Volume II PROCEEDINGS · -b- Message from the Chairman of TASP 2016 Dear Participants, On behalf of the Organizing Committee, I would like to welcome you to The 1st International

July 26-29, 2016

Ambassador Hotel, Bangkok, Thailand

“Integrated Approach in Advanced Animal Science and Innovation Technology”

1st International Conference on

Tropical Animal Science and Production (TASP 2016)

T A S P 2 0 1 6

PROCEEDINGS

Jointly organized by

Volume II

ISBN: 978-974-533-710-7

a

Volume II

Proceedings

1st International Conference on

Tropical Animal Science and Production

(TASP 2016)

“Integrated Approach in Advanced Animal Science

and Innovation Technology”

July 26-29, 2016

Ambassador Hotel, Thailand

Jointly organized by:

Suranaree University of Technology

Rajamangala University of Technology Isan

Nakhon Ratchasima Rajabhat University

Thailand Research Fund

a

Welcome Letter from the Rector of SUT

Dear Participants,

I would like to welcome all of you who have come to Bangkok,

Thailand, from all over the world. We are proud and honored to host

the 1st International Conference on Tropical Animal Science and

Production (TASP 2016). This year, Suranaree University of

Technology is celebrating its 26th Anniversary. Over the past 26 years,

we are proud to be consistently ranked as one of Thailand’s leading

universities, with a strong reputation for research excellence and

impact. This Conference is certainly a wonderful addition to our

calendar of 26th anniversary celebration activities.

The theme of this conference “Integrated Approach in Advanced Animal Science and

Innovation Technology” is appropriate in view of the orientation of technological and

institutional changes in such a manner as to ensure the attainment and continued satisfaction of

human needs for present and future generations.

If you take a look at the conference program, you will agree that this conference is going to be

busy and productive. There is a lot to offer for a four-day event. I would like to take this

opportunity to thank the Conference Organizing Committee for their diligent work. I would

also like to thank participants, especially those of you coming from abroad, for joining us and

sharing your valuable experience and ideas. I hope this conference provides you with a forum

to exchange scientific ideas, inspire new research, and new contacts for closer co-operation, so

that we can, together, envision the future of a promising development of the animal science and

production in the Tropic and all over the world.

Last but not least, I sincere thank our co-hosts, namely, Rajamangala University of Technology

Isan (RMUTI), Nakhon Ratchasima Rajabhat University (NRRU) and The Thailand Research

Fund (TRF), and our sponsors for their immense support in making this conference successful.

In closing, I wish you many splendid ideas, new partnerships and the best of success, and I wish

our visitors from abroad will have a very pleasant stay in the beautiful city of Bangkok.

Sincerely,

Prof. Dr. Prasart Suebka

Rector

-b-

Message from the Chairman of TASP 2016

Dear Participants,

On behalf of the Organizing Committee, I would like to welcome you to

The 1st International Conference on Tropical Animal Science and

Production 2016 (TASP 2016) in Bangkok, Thailand, from 26th to 29th

July 2016. The theme of this conference is “Integrated Approach in

Advanced Animal Science and Innovation Technology” to appropriate

in view of the orientation of technological and institutional changes in

such a manner as to ensure the attainment and continued satisfaction of

human needs for present and future generations. The purpose of the

conference is to provide an opportunity for animal scientists, animal agriculture-crop scientists

and those from related disciplines to discuss and debate research issues relating to aquaculture,

animal behavior and welfare, animal genetics and breeding, animal physiology and

reproduction, animal biotechnology, epigenetics, feed science and technology, livestock

farming system, livestock management, meat science, non-ruminant nutrition, nutrigenomics,

pasture, pet, ruminant nutrition, veterinary and others.

The 1st TASP 2016 is organized by 4 institutes; Suranaree University of Technology (SUT),

Nakhon Ratchasima Rajabhat University (NRRU), Rajamangala University of Technology Isan

(RMUTI) and The Thailand Research Fund (TRF).

I sincerely hope that the papers presented in the 1st TASP 2016 would cover all major areas

animal science and production and yield meaningful information leading to further

strengthening of animal science and production in the tropical areas.

I would like to thank all authors for submitting their works and all members of Local and

International Organizing Committee, reviewers, all co-host institutions, sponsors without the

support of whom a conference of this scale would not have possible, and all individuals who

have contributed greatly to the success of this conference.

Sincerely,

Assoc. Prof. Dr. Pramote Paengkoum

Chairman,

Organizing Committee TASP 2016

-c-

Message from the Chairman of Academic Committee

Dear Participants,

On behalf of Academic Committee, it is my great pleasure to welcome

you to the 1st International Conference on Tropical Animal Science

and Production (TASP 2016) in Bangkok, Thailand.

The conference has been designed to provide an opportunity for animal

scientists, researchers and research scholars to exchange and to share

their experiences, and to promote awareness of the ongoing research

achievements. It also provides the premier interdisciplinary forum for

animal scientists to present their latest research results, ideas,

developments, and applications in all areas of Animal Science and Production. The conference

will feature a program of keynote and plenary speakers, invited speaker and oral sessions,

posters, and workshops.

I would like to express my sincere thanks for all keynote speaker, plenary speakers, invited

speakers and all participants for their contribution to make this scientific meeting a great success

and fruitfulness. It is our great honour to have Prof. Dr. Charan Chantalakhana from Thailand,

Prof. Dr. Metha Wanapat from Thailand, Prof. Dr. Liang Chou Hsia from Taiwan, Assoc. Prof.

Dr. Jowaman Khajarern from Thailand, Prof. Dr. Junichi Takahashi from Japan, Assoc. Prof.

Dr. Pongchan Na-Lampang from Thailand, Dr. Pascal Mermillod from France, Dr. John Moran

from Australia, Prof. Dr. Yang Sheng Lin from China, Prof. Dr. Mongkol Techakumphu from

Thailand, Prof. Dr. Thomas J. Schonenwille from the Netherlands and Prof. Dr. Nguyen Van

Thu from Vietnam, who are the esteemed speakers and specialists on their own fields to joint

us in this conference. I also deepy appreciate the scientific committee, reviewer and editorial

boards for their great contribution to make this conference efficacious.

I hope that you will enjoy the conference and your interaction with your colleagues from many

different countries. Also, I wish that this conference will stimulate a creative exchange of ideas

and opportunity for networking, collaboration, sharing of technical information and the building

of trust relationship internationally. Last but not least, I am confident that you will enjoy your

visit in Bangkok and find a harmonious blend of culture, cuisine, arts, and nice sightseeing

along excellent shopping opportunities.

Yours sincerely,

Asst. Prof. Dr. Sutisa Khempaka

Chairman of Academic Committee

-d-

List of TASP 2016 Committee

International Advisory Committee

Assoc. Prof. Dr. Pongchan Na-Lampang (Thailand) (Advisor)

Assoc. Prof. Dr. Pramote Paengkoum (Thailand) (Chairman)

Asst. Prof. Dr. Sutisa Khempaka (Thailand) (Secretary)

Prof. Dr. Ermias Kebreab (USA)

Prof. Dr. Chris McSweeney (Australia)

Assoc. Prof. Dr. Jatuporn Krajaysri (D.V.M.) (Thailand)

Prof. Dr. Juan Boo Liang (Malaysia)

Asst. Prof. Dr. Pakanit Kupittayanont (D.V.M.) (Thailand)

Prof. Dr. Zhongton Yu (USA)

Assoc. Prof. Dr. Songsak Chumpawadee (Thailand)

Asst. Prof. Dr. Thomas Schonewille (The Netherlands)

Asst. Prof. Dr. Chalermpon Yuangklang (Thailand)

Prof. Dr. Yasuhiro Kawamoto (Japan)

Asst. Prof. Dr. Pipat Lounglawan (Thailand)

Prof. Dr. Pietro Celi (USA)

Assoc. Prof. Dr. Surintorn Boonanuntanasarn (Thailand)

Prof. Dr. Nguyen Van Thu (Vietnam)

Assoc. Prof. Dr. Anut Chantiratikul (Thailand)

Prof. Dr. Hsia Liang Chou (Taiwan)

Dr. Samorn Ponchunchoovong (Thailand)

Dr. Viengsakoun Napasirth (Laos)

Dr. Wittawat Molee (Thailand)

Prof. Dr. Long Ruijun (China)

Asst. Prof. Dr. Amornrat Molee (Thailand)

Organizing Committee of TASP 2016

Advisors

Assoc. Prof. Dr. Pongchan Na-Lampang

Assoc. Prof. Dr. Wisitiporn Suksombat

Chairman

Assoc. Prof. Dr. Pramote Paengkoum

Vice Chairmen

Assoc. Prof. Dr. Jatuporn Krajaysri

Asst. Prof. Dr. Chalermpon Yuangklang

Secretariat

Assoc. Prof. Dr. Surintorn Boonanuntanasarn

Scientific Committee

Asst. Prof. Dr. Sutisa Khempaka

Asst. Prof. Dr. Pipat Lounglawan

-e-

TASP 2016. Integrated Approach in Advanced Animal Science and Innovation

Technology: Proceedings. Volume II: 343 pages.

First Edition: 200 books

ISBN: 978-974-533-710-7

Year 2016

LERDSILP PRINTING 336 Suranaree Sub-district, Muang Nakhon Ratchasima District, Nakhon Ratchasima 30000,

Thailand

The individual contributions in this publication and any liabilities

arising from them remain the responsibility of the authors.

The publisher is not responsible for possible damages, which could

be a result of content derived from this publication.

-f-

Contents

Welcome Letter from the Rector of SUT a

Message from the Chairman of TASP 2016 b

Message from the Chairman of Academic Committee c

List of TASP 2016 Committee d

Animal Behavior and Welfare

FO0065 Noncage housing systems and laying hen welfare: keel bone damage

and feather pecking

M. Petek

2

FP0422 Application of geo informatics for wild elephant habitat in Thap Lan

National Park

A. Srisuwan, and W. Panurak

6

Animal Biotechnology

FO0036 The effect of using Solanum dubium and vinegar as alternatives to

rennet enzyme on sensory characteristics of the white cheese (Gibna

bayda)

Ibrahim Elimam, Mohammed Elawad, and Elgazafey Basheer

12

FO0282 Species specific polymerase chain reaction (PCR) assay for

identification of pig (Sus domesticus) skin in “Rambak” crackers

Y. Erwanto, R. Yuliatmo, Sugiyono, A. Rohman, and Sismindari

16

FP0262 Chromosomal analysis to predict early fertility in bovine bulls

M. F. Saad, A. M. Saeed, and H. A. El-naggar

22

Animal Genetics and Breeding

FO0016 Defining economic values of important traits in one hump camel in

desert areas rearing system

M. Vatankhah

27

FO0072 Cow efficiency in crossbreeding systems as defined by kilogram calf

weaned per large stock unit

M. C. Mokolobate, M. M. Scholtz, F. W. C Neser, and A. Theunissen

31

FO0092 Association between STAT1 gene polymorphism and milk production

traits in Holstein, Jersey and Turkish native cattle breeds

O. Cobanoglu, E. K. Gurcan, S. Cankaya, E. Kul, and H. S. Abaci

35

FO0162 Changes in the cow productivity of the indigenous Afrikaner breed and

its environmental impact

M. M. Scholtz, F. J. Jordaan, M. C. Mokolobate, A. Maiwashe, Z.

King, and F. W.C. Neser

39

FO0244 Production performance of red jungle fowl versus native roosters

under confinement system: a comparative study

Francisco F. Buctot JR.

43

-g-

FO0308 Polymorphisms of GH|EcoRV and GHR|Eco72I loci in Indonesian

indigenous chicken

Isyana Khaerunnisa, Melati Ardiyana, Candra Krisdianto, Sri

Darwati, Tike Sartika, and Cece Sumantri

49

Animal Physiology and Reproduction

FO0084 Influences of location and ecological zones on fresh cow milk

composition from milk vendors in Nigeria

D. Zahraddeen, D. Y. SalihuH. O. Okin-Aminu, and A. A. Mohammed

55

FO0129 Characterization and structure of the mammary gland of rabbit related

to milk production and prolactin blood levels recorded during lactating

period

T. T. Amroun, N. Zerrouki-Daoudi, and M. Charlier

59

FO0152 Reliability of placentome diameter for determination of gestation using

transabdominal ultrasonography in sheep and goats

M. A. Waziri, M. M. Bukar, A. Alapa, and A. Y. Ribadu

64

FO0241 Pregnancy and full-term development of embryos derived from natural

mating and AI following estrous synchronization in swamp buffalo

B. Rosadi, T. Sumarsono, and D. Darmawan

68

Aquaculture

FP0179 Potential aquaponic culture in the Mekong Delta

Vu Ngoc Ut, Nguyen Quoc Linh, and Huynh Truong Giang

72

FP0188 Effect of supplementation of attractants to diets on feeding behavior

and growth performance of white shrimp (Litopenaeus vannamei)

O. Jintasataporn, J. Matas Ballarin, A. Hung, and M. L. He

76

FP0395 The effect of replacement of fishmeal by rice wine residual on growth

performance and intestinal morphology of Thai Panga

K. Taveewannaboon, and S. Ponchunchoovong

80

FP0420 Growth of Thai Climbing Perch (Anabas testudineus) and snakeskin

gourami (Trichogaster pectoralis) in brackish water from saline soil

distribution impact area in Nakhon Ratchasima province

N. Noinumsai, S. Wongma, and P. Ngoaknaiwang

85

FP0428 Microencapsulation of Saccharomyces cerevisiae and its effects on

fish intestinal microbiota

K. Ditthab, S. Boonanuntanasarn, and S. Chaijamrus

94

Feed Science and Technology

FO0117 Effect of beneficial microorganisms supplement diets on growth

performance of post weaned pigs in the Mekong Delta, Vietnam

L. T. Men, N .T. D. Trang, H. M. Tri, V. V. Son, and N. D. Hien

99

FO0320 The feed digestibility of quails as affected by administration of

encapsulated probiotic product

U. Kalsum, F. Wadjdi, and L. Rahardjo

104

FO0360 Treatment of high-tannin madre de cacao (Gliricidia sepium) forage

and crude protein utilization in native goats (Capra hircus)

Hershey P. Mondejar, and Lolito C. Bestil

108

-h-

FO0383 Feed intake, digestibility, growth performances and eating behavior of

Thai native beef cattle fed diets differing in energy density using

cassava pulp with rice straw

T. Gunha, K. Kongphitee, and K Sommart

112

FO0386 Ensilage quality, digestibility and enteric methane emission of the

fermented total mixed ration in Thai native beef cattle

K. Kongphitee, and K. Sommart

116

FP0013 Effect of the incorporation of brewers' grains on growth performance

of broilers

F. Arbouche, R. Arbouche, H. S. Arbouche, and Y. Arbouche

121

FP0193 Effect of functional palatability enhancers with milk cheese flavors on

feed intake and growth performance of weanling piglets

H. F. Wang, X. D. Wang, X. X. Dou, L. Liu, J. L.Wang, and M. L. He

125

FP0197 Effect of functional palatability enhancers with colostrum flavors on

feed intake and growth performance of weanling piglets

F. Li, S. Wu, Y. Zhang, and M. L. He

129

FP0359 Effect of catfish by-products on growth performance of growing

Muscovy ducks

T. L. Nguyen, and T. K. D. Nguyen

133

Livestock Farming System / Pasture

FO0103 Potential benefits to dairy industry productivity through breeding more

digestible tropical grasses utilizing contemporary approaches

B. C. Granzin

138

FO0364 The addition of biogas sludge and cattle waste compost towards

soybean productivity through the use of saturated soil culture (SSC)

technology on tidal swamp

S. A. A. Tani, B. P. Purwanto, M. Ghulahmadi, A. M. Fuah, Salundik,

and W. A. Ridwan

142

FO0384 Husbandry systems of keeping native chickens in tropical areas of

Indonesia

Yusuf L. Henuk, and Darma Bakti

147

FP0014 Effects of phenological stage of flood meadows Northeastern Algeria

on their production and nutritional values

R. Arbouche, Y. Arbouche, H. S. Arbouche, and F. Arbouche

151

FP0015 Effects of phenological of Algerian Northeastern forest permanent

grassland on their production and nutritional value

H. S. Arbouche, R. Arbouche, Y. Arbouche, and F. Arbouche

155

FP0234 Elements that define a livestock system intensive silvopastoral in

Mexico

G. Salas-Razo, D. Ramírez-Martínez, M. Perea-Peña, E. Gutiérrez-

Vázquez, R. Garcidueñas-Piña, and J. Espinoza-Villavicencio

160

FP0367 Analysis of technologies used in the livestock system intensive

silvopastoral in Mexico and the technological changes that could

improve efficiency

G. Salas-Razo, D. Ramírez-Martínez, M. Perea-Peña, E. Gutiérrez-

Vázquez, R. Garcidueñas-Piña, and J. Espinoza-Villavicencio

164

-i-

FP0423 Effects of plant spacing on yields of Napier Pak Chong1 during dry

season (Pennisetum purpureum x Pennisetum americanum)

S. Panatung, S. Boonsari, A. Suntornpithug, and C. Charoenrporn

168

Livestock Management

FO0083 Evaporative cooling has potential to improve performance of Zebu

cattle in tropical environments

Olusola A. Olorunnisomo

173

FO0093 Impediments, interventions and impact assessment for dairy farmers in

Pakistan: A roadmap for improved rural livelihoods

Z. Mustafa, and M. N. M. Ibrahim

177

FO0094 Effect of water and balanced diet on milk production of dairy animals

in Pakistan

M. S. Ashraf, Z. Manzoor, N. Khan, T. Abbas, Z. Mustafa, and M. N.

M. Ibrahim

181

FO0101 Small ruminants supplemental feeding regimes in arid agro-pastoral

systems in Pakistan

A. Razzaq, M. Islam, B. Ann Rischkowsky, M. Louhaichi, and M. N. M.

Ibrahim

185

FO0219 A framework on vulnerability analyses of Southeast Asian countries

on goat husbandry to climate change and food security

S. Ocak, B. Gürer, and O. B. Karaca

189

FP0109 Analysis of productivity factors according to the feeding levels of

automatic milking system installed dairy farms

I. S. Nam, Y. S. Yang, B. M. Heo, W. Y. Kim, J. M. Yeo, K. T. Nam, and

K. M. Chang

194

Meat Science

FO0033 The pH of kid meat influence by diet nutrients

M. Karami

198

FP0216 Effect of white sesame seeds and cayenne pepper on quality in reduced

sodium and low-fat precooked pork patties

Jen-Hua Cheng

201

FP0363 Variations in meat chemical composition and quality characteristics of

different Saudi sheep breeds

G. M. Suliman, A. M. El-Waziry, A. N. Al-Owaimer, E.O.S. Hussein,

H. Metwally, and M. S. Al-Harbi

205

Non Ruminant Nutrition

FO0077 Effect of protein and energy levels on performance and egg quality of

Black-boned layers (Ac chicken) at early first production cycle

Truong Van Phuoc, Nguyen Nhut Xuan Dung, and Luu Huu Manh

209

FO0078 Effect of dietary rice bran oil combination with salmon fish oil on

reproduction performance, fatty acid content of yolk in layers

Le Thanh Phuong, Luu Huu Manh, and Nguyen Nhut Xuan Dung

214

-j-

FO0145 Effect of dietary crude protein levels on nutrient intakes, growth rate

and economic return of Guinea fowls in Mekong delta, Vietnam

T. K. D. Nguyen and V. T. Nguyen

219

FO0265 Effect of dietary supplementation with green tea powder on

performance characteristic, meat organoleptic quality and cholesterol

content of broilers

Nguyen Hoang Thinh, Nguyen Thi Vinh, Bui Huu Doan, and Pham

Kim Dang

224

FO0322 Performance of layers fed diets supplemented with methionine

producing probiotic microorganisms

B. N. Suresh, M. C. Shivakumar, Nagaraj Naik, N. Suma, and L.

Manjunatha

229

FO0327 Performance and production of functional duck’s egg fed diets

containing Indigofera zollingeriana leaves meal and Lemuru fish oil

Sumiati, A. Darmawan, and W. Hermana

233

FO0361 Effects of the mixture of essential oils and organic acids on

performance, carcass yield and internal organs in broilers

Sakine YALÇIN, Handan ESER, Suzan YALÇIN, and İlyas

ONBAŞILAR

238

FP0187 Comparison on effects of two palatability enhancers on growth

performance, intestinal villi and relative taste receptor gene expression

of piglets

T. Huang, C. Xia, J. Zuo, Z. Wang, and M. L. He

241

FP0311 Effect of curcumin powder on productive performance and

digestibility of turkeys

Kraisit Vasupen, Chalermpon Yuangklang, Sasiphan Wongsuthavas,

Benya Saenmahayak, and Smerjai Bureenok

245

FP0338 The effects of methionine and its relationship to tryptophan on feed

intake and growth performance of weaning piglets

W. Jantasin, and L. C. Hsia

248

FP0419 Effects of supplementation of Pro L. in water on production

performance in broiler

P. Sutatta, U. Laedthaisong, and S. Paengkoum

253

FP0425 Reproductive performance and fertility response of laying hens as

affected by dietary energy and antioxidant substance supplementation

P. Pasri, S. Khempaka, W. Molee, A. Molee, N. Gerard, and P.

Mermillod

256

Ruminant Nutrition

FO0128 The effect of concentrate containing a mixture microbes on in vitro

nutrients digestibility and fermentation characteristics of King grass

B. Santoso, T. W. Widayati, B. Tj. Hariadi, and H. Abubakar

261

FO0230 Solid state fermentation of wheat straw on large scale for livestock

feed in Pakistan

S. Faisal, A. Muhammad, and A. S. Chudhry

266

-k-

FO0240 Effect of the level of palm oil decanter meal preserved with cinnamon

bark powder in diet on the rumen environment of cross breed Ettawa

goat

M. Afdal, Jul Andayani, and Hajar Setyaji

270

FO0329 Feed additive of betel leaves meal (Piper betle L.) use on ruminants as

one of methane mitigation efforts

A. Sudarman, I. Y. Marcelina, and A. Jayanegara

273

FO0343 Assessing the potential of oil palm frond juice as animal feed supplements

by determining its nutrients, lignocellulosic and sugar contents

N. D. Rusli, K. Mat, C. H. Hasnita, M. Wan Zahari, K. Azhar, M.

Zamri-Saad, and H. A. Hassim

277

FO0412 Body weight gain and feed efficiency of young thin-tailed sheep raised

under intensive feeding at different level of protein

Ari Prima, Nadlirotun Luthfi, Edy Rianto, and Agung Purnomoadi

283

FP0074 Feedlot performance of South African Nguni cattle fed low and high

energy rations

D. A. Linde, M. M. Scholtz, A. Theunissen, and E. van Marle-Köster

287

FP0254 Differences on biological mechanisms related to residual feed intake in

dairy cows

Y. M. Xi, D. Q. Zhao, L. Li, Z. Y. Han, and G. L. Wang

291

FP0348 Oil enrich omega-3 fatty acid supplementation in Brahman crossbred

fattening steers diets on blood parameter

P. Noosen, P. Lounglawan, and R. Mirattanaphrai

296

Veterinary

FO0086 Study of the changes of serum Ab titer against Newcastle disease virus

at various weeks post vaccination in native chickens

N. Ghaleh Golab Behbahan, and F. Tavan

301

FO0099 Epidemiology, comparative diagnostic options and therapeutic studies

against nematodes infecting range sheep and goats in Pakistan

A. Razzaq, M. Islam, B. Ann Rischkowsky, and M. N. M. Ibrahim

306

FO0121 Identification of major health problems in a breeder goat farm in

Sabah, Malaysia

W. Nieccorita, M. Zamri-Saad, K. Azhar, A. B. M. Zuki, A. H. Hasliza,

and A. Punimin

310

FO0156 Evaluation and use of 2 cow-side tests for on-site detection of ketone

bodies in late gestation does

A. A. Syahirah, H. A. Hassim, A. A. Saharee, S. S. Syed-Hussain, A. F.

M. Azmi, M. Ajat, and R. Mansor

313

FO0357 Investigating the seroprevalence of Ornithobacterium rhinotracheale

infection in broiler in Fars province, Iran

Mohammadjavad Mehrabanpour, and Maryam Ranjabar Bushehri

317

-l-

Others

FO0355 Performance, carcass characteristics and meat composition of broilers

in response to early feed restriction

E. E. Onbaşılar, S. Yalçın, E. Torlak, and P. Özdemir

322

FO0356 Effects of rearing method in the first week on the performance and

meat quality of male and female Pekin ducks

E. Erdem, E. E. Onbaşılar, A. Kocakaya, and S. Yalçın

325

FO0362 Red meat consumption of children aged 12-23 months in Turkey

S. Songül Yalçın, Eda Köksal, Gülden Pekcan, and Başak Tezel

327

FP0276 Amylase, protease and xylanase production from Aspergillus niger by

solid state fermentation using cassava pulp and corncob as substrat

Kunthika, V., P. Pianpumepong, and S.Wongsutthawas

331

FP0340 Changes in composition of awassi and saanen colostrum during

postpartum

Oya Berkay Karaca, and Sezen Ocak

335

FP0417 Phenolic content and antioxidant activities in sunflower sprout

V. Tanamool, S. A. Kohnhorst, and A. Chatkon

340

International Conference on Tropical Animal Science and Production (TASP 2016) st1 July 26-29, 2016 Ambassador Hotel, Thailand

[16]

- FO0282 -

Species specific polymerase chain reaction (PCR) assay for identification of

pig (Sus domesticus) skin in “Rambak” crackers

Y. Erwanto*1,3, R. Yuliatmo1, Sugiyono2, A. Rohman3,4, and Sismindari4

1Faculty of Animal Sciences, Gadjah Mada University, Yogyakarta, 55281, Indonesia; 2Faculty

of Veterinary Medicine, Gadjah Mada University, Yogyakarta, 55281 Indonesia; 3Research

Center of Halal Products, Gadjah Mada University, Yogyakarta, 55221, Indonesia; 4Department

of Pharmaceutical Chemistry, Faculty of Pharmacy, Gadjah Mada University, Yogyakarta,

55221, Indonesia

Abstract

“Rambak” is the animal skin crackers product which one of the popular food products derived from

the animal by-products in Indonesia. For the religion reasoning the presence of pig derivatives in

any food products is prohibited for Moslem community. For this reason, the analytical methods

offering accurate and sensitive results are highly needed in order to assure the halal authenticity of

food. The objectives of this research is to developed the pig species specific primer for

identification of specific pig deoxyribonucleic acid (DNA) in “Rambak” cracker using polymerase

chain reaction ( PCR) analytical methods. This research used were four kinds of animal skin

crackers in individual or mixture samples. Skin mixture were prepared and divided into nine

formulas as follows 1) 100% pig; 2) pig 90%: cattle 10%; 3) pig 75%: cattle 25%; 4) pig 50%:

cattle 50%; 5) pig 25%: cattle 75%; 6) pig 10%: cattle 90%; 7) pig 1%: cattle 99%; 8) pig 0.1%:

cattle 99,9% ; and 9) 100% cattle. The same formula also prepared in mixture between pig skin

with buffalo and goat skin. DNA were isolated from the cracker products and applied in PCR using

perimer specific. The optimized PCR assay was subsequently validated for its specificity with

deoxyribonucleic acid (DNA) extracted from cattle, buffalo, goat and pig in individual or mixture

samples. Results showed the primers designed generated specific fragment of 510 bp length for pig

cytochrome b DNA. The specificity of the primers was tested on four animal species including pig,

cattle, buffalo and goat species. Analysis of experimental mixture meat demonstrated that 0.1% of

pig tissues could be detected using specific primer. The specificity of pig-specific PCR provides a

valuable tool for identification of pig skin and to avoid its fraudulent substitution and adulteration.

Keywords: pig species, identification, primer specific, cytochrome b gen

*Corresponding author: [email protected]

Introduction

“Krupuk Rambak” or Rambak cracker, is one of the traditional popular food in Indonesia,

made from thick skin of domestic animal. This food is traditionally hand made with traditional

process. One of the interesting issues in food industry is the authenticity of food products. The

quality assurance of food product can involve the alteration of the true labeling of food ingredients,


[17]

in which the high value of raw materials are substituted by cheaper materials with similar

biochemical properties. Cattle or buffalo hide are expensive and can be altered with cheaper skin

such as pig skin.

The presence of pig derivatives such as pig skin, lard, pork and porcine gelatin in any food

products is a serious matter for moslem community (Mursyidi, 2013) , and also for Judaism and

Hinduism because some forbid their followers to consume any foods containing porcine and its

derivatives (Regenstein et al., 2003) . Therefore, it is necessary to evaluate the presence of these

pig derivatives in any products using some instrumental techniques. Some previous techniques

used are differential scanning calorimetry (Nurrulhidayah et al., 2015; Marikkar et al., 2012) ,

Fourier transform infrared (FTIR) spectroscopy (Rohman et al., 2011), gas chromatography-mass

spectrometry (Nizar et al., 2013) , high pressure liquid chromatography ( Saeed et al., 1989;

Marikkar et al., 2005) , electronic nose (Nurjuliana et al., 2011; Mansor et al., 2011) , and proton

nuclear magnetic resonance (Fang et al., 2013). Some of these methods are too laborious and time

consuming, consequently, an analytical technique offering rapid and reliable must be used. One of

the promising methods suitable for routine analysis is polymerase chain reaction- restriction

fragment lenght polymorphism (PCR-RFLP).

In the previous research we already developed the new technique based on the PCR- RFLP

technique for meat products determination using cytochrome b gen using universal primer and

resulted 360 bp DNA fragment. The determination was continued with restriction fragment length

polymorphism using BseD I restriction enzyme and able to cleave amplicon of cytochrome b gen

into 221 bp and 138 bp (Erwanto et al., 2012). This technique has been applied in detection of pig

derivative contamination in commercial meatball of Indonesia local market and showed positive

pig meat contamination of nine from thirty- nine samples ( Erwanto et al., 2014) . PCR- RFLP

technique has been established to determine of meat species contamination but still time consuming

and need two stage methods, first the amplification of gen target with universal primer and the

second, cleave PCR products using restriction enzyme. Therefore, in this study we developed the

species specific primer for distinguish of pig derivative particle based on cytochrome b gen in food

derivative products from animal skin.

Materials and Methods

Sample preparation and DNA extraction

Individual skin crackers samples of pig, buffalo, cattle and goat were obtained from local

market in Yogyakarta, Indonesia. Samples was prepared in laboratory with separate equipment to

prevent cross contamination. DNA was extracted and purified from skin cracker samples using

Sambrook et al. (1989) methods with slight modification. Approximately 30 mg of skins were

blended using a commercial blender and placed in a 1. 5 mL microcentrifuge tube. A- 500 μL of

Tris- EDTA- NaCl (TEN) buffer, 10 μL Proteinase K and 50 μL 10% Sodium Dodecyl Sulfate

(SDS) were added and mixed by vortexing and was incubated at 42°C in a water bath overnight.

After overnight incubation, 50 μL5 M NaCl, 400 μL phenol and 400 μL chloroform- isoamyl-

alcohol was added to the mixture and then incubated at 37°C for 1 h and centrifuged at 3,000 rpm

for 5 min. The supernatant was taken and put in a new microcentrifuge tube and mixed by vortexing

for 30 s, then 50 μL 5 M NaCl and 1 mL absolute ethanol were added, mixed vigorously and

incubated at –20°C for 1 h. The DNA was precipitated from the mixture after centrifuging at 8,000

rpm for 5 min, 4°C and the supernatant discarded. The 70% ethanol was gently poured off then

resuspended DNA for a second precipitation. The solution was then placed at room temperature

overnight. The resulting pellet was added with 50 mL Tris-EDTA (TE) buffer and stored at –20°C


[18]

for further analysis. Skin cracker samples were prepared by mixing of pig in buffalo, pig in cattle,

and pig in goat at a final concentration of pig at 0; 0.1; 1; 10; 25; 50; 75; 90 and 100%.

Design of pig specific primer

The nucleotide sequences of mitochondrial Cyt b genes from sus scrofa (pig) stored in

Genbank (www. ncbi. nig. gov) were taken and aligned using Primer3 website

(www.primer3.ut.ee/ ) . The set of primers used for amplification consisted of Cyt b-FW and

Cyt b-REV oligonucleotides as follows:

Cyt b FW 5’-CCC AGC CCC CTC AAA CAT CTC A-3’,

Cyt b REV 5’-ATG TAC GGC TGC GAG GGC GGT-3’.

Amplification of the Cyt b gene was performed in a final volume of 25 μL containing 100 ng

of extracted DNA, 20µL DreamTaq Green PCR master Mix ( Thermo Fisher Scientific) and 20

µmol of each primer. Amplification was performed with a thermal cycler according to the

following PCR step-cycle program: Pre-denaturation at 94°C for 2 min to completely denature the

DNA template, followed by 40 cycles of denaturation at 95°C for 36 s, annealing at 53°C for 73 s.

and extension at 72°C for 84 s. Final extension at 72°C for 3 min followed the final cycle for

complete synthesis of elongated DNA molecules. Six microliters of PCR products were mixed with

1 μL DNA Loading Dye (Thermo Fisher Scientific), then were electrophoresed at constant voltage

( 100 V) on 2% agarose gel ( TopVision, Thermo Fisher Scientific) for about an hour in 1×TBE

buffer, pH 8. 0 and stained by ethidium bromide. A- 100 bp DNA ladder ( GeneRuller, Thermo

Fisher Scientific) was used as size reference.

Results and Discussion

Total genom DNA form the various animal skin species was isolated using Sambrook et al.

( 1989) method with slight modification. During processing proteins are denaturalised due to the

time and temperatures of process and DNA should be employed for species identification

purposes. Therefore the susceptibility of the determination of species in food products depend on

the susceptibility in DNA extraction, without the extracted DNA the measurement of the detection

could not be continued.

DNA genom of four species could be successfully isolated (data not shown) although

there were some smear in goat skin sample. The quality of DNA and the level content of pig in

the crackers mix samples is important data for verifying the sensitivity of the method.

To identify pig skin in a large number of both processed and unprocessed skin crackers, we

carried out specific pig PCR amplification. With this fragment, pig has been detected in mixtures

treated with cattle, buffalo and goat skin crackers. Some commercial products were analyzed which

normally have cattle, buffalo or pig in their crackers product including in fresh skin crackers or

various processed skin crackers. Using the specific PCR amplification, we detected pig in various

concentration. Pig was detected until 0.1% contamination in mixture with cattle (Figure 1), buffalo

(Figure 2) or goat (Figure 3). In our some previous research showed the PCR-RFLP able to detect

pig DNA in heated meat mixtures at levels less than 1% based on fragment of the cytochrome b

DNA (Erwanto et al. , 2012) and Matsunaga et al. (1999) detected 250 pg of beef DNA, in DNA

mixtures. In raw pork and beef mixtures, Calvo et al. (2001) detected up to 0.01% beef in pork.

The choice of the technique to be applied is based on the results of sequence alignments.

Specific PCR seems to be best as a ‘ routine’ test, because it is easy, rapid and allows the

discrimination of several species at the same time (multiplex PCR), as well as the analysis of mixed

http://www.ncbi.nig.gov/

http://www.primer3.ut.ee/


[19]

matrices. It is considered to be a robust method, mainly in comparison with other methods based

on the analysis of single nucleotide polymorphisms (Bottero and Dalmaso, 2011).

Figure 1. PCR products of cyt b specific gene from different skin product separated by 2% high-

resolution agarose gel electrophoresis. M: Marker; 1: 100% pig skin; 2: pig 90%: buffalo 10%; 3:

pig 75%: buffalo 25%; 4: pig 50%: 50% buffalo; 5: pig 25%: 75% skin; 6: pig 10%: buffalo 90%;

7: pig 1%: 99% buffalo; and 8: pig 0.1% buffalo 99.9%; 9: 100% buffalo skin.


resolution agarose gel electrophoresis. M: Marker; 1: 100% pig skin; 2: pig 90%: cattle 10%; 3:

pig 75%: cattle 25%; 4: pig 50%: 50% cattle; 5: pig 25%: 75% cattle; 6: pig 10%: cattle 90%; 7:

pig 1%: 99% cattle; and 8: pig 0.1% cattle 99.9%; 9: 100% cattle skin.


resolution agarose gel electrophoresis. M: Marker; 1: 100% pig skin; 2: pig 90%: goat 10%; 3: pig


[20]

75%: goat 25%; 4: pig 50%: 50% goat; 5: pig 25%: 75% goat; 6: pig 10%: goat skin 90%; 7: pig

skin 1%: 99% goat; and 8: pig skin 0.1% goat skin 99.9; 9: 100% cattle skin.

Conclusion

Specific PCR amplification of this fragment is a powerful technique for the identification

of pig contamination, due to its simplicity, specificity, and sensitivity (with 35 amplification cycles

we can detect 0. 1 % pig skin) . However, further research would be needed in order to develop a

quantitative method since this conventional PCR analysis is only qualitative.

Acknowledgment

This research was funding by project grant from the directorate of higher education,

Ministry of Higher Education, Research and Technology, with contract No. 159/LPPM/2015.

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