Nur-Illiani, H., & Amiruddin, M. B. D. E. (2000). Sensitivity analyses of decision H. (2013). Fish diversity in north Selangor rules in World Conservation Union (IUCN) Red

454 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

List criteria using Australian plants. Biological vertebrate. Proceedings of the Royal Society B, Conservation, 94(3), 311–319. 273(1589), 895–899.

Keith, D. A., McCarthy, M. A., Regan, H., Regan, Kottelat, M., & Ng, P. K. L. (2005). Diagnoses of T., Bowles, C., Drill, C., ... & Ruckelshaus, M. six new species of Parosphromenus (Teleostei: (2004). Protocols for listing threatened species Osphronemidae) from Malay Peninsular and can forecast extinction. Ecology Letters, 7(11), Borneo, with notes on other species. The Raffles 1101-1108. Bulletin of Zoology, 13, 101–113.

Khairul-Adha, A. R., Siti, K. D., Siti, S. S., Aziz, A., Kottelat, M., & Whitten, A. J. (1996). Freshwater Yuzine, E., & Eza, R. I. (2009). Freshwater fish fishes of western Indonesia and Sulawesi: diversity and composition in Batang Kerang Additions and corrections. Hong Kong: Periplus. floodplain, Balai Ringin, Sarawak. Pertanika Kottelat, M., & Widjanarti, E. (2005). The fishes of Journal of Tropical Agricultural Science, 32(1), Danau Sentarum National Park and the Kapuas 7–16. Lakes area, Kalimantan Barat, Indonesia. The Kindler, J. (1998). Linking ecological and development Raffles Bulletin of Zoology, 13, 139–173. objectives: Trade-offs and imperatives. Koupal, K. D., Satterfield, J. R., & Flickinger, S. A. Ecological Applications, 8(3), 591–600. (1997). Comparative gear selectivity for male Kocovsky, P. M., Gowan, C., Fausch, K. D., & Riley, walleyes and influence of method of capture S. C. (1997). Spinal injury rates in three wild on resultant hatching success. The Progressive trout populations in Colorado after eight years Fish-Culturist, 59(3), 218–221. of backpack electrofishing. North American Krebs, C. J. (2009). Ecology: The Experimental Journal of Fisheries Management, 17(2), Analysis of Distribution and Abundance (6th ed). 308–313. Benjamin Cummings. San Francisco. Koh, L. P. & Ghazoul, J. (2010). A matrix-calibrated Lamarque, P. (1990). Fishing With Electricity: species–area model for predicting biodiversity Applications in Freshwater Fisheries losses due to land-use change. Conservation Management. In I. G. Cowx, & P. Lamarque Biology, 24(4), 994–1001. (eds.), Fishing News Books. Oxford, England. Kottelat, M. (1996). The identity of Puntius Langner, A., Miettinen, J., & Siegert, F. (2007). Land eugrammus and diagnoses of two new species cover change 2002–2005 in Borneo and the role of striped barbs (Teleostei: Cyprinidae) from of fire derived from MODIS imagery. Global Southeast Asia. The Raffles Bulletin of Zoology, Change Biology, 13(11), 2329–2340. 44(1), 301–316. Langner, A. & Siegert, F. (2009). Spatiotemporal fire Kottelat, M. (2013). The fishes of the inland waters of occurrence in Borneo over a period of 10 years. southeast Asia: A catalogue and core bibliography Global Change Biology, 15(1), 48–62. of the fishes known to occur in freshwaters, mangroves and estuaries. The Raffles Bulletin of Lee, K. Y. (2001). Fish community of the north Zoology, Supplement No. 27, 1–663. Selangor peat swamp forest. (M.Sc. thesis). University of Malaya, Kuala Lumpur, Malaya. Kottelat, M., Britz, R., Tan, H. H., & Witte, K. E. (2006). Paedocypris, a new genus of southeast Lee, P. G., & Ng, P. K. L. (1994). The systematics Asian cyprinid fish with a remarkable sexual and ecology of snakeheads (Pisces: Channidae) dimorphism, comprises the world’s smallest in Peninsular Malaysia and Singapore. Hydrobiologia, 285(1-3), 59–74.

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 455 Sule, H. A., Ismail, A. and Amal, M. N. A.

Lim, R. P., Furtado, J. I., & Morley, R. J. (1982). Mizuno, N., & Furtado, J. I. (1982). Ecological notes General description of Tasek Bera. In J.I. on fishes. In J.I. Furtado & S. Mori (Eds.), Furtado, & S. Mori (Eds.), Tasek Bera (pp. 7-54). Tasek Bera (pp. 321-354). Dordrecht, Springer Monographiae Biologicae. Dordrecht: Springer Netherlands. Netherlands. Muchlisin, Z. A., Akyun, Q., Rizka, S., Fadli, N., Lo, J., & Parish, F. (2013). Peatlands and climate Sugianto, S., Halim, A., & Siti-Azizah, M. N. change in Southeast Asia. ASEAN Peatland (2015). Ichthyofauna of Tripa peat swamp forest, Forests Project and Sustainable Management of Aceh province, Indonesia. Check List, 11, 1–9. Peatland Forests Project. Muth, R. T. & Ruppert, J. B. (1997). Effects of Low, K. S., & Balamurugan, G. (1989). A Preliminary electrofishing fields on captive embryos and Hydrological Investigation of the North Selangor larvae of razorback sucker. North American Peat Swamp Forest. Asean Wetland Bureau/ Journal of Fisheries Management, 17(1), WWF Malaysia. Kuala Lumpur. 160–166.

Lowe-McConnell, R. H. (1975). Fish Communities in Ng, H. H. (2002). The identity of Mystus nigriceps Tropical Freshwater. Longham Inc. New York. (Valenciennes in Cuvier & Valenciennes, 1840), with the description of a new bagrid catfish Mace, G. M., Collar, N. J., Gaston, K. J., Hilton- (Teleostei: Siluriformes) from southeast Asia. Taylor, C., Akçakaya, H. R., Leader-Williams, The Raffles Bulletin of Zoology, 50(1), 161–168. N., Milner-Gulland, E. J., & Stuart, S. N. (2008). Quantification of extinction risk: IUCN’s system Ng, H. H. & Kottelat, M. (1998). Hyalobagrus, a new for classifying threatened species. Conservation gennus of miniature bagrid catfish from southeast Biology, 22(6), 1424–1442. Asia (Teleostei: Siluriformes). Ichthyological Exploration of Freshwaters, 9, 335–346. Mace, G. M., & Lande, R. (1991). Assessing extinction threats: Toward a reevaluation of IUCN Ng, H. H. & Tan, H. H. (1999). The fishes of Endau threatened species categories. Conservation Drainage, Peninsular Malaysia with descriptions Biology, 5(2), 148–157. of two new species of (Teleostei: Akysidae, Bagridae). Zoological Studies, 38(3), Martin-Smith, M. K. & Tan, H. H. (1998). Diversity of 350–366. freshwater fishes from eastern Sabah: Annotated checklist for Danum Valley and a consideration Ng, P. K. L. & Lim, K. K. P. (1991). The identity of of inter- and intra-catchment variability. The Ophicephalus cyanospilos Bleeker from Sumatra Raffles Bulletin of Zoology, 46(2), 573–604. and a new record of Channa bankanensis (Bleeker) from Peninsular Malaysia (Pisces: Miettinen, J., Shi, C., & Liew, S. C. (2012). Two Channidae). The Raffles Bulletin of Zoology, decades of destruction in southeast Asia’s peat 39, 119–130. swamp forests. Frontiers in Ecology and the Environment, 10(3), 124–128. Ng, P. K. L., Tay, J. B., & Lim, K. K. P. (1994). Diversity and conservation of blackwater fishes Mitton, C. J. A., & McDonald, D. G. (1994). in Peninsular Malaysia, particularly in the north Consequences of pulsed DC electrofishing and Selangor peat swamp forest. Hydrobiologia, air exposure to rainbow trout (Oncorhynchus 285(1-3), 203–218. mykiss). Canadian Journal of Fisheries and Aquatic Sciences, 51(8), 1791–1798.

456 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) A Review of the Ichthyofauna of Malaysian Peat Swamp Forest

Ng, P. K. L., Tay, J. B., Lim, K. K. P., & Yang, C. M. Rainboth, W. J., Vidthayanon, C., & Mai, D. Y. (1992). The conservation of the fish and other (2012). Fishes of the greater Mekong ecosystem aquatic fauna of the north Selangor peat swamp with species list and photographic atlas. forest and adjacent areas. Kuala Lumpur: AWB Miscellaneous Publications, Museum of Publication No. 81. Zoology, University of Michigan.

Ng, T. P. & Shamsudin, I. (2001). Common trees in Renato, A. M., Benedito, S., Amaralc, D., & peat swamp forests of Peninsular Malaysia. Oyakawady, O. T. (2000). Spatial and temporal Research Pamphlet No. 124. Forest Research patterns of diversity and distribution of the upper Institute Malaysia (FRIM). Kepong, Selangor. Juruá river fish community (Brazilian amazon). Environmental Biology of Fishes, 57(1), 25–35. Page, S. E., Hoscilo, A., Langner, A., Tansey, K., Siegert, F., Limin, S., & Rieley, J. O. (2009). Reynolds, J. B., & Kolz, A. L. (1993). Electrofishing. Tropical peatland fires in southeast Asia. In M.A. In V.Z. Alexander, D. L. Parrish, & T.M. Cochrane (Ed.), Tropical Fire Ecology: Climate Sutton (Eds.), Fisheries Technique. Bethesda, Change, Land Use and Ecosystem Dynamics (pp. Maryland: American Fisheries Society. 263–287). USA: Springer. Rezawaty, K. (2004). Taburan ikan di tiga batang Page, S. E., Rieley, J. O., & Banks, C. J. (2011). sungai di Paya Beriah. B.Sc. thesis, Universiti Global and regional importance of the tropical Sains Malaysia, Pulau Pinang. peatland carbon pool. Global Change Biology, Roberts, T. R. (1993). The freshwater fishes of Java, as 17(2), 798–818. observed by Kuhl & van Hasselt in 1820–1823. Parenti, L. R. & Lim, K. K. P. (2005). Fishes of Zoologische Verhandelingen, 285(1), 1–94. the Rajang Basin, Sarawak, Malaysia. The Rydin, H., & Jeglum, J. K. (Eds.) (2006). The Biology Raffles Bulletin of Zoology, Supplement No. 13, of Peatlands. Oxfor: Oxford University Press. 175–208. Sade, A., & Biun, H. (2012). The ichthyofauna of Parish, F. (2002). Overview on peat, biodiversity, Maliau Basin buffer zone at Maliau Basin climate change and fire. In F. Parish, E. conservation area, Sabah, Malaysia. Journal of Padimanabhan, C.L. Lee, & H.C. Thang (eds.), Tropical Biology and Conservation, 9, 105–113. Prevention and Control of Fire in Peatlands, 19-21 March 2002. Global Environmental Centre Shah, A. S. R. M., Zarul, H. H., Chan, K. Y., Zakaria, and Forestry Department, Peninsular Malaysia. R., Khoo, K. H., & Mashhor, M. (2006). A recent Cetaktama, Kuala Lumpur, Malaysia. survey of freshwater fishes of the Paya Beriah peat swamp forest, north Perak, Malaysia. Jurnal PIU-SERT. (2013). Studi ilmiah untuk rehabilitasi Biosains, 17(1), 51–64. dan pengelolaan hutan gambut Rawa Tripa. In Stakeholders Workshop of Tripa Peat Swamp Sharber, N. G. & Black, J. S. (1999). Epilepsy as a Forest Management. Banda Aceh: Unsyiah– unifying principle in electrofishing theory: A Satgas Redd/UKP4. proposal. Transactions of the American Fisheries Society, 128(4), 666–671. Posa, M. R. C., Wijedasa, L. S., & Corlett, R. T. (2011). Biodiversity and conservation of tropical Shiraishi, Y., Mizuno, N., Nagai, M., Yoshimi, M., peat swamp forests. BioScience, 61(1), 49–57. & Nishiyama, K. (1972). Studies on the diel activity and feeding habit of fishes at Lake Bera, Prentice, C. & Parish, D. (1992). Conservation of peat Malaysia. Japanese Journal of , swamp forest: A forgotten ecosystem. Malayan 19(4), 295–306. Nature Journal, 45, 128–144.

Pertanika J. Trop. Agric. Sci. 39 (1): 421 - 458 (2016) 457 Sule, H. A., Ismail, A. and Amal, M. N. A.

Siow, R., Ramli, M. N., Shakori, M. H. M., & Asmuni, on ICONCEES, 3. United Nations Development M. (2013). Fish fauna assemblages and their Programme. Kuala Lumpur, Malaysia. distribution patterns in the north Selangor Van der Werf, G. R., Morton, D. C., DeFries, R. S., peat swamp forest. Proceedings of the Seminar Olivier, J. G. J., Kasibhatla, P. S., Jackson, R. B., on Scientific Biodiversity Expedition, North Collatz, G. J., & Randerson, J. T. (2009). CO2 Selangor Peat Swamp Forest, 28th November emissions from forest loss. Nature Geoscience, 2013, Quality Hotel, Shah Alam, Selangor, 2(11), 737–739. Malaysia. pp.77–82. Wantzen, K. M., Yule, C. M., Mathooko, J. M., & Tabacchi, E., Platy-Tabacchi, A. M., & Decamps, O. Pringle, C. M. (2011). Organic matter processing (1998). Continuity and discontinuity of riparian in tropical streams. Tropical Stream Ecology, vegetation along a fluvial corridor. Landscape 1, 44-65. Ecology, 5(1), 9–20. Welcomme, R. L. (1979). Fisheries Ecology of Tan, H. H., & Kottelat, M. (2009). The fishes of Floodplain Rivers. London: Longman. Batang Hari drainage, Sumatra, with description of six new species. Ichthyological Exploration Whitmore, T. C. (1984). Tropical Rain Forests of the of Freshwaters, 20(1), 13–69. Far East. Clarendon Press, Oxford.

Tan, H. H., & Lim, K. K. P. (2013). Three new Whitmore, T. C. (1988). Forest types and forest species of freshwater halfbeaks (Teleostei: zonation. In Earl of Cranbrook (ed.), Key Zenarchopteridae: Hemirahamphodon) from Environments: Malaysia. Elsevier. Borneo. The Raffles Bulletin of Zoology, 61(2), Wright, J. P., & Flecker, A. A. (2004). Deforesting the 735–747. riverscape: The effects of food on fish diversity Taskforce REDD. (2012). Visi dan Misi Pemulihan in a Venezuelan piedmont stream. Biological Kondisi Lahan Gambut Rawa Tripa. Initial Conservation, 120(3), 443–451. Report Satgas REDD/UKP4, Jakarta. Wyatt-Smith, J. (1959). Peat swamp forests in Malaya. Thompson, K. G., Bergersen, E. P., & Nehring, R. Forester, 22(1), 5–31. B. (1997). Injuries to brown trout and rainbow Wyatt-Smith, J. (1964). A preliminary vegetation map trout induced by capture with pulsed direct of Malaya with description of the vegetation current. North American Journal of Fisheries types. Journal of Tropical Geography, 18, Management, 17(1), 141–153. 200–213. Thompson, K. G., Bergersen, E. P., Nehring, R. B., Yule, C. M. (2010). Loss of biodiversity and ecosystem & Bowden, D. C. (1997). Long-term effects of functioning in Indo-Malayan peat swamp forests. electrofishing on growth and body condition of Biodiversity and Conservation, 19(2), 393–409. brown trout and rainbow trout. North American Journal of Fisheries Management, 17(1), Zakaria, R., Mansor, M., & Ali, A. B. (1999). Swamp- 154–159. riverine tropical fish population : A comparative study of two spatially isolated freshwater Torrres, A. G. (2000). Labiobarbus lineatus. Fish ecosystems in Peninsular Malaysia. Wetlands Base Online. Retrieved from www.fishbase.se/ Ecology and Management, 6(4), 261–268. summary/60498. Zakaria-Ismail, M. (1990). Two-spotted catfish. UNDP. (2006). Malaysia’s Peat Swamp Forests: Nature Malaysiana, 15(3), 88–89. Conservation and Sustainable Use. International Journal of Integrated Engineering – Special Issue

458 Pertanika J. Trop. Agric. Sci. 39 (4) 421 - 458 (2016) Pertanika J. Trop. Agric. Sci. 39 (4): 459 - 482 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Review Article Impact of Heat Stress on Immune Responses of Livestock: A Review

Sophia Inbaraj1, Veerasamy Sejian2*, Madiajagan Bagath2 and Raghavendra Bhatta2 1ICAR-Central Island Agricultural Research Institute, Portblair, Andaman and Nicobar Islands, 744101, India 2ICAR-National Institute of Animal Nutrition and Physiology, Adugodi, Bangalore-560 030, India

ABSTRACT Climate change acts as a major threat to climate sensitive sectors such as agriculture and animal husbandry. This change in climate will be a greatest challenge to about 1.3 billion population who depends on animal husbandry as their livelihood. Heat stress is considered as one of the primary factors that imposes negative impacts on production and reproduction in farm animals. In addition, it also alters the immune functions of the animal and makes them susceptible to infectious diseases. Based on the duration exposure, heat stress either enhances or suppresses the immune functions in farm animals. The stress signal acts mainly through hypothalamo pituitary adrenal (HPA) axis to modulate the immune response. Generally, it is considered that heat stress acts to shift the adaptive immune function from cell mediated to humoral immunity and thus weakens the animal immune function. Another aspect of this climatic change is the threat of emerging and re-emerging pathogens and disease vectors for which livestock needs fine-tuned immune system to fight against naïve pathogens. Thus, the heat stress-immune system interactions need to be studied thoroughly in order to introduce various management and nutritional strategies to alleviate the ill- effects of heat stress in farm animals.

Keywords: Climate change, Heat stress, Immunity, ARTICLE INFO Livestock, HPA axis, Pathogens Article history: Received: 6 November 2015 Accepted: 21 July 2016 INTRODUCTION E-mail addresses: [email protected] (Sophia Inbaraj), Livestock are called as the living bank for [email protected] (Veerasamy Sejian), [email protected] (Madiajagan Bagath), farmers. They contribute about 53 percent [email protected] (Raghavendra Bhatta) * Corresponding author of world agricultural GDP (World Bank,

ISSN: 1511-3701 © Universiti Putra Malaysia Press Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

2009), and also to the economy by means potential and environmental influence. of milk, meat, hide, eggs, drought power, Even though host-pathogen interactions are manure etc. Apart from that, livestock also essential for clinical disease development, the provide employment to 1.3 billion world environment also plays an equally important population (FAO, 2009). The current world role in modifying the host and pathogen population of 7.3 billion is expected to reach factors (Kelley, 2004). Due to global 8.5 billion by 2030, 9.7 billion in 2050 and warming, abrupt climatic conditions like 11.2 billion in 2100 (UN, 2015) and about storms, droughts, floods and extreme hot and significant proportion of the population cold temperatures are prevailing around the in developing countries will migrate to globe. With the changing climatic scenario, towns leading to increased urbanisation of the frequency and duration of exposure 56.9% by 2025 (UNFPA, 2008). Ultimately, of livestock to abiotic and biotic stressors urbanisation will increase the standard of increases. Current incidences of global living of people, while the demand for high climatic change lead to the evolution of quality protein will also increase (Steinfeld new concepts in host-microbe relationship. et al., 2006). Henceforth, the concept of High temperatures, accompanied by high animal husbandry currently changes with relative humidity, favour the survival and major focus on food animal production. multiplication of animal disease vector Animal protein is a cheap source of high like ticks, fleas, tabanid flies, etc.; thus, the quality protein with essential vitamins and risk of spread of vector borne diseases also micronutrients. Thus, to ensure nutritional increases (Wittman & Baylis, 2000). The security to the current growing population, intercontinental spread of blue tongue virus animal protein seems to be inevitable. In by Culicoides sps. is a typical example (Rao order to provide quality animal meat to the et al., 2012). Furthermore, the increasing consumers’ fork, the farm animals should be trend of international trade also favours the healthy in terms of physiology, temperament introduction of new disease species, which and immunity. the host immune system has never been However, there are various stressors, exposed. Introduction of bovine spongiform both biotic and abiotic, which challenge encephalopathy (BSE) through bovine offals the animal’s wellbeing. Temperature, (Pattison, 1998) and introduction of porcine solar radiation, photoperiod, humidity, respiratory and reproductive syndrome geographical location, nutrition and socio- (PRRS) in swine in India are few examples economic signals are the major abiotic of these. stressors Microbes such as bacteria, viruses, Abiotic stressors such as heat and fungi and protozoa, as well as helminths and nutritional stress have a major impact on arthropod vectors, are the biotic stressors. livestock productivity (Sejian et al., 2011). The term clinical disease is an outcome In the changing climate scenario, other of the interaction of host factors, pathogen factors like solar radiation, photoperiod

460 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity and humidity also synergises with the therefore, various nutritional interventions above stressors. Temperature influences need to be taken to ameliorate heat stress the animals’ productive and reproductive mediated immune suppression. traits in a major way. In particular, heat stress is one of the crucial factors affecting STRESS AND IMMUNITY livestock productivity (Rivington et al., Various researchers defined stress as per 2009). Furthermore, the concept of global their observations. It was Seyle in 1946, warming is alarming the planet earth day by who gave the first definition about the day. According to the latest report released animal response to stress. He called it as by IPCC (2013), there is an average of the ‘general adaptation syndrome’. Stress about 1.53°F (0.85ºC) rise in global surface is also defined as the biological response temperature from 1880-2012. Heat stress elicited when an animal perceives a threat to affects animal productive performances like its homeostasis (Moberg & Mench, 2000). milk yield, meat quality and reproductive The threat is the stressor. An animal during performances like age at maturity, ovulation its lifespan experiences non-threatening failure, embryo mortality, etc. (Shinde & stress situations too which did not affect its Sejian, 2013). It also weakens the animal’s health normally. Stress at its minimal level is immune system and makes them more always beneficial. A minimal level of stress prone to diseases. Although this has been called as good stress is required by all living observed by various researchers, the impact organisms to perform a task. Stress is called of heat stress on immune gene expression as bad or stress will become a distress when and process of heat stress mediating it alters the normal biological functions like immune suppression at molecular level production and reproductive performances has not been dealt in detail in livestock. of an animal. Stress becomes important or In order to withstand the existing and needs attention in an animal lifetime in the emerging pathogen challenges, the animal’s point at which its welfare is compromised immune system should be in a right tone. In i.e. its survival, production and reproduction order to mitigate immune suppression by are affected. Thus, stress needs to be an means of various nutritional and hormonal important matter of discussion in livestock interventions, an in depth understanding of production because in a stressed animal, the the immunological pathways affected by energy balance and the body reserves will heat stress and the mechanism by which it be mobilised to counteract or alleviate the is affected is mandatory. stressor. This, in turn, leads to decreased This review deals with the relationship production and reproductive performances. between hypothalamo-pituitary-adrenal axis Furthermore, stress mediated interactions (HPA) and immune axis, the effects of heat with the immune system suppresses its stress on immunity, the mechanism by which normal functions and leads the animal to heat stress affect immune response and a disease-prone state (Moberg & Mench,

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 461 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

2000). Therefore, the type of stresses that the stressors (Mason et al., 1991), genetic the animals encounter and its impact need makeup (Marple et al., 1972), age (Blecha to be studied and documented thoroughly et al., 1983), season and physiological to enhance better animal production in the state. The neuroendocrine system responds changing climatic scenario. In general, mainly through the HPA by the release of animals encounter various stressors glucocorticoids that are normally called as such as thermal stress, production stress, stress hormones (Webster & Glaser, 2008). transportation stress, nutritional stress, They act to adapt the animal to cope up immune stress, stress due to crowding, with the long-term stressors. The immune drought and environmental conditions system responds to stress by enhancement or (Kelley, 1980). In the arid and semi-arid suppression of immune functions (Dhabhar, tropical conditions, animals have to walk 2009). The immune system does not respond considerably long distances which in turn directly to stress but via neuroendocrine leads to walking stress (Sejian et al., 2012; system. The stress related hormones act on Maurya et al., 2012). Currently, the concept the immune cell receptors to modulate the of multiple stresses is evolving in animal immune response. stress physiology. Studies in Malpura sheep of northern India revealed that IMMUNITY multiple stresses had significant influence The immune system functions can be on productive and reproductive parameters broadly classified into: (i) innate immunity (Sejian et al., 2011). In the event of climatic and (ii) adaptive immunity. Innate immunity change, stress due to adverse climatic is the germ line encoded, non-specific, conditions and emerging microbes and preliminary line of defence against the vector – borne diseases are some of which invading pathogens. The first entry of the the animals have to encounter (Epstein et pathogens is prevented at body’s entry al., 1998). site through antimicrobial components in Whenever an animal encounters a mucosa, sweat, tears, saliva, etc. In spite of stressor, the central nervous system first this, if the pathogens enter, the specialised begins to respond by sending signals to any immune receptors called Pathogen of the biological systems viz. behavioural, Recognition Receptors (PRR) will identify autonomic nervous system, neuroendocrine the conserved molecular signatures called system and immune system in order to Pathogen Associated Molecular Patterns alleviate or compensate the threat. It is (PAMPs) present in the pathogens (Janeway not mandatory that all the four biological & Medzhitov, 2002) and recruit leucocytes, systems should respond together whenever particularly neutrophils, chemical mediators a stress occurs. The response of each called pro–inflammatory cytokines, animal to a particular stress condition varies followed by macrophages. They ultimately depending upon its previous exposure to kill the pathogens through reactive oxygen/

462 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity nitrogen species production and engulf them SYMPATHETIC-ADRENAL- up in a process called phagocytosis (Bassett MEDULLARY SYSTEM (SAM) et al., 2003). Their processed fragments are It is the sympathetic trunk of the autonomic presented to another phase of the immune nervous system (ANS) situated in the system for specific immune response called adrenal medulla. It acts by the release of adaptive immune system. Based on the pro– epinephrine and nor epinephrine which are inflammatory cytokines that are released, the responsible for the characteristic flight or adaptive immune response may be either -fright mechanism. This helps the animals antibody mediated or cell mediated. All to overcome the stressor and it is the short these mechanisms will ultimately lead to term adaptation of the animal to acute clearing of pathogens from the host body. stressor. These hormones act to enhance glycogenolysis causing increased glucose THE LINK BETWEEN STRESS AND levels in circulation. The blood glucose will THE IMMUNE SYSTEM reach the stressed organ in order to meet the Sympathetic innervation from brain connects energy requirements and cope up with the the primary lymphoid organs and secondary stressor (Tort & Teles, 2011). lymphoid organs (Ader et al., 1995); thus, the catecholamine receptors in immune cells HYPOTHALAMO-PITUITARY also contribute to immune cell activation ADRENAL AXIS by stress mechanisms. The end product of The core of stress response is believed to HPA axis activation results in glucocorticoid be based on the activation of HPA. The release, which has receptors in almost all key components of this system include: organs including the immune cells (Ader (i) hypothalamus, and (ii) brain stem. et al., 2001). The parvocellular neurons of CRH, arginine vasopressin (AVP) neurons of THE STRESS AXIS paraventricular nuclei of hypothalamus, It includes neural and endocrine organs CRH neurons of paragigantocellular and and its secretory products and hormones parabranchial nuclei of medulla and locus released in response to stress. The HPA axis ceruleus innervate the system (Tort & Teles, and sympathetic-adrenal-medullary system 2011). They sense stressor and get activated (SAM) are the crucial components that to release CRH from paraventricular receive stress signals and act to relieve the nucleus and arginine vasopressin from stress mechanism or adopt the animal to the magnocellular neurons of hypothalamus. stressful condition (Riedemann et al., 2010). They act on chromaffin cells of anterior Figure 1 depicts the interaction between pituitary to release adrenocorticotropic various endocrine axes with the immune hormone (ACTH). ACTH, in turn, acts on system during stress. adrenal cortex to stimulate synthesis and release of steroids leading to cholesterol

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 463 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta uptake. Cholesterol further gets converted HEAT STRESS AS AN IMPORTANT into cortisol and corticosterone. Cortisol is FACTOR AFFECTING IMMUNITY IN LIVESTOCK the major glucocorticoid (GC) secreted in response to stress in mammals. GCs have The process of disease involves three receptors called glucocorticoid receptors factors; namely, host, pathogen and (GRs) in almost all body tissues (de Kloet environment. Environment modulates the & Derijk, 2004). Glucocorticoid receptor host and pathogen interactions in such exists as a complex in the cytoplasm of a way that the pathogen overcomes the most cells including the immune cells. immune barrier of the host and establishes Binding of the hormone – receptor complex itself and henceforth, the outcome is the to Glucocorticoid Responsive Elements disease condition. In calf neonates, the first (GRE), located in the promoter region of 18 hours of post natal life is very crucial the target gene, regulates the expression of as it determines the immune status of the target genes either positively or negatively. animal. During this period, the intestinal Basal levels of GCs are always present in epithelium is permeable to colostral circulation to meet the day-to-day metabolic proteins, particularly immunoglobulins activities. During acute stress, there is and thus passive transfer of immunity occurs stimulation of HPA axis, followed by GC from dam to offspring. Exposing dams and surge. It acts on vital systems to maintain neonates to heat stress, however, has major homeostasis. When the stressor is removed, impacts on the calf’s immunity. Exposing negative feedback mechanism acts to impair heifers to high temperature during late GC secretion, thus, the cycle goes on. pregnancy and early postpartum period not In contrast, chronic stress has long-term only reduces the concentrations of IgG, IgA, increase in glucocorticoids in circulation milk proteins and fatty acids in colostrums which causes deleterious effects on the (Nardone et al., 1997) but also lowers the productive parameters and immunity. During intestinal absorption of immunoglobulins chronic stress, the number of GC receptors (Stott et al., 1976). The low quality of Igs in hippocampus gets reduced. As a result, and reduced intestinal absorption in turn the negative feedback mechanism which lead to calf mortality at high temperature regulates the HPA axis is affected (Webster (Martin et al., 1975). & Cidlowski, 1994). Another reason may be that the chronic stress activates the adrenal HEAT STRESS AND DISEASE OCCURRENCE gland resulting in adrenal hypertrophy and henceforth results in increased and Heat stress directly or indirectly favours prolonged glucocorticoid secretion (Miller disease occurrence in animal host. Directly, & Tyrrell, 1995). high temperature favours the survival of organisms outside the host for a long time. This can be seen in the case of spores of

464 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity

HPA-Hypothalamo-Pituitary-Adrenal; SAM-Sympathetic-Adrenal-Medullary; IFN-Interferon; IL- Interleukin; TNF-Tumor Necrosis Factor; IgG-Immunoglobulin G; T3- Tri-iodo-thyronine; T4-Thyroxine; MHC-Major Histocompatibility Complex; NK Cells- Natural Killer Cells; Th-T Helper Cell

Figure 1. The interaction between various endocrine axes with the immune system during stress

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 465 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

Bacillus anthracis and Clostridium chauvoei HEAT STRESS AND VACCINATION (Hall, 1988), which survive for a long period RESPONSE under high temperature and made available Culling of diseased livestock is not to infect the animals. Indirectly, chronic economically feasible in developing heat stress causes immune suppression in countries of and Africa. Thus, animals and makes them susceptible to regular vaccination seems to be a better diseases. option in order to protect the livestock Moreover, global climatic change has from dreadful diseases like foot-and-mouth created huge modifications on the macro disease (FMD), anthrax, haemorrhagic and microclimate of both the host and the septicaemia (HS), PPR, etc. The effects parasite and also altered certain trends of heat stress on the immune response to of host-microbe interactions, namely: (i) vaccines have been reviewed based on the shortened generation time of microbes which available literature. Varied results have prefer high temperature leading to increased been obtained by various researchers under pathogen population and in turn increased different circumstances. risk of infection, and (ii) population of High temperature combined with high vectors like ticks, flies, midges, fleas relative humidity failed to elicit humoral normally requires high temperature, and immune response to canine distemper hence they increase in number and their vaccine and also failed to protect against feeding frequency also increases. These the virus challenge. /however, the same lead to more chances for transmission of temperature conditions did not affect the pathogen to animal host. The sub-tropical immune response to hepatitis vaccine hot and humid conditions favour the tick (Webstar, 1975). The effect of chronic heat population like Boophilus microplus, stress on FMD vaccination in mice model Haemophysalis bispinosa and Hyalomma revealed that that chronic heat stress had anatolicum (Basu & Bandhopadyay, 2004). adverse effect on cell mediated immune (iii) The extrinsic incubation period reduces response than humoral immune response. due to high temperature has been seen in Th1 based cell mediated immune responses case of Culicoides (Wittman & Baylis, like IgGa production, T cell multiplication, 2000). High ambient temperature combined IFN gamma expression and antigen specific with relative humidity predisposes dairy cytotoxic T lymphocyte activities were cows to clinical mastitis (Singh et al., 1996). affected severely (Hu et al., 2007). The The direct effect of heat compromises the experimental study conducted by the same udder immune system and also favours the team also proved that both humoral and cell housefly population (Sirohi & Michelowa, mediated immune responses to H5N1 avian 2007). influenza vaccine were affected by chronic heat stress. A new concept of up regulation

466 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity of CD4+ CD25+ Foxp3+ T reg cells, with steatohepatitis and decreased productive increased TGF-β, IL-10, decreased Th1, Th2 performance. cell responses, CD8+ T cell proliferation and related cytokines was noticed in the study EFFECT OF HEAT STRESS ON (Meng et al., 2013). IMMUNE RESPONSES Various human studies revealed Heat stress modulates various behavioural activation of latent viruses during stress and physiological parameters in farm conditions (McVoy & Adler, 1989). It was animals and in poultry species which have reported that glucocorticoids released during been discussed in detail by various reviewers stress conditions activated the latent viruses (Lu, 1989; Kadzere et al., 2002; Marai et by directly acting on the viral genome and al., 2006; Yahav, 2009). Various authors also decreasing the immunological memory recorded variable results indicating that response (Glaser et al., 1995). heat stress as either an immune suppressing or immune enhancing factor in animal HEAT STRESS AND GUT HEALTH production. Stress affects both innate and The gastrointestinal tract health is crucial in adaptive immune response in animals. farm animals like cattle, sheep, goat, pigs, etc., as optimum production depends upon HEAT STRESS AND INNATE IMMUNITY efficient feed conversion. The integrity of gastrointestinal tract is essential in order Heat stress reduces the relative weights of to maintain the normal homeostasis of gut lymphoid organs like spleen, thymus and microbiota. During heat stress, however, the cloacal bursa (Aengwanich, 2008). The blood flow to internal organs like intestine mechanical barriers, namely mucosa and gets compromised and peripheral blood skin, act as the first line of defense in innate flow increases to dissipate the internal body immune response. Experiments in poultry heat (Lambert et al., 2002). The decreased revealed that exposure to heat stress caused blood flow leads to ischemia and necrosis mild acute lymphocytic enteritis (Quinteiro- of intestinal epithelial cells (Hall et al., Filho et al., 2010). Chronic heat stress could 1999), decreases tight junctions between affect the integrity of respiratory tract and enterocytes which favour paracellular reduce pulmonary alveolar macrophages entry of bacterial pathogens. Increase in lungs of mice, thus making the animals in intracellular permeability helps in susceptible to Highly Pathogenic Avian translocation of bacteria (Pearce et al., 2013) Influenza or H5N1(Jin et al., 2011). and its antigenic components, particularly NK cells are important components LPS, thus leading to endotoxemia (Pearce of the innate immune system present in et al., 2013). The toxin in turn affects systemic circulation and also in lymphoid the normal liver metabolism, leading to organs like lymph nodes, spleen and bone marrow. They are involved in destruction

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 467 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta of tumour cells and infectious agents like of cytokines responsible for humoral and bacteria, fungi and viruses. Studies in mice cell mediated immune response were revealed that chronic heat stress in mice down regulated in intestine by heat stress. reduced the splenic NK cell cytotoxic Reduction of intestinal immune function functions. The inhibition may be due to enabled bacterial translocation to mesenteric increased glucocorticoid influence in the lymph node (Liu et al., 2012). immune cell (Won & Lin, 1995). The immune profile in PBMC of local HEAT STRESS AND ADAPTIVE Bama miniature pigs exposed to 21 days IMMUNITY of heat stress revealed up regulation of Adaptive immune response collectively the Toll Like Receptors (TLR) 2, 4 which represents the humoral and cell mediated are involved in identification of conserved immune response. Experimental results in molecules of microbes such as lipoprotein farm animals and poultry revealed variable and lipopolysaccharides respectively (Ju adaptive immune responses during heat et al., 2014). Heat stress also up regulated stress. Table 1 represents the adaptive the TLR2, 4 genes in human monocytes immune responses elicited by livestock (Zhou et al., 2005). Studies on the seasonal during heat stress conditions. influences on TLR 1-10 mRNA expression in Black Bengal goats revealed that significant MECHANISM OF HEAT STRESS increases in TLR genes were noticed during IMPACTING LIVESTOCK IMMUNE summer season (Paul et al., 2015). The SYSTEMS pro-inflammatory cytokines, namely IL-6, Various stressors including heat stress IFN-ß, which contributes to innate immune induce the endocrine system to increase response were down regulated by heat stress catecholamines and glucocorticoids. These (Jin et al., 2011). hormones modulate the cytokine release and thereby regulate immune responses. HEAT STRESS AND MUCOSAL IMMUNITY GLUCOCORTICOIDS MEDIATED Heat stress reduces the blood flow to LYMPHOLYSIS intestine, lowers the integrity of intestinal Experiments in poultry revealed that epithelium, causes villi desquamation and decrease in relative weights of immune reduces the villi height and crypt depth (Yu organs like spleen, cloacal bursa and et al., 2010; Yu et al., 2013). Furthermore, thymus. This might be due to glucocorticoid the innate immune components like mucosal induced lympholysis and redistribution of barrier, toll like receptors, secretory Ig lymphocytes from systemic circulation to A, intestinal intraepithelial lymphocytes other organs (Jain, 1993). production (Deng et al., 2012), expression

468 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity

MECHANISM OF INHIBITION OF Recognition Receptors TLR2, TLR4 (Galon INNATE IMMUNITY et al., 2002). Stress induced glucocorticoids act to inhibit the pro-inflammatory cytokines, namely MECHANISM OF INHIBITION OF TNF-α, IL-6, IL-8, which are required ADAPTIVE IMMUNITY to initiate an innate immune response Th1 cells are involved in cell mediated through inhibition of p38 MAPK pathway immune response whereas Th2 cells are which helps in maintaining their stability responsible for humoral immune response. (Abraham et al., 2006). GCs also enhance The modulation of cytokine gene expression IL-10, an anti-inflammatory cytokine which in fact alters the immune function from Th1 is normally found at the end of immune to Th2 and vice versa. Th1 cells secrete response (Marchant et al., 1994). The IFN- γ, IL 2, TNF β which contributes to cytotoxic function of NK cells are inhibited cellular immunity. Th2 cells secrete IL- by catecholamines which act through 4, IL-10 and IL-13, which contribute to adrenergic receptors to elevate cAMP levels humoral immunity. IL-12, in combination that will in turn decrease the cytotoxic with IFN-gamma, converts uncommitted functions (Whalen & Bankhurst, 1990). T helper (Th0) cells to Th1 cells, whereas The hyperthermia mediated suppression cytokines IL-4 and IL-10 induce Th2 cell of NK cell activity is due to the inhibition production. Both Th1 and Th2 cell mediated of perforin and granzyme enzymes (Koga are inhibitory to each other (Elenkov & et al., 2005). GCs upregulates innate Chrousos, 1999). Glucocorticoids acts to immune system via stimulation of Pattern inhibit the release of IL-12 and IFN γ which

Table 1 Effects of heat stress on adaptive immune responses of livestock

Animal Immune Parameters studied Effect of heat stress on References the immune parameter Poultry Humoral immunity Unaltered Regnier et al., 1980 Calves Mitogen stimulation index Unaltered Kelley et al., 1982a Pigs Mitogen stimulation index & Unaltered Bonnette et al., 1990 antibody production Poultry Antibody response Unaltered Donker et al., 1990 Poultry Antibody mediated primary Decreased Thaxton et al., 1968; immune response Subbarao & Glick, 1970 Poultry Humoral immunity Decreased Mashaly et al., 2004 Cattle Cell mediated immunity Unaltered Lacetra et al., 2002 Calves Cell mediated immunity Decreased Kelley et al., 1982b Cattle B and T- cell mitogen Decreased Elvinger et al., 1991 response

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 469 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta are the major cytokines involved in Th1 GENES ASSOCIATED WITH based cell mediated immunity (Elenkov et IMMUNE FUNCTIONS DURING HEAT STRESS al., 1996). Furthermore, the expression of IL-12 receptors in NK cells and Th1 cells The stress related immune responses in are down regulated by glucocorticoids (Wu poultry species revealed that acute stress et al., 1998), and thus the immune function is beneficial to the bird as it the immune get shifted from Th1 to Th2. system. On the other hand, chronic stress Dendritic cells are potent APCs and link shifts the T helper cell response to T the innate and adaptive immune response regulatory cell and also TGF-β, a regulatory as they are involved in phagocytosis and cytokine production, thereby it suppresses antigen presentation. They also express more immune response (Shini et al., 2010). MHC Class II and co-stimulatory molecules Meng et al. (2013) who demonstrated that like CD80, CD83 and CD86 on their cell chronic heat could suppress both Th1 and surface. GCs act to inhibit expression Th2 lymphocyte based immune response of these molecules on DCs and thereby for H5N1 avian influenza virus through prevent its maturation (Girndt et al., 1998). upregulation of CD4+CD25+Foxp3+ Treg In addition, catecholamines, which include cells, immune cytokine TGF-β gene adrenaline and noradrenaline, are also expression. The heat stress signaling released during stress conditions to inhibit pathway was observed to be distinct from the production of IL-12, development of Th1 endotoxemic pathway in mice. It acts cells and Th2 differentiation (Elenkov et al., through HSFs such as HSP 70 and Stress 1996). Treatment with adrenergic agonist Activated Protein Kinase pathways resulting revealed inhibition in IFN γ production by in an early increase in the expression of Th1 cells (Sanders et al., 1997). hsp72, c-fos and c-jun genes. This in turn leads to the expression of a peculiar cytokine MOLECULAR RESPONSES TO HEAT pattern consisting of increased IL-6 and IL- STRESS 10 expression and decreased TNF-α, IL-1β Stress leads to activation of hypothalamic- expression. The pattern recognition receptor, pituitary adrenal axis and ultimately the namely TLR-4, is highly expressed whereas release of glucocorticoids. Glucocorticoids, there was no significant difference in TLR- in normal pulsatile release, enhances the pro- 2 gene expression (Welc et al., 2013). The inflammatory cytokine release. However, endotoxemic pathways differ from the chronic rise in glucocorticoid levels is earlier in which increased expression of inhibitory to majority of immune cytokines. IL-1β, IL-6, TNF-α genes are evident (Lang Glucocorticoid acts in various ways to inhibit et al., 2003). Chronic heat stress could cytokine release. Table 2 represents various influence complement system, a component genomic and non-genomic mechanisms by of the innate immune system in the intestine which GCs suppress cytokine release. of rats (Lu et al., 2011). Furthermore, the

470 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity chemokine signalling pathways and HSP the key cytokine gene to initiate cellular expression were reported to be down immune response was upregulated but IL-2 regulated during heat stress (Liu et al., and IFN-γ, which are also involved in CMI 2014). This might be due to the inhibition of process were down regulated, revealing JAK-STAT signalling pathways, which play differential expression of immune cytokine an important role in induction of immune genes (Ju et al., 2014). Acute heat stress cells to release cytokines and chemokines. exposure in cattle led to up regulation of The JAK-STAT proteins are thermolabile IL-17, a cytokine associated with innate in nature and get decreased during heat immune response up to 48 hrs after heat stress (Nespital & Strous, 2012). Moreover, stress. Moreover, the genes involved in differential expression of adaptive immunity humoral immune response (Cd83, HSPA1A genes were noticed viz. up regulation of & IL1A) showed increased expression HSP-70 genes and down regulation of RT1- during heat stress (Mehla et al., 2014). Table Bb genes, which encode for beta chain of 3 describes the different immune response MHC-class II. Both the genes are involved genes getting expressed during heat stress in antigen presentation to CD4+ T cells and condition in animals. ultimately the CD4+ T cell stimulation gets affected (Liu et al., 2012). Also, heat stress SIGNIFICANCE OF OPTIMUM reduced the expression of MHC class II and NUTRITION FOR IMMUNE FUNCTION UNDER HEAT STRESS co-stimulatory molecules like CD40, CD80 and CD86 by antigen presenting cells, thus The various strategies need to be adopted for delayed DC maturation (Jin et al., 2011). management of animals during heat stress The cytokine genes for both Th1 (IL-2, include shelter management, evaporative IFN-γ) and Th2 (IL-4, IL-10) cell responses cooling systems such as misting, fogging, were down regulated during heat stress (Liu pad cooling, sprinkling or dripping, et al., 2012). Heat stress experiments in modified nutrition strategy and genetic Bama miniature pigs revealed that IL-12, selection of animals for heat tolerant genes.

Table 2 Different mechanisms by which glucocorticoids suppresses cytokine function

General mechanisms involved Cytokines affected References Suppression of cytokine gene IL-1, IL-2, IL-3, IL-8 Smoak & Cidlowski, 2007 transcription Decrease in cytokine secretion IL-1 Destabilization of cytokine IL-1, TNF, GM-CSF mRNAs Inactivation of the released IL-1 cytokine by inducing decoy receptors

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 471 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

TABLE 3 Different immune regulatory genes expressed and their functions during heat stress

Species Immune genes Function Up regulation/ References Down regulation Mice IL-6 Pro-inflammatory cytokine Up regulated Welc et al., IL-10 Anti-inflammatory, Immune 2013 regulatory cytokine TLR-4 PRR for LPS TNF-α Acute inflammatory response Down regulated IL-1β Pro-inflammatory cytokine Cattle IL-17A Innate immunity Up regulated Mehla et al., HSPA1A Induce antibody production & 2014 T cell activation Cd83 Antigen presentation to B lymphocytes IL1A T cell dependant antibody production CXCL5 Chemo attractant for neutrophils Suppressed earlier; induced

FCγR1A Antigen presentation via MHC later class I Poultry IL-1β Pro-inflammatory cytokine Increased during Shini et al., IL-6 Pro-inflammatory cytokine acute stress; 2010 decreased during IL-18 Pro-inflammatory cytokine; chronic stress involved in CMI TGF β Immunoregulatory cytokine Increased during CCL16 Chemo attractant for lymphocytes chronic stress Pigs TLR2, TLR4 Innate immune response Up regulated Ju et al., IL-12 Induction of Th1 based cell Up regulated 2014 IFN γ mediated immune response Down regulated Rats IL-2, IFN γ Th1 based cell mediated immune Down regulated response IL-4, IL-10 Th2 based humoral immune response

472 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity cont’d Table 3 Rats HSPA1A, Up regulated Liu et al., ZBTB16, 2014 CSF2, HPX, IL22RA2, PTDSR, LOC301133, BCL6, MAdCAM1, PPARA, CDK6, VEGFA TNFSF6, Cytokine-cyokine interaction Down regulated MS4A2, CCL5, SPN, CCR5, MM9, IR4, TLR2, CCL4, S100A9, RT2- Bb Rat RGD1561028, Up regulated Lu et al., HSP90AA1, 2011 HSP90AB1, HSPA11, and HSPA8 RT1-Bb, Cytokine-cytokine receptor CCL5, interaction CCR5, Down regulated CXCR3 and Fas IL22RA2 Up regulated CSF2 TNFSF14 Cd55, F3 Complement & coagulation Down regulated cascades Fgb, Up regulated Serpine1, and Serpine 2 Mice IL-10, TGF-b Immunoregulatory cytokines Up regulated Meng et al., 2013 Mice MHC-II, Co-stimulatory molecules Down regulated Jin et al., CD40, CD80, 2011 and CD86 IL-6 and Pro-inflammatory cytokine IFN-β IL-10 Immunoregulatory cytokine Up regulated

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 473 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

The nutrients which enhance immune TRACE MINERALS response in heat stressed animals include Copper is essential for the development of vitamins, minerals, antioxidants, prebiotics, immune organs and antibody production probiotics, synbiotics, etc. (Lukasewycz & Prohaska, 1990). Copper- deficiency was reported to reduce the VITAMINS phagocytic ability of neutrophils and Vitamin A acts to maintain the normal macrophages (Babu & Failla, 1990a; Babu mucosal and epithelial barrier, thereby & Failla, 1990b), IL-2 production and helps to strengthen the mechanical barriers thus lymphocyte proliferation (Bala & which are the components of innate immune Failla, 1992). Keratin acts as a mechanical response (Sordillo et al., 1997). Thus, it is barrier in organs such as mammary gland required for maintaining udder health in and in appendages such as horn, hoof, milch animals. Moreover, supplementation etc. Zinc is involved in various stages of of vitamin A augments the functions of keratinization (Paulrud, 2005). Providing anti-oxidant enzymes like glutathione zinc supplementation in organic form during peroxidase, superoxide dismutase, etc. summer helps in maintaining udder health by (Jin et al., 2014). The supplementation strengthening keratin layer and preventing of vitamin A also results in increased mastitis (Popovic, 2004). Meanwhile, iron pro-inflammatory cytokines like IL-1, helps in the development of immune organs TNF-α , Ig M, Ig G and Ig A, soluble like thymus, spleen, lymph node and cloacal CD4, CD4/CD8 ratio, decreased CD8 (Jin bursa. Experiments with iron-glycine et al., 2014) and aids in immunoglobulin supplementation revealed iron particularly transport proteins production (Tjoelker influences thymus development in animals et al., 1990). Selenium, a trace element, (Feng et al., 2007) and birds (Sun et al., plays an important role in immune function 2015). Chromium supplementation helps in combination with vitamin E. The anti- in the development of primary immune oxidant enzyme, glutathione peroxidase response with increased Ig M and Ig G incorporates selenium within it, thus it is concentration (Moonsie-Shageer & Mowat, indirectly involved in protection of the body 1993). from oxidative stress. Moreover, selenium supplementation in diet decreased the PROBIOTICS incidence of clinical mastitis and somatic Probiotics are mixture of live micro- cell count (Smith et al., 1997). Selenium organisms which are beneficial to animal’s – vitamin E supplementation positively health when given in adequate amounts. influences chemotaxis (Aziz & Klesius, These live organisms occupy the entire 1986), phagocytic ability (Grasso et al., length of the gut to protect against pathogens 1990) and oxidizing property of neutrophils entry and colonisation by a process called (Aziz & Klesius, 1986). “competitive exclusion” or “bacterial

474 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity antagonism” or “bacterial interference” livestock production through emergence (Lloyd et al., 1977) and strenghthen the of different vector borne diseases. This intestinal mechanical barrier. Furthermore, review has thrown light that in addition to probiotics compete with pathogenic bacteria altering the productive and reproductive for nutrition and attachment site. They also performance, heat stress can also alter the produce antimicrobial peptides such as immune functions of animals to bring down colicins and bacteriocins (Lee et al., 2008) their production. The stress hormones – and stimulate cytokine production (Marin cytokine interactions are responsible for et al., 1997). altered immune functions during heat stress. In particular, the highly specific PREBIOTICS adaptive immune mechanisms are affected Prebiotics are said to be undigestible feed by heat stress. In more specific, heat stress ingredients that beneficially affect the deteriorates the cell mediated immune host by selectively stimulating the growth responses. The role of heat stress during or activity of limited number of bacteria critical events in an animal like passive in colon (Gibson & Roberfroid, 1995). transfer of maternal antibodies, vaccination Feeding of fructose-oligosaccharide and and new pathogen challenge has been mannon oligosaccharide increased antibody widely discussed. This reveals that heat responses (White et al., 2002). stress needs to be considered as an important factor compromising animals’ health. SYNBIOTICS Furthermore, various managemental and nutritional interventions were discussed in Synbiotics, a combination of probiotics and detail during such instances to prevent the prebiotics, is supplemented in farm animals. harmful impacts of heat stress on animal’s The necessity for the combination is that welfare. This literature review can serve as a prebiotics acts as nutritional substrate for good reference material for researchers who probiotic organisms, thus helps in improving aim at improving livestock production in the or maintaining the gut health. Synbiotics changing climate scenario by optimising the acts as a stimulant for phagocytosis and immune system functioning in livestock. releases cytokines like TNF-alpha, IFN- gamma and antibody production (Kabir REFERENCES et al., 2004). Synbiotics are reported to increase the antibody response to vaccines Abraham, S. M., Lawrence, T., Kleiman, A., Warden, P., Medghalchi, M., Tuckermann, J., Saklatvala, (Hassanpour et al., 2013). J., & Clark, A. R. (2006). Anti-inflammatory effects of dexamethasone are partly dependent CONCLUSION on induction of dual specificity phosphatase In the global climate change scenario, heat 1. Journal of Experimental Medicine, 203(8), 1883–1889. stress is the predominant stress affecting

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 475 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

Ader, R., Felten, D. L., & Cohen, N. (2001). Bonnette, E. D., Komegay, E. T., Lindemann, M. Psychoneuroimmunology (3rd Ed.). USA, D., & Notter, D. R. (1990). Influence of two Academic Press. supplemental vitamin E levels and weaning age on performance, humoral antibody production Ader, R., Cohen, N., & Felten, D. (1995). and serum cortisol levels of pigs. Journal of Psychoneuroimmunology: Interactions between Animal Science, 68(5), 1346-1353. the nervous system and the immune system. Lancet, 345(8942), 99–103. de Kloet, E. R., & Derijk, R. (2004). Signaling pathways in brain involved in predisposition and Aengwanich, W. (2008). Pathological changes and pathogenesis of stress-related disease: genetic the effects of ascorbic acid on lesion scores of and kinetic factors affecting the MR/GR balance. bursa of Fabricius in broilers under chronic heat Annals of the New York Academy of Sciences, stress. Research Journal of Veterinary Sciences, 1032(1), 14–34. 3(1), 74–78. Deng, W., Dong, X. F., Tong, J. M., & Zhang, Q. Aziz, C. S., & Klesius, P. H. (1986). Depressed (2012). The probiotic Bacillus licheniformis neutrophil chemotactic stimuli in supernatants of ameliorates heat stress induced impairment of ionophore-treated polymorphonuclear leukocytes egg production, gut morphology, and intestinal from selenium deficient goats.American Journal mucosal immunity in laying hens. Poultry of Veterinary Research, 47(1), 148-151. Science, 91(3), 575-582. Babu, U., & Failla, M. L. (1990a). Respiratory Department of economic and social affairs. Affairs. burst and candidacidal activity of peritoneal United Nations. New York. (2015). Retrieved macrophages are impaired in copper-deficient 23 Sep 2015, from http://www.un.org/en/ rats. Journal of Nutrition, 120(12), 1692–1699. development/desa/news/population/2015-report. Babu, U., & Failla, M. L. (1990b). Copper status and html function of neutrophils are reversibly depressed Dhabhar, F. S. (2009). Enhancing versus suppressive in marginally and severely copper-deficient rats. effects of stress on immune function: implications Journal of Nutrition, 120(12), 1700–1709. for immunoprotection and immunopathology. Bala, S., & Failla, M. L. (1992). Copper deficiency Neuroimmunomodulation, 16(5), 300–317. reversibly impairs DNA synthesis in activated T Donker, R. A., Nieuwland, M. G. B., & van der Zijpp, lymphocytes by limiting interleukin 2 activity. A. J. (1990). Heat-stress influences on antibody Proceedings of the National Academy of production in chicken lines selected for high and Sciences, 89(15), 6794–97. low immune responsiveness. Poultry Science, Basset, C., Holton, J., Mahony, R., & Riott, I. (2003). 69(4), 599–607. Innate immunity and pathogen-host interaction. Elenkov, I. J., & Chrousos, G. P. (1999). Stress Vaccine, 21, 2-23. Hormones, Th1/Th2 patterns,Pro/Anti- Basu, A. K., & Bandhyopadhyay, P. K. (2004). The inflammatory Cytokines and Susceptibility effect of season on the incidence of ticks. Bulletin to Disease. Trends in Endocrinology and of Animal Health and Production in Africa, Metabolism, 10(9), 359-368. 52(1), 39–42. Elenkov, I. J., Papanicolaou, D. A., Wilder, R. Blecha, F., Pollmann, D. S., & Nichols, D. A. (1983). L., & Chrousos, G. P. (1996). Modulatory Weaning pigs at an early age decreases cellular effects of glucocorticoids and catecholamines immunity. Journal of Animal Science, 56(2), on human interleukin-12 and interleukin-10 396-400. production: clinical implications. Proceedings of

476 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity

the Association of American Physicians, 108(5), Grasso, P. J., Scholz, R. W., Erskine, R. J., & Eberhart, 374–381. R. J. (1990). Phagocytosis, bactericidal activity and oxidative metabolism of milk neutrophils Elvinger, F., Hansen, P. J., & Natzke, R. P. (1991). from dairy cows fed selenium-supplemented Modulation of function of bovine polymorph and selenium-deficient diets. American Journal nuclear leukocytes and lymphocytes by high of Veterinary Research, 51(2), 269-274. temperature in vitro and in vivo. American Journal of Veterinary Research, 52(10), 1692-8. Hall, D. M., Baumgardner, K. R., Oberley, T. D., & Gisolfi, C. V. (1999). Splanchnic tissues undergo Epstein, P., Diaz, H., Elias, S., Grabherr, G., Graham, hypoxic stress during whole body hyperthermia. N., Martens, W., Thompson, E. M., & Susskind, J. American Journal of Physiology, 276(5), 1195- (1998). Biological and physical signs of climate 1203. change, focus on mosquito borne diseases. Bulletin of the American Meteorological Society, Hall, H. T. B. (1998). Diseases and Parasites 79(3), 409 - 417. of Livestock in the Tropics. Harlow, Essex: Longman Scientific and Technical. FAO. (2009). State of food and agriculture—Livestock in the balance. Rome, FAO. Hassanpour, H., Zamani Moghaddamb, A. K., Khosravi, M., & Mayahi, M. (2013). Effects Feng, J., Ma, W. Q., Xu, Z. R., Wang, Y. Z., & Liu, of synbiotic on the intestinal morphology and J. X. (2007). Effects of iron glycine chelate on humoral immune response in broiler chickens. growth, haematological and immunological Livestock Science, 153(1), 116–122. characteristics in weanling pigs. Animal Feed Science and Technology, 134(3), 261–272. Hu, Y., Jin, H., Du, X., Xiao, C., Luo, D., Wang, B., & She, R. (2007). Effects of chronic heat stress Galon, J., Franchimont, D., Hiroi, N., Frey, G., on immune responses of the foot-and-mouth Boettner, A., Ehrhart-Bornstein, M., Oshea, J. disease DNA vaccination. DNA and Cell Biology, J., Chrousos, G. P., & Bornstein, S. R. (2002). 26(8), 619-626. Gene profiling reveals unknown enhancing and suppressive actions of glucocorticoids on IPCC (Intergovernmental Panel on Climate Change). immune cells. FASEB Journal, 16(1), 61–71. (2013). Climate Change : The Physical Science Basis. Contribution of Working Group I to the Gibson, G. R., & Roberfroid, M. B. (1995). Dietary Fifth Assessment Report of the Intergovernmental modulation of the human colonic microflora: Panel on Climate Change [Stocker, T.F., D. introducing the concept of prebiotics. Journal Qin, G.-K. Plattner, M. Tignor, S.K. Allen, J. of Nutrition, 125(6), 1401–1412. Boschung, A. Nauels, Y. Xia, V. Bex and P.M. Girndt, M., Sester, U., Kaul, H., Hunger, F., & Kohler, Midgley (Eds.)]. United Kingdom and New H. (1998). Glucocorticoids inhibit activation- York, NY, USA: Cambridge University Press, dependent expression of costimulatory molecule Cambridge. 1535 pp. B7–1 in human monocytes. Transplantation, Jain, N. C. (1993). Essentials of veterinary hematology. 66(3), 370–375. USA: Lea and Febiger. Glaser, R., Kutz, L. A., MacCallum, R. C., & Malarkey, Janeway, C. A., & Medzhitov, R. (2002). Innate W. B. (1995). Hormonal modulation of Epstein- immune recognition. Annual Review of Barr virus replication. Neuroendocrinology, Immunology, 20(1), 197–216. 62(4), 356–361.

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 477 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

Jin, Y., Hu, Y., Han, D., & Wang, M. (2011). Kelley, D. W., Greenfield, R. E., Evermann, J. F., Chronic heat stress weakened the innate Parish, S. M., & Perryman, L. E. (1982b). immunity and increased the virulence of highly Delayed-type hypersensitivity, contact pathogenic avian influenza virus H5N1 in sensitivity, and phytohemagglutinin skin test mice. Journal of Biomedicine and Biotechnology. responses of heat- and cold- stressed calves. Retrieved from http:// www.ncbi.nlm.nih.gov/ American Journal of Veterinary Research, 43(5), pubmed/21687549. 775-779.

Jin, L., Yan, S., Shi, B., Bao, H., Gong, J., Guo, X., Koga, T., Harada, H., Shi, T. S., Okada, S., Suico, M. & Li, J. (2014). Effects of vitamin A on the milk A., Shuto, T., & Kai, H. (2005). Hyperthermia performance, antioxidantfunctions and immune suppresses the cytotoxicity of NK cells via functions of dairy cows. Animal Feed Science down-regulation of perforin/granzyme B and Technology, 192, 15–23. expression. Biochemical and Biophysical Research Communications, 337(4), 1319–1323. Ju, X. H., Xu, H. J., Yong, Y. H., An, L. L., Jiao, P. R., & Liao, M. (2014). Heat stress up regulation of Lacetera, N., Bernabucci, U., Ronchi, B., Scalia, Toll-like receptors 2/4 and acute inflammatory D., & Nardone, A. (2002). Moderate summer cytokines in peripheral blood mononuclear cell heat stress does not modify immunological (PBMC) of Bama miniature pigs: an in vivo and parameters of Holstein dairy cows. International in vitro study. Animal, 8(9), 1462-1468. Journal of Biometeorology 46(1), 33-37.

Kabir, S. M. L., Rahman, M. M., Rahman, M. B., & Lambert, G. P., Gisolfi, C. V., Berg, D. J., Moseley, Ahmed, S. U. (2004). The dynamics of probiotics P. L., Oberley, L. W., & Kregel, K. C. (2002). on growth performance and immune response Selected contribution: Hyperthermia-induced in broilers. International Journal of Poultry intestinal permeability and the role of oxidative Science, 3(5), 361–364. and nitrosative stress. Journal of Applied Physiology, 92(4), 1750-1761. Kadzere, C. T., Murphy, M. R., Silanikove, N., & Maltzb, E. (2002). Heat stress in lactating dairy Lang, C. H., Silvis, C., Deshpande, N., Nystrom, G., cows a review. Livestock Production Science, & Frost, R. A. (2003). Endotoxin stimulates in 77(1), 59–91. vivo expression of inflammatory cytokines tumor necrosis factor alpha, interleukin-1beta, 6, and Kelley, K. W. (1980). Stress and immune function: high-mobility-group protein-1 in skeletal muscle. a bibliographic review. Annales de Recherches Shock, 19(6), 538 –546. Veterinaires, 11(4), 445-478. Lee, N. K., Lee, J. Y., Kwak, H. G., & Paik, H. D. Kelley, K. W. (2004). From hormones to immunity: (2008). Perspectives for the industrial use of the physiology of immunology. Brain, Behaviour, bacteriocin in dairy and meat industry. Korean and Immunity, 18(2), 95–113. Journal for Food Science of Animal Resources, Kelley, D. W., Osborne, C. A., Evermann, J. F., 28(1), 1-8. Parish, S. M., & Gaskins, C. T. (1982a). Liu, X., Li, H., Lu, A., Zhong, Y., Hou, X., Wang, N., Effects of chronic heat and cold stressors on Jia, D., Zan, J., Zhao, H., Xu, J., & Liu, F. (2012). plasma immunoglobulin and mitogen-induced Reduction of intestinal mucosal immune function blastogenesis in calves. Journal of Dairy Science, in heat-stressed rats and bacterial translocation. 65(8), 1514-1528. International Journal of Hyperthermia, 28(8), 756- 765.

478 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity

Liu, X., Shi, Y., Hou, X., Wan, C., He, S., Chong, X., Marple, D. N., Aberle, E. D., Forrest, J. C., Blake, W. Liu, M., Li, H., & Liu, F. (2014). Microarray H., & Judge, M. D. (1972). Endocrine responses analysis of intestinal immune-related gene of stress susceptible and stress resistant swine expression in heat-stressed rats. International to environmental stressors. Journal of Animal Journal of Hyperthermia, 30(5), 324-327. Science, 35(3), 576–579.

Lloyd, A. B., Cumming, R. B., & Kent, R. D. Martin, S. W., Schwabe, C. W., & Franti, C. E. (1975). (1977). Prevention of Salmonella typhimurium Dairy calf mortality rate: characteristics of calf infection in poultry by pre-treatment of chickens mortality rates in Tulare County, California. and poults with intestinal extracts. Australian American Journal of Veterinary Research, 36(8), Veterinary Journal, 53(2), 82-87. 1099–1104.

Lu, A., Wang, H., Hou, X., Li, H., Cheng, G., Wang, Mashaly, M. M., Hendricks, G. L., Kalama, M. A. N., Zhu, X., Yu, J., Luan, W., Liu, F., & Xu, J. Gehad, A. E., Abbas, A. O., & Patterson, P. H.. (2011). Microarray analysis of gene expression (2004). Effect of Heat Stress on Production profiles of rat small intestine in response to heat Parameters and Immune Responses of stress. Journal of Biomolecular Screening, 16(6), Commercial Laying Hens. Poultry Science, 655–667. 83(6), 889–894.

Lu, C. D. (1989). Effect of heat stress on goat Mason, W. A., Mendoza, S. P., & Moberg, G. production. Small Ruminant Research, 2(2), P. (1991). Persistent effects of early social 151–162. experience on physiological responsiveness. In A. Ehara, T. Kimura, D. Takenaka, D. & M. Lukasewycz, O. A., & Prohaska, J. R. (1990). The Iwamoto, M. (Ed.), Primatology Today (pp. 469- immune response in copper deficiency. Annals 471). Amsterdam: Elsevier Sciences Publishers. of the New York Academy of Sciences, 587(1), 147–159. Maurya, V. P., Sejian, V., Kumar, K., Singh, G., & Naqvi, S. M. K. (2012). Walking stress Marai, I. F. M., El-Darawany, A. A., Abou-Fandoud, influence on livestock production. In V. Sejian, E. I., & Abdel-Hafez, M. A. M. (2006). Serum S.M.K. Naqvi, T. Ezeji, L. Lakritz, R. Lal blood components during pre-oestrus, oestrus (Eds.), Environmental stress and amelioration and pregnancy phases in Egyptian Suffolk as in livestock production (pp. 75-95). German: affected by heat stress, under the conditions of Springer-Verlag GMbH Publisher. Egypt. Egyptian Journal of Sheep, Goat and Desert Animals Sciences, 1(1), 47–62. McVoy, M. A., & Adler, S. P. (1989). Immunologic evidence for frequent age-related cytomegalovirus Marchant, A., Bruyns, C., Vandenabeele, P., reactivation in seropositive immunocompetent Ducarme, M., Gerard, C., & Delvaux, A. (1994). individuals. Journal of Infectious Diseases, Interleukin-10 controls interferon-γ and tumor 160(1), 1-10. necrosis factor production during experimental endotoxemia. European Journal of Immunology, Mehla, K., Magotra, A., Choudhary, J., Singh, A.K., 24(5), 1167–1171. Mohanty, A. K., Upadhyay, R. C., Srinivasan, S., Gupta, P., Choudhary, N., Antony, B., & Khan, Marin, M. L., Lee, J. H., Murtha, J., Ustunol, Z., F. (2014). Genome-wide analysis of the heat & Pestka, J. J. (1997). Differential cytokine stress response in Zebu (Sahiwal) cattle. Gene, production in clonal macrophage and T-cell lines 533(2), 500-507. cultured with bifidobacteria. Journal of Dairy Science, 80(11), 2713- 2720.

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 479 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

Meng, D., Hu, Y., Xiao, C., Wei, T., Zou, Q., & Pearce, S. C., Mani, V., Boddicker, R. L., Johnson, Wang, M. (2013). Chronic Heat Stress Inhibits J. S., Weber, T. E., Ross, J. W., Rhoads, R. P., Immune Responses to H5N1 Vaccination through Baumgard, L. H., & Gabler, N. K. (2013). Heat Regulating CD4+CD25+Foxp3+ Tregs. BioMed stress reduces intestinal barrier integrity and Research International. Retrieved from http:// favors intestinal glucose transport in growing www.hindawi.com/journals/bmri/2013/160859/ pigs. Plos-One, 8(8), 70215.

Miller, W. L., & Tyrrell, J. B. (1995). The adrenal Popovic, Z. (2004). Performance and Udder Health cortex. In P. Felig, J. D. Baxter, & L. A. Frohman. Status of Dairy Cows influenced by organically (Ed.), Endocrinology and metabolism (pp. 555- bound Zinc and Chromium. Proceedings of 711). USA: McGraw-Hill Inc. the 20th Annual Symposium on Nutritional Biotechnology in the Feed and Food Industries, Moberg, G. P., & Mench, J. A. (2000). The biology of Lexington, KY, USA. animal stress: Basic principles and implications for animal welfare. USA: CABI Pub. Quinteiro-Filho, W. M., Gomes, A. V. S., Pinheiro, M. L., Ribeiro, A., Ferraz-de-Paula, V., Astolfi- Moonsie-Shageer, S., & Mowat, D. N. (1993). Ferreira, C. S., Ferreira, A. J., & Palermo-Neto, Effect of level of supplemental chromium on J. (2010). Heat stress impairs performance performance, serum constituents, and immune and induces intestinal inflammation in broiler status of stressed feeder calves. Journal of chickens infected with Salmonella Enteritidis. Animal Science. 71(1), 232. Avian Pathology, 41(5), 421-427. Nardone, A., Lacetera, N., Bernabucci, U., & Ronchi, Rao, P. P., Hegde, N. R., & Reddy, Y. N. (2012). B. (1997). Composition of colostrum from dairy Intercontinental movement of bluetongue virus heifers exposed to high air temperatures during and potential consequences to trade. Journal of late pregnancy and the early postpartum period. Virology, 86(15), 8341. Journal of Dairy Science, 80(5), 838–844. Regnier, J. A. K., Kelley, W., & Gaskins, C. T. Nespital, T., & Straus, G. J. (2012). The Jak/STAT (1980). Acute thermal stressors and synthesis of signalling pathway is down regulated at febrile antibodies in chickens. Poultry Science, 59(5), temperatures. PLOS One, 7(11), e49374. 985–990. Pattison, J. (1998). The Emergence of Bovine Riedemann, T., Patchev, A. V., Cho, K., & Almeida, Spongiform Encephalopathy and Related O.F. (2010). Corticosteroids: way upstream. Diseases. Emerging Infectious Diseases, 4(3), Molecular Brain, 3(1), 2-20. 390-394. Rivington, M., Matthews, K. B., Buchan, K., Miller, Paul, A., Dangi, S. S., Gupta, M., Singh, J., Thakur, D., & Russell, G. (2009). Investigating climate N., Naskar, S., Nanda, P. K., Mohanty, N., Das, change impacts and adaptation options using A. K., Bandopadhayay, S., Das, B. C., & Sarkar, integrated assessment methods. Aspects of M. (2015). Expression of TLR genes in Black Applied Biology, 93, 85–92. Bengal goat (Capra hircus) during different seasons. Small Ruminant Research, 124, 17–23. Sanders, V. M., Baker, R. A., Ramer-Quinn, D.S., Kasprowicz, D. J., Fuchs, B. A., & Street, N. Paulrud, C. O. (2005). Basic concepts of the bovine E. (1997). Differential expression of the beta2- teat canal. Veterinary Research Communications, adrenergic receptor by Th1 and Th2 clones: 29(3), 215–245. implications for cytokine production and B cell help. Journal of Immunology, 158(9), 4200–4210.

480 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Heat Stress and Immunity

Sejian, V., Maurya, V. P., & Naqvi, S. M. K. (2011). Sordillo, L. M., Shafer-Weaver, K., & DeRosa, D. Effect of thermal stress, restricted feeding and (1997). Immunobiology of the mammary gland. combined stresses (thermal stress and restricted Journal of Dairy Science, 80(8), 1851–1865. feeding) on growth and plasma reproductive Steinfeld, H., Gerber, P., Wassenaar, T., Castel, V., hormone levels of Malpura ewes under semi- Rosales, M., & de Haan, C. (2006). Livestock’s arid tropical environment. Journal of Animal long shadow: environmental issues and options. Physiology and Animal Nutrition, 95(2), 252– Italy: FAO. 258. Stott, G. H., Wiersma, F., Menefee, B. E., & Radwanski, Sejian, V., Maurya, V. P., & Naqvi, S. M. K. (2012). F. R. (1976). Influence of environment on Effect of walking stress on growth, physiological passive immunity in calves. Journal of Dairy adaptability and endocrine responses in Malpura Science, 59(7), 1306–1311. ewes under semi-arid tropical environment. International Journal of Biometeorology, 56(2), Subba Rao, D. S. V., & Glick, B. (1970). 243–252. Immunosuppressive action of heat in chickens. Proceedings of the Society for Experimental Shinde, A. K., & Sejian, V. (2013). Sheep husbandry Biology and Medicine, 133(2), 445-448. under changing climate scenario in India: An overview. Indian Journal of Animal Science, Thaxton, P., Sadler, C.R., & Glick, B. (1968). Immune 83(10), 998–1008. response of chickens following heat exposure or injection with ACTH. Poultry Science, 47(1), Shini, S., Huff, G. R., Shini, A., & Kaiser, P. 264–266. (2010). Understanding stress-induced immunosuppression: Exploration of cytokine and Tjoelker, L. W., Chew, B. P., Tanaka, T. S., & Daniel, chemokine gene profiles in chicken peripheral L. R. (1990). Effect of dietary vitamin A and leukocytes. Poultry Science, 89(4), 841–851. β-carotene on polymorphonuclear leukocyte and lymphocyte function in dairy cows during Singh, K. B., Nauriyal, D. C., Oberoi, M. S., & Baxi, the early dry period. Journal of Dairy Science, K. K. (1996). Studies on occurrence of clinical 73(4), 1017–1022. mastitis in relation to climatic factors. Indian Journal of Dairy Science, 49(8), 534–536. Tort, L., & Teles, M. (2011). The Endocrine Response to Stress-A Comparative View. Basic and Clinical Sirohi, S., & Michaelowa, A. (2007). Sufferer and Endocrinology Up-to-Date, ISBN, 978-953. cause: Indian livestock and climatic change. Climatic Change, 85(3-4), 285-298. UNFPA (United Nations Population Fund). (2008). The State of World Population 2007: unleashing Smith, K. L., Hogan, J. S., & Weiss, W. P. (1997). the potential of urban growth. United Nations Dietary Vitamin E and selenium affect mastitis Population Fund. Retrieved from http://www. and milk quality. Journal of Animal Science, unfpa.org/swp/swpmain.html. 75(6), 1659-1665. United Nations (2015). Department of Economics Smoak, K.A.,& Cidlowski, J.A. (2007). Glucocorticoid and Social Affairs: World population projected Signaling in Health and disease. In del Rey, A., to reach 9.7 billion by 2050. Retrieved from George P. Chrousos, G. P., & Besedovsky, H.O., http://www.un.org/en/development/desa/news/ (Eds.) The hypothalamo-pituitary-Adrenal axis. population/2015-report.html 7, 1-394.

Pertanika J. Trop. Agric. Sci. 39 (1): 459 - 482 (2016) 481 Sophia Inbaraj, Veerasamy Sejian, Madiajagan Bagath and Raghavendra Bhatta

Webster, M. J. I., & Glaser, R. (2008). Stress hormones World Bank. (2009). Minding the stock: bringing public and immune function. Cell Immunology, 252(1), policy to bear on livestock sector development. 16–26. Report no. 44010-GLB.Washington DC.

Webster, J. C., & Cidlowski, J. A. (1994). Wu, C. Y., Wang, K., McDyer, J. F., & Seder, R. A. Downregulation of the glucocorticoid receptor. (1998). Prostaglandin E2 and dexamethasone A mechanism for physiological adaptation to inhibit IL-12 receptor expression and IL-12 hormones. Annals of the New York Academy of responsiveness. Journal of Immunology, 161(6), Sciences, 746(1), 216-220. 2723–2730.

Webstar, A. C. (1975). The adverse effect of Yahav, S. (2009). Alleviating heat stress in domestic environment on the response to distemper fowl: Different strategies. World Poultry Science vaccination. Australian Veterinary Journal, Journal, 65(04), 719–732. 51(10), 488-490. Yu, J., Liu, F., Yin, P., Zhao, H., Luan, W., Hou, X., Welc, S. S., Clanton, T. L., Dineen, S. M., & Leon, L. Zhong, Y., Jia, D., Zan, J., Ma, W., Shu, B., & R. (2013). Heat stroke activates a stress induced Xu, J. (2013). Involvement of oxidative stress cytokine response in skeletal muscle. Journal of and mitogen-activated protein kinase signalling Applied Physiology, 115(8), 1126-1137. pathways in heat stress-induced injury in the rat small intestine. Stress, 16(1), 99–113. Whalen, M. M., & Bankhurst, A. D. (1990). Effects of beta-adrenergic receptor activation, cholera Yu, J., Yin, P., Liu, F., Cheng, G., Guo, K., Lu, A., toxin and forskolin on human natural killer cell Zhu, Z., Luan, W., & Xu, J. (2010). Effect function. Biochemistry Journal, 272(2), 327-331. of heat stress on the porcine small intestine: a morphological and gene expression study. White, L. A., Newman, M. C., Cromwell, G. L., Comparative Biochemistry and Physiology- & Lindemann, M. D. (2002). Brewers dried Part A. Molecular and Integrative Physiology, yeast as a source of mannan oligosaccharides 156(1), 119–128. for weanling pigs. Journal of Animal Science, 80(10), 2619-2628. Zhou, J., An, H., Xu, H., Liu, S., & Cao, X. (2005). Heat shock up-regulates expression of Toll-like Wittmann, E. J., & Baylis, M. (2000). Climate change: receptor-2 and Toll-like receptor-4 in human effects on Culicoides transmitted viruses and monocytes via p38 kinase signal pathway. implications for the UK. The Veterinary Journal, Immunology, 114(4), 522–530. 160(2), 107-117.

Won, S. J., & Lin, M. T. (1995). Thermal stresses reduce natural killer cell cytotoxicity. Journal of Applied Physiology, 79(3), 732-737.

482 Pertanika J. Trop. Agric. Sci. 39 (4) 459 - 482 (2016) Pertanika J. Trop. Agric. Sci. 39 (4): 483 - 490 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Short Communication A Note Comparing the Apparent Metabolisable Energy of Three Oil Sources and their Combination in Broiler Chickens

N. R. Abdulla1,5, T. C. Loh1,2*, H. Akit1, A. Q. Sazili1,3 and H. L. Foo4 1Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 3 Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 5Department of Animal Resource, University of Salah al- Din, Erbil, Kurdistan Region, Iraq

ABSTRACT Apparent metabolisable energy (AME) of palm oil (PO), soybean oil (SO), linseed oil (LO) and blend oil (BO) [PO, SO and LO in a ratio of 4:1:1] were evaluated. A total of 75, 21-day-old birds were a fed corn-soy basal diet and four test diets containing different oil sources (PO, SO, LO and BO), were developed by replacing 60 g/kg of the basal diet for eight days. Differences in the apparent metabolisable energy were found (P<0.05), with the higher values for broiler-fed BO. This study affirmed that BO increases AME of oil enriched with saturated fatty acid in poultry diets.

Keywords: Broiler chicken, blend oil, linseed oil, metabolisable energy, palm oil, soybean oil

INTRODUCTION Broiler diets are supplemented with vegetable oils and animal fats to increase energy concentration and to improve

ARTICLE INFO productivity (Lopez-Bote et al., 1997). Article history: This is because the apparent metabolisable Received: 4 May 2016 Accepted: 11 August 2016 energy (AME) content in oil is thrice higher E-mail addresses: than that of other feedstuff (Mateos & Sell, [email protected] (N. R. Abdulla), [email protected] (T. C. Loh), 1981). Thus, oil is a vital component in [email protected] (H. Akit), [email protected] (A. Q. Sazili), compounding of high-energy broiler diets. [email protected] (H. L. Foo) * Corresponding author As feeding cost accounts for about 65%

ISSN: 1511-3701 © Universiti Putra Malaysia Press N. R. Abdulla, T. C. Loh, H. Akit, A. Q. Sazili and H. L. Foo of total production cost, dietary oils may sources of oil that have been typically used well be used as a way in which broiler in broiler diets, those data were recorded 25 chicken production might enhance growth to 50 years ago. Not only have oil sources performance and improve meat yield at changed since then (composition and quality a reasonably cheaper price (Corzo et al., indices), broilers have also gone through 2005). Other advantages of using fats major heritable change. Consequently, include increased palatability, reduced consistent and current AME data on these dustiness and improved feed texture (Baião sources of oil will allow for accurate & Lara, 2005; Ayed et al., 2015). Among all formulation of the energy content in broiler vegetable oils, soybean oil and oil palm are feeds. Thus, the current study was aimed widely used in the feed industry. Abdulla at assessing the AME in different sources et al. (2015, 2016a and 2016b) observed of oils: Palm oil (PO), soybean oil (SO) differences in dietary sources of palm oil, and linseed oil (LO) when fed to broiler soybean oil and linseed oil and their effect chicks. Also, the study aimed to examine the on growth performance, gut morphology, probable synergism of combination PO, SO composition of fatty acid, oxidative stability and LO in a ratio of 4:1:1. and cholesterol content of breast muscle in broiler chicken. Although previous findings MATERIALS AND METHODS have shown the influence of supplementing Ethical Note different kinds of oils on metabolism of This study was conducted in accordance with fats, growth performance in broiler birds the animal ethics guidelines of the Research as well as the apparent metabolisable Policy of University Putra Malaysia. energy (AME), the information are still not consistent. For instance, Tancharoenrat Birds, Husbandry and Experimental et al. (2013) states that the use of soybean Procedure oil, palm oil and poultry fat had lower One hundred one-day-old Cobb 550 broiler (P<0.05) apparent metabolisable energy birds bought from a commercial farm than tallow while the differences were were fed starter diets (22% crude protein) absent in poultry fat, soybean oil and for 21 days. During the first week, their palm oil. Kavouridou et al. (2008) found temperature was maintained at 35 oC and that the birds consuming linseed oil had a then reduced steadily to about 28 oC until significantly higher percentage of apparent the conclusion of the experiment. The birds metabolisable energy matched with birds fed were vaccinated against Newcastle disease a diet containing palm oil but there was no (ND) and infectious bronchitis (IB) live significant difference from birds fed a diet vaccine (MyVac, Kuala Lumpur, Malaysia) supplemented with soybean and coconut oil. through the intraocular route on day 7 and Although previous works have described 14 of the raising period. The infectious the metabolisable energy (ME) of different bursal disease vaccine (IBD) (MyVac, Kuala

484 Pertanika J. Trop. Agric. Sci. 39 (4) 483 - 490 (2016) Metabolisable Energy of Different Oil Sources in Broiler

Lumpur, Malaysia) was administered on day Apparent Metabolisable Energy Assay 21 through intraocular route. The birds were The AME for PO, SO, LO and blend given feed and water ad-libitum. On day 21, oil (BO) were determined following the birds of similar body weight were picked procedures of Nalle et al. (2011). According at random and allocated to experimental to the procedure, the corn-soybean basal diet units. Three birds per unit (cage) and five was compounded as shown in Table 1 and replicate units were randomly allocated to the test diets, each containing a different oil a basal diet and each of the four test diets: sample, were prepared by substituting 60 g/ Palm oil (PO), soybean oil (SO), linseed oil kg of the basal diet with a different oil. (LO) and blend oil (BO). The blend oil was The DM of samples was determined a combination of PO, SO and LO in a ratio following the standard guidelines of AOAC of 4:1:1. The classic total excreta method (2007). Prior to oven drying, the weight of was used to measure the AME test. The birds all the samples was taken and later placed were fed in mash form for a period of eight in an oven dryer for a period of 24 hours days, with the first four days as an adaptation at a temperature of 105 oC and the weights period. During the last four days, feed were recorded again after half an hour of intake (FI) of each unit was recorded, and cooling in a desiccator. To determine the GE the chicks’ faeces was collected on a daily an adiabatic bomb calorimeter (Gallenkamp basis, collected and weighed within a cage. Autobomb, UK) standardised with benzoic Faeces collected was thoroughly mixed, and acid was used. typical samples were obtained and freeze- The AME was calculated for both the dried (Model 0610, Cuddon Engineering, basal and test diets (different oils) applying Blenheim, New Zealand). Dried fecal the following formula: samples were crushed to pass through a 0.5 mm sieve and kept in airtight plastic AME of diet (kcal/kg) containers at −4 oC for further analyses. Dry = [(FI × GE diet) – (Excreta matter (DM) and gross energy (GE) of the output × GE excreta)] ÷ FI feeds and fecal samples were analysed. AME of oil (kcal/kg) = [(AME of test diet) – (AME basal Determination of Fatty Acid diet × 0.94)] ÷ 0.06 Composition of Oil Sources The total fatty acids were extracted from (Ravindran et al., 2014) the different oils following the protocol of Folch et al. (1957), modified by Ebrahimi et al. (2014) and described by Abdulla et al. (2015).

Pertanika J. Trop. Agric. Sci. 39 (1): 483 - 490 (2016) 485 N. R. Abdulla, T. C. Loh, H. Akit, A. Q. Sazili and H. L. Foo

Table 1 The Basal Diet Compositions (g/kg, as Fed Basis) Used in the AME Digestibility Assays

Ingredient (%) AME assay1 Corn 65.40 Soybean meal 30.38 Mono di calcium phosphate 21% 1.35 Calcium carbonate 1.55 Salt 0.30 L-Lysine 0.30 DL-Methionine 0.20 Vitamin premix2 0.10 Choline chloride 0.10 Toxin binder3 0.15 Anti-oxidant4 0.02 Mineral premix5 0.15 Total, kg 100.00 Calculated Analysis ME (kcal/kg) 2887.00 Crude protein (%) 19.00 Fat (%) 2.66 ME/CP 151.95 Fibre (%) 3.76 Calcium (%) 0.92 Total Phosphorus (%) 0.64 Avail. P for Poultry (%) 0.36 L-Lysine (%) 1.21 DL-Methionine (%) 0.50 1Test diets were prepared by substituting 60 g/kg of the basal diet with palm oil, soybean oil and linseed oil. 2 Supplied per kg diet: Vitamin A 11,494 IU; vitamin D 1,725 IU; vitamin E 40 IU; vitamin K3 2.29 mg; cobalamin 0.05 mg, thiamine 1.43 mg, riboflavin 3.44 mg, folic acid 0.56 mg, biotin 0.05 mg, panthothenic acid 6.46 mg, niacin 40.17 mg, pyridoxine 2.29 mg. 3Toxin binder contains natural hydrated sodium calcium aluminium silicates (HSCAS). 4Antioxidant contains butylated hydroxyanisole (BHA). 5Supplied per kg diet: Fe 120 mg, Mn 150 mg, Cu 15 mg, Zn 120 mg, I 1.5 mg, Se 0.3 mg, Co 0.4 mg.

STATISTICAL ANALYSIS significance for all the analyses was set at Data obtained were subjected to one- 0.05. way ANOVA testing and analysed using the general linear model of SAS (SAS, RESULTS 2007). The significant differences among Table 2 shows the composition of the fatty the treatment means were compared acids of the different oil sources. According using Duncan’s multiple range tests while to the results, the concentration of oleic the Alpha level used for assessment of (C18:1n-9) and palmitic acid (C16:0) in PO

486 Pertanika J. Trop. Agric. Sci. 39 (4) 483 - 490 (2016) Metabolisable Energy of Different Oil Sources in Broiler

Table 2 The Composition of Fatty Acids (% of Total Identified Fatty Acids) of Different Oils1

Fatty acid Palm oil Soybean oil Linseed oil Blend oil SEM8 C12:0 0.34a 0 c 0 c 0.23b 0.06 C14:0 1.00 a 0.09 c 0 d 0.68 b 0.16 C16:0 37.45 a 10.80 c 5.25 d 27.64 b 4.97 C16:1 n-7 0.21 a 0.08 b 0.05 b 0.16 a 0.03 C18:0 4.10 b 4.40 a 3.83 c 4.11 b 0.08 C18:1n-9 44.37 a 25.38 c 20.02 d 37.14 b 3.69 C18:2n-6 12.01 d 52.45 a 15.98 c 19.41 b 6.43 C18:3n-3 0.24 d 6.44 c 54.48 a 10.31 b 8.57 SFA2 42.66 a 15.00 c 9.00 d 32.66 b 5.18 USFA3 56.84 d 84.35 b 90.53 a 67.02 c 5.18 MUSFA4 44.57 a 25.46 c 20.06 d 37.30 b 3.72 PUFAn-35 0.24 d 6.44 c 54.48 a 10.31 b 8.57 PUFAn-66 12.01 d 52.45 a 15.98 c 19.41 b 6.43 n-6: n-3 ratio7 48.75 a 8.14 b 0.29 d 1.88 c 7.52 USFA: SFA 1.32 d 5.51 b 9.96 a 2.05 c 1.25 PUFA: SFA 0.28 d 3.84 b 7.75 a 0.90 c 1.08 1The data are expressed as the percentage of identified fatty acids. 2Total saturated fatty acid= sum of C12:0+C14:0+ C16:0+C18:0. 3Total unsaturated fatty acid= sum of C16:1n-7+ C18:1n-9+ C18:2n-6+C18:3n-3+C20:4n-6+C20:5n-3 +C22:5n-3+C22:6n-3. 4Total monounsaturated fatty acid = sum of C16:1n-7+C18:1n-9. 5 polyunsaturated fatty acid n-3 = sum of C18:3n-3+C20:5n-3+C22:5 n-3+C22:6n-3. 6 polyunsaturated fatty acid n-6 = sum of C18:2n-6+C18:3n- 6+C20:4n-6. 7 polyunsaturated fatty acid n-6: polyunsaturated fatty acid n-3= (C18:2n-6+C18:3n-6+C20:4n-6) ÷ (C18:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3). 8SEM: Standard error of means. was significantly (P<0.05) higher than that (9413 kcal/kg) of AME in comparison with in SO, LO and BO, while LO and SO were PO (8277 kcal/kg), SO (8401 kcal/kg) and significantly higher in terms of α-linolenic LO (8423 kcal/kg). On the other hand, acid and linoleic acid, respectively in there was no significant difference (P>0.05) comparison with PO. The ranking of n-6:n-3 between PO, SO and LO. ratios was in the order of PO>SO>BO>LO. The proportion of total saturated (SFA) DISCUSSION and total mono-unsaturated fatty acids The values of ME in oils is mainly influenced (MUSFA) of PO was higher (P<0.05), while by their ingestion, which is influenced by a total unsaturated fatty acid (USFA) content myriad of factors such as the absence or was lower (P<0.05) compared to those of presence of ester bonds (free fatty acids or SO, LO and BO. tryglycerides), number of double bonds, The apparent metabolisable energy of stretch of the carbonic chain, the type and different sources of oil in broiler chicken is amount of the triglycerides augmented summarised in Figure 1. The result showed in the diets, free fatty acid compositions, that the BO had the highest (P<0.05) value detailed arrangement of SFA and USFA on

Pertanika J. Trop. Agric. Sci. 39 (1): 483 - 490 (2016) 487 N. R. Abdulla, T. C. Loh, H. Akit, A. Q. Sazili and H. L. Foo

PO: Palm oil, SO: Soybean oil, LO: Linseed oil, BO: Blend of palm, soybean and linseed oil in a ratio of 4:1:1. a,b indicate significant differences (p<0.05) between different oil sources. Values are means ± 1 standard error.

Figure 1. Apparent metabolisable energy (AME) for various sources of oil in broiler chicken. the backbone of glycerol, age, sex and the contradict the findings of Kavouridou et al. intestinal flora of birds (Garrett & Young, (2008), who reported that birds fed a diet 1975; Ketels & De Groote, 1989; Leeson supplemented with LO had significantly & Summers, 2001; Nascif et al., 2004). higher AME compared with birds fed a In the present study, although PO had a diet supplemented with PO but this was not higher ratio of USFA to SFA compared to significantly different from birds fed SO. that of SO and LO, the AME in LO and SO The highest AME in BO could be due to the was seen to be similar to that of PO. The utilisation of SFA and this may be improved similarity in the AME of the PO, SO and by the presence of USFA. These results are LO could be due to the low concentration of in agreement with the findings of Garrett and stearic acid (C18:0) in the oils. Stearic acid Young (1975). This interaction is triggered stops the activity of lipase (Van Kuiken & by the excellent blending abilities in USFA Behnke, 1994). Thus, similar concentration (Garrett & Young, 1975). This interaction of acids such as stearic acid in oils may affects the absorption of SFA. However, have similar activity on lipase, resulting in a the utilisation of USFA was not influenced similar AME. This statement agrees with the by changing the USFA:SFA ratio (Garrett report of Tancharoenrat et al. (2013), who & Young, 1975). Ketels and De Groote reported that the AME of PO was similar (1989) reported that oil was used at the to that of SO. However, the present results bird’s maximal capacity if the ratio exceeded

488 Pertanika J. Trop. Agric. Sci. 39 (4) 483 - 490 (2016) Metabolisable Energy of Different Oil Sources in Broiler four. Diets containing saturated fat resulted AOAC. (2007). Official methods of analysis of the in less feed gain ratio than those containing association of official analytical chemists (18th poly unsaturated fats (Zollitsch et al., 1997). ed.). Association of Official Analytical Chemists, Washington D.C., USA. Also, Sanz et al. (2000) posited that the rate of saturation in broiler chickens of dietary Ayed, H. B., Attia, H., & Ennouri, M. (2015). Effect oils was affected by their accumulation of of oil supplemented diet on growth performance and meat quality of broiler chickens. Advanced fats and metabolic use. Techniques in Biology and Medicine, 4(156), 2379–1764. CONCLUSION Baião, N. C., & Lara, L. (2005). Oil and fat in broiler Based on the current results, it appears nutrition. Revista Brasileira De Ciência Avícola, that the AME of PO, SO and LO in broiler 7(3), 129–141. chickens was similar despite differences Corzo, A., Fritts, C. A., Kidd, M. T., & Kerr, B. J. in the fatty acid composition of the oils. In (2005). Response of broiler chicks to essential addition, the present evaluation showed that and non-essential amino acid supplementation blend oil is an attractive way to increase the of low crude protein diets. Animal Feed Science AME of oil that is rich with SFA for poultry and Technology, 118(3), 319–327. by adding oil rich with USFA. Ebrahimi, M., Rajion, M. A., & Goh, Y. M. (2014). Effects of oils rich in linoleic and α-linolenic REFERENCES acids on fatty acid profile and gene expression in goat meat. Nutrients, 6(9), 3913–3928. Abdulla, N. R., Loh, T. C., Akit, H., Sazili, A. Q., & Foo, H. L. (2016a). Effects of dietary oil sources Folch, J., Lees, M., & Sloane-Stanley, G. (1957). A and calcium: Phosphorus levels on growth simple method for the isolation and purification performance, gut morphology and apparent of total lipids from animal tissues. Journal of digestibility of broiler chickens. South African Biological Chemistry, 226(1), 497–509. Journal of Animal Science, 46(1), 42–53. Garrett, R. L., & Young, R. J. (1975). Effect of micelle Abdulla, N. R., Loh, T. C., Akit, H., Sazili, A. Q., Foo, formation on the absorption of neutral fat and H. L., Mohamad, R., Abdul Rahim, R., Ebrahimi, fatty acids by the chicken. Journal Nutrition, M., & Sabow, A. B. (2015). Fatty acid profile, 105(7), 827–838. cholesterol and oxidative status in broiler chicken Kavouridou, K., Barroeta, A., Villaverde, C., breast muscle fed different dietary oil sources Manzanilla, E., & Baucells, M. (2008). Fatty and calcium levels. South African Journal of acid, protein and energy gain of broilers fed Animal Science, 45(2), 153–163. different dietary vegetable oils. Spanish Journal Abdulla, N. R., Loh, T. C., Akit, H., Sazili, A. Q., of Agricultural Research, 6(2), 210–218. Foo, H. L., Kareem, K. Y., Mohamad, R., & Ketels, E., & De Groote, D. (1989). Effect of ratio of Abdul Rahim, R. (2016b). Effects of dietary unsaturated to saturated fatty acids of the dietary oil sources, calcium and phosphorus levels on lipid fraction on utilization and metabolizable growth performance, carcass characteristics energy of added fats in young chicks. Poultry and bone quality of broiler chickens. Journal of Science, 68(11), 1506–1512. Applied Animal Research, 45(1), 423–429.

Pertanika J. Trop. Agric. Sci. 39 (1): 483 - 490 (2016) 489 N. R. Abdulla, T. C. Loh, H. Akit, A. Q. Sazili and H. L. Foo

Leeson, S., & Summers, J. (2001). Minerals. Nutrition Sanz, M., Flores, A., & Lopez-Bote, C. (2000). of the chicken (4th ed.). University Books, The metabolic use of energy from dietary fat Guelph, Ontario, Canada, pp.394–397. in broilers is affected by fatty acid saturation. British Poultry Science, 41(1), 61–68. Lopez-Bote, C. J., Rey, A. I., Sanz, M., Gray, J. I., & Buckley, D. J. (1997). Dietary vegetable SAS. (2007). User’s guide (9.2 ed.). SAS institute. oils and α-tocopherol reduce lipid oxidation in Inc., Cary, NC, USA. rabbit muscle. The Journal of Nutrition, 127(6), Tancharoenrat, P., Ravindran, V., Zaefarian, F., & 1176–1182. Ravindran, G. (2013). Influence of age on the Mateos, G. G., & Sell, J. L. (1981). Influence of fat apparent metabolisable energy and total tract and carbohydrate source on rate of food passage apparent fat digestibility of different fat sources of semipurified diets for laying hens. Poultry for broiler chickens. Animal Feed Science and Science, 60(9), 2114–2119. Technology, 186(3), 186–192.

Nalle, C. L., Ravindran, G., & Ravindran, V. (2011). Van Kuiken, B. A., & Behnke, W. D. (1994). The Nutritional value of peas (Pisum sativum L.) activation of porcine pancreatic lipase by cis- for broilers: Apparent metabolisable energy, unsaturated fatty acids. Biochimica et Biophysica apparent ileal aminoacid digestibility and Acta (BBA)-Lipids and Lipid Metabolism, production performance. Animal Production 1214(2), 148–160. Science, 51(2), 150–155. Zollitsch, W., Knaus, W., Aichinger, F., & Lettner, F. Nascif, C. C. C., Gomes, P. C., Albino, L. F. T., & (1997). Effects of different dietary fat sources Rostagno, H. S. (2004). Determinação dos on performance and carcass characteristics of valores energéticos de alguns óleos e gorduras broilers. Animal Feed Science and Technology, para pintos de corte machos e fêmeas aos 21 66(1), 63–73. dias de idade. Revista Brasileira De Zootecnia, 33(2), 375–385.

Ravindran, V., Abdollahi, M., & Bootwalla, S. (2014). Nutrient analysis, apparent metabolisable energy and ileal amino acid digestibility of full fat soybean for broilers. Animal Feed Science and Technology, 197, 233–240.

490 Pertanika J. Trop. Agric. Sci. 39 (4) 483 - 490 (2016) Pertanika J. Trop. Agric. Sci. 39 (4): 491 - 502 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Stability Analysis of Panicle and Grain Traits of Rainfed Upland Rice in Two Tropical Ecologies of Nigeria

Nassir, Adesola L.* and Alawode, Yetunde O. Department of Crop Production, College of Agricultural Sciences, Olabisi Onabanjo University, Yewa Campus, Ayetoro, Nigeria

ABSTRACT Stability of grain yield in upland rice due to the unpredictability of environmental indices is of important consideration in the development of cultivars adapted to fairly wide cultivation zone. A study was conducted with fifteen upland rice varieties in two locations in South- Western Nigeria to evaluate the contribution of panicle and grain characters to stable grain production. Data collection spanned five environments and was subjected to stability analyses. The effects of genotype, environment and their interaction were significant for all the panicle and grain characters. Broad sense heritability estimate (HB) was moderate for hundred grain weight (62.4) and grain length (58.9) but was generally low for other grain yield traits, particularly grain weight per panicle (11.6) and grain weight per plant (5.6), respectively. Stability variance identified different genotypes as stable for most of the characters. The crossover attribute of AMMI PC 1 however complimented the significant verdict returned by the stability variance though the former also specified the direction of instability. The Yield Stability index (YSi) harnessed the advantages of the two statistics to identify different genotypes as stable for different characters. Thus, there is a need to constitute a pool of genotypes for the evolution of superior synthetic but stable cultivars.

Keywords: AMMI, genotype x environment interaction, grain yield, Oryza sativa, yield-stability index.

INTRODUCTION Genotype by environment (GE) interaction is an important issue in crop improvement ARTICLE INFO efforts, especially considering its importance Article history: Received: 26 September 2015 in the evolution of varieties with appreciably Accepted: 4 July 2016 high and stable grain yield across seasons E-mail addresses: and specific target regions. Plant breeders [email protected] (Nassir, Adesola L.), [email protected] (Alawode, Yetunde O.) are constantly guided by this to define * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press Nassir, Adesola L. and Alawode, Yetunde O. breeding strategies and direction (Ouk et al., for various crops to evaluate interaction 2007; Acuña et al., 2008; Nassir & Ariyo, between genotype and environment 2011). For a genotype to be commercially and hence, stability. This included the successful, it must perform well across computation of stability variance (Shukla, the range of environment likely to be 1972; Kang & Pham, 1991), yield/stability encountered in a target region over the entire biplots (Kempton, 1984), the Additive array of years in which the genotype could Main effect and Multiplicative method be in use. Beyond seasonal and location (AMMI) (McLaren & Chaudhary, 1994), the differences, however, cultivation conditions Genotype plus Genotype-by-Environment within season do transit from one condition Method (GGE) (Yan et al., 2000, 2007; to the other, as dictated by variability in Acuña et al., 2008; Nassir & Ariyo, 2011; moisture and other environmental indices. Acuña & Wade, 2012). The ultimate aim is This, as affirmed by Acuña et al. (2008), to generate conclusions that would guide makes the evaluation of genotypes with breeding direction to develop genotypes with respect to dominant traits necessary in good adaptation to fairly wide environments different environments to guide in efforts within seasons and across regions and aimed at evolving varieties with reasonably cultivation conditions. stable yield. Stability implies that both yield A lot of GE studies have placed emphasis and quality remain somewhat constant, and on identification of megaenvironments. this draws from holding in steady state some Rice production in the upland ecology aspects of morphology and physiology of the however suffers from variation in cultivation crop in question even when other cultivation conditions within and across seasons, factors change. This homoeostatic condition thereby making evaluation of stability must necessarily derive from the stability of necessary, with some emphasis on within the characters that cumulatively determine location environment factors. In most grain yield. cases, genotypes that show most stable Naturally, however, the presence of GE yield appear in the centre of the AMMI interaction makes it difficult to fully realise Biplot and thereby combine stability with the potential of a genotype for a region in average yield (Kempton, 1984; McLaren & which weather varies from year to year. Chaudhary, 1994; Yan et al., 2000; Gauch, When the GE interaction is significant, 2006; Acuña et al., 2008). This average yield the plant and environmental factors that is often a compromise of many plant and play a major role in causing differential environment factors and may not always performance, and their significance in meet the aspiration of farmers, hence the determining desirable breeding strategies, renewed emphasis on combining stability must be carefully considered (Kang & with high grain yield. The combination of Martin 1987; Yan & Hunt, 2001). A number recent techniques for analysing genotype- of approaches have been used overtime by-environment interaction with the Yield-

492 Pertanika J. Trop. Agric. Sci. 39 (4) 491 - 502 (2016) Stability Analysis of Panicle and Grain Traits of Upland Rice

Stability index has been canvassed by Nassir 56-60, WAB 33-25, WAB 96-1-1, WAB and Ariyo (2011). This study consequently 99-1-1and WAB 375-B-5-H2-1 (WAB 375), focused on the evaluation of stability of which are breeding lines being developed grain yield components of upland rice using for improved yield and especially tolerance the Yield-Stability method of Kang and to drought. Pham (1991), along with the AMMI model. Plant Establishment MATERIALS AND METHODS The upland rice varieties were sown in a Study Location nursery and later transplanted onto ploughed This study was conducted at the College upland paddy as soon as rainfall became of Agricultural Sciences, Olabisi Onabanjo steady. There were fifteen plants per single University. The first four plantings were row plot, arranged in a randomised complete done at Ago-Iwoye, Nigeria (3.92oN, block design with three replicates. The 6.95oE) tropical rainforest ecology from plants were separated by 30cm between and 2001 to 2004, either with the early or the late within rows. Similar planting procedure and rains. Two plantings were done at Ayetoro, agronomic practices were carried out for all Nigeria (6.5oN, 5oE); a location with derived the plantings. Only the five internal plants savanna ecology in 2009 and 2010. The for each plot were used for data collection. 2009 planting suffered from severe drought and was unable to produce panicles and Data Collection hence not used in the analysis. Data were collected on each plant included both panicle and reproductive characters, Varieties as described by Anon (1988) following the Fifteen varieties of upland rice were standard evaluation system for rice (SESR). obtained from the African Rice Centre In particular, the data were collected on (formerly West African Rice Development panicle number, panicle length, primary Association, WARDA) the substation branches on panicles, secondary branching, of the International Institute of Tropical spikelets number per panicle, grain weight Agriculture (IITA) Ibadan and were used per panicle, grain weight per plant, 100-grain for the study. The varieties were: ITA 150 weight, grain length and width, and spikelets and OS 6 (which are frequently cultivated fertility. and established in the study region), ITA 257, ITA II7, ITA 315 and ITA 321 (which Data Analysis are improved release varieties), IGUAPE Results were analysed using the means CATETO, LAC 23, IDSA 10 (which are recorded on the characters for each variety. cultivated in other upland ecologies in the Computer analysis of variance (ANOVA) west African sub-region), WAB 35-2-FX was done using the SAS (2000) package. (hereinafter identified as WAB 35), WAB The analysis was based on the five-season

Pertanika J. Trop. Agric. Sci. 39 (1): 491 - 502 (2016) 493 Nassir, Adesola L. and Alawode, Yetunde O.

(year) environment data. Meanwhile, the significant genotype, environment and Additive Main Effect and Multiplicative genotype by environment interaction. Grain Interaction (AMMI) analysis was done to weight per panicle and grain weight per plant obtain Interaction Principal Components had low HB of 11.6 and 5.6, respectively. The

(IPC) using the GENSTAT package Version highest HB estimates of 62.4, 58.9 and 40.0 12. The AMMI PC 1 particularly presents were recorded by 100-grain weight (62.4), the non-crossover attribute of the data and grain length (58.9) and spikelet fertility (40). quantifies the response of genotypes to The significant mean squares for the the trial environments (Yan et al., 2000; genotype effect indicate genetic differences Gauch, 2006; Yan et al., 2007). Broad sense among the varieties for all the characters. heritability estimates HB for characters were Similarly, the significant environmental determined from the ANOVA results using effect implies that the study seasons the methods enumerated by Breese (1969). presented discriminatory conditions for a Stability variance for grain weight genotype by environment (G X E) study. per plant was calculated following the Following other reports on rice (Nassir & procedure established by Shukla (1972). Ariyo, 2005; Acuña et al., 2008; Shrestha In addition, the yield stability index (YSi) et al., 2012), significant differences in was calculated to determine the genotypes, environmental influence are to be expected which have a combination of high yield and when cultivating upland rice over seasons. stability using the procedure of Kang and The significant G X E interaction for all Pham (1991). the characters clearly implies differential genotypic response to different environments RESULTS AND DISCUSSION such that character expression and hence Analysis of Variance and Broad Sense genotypic performance cannot be expected Heritability to be stable over cultivation seasons. The mean squares (MS) and broad sense Hence, upland rice breeding effort must be conscious of this complex scope of heritability (HB) for the panicle characters of the rice genotypes over the five environments interaction of environment with panicle and are presented in Table 1. All the characters grain characters in plant improvement. showed significant genotype, environment Low heritability estimate for a number and genotype by environment interaction. of panicle and grain characters in upland Heritability estimates were quite low with rice has been similarly reported by Nassir the least value of 1.9 for panicle number and and Ariyo (2006). Characters that show low the highest estimate of 18.0 for secondary heritability would require many cycles of branching. hybridisation and selection in a fair range Table 2 shows the mean squares and of environments for meaningful progress. However, gains from selection for hundred broad sense heritability (HB) for grain characters. All the characters also recorded (100) grain weight and grain length is likely

494 Pertanika J. Trop. Agric. Sci. 39 (4) 491 - 502 (2016) Stability Analysis of Panicle and Grain Traits of Upland Rice to be faster, given the moderately high considered as unstable by the stability heritability estimate, and this may impact variance. Similarly, ITA 117 and ITA positively on larger grain weight per plant. 315 had high mean panicle number, and returned stable by the stability variance. GE and Yield-Stability analysis for The two also had positive interaction with panicle and grain characters the environment and were consequently The mean, stability variance (σ2) and AMMI selected by YSi. ITA 257 recorded above PC 1 and yield-stability statistic (YSi) for average panicle number but was the most panicle characters of each genotype over unstable according to σ2 and had the largest the five environments are presented in Table negative interaction with the environment 3. ITA 321 had the highest mean panicle with the largest AMMI PC 1 score of -0.964. number of 6.71 but was considered as The longest panicles were recorded by unstable by the stability variance. However, WAB 375 (27.22cm) and OS 6 (27.19cm). it recorded a near zero, but negative AMMI The two genotypes selected by the YSi PC 1 and was the only one selected by were adjudged as unstable by the stability the YSi statistic among the genotypes variance and had relatively large negative

Table 1

Mean squares and broad sense heritability (HB) for the panicle character of upland rice varieties over five environments

Source of Panicle Panicle length Primary Secondary Spikelets per variation number (cm) branches (No) branching (s) panicle (No) Genotype (G) 4.59* 51.49** 28.32** 1.21** 5948** Environment (E) 305.81** 580.75** 501.90** 9.24** 121737** GE 6.56** 21.62** 6.79** 0.55** 2980** Pooled Error 2.33 5.51 4.17 0.22 1064

HB (%) 1.9 13.5 11.5 18 6.9 * Significant at P< 0.05, ** Significant at P< 0.01

Table 2

Mean squares and broad sense heritability (HB) for the grain characters of upland rice varieties over five environments

Source of Grain Grain Hundred Grain Grain Spikelet Variation weight per weight per grain length width fertility (s) panicle (g) plant (g) weight (g) (mm) (mm) Genotype (G) 4.28** 146.44* 2.40** 1.09** 0.75** 5.94** Environment (E) 39.38** 2914.58** 3.78** 0.88** 4.81** 14.27** GE 2.60** 89.64** 0.36** 0.72** 0.16** 2.89** Pooled Error 1.01 49.38 0.09 0.11 0.61 0.55

HB 11.6 5.6 62.4 58.9 26.7 40.0 * Significant at P< 0.05, ** Significant at P< 0.01

Pertanika J. Trop. Agric. Sci. 39 (1): 491 - 502 (2016) 495 Nassir, Adesola L. and Alawode, Yetunde O.

AMMI PC 1. Indeed, most of the genotypes mean grain width of up to 3mm, large YSi were deemed unstable by σ 2 and also had and were consequently selected. large AMMI PC 1 scores. WAB 33-25 had WAB 99-1-1 had the highest mean the highest YSi value on account of having spikelets number of 175.03 per panicle. It above average panicle length, stable σ2 and had the largest negative AMMI PC 1 score relatively lower PC 1 score. and was also highly unstable by the stability WAB 99-1-1and WAB 375 had the variance. WAB 375 also produced a high highest mean primary and secondary mean spikelets number of 168.2 per panicle, branching scores and were returned unstable and was deemed as stable by σ2. It also had by the stability variance. WAB 375 however the least AMMI PC 1 score and the best YSi. had positive interaction with improving ITA 315, which had a non-significant σ2 and environment in contrast to WAB 99-1-1. OS WAB 35, which had positive interaction 6 had the highest YSi for primary branches. with improving environment, also had high The genotype recorded above average mean YSi values and were therefore selected. primary branching and was considered In term of spikelets fertility, most of the stable by both the σ2 and AMMI PC1. More genotypes were highly unstable by σ2 varieties were considered as stable by σ2 verdict and also had relatively large AMMI for the primary branching than secondary PC 1 scores. ITA 315 had the lowest mean branching. spikelets fertility scores, a non-significant Table 4 presents the mean, stability σ2, as well as a high and positive AMMI variance, AMMI PC 1and the YSi values for PC scores. It also had the best YSi and was grain and spikelets characters. The results consequently selected as a choice genotype indicated that only two genotypes (IDSA 10 for the character. and OS 6) were stable with respect to grain The stability variance, AMMI PC 1 and length. The two had positive interaction with the YSi values for grain yield characters are improving environment. However, only OS shown in Table 5. Nine of the genotypes 6 had above average mean grain length and had mean 100-grain weight above 3.0g. Of was consequently selected by YSi. ITA 150, these, however, only three (IDSA 10, ITA which recorded the mean longest grains 150 and OS 6) had non-significant σ2 and and had a significant σ 2, showed positive large YSi. WAB 33-25 recorded the largest interaction with the high environment with mean panicle grain weight of 4.76g and the an AMMI PC 1 score of 0.42 and was also least non-significant 2σ , which consequently selected by YSi. For grain width, three of the earned it the highest YSi. ITA 315 and WAB genotypes (ITA 321, OS 6, and WAB-96-1- 99 also had high YSi but with lower mean 1) were declared as stable by σ2 and also had panicle grain weight. WAB 35 and WAB small AMMI PC 1 scores. However, only 375 also had high mean panicle weight of the latter two, along with WAB 35-2-FX, 4.71g and 4.70g, respectively, and also with IGUAPE CATETO and WAB 33-25, had significant σ2. Nine of the fifteen genotypes

496 Pertanika J. Trop. Agric. Sci. 39 (4) 491 - 502 (2016) Stability Analysis of Panicle and Grain Traits of Upland Rice + + + + + YSi YSi (s) 0 3 6 10 15 0 1 -3 4 5 5 5 8 8 7 PC 1 -0.29 0.19 0.68 -0.25 -0.26 0.07 0.07 0.35 0.33 0.16 -0.31 0.37 -0.27 -0.96 0.11 2 σ 0.23** 0.04 0.29** 0.13 0.07 0.01 0.02 0.20* 0.12 0.14 0.17* 0.27** 0.21* 0.65** 0.22* Secondary branching (s) Mean 1.83 1.46 2.05 1.85 2.11 1.33 1.40 1.40 1.53 1.65 1.84 1.97 1.95 2.21 1.93 1.81 (0.64) + + + + + + YSi YSi (s) 1 0 3 0 9 12 5 14 13 10 6 3 2 7 9 PC 1 -0.08 -0.75 -0.82 -1.03 -1.28 0.38 0.86 0.05 0.66 -0.56 0.54 1.30 0.32 -0.17 0.56 2 σ 1.87 1.34 2.70 2.63 3.85* 0.66 1.92 0.91 1.28 1.20 0.58 5.38** 0.80 4.52** 4.34** Primary branches 11.51 (2.74) 11.51 Mean 9.69 9.33 10.33 10.75 11.51 12.09 11.24 12.73 12.25 11.53 11.48 11.69 10.17 13.49 14.37 + + + + + YSi YSi (s) -7 4 9 3 -2 -2 4 7 5 0 4 14 -3 -1 8 PC 1 -0.29 0.68 1.01 0.56 1.50 0.74 -0.57 -1.26 -0.85 0.20 -0.26 -0.15 -0.75 1.32 -1.88 2 σ 7.86** 4.03* 4.15* 1.68 8.84** 3.98* 8.48** 9.70** 4.63* 0.96 2.24 1.66 11.52** 18.11** 20.25** Panicle length (cm) Mean 21.18 23.37 24.27 21.83 23.09 21.73 24.13 27.19 23.33 20.98 22.55 24.27 22.95 23.29 27.22 23.43 (3.14) + + + + + YSi YSi (s) -1 0 -5 2 6 3 5 -4 15 14 8 0 12 -2 1 PC 1 -0.84 -1.02 -0.96 0.05 -0.06 1.29 -0.80 1.07 0.55 0.11 -0.09 0.22 -0.63 0.88 0.24 2 Panicle number 3.01** 2.85** 5.66** 0.17 0.62 3.93** 4.28** 2.92** 1.45 0.37 2.29** 0.20 1.29 3.72** 0.01 σ ) and AMMI PC 1 and yield-stability statistic (YSi) for the panicle characters of upland rice genotypes (YSi) for the panicle characters AMMI PC 1 and yield-stability statistic ) and 2 5.53 5.38 5.19 4.99 5.37 5.53 5.87 5.20 6.44 6.05 6.71 4.65 5.57 5.37 4.96 5.52 (2.05) Mean

, selected based on one-third selection from total genotypes. IDSA 150 1TA 257 ITA 35-2-FX WAB 56-60 WAB IGUAPE CATETO LAC 23 0S 6 117 ITA 315 ITA 321 ITA 33-25 WAB 96-1-1 WAB 99-1-1 WAB 375-B-5-H2-1 WAB Grand Mean(LSD) (P<0.05) Genotype Table 3 Table Means, stability variance (σ *,** significant at p<0.05 and 0.01, respectively; +

Pertanika J. Trop. Agric. Sci. 39 (1): 491 - 502 (2016) 497 Nassir, Adesola L. and Alawode, Yetunde O. + + + + + Ysi 8 1 -2 -5 10 12 -1 0 -1 13 3 5 -3 -4 1 PC 1 -0.43 0.30 -0.60 -0.63 0.22 0.02 0.04 -0.37 -0.90 0.47 1.35 0.65 0.24 -0.78 0.42 2 σ 0.37 0.87** 0.62** 0.90** 0.35 0.27 0.43* 1.21** 1.42** 0.44 2.83** 1.14** 0.51** 1.01** 2.06** Spikelets fertility (s) Mean 3.25 3.21 3.55 3.99 2.83 2.49 4.21 3.26 3.43 1.93 2.81 2.37 3.69 3.77 2.92 3.17(1.0) + + + + + Ysi 1 0 3 10 2 0 -6 -1 5 12 2 9 5 8 15 PC 1 1.68 -3.15 -5.87 3.30 -3.17 1.52 4.07 3.60 1.88 -0.77 2.52 2.17 -1.60 -6.02 -0.16 2 σ 254.40 404.19 3073.92** 801.53* 1331.65** 771.14* 1129.74** 944.45* 235.97 216.79 782.09* 499.30 943.46** 2829.36** 683.62 Spikelets per panicle Mean 113.25 107.6 150.23 153.94 150.20 125.47 119.58 125.14 128.91 151.48 136.89 146.89 153.03 175.03 168.20 140.43 (43.8) + + + + + Ysi 5 -4 0 13 -8 12 -3 9 -5 3 5 11 12 -9 6 PC 1 -0.83 -0.39 0.20 -0.08 0.10 -0.03 0.50 0.08 0.12 0.24 -0.09 0.17 -0.01 -0.03 0.06 2 σ 0.27** 0.08** 0.03** 0.01* 0.05** 0.04** 0.19** 0.00 0.05** 0.03** 0.00 0.01* 0.00 0.04** 0.01* Grain width (mm) Mean 2.85 2.71 2.76 3.15 2.40 3.09 2.50 2.79 2.62 2.82 2.71 3.00 2.84 2.40 2.80 2.76 (0.16) + + + + YSi 5 9 7+ 6 7 1 3 13 8 -8 -2 6 -7 -9 2 PC 1 0.10 0.43 -0.62 -0.25 -0.55 0.32 -0.24 0.23 0.01 0.01 0.23 0.64 -0.98 0.38 -0.26 2 σ 0.00 0.27** 0.10* 0.14* 0.63** 0.15** 0.15** 0.05 0.17** 0.17** 0.17** 0.33** 1.00** 0.16** 0.07* Grain length (mm) Mean 8.86 9.32 9.00 9.00 9.25 8.84 8.91 9.11 9.31 8.57 8.90 9.14 8.66 8.39 8.80 8.94 (0.12) ), AMMI PC 1 and yield-stability statistic (YSi) for the grain and spikelets characters of upland rice genotypes the grain and spikelets AMMI PC 1 and yield-stability statistic (YSi) for ), 2

Genotypes IDSA 150 1TA 257 ITA 35-2-FX WAB 56-60 WAB IGUAPE CATETO LAC 23 0S 6 117 ITA 315 ITA 321 ITA 33-25 WAB 96-1-1 WAB 99-1-1 WAB 375-B-5-H2-1 WAB Grand Mean(LSD) (P<0.05) , selected based on one-third selection from total genotypes. Table 4 Table Stability variance (σ *,** significant at p<0.05 and 0.01, respectively. +

498 Pertanika J. Trop. Agric. Sci. 39 (4) 491 - 502 (2016) Stability Analysis of Panicle and Grain Traits of Upland Rice recorded above mean grain weight per plant 100-grain weight were absorbed by the with the highest grain weight per plant of stable and above average performance of 24.58g by WAB 96-1-1, followed by ITA most of the other characters. Noteworthy, 315 with 20.36g. The two genotypes were however is the advantage posed by high however deemed unstable by σ2 although panicle number by ITA 321, the longest and the latter was eventually selected by YSi. most branched panicle by WAB 375 and the WAB 99-1-1, WAB 33 and ITA 117 had the longest grains and largest 100-grain weight next best grain weight of 19.24g, 18.59g and by ITA 150. These genotypes and others 17.26g respectively in that order per plant identified by superior character expression and were selected by YSi. can for a pool of genotypes for the evolution The significant stability variance of of synthetic genotypes for overall increase many of the genotypes for the panicle and grain yield. grain characters underscores the necessity Generally, the stability variance and to shift focus away from yield alone in the AMMI PC 1 appeared to be consistent genotype – environment and stability to a reasonable extent in the value returned analysis. It would appear that there is a as a measure of stability of genotypes for necessity to attain a converging compromise different characters. However, AMMI involving the panicle characters in the PC 1 is only a fraction of the of the GE development of high quality phenotypic component of the AMMI 1 values and expression, eventually in terms of yield would not be expected to capture the entire and yield stability. This partly explains interaction as much as the stability variance. why the YSi, which attempts to adopt a While σ 2 does not give the direction and compromise between higher and stable trait manner of instability, the non-crossover expression, selected different genotypes for attribute AMMI PC 1 (Yan & Hunt, 2001; many characters (Kang & Pham, 1991). Samonte, 2005) helps to complement the The instability of ITA 257 for grain weight decision on stability by specifying the per plant, for instance, appeared to be the manner of genotype reaction to improving cumulative effect of the instability of most (or declining) environment. The selection of the characters with the exception of of different genotypes by the YSi for the primary branches and grain width. Similarly, characters attests to the need to concentrate WAB 96-1-1, which was the most unstable traits for higher expression. For instance, in terms of grain weight per plant, was also WAB 33-25 can be improved for the panicle unstable for the characters except panicle number without sacrificing its stability number, primary branches and grain width. in respect of grain production. Similarly, Conversely, ITA 357, which was unstable ITA 315 would possibly have higher grain for grain weight per plant, was stable for weight per plant through a carefully planned most of the characters. It would seem that improvement in primary and secondary the instability of grain length, width and branching and spikelets number per panicle.

Pertanika J. Trop. Agric. Sci. 39 (1): 491 - 502 (2016) 499 Nassir, Adesola L. and Alawode, Yetunde O. + + + + + YSi YSi 4 0 -5 10 5 9 1 2 11 11 7 13 8 14 8 IPC 1 -0.26 0.22 0.17 -0.17 0.42 -0.12 -0.52 0.07 0.40 -0.05 -0.02 0.02 -0.11 0.16 -0.19 2 σ 10.29 10.03 65.24** -1.30 6.98 24.82 14.71 9.48 5.59 32.18* 9.27 8.08 200.45** 12.38 39.99* Grain weight per plant (g) Mean 16.74 11.31 15.86 17.40 16.96 17.32 12.78 14.28 17.56 20.36 17.24 18.59 24.58 19.24 18.28 17.2 (9.44) + + +

+ + 8 1 1 7 -4 2 -8 5 -6 13 -1 16 3 12 6 YSi YSi 0.29 -0.64 -0.71 -0.68 -0.91 0.12 0.72 0.08 0.65 -0.45 0.46 -0.12 0.82 -0.07 0.43 IPC 1 2 0.49 0.57 0.86** 1.24** 1.30** 1.75** 0.92** 0.26 1.25** 0.51 0.78* 0.04 1.27** 0.53 1.24** σ Panicle grain weight (g) 4.12 3.22 4.23 4.71 3.96 4.25 2.88 4.01 3.69 4.39 3.74 4.76 4.32 4.36 4.70 4.09 (1.35) Mean + + + + + 14 16 6 11 1 5 1 11 2 -2 3 4 -1 -8 -4 YSi YSi IPC 1 0.16 0.10 0.03 0.08 -0.10 -0.55 0.02 -0.23 -0.29 0.15 -0.19 0.80 -0.59 -0.10 0.49 2 σ 0.05 0.03 0.08* 0.08* 0.07* 0.27** 0.07 0.07 0.05 0.17** 0.06 0.43** 0.21** 0.10** 0.18** 100-grain weight (g) Mean 3.61 3.68 3.26 3.26 3.04 3.60 2.57 3.34 2.59 2.59 2.80 3.36 3.11 2.56 2.99 3.15 (1.31) ), AMMI PC 1and yield-stability statistic (YSi) for the grain yield characters of upland rice genotypes the grain yield characters AMMI PC 1and yield-stability statistic (YSi) for ), 2

Genotypes IDSA 150 1TA 257 ITA 35-2-FX WAB 56-60 WAB IGUAPE CATETO LAC 23 0S 6 117 ITA 315 ITA 321 ITA 33-25 WAB 96-1-1 WAB 99-1-1 WAB 375-B-5-H2-1 WAB Grand Mean(LSD) (P<0.05) , selected based on one-third selection from total genotypes. Table 5 Table Stability variance (σ *,** significant at p<0.05 and 0.01, respectively. +

500 Pertanika J. Trop. Agric. Sci. 39 (4) 491 - 502 (2016) Stability Analysis of Panicle and Grain Traits of Upland Rice

CONCLUSION Breese, E.L. (1969). The measurement and significant of genotype- environment interactions in rice. Stability analysis in rice would continue to Heredity, 24(1), 27-44. attract attention as different agro ecological zones present variable environmental Gauch, H. G. (2006). Statistical Analysis of Yield Trials by AMMI and GGE. Crop Science, 46(4), conditions. The need to accumulate 1488–1500. doi:10.2135/cropsci2005.07-0193 desirable genes into genotypes so as to have appreciable plastic response that would Kang, M.S., & Martin, F. A. (1987). A review of important aspect of genotype-environmental assure adequate character expression from interactions and practical suggestions for intra- and inter-ecological variations is sugarcane breeders. Journal of American Society quite germane. The use of simultaneous of Sugarcane Technology, 7, 36-38. selection for higher phenotypic expression Kang, M. S., & Pham, H. N. (1991). Simultaneous and stability resulted in the selection of selection for high yielding and stable crop genotypes with significant stability variance. genotypes. Agronomy Journal, 83(1), 161-165. From the practical point of view, the YSi Kempton, R. A. (1984). The use of biplots in statistics is useful in identifying genotypes interpreting variety by environment interaction. with high and stable expression for different Journal of Agricultural Science, 103(1), 123- characters. The complimentary role of 135.

AMMI PC 1 further shapes the decision on Lafitte, H. R., Yongsheng, G., Yan, S., & Li, Z-K. the genotypes that are compatible to season- (2007). Whole plant responses, key processes, or location-based improving or declining and adaptation to drought stress: the case of rice. environmental conditions, particularly in Journal of Experimental Botany, 58(2), 169-175. upland rice cultivation. McLaren, C. G. & Chaudhary, R. C. (1994). Use of additive main effects and multiplicative REFERENCES interaction models to analyse multilocation rice variety trials. 1994 FCSSP Conference, Puerton Acuña, B.T.L. & Wade, L. J. (2012). Genotype × Princesa, Palawan, . environment interactions for root depth of wheat. Field Crops Research, 137, 117-125. Nassir, A. L. & Ariyo, O. J. (2005). Genotype x Environment stability analysis of grain yield Acuña B.T. L., Lafitte, H. R., & Wade, L. J. (2008). of rice Oryza sativa (L.) Tropical Agriculture, Genotype x environment interactions for 82(4), 269-276. grain yield of upland rice backcross lines in diverse hydrological environments. Field Crops Nassir, A. L., & Ariyo, O. J. (2006). Plant character Research, 108(2), 117-125. correlations and path analysis of grain yield in rice (Oryza sativa). Journal of Genetics and Anonymous (1988). Standard evaluation system Breeding, 60(3), 161-172. for rice. International Rice Research Institute, Philippine. Nassir, A. L., & Ariyo, O. J. (2011). Genotype x Environment Interaction and Yield-Stability Ariyo, O. J. (1998). Use of additive main effects Analyses of Rice Grown in Tropical Inland and multiplicative interaction model to analyse Swamp. Notulae Botanicae Horti Agrobotanici multilocation soybean varietal trials. Journal of Cluj-Napoca, 39(1), 220-225 Genetics and Breeding, 53, 129-134.

Pertanika J. Trop. Agric. Sci. 39 (1): 491 - 502 (2016) 501 Nassir, Adesola L. and Alawode, Yetunde O.

Ouk, M., Basnayake, J. Tsubo, M., Fukai, S., & Shrestha, S., Asch, F., Dusserre, J., Ramanantsoanirina, Fischer, K. S. (2007). Genotype-by-environment A., & Brueck, H. (2012). Climate effects on yield interactions for grain yield associated with water components as affected by genotypic responses availability at flowering in rainfed lowland rice. to variable environmental conditions in upland Field Crops Research, 101(2), 145-154. rice systems at different altitudes. Field Crops Research, 134, 216–228. Pantuwan, G., S. Fukai, M. Cooper, S., Rajatasereekul & O’Toole, J. C. (2002). Yield response of rice Shukla, G. K. (1972). Some statistical aspect of (Oryza sativa) genotypes to drought under partitioning genotype environmental component rainfed lowlands: Selection of drought resistant of variability. Heredity, 29, 237 – 245. genotypes. Field Crops Research, 73(2), 169 Yan W., Hunt, L. A., Sheng, Q., & Szlavnics, – 180. Z. (2000). Cultivar Evaluation and Mega- Samonte, S.O.P.B., Wilson, L. T., McClung, A. M., Environment Investigation Based on the GGE & Medley, J. C. (2005). Targeting Cultivars Biplot. Crop Science, 40(3), 597–605. onto Rice Growing Environments Using AMMI Yan, W., Kang, M. S., Ma, B., Woods, S., & and SREG GGE Biplot Analyses. Crop Science, Cornelius, P. S. (2007). GGE Biplot vs. AMMI 45(6), 2414-2424. Analysis of Genotype-by-Environment Data. SAS Institute. (2000). SAS/STAT Software Release. Crop Science, 47(2), 643–655. doi: 10.2135/ Cary, NC, USA. SAS Institute Inc. cropsci2006.06.0374.

502 Pertanika J. Trop. Agric. Sci. 39 (4) 491 - 502 (2016) Pertanika J. Trop. Agric. Sci. 39 (4): 503 - 513 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

The Effect of Cultivation Techniques on the Growth Rate of Marine Microalgae Species from the Coastal Waters of Kudat, Sabah, Malaysia

Nurzafirah Mazlan* and Ridzwan Hashim Department of Biomedical Science, Kulliyyah of Allied Health Science, International Islamic University Malaysia, Jalan Sultan Ahmad Shah, 25200 Kuantan, Pahang, Malaysia

ABSTRACT This study evaluates the growth of five species of marine microalgae;Chaetoceros muelleri, Isochrysis galbana, Pavlova lutheri, Nitzschia acicularis and Navicula sp., isolated from coastal waters of Kudat in Sabah, Malaysia, using different cultivation techniques for mass outdoor culture. The microalgae were locally isolated and identified based on their morphology. The growth of the microalgae was compared between carboy and polythene bag culture. Results showed that cell count for all species was significantly higher in the polythene bag culture compared to carboy culture (p < 0.05). The time to harvest was also shorter (2-3 days) compared to carboy culture. In particular, Chaetoceros muelleri produced the highest cell count of 87 X 106 cells/ml and shortest time of 2 - 3 days to harvest. There were significant differences (p< 0.05) in cell count between indoor and outdoor mass culture, and these suggest the feasibility to culture the microalgae outdoor, reduce hatchery operation cost and save time.

Keywords: Carboy, microalgae, mass culture, polythene bag

INTRODUCTION industries. The usage of microalgae as food Microalgae are the primary producer of for rearing larvae and juveniles has been the food chain and have become the main extensively reported (Laing, 1991; Martinez- source of food and feed in many aquaculture Fernandez & Souchgate, 2007; Galley et al., 2009). The larval and juvenile stages signify

ARTICLE INFO the importance of nutrition to ensure proper Article history: growth, survival rate and hence, successful Received: 28 September 2015 Accepted: 27 May 2016 hatchery operations. High mortalities E-mail addresses: during larviculture of marine animals such [email protected] (Nurzafirah Mazlan), [email protected] (Ridzwan Hashim) as fish, shellfish and echinoderms remain a * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press Nurzafirah Mazlan and Ridzwan Hashim major bottleneck in aquaculture that limits effort to replace the live algae by artificial production (Dhert et al., 2001). Many factors feeds, marine scientists are still dependent contribute to the high mortality cases such on the production and use of microalgae as diseases, parasites and food availability. as live food for commercial aquaculture Good diet requirement has been proven as industries (Albentosa et al., 2002; Enes the key factor in overcoming these problems & Borges, 2003; Bonaldo et al., 2005; (Rainuzzo et al., 1997). Espinosa & Allam, 2006; Chomaco et al., Culturing of live algae is considered 2007). necessary as feed in the hatcheries. However, Most algae cultivation can be described the process can be costly especially for as either open or closed system. Closed the indoor culture (Cotteau & Sorgeloos, system generally consumes more energy 1992). Feeding mixed algal diet consisting and requires a large capital investment but of two or more algae culture has been delivers a relatively high biomass yield. shown to produce optimal growth and In contrast, open algae growth ponds have development of larvae and juveniles lower energy requirement but produce less compared to monoculture (Ivy & Giraspy, biomass for the same area. Open ponds 2006). However, many of the microalgae can also be affected by contamination have been proven difficult to culture in and evaporation losses (Lardon et al., sizeable quantities necessary for large scale 2009; Jorquera et al., 2010; Stephenson hatchery operations due to contamination, et al., 2010). Various physiological and genetic unstability, light limitation and technological approaches have been weather, which subsequently compromise proposed and investigated for maximising its suitability for commercial applications. productivity in mass algal culture system. It is important to identify the specific diet Numerous, more or less sophisticated requirements to determine which microalgae systems have also been developed for species to be cultured in the hatchery. culturing some 40 algal species to feed Commercial hatcheries have consistently larvae and zooplankton organisms. Many used reliable species such as Isochrysis studies have proven that a large scale galbana, Pavlova leutheri and Chaeteceros production of monospecific algae would be calcitrans. These species have been feasible (Chaumont, 1993). extensively studied for their biochemical Cultivation technique is crucial in composition, as well as nutritional value. determining the growth of algae culture. Microalgae can be used as live feed While popular indoor algae culture utilises or processed into artificial feed. So far, carboy as the culture recipient, polythene no completely satisfactory alternative has bag also shows promising results with been found as compared to the use of live better growth rates and shorter duration microalgae in aquaculture. In spite of all (Sipauba-Tavares et al., 2011). Thus, there

504 Pertanika J. Trop. Agric. Sci. 39 (4) 503 - 513 (2016) The Effect of Cultivation Techniques on the Growth Rate of Marine Microalgae Species is a significant difference in the cultivation 2008; Pereira et al., 2014). The species of technique during indoor algae cultivation Chaetoceros muelleri, Isochrysis galbana, between carboy and polythene bag culture. Pavlova lutheri, Nitszhia acicularis and The aim of the present study was to Navicula sp. were inoculated into fresh f/2 isolate the microalgae from the coastal medium and used as inoculum for further waters of Kudat in Sabah, Malaysia, and studies. All the cultures were maintained subsequently determine the effects of at 25 ± 1 °C on a 12 h/12 h light/dark cycle cultivation techniques on the growth of with 1400 lux of light intensity. marine microalgae. Monoalgae Culture METHODS The seawater used for culturing diatoms was Isolation and Identification of filtered through sand filter and subsequently Microalgae through 1 micron sediment filter cartridge Water and rock samples were collected using and then treated with sodium hypochlorite transparent bottle from the coastal waters for 12 h to kill microorganisms. The sodium of Kudat, Sabah, Malaysia (6°49’12.1”N, hypochlorite in the seawater was neutralised 116°51’37.5”E) on May 13, 2012 and July with sodium thiosulphate before use. Non- 15, 2012, respectively. The samples were axenic monocultures of the microalgae sieved using 50 µm mesh net to eliminate species were cultivated in f/2 medium coarse particles and species such as sand (Guillard, 1975) with an addition of meta- and zooplankton. silicate for diatom species in progressive The mixed algal samples were cultured volumes of 10 ml, 150 ml, 1 L. The size of in a 500 ml shake flasks filled with 400 ml inoculum was adjusted to 1.0 X 104 cells/ml of a standard f/2 medium (Guillard, 1975), for all the microalgae species. with an addition of meta-silicate for diatoms species. After 10 days, the microalgae Algal Culture in Polycarbonate Carboy were identified using stereomicroscope and Polythene Bag and isolated from other algal species Monospecific diatom culture of 2 L was using capillary tube method (Blanco et al., inoculated in either 20 L polycarbonate 2008; Debenest et al., 2009). The isolated carboy or 20 L polythene bag filled with f/2 microalgae were identified morphologically medium (Guillard, 1975), with an addition based on size, form and colour. Identification of meta-silicate for diatom species. results were verified by comparing with three The culture from carboy of 20 L was books, namely, ‘The Diatoms: Biology and inoculated in transparent fiberglass tank Morphology of the Genera’, ‘Marine Algae: filled with 100 L f/2 medium and it was Biodiversity, Taxonomy, Environmental subsequently transferred into an outdoor Assessment, and Biotechnology’ and tank filled with 1000 L f/2 medium. ‘Algae’ (Round et al., 1990; Graham et al.,

Pertanika J. Trop. Agric. Sci. 39 (1): 503 - 513 (2016) 505 Nurzafirah Mazlan and Ridzwan Hashim

The 20 L culture in the polythene bag, with long, thin spines. Navicula sp. was however, was combined with another 5 sets characterised by valves with bilateral of similar polythene bag culture to make up symmetry and a well-developed raphe 100 L culture and transferred directly into an system. Nitzschia acicularis had extremely outdoor tank filled with 1000 L medium. The long and narrow valves, which were lightly differences of algal growth in both methods silicified.Pavlova lutheri was characterised were recorded. based on the spherical shape, with two Continuous aeration was provided. flagellae and golden brown colour of the cell. Cell concentration was counted using a Their sizes were about 3-5 µm. Isochrysis haemocytometer and expressed in cells/ml. galbana had the same morphology as Pavlova lutheri but the size was bigger than Statistical Analysis Pavlova lutheri, i.e. about 5-6 um length and Data were analysed using one-way analysis 2-4 µm wide. of variance (ANOVA), SPSS version 17 (SPSS Inc., Chicago, IL, USA). Significant Mass Culture of Microalgae differences were determined at 0.05 level Isolated microalgae were cultured in of probability. progressive volume of 10 ml, 150 ml and 1 L culture. All the cultures were kept indoor RESULTS at the temperature of 25°C. After 5 to 7 Isolation and Identification of days of incubation period in 1 L shake flask, Microalgae the samples were collected and counted There were 5 species of microalgae isolated using a haemocytometer. Table 1 shows from the mixed culture sample collected the cell count for the 5 species of isolated from the coastal area of the sea cucumber microalgae. The cell for Isochrysis galbana 6 hatchery. The species were identified based showed the highest count of 48 X 10 cells/ on their morphological characteristics. ml, while Navicula sp. showed the lowest 6 Diatoms have a characteristic that they count with an average of 10 X 10 cells/ are enclosed within a cell wall made of ml compared to the other four species of silica. Chaetoceros muelleri had frustules microalgae. Meanwhile, Pavlova lutheri

Table 1 Cell count for 5 species of microalgae measured after 5 to 7 days of incubation period in 1 L culture.

Species Temperature Cell Count Incubation period Volume (°C) (cells/ml) (day) (L) Isochrysis galbana 25.0 ± 1.0 48 X 106 ± 43 X 105 5 - 7 1 Pavlova lutheri 25.0 ± 1.0 34 X 106 ± 33 X 105 5 - 7 1 Chaetoceros muelleri 25.0 ± 1.0 33 X 106 ± 54 X 105 5 - 7 1 Navicula sp. 25.0 ± 1.0 10 X 106 ± 12 X 105 5 - 7 1 Nitzschia acicularis 25.0 ± 1.0 16 X 106 ± 12 X 105 5 - 7 1

506 Pertanika J. Trop. Agric. Sci. 39 (4) 503 - 513 (2016) The Effect of Cultivation Techniques on the Growth Rate of Marine Microalgae Species and Chaetoceros muelleri had an average of 55 X 106 cells/ml, while Nitzschia count of 34 X 106 and 33 X 106 cells/ml, acicularis recorded the lowest cell count, respectively. Low cell count was also with the average of 14 X 106 cells/ml. recorded on Nitzschia acicularis with 16 X Isochrysis galbana was significantly higher 106 cell/ml. than other species (p < 0.05) in the carboy Two litres of monoalgal cultures from culture. However, in the polythene bag the shake flask were used as the starter to culture, Chaetoceros muelleri had the inoculate 20 L carboy or polythene bag highest cell count of average 87 X 106 cells/ culture. The cultures were left to grow for ml [F(4,10) = 99.6, p = 0.000)]) compared 3 to 5 days indoor with temperature ranging to other culture. Meanwhile, Nitzschia from 24 – 26°C before they were used as acicularis showed the lowest cell count in starter culture for the mass outdoor culture. the polythene bag culture as well, with an Table 2 and Table 3 show the cell count of average of 25 x 106 cells/ml. There were the 5 species of microalgae in carboy or also increases in the cell count of Nitzschia polythene bag culture respectively during acicularis and Navicula sp., although the day of harvest. not as much as Chaetoceros muelleri and Isochrysis galbana recorded the highest Isochrysis galbana. The highest cell count cell count in carboy culture with the average for Nitzschia acicularis was in the polythene

Table 2 Cell count for 5 species of microalgae measured after 3 to 5 days of incubation period in 20 L carboy culture.

Species Temperature Cell Count Incubation period Volume (°C) (cells/ml) (day) (L) Isochrysis galbana 25.0 ± 1.0 55 X 106 ± 12 X 105 3 - 4 20 Pavlova lutheri 25.0 ± 1.0 37 X 106 ± 22 X 105 3 - 4 20 Chaetoceros muelleri 25.0 ± 1.0 42 X 106 ± 21 X 105 3 - 4 20 Navicula sp. 25.0 ± 1.0 21 X 106 ± 37 X 105 3 - 5 20 Nitzschia acicularis 25.0 ± 1.0 14 X 106 ± 16 X 105 3 - 5 20

Table 3 Cell count for 5 species of microalgae measured after 2 to 3 days of incubation period in 20 L polythene bag culture.

Species Temperature Cell Count Incubation period Volume (°C) (cells/ml) (day) (L) Isochrysis galbana 25.0 ± 1.0 80 X 106 ± 16 X 105 2 - 3* 20 Pavlova lutheri 25.0 ± 1.0 47 X 106 ± 29 X 105 2 - 3* 20 Chaetoceros muelleri 25.0 ± 1.0 87 X 106 ± 24 X 105 2 - 3* 20 Navicula sp. 25.0 ± 1.0 33 X 106 ± 24 X 105 2 - 3* 20 Nitzschia acicularis 25.0 ± 1.0 25 X 106 ± 21 X 105 2 - 3* 20 *All cultures show a significant difference at p <0.05 when compared to carboy culture

Pertanika J. Trop. Agric. Sci. 39 (1): 503 - 513 (2016) 507 Nurzafirah Mazlan and Ridzwan Hashim culture with average cell of 25 X 106 cells/ to indoor culture, this may be due to changes ml while Navicula sp. recorded an average in environment such as temperature and of 33 x 106 cells/ml. Meanwhile, Pavlova light. Table 4 and Table 5 present the cell lutheri recorded an average of 37 X 106 count for the 5 species of microalgae using cells/ml in the carboy culture and 47 X 106 inoculum from carboy and polythene bag, cells/ml in polythene bag culture. All the respectively. cultures showed a significant (p < 0.05) Nitzschia acicularis had the lowest increase in their cell count when cultured cell count, while Chaetoceros muelleri in polythene bag. grew well in outdoor mass culture. The The time taken to harvest the cultures Chaetoceros muelleri sample from outdoor was also faster in polythene bag compared mass culture tank using carboy culture as to the carboy. It took about 3 to 4 days inoculum recorded an average cell count of to harvest Isochrysis galbana, Pavlova 49 X 106 cells/ml, which was significantly lutheri and Chaetoceros muelleri in carboy higher [F(4,10) = 117.7, p = 0.000)] than culture, whereby Navicula sp. and Nitzschia the other microalgae culture and indoor acicularis required longer period of 3 to 5 culture as well. Following that, Isochrysis days to harvest. Meanwhile in the polythene galbana showed a high cell count of an bag culture, it took an average of 2 to 3 average of 47 X 106 cells/ml. Navicula sp. days for all the microalgal species to be and Nitzschia acicularis recorded a low harvested. cell count with an average of 20 X 106 and One hundred litres of culture from 12 X 106 cells/ml, respectively. However, carboy or polythene bag was used as the amount was considered as feasible for inoculum for outdoor mass culture with an outdoor mass culture. There were no volume of 1 m3 and temperature ranging significant differences between the outdoor from 30 to 32°C. Cell count was taken on and indoor cultures, except for Isochysis the day of harvest. All microalgae culture galbana (p <0.05). showed a slightly lower cell count compared

Table 4 Mean temperature (°C) and cell count (cells/ml) for 5 species of microalgae measured at the harvest time of the outdoor algae culture using microalgae cultured in carboy as inoculum.

Species Temperature Cell Count Time taken Volume °(C) (cells/ml) to harvest (h) (m3) Isochrysis galbana 31.5 ± 1.5 47 X 106 ± 21 X 105 60 - 72 1 Pavlova lutheri 31.3 ± 1.3 34 X 106 ± 17 X 105 60 - 72 1 Chaetoceros muelleri 31.2 ± 0.6 49 X 106 ± 29 X 105 60 -72 1 Navicula sp. 31.7 ± 0.5 20 X 106 ± 24 X 105 60 - 72 1 Nitzschia acicularis 31.7 ± 0.5 12 X 106 ± 12 X 105 60 - 72 1

508 Pertanika J. Trop. Agric. Sci. 39 (4) 503 - 513 (2016) The Effect of Cultivation Techniques on the Growth Rate of Marine Microalgae Species

Table 5 Mean temperature (°C) and cell count (cells/ml) for 5 species of microalgae measured at the harvest time of the outdoor algae culture using microalgae cultured in polythene bag as inoculum.

Species Temperature Cell Count Time taken Volume (m3) (°C) (cells/ml) to harvest (h) Isochrysis galbana 31.3 ± 1.3 73 X 106 ± 27 X 105* 60 - 72 1 Pavlova lutheri 31.7 ± 0.5 40 X 106 ± 12 X 105 60 - 72 1 Chaetoceros muelleri 31.3 ± 1.3 89 X 106 ± 42 X 105* 60 - 72 1 Navicula sp. 31.7 ± 0.5 26 X 106 ± 22 X 105 60 - 72 1 Nitzschia acicularis 31.5 ± 1.5 15 X 106 ± 12 X 105 60 - 72 1 *Cultures show significant difference (p<0.05) compared to culture using carboy as inoculum.

From the tank of outdoor mass culture DISCUSSION using polythene bag culture as inoculum, the The advantageous effects of dietary algae cell count for Chaetoceros muelleri was 89 X are their contributions to minerals, dietary 6 10 cells/ml, which was significantly higher fibre, carotenoids, chemical feeding [F(1,4) = 124.1, p = 0.000)] than the sample attractants, vitamins and synergistic effects from the tank of outdoor mass culture with with vitamins and antioxidants. Microalgae carboy as inoculum and its indoor culture. are widely used in aquaculture due to their All the cultures were generally higher in cell easy culture and fast growth. They have count compared to the cultures grown in also been used as sources of essential fatty the outdoor mass tank using carboy culture acids. There were 5 species of commonly as inoculum. Nitzschia acicularis had the used microalgae in the aquaculture 6 lowest cell count of an average of 15 X 10 industries isolated and identified from a cells/ml. There were significant differences sea cucumber hatchery in Kudat, Sabah, (p < 0.05) in the cell growth for Isochrysis namely, Isochrysis galbana, Pavlova lutheri, galbana [F(1,4) = 108.6, p = 0.000)] and Chaetoceros muelleri, Navicula sp. and Chaetoceros muelleri [F(1,4) = 124.1, p Nitzschia acicularis. = 0.000)] between the indoor and outdoor Isochrysis galbana is generally cultures, while Pavlova leutheri, Navicula considered the best food for bivalves larvae. sp. and Nitzschia acicularis showed no Isochrysis galbana is small, naked, free- significant difference between the indoor living motile flagellates, which are easily and outdoor cultures (p >0.05). ingestible by small (larval) invertebrate The time taken to harvest the outdoor (Martines-Fernandez et al., 2004). Pavlova mass cultures was about 60 – 70 hours after lutheri has the same morphological features inoculation, depending on the cell count and with Isochrysis galbana, but they differ in demand of the species cultured such as the terms of size, whereby Pavlova lutheri cells sea cucumber larvae and juvenile. are bigger than those of Isochrysis galbana.

Pertanika J. Trop. Agric. Sci. 39 (1): 503 - 513 (2016) 509 Nurzafirah Mazlan and Ridzwan Hashim

Based on the results the monoalgal slightly lower yields were observed in the cultures were successfully cultured indoor outdoor culture compared to indoor culture. and outdoor. The algal cultures were species All the microalgae cultures in the specific. Each species carries specific polythene bag have a higher cell count characteristics and requirement for their compared to the carboy culture. Lin et growth (Cobelas et al., 1998). There were al. (2007) reported that light is the main significant differences between the indoor factor that interferes with the growth of and outdoor cultures for Pavlova lutheri, microalgae. Polythene bag has better Navicula sp. and Nitzschia acicularis light penetration compared to carboy. from the polythene bag cultures, as well as Furthermore, the surface area is wider in Isochysis galbana from carboy cultured. polythene bag compared to carboy which However, there were no differences in the has round surface. All the five species of rest of the species cultures. These results microalgae showed the same growth trend showed that the mass outdoor cultures for when the carboy and polythene bag were these species were feasible. compared. The usage of polythene bag as There were slight decreases in the cell culture recipient has proven to be efficient number of a few species of microalgae in with good growth rates and shorter duration this study when they were being transferred (Sipauba-Tavares et al., 2011). Furthermore, outdoor. Numerous studies have shown it incurs low cost and requires smaller space that the biochemical composition and in the laboratory. physiological status of microalgae cells are altered by different environmental CONCLUSION factors (Alonso et al., 2000; Sanchez et This study showed that the mass outdoor al., 2000; Renaud et al., 2002; Berges et cultures of five species of microalgae al., 2002; Tzovenis et al., 2003). Optimum isolated from Kudat in Sabah, Malaysia temperatures for diatoms range from 19°C are feasible. Furthermore, there were to 27°C; thus, the high temperature outdoor significant differences between the indoor may have killed the cells or delay the growth. and outdoor cultures (p < 0.05). All the However, there were also reports indicating microalgae cultures in polythene bag that most algae can tolerate high temperature showed significant differences (p < 0.05) up to 36°C. This further explains why a compared to those of the carboy culture, few species of microalgae grew so well in suggesting that culturing using polythene outdoor culture. Furthermore, the presence bag is more efficient, resulting in a shorter of contaminants is unavoidable in outdoor harvest period. Future studies need to culture. Culturing microalgae in outdoor optimise the growth parameter of the mass environment can achieve higher yields, outdoor culture further by studying other leading to a reduction of operating cost factors such as light, temperature, genetic compared to the cultures cultivated indoors stability and weather. (Elias et al., 2005), however in this study,

510 Pertanika J. Trop. Agric. Sci. 39 (4) 503 - 513 (2016) The Effect of Cultivation Techniques on the Growth Rate of Marine Microalgae Species

REFERENCES Debenest, T., Silvestre, J., Coste, M., Delmas, F., & Pinelli, E. (2009). A new cell primo- Albentosa, M., Perez-Camacho, A., Fernandez-Reriz, culture method for freshwater benthic diatoms M. J., & Labarta, U. (2002). Wheatgerm flour in communities. Journal of Applied Phycology, diets for Manila clam, Tapes philipinarum spat. 21(1), 65 – 73. Aquaculture, 212(1-4), 335 – 345. Dhert, P., Rombaut, G., Suantika, G., & Sorgeloos, Alonso, D. L., Belarbi, E. H., Fernandez Sevilla, J. M., P. (2001). Advancement of rotifer culture and Rodriguez Ruiz, J., & Grima, E.M. (2000). Acyl manipulation techniques in Europe. Aquaculture, lipid composition variation related to culture age 200(1), 129-146. and nitrogen concentration in continuous culture of the microalgea Phaeodactylum tricornutum. Enes, P. & Borges, M. T. (2003). Evaluation of Phytochemistry, 54(5), 461–471. microalgae and industrial cheese whey as diets for Tapes decussatus (L.) seed: effect on water Alvarez-Cobelas, M., Reynolds, C. S., Sanchez- quality, growth, survival and filtration rate. Castillo, P., & Kristiansen, J. (1998). Aquaculture Resource, 34(4), 299 – 309. Phytoplankton and trophic Gradients. Belgium: Kluwer Academic Publishers. Espinosa, E. P. & Allam, B. (2006). Comparative growth and survival of juvenile hard clams, Berges, J. A., Varela, D. E., & Harrison, P. J. (2002). Mercenaria mercenaria, fed commercially Effects of temperature on growth rate, cell available diets. Zoological Biology, 25(6), composition and nitrogen metabolism in the 513–525. marine diatom Thalassiosira pseudonana (Bacillariophyceae). Marine Ecology Progress Galley, T. H., Batistia, F. M., Braithwaite, R., King, Series, 225, 139–146. J., & Beaumont, A. R. (2009). Optimization of larval culture of the mussel Mytilus edulis (L). Blanco, S., Alvarez, I., & Cejudo, C. (2008). A Aquaculture international, 18(3), 315 – 325. test on different aspect of diatoms processing techniques. Journal of Applied Phycology, 20(4), Graham, J. E., Wilcox, L. W., & Graham, L. E. 445 – 450. (2008). Algae (Second Edition). USA: Benjamin Cummings. Bonaldo, A., Badiani, A., Testi, S., Corso, G., Mordenti, A. L., & Gatta, P. P. (2005). Use Guillard, R. R. L. (1975). Culture of phytoplankton of centrifuged and preserved microalgae for feeding marine invertebrates. In W. L. Smith from feeding juvenile Manila clam (Tapes & H. H. Chanley (Eds.), Culture of Marine philipinarum): effects on growth and fatty acid Invertebrates Animals (p). New York: Plenum composition. Italian Journal of Animal Science, Press. 4(4), 375 – 384. Ivy, G., & Giraspy, D. A. B. (2006). Development of Chaumont, D. (1993). Biotechnology of algal biomass large scale of hatchery production techniques production: a review for systems of outdoor for the commercially important seacucumber mass culture. Journal of Applied Phycology, 5, Holothuria scabra var.versicolor (Conand, 1986) 593 – 604. in Queensland, Australia. SPC Beche-de-mer Information Bulletin, 24, 28 – 34. Cotteau, P., & Sorgeloos, P. (1992). The use of algal substitutes and the requirement for the live Jorquera, O., Kiperstok, A., Sales, E. A., Embirucu, algae in the nursery and the hatchery rearing of M., & Ghirardi, M. L. (2010). Comparative the bivalve mollusks: an international survey. energy life cycle analysis of microalgal biomass Journal of Shellfish Research, 11(2), 467 – 476.

Pertanika J. Trop. Agric. Sci. 39 (1): 503 - 513 (2016) 511 Nurzafirah Mazlan and Ridzwan Hashim

production in open ponds and photobioreactors. Pereira, L., Patricio, J. and Neto, J. M. (2014). Marine Bioresource Technology, 101(4), 1406 – 1413. Algae: Biodiversity, Taxonomy, Environmental Assessment, and Biotechnology. USA: CRC Kaplan, D., Cohen, Z., & Abeliovich, A. (1986). Press Inc. Optimal growth condition for Isochrysis galbana. Biomass, 9(1), 37 – 48. Perez Camacho, J. M., Salinas, C., Delgado, M., & Fuertes, C. (2007). Use of single cell detritus Kociolek, P. (2011). Nitzschia acicularis. In Diatoms (SCD) produced from Laminaria saccharina in of the United States. Retrieved July 23, 2014, the feeding of the clam Ruditapes decussatus from http://westerndiatoms.colorado.edu/taxa/ (Linnaeus, 1758). Aquaculture, 266(1), 211 – species/nitzschia_acicularis 218. Laing, I. (1991). Cultivation of Marine: Unicellular Rainuzzo, J. R., Reitan, K. I., & Olsen, Y. (1997). The algae. Laboratory leaflet. Ministry of Agriculture, significance of lipids at early stages of marine Fisheries and Food Directorate of Fisheries fish: a review.Aquaculture, 155(1), 103-115. Research, Lowestoft. Renaud, S. M., Thinh, L. V., Lambrinidis, G., & Parry, Lardon, L., Helias, A., Sialve, B., Steyer J. P., & D. L. (2002). Effect of temperature on growth, Bernard, O. (2009). Life cycle assessment chemical composition and fatty acid composition of biodiesel production from microalgae. of tropical Australian microalgae grown in batch Environmental Science and Technology, 43(17), cultures. Aquaculture, 211(1–4), 195–214. 6475 – 6481. Round, F. E., Crawford, R. M., & Mann, D. G. (1990). Lin, Y. H., Cheng, F. L., Tsao, C. Y., & Lev, J. Y. The Diatoms: Biology and Morphology of the (2007). Influence of growth phase and nutrient Genera. Cambridge: Cambridge University source on fatty and composition of Isochrysis Press. galbana CCMP 1324 in batch photoreactor. Biochemical Engineering Journal, 37(2), 166 Sanchez, S., Martinez, M.E., & Espinola, F. (2000). – 176. Biomass production and biochemical variability of the marine microalga Isochrysis galbana Lopez Elias, J. A., Voltolina, D., Enriquez Ocana, in relation to culture medium. Biochemical F., & Gallegos Simental, G. (2005). Indoor and Engineering Journal, 6(1), 13–18. outdoor of the mass production of the diatom Chaetoceros muelleri in Mexican commercial Sipauba-Tavares, L. H., Millan, R. N., Almeida hatchery. Aquaculture Engineering, 33(3), 181 Berchielli, F., & Souza Braga, F. M. (2011). – 191. Use of alternative media and different types of recipients in a laboratory culture of Martinez-Fernandes, E., Acosta Salmon, H., & Rangel Ankistrodesmus gracilis (Reinsch) Korshikov Davalos, C. (2004). Ingestion and digestion of (Chlorophyceae). Acta Scientiarium. Biological 10 species of microalgae by winged pearl oyster Sciences, 33(3), 247 – 253. Pteria sterna (Gould, 1851) larvae. Aquaculture 230(1), 417 -423. Spaulding, S., & Edlund, M. (2008). Chaetoceros. In Diatoms of the United States. Retrieved July 23, Martinez-Fernandez, E., & Southgate, P. C. (2007). 2014, from http://westerndiatoms.colorado.edu/ Use of tropical microalgae as food for larvae of taxa/genus/Chaetoceros the black-lip pearl oyster Pinctada margaritifera. Aquaculture, 263(1), 220 – 226.

512 Pertanika J. Trop. Agric. Sci. 39 (4) 503 - 513 (2016) The Effect of Cultivation Techniques on the Growth Rate of Marine Microalgae Species

Stephenson, A. L., Kazamia, E., Dennis, J. S., Howe, Tzovenis, I., De Pauw, N., & Sorgeloos, P. (2003). C. J., Scott, S. A., & Smith, A. G. (2010). Life Optimisation of T-Iso biomass production rich cycle assessment of potential algal biodiesel in essential fatty acids: I. Effect of different light production in the United Kingdom: a comparison regimes on growth and biomass production. of raceways and air lift tubular bioreactors. Aquaculture, 216(1–4), 203–222. Energy Fuel, 24(7), 4062 – 4077.

Pertanika J. Trop. Agric. Sci. 39 (1): 503 - 513 (2016) 513

Pertanika J. Trop. Agric. Sci. 39 (4): 515 - 539 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Production and Characterisation of Cellulase from Solid State Fermentation of Rice Straw by Trichoderma harzianum SNRS3

Rahnama, N.1,2#, Shah, U. K. M.2,3,5*, Foo, H. L.2,4, Rahman, N. A. A.2,5 and A. B. Ariff 2,5 1Department of Biological Sciences, Urmia Branch, Islamic Azad University, Urmia, Iran 2Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 3Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 4Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 5Bioprocessing and Biomanufacturing Research Centre, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

ABSTRACT Research on production and the use of cellulase and xylanase by commercial microbial strains is widely reported. However, research on production of cellulase and xylanase by local isolates of Trichoderma harzianum known as potential cellulase producers is still very limited. T. harzianum SNRS3 was used for cellulase and xylanase production from rice straw under solid state fermentation. Our study revealed that unlike Trichoderma sp. that is normally associated with low amounts of β-glucosidase, insufficient to perform an efficient hydrolysis,T. harzianum SNRS3 could be considered as a potential β-glucosidase producer, but not an efficient xylanase producer. As a result of storage of the crude cellulase at room temperature, β-glucosidase activity only decreased to above 80% of its original activity at the end of the 3rd week of storage. The crude cellulase produced by T. harzianum SNRS3 could be industrially applied as the enzyme is still highly active at 60°C and over a wide range of acidic pH.

ARTICLE INFO Keywords: Trichoderma, Aspergillus, rice straw, Article history: Received: 16 December 2015 cellulase, solid state fermentation, characterisation Accepted: 21 July 2016

E-mail addresses: [email protected] (Rahnama, N.) INTRODUCTION [email protected] (Shah, U. K. M.) [email protected] (Foo, H. L.) Plant biomass is composed primarily [email protected] (Rahman, N. A. A.) [email protected] (A. B. Ariff) of cellulose, hemicelluloses, and lignin * Corresponding author # Author’s Current Affiliation (Kuhad et al., 1997; Carpita et al., Department of Biological Sciences, Urmia Branch, Islamic Azad 2001). Lignocellulosic waste materials University, Urmia, Iran

ISSN: 1511-3701 © Universiti Putra Malaysia Press Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff obtained from energy crops, wood and industry. Cellulases are employed in cotton agricultural residues represent the largest processing, paper recycling, as detergent global renewable reservoir of potentially enzymes, in juice extraction, and as animal fermentable carbohydrates (Mtui & feed additives apart from their immense use Nakamura, 2005; Talebnia et al., 2010). in the hydrolysis of lignocellulosic biomass Therefore, Lignocellulosic wastes are as a prerequisite for biofuel production regarded as attractive substrates for the (Wilson, 2009; Singhania et al., 2010). The production and recovery of a large number use of lignocellulosic biomass-derived sugar of value-added products such as enzymes for biofuel production has been shown to (Mtui & Nakamura., 2005). be promising (Park et al., 2012). In fact, Having occupied 2% of the world’s cellulase will become the largest volume cultivated area; rice ranks the second industrial enzyme, in case fermentation- most major crop worldwide. Rice is an derived fuel such as bioethanol, and extensive crop of Asia and Southeast biobutanol could replace the current fossil- Asia, which dominates tropical and sub- based transportation fuel (Wilson, 2009). tropical belts (Devendra & Thomas, Solid state fermentation (SSF) is known 2002; Leff et al., 2004). Rice straw is an as the fermentation whereby an insoluble abundant lignocellulosic waste in the world substrate is fermented in the absence or with several characteristics that make almost absence of free moisture (Chahal, it an appropriate feed stock for biofuel 1985). Submerged fermentation (SmF) production (Binod et al., 2010). Among the and SSF are both the common techniques lignocellulosic crop residues, rice straw is applied for production of cellulases. SSF the largest biomass feedstock in the world is advantageous over SmF due to the (Talebnia et al., 2010). production of the enzymes at a higher yield A variety of microorganisms such and higher pH or temperature stability as bacteria and fungi are plant biomass (Holker & Lenz, 2005; Singhania et al., decomposers in nature. This makes them 2009; Saqib et al., 2010). interesting sources for enzyme discovery Cellulases are the most extensively (Allgaier et al., 2010). Lignocellulosic studied multiple enzyme complexes. As enzymes are important commercial products hydrolytic enzymes, cellulases cleave the of lignocellulosic wastes bioprocessing used β-1,4- glucosidic bonds in the cellulose in many industrial applications including structure (Singhania et al., 2010). The chemicals, fuel, food, brewery and wine, major cellulase components include animal feed, textile and laundry, pulp and cellobiohydrolases (exo-1,4-β-D-glucanase, paper and agriculture (Howard et al., 2003; CBH), endoglucanases (endo-1,4-β-D- Rahnama et al., 2014). Currently, cellulases glucanase, EG) and β-glucosidase (Hong rank as the third largest volume of industrial et al., 2001; Li et al., 2006; Sun et al., enzyme with a wide range of applications in 2008; Singhania et al., 2010). Nonetheless,

516 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

β-glucosidase does not act upon cellulose conditions are mild, and substrate loss chain directly. Instead, β-glucosidase is and generation of side products are almost responsible for the cleavage of cellobiose negligible (Kulkarni et al., 1999). As the into two glucose monomers and this xylan molecule contains several substituted characteristic of β-glucosidase is of groups and side chains synergistic action great significance since accumulation of of multiple hydrolytic enzymes might be cellobiose causes a product inhibition on essential for the molecule to be completely both cellobiohydrolase and endoglucanase. hydrolysed (Dhiman et al., 2008). Xylanases Therefore, in addition to glucose production, have many biotechnological applications β- glucosidase reduces product inhibition by and are therefore categorised among the cellobiose, which in turn causes cellulolytic most important enzymes in industry. enzymes to function more efficiently Xylanases have numerous applications in (Workman & Day, 1982; Lymar et al., 1995; biotechnology, and are widely used in food, Joo et al., 2009). Hence, β-glucosidase is animal feed, paper, and pulp industries, as the rate-limiting factor in the enzymatic well as in bioconversion of hemicelluloses hydrolysis of cellulose (Lymar et al., 1995; to value-added products (Dhiman et al., Fadel, 2000; Elyas et al., 2010). Cellulases 2008; Chapla et al., 2010). with low levels of β-glucosidase hydrolyse Since understanding the action of cellulose slowly, and in such cases, the cellulolytic enzymes under different addition of β-glucosidase enhances cellulose conditions is of great importance, hydrolysis (Johnson et al., 1982; Lymar et many researchers have focused on the al., 1995; Fadel, 2000; Elyas et al., 2010). characterisation of cellulase in terms Unfortunately, the majority of cellulolytic of various physio-chemical parameters fungi, including hypercellulase-producing including temperature and pH (Farinas et mutants of Trichoderma reesei show low al., 2010). production of β- glucosidase (Saha et al., This study provides a comparison 1994; Skory & Freer, 1995; Riou et al., between cellulase production from rice 1998; Elyas et al., 2010). straw under SSF by the local T. harzianum Xylanases, the xylan degrading SNRS3 and A. niger ATCC 6275 as a model enzymes, are present everywhere and are fungus. Crude cellulase enzymes produced diverse by nature (Collins et al., 2005). by T. harzianum SNRS3 were characterised Xylan hydrolytic enzymes have been in terms of physio-chemical parameters such reported mainly from bacteria (Gilbert & as optimum temperature, and pH. Thermal Hazlewood, 1993), fungi (Jin et al., 2012) stability and the effects of storage at room and yeast (Hrmova et al., 1984; Liu et al., temperature (28ºC), 4ºC, −20ºC, and −40ºC 1998). Microbial xylanases are preferable on the enzyme activity have also been since they are highly specific, reaction studied.

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 517 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff

MATERIALS AND METHODS kept at 65% (w/v). Mandels medium (1 L) Substrate Preparation and contained 1.4 g (NH4)2SO4, 2 g KH2PO4, Pretreatment 0.63 g urea, 0.3 g CaCl2, 0.3 g MgSO4. Rice straw was obtained from a paddy field 7H2O, 1 mL Trace elements, 0.75 g peptone, in Sekinchan, Selangor, Malaysia. The dried and 2 mL Tween 80. The pH of the medium rice straw was ground to 2 mm lengths using was adjusted to 5. The flasks were then an electric grinder (Model CW-1, Hsiang incubated at 30°C prior to the extraction Tai, Taiwan), and kept in a cold room at 4°C of crude enzyme. The extraction of crude prior to use. enzyme mixture was carried out by adding 30 mL of 50 mM citrate buffer (pH 4.8) into Microorganism and Inoculum each flask, followed by agitation for 30 min Preparation at 150 rpm and 30°C. The mixture was then o A local isolate of T. harzianum SNRS3 centrifuged at 4 C and 1000 × g for 10 min. (isolated from rice straw collected from a The supernatant was filtered and kept at °C4 rice field in Sekinchan, Selangor, Malaysia) prior to use (Rahnama et al., 2013). and A. niger ATCC 6275 (used as model fungus) were used as inoculum, respectively. Crude Cellulase Enzyme Characterisation The fungal spores were kept in 30% (v/v) glycerol at minus 20°C. Reactivation of The crude cellulase enzyme produced the spores was performed by growing on from rice straw by T. harzianum SNRS3 Potato Dextrose Agar (PDA) for 7 to 9 days. was characterised in terms of temperature Spore suspension was freshly prepared prior and pH optima, thermal stability and to fermentation experiment by washing storage stability at various temperatures the agar surface with sterilised distilled [room temperature (28°C, 4°C, −20°C, and water. The spores were then quantified and −40°C)]. In order to study the optimum adjusted to 1×106 spores mL-1 by using a incubation temperature for the crude haemocytometer (Rahnama et al., 2013). cellulase activity, the reaction mixtures were reacted in a temperature range of 40- Fermentation 90°C and incubated for 1 h for FPase and for 30 min for CMCase and β-glucosidase; the Cellulase enzyme was produced by solid three major components of cellulases that act state fermentation. A series of 250 mL synergistically for the complete hydrolysis Erlenmeyer flasks with cotton stoppers were of cellulose. It was then followed by the autoclaved and used for the production and assay under the standard assay conditions at collection of the enzymes. Three grams of pH4.8, as described in detail in the analytical untreated rice straw was placed in different procedure. CMCase activity was determined flasks. Mandels medium (Mandels et al., by estimating the reducing sugars produced 1974) was added to each flask containing from 1% (w/v) carboxymethylcellulose, the rice straw, and the moisture content was

518 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation whereas FPase activity was determined by from 1% (w/v) carboxymethylcellulose, measuring the reducing sugars released from whereas Filter Paperase (FPase) activity Whatman filter paper No.1. For β-glucosidase was determined by measuring the reducing assay, the ρ-nitrophenol liberated from sugars released from Whatman filter paper ρ-nitrophenyl-β-D- glucopyranoside (PNPG) No.1. The liberated reducing sugars were was determined spectrophotometrically measured using the DNS method (Miller, (Wood & Bhat, 1988). 1959). One unit of CMCase or FPase activity The effect of pH on the activity of was defined as the amount of enzyme that crude cellulase was determined in a pH liberated 1 µmol reducing sugars/min under range of 2.5-7.5 by using citric acid- assay conditions and expressed as a unit of disodium hydrogen phosphate (McIlvaine) enzyme activity per gram fermented dry buffer solutions, pH 2.6-7.6. The activity substrate (U/g). was measured under the standard assay For FPase, substrate blank contained 2.0 conditions. In order to study the crude mL of 50 mM sodium citrate buffer (pH 4.8) enzyme thermal stability, the crude enzyme in the presence of Whatman No.1 filter paper was incubated at 50 and 60°C. The crude as the substrate. Conversely, the enzyme enzyme solution in the screw-capped blank contained 1.8 mL of 50 mM sodium glass tubes was withdrawn periodically citrate buffer (pH 4.8), 0.2 mL of the crude at the time intervals of 30 min, 1 h, 2 h, 3 enzyme mixture in the absence of Whatman h, and 4 h. It was then cooled on ice and filter paper, whilst the test was with the the residual enzyme activity was assayed presence of filter paper. The reactions were under standard assay conditions. In order to carried out for 1 h at 40ºC. investigate the crude enzyme stability over As for CMCase assay, 0.2 mL of the storage, the crude cellulase was aliquoted crude enzyme was added to 1.8 mL of 1% in four bottles. The bottles were each kept (w/v) carboxymethylcellulose in 50 mM in different temperatures including room sodium citrate buffer (pH 4.8), and incubated temperature (28°C), 4°C, −20°C, and at 40ºC for 30 min. The enzyme blank –40°C. The enzyme activity was assayed contained 0.2 mL of the enzyme and 1.8 ml under the standard assay conditions on a 50 mM pH 4.8 sodium citrate buffer only weekly basis for one month and a monthly [without 1% (w/v) carboxymethylcellulose], basis up to two months. while for the substrate blank, 0.2 ml sodium citrate buffer (pH 4.8) was used instead of Analytical Procedure the crude enzyme. Crude cellulase activity was assayed For β-glucosidase assay, the reaction according to the standard method (Wood mixture, which consisted of 0.2 mL crude & Bhat, 1988). Carboxymethylcellulase enzyme added to 2.0 mL of 0.5 mM (CMCase) activity was determined by ρ-nitrophenyl-β-D- glucopyranoside in 50 estimating the reducing sugars produced mM sodium citrate buffer (pH 4.8) was incubated at 40°C for 30 min. The reaction

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 519 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff was stopped by the addition of 2.0 mL of 1 1951) was used with bovine serum albumin

M sodium carbonate (Na2CO3) immediately as a standard. after the incubation time (Wood & Bhat, The impacts of temperature, pH, and 1988). One unit of β-glucosidase was storage on crude cellulase enzyme activity defined as the amount of enzyme that were expressed as relative activity that liberated 1 µmol ρ-nitrophenol/min under is a percentage of the maximum activity. assay conditions and expressed as a unit Thermal stability of the crude cellulase was of enzyme activity per gram fermented expressed as the residual activity that is a dry substrate (U/g). The substrate blank percentage of the original activity. contained 0.2 mL of 50 mM sodium citrate buffer (pH 4.8) instead of the crude enzyme, Statistical Analysis while enzyme blank was prepared by The data were analysed by using one- adding 0.2 mL of crude enzyme into the way analysis of variance (ANOVA). sodium citrate buffer only (i.e., without Meanwhile, t Tests (LSD) was used to ρ-nitrophenyl-β-D- glucopyranoside). compare the difference of means among The xylanase activity was assayed by treatment groups. Differences of p<0.05 estimating the reducing sugars released were considered significant. from 1% (w/v) Birchwood xylan (Dong et al., 1992). The reaction was carried out by RESULTS AND DISCUSSION adding 0.2 mL of the crude enzyme to 1.8 Cellulase from T. harzianum SNRS3 and mL of 1% (w/v) Birchwood xylan in 50 mM A. niger ATCC 6275 as a Model Fungus sodium citrate buffer pH 4.8 and incubated at Cellulase production from local T. harzianum 40ºC for 30 min. As for the substrate blank, SNRS3 was compared to that by A. niger 0.2 mL of 50 mM sodium citrate buffer (pH ATCC 6275 as a model fungus. Untreated 4.8) was added to replace the crude enzyme, rice straw was used as the fermentation while for the enzyme blank, the sodium substrate for both fungi. Figure 1 presents citrate buffer (pH 4.8) did not contain the production of FPase by both fungi any xylan. One unit of xylanase activity studied over a period of 10 days. FPase was defined as the amount of enzyme that production by A. niger and T. harzianum liberated 1 µmol reducing sugars/min under was not significantly different (p>0.05) assay conditions and expressed as a unit of during the first 5 days of fermentation. enzyme activity per gram fermented dry However, on day 6 of fermentation, FPase substrate (U/g). Reducing sugars released as production by A. niger was significantly a result of the reaction of the enzyme (FPase, higher (p<0.05) than that by T. harzianum CMCase, and xylanase) and the substrate and the maximum activity of FPase was was determined by using DNS method. obtained at 7.06 U/g substrate and 6.25 U/g For the soluble protein concentration substrate using A. niger ATCC 6275 and T. analysis, a modified method (Lowry et al., harzianum SNRS3, respectively.

520 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

CMCase and β-glucosidase production 2005). According to the results of this study, profiles were also studied over a period of however, T. harzianum SNRS3 proved to 10 days. A comparison between the CMCase be a better β-glucosidase producer than A. and β-glucosidase production from rice straw niger ATCC 6275. by T. harzianum SNRS3 and A. niger ATCC In comparison, a previous study on SSF 6275 is presented in Figure 2. The CMCase of rice straw has reported the production production by A. niger was significantly of FPase (480.48 U/g substrate), CMCase higher (p<0.05) than that by T. harzianum (363.72 U/g substrate), and β-glucosidase between days 1 and 3 of fermentation. (16.37 U/g substrate) by A. terreus ATTC However, on day 6, the CMCase obtained 74135, and the production of 7.85 and (111.31 U/g substrate) was significantly 11.73 U/g substrate of FPase and CMCase higher (p<0.05) than the maximum CMCase respectively, by A. niger (Jahromi et al., production (86.35 U/g substrate) when A. 2011). On the other hand, using A. niger niger was used. β-glucosidase production ATCC 6275 and palm cake under SSF, in T. harzianum was the highest on days 23.8 U/g substrate CMCase was produced 7 and 8 of fermentation (p<0.05), with a (Prasertsan et al., 1997). yield of 173.18-173.71 U/g. However, the The xylanase production profile highest production of β-glucosidase by A. was studied over a period of 10 days. A niger ATCC 6275 only gave an activity comparison between the xylanase production of 17.41 U/g (Day 7). Trichoderma sp. from rice straw using T. harzianum SNRS3 is normally associated with insufficient and A. niger ATCC 6275 is presented in production of β-glucosidase to perform Figure 3. As demonstrated in Figure 3, efficient hydrolysis, whereas Aspergillus the xylanase production was increased sp. has been reported as the most efficient significantly (p<0.05) when A. niger ATCC producer of β-glucosidase (Wen et al., 6275 was used as the inoculum for enzyme

Figure 1. FPase from T. harzianum SNRS3 and A. niger ATCC 6275. Values are means of 3 replicates ± SD. Closed symbols represent: T. harzianum SNRS3; Open symbols represent: A. niger ATCC 6275

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 521 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff

Figure 2. CMCase and β-glucosidase from T. harzianum SNRS3 and A. niger ATCC 6275. Values are means of 3 replicates ± SD. Symbols represent: (▲) CMCase activity; (●) β-glucosidase activity. Closed symbols represent: T. harzianum SNRS3; Open symbols represent: A. niger ATCC 6275

Figure 3. Xylanase from T. harzianum SNRS3 and A. niger ATCC 6275. Values are means of 3 replicates ± SD. Closed symbols represent: T. harzianum SNRS3; Open symbols represent: A. niger ATCC 6275 production. On day 7 of fermentation, xylanase production of 6,166 U/g substrate xylanase was produced at the activity of was obtained. Table 1 shows cellulase and 433.75 U/g substrate by T. harzianum xylanase production by different fungi SNRS3, as compared to 2378.64 U/g grown on various agricultural wastes under substrate obtained by A. niger ATCC 6275. solid state fermentation. Among the xylanolytic fungi, Aspergillus The profiles of extracellular protein is known as an efficient and high xylanase production by T. harzianum SNRS3 and producer (Fang et al., 2010). A. terreus A. niger ATCC 6275 grown on untreated ATCC 74135 was cultivated on untreated, rice straw studied over a period of 10 days ground rice straw under SSF and a very high were also monitored. Figure 4 indicates that

522 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

Figure 4. Extracellular protein profile ofT. harzianum SNRS3 and A. niger ATCC 6275. Values are means of 3 replicates ± SD. Closed symbols represent: T. harzianum SNRS3; Open symbols represent: A. niger ATCC 6275 protein concentration increased significantly NCIM NO-923 (Das & Ghosh, 2009) and with time for T. harzianum (p<0.05), giving Penicillium funiculosum (Karboune et al., a higher protein concentration of 4.78 mg/ 2008) with an optimum temperature of 50°C mL on day 6 of fermentation compared to and 60°C, respectively. It is worth noting the maximum protein concentration of 2.43 that FPase produced from T. harzianum mg/mL obtained on day 6 of fermentation SNRS3 can remain almost up to 100% for A. niger. active at 60°C, and this characteristic could be considered as a major advantage. Characterisation of Crude Cellulase by Unlike FPase that exhibited almost T. harzianum SNRS3 a similar optimum temperature plateau Effect of Temperature on Crude between 50-60°C, CMCase showed a Cellulase Activity different trend and the relative activity dropped sharply between 50-60°C. Similar Figure 5 illustrates the effects of temperature to CMCase produced by T. harzianum on the activity of crude cellulase in a SNRS3, CMCase produced by Penicillium temperature range of 40-90°C.The notatum NCIM NO-923 (Das & Ghosh, temperature profile of the enzyme shows 2009) and Streptomyces transformant T3-1 an optimal temperature of 50°C for FPase, (Jang & Chen, 2003) has been reported to CMCase, and β-glucosidase produced by be optimally active at 50°C. However, a T. harzianum SNRS3. The temperature lower optimal temperature of 40°C has been profile of FPase illustrates an optimum reported for endoglucanase produced by temperature plateau ranging from 50-60°C. Aspergillus niger Z10 (Coral et al., 2002). The same feature has been reported for The highest activities of CMCase from FPase produced by Penicillium notatum Penicillium sp. CR-316 have been reported

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 523 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff at 65°C (Picart et al., 2007). Whereas, of its maximum activity in a broad pH CMCase from T. aurantiacus (Kalogeris et range of 3.5-6.5, whereas CMCase retained al., 2003a) and Bacillus sp. (Rastogi et al., almost above 70% of its maximum activity 2010) have been reported to be optimally in the pH range of 3.5-7.0. However, active at 75°C. β-glucosidase showed the highest activity Similar to β-glucosidase from at pH 5.0 and remained highly active in a T. harzianum SNRS3, Stachybotrys sp narrow pH spectrum of 4.5-5.5. β-glucosidase have also been reported to As shown in Figure 6, the pH profile of be optimally active at 50°C (Amouri & the three components of the crude cellulase Gargouri, 2006). Interestingly, a closer showed an increasing trend for the activity look at β-glucosidase temperature profile with the rise in pH value. In particular, the revealed that at 60°C, β-glucosidase activity activity of FPase and CMCase increased was still above 70% of its maximum activity. sharply in the pH range between 2.5-4.0. This is considered an advantage of the For β-glucosidase, however, the activity crude cellulase enzyme produced by local increased drastically in the pH range T. harzianum SNRS3. Its ability to retain between 3.5-4.50. Based on a relative high activity at 60°C, when other cellulases activity of 100% for β-glucosidase at pH 5.0, are inactivated, is an important characteristic the activity decreased to 12.2 %, and 8.1 % for cellulases as industrial enzymes. Table 2 at pH3.5 and pH7, respectively. summarises the optimum temperature of the crude cellulase produced by T. harzianum Thermal Stability of Crude Cellulase SNRS3 and various other microorganisms. The results of thermostability of the crude cellulase produced by T. harzianum SNRS3 Effects of pH on Crude Cellulase at 50 and 60°C are presented in Figure 7. As Activity expected and can be observed, the higher As illustrated in Figure 6, cellulolytic enzyme the temperature, the higher the activity of complex system produced by T. harzianum the enzyme is likely to be lost. As shown SNRS3 displayed cellobiohydrolase, in Figure 7, 30 min incubation of the crude endoglucanase, and β-glucosidase activities enzyme at 50°C resulted in a reduction in over a broad range of pH. This characteristic the activity of FPase to around 70% of its of the crude cellulase by T. harzianum original activity. However, incubation at SNRS3 is considered as an advantage for 60°C for 30 min reduced the FPase activity cellulases that are important industrial to almost 40% of its initial activity. It is enzymes. However, the cellulose degrading worth noting that increasing the incubation enzymes are highly active in the acidic period up to 4 h did not further decrease the region (Table 2). Depending on the type of enzyme activity. cellulase, the pH-activity profiles obtained were different. FPase retained above 50%

524 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

Unlike CMCase produced by Effects of Storage on Crude Cellulase Penicillium notatum NCIM NO-923 (Das Activity & Ghosh, 2009) and Penicillium citrinum Results of the effects of storage on the (Dutta et al., 2008) that proved to be more activity of crude cellulase enzyme are themostable at 50 and 60°C than FPase, depicted in Figures 8.A, 8.B, and 8.C. CMCase produced by T. harzianum SNRS3 Regardless of the storage temperature showed less thermal stability compared to of the crude enzyme, the FPase activity was FPase. The incubation of the crude cellulase almost stable within the first week (Figure at 50 and 60°C for 30 min caused the activity 8.A), and only at room temperature, a slight of CMCase to drop to almost 40%, and decrease occurred in the FPase activity from 30% of its original activity, respectively. 100% up to 94%. Meanwhile, the activity At 50°C, however, and with the increase of FPase for the crude samples kept at 4°C, in the incubation time up to 4 h, no more − 20°C, and −40°C almost remained stable reduction in the enzyme activity was within the 2nd week, but the activity dropped detected. Consequently, in the incubation to 77% at room temperature. After weeks 3 period range of 30 min to 4 h, a thermal and 4 at 4°C, −20°C, and −40°C, the activity stability plateau was observed. Meanwhile of FPase slightly decreased. However, at at 60°C, increasing the incubation time of room temperature, the FPase activity of the crude enzyme for up to 4 h steadily the crude enzyme decreased drastically at decreased the CMCase activity to less than the end of week 4 and reached 27% of its 15% of its initial activity. original activity. Interestingly, the storage of As depicted in Figure 7, β-glucosidase crude enzyme at room temperature showed exhibited above 50% of its original activity that FPase activity was still almost 60% of after incubation at 50°C for 30 min and after its original activity at the end of week 3. that, any longer incubation of the enzyme up The FPase activity was still above 85% of to 4 h did not have a significant effect on the its original activity after keeping the crude residual activity of the enzyme. However, enzyme for 3 weeks at 4°C, −20°C, and the enzyme was found to be less stable at −40°C. 60°C and after 2-h incubation, and that As shown in Figure 8.B, CMCase almost all activities were lost probably due proved to be more sensitive to storage to the enzyme denaturation. Table 2 provides temperature compared to FPase. At room a comparison between thermostability of the temperature, the CMCase activity dropped crude cellulase produced by T. harzianum sharply from 71% after week 1 to merely SNRS3 and that of cellulase by various 25% and 7% at the end of weeks 3 and 4, microorganisms. respectively. However, CMCase retained 60% of its original activity at the end of the 2nd week of storage at room temperature. The effect of storage of the crude enzyme at

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 525 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff

Table 1 Cellulase and Xylanase Production by Different Fungi Grown on Various Agricultural Wastes under Solid State Fermentation

Enzyme source Carbon source Enzyme activities (U/g dry substrate) References FPase CMCase β-glucosidase Xylanase Aspergillus niger Palm cake _ 23.8 _ 282.9 (Prasertsan et ATCC 6275 al., 1997) Aspergillus niger Rice straw 19.5 129 100 5070 (Kang et al., KK2 2004) Aspergillus terreus Corn stover 243 581 128 _ (Gao et al., M11 2008) Aspergillus terreus Palm oil fiber _ _ _ 115,269 (Suvarna MTCC 8661 Lakshmi et al., 2009) Aspergillus terreus Rice straw 480.48 363.72 16.37 6,166.01 (Jahromi et al., ATCC74135 2011) Aspergillus ustus Wheat bran 3.78 11.84 60.00 615.26 (Shamala &Sreekantiah, 1986) Aspergillus ustus Rice straw 5.82 12.58 15.82 740 (Shamala & Sreekantiah, 1986) Myceliophthora sp. Corn cob 0.31 11.38 5. 49 411.6 (Badhan et al., IMI 387099 2007) Myceliophthora sp. Bagasse 0.70 6.62 2.01 620.1 (Badhan et al., IMI 387099 2007) Myceliophthora sp. Wheat bran 0.74 26.6 3.83 128.9 (Badhan et al., IMI 387099 2007) Myceliophthora sp. Wheat straw 1.37 30.8 6.78 656.6 (Badhan et al., IMI 387099 2007) Myceliophthora sp. Rice straw 2.44 32.9 7.48 900.2 (Badhan et al., IMI 387099 2007) Thermoascus Wheat straw 4.3 956 46.1 2973 (Kalogeris et aurantiacus al., 1999) Trichoderma Wheat straw _ 1572 101.6 _ (Kalogeris et aurantiacus al., 2003a) Trichoderma Wheat straw 5.5 1709 79 4490 (Kalogeris et aurantiacus al., 2003b) Trichoderma Wheat bran 94 287.3 184 _ (Li et al., koningi F244 2004) Trichoderma Wheat bran and _ _ _ 592.7 (Azin et al., longibrachiatum wheat straw 2007) Trichoderma reesei Rice bran 2.314 _ _ _ (Latifian et al., MCG77 2007) Trichoderma reesei Corncob 158 _ _ _ (Xia & Cen, ZU-02 1999) Aspergillus niger Rice straw 7.06 86.35 17.41 2378.64 Present study ATCC 6275 Trichoderma Rice straw 6.25 111.31 173.71 433.75 Present study harzianum SNRS3

526 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

Figure 5. The effects of temperature on the activity of crude cellulase by T. harzianum SNRS3 from rice straw in SSF. Values are means of 3 replicates ±SD. A: FPase, B: CMCase, C: β-glucosidase

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 527 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff

Figure 6. The effects of pH on the activity of crude cellulase by T. harzianum SNRS3 from rice straw in SSF. Values are means of 3 replicates ± SD. A: FPase B: CMCase C: β-glucosidase

528 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

Figure 7. Thermostability of FPase (A), CMCase (B), and β-glucosidase (C) by T. harzianum SNRS3 from rice straw in SSF. Values are means of 3 replicates ± SD. Closed symbols represent: Residual activity at 60°C; Open symbols represent: Residual activity at 50°C

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 529 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff

Figure 8. The effects of storage on the activity of crude cellulase by T. harzianum SNRS3 from rice straw in SSF. Values are means of 3 replicates ± SD. A: FPase B: CMCase C: β-glucosidase

530 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation Dutta et al., 2008) Dutta et al., 2008) References ( (Karboune et al., 2008) (Das & Ghosh, 2009) (Macris, 1984) (Saqib et al., 2010) (Coral et al., 2002) (Gao et al., 2008) (Rastogi et al., 2010) (Heidorne et al., 2006) (Rastogi et al., 2010) (Picart et al., 2007) ) (Karboune et al., 2008) (Das & Ghosh, 2009) (Grigorevski de Lima et al., 2005) (Jang & Chen, 2003) 7% loss of 7% loss of °C after 15 min °C after 1 day; °C after 2 days °C after 1 day; 97% activity remained °C after 1 day; 97% activity remained ºC; at 50 and 60ºC FPase less ºC; at 50 and 60ºC FPase less °C after 3 h; lost 75% of activity at ºC °C after 5 days; 98% of activity Thermostability less thermostable than CMCase thermostable at up to 55°C stable at 40 thermostable - - above 40% of activity at 90 highly stable; retained 65% of activity after 6 h Stable at 50 at 60°C after 1 day rapid loss of activity at 40 and 50°C 100% active at 50 and 60 activity at 60 stable at 60 65°C after 1 h stable at 50-70°C after incubation for 2 h thermostable at up to 55°C stable at 40 stable at 50°C after 1 h; 40% of activity remained after 2 h; 20% of activity remained 8 h stable at 50 remained at 50°C after 7 days; half-life 15 h 60°C ; half-life 5 h at 70°C pH optimum 6.5 4.0-5.0 4.0 5.0-6.0 - 4.5 and 7.5 2.0 5.0 3.5-5.0 5.0 4.5 5.5 and 8 4.0 4.0 5.0 7.0-8.0 Temperature Temperature optimum (ºC) - 60 50 55-60 64.3 40 70 75 60 70 65 - 60 50 50-60 50 Enzyme FPase FPase FPase CMCase CMCase CMCase CMCase CMCase CMCase CMCase CMCase CMCase CMCase CMCase CMCase CMCase NCIM NCIM NCIM M11 Z10 transformant transformant sp. sp. CR-316 sp.

Microorganism Penicillium citrinum Penicillium funiculosum Penicillium notatum NO-923 Alternaria alternate fumigatus Aspergillus niger Aspergillus terreus Aspergillus Bacillus Ceriporiopsis subvermispora Geobacillus Penicillium Penicillium citrinum Penicillium funiculosum Penicillium notatum NO-923 drozdowiczii Streptomyces Streptomyces T3-1 Table 2 Table Microorganisms by Various pH, and Thermostability of Celluase Produced Optimum Temperature,

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 531 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff (Kalogeris et al., (Kalogeris et al., 2003a) (Macris, 1984) et al., 1992) (Watanabe (Saha et al., 1994) (Gao et al., 2008) (Hayashi et al.,1993) (Leite et al., 2007) (Heidorne et al., 2006) (Chen et al., 2010) (Karboune et al., 2008) (Amouri & Gargouri, 2006) (Leite et al., 2007) (Kalogeris et al., 2003a) (Farinas et al., 2010) , and 10 , and 10 °C °C °C after12 h; 50% of °C after12 h; 50% of , 1.8 h at 65 °C °C; half-life 1 day at 70°C °C; half-life 2.5 days at 70°C °C °C half-life 42 min at 80 Half-life 3.5 days at 60 min at 70 - - highly thermo stable; retained 53% of original activity after 6 h stable at 80°C for 15 min retained 98% of activity after 1 h incubation at 75 rapid loss of activity at 40 and 50°C 96% of activity remained at 50 activity remained at 70°C after 4 h stable at 25-40 - retained 98% of activity after 1 h at 70 half-life 18 min at 80 - 3.5 and 4 4.5-5.0 4.5 - 3.0 4.0 4-4.5 3.5-5.0 4.5-5.0 4.5 5.0 4.5 4.5 4.0-5.5 75 70-75 55 75 70 80 80 60 65-70 60 50 70 80 35-60 -glucosidase -glucosidase -glucosidase CMCase β-glucosidase β β-glucosidase β-glucosidase β-glucosidase β-glucosidase β-glucosidase β-glucosidase β-glucosidase β-glucosidase β-glucosidase β Crude cellulase M11 sp. sp. Table 2 Table T. aurantiacus T. Alternaria alternata niger Aspergillus pullulans Aspergillus terreus Aspergillus Aureobasidium pullulans Aureobasidium Ceriporiopsis subvermispora Penicillium decumbens Penicillium funiculosum Stachybotrys Thermoascus aurantiacus aurantiacus T. niger Aspergillus cont’d

532 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation (Machado de Castro et (Machado de Castro et al., 2010) (Machado de Castro et al., 2010) (Machado de Castro et al., 2010) Present study of activity of activity °C ºC corresponding ºC corresponding , °C after 30 min and up to 4 h, 70% of activity °C after 30 min and up to 4 h, 70% of activity °C after 30 min up to 4 h, 40% of activity °C after 30 min up to 4 h, > 50% - rapid thermal denaturation at 60 to half-life < 1 h - At 50 remained, at 60°C after 30 min and up to 4 h, 40% of activity remained At 50 remained; at 60°C after 30 min 30% of activity remained and after 4 h < than 15% of activity remained At 50 remained; inactivated after 2h at 60 4.9-5.2 5.1-5.3 4.5-5.5 4.0-5.0 5.0-5.5 5.0 47- 54 52-57 50 50 54-57 50 Crude Crude cellulase Crude cellulase Crude cellulase FPase CMCase β-glucosidase

RutC30

SNRS3 Table 2 Table Aspergillus niger Aspergillus ATCC-16404 harzianum Trichoderma IOC-4038 reesei Trichoderma harzianum T. cont’d

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 533 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff

4°C, −20°C, and −40°C was almost similar. β-glucosidase at a much higher activity After week 1, the activity first decreased to (173.71 U/g substrate) compared to 17.41 almost above 75% for the three samples kept U/g substrate β-glucosidase by A. niger at 4°C, −20°C, and −40°C. It is worth noting ATCC 6275, belonging to a genus reported as that storage at 4°C for 1 month caused the the most efficient producer of β-glucosidase. CMCase activity to drop to almost 65% of However, T. harzianum SNRS3 was not an its initial activity. efficient xylanase producer (433.75 U/g Similarly, the β-glucosidase activity substrate) compared to A. niger ATCC 6275 was also affected by storage temperature (2378.64 U/g substrate). FPase showed and duration (Figure 8C). Storing the crude an optimum temperature plateau in the enzyme at room temperature caused the temperature range of 50 to 60ºC, indicating activity to drop sharply from 82% at the that this enzyme can remain active almost up end of week 3 to 4% at the end of week 4. to 100% at 60ºC. At 60ºC, the β-glucosidase However, the β-glucosidase activity was activity was still above 70% of its maximum only slightly affected when the enzyme activity. This is a significant characteristic was kept at 4°C, −20°C, and −40°C for of cellulases with a wide range of industrial one month. Like FPase, β- glucosidase was applications. Cellulose degrading enzymes also proven to be almost stable for the first were highly active in the acidic region and three weeks of storage at room temperature. could be mostly applied over a wide range The β- glucosidase activity only decreased of acidic pH. to above 80% of its original activity at the end of the 3rd week of storage at room ACKNOWLEDGEMENTS temperature. The authors would like to extend their It is worth noting that after keeping gratitude for the financial support generously the crude cellulase for 2 months at 4ºC, provided by the Ministry of Science, −20ºC and −40ºC, CMCase retained almost Technology and Innovation (MOSTI), above 60% of its original activity. However, Malaysia, through the grant NO. 02-01-04- FPase and β- glucosidase remained active, SF1036. i.e. above 80% and 90% of their original activity, respectively. REFERENCES Allgaier, M., Reddy, A., Park, J.I., Ivanova, N., CONCLUSION D’haeseleer, P., Lowry, S., & Hugenholtz, Unlike Trichoderma sp. that are normally P. (2010). Targeted discovery of glycoside associated with the production of low hydrolases from a switchgrass-adapted compost community. PLoS One, 5(1), e8812. amount of β-glucosidase for an efficient hydrolysis, T. harzianum SNRS3 was shown Amouri, B., & Gargouri, A. (2006). Characterization to be a potential β-glucosidase producer. of a novel β-glucosidase from a Stachybotrys strain. Biochemical Engineering Journal, 32(3), Meanwhile, T. harzianum SNRS3 produced 191–197.

534 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

Azin, M., Moravej, R., & Zareh, D. (2007). Production Collins, T., Gerday, C., & Feller, G. (2005). Xylanases, of xylanase by Trichoderma longibrachiatum xylanase families and extremophilic xylanases. on a mixture of wheat bran and wheat straw: FEMS Microbiology Reviews, 29(1), 3-23. Optimization of culture condition by Taguchi Coral, G., Arikan, B., Nisa Unaldi, M., & method. Enzyme and Microbial Technology, Guivenmez, H. (2002). Some properties of 40(4), 801-805. crude carboxymethyl cellulase of Aspergillus Badhan, A. K., Chadha, B. S., Kaur, J., Saini, niger Z10 wild-type strain. Turkish Journal of H. S., & Bhat, M. K. (2007). Production of Biology, 26(4), 209-213. multiple xylanolytic and cellulolytic enzymes Das, A., & Ghosh, U. (2009). Solid-state fermentation by thermophilic fungus Myceliophthora sp. of waste cabbage by Penicillium notatum NCIM IMI 387099. Bioresource Technology, 98(3), NO-923 for production and characterization of 504-510. cellulases. Journal of Scientific and Industrial Binod, P., Sindhu, R., Singhania, R. R., Vikram, Research, 68(8), 714-718. S., Devi, L., Nagalakshmi, S., Kurien, N., Devendra, C., & Thomas, D. (2002). Smallholder Sukumaran, R. K., & Pandey, A. (2010). farming systems in Asia. Agricultural Systems, Bioethanol production from rice straw: An 71(1), 17-25. overview. Bioresource Technology, 101(13), 4767–4774. Dhiman, S. S., Sharma, J., & Battan, B. (2008). Industrial applications and future prospects of Carpita, N., Tierney, M., & Campbell, M. (2001). microbial xylanases: A review. BioResources, Molecular biology of the plant cell wall: 3(4), 1377-1402. searching for the genes that define structure, architecture and dynamics (pp. 1-5). Netherlands: Dong, W. K., Tae, S. K., Young, K. J., & Jae, K. Springer. L. (1992). Adsorption kinetics and behaviors of cellulase components on microcrystalline Chahal, D. S. (1985). Solid-state fermentation with cellulose. Journal of fermentation and Trichoderma reesei for cellulase production. bioengineering, 73(6), 461-466. Applied and Environmental Microbiology, 49(1), 205-210. Dutta, T., Sahoo, R., Sengupta, R., Ray, S.S., Bhattacharjee, A., & Ghosh, S. (2008). Novel Chapla, D., Divecha, J., Madamwar, D., & Shah, A. cellulases from an extremophilic filamentous (2010). Utilization of agro-industrial waste for fungi Penicillium citrinum production and xylanase production by Aspergillus foetidus characterization. Journal of Industrial MTCC 4898 under solid state fermentation and Microbiology and Biotechnology, 35(4), 275– its application in saccharification. Biochemical 282. Engineering Journal, 49(3), 361-369. Elyas, K. K., Mathew, A., Sukumaran, R. K., Chen, M., Qinb, Y., Liua, Z., Liua, K., Wangc, F., & Manzur Ali, P. M., Sapna, K., Ramesh Kumar, Qua, Y. (2010). Isolation and characterization S., & Rekha Mol, K. R. (2010). Production, of a β-glucosidase from Penicillium decumbens optimization and properties of beta glucosidases and improving hydrolysis of corncob residue by from a marine fungus Aspergillus-SA 58. New using it as cellulase supplementation. Enzyme Biotechnology, 27(4), 347-351. and Microbial Technology, 46(6), 444–449.

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 535 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff

Fadel, M. (2000). Production physiology of cellulases Holker, U., & Lenz, J. (2005). Solid state fermentation- and β-glucosidase enzymes of Aspergillus niger are there any biotechnological advantages? grown under solid state fermentation. Online Current Opinion in Microbiology, 8(3), 301-306. Journal of Biological Sciences, 1(5), 401-411. Hong, J., Tamaki, H., Akiba, S., Yamamoto, K., & Fang, T. J., Liao, B. C., & Lee, S. C. (2010). Kumaga, H. (2001). Cloning of a gene encoding Enhanced production of xylanase by Aspergillus a highly stable endo-β-1,4-glucanase from carneus M34 in solid-state fermentation with Aspergillus niger and its expression in yeast. agricultural waste using statistical approach. New Journal of Bioscience and Bioengineering, Biotechnology, 27(1), 25-32. 92(5), 434-441.

Farinas, C. S., Loyo, M. M., Junior, A. B., Tardioli, Howard, R. L., Abotsi, E., Jansen van Rensburg, P. W., Neto, V. B., & Couri, S. (2010). Finding E. L., & Howard, S. (2003). Lignocelluloses stable cellulase and xylanase: evaluation of the biotechnology: issues of bioconversion synergistic effect of pH and temperature. New and enzyme production. African Journal of Biotechnology, 27(6), 810-815. Biotechnology, 2(12), 602-619.

Gao, J., Weng, H., Zhu, D., Yuan, M., Guan, F., & Xi, Hrmova, M., Biely, P., Vrsanska, M., & Petrakova, Y. (2008). Production and characterization of E. (1984). Induction of cellulose- and cellulolytic enzymes from the thermoacidophilic xylan-degrading enzyme complex in the fungal Aspergillus terreus M11 under solid- yeast Trichosporon cutaneum. Archives of state cultivation of corn stover. Bioresource Microbiology, 138(4), 371-376. Technology, 99(16), 7623–7629. Jahromi, M. F., Liang, J. B., Rosfarizan, M., Goh, Y. M., Gilbert, H. J., & Hazlewood, G. P. (1993). Bacterial Shokryazdan, P., & Ho, Y. W. (2011). Efficiency cellulases and xylanases. Journal of General of rice straw lignocelluloses degradability by Microbiology, 139(2), 187-194. Aspergillus terreus ATCC 74135 in solid state fermentation. African Journal of Biotechnology, Grigorevski de Lima, A. L., Pires do Nascimento, R., 10(21), 4428-4435. Pinto da Silva Bonb, E., & Rodrigues Coelho, R. R. (2005). Streptomyces drozdowiczii cellulase Jang, H. D, & Chen, K. S. (2003). Production and production using agro-industrial by-products characterization of thermostable cellulases and its potential use in the detergent and textile from Streptomyces transformant T3-1. World industries. Enzyme and Microbial Technology, Journal of Microbiology and Biotechnology, 37(2), 272-277. 19(3), 263–268.

Hayashi, S., Matsumoto, K., Wada, Y., Takasaki, Y., Jin, N., Ma, S., Liu, Y., Yi, X., He, R., Xu, H., Qiao, & Imada, K. (1993). Stable β-glucosidase from D. R., & Cao, Y. (2012). Thermophilic xylanase Aureobasidium. Letters in Applied Microbiology, production by Aspergillus niger in solid state 17(2), 75-77. fermentation using wheat straw and corn cob. African Journal of Microbiology Research, 6(2), Heidorne, F. O., Magalhaes, P. O., Ferraz, A. L., & 2387-239. Milagres, A. M. F. (2006). Characterization of hemicellulases and cellulases produced by Johnson, E. A., Reese, E. T., & Demain, A. L. (1982). Ceriporiopsis subvermispora grown on wood Inhibition of Clostridium thermocellum cellulase under biopulping conditions. Enzyme and by end products of cellulolysis. Journal of Microbial Technology, 38(3), 436–442. Applied Biochemistry, 4, 64-71.

536 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

Joo, A. R., Jeya, M., Lee, K. M., Sim, W. I., Kim, J. S., xylanases. FEMS Microbiological Reviews, Kim, I. W., Kim, Y. S., Oh, D. K., Gunasekaran, 23(4), 411-456. P., & Lee, J. K. (2009). Purification and Latifian, M., Hamidi-Esfahani, Z., & Barzegar, characterization of a β-1,4-glucosidase from a M. (2007). Evaluation of culture conditions newly isolated strain of Fomitopsis pinicola. for cellulase production by two Trichoderma Applied Microbiology and Biotechnology, 83(2), reesei mutants under solid-state fermentation 285-294. conditions. Bioresource Technology, 98(18), Kalogeris, E., Fountoukides, G., Kekos, D., & Macris, 3634-3637. B. J. (1999). Design of a solid-state bioreactor Leff, B., Ramankutty, N., & Foley, J. A. (2004). for thermophilic microorganisms. Bioresource Geographic distribution of major crops across Technology, 67(3), 313-315. the world. Global Biogeochemical Cycles, Kalogeris, E., Christakopoulos, P., Katapodis, P., 18(1), 1-27. Alexiou, A., Vlachou, S., Kekos, D., & Macris, Leite, R. S., Gomes, E., & Roberto da Silva B. J. (2003a). Production and characterization (2007). Characterization and comparison of of cellulolytic enzymes from the thermophilic thermostability of purified β-glucosidases from fungus Thermoascus aurantiacus under solid a mesophilic Aureobasidium pullulans and a state cultivation of agricultural wastes. Process thermophilic Thermoascus aurantiacus. Process Biochemisrtry, 38(7), 1099-1104. Biochemistry, 42(7), 1101–1106. Kalogeris, E., Iniotaki, F., Topakas, E., Li, Y. H., Ding, M., Wang, J., Xu, G. J., & Christakopoulos, P., Kekos, D., & Macris, Zhao, F. (2006). A novel thermoacidophilic B.J. (2003b). Performance of an intermittent endoglucanase, Ba-EGA, from a new cellulose- agitation rotating drum type bioreactor for solid- degrading bacterium, Bacillus sp. AC-1. Applied state fermentation of wheat straw. Bioresource Microbiology and Biotechnology, 70(4), 430- Technology, 86(3), 207–213. 436. Kang, S. W., Park, Y. S., Lee, J. S., Hong, S. I., & Li, P. J., Jing, D. B., Zhou, Q. X., & Zhang, C. G. Kim, S. W. (2004). Production of cellulases (2004). Optimization of solid fermentation of and hemicellulases by Aspergillus niger KK2 cellulase from Trichoderma koningii. Journal of from lignocellulosic biomass. Bioresource Environmental Sciences, 16(5), 816–820. Technology, 91(2), 153-156. Liu, W., Zhu, W., Lu, Y., Kong, J., & Ma, G. Karboune, S., Geraert, P. A., & Kermasha, S. (2008). (1998). Production, partial purification and Characterization of selected cellulolytic activities characterization of xylanase from Trichosporon of multi-enzymatic complex system from cutaneum SL409. Process Biochemistry, 33(3), Penicillium funiculosum. Journal of Agricultural 331-336. and Food Chemistry, 56(3), 903–909. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Kuhad, R. C., Singh, A., & Eriksson, K. E. L. Randall, R. J. (1951). Protein measurement with (1997). Microorganisms and enzymes involved the folin phenol reagent. Journal of Biological in the degradation of plant fiber cell walls. In Chemistry, 193(1), 265-275. Biotechnology in the pulp and paper industry (pp. 45-125). Berlin Heidelberg: Springer. Lymar, E. S., Li, B., & Renganathan, V. (1995). Purification and characterization of a cellulose- Kulkarni, N., Shendye, A., & Rao, M. (1999). binding β-glucosidase from cellulose-degrading Molecular and Biotechnological aspects of

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 537 Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. A. A. and A. B. Ariff

cultures of Phanerochaete chrysosporium. characterization. Letters in Applied Microbiology, Applied and Environmental Microbiology, 61(8), 45(1), 108-113. 2976–2980. Prasertsan, P., H-Kittikul, A., Kunghae, A., Maneesri, Machado de Castro, A., Rodrigues Pedro, K. C. N., J., & Oi, S. (1997). Optimization for xylanase Cunha da Cruz, J., Costa Ferreira, M., Ferreira and cellulase production from Aspergillus niger Leite, S .G., & Pereira, Jr. N. (2010). Trichoderma ATTC 6275 in palm oil mill wastes and its harzianum IOC-4038: A promising strain for application. World Journal of Microbiology and the production of a cellulolytic complex with Biotechnology, 13(5), 555-559. significant β-glucosidase activity from sugarcane Rahnama, N., Mamat, S., UmiKalsom, M. S., Foo H. bagasse cellulignin. Applied Biochemistry and L., Nor Aini, A. R., & Arbakariya, B. A. (2013). Biotechnology, 162(7), 2111–2122. Effect of alkali pretreatment of rice straw on Macris, B. J. (1984). Production and characterization cellulase and xylanase production by local of cellulase and β-glucosidase from a mutant of Trichoderma harzianum SNRS3 under solid state Alternaria alternate. Applied and Environmental fermentation. BioResources, 8(2), 2881-2896. Microbiology, 47(3), 560-565. Rahnama, N., Foo, H. L., Rahman, N. A. A., Ariff, Mandels, M., Hontz, I., & Nystrom, J. (1974). A., & Shah, U. K. M. (2014). Saccharification of Enzymatic hydrolysis of waste cellulose. rice straw by cellulase from a local Trichoderma Biotechnology and Bioengineering, 16(11), harzianum SNRS3 for biobutanol production. 1471-1493. BMC Biotechnology, 14(1), 103.

Miller, G. L. (1959). Use of dinitrosalicylic acid for Rastogi, G., Bhalla, A., Adhikari, A., Kenneth, M., determination of reducing sugar. Analytical Bischoff, K. M., Hughes, S. R., Christopher, Chemistry, 31(3), 426-428. L. P., Rajesh, K., & Sani, R. K. (2010). Characterization of thermostable cellulases Mtui, G., & Nakamura, Y. (2005). Bioconversion of produced by Bacillus and Geobacillus strains. lignocellulosic waste from selected dumping Bioresource Technology, 101(22), 8798–8806. sites in Dar es Salaam Tanzania. Biodegradation, 16(6), 493–499. Riou, C., Salmon, J. M., Vallier, M. J., Gunata, Z., & Barre, P. (1998). Purification, characterization, Ong, L. G. A., Chuah, C., & Chew, L. A. (2010). and substrate specificity of a novel highly glucose- Comparison of sodium hydroxide and potassium tolerant β-glucosidase from Aspergillus oryzae. hydroxide followed by heat treatment on rice Applied and Environmental Microbiology, straw for cellulase production under solid state 64(10), 3607-3614. fermentation. Journal of Applied Sciences, 10(21), 2608-2612. Saha, B. C., Freer, S. N., & Bothast, R. J. (1994). Production, purification, and properties of a Park, J. I., Steen, E. J., Burd, H., Evans, S. S., thermostable β-glucosidase from a colour variant Redding-Johnson, A. M., Batth, T., & Gladden, strain of Aureobasidium pullulans. Applied and J. M. (2012). A thermophilic ionic liquid-tolerant Environmental Microbiology, 60(10), 3774- cellulase cocktail for the production of cellulosic 3780. biofuels. PLoS One, 7(5), e37010. Saqib, A. A. N., Hassan, M., Farooq khan, N., & Picart, P., Diaz, P., & Pastor F. I. J. (2007). Cellulases Baig, S. (2010). Thermostability of crude from two Penicillium sp. strains isolated endoglucanase from Aspergillus fumigatus from subtropical forest soil: Production and grown under solid state fermentation (SSF)

538 Pertanika J. Trop. Agric. Sci. 39 (4) 515 - 539 (2016) Trichoderma Cellulase Production and Characterisation

and submerged fermentation (SmF). Process Talebnia, F., Karakashev, D., & Angelidaki, I. (2010). Biochemistry, 45(5), 641-646. Production of bioethanol from wheat straw: An overview on pretreatment, hydrolysis and Shamala, T. R., & Sreekantiah, K. R. (1986). fermentation. Bioresource Technology, 101(13), Production of cellulases and D-xylanase by some 4744-4753. selected fungal isolates. Enzyme and Microbial Technology, 8(3), 178-182. Watanabe, T., Sato, T., Yoshioka, S., Koshijima, T., & Kuwahara, M. (1992). Purification and properties Singhania, R. R., Patel, A. K., Soccol, C. R., & of Aspergillus niger β-glucosidase. European Pandey, A. (2009). Recent advances in solid-state Journal of Biochemistry, 209(2), 651 -659. fermentation. Biochemical Engineering Journal, 44(1), 13-18. Wen, Z., Liao, W., & Chen, S. (2005). Production of cellulase/β-glucosidase by the mixed fungi Singhania, R. R., Sukumaran, A. K., Patel, A. K., culture Trichoderma reesei and Aspergillus Larroche, C., & Pandey, A. (2010). Advancement phoenicis on dairy manure. Process Biochemistry, and comparative profiles in the production 40(9), 3087-3094. technologies using solid-state and submerged fermentation for microbial cellulases. Enzyme Wilson, D. B. (2009). Cellulases and biofuels. Current and Microbial Technology, 46(7), 541-549. Opinion in Biotechnology, 20(3), 295–299.

Skory, C. D., & Freer, S. N. (1995). Cloning and Wood, T. M., & Bhat, K. M. (1988). Measuring characterization of a gene encoding a cell-bound, cellulase activities. Methods in Enzymology, extracellular β-glucosidase in the yeast Candida 160, 87-112. wickerhamii. Applied and Environmental Workman, W. E., & Day, D. F. (1982). Purification Microbiology, 61(2), 518-525. and properties of β-glucosidase from Sun, W. C., Cheng, C. H., & Lee, W. C. (2008). Protein Aspergillus terreus. Applied and Environmental expression and enzymatic activity of cellulases Microbiology, 44(6), 1289-1295. produced by Trichoderma reesei Rut C-30 on rice Xia, L., & Cen, P. L. (1999). Cellulase production by straw. Process Biochemistry, 43(10), 1083-1087. solid state fermentation on lignocellulosic waste Suvarna Lakshmi, G., Rao, C. S., Rao, R. S, Hobbs, from the xylose industry. Process Biochemistry, P. J., & Prakasham, R. S. (2009). Enhanced 34(9), 909–912. production of xylanase by a newly isolated Aspergillus terreus under solid state fermentation using palm industrial waste: A statistical optimization. Biochemical Engineering Journal, 48(1), 51-57.

Pertanika J. Trop. Agric. Sci. 39 (1): 515 - 539 (2016) 539

Pertanika J. Trop. Agric. Sci. 39 (4): 541 - 550 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Effect of Higher Density Planting on Irrigation Water Use and Yield of Sugarcane

D. Khodadadi Dehkordi Department of Water Engineering and Sciences, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran

ABSTRACT Sugarcane is one of the important crops that needs plentiful water in tropical and semi- tropical regions. This crop is widely grown in Khuzestan province of Iran. In this study, triple-row planting of sugarcane was evaluated and compared to double-row planting as currently being practiced in the region. Results showed that in spite of the higher density planting in triple-row planting, there was no significant difference between triple-row planting and double-row planting in terms of leaf sheath moisture, leaf nitrogen, ridge EC, qualitative situations of cultivar, plant height and stem diameter. However, the number of shoots and stems per m2 in the triple-row planting were found to be higher than in the double-row planting and the difference between them was significant at 5% level. In addition, water application efficiency, water use efficiency (WUE), mean yield of sugarcane and sugar content in triple-row planting were higher than double-row planting.

Keywords: Sugarcane, triple-row planting, double-row planting

INTRODUCTION is supplied from sugarcane production Sugarcane is one of the most important crops and the rest is produced from sugar beet. The favourable temperature for growing in Southwest Iran. Sugarcane is a C4, a high biomass crop which requires large amount is between 30-34°C and the favourable of water for maximum production. About relative humidity is between 60-80%. 60 percent of the world’s sugar content These conditions are available in Khuzestan province that is located in southwest Iran. The current production of sugarcane in Iran ARTICLE INFO is about 7 million t year-1 and the average Article history: -1 Received: 8 October 2015 of sugarcane yield is about 70 t ha and Accepted: 23 Jun 2016 cultivated area is about 100000 ha (Anon, E-mail addresses: [email protected], [email protected] 2011). Van der Merwe et al. (2003) reported (D. Khodadadi Dehkordi) * Corresponding author that the water use efficiency of sugarcane

ISSN: 1511-3701 © Universiti Putra Malaysia Press D. Khodadadi Dehkordi in alternate furrow irrigation method was improved sucrose content and commercial better than other irrigation methods with cane sugar compared to seeding density of sugar yield of 14.5 t ha-1 obtained from that 75000 setts ha-1. Patel et al. (2005) reported method. Meanwhile, Sheini-Dashtgol (2007) that higher seeding densities increased reported that alternate furrow irrigation the commercial cane sugar. The current method consumed less water than current cultivation of sugarcane in Iran is in the irrigation methods. Noori et al. (2008) form of double-row planting by hilling up reported that by decreasing furrow depth, activities. Thus, this research was conducted water application efficiency of sugarcane to investigate the effects of planting density increased. Bull and Bull (2000) reported on variation of water application efficiency, that a method for increasing sugarcane water use efficiency and crop yield. The production was higher density planting, in purposes of this study were to introduce the form of double-row planting. Seeruttun the triple-row planting of sugarcane and and Ismael (2003) and Ismael et al. (2007) compare its advantages with those of the showed that sugarcane production increased double-row planting in Iran. with double-row planting. Songsri et al. (2009) reported that water application MATERIALS AND METHODS efficiency of sugarcane can be increased Geographical Location and Weather by having an extended root system in Characteristics drought stress conditions. Smith et al. This study was conducted in farms located (2005) reported that sugarcane cultivars in the tropical climate of northwest Shush that have extended root systems, are more County in the Khuzestan province of Iran. resistant to drought stress. There is a Two different farms (labelled as A-01 farm contradiction regarding the effect of row and B-01 farm, respectively) were selected spacing and seeding densities on the quality for the purpose of this experiment. Triple- parameters such as Brix, sucrose contents, row planting was done in A-01 farm and juice extraction and commercial cane sugar, double-row planting was done in B-01 farm. etc. (Sharar et al., 2000; Asokan et al., 2005; The irrigation water of the farms is from El-Geddawy et al., 2005; Pawar et al., 2005). Karkheh River. This research was carried Nevertheless, most of the studies agreed that out from October 2014 to October 2015. sugarcane quality was not affected by row Some weather parameters and Karkheh spacing and seeding densities (Asokan et al., River EC are shown in Table 1. 2005; El-Geddawy et al., 2005). In contrast, Pawar et al. (2005) reported that wider row Soil Characteristics and Sugarcane spacing improved the sucrose contents Variety and commercial cane sugar percentage. Composite samples of 5 random points were Similarly, according to Sharar et al. (2000), taken from 0-30, 30-60, 60-100, 100-150 -1 higher seeding density (100000 setts ha ) and 150-200 cm depths of the cultivated

542 Pertanika J. Trop. Agric. Sci. 39 (4) 541 - 550 (2016) Effect of Higher Density Planting on Irrigation Water Use and Yield of Sugarcane

Table 1 Some weather parameters and Karkheh River EC

Month Mean Mean Mean of Mean Mean Sum Sum of EC of (2014- of min of max temperature of min of max of sunshine Karkheh 2015) temperature temperature (oC) humidity humidity rainfall hours River (oC) (oC) (%) (%) (mm) (dS m-1) October 20.0 39.1 29.8 17.7 58.3 0 287.60 1.69 November 14.4 28.1 20.3 37.1 75.7 25.8 178.60 1.37 December 5.2 20.9 13.1 35.5 81.2 0 260.36 1.40 January 6.4 20.9 13.4 43.6 83.5 12.5 178.60 1.39 February 6.1 19.9 13.1 40.4 78.2 28.4 174.27 1.46 March 9.0 23.1 16.0 30.3 70.1 29 210.10 1.78 April 15.3 31.1 23.2 33.1 80.0 5.5 215.07 1.67 May 21.7 39.8 29.9 28.7 65.0 2.5 246.78 1.72 June 24.8 45.2 35.0 19.0 49.0 0 300.38 2.18 July 26.6 47.0 36.7 20.6 52.6 0 356.47 2.57 August 27.4 48.6 38.0 21.5 54.8 0 352.20 2.68 September 23.3 44.4 33.7 24.3 68.1 0 332.60 2.51 October 19.2 39.4 29.1 33.6 76.3 0 278.45 2.49

Table 2 Some physical and chemical properties of the soil before planting test

- - 2+ 2+ Depth (cm) Texture EC pH Hco3 CL Mg Ca (meq OM (dS m-1) (meq l-1) (meq l-1) (meq l-1) l-1) (%) 0-30 Silt Loam 2.00 8.27 0.10 1.4 13.6 13.5 0.51 30-60 Silt Loam 2.68 8.11 0.05 3.2 22.5 16.1 0.41 60-100 Silt Loam 2.74 8.10 0.06 7.5 21.1 17.3 0.25 100-150 Silt Loam 2.86 8.00 0.00 8.1 16.4 21.2 0.31 150-200 Silt Loam 3.03 8.04 0.05 7.6 17.1 21.2 0.31 land in the farms. The results are presented cuttings (see Figure 1). Space between in Table 2. The sugarcane variety used in this the rows in this step was 50 cm, whereas study was CP69-1062. It is a middle-mature the depth of small furrows was 14 cm; the variety tolerant to drought stress and suitable width of ridge in the triple-row planting was for cultivation in subtropical regions. This 130 cm, the width of ridge in the double- variety is sensitive to cold weather with row planting was 90 cm and the length of acceptable yield and sugar content. the furrows was 50 m (see Figures 3 and 4). After the furrower activities, cuttings Farming operations of sugarcane were put in small furrows A special furrow maker was used to create (Figure 2). Then, covering activities were three small furrows for the sugarcane done by using special covering apparatus.

Pertanika J. Trop. Agric. Sci. 39 (1): 541 - 550 (2016) 543 D. Khodadadi Dehkordi

Meanwhile, irrigation was done based on done according to Duncan’s multiple range soil moisture measurement at FC, PWP test (P < 0.05). and readily available moisture by sampling of root zone soil. Fertilisers were given RESULTS AND DISCUSSION according to the current fertilizer schedules Evaluation of Leaf Sheath Moisture and of the sugarcane farms. A-01 farm (triple- Leaf Nitrogen row planting) and B-01 farm (double-row Leaf sheath moisture content for both farms planting) were cultivated simultaneously was found to tbe equal and there was no and all the cultivation activities were significant difference between them (Table equal for both of farms. Moreover, the 3). In spite of the higher density planting measurements of ridge soil EC, leaf nitrogen in the triple-row planting, the leaf sheath and leaf sheath moisture content were moisture content was equal to that of the simultaneously done for both the farms. The double-row planting. number of cuttings in the triple-row planting was about 23000 per ha and this was about Table 3 15000 per ha in the double-row planting. Percent of leaf sheath moisture of Sugarcane in triple-row planting and double-row planting Besides, in A-01 farm (triple-row planting), three furrows with three Leaf sheath moisture Date replications were evaluated, and generally, Double-row Triple- row planting planting nine furrows including three groups of 2015/27/03 81.01 80.21 triplex furrows were therefore evaluated. 2015/10/04 80.31 80.34 These conditions were repeated exactly 2015/24/04 81.02 79.85 for B-01 farm (double-row planting). The 2015/06/05 79.75 79.52 comparison between two cultivations was 2015/20/05 80.42 79.01 according to the average data of nine furrows 2015/11/06 78.15 78.46 in every farm. The means comparison was 2015/24/06 78.84 78.03

Figure 1. Triple-row furrower

544 Pertanika J. Trop. Agric. Sci. 39 (4) 541 - 550 (2016) Effect of Higher Density Planting on Irrigation Water Use and Yield of Sugarcane

Figure 2. The sugarcane cuttings in triple-row planting

Figure 3. The width of ridge in triple-row planting

Figure 4. The width of ridge in double-row planting

Pertanika J. Trop. Agric. Sci. 39 (1): 541 - 550 (2016) 545 D. Khodadadi Dehkordi

Leaf nitrogen in both farms was found with the height of ridge in the double-row to be equal and there was no significant planting (25 cm). In other words, the lesser difference between them (Table 4). In height of ridge in the triple-row planting spite of the higher density planting in caused better entrance of water in the ridge triple-row planting, the leaf nitrogen was and reduced ridge soil EC. nearly equivalent to that of the double-row planting. Thus, it was not necessary to apply Evaluation of Qualitative Parameters of more nitrogen fertiliser for the triple-row CP69-1062 Cultivar planting (higher density planting) compared As shown in Table 5, there was no significant with the double-row planting. difference between the qualitative parameters of CP69-1062 cultivar in both farms (Table Table 4 Percent of leaf nitrogen in triple-row planting and 5). Therefore, sugarcane can be cultivated double-row planting in the farm with higher density although its quality parameters can be stable. Leaf nitrogen (%) Date Double-row Triple-row planting planting Evaluation of Plant Height and Stem 2015/27/03 2.05 2.19 Diameter 2015/10/04 2.22 2.00 The height of plant for both farms was 2015/24/04 2.03 1.94 observed to be nearly equal and there was 2015/06/05 2.02 1.75 no significant difference between them, 2015/20/05 2.08 1.78 whereas the tallest plant was relevant to 2015/11/06 1.61 1.56 2015/24/06 1.52 1.50 the double-row planting (313 cm) and the shortest was relevant to the triple-row planting (303.8 cm) numerically. Hence, Evaluation of the Ridge Soil EC in the triple-row planting with higher The soil EC of the ridge for both the farms density planting, the height of plant was not was nearly equal and there was no significant significantly shorter than that of the double- difference between them. The soil EC of row planting (see Figure 5). the ridge for the double-row planting was Similarly, the diameter of sugarcane -1 -1 4.1 dS m and this was 4.03 dS m for the stem for both the farms was nearly equal, and triple-row planting. Thus, despite the wider there was no significant difference between ridge which allows more salt to enter in the them. The largest diameter (2.28 cm) was triple-row planting compared to that of the associated with the double-row planting and double-row planting, the soil EC of the ridge the smallest diameter (2.07 cm) was noted for the triple-row planting was nearly equal for the triple-row planting numerically. with that of the double-row planting. This Thus, in higher density planting, the stem might be due to the less height of ridge in the diameter was not significantly smaller than triple-row planting (14 cm) in comparison that of the double-row planting.

546 Pertanika J. Trop. Agric. Sci. 39 (4) 541 - 550 (2016) Effect of Higher Density Planting on Irrigation Water Use and Yield of Sugarcane

Table 5 Qualitative parameters of CP69-1062 cultivar in triple-row planting and double-row planting

Farm Pol (%) Brix (%) Purity (%) Purity Quality Refined Coefficient Ratio Sugar (%) Triple-row planting 16.50 18.96 90.25 130.45 7.66 10.36 Double-row planting 16.55 18.68 90.26 130.46 7.64 10.39 Pol: The juice sucrose percent or Pol percent is the actual cane sugar present in the juice. Brix: Juice Brix refers to the total solids content present in the juice expressed in percentage. Purity coefficient: It refers to the percentage of sucrose present in the total solids content in the juice. Purity Percentage = (Pol Brix-1) 100

Number of Shoots and Stems Moreover, only strong stems were included The difference between the number of in the counts. Therefore, the number of shoots per m2 in the triple-row planting stems was less than the number of shoots. in comparison with double-row planting was significant at 5% level. The number Evaluation of Water Application Efficiency and Water use Efficiency of shoots in the triple-row planting was (WUE) 385.83, while the number of shoots in the The mean of water application efficiency double-row planting was 334.15. Hence, the in the triple-row planting was about 49.6%, number of shoots in the triple-row planting which was higher than the water application was higher than the number of shoots in efficiency of 45% in the double-row the double-row planting because of higher planting. The lower down-slope of A-01 number of cuttings. farm (triple-row planting) in comparison Meanwhile, the difference in the number with B-01 farm (double-row planting) had of stems per m2 in the triple-row planting in probably caused more water application comparison with the double-row planting efficiency in the triple-row planting. Figure was significant at 5% level. The number of 6 shows the variation of water application stems per m2 in the triple-row planting was efficiency in the period of 4 months. 265.42, while the number of stems per m2 in Water use efficiency in the triple-row the double-row planting was 208.25. Hence, planting was more than WUE in double-row the number of stems per m2 in the triple-row planting (Table 6). This is because less water planting was higher than the number of was consumed and more sugarcane and stems per m2 in double-row planting because sugar yield were produced in the triple-row of bigger number of cuttings and also the planting compared to that of the double-row higher number of shoots in the unit area. planting.

Pertanika J. Trop. Agric. Sci. 39 (1): 541 - 550 (2016) 547 D. Khodadadi Dehkordi

Table 6 Water use efficiency (WUE) in triple-row planting and double-row planting

Farm The volume The mean The mean of WUE of produced WUE of of consumed of produced produced cane Sugarcane (kg m-3) produced cane water (m3 ha-1) Sugarcane (t ha-1) sugar (t ha-1) sugar (kg m-3) Triple- row 26623.53 138.45 14.05 5.20 0.528 planting Double-row 29673.46 119 12.55 4.01 0.423 planting

Figure 5. The height of sugarcane in both farms

Figure 6. Water application efficiency in triple-row planting

548 Pertanika J. Trop. Agric. Sci. 39 (4) 541 - 550 (2016) Effect of Higher Density Planting on Irrigation Water Use and Yield of Sugarcane

EVALUATION OF THE MEAN YIELD shoots and number of stems per m2 in the OF SUGARCANE AND SUGAR triple-row planting than the double-row CONTENT planting was significant at 5% level. The The mean yields of sugarcane and sugar numbers of shoots and stems per m2 in the content in triple-row planting were 138.45 triple-row planting were higher because of t ha-1 and 14.05 t ha-1, respectively. These the increased numbers of cuttings and in the were numerically higher than the mean unit area that were higher than the numbers yields of sugarcane and sugar content in the of shoots and stems per m2 in the double- double-row planting of 119 t ha-1 and 12.55 row planting. Besides, water application t ha-1, respectively, although the difference efficiency, water use efficiency (WUE) and between them was not significant. The the mean yield of sugarcane and sugar in the reason was that the higher number of shoots triple-row planting were also higher than due to the increase in the number of cuttings those of the double-row planting, although in the triple-row planting compared to that the difference between them was not of the double-row planting. It is important significant. Actually, we can use yield data to explain that in the end of growing season from the triple-row planting to increase the of sugarcane, all the nine furrows in every mean yield of sugarcane and sugar in spite farm were harvested and the mean weight of higher density planting in comparison to of stems was considered as the sugarcane the double-row planting and also increase yield. Then, the harvested stems were sent to water application efficiency and water use laboratory to determine the qualitative and efficiency of sugarcane and cultivate more quantitative characteristics of sugarcane. lands in arid and semi-arid regions with After extracting stems by using press lower water storage requirement. apparatus, the qualitative characteristics of sugarcane were identified by Saccharimeter REFERENCES and Polarimeter, followed by determining Annual Report of Sugarcane Research Centre. (2011). the other characteristics. Sugarcane development company. Ahvaz, Iran. (In Farsi).

CONCLUSION Asokan, S., Murthi, A. N., & Mahadevaswamy, M. The research work of the current study (2005). Effect of nitrogen levels and row spacing showed that in spite of higher density on yield, commercial cane sugar percentage and planting, there was no significant difference nitrogen uptake in different sugarcane varieties. Sugar Technology, 7(2-3), 44-47. between the triple-row planting and double- row planting on the ridge in terms of leaf Bull, T. A., & Bull, J. K. (2000). High density planting sheath moisture, leaf nitrogen, ridge soil as an economic production strategy: (b) theory and trial results. Proceedings of the Australian EC, quality parameters of the sugarcane Society of Sugar Cane Technology, 22, 104-112. cultivar, plant height and stem diameter. However, the difference in the number of El-Geddawy, I. H., Ahmed, Z. A., & Ahmed, A. M. (2005). Seeding rates and number of hoeing in

Pertanika J. Trop. Agric. Sci. 39 (1): 541 - 550 (2016) 549 D. Khodadadi Dehkordi

relation to yield and quality of sugarcane variety Seeruttun, S., & Ismael F. (2003). Evaluation of high G 85-37. The Egyptian Journal of Agricultural density planting (HDP) in Mauritius: preliminary Research, 83, 1225-1235. results. Revue agric sucr Ile Maurice, 80(3/1-3), 258-263. Ismael, F., Seeruttun, S., Barbe, C., & Gaungoo A. (2007). Improving cane productivity with Songsri, P., Jogloy, S., Holbrook, C. C., Kesmala, T., dual row planting in Mauritius. Proceedings Vorasoot, N., Akkasaeng C., & Patanothai, A. - International Society of Sugar Cane (2009). Association of root, specific leaf area Technologists, 26, 220-228. and SPAD chlorophyll meter reading to water use efficiency of peanut under different available Noori, M., Boroomand Nasab, S., & Kashkuli, H. soil water. Agricultural Water Management, A. (2008). Evaluation of furrow shape effect on 96(5), 790-798. irrigation efficiency and water use efficiency in sugarcane farms of Karoun Agro-Industrial. The Smith, D. M., Inman-Bamber, N. G., & Thorburn P. second National Conference on Irrigation and J. (2005). Growth and function of the sugarcane Drainage Networks Management. p.7. (In Farsi). root system. Field Crops Research, 92(1), 169- 183. Pawar, M. W., More, D. B., Amodkar, V. T., & Joshi S. (2005). Effect of intersettling spacing on Sharar, M. S., Ayub, M., Choudhry, M. A., Amin, sugarcane yield and quality. Sugar Technology, M. M. Z., & Khalid, M. M. (2000). Morpho- 7(1), 87-89. qualitative traits of sugarcane (Saccharum officinarumL.) varieties as influenced by seeding Patel, C. L., Patel, N. B., Pandva, H. V., Mali, S. C., density. Pakistan Journal of Biological Sciences, & Patel, M. N. (2005). Response of sugarcane 3(7), 1156-1157. genotypes to planting geometry and seed rates. Indian Sugar, 55(3), 23-28. Van der Merwe, J., Groenewald, S., & Botha, F. C. (2003). Isolation and evaluation of a Sheini-Dashtgol, A. (2007). Effect of alternate furrow developmentally regulated sugarcane promoter. irrigation on water application efficiency, water Proceeding of South African Sugar Technology use efficiency and qualitative and quantitative Association, 77, 146-149. situations of sugarcane in south of Ahvaz city. Journal of Agricultural Sciences and Natural Resources, 49, 45-57. (In Farsi).

550 Pertanika J. Trop. Agric. Sci. 39 (4) 541 - 550 (2016) Pertanika J. Trop. Agric. Sci. 39 (4): 551 - 563 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Effects of Feeding Different Levels of Low Crude Protein Diets with Different Levels of Amino Acids Supplementation on Layer Hen Performance

M. Tenesa1, T. C. Loh1,4, H. L. Foo2,3*, A. A. Samsudin1, R. Mohamad2 and A. R. Raha2 1Department of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 3Institute of Biological Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 4Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

ABSTRACT The use of synthetic amino acids (methionine, lysine, threonine) makes the formulation of diets with minimum levels of crude protein possible. The objective of this study was to determine the effects of low crude protein diet with amino acids supplementation on the performance, egg production, small intestine villus height and faecal LAB and ENT count in laying hens. A total of 144 16-week old layer hens of Hisex brown were assigned to four dietary treatments: 17.5% CP (control); (ii) 17.5% CP; (iii) 17% CP; and (iv) 16.5% CP supplemented with amino acids. Treatment group supplemented 17% CP was significantly higher (P<0.05) in egg production than other groups. Nonetheless, there were no significant differences (P>0.05) in egg quality. Results showed significant differences (P<0.05) in both villi height and crypt depth in duodenum, jejunum and ileum. The treatment supplemented with 17% CP showed a significant reduction (P<0.05) in faecal pH compared to the other groups. There was a significant different (P<0.05) in LAB and ENT count and LAB/ENT ratio among the dietary treatments. In conclusion, 17% CP treatment supplemented with amino acids demonstrated the best effects in ARTICLE INFO improving the hens’ egg production, small Article history: Received: 29 October 2015 intestine villus height and even promoting Accepted: 27 May 2016 beneficial effects of faecal microflora. E-mail addresses: [email protected] (M. Tenesa), [email protected] (T. C. Loh), Keywords: Methionine, lysine, egg production, egg [email protected], [email protected] (H. L. Foo), [email protected] (A. A. Samsudin), quality, microflora, morphology [email protected] (R. Mohamad), [email protected] (A. R. Raha) * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press M. Tenesa, T. C. Loh, H. L. Foo, A. A. Samsudin, R. Mohamad and A. R. Raha

INTRODUCTION high ambient temperature (Zeweil, 2011). Inclusion of synthetic amino acid in poultry Laudadio et al. (2012a) also stated that rations is becoming very common in the dietary protein level reduction in the poultry industry as chickens can only broilers’ rations under hot environmental utilise approximately 40% of the dietary conditions could be advantageous compared protein (NRC, 1994; Saki et al., 2012) to the conventional feeding programmes. and it has been proven that decreasing Thus, it has become more evident that crude protein level in the diet has many synthetic amino acid inclusion in diets advantages. However, commercial amino allows nutritionists to further decrease crude acid must be supplemented to meet the protein while more effectively meeting requirements of limiting amino acid as the the birds’ amino acid requirements for dietary protein is decreased (Mousavi et al., maintenance and tissue accretion (Gheisar 2013). In corn soybean meal diets for laying et al., 2011). Several investigators have hens, methionine, lysine and threonine are reported that the requirements for certain generally the first and next limiting amino essential amino acids for laying hens (Gheisar et al., 2011). Numerous studies increase with the increase in dietary level on low crude protein diet with amino acid of protein (Adeyemo et al., 2012). supplementation have been conducted Commercial amino acids must be on various classes of poultry essential used in order to meet the requirements of amino acids such as methionine, lysine, limiting amino acids due to the dilution threonine and tryptophan that have become of amino acids as the dietary protein is economically available in recent years due reduced. Inadequate knowledge about the to technological advances (Yakout, 2010). essential amino acid requirements of laying Recent reports indicated that promising hens, essential amino acid content of feed results in terms of maintaining birds’ ingredients, digestibility and bioavailability performance and maximising profitability of amino acids in feed ingredients and proper can be obtained by the use of low protein ratio between essential amino acids in low diets with supplementation of amino acid protein diets may have been the reasons for laying hens (Zeweil et al., 2011; Kashani for the inferior performance of hens fed a et al., 2014). low protein, amino acid-supplemented diet According to Rao et al. (2013), low (Mousavi et al., 2013). A few studies have protein diet with supplementation of been done with layer hen fed with 17.5% synthetic amino acid is able to minimise CP as control and 16.5 % CP for lower nitrogen excretion, production cost, level in early laying phase. The current trial intestinal disorder and amino acid excess. was carried out to determine how much Reducing dietary crude protein increased dietary crude protein could be reduced, the efficiency of utilisation of dietary crude while supplementing diets with constant protein and improved poultry tolerance to amount of synthetic amino acids such as

552 Pertanika J. Trop. Agric. Sci. 39 (4) 551 - 563 (2016) Effects of Low CP, AA Supplementation on Layer Performance

DL-methionine, L-lysine and L-threonine intake/egg mass. Live body weight gain on production performance, egg quality, (LBWG) was calculated as the difference villus height and crypt depth and faecal pH, between the initial and final body weights. LAB and ENT count of commercial laying At the end of the experiment, twelve birds of hens during production phase, i.e. from 20 each treatment were randomly selected and to 32 weeks of age. slaughtered for digesta and small intestine samples for further analysis. MATERIALS AND METHODS A total of 30 eggs per treatment were In present experimental study, 16 week-old randomly collected and used to determine birds were kept in Ladang 2 Poultry Unit, the egg quality. These measurements Universiti Putra Malaysia, Serdang. A total involved Haugh unit, eggshell thickness, of 144 Hisex Brown birds were randomly eggshell weight, yolk percentage and divided into four treatment groups with 6 albumen percentage. The Haugh unit was replications of 36 birds per treatment. The calculated using the egg analyser ORKA feeding period lasted for 17 weeks, starting EggAnalyzer®. Eggshell thickness was from the time when the birds were 16 measured in micrometer (μm) using a weeks of age and ending when they were digimatic micrometre on the large end, 32 weeks of age, with the addition of four equatorial region and small end. Meanwhile, weeks for acclimatisation purpose. Four albumen percentage was calculated as dietary treatments consisted of: (T1) 17.5% albumen weight over egg weight meanwhile CP (without supplementation of lysine and yolk percentage was calculated as yolk threonine; (T2) 17.5% CP diet; (T3) 17% CP weight over egg weight. and (T4) 16.5% CP diets supplemented with Different sections of small intestine were methionine, lysine and threonine. Diets were obtained for the morphometric analysis, as formulated to meet or exceed the minimum described by Choe et al. (2012). Segments amino acid standards, as recommended by of about 5cm long were removed from Hisex Brown Management Guide (2008), the small intestines (duodenum, jejunum and water was provided ad-libitum. The and ileum) at the following locations: (i) compositions of the basal diet are shown from gizzard outlet to the end of pancreatic in Table 1. loop, (ii) segment between pancreatic The individual feed intake was recorded loop and Meckel’s diverticulum, and (iii) weekly, while feed conversion ratio and segment between Meckel’s diverticulum live body weight gain were calculated at and ileo-caecal junction was removed. The the end of the study. Eggs from individual intestinal segments were flushed with 10% cages were collected daily and weighed. neutral buffered formalin solution and then The hen/day egg production and feed used for the morphometric analysis. Then, conversion ratio were calculated as the the segments were fixed in 10% neutral rate of production per hen per day and feed buffered formalin solution overnight.

Pertanika J. Trop. Agric. Sci. 39 (1): 551 - 563 (2016) 553 M. Tenesa, T. C. Loh, H. L. Foo, A. A. Samsudin, R. Mohamad and A. R. Raha

Table 1 Ingredients and composition of the experimental diets

Dietary Treatment1 Parameter T1 T2 T3 T4 Crude Protein (%) 17.5 17.5 17.0 16.5 Corn 46.30 46.34 47.00 47.24 Crude Palm Oil 3.45 3.45 3.44 3.52 Wheat Pollard 11.80 11.97 12.45 12.77 Soy Bean Meal 22.86 22.67 21.75 21.64 Fish Meal 4.30 4.34 4.00 3.24 Dicalcium Phosphate 0.80 0.79 0.85 0.99 Calcium Carbonate 8.00 8.00 8.00 8.00 Vitamin Premix2 0.55 0.55 0.55 0.55 Mineral Premix3 0.55 0.55 0.55 0.55 Salt 0.06 0.06 0.06 0.08 Antioxidant 0.55 0.55 0.55 0.55 Toxin Binder 0.55 0.55 0.55 0.55 L-Lysine 0.00 0.01 0.04 0.07 DL-Methionine 0.15 0.16 0.16 0.17 L-Threonine 0.00 0.01 0.03 0.05 Calculated Analysis 4 (%) Crude Protein 17.5 17.5 17.0 16.5 Energy kcal/Kg 2811 2811 2811 2811 Fat 5.65 5.67 5.66 5.61 Fiber 3.61 3.68 3.68 3.68 Calcium 3.62 3.63 3.61 3.66 Available Phosphorus 0.43 0.43 0.43 0.43 Arginine 1.10 1.09 1.05 1.02 Lysine 0.97 0.98 0.97 0.98 Methionine + Cysteine 0.73 0.74 0.73 0.73 Methionine 0.46 0.47 0.46 0.47 Threonine 0.64 0.65 0.65 0.65 Tryptophan 0.20 0.20 0.20 0.19

1 T1: 17.5% CP without essential amino acid supplementation (negative control); T2: 17.5% CP with amino acid supplementation (positive control); T3: 17% CP with amino acid supplementation; T4: 16.5% CP with amino acid supplementation. 2 Provided per kg diet: Fe 100mg; Mn 110mg; Cu 20mg; Zn 100mg; 12mg; Se 0.2mg; Co 0.6mg. 3 Provided per kg diet: Vitamin A 6,667 IU; Vitamin D 1,000 IU; Vitamin E 23 IU; Vitamin K3 1.33mg; Cobalamin 0.03mg; Thiamin 0.83mg; Riboflavin 2mg; Folic Acid 0.33mg; Biotin 0.03mg; Pantothenic Acid 3.75mg; Niacin 23.3mg; Pyridoxine 1.33mg. 4 The diets were formulated using Feedlive International Software (Thailand).

554 Pertanika J. Trop. Agric. Sci. 39 (4) 551 - 563 (2016) Effects of Low CP, AA Supplementation on Layer Performance

Intestinal samples were then excised, samples were repeated in triplicates and dehydrated in a tissue processing machine the LAB/ENT ratio was also calculated. (Leica Microsystems K. K., Tokyo, Japan) One gram of fresh faecal sample was mixed and embedded in paraffin wax. Sections of homogeneously with 9ml of deionised 4 mm were cut from each of the sample, distilled water in a sterile tube. The pH fixed on slides, stained with haematoxylin value was measured using a Mettler-Toledo and eosin, mounted and examined under pH meter with a glass electrode (Mettler- light microscopes. The distance from the Toledo, England). The faecal pH values tip of the villus to the villus crypt junction were determined as stated by Choe et al. represents villus height, while crypt depth (2012). was defined as the depth of the invagination All the collected data were subjected between adjacent villi. The villi height and to SAS statistical analysis. The means and crypt depth were measured using an image their standard errors were computed using analyser. One Way Analysis of Variance (ANOVA). Fresh faecal droppings were collected Duncan’s Multiple Range Test System was aseptically once a week using a sterile plastic used to compare the significant difference bag from every treatment group and stored between the treatments at P<0.05. The in the chiller prior to the laboratory analysis. results were expressed as the mean ± The faecal lactic acid bacteria (LAB) and standard error of mean. Enterobacteriaceae (ENT) population were determined using the method described by RESULTS Foo et al. (2003) and Loh et al. (2010). The growth performance for the layer hen The faecal samples were diluted in sterile fed low crude protein diet supplemented peptone water to 10% (w/v) and left at room with commercial amino acid is shown in temperature for an hour prior to tenfold Table 2. In particular, the LBWG and egg serial dilutions (v/v). Enumerations of LAB weight of hens had no significant differences were carried out on Lactobacillus agar DE (P>0.05) when fed with different levels of Man, ROGOSA and SHARPE (MRS-agar, low crude protein diets. Treatment 3 (17% Merck, KgaA, Darmstadt, Germany). The CP) had significantly higher (P<0.05) egg plates were incubated anaerobically at production and egg mass than other groups. 30ºC for 48 hours. ENT were spread plated As for the feed intake, there were significant and enumerated on Eosin-methylene-blue differences (P<0.05) among the treatment lactose sucrose agar plates (EMB-agar, groups. T4 had the lowest feed intake and Merck, KgaA, Darmstadt, Germany) and T1 had the highest feed intake. FCR was incubated aerobically for 24 hours at 37ºC. significantly different (P<0.05) among the The number of colony forming units (CFU) treatments, with T3 having the lowest FCR was expressed as the base 10 logarithm at 1.79. of CFU (log10 CFU) per gram. All the

Pertanika J. Trop. Agric. Sci. 39 (1): 551 - 563 (2016) 555 M. Tenesa, T. C. Loh, H. L. Foo, A. A. Samsudin, R. Mohamad and A. R. Raha

The egg quality of layer hen fed with diet with amino acid supplementation are low crude protein diet supplemented with presented in Table 4. The results showed amino acids is shown in Table 3. There were significant differences (P<0.05) in both no significant differences (P>0.05) in Haugh villi height and crypt depth in duodenum, units, yolk and albumen weight percentage, jejunum and ileum. Duodenal, jejunum eggshell weight and thickness among the and ileum villi height in T1 and T3 were treatment groups. significantly higher (P<0.05) than T2 and The intestinal villus height and crypt T4. The duodenal, jejenum and ileum crypt depth for birds fed with low crude protein depth showed similar results as the villi

Table 2 Production performance of layer hen at different levels of crude protein diets with amino acid supplementation

Dietary Treatment1 Parameter T1 T2 T3 T4 HDEP2 (%) 82.77 ± 0.04b 75.76 ± 0.05c 87.79 ± 0.01a 82.77 ± 0.04b Egg weight (g) 55.15 ± 0.64 54.99 ± 0.65 55.44 ± 0.59 55.50 ± 0.56 Egg mass (g/hen/day) 45.65 ± 0.02c 41.66 ± 0.03d 48.68 ± 0.01a 45.94 ± 0.02b Feed intake (g/hen/day) 87.67 ± 0.02 a 85.55 ± 0.05c 87.04 ± 0.04b 82.94 ± 0.04d LBWG3 (kg) 0.33 ± 0.03 0.29 ± 0.02 0.28 ± 0.03 0.28 ± 0.02 FCR4 1.92 ± 0.01b 2.06 ± 0.01a 1.79 ± 0.01d 1.81 ± 0.01c abcd Means in the same row not sharing a common superscript are significantly different (P<0.05). 1T1: 17.5% CP without supplemented essential amino acid (negative control) T2:17.5% CP with amino acid supplementation (positive control) T3: 17% CP with amino acid supplementation T4: 16.5% CP with amino acid supplementation 2HDEP: Hen day egg production 3LBWG: Live body weight gain 4FCR: Feed conversion ratio

Table 3 Egg quality of layer hen at different levels of crude protein diets with amino acid supplementation

Dietary Treatment1 Parameter T1 T2 T3 T4 Haugh units 50.31 ± 0.86 50.66 ± 0.78 50.60 ± 0.73 50.47 ± 0.92 Yolk weight (%) 25.53 ± 0.55 25.60 ± 0.09 26.68 ± 0.59 27.30 ± 0.92 Albumen weight (%) 65.43 ± 0.92 65.76 ± 0.28 64.01 ± 0.93 63.86 ± 0.68 Eggshell weight (g) 5.26 ± 0.03 5.10 ± 0.01 5.28 ± 0.15 4.87 ± 0.09 Eggshell thickness (mm) 0.59 ± 0.01 0.63 ± 0.01 0.59 ± 0.03 0.58 ± 0.02

1T1: 17.5% CP without supplemented essential amino acid (negative control) T2:17.5% CP with amino acid supplementation (positive control) T3: 17% CP with amino acid supplementation T4: 16.5% CP with amino acid supplementation

556 Pertanika J. Trop. Agric. Sci. 39 (4) 551 - 563 (2016) Effects of Low CP, AA Supplementation on Layer Performance height, whereby the values of T1 and T3 treatment groups. T3 also had the lowest were significantly higher (P<0.05) than T2 (P<0.05) ENT counts. As for the LAB/ENT and T4. For VH/CD ratio, however, there ratio, T3 had the highest ratio among all the were no significant differences (P>0.05) in treatment groups. duodenum, jejunum and ileum between all the groups. DISCUSSION The faecal pH, LAB and ENT counts are The egg weight and live body weight gain presented in Table 5. The lowest (P<0.05) (LBWG) were not significantly different faecal pH was observed in T3 and T4 among the treatment groups. This finding compared to the rest of the treatment groups. suggests that low protein is well fortified However, the treatment supplemented with essential amino acids and has adequate with 17% CP showed the lowest reduction level of total nitrogen that can maintain egg (P<0.05) in faecal pH compared to the other weight. Zou and Wu (2005) reported that the groups. There was a significant difference no response of egg weight to protein might (P<0.05) in the LAB and ENT count be due to over consumption of feed of hens and LAB/ENT ratio between the dietary fed with low protein diet. In addition, a study treatments. T3 showed significantly higher conducted by Khajali et al. (2007) revealed (P<0.05) LAB counts compared with other that egg weight was not significantly

Table 4 Villus height and crypt depth of layer hen at different levels of crude protein diets with amino acid supplementation

Dietary Treatment1 Parameter T1 T2 T3 T4 Villi height, µm Duodenum 668.59 ± 44.15a 149.99 ± 7.81b 726.29 ± 91.59a 182.55 ± 16.25b Jejunum 496.17 ± 31.80 a 107.24 ± 7.92 b 433.71 ± 52.87 a 127.86 ± 14.19 b Ileum 361.00 ± 21.88a 81.01 ± 3.00b 317.73 ± 36.44a 111.78 ± 14.60b Crypt depth, µm Duodenum 123.24 ± 11.17a 23.86 ± 2.31b 138.07 ± 15.27a 31.61 ± 4.30b Jejunum 105.32 ± 5.91a 29.93 ± 1.37b 110.69 ± 11.03a 29.70 ± 6.22b Ileum 90.51 ± 6.23a 21.70 ± 1.64b 74.18 ± 6.70a 24.12 ±3.59b Villi height to crypt depth ratio Duodenum 2.87 ± 0.74 2.98 ± 0.64 3.75 ±1.31 3.38 ± 1.04 Jejunum 2.43 ± 0.57 2.31 ± 0.47 2.22 ± 0.70 2.70 ± 0.83 Ileum 2.20 ± 0.53 2.47 ± 0.52 2.27 ± 0.71 2.99 ± 1.05 ab Means in the same row not sharing a common superscript are significantly different (P<0.05). 1T1: 17.5% CP without supplemented essential amino acid (negative control) T2:17.5% CP with amino acid supplementation (positive control) T3: 17% CP with amino acid supplementation T4: 16.5% CP with amino acid supplementation

Pertanika J. Trop. Agric. Sci. 39 (1): 551 - 563 (2016) 557 M. Tenesa, T. C. Loh, H. L. Foo, A. A. Samsudin, R. Mohamad and A. R. Raha

Table 5 Faecal pH and faecal LAB and ENT count of layer hen at different levels of crude protein diets with amino acid supplementation

Dietary Treatment1 Parameter T1 T2 T3 T4 Faecal pH 6.83 ± 0.09a 6.68 ± 0.05a 6.37 ± 0.06b 6.46 ± 0.04b LAB counts (log CFU/g) 5.71 ± 0.09c 5.91 ± 0.07b 6.31 ± 0.04a 6.03 ± 0.01b ENT counts (log CFU/g) 5.82 ± 0.02a 5.75 ± 0.01b 5.61 ± 0.02c 5.47 ± 0.03d LAB/ENT ratio 0.98 ± 0.01c 1.03 ± 0.01b 1.12 ± 0.01a 1.10 ± 0.01a abcd Means in the same row not sharing a common superscript are significantly different (P<0.05). 1T1: 17.5% CP without supplemented essential amino acid (negative control) T2:17.5% CP with amino acid supplementation (positive control) T3: 17% CP with amino acid supplementation T4: 16.5% CP with amino acid supplementation

affected by feeding reduced crude protein (Banarjee et al., 2013). Results of this diet. This result is in agreement with a experiment are also in disagreement with study conducted by Yakout (2010), which those reported by Liu et al. (2004) and Wu et observed no difference in egg weight and al. (2005), who claimed that reducing dietary body weight gain when birds were fed protein would reduce egg production. with low crude protein diets supplemented In both these diets supplemented with with amino acid. Meanwhile, no significant amino acids in this study, there were changes were observed in the initial, final increases in the feed intake when birds were and change in body weight of hens at 21 fed with 17% CP compared with those fed to 33 weeks of age fed different dietary with 17.5% CP diet supplemented with treatments, indicating that different dietary amino acid. Our results are in agreement with treatments with higher levels of protein Kidd et al. (2001), who reported that 20% and methionine are not sufficient to make CP diet with amino acids supplementation alteration in the body weights (Kumar et fed to broilers had significant increase al., 2012). In the current study, higher hen in feed intake than those fed a diet with day egg production was observed in the 23% CP. Feed intake was increased when T3 (17% CP) treatment group compared reduced protein diets were fed to chickens. to other dietary groups. This could be due This happened as the result of an amino to the basic amino acid and dietary protein acid “appetite” that occurred when feeding fulfilment in the dietary treatment. This amino acids marginal diets. In terms of was supported by a previous research that FCR, 17% CP had significantly lower value indicated better performance was observed compared to hens fed with higher level in animals fed with the low dietary crude of dietary protein treatments. According protein supplemented with amino acid to Novak et al. (2006), feed efficiency of

558 Pertanika J. Trop. Agric. Sci. 39 (4) 551 - 563 (2016) Effects of Low CP, AA Supplementation on Layer Performance birds improved as dietary protein decreased. Small intestine is the predominant site for Variation in FCR could also be attributed to protein digestion. Dietary protein not only the differences in egg mass as the resultant plays a major role in poultry growth and effects of feed intake (Adeyemo et al., reproductive performance, but also on the 2012). In a study conducted by Kumar et al. gastrointestinal tract features (Laudadio (2012), FCR was similar in hens fed medium et al., 2012). Maximum digestion and (16%) or high (18%) protein diets during 20 absorption are believed to occur as the to 43 weeks of age. villus height to crypt depth ratio increases In our study, egg qualities are not in weaned pigs. The increased villus height affected by reducing dietary crude protein. to crypt depth ratio produced an intestinal This was supported by a previous study structure more oriented to absorption (Xu et (Adeyemo et al., 2012), which revealed al., 2012). Villi are important structures in egg qualities were not affected by reducing the small intestine that involves mainly in protein level in poultry ration. There were nutrient absorption. Therefore, an increased no differences observed in Haugh units in villus height would increase the surface the early phase (20-43 weeks of age) of the area for nutrient absorption (Choe et al., production cycle when feeding low protein 2012). Lengthening of villi may increase diets (Novak et al., 2006). There was no total luminal villus absorptive area and effect of lysine on the variables Haugh subsequently result in satisfactory digestive unit, yolk and albumen percentage, as enzyme action and higher transport of reported by Torki et al. (2014), Figueiredo nutrients at the villus surface. However, et al. (2012) and Jardim et al. (2008, 2010). deeper crypt indicates faster tissue turnover Eggshell percentage and thickness were not and high demand for the renewal of the influenced by the dietary treatments, which villus, which suggests that the host’s could be explained by the fact that eggshell intestinal response mechanism is trying synthesis requires only a small amount of to compensate for normal sloughing or protein (Praes et al., 2014). atrophy of villi due to inflammation from The dietary nutrient utilisation pathogens and their toxins (Incharoen et efficiency is affected by gastrointestinal tract al., 2010). The villus height to crypt depth development and can be assessed through ratio is a very useful measure to estimate the measurements of villi height and crypt absorption capacity of the small intestine. depth (Swatson et al., 2002). Meanwhile, Higher villus height to crypt depth ratio is Tufarelli et al. (2010) found that the changes a positive aspect as it results in a decreased in gut morphology influenced the nutrient turnover of the intestinal mucosa and leads utilisation and performance of rabbits. The to lower maintenance requirement, which intestinal villi and cells in chickens are ultimately leads to a higher animal growth affected by dietary components (Incharoen rate (Tufarelli et al., 2010; Laudadio et al., & Yamauchi, 2009; Incharoen et al., 2009). 2012).

Pertanika J. Trop. Agric. Sci. 39 (1): 551 - 563 (2016) 559 M. Tenesa, T. C. Loh, H. L. Foo, A. A. Samsudin, R. Mohamad and A. R. Raha

The faecal pH, LAB count, ENT count CONCLUSION and LAB to ENT ratios were significantly The findings of this study indicate that different among the treatment groups. The reducing dietary protein level supplemented decrease in the faecal pH with low CP with essential amino acids in layer regimens provides a more favourable pH ration could be advantageous compared environment for digestive activity, allowing to conventional feeding programmes. for greater digestion and absorption of Furthermore, the diet containing 17% CP nutrients, particularly in the small intestine. with addition of synthetic amino acids offers The acidic environment favours growth of an advantage on improving the hen/day egg LAB. Results for the ENT count showed a production and shows positive effects such clear alteration of faecal microflora in low as increased small intestine villus height level of CP. It has been proven that increased and promoted beneficial effects of faecal LAB count could decrease ENT count (Loh microflora. These findings indicate that et al., 2007; Choe et al., 2012). According the response of layers to lowered levels of to Loh et al. (2007), LAB has antagonistic dietary crude protein can be used in feeding effect on harmful bacteria either in vitro or layer hens. in vivo studies. These beneficial bacteria have been shown to be able to reduce REFERENCES the faecal ENT count. Intestinal LAB is Adeyemo, G. O., Abioye, S. A., & Aderemi, F. A. able to produce antimicrobial compounds (2012). The effect of varied dietary crude protein such as bacteriocin and organic acids, levels with balanced amino acids on performance which are bacteriostatic against pathogenic and egg quality characteristics of layer at first bacteria. The undissociated organic acid laying phase. Food and Nutrition Sciences, 3(4), decreases the pH when it passes through. 526-529. The LAB competes against enteropathogens Choe, D. W., Loh, T. C., Foo, H. L., Hair Bejo, M., for nutrient, binding and receptor sites. & Awis, Q. S. (2012). Egg production, faecal They have strong inhibitory effects in pH and microbial population, small intestine morphology, and plasma and yolk cholesterol in preventing the adherence, establishment laying hens given liquid metabolites produced by and replication of pathogenic bacteria. The Lactobacillus plantarum strains. British Poultry LAB is important component for a balanced Science, 53(1), 106 - 115. microflora in the gastrointestinal tract. In Figueiredo, G. O., Bertechini, A. G., Fassani, E. J., this study, 17% CP has beneficial effects on Rodrigues, P. B., Brito, J. A. G., & Castro, S. F. laying hens, and this is most likely due to the (2012). Performance and egg quality of laying optimal intestinal environment as confirmed hens fed with dietary levels of digestible lysine by the faecal pH (Laudadio et al., 2012). and threonine. Brazillian Journal of Veterinary and Animal Sciences, 64(3), 743-750.

560 Pertanika J. Trop. Agric. Sci. 39 (4) 551 - 563 (2016) Effects of Low CP, AA Supplementation on Layer Performance

Foo, H. L., Loh, T. C, Law, F. L., Lim, Y. Z., Kufli, Kashani, S., Mohebbifar, A., Habibian, M., & Torki, C. N., & Rusul, G. (2003). Effects of feeding M. (2014). Effects of phytase supplementation Lactobacillus plantarum I-UL4 isolated form of low protein diets on performance, egg quality Malaysian Tempeh on growth performance, traits and bloodbiochemical parameters of laying faecal flora and lactic acid bacteria and plasma hens. Annual Research and Review in Biology, cholesterol concentrations in post-weaning rats. 4(4), 684-698. Journal of Food Science and Biotechnology, Khajali, F., Faraji, M., & Saeid, K.D. (2007). 12(4), 403 - 408. Effects of reduced- protein diets at constant Gheisar, M. M., Foroudi, F., & Shad, A. G. (2011). total sulphur amino acids: lysine ratio on Effect of using L-Threonine and reducing dietary pullet development and subsequent laying hen levels of crude protein on egg production in performance. American Journal of Animal and layers. Iranian Journal of Applied Animal Veterinary Sciences, 2(4), 89-92. Science, 1(1), 65-68. Kidd, M. T., Gerard, P. D., Heger, J., Kerr, B. J., Incharoen, T., & Yamauchi, K. (2009). Production Rowe, D., Sistani, K., & Burnham, D. J. (2001). performance, egg quality and intestinal histology Threonine and crude protein responses in broiler in laying hens fed dietary dried fermented ginger. chicks. Animal Feed Science and Technology, International Journal of Poultry Science, 8(11), 94(1), 57-64. 1078-1085. Kumar, S., Tyagi, P. K., Prasad, Y., Mandal, A. B., Incharoen, T., Khambualai, O., & Yamauchi, K. Tyagi, P. K., & Deo, C. (2012). Influence of (2009). Performance and histological changes of methionine and protein supplementation in the intestinal villi in chickens fed dietary natural corn-soya and wheat-soya based diets on laying zeolite including plant extract. Asian Journal of hens performance, egg size and egg quality in Poultry Science, 3(2), 42-50. early stage of egg production. Indian Journal of Poultry Science, 47(1), 54-59. Incharoen, T., Yamauchi, K., Erikawa, T., & Gotoh, H. (2010). Histology of intestinal villi and epithelial Laudadio, V., Passantino, L., Perillo, A., Lopresti, cells in chickens fed low crude protein or low G., Passantino, A., Khan, R. U., & Tufarelli, V. crude fat diets. Italian Journal of Animal Science, (2012). Productive performance and histological 9(4), 429-434. features of intestinal mucosa of broiler chickens fed different dietary protein levels. Poultry Jardim, F. R. M., Stringhni, J. H., Andrade, M. A., Science, 91(1), 265-270. Nunes, A. B., Leandro, N. S. M., Cafe, M. B., & Leandro, N. S. M. (2008). Egg quality, Laudadio, V., Dambrosio, A., Normanno, G., Khan, blood biochemical parameters and reproductive R. U., Naz, S., Rowghani, E., & Tufarelli, V. tract development for Lohmann LSL hens fed (2012a). Effect of reducing dietary protein increasing levels of digestible lysine. Acta level on performance responses and some Scientiarum. Animal Sciences, 30(1), 25-31. microbiological aspects of broiler chickens under summer environmental conditions. Avian Jardim, F. R. M., Stringhni, J. H., Andrade, M. A., Biology Research, 5(2), 88-92. Cafe, M. B., Leandro, N. S. M., & Carvalho, F. B. (2010). Levels of digestible lysine for Hy- Liu, Z., Wu, G., Bryant, M. M. & Roland Sr, Line W-36 hens in production period. Brazillian D. A. (2004). Influence of added synthetic Journal of Animal Science, 39(4), 787-795. lysine for first phase second cycle commercial leghorns with the methionine cysteine/lysine

Pertanika J. Trop. Agric. Sci. 39 (1): 551 - 563 (2016) 561 M. Tenesa, T. C. Loh, H. L. Foo, A. A. Samsudin, R. Mohamad and A. R. Raha

ratio maintained at 0.75. International Journal Saki, A. A., Harsini, N. R., Tabatabaei, M. M., of Poultry Science, 3(3), 220-227. Zamani, P., & Haghight, M. (2012). Estimates of methionine and sulfur amino acid requirements Loh, T. C., Law, F. L., Foo, H. L., Goh, Y. M., & for laying hens using different models. Brazilian Zulkifli, I. (2007). Effects of feeding a fermented Journal of Poultry Science, 14(3), 159-232. product on egg production, faecal microflora and faecal pH in laying hens. Journal of Animal and Swatson H., Gous,R., Iji, P., & Zarrinkalam, R. Feed Sciences, 16(3), 452-462. (2002). Effect of dietary protein level, amino acid balance and feeding level on growth, Loh, T. C., Thanh, N. T., Foo, H. L., Bejo, M. H., gastrointestinal tract, and mucosal structure of & Azhar, B. K. (2010). Feeding of different the small intestine in broiler chickens. Animal levels of metabolite combinations produced by Research, EDP Sciences, 51(6), 501-515. Lactobacillus plantarum on growth performance, faecal Microflora, volatile fatty acids and villi Torki, M., Mohebbifar, A., Ghasemi, H. A., & Zardast, height in broilers. Animal Science Journal, A. (2014). Response of laying hens to feeding 81(2), 205-214. low-protein amino acid-supplemented diets under high ambient temperature: performance, Mousavi, S. N., Khalaji, K., Jirdehi, A. G., & Foroudi, egg quality, leukocyte profile, blood lipids, F. (2013). Investigation on the effects of dietary and excreta pH. International Journal of protein reduction with constant ratio of digestible Biometeorology, 59(5), 575-584. sulfur amino acids and threonine to lysine on performance, egg quality and protein retention Tufarelli, V., Desantis, S., Zizza, S., & Laudadio, in two strains of laying hens. Italian Journal of V. (2010). Performance, gut morphology and Animal Science, 12(2), 9-15. carcass characteristics of fattening rabbits as affected by particle size of pelleted diets. NRC. National Research Council. (1994). Nutrient Archives of Animal Nutrition, 64(5), 373-382. requirements of poultry (9th ed.). National Academy Press Washington, D. C. Wu, G., Liu, Z., Bryant, M. M., & Roland, Sr. D. A. (2005). Performance comparison and nutritional Novak, C., Yakout, H. M., & Scheideler, S. E. (2006). requirements of five commercial layers strains The effect of dietary protein level and total in phase IV. International Journal of Poultry sulfur amino acid:lysine ratio on egg production Science, 4(4), 182-186. parameters and egg yield in Hy-Line W-98 Hens. Poultry Science, 85(12), 2195-2206. Xu, F. Z., Li, L. M., Liu, H. J., Zhan, K., Qian, K., Wu, D., & Ding, X. L. (2012). Effects Praes, M. F. F. M., Junqueira, O. M., Pereira, A. A., of fermented soybean meal on performance, Filardi, R. S., Duarte, K. F., Sgavioli, S., Alva, J. serum biochemical parameters and intestinal C. R., & Domingues, C. H. F. (2014). High-fibre morphology of laying hens. Journal of Animal diets with reduced crude protein for commercial and Veterinary Advances, 11(5), 649-654. layers. Brazillian Journal of Poultry Science, 16(2), 43-50. Yakout, H. M. (2010). Effects of reducing dietary crude protein with amino acids supplementation Rao, R. S. V, Kumari, K. N. R., Latha, T. S., Raju, M. on performance of commercial white leghorn L. N., & Panda, A. K. (2013). Influence of lysine layers during late production period. Egypt levels on performance of layers with sub optimal Poultry Science, 30(4), 975-988. protein in diet. International Journal of Food, Agriculture and Veterinary Sciences, 3(1), 17-25.

562 Pertanika J. Trop. Agric. Sci. 39 (4) 551 - 563 (2016) Effects of Low CP, AA Supplementation on Layer Performance

Zeweil, H. S., Abdalah, A. A., Ahmed, M. H., Marwa, Zou, S. G., & Wu, Y. Z. (2005). Effects of Protein R. S., & Ahmed. (2011). Effect of different level and Supplemental Fat on Performance of Laying of proteins and methionine on performance of Hens. International Journal of Poultry Science, Baheij laying hens and environmental pollution. 4(12), 986-989. Egypt Poultry Science, 31(2), 621-639.

Pertanika J. Trop. Agric. Sci. 39 (1): 551 - 563 (2016) 563

Pertanika J. Trop. Agric. Sci. 39 (4): 565 - 583 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Characterisation of Plant Growth-Promoting Bacteria from Kacip Fatimah (Labisia pumila) under Natural Tropical Forest

Yasmi, A.1, Radziah, O.1,2*, Hawa, ZE. J.3, Zainal Abidin, M. A.4 and Panhwar, Q. A.2, 5 1Department of Land Management, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 2Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 3Department of Crop Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 4Department of Plant Protection, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia 5Soil and Environmental Sciences Division, Nuclear Institute of Agriculture Tandojam, Sindh, Pakistan

ABSTRACT Medicinal plants play a major role in many cultures, not only as medicines, but also as trade commodities to fulfil the demands of distant markets. A study was conducted to characterise growth-promoting bacteria from two varieties of Kacip Fatimah (Labisia pumila) found at two different locations, Sungai Tekala, Semenyih, Selangor for Labisia pumila var. lanceolata and Bukit Slim Permanent Forest Reserve, Perak for Labisia pumila var. alata. Soil and plant samples were taken for the physico-chemical analyses and characterisation of the indigenous plant growth-promoting bacteria (PGPB). Both Labisia varieties were found to thrive in areas with quite similar soil chemical and physical properties in natural forest environments except for altitude, where Labisia pumila var. alata was found in higher elevation compared to Labisia pumila var. lanceolata. The soil in both places was found to be slightly acidic and low in nutrient content. Total bacterial population found on var. alata was higher than in var. lanceolata and the highest population was found in the root endosphere (8.68 × 107 cfu g-1 soil). Morphologically-isolated bacteria were circular in shape, with flat/raised elevation, entire margin, moist texture and smooth and glistening surface but varied in colour and size. Most of the bacterial strains showed several plant- growth promoting traits like plant-growth hormones (indole acetic acid (IAA)), N2 fixation and P solubilisation activities and beneficial ARTICLE INFO Article history: enzymes. Two of the bacterial isolates Received: 14 January 2016 Accepted: 29 August 2016 showing most of the beneficial properties

E-mail addresses: were identified asExiguobacterium sp. and [email protected] (Yasmi, A.), Stenotrophomonas sp. These PGPB have [email protected] (Radziah, O.), [email protected] (Hawa, ZE. J.), the potential to enhance the growth of Kacip [email protected] (Zainal Abidin, M. A.), [email protected] (Panhwar, Q. A.) Fatimah. * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A.

Keywords. Labisia pumila, bacterial population, affected by soil physical and chemical beneficial properties, tropical forest, medicinal plant properties, organic matter and P content, including cultural practices (Kim et al., INTRODUCTION 1998). Microorganisms play an important role in Several plant growth-promoting agriculture by supplying nutrients to plants bacteria (PGPB) have been known to and reducing the demand of chemical enhance growth of numerous plant species fertilisers (Cakmakci et al., 2006). Soil including some medicinal plants. Mixed microbes affect soil fertility through their bacterial inoculations were reported to have influence on organic matter turnover, mineral a higher success in improving plant growth immobilisation and dissolution and soil as they provide more balanced nutrition. aggregation (Davis & Abbott, 2006). Several They significantly improved the adsorption bacteria have ability to fix atmospheric N2 of nitrogen, phosphorus and other mineral and are known as diazotrophs (Ladha & nutrients (Bashan & de-Bashan, 2005). Reddy, 2000; Osivand et al., 2009). Poorly Kacip Fatimah (Labisia pumila) is one soluble P in soil can be solubilised by of the most popular traditional medicinal microorganisms and converted to soluble herbs found in the natural forests of forms by the process of acidification, Malaysia, Indonesia, Thailand, Indochina, chelation and exchange reactions (Chung Philippines and New Guinea (Wiart & et al., 2005). Soil microorganisms such as Wong, 2002; Ong, 2004). Kacip Fatimah the phosphate-solubilising bacteria (PSB) belongs to the family Myrsinaceae, and and arbuscular mycorrhizal fungi have the is a small wild sub-herbaceous plant with ability to mobilise P in soil and reduce inputs creeping stems growing naturally on the of chemical fertilisers (Arpana & Bagyaraj, forest floor of 20-1500 m altitude. Malaysia 2007). Species of Pseudomonas, Bacillus, is listed as the twelfth most bio-diverse Rhizobium, Burkholderia, Achromobacter, nation in the world (Foster, 2009) with a Agrobacterium, Microccocus, Aereobacter, diverse flora and fauna that are important for Flavobacterium and Erwinia are well- the phyto-pharmaceutical, phytocosmetic known as plant growth-promoting bacteria and nutraceutical industries. Herbal industry (PGPB). is one of the important industries in the Higher populations of microorganisms development of health food. Currently, there occur in the rhizosphere than non- is growing interest in medicinal plants as rhizosphere soil and they are metabolically sources of new pharmaceutical products more dynamic than those from other sources in Malaysia. Malaysia stands to gain from (Vazquez et al., 2000). Microorganisms strong growth of natural herbal products. are found everywhere in soils with various The growth rate of the herbal industry in diversities in their functionalities. The Malaysia is estimated at 15% per annum population of these microorganisms is and the industry is projected to have an

566 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest estimated market of RM15 billion and PGPB on the growth of Kacip Fatimah. RM29 billion, respectively, for the years The use of these beneficial bacteria may 2015 and 2020 (ECERDC, 2011). enhance sustainable production and reduce Kacip Fatimah is a popular herb dependency on chemical fertilisers. The among local women (Ezumi et al., 2007); literature records many beneficial effects it is frequently consumed by women of of PGPB in various crops but few studies reproductive age. It has been widely used have been conducted on Kacip Fatimah. by many generations for various ailments, Hence, the present study was carried out to especially those associated with childbirth isolate PGPB from the roots and shoots of and the feminine reproductive system. It Kacip Fatimah (Labisia pumila) obtained has essential astringent, antibacterial and from natural forests and to characterise their antifungal properties due to the presence of plant-growth promotion traits. rich natural phytoestrogens. Other bioactive phytochemical contents are flavonoid (rutin, MATERIALS AND METHODS quercetin, kaempferol, myricetin), phenols Plant and Soil Sampling (gallic acid, pyrogallol, caffeic acid), Kacip Fatimah plants were sampled from saponin, iron, benzoquinoid derivatives, two different locations i.e. Sungai Tekala, alkenylresorcinols, triperpenoid and other Semenyih, Selangor for Labisia pumila var. antioxidative compounds consisting of lanceolata and Bukit Slim Permanent Forest β-carotene, vitamin C and anthocyanin Reserve, Perak for Labisia pumila var. alata. (Foster, 2009; Norhaiza et al., 2009; Karimi A total of 20 plants from each location were et al., 2011). There is an increased demand sampled together with the adhered soil for for Kacip Fatimah but the plant source in bacterial isolation. Soil samples at a depth natural forests are becoming scarce due of 15 cm were taken using an auger for to extensive harvesting. There are a few nutrient analysis. efforts to propagate the plant using seeds, leaves or root cuttings, but the growth rate Locations and Climatic Conditions in unnatural habitats is very slow and not profitable. In general, this plant thrives best The location and climatic conditions of the in shady areas and non-waterlogged humus- natural forest areas where Kacip Fatimah rich soil. (Labisia pumila) were found were not Previous studies showed that PGPB can significantly different. Labisia pumila var. stimulate plant growth directly or indirectly lanceolata was found at elevation 66-116 in various ways through manipulation m above sea level while Labisia pumila of their surrounding environment to var. alata was found at the higher elevation support growth or through secretion of of 266-356 m (Table 1). The light intensity growth-promoting substances that enhance and relative humidity of the area for var. plant growth. Limited studies have been alata was slightly higher than that for the conducted on the beneficial effects of location for var. lanceolata but the soil and

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 567 Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A. leaf temperatures were higher in area of var. Enumeration and Isolation of PGPB lanceolata than that of var. alata. from Soil and Plant Bacteria were isolated from the non- Determination of Soil Chemical rhizosphere (bulk soil), rhizosphere and Properties endosphere (root, stem and leaf) of Kacip The pH of soil at ratio of 1:25 (soil:water) Fatimah plants using serial dilution and was determined using Beckman Digital plate count method (Johnson & Curl, pH meter. Nitrogen concentration in soil 1972). Colony morphology of bacteria on a was determined using Kjeldahl method nutrient agar plate (NA) was observed and (Bremner, 1996). The available P was characterised according to shape, elevation, determined by the method of Bray and Kurtz margin, texture, colour, surface and size (1945), while exchangeable K and cation within seven days of incubation. exchange capacity (CEC) were determined using the modified leaching method of Determination of Gram Reaction Chapman (1965). (Gram Staining and KOH Test) The Gram reaction was determined using Determination of Moisture in Soil potassium hydroxide (KOH) test (Halebian Ten grams of fresh soil were placed in a et al., 1981) and the Gram-staining modified crucible and dried in an oven at 105 oC until method of Hucker (1921). In a KOH test, a constant weight was obtained. The soil Gram-negative bacteria turn viscous/ moisture content was expressed as follows: mucoid, while there is no change in Gram- positive bacteria. Soil moisture % = Weight of water × 100 Dry weight of soil

Table 1 Chemical Properties of Soil Surrounding Kacip Fatimah Plant at 15cm Depth

Locationa Soil Characteristics Sungai Tekala Permanent Reserve Forest Bukit Slim (L.pumila var.lanceolata) (L.pumila var. alata) pH 4.00 ± 0.03 4.42 ± 0.43 Total nitrogen (%) 1.03 ± 0.04 0.99 ± 0.7 Extractable phosphorus (mg 5.23 ± 0.5 2.21 ± 0.18 kg-1) Exchangeable potassium (mg 20.68 ± 2.13 21.26 ± 1.49 kg-1) Cation exchange capacity 18.09 ± 1.42 10.27 ± 0.48 -1 (cmolc kg ) Soil moisture (%) 37.51 ± 5.13 35.08 ± 1.36 aAverage of five samples ± standard error of the mean

568 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest

Screening for Phosphate Solubilising 1 m of sodium chloride (NaCl). A positive Activity reaction was indicated by a visible halo zone The ability of bacterial isolates to solubilise around the bacterial colonies. phosphate was determined using the National Botanical Research Institute’s phosphate Screening for Starch Hydrolysis growth medium (NBRIP) modified method Starch hydrolysis of bacterial isolates was (Nautiyal, 1999). Four drops of 20 µl of carried out using the method of Seeley and bacteria from a nutrient broth was placed Dain (1960). The bacterial isolates were on a plate containing the NBRIP medium. incubated in a 250-ml Erlenmeyer flask The plate was incubated at a temperature of containing 100 ml of a nutrient broth (NB) o o 28 C±2 C for 24 hours. Positive growth for 24 hours before inoculating a starch agar was observed through the visible halo zone plate consisting of 5 g L-1 of starch, 14g surrounding the bacterial colony. L-1 of nutrient agar (NA) and a teaspoon of agar powder. After incubation for three Screening for Potential Nitrogen to five days at a temperature of 28oC ± Fixation Ability 2oC, the plate was flooded with a dilute The ability of the isolated bacteria to fix iodine solution (Gram’s iodine solution). nitrogen was qualitatively determined using The iodine was allowed to remain in the an N-free solid malate medium as used in the medium for 30 seconds and then decanted. modified method of Dobereiner and Pedrosa Hydrolysis activity was indicated by a clear (1987). Nitrogen fixing is indicated by a zone around the growth. change in agar colour from green to blue. Screening for Catalase Production Screening for Hydrolytic Enzyme Catalase production of bacterial isolates Reaction was determined according to the modified The ability of the isolates to produce method of Duke and Jarvis (1972). The enzymes viz. cellulase and pectinase was bacterial isolates were incubated in 250- determined using a carboxy methyl cellulose ml Erlenmeyer flasks containing a 100-ml (CMC) and citrus pectin media, respectively. nutrient broth (NB) for 24 hours before Two grams of substrate (CMC/citrus pectin) streaking on nutrient agar (NA) plates. were added into 1 l of nutrient agar (NA). The plates were incubated for 24-48 hours Bacterial cultures were dropped on a media at a temperature of 28 oC±2oC. Three or plate containing CMC and citrus pectin four drops of 3% hydrogen peroxide were substrates. The plates were incubated for added over the entire surface of the bacterial 24 hours at a temperature of 28 oC±2oC culture. Catalase production was determined before staining with 0.1 % Congo red for 30 by the presence of bubbling or foaming on minutes. The plates were then washed with the culture.

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 569 Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A.

Determination of Indole-Acetic Acid sample lysate was clear. For DNA binding, (IAA) Production 200µl of absolute ethanol was added to the IAA production by bacterial isolates was sample lysate and immediately vortexed for determined using the colorimetric method, 10 seconds. All the mixture was transferred modified from Gordon and Weber (1951). to the GD column and placed in a 2-ml The bacterial isolates were incubated in collection tube and centrifuged at 12 000 250-ml Erlenmeyer flasks containing a rpm for 2 minutes. The 2-ml collection 100-ml nutrient broth (NB) with addition tube containing the flow-through was of 0.2% of L-tryptophan. The bacterial discarded and the GD column was placed cultures were grown on a shaker for 24 in a new 2-ml collection tube. For DNA hours at a temperature of 28 oC±2 oC. Two washing, 400µl of WI buffer was added to mm of bacterial cultures were placed in the GD column and centrifuged at 12 000 an Eppendorf tube and centrifuged for rpm for 30 seconds. The flow-through was 7 minutes at 7000 rpm. One ml of the discarded and the GD column was placed supernatant from each isolate was mixed back in the 2-ml collection tube before with 2 ml of Salkowsky’s reagent. The 600 µl of wash buffer (ethanol added) was colour absorbance was determined using added to the GD column. The sample was a spectrophotometer at 535 ηm after 20-25 centrifuged at 12 000 rpm for 30 seconds. minutes. The flow-through was discarded and the GD column was replaced in the 2-ml collection Strain Identification Using Polymerase tube before being centrifuged again at 12 Chain Reaction (PCR) 000 rpm for 3 minutes to dry the column Bacterial identification was determined matrix. For DNA elution, the dried GD using 16S rRNA gene partial sequence column was transferred to a clean 1.5-ml analysis. DNA for bacterial isolates was microcentrifuge tube before 100 µl of extracted using the Genomic DNA Mini pre-heated elution buffer was added to the Kit (blood cultured cell) Bacteria Protocol centre of the column matrix. The mixture (GENEAID). The cell harvesting/pre-lysis was allowed to stand for 3 minutes until the for the isolates was conducted following the elution buffer was absorbed by the matrix Gram-negative bacteria procedure. Cultured before being centrifuged at 12 000 rpm for bacterial cells was transferred to a 1.5-ml 30 seconds to elude the purified DNA. For microcentrifuge tube and centrifuged for 1 sample mix preparations, Dream TAq Green minute at 10 000 rpm. Then, 200µl of GT PCR master mix-fermentas was used. The Buffer was added and the cell pellet was PCR amplification was carried out using suspended using a vortex. For the lysis the MJ Mini Personal Thermal Cycler, step, 200µl of GB Buffer was added to the Bio-Rad. The universal primer-F used was sample and vortexed for 5 seconds. The 27f, AGAGTTTGATCMTGGCTCAG sample was incubated at 70 ºC until the while the primer-R was 1492r,

570 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest

GGTTACCTTGTTACGACTT. The DNA broth with adjusted pH 3, pH 4, pH 5, pH was visualised using gel electrophoresis 6, pH 7, pH 8 and pH 9 accordingly on under UV light in a UV transluminater. The the orbital shaker. Two ml of the nutrient PCR products were purified using a GeneJet broth was taken after 12 hours, 24 hours PCR Purification Kit and the purified DNA and 36 hours of culturing and put into was stored at -20 ºC before being sent to Eppendorf tubes. The Eppendorf tubes were 1stBase DNA Sequencing Division for centrifuged at 7000 rpm for 7 minutes. The sequencing. The 16s rDNA gene sequence supernatant was taken out, leaving only the was compared with a nucleotide database bacterial cells. The cells were then washed of the National Centre for Biotechnology with 0.85% of phosphate buffered saline Information (NCBI) GenBank database (PBS). The final solution was read using a using the Basic Local Alignment Search spectrophotometer at 600 nm for an optical Tool (BLAST) (Altschul et al., 1990) at density reading. http://blast.ncbi.nih.gov/Blast.cgi. RESULTS Determination of Growth pH for Chemical Properties Bacterial Strains Different species of Labisia pumila were The bacterial strains were tested to determine sampled from two different locations. the suitable pH condition for growth. The Table 2 shows that the two species could strains were cultured in 100-ml Erlenmeyer grow in slightly acidic soil at pH 4.00±0.03 flasks containing a sterilised nutrient broth (var. lanceolata) and 4.42±0.43 (var. adjusted to pH 3, pH 4, pH 5, pH 6, pH 7, alata). For the soil nutrient content, total pH 8 and pH 9 for 24 hours at 28±2 oC on soil nitrogen in surrounding soil was not an orbital shaker. A ten-fold serial dilution significantly different for both species, was made and total bacterial population was which was 1.03±0.04 % for the area with determined using the drop-plate technique var. lanceolata sampled from Sungai Tekala and was calculated as follows: and 0.99±0.7 % for var. alata sampled from cfu = (Mean plate count) (Dilution factor) the Permanent Reserve Forest Bukit Slim. 0.1 ml Extractable phosphorus of var. lanceolata soil was higher (5.23±0.5 mg kg-1) than in Determination of Optimum Growth of var. alata soil but exchangeable potassium Bacterial Strains was higher in var. alata. (21.26 mg kg-1). The bacterial strains were tested to determine Cation exchange capacity in var. lanceolata optimum cell growth. The strains were soil was also greater at 18.09±1.42 cmolc -1 -1 cultured in 250-ml Erlenmeyer flasks kg compared to 10.27±0.48 cmolc kg for containing a 100-ml sterilised nutrient var. alata.

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 571 Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A.

Table 2 Location and Climatic Conditions of Kacip Fatimah (Labisia pumila) in a Natural Forest

Species Location and climatic conditions Light Leaf Relative Elevation Temperature N ( ) E ( ) intensity temperature humidity (m) (oC) (µmol m-2S-1) (oC) (%) ⁰ ⁰ L.pumila var. 3.0598- 101.8718- 66-116 10.2 ± 1.56 29.58 ± 0.25 27.44 ± 0.09 80.6 ± 0.75 lanceolata 3.0605 101.8722 (Sungai Tekala) L.pumila var. 3.9658- 101.4223- 266-356 11.4 ± 1.72 27.66 ± 0.11 25.62 ± 0.18 82.62 ± 0.49 alata 3.9671 101.4226 (Permanent Reserve Forest Bukit

Slim ) aAverage of five samples ± standard error of the mean

Total Bacterial Population medium except for one of the isolates, Total bacterial population of var. alata was which was KLR 06. Out of 20 isolates, only higher than var. lanceolata in most plant five strains were able to solubilise calcium parts except in phyllosphere (Table 3). The phosphate and none of the strains were able bacterial populations were highest in the to synthesise either cellulase or pectinase. root endosphere (8.68 × 107) for both species However, 15 of the strains isolated were and the lowest was in the non-rhizosphere able to synthesise catalase enzyme while of var. lanceolata and in phyllosphere of half of the strains were able to hydrolyse var. alata. starch (Figure 1). There were 17 bacterial strains isolated Plant Growth-Promoting Traits of from which six were isolated from the Bacterial Isolates rhizosphere, six from the roots, four from There were 20 bacterial strains isolated the soil and one from leaf of Kacip Fatimah from the soil and Kacip Fatimah plants (L. pumila var. alata). Results of the Gram obtained from Sungai Tekala, Selangor reaction showed that nine isolates were for var. lanceolata (Table 4). Six of them Gram-positive and eight were Gram- were isolated from the stem, five from the negative. All the bacteria were able to grow root, seven from the rhizosphere and two in the N-free solid malate medium except from the soil. Results of the Gram reaction two of the isolates (KAH03 and KAR18). showed that 16 isolates were Gram-positive Nine of the isolates were able to solubilise and four were Gram-negative. All bacteria calcium phosphate while the rest showed were able to grow in the N-free solid malate a negative result. For hydrolytic enzyme reaction, six of the isolates were positive for

572 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest

Table 3 Total Bacterial Populations from Non-Rhizosphere, Rhizosphere and Endosphere of Kacip Fatimah Total bacterial population (cfug-1 soil plant-1 dw)a Soil/plant parts Sungai Tekala Permanent Reserve Forest Bukit Slim (L. pumila var. lanceolata) (L. pumila var. alata ) Non-rhizosphere 1.04 × 104d 4.28 × 105c Rhizosphere 2.53 × 106b 3.28 × 107a Root endosphere 3.14 × 106b 8.68 × 107a Stem endosphere 2.94 × 104d 1.86 × 105c Phyllosphere 1.13 × 106b 1.57 × 105c aAverage of 19 samples Means within the same column followed by the same letters are not significantly different atP >0.05.

Table 4 Plant Growth Promoting Trait of Bacterial Isolates in Kacip Fatimah (L. pumila var. lanceolata)

Bacterial Gram N2-fixation ability Phosphate Hydrolytic enzyme Starch Catalase strain reaction (N- free solid solubilising reaction hydrolysis production malate medium) activity CMC Citrus pectin KLS01 + + - - - + + KLS02 + + - - - + + KLS03 - + + - - - + KLR05 + + - - - - - KLR06 + - - - - + + KLR07 + + - - - + - KLS09 + + - - - + + KLR10 - + + - - - + KLR11 + + - - - + - KLH12 - + + - - - + KLH13 - + + - - - + KLS14 + + - - - - - KLH19 + + - - - - - KLT22 + + - - - - + KLT23 + + - - - + + KLH24 + + - - - + + KLH25 + + + - - - + KLH27 + + - - - + + KLH29 + + - - - - + KLS35 + + - - - + +

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 573 Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A. both the carboxymethyl cellulase (CMC) and except for one strain (KAT14). The highest citrus pectin test but one isolate (KAR18) production was 5.84 µg/ml from KAH19, showed positive for carboxymethyl cellulase while the lowest was 1.14 µg/ml from (CMC) and negative for citrus pectin. Nine KAR12 isolated from L. pumila var. alata isolates produced catalase and 11 isolates (Figure 3). could hydrolyze starch (Table 5 and Figure 1). Bacterial Identification Using Polymerase Chain Reaction (PCR) Indole-Acetic Acid (IAA) Production Two of the isolates (KAR12 and KAR23) All the strains isolated from var. lanceoalata that possessed the most beneficial traits were able to produce IAA. The highest were taken for identification, while similar production was 5.77 µg/ml from KL S14, characteristics possessed by the isolates while the lowest was 1.45 µg/ml from were not chosen for isolation, such as KAT KL T24 strains isolated from L. pumila 13. Both are from L. pumila var. alata var. lanceolata (Figure 2). However, the sampled from the Permanent Forest Reserve bacterial strains isolated from the L. pumila Bukit Slim, Perak. BLAST output of isolate var. alata were also able to produce IAA, KAR12 after the 16s rRNA gene partial

Table 5 Plant Growth Promoting Trait of Bacterial Isolates in Kacip Fatimah (L. pumila var. alata)

Bacterial Gram N2-fixation ability Phosphate Hydrolitic enzyme Starch Catalase strain reaction (N- free solid solubilising reaction hydrolisis production malate medium) activity CMC Citrus pectin KAL01 + + + - - + - KAH02 + + - - - + + KAH03 + - + + + + - KAH05 - + + - - - - KAH06 + + - - - + + KAR08 - + + - - - - KAR09 - + - - - - + KAR12 + + + + + + + KAT13 + + + + + + + KAT14 + + - - - + - KAT15 - + - + + - + KAT16 + + + + + - - KAR17 - + - - - + + KAR18 + - - + - + - KAH19 - + + - - + + KAR20 - + - - - - + KAR23 - + + + + + -

574 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest sequence PCR procedure showed that the Growth of Bacterial Strains at Different strain belonged to Exiguobacterium sp. pH NIU-K2 (Table 6), while isolate KAR23 Growth of both bacterial strains was belonged to Stenotrophomonas sp. Y06 significantly affected by culture pH. The (Table 6) with 100% and 99% similarity, strains were able to grow at a pH range of 5-9 respectively. and could not grow at pH 3 (Figure 4). After 24 hours, growth of Exiguobacterium sp.

Table 6 BLAST Output of Isolates KAR12 and KAR23 (Sequences Producing Significant Alignment)

Isolates Accession Description Max Total Query E Max score score coverage value ident KAR12 HQ385945.1 Exiguobacterium sp. NIU- 939 1848 100% 0.0 100% K216S ribosomal RNA gene, partial sequence HQ385944.1 Exiguobacterium sp. NIU- 939 1848 100% 0.0 100% K416S ribosomal RNA gene, partial sequence HM047519.1 Exiguobacterium acetylicum 939 1848 100% 0.0 100% strain KNUC604 16S ribosomal RNA gene, partial sequence JF227852.1 Uncultured bacterium clone 939 1848 100% 0.0 100% ncd2602b09c1 16S ribosomal RNA gene, partial sequence JF71564.1 Uncultured bacterium clone 939 1848 100% 0.0 100% ncd1967d06c1 16S ribosomal RNA gene, partial sequence KAR23 JF220295.1 Uncultured bacterium clone 1064 2107 99% 0.0 99% ncd2628f03c1 16S ribosomal RNA gene, partial sequence JF163965.1 Uncultured bacterium clone 1064 2107 99% 0.0 99% ncd1904d07c1 16S ribosomal RNA gene, partial sequence HM161884.1 Stenotrophomonas sp. 1064 2107 99% 0.0 99% Y06(2010) 16S ribosomal RNA gene, partial sequence HM161883.1 Stenotrophomonas sp. 1064 2107 99% 0.0 99% Y05(2010) 16S ribosomal RNA gene, partial sequence HM161882.1 Stenotrophomonas sp. 1064 2107 99% 0.0 99% Y04(2010) 16S ribosomal RNA gene, partial sequence HM161866.1 Uncultured bacterium clone 1064 2107 99% 0.0 99% hdb_SIPB653 16S ribosomal RNA gene, partial sequence

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 575 Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A. -fixation ability on N-free solid malate medium. C (i) Positive result and C (ii) Negative result of phosphate solubilisation on medium. C (i) Positive result and -fixation ability on N-free solid malate 2 Plant-growth promoting trait of bacterial isolates in agar plates. A (i) Positive result, and A (ii) Negative result of Gram staining. B (i) Positive A (i) Positive result, and A Plant-growth promoting trait of bacterial isolates in agar plates. Figure 1. Figure and B (ii) Negative result of N media plate. D, Negative result (left) and positive (right) of catalase production on nutrient agar (NA) E, negative NBRIP result (right) of starch hydrolysis on agar media plate. F (i) positive and (ii) negative hydrolytic enzyme citrus pectin (CMC) plate.

576 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest

Figure 2. Indole-acetic acid (IAA) production by bacterial strains isolated from L. pumila var. lanceolata. Bars indicate standard error, n=3.

Figure 3. Indole-acetic acid (IAA) production by bacterial strains isolated from L. pumila var. alata. Bars indicate standard error, n=3.

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 577 Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A.

Figure 4. Cell growth of Exiguobacterium sp. (a) and Stenotrophomonas sp. (b) at different pH of culture solution. Bars indicate standard error, n=3. was optimum at pH 8 (Figure 4a). However, The bacterial population in most of the the growth of Stenotrophomonas sp. was plant parts except for phyllosphere was optimum at pH 6 and the highest growth higher in var. alata than in var. lanceolate, was observed after 24 hours culturing whereas the population in phyllosphere (Figure 4b). for var. lanceolata was higher than for var. alata perhaps because the leaf surface of DISCUSSION var. lanceolata was greater than that of var. The bacterial populations of Kacip Fatimah alata. Variety lanceolata lives on a higher differed between plant varieties and the surrounding where the temperature is 29.58 location from which they were collected. C±0.25 C) and leaf temperature is 27.44 ⁰ ⁰

578 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest

C±0.09 C as compared to 25.62 C±0.18 the enzyme, catalase. Catalase-positive ⁰C of var.⁰ alata. Higher leaf temperature⁰ organisms use catalase to deactivate may⁰ increase leaf dehydration, reduce peroxide radicals, thus allowing them to water availability and restrict phyllosphere survive unharmed within the host (Rao et bacterial populations as the bacterial al., 2003). populations within leaves are strongly Bacterial strains can directly influence correlated with water availability. Leaf shoot development by secreting IAA as water availability may have direct impact on a plant-growth promoting hormone. The bacterial growth as it can restrict bacterial IAA is one of the most physiologically growth (Freeman & Beattie, 2009). Most active auxins and several microorganisms of the bacterial strains were Gram-positive including PGPB have the ability to produce and showed the ability to grow under IAA through the L-tryptophan metabolism. fixed-nitrogen limiting conditions (N-free The IAA-producing bacteria use this solid medium), indicating that there was phytohormone to interact with plants as strong possibility that the strains could fix part of the colonisation strategy, including nitrogen. Nitrogen fixation is a biological phytostimulation and basal plant defense reaction where atmospheric N2 is converted mechanisms (Spaepen et al., 2007). Several into NH3; this is carried out by nitrogenase studies showed that IAA-producing bacteria enzyme complex, which is activated under promote plant growth and yield (Tsavkelova nitrogen-limiting conditions (Ladha & et al., 2007; Giongo et al., 2007). However, Reddy, 2000; Naher et al., 2011). The the growth of both bacterial strains was bacteria isolated from Kacip Fatimah could significantly affected by the pH culture. be said to be diazotrophs, which have the Generally ,strains were able to grow at the ability to fix nitrogen from the atmosphere pH range of 5-9 and could not grow at the and supply it to plants. low pH level of pH 3. Similar findings were Phosphate-solubilising bacteria (PSB) reported by Small et al. (1994). produce organic acids to solubilise inorganic The bacterial strain that possessed insoluble phosphorus. The starch hydrolysis the most beneficial traits tested was test indicated the ability of bacterial strains Exiguobacterium sp., which has been to produce certain exoenzymes, including characterised as a Gram-positive, facultative α-amylase, amylomaltase and α-glucosidase anaerobe that can grow profusely under (Hii et al., 2012). The exoenzymes are able aerobic conditions (Tatiana et al., to degrade starch molecules into subunits 2014). Several researchers found that that can be utilised as a source of carbon Exiguobacterium strains possess unique and energy for growth (Mutalib et al., 2012). properties for application in biotechnology, The catalase test was carried out to bioremediation, industry and agriculture determine the ability of bacterial cultures (Lee et al., 2009; Margesin & Feller, 2010; to degrade hydrogen peroxide by producing Kumar et al., 2010). This genus can also

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 579 Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A. survive in a wide range of temperatures from study also showed that there is a diverse -12 to 55 oC and environmental conditions growth of microorganisms in Kacip Fatimah and have been isolated from different plants. Most of the bacterial strains isolated habitats (Vishnivetskaya & Kathariou, 2005; from Labisia pumila showed plant growth- Vishnivetskaya et al., 2007). Hence, these promoting traits and several possessed two strains were able to survive well in a multiple traits. Two of the bacterial isolates wide range of temperatures in the Permanent showing most of the beneficial properties Reserve Forest Bukit Slim. Earlier studies were identified asExiguobacterium sp. and also showed that Exiguobacterium sp. can Stenotrophomonas sp. These PGPB have influence growth and nutrient uptake of the potential to enhance growth of Kacip seedlings, improve yield and enhance the Fatimah for future plant propagation. content of secondary metabolites of a salt- tolerant plant and wheat (Selvakumar et al., ACKNOWLEDGEMENTS 2010; Bharti et al., 2013). We wish to acknowledge Universiti Putra The other bacterial strain identified was Malaysia and the Ministry of Education Stenotrophomonas sp., which is a Gram- under Skim Latihan Akademik Bumiputera negative, aerobic bacterial strain that can (SLAB) for financial and technical support. be found in a wide range of environments particularly in close association with soil REFERENCES and plants (Kim et al., 2010; Ramos et Arpana, J., & Bagyaraj, D. J. (2007). Response of al., 2011). Stenotrophomonas sp. has been kalmegh to an arbuscular mycorrhizal fungus and isolated from various plants such as coffee a plant growth promoting rhizomicroorganism (Nunes & de Melo, 2006), dune grass at two levels of phosphorus fertilizer. (Dalton et al., 2004) and medicinal herb American-Eurasian Journal of Agriculture and (Lata et al., 2006). This strain can survive in Environmental Science, 2(1), 33–38. a broad range of temperatures from 4-35 oC, Bharti, N., Yadav, D., Barnawal, D., Maji, D., & Kalra, has potential to be used for bioremediation, A. (2013). Exiguobacterium oxidotolerans, phytoremediation, biocontrol and also as a halotolerant plant growth promoting rhizobacteria, improves yield and content of a plant-growth promoter (Liu et al., 2007; secondary metabolites in Bacopa monnieri (L.) Ryan et al., 2009). Pennell under primary and secondary salt stress. World Journal of Microbiol Biotechnology, CONCLUSION 29(2), 379–387.

Two varieties of Kacip Fatimah were Bray, R. H., & Kurtz, L. T. (1945). Determination of found to thrive in areas with quite similar total organic and available forms of phosphorus. soil chemical and physical properties in Soil Science, 59(1), 39–45. natural forest environments except for the Bremner, J. M. (1996). Nitrogen – Total. In Sparks, altitude where var. alata was found in higher D. L., Page, A. L., Helmke, P. A., Loeppert, R. elevation compared to var. lanceolata. The H., Soltanpour, P. N., Tabatabai, M. A., ... &

580 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest

Sumner, M. E. (Eds.). Methods of soil analysis, East Coast Economic Region Development Council. part 3 – Chemical methods (pp. 1085–1121). (2011). Investment and business opportunities Madison: Soil Science Society of America Inc. in the herbal and biotechnology cluster development in east coast economic region Cakmakci, R., Donme, F., Aydin, A., & Sahin, (ECER). Investment prospectus. Retrieved on F. (2006). Growth promotion of plants by 2012, July 11 from http://www.ecerdc.com.my/ plant growth promoting rhizobacteria under ecerdc/pdf/HerbalBiotech.pdf greenhouse and two different field soil conditions. Soil Biology and Biochemistry, Ezumi, M. F. W., Siti Amrah, S., Suhaimi, A. W. M., & 38(6), 1482–1487. Mohsin, S. S. J. (2007). Evaluation of the female reproductivity toxicity of aqueous extract of Chapman, H. D. (1965). Cation-exchange capacity. In Labisiapumila var. alata in rats. Indian Journal Alexander, M., Page, A., Miller, R., & Keeney, of Pharmacology, 39(1), 30–32. D. (Eds.). Methods of soil analysis – Chemical and microbiological properties (pp.891–901). Foster, S. (2009). Balancing nature and wellness, Agronomy 9. Malaysian traditions of ‘ramuan’ – The history, culture, biodiversity and scientific assimilation Chen, Y. P., Rekha, P. D., & Arun, A. B. (2006). of medicinal plants in Malaysia (Electronic Phosphate solubilizing bacteria from subtropical version). Journal of Tropical Medicinal Plants, soil and their tricalcium phosphate solubilizing 11(1), 1-11. abilities. Applied soil ecology, 34(1), 33–41. Freeman, B. C., & Beattie, G. A. (2009). Bacterial Chung, H., Park, M., Madhaiyan, M., Seshadri, S., growth restriction during host resistance to Song, J., Cho, H., & Sa, T. (2005). Isolation Pseudomonas syringae is associated with leaf and characterization of phosphate-solubilizing water loss and localized cessation of vascular bacteria from the rhizosphere of crop plants of activity in Arabidopsis thaliana. Molecular Korea. Soil Biology and Biochemistry, 37(10), plant-microbe interactions, 22(7), 857–867. 1970–1974. Giongo, A., Beneduzi, A., Ambrosini, A., Vargas, L. Dalton, D. A., Kramer, S., Azios, N., Fusaro, S., K., Stroschein, M. R., Eltz, F. L. … & Passaglia, Cahill, E., & Kennedy, C. (2004). Endophytic L. M. P. (2007). Plant growth promoting nitrogen fixation in dune grass (Ammophila bacteria isolated from the rhizosplane of Lupinus arenaria and Elymus mollis) from Oregon. FEMS albescens H. et Arn. Congresso Brasileiro de Microbiology Ecology, 49(3), 469–479. Ciência do Solo, 2007, Gramado. Resumos do Davis, J., & Abbott, L. (2006). Soil fertility in organic XXXI Congresso Brasileiro de Ciência do Solo. farming systems. In Kristiansen, P., Taji, A., & Porto Alegre: SBCS – Nucleo Regional Sul. Reganold, J. P. (Eds.). Organic agriculture: A Gordon, D. A., & Weber, R. P. (1951). Colorimetric global perspective (pp. 25–51). Ithaca, New estimation of indole-3-acetic acid. Plant York: Comstock Publishing Associates, USA. Physiology, 26(1), 192–195. Dobereiner, J., & Pedrosa, F. O. (1987). Nitrogen- Halebian, S., Harris, B., Finegold, S. M., & Rolfe, R. D. fixing bacteria in non-leguminous crop plants. (1981). Rapid method that aids in distinguishing Madison, Wisconsin: Science Tech. Inc.. Gram-positive from Gram-negative anaerobic Duke, P. B., & Jarvis, J. D. (1972). The catalase test – bacteria. Journal of Clinical Microbiology, A cautionary tale. Journal of Medical Laboratory 13(3), 444–448. Technology, 29(2), 203–204.

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 581 Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A.

Hii, S. L., Tan, J. S., Ling, T. C., & Ariff, A. (2012). Lee, S. H., Chung, C. W., Yu, Y. J., & Rhee, Y. H. Role in starch hydrolysis and potential industrial (2009). Effect of alkaline protease-producing applications. Enzyme Research, 1, 1–14. Exiguobacterium sp. YS1 inoculation on the community of waste activated sludge. Hucker, G. J. (1921). A new modification and Bioresource Technology, 100(20), 4597–4603. application of the Gram stain. Journal of Bacteriology, 6(4), 396–397. Liu, Z, Yang, C., & Qiao, C. (2007). Biodegradation of p-nitrophenol and 4-chlorophenol by Johnson, L. F., & Curl, E. A. (1972). Methods for Stenotrophomonas sp. FEMS Microbiol Letters, research on the ecology of soil borne plant 277(2), 150–156. pathogens (pp.178). Minneapolis, USA: Burgess Publishing Co. Margesin, R., & Feller, G. (2010). Biotechnological applications of psychrophiles. Environmental Karimi, E., Hawa, Z. E. J., & Sahida A. (2011). Technology, 31(8-9), 835–844. Phytochemical analysis and antimicrobial activities of methanolics extracts of leaf, stem Mutalib, A .A., Radziah, O., Shukor, Y., & Naher, and root from different varieties of Labisia U. A. (2012). Effect of nitrogen fertilizer on pumila Benth. Molecules, 16(6), 4438–4450. hydrolytic enzyme production, root colonization, N metabolism, leaf physiology and growth of rice Kim, H. B., Srinivasan, S., Sathiyaraj, G., Quan, L. inoculated with Bacillus sp. (Sb42). Australian H., Kim, S. H., Bui, T. P. N., Liang, Z. Q., Kim, Journal of Crop Science, 6(9), 1383–1389. Y. J., & Yang, C. (2010). Stenotrophomonas ginsengisoli sp. nov., isolated from a ginseng Naher, U. A., Radziah, O., Shamsuddin, Z. Hj., field. International Journal of Systematic and Halimi, M. S., Razi, M. I., & Rahim, K. A. Evolutionary Microbiology, 60(7), 1522–1526. (2011). Effect of root exuded specific sugars on biological nitrogen fixation and growth Kumar, A., Bhargava, P., & Rai, L. C. (2010). Isolation promotion in rice (Oryza sativa). Australian and molecular characterization of phosphate Journal of Crop Science, 5(10), 1210–1217. solubilizing Enterobacter and Exiguobacterium species from paddy fields of Eastern Uttar Nautiyal, C. S. (1999). An efficient microbiological Pradesh India. African Journal of Microbiology growth medium for screening phosphate- Research, 4(9), 820–829. solubilizing microorganisms. FEMS Microbiol Letters, 170(1), 265–270. Ladha, J. K., & Reddy, P. M. (2000). The quest for nitrogen fixation in rice. Proceedings of Norhaiza, M., Maziah, M., & Hakiman, M. (2009). Third Working Group Meeting on Assessing Antioxidative properties of leaf extracts of Opportunities for Nitrogen Fixation in Rice, popular Malaysian herbs, Labisia pumila. 1999, Aug 9-12 (p. 354). LosBanos, Laguna, Journal of Medicinal Plants Research, 3(4), Philippines. International Rice Research Institute. 217–223.

Lata, H., Li, X. C., Silva, B., Moraes, R. M., & Halda- Nunes, F. V., & de Melo, I. S. (2006). Isolation and Alija, L. (2006). Identification of IAA-producing characterization of endophytic bacteria of coffee endophytic bacteria from micropropagated plants and their potential in coffee degradation. Echinacea plants using 16s rRNA sequencing. WIT transactions on Biomedicine and Health. Plant Cell, Tissue and Organ Culture, 85(3), Environ Toxicology, 10, 293–297. 353–359.

582 Pertanika J. Trop. Agric. Sci. 39 (4) 565 - 583 (2016) Characterisation of PGPB from Natural Tropical Forest

Ong, H .C. (2004). Tumbuhan Liar: Khasiat Small, P., Blankenhom, D., Welty, D., Zinser, E. Ubatan dan Kegunaan Lain. Kuala Lumpur: & Slonczewski, J. L. (1994). Acid and base Perpustakaan Negara Malaysia. resistance in Escherichia coli and Shigella flexneri: Role of rpoS and growth pH. Journal Osivand, M., Azizi, P., Kavoosi, M., Davatgar, N.., of bacteriology, 176(6), 1729–1729. & Razavipour, T. (2009). Increasing phosphorus availability from rock phosphate using organic Tatiana, A. V., Chauhan, A., Layton, A. C., Pfiffner, matter in rice (Oryza sativa L.). availability S. M., Huntemann, M., Copeland, A. … Woyke, from rock phosphate. Philippine Agricultural T. (2014). Draft genome sequences of 10 Scientist, 92(3), 301–307. strains of the genus Exiguobacterium. Genome Announcements, 2(5), e01058-14. Ramos, P. L., van Trappen, S., Thompson, F. L., Rocha, R. C., Barbosa, H. R., de Vos, P., & Tsavkelova, E. A., Cherdyntseva, T. A., Klimova, S. Moreira-Filho, C. A. (2011). Screening for Y., Shestakov, A. L., Botina, S. G., & Netrusov, endophytic nitrogen-fixing bacteria in Brazilian A. L. (2007). Orchid-associated bacteria produce sugar cane varieties used in organic farming indole-3-acetic acid, promote seed germination and description of Stenotrophomonas pavanii and increase their microbial yield in response sp. nov. International Journal of Systematic and to exogenous auxin. Archives of Microbiology, Evolutionary Microbiology, 61(4), 926–931. 188(6), 655–664.

Rao, S. P. S., Yamada, Y., & Leung, K. Y. (2003). Vazquez, P., Holguin, G., Puente, M., Cortes, A. E. A major catalase (KatB) that is required for & Bashan, Y. (2000). Phosphate solubilizing

resistance to H2O2 and phagocyte-mediated microorganisms associated with the rhizosphere killing in Edwarsiella tarda. Microbiology, of mangroves in a semi-arid coastal lagoon. 149(9), 2635–2644. Biology and Fertility of Soils, 30(5-6), 460–468.

Ryan, R. P., Monchy, S., Cardinale, M., Taghavi, Vishnivetskaya, T. A., Siletzky, R., Jefferies, N., S., Crossman, L., Avison, L. B. … Dow, J. M. Tiedje, J. M., Katharious, S. (2007). Effect of low (2009). The versatility and adaptation of bacteria temperature and culture media on the growth and from the genus Stenotrophomonas. Nature freeze-thawing tolerance of Exiguobacterium Reviews Microbiology, 7(7), 514–525. strains. Cryobiology, 54(2), 234–240.

Seeley, H. W., & Dain, J. A. (1960). Starch hydrolyzing Wiart, C., & Wong, F. K. (2002). Medicinal Plants of streptococci. Journal of Bacteriology, 79(2), Southeast Asia (2nd ed.). Petaling Jaya: Prentice 230–235. Hall, Kuala Lumpur, Malaysia.

Selvakumar, G., Kundus, S., Joshi, P., Nazim Gupta, A. Yahya, A., & Azawi, S. K. A. (1998). Occurrence of D., & Gupta, H. S. (2010). Growth promotion of phosphate solubilizing bacteria in some Iranian wheat seedlings by Exiguobacterium acetylicum soils. Plant and Soil, 117(1), 135–141. 1P (MTCC8707), a cold tolerant bacterial strain from the Uttarakhand Himalayas. Indian Journal of Microbiology, 50(1), 50–56.

Pertanika J. Trop. Agric. Sci. 39 (1): 565 - 583 (2016) 583

Pertanika J. Trop. Agric. Sci. 39 (4): 585 - 595 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Impact of Daily Supplement of Probiotic on the Production Performance of Akar Putra Chickens

Hasan, S. A. Jawad1,4*, Lokman, I. H.1, Saad, A. Naji2, Zuki, A. B. Z.1 and Kassim, A. B.3 1Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2Department of Animal Resources, Faculty of Agriculture, University of Al-qadisiya, Iraq. 3Department of Animal Sciences, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4Department of Animal Production, Faculty of Agriculture, Baghdad University, Iraq

ABSTRACT This study was conducted to investigate the effects of prepared probiotic (PP) on the live body weight, weight gain, feed intake and feed conversion ratio in Malaysian chicken (Akar Putra). A total of 72 day-old Akar Putra chicks were reared for 12 weeks and randomly assigned to three dietary treatments (24 chicken/treatment), with 3 replications for each (8 chicken/replicate). The treatments consisted of a control group (T1), and the supplemented diet with probiotic in the second treatment was prepared at the rate 1:1 (1 kg of commercial broiler feed + 1 g PP). While the rate was 1:2 (1 kg of commercial broiler feed + 2 g PP) in the third treatment. Supplementing probiotic in both rates revealed significant improvement in terms of males’ and females’ growth rates, final live body weight, weight gain and feed conversion ratio. Based on the research findings, the best results were obtained when chickens received 1 g PP in males and 2 g in females.

Keywords: Akar Putra chicken, probiotic, production performance

INTRODUCTION Akar Putra is a Malaysian chicken species ARTICLE INFO Article history: that is characterised by a slow growth rate Received: 15 December 2015 compared to modern broiler chicken (Jawad Accepted: 27 May 2016

E-mail addresses: et al., 2015). Since 2006, antibiotics has [email protected] (Hasan, S. A. Jawad), been banned for use as feed additives by [email protected] (Lokman, I. H.), [email protected] (Saad, A. Naji), the European Union because the continued [email protected] (Zuki, A. B. Z.), [email protected] (Kassim, A. B.) use results in common problems such as * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press Hasan, S. A. Jawad, Lokman, I. H., Saad, A. Naji, Zuki, A. B. Z. and Kassim, A. B. development of drug resistant bacteria, the benefits of probiotic utilisation on imbalance of normal microflora and drug productive indices (Cavazzoni et al., 1998; residues in animal products (Chen et al., Jin et al., 1998). Dhama et al. (2012) and 2009). This necessitates the need for other Al-Gharawi (2012) also reported that use alternatives like probiotics. Probiotics of probiotics, including Lactobacillus sp., have become important as replacement improved the growth performance, feed feed additives (Steiner, 2006). A probiotics efficiency, immunity parameters and disease is a live microbial feed supplement that resistance. The major probiotic strains beneficially affects the host animal by include Lactobacillus, Saccharomyces, improving its intestinal microbial balance. Streptococcus and Aspergillus (Tannock et Probiotics have been classified as Generally al., 2001). At present, Bacillus, Lactobacillus Recognised as Safe (GRAS) by Food and and Saccharomyces are the major strains Drug Administration (FDA). The concept commonly used in broilers (Zhang et al., of their use relates to maintaining the 2005; Chen et al., 2009). equilibrium of intestinal microflora by The mixture of Lactobacillus acidophilus, the addition of beneficial microorganisms Bacillus subtilis, Bifidobacterium and (Goldin, 1998). Many studies have reported Saccharomyces cerevisia was used as

Table 1 Composition of basal diet

Basal Diet Items 1 to 22 d 23 to 84 d Corn 44.9 53.10 Wheat 18.0 15 Soybean meal (45%) 33 27 Mineral and vitamin premix 1 1 Oil 2 3 Limestone 0.8 0.6 Dicalcium phosphate 0.3 0.3 Total 100 % 100 % Calculated analysis Crude protein (%) 21.92 19.70 Metabolism energy (kilo calorie per kg. Diet) 2990 3100 Calcium (%) 0.93 0.85 Phosphorus (%) 0.48 0.45 Methionine (%) 0.55 0.50 Lysine (%) 1.35 1.25 Methionine + Cysteine (%) 0.85 0.91 Folic acid 1.1 1.2

Calculated analysis according to NRC (1984).

586 Pertanika J. Trop. Agric. Sci. 39 (4) 585 - 595 (2016) Probiotic Effects on Production Performance of Akar Putra Chickens probiotic for the first time by Lokman et months (15th December 2014 to 15th March, al. (2015) when one and two gram of the 2015). A total of 72 one-day old Akar Putra prepared probiotic was fermented with chicks were randomly assigned as (CRD) the daily feed of Akar Putra chicken. chicks in the three experimental groups were The authors reported that noticeable fed, as follows: enhancement in the production parameters T1: Control group fed on dry feed was obtained especially for 2g of the (without probiotic supplementation). probiotic. Moreover, a few studies have T2: Fed on supplemented diet prepared investigated the production effectiveness at the rate 1:1 (1 kg of commercial broiler of adding a mixture of probiotics in the feed+ 1 g PP). chicken diet. Thus, the present study T3: Fed on supplemented diet prepared was undertaken to evaluate the effects of at the rate 1:2 (1 kg of commercial broiler daily supplementing probiotic including feed+ 2 g PP). Lactobacillus acidophilus, Bacillus subtilis, Each treatment group was replicated Bifidobacterium and Saccharomyces three times with 8 chicks per replicate. The cerevisia, with feed (without fermentation) birds were housed in the battery cages with on the production performance of Malaysian eight birds (4 males and 4 females) per chicken (Akar Putra). pen (5”x 4”x1.5”). Since the chicks were reared in the open house, stable temperature, MATERIALS AND METHODS humidity and constant light schedule were Preparation of Probiotic therefore provided, along with ad libitum access of water and feed throughout the Feed shown in Table 1 was offered ad experiment. It is important to note that no libitum the same diets (1–13 days: starter; vaccination was used during the whole 14 day-slaughter: finisher) with continuous experiment period. provision of water. Prepared probiotic (PP) was made at Sampling Procedure and Analytic Universiti Putra Malaysia (UPM). Each one Methods g of PP contained at least 109 CFU (Colony Body weight, weekly weight gain, feed Forming Unit) of Lactobacillus acidophilus, intake and feed conversion ratio for the Bacillus subtilis, Bifidobacterium and at males and females were recorded separately least 108 CFU of Saccharomyces cerevisia. from week 1 until week 12. Growth rate was calculated at the marketing age based Chicken Husbandry and Experimental Design on the formula proposed by Brody (1945). In the same regard, the variation ratio of The experiment was carried out at the the production performance parameters was poultry farm of the Faculty of Veterinary calculated based on the formula, which was Medicine in Universiti Putra Malaysia mentioned by Jawad et al. (2015). (UPM), Malaysia, for the period of three

Pertanika J. Trop. Agric. Sci. 39 (1): 585 - 595 (2016) 587 Hasan, S. A. Jawad, Lokman, I. H., Saad, A. Naji, Zuki, A. B. Z. and Kassim, A. B.

Statistical Analysis observed in T2 for males (1503.3g) and T3 Data generated from the present experiment for females (1274.7g). These findings are the were subjected to statistical analysis using opposite to the results described by Ahmad the General Linear Model (GLM) procedure (2004), and Yousefi and Karkoodi (2007). of SAS (2001) statistical software package. These authors reported that the production When significant differences were noted, parameters were not affected by the dietary means were compared by using Duncan’s probiotic and yeast supplementation. multiple range tests (1955). Alternatively, the results consistently showed that natural feed additives such as RESULTS AND DISCUSSION probiotic are very important materials that can improve, growth rate, daily weight gain, The effects of diet supplementation with feed efficiency utilisation and productive probiotic at the rate of (1 and 2g PP: 1 Kg performance (Wysong, 2003). food) on the mean weekly body weight (g) Total feed intake in males did not of males and females Akar Putra chicks significantly differ between the groups reared to 12 weeks of age are presented receiving probiotic and the control group in Tables 2 and 3. Supplementing 1 and (Table 4), corroborating some previous 2 g of PP caused improvement (P<0.01, results reported for feed intake at 21 days P<0.05) in the final body weight of males (Sato et al., 2002) and at 42 days of age and females chicken. The best results were (Mohan et al., 1996). Nevertheless, total

Table 2 Effects of diet supplementation with probiotic at the rate of (1 and 2g PP: 1 Kg food) on mean weekly body weight (g) and growth rate gauge of males Akar Putra chicks reared to 12 weeks.

Treatments Week T1 T2 T3 1 62±2.887 62.333±2.728 63.667±2.963 2 104±2.887b 128±2.082a 126.333±2.333a 3 150±4.041b 217±3.215a 210.333±3.48a 4 277±6.928b 303.333±4.807a 306±5.292a 5 345±11.547b 443±9.074a 443.333±10.138a 6 499±14.434b 582.667±12.468a 609±13.577a 7 610±9.815c 737.333±9.244b 772.667±8.686a 8 869±11.547b 890±10.693b 988±9.866a 9 1037.667±15.103 1033±16.197 1059±15.373 10 1165±19.053 1157.333±18.478 1219±18.193 11 1290±20.207 1323.333±18.782 1324±19.348 12 1390±20.785b 1503.333±19.359a 1484±19.925a Growth Rate 190.455±0.178b 191.837±0.242a 191.132±0.251ab • Mean values with common superscript in row differ significantly (P < 0.01). • Mean values at weeks 4 and 12 differ significantly (P<0.05). • The values of growth rate differ significantly (P<0.05).

588 Pertanika J. Trop. Agric. Sci. 39 (4) 585 - 595 (2016) Probiotic Effects on Production Performance of Akar Putra Chickens

Table 3 Effects of diet supplementation with probiotic at the rate of (1 and 2g PP: 1 Kg food) on mean weekly body weight (g) and growth rate gauge of females Akar Putra chicks reared to 12 weeks.

Treatments Week T1 T2 T3 1 61.667±3.756 63.733±3.813 65±4.041 2 104.2±3.062b 129.667±3.48a 128±3.786a 3 178.3±4.304b 218.333±4.333a 211.667±4.631a 4 276.667±6.642b 305.333±6.36a 307±6.083a 5 344.667±11.26b 444.667±10.414a 444.333±10.975a 6 468.333±13.86 517.667±14.146 507±14.434 7 516.667±9.528b 640±9.815a 626±8.963a 8 624.267±11.779c 810±11.547a 741.667±11.26b 9 714.667±17.61b 856±17.898a 863±17.039a 10 815.333±18.478b 968.333±17.629a 978±19.053a 11 876.667±19.919b 1075±20.207a 1124±19.348a 12 937.333±20.21c 1201±20.785b 1274.667±20.497a Growth Rate 186.155±0.522b 189.405±0.481a 189.328±0.52a • Mean values with common superscript in row differ significantly (P < 0.01). • Mean values at week 4 differ significantly (P<0.05).

Table 4 Effects of diet supplementation with probiotic at the rate of (1 and 2g PP: 1 Kg food) on weekly feed consumption (g) of males Akar Putra chicks reared to 12 weeks.

Treatments Week T1 T2 T3 1 44±4.041 44.333±3.48 43.667±3.756 2 82±2.887b 110.167±3.032a 105.667±2.603a 3 126±6.928b 174.333±6.36a 140±6.083b 4 196±5.196c 219±4.359b 254.333±4.631a 5 270±6.928a 254.333±4.807ab 242.667±5.044b 6 269±9.815b 218.333±9.244c 348±8.963a 7 407±11.547b 406±10.693b 504.333±10.975a 8 410±13.279c 279±12.423b 534.333±12.706a 9 500±12.124b 627.667±10.99a 352.333±11.552c 10 440±14.434b 386±13.577c 585.667±14.146a 11 534±16.166a 425.333±15.592b 375±15.308b 12 507±15.588a 410.333±15.015b 484±14.731a Total 3785±118.934 3554.833±109.406 3970±110.387 • Mean values with common superscript in row differ significantly (P < 0.01). • Mean values at weeks 5 and 12 differ significantly (P<0.05).

Pertanika J. Trop. Agric. Sci. 39 (1): 585 - 595 (2016) 589 Hasan, S. A. Jawad, Lokman, I. H., Saad, A. Naji, Zuki, A. B. Z. and Kassim, A. B.

Table 5 Effects of diet supplementation with probiotic at the rate of (1 and 2g PP: 1 Kg food) on weekly feed consumption (g) of females Akar Putra chicks reared to 12 weeks.

Treatments Week T1 T2 T3 1 44.1±4.128 44.667±3.756 44±4.041 2 82.3±3.15b 109.667±2.603a 106±2.887a 3 125.333±6.36b 174.667±6.642a 141±6.928b 4 195.667±4.91c 220±5.196b 253.667±4.096a 5 230.667±6.642 255.667±5.812 245±6.928 6 276.333±9.244a 244±8.963a 183±9.815b 7 248.333±10.975c 453.667±10.414a 304.667±9.597b 8 289.667±12.991b 433.667±11.319a 202±12.423c 9 266.667±11.837b 90.333±10.713c 426.333±11.552a 10 357.667±14.146b 501.333±13.017a 301±13.577c 11 260±15.308b 373±14.468a 374.667±15.026a 12 307.333±14.17b 400.333±13.346a 320.333±12.548b Total 2684.067±113b 3301±106.209a 2901.667±109.266b • Mean values with common superscript in row differ significantly (P < 0.01). • The total feed consumption value differ significantly (P<0.05). feed intake was slightly higher when 1 g gain indexes since almost the treatments probiotics were administered in females had similar feed intake. These findings are (Table 5), thus corroborating the previous similar to the results described by Jin et al. finding by Lokman et al. (2015). (1998), Besnard et al. (2000), and Ayanwale Tables 6 and 7 show that superiority in et al. (2006). The authors reported worse the weight gain for birds receiving probiotics feed conversion in the control group when than the control group starting from the compared to groups of broilers, and turkeys starter phase (1-21 days). These findings fed probiotics based on Lactobacillus sp are in contrast to the results reported in and Saccharomyces cerevisiae in the diets, previous trials by Fethiere and Miles (1987), respectively. Maiorka et al. (2001) and Sato et al. (2002). Birds fed probiotics had lower feed Moreover, the distinction continued from intake (p<0.01) associated to improve the the growing period until the marketing age. feed conversion in almost the evaluated Noticeable (p<0.01) enhancement in periods (p<0.01), which were decisive to the total feed conversion ratio was observed result in the high weight gain (p<0.01) in supplementing 1 g PP with diet in males seen in these birds. Although the significant and 2 g PP with diet in females. This differences in performance were observed improvement in feed conversion ratio was between these groups in the finishing phase the principal reason to improve the weight (36-84 days), the increase (p<0.05) in the

590 Pertanika J. Trop. Agric. Sci. 39 (4) 585 - 595 (2016) Probiotic Effects on Production Performance of Akar Putra Chickens

Table 6 Effects of diet supplementation with probiotic at the rate of (1 and 2g PP: 1 Kg food) on weekly weight gain of males Akar Putra chicks reared to 12 weeks.

Treatments Week T1 T2 T3 1 28±1.732 31±1.732 30±1.732 2 42±0c 65.667±0.667b 62.667±0.667a 3 46±1.155c 89±1.155b 84±1.155a 4 127±2.887a 86.333±1.667c 95.667±1.856b 5 68±4.619b 139.667±4.333a 137.333±4.91a 6 154±2.887b 139.667±3.48c 165.667±3.48a 7 111±4.619b 154.667±3.283a 163.667±4.91a 8 259±1.732a 152.667±1.453c 215.333±1.202b 9 168.667±3.844a 143±5.508b 71±5.508c 10 127.333±4.372b 124.333±2.404b 160±3.055a 11 125±1.155b 166±0.577a 105±1.155c 12 100±0.577c 180±0.577a 160±0.577b Total 1356±19.63b 1472±18.475a 1450.333±18.765a • Mean values with common superscript in row differ significantly (P < 0.01). • The total weight gain value differ significantly (P<0.05).

Table 7 EFFECTS of diet supplementation with probiotic at the rate of (1 and 2g PP: 1 Kg food) on weekly weight gain of females Akar Putra chicks reared to 12 weeks.

Treatments Week T1 T2 T3 1 28±1.732 31±1.732 30±1.732 2 42.533±0.742c 65.933±0.581a 63±0.577b 3 74.1±1.242c 88.667±0.882a 83.667±0.882b 4 98.367±2.36a 87±2.082b 95.333±1.667a 5 68±4.619b 139.333±4.055a 137.333±4.91a 6 123.667±2.603a 73±3.786b 62.667±3.48b 7 48.333±4.333b 122.333±4.333a 119±5.508a 8 107.6±2.272c 170±1.732a 115.667±2.333b 9 90.4±5.839b 46±6.351c 121.333±5.783a 10 100.667±0.882b 112.333±0.882a 115±2.082a 11 61.333±1.453c 106.667±2.728b 146±0.577a 12 60.667±0.333c 126±0.577b 150.667±1.202a Total 903.667±18.187c 1168.267±18.707b 1239.667±18.187a • Mean values with common superscript in row differ significantly (P < 0.01). • Mean values at week 4 differ significantly (P<0.05).

Pertanika J. Trop. Agric. Sci. 39 (1): 585 - 595 (2016) 591 Hasan, S. A. Jawad, Lokman, I. H., Saad, A. Naji, Zuki, A. B. Z. and Kassim, A. B.

Table 8 Effects of diet supplementation with probiotic at the rate of (1 and 2g PP: 1 Kg food) on weekly feed conversion ratio (g .feed/ g .gain) of males Akar Putra chicks reared to 12 weeks.

Treatments Week T1 T2 T3 1 1.566±0.048 1.426±0.035 1.451±0.043 2 1.952±0.069a 1.679±0.061b 1.687±0.058b 3 2.735±0.082a 1.957±0.046b 1.665±0.05c 4 1.543±0.006c 2.537±0.02b 2.659±0.015a 5 3.994±0.17a 1.822±0.024b 1.769±0.029b 6 1.746±0.031b 1.562±0.029c 2.1±0.013a 7 3.688±0.258a 2.63±0.123b 3.091±0.16ab 8 1.582±0.041c 1.826±0.064b 2.481±0.046a 9 2.965±0.031c 4.396±0.094b 4.998±0.234a 10 3.456±0.046b 3.103±0.056c 3.66±0.033a 11 4.27±0.09a 2.562±0.09c 3.569±0.107b 12 5.069±0.127a 2.279±0.076c 3.024±0.081b Total 2.79±0.047a 2.414±0.044b 2.736±0.041a • Mean values with common superscript in row differ significantly (P < 0.01). • Mean values at weeks 2 and 7 differ significantly (P<0.05).

Table 9 Effects of diet supplementation with probiotic at the rate of (1 and 2g PP: 1 Kg food) on weekly feed conversion ratio (g .feed/ g .gain) of females Akar Putra chicks reared to 12 weeks.

Treatments Week T1 T2 T3 1 1.569±0.051 1.436±0.042 1.461±0.051 2 1.938±0.105 1.664±0.05 1.683±0.055 3 1.689±0.058b 1.969±0.056a 1.684±0.066b 4 1.989±0.005c 2.529±0.014b 2.661±0.016a 5 3.41±0.135a 1.836±0.012b 1.785±0.013b 6 2.233±0.028c 3.348±0.055a 2.921±0.016b 7 5.263±0.7a 3.724±0.217b 2.579±0.201b 8 2.689±0.065a 2.55±0.041a 1.744±0.073b 9 2.958±0.062b 1.974±0.041c 3.521±0.074a 10 3.551±0.11b 4.464±0.131a 2.615±0.074c 11 4.232±0.152a 3.495±0.063b 2.566±0.098c 12 5.064±0.21a 3.176±0.092b 2.125±0.068c Total 2.968±0.066a 2.824±0.046a 2.339±0.054b • Mean values with common superscript in row differ significantly (P < 0.01). • Mean values at weeks 3 and 7 differ significantly (P<0.05).

592 Pertanika J. Trop. Agric. Sci. 39 (4) 585 - 595 (2016) Probiotic Effects on Production Performance of Akar Putra Chickens growing rate was enough to positively the growth in chickens are too complex to influence the performance of birds fed be explained only under univariate analysis probiotics in the total period of rearing because all related traits are biologically (1-84 days). Similar results were obtained correlated due to the pleiotropic effect of when fermented feed with probiotic in a dry genes and linkage of loci (Udeh & Ogbu, form was used as a daily diet of Akar Putra 2011). Consequently, and based on the view chickens (Lokman et al., 2015). The results point of animals genetic and improvements, of that experiment revealed remarkably the principal components such as growth significant (P<0.01) enhancement for rate and live body weight are simultaneously supplementing treatments than the control considered as a group of attributes, which group in all of the males’ and females’ may be used for selection purpose (Pinto et body weight, weight gain, feed intake al., 2006). and feed conversion ratio measurements. Based on the results of this research, it Furthermore, the best results were obtained can be concluded that supplementing 1 and in the chickens fed on dry feed mixture 2g of prepared probiotic caused dependent with 1g of probiotic. Moreover, such results improvement of the production performance corroborate the findings of Santoso et al. in Akar Putra chickens. Furthermore, the (1995), Yeo and Kim (1997), and Cavazzoni best results were obtained when chickens et al. (1998), but are nevertheless opposite received 1 g PP in males and 2 g in females. to those reported by Buenrostro and Kratzer The prominent influence of the probiotic (1983). was shown in the live body weight, as well With regard to the growth rate criteria as the growth rate traits. (see Tables 2 and 3), the males and females of T2 and T3 treatments outperformed the ACKNOWLEDGEMENTS control group in the growth rate criteria This research was supported by Universiti values. The males’ growth rate variations Putra Malaysia in 2015. The authors wish ratio of T2 and T3 than the males in control to thank Prof. Saad A. Naji, Poultry Science group was 1.315% and 0.486% respectively. scientist, Department of Animal Resources, Meanwhile, the variation ratios of the Dean of the Agriculture Faculty, University growth rate in females were 1.898% for of Al-qadisiya (Iraq), for the technical T2 and 1.335% for T3. Genetic and non- assistance. genetic factors are controlling growth trait in animals (Selvaggi et al., 2015). Growth in REFERENCES the domestic chicken is commonly measured Ahmad, I. (2004). Effect of probiotic (Protextin) by body weight and body conformation, on growth of broilers with special reference to which are the most important parameters. the small intestinal crypt cells proliferation. The techniques included in the control for (Msc. Dissertation). Department of Centre Biotechnology, University of Peshawar.

Pertanika J. Trop. Agric. Sci. 39 (1): 585 - 595 (2016) 593 Hasan, S. A. Jawad, Lokman, I. H., Saad, A. Naji, Zuki, A. B. Z. and Kassim, A. B.

Al-Gharawi, J. K. M. (2012). Effect of in ova probiotic Fethiere, R., & Miles, R.D. (1987). Intestinal-tract injection on production and immunological weight of chicks fed an antibiotic and probiotic. traits and intestinal flora of broilers. (Ph. D Nutrition Reports International, 36(6), 1305- Dissertation). Department of Animal Science, 1309. University of Baghdad. Iraq. Goldin, B. R. (1998). Health benefits of probiotics. Ayanwale, B. A., Kpe, M., & Ayanwale, V. A. (2006). The British Journal of Nutrition, 80(4), S203-7. The effect of supplementing Saccharomyces Jawad, H. S., Idris, L. H. B., Naji, S. A., Bakar, M. cerevisiae in the diets on egg laying and egg B., & Kassim, A. B. (2015). Partial Ablation quality characteristics of pullets. International of Uropygial Gland Effect on Production Journal of Poultry Science, 5(8), 759-763. Performance of Akar Putra Chicken. International Brody, S. (1945). Bioenergetics and growth; with Journal of Poultry Science, 14(4), 213-221.‏ special reference to the efficiency complex in Jin, L. Z., Ho, Y. W., Abdullah, N., & Jalaludin, S. domestic animals. New York: Herman Frasch (1998). Growth performance, intestinal microbial Foundation. populations, and serum cholesterol of broilers fed Besnard, J., Auclair, E., & Larbier, M. (2000). Effect of diets containing Lactobacillus cultures. Poultry yeast supplementation on productive parameters Science, 77(9), 1259-1265.‏ of turkeys. Proceedings of the World’s Poultry Lokman, I. H., Jawad, S. H., Zuki, A. B. Z., & Congress, August 20-24, 2000, Montreal, Kassim, A. B. (2015). Effect of Dry Probiotic Canada. Supplemented Fermented Feed on Production Buenrostro, J. L., & Kratzer, F. H. (1983). Effect of Performance of Akar Putra Chicken. International Lactobacillus inoculation and antibiotic feeding Journal of Poultry Science, 14(7), 420-426. of chickens on availability of dietary biotin. Maiorka, A., Santin, E., Sugeta, S. M., Almeida, J.G., Poultry Science, 62(10), 2022-2029. & Macari, M. (2001). Utilization of prebiotics, Cavazzoni, V., Adami, A., & Castrovilli, C. (1998). probiotics or symbiotics in broiler chicken Performance of broiler chickens supplemented diets. Revista Brasileira de Ciência Avícola, with Bacillus coagulans as probiotic. British 3(1), 75-82. Poultry Science, 39(4), 526-529. Mohan, B., Kadirvel, R., Natarajan, A., & Bhaskaran, Dhama, K., Latheef, S. K., Mani, S., Samad, H. M. (1996). Effect of probiotic supplementation A., Karthik, K., Tiwari, R., ... & Laudadio, V. on growth, nitrogen utilisation and serum (2015). Multiple beneficial applications and cholesterol in broilers. British Poultry Science, modes of action of herbs in poultry health and 37(2), 395-401. production-A review. International Journal of Pinto, L. F. B., Packer, I. U., De Melo, C. M. R., Pharmacology, 11(3), 152-176. Ledur, M. C., & Coutinho, L. L. (2006). Principal Chen, K. L., Kho, W. L., You, S. H., Yeh, R. H., Tang, components analysis applied to performance and S. W., & Hsieh, C.W. (2009). Effects of Bacillus carcass traits in the chicken. Animal Research, subtilis var. natto and Saccharomyces cerevisiae 55(5), 419-425. mixed fermented feed on the enhanced growth Santoso, U., Tanaka, K., & Ohtani, S. (1995). Effect performance of broilers. Poultry Science, 88(2), of dried Bacillus subtilis culture on growth, 309-315. body composition and hepatic lipogenic enzyme Duncan, D. B. (1955). Multiple ranges test and activity in female broiler chicks. British Journal Multiple F – test. Biometrics, 11(1), 1-42. of Nutrition, 74(4), 523-529.

594 Pertanika J. Trop. Agric. Sci. 39 (4) 585 - 595 (2016) Probiotic Effects on Production Performance of Akar Putra Chickens

Sato, R. N. Loddi, M. M. & Nakaghi, L. S. O. (2002). Wysong, D. L. (2003). Rationale for Probiotic Antibiotic use and / or probiotics as growth Supplementation. Retrieved from http://www. promoters in starter diets of chickens. Journal wysong.net/PDFs/probiotic.pdf. of Poultry Science, 4, 37. Yousefi, M., & Karkoodi, K. (2007). Effect of SAS. (2001). SAS users guide. Statistics version 6.12. probiotic Thepax® and Saccharomyces SAS institute, Inc, Cary, NC. cerevisiae supplementation on performance and egg quality of laying hens. International Journal Steiner, T. (2006). Managing gut health. Natural of Poultry Science, 6(1), 52-54. growth promoters as a key to animal performance. Nottingham University Press. Yeo, J., & Kim, K. I. (1997). Effect of feeding diets containing an antibiotic, a probiotic, or yucca Selvaggi, M., Laudadio, V., Dario, C., & Tufarelli, extract on growth and intestinal urease activity in V. (2015). Modelling Growth Curves in a broiler chicks. Poultry Science, 76(2), 381-385. Nondescript Italian Chicken Breed: an Opportunity to Improve Genetic and Feeding Zhang, A. W., Lee, B. D., Lee, S. K., Lee, K. W., Strategies. Journal of Poultry Science, 52, An, G. H., Song, K. B., & Lee, C. H. (2005). 288-294. Effects of yeast (Saccharomyces cerevisiae) cell components on growth performance, meat Tannock, G. W., Fuller, R., Sullivan, D. O., quality, and ileal mucosa development of broiler Svensson, J., Kullen, M. J., Klaenhammer, T. chicks. Poultry Science, 84(7), 1015-1021.‏ R., McCracken, V. J., ... & Crittenden, R.G. (2001). Probiotic: acritical review (3rd ed.). USA: Horizon Scientific Press.

Udeh, I., & Ogbu, C. C. (2011). Principal component analysis of body measurements in three strains of Broiler chicken. Science World Journal, 6(2), 11-14.

Pertanika J. Trop. Agric. Sci. 39 (1): 585 - 595 (2016) 595

Pertanika J. Trop. Agric. Sci. 39 (4): 597 - 607 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

The Effects of Fermented Feed on Broiler Production and Intestinal Morphology

Saad A. Naji1, I. F. B. Al-Zamili2, Hasan, S. A. Jawad3, 4* and J. K. M. Al-Gharawi2 1Department of Animal Resource, Faculty of Agriculture, Al-Qadisia University, Iraq 2Department of Animal Resource, Faculty of Agriculture, Al-Mthanna University, Iraq 3Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4Department of Animal Production, Faculty of Agriculture, Baghdad University, Iraq

ABSTRACT The present experiment was conducted to evaluate the effects of wet feed and fermented feed on the intestinal morphology and histology of broiler chicks. A total of 360 one day old Ross 308 broiler chicks were randomly assigned (CRD) into six treatment groups. Chicks were fed: (T1) Control group of dry feed; (T2) Fed on wet feed (1:1, feed: water); (T3) 25% fermented feed + 75% dry feed; (T4) 50% fermented feed + 50% dry feed; (T5) 75% fermented feed + 25% dry feed and (T6) 100%, fermented feed throughout the experimental period. Each treatment group was replicated three times using 20 chicks per replicate. The chicks were raised at a temperature and in humidity controlled room with a 24-h. constant light and had ad. libitum access to water and feed throughout the experimental period which lasted for six weeks. The results showed that all diets containing fermented feed, especially 100%, had dependent effects on the evaluated production characteristics. The feeds had significantly (P < 0.05) increased the relative weight and length of the small intestine. Likewise, it raised the villi height, crypt depth and the percentage of the villi height to crypt depth in duodenum, jejunum and ileum increased. In conclusion, the results of the current experiment indicated that fermented feed with probiotic would be beneficial economically since the broiler feed conversion ratio had been improved (2.4% in T2; 4.1% in T3; 5.3% in T4; 5.3% in T5 and 7.7%

ARTICLE INFO in T6) as a consequence to enhance their Article history: intestinal morphology. Received: 15 December 2015 Accepted: 21 July 2016 Keywords: Fermented feed, broiler, intestinal morphology, E-mail addresses: [email protected] (Saad A. Naji), histology [email protected] (I. F. B. Al-Zamili), [email protected] (Hasan, S. A. Jawad), [email protected] (J. K. M. Al-Gharawi) * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press Saad A. Naji, I. F. B. Al-Zamili, Hasan, S. A. Jawad and J. K. M. Al-Gharawi

INTRODUCTION use of antibiotics has resulted in common The main concept of fermenting feed with problems such as the development of drug- probiotic is to improve the quality of feed. resistant bacteria, imbalance of normal That application was practiced by Lokman microflora and drug residues in animal et al. (2015) when one and two grams of products (Chen et al., 2009). Since 2006, prepared probiotic was fermented with antibiotics have been banned for use as the daily feed of local Malaysian chicken feed additives in the European Union. (Akar Putra). The authors reported that Probiotics has therefore become important noticeable enhancement in the production as the replacement of feed additives (Steiner, parameters especially when 2 grams of 2006). It is a live microbial feed supplement probiotic was used. Fermentation process that beneficially affects the host animal could improve the nutritive value of the by improving its intestinal microbial soybean (Mathivanan et al., 2006), copra balance. After feeding of probiotics, meals (Hatta & Sundu, 2009) and tofu waste improvements in growth performance, (Rasud, 2009). Fermented feed influenced feed efficiency, immunity parameters and the bacterial ecology of gastrointestinal tract disease resistance have been reported (Al- and reduced the level of Enterobacteriaceae Gharawi, 2012). The major probiotic strains in the different parts of gastrointestinal tract include Lactobacillus, Saccharomyces, in pigs (Winser et al., 2001) and broiler Streptococcus and Aspergillus (Tannock et chicks (Heres et al., 2003). Lactobacilli al., 2001). At present, Bacillus, Lactobacillus and yeast in the kefir, which supplemented and Saccharomyces are the major strains in drinking water, significantly increased applied in broilers (Zhang et al., 2005; Chen the population of Lactobailli spp. and et al., 2009). total aerobic bacteria, thus decreasing Since very few investigations on the the population of Enterobactciaceae and fermentation of broiler feed with probiotic coliform in the goose intestine (Yaman et have been done, therefore, the present study al., 2006). Primarily fermented feed caused was undertaken to evaluate the effects a reduction of pathogenic bacteria, including of fermented feed and fermented feed Salmonella and Campylobacter in the with probiotic on the broiler production digestive tract, most particularly in the crop performance, as well as the intestinal and gizzard. Since the crop often ruptures morphology and histology of broiler during slaughter, the decrease level of chicken. pathogens in this area, in particular, makes contamination of meat less likely (Donkor MATERIALS AND METHODS et al., 2006). Preparation of the Fermented Feed Antibiotic has been used as feed additive A commercial broiler starter and finisher diet to improve growth performance and control (Table 1) was purchased from local markets. disease in animals. However, the continued The chicks were fed the starter diet during

598 Pertanika J. Trop. Agric. Sci. 39 (4) 597 - 607 (2016) Fermented Feed Effects on Broiler Production Performance the first three weeks, followed by the finisher T3: 25% fermented feed + 75% dry diet until 6 weeks. feed. Whole starter and finisher feed were T4: 50% fermented feed + 50% dry fermented with and without the addition feed. of probiotic. The feed was moistened with T5: 75% fermented feed + 25% dry water (1 liter water: 1 kg feed), placed in a feed. plastic tray and inoculated with prepared T6: 100% fermented feed. probiotic (PP) at the rate 10 grams of PP for Each treatment group was replicated each one kilogram of feed. Then, the plastic three times with 20 chicks per replicate. trays were kept and incubated in a small The chicks were reared in battery cages room for 48 h. at 37±2ºC. (1.5 × 1.0 m) with four tiers. The chicks The PP was purchased from laboratory were raised in a temperature and humidity of poultry technology at the Agriculture controlled room with a 24-h. constant light Faculty, University of Baghdad. According and had ad. libitum access to water and feed to the manufacture information label, throughout the experiment. each gram of PP contained at least 109 cfu of Lactobacillus acidophilus, Bacillus Sampling Procedure and Analytic subtilis, Bifidobacterium and at least 108 Methods cfu of Saccharomyces cervisia. Fermented Production parameters, which included final feed was characterised by high lactic acid body weight and weekly feed conversion concentration (up to 260 mmol/ kg feed) ratio, were recorded. At the end of the and moderate amounts of acetic acid (20- feeding trial, six birds per treatment were 30 mmol/ kg feed), high number of lactic selected at random and slaughtered for acid bacteria (Log 9-10 cfu/ g. feed) and sampling. The intestinal parts’ weight and pH of approximately 4.5-5.0, as described length and relative parts; weight and length by Cutlure et al. (2005). were calculated according to live body weight. Broiler Husbandry and Experimental The intestinal parts were separated Design carefully; duodenal loop, jejunum the The experiment was carried out at the middle sections of the intestine between the poultry research farm in the Faculty of duodenum and mackles diverticulum, and Agriculture, University of Al-Mothanna. ileum, which are located between mackles A total of 360 one-day-old Ross308 broiler diverticulum to the ileocecal junction. The chicks were randomly assigned (CRD) into cecum was also separated from the site of six experimental groups, which were daily ileocecal junction and their relative weight fed, as follows: and length were calculated. Samples of the T1: Control group fed on dry feed. intestinal parts were taken for histological T2: Fed on wet feed (1:1, feed: water). study. The samples were fixed at 10%

Pertanika J. Trop. Agric. Sci. 39 (1): 597 - 607 (2016) 599 Saad A. Naji, I. F. B. Al-Zamili, Hasan, S. A. Jawad and J. K. M. Al-Gharawi

Table 1 Composition of basal diet.

Basal Diet Items 1 to 22 d 23 to 35 d Corn 44.9 53.10 Wheat 18.0 15 Soybean meal (45%) 33 27 Mineral and vitamin premix 1 1 Oil 2 3 Limestone 0.8 0.6 Dicalcium phosphate 0.3 0.3 Total 100 % 100 % Calculated analysis Crude protein (%) 21.92 19.70 Metabolism energy (kilo calorie per kg. Diet) 2990 3100 Calcium (%) 0.93 0.85 Phosphorus (%) 0.48 0.45 Methionine (%) 0.55 0.50 Lysine (%) 1.35 1.25 Methionine + Cysteine (%) 0.85 0.91 Folic acid 1.1 1.2 Produced by Ghadeer Babylon, calculated analysis according to NRC (1994). buffered formalin and embedded in paraffin. Statistical Analysis Three micron sections were microtome cut Data generated from the present experiment and stained with haematoxylin and eosin. were subjected to statistical analysis Slides were measured by light microscopy using the GLM procedure of SAS (SAS, to measure crypt depth, villi height and villi 2001) statistical software package. When height/ crypt depth. Measurements of villus significant differences were noted, means height and crypt depth were taken only were compared using Duncan’s multiple from the sections where the plane of section range tests (Duncan, 1955). ran vertically from the top of villus to the base of an adjacent crypt. Values presented RESULTS are means from the seven samples of villi There was noticeable significant interaction measured from the tip to the crypt mouth for using fermented feeds with probiotic and seven associated crypts measured from compared to the control group. The results the crypt mouth to the base (Xu et al., 2003). revealed that using 25%, 50%, 75% and 100% fermented feed with prepared probiotic (PP) in the daily feed had dependent effects on the evaluated characteristics. Chicken in

600 Pertanika J. Trop. Agric. Sci. 39 (4) 597 - 607 (2016) Fermented Feed Effects on Broiler Production Performance

T6 achieved higher (P < 0.01) body weight with birds fed on the control dry feed. As (2092.87 g), followed by T5 (1971.44 g), T4 the percentages of the fermented feed in (1901.99 g), T3 (1862.97 g), T2 (1799.88 the diet increased, these parameters were g) and the control (1700.30 g). Meanwhile, significantly (P ≤ 0.05) increased. chicken received fermented feed with PP Dietary treatment had a significant (P ≤ exhibited lower (P < 0.05) feed conversion 0.05) effect on villus height, crypt depth, and ratio than the control group at the end of the villus height to the crypt depth ratio in the experiment (Table 2). duodenum (Table 5), jejunum (Table 6) and Morphological measurements of the ileum (Table 7). Birds fed on the fermented duodenum, jejunum, ileum and cecum are feed had higher (P ≤ 0.05) villi height and shown in Table 3 and Table 4. Birds fed on crypt depth in the duodenum, jejunum, and fermented feed had higher (P ≤ 0.05) relative ileum than birds fed control dry feed. These weight and relative length of duodenum, birds also had a higher (P ≤ 0.05) villi high to jejunum, ileum and cecum when compared the crypt depth ratio in all these three parts.

TABLE 2 The effect of 25%, 50%, 75% and 100% fermented feed with probiotic on the feed conversion ratio FCR (g food/ g weight gain) of broiler.

Week Treatments Mean FCR 1 2 3 4 5 T1 1.57± 0.01a 1.60± 0.03a 1.66± 0.03a 1.73± 0.02a 1.87± 0.03a 1.69± 0.04a T2 1.54±0.02ab 1.57± 0.02ab 1.63± 0.03ab 1.70± 0.04ab 1.80± 0.02b 1.65± 0.02b T3 1.52± 0.03ab 1.55± 0..01b 1.61± 0.01b 1.67± 0.01bc 1.75± 0.03c 1.62± 0.03b T4 1.51± 0.01b 1.54± 0.03b 1.60± 0.03bc 1.65± 0.03c 1.72± 0.02c 1.60± 0.02b T5 1.51± 0.02b 1.54± 0.02b 1.59± 0.01bc 1.64± 0..01cd 1.70± 0.01c 1.60± 0.03b T6 1.49± 0.02b 1.52± 0.03b 1.56± 0.02c 1.60± 0.02d 1.65± 0.03d 1.56± 0.01c Significant * * * * * * level T1: Control group fed on dry feed. T2: fed on wetting feed (1:1, feed: water). T3: 25% fermented feed + 75% dry feed. T4: 50% fermented feed + 50% dry feed. T5: 75% fermented feed + 25% dry feed. T6: 100% fermented feed throughout the experimental period. Mean values with different superscripts (a,d) in columns differ significantly (P < 0.05).

Pertanika J. Trop. Agric. Sci. 39 (1): 597 - 607 (2016) 601 Saad A. Naji, I. F. B. Al-Zamili, Hasan, S. A. Jawad and J. K. M. Al-Gharawi

Table 3 The effect of 25%, 50%, 75% and 100% fermented feed with probiotic on the relative weight of the small intestine and cecum (%) of broiler.

The relative weight Treatments The small Duodenum jejunum Ileum Cecum intestine T1 3.7±0.031d 0.5± 0.007d 1.46± 0.021d 1.7± 0.018d 0.45± 0.004c T2 4.1± 0.026c 0.69 ± 0.005c 1.5± 0.012c 1.84± 0.022c 0.50± 0.005c T3 4.80± 0.022b 0.84± 0.008b 1.8± 0.021b 2.0± 0.020b 0.57± 0.006b T4 4.90± 0.020b 0.8± 0.007b 1.8± 0.023b 2.13± 0.018b 0.6± 0.005b T5 4.96 ± 0.015a 0.8 ± 0.016b 1.9± 0.010b 2.16± 0.022b 0.6± 0.007b T6 5.7± 0.008a 1.14± 0.007a 2.17± 0.009a 2.4± 0.011a 0.72± 0.009a Significant * * * * * level T1: Control group fed on dry feed. T2: fed on wetting feed (1:1, feed: water). T3: 25% fermented feed + 75% dry feed. T4: 50% fermented feed + 50% dry feed. T5: 75% fermented feed + 25% dry feed. T6: 100% fermented feed throughout the experimental period. Mean values with different superscripts (a,d) in columns differ significantly (P ≤ 0.05).

Table 4 The effect of 25%, 50%, 75% and 100% fermented feed with probiotic on the relative length of the small intestine and cecum (cm/ 100 g body weight) of broiler.

The relative weight Treatments The small Duodenum jejunum Ileum Cecum intestine T1 9.97± 0.14c 1.66± 0.19d 3.96±0.40c 4.35 ± 0.42c 1.01 ± 0.1c T2 10.1± 0.18c 1.70 ± 0.21c 4.02± 0.43c 4.41 ± 0.47c 1.04 ± 0.11c T3 10.8±0.16b 1.86± 0.19b 4.18± 0.47b 4.78± 0.51b 1.14 ±0.13b T4 10.9± 0.21b 1.89± 0.17b 4.22± 0.37b 4.80± 0.43b 1.15 ±0.11b T5 10.9±0.16b 1.91± 0.2b 4.24± 0.5b 4.83± 0.48b 1.21 ± 0.17b T6 11.3± 0.21a 2.07 ± 0.18a 4.42 ± 0.22a 4.96 ± 0.32a 1.34 ± 0.14a Significant * * * * * level T1: Control group fed on dry feed. T2: fed on wetting feed (1:1, feed: water). T3: 25% fermented feed + 75% dry feed. T4: 50% fermented feed + 50% dry feed. T5: 75% fermented feed + 25% dry feed. T6: 100% fermented feed throughout the experimental period. Mean values with different superscripts (a,d) in columns differ significantly (P ≤ 0.05).

602 Pertanika J. Trop. Agric. Sci. 39 (4) 597 - 607 (2016) Fermented Feed Effects on Broiler Production Performance

Table 5 The effect of 25%, 50%, 75% and 100% fermented feed with probiotic on villus height, crypt depth (μm) and the ratio of villus height to crypt depth in the duodenal of broiler.

Villus height Crypt depth The ratio of villus height to Treatments (μm) (μm) crypt depth T1 117.42 ±1.15c 15.35 ± 0.18c 7.65 ± 0.05c T2 122.33 ± 2.06c 15.59 ± 0.21c 7.85 ± 0.05c T3 141.16 ± 1.23b 17.52 ± 0.19b 8.06 ± 0.04b T4 146.21 ± 2.33b 17.72 ±0.18b 8.25 ± 0.08b T5 150.05 ± 1.17b 17.97 ± 0.2b 8.35 ± 0.07b T6 166.36 ± 1.42a 18.82 ± 0.18ab 8.84 ± 0.05a Significant level * * * T1: Control group fed on dry feed. T2: fed on wetting feed (1:1, feed: water). T3: 25% fermented feed + 75% dry feed. T4: 50% fermented feed + 50% dry feed. T5: 75% fermented feed + 25% dry feed. T6: 100% fermented feed throughout the experimental period. Mean values with different superscripts (a,c) in columns differ significantly (P ≤ 0.05).

Table 6 The effect of 25%, 50%, 75% and 100% fermented feed with probiotic on villus height, crypt depth (μm) and the ratio of villus height to crypt depth in the jejunum of broiler.

Villus height Crypt depth The ratio of villus height to Treatments (μm) (μm) crypt depth T1 101.12 ±1.07c 14.32 ± 0.15c 7.06 ± 0.09c T2 103.67 ± 1.12c 14.44 ± 0.12c 7.18 ± 0.07c T3 113.22 ± 1.61b 15.18 ± 0.13b 7.46 ± 0.06b T4 115. 17 ± 1.22b 15.25 ± 0.21b 7.55 ± 0.05b T5 117.20 ± 1.16b 15.37 ± 0.2b 7.62± 0.06b T6 132. 18 ± 1.36a 16.41 ± 0.19a 8.05 ± 0.08a Significant level * * * T1: Control group fed on dry feed. T2: fed on wetting feed (1:1, feed: water). T3: 25% fermented feed + 75% dry feed. T4: 50% fermented feed + 50% dry feed. T5: 75% fermented feed + 25% dry feed. T6: 100% fermented feed throughout the experimental period. Mean values with different superscripts (a,c) in columns differ significantly (P ≤ 0.05).

Pertanika J. Trop. Agric. Sci. 39 (1): 597 - 607 (2016) 603 Saad A. Naji, I. F. B. Al-Zamili, Hasan, S. A. Jawad and J. K. M. Al-Gharawi

Table 7 The effect of 25%, 50%, 75% and 100% fermented feed with probiotic on villus height, crypt depth (μm) and the ratio of villus height to crypt depth in the ileum of broiler.

Villus height Crypt depth The ratio of villus height to Treatments (μm) (μm) crypt depth T1 41.14 ±0.53c 8.62 ±0.79c 4.77 ± 0.05c T2 41.77 ± 0.6c 8.70 ± 0.68c 4.80 ± 0.04c T3 50.18 ± 0.54b 9.81 ± 0.72b 5.12 ± 0.06bc T4 51.25 ± 0.65b 9.87 ± 0.71b 5.19 ± 0.07b T5 53.09 ±0.49b 10.11 ± 0.81b 5.25 ± 0.06ab T6 61.55 ± 0.56a 11.04 ± 0.64a 5.58 ± 0.05a Significant level * * * T1: Control group fed on dry feed. T2: fed on wetting feed (1:1, feed: water). T3: 25% fermented feed + 75% dry feed. T4: 50% fermented feed + 50% dry feed. T5: 75% fermented feed + 25% dry feed. T6: 100% fermented feed throughout the experimental period. Mean values with different superscripts (a,c) in columns differ significantly (P ≤ 0.05).

DISCUSSION was not affected by the dietary probiotic and Dependent effects on production parameters yeast supplementation. Some researchers were achieved by using 25%, 50%, 75% reported that when chicks were housed in a and 100% fermented feed with prepared clean environment, probiotic did not affect probiotic (PP) in the daily broiler feed. The performance (Anderson et al., 1999). positive effects of probiotics to the Akar Putra In the current study, the morphology and chicken also used Lactobacillus acidophilus, histology parameters of the digestive tract Bacillus subtilis, Bifidobacterium and parts were improved in the broiler chicks Saccharomyces cervisia with dry fermented feeding on fermented feed than the broilers feed (Lokman et al., 2015) and wet feeding on control diet. These results are fermented feed (Jawad et al., 2016). This similar to those reported by Chaing et al. result could be strongly supported when the (2010) and Xu et al. (2012). worst feed conversion ratio was reported for The villus height to crypt depth is a very the control group compared to the groups useful measure to estimate the absorption of layer and turkeys fed probiotics based capacity of the small intestine. Maximum on Lactobacillus sp. and Saccharomyces digestion and absorption are believed to cerevisiae in the diet (Besnard et al., 2000; occur as villus height to the crypt depth ratio Ayanwale et al., 2006; Lokman et al., 2015). increased (Chaing et al., 2010). Changes in Adversely, Mutus et al. (2006) did not find the intestinal morphology such as reduced any influence of probiotic to feed conversion villus height and deeper crypt may also ratio. Furthermore, Ahmad (2004), and indicate the presence of toxins (Xu et Yousefi and Karkoodi (2007) reported that al., 2003). In the present study, increased broiler feed intake and feed conversion ratio villus height and increased villus height

604 Pertanika J. Trop. Agric. Sci. 39 (4) 597 - 607 (2016) Fermented Feed Effects on Broiler Production Performance to the crypt depth ratio were observed in to the crypt depth ratio, as well as higher the broilers feeding on fermented feed body weight and improvement in the feed compared with unfermented feed. The conversion ratio. increased villi height and villus height to the crypt depth ratio might be associated with REFERENCES the increased number of beneficial bacteria Al-Gharawi, J. K. M. (2012). Effect of in ovo probiotics like Lactobcilli, Bifidobacterium, Bacillus injection on production and immunological traits subtilis and Sacchromyces cervisia (Xu et al., and intestinal flora of broilers. (Ph.D. thesis). 2003; Naji & Al-Mosawi, 2014). Fermented Agriculture College. Baghdad University, Iraq. feed was characterised by a high number of Ayanwale, B. A., Kpe, M., & Ayanwale, V. A. (2006). lactic acid bacteria (Log 9-10 cfu/ g feed) and The effect of supplementing Saccharomyces pH of approximately 4.5-5.0, as described cerevisiae in the diets on egg laying and egg quality characteristics of pullets. International by Cutlure et al. (2005). The increased Journal of Poultry Science, 5(8), 759-763. villus height and villus height to the crypt depth ratio produced an intestinal structure Ahmad, I. (2004). Effect of probiotic (Protexin) on the growth of broilers with special reference to the more oriented to digestion, with improved small intestinal crypt cells proliferation. (Master absorptive and hydrolysis potential, as well of Philosophy Thesis). Centre of Biotechnology, as fewer nutrients requirement for intestinal University of Peshawar. maintenance. Thus, the intestinal structures Anderson, D. B., McCracken, V. J., Aminovi, R. I., of duodenum, jejunum and ileum are more Simpson, J. M., Mackie, R. I., Verstegen, M. W. favourable for the bird and may help to A. & Gaskins, H. R. (1999). Gut microbiology explain the improvements in weight gain and growth-promoting antibiotics in swine. Pig and feed conversion (Feng et al., 2007a). The News and Information, 20, 115-122. good results obtained from the solid state Besnard, J., Auclair, E., & Larbier, M. (2000). Effect of fermentation feed might be attributed to the yeast supplementation on productive parameters higher production of secondary microbial of turkeys. In World’s Poultry Science Congress. metabolites during fermentation. These Chen, K. L., Kho, W. T., Yeu, S. H., Yeh, R. H., Tang metabolites include organic acid (lactic S. W., & Hsieh, C. W. (2009). Effect of Bacillus acid) produced by Lactobacilli, enzymes subtilis var. natto. and sacchromyces cervesia (amylase and protease) and antimicrobial mixed fermented feed on the enhanced growth substances like iturin and surfactin produced performance of broiler. Poultry Science, 88(2), 309-315. by Bacillus subtilis bacteria during solid fermentation (Feng et al., 2007b). Cutlure, S. A., Rasmussen, M. A., Hensley, M. J., & Conclusively, the results of this study Scanes, G. G. (2005). Effect of Lactobacilli and Lactose on Salmonella typhimurium colonization indicated that using fermented feeds with and microbial fermentation the crop of the young probiotic caused significant improvement turkey. British Poultry Science, 46(6), 702-716. in the intestinal structure of the broilers, Chaing, W. Q., Lu, X. S., Piao, J. K., Gong, L., & including villus height and villus height Thacher, P. A. (2010). Effect of feeding solid

Pertanika J. Trop. Agric. Sci. 39 (1): 597 - 607 (2016) 605 Saad A. Naji, I. F. B. Al-Zamili, Hasan, S. A. Jawad and J. K. M. Al-Gharawi

state fermented rapeseed meal on performance, Mathivanan, R. P., Selvaraj, & Nanjappan, K. (2006) Nutritional digestibility, intestinal ecology and feeding of fermented soybean mecel on broiler intestinal Morphology of broiler chickens. performance. International Journal of Poultry Asian–Australian Journal of Animal Science, Science, 5(9), 866 – 872. 23(2), 263 – 271. Mutus, R., Kocabaǧli, N., Alp, M., Acar, N., Eren, Donkor, O. N., Henriksson, A., Vasilijevic, T., & M., & Gezen, S. S. (2006). The effect of Shah, N. P. (2006). Effect of acidification on the dietary probiotic supplementation on tibial bone activity probiotic in yoghurt during cold storage. characteristics and strength in broilers. Poultry International Dairy Journal, 16(10), 1181-1189. Science, 85(9), 1621-1625.

Duncan, D. B. (1955). Multiple ranges test and Mahdavi, A. H., Rahmani, H. R. & Pourreza, J. (2005). Multiple F – test. Biometrics, 11(1), 1-42. Effect of probiotic supplements on egg quality and laying hen’s performance. International Feng, J. X., Liu, X., & Lu, Y. P. (2007a). Effects of Journal of Poultry Science, 4(4), 488-492. Aspergillus oryza fermented soybean meal on growth performance and plasma biochemical Naji, S. A., & Al-Mosawi, I. F. B. (2014). The Effect parameters in broilers. Animal Feed Science and of Diet Fermentation by Iraqi probiotic on Technology, 134(3), 235-242. Logarithmic number of bacteria and yeast in laboratory. Al-Mothanna Journal, 2(1), 217-223. Feng, J. X., Zu, Z. R., & Liu, J. X. (2007b). Effect of fermented soybean meal on digestive enzyme Rasud, E. (2009). Pengaruh fermentasi ampas tahu activities and intestinal morphology in broilers. supplementasi enzim dalam ransum terhadap Poultry Science, 86(6), 1149-1154. indeks performance. (Thesis, S2). Universidas Tadulako. Palu. Hasan S. A. Jawad, Lokman, I. H., Saad A. Naji, Zuki, A. B. Z., & Kassim, A. B. (2016). Effects of Steiner, T. (2006). Managing gut health. Natural Dietary Supplementation of Wet Fermented Feed growth promoters as a key to animal performance. with Probiotic on the Production Performance of Nottingham, UK: Nottingham University Press. Akar Putra Chicken. Asian Journal of Poultry SAS, (2001). SAS users guide. Statistics version 6.12. Science, 10(2), 72-77. SAS institute, Inc, Cary, NC. Hatta, U. H., & Sundu B. B. (2009). Effect of copra Tannock, G. W., Fuller, R., Sullivan, D. J., Svensson, meal fermented by Aspergillus Niger and M., Kullen, J., Klaenhammer, T. R., McCracken, trichoderma spp on performance of broilers. V. J., Gaskins, H. R., Mercenier, A., Reid, G., & International Seminar and Animal Industry Crittenden, R. G. (2001). Probiotic: a critical Bogor, pp.23-42. review (3th ed). USA: Horizon Scientific Press. Heres, L., Wagenaar, J. A., Van-Knapen, F., & Urlings, Winsen, L., Urlings, A., Lipmand, L.J., & Van-Kripen, B. (2003). Passage of salmonella through the F. (2001). Effect of fermented feed on the group and gizzard of broiler chickens fed with microbial population of the gastrointestinal tracts fermented liquid feed. Avian Pathology, 32(2), of pigs. Apply and Environmental Microbiology, 173 – 181. 67(7), 3071-3076. Lokman, I. H., Jawad, S. H., Zuki, A. B. Z., & Xu, Z. R., Ch, U., & Wang, Q. (2003). Effect of dietary Kassim, A. B. (2015). Effect of Dry Probiotic fructooligosaccharide on digestive enzyme Supplemented Fermented Feed on Production activities, intestinal microflora and morphology Performance of Akar Putra Chicken. International of male broilers. Poultry Science, 82(6), 648-654. Journal of Poultry Science, 14(7), 420-426.

606 Pertanika J. Trop. Agric. Sci. 39 (4) 597 - 607 (2016) Fermented Feed Effects on Broiler Production Performance

Xu, F.Z., Li, L.M., Lin, H.J., Zhin, K., Oian, D.W., & Yousefi, M. & Karkoodi, K. (2007). Effect of probiotic Ding, X.L. (2012). Effect of fermented soybean Thepax® and Saccharomyces cerevisiae meal on performance, serum Biochemical supplementation on performance and egg quality parameters and Intestinal morphology of of laying hens. International Journal of Poultry laying Hens. Journal of Animal and Veterinary Science, 6(1), 52-54. Advances, 11(5), 649 – 654. Zhang, A.W., Lee, B.D., Lee, K.W., Song, Yaman, L.A., Ulukanli, Z., & Unal, Y. (2006). The G.H., & Lee, C.H. (2005). Effects of yeast effect of fermented probiotics, the kefir, on (Saccharomyses servisae) cell components on intestinal flora of poultry domesticated geese growth performance, meat quality and ileal (Anser anser). Revue de médecine vétérinaire, mucosa development of broiler chicks. Poultry 157(7), 371-386. Science, 84(7), 1015-1021.

Pertanika J. Trop. Agric. Sci. 39 (1): 597 - 607 (2016) 607

Pertanika J. Trop. Agric. Sci. 39 (4): 601 - 615 (2016)

TROPICAL AGRICULTURAL SCIENCE

Journal homepage: http://www.pertanika.upm.edu.my/

Characteristics and Potential Usage of Dissolved Silica in Rice Cultivation in Sumani Watershed, Sumatra, Indonesia

Hiroaki Somura1*, Darmawan2, Kuniaki Sato1, Makoto Ueno1, Husnain3, Aflizar4 and Tsugiyuki Masunaga1 1Shimane University, Faculty of Life and Environmental Science, 1060 Nishikawatsu, Matsue, Shimane 6908504, Japan 2Andalas University, Faculty of Agriculture, Padang 25163, West Sumatra, Indonesia 3Indonesian Soil Research Institute, Bogor 16114, West Java, Indonesia 4State Polytechnic Payakumbuh for Agriculture, Payakumbuh 26271, West Sumatra, Indonesia

ABSTRACT Research on watershed silica dynamics in Indonesia has been sparse as most of the focus on water environment has centred on suspended sediments, nitrogen and phosphorous. Thus, Si concentrations in rivers and their seasonal and spatial variations are not well understood. Silicon helps rice plants to overcome abiotic and biotic stresses by preventing lodging and increasing resistance against pests and diseases. Rice is one of the more important crops in the country, and information on Si concentrations in rivers is useful because river water is a primary irrigation source. In this study, we conducted a preliminary research on temporal and spatial variations in dissolved Si (DSi) concentrations at watershed scale to help achieve an efficient use of Si resources through irrigation water management. The Sumani Watershed, located approximately 50 km east of Padang City in West Sumatra, Indonesia, was selected as the target area. Lake Dibawah lies on the upstream end of the watershed, and water is discharged from the watershed into Lake Singkarak. The results verified that Lake Dibawah had a dam effect of naturally reducing DSi concentrations in water. In addition, the average DSi concentration from the samples obtained from rivers, small channels, and ditches from October ARTICLE INFO 2013 to December 2014 did not show Article history: Received: 18 January 2016 strong seasonal patterns at each site but Accepted: 4 July 2016 revealed clear spatial differences among E-mail addresses: sub-watersheds linked to the groundwater [email protected] (Hiroaki Somura), [email protected] (Darmawan), from Mt. Talang. The watershed has a high [email protected] (Kuniaki Sato), [email protected] (Makoto Ueno), capability of supplying DSi to paddy fields [email protected] (Husnain), [email protected] (Aflizar), via irrigation water. [email protected] (Tsugiyuki Masunaga) * Corresponding author

ISSN: 1511-3701 © Universiti Putra Malaysia Press Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga

Keywords: Irrigation water, lake watershed, DSi of available Si decreased from 707±269 to dynamics, sustainable rice production, humid tropical 575±260 kg ha−1 in the 0–20-cm soil layer zone and from 3,121±1,668 to 2,755±1,576 kg ha−1 in the 0–100-cm soil layer between INTRODUCTION 1970 and 2003, especially in the upper Rice is a staple crop for over half of the paddy fields. Additionally, Husnain et al. world’s population and it is grown on nearly (2010) found that long-term fertilisation every continent (Seyfferth et al., 2013). imbalances in the intensive rice-farming Rice production has been increasing since system led to surpluses of nutrients such the mid-1960s, with the implementation of as N and P and deficits of K and Si. This new rice cultivation systems known as green occurred because between 230 and 470 kg revolution technology (Pingali, 2012). By ha−1 of Si was removed during rice harvest, 2013, rice crops covered an area of about while only 75–120, 20–25, and 23–257 165 million ha for an annual production of kg ha−1 of N, P, and K, respectively, were 741 million tons (FAO, 2013). removed (Yoshida et al., 1981; Dobermann Rice is a typical silicon accumulator et al., 1996a,b; Casman et al., 1997). A lack because of the high capability of its roots to of Si available to plants may have adverse take up Si from soil (Mitani & Ma, 2005). effects on rice yield by decreasing resistance Silicon is an important beneficial nutrient to lodging, diseases and pests (Winslow, for healthy and competitive growth of all 1992). cereals including rice (Brunings et al., 2009). In Japan, silicate fertiliser containing Beneficial nutrients are equally important as several types of slag is applied to paddy macronutrients like nitrogen and phosphorus fields to improve Si uptake in rice plants, (Ahmad et al., 2013), and balancing these although the application rate per unit area of nutrients in rice cultivation can enhance the paddy field is being gradually decreased to quality and yield of the crop (Ma, 2004). reduce production costs (Ma & Takahashi, Thus, many studies have investigated the 2002). In South and Southeast Asia, where functions and mechanisms of Si in terms of most of the world’s rice is grown, the straw plant pathology, plant physiology, as well as and husks are typically removed from soil science and plant nutrition (Idris, 1975; the field and used for various purposes, Hso-Freng & Yann-Shee, 1978; Richmond including animal fodder, fuel for stoves or & Sussman, 2003; Ma et al., 2006; Kraska burning (Savant et al., 1997). Since most & Breitenbeck, 2010; Dufey et al., 2014; Si taken up by rice is found within the Makabe-Sakaki et al., 2014). straw and husk, the removal of rice straw Several studies have indicated that accelerates soil desilication with no return continuous rice cultivation affects the of Si via biocycling (Seyfferth et al., 2013). silica content of soil. Darmawan et al. However, the supply of Si from (2006) reported that the average content irrigation water may slow the rate of Si depletion (Drmawan et al., 2006). Imaizumi

610 Pertanika J. Trop. Agric. Sci. 39 (4) 609 - 623 (2016) Characteristics and Potential Usage of Dissolved Silica, Indonesia and Yoshida (1958) reported that irrigation end of the watershed, and water flows from water supplied approximately 30% of the watershed into Lake Singkarak. The the Si taken up by rice in a paddy field in average annual precipitation is 2,201 mm Japan. Silicon is the second most abundant (Farida et al., 2005), and the watershed element in the Earth’s crust (Conley, 2002). lies between 338 and 2,739 m.a.s.l. The Silicon in rivers, reservoirs, and lakes watershed is situated in a humid tropical mainly originates from rock weathering, zone (Aflizar et al., 2010a), with average a process enhanced by high temperatures, annual temperatures ranging from 19 to moisture and active vegetation (Cochran 30°C, and varying along an altitudinal & Berner, 1996; Conley, 2002; Humborg gradient. The average annual humidity et al., 2006; Billen and Garnier, 2007), ranges from 78.1 to 89.4% (Aflizar et al., while direct input of Si through urban or 2010b). industrial wastewater is minor (Sferratore The various land uses of the watershed et al., 2006; Garnier et al., 2006). Thus, the are summarised according to the GIS data management of irrigation water in terms of the Ministry of Forestry in Indonesia of Si and macronutrient supply and water (2010) in Table 1. This range of land uses quantity may help farmers to stabilise rice includes primary and secondary forests, quality and production. tree crop gardens (mixed gardens, coconuts Indonesia is the third largest producer of and tea gardens), vegetable gardens, sawah, rice in the world (FAO, 2013), and because bushes (shrubs, grasses, and alang-alang of the importance of macronutrients for rice or Imperata cylindrica) and settlements growth, Si in irrigation water has received (Aflizar et al., 2010b). Asawah is a levelled limited attention. Research on DSi dynamics and bounded rice field with an inlet and in the watersheds of Sumatra Island has not outlet for irrigation and drainage (Wakatsuki yet been conducted. In this study, therefore, et al., 2009). In a mixed garden, perennial we investigated temporal and spatial crops, primarily trees such as coconut, variations in DSi concentrations at the clove, coffee, teak, mahogany, avocado, watershed scale to improve the efficiency of melinjo (Gnetum gnemon), rubber and Si resource use for rice cultivation through cinnamon are planted in combination with irrigation water management. annual crops (Karyono, 1990). Chilies (Capsicum annum L.), onions (Allium STUDY AREA cepa L.), soybeans (Glycine max L.), corn Sumani Watershed is located approximately (Zea mays L.) and sweet potatoes (Ipomea 50 km east of Padang City in West Sumatra, batatas L.) are the major crops in vegetable Indonesia (Figure 1) and covers about 586 gardens (Aflizar et al., 2010b). km2. Lake Dibawah lies on the upstream

Pertanika J. Trop. Agric. Sci. 39 (1): 609 - 623 (2016) 611 Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga

Figure 1. Location of the Sumani Watershed and sampling sites

Table 1 Landuse characteristics of Sumani Watershed

Forests Tree crop Vegetable Paddy fields/ Settlements Water gardens gardens sawah Sub-watershed I 9% 1% 37% 40% 3% 10% Sub-watershed II 27% 15% 4% 51% 3% 0% Sub-watershed III 24% 10% 2% 48% 16% 0% Sub-watershed IV 57% 15% 12% 14% 2% 0% Sub-watershed V 16% 18% 36% 26% 5% 0% Sub-watershed VI 0% 82% 0% 14% 4% 0% Source: Ministry of Forestry, Republic of Indonesia (2010)

612 Pertanika J. Trop. Agric. Sci. 39 (4) 609 - 623 (2016) Characteristics and Potential Usage of Dissolved Silica, Indonesia

METHODOLOGY for R (The R Foundation for Statistical Field sampling was conducted once per Computing, Vienna, Austria). month between October 2013 and December 2014 (with the exception of March 2014). RESULTS AND DISCUSSION Water samples from rivers, irrigation Roles of Lake Dibawah in DSi channels and ditches, and a paddy field were concentration collected from 23 sites. Due to the lack of Lake Dibawah, which is located on the detailed information on river locations and upstream end of the watershed, is primarily low road accessibility in the area, sampling precipitation- and spring-fed and it provides points were increased with knowledge the main water source for streams. A portion gained throughout the field investigation in of the lake’s water is allocated for irrigation this study. Thus, sampling occurred at only via small channels at the outlet of the lake. 11 sites in October 2013, 16 sites between Of the small ditches along the lake, one November 2013 and January 2014, and 22 from Mt. Talang was selected as a regular sites in June and July 2014. All the sites sampling site because of its accessibility were sampled in the remaining months, (site no. 1). Figure 2 compares the DSi except for March 2014 when road and concentration at this site with that at the weather conditions were not suitable for any outlet of the lake (site no. 2). The average fieldwork. In total, 287 water samples were DSi concentrations were 35.8 mg L−1 (n = collected. Of these, 263 were collected from 14) at site no. 1 and 6.21 mg L−1 (n = 14) rivers, channels and ditches. Additional at site no. 2. The standard deviations were samples were obtained from a paddy field 1.75 and 0.41 mg L−1 at site nos. 1 and 2, (site no. 8) and spring water (site no. 15). respectively. The concentrations at both sites Surface waters of paddy fields were also were relatively stable during the research sampled at five sites downstream, eight sites period from month to month. A comparison midstream, and five sites upstream in the of the two sites indicated that water with watershed in November 2013. high DSi concentrations flowed into the The water samples were filtered through lake, while water with low concentrations a 0.45-μm membrane filter (Advantec was discharged from the lake (p < 0.001). Dismic-25CS, Japan). The concentration of It has been previously reported that DSi was measured using inductively coupled the construction of large dams leads to plasma atomic emission spectroscopy (ICP- decreased input of Si into coastal zones AES, Shimadzu ICPE-9000). because of increased water residence times All the statistical analyses were and net losses of Si to the sediments within performed with EZR (Saitama Medical reservoirs (Conley et al., 1993; Garnier Centre, Jichi Medical University, Saitama, et al., 1999; Friedl et al., 2004; Koch et Japan), which is a graphical user interface al., 2004; Ahearn et al., 2005; Humborg

Pertanika J. Trop. Agric. Sci. 39 (1): 609 - 623 (2016) 613 Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga

Figure 2. Average DSi concentrations and standard deviations. Statistical analysis was performed using Welch t-Test against site nos. 1 and 2. In addition, Kruskal-Wallis Test with the Bonferroni adjustment was applied against site nos. 2, 3 and 4. **p < 0.01, ***p < 0.001. et al., 2006). Development, such as dam research period and exceeded 10 mg L−1 in construction or concrete canal installation all, except for four months (November 2013 within a watershed, was found to result in and May, November, and December 2014). a decrease in Si concentrations in irrigation In addition, at site no. 4, which is also located water (Ma and Takahashi, 2002). Thus, about 2 km downstream of the lake outlet the lake naturally becomes akin to a dam and close to sampling site no. 3, containing reservoir, and dilution through precipitation, a small ditch flowing through an upland consumption of Si through diatom growth, field, the DSi concentrations averaged and subsequent settling of diatom frustules 23.4 mg L−1, with a maximum of 26.1 mg in the lake might occur. L−1 and a minimum of 20.5 mg L−1. These These natural functions may have relatively high concentrations exhibited adverse effects on rice, sugarcane and little variation, with a standard deviation other tall plants because of reduction of of 1.78 mg L−1. These results pointed to a Si levels in irrigation water. However, the possible Si source for river water between concentrations of DSi 2 km downstream of site nos. 2 and 3. As the DSi concentrations the lake outlet (site no. 3) were higher than were consistently higher at site no. 4 than at site no. 2 (p < 0.001). These differences site no. 3 (p < 0.01), agricultural fields were varied from 1.37 to 19.4 mg L−1 during the also likely to contribute Si to the water.

614 Pertanika J. Trop. Agric. Sci. 39 (4) 609 - 623 (2016) Characteristics and Potential Usage of Dissolved Silica, Indonesia

Variations in the DSi concentrations two groups based on the DSi concentration The average DSi concentration for the levels. The group with lower concentrations whole watershed, obtained from river, included sub-watersheds I and IV, and that channel and ditch samples was 18.0 mg with higher concentrations included sub- L−1 (n = 263). The concentrations varied watersheds II and III. Sub-watershed I had −1 over 4.2–38.3 mg L−1 during the research an average concentration of 13.7 mg L and −1 period, with a standard deviation of 7.1 mg a standard deviation of 5.00 mg L , with L−1. Husnain et al. (2010) reported that the measurements frequently falling between 10 −1 DSi concentration of irrigation water in and 15 mg L . The average concentration −1 the Citarum Watershed of Java, Indonesia, in sub-watershed IV was 17.0 mg L , and −1 was 13.64±3.13 mg L−1 (n = 15) from the standard deviation was 4.62 mg L . September 2006 to November 2007, with Measurements were concentrated between −1 monthly sampling. Thus, the average DSi 15 and 20 mg L . On the other hand, in concentrations of the Sumani and Citarum sub-watersheds II and III, the average DSi −1 Watersheds exhibited similar tendencies. concentrations were 26.2 and 24.0 mg L , Figure 3 provides a histogram and standard respectively. Measurements between 25 −1 statistical values of DSi concentrations. For and 30 mg L were most frequent in sub- this analysis, the samples at site nos. 1, 8, watershed II, and those from 20 to 25 mg −1 and 15 were excluded. This is because water L were most frequent in sub-watershed III. at site no. 1 flows into the lake, where high The DSi concentrations in sub-watershed DSi concentrations appeared to be diluted. V were determined by inflow quantity and Water is then discharged from the lake as quality from sub-watersheds I through IV. river water and irrigation water. Thus, the In this study, as river discharge was not concentrations at sampling site no. 1 were observed, the impact ratios of each sub- not influenced by downstream water quality watershed against the concentration in in sub-watershed I. Additionally, the samples sub-watershed V were unknown. In sub- at site nos. 8 and 15 were not collected from watershed V, the average DSi concentration −1 a river or small ditch, but rather from the was 17.6 mg L , and the standard deviation −1 surface water on a paddy field and spring was 2.0 mg L . Measurements tended −1 water, respectively. In order to compare to fall between 15 and 20 mg L . The DSi characteristics among sub-watersheds, concentrations in sub-watershed V were four independent sub-watersheds (sub- similar with those in sub-watershed IV, but watersheds I through IV) were selected. fluctuations were smaller in sub-watershed In this comparison, sub-watershed V was V than in sub-watershed IV. excluded because its water was influenced to In order to investigate possible sources an unknown degree by water from the other of differences in the DSi concentrations four sub-watersheds. For this discussion, the among the sub-watersheds, we examined individual sub-watersheds were divided into groundwater. For the preliminary analysis,

Pertanika J. Trop. Agric. Sci. 39 (1): 609 - 623 (2016) 615 Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga

Figure 3. Histogram and statistical values of DSi concentrations among sub-watersheds, excluded site nos. 1 (Inflow water to Lake Dibawa), 8 (Surface water of a paddy field), and 15 (Spring water). Kruskal-Wallis Test with the Bonferroni adjustment was applied against sub-watersheds I to IV. p < 0.001 in sub-watershed I vs. sub-watersheds II and III, and in sub-watershed IV vs. sub-watersheds II and III. No statistically significant difference was detected between sub-watersheds II and III.

616 Pertanika J. Trop. Agric. Sci. 39 (4) 609 - 623 (2016) Characteristics and Potential Usage of Dissolved Silica, Indonesia

Figure 4. DSi concentrations in the surface water of paddy fields. Box=25th and 75th percentiles; bar=minimum and maximum values. Kruskal-Wallis Test with the Bonferroni adjustment, *p < 0.05, **p < 0.01.

DSi concentrations in spring water upstream was 62.1 mg L−1 (n = 10), with a standard of sub-watershed II were analysed on-site in deviation of 2.8 mg L−1. This finding February 2014 using the pack test (DPM- indicated that water with high concentrations

SiO2). The average concentration was 72.1 of DSi was steadily discharged as spring mg L−1 (n = 2), indicating that spring water water with little variation. might be a source of Si in the area. Thus, Based on the consistently high DSi to examine variations in DSi concentration concentrations at site nos. 1 and 15, it was in spring water (groundwater), the highly concluded that water from Mt. Talang accessible spring water of site no. 15 provided a source of Si through groundwater located in sub-watershed III was sampled. to surface water within the watershed. The averaged DSi concentration of this site

Pertanika J. Trop. Agric. Sci. 39 (1): 609 - 623 (2016) 617 Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga

Spatial Distribution of DSi watersheds II and III, and was allocated to concentrations in the Surface Water of the paddy fields. Paddy Fields Moreover, the DSi concentrations of The DSi concentrations in the surface water surface water at a regular sampling site (no. of upstream through downstream paddy fields 8) exhibited a trend that is similar to that in the watershed were studied in November of the upstream sampling from November 2013 (see Figure 4). The water samples were 2013. The concentration at this site was collected along the longitudinal (A-A’) line relatively low, with an average of 10.1 mg from Lake Dibawah (upstream) to Lake L−1. The DSi levels seemed to be lower Singkarak (downstream). As variations in during the rainy season and higher during the growth stages of rice on each paddy field the dry season, especially in June and July. might affect DSi concentrations of surface Although the cause of this phenomenon water, the water samples were collected is not quite clear, the dilution of surface under conditions similar to those before water by precipitation in the rainy season and after transplanting of rice seedlings and the long retention time of water in the and during the growing stages as much as soil during the dry season might provide an possible. The upstream sampling sites were explanation. located in sub-watershed I, midstream sites in sub-watershed III and downstream sites Si Supply from Irrigation Water in sub-watersheds V and VI. The average Irrigation water may provide an important DSi concentrations were 9.8, 15.5, and source of Si for rice. Thus, the amount of 16.4 mg L−1 for upstream, midstream and Si delivered to a paddy field via irrigation downstream sites, respectively. Most of the was estimated. Ma and Takahashi (2002) DSi concentrations between 5 and 10 mg reported that paddy soil was irrigated with L−1 occurred in upstream paddy fields, while an average of 1,400 mm (14,000 m3 ha−1) those between 15 and 20 mg L−1 occurred of water during the single growth period of more frequently downstream. For the rice. Average Si concentrations from 380 midstream fields, values were concentrated rivers in Japan ranged from 4.0 to 19.5 mg between 10 and 20 mg L−1. Along the A-A’ L−1, with an overall average of 10.1 mg L−1. line, there were clear differences in these From these results, Si supply from irrigation intervals between upstream and downstream water in Japan was estimated to be 141.4 kg sites (p < 0.05). Our findings indicated that ha−1, or about 30% of the crop requirement. water with relatively low DSi concentrations In this study, the amount of irrigation water from Lake Dibawah provided irrigation was also assumed to be 1,400 mm because water via small channels for upstream areas. of the lack of information on irrigation water Downstream, river water mixed with water levels in the area. DSi load to the paddy with high DSi concentrations from sub- fields was then estimated from this irrigation

618 Pertanika J. Trop. Agric. Sci. 39 (4) 609 - 623 (2016) Characteristics and Potential Usage of Dissolved Silica, Indonesia water value for an average of 238±81.2 kg reduced mineral toxicity, improvement of ha−1 per rice cultivation period (Table 2), nutrient imbalance, enhanced drought and accounting for 53.6±18.3% of the Si taken frost tolerance, and improved grain yield up by rice. (Deren et al., 1994; Kim et al., 2002; Ma, On average, 141.4 kg ha−1 of DSi 2004). Therefore, aggressively introducing is supplied by irrigation water in Japan, Si via irrigation water may enhance the and 30±15 kg ha−1 of DSi is supplied by stability of rice production and quality in irrigation in France (Desplanques et al., the region. 2006). Thus, the Sumani Watershed has a higher capacity for supplying Si to paddy CONCLUSION fields via irrigation. DSi loads varied among The dynamics of DSi concentrations in the sub-watersheds. The highest DSi load Sumani Watershed were investigated for occurred in sub-watershed II at 366.8±58.8 the first time between October 2013 and −1 kg ha . The spring water at site no. 15 had a December 2014. From this research, it was −1 DSi load of 869.4±39.2 kg ha , constituting found that Lake Dibawah had a dam effect approximately 195.8±8.8% of the crop of naturally reducing DSi concentrations in requirement. Thus, Si input via irrigation the water, based on a comparison between could increase with the efficient introduction inflow and outflow concentrations. Further, of spring water into paddy fields. Numerous it was revealed that the average DSi beneficial effects of Si have been reported; concentration of samples obtained from these include prevention of lodging (falling rivers, small channels and ditches was over), increased photosynthetic activity, 18.0 mg L−1, with the levels varying from increased insect (e.g., brown planthopper) 4.2 to 38.3 mg L−1 during the research and disease (e.g., rice blast) resistance, period. Among the sub-watersheds, there

Table 2 DSi loads supplied by irrigation and spring water in Sumani Watershed. The load was assumed as 1,400 mm irrigation water per cultivation period.

DSi load DSi concentration Standard deviation Sample (kg ha−1) (mg L−1) (mg L−1) number Whole watershed 238.0±81.2 17.0 a 5.8 249 Sub-watershed I 191.8±70.0 13.7 b 5.0 120 Sub-watershed II 366.8±58.8 26.2 4.2 20 Sub-watershed III 336.0±39.2 24.0 c 2.8 24 Sub-watershed IV 238.0±64.4 17.0 4.6 20 Sub-watershed V 246.4±28.0 17.6 2.0 65 Spring water (no. 15) 869.4±39.2 62.1 2.8 10 a verage value of DSi concentrations in the watershed, excluding site nos. 1, 8, and 15. b average value of DSi concentrations in sub-watershed I, excluding site nos. 1 and 8. c Average value of DSi concentrations in sub-watershed III, excluding site no. 15.

Pertanika J. Trop. Agric. Sci. 39 (1): 609 - 623 (2016) 619 Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga were significant differences in the DSi KAKENHI Grant-in-Aid for Scientific concentrations, with higher concentrations Research (B), grant no. 24405047. The in sub-watersheds II and III than other sub- authors would like to express our gratitude watersheds. Based on an analysis of spring to Ms. Lilian Safitri, Mr. Sapuranda Siregar, water, the differences in DSi concentrations Mr. Maulia Septiza, and Mr. Ikhsanul Adli among sub-watersheds may be closely for their support during the course of this related to the groundwater from Mt. Talang. study. In addition, it was understood that spatial variation in DSi concentration existed REFERENCES upstream to downstream in the surface water Aflizar, Saidi, A., Husnain, Ismawardi, Bambang of paddy fields. Then, by using the measured Istijono, Harmailis, Somura, H. Wakatsuki, T., DSi concentrations and a literature review, & Masunaga, T. (2010a). A land use planning it was determined that more than 50% of recommendation for the sumani watershed, West Sumatra, Indonesia. Tropics, 19(1), 43-51. Si taken up by rice can be provided from irrigation water in the area based on the Aflizar, Saidi, A., Husnain, Indra, R., Darmawan, average DSi concentration value. Moreover, Harmailis, Somura, H., Wakatsuki, T., & Masunaga, T. (2010b). Soil erosion characterization in from the highest average DSi concentration an agricultural watershed in west Sumatra, −1 (26.2±4.2 mg L ) from sub-watershed II, Indonesia. Tropics, 19(1), 29-42. up to 69-96% of Si taken up by rice may be Ahearn, D. S., Sheibley, R. W., & Dahlgren, R. A. (2005). supplied from the irrigation water. Effects of river regulation on water quality in Although the total Si absorbed by rice the lower Mokelumne River, California. River in a growing season is easily determined, Research and Applications, 21, 651-670. no study has yet investigated which Si Ahmad, A., Afzal, M., Ahmad, A. U. H., & Tahir, M. source (soil or irrigation water) is the most (2013). Effect of Foliar Application of Silicon critical for the absorption of Si during a on Yield and Quality of Rice (Oryza Sativa L). rice cultivation period. Additionally, the Cercetari agronomice in Moldova, 46(3), 21–28. relationship between the spatial distribution Billen, G. & Garnier, J. (2007). River basin nutrient of Si concentrations and rice quality in this delivery to the coastal sea: Assessing its potential watershed is not yet understood. Thus, to sustain new production of non-siliceous algae. such research should be conducted as the Marine Chemistry, 106(1-2), 148-160. next step to help produce high-quality rice Brunings, A. M., Datnoff, L. E., Ma, J. F., Mitani, N., through the efficient allocation of irrigation Nagamura, Y., Rathinasabapathi, B., & Kirst, water. M. (2009). Differential gene expression of rice in response to silicon and rice blast fungus ACKNOWLEDGEMENTS Magnaporthe oryzae. Annals of Applied Biology, 155(2), 161–170. This work was supported by the Japan Casman, K. G., Peng, S., & Doberman, A. (1997). Society for the Promotion of Science (JSPS) Nutritional physiology of the rice plants and

620 Pertanika J. Trop. Agric. Sci. 39 (4) 609 - 623 (2016) Characteristics and Potential Usage of Dissolved Silica, Indonesia

productivity declined of irrigated rice systems Phosphorus. Nutrient Cycling in Agroecosystems, in the tropics. Soil Science and Plant Nutrition, 46(2), 111-125. 43, 1101-1106. Dufey, I., Gheysens, S., Ingabire, A., Lutts, S., Cochran, M. F., & Berner, R. A. (1996). Promotion & Bertin, P. (2014). Silicon Application in of chemical weathering by higher plants: field Cultivated Rices (Oryza sativa L and Oryza observations on Hawaiian basalts. Chemical glaberrima Steud) Alleviates Iron Toxicity Geology, 132, 71–77. Symptoms through the Reduction in Iron Concentration in the Leaf Tissue. Journal of Conley, D. J., Schelske, C. L., & Stoermer, E. F. (1993). Agronomy and Crop Science, 200(2), 132–142. Modification of the biogeochemical cycle of silica with eutrophication. Marine Ecology. Farida, Jeanes, K., Kurniasari, D., Widayati, A., Progress Series 101(1-2), 179-192. Ekadinata, A., Hadi, D. P., Joshi, L., Suyamto, D., & van Noordwijk, M. (2005). Rapid Conley, D. J. (2002). Terrestrial ecosystems and the Hydrological Appraisal (RHA) of Singkarak global biogeochemical silica cycle. Global Lake in the context of Rewarding Upland Biogeochemical Cycles, 16(4), 1121. Poor for Environmental Services (RUPES). Darmawan, Kyuma, K., Saleh, A., Subagjo, H., Retrieved from http://www.researchgate.net/ Masunaga, T., & Wakatsuki, T. (2006). Effect publication/268001200_Rapid_Hydrological_ of long-term intensive rice cultivation on the Appraisal_%28RHA%29_of_Singkarak_Lake_ available silica content of sawah soils: Java in_the_context_of_Rewarding_Upland_Poor_ Island, Indonesia. Soil Science and Plant for_Environmental_Services_%28RUPES%29 Nutrition, 52(6), 745-753. Food and Agriculture Organization of the United Deren, C. W., Datnoff, L. E., Snyder, G. H., & Martin, Nations (FAO). FAOSTAT database. Available F. G. (1994). Silicon Concentration, Disease at http://faostat3.fao.org/home/E. (2015.09.03) Response, and Yield Components of Rice Friedl, G., Teodoru, C., & Wehrli, B. (2004). Is the Iron Genotypes Grown on Flooded Organic Histosols. Gate I reservoir on the Danube River a sink for Crop Science, 34(3), 733-737. dissolved silica? Biogeochemistry, 68(1), 21–32. Desplanques, V., Cary, L., Mouret, J.-C., Trolard, F., Garnier, J., Leporcq, B., Sanchez, N., & Philippon, X. Bourrié, G., Grauby, O., & Meunier, J.-D. (2006). (1999). Biogeochemical mass-balances (C, N, P, Silicon transfers in a rice field in Camargue Si) in three large reservoirs of the Seine Basin (France). Journal of Geochemical Exploration, (France). Biogeochemistry, 47(2), 119-146. 88(1–3), 190-193. Garnier, J., Sferratore, A., Meybeck, M., Billen, G., Dobermann, A., Sta Cruz, P. C., & Casman, K. G. & Dürr, H. (2006). Modelling silica transfer (1996a). Fertilizer inputs, nutrient balance, processes in river catchments. Chapter 10. In and soil nutrient-supplying power in intensive, Ittekkot, V., Unger, D., Humborg, C., & An, N. irrigated rice systems. I. Potassium uptake and T., The Silicon Cycle. Human Perturbations and K balance. Nutrient Cycling in Agroecosystems, Impacts on Aquatic Systems (pp. 139-162). Scope 46(1), 1-10. 66, Island Press. Dobermann, A., Sta Cruz, P. C., Advianto, M. A., Humborg, C., Pastuszak, M., Aigars, J., Siegmund, & Pampolino, M. F. (1996b). Fertilizer inputs, H., Morth, C. M., & Ittekot, V. (2006). Decreased nutrient balance and soil nutrient supplying silica land–sea fluxes through damming in power in intensive, irrigated rice systems. III. the Baltic Sea catchment - Significance of

Pertanika J. Trop. Agric. Sci. 39 (1): 609 - 623 (2016) 621 Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga

particle trapping and hydrological alterations. Ma, J. F., & Takahashi, E. (2002). Soil, Fertilizer, and Biogeochemistry, 77(2), 265–281. Plant Silicon Research in Japan. Amsterdam: Elsevier. Husnain, Masunaga, T., & Wakatsuki, T. (2010). Field assessment of nutrient balance under intensive Ma, J. F. (2004). Role of silicon in enhancing the rice-farming systems, and its effects on the resistance of plants to biotic and abiotic stresses. sustainability of rice production in Java Island, Soil Science and Plant Nutrition, 50(1), 11-18. Indonesia. Journal of Agricultural, Food, and Ma, J. F., Tamai, K., Yamaji, N., Mitani, N., Konishi, S., Environmental Science, 4(1), 1-11. Katsuhara, M., Ishiguro, M., Maruta, Y., & Yano, Hso-Freng, Y., & Yann-Shee, C. (1978). Effect of M. (2006). A silicon transporter in rice. Nature, available silicon in paddy soil on the growth 440, 688-691. of rice plants. Botanical Bulletin of Academia Makabe-Sakaki, S., Kakuda, K., Sakaki, Y., & Ando, Sinica, 19, 125-137. H. (2014). Effects of slag silicate fertilizer on Idris, Md., Hossain, M. M., & Choudhury, F. A. (1975). silicon content of rice plants grown in paddy The effect of silicon on lodging of rice in fields on the Shounai Plain, Yamagata, Japan. presence of added nitrogen. Plant and Soil, Soil science and plant nutrition, 60(5), 708-721. 43(1-3), 691-695. Mitani, N., & Ma, J. F. (2005). Uptake system of Imaizumi, K., & Yoshida, S. (1958). Edaphological silicon in different plant species. Journal of studies on silicon supplying power of paddy Experimental Botany, 56(414), 1255-1261. fields. Bulletin of the National Institute of Pingali, P. L. (2012). Green Revolution: Impacts, Agricultural Sciences, Series B (Soils and limits, and the path ahead. Proceedings of Fertilizers), Nishigara, Tokyo, Japan. 8, pp. 261- the National Academy of Sciences, 109(31), 304 (in Japanese with English summary). 12302–12308. Karyono. (1990). Home garden in Java: their structure Richmond, K. E. & Sussman, M., (2003). Got silicon? and function. In K.& M (Eds. Tropical Home The non-essential beneficial plant nutrient. Garden (pp. 138-146). Tokyo: The United Current Opinion in Plant Biology, 6(3), 268-272. Nations University Press. Savant, N. K., Snyder, G. H., & Datnoff, L. E. Kim, S. G., Kim, K. W., Park, E. W., & Choi, D. (2002). (1997). Silicon management and sustainable rice Silicon-induced cell wall fortification of rice production. In D. L. Sparks (Ed.), Advances in leaves: A possible cellular mechanism of Agronomy (pp. 151-199). San Diego: Academic enhanced host resistance to blast. Phytopathology, Press. 92(10), 1095-1103. Seyfferth, A. L., Kocar, B. D., Lee, J. A., & Fendorf, Koch, R. W., Guelda, D. L., & Bukaveckas, P. A. (2004). S. (2013). Seasonal dynamics of dissolved Phytoplankton growth in the Ohio, Cumberland silicon in a rice cropping system after straw and Tennessee Rivers, USA: inter-site differences incorporation. Geochimica et Cosmochimica in light and nutrient limitation. Aquatic Ecology, Acta, 123, 120-133. 38(1), 17-26. Sferratore, A., Garnier, J., Billen, G., Conley, D. Kraska, J. E., & Breitenbeck, G. A. (2010). Survey of J., & Pinault, S. (2006). Diffuse and Point the silicon status of flooded rice in Louisiana. Sources of Silica in the Seine River Watershed. Agronomy Journal, 102(2), 523-529. Environmental Science and Technology, 40(21), 6630-6635.

622 Pertanika J. Trop. Agric. Sci. 39 (4) 609 - 623 (2016) Characteristics and Potential Usage of Dissolved Silica, Indonesia

Wakatsuki, T., Buri, M. M., & Oladele O. I. (2009). Yoshida, S., Ohnisi, Y., & Kitagishi, K. (1981). Chemical West African rice green revolution by sawah forms, mobility and deposition of silicon in rice eco-technology and the creation of African plant. Soil Science and Plant Nutrition, 8(3), SATOYAMA systems. Kyoto Working Papers on 107-113. Area Studies: G-COE Series, 61, 1-30.

Winslow, M. D. (1992). Silicon, disease resistance, and yield of rice genotypes under upland cultural conditions. Crop Science, 32(5), 1208-1213.

Pertanika J. Trop. Agric. Sci. 39 (1): 609 - 623 (2016) 623

REFEREES FOR THE PERTANIKA JOURNAL OF TROPICAL AGRICULTURAL SCIENCE VOL. 39(4) NOV. 2016

The Editorial Board of the Journal of Tropical Agricultural Science wishes to thank the following:

Aileen Shau Hwai Tan Mohd Amin Mohd Soom (USM, Malaysia) (UMS, Malaysia)

Amir Shah Ruddin Md Sah Monjurul Alam Mondal (USM, Malaysia) (Bangladesh Institute of Nuclear Agriculture, Bangladesh)

Catherine Yule Muhd Danish Daniel Abdullah (Monash University, Malaysia) (UMT, Malaysia)

Gustavo Ciniciato Najiah Musa (University of Cambridge, UK) (UMT, Malaysia)

Halimi Mohd Saud Narayanan Manivannan (UPM, Malaysia) (Tamil Nadu Agricultural University, India)

Hardev Singh Sandhu Norhani Abdullah (University of Florida, USA) (UPM, Malaysia)

Hazim Al-Daraji Prakash Chandra Shrivastava (University of Baghdad, IRAQ) (Govind Ballabh Pant University of Agriculture & Technology, India) Ippala Janardhan Reddy (National Institute of Animal Nutrition and Physiology, India) Riza G. Abilgos-Ramos (Philippine Rice Research Institute, Phillippine) Joseph Bagyaraj (Centre for Natural Biological Resources and Community Takaomi Arai Development (CNBRCD), India) (UMT, Malaysia)

Lipismitha Samal Veerasamy Sejian (Orissa University of Agriculture and Technology, India) (National Institute of Animal Nutrition and Physiology, India)

Lokman Hakim Idris Vincenzo Tufarelli (UPM, Malaysia) (University of Bari Aldo Moro, Italy)

Mabry McCray Wan Abdullah Wan Nadiah (University of Florida, USA) (USM, Malaysia)

Mahmoud Tanekhy Wan Zahari Mohamed (Alexandria University, Egypt) (UMK, Malaysia)

UMK - Universiti Malaysia Kelantan UPM - Universiti Putra Malaysia UMS - Universiti Malaysia Sabah USM - Universiti Sains Malaysia UMT - Universiti Malaysia Terengganu

While every effort has been made to include a complete list of referees for the period stated above, however if any name(s) have been omitted unintentionally or spelt incorrectly, please notify the Chief Executive Editor, UPM Journals at [email protected]. Any inclusion or exclusion of name(s) on this page does not commit the Pertanika Editorial Office, nor the UPM Press or the University to provide any liability for whatsoever reason.

Pertanika Journals Our goal is to bring high quality research to the widest possible audience

INSTRUCTIONS TO AUTHORS (Manuscript Preparation & Submission Guide) Revised: June 2016

Please read the Pertanika guidelines and follow these instructions carefully. Manuscripts not adhering to the instructions will be returned for revision without review. The Chief Executive Editor reserves the right to return manuscripts that are not prepared in accordance with these guidelines.

MANUSCRIPT PREPARATION

Manuscript Types Pertanika accepts submission of mainly four types of manuscripts for peer-review.

1. Regular article Regular articles are full-length original empirical investigations, consisting of introduction, materials and methods, results and discussion, conclusions. Original work must provide references and an explanation on research findings that contain new and significant findings. Size: Generally, these are expected to be between 6 and 12 journal pages (excluding the abstract, references, tables and/or figures), a maximum of 80 references, and an abstract of 100–200 words.

2. Review article These report critical evaluation of materials about current research that has already been published by organizing, integrating, and evaluating previously published materials. It summarizes the status of knowledge and outline future directions of research within the journal scope. Review articles should aim to provide systemic overviews, evaluations and interpretations of research in a given field. Re-analyses as meta-analysis and systemic reviews are encouraged. The manuscript title must start with “Review Article:”.

Size: These articles do not have an expected page limit or maximum number of references, should include appropriate figures and/or tables, and an abstract of 100–200 words. Ideally, a review article should be of 7 to 8 printed pages.

3. Short communications They are timely, peer-reviewed and brief. These are suitable for the publication of significant technical advances and may be used to:

(a) report new developments, significant advances and novel aspects of experimental and theoretical methods and techniques which are relevant for scientific investigations within the journal scope; (b) report/discuss on significant matters of policy and perspective related to the science ofthe journal, including ‘personal’ commentary; (c) disseminate information and data on topical events of significant scientific and/or social interest within the scope of the journal. The manuscript title must start with “Brief Communication:”.

Size: These are usually between 2 and 4 journal pages and have a maximum of three figures and/or tables, from 8 to 20 references, and an abstract length not exceeding 100 words. Information must be in short but complete form and it is not intended to publish preliminary results or to be a reduced version of Regular or Rapid Papers. 4. Others Brief reports, case studies, comments, concept papers, Letters to the Editor, and replies on previously published articles may be considered.

PLEASE NOTE: NO EXCEPTIONS WILL BE MADE FOR PAGE LENGTH.

Language Accuracy Pertanika emphasizes on the linguistic accuracy of every manuscript published. Articles must be in English and they must be competently written and argued in clear and concise grammatical English. Contributors are strongly advised to have the manuscript checked by a colleague with ample experience in writing English manuscripts or a competent English language editor.

Author(s) must provide a certificate confirming that their manuscripts have been adequately edited. A proof from a recognised editing service should be submitted together with the cover letter at the time of submitting a manuscript to Pertanika. All editing costs must be borne by the author(s). This step, taken by authors before submission, will greatly facilitate reviewing, and thus publication if the content is acceptable.

Linguistically hopeless manuscripts will be rejected straightaway (e.g., when the language is so poor that one cannot be sure of what the authors really mean). This process, taken by authors before submission, will greatly facilitate reviewing, and thus publication if the content is acceptable.

MANUSCRIPT FORMAT The paper should be submitted in one column format with at least 4cm margins and 1.5 line spacing throughout. Authors are advised to use Times New Roman 12-point font and MS Word format.

1. Manuscript Structure Manuscripts in general should be organised in the following order:

Page 1: Running title This page should only contain the running title of your paper. The running title is an abbreviated title used as the running head on every page of the manuscript. The running title should not exceed 60 characters, counting letters and spaces.

Page 2: Author(s) and Corresponding author information. This page should contain the full title of your paper not exceeding 25 words, with name(s) of all the authors, institutions and corresponding author’s name, institution and full address (Street address, telephone number (including extension), hand phone number, and e-mail address) for editorial correspondence. First and corresponding authors must be clearly indicated.

The names of the authors may be abbreviated following the international naming convention. e.g. Salleh, A.B.1, Tan, S.G2*., and Sapuan, S.M3.

Authors’ addresses. Multiple authors with different addresses must indicate their respective addresses separately by superscript numbers: George Swan1 and Nayan Kanwal2 1Department of Biology, Faculty of Science, Duke University, Durham, North Carolina, USA., 2Office of the Deputy Vice Chancellor (R&I), Universiti Putra Malaysia, Serdang, Malaysia.

A list of number of black and white / colour figures and tables should also be indicated on this page. Figures submitted in color will be printed in colour. See “5. Figures & Photographs” for details.

Page 3: Abstract This page should repeat the full title of your paper with only the Abstract (the abstract should be less than 250 words for a Regular Paper and up to 100 words for a Short Communication), and Keywords.

Keywords: Not more than eight keywords in alphabetical order must be provided to describe the contents of the manuscript. Page 4: Introduction This page should begin with the Introduction of your article and followed by the rest of your paper.

2. Text Regular Papers should be prepared with the headings Introduction, Materials and Methods, Results and Discussion, Conclusions, Acknowledgements, References, and Supplementary data (if avavailble) in this order.

3. Equations and Formulae These must be set up clearly and should be typed double spaced. Numbers identifying equations should be in square brackets and placed on the right margin of the text.

4. Tables All tables should be prepared in a form consistent with recent issues of Pertanika and should be numbered consecutively with Roman numerals. Explanatory material should be given in the table legends and footnotes. Each table should be prepared on a new page, embedded in the manuscript.

When a manuscript is submitted for publication, tables must also be submitted separately as data - .doc, .rtf, Excel or PowerPoint files- because tables submitted as image data cannot be edited for publication and are usually in low-resolution.

5. Figures & Photographs Submit an original figure or photograph. Line drawings must be clear, with high black and white contrast. Each figure or photograph should be prepared on a new page, embedded in the manuscript for reviewing to keep the file of the manuscript under 5 MB. These should be numbered consecutively with Roman numerals.

Figures or photographs must also be submitted separately as TIFF, JPEG, or Excel files- because figures or photographs submitted in low-resolution embedded in the manuscript cannot be accepted for publication. For electronic figures, create your figures using applications that are capable of preparing high resolution TIFF files. In general, we require300 dpi or higher resolution for coloured and half-tone artwork, and 1200 dpi or higher for line drawings are required.

Failure to comply with these specifications will require new figures and delay in publication. NOTE: Illustrations may be produced in colour at no extra cost at the discretion of the Publisher; the author could be charged Malaysian Ringgit 50 for each colour page.

6. References References begin on their own page and are listed in alphabetical order by the first author’s last name. Only references cited within the text should be included. All references should be in 12-point font and double-spaced.

NOTE: When formatting your references, please follow the APA reference style (6th Edition). Ensure that the references are strictly in the journal’s prescribed style, failing which your article will not be accepted for peer-review. You may refer to the Publication Manual of the American Psychological Association for further details (http://www.apastyle.org/). 7. General Guidelines Abbreviations: Define alphabetically, other than abbreviations that can be used without definition. Words or phrases that are abbreviated in the introduction and following text should be written out in full the first time that they appear in the text, with each abbreviated form in parenthesis. Include the common name or scientific name, or both, of animal and plant materials.

Acknowledgements: Individuals and entities that have provided essential support such as research grants and fellowships and other sources of funding should be acknowledged. Contributions that do not involve researching (clerical assistance or personal acknowledgements) should not appear in acknowledgements.

Authors’ Affiliation: The primary affiliation for each author should be the institution where the majority of their work was done. If an author has subsequently moved to another institution, the current address may also be stated in the footer.

Co-Authors: The commonly accepted guideline for authorship is that one must have substantially contributed to the development of the paper and share accountability for the results. Researchers should decide who will be an author and what order they will be listed depending upon their order of importance to the study. Other contributions should be cited in the manuscript’s Acknowledgements.

Copyright Permissions: Authors should seek necessary permissions for quotations, artwork, boxes or tables taken from other publications or from other freely available sources on the Internet before submission to Pertanika. Acknowledgement must be given to the original source in the illustration legend, in a table footnote, or at the end of the quotation.

Footnotes: Current addresses of authors if different from heading may be inserted here.

Page Numbering: Every page of the manuscript, including the title page, references, tables, etc. should be numbered.

Spelling: The journal uses American or British spelling and authors may follow the latest edition of the Oxford Advanced Learner’s Dictionary for British spellings.

SUBMISSION OF MANUSCRIPTS Owing to the volume of manuscripts we receive, we must insist that all submissions be made electronically using the online submission system ScholarOne™, a web-based portal by Thomson Reuters. For more information, go to our web page and click “Online Submission”. Submission Checklist 1. MANUSCRIPT: Ensure your MS has followed the Pertanika style particularly the first four pages as explained earlier. The article should be written in a good academic style and provide an accurate and succinct description of the contents ensuring that grammar and spelling errors have been corrected before submission. It should also not exceed the suggested length.

COVER LETTER: All submissions must be accompanied by a cover letter detailing what you are submitting. Papers are accepted for publication in the journal on the understanding that the article is original and the content has not been published either in English or any other language(s) or submitted for publication elsewhere. The letter should also briefly describe the research you are reporting, why it is important, and why you think the readers of the journal would be interested in it. The cover letter must also contain an acknowledgement that all authors have contributed significantly, and that all authors have approved the paper for release and are in agreement with its content.

The cover letter of the paper should contain (i) the title; (ii) the full names of the authors; (iii) the addresses of the institutions at which the work was carried out together with (iv) the full postal and email address, plus telephone numbers and emails of all the authors. The current address of any author, if different from that where the work was carried out, should be supplied in a footnote.

The above must be stated in the cover letter. Submission of your manuscript will not be accepted until a cover letter has been received 2. COPYRIGHT: Authors publishing the Journal will be asked to sign a copyright form. In signing the form, it is assumed that authors have obtained permission to use any copyrighted or previously published material. All authors must read and agree to the conditions outlined in the form, and must sign the form or agree that the corresponding author can sign on their behalf. Articles cannot be published until a signed form (original pen-to-paper signature) has been received.

Please do not submit manuscripts to the editor-in-chief or to any other office directly. Any queries must be directed to the Chief Executive Editor’s office via email [email protected] .

Visit our Journal’s website for more details at http://www.pertanika.upm.edu.my/home.php.

HARDCOPIES OF THE JOURNALS AND OFF PRINTS Under the Journal’s open access initiative, authors can choose to download free material (via PDF link) from any of the journal issues from Pertanika’s website. Under “Browse Journals” you will see a link, “Current Issues” or “Archives”. Here you will get access to all current and back-issues from 1978 onwards.

The corresponding author for all articles will receive one complimentary hardcopy of the journal in which his/her articles is published. In addition, 20 off prints of the full text of their article will also be provided. Additional copies of the journals may be purchased by writing to the Chief Executive Editor.

Characterization of Plant Growth-Promoting Bacteria from Kacip Fatimah 565 (Labisia pumila) under Natural Tropical Forest Yasmi, A., Radziah, O., Hawa, ZE. J., Zainal Abidin, M. A. and Panhwar, Q. A.

Impact of Daily Supplement of Probiotic on the Production Performance 585 of Akar Putra Chickens Hasan, S. A. Jawad, Lokman, I. H., Saad, A. Naji, Zuki, A. B. Z. and Kassim, A. B.

The Effects of Fermented Feed on Broiler Production and Intestinal 597 Morphology Saad A. Naji, I. F. B. Al-Zamili, Hasan, S. A. Jawad and J. K. M. Al-Gharawi

Characteristics and Potential Usage of Dissolved Silica in Rice Cultivation 609 in Sumani Watershed, Sumatra, Indonesia Hiroaki Somura, Darmawan, Kuniaki Sato, Makoto Ueno, Husnain, Aflizar and Tsugiyuki Masunaga NOV. 2016 VOL. 39 (4) NOV.

Pertanika JTAS Vol. 39 (4) Nov. 2016 i 503 459 491 483 393 421 551 541 515

ield of

Y

A., and

Review A A.

Three Oil gy of r

ater Use and

W

A. raits of Rainfed Upland Rice in raits of Rainfed Upland Rice Review A. Samsudin, R. Mohamad and aters of Kudat, Sabah, Malaysia T

etunde O. A

A. Q. Sazili and H. L. Foo Contents W

A.

Akit, richoderma harzianum SNRS3 T Amal, M. N. Vol. 39 (4) Nov. 2016 Nov. 39 (4) Vol. Alawode, Y echniques on the Growth Rate of Marine echniques on the Growth Rate of Marine Acids Supplementation on Layer Hen di T

A. and Apparent Metabolisable Ene ferent Levels of Low Crude Protein Diets with eerasamy Sejian, Madiajagan Bagath and eerasamy Sejian, Madiajagan Bagath and f . C. Loh, H. the Amino

. C. Loh, H. L. Foo, A., Ismail, their Combination in Broiler Chickens Adesola L. and

daus-Nawi and M. Zamri-Saad , Abdulla, T Articles enesa, T Analysis of Panicle and Grain Pertanika Journal of Tropical Agricultural Science Tropical of Journal Pertanika

Articles fect of Cultivation ropical Ecologies of Nigeria [email protected] T Sophia Inbaraj, V Raghavendra Bhatta Nassir N. R. M. Fir Sule, H. Nayan Deep S. Kanwal M. T A. R. Raha D. Khodadadi Dehkor Rahnama, N., Shah, U. K. M., Foo, H. L., Rahman, N. Rahnama, N., Shah, U. K. M., Foo, H. L., A. B. Ariff eword ferent Levels of fects of Feeding Di fect of Higher Density Planting on Irrigation Note Comparing Review of the Ichthyofauna of Malaysian Peat Swamp Forest Review of the Ichthyofauna wo Tel: +603 8947 1622 / 1619 Tel: Pertanika Editorial O ce, Journal Division Editorial O ce, Journal Pertanika (R&I) Chancellor Vice the Deputy O ce of II Tower IDEA Floor, 1st Centre UPM-MTDC Technology Malaysia Putra Universiti 43400 UPM Serdang Selangor Darul Ehsan Malaysia http://www.pertanika.upm.edu.my/ E-mail: Microalgae Species from the Coastal Short Communication A Impact of Heat Stress on Immune Responses of Livestock: Impact of Heat Stress The Ef Sources and T Regular Stability A Review of Fish Immunity: Major Components Performance Ef Dif Production and Characterisation of Cellulase from Solid State Production and Characterisation of Cellulase Fermentation of Rice Straw by Sugarcane Ef For